2009 Annual Report 1a.Objectives (from AD-416) To develop sensitive and reliable detection methods for bacterial pathogens infecting herbaceous ornamental crops. These methods are intended to work as well with plant material as they do with in vitro cultures. 1b.Approach (from AD-416) Develop reagents, sampling procedures and protocols for the sensitive and reliable detection and differentiation of the plant pathogenic bacteria Agrobacterium and Rhodococcus. Develop PCR primer pairs that can distinguish between virulent isolates of A. tumefaciens and R. fascians from tissue incubated in broth or from fresh plant tissue. Determine appropriate tissue to sample to maximize detection of the pathogens. Determine how long R. fascians remains viable on plant surfaces. Inoculate indicator plants with R. fascians and re-isolate over time to determine fate of populations. 3.Progress Report The objective of this project is to develop a LAMP (Loop-mediated isothermal AMPlification) assay that can distinguish between virulent and avirulent isolates of Rhodococcus fascians and which does not cross react with other bacteria. Initially, 20 sets of primers were screened for amplification of DNA extracted from virulent R. fascians. One primer set (FASR) was chosen that amplifies ‘fasR’, a virulence gene necessary for symptom development. Sequencing of the LAMP products confirmed that the primer set was amplifying fasR. Specificity of the primers was tested against 25 virulent and avirulent isolates of R. fascians. All six avirulent strains tested negative for FASR-LAMP, while 17 known virulent strains tested positive. In addition, nine other bacterial plant pathogens were tested. All non-Rhodococcus strains were negative for FASR-LAMP. We are continuing to test this assay against R. fascians and other bacterial isolates. In conducting PCR, we found that LAMP amplification forms a precipitate that is dependent on the concentration of magnesium ions. We optimized the conditions of the assay for maximum precipitation production and sensitivity. The sensitivity of the assay was evaluated using DNA from bacterial cultures and bacteria extracted with petunia leaf tissue. The FASR-LAMP appeared more sensitive than PCR, detecting approximately 100 cfu per reaction when DNA was extracted directly from bacteria. FASR-LAMP had sensitivity equal to that of PCR, detecting approximately 1,000 cfu per reaction, when bacterial and plant DNA were extracted together. Use of additional loop primers to increase sensitivity is currently being tested. FASR-LAMP demonstrates sufficient specificity and sensitivity to permit its use as a diagnostic tool for detecting virulent strains of R. fascians. Bacteria can be successfully detected in plant tissues extracted with Qiagen mini plant kits and then assayed with our primers. The only equipment needed is a heat block (or water bath) and a microcentrifuge. Amplification can be evaluated by eye, or if additional sensitivity is required, by the addition of ‘picogreen’ reagent, which causes the positive reaction to fluoresce under a UV lamp. The LAMP assay allows sensitive and specific detection of R. fascians in plant material in a matter of hours. Research activities under this agreement were monitored by e-mails and reports.