Progress 10/01/08 to 10/01/13

Outputs
Target Audience:Citrus industry General public-citrus hobbyists enthusiasts State and Federal regulatory agencies Citrus researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Extension of citrus pathogens and varieties related information for the education of the industry and the public. The CCPP collects fruit evaluation data from field propagations during 2-3 week intervals just prior to and during fruit maturity. All fruit evaluation data are presented in the CCPP web site at: http://ccpp.ucr.edu/variety/index.html. The CCPP hosts annual Foundation Block Field Days which were very well attended. During this field day the entire foundation collection, cultivar bank, and experimental plots were available for viewing and fruit tasting. Fruit from CCPP trees was also made available for the annual Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Introduction of propagative materials of citrus varieties in the state of California. Between October 2008 and September 2013, the CCPP received 58 citrus introductions (IPPN 438-495) at its renovated Rubidoux Quarantine Facilities. Sources of 49 introductions (23 public domain and 26 proprietary) were successfully established and disease testing and therapy procedures were initiated. Disease testing and pathogen elimination. Between October 2008 and September 2013, the CCPP performed 65 Variety Index tests of public domain and proprietary varieties (VI 777-841) as well as citrus experimental materials (e.g. citrus breeding program, positive disease controls). As a result, 50 citrus introductions became available to the citrus industry and researchers. The public domain citrus varieties (Table 1) have been deposited in the CCPP Foundation Block at the Lindcove Research and Extension Center (LREC). Table 1. Public domain citrus varieties completed VI index between October 2008 and September 2013 VI Variety Origin 777 Primasole IVIA, Spain 778 Fukumoto navel IVIA 364 IVIA, Spain 779 Miyamoto Satsuma IVIA, Spain 781 Iwasaki Satsuma IVIA, Spain 785 Kinnow LS Mandarin CPB, UCR 792 Bitters C-22 trifoliate hybrid CPB, UCR 793 Carpenter C-54 trifoliate hybrid CPB, UCR 794 Furr C-57 trifoliate hybrid CPB, UCR 795 Rush navel Porterville, CA 797 Ueno Satsuma IVIA 406 IVIA, Spain 798 Maltaise Demi Sanguine Blood Orange IVIA 393 IVIA, Spain 799 Louisiana Early Satsuma (LA Early) LSU, Louisiana 803 Femminello Siracusano White Flower Lemon Italy 804 Kao Nun Pummelo Thailand 805 Fukushu Kumquat USDA, Riverside 806 C. sunki USDA, Riverside 825 Early St. Ann Satsuma LSU, Louisiana 835 Santa Teresa Lemon (W&N) Bakersfield, CA 836 Fukushu Kumquat (W&N) Bakersfield, CA IVIA: Instituto Valenciano de Investigaciones Agrarias, CPB: Citrus Breeding Program-University California Riverside, LSU: Louisiana State University, and USDA: United States Department of Agriculture-National Clonal Germplasm Repository for Citrus and Dates. Maintenance and continuous disease testing of the established trees sources for citrus propagative materials. When the new citrus introductions have successfully completed the VI index and are released from the State and Federal quarantines, they are then moved to the CCPP Foundation Block at LREC for budwood distribution and horticultural evaluation. The movement of citrus germplasm from the Riverside Quarantine Facilities to the LREC Foundation Block is done in accordance with the CDFA and USDA regulations for diseases and pests quarantines (Permit No. QC 1353: Permit For The Intrastate Movement Of ACP/CTV/GWSS Host Propagative Material/Plants). The CCPP Foundation Block trees are carefully evaluated several times during each year for trueness-to-type, fruit quality and overall health and growth characteristics. All CCPP trees at LREC are tested annually for tristeza for the life of the tree. Trees registered for budwood distribution receive additional tests as required by the CDFA regulations (Section 3701, Citrus Nursery Stock Pest Cleanliness Program). Distribution of true-to-type citrus propagative material for the needs of the California citrus industry and researchers. Between October 2008 and September 2013 the CCPP executed 581 orders and distributed 149,095 buds of 290 different varieties. Mandarins represented approximately 35% of the budwood demand. Navel oranges, lemons, and specialty varieties represented 14%, 12%, and 9% of the distributed budwood, respectively.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, J., Boubourakas, I.N., Voloudakis, A.E., Agorastou, T., Magripis, G., Kyriakopoulou, P.E., Vidalakis, G. 2013. Identification and Characterization of Viroids in the Greek National Citrus Germplasm Collection Reveals Novel Viroid Variants and Threats to the Industry. European Journal of Plant Pathology. Vol. 137: p.17-27.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus Huanglongbing disease. Molecular Plant. Vol. 6: p.301-310.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: A. Citrus Clonal Protection Program (CCPP) The CCPP is primarily supporting the California citrus industry. However, since 2010 CCPP is playing a national role as part of the National Clean Plant Network (NCPN). CCPP is also distributing budwood to quarantine programs of countries around the world. During 2012, the CCPP received 16 varieties for introduction into California. Sources of the newly introduced varieties were established under quarantine at the Rubidoux facilities and disease testing or VI index and therapy procedures were initiated. VI index is the most comprehensive testing including inoculation of 60 seedlings and extensive laboratory testing. In 2012, the CCPP also tested 1800 samples from scion and seed registered source trees of California citrus nurseries. In 2012, the CCPP executed 103 orders, for 221 varieties-VIs, and total of 28,531 buds. The Foundation Block Field Day was hosted in March and fruit from CCPP trees was made available for the Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. B. Study of pathogenic and host regulating graft transmissible agents of citrus The population and molecular dynamics of Citrus tristeza virus in California for the past decades were studied and presented in an international conference and an invited abstract in Frontiers in Virology. The protocols of the previously developed molecular method for universal detection of citrus viroids (US patent application 61/556634) and the automated nucleic acid extraction method, also developed in my laboratory were finalized and approved for use for use in the Citrus Nursery Stock Pest Cleanliness Program, on September 10, 2012-Permit No. QC 1354 by the CDFA. We developed a novel diagnostic method for citrus stubborn disease using a secreted protein of Spiroplasma citri (causal agent) as the detection marker. Because S. citri resides in the phloem, secreted proteins could disperse through the plant transportation system, thereby facilitating detection. We identified S. citri proteins that are secreted in high levels in the presence of phloem extract by mass spectrometry. We then generated antibodies against one of these proteins (ScCCPP1) and used it in direct tissue imprint detection assays. Remarkably, ScCCPP1 antibody robustly detected S. citri-infected trees, in a manner that at certain cases was even more reliable than quantitative PCR. Furthermore, using an experimental protocol for mechanical inoculation of S. citri, we successfully traced S. citri during infection development using both PCR- and ScCCPP1 antibody-based methods. In conclusion, ScCCPP1 antibody holds the promise of developing robust immunoassay-based diagnostic methods for citrus stubborn disease. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A. Citrus Clonal Protection Program. Continued availability of disease tested propagation material from the CCPP is essential for the research projects conducted by the University and it is extremely important for the sustainability of the citrus industry. In 2012, the NCPN supported the CCPP activities with approximately $820,000. The majority of those funds were used for the upgrades of the CCPP Citrus Quarantine Facilities (one of the three of its kind in USA) in order to face the threat of the invasive species Asian citrus psyllid and the vectored disease Huanglongbing, one of the most catastrophic diseases of citrus. The combination of the NCPN funds with UCR, citrus industry and private funds resulted on all appropriate upgrades and renovations for CCPP to be CDFA & USDA certified and under compliance for operation within three different pest quarantines as of August 2012. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Seven distinct viroid species representing four genera of the Pospiviroidea family have been identified in citrus. Currently, the standard molecular citrus viroid detection method is the conventional RT-PCR. The conventional RT-PCR requires seven different protocols with seven different sets of primers for the detection of all known citrus viroids as well as gel electrophoresis for visualization of the results. Reverse Transcription followed by Quantitative Real Time PCR (RT-qPCR)is one of the latest technologies in the field of pathogen detection. We developed two sets of degenerate primers and their respective SYBR Green RT-qPCR protocols capable of detecting all known citrus viroids in two reactions. This method revolutionized the California program for nursery owned citrus germplasm source trees which since May 2010, has been mandatory and approximately 7000 new samples required immediate viroid testing. The newly developed method was able to complete viroid testing in two seasons. This method can also benefit private and public diagnostic labs or plant pathology clinics as well as citrus germplasm programs worldwide. Citrus worldwide is facing threats from insect-transmitted and phloem-limited bacterial diseases such as huanglongbing (HLB) and citrus stubborn disease(CSD). Management of such diseases is difficult, expensive, heavily based on eradication and insecticide treatments for vector control. The success of any disease management program is based on early pathogen detection. The most commonly used diagnostic or survey methods are PCR based and require the presence of bacterial cells or DNA on the tested sample for positive diagnosis. This can be problematic in the case of citrus since only a few leaves or stems from a whole tree and eventually a few mg of tissue are processed for DNA extraction while the titer and distribution of the pathogen within trees, types of tissue and disease progression is variable. Our newly developed diagnostic method of CSD using secreted proteins as the detection markers addresses the issues of low titer and uneven distribution in the plant host but most importantly it can be adapted for the diagnosis of ecologically similar diseases such as HLB.

Publications

• Vidalakis, G., et al., (contributor) 2012. Diseases. Integrated Pest Management for Citrus Third Edition. Editors: University of California Statewide Integrated Pest Management Program. University of California Agricultural and Natural Resources (UC ANR) Publication 3303. p.39-84. 270p.
• Wang, J., Bozan, O., Kwon , S.J., Rucker , T.R., Thomas , C.T., Yokomi, R.K., Lee, R.F., Folimonova, S., Vidalakis, G. 2012 Molecular diversity of Citrus tristeza virus strains collected over the past 50 years and maintained in planta collections in California. International Citrus Congress. November 18-23. Valencia, Spain. Conference,Meeting 11/18/2012. Accepted 07/26/2012).
• Shi, J.S., Pagliaccia, D., Morgan , R.M., Ma, W., Vidalakis, G. 2012 Serological detection of Spiroplasma citri using a bacterial secreted protein as the detection marker. International Citrus Congress. November 18-23. Valencia, Spain. Conference,Meeting Date: 11/18/2012. (Accepted 07/31/2012).
• Wang, J., Bozan , O., Kwon , S.J., Rucker , T.R., Yokomi, R.K., Lee, R.F., Folimonova, S., Greer, G.D., Krueger, R.R., Bash, J.A., Vidalakis, G. 2012. Past and Future of a Century Old Citrus Tristeza Virus Collection-A California Citrus Germplasm Tale. Frontiers in Virology Closteroviridae. Editor(s): Flores R., Moreno P., Falk B., Martelli P. G. (Accepted 09/03/2012).
• Karp, D., Kahn, T., Siebert, T., Krueger, R.R., Lee, R.F., Vidalakis, G. 2012. Unforbidden Fruits: Preventing Citrus Smuggling by Introducing Varieties Culturally Significant to Ethnic Communities. Citrograph. Vol. 3,5-Sept,Oct p.40-48. Website: www.citrusresearch.org/current-citrograph

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: A. Citrus Clonal Protection Program (CCPP). The CCPP is primarily supporting the California citrus industry. However, since 2010 CCPP is playing a national role as part of the National Clean Plant Network (NCPN). CCPP is also distributing budwood to quarantine programs of countries around the world. During 2011, the CCPP received four proprietary and four public domain varieties for introduction and/or release into California. Sources of the newly introduced varieties were established under quarantine at the Rubidoux facilities and disease testing or VI index and therapy procedures were initiated. VI index is the most comprehensive testing including inoculation of 60 seedlings and extensive laboratory testing. In 2011 the CCPP also tested 2700 samples from scion and seed registered source trees of 38 citrus nurseries. In 2011, the CCPP executed 111 orders, for 220 varieties VIs, and total of 28,965 buds. The Foundation Block Field Day was hosted in February and fruit from CCPP trees was made available for the Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. B. Study of pathogenic and host regulating graft transmissible agents of citrus, The studies of population and evolutionary dynamics of Citrus exocortis viroid at the level of a single cell, young seedlings arid mature plants were published in Virology 417(2):400-409. In 2011, we developed a molecular method for universal detection of citrus viroids (US patent application 61/556634). This method is coupled with an automated nucleic acid extraction method, also developed in my laboratory during 2011, and provides a powerful, efficient, high though put, and economic tool for detection of seven citrus viroid species in two reactions of one step reverse transcription quantitative polymerase chain reaction (RT-qPCR). During 2011, we studied the genetic profile of emerging California isolates of the Citrus tristeza virus (CTV), the main threat to the citrus budwood production in California. The characterization of unknown viruses of citrus via the isolation of dsRNA and deep sequencing is progressing with the identification of uinbraviruses and luteoviruses in the case of yellow vein disease whereas the same luteovirus species was identified in the vein enation infected material. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A. Citrus Clonal Protection Program. Continued availability of disease tested propagation material from the CCPP is essential for the research projects conducted by the University and it is extremely important for the sustainability of the citrus industry. In 2011, the NCPN supported the CCPP activities with approximately $655,000. The majority of those funds were used for the upgrades of the CCPP Citrus Quarantine Facilities (one of the three of its kind in USA) in order to face the threat of the invasive species Asian citrus psyllid and the vectored disease l-fuanglongbing, one of the most catastrophic diseases of citrus. The combination of the NCPN funds with UCR, citrus industry and private funds resulted in the establishment of the "Delfino Family Plant Laboratory" at the Rubidoux CCPP Citrus Quarantine Facilities in 11/8/2011. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Citrus exocortis viroid (CEVd) populations exist as heterogeneous variants in plant hosts. We studied the CEVd populations in citrus protoplasts, seedlings and mature citrus plants. Selected variants were evaluated for their pathogenicity in three CEVd hosts, replication efficacy in citrus protoplasts and systemic accumulation In citron seedlings. For accurate quantification of replication, we developed a SYBR green based quantitative PCR assay. Sequence variants had a range of phenotypes including loss of infectivity, significantly higher levels of replication and altered systemic accumulation. Seven distinct viroid species representing four genera of the Pospiviroidea family have been identified in citrus. Currently, the standard molecular citrus viroid detection method is the conventional RT-PCR and several protocols have been developed. However, the conventional RT-PCR requires seven different protocols with seven different sets of species specific primers for the detection of all known citrus viroids as well as gel electrophoresis for visualization of the results. Reverse Transcription followed by Quantitative Real Time PCR is one of the latest technologies in the field of pathogen detection. We developed two sets of degenerate primers and their respective SYBR Green RT-qPCR protocols capable of detecting all known citrus viroids in two reactions. This method will simply revolutionize the California program for nursery owned citrus germplasm source trees. Currently the viroid detection is based on bio-indexing - its capacity is 800 samples per year. In May 2010, the program became mandatory and as of 0l-2012 all nursery owned source trees have to be inside insect protective structures. These two events generated approximately 7000 new samples requiring immediate viroid testing. It would have taken more than 8 years to test these samples with bio-indexing. The newly developed method was able to complete this task in two testing seasons. This method can also benefit private and public diagnostic labs or plant pathology clinics as well as specialized citrus germplasm quarantine and certification or registration programs since citrus germplasm importation is prohibited in most citrus producing countries of the world including USA.

Publications

• 1. Hajeri, S., C. Ramadugu, K. Manjunath, J. Ng, R. Lee & G. Vidalakis. 2011. In vivo generated Citrus exocortis viroid progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity. Virology, 417: 400-409.
• 2. R. K. Yokorni, G. Vidalakis, and M. Saponari. 2011. Molecular analysis among MCA 13-reactive isolates reveals a rapid strategy for assessment of Citrus tristeza virus severity. Acta Horticulturae. Accepted 02/09/20 11, pgs. 6.
• 3. Francesco DiScrio, John W. Randles, Ricardo Flores, Robert A. Owens, Temo Sano, SIii-Fang Li, Georgios Vidalakis, and Vicente PalIas, Viroids. In: Andrew M.Q. King, Michael J. Adams, Eric B. Carstens, and Elliot J. Lefkowitz, editors, Virus Taxonomy. Oxford: Elsevier, 2011, pp. 122 1-1234.
• 4. A. Adcsemoye, A, Estilen, J, Mayorquin, C, Roper, P. Wang, G. Vidalakis, C. Kalisen and N. O Connell. 2011. Understanding foamy bark rot of Fukumoto navel. Citrograph, 7: 26-28.
• 5. R. Lee, M. Keremane, C. Ramadugu, G. Vidalakis, M. Roose, S. 1-lalbert, J. L. Rodrigues and S. Lopes. 2011. Huanglongbing: Development of information needed for avoidance/management. Citrograph, 7: 34-40.
• 6. T. Siebert, G. Vidalakis, R. Krueger, J. Bash and T. Kahn. 2011. Background and initial evaluations of recently introduced cultivars distributed by the Citrus Clonal Protection Program. Citrograph, 4: 28-35.
• 7. A. Eskalen, G. Vidalakis, N. O Connell. 2011. Occurrence of citais quick decline in California. APS/IAPPS Joint meeting. August 6-10, Honolulu, Hawaii.
• 8. S. Rajen, J. Ng, C. Ramadugu, M. Keremane, R. Lee, G. Vidalakis. 2011. Study of Citrus exocortis viroid replication in citrus protoplasts. APS/IAPPS Joint meeting. August 6-10, Honolulu, Hawaii.
• 9. A. Adesemoyc, A. Eskalen, N. O Connell, G. Vidalakis, P. Wang, C. Roper. 2011. Foamy bark rot of Fukurnoto navel: A condition with etiology not yet understood. APS/EAPPS Joint meeting. August 6-10, Honolulu, Hawaii.
• 10. S. Kanrar, M. Afunian, G. Greer, I. Tzanetakis, G. Vidalakis. 2011. Viruses associated with yellow vein and vein enation disease of citrus. APS/IAPPS Joint meeting. August 6-lO, Honolulu, Hawaii.
• 11. G. Vidalakis. 2011. Citrus viral and prokaryotic pathogens, new discoveries arid development of novel detection methods relevant to citrus germplasm quarantine and registration programs. 9th International Congress of Citrus Nurserymen, Tucumon, Argentina, June 13-16.
• 12. G. Vidalakis. 2011. Dwarfing of citrus on Poncirus trifoliata and P. trifoliate hybrid rootstocks using TransmissibIe small nuclear ribonueleic acids (TsnRNAs). 9th International Congress of Citrus Nurserymen, Tucurnan, Argentina, June 13-16.
• 14. K. Manjunath, M. Irey, C. Ramadiigu, R.F. Lee, C. LeVesque, B. Brady, M. Polek, H. Lin, 13. Civerolo, M. Afunian, and G. Vidatakis. 2011. Comparison of different extraction and assay protocols in different laboratories to develop a standardized assay for detection of hnanglongbing-associated bacteria from psyllids. 2nd International Research Conference on Huanglongbing. January 10-14, Orlando, Florida.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: A. Citrus Clonal Protection Program-CCPP. The CCPP is primarily supporting the California citrus industry. However, states such as Texas and Arizona are depended on CCPP for propagative material. CCPP is also distributing budwood to quarantine programs of countries around the world. During 2010, the CCPP received one proprietary and 5 public domain varieties for introduction and/or release into California. Sources of the newly introduced varieties were established under quarantine at the Rubidoux facilities and disease testing or VI index and therapy procedures were initiated. VI index is the most comprehensive testing including inoculation of 60 seedlings and extensive laboratory testing. In 2010, the CCPP also tested 660 samples from scion and seed registered source trees of 33 citrus nurseries. In 2010, 8 new varieties (3 from Spain, 1 from Italy, 1 from Thailand, and 3 from the USA) were released from quarantine and to the public domain. The CCPP budwood online ordering system performed well while the ordering history of each user was updated with information prior to the lunch of the system in 2007. In 2010, the CCPP executed 135 orders, for 236 varieties-VIs, and total of 24,621 buds with the Mandarins representing approximately 35percent of the budwood demand double of that of Navel oranges. Fruit evaluation data were collected from field plantings and the process of posting in the new dynamic CCPP site was continued. The Foundation Block Field Day was hosted in December and fruit from CCPP trees was made available for the Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A. Citrus Clonal Protection Program-CCPP. Continued availability of disease tested propagation material from the CCPP is essential for the variety of research projects conducted by the University and it is extremely important for the protection of California's citrus industry. In 2010, the CCPP entered officially the National Clean Plant Network (NCPN) for Specialty Crops and Georgios Vidalakis was elected as Chair of the Citrus CPN that includes all citrus producing states. In 2010, the NCPN supported the CCPP activities with approximately 850,000. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Citrus exocortis viroid (CEVd) populations exist as heterogeneous variants in plant hosts. We studied the CEVd populations in citrus protoplasts, seedlings and mature citrus plants. Selected variants were evaluated for their pathogenicity in 3 CEVd hosts, replication efficacy in citrus protoplasts and systemic accumulation in citron seedlings. For precise quantification of replication, we developed a SYBR green based quantitative PCR assay. Sequence variants had a range of phenotypes including loss of infectivity, notably higher levels of replication and altered systemic accumulation. The term transmissible small nuclear ribonucleic acids(TsnRNAs) describes well-characterised viroid RNA species that do not induce any pronounced disease syndromes in specific citrus hosts, but rather act as regulatory genetic elements modifying tree performance. Trifoliate rootstock interaction with Tsn-RNA IIIb reduces tree canopy volume and enhances fruit size. Preliminary field trials however, indicated that the rootstock Carrizo citrange required a mixture of the Tsn-RNA IIIb, IIa, and Ia for tree size reduction. Although Ia is the critical factor for tree size reduction on Carrizo, IIIb and/or IIa is necessary to significantly reduce the canopy volume. Tsn-RNA IIIb and IIa mix had no effect on tree volume. The canopy volume of 13-yearold navel orange trees on Poncirus trifoliata rootstock treated with TsnRNA-IIIb was reduced by 0.45 and 0.53 in standard-density (6m x 6.7 m) or high-density (3m x 6.7 m) plantings, respectively. The total yield of 8 consecutive harvests was not affected significantly by the TsnRNA-IIIb treatments or the two planting densities. However, the yield per land surface unit (Y/LSU) was increased by 97.5percent for the high-density plantings over the standard-density plantings of the untreated controls. The Y/LSU of the TsnRNA-IIIb treated navel orange trees in the standard-density planting was reduced by 0.327. The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the family Alpha- and Beta-flexiviridae. The DMV genome has 8747 nucleotides (nt) excluding the 3' poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5' and 541 nt at 3' termini. Protein database searches showed 0.98-0.99 matches of DMV ORFs with Citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis, based on RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus.

Publications

• G. Vidalakis, D. Pagliaccia, J. A. Bash, M. Afunian, & J. S. Semancik. 2010. Effects of Citrus dwarfing viroid, a transmissible small nuclear RNA, on tree size and scion performance specific to Poncirus trifoliata rootstock with applications for high density planting. Annals of Applied Biology- Submitted 08/02/10, AAB-2010-0241, Accepted with revision 09/19/10, Published online 12/19/10, DOI:10.1111/j.1744-7348.2010.00454.x.
• S. Hajeri, C. Ramadugu, M. Keremane, G. Vidalakis, and R. Lee. 2010. Nucleotide Sequence and Genome Organization of Dweet mottle virus and Its Relationship to Members of the Family Betaflexiviridae. Archives of Virology. 155:1523-1527.
• G. Vidalakis, D. Pagliaccia, J.A. Bash & J.S. Semancik. 2010. Effects of mixtures of citrus viroids as Transmissible small nuclear RNA (TsnRNA) on tree dwarfing and commercial scion performance on Carrizo citrange rootstock. Annals of Applied Biology. 157:415-423.
• G. Vidalakis, D. Gumpf, M. Polek, and J. Bash. 2010. The California Citrus Clonal Protection Program. In: The Citrus Manual (in preparation). UC ANR Publication. Accepted 07/23/2010, pgs. 28.
• Vidalakis G., da Graca J. V., Dixon W. N., Ferrin D., Kesinger M., Krueger R. R., Lee R. F., Melzer M. J., Olive J., Polek M., Sieburth P. J., Williams L. L., Wright G. C. 2010. Citrus quarantine, sanitary and certification programs in the USA. Prevention of introduction and distribution of citrus pests. Part 2 certification schemes and national programs and efforts. Citrograph, 4:27-39.
• Vidalakis G., da Graca J. V., Dixon W. N., Ferrin D., Kesinger M., Krueger R. R., Lee R. F., Melzer M. J., Olive J., Polek M., Sieburth P. J., Williams L. L., Wright G. C. 2010. Citrus quarantine, sanitary and certification programs in the USA. Prevention of introduction and distribution of citrus pests. Part 1-quarantine and introduction programs. Citrograph, 3:26-35.
• B. Grafton-Cardwell, G. Vidalakis, and R. Copeland. 2010. University of California Lindcove Research and Extension Center celebrates its 50th Anniversary. Citrograph, 2:8-11.
• A. Eskalen, G. Vidalakis, and Neil O Connell. 2010. Citrus quick decline: a disease complex. Citrograph, 1:22-23.
• R. Krueger and G. Vidalakis. 2010. National Clean Plant Network (NCPN) Citrus Clean Plant Network Formed. International Organization of Citrus Virologists Newsletter, May 2010.
• T. Siebert, R. Krueger, T. Kahn, J. Bash and G. Vidalakis. 2010. Descriptions of new varieties recently distributed from the Citrus Clonal Protection Program. Citrograph, 2:20-26.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: A.Citrus Clonal Protection Program-CCPP. The CCPP is primarily supporting the CA citrus industry. However, states such as TX and AZ are depended on CCPP for propagative matrl. CCPP is also distributing budwood to quarantine programs of countries around the world. During 2009, the CCPP received 9 proprietary and 3 public domain varieties for introduction and/or release into CA. Sources of the newly introduced varieties were established under quarantine at the Rubidoux facilities and disease testing or VI index and therapy procedures were initiated. VI index is the most comprehensive testing including inoculation of 60 seedlings and extensive laboratory testing. In addition, during 2009 the CCPP established a real time qPCR testing facility for the Huanglongbing disease of citrus. This lab will supplement and greatly enhance the VI index of the CCPP and will provide the opportunity for the development and employment of new lab tests for the detection of other citrus pathogens. In 2009, the CCPP also tested 644 samples from scion and seed registered source trees of 21 citrus nurseries. In 2009, 8 new varieties (3 from Spain, 1 from Italy, 1 from Thailand, and 3 from the USA) were released from quarantine and to the public domain. The CCPP budwood online ordering system performed well while the ordering history of each user was updated with information prior to the lunch of the system in 2007. In 2009, CCPP distributed approximately 34,500 buds (2008: 30,500, 2007: 24,500) with the Mandarins representing approx. 34% of the budwood demand double of that of Navel oranges. Fruit evaluation data were collected from field plantings and the process of posting in the new dynamic CCPP site was initiated. The Foundation Block Field Day was hosted in Dec. and fruit from CCPP trees was made available for the Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Study of population evolutionary dynamics of CEVd at the level of a single cell: Experiments with CEVd mutants were concluded and a manuscript is in prep. Yield, fruit quality, and tree size data were collected and analyzed from 3 Tsn-RNAs field trials. Effects on Carrizo citrange rootstock close spaced P. trifoliata rootstock, and screening of trifoliate hybrids dwarfed by Tsn-RNA. The Carrizo trial/manuscript was submitted to AAB and was accepted for publication after revision in Feb.2010. Yield, fruit quality, and tree size data from the other two trials are analyzed and two manuscripts are in prep. During 2009, the complete genome sequence of the Dweet mottle virus was determined and its relationship with other members of the Flexiviridae family was investigated.We initiated a new project for the characterization of unknown viruses of citrus via the isolation of dsRNA and deep sequencing. Progress was made for the isolation of dsRNA and cloning was initiated. A visiting student from Italy completed a garlic virus study in my lab and his work was submitted (12/2009) and is currently under review in Archives of Virology. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A. Citrus Clonal Protection Program-CCPP. Continued availability of disease tested propagation material from the CCPP is essential for the variety of research projects conducted by the University and it is extremely important for the protection of California's citrus industry. Recent national initiatives such as the National Clean Plant Network (NCPN) for Specialty Crops emphasizes the importance of programs such as CCPP which helps to maintain California and U.S. in the forefront of high quality fruit production fulfilling the demand for the highest quality propagation material for the newest and classic varieties. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Trifoliate rootstock interaction with Tsn-RNA IIIb reduces tree canopy volume and enhances fruit size. Preliminary field trials however, indicated that the rootstock Carrizo citrange required a mixture of the Tsn-RNA IIIb, IIa, and Ia for tree size reduction. Although Ia is the critical factor for tree size reduction on Carrizo, IIIb and/or IIa is necessary to significantly reduce the canopy volume. Tsn-RNA IIIb and IIa mix had no effect on tree volume. The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the family Alpha- and Beta-flexiviridae. The DMV genome has 8747 nucleotides (nt) excluding the 3' poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5' and 541 nt at 3' termini. ORF1 potentially encoding a 227.48 kDa polyprotein, which has methyltransferase, oxygenase, endo-peptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25kDa while ORF3 is the coat protein of 40.69 kDa polypeptide. Protein database searches showed 98-99% matches of DMV ORFs with Citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis, based on RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. Molecular and evolutionary characterization of viruses detected in Italian garlic varieties. Garlic plants from inflorescence produced bulbils of two Italian varieties as well as commercial bulbs from diverse regions of garlic production were tested for viruses using biological, serological, and molecular methods. Onion yellow dwarf virus (OYDV), Leek yellow stripe virus, Garlic virus X, and Garlic common latent virus were detected in single and mixed virus infections in both types of garlic germplasm. Phylogenetic and genealogical analysis indicated that the geographic origin, type of germplasm, or garlic variety was not a strong segregating factor during virus evolution. The intra-population variability expected for RNA viruses due to error prone replication was documented. Novel nucleotide and amino acid sequence patterns were identified for all viruses. For a California and Argentina OYDV isolate a cluster of 48-49 nucleotides with a predicted hairpin secondary structure and an amino acid signature unique to Chenopodium quinoa, an uncommon host of the virus, were recognized respectively.

Publications

• Vidalakis, G., J. V. da Graca, W. N. Dixon, M. Kesinger, R. R. Krueger, R. F. Lee, M. Polek, P. J. Sieburth, L. L. Williams, and G. C. Wright. 2009. Quarantine, sanitary and certification programs to prevent citrus quarantine pests in the USA. International workshop on citrus quarantine pests. Villahermosa, Tabasco, Mexico, July 27-31.
• M. Afunian, L. Parrano, D. Pagliaccia, and G. Vidalakis. 2009. Detection and characterization of virus mix in Italian garlic grown from inflorescence produced bulbils. Submitted, Archives of Virology 04 Dec 2009 AVIROL-D-09-00554.
• Kahn, T. and Vidalakis, G. 2009. The University of California-Riverside Citrus Variety Collection and Citrus Clonal Protection program Websites Provide Information, Photographs, Fruit Quality data and More. University of California Cooperative Extension, Topics in Subtropics Newsletter, Volume 7, No. 3. July-September 2009.
• Siebert T., Bier, O., Karp, D. Vidalakis, G., and Kahn, T. 2009. "Valentine" A Recently Released Anthocyanin-pigmented Pummelo Hybrid Developed at eh University of California, Riverside. University of California Cooperative Extension, Topics in Subtropics Newsletter, Volume 7, No. 3. July-September 2009.
• Changyong, Z. and Vidalakis, G. 2009. News from CRI, CAAS on the 11th International Citrus Congress Held in Wuhan P.R. China. International Organization of Citrus Virologists Newsletter, April 2009.
• Vidalakis, G. 2009. Asian citrus psyllid and Huanglongbing-A snapshot of the current status in California and U.S.A. International Organization of Citrus Virologists Newsletter, April 2009.
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2008. Observations on Field Performance of Tsn-RNA Dwarfed Poncirus trifoliata Hybrids. Proc. Int. Soc. Citriculture (in press).
• Vidalakis, G. 2009 (contributor). Plant Pathology. In Integrated Pest Management for Citrus. 3rd ed. (in preparation). University of California Statewide Integrated Pest Management Program. UC ANR Publication 3303. Accepted.
• Kahn, T. and Vidalakis, G. 2009. Citrus Scion Cultivars. In: The Citrus Manual (in preparation). UC ANR Publication xxxx. Accepted.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: A. Citrus Clonal Protection Program-CCPP. The CCPP is primarily supporting the California citrus industry. However, states such as Texas and Arizona are depended on CCPP for propagative material. CCPP is also distributing budwood to quarantine programs of countries around the world. In 2008, 14 varieties completed therapy and VI index. VI index is the most comprehensive testing including inoculation of 60 seedlings and extensive laboratory testing. From the 14 varieties released from quarantine 5 were produced by the citrus breeding program at UCR, 2 by USDA, and 1 belonged in a transgenic series produced for research at UCD. Six varieties were introduced from P.R. China, Spain, Cayman Islands, New Zealand, and Puerto Rico. The budwood tree sources were evaluated for trueness-to-type and fruit quality and were diseases tested as required by CA's registration program. Approximately 30,500 buds were distributed with Mandarins representing 30% of the distribution, triple of that of Navel oranges, Grapefruit/Pummelos, Specialty varieties, and Limes. Lemons were second with 20% of the distributed buds. Fruit evaluation data were collected from field plantings and posted online, the Foundation Block Field Day was hosted in December and fruit from CCPP trees was made available for the Lindcove fruit display for growers and homeowners, the World Ag Expo, the Citrus Mutual Citrus Showcase, and other events of this type. B. Study of pathogenic and host regulating graft transmissible agents of citrus. Study of population evolutionary dynamics of CEVd at the level of a single cell: A protocol for the development of citrus protoplasts and the inoculation with RNA transcripts was developed. Experiments with CEVd mutants are in progress. Identification of RNA citrus responses to S. citri infections and the identification of secretion systems and proteins involved in the pathogenesis of the stubborn disease: A protocol for mechanical inoculation of citrus seedlings with culture of S. citri was developed (reported in the International Citrus Congress-ICC in China) and extraction of citrus produced small RNAs was performed. Libraries of the extracted RNAs are constructed. Profiles of S. citri secreted proteins have been identified and compared with protein profiles induced by citrus phloem extracts. Characterization of specific proteins and producing genes is under way. Citrus variants of the HSVd as possible agents of the citrus gummy bark disease: This study was initiated in 2003. Bark and wood examination did not reveal any symptoms. We will monitor the trees for the next 5-6 years since the disease many times requires more than nine years to develop. Yield, fruit quality, and tree size data were collected and analyzed from 3 Tsn-RNAs field trials. Effects on Carrizo citrange rootstock, close spaced P. trifoliata rootstock, and screening of trifoliate hybrids dwarfed by Tsn-RNA. Results were presented in the ICC, in China. Field performance of clementine mandarins in the San Joaquin Valley: Yield, fruit quality, and tree size data were collected. One more year of data is required for a final publication. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A. Citrus Clonal Protection Program-CCPP. Continued availability of disease tested propagation material from the CCPP is essential for the variety of research projects conducted by the University and it is extremely important for the protection of California's citrus industry. Recent national initiatives such as the National Clean Plant Network (NCPN) for Specialty Crops emphasizes the importance of programs such as CCPP which helps to maintain California and U.S. in the forefront of high quality fruit production fulfilling the demand for the highest quality propagation material for the newest and classic varieties. B. Study of pathogenic and host regulating graft transmissible agents of citrus. It has been reported that mechanical inoculation of S. citri is not possible. We reported infection of citrus by mechanical inoculation with culture of S. citri. The culture was subjected to low speed centrifugation and re-suspension in small amount of medium to for highly concentrated inoculum. For inoculation a slit was made in young citrus seedlings and the wound was opened by forcing the bark to each side. 20ul of inoculum was placed into the wound. Trifoliate rootstock interaction with Tsn-RNA IIIb reduces tree canopy volume and enhances fruit size. Preliminary field trials however, indicated that the rootstock Carrizo citrange required a mixture of the Tsn-RNA IIIb, IIa, and Ia for tree size reduction. Although Ia is the critical factor for tree size reduction on Carrizo, IIIb and/or IIa is necessary to significantly reduce the canopy volume. Tsn-RNA IIIb and IIa mix had no effect on tree volume. Yield, fruit grade, and quality of Parent navel/Carrizo, were not affected significantly. Four P. trifoliata hybrids were used as seedlings and rootstocks for Parent navel orange and Oroblanco. Tree volume of X639 seedlings infected with Tsn-RNA mixture was significantly reduced in comparison with the controls. On the contrary, the Tsn-RNA mixture had no effect on the tree volume of the Citrumelo 1452, Citremon 1449, and Trifeola. Similarly, the volume of Parent navel and Oroblanco was not altered by the Tsn-RNAs infected rootstocks. The Oroblanco yield growing on Citremon rootstock infected with Tsn-RNA IIIb and Tsn-RNA mixture was increased by 39 and 30% respectively. The production of Tsn-RNAs infected Parent navel on Citrumelo 1452 and Trifeola increased from a minimum 14% (Citrumelo+IIIb) to a maximum 77% (Trifeola+mixture).Tsn-RNA IIIb significantly reduced the canopy volume of the navel orange on the trifoliate rootstock by an average of 44 and 53% on the standard and high density planting respectively. Total yield was not significantly different between the two planting densities of the dwarfed trees or the controls. However, the high density planting block had an increased yield over the standard planting and the controls by 66 and 11% respectively. Fruit grade and size of the dwarfed trees were not significantly different between the two planting densities or the controls. An 18-35% increase of the fruit production per canopy volume and a 5-10% increase in the higher commercial value fruit were determined for the Tsn-RNA dwarfed navel trees.

Publications

• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2007. Transmissible Small Nuclear RNA Dwarfing of Commercial Citrus on Carrizo Citrange Rootstock. Proc.17th Conf. IOCV, IOCV, Riverside, CA. (in press)
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2008. Tsn-RNA Dwarfing and High Density Planting of Citrus on Poncirus trifoliata rootstock. Proc. Int. Soc. Citriculture (in press)
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2008. Tsn-RNA Dwarfing and High Density Planting of Citrus on Poncirus trifoliata rootstock. Proc. 11th Int. Citrus Congress, 26-30 October, Wuhan, China.
• G. Vidalakis, J. Bash, J. Diaz, G. Greer, and R. Serna Citrus Clonal Protection Program-CCPP. Serving the California Citrus Industry over 50 Years. 2007. Centennial Symposium, Citrus Research Center-Agricultural Experiment Station, 15 February, University of California, Riverside.
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2007. Transmissible Small Nuclear RNA Dwarfing of Commercial Citrus on Carrizo Citrange Rootstock. 17th Conference of the International Organization of Citrus Virologists, 22-26 October, Adana, Turkey.
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2008. Observations on Field Performance of Tsn-RNA Dwarfed Poncirus trifoliata Hybrids. Proc. Int. Soc. Citriculture (in press)
• G. Vidalakis, J. A. Bash, and J. S. Semancik. 2008. Observations on Field Performance of Tsn-RNA Dwarfed Poncirus trifoliata Hybrids. 11th Int. Citrus Congress, 26-30 October, Wuhan, China.
• Georgios Vidalakis. 2007. Citrus Clonal Protection Program (CCPP). Page 60-61. In: Citrus Research Board 2007 Annual Report.
• Georgios Vidalakis. 2007. Citrus Clonal Protection Program (CCPP). Page 60-61. In: Citrus Research Board 2007 Annual Report.
• Georgios Vidalakis. 2008. From the Past and Present Secretaries. Editor: International Organization of Citrus Virologists Newsletter, June 2008. Georgios Vidalakis. 2008. From the Past and Present Secretaries. Editor: International Organization of Citrus Virologists Newsletter, June 2008.
• I. N. Boubourakas, G. E. Vidalakis, A. E. Voloudakis, T. Agorastou, G. Magripis and P. E. Kyriakopoulou. 2007. Pilot survey of citrus mother trees in Greece for the presence of viruses and viroids. Proc.17th Conf. IOCV, IOCV, Riverside, CA. (in press)
• I. N. Boubourakas, G. E. Vidalakis, A. E. Voloudakis, T. Agorastou, G. Magripis and P. E. Kyriakopoulou. 2007. Pilot survey of citrus mother trees in Greece for the presence of viruses and viroids. 17th Conference of the International Organization of Citrus Virologists, 22-26 October, Adana, Turkey.
• M. Afunian, H. Jin, W. Ma, and G. Vidalakis. 2008. Mechanical inoculation of citrus with culture of Spiroplasma citri, the causal agent of stubborn disease. 11th Int. Citrus Congress, 26-30 October, Wuhan, China.
• M. Keremane, C. Ramadugu, M. Luque-Williams, G. Vidalakis and R. Lee 2007. The Threat of Huanglongbing (Citrus Greening Disease) for California. Centennial Symposium, Citrus Research Center-Agricultural Experiment Station, 15 February, University of California, Riverside.
• R.K. Yokomi, M. Polek, E.E. Grafton-Cardwell, G. Vidalakis, N. O Connell, and M. Saponari. 2007. Rapid assessment of the Citrus tristeza virus isolates detected in spring 2007 at the Lindcove Research and Extension Center, Exeter, California. Proc.17th Conf. IOCV, IOCV, Riverside, CA. (in press)
• Subhas Hajeri, J. Ng, and G. Vidalakis. 2007. Study of the intra-population profile of Citrus exocortis viroid in protoplasts of suspension-cultured tobacco and citrus cells. Phytopathology 97:S44. APS/SON Joint Meeting, San Diego, California, USA.