CALIFORNIA CITRUS SHOWCASE

Transcription

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Citrograph Vol. 7, No. 2 | Spring 2016
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2016
This lovely work is a detail from
“Orange Grove of the Valley” (seen
in full at left), a 3’ x 4’ original oil on
canvas by Betty Berk, an expressionist
painter from Visalia, California. The
Dinuba High School art teacher
strives to assist schools and the
community in understanding the
important role that the arts play in
people’s daily lives. She developed
and serves as chair of the annual Dinuba Festival of the Arts and has
won numerous awards for her work. The original artwork on the Citrograph cover is
one of four Berk commissions displayed at the Citrus Research Board headquarters
in Visalia. An on-line gallery featuring more of her paintings may be viewed at www.
bettyberk.com. Her permanent gallery may be found at “The Art Hub,” 2024 N. Van
Ness Blvd., Fresno, CA 93704.
“Orange Grove of the Valley”
©2014 Betty Berk.
IN THIS ISSUE
SPRING 2016 | VOLUME 7 • NUMBER 2 THE OFFICIAL PUBLICATION OF THE CITRUS RESEARCH BOARD
8 EDITORIAL: COMMITTED TO THE FIGHT
GARY SCHULZ
10 CHAIRMAN’S VIEW: OPPORTUNITY IS OURS
RICHARD BENNETT
12 MUTUALLY SPEAKING: TRADE TO BECOME MORE
DIFFICULT
JOEL NELSEN
16 INDUSTRY VIEWS: “HOW CAN WE MINIMIZE THE HLB
THREAT TO THE CALIFORNIA CITRUS INDUSTRY?”
MOJTABA MOHAMMADI, PH.D.
20 EDUCATIONAL AND THOUGHT-PROVOKING
ALYSSA HOUTBY AND IVY LEVENTHAL
24 IMMEDIATE ACTION IS NEEDED
BETH GRAFTON-CARDWELL, PH.D., ET AL.
28 HOPE FOR THE FUTURE OF CITRUS
YINDRA DIXON
34 CALIFORNIA CITRUS THREATS
LAURYNNE CHETELAT, ET AL.
40 PRE- AND POST-HARVEST FUNGICIDES FOR MANAGING
SEPTORIA SPOT
JAMES ADASKAVEG, PH.D., AND HELGA FÖRSTER, PH.D.
46 DEVELOPING RESISTANCE TO HLB
CHANDRIKA RAMADUGU, PH.D., ET AL.
52 DEVELOPMENT OF AN ACP MANAGEMENT PLAN FOR
ORGANIC CITRUS
JAWWAD A. QURESHI, PH.D., AND PHILIP A STANSLY, PH.D.
60
60 A MICROBIOTA-BASED APPROACH TO CITRUS TREE
HEALTH
JOHAN LEVEAU, PH.D., AND PHILIPPE ROLSHAUSEN, PH.D.
64 AN INTEGRATED BIOLOGICAL APPROACH TO FULLER
ROSE BEETLE CONTROL
EDWIN LEWIS, PH.D., AND AMANDA HODSON, PH.D.
5
THE MISSION OF THE CITRUS RESEARCH BOARD:
ENSURE A SUSTAINABLE
CALIFORNIA CITRUS INDUSTRY FOR
THE BENEFIT OF GROWERS BY
PRIORITIZING, INVESTING IN AND
PROMOTING SOUND SCIENCE.
CITRUS RESEARCH BOARD MEMBER LIST
BY DISTRICT 2015-2016 (TERMS EXPIRE JULY 31)
District 1 – Northern California
MemberExpires
Toby Maitland-Lewis 2016
Jack Williams
2016
Donald Roark
2016
Dan Dreyer
2016
Jim Gorden
2017
Greg Galloway
2017
Joe Stewart
2017
Franco Bernardi
2017
MemberExpires
Kevin Olsen 2017
Etienne Rabe
2018
John Konda
2018
Keith Watkins
2018
Jeff Steen
2018
Richard Bennett 2018
Justin Brown
2018
District 2 – Southern California – Coastal
MemberExpires MemberExpires
John Gless III
2017 Alan Washburn
2018
Mike Perricone
2017
District 3 – California Desert
MemberExpires MemberExpires
Mark McBroom 2016 Craig Armstrong
2016
Public Member
Member Expires
Vacant
2018
CALENDAR OF
EVENTS 2016
March 3
California Citrus Showcase sponsored
by California Citrus Mutual (CCM), Visalia
Convention Center, Visalia, California. For
more information, contact CCM at
(559) 592-3790.
March 9
CPDPP Board Meeting,
Riverside/San Bernardino, California. For
more information, contact CDFA at
(916) 403-6652.
March 22-24
CRB Board meeting and research project
updates, Visalia, California.
For more information, contact the CRB at
(559) 738-0246.
April 12-13
Post-harvest Conference, Embassy Suites
Mandalay Beach, Oxnard, California. For
more information, contact the CRB at
(559) 738-0246.
May 11
CPDPP Board Meeting, Ventura, California.
For more information, contact CDFA at
(916) 403-6652.
May 19
CRB Research Priority Screening
Committee Meeting, Visalia, California.
For more information, contact the CRB at
(559) 738-0246.
Citrus Research Board | 217 N. Encina St., Visalia, CA 93291 | PO Box 230, Visalia, CA 93279
(559) 738-0246 | FAX (559) 738-0607 | E-Mail Info@citrusresearch.org | www.citrusresearch.org
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7
EDITORIAL
BY GARY SCHULZ
COMMITTED TO
THE FIGHT
A
s I write my first editorial for the Citrus Research Board’s Citrograph, I tend to
reflect on my first four months as president of the CRB. I want to say thank you
to the Board of Directors for giving me the opportunity to lead such a fine
organization, with such an outstanding staff, and with tremendous
leaders serving in key positions. The California citrus industry is
huge, but it has its challenges. Citrus greening or huanglongbing
(HLB) is about as serious as they come, and I am here to tell you
that our organization is committed with its research dollars to
fight HLB for every acre of California citrus.
Our Board has paid attention and learned from the lessons
of producers in Florida and Texas. Together with allies such
as California Citrus Mutual, the California Citrus Growers
Association, the packinghouses and others, the industry has
created organizations such as the California Citrus Quality
Council, the Citrus Clonal Protection Program and the Citrus
Pest and Disease Prevention Program. All of this collaboration
8
Gary Schulz
Call The
Agri-Business
Insurance Specialists
Farms - Ranches
Crops
Packing Houses
Nursery
Land Developers
Produce Brokers
Tree Trimmers
Chemical Applicators
Pump & Well
makes our industry more productive and more of a
problem-solving industry.
I want to say thanks to interim presidents Ed Civerolo and
Jim Rudig, the entire Board and the warm, friendly staff
for a gracious welcome to the organization. The citrus
producers, who I have enjoyed meeting and getting to
know, are great people; and I appreciate their sincerity,
generosity and creativity.
In closing, I am reminded of the slogan of my friends at This
Week in Agribusiness, Orion Samuelson and Max Armstrong,
when they refer to production agriculture as “America’s
Most Essential Industry.” Here at the Citrus Research Board,
we intend to do our part in leading our industry in these
challenging times.
Gary Schulz is the president of the Citrus Research Board.
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9
CHAIRMAN’S VIEW
BY RICHARD BENNETT
“A single CLas-positive female
theoretically could lead to as many
as one billion CLas-innoculative
positive psyllids in several
generations of offspring.”
OPPORTUNITY IS OURS
T
he opportunity is ours to give the California citrus industry
precious, necessary time to soldier on while our focused
scientific researchers seek the best tools to enable us to forge
a viable future. Although the key to survival is ours, it must be
guided by leadership defining the “allocation of resources.” Our
main issue is finding the bacterium that causes huanglongbing
(HLB), which will require us to fund Early Detection Technologies
(EDTs) to eliminate that bacterium.
The answer to maintaining a productive industry as long as
possible centers on controlling the spread of HLB. Mike Irey,
Director of Research at Southern Gardens Citrus in Florida, believes
he controls the psyllid close to 99 percent, but still has a three to
five percent spread of the bacterium. Susan Halbert, Ph.D., Florida
Department of Agriculture, states that a female Asian citrus psyllid
(ACP) can produce up to several hundred eggs during her spring to
fall lifetime. Therefore, a single CLas-positive female theoretically
could lead to as many as one billion CLas-innoculative positive
psyllids in several generations of offspring.
The future of our industry is directly connected to the discovery
and elimination of infected trees. The California Department
10
Richard Bennett
of Food and Agriculture (CDFA) utilizes a protocol called a
polymerase chain reaction (PCR). This scientific instrument
does not detect the bacterium for upwards of four to seven
years following infection. PCR will detect the bacterium, but
the single branch/limb of flush needs to be sampled for this
protocol to work. The whole tree takes considerable time for
the bacterium to spread, which is why there is such a long
latency period for the disease symptoms to be manifested.
Additionally, PCR protocol will produce a very large number
of false negatives until sufficient levels of the bacterium are
present and the lucky or unlucky branch is tested. This literally
can be years!
The research of David Bartels, Ph.D., demonstrates low-level
bacterium-inoculative ACP to be the ”coal mine canary” that
indicates where positive trees are highly probable. Bartels is
finding widespread areas of Southern California, including
Riverside, with probable HLB-positive trees. The area of the
San Gabriel HLB-positive trees was indicated in the Transect
Study, a project funded by the California Pest and Disease
Prevention Program (CPDPP) to detect HLB-positive trees,
conducted by the Early Detection Technology scientific team.
The most advanced EDTs should be incorporated to further
screen for infected trees in areas identified by Bartels. These
same EDTs will be available for use in our own orchards to
identify non-PCR positive trees (trees infected with the HLB
bacterium, but the single limb of infection not detected) in
the future. These EDT specialists are utilizing the knowledge
already developed in the human science field.
The Florida citrus industry did not respond in a timely manner
to the spread of ACP and did not act on the HLB bacterium
introduction. That state is primarily a juice business. Juice now
is being chemically altered to mask the HLB flavor. However,
California is predominantly a fresh fruit business, so this is not
an option. Fresh market consumers will make their decisions
quickly when they eat their first HLB-infected fruit. We cannot
afford to wait for this to happen. The opportunity is ours to
utilize the available science to keep our industry profitable
for as long as possible while additional management and
eradication tools are sought.
Richard Bennett is the chairman of the Citrus Research
Board.
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MUTUALLY SPEAKING...
BY JOEL NELSEN
On a visit to the Chongqing Terminal Market,
we discovered hand-packed mandarin oranges.
TRADE TO BECOME
MORE DIFFICULT
F
or many years, the concept of world trade for our industry was relatively benign.
The major stumbling block was the level of tariffs, which created expense at the
store level for overseas consumers.
But today, the ability to export is being impacted by currency manipulation,
phytosanitary issues (real or imagined), food safety retribution and
political agendas unrelated to the selling of our product. Indonesia
shuts down trade to protect domestic producers and additionally
wants food safety assurances. Vietnam expresses concern about
farming and packing operations. Korea seeks protection from
two insect pests (scale and Fuller rose beetle) almost to the point
that zero risk is the stated objective. And then there is China!
Right now, China wants assurance that groves are not infested
with disease or pests – ever. Brown rot occurs around the
world, except in China, evidently. Chinese officials shut
down the industry because of one percent detection rate.
(Allegedly: was it one container, one carton or one piece of
fruit? Who knows?)
The Food Safety Modernization Act (FSMA) is creating
retribution among a few partners. In our case, it seeks even
more perfection from an industry with a perfect record. Offshore producers are required to comply. “Good,” you say, as all
12
Joel Nelsen
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should be on the same level playing field. Then, Indonesia
and Southeast Asian officials seek audits on our products
prior to export. As California Citrus Mutual (CCM) feared and
stated, we are sliding down another slippery slope of trade
barriers, and the end is not near.
Maybe this column should be entitled “Trade is Difficult”
rather than talking in futuristic terms.
It was more than 50 years ago when then President Dwight
Eisenhower uttered this famous phrase: “Farming is easy
when a desk is your farm and the pencil is your plow and you
reside 1,000 miles away” – or something like that.
Simply stated, there comes a tipping point when satisfying
export demands does not result in the lucrative revenues
necessary to offset the barriers. Alternatively, the revenue
success of the past decade is directly correlated to the
amount of tonnage on the domestic market and that
exported. Imagine if all or some of the export tonnage
ended up in the domestic scene. Who knows? You could
have a port strike that shuts down exports and results in an
estimated $120 million value of lost opportunity. Hmm, that
did just happen, and the year-end results were not pretty.
Bottom line: as more countries engage in the trade of citrus,
they are going to make our risk greater and our troubles
larger. They’ll want to export into the United States from
heavily-pest and disease-infested areas without taking
appropriate mitigation steps. There will be a promise of
adherence, but verification will be the real issue. There will
be harassment of our products as we seek entry or seek to
sustain entry.
Each grower will need to discern where the tipping point
occurs. That’s the logical approach, and weaving logic into
an illogical arena will be the challenge. China negotiations
this past fall are a case in point. The USDA and China had a
number of commodities on the agenda. We felt confident
going into the discussion that Plan A and Plan B would
achieve the desired objective; but the Chinese were illogical
in their demands to the point that a frustrated USDA team
sought input for Plan C.
It was so illogical that three commodities that were supposed
to be “slam dunks” were thwarted in the effort to achieve
success. Together, the USDA and the citrus industry were
able to open the door, but for how long is the question.
We actually wrote, if you recall, in a November Market
Memo, that growers, shippers and marketers needed to have
an alternative plan for exports because we were not at all
confident the Chinese market would remain open.
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Notwithstanding displays of hot sauces in street markets, the negotiations were equally hot with little in the way of mild.
There are three methods in which
your industry seeks to influence trade
policy decisions. One is to have a solid
working relationship with USDA/APHIS/
FAS and above. We already have this
true partnership. Second is a seat on an
agricultural trade advisory committee
that addresses overarching policy calls
and, therefore, significant interaction
with the Secretary of Agriculture and
the U.S. Trade Representative. Rayne
Thompson occupies a seat on that
committee. Third is a seat on the fresh
fruit and vegetable trade advisory
committee that is specific to the
horticultural industry. It is a counterweight to commodity and animal
influence at the agencies and is very
Not surprisingly, pomelos are a popular item in China. Bags of the large citrus fruit are available on
specific in its approach to proposed
pallets throughout the market.
trade agreements and/or problems.
I have been fortunate to be a member
Indonesia gets its needs for food safety and phytosanitary for more than 12 years and am humbled that the 22-person
concerns addressed and then announces as we begin our committee asked me to chair the effort last fall.
harvest that only a limited amount of fruit via a limited amount
of exporters will be allowed into their country. The logical We will have opportunities to enhance trade and seats at the
approach to export success is going to be challenged for the table to thwart challenges; just don’t expect any logic. It has
foreseeable future in my estimation. Why? Because they can! become a political game of leverage.
Joel Nelsen is the president of California Citrus Mutual.
14
15
INDUSTRY VIEWS
MOJTABA MOHAMMADI
Heavy fruit drop results when
trees are infected with HLB.
We recently asked two renowned researchers, an
entomologist and a plant disease epidemiologist, the
following question:
HOW CAN WE MINIMIZE
THE HLB THREAT TO
THE CALIFORNIA CITRUS
INDUSTRY?
16
David W. Bartels, Ph.D.
Entomologist, USDA-APHIS
Plant Protection & Quarantine
Mission Laboratory
Edinburg, Texas
One of my main research areas is
analyzing the survey data of Asian
citrus psyllid (ACP) and leaf tissue
samples to understand what the
‘Candidatus Liberibacter asiaticus’
(CLas) diagnostic reports from
the laboratories are telling us. The
quantitative PCR protocol has very
high sensitivity and specificity for
(and provides a continuous measure
[Ct-values] of ) the amount of CLas
present. We impose a regulatory
threshold on Ct-values, below which we can confirm the
presence of CLas without question. We cannot reliably confirm
CLas presence above this threshold with molecular techniques
at this point. Therefore, I started analyzing the spatial pattern
of the Ct-values across the landscape to determine if there
may be an underlying biological process, such as clustering
of samples around a known positive tree, and we could
derive some information from these inconclusive Ct-values.
Currently, the results indicate clustering of Ct-values of psyllid
samples in some areas and not just a random distribution. My
work focuses on the full range of Ct-values, and how we may
be able to utilize data from large scale survey efforts to predict
locations that have huanglongbing (HLB)-infected plants.
I am using Geographic Information Systems (GIS) software
and spatial analysis methods to predict those high risk areas,
which should help target our plant tissue surveys.
EARLY DETECTION
TECHNOLOGIES
There are some very interesting ideas and data supporting
the various pre-symptomatic or early detection technologies
that are being developed. It reminds me of some of the
research that I have done in the past on using hyperspectral
remote sensing to differentiate spectral signatures of plant
species. That work was looking into whether or not we could
distinguish ash trees from other species of northern
hardwoods because at that time, emerald ash borer beetle
(EAB) was invading the U.S. The most important thing I
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17
1/22/16 2:13 PM
HLB-infected fruit is often misshapen.
learned from that project was that you must have a massive
data base underlying your detection efforts so you can
understand which variables are important and contribute to
your results.
A big issue that I believe needs to be addressed in the early
detection technology research is developing an understanding
of how different soil types, varieties of citrus in different
environments, and various stressors such as citrus canker, citrus
leprosis, citrus variegated chlorosis (CVC), citrus tristeza virus
(CTV), HLB, etc., affect citrus trees. What we need to understand
is whether there are enough differences in the plant responses
detected by these early detection methodologies across
many different environments to differentiate HLB from other
sources of plant stress. The question I have is how much of
the underlying reference database we have to go along with
these detection technologies. This information will be critical
before we can reliably use these methods to any great extent
in different areas across Florida, Texas and California.
CONTROL STRATEGIES
As far as control strategies are concerned, growers can
implement area-wide management (AWM) techniques across
large areas. The biggest issue with AWM is how to deal with
dooryard citrus, because in many growing areas in the U.S.
we have a fractured environment where commercial citrus
is surrounded by dooryard trees. These trees are typically
poorly managed and provide a refuge for ACP. We definitely
have the conventional and organic pesticides to control the
psyllid populations to very low levels, but it takes highly
coordinated management across a large area to do it well.
The nuPsyllid project is working to develop a psyllid that does
not transmit CLas. If they are successful and also can develop
driver mechanisms to push that trait into the current psyllid
population, we could begin rearing these nuPsyllids. This
technology would completely change the management of
ACP and with a lot of hard work and some luck, may be in
place before HLB shows up in the Central Valley of California.
18
LESSONS FROM FLORIDA AND TEXAS
Some of the lessons I think should be learned from the Florida
and Texas experience include immediate removal of diseased
citrus trees as soon as they are confirmed positive for HLB.
Start early in educating the growers on the three pillars of
disease management: using pathogen-free nursery stock,
AWM for controlling psyllids, and rouging of known inoculum
to prevent a build-up of inoculum pressure. I also believe that
as much effort needs to go into commercial citrus surveys as
is being done in dooryard citrus. Texas has learned that while
we were busy surveying our dooryard citrus, HLB was being
spread rapidly through our commercial citrus.
Neil McRoberts, Ph.D.
Associate Professor
Department of Plant Pathology
University of California
Davis, California
We clearly are making progress
on tackling HLB on several fronts.
For example, when we started a
nuPsyllid project within the last
three years, the team responsible
for rearing the psyllids, releasing
and trapping them (so that we
can track whether they were
successful in invading natural
psyllid populations) said their
major obstacle was the lack of
clear work on whether or not there
was an olfactory signal that lured psyllids to the infected trees.
A couple of years later, Christina Davis, Ph.D., at UC Davis and
Lukasz L. Stelinski, Ph.D., have made good progress on that
topic. Success where there was practically no progress as
recently as two years ago offers some signs of hope.
THE HLB PATHOSYSTEM
There is a lot of very detailed mechanistic understanding of
how the vector and the pathogen ‘Candidatus Liberibacter
asiaticus’ (CLas) interact and how the pathogen interacts with
the host plant. I am not sure if that has yet resulted in practical
outcomes, but because of the level of effort being expended,
there soon could be a major step forward.
The work of Tim Gottwald’s group in Florida, contributions
from researchers in the UK, results from Matt Dougherty’s
group at UC Riverside, and the research David Bartels has
done with APHIS in Texas on the analysis of psyllid and tree
samples are all combining to provide a very clear idea of how
the vector spreads, the dynamics of CLas spread and how the
disease development follows along on the back of that vector
spread.
EARLY DETECTION
TECHNOLOGIES
The issue surrounding early detection methods is incredibly
important. We know from how this disease has spread
elsewhere and also from comparisons with other vectored
diseases that the disease spread is not really tied to the
absolute number of vectors, but to the proportion of the
vector population infested with CLas. Vector suppression can
only get us so far. Even with a small number of vectors present,
if we kill 95 percent of them, five percent of the huge number
of psyllids that can build up in an area is still very sizeable. If,
half of those are infested, there still would be enough vectors
for effective CLas transmission resulting in increased disease
occurrence. If we want to stop disease spread, it is not enough
to suppress the vector. I am not saying we should not do any
vector management, but it won’t stop HLB’s spread. That’s
why it is so important to start tackling inoculum sources, and
that’s why early detection methods matter.
Also, it is not just about finding the disease earlier, it is the scale
at which detection works and the ease with which it works
that matter for practical application. We need methods to
detect the disease when it is pre-symptomatic and preferably
operate at a whole-tree scale. Because of the sub-sampling
problems we can have with PCR and similar methodologies,
there always will be problems with sub-sampling from large
trees. Therefore, being able to tell if the whole tree is infected
from the outside using an electronic nose, dogs or another
technique, are key pieces in the technological tool kit we need
to get ahead of the disease.
If we had something robust and reliable that was effective as
a pre-symptomatic early detection method, we could think
about putting that together with David Bartels’s analysis
of potential HLB hot spot detection and the ARS risk-based
analysis for mapped grid squares. Then we begin to have tools
that might let us hunt for the disease effectively. Find the
infected individuals, get them out of the population, and then
we can start getting the inoculum level down and cut HLB
spread dynamics out completely. As an epidemiologist, that is
where I’d like to see efforts headed in the next few years.
CONTROL STRATEGIES
Regarding vector control in commercial groves, it is clear from
the Florida experience that effective control requires an areawide control program or cooperative management. One issue
with that is effective monitoring for pesticide resistance. Since
there will be a lot of pesticide use and a limited number of
products will be involved, it will be important to keep an eye
on any signs of resistance.
On the disease side, there is promising technology. Some
protein-based and therapeutic technologies and some
plant transformation approaches offer cause for optimism.
However, they do raise the problematic issue of geneticallymodified (GM) technology. I do not know enough about the
conventional breeding program to know whether there are
signs of hope on conventional selection, wild crossing or any of
those types of techniques that might not require GM licenses.
I’d guess the odds are about 70 percent that we will be looking
at GM solutions. As a long-term goal, breeding host resistance
from citrus or close relatives would offer a sustainable and
non-GM solution. It’s worth looking at any non-GM techniques
that do not require high levels of regulatory oversight and do
not spark a public level of alarm.
LESSONS FROM FLORIDA
We have learned from Florida. For instance, diseased plant
materials were moved around the state from nurseries for
commercial sale, and we know transport corridors were major
factors in spreading the disease. In some instances in Florida
where there was a lot of interface between urban and rural
areas, those interfaces were reservoirs for psyllids to retreat
to when growers sprayed. However, ACP can reinvade from
those interfaces.
In northern California, we do not have as much of that type
of interface. Southern California is a little different. Learning
lessons how about to manage that kind of problem is really
important. The Florida experience has given us a check-list of
risk factors to consider for California. We should ask if each of
these risk factors is likely to be important. If so, at least we still
have time to take an action on them.
Mojtaba Mohammadi, Ph.D., is an associate scientist with
the Citrus Research Board in Visalia, California, where he
also serves as associate science editor of Citrograph.
19
Citrus Showcase attendees networking with trade show exhibitors.
The Fruit Growers Supply fruit wash demonstration is a popular attraction
on the trade show floor. Fruit Growers Supply is a longstanding Showcase
supporter and trade show exhibitor.
EDUCATIONAL AND
THOUGHT-PROVOKING
Don’t Miss the 2016 Citrus Showcase
Alyssa Houtby and Ivy Leventhal
The opportunity is fast approaching to spend a day immersed
in informative and original workshops geared toward grower
education at the 2016 Citrus Showcase, which will take place
at the Visalia Convention Center on March 3.
You can join approximately 1,000 of your fellow industry
members in attending this free series of sessions hosted by
California Citrus Mutual (CCM). For the third consecutive year,
CCM will team with the Citrus Research Board (CRB) to offer
workshops on the major issues facing the California citrus
industry. “We are pleased to participate in this important
grower event,” said CRB President Gary Schulz. “It’s the perfect
opportunity to listen, learn and network.”
According to CCM President Joel Nelsen, “The Citrus Showcase
is the single largest educational forum for citrus growers in
California. Each year, we program informative workshops and
bring in expert speakers on issues affecting our industry. This
year is no exception. With the challenges associated with water,
labor regulations, the Asian citrus psyllid and Huanglongbing,
it is imperative that growers are kept informed.”
20
CCM and CRB will hold simultaneous workshops in the
morning on topics including the Irrigated Lands Regulatory
Program, early detection of HLB, using social media to your
business advantage and citrus breeding in the age of HLB.
Labor laws and regulations will be discussed in the afternoon.
A tradeshow will be open between all sessions. During the
luncheon, the keynote address will feature speakers from
Florida and Texas discussing “Huanglongbing: Lessons from
the Frontline.” A complete schedule of the day’s events is
available on the facing page.
The Showcase, originally known as “Citrus Expo,” is now in
its 22nd year. The continental breakfast (sponsored by Mary
Roach Insurance Agency) and workshops (sponsored by
Genesis Nurseries) are free to all attendees, while there is a
small charge to attend the luncheon and keynote speech. To
obtain more information and/or to register for the Showcase,
please contact CCM at 559-592-3790.
Alyssa Houtby is the director of public affairs at California
Citrus Mutual. Ivy Leventhal is the managing editor of
Citrograph.
THURSDAY, MARCH 3 • VISALIA CONVENTION CENTER
2016 CITRUS SHOWCASE SCHEDULE
Continental Breakfast beginning at 7:30 AM
Sponsored by Mary Roach Insurance Agency
consumers would like nothing more than the simple truth about
why you do what you do. Don’t let activists tell your story for you
because chances are you won’t like what they have to say.
Workshops Sponsored by Genesis Nurseries,
Prudential Ag Investments & Wilbur Ellis
The growing world of social media can be daunting, but also a
valuable tool to make our voices heard.
8:00-9:00 AM Workshop Sessions A & B
A - 2016 and Beyond: Grower Requirements under the Irrigated
Lands Regulatory Program (ILRP)
Beginning in 2016, growers in the Tulare Lake Basin will be
required to have a Nutrient Management Plan on file. In 2017,
growers must begin submitting an annual Nitrogen Summary
Report to their Third Party Coalitions under the ILRP. California
Citrus Mutual has worked closely with the Third Party Coalitions
and the Regional Water Quality Control Board over a period of
several years to establish reporting requirements that growers
can comply with and that satisfy the Regional Board’s data
requirements. Amended requirements and shifting deadlines
have created a lot of confusion. This workshop will clear up the
confusion and provide growers the information and resources they
need to be sure they are in compliance. Casey Creamer,
Coordinator, Kings River Water Quality Coalition , will be providing
the most current information on the implementation of the on farm
requirements and will go through a step by step timeline of what
growers need to know and do now and going forward.
B – “Early Detection of Huanglongbing with the Best Science
Available” By Dr. Wenbo Ma
Dr. Ma is Associate Professor and Associate Plant Pathologist at
UC-Riverside, focusing on molecular plant-pathogen interactions.
Her presentation, sponsored by the Citrus Research Board, will
provide an update on her research to develop an Early Detection
Technology that will give citrus growers the ability to identify
positive HLB trees much earlier than current science.
9:00 AM – 10:00 AM Tradeshow Open
10:00 AM – 11:00 AM Workshop Sessions C & D
C - The “Social” Farmer
Facebook, Twitter, Instagram…like it or not, social media is here to
stay. Fortunately for farmers, people WANT to hear from you. This
workshop will delve into how you, the citrus grower, can leverage
social media to tell your story and correct the misperceptions
spread by activists about how you farm. The majority of
Attend this workshop to learn more about this social media “stuff”
and HOW you can use it, and WHY you should be a “social” farmer.
D – “21st Century Citrus Breeding in the Age of Huanglongbing”
By Ed Stover, Research Horticulturist, USDA-ARS, Ft. Pierce,
Florida
Dr. Stover participated in a Scientific Research Review Panel
empowered by the Citrus Research Board in 2015 to evaluate
citrus variety breeding programs currently conducted at the
Lindcove Research and Extension Center and at UC-Riverside. In
this presentation, he will provide growers a summary of the
findings and relate additional research being conducted in Florida,
Texas and other citrus producing regions.
11:00 AM – 12:00 PM Tradeshow Open
12:00 PM – 1:30 PM Luncheon
Huanglongbing: Lessons from the Frontline
Citrus growers in Florida, and now Texas, are on the frontline in the
fight against the Asian citrus psyllid and Huanglongbing. Attend
the 2016 Showcase luncheon to hear from growers in Texas and
Florida on their experiences farming in the midst of
Huanglongbing infestation. This moderated discussion will also
include perspectives from Florida Citrus Mutual and Texas Citrus
Mutual presidents Mike Sparks and Dale Murden. They will discuss
the challenges their industries have faced and how California can
avoid a similar fate.
1:30 PM – 2:30 PM Trade Show Open
2:30 PM – 3:30 PM Workshop Session E
Labor Laws and Regulations: What you need to know in 2016
The Federal and State courts have redefined joint liability and
there will be impacts to employers in the citrus industry. It is
imperative that all parties involved in an employment contract
comply with the laws and regulations in the same manner. To
meet this challenge, the citrus industry lead by CCM has created
the California Agricultural Labor Association (CALA) which will
serve FLCs, growers, and packinghouses alike. In this session, you
will hear about the most up-to-date changes to CA labor laws and
the benefits of an organized agricultural employer association.
www.CitrusResearch.org | Citrograph Magazine 21
FOR MORE INFORMATION, PLEASE CONTACT
CALIFORNIA CITRUS MUTUAL AT 559.592.3790
22
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Scan for crop advice &
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23
The Summit’s afternoon session addressed the question, “Are HLB early detection technologies viable for the California citrus industry?” Featured were (left
to right) Mary Palm, Ph.D., Leader of the USDA’s HLB Multi-Agency Coordination Group; Victoria Hornbaker, CDFA Citrus Program Manager; Cheryl Blomquist,
Ph.D., CDFA Senior Plant Pathologist; Philip Berger, Ph.D., USDA APHIS PPQ Executive Director of Science and Technology; Ed Civerolo, Ph.D., CRB Advisor and
Citrograph Interim Executive Editor; Robert Atkins, Statewide Coordinator, CPDPP; and Richard Bennett, CRB Chairman.
IMMEDIATE ACTION IS NEEDED
Summary of the HLB Summit Morning Session
Beth Grafton-Cardwell, Mike Irey, David Bartels, Carolyn Slupsky and Neil McRoberts
O
n December 1, 2015, a meeting about huanglongbing
(HLB) was conducted by the Citrus Research Board and
California Citrus Mutual at the Visalia Convention Center.
The conference was attended by nearly 250 citrus industry
members. The goal of the morning session was to provide upto-date information on the devastation that HLB is causing
the Florida citrus industry, discuss HLB finds and the potential
for spread in California, describe new technologies to detect
the disease and make recommendations to the citrus industry
for moving forward.
Beth Grafton-Cardwell, Ph.D., Director of the Lindcove
Research and Extension Center, moderated the session. She
began by describing the symptoms of HLB and showed a
map of the HLB-infected trees removed in southern California.
While to date, only 11 trees infected with the bacterium that is
associated with HLB have been identified, it is likely there are
more infected trees in California. The spread of ‘Candidatus
Liberibacter asiaticus’ (CLas) from tree to tree is very
rapid, because the Asian citrus psyllid (ACP) vector lays its
eggs in the same place it feeds and infects the plant. When
nymphs hatch, they feed on the localized bacterial infection
and take the bacteria with them when they molt into adults
and fly away. Psyllid control is a temporary, but important
strategy to buy time for scientists to find a cure for the disease.
Grafton-Cardwell emphasized that management focus
needs to change from ACP to HLB, and the citrus industry
needs to lead efforts to prevent man-made movement of
24
psyllids that are potentially carrying CLas, the bacterium
associated with HLB.
Mike Irey, Director of Research and Business Development,
Southern Gardens Citrus in Florida, provided information
on the devastation that HLB has caused in that state. It is
estimated that 100 percent of Florida groves have some
level of HLB infection. He reported that costs are double
to triple what they were 10 years ago due to increased
application of pesticides for ACP, nutritionals, tree removal,
etc. Additionally, the state is experiencing a 50 percent
reduction in yields in spite of these much greater inputs. In
Florida, they are finding that maintaining optimal tree health
is vital in the presence of HLB, but even with optimum tree
health, growers continue to have production losses due to
HLB. Growers must minimize tree stress of any type – i.e.,
Phytophthora, bicarbonates in water, frost, etc. It takes very
few psyllids to spread the disease-associated bacterium, and
it is very difficult to completely eliminate psyllids from the
groves. However, growers cannot relax ACP control, because
high psyllid numbers will infect trees with HLB-associated
bacterium at a much faster rate than low psyllid numbers. The
more infection sites a tree has, the faster the tree expresses
the disease.
Irey recommended that California should stay in HLB
eradication mode as long as possible because it is
very difficult to control the disease once it becomes
established. He stated that “a little pain on the front end
can buy you a lot of time and increased profits on the back
end.” Early HLB detection is the key to getting ahead of the
disease spread. Having a large psyllid and plant sampling
volume (large number of samples, wide area tested, etc.) is
the most important factor to maximize detection of HLB. He
recommended that Californians utilize both validated tests
and new technologies – not relying on just one or the other.
David Bartels, Ph.D., Entomologist, USDA APHIS PPQ, Mission
Laboratory, Texas, described HLB survey efforts going on in
Texas and California to improve the detection of infected trees.
Currently, PCR (polymerase chain reaction) is the primary tool
for detecting the CLas in psyllids and citrus plants. There
are two types of PCR being utilized, conventional PCR and
quantitative PCR (qPCR). Both Texas and California are using
qPCR for processing samples, because this method can
rapidly process very large numbers of samples and potentially
detect lower amounts of bacterial DNA in samples. However,
conventional PCR provides the regulatory confirmation of
HLB infections since the resulting product can be sequenced
to provide a DNA match. Texas qPCR psyllid testing showed
a shift in psyllid sample results from suspect (Ct-values 3339) to clearly positive (Ct-values < 32) over a two-year period;
then one to two years later, many trees with HLB disease were
detected.
If the disease progresses similarly in California, we should
expect to see additional clearly positive psyllids in the
coming year. To act conservatively and get ahead of the
disease spread, the California citrus industry needs to follow
up on the areas of California that have had ACP with Ctvalues in the suspect 33-39 range and test more psyllids
and trees in those areas. Testing ACP samples is extremely
useful for locating regions with HLB infection, since psyllids
are accumulating bacteria as they feed on infected trees.
A CLas-positive adult psyllid doesn’t tell us exactly which
tree is positive, because the adults move around, but it
tells us that the bacterium is in the area. Because adult
psyllids tend to be on the borders, citrus growers should
initially focus their HLB detection efforts on the borders
of their orchards.
Carolyn Slupsky, Ph.D., Professor, Department of Nutrition and
Department of Food Science and Technology at the University
of California, Davis, discussed the early detection technologies
(EDTs) being developed to detect HLB and why growers should
utilize them in addition to PCR. To contain the disease, we
need to utilize all of the detection technologies available
to us, some of which are direct detection of CLas, and some of
which are indirect detection of the HLB-associated bacterium.
Direct detection technologies (such as PCR and antibody
tests) detect the presence of the bacterium or its by-products.
The amount of bacteria or bacterial products in these
samples must exceed a specific threshold for the test to be
determined positive. In the case of HLB, the bacterium is
often not distributed evenly throughout the tree, and thus,
sampling can be an issue – choose the wrong plant tissue, and
one will miss the bacteria. For this reason, direct detection
technologies can result in “false negatives.”
Indirect detection technologies (such as soluble metabolites,
volatile metabolites [which encompasses testing with
instrumentation or testing with dogs], protein, small RNAs,
spectral imaging and microbial communities) detect
changes in the tree that are part of its defense against
the HLB infection. Because that defense response occurs
throughout the tree, indirect detection methods yield more
“true positives” than direct detection, and have the potential
to detect infection months to years prior to visible symptoms.
However, these tests may result in some “false positives,” since
other conditions in the plant could mimic infection by CLas. A
combination of several early detection technology (EDT)
tests would improve confidence in the results and provide
more timely information on the status and spread of the
disease in California.
25
The room was packed for the recent HLB Summit.
Neil McRoberts, Ph.D., Associate Professor in the Department
of Plant Pathology at the University of California, Davis, used
an epidemiological model approach to show how factors that
influence HLB disease spread are related. The California and
Arizona citrus industries are focused on reducing the rate of
new infections to limit bacterial inoculum by lowering psyllid
densities and surveying for infected trees. However, psyllid
suppression will slow down the rate of disease spread, but
not stop it completely. To shut down HLB disease spread,
it is imperative to find and remove infected trees quickly.
However, there are technical problems with achieving highly
accurate early diagnosis, so acceptance and removal of “false
positives” is a reality.
In the meantime, while early detection is still in development,
it is important to avoid contributing to the HLB problem:
respect quarantines, get involved in Psyllid Management
Areas (PMAs), start monitoring groves for disease and
motivate complacent neighbors. Citrus growers need to
take the initiative to test trees in their groves and remove
suspected HLB-positives, using whatever diagnostic tools
are available. This activity does not need to await regulatory
confirmation of positives. Thanks to lessons learned at
Florida’s expense, the opportunity exists to get ahead of HLB
in California, provided the appropriate cooperation occurs
within the industry, and between the industry and regulatory
agencies, so that resources are appropriately allocated.
Immediate action is needed. Many of the important
problems in dealing with HLB aren’t caused by HLB or ACP,
but by people.
Beth Grafton-Cardwell, Ph.D., is an IPM specialist and
research entomologist with the Department of Entomology
at the University of California, Riverside, and also Director
of the Lindcove Research and Extension Center. Mike
Irey is Director of Research and Business Development at
Southern Gardens Citrus in Florida. David Bartels, Ph.D.,
is an entomologist with the USDA APHIS PPQ in Mission
Laboratory, Texas. Carolyn Slupsky, Ph.D., is a professor
in the Department of Food Science and Technology at the
University of California, Davis. Neil McRoberts, Ph.D., is an
associate professor in the Department of Plant Pathology at
the University of California, Davis.
26
CLas (HLB) Detection
Technology Descriptions
Direct HLB Detection Methods (detects CLas using
psyllids or plant tissue)
• Polymerase Chain Reaction (PCR)
- Conventional PCR detects DNA of the CLas
bacteria and products that can be sequenced
- Quantitative PCR (qPCR) detects CLas DNA using
light and measures quantity of DNA
• Antibody tests use antibodies that react with CLasderived proteins present in the phloem of the plant
Indirect HLB Detection Methods (tests the
responses of the tree to infection by CLas bacteria)
• Soluble metabolites measure the response of the
tree to infection by CLas bacteria using metabolites
extracted from plant tissue
• Volatile organic compound (VOC) detection measures
the response of the tree to infection through a VOC
profile
- Electronic sniffer technology
- Canine detection
• Protein tests measure the response of the tree to
infection using host plant proteins extracted from
plant tissue
• Small RNAs measure the response of the tree to
infection using small RNAs extracted from plant tissue
• Optical imaging measures the response of the tree to
infection through measurement of reflectance spectra
• Bacterial communities use qPCR to measure changes
in the bacterial communities in the phyllosphere
(above-ground portion of the tree) or roots as a
consequence of infection
27
About 300 biocontrol agents (Tamarixia radiata)
are introduced into the ACP foliage-infested
cage to begin the mass-production process
(Photo credit: Dan Flores)
HOPE FOR THE FUTURE
OF CITRUS
Early Detection, Sustainability, Treatment and Vector Control
Yindra Dixon
H
uanglongbing (also known as HLB or citrus greening) is
a devastating citrus disease caused by the bacterium
‘Candidatus Liberibacter asiaticus’ (CLas). Investment in HLB
research is a pivotal need for citrus growers across the country.
The Huanglongbing Multi-Agency Coordination (HLB MAC)
Group, a unique public-private partnership that leverages
the breadth of government resources, the depth of the citrus
industry’s experience and the expertise of top researchers
nationwide, was created in 2013 by the U.S. Department of
Agriculture (USDA) to fund technology to reduce the impact of
HLB. In just two years, the HLB MAC Group has advanced latestage research to curb the spread of HLB and the Asian citrus
psyllid (ACP). With only $20 million in funding, the group has
mobilized research to produce solutions in four focus areas:
early detection, sustainability, treatment and vector control.
EARLY DETECTION
The early detection projects funded through the HLB MAC
Group included optimizing an antibody-based detection
protocol, similar to tests used to detect some human
28
diseases; a standardized root sampling protocol; and canine
HLB detection. Of these three, canine detection achieved
exceptional results in just one year.
Tim Gottwald, Ph.D., a scientist from the USDA’s Agricultural
Research Service in Florida, is working with Peggy Heiser
from Coast to Coast to train dogs to detect HLB-affected trees
(Citrograph: Fall 2014). The trees are inoculated using both
grafting (putting an infected scion on rootstock) and psyllids
(allowing CLas-positive psyllids to feed on a tree). Gottwald
tested a robust set of variables that affect detection, including:
• symptomatic trees at various ages;
• asymptomatic but CLas-positive trees as confirmed by
polymerase chain reaction (PCR) tests;
• asymptomatic trees with such a low level of the bacterium
that PCR tests are negative;
• residential and commercial growing environments;
Disease detection dogs trained to identify HLB-positive citrus trees. (Photo credit: Tim Gottwald, Ph.D., USDA-ARS)
• multiple citrus species, including lemon, lime, grapefruit
and sweet orange;
• various wind conditions; and
• different geographical locations, including Texas and Florida.
In field tests using different variables, the dogs can detect HLB
with 99.43 percent accuracy. Additionally, the dogs can find
the tree with the highest concentration of the HLB bacterium,
which may represent the first infected tree in that grove. This
detection skill could become a way for the dogs to identify
the source of an infection, something no other early detection
method can do. Disease detection dogs show great promise
as a reliable tool for growers and in residential settings in
Texas and California.
TREATMENT
The hottest new treatment for HLB-affected citrus plants is
thermotherapy. Thermotherapy is the application of heat
(typically steam) to an HLB-affected citrus tree to reduce the
impact of HLB infection and to extend the productive life of
these trees. Reza Ehsani, Ph.D., at the University of Florida
developed an in-field thermotherapy technology that he
was able to bring to commercial scale through funding from
the HLB MAC Group. The project has resulted in a licensing
agreement with Premier Energy Technology, Inc., who lauded
the improvement in fruit size, weight and quality from
thermally-treated trees only one season after treatment.
How does it work? Most thermotherapy applications utilize
steam tents over the trees that apply uniform heat throughout
the tree. By steaming the trees for a specific temperature and
duration, the results are new flush (plant growth) and greater
fruit weight, compared to unhealthy trees infected with CLas.
Thermotherapy is a critical tool for growers in Florida where
more than 95 percent of all commercial citrus groves are
HLB-affected. Two companies developing thermotherapy
machinery using the HLB MAC funded technology are
expected to treat more than one million trees in early 2016.
VECTOR CONTROL
One of the ways to reduce spread of the HLB bacterium is
to reduce the population of the ACP, which transmits CLas.
Tamarixia radiata, a biocontrol agent for ACP, is a vector
control available for use in residential areas and as part of an
29
Field insectary cage created by David Ways, Skeeta, Inc. (Photo Credit: Dan Flores)
organic control program. The HLB MAC group invested in multiple biocontrol projects that have increased the rearing capacity
of T. radiata to nearly three times what it was just a year ago. Researchers reported parasitism rates up to 70 percent in some
regions resulting in a corresponding 85 percent reduction rate in the vector population. The HLB MAC Group also provided
funds for production and release of a second biocontrol agent in California.
In-field thermotherapy system in operation (photos courtesy of Reza Ehsani, Ph.D., University of Florida).
30
Comparison of roots at various stages of HLB infection (Photo credit: Evan Johnson, Ph.D., UFC-IFAS-CREC)
In Texas, the public engaged directly in biocontrol by offering
their lemon or lime trees for mass production of T. radiata.
Cages are placed over the ACP-infested tree, and the biocontrol
agent is then released into the enclosure. USDA’s Daniel
Flores, Ph.D., developed this innovative approach that allows
the biocontrol wasp to mass reproduce in the cage, yielding
up to 12,000 wasps per cage. After the cage is removed, the
biocontrol parasitoids can freely move to other trees.
The entrepreneur who designed the cages, David Ways of
Skeeta, Inc., worked with the team at the USDA-APHIS-Center
for Plant Health Science and Technology laboratory in Mission,
Texas, to create, test and redesign the cages to accommodate
different tree sizes and to increase portability. It resulted in
two designs that can be adjusted for various tree sizes and fold
up to the size of a king-size pillow. The cages cost an average
of $1,500 per unit and provide an effective, reusable tool most
suitable for dooryard citrus in urban areas.
SUSTAINABILITY
The HLB MAC Group funded sustainability projects that varied
from cultural practices like integrated management to rapid
propagation of resistant rootstocks. Each method tested the
best approach to improving and ensuring the long-term
health of non-infected and infected trees. With only two years
of research, assessing long-term efficacy of sustainability
practices is a long process.
While the HLB bacterium is transmitted by ACP feeding on the
foliage, CLas quickly moves to the root system causing damage
that impairs root uptake of water and nutrients into the tree.
Jim Graham, Ph.D., a soil microbiologist at the University of
Florida Citrus Research and Education Center, discovered
an immediate 30-50 percent loss of roots in HLB-infected
trees, which occurs even before trees are symptomatic. With
HLB MAC Group funding, he was able to identify a solution
to improve root density and overall tree health in Florida
groves — soil acidification.
Root health can greatly affect a citrus tree’s susceptibility to
various pathogens, including CLas. To improve root health,
Graham prescribes measuring the bicarbonates in the
irrigation water, as well as measuring pH in the root zone to
assess the need to acidify the rhizosphere soil. The optimal soil
pH is 6.5 or lower, depending on the rootstock. Acidification
31
HLB MAC Biocontrol Research Group.(Photo credit: Deborah Millis) First Row (L to R): Grace Radabaugh, Erica Kistner, Mark Hoddle, Richard Stouthamer,
Kenneth Bloem; Second Row (L to R): Matt Ciomperlik, Raina King, Andrew Chow, Brian Taylor, Mary Palm, Raju Pandey , Dan Flores, Gregory Simmons; Third
Row (L to R): Janet Fults, Greg Miller, David Ragsdale, Chris Kerr, Mamoudou Setamou, Kevin Heinz, Trevor Smith, Eric Rohrig
in combination with fertigation, which supplies nutrients and
water in small amounts more frequently, improves root density
and root health. These management practices lead to a better
balance of nutrients in the tree and visual improvements in
tree vigor by the following year. While aggressive ACP control
and other mitigation techniques for HLB should continue to
be used, soil acidification shows promise for citrus growers
by identifying the root cause for loss of tree health and
susceptibility
to biotic and abiotic
stresses.
AWPCitrographFINAL.pdf
1
6/17/14
1:14 PM
WHAT’S NEXT FOR THE HLB MAC
GROUP?
The HLB MAC Group has had multiple successes in its short
history:
• Successful engagement of a cross-jurisdictional group.
The HLB MAC Group comprises representatives from multiple
federal agencies, state departments of agriculture, industry
groups, researchers, growers and scientists. Despite divergent
interests, the Group successfully focused on near-term tools
and solutions to combat HLB and restore the citrus industry’s
production levels.
• Successful allocation of $20 million to fund near-term
solutions to combat the effects of HLB and ACP. In addition,
to the projects listed above, the HLB MAC Group funded 27
projects over two years beginning in 2014.
• Creation of a national collaboration of biocontrol
researchers. The researchers collaborated to create
standardization of measurement, reduce duplication of
research efforts and gain a better understanding of the
differences in type, climate-sensitivity and efficacy of
biocontrol agents.
C
M
Y
CM
MY
Going in to the third year, the HLB MAC Group plans to
continue to identify projects that will directly impact the
effects and spread of HLB, controlling the bacterium and the
vector. The Group is developing a repository of citrus-related
research for easy global access by researchers, growers and
regulators. The outlook for citrus production seems grim in
Florida, according to some recent reports, but there is hope
from HLB MAC-funded tools available today to reduce the
proliferation of the Asian citrus psyllid and the spread of
huanglongbing.
CY
CMY
K
32
Yindra Dixon is a public affairs specialist for the Animal
Plant Health Inspection Service (APHIS), a division of the
U.S. Department of Agriculture (USDA) responsible for
citrus pests and diseases.
33
A tree showing citrus stubborn
disease symptoms. (photo
courtesy of Ray Yokomi, USDA,
ARS, Parlier, California)
CALIFORNIA CITRUS THREATS
Laurynne Chetelat, Elizabeth Chin, Darya Mishchuk and Carolyn Slupsky
INTRODUCTION
There are three major, economically important infectious diseases of citrus that have similar symptoms and
epidemiology. Among them, huanglongbing (HLB or citrus greening disease) is causing the greatest loss of
fruit yield and tree decline worldwide, but citrus stubborn disease (CSD) and Citrus tristeza virus (CTV) may
also cause notable fruit loss depending on host, pathogen and environmental factors. In the United States,
orange and grapefruit production have been diminishing largely due to HLB; approximately 28 percent and
26 percent less oranges and grapefruit, respectively, will be produced in 2015 compared to 2010-11 (USDA
Foreign Agricultural Service, Citrus: World Markets and Trade report, July 2015) which equates to losses of
approximately 2.2 million metric tons of orange and 298,000 metric tons of grapefruit. Despite such big
economic impacts, there is still no efficient way to stop the spread and manage these citrus diseases. We
outline below, and in Table 1 and Figure 1, general information about HLB, CSD and CTV. 34
Figure 1. Comparison of the leaf and fruit symptoms of citrus infected with HLB, CSD or CTV, and leaves from zinc, iron and magnesium deficient citrus trees.
HUANGLONGBING (HLB) is the most devastating citrus disease
worldwide, affecting all commercial cultivars. In the U.S., it currently
is found in Florida, Texas and Southern California. HLB is associated
with three bacterial species: ‘Candidatus Liberibacter asiaticus,’
‘Ca. Liberibacter americanus’ and ‘Ca. Liberibacter africanus’ – each
species named after its continent of emergence. In the U.S. and
globally, ‘Ca. Liberibacter asiaticus’ (CLas) is the most prevalent of
the three species. The term ‘Candidatus’ designates that the species
is unculturable on artificial nutrient media1 in the laboratory, which
makes it difficult to study. CLas produces the most severe symptoms
and is heat-tolerant, while ‘Ca. L. africanus’ is heat-sensitive and
does not induce well at high elevations. ‘Ca. L. americanus’ induces
symptoms with similar severity to CLas, but like ‘Ca. L. africanus’, it is
heat-intolerant.
Despite these differences, all three species are limited to the phloem2
sieve tubes (Laflèche and Bové 1970a, Ding et al. 2015) of their
host citrus plant and are not uniformly present throughout the
tree; additionally, the pathogens’ population also can vary with the
seasons. Nonetheless, HLB bacteria are consistently associated with
symptoms of yellow shoots, blotchy mottle and corked veins on
the leaves that are unevenly distributed throughout the tree. Visual
identification of HLB is difficult as these symptoms may resemble
zinc or nutrient deficiencies or environmental stresses, and may
vary by season, rootstock and scion. The fruit may be small, green
and lopsided, and ripen in the reverse direction from the stylar3. If
infected trees are left in the field, their fruit production will decrease,
and much of the fruit will drop before fully ripening. Advanced stages
of infection involve twig dieback, reduced tree size and premature
tree death. Symptoms may appear months or years after initial
infection. This incubation period is especially problematic, as it allows
for infected trees without visual symptoms to remain unidentified
and serve as sources for pathogen spread. Identifying asymptomatic
diseased trees is, therefore, imperative to curbing HLB spread.
CLas is spread by the Asian citrus psyllid (ACP). This phloem-feeding
insect acquires the pathogen by feeding on infected plants and
transmits the pathogen to healthy plants by depositing it into the
phloem. Therefore, identifying CLas-infected trees and controlling
ACP populations are critical for preventing spread of the disease.
CITRUS STUBBORN DISEASE (CSD) occurs in arid and semi-arid
regions and has only been found in Southern California and Arizona
in the U.S. It is caused by the bacterium Spiroplasma citri. Like CLas,
S. citri also exclusively resides in the sieve tubes (Bové 2003, Laflèche
and Bové 1970b) of host plants. Its distribution in the tree is sporadic
and influenced by seasons and temperature. Symptoms of CSD are
most prominent during the hot summer months (when bacterial titer
35
Table 1. Summary of information about three citrus diseases (HLB, CSD and CTV).
is highest) and are similar to those of HLB and zinc deficiency: green
stripes on yellow leaves, lopsided fruit, reduced fruit production and
stunted growth. Though CSD does not cause early tree death, it can
lead to considerable losses in fruit quality and production, especially
in young trees. Some cultivars such as lemons, limes, trifoliate orange
and trifoliate hybrids, are tolerant to CSD. Like HLB-diseased trees,
CSD-diseased trees may not present symptoms for months to years.
S. citri is transmitted by leafhoppers (Circulifer tenellus and
Scaphytopius nitridus), which infrequently feed on citrus. Still, this
irregular feeding is adequate to infect a tree. Pest management is
not enough to prevent CSD, as spraying insecticides and removing
host plants for leafhoppers are not remarkably effective at reducing
leafhopper numbers. This is because leafhoppers migrate from the
valley to the foothills in winter months and feed on a wide variety
of plants, including weeds in the mustard family that carry the
CSD pathogen. Another option for preventing CSD is removal of
inoculum4 sources by destroying weeds harboring S. citri in groves.
Of course, this method will not destroy infected plants outside of the
grove, which constantly serve as sources of inoculum for invading
leafhoppers. One of the additional ways to manage this disease may
be timely identification and management of the infected trees and
areas containing the pathogen.
CITRUS TRISTEZA is caused by Citrus tristeza virus (CTV) and is
present in all citrus growing regions of the globe, including the U.S.
36
Like CLas and S. citri, CTV resides in the phloem, infecting the sieve
tubes, companion cells, and parenchyma cells (Dawson et al. 2013)
and causes a range of symptoms that sometimes leads to tree death.
Generally, CTV infection is associated with a decrease in fruit size,
leaf chlorosis5, corked leaf veins, stem pitting, twig dieback, reduced
growth and collapsing root system. The severity of symptoms
depends on the strain of CTV, the species and cultivar of citrus, and
environmental factors.
CTV pathogenesis generally falls into one of the three types: quick
decline, stem pitting or seedling yellows. Quick decline occurs when
a virulent strain of CTV infects a sweet orange scion tree on a sour
orange rootstock, preventing transport of photosynthates6, water
and nutrients between the canopy and the roots, thereby killing
the tree. Symptoms may take years to appear, but often arise more
quickly during hot, dry seasons because the lack of water stresses the
roots and further dehydrates the leaves and fruit. Stem pitting caused
by CTV occurs when a virulent CTV strain infects any combination
of rootstock and scion, and ultimately causes deep, long pits
underneath the bark. Consequently, leaves become chlorotic (have
reduced or lost green color), fewer and poorer fruit are produced, and
the tree halts growth. CTV can cause yellowing in seedlings, but also
can affect field trees that are top-worked7 with infected grapefruit or
lemon budwood. Fortunately, trifoliate rootstocks provide tolerance
to CTV.
Table 2. Helpful resources about citrus diseases.
CTV is vectored8 by many aphids, but the brown citrus aphid (BrCA,
Toxoptera citricida) transmits CTV most efficiently. Although, BrCA
can only transmit CTV within 24 hours after acquisition, it is still a
successful vector because it acquires CTV within an hour of feeding.
The wide distribution of CTV is mostly attributable to sharing of CTVinfected budwood. Hence, of particular importance for preventing
CTV spread is the use of certified pathogen-free budwood, as well as
prompt identification and removal of infected trees. Clean, pathogenfree, certified citrus budwoods can be acquired by contacting Citrus
Clonal Protection Program (CCPP) at UC Riverside: http://ccpp.ucr.
edu/.
PERSPECTIVES
These diseases are severe and will have considerable economic
repercussions if allowed to persist. While each disease is caused
by a different pathogen, their symptoms have many overlapping
characteristics not only with themselves, but other conditions such
as nutrient deficiencies (Figure 1). All three of these pathogens
reside in the phloem sieve tubes, and symptom manifestation can
sometimes take years after initial infection. Furthermore, these
pathogens do not evenly distribute themselves throughout the
tree. Their population and symptom expression are influenced by
changes in temperature with seasons. Detection methods that rely
directly on the pathogen’s presence may, therefore, be unreliable,
as they may not be sensitive enough to detect the pathogen at
low concentrations. Some additional helpful resources about these
citrus diseases are summarized in Table 2.
Their insect vectors facilitate dispersion of these pathogens; so
widespread pest management techniques are crucial for reducing
pathogen and disease spread. In many cases, however, pest
management alone is not sufficient to effectively reduce incidences
of these devastating citrus diseases. By the time the insect vector
has been trapped or killed, the pathogen already has been spread.
Early identification of infected trees will allow for intervention and
containment of infection.
One of the new promising solutions to help combat citrus diseases
is development and use of early detection technologies (EDTs). We
previously have described some of these EDTs in our Citrograph
article published in Winter 2014 (Chin et al. 2014). Many of these
EDTs are indirect detection technologies, detecting plant response
to infection, which may allow for detection of infection earlier than
direct methods such as quantitative polymerase chain reaction
(qPCR). The Citrus Research Board (CRB) is funding several studies to
determine the sensitivity and specificity of EDTs to detect HLB and
other citrus diseases including CSD and CTV.
Acronyms & Abbreviations
HLB = Huanglongbing
CSD = Citrus Stubborn Disease
CTV = Citrus Tristeza Virus
CLas = ‘Candidatus Liberibacter asiaticus’
ACP = Asian Citrus Psyllid
BrCA = Brown Citrus Aphid
EDTs = Early Detection Technologies
37
Understanding the shared and unique characteristics of these
citrus diseases, as well as the limitations and successes of current
management programs, is key for improving eradication efforts.
Widespread use of EDTs will help with effective management and
eradication of citrus diseases.
Laurynne Chetelat and Elizabeth Chin are graduate students,
Darya Mishchuk, Ph.D., is a staff research associate, and Carolyn
Slupsky, Ph.D., is a professor at the University of California, Davis,
Department of Food Science and Technology.
Laflèche, D., and Bové, J.M. 1970b. Mycoplasmes dans les agrumes
atteints de “greening”, de “stubborn” ou de maladies similaires. Fruits
25:455-465.
Glossary
Unculturable: a microorganism that is unable
to extensively propagate in controlled laboratory
conditions, outside of an organism.
1
Phloem: the plant vascular tissue that transports
sugars and other products of metabolism from the
leaves to all other parts of the plant. The phloem is
composed of the innermost sieve tubes, neighboring
companion cells, and outer parenchyma cells.
2
References
Bové, J.M., Renaudin, J., Saillard, C., Foissac, X. and Garnier, M. 2003.
Spiroplasma citri, a plant pathogenic Mollicute: relationships with
its two hosts, the plant and the leafhopper vector. Annual Review of
Phytopathology 41:483-500.
Stylar: the side of the fruit that is polar opposite the
stem attachment.
3
Chin, E., Mishchuk, D.O., Bruce, J., Cilia, M., Coaker, G., Davis, C., Jin,
H., Ma, W., Sellar, G., LeVesque, C., Godfrey, K. and Slupsky, C.M. 2014.
An interdisciplinary approach to combat HLB: research in UC Davis’
Contained Research Facility. Citrograph 5(1):28 – 34.
Dawson, W.O., Garnsey, S.M., Tatineni, S., Folimonova, S.Y., Harper, S.J.
and Gowda, S. 2013. Citrus tristeza virus-host interactions. Frontiers in
Microbiology 4(88):1-10.
Inoculum: the collection of a microorganism that can
be used to infect another host.
4
Chlorosis/chlorotic: unusual loss of green color in
leaves.
5
Photosynthates: products of photosynthesis (e.g.
sugars).
6
Ding, F., Duan, Y., Paul, C., Brlansky, R.H. and Hartung, J.S. 2015.
Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in
citrus and periwinkle by direct tissue blot immuno assay with an antiompA polyclonal antibody. PLoS ONE 10(5):e0123939.
Laflèche, D., and Bové, J.M. 1970a. Structures de type mycoplasma
dans les feuilles d’orangers atteints de la maladie du greening.
Comptes Rendus de l’Académie des Sciences Paris 270:1915-1917.
Top-working: the process of grafting a new cultivar
onto an already established tree with a developed root
system.
7
Vector: a small organism, such as an insect, that
carries a pathogen from host-to-host.
8
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CRB-FUNDED RESEARCH PROGRESS REPORT
Bar = 3 mm
A
B
Figure 1. (A) Navel orange fruit with symptoms of Septoria spot. (B) Close-up of lesion with whitish spore tendrils (arrows) exuding from pycnidia (sporulation).
(C) Lesion treated with an anti-sporulation fungicide with no sporulation present.
PRE- AND POST-HARVEST
FUNGICIDES FOR MANAGING
SEPTORIA SPOT
James Adaskaveg and Helga Förster
S
eptoria spot, caused by Septoria citri, is a fungal disease
that causes twig dieback, as well as leaf and fruit spots on
many citrus species worldwide. In some countries, however,
the disease has not been detected; thus, S. citri is a regulated
quarantine pathogen in these locations. Although Septoria
spot is considered a minor disease in the irrigated, lowrainfall citrus production systems of California and generally
occurs at a low incidence, the disease has prevented trade in
economically important markets such as Korea, due to the
detection of the pathogen in fruit upon arrival. One objective
40
of this research project is to develop new pre-harvest fungicide
treatments to prevent fruit infections of S. citri in the orchard.
Historically, copper applied as copper sulfate or as fixed
neutral formulations in a mixture with zinc sulfate and lime
has been a proven management strategy for reducing the
incidence of Septoria spot. Under California conditions, copper
applications generally are done as preventive treatments
before the winter rains at the beginning and during the harvest
season. In winters with low rainfall, one or two applications
Fungicides also were selected based on their potential for
obtaining tolerances and Maximum Residue Levels (MRLs)
in international markets. For example, polyoxin-D is a biofungicide and is exempt from tolerance in the United States;
whereas azoxystrobin is one of the most widely registered
fungicides in the world. Chlorothalonil has MRLs on citrus
in some countries. Therefore, this multi-site mode of action
fungicide also was evaluated and selected for registration on
citrus in the United States.
EFFECTIVENESS OF PRE-HARVEST
FUNGICIDES FOR DISEASE PREVENTION
Two to three field trials in each of the last five years (2011–2015)
were conducted at locations in Fresno County with consistent
occurrences of Septoria spot (i.e., disease developed in the
untreated control at high incidence). A single application
was done in November or January/early February, or two
applications were made at both timings.
C
Bar = 3 mm
have been highly effective. In winters with high rainfall, two
to three applications are required to manage the disease. This
has caused concern about the overuse of copper. Copper is a
metallic element that may accumulate in soils, potentially run
off in surface waters from the orchard or possibly contaminate
water supplies. It also may cause phytotoxicity when excess
amounts are applied. Overuse further may lead to resistant
populations of fungal or bacterial pathogens of citrus.
Therefore, there is a need to develop alternative non-copperbased compounds that can be rotated or mixed with copper
to reduce the overall amount of copper used per season.
NEW FUNGICIDES
New single-site, mode-of-action fungicides have been
developed by agrochemical companies around the world for
managing crop diseases. Based on their mode of action, these
materials are placed into chemical groups by the Fungicide
Resistance Action Committee (FRAC), a multi-company
organization with scientists who specialize in each of the
chemical groups.
New products represent an opportunity to select the most
effective ones for managing Septoria spot. We evaluated
the active ingredients from different groups with the goal
of eventually utilizing pre-mixtures or rotations of different
modes of action to reduce the selection of resistant subpopulations of the pathogen to any one compound.
Products evaluated included copper formulations with
reduced metallic copper equivalent (MCE) content (e.g.,
Kocide 3000, Badge X2 or SC, Cueva), Bravo WeatherStik,
Abound, Quadris Top, Luna Sensation, Merivon/Priaxor and
Tavano. Fruit were evaluated for disease in the spring of
each year. Lesions were verified as being caused by S. citri by
culturing the fungal pathogen or by using the polymerase
chain reaction (PCR) method of the Navel and Valencia Exports
to Korea (NAVEK) program. Septoria spot incidence varied
among years ranging from three to five percent during lowdisease years to 38 to 46 percent in high-disease years.
Our research demonstrates the effectiveness of new copper
fungicides (Table 1) used similar to traditional applications
with zinc oxide and hydrated lime or with hydrated lime alone
(similar to a Bordeaux mixture, but with a fixed copper instead
of copper sulfate). Therefore, we can manage the disease with
less metallic copper than using traditional formulations that
required higher rates of MCE.
Copper fungicides also are important for managing other
citrus diseases such as brown rot caused by Phytophthora
species. Keeping this multi-site mode of action group of
fungicides available is essential to the citrus industry in the
years to come. Still, having rotational products is important
to prevent overuse of copper products. This will prevent
phytotoxicity to trees and minimize risks of environmental
contamination of water-sheds and soil from orchard run-off
water.
In multiple trials over several years, we also showed that new
fungicides for the U.S. citrus industry, such as azoxystrobin
(Abound), chlorothalonil (e.g., Bravo WeatherStik), polyoxin-D
41
Table 1. Summary of pre- and post-harvest fungicides for managing Septoria spot of citrus.
FRAC
Usage
Fungicide*
Pre-‐harvest
Azoxystrobin
Chlorothalonil
Copper
Polyoxin-‐D
Azoxystrobin/difenoconazole
Azoxystrobin + chlorothalonil
Fluxapyroxad/pyraclostrobin
Fluopyram/trifloxystrobin
Post-‐harvest
Fludioxonil
Imazalil
Propiconazole
Pyrimethanil
TBZ
Azoxystrobin/fludioxonil
Registration International Rating***
Anti-‐
Prevention sporulation
11
Yes
Yes
++
+++
M5
Pending
Some
++
+
M1
Yes
Yes
+++
-‐-‐-‐
19
Yes
No
+++
-‐-‐-‐
3-‐Nov
Yes
Some
+++
+++
11 + M5 Yes/pending Yes/pending
+++
+++
7/11
Pending
Pending
+++
+++
7/11
Pending
Pending
+++
+++
Group**
Status
MRLs
12
3
3
9
1
11/12
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Some
Yes
Yes
Yes
NA
NA
NA
NA
NA
NA
++
+
+++
-‐-‐-‐
++
+++
* - Pre-harvest fungicides evaluated include Kocide 3000, Badge X2, Cueva (coppers), Abound (azoxystrobin), Quadris Top (difenoconazole/azoxystrobin),
* -‐ Pre-‐harvest fungicides evaluated Priaxor
include Kocide 3000, Badge X2, and
Cueva coppers), Abound (azoxystrobin), Quadris fungicides
Top Tavano
(polyoxin-D),
Bravo (chlorothalonil),
(fluxapyroxad/pyraclostrobin),
Luna(Sensation
(fluopyram/trifloxystrobin).
Postharvest
evaluated include: Graduate (fludioxonil), Graduate A+ (fludioxonil/azoxystrobin), Mentor (propiconazole), Penbotec (pyrimethanil), Deccocil (imazalil), and
(difenoconazole/azoxystrobin), Tavano polyoxin-‐D), ravo those
(chlorothalonil), riaxor (fluxapyroxad/pyraclostrobin), and Luna Alumni
(TBZ). Fungicide combinations with
a “/” (are
premixtures,Bwhile
with a “+” are P
tank
mixtures.
Sensation (fluopyram/trifloxystrobin). Postharvest fungicides evaluated include: Graduate (fludioxonil), Graduate A+ ** - Fungicide Resistance Action Committee (FRAC) Groups represent distinct modes of action. Numbers are single-site modes of action. Numbers preceeded by
(fludioxonil/azoxystrobin), entor Penbotec (pyrimethanil), Deccocil (imazalil), and Alumni (TBZ). Fungicide the
letter “M” are multi-site modeMof
action(propiconazole), fungicides.
combinations with a "/" are premixtures, while those with a "+" are tank mixtures.
*** - Rating: +++ = high effectiveness; ++ = moderate effectiveness; + = low effectiveness; and --- = ineffective. NA = not applicable (post-harvest fungicides do
not
infections
in fruit that
were
established in
injuriesGsuch
as “ice
mark” in d
the
field).modes of action. Numbers are single-‐site modes of ** eradicate
-‐ Fungicide Resistance Action Committee (FRAC) roups represent istinct action. Numbers preceeded by the letter "(Luna
M" are multi-‐site and
mode oAzoxystrobin
f action fungicides.
(Tavano/Oso),
fluopyram/trifloxystrobin
Sensation)
also was very effective, but we down-rated it
fluxapyroxad/pyraclostrobin
(Priaxor),+as
as relatively
new to+ a= “two
plus” rankingand because
we do not
want
to
*** -‐ Rating: +++ = high effectiveness; + =well
moderate effectiveness; low effectiveness; -‐-‐-‐ = ineffective. NA = not growers
applicable products
such
as
azoxystrobin/difenoconazole
(Quadris
Top),
use
this
fungicide
by
itself.
We
identified
and
helped
register
(post-‐harvest fungicides do not eradicate infections in fruit that were established in injuries such as "ice mark" in the field).
are highly effective in preventing and managing the disease. azoxystrobin in a pre-mixture with fludioxonil as a post-harvest
treatment (e.g., Graduate A+). To protect the fungicide in a
resistant management strategy, we tank-mixed the fungicide
with chlorothalonil or used it in the pre-mixture, Quadris Top,
where these treatments were ranked as highly effective.
Glossary
Phytotoxicity: Toxic effects of a chemical compound
on plant growth. Damage to plant due to toxicity
may be caused by trace metals, salinity, pesticides,
herbicides or plant-produced chemicals.
Fruiting structure: A specialized spore-producing
structure formed by fungal mycelia on plant surface.
Mycelial development: Extensive growth of fungal
mycelia or thread-like filaments on a synthetic agar
medium, in soil or on plant surface. Mycelia act by
absorbing nutrients and giving rise to spores.
Conidia (conidium – singular): Asexual fungal spores
produced on mycelium. Spores can be dispersed by
wind or rain splash in nature resulting in new disease
cycle on susceptible host plants under favorable
climatic conditions.
42
A summary of the pre-harvest fungicides evaluated is shown
in Table 1. Efficacy data from this research project has helped
or is helping in the registration of these fungicides on citrus.
Quadris Top was the first of the new products that has MRLs
in the United States and Korea. We are working with the
registrant and the California Citrus Quality Council (CCQC)
to obtain MRLs in all the major export markets of California
citrus. Polyoxin-D (e.g., Tavano) was registered in the United
States in early 2015 on citrus. This fungicide is a fermentation
product with very low toxicity and impact on the environment.
The United States Environmental Protection Agency (EPA)
classified it as a biopesticide with exemption from tolerance
status in the U.S. Still, international MRLs have to be pursued
with our trade partners.
ANTI-SPORULATION ACTIVITY OF PRE- AND
POST-HARVEST FUNGICIDES
Quarantine inspection programs are based on symptoms and signs of the
disease. Following the current United States-Korea agreement, identification of
Septoria spot is based on disease symptoms that include fruiting structures (i.e.,
pycnidia) and spores. Therefore, anti-sporulation fungicides that prevent pycnidia
formation and subsequent spore production in established infections are part of
an integrated (“systems”) approach for managing the disease.
Post-harvest fungicides were included in these evaluations. Fungicides that are
applied after harvest in the packinghouse are not effective in eradication of fruit
infections that were established in the field in injuries such as “ice mark.” Thus,
ratings of post-harvest fungicides as preventive treatments were not applicable.
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Cultural and fruit assays were done to determine the effectiveness of fungicides
in inhibiting sporulation. For cultural studies, agar plates were inoculated with
S. citri, incubated for two days to allow mycelial development and then treated
with fungicide solutions for 30 min. After seven days, plates were evaluated for
the presence of pycnidia and the amount of conidia in standardized areas. In fruit
assays, fruit were inoculated with S. citri, incubated until lesions began to develop,
dip treated with fungicides and then incubated until sporulation developed on
the control fruit.
Several fungicides with high anti-sporulation activity were identified. Among
those registered (or in registration) for pre-harvest use on citrus, only products
containing a Quinone outside inhibitor (QoI) compound (e.g., Abound, Quadris
Top, Priaxor, Luna Sensation) were highly effective (Table 1). Among post-harvest
fungicides, the QoI-containing Graduate A+, as well as propiconazole (Mentor)
inhibited sporulation of fruit lesions effectively. Thiabendazole (TBZ, Alumni)
and fludioxonil (Graduate) were moderately effective; whereas imazalil and
pyrimethanil (Penbotec) were only slightly or not effective, respectively.
SUMMARY
Septoria spot remains an important disease for the California citrus industry
to manage due to its quarantine status in valuable export markets. Options for
chemical management of the disease have expanded based on our research
project over the last several years. Whereas copper products were the only
effective field treatment in the past, several newer classes of fungicides have
been found to be equally effective. These can be applied in rotations with copper
products to reduce overall copper use and subsequently reduce the risk of
phytotoxicity and possible contamination of the environment.
Some products (e.g., Abound, Quadris Top, Bravo WeatherStik) were evaluated
in low and high rainfall years; whereas the performance of some of the newer
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45
DEVELOPING RESISTANCE
TO HLB
Chandrika Ramadugu, Manjunath L. Keremane, Thomas G. McCollum, David G. Hall
and Mikeal L. Roose
PROJECT SUMMARY
We have identified resistance and tolerance to huanglongbing (HLB) in many citrus relatives in a six-year long
field trial involving 886 trees. After confirming the response of selected accessions to disease challenge in
controlled greenhouse trials, we conducted citrus breeding using HLB resistant/tolerant citrus relatives. Thirtythree types of novel hybrid genotypes are now under evaluation in Florida. Promising hybrids will be useful as:
a) disease tolerant rootstocks;
b) approach grafts with the possibility of disease remediation; and,
c) breeding material to develop HLB tolerant scions by further crosses.
Long-term solutions for HLB will be possible with disease tolerant/resistant citrus cultivars.
46
Despite the cultivation of innumerable
varieties, the genetic diversity of
commercial citrus is low because
nearly all varieties within groups such
as oranges, Clementines, lemons
and grapefruit have nearly identical
genomes. Vulnerability to new diseases
is common in such situations. Wild
germplasm of crop relatives is often a
good resource for enhancing germplasm
and for developing new breeding lines
with improved characteristics. A good
example is the development of superior
rootstocks for apples, peaches and pears
using wild relatives. These rootstocks
currently are being used for a wide
variety of stone fruits (Guajardo et al.,
2015). Resistance to the grassy stunt virus
of rice cultivars was obtained in the1970s
from Oryza nivara, a wild progenitor of
cultivated rice. This resistance is now
bred into several rice cultivars emanating
from the International Rice Research
Institute in the Philippines (Ford-Lloyd et
al., 2011).
Citrus cultivation and cultural practices
have been altered in the past because
of disease problems. Since tristeza
disease became a serious problem in
the western hemisphere, trifoliates
and hybrids of trifoliate orange and
citrus largely replaced sour orange
rootstocks. Many bigeneric hybrids with
tolerance to biotic and abiotic factors
like Citrus tristeza virus, Phytophthora
and cold hardiness were developed.
The evaluation of trifoliate hybrids and
Figure 1. Leaf samples from selected susceptible, tolerant (detectable bacteria, little or no HLB symptoms)
selection of rootstock types for different
and resistant cultivars from the field trial in Florida.
situations was a time-consuming
Citrus industries all over the world are currently looking for process. Currently, trifoliate hybrid rootstocks are considered
solutions to a bacterial disease known as huanglongbing (HLB valuable in many citrus growing regions.
or citrus greening). Found in Asia more than a century ago,
the disease is relatively new to the western hemisphere and At the onset of the current HLB epidemic in Florida, a
is thought to be caused by a gram-negative, phloem-residing, three-pronged approach was recommended for disease
fastidious bacterium known as ‘Candidatus Liberibacter management: planting pathogen-free nursery trees, vector
asiaticus’ (CLas). It is primarily transmitted by the Asian citrus control and removal of infected trees. While all these
psyllied (ACP from an infected citrus plant to a healthy one. measures are still valuable, they are not sufficient for survival
In Florida, where HLB is prevalent, citrus production has been of the citrus industry. Long-term solutions are needed for
decreasing at an alarming rate (http://www.nass.usda.gov/ successful cultivation of citrus. Rendering the psyllid vector
Statistics_by_State/Florida/Publications/Citrus/cit/2015-16/ incompetent to carry the pathogen is one research approach
cit1015.pdf). The disease also is spreading in Texas and has being pursued by the “nuPsyllid” project. A more traditional
been found at two locations in California. Because of the dire approach involves rendering the plant incapable of harboring
consequences of HLB, there is an urgent need to find practical the pathogen.
solutions if citrus production is to continue.
47
Figure 2. Australian limes. A: Australian finger lime tree (Microcitrus australasica). B: A mature fruit (bar represents 1 cm), longitudinal and cross section of the
fruit (not to scale). C: Australian Desert lime tree (Eremocitrus glauca). D: Mature fruits (bar represents 1 cm), longitudinal and cross section of the fruit (not to
scale).
IDENTIFYING SOURCES OF HLB
RESISTANCE
Learning from other horticultural crops, it is likely that wild
relatives of cultivated crops often do possess resistance. This
valuable resource can be harnessed for developing resistance
to certain diseases in the cultivated crop. In an effort to identify
sources of resistance, we conducted a field trial in Fort Pierce,
Florida (CRB grant #5300-123 awarded in 2009 to Richard Lee
and many scientists in the present project), using 91 accessions
and a total of approximately 886 trees. We included many
common citrus cultivars and several other related genera in
48
this field experiment. Now in its sixth year, the field trial has
proven to be very useful in identifying sources of resistance.
Most citrus types and trifoliates that were included in the trial
were at least somewhat susceptible to HLB. However, several
citrus relative genera remained totally resistant (meaning
no detectable Liberibacter bacteria most of the time, except
for occasional transient replication, and no HLB symptoms)
after six years in the disease-ridden field. Among them, two
Australian genera, Microcitrus and Eremocitrus, along with
some of their natural hybrids with Citrus were of special
interest since these two genera are sexually compatible with
citrus and more importantly, because of the demonstrated
inheritance of such resistance in natural citrus hybrids.
Resistance in citrus relatives that are
sexually compatible with Citrus is
especially valuable since it is possible
to generate putative resistant types.
The field resistance was further
evaluated under controlled conditions
in the greenhouse. Selected natural
citrus hybrids with Australian citrus
genera in their parentage were
challenged by inoculations using CLas
positive psyllids. Psyllids were fed for
15 days on selected plants, thereby
exposing the plants to the pathogen
via psyllid feeding. Under these
circumstances of no-choice feeding,
it was determined that the resistance
observed in the field is real and also
that it is heritable.
BREEDING FOR HLB
RESISTANCE
Armed with this knowledge, we
are hoping to breed HLB resistance
traits into Citrus. Intergeneric crosses
between citrus and other relatives
have been reported in the literature
(Barrett, 1977; Iwamasa et al.,
1988). Figure 2 shows pictures of
trees and fruit of Australian Finger
lime (Microcitrus australasica) and
Australian desert lime (Eremocitrus
glauca), two promising plants selected
as male parents for the crosses.
Literature reports of putative HLBresistant Citrus and Poncirus cultivars
describe improved cultivars that have
a delay in the time it takes to develop
symptoms and generally have a
lower level of accumulation of the
HLB-asssociated CLas in comparison Figure 3. Leaf morphology of representative hybrids and some parental types. The bars located to the upper
right of each leaf represent 1 cm.
to commercial cultivars. While this is
desirable until a better alternative is
available, it will be extremely valuable
to develop more resistant types. If the HLB resistance or genera and generated about 1,000 hybrid seeds so far. Thirtyhigh level of field tolerance observed in our field trial can be three crosses yielded viable seeds (Table 1). Since many of these
transferred to cultivated citrus, we may have disease tolerant are zygotic seedlings, each plant is expected to be genetically
varieties.
unique. Figure 3 shows leaf morphologies of certain parental
types and representative hybrids. Leaves from two of the
We have now conducted about 3,500 wide crosses involving hybrid seedlings, 1164.1 and 1164.2, included in this figure
citrus and HLB-resistant/-tolerant Australian citrus relative are obtained from two different seeds of the same pollinated
49
Table 1. Seed parents and pollen parents used for generating viable
seeds from 33 crosses.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Seed parent
Australian Finger lime
Encore mandarin
Encore mandarin
Encore mandarin
Encore mandarin
Encore mandarin
Fallglo mandarin
Fallglo mandarin
Fallglo mandarin
Flying Dragon trifoliate
Fortune mandarin
Fortune mandarin
Fortune mandarin
Fortune mandarin
Pomeroy trifoliate
Pomeroy trifoliate
Pomeroy trifoliate
Rich 16-6 trifoliate
Temple tangor
Temple tangor
Webber-Fawcett trifoliate
Wilking mandarin
Wilking mandarin
Wilking mandarin
Wilking mandarin
Wilking mandarin
Wilking mandarin
Pollen parent
C146 trifoliate
Carrizo
Eremolemon
Hirado Buntan pummelo
Simmons trifoliate
Australian Desert lime
Eremolemon
Microcitrus inodora
Sydney hybrid
Microcitrus inodora
Australian Round lime
Microcitrus inodora
Microcitrus inodora
Sydney hybrid
Microcitrus inodora
Eremolemon
Microcitrus inodora
Sydney hybrid
Figure 4. Distinguishing hybrids based on a short sequence of a nuclear gene.
Mandarins have a gap (deletion) of eight bases in this region. A Mandarin X
Microcitrus will have both parental genotypes as shown.
50
fruit. The difference in leaf shape, margin,
thorn characters, etc. indicates the
diversity involved. In some hybrids, the
pollen parent can be easily identified
by leaf morphology. In situations where
the distinction is not as clear, we have
sequenced a small fragment of a nuclear
gene and identified the parents based
on characteristic patterns obtained.
EVALUATION OF
RESISTANCE
The most important objective of this
project is evaluation of hybrids for HLB
resistance. Experiments are in progress
in Fort Pierce, Florida, to challenge
hybrid plants with psyllid feeding. Since
each hybrid seedling may be unique, we
are in the process of creating duplicate
plants for all hybrids before exposing
them to the psyllid. The hybrids that
show resistance can be utilized in three
different ways: 1) as disease resistant
rootstocks; 2) as approach grafts capable
of imparting resistance to the scion; and,
3) for development of disease resistant
scions. Figure 5 gives an overview of
the strategies and expectations of the
breeding activities.
Figure 5. Breeding strategies and expectations.
In any breeding program, it is
very valuable to identify markers
associated with valued traits. We
will analyze the populations with
susceptible and resistant phenotypes
and associate them with either
morphological, anatomical or genetic
markers to facilitate further selection
of potentially useful types.
CRB Research Project #5200-147A
Chandrika Ramadugu, Ph.D., is an
associate project scientist at the
University of California Riverside
and is the principal investigator
(PI) on the project. Manjunath L.
Keremane, Ph.D., (co-PI) is a plant
pathologist at the USDA Date and
Citrus
Germplasm
Repository,
Riverside, California. Mikeal L.
Roose, Ph.D., (co-PI) is a geneticist
at the University of California
Riverside. Thomas G. McCollum,
Ph.D., (plant physiologist) and David
G. Hall, Ph.D., (entomologist) are coPIs working at the US Horticultural
Research Laboratory, Fort Pierce,
Florida.
References
Barrett, H.C. 1977. Intergeneric
hybridization of citrus and other
genera in citrus cultivar improvement.
Proc. Int. Soc. Citriculture 2:586-589.
Ford-Lloyd,
B.V.,
Schmidt,
M.,
Armstrong, S.J., Barazani, O., Engels,
J. et al. 2011. Crop wild relatives –
undervalued, underutilized and under
threat? Bioscience 61:559-565.
Guajardo, V., Hinrichsen, P. and Munoz,
C. 2015. Breeding rootstocks for
Prunus species: Advances in genetic
and genomics of peach and cherry as
a model. Chilean J. Agric. Res.75:17-27.
Iwamasa, M., Nito, N. and Ling,
J-T. 1988. Intra- and intergeneric
hybridization in the orange subfamily,
Aurantioideae. Goren R and Mendel K
(eds) Proc. 6th Int. Citrus Congress, Tel
Aviv, Israel, 123–130.
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51
Photo 1. Asian citrus psyllid adult
(Photo by J. Lewis)
DEVELOPMENT OF AN ACP
MANAGEMENT PLAN FOR
ORGANIC CITRUS
Jawwad A. Qureshi and Philip A. Stansly
SUMMARY
Control of the Asian citrus psyllid (ACP), vector for the phloem-limited bacterium ‘Candidatus Liberibacter
asiaticus’ (CLas) associated with huanglongbing (HLB or citrus greening disease) in all habitats – including
organic citrus – is critical for area-wide management of this vector-disease complex and sustainable citrus
production. Organic citrus is produced in California, as well as in Florida and Texas.
We evaluated the impact of three separate organic programs – organic insecticides applied alone (Program
1) or with horticultural mineral oil (Program 2) and insecticidal soap (Program 3) – compared with one
conventional program on populations of ACP and beneficial insects in bearing citrus trees during dormant
and growing seasons in southwest Florida. During the dormant winter season, Pyganic alone or with 435
oil or M-pede applied in November, December and January, and Danitol applied in November and January
52
all significantly reduced ACP through the first week of March. This was when ACP adult numbers started to
escalate with the organic programs while still held to the 0.1 per tap sample threshold in the conventional
program. Pyganic with M-pede or 435 oil performed better than Pyganic alone. Six and five applications in the
organic and conventional programs, respectively, were made during the growing season. Organic Programs
2 and 3 with oil or soap, respectively, used 50 percent less insecticides, while providing better control than
Program 1 with insecticides only. However, ACP population was reduced more in the conventional program.
Lacewings, spiders, ants and lady beetles were observed in all programs that also may have contributed to ACP
reduction. Tamarixia radiata was released in all programs, but more were recovered from ACP nymphs in the
trees from the organic program compared to the conventional program.
Significant effects of organic insecticides with 435 oil or M-pede on ACP indicate potential use in all citrus,
including where conventional products may not be appropriate. In the coming cycle, we will repeat and extend
these studies to confirm results, include additional products and evaluate for impacts on ACP, other pests and
beneficial insects.
BACKGROUND
‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited
bacterium known to be associated with HLB, is vectored by
ACP (Photo 1). The ACP adult is responsible for spreading CLas
through its movement, whereas nymphs primarily acquire the
bacterium. Therefore, it is important to control both life stages.
Predatory beneficial insects generally are larger than their prey
and kill or consume more than one type of prey. Lady beetles,
lacewings, spiders and ants attack ACP, citrus leafminer (CLM),
thrips, aphids and other insect pests, and thus are important
for overall citrus pest management.
The small parasitic wasp, Tamarixia radiata, contributes to ACP
control through both feeding and parasitization of nymphs
(Photo 2). The female lays her egg under
the body of the mid-age nymph. Upon
hatching, the developing larva consumes
the body contents of the host and finally
pupates inside the remaining “mummy.” T.
radiata is now mass-produced and released
in Florida, California and Texas to control
ACP.
Citrus trees go through periods of dormancy
during cold or dry weather, producing little
or no new growth. Adult ACP living on
these trees need to wait for new growth
to emerge and lay eggs. Predators and
parasitic wasps also are attracted to these
young shoots or flush where they are
searching for their prey. Therefore, applying
sprays of broad-spectrum insecticides prior
to flush reaps the maximum benefit in
suppressing adult ACP while conserving
key beneficial insects. Insecticidal sprays
made during winter, before bud break, in
Florida are commonly known as dormant sprays. The aim
is to reduce psyllid entry into spring flush and, therefore,
subsequent reproduction during the growing season.
In this project, we are focused on developing holistic ACP
management programs for organic citrus, which is grown
more in California than any other state. Findings will be
useful for organic growers to manage ACP in their groves
and contribute to its area-wide management by reducing the
spread to conventional citrus and other habitats. Conservation
of naturally-occurring populations of beneficial insects and
augmentation of T. radiata will be useful for ACP control across
habitats. In contrast, synthetic chemicals are expensive and
not always welcomed in residential areas that may be suitable
for organic products.
Photo 2: A female Tamarixia radiata laying egg on an ACP nymph. (Photo by J. Lotz).
53
Table 1. Insecticides, rates, manufacturer and timing of spray applications in organic and conventional programs made using final spray volume
of 100 gallons per acre.
RESEARCH OBJECTIVES
1
Determine the effectiveness of the organic insecticide
Pyganic® (natural pyrethrum) to suppress ACP during
dormant winter months in comparison with Danitol®
(synthetic pyrethroid extensively used for ACP control) as a
conventional grower standard.
54
2
Evaluate rotations of organic products potentially effective
against ACP for impact on ACP and its natural enemies
during the growing season.
3
Release and evaluate Tamarixia radiata to determine the
feasibility of parasitoid use in conjunction with insecticides.
DESIGN, TREATMENTS
AND SAMPLING
PROCEDURES
One study site consists of a 22-acre block
of mature Valencia oranges in Hendry
County, Florida. The block was divided
into 20 plots each with three to five rows
and 50 trees distributed among three
organic programs, one conventional
program (Table 1) and one untreated
control in a randomized complete block
design experiment with four replicates.
Organic insecticides alone (Program 1)
or rotated with 435 oil (Program 2) and
M-pede (Program 3) were evaluated.
Synthetic insecticides were evaluated in
the conventional program (Table 1).
Photo 3. Durand Wayland AF100-32 air blast speed sprayer (Photo by J. Qureshi).
Treatments included:
Organic program 1: Nine treatments
using seven insecticides (Pyganic, AzaDirect, Grandevo, Azera, Venerate,
Entrust and Surround);
using four insecticides and horticultural
mineral oil (Pyganic, Aza-Direct, Azera,
Entrust and 435 oil);
using four insecticides and insecticidal
soap (Pyganic, Aza-Direct, Azera,
Entrust and M-pede); and Conventional
program: Seven treatments using six
insecticides (Danitol, Closer, Movento,
Micromite, Imidan and Dimethoate),
Photo 4. Demonstration of the stem tap sampling method and resulting adult psyllids (Photo by P.
Stansly).
Horticultural mineral oil (HMO) “FL 435-66,” is a narrow-range
petroleum-based oil. M-pede is an insecticidal soap that
contains potassium salts of fatty acids. Both also provide
significant reduction in ACP when applied alone. They were
used at two percent of the total application volume which
is 100 gallons of water per acre sprayed by ground using a
Durand Wayland AF100-32 air blast speed sprayer (Table 1,
Photo 3).
Pyganic was applied in November 2014, December 2014
and January 2015 in all three organic programs, either alone
(Program 1) or with 435 oil (Program 2) or M-pede (Program 3).
Danitol intended for January application in the conventional
program also was used in November to reduce the spread of
ACP to other programs, considering high populations in 2015
compared to the previous year. A block of younger Hamlin
orange trees was also used to evaluate the organic plus 435 oil
and conventional programs compared to an untreated check
using methods described for the Valencia block.
Details of insecticides used during growing season are
provided in Table 1. T. radiata colonies were maintained
at the Southwest Florida Research and Education Center
(SWFREC) in Immokalee and the Division of Plant Industry
(DPI) in Gainesville, Florida. A total of 92,821 T. radiata wasps
were released in the Valencia block from May 2014–June 2015.
ACP adult and predator populations were monitored using the
stem tap sampling method (Qureshi and Stansly 2014, Photo
4). At each evaluation, 36 trees were sampled per treatment
using 144 tap samples. Treatment means were separated by
Least Significant Difference (LSD) test when the main effect
was significant at p= 0.05. We used a threshold of 0.1 adults
per tap sample (10 adults in 100 tap samples) to trigger a spray
during the growing season, considering the high incidence
of HLB in Florida. We also took suction samples using a leaf
blower operating in reverse to sample for predators (Qureshi
and Stansly 2014, Photo 5).
55
ACP Adults/tap sample 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Untreated Pyganic 5.0 EC (17 oz/ac) Pyganic 5.0 EC (17 oz/ac) + 435 oil (2%) Pyganic 5.0 EC (17 oz/ac) + M-‐pede (2%) Danitol 2.4 EC (16 oz/ac) 11/14/14 12/4/14 12/12/14 12/24/14 1/6/15 1/20/15 1/27/15 2/10/15 2/24/15 3/3/15 Sampling date Figure 1. Density estimates of ACP populations (mean± SE) in organic and conventional control programs in a Valencia orange block. Pyganic alone and with M-pede
or 435 oil was applied on November 11, December 10, January 12, and Danitol on November 11 and January 12. Arrows indicate spray applications except with
asterisk when Danitol was not sprayed.
Shoots containing three to five instar nymphs were collected
in June, July, August, September and October 2014 and in
March and June 2015. These shoots were held under ventilated
cylinders in the laboratory to allow for the emergence of adult
psyllids or T. radiata to estimate percentage of ACP nymphs
parasitized.
RESEARCH FINDINGS
ACP CONTROL IN DORMANT WINTER SEASON
Mean no. per suc-on sample Adults averaged 0.2 or more per tap sample in the Valencia
block before the start of the dormant application. After the
first spray on November 11, adults remained significantly
fewer in all treatments through the second application on
December 10 made only in organic programs (Table 1).
Reduction with Pyganic plus 435 oil or M-pede averaged 7379 percent, significantly more than 46 percent with Pyganic
alone, but not different from 85 percent with Danitol (Figure
1). A similar trend of ACP suppression persisted after the
second application in organic programs. A significant drop
in populations, including control, was observed in the first
week of January. On January 12, applications were made in all
programs. An average reduction of 76 percent with Pyganic
alone, 77 percent with Pyganic plus 435 oil, 95 percent
with Pyganic plus M-pede, and 100 percent with Danitol
was observed for one month after application. Significant
treatment effects were observed through the first week of
March when ACP adult numbers exceeded 0.1 per tap sample
in organic programs (Figure 1).
1.2 Untreated Organic insec=cide Organic insec=cide with 435 oil Organic insec=cide with M-‐pede 1 0.8 0.6 0.4 0.2 0 Lacewings Spiders Ants Predatory group Lady beetles Figure 2. Populations of different predatory groups of beneficial insects (mean± SE) in the organic and conventional control programs in a Valencia orange block.
56
Photo 5. Suction sampling (Photo by J. Qureshi).
ACP adults averaged less than 0.2 per tap sample before
dormant sprays in the Hamlin block. Significant reduction was
more apparent after application on January 12. Pyganic plus
435 oil lasted through February 24 and with Danitol through
March 3, reduction averaging 76 percent and 85 percent,
respectively.
ACP CONTROL IN GROWING SEASON
Between March 10 and July 7, 2015, six and five treatments
were applied in organic and conventional programs,
respectively (Table 1).
Aza-Direct alone and with 435 oil or M-pede and Closer alone
sprayed on March 10 provided a significant reduction in ACP
adults through March 24 averaging 75 percent, 71 percent,
92 percent and 97 percent, respectively. Only Aza-Direct
plus M-pede and Closer reduced adults to 0.1 per tap sample.
Application of Grandevo, 435 oil, M-pede and Movento all
made alone on April 1 provided 54 percent, 69 percent, 69
percent and 82 percent reductions, respectively, but did not
reduce adults to the desired threshold. Follow-up applications
of Azera alone and with 435 oil or M-pede and Micromite alone
made on April 14 provided adult reductions of 41 percent, 69
percent, 82 percent and 83 percent, respectively, through May
5. Only Azera plus M-pede and Micromite reduced adults to
0.1 per tap sample on May 5.
Reductions of 45 percent, 77 percent, 68 percent and 99
percent were observed for about two weeks with microbial
insecticide Venerate, 435 oil, M-pede and Imidan, respectively,
all applied alone on May 8; but only Imidan held ACP at 0.1 per
tap sample. Application made on May 27 of Entrust alone and
with 435 oil or M-pede provided reductions of 53 percent, 84
percent and 72 percent, respectively, for about three weeks.
Although no application was made in the conventional
program on May 27, an average reduction of 87 percent and
0.1 adults per tap sample indicated a prolonged effect from
Imidan applied on May 8.
BIOLOGICAL CONTROL
Green lacewings were the most abundant predator in all
treatments (Figure 2, Photo 6). Lady beetles were rare
(Figure 2). Spotless lady beetle (Cycloneda sanguinea – Photo
7) and ashy-gray lady beetle (Olla v-nigrum – Photo 8) were
57
the species most commonly observed.
Spiders and ants also were present in all
treatments (Figure 2).
From June to October 2014, average
parasitism rates of 20 ± 3 percent (13-29
percent), 20 ± 11 percent (6-69 percent),
11 ± 7 percent (0-40 percent), 4 ± 4
percent (0-19 percent) and 2 ± 2 percent
(0-10 percent) were observed in the
untreated, Organic Programs 1, 2 and 3
(at that time with vegetable oil Citrus-Soy
instead of M-pede) and the Conventional
Program, respectively. Nymphs were most
easily available from untreated plots,
and parasitism rates more consistent
compared to treated plots.
Photo 6. Green lacewing predator of ACP and other pests (Photo by the University of Florida)
In March 2015, parasitism averaged 31 ± 6
percent in the untreated control, 40 ± 10
percent in the Organic 1, 23 ± 8 percent in
Organic 2 and 10 ± 10 percent in Organic
3. Fewer nymphs were available and none
were parasitized in the conventional
program. Parasitism rates in June were
also more in organic programs than
conventional program. These findings
suggest that T. radiata was able to
contribute to ACP control, particularly in
organic programs.
References
Qureshi, J. A., and Stansly, P. A. 2014.
Development of an Asian citrus psyllid
management plan for organic citrus.
Citrograph 5 (4):36-45.
Photo 7. Adults of Cycloneda sanguinea feeding on ACP nymphs (Photo by J. Qureshi)
Acknowledgements
We would like to thank the Citrus Research
Board for funding this research.
CRB Research Project #5500-189E
Jawwad A. Qureshi, Ph.D., is a research
associate professor of entomology, and
Philip A. Stansly, Ph.D., is a professor of
entomology, both with the University
of Florida-Institute of Food and
Agricultural Sciences at the Southwest
Florida Research and Education Center
in Immokalee, Florida.
Photo 8. Larva and adult of Olla v-nigrum feeding on ACP nymphs (Photo by P. Stansly)
58
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Figure 1: Jackson grapefruit trees differentially impacted by HLB. The tree to the left (A) looks healthier than the tree to the right (B). The trees are genetically
identical and of the same age, so it may be suggested that one of several factors accounting for the different appearances of these trees is that a beneficial
microbiota is protecting tree ‘A’ from CLas-induced symptoms, whereas tree ‘B’ is succumbing to CLas. Pictures were taken at the USDA Fort Pierce research
station in Florida (P.E Rolshausen photos).
A MICROBIOTA-BASED
APPROACH TO CITRUS TREE
HEALTH
Johan Leveau and Philippe Rolshausen
PROJECT SUMMARY
The goal of this CRB-funded research project is to describe, by DNA-based technology, the microbial
communities (also known as microbiota) that associate with healthy and huanglongbing (HLB)-affected citrus
trees from different plant tissues, geographical locations and under different management regimes. We
will be mining this database for correlations between microbiota and metadata to incorporate into models,
experiments and possibly products aimed at increased HLB tolerance, earlier diagnosis and/or more efficient
management. In this first of a series of articles, we provide a primer on:
• plant microbiota,
• complex relationships between HLB and the citrus tree microbiota, and
• objectives, expected outcomes and preliminary first-year results of our research project.
In subsequent articles, we will provide updates on the project’s progress.
60
HLB is one of the most destructive diseases of citrus worldwide.
In the United States, it results from phloem colonization by
the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) and
is vectored by the Asian citrus psyllid (ACP) Diaphorina citri.
Since its discovery in Florida in 2005, HLB has significantly
impacted the economic vitality of the Florida citrus industry
and quickly spread to the western U.S. and Mexico. No costeffective methods for early detection or cure of the disease
have been discovered yet, and this remains a major hurdle
in combatting HLB. In our project, we use DNA-based
methodology to establish principles of how trees impacted by
HLB may be recognized early, and possibly remedied, based
on their associated microbiota.
PLANT MICROBIOTA
Plants and trees are not sterile: they host large and diverse
communities of microscopically small organisms (bacteria,
fungi and viruses) on (epiphytically) and in (endophytically)
their leaves, stems, roots and other tissues. We refer to
these communities as the plant-associated microbiota,
plant microbiome or phytobiome. The most familiar and
best-studied representatives of these communities are the
pathogens, i.e., microorganisms such as CLas that have
evolved to infect their host, impede normal plant functioning
and cause disease. Other members of the plant microbiota
include those that are beneficial to their host, for example,
root-associated mycorrhizal fungi that sequester phosphate
from the soil and share it with their host, or rhizobacteria that
fix atmospheric nitrogen or stimulate plant defenses against
pathogens.
Despite their potential to seriously impact plant health and
function, pathogens and beneficials combined make up
a relatively small proportion of a typical plant-associated
microbiota. The large majority of microorganisms that
colonize plants is composed of commensals. They exploit the
plant as a substrate (as a habitat to attach to and thrive in and
as a source of nutrients), but in doing so are not demonstrably
harmful or helpful to the plant.
New technologies, especially those based on DNA profiling
of host-associated microbial communities, have greatly
facilitated the analysis of plant microbiota in terms of
their composition and function. Such analyses are rapidly
accumulating in the scientific literature and have led to
interesting and novel insights. For example, it is becoming
increasingly clear that the host plant plays an active role in
selecting specific microbes from soil or air to colonize its
roots, leaves or other plant parts. Another driving factor of
plant microbiota composition is the environment, either as a
source of microorganisms (e.g. soil and air) or as a modifier of
microbial activity, whether it is natural (rain, temperature) or
human-imposed (irrigation, fertilization). Possibly yet another
Figure 2. Symptoms of HLB on Jackson grapefruit showing characteristic
blotchy mottle leaves. Pictures were taken at the USDA Fort Pierce research
station in Florida (P.E Rolshausen photo).
driver of plant microbial community structure is plant disease,
or in broader terms, infection with a plant pathogen. We are
interested in exploiting this phenomenon in the context of
HLB and to develop microbiota-based diagnostics for early
(pre-symptom) detection of CLas. Also, because microbial
community structure may impact the establishment of
pathogens in or on plants to the point that disease is delayed
or prevented, we are interested in knowing what such a
‘protective’ microbiota would look like and use it as a major
point of departure for finding practical solutions to manage
HLB (Figure 1A).
HUANGLONGBING
The ‘Candidatus’ label of CLas identifies this bacterium as an
unculturable or ‘yet-to-be-cultured’ organism. The inability
to grow CLas in the lab has greatly hampered the conclusive
demonstration by classical methods that CLas is the causative
agent of HLB. However, consistent association between
disease symptoms and CLas presence (so-called Koch’s
first postulate) has been demonstrated using different
culture-independent, DNA-based methods. For example,
metagenomic sequencing exposed CLas to be the most
abundant bacterial species in phloem tissue from Florida
citrus trees with HLB symptoms.
The HLB disease cycle starts with the feeding of a CLas-infected
ACP on young citrus leaves, thus introducing CLas into the
phloem of the tree. The bacterium also may be introduced by
graft inoculation of CLas-infected material onto a healthy tree.
CLas resides and replicates in the phloem, and moves through
the vascular system site of infection to other parts of the plant,
including the root system. From there, CLas may move back
into the foliage, where it becomes available for pick-up by
psyllids and spread to another tree. Characteristic of HLB is
the long latency period between the time of infection and the
61
trees. A descriptive and quantitative
appreciation for these interactions
may reveal new and complementary
methods of not only early disease
detection, but also the identification of
specific members of the leaf and root
microbiota that prevent or mitigate
infection with or establishment of the
HLB pathogen.
YEAR ONE PROJECT
PROGRESS
We are using DNA-based methodology
to survey the epiphytic and
endophytic microbiota that associate
Figure 3. Principle Coordinate Analysis (PCoA) plot showing the differences in microbial community
composition among leaf surface samples from Lisbon citrus trees grown in the Contained Research Facility
with the leaves, roots and stems of
(CRF) at UC Davis. Each data point represents a sample, and the closer two points are to each other, the
citrus trees from greenhouse and field
more similar their microbial community composition is. The data points are colored by the inoculation status
environments located in California,
of the tree from which the samples came: blue is uninoculated, red is CLas-inoculated (by graft). Data and
figure are courtesy of Nilesh Maharaj (Leveau lab).
Texas and Florida. Specifically, we are
mining the variation in those microbial
appearance of CLas-induced symptoms, which include the communities as a function of time, location, management
blotchy mottling of leaves (Figure 2) and the development practices and disease symptoms, with the goal to extract from
of small, misshapen poorly-colored and bitter fruit and these data consistent associations that have practical use.
eventually, death of the tree (Figure 1B).
This effort is generating a database that will be minable by
researchers and citrus growers for links between microbiota,
Much of the ongoing HLB research is aimed toward a better tree and environment in the context of orchard management.
mechanistic understanding of the interactions between CLas, We also hope to identify organisms that could be used as
ACP and the citrus tree, in order to come up with practical biomarkers for HLB diagnosis and potential biocontrol agents
strategies to manage the disease through prevention, early that could be used to deter the establishment of the disease.
detection and/or intervention. The current gold standard for
CLas detection is based on the polymerase chain reaction In the first year of the project (2014-2015), we received
(PCR) using CLas-specific primer pairs. However, the spotty and processed hundreds of citrus tree samples (leaf, root,
distribution of CLas in a single tree may make it easy to budwood). Of these, 100+ samples came from the UC Davis
miss CLas, resulting in false-negative PCR outcomes. Several Contained Research Facility (CRF), more specifically, from the
alternatives to PCR are in the works, which are based not tail end of a collaborative, CRB-funded experiment aimed at
on the (direct) detection of the bacterial pathogen, but detecting alterations in the transcriptome, metabolome and
on measuring the (indirect) effects of CLas on its host. For microbiome of greenhouse-grown citrus trees that were
example, CLas infection of citrus trees has been shown experimentally inoculated with CLas by grafting. Preliminary
to induce changes in the genes that are expressed in the analysis of the data on bacteria and fungi from these samples
tree (the transcriptome), the proteins that are synthesized revealed several important and interesting insights. A key
(the proteome), and the chemicals that are produced (the observation was that the microbiota of HLB-inoculated and
metabolome). Some of these alternative detection methods, uninoculated trees were different (Figure 3) and that this
for example, those that quantify volatiles emitted from the difference correlated with the presence/absence of single
tree foliage, appear to perform better than the traditional microbial taxa such as Burkholderia and Aspergillus, which
PCR-based method because they suffer much less from the would make very promising candidates as biomarkers for Clas
problem of false-negatives.
infection. We are currently verifying these findings and will
link them to microbial data that will be collected from field
While most of the citrus tree microbiota are unlikely to samples.
interact directly with phloem-limited CLas, we can definitely
predict the existence of indirect interactions. As an example, In addition, about 200 California samples originated from
the microbial community structure on or in plants may alter in AgOps at UC Riverside, the Lindcove Research and Extension
response to the changes in the plant transcriptome, proteome Center, the Citrus Clonal Protection Program and from
and metabolome after initial infection. Some evidence already two commercial orchards located in the Central Valley. The
exists for a change in composition and function in the microbial remaining samples came from Texas A&M Kingsville Citrus
communities on roots of CLas-infected versus uninfected Center, Paramount Citrus orchards in Texas and the USDA
62
research center in Florida. We identified several endophytic
fungal (e.g. Alternaria, Fusarium, Rhizoctonia) and bacterial
(e.g. Bacillus, Streptomyces, Pseudomonas) taxa associated with
roots and vascular tissues of citrus trees.
Overall, our preliminary results suggest that citrus tissue type,
sampling location and disease status influenced microbial
community composition. As we collect more samples and
accumulate more data, we will be able to build correlations
between different variables, such as CLas titer, and
abundance, presence/absence of individual taxonomic group.
This approach will help identify organisms that compose the
disease-informative and/or protective microbiota and could
be utilized for HLB management.
Johan Leveau, Ph.D., is an associate professor in the
Department of Plant Pathology, University of California,
Davis. Philippe Rolshausen, Ph.D., is a cooperative extension
specialist for Subtropical Crops at the Department of Botany
and Plant Sciences, University of California, Riverside.
A longer version of this article may be found on our web
sites: http://leveau.ucdavis.edu and http://ucanr.edu/sites/
Rolshausen.
Collaborators on this project are Carolyn Slupsky, Ph.D., (UC
Davis); James Borneman, Ph.D., Georgios Vidalakis, Ph.D.,
and Caroline Roper, Ph.D., (all UC Riverside); John da Graça,
Ph.D., (Texas A&M University, Kingsville Citrus Center); Ed
Stover, Ph.D., (USDA-ARS, Fort Pierce, Florida); and Craig
Kallsen (Farm Advisor, Kern County).
References
Sagaram, U.S., DeAngelis, K.M., Trivedi, P., Andersen, G.L.,
Lu, S.-E. and Wang, N. 2009. Bacterial diversity analysis of
Huanglongbing pathogen-infected citrus, using PhyloChip
arrays and 16S rRNA gene clone library sequencing. Applied
and Environmental Microbiology 81:1566-1574.
Trivedi, P., He, Z., Van Nostrans, J.D., Albrigo, G., Zhou, J. and
Wang, N. 2012. Huanglongbing alters the structure and
functional diversity of microbial communities associated with
citrus rhizosphere. The ISME Journal 6:363-383.
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63
CRB-FUNDED FINAL RESEARCH REPORT
Fuller rose beetle (photo courtesy of Paulo A. V.
Borges Azorean Biodiversity Group, CITA-A)
AN INTEGRATED BIOLOGICAL
APPROACH TO FULLER ROSE
BEETLE CONTROL
Edwin Lewis and Amanda Hodson
SUMMARY
The Fuller rose beetle (FRB) is a flightless weevil commonly found in California citrus. Neither the adults nor
the larvae cause economically important direct damage. However, starting in January 2014, FRB management
became important due to new importation requirements from California’s most important export market, the
Republic of Korea. Korea currently fumigates imported California navel oranges with methyl bromide when
they arrive at Korean ports of entry to kill any FRB eggs that may be attached to the fruits. Due to the high
risks of worker exposure and environmental concerns associated with methyl bromide, Korea plans to eliminate
this material from use, thus transferring the responsibility of controlling FRB eggs on fruit to the California
citrus industry. The elimination of eggs from fruit is a difficult goal that will require more than a single tactic to
manage this insect.
64
Our goal was to develop tools for FRB management in citrus to satisfy quarantine requirements employing
beneficial nematodes such as entomopathogenic nematodes1 (EPNs). We conducted a series of laboratory,
greenhouse and field trials to determine which EPN species would be most effective and what rate of
application would produce the best levels of control. Two different tactics of application have reduced FRB
populations in citrus; monthly (June, July and August) applications of a mixture of two EPN species in a single
product and one application of Steinernema riobrave in March at twice the recommended rate.
INTRODUCTION
The FRB Naupactus godmani is a flightless weevil that causes
damage to horticultural plants such as citrus, persimmon,
apple, peach, plum, apricot, strawberry, raspberry and
blackberry (Chadwick 1965).
It is widely distributed
throughout the world. Weevils have a thelytokous life
cycle, which means that fertile females are produced from
unfertilized eggs, thus no mating is required for reproduction.
All FRB are females. There is one generation per year. Their
emergence occurs throughout the summer, and they live for
more than eight months. Thus, adults are present throughout
most of the year. We found that peak emergence of adults
occurs in July and August, but persists through October and
November at reduced rates. The larvae take six to ten months
to develop into pupae. About 1.5 months later, they emerge
as adults (UC IPM).
FRB are commonly found in California citrus orchards. Neither
the adults nor the larvae cause economically important direct
damage to the citrus plants or fruit. However, they became
an issue in the United States after 1985, because Japanese
quarantine inspectors detected FRB eggs on imported citrus
fruit (Haney et al. 1987). Thereafter, mandatory fumigation
was imposed on the entire shipment if any eggs were
detected. Starting in January 2014, FRB management became
more important due to new importation requirements from
California’s most important export market, the Republic of
Korea (Western Farm Press, March 19, 2013). Because of the
past use of fumigants in export markets, significant research
efforts toward developing management programs in citrus
for this insect are limited. Different integrated biological
approaches such as nematodes, biopesticides, etc. are needed
to control this insect pest.
EPNs are widely-distributed, commercially-available insect
parasites. They kill their invertebrate hosts with the aid of
mutualistic bacteria2 that are carried in their gut. When
the nematodes enter the body of the insect, they release
the bacteria, and then develop by feeding on the bacteria
which grow on host tissue (Kaya and Gaugler 1993; Gaugler
2002). EPNs have been, and continue to be, incorporated
into integrated pest management (IPM) programs in various
systems (Shapiro-Ilan et al. 2002). For example, EPN use
has expanded against pests such as the navel orangeworm
(Amyelois transitella) in pistachio and the pecan weevil
(Curculio caryae) in pecans (Siegel et al. 2006).
We combined biological control approaches using EPNs
and biopesticides to reduce adult and larval populations
of FRB. Efficacy of single or combined applications of some
biopesticides, such as Grandevo, MyCotrol-O and Safer
Brand Bioneem, against FRB adults also was evaluated in the
laboratory and/or greenhouse.
TEST MATERIALS
The EPNs Heterorhabditis bacteriophora, Steinernema riobrave
and S. carpocapsae were selected for the assays because they
have foraging behaviors that are suited to finding either
adult or larval FRB. The commercial bioinsecticide, Grandevo
(developed and distributed by Marrone Bio Innovations, Inc.
Davis, California) is composed of toxins from the bacterium,
Chromobacterium subtsugae strain PRAA4-1T, and is intended
to control a broad spectrum of chewing and sucking insects
and mites. MyCotrol-O is another biological insecticide based
on the fungus, Beauveria bassiana strain GHA (improved by
BioWorks Inc., New York). Safer Brand Bioneem (Woodstream
Corporation, Pennsylvania) is formulated with Azadirachtin,
a natural insect growth regulator extracted from the neem
seed. The recommended rates listed on the labels of all EPN
and bioinsecticide products were applied in the assays unless
otherwise stated.
LABORATORY AND GREENHOUSE
ASSAYS AGAINST FRB ADULTS
Efficacy of three biopesticides (Grandevo, MyCotrol-O and
Safer Brand Bioneem) and one EPN species (Steinernema
carpocapsae) was tested against FRB adults in the laboratory.
In the Grandevo assays, young citrus plants were sprayed
with the recommended rate for field applications. FRB adults
were tested individually in 12-well tissue culture plates at
room temperature. The Grandevo-sprayed leaves from
citrus were served to the insects in one cm2 pieces. Two trials
were conducted, and ten FRB were tested in each trial. Fresh
leaves were provided every three days. Mortality was scored
after 15 days. MyCotrol-O and Safer Brand BioNeem tests
were conducted in nine cm diameter Petri dishes lined with
65
lined with filter paper. Nematodes
were applied at rates of 25, 50 and 100
per cm2, but little control was achieved
(Figure 2). Efficacy of this nematode
species and the other two pathogen
products against FRB was poor, but
BioNeem promises to be useful.
FIELD ASSAYS AGAINST
FRB LARVAE AND ADULTS
Figure 1. Average (± SEM) survival (%) of Fuller rose beetle (Naupactus godmani) adults after single
or combined biopesticides application. Different letters above bars indicate significant differences at
p<0.05 . (P) indicates that Mycotrol-O plus Bioneem were applied to filter paper and (L) indicates that
the combined treatments were applied to leaves.
Two field trials are summarized here.
The first field trial was conducted
during 2015 in early spring (March) for
larvae in the soil. Steinernema riobrave
and
Heterorhabditis bacteriophora,
were applied at two rates; one billion
infective juveniles (IJs)/acre, which is
the application rate recommended for
most field applications, and two billion
IJs/acre. EPNs were applied by hand
using a watering can for this trial.
Treatments were applied to a five-meter
radius circle around each tree, which is
approximately the area that is reached
by a microjet sprinkler in this irrigation
system. In total, this test included four
treatments (two EPNs x two rates) plus
an untreated control; and seven trees
were treated per treatment for a total of
35 trees.
Efficacy of nematodes was first evaluated
by monitoring FRB adult emergence
using Tedder’s traps between August
and October in 2014. Three Tedder’s
traps were set under the trees and
checked every ten days. Captured
FRB adults were recorded (Figure 3).
In 2015, we also recorded damage to
newly-emerged leaves as an index of
FRB populations, since there were so few
Figure 2 . Average (± SEM) survival (%) of Fuller rose beetle (Naupactus godmani) after exposure to
adults caught. Significantly less damage
different numbers of Steinernema carpocapsae infective juvenile nematodes (IJ) in plastic containers in
was measured in the plots treated with
greenhouse. Different letters above bars indicate significant differences at p<0.05.
S. riobrave at the two billion per acre
filter paper. The products were sprayed either onto the filter application rate compared to controls (p=0.03) (Figure 4).
paper (Mycotrol-O) or to leaves (Bioneem), and application to
both surfaces were tested and compared for the combined In the summer of 2015, the second field experiment was
treatment. After applications, one FRB was added to each dish. designed to test the efficacy of a commercially available,
Fresh leaves were provided for all treatments. Ten adults were OMRI-certified product, Grubguard— a mixture of two EPN
used for each treatment, and the experiment was repeated species, S. carpocapsae and H. bacteriophora. The rationale
twice. The treatments with BioNeem were most efficacious in was that the S. carpocapsae IJs would likely infect adults, while
the H. bacteriophora IJs would infect larvae and pupae.
killing FRB (Figure 1).
The efficacy of the EPN S. carpocapsae against FRB adults
was tested in the laboratory and greenhouse in Petri dishes
66
The experiment consisted of four randomized blocks (of 10
trees in two rows) in the center of an organically managed
citrus orchard. Within the two paired
rows, one tree at each point was selected
to receive Grubguard application. The
nematode mix was applied at a rate
of 25 nematodes/cm2 or 3.6 million
nematodes per tree in 500 mL water.
Nematodes were applied within a threefoot radius of the tree trunk focusing
on areas near the irrigation emitters.
After nematodes were applied, another
1,000 mL of water was applied to help
nematodes penetrate the soil. Each
control tree received 2,500 mL of water
in the same manner as the treated trees.
The product was applied three times –
on July 22, August 13 and September 7,
2015.
Levels of FRB damage were similar
between the treatment and controls
early in the season, and there were no
differences in leaf damage between
treatment and control trees before
application. In October, after three
applications of Grubguard, leaf area
lost to damage was 44 percent lower
in treated trees compared to controls,
although this difference was not
statistically significant (Figure 5).
Figure 3. Average (± SEM) number of Fuller rose beetle (Naupactus godmani) caught in the Tedder’s
traps after nematode application in the field . No significant differences in FRB populations were found
among the treatment plots. Hb = Heterorhabditis bacteriophora and Sr = Steinernema riobrave.
We also tested the relationship
between damage caused on leaves by
FRB and soil texture to see if certain
soil characteristics favored larval
development. Damage per unit of leaf
area decreased with coarse sand (Rho=0.42, p=0.06) and increased with the
silt content (Rho=0.38, p=0.09). Since
nematodes were observed to infect at
least the adult FRB, and nematodes are
known to prefer sandy soils, it could Figure 4. The average (± SEM) damaged area (mm2) measured from 10 leaves/tree due to Fuller rose
be that populations of natural enemies beetle (Naupactus godmani) after nematode application in the field . Hb = Heterorhabditis bacteriophora
are limiting FRB distribution in sandier and Sr = Steinernema riobrave. C = Control.
areas. This relationship may be useful
in designing scouting strategies. The distribution of FRB is bacteriophora and S. carpocapsae applied three times through
highly aggregated. Perhaps testing the soil and concentrating the period of FRB eclosion3 caused a 44 percent decrease in
scouting efforts on areas where the soil has low levels of foliar damage, although there was high variation between
coarse sand would increase efficiency. This relationship needs trees. A more effective treatment combination might be
S. carpocapsae (targeting adults) and S. riobrave (targeting
to be confirmed before using it widely.
larvae and pupae). Another complementary tactic would be
periodic foliar application of Bioneem oil to deter feeding and
oviposition during the summer and early fall. These three
A potential integrated management plan, based on these methods used over an entire season may significantly reduce
studies, would include three management tactics and a even very high FRB populations.
refined sampling strategy. We found that field application
of S. riobrave in the early spring can decrease the amount of Populations of FRB are very highly aggregated. Thus, either
foliar damage significantly if applied at the rate of two billion careful scouting must be conducted, or many uninfested areas
IJs per acre. Further, applications of a combination of H. of a citrus planting will be treated. We have observed that
CONCLUSIONS
67
Figure 5. Average (± SEM) damaged area (mm2) measured from 10 leaves/tree due to Fuller rose beetle (Naupactus godmani) after three applications of
Grubgaurd in the field.
more FRB foliar damage occurred in areas with higher levels
of silt. In other words, very sandy soils have lower populations
of FRB. This finding may help target scouting efforts to the
areas most likely to be infested.
Edwin Lewis, Ph.D., and Amanda Hodson, Ph.D., are in
the Department of Entomology and Nematology at the
University of California, Davis.
References
Adams, B. J. and Nguyen, K. B. 2002. Taxonomy and systematics,
pp. 1-34. In R. Gaugler (ed.), Entomopathogenic Nematology.
New York, NY, CABI.
Chadwick, C.E. 1965. A review of Fuller’s rose weevil
(Pantomorus cervinus Boheman) (Coleoptera, Curculionidae).
Journal of Entomological Society of Australia (N.S.W.) 2:10-20.
Gaugler, R. (ed.) 2002. Entomopathogenic Nematology. New
York, NY, CABI.
Grewal, P.S., Ehlers, R.-U. and Shapiro-Ilan, D. I. 2005. Nematodes
as Biocontrol Agents. CABI Publishing, Wallingford, UK.
Kaya, H. K. and Gaugler, R. 1993. Entomopathogenic
Nematodes. Annual Review of Entomology 38:181-206.
Shapiro-Ilan, D. I. 2001a. Virulence of entomopathogenic
nematodes to pecan weevil larvae Curculio caryae (Coleoptera:
Curculionidae) in the laboratory. Journal of Economic
Entomology 94:7-13.
Shapiro-Ilan, D. I. 2001b. Virulence of entomopathogenic
nematodes to pecan weevil adults (Coleoptera: Curculionidae).
Journal of Entomological Science 36:325-328.
Shapiro-Ilan, D. I., Gouge, D. H., and Koppenhofer, A. M.
2002. Factors affecting commercial success: case studies
in cotton, turf, and citrus, pp. 333-355. In R. Gaugler (ed.),
Entomopathogenic Nematology. New York, NY, CABI.
Siegel, J.P., Lacey, L. A., Higbee, B. S., Noble, P., and Fritts Jr,
R. 2006. Effect of application rates and abiotic factors on
Steinernema carpocapsae for control of overwintering navel
orangeworm (Lepidoptera: Pyralidae, Amyelois transitella) in
pistachios. Biological Control 36:324-330.
UC IPM Online. www.ipm.ucdavis.edu/PMG/r107300311.html.
Western Farm Press. 2013. Beetle threatens California Navel
exports to Korea. Mar. 29, 2013. http://westernfarmpress.com/
orchard-crops/beetle-threatens-california-navel-exportskorea.
Glossary
Entomopathogenic nematodes (EPNs): A group
of nematodes that live parasitically inside and kill
infected insect hosts. EPNs occupy a specialized
biological control niche in that they specifically infect
only insects.
1
Mutualistic bacteria: Bacteria associated with
different organisms (e.g., plants); and in that
relationship, both benefit from the activity of the other.
2
Eclosion: Emerging from the pupal case, or hatching
from the egg.
3
68
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TREE SHAKERS:
FRESNO - Jeremy DeBoer - Sales (209) 480-2863
HUGHSON - Billy Ashby - Sales (209) 613-9454
CHICO - Jared Kinney - Sales (530) 570-4909
Chad Hymel - Sales 559-909-0008 chadhymel@pdi-wind.com
Erik Nelson - Sales 559-731-9708 eriknelson@pdi-wind.com
Jeff Thorning - Sales 559-972-9937 jeffthorning@pdi-wind.com
Randy Quenzer - Sales 559-805-8254 randyquenzer@pdi-wind.com
s,
g almond
in
t
s
e
v
r
a
you’re h
rd-Rite
Whether r pecans, Orcha eeds.
o
n
walnuts, haker to fit your
s
makes a
PDI Wind Machine
24 Hour Emergency Service
559-564-3114 Woodlake, CA
,
R
.
BREEZE
• Sales
• Service
•
•
•
•
R
New & Used
Portable
Stationary
Tropic-Breeze
THE
ORCHARD-RITE®
BLADE:
• Solid Fiberglass
• High Efficiency Design
• Proven Reliable & Safe
• 10 Yr. Extended
Warranty Available*
• No Wind Exclusion
The Bullet and
The Monoboom:
• CAT® C6.6
• CAT® C4.4
Automated
Features*:
• Auto Shake
• Set Throttle
• Reversible Fan
• Plus Much More
*CAT® C4.4 ONLY
w
o
P
.
e
Pur
.
l
u
f
r
e
ce
n
a
m
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o
Perf
AUTO-START:
Standard
Engines:
FORD® V-10
•
•
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•
“OrchardRite stands behind their
equipment, and is constantly
making improvements
toward driver comfort and ease
of maintenance. Today, this is the only
machine to buy.”
Orchard-Rite
®
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EPA Certified
LPG
Vapor Propane
Natural Gas
CAT® C6.6
• Tier III
• Turbo-Charged
• Diesel
• Start & Shut
Down at Proper
Temp.
• Saves Fuel & Labor
• Most Reliable
in the Industry
• Most Purchased
Accessory
GEARBOXES:
• Ductile Iron
• Manufactured
In-House
• Extended
Warranty
Available*
*Pursuant to the terms
& conditions of the
Orchard-Rite extended
warranty agreement.
www.CitrusResearch.org | Citrograph Magazine 69
www.orchard-rite.com
70
THAT’S HOW MOVENTO INSECTICIDE MAKES ORANGES FEEL.
Movento® insecticide delivers powerful two-way systemic action that moves throughout the
tree to protect the parts pests seek most, from new shoot growth to roots. This results in
long-lasting, reliable protection against above- and below-ground pests, including Asian citrus
psyllid, red scale and nematodes. With Movento as part of your ongoing pest management
program, you’ll have stronger, healthier trees that produce a higher quality crop year over year.
For more information, contact your retailer or Bayer representative or visit www.Movento.us.
Bayer CropScience LP, 2 TW Alexander Drive, Research Triangle Park, NC 27709. Always read and follow label instructions. Bayer, the Bayer Cross, and Movento are registered trademarks
of Bayer. For additional product information, call toll-free 1-866-99-BAYER (1-866-992-2937) or visit our website at www.BayerCropScience.us.
CR0114MOVENTA081V00R0
71
72

CALIFORNIA CITRUS SHOWCASE

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