2008 Annual Report 1a.Objectives (from AD-416) The overall goal of this proposed research project is to create a system to assist identification, differentiation and fingerprinting of Ca. L. spp. 1. Analyze available phenotypic and genotypic data to estimate population diversity of Ca. L. spp.; 2. Characterize population diversity of Ca. L. spp. based on current genomic information by using a large number of Ca. L. spp. strains; and 3. Search for new phenomic and genomic information and their applications in Ca. L. spp. differentiation and fingerprinting. 1b.Approach (from AD-416) HLB bacterial DNA will be obtained through collaboration. In phase 1, an extensive literature database on HLB will be constructed. Specific emphasis will be on linking available phenotypic data to more recent genomic discoveries. The second phase is to explore the use of single nucleotide polymorphisms (SNPs) to cluster Ca. L. asiaticus strains. Correlations will be built to link SNPs to geographic and host origins, and other phenotypes such as symptomatology. Efforts will be made to isolate and characterize new DNA sequences from the Ca. L. asiaticus genome for SNP analysis. New data will be added to increase sensitivity and accuracy of the HLB pathogen fingerprinting system. Documents Trust with California Citrus Research Board. Log 34658. 3.Progress Report This study focuses on identification of putative pathogens associated with citrus Huanglongbing (HLB or yellow shoot disease) and documentation of their geographical origins. Citrus HLB is highly destructive to citrus production worldwide. Understanding the etiology of HLB is critical for managing the disease. HLB is currently associated with infection by “Candidatus Liberibacter asiaticus” around the world, including China. However, the causal reltionship of the bacterium with HLB has not been proven. In addition, other plant pathogens may also be involved in HLB. Two surveys were performed in Guangdong Province, P. R. China. Citrus samples (141) showing typical symptoms of HLB from 11 different cities were collected. PCR using phytoplasma-specific primer sets fU5/rU3 nested with primer set P1/P7 identified 110 (78.0%) positive samples. A 1,785-bp amplicon was obtained with primer set P1/P7. Analysis showed a 100% identity of this sequence in the region of 16S rDNA and 16S-23S intergenic transcribed spacer to three strains of “Candidatus Phytoplasma asteri” [onion yellows (Japan), aster yellows ‘watercress’ (Hawaii), and valeriana yellows (Lithuania)]. Of the 141 samples, 89 (63.1%) samples were positive for “Ca. Liberibacter asiaticus”. When mixed infection was considered, 69 (48.9%) of the samples were positive for both phytoplasma and “Ca. L. asiaticus”. Transmission electron microscopy (TEM) showed low titers of both walled and wall-less bodies in the phloem sieve tubes of HLB citrus. When HLB-associated bacteria were transmitted from a symptomatic citrus to periwinkle (Catharanthus roseus (L.) G. Don.) via dodder (Cuscuta campestris Yunck), both phytoplasma and “Ca. L. asiaticus” were detected from the affected periwinkle. In addition to yellowing/mottling, the infected periwinkle showed symptoms of virescence and phyllody which are commonly associated with phytoplasmal diseases. TEM analysis of affected periwinkle revealed variably shaped wall-less organisms (characteristic of phytoplasmas) filling some phloem sieve tubes. Walled bacteria were found only in low abundance. This study showed that in addition to “Ca. L. asiaticus”, a phytoplasma related to “Ca. P. asteri” may also be detected in citrus showing HLB symptoms in Guangdong. Project personnel and management. Research progress was monitored by regular conference calls, email exchanges, phone calls, reports, and writing. Research findings were presented in the annual meetings of the American Phytopathological Society and in California citrus research board annual meeting with posters and oral presentations. A manuscript has been prepared for publication in a refereed journal.