crb citrograph mag q1 2016 final web

www.CitrusResearch.org | Citrograph Magazine 1

MEET CRB’S NEWLEADERSHIP

TEAM

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

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8 CHAIRMAN’S VIEW RICHARD BENNETT

12 BOARD SELECTS NEW EXECUTIVE LEADERSHIP TEAM RICHARD BENNETT

14 INDUSTRY VIEWS – “WHAT PROGRESS ARE WE MAKING ON HLB?” MOJTABA MOHAMMADI, PH.D.

18 WHERE 2016 CRB-FUNDED RESEARCH IS HEADEDMOJTABA MOHAMMADI, PH.D.

22 MEET YOUR BOARD MEMBERS IVY LEVENTHAL

24 HLB DETECTIONS IN SAN GABRIEL VICTORIA HORNBAKER AND LUCITA KUMAGAI

30 HLB IMPACT ON CITRUS ROOT HEALTH AND INTERACTION WITH PHYTOPHTHORA JAMES GRAHAM, PH.D.

34 MONITORING FOR ACP RESISTANCE TO PESTICIDES JOSEPH MORSE, PH.D, ET AL.

40 MATURE CITRUS PROPAGATION IN RITA® BIOREACTORS YOSVANIS ACANDA AND JANICE ZALE, PH.D.

46 NOT ALL PSYLLIDS ARE CREATED EQUAL MICHELLE CILIA, PH.D., ET AL.

52 COMPLEX CITRUS LURES TO TRAP AND CONTROL ACP XAVIER MARTINI, PH.D., ET AL.

56 FOUNDER LINES FOR IMPROVED CITRUS BIOTECHNOLOGY MARIA OLIVEIRA, PH.D., ET AL.

60 CITRUS DISEASE RESPONSE TO HLB INFECTION JESSICA FRANCO, ET AL.

64 DEVELOPMENT OF LOW-SEEDED CITRUS BY MUTATION BREEDING MIKEAL ROOSE, PH.D., ET AL.

IN THIS ISSUEWINTER 2016 | VOLUME 7 • NUMBER 1 THE OFFICIAL PUBLICATION OF THE CITRUS RESEARCH BOARD

40

On The Cover:This autumn, Richard Bennett (left) was elected chairman of the Citrus Research Board (CRB), and Gary Schulz was hired as the CRB’s 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 “Chairman’s View” on page 8.

MEET CRB’S NEW

LEADERSHIPTEAM


CALENDAR OFEVENTS 2016

CITRUS RESEARCH BOARD MEMBER LIST BY DISTRICT 2015-2016 (TERMS EXPIRE JULY 31)

THE MISSION OF THE CITRUS RESEARCH BOARD:ENSURE A SUSTAINABLE CALIFORNIA CITRUS INDUSTRY FOR THE BENEFIT OF GROWERS BY PRIORITIZING, INVESTING IN AND PROMOTING SOUND SCIENCE.

District 1 – Northern California

District 2 – Southern California – Coastal

Member ExpiresToby Maitland-Lewis 2016Jack Williams 2016Donald Roark 2016Dan Dreyer 2016Jim Gorden 2017Greg Galloway 2017Joe Stewart 2017Franco Bernardi 2017

Member ExpiresKevin Olsen 2017Etienne Rabe 2018John Konda 2018Keith Watkins 2018Jeff Steen 2018Richard Bennett 2018Justin Brown 2018

Member ExpiresJohn Gless III 2017Mike Perricone 2017

Member ExpiresAlan Washburn 2018

Member ExpiresMark McBroom 2016

Member ExpiresCraig Armstrong 2016

District 3 – California Desert

Public MemberMember ExpiresVacant 2018

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 [email protected] | www.citrusresearch.org

January 13CPDPP Board Meeting, Visalia/Exeter, California. For more information, contact CDFA at (916) 403-6652.

January 27Annual UC Riverside Citrus Day, Riverside, California. Up-to-date research information, field tours and variety tasting.

January 28CRB Board Meeting, Riverside, California. For more information, contact the CRB at (559) 738-0246.

February 9-11World Ag Expo, International Agri-Center, Tulare, California. For more information, visit www.worldagexpo.com.

March 3California Citrus Mutual Citrus Showcase, Visalia Convention Center, Visalia, California. For more information, contact California Citrus Mutual at (559) 592-3790.

March 9CPDPP Board Meeting, Riverside / San Bernardino, California. For more information, contact CDFA at (916) 403-6652.


OUR #1 GOAL: CONTROLLING HLBThe Ultimate and Only Objective

CHAIRMAN’S VIEW BY RICHARD BENNET T

Richard Bennett

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. That’s the bottom line. This disease is the ultimate threat to our survival.


EXISTING THREE-PRONGED STRATEGYThe 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. Florida’s 2015-16 orange crop is expected to be the smallest in half a century. The heavy use of insecticides to suppress the ACP vector populations is not fully effective, which means psyllids are feeding on an increasingly greater number of likely CLas-infected, yet asymptomatic, trees.

California has adopted this three-prong strategy and, like Florida, relies on regulatory agency data collection, analysis and diagnostic efforts. However, the effectiveness of these approaches is restricted by tight protocols and limited resources. Our industry also has invested heavily in scientific research to assist us in extending our existence long enough to find a solution. California needs to immediately support the high-throughput investment of early detection technology devices.

REALIZING THE THREATThe 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

OUR #1 GOAL: CONTROLLING HLBThe Ultimate and Only Objective

Trees with dropped fruit, showing symptoms of HLB.


state’s 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 California’s productive citrus capacity.

This devastating disease already has manifested itself in Southern California. Collectively, we must formulate an immediate strategy.

HLB SUMMITOn December 1, the industry brought key forces together for an annual HLB Summit here in California. It differed from the meeting held at the University of California-Davis this past autumn in that it added in the research and experiences of Florida to develop a comprehensive grower action plan. The morning session clearly demonstrated the urgent and immediate need for investment in Early Detection Technologies.

In September, the Citrus Research Board (CRB) developed a format to assemble leading scientists and industry leaders from Florida together with us to develop a blueprint to combat HLB right now. The best and most experienced minds currently battling this disease in Florida presented their thoughts on developing short-, medium- and long-term strategies to move forward. Scientists who can predict infection movement and detect the bacteria in a pre-symptomatic stage shared research findings.

This one-day session encouraged attendance from a representative cross-section of industry stakeholders (nurseries, growers, marketers and packers/shippers), as well as researchers at the forefront of observation/modeling, diagnosis and treatment of vector populations and the disease.

THE HLB SUMMIT HAD THREE MAIN OBJECTIVES: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

predictive processes and organizational capabilities to combat the disease along with broad, but achievable, strategic directives with two over-arching goals: a. Extend the economic life viability of existing trees in the ground. b. Shorten the time to development and commercialization of a cure and/or protectant.

FOUR KEY COMPONENTS EMERGED AS A RESULT OF THE SUMMIT:

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.

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This 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 Board’s annual meeting, new executive officers were elected.

We are pleased to introduce Gary Schulz as the CRB’s president. Gary has an impressive resumé 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 non-profit and quasi-governmental agricultural business organizations. Having demonstrated his ability to

BOARD SELECTS NEW EXECUTIVE LEADERSHIP TEAM

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

Richard Bennett


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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.

Gary’s 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 CRB’s fiduciary responsibility and promoting the long-term sustainability of California’s citrus industry.

I am the owner of Bennett Farms in Exeter and am a citrus industry veteran. Right now, I am committed to leading an effort dedicated to finding a solution for HLB (see accompanying article). I know I also speak for Gary, Dan and Toby in sharing with you that we look forward to working hard on your behalf by strategically focusing your funding on research that will enable your businesses to survive and thrive.

Richard Bennett is the chairman of the Citrus Research Board.


WHAT PROGRESS ARE WE MAKING ON HLB?

INDUSTRY VIEWS MOJTABA MOHAMMADI

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)?”


Greg McCollum, Ph.D.Research Plant Physiologist, USDA-ARS, Fort Pierce, Florida

The confirmation of ‘Candidatus Liberibacter asiaticus’ (CLas) in the state has been a real wake-up call. HLB has been found in two different locations in California. There is evidence that the problem is spreading, which means that action needs to be taken sooner rather than later.

HLB is an enemy, but once the enemy has been identified, you can begin the battle. A lot of novel, very interesting work is being done on detection technologies that are alternatives to PCR. They hold great promise if we can get closer to whole tree detection, rather than a very small fraction of tree detection. To prevent or slow the development of an HLB epidemic, it is essential to confirm infections as early as possible and take action immediately. Because trees can be infected with CLas for a lengthy time prior to the appearance of HLB symptoms, detection of the pathogen is challenging. Although PCR is very reliable in detecting CLas, if trees are not symptomatic, it is difficult to determine where to collect diagnostic samples. Early detection means that infections can be found prior to trees developing visible symptoms. That is crucial if HLB is to be controlled in California.

In Florida, the situation got out of control because symptoms were not apparent. As soon as a single tree was found and testing for CLas began, we found the pathogen was everywhere. California is ahead of the curve on that and hopefully can stay that way. I wish there was a significant breakthrough in therapeutics or control in general, but we are not there yet. Currently, the standard three-pronged approach (controlling psyllids, removing infected trees and only planting clean nursery stock) is still the most important management strategy.

DISEASE CONTROL STRATEGIESBreeding and transgenic (introduction of DNA from another source) approaches really are going to be the ultimate

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. We’ve had a breeding program at the ARS for more than 100 years, and new varieties have come out of that program. It is a very slow process that takes about 30 years from hybridization to a variety being released. A rootstock is a bit faster proposition. The advantage is that you maintain the same scion, but impart resistance through the rootstock, which would be a great benefit. Some rootstocks that have been evaluated in Florida appear to have a less rapid rate of decline than others, which holds some promise.

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

BIOLOGICAL CONTROLIn Florida, before HLB, pests were managed almost exclusively through biocontrol. Minimal use of insecticides and the interactions among various pests resulted in good biocontrol. If you do not have enough of the pest that you are trying to control, the population of biocontrol organisms will decline, so there is a cyclic nature. With residential trees, people will be much more amenable to releasing wasps in their backyard than commercial insecticide spraying.

FUNDING RESEARCH AREASNew 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.


Matt Daugherty, Ph.D.Extension Specialist, Department of Entomology, University of California, Riverside, California

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

There have been significant areas of progress on HLB in

recent years; one of which is developing a range of early detection technologies that we hope will soon be available for regulators and growers to begin folding into monitoring programs. Earlier detection of infected trees is critical to narrowing the potential for pathogen acquisition and spread during the asymptomatic phase.

Another area of progress is that we are getting a much better handle on how the vector and the HLB-associated Liberibacter spread in the landscape, what drives their movement and what aspects of local landscape and environmental conditions influence whether the psyllid is likely to be there. This information feeds into risk modeling that provides a better picture of where to look for not only the ACP, but also the disease. By refining how we identify early cases of disease, we can hopefully mitigate some impacts. That process has been going on for years, but it is becoming more defined.

PUBLIC AWARENESSAs 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. We’re 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.

BIOLOGICAL CONTROLDisease 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. We’re learning more about how to increase its chances of successfully slowing disease spread. This will likely involve coordinating with homeowner education programs, emphasizing the need to manage a variety of species, including Argentine ants, to maximize the effectiveness of biocontrol agents.

FLORIDA EXPERIENCEWe’ve 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 they’ve proven burdensome for certain people and industries) are at least part of the reason that the pace of the ACP and HLB situation in California has to date been very different from Florida.

Manjunath Keremane, Ph.D.Research Plant Pathologist, USDA-ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, California

The California citrus industry is concerned about recent finds of HLB-positive citrus trees reported in some residential areas of Los Angeles County. At this time, containment and eradication are our main goals in Southern California. Early detection of ‘Candidatus Liberibacter asiaticus’ (CLas) in psyllids and affected trees, in addition to effective

eradication of compromised citrus trees, can help prevent this devastating disease from spreading in California.


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

DISEASE CONTROL STRATEGIESA long-term solution to control HLB is to develop cultivars with resistance to the disease. Currently, two approaches are being pursued – transgenic and conventional breeding. We have chosen a non-transgenic approach because of the ease associated with field-testing and the release of promising disease-resistant cultivars to the citrus community. Approval by the FDA and EPA will not be needed since the hybrids are a result of natural breeding. Even though the approach is time consuming initially, the advantages associated with conventional breeding are appealing.

FLORIDA EXPERIENCEThe lessons learned from Florida’s 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 Florida’s 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.


Funding 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

greening) is considered the number one enemy, followed by those caused by viruses, pre- and post-harvest 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,

WHERE 2016 CRB-FUNDED RESEARCH IS HEADEDMojtaba Mohammadi


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 pre-symptomatic 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 real-time 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.


Tab

le 1

: 201

5-20

16 R

esea

rch

Pro

ject

s

Printed: 11/24

/15 2:00

PM

Table 1: 2015-‐2016 Research Projects

NUMBER

TITLE

PRINCIPAL

INVESTIGATOR

AFFILIATION

BOARD

APP

ROVED

BUDGET

5100 -‐ Prod

ucGon

Efficiency

Con0

nuing Projects

5100

-‐146

Novel the

rapy of h

igh-‐priority citrus diseases

Gup

taLos Alamos

67,500

$

5100

-‐150

OpH

mizaH

on of w

ater and

nitrate app

licaH

on efficien

cy fo

r citrus trees

Kand

elou

sUC Davis

108,05

9$

5100

-‐152

Iden

HficaHo

n of key com

pone

nts in HLB using

effectors as prob

es (co-‐sponsor with CR

DF)

Ma

UC Riverside

22,290

$

New

Propo

sals

5100

-‐153

Real Tim

e PC

R co-‐detecHo

n of Candidatus Libe

riba

cter spe

cies and

Spiroplasma citri

Pagliaccia

UC Riverside

21,000

$

5100

-‐154

Citrus dwarfin

g of com

mercial varieHe

s using TsnR

NAs

Vidalakis

UC Riverside

6,53

5$

5100

-‐155

Citrus rhizobiom

es and

tree prod

ucHv

ity in respo

nse to soil m

anipulaH

ons

Leveau

UC Davis

86,276

$

5200 -‐ New

VarieGes

Con0

nuing Projects

5200

-‐141

The de

velopm

ent of novel Blood

and

Cara cara like citrus varieH

esTh

omson

USD

A-‐ARS

129,20

2$

5200

-‐142

UHlizaH

on of fou

nder line

s for im

proved

citrus biotechn

olog

y via RM

CETh

omson

USD

A-‐ARS

115,10

6$

5200

-‐144

Develop

men

t of con

sumer-‐frien

dly tran

sgen

ic citrus plants with po

tenH

al broad

spe

ctrum resistance

Louzad

aTexas A&M

110,46

9$

5200

-‐146

Rapid cycling plant bree

ding

in citrus

Moo

reUniversity of Florida

103,71

2$

5200

-‐147

Evalua

Hon of hybrids of citrus an

d citrus relaH

ves for hu

anglon

gbing (HLB) toleran

ce/resistance

Ramad

ugu

UC Riverside

73,333

$

5200

-‐148

Microprop

agaH

on of m

ature citrus in te

mpo

rary im

mersion

bioreactors

Zale

University of Florida

22,872

$

5200

-‐149

Stream

lining the introd

ucHo

n of licensed

citrus varieH

es into Califo

rnia: A

case stud

y -‐ Florida

Vidalakis

UC Riverside

5,00

0$

5200

-‐201

CORE

: integrated citrus breed

ing an

d evalua

Hon for Ca

lifornia

Roose

UC Riverside

627,30

7$

New

Propo

sals

5200

-‐150

OpH

mizing sensory qu

ality an

d consum

er accep

tance of citrus fruit throug

h ho

rHcultural pracHces

Guina

rdUC Davis

101,11

4$

5300 -‐ Vectored Diseases

Con0

nuing Projects

5300

-‐131

Iden

HficaHo

n an

d characterizaHo

n of HLB-‐in

duced sm

all R

NAs an

d mRN

As

JinUC Riverside

146,40

6$

5300

-‐150

Biom

arkers fo

r de

tecHon

of Liberibacter infecHon

in citrus tree

s throug

h 1H

-‐NMR ba

sed metab

olom

ics

Slup

sky

UC Davis

116,43

7$

5300

-‐151

A pha

ge/proph

age-‐ba

sed PC

R system

for sensiHve and

spe

cific detecHo

n of “Ca

ndidatus Liberibacter asiaHcus” an

d Spiroplasm

a citri

Chen

USD

A-‐ARS

NCE

*53

00-‐155

Using

mass spectrom

etry te

chno

logies to

develop

novel m

anagem

ent strategies fo

r citrus insect vector-‐bo

rne pathogen

sCilia

Boyce Th

ompson

InsHtute

183,71

3$

5300

-‐156

The citrus green

ing bibliograp

hical datab

ase

Stan

sly


18,114

$

5300

-‐158

ConstrucHo

n of the

clone

d infecHou

s cD

NA of C

itrus tristeza virus (C

alifo

rnia isolate)

Ng

UC Riverside

94,365

$

5300

-‐160

Iden

Hfying

and

cha

racterizing citrus ta

rgets from

Candidatus Libe

riba

cter asiaH

cus

Coaker

UC Davis

106,97

4$

5300

-‐161

Infrastructure sup

port fo

r research on de

tecHon

and

man

agem

ent of HLB and

ACP

God

frey

UC Davis

100,86

4$

5300

-‐163

Not all psyllids are created eq

ual: Why do some tran

smit Liberibacter an

d othe

rs do no

t?Cilia

Boyce Th

ompson

InsHtute

146,27

5$

5300

-‐164

A m

icrobiota-‐ba

sed ap

proa

ch to

citrus tree

health

Rolsha

usen

/Leveau

UC Riverside & UC Davis

151,65

3$

5300

-‐165

Develop

men

t of m

ature bu

dwoo

d tran

sformaH

on te

chno

logy

Thom

son

USD

A

2,50

4$

5300

-‐168

Use of d

igita

l PCR

for im

proved

early detecHo


riba

cter asiaH

cus infecHon

in citrus an

d ACP

McCollum

USD

A-‐ARS

42,500

$

New

Propo

sals

5300

-‐169

ArHficial m

icroRN

A-‐based

targeH

ng of the

Asian

citrus psyllid fo

r HLB m

anagem

ent

Falk

UC Davis

85,000

$

5300

-‐170

Develop

a novel ta

rget-‐basis of a

nH-‐virulen

ce strategy for controlling

HLB

Lin

USD

A-‐ARS

72,424

$

5300

-‐171

Photosynthate-‐respon

sive polym

eric nan

o-‐carriers fo

r ph

loem

-‐spe

cific delivery in the

treatmen

t of HLB

Sumerlin


117,12

8$

5300

-‐172

Develop

men

t of PCR

-‐based

diagn

osHc to

ols for de

tecHon

and

differen

HaHo

n of citrus leprosis-‐associated viruses

Schn

eide

rUSD

A-‐ARS, FDWSR

U56

,428

$

5300

-‐173

Effect of m

ixed

infecHon

s of plant patho

gens on de

tecHon

of H

LB using

two early de

tecHon

metho

dsGod

frey

UC Davis

214,73

7$

5300

-‐174

Establish a system

to infect and

maintain Nico:

ana benthamiana

and

citrus with the recombina

nt CTV

Yokomi

USD

A-‐ARS

94,950

$

5300

-‐175

EDT expe

rimen

tLeVe

sque

CRB

254,61

9$

5300

-‐176

Flush & Nypmh expe

rimen

tMcCollum

USD

A-‐ARS

113,00

0$

Sub-‐Awards

5300

-‐154

Risk-‐based

decision making in the

man

agem

ent of hua

nglong

bing

(CPD

PP)

Gom

wald

USD

A-‐ARS

180,60

1$

5300

-‐162

DetecHo


riba

cter in citrus in Haciend

a Heights and

other areas of C

alifo

rnia (C

PDPP

)Slup

sky

UC Davis

55,773

$

5050

-‐041

Using

the

internet to

train citrus ho

bbyists to order bud

woo

d from

CCP

P an

d no

t to spread HLB (C

PDPP

)Willey

Fruitm

entor

20,000

$

5400 -‐ Non

-‐Vectored and Post-‐harvest Diseases

Con0

nuing Projects

5400

-‐103

Evalua

Hon of new

post-‐ha

rvest treatm

ents to

red

uce po

st-‐harvest decays in packing

house op

eraH

ons

Ada

skaveg

UC Riverside

61,000

$

5400

-‐119

Disease fo

recasHng

and

man

agem

ent of Sep

toria spot of citrus

Ada

skaveg

UC Riverside

53,000

$

5400

-‐148

Epidem

iology and

man

agem

ent of Phytoph

thora diseases of citrus in Califo

rnia

Ada

skaveg

UC Riverside

134,00

0$

5400

-‐149

Breaking

criHcal pest-‐related trad

e ba

rriers fo

r Ca

lifornia citrus exports

Walse

USD

A-‐ARS

40,576

$

5400

-‐150

Control of p

ost-‐ha

rvest diseases of citrus

Xiao

USD

A-‐ARS

73,557

$

Sub-‐Award

5050

-‐010

Breaking


e ba

rriers fo

r Ca

lifornia citrus exports (TASC)

Walse

USD

A-‐ARS

444,23

5$

5500 -‐ Pest M

anagem

ent

Con0

nuing Projects

5500

-‐189

OpH

mizing chem

ical con

trol of A

sian

citrus psyllid in Califo

rnia

Morse

UC Riverside

116,66

0$

5500

-‐189

EDevelop

men

t of an Asian

citrus psyllid (A

CP) m

anagem

ent plan

for organic citrus

Qureshi


66,565

$

5500

-‐191

Host specificity te

sHng

of D

iaphorencyrtus aligarhensis

Hod

dle

UC Riverside

206,40

7$

5500

-‐194

Release an

d mon

itoring

of Tam

arixia radiata and

phe

nology of A

sian

citrus psyllid in Sou

thern Ca

lifornia

Hod

dle

UC Riverside

231,35

8$

5500

-‐197

Impa

ct of residen

t pred

ator spe

cies on control of A

sian

citrus psyllid pop

ulaH

ons

Stou

tham

erUC Riverside

NCE

*55

00-‐205

Toxicity of syntheH

c an

d organic insecHcide

s to Tam

arixia radiata, ecto-‐pa

rasitoid of A

sian

citrus psyllid

Qureshi


69,642

$

5500

-‐206

Develop

men

t of new

trapp

ing an

d control m

etho

ds fo

r ACP

based

on complex citrus lure blend

sStelinski


100,74

0$

5500

-‐501

CORE

: IPM

program

Grapon

-‐Cardw

ell

UC Riverside

420,15

5$

New

Propo

sals

5500

-‐208

Effects of A

CP cover sprays against fruit flies (Tep

hriHda

e) and

the

ir natural ene

mies

Vargas

USD

A-‐ARS

30,000

$

Sub-‐Awards

5500

-‐196

Biolog

ical con

trol of A

sian


rnia (C

PDPP

)Stou

tham

erUC Riverside

181,74

5$

6310

Contract rearing

of Tam

arixia

TBD

TBD

250,00

0$

6320

/632

1Develop

men

t of m

ass-‐rearing metho

ds fo

r the pa

rasitoid, Tam

arixia radiata, to supp

ort classical biological con

trol

Stou

tham

er & Hod

dle

UC Riverside

248,40

4$

NCE* represents a No Co

st Extension where fu

nds for project comple:

on were approved in the previous fiscal year.

TOTAL

6,70

2,28

4$


Printed: 11/24

/15 2:00

PM

Table 1: 2015-‐2016 Research Projects

NUMBER

TITLE

PRINCIPAL

INVESTIGATOR

AFFILIATION

BOARD

APP

ROVED

BUDGET

5100 -‐ Prod

ucGon

Efficiency

Con0

nuing Projects

5100

-‐146

Novel the

rapy of h

igh-‐priority citrus diseases

Gup

taLos Alamos

67,500

$

5100

-‐150

OpH

mizaH

on of w

ater and

nitrate app

licaH

on efficien

cy fo

r citrus trees

Kand

elou

sUC Davis

108,05

9$

5100

-‐152

Iden

HficaHo

n of key com

pone

nts in HLB using

effectors as prob

es (co-‐sponsor with CR

DF)

Ma

UC Riverside

22,290

$

New

Propo

sals

5100

-‐153

Real Tim

e PC

R co-‐detecHo


riba

cter spe

cies and

Spiroplasma citri

Pagliaccia

UC Riverside

21,000

$

5100

-‐154

Citrus dwarfin

g of com

mercial varieHe

s using TsnR

NAs

Vidalakis

UC Riverside

6,53

5$

5100

-‐155

Citrus rhizobiom

es and

tree prod

ucHv

ity in respo

nse to soil m

anipulaH

ons

Leveau

UC Davis

86,276

$

5200 -‐ New

VarieGes

Con0

nuing Projects

5200

-‐141

The de

velopm

ent of novel Blood

and

Cara cara like citrus varieH

esTh

omson

USD

A-‐ARS

129,20

2$

5200

-‐142

UHlizaH

on of fou

nder line

s for im

proved

citrus biotechn

olog

y via RM

CETh

omson

USD

A-‐ARS

115,10

6$

5200

-‐144

Develop

men

t of con

sumer-‐frien

dly tran

sgen

ic citrus plants with po

tenH

al broad

spe

ctrum resistance

Louzad

aTexas A&M

110,46

9$

5200

-‐146

Rapid cycling plant bree

ding

in citrus

Moo

reUniversity of Florida

103,71

2$

5200

-‐147

Evalua

Hon of hybrids of citrus an

d citrus relaH

ves for hu

anglon

gbing (HLB) toleran

ce/resistance

Ramad

ugu

UC Riverside

73,333

$

5200

-‐148

Microprop

agaH

on of m

ature citrus in te

mpo

rary im

mersion

bioreactors

Zale


22,872

$

5200

-‐149

Stream

lining the introd

ucHo

n of licensed

citrus varieH

es into Califo

rnia: A

case stud

y -‐ Florida

Vidalakis

UC Riverside

5,00

0$

5200

-‐201

CORE

: integrated citrus breed

ing an

d evalua

Hon for Ca

lifornia

Roose

UC Riverside

627,30

7$

New

Propo

sals

5200

-‐150

OpH

mizing sensory qu

ality an

d consum

er accep

tance of citrus fruit throug

h ho

rHcultural pracHces

Guina

rdUC Davis

101,11

4$

5300 -‐ Vectored Diseases

Con0

nuing Projects

5300

-‐131

Iden

HficaHo

n an

d characterizaHo

n of HLB-‐in

duced sm

all R

NAs an

d mRN

As

JinUC Riverside

146,40

6$

5300

-‐150

Biom

arkers fo

r de

tecHon

of Liberibacter infecHon

in citrus tree

s throug

h 1H

-‐NMR ba

sed metab

olom

ics

Slup

sky

UC Davis

116,43

7$

5300

-‐151

A pha

ge/proph

age-‐ba

sed PC

R system

for sensiHve and

spe

cific detecHo

n of “Ca

ndidatus Liberibacter asiaHcus” an

d Spiroplasm

a citri

Chen

USD

A-‐ARS

NCE

*53

00-‐155

Using

mass spectrom

etry te

chno

logies to

develop

novel m

anagem

ent strategies fo

r citrus insect vector-‐bo

rne pathogen

sCilia

Boyce Th

ompson

InsHtute

183,71

3$

5300

-‐156

The citrus green

ing bibliograp

hical datab

ase

Stan

sly


18,114

$

5300

-‐158

ConstrucHo

n of the

clone

d infecHou

s cD

NA of C

itrus tristeza virus (C

alifo

rnia isolate)

Ng

UC Riverside

94,365

$

5300

-‐160

Iden

Hfying

and

cha

racterizing citrus ta

rgets from

Candidatus Libe

riba

cter asiaH

cus

Coaker

UC Davis

106,97

4$

5300

-‐161

Infrastructure sup

port fo

r research on de

tecHon

and

man

agem

ent of HLB and

ACP

God

frey

UC Davis

100,86

4$

5300

-‐163

Not all psyllids are created eq

ual: Why do some tran

smit Liberibacter an

d othe

rs do no

t?Cilia

Boyce Th

ompson

InsHtute

146,27

5$

5300

-‐164

A m

icrobiota-‐ba

sed ap

proa

ch to

citrus tree

health

Rolsha

usen

/Leveau

UC Riverside & UC Davis

151,65

3$

5300

-‐165

Develop

men

t of m

ature bu

dwoo

d tran

sformaH

on te

chno

logy

Thom

son

USD

A

2,50

4$

5300

-‐168

Use of d

igita

l PCR

for im

proved

early detecHo


riba

cter asiaH

cus infecHon

in citrus an

d ACP

McCollum

USD

A-‐ARS

42,500

$

New

Propo

sals

5300

-‐169

ArHficial m

icroRN

A-‐based

targeH

ng of the

Asian

citrus psyllid fo

r HLB m

anagem

ent

Falk

UC Davis

85,000

$

5300

-‐170

Develop

a novel ta

rget-‐basis of a

nH-‐virulen

ce strategy for controlling

HLB

Lin

USD

A-‐ARS

72,424

$

5300

-‐171

Photosynthate-‐respon

sive polym

eric nan

o-‐carriers fo

r ph

loem

-‐spe

cific delivery in the

treatmen

t of HLB

Sumerlin


117,12

8$

5300

-‐172

Develop

men

t of PCR

-‐based

diagn

osHc to

ols for de

tecHon

and

differen

HaHo

n of citrus leprosis-‐associated viruses

Schn

eide

rUSD

A-‐ARS, FDWSR

U56

,428

$

5300

-‐173

Effect of m

ixed

infecHon

s of plant patho

gens on de

tecHon

of H

LB using

two early de

tecHon

metho

dsGod

frey

UC Davis

214,73

7$

5300

-‐174

Establish a system

to infect and

maintain Nico:

ana benthamiana

and

citrus with the recombina

nt CTV

Yokomi

USD

A-‐ARS

94,950

$

5300

-‐175

EDT expe

rimen

tLeVe

sque

CRB

254,61

9$

5300

-‐176

Flush & Nypmh expe

rimen

tMcCollum

USD

A-‐ARS

113,00

0$

Sub-‐Awards

5300

-‐154

Risk-‐based

decision making in the

man

agem

ent of hua

nglong

bing

(CPD

PP)

Gom

wald

USD

A-‐ARS

180,60

1$

5300

-‐162

DetecHo


riba

cter in citrus in Haciend

a Heights and

other areas of C

alifo

rnia (C

PDPP

)Slup

sky

UC Davis

55,773

$

5050

-‐041

Using

the

internet to

train citrus ho

bbyists to order bud

woo

d from

CCP

P an

d no

t to spread HLB (C

PDPP

)Willey

Fruitm

entor

20,000

$

5400 -‐ Non

-‐Vectored and Post-‐harvest Diseases

Con0

nuing Projects

5400

-‐103

Evalua

Hon of new

post-‐ha

rvest treatm

ents to

red

uce po

st-‐harvest decays in packing

house op

eraH

ons

Ada

skaveg

UC Riverside

61,000

$

5400

-‐119

Disease fo

recasHng

and

man

agem

ent of Sep

toria spot of citrus

Ada

skaveg

UC Riverside

53,000

$

5400

-‐148

Epidem

iology and

man

agem

ent of Phytoph

thora diseases of citrus in Califo

rnia

Ada

skaveg

UC Riverside

134,00

0$

5400

-‐149

Breaking


e ba

rriers fo

r Ca

lifornia citrus exports

Walse

USD

A-‐ARS

40,576

$

5400

-‐150

Control of p

ost-‐ha

rvest diseases of citrus

Xiao

USD

A-‐ARS

73,557

$

Sub-‐Award

5050

-‐010

Breaking


e ba

rriers fo

r Ca

lifornia citrus exports (TASC)

Walse

USD

A-‐ARS

444,23

5$

5500 -‐ Pest M

anagem

ent

Con0

nuing Projects

5500

-‐189

OpH

mizing chem

ical con

trol of A

sian


rnia

Morse

UC Riverside

116,66

0$

5500

-‐189

EDevelop

men

t of an Asian

citrus psyllid (A

CP) m

anagem

ent plan

for organic citrus

Qureshi


66,565

$

5500

-‐191

Host specificity te

sHng

of D

iaphorencyrtus aligarhensis

Hod

dle

UC Riverside

206,40

7$

5500

-‐194

Release an

d mon

itoring

of Tam

arixia radiata and

phe

nology of A

sian

citrus psyllid in Sou

thern Ca

lifornia

Hod

dle

UC Riverside

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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 hard-working Board members who volunteer significant portions of their time for the betterment of the citrus community. In this issue, you’ll meet three of the most recent appointees.

MEET YOUR BOARD MEMBERS

Ivy Leventhal

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 Non-Vectored Disease and Post-Harvest.

“My experience on the Board so far has been interesting, amusing,

confusing and at times overwhelming,” Galloway said. “I have come to realize how little I know and how truly open I am to new ideas and concepts. Our industry has many exceptionally bright people working on a tremendous number of research projects. These scientists are compassionate about their work, while patient with those of less intelligence. I feel blessed to be allowed into their sand box.

“Our industry is being bombarded with many challenges,” he continued. “Trade partners are placing quality demands that require significant production and processing adjustments. Neighboring growing regions are experiencing the wrath of a crippling, perhaps apocalyptic disease. We are on high alert regarding the invasive nature of this disease and have spread a very wide research net.

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 CRB’s 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. “It’s not going to be an easy fight, but I think we will be able to beat this thing out if we all work together.”

The San Juan Capistrano native who now lives in Temecula is a relative newcomer to the California citrus industry. For the past several years, he has served as the operations manager for Pauma Ranches in Pauma Valley, responsible for citrus and avocado acreage and staff.

Perricone and his wife have one son. When not working or fulfilling his CRB Board duties, the D.A.R.E. America volunteer enjoys the fast sport of roller hockey and spending time with his family.

The newest over-all Board member, representing District One and elected in 2015, is Keith Watkins. As Vice President of Farming and Field Operations for Bee Sweet Citrus in Fowler, he is charged with overseeing approximately 10,000 acres and is responsible for land acquisition, investor relations and financial oversight. His son, Matthew, works with him on Bee

Sweet’s 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 other’s 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 CRB’s 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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HLB DETECTIONS IN SAN GABRIELWhere are we now?Victoria Hornbaker and Lucita Kumagai


In April 2012, the first California detection of huanglongbing (HLB), also known as citrus greening, was confirmed in a tree in Hacienda

Heights. It wasn’t 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, here’s the sequence of events that occurred in San Gabriel in the continuing battle against this devastating citrus disease.

SAN GABRIEL HLB TIMELINETREE 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 real-time PCR, conventional PCR and DNA sequencing. The tissue was sent to USDA for confirmation, which was received on July 9. With the homeowner’s permission, the tree was first treated with a foliar insecticide and then removed on July 10. CDFA also surveyed and took samples of ACP and plant tissue on all adjacent properties and applied foliar and systemic treatments (with homeowner permission). The state agency announced the HLB detection to the public later that day.

As a result of the first find, CDFA began activities to place a quarantine for HLB in the San Gabriel area, consisting of a five-mile radius around the find site, totaling 87 square miles. CDFA then began survey and treatment activities on all HLB host plants within 800 meters of the find site. By taking those essential steps, the critical reservoir of disease and its vectors (ACP) were in the process of being removed.

On July 13, CDFA Pest Exclusion staff began survey activities of production and retail nurseries in the proposed quarantine area, placing all host plants on hold.

CDFA crew removing HLB-positive lime tree in the San Gabriel area.


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. CDFA’s lab ran samples to determine if the disease had spread to other citrus trees in the area.

To inform the public and particularly the neighborhood, a public meeting was held the evening of July 16 and was very well attended. Assemblyman Ed Chau was in attendance, as was Kurt Floren, the Los Angeles County Agricultural Commissioner.

Following the neighboring finds, notices were delivered to residents to notify them that insecticide treatments would begin on July 20.

TREES 3-4 (JULY 22, 2015)CDFA confirmed two additional HLB-positive trees, a Mandarin and a calamondin, on the same street as the initial tree. Both trees were treated and removed, with homeowner permission on July 22.

CDFA Pest Exclusion staff continued working with the USDA to survey approximately 90 nurseries and garden centers,

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.

HLB quarantine map in Southern California, including the single 2012 Hacienda Heights detection and the 10 San Gabriel finds in the summer of 2015.

2015 Huanglongbing (HLB)Quarantine Map


With the strategy set by the citrus industry and immediate response by CDFA survey and treatment crews, no other San Gabriel samples have been confirmed for HLB at the time of this printing.

CONTINUING THE FIGHT IN THE GROVEThis is a wake-up call for all growers, particularly those in Southern California. Growers should be participating in an area-wide management program to control ACP populations in commercial groves. With HLB in California, ACP management becomes even more crucial. Review the best management practices at www.citrusinsider.org/resources, and employ them in your groves. Also, see the flyer on page 28. The Citrus Pest and Disease Prevention Program (CPDPP) and CDFA encourage growers to regularly survey their own groves for signs of ACP and HLB. CPDPP will continue to trap for ACP in commercial groves throughout the state.

IN THE BACKYARDThrough CPDPP’s 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.

BY THE NUMBERS

Total ACP samples collected from San Gabriel, including expansion areas: 1,504

Total plant samples collected from San Gabriel, including expansion areas: 6,735

Confirmed positive ACP samples: 4

Confirmed positive plant samples: 10

References:www.citrusinsider.org/resourceshttp://www.cdfa.ca.gov/phpps/acp

Victoria Hornbaker is with the Citrus Pest and Disease Prevention Program, where she serves as citrus program manager. Lucita Kumagai is with the Plant Pest Diagnostics Branch at the California Department of Food and Agriculture, where she serves as senior plant pathologist.

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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 dedicat-ed to keeping you informed about psyllid finds and proper treatment protocol. Know who your Grower Liaison is and contact them with any questions about how best to protect your citrus from ACP and HLB.

Sample and Treat – Follow UC recommended guidelines for sampling and treating for the Asian citrus psyllid.

Work with your neighbors – Area-wide management of the Asian citrus psyllid is a strategy where growers in a specific area coordinate management efforts to maximize the impact on psyllid populations. Psyllid Management Areas (PMA) are being formed now so if the time comes to implement an area-wide strategy, growers in the region are ready.

Stay Informed – The Citrus Insider website is your resource for all information pertaining to the Asian citrus psyllid and HLB. Visit www.citrusinsider.org for more information about psyllid finds in your area, best practices, treatment recommendations, area-wide treatment protocol, maps, and more. You can also sign up to have regional alerts sent to your email address.

We must all do our part to protect our trees, our industry, and our way of life.

Young Asian citrus psyllids are yellow and produce a white, waxy substance.

Asian citrus psyllids are brown, aphid-like insects that feed on the leaves and stems of citrus trees.

HLB-infected trees will die.

CitrusInsider.org


Huanglongbing (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.

2) Every country in the Americas adjacent to a CLas-positive nation detects HLB within five years or less (e.g., Texas and California proximal to Mexico, and Argentina proximal to Brazil).

3) In areas where HLB is well established, the disease causes unprecedented increases in production costs, crop loss and reduction of internal and external fruit quality.

While leaf symptoms may resemble nutritional stress or deficiencies (e.g., zinc and iron), these symptoms actually are due to disruption of carbohydrate metabolism and

HLB IMPACT ON CITRUS ROOT HEALTH AND INTERACTION WITH PHYTOPHTHORAJames Graham

Figure 1A


allocation as a result of plugging and necrosis of phloem, the plant’s sugar conducting system. Because of carbohydrate disruption, there is significant loss of fibrous roots, mottling and yellowing of leaves (Figure 1A) and shoots, defoliation, dieback, tree decline and excessive fruit drop (Figure 1B). Fruit are small and misshapen with aborted seeds, are abnormally colored and contain off-flavored juice low in brix. In the 10 years following the detection of HLB, Florida citrus production has dropped 50 percent with no sign that the rate of crop loss is slowing (http://www.nass.usda.gov/Statistics_by_State/Florida/Publications/Citrus/cit/2015-16/cit1015.pdf).

At the University of Florida’s 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. Johnson’s 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 70-80 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.

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.


Importantly, the combination of CLas infection and bicarbonate stress increases susceptibility to root pests and pathogens including Phytophthora species. The causal species of Phytophthora diseases in Florida citrus are Phytophthora nicotianae (parasitica), the most common cause of foot rot (gummosis) and root rot, and P. palmivora, which most often is the cause of brown rot of fruit and root rot in poorly drained soils with high water tables. Wet conditions favor root infection cycles of Phytophthora species.

Susceptibility of fibrous roots is highest during very wet to very dry cycles (spring and fall). Extreme wetting and drying promotes root exudation (release of organic chemicals), which attracts zoospores. CLas-infected roots exude more sugars than healthy roots, which increases zoospore infection. Evidence for greater incidence of this interaction comes from both greenhouse trials and grove surveys. Phytophthora nicotianae populations initially increased in potting soil at two, eight and 14 months post-inoculation in HLB-infected trees compared to un-infected controls. This was followed by a decline after a major loss of fibrous roots mainly due to CLas infection.

Survey data from Florida groves suggest a resistance-breaking interaction of CLas with Phytophthora species. Syngenta Crop Protection has conducted a statewide survey of Phytophthora species that has spanned over two decades, covers all production areas and is largely driven by grower requests. The survey results serve as an indicator of emerging root disease trends. Comparison of the survey data for seasons since HLB became widespread in Florida groves shows a strong trend toward higher incidence of damaging Phytophthora populations coincident with the rise in HLB disease incidence from 2008-2011 (Figure 2A). Since then, there has been a strong downturn in 2013 and recovery of the populations in 2014 associated with biennial fluctuations of fibrous root density as HLB trees continue to decline (Figure 2B). The survey results have served to heighten grower concern for the

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 tree’s response to fungicides for prevention of root loss, because the bacterial infection is the major contributor to damage of co-infected 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 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).


to sustain root health of HLB trees. Details for management are found in the Florida Citrus Pest Management Guide (www.crec.ifas.ufl.edu/extension/pest/). Assessment of Phytophthora disease is based on a soil propagule assay that measures population density in the rhizosphere in the wetted zone. If counts exceed 10-20 propagules per cm3 of soil volume, the following rotation of fungicides may be recommended: fosethyl-al or phosphite after spring shoot flush, mefenoxam after spring-early summer rains begin, fosethyl-al or phosphite after midsummer shoot flush, and mefenoxam after fall shoot flushes. The timing of soil application is for protection of root flushes that follow shoot flushes in the tree life cycle (phenology).

James Graham, Ph.D., is a professor of soil microbiology at the Citrus Research and Education Center (CREC), University of Florida, Lake Alfred, Florida.

The author thanks Davis Citrus Management and Syngenta Crop Protection for generously sharing data cited in the article and the Citrus Research and Development Foundation (CRDF) for grant support.

ReferencesGraham, 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 huanglongbing-affected trees prior to appearance of foliar symptoms. Plant Pathol. 63:290-298.

GlossaryRoot exudation: Roots of higher plants release organic compounds (sugars, amino acids, lipids, vitamins, etc.) that may serve as chemical attractants or repellents to a particular microbial community in rhizosphere.

Propagule: Fungal spores or bacterial cells that transmit a disease.

Rhizosphere: Zone (few mm in thickness) surrounding the plant root system where plant, microorganisms and soil come together influencing root chemistry and biology.

Tree phenology: Plant life cycle events that are influenced by seasonal variations in climate and elevation.

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.

A B


CRB-FUNDED RESEARCH PROGRESS REPORT

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.

MONITORING FOR ACP RESISTANCE TO PESTICIDESBaseline Levels Established for 12 Pesticides

Figure 1. Adult ACP on a curry plant in a cage provided by the UCR Tamarixia radiata program.


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 re-invade 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.

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.

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

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 biocontrol-rearing program. In addition, Project 5500-196 provided ACP nymphs and adults to other research projects, such as for this baseline resistance-monitoring project.

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.


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 µl droplet to each psyllid, we decided to use 0.8 µl, 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

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 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.

Figure 5. Adults in a vial are knocked out with carbon dioxide.


[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

Figure 7. Dead ACP adults on the leaf in a Petri dish when the bioassay is evaluated 24 hours after micro-applicator treatment.

Figure 6. A 0.8 µl 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.


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

RESULTS AND DISCUSSIONProbit 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 µl 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

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

AcknowledgementsWe thank the Citrus Research Board for funding this research in part and also the chemical companies who provided technical product for testing. We thank Jan Hare, Lisa Forster and others in the Richard Stouthamer laboratory, who provided ACP for testing under CRB Project No. 5500-196. We also thank Alan Urena, Lindsay Robinson and Janine Almanzor for technical assistance.


Literature Grafton-Cardwell, E.E., L.L Stelinski and P.A. Stansly. 2013. Biology and management of Asian citrus psyllid, vector of huanglongbing pathogens. Annual Review of Entomology 58: 413-432.

Lee, J.A., S.E. Halbert, W.O. Dawson, C.J. Robertson, J.E. Keesling and B.H. Singer. 2015. Asymptomatic spread of huanglongbing and implications to disease control. Proceedings of the National Academy of Sciences, 112(24): 7605-7610. Available at: www.pnas.org/cgi/doi/10.1073/pnas.1508253112.

Tiwari, S., R.S. Mann, M.E. Rogers and L.L. Stelinski. 2011. Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Management Science 67: 1258-1268.

Tiwari, S., L.L. Stelinski and M.E. Rogers. 2012. Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid. Journal of Economic Entomology 105(2): 540-548.

Tiwari, S., N. Killiny and L.L. Stelinski. 2013. Dynamic insecticide susceptibility changes in Florida populations of Diaphorina citri (Hemiptera: Psyllidae). Journal of Economic Entomology 106(1): 393-399.

Table 1. Baseline susceptibility of Asian citrus psyllid adults to 12 insecticides listed by class of chemistry.Ratio of

Pesticide ACP Field 1,000*Pesticide trade Chi- LD50 LD95 Typical use rate LD95 to

IRAC common name square (ng AI/ (ng AI/ ACP field converted to field use

Classa name (company) Bioassay test dates Nb statistic Slope ± SE insect)c 95% Cid insect)c 95% Cid use rate mg AI/litere ratef

Class 1A (carbamates)

formetanate Carzol 92 SP 5-8-13; 6-5-13; 7-5-13

570 0.1716 6.0917 ± 0.4096 32.2114 a 30.4832-33.9718 59.9828 a 55.2013-66.4935 1.25 lb/a 689.1 87.0

(Gowan)formetanate, Round 2 4-8-14; 5-13-14 420 0.4607 4.9094 ± 0.3857 34.7369 a 32.2420-37.4984 75.1332 a 66.0530-88.8990

Class 1B (organophosphates)

chlorpyrifosLorsban Advanced 3.755 EC

10-2-13; 11-13-13; 11-20-13

354 0.1973 4.2610 ± 0.4286 10.3481 a 9.3399-11.5586 25.1703 a 20.8504-32.9151 6 pts/a 1,687.5 14.9

(Dow)

chlorpyrifos, Round 2

4-24-14; 5-12-14; 6-11-14; 7-8-14; 7-9-14; 7-23-14

932 0.8716 5.0234 ± 0.2744 15.0516 b 14.2124-15.9941 31.9907 a 28.8779-36.1855

Class 3A (pyrethroids)fenpropathrin Danitol 2.4 EC 10-24-12; 1-9-13 251 0.7529 2.1990 ± 0.2313 4.1845 a 3.3012-5.5469 23.4234 a 15.1553-44.1153 16 fl oz/a 179.8 130.3

(Valent)fenpropathrin, Round 2 8-7-13 302 0.4637 4.5397 ± 0.4456 7.6217 b 6.8886-8.4483 17.5543 a 14.8621-22.1481

zeta-cypermethrin Mustang 1.4 EC 2-27-13; 3-13-13 394 0.2889 1.8723 ± 0.2000 0.5044 a 0.3688-0.6429 3.8133 a 2.8392-5.7455 4.3 oz/a 30.2 126.3(FMC)

zeta-cypermethrin, Round 2 8-12-14; 8-28-14 609 0.2302 2.8376 ± 0.2071 1.0716 b 0.9764-1.1777 4.0709 a 3.3606-5.2209Class 4A (Neonicotinoids)

imidacloprid Admire Pro 4.6 EC 8-15-12; 9-26-12; 10-10-12

565 0.1067 2.5470 ± 0.2165 0.6181 a 0.5449-0.7086 2.7342 b 2.0953-3.9257 7 fl oz/a 150.7 18.1

(Bayer)imidacloprid, Round 2 7-17-13 245 0.7033 5.6308 ± 0.6922 0.7270 a 0.6555-0.8053 1.4244 a 1.2169-1.8093

thiamethoxam Actara 25% WDG 1-16-13; 1-30-13 443 0.2905 4.6172 ± 0.4255 0.4234 a 0.3899-0.4612 0.9616 a 0.8252-1.1880 5.5 oz/a 51.5 18.7(Syngenta)

thiamethoxam, Round 2 7-24-13 242 0.6549 5.9113 ± 0.6075 0.3382 a 0.3504-0.4145 0.7248 a 0.6403-0.8624Class 4C (nicotinic acetylcholine receptor agonists)

sulfoxaflor Sequoia 2 EC 9-18-13; 10-9-13; 11-25-13

623 0.2212 4.1400 ± 0.2826 3.1617 a 2.8722-3.4624 7.8919 a 6.9625-9.2009 5.75 fl oz/a 53.8 146.6

(Dow)sulfoxaflor, Round 2 4-15-14 262 0.8370 5.8523 ± 0.6248 3.4192 a 3.1254-3.7240 6.5312 a 5.7362-7.8553

Class 4D (butenolide)

flupyradifuronegSivanto 1.67 EC (Bayer)

8-7-14; 8-14-14; 8-27-14

782 0.3959 2.8398 ± 0.1763 15.1775 13.7792-16.6333 57.6000 49.4197-69.5275 14 fl oz/a 131.1 439.4

Class 5 (spinosyns)

spinetoram Delegate 25% WG 9-11-13; 9-24-13; 1-8-14

372 0.9544 3.5879 ± 0.3157 0.8561 a 0.7556-0.9799 2.4603 a 1.9863-3.2843 6 oz/a 56.2 43.8

(Dow)spinetoram, Round 2 4-18-14; 5-7-14 602 0.2280 3.7880 ± 0.2726 0.9720 a 0.9025-1.0504 2.6416 a 2.2712-3.2108

Class 6 (avermectins, milbemycins)abamectin Agri-Mek 0.7 SC 4-3-13; 4-7-13 462 0.1377 2.7986 ± 0.2262 0.6157 a 0.5352-0.7049 2.3830 a 1.9248-3.1477 4.25 fl oz/a 13.9 171.1

(Syngenta)abamectin, Round 2 9-4-13 313 0.7538 4.1526 ± 0.6214 1.4750 b 1.2979-1.7861 3.6720 a 2.7200-6.2713

Class 21 (mitochondrial complex I electron transport inhibitors)tolfenpyrad Bexar 1.31 EC 7-2-12; 8-1-12 306 0.1102 4.0289 ± 0.3676 2.6759 a 2.3916-2.9824 6.8508 a 5.7954-8.5838 27 fl oz/a 165.6 41.4

(Nichino)

tolfenpyrad, Round 27-31-13; 7-30-14; 8-6-14

414 0.1640 5.4498 ± 0.4475 2.5559 a 2.3747-2.7364 5.1211 a 4.6137-5.8671

Class 28 (ryanodine receptor modulators)cyantraniliprole Exirel 0.83 EC 5-22-13; 8-14-13 336 0.2577 2.3040 ± 0.2815 0.3602 a 0.2857-0.4282 1.8642 a 0.1303-2.7977 4.5 fl oz/a 17.5 106.6

(DuPont)cyantraniliprole, Round 2 4-1-14 314 0.2684 2.8436 ± 0.3081 0.3294 a 0.2808-0.3781 1.2478 a 0.9919-1.7434

a Insecticide Resistance Action Committee class (www.irac-online.org); pesticides in the same class have similar modes of action and thus, cross resistance within the class is likely.b N = total number of insects tested at various bioassay rates, excluding control insects.c 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.d 95% Confidence Interval (fiducial limits).e To convert the listed Typical ACP field use rate to mg AI/liter, we assumed field use of 200 GPA spray volume.f Data in this column are the ratio of 1,000 * LD95 in ng AI applied to each insect (in the 0.8 µl droplet) to the field pesticide use rate in units of mg AI per liter assuming application at 200 GPA.g Technical material received late so no Round 1 test was done with Sivanto; field use rate ratio is based on the Round 2 LC95.

Table 1. Baseline susceptibility of Asian citrus psyllid adults to 12 insecticides listed by class of chemistry.


MATURE CITRUS PROPAGATION IN RITA® BIOREACTORSYosvanis Acanda and Janice Zale


SUMMARYThis 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® (Récipient à 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.

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


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 disease-tolerant, 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.

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.

Figure 1. A temporary immersion RITA® bioreactor for plant propagation.


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.

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

A

A

B

B

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.

Figure 4. Bioreactor-grown Carrizo shoots. A) Carrizo shoots in the bioreactor. B) Primary Carrizo shoots with developing axillary shoots.


number of shoots per cutting, the size of the shoots and the weight of the total biomass in each bioreactor.

We also have tested different plant growth regulators to promote rooting with promising preliminary results in Carrizo and Valencia (Figure 5). In the past, rooting scion has been problematic. If the majority of regenerated shoots prove recalcitrant to rooting, scions will be micro-grafted onto rootstock similar to nursery practices.

In summary, our results thus far suggest that the RITA® bioreactors are promising systems for mature citrus propagation. It seems that the continual changes to the atmosphere in the bioreactor might decrease ethylene gas and its adverse effects on shoot growth, enhancing growth and development. However, it remains to be determined whether the shoots can survive in the bioreactors after excision from stem cuttings. After optimizations of the plant propagation in bioreactors, the genetic stability and maturity of the propagules will be determined using biotechnological instrumentation and techniques, respectively.

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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.


NOT ALL PSYLLIDS ARE CREATED EQUAL Combining genetics and proteomics to understand the basis of ACP vector competencyMichelle Cilia, John Ramsey, Angela Kruse, Richard Johnson, Michael MacCoss, Robert Shatters and David Hall


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.


PROJECT SUMMARYThe 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 Florida’s annual $9 billion industry1.

The need for novel and effective HLB management strategies is urgent, as the HLB-associated Liberibacter and vector are spreading beyond Florida. The urgent need for control strategies was affirmed in July 2015 with the discovery of 10 infected trees in San Gabriel, California, marking the first confirmed report of HLB in the state since a single infected tree was discovered in Hacienda Heights in 2012. HLB management options currently are limited to ineffective strategies to control ACP vectors and early disease detection, and the disease is a death sentence for an infected citrus tree. Control of CLas transmission by the ACP represents a promising new control strategy.

Our new project is focused on comparing populations of the ACP that vary in their ability to transmit CLas. This will enable us to understand the molecular interactions that occur during CLas transmission. These interactions are challenging to study because there are many organisms interacting with one another (Figure 1).

In this article, we briefly describe each of the biological players and our overall experimental approach. Finally, we explain how our results may be used to help the growers in California and elsewhere to better manage the disease. Our team is well poised to achieve our objectives and to translate knowledge into novel management strategies for the citrus industry. Our preliminary data already have led to the identification of potential targets for ACP control using para-transgenic and ribonucleic acid (RNA) interference technologies being developed in other laboratories.

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

Vector competence also may be related to the ability of CLas to survive in the insect hemolymph, either by evading insect immune defenses or by manipulating the ACP immune system to be more permissive for CLas. Genome sequencing results have shown that the ACP lacks a complete insect immune system. The ACP symbionts, described below, may aid in the ACP immune response to CLas.

Bacterial Symbionts. ACP harbors beneficial bacteria, called symbionts, in a specialized, interspecies organ called the bacteriome. The bacteriome depicted in Figure 1 is an interpretative, not-to-scale schematic of interactions among ACP, CLas, bacteriophage and endosymbionts based on a figure published in a paper from Nakabachi and colleagues4. The two primary ACP symbionts are ‘Candidatus Carsonella rudii DC,’ a putative nutritional symbiont, and ‘Candidatus Profftella armature,’ a putative defensive symbiont. Profftella is an ACP-specific symbiont. They have been found in every ACP population studied to date. Carsonella cells are located within the cells on the border of the bacteriocytes. Profftella cells are located in the center. A secondary symbiont, called Wolbachia, is also found in the ACP – this symbiont has been found in a range of insect tissues in addition to the bacteriome.


Collectively, the bacterial symbionts of the ACP are referred to as the insect microbiota. The role of the microbiota in ACP-CLas 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 to increase their own chances of transmission during feeding. Many hemipterans are among the insect world’s most prolific vectors of plant pathogens.

Bacteriophage. At least some isolates of CLas harbor bacteria-infecting viruses called bacteriophages. Although they are presumed to remain inactive in the ACP, bacteriophage may increase the virulence of CLas or may even be used as a suicide switch to kill CLas.

Host plants of the ACP and CLas. The host plants are also other players. Some plants in the Rutaceae are hosts of both CLas and the ACP, while others are hosts for the ACP only. In HLB-susceptible citrus varieties, the ACP can efficiently acquire CLas. In infected Murraya spp., a citrus relative, CLas titers are much lower than in citrus; and the ACP acquires four orders of magnitude less CLas from infected Murraya than from CLas-infected citrus.9 Understanding how each of the players interacts with the others is a critical part of learning how to efficiently manage the disease and to prevent CLas transmission. The molecular approach our team is utilizing will enable us to understand novel ways to prevent CLas from using the ACP to go commit its next crime.

EXPERIMENTAL APPROACHGenetics. 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. For example, in extreme south Florida where citrus is not grown commercially, ACP populations associated with orange jasmine might be less efficient at acquiring and transmitting CLas than ACP associated with citrus in central Florida.

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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-ACP-symbiont 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.

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).


RESULTS AND APPLICATIONS At the International Research Conference on HLB in Orlando, Florida, in February 2015, our team presented proteomics data comparing healthy ACP with those harboring CLas. Our data show that once the ACP acquires CLas, interactions with the symbionts, in particular Profftella, are drastically changed. The data also indicate that CLas may be a pathogen of the ACP as well, and that Profftella may be helping to defend the ACP against CLas infection.

We are further exploring these results by measuring small RNA molecules produced by the ACP. These small RNA molecules will give us additional clues into CLas pathogenicity in the ACP. This idea, which is supported by our data, represents a totally novel means for insect control: if we understood the molecular interactions occurring among the ACP, CLas and Profftella, we could specifically increase CLas virulence in the ACP and not the tree. In other words, we hypothesize that we can infect the insect with CLas to the point where it kills the ACP. The success of this approach is based on our ability to understand how the ACP defends itself against CLas and to develop a method to block that defense mechanism. Ultimately, our research could lead to a solution to the HLB problem. More details on this research can be found in a recently published PLOS ONE article: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140826

AcknowledgementsThe authors thank Jackie Mahoney (Cilia Lab, Boyce Thompson Institute) for technical assistance and are grateful to the Citrus Research Board for funding.

About the authorsMichelle 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.

References1. 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.

3. Ammar,E-D., R.G. Shatters Jr., and D.G. Hall. 2011. Localization of Candidatus Liberibacter asiaticus, associated with citrus huanglongbing disease, in its psyllid vector using fluorescence in situ hybridization. J. Phytopathol. 159: 726-734.

4. Nakabachi, A., R. Ueoka, K. Oshima, R. Teta, A. Mangoni, M. Gurgui, N.J. Oldham, G. van Echten-Deckert, K. Okamura, K. Yamamoto, H. Inoue, M. Ohkuma, Y. Hongoh, S.Y. Miyagishima, M. Hattori, J. Piel, and T. Fukatsu. 2013. Defensive bacteriome symbiont with a drastically reduced genome. Curr. Biol. 23(15):1478-84.

5. Fagen, J.R., A. Giongo, C.T. Brown, A.G. Davis-Richardson, K.A. Gano, and E.W. Triplett. 2012. Characterization of the relative abundance of the citrus pathogen ‘Ca. Liberibacter asiaticus’ in the microbiome of its insect vector, Diaphorina citri, using high throughput 16S rRNA sequencing. Open Microbiol. J. 6:29-33.

6. Gerardo, N.M., B. Altincicek, C. Anselme, H. Atamian, S.M. Barribeau, M. de Vos, E.J. Duncan, J.D. Evans, T. Gabaldon, M. Ghanim, A. Heddi, I. Kaloshian, A. Latorre, A. Moya, A. Nakabachi, B.J. Parker, V. Perez-Brocal, M. Pignatelli, Y. Rahbe, J.S. Ramsey, C.J. Spragg, J. Tamames, D. Tamarit, C. Tamborindeguy, C. Vincent-Monegat, and A. Vilcinskas. 2010. Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biol. 11(2): R21.

7. Zhang, C.R., S. Zhang, J. Xia, F.F. Li, W.Q. Xia, S.S. Liu, and X.W. Wang. 2014. The immune strategy and stress response of the Mediterranean species of the Bemisia tabaci complex to an orally delivered bacterial pathogen. PLOS One, 9(4): e94477.

8. Nachappa, P., J. Levy, and C. Tamborindeguy. 2012. Transcriptome analyses of Bactericera cockerelli adults in response to ‘Candidatus Liberibacter solanacearum’ infection. Mol. Genet. Genomics. 287(10):803-817.

9. Walter, A.J., Y. Duan, and D.G. Hall. 2012. Titers of ‘Candidatus Liberibacter asiaticus’ in Murraya paniculata and Murraya-reared 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.


SUMMARYMonitoring 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.

COMPLEX CITRUS LURES TO TRAP AND CONTROL ACPXavier Martini, Alexander A. Aksenov, Cristina E. Davis and Lukasz L. Stelinski


Unbaited yellow traps are widely used in California.


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 sub-optimal, 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 behavior-modifying 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


trees (Figure 2). Lure development using this strategy could provide several new tools for ACP management, including more effective lures for trapping, attract-and-kill devices and disruption of ACP host plant finding.

Our specific goal has been to develop more potent lure formulations (e.g., season-specific or independent blends) while reducing the number of chemical components. We are working to optimize the ratio and number of the most attractive components, while considering the economics of practical applications. For this, we have been conducting subtraction assays to find the most salient blend of chemicals that elicit attraction of ACP, so as to create a cost-effective and practical tool for ACP monitoring. Field deployment and evaluation of possible practical tools are currently underway.

During the previous year, we developed an attractive lure for ACP that appears to be economically practical for field deployment. However, the majority of our research has been conducted under laboratory conditions. More field testing is necessary to determine whether the lure is effective when deployed within citrus groves. This research is currently underway. We also have begun collaborating with the industry to develop a field-ready dispenser. Field verification is the final necessary step before this technology can be developed and eventually marketed by the commercial pest management industry. We are interested in further improving the attractiveness of this lure. This research has been a collaborative effort between chemists, engineers and entomologists.

ReferencesAksenov, A.A., X. Martini, W. Zhao, L.L. Stelinski and C.E. Davis. 2014. Synthetic blends of volatile, phytopathogen-induced odorants can be used to manipulate vector behavior. Frontiers in Ecology and Evolution. 2: 78. doi: 10.3389/fevo.2014.00078.

Mann, R.S., J.G. Ali, S.L. Hermann, S. Tiwari, K.S. Pelz-Stelinski, H.T. Alborn and L.L. Stelinski. 2012. Induced release of a plant defense volatile ‘deceptively’ attracts insect vectors to plants infected with a bacterial pathogen. PLoS Pathogens. 8(3): e1002610.

Martini, X., M. Hoffmann, M.R. Coy, L.L. Stelinski and K.S. Pelz-Stelinski. 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 California-Davis; and Lukasz L. Stelinski, Ph.D., is an associate professor in the Entomology and Nematology Department at the University of Florida.

Figure 2. Quantifying psyllid behavior in the laboratory.


FOUNDER LINES FOR IMPROVED CITRUS BIOTECHNOLOGYMaria Oliveira, Ed Stover and James Thomson


On 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

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.


Single copy lines provide a unique site in the genome for targeting additional novel transgenes. (A single copy insert indicates that a single transgenic construct was inserted into the citrus.)

The Founder Lines contain the recognition sites (unique DNA sequences) for specific enzymes capable of cutting and pasting DNA without the gain or loss of genetic material. In Figure 1, these sites are designated attP and Res for their respective enzyme partners, Bxb1 and CinH. In between these sites are marker genes that allow one to select for the presence of the Founder Line DNA in the genome using the antibiotic, kanamycin (nptII). Also included is the visual marker DSRed, which allows one to “see” the DNA once it is incorporated into the genome.

Seen in Figure 2 are Founder Lines initially isolated from tissue culture where the transgenic DNA landed in the genome that will determine how effectively it expresses the color red. This system was designed so that the initial selectable markers would be removed from the genome once targeting was achieved using the recombinase technology. It also would allow stacking of more than one gene into the genome. Multiple genes can be inserted all at once or individually over time, proving flexibility and optimization.

Why would so many genes be needed? Many metabolic pathways that produce flavor and/or pigments require multiple enzymes to produce the desired trait. Additionally, this makes it possible to take an existing transgenic – for example, Tango, with a gene for citrus greening resistance – and add an additional desirable trait, knowing it will be highly expressed.

Also seen is the codA selection gene. This is an odd gene that allows one to select for its absence. In other words, removal of this gene from the genome can be detected to verify DNA exchange; this is a crucial element to the RMCE targeting

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 non-toxic compound into a toxic one. So when an RMCE targeting

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.

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).


event is attempted, plants are screened in the presence of this non-toxic compound. If they survive, the RMCE process was successful and the DNA swap completed as desired. Results for the codA selection strategy are seen above in Figure 3.

Creation of these initial Founder Lines is just the midpoint of the project. Currently, only Carrizo and sweet orange (Valencia and Hamlin) Founder Lines are in the greenhouse and available for RMCE testing. Founder Lines are underway for more commercially relevant scion cultivars. We have small plants of Mexican Lime, Cocktail grapefruit, Tango, Limoneira 8A, Valencia, Sidi Aissa Clementine and W. Murcott.

Another important aspect of this project is transformation, which is the ability to get DNA into the plant cell. For many commercially important cultivars, we cannot use standard seedling transformation, since the seeds are likely to be hybrids of unknown quality. This requires us to develop a transformation technique from fully mature citrus tissue (see below), which also should reduce the time from transformation to fruit production. In addition, once a Founder Line is created, it needs to be capable of transformation again and again to stack additional genes, even as it ages. There are multiple hurdles to this process such as tissue physiological state, the type of cultivar and its health. Aspects being investigated include hormones,

salts, media matrix, timing, light and temperature. To date, our lab has had success in transforming mature plant tissue from multiple cultivars including Carrizo, Navel, Tango and Limoneira 8A (Figure 4).

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 on plant growth as seen for the wild type plant. However, Founder Line KCN3.2, which contains the codA gene, shows dramatic growth retardation. 5-Fluorocytosine is converted to 5-Florouracil in the presence of codA and acts to inhibit DNA transcription, which in turn leads to cell death.

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.


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.

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GlossaryTransgene: Any gene brought into the genome by means other than traditional breeding.

Recombinase: Enzymes that are capable of cutting and pasting DNA without the gain or loss of genetic material. Further, these enzymes recognize and only manipulate very specific short DNA sequences (recognition sites), which can be added to larger DNA molecules (genome) for the cutting and pasting process.


SUMMARY The second year of this Citrus Research Board-funded research project focused on studying the in planta distribution of “Candidatus Liberibacter asiaticus” (CLas), the huanglongbing (HLB)-associated bacterium, and citrus responses to HLB infection. Using graft-inoculated navel trees, we found CLas to be in greater titer and more evenly distributed in feeder roots than in young and mature leaves over time using quantitative real-time polymerase chain reaction (qPCR). Through mass spectrometry, we also identified citrus proteins that were differentially expressed during infection. These proteins have the potential to serve as markers for HLB, as well as targets that can be manipulated for disease control. By understanding how CLas manipulates its host, better management strategies can be implemented.

CITRUS DISEASE RESPONSE TO HLB INFECTION


Jessica Franco, Deborah Pagliaccia, Wenbo Ma and Gitta Coaker

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.


INTRODUCTIONIn recent years, the citrus industry has been plagued by HLB, a devastating citrus disease. In the United States, HLB-associated CLas is spread by the Asian citrus psyllid (ACP), a piercing sucking insect, which carries the HLB-associated bacterium, CLas. ACP feeds on the phloem of citrus, which is 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 currently rely on infected tree removal and pesticides targeting ACP. HLB has severely impacted the citrus industry in Florida, and the disease has expanded to Texas and California. In California, the first PCR-positive for CLas tree was detected in Hacienda Heights in Southern California in 2012. In the summer of 2015, additional trees in Southern California were 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

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.

RESULTSIn order to study how CLas manipulates citrus, we performed a graft inoculation experiment in the University of California (UC) Davis Contained Research Facility (CRF), a quarantine greenhouse. The CLas strain used for inoculating citrus corresponds to that identified in the first PCR-positive tree found in California

in 2012. Eleven young navel orange trees grafted on Carrizo rootstock were graft-inoculated or mock-inoculated. Samples from young leaves, mature leaves and feeder roots were taken on a monthly basis for eight months (Figure 1). DNA was extracted to detect the probable presence of CLas by qPCR. Our results indicate that CLas DNA detection is more consistent in feeder roots, than in young or mature leaves (Figure 2). CLas DNA detection occurred in the feeder roots as early as the first month post-inoculation. Furthermore, feeder root detection of CLas DNA was consistently detected over time compared to those in aerial parts of the trees (Figure 2).

We currently are repeating these experiments based on psyllid inoculations at the UC Davis CRF. CLas is well known to be unevenly distributed in citrus, making detection challenging. The results from this and subsequent experiments can help guide PCR-based detection strategies.

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.

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Feeder Root Young Leaves Mature Leaves

Figure 2. Navel orange trees were assayed for the presence of CLas in young and mature leaves and feeder roots using real time qPCR over a nine-month time period. A total of six navel trees were graft-inoculated onto Carrizo rootstock in a greenhouse and monitored for HLB infection. One tree did not become infected by CLas.


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.

Future experiments will determine if these citrus proteases are targeting CLas proteins in response to infection and whether they may contribute to HLB development. Furthermore, because these proteases are highly expressed during infection, they hold promise as a biomarker to facilitate HLB detection.

Selected Literature:1. Wang N, Trivedi P. 2013. Citrus huanglongbing: a newly relevant disease presents unprecedented challenges. Phytopathology103(7):652-65.

2. Van der Hoorn RA. 2008. Plant proteases: from phenotypes to molecular mechanisms. Annual Review of Plant Biology. 59:191-223.

CRB Project: 5300-160

Jessica Franco, is a graduate student in the Plant Pathology Department at the University of California, Davis. Deborah Pagliaccia, Ph.D., is an assistant project scientist in the Plant Pathology and Microbiology Department at the University of California, Riverside. Wenbo Ma, Ph.D., is an associate professor in the Plant Pathology and Microbiology Department at the University of California, Riverside. Gitta Coaker, Ph.D., is an associate professor in the Plant Pathology Department at the University of California, Davis.

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.

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Sub+lisin Protease

Cysteine Protease 1

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Aspar+c Protease 1

Aspar+c Protease 2

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Sub9lisin, Cysteine and Aspar9c-‐like proteases are highly expressed during CLas infec9on in Navel

Uninfected Infected *

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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.


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DEVELOPMENT OF LOW-SEEDED CITRUS BY MUTATION BREEDINGWhat Growers Should Expect from Tango and Other Varieties


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


The University of California, Riverside (UCR) Citrus Breeding Program is focused on development of new citrus cultivars that are adapted to California conditions and that fit known or future market windows. We develop new varieties using hybridization and mutation to create variation, evaluate the initial trees and then select some as potential varieties that justify more detailed evaluation.

Trials of these possible new varieties are established in several locations that represent California citrus production areas. These trials are evaluated for several years, and then decisions about release are made. In this article, we describe how low-seeded selections of existing varieties or hybrids are developed, summarize current activities and then summarize data on seed counts in many trials of Tango to illustrate the level of variation in seed content that growers may encounter in such varieties.

MUTATION BREEDING PROCESSTo 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.

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.


STATUS REPORTThe 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 don’t get too excited about them yet). Release of any of these is at least five years in the future.

SEED CONTENT IN TANGO MANDARINTango is certainly the most successful citrus cultivar developed by mutation breeding. It was first identified as a very low-seeded selection in 1998 at UCR, and was propagated and established in evaluation trials at numerous locations throughout the California citrus-producing area in the early and mid-2000s. Additional trials were established after the release of Tango, including several rootstock trials. Since that time, regular collections of fruit samples from various trials have been evaluated for seed count and fruit quality, with the total number of fruit evaluated for seed count now exceeding 24,000.

To date, Tango mandarin fruit have exhibited very low seed counts in virtually all of the more than 1,000 trees planted in the trials. Although seed counts are consistently low, they do vary among locations and among years for the same trial. Here we present a detailed summary of seed counts in Tango fruit that should help growers to have realistic expectations about seed content in Tango, particularly in locations where other varieties that produce abundant, viable pollen are planted nearby.

Figure 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.


TRIAL SITESSeven 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 cross-pollination 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-Cardwell’s 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 grower’s 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

Figure 3. Porterville rootstock trial site showing adjacent plantings that can serve as pollen sources. Image: Google Earth, June 15, 2011.


W. Murcott, satsuma and navel orange. Cross-pollination pressure is expected to be low.

DATA COLLECTION METHODSSeed 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.

RESULTSSeed 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).

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.

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.

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.

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.”


We saw similar variation among years and locations in the number of seeds per fruit in the fruit quality samples that were cut in the lab (Figure 7). Since seed content often differs in successive years while the surrounding pollen source trees remain unchanged, much of the variation is likely attributable to weather during flowering, the duration of flowering and 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

pollen pressure such as at the Porterville and the scion variety trial sites.

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, and no fruit had more than three seeds. Tango trees at Rocky Hill, South Coast, UCR and Lindcove also have produced single fruit with more than five seeds. We

are investigating the origin of tree 9-29 at the molecular level using markers that distinguish Tango and W. Murcott, but do not yet have a definitive explanation. This tree does, however, represent a single tree out of the more than 1,000 Tango trees in trials, none of which have the same characteristics. From this, it appears that genetic or environmental factors can cause Tango trees to produce seedy fruit, but this is a very rare event.

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

Figure 7. Seed content in fruit quality samples of Tango fruit from nine trials. Year effect (*) indicates 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 range was 60 to 1,053. The symbol “ns” denotes “not significantly different.”


CONCLUSIONSTango 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.

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.

AcknowledgementsWe 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.

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