2011 Annual Report 1a.Objectives (from AD-416) Develop cost-effective and highly sensitive methods to detect huangblongbing (HLB) and multiplex it with detection of other regulated pathogens, thus, improving the cost/benefit ratio per marker per pathogen being diagnosed. 1b.Approach (from AD-416) 1. Establish collections of citrus pathogens including CTV, CVV (Xylella fastidiosa), CPsV, Pospiviroids (CEVd, CVd-IV), Hostuviroids (CVd-IIa, CVd-IIb, CVd-IIc), stubborn (Spiroplasma citri), huanglongbing (HLB, “Candidatus” Liberibacter asiaticus, “C.” L. africanus), Leprosis, and Citrus leaf blotch virus (Dweet mottle) and Tatterleaf; 2. Establish reliable real-time PCR detection of these pathogens; 3. Establish rapid extraction of pathogen nucleic acid from target tissue; 4. Use results of computational design for oligonucleotides for specific identification of the panel of citrus pathogens from LANL collaborators, assemble fluorescent dye to oligo markers and employ Luminex instrument for detection by MOL-PCR; and 5. Test automated high-throughput nucleic acid extraction machines for target oligo acquisition from citrus tissue. 3.Progress Report This Grant is between ARS and the University of California, Riverside in support of Objective 3 of the parent project, 5302-22000-009-00D. The goal is to develop efficient and cost-effective extraction and purification of citrus pathogens or pathogen-derived nucleic acids for pathogen detections by Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). A postdoc was hired at the University of California, Riverside. Two sets of group-specific primers were developed for detection of citrus viroids by real-time Reverse-Transcription Polymerase Chain Reaction: Group 1 primer set detects CVd-I, III, V, VI; Group 2 primer set detects CVd-II, CVd-IV, CEVd. The primer sets work well and effort is underway to optimize total RNA extraction. A database of 6 full-length sequences of citrus viroid species was assembled (CVd-I, CVd-II, CVd-III, CVd-IV, CVd-V, CVd-VI) and sent to the cooperator at Los Alamos National Laboratory for design of oligonucleotides for MOL-PCR. Research activities were monitored by the lead scientist through email and telephone communication and site visits with the cooperator.