Location: Floral and Nursery Plants Research Unit
2012 Annual Report
The objectives of this research are:.
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.