IFB_Summer_v1.pdf

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CitrographSummer 2013

SavoringCitrus

Givaudans TasteTrekCitrusexplores new scent

and favor trends


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3/48Summer 2013 Citrograph3

IN THIS ISSUE

Citrograph is published quarterly by the Citrus Research Board, 217 N. Encina, Visalia, CA 93291. Citrograph is sent to allCaliornia citrus producers courtesy o the Citrus Research Board. I you are currently receiving multiple copies, or would like

to make a change in your Citrograph subscription, please contact the publication ofce (above, let).Every eort is made to ensure accuracy in articles published by Citrograph; however, the publishers assume no responsibility

or losses sustained, allegedly resulting rom ollowing recommendations in this magazine. Consult your local authorities.

The Citrus Research Board has not tested any o the products advertised in this publication, nor has it verifed any o the

statements made in any o the advertisements. The Board does not warrant, expressly or implicitly, the ftness o any product

advertised or the suitability o any advice or statements contained herein.

An Ofcial Publication o the Citrus Research Board

PUBLICATION OFFICE

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Single Copies: $1.501-Year Subscription: $15.00

2-Year Subscription: $28.00

SUBSCRIPTIONS

Send Subscription Requests To:Citrus Research BoardP.O. Box 230, Visalia, CA 93279

P.O. Box 230Visalia, CA 93279Phone: 559-738-0246

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Canadian & Foreign:



Louise Fisher, Managing Editor

Dr. MaryLou Polek, Chief Science Editor

Dr. Mary Lu Arpaia

Ted Batkin

Richard Bennett

Franco Bernardi

Dan DreyerDr. Ben Faber

Jim Gorden

Julia Inestroza

EDITORIAL BOARD

4Editorial

8CRB welcomes new president andcitrus veteran Ken Keck

12Huanglongbing: Why is HLB such aormidable threat?

15 Bringing resh perspectives tocitrus avors

18 Comparing orange juice rom healthy

vs. HLB-aected trees223rd Annual Citrus Health Research

Forum: Vision or the Future

28 Improving trapping systems or earlydetection and eradication o ruities in Caliornia

36 Assessing actors inuencing the post-

harvest quality o Caliornia mandarins40 Citrus Roots: Caliornia citrus

growers enjoyed early love aairwith vehicles

44 Meet Che Nathan Lyon

SCIENCE REVIEW PANEL

Dr. Mary Lu Arpaia

James A. Bethke

Dr. Abhaya Dandekar

Dr. Akif Eskalen

Dr. Stephen GarnseyDr. Joseph Smilanick

Editorial services provided by Ivy Leventhal,principal of Ivy Leventhal Writing Services, Brandon, FL.

FAX: 559-738-0607

Web Site:http://www.citrusresearch.org

SUMMER 2013 VolUME 4 NUMbER 4


4/484Citrograph Summer 2013

EDITORIAL BY KEN KECK, President

Comparing Oranges to OrangesIm amazed by the

beautiul, lush canopies

o citrus, which are a joy

to behold, particularly

next to what Florida

citrus trees lamentablyhave been enduring this

past decade.

Now that Ive been on board at the Citrus Research Board

(CRB) three months, Ive had a chance to catch my breath

and reect on my frst impressions o the incredible indus-

try out here in Caliornia. What Ive learned is that the

2,631-mile distance between Lakeland and Visalia is a good metaphor

or the dierences between the Sunshine State and Golden State cit-

rus industries.

Unlike Florida, where the acreage has been somewhat com-

pressed by the reeze-line, Im struck by how spread out the Calior-nia industry is all the way rom the Mexican border to Madera, a

distance o nearly 600 miles. Not only that, but the trees are so much

healthier and better looking out here. Im amazed by the beautiul,

lush canopies o citrus, which are a joy to behold, particularly next

to what Florida citrus trees lamentably have been enduring this past

decade.

Im urther impressed by the healthy liestyle in Caliornia, which

defnitely is better or the body and soul. There are mountains, ocean

and microclimates galore. Its a great place to be outdoors.

Ive also received an education in diering citrus practices. For ex-ample, I was in a meeting not long ago where we were discussing po-

tential methods to get rid o Fuller Rose Beetle eggs on citrus, which

Koreans do not want to see on their imported ruit. Someone suggest-

ed CRB-unded research to use nematodes under the canopy as a

bio-control method to eliminate the beetles. I was horrifed. In Flor-

ida, nematodes are tremendous pests that oten destroy tree roots

and root hairs, causing the decline and death o citrus groves. To

deliberately distribute them under trees let me speechless. It was

then explained to me that there are certain Caliornia nematodes

that are benefcial and can be used to target certain kinds o insects.I realized I still have a lot to learn about the dierences between

growing citrus on the West Coast and the East Coast!

Thats not to say that there arent commonalities. Whether its

Caliornia or Florida, Ive ound that American citrus growers and

other industry members are known or their hard work, dedication,

perseverance and justifable pride in the worlds best-tasting, saest

and most wholesome citrus.l

To learn more about Ken and where he hopes to lead the Citrus

Research Board, see CRB welcomes new president and citrus veteran

Ken Keck on page 8 o this issue.



District 2 Southern California Coastal

Member Alternate

Earl Rutz Alan Washburn

Joe Barcinas John C. Gless

District 1 Northern California

Member Alternate

Toby Maitland-Lewis Jack Williams

Donald Roark Dan Dreyer

Jim Gorden Dan Galbraith

Joe Stewart Franco Bernardi

Etienne Rabe John Konda

John Richardson Jeff Steen

Kevin Olsen Tommy ElliottRichard Bennett Justin Brown

CITRUS RESEARCH BOARD MEMBER LIST BY DISTRICT 2012-2013

Citrus Research Board

217 N Encina, Visalia, CA 93291PO Box 230, Visalia, CA 93279

(559) 738-0246

FAX (559) 738-0607

E-Mail [email protected]

District 3 California Desert

Member Alternate

Mark McBroom Craig Armstrong

Public Member

Member Alternate

Ed Civerolo Steve Garnsey

The Mission of the Citrus Research Board:

Develop knowledge and build systems for grower vitality.Focus on quality assurance, clonal protection, production research,

variety development, and grower/public education.

DO YOU KNOW...?

CALENDARSeptemer 25 ACP/Hlb Grwer Meeting. Sunkist Center CA Citrus

State Histric Park. Riverside, CA. Fr inf, cntact

Victria Hrnaker at (916)403-6652.

Septemer 26 Citrus Pest and Disease Preventin Cmmittee

(CPDPC). Riverside, CA. Fr inf, cntact Victria

Hrnaker at (916)403-6652.

Septemer 26 Caifrnia Citrus Quaity Cunci (CCQC). Krean

Exprt Requirements. Visaia Cnventin Center. Fr

inf, cntact CCQC at (530) 885-1894.

octer 8 lemn Fied Day. Cachea Vaey ARS (CVARS). Fr

inf, g t www.citrusvariety.ucr.edu

octer 10 Caifrnia Citrus Quaity Cunci (CCQC) Cnference.

Visaia Cnventin Center. Fr inf, cntact CCQC at(530) 885-1894.

Nvemer 5-7 Caifrnia Citrus Nursery Sciety (CCNS)

Cnference. la Quinta, CA. Fr inf, cntact Tm

Den at [email protected]

Nvemer 12 Mandarin Fied Tur. lindcve REC, Exeter, CA. Fr

mre, g t www.ucanr.edu/sites/indcve

Why is fsh served with lemon? (Go to page34 for the answer.)


6/48


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The Citrus Research Board (CRB) warmly welcomedcitrus industry veteran and leader Ken Keck as its newpresident on June 1. Keck replaced retiring President

Ted Batkin, who had served at the helm o the organizationor the past 20 years.

According to CRB Chairman Earl Rutz, As an industry,we are acing some serious threats, most notably the contin-ued identifcation and spread o the Asian citrus psyllid andHuanglongbing (ACP/HLB) in our state. Its a complex andchallenging time, but we believe Ken has the skills to takeus orward.

With the 3,500 citrus growers in Caliornia continuing

CRB welcomes new presidentand citrus veteran Ken Keck

Ken Keck and the CRB delegation, led by Dr. Harold Browning o the Citrus Research and Development Foundation, recentlytoured and examined a Florida citrus grove or visual symptoms o HLB. Aterward, a close and mandatory inspection by thegroup ound several psyllids that had attempted to hitchhike a ride on Richard Bennetts yellow shirt.

to invest in the research necessary to aid the states $2 bil-lion+ citrus industry and battling potential threats to theirgroves and livelihoods, Keck proved a logical choice to takethe reins. He has deep-rooted expertise representing citrusgrowers in a governmental, legal and regulatory ramework.His thorough industry knowledge includes experience infghting ACP/HLB in Florida while at the Florida Depart-ment o Citrus (FDOC), a state agency charged with pro-moting Florida citrus products that is fnanced primarily bygrowers through a box tax. Keck served as general counseland executive director o the FDOC rom 2006-2012 andas director o government aairs and general counsel rom



2002-2006. Prior to that, he was director o legislative andregulatory aairs with Florida Citrus Mutual rom 1999-2002.

Kens track record, enthusiasm and alignment with theperspective o Caliornia growers will make him an excellentasset to the CRB, said Rutz.

Among Kecks accomplishments with the FDOC, hesecured an average o $7 million annually in ederal and

state appropriations or programs; commissioned a NationalAcademies study, resulting in the establishment o a dedi-cated $10 million annual disease research, development andcommercialization oundation; and achieved a range o $3-$5 return to growers or every marketing dollar expended.Keck achieved these results not only because o his tirelesswork ethic, but also rom his understanding o what citrusgrowers ace.

Im a third-generation grower in Florida, so I know thenature o the business. I know what it means to depend onthe harvest each year. I know what it eels like to have yourlivelihood threatened, and I want to use this understanding

and my experiences in Florida to beneft the Caliorniaindustry.However, Keck emphasized that he has not come on

board as someone who has all the answers. He vividly re-calls a sign behind the cash register at the local auto partsstore in Lake Placid, Florida, where he grew up. The signread, THERE WILL BE A $10 SURCHARGE FOR YOUTELLING US HOW MUCH BETTER THINGS ARE UPNORTH. Many years later, he still recalls that sign as areality check in humbleness.

Viewing the ChallengesHLB is our short- and medium-term challenge, Keck

said. The biggest threat and the biggest shame would be iwe did not learn the lessons o what happened in Floridaand Texas. Unortunately, its a matter o when, not i, HLBwill make its next appearance ollowing the fnd in HaciendaHeights. Will our growers understand the critical need to getrid o trees once the frst twig shows up with a visual symp-tom or i technology uncovers a problem even beore symp-toms appear? A tree must be removed in its entirety right

then and there. Keck said he believes that Caliornians aretaking the looming specter o HLB very seriously.

The water shortage is viewed by the new CRB presidentas a long-term challenge. With an average rainall o fveinches in many growing areas, the big question is whetherCaliornia groves can manage on their water allotments indrought years when they must cut back on optimal wateringlevels, he said.

Additionally, Keck sees phytosanitary barriers as a large

ongoing challenge to the industry, citing the example o Chi-nas current ban on Caliornia citrus imports.

The threat o not learning the ACP/

HLB lessons rom Florida and Texas is

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Welcoming the OpportunitiesThe threat o not learning the ACP/HLB lessons rom

Florida and Texas is also the biggest opportunity, Keck said.Specifc to the CRBs core competencies and mission,

Keck sees a tremendous opportunity to prove that geocen-tric research need not rule the day. Caliornia and Floridacan work together - coordinating, cooperating, prioritizingand unding, particularly in the ace o ACP/HLB, he said.Lets decide on commonalities and set aside respective potso money to jointly issue proposals, vote, und and listen tothe fnal reports. We can make one plus one equal three. Thiscould be handled with our respective existing inrastructuresand could involve cooperating university researchers.

Path to StreamliningOne o the frst things that struck Keck when he took the

reins o the CRB was the sheer number o meetings 42 inthe previous fscal year alone that resulted in 126 noticesbeing sent to the industry. We have so many dedicated, un-paid participants in our industry who spend hours traveling

to and attending these gatherings, he said. They then returnhome to attend to their groves and their real jobs. While theindustry appreciates its democracy, I believe we can makethis process more efcient and more manageable or volun-teers who serve on our board and committees.

As an administrator, Keck wants to ocus eorts on pro-grams most critical to Caliornia growers competitiveness.

He believes the way to attain this efciency is to stream-line and specifcally ocus on aligning committees with theCRBs mission. We need to become an organization thatunderstands its fnite resources in terms o mandate, corecompetencies and dollars, he explained. The CRB is cur-rently doing and unding a lot o non-core activities. Wehave to narrow and refne our strategic plan and then alignour mission, personnel and scientifc review process againstthat plan.

To start on the path to achieving this goal, board mem-

Julie and Ken Keck, accompanied by Fletcher and Guinness,inspect the groves.

We have to narrow and refne our

strategic plan and then align our

mission, personnel and scientifcreview process against that plan.

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bers recently traveled to Florida to meet with the Citrus Re-search and Development Foundation board to study ways tostreamline. Keck believes that the majority o the industrywill welcome this ocus and resulting efciencies.

Looking at ROIAnother way to create urther efciency, Keck believes,

is through economic analyses o the research categoriesand areas that have been unded in the past. We need tolook at the return on investment (ROI), he said, althoughthis could be difcult. Is there a way to express the ROI onresearch to our constituencies? This would give our Boardtools to guide the unding parameters.

Keck also thinks that ROI analyses would provide anadditional metric or growers to measure CRBs investmentsin research. It might also encourage State and Federal leg-islators and policy-makers to direct incremental unds to cit-rus research by demonstrating the positive return potential.

There should be a relationship between an ROI analy-sis and unding levels, whether those unds are derived rom

growers or government, Keck said. While he believes thatgrower unding should ow at least at the present level, heeels that i Caliornia and U.S. ofcials could see the con-sequences o ACP/HLB, there could be the opportunity ormore general revenues to ow into the research coers.

We need to dramatically demonstrate the need -through pictures and video, yes, - but also by way o soundeconomic impact analysis, Keck said.

The new president has his work cut out or him, but herelishes the upcoming challenges and the opportunity tohelp protect the Caliornia citrus industry while enhancingthe competitiveness o its growers.l

Meet Ken Keck

BirthplaceLake Wales, Florida

EducationBachelors degree in Spanish rom Stetson Univer-sity in Florida and Juris Doctorate in law rom Wid-ener University School o Law in Delaware

FamilyWie Julie; two high-maintenance aux-Pomera-nians - Fletcher and Guinness; and Kenya, the cat

Citrus rootsThird-generation grower with groves in Lake Placid,Florida

Outside interestsNever mastering the game o gol; learning aboutCaliornia geology

On his nightstandAssembling California by John McPhee (also authoro Oranges)

While learning about Caliornia topography, Keck seeswhat its really like to try to push a rock up a hill.



Huanglongbing (HLB), akayellow shoot disease or citrusgreening disease, is by ar the

most severe disease o citrus in the

world.The HLB-associated pathogens are

three species o a astidious (see KeyTerms), phloem-limited bacteria inthe genus Liberibacter. These bacteriacannot live outside the phloem cells ocitrus or the organs o the insect vector,which can transmit the bacteria roman inected plant to a healthy plant. Inaddition to the insect vector, the Asiancitrus psyllid (ACP), or Diaphorinacitri, HLB can be transmitted by grat-

ing with inected plant propagatingmaterials.

Despite over two decades o re-

Huanglongbing:

Why is HLB sucha ormidable threat?MaryLou Polek

Fig. 1. ROOT MASS LOSS: Scientists inFlorida reported that 30 to 40 percento the root mass is lost beore visual

symptoms are observed in the leavesor beore leaves test positive using thepolymerase chain reaction (PCR) assay.

Fig. 2. NUTRIENT DEFICIENCY: The tree may look as though it is suering romzinc or other nutrient deciencies.

search and hundreds o combinationso growth media, HLB-associatedbacteria have defed laboratory culture.This makes lie difcult or scientistsconducting research on the disease be-cause they must maintain living plants

inected with the Liberibacterbacteria.Federal and state regulations mandatethat these plants must be containedwithin protective structures. This addsto cost and efciency o projects lookingor control solutions.

I this pathogen is so difcult to grow,how can it be so devastating?

Once the bacterium gets inside theplant, the plant cannot be cured o it. Itwill continue to multiply and circulate

throughout the entire plant.First, the bacterial cells go down intothe roots and kill them. Scientists inFlorida have reported that 30 to 40 per-cent o the root mass is lost beore visualsymptoms are observed in the leavesor beore leaves test positive using thepolymerase chain reaction (PCR) assay,which is the USDA-APHIS approvedprotocol or Liberibacter detection(Figure 1).

I the tree is already aected byPhytophthora root rot or nematodes,

the eects o a multiple inection withHLB bacteria are synergistic, and thesymptoms are more severe than i theplant was inected by only one o thesepathogens.

As the disease progresses, the patho-gen reproduces and travels through theplants phloem cells back up into thescion. Here the bacteria plug up theplants vascular system, starving the hostcells o necessary water and nutrients.Subsequently, growth o the scion is hin-

dered and leaves are decreased in size,become yellow and drop. The tree maylook as though it is suering rom zincor other nutrient defciencies (Figure 2).

In Caliornia, we are accustomedto our citrus trees being very lush anddense. In parts o the world where theimpacts o HLB are severe, citrus treesare not vigorous and have sparse oliage(Figure 3). Trees may suer branch orstem dieback.

When they are drought-, moisture-or temperature-stressed, HLB-aected

trees have a tendency to drop their ruitprematurely. Many Florida growers



Fig. 3. SPARSE FOLIAGE: In parts o the world where the impacts o HLB aresevere, citrus trees are not vigorous and have sparse oliage.

were horrifed this past year when theysuered rom an unprecedented ruitdrop attributed primarily to HLB. Al-though trees can certainly die rom thisdisease, they mainly become so debili-tated and unproductive that a grower

or homeowner readily removes them.Perhaps the worst symptoms o all

are those that aect the ruit since,ater all, its the ruit that growers areproducing or income. Not all ruit onan inected tree show visible symptoms.Those that do are generally decreasedin size and misshapen (Figure 4). InSouth Arica, aected ruit tend tostay green at the bottom end, thus thename citrus greening disease. Fruitquality is greatly reduced with a bit-

ter or insipid avor. In Florida, wheresome 90 percent o harvested citrusis processed, aected ruit ruins thetaste and sweetness o orange juice.Caliornias citrus reputation is basedon its production o high-quality tableor resh ruit. This reputation will belost i HLB becomes established in ourcommercial citrus.

Those who attended the CaliorniaCitrus Conerence in Porterville lastOctober will remember the taste test-ing conducted by Drs. Mary Lu Arpaia

and Elizabeth Baldwin. Panelists weregiven juice samples at random that Dr.Baldwin brought rom Florida. Somewere rom known HLB-inected treesand others were rom healthy trees.

My initial reaction to my sampleswas that it was not the best-tasting

juice, but it did not seem that bad. Iwas disappointed, thinking I had notreceived the HLB juice. However, a-ter several minutes, I had a distinctlybad and somewhat metallic taste in my

mouth. The unpleasant taste lasted orabout an hour.I HLB becomes established in

Caliornia, it will change citrus growingas we know it. Gone will be the lush,dark green-leaved trees, the tasty, sweet,perectly-shaped ruit that Caliornia isknown or, and gone will be the beau-tiul citrus trees in our groves and inCaliornians backyards.

How do I know i my tree has HLB?It is not easy to diagnose a tree

positive or HLB. Visual symptomso the canopy can resemble other dis-

eases, nutrient defciencies, toxicitiesor improper horticultural practices.It may take months or even years orsymptoms to develop ater the treebecomes inected.

The most accurate method o de-

tection is perormed in a laboratoryby qualifed individuals. Lea and/or

root samples are collected, and theDNA is extracted. The assay method,polymerase chain reaction (PCR), isused to look or unique segments obacterial DNA.

Although this is the approved

detection by the USDA and CDFA,this method has some limitations. The

Fig. 4. GREEN FRUIT: In South Arica, aected ruit tends to stay green at thebottom end, thus the name citrus greening disease.



bacterium is not evenly distributedthroughout the plant; thereore, i thesample collected does not happen tocontain the bacterium, a alse negativetest may result. The bacterial populationappears to be more consistent withinroot tissue, but this type o sample col-lection is more difcult, time consuming,and somewhat destructive.

The Citrus Research Board hasunded several projects in the past ewyears with the objective o developingmethods that are able to detect the pres-ence o HLB-associated bacteria muchsooner than the currently accepted PCRmethod.

Several research groups have madegood progress, and their platorms arecurrently undergoing evaluation. In-stead o testing or the actual bacteria,these assays detect proteins secreted by

the bacteria or responses o the inectedplants to the presence o the bacteria.Inection by a pathogen induces sev-eral types o plant responses, includingunique volatile organic compounds(VOCs), small RNAs and unique me-

Key Terms

Fastidious: Describes organ-

isms that cannot be cultured

on artifcial nutrient media and/

or under laboratory conditions.

They can only survive in specifc

environments (e.g., within the

cells o their host).

Phloem: Plants have a vascular

system that is composed o the

xylem, which conducts water

rom the roots upward to the can-

opy and the phloem. The phloem

conducts nutrients rom the roots

upward to the canopy and carbo-

hydrates produced by photosyn-

thesis in the leaves downward or

storage in the roots.

Incubation period: The time

interval between plant inection

and the appearance o disease

symptoms.

Latency period: The time inter-

val between plant inection and

when the pathogen can be trans-

mitted to other plants or acquired

by an insect vector.

Small RNAs: small molecules,

typically 22-24 nucleotides in

length o Ribonucleic Acid. Many

small RNAs do not code or pro-

teins, but instead exercise controlover those RNAs that do, thereby

altering gene expression.

tabolomic compounds. Scientists arecapitalizing on these responses and haveound these compounds to be moreevenly distributed throughout the plantthan the pathogen. These improved de-tection assays will be available or usein one to two years.

What can be done to protect our

citrus?Regulation o the importation o

propagating materials prevents theintroduction o bacteria associated withHLB or greening through that mecha-nism, but there are other pathways --the illegal importation o budwood, andthe traveler or immigrant who unknow-ing packs citrus material rom inectedsources. The message or everyone,whether planting a commercial grove ora citrus tree in a backyard, is to always

buy the trees rom a reputable nurserythat obtains their propagation materi-als rom a certifed, disease-ree source.

Where the disease is endemic, itmay be controlled by the reductiono inoculum sources (eradication o

inected trees) and insect control bypesticide application.

In Caliornia, the citrus industry andthe Caliornia Department o Food andAgriculture (CDFA) have partneredand are conducting a survey to look ortrees inected with HLB-associated bac-

teria. I you are a grower or have citrustrees in your backyard, be vigilant andrequently look or the ACP vector. Forthe beneft o uture generations, pleaseallow agriculture inspectors to collectlea or root samples rom your citrustrees. I the disease is out there and itcan be ound early, beore the Asiancitrus psyllid can spread the pathogento other trees, we have a good chanceto win the battle against this devastat-ing disease.

MaryLou Polek, Ph.D is the Vice

President, Science & Technology or the

Caliornia Citrus Research Board.l



On a crisp, cool day in January, a team rom GivaudanFlavors joined Dr. Tracy Kahn to tour the CitrusVariety Collection on the campus o the University

o Caliornia, Riverside (UCR). In collaboration with the Uni-versity, the team was participating in a Givaudan-sponsoredTasteTrekCitrus. What is TasteTrek? One needs to start atthe beginning.

Citrus is the worlds avorite avor and aroma. Whetherit is home ragrances or beverages, people around the globelove the upliting rereshment o citrus. To satisy consum-ers constant desire or new taste experiences, researcherscan look to Mother Nature to inspire tomorrows top citrusavors.

Getting the Givaudan avorists out o their labs and intothe world to discover sources o avor inspiration lets thempersonally experience new tastes, trends and ingredients, ex-panding their thinking on how to help ood and beveragebrands appeal to todays global consumers. Its this go tothe source philosophy that drives the TasteTrek program.

Since 2006, Givaudan has partnered with the University

o Caliornia Riverside Citrus Variety Collection to exploresome o their more than 1,080 unique and globally diversecitrus varieties. Exploration o the ruit is a total sensoryexperience. Through sight, smell, touch and taste, each ruitis experienced entirely, inside and out, to identiy extraordi-nary avor characteristics that could lead to new ideas, avormolecules or ingredients (Fig. 1).

On a recent day at the Citrus Variety Collection (CVC),the Trek team consisted o 8-12 Flavor Chemists, AnalyticalChemists, Beverage Applications and Marketing Specialists,whose primary goals were to fnd ruit with unique avorattributes, evaluate what makes it special and determine

how the avor profle can best be applied to a beverage. TheGivaudan team works with all types o products rom juicedrinks to carbonated sot drinks to avored waters to lowalcohol avored beers and malt spirits. They consider markettrends and consumer appeal to determine what avors mightbe the best ft or urther development.

Prior to visiting the Collection, the sta at Givaudan hasworked with Dr. Tracy Kahn, Curator to develop a list o in-teresting ruits. Dr. Kahn created a map o the locations othe specifc trees the group wanted to visit in the grove. Shebegan the day by sharing a ew varieties, recently developedby the UCR citrus breeding program, that she thought mightbe o interest.

The team set o on their Trek, supply wagon in tow, tosample and taste mandarin varieties and hybrids. Some o

Bringing resh perspectivesto citrus avors

Givaudan Collaborates with University of California, Riverside on TasteTrek Citrus

By Jennier Haggard and Dawn Streich

Fig. 1. Givaudan Flavorist Roberto Terreros knows thataroma is critical to favor development.

Fig. 2. Headspace capture o a lemon in progress.



Fig. 3. Details are taken on all attributes of a fruit as it istested.

the more interesting mandarin types tasted were SeedlessKishu, with its juicy, delicate avor; UCR-developed GoldNugget with its sweet, sugary profle; the complex Indio-mandarinquat, which combines the best o mandarin andkumquat oral notes; and Tango, a variety released by UCRin recent years or commercial propagation, with a proflethat has broad consumer appeal.

Once the team identifed the ruits with the most uniqueprofles, they chose a tree or headspace capture, which en-tailed taking a plug out o one resh ruit still hanging on thetree, and inserting a scent-capturing device connected to acomputer (Fig. 2). This set-up allowed capture o the reshruit scent and ed the inormation directly to a computerthat analyzed the aroma profle. The results o this headspace

Look deep into nature, and then you will understand everything better. -Albert Einstein

Founded more than 200 years ago, Givaudan FlavorsCorp. is the leading company in the favor and ra-grance industry, supplying 25 percent o the worldsfavors and scents to everything rom soda to abricsotener. Givaudan is committed to understandingeverything about citrus favors what makes citrusunique, why consumers across geographic, social andcultural demographics are drawn to citrus and whatis the next big citrus favor. Rooted in its rich his-

tory o citrus expertise, Givaudans program ocuseson innovative new technology and creative vision.

gering on their taste buds helps to build in the unique nuanc-es only the resh ruit provides. Fine tuning is then done inthe lab ater they return. In the last 13 years o working withUCR, Givaudan has evaluated nearly 150 ruits and createdmany distinctive avor varietals ideal or juice drinks, sportsdrinks, carbonated sot drinks and beyond.

TasteTrek Citrus has been a stimulus and source oinnovation or many key Givaudan citrus initiatives. Whena Japanese customer requested in-depth understanding oresh graperuit and insight or authentic avor design, theteam worked with the CVC over several months to collectruit to capture the sensory profles and chemical compo-sition o Marsh graperuit juice throughout ripening. In astudy that spanned six months, Givaudan gained valuablelearning about the evolution o the woodiness character,emergence o berry notes and changes in complexity andbalance o the graperuit avor as it matured. As a result,

a collection o true to ruit seasonal avors was designedto mimic graperuits ripening, starting in early season anddeveloping through mid, peak and late season.

When the Givaudan citrus team in North America be-gan to see the growing popularity o lemonade, they usedtheir broad understanding o lemon and lime varietalsrom the grove to design a diverse range o gold standardavors, which were sensory and consumer tested, includingthe Meyer, Amber, LoPorto and Variegated Pink FleshedEureka lemons, and Millsweet Limetta, etc. Among thesewhite space avors, pushing the boundaries rom tradi-tional lemonade to potential signature status, Amber lemonwas the it avor. A combination o Amber lemon and Mills-weet Limetta was a hit with its lemon-lime and candy notescomplimented by nuances o orange, graperuit and blackpepper. In a close second, Amber lemon combined with Var-iegated Pink Fleshed Eureka lemon was extremely popularbecause o its unusual lemon lime profle with orange pithand jammy raspberry notes.

What is next in citrus? The possibilities are endless.Bringing together avor expertise and collaboration be-tween Givaudan and the UCR Citrus Variety Collection, the

journey continues to discover and bring the best o naturescitrus palette to consumers taste palettes.

Jennier Haggard is Head o Global Marketing at Givau-

dan Flavors Corp. Dawn Streich is Citrus Product Manageror Sweet Global Business Development at Givaudan.l

capture technique, along with sensory and other chemicalanalyses, provide technical insight into the chemical compo-nents responsible or intriguing avor notes (Fig. 3).

Once back in the lab, sensory profling, using SenseItdescriptive avor language, enables the team to quantiyand characterize the presence, quality and intensity o di-erent avor attributes. GC/MS analysis o the juice and peeloil allows them to link the sensory attributes to the natural

chemical(s) responsible or the avor. From these chemical-sensory discoveries, Givaudan scientists identiy potentiallynew avor materials that they can either synthesize or natu-rally recover and add to the avor creation palette.

The true art o translation rom citrus ruit to avorcomes rom the inspired minds o skilled avorists and theuse o Givaudans proprietary avor creation tool, the Virtu-al Aroma Synthesizer (VAS). The portable miniVAS toolcombines the human perception o smell, combining variousscents or keys into a single olactive profle, and precise in-strumentation to transorm desired aroma profles into fn-ished avor ormulas. It allows avorists to get 80 percento the way to their target avor while still in the UCR CVC

grove. Creating the avor while the excitement o being inthe grove is still resh in their minds, the taste o the ruit lin-


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Comparing orange juicerom healthy vs. HLB-aected treesA look at the chemical and sensory favor dierences

Fig. 1. A comparison o ruit rom healthy trees vs. HLB-aected trees.

Greening or Huanglongbing(HLB) disease o citrus isthought to be caused by a bac-

teria, Candidatus Liberibacter asiaticus(CLas) and is vectored by the Asiancitrus psyllid, Diaphorinacitri. The dis-ease can cause rapid decline o citrustrees, eventually killing them within 5-10years. This devastating disease o citrus

has been reported to result in citrus ruitand juice that is sour, bitter and gener-ally o-avored.

Spread o the DiseaseSadly, this disease was preceded by

citrus canker in Florida, or which a pro-gram o eradication was conducted. As

a result, thousands o trees were pulleddue to an epidemiological study showingthat the canker bacteria, disseminated

Elizabeth A. Baldwin by wind-driven rain, could inect treesin a 1,900 square oot radius. Ater thehurricanes o the 2004-2005 seasons,the disease spread statewide, and theeradication program was abandoned.

Unortunately, HLB was identifedin 2005, and citrus growers, having pulledtrees or naught or canker disease, were

not in the mood to pull out even more.Thereore, removing diseased trees wasnot enthusiastically pursued or a dis-ease that was ar worse than canker, onewhich presented more o a blemish issueor resh ruit and only caused slow treedecline over many years i let untreated.

Consequently, HLB disease has pro-gressed throughout the state o Floridaas growers have become more and morereluctant to pull out diseased trees.Rather, a proposition o using oliarnutritional sprays, which oten include

systemic acquired resistance compounds(SARs) to boost plant deense mecha-nisms, has been adopted to compensateor the disease symptoms. Althoughthere are conicting results, these sprayprograms have worked to some extent inreversing tree symptoms. They now havebeen adapted by many o the industrygrowers who either are reluctant or can-not aord tree removal. The situation iscomplicated by the act that trees do notshow symptoms or HLB immediately,

thus being symptomless and inectingother trees or an unknown amount otime. More nutrition cannot but help thesick trees, but it is not known whethernutritional sprays that partly alleviatetree HLB disease symptoms also aectruit o-avor symptoms, and whetheror not these treatments aect theamount o the assumed causal agent,CLas, in the ruit.

Exploring the DierencesOur laboratory compared juice

made rom ruit harvested rom HLB-inected trees to juice made rom ruit


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Fruit used in studyHealthy HLBa HLBs

Fig. 2. A look at the outside and inside o healthy citrus compared to that rom anHLBa-aected tree and an HLBs-inected tree.

harvested rom healthy trees. We urthersegregated the ruit rom HLB-inectedtrees into ruit symptomatic or the dis-ease (HLBs that are small, green andlopsided) and ruit that looked normalor asymptomatic or the disease (HLBa).(See Figs. 1 and 2.) We did both chemi-cal and sensory analyses and ound thatHLBa ruit (that do not show HLB

symptoms) produce juice that tastessimilar to juice rom ruit harvestedrom healthy trees, while the juice romsymptomatic HLBs ruit are less sweetand more bitter, among other o-avors.This correlated with chemical analysesshowing the HLB ruit juice, especially

or HLBs, has lower sugars, sometimeshigher acids and higher levels o thebitter compounds limonin and nomilin,as well as other secondary metabolitessuch as phenolic compounds. These com-pounds are actually healthy or humans,but can impart o-avor above certainconcentrations. The amount o the bit-ter compounds limonin and nomilin inHLB juice was puzzling since the levelso these compounds were below thosereported to be detectable by humans.

We then conducted a study othreshold determination (amount o acompound that is detectable) by spik-ing orange juice with dierent levels

o these bitter compounds, and testedspiking juice with limonin and nomilinalone and in combination, with or with-out added sugars or acids. These spiked

juices were served to panelists to deter-mine the concentration at which theycould detect bitterness. We ound thatthe combination o the two bitter com-pounds was synergistic in the panelistsperception, demonstrating that togetherthey imparted detectable bitterness atthe levels they are ound in HLB juice.Higher sugar levels helped to mask theperception o bitterness. Thereore, HLB

juice that has less sugars and higherlevels o limonin and nomilin will tastemore bitter.

We also tested the amount o HLBsjuice that could be blended with normaljuice beore panelists could detect adierence and ound that 25 percent

o HLBs juice would be required toblend with healthy juice or panelists tonotice a dierence. This is good newssince most HLBs ruit all o the tree. Ipicked, they should be graded out basedon size and, thereore, should not enterthe juice stream in any large volume.

Testing TreatmentsOver the past our years, our labora-

tory has been studying three dierentnutritional-SAR oliar treatments incomparison to conventional orange tree

culture and spray regimes to see whetherthese nutritionalSAR treatments,which mitigate tree disease symptoms,would also mitigate ruit o-lavorsymptoms. Most growers, other thanorganic growers, implement pesticidespray programs to control the HLB

GLOSSARY

HLBa Fruit that is harvestedrom an HLB-inected tree and does

not show HLB ruit symptoms. Itis not small, green and lopsided orasymmetrical, but looks like a nor-mal healthy ruit and is, thereore,asymptomatic.

HLBs Fruit that is harvestedrom an HLB-inected tree and doesshow HLB ruit symptoms. It maybe small, green and/or lopsided andis, thereore, symptomatic or thedisease. (See Fig. 3)

SARs Systemic acquired re-sistance, meaning compounds that

induce plant resistance.



disease psyllid vector, and such was thecase in our studies.

At frst, results showed no dier-ences o nutritional-SAR sprays on miti-gation o the HLB-induced o-avor,especially or symptomatic HLBs ruit

juice. However, in the latter years, it ap-peared that one o the nutritional-SARoliar treatments was showing some e-ect by making the juice sweeter. Furtherseasons o study are warranted since theeect o oliar nutritional-SAR treat-ments may take years to reach ull eect.

Additionally, we developed a pro-cedure to measure the amount o CLaspathogen DNA in orange juice. Wedid this or ruit harvested rom treesreceiving the oliar nutritional-SARsprays compared to conventionallytreated trees. We saw that juice maderom HLBs ruit exhibited higher CLasbacteria titer than juice made rom

HLBa ruit, which had higher titer thanjuice made rom healthy ruit . Yet wedid not see signifcant dierences dueto nutritional treatments, except or onetreatment in one year or Hamlins, butnot or Valencias. Again, more years ostudy are warranted.

Introducing Enose, EtongueFinally, looking or a way to objec-

tively screen juice or quality, whetherdue to HLB, variety, season, harvestdate, etc., we looked at the possibil-ity o using technology that crudelymimics the human olactory system(electronic nose or enose) and tastesystem (electronic tongue or etongue).The olactory bulb receptors and tonguereceptors non-selectively bind odoror taste volatiles or soluble chemicals,respectively, such that one receptor maybind more than one compound, and one

compound may bind more than one re-ceptor. This allows or unlimited patternpossibilities. These receptors then sendsignals to the brain, and the brain makesa decision based on pattern recognition(good orange juice vs. bad orange juice;orange juice vs. apple juice, etc.).

The enose and etongue have sensorsthat interact with volatile odor mol-ecules or soluble chemicals, respectively,in a ood in a non-selective manner,allowing many pattern possibilities,and send signals to a computer thathas pattern recognition sotware. Thissotware does multivariate statistics todiscriminate between samples, such as

juice made rom healthy HLBa or HLBsruit. Using this technology and judgingby sensory panels that smell and tasteorange juice, we can tell that there aremore dierences in healthy juice versus

HLB juice due to solutes than due toaroma volatiles, but there are dierencesin aroma volatiles, as well. Nevertheless,the etongue does a good job (better thanthe enose) at discriminating betweenhealthy HLBa and HLBs juice.

In conclusion, HLB disease doesaect orange juice (and orange ruit)avor, but this is more pronounced orthe HLBs ruit. Avoiding these in theresh market and processed juice plantsshould eliminate most o the HLB-induced o avor. For the processedorange juice industry, blending withhealthy juice will mask most o-avor,but or the resh ruit industry, theseruit will bring down the quality o theproduct.

Elizabeth A. Baldwin, Ph.D, is a

Supervisory Research Horticulturist in

Citrus & Other Subtropical Products at

the USDA-ARS Horticultural Research

Laboratory in Ft. Pierce, Florida.l

Fig. 3. These Hamlins exhibit classic HLBs characteristics.

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INTRODUCTIONThe third annual Citrus Health Re-

sponse Program (CHRP) Citrus HealthResearch Forum (CHRF) was held Au-gust 27-30, 2012, in Ft. Collins, Colorado.As in the past, the meeting was spon-sored by the national CHRP, CaliorniaCitrus Mutual, Citrus Research Board(Caliornia), Florida Citrus Mutual andTexas Citrus Mutual. This annual meet-

ing is industry-driven or the over-allpurpose o ensuring a productive andthriving citrus industry that is currentlythreatened by Huanglongbing (HLB)and the Asian citrus psyllid (ACP). Theorum brings together representativeso the United States citrus industry (in-cluding citrus organizations, growers andprocessors), researchers rom the U.S.and Mexico , as well as State and Federalregulatory personnel to exchange anddiscuss current inormation about citrus,HLB and ACP. They also identiy needs

and priorities in order to develop newand increased knowledge, innovativeand improved technology and eectivestrategies to manage HLB and ACP.

BACKGROUNDIn 2009, an outcome-based research

approach was adopted by the CHRP atthe national level to mitigate the impactso HLB and ACP. Three solution-basedresearch outcomes were identifed, andthree Outcome Groups (OGs) were es-

tablished to ocus on them. The missionso the OGs are to: keep ACP-infested and HLB-

aected groves productive (OG1) slow or prevent the spread of ACP

and HLB (OG2) keep citrus free of ACP and HLB

(OG3)The OGs, along with the Science and

Technology Committee (STC) o theCHRP, acilitate and enhance:

discussion of current research HLBand ACP eorts;

collaboration and coordination ofresearch among scientists;

3rd Annual Citrus Health Research Forum:

Vision or the Future

identication of potential productso current research and critical mile-stones to deliver those products;

outreach efforts; communication among research-

ers; and communication of research prog-

ress to State and Federal regulators,growers and processors.

The purpose o the annual CHRF

conerence is to: review progress in relevant re-search areas in each Outcome Group;

identify research gaps that need tobe bridged to acilitate progress toward

topic-driven discussions; oral and poster presentations re-

garding research progress and currentongoing research;

Vision for the Future; and reviewing and developing Out-

come Group priorities.The inormation presented and dis-

cussed at the 2012 Research Forum in Ft.Collins is currently being incorporated

into a CHRP HLB-ACP Action Plan.

GROWER PERSPECTIVEParticular emphasis was placed on

receiving growers input on the researchprogress made by the scientists as well asthe research priorities that ought to beset by the whole community includingthe researchers, regulators and industry.Thereore, the orum started on the frstday with a session on the citrus growersperspectives in which representativesrom Caliornia, Texas and Florida

expressed their views. The idea was tocapture three main points:

1. What kind o problems are thegrowers acing?

2. What surveillance and controlmeasures are they taking?

3. What other surveillance andcontrol measures do they need in theuture to maintain a productive andeconomically viable citrus industry?

The views o the growers rom thethree states varied since the status o

HLB and ACP is quite dierent in eachstate, and each has its own industry goals.For Caliornia, the goal is to producehigh-value and high-quality citrus ruitor domestic and oreign markets. Thethreat o HLB-associated CandidatusLiberibacter species inection in Cali-ornia is looming, largely due to the rapidgrowth and expansion o ACP popula-tions in Southern Caliornia. VigorousACP trapping and the testing or Liberi-bacters, especially Ca. L. asiaticus, arecurrently underway. However, chemical

control is proving to be insufcient orrobust ACP control. A biological control

the overall goal o eectively managingand mitigating the impacts o HLB andACP;

ensure the continuation of themomentum o research progress; and

encourage communication among

researchers, growers and processors.Additional detailed backgroundinormation about the annual CHRFsis presented in the July/August 2010Citrograph article, First Annual CitrusHealth Research Forum urthers eortsagainst ACP/HLB and the November/December 2011 Citrograph article, 2ndAnnual CHRP Research Forum: Actionitems will ocus on host resistance toHLB and preventing transmission othe bacteria.

Highlights o the 3rd Annual CHRF

included: grower perspectives;

Ed Civerolo and MaryLou Polek



program or ACP also is being devel-oped. In Caliornia, removal o Ca. L.asiaticus inected trees is mandatory asper Secretary Ross. Fortunately, to dateonly one such tree in Southern Caliorniahas been confrmed, and that backyardtree has been removed.

The Caliornia growers identifed theollowing needs or the uture :

Improved and uniform ACPsurveillance and control measuresacross all groves

Accurate platforms for detectiono Ca. L. asiaticus in insect and citrussamples, and pre-symptomatic detec-tion and diagnosis o HLB

Tools for eliminating Ca. L.asiaticus rom citrus trees

HLB-resistant citrus cultivars forlong-term protection

In Texas, the HLB situation is moreserious than in Caliornia. So, ar twogroves have been reported to containHLB-aected trees, and those trees havebeen removed. Areawide ACP manage-ment has been signifcantly ramped upsince the HLB detection. More than 85percent o all growers are cooperating in

the areawide treatment program.Texas growers identifed the ollow-

ing needs or the uture:

Robust and effective ACP sur-veillance and control throughout theyear

ACP bio-control, possibly withthe aid o a predator

Early and pre-symptomatic di-agnosis o HLB

HLB-resistant transgenic citrus

In Florida, most o the citrus treesare already inected with Ca. L. asiati-cus and are showing HLB symptoms.Thereore, it is too late or ACP con-trol alone to eectively manage HLB.Accordingly, HLB mitigation is thetop priority. A combined use o ACPcontrol and nutritional applications hasbeen useul; but nutritional approacheshave not been validated scientifcally.Research ocused on developing HLB-resistant transgenic citrus has producedpromising results and may lead to along-term solution or HLB protection.However, Florida cannot wait or sucha long-term solution without protectingthe already inected trees.

The Florida industry identifed theollowing needs or the uture:

Therapeutics to kill or suppressCandidatus Liberibacter asiaticas

Effective and sustainable ACPcontrol

A new ACP with reduced vectorcapacity to protect new trees

Advanced production systemsto push economic return into earlieryears

The Growers session concludedwith a Q&A period regarding industryconcerns about the use o HLB-resistanttransgenic citrus. It was generally agreedby all o the grower panel members thatacceptance would not be a problem oncethe product is shown to be eective andready or implementation. There was aquestion about what is the best nutri-tional program. Although it was hardto pinpoint a single eective nutritionalprogram, one panel member elt thatnutrients are being applied at an exces-sive rate in some cases. Finally, there wasa question about how long trees mustsurvive to be proftable. The consensuswas that it depends on the return per

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unit. It is possible to generate proft rom8-year-old trees and good profts rom 10to 12-year-old trees.

TOPIC-DRIVEN DISCUSSIONSThe Growers session set the stage

or the topic-driven discussions by thethree OGs, which were introduced bykey speakers and ocused on the select-

ed science and technology approachesnecessary to address growers needs.Each speaker emphasized several keypoints, namely:

How is the chosen topic relevantto the mission o one or more outcomegroups?

How do the science and technologyapproaches in a given topic area meetthe grower needs?

What advances are needed to meetgrower needs in a timely and eectivemanner?

Outcome Group 1 Topics.Cultivation o Liberibacter in-vitro

So ar, it has not been possible to cul-ture Liberibacter in the laboratory; how-ever, the ability to culture Liberibacterwould be helpul in many ways. First, itwould be possible to directly inoculate

citrus with Liberibacter and show thatit causes HLB symptoms (i.e., provingKochs hypothesis). Second, it would bepossible to screen a large library o drugsand natural products that inhibit thegrowth or kill the Liberibacter, at leastin the culture. Third, it would be possibleto perorm genetic manipulations toidentiy the key Liberibacter genes that

acilitate the bacteriums colonizationspecifcally in the citrus phloem.

Several advances have been madethat provide important clues:

Different articial nutrient mediaormulations, especially those with citrus

juice, look promising. Including insect gut cells in the

media with Liberibacter may allow ora short-term culture.

The observation that seed coatsdisplay high Liberibacter titer maylead to the development o sustainableculture in vitro.

The discovery and genome se-quencing o L. crescens marks an im-portant progress, because cultures oL. crescens have been maintained ormore than 10 years. This bacterium isclosely related to the HLB-associatedLiberibacter species.

Comparative analysis of metabolicpathways o the known Liberibactergenomes may shed light on the appro-priate combination o metabolites andnutrients that are suitable or maintain-ing live cultures o the HLB-associatedbacteria.

HLB diagnosis and Liberibacter

detectionDiagnosis o HLB and detection

o Liberibacter are critical to diseasesurveillance. While visual symptoms willalways be important, by the time visualsymptoms appear, the bacteria may havealready spread long distances, makingit difcult to eradicate inected trees.Liberibacter-specifc molecular markersare potentially more rapid and reliable.There are three parts to molecular diag-nosis and detection:

A reliable set of markers is needed. Appropriate reagents and assay

systems need to be developed. A platform that can perform the

assay or detection or diagnosis needsto be designed.

It appears that a speciic set ovolatile organic compounds or a specifcmicroRNA may report early stages o

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HLB inection. In addition, single chainantibodies (scFvs) have been generatedagainst six Liberibacter proteins thatindicate Liberibacter inection. ThesescFvs can be used in an immunoassayor Liberibacter detection. A lateralow platorm has been designed or aDNA sandwich hybridization assay todetect specifc Liberibacter genes. This

platorm combines sample preparation,DNA amplifcation and signal read-outand takes only hal-an-hour or patho-gen detection. There is also a ow-basedluminex platorm that uses multiplexedoligonucleotide ligation-PCR (MOL-PCR) to detect Liberibacter signaturegenes. This platorm is capable o han-dling 2,500 samples in eight hours.

HLB therapeuticsDevelopment o therapeutics that can

suppress the disease and/or reduce or to-tally clear Liberibacter rom the phloemis an extremely high priority. There aretwo important aspects: what the natureo the therapy is, and how it can be deliv-ered. Several types o therapy are beingtried, including nutritional and thermal;however, their eectiveness is yet to bedetermined. A combination o antibiotic

therapy and targeted phloem deliveryshows promise. Delivery o transgenes byCTV oers a route or getting resistancegenes in existing citrus trees.

Areawide pest managementprograms (AWPMPs)

A successul AWPMP requires: 1) ac-tive participation o the growers, 2) ull

engagement o the public sector, and 3)availability and application o eectivetools. Examples o successul AWPMPinclude mosquito and malaria control indierent parts o the world, and controlo glassy-winged sharpshooters (GWSS)as part o integrated pest managemento Pierces disease o grapevines. Suchmeasures are also applicable or ACPcontrol in citrus orchards. It was notedthat there is no eective pheromoneor ACP trapping. Although there areeective pesticides, the efcacy o somematerials needs to be validated. Thereare signifcant challenges to maintain asuccessul AWPMP or ACP:

Groves are often intermingled withresidential areas.

Growers must be convinced to usenew and more eective materials andabandon the cheaper (albeit less eec-

tive) ones they have in hand. For each area, a treatment coordi-

nator is needed. An appropriate grower as point-of-

contact or the coordinating eort mustbe identifed.

Technology or modiying Bt toxinsagainst specifc pests

Although toxins derived rom thebacterium Bacillus thuringiensis (Bt)have been used successully or themanagement o some insect pests, Bttoxins have not been eective againstthe agriculturally important Hemiptera,including the ACP. A report was hearddescribing a new technology that modi-fed Bt toxins to improve their toxicityagainst aphids, also members o theHemiptera. By improving the binding othe Bt toxin to the aphid gut membrane,the toxicity was increased. A similar ap-proach was envisioned or ACP.

Chemical ecology tools to prevent orslow the spread o ACP and HLB

Insect behavioral mechanisms andchemical ecological patterns provideclues to create and optimize tools orthe eective monitoring and manage-

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ment o ACP. The specialized pheromonelure application technology (SPLAT) iscompatible with the standard insecticidespray technologies currently being usedby the industry. The combination oinsecticides and semiochemicals, suchas pheromones, has the potential toproduce remarkable synergy in ACPcontrol. So ar, an eective pheromone

as a lure or ACP is yet to be identifed.Several studies have, however, identifedand tested potential chemical attractantsor ACP. These include petitgrain oil (dis-tilled rom sour orange), Titan+limonenecombination, and vanilla extract, amongothers. New monitoring lures that utilizespecifc plant volatile blends are in theirfnal stages o feld testing. Monitoringtraps baited with one o these lures havereliably resulted in increased ACP trapcatch when compared to unbaited traps.

Such attractants might be good can-didates or semiochemical-based attractand kill technologies. Repellants couldhave a role in ACP management: a veryinexpensive compound, dimethyl disul-fde (DMDS), ormulated in SPLAT hasbeen shown to be an eective ACP re-pellant in the feld. Interestingly, methylsalicylate, as released by HLB-aectedcitrus, has a peculiar property in that itis attractive to ACP. However, HLB-aected citrus plants have an inherentlylow nutritional value that, ater a ew

days, orces the resident ACP to move onto neighboring, uninected citrus plantsrom which they are able to obtain betternutrition. This sequence o behaviors a-vors an increased rate o HLB inectionor re-inection within a grove. It wouldbe beneicial to the citrus industry ilong-lasting ormulations were created toefciently disrupt this semiochemically-driven cycle o HLB inection.

Outcome Group 3 Topics

Model systems or rapid screening otransgenesModel systems are primarily chosen

or screening transgenes that clear Li-beribacter rom citrus or inhibit develop-ment o HLB symptoms. Model systemsare also useul to obtain clues about hostresponses when inected. Although theydo not exactly mimic citrus inection,model systems allow rapid screening otransgenes in a month or two as opposedto years i studied in citrus. The sameis true or host response studies. The

potato/zebra chip (ZC) pathosystem,including Candidatus Liberibacter so-

lanacearum, the potato psyllid, and thepotato host, can serve as a good modelsystem or HLB as the ull range o dis-ease symptoms appears in weeks. Thepotato system has been applied to screen-ing anti-Liberibacter deensins, as wellas or discovering potato proteins thatare specifcally induced upon inection.Tomato and tobacco are other model

hosts that have been used or screeninganti-Liberibacter deensins rom spinach.Finally, in vitro cultures oLiberibactercrescens, a close relative o the HLB-associated bacteria, have been used toscreen potent antimicrobial peptides.

Functional and comparativegenomics o Liberibacter

Comparative genome analysis o theCa. L. asiaticus (CLas), Ca. L. america-nus (CLam) and Ca. L. solanacearum

(CLso) revealed subtle but distinct di-erences in the three genomes. In addi-tion, the CLas, CLso, and CLam genomesall contain genome sequences o twobacteriophages (phages), but dier intheir copy number. These phages appar-ently become lytic, killing Liberibacter,in citrus but not in psyllids. Investiga-tion is underway to determine whetherphages can be used to manage HLB.

Host response studies ordiscovery o pre-symptomatic HLB

biomarkers and characterization oHLB tolerance/resistance

Host plant responses to Liberibacterinection may allow or the early iden-tifcation o HLB prior to the develop-ment o visible symptoms. Moleculesproduced by the plant in response toLiberibacter inection include smallRNA, proteins, metabolite markers andvolatile organic compounds (VOCs).

Use o psyllid genome inormation

or HLB biocontrolLike any other diseases associatedwith vector-borne pathogens, there aretwo potential ways to prevent HLBby targeting the psyllid. One strategyinvolves blocking the psyllid rom ac-quiring and/or transmitting Liberibacter,whereas the other involves killing psyl-lids. Development o both strategies isimmensely helped by complete knowl-edge o the psyllid genome, which pro-vides inormation on pathways relevantto Liberibacter transmission and the

psyllid lie cycle. For example, ACP canbe rendered unable to transmit Liberib-

acter by modiying the specifc receptorthat acilitates bacterial movement inthe psyllid gut. It has been shown thatspecifc double-stranded RNA (dsRNA)acting on actin is eective as a geneticpesticide or GWSS. A similar approachcan be applied or killing psyllids. An-other interesting approach involvesidentifcation o endosymbiotic viruses

in the psyllid and engineering o one ormore o these viruses to express RNAior peptides that are toxic to ACP.

POSTER SESSIONApproximately 30 posters were

presented, which described research andtechnology areas relevant to the mis-sions o the three outcome groups. Allparticipants had the opportunity to viewthe posters. In addition, the presentershad the time and opportunity to explain

their results and answer questions.

VISION FOR THE FUTUREA departure rom the previous

CHRFs was a session in which twodistinguished scientists (Dr. GeorgeBruening, Proessor Emeritus, Depart-ment o Plant Pathology, University oCaliornia-Davis; and Dr. Gary Secor,Proessor Emeritus, Department oPlant Pathology, North Dakota StateUniversity) outside o the HLB-ACP re-search community presented their per-

spectives on the research progress beingmade and what novel approaches mightbe considered. Based on their reviews othe current progress and uture needs aspresented during the grower session bythe topic-driven speakers and posters,each made several recommendations.

Dr. Secors Recommendations: Continue gene-silencing work

or blocking transmission o Liberib-acter and killing psyllids.

Continue work toward makingLiberibacter culturable. Combineadvances made in cultural, biologicaland chemical methods in HLB man-agement into actionable practices inthe feld.

Combine bio-control methodswith eective delivery systems.

Invest in studying psyllid epide-miology and in developing methodsor monitoring psyllid reservoirs.

Explore the possibility of acitrus cell culture system, which mayallow rapid screening o potential



HLB-inhibitory small molecules andbiologics.

Examine the correlation (if any)between the extent o Liberibacterspread in the tree and disease latency.

Dr. Bruenings Recommendations: Focus on accurate, sensitive,

specifc and high throughput detec-tion platorms that are reproducibleor various seasons, climates, times oday, citrus cultivars, etc. (specifcallyemphasize pre-symptomatic diagnosiso HLB).

Fulll the promise of volatileorganic compound (VOC) detectionby high throughput sampling with theaid o a validated set o HLB-specifcmarkers.

Use high throughput combina-torial approaches to discover novel

attractants/repellents or pheromonelure application technology (SPLAT).

Continue work on developingtransgenic citrus by clearing Liberib-acter or by engineering host deense.

Continue work on blockingLiberibacter transmission or killingpsyllids using RNAi and peptides.

For HLB resistance, stack two ormore transgenes that target the sameLiberibacter gene but by dierentmechanisms.

CONCLUDING SESSIONReports by each o the three OGs

were presented that summarized re-search priorities or each group andincluded a brie discussion o the actionitems. These will orm the basis o a co-hesive CHRP Action Plan that is underdevelopment and will address the:

1. status o surveillance and controlo ACP and HLB;

2. high-priority actions needed toaccomplish the overall goal; and

3. research and technology advancesneeded to accomplish the goal.

The missions and research prioritiesidentifed by the three OGs at the 2012CHRF are presented below.

Outcome Group 1Mission:

Keep ACP-inested and HLB-aected groves productive

Research Priorities: Pre-symptomatic diagnosis of

HLB and Liberibacter detection inyoung trees

Liberibacter culture in vitro(needed or screening therapeuticsand or identiying virulence markers)

Therapeutics to keep affectedgroves productive

Effects of HLB and HLB man-agement practices on citrus productsthat impact consumer acceptance


Slow or prevent the spread o ACPand HLB

Research Priorities: Biology of psyllid and psyllid-

Liberibacter interactions Areawide management pro-

grams Biological control of psyllid Study of the effectiveness of

ush management

Getting new citrus plantings intoproduction

Studies on environmental factorsand their eect on HLB symptoms

Diagnostics for early stage in-ection


To keep citrus ree o ACP andHLB

Research Priorities:

Pre-symptomatic detection ofHLB

Breeding transgenic and conven-tional resistance to HLB

Functional genomics of Liberib-acter to improve detection and control

Functional biology of ACP toimprove control measures

GROWERS ASSESSMENT OFTHE 3RD CHRF

Generally, growers were impressed

wi t h what t heyheard. They notedsigniicant coop-eration among theresearchers. How-ever, there is roomor improvement,which would par-ticularly help inavoiding unneces-sary duplication oresearch. All thegrowers empha-

sized translating re-search and technol-

ogy innovations into practical solutionsand implementing these solutions in atimely manner or surveillance and man-agement o HLB. It was noted that earlydiagnosis o disease is not only criticalor industries in Caliornia and Texas,but also or the Florida industry whenit comes to young trees. For Florida, thehighest priorities are: 1) psyllid control,

which involves blocking acquisitionand transmission o Liberibacter, andinsect killing; and 2) development otherapeutics or eective HLB controlor suppression. One suggestion wasto use antibiotics in combination withother therapeutics. While pre-symptom-atic HLB diagnosis and development oHLB-resistant citrus are tremendouslyvaluable, it was suggested that concertedeorts in these two areas would speedup the transition rom the discovery to

the application phase.

FINAL WRAP-UP SESSIONIndustry representatives rom Cali-

ornia, Texas and Florida provided theirthoughts on the CHRP Forum. All othem emphasized that researchers mustalways keep the growers in mind andcooperate with each other as much aspossible. They stressed that it is impor-tant or the scientists to envision howtheir research can be applied and howit would help the growers.

4th CHRFThe 4th CHRF is scheduled to be

held October 15-17, 2013, in Denver,Colorado.

Ed Civerolo is a Plant Pathologist,

USDA/ARS, Department o Plant

Pathology, University o Caliornia at

Davis. MaryLou Polek, Ph.D., serves

as the Caliornia Citrus Research

Boards Vice President o Science and

Technology. l

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Roger I. Vargas, David Haviland, Ben Faber, John Kabashima, Beth Grafton-Cardwell and Joseph G. Morse

Research Project Progress Report

CRB-Funded Research Report

Improving trapping systems orearly detection and eradication oruit ies in Caliornia

Fig 3. Melon fy, Bactroceracucurbitae.

Fig 1. Mediterranean ruit fy, Ceratitiscapitata.

Fig. 2. Oriental ruit fy, Bactroceradorsalis.

IntroductionTrue ruit ies (Diptera: Tephri-

tidae) include some o the most eco-nomically important pests attackingsot ruits worldwide, including cit-rus. From an economic perspective,they 1) inict extensive direct dam-age to ruits and eshy vegetables,2) cause quarantine restrictions oninested areas, 3) require that com-mercial ruits undergo protectiveand postharvest treatment prior toexport, and 4) provide a source ortheir introduction into other partso the world when established in a

country.Some o the most notorious

members o the amily Tephritidaeare the Mediterranean ruit ly,Ceratitis capitata (Wiedemann)(Fig. 1), oriental ruit y, Bactroceradorsalis (Hendel) (Fig. 2) and itsrelatives, Queensland ruit ly, B.tryoni (Froggatt), peach ruit y, B.zonata (Saunders), and melon y,B. cucurbitae (Coquillett) (Fig. 3).The recent alarming spread o ruit

lies worldwide [e. g., B. invadensDrew, Tsutsura, and White) intoArica and carambola ruit ly, B.carambolae Drew and Hancock,into South America] can be attrib-uted to increased: 1) production oruits and vegetables worldwide, 2)global trade o ruits, vegetables andplants between countries, 3) cross-border movement o people andtheir possessions between nearbycountries, and 4) air travel withbaggage containing inested ruits.

The introduction oB. invadens intoArica has been particularly trou-

bling. Discovered in 2003, it quicklyspread rom the east to west Arica,

inesting over 10 countries and evendisplacing many o the Ceratitis sppnative to the continent.

The continued threat o ruit yestablishment in the United Stateshas resulted in large survey and de-tection programs in Caliornia andFlorida. Early detection methodsor accidental introductions o ruitlies involve deployment o largenumbers o traps baited with highlyattractive male specifc lures methyleugenol (ME; 4-allyl-1,2-dimethoxy-

benzene-carboxylate), trimedlure(TML: tert-butyl 4- and 5-chloro-cis- and trans-2-methylcyclohexane-1-carboxylate), and cue-lure [C-L;4-(p-acetoxyphenyl)-2-butanone],to detect oriental ruit ly, Medi-terranean ruit ly, and melon ly(and their relatives), respectively.Eradication treatments against ex-otic ruit ies that are accidentallyintroduced rom various parts o theworld into Caliornia are very costly.

For example, due to continuous in-troductions, current annual costs toexclude Mediterranean ruit y romCaliornia total over $15 million.Annual introductions oBactroceraspp. oten result in temporary traderestrictions and associated areawideeradication treatments, lasting upto nine months beore movemento agricultural commodities canresume without postharvest treat-ments (http://www.cda.ca.gov).

The Caliornia Department o

Food and Agriculture (CDFA) di-rects continuous programs to detect


29/48

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C I T R U S A V O C A D O S O L I V E S

and eradicate invasive Bactrocera ruities. From 1960 to 2012, nine dierentBactrocera species have been detected,with Oriental ruit y detected mostrequently (126 times). There havebeen 140 eradication programs with 25quarantines. Most o these programshave occurred in southern Caliorniawhere the usual number o detection

traps deployed is fve Jackson traps (JT)(TML, ME, and C-L) and fve McPhailtraps (MPT) (Torula Yeast Solution)per mi2 (2.59 km2). Currently, a sepa-rate Jackson trap is deployed or TML,ME, and C-L. Total number o sites inoperation is approximately 25,000 orthe Los Angeles area and over 30,000or the entire state.

Current methods involve trappingsystems used in association with liquidinsecticide ormulations such as naled(Dimethyl 1,2-dibromo-2,2-dichloroeth-yl phosphate) which pose environmentaland worker saety concerns. Throughthe Hawaii Area-Wide Pest Manage-ment program (AWPM), solid singleand double lure insecticidal waers andsolid lure plugs and cones were identi-fed as promising substitutes or trapsbaited with liquid ME or CL and naled.

Fig. 4. This is a TMR dispenser in aJackson trap.

Wk Mallet TMR Liquid ME (6ml)with naled (1%)on wick

1 55.5 22.9 59.3 20.3

2 62.9 27.6 52.5 18.5

3 54.1 18.2 65.7 29.9

4 45.1 15.8 57.5 23.1

5 67.8 39.0 79.7 45.5

6 76.3 47.0 78.4 48.3

7 13.9 1.7 13.9 2.5

8 8.5 2.6 7.3 1.9

Grand meansem 55.2 9.5a 61.4 10.1a

Mean numer f ies captured per treatment ver

time is nt signicanty different at the P = 0.05 eve,Prc GlM (SAS 2009) (Treatment: F = 0.20; df = 2,254; P = 0.658).

Table 2. Weekly male oriental ruit fy

captures (mean sem) in Jackson traps

with two lure dispensers hung in trees

at six orchards (eight sites), near Kona,

HI, 13 December 2011-7 February 2012.

Wk Mallet TMR C-L liquid (6ml)with naled (5%)on wick

1 8.4 2.6 17.4 5.3

2 10.2 3.0 10.3 3.4

3 8.3 2.1 10.7 3.5

4 9.7 3.8 9.1 3.5

5 11.4 3.5 10.8 3.8

6 15.4 1.2 7.9 2.2

7 7.8 2.6 4.0 0.8

8 2.1 0.6 3.4 1.0

Grand mean sem 10.7 1.4a 10.9 1.5a

Table 1. Weekly male melon fy captures

(mean sem) in Jackson traps with

two lure dispensers hung in trees at six

orchards (eight sites), near Kona, HI, 13

December 2011-7 February 2012.

Mean numer f ies captured per treatment ver

time is nt signicanty different at theP

= 0.05 eve,Prc GlM (SAS 2009) (Treatment: F = 0.11; df = 1,254; P= 0.743).

Results rom recent studies providepromising evidence or eliminating theuse o hard to handle liquid lures andhazardous organophosphate insecticidesby consolidating three lures into onesolid single waer with an insecticide(Fig. 4). Besides the environmentalbenefts, the possibility o consolidatingdetection traps would reduce labor costsassociated with deployment o numer-ous traps, which is the biggest expenseo large survey programs.

The overall objective o this CitrusResearch Board-unded study is to eval-uate and develop a convenient, eectiveand saer means to use male lures andinsecticides or improved detection andpotentially improved male annihilationo invasive ruit ies. Incorporating a

single multi-lure dispenser to eectivelydetect or suppress multiple ruit y spe-

cies in large trapping programs coulddrastically reduce costs associated withlabor and materials while improvingsaety and convenience rom a workersstandpoint. Likewise when a detectionoccurs, high densities o traps are placedaround the discovery site, not only serv-ing or delineation o the fnd, but moreimportantly as a mass trapping eradica-

tion technique oten reerred to as themale annihilation technique (MAT).

Evaluations are being conductedthrough the Agricultural ResearchService (ARS), U.S. Department oAgriculture (USDA) and Pacifc BasinAgricultural Research Center (PBARC)in Hilo, HI, in cooperation with theUniversity o Caliornia and Farma TechInternational (North Bend, WA). Twobioassay study sites were selected in Ha-waii because o the combined presenceo oriental, Mediterranean, and melonies. Sites on the leeward side o HawaiiIsland involved six separate avocado or-chards (between 191145 N, 1555159W and 19323 N, 1555523W, at anaverage elevation o 466 m) and sites onKauai Island were in a large (ca. 1,400ha) commercial coee [Coffea arabicaL. cv. Arabica (Rubiaceae)] plantationin Numila (215436 N, 1593254 W,at an average elevation o 125 m).

Experiment 1: Evaluation o Stan-

dard Individual Jackson Traps againstTriple Lure Jackson Trap on Hawaii



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Wk Mallet TMR TML plug (2g)w/ DDVP strip

1 9.4 5.9 16.5 10.1

2 8.2 6.8 8.4 5.9

3 23.4 18.8 25.6 21.6

4 32.5 20.5 16.5 10.2

5 67.6 47.0 53.9 41.1

6 40.8 27.5 30.6 20.7

7 37.4 32.6 11.5 7.9

8 27.5 22.1 10.0 5.5

Grand mean sem 28.9 8.1a 22.7 6.8a

Table 3. Weekly male Mediterranean ruit

fy captures (mean sem) in Jackson traps

with two lure dispensers hung in trees at

six orchards (eight sites), near Kona, HI, 13

December 2011-7 February 2012.

Mean numer f ies captured per treatment vertime is nt signicanty different at the P= 0.05 eve,

Prc GlM (SAS 2009) (Treatment: F = 0.18; df = 1,254; P= 0.6684).

Island. Studies were conducted rom13 Dec 2011 7 Feb 2012 in the Konaarea o Hawaii Island (during coeeand avocado season). Treatments wereplaced inside Jackson traps with a stickyinsert as ollows: 1) Mallet TMR Trap,2) ME solution (6ml) with 1% (AI)naled on a cotton wick, 3) C-L solution(6 ml) with 5% (AI) naled on a cotton

wick, and 4) 2g TML plug. Treatments2, 3 and 4 represent the standard treat-ments currently used in survey arraysin Caliornia or detection o ME, C-Land TML responding ies, respectively.Treatments were weathered in place andtraps were serviced every seven days oreight consecutive weeks with a total oeight replicates (eight blocks) with ourtreatments each.

Experiment 2: Male Annihilation

Evaluations o Fresh Mallet TMR and

Mallet CMR at Kauai Coee Plantation.Studies were conducted rom 19 Nov -26 Nov 2010 (during coee harvest whenMediterranean ruit y numbers werehighest). Treatments were placed inAWPM traps as ollows: 1) Mallet TMR(23% ME, 15.9% TML, 15.9% RK, 3.6%DDVP), 2) Mallet CMR (29.9% ME,4.7% benzyl acetate, 11.7% RK, 12.6%ceralure, 2.8% DDVP), 3) 10g ME cone(Scentry Biologicals) + insecticidal strip(Hercon Vaportape II, DDVP), 4) 2g

C-L plug (Scentry Biologicals) + insec-ticidal strip and 5) 2g TML plug (Scentry

Biologicals) + insecticidal strip. Thesetreatments represented current solidlures identifed through the Hawaii FruitFl

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