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Citrograph Vol. 7, No. 1 | Winter 2016

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Citrus Research Board

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Visalia, CA 93279
P: (559) 738-0246
F: (559) 738-0607
Ed Civerolo, Ph.D., Interim Executive Editor
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Vol. 7,
1 expressed
| Winter
written consent of the publisher.

























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.








MIKEAL ROOSE, PH.D., ET AL. | Citrograph Magazine




District 1 Northern California
Member Expires
Toby Maitland-Lewis 2016
Jack Williams
Donald Roark
Dan Dreyer
Jim Gorden
Greg Galloway
Joe Stewart
Franco Bernardi

Member Expires
Kevin Olsen
Etienne Rabe
John Konda
Keith Watkins
Jeff Steen
Richard Bennett
Justin Brown

District 2 Southern California Coastal

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

Member Expires Member Expires
Mark McBroom
2016 Craig Armstrong

Public Member
Member Expires

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
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,
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 |

Citrograph Vol. 7, No. 1 | Winter 2016 | Citrograph Magazine



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

Citrograph Vol. 7, No. 1 | Winter 2016

Richard Bennett

Trees with dropped fruit, showing symptoms of HLB.


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,
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


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 | Citrograph Magazine

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.






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
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




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

Citrograph Vol. 7, No. 1 | Winter 2016


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. | Citrograph Magazine


From left to right, CRB Secretary/Treasurer Toby Maitland-Lewis, Chairman Richard Bennett, President Gary Schulz and Vice-Chairman Dan Dreyer.


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


Citrograph Vol. 7, No. 1 | Winter 2016

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

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11/18/15 4:30 PM | Citrograph Magazine




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)?


Citrograph Vol. 7, No. 1 | Winter 2016

Greg McCollum, Ph.D.

Research Plant Physiologist,
USDA-ARS, Fort Pierce,

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

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.


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.


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.


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.


Breeding and transgenic (introduction of DNA from another

source) approaches really are going to be the ultimate | Citrograph Magazine


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.


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.


Citrograph Vol. 7, No. 1 | Winter 2016


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.


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,

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.


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.


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.


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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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


Citrograph Vol. 7, No. 1 | Winter 2016

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. | Citrograph Magazine



Citrograph Vol. 7, No. 1 | Winter 2016







New Proposals
Real Time PCR co-detecHon of Candidatus Liberibacter species and Spiroplasma citri
Citrus dwarng of commercial varieHes using TsnRNAs
Citrus rhizobiomes and tree producHvity in response to soil manipulaHons

Con0nuing Projects
The development of novel Blood and Cara cara like citrus varieHes
UHlizaHon of founder lines for improved citrus biotechnology via RMCE
Development of consumer-friendly transgenic citrus plants with potenHal broad spectrum resistance
Rapid cycling plant breeding in citrus
EvaluaHon of hybrids of citrus and citrus relaHves for huanglongbing (HLB) tolerance/resistance
MicropropagaHon of mature citrus in temporary immersion bioreactors
Streamlining the introducHon of licensed citrus varieHes into California: A case study - Florida
CORE: integrated citrus breeding and evaluaHon for California

New Proposals
OpHmizing sensory quality and consumer acceptance of citrus fruit through horHcultural pracHces

Con0nuing Projects
IdenHcaHon and characterizaHon of HLB-induced small RNAs and mRNAs
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
Using mass spectrometry technologies to develop novel management strategies for citrus insect vector-borne pathogens
The citrus greening bibliographical database
ConstrucHon of the cloned infecHous cDNA of Citrus tristeza virus (California isolate)
IdenHfying and characterizing citrus targets from Candidatus Liberibacter asiaHcus
Infrastructure support for research on detecHon and management of HLB and ACP
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
ArHcial microRNA-based targeHng of the Asian citrus psyllid for HLB management
Develop a novel target-basis of anH-virulence strategy for controlling HLB
Photosynthate-responsive polymeric nano-carriers for phloem-specic delivery in the treatment of HLB

5300 - Vectored Diseases

5200 - New VarieGes



Con0nuing Projects
Novel therapy of high-priority citrus diseases
OpHmizaHon of water and nitrate applicaHon eciency for citrus trees
IdenHcaHon of key components in HLB using eectors as probes (co-sponsor with CRDF)

5100 - ProducGon Eciency


Table 1: 2015-2016 Research Projects

Table 1: 2015-2016 Research Projects

UC Davis
University of Florida

UC Riverside
UC Davis
Boyce Thompson InsHtute
University of Florida
UC Riverside
UC Davis
UC Davis
Boyce Thompson InsHtute
UC Riverside & UC Davis

UC Davis

Texas A&M
University of Florida
UC Riverside
University of Florida
UC Riverside
UC Riverside

UC Riverside
UC Riverside
UC Davis

Los Alamos
UC Davis
UC Riverside


$ 85,000

$ 72,424

$ 117,128

$ 146,406

$ 116,437

$ 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

BUDGET | Citrograph Magazine




Con0nuing Projects
EvaluaHon of new post-harvest treatments to reduce post-harvest decays in packinghouse operaHons
Disease forecasHng and management of Septoria spot of citrus
Epidemiology and management of Phytophthora diseases of citrus in California
Breaking criHcal pest-related trade barriers for California citrus exports
Control of post-harvest diseases of citrus



Stouthamer & Hoddle

New Proposals
Eects of ACP cover sprays against fruit ies (TephriHdae) and their natural enemies

Biological control of Asian citrus psyllid in California (CPDPP)
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.


Con0nuing Projects
OpHmizing chemical control of Asian citrus psyllid in California
5500-189E Development of an Asian citrus psyllid (ACP) management plan for organic citrus
Host specicity tesHng of Diaphorencyrtus aligarhensis
Release and monitoring of Tamarixia radiata and phenology of Asian citrus psyllid in Southern California
Impact of resident predator species on control of Asian citrus psyllid populaHons
Toxicity of syntheHc and organic insecHcides to Tamarixia radiata, ecto-parasitoid of Asian citrus psyllid
Development of new trapping and control methods for ACP based on complex citrus lure blends
CORE: IPM program

5500 - Pest Management

Breaking criHcal pest-related trade barriers for California citrus exports (TASC)

5400 - Non-Vectored and Post-harvest Diseases


Risk-based decision making in the management of huanglongbing (CPDPP)
DetecHon of Candidatus Liberibacter in citrus in Hacienda Heights and other areas of California (CPDPP)
Using the internet to train citrus hobbyists to order budwood from CCPP and not to spread HLB (CPDPP)



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
ArHcial microRNA-based targeHng of the Asian citrus psyllid for HLB management
Develop a novel target-basis of anH-virulence strategy for controlling HLB
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
Eect of mixed infecHons of plant pathogens on detecHon of HLB using two early detecHon methods
Establish a system to infect and maintain Nico:ana benthamiana and citrus with the recombinant CTV
EDT experiment
Flush & Nypmh experiment


UC Riverside
UC Riverside


UC Riverside
University of Florida
UC Riverside
UC Riverside
UC Riverside
University of Florida
University of Florida
UC Riverside


UC Riverside
UC Riverside
UC Riverside

UC Davis

UC Davis
University of Florida
UC Davis

$ 181,745

$ 250,000

$ 248,404

$ 30,000

$ 116,660

$ 66,565

$ 206,407

$ 231,358

$ 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

Boyce Thompson InsHtute

UC Riverside & UC Davis


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
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.


Citrograph Vol. 7, No. 1 | Winter 2016

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.


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
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.


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Where are we now?

Victoria Hornbaker and Lucita Kumagai


Citrograph Vol. 7, No. 1 | Winter 2016

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


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
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. | Citrograph Magazine


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
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,


Citrograph Vol. 7, No. 1 | Winter 2016

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.



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., 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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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

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

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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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 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.


Citrograph Vol. 7, No. 1 | Winter 2016 | Citrograph Magazine


Figure 1A


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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
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 (
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. | Citrograph Magazine


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
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


Citrograph Vol. 7, No. 1 | Winter 2016

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. 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
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.


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.

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
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. | Citrograph Magazine



Figure 1. Adult ACP on a curry plant in a cage

provided by the UCR Tamarixia radiata program.


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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. | Citrograph Magazine


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


Citrograph Vol. 7, No. 1 | Winter 2016

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 | Citrograph Magazine


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.


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


Citrograph Vol. 7, No. 1 | Winter 2016

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


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

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
LD95 to
field use

mg AI/litere








4.3 oz/a



7 fl oz/a



5.5 oz/a



5.75 fl oz/a





6 oz/a



4.25 fl oz/a



27 fl oz/a



4.5 fl oz/a



ACP field
use rate

statistic Slope SE



6.0917 0.4096

32.2114 a 30.4832-33.9718

59.9828 a

55.2013-66.4935 1.25 lb/a


4.9094 0.3857

34.7369 a 32.2420-37.4984

75.1332 a



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


5.0234 0.2744

15.0516 b 14.2124-15.9941

31.9907 a




2.1990 0.2313

4.1845 a


23.4234 a

15.1553-44.1153 16 fl oz/a



4.5397 0.4456

7.6217 b


17.5543 a


2-27-13; 3-13-13



1.8723 0.2000

0.5044 a


3.8133 a


8-12-14; 8-28-14



2.8376 0.2071

1.0716 b


4.0709 a


8-15-12; 9-26-12;



2.5470 0.2165

0.6181 a


2.7342 b




5.6308 0.6922

0.7270 a


1.4244 a


Actara 25% WDG 1-16-13; 1-30-13

thiamethoxam, Round 2
Class 4C (nicotinic acetylcholine receptor agonists)
9-18-13; 10-9-13;
Sequoia 2 EC
sulfoxaflor, Round 2
Class 4D (butenolide)
Sivanto 1.67 EC
8-7-14; 8-14-14; 8782
Class 5 (spinosyns)
9-11-13; 9-24-13; 1372
Delegate 25% WG
4-18-14; 5-7-14
spinetoram, Round 2


4.6172 0.4255

0.4234 a


0.9616 a



5.9113 0.6075

0.3382 a


0.7248 a



4.1400 0.2826

3.1617 a


7.8919 a



5.8523 0.6248

3.4192 a


6.5312 a



2.8398 0.1763




49.4197-69.5275 14 fl oz/a


3.5879 0.3157

0.8561 a


2.4603 a



3.7880 0.2726

0.9720 a


2.6416 a



2.7986 0.2262

0.6157 a


2.3830 a



4.1526 0.6214

1.4750 b


3.6720 a



4.0289 0.3676

2.6759 a


6.8508 a



5.4498 0.4475

2.5559 a


5.1211 a



2.3040 0.2815

0.3602 a


1.8642 a



2.8436 0.3081

0.3294 a


1.2478 a





Class 1A (carbamates)

Bioassay test dates N

5-8-13; 6-5-13; 7-5570

4-8-14; 5-13-14
formetanate, Round 2
Class 1B (organophosphates)
Lorsban Advanced 10-2-13; 11-13-13;
3.755 EC

Carzol 92 SP

chlorpyrifos, Round 2
Class 3A (pyrethroids)
Danitol 2.4 EC
fenpropathrin, Round 2
zeta-cypermethrin Mustang 1.4 EC
zeta-cypermethrin, Round 2
Class 4A (Neonicotinoids)

Admire Pro 4.6 EC

imidacloprid, Round 2

10-24-12; 1-9-13


Class 6 (avermectins, milbemycins)

Agri-Mek 0.7 SC
4-3-13; 4-7-13
abamectin, Round 2
Class 21 (mitochondrial complex I electron transport inhibitors)
Bexar 1.31 EC
7-2-12; 8-1-12
7-31-13; 7-30-14; 8414
tolfenpyrad, Round 2
Class 28 (ryanodine receptor modulators)
Exirel 0.83 EC
5-22-13; 8-14-13
cyantraniliprole, Round 2

(ng AI/

(ng AI/
95% Ci


95% Ci

Insecticide Resistance Action Committee class (; 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.


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:
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:

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. | Citrograph Magazine



An experiment with 18 RITA bioreactors to propagate mature Valencia and Carrizo plants.


Yosvanis Acanda and Janice Zale


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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. | Citrograph Magazine


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


Citrograph Vol. 7, No. 1 | Winter 2016 | Citrograph Magazine


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

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.

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Citrograph Vol. 7, No. 1 | Winter 2016 | Citrograph Magazine



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.



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


Citrograph Vol. 7, No. 1 | Winter 2016


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.


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. | Citrograph Magazine


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
own chances
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.


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





There also could be genetic variability in vector efficiency

among geographically isolated ACP populations.
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.


Citrograph Vol. 7, No. 1 | Winter 2016

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. | Citrograph Magazine



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:


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.


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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.
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. 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. | Citrograph Magazine




Unbaited yellow traps are widely used in California.

Xavier Martini, Alexander A. Aksenov, Cristina E. Davis and Lukasz L. Stelinski


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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 | Citrograph Magazine


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


Citrograph Vol. 7, No. 1 | Winter 2016


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):
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. | Citrograph Magazine





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


Citrograph Vol. 7, No. 1 | Winter 2016

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
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
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 | Citrograph Magazine


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
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.


Citrograph Vol. 7, No. 1 | Winter 2016

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.

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


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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.


Jessica Franco, Deborah Pagliaccia, Wenbo Ma and Gitta Coaker


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.


Citrograph Vol. 7, No. 1 | Winter 2016

PCR + Navel Trees


Number of PCR + Trees


Feeder Root
Young Leaves

Mature Leaves


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.


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
navel orange trees grafted on Carrizo
In recent years, the citrus industry has been plagued by
or mock-inoculated. Samples
HLB, a devastating citrus disease. In the United States, HLBfrom
and feeder roots were
associated CLas is spread by the Asian citrus psyllid (ACP), a
(Figure 1). DNA
piercing sucking insect, which carries the HLB-associated
of CLas by
bacterium, CLas. ACP feeds on the phloem of citrus, which is
is more
also where CLas resides. Infected trees are characterized by
hard, misshapen, bitter fruit with yellowing of their shoots and
leaves. Trees do not present symptoms until up to two years
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.


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. | Citrograph Magazine


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



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
highly expressed during infection, they hold
promise as a biomarker to facilitate HLB

Selected Literature:


1. Wang N, Trivedi P. 2013. Citrus

huanglongbing: a newly relevant disease
presents unprecedented challenges.


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


and also can be targeted by pathogen

effectors to facilitate infection.

Citrograph Vol. 7, No. 1 | Winter 2016

2. Van der Hoorn RA. 2008. Plant

proteases: from phenotypes to molecular
mechanisms. Annual Review of Plant Biology.
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 | Citrograph Magazine




What Growers Should Expect from Tango and Other Varieties

M. L. Roose, T. E. Williams and C. T. Federici


Citrograph Vol. 7, No. 1 | Winter 2016

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.


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. | Citrograph Magazine


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.


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


Citrograph Vol. 7, No. 1 | Winter 2016


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
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.


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 | Citrograph Magazine


W. Murcott, satsuma and navel orange.

Cross-pollination pressure is expected to
be low.


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.


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.


Citrograph Vol. 7, No. 1 | Winter 2016

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

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
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
origin of tree 9-29 at the molecular level
We saw similar variation among years and locations in the
Tango and W. Murcott, but do
number of seeds per fruit in the fruit quality samples that were
This tree does, however,
cut in the lab (Figure 7). Since seed content often differs in
than 1,000 Tango trees
successive years while the surrounding pollen source trees
characteristics. From
remain unchanged, much of the variation is likely attributable
factors can
to weather during flowering, the duration of flowering and
a very rare
the prevalence of bees. As expected, sites surrounded by
trees that produce little viable pollen, such as Orosi and UCR
10K, tend to have lower seed content than sites with more | Citrograph Magazine



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.


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.


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Citrograph Vol. 7, No. 1 | Winter 2016 | Citrograph Magazine



Citrograph Vol. 7, No. 1 | Winter 2016

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