1a.Objectives (from AD-416)
To determine if it is possible to develop molecular assays with increased sensitivity which are reliable for detection of R. fascians and A. tumefaciens in asymptomatic tissues.
1b.Approach (from AD-416)
We will examine all published sequences of both pathogens to find gene regions associated with virulence that are most highly conserved to develop primers reliable for detection. Optimization of the assay will follow, and may involve developing better extraction protocols, a nested PCR assay, a quantitative real-time PCR assay or using other enhancements to the current assays.
The objective of this research is to increase the sensitivity of our ability to detect Agrobacterium tumefaciens and Rhodococcus fascians in asymptomatic infected plant material. Because of its simplicity and sensitivity, we proposed using an enrichment phase, followed by a PCR assay. We are also looking at the genome of one of our pathogenic isolates to determine if there are areas that could be exploited for control and/or improved detection methods. Due to personnel turn-over and contamination of both bacterial cultures and our in-vitro tissue culture plantlets, progress on the enrichment and detection project has been slow. Preliminary results show that 24 hours after inoculation enrichment can detect both R. fascians (6 of 8 plants) and A. tumefaciens (5 of 8 plants) at a rate of less than 10 colony forming units. In contrast, detection with a conventional PCR assay was 1 of 8 and 2 of 8 plants for R. fascians and A. tumefaciens, respectively. These studies are ongoing. With a cooperator at Oregon State Univeristy, we have been working on creating a draft sequence of the genome of R. fascians isolate A44a, a known virulent isolate we obtained from an infected Veronica spicata ‘Minuet’ nursery plant. Illumina technology was used to generate the sequences. The reads have been assembled and compared with those of three isolates of R. fascians, including D-188 on which most work on R. fascians has been done, which was sequenced by colleagues in Ghent, Belgium. We are currently in the process of confirming the assemblies and comparing D-188 to A44a. There appears to be a low degree of similarity of contigs between the two isolates, which indicates a great deal of variability within the species. Annotation of the genome will follow once the assemblies have been confirmed.
Improved detection of R. fascians and A. tumefaciens would allow assaying plant material at a stage prior to symptom development, allowing propagation with confidence. This work will continue. The genome sequence will be the first documented for R. fascians and will provide valuable insights into the biochemical and biological strategies of the bacterium.
Research activities under this agreement were monitored by e-mails and reports.