Location: Floral and Nursery Plants Research2012 Annual Report
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.
3. Progress Report:
Disease control should begin with sanitation and clean plant material. Using tissue culture (TC)-derived plants is often recommended as a means of avoiding disease. However, we have detected both Agrobacterium tumefaciens and Rhodococcus fascians in TC plantlets in vitro. The ability of R. fascians to persist in soil is poorly understood. Nurseries with R. fascians problems often experience recurring incidences of the pathogen, in spite of rouging infected plants. It is possible that contaminated soil is acting as a reservoir of inoculum in affected nurseries. The objectives of this research are: 1) to determine if it is possible to detect A. tumefaciens or R. fascians in asymptomatic inoculated TC material (a continuation of work initiated last year); and 2) to determine whether R. fascians can be detected in soils of varying composition, and at what inoculum levels. Bio-PCR was used to determine if A. tumefaciens and R. fascians could be detected in TC plants two weeks after inoculation (10 µl inoculum/plant) at three cell concentrations. Tissue from inoculated Oenothera speciosa plants was incubated in quadruplicate in enrichment broth for each of 24, 48, and 72 h, then assayed in duplicate with PCR. Each experiment was performed 3 times. We could detect R. fascians at 48 h in all 3 experiments in at least 1 plant; inoculum was between 45 and 68 cfu/10 µl. Detection increased after 72 h to 2 of 4 plants. Results for A. tumefaciens were similar, with lower inoculum levels (approx. 10 or 20 cfu/10 µl), except the bacterium was detected in 3/4 plants after 48 h and 4/4 plants at 72 h. Assaying tissue culture plantlets for the presence of both species of bacteria is feasible when there is a question of contamination. For objective 2, three soil types were used, and each was inoculated with R. fascians at 4 concentrations. Inoculated soils were extracted after 3 d and assayed with PCR. The soils were air dried, extracted, and assayed again. Inoculum concentration at time of extraction was determined by dilution plating/colony counting. The experiments were performed 3 times. R. fascians could be detected when soil was inoculated at between 1-6 x 103 cfu/ml in moist soil. Bacteria could not be recovered from dry soils when plated, but could be detected using PCR. Experiments are ongoing. Preliminary results show R. fascians may not be long-lived in dry soils, and that nursery soils can be assayed for the presence of the bacteria.