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2016
24
IN THIS ISSUE
WINTER 2016 | VOLUME 7 NUMBER 1

THE OFFICIAL PUBLICATION OF THE CITRUS RESEARCH BOARD
8 CHAIRMANS VIEW
RICHARD BENNETT
12 BOARD SELECTS NEW EXECUTIVE LEADERSHIP TEAM

RICHARD BENNETT
14 INDUSTRY VIEWS WHAT PROGRESS ARE WE MAKING

ON HLB?
MOJTABA MOHAMMADI, PH.D.
18 WHERE 2016 CRB-FUNDED RESEARCH IS HEADED
MOJTABA MOHAMMADI, PH.D.
22 MEET YOUR BOARD MEMBERS

IVY LEVENTHAL
40
24 HLB DETECTIONS IN SAN GABRIEL

VICTORIA HORNBAKER AND LUCITA KUMAGAI
30 HLB IMPACT ON CITRUS ROOT HEALTH AND INTERACTION

WITH PHYTOPHTHORA
JAMES GRAHAM, PH.D.
34 MONITORING FOR ACP RESISTANCE TO PESTICIDES

JOSEPH MORSE, PH.D, ET AL.
40 MATURE CITRUS PROPAGATION IN RITA BIOREACTORS

YOSVANIS ACANDA AND JANICE ZALE, PH.D.
46 NOT ALL PSYLLIDS ARE CREATED EQUAL

MICHELLE CILIA, PH.D., ET AL.
52 COMPLEX CITRUS LURES TO TRAP AND CONTROL ACP

XAVIER MARTINI, PH.D., ET AL.
M EE T CR B S NE W
L E A D E R S H IP
TEAM
On The Cover:
This autumn, Richard Bennett (left) was

elected chairman of the Citrus Research
Board (CRB), and Gary Schulz was hired
as the CRBs new president. Also elected to
the Executive Board were Dan Dreyer as
vice-president and Toby Maitland-Lewis as
secretary-treasurer. For more information
on the new executive leadership team, see
Chairmans View on page 8.
56 FOUNDER LINES FOR IMPROVED CITRUS BIOTECHNOLOGY

MARIA OLIVEIRA, PH.D., ET AL.
60 CITRUS DISEASE RESPONSE TO HLB INFECTION

JESSICA FRANCO, ET AL.
64 DEVELOPMENT OF LOW-SEEDED CITRUS BY MUTATION

BREEDING
MIKEAL ROOSE, PH.D., ET AL.
72 NOVEL THERAPY OF HIGH-PRIORITY CITRUS DISEASES

HAU NGUYEN, PH.D., ET AL.
76 FIGHTING HLB WITH A CITRUS TRISTEZA VIRUS-BASED

VECTOR
JAMES NG, PH.D., ET AL.
82 DEVELOPMENT OF AN ACP MANAGEMENT PLAN FOR

ORGANIC CITRUS
JAWWAD A. QURESHI, PH.D., AND PHILIP A STANSLY, PH.D.
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
Member Expires
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
Member Expires
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

Member Expires Member Expires
John Gless III
2017 Alan Washburn
2018
Mike Perricone
2017
District 3 California Desert

Member Expires Member Expires
Mark McBroom
2016 Craig Armstrong
2016
Public Member
Member Expires
Vacant
2018
CALENDAR OF
EVENTS 2016
January 13
CPDPP Board Meeting, Visalia/Exeter,
California. For more information, contact
CDFA at (916) 403-6652.
January 27
Annual UC Riverside Citrus Day,
Riverside, California. Up-to-date research
information, field tours and variety
tasting.
January 28
CRB Board Meeting, Riverside, California.
For more information, contact the CRB at
(559) 738-0246.
February 9-11
World Ag Expo, International Agri-Center,
Tulare, California. For more information,
visit www.worldagexpo.com.
March 3
California Citrus Mutual Citrus Showcase,
Visalia Convention Center, Visalia,
California. For more information, contact
California Citrus Mutual at
(559) 592-3790.
March 9
CPDPP Board Meeting,
Riverside / San Bernardino, California.
For more information, contact CDFA at
(916) 403-6652.
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
CHAIRMANS VIEW
BY RICHARD BENNETT
OUR #1 GOAL:
CONTROLLING HLB
The Ultimate and Only Objective
The huanglongbing (HLB) threat to the California citrus

industry is very real. If your tree becomes infected, the fruit
will become unmarketable and the tree will die. Thats
the bottom line. This disease is the ultimate threat to our
survival.
Richard Bennett
Trees with dropped fruit, showing symptoms of HLB.
EXISTING THREE-PRONGED STRATEGY
The current three-pronged strategy to fight HLB (a) keeping

nursery stock clean, (b) suppressing Asian citrus psyllid (ACP)
populations and (c) removing diseased trees upon regulatory
confirmation of the presence of Candidatus Liberibacter
species (Ca. L. asiaticus or CLas) has clearly failed to stem
the spread and progression of the disease in Florida, Texas and
California, according to the recent research of David Bartels,
Ph.D., an entomologist at the USDA APHIS PPQ Mission
Laboratory in Texas. Citrus quality in those states is declining,
and production in many groves has fallen by more than an
astounding 50 percent. Floridas 2015-16 orange crop is
expected to be the smallest in half a century. The heavy use of
insecticides to suppress the ACP vector populations is not fully
effective, which means psyllids are feeding on an increasingly
greater number of likely CLas-infected, yet asymptomatic,
trees.
California has adopted this three-prong strategy and, like
Florida, relies on regulatory agency data collection, analysis
and diagnostic efforts. However, the effectiveness of these
approaches is restricted by tight protocols and limited
resources. Our industry also has invested heavily in scientific
research to assist us in extending our existence long enough
to find a solution. California needs to immediately support the
high-throughput investment of early detection technology
devices.
REALIZING THE THREAT
The most current research indicates that the use of

antimicrobials and other treatment regimens (such as thermal
therapy) to extend the productive life of infected trees is
commercially impractical, at best palliative, and at worst only
serves to keep infected trees in the ground longer. Some
progress has been made in developing cost-effective early
detection technologies, but there has been little industry
discussion about implementation specifics. After more than
15 years and nearly a quarter of a billion dollars of research,
marginal progress has been made in developing a long-term
cure for HLB or an HLB-resistant rootstock.
Ignoring the costs associated with the destruction of grower
balance sheets, the cost to manage the spread of ACP in
California assuming 200,000 acres and at least an additional
two to five dedicated pesticide sprays per year could reach
$50-120 million annually or more ($250-600 per acre) within
the next five years. This equates to $.30-.70 per carton on an
850-carton per acre grove. Then, there is enhanced nutrition.
From what we know to date, the best programs are costing
more than $500 per acre per year. Again, assuming 200,000
acres, the additional cost to our growers is at least $100
million per annum. Therefore, the total cost to the California
citrus industry could be $220 million annually. The cost to the
states growers could total $750-1,100 per acre per year, or

about $1.30 per carton on 850 cartons per acre production.
Even then, such expenditures will not stop the spread of HLB
in our orchards. It may slow the destruction of diseased trees,
but any fresh fruit infected with HLB that is sold marks the
beginning of the end for us in the marketplace.
It should be evident that sticking with the current strategy
and facing the unintended, but likely, consequences of
encouraging growers to hunker down, rely primarily on
bug containment, remove only regulatory-confirmed
and/or symptomatic trees and wait for the commercialization
of a magic bullet will only shackle growers with increasingly
higher farming costs and lead to the inexorable collapse of
Californias productive citrus capacity.
This devastating disease already has manifested itself in
Southern California. Collectively, we must formulate an
immediate strategy.
predictive processes and organizational capabilities to

combat the disease along with broad, but achievable, strategic
directives with two over-arching goals:

a. Extend the economic life viability of existing trees
in the ground.

b. Shorten the time to development and
commercialization of a cure and/or protectant.
FOUR KEY COMPONENTS EMERGED AS A RESULT OF THE

SUMMIT:
1.
2.
HLB SUMMIT
On December 1, the industry brought key forces together

for an annual HLB Summit here in California. It differed from
the meeting held at the University of California-Davis this
past autumn in that it added in the research and experiences
of Florida to develop a comprehensive grower action plan.
The morning session clearly demonstrated the urgent
and immediate need for investment in Early Detection
Technologies.
In September, the Citrus Research Board (CRB) developed a
format to assemble leading scientists and industry leaders from
Florida together with us to develop a blueprint to combat HLB
right now. The best and most experienced minds currently
battling this disease in Florida presented their thoughts on
developing short-, medium- and long-term strategies to move
forward. Scientists who can predict infection movement
and detect the bacteria in a pre-symptomatic stage shared
research findings.
This one-day session encouraged attendance from a
representative cross-section of industry stakeholders
(nurseries, growers, marketers and packers/shippers), as well
as researchers at the forefront of observation/modeling,
diagnosis and treatment of vector populations and the
disease.
THE HLB SUMMIT HAD THREE MAIN OBJECTIVES:
1.
2.
3.
10
Raise key stakeholder awareness of the urgency, scope

and magnitude of the threat.
Provide a forum for open, robust discussion of (a)
disease progression and the effectiveness of current
HLB strategies in Florida, Texas and Brazil, (b) the current
state of HLB-related research and (c) the limitations of
existing disease diagnostics, treatments and strategies.
Achieve consensus on the need for a non-regulatory,
industry-driven approach that includes institutionalized
3.
4.
Institutionalized Process a transparent and highly

interactive, managed process that goes beyond
simply coordinating sample collection/analysis efforts
and recommending psyllid control action plans. It
incorporates regular review and possibly in-field testing
and deployment of early detection technologies,
HLB-infected tree removal and broad industry outreach.
War Room an interdisciplinary panel of researchers,
industry players and regulatory representatives that
meets after each data collection/analysis cycle to
do a situation assessment and create and amend
ACP/HLB remediation action plans within the context of
the latest research and organizational capabilities.
HLB Task Force a small accountable organization
tasked with managing the process.
Infrastructure includes CDFA, other regulatory and
non-regulatory resources and capabilities.
Our whole industry needs to unite to develop this working

plan. California Citrus Mutual and other industy leaders
initiated bringing growers together to create a battle strategy
to attack these devastating bacteria.
HLB will obliterate our industry unless we control our fate on
a unified basis. Every grower and every orchard owner must
understand this has to be a combined effort. Each tree that is
infected with this bacterium must be eliminated immediately.
The bacterium has to be controlled at the earliest stage of
infection. This is not a disease we can attack tomorrow when
we know a tree is infected today. Our industry must stand
together and move forward aggressively.
Florida has the capability of chemically altering orange juice to
mask the HLB flavor change. However, the California industry
sells a single piece of unaltered fruit to each consumer. With
fresh fruit, we cannot mask the flavor change. When we, as
growers, start delivering HLB-affected fruit to consumers,
California will lose its industry. The flavor change that the
HLB-infected tree imparts to the fruit is dramatic, and neither
your family nor our consumers will accept it.
If we allow the HLB bacteria to infect our trees, consumers
will abandon California oranges, mandarins, grapefruit and
lemons, and our citrus industry will be lost.
Richard Bennett is the chairman of the Citrus Research
Board.
11
From left to right, CRB Secretary/Treasurer Toby Maitland-Lewis, Chairman Richard Bennett, President Gary Schulz and Vice-Chairman Dan Dreyer.
BOARD SELECTS NEW

EXECUTIVE LEADERSHIP TEAM
Richard Bennett
his past September, the Citrus Research Board (CRB) instituted some key leadership changes. First,
after a lengthy and thorough search, we hired a new president for the organization. Shortly thereafter,
at the Boards annual meeting, new executive officers were elected.
We are pleased to introduce Gary Schulz as the CRBs president. Gary has an impressive resum that
seems tailor-made for his new responsibilities of leading the agency through the challenging period
we now face. He is a senior executive with an extensive, focused background in member-driven nonprofit and quasi-governmental agricultural business organizations. Having demonstrated his ability to
12
lead strategic planning in order to solve industry issues, Gary

also has proven to be a tremendous team builder. These are all
skills that will serve him well as our new president.
Garys most recent post was as president and CEO of the
California Association of Pest Control Advisers and California
Certified Crop Advisors, a trade association of 3,000 members
and eight staff with a 21-member board of directors. Before
that, he served for a number of years as the president and
general manager of the Raisin Administrative Committee and
CEO of the California Raisin Marketing Board. Among other
prior positions, our new president also was general manager
of International Agri-Center and World Ag Expo for 15 years.
We know that Gary will be a welcome addition to our
organization, and we are very happy to have such a
knowledgeable agriculture industry veteran at the helm.
In addition to Gary, we have new executive officers in place
on the Board following annual elections. I am serving as your
chairman of the Board, Dan Dreyer is your new vice-chairman
and Toby Maitland-Lewis is your secretary/treasurer.
Dan, a third generation California family farmer, is the Northern

Tulare County grower liaison for the CPDPC in addition to
being the manager of Agriculture Services in Exeter. As a
member of the CRB Board, he is dedicated to outreach and
communicating research outcomes to the industry.
Toby, the chief financial officer of Sun Pacific in Exeter, has
worked in citrus for a decade. He has served on the CRB Board
since 2013 and is primarily interested in the CRBs fiduciary
responsibility and promoting the long-term sustainability of
Californias citrus industry.
I am the owner of Bennett Farms in Exeter and am a citrus
industry veteran. Right now, I am committed to leading an
effort dedicated to finding a solution for HLB (see accompanying
article). I know I also speak for Gary, Dan and Toby in sharing
with you that we look forward to working hard on your behalf
by strategically focusing your funding on research that will
enable your businesses to survive and thrive.
Richard Bennett is the chairman of the Citrus Research
Board.
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13
11/18/15 4:30 PM
INDUSTRY VIEWS
MOJTABA MOHAMMADI
WHAT PROGRESS ARE

WE MAKING ON HLB?
During the HLB Research Summit held at the University of California, Davis, September 9-10,
2015, several well-known research experts were asked their thoughts on What progress
are we making on huanglongbing (HLB)?
14
Greg McCollum, Ph.D.

Research Plant Physiologist,
USDA-ARS, Fort Pierce,
Florida
The confirmation of Candidatus

Liberibacter asiaticus (CLas) in
the state has been a real wakeup call. HLB has been found
in two different locations in
California. There is evidence
that the problem is spreading,
which means that action needs
to be taken sooner rather than
later.
HLB is an enemy, but once the

enemy has been identified, you can begin the battle. A lot
of novel, very interesting work is being done on detection
technologies that are alternatives to PCR. They hold great
promise if we can get closer to whole tree detection, rather
than a very small fraction of tree detection. To prevent or slow
the development of an HLB epidemic, it is essential to confirm
infections as early as possible and take action immediately.
Because trees can be infected with CLas for a lengthy time
prior to the appearance of HLB symptoms, detection of the
pathogen is challenging. Although PCR is very reliable in
detecting CLas, if trees are not symptomatic, it is difficult
to determine where to collect diagnostic samples. Early
detection means that infections can be found prior to trees
developing visible symptoms. That is crucial if HLB is to be
controlled in California.
In Florida, the situation got out of control because symptoms
were not apparent. As soon as a single tree was found
and testing for CLas began, we found the pathogen was
everywhere. California is ahead of the curve on that and
hopefully can stay that way. I wish there was a significant
breakthrough in therapeutics or control in general, but we
are not there yet. Currently, the standard three-pronged
approach (controlling psyllids, removing infected trees and
only planting clean nursery stock) is still the most important
management strategy.
solutions. I lean more toward transgenics than conventional

breeding simply because of the time factor. We know it is
feasible with transgenics to develop trees that will likely be
resistant to a host of different diseases. Conventional breeding
is attractive. Weve had a breeding program at the ARS for
more than 100 years, and new varieties have come out of that
program. It is a very slow process that takes about 30 years
from hybridization to a variety being released. A rootstock is
a bit faster proposition. The advantage is that you maintain
the same scion, but impart resistance through the rootstock,
which would be a great benefit. Some rootstocks that have
been evaluated in Florida appear to have a less rapid rate of
decline than others, which holds some promise.
FLORIDA EXPERIENCE
The number one lesson is be vigilant; you must constantly

seek symptoms and constantly assay as many samples as
possible. It is really a numbers game. The public must be
educated. It is amazing that with the extent of HLB in Florida, I
have seen people who have lived there their whole lives who
are not even aware of the problem. The outreach efforts I have
seen here in this meeting are really important, especially with
the huge number of residential trees in California. Ensuring
that the public is aware of this problem, educating them as to
what they can do to help solve it and encouraging them to do
so will go a long way.
BIOLOGICAL CONTROL
In Florida, before HLB, pests were managed almost exclusively

through biocontrol. Minimal use of insecticides and the
interactions among various pests resulted in good biocontrol.
If you do not have enough of the pest that you are trying to
control, the population of biocontrol organisms will decline,
so there is a cyclic nature. With residential trees, people will
be much more amenable to releasing wasps in their backyard
than commercial insecticide spraying.
FUNDING RESEARCH AREAS
New detection technologies could provide a real advantage

over PCR, though none have been validated. Therapeutics of
any kind would be of tremendous value, however, we do not
have any promising ones at the moment.
DISEASE CONTROL STRATEGIES
Breeding and transgenic (introduction of DNA from another

source) approaches really are going to be the ultimate
15
Matt Daugherty, Ph.D.

Extension Specialist,
Department of Entomology,
University of California,
Riverside, California
One of the main challenges

with HLB management has
been that it takes citrus trees
a long time to show disease
symptoms after they become
infected. If not removed, the
infected trees serve as new
sources of inoculum for disease
spread in the following years.
There have been significant

areas of progress on HLB in
recent years; one of which is developing a range of early
detection technologies that we hope will soon be available
for regulators and growers to begin folding into monitoring
programs. Earlier detection of infected trees is critical to
narrowing the potential for pathogen acquisition and spread
during the asymptomatic phase.
Another area of progress is that we are getting a much better
handle on how the vector and the HLB-associated Liberibacter
spread in the landscape, what drives their movement
and what aspects of local landscape and environmental
conditions influence whether the psyllid is likely to be there.
This information feeds into risk modeling that provides a
better picture of where to look for not only the ACP, but also
the disease. By refining how we identify early cases of disease,
we can hopefully mitigate some impacts. That process has
been going on for years, but it is becoming more defined.
PUBLIC AWARENESS
As an extension specialist, another area of progress I should

note is increased awareness of this problem. There are so
many people in California affected one way or another by the
psyllid or the disease. Were lucky to have a very large network
of people at UC, in industry and at state and federal agencies
tackling different aspects of increasing public and stakeholder
education. Such education programs are critical to promoting
early disease and insect finds and widespread adoption of
control measures. For areas like the Central Valley, hopefully
by the time HLB does arrive, growers and the general public
will be in a much better position to adopt coordinated and
aggressive control measures during the early phase when
eradication is most feasible.
16
BIOLOGICAL CONTROL
Disease in urban neighborhoods in Southern California is

incredibly challenging to manage effectively, let alone to try
to eradicate. We absolutely are relying on biocontrol as an
important strategy in these areas. Were learning more about
how to increase its chances of successfully slowing disease
spread. This will likely involve coordinating with homeowner
education programs, emphasizing the need to manage a
variety of species, including Argentine ants, to maximize the
effectiveness of biocontrol agents.
FLORIDA EXPERIENCE
Weve learned a lot from the HLB situation in Florida. One of the
most important lessons for us early on was the role of human
transportation in the spread of psyllids and disease. This
lesson led directly to steps being taken in California, including
the establishment of quarantines, to ensure that people
are not moving around infested or infected plant material.
Similarly, regulations were put in place in California to make
sure that nursery plants are not sources of HLB spread. I am
fairly confident that these measures (although theyve proven
burdensome for certain people and industries) are at least
part of the reason that the pace of the ACP and HLB situation
in California has to date been very different from Florida.
Manjunath Keremane, Ph.D.

Research Plant Pathologist,
USDA-ARS, National Clonal
Germplasm Repository for
Citrus and Dates, Riverside,
California
The California citrus industry is

concerned about recent finds
of HLB-positive citrus trees
reported in some residential
areas of Los Angeles County.
At this time, containment
and eradication are our main
goals in Southern California.
Early detection of Candidatus
Liberibacter asiaticus (CLas)
in psyllids and affected
trees, in addition to effective
eradication of compromised citrus trees, can help prevent this
devastating disease from spreading in California.
EARLY DETECTION TECHNOLOGIES
In order to achieve this goal, the Citrus Research Board

is currently funding research projects on early detection
technologies. With funding from the USDA National Institute
of Food and Agriculture, our group is trying to develop
technologies that can facilitate detection of HLB through
encouraging field-testing by growers, extension agents and
any public members interested in monitoring the disease.
The rationale is that if a single laboratory is expected to
conduct all the required tests, the task will be onerous.
Involving the public sector in this process will enable the
state to focus its resources on high-risk areas. Large-scale
testing by many will facilitate early detection leading to
exclusion and eventual suppression of the disease.
DISEASE CONTROL STRATEGIES
A long-term solution to control HLB is to develop cultivars

with resistance to the disease. Currently, two approaches are
being pursued transgenic and conventional breeding. We
have chosen a non-transgenic approach because of the ease
associated with field-testing and the release of promising
disease-resistant cultivars to the citrus community. Approval
by the FDA and EPA will not be needed since the hybrids
are a result of natural breeding. Even though the approach
is time consuming initially, the advantages associated with
conventional breeding are appealing.
FLORIDA EXPERIENCE
The lessons learned from Floridas HLB experience have

been useful in defining the regulatory process required for
HLB suppression. Since 2005, we have been developing
methodologies to conduct psyllid testing for the presence
of CLas. This is now used as an early indicator for the
existence of HLB. Testing the psyllid vector would give us
a lead-time of two to four years prior to the appearance of
HLB symptoms in the plants.
Big box retail stores in Florida marketing ornamental citrus
trees contributed to the quick spread of HLB throughout
the state. California has regulations in place to avoid such
incidences. In Florida, it was demonstrated that trucks
transporting citrus acted as carriers of disease-spreading
psyllids. Groves along the highway were the first to be
affected by HLB. At present, California has strict regulations
for trucks transporting processed or unprocessed citrus.
Numerous guidelines were established based on Floridas
HLB experiences. This has helped slow the spread of HLB
in California. Additionally, our outreach program seems to
be working efficiently in disseminating information and
educating the public regarding HLB-related issues.
Mojtaba Mohammadi, Ph.D., is an associate scientist with
the Citrus Research Board in Visalia, California, where he
also serves as the associate science editor of Citrograph.
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WHERE 2016 CRB-FUNDED

RESEARCH IS HEADED
Mojtaba Mohammadi
unding research to solve problems associated

with commercial citrus production in California
is the principal mission of the Citrus Research Board
(CRB). Science and technology have been helping
and will continue to assist California citrus growers
in achieving their long-term goals of productivity,
efficiency, competitiveness and sustainability.
Among diseases threatening the California citrus
industry today, huanglongbing (HLB or citrus
18
greening) is considered the number one enemy,

followed by those caused by viruses, pre- and postharvest fungi and root pathogens. HLB is a destructive,
century-old citrus disease that has just spread in the
U.S. within the past decade. It is associated with a
phloem-limited bacterium, Candidatus Liberibacter
asiaticus (CLas), which is transmitted from an
infected plant to a healthy one primarily by the Asian
citrus psyllid (ACP or Diaphorina citri Kuwayama) and
via contaminated budwood propagation. In Florida,
the HLB onslaught has caused serious economic losses to the

citrus industry, which is valued at $9 billion in annual revenues.
Since it takes months or even years for the disease symptoms
to appear, researchers have been working hard to come up
with novel ideas and technologies to detect HLB infection in
pre-symptomatic citrus tissues, so the infected plants can be
removed immediately to prevent further disease spread. Early
detection methods based on citrus biomarkers will continue
to be used in a systems approach to pinpoint hot spots where
the disease might be originating. Citrus transcriptomics (small
and micro RNAs), metabolomics (volatile organic compounds
[VOCs] and metabolites), and proteomics (proteins) currently
are being evaluated to diagnose early HLB infection in presymptomatic tissues in California, Florida and Texas where
the disease has been reported. These promising technologies
need to be fine-tuned, coordinated and validated by other
more sensitive, accurate and specific techniques such as realtime quantitative PCR (qPCR) and digital droplet PCR (ddPCR).
One of the biggest challenges facing plant pathologists today
is to isolate and culture Candidatus Liberibacter species
on an artificial nutrient medium in vitro. Culturing the HLB
bacterium will:
(a) pave the way for better detection and identification of the
bacterium and hence the disease;
(b) facilitate studies on the epidemiology of HLB, which
consequently will foster better disease management
strategies; and
(c) ease high-precision, whole-genome sequencing, leading
the way to better understanding the molecular biology of
host/pathogen/vector interactions that might identify genes
of interest for disease management.
The first and most important line of defense against HLB

is constant monitoring of ACP vector movement and the
HLB-associated Liberibacter spread based on data collected
from trapping and diagnostic analyses of samples using
early detection technologies. Second is taking sanitary and
preventive measures that include elimination of diseased
plants and regular spraying for psyllids in the Central Valley
and the release of parasitic wasps (Tamarixia radiata and
Diaphorencyrtus aligarhensis) to parasitize and kill ACP
nymphal instars on newly-developed leaf flush in Southern
California urban areas. This is accompanied with the use of
pathogen-free nursery stocks, enforcing quarantine measures
and educating the public through effective out-reach
programs. These coordinated actions already have begun in
California and will continue into the future as long as HLB
poses a threat.
Short-term approaches to counteract the debilitating effects
of HLB on the California citrus industry include soil fertigation,
microbial enrichment and effective use of therapeutics
including antimicrobial compounds, thermotherapy, etc.
Long-term approaches taken by research scientists on the
plant side to mitigate the HLB malady include breeding
for disease tolerance or resistance, use of gene silencing
technology (e.g., RNA interference) and development of
transgenic lines with enhanced resistance to HLB infection.
Some of these approaches are still at the infancy stage. Others
are being evaluated in the greenhouse and even in field trials
before being commercialized.
The complete list of 2015-16 approved projects may be found
in Table 1 on the next two pages.
Mojtaba Mohammadi, Ph.D., is an associate scientist with
the Citrus Research Board in Visalia, California, where he
also serves as the associate science editor of Citrograph.
19
20
TITLE
Pagliaccia
Vidalakis
Leveau
Thomson
Thomson
Louzada
Moore
Ramadugu
Zale
Vidalakis
Roose
Guinard
Jin
Slupsky
Chen
Cilia
Stansly
Ng
Coaker
Godfrey
Cilia
Rolshausen/Leveau
Thomson
McCollum
Falk
Lin
Sumerlin
New Proposals
5100-153
Real Time PCR co-detecHon of Candidatus Liberibacter species and Spiroplasma citri
5100-154
Citrus dwarng of commercial varieHes using TsnRNAs
5100-155
Citrus rhizobiomes and tree producHvity in response to soil manipulaHons
Con0nuing Projects
5200-141
The development of novel Blood and Cara cara like citrus varieHes
5200-142
UHlizaHon of founder lines for improved citrus biotechnology via RMCE
5200-144
Development of consumer-friendly transgenic citrus plants with potenHal broad spectrum resistance
5200-146
Rapid cycling plant breeding in citrus
5200-147
EvaluaHon of hybrids of citrus and citrus relaHves for huanglongbing (HLB) tolerance/resistance
5200-148
MicropropagaHon of mature citrus in temporary immersion bioreactors
5200-149
Streamlining the introducHon of licensed citrus varieHes into California: A case study - Florida
5200-201
CORE: integrated citrus breeding and evaluaHon for California
New Proposals
5200-150
OpHmizing sensory quality and consumer acceptance of citrus fruit through horHcultural pracHces
Con0nuing Projects
5300-131
IdenHcaHon and characterizaHon of HLB-induced small RNAs and mRNAs
5300-150
Biomarkers for detecHon of Liberibacter infecHon in citrus trees through 1H-NMR based metabolomics
A phage/prophage-based PCR system for sensiHve and specic detecHon of Candidatus Liberibacter asiaHcus and Spiroplasma citri
5300-151
Using mass spectrometry technologies to develop novel management strategies for citrus insect vector-borne pathogens
5300-155
5300-156
The citrus greening bibliographical database
5300-158
ConstrucHon of the cloned infecHous cDNA of Citrus tristeza virus (California isolate)
5300-160
IdenHfying and characterizing citrus targets from Candidatus Liberibacter asiaHcus
5300-161
Infrastructure support for research on detecHon and management of HLB and ACP
5300-163
Not all psyllids are created equal: Why do some transmit Liberibacter and others do not?
5300-164
A microbiota-based approach to citrus tree health
5300-165
Development of mature budwood transformaHon technology
Use of digital PCR for improved early detecHon of Candidatus Liberibacter asiaHcus infecHon in citrus and ACP
5300-168
New Proposals
5300-169
ArHcial microRNA-based targeHng of the Asian citrus psyllid for HLB management
5300-170
Develop a novel target-basis of anH-virulence strategy for controlling HLB
5300-171
Photosynthate-responsive polymeric nano-carriers for phloem-specic delivery in the treatment of HLB
5300 - Vectored Diseases
5200 - New VarieGes
Gupta
Kandelous
Ma
PRINCIPAL
INVESTIGATOR
Con0nuing Projects
5100-146
Novel therapy of high-priority citrus diseases
5100-150
OpHmizaHon of water and nitrate applicaHon eciency for citrus trees
5100-152
IdenHcaHon of key components in HLB using eectors as probes (co-sponsor with CRDF)
5100 - ProducGon Eciency
NUMBER
Table 1: 2015-2016 Research Projects
Table 1: 2015-2016 Research Projects
UC Davis
USDA-ARS
University of Florida
UC Riverside
UC Davis
USDA-ARS
Boyce Thompson InsHtute
UC Riverside
UC Davis
UC Davis
UC Riverside & UC Davis
USDA
USDA-ARS
UC Davis
USDA-ARS
USDA-ARS
Texas A&M
UC Riverside
UC Riverside
UC Riverside
UC Riverside
UC Riverside
UC Davis
Los Alamos
UC Davis
UC Riverside
AFFILIATION
$ 85,000

$ 72,424

$ 117,128

$ 146,406

$ 116,437

NCE*
$ 183,713

$ 18,114

$ 94,365

$ 106,974

$ 100,864

$ 146,275

$ 151,653

$ 2,504

$ 42,500

$ 101,114

$ 129,202

$ 115,106

$ 110,469

$ 103,712

$ 73,333

$ 22,872

$ 5,000

$ 627,307

$ 21,000

$ 6,535

$ 86,276

$ 67,500

$ 108,059

$ 22,290

BOARD
APPROVED
BUDGET
21
Adaskaveg
Adaskaveg
Adaskaveg
Walse
Xiao
Walse
Con0nuing Projects
5400-103
EvaluaHon of new post-harvest treatments to reduce post-harvest decays in packinghouse operaHons
5400-119
Disease forecasHng and management of Septoria spot of citrus
5400-148
Epidemiology and management of Phytophthora diseases of citrus in California
5400-149
Breaking criHcal pest-related trade barriers for California citrus exports
5400-150
Control of post-harvest diseases of citrus
Sub-Award
5050-010
Vargas
Stouthamer
TBD
Stouthamer & Hoddle
New Proposals
5500-208
Eects of ACP cover sprays against fruit ies (TephriHdae) and their natural enemies
Sub-Awards
5500-196
Biological control of Asian citrus psyllid in California (CPDPP)
6310
Contract rearing of Tamarixia
6320/6321 Development of mass-rearing methods for the parasitoid, Tamarixia radiata, to support classical biological control
Printed: 11/24/15 2:00 PM
NCE* represents a No Cost Extension where funds for project comple:on were approved in the previous scal year.
Morse
Qureshi
Hoddle
Hoddle
Stouthamer
Qureshi
Stelinski
Grapon-Cardwell
Con0nuing Projects
5500-189
OpHmizing chemical control of Asian citrus psyllid in California
5500-189E Development of an Asian citrus psyllid (ACP) management plan for organic citrus
5500-191
Host specicity tesHng of Diaphorencyrtus aligarhensis
5500-194
Release and monitoring of Tamarixia radiata and phenology of Asian citrus psyllid in Southern California
5500-197
Impact of resident predator species on control of Asian citrus psyllid populaHons
5500-205
Toxicity of syntheHc and organic insecHcides to Tamarixia radiata, ecto-parasitoid of Asian citrus psyllid
5500-206
Development of new trapping and control methods for ACP based on complex citrus lure blends
5500-501
CORE: IPM program
5500 - Pest Management
Breaking criHcal pest-related trade barriers for California citrus exports (TASC)
5400 - Non-Vectored and Post-harvest Diseases
Gomwald
Slupsky
Willey
Sub-Awards
5300-154
Risk-based decision making in the management of huanglongbing (CPDPP)
5300-162
DetecHon of Candidatus Liberibacter in citrus in Hacienda Heights and other areas of California (CPDPP)
5050-041
Using the internet to train citrus hobbyists to order budwood from CCPP and not to spread HLB (CPDPP)
Cilia
Rolshausen/Leveau
Thomson
McCollum
Falk
Lin
Sumerlin
Schneider
Godfrey
Yokomi
LeVesque
McCollum
Not all psyllids are created equal: Why do some transmit Liberibacter and others do not?
A microbiota-based approach to citrus tree health
Development of mature budwood transformaHon technology
Use of digital PCR for improved early detecHon of Candidatus Liberibacter asiaHcus infecHon in citrus and ACP
New Proposals
5300-169
ArHcial microRNA-based targeHng of the Asian citrus psyllid for HLB management
5300-170
Develop a novel target-basis of anH-virulence strategy for controlling HLB
5300-171
Photosynthate-responsive polymeric nano-carriers for phloem-specic delivery in the treatment of HLB
Development of PCR-based diagnosHc tools for detecHon and dierenHaHon of citrus leprosis-associated viruses
5300-172
5300-173
Eect of mixed infecHons of plant pathogens on detecHon of HLB using two early detecHon methods
5300-174
Establish a system to infect and maintain Nico:ana benthamiana and citrus with the recombinant CTV
5300-175
EDT experiment
5300-176
Flush & Nypmh experiment
5300-163
5300-164
5300-165
5300-168
UC Riverside
TBD
UC Riverside
USDA-ARS
UC Riverside
UC Riverside
UC Riverside
UC Riverside
UC Riverside
USDA-ARS
UC Riverside
UC Riverside
UC Riverside
USDA-ARS
USDA-ARS
USDA-ARS
UC Davis
Fruitmentor
UC Davis
USDA-ARS
USDA-ARS, FDWSRU
UC Davis
USDA-ARS
CRB
USDA-ARS
$ 181,745

$ 250,000

$ 248,404

$ 30,000

$ 116,660

$ 66,565

$ 206,407

$ 231,358

NCE*
$ 69,642

$ 100,740

$ 420,155

$ 444,235

$ 61,000

$ 53,000

$ 134,000

$ 40,576

$ 73,557

$ 180,601

$ 55,773

$ 20,000

$ 85,000

$ 72,424

$ 117,128

$ 56,428

$ 214,737

$ 94,950

$ 254,619

$ 113,000

$ 146,275

$ 151,653

$ 2,504

$ 42,500

TOTAL $ 6,702,284


UC Riverside & UC Davis
USDA
USDA-ARS
MEET YOUR
BOARD MEMBERS
Ivy Leventhal
The Citrus Research Board (CRB) is governed by 21 dedicated volunteers from a wide variety of backgrounds and
geographical areas of the California citrus industry. Fifteen of the Board members represent northern California; three
are from southern and coastal California; two represent the desert area; and there is one public member.
Periodically, we will introduce you to several of your representatives so that you can learn more about these hardworking Board members who volunteer significant portions of their time for the betterment of the citrus community. In
this issue, youll meet three of the most recent appointees.
Greg Galloway has served on the
Board since 2014 and represents
District One. During the past year,
he has served on a number of
committees Communications,
Production Efficiency, Research
Development and Implementation,
Pest Management and NonVectored Disease and Post-Harvest.
My experience on the Board so
far has been interesting, amusing,
confusing and at times overwhelming, Galloway said. I have come
to realize how little I know and how truly open I am to new ideas
and concepts. Our industry has many exceptionally bright people
working on a tremendous number of research projects. These
scientists are compassionate about their work, while patient with
those of less intelligence. I feel blessed to be allowed into their sand
box.
Our industry is being bombarded with many challenges, he
continued. Trade partners are placing quality demands that require
significant production and processing adjustments. Neighboring
growing regions are experiencing the wrath of a crippling, perhaps
apocalyptic disease. We are on high alert regarding the invasive
nature of this disease and have spread a very wide research net.
22
Our research dollars are prioritized toward preserving our industry,

which is a tall order. The CRB is blessed with a strong, seasoned
Board willing to face industry challenges without reservation. They
are strengthening and raising the bar on management to meet the
requirements of executing the necessary directives. I have no agenda
other than gratitude for the opportunity to serve the industry that
has provided for the livelihood of my family.
A veteran of more than four decades in the citrus industry, Galloway
lives in Porterville, where he is the general manager of Sierra Crest
Agriculture, which has acreage in Tulare and Fresno counties. His work
involves the cultural aspects of citrus and table grape production.
In his free time, the married father of two sons enjoys reading, his RV
and visits to the beach.
The most recent representative
who was elected to District Two in
2014 is Mike Perricone. His areas
of interest are new varieties and
pest management, and he has been
serving on the CRBs New Varieties
and Non-vectored Diseases and
Post-harvest Committees during his
first year.
Leffingwell_Ad.pdf
I think the greatest issue for all of us in the citrus industry is the
threat of HLB, Perricone said. Its not going to be an easy fight,
but I think we will be able to beat this thing out if we all work
together.
The San Juan Capistrano native who now lives in Temecula is a
relative newcomer to the California citrus industry. For the past
several years, he has served as the operations manager for Pauma
Ranches in Pauma Valley, responsible for citrus and avocado
acreage and staff.
Perricone and his wife have one son. When not working or fulfilling
his CRB Board duties, the D.A.R.E. America volunteer enjoys the
fast sport of roller hockey and spending time with his family.
The newest over-all Board
member, representing District
One and elected in 2015, is Keith
Watkins. As Vice President of
Farming and Field Operations
for Bee Sweet Citrus in Fowler,
he is charged with overseeing
approximately 10,000 acres
and is responsible for land
acquisition, investor relations
and financial oversight. His son,
Matthew, works with him on Bee
Sweets farming operations, which has allowed Watkins the time
to serve as a vice president of the Cawelo Water District and a past
president of the Tulare County Farm Bureau, as well as volunteer
on the CRB Board.
In this latter capacity, the Visalia native is a member of the
Pest Control Committee, which dovetails with his interest in
addressing the current major threats facing the industry. HLB
and ACP are dark clouds hanging over us, Watkins said. I want
to help concentrate the CRB in a direction that will focus all of
the research currently being conducted to come up with answers
to those two problems. We need to ensure that the scientists are
talking to each other and building on each others work.
A veteran of more than two decades in the California citrus
industry and an alumnus of the California Agricultural Leadership
Foundation, Watkins stated, We should be focusing on the CRBs
financial responsibility by utilizing the growers money to invest in
areas that will provide good returns to the growers.
When not at work or volunteering in his industry and community,
the married father of three enjoys traveling and family time.
Ivy Leventhal is the managing editor of Citrograph.
6/7/13
2:50 PM
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23
HLB DETECTIONS
IN SAN GABRIEL
Where are we now?
Victoria Hornbaker and Lucita Kumagai
24
CDFA crew removing

HLB-positive lime tree in
the San Gabriel area.
n April 2012, the first California detection of huanglongbing (HLB),

also known as citrus greening, was confirmed in a tree in Hacienda
Heights. It wasnt until July 2015 that the second confirmed California
detection of HLB was found in the San Gabriel area of Los Angeles
County, about 15 miles from Hacienda Heights. Both finds were
residential citrus trees. Prior to the second incident, it appeared
that no news was good news and that the 2012 find was isolated.
However, the 2015 San Gabriel detection has led to nine additional
HLB-positive plant samples. Those detections resulted in the first and
(as of November 2015) only cluster of the disease in California. Now
that the dust has settled, heres the sequence of events that occurred
in San Gabriel in the continuing battle against this devastating citrus
disease.
SAN GABRIEL HLB TIMELINE

TREE 1 (JULY 9, 2015)
The initial HLB-positive tree, a kumquat, was found as a result of the

California Department of Food and Agriculture (CDFA) Risk-Based
HLB Survey and the diligence of the CDFA team. Asian citrus psyllid
(ACP) collected from the find site tested in the HLB-inconclusive range
which triggered a CDFA protocol to revisit the site, collect plant tissue
and additional ACP, if available. The kumquat tree was chlorotic but
the leaves did not present the classic asymmetrical mottling typical
of HLB.
DNA extracted from the plant tissue tested positive for Candidatus
Liberibacter asiaticus, the bacterium associated with HLB, by realtime PCR, conventional PCR and DNA sequencing. The tissue was
sent to USDA for confirmation, which was received on July 9. With
the homeowners permission, the tree was first treated with a foliar
insecticide and then removed on July 10. CDFA also surveyed and
took samples of ACP and plant tissue on all adjacent properties and
applied foliar and systemic treatments (with homeowner permission).
The state agency announced the HLB detection to the public later that
day.
As a result of the first find, CDFA began activities to place a quarantine
for HLB in the San Gabriel area, consisting of a five-mile radius around
the find site, totaling 87 square miles. CDFA then began survey and
treatment activities on all HLB host plants within 800 meters of the
find site. By taking those essential steps, the critical reservoir of disease
and its vectors (ACP) were in the process of being removed.
On July 13, CDFA Pest Exclusion staff began survey activities of
production and retail nurseries in the proposed quarantine area,
placing all host plants on hold.
25
2015 Huanglongbing (HLB)

Quarantine Map
HLB quarantine map in Southern California, including the single 2012 Hacienda Heights detection and the 10 San Gabriel finds in the summer of 2015.
TREE 2 (JULY 15, 2015)
Days later, a lime tree on a neighboring property was

confirmed positive for HLB. Similar to the initial tree, the lime
tree tested positive by PCR and DNA sequencing. The tree was
removed with homeowner permission on July 16. CDFAs lab
ran samples to determine if the disease had spread to other
citrus trees in the area.
To inform the public and particularly the neighborhood, a
public meeting was held the evening of July 16 and was very
well attended. Assemblyman Ed Chau was in attendance,
as was Kurt Floren, the Los Angeles County Agricultural
Commissioner.
Following the neighboring finds, notices were delivered to
residents to notify them that insecticide treatments would
begin on July 20.
TREES 3-4 (JULY 22, 2015)
CDFA confirmed two additional HLB-positive trees, a Mandarin

and a calamondin, on the same street as the initial tree. Both
trees were treated and removed, with homeowner permission
on July 22.
CDFA Pest Exclusion staff continued working with the USDA
to survey approximately 90 nurseries and garden centers,
26
noting that HLB host material was found at 30 of the entities,

and 8,040 plants were placed on hold. All were voluntarily
destroyed except for the 123 plants at one entity, which opted
to construct an insect-resistant screenhouse for the plants.
TREES 5-9 (AUGUST 6, 2015)
Five additional trees were confirmed HLB-positive at three

locations within a block and a half of the previous finds. One
location had three trees, and two locations each had one
infected tree. On August 7, CDFA removed the three trees
on the one property with homeowner consent. By the next
day, CDFA removed the two remaining trees on separate
properties, also with homeowner consent.
As a result of the new find locations, the treatment area was
expanded. A public meeting for the expanded area was held
on August 11. CDFA continued to treat properties where no
contact with the owner could be made, as well as refusal
properties under abatement authority.
TREE 10 (AUGUST 18, 2015)
An additional tree was confirmed HLB-positive less than

half a mile from the initial positive tree, and it was removed
with homeowner consent. Survey and treatment areas were
expanded yet again, adding 900 properties, and an additional
public meeting was held on September 3.
With the strategy set by the citrus industry and immediate response by CDFA
survey and treatment crews, no other San Gabriel samples have been confirmed
for HLB at the time of this printing.
CONTINUING THE FIGHT

IN THE GROVE
This is a wake-up call for all growers, particularly those in Southern California.
Growers should be participating in an area-wide management program to control
ACP populations in commercial groves. With HLB in California, ACP management
becomes even more crucial. Review the best management practices at www.
citrusinsider.org/resources, and employ them in your groves. Also, see the flyer
on page 28. The Citrus Pest and Disease Prevention Program (CPDPP) and CDFA
encourage growers to regularly survey their own groves for signs of ACP and HLB.
CPDPP will continue to trap for ACP in commercial groves throughout the state.
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IN THE BACKYARD
Through CPDPPs support, CDFA survey and treatment crews will remain vigilant
in looking for HLB and treating for the Asian citrus psyllid in residential areas. CDFA
in partnership with the USDA, local agricultural commissioners and the citrus
industry continues to pursue a strategy of controlling the spread of Asian citrus
psyllids while the Citrus Research Board and other researchers work to find a cure
for the disease.
Visalia Citrus Packing Group 813.6 kW DC
BY THE NUMBERS
Total ACP samples collected from San Gabriel, including
expansion areas: 1,504
Total plant samples collected from San Gabriel, including
expansion areas: 6,735
LoBue Citrus 492.2 kW DC
Confirmed positive ACP samples: 4

Confirmed positive plant samples: 10
References:
www.citrusinsider.org/resources
http://www.cdfa.ca.gov/phpps/acp
Victoria Hornbaker is with the Citrus Pest and Disease Prevention Program,
where she serves as citrus program manager. Lucita Kumagai is with the Plant
Pest Diagnostics Branch at the California Department of Food and Agriculture,
where she serves as senior plant pathologist.
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27
Young Asian citrus psyllids are yellow

and produce a white, waxy substance.
Asian citrus psyllids are brown, aphidlike insects that feed on the leaves
and stems of citrus trees.
HLB-infected trees will die.
Growers, your help is needed to protect

California citrus from HLB!
Huanglongbing, or HLB, could be a death sentence for our industry if it is allowed to take hold. We
must work together to suppress, and where possible, eradicate populations of the Asian citrus psyllid
to protect our industry from the devastation caused by HLB.
Here are a few ways you can help:

Talk to your employees and contractors The
Asian citrus psyllid is known for its ability to
hitch hike on plant material so it is critical that
no leaves or plant material leave or enter a field
in order to keep the pest from spreading. Ask
your employees, contractors, and pest control
advisors to follow best practices in the field to
minimize the movement of plant material from
field to field.
Know your Liaison Every citrus producing
region in California has a Grower Liaison dedicated to keeping you informed about psyllid finds
and proper treatment protocol. Know who your
Grower Liaison is and contact them with any
questions about how best to protect your citrus
from ACP and HLB.
Sample and Treat Follow UC recommended
guidelines for sampling and treating for the
Asian citrus psyllid.
Work with your neighbors Area-wide

management of the Asian citrus psyllid is a
strategy where growers in a specific area
coordinate management efforts to maximize
the impact on psyllid populations. Psyllid
Management Areas (PMA) are being formed now
so if the time comes to implement an area-wide
strategy, growers in the region are ready.
Stay Informed The Citrus Insider website
is your resource for all information pertaining
to the Asian citrus psyllid and HLB. Visit
www.citrusinsider.org for more information
about psyllid finds in your area, best practices,
treatment recommendations, area-wide
treatment protocol, maps, and more. You can
also sign up to have regional alerts sent to your
email address.
We must all do our part to protect our trees, our industry, and our way of life.
28
CitrusInsider.org
29
Figure 1A
HLB IMPACT ON CITRUS ROOT

HEALTH AND INTERACTION
WITH PHYTOPHTHORA
James Graham
uanglongbing (HLB), which translates from Chinese

as Yellow Shoot Disease (also referred to as greening),
is the most devastating known disease of citrus. After the
establishment of HLB in a citrus production area, at least three
highly predictable events occur:
1) The citrus industry remains profitable only when the psyllid
vector, Diaphorina citri, and the HLB-associated bacterium,
Candidatus Liberibacter asiaticus (CLas), are stringently
controlled with insecticide sprays and removal of symptomatic
trees, respectively.
30
2) Every country in the Americas adjacent to a CLas-positive

nation detects HLB within five years or less (e.g., Texas and
California proximal to Mexico, and Argentina proximal to
Brazil).
3) In areas where HLB is well established, the disease causes
unprecedented increases in production costs, crop loss and
reduction of internal and external fruit quality.
While leaf symptoms may resemble nutritional stress or
deficiencies (e.g., zinc and iron), these symptoms actually
are due to disruption of carbohydrate metabolism and
Figure 1B
Figure 1. HLB infection symptoms: (A) Leaf mottling symptom on a Hamlin
orange leaf indicative of carbohydrate disruption; (B) HLB-induced fruit drop
in eight-year old Hamlin orange trees on Swingle citrumelo rootstock trees in
October 2015.
allocation as a result of plugging and necrosis of phloem, the

plants sugar conducting system. Because of carbohydrate
disruption, there is significant loss of fibrous roots, mottling
and yellowing of leaves (Figure 1A) and shoots, defoliation,
dieback, tree decline and excessive fruit drop (Figure 1B). Fruit
are small and misshapen with aborted seeds, are abnormally
colored and contain off-flavored juice low in brix. In the 10
years following the detection of HLB, Florida citrus production
has dropped 50 percent with no sign that the rate of crop
loss is slowing (http://www.nass.usda.gov/Statistics_by_State/
Florida/Publications/Citrus/cit/2015-16/cit1015.pdf).
At the University of Floridas Citrus Research and Education
Center (CREC), my research program in collaboration with
Research Assistant Scientist Evan Johnson, Ph.D., has mainly
focused on the belowground impact of systemic CLas infection.

What we have learned about bacterial infection, movement
and root loss has profound implications for how diseased
trees are managed. Initially in field surveys, we measured
30-50 percent loss of fibrous root density before any sign of
aboveground symptoms. Dr. Johnsons detailed investigation
of the early events in bacterial infection revealed that CLas
moves downward to the fibrous roots soon after transmission
in the shoots, is unrestricted in the root system and multiplies
to damaging levels within months of infection. The fibrous
root loss is distributed throughout the root system because
the bacterium does not induce phloem plugging and, at least
initially, does not produce carbohydrate starvation of fibrous
roots. Root loss is followed by the first expression of canopy
symptoms in foliage, premature fruit drop and a proportional
yield loss of 30 percent based on crop loss estimates in Brazil
and Florida. Without management intervention, root turnover
accelerates, and the canopy continues to thin as a result of 7080 percent fibrous root loss.
Paradoxically, root growth is stimulated on HLB-affected
trees even those in advanced decline a sign that HLB trees
are in a survival mode. What these findings tell us is that the
priority is to grow new roots as the tree declines and that
root replacement is expensive, reducing fruit production and
retention. Hence, stimulating extra root growth is not likely
to help and may increase root production at the expense of
fruit. A better approach is to promote regular growth cycles
of roots, increase root lifespan (i.e. reduce root turnover) and,
as much as possible sustain root functioning in water and
nutrient uptake.
Our findings have shifted the focus of HLB management
belowground to practices that increase root health. In the
Florida production system, this begins by minimizing stress
in the micro-sprinkler wetted zone where 80 percent of the
fibrous roots are concentrated. More frequent irrigation cycles
of shorter duration and weekly to bi-weekly fertigation are
recommended to maximize efficiency of water and nutrient
uptake by the reduced fibrous root system.
Monitoring of water quality, as well as quantity, also has
been discovered to be of critical importance. The majority of
Florida groves draw irrigation water from deep wells located
in limestone aquifers. Well-water often has a pH in excess of
7.5 and bicarbonates above 100 ppm. Our surveys established
that bicarbonates reduce fibrous root density and tree yields
where well-water pH exceeds 6.5 and soil pH exceeds 6.2,
especially in groves on Swingle citrumelo and Carrizo citrange,
the rootstocks most sensitive to bicarbonate stress.
31
Figure 2A
Figure 2B
Figure 2. A. Average propagules of Phytophthora nicotianae in rhizosphere soil samples collected in Florida groves between January 2008 and October 2015
(YTD). B. Average dry weight of fibrous roots in soil samples collected between January 2013 and October 2015 (YTD) (data courtesy of J. B. Taylor, Syngenta
Crop Protection).
Importantly, the combination of CLas infection and

bicarbonate stress increases susceptibility to root pests and
pathogens including Phytophthora species. The causal species
of Phytophthora diseases in Florida citrus are Phytophthora
nicotianae (parasitica), the most common cause of foot
rot (gummosis) and root rot, and P. palmivora, which most
often is the cause of brown rot of fruit and root rot in poorly
drained soils with high water tables. Wet conditions favor root
infection cycles of Phytophthora species.
Susceptibility of fibrous roots is highest during very wet to
very dry cycles (spring and fall). Extreme wetting and drying
promotes root exudation (release of organic chemicals),
which attracts zoospores. CLas-infected roots exude more
sugars than healthy roots, which increases zoospore infection.
Evidence for greater incidence of this interaction comes from
both greenhouse trials and grove surveys. Phytophthora
nicotianae populations initially increased in potting soil at
two, eight and 14 months post-inoculation in HLB-infected
trees compared to un-infected controls. This was followed by
a decline after a major loss of fibrous roots mainly due to CLas
infection.
Survey data from Florida groves suggest a resistance-breaking
interaction of CLas with Phytophthora species. Syngenta Crop
Protection has conducted a statewide survey of Phytophthora
species that has spanned over two decades, covers all
production areas and is largely driven by grower requests. The
survey results serve as an indicator of emerging root disease
trends. Comparison of the survey data for seasons since
HLB became widespread in Florida groves shows a strong
trend toward higher incidence of damaging Phytophthora
populations coincident with the rise in HLB disease incidence
from 2008-2011 (Figure 2A). Since then, there has been a
strong downturn in 2013 and recovery of the populations
in 2014 associated with biennial fluctuations of fibrous root
density as HLB trees continue to decline (Figure 2B). The
survey results have served to heighten grower concern for the
32
root health of HLB-affected trees and attention to reduction

of root stresses.
Past research experiences and current Phytophthora data
trends indicate a need for more comprehensive management
of HLB-affected trees. Health of fibrous roots is fundamental
to sustain soil, water and nutrient uptake in marginal soils.
This is to resist fluctuations in soil moisture, root pests and
other adverse conditions. Symptoms of stress intolerance
include off-colored foliage and excessive leaf and fruit drop
in HLB-affected trees, even when trees are managed under
intensive nutrition programs for several seasons. Preliminary
data indicate that the CLas interaction also reduces the trees
response to fungicides for prevention of root loss, because the
bacterial infection is the major contributor to damage of coinfected roots.
Currently, our recommendations are to first manage soil and
water stresses with a balanced application of irrigation and
nutrients to the root system (spoon feeding) and to reduce
soil pH/bicarbonate stress to sustain root function in nutrient
uptake and root longevity. To assess bicarbonate stress,
growers test well-water for pH, bicarbonates, salinity, cations
and anions and periodically check soil pH in the wetted zone. If
bicarbonate stress is indicated, the recommendation is water
conditioning by injection of either sulfuric acid (40 percent)
or N-furic acid (urea plus sulfuric acid) to reduce irrigation
water below 100 ppm bicarbonate, or soil conditioning by
broadcasting sulfur in the wetted zone to reduce soil pH to
6.2 or below. Thus far, acidification has produced a more
balanced leaf nutrient status, especially for calcium (Ca),
magnesium (Mg) and iron (Fe), and improvement in health,
vigor and productivity of HLB trees (Figure 3).
After correcting water and soil stresses, the next priority is
to manage root pest and pathogens. Phytophthora species,
nematodes and weevils should be treated more aggressively
(i.e., use of the full label rates and frequency of applications)
Figure 3. Valencia orange trees on Carrizo citrange rootstock trees before (A) and after (B) 2.5 years of water acidification in a grove irrigated with water high
in bicarbonates. Note more fully expanded leaves and absence of dead twigs in top of the tree canopy after acidification.
to sustain root health of HLB trees. Details for management

are found in the Florida Citrus Pest Management Guide (www.
crec.ifas.ufl.edu/extension/pest/). Assessment of Phytophthora
disease is based on a soil propagule assay that measures
population density in the rhizosphere in the wetted zone.
If counts exceed 10-20 propagules per cm3 of soil volume,
the following rotation of fungicides may be recommended:
fosethyl-al or phosphite after spring shoot flush, mefenoxam
after spring-early summer rains begin, fosethyl-al or phosphite
after midsummer shoot flush, and mefenoxam after fall shoot
flushes. The timing of soil application is for protection of
root flushes that follow shoot flushes in the tree life cycle
(phenology).
James Graham, Ph.D., is a professor of soil microbiology at
the Citrus Research and Education Center (CREC), University
of Florida, Lake Alfred, Florida.
The author thanks Davis Citrus Management and Syngenta
Crop Protection for generously sharing data cited in the article
and the Citrus Research and Development Foundation (CRDF)
for grant support.
References
Graham, J.H., Johnson, E.G., Gottwald, T.R., and Irey, M.S. 2013.
Presymptomatic fibrous root decline in citrus trees caused by
huanglongbing and potential interaction with Phytophthora
spp. Plant Dis. 97:1195-1199.
Johnson, E.G., Wu, J., Bright, D.B. and Graham, J.H. 2014.
Association of Candidatus Liberibacter asiaticus root
infection, but not plugging with root loss on huanglongbingaffected trees prior to appearance of foliar symptoms. Plant
Pathol. 63:290-298.
Glossary
Root exudation: Roots of higher plants release
organic compounds (sugars, amino acids, lipids,
vitamins, etc.) that may serve as chemical attractants
or repellents to a particular microbial community in
rhizosphere.
Propagule: Fungal spores or bacterial cells that
transmit a disease.
Rhizosphere: Zone (few mm in thickness)
surrounding the plant root system where plant,
microorganisms and soil come together influencing
root chemistry and biology.
Tree phenology: Plant life cycle events that are
influenced by seasonal variations in climate and
elevation.
33
CRB-FUNDED RESEARCH PROGRESS REPORT
Figure 1. Adult ACP on a curry plant in a cage

provided by the UCR Tamarixia radiata program.
MONITORING FOR ACP

RESISTANCE TO PESTICIDES
Baseline Levels Established for 12 Pesticides
Joseph Morse, Beth Grafton-Cardwell, Frank Byrne and James Bethke
Many research projects are in progress in Florida, California and elsewhere looking for
long-term solutions to huanglongbing (also known as citrus greening or HLB). In the
interim, area-wide, coordinated chemical control of the Asian citrus psyllid (ACP) is critical
to slowing the spread of HLB in California.
One danger of aggressive ACP chemical control, however, is that resistance can develop
in the psyllids to some of the more effective and persistent classes of chemistries such as
neonicotinoids and pyrethroids (Tiwari et al. 2011, 2012, 2013). We are reporting here on
baseline ACP resistance monitoring designed to determine the susceptibility of a California
ACP population with minimal past exposure to 12 pesticides that will be used for control in
future years.

34
High ACP field populations soon after a treatment are not

always due to resistance - several factors can be at play. In
some cases, high populations of adults are present and reinvade a treated area soon after treatment, especially if a
substantial amount of attractive flush is present. In other
cases, spray timing, choice of chemical or application method
is not ideal. In some situations, natural enemy levels are low in
the treated grove due to past treatment history and thus do
not contribute to pest management. Baseline resistance levels
established in the current study can be used in the future to
differentiate pesticide resistance from other factors leading to
less than expected field control.
and then westward into northwestern Mexico. We assume

these psyllids had little exposure to pesticides in California,
although it is possible that past generations were treated to
some degree in Florida and/or Mexico. Psyllids were reared
under CDFA permit, mostly on curry leaf plants (some citrus
was added to rearing cages) in the UC Riverside Insectary by
the Richard Stouthamer laboratory (CRB Project 5500-196)
and were used mostly for the Tamarixia radiata biocontrolrearing program. In addition, Project 5500-196 provided ACP
nymphs and adults to other research projects, such as for this
baseline resistance-monitoring project.
ACP was first discovered in California in 2008, and it is assumed

that this population migrated north from Mexico after having
moved originally into southeastern Mexico from Florida
In conducting this work, we used six- to seven-day-old adult

ACP, which were provided to us on a curry plant inside a cage
(Figure 1). A small hand-held mouth aspirator was used
to collect about 20 adults into a small plastic vial (Figure
2). Then, a small drop of molten agar was poured onto the
bottom of an 8.5 cm diameter Petri dish; once it solidified, a
piece of grapefruit leaf from the UC Riverside Biocontrol Grove
was dipped into the agar at one end to ensure it would remain
fresh during the entire study. The leaf served as a food source
for the psyllids to feed on (Figure 3). For each trial, we included
three replicates of each pesticide rate, with 20 ACP adults in
Figure 2. Lindsay Robinson aspirates about 20 adult ACP into each vial just
before a baseline resistance micro-applicator test.
Figure 3. A small drop of liquid agar is poured on the bottom of a Petri dish
and allowed to solidify prior to placing part of a clean citrus leaf.
The University of California (UC) Riverside ACP colony used in

these trials was initiated from psyllids collected from a curry
leaf tree (Murraya koenigii) in Azusa, California, on three dates
by David Morgan and the staff of the California Department
of Food and Agriculture (CDFA) Biocontrol Program during
February 24-27, 2012.
35
Figure 4. Three Petri dishes were set up per treatment; these are the acetone controls prior to introduction of about 20 micro-applicator-treated ACP from each
collection vial.
each replicate per dish (Figure 4). Prior to pesticide exposure,

adult ACP were knocked out by exposure to carbon dioxide
for 60 seconds (Figure 5). With practice, so that treatments
could be applied quickly, the adults remained unconscious
long enough to apply a pesticide dose to each psyllid.
The intent of our studies was to use methods similar to those
used by Tiwari et al. (2011) in Florida so that California and
Florida psyllid insecticide resistance data could be compared.
A Burkard Auto Micro-applicator was used to apply small
droplets of technical grade insecticide dissolved in 100
percent acetone to the dorsal (back) surface of individual
adult ACP. The foot pedal activating droplet discharge out of
the micro-applicator syringe made it possible for one person
to hold the 20 ACP on a piece of filter paper, apply a droplet to
each psyllid and then remove them from the syringe tip with
a small paint brush (Figure 6).
In three preliminary trials on June 12, 20 and 28, 2012, we
evaluated the Tiwari et al. (2011) method. Whereas they
applied a 0.2 l droplet to each psyllid, we decided to use
0.8 l, so that we were sure each psyllid received a consistent
exposure before the acetone in the droplet evaporated. In
these preliminary trials, we also (1) evaluated the minimum
exposure to carbon dioxide that was needed and settled on
60 seconds; (2) confirmed that using 100 percent acetone
resulted in acceptable control (acetone only) mortality; and
(3) compared holding treated psyllids for 24 vs. 48 hours
before mortality was assessed, settling on 48 hours (Tiwari et
al. 2011 used 24 hours). Live adult ACP with their characteristic
feeding posture at 45 off the leaf were easy to tell from dead
ACP (Figure 7). If there was any question, a small clean paint
36
Figure 5. Adults in a vial are knocked out with carbon dioxide.
brush was used to prod the psyllid to confirm whether it was

dead or alive.
We conducted a total of 59 micro-applicator bioassays from
July 2, 2012, to August 27, 2014 (details listed in Table 1).
Technical grade insecticides (except formetanate) used in
these trials included abamectin (92 percent active ingredient
Figure 6. A 0.8 l droplet of pesticide in acetone is applied to each ACP adult with a micro-applicator while they are still knocked out with carbon dioxide. The
ACP adult is removed from the micro-applicator syringe tip with a small paint brush.
Figure 7. Dead ACP adults on the leaf in a Petri dish when the bioassay is evaluated 24 hours after micro-applicator treatment.
[AI]; trade name Agri-Mek), chlorpyrifos (97 percent; Lorsban),

cyantraniliprole (97.1 percent; Exirel), fenpropathrin (91.7
percent; Danitol), flupradifurone (99 percent; Sivanto),
formetanate hydrochloride (92 percent; Carzol SP),
imidacloprid (98.8 percent; Admire Pro), spinetoram (84.4
percent; Delegate), sulfoxaflor (97.9 percent; Sequoia),
thiamethoxam (98-100 percent, assumed 98 percent in AI
calculations; Actara), tolfenpyrad (99.5 percent; Bexar),and

zeta-cypermethrin (93.6 percent; Mustang).
Each pesticide (except Sivanto) was tested twice first,
in Round 1 tests and, second, in Round 2. Sivanto prime
was received late enough so that only Round 2 testing was
done. Each material was tested on one to six bioassay dates
37
per round until consistent data were obtained resulting in a

good log pesticide dose probit mortality regression (a Chisquare statistic greater than p=0.05, see Table 1). A total of 33
bioassays were needed for the 11 pesticides tested in Round 1,
and this work was completed from July 2, 2012, to January 8,
2014. Data from six bioassays during Round 1 were discarded
due to high control mortality (14 percent or higher). In the
remaining 27 Round 1 trials, control mortality ranged from
0 - 11.5 percent, averaging 3.6 percent. Round 2 bioassays of
all 12 pesticides were done from July 17, 2013, to August 27,
2014. During Round 2, we discarded data from two bioassays
with high control mortality (16 percent or higher). Data from
24 Round 2 bioassays were used in probit regressions, and
control mortality ranged from 0 - 7.8 percent, with a mean of
3.2 percent.
RESULTS AND DISCUSSION
Probit regressions were done separately for Round 1 and Round

2 bioassays using SAS/STAT software v9.3 (SAS Institute, Cary,
North Carolina). Data are shown in Table 1 with pesticides
listed according to their class of chemistry. LD95 is the Lethal
Dose estimated from the micro-applicator data needed to kill
95 percent of the tested ACP population. Table 1 also lists the
estimated LD95 values as a fraction of the normal ACP field use
rate. This is a somewhat arbitrary comparison as the pesticide
concentration for the LC95 is based on applying technical
grade material in acetone in a 0.8 l droplet to the back of each
psyllid, whereas the field pesticide use rate is based on speed
sprayer application assuming 200 gallons of water per acre.
Some of the older materials, e.g., organophosphates (Lorsban)
and carbamates (Carzol), are used at significantly higher per
acre use rates than many of the newer pesticides.
In general, the estimated LD50 and LD95 values were similar in
Round 1 and Round 2 bioassays. In seven of 11 cases (Actara,
Exirel, Delegate, Admire Pro, Bexar, Sequoia, Carzol), the
LD50 values in Round 1 bioassays were considered the same
as in Round 2 bioassays based on overlap of the 95 percent
confidence intervals (CIs in Table 1).
In the four remaining cases (Agri-Mek, Mustang, Danitol,
Lorsban), the Round 2 LD50 was slightly higher than that
observed in Round 1. In 10 of 11 cases, the Round 1 and
Round 2 LD95 estimates were the same. This was not true only
with Admire Pro. In this case, the Round 2 LD95 was somewhat
lower than that seen in the Round 1 bioassay. Overall, we view
LD values as being similar in Round 1 and Round 2 bioassays,
but for the purpose of future resistance monitoring efforts,
suggest that Round 1 results be used for comparison as these
tests were done relatively soon after the ACP population was
collected from the field. Round 1 tests were done an average
of 14.1 months after field collection (range of five [Bexar] -21
[Sequoia] months; 16 months with Carzol) and Round 2 tests
23.7 months after collection.
In 10 of 11 cases, the Round 2 regression line slope was
steeper than that of the Round 1 slope. The slope of the
38
regression line is considered an indication of how diverse or

uniform the tested population is, with a steeper slope being
indicative of a population with less diversity. For the 10 cases
where Round 2 slopes were steeper than Round 1, the average
slope during Round 1 tests was 3.24, whereas Round 2 slopes
averaged 4.60, an increase of 1.37. For these 10 cases, Round 2
bioassays were started an average of nine months after Round
1 bioassays ended (range of three months with Delegate to 17
months with Mustang). This increase of slopes with increased
time in culture was not unexpected. It is well known that
insect populations become less diverse the longer they are
reared in lab cultures.
We designed this study so that data we generated might be
compared to data developed on Florida ACP populations by
Tiwari et al (2011). Their work was done on a lab population
of ACP that had been in culture since 2000, and testing was
done on that colony and on five field populations during 2009
and 2010. Tiwari et al (2011) studied six of the same pesticides
we examined, and they generated a total of 33 probit
regressions (six bioassays with each of Actara, Admire Pro,
Agri-Mek, Danitol and Lorsban; only the lab colony and two
field populations were examined with Delegate). Comparing
their data with our Round 1 data, 30/33 Florida bioassays gave
LC95 values that agreed with California levels (the exceptions
were one Florida field population treated with Actara and two
treated with Danitol that gave lower LC95s).
We hope that these data will be useful in the future to evaluate
the possible appearance of pesticide resistance in ACP field
populations. We know that many of the pesticides used for
psyllid control are quite persistent and hope that growers
will rotate between different classes of chemistry. Should a
suspected case of resistance appear, we now have a method
of determining whether or not the resistance is real and how
severe it is. Initially, field resistance bioassays might be done
by choosing several discriminating doses, perhaps at the
LD50 and LC95, and testing adult ACP with a micro-applicator
directly after field collection.
Joseph G. Morse, Ph.D., is a professor of entomology, Beth
Grafton-Cardwell, Ph.D., is an extension specialist and
Frank Byrne, Ph.D., is an associate researcher, all in the
Department of Entomology at UC Riverside. James Bethke
is the UC Cooperative Extension Floriculture and Nursery
Farm Advisor for San Diego and Riverside counties.
CRB Project No. 5500-189
Acknowledgements
We thank the Citrus Research Board for funding this research in

part and also the chemical companies who provided technical
product for testing. We thank Jan Hare, Lisa Forster and others
in the Richard Stouthamer laboratory, who provided ACP for
testing under CRB Project No. 5500-196. We also thank Alan
Urena, Lindsay Robinson and Janine Almanzor for technical
assistance.
Table 1. Baseline susceptibility of Asian citrus psyllid adults to 12 insecticides listed by class of chemistry.
Table 1. Baseline susceptibility of Asian citrus psyllid adults to 12 insecticides listed by class of chemistry.
ACP Field
use rate
converted to
Ratio of
1,000*
LD95 to
field use
mg AI/litere
ratef
689.1
87.0
1,687.5
14.9
179.8
130.3
4.3 oz/a
30.2
126.3
7 fl oz/a
150.7
18.1
5.5 oz/a
51.5
18.7
5.75 fl oz/a
53.8
146.6
131.1
439.4
6 oz/a
56.2
43.8
4.25 fl oz/a
13.9
171.1
27 fl oz/a
165.6
41.4
4.5 fl oz/a
17.5
106.6
Typical
ACP field
use rate
Chisquare
statistic Slope SE
insect)
0.1716
6.0917 0.4096
32.2114 a 30.4832-33.9718
59.9828 a
55.2013-66.4935 1.25 lb/a
0.4607
4.9094 0.3857
34.7369 a 32.2420-37.4984
75.1332 a
66.0530-88.8990
0.1973
4.2610 0.4286
10.3481 a 9.3399-11.5586
25.1703 a
20.8504-32.9151 6 pts/a
4-24-14; 5-12-14; 611-14; 7-8-14; 7-9- 932

14; 7-23-14
0.8716
5.0234 0.2744
15.0516 b 14.2124-15.9941
31.9907 a
28.8779-36.1855
251
0.7529
2.1990 0.2313
4.1845 a
3.3012-5.5469
23.4234 a
15.1553-44.1153 16 fl oz/a
302
0.4637
4.5397 0.4456
7.6217 b
6.8886-8.4483
17.5543 a
14.8621-22.1481
2-27-13; 3-13-13
394
0.2889
1.8723 0.2000
0.5044 a
0.3688-0.6429
3.8133 a
2.8392-5.7455
8-12-14; 8-28-14
609
0.2302
2.8376 0.2071
1.0716 b
0.9764-1.1777
4.0709 a
3.3606-5.2209
8-15-12; 9-26-12;
10-10-12
565
0.1067
2.5470 0.2165
0.6181 a
0.5449-0.7086
2.7342 b
2.0953-3.9257
245
0.7033
5.6308 0.6922
0.7270 a
0.6555-0.8053
1.4244 a
1.2169-1.8093
Actara 25% WDG 1-16-13; 1-30-13

443
thiamethoxam
(Syngenta)
242
7-24-13
thiamethoxam, Round 2
Class 4C (nicotinic acetylcholine receptor agonists)
9-18-13; 10-9-13;
623
Sequoia 2 EC
sulfoxaflor
11-25-13
(Dow)
262
4-15-14
sulfoxaflor, Round 2
Class 4D (butenolide)
Sivanto 1.67 EC
8-7-14; 8-14-14; 8782
flupyradifuroneg
(Bayer)
27-14
Class 5 (spinosyns)
9-11-13; 9-24-13; 1372
Delegate 25% WG
spinetoram
8-14
(Dow)
602
4-18-14; 5-7-14
spinetoram, Round 2
0.2905
4.6172 0.4255
0.4234 a
0.3899-0.4612
0.9616 a
0.8252-1.1880
0.6549
5.9113 0.6075
0.3382 a
0.3504-0.4145
0.7248 a
0.6403-0.8624
0.2212
4.1400 0.2826
3.1617 a
2.8722-3.4624
7.8919 a
6.9625-9.2009
0.8370
5.8523 0.6248
3.4192 a
3.1254-3.7240
6.5312 a
5.7362-7.8553
0.3959
2.8398 0.1763
15.1775
13.7792-16.6333
57.6000
49.4197-69.5275 14 fl oz/a
0.9544
3.5879 0.3157
0.8561 a
0.7556-0.9799
2.4603 a
1.9863-3.2843
0.2280
3.7880 0.2726
0.9720 a
0.9025-1.0504
2.6416 a
2.2712-3.2108
0.1377
2.7986 0.2262
0.6157 a
0.5352-0.7049
2.3830 a
1.9248-3.1477
0.7538
4.1526 0.6214
1.4750 b
1.2979-1.7861
3.6720 a
2.7200-6.2713
0.1102
4.0289 0.3676
2.6759 a
2.3916-2.9824
6.8508 a
5.7954-8.5838
0.1640
5.4498 0.4475
2.5559 a
2.3747-2.7364
5.1211 a
4.6137-5.8671
0.2577
2.3040 0.2815
0.3602 a
0.2857-0.4282
1.8642 a
0.1303-2.7977
0.2684
2.8436 0.3081
0.3294 a
0.2808-0.3781
1.2478 a
0.9919-1.7434
IRAC
a
Pesticide
common
name
Pesticide
trade
name
(company)
Class
Class 1A (carbamates)
Bioassay test dates N
5-8-13; 6-5-13; 7-5570

13
(Gowan)
420
4-8-14; 5-13-14
formetanate, Round 2
Class 1B (organophosphates)
Lorsban Advanced 10-2-13; 11-13-13;
354
chlorpyrifos
3.755 EC
11-20-13
(Dow)
formetanate
Carzol 92 SP
chlorpyrifos, Round 2
Class 3A (pyrethroids)
Danitol 2.4 EC
fenpropathrin
(Valent)
fenpropathrin, Round 2
zeta-cypermethrin Mustang 1.4 EC
(FMC)
zeta-cypermethrin, Round 2
Class 4A (Neonicotinoids)
imidacloprid
Admire Pro 4.6 EC
(Bayer)
imidacloprid, Round 2
10-24-12; 1-9-13
8-7-13
7-17-13
Class 6 (avermectins, milbemycins)

Agri-Mek 0.7 SC
462
abamectin
4-3-13; 4-7-13
(Syngenta)
313
9-4-13
abamectin, Round 2
Class 21 (mitochondrial complex I electron transport inhibitors)
Bexar 1.31 EC
306
tolfenpyrad
7-2-12; 8-1-12
(Nichino)
7-31-13; 7-30-14; 8414
tolfenpyrad, Round 2
6-14
Class 28 (ryanodine receptor modulators)
Exirel 0.83 EC
336
cyantraniliprole
5-22-13; 8-14-13
(DuPont)
314
4-1-14
cyantraniliprole, Round 2
a
LD50
(ng AI/
c
LD95
(ng AI/
95% Ci
insect)
95% Ci
Insecticide Resistance Action Committee class (www.irac-online.org); pesticides in the same class have similar modes of action and thus, cross resistance within the class is likely.
N = total number of insects tested at various bioassay rates, excluding control insects.
LD50 or LD90 values for a particular pesticide followed by the same letter are not signficantly different in Round 1 vs. Round 2 tests based on overlap of 95% CI values.
95% Confidence Interval (fiducial limits).
To convert the listed Typical ACP field use rate to mg AI/liter, we assumed field use of 200 GPA spray volume.
Data in this column are the ratio of 1,000 * LD95 in ng AI applied to each insect (in the 0.8 l droplet) to the field pesticide use rate in units of mg AI per liter assuming application at 200 GPA.
Technical material received late so no Round 1 test was done with Sivanto; field use rate ratio is based on the Round 2 LC95.
Literature
Grafton-Cardwell, E.E., L.L Stelinski and P.A. Stansly. 2013.

Biology and management of Asian citrus psyllid, vector of
huanglongbing pathogens. Annual Review of Entomology 58:
413-432.
Lee, J.A., S.E. Halbert, W.O. Dawson, C.J. Robertson, J.E. Keesling
and B.H. Singer. 2015. Asymptomatic spread of huanglongbing
and implications to disease control. Proceedings of the
National Academy of Sciences, 112(24): 7605-7610. Available at:
www.pnas.org/cgi/doi/10.1073/pnas.1508253112.
Tiwari, S., R.S. Mann, M.E. Rogers and L.L. Stelinski. 2011.
Insecticide resistance in field populations of Asian citrus
psyllid in Florida. Pest Management Science 67: 1258-1268.
Tiwari, S., L.L. Stelinski and M.E. Rogers. 2012. Biochemical
basis of organophosphate and carbamate resistance in Asian
citrus psyllid. Journal of Economic Entomology 105(2): 540-548.
Tiwari, S., N. Killiny and L.L. Stelinski. 2013. Dynamic insecticide
susceptibility changes in Florida populations of Diaphorina
citri (Hemiptera: Psyllidae). Journal of Economic Entomology
106(1): 393-399.
39
An experiment with 18 RITA bioreactors to propagate mature Valencia and Carrizo plants.
MATURE CITRUS
PROPAGATION IN RITA
BIOREACTORS
Yosvanis Acanda and Janice Zale
SUMMARY
This project seeks to find a way to propagate mature citrus rapidly, as an alternative
to budding or tissue culture on solid media, while maintaining maturity traits for early
flowering and fruit production. We are utilizing RITA (Rcipient Immersion Temporaire
Automatique) bioreactors to propagate mature citrus, and altering the composition of
the nutrient media and plant growth regulators to generate shoots and roots. Afterwards
the propagated plants will be analyzed at the molecular level and with biotechnological
instrumentation to ensure the maintenance of maturity and genetic integrity.
40
Sometimes age is an advantage. In citrus, juvenility is marked

by thorns and barrenness, while maturity brings fragrant
flowers and delicious fruit. The Mature Citrus Facility at the
University of Florida produces disease-tolerant, mature scion
and rootstock as a service to American scientists, growers and
industry partners.
Due to the current crises in the citrus industries across the
country because of huanglongbing (HLB), it is necessary to
develop tools that will speed the propagation of diseasetolerant, mature citrus that maintain maturity traits.
Propagation using certain types of tissue culture techniques
might induce juvenility, which will delay flowering and
fruit production. Furthermore, propagation on solid tissue
culture media or through traditional budding is relatively
unproductive, time-consuming and labor intensive. The
goals of this project are to develop protocols for the mass
propagation of mature Valencia scion and Carrizo rootstock
by culturing cuttings in liquid media in temporary immersion
bioreactors for shoot proliferation and multiplication. Some
plant tissue culture systems induce undesirable genetic
mutations, so the plantlets must be examined with molecular
techniques to determine whether the genetic fidelity and
maturity traits of the plants are maintained.
Figure 1. A temporary immersion RITA bioreactor for plant propagation.
Bioreactors have been used as a cost-effective alternative for

the mass propagation of plants (e.g. bananas, cedar, coffee
and pineapple). The RITA bioreactor is a very simple system
widely used for commercial propagation of plants (Figure 1).
Figure 2. The initial steps of mature citrus propagation in bioreactors. A) Budstick was surface-sterilized, cut into pieces and plated onto solid media in Petri
dishes. B) Valencia shoots initiated from stem cuttings.
41
Figure 3. Mature citrus shoot growth after excision from stem cuttings. A) Failure of shoot growth in solid medium after excision from the stem cuttings.
Activated charcoal is added to the medium to absorb inhibitory compounds. B) Excised shoots thrive on a carrot nurse tissue culture.
The bioreactor is similar to a hydroponics system in that both use liquid media. However, unlike hydroponics, the bioreactor is
sterile; the plant material has been surface-sterilized; and entire plantlets, embryos or plant tissues are temporarily immersed
in the liquid culture medium for short time periods every few hours. RITA has been shown to reduce the time for propagation,
reduce expenses and improve the quality of the propagules. It was used to propagate cedar, and yields were increased four- to
six-fold in half the time compared to plant propagation in tissue culture on solid media. In addition, plants produced with the
RITA system were hardier and better acclimated.
To be commercially viable, mass propagation of citrus should ensure the genetic fidelity of the mature donor plant. We are
testing and optimizing certain parameters, such as types and sizes of citrus cuttings, the nutrient and growth regulator
composition of the liquid media and the duration and frequency of plant immersion in liquid media.
Figure 4. Bioreactor-grown Carrizo shoots. A) Carrizo shoots in the bioreactor. B) Primary Carrizo shoots with developing axillary shoots.
Our protocol consists of surface sterilization of mature stems (or budsticks), aseptic cutting of the stems into pieces and plating
them on solid nutrient media composed of different plant growth regulators conducive to shoot production (Figures 2A and
2B). It is relatively easy to generate shoots of mature citrus directly from the stem cuttings, but it is difficult to grow after excision
from the stem cutting. Mature shoots appear to produce a high level of ethylene, a gaseous plant growth regulator involved in
plant senescence and fruit ripening, which might cause shoot death. Activated charcoal is used in tissue culture in an attempt
to absorb inhibitory compounds (Figure 3A). Therefore, mature citrus tissue culture is not conducive to plant production in
vitro, because the excised shoots fail to grow and root in culture. In our standard protocol, shoots must be micro-grafted onto
citrus rootstock to survive, which is not always successful. To understand why the shoots die when excised from stem cuttings,
we micro-grafted them onto carrot nurse tissue where they appear more green and vigorous (Figure 3B). Therefore, it appears
that the citrus shoots feed directly from the primary stem cutting and not from the culture medium, or perhaps some growth
regulators are produced in the primary stem cuttings necessary for shoot survival. This research will improve the composition
of the basal culture medium for mature citrus propagation.
Our preliminary results in bioreactors suggest that shoot size and quality are increased compared to growth on solid medium
(Figure 4A and 4B). Currently, we are testing different basal media and growth regulators to optimize shoot growth and
multiplication from axillary buds. The data collected from these experiments will be the percentage of cuttings with shoots, the
42
43
number of shoots per cutting, the size of

the shoots and the weight of the total
biomass in each bioreactor.
We also have tested different plant
growth regulators to promote rooting
with promising preliminary results in
Carrizo and Valencia (Figure 5). In the
past, rooting scion has been problematic.
If the majority of regenerated shoots
prove recalcitrant to rooting, scions will
be micro-grafted onto rootstock similar
to nursery practices.
In summary, our results thus far suggest
that the RITA bioreactors are promising
systems for mature citrus propagation.
It seems that the continual changes to
the atmosphere in the bioreactor might
decrease ethylene gas and its adverse
effects on shoot growth, enhancing
growth and development. However, it
remains to be determined whether the
shoots can survive in the bioreactors
after excision from stem cuttings. After
optimizations of the plant propagation
in bioreactors, the genetic stability
and maturity of the propagules will be
determined using biotechnological
instrumentation
and
techniques,
respectively.
Figure 5. Rooting mature scion and rootstock. Rooted Valencia (left) and Carrizo (right) after the
application of growth regulators in the medium.
Yosvanis Acanda of Vigo, Spain, is a Ph.D. candidate and a senior biologist at the University of Florida, Institute of Food and
Agricultural Sciences (IFAS), Citrus Research and Education Center (CREC). Janice Zale Ph.D. is the Mature Citrus Facility
Coordinator at the University of Florida, IFAS, CREC.
Farm Sales Specialists for Californias Central Valley
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For Brochure Contact:
44
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47.80 acs Avocado Ranch ................................................... $625,000
59.66 acs Woodlake Olives & Navels..................................$1,275,000
69.64 acs Lewis Creek Olives (In Escrow) ...........................$1,250,000
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Roy Pennebaker #0845764 (559)737-0084 or Matt McEwen #01246750 (559)280-0015
45
Figure 1. This project is centered on coupling genetics to proteomics to reveal how the HLB biological players interact at the molecular level during Candidatus
Liberibacter asiaticus (CLas) transmission by the Asian citrus psyllid (ACP), Diaphorina citri. The ACP is the CLas vector, and it harbors other bacteria, called
symbionts, that are beneficial to it. The two primary ACP symbionts include Candidatus Carsonella rudii DC, a putative nutritional symbiont, and Candidatus
Profftella armature, a putative defensive symbiont. Profftella is an ACP-specific symbiont and not found in any other insect species studied to date. This
feature makes the interaction between the ACP and Profftella a prime target for ACP control, because the risks for harm to beneficial insects would be minimal
or none.
Our preliminary data show that Profftella cells inside the ACP respond to CLas acquisition by the insect vector. CLas infects citrus trees, and the ACP vector
carries the bacterium from tree to tree. CLas harbors a virus called phage, and the roles of the phage in transmission are unknown. The host plants also have an
effect on transmission in ways that are just beginning to be understood.
NOT ALL PSYLLIDS ARE

CREATED EQUAL
Combining genetics and proteomics to understand

the basis of ACP vector competency
Michelle Cilia, John Ramsey, Angela Kruse, Richard Johnson,
Michael MacCoss, Robert Shatters and David Hall
46
PROJECT SUMMARY
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economically important pest of citrus and
a vector of Candidatus Liberibacter asiaticus (CLas).1 CLas is a phloem-limited, gram-negative, fastidious
bacterium that is implicated in causing the most serious disease of citrus, huanglongbing (also known as HLB
or citrus greening disease). ACP and HLB have spread to most citrus growing regions worldwide. The disease
threatens the future of Floridas annual $9 billion industry1.
The need for novel and effective HLB management strategies is urgent, as the HLB-associated Liberibacter and
vector are spreading beyond Florida. The urgent need for control strategies was affirmed in July 2015 with the
discovery of 10 infected trees in San Gabriel, California, marking the first confirmed report of HLB in the state
since a single infected tree was discovered in Hacienda Heights in 2012. HLB management options currently
are limited to ineffective strategies to control ACP vectors and early disease detection, and the disease is a
death sentence for an infected citrus tree. Control of CLas transmission by the ACP represents a promising new
control strategy.
Our new project is focused on comparing populations of the ACP that vary in their ability to transmit CLas. This
will enable us to understand the molecular interactions that occur during CLas transmission. These interactions
are challenging to study because there are many organisms interacting with one another (Figure 1).
In this article, we briefly describe each of the biological players and our overall experimental approach. Finally,
we explain how our results may be used to help the growers in California and elsewhere to better manage the
disease. Our team is well poised to achieve our objectives and to translate knowledge into novel management
strategies for the citrus industry. Our preliminary data already have led to the identification of potential targets
for ACP control using para-transgenic and ribonucleic acid (RNA) interference technologies being developed in
other laboratories.
THE BIOLOGICAL PLAYERS
ACP. CLas interactions with ACP tissues determine whether or

not the bacterium will be transmitted to a new tree. Insect
tissues that interact with CLas include the gut, the hemolymph
(insect blood) and the salivary tissues, among others. Studies
have shown that CLas acquisition and transmission are
developmentally regulated. CLas is acquired at higher rates
by ACP nymphs than by adults.2 Transmission barriers to CLas
in adults are thus stronger than in nymphs. Among infected
adults, CLas is less commonly associated with salivary glands
than with the alimentary canal, suggesting that CLas more
easily penetrates the alimentary canal wall than the salivary
gland wall.3 Among ACP with CLas-infected salivary glands,
the titer of CLas is higher in the glands than anywhere
else in the body, suggesting that the pathogen is able to
replicate in the salivary glands once it overcomes barriers
to entry into these organs.3 In addition to a salivary gland
infection/entrance barrier to CLas, there appears to be a
salivary gland escape/exit barrier,3 where the bacterium
can escape the insect tissues to infect a new host plant as a
component of saliva injected into the phloem.
Vector competence also may be related to the ability of CLas

to survive in the insect hemolymph, either by evading insect
immune defenses or by manipulating the ACP immune system
to be more permissive for CLas. Genome sequencing results
have shown that the ACP lacks a complete insect immune
system. The ACP symbionts, described below, may aid in the
ACP immune response to CLas.
Bacterial Symbionts. ACP harbors beneficial bacteria,
called symbionts, in a specialized, interspecies organ called
the bacteriome. The bacteriome depicted in Figure 1 is an
interpretative, not-to-scale schematic of interactions among
ACP, CLas, bacteriophage and endosymbionts based on a
figure published in a paper from Nakabachi and colleagues4.
The two primary ACP symbionts are Candidatus Carsonella
rudii DC, a putative nutritional symbiont, and Candidatus
Profftella armature, a putative defensive symbiont. Profftella is
an ACP-specific symbiont. They have been found in every ACP
population studied to date. Carsonella cells are located within
the cells on the border of the bacteriocytes. Profftella cells are
located in the center. A secondary symbiont, called Wolbachia,
is also found in the ACP this symbiont has been found in a
range of insect tissues in addition to the bacteriome.
47
Collectively, the bacterial symbionts of the ACP are referred

to as the insect microbiota. The role of the microbiota in ACPCLas interactions is not known. The universal presence of the
symbionts in all ACP populations studied thus far, the long
history of co-evolution with the ACP and the existence of a
specialized organ (the bacteriome, where the symbionts are
found) all suggest that these microorganisms play an essential
role in psyllid biology. The microbiota may exert some effects
on the ability of the ACP to transmit Liberibacter; high titers of
Wolbachia symbionts in ACP populations are associated with
increased transmission competence of CLas.5
The pea aphid, Acyrthosiphon pisum, was the first hemipteran
genome to be sequenced. Annotation of predicted genes
revealed a striking reduction in immune-related genes6.
Analysis of additional hemipteran genomes, including that
of a related psyllid, revealed a similar pattern of a predicted
reduction in immune-related genes compared to insects
of other Orders.7,8 The need to maintain high numbers
of symbiotic bacteria in their bodies may have led to the
reduction in immune function in hemipterans, and evolution
of immune-permissive environments in hemipterans may
have occurred due to the increased fitness of insects capable
of harboring a range of beneficial bacterial partners. It is
possible that plant disease-associated bacteria, such as CLas
that are acquired by hemipterans, are capable of exploiting
an immune-suppressed
insect 1to increase
their
own chances
AWPCitrographFINAL.pdf
6/17/14
1:14 PM
of transmission during feeding. Many hemipterans are among
the insect worlds most prolific vectors of plant pathogens.
Bacteriophage. At least some isolates of CLas harbor bacteriainfecting viruses called bacteriophages. Although they are
presumed to remain inactive in the ACP, bacteriophage may
increase the virulence of CLas or may even be used as a suicide
switch to kill CLas.
Host plants of the ACP and CLas. The host plants are also
other players. Some plants in the Rutaceae are hosts of both
CLas and the ACP, while others are hosts for the ACP only.
In HLB-susceptible citrus varieties, the ACP can efficiently
acquire CLas. In infected Murraya spp., a citrus relative, CLas
titers are much lower than in citrus; and the ACP acquires four
orders of magnitude less CLas from infected Murraya than
from CLas-infected citrus.9 Understanding how each of the
players interacts with the others is a critical part of learning
how to efficiently manage the disease and to prevent CLas
transmission. The molecular approach our team is utilizing
will enable us to understand novel ways to prevent CLas from
using the ACP to go commit its next crime.
EXPERIMENTAL APPROACH
Genetics. A genetic component regulating acquisition and

transmission of CLas by ACP may exist. There is already some
evidence that individual ACPs vary with respect to vector
efficiency. Normally, relatively low percentages (1.3 to 12.2
percent) of adult ACP actually transmit CLas, although a much
higher proportion of ACP can be infected with CLas, based
on PCR assays.10, 11 Among psyllids infected with CLas, many
may not be capable of transmitting the HLB-associated CLas,
because the bacterium fails to infect the salivary glands.3
This may be attributed to barriers that block the bacterium
from passing through the wall of the digestive tract into the
hemocoel and/or from the hemocoel into the salivary glands.
Such barriers may be controlled genetically.
Thus, it should be possible to select for a population of
psyllids that is either extremely effective or ineffective in
acquiring and/or transmitting CLas. Differences could exist
in vector efficiency among individuals infesting a given
host plant species within a given geographic area or among
populations infesting different host plant species within a
given geographic area. Citrus is a major host plant of ACP in
Florida, but there are other host plant species grown in Florida
that can be heavily infested by ACP. One prominently-grown
alternate host plant in the urban landscape is orange jasmine
(Murraya exotica); pertinent is whether ACP adults associated
with orange jasmine have the same vector efficiency as those
associated with citrus. Interestingly, CLas is rarely found in
association with either orange jasmine or ACP developing on
jasmine. 9,12
CM
MY
CY
CMY
There also could be genetic variability in vector efficiency

among geographically isolated ACP populations.
For
example, in extreme south Florida where citrus is not grown
commercially, ACP populations associated with orange
jasmine might be less efficient at acquiring and transmitting
CLas than ACP associated with citrus in central Florida.
48
Figure 2. Excised leaf assay for CLas transmission studies. (A) Polypropylene tube for caging psyllids with excised citrus leaf (el). The petiole is inserted into a
microcentrifuge tube (mt) full of water, with Parafilm wrapped around the top of this tube. The caging tube is covered with a piece of fine-mesh screen cloth
under the screw cap, with the flip-top part of the cap removed for ventilation. (B) Caging tubes in a tube-rack (from Ammar et al. 2013).
Discovering a natural population of psyllids incapable

of acquiring or transmitting the pathogen could lead to
new strategies aimed at managing HLB. To the best of our
knowledge, the CLas acquisition and transmission efficiencies
of ACP from California also are unknown.
Our team has started to work on examining these differences
in ACP populations from Florida. Colonies of potentially good
or poor CLas vectors have been established using individual
adult female ACP paired with single males. The ACP pairs were
collected in the wild, introduced onto a potted orange jasmine
plant in a cage maintained in a greenhouse, and allowed to
reproduce. Five such CLas-free iso-female lines have already
been established from individual ACP collected from orange
jasmine in St. Lucie County, Florida. We are using detached
leaf transmission assays (Figure 2), a technique developed
by members of the David Hall lab, for screening various
CLas transmission and acquisition parameters. We expect
to find variability among ACP lines in their ability to acquire
and transmit CLas, perhaps variation that exists in CLas-ACPsymbiont interactions. If an ACP line is found that is superior
at transmitting CLas, it could be used to improve transmission
studies aimed at challenging germplasm against HLB.
Proteomics. Mass spectrometry is the discovery tool that our
team brings to bear on this problem. We use mass spectrometry
to measure proteins, which are peptide molecules that are
critical to all life on Earth, including all the biological players

described above involved in the HLB pathosystem. Prior
to mass spectrometry analysis, we break proteins down
into smaller fragments, called peptides. Then, we use mass
spectrometry to break those peptides down even further into
smaller fragments that are unique signatures. From these
signatures, we learn many things about proteins, including
their identities, modifications, abundance, and even how they
come together to form interactions with one another.
Proteomics is the large-scale study of proteins using mass
spectrometry. Our team is using an approach called discovery
proteomics to peer deep inside the ACP and to characterize
exactly what proteins are different in infected and healthy
insects, and among the ACP colonies that we are developing
with variable transmission efficiencies. Discovery proteomics
enables researchers to ask, What proteins are in my
sample? Although this discovery technique is by no means
comprehensive, it is a powerful approach that will enable us
to discover ACP, endosymbiont and CLas proteins that are
expressed at different levels upon CLas acquisition and among
the different iso-female lines. Proteins that are expressed at
different levels in these different treatment groups inform us
of the molecular interactions that are important during the
acquisition and transmission of CLas by the ACP.
49
RESULTS AND APPLICATIONS
At the International Research Conference on HLB in Orlando,

Florida, in February 2015, our team presented proteomics
data comparing healthy ACP with those harboring CLas. Our
data show that once the ACP acquires CLas, interactions with
the symbionts, in particular Profftella, are drastically changed.
The data also indicate that CLas may be a pathogen of the ACP
as well, and that Profftella may be helping to defend the ACP
against CLas infection.
We are further exploring these results by measuring small RNA
molecules produced by the ACP. These small RNA molecules
will give us additional clues into CLas pathogenicity in the
ACP. This idea, which is supported by our data, represents a
totally novel means for insect control: if we understood the
molecular interactions occurring among the ACP, CLas and
Profftella, we could specifically increase CLas virulence in the
ACP and not the tree. In other words, we hypothesize that
we can infect the insect with CLas to the point where it kills
the ACP. The success of this approach is based on our ability
to understand how the ACP defends itself against CLas and
to develop a method to block that defense mechanism.
Ultimately, our research could lead to a solution to the HLB
problem. More details on this research can be found in a
recently published PLOS ONE article: http://journals.plos.org/
plosone/article?id=10.1371/journal.pone.0140826
Acknowledgements
The authors thank Jackie Mahoney (Cilia Lab, Boyce Thompson

Institute) for technical assistance and are grateful to the Citrus
Research Board for funding.
About the authors
Michelle Cilia, Ph.D., Robert Shatters, Ph.D., and David

Hall, Ph.D., are scientists in the USDA Agricultural Research
Service. John Ramsey, Ph.D., is a postdoctoral associate
in the Cilia lab. Angela Kruse is a Ph.D. plant pathology
student at Cornell University conducting her thesis research
in the Cilia lab. Michael MacCoss, Ph.D., is a professor in
the Department of Genome Sciences at the University of
Washington. Richard Johnson, Ph.D., is a research chemist
working in the MacCoss lab.
References
1. Hall, D.G., M.L. Richardson, E-D. Ammar, and S.E. Halbert.

2012. Asian citrus psyllid, Diaphorina citri, vector of citrus
huanglongbing disease. Entomol. Exp. Appl., 146:207-223.
2. Inoue, H., J. Ohnishi, T. Ito, K. Tomimura, S. Miyata, T. Iwanami,
and W. Ashihara. 2009. Enhanced proliferation and efficient
transmission of Candidatus Liberibacter asiaticus by adult
Diaphorina citri after acquisition feeding in the nymphal stage.
Annal. Appl. Biol. 155(1):29-36.
50
3. Ammar,E-D., R.G. Shatters Jr., and D.G. Hall. 2011. Localization

of Candidatus Liberibacter asiaticus, associated with citrus
huanglongbing disease, in its psyllid vector using fluorescence
in situ hybridization. J. Phytopathol. 159: 726-734.
4. Nakabachi, A., R. Ueoka, K. Oshima, R. Teta, A. Mangoni, M.
Gurgui, N.J. Oldham, G. van Echten-Deckert, K. Okamura, K.
Yamamoto, H. Inoue, M. Ohkuma, Y. Hongoh, S.Y. Miyagishima,
M. Hattori, J. Piel, and T. Fukatsu. 2013. Defensive bacteriome
symbiont with a drastically reduced genome. Curr. Biol.
23(15):1478-84.
5. Fagen, J.R., A. Giongo, C.T. Brown, A.G. Davis-Richardson, K.A.
Gano, and E.W. Triplett. 2012. Characterization of the relative
abundance of the citrus pathogen Ca. Liberibacter asiaticus
in the microbiome of its insect vector, Diaphorina citri, using
high throughput 16S rRNA sequencing. Open Microbiol. J.
6:29-33.
6. Gerardo, N.M., B. Altincicek, C. Anselme, H. Atamian, S.M.
Barribeau, M. de Vos, E.J. Duncan, J.D. Evans, T. Gabaldon,
M. Ghanim, A. Heddi, I. Kaloshian, A. Latorre, A. Moya,
A. Nakabachi, B.J. Parker, V. Perez-Brocal, M. Pignatelli, Y.
Rahbe, J.S. Ramsey, C.J. Spragg, J. Tamames, D. Tamarit, C.
Tamborindeguy, C. Vincent-Monegat, and A. Vilcinskas. 2010.
Immunity and other defenses in pea aphids, Acyrthosiphon
pisum. Genome Biol. 11(2): R21.
7. Zhang, C.R., S. Zhang, J. Xia, F.F. Li, W.Q. Xia, S.S. Liu, and X.W.
Wang. 2014. The immune strategy and stress response of the
Mediterranean species of the Bemisia tabaci complex to an
orally delivered bacterial pathogen. PLOS One, 9(4): e94477.
8. Nachappa, P., J. Levy, and C. Tamborindeguy. 2012.
Transcriptome analyses of Bactericera cockerelli adults in
response to Candidatus Liberibacter solanacearum infection.
Mol. Genet. Genomics. 287(10):803-817.
9. Walter, A.J., Y. Duan, and D.G. Hall. 2012. Titers of Candidatus
Liberibacter asiaticus in Murraya paniculata and Murrayareared Diaphorina citri are much lower than in Citrus and
Citrus-reared psyllids. HortScience 47:1-4.
10. Ammar, E-D., R.G. Shatters, Jr., and D.G. Hall. 2011. Detection
and relative titer of Candidatus Liberibacter asiaticus in
the salivary glands and alimentary canal of Diaphorina citri
(Hemiptera: Psyllidae) vector of citrus huanglongbing disease.
Annal. Entomol. Soc. Amer. 104:526-533.
11. Pelz-Stelinski, K.S., R.H. Brlansky, T.A. Ebert, and M.E. Rogers.
2010. Transmission parameters for Candidatus Liberibacter
asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J. Econ.
Entomol. 103:1531-1541.
12. Walter, A.J., D.G. Hall, and Y. Duan, 2012. Low Incidence of
Candidatus Liberibacter asiaticus in Diaphorina citri and its
host plant Murraya paniculata. Plant Dis. 96:827-832.
51
COMPLEX CITRUS
LURES TO TRAP AND
CONTROL ACP
Unbaited yellow traps are widely used in California.
Xavier Martini, Alexander A. Aksenov, Cristina E. Davis and Lukasz L. Stelinski
SUMMARY
Monitoring of Asian citrus psyllid (ACP), Diaphorina citri, remains critical in California, given
that there is still an effort to quarantine ACP or at least keep it at minimal population
densities. Currently, unbaited yellow sticky traps are the main method for monitoring
psyllid populations, and they are used widely in California. However, the tactic is not
satisfactory without long-range attractants. Although some volatile attractants for ACP
have been developed that do improve effectiveness of monitoring traps, progress has been
slow in implementing such technologies practically. We have worked toward developing
a more potent synthetic lure to improve monitoring and the possibility for effective new
management tactics, such as attract-and-kill devices or disruption of host finding.

52
Figure 1. Flight mill apparatus used to investigate ACP movement.
The use of broad-spectrum insecticides for ACP management

in the U.S. citrus industry has increased dramatically since the
2005 discovery of huanglongbing (HLB) in 2005 in Florida.
Identification of plant-based attractants and development of
effective monitoring and attract-and-kill or disruption devices
may improve ACP management, while concurrently reducing
the need for broad-spectrum insecticide sprays.
The goal of this project has been to develop new and highly
effective lures for monitoring ACP. We are focusing on recent
findings showing that ACP is more attracted to plants that are
infected with Candidatus Liberibacter asiaticus (CLas), the
phloem-dwelling bacterium associated with citrus greening
or HLB, than uninfected plants (Mann et al. 2012). Biologically,
such infected plants act as beacons that attract the ACP
vector. However, since infected plants are nutritionally suboptimal, ACP leaves after initial acquisition of CLas and
spreads it to uninfected trees (Mann et al. 2012). We even
have found that psyllids that acquire CLas tend to move more
than uninfected ones (Martini et al., 2015). We hypothesized
that this complex vector-CLas-host phenomenon has evolved
in this manner so as to maximize the spread of CLas by the
vector. This research was accomplished, in part, by employing
an apparatus called a flight mill (Figure 1) to measure the
flight duration of ACP.
CLas appears to manipulate the host plant defense responses,
which ultimately change vector behavior. Since CLas relies
solely on the ACP vector for its propagation and survival
in nature, these mechanisms may have evolved to spread
efficiently within populations of ACP. Our goal has been to
exploit this system in order to develop a highly effective lure.
Such a lure would be an important tool for an early detection
of ACP; for quarantine programs to predict the need for
spraying insecticides; and also for timing insecticide sprays

effectively instead of relying solely on calendar-based sprays.
During this research, we evaluated previously identified
complex volatile blend(s), as well as formulated and evaluated
novel blends of host-plant attractants for monitoring this
insect. ACP exhibits a strong preference for citrus volatiles and
uses them to find and infest citrus trees. ACP aggregates and
lays eggs exclusively on young unexpanded leaves or flush.
Thus, plant-related chemicals are used, in part, by adults for
plant selection and acceptance for egg laying. Moreover,
infection of citrus plants with CLas renders those trees more
attractive to ACP as compared with non-infected counterparts.
The specific volatiles mediating these interactions have been
identified in our previous reports (Aksenov et al. 2014).
Our research has been based on the documented
phenomenon that CLas infections alter volatile profiles of
plants, rendering infected plants more attractive to the ACP
vector than uninfected plants and thus aiding propagation
of CLas. Mimicking these chemical cues might enable insect
attraction away from the plant or disruption of the host finding
behavior of the vector. However, the practical implications
have not yet been fully explored. Application of behaviormodifying chemicals as a tool for ACP management is still in
its infancy, despite years of previous research.
Previously, we found that it is possible to develop an attractant
comprising a number of compounds in defined abundance
ratios by utilizing information about volatile alterations
to mimic volatile output of citrus plants affected by HLB
(Aksenov et al. 2014). The laboratory-based behavioral testing
of the developed synthetic lure has revealed that it is more
attractive to ACP than odors emanating from uninfected citrus
53
Figure 2. Quantifying psyllid behavior in the laboratory.
trees (Figure 2). Lure development using this strategy could

provide several new tools for ACP management, including
more effective lures for trapping, attract-and-kill devices and
disruption of ACP host plant finding.
Our specific goal has been to develop more potent lure
formulations (e.g., season-specific or independent blends)
while reducing the number of chemical components. We are
working to optimize the ratio and number of the most attractive
components, while considering the economics of practical
applications. For this, we have been conducting subtraction
assays to find the most salient blend of chemicals that elicit
attraction of ACP, so as to create a cost-effective and practical
tool for ACP monitoring. Field deployment and evaluation of
possible practical tools are currently underway.
During the previous year, we developed an attractive lure
for ACP that appears to be economically practical for field
deployment. However, the majority of our research has been
conducted under laboratory conditions. More field testing is
necessary to determine whether the lure is effective when
deployed within citrus groves. This research is currently
underway. We also have begun collaborating with the
industry to develop a field-ready dispenser. Field verification
is the final necessary step before this technology can be
developed and eventually marketed by the commercial
pest management industry. We are interested in further
improving the attractiveness of this lure. This research has
been a collaborative effort between chemists, engineers and
entomologists.
54
References
Aksenov, A.A., X. Martini, W. Zhao, L.L. Stelinski and C.E. Davis.

2014. Synthetic blends of volatile, phytopathogen-induced
odorants can be used to manipulate vector behavior. Frontiers
in Ecology and Evolution. 2: 78. doi: 10.3389/fevo.2014.00078.
Mann, R.S., J.G. Ali, S.L. Hermann, S. Tiwari, K.S. Pelz-Stelinski,
H.T. Alborn and L.L. Stelinski. 2012. Induced release of a plant
defense volatile deceptively attracts insect vectors to plants
infected with a bacterial pathogen. PLoS Pathogens. 8(3):
e1002610.
Martini, X., M. Hoffmann, M.R. Coy, L.L. Stelinski and K.S. PelzStelinski. 2015. Infection of an insect vector with a bacterial
plant pathogen increases its propensity for dispersal. PloS
ONE. 10(6): e0129373. doi:10.1371/journal.pone.0129373.
Xavier Martini, Ph.D., is a postdoctoral associate in the
Entomology and Nematology Department at the University
of Florida; Alexander A. Aksenov, Ph.D., is an assistant
specialist in the Department of Mechanical and Aerospace
Engineering at the University of California-Davis; Cristina E.
Davis, Ph.D., is a professor in the Department of Mechanical
and Aerospace Engineering at the University of CaliforniaDavis; and Lukasz L. Stelinski, Ph.D., is an associate
professor in the Entomology and Nematology Department
at the University of Florida.
55
FOUNDER LINES FOR

IMPROVED CITRUS
BIOTECHNOLOGY
Maria Oliveira, Ed Stover and James Thomson
n October 1, 2011, the Citrus Research Board chose to

fund a unique research project the development of
citrus cultivars specifically for genetic modification (GM).
The objective of this research was to develop genetically
engineered (GE) citrus Founder Lines containing a gene
sequence that will allow the precise insertion of desired traits
using Recombinase-Mediated Cassette Exchange (RMCE)
technology. The RMCE technology uses specific enzymes to
move DNA or transgenes in a predictable manner. Production
of Founder Lines ensures that the inserted transgenes are in a
region of the citrus genome that provides high and consistent
56
transgene activity with a single gene copy and does not

interrupt desirable genes. This research was previously
discussed in the January/February 2013 Citrograph article
entitled Founder Lines for improved citrus biotechnology.
Since the initial inception of this project, a number of goals
have been accomplished. First and foremost is that 335
transgenic plants from Carrizo and 15 transgenic sweet orange
plants have been obtained. Of these, 123 transgenic lines
from Carrizo and all 15 transgenic lines in sweet orange were
confirmed to contain a single copy of the Founder transgene.
Figure 1. Schematic diagram of the landing pad within the Founder Line genome. The attP recognition site is a unique DNA sequence that the recombinase
enzyme Bxb1 uses for DNA integration. The Res recognition site is a unique DNA sequence that the recombinase enzyme CinH uses for DNA excision. Acting
together, these two enzymes can swap out the original DNA between these recognition sites and replace it with new beneficial DNA. The DSRed visual marker
allows one to see the transgenics, while the codA and nptII genes allow for chemical selection of tissue containing the newly inserted DNA.
Single copy lines provide a unique site in the genome for

targeting additional novel transgenes. (A single copy insert
indicates that a single transgenic construct was inserted into the
citrus.)
The Founder Lines contain the recognition sites (unique
DNA sequences) for specific enzymes capable of cutting and
pasting DNA without the gain or loss of genetic material. In
Figure 1, these sites are designated attP and Res for their
respective enzyme partners, Bxb1 and CinH. In between these
sites are marker genes that allow one to select for the presence
of the Founder Line DNA in the genome using the antibiotic,
kanamycin (nptII). Also included is the visual marker DSRed,
which allows one to see the DNA once it is incorporated into
the genome.
Seen in Figure 2 are Founder Lines initially isolated from tissue
culture where the transgenic DNA landed in the genome that
will determine how effectively it expresses the color red. This
system was designed so that the initial selectable markers
would be removed from the genome once targeting was
achieved using the recombinase technology. It also would
allow stacking of more than one gene into the genome.
Multiple genes can be inserted all at once or individually over
time, proving flexibility and optimization.
Why would so many genes be needed? Many metabolic
pathways that produce flavor and/or pigments require
multiple enzymes to produce the desired trait. Additionally,
this makes it possible to take an existing transgenic for
example, Tango, with a gene for citrus greening resistance
and add an additional desirable trait, knowing it will be highly
expressed.
Also seen is the codA selection gene. This is an odd gene that
allows one to select for its absence. In other words, removal
of this gene from the genome can be detected to verify DNA
exchange; this is a crucial element to the RMCE targeting
Figure 2. Initial transgenic citrus Founder Lines screened for expression of

the visual selectable marker DSRed. DsRed or Discosoma sp. red fluorescent
protein has an excitation and emission spectra of 554 and 586 nanometers,
respectively. The color intensity from the KCN3 vector is used to determine
if the transgene (KCN3) landed in a genomic location of high transcriptional
activity. The high activity correlates with strong RED expression. High
transcriptional activity also corresponds to a genomic region that is OPEN
and available for the recombinase to perform their integration and/or
excision reactions (RMCE).
strategy. The RMCE strategy swaps DNA from between the

attP and Res sites using the recombinases Bxb1 and CinH.
The swap will remove the DSRed, nptII and codA genes from
the genome and replace it with DNA of interest. Therefore,
by selecting for the loss of codA, one effectively screens for
the removal of all unneeded selectable markers (i.e., antibiotic
resistance) that are replaced by specific DNA beneficial to
the cultivar. The codA gene works simply by making a nontoxic compound into a toxic one. So when an RMCE targeting
57
Creation of these initial Founder Lines

is just the midpoint of the project.
Currently, only Carrizo and sweet orange
(Valencia and Hamlin) Founder Lines are
in the greenhouse and available for RMCE
testing. Founder Lines are underway
for more commercially relevant scion
cultivars. We have small plants of
Mexican Lime, Cocktail grapefruit,
Tango, Limoneira 8A, Valencia, Sidi Aissa
Clementine and W. Murcott.
Another important aspect of this project
is transformation, which is the ability to
get DNA into the plant cell. For many
commercially
important
cultivars,
we cannot use standard seedling
transformation, since the seeds are likely
to be hybrids of unknown quality. This
requires us to develop a transformation
technique from fully mature citrus tissue
(see below), which also should reduce
the time from transformation to fruit
production. In addition, once a Founder
Line is created, it needs to be capable of
transformation again and again to stack
Figure 3. CodA selection assay. Visualized are a wild type Carrizo line (no codA) in the presence and
absence of the non-toxic compound 5-Fluorocytosine. In the absence of the codA gene, there is little effect additional genes, even as it ages. There
on plant growth as seen for the wild type plant. However, Founder Line KCN3.2, which contains the codA are multiple hurdles to this process
gene, shows dramatic growth retardation. 5-Fluorocytosine is converted to 5-Florouracil in the presence of
such as tissue physiological state, the
codA and acts to inhibit DNA transcription, which in turn leads to cell death.
type of cultivar and its health. Aspects
being investigated include hormones,
event is attempted, plants are screened in the presence of this salts, media matrix, timing, light and temperature. To date,
non-toxic compound. If they survive, the RMCE process was our lab has had success in transforming mature plant tissue
successful and the DNA swap completed as desired. Results from multiple cultivars including Carrizo, Navel, Tango and
for the codA selection strategy are seen above in Figure 3.
Limoneira 8A (Figure 4).
Figure 4. Transgenic citrus lines being produced from mature tissue from Limoneira 8A and Tango cultivars. Green shoots demonstrate tissue competent for
transformation and transgenic production. When the shoots have grown bigger, they will be grafted to rootstock and transferred to the greenhouse for
further molecular evaluation.
58
In summary, the objective of this proposed research is to

implement use of GE citrus Founder Lines containing a
platform to allow the precise insertion of desired traits, via
site-specific recombination (RMCE). In addition to allowing
the targeted integration of transgenes, the system also
enables the removal of unneeded sequences such as
antibiotic resistance marker genes, allowing the generation of
clean (marker-free) GE citrus plants. The system also allows
recycling of a selectable marker, which, in turn, permits
stacking of additional traits into Founder Lines with existing
transgenics. Existing Founder Lines are currently being
evaluated for efficiency of targeted integration and marker
removal, and mature citrus tissue transformation is being fully
developed for a wide range of cultivars to make the Founder
Lines even more useful. Finally, the materials produced from
the research will enable improved citrus biotechnology, which
will benefit U.S. producers in the agriculture marketplace and
will be freely available to other researchers.
Maria Luiza Peixoto de Oliveira Ph.D., and Ed Stover,

Ph.D., are with the USDA-Agricultural Research Service
(ARS) Subtropical Insects and Horticulture Research Unit
in Fort Pierce, Florida. James Thomson, Ph.D., is a research
geneticist with the USDA-ARS Crop Improvement and
Genetics Research Unit in Albany, California.
Glossary
Transgene: Any gene brought into the genome by
means other than traditional breeding.
Recombinase: Enzymes that are capable of cutting
and pasting DNA without the gain or loss of genetic
material. Further, these enzymes recognize and
only manipulate very specific short DNA sequences
(recognition sites), which can be added to larger
DNA molecules (genome) for the cutting and pasting
process.
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59
Figure 1. Young graft-inoculated navel orange trees were used to detect CLas over time. Midrib tissue from young and mature leaves and feeder root tissue
were sampled for DNA to detect CLas and monitor its spatial and temporal distribution using real-time qPCR on a monthly basis. For proteomic analysis, phloem
proteins were extracted from stem tissue to monitor changes in host proteins in response to HLB infection.
CITRUS DISEASE
RESPONSE TO HLB
INFECTION
Jessica Franco, Deborah Pagliaccia, Wenbo Ma and Gitta Coaker
SUMMARY
The second year of this Citrus Research Board-funded research project focused on studying the in planta
distribution of Candidatus Liberibacter asiaticus (CLas), the huanglongbing (HLB)-associated bacterium, and
citrus responses to HLB infection. Using graft-inoculated navel trees, we found CLas to be in greater titer and
more evenly distributed in feeder roots than in young and mature leaves over time using quantitative real-time
polymerase chain reaction (qPCR). Through mass spectrometry, we also identified citrus proteins that were
differentially expressed during infection. These proteins have the potential to serve as markers for HLB, as well
as targets that can be manipulated for disease control. By understanding how CLas manipulates its host, better
management strategies can be implemented.
60
PCR + Navel Trees

6
Number of PCR + Trees
5
4
3
Feeder Root
Young Leaves
Mature Leaves
1
0
2 3 4 5 6 7 8
Months post gra7 inocula;on
secretion system and may be capable

of delivering effectors that manipulate
citrus physiology. Here, we report our
results on characterizing the distribution
and protein changes that occur in citrus
in response to CLas infection.
RESULTS
In order to study how CLas manipulates

citrus, we performed a graft inoculation
experiment in the University of California
Figure 2. Navel orange trees were assayed for the presence of CLas in young and mature leaves and
(UC) Davis Contained Research Facility
feeder roots using real time qPCR over a nine-month time period. A total of six navel trees were graftinoculated onto Carrizo rootstock in a greenhouse and monitored for HLB infection. One tree did not
(CRF), a quarantine greenhouse. The
become infected by CLas.
CLas strain used for inoculating citrus
corresponds to that identified in the
first PCR-positive tree found in California
in
2012.
Eleven
young
navel orange trees grafted on Carrizo
In recent years, the citrus industry has been plagued by
rootstock
were
graft-inoculated
or mock-inoculated. Samples
HLB, a devastating citrus disease. In the United States, HLBfrom
young
leaves,
mature
leaves
and feeder roots were
associated CLas is spread by the Asian citrus psyllid (ACP), a
taken
on
a
monthly
basis
for
eight
months
(Figure 1). DNA
piercing sucking insect, which carries the HLB-associated
was
extracted
to
detect
the
probable
presence
of CLas by
bacterium, CLas. ACP feeds on the phloem of citrus, which is
qPCR.
Our
results
indicate
that
CLas
DNA
detection
is more
also where CLas resides. Infected trees are characterized by
consistent
in
feeder
roots,
than
in
young
or
mature
leaves
hard, misshapen, bitter fruit with yellowing of their shoots and
(Figure
2).
CLas
DNA
detection
occurred
in
the
feeder
roots
as
leaves. Trees do not present symptoms until up to two years
early
as
the
first
month
post-inoculation.
Furthermore,
feeder
post-infection and can die within three to five years. Since
there is no cure after infection, disease mitigation measures root detection of CLas DNA was consistently detected over
currently rely on infected tree removal and pesticides time compared to those in aerial parts of the trees (Figure 2).
targeting ACP. HLB has severely impacted the citrus industry in
Florida, and the disease has expanded to Texas and California. We currently are repeating these experiments based on psyllid
In California, the first PCR-positive for CLas tree was detected inoculations at the UC Davis CRF. CLas is well known to be
in Hacienda Heights in Southern California in 2012. In the unevenly distributed in citrus, making detection challenging.
summer of 2015, additional trees in Southern California were The results from this and subsequent experiments can help
guide PCR-based detection strategies.
found to be PCR-positive for CLas.
INTRODUCTION
The inability to culture CLas in vitro has resulted in significant

challenges for understanding how HLB progresses. CLas
colonization of the phloem leads to changes in sugar
transport, phloem damage and ultimately phloem collapse. In
other bacterial pathogens, one of the most important factors
that enable bacteria to cause disease is the presence of protein
secretion systems that deliver proteins outside of the bacterial
cell and, in some cases, inside plant cells. These secreted
bacterial proteins are called effectors. Based on the genome
sequence, the bacterium possesses the secretory pathway
Despite the importance of HLB, understanding of the cellular

changes that occur in the host in response to CLas is still limited,
particularly at the protein level. Genes are found on DNA; and
when genes are expressed, they are transcribed into RNA, then
translated into protein. Proteins are typically the final products
of gene expression. In order to gain a greater understanding of
how citrus responds to CLas infection, we quantified changes in
protein expression in mock-inoculated and graft-inoculated navel
plants using mass spectrometry.
61
Figure 3. Protein extraction from stem tissue in navel orange. Bark containing phloem tissue was peeled off from the stems, excised into small strips, placed into
a small tube and centrifuged to extract the sap. Sap proteins were analyzed and compared to the control plant.
Expression: Log 2 (MS1 Intensity)
Sub9lisin, Cysteine and Aspar9c-like proteases are

highly expressed during CLas infec9on in Navel
32
30
28
26
Future experiments will determine if these

citrus proteases are targeting CLas proteins
in response to infection and whether they
Uninfected may contribute to HLB development.
Furthermore, because these proteases are
Infected
highly expressed during infection, they hold
promise as a biomarker to facilitate HLB
detection.
Selected Literature:
24
1. Wang N, Trivedi P. 2013. Citrus

huanglongbing: a newly relevant disease
presents unprecedented challenges.
Phytopathology103(7):652-65.
22
20
Sub+lisin Cysteine Cysteine Aspar+c Aspar+c

Protease Protease 1 Protease 2 Protease 1 Protease 2
Figure 4. Expression of various proteases in phloem sap of navel orange graft-inoculated with CLas.
Citrus defense genes (e.g., subtilisin protease, cysteine proteinases 1 and 2, aspartate proteases 1 and
2) were expressed significantly higher in HLB-infected plants than those in the non-inoculated control
plants. The Y-axis shows the relative expression of different host genes . Asterisks indicate significant
differences between infected and non-infected trees.
At ten months post-inoculation, stems were sampled. Crude

navel phloem was extracted by slicing bark into small pieces
and using centrifugation to remove the sap (Figure 3). The
extracted proteins were then subjected to mass spectrometry to
identify those dynamically changing in response to CLas infection.
Approximately 1,300 citrus proteins were identified, among which
the gene expression of 479 was significantly altered compared to
that in mock-inoculated plants. A total of 242 proteins had reduced
expression, while 237 proteins were induced upon CLas infection.
In particular, cysteine, serine and aspartic proteases were among
highly induced proteins in the CLas inoculated trees (Figure 4).
Proteases are enzymes that cleave other proteins. In other plants,
proteases are important components of plant defense responses
62
and also can be targeted by pathogen

effectors to facilitate infection.
2. Van der Hoorn RA. 2008. Plant

proteases: from phenotypes to molecular
mechanisms. Annual Review of Plant Biology.
59:191-223.
CRB Project: 5300-160
Jessica Franco, is a graduate student in the

Plant Pathology Department at the University of California,
Davis. Deborah Pagliaccia, Ph.D., is an assistant project
scientist in the Plant Pathology and Microbiology Department
at the University of California, Riverside. Wenbo Ma, Ph.D., is
an associate professor in the Plant Pathology and Microbiology
Department at the University of California, Riverside. Gitta
Coaker, Ph.D., is an associate professor in the Plant Pathology
Department at the University of California, Davis.
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support documents
63
DEVELOPMENT OF
LOW-SEEDED CITRUS BY
MUTATION BREEDING
What Growers Should Expect from Tango and Other Varieties

M. L. Roose, T. E. Williams and C. T. Federici
64
Figure 1. Irradiator used for inducing mutations in citrus at UCR. Budwood

is placed inside the device and then lowered into a position on a turntable
near the 137Cs radiation source. The budwood is exposed to radiation until the
desired exposure (number of absorbed radiation units) is reached. An elevator
returns the budwood to the upper area for retrieval.
The University of California, Riverside (UCR) Citrus

Breeding Program is focused on development of new
citrus cultivars that are adapted to California conditions
and that fit known or future market windows. We
develop new varieties using hybridization and mutation
to create variation, evaluate the initial trees and then
select some as potential varieties that justify more
detailed evaluation.
Trials of these possible new varieties are established
in several locations that represent California citrus
production areas. These trials are evaluated for several
years, and then decisions about release are made. In
this article, we describe how low-seeded selections of
existing varieties or hybrids are developed, summarize
current activities and then summarize data on seed
counts in many trials of Tango to illustrate the level of
variation in seed content that growers may encounter
in such varieties.
MUTATION BREEDING PROCESS
To develop a low-seeded variety by mutation breeding (a

non-GMO approach), we start with budwood of the existing
variety (or unreleased hybrid) that we obtain from the Citrus
Clonal Protection Program (CCPP). Budwood is sent to UCR
where it is exposed to radiation in a medical irradiator (Figure
1). This device contains a source of the cesium isotope 137Cs,
which emits gamma rays that strike the budwood. If gamma
rays strike chromosomal DNA, they may break the DNA strand.
Plant cells repair the damaged DNA, but the repair mechanisms
sometimes result in deletions or chromosome rearrangements
that can cause sterility by disrupting meiosis, the process that
produces the male (pollen) and female (egg) gametes. The
changes can alter genes leading to changes in other traits,
but these are relatively rare compared to the frequency of
changes in chromosome structure. In a sense, all of the DNA
is the target for mutations that reduce fertility, whereas most
other traits will be changed only by mutations in a few of the
approximately 25,000 genes in citrus.
65
Figure 2. UCR 10K, 13D and 13E trial sites showing adjacent citrus blocks that might serve as pollen sources. 13D and 13E have many fertile citrus varieties.
Image: Google Earth, March 9, 2011.
STATUS REPORT
The UCR breeding program has released low-seeded versions

of several mandarin cultivars, including W. Murcott (Tango),
DaisySL, FairchildLS and KinnowLS. Low-seeded cultivars in
advanced stages of testing include low-seeded forms of Nova,
Encore and Limoneira 8A Lisbon lemon. In some cultivars,
such as Clementines, obtaining low-seeded selections by
mutation breeding seems quite difficult, and we have not yet
been successful. Varieties in which we are currently evaluating
trees from recent irradiation include Sidi Aissa Clementine,
Robinson, Lee, California Honey and Page mandarins, Meyer
lemon, low-thorn (LT) lemon (a local UCR selection), Cocktail
grapefruit and Raspberry Jam, an unreleased pummelo x
blood orange hybrid. Preliminary selections have been made
in Robinson and Ponkan mandarins, Cocktail and Star Ruby
grapefruit, Limoneiro Fino, Walker Lisbon and LT lemons. Some
of these were submitted to the CCPP in 2015, and more may
be submitted in 2015-16 if they remain promising. Typically,
many of these early selections are discarded later as being too
seedy or having other defects (so dont get too excited about
them yet). Release of any of these is at least five years in the
future.
66
SEED CONTENT IN TANGO MANDARIN
Tango is certainly the most successful citrus cultivar developed

by mutation breeding. It was first identified as a very lowseeded selection in 1998 at UCR, and was propagated
and established in evaluation trials at numerous locations
throughout the California citrus-producing area in the early
and mid-2000s. Additional trials were established after the
release of Tango, including several rootstock trials. Since that
time, regular collections of fruit samples from various trials
have been evaluated for seed count and fruit quality, with the
total number of fruit evaluated for seed count now exceeding
24,000.
To date, Tango mandarin fruit have exhibited very low seed
counts in virtually all of the more than 1,000 trees planted
in the trials. Although seed counts are consistently low, they
do vary among locations and among years for the same trial.
Here we present a detailed summary of seed counts in Tango
fruit that should help growers to have realistic expectations
about seed content in Tango, particularly in locations where
other varieties that produce abundant, viable pollen are
planted nearby.
Figure 3. Porterville rootstock trial site showing adjacent plantings that can serve as pollen sources. Image: Google Earth, June 15, 2011.
TRIAL SITES
Seven of the trials evaluated [Arvin, Coachella (CVARS),

Lindcove (LC63), Rocky Hill, Santa Paula, South Coast
(SCREC) and UCR 1B] are locations in which the breeding
program evaluated a range of selections. These trials
were planted between 2002 and 2004 and include 12-20
Tango trees at each location. The trials are similar in that
cross-pollination pressure is high because they are mixed
plantings with many other varieties that produce viable
pollen. The data also include seed counts on the original tree
(called Mother) from which all other Tango trees derive. This is
also in a mixed planting at UCR.
There are three additional Tango trials at UCR (Figure 2). UCR
13D is a rootstock trial for Tango composed of 99 Tango trees
that were planted in 2009. Cross-pollination pressure is high
because this is a mixed planting with many other varieties
that produce viable pollen. UCR 13E includes 26 Tango
trees planted in 2006, also in a mixed block with high crosspollination pressure. UCR 10K is a solid-block of 480 Tango
trees planted in 2008. Cross-pollination pressure is medium
from lemons and other varieties nearby.
Two additional trials located at the UC Lindcove Research

and Extension Center also were studied. LC92 is a single
row of 22 Tango trees planted in 2008. Cross-pollination
pressure is high because the block contains many varieties
with high pollen viability. LC23 is a trial of 912 Tango trees
planted in 2010 as part of Beth Grafton-Cardwells program.
Cross-pollination pressure is likely moderate. Adjacent
plantings are navel orange, but seedy varieties with high
pollen viability are located within about 100 feet.
We also sampled fruit from three Tango rootstock trials planted
in 2008 and 2009. The Porterville rootstock trial (Porterv)
is located about five miles south of Porterville and includes
301 Tango trees planted in 2008. Cross-pollination pressure
is high because the block is surrounded by varieties with
high pollen viability including lemon, pummelo and Cocktail
grapefruit (Figure 3). In 2014, the Cocktail grapefruit trees
were top-worked, but fruit evaluated in 2015 were already
set. Orosi is a rootstock trial with 96 Tango trees planted in
2009. The experimental trees were inter-planted into the
growers 2008 Tango planting. Surrounding blocks contain
navel orange trees, so cross-pollination pressure is low. Arvin
rs is a rootstock trial about 10 miles east of Bakersfield that
includes 288 Tango trees planted in 2009. Adjacent blocks are
67
W. Murcott, satsuma and navel orange.

Cross-pollination pressure is expected to
be low.
DATA COLLECTION
METHODS
Seed counts were obtained using two

protocols. Field-cut seed counts were
obtained by cutting fruit in the field
and counting the number of seeds in
each fruit while adjacent to the sampled
branch. Fruits were either cut into several
slices to visualize all seeds, or seeds in the
stem and stylar ends were dug out with
a knife. An advantage of the field cutting
procedure is that the branch from which
a seedy fruit derives can be sampled
more extensively. Fruit quality samples
were 10-15 fruit picked from one to three
trees and returned to the lab for analysis
of multiple traits, not only seed count.
Seeds were counted after cutting fruit
in half and probing each half of the fruit
with a small scoop.
Figure 4. Number of Tango fruit with various seed counts in 4,224 fruit from fruit quality samples and
19,941 fruit cut in the field. Data collected from 1998 to 2015.
RESULTS
Seed counts were obtained from a total

of more than 24,000 fruit, with 4,224
from fruit quality samples and 19,941
fruit cut in the field. About 65-75 percent
of all fruit had no seeds, and another 20
percent had one seed (Figure 4). Only
about one percent had three or more
seeds. The average number of seeds per
fruit was about 0.30 for field cut fruit and
0.27 for fruit quality samples.
However, considering only these
averages obscures variation among years
and among locations, which likely reflect
effects of other pollen sources, weather
and bee activity on cross-pollination and
seed development. For the field-cut fruit
at UCR, the percentage of fruit having no
seeds ranged from about 42 percent in
UCR 13D in 2015 to 99 percent in UCR
10K in 2012 (Figure 5).
Figure 5. Seed content of Tango fruit in five trials at UCR measured from field-cut fruit between 2004
and 2015. Year effect (*) indicates statistically significant differences between years in the percentage
of fruit from the same field having zero or one seed per fruit compared to fruit having more than one
seed. For UCR 10K, the test included fruit with zero compared to one or more seeds, because the number
of fruit with more than one seed was very low. The significance test is indicated above the most recent
year sampled. The average number of fruit in each sample was 1,078, and the range was 130 to 2,590.
Mother signifies the original Tango from which budwood was collected that eventually was released.
The symbol ns denotes not significantly different.
The percentage of seedless fruit from the same field, but

sampled in different years, also was quite variable. For
example, the percentage of seedless fruit from UCR 10K
ranged from 78 to 99 percent between 2011 and 2015. The
average number of seeds per fruit showed correspondingly
large variation among years and fields, with UCR 13E fruit
ranging from 0.22 to 0.63 seeds per fruit.
68
Similarly large variation was observed in samples from trials

in the San Joaquin Valley and Ventura (Figure 6). In the
Porterville trial, based on samples of five fruit from each
fruiting tree (278 trees in 2012 and 287 trees in 2013), the
percentage of fruit with zero seeds was about 50 percent in
2012, but 84 percent in 2013.
pollen pressure such as at the Porterville

and the scion variety trial sites.
Figure 6. Seed content of Tango fruit in six trials measured from field-cut fruit between 2007 and 2012.
Year effect (*) indicates statistically significant differences between 2012 and 2013 samples from the
Porterville trial in the percentage of fruit having zero or one seed per fruit compared to fruit having
more than one seed. The average number of fruit in each sample was 693, and the range was 142 to
1,432.
Very rarely (fewer than one in 1,000),

Tango fruit have high seed counts
(defined here as six or more seeds).
These cases are discussed here. Such fruit
are so rare that they are not evident in
Figures 4-7. In 2012, we evaluated seed
content of 278 trees in a Tango rootstock
trial near Porterville by counting seeds
in five fruit from each tree that had fruit.
We found that five of 278 trees produced
at least one fruit with six or more seeds,
although the total percentage of seedy
fruit was very small. One tree (9-29)
produced many seedy fruit: the five fruit
sampled had six, six, seven, nine and
ten, and additional fruit also had high
seed counts. In the sample of 1,319 fruit,
excluding this tree, the percentages of
fruit with zero, one, two, three and more
than three seeds were 51, 36, 10, two
and one percent, respectively (Figure
5). The mean number of seeds per
fruit in 2012 was about 0.7, higher than
most previous observations. This field is
surrounded by trees that produce large
amounts of highly viable pollen (Cocktail
grapefruit, pummelo and lemon, Figure
3), and, therefore, is expected to have
higher seed counts than most locations.
In addition, in 2012 on other trees in this
block, we found four fruit with six, seven
or eight seeds and cut additional fruit
from these branches. This revealed one
tree with a single branch having fruit
with three, four, five, seven, seven, 12
and 13 seeds. The other seedy fruit were
found singly on different trees.
In 2013, we again found that tree 9-29

produced seedy fruit, but the average
for other trees in the field dropped to
the normal level of 0.2 seeds per fruit,
Figure 7. Seed content in fruit quality samples of Tango fruit from nine trials. Year effect (*) indicates
and
no fruit had more than three seeds.
statistically significant differences between years in samples from the same trial in the percentage of
fruit having zero or one seed per fruit. The average number of fruit in each sample was 228, and the
Tango trees at Rocky Hill, South Coast,
range was 60 to 1,053. The symbol ns denotes not significantly different.
UCR and Lindcove also have produced
single fruit with more than five seeds. We
are
investigating
the
origin of tree 9-29 at the molecular level
We saw similar variation among years and locations in the
using
markers
that
distinguish
Tango and W. Murcott, but do
number of seeds per fruit in the fruit quality samples that were
not
yet
have
a
definitive
explanation.
This tree does, however,
cut in the lab (Figure 7). Since seed content often differs in
represent
a
single
tree
out
of
the
more
than 1,000 Tango trees
successive years while the surrounding pollen source trees
in
trials,
none
of
which
have
the
same
characteristics. From
remain unchanged, much of the variation is likely attributable
this,
it
appears
that
genetic
or
environmental
factors can
to weather during flowering, the duration of flowering and
cause
Tango
trees
to
produce
seedy
fruit,
but
this
is
a very rare
the prevalence of bees. As expected, sites surrounded by
event.
trees that produce little viable pollen, such as Orosi and UCR
10K, tend to have lower seed content than sites with more
69
CONCLUSIONS
Tango trees nearly always produce fruit with fewer than two
seeds, with the majority of fruit being seedless over a wide
range of sites and years. Sites with high pollen pressure, such
as those adjacent to large plantings of varieties that produce
copious amounts of viable pollen (pummelos, Minneola and
Clementines are examples) are likely to produce fruit with
higher seed counts, with averages of about one seed per
fruit in the worst years. (We have not determined whether
or not the proximity to lemons affect seed content of Tango.)
In such situations, young trees are more likely to have higher
seed counts because bees are more likely to bring pollen
from outside the block when trees are small and have fewer
flowers. On the other hand, solid block plantings (similar to
most commercial plantings) and those isolated from strong
pollen sources may have very low seed counts with 95 percent
or more of fruit being seedless.
Acknowledgements
We would like to thank the Citrus Research Board for funding

this research; several grower-cooperators for managing the
trials and growing trees for the rootstock trials, including Tree
Source Citrus Nursery, Citri-care, Bee Sweet Citrus, Harrison
Smith, Rocky Hill Inc., Johnston Farms, Lyn Citrus (originally
W&N Citrus), Brokaw Nursery; and many lab members who
participated in fruit evaluation.
Mikeal L. Roose, Ph.D., is Professor of Genetics in the
Department of Botany and Plant Sciences at UC Riverside.
Tim Williams, M.S., was Staff Research Associate (now
retired); and Claire T. Federici, Ph.D., is Staff Research
Associate in in the Department of Botany and Plant Sciences
at UC Riverside.
It is important for growers to remember that trees are living

organisms, and the useful variants that we find or discover may
not be perfectly stable (rather like humans). Some occasional
variation in seed content may occur, even in varieties with
outstanding commercial value.
NOW TAKING ORDERS!

All orders grown in
USDA certified pest
exclusionary facilities
Contact Us
(760) 397-4104
Sales@Youngs-Nursery.com
www.Youngs-Nursery.com
70
71
Figure 1. Design of the therapeutic chimeras with

recognition and lysis domains. (A) A schematic
representation of the cell envelope of Gram-negative
bacteria such as Candidatus Liberibacter, Xanthomonas
citri subsp. citri (Xcc) and Xylella fastidiosa (Xf) illustrates
shared structural components (lipopolysaccharide [LPS]
and outer-membrane protein [OMP]) where protein
chimeras bind and break the bacterial cells into small
pieces. (B) Listing of chimeras designed in this project
is shown. The Thionin1-D4E1 and LBP-Thionin2, listed
as chimera A and B, respectively, are currently being
evaluated in expression and efficacy studies. Models of
(C) chimera A (Thionin1-D4E1) and (D) chimera B (LBPThionin2) show recognition (magenta) and cell lysis or
rupturing (red) regions or domains. In both of these
models, an amino acid linker, GSTAPPA (yellow), is used to
join the two domains.
NOVEL THERAPY
OF HIGH-PRIORITY
CITRUS DISEASES
Hau Nguyen, Guixia Hao, Ed Stover and Goutam Gupta
SUMMARY
Huanglongbing (HLB), citrus canker and citrus variegated chlorosis (CVC) are three high-priority citrus diseases,
all caused by a single category (Gram-negative) of bacteria. Any approach that could kill the causative bacteria
when they first infect the tree would be very useful, and the same approach may be able to prevent all three
diseases.
In this project, we lay the foundation of one such approach. We show that the design and delivery of a protein
chimera clears both HLB-associated Candidatus Liberibacter asiaticus (CLas) and canker-causing Xanthomonas
citri subsp. citri (Xcc) and blocks disease symptom development. This chimera has two functional domains
derived from citrus. One domain (called the recognition domain) targets the specific membrane features
conserved in these three bacteria, whereas the other domain (called the lysis domain) creates membrane pores
72
in these bacteria. These two domains are connected

by a flexible linker, which allows synergy in function of
the two domains, thereby facilitating rapid death of
the bacteria. Two routes are chosen for the delivery
of the in planta protein chimeras via transgenic citrus
and nanocapsules. The transgenic citrus lines express
the chimeric protein and provide long-term protection
against bacterial diseases, whereas the nanocapsules
containing the protein chimera potentially can be
applied to the infected trees to clear the bacteria and
block the disease development.
We already have demonstrated the efficacy of a
transgenic citrus expressing a chimera in blocking HLB
and canker disease development. Efficacy studies
with transgenic citrus expressing other chimeras
(expected to possess improved anti-bacterial activity)
are currently in progress. Finally, construction of the
nanocapsules is also underway to deliver the antibacteria protein chimeras.
INTRODUCTION
HLB, canker and CVC are three high-priority bacterial diseases

in citrus1. Although known for a century in East Asia, it was
only recently that HLB was introduced into the Americas,
including the United States and Brazil, two of the top citrusproducing nations2. Typical HLB symptoms are leaves with
blotchy mottling. HLB shortens the lifespan of the trees
and reduces the yield and quality of the fruit. HLB is now
widespread in most citrus-producing areas of Asia, Africa and
most of the Americas. Most citrus cultivars are susceptible to
HLB. However, the disease severity appears to be different in
different cultivars. HLB is associated with an insect-vectored
bacterium. Candidatus Liberibacter asiaticus is a phloemlimited bacterium associated with HLB and is carried by an
insect vector called Asian citrus psyllid (ACP).
In the United States, ACPs carrying CLas have been
encountered in all states where citrus is grown. More than 80
percent of citrus trees in Florida already are infected with CLas.
A small number of citrus trees in Texas also are infected. Only
a few citrus trees with CLas have been found and removed
in California, although ACPs are encountered in a number of
the states citrus growing areas. There currently is no cure for
HLB, nor is there any naturally occurring citrus cultivar that is
resistant to HLB.
Like HLB, citrus canker is an old disease, which originated in
Southeast Asia, but has spread in several citrus-producing
countries. In the United States, citrus canker has been an
issue for some time in Florida3. Citrus canker symptoms

include brown spots with yellow haloes on the leaves. The
disease causes citrus trees to prematurely drop leaves and
fruit. Xanthomonas citri subsp. citri, the bacterium responsible
for the disease, is spread from infected trees to healthy trees
by wind-driven rain or on contaminated tools, clothing and
equipment.
Citrus variegated chlorosis (CVC) first appeared in Brazil in
1987 and has rapidly become one of the most economically
important diseases affecting sweet orange production in
Brazil4. Xylella fastidiosa (Xf-CVC strain), the causal agent of
CVC, is a xylem-limited bacterium vectored by several species
of sharpshooter leafhoppers in the order Hemiptera. Some of
these sharpshooters already are established in Florida. Since
all citrus cultivars in Florida are susceptible to CVC, this disease
can pose a threat to the Sunshine States citrus industry. There
is currently no cure for CVC.
THE PROJECT GOAL AND

HYPOTHESIS
The main goal of this project is to develop the same therapy

that can be applied to HLB, canker and CVC. Although the
mechanisms of disease development by these three gramnegative bacteria are different2, 4, it is our hypothesis that there
are common and conserved features that can be targeted
through a common therapy. In this project, the protein
chimera or fusion proteins are created in such a way that they
have two different functional regions: one for recognizing the
bacterial cells and binding to them and the other for breaking
them down into small pieces. The two regions or domains
(recognition and lysis) target the outer membrane features
that are present in all three bacterial types.
PROGRESS TO DATE
Figure 1 lists different chimeric proteins that we have

designed. The regions that recognize the bacterial cells are
either derived from a citrus protein (called lipid-binding
peptide or LBP) that binds to the lipids (lipopolysaccharide) on
the surface of bacterial outer-membrane5 or a citrus protease
that cleaves the bacterial outer-membrane proteins (OMP)6.
The other regions or domains of the chimeric proteins that
cause destruction or lysis of bacterial cells are derived from
citrus antimicrobial peptides (AMPs). The latter include citrus
Thionins, which are small peptides with dual functions in that
they recognize bacterial outer membrane (LPS) and bind to it,
as well as create numerous holes (pores) in the membrane and
kill the bacterium7.
73
Figure 2. Transgenic citrus (Carrizo) expressing the Thionin1-D4E1 chimera shows protection against both citrus canker and HLB (Collaborator: Ed Stover, USDAARS, Ft. Pierce, Florida). Transgenic citrus expressing the Thionin1-D4E1 chimera is protected after Xcc challenge (A, left panel), whereas nontransgenic citrus
is not (A, right panel). Transgenic citrus shows protection against HLB, (B, left panel) whereas nontransgenic citrus does not (B, right panel). All the citrus trees
were grafted with Liberibacter-infected rough lemon.
In this study, we created three types of chimeric proteins as follows:

A. Thionin1-D4E1 (D4E1 - a synthetic but citrus-friendly
AMP designed by Ed Stover)
B. LBP-Thionin2
C. Serine protease-Thionin
Note that there are several citrus variants of Thionin, LBP and
protease. Thus, different chimeras can be constructed within each
category, thereby allowing us to choose the chimera with the highest
anti-bacterial activity.
Thionin1-D4E1 chimera was the first one that we started working on
three years ago. At that time, the sequence of the citrus genome
was not completed, and hence complete screening of citrus Thionins
was not possible. Therefore, we chose a tobacco Thionin called
Thionin1, which has 70 percent identity with a citrus Thionin (as
observed after the completion of the citrus genome sequencing).
After the availability of the completed citrus genome sequencing8,9,
we designed the LBP-Thionin2 chimera in which both Thionin and
LBP were chosen from the citrus proteome.
Our first objective was to develop transgenic citrus (Carrizo and
Hamlin) that shows resistance to all three citrus diseases HLB, citrus
canker and CVC. This was achieved by transforming citrus plants with
chimeric protein genes such as Thionin1-D4E1 and LBP-Thionin2.
Transgenic plants expressing Thionin1-D4E1 chimeric protein
showed a significant increase in resistance to both citrus canker and
HLB in greenhouse studies (Figure 2). These results validated our
74
hypothesis that the same chimeric proteins can protect citrus plants
against multiple bacterial diseases. Testing of transgenic plants
expressing LBP-Thionin2 chimeric protein for disease resistance is
currently underway in greenhouse.
The second goal of our project was to design nanocapsules
(Figure 310,11) containing the AMPs listed above and deliver them into
citrus plants to combat HLB infection.
We have determined that in planta delivery of nanocapsules will
require a large-scale amount of CTV capsid protein, which we will
obtain by isolating and purifying the protein from the CTV virions.
CONCLUSION
Progress made so far suggests that protein chimera of recognition

and lysis domains provide effective and broad-spectrum protection
against multiple bacterial diseases in citrus. In planta delivery of the
protein chimera enhances the immune defense in citrus.
Our future work will follow the success with the protein chimera,
which will include efficacy testing of other chimeras designed to
show a better activity against citrus diseases.
References
1. Vojnov, A.A., do Amaral, A.M., Dow, J.M., Castagnaro, A.P. and

Marano, M.R. 2010. Bacteria causing important diseases of citrus
utilize distinct modes of pathogenesis to attack a common host.
Applied Microbiology & Biotechnology 87(2):467-477.
Figure 3. Design of two types of lipid-based nanocapsules carrying a small molecule or protein therapeutic agent. A lipid-based inner shell (hydrophobic
interior) contains inhibitory therapeutic agents (A, left panel). An outer shell of Citrus tristeza virus (CTV) capsid protein is used to specifically deliver the
therapeutic agents into the citrus phloem tissue (A, right panel). Inner shell with liposome (hydrophilic interior) contains inhibitory therapeutic agents (B, left
panel). An outer shell of CTV capsid protein is used to specifically deliver the inhibitory therapeutic agents into the citrus phloem tissue (B, right panel).
2. Wang, N. and Trivedi, P. 2013. Citrus huanglongbing: a newly

relevant disease presents unprecedented challenges. Phytopathology
103(7):652-665.
3. Graham, J.H, Gottwald, T.R., Cubero, J. and Achor, D.S. 2004.
Xanthomonas axonopodis pv. citri: factors affecting successful
eradication of citrus canker. Molecular Plant Pathology 5(1):1-15.
4. Beretta, M.J.G., Barthe, G.A., Ceccardi, T.L., Lee, R.F. and Derric, K.S.
1997. A survey for strains of Xylella fastidiosa in citrus affected by
citrus variegated chlorosis and citrus blight in Brazil. Plant Disease
81:1196-1198.
5. Schumann, R.R. 2011. Old and new findings on lipopolysaccharidebinding protein: a soluble pattern-recognition molecule. Biochemical
Society Transactions 39(4):989-993.
6. Galdiero, S., Falanga, A., Cantisani, M., Tarallo, R., Della Pepa
M.E., DOriano V. and Galdiero M. 2012. Microbe-host interactions:
structure and role of Gram-negative bacterial porins. Current Protein
& Peptide Science 13(8):843-54.
7. Pelegrini, P.B. and Franco, O.L. 2005. Plant -thionins: novel
insights on the mechanism of action of a multi-functional class of
defense proteins. International Journal of Biochemistry & Cell Biology
37(11):2239-2253.
8. Wu, G.A. et al. 2014. Sequencing of diverse mandarin, pummelo
and orange genomes reveals complex history of admixture during
citrus domestication. Nature Biotechnology 32(7):656-662.
Hau Nguyen, Ph.D., is a research associate at the NMC Biological

Laboratory in Los Alamos, New Mexico. Guixia Hao, Ph.D., is a
research geneticist at the U.S. Horticultural Research Laboratory
in Ft. Pierce, Florida. Ed Stover, Ph.D., is a research horticulturist
and geneticist at the U.S. Horticultural Research Laboratory in Ft.
Pierce, Florida. Goutam Gupta, Ph.D., is a scientist and leader of
LANL Advanced Therapeutics and Vaccine Initiative, Biosciences,
Los Alamos National Laboratory, Los Alamos, New Mexico.
Glossary
Gram-negative bacteria: Bacteria are divided into
two distinct groups of Gram-negative and Grampositive based on their cell wall architecture. Gramnegative bacteria have an outer membrane with
thinner cell walls and do not retain the crystal violet
stain as opposed to Gram-positive bacteria.
Protein chimera: Chimeric or fusion proteins are
engineered through the joining of two or more
specific genes. The latter are eventually translated
into corresponding proteins, each having a different
functional property or domain to serve a particular
purpose.
In planta: Inside plant cell or tissue
9. Xu, Q. et al. 2013. The draft genome of sweet orange (Citrus sinensis).
Nature Genetics 45(1):59-66.
Transgenic plant: A plant that is genetically modified

by adding and expressing a foreign or native gene(s)
to its genome for the purpose of making it resistant
or tolerant to pests, diseases, drought, salt, etc. or
improving its yield or horticultural quality.
10. Mora-Huertas, C.E., Fessi, H. and Elaissari, A. 2010. Polymer-based

nanocapsules for drug delivery. International Journal of Pharmaceutics
385:113142.
Nanocapsule: Nanoscale shells (10-1000 nanometers)

are made out of a nontoxic polymeric membrane that
encapsulates a therapeutic drug for delivery.
11. Huynh, N.T., Passirani, C., Saulnie, P. and Benoit, J.P. 2009. Lipid
nanocapsules: A new platform for nanomedicine. International
Journal of Pharmaceutics 379:201209.
75

Figure 1. In order to find nucleotide variability
at both ends (3 and 5) of CTV California strain
genome (highlighted in gray), the virus RNA has to
be converted to a double-stranded DNA, first using
a process called reverse transcription (going from
RNA to DNA, rather than from DNA to RNA). In the
second step, nucleotides (T, C, G, A or poly-A tail)
are added to both ends of DNA strands using Rapid
Amplification of cDNA Ends (RACE) technique.
Addition of nucleotides will provide a basis for
sequencing both ends on the two strands (blue and
orange lines).
FIGHTING HLB WITH A

CITRUS TRISTEZA
VIRUS-BASED VECTOR
Heterogeneity in the genome ends of CTV is an important

consideration
James Ng, Angel Chen and Raymond Yokomi
SUMMARY
As California prepares for a potential showdown with huanglongbing (HLB), contemporary strategies that
use low inputs, yet produce high-value control, are needed to manage the disease. With biotechnology, we
can develop Citrus tristeza virus (CTV) into a tool for the protection or treatment of citrus trees. Specifically,
our goal is to engineer an infectious complementary (c)DNA clone of a mild California strain of CTV that
can be developed for the control of HLB (and other disease-causing agents of citrus). Here, we performed
a systematic analysis of the nucleotide variability located at the genome ends of California CTV strains with
76
the T30 and the T36 genotypes an important

consideration in the construction of infectious
cDNA clones, given that nucleotides (nucleic acid
components) in these locations serve essential
biological functions for CTV. We are using knowledge
gained from this analysis to construct the cDNAs for
each of these CTV strains.
In the future, availability of a biologically active
CTV full-length cDNA will provide a template for
manipulation of the CTV genome, thereby facilitating
the rational development and testing of therapeutic
proteins and RNA silencing/interference against HLBassociated Candidatus Liberibacter species and other
citrus pests and pathogens.
THE BIOLOGY OF CTV
Citrus tristeza virus (CTV; a member of the virus family

Closteroviridae) is the causal agent responsible for a number
of citrus maladies characterized by mild to severe disease
symptoms1. Symptomless infection causing no deleterious
effects on citrus can also occur, which appears to be typical
of the interactions between mild CTV strains and most
commercial citrus varieties grown on CTV-resistant and
CTVtolerant rootstocks in California. CTV infects only
phloem tissue located many layers beneath the plant surface.
Experimental transmission can be achieved by grafting, bark
peel inoculation (where a virus preparation is applied to the
inner surface of a partially-peeled bark still attached to the
stem) and slashing the stem of an uninfected citrus plant
with a CTV-contaminated razor blade. The virus also can be
transmitted from plant to plant by several different aphid
species2.
DNA technology has enabled the RNA genomes of many
viruses to be converted to DNA, which is more stable than RNA
and can be engineered for different purposes. For example,
Bill Dawson, Ph.D., at the University of Florida has made the
19.3 kilo base (kb) RNA genome (Figure 1) of a Florida strain
of CTV with the T36 genotype (T36-FL) into an infectious
complementary (c)DNA, which made it possible to engineer
mutations into CTV genes to study their functions3,5.
CTV AS A VIRAL VECTOR
A more practical side to generating the infectious cDNA

clone of CTV is that it also can be developed as a vector, i.e.,
a DNA molecule/genetic element with numerous advantages
for applications in biotechnology that can benefit citrus
production. For example, as with other existing plant virusbased vectors, including the Tobacco mosaic virus (TMV)
and Potato virus X vectors (both of which infect a wide host
range, but not citrus), a CTV vector is a self-replicating entity

within the infected plant. An advantage of using a CTV-based
vector is its stability within the inoculated plants. Existing
data indicate that a CTV vector engineered to express the
jellyfish green fluorescent protein (GFP) can be stably, but not
permanently, maintained in citrus plants for many years6,7.
Furthermore, because the HLB-associated agent Candidatus
Liberibacter asiaticus (CLas) is delivered by its insect vector,
the Asian citrus psyllid (ACP), into (and propagates in) the
phloem, the coincident cohabitation of CTV and CLas in this
location makes it ideal to target the latter for control by using
an engineered CTV vector. CTV/T36-FL already has been
developed and deployed as an RNA interference (RNAi) vector
designed to inhibit the ACP8, as well as for the production of
therapeutic proteins both as a curative and a prophylactic
against CLas and other phloem-residing pathogens.
In light of the emergence of HLB in citrus production areas
across the United States, the breakthrough in constructing
a functional CTV vector is a remarkable achievement that
demonstrates the concerted efforts of the citrus scientific
community in dealing with the problems in the wake of this
devastating disease.
OBJECTIVES
To use a CTV vector for RNAi-mediated silencing of the ACP

or for the expression of therapeutics against CLas, the initial
step involves development of an infectious cDNA clone of the
virus. Because of current EPA, APHIS and CDFA restrictions,
use of CTV-FL in California is not being considered. Rather,
we are developing infectious cDNA clones derived from CTV
strains endemic to California. Our research on constructing
the cDNA clone of CTV has focused on using mild strains that
are widespread but restricted to California e.g., California
strains of CTV with the T30 genotype or its equivalent.
As with many viruses, CTV exists in a host as a population with
one predominant nucleotide (nt) sequence accompanied
by a pool of nt variants9,10. Interestingly, the frequency of nt
variations at the proximal (also called the five-foot) end of
the CTV genome is generally higher than those found at the
distal (also called the three-foot) end9. Because the 5 and
3 ends of viruses are known to play important roles such
as replication, virion assembly and pathogenicity during
viral infection11, the identity of the nts at these locations are
essential considerations for generating the full-length CTV
cDNA.
There are three objectives in this study:
1) Construct the cDNA clone of a mild California strain of CTV.
2) Determine the biological activity/infectivity of the cDNA
77
clone in Nicotiana benthamiana plants

by Agrobacterium tumefaciens agromediated inoculation (agro-inoculation).
3) Prepare CTV from infected N.
benthamiana plants following the
agro-inoculation in Objective 2, and
test its infectivity in citrus plants, as
well as transmission by aphids. This
progress report focuses on Objective 1
and includes most of the work done to
determine the variability in the 5 and 3
ends of CTV populations with the T30
(T30-CA strain) and the T36 (T36-CA
strain) genotypes.
VARIABILITY IN
GENOME ENDS OF
CTV POPULATIONS
Replication of the CTV RNA genome

produces abundant double-stranded
(ds)RNA consisting of a plus (+)-strand
and a complementary minus (-)-strand
form of RNA (Figure 1). We purified
the dsRNAs of T30-CA and T36-CA and
converted them to (+)- and (-)-strands
DNA by two procedures:
1) reverse transcription, and
2) Rapid Amplification of cDNA-Ends
[RACE] (Figure 1).
Figure 2. Nucleotide (nt) variations at 5 end of cDNA clones of CTV strain T30-CA. Only nt variants at
frequencies greater than 20 percent among all the clones sequenced are shown. Numbers at the top of
each bar represent the number of clones with nt sequence shown at the base of that bar over the total
number of clones sequenced. Consensus (conserved) or common nucleotides (nts) are shown in black
letters, variant nts are in red.
Subsequently, the DNA products were

cloned and sequenced to determine
their nt identities. The identities of the
most commonly seen nt variants and the
frequency with which these variant nts
appeared in the cloned DNA are shown
in Figures 2 - 5.
VARIABILITY AT
GENOME ENDS OF
T30-CA
3. Nucleotide variations at 3 end of cDNA clones of CTV strain T30-CA. Only nt variants at
The consensus (conserved) RNA Figure
frequencies greater than 10 percent among all the clones sequenced are shown. Numbers at the top of
sequence at the 5 end of selected each bar represent the number of clones with the nt sequence shown at the base of that bar over the
T30 sequences retrieved from the total number of clones sequenced. Consensus (conserved) or common nts are shown in black letters;
variant nts are in red.
NCBI GenBank database is AAUUUC.
Sequencing of the (+)-strand DNA and
the (-)-strand DNA revealed that the consensus AATTTC (in many other nt variants (data not shown) but the ones shown
DNA, Uracil is replaced by Thymine) sequence was present in in Figure 2 were the most common.
many of the clones sequenced (Figure 2). In several cloned
sequences, one or more extra (variant) nts were observed The 3 end of the T30-CA strain genome contained fewer nt
upstream of this consensus sequence (Figure 2). There were variants than its 5 end. Data obtained from the sequencing
of the (+)-strand DNA and the (-)-strand DNA revealed that
78
Figure 4. Nucleotide variations at 5 end of cDNA clones of CTV strain T36-CA. Only nt variants at frequencies greater than 10 percent among all the clones
sequenced are shown. Numbers at the top of each bar represent the number of clones with the nt sequence shown at the base of that bar over the total
number of clones sequenced. Consensus (conserved) or common nts are shown in black letters; variant nts are in red. Nucleotide sequences that are similar to
the Group 1 consensus sequence, AATTTCTCAAA, and the Group 2 consensus sequence, AATTTCACAAA, are indicated.
AGGTCC was the consensus (Figure 3). All of the T30

genotype sequences retrieved from the NCBI GenBank
database have highly conserved nts at their genomic 3 ends,
and the consensus sequence is AGGUCCA. Sequencing of
the (-)-strand DNA indicated that about 21 percent (five out
of 24) of the sequences contained the last A while another
21 percent (five out of 24) did not (Figure 3). This suggests
that T30-CA strain genome contains a subpopulation with
the AGGUCC sequence and another subpopulation with the
AGGUCCA sequence. There were other nt variants, where one
or more extra nucleotides were observed after the last C in
the consensus AGGTCC (an example of which is AGGTCCG),
but these were detected infrequently (at frequencies < 20
percent as shown in Figure 3).
These results will be used to create the initial nt sequence to
be incorporated in the cDNA clone of a T30 CTV genome from
California.
VARIABILITY AT GENOME ENDS OF

T36-CA
A comparison of selected T36 genotype sequences retrieved

from the NCBI GenBank database indicated that the consensus
genomic 5 end sequence can be placed in two groups: group
1 consensus sequence is AAUUUCUCAAA; group 2 consensus
sequence is AAUUUCACAAA.. In this study, T36-CA sequences

were determined using infected tissues collected from two
California locations Fillmore County and Tulare County.
Sequencing of the (+)- and (-)-strands DNA revealed that the
group 1 consensus sequence (i.e. AATTTCTCAAA) was found
in more than 15 nt variants from the Fillmore County sample
(only one is shown in Figure 4), whereas none was present
in the nt variants from the Tulare County sample (Figure 4).
The nt variants from both the Fillmore and the Tulare samples
have sequences similar (but not identical) to the group 2
consensus sequence (i.e. AATTTCACAAA), except that none
of them has the cytosine (C) at position 8 of the consensus
(Figure 4). The AATTTCAAAA variant (seen more frequently
in the Tulare sample than the Fillmore sample) is most similar
to the group 2 consensus sequence (Figure 4). In many of
the clones sequenced, one or more extra (variant) nts were
observed upstream of this consensus sequence, but variants
shown in Figure 4 were the most common.
As with the T30 genomic sequences, T36 genomic sequences
retrieved from the NCBI GenBank database have highly
conserved nts at their genomic 3 ends, and the consensus
sequence is AGGUCCA.
In this study, data were consistent with AGGUCCA as being
the consensus. For example, of the 21 sequences detected
in the Fillmore and Tulare samples, nine contained AGGTCC
as determined by sequencing the (+)-strand DNA (Figure 5).
79
Figure 5. Nucleotide variations at 3 end of cDNA clones of CTV strain T36-CA. Numbers at the top of each bar represent the number of clones with the nt
sequence shown at the base of that bar over the total number of clones sequenced. Consensus (conserved) nts are written in black letters; variant nts are in red.
Of the 58 sequences originating from the Fillmore and the

Tulare samples, 29 contained the consensus AGGTCCA as
determined by sequencing the (-)-strand DNA (Figure 5). In a
number of cloned sequences, one or more extra nucleotides
were observed after the last A in the consensus AGGTCCA
(an example of which is AGGTCCAT) or after the last C in
AGGTCC (an example of which is AGGTCCG (Figure 5).
These results will be used to create the initial nt sequence to
be incorporated in the cDNA clone of a T36 CTV genome from
California.
Taken together, our analysis of the genomic 5 and 3 ends
of the T30-CA and T36-CA viral dsRNA consistently showed
predominant consensus sequences and a population of
nucleotide variants at the extreme ends. This information is
critical to the success of constructing the full-length cDNA
clones of both the T30 and the T36 CTV strains from California.
Acknowledgements
We thank the Citrus Research Board for funding this research.
James Ng, Ph.D., associate professor of Plant Pathology,
and Angel Chen, Ph.D., assistant specialist, are from the
Department of Plant Pathology and Microbiology at the
University of California, Riverside. Raymond Yokomi,
Ph.D., is a plant pathologist at USDA-Agricultural Research
Service, Parlier, California.
80
References
1. Lee, R.F. and Bar-Joseph, M. 2003. Graft-transmissible
diseases of citrus. p. 607-639. In: G. Loebenstein and G.
Thottappilly (eds.), Virus and Virus-like Diseases of Major Crops
in Developing Countries. Kluwer Academic Publishers, the
Netherlands.
2. Ng, J.C.K. and Zhou, J.S. 2015. Insect vector-plant virus
interactions associated with non-circulative, semi-persistent
transmission: current perspectives and future challenges.
Current Opinion in Virology 15:48-55.
3. Ambrs, S., El-Mohtar, C., Ruiz-Ruiz, S., Pea, L., Guerri, J.,
Dawson, W.O. and Moreno, P. 2011. Agroinoculation of Citrus
tristeza virus causes systemic infection and symptoms in the
presumed nonhost Nicotiana benthamiana. Molecular PlantMicrobe Interactions 24:1119-1131.
4. Tatineni, S., Gowda, S., Boyko, V.P., Albiach-Marti, M.R.,
Mawassi, M., Navas-Castillo, J., Karasev, A.V., Dolja, V., Hilf, M.E.,
Lewandowski, D.J., Moreno, P., Bar-Joseph, M., Garnsey, S.M.
and W.O. Dawson. 1999. An engineered closterovirus RNA
replicon and analysis of heterologous terminal sequences for
replication. Proceedings of the National Academy of Sciences
USA 96:7433-7438.
5. Tatineni, S., Robertson, C.J., Garnsey, S.M. and Dawson,

W.O. 2011. A plant virus evolved by acquiring multiple
nonconserved genes to extend its host range. Proceedings of
the National Academy of Sciences USA 108:17366-17371.
6. El-Mohtar, C. and Dawson, W.O. 2014. Exploring the limits
of vector construction based on Citrus tristeza virus. Virology
448:274-283.
7. Folimonov, A.S., Folimonova, S.Y., Bar-Joseph, M. and
Dawson, W.O. 2007. A stable RNA virus-based vector for citrus
trees. Virology 368:205-216.
8. Hajeri, S., Killiny, N., El-Mohtar, C., Dawson, W.O. and Gowda,
S. 2014. Citrus tristeza virus-based RNAi in citrus plants induces
gene silencing in Diaphorina citri, a phloem-sap sucking insect
vector of citrus greening disease (Huanglongbing). Journal of
Biotechnology 176:42-49.
9. Albiach-Marti, M.R., Mawassi, M., Gowda, S., Tatineni, S., Hilf,
M.E., Shanker, S., Almira, E.C., Vives, M.C., Lpez, C., Guerri, J.,
Flores, R., Moreno, P., Garnsey, S.M. and Dawson, W.O. 2000.
Sequences of Citrus tristeza virus separated in time and space
are essentially identical. Journal of Virology 74(15):6856-6865.
10. Rubio, L., Ayllon, M.A., Kong, P., Fernandez, A., Polek, M.,
Guerri, J., Moreno, P. and Falk, B.W. 2001. Genetic variation of
Citrus tristeza virus isolates from California and Spain: Evidence
for mixed infections and recombination. Journal of Virology
75(17):8054-8062.
11. Dawson, W.O., Garnsey, S.M., Tatineni, S., Folimonova,
S.Y., Harper, S.J., Gowda, S. 2013. Citrus tristeza virus-host
interactions. Frontiers in Microbiology 4:1-10.
Glossary
Infectious complementary (c)DNA clone Many
plant viruses, including CTV, use RNA (as opposed
to DNA) as their genetic material. This RNA can be
converted into DNA, which is complementary to
the RNA (hence the name complementary DNA),
and inserted (cloned) into a piece of autonomously
replicating DNA (called a cloning plasmid) that
exists outside a bacteriums DNA chromosome to
produce many copies of the inserted cDNA. When it
is introduced into a suitable plant, this cloned cDNA
is deemed infectious when it replicates and moves
throughout the plant.
RNA silencing/interference This is an all-natural
gene-inactivation system that allows organisms,
including plants and animals, to regulate their biological
processes, such those involved in development and
in defense against viruses. With biotechnology, RNAi
can be used to inactivate the genes of virtually any
organism that has the RNAi mechanism.
81

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), which is the vector for the phloem-limited bacterium Candidatus
Liberibacter asiaticus (CLas) that is associated with huanglongbing (HLB or 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 all
82
provided significant reduction in 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 threshold in the conventional
program. Pyganic with M-pede performed better than with 435 oil and both performed better than Pyganic
alone. ACP reduction from Pyganic with M-pede was similar to Danitol through the first week of March after
which only the Danitol residual was effective. A total of six applications for the organic programs and five in
the conventional program were made during the growing season. Organic Programs 2 and 3 rotated organic
insecticides with 435 oil or M-pede resulting in a 50 percent reduction in the use of organic insecticides while
providing better control than Program 1 with organic insecticides only.
However, ACP was reduced more in the conventional program. Reduction to 0.1 adults per tap sample was
observed after the treatments of Closer and Aza-Direct plus M-pede in March, Micromite and Azera plus
M-pede in April, and Entrust plus 435 oil and Imidan in May. Lacewings, spiders, ants and ladybeetles were
observed in all treatments that also may have contributed to ACP reduction. Green lacewings were most
abundant. Ladybeetles known for significant predation on ACP were rare, reflecting the negative impact of
increased use of broad-spectrum insecticides to control ACP since the advent of HLB.
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 be repeating and extending these studies to confirm results,
include additional products and evaluate on ACP, other pests and beneficial insects.
BACKGROUND
Candidatus Liberibacter species that are known to be

associated with HLB are vectored by ACP (Photo 1). Both
adults and nymphs of this insect are capable of acquiring
and transmitting Candidatus Liberibacter asiaticus (CLas), a
phloem-limited bacterium associated with HLB. However, the
adult is responsible for spreading CLas through its movement,
whereas the primary role of the nymphs is acquisition of the
bacterium. Therefore, it is important to control
both stages of ACP to reduce the spread of CLas
among habitats including organic citrus.
ACP and HLB are established in Florida and
spreading in California, Texas and other regions.
Monitoring of ACP, beneficial insects, insecticides,
plant nutrition and removal of disease-infected
trees are tools currently used to manage this
vector-disease complex. Beneficial insects such as
predators and parasitoids mainly attack eggs and
nymphs of ACP. Predatory insects generally are
larger than their prey, which they kill or consume.
Predators are more or less generalists, meaning
they consume more than one type of prey and are
thus useful against multiple pests. Insect predators
common in citrus include ladybeetles, lacewings,
spiders and ants. In addition to ACP, they also
attack other pests such as citrus leafminer (CLM),
thrips and aphids, thus playing an important role
in overall citrus pest management.
The small parasitic wasp, Tamarixia radiata , only attacks ACP

and can contribute to its 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
Photo 2: A female Tamarixia radiata laying egg on an ACP nymph. (Photo by J. Lotz).
83
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.
and Texas to control ACP. Reduction in populations of ACP

by predators and T. radiata can play an important role in its
management and ultimately that of HLB, as well. These beneficial
insects are present in the environment at some level and capable
of moving among habitats.
84
ACP adults are primarily controlled through foliar sprays, often

of broad-spectrum insecticides. ACP is attracted to young shoots
or flush for development and reproduction. Therefore, it is
especially important to target adults before trees begin to flush.
Predators and parasitic wasps are also attracted to these shoots
where they are searching for prey such as immature ACP, as well
as citrus leafminer (CLM) and aphids. Therefore, applying broadspectrum insecticides prior to flush reaps the maximum benefit
in suppressing adult ACP while conserving key beneficial insects.
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. 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. Generally, broadspectrum pyrethroids and organophosphates are preferred in
winter followed by selective insecticide chemistries, microbials
and horticultural spray oil during the growing season. Such
programs to manage ACP in conventional citrus are now common
in Florida and are being adopted in California and Texas.
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 areawide management by reducing the spread to conventional citrus
and other habitats. Organic products also will be suitable for
conventional citrus growers as selective options to avoid excessive
use of non-selective insecticides, to limit pesticide resistance and
harm to beneficial insects. 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.
RESEARCH OBJECTIVES
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.
Evaluate rotations of organic products potentially effective

against ACP for impact on ACP and
its natural enemies during the growing
season.
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).
Treatments included:
Organic program 1: Nine treatments using seven insecticides
(Pyganic, Aza-Direct, Grandevo, Azera, Venerate, Entrust and
Surround);
Organic program 2: Nine treatments using four insecticides and
horticultural mineral oil (Pyganic, Aza-Direct, Azera, Entrust and
435 oil);
Organic program 3: Nine treatments 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),
Horticultural mineral oil (HMO) FL 435-66, a narrow-range
petroleum-based oil, is standard in Florida. M-pede is insecticidal
soap that contains potassium salts of fatty acids. Both are
commonly used adjuvants applied with insecticides to enhance
their effect and also provide significant reduction in ACP when
applied alone. Their use is approved for organic citrus. They were
used at two percent of the total application volume when applied
with an insecticide or alone. Recommended rates of insecticides
mixed in 100 gallons of water per acre were sprayed by ground
using a Durand Wayland AF100-32 air blast speed sprayer (Table
1, Photo 3).
Pyrethroids are typically used as (winter) dormant sprays because
of their broad spectrum activity and sensitivity to heat. Pyganic
contains natural pyrethrins, which also are broad spectrum and
break down quickly in sunlight and are, therefore, more suited
for use in winter. Pyganic was applied in November 2014,
December 2014 and January 2015 for dormant season control
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
Photo 3. Durand Wayland AF100-32 air blast speed sprayer (Photo by J. Qureshi).
85
treatments in the dormant season using

methods described for the Valencia
block.
Photo 4. Demonstration of the stem tap sampling method and resulting adult psyllids (Photo by P.
Stansly).
Organic insecticides used during the

growing season included Aza-Direct,
Grandevo, Azera, Venerate, Entrust
and Surround. Aza-Direct is a neembased product that acts as a feeding
deterrent, repellent and insect growth
regulator.
Grandevo and Venerate
are microbial insecticides obtained
from the bacterium Chromobacterium
subtsugae and Burkholderia species,
respectively. Both contain multiple
compounds to create a complex mode
of action. Azera contains two active
ingredients, azadirachtin and pyrethrins,
to enhance its effectiveness. Entrust, an
insecticide that contains spinosad (a
naturally occurring compound found in
the bacterial species Saccharopolyspora
spinosa), is expected to act by contact
and ingestion. Surround is derived
from kaolin clay, a natural mineral that
creates a physical barrier on the foliage
to deter insects. Closer (sulfoxaflor)
was used in March in the conventional
program because it is recommended for
use during bloom when bees and other
beneficial insects are common.
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
2014June 2015.
of ACP in all three organic programs, either alone (Program

1) or with 435 oil (Program 2) or M-pede (Program 3). Danitol
is a synthetic pyrethroid extensively used for ACP control in
conventional citrus. Danitol, intended for January application
in conventional program also was used in November to
reduce the spread of ACP to other programs, considering that
populations at this time were high this year compared to the
previous year.
ACP adult and predator populations were

monitored using the stem tap sampling
method. Samples were collected onto
sheets of 8.5 x 11-inch laminated white paper held on a
clipboard. The clipboard was held horizontally under randomly
selected branches at three to six feet above the ground in the
outer tree canopy. The branches were struck sharply three
times with a short length of PVC pipe (Photo 4). The number of
individual insects that fell onto the paper sheet were identified
and counted.
During the 2014-15 growing season, due to low ACP

populations in the Valencia block, we added another replicated
experiment in a block of younger Hamlin orange trees with
higher psyllid populations to evaluate the organic plus 435 oil
and conventional programs compared to an untreated check.
This block was used again this year to compare these three
This method provides rapid and reproducible information on

psyllid and other pests and beneficial insects important in
making management decisions. Four tap samples were taken
on each of three trees at three randomly selected locations
for a total of nine trees per plot; for a total of 36 trees and 144
samples per treatment. We used a threshold of 0.1 adults per
Photo 5. Suction sampling (Photo by J. Qureshi).
86
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.
tap sample (10 adults in 100 tap samples) to trigger a spray

during the growing season, considering the high incidence of
HLB in Florida. Obviously, no ACP is always the best scenario,
but it is impossible to achieve where ACP is well established.
We also took suction samples using a leaf blower operating
in reverse (Photo 5), which collects more psyllids at low
density, as well as active and other predators such as green
lacewings lady beetles, spiders and ants. Pseudomyrmex ants
are common in Florida. Small colonies nest in twigs, but ants
forage individually, are highly predaceous and do not tend
other insects such as aphids.
Flush density was estimated by placing a one-foot-square
quadrat frame made from PVC pipe at randomly chosen
locations in the outer tree canopy and counting the number
of shoots at the feather stage to recently expanded leaves.
Depending upon availability, 15 randomly selected shoots
were collected from each plot and examined in the laboratory
using a stereoscopic microscope to determine the percentage
of shoots infested with ACP nymphs. Depending upon
availability, shoots containing three to five instar nymphs
were collected in June, July, August, September and October
2014 and in March and June 2015 and brought back to the
laboratory. 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

Adults averaged 0.2 or more per tap sample in the Valencia
block before the start of the first dormant application. After
the first dormant application made on November 11, adults
remained significantly fewer in all treatments compared
to the untreated control through 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 psyllid suppression persisted after the
second application in organic programs, and the impact was
enhanced with Pyganic plus M-pede treatment. 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 from samples taken for one month after
application. Significant effects of treatments in all programs
were observed through the first week of March when ACP
adult numbers were exceeding 0.1 per tap sample in organic
programs; therefore, additional treatment evaluations were
initiated (Figure 1).
ACP adults averaged less than 0.2 per tap sample before
dormant sprays in the Hamlin block. Only Pyganic plus 435
oil and Danitol were evaluated. Treatment effects were more
apparent after the application on January 12. Significant
reduction with Pyganic plus 435 oil lasted through February
24 and with Danitol through March 3, averaging 76 percent
and 85 percent, respectively (Figure 1).
ACP control in growing season
All programs initiated for winter control of ACP continued
into the growing season. Between March 10 and July 7, 2015,
six treatments were applied in organic programs and five in
the conventional programs. All six applications in Program 1
were organic insecticides. Programs 2 and 3 received three
applications of organic insecticides each and three of 435 oil
or M-pede, respectively.
87
Mean no. per suc-on sample
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.
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 all applied alone on May 8, respectively;
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.
Applications made in July are being evaluated.
Biological Control
Green lacewings were the most abundant predator in all
treatments (Photo 6). They attack ACP, CLM, aphids, thrips,
whiteflies and several other pests. Ladybeetles known for
significant predation on ACP were rare (Figure 2). Cycloneda
sanguinea (Photo 7) and Olla v-nigrum (Photo 8) were the
species most commonly observed. Ladybeetle numbers in
Florida have dropped significantly due to the increase in the
use of insecticidal sprays after the advent of HLB.
88
Mostly adults (rather than larvae) of lacewings and ladybeetles

were observed. Adults can fly between habitats and thus
may have moved back into treated plots following sprays.
Spiders and ants also were present in all treatments. Certain
species of both spiders and ants are reported as predators
of ACP, although their roles in suppression of the pest are
as yet unclear. Colonies of ACP nymphs are tended by some
ants, where they are attracted to honeydew produced by the
nymphs. On the other hand, Pseudomyrmex ants forage for
prey individually, do not tend honeydew producers and have
been observed feeding on ACP nymphs. Argentine ants tend
and protect honeydew-producing insects and also have been
observed feeding on ACP nymphs, without discriminating
between those parasitized or not by T. radiata. Thus while ants
do prey on ACP, there is potential for interference between
them and T. radiata.
From June to October 2014, average parasitism 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 Program 1, Organic Program 2, Organic Program 3 (at
that time with vegetable oil Citru-Soy) and the Conventional
Program, respectively. While some individual samples showed
100 percent, 40 percent, 58 percent and 10 percent parasitism
in the Organic Programs 1, 2 and 3, and in the conventional
program, respectively; nymphs were most easily available
from untreated plots, and parasitism rates there were more
consistent compared to treated plots.
In March 2015, parasitism rates averaged 31 6 percent in the
untreated control, 40 10 percent in the Organic Program 1,
23 8 percent in Organic Program 2 and 10 10 percent in
Organic Program 3. Plots designated for conventional control
had fewer nymphs and none were parasitized. Parasitism in
June averaged 17 6 percent in the untreated control, 7
4 percent in Organic Program 1, 14 7 percent in Organic
Program 2 and 12 5 percent in Organic

Program 3. Nymphs were available in
the conventional program but only 3
2 percent were parasitized. These
findings suggest that T. radiata was able to
contribute to ACP control, particularly in
organic programs.
Acknowledgements
We would like to thank the Citrus Research

Board for funding this research.
CRB Research Project #5500-189E
Photo 6. Green lacewing predator of ACP and other pests (Photo by the University of Florida)
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 at
Immokalee, Florida.
Photo 7. Adults of Cycloneda sanguinea feeding on ACP nymphs (Photo by J. Qureshi)
Photo 8. Larva and adult of Olla v-nigrum feeding on ACP nymphs (Photo by P. Stansly)
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MEET CRBS NEW

Metarex 4%* Snail and Slug Bait

Snail damage to orange

Outlasts and outperforms.

The Power is in the Pellet!

Citrograph Vol. 7, No. 1 | Winter 2016

Residual control that goes the distance.

Cleaner. Longer. Alion.

Citrus Research Board

Editorial inquiries - call, email

Reproduction or reuse of any photos and/

WINTER 2016 | VOLUME 7 NUMBER 1

12 BOARD SELECTS NEW EXECUTIVE LEADERSHIP TEAM

14 INDUSTRY VIEWS WHAT PROGRESS ARE WE MAKING

18 WHERE 2016 CRB-FUNDED RESEARCH IS HEADED

MOJTABA MOHAMMADI, PH.D.

22 MEET YOUR BOARD MEMBERS

24 HLB DETECTIONS IN SAN GABRIEL

30 HLB IMPACT ON CITRUS ROOT HEALTH AND INTERACTION

34 MONITORING FOR ACP RESISTANCE TO PESTICIDES

40 MATURE CITRUS PROPAGATION IN RITA BIOREACTORS

46 NOT ALL PSYLLIDS ARE CREATED EQUAL

52 COMPLEX CITRUS LURES TO TRAP AND CONTROL ACP

This autumn, Richard Bennett (left) was

56 FOUNDER LINES FOR IMPROVED CITRUS BIOTECHNOLOGY

60 CITRUS DISEASE RESPONSE TO HLB INFECTION

64 DEVELOPMENT OF LOW-SEEDED CITRUS BY MUTATION

72 NOVEL THERAPY OF HIGH-PRIORITY CITRUS DISEASES

76 FIGHTING HLB WITH A CITRUS TRISTEZA VIRUS-BASED

82 DEVELOPMENT OF AN ACP MANAGEMENT PLAN FOR

www.CitrusResearch.org | Citrograph Magazine

THE MISSION OF THE CITRUS RESEARCH BOARD:

CITRUS RESEARCH BOARD MEMBER LIST

District 2 Southern California Coastal

District 3 California Desert

Citrograph Vol. 7, No. 1 | Winter 2016

www.CitrusResearch.org | Citrograph Magazine

The huanglongbing (HLB) threat to the California citrus

Citrograph Vol. 7, No. 1 | Winter 2016

Trees with dropped fruit, showing symptoms of HLB.

EXISTING THREE-PRONGED STRATEGY

The current three-pronged strategy to fight HLB (a) keeping

REALIZING THE THREAT

The most current research indicates that the use of

states growers could total $750-1,100 per acre per year, or

predictive processes and organizational capabilities to

FOUR KEY COMPONENTS EMERGED AS A RESULT OF THE

On December 1, the industry brought key forces together

THE HLB SUMMIT HAD THREE MAIN OBJECTIVES:

Raise key stakeholder awareness of the urgency, scope

Citrograph Vol. 7, No. 1 | Winter 2016

Institutionalized Process a transparent and highly

Our whole industry needs to unite to develop this working

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BOARD SELECTS NEW

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lead strategic planning in order to solve industry issues, Gary

Dan, a third generation California family farmer, is the Northern

For more information: email jose.lima@wonderful.com or call 559.707.1387

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WHAT PROGRESS ARE

Citrograph Vol. 7, No. 1 | Winter 2016

Greg McCollum, Ph.D.

The confirmation of Candidatus