Citrograph

Summer 2013

Savoring Citrus

Citrograph

Givaudans TasteTrek Citrus explores new scent and flavor trends

Geared for Your Groves

At only 48 inches wide with standard tires, Kubota M Series narrow compact tractors fit right in on hard-working citrus farms. Theyre geared for your groves with powerful 56, 64 or 76 PTO HP Kubota E-CDIS diesel engines, smooth HST transmissions and exclusive Kubota bevel gear bi-speed turn technology for a tighter turn radius than ever before. See one of these authorized Kubota dealerships for more information or to demo an M Series.

Put a Kubota on your team! Visit one of these authorized Kubota dealerships today!
ESCONDIDO Pauley Equipment 1110 West Washington Avenue OXNARD Diamond A Equipment 821 North Rice Avenue PORTERVILLE Farmers Tractor and Equipment 1132 South Main Street THERMAL Torrences Farm Implement 54050 Harrison Street TULARE Linder Equipment 311 East Kern Avenue

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Kubota Tractor Corporation Markets a Full Line of Tractors and Construction Equipment Through a Nationwide Network of Over 1,000 Authorized Dealers. Optional Equipment May be Shown. Kubota Tractor Corporation, 2013

Citrograph
SUMMER 2013 Volume 4 Number 4
Cover photo courtesy of Givaudan Flavors Corp. See article on page 15.

An Official Publication of the Citrus Research Board

SUBSCRIPTIONS
U.S. Single Copies: $1.50 1-Year Subscription: $15.00 2-Year Subscription: $28.00 Canadian & Foreign: 1-Year Subscription: $30.00 2-Year Subscription: $56.00

IN THIS ISSUE

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

4 Editorial 8 CRB welcomes new president and

citrus veteran Ken Keck formidable threat? citrus flavors

PUBLICATION OFFICE
P.O. Box 230 Visalia, CA 93279 Phone: 559-738-0246 FAX: 559-738-0607 Web Site: http://www.citrusresearch.org

12 Huanglongbing: Why is HLB such a

Louise Fisher, Managing Editor Dr. MaryLou Polek, Chief Science Editor EDITORIAL BOARD Dr. Mary Lu Arpaia Ted Batkin Richard Bennett Franco Bernardi Dan Dreyer Dr. Ben Faber Jim Gorden Julia Inestroza SCIENCE REVIEW PANEL Dr. Mary Lu Arpaia James A. Bethke Dr. Abhaya Dandekar Dr. Akif Eskalen Dr. Stephen Garnsey Dr. Joseph Smilanick

15 Bringing fresh perspectives to

18 Comparing orange juice from healthy
vs. HLB-affected trees

22 3rd Annual Citrus Health Research

Forum: Vision for the Future

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

PRODUCTION INFORMATION
Dale Hahn, Design Judy Brent Phone: 630-462-2308 Production Manager 255 38th Avenue Suite P dhahn@farmprogress.com St. Charles, IL 60174 Phone: 630-462-2919 jbrent@farmprogress.com

28 Improving trapping systems for early

detection and eradication of fruit flies in California

36 Assessing factors influencing the post- 40 Citrus Roots: California citrus 44 Meet Chef Nathan Lyon
growers enjoyed early love affair with vehicles

ADVERTISING INFORMATION
Sandy Creighton Ad Sales Manager Phone: (559) 201-9225 screighton@farmprogress.com

harvest quality of California mandarins

ADVERTISING RATES
Rates B/W 2/C 4/C Page..................................$690....... $860...... $1025 2/3 Page Vertical.................540......... 700.......... 875 1/2 Page Vert/Horiz............410......... 580.......... 750 1/3 Page Square/Vert..........285......... 455.......... 620 1/4 Page..............................200 ........ 370.......... 540 1/6 Page Vertical.................140......... 310.......... 480 1/8 Page Horizontal............140......... 310.......... 480
*Frequency discounts: 2X5%, 3X7%, 4X10%
Above rates are gross; 15% discount to recognized agencies.

Citrograph is published quarterly by the Citrus Research Board, 217 N. Encina, Visalia, CA 93291. Citrograph is sent to all California citrus producers courtesy of the Citrus Research Board. If you are currently receiving multiple copies, or would like to make a change in your Citrograph subscription, please contact the publication office (above, left). Every effort is made to ensure accuracy in articles published by Citrograph; however, the publishers assume no responsibility for losses sustained, allegedly resulting from following recommendations in this magazine. Consult your local authorities. The Citrus Research Board has not tested any of the products advertised in this publication, nor has it verified any of the statements made in any of the advertisements. The Board does not warrant, expressly or implicitly, the fitness of any product advertised or the suitability of any advice or statements contained herein.

Summer 2013 Citrograph 3

EDITORIAL

BY KEN KECK, President

Comparing Oranges to Oranges

Im amazed by the beautiful, lush canopies of citrus, which are a joy to behold, particularly next to what Florida citrus trees lamentably have been enduring this past decade.

ow that Ive been on board at the Citrus Research Board (CRB) three months, Ive had a chance to catch my breath and reflect on my first impressions of the incredible industry out here in California. What Ive learned is that the 2,631-mile distance between Lakeland and Visalia is a good metaphor for the differences between the Sunshine State and Golden State citrus industries. Unlike Florida, where the acreage has been somewhat compressed by the freeze-line, Im struck by how spread out the California industry is all the way from the Mexican border to Madera, a distance of nearly 600 miles. Not only that, but the trees are so much healthier and better looking out here. Im amazed by the beautiful, lush canopies of citrus, which are a joy to behold, particularly next to what Florida citrus trees lamentably have been enduring this past decade. Im further impressed by the healthy lifestyle in California, which definitely is better for the body and soul. There are mountains, ocean and microclimates galore. Its a great place to be outdoors. Ive also received an education in differing citrus practices. For example, I was in a meeting not long ago where we were discussing potential methods to get rid of Fuller Rose Beetle eggs on citrus, which Koreans do not want to see on their imported fruit. Someone suggested CRB-funded research to use nematodes under the canopy as a bio-control method to eliminate the beetles. I was horrified. In Florida, nematodes are tremendous pests that often destroy tree roots and root hairs, causing the decline and death of citrus groves. To deliberately distribute them under trees left me speechless. It was then explained to me that there are certain California nematodes that are beneficial and can be used to target certain kinds of insects. I realized I still have a lot to learn about the differences between growing citrus on the West Coast and the East Coast! Thats not to say that there arent commonalities. Whether its California or Florida, Ive found that American citrus growers and other industry members are known for their hard work, dedication, perseverance and justifiable pride in the worlds best-tasting, safest 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 of this issue.
4 Citrograph Summer 2013

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.
CITRUS RESEARCH BOARD MEMBER LIST BY DISTRICT 2012-2013
District 1 Northern California Member Toby Maitland-Lewis Donald Roark Jim Gorden Joe Stewart Etienne Rabe John Richardson Kevin Olsen Richard Bennett Member Earl Rutz Joe Barcinas Alternate Jack Williams Dan Dreyer Dan Galbraith Franco Bernardi John Konda Jeff Steen Tommy Elliott Justin Brown Alternate Alan Washburn John C. Gless District 3 California Desert Member Mark McBroom Public Member Member Ed Civerolo Alternate Craig Armstrong Alternate Steve Garnsey

District 2 Southern California Coastal

Citrus Research Board 217 N Encina, Visalia, CA 93291 PO Box 230, Visalia, CA 93279 (559) 738-0246 FAX (559) 738-0607 E-Mail Info@citrusresearch.org

CALENDAR
September 25 ACP/HLB Grower Meeting. Sunkist Center CA Citrus State Historic Park. Riverside, CA. For info, contact Victoria Hornbaker at (916)403-6652. September 26 Citrus Pest and Disease Prevention Committee (CPDPC). Riverside, CA. For info, contact Victoria Hornbaker at (916)403-6652. September 26 California Citrus Quality Council (CCQC). Korean Export Requirements. Visalia Convention Center. For info, contact CCQC at (530) 885-1894. October 8 Lemon Field Day. Coachella Valley ARS (CVARS). For info, go to www.citrusvariety.ucr.edu October 10 California Citrus Quality Council (CCQC) Conference. Visalia Convention Center. For info, contact CCQC at (530) 885-1894. November 5-7 California Citrus Nursery Society (CCNS) Conference. La Quinta, CA. For info, contact Tom Delfino at tdelfino@earthlink.net November 12 Mandarin Field Tour. Lindcove REC, Exeter, CA. For more, go to www.ucanr.edu/sites/lindcove

DO YOU KNOW...?
Why is fish served with lemon? (Go to page 34 for the answer.)
Summer 2013 Citrograph 5

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Asian Citrus Psyllid, CA_Aug 7-21, 2013 (1,855 records) ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( !
United States Department of Agriculture

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Clemente Island

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Asian Citrus Psyllid, AZ_2013 thru 8-1-13 (84 records) Quarantine for ACP, CA (7/19/2013)

Asian Citrus Psyllid, CA_Aug 7-21, 2013 (1,855 records)

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45,338 sq miles

Asian Citrus Psyllid, Mexico_2013 thru 8-16-13 (356 records) Quarantine for ACP, CA (7/19/2013) 45,338 sq miles

Quarantine for ACP, AZ (5/7/2013) 17,923 sq miles

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USDA, APHIS, PPQ Coordinate-System: Coordinate-System: Data Source: USDA, APHIS, PPQ CA Teale Albers, NAD83 CA Dept ofNAD83 Food & Agriculture GIS Specialist GIS Specialist CA Teale Albers, USDA, APHIS, PPQ 650 Capitol Mall, Suite 6-400 Date Printed: 8/22/2013 650 Capitol Mall, Suite 6-400 USDA, APHIS, IS Sacramento, CA 95814 Time Printed: 09:50 hrs PT Date Printed: 8/22/2013 TeleAtlas Dynamap Sacramento, CA 95814 Time Printed: 09:50 hrs PT

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Quarantine for ACP, AZ (5/7/2013) 17,923 sq miles

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Data Source: These data, and all the information contained therein, have been collected by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS), or by of its cooperators on APHIS behalf, for restricted government purposes only and is the sole property of APHIS. Data may be disseminated on a need-to-know basis CA Dept Food & Agriculture only and must be used for their intended government purpose(s). All information contained within these data are subject to required Federal safeguards and shall only USDA, PPQ be APHIS, shared and/or used consistent with the Trade Secrets Act [18 U.S.C. 1905], the Privacy Act of 1974, as amended [5 U.S.C. 552a], the Freedom of Information Act [5 U.S.C. 552],IS the confidentiality provisions of the Food Security Act of 1985 [7 U.S.C. 2276], Section 1619 of the Food, Conservation, and E nergy Act of 2008 USDA, APHIS, [7 U.S.C. 8791], and other applicable Federal laws and implementing regulations, as well as with the confidentiality or non-disclosure provisions of any other TeleAtlas Dynamap agreement entered into between APHIS and a cooperator. agreement entered into between APHIS and a cooperator.

Map of Asian citrus psyllid ! ( Baja detections, ACP Pima Co California quarantines and ( ! Santa Baja ( ! HLB quarantines ( ! Cruz Co ( ! Sonora California ( ! ( ! in California ( ! ( ! ( ! Sonora and neighboring ( ! ( ! ( ! ( ! ( ! portions of ( ! ( ! ( ! ( ! These data, and all the information contained therein, have been collected by the U.S. Department of Agriculture's Animal and Plant Health Inspecti Arizona and or by its cooperators on APHIS behalf, for restricted government purposes only and is the sole property of APHIS. Data may be disseminated on a n within these data are subject to required Federal safegua only and must be used for their intended government purpose(s). All information contained Mexico through be shared and/or used consistent with the Trade Secrets Act [18 U.S.C. 1905], the Privacy Act of 1974, as amended [5 U.S.C. 552a], the Freedom o [5 U.S.C. 552], the confidentiality provisions of the Food Security Act of 1985 [7 U.S.C. 2276], Section 1619 of the Food, Conservation, and Energy August 21, 2013. [7 U.S.C. 8791], and other applicable Federal laws and implementing regulations, as well as with the confidentiality or non-disclosure provisions of a
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CRB welcomes new president and citrus veteran Ken Keck

he Citrus Research Board (CRB) warmly welcomed citrus industry veteran and leader Ken Keck as its new president on June 1. Keck replaced retiring President Ted Batkin, who had served at the helm of the organization for the past 20 years. According to CRB Chairman Earl Rutz, As an industry, we are facing some serious threats, most notably the continued identification and spread of the Asian citrus psyllid and Huanglongbing (ACP/HLB) in our state. Its a complex and challenging time, but we believe Ken has the skills to take us forward. With the 3,500 citrus growers in California continuing

to invest in the research necessary to aid the states $2 billion+ citrus industry and battling potential threats to their groves and livelihoods, Keck proved a logical choice to take the reins. He has deep-rooted expertise representing citrus growers in a governmental, legal and regulatory framework. His thorough industry knowledge includes experience in fighting ACP/HLB in Florida while at the Florida Department of Citrus (FDOC), a state agency charged with promoting Florida citrus products that is financed primarily by growers through a box tax. Keck served as general counsel and executive director of the FDOC from 2006-2012 and as director of government affairs and general counsel from

Ken Keck and the CRB delegation, led by Dr. Harold Browning of the Citrus Research and Development Foundation, recently toured and examined a Florida citrus grove for visual symptoms of HLB. Afterward, a close and mandatory inspection by the group found several psyllids that had attempted to hitchhike a ride on Richard Bennetts yellow shirt. 8 Citrograph Summer 2013

2002-2006. Prior to that, he was director of legislative and regulatory affairs with Florida Citrus Mutual from 19992002. Kens track record, enthusiasm and alignment with the perspective of California growers will make him an excellent asset to the CRB, said Rutz. Among Kecks accomplishments with the FDOC, he secured an average of $7 million annually in federal and state appropriations for programs; commissioned a National Academies study, resulting in the establishment of a dedicated $10 million annual disease research, development and commercialization foundation; and achieved a range of $3$5 return to growers for every marketing dollar expended. Keck achieved these results not only because of his tireless work ethic, but also from his understanding of what citrus growers face. Im a third-generation grower in Florida, so I know the nature of the business. I know what it means to depend on the harvest each year. I know what it feels like to have your livelihood threatened, and I want to use this understanding and my experiences in Florida to benefit the California industry. However, Keck emphasized that he has not come on board as someone who has all the answers. He vividly recalls a sign behind the cash register at the local auto parts store in Lake Placid, Florida, where he grew up. The sign read, THERE WILL BE A $10 SURCHARGE FOR YOU TELLING US HOW MUCH BETTER THINGS ARE UP NORTH. Many years later, he still recalls that sign as a reality check in humbleness.

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Viewing the Challenges

HLB is our short- and medium-term challenge, Keck said. The biggest threat and the biggest shame would be if we did not learn the lessons of what happened in Florida and Texas. Unfortunately, its a matter of when, not if, HLB will make its next appearance following the find in Hacienda Heights. Will our growers understand the critical need to get rid of trees once the first twig shows up with a visual symptom or if technology uncovers a problem even before symptoms appear? A tree must be removed in its entirety right

The threat of not learning the ACP/ HLB lessons from Florida and Texas is also the biggest opportunity.
then and there. Keck said he believes that Californians are taking the looming specter of HLB very seriously. The water shortage is viewed by the new CRB president as a long-term challenge. With an average rainfall of five inches in many growing areas, the big question is whether California groves can manage on their water allotments in drought years when they must cut back on optimal watering levels, he said. Additionally, Keck sees phytosanitary barriers as a large ongoing challenge to the industry, citing the example of Chinas current ban on California citrus imports.

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The threat of not learning the ACP/HLB lessons from Florida and Texas is also the biggest opportunity, Keck said. Specific to the CRBs core competencies and mission, Keck sees a tremendous opportunity to prove that geocentric research need not rule the day. California and Florida can work together - coordinating, cooperating, prioritizing and funding, particularly in the face of ACP/HLB, he said. Lets decide on commonalities and set aside respective pots of money to jointly issue proposals, vote, fund and listen to the final reports. We can make one plus one equal three. This could be handled with our respective existing infrastructures and could involve cooperating university researchers.

Path to Streamlining
One of the first things that struck Keck when he took the reins of the CRB was the sheer number of meetings 42 in the previous fiscal year alone that resulted in 126 notices being sent to the industry. We have so many dedicated, unpaid participants in our industry who spend hours traveling to and attending these gatherings, he said. They then return home to attend to their groves and their real jobs. While the industry appreciates its democracy, I believe we can make this process more efficient and more manageable for volunteers who serve on our board and committees. As an administrator, Keck wants to focus efforts on programs most critical to California growers competitiveness.

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

bers recently traveled to Florida to meet with the Citrus Research and Development Foundation board to study ways to streamline. Keck believes that the majority of the industry will welcome this focus and resulting efficiencies.

Looking at ROI
Another way to create further efficiency, Keck believes, is through economic analyses of the research categories and areas that have been funded in the past. We need to look at the return on investment (ROI), he said, although this could be difficult. Is there a way to express the ROI on research to our constituencies? This would give our Board tools to guide the funding parameters. Keck also thinks that ROI analyses would provide an additional metric for growers to measure CRBs investments in research. It might also encourage State and Federal legislators and policy-makers to direct incremental funds to citrus research by demonstrating the positive return potential. There should be a relationship between an ROI analysis and funding levels, whether those funds are derived from growers or government, Keck said. While he believes that grower funding should flow at least at the present level, he feels that if California and U.S. officials could see the consequences of ACP/HLB, there could be the opportunity for more general revenues to flow into the research coffers. We need to dramatically demonstrate the need through pictures and video, yes, - but also by way of sound economic impact analysis, Keck said. The new president has his work cut out for him, but he relishes the upcoming challenges and the opportunity to help protect the California citrus industry while enhancing the competitiveness of its growers. l

Meet Ken Keck

Birthplace Lake Wales, Florida Education Bachelors degree in Spanish from Stetson University in Florida and Juris Doctorate in law from Widener University School of Law in Delaware Family Wife Julie; two high-maintenance faux-Pomeranians - Fletcher and Guinness; and Kenya, the cat Citrus roots Third-generation grower with groves in Lake Placid, Florida Outside interests Never mastering the game of golf; learning about California geology On his nightstand Assembling California by John McPhee (also author of Oranges)
While learning about California topography, Keck sees what its really like to try to push a rock up a hill. Summer 2013 Citrograph 11

Huanglongbing:

Why is HLB such a formidable threat?

MaryLou Polek
uanglongbing (HLB), aka yellow shoot disease or citrus greening disease, is by far the most severe disease of citrus in the world. The HLB-associated pathogens are three species of a fastidious (see Key Terms), phloem-limited bacteria in the genus Liberibacter. These bacteria cannot live outside the phloem cells of citrus or the organs of the insect vector, which can transmit the bacteria from an infected plant to a healthy plant. In addition to the insect vector, the Asian citrus psyllid (ACP), or Diaphorina citri, HLB can be transmitted by grafting with infected plant propagating materials. Despite over two decades of re-

search and hundreds of combinations of growth media, HLB-associated bacteria have defied laboratory culture. This makes life difficult for scientists conducting research on the disease because they must maintain living plants infected with the Liberibacter bacteria. Federal and state regulations mandate that these plants must be contained within protective structures. This adds to cost and efficiency of projects looking for control solutions.

If this pathogen is so difficult to grow, how can it be so devastating?

Once the bacterium gets inside the plant, the plant cannot be cured of it. It will continue to multiply and circulate throughout the entire plant. First, the bacterial cells go down into the roots and kill them. Scientists in Florida have reported that 30 to 40 percent of the root mass is lost before visual symptoms are observed in the leaves or before leaves test positive using the polymerase chain reaction (PCR) assay, which is the USDA-APHIS approved protocol for Liberibacter detection (Figure 1). If the tree is already affected by Phytophthora root rot or nematodes, the effects of a multiple infection with HLB bacteria are synergistic, and the symptoms are more severe than if the plant was infected by only one of these pathogens. As the disease progresses, the pathogen reproduces and travels through the plants phloem cells back up into the scion. Here the bacteria plug up the plants vascular system, starving the host cells of necessary water and nutrients. Subsequently, growth of the scion is hindered and leaves are decreased in size, become yellow and drop. The tree may look as though it is suffering from zinc or other nutrient deficiencies (Figure 2). In California, we are accustomed to our citrus trees being very lush and dense. In parts of the world where the impacts of HLB are severe, citrus trees are not vigorous and have sparse foliage (Figure 3). Trees may suffer branch or stem dieback. When they are drought-, moistureor temperature-stressed, HLB-affected trees have a tendency to drop their fruit prematurely. Many Florida growers

Fig. 1. ROOT MASS LOSS: Scientists in Florida reported that 30 to 40 percent of the root mass is lost before visual symptoms are observed in the leaves or before leaves test positive using the polymerase chain reaction (PCR) assay.

Fig. 2. NUTRIENT DEFICIENCY: The tree may look as though it is suffering from zinc or other nutrient deficiencies. 12 Citrograph Summer 2013

were horrified this past year when they suffered from an unprecedented fruit drop attributed primarily to HLB. Although trees can certainly die from this disease, they mainly become so debilitated and unproductive that a grower or homeowner readily removes them. Perhaps the worst symptoms of all are those that affect the fruit since, after all, its the fruit that growers are producing for income. Not all fruit on an infected tree show visible symptoms. Those that do are generally decreased in size and misshapen (Figure 4). In South Africa, affected fruit tend to stay green at the bottom end, thus the name citrus greening disease. Fruit quality is greatly reduced with a bitter or insipid flavor. In Florida, where some 90 percent of harvested citrus is processed, affected fruit ruins the taste and sweetness of orange juice. Californias citrus reputation is based on its production of high-quality table or fresh fruit. This reputation will be lost if HLB becomes established in our commercial citrus. Those who attended the California Citrus Conference in Porterville last October will remember the taste testing conducted by Drs. Mary Lu Arpaia and Elizabeth Baldwin. Panelists were given juice samples at random that Dr. Baldwin brought from Florida. Some were from known HLB-infected trees and others were from healthy trees. My initial reaction to my samples was that it was not the best-tasting juice, but it did not seem that bad. I was disappointed, thinking I had not received the HLB juice. However, after several minutes, I had a distinctly bad and somewhat metallic taste in my mouth. The unpleasant taste lasted for about an hour. If HLB becomes established in California, it will change citrus growing as we know it. Gone will be the lush, dark green-leaved trees, the tasty, sweet, perfectly-shaped fruit that California is known for, and gone will be the beautiful citrus trees in our groves and in Californians backyards.

eases, nutrient deficiencies, toxicities or improper horticultural practices. It may take months or even years for symptoms to develop after the tree becomes infected. The most accurate method of detection is performed in a laboratory by qualified individuals. Leaf and/or

root samples are collected, and the DNA is extracted. The assay method, polymerase chain reaction (PCR), is used to look for unique segments of bacterial DNA. Although this is the approved detection by the USDA and CDFA, this method has some limitations. The

Fig. 3. SPARSE FOLIAGE: In parts of the world where the impacts of HLB are severe, citrus trees are not vigorous and have sparse foliage.

How do I know if my tree has HLB?

It is not easy to diagnose a tree positive for HLB. Visual symptoms of the canopy can resemble other disFig. 4. GREEN FRUIT: In South Africa, affected fruit tends to stay green at the bottom end, thus the name citrus greening disease. Summer 2013 Citrograph 13

bacterium is not evenly distributed throughout the plant; therefore, if the sample collected does not happen to contain the bacterium, a false negative test may result. The bacterial population appears to be more consistent within root tissue, but this type of sample collection is more difficult, time consuming, and somewhat destructive. The Citrus Research Board has funded several projects in the past few years with the objective of developing methods that are able to detect the presence of HLB-associated bacteria much sooner than the currently accepted PCR method. Several research groups have made good progress, and their platforms are currently undergoing evaluation. Instead of testing for the actual bacteria, these assays detect proteins secreted by the bacteria or responses of the infected plants to the presence of the bacteria. Infection by a pathogen induces several types of plant responses, including unique volatile organic compounds (VOCs), small RNAs and unique me-

tabolomic compounds. Scientists are capitalizing on these responses and have found these compounds to be more evenly distributed throughout the plant than the pathogen. These improved detection assays will be available for use in one to two years.

Key Terms
Fastidious: Describes organisms that cannot be cultured on artificial nutrient media and/ or under laboratory conditions. They can only survive in specific environments (e.g., within the cells of their host). Phloem: Plants have a vascular system that is composed of the xylem, which conducts water from the roots upward to the canopy and the phloem. The phloem conducts nutrients from the roots upward to the canopy and carbohydrates produced by photosynthesis in the leaves downward for storage in the roots. Incubation period: The time interval between plant infection and the appearance of disease symptoms. Latency period: The time interval between plant infection and when the pathogen can be transmitted to other plants or acquired by an insect vector. Small RNAs: small molecules, typically 22-24 nucleotides in length of Ribonucleic Acid. Many small RNAs do not code for proteins, but instead exercise control over those RNAs that do, thereby altering gene expression.

What can be done to protect our citrus?

Regulation of the importation of propagating materials prevents the introduction of bacteria associated with HLB or greening through that mechanism, but there are other pathways -the illegal importation of budwood, and the traveler or immigrant who unknowing packs citrus material from infected sources. The message for everyone, whether planting a commercial grove or a citrus tree in a backyard, is to always buy the trees from a reputable nursery that obtains their propagation materials from a certified, disease-free source. Where the disease is endemic, it may be controlled by the reduction of inoculum sources (eradication of

infected trees) and insect control by pesticide application. In California, the citrus industry and the California Department of Food and Agriculture (CDFA) have partnered and are conducting a survey to look for trees infected with HLB-associated bacteria. If you are a grower or have citrus trees in your backyard, be vigilant and frequently look for the ACP vector. For the benefit of future generations, please allow agriculture inspectors to collect leaf or root samples from your citrus trees. If the disease is out there and it can be found early, before the Asian citrus psyllid can spread the pathogen to other trees, we have a good chance to win the battle against this devastating disease. MaryLou Polek, Ph.D is the Vice President, Science & Technology for the California Citrus Research Board. l
14 Citrograph Summer 2013

Bringing fresh perspectives to citrus flavors

Givaudan Collaborates with University of California, Riverside on TasteTrek Citrus
By Jennifer Haggard and Dawn Streich
n a crisp, cool day in January, a team from Givaudan Flavors joined Dr. Tracy Kahn to tour the Citrus Variety Collection on the campus of the University of California, Riverside (UCR). In collaboration with the University, the team was participating in a Givaudan-sponsored TasteTrekCitrus. What is TasteTrek? One needs to start at the beginning. Citrus is the worlds favorite flavor and aroma. Whether it is home fragrances or beverages, people around the globe love the uplifting refreshment of citrus. To satisfy consumers constant desire for new taste experiences, researchers can look to Mother Nature to inspire tomorrows top citrus flavors. Getting the Givaudan flavorists out of their labs and into the world to discover sources of flavor inspiration lets them personally experience new tastes, trends and ingredients, expanding their thinking on how to help food and beverage brands appeal to todays global consumers. Its this go to the source philosophy that drives the TasteTrek program. Since 2006, Givaudan has partnered with the University of California Riverside Citrus Variety Collection to explore some of their more than 1,080 unique and globally diverse citrus varieties. Exploration of the fruit is a total sensory experience. Through sight, smell, touch and taste, each fruit is experienced entirely, inside and out, to identify extraordinary flavor characteristics that could lead to new ideas, flavor molecules or ingredients (Fig. 1). On a recent day at the Citrus Variety Collection (CVC), the Trek team consisted of 8-12 Flavor Chemists, Analytical Chemists, Beverage Applications and Marketing Specialists, whose primary goals were to find fruit with unique flavor attributes, evaluate what makes it special and determine how the flavor profile can best be applied to a beverage. The Givaudan team works with all types of products from juice drinks to carbonated soft drinks to flavored waters to low alcohol flavored beers and malt spirits. They consider market trends and consumer appeal to determine what flavors might be the best fit for further development. Prior to visiting the Collection, the staff at Givaudan has worked with Dr. Tracy Kahn, Curator to develop a list of interesting fruits. Dr. Kahn created a map of the locations of the specific trees the group wanted to visit in the grove. She began the day by sharing a few varieties, recently developed by the UCR citrus breeding program, that she thought might be of interest. The team set off on their Trek, supply wagon in tow, to sample and taste mandarin varieties and hybrids. Some of

Fig. 1. Givaudan Flavorist Roberto Terreros knows that aroma is critical to flavor development.

Fig. 2. Headspace capture of a lemon in progress. Summer 2013 Citrograph 15

Look deep into nature, and then you will understand everything better. -Albert Einstein
the more interesting mandarin types tasted were Seedless Kishu, with its juicy, delicate flavor; UCR-developed Gold Nugget with its sweet, sugary profile; the complex Indiomandarinquat, which combines the best of mandarin and kumquat floral notes; and Tango, a variety released by UCR in recent years for commercial propagation, with a profile that has broad consumer appeal. Once the team identified the fruits with the most unique profiles, they chose a tree for headspace capture, which entailed taking a plug out of one fresh fruit still hanging on the tree, and inserting a scent-capturing device connected to a computer (Fig. 2). This set-up allowed capture of the fresh fruit scent and fed the information directly to a computer that analyzed the aroma profile. The results of this headspace gering on their taste buds helps to build in the unique nuances only the fresh fruit provides. Fine tuning is then done in the lab after they return. In the last 13 years of working with UCR, Givaudan has evaluated nearly 150 fruits and created many distinctive flavor varietals ideal for juice drinks, sports drinks, carbonated soft drinks and beyond. TasteTrek Citrus has been a stimulus and source of innovation for many key Givaudan citrus initiatives. When a Japanese customer requested in-depth understanding of fresh grapefruit and insight for authentic flavor design, the team worked with the CVC over several months to collect fruit to capture the sensory profiles and chemical composition of Marsh grapefruit juice throughout ripening. In a study that spanned six months, Givaudan gained valuable learning about the evolution of the woodiness character, emergence of berry notes and changes in complexity and balance of the grapefruit flavor as it matured. As a result, a collection of true to fruit seasonal flavors was designed to mimic grapefruits ripening, starting in early season and developing through mid, peak and late season. When the Givaudan citrus team in North America began to see the growing popularity of lemonade, they used their broad understanding of lemon and lime varietals from the grove to design a diverse range of gold standard flavors, which were sensory and consumer tested, including the Meyer, Amber, LoPorto and Variegated Pink Fleshed Eureka lemons, and Millsweet Limetta, etc. Among these white space flavors, pushing the boundaries from traditional lemonade to potential signature status, Amber lemon was the it flavor. A combination of Amber lemon and Millsweet Limetta was a hit with its lemon-lime and candy notes complimented by nuances of orange, grapefruit and black pepper. In a close second, Amber lemon combined with Variegated Pink Fleshed Eureka lemon was extremely popular because of its unusual lemon lime profile with orange pith and jammy raspberry notes. What is next in citrus? The possibilities are endless. Bringing together flavor expertise and collaboration between Givaudan and the UCR Citrus Variety Collection, the journey continues to discover and bring the best of natures citrus palette to consumers taste palettes. Jennifer Haggard is Head of Global Marketing at Givaudan Flavors Corp. Dawn Streich is Citrus Product Manager forSweet Global Business Development at Givaudan.l Founded more than 200 years ago, Givaudan Flavors Corp. is the leading company in the flavor and fragrance industry, supplying 25 percent of the worlds flavors and scents to everything from soda to fabric softener. Givaudan is committed to understanding everything about citrus flavors what makes citrus unique, why consumers across geographic, social and cultural demographics are drawn to citrus and what is the next big citrus flavor. Rooted in its rich history of citrus expertise, Givaudans program focuses on innovative new technology and creative vision.

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

capture technique, along with sensory and other chemical analyses, provide technical insight into the chemical components responsible for intriguing flavor notes (Fig. 3). Once back in the lab, sensory profiling, using SenseIt descriptive flavor language, enables the team to quantify and characterize the presence, quality and intensity of different flavor attributes. GC/MS analysis of the juice and peel oil allows them to link the sensory attributes to the natural chemical(s) responsible for the flavor. From these chemicalsensory discoveries, Givaudan scientists identify potentially new flavor materials that they can either synthesize or naturally recover and add to the flavor creation palette. The true art of translation from citrus fruit to flavor comes from the inspired minds of skilled flavorists and the use of Givaudans proprietary flavor creation tool, the Virtual Aroma Synthesizer (VAS). The portable miniVAS tool combines the human perception of smell, combining various scents or keys into a single olfactive profile, and precise instrumentation to transform desired aroma profiles into finished flavor formulas. It allows flavorists to get 80 percent of the way to their target flavor while still in the UCR CVC grove. Creating the flavor while the excitement of being in the grove is still fresh in their minds, the taste of the fruit lin16 Citrograph Summer 2013

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Comparing orange juice from healthy vs. HLB-affected trees

A look at the chemical and sensory flavor differences
Elizabeth A. Baldwin
reening or Huanglongbing (HLB) disease of citrus is thought to be caused by a bacteria, Candidatus Liberibacter asiaticus (CLas) and is vectored by the Asian citrus psyllid, Diaphorina citri. The disease can cause rapid decline of citrus trees, eventually killing them within 5-10 years. This devastating disease of citrus

has been reported to result in citrus fruit and juice that is sour, bitter and generally off-flavored.

Spread of the Disease

Sadly, this disease was preceded by citrus canker in Florida, for which a program of eradication was conducted. As a result, thousands of trees were pulled due to an epidemiological study showing that the canker bacteria, disseminated

by wind-driven rain, could infect trees in a 1,900 square foot radius. After the hurricanes of the 2004-2005 seasons, the disease spread statewide, and the eradication program was abandoned. Unfortunately, HLB was identified in 2005, and citrus growers, having pulled trees for naught for canker disease, were not in the mood to pull out even more. Therefore, removing diseased trees was not enthusiastically pursued for a disease that was far worse than canker, one which presented more of a blemish issue for fresh fruit and only caused slow tree decline over many years if left untreated. Consequently, HLB disease has progressed throughout the state of Florida as growers have become more and more reluctant to pull out diseased trees. Rather, a proposition of using foliar nutritional sprays, which often include systemic acquired resistance compounds (SARs) to boost plant defense mechanisms, has been adopted to compensate for the disease symptoms. Although there are conflicting results, these spray programs have worked to some extent in reversing tree symptoms. They now have been adapted by many of the industry growers who either are reluctant or cannot afford tree removal. The situation is complicated by the fact that trees do not show symptoms for HLB immediately, thus being symptomless and infecting other trees for an unknown amount of time. More nutrition cannot but help the sick trees, but it is not known whether nutritional sprays that partly alleviate tree HLB disease symptoms also affect fruit off-flavor symptoms, and whether or not these treatments affect the amount of the assumed causal agent, CLas, in the fruit.

Exploring the Differences

Fig. 1. A comparison of fruit from healthy trees vs. HLB-affected trees. 18 Citrograph Summer 2013

Our laboratory compared juice made from fruit harvested from HLBinfected trees to juice made from fruit

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harvested from healthy trees. We further segregated the fruit from HLB-infected trees into fruit symptomatic for the disease (HLBs that are small, green and lopsided) and fruit that looked normal or asymptomatic for the disease (HLBa). (See Figs. 1 and 2.) We did both chemical and sensory analyses and found that HLBa fruit (that do not show HLB symptoms) produce juice that tastes similar to juice from fruit harvested from healthy trees, while the juice from symptomatic HLBs fruit are less sweet and more bitter, among other off-flavors. This correlated with chemical analyses showing the HLB fruit juice, especially

for HLBs, has lower sugars, sometimes higher acids and higher levels of the bitter compounds limonin and nomilin, as well as other secondary metabolites such as phenolic compounds. These compounds are actually healthy for humans, but can impart off-flavor above certain concentrations. The amount of the bitter compounds limonin and nomilin in HLB juice was puzzling since the levels of these compounds were below those reported to be detectable by humans. We then conducted a study of threshold determination (amount of a compound that is detectable) by spiking orange juice with different levels

Healthy

Fruit used in study

HLBa

HLBs

of these bitter compounds, and tested spiking juice with limonin and nomilin alone and in combination, with or without added sugars or acids. These spiked juices were served to panelists to determine the concentration at which they could detect bitterness. We found that the combination of the two bitter compounds was synergistic in the panelists perception, demonstrating that together they imparted detectable bitterness at the levels they are found in HLB juice. Higher sugar levels helped to mask the perception of bitterness. Therefore, HLB juice that has less sugars and higher levels of limonin and nomilin will taste more bitter. We also tested the amount of HLBs juice that could be blended with normal juice before panelists could detect a difference and found that 25 percent of HLBs juice would be required to blend with healthy juice for panelists to notice a difference. This is good news since most HLBs fruit fall off the tree. If picked, they should be graded out based on size and, therefore, should not enter the juice stream in any large volume.

Testing Treatments
Over the past four years, our laboratory has been studying three different nutritional-SAR foliar treatments in comparison to conventional orange tree culture and spray regimes to see whether these nutritionalSAR treatments, which mitigate tree disease symptoms, would also mitigate fruit off-flavor symptoms. Most growers, other than organic growers, implement pesticide spray programs to control the HLB

GLOSSARY
HLBa Fruit that is harvested from an HLB-infected tree and does not show HLB fruit symptoms. It is not small, green and lopsided or asymmetrical, but looks like a normal healthy fruit and is, therefore, asymptomatic. HLBs Fruit that is harvested from an HLB-infected tree and does show HLB fruit symptoms. It may be small, green and/or lopsided and is, therefore, symptomatic for the disease. (See Fig. 3) SARs Systemic acquired resistance, meaning compounds that induce plant resistance.

Fig. 2. A look at the outside and inside of healthy citrus compared to that from an HLBa-affected tree and an HLBs-infected tree. 20 Citrograph Summer 2013

Fig. 3. These Hamlins exhibit classic HLBs characteristics.

disease psyllid vector, and such was the case in our studies. At first, results showed no differences of nutritional-SAR sprays on mitigation of the HLB-induced off-flavor, especially for symptomatic HLBs fruit juice. However, in the latter years, it appeared that one of the nutritional-SAR foliar treatments was showing some effect by making the juice sweeter. Further seasons of study are warranted since the effect of foliar nutritional-SAR treatments may take years to reach full effect. Additionally, we developed a procedure to measure the amount of CLas pathogen DNA in orange juice. We did this for fruit harvested from trees receiving the foliar nutritional-SAR sprays compared to conventionally treated trees. We saw that juice made from HLBs fruit exhibited higher CLas bacteria titer than juice made from

HLBa fruit, which had higher titer than juice made from healthy fruit . Yet we did not see significant differences due to nutritional treatments, except for one treatment in one year for Hamlins, but not for Valencias. Again, more years of study are warranted.

Introducing Enose, Etongue

Finally, looking for a way to objectively screen juice for quality, whether due to HLB, variety, season, harvest date, etc., we looked at the possibility of using technology that crudely mimics the human olfactory system (electronic nose or enose) and taste system (electronic tongue or etongue). The olfactory bulb receptors and tongue receptors non-selectively bind odor or taste volatiles or soluble chemicals, respectively, such that one receptor may bind more than one compound, and one

compound may bind more than one receptor. This allows for unlimited pattern possibilities. These receptors then send signals to the brain, and the brain makes a decision based on pattern recognition (good orange juice vs. bad orange juice; orange juice vs. apple juice, etc.). The enose and etongue have sensors that interact with volatile odor molecules or soluble chemicals, respectively, in a food in a non-selective manner, allowing many pattern possibilities, and send signals to a computer that has pattern recognition software. This software does multivariate statistics to discriminate between samples, such as juice made from healthy HLBa or HLBs fruit. Using this technology and judging by sensory panels that smell and taste orange juice, we can tell that there are more differences in healthy juice versus HLB juice due to solutes than due to aroma volatiles, but there are differences in aroma volatiles, as well. Nevertheless, the etongue does a good job (better than the enose) at discriminating between healthy HLBa and HLBs juice. In conclusion, HLB disease does affect orange juice (and orange fruit) flavor, but this is more pronounced for the HLBs fruit. Avoiding these in the fresh market and processed juice plants should eliminate most of the HLBinduced off flavor. For the processed orange juice industry, blending with healthy juice will mask most off-flavor, but for the fresh fruit industry, these fruit will bring down the quality of the product. 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

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3rd Annual Citrus Health Research Forum:

Vision for the Future

Ed Civerolo and MaryLou Polek
identification of potential products of current research and critical milestones to deliver those products; outreach efforts; communication among researchers; and communication of research progress to State and Federal regulators, growers and processors. The purpose of the annual CHRF conference is to: review progress in relevant research areas in each Outcome Group; identify research gaps that need to be bridged to facilitate progress toward

INTRODUCTION
The third annual Citrus Health Response Program (CHRP) Citrus Health Research Forum (CHRF) was held August 27-30, 2012, in Ft. Collins, Colorado. As in the past, the meeting was sponsored by the national CHRP, California Citrus Mutual, Citrus Research Board (California), Florida Citrus Mutual and Texas Citrus Mutual. This annual meeting is industry-driven for the over-all purpose of ensuring a productive and thriving citrus industry that is currently threatened by Huanglongbing (HLB) and the Asian citrus psyllid (ACP). The forum brings together representatives of the United States citrus industry (including citrus organizations, growers and processors), researchers from the U.S. and Mexico , as well as State and Federal regulatory personnel to exchange and discuss current information about citrus, HLB and ACP. They also identify needs and priorities in order to develop new and increased knowledge, innovative and improved technology and effective strategies to manage HLB and ACP.

topic-driven discussions; oral and poster presentations regarding research progress and current ongoing research; Vision for the Future; and reviewing and developing Outcome Group priorities. The information presented and discussed at the 2012 Research Forum in Ft. Collins is currently being incorporated into a CHRP HLB-ACP Action Plan.

GROWER PERSPECTIVE
Particular emphasis was placed on receiving growers input on the research progress made by the scientists as well as the research priorities that ought to be set by the whole community including the researchers, regulators and industry. Therefore, the forum started on the first day with a session on the citrus growers perspectives in which representatives from California, Texas and Florida expressed their views. The idea was to capture three main points: 1. What kind of problems are the growers facing? 2. What surveillance and control measures are they taking? 3. What other surveillance and control measures do they need in the future to maintain a productive and economically viable citrus industry? The views of the growers from the three states varied since the status of HLB and ACP is quite different in each state, and each has its own industry goals. For California, the goal is to produce high-value and high-quality citrus fruit for domestic and foreign markets. The threat of HLB-associated Candidatus Liberibacter species infection in California is looming, largely due to the rapid growth and expansion of ACP populations in Southern California. Vigorous ACP trapping and the testing for Liberibacters, especially Ca. L. asiaticus, are currently underway. However, chemical control is proving to be insufficient for robust ACP control. A biological control

BACKGROUND
In 2009, an outcome-based research approach was adopted by the CHRP at the national level to mitigate the impacts of HLB and ACP. Three solution-based research outcomes were identified, and three Outcome Groups (OGs) were established to focus on them. The missions of the OGs are to: keep ACP-infested and HLBaffected 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) of the CHRP, facilitate and enhance: discussion of current research HLB and ACP efforts; collaboration and coordination of research among scientists;
22 Citrograph Summer 2013

the overall goal of effectively managing and mitigating the impacts of HLB and ACP; ensure the continuation of the momentum of research progress; and encourage communication among researchers, growers and processors. Additional detailed background information about the annual CHRFs is presented in the July/August 2010 Citrograph article, First Annual Citrus Health Research Forum furthers efforts against ACP/HLB and the November/ December 2011 Citrograph article, 2nd Annual CHRP Research Forum: Action items will focus on host resistance to HLB and preventing transmission of the bacteria. Highlights of the 3rd Annual CHRF included: grower perspectives;

program for ACP also is being developed. In California, removal of Ca. L. asiaticus infected trees is mandatory as per Secretary Ross. Fortunately, to date only one such tree in Southern California has been confirmed, and that backyard tree has been removed. The California growers identified the following needs for the future : Improved and uniform ACP surveillance and control measures across all groves Accurate platforms for detection of Ca. L. asiaticus in insect and citrus samples, and pre-symptomatic detection and diagnosis of HLB Tools for eliminating Ca. L. asiaticus from citrus trees HLB-resistant citrus cultivars for long-term protection In Texas, the HLB situation is more serious than in California. So, far two groves have been reported to contain HLB-affected trees, and those trees have been removed. Areawide ACP management has been significantly ramped up since the HLB detection. More than 85 percent of all growers are cooperating in

the areawide treatment program. Texas growers identified the following needs for the future: Robust and effective ACP surveillance and control throughout the year ACP bio-control, possibly with the aid of a predator Early and pre-symptomatic diagnosis of HLB HLB-resistant transgenic citrus In Florida, most of the citrus trees are already infected with Ca. L. asiaticus and are showing HLB symptoms. Therefore, it is too late for ACP control alone to effectively manage HLB. Accordingly, HLB mitigation is the top priority. A combined use of ACP control and nutritional applications has been useful; but nutritional approaches have not been validated scientifically. Research focused on developing HLBresistant transgenic citrus has produced promising results and may lead to a long-term solution for HLB protection. However, Florida cannot wait for such a long-term solution without protecting the already infected trees.

The Florida industry identified the following needs for the future: Therapeutics to kill or suppress Candidatus Liberibacter asiaticas Effective and sustainable ACP control A new ACP with reduced vector capacity to protect new trees Advanced production systems to push economic return into earlier years The Growers session concluded with a Q&A period regarding industry concerns about the use of HLB-resistant transgenic citrus. It was generally agreed by all of the grower panel members that acceptance would not be a problem once the product is shown to be effective and ready for implementation. There was a question about what is the best nutritional program. Although it was hard to pinpoint a single effective nutritional program, one panel member felt that nutrients are being applied at an excessive rate in some cases. Finally, there was a question about how long trees must survive to be profitable. The consensus was that it depends on the return per

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unit. It is possible to generate profit from 8-year-old trees and good profits from 10 to 12-year-old trees.

TOPIC-DRIVEN DISCUSSIONS
The Growers session set the stage for the topic-driven discussions by the three OGs, which were introduced by key speakers and focused on the selected science and technology approaches necessary to address growers needs. Each speaker emphasized several key points, namely: How is the chosen topic relevant to the mission of one or more outcome groups? How do the science and technology approaches in a given topic area meet the grower needs? What advances are needed to meet grower needs in a timely and effective manner?

Outcome Group 1 Topics.

Cultivation of Liberibacter in-vitro
So far, it has not been possible to culture Liberibacter in the laboratory; however, the ability to culture Liberibacter would be helpful in many ways. First, it would be possible to directly inoculate

citrus with Liberibacter and show that it causes HLB symptoms (i.e., proving Kochs hypothesis). Second, it would be possible to screen a large library of drugs and natural products that inhibit the growth or kill the Liberibacter, at least in the culture. Third, it would be possible to perform genetic manipulations to identify the key Liberibacter genes that facilitate the bacteriums colonization specifically in the citrus phloem. Several advances have been made that provide important clues: Different artificial nutrient media formulations, especially those with citrus juice, look promising. Including insect gut cells in the media with Liberibacter may allow for a short-term culture. The observation that seed coats display high Liberibacter titer may lead to the development of sustainable culture in vitro. The discovery and genome se quencing of L. crescens marks an important progress, because cultures of L. crescens have been maintained for more than 10 years. This bacterium is closely related to the HLB-associated Liberibacter species.

Comparative analysis of metabolic pathways of the known Liberibacter genomes may shed light on the appropriate combination of metabolites and nutrients that are suitable for maintaining live cultures of the HLB-associated bacteria.

HLB diagnosis and Liberibacter detection

Diagnosis of HLB and detection of Liberibacter are critical to disease surveillance. While visual symptoms will always be important, by the time visual symptoms appear, the bacteria may have already spread long distances, making it difficult to eradicate infected trees. Liberibacter-specific molecular markers are potentially more rapid and reliable. There are three parts to molecular diagnosis 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 for detection or diagnosis needs to be designed. It appears that a specific set of volatile organic compounds or a specific microRNA may report early stages of

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HLB infection. In addition, single chain antibodies (scFvs) have been generated against six Liberibacter proteins that indicate Liberibacter infection. These scFvs can be used in an immunoassay for Liberibacter detection. A lateral flow platform has been designed for a DNA sandwich hybridization assay to detect specific Liberibacter genes. This platform combines sample preparation, DNA amplification and signal read-out and takes only half-an-hour for pathogen detection. There is also a flow-based luminex platform that uses multiplexed oligonucleotide ligation-PCR (MOLPCR) to detect Liberibacter signature genes. This platform is capable of handling 2,500 samples in eight hours.

therapy and targeted phloem delivery shows promise. Delivery of transgenes by CTV offers a route for getting resistance genes in existing citrus trees.

Areawide pest management programs (AWPMPs)

A successful AWPMP requires: 1) active participation of the growers, 2) full engagement of the public sector, and 3) availability and application of effective tools. Examples of successful AWPMP include mosquito and malaria control in different parts of the world, and control of glassy-winged sharpshooters (GWSS) as part of integrated pest management of Pierces disease of grapevines. Such measures are also applicable for ACP control in citrus orchards. It was noted that there is no effective pheromone for ACP trapping. Although there are effective pesticides, the efficacy of some materials needs to be validated. There are significant challenges to maintain a successful AWPMP for ACP: Groves are often intermingled with residential areas. Growers must be convinced to use new and more effective materials and abandon the cheaper (albeit less effec-

tive) ones they have in hand. For each area, a treatment coordinator is needed. An appropriate grower as point-ofcontact for the coordinating effort must be identified.

Technology for modifying Bt toxins against specific pests

Although toxins derived from the bacterium Bacillus thuringiensis (Bt) have been used successfully for the management of some insect pests, Bt toxins have not been effective against the agriculturally important Hemiptera, including the ACP. A report was heard describing a new technology that modified Bt toxins to improve their toxicity against aphids, also members of the Hemiptera. By improving the binding of the Bt toxin to the aphid gut membrane, the toxicity was increased. A similar approach was envisioned for ACP.

HLB therapeutics
Development of therapeutics that can suppress the disease and/or reduce or totally clear Liberibacter from the phloem is an extremely high priority. There are two important aspects: what the nature of the therapy is, and how it can be delivered. Several types of therapy are being tried, including nutritional and thermal; however, their effectiveness is yet to be determined. A combination of antibiotic

Chemical ecology tools to prevent or slow the spread of ACP and HLB
Insect behavioral mechanisms and chemical ecological patterns provide clues to create and optimize tools for the effective monitoring and manage-

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ment of ACP. The specialized pheromone lure application technology (SPLAT) is compatible with the standard insecticide spray technologies currently being used by the industry. The combination of insecticides and semiochemicals, such as pheromones, has the potential to produce remarkable synergy in ACP control. So far, an effective pheromone as a lure for ACP is yet to be identified. Several studies have, however, identified and tested potential chemical attractants for ACP. These include petitgrain oil (distilled from sour orange), Titan+limonene combination, and vanilla extract, among others. New monitoring lures that utilize specific plant volatile blends are in their final stages of field testing. Monitoring traps baited with one of these lures have reliably resulted in increased ACP trap catch when compared to unbaited traps. Such attractants might be good candidates for semiochemical-based attract and kill technologies. Repellants could have a role in ACP management: a very inexpensive compound, dimethyl disulfide (DMDS), formulated in SPLAT has been shown to be an effective ACP repellant in the field. Interestingly, methyl salicylate, as released by HLB-affected citrus, has a peculiar property in that it is attractive to ACP. However, HLBaffected citrus plants have an inherently low nutritional value that, after a few days, forces the resident ACP to move on to neighboring, uninfected citrus plants from which they are able to obtain better nutrition. This sequence of behaviors favors an increased rate of HLB infection or re-infection within a grove. It would be beneficial to the citrus industry if long-lasting formulations were created to efficiently disrupt this semiochemicallydriven cycle of HLB infection.

lanacearum, the potato psyllid, and the potato host, can serve as a good model system for HLB as the full range of disease symptoms appears in weeks. The potato system has been applied to screening anti-Liberibacter defensins, as well as for discovering potato proteins that are specifically induced upon infection. Tomato and tobacco are other model hosts that have been used for screening anti-Liberibacter defensins from spinach. Finally, in vitro cultures of Liberibacter crescens, a close relative of the HLBassociated bacteria, have been used to screen potent antimicrobial peptides.

acter by modifying the specific receptor that facilitates bacterial movement in the psyllid gut. It has been shown that specific double-stranded RNA (dsRNA) acting on actin is effective as a genetic pesticide for GWSS. A similar approach can be applied for killing psyllids. Another interesting approach involves identification of endosymbiotic viruses in the psyllid and engineering of one or more of these viruses to express RNAi or peptides that are toxic to ACP.

POSTER SESSION
Approximately 30 posters were presented, which described research and technology areas relevant to the missions of the three outcome groups. All participants had the opportunity to view the posters. In addition, the presenters had the time and opportunity to explain their results and answer questions.

Functional and comparative genomics of Liberibacter

Comparative genome analysis of the Ca. L. asiaticus (CLas), Ca. L. americanus (CLam) and Ca. L. solanacearum (CLso) revealed subtle but distinct differences in the three genomes. In addition, the CLas, CLso, and CLam genomes all contain genome sequences of two bacteriophages (phages), but differ in their copy number. These phages apparently become lytic, killing Liberibacter, in citrus but not in psyllids. Investigation is underway to determine whether phages can be used to manage HLB.

VISION FOR THE FUTURE

A departure from the previous CHRFs was a session in which two distinguished scientists (Dr. George Bruening, Professor Emeritus, Department of Plant Pathology, University of California-Davis; and Dr. Gary Secor, Professor Emeritus, Department of Plant Pathology, North Dakota State University) outside of the HLB-ACP research community presented their perspectives on the research progress being made and what novel approaches might be considered. Based on their reviews of the current progress and future needs as presented during the grower session by the topic-driven speakers and posters, each made several recommendations.

Host response studies for discovery of pre-symptomatic HLB biomarkers and characterization of HLB tolerance/resistance
Host plant responses to Liberibacter infection may allow for the early identification of HLB prior to the development of visible symptoms. Molecules produced by the plant in response to Liberibacter infection include small RNA, proteins, metabolite markers and volatile organic compounds (VOCs).

Dr. Secors Recommendations:

Continue gene-silencing work for blocking transmission of Liberibacter and killing psyllids. Continue work toward making Liberibacter culturable. Combine advances made in cultural, biological and chemical methods in HLB management into actionable practices in the field. Combine bio-control methods with effective delivery systems. Invest in studying psyllid epidemiology and in developing methods for monitoring psyllid reservoirs. Explore the possibility of a citrus cell culture system, which may allow rapid screening of potential

Outcome Group 3 Topics

Model systems for rapid screening of transgenes
Model systems are primarily chosen for screening transgenes that clear Liberibacter from citrus or inhibit development of HLB symptoms. Model systems are also useful to obtain clues about host responses when infected. Although they do not exactly mimic citrus infection, model systems allow rapid screening of transgenes in a month or two as opposed to years if studied in citrus. The same is true for host response studies. The potato/zebra chip (ZC) pathosystem, including Candidatus Liberibacter so26 Citrograph Summer 2013

Use of psyllid genome information for HLB biocontrol

Like any other diseases associated with vector-borne pathogens, there are two potential ways to prevent HLB by targeting the psyllid. One strategy involves blocking the psyllid from acquiring and/or transmitting Liberibacter, whereas the other involves killing psyllids. Development of both strategies is immensely helped by complete knowledge of the psyllid genome, which provides information on pathways relevant to Liberibacter transmission and the psyllid life cycle. For example, ACP can be rendered unable to transmit Liberib-

HLB-inhibitory small molecules and biologics. Examine the correlation (if any) between the extent of Liberibacter spread in the tree and disease latency.

Dr. Bruenings Recommendations:

Focus on accurate, sensitive, specific and high throughput detection platforms that are reproducible for various seasons, climates, times of day, citrus cultivars, etc. (specifically emphasize pre-symptomatic diagnosis of HLB). Fulfill the promise of volatile organic compound (VOC) detection by high throughput sampling with the aid of a validated set of HLB-specific markers. Use high throughput combinatorial approaches to discover novel attractants/repellents for pheromone lure application technology (SPLAT). Continue work on developing transgenic citrus by clearing Liberibacter or by engineering host defense. Continue work on blocking Liberibacter transmission or killing psyllids using RNAi and peptides. For HLB resistance, stack two or more transgenes that target the same Liberibacter gene but by different mechanisms.

Liberibacter culture in vitro (needed for screening therapeutics and for identifying virulence markers) Therapeutics to keep affected groves productive Effects of HLB and HLB management practices on citrus products that impact consumer acceptance

Outcome Group 2 Mission:

Slow or prevent the spread of ACP and HLB

Research Priorities:
Biology of psyllid and psyllidLiberibacter interactions Areawide management pro grams Biological control of psyllid Study of the effectiveness of flush management Getting new citrus plantings into production Studies on environmental factors and their effect on HLB symptoms Diagnostics for early stage infection

ogy innovations into practical solutions and implementing these solutions in a timely manner for surveillance and management of HLB. It was noted that early diagnosis of disease is not only critical for industries in California and Texas, but also for the Florida industry when it comes to young trees. For Florida, the highest priorities are: 1) psyllid control, which involves blocking acquisition and transmission of Liberibacter, and insect killing; and 2) development of therapeutics for effective HLB control or suppression. One suggestion was to use antibiotics in combination with other therapeutics. While pre-symptomatic HLB diagnosis and development of HLB-resistant citrus are tremendously valuable, it was suggested that concerted efforts in these two areas would speed up the transition from the discovery to the application phase.

FINAL WRAP-UP SESSION

Industry representatives from California, Texas and Florida provided their thoughts on the CHRP Forum. All of them emphasized that researchers must always keep the growers in mind and cooperate with each other as much as possible. They stressed that it is important for the scientists to envision how their research can be applied and how it would help the growers.

Outcome Group 3 Mission:

To keep citrus free of ACP and HLB

Research Priorities:
Pre-symptomatic detection of HLB Breeding transgenic and conventional resistance to HLB Functional genomics of Liberibacter to improve detection and control Functional biology of ACP to improve control measures

CONCLUDING SESSION
Reports by each of the three OGs were presented that summarized research priorities for each group and included a brief discussion of the action items. These will form the basis of a cohesive CHRP Action Plan that is under development and will address the: 1. status of surveillance and control of ACP and HLB; 2. high-priority actions needed to accomplish the overall goal; and 3. research and technology advances needed to accomplish the goal. The missions and research priorities identified by the three OGs at the 2012 CHRF are presented below.

4th CHRF
The 4th CHRF is scheduled to be held October 15-17, 2013, in Denver, Colorado. Ed Civerolo is a Plant Pathologist, USDA/ARS, Department of Plant Pathology, University of California at Davis. MaryLou Polek, Ph.D., serves as the California Citrus Research Boards Vice President of Science and Technology. l

GROWERS ASSESSMENT OF THE 3RD CHRF

Outcome Group 1 Mission:

Keep ACP-infested and HLBaffected groves productive

Research Priorities:
Pre-symptomatic diagnosis of HLB and Liberibacter detection in young trees

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CRB-Funded Research Report

Research Project Progress Report

Improving trapping systems for early detection and eradication of fruit flies in California
Roger I. Vargas, David Haviland, Ben Faber, John Kabashima, Beth Grafton-Cardwell and Joseph G. Morse Introduction
True fruit flies (Diptera: Tephritidae) include some of the most economically important pests attacking soft fruits worldwide, including citrus. From an economic perspective, they 1) inflict extensive direct damage to fruits and fleshy vegetables, 2) cause quarantine restrictions on infested areas, 3) require that commercial fruits undergo protective and postharvest treatment prior to export, and 4) provide a source for their introduction into other parts of the world when established in a country. Some of the most notorious members of the family Tephritidae are the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Fig. 1), oriental fruit fly, Bactrocera dorsalis (Hendel) (Fig. 2) and its relatives, Queensland fruit fly, B. tryoni (Froggatt), peach fruit fly, B. zonata (Saunders), and melon fly, B. cucurbitae (Coquillett) (Fig. 3). The recent alarming spread of fruit flies worldwide [e. g., B. invadens Drew, Tsutsura, and White) into Africa and carambola fruit fly, B. carambolae Drew and Hancock, into South America] can be attributed to increased: 1) production of fruits and vegetables worldwide, 2) global trade of fruits, vegetables and plants between countries, 3) crossborder movement of people and their possessions between nearby countries, and 4) air travel with baggage containing infested fruits. The introduction of B. invadens into Africa has been particularly trou28 Citrograph Summer 2013

Fig 1. Mediterranean fruit fly, Ceratitis capitata.

Fig. 2. Oriental fruit fly, Bactrocera dorsalis.

Fig 3. Melon fly, Bactrocera cucurbitae.

bling. Discovered in 2003, it quickly spread from the east to west Africa, infesting over 10 countries and even displacing many of the Ceratitis spp native to the continent. The continued threat of fruit fly establishment in the United States has resulted in large survey and detection programs in California and Florida. Early detection methods for accidental introductions of fruit flies involve deployment of large numbers of traps baited with highly attractive male specific lures methyl eugenol (ME; 4-allyl-1,2-dimethoxybenzene-carboxylate), trimedlure (TML: tert-butyl 4- and 5-chlorocis- and trans-2-methylcyclohexane1-carboxylate), and cue-lure [C-L; 4-(p-acetoxyphenyl)-2-butanone], to detect oriental fruit fly, Mediterranean fruit fly, and melon fly (and their relatives), respectively. Eradication treatments against exotic fruit flies that are accidentally introduced from various parts of the world into California are very costly. For example, due to continuous introductions, current annual costs to exclude Mediterranean fruit fly from California total over $15 million. Annual introductions of Bactrocera spp. often result in temporary trade restrictions and associated areawide eradication treatments, lasting up to nine months before movement of agricultural commodities can resume without postharvest treatments (http://www.cdfa.ca.gov). The California Department of Food and Agriculture (CDFA) directs continuous programs to detect

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and eradicate invasive Bactrocera fruit flies. From 1960 to 2012, nine different Bactrocera species have been detected, with Oriental fruit fly detected most frequently (126 times). There have been 140 eradication programs with 25 quarantines. Most of these programs have occurred in southern California where the usual number of detection traps deployed is five Jackson traps (JT) (TML, ME, and C-L) and five McPhail traps (MPT) (Torula Yeast Solution) per mi2 (2.59 km2). Currently, a separate Jackson trap is deployed for TML, ME, and C-L. Total number of sites in operation is approximately 25,000 for the Los Angeles area and over 30,000 for the entire state. Current methods involve trapping systems used in association with liquid insecticide formulations such as naled (Dimethyl 1,2-dibromo-2,2-dichloroethyl phosphate) which pose environmental and worker safety concerns. Through the Hawaii Area-Wide Pest Management program (AWPM), solid single and double lure insecticidal wafers and solid lure plugs and cones were identified as promising substitutes for traps baited with liquid ME or CL and naled.

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

Results from recent studies provide promising evidence for eliminating the use of hard to handle liquid lures and hazardous organophosphate insecticides by consolidating three lures into one solid single wafer with an insecticide (Fig. 4). Besides the environmental benefits, the possibility of consolidating detection traps would reduce labor costs associated with deployment of numerous traps, which is the biggest expense of large survey programs. The overall objective of this Citrus Research Board-funded study is to evaluate and develop a convenient, effective and safer means to use male lures and insecticides for improved detection and potentially improved male annihilation of invasive fruit flies. Incorporating a single multi-lure dispenser to effectively detect or suppress multiple fruit fly spe-

cies in large trapping programs could drastically reduce costs associated with labor and materials while improving safety and convenience from a workers standpoint. Likewise when a detection occurs, high densities of traps are placed around the discovery site, not only serving for delineation of the find, but more importantly as a mass trapping eradication technique often referred to as the male annihilation technique (MAT). Evaluations are being conducted through the Agricultural Research Service (ARS), U.S. Department of Agriculture (USDA) and Pacific Basin Agricultural Research Center (PBARC) in Hilo, HI, in cooperation with the University of California and Farma Tech International (North Bend, WA). Two bioassay study sites were selected in Hawaii because of the combined presence of oriental, Mediterranean, and melon flies. Sites on the leeward side of Hawaii Island involved six separate avocado orchards (between 191145 N, 1555159 W and 19323 N, 1555523W, at an average elevation of 466 m) and sites on Kauai Island were in a large (ca. 1,400 ha) commercial coffee [Coffea arabica L. cv. Arabica (Rubiaceae)] plantation in Numila (215436 N, 1593254 W, at an average elevation of 125 m). Experiment 1: Evaluation of Standard Individual Jackson Traps against Triple Lure Jackson Trap on Hawaii

CITRUS AVOCADOS OLIVES

Table 1. Weekly male melon fly 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

Table 2. Weekly male oriental fruit fly 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 Liquid ME (6ml) with naled (1%) on wick

1 2 3 4 5 6 7 8

8.4 2.6 10.2 3.0 8.3 2.1 9.7 3.8 11.4 3.5 15.4 1.2 7.8 2.6 2.1 0.6

17.4 5.3 10.3 3.4 10.7 3.5 9.1 3.5 10.8 3.8 7.9 2.2 4.0 0.8 3.4 1.0

1 2 3 4 5 6 7 8

55.5 22.9 62.9 27.6 54.1 18.2 45.1 15.8 67.8 39.0 76.3 47.0 13.9 1.7 8.5 2.6

59.3 20.3 52.5 18.5 65.7 29.9 57.5 23.1 79.7 45.5 78.4 48.3 13.9 2.5 7.3 1.9

Grand mean sem 10.7 1.4a 10.9 1.5a

Mean number of flies captured per treatment over time is not significantly different at the P = 0.05 level, Proc GLM (SAS 2009) (Treatment: F = 0.11; df = 1, 254; P = 0.743).

Grand mean sem 55.2 9.5a 61.4 10.1a

Mean number of flies captured per treatment over time is not significantly different at the P = 0.05 level, Proc GLM (SAS 2009) (Treatment: F = 0.20; df = 2, 254; P = 0.658).

30 Citrograph Summer 2013

Island. Studies were conducted from 13 Dec 2011 7 Feb 2012 in the Kona area of Hawaii Island (during coffee and avocado season). Treatments were placed inside Jackson traps with a sticky insert as follows: 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. Treatments 2, 3 and 4 represent the standard treatments currently used in survey arrays in California for detection of ME, C-L and TML responding flies, respectively. Treatments were weathered in place and traps were serviced every seven days for eight consecutive weeks with a total of eight replicates (eight blocks) with four treatments each. Experiment 2: Male Annihilation Evaluations of Fresh Mallet TMR and Mallet CMR at Kauai Coffee Plantation. Studies were conducted from 19 Nov 26 Nov 2010 (during coffee harvest when Mediterranean fruit fly numbers were highest). Treatments were placed in AWPM traps as follows: 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) + insecticidal strip and 5) 2g TML plug (Scentry

Biologicals) + insecticidal strip. These treatments represented current solid lures identified through the Hawaii Fruit Fly Area-Wide program with potential for MAT applications. Five replicates (five blocks with five treatments each) were conducted.

Results
Experiment 1: Evaluation of Solid Cones and Plugs with Insecticidal Strip, Liquid Lure and Insecticide Solution, and Triple Lure and Insecticide Dis-

penser in Jackson Traps with Sticky Inserts. Captures of melon fly in Jackson traps with TMR wafers were not significantly different (P > 0.05) than those with liquid C-L (Table 1). Captures of oriental fruit fly in Jackson traps with TMR wafers were not significantly different (P > 0.05) than those with liquid ME (Table 2). In initial trials, captures of Mediterranean fruit fly in Jackson traps with Mallet TMR wafers were not significantly different (P > 0.05) than those with the TML plugs (Table 3). In

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Table 3. Weekly male Mediterranean fruit fly 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 TML plug (2g) w/ DDVP strip

1 2 3 4 5 6 7 8 Grand mean sem

9.4 5.9 16.5 10.1 8.2 6.8 8.4 5.9 23.4 18.8 25.6 21.6 32.5 20.5 16.5 10.2 67.6 47.0 53.9 41.1 40.8 27.5 30.6 20.7 37.4 32.6 11.5 7.9 27.5 22.1 10.0 5.5 28.9 8.1a 22.7 6.8a

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Mean number of flies captured per treatment over time is not significantly different at the P = 0.05 level, Proc GLM (SAS 2009) (Treatment: F = 0.18; df = 1, 254; P = 0.6684).

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Summer 2013 Citrograph 31

summary, there were few differences for all three groups on the basis of attraction and capture with the TMR wafer and the current standard traps used in California. Experiment 2: Male Annihilation Evaluations of Fresh Mallet TMR and Mallet CMR at Kauai Coffee Plantation. Comparison of Mallet TMR and Mallet CMR for captures of oriental fruit fly, Mediterranean fruit fly, and

melon fly were not significantly different (P > 0.05). As a result, Mallet CMR wafers were not used in subsequent experiments due to the higher production costs associated with producing ceralure when compared to TML. Mallet TMR and Mallet CMR performed equally as well or better than the solid lure cones or plugs with DDVP strips. ME and C-L are commonly used in male annihilation eradication programs. Of particular

interest based on these results would be the application of TML to areawide management of Mediterranean fruit fly.

Discussion
Solid Mallet TMR (trimedlure [TML], methyl eugenol [ME], raspberry ketone [RK]) wafers and Mallet CMR (ceralure, ME, RK, benzyl acetate) wafers impregnated with DDVP insecticide were evaluated in traps as potential

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32 Citrograph Summer 2013

detection and male annihilation devices. Comparisons were made with 1) liquid lure and insecticide formulations, 2) solid cones and plugs with an insecticidal strip, and 3) solid single and double lure wafers impregnated with DDVP for captures of Mediterranean fruit fly, oriental fruit fly and melon fly in Hawaii. Jackson and AWPM bucket traps were tested in avocado orchards near Kona, HI (low to moderate population density) and in a coffee plantation near Eleele, HI (high density population), respectively. In the detection trials near Kona, HI, captures of Mediterranean fruit fly, oriental fruit fly, and melon fly with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in California and Florida detection arrays.

In the male annihilation trials at Eleele, capture of all three species with Mallet TMR were not significantly different from Mallet CMR (P>0.05); therefore, subsequent experiments did not include Mallet CMR due to higher production costs. In these trials, captures in bucket traps with Mallet TMR wafers were equal to any other solid lure (single or double) except the Mallet ME wafer. Results of the Kauai studies also suggest potential suppression applications with TML and ceralure with average captures of over 2,000 Mediterranean fruit flies per trap per day. In summary, a solid Mallet TMR wafer is more convenient to handle, safer and may be used in place of several individual lure and trap systems. Further benefits include potentially reducing costs of large sur-

vey and detection programs in Florida and California, and male annihilation programs in Hawaii and California. Trials of the new TMR wafers have now moved to evaluations in typical citrus growing areas of California. Mallet TMR dispensers are being weathered inside Jackson traps in citrus trees in Tulare, Ventura, Riverside, Kern, and Orange Counties of California (Fig. 5). Two series of trials are being conducted in conditions representative of summer (August-September) and winter (January-March) conditions in California. Each week, weathered wafers are sent to Kauai or Hawaii Island, HI (USDA-ARS-PBARC) for fruit fly bioassays and to Farma Tech International, North Bend, Washington, for chemical analysis using conventional

Fig. 5 .Weathering sites are located in Lindcove, Bakersfield, Ventura, Riverside and Costa Mesa, California. Summer 2013 Citrograph 33

gas liquid chromatography methods. In summary, solid wafers are safer and more convenient to handle and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in California and Florida. Results of our recent research support three possible applications: 1) three individual traps with three solid wafers (TML, Mallet ME, and Mallet RK), 2) two individual traps with two solid wafers (TML and MR), or 3) one trap (Mallet TMR). The Farma Tech Mallet TMR wafers hold the promise of being used in a single trap in place of three traps for TML, ME and C-L/RK detection traps. Further testing needs to be conducted under California weather conditions, and cost-benefit analyses needs to be done on Mallet TMR versus standard trapping systems.

(trimedlure, methyl eugenol, raspberry ketone, and DDVP) dispensers for detection and male annihilation of Ceratitis capitata (Wiedemann), Bactrocera dorsalis (Hendel) and Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) in Hawaii. J. Econ. Entomol. 105: 1557-1565. Vargas R.I., L. Leblanc, J. C. Piero and K. Hoffman. 2013. Male Annihilation. Editors: Shelly, T., Epsky, N. Jang, E.B., Reyes-Flores, J. and Vargas, R.I. Trapping Tephritid Fruit Flies: Lures, Area-Wide Programs, and Trade Implications. Springer Press. (In Review).

Acknowledgments:
We thank Steven Souder, Russell Ijima, Yu Nakane, Dominique EasterGreen, and Jacqueline De La Garza (Pacific Basin Agricultural Research Center, USDA-ARS, Hilo, HI) for assistance in summarizing and collecting field data. We also thank Greg Williams for allowing access to the Kauai Coffee Plantation, Ken Love for coordinating sites on Hawaii Island, and research support from Marc Meisner. Finally, we thank the California Citrus Research Board and IR4 for financial support. Roger Vargas, Ph.D., is a Research Entomologist at the U.S. Pacific Basin Agricultural Research Center in Hilo, Hawaii. David Haviland is the Entomology and Pest Management Farm Advisor for UC Cooperative Extension in Kern County. Ben Faber, Ph.D., is the Farm Advisor for UC Cooperative Extension in Ventura County. John Kabashima, Ph.D., is the Environmental Horticulture Advisor for UC Cooperative Extension in Orange County. Beth Grafton-Cardwell, Ph.D., is an Extension Specialist and Research Entomologist, and Joseph Morse, Ph.D., is a Professor of Entomology. Both are members of the Department of Entomology, UC Riverside. CRB funded project reference number 5500-193. l

Further reading
[CDFA] California Department of Food and Agriculture. 2010. Insect trapping guide, 12th ed. Sacramento, CA. [FDACS] Florida Department of Agriculture and Consumer Services. 2004. Florida fruit fly detection manual. Division of Plant Industry, FDACS, Gainesville, FL. Vargas, R. I., N. Miller, and J. D. Stark. 2003. Field trials of spinosad as a replacement for naled, ddvp, and malathion in methyl eugenol and cuelure bucket traps to attract and kill male oriental fruit flies and melon flies (Diptera: Tephritidae). Hawaii. J. Econ. Entomol. 96: 1780-1785. Vargas, R.I., R.F.L. Mau, J.D. Stark, J.C. Pinero, L. Leblanc, and S.K. Souder. 2010. Evaluation of methyl eugenol and cue-lure traps with solid lure and insecticide dispensers for fruit fly monitoring and male annihilation in the Hawaii area wide pest management program. J. Econ. Entomol. 103: 409-415. Vargas, R. I., R. F. L. Mau, E. B. Jang, and L. Wong. 2010. Area-Wide Fruit Fly Integrated Pest Management Program: A Model System. pp-171-177, In: Editors: B. Sabater-Munoz, V. Navarro Llopis and A. Urvaneja Garcia, Proceedings of the 8th International Symposium on Fruit Flies of Economic Importance. Laimprenta CG, Valencia, Spain, 415 pp. Vargas, R.I., S.K. Souder, B. Mackey, P.J. Cook, J.G. Morse, and J.D. Stark. 2012. Field trials of solid triple lure

To optimize fertigation practices, it is essential that irrigation is applied and fertilizers injected at the optimal concentration, place, and time to ensure that deposition patterns coincide with maximal root uptake.
34 Citrograph Summer 2013

THE ANSWER
(From Page 5, Do You Know?) The custom of serving a slice of lemon with fish dates back to the Middle Ages. It was believed that if a person accidentally swallowed a fish bone, the lemon juice would dissolve it.

CRB-Funded Research Report

Research Project Final Report

Assessing factors influencing the postharvest quality of California mandarins

Variability in waxing-induced ethanol and aroma volatile production among mandarin varieties
Mary Lu Arpaia, S. Collin, J. Sievert, UC Riverside, David Obenland, USDA-ARS, Parlier
e have been interested in studying the role of fruit- ate large numbers of varieties. Fermentation products such handling practices on the eating quality of manda- as ethanol have been often implicated as potential causes rins. Understanding the role of handling practices for postharvest flavor loss in citrus, are easy to measure and is important since mandarins are increasing in consumer could be suitable markers indicating poor flavor developpopularity, but are known to be prone to the development of ment. In this study, as part of our Citrus Research Board off-flavors during handling and storage. project on mandarin quality, we evaluated the usefulness of This change in flavor is one of the most important factors this type of screening by evaluating a number of mandarin that limit mandarin postharvest quality. Researchers have varieties for the ability to produce and accumulate ethanol identified parameters that influence off-flavor development, in response to either a nitrogen atmosphere or waxing and including storage time and temperature and composition of storage and then determined the relationship of ethanol prothe wax coating. Along with others, we have demonstrated duction to the synthesis of other aroma volatiles. that extensive alterations in the aroma volatile profile ocWe harvested Kuno Wase, Kawano Wase and Dobashi cur during storage, including large increases in alcohols and Beni Satsuma mandarins (Citrus unshui Marcovitch), from esters and decreases in aldehydes and terpenes that are be- research plots at the University of California Lindcove Relieved to be major reasons for the poor flavor quality. The search and Education Center (LCREC) during the time for origin of these compounds is not entirely known, but many commercial maturity of these varieties. The fruit were transof these changes are believed to occur as a result of the wax ported to the United States Department of Agriculture in applied during packing, which leads to low oxygen levels Parlier, California, (Parlier USDA) where they were left at within the fruit and enhanced alcohol and ester production. Although it has 300 disadvantages, the use of some type of 2 coating is needed to prevent water loss R = 0.029 and to provide a more pleasing exterior 250 appearance to the consumer. Potential means to prevent or minimize the occurrence of off-flavor in 200 mandarins include both changes in postharvest handling techniques and in the 150 development of mandarins that are less susceptible to flavor alteration during storage. Variation in aroma volatile pro100 duction among mandarin varieties has been previously reported by researchers, Kuno Wase but prior studies focused on the volatile 50 Kawano Wase profiles at harvest and did not investigate Dobashi Beni postharvest effects. 0 It would be useful to determine the 0 50 100 150 200 250 300 350 400 degree of genetic variability that exists in current varieties and breeding populaHeadspace ethanol ( g/kg) tions of mandarins for postharvest flavor changes, but this is complicated by the Fig. 1. Correlation between headspace (measured above the intact fruit in a sealed time-consuming nature of flavor volatile jar) ethanol and juice ethanol from the same fruit of three mandarin varieties analysis, which makes it difficult to evalu- exposed to two hours of nitrogen. Each point indicates an individual fruit.
36 Citrograph Summer 2013

Juice ethanol (mg/L)

ambient laboratory temperature (~68F) overnight. The following day, the fruit were placed individually into one-gallon jars, which were flushed with nitrogen gas for ten minutes and then sealed. After two hours in nitrogen, a gas sample was withdrawn from the closed jar and analyzed for ethanol. Previously, a period of two hours in nitrogen was found to induce the accumulation of ethanol in the closed jars that was readily measureable while no ethanol was detectable when the nitrogen was replaced with air. The methodology of using nitrogen to induce ethanol accumulation was tested in this study as a potential means to rapidly screen large numbers of mandarin varieties for the rate of ethanol production under low oxygen. Since it was necessary that this method accurately predict the concentration of internal ethanol, each fruit was juiced and an aliquot of the juice quantified for ethanol based upon the conversion of ethanol to acetaldehyde by the enzyme alcohol dehydrogenase. This quantification method was previously demonstrated to give the same results as would be obtained by other commonly used means for analysis of the juice. To make a comparison to the postharvest processing that would occur commercially, eight additional mandarin varieties (W. Murcott, Gold Nugget, Temple, Pixie, Ellendale, Minneola, Orlando and Sue Linda) were obtained from LREC, transported to the Parlier USDA ARS research facility and evaluated for the ability to produce ethanol when the fruit were exposed to nitrogen gas for a period of two hours as described above or when waxed and stored to simulate commercial packing and marketing. The first four varieties listed are classified as true mandarins (Citrus reticulata Blanco) and the final four as Citrus x tangelo hybrids. Waxing was conducted manually by dipping the fruit into undiluted Fruit-APeel C carnauba wax (Fruit Growers Supply, Sherman Oaks, California) and wiping off the excess wax using a gloved hand. These fruit were then stored for either one week at 68F or two weeks at 41F followed by one week at 68F.
1200

Following nitrogen gas exposure, or waxing and storage, the fruit were peeled, individually juiced and assayed for ethanol using the Genzyme assay kit. Juice from two high ethanol producers (Pixie and Minneola) and two low producers (W. Murcott and Gold Nugget) was saved for subsequent aroma volatile analysis. Ten fruit were utilized per treatment for both the nitrogen gas and waxing tests, with fruit being individually measured for ethanol concentration. Volatile samples were analyzed following standard protocols. We were interesting in examining whether a relationship existed between ethanol production and aroma volatile concentration for each of the mandarin varieties tested. Each storage treatment within a variety was measured with six replicates, with each replicate being the pooled juice from two different fruit.

Mandarin varieties vary in the amount of accumulated ethanol

Exposing the different mandarin varieties to nitrogen gas for two hours, followed by the measurement of ethanol inside the sealed jars, potentially offered a rapid and easy means of screening large numbers of mandarin varieties for the ability to produce ethanol and other off-flavor components. These ethanol measurements were, however, found to relate very poorly with the actual ethanol content of the fruit as determined by direct measurements of the juice (Fig. 1). The reasons for this discrepancy are unclear, but could be due to fruit-to-fruit differences in the ability of ethanol to diffuse through the peel. Such differences have been noted between grapefruit and Murcott mandarins, but previously not between individual mandarin fruit. This result indicated that ethanol measurements for comparisons among citrus varieties, or even between individual fruit, needed to be made using juice rather than intact fruit. As an additional test of the ethanol screening methodology, eight diverse varieties of mandarins were waxed to
a a a a

Ethanol concentration (mg/L)

Ethanol concentration (mg/L)

1000 800 600 400 200 0

Initial 2 h Nitrogen

3000

Initial 1 wk 20C 2wk 5C + 1 wk 20C

2000
a

aa a a b b a b a

a a b b

1000
c

a b

0
Pi xi e

M ur co tt ol d Nu gg et Te m pl e O rla nd o M inn eo la El len da le Su eL ind a G

M ur co tt ol d Nu gg et Te m pl e O rla nd o M inn eo la El len da le Su eL ind a

Fig. 2. Comparison of ethanol concentration measured from the juice of eight varieties of mandarins at harvest (initial) or subjected to a two-hour exposure in nitrogen. Each column represents the mean of 10 fruit that were individually measured with the bar indicating standard error. Star indicates a significant difference (P 0.05) from initial within variety.

Fig. 3. Comparison of ethanol concentrations measured from the juice of eight varieties of mandarins at harvest (initial) to juice from fruit that had been waxed and stored at either one week 20C or two weeks 5C + one week 20C. Each column represents the mean of 10 fruit that were individually measured with the bar indicating standard error. Columns within a variety with different letters are significantly different (P 0.05). Summer 2013 Citrograph 37

Pi xie

simulate commercial conditions and compared to fruit that had received a two-hour treatment in nitrogen. Although this treatment had been found in prior testing to be effective in inducing the accumulation of ethanol in the sealed jars containing intact mandarin fruit in other varieties, only in W. Murcott and Orlando was the ethanol present in the juice of the treated fruit significantly different (P 0.05) than that in the control fruit (Fig. 2). In contrast, waxing and storage greatly enhanced ethanol in every mandarin variety, with the additional time in cold storage not having impact on any but W. Murcott (Fig 3). Clearly, a two-hour nitrogen treatment did not mimic waxing in these cases and is not suitable as part of a quick screen for ethanol production capability. It is possible that a longer duration of nitrogen exposure as was previously used in mandarins to initiate ethanol production would be useable, but this was not evaluated. What was noteworthy in this portion of the study, however, was that there were large and significant differences (P 0.05) observed among these mandarin types in both the amount of ethanol at harvest and following storage. Assays of aroma volatiles were performed on the two varieties with the lowest ethanol concentrations (W. Murcott

and Gold Nugget) after waxing and storage and compared to two varieties with much higher concentrations (Minneola and Pixie) to evaluate the relationship between differential amounts of ethanol production and aroma volatile content (Table 1). Given their more consistent changes due to storage, only alcohols, esters, ketones and aldehydes are shown in this table, although other compounds were identified and quantified. Most of the evaluated compounds increased in amount during storage with the exception of a few of the alcohols and aldehydes. Previous research showed that high levels of ethanol can enhance ester production in apples. The presence of esters was correlated with off-flavors and, therefore, may be involved in the reduction of eating quality following storage. Esters are compounds with fruity aromas that are not directly objectionable, but which could together act to greatly alter flavor. Changes in the concentration of certain esters or the formation of others in response to storage can play a role in an individual having a positive or negative experience when eating a fruit. It has been shown that only very low concentrations of certain esters are sufficient to have an impact on flavor. It is worth noting that a number of the

Table 1. Concentrations (mg/L)a of aroma volatiles present in mandarins immediately after harvest and following waxing and three weeks (two weeks 5C + one week 20C) of storage, tr = trace amount. Storage time (wks) Compound W. Murcott Gold Nugget Minneola Pixie Alcohols 0 3 0 3 0 3 0 3 Ethanol (mg/L) 58 595* 322 365 267 2364* 239 1938* 3-Methylbutanol tr tr tr tr tr 72* tr 75* 2-Methyl-1-butanol tr tr tr tr tr 42* tr 44* Linalool 49 31 254 34* 131 42* 122 60* 4-Terpineol 26 29 104 22* 52 25* 41 24* Esters Ethyl acetate 0 720* 1753 2922* 55 1397* 487 5812* Ethyl propanoate tr 36* 92 201* 6 43* 36 320* Ethyl isobutyrate tr tr tr 8* tr 11* tr 73* Ethyl butanoate tr tr 18 37* tr 21* tr 466* Ethyl 2-methylbutanoate tr tr tr 6* 1 30* tr 209* 3-Methylbutyl acetate tr tr tr tr tr 10* tr 9* 2-Methylbutyl acetate tr tr tr tr tr 7* tr 8* Octyl acetate tr 17* 256 60* 118 101 185 106 Decyl acetate 37 30 102 19* 52 53 64 34 Ketones 1-Penten-3-one 8 20* 7 7 6 8 6 8* Carvone 15 52* tr 12* 8 8 15 8 Aldehydes Pentanal 20 121* 9 11* 8 19* 9 15* Hexanal 157 1675* 20 24 36 62 28 19 E-2-Hexenal 8 10 27 21 17 13 18 47* Heptanal 24 187* 17 16 13 26* 15 19 Octanal 55 103* 179 48* 171 65* 386 66* E-2-Octenal 13 137* tr tr 9 9 8 6 Nonanal 37 80* 83 44* 61 46 106 47 E-2-Nonenal 10 61 tr tr 4 6 tr tr Decanal 0 40* 529 38* 137 45* 223 54*
*Star after value indicates statistical significance between initial (0 week) and three weeks storage within a variety.

38 Citrograph Summer 2013

esters that greatly increased due to storage, and especially so in the high ethanol producers such as ethyl isobutyrate, ethyl butanoate and ethyl 2-methyl butanoate, have relatively low odor thresholds (1 g/L or less) and require only low concentrations to have an impact on flavor. Certain alcohols, such as 3-methylbutanol and 2-methylbutanol, were only present in high ethanol producers that had been stored. These alcohols both have a malty aroma, but have higher odor thresholds (1,000 g/L and 320 g/L, respectively) than the esters. W. Murcott mandarins were unique from the other mandarin varieties examined in that they accumulated far greater amounts of ketones and aldehydes as a result of storage (Table 1).

What we can conclude

The mandarin varieties had substantial differences in both the amount of ethanol present in freshly-harvested fruit and in the amount produced in response to waxing and storage. These differences in ethanol concentrations were associated with enhanced accumulation of other alcohols and esters that likely influence flavor. Even though this study needs to be repeated in different locations and in multiple years to fully substantiate these differences, the study does illustrate the potential utility of screening for ethanol production to estimate the degree of postharvest accumulation of other esters and alcohols that could contribute to off-flavor. It also opens the possibility to use this screening approach to more fully understand the importance of the role of fermentative metabolism and ethanol accumulation to flavor loss during mandarin storage.

Change in apple fruit volatiles after storage in atmospheres inducing anaerobic metabolism. J. Agric. Food Chem. 39:1602-1605. Miyazaki, T., Plotto, A., Goodner, K., and Gmitter, F.G. 2011. Distribution of aroma volatile compounds in tangerine hybrids and proposed inheritance. J. Sci. Food Agric. 91:449-460. Obenland, D., Collin, S., Mackey, B., Sievert, J., and Arpaia, M.L. 2011. Storage temperature and time influences sensory quality of mandarins by altering soluble solids, acidity and aroma volatile composition. Postharvest Biol. Technol. 59:187-193. Porat, R., Weiss, B., Cohen, L., Daus, A., and Biton, A. 2005. Effects of polyethylene wax content and composition on taste, quality, and emission of off-flavor volatiles in Mor mandarins. Postharvest Biol. Technol. 38:262-268. Rudell, D.R., Mattinson, D.S., Mattheis, J.P., Wyllie, S.G., and Fellman, J.K. 2002. Investigations of aroma volatile biosynthesis under anoxic conditions and in different tissues of Redchief Delicious apple fruit (Malus domestica Borkh.). J. Agric. Food Chem. 50:2627-2632. Shi, J.X., Goldschmidt, E.E., Goren, R., and Porat, R. 2007. Molecular, biochemical and anatomical factors governing ethanol fermentation metabolism and accumulation of off-flavors in mandarins and grapefruit. Postharvest Biol. Technol. 46:242-251. Shi, J.X., Porat, R., Goren, R., and Goldschmidt, E.E. 2005. Physiological responses of Murcott mandarins and Star Ruby grapefruit to anaerobic stress conditions and their relation to fruit taste, quality and emission of off-flavor volatiles. Postharvest Biol. Technol. 38:99-105. Tietel, Z., Bar, E., Lewinsohn, E., Feldmesser, E., and Fallik, E. 2010. Effects of wax coatings and postharvest storage on sensory quality and aroma volatiles composition of Mor mandarins. J. Sci. Food Agric. 90:995-1007. l

This projects benefit to California citrus growers

This project continues our studies on mandarin fruit quality. Our goal is to provide recommendations to growers, packers and fruit breeders on proper handling practices for mandarin varieties. Since there are numerous mandarin varieties, it would be useful to develop quick screening methods to assess new material. As marker-assisted breeding and other molecular techniques are further developed, this knowledge will contribute to enhance efficiency in screening mandarin breeding populations.
We would like to thank P. Neipp for his assistance with this project. This work was partially funded by a grant from the California Citrus Research Board. Mary Lu Arpaia, Ph.D., is a UC Cooperative Extension Subtropical Horticulturist based at the Kearney Ag Center in Parlier, California, and is a member of the Department of Botany and Plant Sciences at UC Riverside. David Obenland, Ph.D., is a plant physiologist, USDA Agricultural Research Sciences Center in Parlier, California. Sue Collin and Jim Sievert are Staff Research Associates at the UC Kearney Ag Center. Baldwin, E.A., Nisperos-Carriedo, M., Shaw, P.E., and Burns, J.K. 1995. Effect of coatings and prolonged storage conditions on fresh orange flavor volatiles, degrees brix, and ascorbic acid levels. J. Agric. Food Chem. 43:1321-1331. Hagenmaier, R.D. 2002. The flavor of mandarin hybrids with different coatings. Postharvest Biol. Technol. 24:79-87. Mattheis, J.P., Buchanan, D.A., and Fellman, J.K. 1991.

Acknowledgements

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Summer 2013 Citrograph 39

Citrus Roots

Preserving Citrus Heritage Foundation

California citrus growers enjoyed early love affair with vehicles

Richard H. Barker

he Roaring Twenties were a boom time for California citrus growers. During the decade, grower returns averaged $2.99 per box (roughly the equivalent of $40.42* in 2013 dollars). One acre of 20 x 20 trees contained approximately 100 trees, with each one yielding seven boxes of fruit. Therefore, returns on 10 acres would have been $20,930, which would translate to a whopping $282,940 today. What to do with all of that profit? Growers invested heavily in vehicles a truck for the grove, perhaps
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a tank truck for smudge oil and/or water, a tractor and one or two family cars. By the early 1930s, the 13,000 members of the Exchange each averaged 3.5 motor vehicles, which totaled 45,500 for the industry as a whole. At a time when the minimum wage was 33 cents ($4.62 in 2013 dollars; todays minimum wage in California actually is $8.00) and vehicle ownership was still considered a luxury, it is very likely that California citrus growers owned more cars and trucks than any other occupational category. Lets take a look back at some of the classic cars that might have been bought with California citrus dollars. l
*This article contains prices and costs from the 1920s and 1930s. All conversions to 2013 cost equivalents are provided by the Citrus Research Board based on the Consumer Price Index calculator (as of July 31, 2013) from the U.S. Bureau of Labor Statistics.

40 Citrograph Summer 2013

1925-$3,185 ($42,514)

1924-$640 ($8,743)

1924-$685 ($9,358)

1923-$2,485 ($33,947) Summer 2013 Citrograph 41

The romance with the orange is compelling.

1921-$1,3

95 ($18,20

5)
1927-$1,3 7 5 ($18,459)

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Citrus Powered the Economy of Orange County for over a half century Induced by a Romance

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42 Citrograph Summer 2013

The Pomona Progress wrote that for the 1924-25 season, growers received a return of 25 percent based on their invested capital in citrus. The following season, they received an 18 percent return. These were golden returns that gave growers the ability to purchase their motor vehicles and tractors.

Summer 2013 Citrograph 43

Celebrating Citrus

Meet Chef Nathan Lyon

California citrus growers grow the best-tasting citrus!

alifornia citrus can be enjoyed in a wide variety of ways fresh, juiced or as an ingredient in a delicious recipe. One food expert who appreciates its many great-tasting applications is the popular Chef Nathan Lyon. Perhaps Im biased, having worked at Los Angeles farmers markets for more than a decade, Chef Lyon said, but California citrus growers grow the best-tasting citrus! The variety of citrus on offer is always changing throughout the year, and with that vast selection comes an amazing spectrum of delicious flavors. In fact, one specific Southern California grower, Bernard Ranches, has spoiled me for life with their tasty citrusthank goodness! The chef knows whereof he speaks. Familiar to television viewers throughout the country for his simple and innovative cuisine featuring fresh, seasonal, local ingredients, he is the host of Good Food America (Veria Living), as well as chef and co-host of Growing A Greener World (PBS). He received an Emmy nomination this year for Outstanding Culinary Host. Chef Lyon also recently came out with his first cookbook, Great Food Starts Fresh, featuring 135 recipes broken down into five seasons: spring, summer, autumn, winter, and...chocolate! The cookbook already has been quoted by renowned culinary names such as Graham Kerr, Jamie Oliver and Curtis Stone. Alice Waters, the international governor of Slow Food and owner of the famed Chez Panisse in Berkeley, California, said, Nathan is an exuberant force for good in the sustainable food movement. Through his fresh, simple recipes, he teaches the important truth that we are happiest and healthiest when we are eating real food made from seasonal and local ingredients. After graduating from James Madison University with a Bachelor of Science in Health Science and a minor in Public Health, the future chef backpacked his way across Europe, learning about local customs, culture and cuisine. His eureka moment

occurred in an outdoor market near Florence, Italy, when an old woman, puzzled by the massive amount of produce he was purchasing, asked, Why are you buying so much food? Why not just buy fresh every day? Lyon immediately dumped out half his basket and began contemplating those simple questions. It was in that market, clutching a wheel of cheese, that he discovered his truth: great food starts fresh. Laden with ideas and information, Lyon headed home, eventually opting for culinary school at Le Cordon Bleu in Los Angeles, where he earned his Culinary Arts degree. Since that time, he has worked in many restaurants, both in and out of the kitchen. He also was the creator and host of A Lyon in the Kitchen (Discovery Health and Fit TV), among the final four on the second season of The Next Food Network Star, and appeared as a guest chef and expert on Home Made Simple (TLC) and Real Simple Real Life (TLC). Through it all, Chef Lyon has found citrus to be one of his favorite ingredients. Using California citrus makes my job as a chef far easier because great produce makes for amazing meals, he explained. In fact, with such a wide variety of citrus here in California, the culinary applications are almost endless! I use the fresh juice for salad dressings, to brighten soups and round out reduction sauces. Ill section out the citrus for a wonderful variety of salads and use the zest for flavoring slowcooked stews, sprinkled over grilled chicken or baked fish or my favoritefor flavoring delicious desserts. Chef Lyon is delighted to share three of his delicious recipes with the people whove made the flavor possible the California citrus growers. l
To learn more about Chef Lyons new cookbook, Great Food Starts Fresh, or to order a copy, visit www.chefnathanlyon.com. Also, check your TV listings to see when Growing a Greener World airs on your local PBS station.

44 Citrograph Summer 2013

Raw Kale Salad with Feta, Pine Nuts, and Cranberries

Yield: 4 servings Salad: 1 bunch curly kale cup pine nuts cup toasted walnuts, chopped roughly cup toasted almonds, chopped roughly cup pumpkin seeds (pepitas)  cup dried cranberries, preferably unsweetened 3 large oranges cup crumbled feta cheese Kosher salt, to taste Freshly ground black pepper, to taste Vinaigrette:  1 medium shallot, peeled and diced finely (3 tablespoons) 1 tablespoon red wine vinegar  3 tablespoons freshly squeezed orange juice (from the oranges) 1/3 cup extra-virgin olive oil 1. Strip the tender leaves from the tough stems of the kale; discard the stems. Rinse and dry the leaves, then roll them up and slice into thin strips. You should have approximately 5 packed cups of kale ribbons. 2. Cut the peel and pith off the oranges, then segment them by cutting between the dividers. Discard any seeds, but reserve the juice for the vinaigrette.

3. In a medium container with a tight fitting lid, combine the vinaigrette ingredients, close the lid tightly, and shake well to combine. Or, whisk to combine the ingredients in a medium-sized bowl. 4. Add the kale, nuts, seeds, cranberries, orange segments, and crumbled feta to a large serving bowl. Drizzle half the vinaigrette over the salad and toss to combine. Season to taste with salt and pepper, adding more vinaigrette if needed. Note: Other optional ingredients that go wonderfully in kale salads are sunflower seeds, diced avocado, and diced apples.

Fresh Date Pecan Bread

Yield: 1 - 9-inch pie (6 servings)  Nonstick spray or unsalted butter, to prepare the pie pan  cup plus 2 tablespoons unbleached all-purpose flour, plus more for dusting  cup dark brown sugar  teaspoon ground cinnamon 1 /8 teaspoon kosher salt  2 teaspoons unsweetened cocoa powder  1 tablespoon orange zest (zest of a large orange), grated on a Microplane  teaspoon baking powder  1 cup roughly chopped dates, pits removed (approximately 30 dates)  2 tablespoons 2% or whole milk  1 cup raw pecan halves, chopped roughly  3 large eggs 1. Adjust two oven racks to the middle position, then preheat the oven to 350F. 2. Spray or butter a 9-inch pie pan, then dust with flour, tapping out any excess. 3. Mix together the flour, brown sugar, cinnamon, salt, zest, cocoa and baking powder in a medium-sized bowl. 4. Add the dates, milk, eggs, and nuts, and mix very well. Get in there with some elbow grease, and really stir until well combined. Itll be thick and chunky, but thats a good thing. 5. Pour the date batter into the prepared pie pan and give it a little shake to distribute the ingredients evenly, or use the back of a spatula to level off the batter. 6. Place the pie pan on a sheet pan and transfer to the oven. 7. Bake, uncovered, 40 to 45 minutes, until the top is light to golden brown and the center springs back when pressed. 8. Remove from the oven and allow to cool in the pan. Once cool, remove from the pie pan, slice, and serve.
Summer 2013 Citrograph 45

Roasted Golden Beet Salad with Frise, Goat Cheese, and Candied Nuts
Yield: 6 to 8 servings Beets: 3 pounds golden beets  3 tablespoons extra-virgin olive oil, divided  teaspoon kosher salt, plus more to taste 1/3 cup water  1 tablespoon freshly squeezed lemon juice  Freshly ground black pepper, to taste Salad:  1 large head frise, rinsed, dried, and chopped or torn into bite-size pieces 3 medium oranges  4 ounces goat cheese (chvre) (about cup) Candied Walnuts or Pecans for serving Vinaigrette: 2 tablespoons red wine vinegar 4 tablespoons extra-virgin olive oil  3 tablespoons freshly squeezed orange juice (from the oranges) 1 medium shallot peeled and diced small (3 tablespoons) Kosher salt, to taste Freshly ground black pepper, to tast 1. Adjust an oven rack to the middle position, then preheat the oven to 350F. 2. Cut off the greens half an inch above the beets. Scrub the beets. 3. Cut the peel and pith off the oranges, then segment them by cutting between the dividers. Discard any seeds, but reserve the juice for the vinaigrette. 4. In a roasting pan large enough to hold the beets in one layer, add the beets, then drizzle with 1 tablespoons of olive oil, the salt and water. Cover tightly with aluminum foil, place on a

sheet pan and roast in the oven for approximately 50 minutes to an hour, or until the beets are easily pierced with the tip of a knife. Remove from the oven, remove the foil and allow to cool. 5. When the beets are cool enough to handle, using a not-soimportant hand cloth, wipe the beet skins off and discard them. Know this, though: even in your sweetest of dreams, you will most likely never wash the red stains out of that hand cloth. 6. Cut the beets into wedges, place them in a bowl, then combine 2 tablespoons of olive oil and 1 tablespoon lemon juice in a cup and drizzle over the beets. Fold to combine. Season to taste with salt and pepper. 7. In a medium container with a tight fitting lid, combine the vinaigrette ingredients, close the lid tightly, and shake well to combine. Or, whisk to combine the ingredients in a mediumsized bowl. 8. Gently toss the frise and orange segments with your hands, then dress with half the vinaigrette. Season to taste with salt and pepper and additional vinaigrette to taste. 9. Divide the salad among 4 plates, then garnish with the beets, candied nuts, and goat cheese, divided equally.

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.67 ac lot, with a view Lindsay area. ..............................................$50,000 1.44 acs Exeter Commercial Building..........................................$250,000 2.62 acs Exeter Cold Storage Facility, High Quality...................$1,975,000 4.2 acs Exeter/Farmersville Area Homesite..................................$149,000 4.68 acs Almonds & Homesite, Kingsburg.................................. $150,000 6.48 acs Turnkey Citrus Packing/Cold Storage......................... $1,950,000 9.72 acs Cutler Area Cold Storage/Offices................................$2,399,000 10 acs Strathmore Area Citrus (SOLD)........................................ $120,000 14.92 acs Lemon Cove Navels & Home Site (SOLD). ....................$285,000 15.98 ac Lindsay Development Potential (SOLD) ........................$280,000 20 acs Porterville Ranch & Home (SOLD).....................................$405,000 20 acs Lindsay-Strathmore Area Citrus & Residence...................$595,000 20 acs Orange Cove Navels & Home (In Escrow).........................$450,000 20.18 acs Sanger Citrus/Residence.............................................$875,000 26.16 acs Lindsay Mandarins (SOLD)..........................................$425,000 30 acs Seville Area Open.............................................................$210,000 37.49 acs Cutler Area Citrus........................................................$828,000 40 acs Valencia Oranges & Open (In Escrow) . ............................$360,000 40.16 acs Tulare County Olives (SOLD)........................................$595,000 57.24 acs Navel Oranges (in Escrow...........................................$745,000 59.98 acs American Ave Citrus & Olives (SOLD). ..........................$900,000 90.30 acs Cutler Area Navels...................................................$1,080,000 127.33 acs Orange Cove Citrus Ranch with Custom Home.......$2,500,000 158.41 acs Orosi Organic Citrus...............................................$1,425,000 171.08 acs Stone Corral Area Open (SOLD)..............................$1,625,260 236.4 acs Citrus Ranch (SOLD). ................................................$3,000,000 320 acs South County Citrus Ranch. .........................................$5,120,000 498 acs Antelope Valley Ranch.................................................$3,200,000 604.37 acs Hills Valley Ranch.................................................. $1,725,000

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46 Citrograph Summer 2013

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