Genetic Diversity of Internal Transcribed Spacer Region of

International Journal of Genetics 5(1): 01-06, 2015
ISSN 2222-1301
© IDOSI Publications, 2015
DOI: 10.5829/idosi.ijg.2015.5.1.9378
Genetic Diversity of Internal Transcribed Spacer Region of
Filamentous Fungi Isolated from Wheat in Lagos State, Nigeria
R.M. Kolawole, 2B.T. Thomas, 1,3J.B. Folorunso and 1A. Oluwadun
1,2
Department of Medical Microbiology and Parasitology, Olabisi
1
Onabanjo University, Sagamu, Ogun State, Nigeria
2
Department of Cell Biology and Genetics, University of Lagos, Akoka, Lagos, Nigeria
3
Department of Medical Laboratory, Health Services Directorate,
Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria
1
Abstract: The high prevalent rate of fungal contamination in wheat circulating in Lagos State, Nigeria
necessitated the need to understand the diversity of Internal transcribed spacers of the ribosomal DNA region
of the isolated filamentous fungi. Results obtained reveals inter specific variation among the isolated organisms
with limited intra specific variability. It can therefore be inferred based on findings that ITS analysis could
provide taxonomic evidence for the isolated organisms but may not be informative enough to score diversity.
Key words: Genetic
Diversity
ITS
Filamentous Fungi
INTRODUCTION
Wheat
RAPD, RFLPs, DNA hybridization, AFLP and DNA
sequences [12-17]. Endophytic isolates of A. alternata,
however, have also been investigated using random
amplified microsatellite (RAMS) markers which was
originally used to measure genetic diversity of plants and
animals [18], it has also been applied in studies of fungi
[18-20] and particularly endophytic fungi. For examples,
Burgess et al. [21] clearly distinguished between
morphotypes of Sphaeropsis sapinea, a fungal
endophyte of Pinus spp., using RAMS markers.
Grünig et al.[22] have also differentiated between the dark
septate endophytic fungi Phialocephala fortinii and type
I of a non-sporulating mycelium, which had the same
allozyme phenotype.
With several molecular methods being developed,
many species-specific primers targeting several genomic
regions such as satellite DNA [23,24], the heat shock
protein 70 gene [25-26], the topoisomerase I gene [27] and
internal transcribed spacer (ITS) regions of ribosomal
DNA (rDNA) [28,29] or by using nonspecific primers for
polymerase chain reaction (PCR) amplification of the ITS
regions and subsequent analysis with restriction fragment
length polymorphism (RFLP) [30,31]. The ITS regions are
located between the repeating array of nuclear 18S, 5.8S
and 28S ribosomal RNA genes and have 100-200
copies/genome.
Filamentous fungi are well known for the production
of natural toxic compounds (mycotoxins) in several
agricultural products [1]. These fungal contaminants
causes biochemical deterioration and losses in nutritional
quality [2,3]. They are also responsible for substantial
effects in stored food stuffs including discoloration,
production of off-odours, deterioration in technological
quality [4,5] and mycotoxin contamination [6-8].
These fungi are gaining popularity in developing and
under developed countries of the world due to their
association with significant agricultural losses. Like other
toxigenic organisms, these organisms produces
secondary metabolites that are immunotoxic, genotoxic,
carcinogenic, hepatotoxic among other effects [9-10].
Until about two decades ago, most studies of natural
fungal strains and populations focused on phenotypic
differences in morphology and physiology and when
possible, mating. However, these phenotypic features are
often difficult to observe, quantify, standardize and/ or
analyze. In recent years, there has been substantial
progress in the development of innovative methods to
analyze fungi (and other organisms) at the molecular level
[12]. For example, the genetic variation of saprobic and
pathogenic A. alternata isolates was assessed using
Corresponding Author: Thomas Benjamin Thoha, University of Lagos, Akoka, Lagos, Nigeria. E-mail: benthoa2013@gmail.com.
1
Intl. J. Genet., 5(1): 01-06, 2015
These regions are rapidly evolving and thus have
been routinely used in species-level phylogeny in a
wide range of organisms, including nematodes [32].
In B. xylophilus, the intraspecific variation of the ITS
regions has been used to study the genetic structure of
isolates from various regions of the world through
sequencing [33-36] and RFLP analysis [30]. Intraspecific
diversity between virulent and avirulent B. xylophilus
isolates has been detected using RFLP analysis with the
enzyme HhaI [37-38]. ITS genetic diversity has been
reported within species and individuals from a diverse
range of eukaryotes, including insects [39,40], marine
sponges [41,42], fungi [43,44] and nematodes such as
Meloidogyne [45], Brugia [46], Ascaris [47] and
Pratylenchus [48]. However, to our knowledge, no data
on ITS intra and inter isolate and intra-individual diversity
in filamentous fungi from wheat in Lagos, Nigeria have
been published. In the present research, the ITS rDNA
regions was used to study the genetic diversity of
filamentous fungi isolated from wheat circulating in Lagos
State, Nigeria.
was directly sequenced using an Automatic Sequencer
3730xl under Big DyeTM terminator cycling conditions at
International Institute of Tropical Agriculture, Ibadan,
Oyo State, Nigeria.
Sequence and Statistical Analysis: Alignments and
determination of consensus sequences were carried out
using BioEdit [50]. Homologous sequences in the
databases were searched using the Basic Local Alignment
Search Tool [51] while phylogenetic tree was constructed
using MEGA software version 7.0. The statistical
procedure for phylogenetic tree construction used was
the Unweighted Pair Group Method with Arithmetic Mean
(UPGMA) while 10000 bootstrap replications was carried
out for the test of phylogeny in a maximum composite
likelihood pattern involving uniform substitution of the
nucleotides. The substitution techniques includes
transition and transversion. Also, the pattern of
substitution selected among lineages was homogenous.
All missing data and gaps were completely deleted in an
analysis involving only the first second and third codon
only.
MATERIALS AND METHODS
RESULTS
Fungal Strains: The fungal strains used in this study was
isolated in our previous study [49] and maintained on
Sabouraud dextrose agar at 25°C. The isolates were kept
at 4°C until needed.
A total of twenty three strains of filamentous
fungi belonging to six different genera and eight species
were used for this study. These species of fungi were
identified as Aspergillus fumigatus, Aspergillus flavus,
Aspergillus niger, Fusarium solani, Trichoderma
atroviride, Rhizopus stolonifer, Alternaria altenata and
Penicillium verrucossum. When these isolates were
characterized using the ITS region of the studied fungi, it
was observed that the strains of filamentous fungi
isolated from wheat shows both intra and inter genera
similarities. As shown in this dendogram, some of the
fungal strains shares relationship both within and
between genera. Also, two strains of Alternaria
alternata, Fusarium solani, Trichoderma atroviridae and
Aspergillus fumigatus were found sharing hundred
percent similarity with each other while Rhizopus
stolonifers, Penicillium verrucosum, Trichoderma
atroviridae and Alternaria alternata have 33%, 30%,
37% and 19% similarities with a strain of Penicillium
verrucosum, Aspergillus fumigatus and Aspergillus niger
respectively. Also, the number of clusters obtained from
the dendogram is a function of the percentage of
truncation as higher percentage of truncation was
found to be directly proportional to higher numbers of
clusters.
DNA Extraction and Amplification of ITS Regions:
Fungal DNA was extracted using a DNeasy® Blood and
Tissue Mini kit (Qiagen, Germany) following manufacturer
instructions. The ITS rDNA regions containing partial
ITS1 and ITS 4 sequences were amplified with PCR using
50 ng extracted DNA and 1 U Dream Taq DNA
polymerase (Fermentas, USA) in 1X Dream Taq buffer, 0.2
mM of each deoxyribonucleotide triphosphates and 1 ìM
primers ITS1 (5´TCC GTA GGT GAA CCT TGC GG 3´) and
ITS4 (5´TCC TCC GCT TAT TGA TAT GC 3´). The PCR
reaction mix contained 0.5 ìM of each primer, 10 ìM
deoxynucleotides, 1.5 mM MgCl2 and 1 x buffer
(Promega). The suspension was heated at 95°C for 15 min
in a PE 2400 (Perkin Elmer) thermocycler. One unit of the
Taq Polymerase (Promega) was then added to each tube.
PCR conditions were as follows: 35 cycles of denaturing
at 94°C for 1 min; annealing at 55.5°C for 2 min and
extension at 72°C for 2 min; and final extension at 72° for
10 min. The resulting amplification products were purified
using a QIAquick® PCR Purification Kit (Qiagen)
according to manufacturer instructions while both strands
2
Intl. J. Genet., 5(1): 01-06, 2015
100
99
19
17
100
3
99
84
100
TA2
FS2
AN3
45
AFL2
AFL3
60
38
PV3
RS1
RS2
63
3
FS1
FS3
AA
AFU1
AN1
TA3
TA1
37
3
the highly ubiquitous nature of the isolated fungi. These
organisms might have being dispersed by wind. Indeed,
most are common soil saprophyte in many environments
[58,59]. Alternatively, if the isolated fungi is endemic to
wheat in Lagos, it may be more common in soils and on
vegetation around the collection areas such that strongly
directional winter east winds could potentially transport
conidia from the environment into the wheat. Any efforts
taken to control fungal infections of wheat should bear in
mind the genetic diversity found in this study and the
possibility that surrounding soils and vegetation and
prevailing winds may be serving as sources of inoculum
of this apparently common fungi. Before any control
measure can be put in place, efforts should be made to
identify all means of conidia dispersal and sources of
inoculum, as well as the identity of the pathogen relative
to the known phylogeny of this group [58]. Given the
inherent difficulties in attempting to control such a
potentially common and ubiquitous pathogen, further
studies should address whether this fungus really is
reducing plant recruitment at higher than normal levels.
The results outlined in this study shows that while
interspecific variation of the isolated fungi ITS region is
relatively high, intraspecific variability is very limited and
that ITS analysis has potential for developing species
level markers for many, but not necessarily all, filamentous
fungi species. It appears that ITS analysis is a potent tool
for the taxonomic study of fungi, with the minute amounts
of DNA required and the high reproducibility of this
procedure making it an ideal method both for studying
population heterogeneity in the field and the identification
and monitoring of specific strains introduced into the soil.
AA2
AA3
AN2
PV2
AFU2
15
30
100
AFU3
PV1
33
2.0
1.5
RS3
1.0
0.5
0.0
DISCUSSION AND CONCLUSION
The genetic variation of filamentous fungi population
isolated from wheat in Lagos, Nigeria was investigated
using ITS1 and ITS 4 primers. The inter and intra genera
similarities and diversities observed in the present study
was comparable with the other studies using the same
technique [18-20]. This observation confirmed that
ITS1/ITS4 primers are not fungi specific [52,53].
However, in some cases, heterologous DNA from the
plant material did not interfere with PCR amplification
[54-56]. Glen et al.[57] tested six primers pairs (targeting
three nuclear and three mitochondrial regions) for
specificity, sensitivity and species discrimination on
identified collections of fungi. Two sets of these primers,
one newly designed and targeting the ITS region and the
other amplifying a ribosomal DNA fragment of the large
mitochondrial subunit met the requirements of high
specificity and sensitivity, amplifying DNA from a broad
range of the larger basidiomycetes, with no amplification
of plant, bacterial or ascomycete DNA. These specific
primers discriminated fungi to species level for 91 fungal
species from 28 families and are a potential practical
PCR-RFLP tool for identifying basidiomycetes in plants
from field samples. The little variation observed in the
studied filamentous fungi may however be explained by
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