citrograph winter 2016a

7/24/2019 Citrograph Winter 2016a

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www.CitrusResearch.org | Citrograph Magazine 1

MEET CRBS NEW

LEADERSHIPTEAM


2/902 Citrograph Vol. 7, No. 1 | Winter 2016

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Citrographis published quarterly by the Citrus Research Board,217 N. Encina, Visalia, CA 93291. If you are

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12

18

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8 CHAIRMANS VIEW

RICHARD BENNETT

12 BOARD SELECTS NEW EXECUTIVE LEADERSHIP TEAM

RICHARD BENNETT

14 INDUSTRY VIEWS WHAT PROGRESS ARE WE MAKING

ON HLB?

MOJTABA MOHAMMADI, PH.D.

18 WHERE 2016 CRB-FUNDED RESEARCH IS HEADED

MOJTABA MOHAMMADI, PH.D.

22 MEET YOUR BOARD MEMBERS

IVY LEVENTHAL

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 RITABIOREACTORS

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.

72 NOVEL THERAPY OF HIGH-PRIORITY CITRUS DISEASES

HAU NGUYEN, PH.D., ET AL.

76 FIGHTING HLB WITH A CITRUS TRISTEZA VIRUS-BASED

VECTOR

JAMES NG, PH.D., ET AL.

82 DEVELOPMENT OF AN ACP MANAGEMENT PLAN FOR

ORGANIC CITRUS

JAWWAD A. QURESHI, PH.D., AND PHILIP A STANSLY, PH.D.

IN THIS ISSUE WINTER 2016 | VOLUME 7 NUMBER 1THE 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 CRBs new president. Also elected to

the Executive Board were Dan Dreyer as

vice-president and Toby Maitland-Lewis as

secretary-treasurer. For more information

on the new executive leadership team, see

Chairmans View on page 8.

MEETCRBSNEW

LEADERSHIPTEAM



CALENDAR OFEVENTS 2016

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

THE MISSION OF THE CITRUS RESEARCH BOARD:

ENSURE A SUSTAINABLECALIFORNIA CITRUS INDUSTRY FORTHE BENEFIT OF GROWERS BYPRIORITIZING, INVESTING IN ANDPROMOTING 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 2017

Joe Stewart 2017Franco Bernardi 2017

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

Justin 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 Member

Member Expires

Vacant 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 researchinformation, field tours and varietytasting.

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, contactCalifornia Citrus Mutual at

(559) 592-3790.

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


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OUR #1 GOAL:CONTROLLING HLBThe Ultimate and Only Objective

CHAIRMANSVIEW BY RICHARD BENNETT

Richard Bennett

The huanglongbing (HLB) threat to the California citrusindustry is very real. If your tree becomes infected, the fruitwill become unmarketable and the tree will die. Thatsthe bottom line. This disease is the ultimate threat to our

survival.


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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 Liberibacterspecies (Ca. L. asiaticus or CLas) has clearly failed to stem

the spread and progression of the disease in Florida, Texas andCalifornia, according to the recent research of David Bartels,Ph.D., an entomologist at the USDA APHIS PPQ Mission

Laboratory in Texas. Citrus quality in those states is declining,and production in many groves has fallen by more than an

astounding 50 percent. Floridas 2015-16 orange crop isexpected to be the smallest in half a century. The heavy use of

insecticides to suppress the ACP vector populations is not fullyeffective, 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, likeFlorida, relies on regulatory agency data collection, analysis

and diagnostic efforts. However, the effectiveness of theseapproaches is restricted by tight protocols and limited

resources. Our industry also has invested heavily in scientificresearch to assist us in extending our existence long enoughto find a solution. California needs to immediately support the

high-throughput investment of early detection technologydevices.

REALIZING THE THREATThe most current research indicates that the use ofantimicrobials and other treatment regimens (such as thermaltherapy) to extend the productive life of infected trees iscommercially impractical, at best palliative, and at worst onlyserves to keep infected trees in the ground longer. Some

progress has been made in developing cost-effective earlydetection technologies, but there has been little industry

discussion about implementation specifics. After more than15 years and nearly a quarter of a billion dollars of research,

marginal progress has been made in developing a long-termcure for HLB or an HLB-resistant rootstock.

Ignoring the costs associated with the destruction of growerbalance sheets, the cost to manage the spread of ACP in

California assuming 200,000 acres and at least an additionaltwo to five dedicated pesticidesprays per year could reach

$50-120 million annually or more ($250-600 per acre) withinthe next five years. This equates to $.30-.70 per carton on an850-carton per acre grove. Then, there is enhanced nutrition.

From what we know to date, the best programs are costingmore than $500 per acre per year. Again, assuming 200,000

acres, the additional cost to our growers is at least $100million per annum. Therefore, the total cost to the California

citrus industry could be $220 million annually. The cost to the

Trees with dropped fruit, showing symptoms of HLB.



states growers could total $750-1,100 per acre per year, orabout $1.30 per carton on 850 cartons per acre production.

Even then, such expenditures will not stop the spread of HLBin 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 strategyand facing the unintended, but likely, consequences of

encouraging growers to hunker down, rely primarily onbug containment, remove only regulatory-confirmedand/or symptomatic trees and wait for the commercialization

of a magic bullet will only shackle growers with increasinglyhigher farming costs and lead to the inexorable collapse of

Californias productive citrus capacity.

This devastating disease already has manifested itself inSouthern California. Collectively, we must formulate animmediate strategy.

HLB SUMMITOn December 1, the industry brought key forces togetherfor an annual HLB Summit here in California. It differed fromthe meeting held at the University of California-Davis thispast 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 DetectionTechnologies.

In September, the Citrus Research Board (CRB) developed aformat 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 currentlybattling this disease in Florida presented their thoughts ondeveloping short-, medium- and long-term strategies to moveforward. Scientists who can predict infection movement

and detect the bacteria in a pre-symptomatic stage sharedresearch findings.

This one-day session encouraged attendance from a

representative cross-section of industry stakeholders(nurseries, growers, marketers and packers/shippers), as wellas researchers at the forefront of observation/modeling,

diagnosis and treatment of vector populations and thedisease.

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 currentHLB strategies in Florida, Texas and Brazil, (b) the current

state of HLB-related research and (c) the limitations ofexisting 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 tocombat the disease along with broad, but achievable, strategic

directives with two over-arching goals: a.Extend the economic life viability of existing treesin the ground.

b. Shorten the time to development andcommercialization of a cure and/or protectant.

FOUR KEY COMPONENTS EMERGED AS A RESULT OF THE

SUMMIT:Institutionalized Process a transparent and highlyinteractive, managed process that goes beyondsimply coordinating sample collection/analysis efforts

and recommending psyllid control action plans. Itincorporates 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 thatmeets after each data collection/analysis cycle to

do a situation assessment and create and amend

ACP/HLB remediation action plans within the context ofthe latest research and organizational capabilities.HLB Task Force a small accountable organization

tasked with managing the process.Infrastructure includes CDFA, other regulatory andnon-regulatory resources and capabilities.

Our whole industry needs to unite to develop this working

plan. California Citrus Mutual and other industy leadersinitiated 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 mustunderstand 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 whenwe know a tree is infected today. Our industry must standtogether and move forward aggressively.

Florida has the capability of chemically altering orange juice to

mask the HLB flavor change. However, the California industrysells a single piece of unaltered fruit to each consumer. With

fresh fruit, we cannot mask the flavor change. When we, asgrowers, start delivering HLB-affected fruit to consumers,

California will lose its industry. The flavor change that theHLB-infected tree imparts to the fruit is dramatic, and neitheryour family nor our consumers will accept it.

If we allow the HLB bacteria to infect our trees, consumers

will abandon California oranges, mandarins, grapefruit andlemons, and our citrus industry will be lost.

Richard Bennett is the chairman of the Citrus Research

Board.

1.2.

3.

1.

2.

3.4.


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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 Boards annual meeting, new executive officers were elected.

We are pleased to introduce Gary Schulz as the CRBs president. Gary has an impressive resum that

seems tailor-made for his new responsibilities of leading the agency through the challenging periodwe 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 NEWEXECUTIVE 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 allskills that will serve him well as our new president.

Garys most recent post was as president and CEO of the

California Association of Pest Control Advisers and CaliforniaCertified 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 placeon the Board following annual elections. I am serving as your

chairman of the Board, Dan Dreyer is your new vice-chairman

and Toby Maitland-Lewis is your secretary/treasurer.

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

Tulare County grower liaison for the CPDPC in addition to

being the manager of Agriculture Services in Exeter. As a

member of the CRB Board, he is dedicated to outreach and

communicating research outcomes to the industry.

Toby, the chief financial officer of Sun Pacific in Exeter, has

worked in citrus for a decade. He has served on the CRB Board

since 2013 and is primarily interested in the CRBs fiduciary

responsibility and promoting the long-term sustainability of

Californias citrus industry.

I am the owner of Bennett Farms in Exeter and am a citrus

industry veteran. Right now, I am committed to leading an

effort dedicated to finding a solution for HLB (see accompanying

article). I know I also speak for Gary, Dan and Toby in sharing

with you that we look forward to working hard on your behalf

by strategically focusing your funding on research that will

enable your businesses to survive and thrive.

Richard Bennett is the chairman of the Citrus Research

Board.



WHAT PROGRESS AREWE MAKING ON HLB?

INDUSTRYVIEWS 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 progressare we making on huanglongbing (HLB)?


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Greg McCollum, Ph.D.

Research Plant Physiologist,USDA-ARS, Fort Pierce,Florida

The confirmation of Candidatus

Liberibacter asiaticus (CLas) inthe state has been a real wake-

up call. HLB has been foundin two different locations in

California. There is evidencethat the problem is spreading,which means that action needs

to be taken sooner rather thanlater.

HLB is an enemy, but once the

enemy has been identified, you can begin the battle. A lotof novel, very interesting work is being done on detectiontechnologies that are alternatives to PCR. They hold great

promise if we can get closer to whole tree detection, ratherthan a very small fraction of tree detection. To prevent or slow

the development of an HLB epidemic, it is essential to confirminfections as early as possible and take action immediately.

Because trees can be infected with CLas for a lengthy timeprior to the appearance of HLB symptoms, detection of thepathogen is challenging. Although PCR is very reliable in

detecting CLas, if trees are not symptomatic, it is difficultto determine where to collect diagnostic samples. Early

detection means that infections can be found prior to treesdeveloping 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 foundand testing for CLas began, we found the pathogen was

everywhere. California is ahead of the curve on that andhopefully can stay that way. I wish there was a significant

breakthrough in therapeutics or control in general, but weare not there yet. Currently, the standard three-prongedapproach (controlling psyllids, removing infected trees and

only planting clean nursery stock) is still the most importantmanagement strategy.

DISEASE CONTROL STRATEGIESBreeding and transgenic (introduction of DNA from anothersource) 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 beresistant to a host of different diseases. Conventional breedingis attractive. Weve had a breeding program at the ARS for

more than 100 years, and new varieties have come out of thatprogram. It is a very slow process that takes about 30 yearsfrom hybridization to a variety being released. A rootstock is

a bit faster proposition. The advantage is that you maintainthe same scion, but impart resistance through the rootstock,

which would be a great benefit. Some rootstocks that havebeen 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 constantlyseek symptoms and constantly assay as many samples as

possible. It is really a numbers game. The public must beeducated. It is amazing that with the extent of HLB in Florida, I

have seen people who have lived there their whole lives whoare not even aware of the problem. The outreach efforts I have

seen here in this meeting are really important, especially withthe huge number of residential trees in California. Ensuringthat the public is aware of this problem, educating them as to

what they can do to help solve it and encouraging them to doso will go a long way.

BIOLOGICAL CONTROLIn Florida, before HLB, pests were managed almost exclusivelythrough 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 willbe much more amenable to releasing wasps in their backyard

than commercial insecticide spraying.

FUNDING RESEARCH AREASNew detection technologies could provide a real advantageover PCR, though none have been validated. Therapeutics ofany 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 challengeswith HLB management has

been that it takes citrus treesa long time to show disease

symptoms after they becomeinfected. If not removed, the

infected trees serve as newsources of inoculum for diseasespread in the following years.

There have been significant

areas of progress on HLB inrecent years; one of which is developing a range of early

detection technologies that we hope will soon be availablefor regulators and growers to begin folding into monitoringprograms. Earlier detection of infected trees is critical to

narrowing the potential for pathogen acquisition and spreadduring the asymptomatic phase.

Another area of progress is that we are getting a much better

handle on how the vector and the HLB-associated Liberibacterspread in the landscape, what drives their movementand 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 alsothe disease. By refining how we identify early cases of disease,

we can hopefully mitigate some impacts. That process hasbeen 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 somany people in California affected one way or another by the

psyllid or the disease. Were lucky to have a very large networkof 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 promotingearly disease and insect finds and widespread adoption ofcontrol measures. For areas like the Central Valley, hopefullyby the time HLB does arrive, growers and the general public

will be in a much better position to adopt coordinated andaggressive control measures during the early phase when

eradication is most feasible.

BIOLOGICAL CONTROLDisease in urban neighborhoods in Southern California isincredibly challenging to manage effectively, let alone to try

to eradicate. We absolutely are relying on biocontrol as animportant strategy in these areas. Were learning more abouthow to increase its chances of successfully slowing disease

spread. This will likely involve coordinating with homeownereducation programs, emphasizing the need to manage a

variety of species, including Argentine ants, to maximize theeffectiveness of biocontrol agents.

FLORIDA EXPERIENCEWeve learned a lot from the HLB situation in Florida. One of themost important lessons for us early on was the role of human

transportation in the spread of psyllids and disease. Thislesson led directly to steps being taken in California, includingthe 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 amfairly confident that these measures (although theyve proven

burdensome for certain people and industries) are at leastpart of the reason that the pace of the ACP and HLB situationin California has to date been very different from Florida.

Manjunath Keremane, Ph.D.Research Plant Pathologist,USDA-ARS, National ClonalGermplasm Repository forCitrus and Dates, Riverside,California

The California citrus industry isconcerned about recent finds

of HLB-positive citrus treesreported in some residential

areas of Los Angeles County.At this time, containmentand eradication are our main

goals in Southern California.Early detection of Candidatus

Liberibacter asiaticus (CLas)in psyllids and affected

trees, in addition to effectiveeradication of compromised citrus trees, can help prevent thisdevastating disease from spreading in California.


17/90


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EARLY DETECTION TECHNOLOGIESIn order to achieve this goal, the Citrus Research Boardis currently funding research projects on early detection

technologies. With funding from the USDA National Instituteof Food and Agriculture, our group is trying to developtechnologies that can facilitate detection of HLB through

encouraging field-testing by growers, extension agents andany public members interested in monitoring the disease.

The rationale is that if a single laboratory is expected toconduct all the required tests, the task will be onerous.

Involving the public sector in this process will enable thestate to focus its resources on high-risk areas. Large-scale

testing by many will facilitate early detection leading toexclusion 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 arebeing pursued transgenic and conventional breeding. We

have chosen a non-transgenic approach because of the easeassociated with field-testing and the release of promising

disease-resistant cultivars to the citrus community. Approvalby the FDA and EPA will not be needed since the hybridsare a result of natural breeding. Even though the approach

is time consuming initially, the advantages associated withconventional breeding are appealing.

FLORIDA EXPERIENCEThe lessons learned from Floridas HLB experience havebeen useful in defining the regulatory process required for

HLB suppression. Since 2005, we have been developing

methodologies to conduct psyllid testing for the presenceof CLas. This is now used as an early indicator for the

existence of HLB. Testing the psyllid vector would give usa 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 citrustrees contributed to the quick spread of HLB throughout

the state. California has regulations in place to avoid suchincidences. In Florida, it was demonstrated that truckstransporting citrus acted as carriers of disease-spreading

psyllids. Groves along the highway were the first to beaffected by HLB. At present, California has strict regulations

for trucks transporting processed or unprocessed citrus.Numerous guidelines were established based on Floridas

HLB experiences. This has helped slow the spread of HLBin California. Additionally, our outreach program seems tobe 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 associatedwith commercial citrus production in Californiais the principal mission of the Citrus Research Board(CRB). Science and technology have been helping

and will continue to assist California citrus growersin 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 aphloem-limited bacterium, Candidatus Liberibacterasiaticus (CLas), which is transmitted from an

infected plant to a healthy one primarily by the Asiancitrus psyllid (ACP or Diaphorina citri Kuwayama) and

via contaminated budwood propagation. In Florida,

WHERE 2016 CRB-FUNDEDRESEARCH IS HEADEDMojtaba Mohammadi


19/90


the HLB onslaught has caused serious economic losses to thecitrus 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 upwith novel ideas and technologies to detect HLB infection in

pre-symptomatic citrus tissues, so the infected plants can beremoved immediately to prevent further disease spread. Earlydetection methods based on citrus biomarkers will continue

to be used in a systems approach to pinpoint hot spots wherethe disease might be originating. Citrus transcriptomics (small

and micro RNAs), metabolomics (volatile organic compounds

[VOCs] and metabolites), and proteomics (proteins) currentlyare being evaluated to diagnose early HLB infection in pre-symptomatic tissues in California, Florida and Texas wherethe disease has been reported. These promising technologies

need to be fine-tuned, coordinated and validated by othermore 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 todayis to isolate and culture Candidatus Liberibacter specieson an artificial nutrient medium in vitro. Culturing the HLB

bacterium will:

(a)pave the way for better detection and identification of thebacterium and hence the disease;

(b) facilitate studies on the epidemiology of HLB, whichconsequently will foster better disease management

strategies; and

(c) ease high-precision, whole-genome sequencing, leadingthe way to better understanding the molecular biology of

host/pathogen/vector interactions that might identify genesof interest for disease management.

The first and most important line of defense against HLBis constant monitoring of ACP vector movement and the

HLB-associated Liberibacter spread based on data collectedfrom trapping and diagnostic analyses of samples using

early detection technologies. Second is taking sanitary andpreventive measures that include elimination of diseased

plants and regular spraying for psyllids in the Central Valleyand the release of parasitic wasps (Tamarixia radiata andDiaphorencyrtus aligarhensis) to parasitize and kill ACP

nymphal instars on newly-developed leaf flush in SouthernCalifornia urban areas. This is accompanied with the use of

pathogen-free nursery stocks, enforcing quarantine measures

and educating the public through effective out-reachprograms. These coordinated actions already have begun inCalifornia and will continue into the future as long as HLBposes 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 theplant side to mitigate the HLB malady include breeding

for disease tolerance or resistance, use of gene silencing

technology (e.g., RNA interference) and development oftransgenic 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 trialsbefore being commercialized.

The complete list of 2015-16 approved projects may be foundin Table 1on 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.


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The Citrus Research Board (CRB) is governed by 21 dedicated volunteers from a wide variety of backgrounds andgeographical areas of the California citrus industry. Fifteen of the Board members represent northern California; threeare 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. Inthis issue, youll meet three of the most recent appointees.

MEET YOURBOARD 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. Thesescientists 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. Theyare strengthening and raising the bar on management to meet the

requirements of executing the necessary directives. I have no agenda

other than gratitude for the opportunity to serve the industry that

has provided for the livelihood of my family.

A veteran of more than four decades in the citrus industry, Galloway

lives in Porterville, where he is the general manager of Sierra Crest

Agriculture, which has acreage in Tulare and Fresno counties. His work

involves the cultural aspects of citrus and table grape production.

In his free time, the married father of two sons enjoys reading, his RV

and visits to the beach.

The most recent representative

who was elected to District Two in

2014 is Mike Perricone. His areas

of interest are new varieties and

pest management, and he has been

serving on the CRBs New Varieties

and Non-vectored Diseases and

Post-harvest Committees during his

first year.


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I think the greatest issue for all of us in the citrus industry is the

threat of HLB, Perricone said. Its not going to be an easy fight,

but I think we will be able to beat this thing out if we all work

together.

The San Juan Capistrano native who now lives in Temecula is a

relative newcomer to the California citrus industry. For the past

several years, he has served as the operations manager for Pauma

Ranches in Pauma Valley, responsible for citrus and avocado

acreage and staff.

Perricone and his wife have one son. When not working or fulfilling

his CRB Board duties, the D.A.R.E. America volunteer enjoys the

fast sport of roller hockey and spending time with his family.

The newest over-all Board

member, representing District

One and elected in 2015, is Keith

Watkins. As Vice President of

Farming and Field Operations

for Bee Sweet Citrus in Fowler,

he is charged with overseeingapproximately 10,000 acres

and is responsible for land

acquisition, investor relations

and financial oversight. His son,

Matthew, works with him on Bee

Sweets farming operations, which has allowed Watkins the time

to serve as a vice president of the Cawelo Water District and a past

president of the Tulare County Farm Bureau, as well as volunteer

on the CRB Board.

In this latter capacity, the Visalia native is a member of the

Pest Control Committee, which dovetails with his interest in

addressing the current major threats facing the industry. HLBand ACP are dark clouds hanging over us, Watkins said. I want

to help concentrate the CRB in a direction that will focus all of

the research currently being conducted to come up with answers

to those two problems. We need to ensure that the scientists are

talking to each other and building on each others work.

A veteran of more than two decades in the California citrus

industry and an alumnus of the California Agricultural Leadership

Foundation, Watkins stated, We should be focusing on the CRBs

financial responsibility by utilizing the growers money to invest in

areas that will provide good returns to the growers.

When not at work or volunteering in his industry and community,

the married father of three enjoys traveling and family time.

Ivy Leventhal is the managing editor of Citrograph.

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HLB DETECTIONS

IN SAN GABRIELWhere are we now?Victoria Hornbaker and Lucita Kumagai


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In April 2012, the first California detection of huanglongbing (HLB),also known as citrus greening, was confirmed in a tree in HaciendaHeights. It wasnt until July 2015 that the second confirmed California

detection of HLB was found in the San Gabriel area of Los AngelesCounty, about 15 miles from Hacienda Heights. Both finds were

residential citrus trees. Prior to the second incident, it appearedthat no news was good news and that the 2012 find was isolated.However, the 2015 San Gabriel detection has led to nine additional

HLB-positive plant samples. Those detections resulted in the first and(as of November 2015) only cluster of the disease in California. Now

that the dust has settled, heres the sequence of events that occurredin 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-BasedHLB 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 tissueand additional ACP, if available. The kumquat tree was chlorotic but

the leaves did not present the classic asymmetrical mottling typicalof 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 wassent to USDA for confirmation, which was received on July 9. With

the homeowners permission, the tree was first treated with a foliarinsecticide and then removed on July 10. CDFA also surveyed and

took samples of ACP and plant tissue on all adjacent properties andapplied 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 quarantinefor HLB in the San Gabriel area, consisting of a five-mile radius around

the find site, totaling 87 square miles. CDFA then began survey andtreatment activities on all HLB host plants within 800 meters of the

find site. By taking those essential steps, the critical reservoir of diseaseand its vectors (ACP) were in the process of being removed.

On July 13, CDFA Pest Exclusion staff began survey activities ofproduction and retail nurseries in the proposed quarantine area,

placing all host plants on hold.

CDFA crew removingHLB-positive lime tree inthe 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 limetree tested positive by PCR and DNA sequencing. The tree wasremoved with homeowner permission on July 16. CDFAs lab

ran samples to determine if the disease had spread to othercitrus trees in the area.

To inform the public and particularly the neighborhood, a

public meeting was held the evening of July 16 and was verywell attended. Assemblyman Ed Chau was in attendance,as was Kurt Floren, the Los Angeles County Agricultural

Commissioner.

Following the neighboring finds, notices were delivered toresidents 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 Mandarinand a calamondin, on the same street as the initial tree. Both

trees were treated and removed, with homeowner permissionon July 22.

CDFA Pest Exclusion staff continued working with the USDAto 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 optedto construct an insect-resistant screenhouse for the plants.

TREES 5-9 (AUGUST 6, 2015)Five additional trees were confirmed HLB-positive at threelocations within a block and a half of the previous finds. One

location had three trees, and two locations each had oneinfected tree. On August 7, CDFA removed the three treeson the one property with homeowner consent. By the next

day, CDFA removed the two remaining trees on separateproperties, 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 heldon August 11. CDFA continued to treat properties where nocontact 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 thanhalf a mile from the initial positive tree, and it was removed

with homeowner consent. Survey and treatment areas wereexpanded 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


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With the strategy set by the citrus industry and immediate response by CDFA

survey and treatment crews, no other San Gabriel samples have been confirmedfor HLB at the time of this printing.

CONTINUING THE FIGHTIN THE GROVE

This is a wake-up call for all growers, particularly those in Southern California.

Growers should be participating in an area-wide management program to controlACP populations in commercial groves. With HLB in California, ACP managementbecomes 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 CDFAencourage 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 CPDPPs support, CDFA survey and treatment crews will remain vigilant

in looking for HLB and treating for the Asian citrus psyllid in residential areas. CDFA in partnership with the USDA, local agricultural commissioners and the citrus

industry continues to pursue a strategy of controlling the spread of Asian citruspsyllids while the Citrus Research Board and other researchers work to find a curefor the disease.

BY THE NUMBERS

Total ACP samples collected from San Gabriel, includingexpansion 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/resources

http://www.cdfa.ca.gov/phpps/acp

Victoria Hornbaker is with the Citrus Pest and Disease Prevention Program,

where she serves as citrus program manager. Lucita Kumagai is with the Plant

Pest Diagnostics Branch at the California Department of Food and Agriculture,

where she serves as senior plant pathologist.

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Growers, your help is needed to protectCalifornia 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 psyllidto 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 fromfield 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 onpsyllid 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.orgfor 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 yellowand produce a white, waxy substance.

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

HLB-infected trees will die.

CitrusInsider.org


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Huanglongbing (HLB), which translates from Chinese

as Yellow Shoot Disease (also referred to as greening),is the most devastating known disease of citrus. After theestablishment 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 symptomatictrees, respectively.

2) Every country in the Americas adjacent to a CLas-positive

nation detects HLB within five years or less (e.g., Texas andCalifornia proximal to Mexico, and Argentina proximal toBrazil).

3) In areas where HLB is well established, the disease causesunprecedented increases in production costs, crop loss and

reduction of internal and external fruit quality.

While leaf symptoms may resemble nutritional stress ordeficiencies (e.g., zinc and iron), these symptoms actually

are due to disruption of carbohydrate metabolism and

HLB IMPACT ON CITRUS ROOTHEALTH AND INTERACTIONWITH PHYTOPHTHORA

James Graham

Figure 1A


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allocation as a result of plugging and necrosis of phloem, the

plants sugar conducting system. Because of carbohydratedisruption, 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 abnormallycolored and contain off-flavored juice low in brix. In the 10years following the detection of HLB, Florida citrus production

has dropped 50 percent with no sign that the rate of croploss is slowing (http://www.nass.usda.gov/Statistics_by_State/

Florida/Publications/Citrus/cit/2015-16/cit1015.pdf).

At the University of Floridas Citrus Research and EducationCenter (CREC), my research program in collaboration withResearch Assistant Scientist Evan Johnson, Ph.D., has mainly

focused on the belowground impact of systemic CLas infection.What we have learned about bacterial infection, movementand root loss has profound implications for how diseased

trees are managed. Initially in field surveys, we measured30-50 percent loss of fibrous root density before any sign of

aboveground symptoms. Dr. Johnsons detailed investigationof the early events in bacterial infection revealed that CLas

moves downward to the fibrous roots soon after transmissionin the shoots, is unrestricted in the root system and multipliesto damaging levels within months of infection. The fibrous

root loss is distributed throughout the root system becausethe 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 canopysymptoms in foliage, premature fruit drop and a proportionalyield loss of 30 percent based on crop loss estimates in Brazil

and Florida. Without management intervention, root turnoveraccelerates, 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 treesare in a survival mode. What these findings tell us is that the

priority is to grow new roots as the tree declines and thatroot replacement is expensive, reducing fruit production andretention. Hence, stimulating extra root growth is not likely

to help and may increase root production at the expense offruit. 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 theFlorida production system, this begins by minimizing stress

in the micro-sprinkler wetted zone where 80 percent of thefibrous roots are concentrated. More frequent irrigation cycles

of shorter duration and weekly to bi-weekly fertigation are

recommended to maximize efficiency of water and nutrientuptake 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 ofFlorida groves draw irrigation water from deep wells located

in limestone aquifers. Well-water often has a pH in excess of7.5 and bicarbonates above 100 ppm. Our surveys establishedthat 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 Hamlinorange leaf indicative of carbohydrate disruption; (B) HLB-induced fruit dropin eight-year old Hamlin orange trees on Swingle citrumelo rootstock trees inOctober 2015.



Importantly, the combination of CLas infection andbicarbonate stress increases susceptibility to root pests and

pathogens including Phytophthoraspecies. The causal speciesof Phytophthora diseases in Florida citrus are Phytophthoranicotianae (parasitica), the most common cause of foot

rot (gummosis) and root rot, and P. palmivora, which mostoften is the cause of brown rot of fruit and root rot in poorly

drained soils with high water tables. Wet conditions favor rootinfection cycles of Phytophthoraspecies.

Susceptibility of fibrous roots is highest during very wet tovery 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 fromboth greenhouse trials and grove surveys. Phytophthoranicotianae populations initially increased in potting soil attwo, eight and 14 months post-inoculation in HLB-infected

trees compared to un-infected controls. This was followed bya decline after a major loss of fibrous roots mainly due to CLas

infection.

Survey data from Florida groves suggest a resistance-breakinginteraction of CLas with Phytophthoraspecies. Syngenta CropProtection 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. Thesurvey results serve as an indicator of emerging root diseasetrends. Comparison of the survey data for seasons since

HLB became widespread in Florida groves shows a strongtrend toward higher incidence of damaging Phytophthorapopulations coincident with the rise in HLB disease incidence

from 2008-2011 (Figure 2A). Since then, there has been astrong downturn in 2013 and recovery of the populations

in 2014 associated with biennial fluctuations of fibrous rootdensity 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 reductionof root stresses.

Past research experiences and current Phytophthora datatrends indicate a need for more comprehensive management

of HLB-affected trees. Health of fibrous roots is fundamentalto sustain soil, water and nutrient uptake in marginal soils.

This is to resist fluctuations in soil moisture, root pests andother adverse conditions. Symptoms of stress intoleranceinclude off-colored foliage and excessive leaf and fruit drop

in HLB-affected trees, even when trees are managed underintensive nutrition programs for several seasons. Preliminary

data indicate that the CLas interaction also reduces the treesresponse 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 andwater stresses with a balanced application of irrigation and

nutrients to the root system (spoon feeding) and to reducesoil 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. Ifbicarbonate stress is indicated, the recommendation is waterconditioning 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 bybroadcasting sulfur in the wetted zone to reduce soil pH to6.2 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 Phytophthoranicotianaein 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, SyngentaCrop Protection).


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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 Phytophthoradisease 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 phosphiteafter midsummer shoot flush, and mefenoxam after fall shootflushes. 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 SyngentaCrop 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 Phytophthoraspp. Plant Dis. 97:1195-1199.

Johnson, E.G., Wu, J., Bright, D.B. and Graham, J.H. 2014.

Association of Candidatus Liberibacter asiaticus rootinfection, 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 releaseorganic compounds (sugars, amino acids, lipids,

vitamins, etc.) that may serve as chemical attractantsor 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 influencingroot chemistry and biology.

Tree phenology:Plant life cycle events that areinfluenced 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 highin 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 theinterim, area-wide, coordinated chemical control of the Asian citrus psyllid (ACP) is criticalto slowing the spread of HLB in California.

One danger of aggressive ACP chemical control, however, is that resistance can developin the psyllids to some of the more effective and persistent classes of chemistries such asneonicotinoids and pyrethroids (Tiwari et al. 2011, 2012, 2013). We are reporting here onbaseline 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 infuture years.

MONITORING FOR ACPRESISTANCE TO PESTICIDESBaseline Levels Established for 12 Pesticides

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


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High ACP field populations soon after a treatment are notalways 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 othercases, spray timing, choice of chemical or application method

is not ideal. In some situations, natural enemy levels are low inthe treated grove due to past treatment history and thus donot contribute to pest management. Baseline resistance levels

established in the current study can be used in the future todifferentiate pesticide resistance from other factors leading to

less than expected field control.

The University of California (UC) Riverside ACP colony used inthese trials was initiated from psyllids collected from a curryleaf tree (Murraya koenigii) in Azusa, California, on three dates

by David Morgan and the staff of the California Departmentof Food and Agriculture (CDFA) Biocontrol Program during

February 24-27, 2012.

ACP was first discovered in California in 2008, and it is assumedthat this population migrated north from Mexico after havingmoved originally into southeastern Mexico from Florida

and then westward into northwestern Mexico. We assumethese psyllids had little exposure to pesticides in California,

although it is possible that past generations were treated tosome degree in Florida and/or Mexico. Psyllids were reared

under CDFA permit, mostly on curry leaf plants (some citruswas 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 thisbaseline 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 usedto collect about 20 adults into a small plastic vial (Figure

2). Then, a small drop of molten agar was poured onto thebottom of an 8.5 cm diameter Petri dish; once it solidified, a

piece of grapefruit leaf from the UC Riverside Biocontrol Grovewas dipped into the agar at one end to ensure it would remain

fresh during the entire study. The leaf served as a food sourcefor the psyllids to feed on (Figure 3). For each trial, we includedthree replicates of each pesticide rate, with 20 ACP adults in

Figure 2. Lindsay Robinson aspirates about 20 adult ACP into each vial justbefore a baseline resistance micro-applicator test.

Figure 3. A small drop of liquid agar is poured on the bottom of a Petri dishand 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 dioxidefor 60 seconds (Figure 5). With practice, so that treatments

could be applied quickly, the adults remained unconsciouslong 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 andFlorida psyllid insecticide resistance data could be compared.A Burkard Auto Micro-applicator was used to apply small

droplets of technical grade insecticide dissolved in 100percent acetone to the dorsal (back) surface of individual

adult ACP. The foot pedal activating droplet discharge out ofthe micro-applicator syringe made it possible for one person

to hold the 20 ACP on a piece of filter paper, apply a droplet toeach psyllid and then remove them from the syringe tip with

a small paint brush (Figure 6).

In three preliminary trials on June 12, 20 and 28, 2012, we

evaluated the Tiwari et al. (2011) method. Whereas they

applied a 0.2 l droplet to each psyllid, we decided to use0.8 l, so that we were sure each psyllid received a consistentexposure before the acetone in the droplet evaporated. In

these preliminary trials, we also (1) evaluated the minimumexposure to carbon dioxide that was needed and settled on60 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 etal. 2011 used 24 hours). Live adult ACP with their characteristic

feeding posture at 45 off the leaf were easy to tell from deadACP (Figure 7). If there was any question, a small clean paint

brush was used to prod the psyllid to confirm whether it wasdead 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 inthese 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 eachcollection vial.

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


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[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 (84.4percent; Delegate), sulfoxaflor (97.9 percent; Sequoia),

thiamethoxam (98-100 percent, assumed 98 percent in AI

calculations; Actara), tolfenpyrad (99.5 percent; Bexar),andzeta-cypermethrin (93.6 percent; Mustang).

Each pesticide (except Sivanto) was tested twice first,

in Round 1 tests and, second, in Round 2. Sivanto primewas 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 l droplet of pesticide in acetone is applied to each ACP adult with a micro-applicator while they are still knocked out with carbon dioxide. TheACP 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 thanp=0.05, seeTable 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 theremaining 27 Round 1 trials, control mortality ranged from

0 - 11.5 percent, averaging 3.6 percent. Round 2 bioassays ofall 12 pesticides were done from July 17, 2013, to August 27,

2014. During Round 2, we discarded data from two bioassayswith high control mortality (16 percent or higher). Data from24 Round 2 bioassays were used in probit regressions, and

control mortality ranged from 0 - 7.8 percent, with a mean of3.2 percent.

RESULTS AND DISCUSSIONProbit regressions were done separately for Round 1 and Round2 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 LethalDose estimated from the micro-applicator data needed to kill

95 percent of the tested ACP population. Table 1also lists theestimated LD95 values as a fraction of the normal ACP field use

rate. This is a somewhat arbitrary comparison as the pesticideconcentration for the LC95 is based on applying technical

grade material in acetone in a 0.8 l droplet to the back of eachpsyllid, whereas the field pesticide use rate is based on speedsprayer 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 inRound 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 sameas 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 andRound 2 LD95 estimates were the same. This was not true onlywith 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 wascollected from the field. Round 1 tests were done an averageof 14.1 months after field collection (range of five [Bexar] -21

[Sequoia] months; 16 months with Carzol) and Round 2 tests23.7 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 beingindicative of a population with less diversity. For the 10 cases

where Round 2 slopes were steeper than Round 1, the averageslope during Round 1 tests was 3.24, whereas Round 2 slopesaveraged 4.60, an increase of 1.37. For these 10 cases, Round 2

bioassays were started an average of nine months after Round1 bioassays ended (range of three months with Delegate to 17

months with Mustang). This increase of slopes with increasedtime in culture was not unexpected. It is well known that

insect populations become less diverse the longer they arereared in lab cultures.

We designed this study so that data we generated might becompared to data developed on Florida ACP populations by

Tiwari et al (2011). Their work was done on a lab populationof ACP that had been in culture since 2000, and testing was

done on that colony and on five field populations during 2009and 2010. Tiwari et al (2011) studied six of the same pesticideswe 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 twofield populations were examined with Delegate). Comparingtheir data with our Round 1 data, 30/33 Florida bioassays gave

LC95 values that agreed with California levels (the exceptionswere one Florida field population treated with Actara and twotreated 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 fieldpopulations. We know that many of the pesticides used for

psyllid control are quite persistent and hope that growerswill rotate between different classes of chemistry. Should asuspected case of resistance appear, we now have a method

of determining whether or not the resistance is real and howsevere it is. Initially, field resistance bioassays might be done

by choosing several discriminating doses, perhaps at theLD50 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 technicalproduct for testing. We thank Jan Hare, Lisa Forster and othersin the Richard Stouthamer laboratory, who provided ACP for

testing under CRB Project No. 5500-196. We also thank AlanUrena, Lindsay Robinson and Janine Almanzor for technical

assistance.


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LiteratureGrafton-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 Entomology58:413-432.

Lee, J.A., S.E. Halbert, W.O. Dawson, C.J. Robertson, J.E. Keesling

and B.H. Singer. 2015. Asymptomatic spread of huanglongbingand implications to disease control. Proceedings of theNational 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 citruspsyllid in Florida. Pest Management Science67: 1258-1268.

Tiwari, S., L.L. Stelinski and M.E. Rogers. 2012. Biochemicalbasis of organophosphate and carbamate resistance in Asian

citrus psyllid.Journal of Economic Entomology105(2): 540-548.

Tiwari, S., N. Killiny and L.L. Stelinski. 2013. Dynamic insecticidesusceptibility 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 LD95to

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

Classa name (company) Bioassay test dates N

b statistic Slope SE insect)c

95% Cid

insect)c

95% Cid use rate m AI/liter

erate

f

Class 1A (carbamates)

formetanateCarzol 92 SP

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

13570 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)

chlorpyrifos

Lorsban 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 3.3606-5.2209

Class 4A (Neonicotinoids)

imidaclopridAdmire Pro 4.6 EC

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

10-10-12565 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 0.6403-0.8624

Class 4C (nicotinic acetylcholine receptor agonists)

sulfoxaflorSequoia 2 EC

9-18-13; 10-9-13;

11-25-13623 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)

flupyradifuroneg

Sivanto 1.67 EC

(Bayer)

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

27-14782 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)

spinetoramDelegate 25% WG

9-11-13; 9-24-13; 1

8-14372 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 2

7-31-13; 7-30-14; 8

6-14414 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 1.864

citrograph winter 2016a

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