Editor s Note: This is Part 1 of a

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1 Prevention of Introduction and Distribution of Citrus Diseases Part 1 Citrus quarantine and introduction programs Georgios Vidalakis 1, John V. da Graça 2, Wayne N. Dixon 3, Donald Ferrin 4, Michael Kesinger 5, Robert R. Krueger 6, Richard F. Lee 6, Michael J. Melzer 7, John Olive 8, MaryLou Polek 9, Peggy J. Sieburth 5, Lisa L. Williams 10, and Glenn C. Wright 11 Editor s te: This is Part 1 of a two-part article discussing the major USA citrus germplasm programs and other national efforts to harmonize the protocols for citrus germplasm movement. This first part describes the different citrus quarantineintroduction programs of the USA. Part 2, which is scheduled for the next issue, will describe the different certification schemes as well as national efforts and programs for citrus propagative material. Background Citrus germplasm originated in Australasia, the Far East, and Africa; thus all citrus grown in the New World was imported. This importation of citrus also resulted in importation of grafttransmissible pathogens of citrus capable of causing destructive epidemics such as the tristeza quick decline that killed more than 60 million trees worldwide. One of the main reasons for the citrus industry s survival and prosperity through the decades is the early establishment of citrus germplasm programs, such as California s Citrus Clonal Protection Program, that provide a safe mechanism of introduction of new varieties via disease testing and therapy and distribution of high-quality diseasetested citrus propagative materials. Introduction Citrus germplasm has moved from its geographic origin in the Australasia (Pangea, c. 20 Ma), Far East, and Africa, May/June 2010 bringing with it graft-transmissible diseases to all the citrus growing areas of the world. In spite of the plethora of such diseases, the citrus industry has survived through the decades and prospered in many countries including the USA. This is a direct result of the establishment of citrus germplasm programs that provide a safe mechanism of introduction of new varieties via disease testing and therapy and that distribute high-quality disease-tested propagative material to the industry. The basic protocols for the detection and elimination of graft-transmissible pathogens of citrus are well defined and have been known for many decades. Similarly, the budwood distribution mechanisms with disease re-tested mother trees, increase blocks, and fruit evaluation for horticultural truenessto-type are also well defined and in use in many citricultural countries around the world. In the USA, the main citrus producing states are California, Arizona, Texas and Florida. Even though the protocols and technology for the citrus germplasm programs are common across the board, each of these states has developed and adjusted their quarantine-introduction and budwood distribution-certification programs to the needs (size, type, available resources, involved agencies, disease or pest pressures etc) of their specific industries. In California, the Citrus Clonal Protection Program handles the majority of quarantine-introduction and budwood distribution-certifications. In Florida, two different programs the Citrus Germplasm Introduction Program and the Bureau of Citrus Budwood Registration handle the quarantine-introduction and budwood distribution-certification, respectively. Arizona and Texas do not have a quarantine-introduction program. They acquire pathogen tested citrus budwood from California and Florida, and they maintain, re-test and distribute budwood to their industries via distribution-certification programs, namely the Arizona Certified Budwood Program and the Texas Citrus Budwood Certification Program. Overall, the different USA programs presented here involve multiple agencies or entities (university, state and federal government, and industry) in order to have the necessary funds, scientific knowledge, infrastructure, and regulatory basis for successful operation. In the United States, the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), while based in California, actually serves the entire USA and distributes materials free of charge to qualified scientists and certification programs around the globe. The NCGRCD is not focused only on citrus varieties with commercial interest, like most of the state operating programs, but it also preserves, in general, the germplasm of citrus and citrus relatives. The control of graft-transmissible

2 diseases of citrus is based primarily on preventative measures. The most important one is the use of healthy propagating material through strict quarantine and the implementation of in-country certification schemes for the provision of adequate supplies of disease-tested, trueto-type citrus germplasm. In the USA, such programs started approximately 60 years ago in the main citrus-producing states. Today, California, Arizona, Texas, Louisiana, Alabama, Florida and Hawaii have developed, or are in the process of developing, different citrus quarantineintroduction and budwood-certification programs adjusted to the needs of their citrus industries based on the type (e.g., fresh fruit, juice, ornamental), size, available resources, growing conditions, disease and pest pressures, and participants or collaborators (state, federal, university, private etc.). California The California Citrus Clonal Protection Program (CCPP) has its roots in the 1930s when the original discovery of the viral nature of the graft-transmissible disease citrus psorosis by Dr. H. Fawcett at the University of California Citrus Experiment Station in Riverside triggered the establishment of the Psorosis Free Program. The CCPP was officially established in 1956 as the Citrus Variety Improvement Program (CVIP) after the request of the citrus industry to the University of California. In 1977, the CVIP was restructured and renamed to CCPP and today stands as a cooperative program between the University of California Riverside (UCR, Department of Plant Pathology & Microbiology), the California Department of Food and Agriculture (CDFA), the United States Department of Agriculture s Animal and Plant Health Inspection Service (USDA-APHIS), and the citrus industry of the state of California represented by the California Citrus Nursery Board (CCNB) and the Citrus Research Board (CRB). The CCPP has a staff director, three staff research associates, and one to three nursery technicians and operates at three locations: the Rubidoux Quarantine Facility in downtown Riverside, the Citrus Diagnostic Laboratory on the UCR campus, and the Foundation and Evaluation Blocks at the Lindcove Research and Extension Center (LREC), Exeter, CA. The CCPP is supported almost exclusively by the CRB (a grower s box tax research marketing order founded in 1968) while UCR offers infrastructural support and scientific expertise. The CCNB contributes to the CCPP operations via the program of Cooperative Registration Testing of Nursery Owned Citrus Scion and Seed Source Trees. A committee of approximately 12 industry members (growers and nurserymen) supports the CCPP activities. The purpose of the CCPP is to provide a safe mechanism for the introduction into California of citrus varieties from any citrus-growing area of the world for research, variety improvement, or for use by the commercial industry of the state. The introduction mechanism includes disease diagnosis and pathogen elimination followed by maintenance and distribution of true-to-type primary citrus propagative material of the commercially important scion and rootstock varieties. We will describe these functions in detail only for the CCPP since in principal they are used in all the other programs described here. A. Introduction of citrus varieties under quarantine The citrus quarantine in California is a cooperative venture involving federal, state, and county departments of agriculture and the University of California (UC). The federal government represented by the USDA-APHIS-PPQ (Plant Protection and Quarantine) is concerned with citrus pest exclusion from foreign sources entering the U.S. There are also instances where federal quarantines exist between states, which likewise govern the movement of citrus. The director of the CCPP has a USDA-APHIS-PPQ issued permit to import citrus budwood from foreign countries (one of the only three permits for citrus introduction into the USA). There are specific stipulations spelled out on this permit regarding handling and treatment of materials in quarantine which must be followed when citrus material enters the Riverside CCPP Quarantine Facilities at Rubidoux. These stipulations are also required by the State of California and are enforced by the CDFA. The State of California Agricultural Code (Cal. Admin. Code tit. 3, 3250 Citrus pests exterior quarantine) clearly states that all citrus budwood is prohibited from entry into California unless authorized under a CDFA and/ or USDA permit such as the one issued to the CCPP director. All citrus germplasm entering the USA first goes through the federal quarantine facility in Beltsville, Maryland. Upon arrival, the budwood is visually inspected for the presence of insect, fungal Fig.1. The Citrus Clonal Protection Program quarantine facilities at the University of California Riverside. Glasshouse for the biological indexing of graft-transmissible diseases of citrus (A), screenhouse for the maintance of the positive control collection and the production of field trees (B), laboratory for diagnostics and the therapeutic protocols (C), the interior of the screenhouse with field trees (D), and the interior of the glasshouse with different developmental stages of citrus bio-indicators (E). May/June 2010

3 Fig. 2. Procedure and timeline for the introduction of new varieties (including proprietary varieties) into California via the Citrus Clonal Protection Program (CCPP). May/June 2010

4 pests or other potentially bio-hazardous materials (e.g. soil). It is then sent to the CCPP Rubidoux Quarantine Facility in Riverside. The Rubidoux Facility consists of approximately 5,000 sq. ft. (~450 m 2 ) of insect-proof greenhouse with temperature and light controls that are required for biological indexing, 9,000 sq. ft. (~850 m 2 ) of screenhouse, and a modular office and laboratory area. The Rubidoux Quarantine Facility is located within the city of Riverside adjacent to the original location of the Citrus Experiment Station (1907) and isolated from the nearest commercial citrus and University experimental orchards by about 3 miles (~5 km) (Fig. 1). The CCPP quarantine and campus laboratory facilities are part of the space allocated by the Department of Plant Pathology & Microbiology of the College of Natural and Agricultural Sciences in the University of California Riverside. Typically, when a new import is received by CCPP at the Rubidoux Quarantine Facility, propagations are made onto rough lemon rootstocks to preserve the budline and to produce budwood for future index and/or therapy. These propagations are placed in a warm greenhouse for maximum and rapid growth. In addition, some of the imported budwood is cultured in vitro and the meristems of the produced young flushes are used for in vitro micro-propagations following the shoot-tip-micrografting procedure. The remaining portion of the original imported budwood is used to graft inoculate indicator seedlings in a screening index called a pre-index. Disease symptoms in the pre-index plant indicators will indicate if the import budline is infected with tristeza virus, vein enation virus, psorosis and psorosisrelated pathogens or citrus viroids (Fig. 2, 1A & B). A very high percentage of newly imported budwood arrives infected with one or more of these budtransmissible diseases. B. Disease diagnosis - Pathogen detection The CCPP program of importation, production and distribution of diseasefree propagative materials is based on a comprehensive indexing, or testing, program to detect graft-transmissible diseases, which may arrive in an imported budline. Graft-transmissible diseases may be caused by viruses, viroids or other pathogens (bacteria, phytoplasmas) and are vegetatively transmitted with an infected budline. Graft-transmissible diseases can seriously affect fruit quality, production, tree health and longevity. Additionally, diseases from infected field propagations may be spread to neighboring orchards of healthy trees by insects or farming equipment. Detection of graft-transmissible diseases of citrus is based primarily upon biological indexing, which is accomplished by grafting tissue of the imported budline onto citrus indicator seedlings. Specific indicator seedlings are used to detect specific diseases. Indicator varieties have been selected for sensitivity to diseases and ability to express symptoms. In each index, adequate positive controls, or disease-infected seedlings along with healthy control seedlings of each indicator variety, are held under the same environmental conditions as test seedlings. Controls are used as a comparison with the test source and also as confirmation Fig. 3. Biological indexing of graft-transmissible diseases of citrus. Young growth of plant indicator (approx. 4-6 weeks post inoculation) will be observed for months for symptom development (A), graft inoculation of plant indicator (B). T-cut and budwood chip (inoculum) insertion (B i), wrapping with budding tape (B ii), and healing with alive inoculum, 3-4 weeks later (B iii). Reactions of citrus bio-indicators on wood (C), branches (D), and leaves (E). (C) Wood of healthy citrus (C i), gumming and wood coloration reaction of Pearson s special mandarin to the cachexia viroid (C ii), and stem pitting reaction of sweet orange to the tristeza virus (C iii). (D) Branch of healthy citrus (D i), severe leaf epinasty reaction of Etrog citron Arizona 861-S-1 to the exocortis viroid (D ii), leaf bending and mild leaf epinasty of Etrog citron Arizona 861-S-1 to the bent leaf viroid (D iii), leaf drooping reaction of Etrog citron Arizona 861-S-1 to the dwarfing viroid (D iv), stem, petiole, and mid vein necrosis reaction of Etrog citron Arizona 861-S-1 to mixed infection of citrus viroids (D v), leaf cupping reaction of Mexican lime to the tristeza virus (D vi), shock reaction of Dweet tangor to the psorosis virus (D vii), and leaf curling and chlorosis of lemon due to fetal yellows disease (pathogen unknown) (D viii). (E) Leaf of healthy citrus (E i), corky vein reaction to the tristeza virus (E ii), leaf deformation reaction of sour orange to the infectious variegation virus (E iii), yellow vein reaction of Etrog citron Arizona 861-S-1 due to yellow vein disease (pathogen unknown) (E iv), tatter leaf reaction of Carrizo citrange to the tatter leaf virus (E v), young leaf pattern reaction of Dweet tangor to the leaf blotch virus (E vi), vein clearing (E vii) and water soaked (E viii) reaction of the top and lower part of a Mexican lime leaf respectively to the tristeza virus, vein enation reaction of Mexican lime due to vein enation disease (pathogen unknown) (E ix). May/June 2010

5 that environmental conditions in the greenhouse are optimal for plant growth and symptom expression (Fig. 3). Laboratory tests are also part of the disease diagnosis process. Examples of complementary laboratory techniques to the biological testing are the enzyme linked immunosorbent assay (ELISA) used for the detection of the tristeza virus; sequential polyacrylamide gel electrophoresis (spage), polymerase chain reaction (PCR); and hybridization used for the detection of citrus viroids; and culture in growth media used for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease (Fig. 4). Pathogen detection or indexing occurs at different times and steps of the introductory procedure. One small scale testing (pre-index) takes place at the time of introduction (see section A) (Fig. 2, 1A & 1B). The results of the pre-index indicate the need and type of therapy necessary (see section C) (Fig.2, 2A & 2B). The success of the therapy is assessed by another small scale preindexing (biological and/or laboratory testing) (Fig. 2, 3A & 3B). If all results indicate that therapy was successful, then the budline enters a full scale VI index. The VI testing lasts approximately 12 months and includes bio-indexing onto a host range of some 60 indicator seedlings including a broad range of negative and positive disease controls (mild, moderate, and severe isolates) and laboratory testing (i.e. ELISA, spage, PCR, culturing) (Fig.2, 4 and Fig. 3 and 4). C. Pathogen elimination-therapy If the introductory pre-index indicates the presence of disease(s) or pathogen(s), the budline must be subjected to therapy procedures that can eliminate the disease(s) agent(s). The CCPP employs two methods of therapy: thermaltherapy (aka thermotherapy), and shoot-tip-micrografting (Fig. 2, 2A & 2B). UCR and CCPP have been instrumental in the development, validation and employment of both techniques since the early 70s. Thermaltherapy or heat treatment is performed by taking buds from the infected budline and grafting them onto citrange (sweet x trifoliate orange) seedlings. The infected bud grafted onto each seedling is tightly and completely wrapped with budding tape so that the bud will not flush during thermaltherapy. The citrange seedlings, each with an infected bud grafted on it, are placed into a hot greenhouse with temperatures maintained at C daytime and 25 C nighttime for preconditioning to high temperatures for 30 days. Following preconditioning, the seedlings are placed into a controlled temperature chamber which is set for 16-hour days at 40 C and 8-hour nights set at 30 C. Plants are maintained in the thermaltherapy chamber for a period of 3 months. Upon removal of the plants from the temperature chamber, the buds are unwrapped (Fig. 5). The rootstock seedling is lopped over, and the top of the seedling is pushed into the potting soil such that the grafted bud will become the terminal bud. The plants are then placed in the greenhouse until sufficient budwood growth is produced from the Fig. 5. Thermal therapy heat chamber. Fig. 4. Citrus Clonal Protection Program laboratory diagnostic techniques for graft-transmissible pathogens of citrus. Enzyme-linked immunosorbent assay (ELISA) microtiter plate reader (A) and plate with yellow positive reactions (B), ethidium bromide stained agarose gel under ultraviolate light for the visualization of DNA products of conventional polymerase chain reaction (PCR) (C), quantitative real time PCR equipment (D), silver stained gel after sequential polyacrylamide gel electrophoresis (spage) for the detection of viroid and viroid-like RNA molecules (E), hybridization against DIG-labeled probes for the detection of viral nucleic acids (F), and microscopic observation of culture of prokaryotic pathogens (in this case Spiroplasma citri) (G). May/June 2010

6 grafted bud for further indexing. This method is effective against most citrus viruses; however, is not effective against viroids, and its effect on citrus tatter leaf virus has been reported to be variable. All imports received by CCPP originating outside the U.S. are routinely subjected to thermaltherapy as a precaution. Shoot-tip-micrografting (STG) is the other form of disease clean-up therapy employed by the CCPP. Some pathogens, particularly the citrus viroids (e.g. exocortis, cachexia), are difficult or impossible to eliminate by thermaltherapy and are much more readily eliminated by STG. STG is a procedure where several new growth tips slightly less than 1cm in length are taken from one of the original infected import propagations. Under a dissecting microscope, an apical meristem of about 0.15 mm, barely visible to the naked eye, is removed from the infected growth tip and grafted onto a seedling grown in vitro. If small enough when removed from the growth tip, the apical meristem is not yet developed enough to contain the pathogen, and therefore the disease will not be present in the micro-grafted propagation. STG propagations are returned to glass tubes and placed under light in a culture chamber. When the scion of the micrografted propagation reaches about 2 cm, it is regrafted onto a clean rough lemon seedling and moved to the greenhouse. STG is effective against all graft-transmissible agents including viroids (Fig. 6). The two therapy techniques thermotherapy and shoot-tip-grafting are complimentary, providing a flexible system for pathogen elimination bypassing the limitations (thermotherapy ineffective for viroids) and/or practical restrictions (STG long growth-regeneration time, specialized equipment, in vitro contaminants, and possible rootstockscion incompatibilities) that one method may have. Following any therapy procedure, all propagations produced during therapy must go through indexing again to determine their disease status. If the subsequent indexing indicates that a disease is still present, then the plant material must be subjected again to therapy. This cycle of therapy and testing continues until all tests are negative. When propagation of a budline tests negative in a pre-indexing following therapy, it may then enter the full scale VI index (see section B) (Fig.2, 4). If a budline is shown to be free of Fig.6. Shoot-tip-grafting. Preparation of shoot tip from the source plant (A i) and excision of apical meristem (2-3 leaf primordia barely visible with naked eye) (A ii). Preparation of rootstock seedling, seed germination in vitro (B i) and removal of portion of shoot and root growth (B ii). Necessary tools (scalpels, scissors, and forceps) for shoot-tip-grafting (C i) and dissecting microscope (C ii). Inverted T-cut on rootstock seedling under the dissecting microscope and placement of the apical meristem in contact with cambial tissue ((D I and ii). In vitro growth of shoot-tip grafted plants in liquid media (E i) and apical meristem growth after shoot-tip-grafting (E ii). known diseases in the VI Index, it is then considered ready for release from quarantine (Fig.2, 5) (see section D). D. Quarantine release When an introduced budline has tested negative for all known budtransmissible diseases in the VI index, the CCPP then applies for its release from both state and federal quarantine. The CCPP must first obtain release from CDFA by outlining the testing procedures and test results. Once released by the State of California, an application for federal quarantine release is sent to USDA/APHIS containing the testing information and a copy of the letter of approval by the State of California for release from quarantine. The distribution of citrus material that has been released from quarantine is also a highly regulated and carefully executed procedure that involves close interaction between CDFA, CCPP, and citrus nurserymen and growers (Fig. 2, 5). The above described introductory procedure and release from quarantine is available to private entities that wish to import patented or other proprietary varieties into California. The propagator/owner signs an agreement with UCR for the recovery of the cost of the testing and therapy procedures (currently set at $10,000), and when the variety is released from quarantine it is delivered to the owner and is not maintained in the CCPP Foundation Blocks (see section E). E. Maintenance The Lindcove Foundation and Evaluation Block The newly introduced varieties that have been pathogen tested and found clean and released from state and federal quarantine are propagated at Rubidoux on suitable rootstocks for field planting in the Foundation and Evaluation Block. The CCPP Foundation and Evaluation Block is located at the University of California Lindcove Research and Extension Center near Exeter in the San Joaquin Valley of California. This is a field planting of about 20 acres and now contains over 1,200 trees and over 300 different scion and rootstock varieties. The Foundation and Evaluation Block is planted on fumigated soil and has a wide planting distance between rows and trees to allow for better visual evaluation of each tree. Each tree of the Foundation and Evaluation Block is examined several times each year by CCPP and interested University and industry people for horticultural trueness-to-type, fruit quality, freedom from budsports and chimeras, spontaneous genetic disorders, and symptoms of disease. Until 2007, each tree was annually re-indexed biologically and by ELISA for tristeza and was tested up to three more times by ELISA during the May/June 2010

7 annual budwood distributions. Any tree showing abnormal growth characteristics or which tests positive for disease is immediately removed from the block, and the soil is fumigated prior to planting of another tree. In 2006 and 2007, a significant number of trees in the Lindcove station tested positive for tristeza, and some of them were located in the CCPP Foundation and Evaluation Block. This epidemic was the result of increasing natural spread of the disease in the commercial orchards surrounding the Lindcove station. Therefore, after almost 50 years of budwood distribution from the outdoor field block, the CCPP began budwood distribution exclusively from the Protected Foundation Block. The outdoor block is now used only as a trueness-to-type Evaluation Block for trees propagated from the Protected Foundation Block. Testing for tristeza continues annually with ELISA, and tristeza-infected trees continue to be removed. For an overview of the budwood movement after quarantine release, see Figure 7. The Lindcove Protected Foundation Block When CTV-infected tree removal in the commercial citrus surrounding Lindcove ended in 1998, the CRB sponsored the construction of a 40,000 sq. ft. (~3,700 m 2 ) protective screenhouse, completed in two phases between 1998 and In 2007, after positive tristeza findings Fig. 8. Panoramic view of the Citrus Clonal Protection Program Protected Foundation Blocks (PFB) and laboratory facilities at the Lindcove Research and Extension Center in Tulare County (A, figure was edited by Toan Khuong), the first screenhouse (40,000 sq, ft, ~3,700 m 2 ) constructed between 1998 and 1999 (B), the second screenhouse (30,000 sq. ft. ~2,800 m 2 ) completed in 2010 (C), the interior of the PFB with both container (D) and in-ground planted trees (E). May/June 2010 Fig. 7. Schematic representation of citrus budwood movement through quarantine and distribution to the California industry via the Citrus Clonal Protection Program (CCPP). in the Foundation and Evaluation Block, the screened Protected Foundation Block became the primary source of budwood. In 2008, the CRB committed the necessary funds for the expansion of the protected block by 30,000 sq. ft. (~2,800 m 2 ). Construction was completed in 2010, and tree planting began in the spring of 2010.Today, the Protected Foundation Block contains 750 trees, representing approximately 400 varieties (Fig. 8). The CCPP maintains both potted and in-ground trees inside these structures for the varieties that traditionally have high budwood demand. F. Registration of trees and budwood availability Prior to the distribution of any budwood from Foundation Block trees, they must be registered as budwood source trees with the CDFA. Registration by CDFA requires that trees be tested for tristeza, viroids and psorosis. If these tests are all negative, the tree is then assigned a CDFA registration number which must accompany any budwood distributed from any foundation tree. In order to remain as registered budwood sources, the CCPP re-indexes the Protected Foundation Block trees annually for tristeza, every three years for citrus viroids, and every five years for psorosis. In 2010, regulations for a mandatory Citrus Nursery Stock Pest Cleanliness Program were filed as an emergency action, based on the authority conveyed in Food and Agricultural Code, Sections This new program includes testing for additional diseases such as Huanglongbing (HLB). Most importantly, it also contains provisions to include exotic or emerging diseases as they become a threat to the budwood distribution scheme and to approve new, robust, and economic diagnostic technologies as they are developed. Only when the process of registering

8 source trees is completed can budwood from the CCPP Lindcove Foundation Blocks be distributed to the California nurserymen and growers. Limited quantities of budwood are available, and recipients normally use Foundation Block budwood to produce their own nursery- or grower-owned registered trees or nursery increase blocks, which are also regulated by CDFA. There is also provision for the distribution of limited amounts of Early Release budwood from containergrown trees of some selected newer varieties maintained in the protected screenhouse at Lindcove. Early Release variety trees have not fruited but have otherwise undergone all the required indexing and are registered with CDFA. Recipients of small lots of Early Release budwood understand that the fruiting characteristics of that particular budline have not yet been evaluated by CCPP. A waiver of liability for budwood that may not be horticulturally true-to-type or may contain budsports must be signed prior to receiving Early Release budwood. Budwood from the CCPP Protected Foundation Block is cut three times per year: January, June and September. The CCPP primarily serves the California citrus nurserymen and growers, but clients outside the state can request budwood. Budwood cut dates, order forms, prices, and other information is available at Letters are sent out prior to each cut and announce the specific date of the cut as well as a list of available varieties. Fig. 9. Schematic representation of Florida s citrus budwood introduction program-citrus Germplasm Introduction Program (CGIP). ELISA: enzyme-linked immunosorbent assay, CTV: Citrus tristeza virus, TLCSV: Tatter leaf citrange stunt virus, S. citri: Spiroplasma citri, X. fastidiosa: Xylella fastidiosa, PCR: polymerase chain reaction, rt: reverse transcription, CLBV: Citrus leaf blotch virus, CpsV: Citrus psorosis virus, spage: sequential polyacrylamide gel electrophoresis, dsrna: double stranded RNA, CG: Concave gum, CVV: Citrus variegated virus. G. Outreach The CCPP makes all the information related to budwood distribution, variety evaluation, citrus disease and management issues available to the public via the web and publication in agricultural magazines (for recently distributed citrus varieties see Citrograph March/April 2010 pages 20-26), peer-reviewed journals, and scientific conferences. Yearly Foundation-Evaluation Block inspections or walk-throughs are scheduled by the CCPP for the benefit of the citrus nursery personnel and interested growers. The CCPP also is present at many local and international agricultural shows with booths displaying the citrus varieties of its collection and distributing information as to the program and its objectives. For more information on CCPP visit: or contact Georgios Vidalakis, [email protected] or John Bash, [email protected] Tel: Department of Plant Pathology and Microbiology University of California Riverside, CA U.S.A. Florida In 1975, the Florida Department of Agriculture and Consumer Services (FDACS) Bureau of Citrus Budwood Registration in Winter Haven began removing graft-transmissible pathogens from Florida-grown citrus using shoottip-grafting and heat therapy. However, there was no approved method of importation for new citrus varieties into Florida, which most likely contributed to May/June 2010

9 Table 1. Comparative summary of the citrus quarantine programs in USA Variety Introduction Therapy Commercial Foundation Blocks under Quarantine Budwood Distribution Re-testing California Citrus Clonal Protection Program (CCPP) Yes Yes Yes Yes Florida Citrus Germplasm Introduction Program (CGIP) Yes Yes N/A Citrus Nursery Stock Certification Program Yes Yes Yes Arizona Certified Budwood Program Yes Yes Texas Texas A & M University-Kingsville Citrus Center Program Yes Yes Yes Louisiana Louisiana State University N/A Agricultural Center Citrus Research Station material from other programs Alabama Auburn University N/A Alabama Agricultural Experiment Station material from other programs Hawaii University of Hawaii N/A Department of Plant and Environmental material from other programs Protection Sciences N/A: not applicable the illegal introduction of citrus varieties into that state. In one budwood importation case, more than 210 acres or 95,000 trees were found as grove plantings and nursery stock infected with viral diseases exotic to Florida. Aware of the risk associated with the introduction of out-of-state budwood, FDACS proposed the establishment of an indexing program to be conducted in quarantine greenhouses and laboratories where foreign and domestic citrus germplasm could be shoot-tip grafted, indexed, and released as healthy budwood. By 1976, a plant quarantine facility with two indexing greenhouses (760 sq ft ~70 m 2 ) and adjoining laboratory space was constructed by the FDACS Bureau of Plant Pathology in Gainesville, Florida. Two more greenhouses were added in 1980, and for the next five years, three employees worked part-time cleaning-up and indexing citrus budwood, maintaining a maximum of five citrus selections and releasing one healthy variety every other year. Recognizing the need to increase the capacity of the introduction program, additional greenhouse space (3,000 sq ft ~280 m 2 ) and an office/headhouse facility (560 sq ft ~50 m 2 ) were constructed in Backup May/June 2010 foundation facilities (screenhouse and office/headhouse) are scheduled to be built in the near future. Today, the Citrus Germplasm Introduction Program (CGIP) can handle 30 foreign and domestic varieties, with up to 10 new imports each year, as well as numerous Florida-grown field-selections in various stages of therapy and indexing. The program currently consists of three full-time employees who are crosstrained in duties and responsibilities: one manager who directs the program, compiles reports, maintains records and reads citrus indicators, one biologist who performs shoot-tip grafting, conducts laboratory-based testing and transmission electron microscopy, and one greenhouse technician who is responsible for greenhouse maintenance, pesticide application, plant propagations and graft inoculations. Additional program direction and decision-making is provided by the FDACS Division of Plant Industry s (DPI) Assistant Director. Funding for the program is primarily provided by the state of Florida while additional funds are provided by national programs (e.g. Citrus Health Response Program (CHRP) and industry research grants. New varieties for introduction into Florida are chosen from requests submitted by individuals, companies and researchers. All requests must be approved by a citrus advisory committee and the Director of DPI. Importation of foreign citrus budwood is restricted by the USDA and requires a non-transferrable departmental permit issued to the program manager of the introduction program. This is the second of the three permits that allow introduction of citrus germplasm in the USA. Once budwood is received, it undergoes therapy and indexing to produce plants with no detectable pathogens. The process of introduction requires 18 months to five or more years depending on budwood quality and vigor, and then approval for release from the Citrus Budwood Technical Advisory Committee, DPI Director, and USDA (Fig. 9). Between 2003 and 2008, a total of 138 selections were released from the program. In addition, more than 250 field selections of the University of Florida (UF) breeding program were submitted to CGIP that were either infected with severe strains of CTV or exposed to citrus canker or Huanglongbing (citrus greening). For more information on the Citrus Germplasm Introduction Program in

10 Florida visit: germplasm/protocol.html or contact Lisa L. Williams, Tel: ext 498 Citrus Germplasm Introduction Program FDACS DPI 1911 SW 34th St. Gainesville, FL U.S.A. Arizona, Texas, Louisiana, Alabama, and Hawaii The states of Arizona, Texas, Louisiana, Alabama and Hawaii do not have a Quarantine-Introduction program. Citrus budwood is distributed to these states from the CCPP, NCGRCD and/or the Florida Bureau of Citrus Budwood. In the case of Texas, testing and therapy protocols are employed for some local citrus selections. Authors affiliation information 1 Citrus Clonal Protection Program, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA, [email protected] 2 Texas A & M University-Kingsville, Citrus Center, Weslaco, TX 78596, USA, [email protected] 3 Florida Department of Agriculture and Consumer Services. PO Box , Gainesville, FL, [email protected] 4 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, 302 Life Science, Baton Rouge, LA 70803, USA, DFerrin@ agctr.lsu.edu 5 Florida Department of Consumer Services, Bureau of Citrus Budwood Registration, 3027 Lake Alfred Rd. (US 17), Winter Haven, FL 33881, USA, kesingm@doacs. state.fl.us & [email protected] 6 USDA-ARS National Clonal Germplasm Repository for Citrus & Dates, Riverside, CA 92507, USA., robert.krueger@ars. usda.gov & [email protected] 7 Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii, 3050 Maile Way, Honolulu, HI 96822, USA, [email protected] 8 Alabama Agricultural Experiment Station, Auburn University, 411 rth McGregor Ave., P.O. Box 8276, Mobile, AL 36689, USA, [email protected] 9 Citrus Research Board, 323 W. Oak, P.O. Box 230, Visalia, CA 93279, marylou@ citrusresearch.org 10 Citrus Germplasm Introduction Program, Division of Plant Industry, 1911 SW 34th St. Gainesville, FL, 32608, USA, [email protected] 11 University of Arizona, Yuma Agriculture Center, 6425 W. 8th Street, Yuma, AZ 85364, USA, [email protected] Selected literature Bash J The California Citrus Clonal Protection Program. Pages in: Proceedings of the 5th ISCN International Congress. Calavan C.E., Mather S.M., and McEachern E.H Registration, certification, 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 University of California, Division of Agricultural Sciences. Calavan EC, CN Roistacher, and EM Nauer Thermotherapy of citrus for inactivation of certain viruses. Plant Disease Rep 56: Frison, E. A., and Taber, M. M. (eds.) FAO/IBPR Technical guidelines for the safe movement of citrus germplasm. FAO, Rome. 50 pages. Gumpf D. J., Bash J., Greer G., Diaz J., Serna R. and J. Semancik The California Citrus Clonal Protection Program. Proc. Int. Soc. Citriculture, Gumpf, D. J Citrus quarantine, California. pp In: R. P. Kahn and S. B. Mathur (eds.) Plant pest and pathogen exclusion: containment facilities and safeguards. American Phytopathological Society Press. Hiltabrand W.F Certification program for maintenance of virus-free propagation sources of citrus in California. In: Wallace J. M. (ed.) Citrus virus diseases. pp Univ. Calif. Div. Agr. Sci., Berkeley. Kahn T.L., Krueger R.R., Gumpf D.J., Roose M.L., Arpaia M.L., Batkin T.A., Bash J.A., Bier O.J., Clegg M.T., Cockerham S.T., Coggins C.W. Jr., Durling D., Elliott G., Mauk P.A., McGuire P.E., Orman C., Qualset C.O., Roberts P.A., Soost R.K., Turco J., Van Gundy S.G., and Zuckerman B Citrus Genetic Resources in California. Analysis and Recommendations for Long-Term Conservation. Report of the Citrus Genetic Resources Assessment Task Force. Report. 22. Genetic Resources Conservation Program. Division of Agriculture and Natural Resources. University of California. full.pdf Lee, R. F Certification programs for citrus. Pgs In: Management of Fruits and Vegetable Diseases. Diagnosis and Management. Vol I. (Naqvi, S. A. M. H., Ed.) Kluwer Academic Publishers, The Netherlands. Lee, R. F., P. Lehmann and L. Navarro Nursery practices, budwood and rootstock certification programs. In: Citrus Health Guide. L. W. Timmer and L. Duncan, Eds. APS Press, Minn. Pgs Nauer E. M., E. C. Calavan, C. N. Roistacher, R. L. Blue and J. H. Goodale The Citrus Variety Improvement Program in California. California Citrograph, 52: 133, 142, 144, 146, 148, 151, 152. Navarro, L., Roistacher, C. N., Murashige, T Improvement of shoot tip grafting in vitro for virus-free citrus. J. Am. Soc. Hortic. Sci. 160: Reuther W A program for establishing and maintaining virus-free citrus stock. In: Wallace J. M. (ed.) Citrus virus diseases. pp Univ. Calif. Div. Agr. Sci., Berkeley. Reuther W The Citrus Clonal Protection Program. Calif. Agriculture. 35: Reuther W., Calavan E. C., Nauer E. M. and Roistacher C. N The California citrus variety improvement program after twelve years. pp In: Proc. 5th Conf. Int. Organ. Citrus Virologists. IOCV, Riverside. Roistacher, C. N Graft transmissible diseases of citrus. Handbook for detection and diagnosis. FAO, Rome Rucks P Quality tree program for Florida citrus. Proc. Fla. State Hort. Soc. 107:4-8. Skaria, M., C. J. Kahlke, N. Solis-Gracia and R. Prewett Virus-free citrus budwood production and tristeza management program in Texas through industry partnership. Subtrop. Plant Sci. 49: 1-7. Wallace J.M. and Drake R.J An indexing program to avoid viruses in citrus introduced into the United States. In: Wallace J. M. (ed.) Citrus virus diseases. pp Univ. Calif. Div. Agr. Sci., Berkeley. Wallace, J. M Virus and viruslike diseases. In Reuther W., Calavan C. E., and Carman G. E. (eds.). The citrus industry Vol. IV. Crop protection. Chapter 2, pages University of California, Division of Agricultural Sciences. Jul 9 Aug 26 CRB-UCCE Citrus Research Grower Seminar Santa Paula, CA CRB-UCCE Citrus Research Grower Seminar Exeter, CA Sept CRB Research Proposals CRB Board Meeting Bakersfield, CA Oct 19 Oct 29 CRB Annual Meeting Exeter, CA CRB-UCCE Citrus Research Grower Seminar Orland, CA May/June 2010

11 Citrus Quarantine, Sanitary, and Certification Programs in the USA Prevention of Introduction and Distribution of Citrus Diseases Part 2 Certification schemes and national programs Highlights of new California citrus nursery regulations Georgios Vidalakis 1, John V. da Graça 2, Wayne N. Dixon 3, Donald Ferrin 4, Michael Kesinger 5, Robert R. Krueger 6, Richard F. Lee 6, Michael J. Melzer 7, John Olive 8, MaryLou Polek 9, Peggy J. Sieburth 5, Lisa L. Williams 10, and Glenn C. Wright 11 Editor s te: The major USA citrus quarantine-introduction germplasm programs were presented in Part I CITRUS QUARANTINE AND IN- TRODUCTION PROGRAMS (Citrograph May/June 2010, 3: 26-35). In this issue, we are presenting the various certification schemes and national programs and efforts to harmonize the protocols for citrus germplasm movement and use. Parent/Foundation Trees: Trees receive a high level of evaluation testing and are maintained by the certification program (CCPP, FDACS-CBR) in a Foundation Block. These are the sources of registered budwood provided to nurseries which are able to multiply or increase the material to produce clean nursery stock. Foundation Block: A group of parent/foundation trees maintained by a certification program (CCPP, FDACS-CBR) from which registered budwood is cut and distributed to nurseries. Registered Mother Trees: Production nurseries obtain budwood cut from registered foundation trees and use it to start source trees for their propagation of nursery stock. These trees must successfully meet state testing regulations for known graft-transmissible pathogens. Increase Blocks: Means by which production nurseries rapidly produce lots of propagation material for nursery tree production; bud eyes are cut from registered sources and grafted onto rootstock seedlings. Introduction Citrus germplasm originated in Australasia, the Far East, and Africa thus, all citrus grown in the New World was imported. Unfortunately, accompanying this germplasm were graft-transmissible pathogens of citrus capable of causing destructive epidemics such as the tristeza quick decline that killed more than 60 million trees worldwide. One of the main reasons for the cit- rus industry s survival and prosperity through the decades is the early establishment of citrus germplasm programs, such as California s Citrus Clonal Protection Program, that provide a safe mechanism of introduction of new varieties. By means of disease testing and therapy, they ensure the distribution of high quality, disease tested citrus propagative materials. After citrus germplasm has been through the basic protocols for the detection and elimination of grafttransmissible pathogens (for details see Citrograph May/June 2010, 3: 26-35), it is ready to enter the budwood distribution process which includes the continuous testing of mother trees and increase blocks, as well as fruit evaluation for horticultural trueness-to-type. In the USA, the different citrus producing states have developed and adjusted their budwood distributioncertification programs to the needs (size, type, available resources, involved agencies, disease or pest pressures etc) of their specific industries. In general, the California Citrus Clonal Protection Program (CCPP) handles both the quarantine-introduction and budwood distributioncertification. In Florida, two different programs the Citrus Germplasm Introduction Program and the Bureau of Citrus Budwood Registration handle the quarantine-introduction and budwood distribution-certification, respectively. Arizona, Texas, Louisiana, Alabama, and Hawaii acquire pathogen-tested citrus budwood from California and Florida, and they maintain, re-test and distribute budwood to their industries via distribution-certification programs (Table 1). The fundamental need for the use of July/August 2010 Citrograph 27

12 pathogen-tested citrus propagative material is well understood and accepted both nationally and internationally. In the United States, the USDA ARS National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), while based in California, actually serves the entire USA and distributes materials free of charge to qualified scientists and certification programs around the globe. The NCGRCD is not focused only on citrus varieties with commercial interest, like most of the state operating programs, but it also preserves, in general, the germplasm of citrus and citrus relatives. This material is the basis for citrus breeding programs. In order to facilitate the proper exchange of the genetic material between the state and national citrus programs, the development of a National Citrus Germplasm Passport Program (NC- GPP) is currently in progress. This program will have two benefits. First, it will provide a safe pathway for the different citrus breeding programs in the USA to exchange material; and second, it will set a minimum national standard for citrus germplasm programs that can be used with international organizations such as the rth American Plant Protection Organization (NAPPO) bypassing the issue of multiple state standards. The most recent development for citrus germplasm programs at the national level comes from a collaborative effort of the three agencies of the USDA: the Animal and Plant Health Inspection Service (APHIS), the Agricultural Research Service (ARS), and the Cooperative State Research, Education, and Extension Service (CSREES). The mission of the newly developed National Clean Plant Network (NCPN) is to provide high quality asexually propagated plant material free of targeted plant pathogens and pests that cause economic loss to protect the environment and ensure the global competitiveness of specialty crop producers. In the case of citrus, all the state and national citrus germplasm programs have been welcomed in the NCPN. A national organizational group for citrus, the Citrus Clean Plant Network (CCPN), was established on March 23, Table 1. Comparative summary of the citrus quarantine and certification programs in USA Mandatory Nursery Budwood Program Variety Introduction under Quarantine Therapy California Yes Citrus Clonal Protection Program (CCPP) Budwood Distribution-Certification Schemes California The California certification program is one of the oldest in the world, having its origins in the 1930s Psorosis Free Program. Currently, it is a cooperative effort between the University of California Riverside (UCR), the California Department of Food and Agriculture (CDFA), and the California Citrus Nursery Board (CCNB). All citrus trees produced for sale in California must be propagated from mother source trees registered with the CDFA. There are over 4,000 registered trees representing approximately 300 different varieties in the CCPP and commercial citrus nurseries (approximately 60). During the past five years, the CCPP distributed approximately 175,000 buds from 285 different citrus varieties from its registered trees in the Foundation and Evaluation Block at the Lindcove Research and Extension Center in Central California (Fig. 1), while nurseries produced approximately 18 million trees (between 3 and 4 million trees per year). Prior to May 17, 2010, the Cali- Table 1. Comparative summary of the citrus quarantine and certification programs in USA Commercial Budwood Distribution Foundation Blocks Re-testing Yes Yes Yes Yes Arizona Certified Budwood Program Yes Yes Texas Texas A & M University-Kingsville Citrus Center Program Yes Yes Yes Yes Louisiana Louisiana State University Agricultural Center Citrus Research Station material from other programs N/A Alabama Auburn University, Alabama Agricultural Experiment Station material from other programs N/A Florida Citrus Germplasm Introduction Program (CGIP) Citrus Nursery Stock Certification Program Yes Yes Yes N/A Yes Yes Yes Hawaii Department of Plant and Environmental Protection Sciences University of Hawaii material from other programs N/A National N/A NCGRCD Yes Yes N/A: not applicable 28 Citrograph July/August 2010 free of charge worldwide N/A retested protected collection

13 fornia registration program (active since 1962) included annual mandatory testing for the Citrus tristeza virus (CTV) and voluntary testing for the psorosis (Citrus psorosis virus, CPsV) and psorosis-like diseases and citrus viroid diseases every five years. The mother seed tree sources were tested for the psorosis and psorosis-like diseases every six years, also on a voluntary basis. The mandatory CTV testing is a requirement of the CTV State Interior Quarantine 3407 which also regulates the movement of citrus plants. Increase blocks propagated from registered source trees could be used for the propagation of certified trees for commercial groves for 18 months without any additional testing and for 24 months with one CTV test. After 18 (or 24) months, the increase block must be destroyed or planted as trees. New registered budwood must be obtained to produce new increase blocks. The cost of testing was estimated at $20 per registered tree, and it was subsidized by the CCNB at 40-50% to encourage nurseries to participate in the voluntary program. On May 17, 2010, regulations for a mandatory Citrus Nursery Stock Pest Cleanliness Program were filed as an emergency action, based on the authority conveyed in Food and Agricultural Code, Some highlights of the new registration program ( ) include: 1. Section allows for the registration of scion mother trees, not previously registered, until July 1, 2011 providing they have tested negative for CTV and Candidatus Liberibacter spp., the causal agents of tristeza and huanglongbing, respectively. 2. Other than described above (Item 1), scion mother trees and seed trees will qualify for registration only if they were propagated from CCPP sources or material that has been tested by the CCPP or from registered scion mother trees. Registered rootstock trees shall be from registered seed sources or propagated vegetatively from registered sources ( ). 3. Registered increase trees, maintained in CDFA approved insect resistant structures, may be used as a source of propagative materials for a period of 36 months from date of propagation with no additional testing. The cutting period may be extended for an additional 24 months provided that the Figure 1 Five main commercial Texas varieties selected and shoot tip grafted. Foundation Block Tested annually for CTV and HLB every 4 years for other viruses/viroids Potted plants maintained in small screenhouse Potted collection in large screenhouse Planned Increase block of commonly ordered commercial and non commercial varieties Tested annually for CTV (Plan to be under screen in 2010) Buds supplied to nurseries for direct grafting onto rootstocks (Mandatory for budwood of main commercial varieties to come from the program) rest of the testing requirements are met ( ). 4. Scion mother and rootstock source trees may be field grown until January 1, To be eligible for registration after that date, scion mother trees shall be maintained in CDFA approved insect resistant structures ( ). 5. Increase trees may be field grown until January 1, To be eligible for registration after that date, increase trees shall be maintained in CDFA approved insect resistant structures ( ). 6. Each insect resistant structure shall be approved and inspected by the CDFA prior to planting or moving stock into it. Structures under construction or completed prior to the adoption of these regulations may be grandfathered in provided they meet specific performance standards ( ). 7. Testing activities shall be conducted by the CDFA or its authorized agents which includes, the CCPP, the Central California Tristeza Eradication Agency and the Citrus Research Board ( ). 8. Registered scion mother trees, maintained in a CDFA approved insect resistant structure whose scion parent material was sourced directly from the CCPP and not subsequently field planted shall be tested as follows ( ): a. CTV and Ca. L. spp.: Trees shall be tested prior to the first use as a propagative source, but no later than three years from the date of propagation. Thereafter, the trees shall be tested each year in which they are to be used as propagative sources and shall not have a testing gap of three or more years. Pathogen free budwood of 100 noncommercial varieties obtained from CCCP Potted collection being established in large screenhouse in Weslaco and smaller backup collection in N. Texas July/August 2010 Citrograph 29

14 b. Viroids: i) Until January 1, 2013, the trees shall be tested no later than three years from date of propagation. ii) After January 1, 2013, the trees shall be tested prior to their first use as a propagative source, and at least once every three years thereafter. iii) After January 1, 2016, trees maintained in insect resistant structures in which no trees have tested positive for viroids shall be tested at least once every six years. Should any tree within the structure subsequently test positive, all scionmother trees within the structure shall be tested at least once every Figure 2 30 Citrograph July/August 2010 three years until all trees within the structure have had two consecutive negative tests. c. Citrus psorosis virus (CPsV) and psorosis-like diseases: Trees shall be tested at least once every six years. 9. Trees registered for seed production shall be tested once every six years for CPsV A & B and citrus leaf blotch virus (CLBV), and beginning January 1, 2013, annually for Ca. L. spp. ( ). 10. Registered seed trees maintained in a CDFA approved insect resistant structure that also contains scion source trees shall be tested i) every three years for CTV, once every three years for viroids, once every six years for CPsV A & B and CLBV, and ii) annually for Ca. L. spp. after January 1, 2013 ( ). 11. Increase tree(s) may be used as propagative sources for a maximum of five years from date of propagation, provided that they are tested for CTV and Ca. L. spp. between 24 and 36 months from date of propagation and annually thereafter ( ). 12. CDFA may approve or require the substitution or addition of other tests which are of equal or better reliability in detecting the diseases and disease agents of concern ( ). 13. The cost of the new registration program includes; an annual application fee of $200, $25-35 per scion mother or seed tree, and additional fees for the testing of increase trees, ( ). The CDFA-approved diagnostic tests include: ELISA for CTV, PCR for Ca. L. spp., and bio-indicator hosts Etrog citron (Citrus medica, Arizona 861-S1) and Dweet tangor (C. reticulata x C. sinensis maintained in the greenhouse for citrus viroids, CPsV A & B, and CLBV. The CDFA has also approved the molecular test of imprint hybridization for the detection of the citrus viroid II (Hop stunt viroid) as it causes a very mild reaction in Etrog citron, and diagnosis may be inaccurate with only bio-indexing. The testing is performed mainly by the CCPP, but the CDFA and CDFA-approved laboratories are also involved. The documents used for the above summary can be viewed in their entirety at: regs_cns.html#cns The CDFA has held several scoping meetings with the industry that have shaped the new regulations. The revisions are open for public comments for a 30-day period. Comments may be submitted by mail, facsimile (FAX) at or by to smccarthy@ cdfa.ca.gov. The written comment period closes at 5:00 p.m. on Sept.13, CDFA will consider only comments received at the Department offices by that time. Submit comments to: Susan McCarthy Department of Food and Agriculture Plant Health and Pest Prevention Services 1220 N Street, Room 210 Sacramento, California 95814

15 Figure 3 Florida Citrus Nursery Regulations All citrus nursery propagation must occur in enclosed greenhouse structures Entryways must be double entry with forced All openings, fans, coolers, vents must be sealed air exiting the first door as it is opened. with insect exclusion screen. Air curtain Personnel and equipment must be decontaminated prior to greenhouse entry Budwood sources must meet the same standards as the propagating greenhouses Clippers, budding knives must be sterilized with household bleach between each source tree All nusery propagations must be submitted on a Bud Cutting Report Other regulations pertaining to nursery stock production can be found in rule chapter 5B-62 FAC. and in the citrus Nursery Stock Certification Manual

16 Two public hearings are scheduled. The first is Sept. 15, 2010 in the auditorium of the Tulare County Agricultural Center (4437 Laspina Street, Tulare), and the second is on Sept. 17, 2010 in the Rummonds Training Room of the Coachella Valley Water District ( Avenue 52, Coachella). Finalization of the revised regulations is intended for the middle of vember For more information about the California citrus registration program, you may also contact: Mike Colvin, Tel: California Department of Food and Agriculture Nursery, Seed, and Cotton Programs 1220 N Street, Room A-372 Sacramento, CA U.S.A. 32 Citrograph July/August 2010 Florida Florida s Citrus Nursery Stock Certification Program is administered by 16 staff employees of the Bureau of Citrus Budwood Registration, which is funded by a grower box tax. The Bureau of Citrus Budwood Registration is part of the Florida Department of Agriculture and Consumer Services, Division of Plant Industry. The objective of the nursery stock program is to assist growers and nurserymen in the production of horticulturally superior citrus nursery stock that is believed to be free of viruses and other graft-transmissible diseases. The bureau was established in 1953, initially to deal with psorosis and cachexia diseases and later included testing for exocortis disease. The budwood program remained voluntary for 44 years until 1997 when it became mandatory to test all propagation sources for CTV annually and called for recurring indexing (every 6 years) for citrus viroids. The Florida budwood program is headquartered in Winter Haven in Polk County with office, laboratory and greenhouse facilities. A four-acre citrus arboretum is located on site along with a nearby 80-acre (~ 32 hectares) field evaluation grove located at Dundee. Budwood for distribution to growers is kept in 1.8 acres (~ 0.7 hectares) of protected greenhouses outside of the commercial citrus industry at Chiefland in Levy County. In 2008, the Foundation Grove was moved from Winter Haven into protected screenhouses in Chiefland (screenhouses 80,000 sq ft ~0.75 hectares and office/headhouse facility (4,000 sq ft ~370 m2). Backup foundation facilities (screenhouse and office/headhouse) are scheduled to be built. The Florida program offers 321 different clonal selections from its Chiefland foundation greenhouses and distributed over 130,000 budeyes in Foundation budwood is used to establish nursery scion and increase blocks in commercial nurseries. The selection of high quality parent trees and the development of nucellar selections improved the quality of fruit along with the production per acre (Fig. 2). Additional important program functions are the supervision of all citrus nursery propagations and the maintenance of detailed records to retain positive clonal identity of registered, pathogen-tested nursery trees offered to Florida citrus growers. All protected enclosures at citrus nurseries are inspected every 30 days for structural integrity. Budwood source trees are tested by biological and molecular methods to detect grafttransmissible pathogens detrimental to tree performance (Fig.3). A four-person laboratory staff runs qpcr testing for Ca. las, CTV, CLBV, Citrus tatterleaf virus, and viroid diseases. Two greenhouse technicians conduct biological indexing on greenhouse grown indicator plants. Testing of all nursery stock is conducted annually for pathogens vectored by insects and all others at regular intervals. Because of the many disease challenges, the Florida Citrus Nursery Stock Certification Program in 2007 began to require all propagation of citrus to be done in protected greenhouse structures (Fig. 4). All budwood sources have to likewise be maintained in protected greenhouse structures with double entryways, positive air pressure and other safeguarding mechanisms. Forty-four commercial citrus nurseries produce approximately 3.5 million trees per year. Today, Florida growers struggle with vectored diseases such as HLB and tristeza. HLB is one of the worst diseases of citrus and has the potential to devastate a citrus industry. Since the introduction of the brown citrus aphid in 1995, stem-pitting strains of CTV have been found in Florida, and these more virulent forms threaten trees regardless of rootstock. Other major exotic diseases such as citrus variegated chlorosis and citrus leprosis from Central and South America pose a risk if they become established in Florida. New and existing diseases threaten to limit citrus production and may mean the difference in making a profitable living or bankruptcy. For more information on the Florida budwood program visit: budwood/index.html chapterhome.asp?chapter=5b-62 budwood/docs/citrus_nursery_ Stock_Certification_Manual_ pdf or contact Michael Kesinger, kesingm@doacs. state.fl.us Bureau of Citrus Budwood Registration 3027 Lake Alfred Rd. (US 17) Winter Haven, FL Tel: Arizona The Arizona Certified Budwood Program was created in the 1960s with a focus on CTV and viroids (Exocortis and Cachexia/Xyloporosis). The program is located in Yuma, Arizona, and it is a collaborative effort between the Yuma County Citrus Pest Abatement District (YCCPAD), the University of Arizona, and the Arizona Crop Improvement Association. The program consists of one office and an in-ground, unprotected budwood block holding approximately 100 varieties that also functions as the University of Arizona variety collection. The program employs one manager and one half-time employee and has additional support of volunteers. Funding of this program is through the YCCPAD that collects a fee from budwood sales, funds generated from the sale of fruit to local vendors, and in kind contributions from the University of Arizona.. The state has a very small number of nurseries and therefore limited need for budwood. There are only four citrus nurseries; three are wholesale only, and one is both wholesale and retail. Some nurseries in the Phoenix area that sell predominantly landscape plants also grow citrus, but in most cases they get their trees from one of the four wholesale nurseries and simply grow them out to larger size. In addition, much of the

17 Figure 4 The Florida Citrus Budwood Registration Program Parent Tree Selection Pathogen Indexing and Clean up Clonal Propagation 1 Candidate Tree Selected 2 Budwood Foundation Scion Planting Pathogen Detection 3 Foundation Trees planted of each Pathogen free selection Nursery Increase Block 4 Foundation budwood used to start Scion Planting in enclosed greenhouse at nursery site Commercial Nursery Propagations 5 Increase Block started from Scion Trees 6 Commercial trees propagated from Increase Block Commercial Citrus Grove 7 Sale of trees to Commercial Groves July/August 2010 Citrograph 33

18 landscape citrus is supplied by the bigbox stores that buy their trees from CA nurseries. The largest wholesale citrus nursery in the state just constructed a screenhouse for indoor production. Since most nurseries obtain their budwood from their own increase trees, and the majority of the budwood needed for Arizona comes from California nurseries and the CCPP, only approximately 500 buds are distributed from the University block per year. Due to this small number, there is no systematic disease testing. The block is occasionally tested for tristeza and citrus viroids, and so far there has been no positive finding. The budwood program in Arizona operates on a voluntary basis. For more information for the Arizona program you can contact John Loghry, [email protected] Yuma County Citrus Pest Abatement District 2186 W. County 15th Street Somerton, AZ Tel: Texas The high incidence of psorosis disease prompted the Texas citrus industry to initiate a voluntary virus-free program based on California s Psorosis Free Program in The program was voluntary, but even so, nearly 600 trees were certified. By 1959, these budwood sources had produced 1.6 million trees or 23% of the total. By the early 1960s, over 90% of new citrus trees were from psorosis-free sources. Unfortunately, the program gradually became ineffective and was abandoned by the 1960s. A similar program in the 1980s was short-lived. The discovery of CTV in nursery trees in 1992, the prevalence of sour orange rootstock, and the presence of viroids and viruses which affect alternative rootstocks were catalysts for a mandatory virus-free budwood program. Today, Texas A & M University- Kingsville Citrus Center operates the program under the authority of the Texas Department of Agriculture (TDA), whose Commissioner appoints a 7-member advisory committee which meets 3-to-4 times a year. The program facilities are located at the Texas A&M Citrus Center in Weslaco, Hidalgo Co and include one greenhouse, two screenhouses, and fenced foundation (FB) and increase blocks (IB). A greenhouse in Stephenville, in Erath Co. (70 miles SW of Dallas-Ft Worth) is being renovated for a back-up foundation collection also planned to be part of the program in the near future, and the increase trees are to be covered with insect-resistance structures by the end of Program personnel include a manager, 2 nursery workers, 2 field workers, and 1 lab technician. The program is funded by budwood sales, the Texas Citrus Producer s Board, under a USDA-APHIS cooperative agreement, and the University. When the decision was made for the mandatory budwood program, a foundation block was started with selected trees of the commercially important varieties of grapefruit and sweet orange. These were indexed for several viruses and viroids, subjected to shoot-tipgrafting and re-indexed. Other varieties were subsequently imported from the California CCPP and from the FDACS Bureau of Citrus Budwood Registration. There are now over 80 varieties in the foundation block, and over one million buds have been supplied to nurseries since All trees in the FB and IB are tested for CTV by ELISA annually, and testing for other pathogens occurs every 4-to-5 years. The program is enforced now by Texas State law. Fruit characteristics of the commercial varieties in the FB are July/August 2010

19 Figure 5 Florida Parent Tree Entry and Indexing Parent Selected For: Outstanding horticultural traits, yield, absence of disease, environmental adaptation and fruit attributes such as: solids, % juice, color, size, seedlessness, etc. Parent Tree 1 Selection of Candidate Tree Pathogens Tested For: Citrus Greening Citrus Tristeza Virus Exocortis Viroid Cachexia Viroid Other Citrus Viroids Citrus Leaf Blotch Virus Psorosis Virus Citrus Tatter leaf Virus Budsticks Propagation Bioindexing Laboratory Prepared for STG Source for STG & testing Inoculation of indicators PCR and ELISA testing 2 Budwood harvested from candidate to set up procedures above: Positive test results used to develop clean-up strategy. Shoot-tip grafting (STG) may require thermotherapy for eliminating some pathogens and cooler temperatures for others. Plants with mixed infections may take considerably longer to clean up as one pathogen may have to be eliminated at a time. 3 Testing results reviewed 4 Shoot-tip Grafting for pathogen clean-up Successful shoot-tips are propagated and grown out and tested for pathogens detected in step 2 above. 5 Follow up testing of STG Clean budwood sources maintained in greenhouses located at Chiefland 6 Tested STG multiplied for fruit check and budwood sources 7 Fruiting out to determine trueness to type 8 Budwood distributed to nurseries for establishing budwood sources July/August 2010 Citrograph 35

20 evaluated annually, and when sufficient budwood from true-to-type trees is available, the state-mandated requirements will come into force. Since there is no current expansion or replanting of commercial citrus in the Rio Grande Valley of South Texas, a growing proportion of budwood is for the homeowner market, especially in East Texas (Fig. 5). For more information in the Texas program you can contact John Watson, [email protected] 312 N. International Blvd Weslaco TX Tel: Louisiana Louisiana currently has no program to provide pathogen-tested foundation materials to growers, and there are no plans to replace the LSU AgCenter Citrus Research Station in Port Sulfur, LA that was destroyed by hurricane Katrina in Growers and nurserymen, therefore, rely on the programs of other states as a source of pathogentested budwood. With the discovery of Huanglongbing (HLB) in Louisiana in 2008, the need for Louisiana nurserymen and growers to have access to clean budwood is crucial to the survival of the citrus industry here. The focus of the commercial Louisiana citrus industry is primarily on small-scale production of satsumas (375 acres), navel oranges (418 acres), and other assorted citrus (48 acres) for local markets. However, backyard citrus continues to be an equally significant portion of the Louisiana citrus industry. Additionally, we have a small citrus nursery industry located primarily in Plaquemines Parish that supplies plant materials to commercial growers and homeowners throughout the Gulf Coast region from Louisiana to Alabama. For more information in the Louisiana program you can contact Donald Ferrin, [email protected]. edu Louisiana State University Agricultural Center 302 Life Science Baton Rouge, LA Tel: Citrograph July/August 2010 Alabama Alabama is in the early stages of establishing a program for obtaining clean propagative material and establishing a foundation planting to maintain propagation materials of common commercial varieties and varieties unique to the region. t only is there the need for clean propagation material, but there is also a need to educate both growers and home gardeners of the need to keep these diseases out of the state. The citrus industry in Alabama currently is relatively small (~15,000 trees) and predominately Satsuma varieties, but is rapidly expanding to meet the demand of regional markets. In addition, there are citrus nurseries in the state that are projecting the production of 25,000 trees/yr for retail and wholesale markets. The establishment of a clean plant source to serve the industry in this state and the Southeast is crucial at this time. The growing areas for citrus are in close proximity to states with quarantined diseases, and plant material can be easily moved across state lines without detection. For more information in the Alabama program you can contact John Olive, [email protected] P.O. Box rth McGregor Ave. Mobile, AL Tel: Hawaii Commercial citrus production in Hawaii started the early 1800 s when island-grown fruits were exported throughout the Pacific Rim. The introduction of several citrus pests and diseases, however, has eliminated exportation due to quarantines and has relegated citrus to its current status as a minor agricultural commodity. Currently, only a few hundred acres are devoted to commercial citrus production, representing approximately 3-5% of the citrus sold in Hawaii. Most of these farms are small, family operations with less than five acres dedicated to citrus. Dooryard citrus trees, however, are very common in Hawaii and may exceed the number of commercial trees. Hawaii does not have a program to distribute clean propagative materials to growers. Some nurseries import certified budwood from states with programs in place such as California. Due to the high incidence (~75%) of Citrus tristeza virus in Hawaii, other nurseries have identified trees with mild strains of the virus and use budwood from these trees to cross-protect against the more severe strains. The recent discovery of the Asian citrus psyllid (Diaphorina citri) in Hawaii has been cause for concern that Huanglongbing (HLB) is also present. To date, no samples from Hawaii have tested positive for any of the three Candidatus Liberibacter species known to be responsible for this disease. Attempts are being made to establish an in-state program for the detection of these bacteria. The establishment of such a program may allow detection of other graft-transmissible pathogens of citrus to be added in the future. For more information for the Hawaii program contact Michael J Melzer, melzer@hawaii. edu Department of Plant and Environ mental Protection Sciences College of Tropical Agriculture and Human Resources 3050 Maile Way, Room 310 University of Hawaii Honolulu, HI Tel: National Efforts USDA-ARS National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) The National Plant Germplasm System (NPGS) was established in 1974 as an umbrella system which incorporated genetic resource-related activities that had previously been part of a wide range of agencies within and outside of USDA-ARS. Its goal is to collect, evaluate, maintain, and preserve plant genetic resources. The stated mission is to facilitate and encourage the free exchange of genetic resources. The National Clonal Germplasm Repository for Citrus and Dates (NC- GRCD) was established in 1987 as a subset of the NPGS that focuses on two commodities, citrus and dates. It is located on the UCR campus to take advantage of the CCPP, the Citrus Variety Collection (CVC), and other citrus germplasm resources available at the UCR Citrus Experiment Station. The NCGRCD is a cooperative effort between USDA-ARS, USDA-APHIS, the Agricultural Experiment Station at UCR, and CDFA. The program is financially supported by federal funds and research grants. Today, the NCGRCD consists of six personnel (one leader researcher/plant pathologist, one curator/horticulturist, three technicians, and one secretary)

21 working at the facilities. The facilities include a 500 sq ft (~ 47 m 2 ) laboratory, a 6,000 sq ft (~ 560 m 2 ) greenhouse for disease indexing and citrus propagation, and an additional 4,800 sq ft (~ 445 m 2 ) for plant maintenance. Approximately 400 accessions have been tested and found free of graft-transmissible pathogens. These serve as the primary source of budwood for distribution. They are housed in a 16,000 sq ft (~ 1500 m 2 ) protective screenhouse. The mission of the NCGRCD is to acquire, preserve, distribute, and evaluate genetic diversity within Citrus, the 32 related genera, and date palms and their relatives and to do research which supports these objectives. There are several ways that the NC- GRCD obtains new virus-free genetic resources. The NCGRCD acquires citrus selections that have been released from quarantine via the CCPP. The NCGRCD moves accessions from the CVC into the screenhouse collection by an internal quarantine process similar to the CCPP as outlined previously (i.e. indexing and therapy). Finally, the NC- GRCD holds the third and final permit for the introduction of citrus germplasm in to the USA under quarantine (via USDA Beltsville, Maryland). This way, state Quarantine-Introduction Programs such as the CCPP and CGIP can focus on the commercially important varieties while the NCGRCD can focus on citrus and citrus relatives that have no immediate commercial potential but may be useful as genetic resources for disease resistance, fruit quality, scion and rootstock breeding and development. Figure 6 The NCGRCD distributes materials free of charge to qualified scientists and certification programs. Exchange of citrus genetic resources is highly regulated. Most citrus-producing countries have restrictions on the introduction of citrus materials to prevent the concurrent introduction of new pathogens or strains of pathogens. Most countries will accept only pathogen-tested citrus budwood. Therefore, the NCGRCD is using all of the available indexing and therapy techniques necessary to maintain disease-free citrus genetic material (Fig. 6). For more information regarding the NCGRCD you can visit: (NC- GRCD administrative homepage), and acc_queries.html (Search GRIN for citrus or date germplasm) or contact Richard F Lee, Richard.Lee@ARS. USDA.GOV and Robert R Krueger, robert. [email protected] USDA-ARS National Clonal Germplasm Repository for Citrus & Dates 1060 Martin Luther King Blvd Riverside, California USA Tel: National Citrus Germplasm Passport Program (NCGPP) In 2006, representatives of the three largest citrus germplasm programs in the U.S. met with other citrus stakeholders in Florida to lay the groundwork for a harmonization of the various state and federal citrus germplasm introduction and indexing programs. Since that initial meeting, a number of teleconferences and visits to the programs in California and Florida have taken place to further develop the concept and procedures for a National Citrus Germplasm Passport Program (NCGPP). Key participants in the development of the passport system are the Citrus Germplasm Programs: Citrus Clonal Protection Program (California), Citrus Germplasm Introduction Program (Florida), and the National Clonal Germplasm Repository for Citrus and Dates (National). Other participants and supporters of this effort are the Florida Department of Agriculture and Consumer Services, the California Department of Food and Agriculture, Citrus Research Board, and the USDA- APHIS Plant Protection and Quarantine program within APHIS. The proposed passport program will expedite the movement of citrus germplasm from one U.S. citrus germplasm program to another and will help to set national standards for the U.S. citrus germplasm programs. The NCGPP will provide a safe mechanism for the exchange of citrus varieties by setting the minimal standards for introduction, shipping preparation, core disease testing, and therapy. (Individual programs can always conduct additional assays and disinfecting steps if desired.) (Table 2). Typically, two or three years are required to index and provide therapy to a new germplasm regardless of its source. However, using a minimal standard of indexing and therapy procedures, citrus The first source trees moved in to the expanded CCPP Protected Foundation Block at Lindcove Research and Extension center (LREC) August 2010 and are ready to service the budwood needs of California s citrus industry. Photo by Therese Kapaun, LREC. "The first source trees moved in to the expanded CCPP Protected Foundation Block at Lindcove Research and Extension center (LREC) August 2010 and are ready to service the May/June 2010 Citrograph 37

22 germplasm accompanied by a passport would provide the needed transparency to move citrus germplasm from one state to another in an expeditious manner and limit the amount or need of additional re-testing for graft-transmissible pathogens. By adhering to these safeguards, the period of time from shipment to use by the industry and the risk of pest and pathogen introduction will be minimized. To ensure the success of a national citrus budwood passport program, the participating state departments of Table 2. Core testa for the National Citrus Germplasm Passport Program, USA DISEASE PATHOGEN CORE TEST 1 agriculture, certification programs and the USDA-APHIS must work cooperatively and agree on and implement adequate safeguards for the protection of the United States citrus industry. Currently, the only facilities that are authorized to receive citrus germplasm Tristeza Citrus tristeza virus (CTV) Mexican lime 2, ELISA 3 Psorosis A & B Citrus psorosis virus (CPsV) Sweet orange, Dweet tangor Concave gum Unknown Sweet orange, Dweet tangor Sour Infectious Variegation Citrus variegation virus orange Leaf rugose Citrus leaf rugose virus Crinkly leaf Citrus crinkly leaf virus Etrog citron, Eureka lemon DISEASE PATHOGEN CORE TEST 1 Tatter leaf-citrange stunt Apple stem grooving virus (ASGV) syn. Citrus tatter leaf virus (CTLV) Tristeza Citrus tristeza virus (CTV) Mexican lime 2, ELISA 3 Psorosis A & B Citrus psorosis virus (CPsV) Sweet orange, Dweet tangor Concave gum Unknown Sweet orange, Dweet tangor Infectious Variegation Rusk citrange/rl 4, Citrus excelsa Leaf blotch Citrus leaf blotch virus (CLBV) Dweet tangor RT-PCR 5 Dweet mottle Exocortis Cachexia and Cachexia various citrus growth abnormalities Huanglongbing (Citrus greening) Citrus variegation virus Sour orange Leaf rugose Citrus leaf rugose virus Crinkly leaf Citrus crinkly leaf virus Etrog citron, Eureka lemon Tatter leaf-citrange stunt Dweet mottle virus (DMV) Apple stem grooving virus (ASGV) syn. Citrus tatter leaf virus (CTLV) Rusk citrange/rl 4, Citrus excelsa Citrus exocortis viroid (CEVd) Hop stunt viroid (HSVd) HSVd citrus variants: CVd-IIb & -IIc: Cachexia Leaf blotch Citrus leaf blotch virus (CLBV) Dweet tangor RT-PCR 5 Dweet mottle Exocortis and various citrus growth abnormalities Huanglongbing (Citrus greening) Dweet mottle virus (DMV) Citrus exocortis viroid (CEVd) Hop stunt viroid (HSVd) HSVd citrus variants: CVd-IIb & -IIc: Cachexia Etrog citron, Arizona 861- S1/RL 6, spage 7, 8 Five citrus viroids species Candidatus Liberibacter sp. Five citrus viroids species Candidatus Liberibacter sp. Sweet orange 9, PCR 10, Source plant observation 11 Citrus variegated chlorosis Xylella fastidiosa PCR 12 Citrus variegated chlorosis Xylella fastidiosa PCR 12 Etrog citron, Arizona 861- S1/RL 6, spage 7, 8 Sweet orange 9, PCR 10, Source plant observation 11 Stubborn Spiroplasma citri Culture Spiroplasma citri 13 Culture 13 1 Core tests are included in the NAPPO listed tests for citrus germplasm. Preferable test is highlighted 1 Core 2 Indexing on seedlings of the indicated host at cool temperatures (24-27 tests are included in the NAPPO listed tests for citrus o C)unless otherwise described. germplasm. Preferable test is highlighted 2 3 Enzyme Linked Immunosorbent Assay. Indexing 4 Rough on lemon seedlings rootstock. of the indicated host at cool temperatures (24-27 o C)unless otherwise described. 3 Enzyme 5 Reverse Linked Transcription Immunosorbent Polymarase Chain Assay. Reaction. 4 6 Warm test (28-40 o C). Rough 7 lemon rootstock. 5 Sequential Polyacrylamide Gel Electrophoresis. Reverse 8 On tissue Transcription from greenhouse Polymarase grown graft inoculated Chain citron, Reaction. preferably from warm test. 9 6 Sweet orange grown Warm test (28-40 o in both warm (Asian-HLB) and cool (African-HLB). American-HLB is under investigation, will C). 7 adjust accordingly when results will be available. Sequential 10 If possible Polyacrylamide with all different set of Gel primers Electrophoresis. for Asian, African, & American HLB. Otherwise state the test that was 8 performed. On tissue 11 Source from plant must greenhouse come from a trusted, grown protected, graft inoculated facility (i.e. Florida: citron, CGIP/CBR, preferably California: from CCPP warm or NCGRCD test. 9 Sweet screen orange houses). grown A minimum both of 18 warm months of (Asian-HLB) observation for leaf and and/or cool fruit (African-HLB). symptoms. American-HLB is under investigation, will adjust 12 Use of generic primers for the species. accordingly when results will be available Culture from tissue of the source tree after maintained a minimum of 3 months in elevated greenhouse If possible temperatures. with all different set of primers for Asian, African, & American HLB. Otherwise state the test that was performed Citrograph July/August 2010 Source plant must come from a trusted, protected, facility (i.e. Florida: CGIP/CBR, California: CCPP or NCGRCD

23 are the Citrus Clonal Protection Program at the Rubidoux Quarantine Facility in Riverside, CA, the USDA-ARS National Germplasm Repository for Citrus and Dates facility also in Riverside, CA, and the Citrus Germplasm Introduction Program in Gainesville, FL. The National Clean Plant Network (NCPN) The NCPN was established under a provision of the 2008 USA Farm Bill. The NCPN facilitates the partnership of clean plant centers for diagnosis and pathogen elimination for the production and maintenance of clean propagative plant material in sites located throughout the US. The NCPN is focused on the protection of US specialty crops, such as grapes, nuts, apples, peaches, citrus and other fruits, from the spread of economically harmful plant pests and diseases. The NCPN is a collaborative effort among three USDA agencies: the Animal and Plant Health Inspection Service (APHIS) for quarantine and regulatory programs, the Agricultural Research Service (ARS) for technology and germplasm issues, and the Cooperative State Research, Education, and Extension Service (CSREES) for outreach and partnership initiatives. However, the NCPN includes in its function representatives from the nursery industry, the grower community, the National Plant Board (NPB) and other state regulatory agencies, and the land-grant university system. The mission of NCPN is to provide high-quality asexually propagated plant material free of targeted plant pathogens and pests that cause economic loss, to protect the environment and ensure the global competitiveness of specialty crop producers. To achieve this mission, the NCPN will fund the organizational structure; germplasm program operations; the development of methods and technologies for disease and pest testing and elimination; the extension, education, and outreach; and, the review and quality assurance of the regional centers of the program. NCPN s regional centers of excellence are recognized leaders in the introduction of the highest quality, regionally adapted, true-to-type propagative plant materials that are free of targeted plant pathogens and pests. Starting in 2007 citrus has been invited to the NCPN functions, and finally in March of 2010 all the state and national citrus germplasm programs were officially recognized as regional centers of excellence, and the Citrus CPN charter and Governing Body were finalized. In June of 2010 the USA citrus programs submitted a request for over a million dollars to NCPN for research, facilities and infrastructure upgrades, personnel, and extension outreach. Participants have been notified that funding has been awarded, but at the time of this writing the level has not been publicized. For additional information regarding the NCPN you can visit: and health/ncpn/index.shtml or contact Erich Rudyj, Erich.S.Rudyj@aphis. usda.gov Coordinator - National Clean Plant Network (NCPN) USDA, APHIS, PPQ Plant Health Programs Plant Safeguarding and Pest Identification (PSPI) 4700 River Road, Unit 52 Riverdale, MD USA Authors affiliation information 1 Citrus Clonal Protection Program, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA, [email protected] 2 Texas A & M University-Kingsville, Citrus Center, Weslaco, TX 78596, USA, [email protected] 3 Florida Department of Agriculture and Consumer Services, Division of Plant Industry. PO Box , Gainesville, FL, 32614, [email protected] 4 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, 302 Life Science, Baton Rouge, LA 70803, USA, [email protected]. edu 5Florida Department of Agriculture and Consumer Services, Bureau of Citrus Budwood Registration, 3027 Lake Alfred Rd. (US 17), Winter Haven, FL 33881, USA, [email protected] & sieburp@doacs. state.fl.us 6 USDA-ARS National Clonal Germplasm Repository for Citrus & Dates, Riverside, CA 92507, USA., robert.krueger@ars. usda.gov & [email protected] 7 Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii, 3050 Maile Way, Honolulu, HI 96822, USA, [email protected] 8 Alabama Agricultural Experiment Station, Auburn University, 411 rth McGregor Ave., P.O. Box 8276, Mobile, AL 36689, USA, [email protected] 9 Citrus Research Board, 323 W. Oak, P.O. Box 230, Visalia, CA 93279, marylou@ citrusresearch.org 10 Florida Department of Agriculture and Consumer Services, Citrus Germplasm Introduction Program, 1911 SW 34th St. Gainesville, FL, 32608, USA, willial1@doacs. state.fl.us 11 University of Arizona, Yuma Agriculture Center, 6425 W. 8th Street, Yuma, AZ 85364, USA, [email protected] Selected literature Bash J The California Citrus Clonal Protection Program. Pages in: Proceedings of the 5th ISCN International Congress. Gottwald T. R., Polek M., and Riley K. M Citrus Tristeza Virus History, Present Incidence, and Spatial Distribution of Citrus tristeza virus in the California Central Valley. Pages in: Proc. 15th Conf. Int. Organ. Citrus Virologists. IOCV, Riverside. Gumpf D. J., Bash J., Greer G., Diaz J., Serna R. and J. Semancik The California Citrus Clonal Protection Program. Proc. Int. Soc. Citriculture, Hiltabrand W.F Certification program for maintenance of virus-free propagation sources of citrus in California. In: Wallace J. M. (ed.) Citrus virus diseases. pp Univ. Calif. Div. Agr. Sci., Berkeley. Kahlke C. J., Watson J. W., Solís-Gracia N., Skaria M., and da Graça J. V The Texas Citrus Budwood Certification Program. Pages in: Proc. 16th Conf. Int. Organ. Citrus Virologists. IOCV, Riverside. Krueger R.R The California citrus certification program. Pages in: Proceedings of the 5th ISCN International congress. Lee, R. F., P. Lehmann and L. Navarro Nursery practices, budwood and rootstock certification programs. In: Citrus Health Guide. L. W. Timmer and L. Duncan, Eds. APS Press, Minn. Pgs Reuther W The Citrus Clonal Protection Program. Calif. Agriculture. 35: Reuther W., Calavan E. C., Nauer E. M. and Roistacher C. N The California citrus variety improvement program after twelve years. pp In: Proc. 5th Conf. Int. Organ. Citrus Virologists. IOCV, Riverside. Rucks P Quality tree program for Florida citrus. Proc. Fla. State Hort. Soc. 107:4-8. Skaria, M., C. J. Kahlke, N. Solis-Gracia and R. Prewett Virus-free citrus budwood production and tristeza management program in Texas through industry partnership. Subtrop. Plant Sci. 49: 1-7. l July/August 2010 Citrograph 39