2008 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 Use of DNA extraction directly from plant tissue Our plant wash procedure for detection of Agrobacterium tumefaciens and Rhodococcus fascians was an effective procedure, but cumbersome. We moved to DNA extraction directly from plant sap using the FastDNA Spin Kits from MP Biomedical. We assayed 93 naturally infected samples from 34 genera. Sixty-six samples were processed for R. fascians; 15 were DNA only. Forty seven plant samples have been completed. Of the samples that were PCR positive, 3 (6.4%) were culture negative indicating potential false positives with PCR. However, all 3 samples showed symptoms of R. fascians infection, and the inability to recover bacteria may have been a sampling error. Of the 28 samples that were PCR negative, 4 were culture positive, indicating 8.5% gave a false negative reaction with PCR. We received 27 samples for crown gall diagnosis. Of these, 25 have been completed, and there was 100% agreement between the PCR and culture results. Faster virulence determination The standard plant traditionally used for virulence testing of R. fascians is garden pea or sweet pea, with symptoms developing in 4 weeks. We have developed a system using Oenothera speciosa in tissue culture and can now test virulence of recovered bacteria in two weeks. Loop-Mediated Isothermal Amplification (LAMP) Assay Our PCR assay for R. fascians requires special and expensive facilities. We wanted a sensitive, specific assay which does not require specialized equipment. The LAMP assay is a novel nucleic acid amplification method which is highly sensitive and can be completed in an hour. Results are visualized by a simple visual examination of the reaction mixture. We are in the process of developing a LAMP assay for R. fascians. Impact of research 1. Our development of sensitive and accurate primers for both R. fascians and A. tumefaciens can be used with direct tissue DNA extractions, and samples can be conclusively diagnosed in a matter of days, versus weeks, allowing nursery operators to rapidly make decisions about questionable plant material. 2. In the process of testing the direct tissue DNA extractions, we identified five new hosts of R. fascians. This discovery adds to the ever-broadening list of susceptible hosts of this pathogen and allows growers to be aware if their material is at risk of infection. 3. Development of a LAMP assay for R. fascians will allow growers to perform their own diagnostic assays using only simple equipment. They will be able to directly screen their own material, whether new introductions, elite stock, or production plants. Research activities under this agreement were monitored by e-mails and reports.