Fungal pathogens of food and fibre crops - CBS

Transcription

The CBS taxonomy series “Studies in Mycology” is issued as individual booklets. Regular subscribers receive each issue automatically. Prices of backvolumes are specified below.
For more information and ordering of other CBS books and publications see www.cbs.knaw.nl and www.studiesinmycology.org.
Studies in Mycology 79 (September 2014)
ISSN 0166-0616
Fungal pathogens of food and fibre crops
Pedro W. Crous and Johannes Z. Groenewald, editors
79 Crous PW, Groenewald JZ (eds) (2014). Fungal pathogens of food and fibre crops. 288 pp., € 65.00
78 Samson RA, Visagie CM, Houbraken J (eds) (2014). Species diversity in Aspergillus, Penicillium and Talaromyces. 451 pp., € 75.00
77 Stadler M, Læssøe T, Fournier F, Decock C, Schmieschek B, Tichy H-V, Peršoh D (2014). A polyphasic taxonomy of Daldinia (Xylariaceae). 143 pp.,
€ 60.00
76 Phillips AJL, Slippers B, Groenewald JZ, Crous PW (eds) (2013). Plant pathogenic and endophytic Botryosphaeriales known from culture. 167 pp.,
€ 65.00
75 Crous PW, Verkley GJM, Groenewald JZ (eds) (2013). Phytopathogenic Dothideomycetes. 406 pp., € 70.00
74 Dijksterhuis J, Wösten H (eds) (2013). Development of Aspergillus niger. 85 pp., €40.00
73 Damm U, Cannon PF, Crous PW (eds) (2012). Colletotrichum: complex species or species complexes? 217 pp., € 65.00
72 Bensch K, Braun U, Groenewald JZ, Crous PW (2012). The genus Cladosporium. 401 pp., € 70.00
71 Hirooka Y, Rossmann AY, Samuels GJ, Lechat C, Chaverri P (2012). A monograph of Allantonectria, Nectria, and Pleonectria (Nectriaceae, Hypocreales,
Ascomycota) and their pycnidial, sporodochial, and synnematous anamorphs. 210 pp., € 65.00
70 Samson RA, Houbraken J (eds) (2011). Phylogenetic and taxonomic studies on the genera Penicillium and Talaromyces. 183 pp., € 60.00
69 Samson RA, Varga J, Frisvad JC (2011). Taxonomic studies on the genus Aspergillus. 97 pp., € 40.00
68 Rossman AY, Seifert KA (eds) (2011). Phylogenetic revision of taxonomic concepts in the Hypocreales and other Ascomycota - A tribute to Gary J.
Samuels. 256 pp., € 65.00
67 Bensch K, Groenewald JZ, Dijksterhuis J, Starink-Willemse M, Andersen B, Summerell BA, Shin H-D, Dugan FM, Schroers H-J, Braun U, Crous PW
(2010). Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). 96 pp., € 40.00
66 Lombard L, Crous PW, Wingfield BD, Wingfield MJ (2010). Systematics of Calonectria: a genus of root, shoot and foliar pathogens. 71 pp., € 40.00
65 Aveskamp M, Gruyter H de, Woudenberg J, Verkley G, Crous PW (2010). Highlights of the Didymellaceae: A polyphasic approach to characterise
Phoma and related pleosporalean genera. 64 pp., € 40.00
64 Schoch CL, Spatafora JW, Lumbsch HT, Huhndorf SM, Hyde KD, Groenewald JZ, Crous PW (2009). A phylogenetic re-evaluation of Dothideomycetes.
220 pp., € 65.00
63 Jaklitsch WA (2009). European species of Hypocrea. Part I. The green-spored species. 93 pp., € 40.00
62 Sogonov MV, Castlebury LA, Rossman AY, Mejía LC, White JF (2008). Leaf-inhabiting genera of the Gnomoniaceae, Diaporthales. 79 pp., € 40.00
61 Hoog GS de, Grube M (eds) (2008). Black fungal extremes. 198 pp., € 60.00
60 Chaverri P, Liu M, Hodge KT (2008). Neotropical Hypocrella (anamorph Aschersonia), Moelleriella, and Samuelsia. 68 pp., € 40.00
59 Samson RA, Varga J (eds) (2007). Aspergillus systematics in the genomic era. 206 pp., € 65.00
58 Crous PW, Braun U, Schubert K, Groenewald JZ (eds) (2007). The genus Cladosporium and similar dematiaceous hyphomycetes. 253 pp., € 65.00
57 Sung G-H, Hywel-Jones NL, Sung J-M, Luangsa-ard JJ, Shrestha B, Spatafora JW (2007). Phylogenetic classification of Cordyceps and the
clavicipitaceous fungi. 63 pp., € 40.00
56 Gams W (ed.) (2006). Hypocrea and Trichoderma studies marking the 90th birthday of Joan M. Dingley. 179 pp., € 60.00
55 Crous PW, Wingfield MJ, Slippers B, Rong IH, Samson RA (2006). 100 Years of Fungal Biodiversity in southern Africa. 305 pp., € 65.00
54 Mostert L, Groenewald JZ, Summerbell RC, Gams W, Crous PW (2006). Taxonomy and Pathology of Togninia (Diaporthales) and its Phaeoacremonium
anamorphs. 115 pp., € 55.00
53 Summerbell RC, Currah RS, Sigler L (2005). The Missing Lineages. Phylogeny and ecology of endophytic and other enigmatic root-associated fungi.
252 pp., € 65.00
52 Adams GC, Wingfield MJ, Common R, Roux J (2005). Phylogenetic relationships and morphology of Cytospora species and related teleomorphs
(Ascomycota, Diaporthales, Valsaceae) from Eucalyptus. 147 pp., € 55.00
51 Hoog GS de (ed.) (2005). Fungi of the Antarctic, Evolution under extreme conditions. 82 pp., € 40.00
50 Crous PW, Samson RA, Gams W, Summerbell RC, Boekhout T, Hoog GS de, Stalpers JA (eds) (2004). CBS Centenary: 100 Years of Fungal Biodiversity
and Ecology (Two parts). 580 pp., € 105.00
49 Samson RA, Frisvad JC (2004). Penicillium subgenus Penicillium: new taxonomic schemes, mycotoxins and other extrolites. 253 pp., € 55.00
48 Chaverri P, Samuels GJ (2003). Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. 113 pp., € 55.00
47 Guarro J, Summerbell RC, Samson RA (2002). Onygenales: the dermatophytes, dimorphics and keratin degraders in their revolutionary context. 220
pp., € 55.00
46 Schroers HJ (2001). A monograph of Bionectria (Ascomycota, Hypocreales, Bionectriaceae) and its Clonostachys anamorphs. 214 pp., € 55.00
45 Seifert KA, Gams W, Crous PW, Samuels GJ (eds) (2000). Molecules, morphology and classification: Towards monophyletic genera in the Ascomycetes.
200 pp., € 55.00
44 Verkley GJM (1999). A monograph of the genus Pezicula and its anamorphs. 180 pp., € 55.00
43 Hoog GS de (ed.) (1999). Ecology and evolution of black yeasts and their relatives. 208 pp., € 55.00
42 Rossman AY, Samuels GJ, Rogerson CT, Lowen R (1999). Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes). 248
pp., € 55.00
41 Samuels GJ, Petrini O, Kuhls K, Lieckfeldt E, Kubicek CP (1998).The Hypocrea schweinitzii complex and Trichoderma sect. Longibrachiatum. 54 pp.,
€ 35.00
Volume 79, September 2014
Studies in Mycology (ISSN 0166-0616)
H O S T E D BY
CBS-KNAW Fungal Biodiversity Centre,
Utrecht, The Netherlands
An institute of the Royal Netherlands Academy of Arts and Sciences
For a complete list of the Studies in Mycology see www.cbs.knaw.nl.
ELSEVIER
SIMYCO_v79_iC_COVER.indd 1
21-11-2014 11:20:38
Executive Editor
Prof. Dr. Dr. h.c. Robert A. Samson, CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail:
r.samson@cbs.knaw.nl
Managing Editor
Prof. Dr. Pedro W. Crous, CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: p.crous@
cbs.knaw.nl
Assisting Editor
Manon van den Hoeven-Verweij, CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail:
m.verweij@cbs.knaw.nl
Scientific Editors
Prof. Dr. Dominik Begerow, Lehrstuhl für Evolution und Biodiversität
der Pflanzen, Ruhr-Universität Bochum, Universitätsstr. 150,
Gebäude ND 44780, Bochum, Germany. E-mail: dominik.begerow@
rub.de
Dr. Amy Y. Rossman, Rm 246, Bldg 010A Barc-West, Systematic
Botany & Mycology Laboratory, Beltsville, MD, U.S.A. 20705. E-mail:
amy@nt.ars-grin.gov
Prof. Dr. Uwe Braun, Martin-Luther-Universität, Institut für Biologie,
Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21,
D-06099 Halle, Germany. E-mail: uwe.braun@botanik.uni-halle.de
Dr. Keith A. Seifert, Research Scientist / Biodiversity (Mycology
and Botany), Agriculture & Agri-Food Canada, KW Neatby Bldg, 960
Carling Avenue, Ottawa, ON, Canada K1A OC6. E-mail: seifertk@
agr.gc.ca
Dr. Paul Cannon, CABI and Royal Botanic Gardens, Kew, Jodrell
Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9
3AB, U.K. E-mail: p.cannon@kew.org
Prof. Dr. Hyeon-Dong Shin, Division of Environmental Science &
Ecological Engineering, Korea University, Seoul 136-701, Korea.
E-mail: hdshin@korea.ac.kr
Prof. Dr. Lori Carris, Associate Professor, Department of Plant
Pathology, Washington State University, Pullman, WA 99164-6340,
U.S.A. E-mail: carris@mail.wsu.edu
Dr. Roger Shivas, Biosecurity Queensland, Department of
Agriculture, Fisheries and Forestry, GPO Box 267, Brisbane, Qld
4001, Australia. E-mail: Roger.Shivas@daff.qld.gov.au
Prof. Dr. David M. Geiser, Department of Plant Pathology, 121
Buckhout Laboratory, Pennsylvania State University, University Park,
PA, U.S.A. 16802. E-mail: dgeiser@psu.edu
Prof. Dr. Marc Stadler, Head of Department Microbial Drugs,
Helmholtz-Centre for Infection Research, Inhoffenstrasse 7, 38124
Braunschweig, Germany. E-mail: marc.stadler@helmholtz-hzi.de
Dr. Johannes Z. Groenewald, CBS-KNAW Fungal Biodiversity
Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail:
e.groenewald@cbs.knaw.nl
Prof. Dr. Jeffrey K. Stone, Department of Botany & Plant Pathology,
Cordley 2082, Oregon State University, Corvallis, OR, U.S.A. 973312902. E-mail: stonej@bcc.orst.edu
Prof. Dr. David S. Hibbett, Department of Biology, Clark University,
950 Main Street, Worcester, Massachusetts, 01610-1477, U.S.A.
E-mail: dhibbett@clarku.edu
Dr. Richard C. Summerbell, 27 Hillcrest Park, Toronto, Ont. M4X
1E8, Canada. E-mail: summerbell@aol.com
Dr. Lorelei L. Norvell, Pacific Northwest Mycology Service, 6720 NW
Skyline Blvd, Portland, OR, U.S.A. 97229-1309. E-mail: llnorvell@
pnw-ms.com
Prof. Dr. Alan J.L. Phillips, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Quinta de Torre, 2829-516 Caparica,
Portugal. E-mail: alp@mail.fct.unl.pt
Prof. Dr. Brett Summerell, Royal Botanic Gardens and Domain
Trust, Mrs. Macquaries Road, Sydney, NSW 2000, Australia. E-mail:
brett.summerell@rbgsyd.nsw.gov.au
Prof. Dr. Ulf Thrane, Department of Systems Biology, Center
for Microbial Biotechnology, Technical University of Denmark,
Søltofts Plads 221, DK-2800 Kgs. Lyngby, Denmark. E-mail: ut@
bio.dtu.dk
ISBN/EAN: 978-94-91751-01-1
Cover: Left column, Early blight of tomato, caused by Alternaria solani. Right column, resin exudation from the stem
of Acacia mearnsii in South Africa caused by Ceratocystis albifundus. Central top row, conidiophores and conidia of
Bipolaris heveae; ascomata of Ceratocystis fimbriata on wood with masses of ascospores emerging from their necks.
Central bottom row, conidiophores and conidia of Xenopyricularia zizaniicola; conidiophores and conidia of Alternaria
solani.
SIMYCO_v79_iC_COVER.indd 2
21-11-2014 11:20:28
STUDIES IN MYCOLOGY
Volume 79 / September 2014
edited by
Pedro W. Crous
and
Johannes Z. Groenewald
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
CBS-KNAW Fungal Biodiversity Centre,
Utrecht, The Netherlands
An institute of the Royal Netherlands Academy of Arts and Sciences
AIMS AND SCOPE: STUDIES IN MYCOLOGY
The CBS Fungal Biodiversity Centre – an institute of the Royal Netherlands Academy of Arts and Sciences (KNAW) and situated
in Utrecht, The Netherlands – maintains a world-renowned collection of living filamentous fungi, yeasts and bacteria. The institute’s
research programs principally focus on the taxonomy and evolution of fungi as well as on functional aspects of fungal biology and
ecology, incorporating molecular and genomics approaches. The CBS employs circa 70 personnel, among whom circa 24 scientists.
“Studies in Mycology” is an international journal which publishes systematic monographs of filamentous fungi and yeasts, and special
topical issues related to all fields of mycology, biotechnology, ecology, molecular biology, pathology and systematics. The first issue
was published in 1972.
“Studies in Mycology” is an open access journal that is freely available on the internet, but is also issued as individual booklets,
each priced according to size. Regular subscribers receive each hardcopy issue automatically upon publication. Become a regular
subscriber to the hardcopy version by sending your request to info@cbs.knaw.nl. Single hardcopies can be bought through the CBS
Web shop. A 20% reduction on the list price applies only when 10 or more copies of a specific publication are bought at the same
time. The publication fee is € 1000 per paper.
Authors who intend to submit monographs or topical issues should contact the Executive Editor in advance. Authors are obliged to
meet the requirements as set out in our Instructions for Authors.
Notice
No responsibility is assumed by Studies in Mycology nor Elsevier for any injury and/or damage to persons or property as a matter of
product liability, negligence, or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in
the material herein.
Although all advertising material is expected to conform to ethical standards, inclusion in this publication does not constitute a
guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer.
Copyright © 2014, CBS-KNAW Fungal Biodiversity Centre. Production and hosting by ELSEVIER B.V. Peer review under
responsibility of CBS-KNAW Fungal Biodiversity Centre.
available online at www.studiesinmycology.org
STUDIES
IN
MYCOLOGY 79: iii.
Studies in Mycology
EDITORIAL
Emerging and established diseases caused by fungi pose a
serious threat to biodiversity as well as global food and fibre
supply. Although there are several major groups of pathogens,
the present volume focuses exclusively on plant pathogenic
fungi. Presently more than 800 million people do not have
adequate food, and at least 10 % of global food production is lost
due to plant disease (Christou & Twyman 2004). Likewise, fungi
also play a major role in tree disease, leading to significant loss
in timber and pulp production (Wingfield et al. 2001).
As many different genera of phytopathogenic fungi play a role
in plant disease, its impossible to treat all in a single issue of
Studies in Mycology, and hence only a few can be dealt with
here. One of these genera is Bipolaris (= Cochliobolus;
Pleosporaceae), which has species that are commonly associated with leaf spots, leaf blights, root and foot rots, and other
disease symptoms of high value field crops in the Poaceae,
including rice, maize, wheat and sorghum. Their global distribution may result from the transfer of agricultural commodities
including plants and seeds across geographical borders. Lack of
ex-type or authenticated sequences in public databases is a
drawback in the accurate molecular identification and detection
of Bipolaris species, since the names are the key to accessing
accumulated knowledge (see Manamgoda et al. 2014)
Species of Colletotrichum (= Glomerella; Glomerellaceae) are
commonly associated with anthracnose diseases of crops in
tropical and subtropical regions. This species-rich genus has a
wide host range, and taxa on important crops such as clover,
alfalfa, cowpea and lentil, are difficult if not impossible to identify
based solely on morphological characters (see Damm et al.
2014). Pestalotiopsis species (= Pestalosphaeria; Amphisphaeriaceae) are commonly isolated as endophytes, but also
include phytopathogens that cause a variety of post-harvest
diseases, fruit rots and leaf spots, as well as other emerging
diseases (see Maharachchikumbura et al. 2014). Similar to
Pestalotiopsis, the genus Alternaria (= Lewia; Pleosporaceae) is
also omnipresent, causing disease on a range of agriculturally
important crops. The revision of the species of Alternaria associated with diseases of potato, tomato, sweet potato and onion, is
therefore of huge economic importance (see Woudenberg et al.
2014).
Rice is currently the world’s most widely consumed staple
food. Rice blast (Pyricularia oryzae) results in losses of 10–30 %
of this crop each year (Talbot 2003). Several Pyricularia pathogens (magnaporthe-like sexual morphs; Pyriculariaceae), and
many newly introduced pyricularia-like genera also occur on
other cereals, further affecting global yield of field crops. Species
of Nakataea (= Magnaporthe) and Gaeumannomyces (harpophora-like asexual morphs), however, cluster in the Magnaporthaceae (see Klaubauf et al. 2014).
The genus Ceratocystis sensu lato (Ceratocystidaceae) includes serious plant pathogens, significant insect symbionts and
agents of timber degradation that result in substantial economic
losses. In recent years it has become very obvious that this
genus incorporates a wide diversity of very different fungi.
Results obtained by De Beer et al. (2014) made it possible to
distinguish seven major groups for which generic names have
been chosen and descriptions are either provided or emended.
This major revision of the generic boundaries in Ceratocystis s.
lat. will provide a stable platform to facilitate future research on
this important group of fungi, including distantly related species
aggregated under this name.
Given the breadth of scope of the current volume of Studies in
Mycology, covering pathogens in a range of genera, including
Alternaria, Bipolaris, Ceratocystis, Colletotrichum, Pestalotiopsis
and Pyricularia, many which have members that are known to
include endophytic phases in their life cycles, it is clear that they
represent a major challenge to international trade in agricultural
and forestry produce. Although it remains difficult, if not impossible, to combat or contain that which you cannot see or
recognise, one of our aims was to define DNA barcodes that
would reliably distinguish the taxa treated. Armed with this
knowledge, it is our hope that agricultural and forest pathologists
would be better equipped to recognise these pathogens,
enabling them to come up with better disease control strategies,
as well as more efficient mechanisms for pathogen detection.
The Editors
September 2014
REFERENCES
Christou P, Twyman RM (2004). The potential of genetically enhanced plants to
address food insecurity. Nutrition Research Reviews 17: 23–42.
Talbot NJ (2003). On the trail of a cereal killer: exploring the biology of Magnaporthe grisea. Annual Review of Microbiology 57: 177–202.
Wingfield MJ, Slippers B, Roux J, et al. (2001). Worldwide movement of exotic
forest fungi, especially in the tropics and the Southern Hemisphere.
BioScience 51: 134–140.
Published online 25 November 2014
Hard copy: September 2014
Peer review under responsibility of CBS-KNAW Fungal Biodiversity Centre.
iii
CONTENTS
J.H.C. Woudenberg, M. Truter, J.Z. Groenewald, and P.W. Crous. Large-spored Alternaria pathogens in section Porri disentangled ............. 1
U. Damm, R.J. O’Connell, J.Z. Groenewald, and P.W. Crous. The Colletotrichum destructivum species complex – hemibiotrophic
pathogens of forage and field crops ................................................................................................................................................................. 49
S. Klaubauf, D. Tharreau, E. Fournier, J.Z. Groenewald, P.W. Crous, R.P. de Vries, and M.-H. Lebrun. Resolving the polyphyletic
nature of Pyricularia (Pyriculariaceae).............................................................................................................................................................. 85
S.S.N. Maharachchikumbura, K.D. Hyde, J.Z. Groenewald, J. Xu, and P.W. Crous. Pestalotiopsis revisited ............................................... 121
Z.W. de Beer, T.A. Duong, I. Barnes, B.D. Wingfield, and M.J. Wingfield. Redefining Ceratocystis and allied genera .................................. 187
D.S. Manamgoda, A.Y. Rossman, L.A. Castlebury, P.W. Crous, H. Madrid, E. Chukeatirote, and K.D. Hyde. The genus Bipolaris .............. 221
STUDIES
IN
MYCOLOGY 79: 1–47.
Large-spored Alternaria pathogens in section Porri disentangled
J.H.C. Woudenberg1,2*, M. Truter3, J.Z. Groenewald1, and P.W. Crous1,2,4
1
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, Netherlands; 2WUR, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB
Wageningen, Netherlands; 3ARC-Plant Protection Research Institute, P. Bag X134, Queenswood 0121, South Africa; 4Forestry and Agricultural Biotechnology
Institute (FABI), University of Pretoria, Pretoria 0002, South Africa
*Correspondence: J.H.C. Woudenberg, j.woudenberg@cbs.knaw.nl
Abstract: The omnipresent fungal genus Alternaria was recently divided into 24 sections based on molecular and morphological data. Alternaria sect. Porri is the largest
section, containing almost all Alternaria species with medium to large conidia and long beaks, some of which are important plant pathogens (e.g. Alternaria porri, A.
solani and A. tomatophila). We constructed a multi-gene phylogeny on parts of the ITS, GAPDH, RPB2, TEF1 and Alt a 1 gene regions, which, supplemented with
morphological and cultural studies, forms the basis for species recognition in sect. Porri. Our data reveal 63 species, of which 10 are newly described in sect. Porri, and
27 species names are synonymised. The three known Alternaria pathogens causing early blight on tomato all cluster in one clade, and are synonymised under the older
name, A. linariae. Alternaria protenta, a species formerly only known as pathogen on Helianthus annuus, is also reported to cause early blight of potato, together with
A. solani and A. grandis. Two clades with isolates causing purple blotch of onion are confirmed as A. allii and A. porri, but the two species cannot adequately be
distinguished based on the number of beaks and branches as suggested previously. This is also found among the pathogens of Passifloraceae, which are reduced from
four to three species. In addition to the known pathogen of sweet potato, A. bataticola, three more species are delineated of which two are newly described. A new
Alternaria section is also described, comprising two large-spored Alternaria species with concatenate conidia.
Key words: Alternaria, Early blight of potato, Early blight of tomato, Leaf and stem blight of sweet potato, Multi-gene phylogeny, Purple blotch of onion.
Taxonomic novelties: New species: Alternaria alternariacida Woudenb. & Crous, A. carthamicola Woudenb. & Crous, A. catananches Woudenb. & Crous, A. citrullicola
Woudenb. & Crous, A. conidiophora Woudenb. & Crous, A. deserticola Woudenb. & Crous, A. ipomoeae M. Truter, Woudenb. & Crous, A. neoipomoeae M. Truter,
Woudenb. & Crous, A. paralinicola Woudenb. & Crous, A. sennae Woudenb. & Crous; New section in Alternaria: sect. Euphorbiicola Woudenb. &
Crous; Typifications (basionyms): Epitypifications: Alternaria bataticola W. Yamam., Cercospora crassa Sacc., Macrosporium porri Ellis, M. ricini Yoshii,
Sporidesmium scorzonerae Aderh.; Neotypification: Sporidesmium exitiosum var. dauci J.G. Kühn.
Published online 16 October 2014; http://dx.doi.org/10.1016/j.simyco.2014.07.003. Hard copy: September 2014.
Studies in Mycology
INTRODUCTION
Alternaria is an important fungal genus with a worldwide distribution. This hyphomycetous ascomycete with phaeodictyospores includes saprophytic, endophytic and pathogenic species,
which can be plant pathogens, post-harvest pathogens or human
pathogens (Thomma 2003). The genus Alternaria was recently
divided into 24 sections (Woudenberg et al. 2013) based on
molecular and morphological data, which followed the recent
initiative to divide Alternaria into sections (Lawrence et al. 2013).
Alternaria sect. Porri is the largest section, containing almost all
Alternaria species with medium to large conidia and long beaks.
Among them are some important plant pathogens, such as
Alternaria bataticola, A. porri, A. solani and A. tomatophila.
Alternaria bataticola causes leaf petiole and stem blight of sweet
potato in tropical and sub-tropical regions. The disease is most
severe in East and Central Africa, with yield losses of over 70 %
reported (Osiru et al. 2007). Alternaria porri causes purple blotch
of onion, a very destructive disease of onions worldwide. The
disease causes a significant reduction in seed and bulb yield,
with seed losses of up to 100 % (Abo-Elyousr et al. 2014).
Alternaria solani is the causative agent of early blight of potato.
This very common disease, which can be found in most potato-
growing countries, can cause considerable defoliation. The disease typically reduces yields by ~20 %, but yield reductions of up
to 80 % have been reported (Horsfield et al. 2010). Alternaria
tomatophila is known for causing early blight of tomato, attacking
the leaves, stems and fruit. This airborne pathogen has spread
worldwide, mainly affecting field crops. When left untreated the
damage can result in plant defoliation in excess of 60 % (Zitter &
Drennan 2005).
The identification of these species has been problematic for
many years, with every large-spored Alternaria found on
Solanaceae commonly being identified as A. solani. This
assumption changed with the treatment of Alternaria species on
Solanaceae, in which Simmons (2000) distinguished 22 Alternaria
and Nimbya species on solanaceous hosts on the basis of
morphology. On potato, Simmons described the large-spored,
long-beaked species A. grandis and A. solani, while on tomato
he described A. tomatophila, A. cretica and A. subcylindrica. The
distinction between potato and tomato pathogens was supported
by subsequent molecular studies and chemotaxonomy (Andersen
et al. 2008, Rodrigues et al. 2010, Brun et al. 2013, Gannibal et al.
2014).
The taxonomy of Alternaria species on Allium is also confused.
Macrosporium porri was first described as pathogen of Allium
Copyright © 2014, CBS-KNAW Fungal Biodiversity Centre. Production and hosting by ELSEVIER B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/3.0/).
1
WOUDENBERG
ET AL.
(Cooke & Ellis 1879), followed by Alternaria allii (Nolla 1927). Both
species were later synonymised (Angell 1929) and the name
changed to Alternaria porri (Cifferi 1930). The name A. allii was
resurrected by Simmons in his identification manual (2007) where
he described five large-spored, long-beaked species from Allium,
which he could distinguish based on morphology. Large-spored
Alternaria from sweet potato were mostly identified as
A. bataticola, even if the isolates from some studies (Osiru et al.
2008, Narayanin et al. 2010) showed morphological differences
compared with the description of Simmons (2007).
In the present study we aim to use a molecular approach to
delineate the medium- to large-spored Alternaria species with
long beaks in sect. Porri. A multi-locus analysis based on five
partial gene regions, the internal transcribed spacer regions 1
and 2 and intervening 5.8S nrDNA (ITS), glyceraldehyde-3phosphate dehydrogenase (GAPDH), RNA polymerase second
largest subunit (RPB2), translation elongation factor 1-alpha
(TEF1) and the Alternaria major allergen gene (Alt a 1), was
performed. All available ex-type and representative isolates of
medium to large-spored, long-beaked species described in
Simmons (2007) were included in this study. The present multilocus analysis supplemented with morphological and cultural
data forms the basis for species recognition in sect. Porri.
MATERIALS AND METHODS
Isolates
One hundred eighty-three Alternaria strains including 116 extype or representative strains present at the Centraalbureau
voor Schimmelcultures (CBS), Utrecht, the Netherlands were
included in this study (Table 1). With “representative isolate” we
refer to the strains used to describe the species based on
morphology in Simmons (2007). Freeze-dried strains were
revived in 2 mL malt/peptone (50 % / 50 %) and subsequently
transferred to oatmeal agar (OA, Crous et al. 2009). Strains
stored in the liquid nitrogen collection of the CBS were transferred to OA directly from the −80 °C storage.
PCR and sequencing
DNA extraction was performed using the UltraClean Microbial DNA
isolation kit (Mobio laboratories, Carlsbad, CA, USA), according to
the manufacturer's instructions. The ITS region was amplified with
the primers V9G (de Hoog & Gerrits van den Ende 1998) and ITS4
(White et al. 1990), the GAPDH region with gpd1 and gpd2 (Berbee
et al. 1999) the RPB2 region with RPB2–5F2 (Sung et al. 2007)
and fRPB2–7cR (Liu et al. 1999), the TEF1 gene with the primers
EF1-728F and EF1-986R (Carbone & Kohn 1999) or EF2
(O'Donnell et al. 1998) and the Alt a 1 region with the primers Alt-for
and Alt-rev (Hong et al. 2005). The ITS, GAPDH, RPB2 and TEF1
PCRs were performed as described in Woudenberg et al. (2013).
The reaction mixture for the Alt a 1 PCR consisted of 1 μL genomic
DNA, 1 × NH4 reaction buffer (Bioline, Luckenwalde, Germany),
3 mM MgCl2, 20 μM of each dNTP, 0.2 μM of each primer and 0.25
U BIOTAQ DNA polymerase (Bioline). Conditions for PCR
2
amplification consisted of an initial denaturation step of 5 min at
94 °C followed by 40 cycles of 30 s at 94 °C, 30 s at 55 °C and 60 s
at 72 °C and a final elongation step of 7 min at 72 °C. The PCR
products were sequenced in both directions using the PCR primers
and the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA, USA), and analysed with an ABI
Prism 3730XL Sequencer (Applied Biosystems) according to the
manufacturer's instructions. Consensus sequences were
computed from forward and reverse sequences using the BioNumerics v. 4.61 software package (Applied Maths, St-MartensLatem, Belgium). All newly generated sequences were deposited
in GenBank (Table 1).
Phylogenetic analysis
Multiple sequence alignments were generated with MAFFT v. 7
(http://mafft.cbrc.jp/alignment/server/index.html), and adjusted by
eye where necessary. Bayesian inference and Maximum Likelihood analyses were performed on both the individual sequence
datasets as well as the concatenated datasets as described in
Woudenberg et al. (2013), with the sample frequency set to 1000
instead of 100 in the Bayesian analysis. For the TEF1 partition an
online tool (http://www.hiv.lanl.gov/content/sequence/findmodel/
findmodel.html) suggested the K2P model with a gamma-rate
variation as nucleotide substitution model, and for the remaining
four partitions the TrN model with gamma-distributed rate variation.
Sequences from the type species of the phylogenetically closest
section, sect. Gypsophilae, A. gypsophilae (Woudenberg et al.
2013), were used as outgroup. The resulting trees were printed
with TreeView v. 1.6.6 (Page 1996) and the alignments and trees
deposited into TreeBASE (http://www.treebase.org).
Taxonomy
Cultures were incubated on potato carrot agar (PCA, Crous et al.
2009) and synthetic nutrient-poor agar (SNA, Nirenberg 1976)
plates at moderate temperatures (~22 °C) under CoolWhite
fluorescent light with an 8 h photoperiod. After 7 d the growth rates
were measured and the colony characters noted. Colony colours
were rated according to Rayner (1970). Morphological descriptions were made for isolates grown on SNA with a small piece
of autoclaved filter paper placed onto the agar surface to enhance
sporulation. When sporulation occurred, the sellotape technique
was used for making slide preparations (Schubert et al. 2007) with
Titan Ultra Clear Tape (Conglom Inc., Toronto, Canada) and
Shear's medium as mounting fluid. The 95 % confidence intervals
were derived from measurements of 30 structures, with extremes
given in parentheses. Photographs of characteristic structures
were made with a Nikon Eclipse 80i microscope equipped with a
Nikon digital sight DS-Fi1 high definition colour camera, using
differential interference contrast (DIC) illumination and the Nikon
software NIS-Elements D v. 3.00. Adobe Bridge CS5.1 and Adobe
Photoshop CS5 Extended, v. 12.1, were used for the final editing
and photographic preparation. Colonies which did not sporulate
after 7 d were checked for sporulation up to 3 wk; after this period
they were noted as sterile. Nomenclatural data were deposited in
MycoBank (Crous et al. 2004).
www.studiesinmycology.org
Alternaria azadirachtae
Azadirachta indica, leaf spot
T
R
CBS 116444; E.G.S. 46.195;
BRIP 25386(ss1)
CBS 116445; E.G.S. 46.196;
BRIP 25386(ss2)
Ipomoea batatas, leaf and
stem lesion
Azadirachta indica, leaf spot
Australia, Queensland
South Africa, Mpumalanga
South Africa, Gauteng
Ipomoea batatas, stem lesion
PPRI 11848
USA, Hawaii
PPRI 11971
Passiflora edulis
Anoda cristata, leaf
USA, Hawaii
T
T
New Zealand, Auckland
Argyroxiphium sp.
CBS 594.93; E.G.S. 29.016;
QM 9046
CBS 117222; E.G.S. 35.122
Alternaria argyroxiphii
PPRI 12376
R
CBS 117129; E.G.S. 50.091
Anagallis arvensis, leaf spot
R
CBS 117128; E.G.S. 42.074
Denmark, Copenhagen
UK, England
Vanuatu
USA, Massachusetts
Italy
Denmark
Puerto Rico
Canada, Saskatchewan
USA, Illinois
Seychelles
Locality
Solanum lycopersicum, fruit
Allium cepa, seed
CBS 101004
T
(T)
R
Euphorbia esula, stem lesion
Allium cepa, leaf spot
CBS 105.51; ATCC 11078;
IMI 46816; CECT 2997
CBS 107.44
Alternaria aragakii
Alternaria solani
Alternaria anodae
Alternaria
alternariacida sp. nov.
Alternaria anagallidis
CBS 121345; E.G.S. 45.018
Allium cepa var. viviparum,
floral bract
Allium cepa, leaf
CBS 116701; E.G.S. 33.134
Alternaria vanuatuensis
Allium porrum, leaf
CBS 109.41; CBS 114.38
CBS 225.76
Alternaria porri
Alternaria porri
T
T
Ageratum houstonianum
CBS 107.28; E.G.S. 48.084
R
Acalypha indica
Alternaria porri
Alternaria agripestis
Alternaria agerati
T
Status2 Host / Substrate
Alternaria allii
Strain number1
CBS 541.94; E.G.S. 38.100;
IMI 266969
CBS 117221; E.G.S. 30.001;
QM 9369
CBS 577.94; E.G.S. 41.034
Old name
Alternaria acalyphicola
Name
Table 1. Isolates used in this study and their GenBank accession numbers. Bold accession numbers were generated in other studies.
KJ718116
KJ718115
KJ718114
KJ718113
KJ718112
KJ718111
KJ718110
KJ718109
KJ718108
KJ718107
KJ718106
KJ718105
KJ718104
KJ718103
KJ718102
KJ718101
KJ718100
KJ718099
KJ718098
KJ718097
ITS
KJ717968
KJ717967
KJ717966
KJ717965
JQ646350
KJ717964
KJ717963
KJ717962
KJ717961
KJ717960
JQ646338
KJ717959
KJ717958
KJ717957
KJ717956
KJ717955
KJ717954
JQ646356
KJ717953
KJ717952
KJ718636
KJ718635
KJ718634
KJ718633
KJ718632
KJ718631
KJ718630
KJ718629
KJ718628
KJ718627
KJ718626
KJ718625
KJ718624
KJ718623
KJ718622
KJ718621
KJ718620
KJ718619
KJ718618
KJ718617
GAPDH Alt a 1
KJ718279
KJ718278
KJ718277
KJ718276
KJ718275
KJ718274
KJ718273
KJ718272
KJ718271
RPB2
KJ718290
KJ718289
KJ718288
KJ718287
KJ718286
KJ718285
KJ718284
KJ718283
KJ718282
KJ718281
(continued on next page)
KJ718464
KJ718463
KJ718462
KJ718461
KJ718460
KJ718459
KJ718458
KJ718457
KJ718456
KJ718455
EU130544 KJ718280
KJ718454
KJ718453
KJ718452
KJ718451
KJ718450
KJ718449
KJ718448
KJ718447
KJ718446
TEF1
GenBank accesion numbers
LARGE-SPORED ALTERNARIA
PATHOGENS
3
4
Alternaria catananches sp. nov.
Alternaria cassiae
Alternaria carthamicola
Alternaria carthami
Alternaria calendulae
Alternaria blumeae
Alternaria bataticola
Name
Table 1. (Continued).
Ipomoea batatas, leaf and
stem lesion
Ipomoea batatas, leaf lesion
T
CBS 116119; E.G.S. 47.112;
IMI 286317; IMI 392448
CBS 117224; E.G.S. 40.121
CBS 117369; E.G.S. 50.166
Alternaria sauropodis
Alternaria hibiscinficiens
R
Catananche caerulea
Hibiscus sabdariffa, leaf
Senna obtusifolia, leaf spot
(T)
(T)
Senna obtusifolia, diseased
seedling
Sauropus androgynus
R
Carthamus tinctorius
Carthamus tinctorius, leaf spot
(R)T
R
CBS 137456; PD 013/05703936 T
CBS 117092; E.G.S. 37.057;
IMI 276943
CBS 478.81; E.G.S. 33.147
Helianthus annuus, leaf
Carthamus tinctorius, leaf
Calendula officinalis, leaf spot
Netherlands
Fiji
Brazil, Federal District
Malaysia, Sarawak
USA, Mississippi
Iraq
USA, Montana
Italy, Perugia
Japan, Tokyo
Calendula officinalis, leaf
Rosa sp., leaf spot
Germany
Thailand, Yala Province
Brazil, Esperito Santo
South Africa, Kwazulu-Natal
South Africa, Kwazulu-Natal
Calendula officinalis, leaf spot
Blumea aurita
Phaseolus vulgaris, leaf spot
(T)
R
CBS 116650; E.G.S. 30.142;
QM 9561
CBS 635.80
CBS 116440; E.G.S. 43.143;
IMI 366164
CBS 117091; E.G.S. 31.037
(T)
CBS 116439; E.G.S. 42.197
T
(R)
CBS 117364; E.G.S. 40.149;
ATCC 201357
CBS 224.76; ATCC 38903;
DSM 63161; IMI 205077
CBS 101498
PPRI 11934
CBS 117215; E.G.S. 39.116
PPRI 11931
PPRI 11930
Ipomoea batatas, leaf spot
R
Japan, Tokyo
Ipomoea batatas, leaf spot
Ipomoea batatas
R
CBS 117095; E.G.S. 42.157;
IMI 350492; BRIP 19470a
CBS 117096; E.G.S. 42.158;
BRIP 19470b
PPRI 10502
Japan
Ipomoea batatas
Locality
T
CBS 531.63; IFO 6187;
MUCL 28916
CBS 532.63
Strain number1
Alternaria carthami
Alternaria heliophytonis
Alternaria rosifolii
Alternaria brasiliensis
Old name
KJ718139
KJ718138
KJ718137
KJ718136
KJ718135
KJ718134
KJ718133
KJ718132
KJ718131
KJ718130
KJ718129
KJ718128
KJ718127
KJ718126
KJ718125
KJ718124
KJ718123
KJ718122
KJ718121
KJ718120
KJ718119
KJ718118
KJ718117
ITS
KJ717989
KJ717988
KJ717987
KJ717986
KJ717985
KJ717984
KJ717983
KJ717982
KJ717981
KJ717980
KJ717979
KJ717978
KJ717977
AY562405
KJ717976
KJ717975
KJ717974
KJ717973
KJ717972
KJ717971
KJ717970
KJ717969
JQ646349
KJ718657
KJ718656
KJ718655
KJ718654
KJ718653
KJ718652
KJ718651
KJ718650
KJ718649
KJ718647
KJ718646
KJ718645
KJ718648
AY563291
KJ718644
KJ718643
KJ718642
KJ718641
KJ718640
KJ718639
KJ718638
KJ718637
JQ646433
GAPDH Alt a 1
KJ718487
KJ718486
KJ718485
KJ718484
KJ718483
KJ718482
KJ718481
KJ718480
KJ718479
KJ718478
KJ718477
KJ718476
KJ718475
KJ718474
KJ718473
KJ718472
KJ718471
KJ718470
KJ718469
KJ718468
KJ718467
KJ718466
KJ718465
TEF1
KJ718313
KJ718312
KJ718311
KJ718310
KJ718309
KJ718308
KJ718307
KJ718306
KJ718305
KJ718304
KJ718303
KJ718302
KJ718301
KJ718300
KJ718299
KJ718298
KJ718297
KJ718296
KJ718295
KJ718294
KJ718293
KJ718292
KJ718291
RPB2
WOUDENBERG
ET AL.
Alternaria dauci
Alternaria cyamopsidis
Alternaria cucumerina
Alternaria crassa
Alternaria conidiophora sp. nov.
Alternaria citrullicola sp. nov.
Alternaria poonensis
Alternaria cichorii
Alternaria loofahae
Alternaria capsici
R
R
CBS 117226; E.G.S. 44.197;
BRIP 23060
CBS 364.67; E.G.S. 17.065;
QM 8575
CBS 117219; E.G.S. 13.120;
QM 8000
CBS 111.38
(R)
R
CBS 117100; E.G.S. 47.138
CBS 117099; E.G.S. 47.131
Coriandrum sativum, seedling
Daucus carota, seed
Daucus carota, leaf spot
Daucus carota, commercial seed
R
R
CBS 117097; E.G.S. 46.006
CBS 117098; E.G.S. 46.152
Cichorium intybus var.
foliosum, leaf spot
Daucus carota, seed
Daucus carota, leaf spot
CBS 345.79; LEV 14814
CBS 477.83; CBS 721.79;
PD 79/954
CBS 101592
Daucus carota, seed
Cyamopsis tetragonoloba,
leaf spot
Cyamopsis tetragonoloba,
leaf spot
Daucus carota, seed
Cucumis melo, leaf spot
Cucumis melo, leaf spot
Luffa acutangula
Datura stramonium, leaf spot
CBS 106.48
T
R
R
(T)
CBS 117225; E.G.S. 41.127
R
CBS 122590; E.G.S. 44.071
CBS 116114; E.G.S. 35.123
R
CBS 116648; E.G.S. 50.180
USA, Indiana
Puerto Rico
USA, California
New Zealand
KJ718165
KJ718164
KJ718163
KJ718658
AY563298
KJ718665
KJ718664
KJ718663
KJ718662
KJ718661
KJ718660
KJ718659
KJ718496
KJ718495
KJ718494
KJ718493
KJ718492
KJ718491
KJ718490
KJ718489
KJ718488
TEF1
KJ718667
KJ718666
KJ718499
KJ718498
KJ718009
KJ718008
KJ718007
KJ718006
KJ718005
KJ718004
KJ718003
KJ718002
KJ718001
KJ718000
JQ646348
KJ718011
KJ718010
KJ718680
KJ718679
HE796726
KJ718678
KJ718677
KJ718676
KJ718675
KJ718674
KJ718673
KJ718672
KJ718671
KJ718670
KJ718669
KJ718668
KJ718335
KJ718334
KJ718333
KJ718332
KJ718331
KJ718330
KJ718329
KJ718328
KJ718327
KJ718326
KJ718325
KJ718324
KJ718323
KJ718322
KJ718321
KJ718320
KJ718319
–
KJ718318
KJ718317
KJ718316
KJ718315
KJ718314
RPB2
KJ718338
KJ718337
KJ718336
KJ718513
KJ718512
KJ718511
KC584651 KC584392
KJ718510
KJ718509
KJ718508
KJ718507
KJ718506
KJ718505
KJ718504
KJ718503
KJ718502
KJ718501
GQ180072 GQ180088 KJ718500
KJ717999
KJ717998
GQ180073 GQ180089 KJ718497
AY562408
KJ717997
KJ717996
KJ717995
KJ717994
KJ717993
KJ717992
KJ717991
KJ717990
GAPDH Alt a 1
KC584192 KC584111
KJ718162
Netherlands
USA, California
KJ718161
Netherlands, Limburg
KJ718160
KJ718159
New Zealand, Ohakune
KJ718158
–
KJ718157
KJ718156
KJ718155
KJ718154
KJ718153
KJ718152
KJ718151
KJ718150
KJ718149
KJ718148
KJ718147
KJ718146
KJ718145
KJ718144
KJ718143
KJ718142
KJ718141
KJ718140
ITS
Italy
USA, Georgia
USA, Maryland
USA, Indiana
USA, Hawaii
USA, Indiana
USA, Indiana
Nicandra physalodes
CBS 116647; E.G.S. 46.013
R
Australia
Capsicum annuum
(T)
USA, Wisconsin
Cyprus
CBS 109160; E.G.S. 45.075;
IMI 262408; IMI 381021
CBS 109162; E.G.S. 46.014
Netherlands
–
Datura sp., leaf spot
T
Cyprus
Greece
Cyprus
USA, California
Locality
Citrullus vulgaris, fruit
CBS 110.38
T
T
Cirsium arvense, stem lesion
Cichorium endivia
R
T
Cichorium intybus, leaf spot
Centaurea solstitialis, leaf spot
T
T
CBS 103.18
CBS 103.32; VKM F-1881;
Nattrass No. 190
CBS 137457
CBS 102.33; E.G.S. 07.017;
QM 1760
CBS 117218; E.G.S. 52.046;
IMI 225641
CBS 113261; E.G.S. 41.136
Alternaria cirsinoxia
CBS 116446; E.G.S. 47.119
Strain number1
Alternaria cichorii
Old name
Alternaria centaureae
Name
PATHOGENS
5
6
(T)
CBS 109161; E.G.S. 45.113
CBS 109164; E.G.S. 46.188
CBS 116438; E.G.S. 41.057
CBS 116441; E.G.S. 45.108
CBS 116704; E.G.S. 44.074
Alternaria subcylindrica
Alternaria cretica
Alternaria cucumericola
Alternaria tabasco
Alternaria tomatophila
CPC 21620
(T)
CBS 109156; E.G.S. 42.156
(R)
(T)
(T)
CBS 107.61
Alternaria solani
(T)
CBS 108.53
Alternaria solani
T
R
CBS 117360; E.G.S. 43.009
CBS 105.41; E.G.S. 07.016
CBS 483.90; E.G.S. 39.070
Alternaria linariae
T
CBS 133855; CCM 8361
T
T
T
Alternaria limicola
PPRI 8988
CBS 107.41; E.G.S. 07.025;
IMI 264349
CBS 219.79
R
–
Solanum lycopersicum, leaf spot
Capsicum frutescens, leaf spot
Cucumis sativus, leaf spot
Solanum lycopersicum var.
cerasiforme, leaf spot
Thailand, Chiang Mai
USA, Indiana
USA, Louisiana
New Zealand
Greece, Crete
USA, Louisiana
USA, Indiana
–
Belgium
–
Denmark
Mexico, Jalisco
Mexico, Colima
Slovakia
Ethiopia
Netherlands
USA, Louisiana
USA, Hawaii
USA, Florida
USA, Pennsylvania
USA, Pennsylvania
New Zealand, Gisborne
New Zealand, Gisborne
New Zealand
Italy
Italy
Namibia
Locality
Linaria maroccana, seedling
Citrus sp.
Citrus aurantiifolia, leaf spot
Fumana procumbens, seed
Ipomoea batatas, stem
Ipomoea batatas, stem and petiole
Gypsophila elegans, seed
Euphorbia hyssopifolia
Euphorbia pulcherrima
CBS 133874; E.G.S. 38.191
CBS 119410; E.G.S. 41.029
Solanum tuberosum, leaf spot
R
Echinacea sp., leaf lesion
CBS 198.86; E.G.S. 38.082
CBS 116695; E.G.S. 44.108
R
T
CBS 109158; E.G.S. 44.106
CBS 116118; E.G.S. 46.082
Echinacea sp., leaf lesion
Dichondra sp., leaf
R
T
CBS 116117; E.G.S. 46.081
CBS 117127; E.G.S. 40.057
Dichondra repens, leaf spot
T
CBS 200.74; E.G.S. 38.008
desert soil
CBS 346.79
T
T
CBS 199.74; E.G.S. 38.007
CBS 110799
Strain number1
Alternaria jesenskae
Alternaria ipomoeae sp. nov.
Alternaria gypsophilae
Alternaria euphorbiicola
Alternaria grandis
Alternaria echinaceae
Alternaria deserticola sp. nov.
Alternaria dichondrae
Old name
Name
KJ718019
KJ718018
KJ718017
KJ718070
JQ646341
KJ718016
KJ718015
KJ718014
KJ718013
KJ718012
JQ646357
KJ718077
KJ718189
KJ718188
KJ718187
KJ718186
KJ718185
KJ718184
KJ718183
KJ718182
KJ718181
KJ718180
KJ718179
KJ718178
KJ718177
KJ718176
KJ718175
KJ718030
KJ718029
KJ718028
KJ718027
JQ646342
JQ646345
JQ646347
KJ718026
KJ718025
KJ718024
KJ718023
JQ646329
KJ718022
KJ718021
KJ718020
KJ718530
KJ718529
KJ718698
KJ718697
KJ718696
KJ718695
JQ646426
JQ646429
KJ718347
KJ718346
KJ718357
KJ718356
KJ718355
KJ718354
KJ718353
KJ718352
KJ718351
KJ718350
KJ718349
KJ718348
KJ718536
KJ718535
KJ718534
KJ718533
KJ718362
KJ718361
KJ718360
KJ718359
EU130545 KJ718358
KJ718532
GQ180101 KJ718531
KJ718694
KJ718693
KJ718528
KJ718527
–
KJ718525
KJ718524
KJ718523
KJ718526
KJ718692
KJ718416
KJ718345
KC584660 KC584401
JQ646413
KJ718691
KJ718690
KJ718689
KJ718688
KJ718522
KJ718521
–
KJ718587
KJ718520
KJ718687
KJ718344
KJ718343
KJ718342
KJ718341
KJ718340
KJ718339
KJ718424
RPB2
EU130547 KJ718414
KJ718519
KJ718518
KJ718517
KJ718516
KJ718515
KJ718514
KJ718595
TEF1
KJ718686
KJ718748
JQ646425
KJ718685
KJ718684
KJ718683
KJ718682
KJ718681
JQ646441
KJ718755
GAPDH Alt a 1
KC584199 KC584118
KJ718174
KJ718173
KJ718172
KJ718241
KJ718239
KJ718171
KJ718170
KJ718169
KJ718168
KJ718167
KJ718166
KJ718249
ITS
WOUDENBERG
ET AL.
Alternaria porri
Alternaria macrospora
Ipomoea batatas, stem
PPRI 11845
Alternaria pipionipisi
Alternaria passiflorae
Alternaria paralinicola sp. nov.
Alternaria obtecta
CBS 116120; E.G.S. 47.198
(T)
R
R
CBS 117102; E.G.S. 51.165
CBS 117103; E.G.S. 52.032
Alternaria hawaiiensis
CBS 116115; E.G.S. 40.096;
IMI 340950
CBS 117365; E.G.S. 42.048
CBS 134265; E.G.S. 42.047
Alternaria obtecta
Alternaria obtecta
Alternaria gaurae
Passiflora edulis, fruit
(R)
T
(T)
R
CBS 629.93; E.G.S. 16.150;
QM 8458
CBS 630.93; E.G.S. 29.020;
QM 9050
CBS 116333; E.G.S. 50.121
Euphorbia pulcherrima, leaf
Cajanus cajan, seed
Passiflora caerulea, leaf spot
Passiflora ligularis, fruit spot
Gaura lindheimeri, leaf
Passiflora edulis
Passiflora edulis
Capsicum frutescens, leaf
CBS 166.77
Alternaria solani
Linum usitatissimum, seed
Galinsoga parviflora, leaf
Citrus sp., dry leaf
CBS 116652; E.G.S. 47.157;
DAOM 225747
CBS 113.38
(R)T
R
T
Alternaria linicola
CBS 134278; E.G.S. 42.064
CBS 117367; E.G.S. 42.063
PPRI 12171
Alternaria novae-guineensis
Solanum viarum, leaf spot
T
PPRI 13903
CBS 109163; E.G.S. 46.151
Ipomoea batatas
PPRI 11847
T
USA, Georgia
Richardia scabra, floral bract
R
USA, California
USA, California
India
USA, Hawaii
New Zealand
New Zealand, Waitakere
Australia, South Queensland
Canada, Manitoba
USA, California
USA, California
Papua New Guinea
Puerto Rico
South Africa, Mpumalanga
South Africa, North West
USA, Georgia
Richardia scabra, floral bract
T
Ipomoea batatas
USA, Montana
USA, Arizona
Nigeria
Locality
Cirsium arvense
T
Gossypium barbadense
CBS 117228; E.G.S. 50.190;
ATCC 58172
CBS 121343; E.G.S. 44.112;
IMI 257563
CBS 712.68; ATCC 18515;
IMI 135454; MUCL 11722;
QM 8820; VKM F-2997
CBS 713.68; ATCC 18517;
IMI 135455;
MUCL 11715; QM 8821
PPRI 8990
T
Gossypium sp.
CBS 106.29
Strain number1
Alternaria nitrimali
Alternaria neoipomoeae sp. nov.
Alternaria multirostrata
Alternaria montanica
Old name
Name
KJ718216
KJ718215
KJ718214
KJ718213
KJ718212
KJ718211
KJ718210
KJ718209
KJ718208
KJ718207
KJ718206
KJ718205
KJ718204
KJ718203
KJ718202
KJ718201
KJ718200
KJ718199
KJ718198
KJ718197
KJ718196
KJ718195
KJ718194
KJ718051
KJ718050
KJ718049
KJ718048
KJ718047
KJ718046
JQ646352
KJ718045
KJ718044
JQ646353
KJ718043
KJ718042
KJ718041
KJ718040
KJ718039
JQ646358
KJ718038
KJ718037
KJ718036
KJ718035
KJ718034
JQ646362
KJ718033
KJ718724
KJ718723
KJ718722
KJ718721
KJ718720
KJ718719
KJ718718
KJ718717
KJ718716
JQ646437
KJ718715
KJ718714
KJ718713
KJ718712
KJ718711
KJ718710
KJ718709
KJ718708
KJ718707
KJ718706
KJ718705
KJ718704
KJ718703
KJ718702
KJ718701
GAPDH Alt a 1
KJ718032
KC584204 KC584124
KJ718193
ITS
KJ718366
RPB2
KJ718367
KJ718389
KJ718388
KJ718387
KJ718386
KJ718385
KJ718384
KJ718383
KJ718382
KJ718381
KJ718380
KJ718379
KJ718378
KJ718377
KJ718376
KJ718375
KJ718374
KJ718373
KJ718372
KJ718371
KJ718370
KJ718369
KJ718562
KJ718561
KJ718560
KJ718559
KJ718558
KJ718557
KJ718556
KJ718555
KJ718554
KJ718553
KJ718552
KJ718551
KJ718550
KJ718549
KJ718548
KJ718547
KJ718546
KJ718545
KJ718544
KJ718543
KJ718542
EU130546 KJ718368
KJ718541
KC584668 KC584410
KJ718540
TEF1
PATHOGENS
7
8
Russia, Vladivistok
Russia, Vladivistok
Silybum marianum, leaf
Kiribati, Phoenix Islands
CBS 134093; VKM F-4117
Sida fallax, leaf spot
India
Egypt
India, Uttar Pradesh
UK, Derbyshire
CBS 134094; VKM F-4118
T
T
Sesamum indicum, seedling
Sesamum indicum
Senna corymbosa, leaf
Linum usitatissimum, seed
Russia, Vladivistok
Alternaria silybi
R
(R)T
(R)
Netherlands, Reusel
UK, Scotland
USA, California
USA, Virginia
Italy, Sardinia
Japan
Israel
USA, California
New Zealand, Hastings
Israel
Israel
USA, California
New Zealand
New Zealand, Levin
USA, New York
USA, New York
USA, Nebraska
Locality
CBS 134092; VKM F-4109
Alternaria sidae
Alternaria cassiae
CBS 115264; CBS 117214;
E.G.S. 13.027
CBS 117730; E.G.S. 12.129
Alternaria sesami
Alternaria sennae sp. nov.
CBS 116703; E.G.S. 36.110;
IMI 274549
CBS 477.81; E.G.S. 34.030;
IMI 257253
CBS 240.73
Scorzonera hispanica, leaf spot
Alternaria linicola
Linum usitatissimum
CBS 478.83; E.G.S. 38.011
R,T
CBS 103.46; Elliot No. 45-190C
Alternaria linicola
Alternaria scorzonerae
Ricinus communis
T
R
CBS 117361; E.G.S. 06.181
CBS 117366; E.G.S. 42.061
Ricinus communis
Ricinus communis
Ranunculus asiaticus, seed
Solanum tuberosum
CBS 353.86
T
T
T
(R)
(R)
Helianthus annuus, leaf spot
Helianthus annuus, leaf spot
Solanum tuberosum, tuber
Hordeum vulgare, seed
Solanum lycopersicum, fruit rot
Alternaria rostellata
Alternaria ricini
CBS 116330; E.G.S. 38.039;
IMI 285697
CBS 215.31
Alternaria ranunculi
E.G.S. 45.053
CBS 119411; E.G.S. 42.060
Alternaria pseudorostrata
Alternaria solani
E.G.S. 42.122;
R
E.G.S. 45.024;
Alternaria pulcherrimae
R
E.G.S. 45.023;
CBS 116696;
IMI 372955
CBS 116697;
IMI 372957
CBS 121342;
IMI 310506
CBS 135189;
(T)
(R)
CBS 116651; E.G.S. 45.020
Alternaria hordeiseminis
Alternaria solani
Alternaria protenta
R
R,T
CBS 116699; E.G.S. 48.152
Allium cepa, leaf
(R)
CBS 116649; E.G.S. 17.082;
QM 8613
CBS 116698; E.G.S. 48.147
Alternaria solani
Alternaria allii
Alternaria porri
Strain number1
CBS 347.79; E.G.S. 44.091;
LEV 14726; ATCC 38569
CBS 116437; E.G.S. 32.076
Old name
Name
KJ718052
KJ718055
KJ718054
KJ718053
KJ718235
KJ718234
KJ718233
KJ718232
JF780939
KJ718231
KJ718230
KJ718192
KJ718191
KJ718190
KJ718229
KJ718228
KJ718227
KJ718226
KJ718225
JN383483
KJ718224
KJ718223
KJ718222
KJ718221
KJ718726
KJ718566
KJ718565
KJ718564
KJ718065
KJ718064
KJ718063
KJ718062
KJ718061
JQ646343
JQ646344
KJ718031
JQ646334
JQ646363
JQ646332
KJ718060
JQ646331
KJ718059
KJ718058
AY562406
KJ718393
KJ718392
KJ718391
KC584679 KC584421
KJ718390
RPB2
KJ718731
KJ718730
JQ646419
KJ718569
KJ718568
KJ718567
KJ718742
KJ718741
KJ718740
KJ718739
KJ718738
KJ718737
JQ646428
KJ718700
KJ718699
JQ646447
KJ718736
KJ718735
KJ718734
KJ718733
KJ718732
AY563295
KJ718365
KJ718364
KJ718363
KJ718402
KJ718401
KJ718400
KJ718399
KJ718398
KJ718581
KJ718580
KJ718579
KJ718578
KJ718577
KJ718576
KJ718409
KJ718408
KJ718407
KJ718406
KJ718405
KJ718404
EU130543 KJ718403
KJ718539
KJ718538
KJ718537
KJ718575
KJ718574
KJ718573
KJ718572
KJ718571
KC584680 KC584422
KJ718397
KJ718396
KJ718395
KJ718394
GQ180097 KC584688 KC584430
KJ718729
KJ718728
KJ718727
KJ718563
TEF1
GQ180082 GQ180098 KJ718570
KJ718057
KJ718056
JQ646335
KC584217 KC584139
KJ718220
KJ718219
KJ718218
KJ718725
GAPDH Alt a 1
DQ323700 KC584132
KJ718217
ITS
WOUDENBERG
ET AL.
CBS 111.44; E.G.S. 07.029;
QM 1772
CBS 109157; E.G.S. 44.098
R
Tagetes erecta, seed
Tagetes sp., seed
R
R
R
CBS 479.81; E.G.S. 33.081;
GST 556
CBS 480.81; E.G.S. 33.184
CBS 117217; E.G.S. 44.045
T
R
CBS 631.93; E.G.S. 39.126
CBS 117216; E.G.S. 39.125
T
(T)
CBS 116116; E.G.S. 43.074
CBS 122597
T
Tagetes sp., seed
CBS 116331; E.G.S. 41.073;
BRIP 14963
CBS 120986; E.G.S. 51.075
Tagetes sp., seed
CBS 298.79; GST AM3
Tillandsia usneoides
Thunbergia alata
Allium cepa, leaf
Thunbergia alata, leaf spot
Tagetes sp., leaf spot
Stevia rebaudiana, leaf spot
CBS 117362; E.G.S. 37.019;
IFO 31182
CBS 297.79; GST AM2
Allium ascalonicum, leaf spot
Solanum nigrum, leaf spot
Alternaria tropica
Alternaria iranica
Glyceria maxima, leaf spot
Beta vulgaris, leaf spot
T
USA, Washington
KJ718240
KJ718238
New Zealand, Waikato
USA, Florida
USA, Florida
New Zealand
Iran, Miandoab
USA, Ohio
USA, South Carolina
UK, England
UK
UK
Japan, Kagawa
Japan, Kagawa
Japan, Kagawa
New Zealand, Hastings
New Zealand, Canterbury
KJ718262
KJ718261
KJ718260
KJ718259
KJ718258
KJ718257
KJ718256
KJ718255
KJ718745
KJ718744
KJ718743
KJ718081
KJ718080
KJ718079
JQ646339
KJ718078
KJ718076
KJ718075
KJ718074
KJ718073
KJ718072
KJ718071
JQ646360
KJ718069
KJ718089
KJ718088
KJ718087
KJ718086
KJ718085
KJ718084
KJ718083
KJ718082
KJ718769
KJ718768
KJ718767
KJ718766
KJ718765
KJ718764
KJ718763
KJ718762
KJ718761
KJ718760
KJ718759
KJ718758
KJ718757
KJ718756
KJ718754
KJ718753
KJ718752
KJ718751
KJ718750
KJ718749
JQ646444
KJ718747
GQ180080 KJ718746
KJ718068
KJ718067
KJ718066
GAPDH Alt a 1
KJ718429
KJ718428
KJ718427
KJ718426
KJ718425
KJ718423
KJ718422
KJ718421
KJ718420
KJ718419
KJ718418
KJ718417
KJ718415
KJ718413
KJ718412
KJ718411
KJ718410
RPB2
KJ718437
KJ718436
KJ718435
KJ718434
KJ718433
KJ718432
KJ718431
KJ718430
KJ718608
KJ718607
KJ718606
KJ718605
KJ718604
KJ718603
KJ718602
KJ718601
KC584692 KC584434
KJ718600
KJ718599
KJ718598
KJ718597
KJ718596
KJ718594
KJ718593
KJ718592
KJ718591
KJ718590
KJ718589
KJ718588
KJ718586
KJ718585
KJ718584
KJ718583
KJ718582
TEF1
KC584221 KC584143
KJ718254
KJ718253
KJ718252
KJ718251
KJ718250
KJ718248
KJ718247
KJ718246
KJ718245
KJ718244
KJ718243
New Zealand, New Plymouth KJ718242
New Zealand
Y17070
KJ718237
Italy
KJ718236
–
ITS
–
Locality
Petroselinum crispum, stunted plant New Zealand, Taranaki
CBS 632.88; IFO 31183
(T)
(T)
Cyphomandra betacea, fruit
Solanum nigrum, leaf spot
CBS 631.88; IFO 31212
CBS 121347; E.G.S. 46.052
CBS 117101; E.G.S. 51.032
Alternaria beticola
Alternaria ascaloniae
(T)
CBS 116334; E.G.S. 51.107
CBS 116447; E.G.S. 47.196
Alternaria glyceriae
(T)
Alternaria herbiculinae
(T)
R
CBS 113403; E.G.S. 51.106;
CPC 10620
CBS 116332; E.G.S. 49.180
Vicia faba
R
(T)
Ageratum houstonianum, seed
(T)
–
Solanum aviculare, leaf spot
CBS 106.21
CBS 111.41
Strain number1
CBS 116442; E.G.S. 46.162;
ICMP 10242
Alternaria cyphomandrae CBS 109155; E.G.S. 40.058
Alternaria viciae-fabae
Alternaria danida
Old name
Alternaria tillandsiae
Alternaria thunbergiae
Alternaria tagetica
Alternaria steviae
Alternaria solani-nigri
Alternaria solani
Name
PATHOGENS
9
10
Zinnia elegans
Zinnia elegans
Zinnia sp., seed
Zinnia sp., seed
Zinnia elegans, leaf spot
CBS 117.59
CBS 108.61
CBS 299.79
CBS 300.79
CBS 117223; E.G.S. 44.035
UK
KJ718270
KJ718269
KJ718268
KJ718267
–
UK
KJ718266
KJ718265
KJ718264
KJ718263
ITS
Italy, Sardinia
Netherlands
Hungary
Venezuela, Maracay
Locality
KJ718096
KJ718095
KJ718094
KJ718093
KJ718092
KJ718091
JQ646361
KJ718090
KJ718777
KJ718776
KJ718775
KJ718774
KJ718773
KJ718772
KJ718771
KJ718770
GAPDH Alt a 1
KJ718438
KJ718439
KJ718440
KJ718441
KJ718442
KJ718443
KJ718444
KJ718445
KJ718609
KJ718610
KJ718611
KJ718612
KJ718613
KJ718614
KJ718615
KJ718616
RPB2
TEF1
1
ATCC: American Type Culture Collection, Manassas, VA, USA; BRIP: Queensland Plant Pathology Herbarium, Queensland, Australia; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht,
Netherlands; CCM: Czech Collection of Microorganisms, Brno, Czech Republic; CECT: Spanish Type Culture Collection, Valencia, Spain; CPC: Personal collection of P.W. Crous, Utrecht, Netherlands; DAOM: Canadian Collection of Fungal Cultures,
Ottawa, Canada; DSM: German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany; E.G.S.: Personal collection of Dr. E.G. Simmons; Elliott: Personal collection of M.E. Elliott; GST: Personal collection of G.S.
Taylor; ICMP: International Collection of Micro-organisms from Plants, Auckland, New Zealand; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IMI: Culture collection of CABI Europe UK Centre, Egham UK; LEV: Plant Health and
Diagnostic Station, Levin, New Zealand; MUCL: (Agro)Industrial Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Louvain-la Neuve, Belgium; Nattrass: Personal collection of R.M. Nattrass; PD: Plant
Protection Service, Wageningen, Netherlands; PPRI: ARC-Plant Protection Research Institute, Roodeplaat, South Africa; QM: Quarter Master Culture Collection, Amherst, MA, USA; VKM: All-Russian Collection of Microorganisms, Moscow, Russia.
2
T: ex-type strain; R: representative strain; Letters between parentheses refer to synonymised species names; Bold letters are designated in this study.
R
Zinnia sp., leaf
CBS 107.48
Phaseolus vulgaris, leaf spot
Callistephus chinensis, seed
T
CBS 118.44
CBS 116121; E.G.S. 48.065
Strain number1
Alternaria venezuelensis
Old name
Alternaria zinniae
Name
WOUDENBERG
ET AL.
RESULTS
Phylogeny
Because the amplification/sequencing of the RPB2 region of CBS
137457 and the Alt a 1 region of CBS 119410 and CBS 117360
failed, these genes were included as missing data in the combined
analysis of these isolates. The topologies of the trees obtained from
the RAxML and Bayesian analyses were overall congruent,
resulting in identical species-clades. The phylogenies of the singlegene trees were congruent with one exception, CBS 137456, which
swapped between clusters with the different genes used, resulting
in a somewhat distorted picture in the combined analysis. The
aligned sequences of the ITS (538 characters), GAPDH (581
characters), RPB2 (772 characters), TEF1 (355 characters) and Alt
a 1 (476 characters) gene regions of the 183 included Alternaria
strains had a total length of 2 722 characters, with respectively 77,
111, 134, 141 and 131 unique site patterns. After discarding the
burn-in phase trees, the Bayesian analysis resulted in 7 502 trees
from which the 50 % majority rule consensus tree and posterior
probabilities were calculated. The multi-gene phylogeny of section
Porri (Fig. 1) divided the isolates in 62 species (clades) and one
new Alternaria section. The species A. euphorbiicola and
A. limicola, previously assigned to sect. Porri (Lawrence et al. 2013,
Woudenberg et al. 2013), form a sister-clade to sect. Porri, here
described as Alternaria sect. Euphorbiicola sect. nov. A Bayesian
phylogeny based on the GAPDH, RPB2 and TEF1 sequences of
representative isolates of the closely related sections in Alternaria
(sequences obtained from Woudenberg et al. 2013) was constructed for comparison, with A. brassicicola CBS 118699 from
sect. Brassisicola, as outgroup (Fig. 2).
Fig. 1. Bayesian 50 % majority rule consensus tree based on the ITS, GAPDH, RPB2, TEF1 and Alt a 1 sequences of 183 Alternaria strains. The Bayesian posterior probabilities > 0.75 (PP) and RAxML bootstrap support values > 65 (ML) are given at the
nodes (PP/ML). Thickened lines indicate a PP of 1.0 and ML of 100. Species names between parentheses represent synonymised species names. Ex-type strains are indicated with T and representative strains with R. Novel species names are printed in bold
face. The tree was rooted to A. gypsophilae (CBS 107.41).
PATHOGENS
11
Fig. 1. (continued).
WOUDENBERG
12
ET AL.
PATHOGENS
Fig. 1. (continued).
13
WOUDENBERG
ET AL.
Fig. 2. Bayesian 50 % majority rule consensus tree based on the GAPDH, RPB2 and TEF1 sequences of 41 Alternaria strains. The Bayesian posterior probabilities (PP) are
given at the nodes. Thickened lines indicate a PP of 1.0. The tree was rooted to A. brassicola (CBS 118699).
14
Taxonomy
At the onset of this study, Alternaria sect. Porri contained 82
Alternaria species. After extensive phylogenetic analyses and
morphological examination we now recognise 63 species in this
section (Table 2), of which 10 are newly described. Twenty-seven
species names are reduced to synonymy (Table 2). All isolates
where taxonomic changes were found based on the multi-gene
phylogeny were studied morphologically; photo plates of these
species are included. Type details are only listed when typification is proposed.
Section Porri D.P. Lawr., Gannibal, Peever & B.M. Pryor,
Mycologia 105: 541. 2013
Type species: Alternaria porri (Ellis) Cif.
Section Porri is characterised by broadly ovoid, obclavate,
ellipsoid, subcylindrical or obovoid, medium to large conidia,
disto- and euseptate, solitary or in short chains, with a
simple or branched, long to filamentous beak. Conidia
contain multiple transverse and longitudinal septa and are
slightly constricted near some transverse septa. Secondary
conidiophores can be formed apically and/or laterally.
Species in sect. Porri
Alternaria acalyphicola E.G. Simmons, Mycotaxon 50:
260. 1994.
Material examined: Seychelles, from Acalypha indica (Euphorbiaceae), before
Apr. 1982, C. Kingsland, culture ex-type of A. acalyphicola CBS 541.94 = E.G.S.
38.100 = IMI 266969.
Notes: Alternaria acalyphicola is closely related to A. ricini, with
only 1 nt difference in three out of the five genes sequenced;
RPB2, TEF1 and GAPDH. Based on this single isolate, the data
is inconclusive to support the synonymy of these two species.
Alternaria agerati E.G. Simmons, Mycotaxon 65: 63.
1997.
= Alternaria agerati Sawada, Rep. Dept. Agric. Gov. Res. Inst. Formosa 86:
165. 1943. (nom. inval., Art. 36.1)
Material examined: USA, Illinois, Springfield, from Ageratum houstonianum
(Asteraceae) in a commercial greenhouse, Nov. 1968, J.L. Forsberg, representative isolate of A. agerati CBS 117221 = E.G.S. 30.001 = QM 9369.
Alternaria agripestis E.G. Simmons & K. Mort., Mycotaxon 50: 255. 1994.
Material examined: Canada, Saskatchewan, Maxim, from infected stem of
Euphorbia esula (Euphorbiaceae), 9 Jul. 1992, P. Harris, culture ex-type of
A. agripestis CBS 577.94 = E.G.S. 41.034.
Alternaria allii Nolla, Phytopathology 17: 118. 1927. Fig. 3.
= Alternaria vanuatuensis E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 260. 2007.
Materials examined: Denmark, from seed of Allium cepa (Amaryllidaceae),
1937, P. Neergaard, CBS 109.41 = CBS 114.38. Italy, from leaf of Allium
porrum (Amaryllidaceae), 1974, H. Nirenberg, CBS 225.76. Puerto Rico, from
PATHOGENS
leaf of Allium cepa, before 1928, J.A.B. Nolla, culture ex-type of A. allii CBS
107.28 = E.G.S. 48.084. USA, Massachusetts, Hadley, from floral bract of
Allium cepa var. viviparum, 13 Jul. 1980, E.G. Simmons, representative of
A. allii CBS 116701 = E.G.S. 33.134. Vanuatu, from leaves of Allium cepa,
1996, C.F. Hill, culture ex-type of A. vanuatuensis CBS 121345 = E.G.S
45.018.
Notes: Simmons (2007) designated the lectotype of A. allii as
Nolla (1927), loc. cit., Pl. III, fig. 11–19, based on the absence of
original Nolla specimens. In our study, however, we managed to
uncover an original specimen, CBS 107.28, which was deposited
in the CBS by J.A.B. Nolla in December 1927 as his “A. allii sp.
nov.”, just after he published the new species description. We
therefore recognise this isolate as the ex-type strain of A. allii.
Isolate CBS 116701 did not sporulate after 3 wk of cultivation on
SNA.
Alternaria alternariacida Woudenb. & Crous, sp. nov.
MycoBank MB808990. Fig. 4.
Etymology: Named after its ability to produce high amounts of
alternaric acid.
Alternaria alternariacida differs from the ex-type isolate of
its closest phylogenetic neighbour A. silybi (CBS 134092)
based on alleles in three loci (positions derived from
respective alignments of the separate loci deposited in
TreeBASE): ITS position 386 (T), 497 (T), 498 (T); TEF1
position 3 (T), 18 (T); Alt a 1 position 205 (C), 336 (T), 339
(A), 350 (C), 404 (T), 408 (G).
Sporulation is atypical. Primary conidiophores solitary, simple,
straight to slightly curved, septate, pale brown with a subhyaline tip, (52 –)73–93(–155) × (4–)5–6(–7) μm, bearing a
single, darkened, apical conidiogenous locus. Conidia solitary
or in unbranched chains of 2(–3) conidia, conidium body pale
olive-brown, smooth-walled, narrowly ovoid, solitary, noncatenulate, and secondary conidia (33 –)44–49(–56) × (5–)
7–8( –9) μm, with (3–)5–6(–8) transverse eusepta and no
longitudinal septa; primary conidia in total (85–)
99–111(–121) × (6–)7–8( –10) μm. The conidial body can be
slightly constricted near the septa. The conidium body gradually tapers into mostly an aseptate, single, unbranched beak,
but branched beaks do occur; apical and multiple lateral
secondary conidiophores can also occur. Beaks (47−)
129−257(−610) μm long, ca. 2 μm wide throughout their
length. Sexual morph not observed.
Culture characteristics: After 7 d cultures on SNA flat, fimbriate,
white; aerial mycelium sparse, white, colonies reaching
25−30 mm diam; cultures on PCA flat, entire, olivaceous in the
centre with three olivaceous concentric circles and a buff to white
margin; aerial mycelium fine, felty, white, colonies reaching
50 mm diam; reverse with four olivaceous concentric circles.
Material examined: UK, England, from fruit of Solanum lycopersicum (Solanaceae), 1946, P.W. Brian (holotype CBS H-21734, culture ex-type CBS
105.51 = ATCC 11078 = IMI 46816 = CECT 2997 = IBPG 14 = BRL408).
Note: The atypical sporulation of the single isolate of
A. alternariacida, which is over 60 yr old, resulted in our decision
to include sequence data in the species description.
15
WOUDENBERG
ET AL.
Table 2. Current species within Alternaria sect. Porri and their host / substrate.
Species name
Synonymised names (this study)
Host / Substrate
Euphorbiaceae (Acalypha indica)
Alternaria agerati
Asteraceae (Ageratum houstonianum)
Alternaria agripestis
Euphorbiaceae (Euphorbia esula)
Alternaria allii
Alternaria vanuatuensis
Amaryllidaceae (Allium cepa, A. porrum)
Alternaria alternariacida
Solanaceae (Solanum lycopersicum)
Alternaria anagallidis
Primulaceae (Anagallis arvensis)
Alternaria anodae
Malvaceae (Anoda cristata)
Alternaria aragakii
Passifloraceae (Passiflora edulis)
Alternaria argyroxiphii
Asteraceae (Argyroxiphium sp.), Convolvulaceae (Ipomoea batatas)
Alternaria azadirachtae
Meliaceae (Azadirachta indica)
Alternaria bataticola
Convolvulaceae (Ipomoea batatas)
Alternaria blumeae
Alternaria brasilliensis
Asteraceae (Blumea aurita), Fabaceae (Phaseolus vulgaris)
Alternaria calendulae
Alternaria rosifolii
Asteraceae (Calendula officinalis), Rosaceae (Rosa sp.)
Alternaria carthami
Alternaria heliophytonis
Asteraceae (Carthamus tinctorius, Helianthus annuus)
Alternaria carthamicola
Alternaria cassiae
Asteraceae (Carthamus tinctorius)
Alternaria hibiscinficiens
Alternaria sauropodis
Alternaria catananches
Fabaceae (Senna obtusifolia), Malvacea (Hibiscus sabdariffa), Phyllanthaceae
(Sauropus androgynus)
Asteraceae (Catananche caerulea)
Alternaria centaureae
Asteraceae (Centaurea solstitialis)
Alternaria cichorii
Asteraceae (Cichorium endivia, C. intybus)
Alternaria cirsinoxia
Asteraceae (Cirsium arvense)
Alternaria citrullicola
Cucurbitaceae (Citrullus lanatus)
Alternaria conidiophora
Unknown
Alternaria crassa
Alternaria capsici
Alternaria loofahae
Alternaria cyamopsidis
Alternaria dauci
Solanaceae (Capsicum annuum, Datura stramonium, Nicandra physalodes)
Cucurbitaceae (Cucumis melo, Luffa acutangula)
Fabaceae (Cyamopsis tetragonoloba)
Alternaria poonensis
Apiaceae (Daucus carota, Coriandrum sativum), Asteraceae (Cichorium intybus)
Alternaria deserticola
Soil
Alternaria dichondrae
Convolvulaceae (Dichondra sp., D. repens)
Alternaria echinaceae
Asteraceae (Echinacea sp.)
Alternaria grandis
Solanaceae (Solanum tuberosum)
Alternaria ipomoeae
Alternaria jesenskae
Alternaria linariae
Cistaceae (Fumana procumbens)
Alternaria cretica
Alternaria cucumericola
Cucurbitaceae (Cucumis sativus), Scrophulariaceae (Linaria maroccana),
Solanaceae (Capsicum frutescens, Solanum lycopersicum)
Alternaria subcylindrica
Alternaria tabasco
Alternaria macrospora
Malvaceae (Gossypium sp., G. barbadense)
Alternaria montanica
Asteraceae (Cirsium arvense)
Alternaria multirostrata
Rubiaceae (Richardia scabra)
Alternaria neoipomoeae
Alternaria nitrimali
Solanacaea (Solanum viarum)
Alternaria novae-guineensis
Asteraceae (Galinsoga parviflora), Rutaceae (Citrus sp.)
Alternaria obtecta
Euphorbiaceae (Euphorbia pulcherrima)
Alternaria paralinicola
Alternaria passiflorae
Linaceae (Linum usitatissimum)
Alternaria gaurae
Alternaria hawaiiensis
Onagraceae (Gaura lindheimeri), Passifloraceae (Passiflora edulis,
P. caerulea, P. ligularis), Solanaceae (Capsicum frutescens)
Alternaria pipionipisi
Euphorbiaceae (Euphorbia pulcherrima), Fabaceae (Cajanus cajan)
Alternaria porri
Amaryllidaceae (Allium cepa, A. porrum)
16
PATHOGENS
Species name
Synonymised names (this study)
Host / Substrate
Alternaria protenta
Alternaria hordeiseminis
Asteraceae (Helianthus annuus), Euphorbiaceae (Euphorbia pulcherrima),
Gramineae (Hordeum vulgare), Solanaceae (Solanum lycopersicum, S. tuberosum)
Alternaria pulcherrimae
Alternaria pseudorostrata
Alternaria ranunculi
Ranunculaceae (Ranunculus asiaticus)
Alternaria ricini
Euphorbiaceae (Ricinus communis)
Alternaria rostellata
Alternaria scorzonerae
Alternaria linicola
Asteraceae (Sorzonerae hispanica), Linaceae (Linum usitatissimum)
Alternaria sennae
Fabaceae (Senna corymbosa)
Alternaria sesami
Pedaliaceae (Sesamum indica)
Alternara sidae
Malvaceae (Sida fallax)
Alternaria silybi
Asteraceae (Silybum marianum)
Alternaria solani
Alternaria danida
Alternaria solani-nigri
Alternaria ascaloniae
Alternaria viciae-fabae
Alternaria beticola
Asteraceae (Ageratum houstonianum), Fabaceae (Vicia faba), Solanaceae
(Solanum aviculare, S. tuberosum)
Amaryllidaceae (Allium ascalonicum), Apiaceae (Petroselinum crispum),
Chenopodiaceae (Beta vulgaris), Gramineae (Glyceria maxima),
Solanaceae (Cyphomandra betacea, Solanum nigrum)
Alternaria cyphomandrae
Alternaria glyceriae
Alternaria herbiculinae
Alternaria steviae
Asteraceae (Stevia rebaudiana)
Alternaria tagetica
Asteraceae (Tagetes sp., T. erecta)
Alternaria thunbergiae
Alternaria iranica
Acanthaceae (Thunbergia alata), Amaryllidaceae (Allium cepa)
Alternaria tillandsiae
Bromeliaceae (Tillandsia usneoides)
Alternaria tropica
Passifloraceae (Passiflora edulis)
Alternaria venezuelensis
Fabaceae (Phaseolus vulgaris)
Alternaria zinniae
Asteraceae (Callistephus chinensis, Zinnia sp., Z. elegans)
Alternaria anagallidis A. Raabe, Hedwigia 78: 87. 1939.
Materials examined: Denmark, Copenhagen, from Anagallis arvensis (Primulaceae), before Mar. 1944, P. Neergaard, CBS 107.44. New Zealand, Auckland,
Lynfield, from Anagallis arvensis, 4 May 1998, C.F. Hill, CBS 101004; Auckland,
Lynfield, from Anagallis arvensis, 28 Jun. 1995, C.F. Hill, representative isolate of
A. anagallidis CBS 117128 = E.G.S. 42.074; Auckland, from leaf spot of Anagallis
arvensis, Jan. 2002, C.F. Hill, representative isolate of A. anagallidis CBS
117129 = E.G.S. 50.091.
Notes: Isolate CBS 107.44 differs on 6 nt positions in its RPB2
sequence from the other three A. anagallidis isolates included in
this study. Because CBS 107.44 still clusters closest to the other
A. anagallidis isolates, and since these isolates, from a single
host species, form a distinct clade from all other Alternaria spp.,
we retained the name A. anagallidis for this isolate.
Alternaria anodae E.G. Simmons, Mycotaxon 88: 198.
2003.
Material examined: South Africa, Gauteng Province, Pretoria, ARC-Roodeplaat
VOPI, from leaves of Anoda cristata (Malvaceae), 12 Jan. 2012, A. Thompson,
PPRI 12376.
Alternaria aragakii E.G. Simmons, Mycotaxon 46: 181.
1993.
Material examined: USA, Hawaii, from Passiflora edulis (Passifloraceae), before
Oct. 1968, M. Aragaki, culture ex-type of A. aragakii CBS 594.93 = E.G.S.
29.016 = QM 9046.
Alternaria argyroxiphii E.G. Simmons & Aragaki, Mycotaxon 65: 40. 1997.
Materials examined: South Africa, Gauteng Province, Pretoria, ARCRoodeplaat VOPI, from stem lesion of Ipomoea batatas (Convolvulaceae), 20
Apr. 2005, A. Thompson, PPRI 11848; Mpumalanga Province, Marble Hall, from
stem and leaf lesion of Ipomoea batatas, 22 Nov. 2011, A. Thompson, PPRI
11971. USA, Hawaii, Maui, Haleakala, from Argyroxiphium sp. (Asteraceae),
1969, M. Aragaki, culture ex-type of A. argyroxiphii CBS 117222 = E.G.S.
35.122.
Note: The host range of A. argyroxiphii is not restricted to
Argyroxiphium, but has been broadened with the inclusion of two
isolates from Ipomoea batatas (Convolvulaceae).
Alternaria azadirachtae E.G. Simmons & Alcorn, CBS
Biodiversity Ser. (Utrecht) 6: 218. 2007.
Materials examined: Australia, Queensland, Tewantin, from Azadirachta indica
(Meliaceae), 20 Jul. 1998, A. Bradley, culture ex-type of A. azadirachtae CBS
116444 = E.G.S. 46.195 = BRIP 25386 (ss1); additional strain from the same
source, CBS 116445 = E.G.S. 46.196 = BRIP25386 (ss2).
Alternaria bataticola W. Yamam., Trans. Mycol. Soc.
Japan 2(5): 89. 1960.
= Macrosporium bataticola Ikata, Agric. Hort. (Tokyo) 22: 241. 1947 (nom.
inval., Art. 36.1).
Type: (Lectotype, designated in Simmons 2007) S. Ikata, Agric.
& Hort. 22: 241. fig. 1. 1947.
17
WOUDENBERG
ET AL.
Fig. 3. Alternaria allii: conidia and conidiophores. A–C. CBS 107.28. D–E. CBS 109.41. F–H. CBS 225.76. I–L. CBS 121345. Scale bars = 10 μm.
Materials examined: Australia, Queensland, Walkamin, from leaf spot of Ipomoea
batatas (Convolvulaceae), 5 Jul. 1991, collector unknown, representative isolate
of A. bataticola CBS 117095 = E.G.S. 42.157 = IMI 350492 = BRIP 19470a;
additional strain from the same source CBS 117096 = E.G.S. 42.158 = BRIP
19470b. Japan, Tokyo, from Ipomoea batatas, before Nov. 1963, collector unknown, CBS 532.63; from Ipomoea batatas, before Nov. 1963, collector unknown
(epitype designated here CBS H-21743, MBT178114, culture ex-epitype CBS
531.63 = IFO 6187 = MUCL 28916). South Africa, Gauteng Province, Pretoria,
ARC-Roodeplaat VOPI, from leaf and stem lesion of Ipomoea batatas, 16 Jun.
2010, M. Truter, PPRI 10502; Kwazulu-Natal Province, Empangeni, from leaf
lesion of Ipomoea batatas, 4 Jul. 2011, A. Thompson, PPRI 11930; Kwazulu-Natal
Province, Empangeni, from leaf lesion of Ipomoea batatas, 4 Jul. 2011, A.
Thompson, PPRI 11931; Gauteng Province, Pretoria, ARC-Roodeplaat VOPI,
from leaf lesion of Ipomoea batatas, 12 Jan. 2012, A. Thompson, PPRI 11934.
Alternaria blumeae E.G. Simmons & Sontirat, Mycotaxon
65: 81. 1997. Fig. 5.
18
= Alternaria brasiliensis F.M. Queiroz, M.F.S. Muniz & M. Menezes, Mycopathologia 150: 63. 2001.
Materials examined: Brazil, Espirito Santo, from leaf spot of Phaseolus vulgaris
(Fabaceae), 1989, F.M. Queiroz, representative isolate of A. brasiliensis CBS
117215 = E.G.S. 39.116. Thailand, Yala Province, Amphoe Muang, from Blumea
aurita (Asteraceae), 18 Jan. 1992, P. Sontirat, culture ex-type of A. blumeae CBS
117364 = E.G.S. 40.149 = ATCC 201357.
Notes: By synonymising A. brasiliensis with A. blumeae, the host
range of this taxon has expanded to include Phaseolus vulgaris.
The five sequenced genes are 100 % identical between the two
examined specimens.
Alternaria calendulae Ondrej, Cas.
Slez. Mus., Ser. A,
Hist. Nat. 23: 150. 1974. Fig. 6.
PATHOGENS
Fig. 4. Alternaria alternariacida sp. nov. CBS 105.51: A–H. Conidia and conidiophores. Scale bars = 10 μm.
Fig. 5. Alternaria blumeae: conidia and conidiophores. A–D. CBS 117364. E–H. CBS 117215. Scale bars = 10 μm.
19
WOUDENBERG
ET AL.
Fig. 6. Alternaria calendulae: conidia and conidiophores. A–C. CBS 224.76. D–E. CBS 101498. F–H. CBS 116650. I–L. CBS 116439. Scale bars = 10 μm.
= Alternaria calendulae W. Yamam. 1939 (nom. nud.).
= Macrosporium calendulae Nelen, Bull. Centr. Bot. Gard. (Moscow) 35: 90.
1959 (nom. inval., Art. 36.1).
= Macrosporium calendulae Nelen, Bot. Mater. Otd. Sporov. Rast. Bot. Inst.
Akad. Nauk S.S.S.R. 15: 144. 1962.
= Alternaria calendulae Nirenberg, Phytopathol. Z. 88: 108. 1977 (nom.
illegit., Art. 53.1).
= Alternaria rosifolii E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 192. 2007.
Materials examined: Germany, former West-Germany, from leaf spot of
Calendula officinalis (Asteraceae), 1974, H. Nirenberg, culture ex-type of
A. calendulae Nirenberg CBS 224.76 = ATCC 38903 = IMI 205077 = DSM
63161. Japan, Tokyo, from leaf spot of Calendula officinalis, before 1964,
representative isolate of A. calendulae CBS 116650 = E.G.S. 30.142 = QM
9561. New Zealand, Auckland, Kumeu, from leaf spot of Calendula officinalis,
Oct. 1998, C.F. Hill, CBS 101498; Auckland, Mount Albert, from leaf of Rosa sp.
20
(Rosaceae), before Feb. 1995, C.F. Hill, culture ex-type of A. rosifolii CBS
116439 = E.G.S. 42.197.
Note: By synonymising A. rosifolii with A. calendulae, the host
range of this taxon has expanded to include Rosa.
Alternaria carthami S. Chowdhury, J. Indian Bot. Soc. 23:
65. 1944. Fig. 7.
= Macrosporium anatolicum A. Savul., Bull. Sect. Sci. Acad. Roumaine 26:
709. 1944.
= Alternaria heliophytonis E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6:
206. 2007.
Materials examined: Canada, Saskatchewan, Saskatoon, from leaf of Helianthus
annuus (Asteraceae), 26 Aug. 1993, C. Jasalavich, culture ex-type of
A. heliophytonis CBS 116440 = IMI 366164 = E.G.S. 43.143. Italy, Perugia, from leaf
PATHOGENS
Fig. 7. Alternaria carthami: conidia and conidiophores. A–D. CBS 117091. E–H. CBS 116440. Scale bars = 10 μm.
of Carthamus tinctorius (Asteraceae), before Nov. 1980, A. Zazzerini, CBS 635.80.
USA, Montana, Sidney, from leaf spot of Carthamus tinctorius, 11 Jul. 1973, E.E.
Burns, representative isolate of A. carthami CBS 117091 = E.G.S. 31.037.
Notes: Isolate CBS 635.80 did not sporulate after 3 wk cultivation
on SNA. By synonymising A. heliophytonis with A. carthami, the
host range of this taxon has expanded to include Helianthus
annuus (Asteraceae).
Alternaria carthamicola Woudenb. & Crous, sp. nov.
Etymology: Named after the host genus from which it was
collected, Carthamus.
Primary conidiophores solitary or in small groups, simple,
straight to slightly curved, septate, pale to dark brown with a
subhyaline tip, (33–)55–71(–108) × 5–6(–7) μm, bearing a
single, darkened, apical conidiogenous locus, but may produce
geniculate conidiogenous extensions. Conidia solitary, rarely in
chains of two conidia, conidium body pale olive-brown, mostly
smooth-walled but sometimes ornamented at the base, ovoid,
(39–)58–64(–82) × (13–)15–16(–17) μm; with (5–)6–7(–9)
transverse and (1–)3(–4) longitudinal septa. Dark coloured
eusepta can be formed during development; the conidial body is
slightly constricted near the transverse septa. Conidia mostly
have a septate, single to double filamentous beak, triple beaks
are observed but not common, apical secondary conidiophores
can be formed. Beaks (40–)158–186(–219) μm long, ca. 2 μm
diam throughout their length and 4 μm at the base. Sexual morph
not observed.
Culture characteristics: After 7 d cultures on SNA flat, rhizoid,
white to opaque; aerial mycelium sparse, white, floccose, colonies reaching 55–60 mm diam; cultures on PCA flat, entire,
olivaceous with three clear concentric circles; aerial mycelium
fine, felty, olivaceous to olivaceous-grey, colonies reaching
65–70 mm diam; reverse shows four olivaceous concentric
circles with an buff edge.
Material examined: Iraq, from Carthamus tinctorius (Asteraceae), 10 Apr. 1983,
M.M. Elsahookie (holotype CBS H-21735, culture ex-type CBS 117092 = IMI
276943 = E.G.S. 37.057).
Notes: The new species A. carthamicola, originally identified as
A. carthami, differs only on 9 nt positions in its RPB2 sequence
from the other two A. carthami strains studied. Based on its
RPB2 sequence it clusters with A. linicola.
Alternaria cassiae Jurair & A. Khan, Pakistan J. Sci.
Industr. Res. 3: 72. 1960. Fig. 9.
= Alternaria hibiscinficiens E.G. Simmons & C.F. Hill, Mycotaxon 88: 205.
2003.
= Alternaria sauropodis E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6:
340. 2007.
Materials examined: Brazil, Federal District, from leaf spot of Senna obtusifolia
(Fabaceae), May 1990, G. Fiqueiredo, representative isolate of A. cassiae CBS
117224 = E.G.S. 40.121. Fiji, from leaf of Hibiscus sabdariffa (Malvaceae), Jun.
21
WOUDENBERG
ET AL.
Fig. 8. Alternaria carthamicola sp. nov. CBS 117092: A–L. Conidia and conidiophores. Scale bars = 10 μm.
2002, C.F. Hill, culture ex-type of A. hibiscinficiens CBS 177369 = E.G.S. 50.166.
Malaysia, Sarawak, Kuching, from Sauropus androgynus (Phyllanthaceae), 25
Apr. 1984, T.K. Kieh, culture ex-type of A. sauropodis CBS 116119 = IMI
286317 = IMI 392448 = E.G.S. 47.112. USA, Mississippi, Stoneville, from
diseased seedling of Senna obtusifolia, before Oct. 1980, H.L. Walker, representative isolate of A. cassiae CBS 478.81 = E.G.S. 33.147.
Notes: Isolate CBS 478.81 did not sporulate after 3 wk incubation
on SNA. By synonymising A. hibiscinficiens and A. sauropodis
with A. cassiae, the host range of this taxon has expanded to
include Sauropus androgynus (Euphorbiaceae) and Hibiscus
sabdariffa (Malvaceae).
Alternaria catananches Woudenb. & Crous, sp. nov.
22
Etymology: Named after its host genus from which it was isolated, Catananche.
Primary conidiophores solitary, simple, straight to curved, septate,
pale brown, (31–)54–67(–94) × (5–)6(–7) μm, bearing a single,
darkened, apical conidiogenous locus, but may produce geniculate
conidiogenous extensions. Conidia solitary, conidium body pale
olive-brown, ornamented in lower half of the conidium, narrowly
ovoid, (26–)37–43(–57) × (7–)8–9(–11) μm, with (2–)4(–6)
transverse septa and no longitudinal septa. Some darker coloured
eusepta can be formed during development. The conidium body
gradually tapers into a single, septate, unbranched beak; basal
lateral secondary conidiophores can be formed. Beaks (77–)
PATHOGENS
Fig. 9. Alternaria cassiae: conidia and conidiophores. A–D. CBS 116119. E–H. CBS 117224. I–L. CBS 117369. Scale bars = 10 μm.
126–160(–260) μm long, ca. 2 μm diam throughout their length.
Sexual morph not observed.
Culture characteristics: After 7 d cultures on SNA flat, entire/
fimbriate, olivaceous around agar plug, white; aerial mycelium
felty, white to olivaceous, colonies reaching 10–15 mm diam;
cultures on PCA flat, erose, grey-olivaceous; aerial mycelium
fine felty, olivaceous-grey; colonies reaching 25 mm diam;
reverse identical.
Material examined: Netherlands, from Catananche caerulea (Asteraceae), 11
Dec. 2013, N. Troost-Riksen (holotype CBS H-21736, culture ex-type CBS
137456 = PD 013/05703936).
Notes: Alternaria catananches seems closely related to the
A. cichorii isolates in the multi-gene phylogeny, but this is
probably caused by long-branch attraction and incongruency
between the different gene trees. Based on the ITS sequence it
is identical to A. jesenskae, with RPB2 it is identical to
A. cirsinoxia, with TEF1 it clusters with A. cichorii/A. cirsinoxia/
A. carthami and with Alt a 1 it is identical to A. cichorii CBS
102.33, A. alternariacida and A. scorzonerae. Only its GAPDH
sequences make it distinct from all other Alternaria species.
Although the multi-gene tree does not provide strong support for
separating it from the A. cichorii isolates, based on the individual
gene sequences it is described here as a new Alternaria species.
23
WOUDENBERG
ET AL.
Fig. 10. Alternaria catananches sp. nov. A–B. Disease symptoms on Catananche caerulea (photo's K.-H. Nugteren, Florensis B.V., Netherlands). C–L. CBS 137456: conidia
and conidiophores. Scale bars = 10 μm.
Alternaria centaureae E.G. Simmons, CBS Biodiversity
Ser. (Utrecht) 6: 236. 2007.
Specimen examined: USA, California, Sacramento, from Centaurea solstitialis
(Asteraceae), Feb. 1999, D. Fogle, culture ex-type of A. centaureae CBS
116446 = E.G.S. 47.119.
Alternaria cichorii Nattrass, First List of Cyprus Fungi: 29.
1937.
≡ Alternaria porri f. sp. cichorii (Nattrass) T. Schmidt, Pflanzenschutzberichte 32: 181. 1965.
≡ Macrosporium cichorii (Nattrass) Gordenko, Mikol. Fitopatol. 9: 241.
1975.
24
Materials examined: Cyprus, from leaf spot of Cichorium intybus (Asteraceae),
1933, R.M. Nattrass (holotype IMI 1007, culture ex-type CBS 102.33 = E.G.S.
07.017 = QM 1760). Greece, Attica, from Cichorium endivia (Asteraceae), 24
Feb. 1978, S.D. Demetriades, representative isolate of A. cichorii CBS
117218 = E.G.S. 52.046 = IMI 225641.
Notes: Strain CBS 102.33 was deposited in Aug. 1933 in the
CBS by R.M. Nattrass as A. cichorii sp. nov., with the remark that
the description of the new species was in preparation. The holotype was subsequently deposited in IMI (IMI 1007) which
consists of a dried herbarium specimen. In the present study we
link CBS 102.33 as ex-type of A. cichorii to IMI 1007. The two
isolates used in this study, CBS 102.33 and CBS 117218, differ
only on 7 nt positions in their Alt a 1 sequence. Unfortunately
CBS 102.33 is sterile, which does not provide additional
PATHOGENS
Fig. 11. Alternaria citrullicola sp. nov. CBS 103.32: A–H. Conidia and conidiophores. Scale bars = 10 μm.
Alternaria cirsinoxia E.G. Simmons & K. Mort., Mycotaxon 65: 72. 1997.
white to opaque with primrose sections near the edge; aerial
mycelium sparse, fine felty, colonies reaching 45–50 mm diam;
cultures on PCA flat, entire, olivaceous with three unclear
concentric circles; aerial mycelium is sparse, pale olivaceousgrey, colonies reaching 50–55 mm diam; reverse shows
olivaceous-buff to olivaceous rings.
Material examined: Canada, Saskatchewan, Watrous, from stem lesion and top
dieback of Cirsium arvense (Asteraceae), 5 Aug. 1993, K. Mortensen, culture extype of A. cirsinoxia CBS 113261 = E.G.S. 41.136.
Material examined: Cyprus, from fruit of Citrullus lanatus (Cucurbitaceae), before
Jul. 1932, R.M. Nattrass (holotype CBS H-21742, culture ex-type CBS
103.32 = VKM F-1881).
Alternaria citrullicola Woudenb. & Crous, sp. nov.
Alternaria conidiophora Woudenb. & Crous, sp. nov.
collected, Citrullus.
Etymology: Named after its characteristically long, thick,
conidiophores.
Primary conidiophores solitary, simple, straight or sometimes
curved, septate, pale brown with a subhyaline tip, (28–)
35–52(–73) × (3–)4(–5) μm, bearing a single, darkened, apical
conidiogenous locus. Conidia mostly solitary but chains of two
conidia can occur, conidium body pale olive-brown, smoothwalled, narrowly ovoid, (28–)35–41(–56) × (6–)8(–10) μm; with
(3–)5–6(–9) transverse distosepta and 0–1(–2) longitudinal
septa. Conidia have a single, aseptate, unbranched filamentous
beak; apical secondary conidiophores can be formed. Beaks
(72–)178–232(–324) μm long, ca. 2 μm diam throughout their
Primary conidiophores solitary, simple, mostly straight but
sometimes curved, septate, dark brown with a subhyaline tip,
(46–)89–105(–152) × (6–)7(–8) μm, bearing a single to multiple, darkened, long geniculate conidiogenous loci. Conidia
solitary, conidium body olive-brown, smooth-walled, narrowly
ovoid, (30–)45–52(–66) × (10–)12–13(–18) μm, with (2–)
6–7(–9) transverse septa and (0–)1–2(–4) longitudinal septa.
Darker coloured eusepta are formed during development. The
conidial body is slightly constricted near the transverse septa.
Conidia have a single, septate, unbranched, filamentous beak;
basal, lateral secondary conidiophores can be formed. Beaks
information to support them as being two different species.
Furthermore, the time difference of 45 yr between isolation of the
two strains led to the decision to retain them as one species for
now, pending fresh collections.
25
WOUDENBERG
ET AL.
Fig. 12. Alternaria conidiophora sp. nov. CBS 137457: A–H. Conidia and conidiophores. Scale bars = 10 μm.
(49–)117–138(–186) μm long; ca. 2 μm diam throughout their
Culture characteristics: After 7 d cultures on SNA flat, fimbriate to
rhizoid, white to opaque; aerial mycelium felty, white, colonies
reaching 55–60 mm diam; cultures on PCA flat, entire, greyolivaceous with two concentric circles; aerial mycelium wooly,
pale olivaceous-grey, colonies reaching 55–60 mm diam;
reverse identical.
Material examined: Netherlands, from unidentified host, Jul. 2011, U. Damm
(holotype CBS H-21737, culture ex-type CBS 137457).
Alternaria crassa (Sacc.) Rands, Phytopathology 7: 337.
1917. Fig. 13.
Basionym: Cercospora crassa Sacc., Michelia 1(no. 1): 88. 1877.
= Macrosporium solani Cooke, Grevillea 12: 32. 1883. (non M. solani Ellis &
Martin, 1882)
= Cercospora daturae Peck, Rep. New York State Mus. Nat. Hist. 35: 140.
1884.
= Macrosporium cookei Sacc., Syll. Fungorum 4: 530. 1886. (nom. nov. in
Saccardo for M. solani Cooke, 1883, non M. solani Ellis & Martin, 1882)
€
€
≡ Alternaria cookei (Sacc.) Bremer, Iʂmen, Karel, Ozkan
& M. Ozkan,
Istanbul Üniv. Fak. Mecm., B. 13: 42. 1948.
= Macrosporium daturae Fautrey, Rev. Mycol. (Toulouse) 16: 76. 1894.
≡ Alternaria daturae (Fautrey) Bubak & Ranoj., Fungi Imperf. Exsicc.
Fasc. 14: 694. 1911.
= Alternaria capsici E.G. Simmons, Mycotaxon 75: 84. 2000.
Type: (Lectotype, designated in Simmons 2000) PAD, Cercospora crassa, Datura stramonium, S. [elva] 0 76. 10.
26
Materials examined: Australia, from Capsicum annuum (Solanaceae), May 1981,
D. Trimboli, culture ex-type of A. capsici CBS 109160 = IMI 262408 = IMI
381021 = E.G.S 45.075. Cyprus, Famagusta, from leaves of Datura stramonium
(Solanaceae), Jan. 1936, R.M. Nattrass (epitype designated here CBS H-21744,
MBT178115, culture ex-epitype CBS 110.38). New Zealand, Auckland, from leaf
spot of Datura stramonium, 2002, C.F. Hill, representative isolate of A. crassa CBS
116448 = E.G.S. 50.180. USA, Indiana, Montgomery County, Nicandra physalodes
(Solanaceae), 5 Sep. 1997, E.G. Simmons, CBS 109162 = E.G.S. 46.014; Indiana,
from leaf spot of Datura stramonium, 5 Sep. 1997, E.G. Simmons, representative
isolate of A. crassa CBS 116447 = E.G.S. 46.013; Indiana, Montgomery County,
from leaf spot of Datura stramonium, 1 Aug. 1996, E.G. Simmons, representative
isolate of A. crassa CBS 122590 = E.G.S. 44.071; Wisconsin, Madison, from leaf
spot of Datura sp., before Apr. 1918, R.D. Rands, CBS 103.18.
Notes: Isolates CBS 110.38 and CBS 116647 did not sporulate
after 3 wk incubation on SNA. By synonymising A. capsici with
A. crassa, the host range of this taxon expanded to include
Capsicum annuum, which also belongs to the Solanaceae.
Alternaria cucumerina (Ellis & Everh.) J.A. Elliott, Amer.
J. Bot. 4: 472. 1917. Fig. 14.
Basionym: Macrosporium cucumerinum Ellis & Everh., Proc.
Acad. Nat. Sci. Philadelphia 47: 440. 1895.
= Alternaria loofahae E.G. Simmons & Aragaki, CBS Biodiversity Ser.
(Utrecht) 6: 316. 2007.
Materials examined: Australia, Queensland, from leaf spot of Cucumis melo
(Cucurbitaceae), Oct. 1996, R. O’Brien, representative isolate of A. cucumerina
CBS 117226 = E.G.S. 44.197 = BRIP 23060. USA, Hawaii, Oahu, Waialua, from
Luffa acutangula (Cucurbitaceae), 1971, M. Aragaki, culture ex-type of
A. loofahae CBS 116114 = E.G.S. 35.123; Indiana, Knox County, from leaf spot of
Cucumis melo, 1993, R.X. Latin, representative isolate of A. cucumerina CBS
117225 = E.G.S. 41.127.
PATHOGENS
Fig. 13. Alternaria crassa: conidia and conidiophores. A–D. CBS 109162. E–H. CBS 116648. I–L. CBS 119160. Scale bars = 10 μm.
Notes: The species clade for A. cucumerina does not have a
clear support in the multi-gene phylogeny. CBS 117225 and CBS
117226 differ only on 2 nt in their RPB2 sequence, while the extype of A. loofahae (CBS 116114) differs on 1 nt from both
A. cucumerina isolates in RPB2 and on 1 nt in Alt a 1. This internal variation in the two A. cucumerina isolates and the identical host family, Cucurbitaceae, with A. loofahae, supported the
synonymy of A. loofahae. By synonymising A. loofahae with
A. cucumerina, the host range of this taxon expanded to include
Luffa acutangula.
Alternaria cyamopsidis Rangaswami & A.V. Rao, Indian
Phytopathol. 10: 23. 1957.
≡ Alternaria cucumerina var. cyamopsidis (Rangaswami & A.V. Rao)
E.G. Simmons, Mycopathol. Mycol. Appl. 29: 131. 1966.
Materials examined: USA, Georgia, from leaf spot of Cyamopsis tetragonoloba
(Fabaceae), Jul. 1961, G. Sowell, representative isolate of A. cyamopsidis CBS
117219 = E.G.S. 13.120 = QM 8000; Maryland, Beltsville, from leaf spot of
Cyamopsis tetragonoloba, 1964, R.G. Orellana, representative isolate of
A. cyamopsidis CBS 364.67 = E.G.S. 17.065 = QM 8575.
Alternaria dauci (J.G. Kühn) J.W. Groves & Skolko,
Canad. J. Res., Sect. C, Bot. Sci. 22: 222. 1944. Fig. 15.
Basionym: Sporidesmium exitiosum var. dauci J.G. Kühn, Hedwigia 1: 91. 1855.
≡ Polydesmus exitiosus var. dauci (J.G. Kühn) J.G. Kühn, Die Krankheiten der Kulturgew€achse, ihre Ursachen und ihre Verhütung: 165.
1858.
≡ Macrosporium dauci (J.G. Kühn) Rostr., Tidsskr. Landoekon. ser. 5, 7:
385. 1888.
≡ Alternaria brassicae var. dauci (J.G. Kühn) Lindau, Rabenhorst‘s
Kryptog.-Fl., Edn 2 (Leipzig) 1(9): 260. 1908.
27
WOUDENBERG
ET AL.
Fig. 14. Alternaria cucumerina: conidia and conidiophores. A–D. CBS 117225. E–H. CBS 117226. I–L. CBS 116114. Scale bars = 10 μm.
≡ Alternaria porri f. sp. dauci (J.G. Kühn) Neerg, Danish species of
Alternaria & Stemphylium: 252. 1945.
= Macrosporium carotae Ellis & Langl., J. Mycol. 6: 36. 1890.
≡ Alternaria carotae (Ellis & Langl.) J.A. Stev. & Wellman, J. Wash.
Acad. Sci. 34: 263. 1944.
= Alternaria poonensis Ragunath, Mycopathol. Mycol. Appl. 21: 315. 1963.
Type: (Lectotype, designated in Simmons 1995) B, ms. spec.
Sporidesmium exitiosum var. dauci Kühn, Leg. Gross Krausche
p. Bunzlau, Jul. Kühn.
Materials examined: Italy, from seed of Daucus carota (Apiaceae), Sept. 1937, P.
Neergaard (neotype designated here CBS H-21745, MBT178116, culture exneotype CBS 111.38). Netherlands, Limburg, Horst, from leaf spot in Cichorium
intybus var. foliosum (Asteraceae), 1979, W.M. Loerakker, CBS 477.83 = CBS
721.79 = PD 79/954; from seed of Daucus carota, 1993, S&G Seeds, CBS
28
101592. New Zealand, from leaf spot of Daucus carota, Mar. 1998, C.F. Hill,
representative isolate of A. dauci CBS 117098 = E.G.S. 46.152; Ohakune, from
leaf spot of Daucus carota, before Jul. 1979, G.F. Laundon, CBS 345.79 = LEV
14814. Puerto Rico, from seedling of Coriandrum sativum (Apiaceae), 1999, W.
Almodovar, representative isolate of A. poonensis CBS 117100 = E.G.S. 47.138.
Unknown, from seed of Daucus carota, Jan. 1948, J.W. Groves, CBS 106.48.
USA, California, from commercial seed of Daucus carota, Nov. 1994, B.M. Pryor,
representative isolate of A. dauci CBS 117097 = E.G.S. 46.006; California, Kern
County, from seed of Daucus carota, 1999, D. Fogle, representative isolate of
A. dauci CBS 117099 = E.G.S. 47.131.
Notes: The indicated lectotype cannot be traced in B, and appears to be lost. We therefore designate CBS 111.38 as neotype.
The isolates CBS 111.38, CBS 345.79 and CBS 101592 did not
sporulate after 3 wk incubation on SNA.
PATHOGENS
Fig. 15. Alternaria dauci. A. Disease symptoms on Daucus carota. B–L. Conidia and conidiophores. B–C. CBS 117097. D–F. CBS 117098. G–I. CBS 117099. J–L. CBS
117100. Scale bars = 10 μm.
Alternaria deserticola Woudenb. & Crous, sp. nov.
MycoBank MB808996.
Etymology: Named after the substrate from which it was isolated,
namely desert soil.
Culture sterile
Alternaria deserticola differs from the ex-type strain of its
closest phylogenetic neighbour A. thunbergiae (CBS
116331) based on alleles in all five loci (positions derived
from respective alignments of the separate loci deposited in
TreeBASE): ITS position 165 (−), 373 (T), 381 (C), 383 (C),
488 (A); GAPDH position 484 (T); RPB2 position 76 (C), 88
(T), 91 (T), 139 (C), 211 (T), 316 (T), 490 (C), 496 (A), 646
(T), 670 (C), 671 (T), 673 (A), 760 (G); TEF1 position 37 (C),
49 (G), 197 (A), 223 (A), 274 (T), 277(–), 311(T); Alt a 1
position 10 (C), 209 (A), 210 (T), 220 (G), 322 (T), 452 (G).
Culture characteristics: After 7 d cultures on SNA flat, rhizoid,
olivaceous-buff; aerial mycelium absent, colonies reaching
55 mm diam; cultures on PCA flat, entire, five grey-olivaceous
concentric circles; aerial mycelium sparse, colonies reaching
75–80 mm diam; reverse shows five olivaceous-grey rings.
29
WOUDENBERG
ET AL.
Fig. 16. Alternaria grandis: conidia and conidiophores. A–D. CBS 109158. E–H. CBS 116695. Scale bars = 10 μm.
Material examined: Namibia, from desert soil, 2001, M. Christensen (holotype
CBS H-21738, culture ex-type CBS 110799).
Note: The clear phylogenetic distinction of the sterile culture of
A. deserticola from all other strains included in this study, resulted in
our decision to describe this species based on sequence data only.
Alternaria dichondrae Gambogi, Vannacci & Triolo,
Trans. Brit. Mycol. Soc. 65(2): 323. 1975.
Materials examined: Italy, Pisa, from leaf spot of Dichondra repens (Convolvulaceae), Mar. 1974, P. Gambogi, ex-isotype of A. dichondrae CBS
199.74 = E.G.S. 38.007; Pisa, from leaf spot of Dichondra repens, Mar. 1974, P.
Gambogi, living lectotype of A. dichondrae CBS 200.74 = E.G.S. 38.008. New
Zealand, from leaf spot of Dichondra repens, before 1979, G.F. Laundon, CBS
346.79; Auckland, Lynfield, from leaf of Dichondra sp., Apr. 1991, C.F. Hill,
representative isolate of A. dichondrae CBS 117127 = E.G.S. 40.057.
Note: Simmons (2007) designated a lectotype with ex-lectotype
strain (CBS 200.74), as he found the ex-isotype strain (CBS
199.74) to be sterile.
Alternaria echinaceae E.G. Simmons & C.F. Hill, CBS
Materials examined: New Zealand, Gisborne, Makaraka, from leaf of Echinacea
sp. (Asteraceae), Jan. 1998, C.F. Hill, culture ex-type of A. echinaceae CBS
116117 = E.G.S. 46.081; Gisborne, Makaraka, from leaf of Echinacea sp., Jan.
1998, C.F. Hill, representative isolate of A. echinaceae CBS 116118 = E.G.S.
46.082.
30
Alternaria grandis E.G. Simmons, Mycotaxon 75: 96.
2000. Fig. 16.
Materials examined: USA, Pennsylvania, Centre County, from leaf lesion of
Solanum tuberosum (Solanaceae), Sep. 1966, B.J. Christ, culture ex-type of
A. grandis CBS 109158 = E.G.S. 44.106; Pennsylvania, Clarion County, from leaf
spot of Solanum tuberosum, Sep. 1966, B.J. Christ, representative isolate of
A. grandis CBS 116695 = E.G.S 44.108.
Notes: Although A. grandis differs by only 1 nt in its GAPDH
sequence from A. solani, we retain it as a distinct species.
Conidia of A. grandis are substantially larger than those of
A. solani, and a recently published study could separate A. solani
(CBS 109157) and A. grandis (CBS 109158) based on partial
calmodulin gene sequence data (Gannibal et al. 2014).
Alternaria ipomoeae M. Truter, Woudenb. & Crous, sp.
nov. MycoBank MB808997. Fig. 17.
Etymology: Named after the host genus on which it occurs,
Ipomoea.
Primary conidiophores simple to branched, straight to slightly
curved, septate, pale brown, (10–)51–73(–145) × (4–)5 μm,
bearing a single to multiple, darkened, geniculate conidiogenous
loci. Conidia mostly solitary but chains of two conidia can occur,
conidium body olive-brown, smooth-walled with ornamented
base, long ellipsoid to obclavate, (53–)60–65(–76) × (9–)
12(–15) μm, with (6–)8–9(–12) transverse septa and (0–)2(–3)
longitudinal septa. Up to four dark coloured eusepta can be
PATHOGENS
Fig. 17. Alternaria ipomoeae sp. nov. CBS 219.79: A–L. Conidia and conidiophores. Scale bars = 10 μm.
formed during development; the conidial body is constricted near
these eusepta. Conidia have a septate, single to double, filamentous beak; apical and lateral secondary conidiophores can
be formed. Beaks (47–)136–162(–221) μm long, single beaks
generally longer than multiple beaks, ca. 2 μm diam throughout
their length, and approx. 3 μm diam at the base. Sexual morph
not observed.
Materials examined: Ethiopia, from black lesions of Ipomoea batatas (Convolvulaceae), Jun. 1978, A.H.C. van Bruggen (holotype CBS H-21739, culture
ex-type CBS 219.79). South Africa, Gauteng Province, Pretoria, ARCRoodeplaat VOPI, from stem lesions of Ipomoea batatas, 16 Nov. 2006, C.D.
Narayanin (paratype PREM 60979, culture ex-paratype PPRI 8988).
Culture characteristics: After 7 d cultures on SNA are flat,
fimbriate, white; aerial mycelium sparse, felty, white, colonies
reaching 50 mm diam; cultures on PCA flat, entire, greyolivaceous with some darker sections; aerial mycelium fine
felty, pale olivaceous-grey, colonies reaching 65–70 mm diam;
reverse identical.
Material examined: Slovakia, district of the village Muzla, Podunajska nizina
lowland, from seeds of Fumana procumbens (Cistaceae), Aug. 1999, P. Elias jr.,
culture ex-type of A. jesenskae CBS 133855 = CCM 8361.
Alternaria jesenskae Labuda, P. Elias & Sterfl., Microbiol.
Res. 163: 209. 2008.
Alternaria linariae (Neerg.) E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 677. 2007. Fig. 18.
31
WOUDENBERG
ET AL.
Fig. 18. Alternaria linariae. A. Disease symptoms on Solanum lycopersicum. B–P. Conidia and conidiophores. B–C. CBS 105.41. D–F. CBS 109161. G–H. CBS 107.61. I–J.
CBS 109156. K–L. CBS 109164. M–N. CBS 116438. O–P. CBS 116441. Scale bars = 10 μm.
32
Basionym: Alternaria anagallidis var. linariae Neerg., Danish
species of Alternaria & Stemphylium: 297. 1945.
= Alternaria cretica E.G. Simmons & Vakal., Mycotaxon 75: 64. 2000.
= Alternaria subcylindrica E.G. Simmons & R.G. Roberts, Mycotaxon 75: 62.
2000.
= Alternaria tomatophila E.G. Simmons, Mycotaxon 75: 53. 2000.
= Alternaria cucumericola E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 210. 2007.
= Alternaria tabasco E.G. Simmons & R.G. Roberts, CBS Biodiversity Ser.
(Utrecht) 6: 158. 2007.
Materials examined: Belgium, host unknown, before Mar. 1961, R. Sys, CBS
107.61. Denmark, from seedling of Linaria maroccana (Scrophulariaceae), 13
Nov. 1940, P. Neergaard, culture ex-type of A. linariae CBS 105.41 = E.G.S.
07.016. Greece, Crete, Heraklio, from leaf spot of Solanum lycopersicum
(Solanaceae), 1997, D.J. Vakalounakis, culture ex-type of A. cretica, CBS
109164 = E.G.S. 46.188. New Zealand, Northland, Kerikeri, from leaf spot of
Cucumis sativus (Cucurbitaceae), Mar. 1993, C.F. Hill, culture ex-type of
A. cucumericola CBS 116438 = E.G.S. 41.057. Thailand, Chiang Mai, Royal
project, from leaf spot of Solanum lycopersicum, 5 Nov. 2012, P.W. Crous,
CPC 21620. Unknown, host unknown, before Apr. 1953, P.W. Brian, CBS
108.53 = No. 408P. USA, Indiana, Montgomery County, from leaf spot of
Solanum lycopersicum, 23 Aug. 1995, E.G. Simmons, culture ex-type of
A. tomatophila CBS 109156 = E.G.S. 42.156; Indiana, from leaf lesion of
Solanum lycopersicum, Aug. 1996, E.G. Simmons, representative isolate of
A. tomatophila CBS 116704 = E.G.S. 44.074; Louisiana, Baton Rouge, Louisiana State University Burden Research Plantation, from leaf lesion of Solanum lycopersicum var. cerasiforme, 2 Jul. 1997, R.G. Roberts, culture extype of A. subcylindrica CBS 109161 = E.G.S. 45.113; Louisiana, Avery Island, from leaf spot of Capsicum frutescens (Solanaceae), 1 Jul. 1997, R.G.
Roberts, culture ex-type of A. tabasco CBS 116441 = E.G.S 45.108 = R.G.R.
97-52.
Notes: By synonymising A. cretica, A. cucumericola,
A. subcylindrica, A. tabasco and A. tomatophila with A. linariae, the
broad host range of this taxon now consists of Solanaceae,
Cucurbitaceae and Scrophulariaceae species. The isolates CBS
108.53 and CBS 116704 did not sporulate on SNA after 3 wk of
incubation.
Alternaria macrospora Zimm.,
Deutsch-Ostafrika 2: 24. 1904.
Ber.
Materials examined: USA, Georgia, Tifton, from floral bract of Richardia scabra
(Rubiaceae), 1967, C.R. Jackson, culture ex-type of A. multirostrata CBS
712.68 = ATCC 18515 = IMI 135454 = MUCL 11722 = QM 8820 = VKM-F2997;
Georgia, Tifton, from floral bract of Richardia scabra, 1967, C.R. Jackson,
representative isolate of A. multirostrata CBS 713.68 = ATCC 18517 = IMI
135455 = MUCL 11715 = QM 8821.
Alternaria neoipomoeae M. Truter, Woudenb. & Crous,
sp. nov. MycoBank MB808998. Fig. 19.
Etymology: Named after its close phylogenetic relationship to
A. ipomoeae.
Primary conidiophores solitary, simple, straight to slightly curved,
septate, pale brown, (10–)23–59(–111) × (4–)5 μm, bearing a
single, darkened, apical conidiogenous locus, which may produce
1–2 geniculate conidiogenous extensions. Conidia are mostly
solitary but chains of two conidia can occur, conidium body olivebrown, smooth-walled with ornamented base, long ellipsoid to
obclavate, (52–)66–77(–93) × (12–)14–16(–18) μm, with (7–)
9(–12) transverse and (2–)3–4(–5) longitudinal septa. Up to four
dark coloured eusepta can be formed during development; the
conidial body is constricted near these eusepta. Conidia mostly
have a septate, single to double, filamentous beak, triple beaks are
observed but not common; apical and lateral secondary conidiophores can be formed. Beaks (54–)104–136(–200) μm long,
ca. 2 μm diam throughout their length, and approx. 3 μm diam at the
base. Sexual morph not observed.
white to opaque; aerial mycelium sparse, fine felty, white, colonies reaching 60−65 mm diam; cultures on PCA flat, entire,
grey-olivaceous with 2 dark and one lighter concentric circles
and a pale olivaceous edge; aerial mycelium fine felty, pale
olivaceous-grey, colonies reaching 55–60 mm diam; reverse
four olivaceous-grey rings.
Land-Forstw.
≡ Macrosporium macrosporum (Zimm.) Nishikado & Oshima, Agric.
Res. (Kurashiki) 36: 391. 1944.
= Sporidesmium longipedicellatum Reichert, Bot. Jahrb. Syst. 56: 723. 1921.
≡ Alternaria longipedicellata (Reichert) Snowden, Rep. Dept. Agric.
Uganda: 31. 1927 [1926].
Materials examined: Nigeria, from Gossypium sp. (Malvaceae), May 1929, Jones,
CBS 106.29. USA, Arizona, from Gossypium barbadense (Malvaceae), before
1984, P.J. Cotty, culture epitype of A. macrospora CBS 117228 = E.G.S.
50.190 = ATCC 58172.
Notes: Isolate CBS 106.29 was preserved in the CBS collection
as A. porri, but did not sporulate since 1978. Based on our
molecular data this isolate belongs to A. macrospora, which,
based on the same host, seems plausible.
Alternaria montanica E.G. Simmons & Robeson, CBS
Material examined: USA, Montana, from Cirsium arvense (Asteraceae), before
Apr. 1981, D.J. Robeson, culture ex-type of A. montanica CBS 121343 = E.G.S.
44.112 = IMI 257563.
Alternaria multirostrata E.G. Simmons & C.R. Jacks.,
Phytopathology 58: 1139. 1968.
PATHOGENS
Materials examined: South Africa, Gauteng Province, Pretoria, ARC-Roodeplaat
VOPI, from stem lesion of Ipomoea batatas (Convolvulaceae), 8 Jun. 2011, A.
Thompson (holotype PREM 60981, culture ex-type PPRI 11845); North-West
Province, Brits, from Ipomoea batatas, 25 Oct. 2007, C.D. Narayanin (paratype PREM 60982, culture ex-paratype PPRI 8990); Mpumalanga Province,
Kwamahlanga, from Ipomoea batatas, between 2006 and 2008, C.D. Narayanin
(paratype PREM 60983, culture ex-paratype PPRI 11847); Gauteng Province,
Pretoria, ARC-Roodeplaat VOPI, from leaf lesion of Ipomoea batatas, Oct. 2013,
A. Thompson (paratype PREM 60984, culture ex-paratype PPRI 13903).
Alternaria nitrimali E.G. Simmons & M.E. Palm, Mycotaxon 75: 93. 2000.
Material examined: Puerto Rico, Luquillo, from leaf spot of Solanum viarum
(Solanaceae), 26 Feb. 1998, USDA-APHIS, culture ex-type of A. nitrimali CBS
109163 = E.G.S 46.151.
Alternaria novae-guineensis E.G. Simmons & C.F. Hill,
CBS Biodiversity Ser. (Utrecht) 6: 350. 2007.
Materials examined: Papua New Guinea, from dried leaf of Citrus sp. (Rutaceae)
imported to New Zealand, 1999, C.F. Hill, culture ex-type of A. novae-guineensis
CBS 116120 = E.G.S. 47.198. South Africa, Gauteng, Pretoria, ARC-Roodeplaat
VOPI, from leaves of Galinsoga parviflora (Asteraceae), 12 Jan. 2012, A.
Thompson, PPRI 12171.
Alternaria obtecta E.G. Simmons, Mycotaxon 50: 250.
1994.
33
WOUDENBERG
ET AL.
Fig. 19. Alternaria neoipomoeae sp. nov. A. Disease symptoms on Ipomoeae batatas (Photo A.H. Thompson, ARC, South Africa). B–L. PPRI 11845: conidia and
conidiophores. Scale bars = 10 μm.
Materials examined: USA, California, Encinitas, from leaf of Euphorbia pulcherrima (Euphorbiaceae), Nov. 1994, C.F. Hill, representative isolate of
A. obtecta CBS 117367 = E.G.S. 42.063; California, Encinitas, from Euphorbia
pulcherrima (Euphorbiaceae), Nov. 1994, C.F. Hill, CBS 134278 = E.G.S. 42.064.
Alternaria paralinicola Woudenb. & Crous, sp. nov.
Etymology: Named after its close phylogenetic relationship to
A. linicola.
septate, pale brown, (39–)64–82(–133) × (4–)5–6 μm, bearing a
34
single, darkened, apical conidiogenous locus, but may produce
geniculate conidiogenous extensions. Conidia are mostly
solitary but chains of two conidia can occur, conidium body
pale olive-brown, smooth-walled, narrowly ovoid, (31–)
39–44(–58) × (8–)10–11(–15) μm, with (3–)5–6(–8) transverse
septa and 0–1(–2) longitudinal septa. Dark coloured eusepta are
formed during maturation. The conidial body is slightly constricted
near the transverse septa. Some transverse blocks of cells can
have a conspicuously different width in comparison with neighbouring segments, resulting in specific shape of the conidium body.
Conidia mostly have a single, aseptate, unbranched, filamentous
beak; double beaks are observed but not common; apical or lateral
secondary conidiophores can be formed. Beaks (61–)
PATHOGENS
Fig. 20. Alternaria paralinicola sp. nov. CBS 116652: A–L. Conidia and conidiophores. Scale bars = 10 μm.
114–135(–169) μm long, ca. 2 μm diam throughout their length.
Sexual morph not observed.
white to opaque; aerial mycelium sparse, white, colonies
reaching 70–75 mm diam; cultures on PCA flat, entire, greyolivaceous with four olivaceous clear concentric circles; aerial
mycelium is fine felty, olivaceous, colonies reaching 70 mm diam;
reverse shows five grey-olivaceous concentric circles.
Material examined: Canada, Manitoba, from seeds of cultivated Linum usitatissimum (Linaceae), 1996, M.E. Corlett (holotype CBS H-21740, culture extype CBS 116652 = E.G.S. 47.157 = DAOM 225747).
Note: Alternaria paralinicola, which was originally identified as
A. linicola, differs on 16 nt positions in its RPB2 sequence from
the other two A. linicola strains studied. Based on its RPB2
sequence it clusters with A. passiflorae.
Alternaria passiflorae J.H. Simmonds, Proc. Roy. Soc.
Queensland. 49: 151. 1938. Fig. 21.
= Alternaria hawaiiensis E.G. Simmons, Mycotaxon 46: 184. 1993.
= Alternaria gaurae E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 188. 2007.
Materials examined: New Zealand, from fruit of Passiflora edulis (Passifloraceae), 6 Feb. 1963, F.J. Mortin, representative isolate of A. passiflorae CBS
629.93 = E.G.S. 16.150 = QM 8458; Auckland, from fruit spot of Passiflora
ligularis (Passifloraceae), Apr. 2004, C.F. Hill, representative isolate of
A. passiflorae CBS 117102 = E.G.S. 51.165; Auckland, from leaf spot of
35
WOUDENBERG
ET AL.
Fig. 21. Alternaria passiflorae: conidia and conidiophores. A–B. CBS 117102. C–D. CBS 117103. E–F. CBS 116333. G–H. CBS 166.77. I–J. CBS 630.93. K–L. CBS 629.93.
Scale bars = 10 μm.
Passiflora caerulea (Passifloraceae), Jul. 2004, C.F. Hill, representative isolate of
A. passiflorae CBS 117103 = E.G.S. 52.032; Auckland, from leaf spot of Gaura
lindheimeri (Onagraceae), May 2002, C.F. Hill, culture ex-type of A. gaurae CBS
116333 = E.G.S. 50.121; Waitakere, from leaf of Capsicum frutescens (Solanaceae), May 1975, CBS 166.77. USA, Hawaii, from Passiflora edulis, before
Oct. 1968, M. Aragaki, culture ex-type of A. hawaiiensis CBS 630.93 = E.G.S.
29.020 = QM 9050.
Materials examined: India, Andhra Pradesh, Hyderabad, from seed of Cajanus
cajan (Fabaceae), before Feb. 1990, K.M. & Ch. Reddy, culture ex-type of
A. pipionipisi CBS 116115 = E.G.S. 40.096 = IMI 340950. USA, California,
Encinitas, from Euphorbia pulcherrima (Euphorbiaceae), Sep. 1994, C.F. Hill,
CBS 134265 = E.G.S. 42.047; California, Encinitas, from Euphorbia pulcherrima,
Sep. 1994, C.F. Hill, representative isolate of A. obtecta CBS 117365 = E.G.S.
42.048.
Notes: By synonymising A. gaurae with A. passiflorae, and
including CBS 166.77, formerly identified as A. solani, the host
range of A. passiflorae has broadened to include Gaura sp.
(Onagraceae) and Capsicum frutescens (Solanaceae).
Alternaria porri (Ellis) Cif., J. Dept. Agric. Porto Rico 14:
30. 1930 [1929]. Fig. 22.
Alternaria pipionipisi E.G. Simmons, CBS Biodiversity
Ser. (Utrecht) 6: 302. 2007.
36
Basionym: Macrosporium porri Ellis, Grevillea 8 (no. 45): 12.
1879.
≡ Alternaria porri (Ellis) Sawada, Rep. Dept. Agric. Gov. Res. Inst.
Formosa, 61: 92. 1930.
PATHOGENS
Fig. 22. Alternaria porri: conidia and conidiophores. A–D. CBS 116698. E–H. CBS 116699. I–L. CBS 116649. Scale bars = 10 μm.
Type: (Lectotype, designated in Simmons 2007) NY, Ellis
Collection: on leaves of Allium porrum, Newfield, N.J. Sept. 78.
= Alternaria hordeiseminis E.G. Simmons & G.F. Laundon, CBS Biodiversity
Ser. (Utrecht) 6: 150. 2007.
Materials examined: USA, Nebraska, Lincoln, from leaf of Allium cepa (Amaryllidaceae), 1965, D.S. Meredith, representative isolate of A. allii CBS
116649 = E.G.S. 17.082 = QM 8613; New York, Ithaca, from leaf of Allium cepa,
1996, M.J. Ya~nes Morales, representative isolate of A. porri CBS 116698 = E.G.S.
48.147; New York, Orange County, from leaf of Allium cepa, 1996, M.J. Ya~nes
Morales (epitype designated here CBS H-21746, MBT178117, culture exepitype CBS 116699 = E.G.S. 48.152).
Materials examined: Australia, Queensland, Brisbane, Chapel Hill, from Euphorbia
pulcherrimae (Euphorbiaceae), 25 Aug. 1986, J.L. Alcorn, representative isolate of
A. pulcherrimae CBS 121342 = E.G.S. 42.122 = IMI 310506. Israel, from Helianthus annuus (Asteraceae), 1996, collector unknown, representative isolate of
A. protenta CBS 116697 = E.G.S. 45.024 = IMI 372957; from Helianthus annuus,
1996, collector unknown, representative isolate of A. protenta CBS
116696 = E.G.S. 45.023 = IMI 372955. New Zealand, Hastings, from Solanum
tuberosum (Solanaceae), Mar. 1997, C.F. Hill, representative isolate of A. solani
CBS 135189 = E.G.S. 45.053; Levin, from fruit rot of Solanum lycopersicum
(Solanaceae), before Jul. 1979, G.F. Laundon, CBS 347.79 = E.G.S.
44.091 = ATCC 38569 = LEV 14726; Palmerston North, from seed of Hordeum
vulgare (Gramineae), Jul. 1977, G.F. Laundon, culture ex-type of A. hordeiseminis
CBS 116437 = E.G.S. 32.076 = CBS 116443 = E.G.S. 46.163. USA, California,
Alternaria protenta E.G. Simmons, Mycotaxon 25: 207.
1986. Fig. 23.
= Alternaria pulcherrimae T.Y. Zhang & J.C. David, Mycosystema 8-9: 110.
1996.
37
WOUDENBERG
ET AL.
Fig. 23. Alternaria protenta: conidia and conidiophores. A–B. CBS 116696. C–D. CBS 116697. E–G. CBS 116643. H–J. CBS 116651. K–M. CBS 121342. N–P. CBS 347.79.
38
Siskiyou, from Solanum tuberosum, 1996, D. Fogle, representative isolate of
A. solani CBS 116651 = E.G.S. 45.020.
Notes: By synonymising A. pulcherrimae and A. hordeiseminis
with A. protenta and including three isolates formerly identified as
A. solani (CBS 347.79, 116651 and 135189), the host range of
A. protenta has expanded extensively. It now comprises plants
from the Asteraceae, Euphorbiaceae, Gramineae and Solanaceae. Based on molecular (and morphological) data,
A. protenta is closely related to A. solani, and these two species
can only be distinguished based on 9 nt differences in their RPB2
sequences (see RPB2 alignment in TreeBASE).
Alternaria pseudorostrata E.G. Simmons, Mycotaxon 57:
398. 1996.
Material examined: USA, California, Encinitas, from Euphorbia pulcherrimae
(Euphorbiaceae), Dec. 1994, C.F. Hill, culture ex-type of A. pseudorostrata CBS
119411 = E.G.S. 42.060.
Alternaria ranunculi E.G. Simmons, CBS Biodiversity
Ser. (Utrecht) 6: 212. 2007.
Material examined: Israel, Palestine, from seed of Ranunculus asiaticus
(Ranunculaceae), 10 Apr. 1984, collector unknown, culture ex-type of
A. ranunculi CBS 116330 = E.G.S. 38.039 = IMI 285697.
Alternaria ricini (Yoshii) Hansf., Proc. Linn. Soc. Lond. : 53.
1943.
Basionym: Macrosporium ricini Yoshii, Bult. Sci. Fak. Terk. Kjusu
Imp. Univ. 3(4): 327. 1929.
Type: (Lectotype, designated in Simmons 1994) BPI 445446,
Macrosporium ricini, Japan, Fukuoka, Ricinus communis, July
1928.
Materials examined: Italy, Sardinia, Sasseri, from Ricinus communis (Euphorbiaceae), before Aug. 1986, J.A. von Arx, CBS 353.86. Japan, Ricinus communis, deposited Feb. 1931 by K. Nakata (epitype designated here CBS H21747, MBT178118, culture ex-epitype CBS 215.31). USA, Virginia, Holland,
from leaf of Ricinus communis, 9 Aug. 1954, C.A. Thomas, representative isolate
of A. ricini CBS 117361 = E.G.S. 06.181.
Alternaria rostellata E.G. Simmons, Mycotaxon 57: 401.
1996.
PATHOGENS
Scotland, from Linum usitatissimum (Linaceae), 22 Nov. 1945, J.W. Groves, CBS
103.46; Derbyshire, from seed of Linum usitatissimum, 1983, C. Nicholls,
representative isolate of A. linicola CBS 116703 = E.G.S. 36.110 = IMI 274549.
Notes: None of the three isolates sporulated on SNA or PCA
after 3 wk of incubation, also not after scarification. Corlett &
Corlett (1999) already stated that, after sub-cultivation,
A. linicola sporulates poorly, or not at all. By synonymising
A. linicola with A. scorzonerae, the host range of A. scorzonerae
is expanded to include Linum usitatissimum (Linaceae).
Alternaria sennae Woudenb. & Crous, sp. nov. MycoBank MB809000. Fig. 24.
Etymology: Named after the host genus on which it occurs,
Senna.
septate, dark brown with a hyaline tip, (43–)67–81(–108) × (5–)
6(–7) μm, bearing a single, darkened, apical conidiogenous locus,
but may produce geniculate conidiogenous extensions. Conidia
solitary, conidium body pale olive-brown, smooth-walled, narrowly
ovoid, (46–)55–62(–69) × (8–)10–12(–14) μm, with (7–)
7–8(–10) transverse distosepta and (1–)2–3(–4) longitudinal
septa. The conidial body can be slightly constricted near some
transverse septa. Conidia have a single, aseptate, filamentous
beak, which occasionally branches once; basal lateral secondary
conidiophores can be formed. Beaks (38–)99–163(–314) μm
long, ca. 2 μm diam. Sexual morph not observed.
white to opaque with two olivaceous concentric circles; aerial
mycelium sparse, white, floccose, colonies reaching 35−40 mm
diam; cultures on PCA flat, undulate, white with grey-olivaceous
zones; aerial mycelium felty, pale olivaceous-grey, colonies
reaching 50–55 mm diam; reverse with pale olivaceous-grey
zones.
Material examined: India, Uttar Pradesh, Gorakhpur, from leaf of Senna corymbosa (Fabaceae), 10 Apr. 1981, R.P. Verma (holotype CBS H-21741, culture
ex-type CBS 477.81 = E.G.S. 34.030 = IMI 257253).
Alternaria sesami (E. Kawam.) Mohanty & Behera, Curr.
Sci. 27: 493. 1958.
Basionym: Macrosporium sesami E. Kawam., Fungi 1: 27. 1931.
Material examined: USA, California, Encinitas, from leaf of Euphorbia pulcherrimae (Euphorbiaceae), Jan. 1995, C.F. Hill, culture ex-type of A. rostellata CBS
117366 = E.G.S. 42.061.
Alternaria scorzonerae (Aderh.) Loer., Netherlands J. Pl.
Pathol. 90(1): 37. 1984.
Basionym: Sporidesmium scorzonerae Aderh., Arbeiten Kaiserl.
Biol. Anst. Land-Forstw. 3: 439. 1903.
= Alternaria linicola J.W. Groves & Skolko, Canad. J. Res., Sect. C, Bot. Sci.
22: 223. 1944.
= Alternaria linicola Neerg, Danish species of Alternaria & Stemphylium: 302.
1945. (nom. illegit., Art. 53.1)
Type: (Lectotype, designated in Simmons 1997) Aderhold, Arbeiten
Kaiserl. Biol. Anst. Land-Forstw. 3: 440. fig. w/o number. 1903.
Materials examined: Netherlands, Reusel, from leaf spot of Scorzonera hispanica (Asteraceae), 1982, W.M. Loerakker (epitype designated here CBS H21748, MBT178119, culture ex-epitype CBS 478.83 = E.G.S. 38.011). UK,
Materials examined: Egypt, from Sesamum indicum (Pedaliaceae), 1972, S.B.
Mathur, CBS 240.73. India, from seedlings of Sesamum indicum, Dec. 1959, E.E.
Leppik, representative isolate CBS 115264 = CBS 117214 = E.G.S. 13.027.
Alternaria sidae E.G. Simmons, Mycotaxon 88: 202.
2003.
Material examined: Kiribati, Phoenix islands, Canton Island, from leaf spot of Sida
fallax (Malvaceae), 11 Feb. 1958, O. & I. Degener, culture ex-type of A. sidae CBS
117730 = E.G.S. 12.129.
Alternaria silybi Gannibal, Mycotaxon 114: 110. 2011.
Materials examined: Russia, Vladivostok, Trudovoe, from leaf lesion of Silybum
marianum (Asteraceae), 1 Sep. 2006, Ph. B. Gannibal, culture ex-type of A. silybi
CBS 134092 = VKM F-4109; Vladivostok, Trudovoe, from leaf lesion of Silybum
marianum, 1 Sep. 2006, Ph. B. Gannibal, CBS 134094 = VKM F-4118; Vladivostok,
39
WOUDENBERG
ET AL.
Fig. 24. Alternaria sennae sp. nov. CBS 477.81: A–L. Conidia and conidiophores. Scale bars = 10 μm.
Botanical Garden-Institute, from leaf lesion of Silybum marianum, 6 Sep. 2006, Ph. B.
Gannibal, CBS 134093 = VKM F-4117.
Alternaria solani Sorauer, Z. Pflanzenkrankh. Pflanzenschutz 6: 6. 1896. Fig. 25.
= Macrosporium solani Ellis & G. Martin, Amer. Naturalist 16(12): 1003. 1882
(non M. solani Cooke, 1883)
≡ Alternaria solani (Ellis & G. Martin) L.R. Jones & Grout, Vermont
Agric. Exp. Sta. Annual Rep. 9: 86. 1899. (nom. illegit., Art. 53.1)
≡ Alternaria americana Sawada, Rep. Dept. Agric. Gov. Res. Inst.
Formosa 51:117. 1931. (nom. nov. for A. solani (Ellis & G. Martin) L.R.
Jones & Grout (1899), non A. solani Sorauer (1896))
≡ Alternaria porri f. sp. solani (Ellis & G. Martin) Neerg, Danish species
of Alternaria & Stemphylium: 260. 1945.
= Sporidesmium solani-varians Va~nha, Naturwiss. Z. Forst- Landw. 2: 117.
1904.
40
= Alternaria danida E.G. Simmons, Mycotaxon 65: 78. 1997.
= Alternaria viciae-fabae E.G. Simmons & G.F. Laundon, CBS Biodiversity
Ser. (Utrecht) 6: 234. 2007.
Materials examined: Italy, from seed of Ageratum houstonianum (Asteraceae), 27
Aug. 1941, P. Neergaard, culture ex-type of A. danida CBS 111.44 = E.G.S.
07.029 = QM 1772. New Zealand, from Vicia faba (Fabaceae), Jun. 1979, G.F.
Laundon, culture ex-type of A. viciae-fabae CBS 116442 = E.G.S. 46.162 = ICMP
10242. Unknown, from leaf spot of Solanum aviculare (Solanaceae), before May
1941, P. Neergaard, CBS 111.41; unknown host, before Nov. 1921, isolated by
Künkel, CBS 106.21. USA, Washington, Douglas County, from leaf spot of Solanum tuberosum (Solanaceae), 25 Aug. 1996, E.G. Simmons, representative isolate
of A. solani CBS 109157 = E.G.S. 44.098.
Notes: By synonymising A. danida and A. viciae-fabae with
A. solani, the host range of this pathogen has expanded to
PATHOGENS
Fig. 25. Alternaria solani. A. Disease symptoms on Solanum tuberosum (Photo J.E. van der Waals, University of Pretoria, South Africa). B–H. Conidia and conidiophores.
B–D. CBS 109157. E–H. CBS 116442. Scale bars = 10 μm.
include Asteraceae and Fabaceae host plants. The isolates CBS
106.21 and CBS 111.44 did not sporulate after 3 wk of incubation
on SNA (both were already labelled as sterile in the CBS
collection database). Isolate CBS 111.41 did sporulate, but the
spore formation was atypical.
Alternaria solani-nigri R. Dubey, S.K. Singh & Kamal [as
“solani-nigrii”], Microbiol. Res. 154: 120. 1999. Fig. 26.
= Alternaria cyphomandrae E.G. Simmons, Mycotaxon 75: 86. 2000.
= Alternaria ascaloniae E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 168. 2007.
= Alternaria beticola E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 170. 2007.
= Alternaria glyceriae E.G. Simmons & C.F. Hill, CBS Biodiversity Ser.
(Utrecht) 6: 148. 2007.
= Alternaria herbiculinae E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6:
166. 2007.
Materials examined: New Zealand, Canterbury, Ashburton, from leaf lesion of
Beta vulgaris (Chenopodiaceae), Jul. 1999, B. Alexander, culture ex-type of
A. beticola CBS 116447 = E.G.S. 47.196; Hastings, from leaf spot of Allium
ascalonicum (Amaryllidaceae), Oct. 1997, C.F. Hill, culture ex-type of
A. ascaloniae CBS 121347 = E.G.S 46.052; New Plymouth, from fruit of
Cyphomandra betacea (Solanaceae), May 1991, C.F. Hill, culture ex-type of
A. cyphomandrae CBS 109155 = E.G.S. 40.058; Taranaki, Otaki, from stunted
Petroselinum crispum (Apiaceae), 14 Jun. 2001, J.B. Wong, culture ex-type of
A. herbiculinae CBS 116332 = E.G.S. 49.180; Waikato, Kopuku, from leaf spot of
Glyceria maxima (Gramineae), Apr. 2003, C.F. Hill, culture ex-type of
A. glyceriae CBS 116334 = E.G.S. 51.107; Waikato, Whangamarino swamp, from
leaf spot of Solanum nigrum (Solanaceae), 21 Jun. 2003, C.F. Hill, representative
isolate of A. solani-nigri CBS 113403 = E.G.S. 51.106 = CPC 10620; Waikato,
Whangamarino swamp, from leaf spot of Solanum nigrum, 6 Feb. 2003, C.F. Hill,
representative isolate of A. solani-nigri CBS 117101 = E.G.S. 51.032.
Notes: By synonymising these five Alternaria species with
A. solani-nigri, this becomes a species with a broad host range
found on Amaryllidaceae, Apiaceae, Chenopodiaceae, Gramineae and Solanaceae. All studied specimens originate from New
Zealand, but the holotype of A. solani-nigri was described from
India. The five sequenced genes are 100 % identical between all
the specimens studied.
Alternaria steviae Ishiba, T. Yokoy. & Tani, Ann. Phytopathol. Soc. Japan 48(1): 46. 1982.
Materials examined: Japan, Kagawa, Kida-gun, Miki-cho, Ikenobe, from leaf spot
of Stevia rebaudiana (Asteraceae), CBS 631.88 = IFO 31212; Kagawa, Kida-gun,
Miki-cho, Ikenobe, from leaf spot of Stevia rebaudiana, Jun. 1980, CBS
632.88 = IFO 31183; Kagawa, Zentsuji, Harada-cho, from leaf spot of Stevia
rebaudiana, Aug. 1978, C. Ishiba, culture ex-type of A. steviae CBS
117362 = IFO 31182 = E.G.S. 37.019.
Alternaria tagetica S.K. Shome & Mustafee, Curr. Sci. 35:
370. 1966.
Materials examined: UK, from seed of Tagetes sp. (Asteraceae), before May
1979, G.S. Taylor, CBS 297.79; from seed of Tagetes sp., before May 1979, G.S.
Taylor, CBS 298.79; England, Manchester, from seed of Tagetes erecta (Asteraceae), before Apr. 1980, G.S. Taylor, representative isolate of A. tagetica CBS
479.81 = E.G.S. 33.081. USA, Ohio, Butler County, Oxford, from leaf of cultivated
Tagetes sp., 14 Jun. 1996, M.A. Vincent, representative isolate of A. tagetica CBS
117217 = E.G.S 44.045; South Carolina, Clemson, from seed of Tagetes sp.,
before Mar. 1981, E. Smallwood Hotchkiss, representative isolate of A. tagetica
CBS 480.81 = E.G.S. 33.184.
41
WOUDENBERG
ET AL.
Fig. 26. Alternaria solani-nigri: conidia and conidiophores. A–B. CBS 113403. C–D. CBS 116447. E–G. CBS 109155. H–I. CBS 116334. J–K. CBS 121347. L–M. CBS
116332. N–P. CBS 117101. Scale bars = 10 μm.
42
PATHOGENS
Fig. 27. Alternaria thunbergiae: conidia and conidiophores. A–C. CBS 116331. D–E. CBS 122597. F–H. CBS 120986. Scale bars = 10 μm.
Alternaria thunbergiae E.G. Simmons & Alcorn, CBS
Biodiversity Ser. (Utrecht) 6: 136. 2007. Fig. 27.
= Alternaria iranica E.G. Simmons & Ghosta, CBS Biodiversity Ser. (Utrecht)
6: 122. 2007.
Materials examined: Australia, Queensland, Brisbane, Chapel Hill, from leaf spot
of Thunbergia alata (Acanthaceae), 6 Feb. 1986, J.L. Alcorn, culture ex-type of
A. thunbergiae CBS 116331 = E.G.S. 41.073 = BRIP 14963. Iran, Miandoab,
from leaf of Allium cepa (Amaryllidaceae), 13 Sep. 2001, Y. Ghosta, culture extype of A. iranica CBS 120986 = E.G.S. 51.075. New Zealand, Auckland,
Mangere, Tidal Road, from Thunbergia alata, 4 Jun. 2001, C.F. Hill, CBS 122597.
Notes: By synonymising A. iranica with A. thunbergiae, the host
range of this taxon has expanded to include Allium cepa. The five
sequenced genes are 100 % identical between the ex-type
strains of A. thunbergiae and A. iranica. As both species were
originally described in the same publication, there is no case for
nomenclatural priority. Therefore we chose to synonymise
A. iranica under A. thunbergiae because A. thunbergiae is more
commonly used in literature (Leahy 1992, Melo et al. 2009).
Alternaria tillandsiae E.G. Simmons & C.F. Hill, CBS
Material examined: USA, from Tillandsia usneoides (Bromeliaceae), Dec. 1995,
B. Milnes, culture ex-type of A. tillandsiae CBS 116116 = E.G.S. 43.074.
Alternaria tropica E.G. Simmons, Mycotaxon 46: 187.
1993.
Materials examined: USA, Florida, Homestead, from fruit of Passiflora edulis
(Passifloraceae), May 1990, R.T. McMillan Jr., culture ex-type of A. tropica CBS
631.93 = E.G.S. 39.126; Florida, Homestead, from fruit of Passiflora edulis, May
1990, R.T. McMillan Jr., representative isolate of A. tropica CBS 117216 = E.G.S.
39.125.
Alternaria venezuelensis E.G. Simmons & Rumbos, CBS
Material examined: Venezuela, Maracay, from leaf spot of Phaseolus vulgaris
(Fabaceae), before Oct. 1999, R. Rumbos, culture ex-type of A. venezuelensis
CBS 116121 = E.G.S. 48.065.
Alternaria zinniae M.B. Ellis, Mycol. Pap. 131: 22. 1972.
= Alternaria zinniae H. Pape, Angew. Bot. 24: 61. 1942. (nom. inval., Art.
36.1)
Materials examined: Hungary, from seed of Callistephus chinensis (Asteraceae),
12 Aug. 1942, P. Neergaard, CBS 118.44. Italy, Sardinia, Sasseri, from Zinnia
elegans (Asteraceae), 18 Oct. 1958, U. Prota, CBS 117.59. Netherlands, Huizum, from leaf of Zinnia sp., 27 Jul. 1948, A. Jaarsveld, CBS 107.48. New
Zealand, Auckland, Royal Oak, from leaf spot of Zinnia elegans, May 1996, C.F.
Hill, representative isolate of A. zinniae CBS 117223 = E.G.S. 44.035. UK, from
seed of Zinnia sp., 1979, G.S. Taylor, CBS 299.79; from seed of Zinnia sp., 1979,
G.S. Taylor, CBS 300.79. Unknown, from Zinnia elegans, summer 1961, Smith,
CBS 108.61.
Section Euphorbiicola Woudenb. & Crous, sect. nov.
MycoBank MB809001. Fig. 28
Type species: Alternaria euphorbiicola E.G. Simmons &
Engelhard.
43
WOUDENBERG
ET AL.
Fig. 28. Alternaria section Euphorbiicola: conidia and conidiophores. A–G. Alternaria limicola. H–P. Alternaria euphorbiicola. A–D. CBS 117360. E–G. CBS 483.90. H–J. CBS
198.86. K–M. CBS 119410. N–P. CBS 133874. Scale bars = 10 μm.
44
Section Euphorbiicola is characterised by ovoid, obclavate,
medium to large conidia that are disto- and euseptate, in
short to moderately long chains, with no or a simple long
beak in the terminal conidia. Conidia contain multiple
transverse and some longitudinal septa and are slightly
constricted near some transverse septa. Short to long,
broad, apical, and sometimes lateral, secondary conidiophores are formed.
Note: The new Alternaria sect. Euphorbiicola can be easily
distinguished from sect. Porri based on the formation of conidia
in chains in sect. Euphorbiicola.
Alternaria euphorbiicola E.G. Simmons & Engelhard,
Mycotaxon 25: 196. 1986.
≡ Macrosporium euphorbiae Reichert, Bot. Jahrb. Syst. 56: 723. 1921.
Non Macrosporium euphorbiae Bartholomew 1908. (nom. illegit., Art
53.1).
Materials examined: USA, Florida, from Euphorbia pulcherrima (Euphorbiaceae),
1985, A.W. Engelhard, CBS 198.86 = E.G.S. 38.082; Hawaii, Oahu, from
Euphorbia pulcherrima, Mar. 1984, M. Aragaki, representative isolate CBS
119410 = E.G.S. 41.029; Louisiana, from Euphorbia hyssopifolia (Euphorbiaceae), 1986, L. Walker, CBS 133874 = E.G.S 38.191.
Alternaria limicola E.G. Simmons & M.E. Palm, Mycotaxon 37: 82. 1990.
Materials examined: Mexico, Colima, from leaf of Citrus aurantiifolia (Rutaceae),
May 1989, M. Palm, culture ex-type of A. limicola CBS 483.90 = E.G.S. 39.070;
Jalisco, from Citrus sp., Sep. 1995, M. Palm, representative isolate CBS
117360 = E.G.S. 43.009.
DISCUSSION
In the present phylogenetic study aiming to delimit Alternaria
species in sect. Porri, we reduced the 82 known morphospecies
in this section to 63 based on our polyphasic approach. Some
important plant pathogens have now been assigned to specific
clades in the phylogenetic tree and correlated with their distinct
morphology, which will aid plant pathologists to identify their
newly collected isolates.
The 10 isolates named A. solani at the onset of this study
cluster within five different species-clades, and only three of them
retain the name A. solani. This is not surprising, as almost all
large-spored, narrow-beaked Alternaria strains hitherto isolated
from Solanaceae were called A. solani, following the concept of
M.B. Ellis (1971). Simmons (2000) already noted that early blight
of tomato is actually caused by A. tomatophila rather than
A. solani, and also described two additional species on tomato,
A. cretica and A. subcylindrica. These tomato pathogens all
cluster in one clade based on our phylogenetic analysis, which
also includes the ex-type strain of A. linariae. The basionym of
A. linariae, A. anagallidis var. linariae, is the oldest name in this
cluster, which therefore applies to this clade mainly represented
by tomato pathogens. When Neergaard (1945) described this
species he found the fungus on seeds and seedlings with
damping-off symptoms from Linaria marroccana (Scrophulariaceae), Antirrhinum majus (Scrophulariaceae) and on a healthy
seedling of Papaver rhoeas (Papaveraceae). His pathogenicity
tests (Neergaard 1945) showed that A. linariae could also attack
PATHOGENS
Brassica oleracea (Brassicaceae), Solanum lycopersicum (Solanaceae), Lactuca sativa (Asteraceae), Godetia hybrida (Onagraceae), Nicotiana affinis (Solanaceae) and Papaver
paeoniflorum (Papaveraceae), indicating a very broad host
range. The isolates included in this study also show that, besides
its broad host range, A. linariae is also widespread, found in
Europe, USA, New Zealand and Asia. Three other isolates
formerly identified as A. solani, including a former representative
isolate used by Simmons (2007), cluster with A. protenta, an
Alternaria species originally described from Helianthus annuus
(Asteraceae). CBS 116651 is mentioned as a representative
strain of A. solani by Simmons (2007), but he later expressed
doubt as to the identity of this isolate (Simmons pers. comm.).
The host range of A. protenta has expanded extensively, now
comprising plants from the Asteraceae, Euphorbiaceae, Gramineae and Solanaceae. A pathogenicity test performed on
A. protenta isolated from sunflower seed (Wu & Wu 2003)
concluded that sunflower was the only susceptible host among
the 10 host plants tested. One of the host plants tested was
Solanum lycopersicum, which we include as host of A. protenta.
However, the authors did not clearly state how the A. protenta
isolates, which they only found on seed of one out of seven
cultivars of sunflower seeds tested, were identified. The manuscript also lacks molecular data, which could affirm their identification of A. protenta. To our knowledge, no pathogenicity tests
have thus far been performed with the species synonymised
under A. protenta, A. hordeiseminis or A. pulcherrimae. Based on
molecular and morphological data, A. protenta is closely related
to A. solani, and these two species can only be distinguished by
the 9 nt differences in their RPB2 sequences. To confirm the
potato pathogen clade, called A. solani, we sequenced the
RPB2 region of multiple isolates collected from Solanum
tuberosum, which are present in E.G. Simmons collection, now
deposited at the CBS. Almost all (22/24 strains) cluster within the
now recognised A. solani species clade (data not shown). The
ex-type strain of A. danida (CBS 111.44), now a synonym of
A. solani, was originally deposited in the CBS collection by
P. Neergaard as A. porri f. sp. solani. Pathogenicity tests performed on this strain (Neergaard 1945) showed that it could
attack hosts from several plant families [e.g. Allium cepa
(Amaryllidaceae), Brassica oleracea (Brassicaceae), Solanum
lycopersicum (Solanaceae) and Lactuca sativa (Asteraceae)],
indicating a very broad host range. Our sequences of A. danida
differ from those deposited in GenBank by Lawrence et al.
(2013), and therefore we repeated the cultivation and DNA
extraction to confirm our results and the resulting synonymy with
A. solani. Although the other large-spored, long-beaked Alternaria species described from potato, A. grandis (Simmons 2000),
differs only by 1 nt in its GAPDH sequence (position 99, T instead
of C, see locus alignment in TreeBASE) within the 2 722 positions used in the phylogeny, we did not synonymise A. grandis
under A. solani. The two isolates included, CBS 109158 and CBS
116695, have substantially larger conidia than the other A. solani
isolates, and a recently published study revealed that A. solani
(CBS 109157) and A. grandis (CBS 109158) differ on 8 out of
770 nt in their calmodulin sequence (Gannibal et al. 2014).
The oldest large-spored onion pathogens, A. porri and
A. allii, form two closely related but distinct clades, which only
differ based on 8 nt in their RPB2 sequences (see locus
alignment in TreeBASE). The three newer species described
from Allium, A. ascaloniae, A. iranica and A. vanuatuensis
(Simmons 2007), are all synonymised with other species.
45
WOUDENBERG
ET AL.
Alternaria ascaloniae is synonymised under A. solani-nigri, a
species with a broad host range, mainly found in New Zealand.
To our knowledge, no pathogenicity tests have been performed
with the species now placed in synonomy with A. solani-nigri,
which could affirm the broad host range for this species.
Alternaria iranica is synonymised under A. thunbergiae, known
as the causative agent of Alternaria leaf spot on Thunbergia
(Leahy 1992), reported from Australia, USA and Brazil. Alternaria vanuatuensis clusters in the Allium clade, comprising
A. allii and A. porri. Based on the sequence data generated
here, it is synonymised under A. allii. According to Simmons
(2007), the conidia of A. allii are distinguishable from those
of A. porri and other large-spored species known on Allium,
based on their multiple beaks and branches. However, the
representative isolates of A. allii used by Simmons (2007) do
not cluster in a single clade; CBS 116649 clusters with the two
A. porri representative isolates. On the other hand,
A. vanuatuensis is described as a single-beaked species, but
clusters with the A. allii isolate deposited in the CBS collection
by J.A.B. Nolla on 27 December 1927 as A. allii sp. nov. (CBS
107.28, recognised as the ex-type strain here). Simmons obtained this isolate from the CBS in February 2000 (E.G.S.
48.084), but was unable to induce sporulation. We observed
few conidia, but these were only single-beaked. Unfortunately
we could not induce CBS 116701 to sporulate, which leaves us
at odds with Simmons's notes, with only single- to doublebeaked conidia in the A. allii clade, and double- to triplebeaked conidia in the A. porri clade. The number of beaks
and branches from the Allium isolates therefore is not suitable
to make a distinction between the two major Allium species.
The species can be easily differentiated on the basis of
sequence data of the RPB2 gene region generated in this
study.
Based on morphology, four large-spored Alternaria species
with long beaks were described as Passifloraceae pathogens.
Our phylogeny only supports three of these: A. tropica,
A. aragakii and the more common A. passiflorae. The fourth
species, A. hawaiiensis, is synonymised under A. passiflorae
based on sequence data. Simmons (2007) described
A. hawaiiensis as a new species lacking multiple beaks, which is
a characteristic of A. passiflorae. Our sequence data led us to
conclude that this characteristic is not suitable for species delimitation, which we also concluded from the data of the onion
pathogens, A. allii, A. vanuatuensis and A. porri. The clustering
of two isolates within our A. passiflorae clade, which originate
from different host families (Onagraceae and Solanaceae),
renders A. passiflorae as unspecific to Passifloraceae.
An ongoing study in South Africa on sweet potato pathogens reveals multiple Alternaria species on this host associated
with blight symptoms on leaves, petioles, and stems. In addition
to the known pathogen of sweet potato, A. bataticola, three other
pathogenic species are delineated of which two are newly
described as A. ipomoeae and A. neoipomoea. A new unknown
Alternaria pathogen, causing sweet potato stem blight in
Ethiopia, was reported by van Bruggen in 1984. This isolate
(CBS 219.79) was sent to the CBS for identification, but the
author did not agree with the morphological identification made at
that time as A. cucumerina, a name under which it was still
stored in the CBS collection. Our data indicate that this pathogen,
which also is found in stem lesions of Ipomoea batatas in South
Africa, should be recognised as a new species, now named
A. ipomoeae. Most isolates from South Africa however cluster in
46
a clade close to A. ipomoeae, now named A. neoipomoea, which
can clearly be distinguished from A. ipomoeae morphologically
and by sequence data. Two more isolates from sweet potato in
South Africa are identified as A. argyroxiphii, an Alternaria
species originally described from Argyroxiphium sp. This finding
is a new host report for A. argyroxiphii, and a first report of the
fungus from South Africa.
Based on the sequence data generated in this study,
A. euphorbiicola and A. limicola clearly separate from the other
species in sect. Porri (Fig. 1). This separation is supported by
morphological differences, and we therefore propose the new
section, sect. Euphorbiicola. However, when we examined the
phylogeny displaying the neighbouring sections of sect. Porri
(Fig. 2), questions arose concerning sect. Gypsophilae and sect.
Radicina. These two sections display almost similar branch
length differences within the respective sections, comparable to
what sect. Porri displays with sect. Euphorbiicola. An additional
character of sect. Gypsophilae and sect. Radicina is that the
species within these sections share the same host family,
respectively Caryophyllaceae and Apiaceae. We therefore
choose to retain these sections at present, but additional molecular and morphological studies could eventually lead to the
recognition of additional sections.
The present polyphasic approach displays the current species delimitation in Alternaria sect. Porri. We recognise 63
Alternaria species in this section with medium to large conidia
and a long (filamentous) beak, which can be distinguished based
on molecular data. Not all species distinctions are 100 % clear
based on DNA data only; nevertheless, we tried to be consistent
in synonymising or not synonymising species: the number of
genes with nt differences and the number of nt differences are
taken into account, together with the morphology, host, country
and time of isolation. All Alternaria isolates currently stored in the
CBS collection, which cluster within sect. Porri based on their
gene sequences, were included in our study. Some species,
however, are under-sampled, which results in some uncertainty
in keeping isolates as separate species or reducing them to
synonymy. Although we attempted to use the available data as
best as possible, with the inclusion of additional isolates some
uncertain species boundaries are bound to be better resolved.
The finding of the third species on potato (A. protenta) is a
good example of the importance of fungal systematics. Multiple
manuscripts report on the high level of genetic variability
observed among A. solani isolates (van der Waals et al. 2004;
Lourenco et al. 2011, Leiminger et al. 2013) and based on
secondary metabolite profiling A. solani isolates cluster in two
distinct groups (Andersen et al. 2008). Furthermore, two genotypes are described based on the cytochrome b gene structure of
A. solani isolates (Leiminger et al. 2014), which is an important
gene in fungicide resistance. However, our study indicates that
previous reports could actually be dealing with three (or more)
different species. Without knowing the correct identity of your
pathogen, many incorrect conclusions can be drawn about diversity, evolutionary mechanisms, host range, and options for
disease control.
ACKNOWLEDGEMENTS
This research was supported by the Dutch Ministry of Education, Culture and
Science through an endowment of the FES programme “Making the tree of life
work”.
REFERENCES
Abo-Elyousr KAM, Abdel-Hafez SII, Abdel-Rahim IR (2014). Isolation of
Trichoderma and evaluation of their antogonistic potential against Alternaria
porri. Journal of Phytopathology 162: 567–574.
Andersen B, Dongo A, Pryor BM (2008). Secondary metabolite profiling of
Alternaria dauci, A. porri, A. solani, and A. tomatophila. Mycological
Research 112: 241–250.
Angell HR (1929). Purple blotch of onion (Macrosporium porri Ell.). Journal of
Agricultural Research 38(9): 467–487.
Berbee ML, Pirseyedi M, Hubbard S (1999). Cochliobolus phylogenetics and the
origin of known, highly virulent pathogens, inferred from ITS and
glyceraldehyde-3-phosphate dehydrogenase gene sequences. Mycologia
91: 964–977.
Bruggen AHC van (1984). Sweet potato stem blight caused by Alternaria sp.: a
new disease in Ethiopia. Netherlands Journal of Plant Pathology 90:
155–164.
Brun S, Madrid H, Gerrits van den Ende AHG, et al. (2013). Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria
dauci and relatives. Fungal Biology 117: 32–40.
Carbone I, Kohn LM (1999). A method for designing primer sets for speciation
studies in filamentous ascomycetes. Mycologia 91: 553–556.
Cifferi R (1930). Phytopathological survey of Santo Domingo, 1925–1929.
Journal of the Department of Agriculture of Porto Rico 14: 5–44.
Cooke MC, Ellis JB (1879). New Jersey fungi. Grevillea 8: 11–16.
Corlett M, Corlett ME (1999). Fungi Canadenses. No. 341. Alternaria linicola.
Canadian Journal of Plant Pathology 21(1): 55–57.
Crous PW, Gams W, Stalpers JA, et al. (2004). MycoBank: an online initiative to
launch mycology into the 21st century. Studies in Mycology 50: 19–22.
Crous PW, Verkley GJM, Groenewald JZ, et al. (eds) (2009). Fungal Biodiversity. CBS laboratory Manual Series 1. CBS-KNAW Fungal Biodiversity
Centre, Utrecht, Netherlands.
Ellis MB (1971). Dematiaceous hyphomycetes. Commonwealth Mycological
Institute, Kew, UK.
Gannibal PB, Orina AS, Mironenko NV, et al. (2014). Differentiation of the
closely related species, Alternaria solani and A. tomatophila, by molecular
and morphological features and aggressiveness. European Journal of Plant
Pathology 139: 609–623.
Hong SG, Cramer RA, Lawrence CB, et al. (2005). Alt a 1 allergen homologs
from Alternaria and related taxa: analysis of phylogenetic content and
secondary structure. Fungal Genetics and Biology 42: 119–129.
Hoog GS de, Gerrits van den Ende AHG (1998). Molecular diagnostics of
clinical strains of filamentous Basidiomycetes. Mycoses 41: 183–189.
Horsfield A, Wicks T, Davies K, et al. (2010). Effect of fungicides use strategies
on the control of early blight (Alternaria solani) and potato yield. Australasian
Plant Pathology 39: 368–375.
Lawrence DP, Gannibal PB, Peever TL, et al. (2013). The sections of Alternaria:
formalizing species-groups concepts. Mycologia 105: 530–546.
Leahy RM (1992). Alternaria leaf spot of Thunbergia. Plant pathology circular
No. 352. Florida Department of Agriculture and Consumer Services, Division
of Plant Industry.
Leiminger JH, Auinger H-J, Wenig M, et al. (2013). Genetic variability
among Alternaria solani isolates from potatoes in Southern Germany
based on RAPD-profiles. Journal of Plant Diseases and Protection 120:
164–172.
Leiminger JH, Adolf B, Hausladen H (2014). Occurence of the F129L mutation in
Alternaria solani populations in Germany in response to QoI application, and
its effect on sensitivity. Plant Pathology 63: 640–650.
Liu YJ, Whelen S, Hall BD (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Molecular Biology and
Evolution 16: 1799–1808.
Lourenço Jr V, Rodrigues TTMS, Campos AMD, et al. (2011). Genetic structure
of the population of Alternaria solani in Brazil. Journal of Phytopathology
159: 233–240.
PATHOGENS
Melo MP, Soares DJ, Araújo JSP, et al. (2009). Alternaria leaf spot, caused by
Alternaria thunbergiae, recorded for the first time on Thunbergia alata from
Brazil. Australasian Plant Disease Notes 4: 23–25.
Narayanin CD, Thompson AH, Slabbert MM (2010). First report of Alternaria
blight of sweet potato caused by Alternaria bataticola in South Africa. African
Plant Protection 16: 7–9.
Neergaard P (1945). Danish species of Alternaria and Stemphylium. Oxford
University Press, London.
Nirenberg HI (1976). Untersuchungen über die morphologische und biologische
Differenzierung in der Fusarium-Section Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem 169:
1–117.
Nolla JAB (1927). A new Alternaria disease of onions (Allium cepa L.). Phytopathology 17(2): 115–132.
O'Donnell K, Kistler HC, Cigelnik E, et al. (1998). Multiple evolutionary origins of
the fungus causing Panama disease of banana: concordant evidence from
nuclear and mitochondrial gene genealogies. Proceedings of the National
Academy of Sciences of the United States of America 95: 2044–2049.
Osiru M, Adipala E, Olanya OM, et al. (2007). Occurrence and distribution of
Alternaria leaf petiole and stem blight on sweetpotato in Uganda. Plant
Pathology Journal 6(2): 112–119.
Osiru MO, Adipala E, Olanya OM, et al. (2008). Leaf petiol and stem blight of
sweet potato caused by Alternaria bataticola in Uganda. Plant Pathology
Journal 7(1): 118–119.
Page RDM (1996). TreeView: an application to display phylogenetic trees on
personal computers. Computer Applications in the Biosciences 12: 357–358.
Rayner RW (1970). A Mycological Colour Chart. Commonwealth Mycological
Institute, Kew, UK.
Rodrigues TTMS, Berbee ML, Simmons EG, et al. (2010). First report of
Alternaria tomatophila and A. grandis causing early blight on tomato and
potato in Brazil. New Disease Reports 22: 28.
Schubert K, Groenewald JZ, Braun U, et al. (2007). Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales) with standardisation of methods for Cladosporium taxonomy and diagnostics. Studies
in Mycology 58: 105–156.
Simmons EG (1994). Alternaria themes and variations (74–105). Mycotaxon 50:
219–270.
Simmons EG (1995). Alternaria themes and variations (112–144). Mycotaxon
55: 55–163.
Simmons EG (1997). Alternaria themes and variations (151–223). Mycotaxon
65: 1–91.
Simmons EG (2000). Alternaria themes and variations (244–286). Species on
Solanaceae. Mycotaxon 75: 1–115.
Simmons EG (2007). Alternaria: an Identification Manual. CBS Biodiversity
Series 6. CBS Fungal Biodiversity Centre, Utrecht, Netherlands.
Sung G-H, Sung J-M, Hywel-Jones NL, et al. (2007). A multi-gene phylogeny of
Clavicipitaceae (Ascomycota, Fungi): identification of localized incongruence
using a combinational bootstrap approach. Molecular Phylogenetics and
Evolution 44: 1204–1223.
Thomma BPHJ (2003). Alternaria spp.: from general saprophyte to specific
parasite. Molecular Plant Pathology 4: 225–236.
Waals JE van der, Korsten L, Slippers B (2004). Genetic diversity among Alternaria
solani isolates from potatoes in South Africa. Plant Disease 88: 959–964.
White TJ, Bruns T, Lee S, et al. (1990). Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a Guide
to Methods and Applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ,
eds). Academic Press, San Diego, California, USA: 315–322.
Woudenberg JHC, Groenewald JZ, Binder M, et al. (2013). Alternaria redefined.
Studies in Mycology 75: 171–212.
Wu HC, Wu WS (2003). Sporulation, pathogenicity and chemical control of
Alternaria protenta a new seedborne pathogen on sunflower. Australasian
Zitter TA, Drennan JL (2005). Shift in performance of fungicides for the control of
tomato early blight. In: Proceedings in the 20th Annual Tomato Disease
Workshop. Ohio State University, Ohio: 28–30.
47
STUDIES
IN
The Colletotrichum destructivum species complex – hemibiotrophic
pathogens of forage and field crops
U. Damm1*, R.J. O'Connell2, J.Z. Groenewald1, and P.W. Crous1,3,4
1
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2UMR1290 BIOGER-CPP, INRA-AgroParisTech, 78850 Thiverval-Grignon,
France; 3Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; 4Wageningen University and Research Centre
(WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
*Correspondence: U. Damm, ulrike.damm@senckenberg.de, Present address: Senckenberg Museum of Natural History Görlitz, PF 300 154, 02806
Görlitz, Germany.
Abstract: Colletotrichum destructivum is an important plant pathogen, mainly of forage and grain legumes including clover, alfalfa, cowpea and lentil, but has also been
reported as an anthracnose pathogen of many other plants worldwide. Several Colletotrichum isolates, previously reported as closely related to C. destructivum, are
known to establish hemibiotrophic infections in different hosts. The inconsistent application of names to those isolates based on outdated species concepts has caused
much taxonomic confusion, particularly in the plant pathology literature. A multilocus DNA sequence analysis (ITS, GAPDH, CHS-1, HIS3, ACT, TUB2) of 83 isolates of
C. destructivum and related species revealed 16 clades that are recognised as separate species in the C. destructivum complex, which includes C. destructivum,
C. fuscum, C. higginsianum, C. lini and C. tabacum. Each of these species is lecto-, epi- or neotypified in this study. Additionally, eight species, namely C. americaeborealis, C. antirrhinicola, C. bryoniicola, C. lentis, C. ocimi, C. pisicola, C. utrechtense and C. vignae are newly described.
Key words: Anthracnose, Ascomycota, Glomerella, Phylogenetics, Systematics.
Taxonomic novelties: New species: Colletotrichum americae-borealis Damm, C. antirrhinicola Damm, C. bryoniicola Damm, C. lentis Damm, C. ocimi Damm,
C. pisicola Damm, C. utrechtense Damm, C. vignae Damm; Typifications: Epitypifications (basionyms): C. destructivum O'Gara, C. fuscum Laubert,
C. higginsianum Sacc., Gloeosporium lini Westerd; Lectotypifications (basionyms): C. fuscum Laubert, Gm. lini Westerd., C. pisi Pat; Neotypification
(basionym): C. tabacum Böning.
Studies in Mycology
INTRODUCTION
Colletotrichum destructivum was originally described as the
causal organism of a disease of clover (Trifolium pratense and
T. hybridum) in the western USA (O'Gara 1915). To date this
species has been reported from more than 30 hosts belonging to
at least 11 plant families, the majority of them being Fabaceae
(especially Trifolium, Medicago, Glycine), but also including
several reports from Poaceae (especially Phalaris, Triticum) and
a few reports from Asteraceae (Chrysanthemum), Convolvulaceae (Cuscuta), Magnoliaceae (Michelia), Menispermaceae (Cocculus), Polygonaceae (Rumex), Solanaceae
(Nicotiana), Lamiaceae (Perilla), Scophulariaceae (Antirrhinum,
Sutera) and Orchidaceae (Bletilla). These reports originate from
18 countries, mainly in North America, Asia and Africa; with
comparatively few reports from Europe, South America and
Oceania (Kawaradani et al. 2008, Tomioka et al. 2011, 2012, Farr
& Rossman 2014).
According to Sutton (1992), conidia of C. destructivum
measure 10–22 × 4–6 μm, are straight to slightly curved,
abruptly tapered to an obtuse apex and a truncate base, while
according to Baxter et al. (1983) they are much narrower,
measuring 16–18 × 3 μm, mostly straight and have tapered
ends.
Since many other Colletotrichum species are also known
from the host plants listed above, there is confusion about the
names applied to different collections. For example, Cannon
et al. (2012) found that half of the ITS sequences of C. trifolii
submitted to GenBank prior to their study, were based on
misidentified strains that actually belonged to the
C. destructivum complex. Many isolates assigned to the
C. destructivum species complex in a preliminary phylogeny
based on ITS and included in this study for further analysis, had
previously been identified as C. coccodes, C. lindemuthianum,
C. trifolii, C. truncatum, C. gloeosporioides or Glomerella cingulata var. cingulata. Further confusion was caused by connecting C. destructivum to the sexual morph Ga. glycines
(Tiffany & Gilman 1954, Manandhar et al. 1986), which was
originally described by Lehman & Wolf (1926) from soybean
stems as the sexual morph of C. glycines. In contrast, von Arx
& Müller (1954) treated Ga. glycines as a form of Ga. cingulata
with large ascospores.
A number of species were observed to have a similar
morphology to C. destructivum and were considered to be
closely related to that species. In the study of Moriwaki et al.
(2002), Japanese Colletotrichum isolates clustered into 20
groups based on ITS1 sequences, which correlated with their
morphology; isolates of C. destructivum, C. fuscum,
C. higginsianum and C. linicola belonged to the same ribosomal
group and were considered as possibly conspecific. Based on
D2 and ITS2 rDNA sequences, Latunde-Dada & Lucas (2007)
found a close relationship among C. destructivum isolates from
Vigna unguiculata and Medicago sativa, C. linicola isolates from
Linum and C. truncatum isolates from Pisum sativum, Vicia faba
49
DAMM
ET AL.
and Lens culinaris, which clustered with C. higginsianum isolates
in their phylogeny. Based on multilocus phylogenies,
C. destructivum was recently delineated as a species complex
with C. fuscum, C. higginsianum, C. tabacum, C. linicola and Ga.
truncata (Cannon et al. 2012, O'Connell et al. 2012). However,
only a few isolates were included in those studies.
The infection strategy of isolates from several hosts of
C. destructivum and related species has been reported as
hemibiotrophic (Bailey et al. 1992, O'Connell et al. 1993, Shen
et al. 2001) and several genes involved in plant infection have
been studied (Huser et al. 2009, Kleemann et al. 2012, Liu
et al. 2013b). To better understand the molecular basis of the
infection process, O'Connell et al. (2012) compared genome
and transcriptome sequence data of C. higginsianum with those
of C. graminicola, a hemibiotrophic species from a different
Colletotrichum species complex. This study revealed that both
species possessed unusually large sets of pathogenicity-related
genes, combining features of both biotrophic and necrotrophic
pathogens. In particular, genes encoding plant cell walldegrading enzymes, proteases and secondary metabolism enzymes are all expanded, similar to necrotrophs, but these fungi
also encode large numbers of effector proteins for host
manipulation, more similar to biotrophs. Transcriptome
sequencing showed that expression of these genes is highly
stage-specific, with most effector and secondary metabolism
genes expressed early during appressorial penetration and
biotrophy, and most plant cell wall-degrading enzymes, proteases and nutrient uptake transporters induced later at the
switch to necrotrophy.
Prior to this study, the phylogenetic relationships of species in
the C. destructivum complex have been studied inadequately using
modern molecular methods. Many species names in this complex
have been applied inconsistently or incorrectly, as there have been
no recent studies of type specimens and few ex-type cultures are
available for sequence analyses. Preliminary results based on
multilocus DNA sequences of a small dataset indicated that isolates from different hosts belonged to several closely related
species. The aim of our study was to recollect, delineate, typify and
characterise the species within the C. destructivum complex,
based on multilocus DNA sequence and morphological data.
Isolates
A total of 83 isolates from the CBS-KNAW Fungal Biodiversity
Centre (CBS), Utrecht, the Netherlands, and other culture collections was studied, most of which had been previously identified as C. destructivum. Type specimens (holo-, lecto-, epi- and
neotypes) of the species studied are located in the fungaria of the
CBS, the US National Fungus Collections (BPI), Beltsville,
Maryland, USA, the Royal Botanic Gardens, Kew, UK, (IMI and
K(M)), and the Botanic Garden and Botanical Museum BerlinDahlem, Freie Universit€at Berlin (B), Germany. All descriptions
are based on ex-holotype, ex-epitype or ex-neotype cultures as
applicable. Features of other isolates or specimens are included
if they deviate from the ex-type cultures. Subcultures of the holo-,
epi- and neotypes as well as all other isolates used for
morphological and sequence analyses are maintained in the
culture collections listed in Table 1.
50
Morphological analysis
To enhance sporulation, autoclaved filter paper and doubleautoclaved stems of Anthriscus sylvestris were placed onto the
surface of synthetic nutrient-poor agar medium (SNA; Nirenberg
1976). SNA and OA (oatmeal agar; Crous et al. 2009) cultures
were incubated at 20 °C under near-UV light with a 12 h
photoperiod for 10 d. Measurements and photomicrographs of
characteristic structures were made according to Damm et al.
(2007). Appressoria were observed on the reverse side of
SNA plates. Microscopic preparations were made in clear lactic
acid, with 30 measurements per structure and observed with a
Nikon SMZ1000 dissecting microscope (DM) or with a Nikon
Eclipse 80i microscope using differential interference contrast
(DIC) illumination.
Colony characters and pigment production on SNA and OA
cultures incubated at 20 °C under near-UV light with a 12 h
photoperiod were noted after 10 d. Colony colours were rated
according to Rayner (1970). Growth rates were measured after 7
and 10 d.
Phylogenetic analysis
Genomic DNA of the isolates was extracted using the method of
Damm et al. (2008). The ITS, GAPDH, and partial sequences of
the chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT)
and beta-tubulin (TUB2) genes were amplified and sequenced
using the primer pairs ITS-1F (Gardes and Bruns 1993) + ITS-4
(White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), CHS354R + CHS-79F (Carbone & Kohn 1999), CYLH3F + CYLH3R
(Crous et al. 2004b), ACT-512F + ACT-783R (Carbone & Kohn
1999) and T1 (O'Donnell & Cigelnik 1997) + Bt-2b (Glass &
Donaldson 1995) or T1 + BT4R (Woudenberg et al. 2009),
respectively. The PCRs were performed in a 2720 Thermal
Cycler (Applied Biosystems, Foster City, California) in a total
volume of 12.5 μL. The GAPDH, CHS-1, HIS3, ACT and TUB2
PCR mixture contained 1 μL 20× diluted genomic DNA, 0.2 μM of
each primer, 1× PCR buffer (Bioline, Luckenwalde, Germany),
2 mM MgCl2, 20 μM of each dNTP, 0.7 μL DMSO and 0.25 U Taq
DNA polymerase (Bioline). Conditions for PCR of these genes
constituted an initial denaturation step of 5 min at 94 °C, followed
by 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C,
and a final denaturation step of 7 min at 72 °C, while the ITS
PCR was performed as described by Woudenberg et al. (2009).
The DNA sequences generated with forward and reverse
primers were used to obtain consensus sequences using Bionumerics v. 4.60 (Applied Maths, St-Marthens-Lathem, Belgium),
and the alignment assembled and manually adjusted using
Sequence Alignment Editor v. 2.0a11 (Rambaut 2002).
To determine whether the six sequence datasets were
congruent and combinable, tree topologies of 70 % reciprocal
Neighbour-Joining bootstrap with Maximum Likelihood distances
(10 000 replicates) with substitution models determined separately for each partition using MrModeltest v. 2.3 (Nylander 2004)
were compared visually (Mason-Gamer and Kellogg 1996). A
maximum parsimony analysis was performed on the multilocus
alignment (ITS, GAPDH, CHS-1, HIS3, ACT, TUB2) as well as
for each gene separately with PAUP (Phylogenetic Analysis
Using Parsimony) v. 4.0b10 (Swofford 2003) using the heuristic
search option with 100 random sequence additions and tree
bisection and reconstruction (TBR) as the branch-swapping
THE COLLETOTRICHUM
DESTRUCTIVUM SPECIES COMPLEX
Table 1. Strains of Colletotrichum spp. studied, with collection details and GenBank accession numbers.
Species
Accession No.1
Host
GenBank No.2
Country
ITS
C. americae-borealis CBS 136232*
GAPDH CHS-1
HIS3
ACT
TUB2
Medicago sativa
USA
KM105224 KM105579 KM105294 KM105364 KM105434 KM105504
CBS 136855
Medicago sativa
USA
KM105225 KM105580 KM105295 KM105365 KM105435 KM105505
ATCC 11869, LARS
373, CPC 18946
Medicago sativa
USA
KM105223 KM105578 KM105293 KM105363 KM105433 KM105503
C. antirrhinicola
CBS 102189*
Antirrhinum majus
New Zealand KM105180 KM105531 KM105250 KM105320 KM105390 KM105460
C. bryoniicola
CBS 109849*
Bryonia dioica
Netherlands
KM105181 KM105532 KM105251 KM105321 KM105391 KM105461
C. destructivum
CBS 114801, AR 4031
Crupina vulgaris
Greece
KM105219 KM105574 KM105289 KM105359 KM105429 KM105499
CBS 119187, AR 4031
C. fuscum
C. higginsianum
Crupina vulgaris
Greece
KM105220 KM105575 KM105290 KM105360 KM105430 KM105500
CBS 128509, LARS 320 Medicago sativa
Canada
KM105214 KM105569 KM105284 KM105354 KM105424 KM105494
CBS 157.83
Serbia
KM105215 KM105570 KM105285 KM105355 KM105425 KM105495
CBS 511.97, LARS 202 Medicago sativa
Medicago sativa
Morocco
KM105216 KM105571 KM105286 KM105356 KM105426 KM105496
CBS 520.97, LARS 709 Medicago sativa
Saudi Arabia
KM105217 KM105572 KM105287 KM105357 KM105427 KM105497
CBS 167.58
Italy
KM105213 KM105568 KM105283 KM105353 KM105423 KM105493
CBS 130238, 5/5/11-1-1 Phragmites
USA
KM105218 KM105573 KM105288 KM105358 KM105428 KM105498
IMI 387103, CPC 18082 Rumex sp.
Korea
KM105221 KM105576 KM105291 KM105361 KM105431 KM105501
CBS 136228*
Trifolium hybridum
USA
KM105207 KM105561 KM105277 KM105347 KM105417 KM105487
CBS 136852
Trifolium hybridum
USA
KM105208 KM105562 KM105278 KM105348 KM105418 KM105488
CBS 136853
Trifolium hybridum
USA
KM105209 KM105563 KM105279 KM105349 KM105419 KM105489
CBS 136229
Trifolium hybridum
USA
KM105211 KM105565 KM105281 KM105351 KM105421 KM105491
CBS 136230
Trifolium repens
USA
KM105210 KM105564 KM105280 KM105350 KM105420 KM105490
CBS 136231
Trifolium repens
USA
KM105212 KM105566 KM105282 KM105352 KM105422 KM105492
Medicago sativa
CBS 149.34
Trifolium sp.
Netherlands
JQ005764
CBS 133704
Digitalis dubia
Netherlands
KM105176 KM105526 KM105246 KM105316 KM105386 KM105456
KM105567 JQ005785
KM105530 JQ005783
JQ005806
CBS 130.57
Digitalis lanata
unknown
JQ005762
Digitalis lutea
Germany
KM105174 KM105524 KM105244 KM105314 KM105384 KM105454
CBS 133702
Digitalis lutea
Netherlands
KM105178 KM105528 KM105248 KM105318 KM105388 KM105458
CBS 133703
Digitalis obscura
Netherlands
KM105175 KM105525 KM105245 KM105315 KM105385 KM105455
CBS 825.68
Digitalis purpurea
Netherlands
KM105177 KM105527 KM105247 KM105317 KM105387 KM105457
CBS 200.54
unknown
Germany
KM105179 KM105529 KM105249 KM105319 KM105389 KM105459
Abc 6-2, CPC 19368
Brassica chinensis
Japan
KM105187 KM105539 KM105257 KM105327 KM105397 KM105467
IMI 349061, CPC
19379*
Brassica chinensis
Trinidad and
Tobago
KM105184 KM105535 KM105254 KM105324 KM105394 KM105464
IMI 349063, CPC 19380 Brassica chinensis
Trinidad and
Tobago
JQ005760
Abo 1-1, CPC 19364
Brassica oleracea
Gemmifera group
Japan
KM105185 KM105537 KM105255 KM105325 KM105395 KM105465
Abp 1-2, CPC 19365
Brassica pekinensis Japan
KM105186 KM105538 KM105256 KM105326 KM105396 KM105466
Abr 2-2, CPC 19369
Brassica rapa
Japan
KM105188 KM105540 KM105258 KM105328 KM105398 KM105468
Abr 3-1, CPC 19370
Brassica rapa
Japan
KM105189 KM105541 KM105259 KM105329 KM105399 KM105469
MAFF 305635,
Abr 1-5, CPC 19366
Brassica rapa
Perviridis Group
Japan
JQ005761
CBS 128508, LARS
889, Kyoto 337-5
Brassica rapa var.
komatsuna
Japan
KM105190 KM105543 KM105260 KM105330 KM105400 KM105470
NBRC 6182, CPC
18944
Brassica sp.
Italy
KM105191 KM105544 KM105261 KM105331 KM105401 KM105471
AR 3-5, CPC 19363
Raphanus sativus
Japan
KM105192 KM105545 KM105262 KM105332 KM105402 KM105472
AR 3-1, CPC 19394
Raphanus sativus
Japan
KM105193 KM105546 KM105263 KM105333 KM105403 KM105473
AR 7-3, CPC 19395
Raphanus sativus
var. sativus
Japan
KM105194 KM105547 KM105264 KM105334 KM105404 KM105474
AR 8-1, CPC 19396
Raphanus sativus
Japan
KM105195 KM105548 KM105265 KM105335 KM105405 KM105475
KM105542 JQ005782
JQ005802
JQ005803
JQ005825
JQ005848
CBS 133701*
KM105536 JQ005781
JQ005804
JQ005827
JQ005823
JQ005824
JQ005846
JQ005844
JQ005845
51
DAMM
ET AL.
Species
C. lentis
C. lini
Accession No.1
Host
GenBank No.2
Country
ITS
GAPDH CHS-1
HIS3
ACT
TUB2
KM105597 JQ005787
JQ005808
JQ005829
JQ005850
CBS 127604, DAOM
235316, CT21*
Lens culinaris
Canada
JQ005766
CBS 127605, DAOM
235317, CT26
Lens culinaris
Canada
KM105241 KM105598 KM105311 KM105381 KM105451 KM105521
CBS 172.51*
Linum
usitatissimum
Netherlands
JQ005765
CBS 505.97, LARS 77
Linum
usitatissimum
Ireland
KM105226 KM105582 KM105296 KM105366 KM105436 KM105506
IMI 103842, CPC 18947 Linum
usitatissimum
UK
KM105227 KM105583 KM105297 KM105367 KM105437 KM105507
IMI 103844, CPC 16816 Linum
usitatissimum
UK
KM105228 KM105584 KM105298 KM105368 KM105438 KM105508
CBS 112.21, LCP
46.621
Linum
usitatissimum
UK
KM105229 KM105585 KM105299 KM105369 KM105439 KM105509
CBS 100569, PD 97/
14304
Nigella sp.
France
KM105230 KM105586 KM105300 KM105370 KM105440 KM105510
IMI 391904, IS320, CPC Raphanus
19382
raphanistrum
Tunisia
KM105232 KM105588 KM105302 KM105372 KM105442 KM105512
CBS 117156
Teucrium
scorodonia
Netherlands
KM105231 KM105587 KM105301 KM105371 KM105441 KM105511
CBS 136856
Medicago sativa
USA
KM105233 KM105589 KM105303 KM105373 KM105443 KM105513
CBS 136857
Taraxacum sp.
USA
KM105239 KM105595 KM105309 KM105379 KM105449 KM105519
CBS 136233
Taraxacum sp.
USA
KM105240 KM105596 KM105310 KM105380 KM105450 KM105520
CBS 136850
Trifolium hybridum
USA
KM105237 KM105593 KM105307 KM105377 KM105447 KM105517
CBS 136851
Trifolium hybridum
USA
KM105238 KM105594 KM105308 KM105378 KM105448 KM105518
CBS 130828
Trifolium repens
Germany
KM105234 KM105590 KM105304 KM105374 KM105444 KM105514
CBS 130829
Trifolium repen
Germany
KM105235 KM105591 KM105305 KM105375 KM105445 KM105515
KM105581 JQ005786
JQ005807
JQ005828
JQ005849
IMI 69991, CPC 20242
Trifolium sp.
New Zealand KM105236 KM105592 KM105306 KM105376 KM105446 KM105516
C. ocimi
CBS 298.94*
Ocimum basilicum
Italy
KM105222 KM105577 KM105292 KM105362 KM105432 KM105502
C. panacicola
C08087
Panax ginseng
Korea
GU935869 GU935849
GU944758
C08061
Panax ginseng
Korea
GU935868 GU935848
GU935791
C08048
Panax ginseng
Korea
GU935867 GU935847
GU944757
C. pisicola
CBS 724.97, LARS 60*
Pisum sativum
USA
KM105172 KM105522 KM105242 KM105312 KM105382 KM105452
C. tabacum
CBS 124249, MUCL
44942
Centella asiatica
Madagascar
KM105206 KM105560 KM105276 KM105346 KM105416 KM105486
N150, CPC 18945*
Nicotiana tabacum
Canada
KM105204 KM105557 KM105274 KM105344 KM105414 KM105484
IMI 50187, CPC 16820
Nicotiana tabacum
India
KM105205 KM105558 KM105275 KM105345 KM105415 KM105485
CBS 161.53
Nicotiana tabacum
Zambia
JQ005763
KM105559 JQ005784
CBS 132693, BRIP
57314, UM01*
Tanacetum
cinerariifolium
Australia
JX218228
JX218243
CBS 132818, BRIP
57315, TAS060-0003
Tanacetum
cinerariifolium
Australia
JX218229
BRIP 57316, TAS0600004
Tanacetum
cinerariifolium
Australia
JX218230
CBS 130243*
Trifolium pratense
Netherlands
KM105201 KM105554 KM105271 KM105341 KM105411 KM105481
CBS 135827
Trifolium pratense
Netherlands
KM105202 KM105555 KM105272 KM105342 KM105412 KM105482
CBS 135828
Trifolium pratense
Netherlands
KM105203 KM105556 KM105273 KM105343 KM105413 KM105483
CBS 501.97, LARS 56*
Vigna unguiculata
Nigeria
KM105183 KM105534 KM105253 KM105323 KM105393 KM105463
IMI 334960, CPC 19383 Vigna unguiculata
Nigeria
KM105182 KM105533 KM105252 KM105322 KM105392 KM105462
CBS 125336
Heracleum sp.
Netherlands
KM105198 KM105551 KM105268 KM105338 KM105408 KM105478
CBS 126510
Heracleum sp.
Netherlands
KM105199 KM105552 KM105269 KM105339 KM105409 KM105479
CPC 18076
Heracleum sp.
Netherlands
KM105200 KM105553 KM105270 KM105340 KM105410 KM105480
C. tanaceti
C. utrechtense
C. vignae
Colletotrichum sp.
52
JQ005805
JQ005826
JQ005847
JX259268
JX218238
JX218233
JX218244
JX259269
JX218239
JX218234
JX218245
JX259270
JX218240
JX218235
THE COLLETOTRICHUM
Species
Accession No.1
Host
GenBank No.2
Country
ITS
Colletotrichum sp.
GAPDH CHS-1
HIS3
ACT
TUB2
CH90-M1, CPC 19361
Matthiola incana
Japan
KM105196 KM105549 KM105266 KM105336 KM105406 KM105476
CH93-M1, CPC 19362
Matthiola incana
Japan
KM105197 KM105550 KM105267 KM105337 KM105407 KM105477
CBS 107.40
Pisum sativum
Russia
KM105173 KM105523 KM105243 KM105313 KM105383 KM105453
*ex-holotype, ex-epitype or ex-neotype culture.
ATCC: American Type Culture Collection, Virginia, USA; BRIP: Plant Pathology Herbarium, Department of Primary Industries, Queensland, Australia; CBS: Culture
collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS;
DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; LARS: Culture
collection of Long Ashton Research Station, Bristol, UK (no longer existing); MAFF: MAFF Genebank Project, Ministry of Agriculture, Forestry and Fisheries, Tsukuba,
Japan; MUCL: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium; NBRC: Culture Collection of the Biological Resource Center, National Institute of Technology
and Evaluation, Kisarazu, Japan; PD: Plantenziektenkundige Dienst, Wageningen, Netherlands.
2
ITS: internal transcribed spacers and intervening 5.8S nrDNA; GAPDH: partial glyceraldehyde-3-phosphate dehydrogenase gene; CHS-1: partial chitin synthase-1 gene;
HIS: partial histone H3 gene; ACT: partial actin gene; TUB2: partial beta-tubulin gene. Sequences generated in this study are emphasised in bold face.
1
algorithm. Alignment gaps were treated as new states and all
characters were unordered and of equal weight. The robustness
of the trees obtained was evaluated by 1 000 bootstrap replications using the same settings as for the parsimony analysis
itself (Hillis & Bull 1993). Tree length, consistency index (CI),
retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the resulting trees. A
Markov Chain Monte Carlo (MCMC) algorithm was used to
generate phylogenetic trees with Bayesian probabilities using
MrBayes v. 3.2.2 (Ronquist & Huelsenbeck 2003) for the combined sequence datasets. Models of nucleotide substitution for
each gene determined by the AIC criterion as implemented in
MrModeltest v. 2.3 were included for each gene partition. The
analyses of two parallel Markov Chain Monte Carlo (MCMC)
runs, each consisting of four chains, were run from random trees
for 100 M generations and sampled every 1 000 generations until
the runs converged with a split frequency of 0.01. The first 25 %
of trees were discarded as the burn-in phase of the analysis and
posterior probabilities determined from the remaining trees. For
additional comparison, a Neighbour-Joining analysis was performed on the multilocus alignment using PAUP with 10 000
bootstrap replications. Sequences derived in this study have
been lodged at GenBank, the alignment and tree in TreeBASE
(www.treebase.org/treebase-web/home.html) (S16069), and
taxonomic novelties in MycoBank (Crous et al. 2004a).
RESULTS
Phylogeny
The six individual datasets did not show any conflicts in tree
topology for the 70 % reciprocal bootstrap trees, which allowed
us to combine them. In the multilocus analyses (gene boundaries
of ITS: 1–560, GAPDH: 571–795, CHS-1: 806–1085, HIS3:
1096–1485, ACT: 1496–1758, TUB2: 1769–2281) of 83 isolates
of C. destructivum and related Colletotrichum species and the
outgroup (C. pisicola CBS 724.97 and Colletotrichum sp. CBS
107.40), 2 281 characters including the alignment gaps were
processed, of which 349 characters were parsimony-informative,
48 parsimony-uninformative and 1 884 constant. After a heuristic
search using PAUP, 14 equally most parsimonious trees were
retained (tree length = 540 steps, CI = 0.828, RI = 0.962,
RC = 0.796, HI = 0.172) of which the first tree is shown in Fig. 1.
The overall topology of all of the equally most parsimonious trees
was similar; they differed only in the position of isolates within the
C. destructivum s. str. clade. The Bayesian analysis was conducted using the following substitution models: dirichlet (1,1,1,1)
state frequency distributions were used for all loci except for
CHS-1 which had a fixed (equal) state frequency distribution; for
ITS the model was HKY with a proportion of invariable sites
allowed, for both GAPDH and CHS-1 the model was HKY with an
equal variation rate across sites, for HIS3 the model was GTR
with a gamma-shaped rate variation across sites, for ACT the
model was HKY with a gamma-shaped rate variation across
sites, and for TUB2 the model was GTR with a proportion of
invariable sites allowed. The Bayesian analysis lasted 1 081 000
generations, after which the split frequency reached less than
0.01; 1 622 trees of the 2 162 trees were used to calculate the
consensus tree and posterior probabilities (PP's; see values
plotted onto Fig. 1).
The analysis resulted in the delineation of seven main clades
and 16 subclades within the C. destructivum species complex,
which we accept as representing different Colletotrichum species. The first main clade (bootstrap support value = 91 %/
Bayesian posterior probability value = 1.00) consists of several
closely related species including C. fuscum (74/1.00),
C. higginsianum (74/1.00), C. vignae (99/1.00), two single isolates clades belonging to C. anthirrhinicola and C. bryoniicola
and five unnamed strains. Colletotrichum utrechtense (78) and
C. panacicola (95/1.00) belonged to the second main clade,
while the third clade only contained one subclade, representing
C. tabacum (100/1.00). Clade four consists of a large number of
C. destructivum s. str. isolates (95/0.99) and a sister clade on a
long branch representing C. ocimi. Clade five consists of two
subclades representing C. americae-borealis (67/0.92) and
C. lini (98/1.00). Clade six is represented by two well-supported
subclades on long branches, C. lentis (100/1.00) and C. tanaceti
(100/1.00). The seventh main clade consists of a long branch
with two single strain clades representing C. pisicola and a
second unidentified species from Pisum and is basal to the rest
of the isolates and was consequently chosen as the outgroup of
the phylogeny.
The consensus tree obtained from the Bayesian analysis and
the NJ tree (not shown) confirmed the tree topology obtained
from the parsimony analysis. Bayesian posterior probability
values mostly agreed with bootstrap support values and are also
plotted on the phylogram (Fig. 1). The individual alignments and
maximum parsimony analyses of the six single genes were
53
DAMM
ET AL.
64
0.98
CBS 133701 Digitalis Germany
CBS 133703 Digitalis NL
74
1.00
86 CBS 200.54 unknown Germany
CBS 130.57 Digitalis unknown
59
CBS 825.68 Digitalis NL
CBS 102189 Antirrhinum NZ
56
CBS 109849 Bryonia NL
99 IMI 334960 Vigna Nigeria
78 1.00 CBS 501.97 Vigna Nigeria
CBS 125336 Heracleum NL
CBS 126510 Heracleum NL
CPC 18076 Heracleum NL
IMI 349061 Brassica Trin Tobago
IMI 349063 Brassica Trin Tobago
Abo 1-1 Brassica Japan
Abp 1-2 Brassica Japan
91
Abc 6-2 Brassica Japan
1.00
63
Abr 2-2 Brassica Japan
0.98
74 Abr 3-1 Brassica Japan
1.00 AR 3-5 Raphanus Japan
AR 3-1 Raphanus Japan
63
MAFF 305635 Brassica Japan
58 CBS 128508 Brassica Japan
96
NBRC 6182 Brassica Italy
0.91
1.00
CH90 M1 Matthiola Japan
CH93 M1 Matthiola Japan
CBS 130243 Trifolium NL
78 CBS 135827 Trifolium NL
80
CBS 135828 Trifolium NL
94
1.00
1.00 95 C08087 Panax Korea
C08061 Panax Korea
1.00
63
C08048 Panax Korea
0.99
N150 Nicotiana France
79
IMI 50187 Nicotinia India
100
CBS 161.53 Nicotiana Zambia
1.00
CBS 124249 Centella Madagascar
CBS 136228 Trifolium USA
69 CBS 136853 Trifolium USA
65
1.00 CBS 136230 Trifolium USA
0.99
CBS 130238 Phragmites USA
61
CBS 149.34 Trifolium NL
84
CBS 511.97 Medicago Morocco
CBS 520.97 Medicago Saudi Arabia
CBS 167.58 Medicago Italy
95
CBS 128509 Medicago Canada
CBS 157.83 Medicago Serbia
0.99
100
CBS 114801 Crupina Greece
1.00
100
CBS 119187 Crupina Greece
1.00
IMI 387103 Rumex Korea
CBS 298.94 Ocimum Italy
ATCC 11869 Medicago USA
67
0.92 99 CBS 136232 Medicago USA
1.00 CBS136855 Medicago USA
CBS 172.51 Linum NL
CBS 505.97 Linum Ireland
100
IMI 103842 Linum UK
1.000.99 IMI 103844 Linum UK
CBS 112.21 Linum UK
CBS100569 Nigella France
CBS 117156 Teucrium NL
98
IMI 391904 Raphanus Tunisia
1.00
63
CBS 136856 Medicago USA
0.99
CBS 130828 Trifolium Germany
CBS 130829 Trifolium Germany
51
IMI 69991 Trifolium NZ
0.98
67 CBS 136850 Trifolium USA
0.97 CBS 136851 Trifolium USA
CBS 136857 Taraxacum USA
CBS 136233 Taraxacum USA
100 CBS 127604 Lens Canada
66
1.00 CBS 127605 Lens Canada
CBS 132693 Tanacetum Australia
0.89
100 CBS 132818 Tanacetum Australia
1.00 BRIP 57316 Tanacetum Australia
CBS 724.97 Pisum USA
CBS 107.40 Pisum Russia
88
1.00
5 changes
100
C. fuscum
C. antirrhinicola
C. bryoniicola
C. vignae
Colletotrichum sp.
1
C. higginsianum
Colletotrichum sp.
C. utrechtense
2
C. panacicola
C. tabacum
3
C. destructivum
4
C. ocimi
C. americae-borealis
5
C. lini
C. lentis
C. tanaceti
C. pisicola
Colletotrichum sp.
6
7
Fig. 1. The first of 14 equally most parsimonious trees obtained from a heuristic search of the combined ITS, GAPDH, CHS-1, ACT, HIS3 and TUB2 sequences alignment of
the Colletotrichum destructivum species complex. Bootstrap support values above 50 % (bold) and Bayesian posterior probability values above 0.90 are shown at the nodes.
Colletotrichum pisicola CBS 724.97 and Colletotrichum sp. CBS 107.40 are used as outgroup. Numbers of ex-holotype, ex-neotype and ex-epitype isolates are emphasised in
bold. Strain numbers are followed by substrate (host genus) and country of origin, NL = Netherlands, NZ = New Zealand, Trin Tobago = Trinidad and Tobago. Main clades are
indicated by blue lines. Branches that are crossed by diagonal lines are shortened by 50 %.
54
THE COLLETOTRICHUM
compared with respect to their performance in species recognition. None of the loci differentiated all clades, but TUB2 provided the highest resolution of the tested loci. All clades are
recognised by using a combination of both TUB2 and GAPDH
sequences; other loci only recognised some of the species.
Some species differ only in one or two nucleotides (see notes
accompanying each species).
Taxonomy
Based on DNA sequence data and morphology, the 83 isolates
studied (Table 1) are assigned to 16 species, including eight
species that are considered to be new to science. All species
studied in culture are characterised below.
Colletotrichum americae-borealis Damm, sp. nov.
Etymology: The species epithet is derived from the region where
the species was collected, North America.
Sexual morph not observed. Asexual morph on SNA. Vegetative
hyphae 1–7.5 μm diam, hyaline, smooth-walled, septate,
branched. Chlamydospores not observed. Conidiomata absent,
conidiophores formed directly on hyphae or on a cushion of pale
brown, angular cells, 3–6.5 μm diam. Setae medium brown,
smooth-walled to finely verruculose, 55–230 μm long, 1–4septate, base cylindrical to conical, 2.5–7.5 μm diam,
tip ± acute to ± rounded. Conidiophores hyaline to pale brown,
smooth-walled, septate, branched, to 40 μm long. Conidiogenous
cells hyaline to pale brown, smooth-walled, cylindrical to
ampulliform, sometimes intercalary (necks not separated from
hyphae by septum), 9.5–24.5 × 3.5–5 μm, opening 1–1.5 μm
diam, collarette 0.5–1 μm long, periclinal thickening observed.
Conidia hyaline, smooth-walled, aseptate, cylindrical to fusoid,
straight to slightly curved, both ends rounded, (13.5–)
15.5–18(–19) × 3.5–4 μm, av. ± SD = 16.6 ± 1.3 × 3.7
± 0.2 μm, L/W ratio = 4.5, conidia of strain ATCC 11869 shorter,
measuring (9.5–)11.5–15.5(–17.5) × (3–)3.5–4(–4.5) μm,
av. ± SD = 13.5 ± 2.2 × 3.8 ± 0.4 μm, L/W ratio = 3.5.
Appressoria not observed, appressoria of strain CBS 136855
single or in loose groups, medium to dark brown, smooth-walled,
ellipsoid, clavate or irregular outline, with an undulate to lobate
margin, (4.5–)6–10.5(–13) × (3.5–)4–7(–10) μm, av. ± SD
= 8.1 ± 2.2 × 5.4 ± 1.5 μm, L/W ratio = 1.5.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on pale brown, angular cells,
4–7 μm diam. Setae medium brown, smooth-walled, 8–250 μm
long, 1–6-septate, base cylindrical to conical, 5–10 μm diam,
tip ± acute to ± rounded. Conidiophores hyaline to pale brown,
smooth-walled, simple or septate and branched, to 30 μm long.
Conidiogenous cells hyaline to pale brown, smooth-walled,
Fig. 2. Colletotrichum americae-borealis (A–N, U–V from ex-holotype strain CBS 136232. O–T from strain CBS 136855). A–B. Conidiomata. C, I. Tip of a seta. D, J. Base of
a seta. E–H, K–N. Conidiophores. O–T. Appressoria. U–V. Conidia. A, C–H, U. from Anthriscus stem. B, I–T, V. from SNA. A–B. DM, C–V. DIC, Scale bars: A = 200 μm,
G = 10 μm. Scale bar of A applies to A–B. Scale bar of G applies to C–V.
55
DAMM
ET AL.
cylindrical to ampulliform, 8.5–19 × 3.5–5.5 μm, opening 1–2 μm
diam, collarette 0.5–1 μm long, periclinal thickening distinct.
Conidia hyaline, smooth-walled, aseptate, cylindrical to fusoid,
straight to slightly curved, both ends rounded, (14.5–)
16.5–18.5(–19.5) × (3–)3.5(–4) μm, av. ± SD = 17.4 ± 1.0
× 3.5 ± 0.2 μm, L/W ratio = 5.0, conidia of strain ATCC 11869
shorter, measuring (8–)13–18(–19.5) × 3–4 μm, av. ± SD = 15.5
± 2.4 × 3.8 ± 0.3 μm, L/W ratio = 4.5.
Culture characteristics: Colonies on SNA flat with entire margin,
hyaline, pale cinnamon in the centre, agar medium, filter paper
and Anthriscus stem partly covered with saffron to dark grey
acervuli, medium and filter paper partly covered with sparse,
whitish aerial mycelium, reverse same colours; growth
21.5–25 mm in 7 d (35–37 mm in 10 d). Colonies on OA flat with
entire margin; buff, rosy buff to saffron, towards the centre saffron
to dark grey acervuli, aerial mycelium lacking, reverse buff to
rosy buff, growth 22.5–25 mm in 7 d (33.5–36 mm in 10 d).
Conidia in mass saffron.
Materials examined: USA, Utah, Bluffdale (near Salt Lake City), from stems of
Medicago sativa, 25 Aug. 2013, U. Damm (CBS H-21661 holotype, culture exholotype CBS 136232); Utah, Bluffdale (near Salt Lake City), from stems of
Medicago sativa, 25 Aug. 2013, U. Damm, culture CBS 136855; Iowa, from
Medicago sativa, collection date and collector unknown, (received from R.
O'Connell, before from F. Uruburu, deposited in ATCC collection by L.H. Tiffany)
culture ATCC 11869 = CPC 18946 = LARS 373.
Notes: The conidial shape of C. americae-borealis is similar to
that of C. lini, but more complex appressoria were observed. In
contrast to most of the other species in this complex, setae were
very abundant. Several species have been described from
Trifolium and Medicago that are discussed under
C. destructivum.
The ITS and GAPDH sequences of C. americae-borealis are
the same as those of C. lini. This species can be distinguished
from other species in this complex by TUB2, CHS-1, HIS3 and
ACT sequences.
Strain ATCC 11869 shows additional differences in CHS-1,
HIS3 and ACT sequences to strains CBS 136232 and CBS
136855, the other two strains of this species studied. We prefer
to treat this strain as C. americae-borealis for the present,
because it has the same host and origin as the other two strains.
This strain was hardly sporulating; appressoria resembled those
of strains CBS 136232 and CBS 136855. Strain ATCC 11869
was deposited in the ATCC collection by L.H. Tiffany, and
apparently belongs to the large collection of Colletotrichum isolates from legumes studied by Tiffany & Gilman (1954). It would
be interesting to include more isolates related to ATCC 11869 in
a future study to determine whether ATCC 11869 and additional
isolates might form a distinct clade or reveal morphological or
biological differences to the ex-type strain of C. americaeborealis.
The closest match in a blastn search with the TUB2 sequence
of strain CBS 136232 was with 99 % identity (1 nucleotide difference) C. linicola (= C. lini) strain CBS 172.51 (GenBank
JQ005849, O'Connell et al. 2012), which is included in this study.
Blastn searches with the ITS sequence of strain CBS 136232
resulted in 100 % matches with sequences of C. destructivum (s.
lat.) strains 1212, MP11 (GenBank KF181248, KF181247, Z.
Wen & Z. Nan, an unpublished study on alfalfa root rot in Gansu,
China), DAOM 179749 from an unknown host (GenBank
EU400143, Chen et al. 2007) and strain Hamedan from clover in
56
Iran (GenBank FJ185789, Zafari & Tarrah 2009), C. linicola (=
C. lini) strain CBS 172.51 (GenBank JQ005765, O'Connell et al.
2012 and GenBank AB046609, Moriwaki et al. 2002), a C. linicola
(= C. lini) isolate from Convolvulus in Turkey (GenBank
EU000060, Tunali et al. 2008), unidentified fungus strains DJJ15
and DY20 from Oxytropis (GenBank JF461333, JF461335, J.
Wang, unpubl. study), C. higginsianum strain IMI 391904 from
Raphanus in Tunisia (GenBank JX499034, Naumann & Wicklow
2013) that is included in this study and re-identified as C. lini,
Colletotrichum sp. isolates 2002 from Holcus (GenBank
FN386304, Sanchez Marquez et al. 2012) and 842 and 865 from
Arabidopsis (GenBank JX982460, JX982461, Garcia et al. 2013),
both the latter reports concerning endophytes isolated in Spain.
Colletotrichum antirrhinicola Damm, sp. nov. MycoBank
MB809399. Fig. 3.
Etymology: The species epithet is derived from its host plant
Antirrhinum.
hyphae 1–10 μm diam, hyaline, some are pale to medium brown,
smooth-walled, septate, branched. Chlamydospores not
observed. Conidiomata absent, conidiophores and setae formed
directly on hyphae. Setae pale to medium brown, verruculose,
30–100 μm long, 1–4-septate, base cylindrical, conical
to ± inflated, 5.5–6.5 μm diam, tip rounded. Conidiophores hyaline, smooth-walled, septate, branched, to 35 μm long. Conidiogenous cells hyaline, smooth-walled, cylindrical to
ampulliform, sometimes intercalary (necks not separated from
hyphae
by
septum),
polyphialides
observed,
8.5–25 × 3.5–5.5 μm, opening 1.5–2 μm diam, collarette 1 μm
long, periclinal thickening distinct. Conidia hyaline, smooth-walled,
aseptate, cylindrical, straight to slightly curved, with one end round
and the other truncate, (14.5–)15.5–19(–23.5) × (3.5–)
4–4.5(–5) μm, av. ± SD = 17.2 ± 1.7 × 4.3 ± 0.3 μm, L/W ratio = 4.0. Appressoria single, medium brown, smooth-walled,
subglobose, ovate to broadly elliptical in outline, with an entire
or undulate margin, (9–)9.5–12(–13.5) × (5–)6–8(–10) μm,
av. ± SD = 10.9 ± 1.3 × 7.0 ± 1.0 μm, L/W ratio = 1.5.
3–8 μm diam. Setae pale to medium brown, smooth-walled,
35–170 μm long, 1–3-septate, base cylindrical to conical,
5.5–6 μm diam, tip ± acute to ± rounded. Conidiophores hyaline,
cells hyaline, smooth-walled, cylindrical to ampulliform,
11–15 × 4–5.5 μm, opening 1–1.5 μm diam, collarette 1 μm
long, periclinal thickening distinct. Conidia hyaline, smoothwalled, aseptate, cylindrical, straight to slightly curved, with
one end round and the other truncate, (13–)
15.5–19(–20) × (3.5–)4–4.5(–5) μm, av. ± SD = 17.3 ± 1.6
× 4.2 ± 0.4 μm, L/W ratio = 4.1.
hyaline to pale rosy-buff, filter paper and agar medium in centre
partly grey, agar medium, filter paper and Anthriscus stem partly
covered with white aerial mycelium, reverse same colours;
growth 21.5–23 mm in 7 d (33–34.5 mm in 10 d). Colonies on
OA flat with entire margin; buff, partly covered with black acervuli
and salmon conidial masses, aerial mycelium lacking, reverse
THE COLLETOTRICHUM
Fig. 3. Colletotrichum antirrhinicola (from ex-holotype strain CBS 102189). A–B. Conidiomata. C, H. Tip of a seta. D, I. Base of a seta. E–G, J–M. Conidiophores. N–S.
Appressoria. T–U. Conidia. A, C–G, T. from Anthriscus stem. B, H–S, U. from SNA. A–B. DM, C–U. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B.
Scale bar of E applies to C–U.
buff, rosy-buff to pale olivaceous-grey, growth 20.5–22 mm in 7 d
(30–31.5 mm in 10 d). Conidia in mass salmon.
Material examined: New Zealand, Auckland, Kingsland, from foliage of Antirrhinum majus, collection date unknown (deposited in CBS collection Sep. 1999
by C.F. Hill, isolated 22 Jul. 1999 by H.M. Dance, Agriquality N2, No. 017), HM
Dance (CBS H-21647 holotype, culture ex-holotype CBS 102189).
Notes: Colletotrichum antirrhinicola is only known from snapdragon (Antirrhinum majus, Scrophulariaceae) in New Zealand.
The species can be identified by its unique GAPDH and ITS
sequences. The HIS3 sequence is the same as that of
C. fuscum, while the ACT sequence is identical with C. fuscum
and C. bryoniicola. Closest match in a blastn search with the ITS
sequence of CBS 102189 with 99 % identity (1 nucleotide difference) was C. fuscum strain DAOM 216112 from an unknown
host (GenBank EU400144, Chen et al. 2007), while the most
similar GAPDH sequences on NCBI GenBank are 96 % identical
to that of CBS 102189. In blastn searches, ACT and HIS3 sequences of CBS 102189 are identical with GenBank JQ005825
and GenBank JQ005804, respectively from C. fuscum CBS
130.57 (O'Connell et al. 2012) that are included here.
Tomioka et al. (2011) reported C. destructivum to cause a
severe anthracnose disease on leaves of A. majus in Japan. ITS
sequences of two strains (MAFF 239947, MAFF 239948) are
available in NCBI GenBank (GenBank AB334521, AB334522);
additionally, ACT, EF1-α, GAPDH, ITS and TUB2 sequences are
available on NIAS GenBank. However, none of these sequences
agree with those of strain CBS 102189 (95–99 % identity); the
strains from Japan therefore probably represent a different
species, most likely in the same species complex.
Stewart (1900a, b) reported a new anthracnose disease of
cultivated snapdragon in the USA as C. antirrhini. The description by Stewart (1900b) indicates the species may belong to the
C. destructivum complex with conidia measuring 16–21 × 4 μm.
The species was regarded as synonym of C. gloeosporioides by
von Arx (1957), and is listed as a synonym of C. coccodes in
Index Fungorum (www.indexfungorum.org, retrieved 20 Aug.
2014). However, as there are apparently several Colletotrichum
species causing anthracnose on this host we refrain from epitypifying this species with a strain from New Zealand instead of
the USA, and rather describe it here as a new species.
Strain CBS 102189 was previously identified as C. fuscum by
C.F. Hill. A strain from the same host and country (IMI 197877),
also identified by C.F. Hill but apparently much earlier, as well as
collections from UK were listed by Sutton (1980) as C. fuscum,
which is closely related to C. antirrhinicola.
Colletotrichum bryoniicola Damm, sp. nov. MycoBank
MB809400. Fig. 4.
Etymology: The species epithet is derived from its host plant
Bryonia.
57
DAMM
ET AL.
Fig. 4. Colletotrichum bryoniicola (from ex-holotype strain CBS 109849). A–B. Conidiomata. C–I. Conidiophores. J–O. Appressorium-like structures. P–Q. Conidia. A, C–F, P.
from Anthriscus stem. B, G–O, Q. from SNA. A–B. DM, C–Q. DIC, Scale bars: A = 100 μm, C = 10 μm. Scale bar of A applies to A–B. Scale bar of C applies to C–Q.
conidiophores formed directly on hyphae. Additionally, structures
formed resembling the basal cushions of acervuli, but lacking
conidiophores, cells angular to roundish, pale brown, 3.5–10 μm
diam. Setae not observed. Conidiophores hyaline to pale brown,
smooth-walled, sometimes septate and branched, to 15 μm long.
Conidiogenous cells rarely observed, hyaline to pale brown,
smooth-walled, cylindrical to conical, 5.5–10 × 3.5–4 μm,
opening 1–1.5 μm diam, collarette 0.5 μm long, periclinal
thickening not observed. Conidia hyaline, smooth-walled, aseptate, cylindrical, straight to slightly curved, with one end round
and the other truncate, (13.5–)15–18.5(–22) × 4–5(–5.5) μm,
av. ± SD = 16.8 ± 1.6 × 4.6 ± 0.4 μm, L/W ratio = 3.6.
Appressoria not observed on the undersurface of the medium.
Appressoria-like structures that possibly function as chlamydospores were observed within the medium. These are single or in
loose groups, pale brown, smooth-walled, subglobose to elliptical
in outline, with an entire or slightly undulate margin, (3.5–)
4–10(–18) × (2.5–)3.5–6.5(–7.5) μm, av. ± SD = 7.1 ± 3.0
× 4.9 ± 1.5 μm, L/W ratio = 1.4.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores formed on pale brown, angular cells, 3–9 μm diam.
Setae not observed. Conidiophores rarely seen, pale brown,
smooth-walled. Conidiogenous cells hyaline to pale brown,
smooth-walled,
doliiform,
ampulliform
to
cylindrical,
58
7–13.5 × 3–5 μm, opening 1–1.5 μm diam, collarette 0.5 μm long,
periclinal thickening not observed. Conidia hyaline, smooth-walled,
aseptate, cylindrical, straight to slightly curved, with one end round
and the other truncate, (16–)17–19.5(–21) × 4.5(–5) μm,
av. ± SD = 18.2 ± 1.1 × 4.5 ± 0.1 μm, L/W ratio = 4.0.
hyaline, agar medium, filter paper and Anthriscus stem partly
covered with white aerial mycelium, reverse same colours;
growth 20.5–21.5 mm in 7 d (29.5–31.5 mm in 10 d). Colonies
on OA flat with entire margin; buff, with cinnamon, olivaceousgrey to iron-grey sectors, aerial mycelium lacking, reverse
same colours, growth 17.5–18.5 mm in 7 d (27.5–29 mm in
10 d). Conidia in mass whitish to pale salmon.
Material examined: Netherlands, Wissenkerke, Camperduin, coord. 35.5/401.6,
from decaying leaves of Bryonia dioica, 27 Aug. 2001, G. Verkley, No. V1114
(CBS H-21663 holotype, culture ex-holotype CBS 109849).
Notes: Colletotrichum bryoniicola differs from closely related
species in ITS, GAPDH, HIS3 and TUB2 sequences by a single
nucleotide in each locus. The ACT sequence is the same as that
of C. fuscum and C. antirrhinicola, the CHS-1 sequence is
identical with that of C. tanaceti. There are no previously
accessioned sequences of a Colletotrichum species from Bryonia in GenBank. With the exception of the ACT and CHS-1 sequences, there are no sequences in GenBank that are identical
to those of C. bryoniicola.
THE COLLETOTRICHUM
Conidia of C. bryoniicola are broader ( 4 μm on SNA, 4.5 μm on Anthriscus stem) than the other species in the
C. destructivum complex, no setae were observed and the
conidiogenous cells are very indistinct.
A species from Bryonia dioica (Cucurbitaceae) was previously described ad interim by Maire (1917), as C. bryoniae.
Although Maire (1917) did not mention any connection of the new
species from Alger (= Algiers), Mauretania (today Algeria) with
C. oligochaetum f. bryoniae Ferraris from B. dioica in Italy
(Ferraris & Massa 1912), that taxon was accepted as an independent species by Saccardo et al. (1931) and cited incorrectly
as C. bryoniae (Ferraris) Maire (1917). As it is based on a forma
of C. oligochaetum that was considered as a synonym of
C. orbiculare by von Arx (1957), C. bryoniae was regarded as a
synonym of C. orbiculare as well. The conidial size is similar to
the strain from the Netherlands, measuring 18–22 × 4–5 μm
(Maire 1917). However, as there are often several Colletotrichum
species within this species complex causing anthracnose on the
same host plants and we have no proof that this species belongs
to this complex, we refrain from epitypifying this species with a
strain from the Netherlands instead of Algeria, and rather
describe it here as a new species. Material of the species from
Algeria has not been examined and living cultures derived from
its type are not available.
Colletotrichum destructivum O'Gara, Mycologia 7: 38.
1915. Fig. 5.
hyphae 1–9 μm diam, hyaline, smooth-walled, septate,
conidiophores and setae formed directly on hyphae or on pale to
medium brown, angular cells, 3–10 μm diam, sometimes
developing to dark brown, round structures on which many setae
and conidiophore-like structures are formed, conidia released as
well, however, most conidiophore-like structures without visible
conidiogenous openings, thick-walled, septate, branched at the
base, up to 70 μm long, very broad and usually broadest at the
tip, cells at the tip measure 6.5–22 × 4–6 μm, surrounded by a
slime sheath. Setae medium brown, smooth-walled to finely
verruculose, sometimes verrucose, 50–180 μm long, 1–3septate, base cylindrical, conical, sometimes ± inflated,
3.5–6 μm diam, tip ± rounded to ± acute. Conidiophores pale to
medium brown, smooth-walled, septate, branched, to 85 μm
long. Conidiogenous cells pale to medium brown, smooth-walled,
elongate-ampulliform to cylindrical, 9.5–17 × 3.5–5 μm, opening
1–1.5 μm diam, collarette 0.5–1 μm long, periclinal thickening
visible. Conidia hyaline, smooth-walled, aseptate, cylindrical,
straight to slightly curved, with both ends ± rounded, (14–)
Fig. 5. Colletotrichum destructivum (from ex-epitype strain CBS 136228). A–B. Conidiomata. C, I. Tip of a seta. D, J. Base of a seta. E–H, K–N. Conidiophores. O–T.
Appressoria. U–V. Conidia. A, C–H, U. from Anthriscus stem. B, I–T, V. from SNA. A–B. DM, C–V. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B.
Scale bar of E applies to C–V.
59
DAMM
ET AL.
14.5–16.5(–18) × 3.5–4(–4.5) μm, av. ± SD = 15.4 ± 0.8 × 3.7
± 0.2 μm, L/W ratio = 4.2. Appressoria single, pale brown,
smooth-walled, clavate, fusiform to ellipsoidal outline, with a
lobate, undulate or crenate margin, (6.5–)10–15.5(–20.5)
× (4.5–)5–8(–10.5) μm, av. ± SD = 12.5 ± 2.7 × 6.7 ± 1.5 μm,
L/W ratio = 1.9, Appressoria of strain CBS 149.34 smaller,
measuring (4–)6–14(–25) × (3.5–)4.5–7.5(–10) μm, av. ± SD
= 9.8 ± 4.1 × 5.9 ± 1.5 μm, L/W ratio = 1.7, strain CBS 149.34
also forms appressorium-like structures inside the medium,
single, medium brown, smooth-walled, subglobose, ovate to
broadly elliptical in outline, with an entire or undulate margin,
(3.5–)5–10(–13) × (3–)3.5–7(–8.5) μm, av. ± SD = 7.6 ± 2.5
× 5.2 ± 1.7 μm, L/W ratio = 1.4.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on a cushion of pale to medium
brown, angular cells, 3–8.5 μm diam that are intermingled and
surrounded by medium brown, thick-walled hyphae,
with ± inflated cells, up to 8.5 μm diam. Setae medium brown,
smooth-walled, towards the tip often verruculose to verrucose,
constricted and slightly wavy, 65–110 μm long, 1–4-septate,
base conical to ± inflated, 4.5–12 μm diam, tip ± rounded
to ± acute. Conidiophores hyaline to pale brown, smooth-walled,
simple or septate and branched, to 25 μm long. Conidiogenous
cells hyaline to pale brown, smooth-walled, cylindrical, doliiform
to ampulliform, 5–14 × 2.5–5.5 μm, opening 1–2 μm diam,
collarette 0.5–1.5 μm long, periclinal thickening distinct. Conidia
hyaline, smooth-walled, aseptate, cylindrical, straight to slightly
curved, with both ends ± rounded, (15–)16–18(–19) × (3–)
3.5–4 μm, av. ± SD = 16.9 ± 1.0 × 3.6 ± 0.2 μm, L/W ratio = 4.7.
hyaline to honey, agar medium and filter paper partly covered by
sparse filty white aerial mycelium; agar medium, filter paper and
Anthriscus stem partly covered with grey to black acervuli,
reverse same colours; growth 23–25 mm in 7 d (36–37.5 mm in
10 d). Colonies on OA flat with entire margin; buff to honey,
almost entirely covered with grey to black acervuli; partly covered
with whitish to grey aerial mycelium, reverse buff to olivaceousgrey, growth 25–27.5 mm in 7 d (37.5–40 mm in 10 d). Conidia
in mass whitish to rosy-buff.
Materials examined: Canada, south-western Ontario, from anthracnose on stems
of Medicago sativa, 1985/86 collector unknown (deposited by R. O'Connell,
before from G.J. Boland, A 22), culture CBS 128509 = LARS 320. Korea, from
Rumex sp., collection date and collector unknown (CBS H-21655, culture ex IMI
387103 = CPC 18082). Netherlands, experimental plot of P.M.L.Tammes, from
stem burn of Trifolium sp., 1 Oct. 1934, P.M.L.Tammes, culture CBS 149.34.
USA, Utah, probably Salt Lake City, on stems and leaves of Trifolium pratense,
30 Jun. 1914, P. J. O'Gara (BPI 397373 holotype); Utah, Salt Lake City, cemetery, from small black spots on petioles of T. hybridum, 24 Aug. 2013, U. Damm
(CBS H-21652 epitype, here designated, MBT178515, culture ex-epitype CBS
136228); Utah, Syracuse (close to Salt Lake City), pasture, from small black
spots on petioles of T. hybridum, 24 Aug. 2013, U. Damm, CBS H-21653, culture
CBS 136229; from Phragmites sp., collection date and collector unknown, CBS
H-21654, culture CBS 130238.
Notes: Colletotrichum destructivum was described by O'Gara
(1915) from stems and petioles of red clover (Trifolium pratense) and alsike clover (T. hybridum) in clover fields in the Salt
Lake Valley, Utah, USA. The species forms minute acervuli,
25–70 μm diam, hyaline conidia with 1–4 guttules that are
straight to slightly curved with rounded apices and bases,
measuring 14–22 × 3.5–5 μm, few to numerous setae that are
straight, curved to flexuous, often nodulose, aseptate to obscure
60
1-septate, subacute to rounded, constricted at the apex,
38–205 μm long and 4.5–7 μm diam at the basis (O'Gara 1915).
Conidia from small acervuli were observed on stems, petioles
and leaves of the holotype specimen (BPI 397373) and measure
(14–)15–19.5(–23.5) × (3–)4–4.5 μm, av. ± SD = 17.2 ± 2.1
× 3.8 ± 0.4 μm, L/W ratio = 4.5; setae were 50–110 μm long
with a cylindrical to ± inflated base, 3.5–8 μm diam and
a ± rounded tip.
The specimen BPI 397373 was collected on 30 June 1914 by
P.J. O'Gara and designated no. 20. The package has a type
stamp and origins from Fungi Utahensis, Herbarium of Department of Agricultural Investigation, American Smelting & Refining
Co., Salt Lake City, Utah, which was the institute where P.J.
O'Gara used to work as stated in the publication. There is an
isotype of this fungus in herbarium NY with the information
“Incorporated herbarium: Garrett Herbarium, University of Utah”.
There is no specimen of C. destructivum available in the Garrett
Herbarium any more; the specimen was apparently sent away
together with many specimens from that herbarium (M. Power, in
lit.). This agrees with a note on the specimen packet stating “Rec.
by Path. Coll. Apr. 8, 1921”; BPI 397373 must therefore be the
holotype.
In order to epitypify C. destructivum, collections of Trifolium
hybridum and Medicago sativa were made in August 2014 from
field as well as urban locations in and around Salt Lake City;
T. pratense was not found in the area. Colletotrichum spp. were
isolated from small black spots on stems, petioles and leaves of
both host plants. Isolates of the C. destructivum species complex
were identified based on morphology. Some of the isolates
grouped with a species that had often been collected from both
host genera worldwide, for which the name C. destructivum is
usually applied. The other isolates belonged to C. lini or a
species closely related to C. lini. Colletotrichum lini contained
both Trifolium and Medicago isolates, the other clade only
Medicago isolates (see C. americae-borealis). It is possible that
O'Gara (1915) collected more than one species as well. However, there is not enough material available of the holotype to
extract DNA for molecular examination. The two species
collected from clover are morphologically very similar. However,
setae of the ex-epitype strain CBS 136228 grown on Anthriscus
stem were often constricted towards the tip, which is in accordance with the observations made by O'Gara (1915). This was
not observed in C. lini.
Several Colletotrichum and Gloeosporium species have been
described from Trifolium and Medicago as already discussed in
Damm et al. (2013). Except for Gm. trifolii and Gm. medicaginis,
these species were described later than C. destructivum and
cannot be considered as possible synonyms of C. destructivum.
Except for C. destructivum and C. trifolii, these names have not
been used since their description. Colletotrichum trifolii was
originally described from T. pratense in the USA (Bain & Essary
1906); the species was epitypified recently and revealed to
belong to the C. orbiculare species complex (Damm et al. 2013)
that is distinct from the species complex studied here (Cannon
et al. 2012). Strain CBS 149.34 was previously identified as
C. trifolii (Nirenberg et al. 2002), but re-identified here as
C. destructivum.
Gloeosporium trifolii described from T. pratense in Albany, NY,
USA (Peck 1879, publ. 1883) forms conidia that measure
15–23 × 4–6.3 μm, which are slightly larger than
C. destructivum. Gloeosporium medicaginis forms acervuli on
Medicago sativa in Kansas, USA, with cylindrical conidia that are
THE COLLETOTRICHUM
subhyaline and mostly narrowed in the middle, measuring
15–20 × 3–4 μm (Ellis & Kellerman 1887). Conidia that are
constricted in the middle have not been observed in this species
complex. We have not studied the type specimens of these
species. However, even if either of these “forgotten” species
belong to the C. destructivum species complex, it would be
difficult to link recent collections to one of them based on
morphology alone.
Colletotrichum sativum, a species described from Vicia sativa
in Louisiana, USA (Horn 1952), was listed as a synonym of
C. destructivum by von Arx (1957). We cannot confirm this
synonymy as no strain from Vicia sativa belonging to this species
complex was studied.
The description and drawing of C. rumicis-crispi from Rumex
crispus in Taiwan described by Sawada (1959) are similar to our
observations made of C. destructivum that includes a strain from
Rumex in Korea (IMI 387103). Colletotrichum rumicis-crispi is
probably a synonym of C. destructivum. However, as we have
not seen the type specimen, we cannot confirm this.
Strain IMI 387103 differs from the other C. destructivum sequences in ACT and TUB2 sequences in one nucleotide each, but
the other loci are identical. In contrast, the ITS sequence of
C. destructivum strain RGT-S12 from R. gmelinii from China
(GenBank HQ674658, Hu et al. 2012) was 99 % identical (2
nucleotides different) with the ITS sequence of the ex-type strain
of C. destructivum (CBS 136228), and strain IMI 387103 from
Rumex, but identical with those of C. higginsianum. This indicates
the occurrence of at least two Colletotrichum species on Rumex.
Manandhar et al. (1986) claimed C. destructivum to be the
asexual morph of Ga. glycines based on morphological comparison of isolates from Glycine that formed a sexual morph
resembling Ga. glycines (Lehman & Wolf 1926) and an isolate
from Medicago that was initially identified as C. destructivum.
The authors apparently assumed that Colletotrichum strains from
legumes are all C. destructivum unless the conidia are curved.
Isolates from both hosts that were included in the study of
Manandhar et al. (1986) were sequenced and confirmed to
belong to the same species that is, however, not closely related
to C. destructivum and belongs in a different species complex (U.
Damm, unpubl. data). Consequently, there is no evidence that
C. destructivum forms a sexual morph.
The hemibiotrophic infection of Medicago sativa by
C. destructivum was observed by Latunde-Dada et al. (1997).
The fungus initially produced large, prominently multilobed
infection structures that were localised within single epidermal
cells of the infected host. Two of the three isolates studied, LARS
202 (= CBS 511.97) and LARS 709 (= CBS 520.97), were
confirmed as C. destructivum s. str. in this study. The third isolate
LARS 319 originated from the same collection (A22) from
Canada as C. destructivum s. str. strain LARS 320 (= CBS
128509), and is also included here. All isolates originated from a
pathogenicity study by Boland & Brochu (1989).
Conidia of C. destructivum are very slightly curved and
appear almost straight, similar to those of C. tabacum, especially
on SNA; however, no dark halo around the penetration pores of
appressoria was observed.
Colletotrichum destructivum can be distinguished by its ITS,
HIS3, ACT and TUB2 sequences, while the GAPDH sequence is
identical to that of C. ocimi that is described as a new species in
this study. Further intraspecific grouping was observed with sequences of all loci studied. Strain IMI 387103 from Rumex in
Korea was the most distant strain from the rest of the
C. destructivum strains that form a cluster with a bootstrap
support value of 84 %. However, we refrained from considering
strain IMI 387103 as a separate species as it forms a strong
cluster with C. destructivum (95/0.99) and only differs in a few
nucleotides from the majority of the C. destructivum strains. In
order to investigate if this represents a distinct species, additional
collections from Rumex should be studied. The three subclades
with a bootstrap support 69 % did not show clear host preferences and did not suggest any further splitting of the species.
ITS sequences of a large number of isolates detected in
blastn searches were identical to the ITS sequence of the exepitype strain (CBS 136228) of C. destructivum:
C. destructivum strains CBS 149.34 from Trifolium in the
Netherlands (GenBank JQ005764, O'Connell et al. 2012) that is
included in this study, Coll-48, Coll-68, Coll-75 and CC 657 from
Medicago in Serbia (GenBank JX908362, JX908363, JX908361,
Vasic, unpubl. data), MAFF 239947 and MAFF 239948 from
Antirrhinum in Japan (GenBank AB334521, AB334522, Tomioka
et al. 2011), MAFF 410037 from Robinia in Japan (GenBank
AB105961, Moriwaki et al. 2002), CGMCC 3.15129 from Bletilla
in China (GenBank JX625174, Tao et al. 2013), uncultured
fungus clone CMH309 from house dust in the USA (GenBank
KF800400, Rittenour et al. 2013), DAOM 196849 from an unknown host (GenBank EU400156, Chen et al. 2007), C. trifolii
isolate UQ349 from Medicago in Australia (GenBank AF451909,
Ford et al. 2004) and CBS 149.34 (GenBank AJ301942,
Nirenberg et al. 2002) and C. cf. gloeosporioides strain AR 4031
(= CBS 119187) from Crupina in Greece (GenBank AY539806,
Berner et al. 2004). Colletotrichum trifolii strain CBS 149.34 and
C. cf. gloeosporioides CBS 119187 are included in this study and
re-identified as C. destructivum s. str.
The TUB2 sequence of CBS 136228 is identical with GenBank JQ005848 from C. destructivum strain CBS 149.43
(O'Connell et al. 2012), 99 % (1 nucleotide difference) identical
with GenBank JX625198 and JX625200 from isolates CGMCC
3.15127 and CGMCC 3.15128 and 99 % identical (3 nucleotides
difference) with GenBank JX625203 from isolate CGMCC
3.15129 from Bletilla in China (Tao et al. 2013).
Colletotrichum fuscum Laubert, Gartenwelt 31: 675.
1927. Fig. 6.
= Colletotrichum digitalis Unamuno, Revista Real Acad. Ci. Madrid. 30:
503. 1933 – Nom. illegit., Art. 53.1
≡ Colletotrichum unamunoi Cash, Syll. fung. (Abellini) 26: 1222. 1972.
hyphae 1–9.5 μm diam, hyaline to pale brown, smooth-walled,
septate, branched. Chlamydospores not observed. Conidiomata absent, conidiophores aggregated directly on pale to
medium brown hyphae or on clusters of irregularly arranged
medium brown hyphae. Setae (one observed) medium brown,
smooth-walled, 71 μm long, 3-septate, base conical, 5.5 μm
diam, tip rounded. Conidiophores hyaline to medium brown,
smooth-walled, simple or septate and branched, to 20 μm long.
Conidiogenous cells hyaline to pale brown, smooth-walled,
ampulliform, doliiform to cylindrical, 7–18.5 × 3.5–6 μm,
sometimes not separated from hyphae by a septum (intercalary)
or opening with collarette formed directly on hyphae, opening
1–1.5 μm diam, collarette 0.5–1 μm long, periclinal thickening
visible. Conidia hyaline, smooth-walled, aseptate, cylindrical,
slightly curved to straight, with one end round and the other
truncate, (16–)16.5–20(–34) × (3.5–)4–4.5(–5.5) μm,
61
DAMM
ET AL.
Fig. 6. Colletotrichum fuscum (from ex-epitype strain CBS 133701). A–B. Conidiomata. C, H. Tip of a seta. D, I. Base of a seta. E–G, J–L. Conidiophores. M–Q. Appressoria.
R–S. Conidia. A, C–G, R. from Anthriscus stem. B, H–Q, S. from SNA. A–B. DM, C–S. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B. Scale bar of
E applies to C–S.
av. ± SD = 18.3 ± 1.9 × 4.1 ± 0.3 μm, L/W ratio = 4.5.
Appressoria single, medium brown, smooth-walled, roundish,
ellipsoidal to clavate in outline, with an lobate (to undulate)
margin, (6–)8.5–14.5(–19) × (6.5–)7–10(–11.5) μm,
av. ± SD = 11.5 ± 2.8 × 8.6 ± 1.5 μm, L/W ratio = 1.3.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on a cushion of medium brown,
angular to roundish cells, 4.5–12.5 μm diam. Setae medium to
dark brown, smooth-walled, 3–160 μm long, 1–3-septate, base
conical, 5–7.5 μm diam, tip ± rounded to ± acute. Conidiophores
hyaline to medium brown, smooth-walled, simple or septate and
branched, to 20 μm long. Conidiogenous cells hyaline to medium
brown,
smooth-walled,
ampulliform
to
conical,
5.5–17.5 × 3–5.5 μm, opening 1–1.5 μm diam, collarette
0.5–1 μm long, periclinal thickening visible. Conidia hyaline,
smooth-walled, aseptate, cylindrical, slightly curved to straight,
with one end round and the other truncate, (16–)
17–19.5(–20.5) × (3.5–)4–4.5(–5) μm, av. ± SD = 18.1 ± 1.4
× 4.1 ± 0.3 μm, L/W ratio = 4.4.
hyaline to pale honey, agar medium, filter paper and Anthriscus
stem partly covered with appressed whitish aerial mycelium,
Anthriscus stem partly covered with black acervuli, reverse same
colours; growth 21–24 mm in 7 d (35–40 mm in 10 d). Colonies
on OA flat with entire margin; buff to rosy-buff with small black
62
spots (not clearly recognisable as conidiomata) towards the
centre, aerial mycelium lacking, reverse buff to rosy-buff, vinaceous buff towards the centre, growth 21–24 mm in 7 d
(31–34 mm in 10 d). Conidia in mass rosy-buff to pale salmon.
Materials examined: Germany, Berlin, Zehlendorf, garden, from leaves of Digitalis purpurea, 1927, R. Laubert [B 70 0021851 (ex BBA acc. 9.1.1980) lectotype, here designated, MBT178720]; Berlin, Zehlendorf, garden, from leaves of
Digitalis purpurea, 1927–1933, R. Laubert (B 70 0021852); Berlin, garden, from
leaf of Digitalis lutea, 2 Aug. 2012, U. Damm (CBS H-21651 epitype, here
designated, MBT178517, culture ex-epitype CBS 133701). Netherlands,
Utrecht, garden, from leaf of Digitalis obscura, 29 Aug. 2012, U. Damm, culture
CBS 133703; Baarn, garden Eemnesserweg 90, from living leaves of Digitalis
purpurea, Nov. 1968, H.A. van der Aa, 944, CBS H-10616, culture CBS 825.68;
Utrecht, from dead stem of Heracleum sp., 12 Aug. 2009, U. Damm, CBS H21666, culture CBS 125336; Utrecht, from dead stem of Heracleum sp., 12 Aug.
2009, U. Damm, CBS H-20404, culture CBS 126510; Utrecht, from dead stem of
Heracleum sp., 12 Aug. 2009, U. Damm, CBS H-20405, culture CPC 18076.
Notes: Colletotrichum fuscum causes anthracnose on some
Digitalis spp. (foxglove) and was reported from the USA (Connecticut, Maryland, Oregon, Pennsylvania, South Dakota),
Poland, Australia, Canada, Germany, England, New Zealand,
Portugal and Czechoslovakia (Farr & Rossman 2014). According
to Sutton (1980), C. fuscum also attacks Antirrhinum majus in
New Zealand and the UK. However, the IMI strain from Antirrhinum majus in New Zealand listed by Sutton (IMI 197877)
belongs to a different species (see C. antirrhinicola). Tomioka
THE COLLETOTRICHUM
et al. (2001) showed C. fuscum caused anthracnose of Nemesia
strumosa in Japan. Based on morphology (and host), this Japanese collection may belong to the C. destructivum complex, but
its identification needs to be confirmed based on molecular data.
Thomas (1951) reports serious damage of Digitalis lanata in
commercial plantings by C. fuscum.
Laubert (1927) described C. fuscum from diseased leaves of
Digitalis purpurea in Berlin with conidia that are 12–24 μm long
and 2–4 μm wide, straight or slightly clavate and then slightly
curved at the narrow end, formed from short crowded conidiophores, setae 8–10 × 45–100 μm with a slightly inflated
base up to 9 μm diam. Two authentic specimens were located in
the fungarium B, both without type designation, of which B 70
0021851 collected by R. Laubert in 1927, was selected as
lectotype of C. fuscum. The collection date of the second
specimen (B 70 0021852) was imprecise (1927–1933); the
specimen might have been collected after the publication of
Laubert's description. Conidia observed on the lectotype specimen measured (15–)17–21.5(–23) × 3.5–5(–5.5) μm,
av. ± SD = 19.3 ± 2.2 × 4.2 ± 0.6 μm, L/W ratio = 4.6 and
resembled those seen in culture.
Several other species have been described on Digitalis.
Gloeosporium digitalis, which was described from leaves of
Digitalis purpurea in Landbohøjskolens Have, Frederiksberg,
Denmark, forms smaller conidia than C. fuscum, measuring
8–10 × 3–4 μm and apparently lacks setae (Rostrup 1899).
Goto (1938) concluded Gm. digitalis to be a different species.
Von Arx (1957) regarded Gm. digitalis as a synonym of Ascochyta digitalis Fuckel.
However, Moesz (1931) combined Gm. digitalis in Colletotrichum on the basis of observations of a fungus on Digitalis
ferruginea from Hungary that more closely resembled C. fuscum
than Gm. digitalis. Goto (1938) regarded this fungus as a form of
C. fuscum, while von Arx (1957) listed this species as a synonym
of C. fuscum and called it C. digitalis Moesz. Colletotrichum
digitalis could also be a different species based on shape and
size of the conidia (cylindrical with blunt ends, measuring
10–15 × 3 μm) and the long conidiophores that are illustrated
(Moesz 1931).
Unamuno (1933) described a Colletotrichum species from
leaves of Digitalis purpurea in Spain and, apparently unaware of
Moesz's combination, called it C. digitalis Unamuno. As this
name is illegitimate (Art. 53.1), Trotter & Cash (1972) gave the
species a new name, C. unamunoi Cash. Based on the
morphological features (conidia 16–22 × 3–3.5 μm, hyaline,
cylindrical, usually straight, sometimes slightly curved, rounded
at both ends, setae 63 × 3.5–4 μm, brown, septate, straight,
curved or flexuous, often nodular; Trotter & Cash 1972), both
Goto (1938) and von Arx (1957) regarded C. digitalis Unamuno
as a synonym of C. fuscum. We have not seen the type of
C. digitalis Unamuno, but agree that this species is most likely a
synonym of C. fuscum that seems to be the common anthracnose pathogen of several Digitalis spp., at least in Europe.
Colletotrichum dematium was reported from Digitalis atropurpurea in UK and Scotland (Kirk & Spooner 1984). We do not
know whether this report refers to C. dematium s. str.; all species
called C. dematium (s. lat.) usually have distinctly curved conidia
and are not closely related with C. fuscum (Damm et al. 2009,
Cannon et al. 2012).
Goodman (1960) discovered the phytotoxin colletotin in three
strains of C. fuscum, one of which was obtained from the CBS
collection and called the “von Arx strain”. This strain is probably
identical to strain CBS 130.57 that was deposited in the CBS
collection in Sep. 1957 by von Arx, listed as forming colletotin
with a reference to R.N. Goodman in the CBS strain database,
and is included in this study.
Moriwaki et al. (2002) noticed the similarity and close relationship of C. fuscum to C. destructivum and assumed them to be
conspecific. Preliminary multilocus phylogenies (O'Connell et al.
2012, Cannon et al. 2012) recently indicated C. fuscum to be a
distinct species, which is confirmed in this study.
The complex appressoria and the conidiogenous cells that
are often ampulliform on the two media tested are diagnostic for
this species. Colletotrichum fuscum is distinguishable by
GAPDH, but has only one nucleotide difference from
C. bryoniicola. The ITS sequence is variable; isolates do cluster,
but one strain (CBS 825.68) sits separately. The CHS-1
sequence is the same as that of C. antirrhinicola, the ACT
sequence the same as that of C. antirrhinicola and C. bryoniicola.
Additionally, unnamed isolates from Heracleum are basal to
C. fuscum, C. antirrhinicola, C. bryoniicola and C. vignae in our
phylogeny, and could represent an additional, currently unidentified species (Fig. 1).
The closest matches with the GAPDH sequence of strain CBS
133701, with 98 % identity (3 nucleotides different), are GenBank
GU935850 and GU935851 from C. higginsianum isolates
C97027 and C97031 (Choi et al. 2011). The closest matches with
the ITS sequence, with 99 % identity (2 nucleotides different)
were C. fuscum strains CBS 130.57 from Digitalis (GenBank
JQ005762, O'Connell et al. 2012), DAOM 216112 (GenBank
EU400144, Chen et al. 2007) and BBA 70535 from Digitalis in
Germany (GenBank AJ301938, Nirenberg et al. 2002).
Colletotrichum higginsianum Sacc., J. Agric. Res.,
Washington 10: 161. 1917. Fig. 7.
branched. Chlamydospores not observed. Conidiomata conidiophores and setae on pale brown, angular cells, 3–9 μm
diam. Setae medium brown, smooth-walled to finely verruculose,
3.5–6 μm diam, tip rounded to ± acute. Conidiophores hyaline,
cells hyaline, smooth-walled, cylindrical, 8–27 × 3.5–4.5 μm,
sometimes intercalary (necks not separated from hyphae by
septum), opening 1–2 μm diam, collarette 1–2 μm long, periclinal thickening distinct. Conidia hyaline, smooth-walled, aseptate, cylindrical, straight to very slightly curved, with one end
rounded and the other truncate, (17–)19–20.5(–21) × (3–)
3.5–4(–4.5) μm, av. ± SD = 19.6 ± 0.9 × 3.7 ± 0.2 μm, L/W
ratio = 5.3; conidia of strain IMI 349063 shorter, measuring
(13.5–)15–19(–21.5) × 3.5–4(–4.5) μm, av. ± SD = 17.0 ± 1.8
× 3.7 ± 0.3 μm, L/W ratio = 4.6.
Appressoria in loose groups, medium brown, smooth-walled,
fusiform, clavate, elliptical or irregular outline, with an entire,
crenate or lobate margin, (5.5–)10–20(–28.5) × (3.5–)
5–9(–12) μm, av. ± SD = 15.0 ± 5.1 × 6.8 ± 2.0 μm, L/W ratio = 2.2; appressoria of strain MAFF 305635 smaller, measuring
(7.5–)9–13(–15) × (3.5–)4.5–6.5(–8) μm, av. ± SD = 11.0
± 1.9 × 5.4 ± 0.9 μm, L/W ratio = 2.0.
63
DAMM
ET AL.
Fig. 7. Colletotrichum higginsianum (from ex-epitype strain IMI 349061). A–B. Conidiomata. C, H. Tip of a seta. D, I. Base of a seta. E–F, J–L. Conidiophores. G. Bases of a
seta and conidiophores. M–R. Appressoria. S–T. Conidia. A, C–G, S. from Anthriscus stem. B, H–R, T. from SNA. A–B. DM, C–T. DIC, Scale bars: A = 100 μm, G = 10 μm.
Scale bar of A applies to A–B. Scale bar of G applies to C–T.
3–7.5 μm diam. Setae medium brown, smooth-walled,
5–12 μm diam, tip ± rounded to ± acute. Conidiophores hyaline
to pale brown, smooth-walled, simple or septate and branched,
to 15 μm long. Conidiogenous cells hyaline to pale brown,
smooth-walled, cylindrical to ampulliform, 8–14 × 3–3.5 μm,
opening 1–1.5 μm diam, collarette 0.5–1 μm long, periclinal
thickening distinct. Conidia hyaline, smooth-walled, aseptate,
cylindrical, straight to very slightly curved, with one end rounded
and the other truncate, (17.5–)18–20(–22) × (3–)3.5–4 μm,
av. ± SD = 19.0 ± 0.9 × 3.6 ± 0.2 μm, L/W ratio = 5.2; conidia of
strain IMI 349063 shorter, measuring (12.5–)15–18(–18.5)
× 3.5–4.5 μm, av. ± SD = 16.5 ± 1.7 × 4.0 ± 0.3 μm, L/W
ratio = 4.1.
covered with salmon to grey acervuli and floccose white aerial
mycelium, filter paper partly pale luteous to pale orange, reverse
same colours; growth 23–24 mm in 7 d (35–37.5 mm in 10 d);
with strain IMI 349063 aerial mycelium lacking and filter paper
partly pale luteous. Colonies on OA flat with entire margin; buff,
saffron to pale orange, partly covered with pale orange, grey to
black acervuli and floccose white aerial mycelium, reverse buff,
salmon to olivaceous-grey, growth 23.5–29 mm in 7 d
(36–39.5 mm in 10 d); with strain IMI 349063 aerial mycelium
64
lacking and slower growth 20.5–22.5 mm in 7 d (30–33.5 mm in
10 d). Conidia in mass saffron to orange.
Materials examined: Japan, Edogawa, Tokyo, from Brassica rapa var. komatsuna,
collection date and collector unknown (isolated 6 Oct. 1980 by H. Horie), culture
MAFF 305635 = Abr 1-5 = CPC 19366, CPC 18943; Edogawa, Tokyo, from
Raphanus sativus, collection date and collector unknown (isolated 21 Oct. 1980),
culture AR 3-1 = CPC 19394; Tateyama, Chiba, from Matthiola incana, collection
date and collector unknown (isolated Oct. 1990), CBS H-21665, culture CH90M1 = CPC 19361; Chikura, Chiba from Matthiola incana, collection date and
collector unknown (isolated Oct. 1990), culture CH93-M1 = CPC 19362. Romania,
Târgu Neamț, garden near Varatec, on leaves of Matthiola incana, 20 Jul. 1952, C.
Sandu-Ville, (GLM-F102751 holotype of C. mathiolae Sandu ex Herbarul
Micologic “C. Sandu-Ville”). Trinidad and Tobago, Trinidad, Wallerfield, from leaf
spot on living leaf of Brassica rapa subsp. chinensis, collection date and collector
unknown (IMI 349061 epitype of C. higginsianum, here designated,
MBT178519, CBS H-21664 isoepitype, culture ex-epitype IMI 349061 = CPC
18941, CPC 19379); Trinidad, from leaf spot on living leaf of Brassica rapa subsp.
chinensis, collection date and collector unknown, culture IMI 349063 = CPC 18942,
CPC 19380. USA, Georgia, experiment, on leaf spots of Brassica rapa, 24 Jul.
1916, B. B. Higgins (no. 340), (BPI 398582 holotype of C. higginsianum).
Notes: Colletotrichum higginsianum is known as the causal organism of anthracnose disease of a wide range of cruciferous
plants (Brassicaceae) and causes mainly leaf spots but also
attacks stems, petioles, seed pods and even roots, and is
especially destructive in the south Atlantic and Gulf states of the
USA (Higgins 1917, Rimmer 2007), but also occurs in the Carribean and south-east Asia (Birker et al. 2009).
THE COLLETOTRICHUM
Higgins (1917) noted that this species was associated with a
leaf spot disease of turnips (Brassica rapa) in various localities in
Georgia, USA and tentatively called it C. brassicae Schulzer &
Sacc. However, Higgins had doubts about this identification and
sent specimens to P.A. Saccardo. In a footnote, Higgins
explained that Saccardo considered that the fungus was a new
species and added Saccardo's species description from the note
he received after his paper was ready for publication.
A specimen of C. higginsianum was located at BPI that was
collected by B.B. Higgins prior to the publication (BPI 398582),
and is therefore considered as the holotype. The specimen
comprises two leaves with leaf spots that agree with the
description and figures in the publication. Conidia of the holotype
are nearly straight, sometimes very slightly curved, measuring
(14–)16–20(–22)
×
(3–)3.5–4.5(–5)
μm,
av. ± SD = 18.1 ± 1.9 × 4.1 ± 0.6 μm, L/W ratio = 4.5. This
agrees with the shape and measurements of the isolates studied
here.
The only species that was described on Brassica prior to
Higgins (1917) is C. brassicae Schulzer & Sacc. (1884), on
Brassica oleracea v. caulocarpa from Vinkovce, Slovenia that
forms curved conidia 19–24 μm long (Schultzer von
Mueggenburg & Saccardo 1884). The ITS sequence (GenBank EU400155) of strain DAOM 116226 identified as
C. brassicae (Chen et al. 2007) is identical to that of the ex-type
strain of C. spaethianum (CBS 167.49) from a study on Colletotrichum species with curved conidia (Damm et al. 2009).
Another strain from a stump of Brassica sp. in the Netherlands
included in the study of Damm et al. (2009) was identified as
C. truncatum, based on sequence similarities with the ex-epitype
strain of that species. It is possible that C. brassicae is synonymous with either C. spaethianum or C. truncatum. We have not
studied the type specimen as we do not consider this species to
be part of the C. destructivum species complex. Colletotrichum
brassicicola was described recently from Brassica; it forms
straight conidia and belongs to the C. boninense species complex (Damm et al. 2012).
A species described from leaf spots on Matthiola incana in
Romania by Sandu-Ville (1959), C. mathiolae, also resembles
C. higginsianum and could be a synonym of this species or
closely related based on similar conidia shape and size. Colletotrichum mathiolae also forms conidia that are straight to slightly
curved, measuring 12–21 × 3–4 μm. The two isolates from
Matthiola in Japan are closely related to C. higginsianum, but do
not have the same GAPDH and HIS3 sequences, which may
explain why they do not form a stable clade in our phylogeny.
Additional isolates from this host, especially from Romania, are
required to determine species boundaries and its affinity to
C. mathiolae.
Colletotrichum higginsianum was regarded as a synonym of
C. gloeosporioides by von Arx (1957), but Sutton (1980, 1992)
considered it as a distinct species based on its conidial
morphology and consistent association with cruciferous hosts.
O'Connell et al. (2004) recognised the similarity and relatedness
with C. destructivum and regarded C. higginsianum as a synonym of C. destructivum based on ITS sequences. Colletotrichum
higginsianum is confirmed as a distinct species in the present
study.
Two isolates included in this study, strain CBS 124249 from
Centella asiatica in Madagascar (Rakotoniriana et al. 2008) and
strain IMI 391904 from Raphanus raphanistrum in Tunisia
(Djebali et al. 2009), which were previously identified as
C. higginsianum, were re-identified as C. tabacum and C. lini,
respectively.
O'Connell et al. (2004) observed the two-stage hemibiotrophic infection process of C. destructivum (re-identified here
as C. higginsianum) from Brassica rapa subsp. chinensis on
Arabidopsis thaliana. They also established an Agrobacteriummediated DNA-transformation system for this fungus. The Arabidopsis-Colletotrichum pathosystem provides a model for molecular analysis of plant-fungal interactions in which both
partners can be genetically manipulated. This pathosystem has
been intensively studied in recent years (Huser et al. 2009,
Ushimaru et al. 2010, Kleemann et al. 2012). The genome and
in planta transcriptome of C. higginsianum strain IMI 349063
were sequenced (O'Connell et al. 2012), and this is one of the
strains included in the present study.
Colletotrichum higginsianum can be identified by TUB2 and
ITS sequences. However, there is only one nucleotide difference
to the TUB2 sequences of the two unnamed strains from Matthiola (CM90-M1 and CM93-M1) and one nucleotide difference to
the ITS sequences of C. tabacum, respectively. Three of the
strains diverge with a further single nucleotide difference to the
other C. higginsianum strains.
The ITS of strain IMI 349061 was identical with those of
C. higginsianum isolates 05131 from Eruca in the USA (GenBank KF550281, Patel et al. 2014), 12-223 (GenBank JX997428,
K.S. Han et al., unpubl. data), C97027 and C00112 (GenBank
GU935870, GU935872, Choi et al. 2011) from Brassica probably
in Korea, IMI 349063 and MAFF 305635 (GenBank JQ005760,
JQ005761 O'Connell et al. 2012, Naumann & Wicklow 2013),
MAFF 305635, MAFF 238563, MAFF 305970, IFO6182 (GenBank AB042302, AB042303, AB105955, AB105957, Moriwaki
et al. 2002) from Brassica, Matthiola and an unknown host,
and except for the last, included in this study, and
C. destructivum isolates RGT-S12, endophyte of Rumex probably in China (GenBank HQ674658, Hu et al. 2012) and CD-hz
01–CD-hz 03 from Vigna in China (GenBank EU070911–
EU070913, Sun & Zhang 2009).
Colletotrichum lentis Damm, sp. nov. MycoBank
MB809921. Fig. 8.
≠ Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore,
Basionym: Vermicularia truncata Schwein., Trans. Amer. Philos.
Soc. 4(2): 230. 1832.
≡ Glomerella truncata (Schwein.) C.L. Armstrong & Banniza, Mycol.
Res. 110: 953. 2006.
Etymology: The species epithet is derived from the host genus
Lens.
hyphae 1.5–11 μm diam, hyaline, smooth-walled, septate,
conidiophores and setae formed directly on hyphae or on or
close to chains or clusters of pale to dark brown, verruculose,
cylindrical to subglobose, cells. Setae pale to medium brown,
smooth-walled, 40–85 μm long, 1–3-septate, base ± inflated,
sometimes constricted at the basal septum, 5–6 μm diam, tip
round. Conidiophores hyaline, smooth-walled, septate,
branched, to 30 μm long. Conidiogenous cells hyaline, smoothwalled, cylindrical to ampulliform, 9–28 × 3.5–5 μm, sometimes
intercalary (necks not separated from hyphae by septum) and
65
DAMM
ET AL.
Fig. 8. Colletotrichum lentis (from ex-holotype strain CBS 127604). A–B. Conidiomata. C, G. Seta. D–F, H–K. Conidiophores. L–Q. Appressoria. R–S. Conidia. A, C–F, R.
from Anthriscus stem. B, G–Q, S. from SNA. A–B. DM, C–S. DIC, Scale bars: A = 100 μm, F = 10 μm. Scale bar of A applies to A–B. Scale bar of F applies to C–S.
sometimes polyphialides observed, opening 1–2 μm diam, collarette 0.5–1 μm long, periclinal thickening observed. Conidia
hyaline, smooth-walled, aseptate straight to slightly curved,
fusiform with ± acute ends, (13–)16–20(–26) × 3–4(–5) μm,
av. ± SD = 18.1 ± 2.0 × 3.5 ± 0.4 μm, L/W ratio = 5.1, conidia of
strain
CBS
127605
shorter,
measuring
(13–)
15–17.5(–19.5) × 3–3.5(–4) μm, av. ± SD = 16.3 ± 1.4
× 3.4 ± 0.2 μm, L/W ratio = 4.8. Appressoria single or in loose
groups, medium brown, smooth-walled, globose, subglobose to
elliptical in outline, with an entire margin, (5–)
5.5–7.5(–9) × (3.5–)4.5–6(–6.5) μm, av. ± SD = 6.4 ± 0.8
× 5.2 ± 0.6 μm, L/W ratio = 1.2.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on hyaline to pale brown, angular
cells, 3.5–9 μm diam. Setae pale brown, smooth-walled,
30–120 μm long, 1–3-septate, base ± inflated, 5–6 μm diam,
tip round. Conidiophores hyaline to pale brown, smooth-walled,
septate, branched, to 20 μm long. Conidiogenous cells hyaline,
smooth-walled, cylindrical to ampulliform, 11–22 × 3.5–5 μm,
opening 1.5–2 μm diam, collarette 0.5–1 μm long, periclinal
thickening distinct. Conidia hyaline, smooth-walled, aseptate,
straight to slightly curved, fusiform with ± acute ends, (15.5–)
17–20(–21.5) × 3–3.5(–4 μm, av. ± SD = 18.6 ± 1.6
× 3.4 ± 0.3 μm, L/W ratio = 5.5.
hyaline, partly pale rosy buff to pale olivaceous grey, agar
66
medium, filter paper and Anthriscus stem partly covered with
floccose white aerial mycelium or aerial mycelium lacking,
reverse same colours; growth 15–18.5 mm in 7 d (23.5–25 mm
in 10 d). Colonies on OA flat with entire margin; surface straw,
pale luteous to amber, partly covered with very short aerial
mycelium and partly covert with black to salmon acervuli, aerial
mycelium lacking, reverse same colours; growth 21–22.5 mm in
7 d (30–34 mm in 10 d). Conidia in mass whitish to salmon.
Materials examined: Canada, Saskatchewan, North Battlefield, from seed, 2001
crop, sample 90812, of Lens culinaris cv. ‘CDA Grandora’, 2001, R.A.A. Morrall
and Discovery Seed Labs (CBS H-21649 holotype of C. lentis, culture exholotype CBS 127604 = DAOM 235316 = CT21, Race Ct1); Saskatchewan,
Moose Jaw, from seed, 2001 crop, sample 91639, of Lens culinaris cv. ‘CDA
Grandora’, 2001, R.A.A. Morrall and Discovery Seed Labs, CBS H-21650, culture
CBS 127605 = DAOM 235317 = CT26, Race Ct0. Romania, Iaşi, on pods and
leaves of Lens culinaris, 30 Jun. 1950, C. Sandu-Ville (GLM-F102752 holotype
of C. savulescui Sandu ex Herbarul Micologic “C. Sandu-Ville”).
Notes: In 1986 and 1987, an anthracnose disease of lentil (Lens
culinaris) was observed in Manitoba, Canada, and identified as
C. truncatum by Morrall (1988). Armstrong-Cho & Banniza
(2006) induced the formation of perithecia by crossing single
conidial isolates of the lentil pathogen in the laboratory. Consequently, they considered these crosses as the sexual morph of
C. truncatum and with the dual nomenclature still in place,
named this sexual morph Glomerella truncata, although
morphological as well as molecular studies (Ford et al. 2004)
THE COLLETOTRICHUM
comparing lentil isolates with “C. truncatum” isolates from soybean, clover, peanut and cocklebur indicated different species
were involved. Latunde-Dada & Lucas (2007) and Gossen et al.
(2009) found isolates from anthracnose of lentil in Canada to be
closely related to C. destructivum. Damm et al. (2009) epitypified
C. truncatum and revealed the lentil pathogen from Canada to be
a different species. In contrast to that species, C. truncatum
forms strongly curved conidia and does not belong to the
C. destructivum complex (Damm et al. 2009). The phylogenetic
relationship between the two species was demonstrated by
O'Connell et al. (2012) and Cannon et al. (2012).
With the adoption of the new International Code of Nomenclature for algae, fungi and plants concerning species names for
morphs with the same epithet introduced prior to 1 January 2013,
the name Ga. truncata will be considered as a new combination
of the previously described C. truncatum and not as a new
species, although it is based on a different type and the two types
are not conspecific (McNeill et al. 2012, Hawksworth et al. 2013).
Consequently, the lentil pathogen from Canada is described as a
new species in this study, C. lentis. As suggested by Hawksworth
et al. (2013), the name Ga. truncata was treated as a formal error
for a new combination and corrected accordingly to Glomerella
truncata (Schwein.) C.L. Armstrong & Banniza.
The two strains studied here, CBS 127604 and CBS 127605,
were crossed to produce the original holotype specimen of “Ga.
truncata”, DAOM 235318, on inoculated sterilised stems of Lens
culinaris, but the latter will have no nomenclatural status under
the new changes to the Code (Hawksworth et al. 2013). As there
is no strain derived from this specimen and an epitypification
would be needed in order to have an ex-type strain, it was not
used as holotype of C. lentis. Instead, CBS 127604 was chosen
for the holotype of C. lentis.
A second species from Lens culinaris, C. savulescui, was
described in Romania by Sandu-Ville (1959). As specimen GLMF102752 is apparently the only specimen of this species from the
Herbarium Mycologicum Moldavicum “Constantin Sandu-Ville”
(www.uaiasi.ro/agricultura/index.php?lang=en&pagina=pagini/
herbarium.html) and no specimen was located elsewhere, it was
considered as the holotype of C. savulescui, although its
collection date was prior to that listed in the publication of SanduVille (1959). Conidia of C. savulescui are hyaline, cylindrical with
both ends rounded, straight or slightly curved, measuring
7.5–18 × 3–4.5 μm, mostly 12–18 × 4 μm. The size of the
conidia found on the holotype is similar to that of C. lentis; the
conidia are also described as straight to slightly curved, which
indicates this species might belong to the C. destructivum species complex. However, C. lentis has conidia that are fusiform
with ± acute ends. The name of this species, as far as we know,
has not been used since its original description.
Additionally, isolates from lentil were also included in the
study of Liu et al. (2013a), identified as C. nigrum. Colletotrichum
nigrum is not closely related to the species treated here and
forms entirely straight conidia.
Armstrong-Cho & Banniza (2006) observed self-sterility of all
isolates tested, while many pairings produced perithecia and
concluded the species to have a homothallic mating system. The
study by Menat et al. (2012) confirmed a bipolar mating system,
however an atypical one, with the HMG box that is part of the
MAT1-2 idiomorph being present in both incompatibility groups.
The sexual morph was described by Armstrong-Cho & Banniza
(2006) as follows “Perithecia were brown-black, superficial,
solitary or in small groups, obpyriform to ovate or ampulliform,
200–520 × 110–320 μm (mean: 350 × 200 μm). Asci were
cylindrical, narrowing slightly at the apex, unitunicate, evanescent, 53–142 × 5–14 μm (mean: 90 × 8 μm), and contained eight
ascospores. Ascospores were hyaline, aseptate, oblong,
12–20 × 5–8 mm (mean: 15.7–6.7 μm).”
Buchwaldt et al. (2004) identified two physiological races of
“C. truncatum” from lentil on the basis of their pathogenicity on a
number of lentil cultivars and germplasm lines in western Canada, designating them Ct0 and Ct1.
The intracellular hemibiotrophic infection of the lentil pathogen was studied by Latunde-Dada & Lucas (2007) and
Armstrong-Cho et al. (2012). This pathosystem was used to
identify secreted effector proteins expressed at the switch from
biotrophy to necrotrophy (Bhadauria et al. 2011) and functional
analysis of a nudix hydrolase effector eliciting plant cell death
(Bhadauria et al. 2012).
Strains that are morphologically similar and molecularly
closely related (based on ITS) to C. lentis were isolated from the
noxious weed scentless chamomile (Tripleurospermum
inodorum) in Canada (Forseille 2007). The potential of this
fungus for biocontrol of scentless chamomile was tested (Peng
et al. 2005, Forseille et al. 2009). In the field, chamomile isolates caused symptoms on its original host but not on lentil or
pea. Forseille et al. (2009) also observed the hemibiotrophic
infection process of this fungus, which might represent a further
species of the C. destructivum complex.
Colletotrichum lentis is characterised by its slightly curved,
fusoid conidia that are gradually tapering to the ± acute ends and
by the ± globose appressoria with an entire margin. It can be
identified by all loci included in this study.
The ITS sequence of strain CBS 127604 matched in a blastn
search with the same sequence (GenBank JQ005766, O'Connell
et al. 2012) and that of “C. truncatum” isolate 9969473 (GenBank
AF451902, Ford et al. 2004) and with 99 % identity (1–3 nucleotides difference) with “C. truncatum” isolates 95S25,
9971646, 95A8, 9970034 from lentil in Canada (GenBank
AF451901, AF451904, AF451900, AF451903, Ford et al. 2004)
and “Ga. glycines” isolate IFO7384 from an unknown host
(GenBank AB057435, Moriwaki et al. 2002). The only matching
TUB2 sequence found in GenBank is that of the same strain
(GenBank JQ005850, O'Connell et al. 2012); all other TUB2
sequences are 95 % identical.
Colletotrichum lini (Westerd.) Tochinai, J. Coll. Agric.
Hokkaido Imp. Univ. 14: 176. 1926. Fig. 9.
Basionym: Gloeosporium lini Westerd., Jaarversl. Phytopathol.
Lab. “Willie Commelin Scholten” 6. 1916 [1915].
= Colletotrichum linicola Pethybr. & Laff. [as ‘linicolum’], Sci. Proc. Roy.
Dublin Soc. 15: 368. 1918.
hyphae 1.5–6 μm diam, hyaline, smooth-walled, septate,
conidiophores formed directly on hyphae. Setae not observed.
Setae of strain IMI 391904 medium brown, smooth-walled to
verruculose, 52–94 μm long, 1–3-septate, base cylindrical to
conical, 3.5–6.5 μm diam, tip rounded. Conidiophores hyaline,
cells hyaline, smooth-walled, cylindrical, 9–32 × 2.5–4.5 μm,
opening 1–1.5 μm diam, collarette 0.5 μm long, periclinal
thickening rarely observed. Conidia hyaline, smooth-walled,
aseptate, fusiform, slightly curved to straight, tapering to the
67
DAMM
ET AL.
Fig. 9. Colletotrichum lini (from ex-epitype strain CBS 172.51). A–B. Conidiomata. C–J. Conidiophores. K–P. Appressoria. Q–R. Conidia. A, C–E, Q. from Anthriscus stem. B,
F–P, R. from SNA. A–B. DM, C–R. DIC, Scale bars: A = 100 μm, C = 10 μm. Scale bar of A applies to A–B. Scale bar of C applies to C–R.
slightly rounded to acute ends, (13–)15–18(–22.5) × (3–)
3.5–4(–4.5) μm, av. ± SD = 16.6 ± 1.6 × 3.8 ± 0.3 μm, L/W
ratio = 4.4, conidia of strain CBS 112.21 are smaller, measuring
(12–)13.5–16.5(–18.5) × (3–)3.5–4.5(–5) μm, av. ± SD = 15.0
± 1.4 × 4.0 ± 0.4 μm, L/W ratio = 3.7, conidia of strain CBS
117156
are
longer,
measuring
(18–)18.5–20(–21)
× 3.5–4(–4.5) μm, av. ± SD = 19.3 ± 0.8 × 3.9 ± 0.2 μm, L/W
ratio = 5.0, the ex-epitype strain CBS 172.51 and of strain CBS
112.21 formed inside SNA agar medium are larger conidia than
on the surface of the medium, those of strain CBS 172.51
measure (23.5–)24–33(–52.5) × 4–4.5(–5) μm, av. ± SD
= 28.6 ± 4.3 × 4.3 ± 0.3 μm, L/W ratio = 6.7. Appressoria single
or in loose groups, pale brown, smooth-walled, ellipsoidal to
subglobose outline, with an entire or undulate margin, (5–)
6.5–10(–12.5) × (4–)4.5–6(–7) μm, av. ± SD = 8.3 ± 1.9
× 5.3 ± 0.9 μm, L/W ratio = 1.6, strain IMI 391904 additionally
formed appressoria-like structures within the mycelium,
measuring (3.5–)5–7.5(–8) × (2.5–)3.5–5.5(–6) μm, av. ± SD
= 6.3 ± 1.2 × 4.5 ± 0.8 μm, L/W ratio = 1.4.
3–8.5 μm diam. Setae not observed. Setae of strain IMI 391904
medium brown, smooth-walled to finely verruculose, 55–210 μm
long, 1–5(–6)-septate, base cylindrical to conical, 3.5–7 μm
diam, tip slightly rounded. Conidiophores hyaline, smooth-walled,
septate, branched, to 40 μm long. Conidiogenous cells
hyaline, smooth-walled, cylindrical to elongate ampulliform,
68
8–22 × 2.5–4 μm, opening 1–2 μm diam, collarette 0.5–1 μm
long, periclinal thickening observed. Conidia hyaline, smoothwalled, aseptate, fusiform, slightly curved to straight, tapering to
the slightly rounded to acute ends, (14.5–)16.5–19.5(–21.5)
× 3.5–4 μm, av. ± SD = 18.0 ± 1.5 × 3.8 ± 0.2 μm, L/W ratio = 4.7,
conidia of strain CBS 112.21 are smaller, measuring (13.5–)
15–17.5(–19.5) × 4–4.5 μm, av. ± SD = 16.3 ± 1.4 × 4.3 ± 0.2 μm,
L/W ratio = 3.8, conidia of strain CBS 117156 are longer (17.5–)
19.5–22.5(–23.5) × (3–)3.5–4(–4.5) μm, av. ± SD = 21.1
± 1.4 × 3.8 ± 0.2 μm, L/W ratio = 5.5.
hyaline to pale luteous, filter paper partly pale luteous, agar medium
and Anthriscus stem partly covered with floccose white aerial
mycelium, reverse same colours; growth 27.5–30 mm in 7 d
(>40 mm in 10 d). Colonies on OA flat with entire margin; buff to
rosy-buff, aerial mycelium lacking, reverse buff, growth 26–29 mm
in 7 d (37.5–>40 mm in 10 d). Conidia in mass not observed.
Materials examined: Germany, Dierhagen-Neuhaus, walkway, from stems and
leaves with black spots of Trifolium repens, 4 Aug. 2010, U. Damm, CBS H21660, culture CBS 130828. Ireland, from Linum usitatissimum, collection date
unknown (isolated by P. Mercer and deposited in CBS collection Feb. 1997 by
J.A. Bailey), P. Mercer, culture CBS 505.97 = LARS 77. Netherlands, from
leaves and stems of Linum sp., collection date and collector unknown (IMI
194722 ex coll. Prof. J. van Westerdijk, lectotype of Gm. lini, here designated,
MBT178721); from seed plants of Linum sp., collection date and collector unknown (IMI 194721 ex coll. Prof. J. van Westerdijk); from seedling disease of
Linum usitatissimum, collection date and collector unknown (deposited in CBS
THE COLLETOTRICHUM
collection Sep. 1951 by Plantenziektenkundige Dienst Wageningen, Nederland,
identified by A.C. Stolk) (CBS H-21657 epitype of Gm. lini, here designated,
MBT178521, culture ex-epitype CBS 172.51); Province Gelderland, Malden,
closed railway nearby gliding-club, from leaf spots of Teucrium scorodonia, 23
Aug. 2004, G. Verkley and M. Starink, V3037, culture CBS 117156. New Zealand, from Trifolium sp., collection date and collector unknown, (history: I.D. Blair,
1957 CABI), culture IMI 69991 = CPC 20242. Tunisia, site Barragage Jjoumine,
from symptoms on a living leaf of Raphanus raphanistrum, collection date and
collector unknown (deposited in IMI collection by Dr. M. Jourdan), CBS H-21658,
culture IMI 391904 = CPC 19382 = IS320. UK, from seedling disease of Linum
usitatissimum, collection date and collector unknown (isolated by G.H. Pethybridge, deposited in CBS collection Aug. 1921 by G.H. Pethybridge), CBS H21656, culture CBS 112.21 = LCP 46.621. USA, Utah, Salt Lake City, cemetery,
from small black spots on petioles of Trifolium hybridum, 24 Aug. 2013, U. Damm,
CBS H-21659, culture CBS 136850; Utah, Bluffdale near Salt Lake City, stems of
Medicago sativa, 25 Aug. 2013, U. Damm, culture CBS 136856.
Notes: Anthracnose has a serious impact on yield and fibre
quality of flax (Linum usitatissimum) and is well-known in Europe,
Asia and America. Flax anthracnose increased in Germany when
flax production was expanding in the 1930s (Rost 1938). The
anthracnose pathogen is seed- and soilborne, causes damping
off of flax seedlings (Rost 1938), and is one of the causal organisms of so-called flax-sick soils (Bolley & Manns 1932).
Van Westerdijk (1916) described the flax anthracnose pathogen in the Netherlands as Gloeosporium lini, citing the genus as
Gloeosporium (Colletotrichum). This name was combined into
Colletotrichum by Tochinai (1926), following the study of several
Japanese collections. Neither the location of the fungus nor a
type was listed by van Westerdijk. Unfortunately, no strain was
preserved in the CBS culture collection. However, two specimens from Van Westerdijk's Gm. lini collections were sent to the
IMI fungarium by von Arx, and the one containing a larger
amount of diseased plant material (IMI 194722) is designated as
lectotype. The specimen includes fusiform, slightly curved to
straight conidia with slightly rounded to acute ends that measure
(14–)16–21(–24) × (3–)3.5–5 μm, av. ± SD = 18.4 ± 2.3
× 4.2 ± 0.7 μm, L/W ratio = 4.4. Sutton (1980) listed the species
from flax as C. lini (Westerd.) Tochinai, but later (Sutton 1992)
followed Dickson's (1956) opinion that the basionym, Gm. lini
Westerd. was probably synonymous with Polyspora lini Laff.
(current name in Species Fungorum: Kabatiella lini) and not a
Colletotrichum. However, the conidia on the lectotype specimen
of Gm. lini agreed both in shape and size with the Colletotrichum
species from Linum we treat in this study.
A Colletotrichum species on Linum in North Dakota, USA, was
studied between 1901 and 1903 by T.F. Manns and also called
C. lini; however, his thesis was never published (Manns & Bolley
1932). The name was taken up by Bolley (1910); however, it is
illegitimate as it is a “nomen nudum”. Bolley & Manns later (1932)
treated the fungus as C. lini Manns et Bolley. Conidia of this
species measure 15–20 × 2–4.5 μm, setae are 70–130 μm long
and 2–4-septate, and the olive brown “chlamydospores” measure
10–15 × 10–12 μm (Bolley & Manns 1932). This agrees with the
observations of the Colletotrichum from Linum in this study and is
probably a synonym. We have not seen the type of this species
and no isolates from Linum in the USA were available to us.
Pethybridge & Lafferty (1918) described C. linicola as the
causal agent of damping off of flax seedlings in Ireland with
conidia measuring 17 × 4 μm and 3-septate setae measuring
150 × 4 μm. This species is most probably a synonym of C. lini
(Westerd.) Tochinai. Both an authentic strain from the UK isolated by G.H. Pethybridge (CBS 112.21) and a strain from Ireland
(CBS 505.97) are included in our study.
Rost (1938) lists C. atramentarium that formed straight conidia on flax in Germany and which is probably a synonym of
C. coccodes (Liu et al. 2011). Wollenweber & Hochapfel (1949)
also identified a collection from stems of Linum from Silesia as
C. atramentarium.
Hahn (1952) examined the infection process of C. lini on
resistant and susceptible flax lines and provided the first
description of bulbous primary hyphae colonising single
epidermal cells. These were subsequently found to be the
characteristic biotrophic infection structures formed by all
members of the C. destructivum species complex examined to
date.
Conidia of C. lini strains from Linum are similar to those of
C. lentis. They are both slightly curved and fusiform, but conidia
of C. lini are more abruptly tapering to the slightly acute ends; this
shape was noticed in the type material (not shown), and very
long conidia were found within the agar medium. In accordance
with the original description and the observations on the type, no
setae were observed on the strains from flax, but none of the
isolates was recently collected.
In contrast to the C. lini strains from Linum, the strains from
Trifolium hybridum, T. repens, Medicago sativa and Taraxacum
sp. formed setae and rather cylindrical conidia with rounded
ends. These strains formed a subclade within C. lini. However,
we refrained from describing these strains as a new species,
because there was only one nucleotide difference in the TUB2
sequence to separate them from the remaining C. linum strains;
the overall sequence variability within C. lini was higher. Moreover, their morphology was similar to strains from Nigella,
Raphanus and Teucrium, which belong to the same subclade as
the strains from Linum. Both subclades contain strains from
multiple hosts.
Colletotrichum lini is distinguishable by CHS-1, HIS3, ACT
and TUB2. The ITS and GAPDH sequences are the same as
those of C. americae-borealis. The sequences of all genes in
strains from Linum, Nigella and Teucrium are identical. The strain
from Raphanus in Tunisia (IMI 391904) with the longest branch
differs only in its GAPDH sequence.
As the sequences are the same, blastn searches with the ITS
sequence of C. lini strain CBS 172.51 resulted in the same
matching sequences as those with the ITS of C. americae-borealis, including isolates from alfalfa, clover, Oxytropis, endophytes from Holcus and Arabidopsis as well as strain IMI 391904
that is included in our study and a strain from Convolvulus in
Turkey. Strain IMI 391904 originated from a study on pathogenic
fungi on wild radish (Raphanus raphanistrum) in northern Tunisia
in order to screen for potential biocontrol agents against this
weed (Djebali et al. 2009). It was previously identified as
C. higginsianum and re-identified as C. lini in this study. The
identification of the strain from field bindweed (Convolvulus
arvensis) in Turkey as C. linicola is based on the ITS sequence
only (Tunali et al. 2008); it was tested to be effective as a potential biocontrol agent against that plant (Tunali et al. 2009).
However, the identity of this strain needs to be confirmed with
sequences of additional loci.
The ITS sequence of C. lini strain Coll-44 from a recent
disease report of anthracnose on Medicago in Serbia (GenBank
JX908364, Vasic et al. 2014) is identical to strains from
C. americae-borealis and C. lini. As the TUB2 sequence (GenBank KJ556347, kindly provided by Tanja Vasic) was identical to
that of C. lini strain CBS 136850 (from Trifolium hybridum, USA),
strain Coll-44 is confirmed as C. lini. Sequences of an isolate
69
DAMM
ET AL.
from our study (CBS 157.83) and several ITS sequences
detected in GenBank (GenBank JX908362, JX908363,
JX908361, Vasic, unpubl. data) are identical to those of
C. destructivum s. str., indicating the occurrence of at least two
species on Medicago in Serbia.
The performance of C. lini strain CBS 112.21 in comparison
with Botryodiplodia malorum in steroid hydroxylations, to improve
the biotransformation of steroids for the pharmaceutical industry,
was studied by Romano et al. (2006).
Colletotrichum ocimi Damm, sp. nov. MycoBank
MB809401. Fig. 10.
name Ocimum.
branched. Chlamydospores not observed. Conidiomata conidiophores and setae formed on pale brown, roundish cells,
5–22 μm diam. Setae medium brown, smooth-walled to verruculose, 43–103 μm long, 1–2-septate, base cylindrical
to ± inflated, 4.5–9.5 μm diam, tip ± rounded to ± acute. Conidiophores hyaline, smooth-walled, septate, branched, to 60 μm
long. Conidiogenous cells hyaline to pale brown, smooth-walled
to verruculose, cylindrical to clavate, sometimes intercalary
(necks not separated from hyphae by septum), often with slime
sheaths, 10.5–24 × 3.5–5.5 μm, opening 1–1.5 μm diam, collarette 0.5–1.5 μm long, periclinal thickening distinct. Conidia
hyaline, smooth-walled, aseptate, straight, cylindrical, with both
ends rounded or one end round and the other truncate, (13.5–)
14.5–15.5(–16.5) × (3.5–)4–4.5 μm, av. ± SD = 15.0
± 0.7 × 4.1 ± 0.2 μm, L/W ratio = 3.7. Appressoria very few,
single, scattered, pale brown, smooth-walled, ellipsoidal, clavate,
subglobose or irregular outline, with a lobate or entire margin,
(6.5–)7–13(–15.5) × (4–)4.5–7.5(–9) μm, av. ± SD = 9.9 ± 2.9
× 6.0 ± 1.3 μm, L/W ratio = 1.6.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on pale brown, verruculose,
roundish cells, 4–17 μm diam. Setae medium brown, verruculose, 30–145 μm long, 1–4-septate, base cylindrical, conical
to ± inflated, 4–7.5 μm diam, tip ± rounded to ± acute. Conidiophores hyaline to pale brown, smooth-walled, septate,
branched, to 25 μm long. Conidiogenous cells hyaline to pale
brown,
smooth-walled,
cylindrical
to
ampulliform,
8–21.5 × 3.5–5 μm, opening 1–1.5 μm diam, collarette 0.5 μm
long, periclinal thickening visible. Conidia hyaline, smoothwalled, aseptate, straight, cylindrical, with both ends rounded
or one end round and the other truncate, (11–)
14–16(–16.5) × (3.5–)4(–4.5) μm, av. ± SD = 14.8 ± 1.0
× 4.0 ± 0.2 μm, L/W ratio = 3.7.
hyaline to cinnamon, agar medium, filter paper and Anthriscus
stem partly covered with grey acervuli, aerial mycelium lacking,
reverse same colours; growth 18–19.5 mm in 7 d (28–29.5 mm
in 10 d). Colonies on OA flat with entire margin; buff to honey,
almost entirely covert with dark grey to black acervuli and salmon
conidial masses, aerial mycelium lacking, reverse rosy buff,
vinaceous buff to pale olivaceous-grey, growth 20.5–22 mm in
7 d (30–31.5 mm in 10 d). Conidia in mass salmon.
70
Material examined: Italy, Riviera Ligure, from a black spot on leaf of Ocimum
basilicum, collection date and collector unknown (deposited in CBS collection
May 1994 by A. Garibaldi, Inst. degli studi di Torino, Depart. di Valorizzazione e
Protezione delle Risore agroforestiali) (CBS H-21646 holotype, culture exholotype CBS 298.94).
Notes: Basil (Ocimum basilicum) is an aromatic culinary herb, for
which flawless leaves are of special importance. Gullino et al.
(1995) reported an outbreak of a new foliar disease of basil
cultivated in greenhouses in northern Italy and consistently isolated a Colletotrichum species. The fungus caused black spots
on stems and leaves of basil; lesions on stems often resulted in
girdling and plant death. One strain (CBS 298.94) was sent to
CBS and identified as Glomerella cingulata var. cingulata (until
recently regarded as the sexual stage of C. gloeosporioides) by
H.A. van der Aa (HA 11925) as indicated in the database of the
CBS culture collection.
This species forms cylindrical, straight conidia with round
ends, reminiscent of species in the C. gloeosporioides complex
(Weir et al. 2012). However, we found that C. ocimi belongs to
the C. destructivum species complex. Gullino et al. (1995) did not
observe a sexual stage of the basil fungus. Apart from the
conidia, C. ocimi differs from the other species in the
C. destructivum complex by its conidiogenous cells that are often
covered by mucoid sheaths.
No species were previously described on Ocimum. Additionally to Gullino et al. (1995), Farr & Rossman (2014) list a few
further reports of Colletotrichum species on basil: C. capsici in
India, C. gloeosporioides in Cambodia and Colletotrichum sp. in
Florida, USA. It is possible that the latter two reports refer to
C. ocimi as well. However, the only sequence of a Colletotrichum
strain from basil in GenBank, which is an ITS sequence of strain
EGJMP 40 probably from India (GenBank KF234012) identified
as C. aotearoa (E.G. Jagan et al., unpubl. data), indeed refers to
a species belonging to the C. gloeosporioides species complex.
This species can be identified by its unique ITS, CHS-1, HIS3,
ACT, and TUB2 sequences. The closest match in a blastn search
with the ITS sequence of strain CBS 298.94 is GenBank
EU400148 from C. lini strain DAOM 183091 (Chen et al. 2007).
No TUB2 sequences were detected in GenBank with >97 %
identity. The GAPDH sequence of C. ocimi is the same as that of
C. destructivum (s. str.).
Colletotrichum panacicola Uyeda & S. Takim., Bull.
Korean Agric. Soc. 14: 24. 1919. MycoBank MB809665.
≡ Colletotrichum panacicola Uyeda & S. Takim., Bull. Agric. Experiment
Stat. Chosen (Korea) 5: 16. 1922. Nom. illegit., Art. 53.1.
Notes: Colletotrichum panacicola, originally described from
Panax ginseng in Korea, has also been reported from China,
eastern Russia and Japan, while anthracnose of American
ginseng (P. quinquefolius) is caused by C. dematium (s. lat.) and
C. coccodes (McPartland & Hosoya 1997). McPartland &
Hosoya (1997) corrected the author citation of the species that
had been described already by Takimoto (1919), but that would
have to be cited as C. panacicola Uyeda & S. Takim. The
confusion was caused by Nakata & Takimoto (1922) who
described the same species again as a new species. Petrak
(1953) cited the species wrongly as C. panacicola Nakata &
S. Takim., which was subsequently taken up by Index Fungorum.
The species was characterised with aseptate, cylindrical,
straight or slightly curved conidia with rounded ends, measuring
17.0–22.1 × 3.4–5.1 μm, pyriform olive coloured appressoria,
THE COLLETOTRICHUM
Fig. 10. Colletotrichum ocimi (from ex-holotype strain CBS 298.94). A–B. Conidiomata. C, G. Tip of a seta. D, H. Base of a seta. E–F, I–K. Conidiophores. L–Q. Appressoria.
R–S. Conidia. A, C–F, R. from Anthriscus stem. B, G–Q, S. from SNA. A–B. DM, C–S. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B. Scale bar of E
applies to C–S.
measuring 14–8 μm and dark olive 1–3-septate setae with acute
paler apices that measure 31–144 × 2.4–8.4 μm (Takimoto
1919, Nakata & Takimoto 1922, both cited by McPartland &
Hosoya 1997). McPartland & Hosoya (1997) were unable to
locate either type or authentic specimens. As the illustration in
Nakata & Takimoto (1922) is not sufficiently diagnostic to act as a
lectotype, the species needs to be neotypified.
Fresh cultures are available as Choi et al. (2011) recently
studied isolates of this species and observed similarity with
C. higginsianum, C. destructivum and C. coccodes. ITS sequences did not distinguish the species from C. higginsianum
and C. destructivum. The inclusion of more genes (ACT, translation elongation factor 1-α, glutamine synthase) clearly showed
this species to be different from the other two (Choi et al. 2011).
Choi et al. (2011) who also showed this species to only infect
Korean ginseng, suggesting it was a distinct taxon.
As there were no isolates available to us, we could not directly
compare the morphology of C. panacicola. However, we included
DNA sequences of three isolates from the study of Choi et al.
(2011) that were retrieved from GenBank in our molecular analyses (only with ITS, GAPDH and ACT), which confirmed
C. panacicola to belong to the C. destructivum complex and to be
a distinct species, although closely related to the newly described
C. utrechtense, which has the same ACT sequence. Colletotrichum panacicola can be identified by ITS and GAPDH sequences; 100 % sequence identities on GenBank were only
found with the C. panacicola sequences from the study of Choi
et al. (2011). Unfortunately, the TUB2 region sequenced by Choi
et al. (2011) was different from the region we studied and could
not be compared to our dataset; the CHS-1 and HIS sequences
of this species were not available for comparison.
Colletotrichum pisicola Damm, sp. nov. MycoBank
MB809403. Fig. 11.
Etymology: The species epithet is derived from the host plant
genus, Pisum.
branched, at some parts pale to medium brown. Chlamydospores
not observed. Conidiomata absent, conidiophores and setae
formed directly on hyphae or aggregated on clusters of pale to
medium brown, roundish cells, 3.5–11 μm diam. Setae few
observed, pale brown, smooth-walled to verrucose, 30–40 μm
long, 1–2-septate, base cylindrical to conical, 4–5 μm diam, tip
round or with a conidiogenous locus. Conidiophores hyaline,
cells hyaline, smooth-walled, cylindrical to ampulliform, sometimes intercalary (necks not separated from hyphae by septum),
11–30 × 2.5–5 μm, opening 1–1,5 μm diam, collarette 1–2,5 μm
long, periclinal thickening visible, sometimes distinct. Conidia
71
DAMM
ET AL.
Fig. 11. Colletotrichum pisicola (from ex-holotype strain CBS 724.97). A–B. Conidiomata. C, G. Tip of a seta. D, H. Base of a seta. E–F, I–M. Conidiophores. N–S.
Appressoria. T–U. Conidia. A, C–F, T. from Anthriscus stem. B, G–S, U. from SNA. A–B. DM, C–S. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B.
Scale bar of E applies to C–S.
hyaline, smooth-walled, aseptate, fusiform, distinctly curved
gradually tapering to the ± acute ends, (11–)
15–21(–29.5) × (3–)3.5–4 μm, av. ± SD = 18.1 ± 2.9
× 3.5 ± 0.2 μm, L/W ratio = 5.2. Appressoria single, pale brown,
smooth-walled, elliptical, clavate to irregular outline, with an entire
or undulate margin, (5.5–)7–11.5(–13.5) × (4–)4.5–6(–6.5) μm,
av. ± SD = 9.3 ± 2.2 × 5.1 ± 0.7 μm, L/W ratio = 1.8.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on pale to medium brown,
roundish to angular cells, 3.5–12 μm diam. Setae pale brown,
smooth-walled to verrucose, 40–55 μm long, 1–2-septate, base
conical, 4–6.5 μm diam, tip rounded. Conidiophores pale brown,
smooth-walled, sometimes septate and branched, to 30 μm long.
Conidiogenous cells pale brown, smooth-walled, ampulliform to
cylindrical, 10.5–24 × 3.5–5.5 μm, opening 1–1.5 μm diam,
collarette 0.5–1 μm long, periclinal thickening observed. Conidia
hyaline, smooth-walled, aseptate, fusoid, distinctly curved,
gradually tapering to the ± acute ends, (12–)
15–20.5(–23.5) × (3–)3.5–4 μm, av. ± SD = 17.8 ± 2.8
× 3.7 ± 0.3 μm, L/W ratio = 4.7.
pale straw, covered short filty whitish aerial mycelium, reverse
pale luteous; growth 6.5–7.5 mm in 7 d (8.5–10 mm in 10 d).
Colonies on OA flat with entire margin; pure yellow to luteous,
with a buff margin, covered with very short aerial mycelium,
72
reverse pale luteous to luteous, growth 12–15 mm in 7 d
(17.5–20 mm in 10 d). Conidia in mass not visible.
Materials examined: Ecuador, Quito, from anthracnose symptoms on pods of
Pisum sp., Jan. 1891, G. Lagerheim (BPI 797146 (ex herbarium N. Patouillard)
lectotype of C. pisi, here designated, MBT178523); Quito, from anthracnose
symptoms on pods of Pisum sativum, Jan. 1892, G. Lagerheim, BPI 399530,
includes slide; Quito, from anthracnose symptoms on pods of Pisum sativum,
Feb. 1892, G. Lagerheim, BPI 399531, includes slide; Quito, from pods of Pisum
sativum, Feb. 1892, G. Lagerheim (No. 2944), BPI 399532, includes slide.
Mexico, intercepted at El Paso, Texas, USA, from anthracnose symptoms on
pods of Pisum sativum, 17 Dec. 1952, J.A. Baker (No. 53856), BPI 399536;
intercepted at Laredo, Texas, USA, from anthracnose symptoms on pods of
Pisum sativum, 19 Nov. 1954, Ragsdale (No. 55077), BPI 399534. USA, Wisconsin, from Pisum sativum, collection date unknown (isolated by H.D. van Etten,
deposited in LARS collection by D.O. TeBeest, No. 403, deposited in CBS
collection Apr. 1997 by J.A. Bailey), H.D. van Etten (CBS H-21644 holotype of
C. pisicola, culture ex-holotype CBS 724.97 = LARS 60 = ATCC 64197 = IMI
317934).
Notes: Patouillard & Lagerheim (1891) described C. pisi from
Pisum sativum in Quito, Ecuador with hyaline, fusoid conidia with
acute ends, straight to curved, measuring 11–13 × 3–4 μm and
setae measuring 60–90 × 6 μm. Three specimens were located
in the BPI fungarium that were collected by G. Lagerheim, but
only one was collected in 1891. This specimen, BPI 797146 that
also originated from the collection of N. Patouillard is designated
as the lectotype of C. pisi in our study. Conidia found on the
THE COLLETOTRICHUM
material are fusiform and mostly ± curved and agree with the
original description of the species: (10–)11.5–15(–16.5) × (3–)
3.5–4(–4.5) μm, av. ± SD = 13.2 ± 1.8 × 3.7 ± 0.4 μm, L/W
ratio = 3.5. Conidia found on BPI 399534 were larger, measuring
(10–)14–17.5(–20) × (3–)3.5–4.5 μm, av. ± SD = 15.6 ± 1.8
× 3.8 ± 0.5 μm, L/W ratio = 4.1. Whether the specimens collected
by G. Lagerheim represent the same species is doubtful.
Among other C. pisi specimens in BPI were two (BPI 399534,
BPI 399536) that originated from Mexico and were intercepted at
the border with the USA. The species on the pea pods and seeds
of these specimens were identified as C. pisi, although considerably longer conidia were noted; for BPI 399536 the measurements were included in the note on the package as
16–22 × 3–5 μm, which agrees with the observations on strain
CBS 724.97 studied here. Hemmi (1921) also observed larger
conidia in material on P. sativum in Japan compared to those of
the original description. He also considered the fungus as C. pisi,
with straight to slightly curved, fusiform conidia with slightly acute
ends. This indicates the presence of at least two Colletotrichum
species with curved conidia on Pisum sativum.
Conidia of strain CBS 724.97 are larger than those of the
lectotype of C. pisi, measuring (11–)15–21(–29.5) × (3–)
3.5–4 μm on SNA compared to (10–)11.5–15(–16.5) × (3–)
3.5–4(–4.5) μm of C. pisi. The species represented by strain
CBS 724.97 is described as new. Regarding conidial size, the
specimens from Mexico and Hemmi's Japanese collection
resemble C. pisicola rather than C. pisi.
The existence of at least two Colletotrichum species is further
supported by the second strain from Pisum sativum included in
this study, strain CBS 107.40 from Russia. This strain is
deposited in CBS as C. pisi and belongs to a species closely
related to C. pisicola (see below Colletotrichum sp. CBS 107.40).
Farr & Rossman (2014) report C. pisi on Pisum sativum in
Brazil, China, Canada, USA (Connecticut, Florida, Georgia,
Hawaii, Iowa, Idaho, Louisiana, Maine, Minnesota, Texas, Wisconsin), USSR, Guatemala, India and the Malay Peninsula.
Hemmi (1921) also reported the species to be common on
P. sativum in Japan. Further species reported on P. sativum (or
P. arvense) include C. dematium from Barbados and Mexico,
C. falcatum from Hawaii, C. gloeosporioides from China, India
and USA (North Carolina), C. lindemuthianum from Chile, China
and Poland, C. truncatum from Pakistan and the USA and
Colletotrichum sp. from Brazil, Malaysia and the USA (Oregon).
Hagedorn (1974) reports widespread and serious local damage
by pea anthracnose in Wisconsin, USA. We cannot prove which
of these reports actually refer to C. pisicola as there are no
isolates available.
Strain CBS 724.97 was regarded as C. truncatum e.g. by
Sherriff et al. (1994), Shen et al. (2001) and Latunde-Dada &
Lucas (2007) and is included in the ATCC collection as
C. dematium f. truncatum. Based on information in the CBS
strain database, this strain was also previously identified as
C. destructivum and as C. pisi.
The two Colletotrichum strains from Pisum represent basal
species in the C. destructivum complex. This was also observed
in preliminary LSU and ITS phylogenies of the genus Colletotrichum, in which they formed a sister clade to the other species
in this complex (U. Damm, unpubl. data). Consequently, the two
species were chosen as outgroup in the phylogeny of the species
complex in this study. Their morphological features are not
typical for this complex: conidia at least of C. pisicola are curved.
However, O'Connell et al. (1993) investigated the hemibiotrophic
infection of Pisum sativum by strain LARS 60 (= CBS 724.97,
C. pisicola) with light and electron microscopy. Both the biotrophic phase and primary hyphae of this fungus were confined
to the first infected epidermal cell, but these hyphae were less
bulbous and more convoluted than those reported for other
members of the C. destructivum species complex.
The identification of a strain from roots of Salix as C. pisi from
Corredor et al. (2012) based on a blastn search on GenBank with
its ITS sequence (Genbank GU934514) is based on another
apparently wrongly identified strain, DAOM 196850 (Chen et al.
2007), that is not a Colletotrichum species; its ITS sequence
(GenBank EU400150) is identical to several Plectosphaerella
cucumerina strains.
Colletotrichum pisicola is characterised by distinctly curved
conidia that gradually taper to the ± acute ends, short and few
pale brown setae with rounded tips. Strain CBS 724.97 is the
slowest growing culture in the species complex studied.
The sequences of all loci studied of C. pisicola strain CBS
724.97 are unique; there is on CHS-1 only a single nucleotide
difference to Colletotrichum sp. strain CBS 107.40 from Pisum in
Russia (see Colletotrichum sp. CBS 107.40). No ITS sequences
with >98 % identity (9 nucleotides different) and no TUB2
sequence with >91% identity were found in GenBank.
Colletotrichum sp. CBS 107.40
branched. Chlamydospores not observed. Conidiomata, conidiophores, conidiogenous cells and Setae not observed. No
sporulation. Appressoria single, scattered, pale brown, smoothwalled, ellipsoidal, clavate to navicular outline, with an entire or
undulate margin, (4.5–)6.5–12(–15) × (3.5–)4.5–7.5(–9.5) μm,
av. ± SD = 9.2 ± 2.7 × 5.9 ± 1.5 μm, L/W ratio = 1.6.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on pale to medium brown,
roundish to angular cells, 4–11.5 μm diam. Chlamydospores not
observed. Conidiomata absent, conidiophores and setae formed
directly on hyphae. Setae not observed. Conidiophores and
conidiogenous cells not observed. Conidia only few observed,
hyaline, smooth-walled, aseptate, ± curved, with slightly acute
ends, 13–16.5 × 3.5–4 μm, mean = 14.9 × 3.8 μm, L/W
ratio = 4.0.
covered with sparse aerial mycelium, reverse same colours;
growth 12.5–14.5 mm in 7 d (18.5–20.5 mm in 10 d). Colonies
on OA flat with entire margin; greenish olivaceous to citrine, with
a straw margin, aerial mycelium lacking, reverse straw to
greenish olivaceous, growth 17.5–19 mm in 7 d (25–28.5 mm in
10 d). Conidia in mass not visible.
Material examined: Russia, Omsk, from Pisum sativum, collection date and
collector unknown (deposited in CBS collection Feb. 1940 by K. Murashkinsky),
CBS H-21645, culture CBS 107.40.
Notes: Stain CBS 107.40 from peas in Russia was deposited as
Macrophoma sheldonii in CBS by K. Murashkinsky. This species
was described by Rodigin (1928) from seeds of Pisum sativum in
Russia as forming cylindrical-ovate, thick-walled conidia,
measuring 10–18 × 5–6 μm that are mass pink and formed in
73
DAMM
ET AL.
spherical to flattened pycnidia. This species, if a Colletotrichum
species at all, is not the same species as strain CBS 107.40, as
conidial shapes and sizes are different. The spherical “pycnidia”
could refer to the closed conidiomata that have been observed in
species of the C. boninense species complex, e.g. C. dacrycarpi
and C. karstii (Damm et al. 2012). Macrophoma sheldonii was
regarded as a synonym of C. lagenarium by Vassiljevski &
Karakulin (1950) and of C. orbiculare by von Arx (1957). Since
we have not seen type material of this fungus, we cannot confirm
this species as a Colletotrichum sp.
After the strain was deposited in CBS, it was re-identified as
C. pisi. The strain was also treated as C. pisi by Nirenberg et al.
(2002), who submitted an ITS sequence to GenBank (GenBank
AJ301940). The conidia of strain CBS 107.40 are shorter than
those of C. pisicola strain CBS 724.97, and more similar to C. pisi
than those of C. pisicola (newly described in this study). However,
we refrain from using this strain to epitypify C. pisi, because the
strain is degenerated, the sporulation almost suppressed, and only
four conidia were observed that might not be typical of the species.
Moreover, the strain was from a different continent than C. pisi.
The sequences of all loci studied are unique for this species,
and different from those of C. pisicola strain CBS 724.97; however, the CHS-1 sequence differs in only one nucleotide from that
of C. pisicola.
There is no match with sequences >97 % identical to our ITS
sequence and no match with sequences >89 % identical to our
TUB2 sequence in GenBank.
Colletotrichum tabacum Böning, Prakt. Bl€att. Pflanzenbau Pflanzenschutz 10: 89. 1932. Fig. 12.
hyphae 1–7 μm diam, hyaline, smooth-walled, septate, branched.
Chlamydospores not observed. Conidiomata absent, conidiophores and setae formed directly on hyphae, sometimes also
on few pale to medium brown, roundish cells, 5–10.5 μm diam.
Setae pale to medium brown, smooth-walled, 65–150 μm long,
1–4-septate, base cylindrical to conical, 4–5.5 μm diam,
tip ± rounded to ± acute. Conidiophores hyaline to pale brown,
cells hyaline to pale brown, smooth-walled, cylindrical to ampulliform, 9–22.5 × 3–4.5 μm, opening 1–1.5 μm diam, collarette
0.5–1 μm long, periclinal thickening observed. Conidia hyaline,
smooth-walled, aseptate, narrowly cylindrical, mostly straight, with
round ends, one of the ends sometimes very slightly bent to one
side, (13.5–)15.5–18.5(–20) × 3–3.5(–4) μm, av. ± SD = 17.0
± 1.4 × 3.4 ± 0.2 μm, L/W ratio = 5.0, conidia of strain CBS 124249
longer, measuring (16–)17–20(–23.5) × 3–3.5. Appressoria
single or in loose groups, medium brown, smooth-walled, clavate,
ellipsoidal or irregular outline, with a lobate to undulate margin,
with a distinct penetration pore with a dark halo, (7–)
8–13(–19) × (4.5–)5.5–8(–10) μm, av. ± SD = 10.4 ± 2.3
× 6.6 ± 1.3 μm, L/W ratio = 1.6.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores and setae formed on a small cushion of hyaline to
pale brown, angular cells, 3–6 μm diam. Setae medium brown,
smooth-walled to finely verruculose, 55–170 μm long, 1–5septate, base cylindrical, 3.5–8.5 μm diam, tip ± rounded
to ± acute. Conidiophores hyaline to pale brown, single or
smooth-walled and septate, branched, to 30 μm long. Conidiogenous cells hyaline to pale brown, smooth-walled, cylindrical
74
to doliiform, 7–16.5 × 3.5–5 μm, opening 1–1.5 μm diam, collarette 0.5–1.5 μm long, periclinal thickening distinct. Conidia
hyaline, smooth-walled, aseptate, narrowly cylindrical, mostly
straight, with round ends, one of the ends sometimes very
slightly bent to one side, (16.5–)17–19(–21) × (3–)3.5(–4) μm,
av. ± SD = 17.8 ± 1.0 × 3.5 ± 0.2 μm, L/W ratio = 5.1.
hyaline to cinnamon, aerial mycelium lacking, reverse same
colours; growth 25–26.5 mm in 7 d (>40 mm in 10 d). Colonies
on OA flat with entire margin; isabelline, honey, buff to rosy buff,
aerial mycelium lacking, reverse buff, vinaceous buff, hazel to
pale olivaceous-grey, colonies of strain CBS 124249 differed
slightly on OA: colonies buff, almost entirely covered with honey,
grey to black acervuli and partly covert with white short filty aerial
mycelium, reverse buff, honey to olivaceous-grey, growth
27.5–29 mm in 7 d (>40 mm in 10 d). Conidia in mass salmon,
conidia of strain CBS 161.53 in mass whitish.
Materials examined: France, from Nicotiana tabacum, collection date and collector unknown (received from R. O'Connell, before from P. Goodwin, before from
M. Maurhofer Bringolf, originally from Novartis as Novartis Isolate 150) (CBS H21669 neotype here designated, MBT178524, culture ex-neotype N150 = CPC
18945). Germany, Middle Franconia, from leaves of Nicotiana rustica, holotype,
presumably lost. India, Rajahnundry, from Nicotiana tabacum, collection date
unknown, B. S. Kadam, culture IMI 50187 = CPC 16820. Madagascar, from
Centella asiatica, collection date and collector unknown (isolated by Rakotoniriana F. 2003), CBS H-21668, culture CBS 124249 = MUCL 44942. Zambia,
from Nicotiana tabacum, collection date and collector unknown (send to CBS
collection Nov. 1953 from Mt. Makulu Research St., Zambia), CBS H-21667,
culture CBS 161.53.
Notes: In the late 1920s anthracnose of tobacco, especially
Nicotiana rustica, was observed in Middle Franconia, Germany.
The pathogen, C. tabacum, differed morphologically from the
previously described C. nicotianae Averna (Böning 1929, 1932).
The fungus formed conidia that measured 15–22 × 4–5 μm in
small open clusters and setae that were 60–90 μm long (Böning
1929). In contrast, C. nicotianae that was described from stems
of N. tabacum in Sao Paulo, Brazil, formed straight to curved
conidia that were larger than those of C. tabacum, measuring
19–32.5 × 8–8.6 μm and turn yellow with age, and setae that
were 60–175 × 8.5 μm long and 3–5-septate (Averna-Sacca
1922). Colletotrichum tabacum forms distinct spots with
necrotic centres on leaves, stems, flowers and seeds and also
causes a seedling disease of tobacco (Böning 1929). The
microscopical features of the isolates studied here agree with
C. tabacum, although the conidia are slightly smaller than those
observed by Böning (1929). Böning (1929, 1932) did not
designate a type, and no type or authentic material could be
located in any fungarium.
Shortly after, an additional species was described by Böning
(1933), Gloeosporium nicotianae that caused blisters and diffuse
browning on leaf surfaces of N. rustica in Königsberg, East
Prussia (today Kaliningrad, Russia), consistently lacked setae
and also exhibited different cultural characteristics. Colletotrichum tabacum formed greenish black cultures with a uniform
grey aerial mycelium vs. Gm. nicotianae with slightly brownish
cultures and floccose aerial mycelium. Conidia of Gm. nicotianae
are on average smaller than those of C. tabacum, measuring
8–18 × 2–5 μm, depending on the substrate and formed
swollen, 12 μm diam cells in chains in the mycelium as well as
sterile pycnidia- or perithecia-like structures (Böning 1933).
THE COLLETOTRICHUM
Fig. 12. Colletotrichum tabacum (from ex-neotype strain N150). A–B. Conidiomata. C, H. Tip of a seta. D, I. Base of a seta. E–G, J–M. Conidiophores. N–S. Appressoria.
T–U. Conidia. A, C–G, T. from Anthriscus stem. B, H–S, U. from SNA. A–B. DM, C–U. DIC, Scale bars: A = 100 μm, E = 10 μm. Scale bar of A applies to A–B. Scale bar of E
applies to C–U.
Based on the description alone it is difficult to confirm whether
C. tabacum and Gm. nicotianae are different species.
Lucas & Shew (1991) concluded C. nicotiniae and C. tabacum
were synonyms of C. gloeosporioides. This was probably based
on von Arx (1957), who listed C. tabacum as synonym of
C. gloeosporioides. Farr & Rossman (2014) cited various reports
of C. nicotianae, C. tabacum, C. destructivum, C. coccodes,
C. gloeosporioides and Colletotrichum sp. from tobacco around
the world. One of the studies cited (Barksdale 1972) includes a
picture and measurements of conidia of C. destructivum from
tobacco that resemble those of C. tabacum. Isolates from Nicotiana used in molecular studies of pathogen-host-interactions are
either called C. nicotianae, or C. destructivum (e.g. Chen et al.
2003; Yang et al. 2010). Based on rDNA ITS sequences and
morphology, Shen et al. (2001) identified strain N150 (here reidentified as C. tabacum) as C. destructivum. As the isolates
studied here were previously identified as C. destructivum,
C. higginsianum, C. gloeosporioides or C. tabaci, many of the
reports listed by Farr & Rossman (2014) might actually refer to
C. tabacum. The few isolates of C. tabacum included in this study
already represent the occurrence of the species on three continents. But to our knowledge, there is no report listed from Germany since Böning (1933).
Shen et al. (2001) discovered the intracellular hemibiotrophic
infection process of C. destructivum (here re-identified as
C. tabacum) strain N150 on tobacco. Shan & Goodwin (2004,
2005) used a GFP-expressing transgenic strain of this fungus
to study rearrangement of host actin microfilaments and nuclei
around biotrophic hyphae. Secondary metabolite production by
C. tabacum (ATCC 11995) was extensively studied by
Gohbara and co-workers during the 1970s, leading to the
identification and structural characterisation of two novel terpenoid phytotoxins, colletotrichin and colletopyrone (Gohbara et al.
1976, 1978).
One of the strains included in this study, CBS 124249 (=
MUCL 44942) was isolated by F. Rakotoniriana from Centella
asiatica in Madagascar and identified as C. higginsianum
(Rakotoniriana et al. 2008). It is re-identified as C. tabacum in
this study. Rakotoniriana et al. (2013) recently described a
species from Centella asiatica in Madagascar, C. gigasporum
that forms larger conidia than C. tabacum and belongs to the
C. gigasporum complex (Liu et al. 2014), confirming that more
Colletotrichum species occur on this host in Madagascar.
Conidia of C. tabacum are narrowly cylindrical with round
ends, one of the ends sometimes slightly bent to one side; the
conidia still appearing straight. Appressoria with a distinct
penetration pore with a dark halo were observed.
Colletotrichum tabacum is distinguished from the other species in the C. destructivum complex by all loci studied, but sequences of some loci only differ with a single nucleotide from its
closest relative. Strain CBS 124249 from Centella differs additionally in CHS-1 and TUB2 sequences from the other three
75
DAMM
ET AL.
strains, but intraspecific variability was also observed with ITS,
GAPDH and ACT.
The closest match in a blastn search with the TUB2 sequence
of strain N150 with 100 % identity was C. tabacum strain CBS
161.53 (GenBank JQ005847, O'Connell et al. 2012). No GAPDH
sequence with <93 % identity was found in GenBank.
Colletotrichum tanaceti M. Barimani, et al., Plant Pathol.
62: 1252. 2013. Fig. 13.
conidiophores and setae formed directly on hyphae. Setae medium brown, smooth-walled to verruculose, 40–140 μm long,
2–4-septate, base cylindrical to conical, 4–5.5 μm diam, tip
rounded to slightly acute. Conidiophores, smooth-walled,
septate, branched, to 75 μm long. Conidiogenous cells hyaline,
smooth-walled, sometimes extending to form new conidiogenous
loci, 15–28 × 3.5–4.5 μm, opening 1–2 μm diam, collarette 1 μm
long, periclinal thickening distinct. Conidia hyaline, smoothwalled, aseptate, cylindrical to slightly clavate, slightly but
distinctly curved with both ends ± rounded, (13–)
14.5–17.5(–19) × (3–)3.5–4(–4.5) μm, av. ± SD = 16.0 ± 1.5
× 3.8 ± 0.3 μm, L/W ratio = 4.2. Appressoria single or in loose
groups, medium brown, smooth-walled, subglobose, to elliptical
in outline, with an entire or undulate margin, (5–)6.5–12(–14.6)
× (3.5–)4.5–7(–10) μm, av. ± SD = 9.1 ± 2.7 × 5.7 ± 1.4 μm, L/
W ratio = 1.6, appressoria of stem CBS 132818 are slightly
larger, measuring (7.5–)8.5–13.5(–16) × (5–)5.5–9(–12) μm,
av. ± SD = 11.0 ± 2.5 × 7.4 ± 1.8 μm, L/W ratio = 1.5.
Asexual morph on Anthriscus stem. Conidiomata, conidiophores
and setae formed on hyaline to pale brown, angular cells,
3–7.5 μm diam. Setae medium brown, smooth-walled to finely
verruculose, 30–165 μm long, 1–4-septate, base cylindrical to
conical, 4–7 μm diam, tip rounded to slightly acute. Conidiophores hyaline to pale brown, smooth-walled, septate,
brown, smooth-walled, cylindrical, sometimes extending to form
new conidiogenous loci, 18–28 × 4–5 μm, opening 1–1.5 μm
diam, collarette 0.5 μm long, periclinal thickening distinct. Conidia
hyaline, smooth-walled, aseptate, cylindrical, slightly but
distinctly curved with both ends ± rounded or one end ± acute,
(12–)16–20.5(–22) × (3–)3.5–4(–4.5) μm, av. ± SD = 18.1
± 2.1 × 3.7 ± 0.3 μm, L/W ratio = 4.9.
hyaline to pale isabelline, filter paper partly yellow, aerial
mycelium lacking, reverse same colours; growth 14–16 mm in
7 d (22.5–25 mm in 10 d). Colonies on OA flat with entire margin,
buff to straw, partly covered with tiny grey to black acervuli, aerial
Fig. 13. Colletotrichum tanaceti (from ex-holotype strain CBS 132693). A–B. Conidiomata. C. Tip of a seta. D, G–I. Conidiophores. E. Base of a seta and conidiophores. F.
Seta. J–N. Appressoria. O–P. Conidia. A, C–E, O. from Anthriscus stem. B, F–N, P. from SNA. A–B. DM, C–P. DIC, Scale bars: A = 100 μm, D = 10 μm. Scale bar of A
applies to A–B. Scale bar of D applies to C–P.
76
THE COLLETOTRICHUM
mycelium lacking, reverse olivaceous-grey, growth 14.5–17 mm
in 7 d (21.5–24.5 mm in 10 d). Conidia in mass whitish to rosybuff.
Materials examined: Australia, northern Tasmania, Scottsdale, from anthracnose
on leaves of Tanacetum cinerariifolium, Aug. 2010, S.J. Pethybridge, culture exholotype CBS 132693 = BRIP 57314 = UM01; Australia, northern Tasmania,
Ulverstone, from Tanacetum cinerariifolium, collection date unknown, S.J.
Pethybridge, living strain CBS 132818 = BRIP 57315 = TAS060-0003.
Notes: Pyrethrum (Tanacetum cinerariifolium, Asteraceae) is a
perennial plant grown for the extraction of pyrethrin insecticides
in Australia, mainly in Tasmania, one of the largest producers of
pyrethrin worldwide (Greenhill 2007). Colletotrichum tanaceti
was recently described as an anthracnose pathogen of pyrethrum in Tasmania and revealed to be closely related to
C. destructivum, C. higginsianum and C. panacicola (Barimani
et al. 2013). This species can be confirmed as distinct in this
study, and can be identified with all loci studied.
Additionally, C. tanaceti is one of the two species in this
complex with distinctly curved conidia. In contrast to C. pisicola,
the conidia are more abruptly tapered towards mostly rounded
ends. In both media, conidiogenous cells were observed that
extended to form new conidiogenous loci (Fig. 14E, H), a feature
common for species in the C. boninense species complex
(Damm et al. 2012) but not elsewhere in the C. destructivum
complex.
Our conidia measurements differ from those given in the
study of Barimani et al. (2013). In that study, conidia on pyrethrum tissue measured on average 30.9 × 5.6 μm, and those on
SNA, 22.5 × 4.1 μm. In contrast, conidia of the same strain on
SNA measured in our study on average 16.0 × 3.8 μm.
This fungus formed perithecia in a mating experiment and is
apparently heterothallic (Barimani et al. 2013). The sexual morph
was described by Barimani et al. (2013) as follows “Perithecia
dark brown, ampulliform with setaceous hairs in ostiole,
becoming erumpent through the epidermis, perithecia ostiolate
measuring 33 × 31 μm in diameter, individual locules measuring
200 × 380 μm (length × width), thick-walled texture. Asci
89.6 ± 2.9 × 10.9 ± 0.4 μm (n = 30), unitunicate, thin-walled,
clavate or cymbiform, stipitate, 8–10 spored. Ascospores
(18–)21.5–22.5(–26.5) × (4–)5.5–6(–7) μm (n > 50),
av. ± SD = 22 ± 1.7 × 5.8 ± 0.7 μm, one-celled, hyaline, smooth,
becoming septate through germination, fusiform and blunt at
both ends (widest at middle and narrower at the ends) or widest
at middle and upper third, many formed within 2 months.”
Barimani et al. (2013) also studied the infection strategy,
which they suggested to be intracellular hemibiotrophic, similar to
that of C. destructivum and C. higginsianum.
Colletotrichum utrechtense Damm, sp. nov. MycoBank
MB809404. Fig. 14.
Fig. 14. Colletotrichum utrechtense (from ex-holotype strain CBS 130243). A–B. Conidiomata. C, F. Tip of a seta. D, H–J. Conidiophores. E. Bases of setae and conidiophores. G. Base of a seta. K–P. Appressoria. Q–R. Conidia. A, C–E, Q. from Anthriscus stem. B, F–P, R. from SNA. A–B. DM, C–R. DIC, Scale bars: A = 100 μm,
D = 10 μm. Scale bar of A applies to A–B. Scale bar of D applies to C–R.
77
DAMM
ET AL.
Etymology: The species epithet is derived from the place where it
was collected, Utrecht, the Netherlands.
hyphae 1–7.5 μm diam, hyaline to pale brown, smooth-walled,
septate, branched. Chlamydospores not observed. Conidiomata absent, conidiophores and setae formed directly on
hyphae. Setae medium brown, smooth-walled to finely verruculose, 95–180 μm long, 2–5-septate, base cylindrical
to ± inflated, 3–6.5 μm diam, tip ± rounded to slightly acute.
Conidiophores hyaline to pale brown, smooth-walled, septate,
brown, smooth-walled, cylindrical to ± inflated, 13–26
× 3–4.5 μm, opening 1–1.5 μm diam, collarette 1–1.5 μm long,
periclinal thickening distinct. Conidia hyaline, smooth-walled,
aseptate, straight to slightly curved, with both ends ± rounded,
17.5–20.5(–23) × 3.5–4(–4.5) μm, av. ± SD = 19.0
± 1.4 × 4.0 ± 0.2 μm, L/W ratio = 4.8. Appressoria single,
sometimes in clusters of two, medium brown, smooth-walled,
navicular, ellipsoidal or irregular in outline, with an lobate or
undulate margin, (7–)10–14.5(–15) × (5–)6.5–9.5(–10) μm,
av. ± SD = 12.2 ± 2.1 × 8.0 ± 1.5 μm, L/W ratio = 1.5,
appressoria of strain CBS 135827 smaller, measuring (6.5–)
7.5–13.5(–19) × (3.5–)4.5–7(–9) μm, av. ± SD = 10.5 ± 3.0
× 5.7 ± 1.3 L/W μm, ratio = 1.8.
Asexual morph on Anthriscus stem. Conidiomata absent,
conidiophores and setae formed directly on hyphae, or rarely on
pale brown, angular cells, 3.5–8 μm diam. Setae medium brown,
basal cell pale brown, smooth-walled to finely verruculose,
75–255 μm long, 2–4-septate, base ± inflated or cylindrical,
3.5–8.5 μm diam, tip slightly rounded to slightly acute. Conidiophores hyaline to pale brown, smooth-walled, simple or
septate and branched, to 20 μm long. Conidiogenous cells hyaline to pale brown, smooth-walled, ellipsoidal to cylindrical,
9–17 × 4–5.5 μm, opening 1–2 μm diam, collarette 1–2 μm
long, periclinal thickening distinct. Conidia hyaline, smoothwalled, aseptate, straight to slightly curved, with both
ends ± rounded, (16.5–)18–20(–21.5) × 3.5–4 μm,
av. ± SD = 19.0 ± 1.0 × 3.7 ± 0.2 μm, L/W ratio = 5.2.
hyaline, pale cinnamon in the centre, filter paper partly pale
olivaceous grey, Anthriscus stem partly covert with filty white
aerial mycelium, reverse same colours; 20–24 mm in 7 d
(35–36.5 mm in 10 d). Colonies on OA flat with entire margin;
buff, pale cinnamon, pale olivaceous grey to olivaceous grey,
with few patches of floccose, whitish, aerial mycelium, reverse
same colours, 22.5–25 mm in 7 d (33.5–39 mm in 10 d). Conidia
in mass whitish to very pale salmon.
Materials examined: Netherlands, Utrecht, from a leaf of Trifolium pratense, 13
Jun. 2011, U. Damm (CBS H-21662 holotype, culture ex-holotype CBS 130243);
Utrecht, from a leaf of T. pratense, 13 Jun. 2011, U. Damm, culture CBS 135827;
Utrecht, from a leaf of T. pratense, 13 Jun. 2011, U. Damm, culture CBS 135828.
Notes: This species is only known from Trifolium pratense in the
Netherlands. Other Colletotrichum species described from this
host are reviewed in the notes under C. destructivum.
The CHS-1, HIS3 and TUB2 sequences are different from all
species included. The ACT sequences are the same as that of
C. panacicola; ITS and GAPDH distinguishes the species from
C. panacicola but the ITS is identical with the unnamed isolates
78
from Heracleum, while the GAPDH sequence is the same as that of
C. higginsianum and the isolates from Heracleum and Matthiola.
In blastn searches the ITS and GAPDH sequences of strain
CBS 130243 were found to be identical to the ITS sequence of
“C. coccodes” strain BBA 71527 from Lupinus in Germany (GenBank AJ301984, Nirenberg et al. 2002) and the GAPDH sequences
of C. higginsianum isolates C97027 and C97031 from Brassica and
Raphanus probably from Korea (GenBank GU935850, GU935851,
Choi et al. 2011). Closest matches in blastn searches with the TUB2
sequences of strain CBS 130243 with 99 % identity (3 nucleotides
different) were C. fuscum CBS 130.57 (GenBank JQ005846,
O'Connell et al. 2012) and Colletotrichum isolates from a study on
ramie (Boehmeria nivea) anthracnose in China (GenBank
JF811024–JF811028, W.X. Xia, unpubl. data).
Colletotrichum vignae Damm, sp. nov. MycoBank
MB809405. Fig. 15.
name Vigna.
conidiophores and setae formed directly on hyphae. Setae hyaline to very pale brown, smooth-walled, wall up to 0.8 μm wide,
3–4.5 μm diam, tip rounded to ± acute. Conidiophores hyaline,
sometimes pale brown, smooth-walled, septate, branched, to
35 μm long. Conidiogenous cells hyaline, sometimes pale brown,
smooth-walled, cylindrical, 12–25 × 3–5 μm, polyphialides
observed, opening 1–1.5 μm diam, collarette 0.5–2 μm long,
periclinal thickening sometimes observed. Conidia hyaline,
smooth-walled, aseptate, old conidia sometimes septate, cylindrical, straight to slightly curved, with one end round and the
other truncate, (12–)14–17.5(–18.5) × (3–)3.5–4(–4.5) μm,
av. ± SD = 15.8 ± 1.6 × 3.8 ± 0.3 μm, L/W ratio = 4.2.
Appressoria not observed on the undersurface of the medium.
Appressoria-like structures that possibly function as chlamydospores were observed within the medium. These are single or in
dense clusters, medium brown, smooth-walled, ellipsoidal, subglobose to clavate outline, with an entire or undulate margin,
because not attached to any surface (4–)4.5–8.5(–12.4)
× (3.5–)4–5(–6.5) μm, av. ± SD = 6.6 ± 2.0 × 4.6 ± 0.6 μm, L/W
ratio = 1.4.
2.5–8 μm diam. Setae pale to medium brown, smooth-walled to
verruculose, very thick-walled (up to 1.5 μm wide), 40–120 μm
long, 1–3-septate, base conical to cylindrical, 5–8.5 μm diam, tip
rounded to slightly acute. Conidiophores hyaline, smooth-walled,
septate, branched, to 60 μm long. Conidiogenous cells hyaline to
pale brown, smooth-walled, cylindrical, 8–35 × 3.5–4(–6.5) μm,
opening 1–1.5 μm diam, collarette 0.5–1 μm long, periclinal
thickening visible. Conidia hyaline, smooth-walled, aseptate,
cylindrical, straight to slightly curved, with one end round to
slightly acute and the other truncate, (10–)12–16.5(–21.5)
× 3.5–4 μm, av. ± SD = 14.2 ± 2.2 × 3.8 ± 0.2 μm, L/W ratio = 3.8. conidia of strain IMI 334960 are shorter, measuring (8–)
9–13(–15.5) × (3.5–)4–4.5(–5) μm, av. ± SD = 11.0 ± 1.8
× 4.3 ± 0.3 μm, L/W ratio = 2.6.
THE COLLETOTRICHUM
Fig. 15. Colletotrichum vignae (from ex-holotype strain CBS 501.97). A–B. Conidiomata. C, G. Tip of a seta. D–E. Conidiophores. F. Base of a seta and conidiophores. H.
Base of a seta. I–L. Conidiophores. M–R. Appressorium-like structures. S–T. Conidia. A, C–F, S. from Anthriscus stem. B, G–R, T. from SNA. A–B. DM, C–T. DIC, Scale
bars: A = 100 μm, F = 10 μm. Scale bar of A applies to A–B. Scale bar of F applies to C–T.
covered with saffron to cinnamon acervuli, aerial mycelium
lacking, reverse same colours; growth 12.5–15 mm in 7 d
(19–22.5 mm in 10 d). Colonies on OA flat with entire margin;
honey to cinnamon, with a buff margin, aerial mycelium lacking,
reverse same colours; growth 13.5–15 mm in 7 d (20–21.5 mm
in 10 d). Colours and growth rate of strain IMI 334960 differed on
OA by being dark grey olivaceous, partly covered with short
white aerial mycelium, reveres dark grey olivaceous to olivaceous grey; growth 17–18.5 mm in 7 d (24.5–26 mm in 10 d).
Conidia in mass saffron.
Materials examined: Nigeria, from Vigna unguiculata, collection date unknown
(deposited in CBS collection Feb. 1997 by J.A. Bailey, isolated by R.A. Skipp, No.
I 57), R.A. Skipp (CBS H-21648 holotype, culture ex-type CBS 501.97 = LARS
56); from Vigna unguiculata, collection date and collector unknown, culture IMI
334960 = CPC 19383.
Notes: The isolates studied here apparently originate from a
study on cowpea diseases in Nigeria by Williams (1975), who
sent isolates to IMI where they were identified as
C. lindemuthianum. In contrast to the species studied here,
C. lindemuthianum belongs to the C. orbiculare species complex
(Damm et al. 2013, Liu et al. 2013a). Judging from information
retrieved from Bailey et al. (1990) these two strains originated
from the same isolate.
Bailey et al. (1990) observed the single-cell hemibiotrophic
infection of cowpea by C. lindemuthianum from cowpea (= C.
vignae) for the first time. They also revealed the morphology,
pathogenicity and host specificity of strain I57 (= LARS 56 = CBS
501.97) to be different from C. lindemuthianum isolates from
Phaseolus vulgaris. Latunde-Dada et al. (1996, 1999) studied the
infection of cowpea by the same strain and by strain LARS 860,
another strain from cowpea in Nigeria. They identified the species as C. destructivum based on similarity of morphological
features and the ITS2-D2 sequences with isolates from Medicago that were confirmed as C. destructivum s. str. in our study.
However, based on the ITS2-D2 phylogeny of the second study
strain, LARS 860 is a different species to LARS 56 (C. vignae),
not belonging to the C. destructivum complex and more closely
related to C. gloeosporioides (s. lat.) strains; the infection process differs considerably and is not hemibiotrophic.
Takimoto (1934) described C. phaseolorum from Vigna
angularis and V. sinensis in Japan. The type of this species was
not designated. Authentic isolates from the two hosts studied by
Damm et al. (2009) are not conspecific, but neither is closely
related to C. vignae. In contrast to C. vignae, this species forms
distinctly curved conidia. Further isolates from Vigna, from
V. unguiculata in Burkina Faso and V. sinensis in Pakistan,
respectively, were recently identified as C. truncatum in the same
multilocus analyses (Damm et al. 2009). Colletotrichum phaseolorum was treated as a synonym of C. gloeosporioides by
79
DAMM
ET AL.
von Arx (1957, wrongly cited as C. phascorum). Shen et al.
(2010) reported anthracnose of mung bean (V. radiata) sprouts
to be caused by C. acutatum (s. lat.) in Taiwan.
Glomerella vignicaulis was described by Tehon (1937) on
Vigna sinensis in Illinois, USA. Tehon never found an asexual
Colletotrichum morph on the host; however, a Cercospora stage
accompanying the perithecia that appeared to arise from the
same mycelium was always observed. If Ga. vignicaulis is a
Colletotrichum species at all, it is unlikely to belong to the
C. destructivum species complex, as in this complex conidia are
dominating; none of the species is known to form a sexual morph
in nature. The two species that are known to form a sexual stage,
C. lentis (= Ga. truncata) and C. tanaceti, are apparently heterothallic and sexual morphs were only observed by crossing
experiments in the laboratory (Armstrong-Cho & Banniza 2006,
Barimani et al. 2013).
Colletotrichum vignae was one of the slowest growing species studied in the C. destructivum complex and the slowest
growing species within the first main clade (Fig. 1). Conidia of
C. vignae are highly variable in length; the few setae observed
were pale brown and thick-walled.
This species can be identified by its ITS, GAPDH, HIS3 and
ACT sequences. Blastn searches with the respective sequences
of strain CBS 501.97 resulted in 99 % identity (a single nucleotide difference) with the ITS sequence of C. fuscum strain
DAOM 216112 (GenBank EU400144; Chen et al. 2007), and
98 % identity with the GAPDH sequences of C. higginsianum
isolates C97027 and C97031 from Brassica and Raphanus,
respectively, probably from Korea (GenBank GU935870 and
GU935873; Choi et al. 2011), and 99 % identity (2 nucleotides
difference) with the HIS3 sequence of C. higginsianum isolate
MAFF 305635 (GenBank JQ005803; O'Connell et al. 2012,
included in this study) and 99 % identity (a single nucleotide
difference) with the ACT sequences of C. fuscum CBS 130.57
(GenBank JQ005825; O'Connell et al. 2012, included in this
study), respectively. The CHS-1 sequences are the same as
those of C. fuscum, C. higginsianum, C. antirrhinicola and the
unnamed isolates from Heracleum and Matthiola.
Sun & Zhang (2009) isolated Colletotrichum from anthracnose lesions on leaves of cowpea in China that they identified as
C. destructivum based on morphology. As the ITS sequences
were the same as those from cruciferous hosts, they concluded
C. higginsianum to be a synonym of C. destructivum. The ITS
sequence from those strains, however, differed in 4 nucleotides
from those of C. vignae.
DISCUSSION
Previous multilocus phylogenies have shown the C. destructivum
species complex was monophyletic, and sister to the combined
C. graminicola and C. spaethianum complexes (Cannon et al.
2012, O'Connell et al. 2012). Based on a multilocus phylogeny
including a large number of isolates from various host plants, we
differentiated several distinct species.
While, C. destructivum, C. lini and C. fuscum are regarded as
separate species, von Arx (1957) listed C. higginsianum and
C. tabacum as synonyms of C. gloeosporioides. However, these
species are not closely related to C. gloeosporioides that belongs
to a different species complex within the genus, and all five were
regarded as distinct species in this study.
80
One characteristic morphological feature of the C. destructivum
species complex is the conidia that are slightly curved due to their
unilaterally tapering ends, which is apparent in most of the species.
However, some species are distinctly curved (C. pisicola, C.
tanaceti), while others are almost straight (especially C. tabacum
and C. ocimi) and reminiscent of C. coccodes or
C. gloeosporioides. The variation between almost straight and
curved conidia in this species complex was one of the reasons for
some isolates having been confused with species belonging to
other species complexes, e.g. Ga. glycines, C. coccodes,
C. truncatum, C. gloeosporioides, C. lindemuthianum or C. trifolii.
Another typical characteristic is the small inconspicuous acervuli
with rather effuse growth that are sometimes difficult to spot on the
host plant. Latunde-Dada & Lucas (2007) observed several species in the C. destructivum complex that formed acervuli with only a
single seta on the host plant. Setae are comparatively short, pale to
medium brown, often smooth-walled with round apices. However,
these features are variable on different culture media and large
distinct acervuli with abundant dark setae may be produced as well,
depending on species, strain, substrate and age of the culture. The
size of conidia and appressoria is also variable within species, and
usually not taxonomically informative for species differentiation.
Sexual morphs were not observed in the cultures used in this
study. As far as we know, C. destructivum s. str. does not form a
sexual morph. The sexual morph linked to it, Ga. glycines, is not
closely related to C. destructivum and belongs to a different
species complex (U. Damm, unpublished results). However,
there are two heterothallic species, C. lentis (as Ga. truncatum
by Armstrong-Cho & Banniza 2006) and C. tanaceti (Barimani
et al. 2013) that form sexual morphs by artificially crossing isolates. In contrast, many species in the C. boninense species
complex are apparently homothallic (Damm et al. 2012).
The most intensively-studied species in this complex are all
serious economic pathogens. The infection strategy of several of
them has been found to be hemibiotrophic. Using light and electron
microscopy, O'Connell et al. (1993), Bailey et al. (1990) and
Latunde-Dada et al. (1996, 1997) investigated the hemibiotrophic
infection of Pisum, Vigna and Medicago, respectively, by isolates
that are shown here to belong to three different species of the
C. destructivum complex, namely C. pisicola, C. vignae and
C. destructivum s. str. The infection processes of C. lini on flax
(Hahn 1952), C. tabacum on tobacco (Shen et al. 2001),
C. higginsianum on Arabidopsis (O'Connell et al. 2004), Ga.
truncata (re-identified here as C. lentis) on lentil (Armstrong-Cho
et al. 2012) and C. tanaceti on Tanacetum (Barimani et al. 2013)
were very similar. The characteristic feature of hemibiotrophy in all
these species is that initial penetration of the fungus by appressoria is followed by an intracellular biotrophic phase associated
with fat, bulbous primary hyphae that invaginate the plasma
membrane of living plant cells. Both the primary hyphae and the
entire biotrophic phase are confined within a single epidermal cell.
Much thinner, filamentous secondary hyphae then develop from
the tips of the primary hyphae to rapidly colonise surrounding
tissues. This morphological transition is associated with a switch to
destructive necrotrophy and the appearance of disease symptoms. The major difference in all other hemibiotrophic Colletotrichum species so far examined (e.g. pathogens from the
C. orbiculare and C. graminicola complexes) is that the primary
hyphae are less bulbous and the biotrophic phase extends into
many host cells (O'Connell et al. 1985, Wharton et al. 2001,
Crouch et al. 2014). Probably all species are hemibiotrophic in
the C. destructivum complex, but this needs confirmation.
THE COLLETOTRICHUM
Latunde-Dada & Lucas (2007) found a close relationship
among isolates of several species in the C. destructivum complex. They also demonstrated that there are three clades within
the genus Colletotrichum containing hemibiotrophic species,
which they called C. orbiculare, C. destructivum-linicola-truncatum (including wrongly identified C. truncatum strains) and
C. cereale-graminicola-sublineolum aggregates. Previously,
hemibiotrophic C. truncatum isolates from different hosts, e.g.
Pisum and Lens, were wrongly identified. These isolates belong
to species in the C. destructivum complex. In contrast,
C. truncatum (= C. capsici) is a different species that does not
belong to this complex (Cannon et al. 2012) and utilises an
infection strategy that is necrotrophic rather than hemibiotrophic
(Pring et al. 1995). More recent studies on the infection process
of C. truncatum on chili leaves and fruits using light microscopy
(Ranathunge et al. 2012) and fluorescence microscopy of
transformants expressing GFP (Auyong et al. 2012) revealed
that an initial subcuticular-intramural endophytic phase was followed by a destructive, necrotrophic phase of colonisation.
Based on the host origins of species for which a large number
of isolates were available, some species appear to be specific to
certain genera or families of herbaceous plants, for example
C. fuscum on Digitalis and C. higginsianum on Brassicaceae
(Fig. 1). In contrast, other species appear to be generalists with
broad host ranges, having been collected from taxonomically
highly divergent plant families, e.g. C. destructivum from Asteraceae, Fabaceae and Polygonaceae, and notably C. lini from
Asteraceae, Brassicaceae, Fabaceae, Lamiaceae, Linaceae and
Ranunculaceae. In contrast, most species in the C. graminicola
complex were restricted to single host species or genera (Crouch
et al. 2009, Crouch 2014). Furthermore, we found that several
host species can be attacked by more than one member of the
C. destructivum complex. For example, Medicago and Trifolium
are each attacked by three different species, while Raphanus and
Pisum are each attacked by two different species (Fig. 1). There is
much evidence that pathogen host range is determined by rapidly
evolving secreted effector proteins that facilitate infection, notably
by suppressing plant immunity (Schulze-Lefert & Panstruga
2011). Comparative genomic analyses of the effector repertoires of “specialist” and “generalist” members of the
C. destructivum complex could thus provide important insights into
the molecular basis of host range within this fungal clade.
Host range has been considered an unambiguous criterion for
delimiting two species (for example Sun & Zhang, 2009). However, the results of pathogenicity tests with species from the
C. destructivum complex are often contradictory. In laboratory
assays with C. higginsianum, Higgins (1917) observed abundant
infection of turnip (Brassica rapa) and radish (Raphanus sativus),
limited leaf spotting on cabbage (Brassica oleracea capitata) and
collards (Brassica oleracea viridis) and no infection of lettuce
(Lactuca sativa). Sun & Zhang (2009) found that C. higginsianum
isolates from cowpea (Vigna unguiculata) infected Arabidopsis
thaliana and some cowpea cultivars, while other cowpea cultivars, lentil (Lens culinaris), Chinese cabbage (Brassica rapa
subsp. pekinensis), and tobacco (Nicotiana tabacum) were all
resistant. In pathogenicity tests by O'Connell et al. (2004),
legume isolates of C. destructivum were unable to infect
A. thaliana, while C. destructivum strain N150 (re-identified as
C. tabacum in this study) infected tobacco, alfalfa (Medicago
sativa), cowpea and Medicago truncatula, but not soybean
(Glycine max) (Shen et al. 2001). In contrast, Manandhar et al.
(1986) regarded C. destructivum as a soybean pathogen.
The contradictory results obtained from pathogenicity tests
may be partly attributed to variation in factors affecting the hostpathogen interaction, for example incubation conditions (humidity and temperature), and variation in the inoculation methods
used, such as detached leaves or intact host tissues. For example,
Liu et al. (2007) found that C. lini could infect detached Arabidopsis leaves but not intact plants, due to senescence of the
detached tissues, associated with impairment of salicylic acid- and
ethylene/jasmonate-dependent host defense responses. A further
problem is that isolates are frequently misidentified or only identified to species complex level. This likely explains the different
results of pathogenicity tests obtained with C. destructivum (s. lat.)
isolates from cowpea. The isolates from cowpea tested by Sun &
Zhang (2009) have ITS sequences that are identical to
C. higginsianum, while the isolate from cowpea included in the
study by O'Connell et al. (2004) is a different species and
described as C. vignae in this study (see notes under C. vignae).
Moreover, fungus-host relationships can also be endophytic in
nature. Thus, many Colletotrichum species were isolated as
symptomless endophytes, including species of the
C. destructivum complex from Holcus (Sanchez Marquez et al.
2012) and Arabidopsis (Garcia et al. 2013) in Spain and from
Rumex (Hu et al. 2012) and Bletilla (Tao et al. 2013) in China. In
conclusion, host range and pathogenicity can only provide indications of the identity of a Colletotrichum species, and should not
be used as criteria for species delimitation or identification.
ACKNOWLEDGEMENTS
We thank the curators and staff of the CABI and CBS culture collections as well as
Dr Jouji Moriwaki (National Agricultural Research Center, National Agriculture and
Food Research Organisation, Joetsu, Japan) and Prof. Dr Paul Taylor (Department
of Agriculture and Food systems, University of Melbourne, Australia) for kindly
supplying isolates for this study. We kindly thank the curators of the fungaria at the
US National Fungus Collections, Beltsville, Maryland, USA, at the Royal Botanic
Gardens in Kew, UK, at the Botanic Garden and Botanical Museum Berlin-Dahlem,
Freie Universit€at Berlin, Berlin, for providing access to and Prof. Dr Viorica C. Iacob
(Herbarium Fitopatologie, Institutul Agronomic, Iaşi, Romania) for donating historical type specimens. Joyce Woudenberg (CBS-KNAW Fungal Biodiversity
Centre, Utrecht, The Netherlands) is thanked for assistance with some of the
molecular data generated in this project. Dr Joost Stalpers (CBS-KNAW Fungal
Biodiversity Centre, Utrecht, The Netherlands) is thanked for providing valuable
nomenclatural advice, Dr Toyozo Sato (National Institute of Agrobiological Sciences, Tsukuba, Japan) for important name and reference corrections and Prof. Dr
Uwe Braun, (Martin-Luther-Universit€at Halle-Wittenberg, Institut für Geobotanik
und Botanischer Garten, Germany) for verifying the Latin names. This research
was supported by the Dutch Ministry of Agriculture, Nature and Food Quality
through an endowment of the FES programme “Versterking infrastructuur Plantgezondheid” and “Making the Tree of Life Work”.
REFERENCES
Armstrong-Cho C, Wang J, Wei Y, et al. (2012). The infection process of two
pathogenic races of Colletotrichum truncatum on lentil. Canadian Journal of
Armstrong-Cho CL, Banniza S (2006). Glomerella truncata sp. nov., the teleomorph of Colletotrichum truncatum. Mycological Research 110: 951–956.
Arx JA von (1957). Die Arten der Gattung Colletotrichum Cda. Phytopathologische Zeitschrift 29: 413–468.
Arx JA von, Müller E (1954). Die Gattungen der amerosporen Pyrenomyceten.
Beitr€age zur Kryptogamenflora der Schweiz 11(1): 1–434.
Averna-Sacca R (1922). Algumas dos malestias cryptogamicas do tobaco
(Nicotiana tabacum). Boletim de Agricultura, S. Paulo 23: 201–268.
Auyong ASM, Ford R, Taylor PWJ (2012). Genetic transformation of Colletotrichum truncatum associated with anthracnose disease of chili by random
insertional mutagenesis. Journal of Basic Microbiology 52: 372–382.
81
DAMM
ET AL.
Bailey JA, Nash C, O'Connell RJ, et al. (1990). Infection process and host
specificity of a Colletotrichum species causing anthracnose disease in
cowpea, Vigna unguiculata. Mycological Research 94: 810–814.
Bailey JA, O'Connell RJ, Pring RJ, et al. (1992). Infection strategies of Colletotrichum species. In: Colletotrichum – biology, pathology and control
(Bailey JA, Jeger MJ, eds). CAB International, Wallingford, UK: 88–121.
Bain SM, Essary SH (1906). A new anthracnose of alfalfa and red clover.
Journal of Mycology 12: 192–193.
Barimani M, Pethybridge SJ, Vaghefi N, et al. (2013). A new anthracnose disease of pyrethrum caused by Colletotrichum tanaceti sp. nov. Plant Pathology 62: 1248–1257.
Barksdale TH (1972). Resistance in tomato to six anthracnose fungi. Phytopathology 62: 660–663.
Baxter AP, Westhuizen GCA van der, Eicker A (1983). Morphology and taxonomy of South African isolates of Colletotrichum. South African Journal of
Botany 2: 259–289.
Berner DK, Eskandari FM, Rossman AY, et al. (2004). First report of anthracnose of Crupina vulgaris caused by a Colletotrichum sp. in Greece. Plant
Disease 88: 1161.
Bhadauria V, Banniza S, Vandenberg A, et al. (2011). EST mining identifies
proteins putatively secreted by the anthracnose pathogen Colletotrichum
truncatum. BMC Genomics 12: 327.
Bhadauria V, Banniza S, Vandenberg A, et al. (2012). Overexpression of a novel
biotrophy-specific Colletotrichum truncatum effector CtNUDIX in hemibiotrophic fungal phytopathogens causes incompatibility with their host
plants. Eukaryotic Cell 12: 2–11.
Birker D, Heindrich K, Takahara H, et al. (2009). A locus conferring resistance to
Colletotrichum higginsianum is shared by four geographically distinct Arabidopsis accessions. The Plant Journal 60: 602–613.
Boland GJ, Brochu LD (1989). Colletotrichum destructivum on alfalfa in Ontario
and cultivar response to anthracnose. Canadian Journal of Plant Pathology
11: 303–307.
Bolley HL (1910). Seed desinfection and crop production. North Dakota
Experiment Station Bulletin 87: 130–166.
Bolley HL, Manns TF (1932). Fungi of flaxseed and flaxsick soil. North Dakota
Experiment Station Bulletin 259: 1–57.
Böning K (1929). Die Brennfleckenkrankheit des Tabaks. Praktische Bl€atter für
Pflanzenbau und Pflanzenschutz 7: 36–40.
Böning K (1932). Die Bek€ampfung der Brennfleckenkrankheit des Tabaks
(Colletotrichum tabacum) durch Beizung des Samens und vorbeugende
Behandlung der Pflanzen mit chemischen Mitteln. Praktische Bl€atter für
Pflanzenbau und Pflanzenschutz 10: 89–106.
Böning K (1933). Über eine zweite Brennfleckenkrankheit des Tabaks, hervorgerufen durch einen Pilz aus der Gattung Gloeosporium Desmaz. et
Mont. Praktische Bl€atter für Pflanzenbau und Pflanzenschutz 10:
253–255.
Buchwaldt L, Anderson KL, Morrall RAA, et al. (2004). Identification of lentil
germplasm resistant to Colletotrichum truncatum and characterization of two
pathogen races. Phytopathology 94: 236–243.
Cannon PF, Damm U, Johnston PR, et al. (2012). Colletotrichum – current
status and future directions. Studies in Mycology 73: 181–213.
Chen N, Goodwin PH, Hsiang T (2003). The role of ethylene during the infection
of Nicotiana tabacum by Colletotrichum destructivum. Journal of Experimental Botany 54: 2449–2456.
Chen YY, Conner RL, Gillard CL, et al. (2007). A specific and sensitive method
for the detection of Colletotrichum lindemuthianum in dry bean tissue. Plant
Disease 91: 1271–1276.
Choi KJ, Kim WG, Kim HG, et al. (2011). Morphology, molecular phylogeny and
pathogenicity of Colletotrichum panacicola causing anthracnose of Korean
ginseng. The Plant Pathology Journal 27: 1–7.
Corredor AH, Rees K van, Vujanovic V (2012). Changes in root-associated
fungal assemblages within newly established clonal biomass plantations
of Salix spp. Forest Ecology and Management 282: 105–114.
Crouch JA (2014). Colletotrichum caudatum s.l. is a species complex. IMA
Fungus 5: 17–30.
Crouch JA, Clarke BB, White JF, et al. (2009). Systematic analysis of the
falcate-spored graminicolous Colletotrichum and a description of six new
species from warm season grasses. Mycologia 101: 717–732.
Crouch JA, O'Connell R, Gan P, et al. (2014). The genomics of Colletotrichum.
In: Genomics of plant-associated fungi and oomycetes: monocot pathogens
(Dean R, Lichens-Park A, Kole C, eds). Springer-Verlag, Berlin, Heidelberg,
Germany: 69–102.
82
Crous PW, Gams W, Stalpers JA, et al. (2004a). MycoBank: an online initiative
to launch mycology into the 21st century. Studies in Mycology 50: 19–22.
Crous PW, Groenewald JZ, Risede JM, et al. (2004b). Calonectria species and
their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415–430.
Crous PW, Verkleij GJM, Groenewald JZ, et al. (eds) (2009). Fungal biodiversity.
CBS laboratory manual series 1. Centraalbureau voor Schimmelcultures,
Utrecht, Netherlands.
Damm U, Cannon PF, Liu F, et al. (2013). The Colletotrichum orbiculare species
complex: important plant pathogens of field crops and weeds. Fungal Diversity 61: 29–59.
Damm U, Cannon PF, Woudenberg JHC, et al. (2012). The Colletotrichum
boninense species complex. Studies in Mycology 73: 1–36.
Damm U, Crous PW, Fourie PH (2007). Botryosphaeriaceae as potential
pathogens of Prunus species in South Africa, with descriptions of Diplodia
africana and Lasiodiplodia plurivora spp. nov. Mycologia 99: 664–680.
Damm U, Mostert L, Crous PW, et al. (2008). Novel Phaeoacremonium species
associated with necrotic wood of Prunus trees. Persoonia 20: 87–102.
Damm U, Woudenberg JHC, Cannon PF, et al. (2009). Colletotrichum species
with curved conidia from herbaceous hosts. Fungal Diversity 39: 45–87.
Dickson JG (1956). Diseases of field crops, 2nd edn. McGraw-Hill, New York,
USA.
Djebali N, Scott JK, Jourdan M, et al. (2009). Fungi pathogenic on wild radish
(Raphanus raphanistrum L.) in northern Tunisia as potential biocontrol
agents. Phytopathologia Mediterranea 48: 205–213.
Ellis JB, Kellerman WA (1887). New Kansas fungi. Journal of Mycology 3:
102–105.
Farr DF, Rossman AY (2014). Fungal Databases, Systematic Mycology and
Microbiology Laboratory, ARS, USDA. Retrieved May 13, 2014, from: http://
nt.ars-grin.gov/fungaldatabases/.
Ferraris T, Massa C (1912). Micromiceti nuovi o rari per la flora micologica
Italiana. Nota prima. Annales Mycologici 10(3): 285–302.
Ford R, Banniza S, Photita W, et al. (2004). Morphological and molecular
discrimination of Colletotrichum truncatum causing anthracnose on lentil in
Canada. Australasian Plant Pathology 33: 559–569.
Forseille L (2007). Molecular and pathological differentiation of Colletotrichum
truncatum from scentless chamomile and legume crops. Thesis (M.Sc.).
Biology Department, Univ. of Saskatchewan, Canada, Saskatchewan,
Saskatoon, 122 pp.
Forseille L, Peng G, Gossen BD, et al. (2009). Further evidence for host
specificity of Colletotrichum truncatum from scentless chamomile. Canadian
Journal of Plant Pathology 31: 301–808.
Garcia E, Alonso A, Platas G, et al. (2013). The endophytic mycobiota of
Arabidopsis thaliana. Fungal Diversity 60: 71–89.
Gardes M, Bruns TD (1993). ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts.
Molecular Ecology 2: 113–118.
Glass NL, Donaldson G (1995). Development of primer sets designed for use
with PCR to amplify conserved genes from filamentous ascomycetes.
Applied and Environmental Microbiology 61: 1323–1330.
Gohbara M, Hyeon S-B, Suzuki A, et al. (1976). Isolation and structure elucidation of colletopyrone from Colletotrichum nicotianae. Agricultural and
Biological Chemistry 40: 1453–1455.
Gohbara M, Kosuge Y, Yamasaki S, et al. (1978). Isolation, structures and
biological activities of colletotrichins, phytotoxic substances from Colletotrichum nicotianae. Agricultural and Biological Chemistry 42: 1037–1043.
Goodman RN (1960). Colletotin, a toxin, produced by Colletotrichum fuscum.
Phytopathological Notes 50: 325–327.
Gossen BD, Anderson KL, Buchwaldt L (2009). Host specificity of Colletotrichum
truncatum from lentil. Canadian Journal of Plant Pathology 31: 65–73.
Goto K (1938). Anthracnose of Digitalis caused by Colletotrichum fuscum
Laubert. Annals of the Phytopathological Society of Japan 8: 1–8.
Greenhill M (2007). Pyrethrum production: Tasmanian – success story. Chronica
Horticulturae 47: 5–8.
Guerber JC, Liu B, Correll JC, et al. (2003). Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns,
mtDNA and intron RFLPs, and mating compatibility. Mycologia 95: 872–895.
Gullino ML, Garibaldi A, Minuto G (1995). First report of ‘black spot’ of basil
incited by Colletotrichum gloeosporioides in Italy. Plant Disease 79: 539.
Hagedorn DJ (1974). Recent pea anthracnose and downy mildew epiphytotics in
Wisconsin. Plant Disease Reporter 58: 226–229.
Hahn H (1952). Das Verhalten resistenter und anf€alliger Leinsorten gegenüber
Colletotrichum lini Manns et Bolley. Phytopathologische Zeitschrift 20:
83–88.
THE COLLETOTRICHUM
Hawksworth DL, McNeill J, de Beer ZW, et al. (2013). Names of fungal species
with the same epithet applied to different morphs: how to treat them. IMA
Fungus 4: 53–56.
Hemmi T (1921). Nachtr€age zur Kenntnis der Gloeosporien. Journal of the
College of Agriculture, Hokkaido Imperial University 9: 305–346.
Higgins BB (1917). A Colletotrichum leafspot of turnips. Journal of Agricultural
Research Washington 10: 157–162.
Hillis DM, Bull JJ (1993). An empirical test of bootstrapping as a method for
assessing confidence in phylogenetic analysis. Systematic Biology 42:
182–192.
Horn NL (1952). A comparative study of two species of Colletotrichum on Vetch.
Phytopathology 42: 670–674.
Hu F, Hou SY, Gao N, et al. (2012). Distribution of endophytic fungi from Rumex
gmelini and similarity analysis of chemical components of the dominant flora.
Academic Journal of Guangdong College of Pharmacy 28: 387–391.
Huser A, Takahara H, Schmalenbach W, et al. (2009). Discovery of pathogenicity genes in the crucifer anthracnose fungus, Colletotrichum higginsianum, using random insertional mutagenesis. Molecular Plant-Microbe
Interactions 22: 143–156.
Kawaradani M, Nishimura A, Moriwaki J, et al. (2008). Anthracnose of Perilla
caused by Colletotrichum destructivum. Japanese Journal of Phytopathology
74: 335–339 (in Japanese).
Kirk PM, Spooner BM (1984). An account of the fungi of Arran, Gigha and
Kintyre. Kew Bulletin 38: 503–597.
Kleemann J, Rincon-Rivera LJ, Takahara H, et al. (2012). Sequential delivery of
host-induced virulence effectors by appressoria and intracellular hyphae of the
phytopathogen Colletotrichum higginsianum. PLoS Pathogens 8(4): e1002643.
Latunde-Dada AO, Bailey JA, Lucas JA (1997). Infection process of Colletotrichum destructivum O'Gara from lucerne (Medicago sativa L.). European
Journal of Plant Pathology 103: 35–41.
Latunde-Dada AO, Lucas JA (2007). Localized hemibiotrophy in Colletotrichum:
cytological and molecular taxonomic similarities among C. destructivum,
C. linicola and C. truncatum. Plant Pathology 56: 437–447.
Latunde-Dada AO, O'Connell RJ, Nash C, et al. (1999). Stomatal penetration of
cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing
latent anthracnose. Plant Pathology 48: 777–785.
Latunde-Dada AO, O'Connell RJ, Nash C, et al. (1996). Infection process and
identity of the hemibiotrophic anthracnose fungus (Colletotrichum destructivum) from cowpea (Vigna unguiculata). Mycological Research 100:
1133–1141.
Laubert R (1927). Ein neuer Digitalis-Sch€adling. Gartenwelt 31: 674–675.
Lehman SG, Wolf FA (1926). Soy-bean anthracnose. Journal of Agricultural
Research 33: 381–390.
Liu F, Cai L, Crous PW, et al. (2013a). Circumscription of the anthracnose
pathogens Colletotrichum lindemuthianum and C. nigrum. Mycologia 105:
844–860.
Liu F, Cai L, Crous PW, et al. (2014). The Colletotrichum gigasporum species
complex. Persoonia 33: 83–97.
Liu F, Hyde KD, Cai L (2011). Neotypification of Colletotrichum coccodes, the
causal agent of potato black dot disease and tomato anthracnose. Mycology
2: 248–254.
Liu G, Kennedy R, Greenshields DL, et al. (2007). Detached and attached
Arabidopsis leaf assays reveal distinctive defense responses against
hemibiotrophic Colletotrichum spp. Molecular Plant-Microbe Interactions 20:
1308–1319.
Liu LP, Zhao D, Zheng L, et al. (2013b). Identification of virulence genes in the
crucifer anthracnose fungus Colletotrichum higginsianum by insertional
mutagenesis. Microbial Pathogenesis 64: 6–17.
Lucas GB, Shew DH (1991). Anthracnose. In: Compendium of tobacco diseases
(Shew DH, Lucas GB, eds). APS Press, St. Paul, Minnesota, USA: 12.
Maire RCJE (1917). Champignons nord-Africains nouveaux ou peu connus.
Bulletin de la Societe d'Histoire Naturelle de l'Afrique de Nord 8: 134–200.
Manandhar JB, Hartman GL, Sinclair JB (1986). Colletotrichum destructivum,
the anamorph of Glomerella glycines. Phytopathology 76: 282–285.
Mason-Gamer RJ, Kellogg EA (1996). Testing for phylogenetic conflict among
molecular data sets in the tribe Triticeae (Gramineae). Systematic Biology
45: 524–545.
McNeill J, Barrie FR, Buck WR, et al. (eds) (2012). International Code of
Nomenclature for algae, fungi, and plants (Melbourne Code). Koeltz Scientific Books, Königstein, Germany [Regnum vegetabile no. 154.].
McPartland J, Hosoya T (1997). Species of Colletotrichum on ginseng (Panax).
Mycotaxon 67: 3–8.
Menat J, Cabral AL, Vijayan P, et al. (2012). Glomerella truncata: another
Glomerella species with an atypical mating system. Mycologia 104: 641–649.
Moesz G (1931). Mycologiai Közlemenyek VIII. Közlemeny. Botanikai
Közlemenyek 28: 161–174.
Moriwaki J, Tsukiboshi T, Sato T (2002). Grouping of Colletotrichum species in
Japan based on rDNA sequences. Journal of General Plant Pathology 68:
307–320.
Morrall RAA (1988). A new disease of lentil induced by Colletotrichum truncatum
in Manitoba. Plant Disease 72: 994.
Nakata K, Takimoto S (1922). Studies on ginseng diseases in Korea. Research
Bulletin of the Encouragement Exemplary Station 5: 1–81 (in Japanese).
Naumann TA, Wicklow DT (2013). Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens. Physiological and Molecular Plant Pathology 82: 1–9.
Differenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem 169:
1–117.
Nirenberg HI, Feiler U, Hagedorn G (2002). Description of Colletotrichum lupini
comb. nov. in modern terms. Mycologia 94: 307–320.
Nylander JAA (2004). MrModeltest v2. Program distributed by the author.
Evolutionary Biology Centre, Uppsala University.
O'Connell RJ, Uronu AB, Waksman G, et al. (1993). Hemibiotrophic infection of
Pisum sativum by Colletotrichum truncatum. Plant Pathology 42: 774–783.
O'Connell R, Herbert C, Sreenivasaprasad S, et al. (2004). A novel ArabidopsisColletotrichum pathosystem for the molecular dissection of plant-fungal interactions. Molecular Plant-Microbe Interactions 17: 272–282.
O'Connell RJ, Bailey JA, Richmond DV (1985). Cytology and physiology of
infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiological Plant Pathology 27: 75–98.
O'Connell RJ, Thon MR, Hacquard S, et al. (2012). Life-style transitions in plant
pathogenic Colletotrichum fungi deciphered by genome and transcriptome
analyses. Nature Genetics 44: 1060–1065.
O'Donnell K, Cigelnik E (1997). Two divergent intragenomic rDNA ITS2 types
within a monophyletic lineage of the fungus Fusarium are nonorthologous.
Molecular Phylogenetics and Evolution 7: 103–116.
O'Gara PJ (1915). New species of Colletotrichum and Phoma. Mycologia 7:
38–41.
Patel JS, Costa de Novaes MI, Zhang S (2014). First report of Colletotrichum
higginsianum causing anthracnose of arugula (Eruca sativa) in Florida. Plant
Disease 98: 1269.
Patouillard NT, Lagerheim G von (1891). Champignons de l'equateur. Bulletin de
la Societe Mycologique de France 7: 158–184.
Peck CH (1879, publ. 1883). Report of the Botanist (1879). Annual Report on
the New York State Museum of Natural History 33: 11–49.
Peng G, Bailey KL, Hinz HL, et al. (2005). Colletotrichum sp: A potential
candidate for biocontrol of scentless chamomile (Matricaria perforata) in
western Canada. Biocontrol Science and Technology 15: 487–511.
Pethybridge GH, Lafferty HA (1918). A disease of flax seedlings caused by a
species of Colletotrichum, and transmitted by infected seed. Scientific
Proceedings of the Royal Dublin Society 15: 359–384.
Petrak F (1953). List of new species and varieties of fungi, new combinations
and new names published 1922–1928. Commonwealth Mycological Institute, Kew, Surrey, UK.
Pring RJ, Nash C, Zakaria M, et al. (1995). Infection process and host range of
Colletotrichum capsici. Physiological and Molecular Plant Pathology 46:
137–152.
Rakotoniriana EF, Munaut F, Decock C, et al. (2008). Endophytic fungi from
leaves of Centella asiatica: occurrence and potential interactions within
leaves. Antonie van Leeuwenhoek 93: 27–36.
Rakotoniriana EF, Scauflaire J, Rabemanantsoa C, et al. (2013). Colletotrichum
gigasporum sp. nov., a new species of Colletotrichum producing long
straight conidia. Mycological Progress 12: 403–412.
Rambaut A (2002). Sequence alignment editor. Version 2.0. University of Oxford,
Oxford, UK.
Ranathunge NP, Mongkolporn O, Ford R, et al. (2012). Colletotrichum truncatum
pathosystem on Capsicum spp: infection, colonization and defence mechanisms. Australasian Plant Pathology 41: 463–473.
Rayner RW (1970). A mycological colour chart. Commonwealth Mycological
Institute, Kew, UK.
Rimmer SR (2007). Anthracnose. In: Compendium of brassica diseases
(Rimmer SR, Shattuck YI, Buchwaldt L, eds). APS Press, St. Paul, Minnesota, USA: 18–19.
Rittenour WR, Ciaccio CE, Barnes CS, et al. (2013). Internal transcribed spacer
rRNA gene sequencing analysis of fungal diversity in Kansas City indoor
environments. Environmental Science: Processes & Impacts 16: 33–43.
83
DAMM
ET AL.
Rodigin MN (1928). About Gloeosporium and Macrophoma on cucurbits. Morbi
plantarum Leningrad 17: 153–154 (in Russian).
Romano A, Romano D, Ragg E, et al. (2006). Steroid hydroxylations with
Botryodiplodia malorum and Colletotrichum lini. Steroids 71: 429–434.
Ronquist F, Huelsenbeck JP (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Rost H (1938). Untersuchungen über einige Krankheiten des Leins in
Deutschland. Angewandte Botanik 20(6): 412–430.
Rostrup E (1899). Mykologiske Meddelelser. VIII. Botanisk Tidsskrift 22:
254–279.
Saccardo PA, Saccardo D, Traverso JB, et al. (1931). Sylloge fungorum: vol. 25.
Padova, Italy.
Sanchez Marquez S, Bills GF, Herrero N, et al. (2012). Non-systemic fungal
endophytes in grasses. Fungal Ecology 5: 289–297.
Sandu-Ville C (1959). Contribuţii la cunoaşterea micromycetelor din R.P.P. In:
Omagui lui Traian Savulescu (Bontea V, Codarcea A, Gheorghiu IS,
Kreindler A, Knechtel W, et al., eds). Editura Academiei Republicii Populare
Romine: 829–844.
Sawada K (1959). Descriptive catalogue of Taiwan (Formosan) Fungi Part XI.
Special Publication, College of Agriculture, National Taiwan University 8:
1–268.
Schulze-Lefert P, Panstruga R (2011). A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation.
Trends in Plant Science 16: 117–125.
Schultzer von Mueggenburg SVM, Saccardo PA (1884). Micromycetes Sclavonici novi. Revue mycologique 6: 78–80.
Shan XC, Goodwin PH (2004). Monitoring host nuclear migration and degradation with green fluorescent protein during compatible and incompatible
interactions of Nicotiana tabacum with Colletotrichum species. Journal of
Phytopathology 152: 454–460.
Shan XC, Goodwin PH (2005). Reorganization of filamentous actin in Nicotiana
benthamiana leaf epidermal cells inoculated with Colletotrichum destructivum and Colletotrichum graminicola. International Journal of Plant Sciences
166: 31–39.
Shen S, Goodwin PH, Hsiang T (2001). Hemibiotrophic infection and identity of
the fungus, Colletotrichum destructivum, causing anthracnose of tobacco.
Mycological Research 105: 1340–1347.
Shen YM, Liu HL, Chang ST, et al. (2010). First report of Anthracnose caused by
Colletotrichum acutatum on mung bean sprouts in Taiwan. Plant Disease 94:
131.
Sherriff C, Whelan MJ, Arnold GM, et al. (1994). Ribosomal DNA sequence
analysis reveals new species groupings in the genus Colletotrichum.
Experimental Mycology 18: 121–138.
Stewart FC (1900a). An anthracnose and a stem rot of Antirrhinum majus. In:
Proceedings of the Section of Botany at the New York Meeting of the
American Association. Science, N.Y., 12: 581.
Stewart FC (1900b). An anthracnose and a stem rot of the cultivated snapdragon. New York Agricultural Experiment Station Bulletin 179: 105–110.
Sun H, Zhang J (2009). Colletotrichum destructivum from cowpea infecting
Arabidopsis thaliana and its identity to C. higginsianum. European Journal of
Sutton BC (1980). The coelomycetes. Fungi imperfecti with pycnidia, acervuli
and stromata. Commonwealth Mycological Institute, Kew, Surrey, England:
1–696.
Sutton BC (1992). The genus Glomerella and its anamorph Colletotrichum. In:
Colletotrichum: biology, pathology and control (Bailey JA, Jeger MJ, eds).
CAB International, Wallingford, UK: 1–26.
Swofford DL (2003). PAUP*. Phylogenetic analysis using parsimony (*and other
methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Takimoto S (1919). Diseases of medicinal plants (3). Bulletin of the Korean
Agricultural Society 14: 24–27 (in Japanese).
Takimoto S (1934). A new anthracnose of Azuki bean. Annals Phytopathological
Society of Japan 5: 21–24.
84
Tao G, Liu ZY, Liu F, et al. (2013). Endophytic Colletotrichum species from
Bletilla ochracea (Orchidaceae), with descriptions of seven new species.
Fungal Diversity 61: 139–164.
Tehon LR (1937). Notes on the parasitic fungi of Illinois – VI. Mycologia 29:
434–446.
Thomas CA (1951). Anthracnose of digitalis. Phytopathology 41: 997–1000.
Tiffany LH, Gilman JC (1954). Species of Colletotrichum from legumes. Mycologia 46: 52–75.
Tochinai Y (1926). Comperative studies on the physiology of Fusarium lini and
Colletotrichum lini. Journal of the College of Agriculture, Hokkaido Imperial
University 14: 171–236.
Tomioka K, Nishikawa J, Moriwaki J, et al. (2011). Anthracnose of snapdragon
caused by Colletotrichum destructivum. Journal of General Plant Pathology
77: 60–63.
Tomioka K, Sato T, Koganezawa H (2001). Anthracnose of Nemesia strumosa
caused by Colletotrichum fuscum. Journal of General Plant Pathology 67:
111–115.
Tomioka K, Sato T, Moriwaki J, et al. (2012). Anthracnose of bacopa caused by
Colletotrichum destructivum. Journal of General Plant Pathology 78:
133–135.
Trotter A, Cash EK (1972). Sylloge fungorum: 26: 1–1563.
Tunali B, Berner DK, Dubin HJ (2008). First report of leaf spot caused by
Colletotrichum cf. linicola on field bindweed in Turkey. Plant Disease 92:
316.
Tunali B, Kansu B, Berner DK (2009). Biological control studies on Convolvulus
arvensis L. with fungal pathogens. Journal of Turkish Phytopathology 38:
1–8.
Unamuno LM (1933). Contribucion al estudio de los hongos micropicos de
Galicia. Revista de la Real Academia de Ciencias Madrid 30: 460–518.
Ushimaru T, Terada H, Tsuboi K, et al. (2010). Development of an efficient gene
targeting system in Colletotrichum higginsianum using a non-homologous
end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer. Molecular Genetics and Genomics 284: 357–371.
Vasic T, Bulajic A, Krnjaja V, et al. (2014). First report of anthracnose on alfalfa
caused by Colletotrichum linicola in Serbia. Plant Disease 98: 1276.
Vassiljevski NI, Karakulin BP (1950). Fungi imperfecti parasitici: Pars II. Melanconiales. Academiae Scientiarum URSS, Moscow and Leningrad, Russia.
Weir B, Damm U, Johnston PR (2012). The Colletotrichum gloeosporioides
species complex. Studies in Mycology 73: 115–180.
Westerdijk J van (1916). Anthracnose van het vlas. In: Jaarverslag van het
Phytopathologisch Laboratorium “Willie Commelin Scholten.”: 6–7.
Wharton PS, Julian AM, O'Connell RJ (2001). Ultrastructure of the infection of
Sorghum bicolor by Colletotrichum sublineolum. Phytopathology 91: 149–158.
fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to
methods and applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ,
eds). Academic Press, San Diego, USA: 315–322.
Williams RJ (1975). Diseases of cowpea (Vigna unguiculata (L.) Walp.) in
Nigeria. PANS Pest Articles & News Summaries 21: 253–267.
Wollenweber HW, Hochapfel H (1949). Beitr€age zur Kenntnis parasit€arer und
saprophytischer Pilze VI. Vermicularia, Colletotrichum, Gloeosporium,
Glomerella und ihre Beziehung zur Fruchtf€aule. Zeitschrift für Parasitenkunde 14: 181–268.
Woudenberg JHC, Aveskamp MM, Gruyter J de, et al. (2009). Multiple Didymella teleomorphs are linked to the Phoma clematidina morphotype. Persoonia 22: 56–62.
Yang H, Wang M, Gao Z, et al. (2010). Isolation of a novel RNA-dependent RNA
polymerase 6 from Nicotiana glutinosa, NgRDR6, and analysis of its
response to biotic and abiotic stresses. Molecular Biology Reports 38:
929–937.
Zafari D, Tarrah S (2009). Characterization of Colletotrichum species from
legume crop plants in Iran. Pests and Plant Diseases 77: 37–57 (in
Persian).
STUDIES
IN
Resolving the polyphyletic nature of Pyricularia (Pyriculariaceae)
S. Klaubauf1,2, D. Tharreau3, E. Fournier4, J.Z. Groenewald1, P.W. Crous1,5,6*, R.P. de Vries1,2, and M.-H. Lebrun7*
1
CBS-KNAW Fungal Biodiversity Centre, 3584 CT Utrecht, The Netherlands; 2Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands; 3UMR BGPI,
CIRAD, Campus International de Baillarguet, F-34398 Montpellier, France; 4UMR BGPI, INRA, Campus International de Baillarguet, F-34398 Montpellier, France;
5
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; 6Wageningen University and Research Centre (WUR),
Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; 7UR1290 INRA BIOGER-CPP, Campus AgroParisTech, F-78850
Thiverval-Grignon, France
*Correspondence: P.W. Crous, p.crous@cbs.knaw.nl; M.-H. Lebrun, marc-henri.lebrun@versailles.inra.fr
Studies in Mycology
Abstract: Species of Pyricularia (magnaporthe-like sexual morphs) are responsible for major diseases on grasses. Pyricularia oryzae (sexual morph Magnaporthe
oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, while Pyricularia grisea (sexual morph Magnaporthe
grisea) is responsible for foliar diseases of Digitaria. Magnaporthe salvinii, M. poae and M. rhizophila produce asexual spores that differ from those of Pyricularia sensu
stricto that has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation. Magnaporthe salvinii was recently allocated to Nakataea, while
M. poae and M. rhizophila were placed in Magnaporthiopsis. To clarify the taxonomic relationships among species that are magnaporthe- or pyricularia-like in
morphology, we analysed phylogenetic relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as
LSU, ITS, RPB1, actin and calmodulin. Species of Pyricularia s. str. belong to a monophyletic clade that includes all P. oryzae/P. grisea isolates tested, defining the
Pyriculariaceae, which is sister to the Ophioceraceae, representing two novel families. These clades are clearly distinct from species belonging to the Gaeumannomyces
pro parte/Magnaporthiopsis/Nakataea generic complex that are monophyletic and define the Magnaporthaceae. A few magnaporthe- and pyricularia-like species are
unrelated to Magnaporthaceae and Pyriculariaceae. Pyricularia oryzae/P. grisea isolates cluster into two related clades. Host plants such as Eleusine, Oryza, Setaria or
Triticum were exclusively infected by isolates from P. oryzae, while some host plant such as Cenchrus, Echinochloa, Lolium, Pennisetum or Zingiber were infected by
different Pyricularia species. This demonstrates that host range cannot be used as taxonomic criterion without extensive pathotyping. Our results also show that the
typical pyriform, 2-septate conidium morphology of P. grisea/P. oryzae is restricted to Pyricularia and Neopyricularia, while most other genera have obclavate to more
ellipsoid 2-septate conidia. Some related genera (Deightoniella, Macgarvieomyces) have evolved 1-septate conidia. Therefore, conidium morphology cannot be used as
taxonomic criterion at generic level without phylogenetic data. We also identified 10 novel genera, and seven novel species. A re-evaluation of generic and species
concepts within Pyriculariaceae is presented, and novelties are proposed based on morphological and phylogenetic data.
Key words: Magnaporthaceae, Magnaporthe, Pyricularia, Pyriculariaceae, Phylogeny, Systematics.
Taxonomic novelties: New families: Ophioceraceae Klaubauf, Lebrun & Crous, Pyriculariaceae Klaubauf, Lebrun & Crous; New genera: Bambusicularia Klaubauf,
Lebrun & Crous, Barretomyces Klaubauf, Lebrun & Crous, Bussabanomyces Klaubauf, Lebrun & Crous, Kohlmeyeriopsis Klaubauf, Lebrun & Crous, Macgarvieomyces
Klaubauf, Lebrun & Crous, Neopyricularia Klaubauf, Lebrun & Crous, Proxipyricularia Klaubauf, Lebrun & Crous, Pseudopyricularia Klaubauf, Lebrun & Crous,
Slopeiomyces Klaubauf, Lebrun & Crous, Xenopyricularia Klaubauf, Lebrun & Crous; New species: Bambusicularia brunnea Klaubauf, Lebrun & Crous,
Pseudopyricularia cyperi Klaubauf, Lebrun & Crous, Pseudopyricularia kyllingae Klaubauf, Lebrun & Crous, Pyricularia ctenantheicola Klaubauf, Lebrun & Crous,
Pyricularia penniseticola Klaubauf, Lebrun & Crous, Pyricularia pennisetigena Klaubauf, Lebrun & Crous, Pyricularia zingibericola Klaubauf, Lebrun & Crous; New
combinations: Barretomyces calatheae (D.J. Soares, F.B. Rocha & R.W. Barreto) Klaubauf, Lebrun & Crous, Bussabanomyces longisporus (Bussaban) Klaubauf,
Lebrun & Crous, Kohlmeyeriopsis medullaris (Kohlm., Volkm.-Kohlm. & O.E. Erikss.) Klaubauf, Lebrun & Crous, Macgarvieomyces borealis (de Hoog & Oorschot)
Klaubauf, Lebrun & Crous, Macgarvieomyces juncicola (MacGarvie) Klaubauf, Lebrun & Crous, Magnaporthiopsis maydis (Samra, Sabet & Hing.) Klaubauf, Lebrun
& Crous, Neopyricularia commelinicola (M.J. Park & H.D. Shin) Klaubauf, Lebrun & Crous, Proxipyricularia zingiberis (Y. Nisik.) Klaubauf, Lebrun & Crous,
Pseudopyricularia higginsii (Luttr.) Klaubauf, Lebrun & Crous, Xenopyricularia zizaniicola (Hashioka) Klaubauf, Lebrun & Crous; Neotypification (basionym):
Pyricularia zizaniicola Hashioka.
INTRODUCTION
The Magnaporthaceae contains several genera that are important plant pathogens of Poaceae, most notably Magnaporthe
(now Nakataea sensu Luo & Zhang 2013), Pyricularia, and
Gaeumannomyces. The family was originally described with six
genera and 20 species, and presently includes 13 genera and
more than 100 species (Cannon 1994, Bussaban et al. 2005,
Thongkantha et al. 2009, Zhang et al. 2011). The family also
includes genera (Ophioceras, Pseudohalonectria, Ceratosphaeria) that occur in aquatic habitats, or on dead plant materials
such as wood (Shearer et al. 1999, Reblova 2006, Huhndorf
et al. 2008, Thongkantha et al. 2009). The Magnaporthaceae
is currently defined by having perithecial ascomata immersed in
host tissue, frequently with long necks, and cylindrical asci that
stain positive in Meltzer's reagent. Ascospores are highly variable in their morphology. Genera with filiform ascospores
(Gaeumannomyces) tend to have simple, pigmented conidiophores with flared collarettes, and curved, aseptate conidia
(harpophora-like). Genera with fusiform ascospores tend to have
pigmented median cells (Nakataea = Magnaporthe), simple,
pigmented conidiophores, or septate, pyriform to obclavate,
pigmented conidia (Pyricularia and related genera).
The present study does not aim to revise all genera in
Magnaporthales (Hernandez-Restrepo et al. unpubl data), but
focuses primarily on species that are pyricularia-like in
morphology. The genus Pyricularia (in reference to the pyriform
shape of its conidia; Bussaban et al. 2005, Murata et al. 2014)
85
KLAUBAUF
ET AL.
includes species that are pathogenic on a wide range of monocot
plants. Of these, Pyricularia oryzae (sexual morph Magnaporthe
oryzae), the causal agent of the rice blast disease of rice, is one
of the most widely distributed diseases of this crop, and is highly
destructive leading to up to 30 % yield loss worldwide (Skamnioti
& Gurr 2009). Pyricularia oryzae isolates from rice are mostly
host-specific and only infect few host plants beside rice (barley
and Lolium) (Ou 1985, Kato et al. 2000, Couch et al. 2005, Tosa
& Chuma 2014). Pyricularia oryzae isolates from other host
plants such as Eleusine, Setaria and Triticum are also hostspecific, and unable to infect rice (Kato et al. 2000, Couch
et al. 2005, Murata et al. 2014, Tosa & Chuma 2014). A close
relative species of P. oryzae is Pyricularia grisea, which is
indistinguishable in conidium, perithecium and ascopore
morphology. Pyricularia grisea isolates from Digitaria were
shown to form a distinct clade by phylogenetic analysis (Kato
et al. 2000, Couch & Kohn 2002, Hirata et al. 2007, FaivreRampant et al. 2008, Choi et al. 2013) and infect crabgrass
(Digitaria), but not other hosts (Mackill & Bonham 1986, Kato
et al. 2000, Tsurushima et al. 2005, Chen et al. 2006, FaivreRampant et al. 2008, Choi et al. 2013). However, some
P. oryzae isolates from rice and other grasses and some
P. grisea isolates from crabgrass showing cross-infectivity on
crabgrass and rice, respectively have been described (Choi et al.
2013). Sexual morphs were reported for P. grisea and P. oryzae.
However, the genus Pyricularia comprises several other species
for which the sexual morph has not yet been discovered. Such
Pyricularia species include P. higginsii pathogenic on Cyperus
(Luttrell 1954, Hashioka 1973), P. zingiberi pathogenic on Zingiber (Kotani & Kurata 1992), P. zizaniaecola pathogenic on
Zizania (Hashioka 1973) and P. commelinicola on Commelina
(Park & Shin 2009). Other notable pathogens from the Magnaporthaceae include Nakataea oryzae, Gaeumannomyces graminis, Magnaporthiopsis poae and M. rhizophila.
The aims of the present study were to determine the phylogenetic relationships among species of Pyricularia compared to
P. oryzae/P. grisea, as well as those taxa now accommodated in
Magnaporthiopsis and Nakataea, using multilocus sequence
analysis. This study allowed defining two novel families,
Ophioceraceae and Pyriculariaceae, as well as novel genera and
species.
Isolates
A global collection of 153 isolates was included in this study
(Table 1). Cultures for morphological observation were inoculated in a three-point position onto the following agar media:
Cornmeal agar (CMA), oatmeal agar (OA), 2 % potato dextrose
agar (PDA) and 2 % malt extract agar (Oxoid) (MEA). All media
were prepared as described previously (Crous et al. 2009,
Samson et al. 2010). Representative isolates were deposited
in the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht,
The Netherlands.
DNA extraction, amplification and sequencing
Fungal cultures were grown on a cellophane disc on MEA to
easily scrape off mycelium. Genomic DNA was extracted using
86
the UltraClean Microbial DNA isolation kit (MoBio Laboratories,
USA), according to the manufacturer’s instructions. Parts of the
following loci were amplified and sequenced: RPB1, partial
RNA polymerase II largest subunit gene; ITS, internal transcribed spacer regions and intervening 5.8S nuclear ribosomal
RNA (nrRNA) gene; LSU, partial nrRNA gene large subunit
(28S); ACT, partial actin gene and CAL, partial calmodulin
gene.
The reactions were performed in 20 μL mixtures containing
1 μL of genomic DNA, 2 mM MgCl2 (Bioline, Germany), 4 μL 5×
Colourless GoTaq® Flexi Buffer (Promega, USA), 80 μM dNTPs
(Promega), 0.2 μM of each primer and 0.10 μL GoTaq® Flexi
DNA Polymerase (Promega).
The primers V9G (de Hoog & Gerrits van den Ende 1998)
and LR5 (Vilgalys & Hester 1990) were used to amplify the
ITS + LSU region by using the following PCR programme: initial
denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C
for 30 s, 52 °C for 30 s, 72 °C for 2 min, and finally an
additional 7 min at 72 °C. The primers ACT-512F and ACT783R were used for actin and CAL-228F and CAL-737R for
calmodulin (Carbone & Kohn 1999). The following PCR programme was used for actin/calmodulin: initial denaturation at
94 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 61/
55 °C for 20 s, 72 °C for 40 s, and final extension at 72 °C for
5 min. For amplification of RPB1 the primers RPB1F and
RPB1R (see Table 2) were designed for the Nakataea/Gaeumannomyces group from unpublished sequence data of eight
P. oryzae strains and one P. grisea strain, as well as public
genomes of P. oryzae 70-15, Magnaporthiopsis poae
ATCC 64411 and Gaeumannomyces graminis var. tritici
R3111a. The following PCR programme was used: initial
denaturation at 94 °C for 5 min, followed by 12 cycles of 94 °C
for 30 s, 57–51 °C (decreasing for 0.5° every cycle) for 20 s,
72 °C for 70 s; 25 cycles of 94 °C for 30 s, 51 °C for 20 s,
72 °C for 70 s; and finally an additional 5 min at 72 °C.
Both strands of the PCR fragments were sequenced with the
BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) using the primers indicated in Table 2. The
products were analysed on an ABI Prism 3730 XL DNA
Sequencer (Applied Biosystems). Contigs were assembled by
using the forward and reverse sequences with the programme
SeqMan from the LaserGene v. 9 package (DNAstar, USA).
Genomic sequences of Cryphonectria parasitica strain
EP155, Gaeumannomyces graminis var. tritici strain R3111a,
P. oryzae strain 70-15 and M. poae strain ATCC 64411 were
retrieved from Broad Institute (www.broadinstitute.org;
G. graminis var. tritici, P. oryzae and M. poae) and JGI Genome
Portal (http://genomeportal.jgi.doe.gov/; C. parasitica) databases
(Dean et al. 2005).
Phylogenetic analyses
Megablast searches of the NCBI's GenBank nucleotide database
were used to supplement the sequence data generated in this
study, especially to populate the overview LSU phylogeny. Sequences were aligned using the online version of MAFFT (http://
mafft.cbrc.jp/alignment/software/) and the alignments were
manually adjusted using MEGA v. 5.2 (Tamura et al. 2011).
Analyses were performed with the individual and combined
datasets to test the robustness of each included locus. Phylogenetic trees were reconstructed by Bayesian Inference (BI)
Avena sativa
Australia: Western Australia
Pennisetum clandestinum
Stenotaphrum secundatum
Australia: New South Wales
USA: Florida
M33
CBS 186.65
M.B. Ellis
Carex rostrata
UK: Wales
R3-111a-1
CBS 388.81
CBS 117.83
M55
–
USA: Washington
CBS 905.73 = DAR 23140
–
CBS 249.29 = IMI 083849
A. Parker
–
USA: Montana
Triticum aestivum
Triticum sp.
Australia: Western Australia
–
Soil in potato field
Triticum aestivum
–
Netherlands: Groningen
–
Triticum aestivum
Netherlands
CBS 247.29
CBS 903.73 = DAR 23471
–
CBS 902.73 = DAR 17502
P. Wong
J. Kuiper
–
CBS 387.81
CBS 352.93 = PD 93/290
–
M.B. & J.P. Ellis
CBS 235.32
–
–
Deschampsia caespitosa,
dead culm and sheath
UK: England
CBS 870.73 = DAR 20999
–
Hordeum vulgare
Ctenanthe sp., stem base
Netherlands: near
Barendrecht
CBS 187.65
–
Netherlands: Flevoland
Oryza sativa
USA: Arkansas
CBS 128780 = CPC 18916
(ex-type)
EP155 = ATCC 38755
CBS 125232 (ex-type)
ATCC 22848
CBS 129274 = CPC 18464
CBMAI 1060 (ex-type)
CBS 133600 = MAFF
240226 = INA-B-93-19(Ph-1J)
CBS 133599 = MAFF
240225 = INA-B-92-45(Ss-1J)
(ex-type)
Culture collection no1
W. Quaedvlieg
N. DePalma
B. Bussaban
R.V. Gessner
P.W. Crous
D.J. Soares
S. Koizumi
S. Koizumi
Collector
KM484843
KM484842
Genome
JF414850
KM484841
KM484840
KM484839
KM484838
JF710374
KM484837
KM484836
KM484835
KM484834
JX134669
KM484833
JX134668
JF951153
Genome
KM484832
JX134666
KM484831
GU294490
AB274436
KM484830
ITS
KM484960
KM484959
Genome
JF414900
KM484958
KM484957
KM484956
KM484955
JF414896
KM484954
KM484953
KM484952
DQ341496
JX134681
DQ341495
JX134680
JF951176
Genome
KM484951
DQ341492
KM485059
KM485058
Genome
JF710445
KM485057
KM485056
KM485055
KM485054
JF710442
KM485053
KM485052
KM485051
KM485050
KM485049
KM485048
JX134722
KM485047
Genome
KM485046
JX134720
KM485045
–
–
KM484950
KM485044
KM485043
RPB1
KM484949
KM484948
LSU
–
–
–
–
–
–
–
–
–
–
Genome
–
Genome
–
–
–
–
–
–
–
–
KM485164
–
–
–
–
–
–
–
–
KM485232
–
–
–
–
KM485163
Genome
–
Genome
KM485231
–
–
AB274483
AB274482
CAL
KM485162
–
AB274450
AB274449
ACT
GenBank Accession no2
THE POLYPHYLETIC NATURE OF
Gaeumannomyces sp.
Gaeumannomyces graminis
var. tritici
var. graminis
Avena sativa, root
Netherlands: Flevoland
var. avenae
Castanea dentata
Phragmites australis, leaves
USA: Connecticut
Netherlands: Utrecht
Cryphonectria parasitica
Deightoniella roumeguerei
Amomum siamense,
leaf endophyte
Thailand: Chiang Mai
Bussabanomyces
longisporus
Calathea longifolia
Spartina alterniflora, leaves
Brazil: Minas Gerais
USA
Buergenerula spartinae
Calathea longifolia
Japan: Aichi
Brazil: Minas Gerais
Phyllostachys bambusoides
Japan: Aichi
Bambusicularia brunnea
Barretomyces calatheae
Sasa sp.
Location
Species
Substrate
Table 1. Collection details and GenBank accession numbers of isolates included in this study (“–” = unknown).
RESOLVING
PYRICULARIA
87
88
Zea mays, root
South Africa
Cynodon dactylon × Cynodon
transvaalensis
–
Zoysia matrella
Australia: South Australia
–
USA: Kansas
Magnaporthiopsis rhizophila
Magnaporthiopsis poae
Magnaporthiopsis maydis
Magnaporthiopsis
incrustans
Cynodon dactylon × Cynodon
transvaalensis
Australia: Queensland
Magnaporthe griffinii
Juncus effusus, leaf spots
Poa pratensis
Poa pratensis
–
Zea mays hybrid
“Ganga Safed 2”
India: Bihar, Messina
Triticum aestivum
Zea mays
India: Rajasthan, Jaipur
USA: New Jersey
Zea mays
India: Rajasthan, Jaipur
USA
Zea mays
Egypt
Juncus effusus, stem base
UK: Scotland
Netherlands
Juncus roemerianus
USA: North Carolina
Macgarvieomyces borealis
Juncus roemerianus
USA: North Carolina
Macgarvieomyces juncicola
Kohlmeyeriopsis medullaris
Triticum aestivum,
seedling
Zea mays, root
South Africa
Germany
Zea mays, root
South Africa
Zea mays, root
Zea mays, root
South Africa
UK: England
Zea mays, root
Canada: Ontario
Harpophora sp.
Zea mays
South Africa
Harpophora radicicola
Substrate
Location
Species
Table 1. (Continued)
CBS 117849 = JK5528S
CBS 118210 = JK5522N
= ATCC MYA-3560
–
–
M51
M23
M47
–
ATCC 64411
–
CBS 664.82
CBS 663.82B
CBS 663.82A
P.J. Landschoot
M.M. Payak
B.S. Siradhana
B.S. Siradhana
CBS 662.82A
–
H.A. Elshafey
M35
TY2
TS99
CBS 610.82
–
P. Toy
A.M. Stirling
G.S. de Hoog
CBS 461.65 (ex-type)
CBS 541.86
–
G.D. MacGarvie
CPC 18689 = Z 426 AJ
CPC 18685 = Z 397 L
–
CBS 350.77 = ATCC
28234 = IMI 187786
CPC 18683 = Z 390 G
–
–
CPC 18682 = Z 383 Y
–
–
CBS 296.53 = MUCL
28970 = TRTC 23660 (isotype
of Phialophora radicicola)
CBS 149.85 = PREM 45754
(isotype of Phialophora
zeicola)
–
R.F. Cain
Collector
JF414834
JF414836
Genome
KM484859
KM484858
KM484857
KM484856
JF414846
JF414843
JQ390312
JQ390311
KM484855
KM484854
KM484853
KM484852
KM484851
KM484850
KM484849
KM484848
KM484847
KM484846
KM484845
KM484844
ITS
–
–
JF414895
JF414846
JF414885
Genome
KM484974
KM484973
KM484972
KM484971
JF710432
JF710433
Genome
KM485075
KM485074
KM485073
KM485072
JF710440
JF710437
–
–
JF414892
KM485071
KM485070
KM485069
KM485068
KM485067
KM485066
KM485065
KM485064
KM485063
KM485062
KM485061
KM485060
RPB1
KM484970
DQ341511
KM484969
KM484968
DQ341497
KM484967
KM484966
KM484965
KM484964
KM484963
KM484962
KM484961
LSU
–
–
–
–
–
–
–
–
–
–
Genome
–
Genome
–
–
–
–
–
KM485240
KM485239
–
–
–
–
–
–
KM485171
KM485170
–
–
–
–
–
KM485238
–
KM485237
KM485236
KM485169
KM485168
KM485167
KM485235
KM485234
–
KM485166
KM485233
CAL
KM485165
ACT
KLAUBAUF
ET AL.
Zingiber mioga
Zingiber mioga
Japan: Hyogo
Japan: Hyogo
Proxipyricularia zingiberis
Dead stem of dicot plant
(probably Urtica dioica)
UK: England
Ophioceras leptosporum
Wood
Hong Kong
Ophioceras dolichostomum
Rotten wood
Rotten wood
China: Yunnan
China: Yunnan
Ophioceras commune
Commelina communis, leaves
South Korea: Hongcheon
Panicum effusum var. effusum,
grass leaves
Commelina communis
Australia: Queensland
Commelina communis
South Korea: Pocheon
Omnidemptus affinis
Commelina communis, leaves
Neopyricularia
commelinicola
Oryza sativa
Oryza sativa
USA: California
USA: Arkansas
Nakataea sp.
CBS 894.70 = ATCC
24161 = HME 2955 (ex-type
of Gaeumannomyces
leptosporus)
–
I. Chuma
CBS 132196 = MAFF
240223 = HYZiM202-1-2
(Z-3J)
CBS 132195 = MAFF
240224 = HYZiM201-1-1-1
(Z-4J)
CBS 114926 = HKUCC
3936 = KM 8
–
I. Chuma
M91
M92
–
–
ATCC 200212 (ex-type)
CBS 128308 = KACC 43081
(ex-type)
CBS 128307 = KACC 44083
CBS 128306 = KACC 43869
CBS 128303 = KACC 44637
V.P. Cooper
H.D. Shin & M.J. Park
M.J. Park
CBS 727.74
CBS 332.53
–
CBS 726.74
R.K. Webster
R.K. Webster
CBS 288.52
Oryza sativa, stem
Oryza sativa
Japan: Takada
USA: California
–
CBS 252.34
CBS 253.34
–
–
–
Oryza sativa, straw
–
CBS 243.76
–
Burma
CBS 202.47
–
–
Oryza sativa
ATCC 44754 = M21 = Roku-2
–
Oryza sativa
Italy
Japan
Nakataea oryzae
Collector
Substrate
Italy
Location
Species
KM484870
KM484869
JX134678
JX134677
JX134676
JX134675
JX134674
FJ850122
FJ850125
FJ850123
KM484868
KM484867
KM484866
KM484865
KM484864
KM484863
KM484862
KM484861
KM484860
JF414838
ITS
KM485088
KM485089
–
JX134732
JX134731
JX134730
JX134729
JX134728
KM485087
KM485086
KM485085
KM485084
KM485083
KM485082
KM485081
KM485080
KM485079
KM485078
KM485077
KM485076
JF710441
RPB1
KM484986
JX134690
JX134689
JX134688
JX134687
JX134686
KM484985
KM484984
KM484983
KM484982
KM484981
KM484980
KM484979
KM484978
KM484977
KM484976
DQ341498
KM484975
JF414887
LSU
–
–
KM485245
KM485244
–
–
–
AB274447
AB274448
–
–
–
–
–
–
–
–
KM485243
KM485242
KM485241
KM485175
KM485174
KM485173
KM485172
–
–
–
–
–
–
–
–
–
–
CAL
–
–
–
–
–
–
ACT
RESOLVING
PYRICULARIA
89
90
Pyricularia oryzae
Pyricularia grisea
CBS 255.38
J.-L. Notteghem
–
BR0032
BR0045
J.-L. Notteghem
–
US0043 = G184
BF0028
B. Valent
PH0055 = Dc88420
J.-L. Notteghem
Romania
Digitaria sp.
USA: Delaware
IRRI
JP0034 = NI980
Triticum sp.
Digitaria ciliaris
Philippines: Sto Tomas,
Batangas
CR0024
–
CBS 128304 = KACC 41641
C.K. Kim
Brazil
Digitaria smutsii
Japan
Triticum sp.
Lolium perenne
South Korea: Suwon
H.K. Sim
Br33
Brazil
Echinochloa crus-galli
var. frumentacea
Korea: Woanju
BR0029
–
GR0002 (ex-type)
GR0001 = Ct-4 = ATCC
200218
PH0054 = Cb8959
CBS 133597 = MAFF
240227 = HYKB202-1-2(K-1J)
(ex-type)
CBS 121934 = 09/2007/1470
PH0053 = Cr88383
CBS 665.79
CBS 133595 = MAFF
240229 = HYCI201-1-1(Ci-1J)
(ex-type)
CBS 303.39 = MUCL 9449
CBS 133594 = MAFF
240222 = HYZiM201-0-1
(Z-2J)
CBS 132355 = MAFF
240221 = HYZiM101-1-1-1
(Z-1J)
J.-L. Notteghem
Paspalum sp.
Digitaria horizontalis
A.C. Pappas &
E.J. Paplomatas
A.C. Pappas &
E.J. Paplomatas
IRRI
I. Chuma
C.F. Hill
IRRI
R. Kenneth
H. Kato
Y. Nisikado
H. Kato
M. Ogawa
Collector
Burkina Faso
Digitaria sanguinalis
Brazil
Ctenanthe oppenheimiana
Greece: Almyros, imported
from Brazil via Netherlands
Brazil: Goias, Goiana
Ctenanthe oppenheimiana
Greece: Almyros, imported
from Brazil via Netherlands
Cyperus brevifolius
Philippines: Los Banos
Pyricularia ctenantheicola
Kyllinga brevifolia
Japan: Hyogo
Pseudopyricularia kyllingae
Typha orientalis, dead leaves
Cyperus rotundus
Philippines: Sto Tomas,
Batangas
New Zealand: Auckland,
Mount Albert
Cyperus rotundus
Israel
Pseudopyricularia higginsii
Cyperus iria
Zingiber officinale
Japan
Japan: Hyogo
Zingiber mioga
Japan: Hyogo
Pseudopyricularia cyperi
Zingiber mioga
Japan: Hyogo
Substrate
Location
Species
KM484889
KM484888
KM484887
KM484886
KM484885
KM484884
KM484883
KM484882
KM484881
AB274430
KM484880
KM484879
KM484878
KM484877
KM484876
KM484875
KM484874
KM484873
KM484872
KM484871
AB274434
AB274433
ITS
KM485105
–
–
KM484999
–
KM485109
KM485108
KM485107
KM485106
KM485104
–
KM484998
KM485103
–
KM485102
KM485101
–
KM484997
–
KM484996
KM485100
KM485099
–
KM484995
KM485098
KM485097
KM485096
KM484994
KM484993
KM484992
KM485095
KM485094
–
KM484991
KM485093
AB818013
KM485092
KM485091
KM485090
RPB1
DQ341512
KM484990
KM484989
KM484988
KM484987
LSU
KM485190
KM485189
DQ240884
KM485188
KM485187
DQ240877
KM485186
KM485185
KM485184
–
DQ240874
KM485183
KM485182
KM485181
AB274451
KM485180
KM485179
KM485178
AB274453
KM485177
AB274446
KM485176
ACT
KM485261
KM485260
DQ240900
KM485259
KM485258
DQ240893
KM485257
KM485256
KM485255
KM485254
DQ240890
KM485253
KM485252
KM485251
AB274484
KM485250
KM485249
KM485248
AB274485
KM485247
KM485246
AB274481
CAL
KLAUBAUF
ET AL.
Guy11 = FGSC 9462
J.-L. Notteghem
Leptochloa chimensis
Paspalum distichum
Rottboellia exalta
Echinochloa colona
Panicum repens
Philippines: Cabanatuan
Philippines
Philippines
PH0035 = Bm8309 = PH0075
PH0079 = GPr8212
PH0077 = Ec8202
PH0063 = ReA8401 = ATCC
62619
PH0062 = Pd8824
PH0060 = LcA8401
PH0051 = Cd88215
KM484919
KM484918
KM484916
KM484915
KM484914
KM484913
KM484912
KM484911
KM484910
KM484909
KM484908
KM484907
KM484906
AF074404
KM484905
KM484904
KM484903
KM484902
KM484901
KM484900
KM484899
KM484898
KM484897
KM484896
KM484895
KM484894
KM484893
KM484892
KM484891
KM484890
ITS
KM485010
KM485121
KM485025
KM485024
KM485022
KM485138
KM485137
KM485135
KM485134
–
KM485021
–
–
KM485133
KM485132
–
KM485020
KM485130
KM485131
KM485019
KM485129
KM485128
–
KM485018
KM485127
KM485126
KM485287
KM485286
KM485284
KM485283
KM485282
KM485281
KM485280
DQ240904
KM485279
KM485278
KM485277
KM485276
KM485275
AF396018
KM485274
AF396024
DQ240898
DQ240901
KM485273
KM485272
KM485271
KM485270
KM485269
KM485268
KM485267
KM485266
KM485265
KM485264
KM485263
KM485262
CAL
KM485214
KM485213
KM485211
KM485210
KM485209
KM485208
KM485207
DQ240888
KM485206
KM485205
AF395964
KM485204
AF395961
KM485203
AF395970
–
KC167438
DQ240882
DQ240885
KM485202
KM485201
KM485200
KM485199
KM485198
KM485197
KM485196
KM485195
KM485194
KM485193
KM485192
KM485191
ACT
KM485125
KM485124
KM485123
KM485122
KM485017
KM485016
KM485015
KM485014
KM485013
KM485012
KM485011
KM485120
KM485119
–
KM485009
KM485118
KM485117
KM485116
KM485115
KM485114
KM485113
KM485112
KM485111
KM485110
RPB1
KM485008
KM485007
KM485006
KM485005
KM485004
KM485003
KM485002
KM485001
KM485000
LSU
J. M. Bonman
IRRI
IRRI
IRRI
IRRI
IRRI
IRRI
Brachiaria mutica
Cynodon dactylon
Philippines: Cabanatuan
PH0014 = PO6-6
JP0040 = NI901
Oryza sativa
–
IRRI
Phalaris arundinacea
Philippines
–
Japan
JP0038 = IN909
JP0039 = NI904
H. Kato
Eragrostis curvula
JP0028 = K76-79
JP0033 = NI859
–
JP0017 = C10
H. Yaegashi
H. Yaegashi
IN0108 = VII-765-1
GN0001
J.-L. Notteghem
J. Kumar
CR0029
FR0013
J.-L. Notteghem
CR0026
CR0021
CR0020
CD0156
C.K. Kim
C.K. Kim
C.K. Kim
C.K. Kim
J.-L. Notteghem
Anthoxanthum odoratum
Eriochloa villosa
Japan
CBS 659.66
–
Japan
Eragrostis curvula
Japan
CBS 658.66
–
CD0067
CBS 657.66
–
J.-L. Notteghem
CBS 433.70
CBS 375.54
CBS 365.52 = MUCL 9451
–
–
Japan
Setaria sp.
Oryza sativa
French Guiana
Eleusine indica
Zea mays
Gabon: Wey
India: Uttar Pradesh
Oryza sativa
Japan
Festuca elalior
Phleum pratense
Côte d'Ivoire: Ferkessedougou
South Korea: Suwon
France: Camargue
Eleusine indica
Côte d'Ivoire: Bouake
South Korea: Suwon
Leersia hexandra
Israel
Panicum miliaceum
Israel
Lolium hybridum
Echinochloa crus-galli
Egypt
South Korea: Yongin
Oryza sativa
–
South Korea: Suwon
–
Oryza sativa, seed
–
–
–
Japan: Nagano
Pyricularia oryzae
Collector
Substrate
Location
Species
RESOLVING
PYRICULARIA
91
92
ML0048
BR0067
J.-L. Notteghem
–
Brazil
Japan: Chiba
Thailand
Reunion
Hong Kong: Discovery Bay
Pyricularia sp.
Pyricularia variabilis
Pyricularia zingibericola
Pyriculariopsis parasitica
Musa sp., leaves
Zingiber officinale
Amomum siamense,
healthy leaves
Leersia oryzoides
Setaria geniculate
Pennisetum glaucum
USA: Tifton
Pyricularia sp.
Pennisetum glaucum
USA: Tifton
J.-L. Notteghem
Pennisetum sp.
Cenchrus echinatus
Mali: Cinzana
Philippines: Plaridel
N. Nishihara
Cenchrus ciliaris
Japan: Kumamoto
H. Kato
K.D. Hyde
CBS 114973 = HKUCC
5562 = Maew HK 1
RN0001
CMUZE0229 = ICMP 14487
–
J.-C. Girard
CBS 133598 = MAFF
305509 = NI919
(Leo-1J) = JP0036
Br37
US0045 = 84P-19
US0044 = 83P-25
PH0047 = Ce88454
ML0036 (ex-type)
CBS 133596 = MAFF
305501 = NI981(Cc-1J)
Br36
BR0093
CD0086
N. Nishihara
S. Igarashi
H. Wells
H. Wells
IRRI
S. Igarashi
Echinochloa colona
Cenchrus echinatus
Brazil: Primeiro de Maio
J.-L. Notteghem
Brazil
Cenchrus echinatus
Digitaria exilis
Brazil: Imperatriz
Mali
ML0031 (ex-type)
J.-L. Notteghem
Pyricularia pennisetigena
CD0180
J.-L. Notteghem
Pennisetum sp.
Pennisetum typhoides
Côte d'Ivoire: Madiani
Digitaria exilis
Mali: Longorola Sikasso
CD0143
J.-L. Notteghem
Côte d'Ivoire: Odienne
BF0017
CBS 376.54 = ICMP
14696 = MUCL 9450 = QM
1092
70-15 = ATCC MYA4617 = FGSC 8958
Côte d'Ivoire: Bouake
J.-L. Notteghem
–
–
VT0032
US0071
Laboratory strain
–
B. Couch
M. Farman
Burkina Faso: Kamboinse
Leersia hexandra
Vietnam: O Mon
RW0012
J.-L. Notteghem
Pyricularia penniseticola
Setaria viridis
USA: Kentucky
Phyllachora graminis
Eleusine coracana
Rwanda: Kunynya
PR0104
PR0067
A. Lima
A. Lima
USA: Iowa
Stenotaphrum secondatum
Portugal
Collector
“Pyricularia parasitica”
Stenotaphrum secondatum
Portugal
Pyricularia oryzae
Substrate
Location
Species
–
–
KM485037
DQ341514
–
–
KM485036
–
KM485157
–
KM485156
–
KM485035
KM485228
–
KM485155
–
KM485225
–
KM485229
–
AB274440
KM485227
KM485226
KM485153
KM485154
–
–
KM485034
KM485224
–
–
KM485152
KM485223
KM485151
KM485033
–
KM485222
KM485221
KM485220
DQ240880
KM485219
DQ240879
DQ240878
–
Genome
KM485218
KM485217
AF395959
KM485216
KM485215
ACT
KM485150
KM485149
KM485148
KM485147
KM485146
KM485145
KM485144
–
Genome
KM485143
KM485142
KM485141
KM485140
KM485139
RPB1
KM485032
–
–
–
–
–
KM485031
KM485030
Genome
KM485029
KM485028
–
KM485027
KM485026
LSU
KM484941
AY265333
KM484940
KM484939
KM484938
KM484937
KM484936
KM484935
KM484934
KM484933
KM484932
KM484931
KM484930
KM484929
KM484928
KM484927
KM484926
KM484925
AY265340
Genome
KM484924
KM484923
KM484922
KM484921
KM484920
ITS
–
KM485297
–
AB274473
AB274474
–
KM485296
KM485295
KM485294
AB274475
KM485293
KM485292
KM485291
–
–
DQ240896
–
DQ240895
DQ240894
–
Genome
KM485290
–
AF396014
KM485289
KM485288
CAL
KLAUBAUF
ET AL.
Zizania latifolia
Zizania latifolia
Japan: Ibaraki
Grass roots; associated with
Phialophora graminicola
UK: England
Japan: Kyoto
UK: England
N. Hayashi
K. Yoshida &
K. Hirata
D. Hornby
D. Hornby
D. Hornby
CBS 132356 = MAFF
240220 = KYZL201-1-1
(Zz-2J)
CBS 133593 = MAFF
240219 = IBZL3-1-1(Zz-1J)
(ex-neotype)
CBS 318.95 = INIFAT C94/
182 (ex-type)
CBS 244.95 = INIFAT C94/
182
R.F. Casta~neda &
M. Saikawa
R.F. Casta~neda
Collector
KM484947
KM484946
KM484945
JX134667
KM484944
KM484943
KM484942
ITS
KM485160
KM485161
–
KM485159
JX134721
KM485158
–
–
RPB1
KM485042
KM485041
DQ341494
KM485040
KM485039
KM485038
LSU
–
–
–
–
KM485230
AB274479
AB274480
–
–
AB274444
–
–
CAL
–
–
ACT
ATCC: American Type Culture Collection, Virginia, U.S.A.; BCC: BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok, Thailand; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The
Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; DAR: Plant Pathology Herbarium, Orange Agricultural Institute, Forest Road, Orange. NSW 2800, Australia; FGSC: Fungal Genetics Stock Center, University of Kansas Medical
Center, KS, U.S.A.; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; ICMP: International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand; IMI: International Mycological Institute, CBIBioscience, Egham, Bakeham Lane, United Kingdom; INIFAT: Alexander Humboldt Institute for Basic Research in Tropical Agriculture, Ciudad de La Habana, Cuba; KACC: Korean Agricultural Culture Collection, National Institute of Agricultural
Biotechnology, Rural Development Administration, Suwon, Republic of Korea; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; MUCL: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium; PD: Plant Protection
Service, nVWA, Division Plant, Wageningen, The Netherlands; PREM: South African National Collection of Fungi (NCF), Mycology Unit, Biosystematics Division, Plant Protection Institute, Agricultural Research Council, Roodeplaat, Pretoria, South
Africa; QM: Quartermaster Research and Development Center, U.S. Army, Massachusetts, U.S.A.
2
ITS: internal transcribed spacers and intervening 5.8S nrDNA; LSU: partial large subunit (28S) of the nrRNA gene operon; RPB1: partial RNA polymerase II largest subunit gene; ACT: partial actin gene; CAL: partial CAL gene. Genome sequences of
C. parasitica strain EP155: JGI Genome Portal; Genome sequences of G. graminis var. tritici strain R3111a, P. oryzae strain 70-15 and M. poae strain ATCC 64411: Broad Institute.
1
Xenopyricularia zizaniicola
UK: England
Nectandra antillana, leaf litter
Cuba: Pinar del Rio
Slopeiomyces
cylindrosporus
Nectandra antillana, leaf litter
Cuba: Pinar del Rio
Rhexodenticula
cylindrospora
Substrate
Location
Species
RESOLVING
PYRICULARIA
93
KLAUBAUF
ET AL.
Table 2. Details of primers used and/or developed for this study.
Sequence (5′ – 3′)
Orientation
Reference
ACT-512F
ATG TGC AAG GCC GGT TTC GC
Forward
Carbone & Kohn (1999)
ACT-783R
TAC GAG TCC TTC TGG CCC AT
Reverse
CAL-228F
GAG TTC AAG GAG GCC TTC TCC C
Forward
CAL-737R
CAT CTT TCT GGC CAT CAT GG
Reverse
ITS4
TCC TCC GCT TAT TGA TAT GC
Reverse
White et al. (1990)
ITS5
GGA AGT AAA AGT CGT AAC AAG G
Forward
White et al. (1990)
V9G
TTA CGT CCC TGC CCT TTG TA
Forward
de Hoog & Gerrits van den Ende (1998)
LR5
TCC TGA GGG AAA CTT CG
Reverse
Vilgalys & Hester (1990)
NL1
GCA TAT CAA TAA GCG GAG GAA AAG
Forward
O'Donnell (1993)
AGA CGA TYG AGG AGA TCC AGT T
ART CCA CAC GCT TAC CCA TC
Forward
Reverse
This study
This study
Locus1 and primer name
Actin
Calmodulin
ITS
LSU
RPB1
RPB1F
RPB1R
1
ACT: partial actin gene; CAL: partial CAL gene; ITS: internal transcribed spacers and intervening 5.8S nrDNA; LSU: partial large subunit (28S) of the nrRNA gene operon;
RPB1: partial RNA polymerase II largest subunit gene.
using MrBayes v. 3.2.2 ((Ronquist et al. 2012); LSU only) and
maximum parsimony (MP) using PAUP v. 4.0b10 (Swofford
2003) for all datasets as described by Crous et al. (2006). To
check the congruency of the individual datasets, a 70 %
neighbour-joining (NJ) reciprocal bootstrap was performed
(Mason-Gamer & Kellogg 1996, Lombard et al. 2010). Novel
sequences derived in this study were lodged at GenBank, and
the alignments and phylogenetic trees in TreeBASE (www.
treebase.org/treebase/index.html).
Morphology
For morphological characterisation, cultures were grown on
synthetic nutrient-poor agar (SNA; Nirenberg 1976), supplemented with autoclaved barley seeds, water agar supplemented
with autoclaved barley seeds and leaves, as well as OA. Plates
were inoculated with agar plugs from cultures growing on MEA,
PDA or OA. Plates were incubated at 23–25 °C under a regime
of 12 h dark/12 h near-ultaviolet light, and examined after 1–3 wk
for sporulation. Observations were made with a Zeiss V20 Discovery stereo-microscope, and with a Zeiss Axio Imager 2 light
microscope using differential interference contrast (DIC) illumination and an AxioCam MRc5 camera and software. Measurements and photographs were made from structures mounted in
clear lactic acid. The 95 % confidence intervals were derived
from 30 observations (×1 000 magnification), with the extremes
given in parentheses. Ranges of the dimensions of other characters are given. Colony diameter and other macroscopic features were recorded after 1 wk of incubation at 25 °C in the dark.
Colony colours were determined using the colour charts of
Rayner (1970). Specimens were deposited in the fungarium at
CBS (CBS H) in Utrecht, and taxonomic novelties in MycoBank
(Crous et al. 2004).
94
RESULTS
DNA phylogeny
We combined the LSU sequences obtained from our Pyricularia/
Magnaporthe species (Table 1) with sequences from NCBI
corresponding to other Pyricularia/Magnaporthe species. The
LSU dataset consists of 99 aligned sequences, including the
outgroup Peziza vesiculosa. It contains 772 characters, of which
336 constitute unique site patterns (BI analysis with the GTR
model, dirichlet (1,1,1,1) state frequency distribution and inverse
gamma-shaped rate variation across sites). 405 characters were
constant, 62 were variable and parsimony-uninformative while
305 were parsimony informative (MP analysis). A maximum of
1 000 equally most parsimonious trees were retained from this
analysis (Tree length = 1 362, CI = 0.438, RI = 0.785 and
RC = 0.343, Fig. 1). The majority of strains clustered in the
Magnaporthales (Thongkantha et al. 2009). However, “Pyricularia” parasitica, based on CBS 376.54, clusters in the Chaetothyriales (Eurotiomycetes) and Rhexodenticulata cylindrospora
(=Pyricularia lauri, Nakataea cylindrospora) is placed incertae
sedis in the Sordariomycetes, but in both the parsimony (69 %
bootstrap support) and Bayesian analyses (posterior probability
of 1.0), this clade is related to Boliniales and Sordariales.
Within Magnaporthales, the different clades were not wellresolved using LSU sequences (Fig. 1). Therefore, LSU was
supplemented with RPB1 sequences to generate a novel
phylogenetic tree restricted to species from Magnaporthales. The
combined LSU/RPB1 dataset consists of 101 aligned sequences
including Cryphonectria parasitica as outgroup. This dataset
contains 1 391 characters, of which the LSU dataset contributed
748 characters and the RPB1 dataset contributed 643 characters; 772 characters were constant, while 131 were variable and
RESOLVING
PYRICULARIA
DQ470948 Peziza vesiculosa
Herpotrichiellaceae
Ophiostomataceae
Sordariales
Calosphaeriaceae
Calosphaeriales
Eurotiomycetes
Chaetothyriales
EU035411 Cladophialophora proteae
FJ358249 Sarcinomyces petricola
KF777234 Phaeococcomyces aloes
70
HQ599589 Exophiala encephalarti
“Pyricularia” parasitica CBS 376.54
71
FJ839641 Brycekendrickomyces acaciae
DQ836904 Ophiostoma stenoceras
AF234836 Ophiostoma floccosum
AF234837 Ophiostoma piceae
100
AY761075 Calosphaeria pulchella
AY761076 Togniniella acerosa
Diaporthaceae
AY346279 Diaporthe phaseolorum
Harknessiaceae
AF408363 Harknessia eucalypti
52
Sordario1.0/100
mycetes
Cryphonectria parasitica EP155
60
AF408339 Cryphonectria havanensis
Cryphonectriaceae
CBS 244.95 Rhexodenticula cylindrospora
CBS 318.95 Rhexodenticula cylindrospora
Incertae sedis
AY083821 Camarops microspora
0.76/81
Boliniaceae
DQ231441 Cornipulvina ellipsoides
0.96/72
Boliniales
AY346267 Camarops ustulinoides
1.0/69
Diaporthales
AF408350 Diaporthe eres
1.0/94
AF286408 Farrowia longicollea
FJ666353 Chaetomidium leptoderma
1.0/84
AF286403 Chaetomium globosum
Chaetomiaceae
Sordariales
65
AF286413 Thielavia cephalothecoides
AY346305 Zopfiella ebriosa
AY587951 Lasiosphaeria ovina
1.0/85
AY545728 Sordaria fimicola
AF286411 Neurospora crassa
Sordariaceae
DQ470980 Gelasinospora tetrasperma
JQ797434 Pseudohalonectria lignicola
0.65/55
1.0/57
AY346299 Pseudohalonectria lignicola
JX066706 Pseudohalonectria lutea
AY346270 Ceratosphaeria lampadophora
EU107297 Pyricularia angulata
CBS 433.70 Pyricularia oryzae
1.0/61
PH0075 Pyricularia oryzae
Legend:
1.0 / 100
>0.95 / >95
0.99/78
Br36 Pyricularia pennisetigena
Magnaporthales
JP0017 Pyricularia oryzae
CBS 133596 Pyricularia pennisetigena
BF0017 Pyricularia penniseticola
CBS 132356 Xenopyricularia zizaniicola
10 changes
Br33 Pyricularia grisea
0.98/94
BR0029 Pyricularia grisea
Fig. 1. The first of 1000 equally most parsimonious trees (Tree length = 1362, CI = 0.438, RI = 0.785 and RC = 0.343) obtained from a maximum parsimony analysis of the
LSU alignment. The bootstrap support values (integers) from 1000 replicates and the posterior probability values (values 1.0) are indicated as numbers at the nodes or as
coloured branches (see legend) and the scale bar represents 10 changes. Thickened branches reflect those branches present in the strict consensus parsimony tree. Families
are highlighted in the horizontal coloured boxes, orders in the vertical coloured boxes and classes are shown to the left of the tree. “Pyricularia” parasitica and Rhexodenticula
cylindrospora are shown in bold text. The tree was rooted to Peziza vesiculosa (GenBank DQ470948).
95
KLAUBAUF
ET AL.
Br37 Pyricularia sp.
CBS 133598 Pyricularia sp.
GR0001 Pyricularia ctenantheicola
RN0001 Pyricularia zingibericola
CBS 303.39 Proxipyricularia zingiberis
CBS 133599 Bambusicularia brunnea
CBS 133600 Bambusicularia brunnea
CBS 128308 Neopyricularia commelinicola
68
CBS 129274 Barretomyces calatheae
0.90
DQ341499 Mycoleptodiscus coloratus
1.0/63
JX134690 Ophioceras leptosporum
JX134689 Ophioceras dolichostomum
1.0/85
M92 Ophioceras commune
CBS 133595 Pseudopyricularia cyperi
CBS 665.79 Pseudopyricularia cyperi
CBS 133597 Pseudopyricularia kyllingae
0.99/88
CBS 121934 Pseudopyricularia higginsii
0.56/85
KF777238 Pyricularia bothriochloae
CBS 128780 Deightoniella roumeguerei
0.84/56
CBS 610.82 Macgarvieomyces juncicola
JX134686 Omnidemptus affinis
CBS 125232 Bussabanomyces longisporus
0.99
CBS 114973 Pyriculariopsis parasitica
CBS 118210 Kohlmeyeriopsis medullaris
0.89
CBS 610.75 Slopeiomyces cylindrosporus
0.81
CBS 388.81 Gaeumannomyces sp.
DQ341492 Buergenerula spartinae
CBS 117.83 Gaeumannomyces sp.
0.72
ATCC 64411 Magnaporthiopsis poae
0.96
0.93
M23 Magnaporthiopsis rhizophila
1.0
M51 Magnaporthiopsis incrustans
0.69
CBS 664.82 Magnaporthiopsis maydis
CBS 332.53 Nakataea oryzae
CBS 252.34 Nakataea oryzae
0.98
JF414887 Nakataea oryzae
0.91
1.0/83
CBS 905.73 Gaeumannomyces graminis var. tritici
M55 Gaeumannomyces graminis var. tritici
CBS 235.32 Gaeumannomyces graminis var. graminis
Legend:
1.0 / 100
>0.95 / >95
10 changes
0.59
R3-111a-1 Gaeumannomyces graminis var. tritici
0.95/81
CBS 350.77 Harpophora sp.
CBS 870.73 Gaeumannomyces graminis var. avenae
0.94/50
CBS 187.65 Gaeumannomyces graminis var. avenae
CBS 296.53 Harpophora radicicola
CPC 18682 Harpophora radicicola
86 CPC 18683 Harpophora radicicola
Fig. 1. (Continued).
96
Magnaporthales (continued)
CBS 461.65 Macgarvieomyces borealis
1.0/60
RESOLVING
PYRICULARIA
Cryphonectria parasitica EP155
100
10 changes
Pyriculariaceae
88
Ophioceraceae
CBS 894.70 Oph. leptosporum
CBS 114926 Oph. dolichostomum
98
Ophioceras
M91
Oph. commune
100
M92 Oph. commune
Barretomyces
CBS 129274 Bar. calatheae
CBS
133599
Bam.
brunnea
100
Bambusicularia
CBS 133600 Bam. brunnea
100
CBS 128780 Dei. roumeguerei
Deightoniella
CBS 461.65 Mac. borealis
80
Macgarvieomyces
CBS 610.82 Mac. juncicola
100 CBS 133597 Pse. kyllingae
68
PH0054 Pse. kyllingae
97
CBS 121934 Pse. higginsii
Pseudopyricularia
CBS 133595 Pse. cyperi
100 CBS 665.79 Pse. cyperi
CBS 128306 Neo. commelinicola
50
100
Neopyricularia
CBS 133594 Proxipyricularia zingiberis
CBS 132355 Proxipyricularia zingiberis
100
Proxipyricularia
63 CBS 132195 Proxipyricularia zingiberis
CBS 303.39 Proxipyricularia zingiberis
CBS 132356 Xen. zizaniicola
Xenopyricularia
100
GR0001 Pyr. ctenantheicola
CBS 133596 Pyr. pennisetigena
81
BR0067 Pyr. pennisetigena
88
BF0017 Pyr. penniseticola
99
BR0029 Pyr. grisea
100
CR0024 Pyr. grisea
CBS 133598 Pyricularia sp.
97
RN0001 Pyr. zingibericola
JP0028 Pyr. oryzae
PH0075 Pyr. oryzae
CBS 658.66 Pyr. oryzae
63
GN0001 Pyr. oryzae
99 CD0156 Pyr. oryzae
BF0028 Pyr. oryzae
Pyricularia
PH0063 Pyr. oryzae
61
PH0062 Pyr. oryzae
PH0079 Pyr. oryzae
CR0020 Pyr. oryzae
PH0077 Pyr. oryzae
VT0032 Pyr. oryzae
74
JP0039 Pyr. oryzae
ATCC MYA-4617 Pyr. oryzae
CD0067 Pyr. oryzae
100
Fig. 2. The first of two equally most parsimonious trees (Tree length = 2483, CI = 0.416, RI = 0.879 and RC = 0.365) obtained from a maximum parsimony analysis of the
combined LSU/RPB1 alignment. The bootstrap support values from 1000 replicates are indicated at the nodes and the scale bar represents the number of changes. Thickened
branches reflect those branches present in the strict consensus tree. Genera are highlighted in the horizontal coloured boxes, families in the vertical coloured boxes and novel
species and families are shown in bold text. The tree was rooted to Cryphonectria parasitica strain EP155.
97
KLAUBAUF
ET AL.
98
Bussabanomyces
Omnidemptus
Kohlmeyeriopsis
Slopeiomyces
Nakataea
Buergenerula
Magnaporthiopsis
Harpophora
Gaeumannomyces
Magnaporthaceae
CBS 125232 Bus. longisporus
ATCC 200212 Omn. affinis
100 CBS 117849 Koh. medullaris
98
CBS 118210 Koh. medullaris
51
CBS 388.81 “Gaeumannomyces” sp.
CBS 609.75 Slo. cylindrosporus
78
100
100
CBS 332.53 Nakataea sp.
CBS 243.76 Nak. oryzae
100
ATCC 44754 Nak. oryzae
100
ATCC 22848 Bue. spartinae
CBS 117.83 “Gaeumannomyces” sp.
100
59 98 M23 Mag. rhizophila
ATCC 64411 Mag. poae
M47 Mag. poae
88
M35 Mag. incrustans
100
M51
Mag. incrustans
100
CBS 662.82A Mag. maydis
64
CBS 663.82A Mag. maydis
100
CBS 663.82B Mag. maydis
92
CBS 664.82 Mag. maydis
100
100 CPC 18682 Har. radicicola
CPC 18683 Har. radicicola
100
CBS 296.53 Har. radicicola
100
CBS 149.85 Har. radicicola
M55 Gae. graminis var. tritici
67
CBS 235.32 Gae. graminis var. graminis
84
94
M33 Gae. graminis var. graminis
98 CBS 902.73 Gae. graminis var. graminis
100
96
R3-111a-1 Gae. graminis var. tritici
79
CBS 870.73 Gae. graminis var. avenae
87
CBS 187.65 Gae. graminis var. avenae
10 changes
61 CBS 249.29 Gae. graminis var. tritici
CBS 905.73 Gae. graminis var. tritici
63
RESOLVING
PYRICULARIA
CBS 129274 Barretomyces calatheae
100 CBS 133599 Sasa sp. Japan
Bambusicularia brunnea
CBS 133600 Phyllostachys bambusoides Japan
100 CBS 128303 Commelina communis South Korea
Neopyricularia commelinicola
CBS 128308 Commelina communis South Korea
CBS 133594 Zingiber mioga Japan
100
100 CBS 132196 Zingiber mioga Japan
78 CBS 132355 Zingiber mioga Japan
CBS 132195 Zingiber mioga Japan
64
CBS 303.39 Zingiber officinale Japan
80
CBS 128780 Phragmites australis Netherlands
Deightoniella roumeguerei
CBS 461.65 Juncus effusus United Kingdom
Macgarvieomyces borealis
86
94
CBS 610.82 Juncus effusus Netherlands
Macgarvieomyces juncicola
75
100 CBS 133597 Kyllinga brevifolia Japan
Pseudopyricularia kyllingae
PH0054 Cyperus brevifolius Philippines
100
CBS 121934 Typha orientalis New Zealand
Pseudopyricularia higginsii
CBS
133595
Cyperus
iria
Japan
100
Pseudopyricularia cyperi
PH0053 Cyperus rotundus Philippines
100 CBS 132356 Zizania latifolia Japan
Xenopyricularia zizaniicola
CBS 133593 Zizania latifolia Japan
BR0093 Echinochloa colona Brazil
ML0036 Pennisetum sp. Mali
100 BR0067 Cenchrus echinatus Brazil
97
Pyricularia pennisetigena
US0045 Pennisetum glaucum USA
CBS 133596 Cenchrus ciliaris Japan
PH0047 Cenchrus echinatus Philippines
100 GR0001 Ctenanthe oppenheimiana Imported into Greece
Pyricularia ctenantheicola
100
GR0002 Ctenanthe oppenheimiana Imported into Greece
CD0143 Digitaria exilis Côte d'Ivoire
100
ML0048 Digitaria exilis Mali
100 CD0086 Pennisetum typhoides Côte d'Ivoire
Pyricularia penniseticola
86
ML0031 Pennisetum typhoides Mali
BF0017 Pennisetum typhoides Burkina Faso
100 CD0180 Pennisetum sp. Côte d'Ivoire
BR0029 Digitaria sanguinalis Brazil
US0043 Digitaria sp. USA
CBS 128304 Echinochloa crus-galli var. frumentacea Korea
100
Pyricularia grisea
PH0055 Digitaria ciliaris Philippines
72
CR0024 Lolium perenne South Korea
51
JP0034 Digitaria smutsii Japan
CBS 133598 Leersia oryzoides Japan
Pyricularia sp.
81
RN0001 Zingiber officinale Réunion
Pyricularia zingibericola
BF0028 Paspalum sp. Burkina Faso
GN0001 Zea mays Gabon
80
JP0028 Eragrostis curvula Japan
BR0032 Triticum sp. Brazil
100
PH0075 Brachiaria mutica Philippines
CD0156 Eleusine indica Côte d'Ivoire
93
PH0035 Brachiaria mutica Philippines
25 changes
Pyricularia oryzae
CR0021 Panicum miliaceum South Korea
CR0020 Phleum pratense South Korea
74 RW0012 Eleusine coracana Rwanda
PH0079 Panicum repens Philippines
PH0014 Oryza sativa Philippines
70 CBS 657.66 Oryza sativa Egypt
CD0067 Leersia hexandra Côte d'Ivoire
Fig. 3. The first of 192 equally most parsimonious trees (Tree length = 2587, CI = 0.563, RI = 0.821 and RC = 0.462) obtained from a maximum parsimony analysis of the
combined ACT/ITS/RPB1 alignment. The bootstrap support values from 1000 replicates are indicated at the nodes and the scale bar represents the number of changes.
Thickened branches reflect those branches present in the strict consensus tree. Species are highlighted in the coloured boxes and ex-type strain numbers and novel species
are shown in bold text. The tree was rooted to Barretomyces calatheae strain CBS 129274.
99
KLAUBAUF
ET AL.
parsimony-uninformative and 488 were parsimony informative
(LSU: 539, 74, 135 characters respectively and RPB1: 233, 57,
353 characters respectively). Two equally most parsimonious
trees were retained from this analysis (Tree length = 2 483,
CI = 0.416, RI = 0.879 and RC = 0.365), the first of which is
shown in Fig. 2. This phylogenetic tree delimited three families,
of which two are described as new (Ophioceraceae, Pyriculariaceae), and 19 genus clades, ten of which represent novel
genera, described in the Taxonomy Section. A further two lineages represent “Gaeumannomyces” spp., but these species
defined clades distinct from other known species of the genus
and are not treated further here.
To improve the resolution of the clades within Pyriculariaceae,
we combined ACT/ITS/RPB1 sequences. The combined dataset
consists of 56 sequences including Barretomyces calatheae as
outgroup, since it defines a clade basal to other species from this
family (Fig. 2). This dataset contains 1 866 characters, of which
the ACT dataset contributed 364 characters, the ITS dataset
contributed 507 characters and the RPB1 dataset contributed 995
characters: 1 018 characters were constant, 118 were variable
and parsimony-uninformative and 730 were parsimony informative (ACT: 94, 34, 236 characters respectively, ITS: 324, 27, 156
characters respectively, and RPB1: 600, 57, 338 characters
respectively). A total of 192 equally most parsimonious trees were
retained from this analysis (Tree length = 2 587, CI = 0.563,
RI = 0.821 and RC = 0.462), the first of which is shown in Fig. 3.
The phylogenetic tree delimited 17 species clades, seven of which
represent novel species described in in the Taxonomy section.
Taxonomy
Magnaporthales Thongk., Vijaykr. & K.D. Hyde, Fungal
Diversity 34: 166. 2009.
Magnaporthaceae P.F. Cannon, Systema Ascomycetum
13: 26. 1994.
Ascomata perithecial, immersed, scattered to separate, globose
to subglobose, black, with long unilateral, cylindrical, black,
periphysate neck; wall of several layers of textura epidermoidea.
Paraphyses hyaline, thin-walled, septate, intermingled among
asci. Asci 8-spored, subcylindrical, unitunicate, short-stipitate or
not, with a large apical ring staining in Meltzer’s iodine reagent.
Ascospores curved to sigmoid, septate, filiform or fusoid, hyaline
to olivaceous, with bluntly rounded ends, lacking sheath.
Mycelium with simple to lobed brown appressoria. Asexual
morphs hyphomycetous, at times formed from sclerotia, with
simple, unbranched or branched conidiophores. Conidiogenous
cells integrated, pigmented, phialidic with collarettes, or denticulate. Conidia hyaline to pale brown, septate to aseptate, variable in shape, straight or curved.
Type genus: Nakataea Hara (= Magnaporthe R.A. Krause & R.K.
Webster)
Type species: Nakataea oryzae (Catt.) J. Luo & N. Zhang
Genera included: Buergenerula, Bussabanomyces, Endopyricularia, Gaeumannomyces, Harpophora, Kohlmeyeriopsis,
Magnaporthiopsis, Nakataea, Omnidemptus, Pyriculariopsis and
Slopeiomyces.
100
Notes: Other than being phylogenetically distinct, the Magnaporthaceae is distinguished from the Pyriculariaceae by their
asexual morphs, which are either phialophora-like, or with falcate
versicoloured conidia on brown, erect conidiophores.
Bussabanomyces Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810195.
Etymology: Named after Dr. B. Bussaban, who collected this
fungus from Chiang Mai, Thailand.
Mycelium consisting of verruculose, pale brown, branched,
septate hyphae. Conidiophores macronematous, rarely
branched, straight, septate, pale brown near the base, subhyaline at the apex. Conidiogenous cells cylindrical, terminal,
denticulate; denticles cylindrical, thin-walled, mostly cut off by a
septum to form a separating cell. Conidia solitary, dry, obclavate,
hyaline to pale brown, smooth, 4(–5)-septate.
Type species: Bussabanomyces longisporus (Bussaban) Klaubauf, Lebrun & Crous
Notes: Morphologically similar to Pyricularia, but distinct in that
conidiophores are usually unbranched, with terminal conidiogenous cells that give rise to 4(–5)-septate, pale brown
conidia.
Bussabanomyces longisporus (Bussaban) Klaubauf,
Lebrun & Crous, comb. nov. MycoBank MB810196.
Basionym: Pyricularia longispora Bussaban, Mycologia 95: 520.
2003.
Illustrations: See Bussaban et al. (2003).
Mycelium consisting of verruculose, pale brown, branched,
septate hyphae, 3–5 μm diam. Conidiophores macronematous,
up to 400 μm long, 3–4.6 μm diam, rarely branched, straight,
septate, pale brown near the base, subhyaline at the apex.
Conidiogenous cells cylindrical, denticulate; each denticle cylindrical, thin-walled, mostly cut off by a septum to form a separating cell. Conidia 47–72 × 5.6–7.6 μm, solitary, dry, obclavate,
hyaline to pale brown, smooth, 4(–5)-septate. (Description from
Bussaban et al. 2003).
Culture characteristics: Colonies on MEA pale olivaceous-grey,
irregularly raised with a hairy edge, velutinous, reaching
2.3–2.4 cm after 1 wk; reverse umber to chestnut. Similar
appearance on CMA and OA with slightly bigger colony diameters, 2.6–3.1 cm. On PDA colonies were olivaceous, with
central tufts. No sporulation was observed.
Material examined: Thailand, Chiang Mai, Doi Suthep-Pui National Park, isolated
as an endophyte from leaves of Amomum siamense, Feb. 2000, B. Bussaban
(holotype BCC11377, culture ex-type CBS 125232).
Harpophora W. Gams, Stud. Mycol. 45: 192. 2000.
Mycelium consisting of olivaceous-brown hyphae, with typical
“runner hyphae” and narrower lateral hyphae. Conidiogenous
cells phialidic, resembling those of Phialophora, solitary on hyphae or aggregated in clusters, faintly pigmented, with a conspicuous, divergent collarette. Conidia borne in slimy heads,
RESOLVING
cylindrical, but prominently curved, hyaline. (Description from
Gams 2000).
Type species: Harpophora radicicola (Cain) W. Gams
Harpophora radicicola (Cain) W. Gams, Stud. Mycol. 45:
192. 2000.
Basionym: Phialophora radicicola Cain, Canad. J. Bot. 30: 340.
1952.
= Phialophora zeicola Deacon & D.B. Scott, Trans. Brit. mycol. Soc. 81:
256. 1983.
≡ Harpophora zeicola (Deacon & D.B. Scott) W. Gams, Stud. Mycol. 45:
192. 2000.
Materials examined: Canada, Ontario, Chatham, on Zea mays, 1950, R.F. Cain,
isotypes of P. radicicola, specimens CBS H-7592, 7593, cultures ex-isotype
CBS 296.53 = MUCL 28970 = TRTC 23660. South Africa, on Zea mays, isotype of P. zeicola, specimens PREM 45754, CBS H-7597, culture ex-isotype
CBS 149.85.
PYRICULARIA
Kohlmeyeriopsis medullaris (Kohlm., Volkm.-Kohlm. &
O.E. Erikss.) Klaubauf, Lebrun & Crous, comb. nov.
MycoBank MB810198.
Basionym: Gaeumannomyces medullaris Kohlm., Volkm.-Kohlm.
& O.E. Erikss., Mycologia 87: 540. 1995.
= Trichocladium medullare Kohlm. & Volkm.-Kohlm., Mycotaxon 53:
349. 1995.
Illustrations: See Kohlmeyer et al. (1995).
Materials examined: USA, North Carolina, Broad Creek, Carteret County, on
Juncus roemerianus, isol. Kohlmeyer JK5528S, deposited by C. Schoch, CBS
117849; North Carolina, Broad Creek, Carteret County, on Juncus roemerianus,
isol. Kohlmeyer JK 5522N, deposited by C. Schoch, CBS 118210.
Magnaporthiopsis J. Luo & N. Zhang, Mycologia 105:
1021. 2013.
Notes: When Gams (2000) introduced the genus Harpophora, it
was assumed to be the asexual morph of Gaeumannomyces.
The latter genus however, clusters apart in the Magnaporthaceae, and has harpophora-like asexual morphs. Furthermore,
based on phylogenetic analyses of several isolates of H. zeicola
from South Africa (Fig. 1), as well as the ex-type isolate of
H. radicicola and H. zeicola, the latter must be reduced to synonymy under the older name H. radicicola.
Plant pathogenic. Ascomata perithecial, solitary or gregarious,
superficial or immersed, globose, with a cylindrical neck, black,
smooth; wall consisting of two layers. Asci unitunicate, clavate,
with a refractive ring. Ascospores fusoid, septate, hyaline or
yellow-brown, smooth, biseriate. Paraphyses hyaline, septate,
branched. Hyphopodia simple. Conidiophores solitary, branched
or not. Conidiogenous cells phialidic, hyaline. Conidia subglobose to ovoid, aseptate, hyaline, smooth. (Description from
Luo & Zhang 2013).
Kohlmeyeriopsis Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810197.
Type species: Magnaporthiopsis poae (Landsch. & N. Jacks.) J.
Luo & N. Zhang
Etymology: Named after Jan Kohlmeyer and Brigitte VolkmannKohlmeyer, who dedicated their careers to studying marine fungi,
and collected this genus in the process.
Notes: Luo & Zhang (2013) introduced Magnaporthiopsis to
accommodate species with black, globose perithecia with long
cylindrical necks, clavate asci with an apical ring, septate, fusoid
ascospores, and a harpophora-like asexual morph.
Ascomata ellipsoid, immersed, ostiolate, dark brown, solitary, with
long cylindrical periphysate necks, lateral or central; wall consisting of 3–4 layers of textura angularis. Paraphyses hyaline,
septate, unbranched. Asci 8-spored, fusoid to cylindrical, short
stipitate, unitunicate, with a large apical ring staining in Meltzer’s
iodine reagent. Ascospores filamentous, tapering towards the
base, indistinctly septate, hyaline, coiled in the ascus, producing
appressoria at germination. Asexual morph trichocladium-like.
Mycelium consisting of pale brown, septate, branched hyphae.
Conidiophores reduced to conidiogenous cells, short, with lateral
branches, giving rise to conidia. Conidia 2-celled, with a brown,
large ellipsoidal, rarely with kidney-shaped apical cell, and 1–2
small, cylindrical or doliiform, pale brown basal cells.
Type species: Kohlmeyeriopsis medullaris (Kohlm., Volkm.Kohlm. & O.E. Erikss.) Klaubauf, Lebrun & Crous
Notes: Gaeumannomyces medullaris was originally described
from dead culms of Juncus roemerianus in North Carolina
(Kohlmeyer et al. 1995). They described it as an aggressive
cellulose decomposer, specific to the marine environment,
commonly forming the trichocladium-like asexual morph in culture (Kohlmeyer & Volkmann-Kohlmeyer 1995). The genus
Gaeumannomyces has harpophora-like asexual morphs, and the
genus Trichocladium is heterogeneous (Seifert et al. 2011), and
genetically unrelated to this fungus, for which a new genus is
introduced.
Magnaporthiopsis maydis (Samra, Sabet & Hing.)
Klaubauf, Lebrun & Crous, comb. nov. MycoBank
MB810225.
Basionym: Cephalosporium maydis Samra, Sabet & Hing.,
≡ Harpophora maydis (Samra, Sabet & Hing.) W. Gams, Stud. Mycol.
45: 192. 2000.
Materials examined: Bihar, Messina, on Zea mays hybrid “Ganga Safed 2”, Mar
1976, M.M. Payak, CBS 664.82. Egypt, on Zea mays, Dec. 1982, H.A. Elshafey,
CBS 662.82A. India, Rajasthan, Jaipur, on Zea mays, Dec. 1982, B.S. Siradhana, CBS 663.82A, CBS 663.82B.
Notes: Gams (2000) introduced the genus Harpophora, based
on H. radiciola for a group of species that are phialophora-like in
morphology, with cylindrical, curved conidia. Harpophora is
however heterogeneous (e.g. Gaeumannomyces has
harpophora-like asexual morphs), and H. maydis clusters with
species of Magnaporthiopsis (see Fig. 2), hence a new combination is introduced to accommodate it.
Nakataea Hara, The diseases of the rice-plant, 2nd ed.:
185. 1939.
= Nakataea Hara, Nippon-gaikingaku: 318. 1936. nom. nud.
Plant pathogenic. Sclerotia spherical to subspherical, black,
formed on the host and in culture. Ascomata perithecial, globose,
101
KLAUBAUF
ET AL.
dark brown, immersed in leaf sheaths; wall consisting of 5–12
layers of thick-walled dark cells; neck frequently protruding from
the leaf tissue. Asci 8-spored, subcylindrical, thin-walled, shortstipitate, deliquescing at maturity, spirally twisted, 3-septate,
slightly constricted at septa, fusiform, curved, granular, with
median cells turning yellowish brown. Conidiophores solitary,
erect, brown, smooth, branched or not, septate, with integrated
terminal conidiogenous cells forming a rachis with several denticles, each separated from the conidiogenous cell by a septum.
Conidia solitary, falcate to sigmoid, smooth, 3-septate, widest in
the middle, end cells hyaline, median cells medium brown.
Type species: Nakataea sigmoidea (Cavara) Hara
Nakataea oryzae (Catt.) J. Luo & N. Zhang, Mycologia
105: 1025. 2013.
Basionym: Sclerotium oryzae Catt., Arch. Triennale Lab. Bot.
Crittog. 1: 10. 1877.
= Helminthosporium sigmoideum Cavara, Mat. Lomb.: 15. 1889.
≡ Nakataea sigmoidea (Cavara) Hara, as “sigmoideum”, Nippongaikingaku: 318. 1936. nom. nud.
≡ Nakataea sigmoidea (Cavara) Hara, as “sigmoideum”, The diseases
of the rice-plant 2nd ed.: 185. 1939.
= Leptosphaeria salvinii Catt., Arch. Labor. Bot. Critt. Univ. Pavia 2, 3:
126. 1879.
≡ Magnaporthe salvinii (Catt.) R.A. Krause & R.K. Webster, Mycologia
64: 110. 1972.
Additional synonyms listed in MycoBank.
Materials examined: Burma, on straw of Oryza sativa, date and collector unknown, CBS 252.34. Italy, no collection details, CBS 202.47; on Oryza sativa, sent
to CBS for identification by Centro di Ricerche sul Riso, Mortara, Italy, Nov 1975,
collector unknown, specimen CBS H-14204, culture CBS 243.76. Japan, on
Oryza sativa, date and collector unknown, ATCC 44754 = M21 = Roku-2; Takada,
on stem of Oryza sativa, date and collector unknown, CBS 288.52. USA, Calivornia, Davis, on Oryza sativa, Dec. 1974, R.K. Webster, specimens CBS H14203; CBS H-14205, cultures CBS 726.74, CBS 727.74. Unknown, CBS 253.34.
Notes: The genus Nakataea (based on N. sigmoidea, described
from rice in Italy) has some similarity to Pyricularia in general
morphology, but differs in having falcate conidia with darker
median cells (Luo & Zhang 2013). Magnaporthe oryzae (=M.
salvinii), the type of Magnaporthe, forms a Nakataea asexual
morph, and hence Luo & Zhang (2013) introduced the combination N. oryzae for this fungus, as the name Nakataea (1939) is
older than Magnaporthe (1972). This decision effectively reduced
Magnaporthe to synonymy under Nakataea. The majority of
species formerly treated as Magnaporthe, fall in the Pyricularia
complex (Murata et al. 2014).
Pyriculariopsis M.B. Ellis, In: Ellis, Dematiaceous
Hyphomycetes (Kew): 206. 1971.
Plant pathogenic. Mycelium consisting of smooth, hyaline to
brown, branched, septate hyphae; hyphae developing chains of
globose, swollen chlamydospores that give rise to black microsclerotia. Conidiophores forming from hyphae or microsclerotia,
solitary, erect, straight or curved, unbranched, medium brown,
thick-walled, smooth, subcylindrical, septate; base bulbous,
lacking rhizoids. Conidiogenous cells integrated, terminal, medium brown, smooth, forming a rachis with several protruding
denticles, and minute marginal frill due to rhexolytic secession.
Conidia solitary, obclavate, smooth, guttulate, 3-septate, two
median cells brown, apical and basal cell olivaceous to
102
subhyaline; hilum truncate, slightly protruding, with marginal frill,
unthickened, not darkened; apex tapering, subacutely rounded,
with persistent mucoid cap.
Type species: Pyriculariopsis parasitica (Sacc. & Berl.) M.B. Ellis
Pyriculariopsis parasitica (Sacc. & Berl.) M.B. Ellis,
Dematiaceous Hyphomycetes (Kew): 207. 1971. Fig. 4.
Basionym: Helminthosporium parasiticum Sacc. & Berl., Revue
mycol., Toulouse 11: 204. 1889.
On SNA on sterile barley seed. Mycelium consisting of smooth,
hyaline to brown, branched, septate hyphae, 3–4 μm diam; hyphae developing chains of globose, swollen chlamydospores that
give rise to black microsclerotia. Conidiophores forming from hyphae or microsclerotia, solitary, erect, straight or curved, unbranched, medium brown, thick-walled, smooth, subcylindrical,
60–180 × 6–8 μm, 3–10-septate; base bulbous, 10–16 μm diam,
lacking rhizoids. Conidiogenous cells 10–50 × 7–8 μm, integrated,
terminal, medium brown, smooth, forming a rachis with several
protruding denticles, 2–4 μm long, 3–5 μm diam, and minute
marginal frill due to rhexolytic secession. Conidia solitary, obclavate, smooth, guttulate, 3-septate, two median cells brown, apical
and basal cell olivaceous to subhyaline, (30–)40–55(–60) × (7–)
8–9(–12) μm; apical cell 18–22 μm long, basal cell 8–11 μm long;
hilum truncate, slightly protruding, 2–3 μm diam with marginal frill,
unthickened, not darkened; apex tapering, subacutely rounded,
with persistent mucoid cap, 2–3 μm diam.
Culture characteristics: Colonies on MEA with white aerial
mycelium, mouse-grey in centre, raised, cottony, round, reaching
up to 5 cm diam after 1 wk; reverse with dark mouse-grey in
centre. Colonies on CMA and OA transparent, with very thin,
spreading mycelium with scattered dark spots of sporulation,
covering full plate after 1 wk. Colonies on PDA transparent with
dark mouse-grey areas, flat, covering plate after 1 wk; reverse
with some dark spots.
Material examined: Hong Kong, Discovery Bay, Lantau Island, on leaves of
Musa sp., 5 Oct. 1999, K.D. Hyde, CBS 114973 = HKUCC 5562 = Maew HK 1.
Notes: The denticles of Pyriculariopsis are similar to those of
Pyricularia. The main difference lies in the conidium pigmentation, septation, and the persistent apical mucoid cap. In Pyricularia conidia are 2-septate, uniformly olivaceous to medium
brown, and the apical mucoid cap is not persistent, leaving the
apex with what appears to be a marginal frill surrounding the
apex (mucoid remnant?), from where the globoid mucoid cap
extended.
Slopeiomyces Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810199.
Etymology: Named after D.B. Slope, who collected this fungus
from cereal roots in Rothamsted Experimental Station, UK.
Perithecia superficial, globose, black, solitary, sometimes 2–3
aggregated, with cylindrical, black, periphysate neck bearing
hyphae; wall consisting of several layers of textura prismatica to
angularis. Paraphyses hyaline, septate, unbranched. Asci 8spored, clavate, straight to curved, with a non-amyloid apical
ring staining in Congo red. Ascospores hyaline, cylindrical to
RESOLVING
PYRICULARIA
Fig. 4. Pyriculariopsis parasitica (CBS 114973). A–G. Conidiophores sporulating on SNA, having a rachis with conidia. H. Arrows indicate conidial median cells with darker
pigmentation. Scale bars = 10 μm.
fusoid, septate, slightly curved, tapering somewhat to base,
forming appressoria at germination. Asexual morph phialophoralike. Conidiogenous cells developing on hyphae, phialidic, subcylindrical to ampulliform with flared collarette, hyaline. Conidia
hyaline, aseptate, apex rounded, pointed towards base, straight
to curved or sigmoid.
Type species: Slopeiomyces cylindrosporus (D. Hornby, Slope,
Gutter. & Sivan.) Klaubauf, Lebrun & Crous
Notes: Slopeiomyces is morphologically similar to Gaeumannomyces in the general morphology of its sexual and asexual
morphs, the production of appressoria, and its ecology, being a
root pathogen of Poaceae (Hornby et al. 1975). The only obvious
morphological difference lies in its ascospores, which are much
shorter and wider than observed in species of Gaeumannomyces. The link between S. cylindrosporus and the asexual
morph originally used in inoculation experiments, Phialophora
radiciola var. graminis, could not be confirmed. Phylogenetically,
however, Slopeiomyces is clearly distinct from Gaeumannomyces (see Fig. 2).
Slopeiomyces cylindrosporus (D. Hornby, Slope, Gutter.
& Sivan.) Klaubauf, Lebrun & Crous, comb. nov. MycoBank MB810200.
Basionym: Gaeumannomyces cylindrosporus D. Hornby, Slope,
Gutter. & Sivan., Trans. Br. mycol. Soc. 69: 21 (1977).
Materials examined: UK, on grass roots, associated with Phialophora graminicola,
Dec. 1975, D. Hornby, cultures ex-type CBS 609.75, CBS 610.75, CBS 611.75.
Ophioceraceae Klaubauf, Lebrun & Crous, fam. nov.
MycoBank MB810201.
Ascomata perithecial, immersed to superficial, scattered to
separate, globose to subglobose, black, with long cylindrical,
black, periphysate neck, pale brown at apex; wall consisting of
several layers of textura angularis. Paraphyses hyaline, thinwalled, septate, intermingled among asci. Asci 8-spored, subcylindrical to narrowly fusoid, unitunicate, short-stipitate or not,
with a large apical ring staining in Meltzer’s iodine reagent. Ascospores curved to sigmoidal, septate, filiform, hyaline to olivaceous, with bluntly rounded ends, lacking sheath.
Type genus: Ophioceras Sacc., Syll. fung. (Abellini) 2: 358. 1883.
Type species: Ophioceras dolichostomum (Berk. & M.A. Curtis)
Sacc., Syll. fung. (Abellini) 2: 358 (1883)
Genus included: Ophioceras.
Notes: Although Ophioceras is morphologically similar to
Gaeumannomyces, the two genera can be distinguished by the
aquatic habit of Ophioceras, occurring on wood and herbaceous
material, versus the plant pathogenic nature of Gaeumannomyces, which has harpophora-like asexual morphs, mycelial
appressoria, and a perithecial peridium of textura epidermoidea
(Walker 1980, Chen et al. 1999). Although the family placement
of Ophioceras was not resolved, the genus was temporarily
added to the Magnaporthaceae (established for nectrotrophic
and hemibiotrophic plant pathogens infecting root and shoots of
103
KLAUBAUF
ET AL.
Poaceae and Cyperaceae; Cannon 1994) awaiting further study
(Shearer 1989, Shearer et al. 1999, Chen et al. 1999). As shown
in the present analyses (Fig. 2) Ophioceras clearly clusters
separate from the Magnaporthaceae in the Magnaporthales, and
hence a separate family, the Ophioceraceae, is introduced to
accommodate it.
Pyriculariaceae Klaubauf, Lebrun & Crous, fam. nov.
MycoBank MB810202.
Ascomata perithecial, immersed, black, with long cylindrical
necks covered in setae. Asci subcylindrical, unitunicate, shortstipitate, with a large apical ring staining in Meltzer's iodine reagent. Paraphyses hyaline, thin-walled, septate, intermingled
among asci. Ascospores septate, fusiform, often with median
cells pigmented, lacking sheath. Asexual morphs hyphomycetous, with simple, branched conidiophores. Conidiogenous cells
integrated, pigmented, denticulate. Conidia hyaline to brown,
transversely septate, apical mucoid appendage rarely present.
Type genus: Pyricularia Sacc.
Type species: Pyricularia grisea Sacc.
Genera included: Bambusicularia, Barretomyces, Deightoniella,
Macgarvieomyces, Neopyricularia, Proxipyricularia, Pseudopyricularia, Pyricularia, Xenopyricularia.
Bambusicularia Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810203.
solitary, erect, straight or curved, unbranched, flexuous to
geniculate, dark brown, finely roughened, 280–500 × 5–7 μm,
5–11-septate; base bulbous, lacking rhizoids, 7–10 μm diam.
Conidiogenous cells 20–120 × 4–6 μm, integrated, terminal and
intercalary, pale brown at apex, intercalary cells medium brown,
finely roughened, with several protruding denticles, 1–2 μm long,
1.5–2 μm diam. Conidia solitary, ellipsoid to obclavate, medium
brown, finely roughened, granular to guttulate, 2-septate, (20–)
21–25(–27) × 10–11(–11.5) μm; apical cell 4–7 μm long, basal
cell 6–9 μm long; hilum truncate, protruding, 0.5–1 μm long,
1.5–2 μm diam.
Culture characteristics: Colonies on MEA white, round, cottony,
slightly raised, reaching 3.8 cm diam after 1 wk; reverse
ochreous. Colonies on PDA transparent with white centre, flat,
round, slightly cottony, reaching up to 3.7 cm after 1 wk, with
diffuse, hairy margin. Colonies on CMA and OA transparent,
smooth, flat, round, reaching up to 3.3 cm diam after 1 wk;
colonies fertile.
Materials examined: Japan, Aichi, on Sasa sp. (Poaceae), 1992, S. Koizumi
[holotype CBS H-21839, culture ex-type CBS 133599 = MAFF 240225 = INA-B92-45(Ss-1J)]; Aichi, on Phyllostachys bambusoides (Poaceae), 1993, S. Koizumi, CBS 133600 = MAFF 240226 = INA-B-93-19(Ph-1J).
Note: Isolate CBS 133600 sporulated poorly, and had slightly
larger conidia than CBS 133599, measuring (23–)
25–30(–34) × (7–)8–9 μm; apical cell 7–11 μm long, basal cell
7–10 μm long.
Barretomyces Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810205.
Etymology: Named after its occurrence on bamboo.
Plant pathogenic. Mycelium consisting of smooth, hyaline,
branched, septate hyphae. Conidiophores solitary, erect, straight
or curved, unbranched, flexuous to geniculate, dark brown, finely
roughened, up to 500 μm long, multi-septate; base bulbous,
lacking rhizoids. Conidiogenous cells integrated, terminal and
intercalary, pale brown at apex, intercalary cells medium brown,
finely roughened, with several protruding denticles. Conidia
solitary, ellipsoid to obclavate, medium brown, finely roughened,
granular to guttulate, 2-septate, hilum truncate, somewhat
protruding.
Type species: Bambusicularia brunnea Klaubauf, Lebrun &
Crous
Notes: The main distinguishing character between Bambusicularia and Pyricularia is in their conidiophore morphology. Conidiophores in Bambusicularia are flexuous, longer, wider and
darker brown than seen in species of Pyricularia. Conidia are
pale brown, but appear to have darker brown septa. The two
genera are also phylogenetically distinct (Figs 2, 3).
Bambusicularia brunnea Klaubauf, Lebrun & Crous, sp.
Etymology: Named after Prof. dr. Robert W. Barreto, in
acknowledgement of his contribution to mycology and plant
pathology in Brazil.
Plant pathogenic. Mycelium consisting of verruculose, pale
brown, branched, septate hyphae. Conidiophores macronematous, rarely branched, straight, septate, pale brown near
the base, subhyaline at the apex. Conidiogenous cells cylindrical,
terminal, denticulate; each denticle cylindrical, thin-walled, mostly
cut off by a septum to form a separating cell. Conidia solitary, dry,
obclavate, basal and terminal cell hyaline to pale brown, median
cell darker brown, smooth, 4(–5)-septate.
Type species: Barretomyces calatheae (D.J. Soares, F.B. Rocha
& R.W. Barreto) Klaubauf, Lebrun & Crous
Notes: Barretomyces calatheae, which is a foliar pathogen of
Calathea longifolia in Brazil (Soares et al. 2011), was originally
described in Pyriculariopsis based on its versicoloured conidia
(with paler basal cell). Furthermore, they noted this species to
have schizolytic secession, and Ellis (1971) defined Pyriculariopsis as having schizolytic secession, in contrast to the rhexolytic secession observed in Pyricularia. We have however found
conidiogenesis to be variable, and not a good taxonomic criterion
in distinguishing these genera.
Etymology: Named after its dark brown conidiophores.
hyaline, branched, septate hyphae, 2–3 μm diam. Conidiophores
104
Barretomyces calatheae (D.J. Soares, F.B. Rocha & R.W.
Barreto) Klaubauf, Lebrun & Crous, comb. nov. MycoBank
MB810206. Fig. 6.
RESOLVING
PYRICULARIA
Fig. 5. Bambusicularia brunnea (CBS 133599). A. Sporulation on sterile barley seed on SNA. B, C. Sporulation on sterile barley leaves. D–H. Conidiophores bearing conidia. I.
Conidia. Scale bars = 10 μm.
Basionym: Pyriculariopsis calatheae D.J. Soares, F.B. Rocha &
R.W. Barreto, Mycol. Prog. 10: 317. 2011.
tapered, 9–12 μm long, basal cell 7–9 μm long; base tapering
prominently to a truncate, protruding hilum, 1–1.5 μm diam.
Leaf spots amphigenous, 0.5–11 cm diam, progressing from
small yellow spots to large, circular to elliptic, grey-brown lesions,
sometimes with a darker centre and with concentric circles, the
outer region being dark-brown, surrounded by a large chlorotic
border; sometimes coalescing, leading to leaf necrosis; disease
symptoms also occurring on leaf petioles, as brown spots. On
SNA medium. Mycelium consisting of smooth, hyaline, branched,
septate hyphae, 2–3.5 μm diam. Conidiophores forming from
hyphae, solitary, erect, straight or curved, unbranched, medium
brown, smooth, 70–160 × 4–6 μm, 2–9-septate. Conidiogenous
cells 20–70 × 5–6 μm, integrated, terminal and intercalary, pale
to medium brown, smooth, forming a rachis with several protruding flat-tipped denticles, 1–3 μm long, 1–2 μm diam. Conidia
solitary, obclavate, smooth, basal and terminal cell hyaline to
pale brown, median cell darker brown, granular to guttulate, 2septate, (19–)28–32(–35) × (5.5–)6–7(–8) μm; apical cell
Culture characteristics: Colonies on MEA white, round, raised,
with a thick, furry texture, reaching 3 cm diam after 1 wk; reverse
cinnamon. Colonies on OA white with a mouse grey centre,
reaching 3.2 cm after 1 wk. Colonies on CMA white to pale
mouse grey, round with entire edge, flat, felty, exuding droplets,
reaching 3.3 cm after 1 wk, sporulating in centre. Colonies on
PDA whitish, transparent with vinaceous-buff centre, irregular in
shape, felty, reaching 2.8 cm after 1 wk.
Materials examined: Brazil, Minas Gerais, Viçosa, ‘Mata do Seu Nico’ on Calathea longifolia (Marantaceae), Dec. 2003, D.J. Soares (holotype VIC 30699,
culture ex-type culture CBMAI 1060); Minas Gerais, Viçosa, on C. longifolia, Aug.
2010, P.W. Crous, CBS 129274 = CPC 18464.
Notes: A microconidial state was observed being similar in
morphology to that reported for P. oryzae (Chuma et al. 2009,
Zhang et al. 2014), and also observed in this study for
105
KLAUBAUF
ET AL.
Fig. 6. Barretomyces calatheae (CBS 129274). A. Leaf spot on Calathea longifolia in Brazil. B–G. Conidiophores bearing conidia. H. Conidia. Scale bars = 10 μm.
P. grisea. The denticles of Barretomyces are different to those of
Pyricularia, in that they are flat-tipped, but with a central pore.
Description and illustration: Constantinescu (1983), Crous et al.
(2011).
Deightoniella S. Hughes, Mycol. Pap. 48: 27. 1952.
Material examined: Netherlands, Utrecht, De Uithof University Campus, intersection of Harvardlaan with Uppsalalaan, on leaves of Phragmites australis
growing along water canals, 14 Dec. 2010, W. Quaedvlieg (holotype of
U. cibiessiae CBS H-20594, cultures ex-type CPC 18917, 18916 = CBS
128780).
= Utrechtiana Crous & Quaedvl., Persoonia 26: 153. 2011.
Plant pathogenic. Conidiophores solitary, erect, aggregated,
brown, smooth, becoming pale brown towards apex, base
swollen, partly immersed in epidermis, but lacking rhizoids, with
circular scar where base of conidiophore is attached to immersed
hyphal network; conidiophore with swellings (twisted growth)
along its axis, swellings coinciding with internal conidiophore
proliferation (percurrently) through conidial scars; lacking transverse septa and reduced to conidiogenous cells (though some
species have a basal septum). Conidiogenous cells integrated
terminal, with truncate and flattened scar; sometimes thickened,
not darkened, nor refractive. Conidia pale brown, ellipsoid to
pyriform, guttulate to granular, finely verruculose, 1-septate slightly
above the conidial median, thin-walled, apex bluntly to acutely
rounded, base obtusely rounded with a flattened, darkened and
thickened hilum that has a central pore, and minute marginal frill.
Type species: Deightoniella africana S. Hughes
Deightoniella roumeguerei (Cavara) Constant., Proc. K.
Ned. Akad. Wet., Ser. C, Biol. Med. Sci. 86(2): 137. 1983.
Fig. 7.
Basionym: Scolicotrichum roumeguerei Cavara (as “roumegueri”), in Briosi & Cavara, Funghi Parass. Piante Colt. od Utili,
Fasc. 5: no. 112. 1890.
= Utrechtiana cibiessia Crous & Quaedvl., Persoonia 26: 153. 2011.
106
Notes: Deightoniella as presently defined is heterogeneous. The
genus Deightoniella (based on D. africana, occurring on leaves
of Imperata cylindrica var. africana; Poaceae) has solitary conidiophores, with conidiogenous cells that rejuvenate percurrently. Deightoniella is distinct from Neodeightoniella, as the latter
does not undergo percurrent rejuvenation, has conidiophores
arranged in fascicles, well-developed apical and intercalary
conidiogenous loci, and conidia with mucoid caps (Crous et al.
2013).
Macgarvieomyces Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810207.
Etymology: Named after Quentin D. MacGarvie, the Scottish
plant pathologist that first named these species.
branched, septate hyphae. Chlamydospores brown, ellipsoid,
arranged in chains. Conidiophores solitary, erect, straight or
curved, mostly unbranched, medium brown, smooth, septate.
Conidiogenous cells integrated, terminal, rarely intercalary, medium brown, smooth, forming a rachis with several protruding
denticles, appearing flat-tipped. Conidia solitary, narrowly
RESOLVING
PYRICULARIA
Fig. 7. Deightoniella roumeguerei (CBS 128780). A. Leaf spot on Phragmites australis. B. Close-up of conidiophores on leaf surface. C–G. Conidiophores bearing conidia. H.
Germinating conidium. I, J. Conidia. Scale bars = 10 μm.
obclavate, hyaline, smooth, granular and guttulate, medianly 1septate; hilum somewhat thickened, not refractive, nor darkened.
Type species: Macgarvieomyces borealis (de Hoog & Oorschot)
Klaubauf, Lebrun & Crous
Notes: MacGarvie described two species occurring on Juncus in
the genus Diplorhinotrichum. de Hoog (1985) treated this genus
as synonym of Dactylaria, but preferred to retain the plant
pathogenic species in Pyricularia. As these taxa are clearly not
congeneric with Pyricularia (Figs 2, 3), a new genus, Macgarvieomyces, is herewith introduced to accommodate them.
Macgarvieomyces borealis (de Hoog & Oorschot) Klaubauf, Lebrun & Crous, comb. nov. MycoBank MB810208.
Basionym: Pyricularia borealis de Hoog & Oorschot (as “boreale”), Stud. Mycol. 26: 114. 1985. (a nom. nov. for D. juncicola
MacGarvie 1965).
≡ Diplorhinotrichum juncicola MacGarvie, Trans. Br. mycol. Soc. 48(2):
269. 1965.
≡ Dactylaria juncicola (MacGarvie) G.C. Bhatt & W.B. Kendr., Canad. J.
Bot. 46: 1257. 1968.
Illustration: de Hoog (1985).
On OA. Conidiophores scattered, pale olivaceous-brown, thickwalled near the base, 7–9 μm diam, tapering towards the apex,
30–70 μm long, 1–3-septate. Conidiogenous cells apical, with
flat-tipped denticles, 2 μm diam, unthickened, not pigmented.
Conidia solitary, 1–4 per conidiogenous cell, subhyaline,
ellipsoid with obtuse apex, tapering in basal cell towards
obconically truncate base, slightly constricted at median septum,
16–17(–40) × 6–9 μm. (Description from de Hoog 1985).
Culture characteristics: Colonies on MEA buff to rosy buff with
entire edge, umbonate to conical colony with somewhat velvety
texture, reaching up to 3.3 cm diam after 2 wk; reverse ochreous
and buff towards the edge. Colonies on CMA and OA transparent
with smooth surface, reaching up to 3.5 cm diam after 2 wk. On
PDA whitish to buff colony with honey centre, irregular outline,
slightly furrowed in centre, reaching up to 3 cm diam after 2 wk;
colony reverse whitish to buff with honey centre. No sporulation
was observed.
Material examined: UK, Scotland, Moorland near Carnwat in Lanarkshire, 275 m
alt. and near East Graigs, Edinburgh, 33 m alt., associated with leaf spots on
Juncus effusus, Apr 1964, G.D. MacGarvie, culture ex-type CBS 461.65.
Macgarvieomyces juncicola (MacGarvie) Klaubauf,
Lebrun & Crous, comb. nov. MycoBank MB810209. Fig. 8.
Basionym: Pyricularia juncicola MacGarvie, Scientific Proc. R.
Dublin Soc., Ser. B 2(no. 16): 155. 1968.
hyaline, branched, septate hyphae, 1.5–2 μm diam. Chlamydospores arranged in intercalary chains, ellipsoid, hyaline to
pale brown, smooth, 5–7 μm diam, frequently giving rise to
conidiophores. Conidiophores solitary, erect, straight or curved,
mostly unbranched, medium brown, smooth, 50–200 × 3–5 μm,
with basal septum, developing additional septum if branched.
107
KLAUBAUF
ET AL.
Fig. 8. Macgarvieomyces juncicola (CBS 610.82). A. Colony sporulating on OA. B–G. Conidiophores and conidia forming on SNA. H. Conidia. Scale bars = 10 μm.
Conidiogenous cells 50–180 × 3–5 μm, integrated, terminal,
rarely intercalary, medium brown, smooth, forming a rachis with
several protruding denticles, 1.5–2 μm long, 1–1.5 μm diam.
Conidia solitary, narrowly obclavate, hyaline, smooth, granular
and guttulate, medianly 1-septate, (17–)25–30(–32) × (4–)
5 μm; hilum somewhat thickened, 1–1.5 μm diam.
with sympodial growth. Conidiogenous cells terminal and intercalary, olivaceous, with denticulate conidiogenous loci, slightly
darkened, and rhexolitic secession. Conidia solitary, formed
sympodially, pyriform to obclavate, narrowed toward tip, rounded
at the base, 2-septate, subhyaline to pale brown, with a distinct
protruding basal hilum, and minute marginal frill.
Culture characteristics: Colonies on MEA isabelline with pale
olivaceous grey central mycelium, slightly raised wool-like texture,
round and hairy edge, reaching up to 2.6 cm after 1 wk; reverse
iron grey. On CMA and OA olivaceous to grey olivaceous, flat,
smooth and velutinous surface, undulate edge. Colonies fertile on
MEA, CMA and OA. Colonies on PDA white with buff centre,
round, flat, fringed edge, reverse white with buff centre.
Type species: Neopyricularia commelinicola (M.J. Park & H.D.
Shin) Klaubauf, Lebrun & Crous
Material examined: Netherlands, on stem base of Juncus effusus, 3 Nov. 1982,
G.S. de Hoog, specimens CBS H-11668; CBS H-1764; CBS H-17648, culture
CBS 610.82.
Note: Macgarvieomyces borealis and M. juncicola can be
distinguished based on conidial dimensions, because conidia of
M. juncicola are on average longer and narrower.
Neopyricularia Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810210.
Etymology: Named after its morphological similarity to
Pyricularia.
Plant pathogenic. Conidiophores solitary or in fascicles, subcylindrical, erect, olivaceous, smooth, rarely branched, septate,
108
Neopyricularia commelinicola (M.J. Park & H.D. Shin)
Klaubauf, Lebrun & Crous, comb. nov. MycoBank
MB810507. Fig. 9.
Basionym: Pyricularia commelinicola M.J. Park & H.D. Shin,
Mycotaxon 108: 452. 2009.
Description: Park & Shin (2009).
Materials examined: South Korea, Hongcheon, Bukbang-ri, 37°480 100 N,
127°510 900 E, on leaves of Commelina communis, 9 Sep. 2007, H.D. Shin & M.J.
Park (holotype KUS (F) 22838, culture ex-type CBS 128308 = KACC 43081);
Hongcheon, on C. communis, 30 June 2009, H.D. Shin & M.J. Park, CBS
128303 = KACC 44637; Pocheon, on C. communis, 29 July 2008, M.J. Park,
CBS 128306 = KACC 43869; Hongcheon, on C. communis, 27 Oct. 2008, H.D.
Shin & M.J. Park, CBS 128307 = KACC 44083.
Notes: Characteristic for this species is its long, flexuous,
branched, pale brown, smooth conidiophores, with a terminal
rachis, with terminal and intercalary conidiogenous cells with
denticle-like loci that are 2–3 μm long and wide, not thickened,
but trapping air (also in conidial hila), so appearing thickened.
RESOLVING
PYRICULARIA
Fig. 9. Neopyricularia commelinicola (CBS 128308). A. Sporulation on sterile barley seed on SNA. B. Conidiophores and conidia. C. Conidia. Scale bars = 10 μm.
Conidia are pyriform to obclavate, subhyaline to pale brown, 2septate, (27–)30–38(–40) × (9–)10–11(–13) μm (on SNA).
Phylogenetically P. commelinicola does not cluster within clades
corresponding to species of Pyricularia s. str. (Figs 2, 3), and
hence a new genus is introduced to accommodate it.
Proxipyricularia Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810211.
Etymology: Named after the fact that it is morphologically similar
to the genus Pyricularia.
Plant pathogenic. Conidiophores solitary or in fascicles, subcylindrical, erect, olivaceous to medium brown, smooth, septate.
Conidiogenous cells terminal and intercalary, pale brown, with
denticulate conidiogenous loci and rhexolitic secession. Conidia
solitary, formed sympodially, pyriform to obclavate, narrowed
toward tip, rounded at the base, 2-septate, subhyaline to pale
brown, with a distinct protruding basal hilum, frequently with
minute marginal frill.
Type species: Proxipyricularia zingiberis (Y. Nisik.) Klaubauf,
Lebrun & Crous
Note: Proxipyricularia is morphologically similar to Pyricularia,
but phylogenetically distinct (Figs 2, 3).
Proxipyricularia zingiberis (Y. Nishik.) Klaubauf, Lebrun
& Crous, comb. nov. MycoBank MB810212. Fig. 10.
Basionym: Pyricularia zingiberis Y. Nishik. (as “Piricularia zingiberi”), Ber. Ohara Inst. Landwirt. Forsch. 1(2): 216. 1917.
On SNA on sterile barley seed. Conidiophores solitary or in
fascicles, subcylindrical, erect, olivaceous to medium brown,
smooth, 2–4-septate, 50–180 × 1.5–4 μm. Conidiogenous cells
terminal and intercalary, pale brown, with denticulate conidiogenous loci and rhexolitic secession. Conidia
14–20(–24) × (5–)6–8(–9.5) μm, apical cell 5–8 μm long,
basal cell 5–7 μm long, solitary, pyriform to obclavate, narrowed
toward tip, rounded at the base, 2-septate, subhyaline to pale
brown, with a distinct protruding basal hilum and marginal frill.
Materials examined: Japan, Hyogo, on Zingiber mioga, 2002, H. Kato, CBS
133594 = MAFF 240222 = HYZiM201-0-1(Z-2J); location unknown, on Zingiber
officinale, Jan 1939, Y. Nisikado, CBS 303.39 = MUCL 9449; Hyogo, on Zingiber
mioga, 2003, I. Chuma, CBS 132195 = MAFF 240224 = HYZiM201-1-1-1(Z-4J);
Hyogo, on Zingiber mioga, 2003, I. Chuma, CBS 132196 = MAFF
240223 = HYZiM202-1-2(Z-3J); Hyogo, on Zingiber mioga, 1990, M. Ogawa,
CBS 132355 = MAFF 240221 = HYZiM 101-1-1-1(Z-1J).
Notes: Proxipyricularia zingiberis is phylogenetically distant (Figs
2, 3) from Pyricularia s. str., although morphologically, it appears
similar, with medium brown conidiophores and a terminal and
intercalary denticulate rachis, and subhyaline, 2-septate, obclavate conidia. Isolates of P. zingiberis from Zingiber mioga and
Z. officinale are able to infect both plants, but not Oryza, Setaria
or Panicum spp. (Nishikado 1917, Kato et al. 2000). Nishikado
(1917) regarded the fungus from Zingiber as genetically distant
from Pyricularia species isolated from rice or other Poaceae, as
well as (Kato et al. 2000) using RFLP patterns and (Hirata et al.
2007) using multilocus sequence analysis.
Pseudopyricularia Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810213.
Pyricularia.
branched, septate hyphae. Conidiophores solitary, erect, straight
or curved, branched or not, medium brown, finely roughened,
septate. Conidiogenous cells integrated, terminal, rarely intercalary, medium brown, finely roughened, forming a rachis with
several protruding, flat-tipped denticles. Conidia solitary, obclavate, pale to medium brown, finely roughened, guttulate, 2-septate;
hilum truncate, slightly protruding, unthickened, not darkened.
Type species: Pseudopyricularia kyllingae Klaubauf, Lebrun &
Crous
Fig. 10. Proxipyricularia zingiberis (CBS 133594). A. Conidiophore forming on
SNA. B. Conidia. Scale bars = 10 μm.
Notes: Several isolates previously identified as representative of
P. higginsii were found to belong to a complex of three related
109
KLAUBAUF
ET AL.
species (Fig. 3) classified into Pseudopyricularia (P. cyperi, P.
kyllingae and P. higginsii). Taxa in this complex are primarily
distinguished from Pyricularia s. str. by having short, determinate, brown conidiophores with an apical rachis with flat-tipped
denticles. It was also based on this character, that Ellis (1976)
originally suspected P. higginsii to represent a species of
Dactylaria.
Notes: The distinguishing character of this species is its conidiophores that are commonly branched, forming a rachis with
flat-tipped denticles. Morphologically it is similar to P. higginsii,
except that conidia are longer and narrower in culture
(26.1–28.6 × 6–6.1 μm; av. 26.1 × 6.1 μm) (Luttrell 1954).
Pseudopyricularia cyperi Klaubauf, Lebrun & Crous, sp.
Basionym: Pyricularia higginsii Luttr., Mycologia 46: 810. 1954.
collected, Cyperus.
Pseudopyricularia higginsii (Luttr.) Klaubauf, Lebrun &
Crous, comb. nov. MycoBank MB810215.
≡ Dactylaria higginsii (Luttr.) M.B. Ellis, Dematiaceous Hyphomycetes
(Kew): 173. 1976.
Material examined: New Zealand, Auckland, Mount Albert, Carrington Road,
UNITEC Technical Institute, on dead leaves of Typha orientalis, 30 Apr. 2007,
C.F. Hill, specimen in PDD, culture CBS 121934.
hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores
solitary, erect, straight or curved to geniculate, branched, medium brown, smooth, 40–100 × 3–4 μm, 1–5-septate. Conidiogenous cells 35–70 × 3–4 μm, integrated, terminal and
intercalary, pale brown, smooth, forming a rachis with several
protruding, flat-tipped denticles, 2–3 μm long, 1.5–2 μm diam.
Conidia solitary, obclavate, medium brown, smooth to finely
roughened, granular and guttulate, 2-septate, (22–)
25–28(–35) × (4–)5(–6) μm; apical cell 12–17 μm long, basal
cell 7–9 μm long; hilum truncate, slightly protruding, 1.5–2 μm
diam, unthickened, not darkened.
Notes: Pyricularia higginsii was originally described from Cyperus sp. in Georgia (Luttrell 1954). Conidiophores were described
as being 3-septate, up to 76 um long, while conidia were 2septate, 17.5–36.5 × 5.3–6.5 μm (av. 28 × 6 μm), in culture
26.1–28.6 × 6–6.1 μm (av. 26.1 × 6.1 μm) (Luttrell 1954).
Species in the Pseudopyricularia higginsii complex are all very
similar based on their conidial dimensions, and fresh collections
from Georgia would be required to resolve the phylogeny of
P. higginsii.
Culture characteristics: Colonies on MEA buff, round, raised,
cottony, reaching up to 1.8 cm diam after 1 wk; reverse ochreous.
On CMA and OA transparent, round to undulate colonies with
smooth surface. Colonies on PDA white, round, diffuse edge,
cottony, reaching up to 2.2 cm diam after 1 wk; reverse buff.
collected, Kyllinga.
Materials examined: Israel, on Cyperus rotundus, date unknown, R. Kenneth,
specimen CBS H-17647, culture CBS 665.79. Japan, Hyogo, on Cyperus iria,
2002, H. Kato (holotype CBS H-21840, culture ex-type CBS 133595).
Philippines, Sto Tomas, Batangas, on Cyperus rotundus, 1983, IRRI collector
unknown, CR88383 (Borromeo et al. 1993) = PH0053.
Pseudopyricularia kyllingae Klaubauf, Lebrun & Crous,
hyaline, branched, septate hyphae, 1.5–2 μm diam. Conidiophores solitary or in fascicles of 2–3, erect, straight or
curved, branched or not, medium brown, finely roughened,
50–80 × 4–6 μm, 1–3-septate. Conidiogenous cells
15–60 × 3–4 μm, integrated, terminal, rarely intercalary, medium
Fig. 11. Pseudopyricularia cyperi (CBS 133595). A. Sporulation on SNA. B–E. Conidiophores. F. Conidia. Scale bars = 10 μm.
110
RESOLVING
PYRICULARIA
Fig. 12. Pseudopyricularia kyllingae (CBS 133597). A. Sporulation on sterile barley seed on SNA. B–G. Conidiophores and conidia. H. Conidia. Scale bars = 10 μm.
brown, finely roughened, forming a rachis with several protruding, flat-tipped denticles, 1–2 μm long, 1–1.5 μm diam. Conidia
solitary, obclavate, pale to medium brown, finely roughened,
guttulate, 2-septate, (23–)27–30(–35) × (5–)6(–7) μm; apical
cell 12–20 μm long, basal cell 9–10 μm long; hilum truncate,
slightly protruding, 1–1.5 μm diam, unthickened, not darkened.
clavate, unitunicate, short-stipitate, with prominent apical ring.
Paraphyses intermingled among asci, unbranched, septate.
Ascospores bi- to multiseriate in asci, hyaline, guttulate, smoothwalled, fusiform, curved with rounded ends, transversely 3septate, slightly constricted at septa.
Type species: Pyricularia grisea Sacc., Michelia 2(no. 6): 20. 1880.
Culture characteristics: Colonies on MEA transparent, funiculate,
reaching up to 6.5 cm diam after 1 wk; reverse ochreous. On
CMA transparent smooth colony, reaching up to 5 cm diam after
1 wk. On PDA transparent colony, plate covering after 1 wk;
transparent reverse.
Materials examined: Japan, Hyogo, on Kyllinga brevifolia, 2003, I. Chuma (holotype CBS H-21841, culture ex-type CBS 133597). Philippines, Los Banos,
Laguna, on Cyperus brevifolius, 1989, IRRI collector unknown, CB8959
(Borromeo et al. 1993) = PH0054.
Note:
Morphologically
similar
to
P.
higginsii
(26.1–28.6 × 6–6.1 μm; av. 26.1 × 6.1 μm sensu Luttrell 1954),
except that conidia of P. kyllingae (23–35 × 5–7 μm; av.
29 × 6 μm) are longer in culture.
Pyricularia Sacc., Michelia 2(no. 6): 20. 1880.
Plant pathogenic. Conidiophores solitary or in fascicles, subcylindrical, erect, brown, smooth, rarely branched, with sympodial proliferation. Conidiogenous cells terminal and intercalary,
pale brown, with denticulate conidiogenous loci and rhexolytic
secession. Conidia solitary, pyriform to obclavate, narrowed toward tip, rounded at the base, 2-septate, hyaline to pale brown,
with a distinct basal hilum, sometimes with marginal frill. Ascomata perithecial, solitary to gregarious, subspherical, brown to
black, base immersed in host tissue, with long neck protruding
above plant tissue; wall consisting of several layers of brown
textura angularis. Asci 8-spored, hyaline, subcylindrical to
Pyricularia ctenantheicola Klaubauf, Lebrun & Crous, sp.
collected, Ctenanthe.
hyaline, branched, septate hyphae, 1.5–2 μm diam. Conidiophores solitary, erect, straight or curved, branched or not,
medium brown, smooth, 70–200 × 3–5 μm, 1–6-septate; base
bulbous, lacking rhizoids, 7–10 μm diam. Conidiogenous cells
40–110 × 3–5 μm, integrated, terminal and intercalary, pale
brown, smooth, with several protruding denticles, 1–2 μm long,
1–1.5 μm diam. Conidia solitary, pyriform to obclavate, pale
brown, finely roughened, granular to guttulate, 2-septate, (19–)
cell 5–7 μm long; hilum truncate, 0.5–1.5 μm long, 1.5–2 μm
diam, unthickened, not darkened.
Culture characteristics: Colonies on MEA white to vinaceous
buff, cottony, with undulating margin, reaching up to 2.7 cm diam
after 1 wk; reverse ochreous to umber. Colonies on CMA pale
luteous, with hazel centre, reaching up to 2.5 cm diam after 1 wk.
Colonies on PDA hazel, with smoke grey tufts, reaching up to
3.5 cm diam after 1 wk; reverse hazel. Colonies on OA reaching
up to 3.5 cm after 1 wk, sporulating abundantly after 1 wk in
the dark.
111
KLAUBAUF
ET AL.
Fig. 13. Pyricularia ctenantheicola (GR0002). A. Sporulation on sterile barley seed on SNA. B. Sporulation on SNA. C–G. Conidiophores and conidia. H. Conidia. Scale
bars = 10 μm.
Materials examined: Greece, Almyros, on Ctenanthe oppenheimiana imported
from Brazil via Netherlands, 1998, A.C. Pappas & E.J. Paplomatas (holotype
CBS H-21842, culture ex-type CBS 138601 = GR0002); ibid., GR0001 = Ct4 = ATCC 200218.
Note: Although the leaf spot disease of Ctenanthe has previously
been reported (Pappas & Paplomatas 1998), the fungus was
never officially named.
Pyricularia grisea Sacc., Michelia 2(no. 6): 20. 1880.
Fig. 14.
Basionym: Ceratosphaeria grisea T.T. Hebert, Phytopathology
61(1): 86. 1971.
= Magnaporthe grisea (T.T. Hebert) M.E. Barr, Mycologia 69(5): 954. 1977.
Materials examined: Brazil, on Digitaria horizontalis, date and collector unknown,
Br33; Goias, Goiana, on Digitaria sanguinalis, 1989, J.-L. Notteghem, BR0029.
Japan, on Digitaria smutsii, date and collector unknown, JP0034 = NI980. Korea,
Woanju, on Echinochloa crus-galli var. frumentacea, date unknown, H.K. Sim,
CBS 128304 = KACC 41641. Philippines, Sto Tomas, Batangas, on Digitaria
ciliaris, 1988, IRRI collector unknown, Dc88420 (Borromeo et al.
1993) = PH0055. South Korea, Suwon, on Lolium perenne, 1991, C.K. Kim,
CR0024. USA, Delaware, on Digitaria sp., 1991, B. Valent, US0043 = G-184.
Note: Isolates of P. grisea were observed to form apical mucilaginous droplets on their macroconidia in culture, as well as
produce microconidia on SNA, as observed previously in
P. oryzae (Chuma et al. 2009, Zhang et al. 2014).
Pyricularia oryzae Cavara, Fung. Long. Exsicc. 1: no. 49.
1892. Fig. 15.
= Magnaporthe oryzae B.C. Couch, Mycologia 94(4): 692. 2002.
112
Materials examined: Brazil, on Triticum aestivum, 1989, J.-L. Notteghem,
BR0032, BR0045. Burkina Faso, on Paspalum sp., 1990, collector unknown,
BF0028 = CBS 138602. C^
ote d'Ivoire, Bouake, on Leersia hexandra, 1983, J.L. Notteghem, CD0067; Ferkessedougou, on Eleusine indica, 1989, J.-L.
Notteghem, CD0156. Egypt, on Oryza sativa, date and collector unknown,
CBS 657.66. France, Camargue, on Oryza sativa, 1988, J.-L. Notteghem,
FR0013. French Guyana, on Oryza sativa, 1978, J.-L. Notteghem,
Guy11 = FGSC 9462. Gabon, Wey, on Zea mays, 1985, J.-L. Notteghem,
GN0001. India, Uttar Pradesh, on Setaria sp., date unknown, J. Kumar, IN0108.
Israel, Masmiah, on Echinochloa crus-galli, date and collector unknown, CBS
658.66; Rishon-le-Zien, on Stenotaphrum secundatum, date and collector unknown, CBS 659.66. Japan, on Eragrostis curvula, 1983, H. Kato, JP0038; on
Eriochloa villosa, date and collector unknown, JP0033; on Phalaris arundinacea,
date and collector unknown, JP0040; on Anthoxanthum odoratum, date and
collector unknown, JP0039; on Eleusine indica, 1974, H. Yaegashi, JP0017; on
Eragrostis curvula, 1976, H. Yaegashi, JP0028; Nagano, host, date and collector unknown, CBS 365.52 = MUCL 9451. Philippines, Los Banos, Laguna,
on Brachiaria mutica, 1983 IRRI collector unknown, BmA8309 (Borromeo et al.
1993) = PH0035 = PH0075; Cabanatuan, Nueva Ecija, on Cynodon dactylon,
1988, IRRI collector unknown, Cd88215 (Borromeo et al. 1993) = PH0051; on
Echinochloa colona, 1982, IRRI collector unknown, PH0077 = Ec8202; Los
Banos, Laguna, on Leptochloa chimensis, 1984, IRRI collector unknown,
Lc8401 (Borromeo et al. 1993) = PH0060; on Oryza sativa, 1980, IRRI collector
unknown, PO6-6 (Wang et al. 1994) = PH0014; on Panicum repens, 1982, J. M.
Bonmam, Pr8212 = PH0079; Cabanatuan, Nueva Ecija, on Paspalum distichum, 1988, IRRI collector unknown, Pd8824 (Borromeo et al.
1993) = PH0062; Los Banos, Laguna, on Rottboellia exalta, 1984, IRRI collector unknown, ReA8401(Borromeo et al. 1993) = PH0063 = ATCC 62619.
Portugal, on Stenotaphrum secondatum, 1992, A. Lima, PR0067, PR0104.
Romania, no further details, CBS 255.38. Rwanda, Kunynya, on Eleusine
coracana, 1990, J.-L. Notteghem, RW0012. South Korea, Suwon, on Festuca
elalior, date unknown, C.K. Kim, CR0029; Suwon, on Lolium hybridum, 1991,
C.K. Kim, CR0026; Suwon, on Phleum pratense, 1991, C.K. Kim, CR0020;
Yongin, on Panicum miliaceum, date unknown, C.K. Kim, CR0021. USA,
^ Môn, on
Kentucky, on Setaria viridis, 1998, M. Farman, US0071. Vietnam, O
Leersia hexandra, 2002, B. Couch, VT0032. Unknown, no collection details,
RESOLVING
PYRICULARIA
Fig. 14. Pyricularia grisea (BR0029). A. Sporulation on sterile barley seed on SNA. B–G. Conidiophores and conidia. H. Macroconidia (arrows indicate apical marginal frill,
which is a remnant of the apical mucoid cap). I. Microconidia. Scale bars = 10 μm.
Fig. 15. Pyricularia oryzae (BF0028). A. Sporulation on sterile barley seed on SNA. B–G. Conidiophores and conidia. H. Conidia. Scale bars = 10 μm.
113
KLAUBAUF
ET AL.
CBS 375.54; on Oryza sativa, date and collector unknown, 70-15 = ATCC MYA4617 = FGSC 8958; laboratory strain, progeny from a cross between strains
with different host specificity, CBS 433.70.
Pyricularia penniseticola Klaubauf, Lebrun & Crous, sp.
Materials examined: Burkina Faso, Kamboinse (Guaga), Pennisetum typhoides,
27 Sept. 1990, J.-L. Notteghem, BF0017. C^
ote d'Ivoire, Bouake, P. typhoides,
1 Dec. 1983, J.-L. Notteghem, CD0086; Odienne, Digitaria exilis, 1 Oct. 1989, J.L. Notteghem, CD0143; Madiani, Pennisetum sp., 17 Oct. 1991, J.-L. Notteghem,
CD0180. Mali, Segou field 2, D. exilis, 17 Oct. 1993, J.-L. Notteghem, ML048;
Longorola Sikasso, on P. typhoides, 14 Sept. 1990, J.-L. Notteghem (holotype
CBS H-21843, culture ex-type ML0031 = CBS 138603).
collected, Pennisetum.
Pyricularia pennisetigena Klaubauf, Lebrun & Crous, sp.
hyaline, branched, septate hyphae, 1.5–2 μm diam. Conidiophores solitary, erect, straight or curved, frequently
branched, medium brown, smooth, 100–350 × 4–6 μm, multiseptate; base bulbous, lacking rhizoids. Conidiogenous cells
40–130 × 3–4 μm, integrated, terminal and intercalary, pale
brown, smooth, forming a rachis with several protruding denticles, 1–2 μm long, 1–1.5 μm diam, with rhexolytic secession.
Conidia solitary, pyriform to obclavate, pale brown, finely
roughened, granular to guttulate, 2-septate, (23–)
cell 7–10 μm long; attenuated towards a truncate hilum,
0.5–1 μm long, 1.5–2 μm diam, with minte marginal frill.
collected, Pennisetum.
Culture characteristics: Colonies on MEA pale olivaceous grey,
cottony, reaching up to 3 cm diam after 1 wk; reverse olivaceousblack. Colonies on CMA reaching up to 3 cm diam after 1 wk.
Colonies on PDA iron-grey, reaching up to 4.5 cm diam after
1 wk, reverse olivaceous-black. Colonies on OA up to 3.6 cm
diam after 1 wk, surface sectoring.
On SNA on sterile barley seeds. Mycelium consisting of smooth,
hyaline, branched, septate hyphae, 1.5–2 μm diam. Conidiophores solitary, erect, straight or curved, unbranched, medium brown, smooth, 60–150 × 4–6 μm, 2–3-septate; base
arising from hyphae, not swollen, lacking rhizoids. Conidiogenous
cells 40–95 × 3–5 μm, integrated, terminal and intercalary, pale
brown, smooth, forming a rachis with several protruding denticles,
0.5–1 μm long, 1.5–2 μm diam. Conidia solitary, pyriform to
obclavate, pale brown, smooth, granular to guttulate, 2-septate,
(25–)27–29(–32) × (8–)9(–10) μm; apical cell 10–13 μm long,
basal cell 6–9 μm long; hilum truncate, protruding, 1–1.5 μm long,
1.5–2 μm diam, unthickened, not darkened.
Culture characteristics: Colonies on MEA cottony to velvety, buff,
smoke grey, with broad white rim, reaching up to 4.8 cm diam
after 1 wk; reverse iron grey with pale margin. Colonies on CMA
buff with grey dots, reaching up to 5.0 cm diam after 1 wk.
Fig. 16. Pyricularia penniseticola (ML0031). A. Sporulation on SNA. B–G. Conidiophores and conidia. H. Conidia. Scale bars = 10 μm.
114
RESOLVING
PYRICULARIA
Fig. 17. Pyricularia pennisetigena (ML0036). A. Sporulation on sterile barley leaf on SNA. B–G. Conidiophores and conidia. H. Conidia. Scale bars = 10 μm.
Colonies on OA buff, reaching up to 5.0 cm diam after 1 wk,
sporulating after 4 d in the dark. Colonies on PDA fuscous black
with grey centre, and broad white rim, flat, erose, reaching up to
5.0 cm diam after 1 wk; reverse brown.
Materials examined: Brazil, on Cenchrus echinatus, date unknown, S. Igarashi,
Br36; Imperatriz, on C. echinatus, 28 Feb. 1990, collector n.a., BR0067; Primeiro
de Maio, on Echinochloa colona, 1 Apr. 1990, H. Kato, BR0093. Japan,
Kumamoto, on Cenchrus ciliaris, 1975, N. Nishihara, CBS 133596 = MAFF
305501 = NI981(Cc-1J). Mali, Cinzana, on Pennisetum sp., 19 Sept. 1990, J.-L.
Notteghem (holotype CBS H-21844, culture ex-type ML0036 = CBS 138604).
Philippines, Plaridel, Bucalan, on Cenchrus echinatus, 1988, IRRI collector
unknown, Ce88454 (Borromeo et al. 1993) = PH0047. USA, Tifton, Pennisetum
glaucum, 1983, H. Wells, US0044 = 83P-25, Tifton, Pennisetum glaucum, 1984,
H Wells, US0045 = 84P-19 (Kang et al. 1995).
Notes: Another forgotten species on this host is P. penniseti
(Prasada & Goyal 1970). Pyricularia penniseti was described as
having conidia that are pyriform and 2-septate,
18.4–36.7 × 7.4–11 μm. In spite of differences in conidial dimensions to P. penniseticola and P. pennisetigena, no cultures
are presently available to determine if it would also be distinct on
a phylogenetic basis.
Pyricularia zingibericola Klaubauf, Lebrun & Crous, sp.
Conidiophores solitary, erect, straight or curved, branched or not,
medium brown, smooth, 100–200 × 4–6 μm, 3–8-septate; base
bulbous, lacking rhizoids, 5–7 μm diam. Conidiogenous cells
45–70 × 3–4 μm, integrated, terminal and integrated, pale
brown, smooth, with several protruding apical denticles,
1–1.5 μm long, 1–2 μm diam. Conidia solitary, pyriform to
obclavate, pale brown, smooth to finely roughened, guttulate, 2septate, (18–)20–23(–25) × (7–)8(–10) μm; apical cell
8–10 μm long, basal cell 5–7 μm long; hilum truncate, protruding, 0.5–1 μm long, 1.5–2 μm diam, unthickened, not
darkened.
Culture characteristics: Colonies on MEA transparent to white
with leaden grey centre, sulcate colony with entire edge, some
irregular tufts, sporulating in centre, reaching up to 4 cm diam
after 1 wk; reverse pale with olivaceous grey centre. Colonies
on OA white with some dark spots, greenish olivaceous in
centre, flat, smooth, cotton-like surface, reaching up to 4.5 cm
diam after 1 wk. Colonies on CMA grey olivaceous to olivaceous black with olivaceous grey centre, entire edge, flat colony, slightly wool-like surface, reaching up to 4 cm diam after
1 wk. Colonies on PDA transparent with some greenish olivaceous parts, white centre, umbonate, powdery surface in
centre, reaching up to 4.5 cm diam after 1 wk; reverse greenish
olivaceous.
collected, Zingiber.
Material examined: Reunion, on Zingiber officinale, J.-C. Girard (holotype CBS
H-21845, culture ex-type RN0001 = CBS 138605).
hyaline, branched, septate hyphae, 1.5–2 μm diam.
Notes: Pyricularia zingibericola, which appears to be unique on
Zingiber, has smaller conidia than P. leersiae (20–)
115
KLAUBAUF
ET AL.
Fig. 18. Pyricularia zingibericola (RN0001). A. Sporulation on SNA. B–F. Conidiophores and conidia. G, H. Conidia. Scale bars = 10 μm.
27(–35) × (7–)8.6(–10) μm, which is also known to occur on
Leersia (Hashioka 1973). Presently no cultures of P. leersiae are
available to facilitate a molecular comparison.
Xenopyricularia Klaubauf, Lebrun & Crous, gen. nov.
MycoBank MB810223.
Pyricularia.
Plant pathogenic. Conidiophores solitary or in fascicles, subcylindrical, erect, medium brown, smooth, flexuous, branched,
with sympodial growth. Conidiogenous cells terminal and
intercalary, pale brown, denticulate conidiogenous loci. Conidia
solitary, formed sympodially, obovoid, narrowed toward tip,
rounded at the base, 2-septate, pale brown, with central cell
appearing slightly darker brown, with a distinct protruding basal
hilum.
Type species: Xenopyricularia zizaniicola (Hashioka) Klaubauf,
Lebrun & Crous
Xenopyricularia zizaniicola (Hashioka) Klaubauf, Lebrun
& Crous, comb. nov. MycoBank MB810224. Fig. 19.
Basionym: Pyricularia zizaniicola Hashioka (as “zizaniaecola”),
Trans. Mycol. Soc. Japan 14(3): 264. 1973.
≡ Pyricularia zizaniicola Hashioka (as “zizaniaecola”), Res. Bull. Fac.
Agr. Gifu Univ. 29: 21. 1970. (nom. nud.)
116
Description and illustration: Hashioka (1973).
Materials examined: Japan, Gifu, on Zizania latifolia, 15 Sep. 1967, Y.
Hashioka (holotype presumably lost); Ibaraki, on Zizania latifolia, 1985, N.
Hayashi, (neotype designated here CBS H-21846, culture ex-neotype CBS
133593 = MAFF 240219 = IBZL3-1-1(Zz-1J)); Kyoto, on Zizania latifolia,
2003, K. Yoshida & K. Hirata, CBS 132356 = MAFF 240220 = KYZL201-11(Zz-2J).
Notes: Xenopyricularia zizaniicola has long, flexuous, pale
brown, branched conidiophores. Conidia are brown, 2-septate,
obovoid, (22–)25–28(–35) × (12–)13(–14) μm (on SNA), with a
small protruding hilum, 0.5–1 μm long, 1 μm diam. Morphologically Xenopyricularia resembles Pyricularia, except that its
conidia are very wide and more obovoid than are typical Pyricularia conidia, and some appear to be irregularly pigmented.
The present culture corresponds very well with the original
description and illustrations provided by Hashioka (1973), who
cited conidia as being (24–)27.7(–33) × (10.5–)13.5(–15.5) μm,
and is therefore designated as neotype.
Another forgotten species on this host is Pyricularia zizaniae
Hara, (as “Piricularia”) Trans. Shizuoka Agric. Soc. 336: 29.
1925. Translated from Japanese: “Leaf spots small, circular, later
elongate, brown, ellipsoid to fusiform, finaly greyish brown with
brown border, 2–8 × 2–6 mm. Caespituli mainly hypophyllous,
sooty-coloured. Conidiophores linear, 60–130 × 2.5–4 μm,
rarely branched, solitary or densely fasciculate, dark brown and
swollen at the base, paler and attenuate toward the apex,
RESOLVING
PYRICULARIA
Fig. 19. Xenopyricularia zizaniicola (CBS 133593). A. Sporulation on sterile barley seed on SNA. B–D. Conidiophores and conidia (arrows indicate conidiogenous loci in D). E,
F. Conidia. Scale bars = 10 μm.
geniculate at the apex. Conidia pyriform to clavate, rounded at
base, attenuate at apex, 1–2-septate, not constricted at septa,
protruding at base, hyaline to pale smoky in colour. Notes: When
it was inoculated onto rice, it was not pathogenic. This disease
was observed in shaded area”. Pyricularia zizaniae has conidia
that are described as being 1–2-septate, (18–)
22(–28) × 7(–10) μm. No cultures are available, however, to
determine if it could represent a second species of
Xenopyricularia.
Sordariales, incertae sedis
Rhexodenticula W.A. Baker & Morgan-Jones, Mycotaxon
79: 363. 2001.
Mycelium immersed and superficial, consisting of branched,
septate, pale brown to brown, smooth hyphae that become
verruculose. Conidiophores solitary, erect, subcylindrical, straight
or curved, unbranched, medium brown, finely verruculose,
septate. Conidiogenous cells integrated, terminal, subclavate,
pale brown, finely verruculose, forming a rachis with several
protruding denticles, and rhexolytic secession. Conidia solitary,
fusoid-ellipsoidal, finely verruculose, medium brown, guttulate, 3septate; base rounded, hilum truncate, slightly protruding, with
minute marginal frill.
Type species: Rhexodenticula cylindrospora (R.F. Casta~neda,
Saikawa & Hennebert) W.A. Baker & Morgan-Jones
Notes: An isolate deposited at CBS as Pyricularia lauri (CBS
244.95, on leaf litter of Nectandra antillana, Cuba) was
morphologically identical to the ex-type isolate of Rhexodenticula
cylindrospora (CBS 318.95, also isolated from leaf litter of
Nectandra antillana, Cuba). Although the phylogenetic position of
the genus is still unclear, it does not belong to the
Magnaporthaceae, but appears to be sister to Boliniales and
Sordariales (Fig. 1).
Rhexodenticula cylindrospora (R.F. Casta~neda, Saikawa & Hennebert) W.A. Baker & Morgan-Jones, Mycotaxon 79: 363. 2001. Fig. 20.
Basionym: Nakataea cylindrospora R.F. Casta~neda, Saikawa &
Hennebert, Mycotaxon 59: 457. 1996.
On SNA on sterile barley seed. Mycelium consisting of finely
verruculose, hyaline, branched, septate hyphae, becoming
brown and verruculose, 2.5–3 μm diam. Conidiophores solitary,
erect, subcylindrical, straight or curved, unbranched, medium
brown, finely verruculose, 40–90 × 4–5 μm, 1–6-septate.
Conidiogenous cells 10–20 × 3–5 μm, integrated, terminal,
subclavate, pale brown, finely verruculose, forming a rachis
with several protruding denticles, 1 μm long and in diam, with
rhexolytic secession. Conidia solitary, fusoid-ellipsoidal, finely
verruculose, medium brown, guttulate, 3-septate, (15 –)
17–19(–20) × (4–)5(–6) μm; base rounded, hilum truncate,
slightly protruding, 1 μm long and diam, with minute marginal
frill.
Culture characteristics: Colonies on MEA mouse-grey, vinaceous
buff at the margin, sulcate, velutinous, reaching up to 1.7 cm
diam after 15 d; reverse isabelline with sepia centre. Colonies on
OA dark mouse-grey with greenish black rim, undulate, funiculose, reaching up to 2.1 cm diam after 15 d. Colonies on PDA
buff to honey, isabelline in centre, undulate, sulcate, reaching up
to 1.5 cm diam after 15 d; reverse buff to honey, isabelline in
centre.
Materials examined: Cuba, Pinar del Rio, leaf litter of Nectandra antillana, 9 Aug.
1994, R.F. Casta~neda, culture ex-type CBS 318.95 = INIFAT C94/182; on leaf
litter of N. antillana, 9 Aug. 1994, R.F. Casta~neda & M. Saikawa, CBS
244.95 = INIFAT C94/182.
117
KLAUBAUF
ET AL.
Fig. 20. Rhexodenticula cylindrospora (CBS 318.95). A. Sporulation on SNA. B–G. Conidiophores and conidia. H, I. Conidia. Scale bars = 10 μm.
DISCUSSION
Prior to this study, the Magnaporthales contained a single family,
the Magnaporthaceae (Thongkantha et al. 2009). However, the
elucidation of Nakataea as older name for Magnaporthe (Luo &
Zhang 2013) justified a reevaluation of the genera included in
this order, as many are quite extreme in their morphology and
ecology. Based on the results of our phylogenetic analyses
(Fig. 2), three clear clades could be distinguished, one corresponding to Magnaporthaceae (based on Nakataea), and two
other clades corresponding to new families, Pyriculariaceae
(based on Pyricularia), and Ophioceraceae (based on Ophioceras). The genus Pseudohalonectria, which clusters basal to
these three families (Fig. 1) is polyphyletic (Thongkantha et al.
2009) and is closely related to species of Ceratosphaeria
(Reblova 2006, Huhndorf et al. 2008, Thongkantha et al. 2009),
but could not be treated due to a lack of cultures. These families
have different ecological characteristics. Magnaporthaceae and
Pyriculariaceae are mainly composed of plant pathogenic species, some of which are of major importance in plant pathology
(Gaeumannomyces, Nakataea and Pyricularia). Ophioceraceae
and Pseudohalonectria (incertae sedis) are mainly composed of
aquatic or wood-associated saprobic species. Magnaporthaceae
is distinguished from the Pyriculariaceae by their asexual
morphs, which are phialophora- or harpophora-like, or with
falcate versicoloured conidia on brown, erect conidiophores in
the case of Magnaporthaceae, and Pyricularia or pyricularia-like,
characterised by pyriform 2-septate conidia and rhexolytic
secession, in the case of Pyriculariaceae. Although Ophioceras
is morphologically similar to Gaeumannomyces, the two genera
118
can be distinguished by the aquatic habit of Ophioceras,
occurring on wood and herbaceous material, versus the plant
pathogenic nature of Gaeumannomyces, which has harpophoralike asexual morphs, mycelial appressoria, and a perithecial
peridium of textura epidermoidea (Walker 1980, Chen et al.
1999). The allocation of Ophioceras to the Magnaporthaceae
has always been seen as a temporary measure, awaiting further
study (Shearer 1989, Shearer et al. 1999). As shown in the
present analyses (Fig. 2), Ophioceras clusters separate from the
Magnaporthaceae and Pyriculariaceae in the Magnaporthales,
and hence a separate family, the Ophioceraceae, had to be
defined for these taxa. Several genera were distinguished in the
Magnaporthaceae in the present study, namely Buergenerula,
Bussabanomyces, Gaeumannomyces, Harpophora, Kohlmeyeriopsis, Magnaporthiopsis, Nakataea, Omnidemptus, Pyriculariopsis and Slopeiomyces. The Pyriculariaceae includes
eight additional genera, namely Bambusicularia, Barretomyces,
Deightoniella, Macgarvieomyces, Neopyricularia, Proxipyricularia, Pseudopyricularia and Xenopyricularia and four novel
Pyricularia species.
Some previously published and rather broadly defined species of Pyricularia and Magnaporthe clustered outside these
families. These include isolate CBS 244.95, which was originally
identified as Pyricularia lauri, and is shown here to represent
Rhexodenticula cylindrospora (incertae sedis) (Fig. 1). In addition, an isolate deposited at CBS as Pyricularia parasitica (CBS
376.54, sterile on SNA) clustered in the Chaetothyriales (Fig. 1),
and sequences of Magnaporthe griffinii (ITS GenBank
JQ390311, JQ390312) proved to be distant to the Sordariomycetes (not included).
RESOLVING
The Magnaporthaceae phylogeny (Fig. 2) provided good
support (BS = 100 %) for several genera that were included in
the analysis, namely Magnaporthiopsis, Nakataea, and two new
genera, Kohlmeyeriopsis (for Gaeumannomyces medullaris),
and Slopeiomyces (for Gaeumannomyces cylindrosporus)
except Gaeumannomyces pro parte. The genus Pyriculariopsis
was omitted from the final analysis however, due to the lack of a
RPB1 sequence.
The Pyriculariaceae phylogenies (Figs 2, 3) delineated Pyricularia from Deightoniella, as well as novel genera such as
Bambusicularia (based on Bambusicularia brunnea), Barretomyces (based on Barretomyces calatheae = Pyriculariopsis
calatheae), Macgarvieomyces (based on Macgarvieomyces
borealis = Pyricularia borealis), Neopyricularia (based on Neopyricularia commelinicola = Pyricularia commelinicola), Proxipyricularia (based on Proxipyricularia zingiberis = Pyricularia
zingiberis), Pseudopyricularia (based on Pseudopyricularia kyllingae), and Xenopyricularia (based on Xenopyricularia
zizaniicola = Pyricularia zizaniicola).
Several new species were introduced in Pyricularia, namely
P. ctenantheicola (occurring on Ctenanthe oppenheimiana in
Greece), P. penniseticola (occurring on Digitaria exilis and
Pennisetum typhoides in West African countries such as Burkina
Faso, Ivory Coast, and Mali), P. pennisetigena (occurring on
Cenchrus ciliaris, Cenchrus echinatus, Echinochloa colona and
Pennisetum glaucum in Brazil, Japan, Mali, Philippines and the
USA), and P. zingibericola (occurring on Zingiber officinale in
Reunion Island). The surprising high number of undescribed
Pyricularia species encountered in this study suggests that
Pyricularia is actually a species-rich genus, and that sampling
leaf spot diseases of different members of Poaceae could reveal
many more novel taxa.
What started out as an investigation into the systematics of
Pyricularia, not only delineated four novel species, but also
several novel pyricularia-like genera. The genus Pyricularia is
defined by having pale brown conidiophores and a terminal and
intercalary denticulate rachis, and subhyaline, 2-septate, pyriform conidia (Yaegashi & Nihihara 1978, Murata et al. 2014).
Surprisingly, the pyriform 2-septate conidial shape was also
found for isolates from Neopyricularia (Fig. 3), whereas other
Pyriculariaceae genera had conidia that varied in shape from
obclavate to more ellipsoid. Other than conidial shape, it appears
that conidial septation also varies among Pyriculariaceae species. Indeed, three species from two related genera (Deightoniella, Macgarviennomyces, Fig. 3) have 1-septate conidia.
Since other related genera (Neopyricularia, Proxypyricularia,
Pseudopyricularia) that are basal to Deightoniella and Macgarviennomyces (Fig. 3), have 2-septate conidia, it is likely that a
common ancestor of these related genera had 2-septate conidia.
Our phylogenetic study showed that the host plant from which
Pyricularia isolates were sampled could not be used as a
taxonomic criterion, since the host range varied depending on
the fungal species. For example, Pyricularia isolates sampled
from infected leaves of Eleusine, Oryza, Setaria and Triticum
were exclusively clustering in the P. oryzae clade (Table 1,
Fig. 3). These isolates are known to be strictly host-specific, and
to have a shared evolutionary origin (Tosa & Chuma 2014). The
genetic groups (sub-species) underlying these host-specific
forms could not be differentiated by the multilocus sequences
used in this study, but were clearly delineated using additional
genetic markers (Borromeo et al. 1993, Kato et al. 2000, Couch
et al. 2005, Hirata et al. 2007, Choi et al. 2013, Saleh et al. 2014).
PYRICULARIA
On the contrary, isolates from host plants such as Cenchrus,
Echinocloa, Lolium, Pennisetum and Zingiber belong to different
Pyricularia clades corresponding to unrelated species. For
example, isolates sampled from infected Pennisetum leaves in
West Africa belong to two unrelated fungal species,
P. pennisetigena and P. penniseticola (Fig. 3). Similarly, isolates
sampled from infected Echinochloa leaves belong to three fungal
species, P. oryzae, P. grisea and P. pennisetigena (Fig. 3). This
could reflect that Echinochloa is infected by different Pyricularia
species, as some P. oryzae isolates from rice are pathogenic to
Echinochloa (Mackill & Bonham 1986, Serghat et al. 2005). It is
therefore clear from this study that some host plants can be
infected by more than one species of Pyricularia.
It would not be fitting to round off a paper on Pyricularia and
Magnaporthe without commenting on the ongoing debate about
generic names. The decision to allocate the rice pathogen
M. salvinii to Nakataea, has reduced Magnaporthe to synonymy
under Nakataea, rendering the family Magnaportaceae without
the genus Magnaporthe. Although the genus Magnaporthe has
proven to be polyphyletic, we would have advocated a different
approach in view of stability for the application of this name in
literature. Likewise, the same can be said for Pyricularia, which
also turned out to be polyphyletic, forming a generic complex.
Although we introduce several genera to address this heterogeneity, Pyricularia can fortunately be retained as a well-defined
genus in the Pyriculariaceae.
ACKNOWLEDGEMENTS
We thank Prof. Yukio Tosa and Prof. Yong-Hwan Lee for providing cultures or
DNA for phylogenetic analysis. We thank the technical staff, Arien van Iperen
(cultures), Marjan Vermaas (photographic plates), and Mieke Starink-Willemse
(DNA isolation, amplification and sequencing), as well as Federico Santoro
and Alessandro Trotta (cultures, DNA isolation, amplification and sequencing) for
their invaluable assistance.
REFERENCES
Borromeo ES, Nelson RJ, Bonman JM, et al. (1993). Genetic differentiation
among isolates of Pyricularia infecting rice and weed hosts. Phytopathology
83: 393–399.
Bussaban B, Lumyong S, Lumyong P, et al. (2003). Three new species of
Pyricularia are isolated as zingiberaceous endophytes from Thailand.
Mycologia 95: 519–524.
Bussaban B, Lumyong S, Lumyong P, et al. (2005). Molecular and morphological characterization of Pyricularia and allied genera. Mycologia 97:
1002–1011.
Cannon PF (1994). The newly recognized family Magnaporthaceae and its interrelationships. Systema Ascomycetum 13: 25–42.
Chen QH, Wang YC, Zheng XB (2006). Genetic analysis and molecular mapping of the avirulence gene PRE1, a gene for host-species specificity in the
blast fungus Magnaporthe grisea. Genome 49: 873–881.
Chen W, Shearer CA, Crane JL (1999). Phylogeny of Ophioceras spp. based on
morphological and molecular data. Mycologia 91: 84–94.
Choi J, Park S-Y, Kim B-R, et al. (2013). Comparative analysis of pathogenicity
and phylogenetic relationship in Magnaporthe grisea species complex. PLoS
One 8: e57196.
Chuma I, Shinogi T, Hosogi N, et al. (2009). Cytological characteristics of
microconidia of Magnaporthe oryzae. Journal of General Plant Pathology 75:
353–358.
Constantinescu O (1983). Deightoniella on Phragmites. Proceedings van de
Koninklijke Nederlandse Akademie van Wetenschappen Section C 86:
137–141.
119
KLAUBAUF
ET AL.
Couch BC, Fudal I, Lebrun M-H, et al. (2005). Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication
with subsequent expansion of pandemic clones on rice and weeds of rice.
Genetics 170: 613–630.
Couch BC, Kohn LM (2002). A multilocus gene genealogy concordant with host
preference indicates segregation of a new species, Magnaporthe oryzae,
from M. grisea. Mycologia 94: 683–693.
Crous PW, Groenewald JZ, Shivas RG, et al. (2011). Fungal Planet Description
Sheets: 69–91. Persoonia 26: 108–156.
Crous PW, Verkley GJM, Groenewald JZ, et al. (2009). Fungal biodiversity. CBS
Laboratory manual 1. CBS-KNAW Fungal Biodiversity Centre, Utrecht, The
Netherlands.
Crous PW, Wingfield MJ, Guarro J, et al. (2013). Fungal Planet description
sheets: 154–213. Persoonia 31: 188–296.
Crous PW, Wingfield MJ, Mansilla JP, et al. (2006). Phylogenetic reassessment
of Mycosphaerella spp. and their anamorphs occurring on Eucalyptus. II.
Dean RA, Talbot NJ, Ebbole DJ, et al. (2005). The genome sequence of the rice
blast fungus Magnaporthe grisea. Nature 434: 980–986.
Ellis MB (1971). Dematiaceous Hyphomycetes. CMI, Kew, England.
Ellis MB (1976). More Dematiaceous Hyphomycetes. CMI, Kew, England.
Faivre-Rampant O, Thomas J, Allegre M, et al. (2008). Characterization of the
model system rice-Magnaporthe for the study of nonhost resistance in cereals. New Phytologist 180: 899–910.
Gams W (2000). Phialophora and some similar morphologically little-differentiated
anamorphs of divergent ascomycetes. Studies in Mycology 45: 187–199.
Hashioka Y (1973). Notes on Pyricularia II. Four species and one variety
parasitic to Cyperaceae, Gramineae and Commelinaceae. Transactions of
the Mycological Society of Japan 14: 256–265.
Hirata K, Kusaba M, Chuma I, et al. (2007). Speciation in Pyricularia inferred
from multilocus phylogenetic analysis. Mycological Research 111: 799–808.
Hoog GS de (1985). Taxonomy of the Dactylaria complex, IV. Dactylaria, Neta,
Subulipora and Scolecobasidium. Studies in Mycology 26: 1–60.
Hoog GS de, Gerrits van den Ende AHG (1998). Molecular diagnostics of
clinical strains of filamentous Basidiomycetes. Mycoses 41: 183–189.
Hornby D, Slope DB, Gutteridge RJ, et al. (1975). Gaeumannomyces cylindrosporus, a new ascomycete from cereal roots. Transactions of the British
Mycological Society 69: 21–25.
Huhndorf SH, Greif M, Mugambi GK, et al. (2008). Two new genera in the
Magnaporthaceae, a new addition to Ceratosphaeria and two new species
of Lentomitella. Mycologia 100: 940–955.
Kang S, Sweigard JA, Valent B (1995). The PWL host specificity gene family in
the blast fungus Magnaporthe grisea. Molecular Plant Microbe Interactions
8: 939–948.
Kato H, Yamamoto M, Yamaguchi-Ozaki T, et al. (2000). Pathogenicity, mating
ability and DNA Restriction fragment length polymorphisms of Pyricularia
populations isolated from Gramineae, Bambusideae and Zingiberaceae
plants. Journal of General Plant Pathology 66: 30–47.
Kohlmeyer J, Volkmann-Kohlmeyer B (1995). Fungi on Juncus roemerianus. I.
Trichocladium medullare sp. nov. Mycotaxon 53: 349–353.
Kohlmeyer J, Volkmann-Kohlmeyer B, Eriksson OE (1995). Fungi on Juncus
roemerianus. 4. New marine ascomycetes. Mycologia 87: 532–542.
Kotani S, Kurata M (1992). Black blotch of ginger rhizome by Pyricularia zingiberi
Nishikado. Annals of the Phytopathological Society of Japan 58: 469–472.
Lombard L, Crous PW, Wingfield BD, et al. (2010). Phylogeny and systematics
of the genus Calonectria. Studies in Mycology 66: 31–69.
Luo J, Zhang N (2013). Magnaporthiopsis, a new genus in Magnaporthaceae
(Ascomycota). Mycologia 105: 1019–1029.
Luttrell ES (1954). An undescribed species of Pyricularia on sedges. Mycologia
46: 810–814.
Mackill AO, Bonham JM (1986). New hosts of Pyricularia oryzae. Plant Disease
70: 125–128.
Mason-Gamer RJ, Kellogg EA (1996). Testing for phylogenetic conflict among
molecular data sets in the tribe Triticeae (Gramineae). Systematic Biology
45: 524–545.
Murata N, Aoki T, Kusaba M, et al. (2014). Various species of Pyricularia
constitute a robust clade distinct from Magnaporthe salvinii and its relatives
in Magnaporthaceae. Journal of General Plant Pathology 80: 66–72.
differenzierung in der Fusarium-Section Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft (Berlin-Dahlem) 169:
1–117.
120
Nishikado Y (1917). Studies on the rice blast fungus, (I). Berichte des Ohara
Instituts für Landwirtschaftliche Forschungen 1: 171–218.
O'Donnell K (1993). Fusarium and its near relatives. In: The fungal holomorph:
mitotic, meiotic and pleomorphic speciation in fungal systematics
(Reynolds DR, Taylor JW, eds). CAB International, Wallingford, UK: 225–233.
Ou SH (1985). Rice diseases. CAB International, Wallingford, UK.
Pappas AC, Paplomatas EJ (1998). Pyricularia leaf spot: a new disease of
ornamental plants of the family Marantaceae. Plant Disease 82: 465–469.
Park MJ, Shin HD (2009). A new species of Pyricularia on Commelina communis. Mycotaxon 108: 449–456.
Prasada R, Goyal JP (1970). A new species of Pyricularia on Bajra. Current
Science 39: 287–288.
Rayner RW (1970). A mycological colour chart. CMI and British Mycological
Society, Kew, Surrey, England.
Reblova M (2006). Molecular systematics of Ceratostomella sensu lato and
morphologically similar fungi. Mycologia 98: 68–93.
Ronquist F, Teslenko M, van der Mark P, et al. (2012). MrBayes 3.2: efficient
Bayesian phylogenetic inference and model choice across a large model
space. Systematic Biology 61: 539–542.
Saleh D, Milazzo J, Adreit H, et al. (2014). South-East Asia is the center of
origin, diversity and dispersion of the rice blast fungus, Magnaporthe oryzae.
New Phytologist 201: 1440–1456.
Samson RA, Houbraken J, Frisvad JC, et al. (2010). Food and indoor fungi.
CBS Laboratory Manual 2. CBS-KNAW Fungal Biodiversity Centre, Utrecht,
The Netherlands.
Seifert KA, Morgan-Jones G, Gams W, et al. (2011). The Genera of Hyphomycetes. CBS Biodiversity Series no. 9. CBS-KNAW Fungal Biodiversity
Centre, Utrecht.
Serghat S, Mradmi K, Touhami AO, et al. (2005). Rice leaf pathogenic fungi on
wheat, oat, Echinochloa phyllopogon and Phragmites australis. Phytopathologia Mediterranea 44: 44–49.
Shearer CA (1989). Pseudohalonectria (Lasiosphaeriaceae), an antagonistic
genus from wood in freshwater. Canadian Journal of Botany 67: 1944–1955.
Shearer CA, Crane JL, Chen W (1999). Freshwater ascomycetes: Ophioceras
species. Mycologia 91: 145–156.
Skamnioti P, Gurr SJ (2009). Against the grain: safeguarding rice from rice blast
disease. Trends in Biotechnology 27: 141–150.
Soares DJ, Rocha FB, Barreto RW (2011). Pyriculariopsis calatheae anam. nov.
a novel fungus from the Atlantic rainforest of Brazil associated with leaf
spots on Calathea sp., with a key of Pyriculariopsis spp. Mycological
Progress 10: 315–321.
Swofford DL (2003). PAUP*. Phylogenetic analysis using parsimony (*and other
methods). Version 4. Sinauer Associates, Sunderland, Massachusetts, USA.
Tamura K, Peterson D, Peterson N, et al. (2011). MEGA5: molecular evolutionary
genetics analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.
Thongkantha S, Jeewon R, Vijaykrishna D, et al. (2009). Molecular phylogeny of
Magnaporthaceae (Sordariomycetes) with a new species Ophioceras chiangdaoense from Dracaena loureiroi in Thailand. Fungal Diversity 34: 157–173.
Tosa Y, Chuma I (2014). Classification and parasitic specialization of blast fungi.
Journal of General Plant Pathology 80: 202–209.
Tsurushima T, Don LD, Kawashima K, et al. (2005). Pyrichalasin H production
and pathogenicity of Digitaria-specific isolates of Pyricularia grisea. Molecular Plant Pathology 6: 605–613.
Vilgalys R, Hester M (1990). Rapid genetic identification and mapping of
enzymatically amplified ribosomal DNA from several Cryptococcus species.
Journal of Bacteriology 172: 4238–4246.
Walker J (1980). Gaeumannomyces, Linocarpon, Ophiobolus and several other
genera of scolecospored ascomycetes and Phialophora conidial states, with
a note on hyphopodia. Mycotaxon 11: 1–129.
Wang GL, Mackill DJ, Bonman JM, et al. (1994). RFLP mapping of genes
conferring complete and partial resistance to blast in a durably resistant rice
cultivar. Genetics 136: 1421–1434.
White T, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: PCR protocol: a guide to
eds). Academic Press, San Diego, CA, USA.
Yaegashi H, Nihihara N (1978). The taxonomical identity of the perfect state of
Pyricularia grisea and its allies. Canadian Journal of Botany 56: 180–183.
Zhang H, Wu Z, Wang C, et al. (2014). Germination and infectivity of microconidia
in the rice blast fungus Magnaporthe oryzae. Nature Communications 5: 4518.
Zhang N, Zhao S, Shen Q (2011). A six-gene phylogeny reveals the evolution of
mode of infection in the rice blast fungus and allied species. Mycologia 103:
1267–1276.
STUDIES
IN
MYCOLOGY 79: 121–186.
Pestalotiopsis revisited
S.S.N. Maharachchikumbura1,2,3, K.D. Hyde1,2,3*, J.Z. Groenewald4, J. Xu1,2, and P.W. Crous4,5,6
1
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, China; 2World
Agroforestry Centre, China & East-Asia Office, 132 Lanhei Road, Kunming 650201, China; 3Institute of Excellence in Fungal Research, School of Science, Mae Fah
Luang University, Chiang Rai 57100, Thailand; 4CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands; 5Forestry and Agricultural
Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; 6Wageningen University and Research Centre (WUR), Laboratory of Phytopathology,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
*Correspondence: K.D. Hyde, kdhyde3@gmail.com
Studies in Mycology
Abstract: Species of Pestalotiopsis occur commonly as plant pathogens, and represent a fungal group known to produce a wide range of chemically novel, diverse
metabolites. In the present study, we investigated 91 Pestalotiopsis isolates from the CBS-KNAW Fungal Biodiversity Centre (CBS) culture collection. The phylogeny of
the Amphisphaeriaceae was constructed based on analysis of 28S nrRNA gene (LSU) sequence data, and taxonomic changes are proposed to reflect more natural
groupings. We combined morphological and DNA data, and segregated two novel genera from Pestalotiopsis, namely Neopestalotiopsis and Pseudopestalotiopsis. The
three genera are easily distinguishable on the basis of their conidiogenous cells and colour of their median conidial cells. We coupled morphological and combined
sequence data of internal transcribed spacer (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) gene regions, which revealed 30 clades
in Neopestalotiopsis and 43 clades in Pestalotiopsis. Based on these data, 11 new species are introduced in Neopestalotiopsis, 24 in Pestalotiopsis, and two in
Pseudopestalotiopsis. Several new combinations are proposed to emend monophyly of Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis.
Key words: Amphisphaeriaceae, New species, Pestalosphaeria, Pestalotia, Phylogeny, Taxonomy.
Taxonomic novelties: New genera: Neopestalotiopsis Maharachch., K.D. Hyde & Crous, Pseudopestalotiopsis Maharachch., K.D. Hyde & Crous; New species:
Neopestalotiopsis aotearoa Maharachch., K.D. Hyde & Crous, N. australis Maharachch., K.D. Hyde & Crous, N. cubana Maharachch., K.D. Hyde & Crous,
N. eucalypticola Maharachch., K.D. Hyde & Crous, N. formicarum Maharachch., K.D. Hyde & Crous, N. honoluluana Maharachch., K.D. Hyde & Crous,
N. javaensis Maharachch., K.D. Hyde & Crous, N. mesopotamica Maharachch., K.D. Hyde & Crous, N. piceana Maharachch., K.D. Hyde & Crous, N. surinamensis
Maharachch., K.D. Hyde & Crous, N. zimbabwana Maharachch., K.D. Hyde & Crous, Pestalotiopsis arceuthobii Maharachch., K.D. Hyde & Crous, P. arengae
Maharachch., K.D. Hyde & Crous, P. australasiae Maharachch., K.D. Hyde & Crous, P. australis Maharachch., K.D. Hyde & Crous, P. biciliata Maharachch., K.D.
Hyde & Crous, P. chamaeropis Maharachch., K.D. Hyde & Crous, P. colombiensis Maharachch., K.D. Hyde & Crous, P. diploclisiae Maharachch., K.D. Hyde &
Crous, P. grevilleae Maharachch., K.D. Hyde & Crous, P. hawaiiensis Maharachch., K.D. Hyde & Crous, P. hollandica Maharachch., K.D. Hyde & Crous, P. humus
Maharachch., K.D. Hyde & Crous, P. kenyana Maharachch., K.D. Hyde & Crous, P. knightiae Maharachch., K.D. Hyde & Crous, P. malayana Maharachch., K.D.
Hyde & Crous, P. monochaeta Maharachch., K.D. Hyde & Crous, P. novae-hollandiae Maharachch., K.D. Hyde & Crous, P. oryzae Maharachch., K.D. Hyde &
Crous, P. papuana Maharachch., K.D. Hyde & Crous, P. parva Maharachch., K.D. Hyde & Crous, P. portugalica Maharachch., K.D. Hyde & Crous, P. scoparia
Maharachch., K.D. Hyde & Crous, P. spathulata Maharachch., K.D. Hyde & Crous, P. telopeae Maharachch., K.D. Hyde & Crous, Pseudopestalotiopsis cocos
Maharachch., K.D. Hyde & Crous, P. indica Maharachch., K.D. Hyde & Crous; New combinations: Neopestalotiopsis asiatica (Maharachch. & K.D. Hyde)
Maharachch., K.D. Hyde & Crous, N. chrysea (Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde & Crous, N. clavispora (G.F. Atk.) Maharachch., K.D. Hyde &
Crous, N. ellipsospora (Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde & Crous, N. foedans (Sacc. & Ellis) Maharachch., K.D. Hyde & Crous, N. magna
(Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde & Crous, N. natalensis (J.F.H. Beyma) Maharachch., K.D. Hyde & Crous, N. protearum (Crous & L. Swart)
Maharachch., K.D. Hyde & Crous, N. samarangensis (Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde & Crous, N. saprophytica (Maharachch. & K.D. Hyde)
Maharachch., K.D. Hyde & Crous, N. steyaertii (Mordue) Maharachch., K.D. Hyde & Crous, N. umbrinospora (Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde
& Crous, Pestalotiopsis brassicae (Guba) Maharachch., K.D. Hyde & Crous, Pseudopestalotiopsis theae (Sawada) Maharachch., K.D. Hyde & Crous.
INTRODUCTION
History of Pestalotia, Pestalotiopsis and
Truncatella
Based on the conidial forms, Steyaert (1949) split Pestalotia
into three genera, namely Pestalotia, Pestalotiopsis and Truncatella. Pestalotia pezizoides is the generic type of Pestalotia,
which was described from leaves and stems of Vitis vinifera
collected in Italy, and is presently not known from culture nor
DNA sequence. Characteristics of the species include 6-celled
conidia with four olivaceous-brown median cells, distoseptate,
hyaline terminal cells and simple or branched appendages
arising from the apex of the apical cell (Fig. 1). Pestalotiopsis
was introduced for species with 5-celled conidia, and Truncatella for those with 4-celled conidia. Pestalotia was retained as
a monotypic genus with a single 6-celled species, P. pezizoides.
Steyaert (1949) subsequently divided Pestalotiopsis into additional sections, namely Monosetulatae, Bisetulatae, Trisetulatae
and Multisetulatae, based on the number of apical appendages.
These sections were further divided into subdivisions based on
concolourous (for those possessing equally pigmented median
cells) or versicolourous conidia (two upper median cells darker
than lowest median cell), fusoid or claviform conidia, branched
or unbranched apical appendages and spatulate or nonspatulate apical appendages. Steyaert (1949) did not retain
Monochaetia as a distinct genus, and placed species with
single apical appendages in section Monosetulatae of Pestalotiopsis, or in Truncatella. Steyaert (1949) provided
121
MAHARACHCHIKUMBURA
ET AL.
Fig. 1. Pestalotia pezizoides (BPI0406483). A–B. Conidiomata on stems of Vitis vinifera. C. Conidiogenous cells. D–E. Conidia. Scale bars = 10 μm.
descriptions of 46 species and Pestalotiopsis guepinii was
considered to be the type species of the newly introduced
genus. Steyaert's (1949) introduction of the genus Pestalotiopsis to accommodate the 5-celled conidial forms of Pestalotia
resulted in appreciable controversy from Moreau (1949) and
Guba (1956, 1961). All expressed disapproval of Steyaert's
classification, which resulted in three different genera instead of
the single genus Pestalotia.
A major revision of Pestalotia sensu lato was published by
Guba (1961) in his “Monograph of Monochaetia and Pestalotia”
in which he described 220 species. Guba (1961) separated
Pestalotia into the sections quadriloculate (4-celled conidia),
quinqueloculatae (5-celled conidia) and sexloculatae (6-celled
conidia). He further subdivided the sections into different categories, mainly on the basis of conidial form, colour, and the
position, and nature of the setulae. Monochaetia was retained as
a distinct genus, but the two novel genera (Pestalotiopsis and
Truncatella) proposed by Steyaert (1949) were synonymised
under Pestalotia. In his support of a single genus Guba (1956)
emphasised that there is no justification for other genera
based on fruiting structure and there was no point in assembling
species with similar numbers of conidial septa into distinct
genera. These characters might be useful only for defining
species. Furthermore, Dube & Bilgrami (1965) favoured Guba's
opinion and pointed out that there is no clear differentiation in
conidial morphology of Pestalotia, Pestalotiopsis and Truncatella.
Therefore, Dube & Bilgrami (1965) considered it to be more
reasonable to retain all species in Pestalotia, instead of three
different genera, which were introduced mainly on the basis of
cell number.
Steyaert (1953a,b, 1961, 1963), however, provided further
evidence in support of splitting Pestalotia, arguing that retention
of Monochaetia as a separate genus based on a solitary character, a single apical appendage, was unwise, while Pestalotiopsis, Truncatella and Pestalotia were distinguished from each
other based on a set of characters. Steyaert (1963) opined that
Monochaetia was an artificial genus, which is incompatible with
modern views of fungal systematics. Sutton (1980) accepted
most of the genera discussed here (Pestalotia, Pestalotiopsis,
Truncatella) which fitted into fairly well-defined groups and are
characterised by acervuli, most with pigmented conidia, with
annellidic conidiogenous cells. Sutton (1980) cited the electron
microscope investigation of Griffiths & Swart (1974a,b), which
examined the conidial wall of Pestalotia pezizoides and two
species of Pestalotiopsis (P. funerea and P. triseta) to support
Steyaert's division of Pestalotiopsis. Griffiths & Swart (1974a,b)
regarded the conidial wall of P. pezizoides as being composed
of three zones (based on electron density and melanisation) and
122
in Pestalotiopsis of 2-layered zones. Until an evaluation of the 5celled Pestalotia species in culture is made, Sutton (1969)
preferred to regard Pestalotia as a monotypic genus. According to the revisions of Steyaert (1949) and Sutton (1969, 1980),
all earlier designated Pestalotia species, except P. pezizoides,
have been transferred to other genera, many to Pestalotiopsis.
Pestalotia valdiviana, P. cornu-cervae, and P. corni were also
included in Pestalotia section sexloculatae (Guba 1961). In his
revision of Pestalotia, Sutton (1969) considered P. valdiviana as
a nomen dubium, P. cornu-cervae was maintained as the type
and only species of Labridella, and P. corni was transferred to
Seiridium. Sutton (1980) identified several problems with the
taxonomy of Pestalotiopsis. Although Steyaert (1949) treated
Pestalotia as a monotypic genus, more than 600 species still
remain in the genus and need reassignment to Monochaetia,
Pestalotiopsis or Truncatella (Sutton 1980). Furthermore, identification of species from culture and the application of names
based on herbarium material as designated by Guba (1961) and
Steyaert (1949, 1953a,b, 1955, 1956, 1961), present a confusing
situation.
Nag Raj (1985, 1993) found it necessary to reassign many
species described in Pestalotia to other genera. However Nag
Raj (1985, 1993) preferred to adopt a broader concept for
Pestalotiopsis to include 3-septate conidial forms. Pestalotiopsis
besseyi, P. casuarinae, P. citrina, P. eupyrena, P. gastrolobi,
P. jacksoniae, P. moorie, P. pestalozzioides, P. puyae,
P. stevensoniii and P. torrendiii are 3-celled conidial forms Nag
Raj (1993) placed in Pestalotiopsis but which actually belong in
Truncatella. Therefore, his view of Pestalotiopsis was far
broader than the actual concept of Steyaert (1949) (Jeewon
et al. 2003). Pestalotiopsis guepinii, the type species of Pestalotiopsis, was described from stems and leaves of Camellia
japonica collected in France, and is characterised by 5-celled
conidia with three concolourous median cells, hyaline terminal
cells and simple or unbranched appendages arising from the
apex of the apical cell (Steyaert 1949). However, Nag Raj
(1985) pointed out that it is essential to re-examine the type
material of Pestalotiopsis and related genera and also consider
the contentious placement of P. guepinii as the generic type of
Pestalotiopsis. Nag Raj (1985) redescribed Pestalotiopsis
maculans and considered it as the generic type of Pestalotiopsis, with P. guepinii as synonym. Hughes (1958) introduced
a new combination for P. maculans, which was originally
described by Corda (1839) as Sporocadus maculans. However,
the new combination introduced by Hughes (1958) lacked a
detailed description of the fungus. Furthermore, there was no
reference to this binomial in the monograph of Guba (1961),
other than reference to a collection of S. maculans listed under
PESTALOTIOPSIS
P. guepinii. Nag Raj (1985) observed the holotype specimen of
S. maculans (PR 155665), which was isolated from Camellia
japonica in Prague, Czech Republic, and clarified that the
morphology of the fungus exactly matched the generic concept
of Pestalotiopsis. Furthermore he observed the isotype
specimen of P. guepinii in BPI, which he compared with
S. maculans and found them to be identical. Therefore Nag Raj
(1985) regarded P. maculans as the correct, older name for
P. guepinii, and the type species of Pestalotiopsis. Based on
morphology and phylogeny, Jeewon et al. (2003) also pointed
out that (based on ITS sequences) P. maculans clusters with
species having concolourous median cells, and that P. karstenii
might be a synonym of P. maculans.
Biology of Pestalotiopsis species
Pestalotiopsis is a species-rich asexual genus with appendagebearing conidia in the Amphisphaeriaceae (Barr 1975, 1990,
Kang et al. 1999, Lee et al. 2006), and is widely distributed
throughout tropical and temperate regions (Bate-Smith &
Metcalfe 1957). Most species in the genus lack sexual
morphs, and presently only 13 sexual morphs have been
recorded in literature, which were previously treated as species
of Pestalosphaeria (Maharachchikumbura et al. 2011). Pestalotiopsis species are common phytopathogens that cause a variety
of diseases, including canker lesions, shoot dieback, leaf spots,
needle blight, tip blight, grey blight, scabby canker, severe
chlorosis, fruit rots and various post-harvest diseases (Fig. 2)
(Crous et al. 2011, Maharachchikumbura et al. 2012, 2013a,b,
REVISITED
Zhang et al. 2012a, 2013). Pestalotiopsis species also reduce
production and cause economic loss in apple, blueberry, coconut, chestnut, ginger, grapevine, guava, hazelnut, lychee,
mango, orchid, peach, rambutan, tea and wax apple due to
disease (Sun & Cao 1990, Sangchote et al. 1998, Xu et al. 1999,
Keith et al. 2006, Joshi et al. 2009, Keith & Zee 2010, Chen et al.
2011, Evidente et al. 2012, Ismail et al. 2013,
Maharachchikumbura et al. 2013a,b,c, Ren et al. 2013).
Pestalotiopsis species are also commonly isolated as endophytes (Watanabe et al. 2010, Maharachchikumbura et al. 2012,
Debbab et al. 2013) and there are numerous reports that these
endophytes produce novel compounds with medicinal, agricultural and industrial applications (Aly et al. 2010, Xu et al. 2010,
2014). Species of Pestalotiopsis are thought to be a rich source
for bioprospecting compared to other fungal genera, and Xu et al.
(2010, 2014) reviewed 130 and 160 different compounds
respectively, isolated from species of Pestalotiopsis. Due to their
ability to switch nutritional-modes, many endophytic and plant
pathogenic Pestalotiopsis species persist as saprobes (Hu et al.
2007, Maharachchikumbura et al. 2012), and have been isolated
from dead leaves, bark and twigs (Ellis & Ellis 1997,
Maharachchikumbura et al. 2013d). Several species have
been recovered from soil, polluted stream water, wood, paper,
fabrics, and wool (Guba 1961). Some species have been
associated with human and animal infections (Sutton 1999,
Monden et al. 2013) and others (e.g. Pestalotiopsis guepinii
and P. microspora) have also been isolated from extreme environments (Strobel et al. 1996, Tejesvi et al. 2007).
Fig. 2. Disease symptoms associated with various species of Pestalotiopsis. A. Leaf spots on Mangifera indica. B. Grey blight on camellia sinensis. C. Leaf blight on camellia
japonica. D. Tip blight on Podocarpus macrophyllus. E. Leaf blotch on Rhododendron sinogrande. F. Shoot dieback on Mangifera indica. G. Guava scab on Psidium guajava. H.
Fruit rot on Syzygium samarangense.
123
MAHARACHCHIKUMBURA
ET AL.
Naming Pestalotiopsis species
Pestalotiopsis species were historically named according to the
host from which they were first observed. In spite of this practise,
many argued that Pestalotiopsis species are generally not hostspecific and are found on a wide range of hosts and substrates
(Jeewon et al. 2004, Lee et al. 2006). Therefore, many of the
traditional host-based species may be spurious. However, species
of Pestalotiopsis display considerable diversity in phenotype, and
group together based on similarities in conidial morphology
(Jeewon et al. 2003, Maharachchikumbura et al. 2012, 2013d).
Conidial characters such as conidial length, width, median cell
length, colour of median cells and length of the apical appendages
appear to be stable characters within Pestalotiopsis (Jeewon et al.
2003, Hu et al. 2007). Previous phylogenetic studies revealed
Pestalotiopsis strains to cluster in three strongly supported clades.
These clades corresponded to three conidial types: those with
pale brown or olivaceous concolourous median cells, those with
versicolourous median cells and those with dark-coloured concolourous median cells (Jeewon et al. 2003, Liu et al. 2010,
Maharachchikumbura et al. 2011, 2012). Steyaert (1949) and
Guba (1961) had previously grouped species with versicolourous
conidia into two groups based on the intensity of colour of the
median cells, namely umber-olivaceous (two upper median cells
umber and lowest median cell yellow-brown) and fuliginousolivaceous (two upper median cells fuliginous, usually opaque,
and lowest median cell pale brown). However, based on multilocus DNA sequence analysis, the division of the versicolourous
group based on colour intensities of the median conidial cell
proved to not be a taxonomically reliable character (Liu et al. 2010,
Maharachchikumbura et al. 2011, 2012).
The sexual state of Pesalotiopsis is Pestalosphaeria, which
was introduced by Barr (1975) with the type species Pestalosphaeria concentrica. This species was isolated from the
grey-brown spots on living leaves of Rhododendron maximum
growing on North Carolina, USA. Pestalosphaeria concentrica is
characterised by immersed, subglobose ascomata and unitunicate, cylindrical asci with a J+ apical ring; ascospores uniseriate in the ascus, ellipsoid, pale dull brown and 2-septate.
The germinated ascospores of Pestalosphaeria concentrica
give rise to the Pestalotiopsis conidial state, P. guepini var.
macrotricha, which contains three median concolourous conidial
cells.
Objectives of study
In the present study we examined 91 Pestalotiopsis strains from
the culture collection of the CBS-KNAW Fungal Biodiversity
Centre, Utrecht, the Netherlands (CBS), which were isolated
from various hosts and geographic origins. Phylogenetic relationships between the strains and other genera in the
Amphisphaeriaceae are resolved based on analysis of 28S
nrRNA gene (LSU) sequence data. The phylogeny resolved
Pestalotiopsis as a distinct clade in Amphisphaeriaceae, with
three well-supported groups that correlated with morphology;
besides Pestalotiopsis, two new genera, Neopestalotiopsis and
Pseudopestalotiopsis are proposed. Various Pestalotiopsis
species known from culture are therefore allocated to Neopestalotiopsis and Pseudopestalotiopsis. Phylogenetic analyses
of combined sequence data of the internal transcribed spacer
regions and intervening 5.8S nrRNA gene (ITS), partial
124
β-tubulin (TUB) and translation elongation factor 1-alpha (TEF)
gene regions supplemented with conidial morphology clarify
species boundaries in the three genera.
Isolates
A total of 91 strains were obtained from the CBS culture
collection. Freeze-dried strains were revived in 2 mL malt/
peptone (50 % / 50 %) and subsequently transferred to Petri
dishes containing oatmeal agar (OA) (Crous et al. 2009). Isolates
of the CBS collection stored in liquid nitrogen at −80 °C were
transferred directly to Petri dishes containing OA.
Morphological analysis
Morphological descriptions were made for isolates grown on
2 % potato dextrose agar (PDA; Crous et al. 2009) under
moderate temperatures (~22 °C) at 12 h daylight. Autoclaved
pine needles were placed on synthetic nutrient-poor agar
(PNA) (Crous et al. 2009) to observe conidiomatal development. Colony colour on PDA was determined with the colour
charts of Rayner (1970). Microscopic preparations were made
in distilled water, with 30 measurements per structure as
observed under a Nikon SMZ1000 dissecting microscope (DM)
or with a Nikon Eclipse 80i compound microscope using differential interference contrast (DIC) illumination. Taxonomic
descriptions and nomenclature were deposited in MycoBank
(Crous et al. 2004).
PCR and sequencing
The UltraClean Microbial DNA Isolation Kit (MoBio laboratories,
Carlsbad, CA, USA) was used to extract genomic DNA from
fungal mycelia. For nucleotide sequence comparisons, the nuclear rDNA operon spanning the 30 end of the 18S nrRNA
gene, the first internal transcribed spacer region, the 5.8S
nrRNA gene, the second internal transcribed spacer region and
the 50 end of the 28S nrRNA gene (ITS), and the partial
β-tubulin (TUB) and partial translation elongation factor 1-alpha
(TEF) genes were amplified using primer pairs LR0R/LR5
(Vilgalys & Hester 1990, Rehner & Samuels 1994), ITS5/ITS4
(White et al. 1990), T1/Bt-2b (Glass & Donaldson 1995,
O'Donnell & Cigelnik 1997), and EF1-728F/EF-2 (O'Donnell
et al. 1998, Carbone & Kohn 1999). Amplification conditions
for LSU, ITS and TEF followed Crous et al. (2013) and for TUB,
Lee et al. (2004).
Sequencing of the PCR amplicons was conducted using
the same primers as those used for the amplification reactions. The sequence products were purified using Sephadex columns (Sephadex G-50 Superfine, Amersham
Biosciences, Roosendaal, Netherlands) and analysed with an
ABI Prism 3730XL Sequencer (Applied Biosystems) according
to the manufacturer's instructions. DNASTAR Lasergene
SeqMan Pro v. 8.1.3 was used to obtain consensus sequences from sequences generated from forward and reverse
primers and these were subsequently lodged with GenBank
(Table 1).
Dead plant
MFLUCC 12-0286; NN0476380*
CBS 114159; STE-U 3017*
MFLUCC 12-0261; NN042855*
MFLUCC 12-0262; NN047037
N. asiatica
N. australis
N. chrysea
Eucalyptus sp.
Pinus brutia
Achras sapota
CBS 299.74
CBS 336.86*
CBS 464.69
N. mesopotamica
Leucospermum
cuneiforme cv. ‘Sunbird’
Picea sp.
CBS 394.48*
CBS 114178; STE-U 1765*
Cocos nucifera
CBS 254.32
N. protearum
Mangifera indica
CBS 225.30
N. piceana
Acacia mollissima
CBS 138.41*
N. natalensis
Pteridium sp.
MFLUCC 12-652; ICMP 20011*
N. magna
Cocos nucifera
Telopea sp.
CBS 114495; STE-U 2076*
CBS 257.31*
Telopea sp.
CBS 111535; STE-U 2078
Dead Formicidae (ant)
N. javaensis
N. honoluluana
Plant debris
CBS 362.72*
CGMCC 3.9202
CBS 115.83
Calliandra haematocephala
CGMCC 3.9178
N. formicarum
Neodypsis decaryi
CGMCC 3.9123*
Eucalyptus globulus
Mangrove plant
CBS 264.37; BBA 5300*
N. foedans
Dead plant materials
MFLUCC 12-0284
N. eucalypticola
Dead plant materials
MFLUCC 12-0283*
Leaf litter
Ardisia crenata
CBS 115113; HKUCC 9136
Magnolia sp.
MFLUCC 12-0281; NN043133*
CBS 600.96; INIFAT C96/44-4*
Magnolia sp.
MFLUCC 12-0280; NN043011
N. ellipsospora
Decaying wood
CBS 447.73
Telopea sp.
Unidentified tree
N. cubana
N. clavispora
Dead leaves
CBS 367.54; ATCC 11763; QM 381*
Neopestalotiopsis aotearoa
Canvas
Culture accession No.1
Species
Host/Substrate
Table 1. Collection details and GenBank accession numbers of isolates includes in this study.
China
Sri Lanka
—
—
—
Ghana
—
Proteaceae
Proteaceae
Pinaceae
Arecaceae
Anacardiaceae
Fabaceae
Sapotaceae
Pinaceae
Myrtaceae
Dennstaedtiaceae
Arecaceae
JX398988
—
KM199357
KF582795
—
—
Zimbabwe
UK
JN712564
KM116266
KM116267
KM116270
Indonesia: Sulawesi
KM116279
JN712498
KM199368
KM199372
KM199371
KM199377
KM199353
—
—
KM199362
KM116271
KM199361
KM199364
—
KM116257
KM199363
KM199358
KM116263
KM116248
KM199344
JX398989
—
KM116255
JX398987
KM199376
JX398981
JX398980
KM199343
—
South Africa
India
Iraq
Turkey
France
Indonesia: Java
USA: Hawaii
USA: Hawaii
Cuba
—
Proteaceae
China
Fabaceae
China
KM116256
—
China
Myrtaceae
Arecaceae
—
Thailand
—
—
China
—
KM116269
KM199347
JX398979
—
KM116253
JX398978
—
Hong Kong
Cuba
—
KM199374
JX398986
JX398985
KM199348
JX398983
KM199369
ITS
KM199542
KM199527
KM199529
KM199535
KM199552
—
KM199555
KM199541
KF582791
KM199543
KM199548
KM199546
KM199517
KM199519
JX399054
JX399055
JX399053
KM199551
JX399046
JX399047
KM199544
KM199521
JX399045
JX399044
KM199539
JX399052
JX399051
KM199537
JX399049
KM199526
TEF
KM199463
KM199453
KM199452
KM199451
KM199466
KM199436
KM199441
KM199435
KF582793
KM199437
KM199457
KM199461
KM199455
KM199444
JX399023
JX399024
JX399022
KM199431
JX399015
JX399016
KM199450
KM199438
JX399014
JX399013
KM199443
JX399021
JX399020
KM199432
JX399018
KM199454
TUB
GenBank accession2
KM116275
—
—
KM116252
—
KM116247
LSU
Myrsinaceae
China
Magnoliaceae
China
China
Magnoliaceae
China
—
—
New Zealand
—
Proteaceae
Location
Family
PESTALOTIOPSIS
REVISITED
125
126
CBS 434.65*
CBS 331.92*
P. arceuthobii
P. arengae
Syzygium sp.
IFRDCC 2397*
MFLUCC 10-146
P. anacardiacearum
On refrigerator door
PVC gasket
ICMP 6088*
Pestalotiopsis adusta
Arenga undulatifolia
Arceuthobium campylopodum
Mangifera indica
Leucospermum cunciforme
cv. ‘Sunbird’
CBS 111495; STE-U 1777*
N. zimbabwana
Soil under Elaeis guineensis
Unidentified plant
CBS 450.74*
MFLUCC 12-0285; NN042986*
N. umbrinospora
Protea eximia
CBS 111494; STE-U 1779
Eucalyptus viminalis
IMI 192475*
Cissus sp.
CBS 361.61
N. surinamensis
Erica gracilis
CBS 323.76
Vitis vinifera
Erica sp.
CBS 266.37; BBA 5087; IMI 083708
CBS 266.80
Cinchona sp.
Achras sapota
CBS 119.75
N. steyaertii
Neopestalotiopsis sp. Clade 26
CBS 360.61
Cocos nucifera
Dune sand
CBS 664.94
CBS 164.42
Camellia sp.
CBS 322.76
Cocos nucifera
CBS 274.29
—
Arecaceae
Santalaceae
Anacardiaceae
Singapore
USA
China
Thailand
Fiji
—
Myrtaceae
Zimbabwe
China
Proteaceae
Suriname
—
Zimbabwe
Australia
Netherlands
France
Germany
India
India
Arecaceae
Proteaceae
Myrtaceae
Vitaceae
Ericaceae
Ericaceae
Vitaceae
Sapotaceae
Guinea
France
Rubiaceae
Netherlands
—
France
Indonesia: Java
KM199342
KC247154
—
KM116207
KM199340
KM199341
JX399007
—
KM116243
JX399006
JX556231
JX398984
KM199351
JX556232
KF582796
KM199355
KM199350
KM199349
KM199352
KM199356
KM199346
KM199367
KM199354
KM199366
KM199375
KM199370
—
JX556249
—
KM116258
JX556250
KM116285
KM116274
KM116262
KM116273
KM116264
KM116265
KM116260
KM116268
KM116254
KM116259
KM116261
KM116250
KM116246
—
KM199373
JX398982
KM116249
—
JQ968609
KM199345
—
KM116251
KM199365
KM199360
KM199359
ITS
KM199426
KM199427
KC247155
JX399038
JX399037
KM199456
JX399019
KM199465
KM199462
KF582794
KM199460
KM199458
KM199515
KM199516
KC247156
JX399071
JX399070
KM199545
JX399050
KM199518
KM199530
KF582792
KM199549
KM199550
KM199547
KM199532
—
KM199459
KM199531
KM199522
KM199520
KM199525
KM199536
KM199534
KM199533
KM199540
KM199528
JX399048
KM199538
JQ968611
KM199556
KM199524
KM199523
TEF
KM199439
KM199440
KM199434
KM199449
KM199446
KM199448
KM199445
KM199442
KM199464
JX399017
KM199433
JQ968610
KM199447
KM199430
KM199429
TUB
GenBank accession2
—
KM116272
KM116245
LSU
—
India
China
Hong Kong
Arecaceae
Theaceae
Arecaceae
Fabaceae
—
Dalbergia sp.
CBS 110.20
CBS 177.25
Fabaceae
Magnoliaceae
Lauraceae
Thailand
Hong Kong
—
Myrtaceae
USA
New Zealand
Location
Paeoniaceae
Rosaceae
Family
Crotalaria juncea
Magnolia sp.
MFLUCC 12-0282; NN047136*
CBS 233.79
Litsea rotundifolia
Syzygium samarangense
CBS 115452; HKUCC 8684
MFLUCC 12-0233*
N. saprophytica
Unidentified tree
CBS 115451; HKUCC 9095
Paeonia suffruticosa
CBS 124745
N. samarangensis
Rosa sp.
CBS 101057*
N. rosae
Host/Substrate
Species
MAHARACHCHIKUMBURA
ET AL.
Arecaceae
—
—
—
Chamaerops humilis
—
CBS 113604; STE-U 3078
CBS 113607; STE-U 3080
CBS 186.71*
CBS 237.38
IFRDCC 2439*
MFLUCC 12-0054; CPC 20280*
IFRD 411-014*
CBS 114127; STE-U 2919*
CBS 114491; STE-U 2215*
CBS 265.33*
P. ericacearum
P. furcata
P. gaultheria
P. grevilleae
P. hawaiiensis
P. hollandica
Diploclisia glaucescens
CBS 115587; HKUCC 10130*
MFLUCC 12-0287; NN0472610*
Diploclisia glaucescens
CBS 115449; HKUCC 9103
CBS 115585; HKUCC 8394
P. diploclisiae
P. diversiseta
Psychotria tutcheri
CBS 118553; CPC 10969*
P. colombiensis
Sciadopitys verticillata
Leucospermum sp.
cv. ‘Coral’
Grevillea sp.
Gaultheria forrestii
Camellia sinensis
Rhododendron delavayi
Rhododendron sp.
Eucalyptus eurograndis
MFLUCC 12-0268; NN0471340*
P. clavata
Buxus sp.
—
—
Camellia japonica
MFLUCC 12-0278
P. chamaeropis
Theaceae
Camellia japonica
MFLUCC 12-0277*
Sciadopityaceae
Myrtaceae
Proteaceae
Ericaceae
Theaceae
Ericaceae
Ericaceae
Menispermaceae
Menispermaceae
Rubiaceae
Myrtaceae
Buxaceae
Theaceae
Theaceae
Brassicaceae
Camellia sinensis
Brassica napus
CBS 443.62
Taxaceae
Proteaceae
CBS 170.26*
Taxus baccata
CBS 790.68
Platanaceae
Proteaceae
Proteaceae
Proteaceae
P. camelliae
Paeonia sp.
CBS 236.38
Brabejum stellatifolium
CBS 119350; CMW 20013
Platanus × hispanica
Protea neriifolia × susannae
cv. ‘Pink Ice’
CBS 114474; STE-U 1769
CBS 124463*
Grevillea sp.
CBS 114193; STE-U 3011*
Proteaceae
Proteaceae
Proteaceae
Family
P. brassicae
P. biciliata
cv. ‘Pink Ice’
CBS 111503; STE-U 1770
Protea sp.
CBS 114141; STE-U 2949
P. australis
Knightia sp.
CBS 114126; STE-U 2896*
P. australasiae
Host/Substrate
Species
Netherlands
USA: Hawaii
Australia
China
Thailand
China
China
Hong Kong
Hong Kong
Hong Kong
Colombia
China
Italy
Italy
—
—
China
China
Turkey
New Zealand
Netherlands
Italy
Slovakia
South Africa
South Africa
South Africa
New Zealand
Location
JX398990
KM116228
KM116239
KM199328
KM199339
KM199300
KC537805
KM116212
—
KC537807
—
JQ683724
JX399009
KM116283
KM199320
—
KM199315
KM199314
KM116242
KM116213
KM116215
KM199307
—
KM116222
KM199324
KM199326
KM199325
KM199323
KM116217
KM116210
KM116211
KM116201
JX399011
JX399010
KM116284
—
KM199379
KM199336
—
KM116225
KM199305
KM199309
KM199308
KM199333
KM199334
KM199332
KM199331
KM199298
KM199297
ITS
KM116235
KM116214
KM116224
KM116209
KM116220
KM116197
KM116200
KM116203
KM116218
LSU
KM199481
KM199514
KM199504
KC537812
JQ683740
KC537814
JX399073
KM199486
KM199483
KM199485
KM199488
JX399056
KM199474
KM199473
KM199472
KM199471
JX399075
JX399074
KM199512
KM199558
KM199507
KM199506
KM199505
KM199476
KM199477
KM199475
KM199557
KM199501
KM199499
TEF
KM199388
KM199428
KM199407
KC537819
JQ683708
KC537821
JX399040
KM199419
KM199417
KM199416
KM199421
JX399025
KM199392
KM199391
KM199390
KM199389
JX399042
JX399041
KM199424
—
KM199400
KM199401
KM199399
KM199384
KM199385
KM199383
KM199382
KM199410
KM199409
TUB
GenBank accession2
PESTALOTIOPSIS
REVISITED
127
128
Raw material from agar-agar
CBS 442.67*
CBS 911.96
P. kenyana
Quercus robur
Taxus baccata
CBS 144.97*
CBS 440.83; IFO 32686
P. monochaeta
China
—
Rhododendron ponticum
Cocos sp.
MFLUCC 12-0258; NN0471350*
CBS 176.25*
CBS 263.33
CBS 264.33
P. rosea
P. scoparia
Pestalotiopsis sp. Clade 33
—
Telopea sp.
cv. ‘Pink Ice’
Telopea sp.
CBS 113606; STE-U 3082
CBS 114137; STE-U 2952
CBS 114161; STE-U 3083*
P. telopeae
Gevuina avellana
CBS 356.86*
P. spathulata
Chamaecyparis sp.
Pinus sp.
Proteaceae
Proteaceae
Proteaceae
Proteaceae
Arecaceae
Ericaceae
Cupressaceae
Pinaceae
Ericaceae
—
IFRDCC 2399*
Rhododendron sinogrande
CBS 393.48*
P. rhododendri
Leucothoe fontanesiana
Fabaceae
Ericaceae
Delonix regia
JX398992
KM116226
KM116205
—
—
JX399005
—
KM116216
China
—
Australia
Australia
Australia
Chile
Indonesia: Sulawesi
Netherlands
KM199316
KM199301
KM199296
—
KM199295
KM199338
KM199322
KM116219
KM116202
KM116236
KM116199
KM116198
KC537804
KM199330
KM199335
China
—
KM199313
KM199312
KM199318
KM199321
KM199299
KM116233
Portugal
KM116231
KM116240
KM116221
KM199304
KM199294
KM116206
KM199337
—
KM199329
KM199327
KM116232
KM116196
KM116229
KM199306
—
KM116238
KM199310
KM199311
KM199303
KM199302
KM199380
JX398993
JX399008
KM199317
KM199319
ITS
KM199403
KM199469
KM199402
KM199423
KM199412
KM199414
KM199393
JX399036
KC537818
KM199422
KM199405
KM199404
KM199415
KM199413
KM199398
KM199397
KM199394
KM199425
KM199387
KM199386
KM199411
JX399027
KM199408
KM199406
KM199396
KM199395
KM199468
JX399028
JX399039
KM199420
KM199418
TUB
GenBank accession2
KM116227
KM116241
KM116204
KM116234
KM116281
—
—
KM116230
KM116208
LSU
Papua New Guinea
Papua New Guinea
Italy
—
Arecaceae
CBS 278.35
Cocos nucifera
CBS 887.96
CBS 265.37; BBA 2820*
Coastal soil
CBS 331.96*
P. portugalica
P. parva
P. papuana
CBS 353.69*
Denmark
—
—
Oryza sativa
CBS 171.26
USA: Hawaii
Australia
Netherlands
Netherlands
Malaysia
China
New Zealand
Poaceae
Proteaceae
CBS 111522; STE-U 2083
Proteaceae
Telopea sp.
CBS 130973*
Taxaceae
Fagaceae
Euphorbiaceae
Apocynaceae
Proteaceae
New Zealand
—
—
Proteaceae
Kenya
Rubiaceae
P. oryzae
Banksia grandis
China
—
Papua New Guinea
Papua New Guinea
Gentianaceae
Hong Kong
—
Location
Aquifoliaceae
Family
P. novae-hollandiae
Macaranga triloba
CBS 102220*
Trachelospermum sp.
MFLUCC 12-0271; NN0471900*
Knightia sp.
CBS 114138; STE-U 2906*
P. malayana
Knightia sp.
CBS 111963; STE-U 2905
Fragraea bodenii
P. linearis
P. knightiae
Coffea sp.
CBS 109350 = MONT 6M-B-3*
P. jesteri
Unidentified tree
MFLUCC 12-0259; NN0476420*
P. intermedia
Unidentified tree
MFLUCC 12-0270; NN0470980*
Soil
CBS 336.97*
P. inflexa
Ilex cinerea
CBS 115450; HKUCC 9100
P. humus
Host/Substrate
Species
KM199500
KM199559
KM199498
KM199513
KM199490
KM199489
KM199478
JX399069
KC537811
KM199510
KM199509
KM199508
KM199492
KM199491
KM199496
KM199494
KM199493
KM199511
KM199480
KM199479
KM199482
JX399058
KM199497
KM199495
KM199503
KM199502
KM199554
JX399059
JX399072
KM199484
KM199487
TEF
MAHARACHCHIKUMBURA
ET AL.
Trachycarpus fortunei
OP068; IFRDCC 2440*
MFLUCC 12-0274; NN0473090*
CBS 272.29*
CBS 459.78*
MFLUCC 12-0055; CPC 20281*
SC011
P. verruculosa
Pseudopestalotiopsis cocos
Ps. indica
Ps. theae
Camellia sinensis
Camellia sinensis
Hibiscus rosa-sinensis
Cocos nucifera
Rhododendron sp.
Theaceae
Theaceae
Malvaceae
Arecaceae
Ericaceae
Thailand
Thailand
India
Indonesia: Java
China
China
China
—
Ericaceae
China
China
—
Arecaceae
China
China
Symplocaceae
Theaceae
China
China
—
Sapotaceae
China
Location
Podocarpaceae
Family
JQ683727
JQ683726
KM199381
KM116282
—
—
JX398996
—
KM199378
JX398999
—
KM116276
JX398998
—
JQ845947
JX399002
—
JX399001
JX399003
—
—
JX399004
—
JX399000
—
KC537809
—
—
ITS
LSU
JQ683711
JQ683710
KM199470
KM199467
—
JX399030
JX399029
JQ845945
JX399032
JX399033
JX399034
JX399035
JX399031
KC537823
TUB
GenBank accession2
JQ683743
JQ683742
KM199560
KM199553
JX399061
—
JX399063
JQ845946
JX399065
JX399066
JX399067
JX399068
JX399064
KC537816
TEF
ATCC: American Type Culture Collection, Virginia, USA; BBA: Institute for Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry (BBA), Germany; CBS: Culture collection of the Centraalbureau
voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CGMCC: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; CMW: Tree Pathology Cooperative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CPC: Culture collection of Pedro Crous, housed at CBS; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; ICMP:
International Collection of Microorganisms from Plants, Auckland, New Zealand; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IFRDCC: International Fungal Research & Development Centre Culture Collection, China; IMI: Culture
collection of CABI Europe UK Centre, Egham, UK; INIFAT: Alexander Humboldt Institute for Basic Research in Tropical Agriculture, Ciudad de La Habana, Cuba; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; NN:
Novozymes, Beijing, China; QM: Quarter Master Culture Collection, Amherst, MA, USA; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa. * = ex-holotype or ex-epitype culture.
2
LSU: large subunit (28S) of the nrRNA gene operon; ITS: internal transcribed spacers and intervening 5.8S nrDNA; TUB: partial beta-tubulin gene; TEF: partial translation elongation factor 1-alpha gene.
1
Unidentified tree
MFLUCC 12-0267; NN0470990
Rhododendron sp.
Sympolocos sp.
MFLUCC 12-0266; NN0469780
Unidentified tree
Schima sp.
MFLUCC 12-0265; NN0469830
MFLUCC 12-0276; NN0469740*
Chrysophyllum sp.
MFLUCC 12-0264; NN0471960
MFLUCC 12-0275; NN0473080
Unidentified tree
MFLUCC 12-0263; NN0470720
P. unicolor
Podocarpus macrophyllus
IFRDCC 2403
P. trachicarpicola
Host/Substrate
Species
PESTALOTIOPSIS
REVISITED
129
MAHARACHCHIKUMBURA
ET AL.
RESULTS
The sequences generated in this study were supplemented with
additional sequences obtained from GenBank (Table 1) based on
blast searches and literature. Multiple sequence alignments were
generated with MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/
index.html); the alignment was visually improved with Mesquite v.
2.75 (Maddison & Maddison 2011) and MEGA v. 5.2.2 (Kumar
et al. 2012) or BioEdit v. 7.0.5.2 (Hall 1999). Three different
datasets were used to estimate three phylogenies: an Amphisphaeriaceae family tree, a combined Neopestalotiopsis and
Pseudopestalotiopsis species tree, and a Pestalotiopsis species
tree. The first tree focuses on the placement and further division of
Pestalotiopsis into two new genera in Amphisphaeriaceae by
using the LSU region. The second and third phylogenetic analyses were produced to show species relationships in Pestalotiopsis, Neopestalotiopsis and Pseudopestalotiopsis based on the
combined datasets (ITS, TUB and TEF). The combined alignments were split between the genera to improve the robustness of
the alignment across the three loci. Phylogenetic analyses of the
sequence data consisted of Bayesian Inference (BI), Maximum
Likelihood (ML) and Maximum Parsimony (MP) analyses of both
the individual data partitions as well as the combined aligned
dataset. Ambiguously aligned regions were excluded from all
analyses and gaps were treated as “fifth character state” in the
parsimony analysis. Suitable models for the Bayesian analysis
were first selected using models of nucleotide substitution for
each gene, as determined using MrModeltest v. 2.2 (Nylander
2004), and included for each gene partition. The Bayesian analyses (MrBayes v. 3.2.1; Ronquist et al. 2012) of four simultaneous
Markov Chain Monte Carlo (MCMC) chains were run from random
trees for 10 000 000 generations and sampled every 1 000 generations. The temperature value was lowered to 0.15, burn-in was
set to 0.25, and the run was automatically stopped as soon as the
average standard deviation of split frequencies reached below
0.01. A maximum likelihood analysis was performed using
raxmlGUI v. 1.3 (Silvestro & Michalak 2011). The optimal ML tree
search was conducted with 100 separate runs, using the default
algorithm of the program from a random starting tree for each run.
The final tree was selected among suboptimal trees from each run
by comparing likelihood scores under the GTR+GAMMA substitution model. The MP analysis was performed with PAUP
(Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford
2003). Trees were inferred by using the heuristic search option
with TBR branch swapping and 1 000 random sequence additions. The maximum number of retained trees were limited to
5 000, branches of zero length were collapsed and all multiple
equally most parsimonious trees were saved. Tree length [TL],
consistency index [CI], retention index [RI], rescaled consistency
index [RC], homoplasy index [HI], and log likelihood [-ln L] (HKY
model) values were calculated. The robustness of the equally
most parsimonious trees was evaluated by 1 000 bootstrap replications (Felsenstein 1985) resulting from a maximum parsimony
analysis, each with 10 replicates of random stepwise addition of
taxa. The Kishino-Hasegawa tests (Kishino & Hasegawa 1989)
were performed to determine whether the trees inferred under
different optimality criteria were significantly different. The
resulting trees were printed with FigTree v. 1.4.0 (http://tree.bio.
ed.ac.uk/software/figtree/) and the layout was done with Adobe
Illustrator CS v. 6. The alignments and trees were deposited in
TreeBASE (www.treebase.org/treebase/index.html).
Phylogeny
130
The LSU alignment was used to resolve the generic placement of
Pestalotiopsis strains in the Amphisphaeriaceae (Fig. 3). The
alignment comprised 74 strains (including the outgroup taxon
Xylaria hypoxylon) and the manually adjusted dataset comprised
807 characters including gaps; the data partition contained 173
unique site patterns. Dirichlet base frequencies and the
GTR+I+G model with inverse gamma-distributed rate were recommended by the MrModeltest analysis and used in the
Bayesian analysis. The Bayesian analysis lasted 1 435 000
generations and the 50 % consensus trees and posterior probabilities were calculated from the 2 154 trees left after discarding
718 trees (the first 25 % of generations) for burn-in (Fig. 3). The
parsimony analysis indicated that 617 characters were constant,
73 variable characters parsimony-uninformative and 117 characters parsimony-informative. After a heuristic search using
PAUP, 125 equally most parsimonious trees were obtained (tree
length = 408 steps, CI = 0.591, RI = 0.871, RC = 0.514,
HI = 0.409). The Bayesian analysis resulted in a tree with the
same topology and clades as the ML and MP trees. The BI, ML
and MP analyses of LSU indicated that Pestalotiopsis comprises
three major monophyletic clades, each supported with high
bootstrap confidence or posterior probability. Species possessing
morphology similar to the type species of Pestalotiopsis
(P. maculans) clustered in one clade designated as Pestalotiopsis s. str. Two well-supported clades clustered outside Pestalotiopsis s. str., for which two new genera, Neopestalotiopsis
and Pseudopestalotiopsis are introduced. In all analyses,
Pseudopestalotiopsis was always sister to Pestalotiopsis and
clustered as a basal sister clade to Neopestalotiopsis. The
species containing versicolourous median cells form a monophyletic clade named Neopestalotiopsis and appear to have
evolved from the Pseudopestalotiopsis lineage, whose members
have concolourous median cells.
Species relationships in Neopestalotiopsis and Pseudopestalotiopsis are shown in Fig. 4. For the combined genes, BI, ML,
and MP consensus trees revealed the same phylogenetic relationships between the significantly supported clades. The
combined ITS, TUB and TEF alignment comprises 59 strains
(including 24 ex-type / ex-epitype strains for species of Neopestalotiopsis, three ex-type / ex-epitype strains for species of
Pseudopestalotiopsis, and Pestalotiopsis trachicarpicola as the
outgroup taxon) and 1 418 characters including gaps with 66,
145 and 180 unique site patterns for ITS, TUB and TEF,
respectively. Suitable models were selected using models of
nucleotide substitution for each gene, as determined using
MrModeltest. The GTR+I model with a proportion of invariable
sites for ITS and the HKY+G model with gamma-distributed rate
model for TUB and the GTR+I+G model with inverse gamma rate
were selected for TEF and included for each gene partition. The
Bayesian analysis lasted 2 585 000 generations and the 50 %
consensus trees and posterior probabilities were calculated from
the 3 880 trees left after discarding 1 293 trees (the first 25 % of
generations) for burn-in (Fig. 4). Among these 1 418 characters
(ITS = 491, TUB = 442 and TEF = 485), 990 were constant, 172
variable characters parsimony uninformative and 256 characters
parsimony-informative. The parsimony analysis resulted in 108
equally most parsimonious trees (tree length = 805 steps,
PESTALOTIOPSIS
-/100
-/64
87/75
0.3
REVISITED
AF132333 Xylaria hypoxylon
AF452038 Arecophila bambusae
AY772015 Funiliomyces biseptatus
AF452029 Amphisphaeria umbrina
89/75
AF452035 Lanceispora sp.
100/100
AF452032 Lanceispora sp.
DQ534037 Monochaetia kansensis
100/100
DQ414531 Seiridium papillatua
53/62
AF382377 Seiridium cardinale
0.95/56
AF382376 Seiridium cardinale
KM116280 Seiridium sp.
KC005810 Seiridium phylicae
57/60
87/67 KC005809 Seiridium phylicae
100/100
AF382385 Truncatella laurocerasi
100/100
AF382383 Truncatella angustata
100/100
AF382382 Truncatella sp.
71/50
DQ278929 Truncatella restionacearum
DQ278928 Truncatella hartigii
85/81
79/74
AF452047 Dyrithiopsis lakefuxianensis
AF382368 Bartalinia lateripes
53/55
60/53
AF382369 Bartalinia laurina
90/74
96/58 AF382367 Bartalinia bischofiae
AB593720 Discosia sp.
86/- AB593708 Discosia pini
AB593705 Discosia artocreas
86/AB593712 Discosia sp.
98/95 JN871212 Seimatosporium eucalypti
JN871209 Seimatosporium eucalypti
53/56
78/83
AB593737 Seimatosporium hypericinum
98/98 68/61 AB593733 Seimatosporium elegans
AB593739 Discostroma fuscellum
AB593727 Discostroma tostum
68/97
AB593735 Seimatosporium glandigenum
57/51
90/64 AB593726 Discostroma fuscellum
CBS 272.29 Pseudopestalotiopsis cocos
EU833969 Pseudopestalotiopsis theae
91/85
KM116278 Pseudopestalotiopsis sp.
MFLUCC 12-0055 Pseudopestalotiopsis theae
KM116277 Pseudopestalotiopsis sp.
63/51
IMI 192475 Neopestalotiopsis steyaertii
CBS 111495 Neopestalotiopsis zimbabwana
CBS 114159 Neopestalotiopsis australis
CBS 254.32 Neopestalotiopsis piceana
96/91 CBS 164.42 Neopestalotiopsis sp.
CBS 115.83 Neopestalotiopsis formicarum
98/99
CBS 111494 Neopestalotiopsis surinamensis
CBS 110.20 Neopestalotiopsis sp.
CBS 336.86 Neopestalotiopsis mesopotamica
CBS 124745 Neopestalotiopsis rosae
CBS 138.41 Neopestalotiopsis natalensis
62/55
CBS 101057 Neopestalotiopsis rosae
100/94 EU715665 Pestalotiopsis sp.
CBS 434.65 Pestalotiopsis arceuthobii
95/89 71/- CBS 356.86 Pestalotiopsis spathulata
CBS 114491 Pestalotiopsis hawaiiensis
CBS 109350 Pestalotiopsis jesteri
CBS 887.96 Pestalotiopsis papuana
CBS 331.92 Pestalotiopsis arengae
CBS 263.33 Pestalotiopsis sp.
CBS 237.38 Pestalotiopsis chamaeropis
59/CBS 118553 Pestalotiopsis colombiensis
KM116223 Pestalotiopsis sp.
-/57 CBS 114138 Pestalotiopsis knightiae
CBS 114137 Pestalotiopsis telopeae
CBS 790.68 Pestalotiopsis biciliata
91/91
CBS 443.62 Pestalotiopsis camelliae
MFLUCC 12-0278 Pestalotiopsis camelliae
MFLUCC 12-0054 Pestalotiopsis furcata
CBS 265.33 Pestalotiopsis hollandica
CBS 102220 Pestalotiopsis malayana
Fig. 3. Consensus phylogramme (50 % majority rule) of 2 154 trees resulting from a Bayesian analysis of the LSU sequence alignment of Neopestalotiopsis, Pestalotiopsis,
Pseudopestalotiopsis and other genera in family Amphisphaeriaceae. Genera are indicated in coloured blocks and red-thickened lines indicate Bayesian posterior probabilities
(PP) above 95 %. RAxML bootstrap support values (MLB) and maximum parsimony bootstrap support values (MPB) are given at the nodes (MLB/MPB). The scale bar
represents the expected number of changes per site. The tree was rooted to Xylaria hypoxylon (GenBank AF132333).
131
MAHARACHCHIKUMBURA
ET AL.
Fig. 4. Consensus phylogramme (50 % majority rule) of 3 880 trees resulting from a Bayesian analysis of the combined (ITS+TUB+TEF) alignment of the analysed Neopestalotiopsis and Pseudopestalotiopsis sequences. Pseudopestalotiopsis is indicated in grey shades and Neopestalotiopsis clades are indicated in yellow and orange coloured
blocks. Clades are numbered to the right of the blocks (1–30). Red-thickened lines indicate Bayesian posterior probabilities (PP) above 95 %. RAxML bootstrap support values
(MLB) and maximum parsimony bootstrap supports (MPB) are given at the nodes (MLB/MPB). Strain accession numbers (sequences derived from ex-type are printed in bold)
are followed by the isolation source (green) and country of origin (brown). The correct species name is indicated to the right of the clade. The scale bar represents the expected
number of changes per site. The tree was rooted to Pestalotiopsis trachicarpicola (OP068).
132
PESTALOTIOPSIS
REVISITED
2x
Neopestalotiopsis saprophyta MFLUCC 12-0282
CBS 109350 Fragraea Papua New Guinea
IFRDCC 2439 Rhododendron China
P. ericacearum
P. arceuthobii
CBS 434.65 Arceuthobium USA
87/100
P. arengae
CBS 331.92 Arenga Singapore
70/78
P. hawaiiensis
CBS 114491 Leucospermum Hawaii
100/100
IFRDCC 2397 Mangifera China
P. anacardiacearum
P. diversiseta
MFLUCC 12-0287 Rhododendron China
89/100
P. spathulata
CBS 356.86 Gevuina Chile
98/100
P. gaultheria
IFRD 411-014 Gaultheria China
CBS 130973 Banksia Australia
84/98
P. camelliae
13
P. inflexa
P. rhododendri
14
15
16
P. monochaeta
17
P. hollandica
P. linearis
18
19
20
21
22
P. chamaeropis
23
P. unicolor
24
P. scoparia
25
P. australis
26
CBS 443.62 Camellia Turkey
98/100 MFLUCC 12-0277 Camellia China
100/100 MFLUCC 12-0278 Camellia China
MFLUCC 12-0270 unidentified tree China
MFLUCC 12-0268 Buxus China
100/100
IFRDCC 2399 Rhododendron China
95/90 CBS 144.97 Quercus Netherlands
92/95
P. clavata
CBS 440.83 Taxus Netherlands
98/80
CBS 265.33 Sciadopitys Netherlands
-/99 CBS 170.26 Brassica New Zealand
MFLUCC 12-0274 Rhododendron China
-/92
100/99
MFLUCC 12-0271 Trachelospermum China
94/99 83/98/97
P. brassicae
P. verruculosa
P. intermedia
CBS 186.71 Chamaerops Italy
CBS 237.38 Unknown Italy
CBS 113604 Unknown Unknown
89/78
77/- CBS 113607 Unknown Unknown
95/- MFLUCC 12-0276 Rhododendron China
CBS 176.25 Chamaecyparis Unknown
CBS 119350 Brabejum South Africa
79/93
83/82
6
7
8
9
P. furcata
P. novae-hollandiae
MFLUCC 12-0054 Camellia Thailand
100/100
1
2
3
4
5
10
11
12
P. portugalica
CBS 393.48 Unknown Portugal
76/96
P. jesteri
100/100
87/93
CBS 111503 Protea South Africa
CBS 114193 Grevillea Australia
CBS 114474 Protea South Africa
0.05
Fig. 5. Consensus phylogramme (50 % majority rule) of 1 120 trees resulting from a Bayesian analysis of the combined (ITS+TUB+TEF) alignment of the analysed Pestalotiopsis isolates. Clades are indicated in coloured blocks. Clades are numbered to the right of the boxes (1–43). Red-thickened lines indicate Bayesian posterior probabilities
(PP) above 95 %. RAxML bootstrap support values (MLB) and maximum parsimony bootstrap supports (MPB) are given at the nodes (MLB/MPB). Strain accession numbers
(sequences derived from ex-type are printed in bold) are followed by the isolation source (white) and country of origin (red). The correct species name is indicated to the right of
the clade. The scale bar represents the expected number of changes per site. The tree is rooted to Neopestalotiopsis saprophytica (MFLUCC 12-0282).
133
MAHARACHCHIKUMBURA
ET AL.
CBS 118553 Eucalyptus Colombia
CBS 336.97 soil Papua New Guinea
P. colombiensis
27
P. humus
28
P. diploclisiae
29
P. malayana
30
P. adusta
31
P. papuana
32
Pestalotiopsis sp.
33
P. rosea
34
P. parva
35
P. grevilleae
36
P. knightiae
37
P. biciliata
38
P. australasiae
39
P. telopeae
40
P. oryzae
41
P. kenyana
42
P. trachicarpicola
43
CBS 115450 Ilex Hong Kong
100/100 70/-
CBS 115449 Psychotria Hong Kong
91/85
CBS 115585 Diploclisia Hong Kong
CBS 115587 Diploclisia Hong Kong
98/97
CBS 102220 Macaranga Malaysia
ICMP 6088 refrigerator door gasket Fiji
MFLUCC 10-0146 Syzygium Thailand
73/75
91/85
94/92
CBS 331.96 coastal soil Papua New Guinea
97/100 CBS 887.96 Cocos Papua New Guinea
CBS 264.33 Cocos Sulawesi
86/98 CBS 263.33 Rhododendron Netherlands
MFLUCC 12-0258 Pinus China
100/100 CBS 265.37 Delonix Unknown
CBS 278.35 Leucothoe Unknown
99/99
CBS 114127 Grevillea Australia
93/92
100/100
CBS 111963 Knightia New Zealand
CBS 790.68 Taxus Netherlands
-/-
97/- CBS 124463 Platanus Slovakia
95/98 CBS 236.38 Paeonia Italy
100/100
CBS 114141 Protea Australia
73/85CBS 114137 Protea Australia
100/99
CBS 113606 Telopea Australia
CBS 114161 Telopea Australia
100/100
CBS 111522 Telopea Hawaii
CBS 353.69 Oryza Denmark
CBS 171.26 Unknown Italy
97/94
100/100
CBS 442.67 Coffea Kenya
CBS 911.96 agar Unknown
IFRDCC 2403 Podocarpus China
OP068 Trachycarpus China
MFLUCC 12-0266 Sympolocos China
MFLUCC 12-0265 Schima China
0.05
134
MFLUCC 12-0264 Chrysophyllum China
PESTALOTIOPSIS
CI = 0.688, RI = 0.810, RC = 0.557, HI = 0.312). Neopestalotiopsis and Pseudopestalotiopsis isolates clustered into
two well-supported clades (BI = 1, ML = 100 and MP = 100).
Furthermore, thirty clades are recognised in Neopestalotiopsis
and discussed here (Fig. 4).
To clarify species boundaries within Pestalotiopsis, a combined alignment of ITS, TUB and TEF contained 96 sequences
(including the outgroup Neopestalotiopsis saprophytica; MFLUCC
12-0282), and 1 519 characters including alignment gaps with
101, 213 and 268 unique site patterns for ITS, TUB and TEF,
respectively (Fig. 5). Dirichlet base frequencies and the GTR+I+G
model with inverse gamma-distributed rate for ITS and HKY+I+G
model with inverse gamma-distributed rate were selected for TUB
and TEF and set in MrBayes. The Bayesian analysis lasted
745 000 generations and the 50 % consensus trees and posterior
probabilities were calculated from the 1 120 trees left after discarding 373 trees (the first 25 % of generations) for burn-in
(Fig. 5). Of the 1 519 characters (ITS = 552, TUB = 463 and
TEF = 504), 890 were constant, 250 variable characters parsimony uninformative and 379 characters parsimony-informative. A
MP analysis yielded 96 equally most parsimonious trees (tree
length = 1 628 steps, CI = 0.596, RI = 0.808, RC = 0.482,
HI = 0.404). The Bayesian analysis resulted in a tree with the
same topology and terminal clades as the ML and MP trees.
Fourty-three clades are recognised and discussed here (Fig. 5).
Taxonomy
Phylogenetic analyses based on the LSU alignment, together with
an appraisal of the literature and morphology, resulted in the
proposal of two novel genera in Amphisphaeriaceae. The new
genera Neopestalotiopsis and Pseudopestalotiopsis, which
segregate off Pestalotiopsis, are proposed based on the types
Neopestalotiopsis protearum and Pseudopestalotiopsis theae,
respectively. Descriptions of the new genera Neopestalotiopsis
and Pseudopestalotiopsis are provided. Based on the results of
ITS, TUB and TEF sequence analyses, 30 internal clades (clades
1–30; Fig. 4) can be distinguished in Neopestalotiopsis; three
clades in Pseudopestalotiopsis (Fig. 4) and 43 clades in Pestalotiopsis (clades 1–43; Fig. 5). Several Pestalotiopsis species are
transferred to Neopestalotiopsis and Pseudopestalotiopsis.
Eleven new species of Neopestalotiopsis are described and one
ex-type re-examined. Two novel species are introduced in
Pseudopestalotiopsis. Twenty-four new species of Pestalotiopsis
are described and illustrated here and two ex-types are reexamined. Based on the molecular phylogeny, several remaining isolates represent unnamed species; these are not treated
further as most of these isolates did not sporulate, or due to lack of
ecological diversity.
Neopestalotiopsis Maharachch., K.D. Hyde & Crous,
gen. nov. MycoBank MB809759.
Pestalotiopsis.
Conidiomata acervular or pycnidial, subglobose, globose,
clavate, solitary or aggregated, dark brown to black, immersed to
erumpent, unilocular or irregularly plurilocular; exuding dark
brown to black conidia in a slimy, globose mass. Conidiophores
indistinct, often reduced to conidiogenous cells. Conidiogenous
REVISITED
cells discrete, cylindrical, ampulliform to lageniform, hyaline,
smooth, thin-walled; conidiogenesis initially holoblastic,
becoming percurrent to produce additional conidia at slightly
higher levels. Conidia fusoid, ellipsoid to subcylindrical, straight
to slightly curved, 4-septate; basal cell conic to subcylindrical,
with a truncate base, hyaline or pale brown to olivaceous, thin
and rugose to smooth-walled; three median cells doliiform, wall
rugose to verruculose, versicoloured, septa darker than the rest
of the cell; apical cell hyaline, conic to cylindrical, thin- and
smooth-walled; with tubular apical appendages, one to many,
filiform or attenuated, flexuous, branched or unbranched; basal
appendage single, tubular, unbranched, centric.
Type species: Neopestalotiopsis protearum (Crous & L. Swart)
Maharachch., K.D. Hyde & Crous (see below).
Notes: Based on LSU sequence data (Fig. 3), Neopestalotiopsis
clusters in Amphisphaeriaceae and is distinct from Pseudopestalotiopsis and Pestalotiopsis, and is best treated as a separate
genus. Liu et al. (2010), based on the length of the ITS alignment,
also revealed that species of Pestalotiopsis cluster in three groups.
The ITS sequence lengths in groups A, B, and C (i.e. Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis) were
480–484 bp, 489–495 bp and 536–540 bp, respectively.
Morphologically Neopestalotiopsis can also be easily distinguished from Pseudopestalotiopsis and Pestalotiopsis by its versicolourous median cells. Furthermore, in Neopestalotiopsis
conidiophores are indistinct and often reduced to conidiogenous
cells. In the key provided by Guba (1961) and Steyaert (1949) the
species in the versicolourous group divided into two subgroups:
umber-olivaceous (two upper median cells umber and lowest
median cell yellow-brown) and fuliginous-olivaceous (two upper
median cells fuliginous, usually opaque, and lowest median cell
pale brown). In his monograph Guba (1961) treated the versicolourous umber-olivaceous group, which comprised 40 species and
the versicolourous fuliginous-olivaceous group, which comprised
56 species. The two groups were differentiated depending on the
intensities of the median cells, while most species have similar
conidial measurements. Jeewon et al. (2003), Liu et al. (2010) and
Maharachchikumbura et al. (2011) concluded that the division of
the versicolourous group based on colour intensities of the median
conidial cell is not a taxonomically good character. Instead of using
two groups, we propose Neopestalotiopsis as a new genus for the
versicolourous group.
Neopestalotiopsis aotearoa Maharachch., K.D. Hyde &
Crous, sp. nov. MycoBank MB809760. Fig. 6.
Etymology: Named after the Maori name (= Aotearoa) for the
country where it was collected, New Zealand.
Conidiomata (on PDA) pycnidial, globose to clavate, solitary or
confluent, embedded or semi-immersed to erumpent, dark
brown, 200–450 μm diam; exuding globose, dark brown to black
conidial masses. Conidiophores indistinct, often reduced to
conidiogenous cells. Conidiogenous cells discrete, subcylindrical
to ampulliform, hyaline, proliferating 2–4 times percurrently,
5–20 × 2–10 μm, apex 2–5 μm diam. Conidia fusoid, ellipsoid,
straight to slightly curved, 4-septate, (19.5–)21–28(–29) × (6–)
6.5–8.5(–9) μm, x ± SD = 24.8 ± 1.6 × 7.7 ± 0.5 μm; basal cell
conic with a truncate base, hyaline, rugose and thin-walled,
135
MAHARACHCHIKUMBURA
ET AL.
Fig. 6. Neopestalotiopsis aotearoa CBS 367.54T. A. Conidiomata sporulating on PNA (pine needle agar). B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia.
4–6.5 μm long; three median cells doliiform, (13–)
14–18(–18.5) μm long, x ± SD = 15.9 ± 1.1 μm, wall verruculose, versicoloured, septa darker than the rest of the cell (second
cell from the base pale brown, 4–6 μm long; third cell honeybrown, 3.5–7 μm long; fourth cell brown, 4–6.5 μm long); apical cell 3.5–5.5 μm long, hyaline, cylindrical to subcylindrical,
thin- and smooth-walled; with 2–3 tubular apical appendages
(mostly 3), arising from the apical crest, unbranched, filiform,
(3–)5–12(–13) μm long, x ± SD = 8.1 ± 1.2 μm; basal
appendage single, tubular, unbranched, centric, 1.5–4 μm long.
Notes: Neopestalotiopsis aotearoa (clade 16; Fig. 4) is described
from a canvas in New Zealand. In the phylogenetic analyses,
N. aotearoa proved to be sister to N. piceana (clade 17; Fig. 4),
but the two species are morphologically easily distinguishable.
Neopestalotiopsis piceana is distinct from N. aotearoa by its
clavate conidia, longer basal, and apical appendages.
Culture characteristics: Colonies on PDA attaining 30–40 mm
diam after 7 d at 25 °C, with undulate edge, pale honey-coloured,
sparse aerial mycelium on the surface with black, gregarious
conidiomata; reverse similar in colour.
Basionym: Pestalotiopsis asiatica Maharachch. & K.D. Hyde,
Fungal Divers. 56: 104. 2012.
Habitat: Saprobe on canvas.
Known distribution: New Zealand.
Material examined: New Zealand, from canvas, Sep. 1954, G.C. Wade (CBS H15765, holotype, ex-type culture CBS 367.54 = ATCC 11763 = QM 381).
136
Neopestalotiopsis asiatica (Maharachch. & K.D. Hyde)
Maharachch., K.D. Hyde & Crous, comb. nov. MycoBank
MB809761.
Material examined: China, Hunan Province, Yizhang County, Mangshan, from
living leaves of unidentified tree, 12 Apr. 2002, W.P. Wu (HMAS047638, holotype; MFLU 12-0422, isotype, ex-type culture NN0476380 = MFLUCC 12-0286).
Note: This species (clade 6; Fig. 4) was treated in detail by
Maharachchikumbura et al. (2012).
PESTALOTIOPSIS
Neopestalotiopsis australis Maharachch., K.D. Hyde &
Etymology: Named after the country where it was collected,
Australia.
Conidiomata pycnidial in culture on PDA, globose to clavate,
solitary or aggregated in clusters, semi-immersed, brown to
black, 100–500 μm diam; exuding globose, dark brown to black
conidiogenous cells. Conidiogenous cells discrete, ampulliform to
lageniform, hyaline, rugose-walled, simple, proliferating 1–3
times percurrently, 5–12 × 2–7 μm, apex 1–2 μm diam. Conidia
fusoid, ellipsoid, straight to slightly curved, 4-septate, (19–)
21–27(–28) × (7–)7.5–9(–9.5) μm, x ± SD = 24.6 ± 1.8
× 8 ± 0.4 μm; basal cell conic with a truncate base, hyaline,
rugose and thin-walled, 3.5–5.5 μm long; three median cells
doliiform, (13–)14–18(–18.5) μm long, x ± SD = 16.1 ± 1 μm,
wall rugose, versicoloured, septa darker than the rest of the cell
(second cell from the base pale brown, 3.5–6.5 μm long; third cell
darker brown, 4–7 μm long; fourth cell brown, 5–6.5 μm long);
REVISITED
apical cell 3–6 μm long, hyaline, subcylindrical to obconic, rugose
and thin-walled; with 3–4 tubular apical appendages (mostly 3),
arising from the apical crest, unbranched, filiform, flexuous, (19–)
21–32(–34) μm long, x ± SD = 26.6 ± 3 μm; basal appendage
single, tubular, unbranched, centric, 3–7 μm long.
diam after 7 d at 25 °C, with lobate edge, pale honey-coloured,
with dense aerial mycelium on the surface with black, concentric
Habitat: On Telopea sp.
Known distribution: Australia.
Material examined: Australia, New South Wales, from Telopea sp., 12 Oct. 1999,
P.W. Crous (CBS H-21773, holotype, ex-type culture CBS 114159 = STE-U 3017).
Notes: Neopestalotiopsis australis (clade 21; Fig. 4) was isolated
from Telopea sp. in New South Wales, Australia. The conidiogenous cells and conidia of N. australis resemble those of the
Fig. 7. Neopestalotiopsis australis CBS 114159T. A. Conidioma sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
137
MAHARACHCHIKUMBURA
ET AL.
two Indian isolates, CBS 266.80 and CBS 119.75 (clade 22; Fig. 4),
which were isolated from Vitis vinifera and Eucalyptus globulus,
respectively. Since there is geographical variation of the two Indian
isolates and a slight distinction in phylogeny, they are tentatively
maintained as Neopestalotiopsis sp. Clade 22 until additional collections and cultures become available. There are various fungal
pathogens recorded from Proteaceae, which is an important plant
family in world floriculture markets (Crous et al. 2011). Neopestalotiopsis and Pestalotiopsis have subsequently been isolated
from several Protea and Leucospermum hosts (Swart et al. 1999),
and intercepted at quarantine inspection points (Taylor 2001).
Neopestalotiopsis australis, N. honoluluana, N. protearum and
N. zimbabwana are recorded from Proteaceae plants. Most of
these species cause leaf spots and tip dieback, and can be easily
identified based on diagnostic morphology and phylogeny.
Neopestalotiopsis chrysea (Maharachch. & K.D. Hyde)
MB809763.
Basionym: Pestalotiopsis chrysea Maharachch. & K.D. Hyde,
Materials examined: China, Guangxi Province, Shangsi, Shiwandashan, Wangle,
dead leaves of unidentified plant, 2 Jan. 1997, W.P. Wu (HMAS042855, holotype; MFLU 12-0411, isotype, ex-type culture NN042855 = MFLUCC 12-0261);
Hunan Province, Yizhang County, Mangshanon, dead plant material, 12 Apr.
2002, W.P. Wu, culture NN047037 = MFLUCC 12-0262.
Neopestalotiopsis clavispora (G.F. Atk.) Maharachch.,
K.D. Hyde & Crous, comb. nov. MycoBank MB809764.
Basionym: Pestalotia clavispora G.F. Atk., Bull. Cornell Univ. 3:
37. 1897.
≡ Pestalotiopsis clavispora (G.F. Atk.) Steyaert, Bull. Jard. bot. Etat
Brux. 19: 335. 1949.
Materials examined: China, Guangxi Province, Shiwandashan, on dead leaves of
Magnolia sp., 28 Dec. 1997, W.P. Wu (HMAS043133 = MFLU 12-0418, epitype,
ex-epitype culture NN043133 = MFLUCC 12-0281); Guangxi Province, Yunnan,
Shiwandashan, on dead leaves of Magnolia sp., 28 Dec. 1997, W.P. Wu, culture
NN043011 = MFLUCC 12-0280. Sri Lanka, decaying wood, 23 Jan. 1973, W.
Gams, culture CBS 447.73. USA, Auburn, Alabama, on fallen leaves of Quercus
rubra, 10 Mar. 1891, F. Atkinson (CUP-A-032389, holotype).
Neopestalotiopsis cubana Maharachch., K.D. Hyde &
present and not flared. Conidia fusoid, ellipsoid, straight to slightly
curved, somewhat constricted at septa, 4-septate, (19–)
20–25(–27) × (7.5–)8–9.5(–10) μm, x ± SD = 23.4 ± 1.4 ×
8.8 ± 0.4 μm; basal cell obconic to conic with a truncate base,
hyaline, rogose and thin-walled, 3–5 μm long; three median cells
doliiform, (13.5–)14–16.5(–17.5) μm long, x ± SD = 15.5
± 0.9 μm, wall rugose, versicoloured, septa darker than the rest of
the cell (second cell from the base pale brown, 4.5–6 μm long; third
cell honey-brown, 4.5–6.5 μm long; fourth cell brown, 4–5.5 μm
long); apical cell 4–5 μm long, hyaline, subcylindrical, thin- and
smooth-walled; with 2–4 tubular apical appendages (mostly 3),
arising from the apical crest, unbranched, filiform, flexuous, (19–)
21–27(–28) μm long, x ± SD = 24 ± 2 μm; basal appendage
diam after 7 d at 25 °C, with lobate edge, pale honey coloured,
with sparse aerial mycelium on the surface with black, gregarious
Habitat: On leaf litter.
Known distribution: Cuba.
Material examined: Cuba, from leaf litter, Jun. 1996, R.F. Casta~neda (CBS H21772, holotype, ex-type culture CBS 600.96 = INIFAT C96/44-4).
Notes: Neopestalotiopsis cubana (clade 19; Fig. 4) is from leaf
litter isolated in Cuba, and forms a sister clade to CBS 164.42
and CBS 360.61, which were isolated from sand dunes in France
and Cinchona sp. in Guinea, respectively. The latter isolates are
morphologically somewhat similar to N. cubana, even though,
due to clear ecological differences we prefer to maintain them as
Neopestalotiopsis sp. Clade 20 until we have obtained more
cultures and collections. Neopestalotiopsis cubana is distinguished from the sister N. saprophytica (clade 18; Fig. 4)
(22–30 × 5–6 μm) by its wider conidia.
Neopestalotiopsis ellipsospora (Maharachch. & K.D.
Hyde) Maharachch., K.D. Hyde & Crous, comb. nov.
MycoBank MB809766.
Basionym: Pestalotiopsis ellipsospora Maharachch. & K.D.
Hyde, Fungal Divers. 56: 112. 2012.
Materials examined: China, Yunnan Province, on dead plant materials, L.D. Guo
(MFLU 12-0420, holotype, ex-type culture MFLUCC 12-0283); Hong Kong, on fruits
of Ardisia crenata, 1 Jan. 2002, unknown collector, culture CBS 115113 = HKUCC
9136. Thailand, Chiang Rai, Tool Kwan, Huay Mesak waterfall, on dead plant
material, 12 Jan. 2010, S.S.N. Maharachchikumbura, culture MFLUCC 12-0284.
Etymology: Named after the country where it was collected, Cuba.
Conidiomata pycnidial in culture on PDA, globose, solitary or
aggregated, embedded or semi-immersed, dark brown to black, up
to 250 μm diam; exuding globose, brown to black conidial masses.
Conidiophores reduced to conidiogenous cells. Conidiogenous
cells discrete, cylindrical to subcylindrical, 5–12 × 2–4 μm, or
ampulliform to lageniform, 3–8 × 1–4 μm, hyaline, smooth-walled,
proliferating 2–4 times percurrently, 5–15 × 2–5 μm, collarette
138
Neopestalotiopsis eucalypticola Maharachch., K.D.
Hyde & Crous, sp. nov. MycoBank MB809767. Fig. 9.
Etymology: Named after the host genus from which it was isolated, Eucalyptus.
Conidiomata (on PDA) pycnidial, globose, solitary or aggregated
in clusters, semi-immersed, brown to black, 100–400 μm diam;
PESTALOTIOPSIS
REVISITED
Fig. 8. Neopestalotiopsis cubana CBS 600.96T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
exuding globose, dark brown conidial masses. Conidiophores
reduced to conidiogenous cells. Conidiogenous cells discrete,
ampulliform to lageniform, hyaline, smooth, thin-walled, simple,
proliferating up to several times percurrently, 3–10 × 2–8 μm,
opening 2–6 μm diam. Conidia fusoid, ellipsoid, straight to
slightly curved, 4-septate, (22–)23–30(–31) × (9–)
7.5–9(–9.5) μm, x ± SD = 26.7 ± 1.3 × 8.3 ± 0.4 μm; basal cell
conic to obconic with a truncate base, hyaline, rugose and thinwalled, 5–7 μm long; three median cells doliiform, (15.5–)
16–19.5(–20) μm long, x ± SD = 17.6 ± 1.1 μm, wall rugose,
versicoloured, septa darker than the rest of the cell (second cell
from the base pale brown, 5–7 μm long; third cell darker brown,
4.5–7.5 μm long; fourth cell darker brown, 5–7 μm long); apical
cell 4.5–7.5 μm long, hyaline, cylindrical to subcylindrical, rugose
and thin-walled; with 1–2 tubular apical appendages, arising as
an extension of the apical cell, unbranched, attenuated, flexuous,
(20–)32–55(–66) μm long, x ± SD = 43 ± 6 μm; basal
appendage single, tubular, unbranched, centric, 6–11 μm long.
diam after 7 d at 25 °C, with smooth edge, white to pale honeycoloured, with sparse aerial mycelium on the surface with black,
gregarious conidiomata; reverse similar in colour.
Habitat: On Eucalyptus globulus.
Known distribution: Unknown.
Material examined: Unknown country, from Eucalyptus globulus, Jun. 1937,
H.W. Wollenweber (CBS H-15658, holotype, ex-type culture CBS 264.37 = BBA
5300).
Notes: Neopestalotiopsis eucalypticola (clade 23; Fig. 4), which
was isolated from Eucalyptus globulus, is phylogenetically and
morphologically well distinguished from all other species in the
genus. The 1–2, long tubular apical appendages, which are
sometimes branched, attenuated, arising as an extension of the
apical cell, notably distinguish N. eucalypticola from other species.
Neopestalotiopsis foedans (Sacc. & Ellis) Maharachch.,
Basionym: Pestalotia foedans Sacc. & Ellis, Michelia 2: 575. 1882.
≡ Pestalotiopsis foedans (Sacc. & Ellis) Steyaert, Bull. Jard. bot. Etat
Brux. 14: 329. 1949.
Materials examined: China, Xinglong, Hainan, on mangrove plant leaves, Apr.
2005, A.R. Liu (MFLU 12-0424, epitype, ex-epitype culture CGMCC 3.9123);
139
MAHARACHCHIKUMBURA
ET AL.
Fig. 9. Neopestalotiopsis eucalypticola CBS 264.37T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
Xinglong, Hainan, on leaves of Calliandra haematocephala, May 2004, A.R. Liu,
culture CGMCC 3.9202; Xinglong, Hainan, on leaves of Neodypsis decaryi, May
2004, A.R. Liu, culture CGMCC 3.9178. USA, Newfield, New Jersey, on decaying
bark of white cedar, Thuja occidentalis, Oct. 1880, Ellis & Harkness (BPI
0405695, holotype).
Neopestalotiopsis formicarum Maharachch., K.D. Hyde
& Crous, sp. nov. MycoBank MB809769. Fig. 10.
Etymology: Named after the insect host family from which it was
isolated, Formicidae.
aggregated in clusters, semi-immersed, brown to black,
200–500 μm diam; exuding globose, dark brown conidial masses.
cells discrete, ampulliform to lageniform, hyaline, smooth, thinwalled, simple, proliferating several times percurrently,
140
3–10 × 2–5 μm, apex 1–3 μm diam. Conidia ellipsoid, straight to
slightly curved, 4-septate, (20–)21–28(–29) × 7.5–9.5 μm,
x ± SD = 24.6 ± 1.4 × 8.6 ± 0.4 μm; somewhat constricted at septa;
basal cell conic to acute with truncate base, rugose and thinwalled, 4.5–6 μm long; three median cells (14–)
15–16.5(–17) μm long, x ± SD = 15.1 ± 1 μm, doliiform, verruculose, versicoloured, brown, septa darker than the rest of the cell
(second cell from base pale brown, 4–6.5 μm long; third cell dark
brown, 4–6 μm long; fourth cell brown, 4.5–6.5 μm long); apical
cell subcylindrical, hyaline, thin- and smooth-walled, 4–5.5 μm
long; with 2–3 tubular apical appendages, arising from the apical
crest, flexuous, unbranched, (20–)23–33(–36) μm long,
x ± SD = 27 ± 4 μm; basal appendage single, tubular, unbranched,
centric, 4–8 μm long.
Culture characteristics: Colonies on PDA reaching 30–40 mm
diam after 7 d at 25 °C, edge undulate, whitish to pale honeycoloured, with moderate aerial mycelium on the surface, with
black, gregarious conidiomata; reverse similar in colour.
PESTALOTIOPSIS
REVISITED
Fig. 10. Neopestalotiopsis formicarum CBS 362.72T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
Habitat: On dead ants and plant debris.
Known distribution: Cuba and Ghana.
Materials examined: Cuba, from plant debris, 1982, sent to CBS for ident. by G.
Arnold (via W. Gams), CBS H-15752, culture CBS 115.83. Ghana, from dead ant
(Formicidae), Nov. 1971, H.C. Evans (CBS H-15661, holotype, ex-type culture
CBS 362.72).
Notes: Neopestalotiopsis formicarum (clade 11; Fig. 4) is a
saprobic species collected from dead ants in Ghana and plant
debris from Cuba. This species is a sister taxon to N. clavispora
and Neopestalotiopsis sp. Clade 10 (clades 12 and clade 10,
respectively; Fig. 4). It differs from N. clavispora in having larger
conidia and longer apical appendages.
Neopestalotiopsis honoluluana Maharachch., K.D. Hyde
Etymology: Named after the city where it was collected, Honolulu
in Hawaii.
solitary or aggregated in clusters, semi-immersed, brown to
black, 100–400 μm diam; exuding globose, dark brown conidial
masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete, subcylindrical to ampulliform, hyaline,
smooth, thin-walled, simple, proliferating up to 3 times percurrently, 5–20 × 2–6 μm, opening 1–3 μm diam. Conidia ellipsoid,
straight to slightly curved, somewhat constricted at septa, 4septate, (21–)24–34(–35) × (7–)7.5–9.5(–10) μm,
x ± SD = 28 ± 2.3 × 8.3 ± 0.6 μm, basal cell obconic with
truncate base, rugose and thin-walled, 4.5–7 μm long; three
median cells (14.5–)15–20(–21) μm long, x ± SD = 17.3
± 1.6 μm, doliiform, rugose, versicoloured, brown to olivaceous
(second cell from base pale brown, 4.5–7 μm long; third cell
darker brown, 4–6.5 μm long; fourth cell brown, 5.5–7.5 μm
long); apical cell subcylindrical, hyaline, thin- and smooth-walled,
4–7.5 μm long; with 3 tubular apical appendages, arising from
the apical crest, flexuous, unbranched, (22–)23–40(–47) μm
long, x ± SD = 32 ± 6.0 μm; basal appendage single, unbranched, centric, 2.5–10 μm long.
141
MAHARACHCHIKUMBURA
ET AL.
Fig. 11. Neopestalotiopsis honoluluana CBS 114495T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
diam after 7 d at 25 °C, edge entire, whitish to pale honeycoloured, with moderate aerial mycelium on the surface, with
Neopestalotiopsis australis was isolated from the same host
genus Telopea, in Australia. Morphologically, however, conidia of
N. australis are smaller and apical appendages are somewhat
shorter.
Habitat: On Telopea sp.
Neopestalotiopsis javaensis Maharachch., K.D. Hyde &
Known distribution: USA (Hawaii).
Etymology: Named after the island where it was collected, Java.
Materials examined: USA, Hawaii, Honolulu, from Telopea sp., 8 Dec. 1998, P.W.
Crous & M.E. Palm (CBS H-21771, holotype, ex-type culture CBS
114495 = STE-U 2076); Waimea, Telopea sp., 8 Dec. 1998, P.W. Crous & M.E.
Palm, culture CBS 111535 = STE-U 2078.
Notes: Neopestalotiopsis honoluluana (clade 24; Fig. 4) is
confined to Telopea sp. in Hawaii, and is a sister taxon to
N. eucalypticola and N. zimbabwana. Neopestalotiopsis eucalypticola differs from N. honoluluana in its longer and fewer apical
appendages. The conidia of N. zimbabwana are smaller and
apical appendages are shorter than those in N. honoluluana.
142
solitary, semi-immersed, dark brown to black, up to 250 μm diam;
exuding dark brown to black conidial masses. Conidiophores
ampulliform to lageniform, hyaline, rugose-walled, proliferating
2–3 times percurrently, 5–25 × 3–10 μm, apex 2–4 μm diam.
Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate,
(24–)25–30(–31) × (6.5–)7–8.5(–9) μm, x ± SD = 27.3 ± 1.6 ×
7.6 ± 0.3 μm; basal cell conic to obconic with a truncate base,
PESTALOTIOPSIS
REVISITED
Fig. 12. Neopestalotiopsis javaensis CBS 257.31T. A. Conidiomata sporulating on PNA. B. Conidioma on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
hyaline, rugose and thin-walled, 4.5–6.5 μm long; three median
cells doliiform, (14.5–)15–18.5(–19) μm long, x ± SD = 17.1
± 1.2 μm, wall rugose, versicoloured, septa darker than the rest
of the cell (second cell from the base pale brown, 5–7 μm long;
third cell brown, 5–7 μm long; fourth cell brown, 5.5–7.5 μm
long); apical cell subcylindrical, hyaline, thin- and smooth-walled,
3.5–5.5 μm long; with 1–3 tubular apical appendages, arising
from the apical crest, unbranched, filiform, 2–10(–18) μm long,
x ± SD = 5.7 ± 3 μm; basal appendage single, tubular, unbranched, centric, 2–4 μm long.
sparse aerial mycelium on the surface with black, gregarious
Habitat: On leaves of Cocos nucifera.
Known distribution: Java.
Material examined: Indonesia, Java, Manado, from leaf of Cocos nucifera,
collection date unknown, R.L. Steyaert (CBS H-15764, holotype, ex-type culture
CBS 257.31).
Notes: Neopestalotiopsis javaensis (clade 28; Fig. 4) was isolated from leaves of coconut in Java. It forms a separate cluster
in the DNA phylogeny, as sister to a species assemblage
including N. foedans, N. mesopotamica and N. rosae. Nestalotiopsis javaensis has relatively larger conidial dimensions when
compared with N. foedans (19–23.5 × 5.5–7 μm)
(Maharachchikumbura et al. 2012). Nestalotiopsis javaensis
differs from N. mesopotamica and N. rosae in having notably
shorter apical appendages (see notes under N. rosae).
Neopestalotiopsis magna (Maharachch. & K.D. Hyde)
MB809772.
Basionym: Pestalotiopsis magna Maharachch. & K.D. Hyde,
Mycol. Prog. 13: 618. 2013.
Material examined: France, Ariege, Rimont, on decaying leaves of Pteridium sp.,
Aug. 2011, K.D. Hyde (MFLU 13-0594, holotype, ex-type culture MFLUCC 120652 = ICMP 20011).
Maharachchikumbura et al. (2013d).
143
MAHARACHCHIKUMBURA
ET AL.
Neopestalotiopsis mesopotamica Maharachch., K.D.
Etymology: Named after the country where the type specimen
was collected, Iraq, hence Mesopotamia.
Conidiomata (on PDA) pycnidial, globose or clavate, aggregated
or confluent, embedded or semi-immersed, black, up to 250 μm
diam; exuding brown to black conidial masses. Conidiophores
cells discrete, cylindrical to subcylindrical, 8–20 × 2–7 μm, hyaline, smooth-walled, proliferating 2–3 times percurrently,
5–18 × 2–4 μm, collarette present and not flared, with prominent
periclinal thickening. Conidia fusoid, ellipsoid, straight to slightly
curved, 4-septate, (25–)26–32(–34) × (7–)7.5–9(–9.5) μm,
x ± SD = 29.6 ± 1.1 × 8 ± 0.4 μm; basal cell conic with a truncate
base, hyaline, rugose and thin-walled, 6–7.5 μm long; three
median cells doliiform, (17–)17.5–20(–21) μm long,
x ± SD = 18.5 ± 1.2 μm, wall rugose, versicoloured, septa darker
than the rest of the cell (second cell from the base pale brown,
5–7.5 μm long; third cell honey brown, 5.5–7.5 μm long; fourth
cell honey brown, 6.5–7.5 μm long); apical cell 4.5–6 μm long,
hyaline, cylindrical to subcylindrical, thin- and smooth-walled;
with 3–4 tubular apical appendages (mostly 3), arising from
the apical crest, unbranched, filiform, flexuous (25–)
28–38(–41) μm long, x ± SD = 33.3 ± 3.2 μm; basal appendage
single, tubular, unbranched, centric, 4–6.5 μm long.
with sparse aerial mycelium on the surface with black, concentric
Habitat: On Achras sapota, Eucalyptus sp. and Pinus brutia.
Known distribution: India, Iraq and Turkey.
Materials examined: India, New Delhi, from Achras sapota, May 1969, unknown
collector, culture CBS 464.69. Iraq, from Pinus brutia, 23 Jun. 1986, sent to CBS
for ident. by A.I. Al-Kinany, Mosul University, Mosul, Iraq (CBS H-15782, holotype, ex-type culture CBS 336.86). Turkey, from Eucalyptus sp., 2 Apr. 1974, G.
Turhan, CBS H-15739 = CBS H-15741, culture CBS 299.74.
Fig. 13. Neopestalotiopsis mesopotamica CBS 336.86T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
144
PESTALOTIOPSIS
Notes: Neopestalotiopsis mesopotamica (clade 29; Fig. 4) forms
a sister group to N. javaensis and N. rosae, and deviates in
having larger conidia and longer apical appendages (see notes
under N. rosae).
Neopestalotiopsis natalensis (J.F.H. Beyma) Maharachch., K.D. Hyde & Crous, comb. nov. MycoBank
MB809774. Fig. 14.
Basionym: Pestalotia natalensis J.F.H. Beyma, Antonie van
Leeuwenhoek 6: 288. 1940.
=Pestalotiopsis natalensis (J.F.H. Beyma) Steyaert, Bull. Jard. bot. Etat
Brux. 19: 344. 1949.
Conidiomata (on PDA) pycnidial, globose, solitary or aggregated,
immersed or semi-immersed, dark brown, 50–150 μm diam.
α-conidiophores indistinct, often reduced to conidiogenous cells.
α-conidiogenous cells discrete, hyaline, rugose, simple, ampulliform, sometimes slightly wide at the base, truncate at apex,
proliferating once or twice, 4–10 × 3–9 μm. α-conidia fusoid,
REVISITED
ellipsoid, straight to slightly curved, 4-septate, (21–)
23–28(–29) × (7.5–)8–10(–10.5) μm, x ± SD = 25.0 ± 1.6
× 9 ± 0.4 μm; basal cell hemispherical, hyaline or slightly brown,
thin- and smooth-walled, 4–7 μm long; three median cells
(15.5–)16–19(–19.5) μm long, x ± SD = 17.5 ± 0.8 μm, concolourous or two upper median cells slightly darker than the
lower median cell, brown, septa darker than the rest of the cell,
and conidium constricted at septum (second cell from the base
5.5–8 μm long; third cell 5.5–8 μm long; fourth cell 5–7 μm
long); apical cell 4–6.5 μm long, hyaline, conic; with 3–5 tubular
apical appendages, arising from the apical crest, unbranched,
(15–)18–32(–35) μm long, x ± SD = 25 ± 4 μm; lacking basal
appendages, when present unbranched, centric, 2–8 μm long.
β-conidiophores 1–2 septate, subcylindrical, hyaline, smooth, up
to 12 μm long or often reduced to conidiogenous cells. β-conidiogenous cells discrete, hyaline, smooth, cylindrical, terminated
in an apex with 1–2 loci which gave rise to β-conidia in a
sympodial arrangement. 5–15 × 2–6 μm. β-conidia (20–)
22–29(–31) × 1–3 μm, x ± SD = 25.6 ± 2 × 1.9 ± 0.2 μm, widest
in the middle, curved, hyaline, apex subobtuse, base truncate.
Fig. 14. Neoestalotiopsis natalensis CBS 138.41T. A. Conidioma sporulating on PNA. B. Conidioma on PDA. C–E. Conidiogenous cells. F–G. β-conidia. H. Beta and alpha
conidia. I–K. α-conidia. Scale bars = 10 μm.
145
MAHARACHCHIKUMBURA
ET AL.
diam after 7 d at 25 °C, with smooth edge, whitish, with sparse
aerial mycelium on the surface; reverse similar in colour. Cultures sporulate poorly on PDA, only few conidiomata can be
seen upon 4 mo of incubation.
Neopestalotiopsis piceana Maharachch., K.D. Hyde &
Habitat: On Acacia mollissima.
Conidiomata (on PDA) pycnidial, globose to clavate, solitary,
semi-immersed, brown to black, 100–300 μm diam; exuding
globose, dark brown to black conidial masses. Conidiophores
ampulliform to lageniform, hyaline, smooth- and thin-walled,
simple, (4–12 × 2–10 μm), apex 2–5 μm diam. Conidia ellipsoid to clavate, straight to slightly curved, 4-septate, (19–)
19.5–25(–26) × (7–)7.5–9(–9.5) μm, x ± SD = 22.1 ± 0.8
× 8.1 ± 0.6 μm; somewhat constricted at septa; basal cell
obconic with truncate base, rugose and thin-walled, 3.5–5.5 μm
long; three median cells (13–)13.5–16(–16.5) μm long,
x ± SD = 15 ± 0.9 μm, doliiform, verruculose, versicoloured,
septa darker than the rest of the cell (second cell from base pale
brown, 4–6 μm long; third cell dark brown, 4.5–6.5 μm long;
fourth cell brown, 5–7 μm long); apical cell obconic, hyaline, thinand smooth-walled, 3–6 μm long; with 3 tubular apical
Known distribution: South Africa.
Material examined: South Africa, KwaZulu-Natal, from Acacia mollissima (black
wattle), Jan. 1941, M.S.J. Ledeboer, ex-type culture CBS 138.41.
Notes: An unusual feature of N. natalensis (clade 2; Fig. 4) is the
presence of a synanamorph in culture. Most species form
β-conidia on the host tissue. Crous et al. (2006) observed α- and
β-conidia in Pestalotiopsis disseminata isolated from Eucalyptus
eurograndis in Colombia. However, α- and β-conidia were only
observed on the original host substrate and not in culture. According to the original description of Van Beyma (1940), the
conidia of N. natalensis are narrower (25–33 × 6–9 μm) and
apical appendages are longer (30–40 μm) than observed here.
Etymology: Named after the host genus from which it was isolated, Picea.
Fig. 15. Neopestalotiopsis piceana CBS 394.48T. A. Conidioma sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
146
PESTALOTIOPSIS
appendages, arising from the apical crest, flexuous, unbranched,
(19–)21–31(–33) μm long, x ± SD = 24.8 ± 3 μm; basal
diam after 7 d at 25 °C, edge entire, whitish to pale honeycoloured, with sparse aerial mycelium on the surface, with
Habitat: On wood of Picea sp., Cocos nucifera and fruit of
Mangifera indica.
Known distribution: Indonesia (Sulawesi) and UK.
Materials examined: Indonesia, Sulawesi, from Cocos nucifera, unknown
collection date and collector, CBS H-15645, culture CBS 254.32. UK, from wood
of Picea sp., Aug. 1948, S.M. Hasan (CBS H-15705, holotype, ex-type culture
CBS 394.48). Unknown country, from fruit of Mangifera indica, Apr. 1930, Levie,
CBS H-15688, culture CBS 225.30.
Notes: Neopestalotiopsis piceana (clade 17; Fig. 4) is characterised by clavate conidia with a long basal appendage. Neopestalotiopsis piceana is sister to N. aotearoa (clade 16; Fig. 4),
which has been described from a canvas in New Zealand. The
two species are distinguishable by TEF (3 bp) sequence data
and not by their ITS and TUB sequences. The species differ by
shape of their conidia and length of their apical appendages (see
notes under N. aotearoa).
Neopestalotiopsis protearum (Crous & L. Swart)
MB809776.
Basionym: Pestalotiopsis protearum Crous & L. Swart, Persoonia 27: 34. 2011.
Material examined: Zimbabwe, Harare, Aveley Farm, on living leaves of Leucospermum cuneiforme cv. ‘Sunbird’, 6 Mar. 1998, L. Swart (PREM 56186,
holotype, ex-type culture CBS 114178 = STE-U 1765).
Note: This species (clade 5; Fig. 4) was treated in detail by Crous
et al. (2011).
Neopestalotiopsis rosae Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Rosa.
Conidiomata (on PDA) pycnidial, globose, solitary, semiimmersed, dark brown to black, 100–300 μm diam; exuding
globose, dark brown, glistening conidial masses. Conidiophores
cells discrete, cylindrical, hyaline, smooth-walled, simple, proliferating 2–4 times percurrently, tapering towards a truncate apex
with visible periclinal thickening, 5–20 × 2–8 μm. Conidia fusoid,
22–37(–29) × (7–)7.5–9.5(–10.5) μm, x ± SD = 24.8 ± 1.5
× 8.5 ± 0.6 μm; basal cell conic to obconic with a truncate base,
hyaline, rugose and thin-walled, 3.5–6 μm long, often with a
short oblique appendage projecting from the base adjoining the
point of attachment of the basal appendage; three median cells
REVISITED
doliiform, (14–)14.5–18(–18.5) μm long, x ± SD = 16 ± 1.1 μm,
wall rugose, versicoloured, septa darker than the rest of the cell
(second cell from the base pale brown, 4.5–6.5 μm long; third
cell honey brown, 5–7 μm long; fourth cell brown, 5–7 μm long);
apical cell 3.5–5.5 μm long, hyaline, cylindrical, thin- and
smooth-walled; with 3–5 tubular apical appendages, not arising
from the apical crest, but each inserted at a different locus in the
upper half of the apical cell, unbranched, filiform, (22–)
24–31(–33) μm long, x ± SD = 27 ± 2.1 μm; basal appendage
diam after 7 d at 25 °C, with lobate edge, pale yellow-coloured,
with moderate aerial mycelium on the surface with black,
concentric conidiomata; reverse similar in colour.
Habitat: On stem of Paeonia suffruticosa and stem lesion in
Rosa sp.
Known distribution: New Zealand and USA.
Materials examined: New Zealand, from stem lesion in Rosa sp., Jul. 1998, J.
Reeve (CBS H-21770, holotype, ex-type culture CBS 101057). USA, Connecticut, Torrington, from stem of Paeonia suffruticosa, 17 May 2007, R.E. Marra,
culture CBS 124745.
Notes: Neopestalotiopsis rosae (clade 27; Fig. 4) was isolated
from a stem lesion in Rosa sp. in New Zealand and stem of
Paeonia suffruticosa in USA, and is morphologically quite
distinct from other taxa in the genus. It has 3–5 tubular apical
appendages, which do not arise from the apical crest; instead
they arise at different regions in the upper half of the apical
cell. Sequences of N. rosae form a sister group to N. javaensis
(clade 28; Fig. 4) and N. mesopotamica (clade 29; Fig. 4), but
N. rosae could be separated from N. javaensis by Bayesian
analysis. However the two clades were supported in the ML
and MP analyses. The two species are separable by TEF
(5 bp) sequence data. There is only a 2-bp difference in
ITS sequence between N. javaensis and N. rosae. Neopestalotiopsis javaensis can be differentiated morphologically
from N. rosae by its long and thin conidia, and shorter apical
appendages. The conidia of N. rosae are wider than those of
N. mesopotamica, and the conidia and apical appendages are
shorter.
Neopestalotiopsis samarangensis (Maharachch. & K.D.
MycoBank MB809778.
Basionym: Pestalotiopsis samarangensis Maharachch. & K.D.
Hyde, Trop. Plant Pathol. 38: 229. 2013.
Materials examined: China, Hong Kong, leaf of unidentified tree, 6 Mar. 2002,
unknown collector, culture CBS 115451 = HKUCC 9095. Thailand, Chiang Mai
Province, Chiang Mai, on fruits of Syzygium samarangense, 20 Jan. 2010, S.S.N.
Maharachchikumbura (MFLU 12-0133, holotype, ex-type culture MFLUCC 120233); ibid., 15 May 2011, S.S.N. Maharachchikumbura, MFLU 12-0134; Chiang
Rai Province, Chiang Rai, 15 Sep. 2011, S.S.N. Maharachchikumbura, MFLU 120135.
Maharachchikumbura et al. (2013b).
147
MAHARACHCHIKUMBURA
ET AL.
Fig. 16. Neopestalotiopsis rosae CBS 101057T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale bars = 10 μm.
Neopestalotiopsis saprophytica (Maharachch. & K.D.
MycoBank MB809780.
juncea in India. Sequences of this taxon form a sister group to
N. protearum (clade 5; Fig. 4). However, due to clear ecological
differences, we retain this isolate as Neopestalotiopsis sp. until
we obtain more collections and cultures for further study.
Basionym: Pestalotiopsis saprophyta Maharachch. & K.D. Hyde,
Materials examined: China, Hong Kong, on fruits of Litsea rotundifolia, 19 Nov.
2001, unknown collector, culture CBS 115452 = HKUCC 8684; Yunnan Province,
Kunming, Kunming Botanical Garden, on leaves of Magnolia sp., 19 Mar. 2002,
W.P. Wu (HMAS047136, holotype; MFLU 12-0419, isotype, ex-type culture
NN047136 = MFLUCC 12-0282).
Material examined: Unknown country, unknown host, Dec. 1920, N.A. Brown,
culture CBS 110.20.
Note: Although phylogenetically slightly distinct (clade 10; Fig. 4),
this culture proved to be sterile, and thus is not treated further.
Material examined: India, from leaf Crotalaria juncea, Feb. 1979, M. Mathur,
culture CBS 233.79.
Materials examined: France, from twig of Camellia sp., Apr. 1976, J. Vegh,
culture CBS 322.76. Indonesia, Java, Cocos nucifera, C.M. Doyer, culture CBS
274.29. Netherlands, from commercial Cocos nucifera imported from Africa, Jan.
1995, A. Aptroot, culture CBS 664.94. Unknown country, from Dalbergia sp.,
unknown collector and collection date, culture CBS 177.25.
Notes: Culture CBS 233.79 (clade 4; Fig. 4) represents a Neopestalotiopsis sp. that was isolated from a leaf of Crotalaria
Notes: Although these isolates (clade 15; Fig. 4) appear to
represent an undescribed species based on phylogenetic data,
148
PESTALOTIOPSIS
due to clear ecological differences of the isolates, we maintain
this clade as Neopestalotiopsis sp. until more cultures and collections are obtained.
Materials examined: France, on dune sand, Mar. 1942, F. Moreau, culture CBS
164.42. Guinea, from young shoot of Cinchona sp. (attacked by Phytophthora
canker), Nov. 1961, J. Chevaugeon, culture CBS 360.61.
Note: Although phylogenetically distinct (clade 20; Fig. 4), both
cultures of this species proved to be sterile, and thus are not
treated further.
Materials examined: India, from Achras sapota, Feb. 1975, H.S. Sohi, culture
CBS 119.75; from berries, leaves and canes of Vitis vinifera, Apr. 1980, H.R.
Reddy, culture CBS 266.80.
Notes: Although phylogenetically and ecologically distinct, these
two isolates (clade 22; Fig. 4) are morphologically similar to
N. australis (clade 21; Fig. 4). Therefore, until more cultures and
collections become available, we prefer to maintain this as
Neopestalotiopsis sp. Clade 22.
Materials examined: France, from Erica gracilis, Aug. 1975, sent to CBS for ident.
by J. Vegh, culture CBS 323.76. Germany, from Erica sp., unknown date, H.W.
Wollenweber, culture CBS 266.37 = BBA 5087 = IMI 083708. Netherlands, from
Cissus sp., unknown collector and collection date, culture CBS 361.61.
Note: See notes under N. zimbabwana.
Neopestalotiopsis steyaertii (Mordue) Maharachch.,
Basionym: Pestalotiopsis steyaertii Mordue, Trans Brit. Mycol.
Soc. 85: 379. 1985.
Material examined: Australia, Australian Capital Territory, Brindabella mountains,
from roots of Eucalyptus viminalis grown in soil, 24 Mar. 1975, G.C. Johnson (extype culture IMI 192475).
Maharachchikumbura et al. (2013d).
REVISITED
± 0.4 μm; basal cell obconic to subcylindrical with a truncate
base, hyaline, rugose and thin-walled, 5–7.5 μm long; three
median cells doliiform, (14.5–)15–17(–17.5) μm long,
x ± SD = 16.5 ± 0.6 μm, wall rugose, versicoloured, septa darker
than the rest of the cell (second cell from the base pale brown,
5.5–6.5 μm long; third cell honey brown, 5–6.5 μm long; fourth
cell brown, 4.5–6 μm long); apical cell 4–5.5 μm long, hyaline,
cylindrical to subcylindrical, thin- and smooth-walled; with 2–3
tubular apical appendages (mostly 3), arising from the apical
crest, unbranched, filiform, flexuous (15–)18–27(–28) μm long,
with dense aerial mycelium on the surface with black, concentric
Habitat: On soil under Elaeis guineensis and leaves of Protea
eximia.
Known distribution: Suriname and Zimbabwe.
Materials examined: Suriname, Brokobaka, from soil under Elaeis guineensis,
Mar. 1974, J.H. van Emden (CBS H-15730, holotype, ex-type culture CBS
450.74). Zimbabwe, Karoi, Glenellen Farm, on living leaves of Protea eximia, 10
Mar. 1998, L. Swart, PREM 56190, culture CBS 111494 = STE-U 1779.
Notes: Neopestalotiopsis surinamensis (clade 3; Fig. 4) was
isolated from soil under Elaeis guineensis (African oil palm) in
Suriname, which is the principal source of palm oil and leaves of
Protea eximia in Zimbabwe. Although phylogenetically closely
related to N. protearum (clade 5; Fig. 4) (Crous et al. 2011), the
two species can be distinguished by their ITS (4 bp) and TEF
(9 bp) sequences, and less easily by their TUB (1 bp) sequences. In morphology, N. surinamensis differs from
N. protearum in having wider conidia, as well as longer and fewer
apical appendages.
Neopestalotiopsis umbrinospora (Maharachch. & K.D.
MycoBank MB809782.
Basionym: Pestalotiopsis umberspora Maharachch. & K.D.
Hyde, Fungal Divers. 56: 121. 2012.
Neopestalotiopsis surinamensis Maharachch., K.D.
Material examined: China, Guangxi Province, Shiwandashan, on dead leaves of
unidentified plant, 30 Dec. 1997, W.P. Wu (HMAS042986, holotype; MFLU 120421, isotype, ex-type culture NN042986 = MFLUCC 12-0285).
Suriname.
Conidiomata (on PDA) pycnidial, globose, mostly aggregated in
clusters, semi-immersed or erumpent, black, up to 350 μm diam;
exuding globose, brown conidial masses. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells
discrete, ampulliform to lageniform, 4–10 × 2–6 μm, hyaline,
smooth-walled, simple, proliferating 2–3 times percurrently, wide
at the base, opening 1–2 μm diam. Conidia fusoid, ellipsoid to
subcylindrical, straight to slightly curved, 4-septate, (23–)
24–28(–29) × (7–)7.5–9(–9.5) μm, x ± SD = 27.7 ± 1 × 8.1
Neopestalotiopsis zimbabwana Maharachch., K.D. Hyde
Zimbabwe.
Conidiomata (on PDA) pycnidial, globose, aggregated or scattered, semi-immersed, black, 150–400 μm diam; exuding
149
MAHARACHCHIKUMBURA
ET AL.
Fig. 17. Neopestalotiopsis surinamensis CBS 450.74T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale
bars = 10 μm.
cells discrete, ampulliform, hyaline, smooth-walled, simple,
proliferating several times percurrently, 5–15 × 3–8 μm, apex
2–5 μm diam. Conidia fusoid, ellipsoid, straight to slightly curved,
4-septate, (22–)23–29(–30) × (6.5–)7–8.5(–9) μm,
x ± SD = 25.3 ± 1.2 × 7.7 ± 0.3 μm; basal cell conic to obconic
with a truncate base, hyaline, rugose and thin-walled,
3.5–5.5 μm long; three median cells doliiform, (15–)
15.5–17.5(–18) μm long, x ± SD = 16.5 ± 0.6 μm, wall rugose,
versicoloured, septa darker than the rest of the cell (second cell
from the base pale brown to pale olivaceous, 4.5–6.5 μm long;
third cell brown to olivaceous, 4.5–6.5 μm long; fourth cell brown
to olivaceous, 5–7 μm long); apical cell 4–6.5 μm long, hyaline,
cylindrical to subcylindrical, rugose and thin-walled; with 2–3
crest, unbranched, filiform, flexuous (18–)23–35(–41) μm long,
x ± SD = 28.6 ± 4 μm; basal appendage single, tubular, unbranched, centric, 3–9.5 μm long.
diam after 7 d at 25 °C, with smooth edge, pale honey-coloured,
150
Habitat: On Leucospermum cuneiforme.
Known distribution: Zimbabwe.
Material examined: Zimbabwe, Banket, Mariondale Farm, on living leaves of
Leucospermum cuneiforme cv. ‘Sunbird’, 9 Mar. 1998, L. Swart (CBS H-21769,
holotype; PREM 56188, isotype, ex-type culture CBS 111495 = STE-U 1777).
Notes: Neopestalotiopsis zimbabwana (clade 25; Fig. 4) occurs
on leaf spots of Leucospermum cuneiforme in Zimbabwe. In our
phylogenetic analyses, N. zimbabwana proved to be allied to
CBS 266.37, CBS 361.61 and CBS 323.76 (clade 26; Fig. 4),
which were isolated from Erica sp. in Germany, Cissus sp. in
Netherlands and Erica gracilis in France, respectively. Even
though the latter isolates have overlapping morphological characters with N. zimbabwana, due to clear geographical differences, we maintain these isolates as Neopestalotiopsis sp.
Clade 26 until we have obtained more collections and cultures.
Neopestalotiopsis protearum (clade 5; Fig. 4) was also identified
PESTALOTIOPSIS
REVISITED
Fig. 18. Neopestalotiopsis zimbabwana CBS 111495T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
as a pathogen on Leucospermum cuneiforme in Zimbabwe.
However, N. protearum and N. zimbabwana are phylogenetically
distinct.
Material examined: China, Yunnan Province, Mangshi, Dehong, on living leaves
of Mangifera indica, Sep. 2011, Y.M. Zhang (IFRD 411-015, holotype, ex-type
culture IFRDCC 2397).
Pestalotiopsis adusta (Ellis & Everh.) Steyaert
Maharachchikumbura et al. (2013c).
Materials examined: Fiji, on refrigerator door PVC gasket, 1 Jun. 1978, E.H.C.
McKenzie (MFLU 12-0425, epitype, ex-epitype culture ICMP 6088 = PDDCC
6088). Thailand, Chiang Rai, on living leaves of Syzygium sp., 6 Feb. 2010,
S.S.N. Maharachchikumbura, culture MFLUCC 10-0146.
Pestalotiopsis arceuthobii Maharachch., K.D. Hyde &
Pestalotiopsis anacardiacearum Y. M. Zhang, Maharachchikumbura & K. D. Hyde
Etymology: Named after the host genus from which it was isolated, Arceuthobium.
solitary or aggregated in clusters, brown to black, semi-immersed,
100–500 μm diam; exuding dark brown conidia in a slimy, globose
151
MAHARACHCHIKUMBURA
ET AL.
Fig. 19. Pestalotiopsis arceuthobii CBS 434.65T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale bars = 10 μm.
mass. Conidiophores mostly reduced to conidiogenous cells,
branched or unbranched, 0–2-septate, hyaline and smooth, up to
10 μm long. Conidiogenous cells discrete, subcylindrical
(3–12 × 1–3 μm) or ampulliform to lageniform (3–10 × 2–6 μm),
hyaline, smooth, thin-walled, proliferating up to 4 times percurrently, collarette present and not flared. Conidia ellipsoid, straight
to slightly curved, somewhat constricted at septa, 4-septate, (21–)
22–25.5(–26) × 6.5–8(–8.5) μm, x ± SD = 24.4
± 1.3 × 7.2 ± 0.5 μm, basal cell obconic with truncate base, rugose
and thin-walled, 5–6 μm long; three median cells (14–)
15–16.5 μm long, x ± SD = 15.6 ± 0.9 μm, doliiform, verruculose,
concolourous, brown (second cell from base 5–6 μm long; third
cell 5.5–6.5 μm long; fourth cell 4.5–6 μm long); apical cell cylindrical, hyaline, thin- and smooth-walled, 4–5 μm long; with 2–3
crest, flexuous, unbranched, (10–)11–14.5(–16) μm long,
x ± SD = 12.8 ± 1.0 μm; basal appendage single, tubular, unbranched, centric, 3–6 μm long.
diam after 7 d at 25 °C, edge entire, whitish to pale honeycoloured, with aerial mycelium on the surface, with black,
152
Habitat: On Arceuthobium campylopodum.
Known distribution: USA.
Material examined: USA, Washington, King County, North Bend, from Arceuthobium campylopodum, Aug. 1965, E.F. Wicker (CBS H-15695, holotype, extype culture CBS 434.65).
Notes: Pestalotiopsis arceuthobii is a distinct species represented
by a single isolate (clade 3; Fig. 5), sister to P. ericacearum (clade
2; Fig. 5). Pestalotiopsis arceuthobii can be distinguished from
P. ericacearum (conidia size = 15–21 × 5–9 μm) by its narrow
conidia (size = 21–26 × 6.5–8.5 μm) as well as short apical appendages (10–16 μm). In P. ericacearum the apical appendages
are longer (19–45 μm), and knobbed.
Pestalotiopsis arengae Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Arenga.
Conidiomata (on PDA) pycnidial, globose or clavate, solitary or
aggregated, semi-immersed, dark brown to black, 200–400 μm
PESTALOTIOPSIS
REVISITED
Fig. 20. Pestalotiopsis arengae CBS 331.92T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
diam; exuding dark brown conidial masses. Conidiophores most
often reduced to conidiogenous cells. Conidiogenous cells
discrete, ampulliform to lageniform, hyaline, smooth, thin-walled,
3–15 × 3–10 μm, proliferating several times percurrently, with
minute periclinal thickenings. Conidia ellipsoid, straight to
slightly curved, slightly constricted at septa, 4-septate, (24–)
25–32(–33) × 7–9.5(–10) μm, x ± SD = 27.6 ± 2 × 8
± 0.4 μm; basal cell conic with a truncate base, rugose and
thin-walled, 4–7 μm long; three median cells (17–)
17.5–21.5(–22) μm long, x ± SD = 19 ± 1.3 μm, doliiform,
verruculose, concolourous, brown, septa darker than the rest of
the cell (second cell from base 5.5–7 μm long; third cell
5.5–8 μm long; fourth cell 6–7.5 μm long); apical cell subcylindrical, hyaline, thin- and smooth-walled, 2.5–4.5 μm long;
with 2–3 tubular apical appendages (mostly 3), arising from the
apical crest, unbranched, filiform, (4–)4.5–11(–12) μm long,
x ± SD = 7.3 ± 1.3 μm; basal appendage single, tubular, unbranched, centric, 1.5–3 μm long.
Habitat: On dead leaves of Arenga undulatifolia.
diam after 7 d at 25 °C, undulate at the margin, white to pale
luteous-coloured, with moderate aerial mycelium on the surface,
with black, gregarious conidiomata; reverse similar in colour.
Etymology: Refers to the broader geographical region (Australia
and New Zealand) where the fungus was isolated.
Known distribution: Singapore.
Material examined: Singapore, Botanical Gardens, from dead leaves of Arenga
undulatifolia, Nov. 1991, W. Gams (CBS H-21768, holotype, ex-type culture CBS
331.92).
Notes: Pestalotiopsis arengae (clade 4; Fig. 5) forms a separate
cluster in the combined phylogeny as basal sister to
P. anacardiacearum (clade 6; Fig. 5) and P. hawaiiensis (clade 5;
Fig. 5), which were isolated from mango from China and Leucospermum sp. from Hawaii, respectively. In morphology,
P. arengae differs from P. anacardiacearum and P. hawaiiensis by
its smaller conidia and shorter apical appendages.
Pestalotiopsis australasiae Maharachch., K.D. Hyde &
Conidiomata pycnidial in culture on PDA, globose, scattered,
semi-immersed, up to 200 μm diam; exuding globose, dark
153
MAHARACHCHIKUMBURA
ET AL.
Fig. 21. Pestalotiopsis australasiae CBS 114126T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
brown to black conidial masses. Conidiophores indistinct, often
reduced to conidiogenous cells. Conidiogenous cells discrete or
integrated, ampulliform or cylindrical, hyaline, minutely verruculose, proliferating 2–4 times percurrently, tapering to a long, thin
neck, 15–50 × 3–9 μm, with flaring collarettes. Conidia fusoid,
24.5–29(–31) × (6–)6.5–8(–8.5) μm, x ± SD = 26 ± 1.4 × 7.5
± 0.2 μm; basal cell obconic to hemispherical, hyaline, verruculose and thin-walled, 5–6.5 μm long; three median cells
doliiform, (15–)15.5–18(–18.5) μm long, x ± SD = 16.7
± 0.7 μm, wall verruculose, concolourous, brown, septa darker
than the rest of the cell (second cell from the base 5–6.5 μm
long; third cell 5.5–7 μm long; fourth cell 5.5–7 μm long); apical
cell 3.5–5 μm long, hyaline, cylindrical to subcylindrical; with 2–3
tubular apical appendages, arising from an apical crest, unbranched, filiform, flexuous, (9–)10–15(–16) μm long,
x ± SD = 12.6 ± 1.7 μm; basal appendage single, tubular, unbranched, centric, 2.5–4.5 μm long.
diam after 7 d at 25 °C, flat with entire edge, whitish, with sparse
154
aerial mycelium on the surface with black, gregarious conidiomata; reverse similar in colour.
Habitat: On Knightia sp. and Protea sp.
Known distribution: Australia and New Zealand.
Materials examined: Australia, New South Wales, from Protea neriifolia × susannae
cv. ‘Pink Ice’, 12 Oct. 1999, P.W. Crous, culture CBS 114141 = STE-U 2949. New
Zealand, from Knightia sp., 2002, P.W. Crous (CBS H-21767, holotype, ex-type
culture CBS 114126 = STE-U 2896).
Notes: Morphologically P. australasiae (clade 39; Fig. 5) is
comparable to P. knightiae (clade 37; Fig. 5), P. parva (clade 35;
Fig. 5) and P. grevilleae (clade 36; Fig. 5), but differs in having
larger conidia when compared to P. parva, and shorter apical
appendages when compared to P. knightiae and P. grevilleae. It
has an overlapping conidial size with P. telopeae (clade 40;
Fig. 5), which causes a leaf spot disease on Telopea spp. Since
the two species are genetically distinct, we maintain them as two
separate species (see notes under P. telopea).
PESTALOTIOPSIS
Pestalotiopsis australis Maharachch., K.D. Hyde &
Australia.
Conidiomata pycnidial in culture on PDA, globose or clavate,
aggregated or scattered, semi-immersed or partly erumpent, dark
brown to black, up to 400 μm diam; exuding globose, dark brown to
black conidial masses. Conidiophores 1–3-septate, sparsely
branched at the base, subcylindrical, hyaline, verruculose, up to
25 μm long. Conidiogenous cells discrete or integrated, ampulliform or cylindrical, hyaline, smooth, proliferating 2–4 times percurrently, 20–60 × 2–6 μm, collarette present and slightly flared.
Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate, (26–)
27–34(–36) × 7–8.5 μm, x ± SD = 30.8 ± 2.1 × 7.7 ± 0.3 μm; basal
cell conic to obconic with a truncate base, hyaline, minutely verruculose and thin-walled, 6–10 × μm long; three median cells
doliiform, (16–)17–21(–21.5) μm long, x ± SD = 19.1 ± 1.2 μm,
REVISITED
wall minutely verruculose, concolourous, brown, septa darker than
the rest of the cell (second cell from the base 5.5–7.5 μm long; third
cell 5.5–7.5 μm long; fourth cell 6–8 μm long); apical cell
4–6.5 × μm long, hyaline, cylindrical to subcylindrical, thin- and
smooth walled; with 2–3 tubular apical appendages (mostly 3),
arising from the apical crest, unbranched, filiform, (11–)
Habitat: On Brabejum stellatifolium, Grevillea sp. and Protea
neriifolia × susannae.
Known distribution: Australia and South Africa.
Fig. 22. Pestalotiopsis australis CBS 114193T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
155
MAHARACHCHIKUMBURA
ET AL.
Materials examined: Australia, New South Wales, from Grevillea sp. 12 Oct.
1999, P.W. Crous (CBS H-21766, holotype, ex-type culture CBS 114193 = STEU 3011). South Africa, KwaZulu-Natal, from Protea neriifolia × susannae cv.
‘Pink Ice’, 15 May 1998, L. Swart, culture CBS 114474 = STE-U 1769; ibid., 15
May 1998, L. Swart, culture CBS 111503 = STE-U 1770; on dead leaves of
Brabejum stellatifolium, 3 Nov. 2000, S. Lee, PREM 59519, culture CBS
119350 = CMW 20013.
Notes: Pestalotiopsis australis (clade 26; Fig. 5) is a distinct
species, which can be isolated from members of Proteaceae.
Pestalotiopsis australis is closely related to P. scoparia (clade 25;
Fig. 5), and is distinguished morphologically from related species
by its large conidia.
Pestalotiopsis biciliata Maharachch., K.D. Hyde & Crous,
Etymology: Name refers to its two basal appendages.
aggregated or scattered, semi-immersed, dark brown to black,
up to 300 μm diam; exuding globose, slimy, dark brown conidial
droplets. Conidiophores sparsely septate and unbranched or
irregularly branched at the base, up to 40 μm long, or reduced to
conidiogenous cells. Conidiogenous cells discrete, cylindrical to
subcylindrical, hyaline, smooth, tapering to a long, thin neck,
10–45 × 2–5 μm, proliferating several times percurrently near
apex, with flaring collarettes. Conidia fusoid, ellipsoid, straight to
slightly curved, 4-septate, (21–)22–28.5(–30) × (5.5–)
6–7.5(–8) μm, x ± SD = 25.3 ± 2 × 6.7 ± 0.3 μm; basal cell
obconic to hemispherical with a truncate base, hyaline, verruculose and thin-walled, 4–7 μm long; three median cells doliiform, (13.5–)14.5–17.5(–18.5) μm long, x ± SD = 16 ± 1.1 μm,
wall verruculose, concolourous, olivaceous, septa darker than
the rest of the cell (second cell from the base 4–6.5 μm long;
third cell 4–7 μm long; fourth cell 4–6.5 μm long); apical cell
3–4.5 μm long, hyaline, subcylindrical, rugose and thin-walled;
the apical crest, unbranched, filiform, (6–)8–18(–20) μm long,
x ± SD = 13.3 ± 3.2 μm; two basal appendages; centric
appendage tubular, 3–8 μm long and excentric appendage
tubular, 1–3 μm long.
Fig. 23. Pestalotiopsis biciliata CBS 124463T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
156
PESTALOTIOPSIS
diam after 7 d at 25 °C, with lobate edge, whitish, with sparse
aerial mycelium on the surface with black, gregarious conidiomata; reverse pale honey-coloured.
Habitat: On Paeonia sp., bark of Platanus × hispanica and Taxus
baccata dry needles.
Known distribution: Italy, Netherlands and Slovakia.
Materials examined: Italy, from Paeonia sp., Jun. 1938, O. Servazzi, culture CBS
236.38. Netherlands, from Taxus baccata dry needles attached to the tree, 23
Oct. 1968, H.A. van der Aa, culture CBS 790.68. Slovakia, Giraltovce, from bark
of Platanus × hispanica, unknown collection date, M. Pastircak (CBS H-21765,
holotype, ex-type culture CBS 124463).
Notes: Pestalotiopsis biciliata (clade 38; Fig. 5) is a species often
having two basal appendages. Pestalotiopsis biciliata overlaps
morphologically with P. trachicarpicola (clade 43; Fig. 5). However, in the phylogenetic analyses it formed a distinct lineage
apart from Pestalotiopsis kenyana (which has wider conidia;
clade 42; Fig. 5) and P. trachicarpicola (clade 43; Fig. 5).
Pestalotiopsis brassicae (Guba) Maharachch., K.D.
Hyde & Crous, comb. nov. MycoBank MB809734. Fig. 24.
REVISITED
Basionym: Pestalotia brassicae Guba, Monograph of Monochaetia and Pestalotia: 245. 1961.
Conidiomata acervular to pycnidial in culture on PDA, globose,
scattered or gregarious and confluent, semi-immersed or erumpent, dark brown to black, up to 500 μm diam; exuding globose,
black conidial masses. Conidiophores septate near base,
branched, subcylindrical, hyaline, up to 10 μm long. Conidiogenous
cells discrete, cylindrical 20–70 × 2–10 μm or ampulliform to
lageniform 4–10 × 3–8 μm, hyaline, smooth-walled, proliferating
2–4 times percurrently, wide at base, collarette present and not
flared, with prominent periclinal thickening. Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate, (29–)30–37(–40)
× (8–)8.5–11(–11.5) μm, x ± SD = 34 ± 2.1 × 9.7 ± 0.7 μm; basal
cell obconic with a truncate base, hyaline, minutely verruculose
and thin-walled, 5–8.5 × μm long; three median cells doliiform to
subcylindrical, (20–)20.5–24.5(–25) μm long, x ± SD = 22.6
± 1.5 μm, wall verruculose, concolourous, but occasionally the two
upper median cells slightly darker than the lower median cell,
brown to olivaceous, septa darker than the rest of the cell (second
cell from the base 5.5–9 μm long; third cell 7–9.5 μm; fourth cell
6–9 μm); apical cell 3.5–7 × μm long, hyaline, cylindrical to subcylindrical, thin- and smooth walled; with 3–5 tubular apical appendages (mostly 4), arising from the apical crest, unbranched,
Fig. 24. Pestalotiopsis brassicae CBS 170.26isoT. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
157
MAHARACHCHIKUMBURA
ET AL.
filiform, flexuous, (27–)28.5–48(–50) μm long, x ± SD = 37
± 5 μm; basal appendage single, tubular, unbranched, centric,
10–25 μm long.
concolourous, but occasionally the two upper median cells are
slightly darker than the lower median cell, brown, septa darker than
the rest of the cell (second cell from the base 4.5–6.5 μm long; third
cell 4.5–6.5 μm long; fourth cell 4.5–6 μm long); apical cell 4–6 μm
long, hyaline, subcylindrical, thin- and smooth walled; with 2–3
tubular apical appendages (mostly 3), arising from the apical crest,
unbranched, filiform, (13–)14.5–23(–24) μm long, x ± SD = 18
± 3.1 μm; basal appendage single, tubular, unbranched, centric,
4–8.5 μm long.
Habitat: On seeds of Brassica napus.
Material examined: New Zealand, from seeds of Brassica napus, May 1926,
G.H. Cunningham (CBS H-7542, isotype, ex-isotype culture CBS 170.26).
Notes: According to the original description of Guba (1961), conidia
of P. brassicae are somewhat smaller (25–32 × 8.5–9.5 μm) and
the apical appendages are shorter (20–35 μm) than in the present
observation. In his monograph Guba placed this species in a group
with species having versicoloured median cells. However, our
phylogenetic analyses (Fig. 5) do not support placing P. brassicae
(clade 19; Fig. 5) within the versicoloured group (genus Neopestalotiopsis; Fig. 4). Pestalotiopsis brassicae formed a sister
group to P. hollandica (clade 18; Fig. 5), which was isolated from
Sciadopitys verticillata in the Netherlands. The latter species is
clearly distinguished from P. brassicae by having wider conidia,
and branched, sub-apically attached apical appendages.
Furthermore, P. brassicae is distinguished from its other closest
phylogenetic neighbour, P. verruculosa (clade 20; Fig. 5)
(28–35 × 9–11 μm) by its larger conidia.
Pestalotiopsis camelliae Y.M. Zhang, Maharachch. &
K.D. Hyde
Materials examined: China, Yunnan Province, Chuxiong, Shuangbai, on living
leaves of Camellia japonica, Jul. 2011, Y.M. Zhang (IFRD OP111, holotype, extype culture MFLUCC 12-0277); ibid., Aug. 2011, IFRD OP131, culture MFLUCC
12-0278. Turkey, Samsun, on leaf of Camellia sinensis, collection date unknown,
O. Orbas, culture CBS 443.62.
Zhang et al. (2012b).
Pestalotiopsis chamaeropis Maharachch., K.D. Hyde &
Etymology: Named after the host genus, Chamaeropis.
Conidiomata pycnidial in culture on PDA, globose, semi-immersed
or partly erumpent, aggregated or scattered, up to 250 μm diam;
exuding globose, dark brown to black conidial masses. Conidiophores 1–3-septate, branched, subcylindrical, hyaline, verruculose, up to 25 μm long. Conidiogenous cells discrete,
cylindrical, hyaline, smooth-walled, proliferating 2–4 times percurrently, 20–50 × 2–5 μm, collarette present and not flared, with
prominent periclinal thickening. Conidia fusoid, ellipsoid, straight
to slightly curved, 4-septate, (21–)22.5–27(–28) × (6–)
7–9(–9.5) μm, x ± SD = 25.2 ± 1.3 × 8 ± 0.4 μm; basal cell obconic
with a truncate base, hyaline, minutely verruculose and thin-walled,
5–6.5 μm long; three median cells doliiform to subcylindrical, (15–)
16–17.5(–18.5) μm long, x ± SD = 16.7 ± 0.8 μm, wall verruculose,
158
Habitat: On leaves of Chamaerops humilis.
Known distribution: Italy.
Materials examined: Italy, Sardinia, Dorgali, from leaf of Chamaerops humilis,
Feb. 1971, H.A. van der Aa (CBS H-15702, holotype, ex-type culture CBS
186.71); unknown collection details (June 1938 deposited in CBS collection), O.
Servazzi, culture CBS 237.38. Unknown locality, unknown collection details,
culture CBS 113604 = STE-U 3078, CBS 113607 = STE-U 3080.
Notes: Clade 23 (Fig. 5) is represented by four isolates of
P. chamaeropis. It differs from related species in having distinctly
wider conidia. Pestalotiopsis chamaeropis forms a separate
cluster in the combined phylogeny, as sister to a group including
P. intermedia (clade 21; Fig. 5) and P. linearis (clade 22; Fig. 5),
which were isolated from dead leaves of an unidentified tree, and
as an endophyte of Trachelospermum sp. respectively, both
collected in China. In 1938, O. Servazzi deposited two isolates
(CBS 237.38 and CBS 236.38) in CBS as authentic strains of
Pestalotia paeoniae. Even though these two isolates had overlapping conidial dimensions, the deposited isolates cluster in
distinct clades (CBS 237.38 in clade 23 and CBS 236.38 in clade
38; Fig. 5) with species having concolourous median cells. According to the description of Guba (1961), P. paeoniae belongs to
the species with versicoloured median cells (presently Neopestalotiopsis; Fig. 4). The reliability of these two “authentic”
strains is thus doubtful, and CBS 237.78 is placed in
P. chamaeropis, and CBS 236.38 in P. biciliata (clade 38; Fig. 5).
Pestalotiopsis clavata Maharachch. & K.D. Hyde
Material examined: China, Yunnan Province, Kunming, Kunming Botanical Garden,
on living leaves of Buxus sp., 19 Mar. 2002, W.P. Wu (HMAS047134, holotype,
MFLU 12-0412, isotype, ex-type culture NN0471340 = MFLUCC 12-0268).
Pestalotiopsis colombiensis Maharachch., K.D. Hyde &
Etymology: Named after the country from where it was collected,
Colombia.
aggregated, semi-immersed, dark brown, 200–400 μm diam;
exuding globose, dark brown, glistening conidial masses. Conidiophores reduced to conidiogenous cells; when present,
PESTALOTIOPSIS
REVISITED
Fig. 25. Pestalotiopsis chamaeropis CBS 186.71T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
septate, unbranched, or irregularly branched, hyaline, thin-walled,
5–12 × 2–6 μm. Conidiogenous cells discrete, cylindrical, proliferating 2–5 times percurrently, tapering to a long, thin neck,
10–50 × 2–8 μm, with prominent periclinal thickening, collarette
present and not flared. Conidia ellipsoid, straight to slightly curved,
4-septate, slightly constricted at septa, (19–)21–27(–28.5)
× 5.5–7.5(–8) μm, x ± SD = 24 ± 1.5 × 6.3 ± 0.5 μm; basal cell
conic to acute with truncate base, minutely verruculose and thinwalled, 5–7.5 μm long; three median cells, (13–)
13.5–16.5(–17) μm long, x ± SD = 15.2 ± 0.8 μm, doliiform, thickwalled, verruculose, concolourous, brown (second cell from base
5–6.5 μm long; third cell 4.5–6 μm long; fourth cell 5–6.5 μm long);
apical cell cylindrical to subcylindrical, hyaline, thin- and smoothwalled, 3.5–5 μm long; with 2–3 tubular apical appendages
(mostly 3), arising from the apical crest, unbranched, filiform, (11–)
13–25(–28) μm, x ± SD = 17.5 ± 3 μm; basal appendage single,
tubular, unbranched, centric, 2–5 μm long.
diam after 7 d at 25 °C, entire at the edge, whitish to pale greycoloured, with dense aerial mycelium on the surface, with black,
Habitat: On living leaves of Eucalyptus eurograndis.
Known distribution: Colombia.
Material examined: Colombia, from living leaves of Eucalyptus eurograndis, 2004,
M.J. Wingfield (CBS H-21764, holotype, ex-type culture CBS 118553 = CPC
10969).
Notes: Pestalotiopsis colombiensis (clade 27; Fig. 5) is a distinct
species represented by a Colombian isolate from Eucalyptus. It
differs from its closest phylogenetic neighbours, P. diploclisiae
(clade 29; Fig. 5) and P. humus (clade 28; Fig. 5) by its longer apical
appendages. Furthermore P. colombiensis is geographically
159
MAHARACHCHIKUMBURA
ET AL.
Fig. 26. Pestalotiopsis colombiensis CBS 118553T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale
bars = 10 μm.
distinct from P. diploclisiae and P. humus, which were isolated from
Hong Kong and Papua New Guinea, respectively.
Pestalotiopsis diploclisiae Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Diploclisia.
Conidiomata pycnidial in culture on PDA, globose, solitary or
aggregated, semi-immersed, black, up to 500 μm diam; exuding
globose, slimy, dark brown, conidial droplets. Conidiophores
often reduced to conidiogenous cells, sparsely septate at the
base and unbranched or branched, up to 20 μm long. Conidiogenous cells discrete, cylindrical to subcylindrical, hyaline,
smooth, simple, proliferating 2–3 times percurrently,
6–20 × 2–5 μm. Conidia fusoid, ellipsoid, straight to slightly
curved, 4-septate, (20–)22–26.5(–28) × 5–6.5(–7) μm,
x ± SD = 24 ± 1.3 × 5.7 ± 0.4 μm; basal cell obconic to subcylindrical with a truncate base, hyaline, rugose and thin-walled,
160
4–6.5 μm long; three median cells doliiform, (13.5–)
14–16(–17) μm long, x ± SD = 15.4 ± 0.9 μm, wall minutely
verruculose, concolourous, pale brown, septa darker than the
rest of the cell (second cell from the base 4.5–6 μm; third cell
4.5–7 μm; fourth cell 4.5–6.5 μm); apical cell 3.5–6 μm long,
hyaline, subcylindrical, thin- and smooth-walled; with 2–4 tubular
apical appendages (mostly 3), arising from the apical crest,
unbranched, filiform, flexuous (10–)13–19(–22) μm long,
x ± SD = 16.6 ± 2.1 μm; basal appendage single, tubular, unbranched, centric, 3–8 μm long.
Habitat: On fruit of Diploclisia glaucescens and Psychotria
tutcheri.
Known distribution: China (Hong Kong).
PESTALOTIOPSIS
REVISITED
Fig. 27. Pestalotiopsis diploclisiae CBS 115587T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Materials examined: China, Hong Kong, Lamma Island, from fruit of Diploclisia
glaucescens, 5 Jul. 2001, K.D. Hyde (CBS H-21763, holotype, ex-type culture
CBS 115587 = HKUCC 10130); ibid., culture CBS 115585 = HKUCC 8394; Mount
Nicholson, from fruit of Psychotria tutcheri, 15 Feb. 2002, K.D. Hyde, culture CBS
115449 = HKUCC 9103.
Notes: Pestalotiopsis diploclisiae (clade 29; Fig. 5) comprises
three isolates originating from Hong Kong. Pestalotiopsis diploclisiae is morphologically very similar to P. colombiensis (clade
27; Fig. 5), but genetically clearly distinct, forming a wellseparated clade. Pestalotiopsis diploclisiae is genetically close
to P. humus (clade 28; Fig. 5), which was isolated from soil in
Papua New Guinea, but can be distinguished by its narrow
conidia and longer apical appendages.
Pestalotiopsis diversiseta Maharachch. & K.D. Hyde
Material examined: China, Yunnan Province, Kunming, Kunming Botanical
Garden, on living leaves of Rhododendron sp., 19 Mar. 2002, W.P. Wu
(HMAS047261, holotype, MFLU 12-0423, isotype, ex-type culture
NN0472610 = MFLUCC 12-0287).
Pestalotiopsis ericacearum Y. M. Zhang, Maharachch. &
K. D. Hyde
Material examined: China, Yunnan Province, Chuxiong, Zixishan, leaf spots on
Rhododendron delavayi, Feb. 2011, Y.M. Zhang (IFRD 410-008, holotype, extype culture IFRDCC 2439).
Note: This species (clade 2: Fig. 5) was treated in detail by
Zhang et al. (2013).
Pestalotiopsis furcata Maharachch. & K.D. Hyde
Material examined: Thailand, Chiang Mai Province, Mae Taeng District, Ban Pha
Deng, Mushroom Research Centre, 19°17.1230 N 98°44.0090 E, on living leaves
of Camellia sinensis, 20 Jan. 2010, S.S.N. Maharachchikumbura (MFLU 12-0112,
holotype, ex-type culture MFLUCC 12-0054 = CPC 20280).
Maharachchikumbura et al. (2013a).
Pestalotiopsis gaultheria Y. M. Zhang, Maharachch. &
K. D. Hyde
161
MAHARACHCHIKUMBURA
ET AL.
Material examined: China, Yunnan Province, Dehong, Mangshi, leaf spots on
Gaultheria forrestii, Sep. 2011, Y.M. Zhang (IFRD 411-014, holotype).
Pestalotiopsis grevilleae Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Grevillea.
Conidiomata pycnidial in culture on PDA, globose, aggregated or
scattered, semi-immersed, dark brown to black, up to 200 μm diam;
releasing globose, dark brown to black conidial masses. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells discrete, cylindrical to subcylindrical, hyaline,
smooth, proliferating 2–3 times percurrently, flared collarette, with
prominent periclinal thickening, 5–25 × 2–8 μm. Conidia fusoid,
22.5–28(–29) × (7–)7.5–9(–9.5) μm, x ± SD = 25.2 ± 1.2 × 8.2
± 0.5 μm; basal cell conic with a truncate base, hyaline, rugose and
thin-walled, 3.5–5.5 μm long; three median cells doliiform, (12.5–)
13–17(–17.5) μm long, x ± SD = 15 ± 1.2 μm, wall verruculose,
concolourous, olivaceous, septa darker than the rest of the cell
(second cell from the base 4.5–6.5 μm; third cell 4.5–6.5 μm; fourth
cell 4–6.5 μm); apical cell 3.5–5.5 μm long, hyaline, cylindrical to
subcylindrical, rugose and thin-walled; with 2–3 tubular apical
appendages (mostly 3), arising from the apical crest, unbranched,
filiform, flexuous (12–)14–26.5(–29) μm long, x ± SD = 19 ± 3 μm;
basal appendage single, tubular, unbranched, centric, 3–8 μm
long.
Habitat: On Grevillea sp.
Fig. 28. Pestalotiopsis grevilleae CBS 114127T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
162
PESTALOTIOPSIS
Material examined: Australia, New South Wales, Sydney, Grevillea sp., 1999, P.W.
Crous (CBS H-21762, holotype, ex-type culture CBS 114127 = STE-U 2919).
REVISITED
Etymology: Named after the island from where it was collected,
Hawaii.
black conidial masses. Conidiophores simple or branched, hyaline,
subcylindrical, smooth-walled, 5–15 × 3–8 μm. Conidiogenous
cells discrete, cylindrical, hyaline, smooth-walled, proliferating 2–4
times percurrently near apex, 20–50 × 3–6 μm, collarette present
and not flared, with prominent periclinal thickening. Conidia fusoid,
27–34.5(–37) × (7–)7.5–10(–10.5) μm, x ± SD = 31.6 ± 2 × 8.7
± 0.6 μm; basal cell conic to obconic with a truncate base, hyaline,
minutely verruculose and thin-walled, 4–8 μm long; three median
cells doliiform to subcylindrical, (19–)19.5–23(–25) μm long,
x ± SD = 21.4 ± 1.2 μm, wall verruculose, concolourous brown,
septa darker than the rest of the cell (second cell from the base
5–8.5 μm; third cell 6.5–9.5 μm; fourth cell 6–9 μm); apical cell
4–7 × μm long, hyaline, cylindrical to subcylindrical, thin- and
smooth-walled; with 2–3 tubular apical appendages (mostly 3),
arising from the apical crest, unbranched, filiform, (14–)
Conidiomata (on PDA) pycnidial, globose, solitary, semi-immersed,
dark brown to black, 200–600 μm diam; exuding globose, brown to
diam after 7 d at 25 °C, with undulate edge, whitish, sparse aerial
Notes: Pestalotiopsis grevilleae (clade 36; Fig. 5) forms a sister
clade to P. knightiae (clade 37; Fig. 5), being distinct from the
latter species in having narrower conidia. Pestalotiopsis grevilleae has overlapping conidial dimensions with P. australasiae
(clade 39; Fig. 5), although their basal cells are distinct. In
P. grevilleae the basal cells are conic, while in P. australasiae
they are obconic to hemispherical. Furthermore, phylogenetic
analyses (Fig. 5) indicate that the two species are genetically
distinct.
Pestalotiopsis hawaiiensis Maharachch., K.D. Hyde &
Fig. 29. Pestalotiopsis hawaiiensis CBS 114491T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–F. Conidiogenous cells. G–L. Conidia. Scale bars = 10 μm.
163
MAHARACHCHIKUMBURA
ET AL.
mycelium on the surface with black, gregarious conidiomata;
reverse similar in colour.
Habitat: On Leucospermum sp.
China (Maharachchikumbra et al. 2013c). However,
P. anacardiacearum differs from P. hawaiiensis by having longer
apical appendages (20–45 μm). Furthermore, the two species
are genetically, geographically and ecologically distinct, and thus
we maintain them as two separate species.
Known distribution: USA (Hawaii).
Material examined: USA, Hawaii, from Leucospermum sp. cv. ‘Coral’, 9 Dec.
1999, P.W. Crous (CBS H-21761, holotype, ex-type culture CBS 114491 = STEU 2215).
Notes: Pestalotiopsis hawaiiensis (clade 5; Fig. 5), known from
Hawaii on Leucospermum sp., has overlapping conidial dimensions with P. anacardiacearum (27–39 × 7–10 μm; clade 6;
Fig. 5), which was isolated from leaves of Mangifera indica in
Pestalotiopsis hollandica Maharachch., K.D. Hyde &
Etymology: Named after the pars pro toto name “Holland” for the
country where it was collected, the Netherlands.
Conidiomata (on PDA) pycnidial, 200–350 μm diam, globose or
clavate, solitary or aggregated, semi-immersed, dark brown to
Fig. 30. Pestalotiopsis hollandica CBS 265.33T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale bars = 10 μm.
164
PESTALOTIOPSIS
black; exuding dark brown conidial masses. Conidiophores
septate, branched at base, sometimes reduced to conidiogenous
cells, hyaline, smooth-walled, up to 30 μm long. Conidiogenous
cells discrete, cylindrical, proliferating 2–5 times percurrently near
apex, tapering to a long, thin neck, collarette present and not flared.
Conidia ellipsoid, straight to slightly curved, 4-septate, slightly
constricted at septa, (25–)25.5–33(–34) × 8.5–10(–10.5) μm,
x ± SD = 28 ± 2 × 9.4 ± 0.3 μm; basal cell conic to obconic with
truncate base, thin-walled, 5–7.5 μm long; three median cells
(16.5–)17–23(–24) μm long; x ± SD = 28 ± 2 × 9.4 ± 0.3 μm,
doliiform, thick-walled, verruculose, concolourous, but occasionally
the two upper median cells slightly darker than the lower median
cell, wall rugose (second cell from base 5–8.5 μm; third cell
6–9 μm; fourth cell 6–8 μm); apical cell conic, hyaline, thin- and
smooth-walled, 3.5–5 μm long; with 1–4 tubular apical appendages, with some branched appendages, arising from the apex of
the apical cell and sometimes from just above the septum separating the apical and subapical cell, 20–40 μm long,
x ± SD = 27 ± 1.5 μm; basal appendage single, tubular, unbranched, centric, 3–9 μm long.
diam. after 7 d at 25 °C, with an undulate edge, whitish to pale
grey-coloured, with dense aerial mycelium on surface, and black,
Habitat: On Sciadopitys verticillata.
Known distribution: Netherlands.
Material examined: Netherlands, Baarn, from Sciadopitys verticillata, Jul. 1933,
A. Punt (CBS H-15703, holotype, ex-type culture CBS 265.33).
Notes: Pestalotiopsis hollandica (clade 18; Fig. 5) differs from all
other related species (clades 17, 19 and 20; Fig. 5) in having
some appendages that arise from different parts of the apical
cell. Pestalotiopsis hollandica differs from P. monochaetioides
(22–30 × 5–10 μm; no culture available for molecular study),
which was isolated from a dead twig of Chamaecyparis lawsoniana in the Netherlands (Guba 1961), by its branched, subapically attached apical appendages.
Pestalotiopsis humus Maharachch., K.D. Hyde & Crous,
Etymology: Name refers to the substrate from which it was
isolated, soil.
Conidiomata pycnidial in culture on PDA, globose, semiimmersed, aggregated or scattered, up to 400 μm diam;
exuding dark brown to black, globose conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells
discrete, cylindrical, hyaline, smooth-walled, simple, proliferating
up to 3 times percurrently, 8–28 × 2–5 μm, apex 1–2 μm diam.
Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate,
constricted at septum, (17–)18.5–22(–23) × 5–7(–7.5) μm,
x ± SD = 20 ± 1.4 × 6 ± 0.4 μm; basal cell obconic to conic with a
truncate base, hyaline, minutely verruculose and thin-walled,
3.5–5.5 μm long; three median cells subcylindrical, (11.5–)
12–14(–14.5) μm long, x ± SD = 12.8 ± 0.8 μm, wall rugose,
concolourous, brown, septa darker than the rest of the cell
(second cell from the base 3.5–5.5 μm long; third cell 3.5–6 μm
REVISITED
long; fourth cell 3.5–5.5 μm long); apical cell 3.5–4.5 × μm long,
hyaline, subcylindrical; with 2–3 tubular apical appendages,
arising from an apical crest, unbranched, filiform, flexuous, (6–)
6.5–12(–13) μm long, x ± SD = 9.0 ± 1.5 μm; basal appendage
Habitat: On fruits of Ilex cinerea and soil.
Known distribution: China (Hong Kong) and Papua New Guinea.
Materials examined: China, Hong Kong, from fruit of Ilex cinerea, 20 Jan. 2002,
K.D. Hyde, culture CBS 115450 = HKUCC 9100. Papua New Guinea, from soil in
tropical rain forest, Nov. 1995, A. Aptroot (CBS H-21760, holotype, ex-type
culture CBS 336.97).
Notes: Clade 28 (Fig. 5) comprises P. humus, isolated from rain
forest soil in Papua New Guinea and fruit of Ilex cinerea in Hong
Kong. Sequences of Pestalotiopsis humus form a sister clade to
P. diploclisiae (clade 29; Fig. 5). Pestalotiopsis diploclisiae differs
from P. humus in conidial morphology, in having narrower conidia
(20–28 × 5–7 μm), and longer apical appendages (10–22 μm).
Pestalotiopsis inflexa Maharachch. & K.D. Hyde
Material examined: China, Hunan Province, Yizhang County, Mangshan, on
living leaves of unidentified tree, 12 Apr. 2002, W.P. Wu (HMAS047098, holotype, MFLU 12-0413, isotype, ex-type culture NN0470980 = MFLUCC 12-0270).
Pestalotiopsis intermedia Maharachch. & K.D. Hyde
Material examined: China, Hubei Province, Shengnongjia, on dead leaves of
unidentified tree, 24 Mar. 2003, W.P. Wu (HMAS047642, holotype, MFLU 120410, isotype, ex-type culture NN0476420 = MFLUCC 12-0259).
Pestalotiopsis jesteri Strobel, J. Yi Li, E.J. Ford & W.M.
Hess, Mycotaxon 76: 260. 2000. Fig. 32.
Conidiomata (on PDA) pycnidial, globose, solitary or aggregated,
immersed, medium to dark brown, 100–450 μm diam; releasing
cells discrete, lageniform to subcylindrical, hyaline, smooth,
proliferating once or twice, 5–20 × 3–7 μm; collarette flared, apex
2–5 μm diam. Conidia fusoid, ellipsoid to subcylindrical, straight to
slightly curved, 4-septate, (21–)22.5–31(–34.5) × 7–9 μm,
x ± SD = 26.8 ± 3 × 8.2 ± 0.2 μm; basal cell narrowly obconic with
a truncate base, hyaline, thin- and smooth-walled, 4.5–6.5 μm
long; three median cells doliiform to subcylindrical, (15.5–)
16–20(–21) μm long, x ± SD = 17.5 ± 1.4 μm, wall rugose,
concolourous, golden brown, septa darker than the rest of the cell
(second cell from the base 4.5–7 μm long; third cell 5.5–7.5 μm
long; fourth cell 5.5–7.5 μm long); apical cell 3.5–7.5 μm long,
165
MAHARACHCHIKUMBURA
ET AL.
Fig. 31. Pestalotiopsis humus CBS 336.97T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale bars = 10 μm.
hyaline, obconic with an acute apex, thin- and smooth-walled;
appendages tubular, attenuated; apical appendage single,
14–25 long; lateral appendages 2–4, arising just above the
septum separating the apical cell and upper median cell, unbranched, 14–25 long; basal appendage single, tubular, unbranched, centric, 4–14 μm long.
diam after 7 d at 25 °C, with undulate edge, whitish, with sparse
Habitat: On bark of Fragraea bodenii.
Known distribution: Papua New Guinea.
Material examined: Papua New Guinea, Southern Highlands, Aluak ambe
village, from bark of Fragraea bodenii, E. Eroli & G. Strobel (deposited in CBS
collection Mar. 2001 by G. Strobel) (MONT Strobel 6T-L-3, holotype, MONT
166
Strobel 6M-B-3 and MONT Strobel 6B-S-4, isotypes, ex-type culture CBS
109350 = MONT 6M-B-3).
Notes: Pestalotiopsis jesteri (clade 1; Fig. 5) is described from
bark of Fragraea bodenii in Papua New Guinea and is wellcharacterised and easily recognisable by the unique appendages attached to the apical cell. The arrangement of apical appendages in P. jesteri is comparable with Pestalotia montellica
(Guba 1961). However, P. jesteri differs from Pestalotia montellica by the presence of knobbed apical appendages.
Furthermore, P. jesteri is a basal species in the species phylogeny (Fig. 5), and forms a lineage distinct from all other
species.
Pestalotiopsis kenyana Maharachch., K.D. Hyde &
Kenya.
PESTALOTIOPSIS
REVISITED
Fig. 32. Pestalotiopsis jesteri CBS 109350. A. Conidioma sporulating on PNA. B. Conidioma on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Conidiomata pycnidial in culture on PDA, globose, scattered,
semi-immersed, black, up to 400 μm diam; exuding globose, dark
brown to black conidial masses. Conidiophores sparsely septate
at base, branched or unbranched, subcylindrical, hyaline, smooth,
up to 15 μm or reduced to conidiogenous cells. Conidiogenous
cells discrete, lageniform to subcylindrical, hyaline, smooth,
proliferating 1–3 times percurrently, 10–25 × 2–5 μm, apex with
minute periclinal thickening and collarette. Conidia fusoid, ellipsoid to subcylindrical, straight to slightly curved, 4-septate, (22–)
23–28(–29) × 7–9 μm, x ± SD = 25.5 ± 1.2 × 8 ± 0.4 μm; basal
cell conic to obconic with a truncate base, hyaline, minutely verruculose and thin-walled, 4–6 μm long; three median cells doliiform, (15–)15.5–18.5(–19) μm long, x ± SD = 17 ± 0.7 μm, wall
verruculose concolourous, brown, septa darker than the rest of the
cell (second cell from the base 4.5–6 μm long; third cell
5.5–7.5 μm long; fourth cell 3.5–4.5 μm long); apical cell
3.5–5.5 μm long, hyaline, subcylindrical, rugose and thin-walled;
with 2–3 tubular apical appendages (mostly 3), arising from the
apical crest, unbranched, filiform, (8–)9–18(–20) μm long,
x ± SD = 14 ± 3 μm; two basal appendages; centric appendage
tubular, flexuous, 3–20 μm long and eccentric appendage tubular,
1–4 μm long.
diam after 7 d at 25 °C, with undulate edge, whitish, with medium
dense aerial mycelium on the surface with black, gregarious
Habitat: On branches of Coffea sp., and raw material from agaragar, kobe 1.
Known distribution: Kenya.
Materials examined: Kenya, from Coffea sp. branch, Oct. 1967, H. Vermeulen
(CBS H-15657, holotype, ex-type culture CBS 442.67). Unknown country, from
raw material from agar-agar, kobe 1 (stips), Sep. 1996, A.K. Johansen, culture
CBS 911.96.
Notes: Pestalotiopsis kenyana (clade 42; Fig. 5) formed a
separate clade in the phylogenetic analyses as sister to
P. trachicarpicola (clade 43; Fig. 5). Both P. kenyana and
P. trachicarpicola often have two basal appendages. Pestalotiopsis kenyana differs from both P. trachicarpicola and P. biciliata
(clade 38; Fig. 5) in having wider conidia (see comparison under
P. biciliata).
167
MAHARACHCHIKUMBURA
ET AL.
Fig. 33. Pestalotiopsis kenyana CBS 442.67T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Pestalotiopsis knightiae Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Knightia.
Conidiomata pycnidial, globose, aggregated or scattered, semiimmersed to erumpent on PDA, dark brown to black,
100–200 μm diam; exuding globose, dark brown to black conidial
masses. Conidiophores indistinct, often reduced to conidiogenous
cells. Conidiogenous cells discrete, ampulliform or lageniform,
hyaline, smooth, simple, proliferating once or twice, wide at
the base, 10–30 × 2–10 μm. Conidia fusoid, ellipsoid,
straight to slightly curved, 4-septate, (21–)22–27(–29) × (8–)
8.5–10.5(–11) μm, x ± SD = 24.8 ± 1.3 × 9.6 ± 0.4 μm; basal cell
obconic to conic with a truncate base, hyaline, thin- and smoothwalled, 3–6.5 μm long; three median cells doliiform, (15.5–)
16–18.5(–19.5) μm long, x ± SD = 17.4 ± 1.2 μm, wall minutely
rugose, concolourous, pale brown, septa darker than the rest of
the cell (second cell from the base 5.5–7 μm long; third cell
168
6–7.5 μm long; fourth cell 5.5–7 μm long); apical cell
3–4.5(–5) μm long, hyaline, cylindrical to subcylindrical; with 2–4
tubular apical appendages (mostly 3), not arising from the apical
crest, but each inserted at a different locus in the upper half of the
cell, unbranched, filiform, (8–)12–20(–23) μm long, x ± SD
= 15 ± 3.9 μm; basal appendage single, tubular, unbranched,
centric, 2.5–7.5 μm long.
Habitat: On Knightia sp.
Materials examined: New Zealand, from Knightia sp., 1999, P.W. Crous (CBS H21759, holotype, ex-type culture CBS 114138 = STE-U 2906); Tamaki, Maori
Village, from Knightia sp., 1999, P.W. Crous, culture CBS 111963 = STE-U 2905.
PESTALOTIOPSIS
REVISITED
Fig. 34. Pestalotiopsis knightiae CBS 114138T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Notes: Pestalotiopsis knightiae (clade 37; Fig. 5) is a species
occurring on Knightia sp. in New Zealand, and is distinct from
other morphologically closely related species (clades 36 and 38;
Fig. 5) based on its DNA phylogeny. It forms a sister clade with
P. grevilleae (clade 36; Fig. 5), and is distinguishable from other
phylogenetically closely related species by its wider conidia.
Pestalotiopsis linearis Maharachch. & K.D. Hyde
Garden, on living leaves of Trachelospermum sp., 19 Mar. 2002, W.P. Wu
NN0471900 = MFLUCC 12-0271).
Pestalotiopsis malayana Maharachch., K.D. Hyde &
Malaysia.
Conidiomata (on PDA) pycnidial, globose, scattered or aggregated, semi-immersed, dark brown to black, up to 400 μm diam;
exuding globose, dark brown to black conidial masses. Conidiophores 2–5-septate, irregular branched, cylindrical, hyaline,
verruculose-walled, up to 50 μm, sometimes reduced to conidiogenous cells. Conidiogenous cells discrete, subcylindrical to
ampulliform, hyaline, smooth, tapering to a long, thin neck,
6–18 × 2–4 μm, proliferating several times percurrently near
apex, with flaring collarettes. Conidia fusoid, ellipsoid, straight to
slightly curved, slightly constricted at septa, 4-septate, (20.5–)
22–29.5(–31) × 5–7.5 μm, x ± SD = 25.6 ± 2 × 6.3 ± 0.4 μm;
basal cell obconic to conic with a truncate base, hyaline, minutely
verruculose and thin-walled, 3.5–7.5 μm long; three median cells
doliiform, 15–18 μm long, x ± SD = 16.5 ± 0.8 μm, wall minutely
verruculose, concolourous, pale brown, septa darker than the
rest of the cell (second cell from the base 4.5–7 μm long; third
cell 4.5–6.5 μm long; fourth cell 5–7 μm long); apical cell
3–6 μm long, hyaline, cylindrical to subcylindrical; with 1–3
tubular apical appendages (mostly 2), arising as an extension of
the apical cell, unbranched, filiform, (11–)11.5–18.5(–19) μm
long, x ± SD = 15.1 ± 1.4 μm; basal appendage single, tubular,
unbranched, centric, 2–5 μm long.
after 7 d at 25 °C, edge rhisoid, white to pale honey-coloured,
conidiomata black, gregarious; reverse of culture same colours.
169
MAHARACHCHIKUMBURA
ET AL.
Fig. 35. Pestalotiopsis malayana CBS 102220T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale bars = 10 μm.
Habitat: On stem of Macaranga triloba colonised by ants.
Known distribution: Malaysia.
Material examined: Malaysia, from stem of Macaranga triloba colonised by ants,
Sep. 1999, W. Federle (CBS H-21758, holotype, ex-type culture CBS 102220).
Notes: Clade 30 (Fig. 5) represents Pestalotiopsis malayana
(CBS 102220), which is characterised by having two apical appendages. Pestalotiopsis malayana formed a distinct lineage in
the phylogenetic analyses from its closely related species
P. adusta (clade 31; Fig. 5), P. papuana (clade 32; Fig. 5) and
Pestalotiopsis sp. Clade 33 (clade 33; Fig. 5). Furthermore,
morphologically P. malayana is well distinguished from allied
species by its larger conidia and longer apical appendages.
Pestalotiopsis monochaeta Maharachch., K.D. Hyde &
Etymology: The name refers to the unique single apical
appendage.
Conidiomata pycnidial in culture on PDA, globose or clavate,
aggregated or scattered, semi-immersed or partly erumpent,
250–500 μm diam; exuding a globose, dark brown to black
170
conidial masses. Conidiophores septate, sparsely branched and
sometimes reduced to conidiogenous cells, hyaline, smoothwalled, up to 50 μm long. Conidiogenous cells discrete or integrated, ampulliform to lageniform (4–12 × 2–4 μm) or cylindrical
(10–60 × 2–8 μm), proliferating 2–4 times percurrently near
apex, tapering to a long, thin neck, collarette present and
not flared. Conidia ellipsoid, straight to slightly curved, 4-septate,
slightly constricted at septa, (25–)27–40(–42) × 7–11
(–11.5) μm, x ± SD = 32.8 ± 3.5 × 9.6 ± 0.6 μm; basal cell conic to
obconic with a truncate base, rugose and thin-walled, 5.5–9.5 μm
long; three median cells (17–)18–25(–26) μm, x ± SD
= 21 ± 2 μm, doliiform, verruculose, concolourous, but occasionally the two upper median cells slightly darker than the lower
median cell (second cell from base 5–8.5 μm long; third cell
7–9 μm long; fourth cell 7–9 μm long); apical cell conic, hyaline,
thin- and smooth-walled, 4–6.5 μm long; with single, central,
tubular apical appendage, unbranched, filiform, (40–)
43–67(–75) μm, x ± SD = 51 ± 6 μm; basal appendage single,
tubular, unbranched, centric, 6–14 μm long.
diam after 7 d at 25 °C, with undulate edge, whitish to pale
yellow-coloured, with dense, with aerial mycelium on surface,
with black, gregarious conidiomata; reverse similar in colour.
PESTALOTIOPSIS
REVISITED
Fig. 36. Pestalotiopsis monochaeta CBS 144.97T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Habitat: On Taxus baccata and endophytic in branches of
Quercus robur.
Etymology: Named after the historical European name, New
Holland or Hollandia Nova, for the country where it was collected,
Australia.
Known distribution: Netherlands.
Materials examined: Netherlands, Baarn, Eemnesserweg, endophytes on
branches of Quercus robur, Jul. 1996, H.A. van der Aa (CBS H-21757, holotype,
ex-type culture CBS 144.97); Baarn, Eemnesserweg 90, from Taxus baccata, 14
Apr. 1983, H.A. van der Aa, CBS H-14560, culture CBS 440.83 = IFO 32686.
Notes: Pestalotiopsis monochaeta (clade 17; Fig. 5) differs from
all other species in the genus in having a single apical
appendage. Pestalotiopsis brassicae (clade 19; Fig. 5),
P. hollandica (clade 18; Fig. 5) and P. verruculosa (clade 20;
Fig. 5) are closely related species, but have conidia with more
than two apical appendages. This species can easily be misidentified as Monochaetia, since it has borderline morphological
features of both genera.
Pestalotiopsis novae-hollandiae Maharachch., K.D.
Conidiomata (on PDA) pycnidial, globose to clavate, solitary to
aggregated, embedded or semi-immersed, dark brown,
200–450 μm diam, exuding a globose, dark brown, glistening
conidial masses. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells discrete, simple, straight to curved, lageniform, smooth, thin-walled, hyaline, 5–20 × 5–10 μm. Conidia
fusoid to ellipsoid, straight to slightly curved, 4-septate, (24–)
25–31(–32) × (7.5–)8–10(–10.5) μm, x ± SD = 28.1 ± 1.6
× 9 ± 0.7 μm; basal cell obconic with truncate base, hyaline or
slightly olivaceous, rugose and thin-walled, 4–7 μm long; three
median cells (16–)16.5–20.5(–21) μm long, x ± SD = 19
± 1.3 μm, doliiform to subcylindrical, verruculose, concolourous,
olivaceous, constricted at the septa (second cell from base
6–8 μm long; third cell 6–7 μm long; fourth cell 5–7 μm long);
apical cell hyaline, conic to cylindrical, hyaline, thin- and smoothwalled, 3–5 μm long; with 3–9 tubular apical appendages,
arising not in an apical crest, but each inserted at a different
171
MAHARACHCHIKUMBURA
ET AL.
Fig. 37. Pestalotiopsis novae-hollandiae CBS 130973T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–J. Conidia. Scale
bars = 10 μm.
locus in the upper half of the cell, unequal in length, some appendages branched, filiform, flexuous, (20–)22–44(–50) μm
long, x ± SD = 31 ± 9 μm; basal appendage single, tubular,
unbranched, centric, 2–5 μm long.
diam after 7 d at 25 °C, undulated at the edge, whitish to pale
yellow-coloured, with dense aerial mycelium on surface, forming
Habitat: On old inflorescence of Banksia grandis.
Material examined: Australia, Perth, Jarrah Forest, from old inflorescence of
Banksia grandis, 2010, W. Gams (CBS H-21756, holotype, ex-type culture CBS
130973).
172
Notes: This species (clade 11; Fig. 5) is characterised by a large
number of apical appendages and in having a short basal
appendage. Species such as P. camelliae (clade 13; Fig. 5) and
P. furcata (clade 12; Fig. 5) have as large a number of apical
appendages as P. novae-hollandiae, but they lack a basal
appendage. Pestalotiopsis novae-hollandiae is sister to
P. portugalica (clade 10; Fig. 5), which has rather smaller conidia
(15–21 × 5–7 μm), and few apical appendages (1–3).
Pestalotiopsis oryzae Maharachch., K.D. Hyde & Crous,
Etymology: Named after the host genus from which it was isolated, Oryza.
aggregated or scattered, dark brown to black, semi-immersed or
PESTALOTIOPSIS
REVISITED
Fig. 38. Pestalotiopsis oryzae CBS 353.69T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
partially erumpent, up to 300 μm diam; releasing globose, dark
brown to black conidial masses. Conidiophores sparsely septate
at base, branched or unbranched, subcylindrical, hyaline,
smooth, up to 20 μm. Conidiogenous cells discrete, ampulliform
to lageniform, hyaline, smooth, proliferating 2–5 times percurrently, 10–25 × 3–7 μm. Conidia fusoid, ellipsoid to subcylindrical, straight to slightly curved, 4-septate, (23–)
24.5–29(–30) × 6–8 μm, x ± SD = 26.9 ± 1.4 × 7 ± 0.2 μm;
basal cell obconic to conic with a truncate base, hyaline, verruculose and thin-walled, 4.5–6.5 μm long; three median cells
doliiform, (14–)16–18.5(–19) μm long, x ± SD = 17 ± 1.3 μm,
wall minutely verruculose, concolourous or middle median cell is
much darker than other cell, olivaceous, septa darker than the
rest of the cell (second cell from the base 5–7 μm; third cell
5.5–7 μm; fourth cell 5–6.5 μm); apical cell 3.5–5 μm long,
hyaline, cylindrical to subcylindrical, thin- and smooth-walled;
the apical crest, unbranched, filiform, flexuous (9–)
diam after 7 d at 25 °C, with undulate edge, convex with papilate
surface, hyaline to pale honey-coloured, with sparse aerial
mycelium on the surface with black, gregarious conidiomata;
reverse pale honey-coloured.
Habitat: On Telopea sp. and seeds of Oryza sativa.
Known distribution: Denmark, Italy and USA.
Materials examined: Denmark, from seeds of Oryza sativa, S.B. Mathur (CBS H15697, holotype, ex-type culture CBS 353.69). Italy, unknown substrate, Dec.
1926, R. Ciferri, culture CBS 171.26. USA, Hawaii, from Telopea sp. (introduced
from Australia), 8 Dec. 1998, P.W. Crous & M.E. Palm, CBS H-21753, culture
CBS 111522 = STE-U 2083.
Notes: Clade 41 (Fig. 5) consists of three isolates of P. oryzae,
including the ex-type strain (CBS 353.69) isolated from seeds of
Oryza sativa from Denmark. Pestalotiopsis oryzae has overlapping conidial characters with P. kenyana (clade 42; Fig. 5) and
P. trachicarpicola (clade 43; Fig. 5). However, P. oryzae is
173
MAHARACHCHIKUMBURA
ET AL.
genetically distinct and has a different geographic distribution
from these two species.
Pestalotiopsis papuana Maharachch., K.D. Hyde &
Papua New Guinea.
Conidiomata pycnidial, globose to clavate, aggregated or scattered, semi-immersed on PDA, dark brown to black,
100–500 μm diam; exuding globose, dark brown conidial
masses. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells discrete, lageniform to
subcylindrical, hyaline, smooth, proliferating once or twice,
4–20 × 2–4 μm; apex with minute periclinal thickening and
flaring collarettes. Conidia fusoid, ellipsoid, straight to slightly
curved, 4-septate, (17–)18–22(–24) × 6–7.5 μm, x ± SD
= 20.5 ± 1.5 × 6.7 ± 0.3 μm; basal cell obconic with a truncate
base, hyaline, verruculose and thin-walled, 3–5 μm long; three
median cells doliiform, 12–15 μm long, x ± SD = 13.6 ± 0.7 μm,
wall verruculose, concolourous, brown, septa darker than the
rest of the cell (second cell from the base 3.5–5.5 μm; third cell
4.5–5.5 μm; fourth cell 4.5–6 μm); apical cell 2–4 μm long,
hyaline, cylindrical to subcylindrical, rugose and thin-walled; with
1–2 tubular apical appendages, arising from the apical crest,
unbranched, filiform, 1.5–7 μm long, x ± SD = 4.1 ± 1 μm; basal
appendage single, tubular, unbranched, centric, 0.5–2 μm long.
diam after 7 d at 25 °C, with undulate edge, pale honey-coloured,
with medium sparse aerial mycelium on the surface with black,
Habitat: On coastal soil and leaves of Cocos nucifera.
Known distribution: Papua New Guinea.
Materials examined: Papua New Guinea, from soil along the coast, Nov. 1995,
A. Aptroot (CBS H-21755, holotype, ex-type culture CBS 331.96); from leaves of
Cocos nucifera (coastal primary forest), 27 Oct. 1995, A. Aptroot, culture CBS
887.96.
Notes: Pestalotiopsis papuana (clade 32; Fig. 5) is genetically
close to P. adusta (clade 31; Fig. 5) and two isolates representing
Pestalotiopsis sp. Clade 33 (clade 33; Fig. 5). The latter two
isolates are unnamed for the present since both cultures were
Fig. 39. Pestalotiopsis papuana CBS 331.96T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
174
PESTALOTIOPSIS
sterile, making morphological comparisons impossible (see
notes under Pestalotiopsis sp. Clade 33). Morphologically,
however, P. papuana is quite distinct from P. adusta in having
larger conidia and shorter apical appendages.
Pestalotiopsis parva Maharachch., K.D. Hyde & Crous,
Etymology: The epithet parva refers to the small conidial size of
this species.
Conidiomata pycnidial, globose, aggregated or scattered, dark
brown to black, semi-immersed on PDA, 100–200 μm diam;
exuding globose, dark brown to black conidial masses. Conidiophores indistinct, often reduced to conidiogenous cells.
Conidiogenous cells discrete, cylindrical to subcylindrical, hyaline, smooth-walled, simple, proliferating 2–3 times percurrently,
5–18 × 2–4 μm, apex 1–1.5 μm diam. Conidia fusoid, straight to
slightly curved, 4-septate, (16–)16.5–20(–21) × 5–7(–7.5) μm,
x ± SD = 18.3 ± 1.2 × 6.2 ± 0.5 μm; basal cell obconic to conic
with a truncate base, hyaline, thin- and smooth-walled, 3–5 μm
REVISITED
long; three median cells doliiform, (10–)10.5–13(–13.5) μm
long, x ± SD = 12.1 ± 1.0 μm, wall minutely verruculose, concolourous, pale brown, septa darker than the rest of the cell
(second cell from the base 3.5–5 μm long; third cell 3.5–4.5 μm
long; fourth cell 4–5 μm long); apical cell (2–)2.5–4 μm long,
hyaline, subcylindrical; with 2–3 tubular apical appendages
(mostly 3), arising from the apical crest, unbranched, (6–)
6.5–12(–13) μm long, x ± SD = 9.0 ± 1.9 μm; basal appendage
Habitat: On Delonix regia and Leucothoe fontanesiana.
Materials examined: Unknown country, from Leucothoe fontanesiana, 1935,
R.P. White (CBS H-15694, holotype, ex-type culture CBS 278.35); from Delonix
regia, H.W. Wollenweber, CBS H-15659, culture CBS 265.37 = BBA 2820.
Fig. 40. Pestalotiopsis parva CBS 278.35T. A. Conidioma sporulating on PNA. B. Conidiomata on PDA. C–D. Conidiogenous cells. E–I. Conidia. Scale bars = 10 μm.
175
MAHARACHCHIKUMBURA
ET AL.
Notes: Pestalotiopsis parva is a distinct species represented by
two isolates (clade 35; Fig. 5). Pestalotiopsis rosea (clade 34;
Fig. 5), which is an endophyte isolated from living leaves of Pinus
sp. in China, is a sister species. Although these two species are
morphologically similar, they differ in having distinctly longer
apical appendages, which are sometimes branched. Furthermore, the reddish colony is unique to P. rosea and this reddish
colour can be seen even in conidiogenous cells and some
conidia.
Pestalotiopsis portugalica Maharachch., K.D. Hyde &
Portugal.
aggregated, black, semi-immersed, 200–400 μm diam; releasing
brown to black, slimy, globose conidial masses. Conidiophores
hyaline, septate, irregularly branched, up to 100 μm in long.
Conidiogenous cells cylindrical, hyaline, smooth, proliferating
2–6 times percurrently, 10–60 × 4–12 μm, collarette present
and not flared, with prominent periclinal thickening. Conidia
fusoid, straight to slightly curved, 4-septate, (14.5–)
15.5–20(–21.5) × 5–7 μm, x ± SD = 17.9 ± 1.6 × 6.0 ± 0.5 μm;
basal cell obconic with a truncate base, hyaline, thin- and
smooth-walled, 2.5–4 μm long; three median cells (9–)
9.5–13.5(–14) μm long, x ± SD = 11.7 ± 1 μm, doliiform to
subcylindrical, with thick verruculose walls, constricted at the
septa, concolourous, pale brown (second cell from base 3–5 μm
long; third cell 3.0–5 μm long; fourth cell 3.5–5 μm long); apical
cell conic to cylindrical, hyaline, thin- and smooth-walled, 2–5 μm
long; 1–3 tubular apical appendages arising from an apical crest
or branched irregular along their length resulting 2–3 branched,
filiform, (8–)9–18(–20) μm long, x ± SD = 14 ± 3 μm; basal
appendage lack or when present single, tubular, unbranched,
centric, 1–4 μm long.
diam after 7 d at 25 °C, edge entire, whitish to pale honeycoloured, aerial mycelium on surface, conidiomata black,
gregarious; reverse similar in colour.
Fig. 41. Pestalotiopsis portugalica CBS 393.48T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C. Conidiogenous cells. D–J. Conidia. Scale bars = 10 μm.
176
PESTALOTIOPSIS
REVISITED
Habitat: Unknown.
Pestalotiopsis rosea Maharachch. & K.D. Hyde
Known distribution: Portugal.
Garden, on living leaves of Pinus sp., 19 Mar. 2002, W.P. Wu (HMAS047135,
holotype, MFLU12 0409, isotype, ex-type culture NN0471350 = MFLUCC 120258).
Material examined: Portugal, unknown host, Jun. 1948, collector unknown (CBS
H-21754, holotype, ex-type culture CBS 393.48).
Notes: Pestalotiopsis portugalica (clade 10; Fig. 5) is a distinct
species in terms of morphology and phylogeny. It differs from its
phylogenetically related species P. camelliae (clade 13; Fig. 5),
P. furcata (clade 12; Fig. 5) and P. novae-hollandiae (clade 11;
Fig. 5) by smaller conidia and fewer apical appendages. Its
conidial size overlaps with P. rosea (17.5–21.8 × 5.7–7 μm; clade
34; Fig. 5), but those two species are phylogenetically distinct.
Pestalotiopsis rhododendri Y.M. Zhang, Maharachch. &
K.D. Hyde
Material examined: China, Yunnan Province, Chuxiong, Zixishan, leaf spots on
Rhododendron sinogrande, May 2011, Y.M. Zhang (IFRD 410-018, holotype, extype culture IFRDCC 2399).
Pestalotiopsis scoparia Maharachch., K.D. Hyde &
Etymology: The epithet scoparia refers to the broom-shaped
apical appendages of this species.
Conidiomata pycnidial, globose, aggregated or scattered, semiimmersed on PDA, dark brown to black, 100–400 μm diam;
exuding globose, dark brown to black conidial masses. Conidiophores indistinct, often reduced to conidiogenous cells.
Conidiogenous cells discrete, cylindrical to subcylindrical, hyaline,
smooth, proliferating up to 3 times, 10–30 × 2–4 μm, with visible
periclinal thickening; collarette slightly flared, up to 3 μm long when
Fig. 42. Pestalotiopsis scoparia CBS 176.25T. A. Conidioma sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
177
MAHARACHCHIKUMBURA
ET AL.
present. Conidia fusoid, ellipsoid, straight to slightly curved, 4septate, (22–)23.5–29(–31) × 6–8.5 μm, x ± SD = 26.3 ± 2
× 7.4 ± 0.3 μm; basal cell hemispherical to obconic with a truncate
base, hyaline, verruculose and thin-walled, 4–6 μm long; three
median cells doliiform, 15.5–19.5 μm long, x ± SD = 17 ± 1 μm,
wall verruculose, concolourous, but occasionally the two upper
median cells darker than the lower median cell, brown, septa
darker than the rest of the cell (second cell from the base
5–6.5 μm long; third cell 5–7 μm long; fourth cell 5.5–7.5 μm
long); apical cell 4.5–6 μm long, hyaline, subcylindrical, rugose
and thin-walled; with 3–5 tubular apical appendages, arising from
the apical crest, unbranched, filiform, (20–)23–35(–42) μm long,
(24–)25–31(–32) × 7.5–9.5 μm, x ± SD = 27.7 ± 2 × 8.6
± 0.3 μm, slightly constricted at septa; basal cell conic to obconic
with a truncate base, rugose and thin-walled, 5–7.5 μm long;
three median cells, (13–)14–19.5(–20) μm long,
x ± SD = 17.1 ± 1.8 μm, doliiform, verruculose, dark brown to
olivaceous, versicoloured (second cell from base pale brown to
olivaceous, 4.5–7 μm, third cell honey brown, 4.5–6 μm long;
fourth cell honey brown, 5.5–7 μm long); apical cell cylindrical,
hyaline, thin and smooth-walled, 5–6 μm long; with 2–5 tubular
apical appendages, arising not in an apical crest, but each
inserted at a different locus in the upper half of the cell, swollen at
the tip, filiform, flexuous, some appendages branched, (17–)
18–24(–25) μm, x ± SD = 21.1 ± 1.7 μm; basal appendage
with medium dense aerial mycelium on the surface with black,
diam after 7 d at 25 °C, with undulate edge, whitish, with dense,
aerial mycelium on surface, conidiomata black, gregarious;
Habitat: On Chamaecyparis sp.
Habitat: On leaf spot on Gevuina avellana.
Known distribution: Chile.
Material examined: Unknown country, from young Chamaecyparis sp. ‘Retinospora’, May 1925, C.M. Doyer (CBS H-21752, holotype, ex-type culture CBS
176.25).
Material examined: Chile, leaf spot on Gevuina avellana, Sep. 1961, unknown
collector (CBS H-21751, holotype, ex-type culture CBS 356.86).
Notes: Pestalotiopsis scoparia (clade 25; Fig. 5) is genetically a
clearly distinct species, forming a separate clade in a sister
position to P. australis (clade 26; Fig. 5) and P. unicolor (clade 24;
Fig. 5). It is well characterised by its rather long broom-shaped,
3–5 apical appendages, long basal appendages and occasionally by having versicoloured median cells.
Pestalotiopsis sp. Clade 33
Materials examined: Indonesia, Sulawesi, from leaf spot in bibit of Cocos sp.,
unknown collection date, P.M.L. Tammes, culture CBS 264.33. Netherlands,
Boskoop, from Rhododendron ponticum, Mar. 1933, W.F. van Hell, culture CBS
263.33.
Note: Although phylogenetically distinct (clade 33; Fig. 5), both
cultures of this species proved to be sterile, and thus are not
treated further.
Pestalotiopsis spathulata Maharachch., K.D. Hyde &
Etymology: The species epithet refers to the knobbed nature of
its apical appendages.
Conidiomata pycnidial, globose, aggregated or scattered, semiimmersed to erumpent or embedded on PDA, dark brown to
black, 100–400 μm diam; exuding globose, dark brown to black
conidial masses. Conidiophores 0–2-septate, branched at base,
subcylindrical, often reduced to conidiogenous cells, hyaline,
smooth-walled up to 20 μm long. Conidiogenous cells discrete,
ampulliform to lageniform or cylindrical, proliferating 2–5 times
percurrently, wide at the base, tapering to a long, thin neck,
5–40 × 2–8 μm, prominent periclinal thickening with flaring
collarettes. Conidia fusoid, straight to slightly curved, 4-septate,
178
Notes: Pestalotiopsis spathulata (clade 8; Fig. 5) is morphologically and phylogenetically distinct (Fig. 5). The two upper median cells in P. spathulata are especially darker than the lower
median cell. This is also found in its sister species P. gaultheria
(clade 9; Fig. 5). Pestalotiopsis gaultheria differs from
P. spathulata in having fewer (–3), and longer apical appendages
(13–54 μm).
Pestalotiopsis telopeae Maharachch., K.D. Hyde &
Etymology: Named after the host genus, Telopea.
Leaf spots on Telopea sp. circular to subcircular, up to 2 cm
diam, amphigenous, pale to medium brown with a broad, dark
brown border, which can be conspicuously raised in some leaf
spots. Conidiomata pycnidial in culture on PDA, globose,
aggregated or scattered, semi-immersed, dark brown to black,
up to 500 μm diam; exuding globose, dark brown to black
conidiogenous cells. Conidiogenous cells discrete, ampulliform
or lageniform, hyaline, smooth, proliferating 2–4 times percurrently, 5–15 × 2–9 μm, collarette present and not flared. Conidia
fusoid, ellipsoid, straight to slightly curved, 4-septate, (24–)
24.5–31(–32) × 6–8 μm, x ± SD = 27 ± 1.5 × 7 ± 0.3 μm; basal
cell obconic, hyaline, verruculose and thin-walled, 4.5–7 μm
long; three median cells doliiform, (15–)16–18.5(–19) μm long,
x ± SD = 17.1 ± 1 μm, wall verruculose, concolourous, brown to
olivaceous (second cell from the base 4.5–7 μm long; third cell
5–7.5 μm long; fourth cell 5–7 μm long); apical cell 3.5–5.5 μm
long, hyaline, subcylindrical; with 2–4 tubular apical appendages
(mostly 3), arising from an apical crest, unbranched, filiform, (7–)
single, tubular, unbranched, centric, 3.5–7 μm long.
PESTALOTIOPSIS
REVISITED
Fig. 43. Pestalotiopsis spathulata CBS 356.86T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
diam after 7 d at 25 °C, with undulate edge, whitish, with dense,
aerial mycelium on surface, conidiomata black, gregarious;
clade. Although no pathogenicity tests were conducted,
P. telopeae is consistently associated with a prominent leaf spot
disease of Telopea in Australia.
Pestalotiopsis trachicarpicola Y.M. Zhang & K.D. Hyde
Habitat: On leaves of Telopea sp.
Materials examined: Australia, New South Wales, Mount Annan, on leaves of
Telopea sp., Aug. 1999, P.W. Crous, JT 975 (CBS H-21750, holotype, ex-type
culture CBS 114161 = STE-U 3083); ibid., JT 975, culture CBS
113606 = STE-U 3082; Protea neriifolia × susannae cv. ‘Pink Ice’, 12 Oct. 1999,
P.W. Crous, culture CBS 114137 = STE-U 2952.
Notes: The two collections of P. telopeae (clade 40; Fig. 5) are
morphologically most similar to P. australasiae (clade 39; Fig. 5),
but differ in having shorter conidiogenous cells. Furthermore, in
the phylogenetic analyses, P. telopeae represents a distinct
Materials examined: China, Hunan Province, Yizhang County, Mangshan, on
living leaves of Schima sp., 12 Apr. 2002, W.P. Wu, culture
NN0469830 = MFLUCC 12-0265; Hunan Province, Yizhang County, Mangshan,
on living leaves of Sympolocos sp., 12 Apr. 2002, W.P. Wu, culture
NN0469780 = MFLUCC 12-0266; Hunan Province, Yizhang County, Mangshan,
on living leaves of unidentified tree, 12 Apr. 2002, W.P. Wu, cultures
NN0470990 = MFLUCC 12-0267, NN0470720 = MFLUCC 12-0263; Yunnan
Province, Dehong, Mangshi, leaf spots on Podocarous macrophyllus, Sep. 2011,
Y.M. Zhang, IFRD 411-018, culture IFRDCC 2403; Yunnan Province, Kunming,
Kunming Botanical Gardens, leaf spots on Trachycarpus fortunei, Mar. 2011, K.D.
Hyde OP068 (IFRD 9026, holotype, ex-type culture IFRDCC 2440); Yunnan
Province, Kunming, on living leaves of Chrysophullum sp., 19 Mar. 2002, W.P.
Wu, culture NN0471960 = MFLUCC 12-0264.
179
MAHARACHCHIKUMBURA
ET AL.
Fig. 44. Pestalotiopsis telopeae CBS 114161T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
Zhang et al. (2012a).
Pestalotiopsis unicolor Maharachch. & K.D. Hyde
Pseudopestalotiopsis Maharachch., K.D. Hyde & Crous, gen.
nov. MycoBank MB809753.
Materials examined: China, Hunan Province, Yizhang County, Mangshan, on
living leaves of Rhododendron sp., 12 Apr. 2002, W.P. Wu (HMAS046974, holotype, MFLU 12-0417, isotype, ex-type culture NN0469740 = MFLUCC 120276); Hunan Province, on living leaves of unidentified tree, 12 Apr. 2002, W.P.
Wu, culture NN0473080 = MFLUCC 12-0275.
Pestalotiopsis verruculosa Maharachch. & K.D. Hyde
Garden, on living leaves of Rhododendron sp., 19 Mar. 2002, W.P. Wu
NN0473090 = MFLUCC 12-0274).
180
Pestalotiopsis.
Conidiomata acervular or pycnidial, subglobose, globose,
clavate, solitary or aggregated, dark brown to black, immersed to
erumpent, unilocular; exuding dark brown to black conidia in a
slimy, globose mass. Conidiophores indistinct, reduced to conidiogenous cells. Conidiogenous cells discrete, cylindrical,
ampulliform to lageniform, hyaline, smooth- and thin-walled;
conidiogenesis initially holoblastic, percurrent proliferations to
produce additional conidia at slightly higher levels. Conidia
fusoid, ellipsoid, subcylindrical, straight to slightly curved, 4septate, slightly constricted at septa; basal cell conical to cylindric with a truncate base; three median cells doliiform,
PESTALOTIOPSIS
concolourous, brown to dark brown or olivaceous, wall rugose to
verruculose, septa darker than the rest of the cell; apical cell
conic to cylindrical, thin- and smooth-walled; with tubular apical
appendages, one to many, filiform or attenuated, flexuous,
branched or unbranched, with or without spatulate tips; basal
appendage single, tubular, unbranched, centric.
Type species: Pseudopestalotiopsis theae (Sawada) Maharachch., K.D. Hyde & Crous (see below).
Notes: In most studies (Jeewon et al. 2003, Liu et al. 2010, Hu
et al. 2007, Maharachchikumbura et al. 2011, 2012), species with
dark concolourous median cells with knobbed apical
REVISITED
appendages formed a distinct clade with high support, which is
defined here as a novel genus, Pseudopestalotiopsis. Partial
LSU sequence data confirm that Pseudopestalotiopsis is
phylogenetically related to Neopestalotiopsis (Fig. 3), but these
genera are also morphologically distinct. In Pseudopestalotiopsis
the three median cells are the same colour (concolourous),
whereas in Neopestalotiopsis these are versicoloured.
Pseudopestalotiopsis cocos Maharachch., K.D. Hyde &
Etymology: Named after the host genus from which it was isolated, Cocos.
Fig. 45. Pseudopestalotiopsis cocos CBS 272.29T. A. Conidioma sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–J. Conidia. Scale bars = 10 μm.
181
MAHARACHCHIKUMBURA
ET AL.
Conidiomata pycnidial, 100–300 μm diam, globose, dark brown,
semi-immersed on host substrate on PDA; exuding black conidia
in a slimy, globose, glistening mass. Conidiophores indistinct,
often reduced to conidiogenous cells. Conidiogenous cells
discrete, hyaline, smooth-walled, simple, filiform, sometimes
slightly wide at the base, truncate at apex, proliferating 2–3 times
percurrently, 12–15 × 1–3 μm. Conidia fusoid, ellipsoid, straight to
slightly curved, 4-septate, constricted at septum, (20–)
21–25(–26.5) × 6–7.5 μm, x ± SD = 23.0 ± 1.6 × 6.5 ± 0.4 μm;
basal cell obconic with a truncate base, hyaline, thin- and smoothwalled, granular, 3.5–5 μm long; three median cells (13.5–)
14–16.5(–17.5) μm long, x ± SD = 15.5 ± 1.2 μm, concolourous,
pale brown, septa darker than the rest of the cell (second cell from
the base 5.5–6.5 μm long; third cell 4.5–5.5 μm long; fourth cell
5.5–6 μm long); apical cell 3.5–5 μm long, hyaline, cylindrical;
with 2–4 tubular apical appendages (mostly 3), arising in an apical
crest, but each inserted at a different locus, flexuous, unbranched,
(12–)14–21(–23) μm long, x ± SD = 17.6 ± 3.2 μm; basal
diam after 7 d at 25 °C, with smooth edge, whitish to grey, with
Habitat: On Cocos nucifera.
Known distribution: Indonesia (Java).
Material examined: Indonesia, Java, Bogor (Buitenzorg), from Cocos nucifera,
unknown collection date, C.M. Doyer (CBS H-15666, holotype, ex-type culture
CBS 272.29).
Notes: Pseudopestalotiopsis cocos is a distinct species based on
its morphology and phylogeny (Figs 3, 4). It can clearly be
differentiated from its sibling species, Ps. indica
(31.5–37 × 6.5–9 μm; Fig. 4) by relatively smaller conidia
(20–26.5 × 6–7.5 μm), and shorter apical appendages
(12–23 μm), whereas in Ps. indica appendages are longer
(30–40 μm). Furthermore, the three median cells in Ps. cocos
are paler in colour than in Ps. indica. This species is sister to a
clade that contains Ps. theae (22–32 × 5–8 μm; Fig. 4) and they
have overlapping morphometric characters. However, in Ps.
theae the apical appendages are knobbed, which is a feature
absent in Ps. cocos.
Pseudopestalotiopsis indica Maharachch., K.D. Hyde &
Fig. 46. Pseudopestalotiopsis indica CBS 459.78T. A. Conidiomata sporulating on PNA. B. Conidiomata on PDA. C–E. Conidiogenous cells. F–K. Conidia. Scale bars = 10 μm.
182
PESTALOTIOPSIS
Etymology: Named after the country where it was collected, India.
aggregated, dark brown, semi-immersed or partly erumpent,
200–500 μm diam; exuding brown to black conidial masses.
cells discrete, ampulliform to lageniform, 5–18 × 2–7 μm, hyaline,
smooth- and thin-walled, sometimes percurrently proliferating
1–2 times, periclinal thickening in the apical region, collarette
present and flared. Conidia fusoid to ellipsoid, straight to slightly
curved, 4-septate, slightly constricted at septa, (31.5–)
32.5–36(–37) × 6.5–9 μm, x ± SD = 34.5 ± 1.6 × 7.5 ± 0.5 μm;
basal cell conic with truncate base, rugose and thin-walled,
5.5–7 μm long; three median cells (19.5–)20–22(–22.5) μm
long, x ± SD = 21.6 ± 1.0 μm, doliiform, verrucose, concolourous,
dark brown, septa darker than the rest of the cell (second cell from
base 6.5–8.5 long; third cell 5.5–8 μm long; fourth cell 6.5–8.5 μm
long); apical cell subcylindrical, hyaline, thin and smooth-walled,
5.5–7 μm long; with 3–4 tubular apical appendages (mostly 3)
arising from the apical crest, flexuous, unbranched, (30–)
33–39(–40) μm long, x ± SD = 35 ± 2.8 μm; basal appendage
diam after 7 d at 25 °C, undulate at the edge, whitish to pale
honey-coloured, with black, gregarious conidiomata; reverse
pale honey-coloured.
Habitat: On Hibiscus rosa-sinensis.
Known distribution: India.
Material examined: India, Bangalore, on Hibiscus rosa-sinensis, Aug. 1978, H.C.
Govindu (CBS H-21749, holotype, ex-type culture CBS 459.78).
Notes: This species is characterised by large conidia
(32.5–36 × 7–8.5 μm) with three median cells that are dark in
colour. It forms a sister group (Fig. 4) with Ps. cocos and Ps.
theae. Pseudopestalotiopsis indica differs from Ps. cocos
(20–26.5 × 6–7.5 μm) and Ps. theae (22–32 × 5–8 μm) in its
larger conidia.
Pseudoestalotiopsis theae (Sawada) Maharachch., K.D. Hyde
& Crous, comb. nov. MycoBank MB809756.
Basionym: Pestalotia theae Sawada, Spec. Report Agric. Exp.
Station Formosa 11: 113. 1915. as “Pestalozzia”
Brux. 19:
≡ Pestalotiopsis theae (Sawada) Steyaert, Bull. Jard. bot. Etat
327. 1949.
Materials examined: Taiwan, Republic of China, Taipei, on living leaves of
Camellia sinensis, 13 Jul. 1908, Y. Fujikiro, det. K. Sawada (BPI 406804,
lectotype). Thailand, Chiang Mai Prov., Mae Taeng Distr., Ban Pha Deng,
Mushroom Research Centre, 19°17.1230 N 98°44.0090 E, 900 m, rainforest, on
living leaves of Camellia sinensis, 20 Jan. 2010, S.S.N. Maharachchikumbura
(MFLU 12-0116, epitype, ex-epitype culture MFLUCC 12-0055 = CPC 20281);
on living leaves of Camellia sinensis, unknown collection date and collector,
culture SC011.
DISCUSSION
Winter (1887) established the Amphisphaeriaceae, which is
characterised by having immersed ascomata in the host and with
REVISITED
dark peridial walls and ascal apices that are usually amyloid
(Barr 1975). The Amphisphaeriaceae is a large heterogeneous
family, which mainly possesses pestalotiopsis-like asexual states
(Jeewon et al. 2002). These conidial forms are generally characterised by septate conidia with filiform apical appendages (Barr
1990, Nag Raj 1993) and with the exception of Bartalinia, Discosia and Monochaetia, most genera are linked to a sexual
morph. Conidial septation appears to be effective in placement of
taxa in genera of Amphisphaeriaceae. Sequence data generated
to date reveal Truncatella, Pestalotiopsis and Seiridium to
represent three distinct genera, which are characterised by 4celled, 5-celled and 6-celled conidia, respectively. However, it
has not been established whether, as defined, Pestalotia differs
from Pestalotiopsis based on molecular evidence. Although they
are clearly distinct in conidial morphology, Pestalotiopsis has 5celled conidia while Pestalotia has 6-celled conidia. From a
phenotypic viewpoint, Pestalotia species are more similar to
Seiridium species, as both have 6-celled conidial forms. The type
species of Pestalotia, P. pezizoides, can be distinguished from
Seiridium species by its more numerous appendages, which are
branched, while in Seiridium appendages are fewer and generally unbranched. However, branched apical appendages typical
of Pestalotia are also found in S. corni and S. venetum (Nag Raj
1993), and thus Pestalotia could potentially prove to be congeneric with Seiridium. Appendage morphology appears to be
highly informative at the species level, even though conidial
appendages alone cannot be used as a useful character for
generic separation (Crous et al. 2012). The monotypic genus
Pestalotia (1839) may therefore be a synonym of Seiridium
(1816), since both genera have similar morphologies. However,
Guba's (1961) treatment of Monochaetia as a distinct genus has
proved valid. LSU phylogenetic analyses reveal Monochaetia to
represent a genus that is distinct from Pestalotiopsis, Seiridium
and Truncatella (Fig 3). However, it is essential to incorporate
molecular data and more taxon sampling in future analyses as
Monochaetia includes 3-, 4-, and 6-celled conidial forms.
Pestalotiopsis species are morphologically diverse in conidial
morphology, and phylogenetic analyses of different gene regions
have established that Pestalotiopsis comprises three distinct
lineages (Jeewon et al. 2003, Maharachchikumbura et al. 2011,
2012). Based on these findings, we divided Pestalotiopsis into
three genera: Pestalotiopsis, Neopestalotiopsis and Pseudopestalotiopsis. However, our phylogenetic analyses disagree with
Nag Raj's (1993) broad concept of Pestalotiopsis, which included
3-celled, and 4-celled conidial forms. All species within Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis contain
only 4-celled conidial forms. Pestalotiopsis maculans, which is
the type species of Pestalotiopsis, commonly occurs on Camellia
and provides a stable generic concept for Pestalotiopsis. In
P. maculans conidiophores are septate, unbranched and often
reduced to conidiogenous cells; conidiogenous cells are
ampulliform to lageniform or cylindrical to subcylindrical phialides, and conidia have concolourous median cells. Neopestalotiopsis has versicolourous median cells with indistinct
conidiophores, while Pseudopestalotiopsis can be distinguished
from Pestalotiopsis by sequence data and generally darkcoloured concolourous median cells with indistinct conidiophores. The three genera can also be roughly assigned to
distinct groups based on the total number of base pairs in the ITS
region.
Pestalotiopsis is a species-rich asexual morph-typified genus
with only 13 known sexual states, as compared to the possible
183
MAHARACHCHIKUMBURA
ET AL.
253 asexual names (Zhang et al. 2012a, Maharachchikumbra
et al. 2013d). Of the 13 sexually reproducing species, nine are
linked to named Pestalotiopsis species and eight have concolourous median cells typical of Pestalotiopsis. Pestalosphaeria
maculiformans is linked to Pestalotiopsis maculiformans
(Marincowitz et al. 2008), which has versicolourous median cells,
hence, belongs in Neopestalotiopsis. However, based on a
megablast search of NCBIs GenBank nucleotide database for
this species (CBS 122683, GenBank EU552147), the closest hits
using the ITS sequence had highest similarity to Pestalotiopsis
(species with concolourous median cells). Therefore, presently
the known asexual states of Pestalosphaeria are Pestalotiopsis
species. Because only one name can be applied to any fungal
species (Hawksworth et al. 2011, Taylor 2011, Wingfield et al.
2012) and since Pestalotiopsis is the oldest and the most
common name; Maharachchikumbura et al. (2011) suggested
that Pestalotiopsis should be adopted for this genus. This has
been followed in subsequent publications and is followed in this
paper (Maharachchikumbura et al. 2012, Zhang et al. 2012a).
Conidial morphology is the most widely used taxonomic
character for inter-specific delineation of Pestalotiopsis (Steyaert
1949, Guba 1961, Nag Raj 1993). However, there are considerable overlapping phenotypic characteristics that make it difficult to segregate morphologically equivocal taxa (Tejesvi et al.
2009). Conidial length and width have been emphasised as
crucial characters for species identification, and many contemporary researchers have used length and width to segregate taxa
(Steyaert 1949, Guba 1961, Mordue 1985). In the present study,
however, species sharing similar conidial dimensions did not
necessarily group together. As an example, P. malayana (clade
30; Fig. 5) and P. biciliata (clade 38; Fig. 5) have similar conidial
dimensions, but cluster in distinct clades. Therefore, the
continued use of conidium length and width in classification for
Pestalotiopsis species is unwise. A similar observation was
made by Dube & Bilgrami (1965) who showed that conidial size
is a homoplasious character and species sharing similar spore
sizes may not be closely related (Jeewon et al. 2003).
Various features/aspects of conidial appendages are taxonomically informative at the species level in many coelomycetous genera (Nag Raj 1993, Crous et al. 2012). The function of
appendages should not be considered in isolation since appendages usually relate to an ecological function linked to spore
dispersal, liberation, deposition and the colonisation of new
substrates or niches (Gregory 1952, Crous et al. 2012).
Watanabe et al. (2000) investigated the conidial adhesion and
germination of Pestalotiopsis neglecta and observed that apical
appendages firmly attached conidia to the substrate during the
infection process. Generally in Neopestalotiopsis, Pestalotiopsis
and Pseudopestalotiopsis apical appendages arise as tubular
extensions and maintain protoplasmic continuity with the
conidium body. Appendage morphology has been widely used in
Pestalotiopsis taxonomy to introduce novel taxa (Steyaert 1949,
Guba 1961, Nag Raj 1993, Maharachchikumbura et al. 2013a,
Zhang et al. 2012b). Among the appendage-bearing coelomycetes, Pestalotiopsis shows high variation in appendage
morphology. These apical appendage characters vary in length
of the apical appendage, appendage number, shape, branched
or unbranched nature, presence or absence of knobbed tips and
the position of the apical appendage attached to the conidial
body.
The ecology of species of Pestalotiopsis is poorly understood,
especially now that species have been recircumscribed using
184
molecular data. There is little data on geographical distribution
and even host range. Since our data set is not robust, it is not
clear whether the geographic influences or hosts range or
allopatry play a key role in species circumscription and delineation. Therefore, much research is needed and it might be
useful to account for substrate, geographic influences, host
ranges, and morphological characters when incorporating molecular sequence data to define species borders within Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis. This
kind of approach has been successfully used in the past to
investigate species in for example Cladosporium (Bensch et al.
2012), Colletotrichum (Damm et al. 2012), Diaporthe (Gomes
et al. 2013) and Teratosphaeriaceae (Quaedvlieg et al. 2014).
Common problems in Pestalotiopsis taxonomy are that new
species (e.g. P. alpiniae, P. oenotherae and P. nelumbinis) have
been defined without accompanying sequence data. In fact, in
2011 there were only four ex-type cultures available for this study
on Pestalotiopsis phylogeny. In the first inclusive phylogenetic
study of Pestalotiopsis, Jeewon et al. (2003) used ITS sequence
data to evaluate the phylogenetic significance of Pestalotiopsis
morphological characters in taxonomy. In differentiating endophytic species of Pestalotiopsis in Pinus armandii and Ribes
spp., Hu et al. (2007) pointed out that the TUB gene better
resolved Pestalotiopsis phylogeny. A combination of both the
TUB and ITS genes gave better phylogenetic resolution, and
they suggested that at least two genes should be used to resolve
the phylogeny of species of Pestalotiopsis. Maharachchikumbura
et al. (2012) tested 10 gene regions to resolve species boundaries in the Pestalotiopsis (actin, calmodulin, glutamine synthase,
glyceraldehyde-3-phosphate dehydrogenase, ITS, LSU, 18S
nrDNA, RNA polymerase II, TEF and TUB). The authors
compared the morphological versus sequence data from each
gene to establish which characters satisfactorily resolved species limits and ITS, TUB and TEF proved to be the better molecular markers. In the present study, phylogenetic species
recognition based on combined ITS, TUB and TEF gene regions
gave a high number of strongly supported nodes at the terminal
clades. In Neopestalotiopsis however, overall branch-length
support values were lower, when compared to Pestalotiopsis.
Future studies of Neopestalotiopsis may require additional loci to
obtain a better separation of species.
The genus Pestalotiopsis has been shown to produce
numerous secondary metabolites with diverse structural features, with antitumour, antifungal, antimicrobial and other activities (Xu et al. 2010, 2014). Three reviews have been recently
published and reveal the chemistry of Pestalotiopsis species and
related genera. Species belonging to these genera are a rich
source for bioprospecting when compared to other fungal genera
(Aly et al. 2010, Xu et al. 2010, 2014). Xu et al. (2010) discussed
130 diverse compounds isolated from species of Pestalotiopsis
in the past 10 years, while Xu et al. (2014) discussed a further
160 compounds. These biochemicals may have significance in
pharmaceutical, agricultural and industrial applications. The
names assigned to Pestalotiopsis species producing novel
compounds lacked a phylogenetic basis (Maharachchikumbura
et al. 2012, 2013c). It would be interesting to establish if
different species of Pestalotiopsis were chemically more creative
than others and also to establish if Neopestalotiopsis and
Pseudopestalotiopsis species are different from Pestalotiopsis
species in this regard.
Pestalotiopsis species are important causal agents of plant
disease (Keith et al. 2006, Joshi et al. 2009, Keith & Zee 2010,
PESTALOTIOPSIS
Chen et al. 2011, Evidente et al. 2012, Maharachchikumbura
et al. 2013a,b,c), chemically highly diverse (Aly et al. 2010, Xu
et al. 2014), extremely common in most habitats (Bate-Smith
& Metcalfe 1957, Jeewon et al. 2004, Maharachchikumbura
et al. 2011) and are fascinating because of their distinct
conidial morphology (Sutton 1980, Nag Raj 1993); thus they are
a remarkable group of fungi that have been well-studied
morphologically
in
the
past
(Steyaert
1949,
Maharachchikumbura et al. 2013a,b). In this study we advance
the understanding of this group using morphology and multilocus
sequence analyses and introduce two new genera, Neopestalotiopsis and Pseudopestalotiopsis, to accommodate seggregates of Pestalotiopsis. Phenotypic analyses of conidial
characters coupled with phylogenetic analyses of sequence data
were used to clarify species boundaries in the three genera.
Although genetic differences exist, several isolates were not
assigned to species because of sterile cultures and lack of data
on geographical differences; thus the data were insufficient to
determine species boundaries in those cases. Sequence data
are provided for 24 species of Neopestalotiopsis, 43 species of
Pestalotiopsis and three species of Pseudopestalotiopsis and
can be used in future studies to increase the understanding of
this group. We predict that future studies will reveal numerous
distinct and new taxa in this generic complex.
ACKNOWLEDGEMENTS
We thank the Kunming Institute of Botany, Chinese Academy of Sciences for
providing facilities and the World Agroforestry Centre, East and Central Asia
office for hosting us, we would also like to thank Humidtropics, a CGIAR
Research Program that aims to develop new opportunities for improved livelihoods in a sustainable environment, for partially funding this work, and the National Research Council of Thailand (grant for Pestalotiopsis No: 55201020008).
We thank the CBS-KNAW Fungal Biodiversity Centre for funding, and the
technical staff, Arien van Iperen (cultures) and Mieke Starink-Willemse (DNA
isolation, amplification, and sequencing) for their invaluable assistance.
REFERENCES
Aly AH, Debbab A, Kjer J, et al. (2010). Fungal endophytes from higher plants: a
prolific source of phytochemicals and other bioactive natural products.
Barr ME (1975). Pestalosphaeria, a new genus in the Amphisphaeriaceae.
Mycologia 67: 187–194.
Barr ME (1990). Prodromus to nonlichenized, pyrenomycetous members of
class Hymenoascomycetes. Mycotaxon 39: 43–184.
Bate-Smith EC, Metcalfe CR (1957). Leucanthocyanins. 3. The nature and
systematic distribution of tannin in dicotyledonous plants. The Journal of the
Linnean Society, Botany 55: 669–705.
Bensch K, Braun U, Groenewald JZ, et al. (2012). The genus Cladosporium.
Beyma FH van (1940). Beschreibung einiger neuer Pilzarten aus dem Centraalbureau voor Schimmelcultures, Baarn (Nederland), VI. Mitteilung.
Antonie van Leeuwenhoek 6: 263–290.
Chen CQ, Zhang B, Yang LN, et al. (2011). Identification and biological characteristics of round leaf spot on blueberry caused by Pestalotiopsis photiniae
(in Chinese). Journal of Northeast Forestry University 39: 95–98.
Corda ACJ (1839). Icones fungorum hucusque cognitorum: 3: 1–55.
Crous PW, Braun U, Hunter GC, et al. (2013). Phylogenetic lineages in Pseudocercospora. Studies in Mycology 75: 37–114.
Crous PW, Summerell BA, Swart L, et al. (2011). Fungal pathogens of Proteaceae. Persoonia 27: 20–45.
REVISITED
Crous PW, Verkley GJM, Christensen M, et al. (2012). How important are
conidial appendages? Persoonia 28: 126–137.
Crous PW, Verkley GJM, Groenewald JZ (2006). Eucalyptus microfungi known
from culture. 1. Cladoriella and Fulvoflamma genera nova, with notes on
some other poorly known taxa. Studies in Mycology 55: 53–63.
Crous PW, Verkley GJM, Groenewald JZ, et al. (eds) (2009). Fungal biodiversity.
CBS laboratory manual series: 1. Centraalbureau voor Schimmelcultures,
Utrecht, Netherlands: 1–269.
Damm U, Cannon PF, Woudenberg JHC, et al. (2012). The Colletotrichum
boninense species complex. Studies in Mycology 73: 1–36.
Debbab A, Aly AH, Proksch P (2013). Mangrove derived fungal endophytes – a
chemical and biological perception. Fungal Diversity 61: 1–27.
Dube HC, Bilgrami KS (1965). Pestalotia or Pestalotiopsis? Mycopathologia et
Mycologia Applicata 29: 33–54.
Ellis MB, Ellis JP (1997). Microfungi on land plants – an identification handbook.
Richmond Publishing, England.
Evidente A, Zonno MC, Andolfi A, et al. (2012). Phytotoxic a-pyrones produced
by Pestalotiopsis guepinii, the causal agent of hazelnut twig blight. The
Journal of Antibiotics 65: 203–206.
Felsenstein J (1985). Confidence limits on phylogenies: an approach using the
bootstrap. Evolution 39: 783–791.
Glass NL, Donaldson GC (1995). Development of primer sets designed for use
with the PCR to amplify conserved genes from filamentous ascomycetes.
Applied and Environmental Microbiology 61: 1323–1330.
Gomes RR, Glienke C, Videira SIR, et al. (2013). Diaporthe: a genus of
endophytic, saprobic and plant pathogenic fungi. Persoonia 31: 1–41.
Gregory PH (1952). Fungus spores. Transactions of the British Mycological
Society 35: 1–18.
Griffiths DA, Swart HJ (1974a). Conidial structure in two species of Pestalotiopsis. Transactions of the British Mycological Society 62: 295–304.
Griffiths DA, Swart HJ (1974b). Conidial structure in Pestalotia pezizoides.
Transactions of the British Mycological Society 63: 169–173.
Guba EF (1956). Monochaetia and Pestalotia vs. Truncatella, Pestalotiopsis and
Pestalotia. Annals of Microbiology 7: 74–76.
Guba EF (1961). Monograph of Pestalotia and Monochaetia. Harvard University
Press, Cambridge.
Hall TA (1999). BioEdit: a user-friendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series
41: 95–98.
Hawksworth DL, Crous PW, Redhead SA, et al. (2011). The Amsterdam
declaration on fungal nomenclature. IMA Fungus 2: 105–112.
Hu HL, Jeewon R, Zhou DQ, et al. (2007). Phylogenetic diversity of endophytic
Pestalotiopsis species in Pinus armandii and Ribes spp.: evidence from
rDNA and β-tubulin gene phylogenies. Fungal Diversity 24: 1–22.
Hughes SJ (1958). Revisiones Hyphomycetum aliquot cum appendice de
nominibus rejiciendis. Canadian Journal of Botany 36: 727–836.
Ismail AM, Cirvilleri G, Polizzi G (2013). Characterisation and pathogenicity of
Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of
mango (Mangifera indica L.) in Italy. European Journal of Plant Pathology
135: 619–625.
Jeewon R, Liew ECY, Hyde KD (2002). Phylogenetic relationships of Pestalotiopsis and allied genera inferred from ribosomal DNA sequences and
morphological characters. Molecular Phylogenetics and Evolution 25:
378–392.
Jeewon R, Liew ECY, Hyde KD (2004). Phylogenetic evaluation of species
nomenclature of Pestalotiopsis in relation to host association. Fungal Diversity 17: 39–55.
Jeewon R, Liew ECY, Simpson JA, et al. (2003). Phylogenetic significance of
morphological characters in the taxonomy of Pestalotiopsis species. Molecular Phylogenetics and Evolution 27: 372–383.
Joshi SD, Sanjay R, Baby UI, et al. (2009). Molecular characterization of
Pestalotiopsis spp. associated with tea (Camellia sinensis) in southern India
using RAPD and ISSR markers. Indian Journal of Biotechnology 8:
377–383.
Kang JC, Hyde KD, Kong RYC (1999). Studies on the Amphisphaeriales. The
Amphisphaeriaceae (sensu stricto). Mycological Research 103: 53–64.
Keith LM, Velasquez ME, Zee FT (2006). Identification and characterization of
Pestalotiopsis spp. causing scab disease of guava, Psidium guajava in
Hawaii. Plant Disease 90: 16–23.
Keith LM, Zee FT (2010). Guava disease in Hawaii and the characterization of
Pestalotiopsis spp. affecting guava. Acta Horticulturae (ISHS) 849: 269–276.
Kishino H, Hasegawa M (1989). Evaluation of the maximum likelihood estimate
of the evolutionary tree topologies from DNA sequence data. Journal of
Molecular Evolution 29: 170–179.
185
MAHARACHCHIKUMBURA
ET AL.
Kumar S, Stecher G, Peterson D, et al. (2012). MEGA-CC: Computing Core of
Molecular Evolutionary Genetics Analysis Program for Automated and
Iterative Data Analysis. Bioinformatics 28: 2685–2686.
Lee S, Crous PW, Wingfield MJ (2006). Pestalotioid fungi from Restionaceae in
the Cape Floral Kingdom. Studies in Mycology 55: 175–187.
Lee S, Groenewald JZ, Crous PW (2004). Phylogenetic reassessment of the
coelomycete genus Harknessia and its teleomorph Wuestneia (Diaporthales), and the introduction of Apoharknessia gen. nov. Studies in
Mycology 50: 235–252.
Liu AR, Chen SC, Wu SY, et al. (2010). Cultural studies coupled with DNA
based sequence analyses and its implication on pigmentation as a phylogenetic marker in Pestalotiopsis taxonomy. Molecular Phylogenetics and
Evolution 57: 528–535.
Maddison WP, Maddison DR (2011). Mesquite: a modular system for evolutionary analysis. Version 2.75. http://mesquiteproject.org.
Maharachchikumbura SSN, Guo LD, Cai L, et al. (2012). A multi-locus backbone
tree for Pestalotiopsis, with a polyphasic characterization of 14 new species.
Maharachchikumbura SSN, Chukeatirote E, Guo LD, et al. (2013a). Pestalotiopsis
species associated with Camellia sinensis (tea). Mycotaxon 123: 47–61.
Maharachchikumbura SSN, Guo LD, Chukeatirote E, et al. (2011). Pestalotiopsis – morphology, phylogeny, biochemistry and diversity. Fungal Diversity 50: 167–187.
Maharachchikumbura SSN, Guo LD, Chukeatirote E, et al. (2013d). Improving
the backbone tree for the genus Pestalotiopsis; addition of P. steyaertii and
P. magna sp. nov. Mycological Progress 13: 617–624.
Maharachchikumbura SSN, Guo LD, Chukeatirote E, et al. (2013b). A destructive
new disease of Syzygium samarangense in Thailand caused by the new
species Pestalotiopsis samarangensis. Tropical Plant Pathology 38: 227–235.
Maharachchikumbura SSN, Zhang YM, Wang Y, et al. (2013c). Pestalotiopsis
anacardiacearum sp. nov. (Amphisphaeriaceae) has an intricate relationship
with Penicillaria jocosatrix, the mango tip borer. Phytotaxa 99: 49–57.
Marincowitz S, Crous PW, Groenewald JZ, et al. (2008). Micro-fungi occurring
on Proteaceae in the fynbos. CBS Biodiversity Series 7: 1–166.
Monden Y, Yamamoto S, Yamakawa R, et al. (2013). First case of fungal keratitis
caused by Pestalotiopsis clavispora. Clinical Ophthalmology 7: 2261–2264.
Mordue JEM (1985). An unusual species of Pestalotiopsis: P. steyaertii sp. nov.
Moreau C (1949). Micomycetes africains. I. Revue de Mycologie, Suppliment
Colonial (Paris) 14: 15–22.
Nag Raj TR (1985). Redisposals and redescriptions in the Monochaetia-Seiridium, Pestalotia-Pestalotiopsis complexes. II. Pestalotiopsis besseyii (Guba)
comb. nov. and Pestalosphaeria varia sp. nov. Mycotaxon 22: 52–63.
Nag Raj TR (1993). Coelomycetous anamorphs with appendage-bearing conidia. Mycologue Publications, Waterloo, Ontario, Canada.
Nylander JAA (2004). MrModeltest v2.2. Program distributed by the author: 2.
Evolutionary Biology Centre, Uppsala University: 1–2.
O'Donnell K, Cigelnik E (1997). Two divergent intragenomic rDNA ITS2 types
within a monophyletic lineage of the fungus Fusarium are nonorthologous.
Molecular Phylogenetics and Evolution 7: 103–116.
O'Donnell K, Kistler HC, Cigelnik E, et al. (1998). Multiple evolutionary origins of
the fungus causing Panama disease of banana: concordant evidence from
nuclear and mitochondrial gene genealogies. Proceedings of the National
Academy of Sciences of United States of America 95: 2044–2049.
Quaedvlieg W, Binder M, Groenewald JZ, et al. (2014). Introducing the
consolidated species concept to resolve species in the Teratosphaeriaceae.
Persoonia 33: 1–40.
Rayner RW (1970). A mycological colour chart. CMI and British Mycological
Society, Kew, UK.
Rehner SA, Samuels GJ (1994). Taxonomy and phylogeny of Gliocladium
analysed from nuclear large subunit ribosomal DNA sequences. Mycological
Research 98: 625–634.
Ren HY, Li G, Qi XJ, et al. (2013). Identification and characterization of Pestalotiopsis spp. causing twig blight disease of bayberry (Myrica rubra Sieb. &
Zucc) in China. European Journal of Plant Pathology 137: 451–461.
Ronquist F, Teslenko M, Mark P van der, et al. (2012). MrBayes 3.2: efficient
Bayesian phylogenetic inference and model choice across a large model
space. Systematic Biology 61: 539–542.
Sangchote S, Farungsang U, Farungsang N (1998). Pre and postharvest
infection of rambutan by pathogens and effect on postharvest treatments. In:
Disease control and storage life extension in fruits (Coates LM, Hofman PJ,
Johnson GI, eds), ACIAR Proceedings, 81: 87–91.
Silvestro D, Michalak I (2011). raxmlGUI: a graphical front-end for RAxML.
Organisms Diversity and Evolution 12: 335–337.
186
Steyaert RL (1949). Contributions a l'etude monographique de Pestalotia de Not.
et Monochaetia Sacc. (Truncatella gen. nov. et Pestalotiopsis gen. nov.).
Bruxelles 19: 285–354.
Bulletin Jardin Botanique Etat
Steyaert RL (1953a). New and old species of Pestalotiopsis. Transactions of the
British Mycological Society 36: 81–89.
Steyaert RL (1953b). Pestalotiopsis from the Gold Coast and Togoland.
Steyaert RL (1955). Pestalotia, Pestalotiopsis et Truncatella. Bulletin Jardin
Botanique Etat
Steyaert RL (1956). A reply and an appeal to Professor Guba. Mycologia 48:
767–768.
Steyaert RL (1961). Type specimens of Spegazzini's collections in the Pestalotiopsis and related genera (Fungi Imperfecti: Melanconiales). Darwinia
(Buenos Aires) 12: 157–190.
Steyaert RL (1963). Complementary informations concerning Pestalotiopsis
guepini (Desmazieres) Steyaert and designation of its lectotype. Bulletin
Jardin Botanique l'Etat
Strobel G, Yang XS, Sears J, et al. (1996). Taxol from Pestalotiopsis microspora,
an endophytic fungus of Taxus wallachiana. Microbiology 142: 435–440.
Sun HT, Cao RB (1990). Identification of Pestalotiopsis parasitized on fruit crops
(in Chinese). Acta Agriculturae University Zhejiangensis 16: 179–185.
Sutton BC (1969). Forest microfungi. III. The heterogeneity of Pestalotia de Not.
section Sexloculatae Klebahn sensu Guba. Canadian Journal of Botany 48:
2083–2094.
Sutton BC (1980). The Coelomycetes. Fungi imperfecti with pycnidia, acervuli
and stromata. Commonwealth Mycological Institute, Kew, Surrey, UK.
Sutton DA (1999). Coelomycetous fungi in human disease. A review: clinical
entities, pathogenesis, identification and therapy. Revista Iberoamericana de
Micología 16: 171–179.
Swart L, Taylor JE, Crous PW, et al. (1999). Pestalotiopsis leaf spot disease of
Proteaceae in Zimbabwe. South African Journal of Botany 65: 239–242.
Swofford DL (2003). PAUP*. Phylogenetic Analysis Using Parsimony (*and other
methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Taylor JE (2001). Proteaceae pathogens: the significance of their distribution in
relation to recent changes in phytosanitary regulations. Acta Horticulturae
545: 253–264.
Taylor JW (2011). One Fungus = One Name: DNA and fungal nomenclature
twenty years after PCR. IMA Fungus 2: 113–120.
Tejesvi MV, Nalini MS, Mahesh B, et al. (2007). New hopes from endophytic fungal
secondary metabolites. Boletín de la Sociedad Química de Mexico 1: 19–26.
Tejesvi MV, Tamhankar SA, Kini KR, et al. (2009). Phylogenetic analysis of
endophytic Pestalotiopsis species from ethnopharmaceutically important
medicinal trees. Fungal Diversity 38: 167–183.
Vilgalys R, Hester M (1990). Rapid genetic identification and mapping of
enzymatically amplified ribosomal DNA from several Cryptococcus species.
Journal of Bacteriology 172: 4239–4246.
Watanabe K, Motohashi K, Ono Y (2010). Description of Pestalotiopsis pallidotheae: a new species from Japan. Mycoscience 51: 182–188.
Watanabe K, Parbery DG, Kobayashi T, et al. (2000). Conidial adhesion and
germination of Pestalotiopsis neglecta. Mycological Research 104: 962–968.
fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to
eds). Academic Press, San Diego, California: 315–322.
Wingfield MJ, De Beer ZW, Slippers B, et al. (2012). One fungus, one name
promotes progressive plant pathology. Molecular Plant Pathology 13:
604–613.
Winter G (1887). Pilze; Ascomyceten. In: Rabenhorst's Kryptogamen-Flora von
Deutschland, Oesterreich und der Schweiz [i–vi] 1(2): 1–928.
Xu J, Ebada SS, Proksch P (2010). Pestalotiopsis a highly creative genus: chemistry
and bioactivity of secondary metabolites. Fungal Diversity 44: 15–31.
Xu L, Kusakari S, Hosomi A, et al. (1999). Postharvest disease of grape caused
by Pestalotiopsis species. Annals of the Phytopathological Society of Japan
65: 305–311.
Xu J, Yang X, Lin Q (2014). Chemistry and biology of Pestalotiopsis-derived
natural products. Fungal Diversity 66: 37–68.
Zhang YM, Maharachchikumbura SSN, McKenzie EHC, et al. (2012a). A novel
species of Pestalotiopsis causing leaf spots of Trachycarpus fortunei.
Cryptogamie Mycologie 33: 1–8.
Zhang YM, Maharachchikumbura SSN, Tian Q, et al. (2013). Pestalotiopsis
species on ornamental plants in Yunnan Province, China. Sydowia 65: 59–74.
Zhang YM, Maharachchikumbura SSN, Wei JG, et al. (2012b). Pestalotiopsis
camelliae, a new species associated with grey blight of Camellia japonica in
China. Sydowia 64: 335–344.
STUDIES
IN
MYCOLOGY 79: 187–219.
Redefining Ceratocystis and allied genera
Z.W. de Beer1*,3, T.A. Duong2,3, I. Barnes2, B.D. Wingfield2, and M.J. Wingfield1
1
Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa; 2Department of Genetics, Forestry and Agricultural Research
Institute (FABI), University of Pretoria, Pretoria 0002, South Africa
*Correspondence: Z.W. de Beer. wilhelm.debeer@fabi.up.ac.za
3
These authors contributed equally to this study.
Studies in Mycology
Abstract: The genus Ceratocystis was established in 1890 and accommodates many important fungi. These include serious plant pathogens, significant insect
symbionts and agents of timber degradation that result in substantial economic losses. Virtually since its type was described from sweet potatoes, the taxonomy of
Ceratocystis has been confused and vigorously debated. In recent years, particulary during the last two decades, it has become very obvious that this genus includes a
wide diversity of very different fungi. These have been roughly lumped together due to their similar morphological structures that have clearly evolved through convergent
evolution linked to an insect-associated ecology. As has been true for many other groups of fungi, the emergence of DNA-based sequence data and associated
phylogenetic inferences, have made it possible to robustly support very distinct boundaries defined by morphological characters and ecological differences. In this study,
DNA-sequence data for three carefully selected gene regions (60S, LSU, MCM7) were generated for 79 species residing in the aggregate genus Ceratocystis sensu lato
and these data were subjected to rigorous phylogenetic analyses. The results made it possible to distinguish seven major groups for which generic names have been
chosen and descriptions either provided or emended. The emended genera included Ceratocystis sensu stricto, Chalaropsis, Endoconidiophora, Thielaviopsis, and
Ambrosiella, while two new genera, Davidsoniella and Huntiella, were described. In total, 30 new combinations have been made. This major revision of the generic
boundaries in the Ceratocystidaceae will simplify future treatments and work with an important group of fungi including distantly related species illogically aggregated
under a single name.
Key words: Ceratocystidaceae, New combinations, Nomenclature, Multigene analyses, Taxonomy.
Taxonomic novelties: New genera: Davidsoniella Z.W. de Beer, T.A. Duong & M.J. Wingf., Huntiella Z.W. de Beer, T.A. Duong & M.J. Wingf; New combinations:
Chalaropsis ovoidea (Nag Raj & W.B. Kendr.) Z.W. de Beer, T.A. Duong & M.J. Wingf., Ch. populi (Kiffer & Delon) Z.W. de Beer, T.A. Duong & M.J. Wingf., Davidsoniella
australis (J. Walker & Kile) Z.W. de Beer, T.A. Duong & M.J. Wingf., D. eucalypti (Z.Q. Yuan & Kile) Z.W. de Beer, T.A. Duong & M.J. Wingf., D. neocaledoniae (Kiffer &
Delon) Z.W. de Beer, T.A. Duong & M.J. Wingf., D. virescens (R.W. Davidson) Z.W. de Beer, T.A. Duong & M.J. Wingf., Endoconidiophora douglasii (R.W. Davidson) Z.W.
de Beer, T.A. Duong & M.J. Wingf., E. fujiensis (M.J. Wingf., Yamaoka & Marin) Z.W. de Beer, T.A. Duong & M.J. Wingf., E. laricicola (Redfern & Minter) Z.W. de Beer,
T.A. Duong & M.J. Wingf., E. pinicola (T.C. Harr. & M.J. Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf., E. polonica (Siemaszko) Z.W. de Beer, T.A. Duong & M.J.
Wingf., E. resinifera (T.C. Harr. & M.J. Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf., E. rufipennis (M.J. Wingf., T.C. Harr. & H. Solheim) Z.W. de Beer, T.A. Duong &
M.J. Wingf., Huntiella bhutanensis (M. van Wyk, M.J. Wingf. & T. Kirisits) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. ceramica (R.N. Heath & Jol. Roux) Z.W. de Beer,
T.A. Duong & M.J. Wingf., H. chinaeucensis (S.F. Chen, M. van Wyk, M.J. Wingf. & X.D. Zhou) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. cryptoformis (Mbenoun & Jol.
Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. decipiens (Kamgan & Jol. Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. inquinans (Tarigan, M. van Wyk & M.J.
Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. microbasis (Tarigan, M. van Wyk & M.J. Wingf) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. moniliformis (Hedgc.)
Z.W. de Beer, T.A. Duong & M.J. Wingf., H. moniliformopsis (Yuan & Mohammed) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. oblonga (R.N. Heath & Jol. Roux) Z.W. de
Beer, T.A. Duong & M.J. Wingf., H. omanensis (Al-Subhi, M.J. Wingf., M. van Wyk & Deadman), Z.W. de Beer, T.A. Duong & M.J. Wingf., H. salinaria (Kamgan & Jol.
Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. savannae (Kamgan & Jol. Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. sublaevis (M. van Wyk & M.J. Wingf.)
Z.W. de Beer, T.A. Duong & M.J. Wingf., H. sumatrana (Tarigan, M. van Wyk & M.J. Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. tribiliformis (M. van Wyk & M.J.
Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf., H. tyalla (Kamgan & Jol. Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf., Thielaviopsis cerberus (Mbenoun, M.J. Wingf. &
Jol. Roux) Z.W. de Beer, T.A. Duong & M.J. Wingf.
Published online 7 November 2014; http://dx.doi.org/10.1016/j.simyco.2014.10.001. Hard copy: September 2014.
INTRODUCTION
Ceratocystis was established in 1890 to accommodate
C. fimbriata, a pathogen causing black rot of sweet potatoes in
the USA (Halsted 1890). The genus now includes many important fungi including important pathogens of plants and the causal
agents of sap stain in timber that are symbiotic associates of
insects (Fig. 1). These fungi have ascomata with round usually
dark bases that are sometimes ornamented. These bases give
rise to long necks terminating in ostiolar hyphae and from which
ascospores exude in slimy masses (Fig. 2). All species have
ascospores surrounded by sheaths, which can be hat-shaped,
ellipsoidal or obovoid and that are either evenly or unevenly
distributed around the spores (Fig. 3). The asexual states of most
species in Ceratocystis are morphologically “chalara”- or “thielaviopsis”-like forms and characterised by simple, tubular conidiogenous cells. These cells, which are phialides, typically taper
towards their apices and produce chains of rectangular conidia
or in some cases dark barrel-shaped secondary conidia (Fig. 3).
Some species produce simple, single-celled or more complex
chlamydospores (Fig. 3) that facilitate a soil-borne life-style.
Since the time of its first discovery, Ceratocystis has been
beset by taxonomic complications and controversy. The first of
these emerged with the description of Ophiostoma in 1919
(Sydow & Sydow 1919). It was set up to accommodate several
Ceratostomella spp., with O. piliferum as type species and
including Ceratostomella moniliformis. Not long thereafter, Melin
& Nannfeldt (1934) disposed additional species in the genus,
187
DE
BEER
ET AL.
Fig. 1. Disease symptoms of plants infected with species of Ceratocystis s.l. A. Eucalyptus wilt in Uruguay caused by C. fimbriata s.l. B. Dying clove trees infected with
C. polychroma in Sulawesi. C. Wilting shoots of Acacia mearnsii in South Africa infected with C. albifundus. D. Ceratocystis wilt of Acacia sp. caused by C. manginecans. E, F.
Wilted shoots and damaged stems of Protea cynaroides in South Africa caused by C. albifundus. G. Resin exudation from the stem of A. mearnsii in South Africa caused by
C. albifundus. H. Fungal mats of C. albifundus on Acacia exuvialis. I. Vascular streaking caused by C. manginecans after wounding. J. Fungal mats of C. albifundus on
A. exuvialis. K. Staining of the wood of Acacia caused by C. albifundus. L. Streaking and stain of mango trees from infections by C. manginecans in Oman. M. Cross section
through a Eucalyptus grandis stump showing streaking caused by C. fimbriata s.l. N. Sweet potato with black rot caused by C. fimbriata s. str. O. Rotted cacao pod infected with
C. ethacetica (now T. ethacetica). P. Ascomata of C. polonica (now E. polonica) in the gallery of the bark beetle Ips typographus.
188
REDEFINING CERATOCYSTIS
AND ALLIED GENERA
189
DE
BEER
ET AL.
including the type species of Ceratocystis, C. fimbriata. These
studies and others (Bakshi 1951, Moreau 1952) resulted in a
long-standing confusion between the two genera. This is largely
because the genera have morphologically similar ascomata
featuring globose bases and generally long necks from which
ascospores exude in slimy masses (Upadhyay 1981). According
to Malloch & Blackwell (1993) the basic construction of the
ascomata may be the result of an adaptation to insect-associated
niches and shows the convergent evolution of fruiting structures
that facilitate insect-borne transport of spores to new environments (Malloch & Blackwell 1993). Interestingly, but adding to the
confusion between them, species of both Ceratocystis and
Ophiostoma have evanescent asci that are seldom seen. Ascospores were confused with conidia when the genera were first
discovered. The fact that both genera include species with hatshaped ascospores re-inforced debate over their relationships
for many years (Van Wyk et al. 1993).
The taxonomic confusion between Ceratocystis and Ophiostoma was finally resolved once DNA sequence data became
available to provide phylogenetic insights into their relatedness.
Hausner et al. (1993a,b) and Spatafora & Blackwell (1994) provided the first phylogenetic trees showing that these genera are
unrelated. A considerable body of evidence has contributed to the
current understanding that Ophiostoma resides in the Ophiostomatales in the Sordariomycetidae and that Ceratocystis is
accommodated in the Ceratocystidaceae (Microascales) in the
Hypocreomycetidae (Reblova et al. 2011, De Beer et al. 2013a).
Importantly, resolution of the taxonomic confusion regarding these
genera has made it possible to study them independently and thus
to better understand their similarities, but also their many very
different ecologies (Seifert et al. 2013).
Once Ceratocystis was clearly recognised as unrelated to
Ophiostoma, an increasingly clear picture emerged of a genus
that included species that were morphologically and ecologically
very distinct from one another. These differences have been
substantially amplified by the discovery of many new and often
cryptic species, revealed through DNA-sequence comparisons
(Wingfield et al. 1996, Witthuhn et al. 1998, Harrington &
Wingfield 1998). For example, perhaps the two best-known
species names within Ceratocystis, C. fimbriata and
C. moniliformis, are now known to represent complexes of many
different species (Van Wyk et al. 2013, Wingfield et al. 2013).
Recognition of these complexes has made it possible to interpret
their very clear differences.
Wingfield et al. (2013) provided the first intensive, phylogenetically based reconsideration of the taxonomy of Ceratocystis.
This study included all available sequence data up to 2006 when
the study was completed, and it clearly exposed five very different
taxonomic groups. These included the species of the C. fimbriata
complex, the C. moniliformis complex, and the C. coerulescens
complex, as well the Thielaviopsis and Ambrosiella complexes,
known only by their asexual states. Importantly, species in these
complexes could easily be separated by their morphological and
ecological differences. The DNA sequence data used merely
reaffirmed the circumscription of the groups. Wingfield et al.
(2013) provided substantial evidence that species in Ceratocystis s. l. should be assigned to discrete genera. They argued that
this would substantially reduce taxonomic confusion among these
very different groups of fungi and importantly, also enhance understanding of their different ecologies.
Wingfield et al. (2013) were not able to place all species of
Ceratocystis s. l. in discrete complexes. Some, such as
C. paradoxa, C. adiposa and C. fagacearum fell away from all
clearly defined species groups. In retrospect, it appears that this
problem stemmed from a lack of sampling and was resolved by
the discovery of additional species that could define complexes
based on these isolated phylogenetic branches. Such a pattern
has become clearly evident from a recent study of a large
collection of isolates that would previously have been identified
as C. paradoxa (Mbenoun et al. 2014a). These isolates have
now been shown to represent a number of very different but
related species that are now recognised as comprising the
C. paradoxa complex. It is, therefore, very likely that other
complexes will emerge in Ceratocystis s. l., as new species are
collected and treated in the future.
Ceratocystis s. l., as it is currently defined includes many
ecologically important fungi (Fig. 1). For example, most species
in the C. fimbriata complex are important and in some cases
devastating plant pathogens (Kile 1993, Wingfield et al. 2013).
These include C. albifundus, a virulent pathogen of Acacia
mearnsii in Africa (Roux & Wingfield 2013), C. cacaofunesta, a
pathogen of cacao in South America (Engelbrecht et al. 2007),
C. platani, an invasive alien pathogen of Platanus trees in
Europe (Gibbs 1981, Ocasio-Morales et al. 2007), and
C. manginecans that has devasted mango (Mangifera indica)
and Acacia mangium trees in the Middle East and south-east
Asia respectively (Van Wyk et al. 2007, Tarigan et al. 2011).
Species in the C. coerulescens complex include associates of
bark beetles (Coleoptera: Scolytinae) as well as important causal
agents of sap-stain in timber (Seifert 1993, Wingfield et al. 1997).
The Thielaviopsis complex includes plant pathogens, while the
Ambrosiella complex comprise obligate associates of ambrosia
beetles (Coleoptera: Scolytinae) (Batra 1967, Kile 1993). Species in the C. moniliformis complex are mostly wound-inhabiting
saprobes or mild pathogens, often causing sap stain in timber
(Hedgcock 1906, Seifert 1993). The members of the C. paradoxa
complex are all pathogens of monocotyledonous plants,
including pineapples and palms (Mitchell 1937, Alvarez et al.
2012, Mbenoun et al. 2014a).
All available evidence shows that Ceratocystis s. l. represents
a suite of morphologically, phylogenetically and ecologically
different fungi. There is no reasonable argument for retaining
them in a unitary genus, and indeed, doing so would result only
in confusion arising from a diminished lack of appreciation of
their dramatic differences. Placing them in discrete genera will
enhance the perception of opportunities to understand these
organisms and, where applicable, to manage or conserve them.
It will provide an improved interpretive framework for analysing
Fig. 2. Morphological features of the ascomata of species of Ceratocystis s.l. A, B. Ascomata of C. albifundus and C. fimbriata respectively, on woody substrates with masses of
ascospores emerging from their necks. C–E. Ascomata showing different morphological features such as light-coloured bases of C. albifundus (CMW4059), pear-shaped
ascomatal bases characteristic of C. pirilliformis (CMW6579), ornamented bases and divergent necks of C. cerberus (now T. cerberus) (CMW 36668). F, G. Apices of
ascomata showing a range of forms of ostiolar hyphae such as long, divergent ostiolar hyphae of C. ethacetica (CMW 36671) (now T. ethacetica) and short, convergent ostiolar
hyphae of C. inquinans (now H. inquinans) (CMW 21106). H, I. Hat-shaped ascospores being released from ostiolar hyphae in C. sumatrana (now H. sumatrana) (CMW 21113)
and C. pirilliformis (CMW 6670). J. Bases of ascomata in the C. moniliformis s.l. complex (now Huntiella) with distinct plates at the bases of the ascomatal necks, and (K, L)
spine-like ornamentations of H. microbasis (CMW 21117) and H. oblonga (CMW 23803) respectively. M. Digitate ornamentations on the ascomatal bases in species residing in
C. paradoxa s.l. (now Thielaviopsis) (CMW 36642).
190
AND ALLIED GENERA
Fig. 3. Sexual and asexual spores in Ceratocystis s.l. A–D. A range of ascospore shapes all with hyaline sheaths and including those that are fusoid [e.g. C. eucalypti (now
D. eucalypti), photo from Kile et al. 1996], hat-shaped (e.g. C. fimbriata, CMW 15049), oblong (e.g. C. paradoxa, now T. paradoxa, CMW 36642) and obovoid (e.g. C. laricicola,
now E. laricicola, CMW 20928). E–H. Simple tubular conidiophores commonly tapering to their apicies, and found in most species of Ceratocystis s.l. E. Flasked-shaped
phialidic conidiophores of T. paradoxa (CMW 36642) releasing obovoid secondary conidia. F. Phialide releasing cylindrical conidia of C. pirilliformis (CMW 6670). G. Chlamydospore of T. basicola (CMW 7068) and H. C. pirilliformis (CMW 6670). I–L. Darkly pigmented, thick-walled aleurioconidia of (I) T. paradoxa (CMW 36642), (J) T. euricoi
(CMW 28537), (K) T. punctulata (CMW 26389) and (L) T. ethacetica (CMW 36671). M, N. Cylindrical and barrel-shaped conidia of C. pirilliformis (CMW 6670). O. Oblong
secondary conidia of T. ethacetica (CMW 36671). P. Secondary conidia of T. punctulata (CMW 26389).
191
DE
BEER
ET AL.
the ecological differences among the species, such as differences in pathogenicity and insect associations, particularly when
complete genome sequences become available for these fungi,
as they have recently done for C. fimbriata s. str., C. moniliformis
s. str. and C. manginecans (Wilken et al. 2013, Van der Nest
et al. 2014).
Revising Ceratocystis s. l. and providing genera to accommodate the well-defined groups in this aggregate genus must be
done in conformity with the principles of the new International
Code for algae, fungi and plants (Melbourne Code) adopted at
the 18th International Botanical Congress (McNeill et al. 2012).
Importantly, this must reflect the One Fungus One Name (1F1N)
principles that originally emerged from the Amsterdam Declaration (Hawksworth et al. 2011 ) and subsequent discussions
(Hawksworth 2011, Norvell 2011, Wingfield et al. 2012). In this
regard, De Beer et al. (2013b) listed six genus names as
possible synonyms of Ceratocystis s. l. One of these names
belongs to a sexual genus Endoconidiophora, originally
described for E. coerulescens (Münch 1907). The five other
names were all considered to denote asexual genera under the
dual nomenclature system: they included Thielaviopsis (Went
1893, type species T. ethacetica), Chalaropsis (Peyronel 1916,
type species Ch. thielavioides), Hughesiella (Batista & Vital
1956, type species Hu. euricoi), Ambrosiella (Von Arx &
Hennebert 1965, type species A. xylebori), and Phialophoropsis (Batra 1967, type species Ph. trypodendri). These names
are available for new generic circumscriptions accommodating
groups currently residing in Ceratocystis s. l.
The major aim of this study was to revise the generic
boundaries for species currently accommodated in Ceratocystis
s. l. This task involved obtaining material from as many species
as possible and applying 1F1N principles. Generating the full
genome sequences for 19 species including representatives of
all the phylogenetic groups in Ceratocystis s. l. provided the
opportunity to screen multiple gene regions to address genuslevel questions. In addition, gene regions from the AFTOL
project (Lutzoni et al. 2004, Hibbett et al. 2007), the ITS barcoding initiative (Schoch et al. 2012), as well as additional barcoding genes from an ongoing project at CBS (Stielow et al.
2014) were used to design Microascales-specific primers and
to select the most appropriate gene regions to clearly resolve
generic boundaries for Ceratocystis s. l.
freeze-dried in 2 mL Eppendorf tubes. The freeze-dried mycelium was submerged in liquid nitrogen, followed by pulverising
the mycelium with a pipette tip. About 10 mg of mycelial “powder”
was used for DNA extraction using PrepMan Ultra Sample
Preparation reagent (Applied Biosystems, Foster City, California)
as described in Duong et al. (2012).
Selection of gene regions and primers
Ten different gene regions [the nuclear ribosomal DNA large
subunit (LSU), the nuclear ribosomal DNA small subunit (SSU),
nuclear ribosomal DNA internal transcribed spacer regions (ITS),
the 60S ribosomal protein RPL10 (60S), beta-tubulin (BT),
translation elongation factor 1-alpha (EF1), translation elongation
factor 3-alpha (EF3), mini-chromosome maintenance complex
component 7 (MCM7), the RNA polymerase II largest subunit
(RPB1), and the RNA polymerase II second largest subunit
(RPB2)] were extracted from 19 Ceratocystis draft genome sequences that included species from all the major clades. The
genome sequences, of which three have been published (Wilken
et al. 2013, Van der Nest et al. 2014), are available at the
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria. Phylogenetic analyses were conducted with
all ten gene regions (data not shown). LSU, 60S, and MCM7
were selected as candidate genes for further investigation
including all the isolates in the study, based on their level of
support at the basal nodes, the ease of amplification and
sequencing, and the popularity of their use in studies of other
fungal lineages.
The ITS region has been widely used in phylogenetic studies
to distinguish between species in Ceratocystis. However, due to
the recent discovery of multiple ITS forms in certain species of
Ceratocystis (Al Adawi et al. 2013, Naidoo et al. 2013), and the
fact that gene regions were chosen that were slightly more
conserved to resolve the genus level questions, the ITS was
intentionally not used in the present study.
Primers LR0R and LR5 (Vilgalys & Hester, 1990) were used
in PCR amplification and sequencing of LSU. Primers
Algr52_412-433_f1 and Algr52_1102_1084_r1 (Stielow et al.
2014) were used for PCR amplification and sequencing of
60S. Based on the sequences obtained from genomes, new
primers Cer-MCM7F (ACICGIGTITCIGAYGTNAAGCC) and
Cer-MCM7R
(TTRGCAACACCAGGRTCACCCAT)
were
designed and used in PCR amplification and sequencing of
MCM7.
Cultures
PCR and sequencing
All cultures used in this study were obtained from the Culture
Collection of the Forestry and Agricultural Biotechnology Institute
(FABI), University of Pretoria, South Africa (CMW) and Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands
(CBS). Single spore or single hyphal-tip cultures were prepared
and maintained on 2 % Malt Extract Agar (MEA). A list of isolates
used in this study is presented in Table 1.
All PCR reactions were done in a total volume of 25 μL. The
reaction mixture consisted of 2.5 μL of 10X PCR reaction buffer,
2.5 mM MgCl2, 200 μM of each dNTP, 0.2 μM of each of the
forward and reverse primers for LSU (1 μM of each primer in
case of degenerate primers for 60S and MCM7), 1 U FastStart
Taq DNA Polymerase (Roche) and 2 μL of genomic DNA solution. The PCR thermal conditions included an initial denaturation
at 96 °C for 5 min, followed by 35 cycles of 95 °C for 30 sec,
55 °C for 30 s, and 72 °C for 60 s, and ended with a final
extension at 72 °C for 8 min. The annealing temperature was set
at 55 °C for all gene regions and all isolates at first. In some
cases where the PCR failed or non-specific amplification was
observed, we experimented with different annealing
DNA extraction
Single spore/single hyphal-tip cultures were inoculated in YM
broth (2 % malt extract, 0.2 % yeast extract) and incubated at
25 °C with shaking for 2–5 d. Mycelium was harvested and
192
C. ecuadoriana
T. ethacetica
C. corymbiicola
C. corymbiicola
C. ecuadoriana
C. colombiana
C. colombiana
C. ethacetica
Endoconidiophora
coerulescens
C. coerulescens
E. douglasii
H. chinaeucensis
C. chinaeucensis
C. douglasii
Thielaviopsis
cerberus
C. cerberus
C. diversiconidia
C. caryae
C. caryae
C. diversiconidia
Ecuador
C. cacaofunesta
C. cacaofunesta
C. curvata
Huntiella
bhutanensis
C. bhutanensis
H. decipiens
C. atrox
C. atrox
C. curvata
C. albifundus
C. albifundus
C. decipiens
Australia
C. adiposa
C. adiposa
Malaysia
Ecuador
USA
Ecuador
South Africa
Colombia
Germany
China
Cameroon
USA
Ecuador
Bhutan
Australia
South Africa
Japan
Indonesia
Ivory Coast
Germany
Germany
A. xylebori
A. hartigii
A. hartigii
Ceratocystis
acaciivora
A. ferruginea
A. ferruginea
USA
Ceratocystis acaciivora
Ambrosiella
beaveri
Ambrosiella beaveri
Country
A. xylebori
New name
Previous name
Ananas comosus
Eucalyptus deglupta
Pseudotsuga taxifolia
Terminalia ivorensis
Eucalyptus saligna
Eucalyptus deglupta
Eucalyptus pilularis
Coffea arabica
Picea abies
Eucalyptus grandis
x E. urophylla
Elaeis guineensis
Carya ovata
Theobromae cacao
Picea spinulosa
Eucalyptus grandis
Acacia mearnsii
Saccharum officiarum
Acacia mangium
Coffea canephora
Acer sp.
Fagus sylvatica
Vitus rotundifolia
Host/substrate
Table 1. Isolates used in the phylogenetic analyses in this study.
A. Johnson; 1952
M.J. Wingfield; 2004
R.W. Davidson; 1951
G. Kamgan Nkuekam &
J. Roux; 2008
G. Kamgan Nkuekam;
2008
M. Marin; 2000
T. Rohde; 1937
M.J. Wingfield &
S.F. Chen; 2006
M. Mbenoun &
J. Roux; 2010
J.A. Johnson; 2001
T.C. Harrington; 2000
T. Kirisits &
D.B. Chhetri; 2001
J. Roux; 1997
T. Miyake; 1934
CMW 4068; CBS 128992
PREM 60961
PREM 60155
BPI 595613
= FP 70703
PREM 60160
PREM 60560
PREM 60154
PREM 60433
CMW 37775; IMI 50560; MUCL 2170
CMW 22092; CBS 124020
CMW 26367; CBS 556.97
CMW 22445; CBS 123013
CMW 30855; CBS 129736
CMW 22432
CMW 29349; CBS 127216
CMW 5751; CBS 121792
CMW 26365; CBS 140.37;
MUCL 9511; C 313; C 695
–
PREM 59434
CMW 24658; CBS 127185
PREM 60735
CMW 36668; CBS 130765
CMW 14808; CBS 115168; C 1827
–
PREM 60770
CMW 14803; CBS 115163; C 1695
CMW 8217; CBS 114289
BPI 843731
PREM 57804
CMW 19385; CBS 120518
–
PREM 59012
CMW 2573; CBS 136.34
CMW 25531; CBS 110.61
–
M. Tarigan; 2005
L. Brader; 1961
–
CMW 25525; CBS 403.82
–
– ; 1970
CMW 22563
CMW 25522; CBS 460.82
–
G. Zimmerman; 1971
PREM 59884
CMW 26179; CBS 121753; DLS 1624
–
D. Six; 2005
Culture collection number(s)1
Herbarium
Specimen1
Collector;
collection year
ex-epitype
ex-holotype
ex-holotype
ex-holotype
ex-holotype
ex-paratype
ex-paratype
ex-holotype
not type
ex-holotype
ex-holotype
original
collection
original
collection
ex-holotype
ex-holotype
not type
not type
ex-holotype
ex-isotype
not type
not type
ex-paratype
Strain
status
KM495514
KM495513
KM495512
KM495511
KM495510
KM495509
KM495508
KM495507
KM495506
KM495504
KM495503
KM495502
KM495501
KM495500
KM495499
KM495498
KM495497
KM495496
KM495495
KM495494
KM495493
KM495492
60S
KM495426
KM495425
KM495424
KM495423
KM495422
KM495421
KM495420
KM495419
KM495418
KM495416
KM495415
KM495414
KM495413
KM495412
KM495411
KM495410
KM495409
KM495408
KM495407
–
KM495406
KM495405
MCM7
KM495337
KM495336
KM495335
KM495334
KM495333
KM495332
KM495331
KM495330
KM495329
KM495327
KM495326
KM495325
KM495324
KM495323
KM495322
KM495321
KM495320
KM495319
KM495318
KM495317
KM495316
KM495315
LSU
GenBank accession
numbers2
AND ALLIED GENERA
193
194
C. neglecta
H. oblonga
C. obpyriformis
H. omanensis
C. papillata
C. obpyriformis
C. omanensis
C. papillata
T. musarum
C. neglecta
H. moniliformopsis
C. moniliformopsis
C. musarum
C. oblonga
H. microbasis
H. moniliformis
C. mangivora
C. mangivora
C. moniliformis
C. manginecans
C. manginecans
C. microbasis
C. mangicola
C. mangicola
E. laricicola
C. laricicola
C. larium
H. inquinans
C. inquinans
C. adiposa
C. harringtonii
C. harringtonii
(= C. populicola)
C. major
E. fujiensis
C. fujiensis
C. larium
C. fimbriatomima
C. fimbriatomima
Japan
C. ficicola
C. fimbriata
C. ficicola
C. fagacearum
C. fagacearum
C. fimbriata
USA
C. eucalypticola
C. eucalypticola
Colombia
Oman
South Africa
South Africa
Colombia
New Zealand
Australia
South Africa
Indonesia
Brazil
Oman
Brazil
Netherlands
Indonesia
UK
Indonesia
Netherlands
Japan
Venezuela
USA
South Africa
Citrus x Tangelo
hybrid
Mangifera indica
Acacia mearnsii
Acacia mearnsii
Eucalyptus grandis
Musa sp.
Eucalyptus obliqua
Eucalyptus grandis
Acacia mangium
Mangifera indica
Prosopis cineraria
Mangifera indica
Air
Styrax benzoin
Larix decidua
Acacia mangium
Populus hybrid
Larix kaempferi
Eucalyptus hybrid
Ipomoea batatas
Ficus carica
Quercus rubra
Eucalyptus sp.
Eucalyptus sieberi
Host/substrate
CMW 17570; CBS 138185
B. Castro; 2001
A. Al Adawi &
M. Deadman; 2003
R.N. Heath; 2006
R.N. Heath; 2006
CMW 8856; CBS 121793
CMW 11056; CBS 118113
PREM 59438
CMW 23808; CBS 122511
–
CMW 23803; CBS 122291
CMW 17808; CBS 121789
CMW 1546; C 907
CMW 9986; CBS 109441
PREM 59796
PREM 59792
PREM 59616
PREM 60962
C. Rodas & J. Roux; 2004
DAR 74608
–
T.W. Canter-Visscher; –
CMW 21117
CMW 10134; CBS 118127
PREM 59872
CMW 27305; CBS 128702
–
PREM 60570
CMW 28908; CBS 127210
CMW 3189; CBS 138.34;
ATCC 11932; MUCL 9518
PREM 60185
CMW 25434; CBS 122512
–
CMW 20928; CBS 100207;
C 181; Redfern 56-10
–
PREM 60193
CMW 21106; CBS 124388
CMW 14789; CBS 119.78; C 995
–
PREM 59866
CMW 1955; CBS 100208; JCM 9810
CMW 24174; CBS 121786
CMW 15049; CBS 141.37
PREM 57513
PREM 59439
–
CMW 38543; MAFF 625119
CMW 2656; C463
–
NIAES 20600
CMW 11536; CBS 124016
CMW 3254; C 639
PREM 60168
DAR 70205
Herbarium
Specimen1
Z.Q. Yuan; 2001
M. van Wyk; 2002
M. Tarigan; 2005
C.J. Rosetto; 2001
A. Al Adawi; 2005
C.J. Rosetto; 2008
F.H. van Beyma; 1934
D. Redfern; 1983
M. Tarigan; 2005
J. Gremmen; 1978
M.J. Wingfield &
Y. Yamaoka; 1997
C.F. Andrus; 1937
Y. Kajitani; 1990
S. Seegmuller; 1991
M. van Wyk &
J. Roux; 2002
M.J. Dudzinski; 1989
Collector;
collection year
ex-holotype
original
collection
ex-holotype
ex-holotype
ex-holotype
ex-epitype
ex-holotype
not type
ex-holotype
ex-holotype
not type
ex-paratype
ex-holotype
ex-holotype
ex-paratype
ex-holotype
original
collection
ex-holotype
ex-holotype
not type
ex-holotype
not type
ex-holotype
ex-holotype
Strain
status
KM495539
KM495538
KM495537
KM495536
KM495535
KM495534
KM495533
KM495532
KM495531
KM495530
KM495529
KM495528
KM495527
KM495526
KM495525
KM495524
KM495523
KM495522
KM495521
KM495520
KM495519
KM495518
KM495516
KM495515
60S
KM495450
KM495449
KM495448
KM495447
KM495446
KM495445
KM495444
KM495443
KM495442
KM495441
KM495440
KM495439
KM495438
–
KM495437
KM495436
KM495435
KM495434
KM495433
KM495432
KM495431
KM495430
KM495428
KM495427
MCM7
KM495362
KM495361
KM495360
KM495359
KM495358
KM495357
KM495356
KM495355
KM495354
KM495353
KM495352
KM495351
KM495350
KM495349
KM495348
KM495347
KM495346
KM495345
KM495344
KM495343
KM495342
KM495341
KM495339
KM495338
LSU
GenBank accession
numbers2
BEER
Australia
Davidsoniella
eucalypti
C. eucalypti
Country
New name
Previous name
DE
ET AL.
USA
Ecuador
South Africa
Indonesia
C. polyconidia
T. punctulata
E. resinifera
E. rufipennis
H. salinaria
H. savannae
C. smalleyi
H. sublaevis
H. sumatrana
C. tanganyicensis
C. thulamelensis
H. tribiliformis
C. tsitsikammensis
H. tyalla
C. variospora
D. virescens
C. radicicola
C. resinifera
C. rufipennis
C. salinaria
C. savannae
C. smalleyi
C. sublaevis
C. sumatrana
C. tanganyicensis
C. thulamelensis
C. tribiliformis
C. tsitsikammensis
C. tyalla
C. variospora
C. virescens
USA
USA
Australia
South Africa
Tanzania
Indonesia
South Africa
South Africa
Canada
Norway
USA
South Africa
Indonesia
Norway
USA
C. polyconidia
C. platani
C. platani
Australia
E. polonica
C. pirilliformis
C. pirilliformis
UK
C. polychroma
E. pinicola
C. pinicola
Cameroon
C. polychroma
T. paradoxa
C. paradoxa
Country
C. polonica
New name
Previous name
Acer saccharum
Quercus alba
Eucalyptus dunnii
Rapanea melanophloeos
Pinus merkusii
Colophospermum
mopane
Acacia mearnsii
Acacia mangium
Terminalia ivorensis
Carya cordiformis
Acacia nigrescens
Eucalyptus maculata
Picea engelmannii
Picea abies
Phoenix dactylifera
Acacia mearnsii
Syzygium aromaticum
Picea abies
Platanus occidentalis
Eucalyptus nitens
Pinus sylvestris
Theobromae cacao
Host/substrate
D. Houston; 1987
J.A. Johnson; 2001
G. Kamgan Nkuekam &
A.J. Carnegie; 2008
G. Kamgan Nkuekam;
2005
M. Mbenoun &
J. Roux; 2010
R.N. Heath &
J. Roux; 2004
M. Tarigan; 2005
E. Smalley; 1993
G. Kamgan Nkuekam &
J. Roux; 2005
G. Kamgan Nkuekam;
2007
H. Solheim; 1992
CMW 20935; CBS 114715; C 1843
CMW 17339; CBS 130772; C 261
–
CMW 28932; CBS 128703
–
BPI 843737
CMW 14276; CBS 121018
CMW 13013; CBS 115866
PREM 59424
PREM 57827
CMW 35972; CBS 131284
CMW 15999; CBS 122294
–
PREM 60828
CMW 21109; CBS 124011
CMW 22449; CBS 122517
CMW 14800; CBS 114724; C 684
CMW 17300; CBS 121151
PREM 59868
PREM 60163
BPI 843722
PREM 59423
CMW 25911; CBS 129733
CMW 11661
PREM 60557
CMW 20931; CBS 100202; C 662
–
CMW 1032; CBS 114.47; MUCL 9526
CMW 23809; CBS 122289
CMW 11424; CBS 115778
DAOM 225449
BPI 596268
H. Solheim; 1986
PREM 59788
D.E. Bliss; –
PREM 57818
CMW 20930; CBS 100205; C791
CMW 14802; CBS 115162; C 1317
–
DAOM 225451
CMW 6579; CBS 118128
CMW 29499; CBS 100199;
C 488; DAOM 225447
CMW 36689; CBS 130761
PREM 57323
DAOM 225447
PREM 60766
Herbarium
Specimen1
R.N. Heath; 2006
E.C.Y. Liew; 2002
H. Solheim; 1990
T.C. Harrington; 1998
J. Gibbs; 1988
M. Mbenoun &
J. Roux; 2010
Collector;
collection year
not type
ex-paratype
ex-holotype
ex-holotype
ex-holotype
ex-holotype
ex-paratype
ex-paratype
ex-paratype
ex-holotype
ex-holotype
ex-holotype
original
collection
ex-holotype
ex-holotype
ex-holotype
ex-holotype
ex-neotype
original
collection
ex-holotype
ex-holotype
ex-epitype
Strain
status
KM495562
KM495561
KM495560
KM495559
KM495558
KM495557
KM495556
KM495555
KM495554
KM495553
KM495552
KM495551
KM495550
KM495549
KM495548
KM495546
KM495545
KM495544
KM495543
KM495542
KM495541
KM495540
60S
KM495472
KM495471
KM495470
KM495469
KM495468
KM495467
KM495466
KM495465
KM495464
KM495463
KM495462
KM495461
–
KM495460
KM495459
KM495457
KM495456
KM495455
KM495454
KM495453
KM495452
KM495451
MCM7
KM495385
KM495384
KM495383
KM495382
KM495381
KM495380
KM495379
KM495378
KM495377
KM495376
KM495375
KM495374
KM495373
KM495372
KM495371
KM495369
KM495368
KM495367
KM495366
KM495365
KM495364
KM495363
LSU
GenBank accession
numbers2
AND ALLIED GENERA
195
196
New name
C. zambeziensis
Chalaropsis sp. 1
Chalaropsis sp. 1
Graphium fabiforme
G. fimbriisporum
G. laricis
G. pseudormiticum
H. chlamydoformis
nom. prov.
H. pycnanthi
nom. prov.
Knoxdaviesia capensis
K. cecropiae
K. proteae
K. serotectus
K. ubusi
D. australis
T. basicola
H. ceramica
T. euricoi
D. neocaledoniae
Previous name
C. zambeziensis
Chalaropsis sp. 1
Chalaropsis sp. 1
Graphium fabiforme
G. fimbriisporum
G. laricis
G. pseudormiticum
Huntiella chlamydoformis
nom. prov.
H. pycnanthi nom. prov.
Knoxdaviesia capensis
K. cecropiae
K. proteae
K. serotectus
K. ubusi
Thielaviopsis australis
T. basicola
T. ceramica
T. euricoi
T. neocaledoniae
New Caledonia
Brazil
Malawi
Netherlands
Australia
South Africa
South Africa
South Africa
Costa Rica
South Africa
Cameroon
Cameroon
South Africa
Austria
France
Madagascar
USA
Belgium
Coffea robusta
Air
Eucalyptus grandis
Lathyrus odoratus
Nothofagus cunninghamii
Insect tunnels in
Euphorbia tetragona
CMW 22738; CBS 130.39;
C 1378; MUCL 9540; RWD E-1
–
R. Dadant; 1948
E.A.F. da Matta; 1956
R.N. Heath &
J. Roux; 2004
CMW 28537; CBS 893.70;
MUCL 1887; UAMH 1382
CMW 3270; CBS 149.83; C 694
–
CMW 15245; CBS 122299; CMW 15251
URM 640
PREM 59808
–
G.A. van Arkel; –
CMW 7068; CBS 413.52
CMW 2333
–
M. Hall; 2001
CMW 36767; CBS 129738
CMW 738; CBS 486.88
CMW 36769; CBS 129742
PREM 60566
PREM 48924
CMW 22991; CCF 3565
CMW 997; CBS 120015
–
PRM 858080
CMW 36916; CBS 131672
CMW 36932; CBS 131674
CMW 503
CMW 5601; CBS 116194;
DAOM 229757; IFFF ICL/MEA/13
CMW 5605; CBS 870.95; MPFN 281-8
PREM 60835
PREM 60837
PREM 51539
DAOM 229757
PFN 1494
CMW 30626; CBS 124921
CMW 22737; CBS 180.75
–
PREM 60310
CMW 35963; CBS 131282
PREM 60826
Herbarium
Specimen1
PREM 60568
J. Roux; 2010
J.A. van der Linde &
J. Roux; 2009
L.J. Strauss; 1985
Protea repens flower
infested with insects
Grow on insect
(Cossonus sp.) found
in Euphorbia ingens
L. Kirkendall &
J. Hulcr; 2005
M. Mbenoun; 2009
M. Mbenoun &
J. Roux; 2009
T. Kirisits &
P. Baier; 1995
M. Morelet; 1992
J. Roux &
R.W. Davidson; 1939
R. Veldeman; 1975
M. Mbenoun &
J. Roux; 2010
Collector;
collection year
Cecropia angustifolia
Protea longifolia
Theobromae cacao
Theobromae cacao
Pinus sp.
Synnemata occuring in
galleries of the bark
beetle Ips cembrae
Ips typographus gallery,
in stump of Picea abies
Dead Adansonia
rubrostipa
Ulmus sp.
Populus sp.
Acacia nigrescens
Host/substrate
ex-holotype
ex-holotype
ex-holotype
not type
not type
ex-holotype
ex-holotype
ex-holotype
ex-holotype
not type
ex-holotype
ex-holotype
ex-holotype
ex-holotype
ex-holotype
ex-holotype
not type
not type
ex-paratype
Strain
status
KM495576
KM495517
KM495575
KM495574
KM495573
KM495572
KM495571
KM495570
KM495569
KM495568
KM495547
KM495505
KM495567
KM495566
KM495565
KM495564
KM495581
KM495580
KM495563
60S
KM495486
KM495429
KM495485
KM495484
KM495483
KM495482
KM495481
KM495480
KM495479
KM495478
KM495458
KM495417
KM495477
KM495476
KM495475
KM495474
KM495491
KM495490
KM495473
MCM7
KM495399
KM495340
KM495398
KM495397
KM495396
KM495395
KM495394
KM495393
KM495392
KM495391
KM495370
KM495328
KM495390
KM495389
KM495388
KM495387
KM495404
KM495403
KM495386
LSU
GenBank accession
numbers2
BEER
South Africa
Country
DE
ET AL.
1
ATCC: American Type Culture Collection, Virginia, U.S.A.; BPI: US National Fungus Collections, Systematic Botany and Mycology Laboratory, Maryland, U.S.A.; C: Culture collection of T.C. Harrington, Iowa State University, U.S.A.; CBS: Culture
collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CCF: Culture Collection of Fungi, Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic; CMW: Culture collection Forestry and
Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; DAR: New South Wales, Plant Pathology Herbarium, Australia; DLS:
Culture collection of D. Six, University of Montana, U.S.A.; FP: Rocky Mountain Forest & Range Experimental Station Herbarium, Fort Collins, Colorado, U.S.A.; IFFF: Culture collection of the Institute of Forest Entomology, Forest Pathology and Forest
Protection (IFFF), University of Natural Resources and Applied Life Sciences, Vienna (BOKU), Vienna, Austria; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, United Kingdom; JCM: Japan Collection of Microorganism, RIKEN BioResource Center, Japan; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; MPFN: Culture collection at the Laboratoire de Pathologie Forestiere, INRA, Centre de Recherches de Nancy, 54280
Champenoux, France; MUCL: Universite Catholique de Louvain, Louvain-la-Neuve, Belgium; NIAES: National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, 305-8604, Japan; PREM: National Collection of Fungi, Pretoria, South
Africa; PRM: Corda Herbarium, Prague, Czech Republic; Redfern: Culture Collection of D.B. Redfern, Forestry Commission, Northern Research Station, Roslin, Midlothian, UK; RWD: Culture collection of R.W. Davidson, Department of Forest and
Wood Sciences, Colorado State University, Fort Collins, Colorado; UAMH: University of Alberta Microfungus Collection and Herbarium, Edmonton, Alberta, Canada; URM: Father Camille Torrend Herbarium-URM (previously University of Recife
Herbarium), Department of Mycology, Universidade Federal de Pernambuco, Recife, Brazil.
2
60S: partial 60S ribosomal protein RPL10 gene; LSU: partial nuclear ribosomal DNA large subunit (28S); MCM7: partial mini-chromosome maintenance complex component 7 gene.
KM495488
KM495489
KM495401
KM495402
KM495578
KM495579
CMW 22732; CBS 136.88
CMW 22736; CBS 148.37; MUCL 6235
–
–
Quercus petraea
Lupinus albus
Ch. ovoidea
Ch. thielavioides
T. ovoidea
T. Thielavioides
Germany
Italy
H. Kleinhempel; 1987
R. Ciferri; 1937
not type
not type
KM495487
KM495400
KM495577
CMW 22733; CBS 354.76; C 1375
–
Firewood
Chalaropsis
ovoidea
T. ovoidea
Netherlands
W. Gams; 1976
not type
MCM7
LSU
60S
Herbarium
Specimen1
Collector;
collection year
Host/substrate
Country
New name
Previous name
Strain
status
GenBank accession
numbers2
AND ALLIED GENERA
temperatures (between 52 °C and 60 °C) until successful
amplification was obtained. Direct sequencing of PCR products
was done using BigDye® Terminator v. 3.1 Cycle Sequencing kit
(Applied Biosystems) with a 1/16 reaction and at 55 °C annealing
temperature for all primers. Sequencing PCR products were
precipitated using the sodium acetate and ethanol precipitation
protocol, followed by fragment separation using an ABI PRISM®
3100 Genetic Analyzer (Applied Biosystems).
Sequences from different gene regions were aligned using an
online version of MAFFT v. 7 (Katoh & Standley 2013). The three
gene regions (LSU, 60S and MCM7) were combined and analysed as a single dataset. Each of the gene regions was also
analysed separately and results were compared with those of the
combined analyses. Maximum parsimony (MP) analyses were
performed in MEGA6 (Tamura et al. 2013) with 1000 bootstrap
replications. The subtree-pruning-regrafting (SPR) algorithm was
selected, and alignment gaps and missing data included.
Maximum likelihood (ML) analyses were done using raxmlGUI
(Silvestro & Michalak 2012) with the GTR+G+I substitution
model selected. Ten parallel runs with four threads and 1000
bootstrap replications were conducted. Bayesian inference (BI)
analyses were performed using MrBayes v. 3.2 (Ronquist et al.
2012) employing the GTR+G+I substitution model. Ten parallel
runs, each with four chains, were conducted. Trees were
sampled at every 100th generation for 5 M generations. After
sampling, 25 % of trees were discarded as a burn-in phase and
posterior probabilities were calculated from all the remaining
trees.
Morphology
Morphological descriptions from the protologues of all species
treated in this study were carefully considered when genera were
redefined. Based on these species descriptions, the most
common characters of all species in a genus were selected and
incorporated in the emended and new genus descriptions. Over
time, different authors often used different terminology describing
similar characters. We aligned the generic descriptions of the
different genera with each other using similar terminology.
RESULTS
Maximum likelihood, BI and MP trees obtained from analyses of
the individual gene regions (Figs 4–6) and the combined datasets (Fig. 7) of the LSU, 60S and MCM7 sequences, consistently
resulted in nine well-supported major lineages. Although trees
derived from individual datasets had different topologies (Figs
4–6), they were not significantly incongruent with the trees obtained from the combined analyses (Fig. 7). This was indicated
by the fact that most major lineages found in the combined
analyses were present in trees resulting from individual datasets.
Only few exceptions were observed in the cases of 60S and LSU
datasets. In one exceptional case, the 60S dataset (Fig. 5)
showed Lineage 6 as split into two clades. In another case, the
LSU tree (Fig. 4) depicted lineage 5 as not being monophyletic,
although isolates belonging to this lineage still grouped relatively
197
DE
BEER
ET AL.
Fig. 4. Bayesian phylogram derived from analyses of the aligned LSU dataset containing 898 characters, of which 164 were parsimony informative. Thick branches represent
BI posterior probabilities 95 %. Bootstrap support values 70 % are indicated at nodes as MP/ML. * = no bootstrap support or bootstrap support values <70 %.
198
AND ALLIED GENERA
Fig. 5. RAxML phylogram derived from analyses of the aligned 60S dataset containing 711 characters, of which 258 were parsimony informative. Thick branches represent BI
posterior probabilities 95 %. Bootstrap support values 70 % are indicated at nodes as MP/ML. * = no bootstrap support or bootstrap support values <70 %.
199
DE
BEER
ET AL.
Fig. 6. Bayesian phylogram derived from analyses of MCM7 dataset containing 628 characters, of which 313 were parsimony informative. Thick branches represent BI
posterior probabilities 95 %. Bootstrap support values 70 % are indicated at nodes as MP/ML. * = no bootstrap support or bootstrap support values <70 %.
200
AND ALLIED GENERA
Fig. 7. Bayesian phylogram derived from analyses of the concatenated dataset (60S, LSU and MCM7) containing 2 237 characters, of which 735 were parsimony informative. Thick
branches represent BI posterior probabilities 95 %. Bootstrap support values 70 % are indicated at nodes as MP/ML. * = no bootstrap support or bootstrap support values <70 %.
201
DE
BEER
ET AL.
close to each other. Neither of these placements, however, was
supported by phylogenetic statistics. Among the three gene regions used, MCM7 proved to be the most informative and
resulted in trees with topologies similar to those obtained from
the combined dataset.
The first of the nine lineages (Figs 4–7), representing the
largest number of species, included C. fimbriata (type species of
Ceratocystis) and 31 other species previously included in the
C. fimbriata complex. The second lineage included CMW 22736,
representing T. thielavioides (type species for Chalaropsis),
T. ovoidea, and two isolates from the USA and Belgium, previously described as T. thielavioides, but clearly distinct from CMW
22736. These two isolates are thus referred to as Chalaropsis sp.
1. The third lineage included C. coerulescens, type species for
Endoconidiophora, and seven species previously considered
part of the C. coerulescens complex. Isolates representing
C. virescens, C. eucalypti, T. australis and T. neocaledoniae
represented the fourth lineage, which did not include a type
species of a previously described genus. Lineage 5 was previously referred to as the C. paradoxa complex, and included
C. ethacetica (type species of Thielaviopsis), C. euricoi,
C. musarum, C. radicicola and the recently described species,
C. cerberus. The sixth lineage was the second largest and
included C. moniliformis s. str. and 17 other species, but contained no type species representing a previously described
genus. Two new species that are currently being described
(Mbenoun et al., unpubl. data) grouped in this lineage, and were
labelled according to provisional species names provided by M.
Mbenoun (unpublished), namely Huntiella chlamydospora nom.
prov. and H. pycnanthi nom. prov. Isolates of Ambrosiella xylebori (type species for Ambrosiella), A. hartigii and A. beaveri
formed a distinct lineage. The last two lineages comprised
Knoxdaviesia and Graphium species used as outgroups in all
analyses.
Five of the 79 species in Ceratocystis s. l. were not accommodated in any of the nine major lineages discussed above (Figs
4–7). Ceratocystis adiposa and C. major had identical sequences in ITS (data not shown), LSU and 60S, and formed a
distinct clade that was most closely related to lineage 7 (representing Ambrosiella). Ceratocystis fagacearum
and
A. ferruginea, although significantly different from each other,
formed a clade of their own separating them from other Ceratocystis and Ambrosiella lineages. The fifth species, T. basicola,
formed a unique lineage distinct from, but related to species in
lineage 2 as its closest relatives.
GENERIC DESCRIPTIONS AND NOMENCLATOR
Phylogenetic data generated in this study revealed seven wellsupported lineages in Ceratocystis s. l. The distinction between these lineages is also supported by morphological and
ecological data for the species in these groups. These lineages
are, therefore, treated as distinct genera. Five of the lineages
incorporate the type species of earlier described genera, and we
thus emend the descriptions of Ambrosiella, Ceratocystis s. str.,
Chalaropsis, Endoconidiophora, and Thielaviopsis, based on the
types and other species accommodated in the lineages. Two
lineages for which existing names are not available are treated
as novel genera, described here as Davidsoniella and Huntiella.
Where necessary, new combinations are provided for the names
202
of species in these genera. Species previously treated in
Ceratocystis, but excluded from the newly defined genera in the
Ceratocystidaceae (Tables 2 and 3), invalidly described species
(Table 4), and homonyms from kingdoms other than the Fungi
(Table 5), are not treated in the nomenclator, but listed in the
tables as indicated.
Ambrosiella Brader ex Arx & Hennebert, Mycopath.
Mycol. Appl. 25: 314. 1965.
?= Phialophoropsis L.R. Batra, Mycologia 59: 1008. 1967. (type species
Ph. trypodendri).
Type species: Ambrosiella xylebori Brader ex Arx & Hennebert,
Mycopath. Mycol. Appl. 25: 314. 1965.
Sexual state not known. Conidiophores phialidic, single to
aggregated in sporodochia, hyaline, unbranched or sparingly
branched, one-celled to septate. Conidia formed in chains or as
terminal aleurioconidia.
Notes: We followed the emended generic description for
Ambrosiella by Harrington et al. (2010), who restricted the genus
to those species belonging to the Microascales. DNA sequence
data is not available for A. trypodendri, type species of Phialophoropsis, which means the synonymy of the latter genus with
Ambrosiella cannot be confirmed for the present. All known
Ambrosiella species are associates of ambrosia beetles.
Ambrosiella beaveri Six, Z.W. de Beer & W.D. Stone,
Antonie van Leeuwenhoek 96: 23. 2009.
Note: Sexual state unknown.
Ambrosiella hartigii L.R. Batra, Mycologia 59: 998. 1967.
Note: Sexual state unknown.
Ambrosiella roeperii T.C. Harr. & McNew, Mycologia 106:
841. 2014.
Notes: Sexual state unknown. Sequences of this newly
described species were not included in our analyses, but
Harrington et al. (2014b) clearly showed that this species groups
within Ambrosiella.
Ambrosiella trypodendri (L.R. Batra) T.C. Harr., Mycotaxon 111: 355. 2010
Basionym: Phialophoropsis trypodendri L.R. Batra, Mycologia
59: 1008. 1967.
Notes: Sexual state unknown. Ambrosiella trypodendri is the type
species of Phialophoropsis (Batra 1967). No cultures are available for this species. However, Harrington et al. (2010) argued
that it is morphologically similar to Ambrosiella and provided a
new combination for it. Seifert has examined the type, and made
a drawing from it that was used to represent this species in The
Genera of Hyphomycetes (Seifert et al. 2011).
Ambrosiella xylebori Brader ex Arx & Hennebert,
Mycopath. Mycol. Appl. 25: 314. 1965.
AND ALLIED GENERA
Table 2. Species previously treated in Ceratocystis, but now excluded from the genus because they were shown to belong to other
genera. More details on each species are presented by De Beer et al. (2013b).
Name in Ceratocystis
Current name
Basionym
C. abiocarpa R.W. Davidson
Grosmannia abiocarpa (R.W. Davidson) Zipfel,
Z.W. de Beer & M.J. Wingf.
Ceratocystis abiocarpa R.W. Davidson
C. adjuncti R.W. Davidson
Ophiostoma adjuncti (R.W. Davidson) Harrington
Ceratocystis adjuncti R.W. Davidson
C. albida (Math.-K€a€arik) J. Hunt
synonym of Ophiostoma stenoceras (Robak) Nannf.
Ophiostoma albidum Math.-K€a€arik
C. allantospora H.D. Griffin
Ophiostoma allantosporum (Griffin) M. Villarreal
Ceratocystis allantospora H.D. Griffin
C. ambrosia Bakshi
Ophiostoma ambrosium (Bakshi) Hausner, J. Reid &
Klassen
Ceratocystis ambrosia Bakshi
C. angusticollis Wright & H.D. Griffin
Ophiostoma angusticollis (Wright & Griffin) M. Villarreal
Ceratocystis angusticollis Wright & H.D. Griffin
C. araucariae Butin
Ophiostoma araucariae (Butin) de Hoog & Scheffer
Ceratocystis araucariae Butin
C. arborea Olchow. & J. Reid
Ophiostoma arborea (Olchow. & J. Reid) Yamaoka &
M.J. Wingf.
Ceratocystis arborea Olchow. & J. Reid
C. aurea (R.C. Rob. & R.W. Davidson) H.P.
Upadhyay
Grosmannia aurea (R.C. Rob. & R.W. Davidson) Zipfel,
Europhium aureum R.C. Rob. & R.W. Davidson
C. bacillospora Butin & G. Zimm.
Ophiostoma bacillosporum (Butin & G. Zimm.)
de Hoog & Scheffer
Ceratocystis bacillospora Butin & G. Zimm.
C. bicolor (R.W. Davidson & Wells) R.W.
Davidson
Ophiostoma bicolor R.W. Davidson & D.E. Wells
Ophiostoma bicolor R.W. Davidson & D.E. Wells
C. brunnea R.W. Davidson
Ophiostoma brunneum (R.W. Davidson) Hausner &
J. Reid
Ceratocystis brunnea R.W. Davidson
C. brunneo-ciliata (Math.-K€a€arik) J. Hunt
Ophiostoma brunneo-ciliatum Math.-K€a€arik
Ophiostoma brunneo-ciliatum Math.-K€a€arik
C. brunneocrinita E.F. Wright & Cain
Graphilbum brunneocrinitum (E.F. Wright & Cain)
Ceratocystis brunneocrinita E.F. Wright & Cain
C. cainii Olchow. & J. Reid
Grosmannia cainii (Olchow. & J. Reid) Zipfel,
Ceratocystis cainii Olchow. & J. Reid
C. californica DeVay, R.W. Davidson & Moller
Ophiostoma californicum (DeVay, R.W. Davidson &
Moller) Hausner, J. Reid & Klassen
Ceratocystis californica DeVay, R.W. Davidson & Moller
C. cana (Münch) Moreau
Ophiostoma canum (Münch) Syd.
Ceratostomella cana Münch
C. capitata H.D. Griffin
synonym of Ophiostoma tenellum (R.W. Davidson)
M. Villarreal
Ceratocystis capitata H.D. Griffin
C. castaneae (Vanin & Solovjev) C. Moreau
Ophiostoma castaneae (Vanin & Solovjev) Nannf.
Ceratostomella castaneae Vanin & Solovjev
C. catoniana (Goid.) C. Moreau
Ophiostoma catonianum (Goid.) Goid.
Ceratostomella catoniana Goid.
C. clavata (Math.) Hunt
Ophiostoma clavatum Math.
Ophiostoma clavatum Math.
C. clavigera (R.C. Rob. & R.W. Davidson) H.P.
Upadhyay
Grosmannia clavigera (R.C. Rob. & R.W. Davidson)
Zipfel, Z.W. de Beer & M.J. Wingf.
Europhium clavigerum R.C. Rob. & R.W. Davidson
C. columnaris Olchow. & J. Reid
Ophiostoma columnare (Olchow. & J. Reid) Seifert &
G. Okada
Ceratocystis columnaris Olchow. & J. Reid
C. concentrica Olchow. & J. Reid
Ceratocystiopsis concentrica (Olchow. & J. Reid)
H.P. Upadhyay
Ceratocystis concentrica Olchow. & J. Reid
C. conicicollis Olchow. & J. Reid
Ceratocystiopsis conicicollis (Olchow. & J. Reid)
H.P. Upadhyay
Ceratocystis conicicollis Olchow. & J. Reid
C. coronata Olchow. & J. Reid
Ophiostoma coronatum (Olchow. & J. Reid) M. Villarreal Ceratocystis coronata Olchow. & J. Reid
C. crassivaginata H.D. Griffin
Grosmannia crassivaginata (H.D. Griffin) Zipfel,
C. crenulata Olchow. & J. Reid
Ophiostoma crenulatum (Olchow. & J. Reid) Hausner & Ceratocystis crenulata Olchow. & J. Reid
J. Reid
C. curvicollis Olchow. & J. Reid
Graphilbum curvicolle (Olchow. & J. Reid)
Ceratocystis curvicollis Olchow. & J. Reid
C. davidsonii Olchow. & J. Reid
Grosmannia davidsonii (Olchow. & J. Reid) Zipfel,
Ceratocystis davidsonii Olchow. & J. Reid
C. denticulata R.W. Davidson
Ophiostoma denticulatum (R.W. Davidson)
Ceratocystis denticulata R.W. Davidson
C. distorta R.W. Davidson
Ophiostoma distortum (R.W. Davidson) de Hoog &
Scheffer
Ceratocystis distorta R.W. Davidson
Ceratocystis cr

Fungal pathogens of food and fibre crops - CBS

Transcription

Similar documents

iSkin - Specifications

Errata

DILEMMA DILEMMA INCIDENT INTERRUPT Admiral McCoy Morn

Friday, February 15, 2013

Pilots in the Air - CBS Studio Center

Baby Daddy: A Family Business

5 TV Shows You Should Watch This Fall

victorias-secret-fashion-show-musical-guests

Tuesday, June 5, 2007 Newsroom: (800) 290-6301

marg helgenberger - Athlon Media Group