citrograph march / april 2010

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March/April 2010Citrograph

CitrographLindcove’s50thAnniversary

March/April 2010 Citrograph 3

IN THIS ISSUE

Citrograph is published bimonthly by the Citrus Research Board, 323 W. Oak Street, 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.

An Official Publication of the Citrus Research BoardMARCH/APRIL 2010 • VoLuMe 1 • NuMbeR 2

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Cover photo: Lindcove’s 50th Anniversary – photo by Sara Davidian

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EDITORIAL BOARD

4 Editorial

6 Industry Views

8 University of California Lindcove Research and Extension Center celebrates its 50th Anniversary

12 CRB 2009Annual Report

16 G. Harold Powell and the defeat of citrus decay, 1904-1912

20 Descriptions of new varieties recently distributed from the Citrus Clonal Protection Program

27 Parasitoid preference for citricola scale in southern California citrus versus San Joaquin Valley citrus

30 Novel immunocapture technology for field deployable nucleic acid-based detection of plant pathogens

32 Identification of Spiroplasma citri secreted proteins as detection markers for citrus stubborn disease

33 Probiotic diet for SIT Medfly

4 Citrograph March/April 2010

EDITORIAL

Industry adopts vehicle to battle pests/diseases

As we begin a new decade, our industry once again shows its age-old ability to work together for the common good. Witnessing the disaster in Florida associated with the Asian citrus psyllid and Huanglongbing, California’s citrus industry leaders, growers and organization directors all saw the need for a vehicle to avoid the catastrophe now occurring in Florida.

Temporarily, that need and vehicle has been our Citrus Research Board (CRB). But the program and effort now being designed and implemented by federal and state government in partnership with industry will become controversial. The program will require additional spraying. It will require the loss of organic production. It will require constant intrusion into residential homes, and it may well cause the elimination of backyard trees and landscape material.

This controversy cannot be shouldered by our Research Board because inevitably it would then become incapacitated to perform its core function – research! Whether it be research to enable our industry to create revenue streams via climate change legislation, research for beneficial bugs, research to help thwart ACP and HLB, or research for new varieties, CRB cannot become embroiled in constant controversy and legal attacks.

It was recognized that another vehicle was needed to specifically address this ACP/HLB threat and similar threats. It was also recognized that our State government was in a fiscal meltdown, and many questioned whether it would be there to help when and if another invasive threatened our ability to produce, harvest and market fresh citrus. Florida didn’t have that vehicle, and they are paying a price. Texas doesn’t have that vehicle, and they are behind the eight ball. Mexico, Brazil and other production areas did not have the industry tool to address challenges such as the ACP/HLB threat.

Thus was borne the legislation to facilitate an industry tool to address pests and diseases specific to the California citrus industry. Dubbed “the 281 committee”, which is the legislation number, the California Citrus Pest and Disease Prevention Committee is now in its early stages. Fourteen growers within our industry have stepped forward to help give birth to this effort.

Those 14 asked a Fresno County producer, Nick Hill, to be their first Chair. They asked a Coachella Valley grower, Craig Armstrong; and a Tulare County producer, Richard Bennett, to also serve on the executive committee. The balance of the 14 represent a cross section of our industry from small to large producer; from PCA experience to the handling of fruit. They are Dan Dreyer, Bob Felts, John Gless, Jim Gorden, Gus Gunderson, Link Leavens, Mark McBroom, George McEwen, James McFarlane, Dr. Etienne Rabe, and Kevin Severns. There are two floating alternates who are Earl Rutz and Don Barioni Jr.

They come from all the production areas of this state, and they come with one goal – to prevent the introduction of Huanglongbing and its spread throughout the citrus industry. Their meetings are public, and their agenda is ambitious. In the next few months they will be discussing an action plan or protocol should HLB be found in this state. They will take the scientific recommendations, add a touch of practicality, and subsequently make recommendations to our Secretary of Agriculture. They’ll determine the best use of industry funds to fill voids in the existing effort. They’ll determine what components of the existing CRB program will transfer to the AB 281 committee. Actually there is a whole lot more, but one can see how ambitious their agenda will become.

The Committee will be staffed by the California Department of Food and Agriculture and it will have the authority to advise the Secretary on specific pest and disease issues. It will have the power to create the funding necessary to protect our valuable industry. The closest vehicle to this tool is the glassy-winged sharpshooter program and advisory group, the Pierce’s Disease Control Program. This new citrus program is far more encompassing and, again, a bit more precedent-setting.

But that’s what we should expect from an industry that provides leadership to California agriculture and the nation’s specialty crop industry. So while it may be a new decade, our industry leadership is once again coming back to the future and creating a mechanism that is sure to be copied.

Next to our citrus fruit, that is the commodity we are best known for! l

California Citrus Mutual (CCM) is a nonprofit, grower-based trade association formed in 1977 “by citrus growers, for citrus growers”. The organization is headquartered in Exeter, California.

BY JOEL NELSEN, President, California Citrus Mutual

They will take the scientific

recommendations, add a touch of

practicality, and subsequently make recom-

mendations to our Secretary of

Agriculture.


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District 2 – Southern California – Coastal

Member AlternateEarlRutz,Pauma Valley AlanWashburn,RiversideWilliamPidduck,Santa Paula JamesFinch,Santa PaulaJoeBarcinas,Riverside KenKelley,Hemet

District 1 – Northern California

Member AlternateAllanLombardi,Exeter KevinSeverns,Orange CoveDonaldRoark,Lindsay DanDreyer,ExeterJimGorden,Exeter DanGalbraith,Porterville JoeStewart,Bakersfield FrancoBernardi,VisaliaEtienneRabe,Bakersfield RichardBennett,VisaliaJohnRichardson,Porterville JeffSteen,StrathmoreKevinOlsen,Pinedale DavidDir,Visalia

CITRUS RESEARCH BOARD MEMBER LIST BY DISTRICT 2009-2010

Citrus Research Board323 W Oak, Visalia, CA 93291PO Box 230, Visalia, CA 93279

(559) 738-0246FAX (559) 738-0607

E-Mail [email protected]

District 3 – California Desert

Member AlternateWilliamStein,Oasis JohnTurco,Indio Public MemberMember Alternate SeymourVanGundy,Riverside SteveGarnsey,Fallbrook

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.


INDUSTRY VIEWS

“What is the hottest topic you are facing at this time?

asks:Citrograph

I would be surprised if any California citrus nursery owner answered anything other than ACP/HLB to this question. We are business as usual on the outside, but inside we are all about the

upcoming changes in our industry. We have begun the physical transformation of our nursery, from field-grown trees to container trees grown in hot houses, protected from the Asian citrus psyllid. We continue to do research on matters of containers, soil mixes, cold frame structure vs. climate-controlled houses and many other questions related to the impending changes. We are evaluating all the options so that we will create the highest quality tree at a reasonable price for our customers. So far the ACP/HLB threat has been a huge challenge that is keeping our entire staff on its toes; however, we at Willits & Newcomb are excited about developing new methods of producing quality citrus trees. – Jackie Maxwell, President, Willits & Newcomb Citrus Nursery

The Asian citrus psyllid/Huanglongbing threat is our greatest focus in our citrus nursery program. At Duarte Nursery our efforts are focused on delivering growers disease-free

citrus trees while operating under conditions of, hopefully distant, future ACP infestations throughout California. We have introduced over 50 commercial varieties of citrus scion and rootstock into in vitro culture for both production and germplasm banking and will continue with another 40 – 50 this year. We have begun screening of our greenhouses. This fall we will begin building our specialized clean citrus propagation facility where laboratory-produced plantlets of both scion and rootstock will be grown into rootstock liners, scion blocks and finished trees under fully protected screened greenhouses near Modesto, CA. It is thought that major inter-regional spread of the HLB in Florida and other southern states was through infected nursery stock plants. We believe that our preparations to maintain an in vitro germplasm bank and to produce HLB-free trees under eventual ACP pressure are very important to the long-term viability of the California citrus industry. In either case, whether genetic cures arise for HLB, California growers will need to protect their current generations of plantings while cycling in newly developed, and currently nonexistent, commercially acceptable resistant varieties. We at Duarte Nursery are preparing for a future when disease-free nursery stock is paramount. – John Duarte, President, Duarte Nursery, Inc.

Citrus nurseries in California have enjoyed growing in the outdoors in the last 100 years in an environment with few diseases that were vectored by insects. Although stubborn dis-

ease and Citrus tristeza virus (CTV) have been endemic in citrus growing areas for years, the incidence in citrus nurseries has been a rare occurrence. The threat of HLB disease vectored by the Asian citrus psyllid (ACP) coupled with the ongoing concern of CTV infection has caused citrus nurserymen to rethink their business plans and evaluate the costs of moving into an insect excluding environment. Greenhouses or screenhouses built to exclude insects have been in use in Brazil for the past 10 years and became mandatory in Florida four years ago. The concept is simple – keep out the insect vector and you keep out the disease. Entry doors are equipped with air curtains that blow on you as you enter, and when an outside door is open, an inside door is closed, which is called a “double entry door system.” All air intakes are covered with screen boxes, and any opening to outside air is either screened or sealed off. In Brazil, some nurseries even require workers to change clothing before entering the facilities to prevent bad bugs from hitchhiking on clothing. The success of these measures proves out in Florida where no ACP have been found in a citrus nursery since they all moved indoors. The cost of these measures is high, from $10-$15 per square foot, so nurseries will soon be measured in square feet not acres. Citrus trees grown in protected environments will have to be smaller but can be as good as the old reliable field-grown ball & burlap tree if 21st century technology in soil media, pot design and environment control are used. Growers can count on quality trees grown in nurseries that are protected from disease, but they will have to adapt to the new look of these trees and the higher cost that will inevitably be required to fund these expensive measures to insure a strong future for the California citrus industry. – Roger Smith, General Manager, TreeSource Citrus Nursery.

8 Citrograph March/April 20108 Citrograph March/April 2010

The University of California Lindcove Research and Exten-sion Center (Lindcove LREC)

celebrated its 50th Anniversary on December 11, 2009. The celebration in-cluded a dedication of the new research laboratory (funded by the University of California) with a ribbon cutting by Associate Vice President of Programs Barbara Allen-Diaz and Lindcove REC Director Beth Grafton-Cardwell. A new screenhouse (funded by the Citrus Re-search Board-CRB) that protects Citrus Clonal Protection Program (CCPP) budwood source trees from pests and diseases was dedicated with a ribbon cutting by Chairman of the CRB Jim Gorden, CRB Vice President of Science & Technology MaryLou Polek, Vice President of UC Agriculture and Natu-ral Resources Dan Dooley, and Director of the CCPP Georgios Vidalakis.

Additional events included the annu-al Fruit Display and Tasting in the Con-ference room, sensory taste testing of citrus fruit by UC Riverside Subtropical

University of California Lindcove Research and Extension Center celebrates its 50th Anniversary

Beth Grafton-Cardwell, Georgios Vidalakis, and Ray Copeland

Director Beth Grafton-Cardwell with Barbara Allen-Diaz, Bill Frost, Shawn Tibor and Luzanne Martin dedicate the new laboratory at Lindcove REC.

Jim Gorden, Dan Dooley, Georgios Vidalakis and MaryLou Polek (left to right) cut the ribbon for the new CCPP screenhouse, funded by the Citrus Research Board.

Horticulture Extension Specialist Mary Lu Arpaia and USDA-ARS researcher Dave Obenland, and a walking tour of the citrus trees in the Lindcove REC demonstration block by UC Riverside Senior Museum Scientist Tracy Kahn.

The history of the Lindcove REC (until 1992 known as Lindcove Field Station) is closely linked to global events such as the end of World War II and sev-eral California citrus industry decisions, such as the petition to the University of California for the development of “variety foundation plantings” which resulted in the establishment of the Citrus Variety Improvement Program (CVIP) in 1957 (today known as the CCPP) and the creation of the CRB in 1968 (Calavan et al., 1978).

Following World War II, southern California became the aviation manu-facturing center for the USA as well as much of the world. The need for skilled labor in the factories plus the ideal subtropical climate acted as a magnet in drawing many of the returning GIs

to the area. The huge influx of people created a need for housing, turning productive farm land into housing tracts. One of the prime areas for new citrus groves in the 1950s was near the foothills in Kern, Tulare and Fresno counties of the San Joaquin Valley, and many south-ern California citrus growers made the migration north.

As the citrus industry grew in the San Joaquin Valley, the need for information concerning citrus production increased. Most of the citrus research in California had been done through the University of California (UC) Citrus Experiment Station located in Riverside, California under conditions quite different than those in Tulare County. In the mid-1950s with leadership from UCCE Tulare County Farm Advisor Karl Opitz and UC Cooperative Extension Director Sheldon Jackson, an advisory committee of citrus growers was formed to find a site in Tulare County that would be suit-able for the establishment of research plots by UC Riverside researchers. A

March/April 2010 Citrograph 9March/April 2010 Citrograph 9

Dedication ceremony of the new CCPP screenhouse.

number of sites were reviewed with the final selection of the Lindcove site which provided researchers an opportunity to look at problems and conduct research in the San Joaquin Valley citrus grow-ing areas.

Initial property for the station was donated by Adna Neil, a citrus nurs-eryman and grower. By the spring of 1959, the grower committee, chaired by citrus grower Roy McLean, turned over the keys to Dan Aldrich, Dean of Agriculture Sciences for the University of California Riverside in a dedication ceremony on the hillside overlooking the current shop and greenhouse areas (Citrograph 1959). Willard Bitters of the Department of Botany and Plant Sci-ences, UC Riverside, was the first acting director of the station. In the first year, an office was constructed, land was lev-eled and irrigations systems installed in preparation for the planting of the first orchards in 1960.

The funding of this off-campus research facility was difficult for the Botany and Plant Sciences Department, and so in 1964 UC Riverside turned over the operation of the Lindcove REC to the statewide UC Agricultural Field Sta-tions. From 1965 to 1987, UCCE Tulare County Farm Advisor Ray Copeland was the Superintendent of the station, and this period was characterized by development of the land and facilities (Citrograph 1985). Land was donated or purchased to bring the station up to its current 175 acres, and research proj-ects expanded to include variety trials,

rootstock trials, and pest management studies.

Land purchased in 1959 was largely dedicated to the establishment of the “primary variety foundation planting” (in addition to three secondary founda-tion plantings in Ventura and Orange counties) (Reuther et al., 1972). In 1961, five years after the establishment of the CCPP (then named CVIP), the first disease-free budwood source trees were moved out of the Riverside CCPP quarantine facilities and in to the Lind-cove REC for planting in what is known today as the “CCPP’s Foundation and Evaluation Blocks”. Since then, 807 different citrus varieties from around the world have completed the rigor-ous “Variety Introduction-VI” disease testing and therapy program under quarantine at the CCPP, and more than 4,000 budwood source trees have been propagated and safely moved for plant-ing at Lindcove REC.

For decades now, the Lindcove REC has served the very important function of maintaining the CCPP budwood tree sources as well as assisting with the trueness-to-type evaluations and budwood distribution to nurseries, growers and researchers in California, Arizona, Texas, Alabama, and several countries around the world. CCPP’s budwood distribution records indicate that 750,000 buds have been distributed from the Lindcove REC-housed CCPP budwood source trees since 1982. Speak-ing conservatively, a citrus nursery can produce 200 field trees from each CCPP

bud within a year. Thus, in the last 28 years the Lindcove REC has been the source of at least 150 million citrus trees worldwide.

Outbreaks of diseases have always been driving forces for major changes in the California citrus industry and research. The widespread occurrence of psorosis in the 1930s and the epidemic of tristeza in the 1940s to the 1960s, in combination with the rapid spread of the exocortis and stubborn disease in the 1950s, formed many of today’s citrus cul-tural practices as well as the current reg-istration and certification program for citrus budwood. In a similar manner, the increased threat of the tristeza disease around the Lindcove REC prompted the CRB to fund the construction of a screenhouse for the establishment of the first CCPP “Protected Foundation Block” in 1998.

Today, the Protected Foundation Block covers 65,000 square feet which contain approximately 1,000 trees rep-resenting 400 different citrus varieties. In 2007, after almost 50 years, the Pro-tected Foundation Block in Lindcove REC replaced the open field trees of the Foundation and Evaluation Blocks as the primary source for budwood distribution. Over the past three years, the Protected Foundation Block at Lindcove REC has fulfilled 400 orders for 280 different varieties that total ap-proximately 100,000 buds.

Since its establishment in 1968, the CRB has provided grant funding for the CCPP program and the majority of


the research projects that take place at Lindcove REC. In the 1990-2000s, when Walter Stutzman and Louis Whitendale were superintendents, the CRB became an especially important partner in the rapid expansion of the facilities of Lindcove REC.

In 1995, the Citrus Research Board, California Citrus Quality Council (CCQC) and a number of citrus indus-try equipment manufacturers provided the funding and equipment to build a packing facility to conduct postharvest fruit quality evaluations. Prior to the construction of this facility, researchers manually counted, sized, and rated the damage of fruit from research trees on tables in the orchard. Manual labor limited the amount of data that could be collected and the number of proj-ects that could be supported each year. With the building of the packline, which includes a high pressure washer, waxer and dryer, sorters, and an electronic eye that can count, size, and grade fruit, researchers are able to rapidly collect data from individual trees in their plots. This equipment provides a much greater depth of knowledge of the effects of scions and rootstocks on fruit size, yield and quality.

The Citrus Research Board also pro-vided funding to build a Fruit Quality Laboratory. The laboratory has equip-ment and staff that allows researchers to measure parameters such as fruit length and width, color, rind thickness, fruit and juice weight, % juice, total soluble solids, % sugar/acid ratio, and % crease and puff -- again, expanding the parameters that researchers can study.

In 2004, the University constructed a conference building at Lindcove REC. The conference building hosts meetings of University and citrus industry organi-zations and hosts educational programs on citrus varieties, pest management, and horticultural practices for citrus growers, PCAs and nurserymen as well as Master Gardeners and the general public. Each December, the conference building hosts a two-day event to display more than 100 varieties of citrus from the Foundation Block for tasting by the growers and the general public. For the growers, this provides them an opportu-nity to evaluate new citrus varieties and helps them make decisions on planting and topworking. For the homeowners, the fruit display educates the public about the large number of varieties of citrus available and the timing of their maturation.

Lindcove REC has always had an impressive collaboration between UC Riverside and UC Davis researchers and extension specialists, UC Cooperative Extension Farm Advisors, and USDA researchers. Lindcove REC currently supports an average of 30 research projects per year covering topics such as freeze damage measurements, volatile organic compound (VOC), measure-ments, pruning, irrigation, variety de-velopment, pesticide treatments for nematodes, insects, mites, and posthar-vest diseases, and economic thresholds for pests. One of the early collaborative research contributions was the develop-ment of techniques for detecting and removing graft transmissible pathogens such as viruses and viroids from citrus

trees in order to support the CCPP budwood distribution program.

Because of its ability to conduct long-term studies under controlled circumstances, Lindcove REC is able to provide the citrus industry and regulatory agencies with information about fruit production and fruit quality on a wide array of rootstock and scion combinations as well as study problems that develop with time. For example, the California Department of Food Agri-culture (CDFA) has been using either directly or indirectly data generated by Lindcove REC studies performed by David Gumpf and Joseph Semancik (Dept. of Plant Pathology, UC River-side) for the determination of maturity and quality standards for harvest and packing of individual varieties as well as the commercial use of two selections of small transmissible RNAs which have been shown to modify the growth of citrus varieties growing on trifoliate orange rootstock. Lindcove REC has played a significant role in the expan-sion of the number of navel varieties grown in the San Joaquin Valley from a few navel varieties (Washington, T.I. and Atwood) to many additional vari-eties (Powell, Fukumoto, Lane Lates, Chislet, Barnfield, and Bonanza). More recently, researchers have been study-ing the many varieties of the wonderful tasting and easy to peel mandarins at Lindcove REC.

Examples of several current re-search projects at Lindcove include the breeding program of Mikeal Roose, the fruit quality evaluation program of Tracy Kahn, and the pesticide evalua-

Director Beth Grafton-Cardwell with retired nurseryman Raul Gonzales and superintendents Walt Stutsman and Ray Copeland (left to right).

Tracy Kahn leads a walking tour of the citrus trees in the demonstration block at Lindcove REC.

Chet Roistacher planting the first citrus tree in the Foundation Block at LREC. Photo by E.C. Calavan


tion program of Beth Grafton-Cardwell. Mikeal Roose of the UC Riverside Dept. of Botany and Plant Sciences plants trees from his scion breeding program at Lindcove REC to determine their growth characteristics on various rootstocks under San Joaquin Valley conditions. In recent years, Dr. Roose has been utilizing irradiation of citrus tissue to create trees that are evalu-ated at Lindcove for qualities such as seedlessness. From this process, he has created several seedless varieties of mandarins, most notably the Tango, a seedless form of the W. Murcott Afourer.

Tracy Kahn, the curator of the Riverside Citrus Variety Collection, began planting trees for evaluation and demonstration of differences in variet-ies at Lindcove REC during 1992. This demonstration block provides San Joa-quin Valley fruit quality and maturation data, which is reported on the website http://www.citrusvariety.ucr.edu/. The demonstration block also provides an excellent teaching tool for the field day events conducted by Dr. Kahn and the tour groups that visit Lindcove.

Beth Grafton-Cardwell, Dept. of En-tomology UC Riverside, has conducted

more than 80 insecticide trials during the past 20 years at Lindcove REC. In these studies, she has evaluated the efficacy and residuality of various rates and tim-ings of insecticides against pests such as California red scale, citricola scale, citrus red mite, katydids, citrus cutworm, citrus peelminer, citrus leafminer, and ants and their selectivity favoring natural enemies. This work could not be done elsewhere because it often includes un-registered insecticides that require crop destruction. Her research is the basis for the UC IPM pest management guide-lines for citrus and for recommendations presented at numerous field days, slide shows and workshops.

The research projects and exten-sion programs at Lindcove REC have educated thousands of citrus growers and nurserymen about the best methods for producing the many varieties of cit-rus grown in the San Joaquin Valley of California. The success of this program is due to the excellent collaboration of the University and USDA project leaders and their staff with the citrus industry. Lindcove REC is toured by visitors from all over the world who marvel at the quality of the trees and the research

programs and view it as a jewel of the California citrus industry.

Beth Grafton-Cardwell is Extension Specialist and Research Entomologist, UC Riverside and Director of Lindcove Research and Extension Center. Georgios Vidalakis is Extension Specialist and Plant Pathologist, UC Riverside, and Director of the Citrus Clonal Protection Program. Ray Copeland, UCCE-retired, served as Superintendent of Lindcove Field Station from 1965-1987.

1959. Lindcove Field Station Dedi-cated. Citrograph 44 (8): 262.

Calavan C.E., Mather S.M., and McEachern E.H. 1978. Registration, cer-tification, and indexing of citrus trees. In Reuther W., Calavan C. E., and Carman G. E. (eds.). The citrus industry Vol. IV. Crop protection. Chapter 3, pages 185-222. University of California, Division of Agricultural Sciences.

Reuther W, Calavan C.E., Naurer E.M., and Roistacher C.N. 1972. The Cali-fornia Citrus Variety Improvement Pro-gram after twelve years. p 271-278 in: The 5th Proc Int Org Citrus Virologists. Univ of Florida Press, Gainesville, Florida USA.

1985. Lindcove at 25: Planning for the Future. Citrograph 70 (6):120-121. l


EXPENSES

RESEARCH PROGRAM

Plant ManagementDMS VoC Sensor for Citrus ............................ 189,641Measuring ozone Removal............................. 176,958

Subtotal Plant Management ......................... 366,599

This past fiscal year (2008-2009) has been a season of change for the Citrus Research Board. With the discovery of the Asian citrus psyllid in 2008, the

Program took on a completely new adventure, that of de-veloping an Early Detection-Rapid Response operational program. In late 2008, the industry approved a change to the authorities for the CRB Program to include an opera-tions function for pest and disease detection and identifica-

tion. This has provided the legal foundation for the addition of an Operations Department.

The audited budget gives the amount of funding to the Operations Department along with the initial functions of the Program. As you can see, con-siderable attention has been given to the new program as a measure of protecting the California citrus industry. The 2008-2009 funding went to the

formation of the field teams, the establishment of the data management system, and the opening of the laboratory in Riverside. By the end of the fiscal period in October, all three systems were up and operating. The current fiscal year has seen an expansion of the field teams to accommodate expanded trapping programs throughout the state.

The research program for the 2008-2009 fiscal year expanded the focus on citrus disease diagnosis and un-derstanding. The California industry is in a unique posi-tion in that the pest is just arrived and the bacterium that causes HLB has not been detected in the state. Therefore, investments in improved detection systems for both the pest and the bacterium have been prudent. The Program continues to create systems that will attract the ACP to the trapping systems through research in the role of volatile organic compounds (VOCs) to be attractive to the pest. This process should lead to a trap that will have a wider range of detection and increase the probability of finding

CRB 2009 Annual Reportbreeding populations early in their development.

Additional areas of research are the continuation of the pest programs including efficacy trials of new compounds, work on biological control programs, and resistance man-agement. IPM continues to be the main driving force in future research. Understanding resistance management will help keep the existing crop protection tools working for the growers as new technologies are developed.

The postharvest investments are aimed at providing tools for maintaining our markets with fruit that is of the highest quality as it reaches the consumer. Additionally the Program includes developing new tools to meet the inter-national marketing requirements and tools for increased efficiency in harvesting.

The Board welcomes your comments and observations to the Citrus Research Program. Please feel free to contact us with your input at any time.

Ted Batkin, President

INCOME

2008 Fund balance (Carryover) ...................... 921,8972008-2009 FY Assessment Income ............. 5,049,591Prior Season Income ...................................... 359,283outside Income .................................................. 6,075Grower Seminar Registration Income ................. 8,058Investment Dividend Income ............................ 27,690

TOTAL FUNDS AVAILABLE ........................... 6,372,594

CITRUS RESEARCH BOARDNovember 1, 2008 through october 31, 2009


Plant Improvement

Citrus Rootstock evaluation .............................. 80,000 Variety evaluation for Trueness ........................ 60,000 New Citrus breeding ...................................... 116,830 evaluation of Strain Trials ................................. 29,595 evaluation of Desert Lemons .............................. 9,802

Subtotal Plant Improvement ...................... 296,227

Biotechnology

Improving Peel Quality in Citrus ....................... 10,000Sweet orange Physical Map ............................ 28,850

Subtotal Biotechnology ................................. 38,850

Plant Pathology

Septoria Spot ................................................... 39,500 HLb Management Systems .............................. 45,000 Small RNA for HLb ........................................... 89,006 Small RNA for Stubborn ................................... 26,739 Lateral Flow Microarray ................................. 180,000 Fingerprinting of HLb ....................................... 35,413 Cultivation and Sequencing of HLb .................. 58,300 evaluation of Field Treatments ........................... 6,400 Investigation of Seedling Yellow ....................... 30,000 Identification of Spiroplasma ............................ 57,500

Subtotal Plant Pathology ............................ 567,858

Entomology

Pest Management Infrastructure .................... 195,000 Chemical Control of Thrips ............................... 75,000 Armored Scale Research .................................. 27,040 Rearing Peelminer Parasitoids .......................... 59,540 Parasitoid of Citricola Scale .............................. 45,789 Nematodes Against Diaprepes ......................... 32,959 Dev. of Attractants/Repellents for ACP ............ 173,625 Probiotic Diet ..................................................... 6,335 Assessment of Systemic Neonicotinoid .......... 156,449

Subtotal Entomology ................................... 771,737

Post Harvest

Treatment evaluation ....................................... 46,500 New Methods for P.H. Decay ............................ 46,000 Robotic Picker ............................................... 100,000 ethyl Formate Studies for bean Thrips .............. 35,900

Subtotal Post Harvest ................................. 228,400

TOTAL RESEARCH PROGRAM .................... 2,269,671

COMUNICATIONS PROGRAM Core Grower education Program ...................... 31,439HLb Task Force .............................................. 124,398 HLb Task Force Activities ...................................... 863 Conferences .................................................... 12,070 HLb/ACP Grower Training ................................. 10,565

TOTAL COMMUNICATIONS PROGRAM .......... 179,335

CITRUS CLONAL PROTECTION PROGRAMCore Citrus Clonal Protection Program ........... 363,255 Facilities Improvement ................................... 109,741 Screenhouse Construction ............................. 159,544

TOTAL CITRUS CLONAL PROTECTION PROGRAM ............................... 632,540

OPERATIONS PROGRAMSalaries – Management ................................... 88,589 benefits – Management ................................... 14,819 Payroll Taxes – Management ............................ 6,777 Travel & Mileage – Management ...................... 17,295 Salaries – ACP Field Staffing ............................ 45,899 benefits – ACP Field Staffing .............................. 4,115 Payroll Taxes – ACP Field Staffing ...................... 3,511 Travel & Mileage – ACP Field Staffing ............... 11,731 Salaries – Laboratory Staffing .......................... 55,208 benefits – Laboratory Staffing .......................... 10,451 Payroll Taxes – Laboratory Staffing .................... 4,223 Travel & Mileage – Laboratory Staffing .............. 4,632 Salaries – Data Management ........................... 40,625 benefits – Data Management ........................... 12,665 Payroll Taxes – Data Management ..................... 3,108 Travel & Mileage – Data Management ............... 2,132 equipment Purchase/Supplies – Data Management .................................... 17,811 equipment Repair & Maintenance – Data Management ......................................... 780 office equipment – Data Management ............... 2,832 Information Services – Data Management ....... 20,372 Supplies – Data Management ............................ 2,464 Telephone – Data Management ......................... 3,008 Rent – Data Management .................................. 7,500 equipment/Supplies – Lab operations ............. 17,273 equipment Repairs & Maintenance – Lab operations ........................................... 7,616 Insurance bonds – Lab operations ........................ 160 office equipment – Lab operations .................... 1,163 Supplies – Lab operations ............................... 33,553 building Re-Model – Lab operations ................ 43,093


Sandy Creighton, Sales Manager Phone: 559-433-9343

E-mail: [email protected]

Whether you're selling tractors or other farm equipment,pickup trucks, irrigation equip-

ment, fertilizer or pesticides...consider the value of your ad dollar in the pages of Citrograph.

Each issue reaches every commercial citrus grower in the states of California and Arizona, plus associ-ated business members affiliated with the citrus industry...the people in charge of purchas-ing. Your advertising message is directed to farm leaders who use vast amounts of goods and services.

Circulation reach-es over 5,000 key decision makers among California and Arizona fresh citrus growers, landowners and industry-involved companies. In the near future, Citrograph will reach the entire United States.

Don’t miss the next issue!

Reach Commercial California &

Arizona Citrus Growers

Contact us today to be included in future issues

of Citrograph

utilities – Lab operations ................................... 4,761 Telephone – Lab operations ............................... 8,970 Rent – Lab operations ..................................... 21,413 equipment/Supplies – Field operations ................. 647 Supplies – Field operations ............................. 41,567 Telephone Field operations ................................ 1,751 equipment Purchases – Transportation .......... 145,153 equipment Repair & Maintenance – Transportation ................................................ 151 Insurance & DMV Fees – Transportation ............. 2,887 Miscellaneous expenses ....................................... 446 Printing ................................................................. 178 office Supplies ................................................ 12,808 other Projects .................................................... 1,089

TOTAL OPERATIONS PROGRAM .................... 725,226

CALIFORNIA CITRUS QUALITY COUNCIL (CCQC)CCQC Administration ...................................... 371,600

TOTAL CALIFORNIA CITRUS QUALITY COUNCIL (CCQC) ........................... 371,600

GENERAL AND ADMINISTRATIVE Audit Fee ........................................................... 7,498 equipment/Supplies Purchased .......................... 6,798 employee benefits ........................................... 87,078 equipment Repair & Maintenance .................... 80,824 equipment Rental .............................................. 5,630 Information Services ........................................ 20,193 Insurance & bonds ........................................... 10,095 office Supplies ................................................ 26,174 Postage ............................................................. 7,842 Printing ............................................................ 18,627 Rent & Storage ................................................ 36,967 Research Consultant .......................................... 5,286 Salaries ......................................................... 345,989 Meeting Costs .................................................. 31,243 Payroll Taxes .................................................... 39,283 Telephone ........................................................ 10,612 Travel & Mileage – Consultant .......................... 5,733 Travel & Mileage – Members ........................... 54,063 Travel & Mileage – Staff ................................... 54,910 CDFA – bureau of Marketing ............................ 55,289 CDFA – Handler Audit ......................................... 6,750

TOTAL GENERAL & ADMINISTRATIVE .......... 916,884

SUMMARY

TOTAL EXPENSES ...................................... 5,095,256

RESERVE AT END OF YEAR ........................ 1,277,338

TOTAL ALL ................................................................. 6,372,594

OPERATIONS PROGRAM...continued


CRB launches special ACP/HLB

survey website

The Citrus Research Board has begun working to detect the Asian citrus psyllid and

Huanglongbing in commercial cit-rus groves across the state. CRB has a crew of nine trappers working in eight counties and has already established over 6,700 ACP trap lo-cations with more being added daily. The CRB diagnostic laboratory in Riverside is testing both insect and plant tissue samples for the pres-ence of the HLB bacterium.

With the help of the University of California, CRB has completed the first phase of constructing a special citrus invasive pest website, and we want you to see it. This will be the principal vehicle for extending the results of the ACP/HLB detection program in com-mercial citrus. In the future, it will be expanded to include additional exotic citrus pests and diseases and become a valued resource for growers, packers, collaborating researchers, regulators, and area-wide pest managers. Despite the preliminary nature of this launch, the site already has many features and func-tions with more to come.

Access to the site will be limited to legitimate stakeholders in the California citrus industry including growers, handlers, researchers, and regulators. Citro-graph readers, feel free to request your own new account at any time.

Access the site at: https://crbcitrussurvey.uckac.edu/viewerThrough the end of May, use Username: DemoGrower

Password: 123EasyDemo*

CRB invites your comments, questions and suggestions. Contact Richard Dunn, CRB’s data, information & management director, by email

to [email protected] or by phone at (559) 738-0246


H. Vincent Moses

G. Harold Powell and the defeat of citrus decay,

1904-1912

The directors of this Foundation

are elated and honored in having

this opportunity to “showcase”

our work through the Citrograph

magazine. Our “Mission” is to el-

evate the awareness of California

citrus heritage through publications,

education, and artistic work. We

are pleased with the response of the

three current university displays: the

University Library-Special Collec-

tions at Cal Poly Pomona, Pomer-

antz Library at Western University,

and our largest display in the John

M. Pfau Library at CSU San Ber-

nardino. We are especially happy to

report that the Foundation’s latest

book has just arrived, titled Citrus

Powered the Economy of Orange

County for Over a Half Century In-

duced by “A Romance”. Please visit

our website… www.citrusroots.com.

Citrus RootsPreserving Citrus Heritage Foundation

By 1900, something was very rot-ten indeed but not in Denmark. Every year since the emergence

of California’s modern citrus enterprise, devastating rot had manifested itself in thousands of refrigerator rail cars laden with California citrus. These cars carried the expanding, extremely lucrative and intensely sought-after orange and lemon crops of thousands of growers.

Growers complained to the USDA and the University of California that they were losing as much as a million dollars a year to decay while their fruit was in transit to New York, New Orleans, Chicago, London (Ontario, Canada) and other distant market destinations. The losses were hurting their bottom line and threatening the future of the entire California citrus enterprise.

Already practitioners of Professor Liberty Hyde Bailey’s scientific agricul-ture and modern business organization, these growers wanted answers, particu-larly to the problem of citrus decay in transit. Many growers thought this situation inherent in the keeping quality of oranges and lemons. They assumed it had to be considered part of doing business. Other growers blamed the rail-roads for mishandling their shipments and sued them constantly in Commerce Court or filed complaints with the I.C.C.

Certain scientifically astute, busi-ness-minded growers in Riverside refused to accept either the inherent weakness argument or the railroad bugaboo theory, believing that the industry could not sustain such losses without long-term impact on the busi-ness. Their persistent demands on the

We are proud of our accomplishments as a volunteer organization, which means each donated dollar works for you at 100% [for we have no salaries, wages, rent, etc.]. All donations are tax deductible for income tax purposes to the full extent allowed by law.

Citrus Roots – Preserving Citrus Heritage Foundation

P.O. Box 4038, Balboa, CA 92661 USA

501(c)(3) EIN 43-2102497

USDA eventually prevailed. In short order, this triumph sealed the future of the industry, ultimately guaranteeing its existence as what Harvard business historian Alfred D. Chandler, Jr. has described as a modern industrial enter-prise, on par with the big meat packer trusts Swift and Armour.

In 1904, orange grower pressure brought youthful, talented and brash G. Harold Powell roaring into the region. One of “Uncle Sam’s missionaries, “ fresh from the USDA’s Bureau of Plant Industry, Powell expected to educate growers in scientific practices. Would he be surprised! Born February 8, 1872 as part of the post-Civil War generation that created the Progressive Movement, Powell grew up on the comfortable and successful commercial apple orchards of his influential father, George T. Powell, a notable Quaker, at Ghent, New York. G. Harold Powell worked his way through Cornell University and took his training in horticulture under the agricultural “rock star” at Cornell, Professor Lib-erty Hyde Bailey, who helped him gain appointment as the Graduate Fellow in Horticulture.

Powell took his Master of Science in Horticulture and soon landed a job with the Bureau of Plant Industry of the United States Department of Agri-culture, Washington, D.C. It was 1901, and Powell reveled in his new work as assistant pomologist assigned to study the problems of apples in cold storage. Within two years the Department pub-lished two of his bulletins, “The Apple in Cold Storage” and “Cold Storage with Special Reference to the Pear and


G. Harold Powell (1872-1922), the tallest man in the photograph, standing between B. A. Woodford, President of the California Fruit Growers Exchange (CFGE) and La Verne grower, and Walter Barnwell, Assistant Freight Agent for the Atchison, Topeka & Santa Fe Railroad, inspecting one of Powell’s USDA citrus pre-cooling and refrigeration experiments sponsored by the CFGE, c1907. Photo courtesy of the Huntington Library.

They launched a revolution in

the handling of fresh produce.

Peach.” They launched a full-blown rev-olution in the handling of fresh produce. His study of barrel spoilage in apples launched all other such investigations of the shelf life of perishable agricultural products in the United States.

As historian Richard Lillard ar-gued, prior to Powell’s apple spoilage experiments, both the USDA and farm-ers, even the commercial ones like his father, had concentrated almost solely on production problems. Then, as a complement to the apple study, Powell demonstrated conclusively why Georgia peaches decayed in transit aboard trains. Consequently, Powell rose rapidly up the ladder of his discipline within the USDA’s Bureau of Plant Industry. Just prior to his first trip to California in 1904, B. T. Galloway, Chief of the Bureau, placed him in charge of the Fruit Storage and Transportation Division.

As Pomologist-in-Charge, Powell and his subordinates pursued studies in ways to obtain more efficient cooling and air flow in refrigerated rail cars. The USDA Yearbook of Agriculture pub-lished the results of his team’s research as “The Handling of Fruit for Transpor-

tation” in 1905. By this time, however, the direction and nature of Powell’s professional life made a radical shift to the great West, placing him in position to ultimately transform the very structure of agriculture in the entire country.

Here is a reason why. The prominent leaders among California orange and lemon growers were previously captains of industry or doctors, lawyers, accountants, bankers, and refugees from other success-ful urban professions. Without prompting from Powell or any oth-er USDA missionary, orange growers under-stood the need to organize for their own interests along modern business lines. While some were skeptical of a pure scientist, Powell soon won them over with his persuasive manner and practi-cal knowledge of horticultural practices.

The rise of sanitary engineering, brought on by Progressive Movement reformers and muck-raking journalists, had stimulated leading growers in River-side, California to seek government help

in finding a solution. In dramatic fash-ion, Powell and his team quickly identi-fied both the source of the decay, blue mold (Penicillium italicum), and the reasons it took hold in citrus shipments. Then they submitted proposals for industrial solutions to the problem, i.e. careful handling from field to shipping crate. Those proposals were adopted

wholesale by growers and packers

As early as April 7, 1905, Powell’s team of agricultural research scientists had gathered enough evidence to provide a searing argu-ment that decay could

be prevented, and that growers brought on most of it through their own faulty practices. April 7th, the date of Powell’s well publicized appearance at a Farm-ers’ Citrus Institute in Riverside, had been eagerly awaited and touted by the regular and agricultural press for several weeks. Editorials predicted that Powell’s work might lead to a revolution in picking and packing methods.

His Institute presentation, in fact,


Washington navel oranges showing blue mold decay, frontispiece, USDA Bureau of Plant Industry Bulletin No. 123, Powell et al, “The Decay of Oranges While in Transit from California.”

produced radical results. It took on piv-otal significance for citriculture practices in California . These practices would never be quite the same afterward. Powell reported to his superior, B. T. Galloway. “The institute which closed yesterday,” he recalled, “is said by the best men here to have been the best one held in southern California.” Set up as a forum for Powell’s preliminary findings, the two-day meeting drew scores of growers, packers, and a large contingent of transportation representatives, both from the railroads and the refrigerator car lines such as Armour.

In one fell swoop, he deftly linked the industry with the scientific prow-ess of the United States Department of Agriculture. From that time on, the industry was his to shape. In 1910, grow-ers summoned Powell back to California to head the Citrus Protective League, a trade association representing 90% of the industry, and the first such entity in agriculture. As manager of the League, he emerged as a potent spokesman for the industry, promulgating his ideology of cooperation wherever and whenever he could.

By means of his established position with President Benjamin Ide Wheeler of

the University of California (previously his wife’s Greek professor at Cornell) and among Washington bureaucrats and congressmen, he furthered ties between the state and federal governments and the growers of California. Lastly, serving as General Manager of the California Fruit Growers Exchange, 1912-1922, Powell insured the future of the industry, and thus the region, by consolidating the Exchange into a full-fledged managerial corporation.

Future chapters will put Powell squarely into the story of California orange growers, their famous business organization, the California Fruit Grow-ers Exchange, and their establishment of the first professional managerial

corporation in American agriculture. These additional articles will tell the story of the arrival of the “Revolution of Corporate Capitalism” to California, and to agriculture. They will describe the introduction of “Country Life” ideology into Southern California via G. Harold Powell. The story will focus on that pivotal period when the corporate and managerial revolution growers led con-solidated their industry into a modern business enterprise, with the ability to shape a regional culture.

Dr. Vince Moses, Owner, VinCate & Associates Museum and Preservation Consultants, and member of the Advi-sory Board, Citrus Roots, Preserving Citrus Heritage Foundation. l

22144 BOSTON AVENUEEXETER, CALIFORNIA 93221

TangoMiho WaseGold Nugget

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onCarrizo Rootstock

559-592-3367 Bus559-592-4158 [email protected]

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


Citrus Roots Preserving Citrus Heritage Foundation

Keeping citrus heritage alive in the minds of those living in California through publications, educational exhibits and artistic works

Citrus Roots...Our Legacy - Volume I Selling the Gold - History of Sunkist® and Pure Gold®

Citrus Roots...Our Legacy - Volume II Citriculture to Citrus Culture

Citrus Roots...Our Legacy - Volume III Our Legacy...Baldy View Entrepreneurs - 25 men & women who left a legacy

All donations are tax deductible for income tax purposes to the full extent allowed by law.

Citrus Roots Series...NEWEST RELEASE!! Citrus Roots...Our Legacy - Volume IV Citrus Powered the Economy of Orange County for over a half century Induced by a “Romance”

By: Rahno Mabel MacCurdy, V.A. Lockabey and others...compiled and edited by R.H. Barker

GOLDHistory of

Sunkist® and Pure Gold®

CITRUS ROOTS . . . OUR LEGACY Volume I of III

Selling the

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American Business Cycles from 1810 to 1978 vs. the Life Span of Twenty-Five Entrepreneurs

by Marie A. Boyd and Richard H. Barker

Including a fold out time line chart of

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CITRUS ROOTS ... OUR LEGACY Volume III of III CITRUS ROOTS ... OUR LEGACY Volume III of III

For ordering information visit our website

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(Fed. Tax ID # 43-2102497)

KINGS

ORANGE

IMPERIAL

SAN DIEGO

San Nicolas Island

MADERA

SAN MATEO

MERCED

FRESNO

SANTACLARA

INYO

SANTA CRUZ

SAN BENITO

MONTEREY TULARE

San Clemente Island

SAN BERNARDINO

KERNSAN LUIS OBISPO

SANTA BARBARA

VENTURA

LOS ANGELES

RIVERSIDE

Santa Cruz IslandSan Miguel IslandSanta Rosa Island

UT

NV

AZ

SonoraBaja California

United States Department of Agriculture

Animal and Plant Health Inspection Service

Date Created: April 5, 2010

Data Source:CA Dept of Food & Agric.AZ Dept of AgricultureUSDA, APHIS, ISTeleAtlas Dynamap

USDA, APHIS, PPQWestern Region GIS Specialist650 Capitol Mall, Suite 6-400Sacramento, CA 95814

The U.S. Department of Agriculture's Animal and Plant Health Inspection Service collected the data displayed for internal agency purposes only. These data may be used by others; however, they must be used for their original intended purpose.0 20 40 60 80 10010

Miles

Asian Citrus Psyllid Cooperative ProjectCalifornia, Arizona, and Baja California

Coordinate System:CA Teale Albers, NAD83

LegendAsian Citrus Psyllid, CA_2010 thru 3-29-10

Asian Citrus Psyllid, CA_2009

Asian Citrus Psyllid, CA_2008

Asian Citrus Pysllid, AZ_2010 thru 3-31-10

Asian Citrus Psyllid, AZ_2009

Asian Citrus Psyllid, Mexico_2010 thru 3-25-10

Asian Citrus Psyllid, Mexico_2009

Asian Citrus Psyllid, Mexico_2008

ACP_Traps_Citrus Research Board_3-25-10

Quarantine for Asian Citrus Psyllid, CA_11-17-09

Quarantine for Asian Citrus Psyllid, AZ_12-7-09

Parks in CANational

State

Local

Map of Asian citrus psyllid detections prior to 3/29/2010 in California and neighboring portions of Arizona and Mexico. This map also indicates the locations of ACP traps installed by the Citrus Research Board prior to 3/25/2010. Traps installed and maintained by other agencies are not displayed.


Descriptions of new varieties recently distributed from the

Citrus Clonal Protection ProgramToni Siebert, Robert Krueger, Tracy Kahn, John Bash and Georgios Vidalakis

‘C54-4-4’ mandarin: Citrus Variety Collection, Riverside, California, 16 Feb 2010. Photo by T. Siebert

“Protected Foundation Block Budwood” is budwood provided from CDFA registered CCPP citrus trees from the LREC screenhouses and is available from the University of California in accordance with the CDFA regulations for cit-rus registration and certification. Protected Foundation Block Budwood is produced by trees grown in pots and in ground under protective screen and is intended for individual nurseries or growers to produce their own registered budwood source trees or for the production of nursery increase blocks from which additional budwood may be harvested in accordance with CDFA (or other appropriate) regulations and used for the production of certified nursery stock. A signed “Waiver and Release” form must accompany all orders for Protected Foundation Block Budwood. The “Waiver and Release” form is available on the CCPP website (http://www.ccpp.ucr.edu).

SRA 337 or C54-4-4 Mandarin (VI 672): First distribution of buds from the CCPP: September 2009

‘C54-4-4’ was selected for introduction to California in 1997 by members of the California Citrus Nurserymen Society (CCNS) during a tour of the INRA-CIRAD Station de Re-cherches Agronomiques in San Giuliano, Corsica, associated with the Congress of the International Citrus Nurserymen’s Society. C54-4-4 is actually a product of California, being a cross of ‘Clementine’ X ‘Murcott’. The cross was actually made at the USDA Horticultural Research Laboratory in

The Citrus Clonal Protection Program (CCPP) is operated through the Department of Plant Pathology and Microbiology at University of

California (UC) Riverside and is funded in large part by The California Citrus Research Board (CRB). The CCPP processes citrus propagative material in two phases. First, during the quarantine phase, citrus bud-wood of potentially important commercial varieties is introduced from any citricultural area, germplasm or breeding program of the world under the authority of a permit which is issued to CCPP by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service in cooperation with the Cali-fornia Department of Food and Agriculture (CDFA). While in quarantine at the Rubidoux Facility in River-side (approximately 2.5-3 years), newly imported vari-eties are tested extensively and any detected pathogens (such as viruses and bacteria that cause the tristeza, exocortis, stubborn, or Huanglongbing (HLB) disease of citrus) are eliminated via therapy. The second phase includes the production of budwood source trees which are moved out of quarantine in Riverside and to the UC Lindcove Research and Extension Center (LREC) in Exeter, California, where the CCPP Protected Foun-dation (screenhouse) and Evaluation Blocks (field) are housed. Trees established in the Evaluation Blocks are evaluated for trueness to type by scientists, growers, and nurserymen and are accessible to the public during field and fruit testing days (aka “walkthroughs”). Trees established in the Protected Foundation Blocks are off limits to the public, they are regularly tested for a vari-ety of pathogens and are registered with the CDFA as budwood source trees.

Over the past several years, many varieties have been through the rigorous “Variety Introduction-VI” disease testing and therapy program under quarantine at the CCPP. Varieties that successfully complete the VI process receive a unique VI identification number that permanently accompanies the budwood that is made available to growers, nurseries, researchers, and others. Little information about many of the CCPP VI variet-ies is accessible to the public, or may take a great deal of effort to find. As a result, the UCR-Citrus Variety Collection (CVC), USDA-National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), and CCPP have compiled information on the 18 most recently dis-tributed varieties.

Protected Foundation Block Budwood


Orlando, Florida, by Dr Phil Reece but the seeds were sent to Dr Joe Furr at the USDA Date and Citrus Station in Indio for testing and development. In 1963, budwood was sent from California to Texas for evaluation by Dr Heinz Wutscher, USDA, Weslaco. The characteristics of ‘C54-4-4’ under Texas conditions is presented in a paper in the Journal of the American Society for Horticultural Science, 103:124-127 (1978). Those results indicate that ‘C54-4-4’ is a late maturing mandarin with uniform exterior color with 20 % granulation. The fruit was large with an average of 22 seeds per fruit. Brix was intermediate in the varieties reported upon, but acid was among the lowest reported. Therefore, the brix/acid ratio was the second highest among the varieties evaluated. Yields were intermediate. This data was based upon harvest in January or February for the years 1969 – 1974. Dr Wutscher introduced this selection into Florida when he transferred to Orlando in 1975, and evaluated it in Florida conditions. In Florida, it proved susceptible to scab (a disease that we do not have in California). Dr Wutscher later made a scab-resistant selec-tion of ‘C54-4-4’ that has recently been released by Florida Division of Plant Industry (DPI) as ‘Furr’ mandarin. Note that this is not the ‘Furr’ trifoliate hybrid rootstock recently released by UC Riverside. In the 1980’s, prior to making the scab-resistant selection, Dr Wutscher sent budwood of ‘C54-4-4’ to Corsica. This is the source of the recent introduction into California and hence does not represent the scab-resistant Florida selection ‘Furr’.

Imperial Mandarin (VI 684): First distribution of buds from the CCPP: January 2008

‘Imperial’ is reported to have originated in Emu Plains, near Sydney, Australia, as a chance hybrid of ‘Mediterranean’ or ‘Willowleaf’ and another mandarin, possibly ‘Emperor’ in approximately 1890. ‘Imperial’ is one of Australia’s most im-portant and long-established mandarin selections. It is widely planted throughout existing mandarin-growing regions, with about 361,000 bearing and 26,000 non-bearing trees in 1999, mostly in Queensland. ‘Imperial’ is an extremely early matur-ing mandarin, equivalent in this regard to ‘Owari’ satsuma. Brix:acid ratio reaches 7:1 around mid-March in the earliest regions of Australia, with later regions reaching this ratio in early June. ‘Imperial’ can be harvested up to July or August in Australia, depending upon the region. Fruit quality is con-sidered good with a good balance of sugar and acid and good internal color. The skin is thin and soft, and although adherent, it peels easily. Juice levels are at least 35 % and fruit are firm when peeled. Granulation is sometimes a problem because of the low juice content. There are usually 4 or less seeds per fruit in both solid and mixed plantings. The fruit is medium sized (or small in heavy crop years). The external color is yellow orange, but is more intense in regions where fall tem-peratures are cool. Fruit generally must be clipped from the tree rather than plucked. De-greening is necessary when fruit are harvested early and the post-harvest life is short at 2 - 4 weeks. ‘Imperial’ responds poorly to heat and cold treatments for pest disinfestation, which makes it unsuitable for export (from Australia). ‘Imperial’ performs adequately on a variety of rootstocks, although incompatibilities have sometimes been noted on ‘Carrizo’ and ‘Troyer’. ‘Cleopatra’ produces high quality fruit. ‘Cleopatra’ and ‘Troyer’ are the most com-monly used rootstocks in Queensland, whereas ‘Carrizo’ is

more popular in southern areas. There is a tendency towards alternate bearing, which sometimes is managed by thinning. The tree’s habit is vigorous and upright. Cross-pollinators may help with set, yield, and size. This variety is currently being evaluated by Toni Siebert and Tracy Kahn. This information was summarized from The Citrus Industry, 1967, I:516; and Saunt, 2000, ‘Citrus Varieties of the World’, pp 65-66. A down-loadable factsheet developed by Australian Citrus Limited is available at http://www.australiancitrusgrowers.com.au/aspdev/resources/documents/ImperialB.pdf

Hickson Mandarin (VI 685): First distribution of buds from the CCPP: June 2008

‘Hickson’ is reported to have originated near the town of Roma, Queensland, Australia, as a sporting limb on ‘Ellendale’ tangor. ‘Hickson’ is similar in many respects to its ‘Ellendale’ progenitor. In Australia, it is a mid-season variety, maturing starting in late June, about 2 weeks earlier than ‘Ellendale’. ‘Hickson’ is considered to hang better than ‘Ellendale’, with the harvest date extending through August. In August, it is usually slightly puffed but the juice content is satisfactory.

The general appearance of the tree is similar to ‘Ellendale’, although there are some differences in leaf shape. The fruit is similar in appearance to ‘Ellendale’ but the rind is not as smooth and it peels more easily. The fruit averages 6 cm X 5 cm, has a slight neck, and a smooth, yellowish-orange rind with some gloss. The orange-fleshed fruit is juicy with good flavor and has 12-15 seeds. ‘Hickson’ is resistant to brown spot and was initially considered a promising alternative to ‘Ellendale’. However, ‘Hickson’ declines when propagated on rough lem-on rootstock probably due to genetic or physiological reasons. It is also susceptible to a crotch rot associated with Phomopsis and in addition does not perform well on trifoliate. For these reasons, it is no longer as popular in Australia. This variety is currently being evaluated by Toni Siebert and Tracy Kahn. Information summarized from: Jorgenson, 1972, Queensland Citrus Bulletin, 1972:23-24; Cox, 1975, NSW Dept of Agri-culture, Bulletin H2.2.6; Broadbent et al, Proceedings, ISC,

Hickson mandarin: Citrus Variety Collection, Riverside, California, 16 Feb 2010. Photo by T. Siebert


1978:207-208; Forsyth et al, 1985, NSW Dept of Agriculture, Agfact H2.1.4.). A factsheet developed by Australian Citrus unlimited is available at http://www.australiancitrusgrowers.com.au/aspdev/resources/documents/HicksonB.pdf.

Sudachi Ichandarin (VI 693): First distribution of buds from the CCPP: June 2007

Thought to be a hybrid of a papeda and a mandarin, ‘Su-dachi’ arose as a chance seedling in the Tokushima Prefecture of Japan, on Shikoku island, where it has traditionally been grown. When harvested young, ‘Sudachi’ is considered to have a distinctive fragrance that is different from ‘Yuzu’. The young fruits are used for cooking while still green, often being incorporated into vinegars or flavoring many different entrees, especially fish. The flavor is now also used in soft drinks and alcoholic beverages. Fruit of ‘Sudachi’ was formally evaluated by Ottillia Bier, Toni Siebert and Tracy Kahn in September and October of years 2003 to 2007 at Riverside, California. Significantly smaller than ‘Yuzu’, the average fruit size has a mean width of 3.8 cm and a height of 3.4 cm. The fruits have an oblate (spherical and flattened at both poles) shape, although some fruits can be round. ‘Sudachi’ has a mean weight per fruit of 27.2 grams. Color break was reached between the first and third week of September. The rind texture is slightly pebbly with a mean thickness of 1.9 mm. The number of seeds per fruit averages 9. The mean juice weight is 9.6 grams and the average juice content is 34.4%, which is fitting as ‘Sudachi’ is primarily used for juice. The juice weight and juice content increased during the sampling dates. The internal flesh color of ‘Sudachi’ in the green stage is light green to green-yellow. ‘Sudachi’ is slightly more acidic than ‘Yuzu’ with an average of 5% citric acid. ‘Sudachi’ trees tend to have a spreading habit of moderate vigor, but can be considered a small to medium

Australian Finger Lime (VI 697): First distribution of buds from the CCPP: June 2007

The ‘Australian finger lime’, a citrus relative also known as Microcitrus australasica, is one of six different species of citrus considered to be native to Australia. This VI is one of 8 different accessions of Microcitrus australasica in the Citrus Variety Collection, and was imported from Sydney, Australia, in 1965. Depending on the type of rootstock used (The CVC has used several: Schaub rough lemon, Cleopatra mandarin,

sized tree, as 26-years-old trees on Carrizo and C-35 citrange rootstocks are only approximately 8 feet tall, with no indica-tions of rootstock-scion incompatibility. Thorns up to 5 mm in length are present in each leaf axil. Leaves are elliptical in shape, with a small winged petiole. The tree canopy has dense branching. (Kawada, K., and Kitagawa, H. “Storage of Sudachi (Citrus sudachi Hort. ex Shirai) in Japan”. Pro. Int. Soc. Citriculture, 1084-1085. 1992. Kawada, K., and Kitagawa, H. “Citriculture, Marketing are Different in Japan”. Fresh Citrus Fruits. Avi Publishing Company: Connecticut. 1986.)

Sudachi ichandarin: Citrus Variety Collection, Riverside, California, 15 Nov 2002. Photo by D. Karp.

Australian Finger Lime: Citrus Variety Collection, Riverside, California, 5 Dec 2008. Photo by D. Karp and T. Siebert.

C-35 citrange, Carrizo citrange, Calamondin, Citrus macro-phylla), the ‘Australian finger lime’ can be a very small (about 5 feet on Schaub rough lemon) to large-sized tree. The leaves are tiny at approximately one-half inch long and the branches become very dense and spiny with about 1 thorn set in every leaf axil. New growth is purple in color and the one-quarter inch wide flowers are white and pink during the main flowering season of February to April. The finger limes are about three inches long and roughly the size of an average person’s index finger, but fruit from juvenile trees can be less than one inch long. The skin of the finger lime is usually a greenish black to very dark purple and thin, but durable. Once the fruit is cut open the tiny round juice vesicles will slowly seep out of the fruit without squeezing, and resemble what we like to call “citrus caviar”. The round vesicles are usually a clear-green, but can be very light pink. The juice is very tart, much like a Mexican lime. Although the tree produces fruit year round, the main fruiting season in California is November-December when the fruit falls off in your hand. Australian finger lime is reported to fetch approximately 40-50 dollars per pound. The fruits are technically edible, but this is not commonly done. Its most common use is as a garnish or flavor component in culinary creations.

Persian Lime SPB-7 (VI 708): First distribution of buds from the CCPP: June 2007

This selection of Persian-type lime is said to be free of the genetic disorder called wood pocket, which is found in many of the large-fruited acid limes. Wood pocket was formerly very common in Florida and caused extensive losses. The industry requested help from researchers and after screening more than 100,000 trees, 10 trees were identified that were appar-ently free of wood pocket, based upon their survival. The current selection (SPB-7) was entered into the program in


1954 as Li-38-1-1-X. This selection was apparently erroneously identified as being wood pocket positive and was dropped but later reinstated and never showed wood pocket symptoms. The current selection was entered into the Florida DPI foundation program about 1961 and was imported into California in the year 2000. The current screenhouse bud source descended from a tree planted in the Haines City Foundation Grove. Because of this budsource, the Florida Persian lime industry became virtually free of wood pocket by the early 1970s. (This information redacted from an email from Mike Kesinger, 06/25/2006) Trees of this selection have been planted at various places in California for evaluation as to the development of wood pocket. One of these locations is the Coachella Valley Agricultural Research Station in Thermal, CA. Since wood pocket develops more rapidly at high temperatures, the trees in Thermal should be the most susceptible to development of wood pocket. More information on these observations will be provided as it becomes available.

Lemonade (VI 734): First distribution of buds from the CCPP: September 2008

‘Lemonade’ is reported to be a sweet lemon hybrid of un-known parentage with a very pleasant taste, and can be readily but not easily peeled. The fruit is small-medium, and not very seedy. The trees are semi-dwarfed (on trifoliate rootstock), but quite productive. The main crop matures in early spring in New Zealand, with much smaller summer crops also occurring. Un-fortunately there is no commercial production in New Zealand, although it is a popular home garden tree. It is susceptible to citrus scab disease; however, in a drier climate this should be less of a problem. (This information redacted from an email from Andrew Harty via Peter Chaires, 12/07/2005) Although the budsource trees are derived from trees at the CCPP that tested negative for all known graft-transmissible diseases, trees of ‘Lemonade’ propagated in Riverside have shown a tendency to develop small brown to black lesions on the bark. The reason for these lesions is currently unknown. ‘Lemonade’ trees propagated at Riverside are not particularly vigorous but the relationships of this to the observed lesions is not known.

Early Release Budwood“Early Release Budwood” is budwood provided from se-

lected cultivars that have been recently out of quarantine and are maintained by the CCPP at the LREC Protected Founda-tion Blocks for the “Early Release” program. These cultivars are grown in pots under protective screen producing limited amounts of budwood. Therefore, supply of Early Release Budwood will be limited. A signed “Waiver and Release” form must accompany all orders for Early Release Budwood. The “Waiver and Release” form is available on the CCPP website (http://www.ccpp.ucr.edu).

Valentine Pummelo Hybrid (VI 597): First distribution of buds from the CCPP: September 2009

‘Valentine’ is the most promising of the seedy pigmented low-acid pummelo hybrids selected by Drs. Soost and Cam-eron in 1986 from a cross of ‘Siamese Sweet’ pummelo x

Persian lime: Citrus Variety Collection, Riverside, California, 3 Nov 2009. Photo by D. Karp and T. Siebert.

Lemonade: Citrus Variety Collection, Riverside, 3 Nov 2009. Photo by D. Karp and T. Siebert.

Valentine pummelo hybrid: Citrus Variety Collection laboratory, Riverside, California. Photo by O. J. Bier.


(‘Ruby’ blood orange x ‘Dancy’ mandarin). It received its name from former Staff Research Associate for the Citrus Variety Collection Ottillia ‘Toots’ Bier, who nicknamed it ‘Valentine’ not only because the fruit matures in mid-February near the Valentine’s Day holiday, but also because often when the fruit is cut lengthwise and turned upside down, the flesh of the fruit resembles a vibrant red heart. ‘Valentine’ fruits are round to somewhat pyriform (pear-shaped). The average fruit size is large with a mean width of 10.8 cm (4.25 inches) and a height (including the neck) of 11.0 cm (4.33 inches). The mean weight per fruit is 531.1 grams (18.7 ounces). Rind color is medium to dark yellow. The rind texture is moder-ately smooth with a mean thickness of 8.8 mm (0.35 inches). Fruit samples from Lindcove generally have a thicker rind than samples from Riverside. The number of seeds per fruit averages 27.6. However, the mean number of seeds per fruit among 36 different 10-fruit samples ranged from 2.6 seeds per fruit to 51 seeds per fruit. The mean juice weight is 201.8 grams (7.1 ounces) and the average juice content is 38.6%. The red flesh color of ‘Valentine’ can be somewhat variable in its distribution and intensity inside the fruit. Color forma-tion first appears in mid-January and becomes more intense in early to mid February when the solids to acid ratio is an average of 16:1. Please see “’Valentine’, A Recently Released Anthocyanin-pigmented Pummelo Hybrid Developed at UC Riverside” from Topics in Subtropics, 2009, 7(3): 2-4, for a more detailed description of this variety.

Xie Shan Satsuma (VI 621): First distribution of buds from the CCPP: June 2007

‘Xie Shan’ was originally imported from the Institute of Subtropical Crops of Zheijiang Academy in China in 1992. Dr. Fred Gmitter, University of Florida, who was responsible for the collection of this variety, reported Xie Shan to be “extremely early ripening in comparison to other Chinese satsumas”. In a California trial, Dr. Thomas Chao, UC Riv-erside, reported that ‘Xie Shan’ developed high brix levels somewhat earlier than other early Satsuma cultivars tested (‘Armstrong’, ‘Miyagawa’, and ‘Chinese S-9’). However, high acid levels kept the sugar/acid ratio within about the same range as the other cultivars. ‘Xie Shan’ and ‘Miyagawa’ were considered the earliest cultivars in this trial. Additionally, it

was reported that ‘Xie Shan’ was completely seedless, easy peeling, and had a unique taste and flavor. Projected harvest in the San Joaquin Valley was mid-September. (Information from Topics in Subtropics, 2005, 3(2): 3-5). This variety is cur-rently being evaluated by Toni Siebert and Tracy Kahn.

China S-9 Satsuma (VI 636): First distribution of buds from the CCPP: June 2007

‘China S-9’ was part of the first of two different sets of several satsumas collected in the Hubei Province of China, by Dr. H. Huang, Auburn University, in 1995 and given to the CCPP for introduction and quarantine. Each selection in the first set was given the “S” signifier and a number, and was described in a letter from Bill Dozier, Auburn University, to the late Dave Gumpf on August 28, 1995. They were selected from fields in Hubei and Sichuan that had been devastated after a very bad freeze. Each selection was from branches on satsuma trees that had survived the freeze, and the three selection characteristics they looked for were cold hardiness, fruit quality and tree vigor. Huang rated the tree vigor and fruit quality of ‘China S-9’ as ‘acceptable’, and cold hardiness as ‘good’. The second set of satsumas were brought back by Dozier in either 1996 or 1998 after he visited these same areas of China where the freeze occurred. Preliminary observations by Thomas Chao at Santa Paula (see citation in previous de-scription) suggested that ‘China S-9’ should mature early in the San Joaquin Valley (about the same time as ‘Miyagawa’ and ‘Xie Shan’). In addition, preliminary observations at LREC showed that ‘China S-9’ has a smoother peel than most other satsumas. This variety is currently being evaluated by Toni Siebert and Tracy Kahn.

Pehrson #3 Valencia Orange and Pehrson #4 Valencia Orange (VI 749 and VI 750): First distribution of buds from the CCPP: September 2008

These VI’s are two of a group of 8 Valencia clones se-lected by University of California Cooperative Extension Specialist Emeritus John Pehrson. These were included in a Valencia Strain Trial established at LREC. In a letter sent by John Pehrson on April 13, 2009, to the CVC, he described the

Xie Shan satsuma: Citrus Variety Collection, Riverside, California, 27 Nov 2007. Photo by T. Siebert.

China S-9 satsuma: Citrus Variety Collection, Riverside, California, 27 Nov 2007. Photo by T. Siebert.


complex history of the Valencia clones. The objective was to salvage propagative material from high production Valencia trees of good quality fruit because urban growth was shrinking Valencia acreage in Orange County, CA. Some of the strains were reported to be from the Smith Family Ranch and the Wagner Family Ranch in Placentia, CA, and the Gilman Grove, Fullerton, CA. Seedlings were grown from collected fruits and budded onto Troyer citrange. These were planted in coopera-tion with a grower in a Brea area oil lease that would not last another 15 years. The time frame was from the late 1950’s to the mid 1960’s for collection, growing and planting. Shortly after planting in Brea, Pehrson was moved from Orange County to Tulare County, so efforts were made to save these selections. With propagating material from the plot Pehrson had estab-lished with the grower in Brea, CA, Ed Nauer cleaned up 8 of the clones in Riverside, CA, and moved them into a Valencia strain trial at LREC in 1986 where they were compared with Campbell, Cutter, Frost, Olinda, and Chapman Valencias. The 8 Valencia clones were put through the VI index as VI’s 747 to 754 with budwood taken directly from the nucellar block in November 2006. ‘Pehrson Valencia #3’ and ‘Pehrson Valencia #4’ were selected as being the most promising out of the 8 clones. ‘Pehrson Valencia #3’ was selected for having good peel color, minimal regreening, having a good yield, and is later maturing. ‘Pehrson Valencia #4’ was selected as having the highest cumulative yield and weight, and a good distribution of fruit size (information from M.L. Arpaia).

FairchildLS Mandarin (VI 762): First distribution of buds from the CCPP: January 2010

‘FairchildLS’ is an irradiated selection of ‘Fairchild’ mandarin developed at UC Riverside. The distinctive trait of ‘FairchildLS’ is that it is considered to be low seeded (2.4 seeds per fruit) despite any cross-pollination. ‘FairchildLS’ fruit are deeply oblate in shape with no neck. The fruit is medium sized for a mandarin (classed as Large by State of California standards) averaging 64 mm (2.5 in.) in diameter and 56 mm (2.2 in.) in height with a very smooth, deep orange rind color. The rind is relatively thin and at maturity is consid-ered moderately easy to peel. The fruit interior has fine flesh texture with 10-11 segments. The fruit are juicy averaging 47% juice with an average weight of 110g. ‘FairchildLS’ matures in winter (late January) and holds its fruit quality characteris-tics through late March. Fruit from trees on Carrizo citrange rootstocks average 12.1-13.0% soluble solids and 0.85-1.17% acid in January increasing to 13.2-14.5% soluble solids with decreasing acid of 0.76-0.93% by mid-March at four trial locations. Fruit from trees on C-35 citrange rootstocks aver-age 11.2-12.8% soluble solids and 0.94-1.23% acid in January increasing to 13.1-14.0% soluble solids with decreasing acid of 0.78-1.01% by mid-March. ‘FairchildLS’ averages 2.4 + 0.6 seeds per fruit in the presence of cross-pollination at all trial locations throughout California compared to 15-25 seeds per fruit for regular ‘Fairchild’. Pollen of ‘FairchildLS’ has very low viability, therefore it has a very low likelihood of causing seeds in other citrus, particularly mandarins, when planted nearby. Fruit production for ‘FairchildLS’ begins in the third year after planting. Four-year-old trees averaged 62-88 lb, and five year old trees averaged 92-108 lbs. Alternate bearing can be a problem by years seven and eight (information from M.L Roose and T.E. Williams). Patent and/or propagation

rights for Fairchild LS Mandarin are held by the Regents of the University of California. Budwood is available only to nurserymen who have a License Agreement for these cultivars.

Tango Mandarin (VI 765): First distribution of buds from

the CCPP: June 2007Tango is a patented (Plant Patent #17863) irradiated selec-

tion of W. Murcott mandarin developed at UC Riverside. Fruit of ‘Tango’ are similar to W. Murcott in all appearance, quality and production characteristics with the exception of seed numbers. ‘Tango’ fruit are deeply oblate in shape with no neck. The fruit is medium sized for a mandarin (classed as Large by State of California standards and size 28 by industry packing standards) averaging 59 mm (2.32 in.) in diameter and 48 mm (1.89 in.) in height with a very smooth, deep orange rind color. The rind is relatively thin and at maturity is easy to peel. The fruit interior has fine flesh texture with 9-10 segments and a semi-hollow axis of medium size at maturity. The fruit are juicy averaging slightly over 50% juice with an average weight of 90.6 g (3.2 oz.). ‘Tango’ matures in winter (late January) and holds its fruit quality characteristics through April into May. Production is excellent averaging 800-900 cartons/acre when planted at densities of 250-300 trees/acre. Fruit from trees on Carrizo and C-35 citrange rootstocks average 11.1-13.1% soluble solids and 0.97-1.19% acid in January increasing to 13.5-15.4% soluble solids with decreasing acid of 0.54-0.82%

FairchildLS mandarin: Photo by M. Roose.

Tango mandarin: Citrus Variety Collection, Riverside, California, 21 Feb 2007. Photo by D. Karp.


in April. ‘Tango’ averages 0.04 + 0.2 seeds per fruit in the pres-ence of cross-pollination at seven trial locations throughout California compared to 11.6-22.6 seeds per fruit for W. Murcott. Pollen of ‘Tango’ has very low viability consequently it has a very low likelihood of causing seeds in other citrus, particularly mandarins, when planted nearby. Like ‘W. Murcott’, trees of ‘Tango’ have a tendency to overbear and therefore need to be regularly pruned to maintain good, but not excessive produc-tion and to maintain fruit size and prevent alternate bearing. Crop yields should be limited to about 150-170 lbs/tree (6-7 mandarin boxes) through a combination of pruning and, if needed, fruit thinning and should be harvested on time, not left on the trees as this can lead to alternate bearing (information from M.L Roose and T.E. Williams). Patent and/or propaga-tion rights for Tango Mandarin are held by the Regents of the University of California. Budwood is available only to nurserymen who have a License Agreement for these cultivars.

Primosole Mandarin (VI 777): First distribution of buds from the CCPP: September 2009

‘Primosole’ mandarin is a hybrid of ‘Carvalhais’ mandarin and ‘Miho wase’ Satsuma produced at the University of Cata-nia, Sicily, in 1980. It is described as being seedless in isolation and matures very early (at the beginning of October in south-ern Italy or early April in Australia). Coastal Fruitgrowers Newsletter (Australia, August 2005) reported, upon release of ‘Primosole’ from quarantine, that it reached maturity about 10 days before Okitsu satsuma. Fruits are oblate in shape and average 150 grams in weight in southern Italy. The trees are said to be vigorous and productive with an open growth habit. They do not exhibit alternate bearing tendencies. The branches do not have spines, and the lanceolate (long, wider in the middle or lance-shaped) leaves tend to fold as if under water stress. According to an article published in Plant Disease, December 2001, pg. 1291, called “Extreme Susceptibility of Pri-mosole Mandarin to Alternaria Fruit Rot in Italy”, ‘Primosole’ is extremely susceptible to Alternaria fruit rot due to growth cracks at the stylar end (blossom end of fruit) and sensitivity to sunburn. Citrogold reports that this variety has orange rind color, a good to large fruit size, and a good to very good crop load. The peel separates easily. The flavor is said to be between satsuma and mandarin. The variety handles degreening well, stores well, handles cold sterilization well, and has a firm rind in comparison to satsumas. It tends to have a ricyness in the core of the fruit that lessens with tree age and characteristic leaf wilting. ‘Primosole’ is a self-incompatible variety, but will pollinize compatible varieties such as clementines. Cross pol-lination is managed by a buffer of 10 rows. It does not require a plant growth regulator to increase fruit set or size. It is very sensitive to strong winds and can defoliate under windy con-ditions. Swingle is reported to not be suitable as a rootstock for ‘Primosole’ mandarin, but it does well on C-35, Carrizo, and Troyer citranges. (Aznar and Fayos, Citricos. Variedades y tecnicas de cultivo. Mundi-Prensa Libros. 2006. pg 128)

Bitters C-22 Citrange (VI 792), Carpenter C-54 Citrange (VI 793), and Furr C-57 Citrange (VI 794): First distribution of buds from the CCPP: September 2009

According to the report provided by Claire Federici, Ricarda Kupper, and Mikeal Roose, “ ‘Bitters’, ‘Carpenter’ and ‘Furr’ trifoliate hybrids: Three New Citrus Rootstocks”

Bitters C-22, Carpenter C-54, and Furr C-57 citrange rootstocks: Photo by M. Roose.

(information can be found at http://plantbiology.ucr.edu/faculty/new%20citrus%20rootstocks%202009.pdf), all three are hybrids of Citrus sunki and Swingle trifoliate orange. John Carpenter and Joe Furr made all three hybrids as part of the USDA breeding program at Indio, CA. Professor W.P. Bitters tested the hybrids for Citrus Tristeza Virus tolerance in a trial established at the South Coast Research and Exten-sion Center in Irvine, CA, in 1966 and 1968. All three hybrids showed good tolerance to the virus. Details about subsequent field trials, soils, management, and results compared to other varieties can be found at http://plantbiology.ucr.edu/faculty/Summary-of-Active-Rootstock-Trials-5-09v5.pdf

• ‘Bitters’ produces a small tree, with high yield per canopy volume. Young trees on this rootstock showed good tolerance to freezing. Fruit quality of late navels was good and granula-tion was no worse than fruit on ‘Carrizo’ or ‘C-35’. It is toler-ant to CTV, moderately tolerant of Phytophthora parasitica, not very tolerant of citrus nematode, and very tolerant of calcareous soil. It was reported that this rootstock is the best candidate in Texas to replace sour orange due to its tolerance of calcareous soil conditions.

• ‘Carpenter’ produces medium to large trees, with good yield. Young trees on this rootstock showed moderate toler-ance to freezing. Fruit quality of late navels was good and granulation was no worse than fruit on ‘Carrizo’ but was slightly worse than on ‘C-35’. It is tolerant to CTV, moderately tolerant to P. parasitica, very tolerant of citrus nematode, and moderately tolerant of calcereous soil.

• ‘Furr’ produces medium to large trees, with good yield. Young trees on this rootstock showed good tolerance to freez-ing. Fruit quality of late navels was good and granulation was no worse than fruit on ‘Carrizo’ or ‘C-35’. It is tolerant to CTV, very tolerant to P. parasitica, very tolerant of citrus nematode, and moderately tolerant of calcareous soil.

Please visit the Citrus Clonal Protection Program website at http://www.ccpp.ucr.edu for more information about how to obtain budwood of these varieties. Registered users of the online budwood ordering system may visit http://ccpp.ucr.edu/budwood/budwood.php. If you are not a registered user you can e-mail [email protected] with your name, address, e-mail, and phone number or call (951) 684 8580 and the CCPP will generate a username and password for you. After becoming a registered user of the budwood ordering system you will also receive announcements about future budwood distributions for other citrus varieties. l


Citricola scale was a key pest in both southern and central Cali-fornia citrus during the first half

of the 20th century. However, it became scarce in southern California citrus dur-ing the late 1930’s coincident to the in-troduction of the exotic parasitoids for black scale. Furthermore, southern Cal-ifornia’s complex of soft scales includes brown soft scale, which provides year round hosts that support a diverse com-plex of parasitoids. This complex has kept citricola scale to near extinction.

By contrast, citricola scale remains a key pest in the San Joaquin Valley (SJV) where brown soft scale populations are absent. Moreover, citricola scale is developing resistance to Lorsban and some of the newer pesticides. This is of real concern. Oil is the most selective pesticide for control of this scale but it only reduces its density when it is ap-plied 1-2 times annually, and there are concerns that oils will affect yield.

Research Project Final Report

Parasitoid preference for citricola scale in southern California citrus versus San Joaquin Valley citrus:

which one or several species should we produce and release?Robert F. Luck, Joseph G. Morse, and Lisa Forster

Fig. 1. A) Citricola scale from SJV foliage moving onto potted citrus at UC Riverside. B) Successful scale transfer to potted citrus. C) Cages used to protect scale from natural enemies during scale growth and development prior to conducting experiments.

CRB Funded Research Reports 2009

The primary justification for this project is based on the hypothesis that the parasitoids present in southern Cali-fornia, coupled with ant control and skirt pruning, have driven citricola scale pop-ulations nearly extinct. However, these parasitoid species are lacking in the SJV. We have identified the parasitoid complex present in southern California and have determined which parasitoid species prefers or utilizes citricola scale. We are now using this information to develop a sustainable, biologically-based pest management program for citricola scale in the SJV.

To determine the species that para-sitize citricola scale, we brought citrus foliage with citricola scale from the SJV to southern California and inoculated them on potted citrus plants. These scales were then used in laboratory and field experiments to identify which southern California parasitoid species attack citricola scale.

Initially we used large potted plants (15 gal.), but we had great difficulty keeping the plants and citricola scales protected from other pests or con-tamination of predators and parasitoids. When we switched to small, 1-gallon pot-ted citrus, we achieved good inoculations with citricola scale, and we were able to keep the plants and scales clean of other pests or predators.

We made cages to fit over a modified tomato trellis and used closed cell foam around the lower quarter of the pot to prevent parasitoid and predator access (Fig. 1). The plants were maintained in shade and watered as needed. Every week, ca. 2 to 6 plants were cleaned and inspected for contaminates (soft scale parasitoids, mealybug, mites, etc.), and they were resealed in their cages. All pots were set in basins of water to preserve moisture and reduce the likeli-hood of ants invading through the drain holes in the pots. Using this method to

A B C


Fig. 2. Complex of parasitoids from brown soft scale in southern California (A = past survey, B = current survey {with citricola scale}).

Fig. 3 Brown soft scale on yucca in the field and closeup of scale.

grow citricola scale on living plants elim-inated the mealybug and Coccophagus contamination that we had experienced with the larger potted citrus.

The design of our field study in-volved both brown soft scale and citri-cola scale (=out-plants). Through past research, we have identified the complex of parasitoids in southern California that attack brown soft scale (Fig. 2a). Moreover, with this particular project, for every citricola scale- infested plant we put out, we also put out brown soft scale as an index to determine what species attacks brown scale but not citricola scale.

Citricola scale plants were placed in the field in basins with water beneath the tree canopy edge. This avoided excessive scale mortality due to high temperatures. Detached yucca leaves infested with brown soft scale were put three to four feet from the citricola scale (Fig. 3). The citricola scale out-plants (and brown soft scale out-plants) were placed in the field and uncaged (to expose the scale) only when the sizes of both scale species were large enough to support parasitoid offspring. We did not evaluate field predation and host feeding of small scale in this study.

Although there seems to be some size variation associated with where the scale settles on the potted tree (e.g., leaf vein, leaf margin, etc.), citricola scale

has a single generation annually, thus scale size is fairly uniform at any given time of the year. All sets of out-plants (brown soft scale & citricola scale) were left in the field for 14 to 18 days before they were re-caged and returned to the laboratory for subsequent evaluation. We used the same sites for this field experiment that we used for our original parasitoid survey.

The same citricola scale-infested plants were used to study parasitoid behavior in the laboratory. All the be-havior studies were conducted on leaves and stems of plants before the plants were used in our field trials. We evalu-ated four species of Metaphycus (M. luteolus, M. angustifrons, M. nr. flavus

and M. stanleyi) for their preference to attack citricola scale. Metaphycus spe-cies are the main parasitoid complex that controls the soft scale pests.

We conducted 35+ replications for each parasitoid species using the scales on leaves and twigs that we removed from the plant just prior to their use in an experiment. All females were mated and fed honey and had not been previ-ously exposed to live scale prior to the experiment. Each female was given five minutes to oviposit (lay eggs), host feed, or reject the scale.

Our initial expectation was that most if not all of the species in this diverse complex of parasitoids that had been identified in southern California on brown soft scale would also attack citricola scale. This was especially true of Metaphycus angustifrons, as it was the most abundant species that we recovered from brown soft scale in our southern California survey. We recovered very similar proportions of M. angustifrons and M. luteolus from the brown soft scale out-plants that ac-companied each citricola scale infested out-plant. This duplicated our original survey and verified our results in this smaller, second survey (Fig. 2a & b).

The behavioral trials clearly showed that M. angustifrons is not well suited to citricola scale and has great difficulty drilling through the epidermis of the scale. The parasitoid primarily host fed on small scale and killed them (Fig. 4). The average number of eggs laid by M. angustifrons was only 0.2 eggs per scale, and no offspring emerged from these scale. This contrasts with the other three species which ranged from 1.47 to 1.8 eggs per scale and yielded female offspring that had a sex ratio ranging

A B


Fig. 6. Parasitoid preference in the field when given the choice of citricola scale versus brown soft scale.

Fig. 5. Average number of eggs laid in citricola scale, and percent of female offspring produced.

Fig. 4. Laboratory experiments of behavior activity: four different Metaphycus species on citricola scale.

from 50% to 74% females (Fig. 5).The field study confirmed what we

observed in our laboratory behavioral experiments. Although we did not re-cover M. nr. flavus or M. stanleyi from our out-plants, M. luteolus dominated the parasitoid species emerging from our citricola scale out-plants, even when M. angustifrons was clearly present and attacking the brown soft scale just 3 feet from the citricola scale (Fig. 6). Evident from this field experiment is the impor-tance of using the correct parasitoid to release against a target pest. Moreover, although Metaphycus species look very similar taxonomically, they each behave quite differently when they parasitize a scale.

In our previous field studies, we detected substantial Coccophagus lycimnia parasitism on the very young scale, scale too small for Metaphycus spp. This suggests that Coccophagus may compliment Metaphycus in sup-pressing citricola scale. This observation in our earlier trials is confirmed in this study. Coccophagus’ representation in the parasitoid complex emerging from citricola scale is only 3%. In brown soft scale it remains below 30%. Moreover, the concern that male Coccophagus hyperparasitoids may interfere with Metaphycus species control of citricola scales seems unlikely.

Dr. Paul Rugman-Jones (UCR - Dr. Richard Stouthamer Laboratory) developed a species-specific identifi-cation tool for both Metaphycus spp. and Coccophagus sp. which is based on differences between each species’ DNA sequences (PCR). He has con-firmed the identity of all of our field specimens. This was critical for this system, as the taxonomy of these para-sitoids (Metaphycus and Coccophagus) historically have been plagued with misidentifications. After reviewing the specimens from our last survey, all of the species identifications have been confirmed genetically and are now be-ing identified taxonomically by a spe-cialist in Metaphycus. Only specimens identified as M. helvolus failed to match the DNA sequence; thus, it is a differ-ent species. Our original concern with the brown soft scale survey was that M. helvolus would not attack brown soft scale, which seems to be the case. We identified one additional Metaphycus species using Dr. Rugman-Jones’ pro-tocols, and we are getting it identified

(e.g., named). We have also identified all four species of Coccophagus (C. lycimnia, C. cowperi, C. rusti, and C. scutellaris) using these molecular tech-niques. The other benefit arising from Dr. Rugman-Jones’ technique is that it gives very rapid results.

Results from this project funded by the Citrus Research Board have allowed us to better understand the parasitoid species complexes attacking soft scale pests in citrus. Clearly it is a very com-plicated system, but the information that we have gained will now allow us

to exploit these parasitoids with refer-ence to their preferences for particular scale species. We are currently seeking USDA funding for a field project to test the proof-of-concept for a sustainable management program for citricola scale in SJVcitrus that we have developed as a result of this CRB project.

Project Leaders Robert F. Luck, Ph.D., and Joseph G. Morse, Ph.D., are Professors of Entomology, University of California Riverside. Lisa Forster is Staff Research Associate, Department of Entomology, UC Riverside. l




Fig. 1. A-B) Laser ablation of nitrocellulose laminated to a polycarbonate substrate can be used to generate fluid flow modulating structures in a planar format. C) Buffer exchange mediating geometries cut in absorbent materials are incorporated into a low-cost plastic housing to provide an easily used sample preparation system.

Research and development at Mesa Tech Internation-al, supported in part by the Citrus Research Board (CRB), is aimed at the simplification of sensitive and

reliable testing for citrus diseases.Currently, molecular tests for devastating citrus pathogens

require sophisticated laboratories and highly trained person-nel resulting in high costs and long turnaround times. Mesa Tech’s approach, however, will provide the same reliability and sensitivity as existing laboratory-based tests in completely self-contained disposable cartridges similar in complexity to the test strips long employed for home pregnancy tests.

Unlike existing test strip approaches based upon antibody binding, known as immunoassays, Mesa’s technology employs far more sensitive and specific nucleic acid-based molecular assays. The ease of use and low cost of Mesa Tech’s approach to nucleic acid-based assays will empower growers with the technology they need to accurately monitor their orchards for such diseases as Huanglongbing (HLB). By placing rapid and reliable tests directly in the hands of growers and regulatory agencies, rapid identification of new and emerging diseases, as well as tracking their movement through ports of entry, will be greatly facilitated.

Although nucleic acid-based assays for pathogen detection and identification offer sensitivity, specificity and resolution, they continue to be relatively elaborate and often costly, limit-ing their utility for deployment under field conditions where

Novel immunocapture technology for field deployable nucleic acid-based detection of plant pathogens

R. Bruce Cary

a supporting laboratory infrastructure is limited or absent. One of the most challenging hurdles to moving nucleic

acid testing from the laboratory to the field has been the extraction of sufficiently pure nucleic acid molecules from complex samples such as leaf and other plant tissues. The biochemical processes upon which DNA and RNA testing hinges are adversely impacted by the remnants of tissue and other contaminants present in crude samples, typically neces-sitating lengthy laboratory processing to obtain highly purified DNA or RNA suitable for test procedures.

With CRB support, Mesa Tech has developed and con-ducted pilot field tests of an exciting new approach to nucleic acid purification. Unlike laboratory methods, Mesa Tech’s approach eliminates the need for instrumentation and user expertise, allowing anyone to isolate readily testable nucleic acids from field-collected plant tissue samples. This break-through technology is now being incorporated into an inex-pensive and disposable handheld unit that integrates nucleic acid purification, amplification and detection within the device without additional user manipulation.

To develop simple nucleic acid sample preparation tech-nologies, three initial project objectives were established. These objectives can be summarized as: 1). Develop buffer solutions capable of disrupting bacterial pathogen cells or virus particles while simultaneously stabilizing their DNA and RNA for isolation and later amplification and detec-


Fig. 2. A lateral flow microarray (LFM) was used to detect amplified CTV RNA from citrus leaf nucleic acids isolated using Mesa Tech’s sample preparation system. Field collected leaf samples were processed at the ranch, and RNA targets indicative of CTV infection were detected on site. Panel (A) is a schematic legend of the LFM layout. Positive controls confirm proper test performance and provide positional markers (green). Negative controls confirm assay specificity (black). The location of diagnostic probes for HLB (Candidatus Liberibacter spp.) and CTV are indicated in beige and red respectively. (B) One of 20 trees examined was negative for CTV by both the Mesa Tech sample preparation method and Qiagen RNeasy, a laboratory-based approach. (C) A representative CTV positive LFM reveals strongly positive spots (blue) at corresponding CTV probe locations on the strip.

tion; 2). Design and build an inexpensive and easily used disposable device to accomplish nucleic acid binding from solutions of disrupted plant tissue; 3). Devise methods and wash solutions to efficiently wash bound nucleic acids to free them of deleterious cell and sample debris; and, 4). Demon-strate that isolated nucleic acids are suitable for subsequent amplification and detection.

To accomplish these goals, Mesa Tech developed a com-pact disposable device and associated system of optimized buffer solutions to isolate nucleic acids from citrus leaf tis-sue. Fig. 1 A-C depicts the fabrication of a key component of the prototype device that controls the sequence and rate of solution movement (patent applied for). This technology allows sample and wash buffers to be added to the device at the same time, yet exhibit optimized flow for sample prepara-tion without user intervention, pumps, valves or other moving parts. This component is incorporated into a plastic housing, shown in Fig. 1C. The user simply adds the sample and two wash buffer solutions to the device; approximately 15 min-utes later, purified nucleic acids are ready for amplification and detection.

To test the ability of the device to yield useful RNA for the detection of Citrus tristeza virus (CTV), Mesa Tech conducted a pilot field study at the citrus ranch of Earl Rutz in Pauma Valley, California. These studies tested 20 randomly selected trees for CTV using both Mesa Tech’s sample preparation system and a widely accepted laboratory-based method (RNeasy, Qiagen, Inc.). Comparisons of Mesa Tech’s methods with traditional laboratory sample preparation approaches resulted in perfect agreement between these two disparate methods.

To accomplish RNA amplification, this study employed a technique that amplifies RNA molecules through a biochemi-cal reaction that can be conducted in a single tube at a single temperature. The single temperature nature of this approach allows target RNA sequences, in this case sequences indicative of the presence of CTV, to be exponentially amplified without the complex instrumentation typically associated with such procedures as polymerase chain reaction (PCR).

Detection of amplified CTV RNA sequences was ac-complished using lateral flow microarrays, a simple yet rapid and sensitive method developed by one of Mesa Tech’s co-founders (the scientific publication describing this method is available for download at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904290/ ). Fig. 2 illustrates the lateral flow microarray detection strips used in this study.

Despite the simplicity and excellent performance char-acteristics of Mesa Tech’s sample processing, amplification and detection methods, these technologies currently exist as independent steps of an analytical procedure. Although our prior work has resulted in adaptations of each requisite pro-cess that are amenable to field deployment, the manipulations required, though simple, remain reliant upon user interactions during the test procedure.

A truly easy to use field assay for nucleic acids will require a level of subsystem integration not currently available. How-ever, the methods we have developed were devised with the longer-term vision of integration into a low-cost disposable device suitable for use under field conditions with little or no end user training. By developing simplified methods for each stage of assay performance, Mesa Tech has greatly facilitated

the ultimate integration of existing component technologies into an easily used sample-to-result diagnostic tool. Indeed, Mesa Tech is now engaged in a significant follow-on effort, supported by the CRB and the USDA, to develop a low-cost and easily used disposable device that integrates all steps of molecular diagnostic testing from sample to result. This system will enable users with no molecular testing expertise to obtain sensitive and specific results without the time and costs typically associated with the use of external laboratory testing.

Project Leader R. Bruce Cary, Ph.D., is Co-founder and Vice President, Mesa Tech International Inc., Santa Fe, New Mexico. l

Note from: MaryLou Polek, CRB Vice President of Science & Technology

The Citrograph will now serve to communicate progress on Citrus Research Board funded projects. This is in lieu of a separate CRB Annual Report as published in previ-ous years. Each issue of the magazine will feature one or two project completion reports and several progress re-ports. I hope you find this format useful and informative.


Project introduction and significanceThe phloem-limited and insect

vector-transmitted citrus stubborn disease, caused by the bacterial patho-gen Spiroplasma citri, has resulted in constant/consistent yield losses and quality reductions to the citrus industry. Depending on the varieties, the reduc-tion on the yields from this disease can be up to 100%.

The currently available diagnostic methods are either labor-intensive or inaccurate due to the uneven distribu-tion, seasonal fluctuation, and the low titer of S. citri in the infected trees. So far, there are no robust, economic and reliable detection tools for citrus stub-born disease, especially for certifica-tion programs. This gap in the current diagnostic capacity for citrus stubborn disease greatly impacts the fundamen-tal control method for the disease, i.e., the use of disease-tested propagative material.

The main goal of this project is to develop a novel serological diagnostic method for citrus stubborn disease by detecting S. citri proteins that are secreted from the pathogen into citrus phloem. Suitable protein markers will be identified from S. citri and then used to generate antibodies for detecting the stubborn disease in citrus trees.

Since secreted proteins are not restricted to the infection sites and can be relatively abundant, they can be used as detection markers which will enable rapid, reliable and cost-efficient detection of citrus stubborn disease. The development of such a novel immunoassay-based diagnostic tool is particularly important for large-scale testing in certification programs, such as the Citrus Clonal Protection Program (CCPP), which provides clean citrus propagative materials to growers, breed-ers, and researchers worldwide. There-fore, this project holds the potential to

Fig. 1. Citrus Stubborn Disease-Leaf and Fruit Symptoms. A) Leaf mottling, B) Lopsided fruit with stylar end greening or color inversion of maturation and, C) Lopsided fruit longitudinal and cross section with aborted seeds.

Progress Report

Identification of Spiroplasma citri secreted proteins as detection markers for citrus stubborn disease

Wenbo Ma and Georgios Vidalakis


significantly benefit the citrus industry not only in California, but also in other citrus-growing areas worldwide.

Thus far, no secreted proteins have been identified from S. citri or any other spiroplasmas. In order to identify suitable secreted proteins as detection markers, we will first carry out a systemic analysis on the secretome (complete repertoire of the secreted proteins) of S. citri. Secreted proteins play an essential role in pathogen infection and disease development.

In addition to directly providing a

robust detection tool for citrus stub-born disease, this project, as the first systemic analysis on the secretome of spiroplasmas, will also fill a gap in our basic understanding on the molecular basis of bacterial pathogenicity. Phloem-limited and insect-transmitted bacte-rial diseases, including citrus stubborn disease and Huanglongbing (HLB) are

among the most devastating citrus dis-eases nowadays. The knowledge on how phloem-limited and insect-transmitted bacterial pathogens cause widespread and severe diseases in citrus is urgently required in order to develop sustainable disease control strategies.

The extensive similarity on the host responses, disease symptoms and pathogen life styles between citrus stubborn disease and HLB lead to the hypothesis that similar virulence fac-tors or strategies might be used by the bacterial pathogens to cause disease in citrus. Therefore, novel knowledge obtained from this project, including the secreted proteins, protein markers, ge-nome sequences and detection tools will provide guidance to the development of detection strategies for HLB. This is especially important because the causal agent of HLB is very difficult to work with at this time. Moreover, since HLB research is not possible at this point in California due to its exotic status, this project is California’s best option to study the genes that might be involved in the pathogenicity of the causal agent of HLB or other ecologically related pathogens such as phytoplasmas.

Progress during November 2008 – October 2009

This project has been funded by the Citrus Research Board since November 2008. During the year of November 2008 through October 2009, we have made significant progress on the identification and systemic analysis of the secretome of S. citri using modern high throughput approaches. Two distinct but comple-mentary approaches are employed: 1) a comparative proteomic approach using mass spectrometry, from which the secreted proteins in the cell-free supernatant of S. citri cell culture are identified; 2) a genomic approach using the next generation genome sequencing

A.

B.

C.


and bioinformatic analyses. Proteins secreted from S. citri cells

in liquid culture grown in the artificial media have been identified using mass spectrometry. We have established and optimized the bacterial culturing proto-cols and the induction conditions using phloem extracts from citrus. We have tried several liquid media and different induction conditions, and have decided to induce the S. citri cells in C3G medium at room temperature for 24 hours. This short induction time allows us to iden-tify early-induced proteins, which are useful for early disease detection. After 24 hours of induction at room tempera-ture, the bacterial cells were removed from the cell culture by centrifugation. Total proteins were extracted from the cell-free supernatant and then ana-lyzed using mass spectrometry – these proteins should be secreted from the S. citri cells. By comparing the secreted protein complements of S. citri cell cul-tures with and without the induction of phloem extracts, we have identified proteins that are highly induced by the phloem extract. Among these proteins, two or three candidates with relatively high abundance are targeted for fur-ther examination as detection markers. The secreted protein profiles were also compared to that of the phloem extract to make sure that we are not looking at any plant proteins.

The protein candidates identified from mass spectrometry are also sub-

jected to bioinformatics analysis to predict the presence of the N-terminal secretion signals. S. citri belongs to the gram-positive bacteria, in which the ma-jor protein secretion system is called the Sec system. Proteins secreted through the Sec system possess a sequence motif at the N-termini which lead the secretion of these proteins. In other words, the presence of the N-terminal secretion signal is a strong hint that the protein is secreted.

Thus far, we have repeated the experiments several times and came up with a short list of secreted protein candidates for further analysis. Some of these candidates are listed in Table 1. These proteins are all highly induced in the presence of citrus phloem extracts. Moreover, all of them are predicted to have a potential N-terminal secretion signal. Therefore, these proteins could be used as detection markers.

Another significant point of progress

we have made during the first year of the grant is that we have successfully established the mechanical transmission of S. citri from pure culture into pine-apple sweet orange (Citrus sinensis var Pineapple) seedlings. Four weeks post inoculation we could detect the systemic infection using PCR. Once we confirm the secretion of some of the candidate proteins, antibodies will be generated to examine the presence of the proteins in citrus. The mechanical transmission of S. citri in citrus is very important to confirm the secretion and evaluate the candidate detection markers.

Project Leader Wenbo Ma, Ph,D., is Assistant Professor, Department of Plant Pathology and Microbiology, University of California Riverside. Co-Project Leader Georgios Vidalakis, Ph.D., is UC Cooperative Extension Specialist and Plant Pathologist, UC Riverside, and Director of the Citrus Clonal Protection Program (CCPP). l

CAK99227 chromosome 0.998 5 1





Table 1. Candidate secreted proteins of Spiroplasma citri identified from mass spectrometry.

# of peptide hits using Mass SpectrometryTrial 1 Trial 2Gene Name Location

Probability of N-terminal secretion signal

California and Florida are at con-siderable risk for the establish-ment of the Mediterranean fruit

fly (MFF) due to their favorable climate and host availability. Any Medfly inva-sion in these or neighboring states trig-gers a sobering reality. Colonization of MFF would result in devastating losses of crops; personal, state, and national income; jobs; and global trade.

We can never afford a Medfly in-vasion, and during “times like these”


Probiotic diet for SIT Medfly


Carol R. Lauzon

management of an infestation could prove ruinous. In light of this, we should be hyper-critical of our existing pest management strategies to safeguard our high-valued products and economy and continue to work toward improv-ing existing management programs and strategies.

Radiation is used to produce adult males that are reproductively sterile. These irradiated males are subsequent-ly introduced into susceptible regions,

Photo by Jack Kelly Clark, University of Cali-fornia, Agriculture and Natural Resources.


Intact DNA (smear) from non-irradiated male Medflies seen in Lanes 1 and 3. Lane 4 shows a DNA ladder. Lanes 5 and 7 contain fragmented DNA (bands) from irradiated male Medflies. DNA fragmentation is characteristic of apoptosis, or programmed cell death.

DNA from irradiated males that fed on a standard diet (white arrow). Notice the bands, or fragments of DNA. DNA from males that fed on flagellin (black arrow). Equal amounts of DNA were used. Thus, there appears to be less fragmentation of DNA for males that consumed flagellar protein.

N

Transmission electron micrograph of a sterile male that fed on flagellar protein. Notice the mitochondria in the act of dividing (arrow) and a nucleus (N) dense with nuclear material. There is no visual indication of apoptosis.

i.e. the Los Angeles Basin, with the in-tent to interrupt the reproductive cycle of any female Medfly that may have invaded the area. This strategy, know as Sterile Insect Technique (SIT) is part of a Preventative Release Program (PRP), and while both SIT and the PRP have proven beneficial for control of Medfly infestations, there are areas within each that can be improved and/or optimized, such as SIT male fitness.

In recent years, we have shown that radiation used in SIT damages the gut of sterile males. This damage likely compromises the performance of sterile males because improvements in sterile male mating performance have been observed after these males con-sumed beneficial gut bacteria (Niyazi et al. 2004).

The bacteria used in those diets, Pantoea agglomerans (formerly, En-terobacter agglomerans) and Klebsi-ella pneumoniae, have been shown to be vertically transmitted, that is, passed from female to her eggs and to emerging adults (Lauzon et al. 2009).

Information regarding the relation-ships between pest fruit flies and these bacteria is dovetailing (i.e. Lauzon 2004). There really is no question now that these bacteria exert positive influ-ences on Medfly physiology, but there are several questions regarding how these bacteria do what they do.

Our research was directed toward a more complete understanding of how bacteria can be used to improve the fitness of SIT Medflies. It utilized new ways of assessing the degree of nontar-get radiation damage (fitness costs) in SIT Medflies. The aim of the work is to find a practical way to facilitate gut repair and/or protect the Medfly gut from radiation damage by enlisting the bacteria themselves or using some microbial product to improve the fit-ness of SIT males. The goal of the work is to strengthen the Preventative Re-lease Program and protect California more soundly from a Medfly invasion. The intent of this short term one-year project was also to determine if suf-ficient evidence could be garnered to

support a larger research plan.In earlier work, our laboratory

found that cells within the midgut of SIT males were apoptotic. Apoptosis, also known as programmed cell death, is a common cellular phenomenon that occurs, within insects for example, to delete unwanted cells, whether they are cells irreversibly damaged, tumor cells, or superfluous (Dorstyn et al. 2002). This process is highly controlled and self-inflicted and in the case of SIT males is due to radiation expo-sure. Visual signs of apoptosis include cell organelles, such as mitochondria, that appear aberrant in shape. Other evidence includes DNA in fragments. Apoptosis can be determined for cells by looking for DNA fragmentation, measuring protein and enzyme levels, and inspecting the cells using electron microscopy, for example.

We have observed that the degree of apoptosis is lessened in SIT males after they ingest the beneficial gut bacterium, P. agglomerans. Bacterial flagella, structures that allow bacteria


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The graph shows the means of Caspase 3 levels in pools of: 1 = nonirradiated factory males; 2 = sterile males on standard diet; 3 = sterile males on standard diet plus flagellar protein. Y axis = absorbance at 405 nm

to swim or move, have been reported to inhibit apoptosis as part of preserving a host cell for pathogen invasion. P. ag-glomerans also possess flagella. Thus, we hypothesized that the flagella of P. ag-glomerans were influencing the amount of apoptosis in irradiated gut cells.

If flagella are the responsible entity for decreasing the amount of apoptosis in the SIT midgut, and thus improving the gut condition, then an important question is: at what point in the apop-totic process are the bacteria inhibiting reactions? Based on earlier work in our laboratory, and part of this research project, we hypothesized that levels of Cytochrome C and Cytochrome C Oxidase from mitochondria or Caspase 3 levels in cell cytoplasm are reduced in SIT males that ingest flagella. DNA fragmentation should be also lessened after ingestion of flagella because those enzymes indirectly affect the integrity of DNA.

Methods: Briefly, fragella from and whole cells of living Pantoea agglo-merans and in the form of an extract were provisioned to irradiated and nonirradiated Medflies through their diet. Similarly, commercially prepared flagellin, the flagellar protein, was also provisioned to the adult flies. After two days, flies were sacrificed and were examined for the amount of DNA fragmentation of their midgut cells, and levels of Cytochrome C Oxidase , Cytochrome C, and Caspase 3, all pro-teins associated with apoptosis. Midgut epithelial cells were also scrutinized visually using transmission electron microscopy (TEM).

Results: Fragmentation of DNA ap-peared less in sterile males that fed on flagellar proteins than sterile males that fed on standard diet lacking flagella or flagellin. Micrographs of irradiated male medflies that fed on preparations containing whole cells of Pantoea ag-glomerans, flagellar extract from P. ag-glomerans, or commercial flagellin did not show evidence of apoptosis. Mito-chondria were captured in the process of dividing, suggestive of normal cell activity. Nuclei were densely stained with nuclear material.

Caspase 3 enzymatic levels were assayed for both sterile and non-irradi-ated factory medflies on diets with and without flagellar protein. Flagellar pro-tein decreased the amount of Caspase 3 activity within irradiated flies, a key

enzyme that is involved in apoptosis.We compared Cytochrome c oxi-

dase levels for sterile and non-irradi-ated factory Medflies using quantita-tive PCR (qPCR) and found Cycle threshold (Ct) levels to differ by as much as 2 points (sterile medflies avg. Ct value, 16.076 vs. nonirradiated avg. Ct value, 18.171). Therefore, the sterile males demonstrated an increase in gene expression for Cytochrome c oxidase beyond that seen in the nonirradiated flies, indicative of apoptosis. Although we found differences that serve as our foundation for the study, we are repeat-ing our experiments currently with the advent of recent technical improve-ments in the assays.

In conclusion, we have found that whole cells of Pantoea agglomerans, flagellar extracts of P. agglomerans and commercially prepared flagellin all improve the cellular status of irradiated male Medflies (not all data shown). Fla-gellar extracts and flagellin improved cellular organelles.

One reason why a probiotic diet has not yet been put into mass rearing is the perceived difficulty in adding a living component to the diet. As our research continues, if flagellar proteins are determined to produce the same ef-fect on sterile males that living bacteria do, and our results suggest they do, then this concern can be eliminated.

While we still consider our data preliminary at this point, we are cau-tiously optimistic that we are mak-ing gains toward understanding the mechanism behind radiation damage improvements for fruit flies that ingest a probiotic diet and that further work is merited. A fuller understanding of

this dynamic should lead to practical improvements in sterile male fitness and strengthening of the Preventative Release Program.

Acknowledgements: I gratefully acknowledge the contributions of CSUEB graduate students Jacque-line Louie and Sima Bahadori; Dr. Susan McCombs of USDA- APHIS, Waimanalo, HI; and Tina Carvalho of the University of Hawaii at Manoa, Honolulu, HI. This research was made possible through the generous support of the Citrus Research Board.

Project Leader Carol Lauzon, Ph.D., is Professor, Department of Biological Sciences, California State University East Bay.

References:Dorstyn, L., Read, S., Cakouros, D.,

Huh, J.R., Hay, B.R., and S. Kumar. 2002. The role of cytochrome c in caspase activation in Drosophila melanogaster cells. J. Cell Biol. 156(6):1089-1098.

Lauzon, C.R. Symbionts of Teph-ritids. In: Insect Symbioses, A CRC publication. Bourtzis and Miller, eds. Feb. 2003. p. 115-130.

Lauzon, C.R., S.D.McCombs and S.E. Potter. 2009 Vertical passage of Enterobacter agglomerans and Kleb-siella pneumoniae in Ceratitis capitata Weidemann, the Mediterranean fruit fly. Ann. Entomol. Soc. Amer. 102(1): 85-95.

Niyazi, N., C.R. Lauzon, and T.E. Shelly. 2004. Effect of probiotic adult diets on fitness components of sterile male Mediterranean fruit flies (Diptera: Tephritidae) under laboratory and field conditions. J. Econ. Entomol. 97: 1570-1580. l

citrograph march / april 2010

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