3.3.46
COMPARISON OF TECHNIQUES FOR SENSITIVE DETECTION OF RALSTONIA SOLANACEARUM IN SOIL

JG ELPHINSTONE1, PM PRADHANANG 2 and RTV FOX3

1Central Science Laboratory, Sand Hutton, York Y04 1LZ, UK; 2Lumle Agricultural Research Centre, PO Box No. 1, Pokhara, Kaski, Nepal; 3School of Plant Sciences, University of Reading, Earley Gate, Reading RG 6 6AS, UK

Background and objective
The ecology of Ralstonia solanacearum race 3 (biovar 2A), the causal pathogen of potato brown rot disease, remains little understood. As a result, fully effective control measures are not available. The limited understanding of pathogen persistence in the environment may be largely due to a lack of sensitive means for its detection. The objective of this study was to compare sensitivity of newly-developed techniques for routine detection of R. solanacearum in soil.

Materials and methods
Samples of natural field soil (10 g each) were inoculated with 1 ml of aqueous suspensions of R. solanacearum ranging from 7.5 x 108 to 7.5 x 102 colony forming units (cfu) per ml. Following incubation, bacteria were extracted by suspending inoculated samples in 100 ml 0.05 M phosphate buffer and shaking vigorously for 2 min. Limits of detection were determined using a modified semi-selective medium (SMSA), a tomato bioassay, an indirect enzyme-linked immunosorbent assay (ELISA) and amplification of pathogen-specific DNA sequences by polymerase chain reactions (PCR) with specific [1] and nested [2] primers. ELISA and PCR tests were performed before and after pathogen enrichment in 1 ml of soil suspension by incubating overnight with 9 ml modified SMSA broth at 28 OC.

Results and conclusion
The bacterium was recovered from all inoculated suspensions following dilution plating on modified SMSA agar. However, only soil-borne populations of 104 cfu per m] or greater were detected by the tomato bioassay. Tomato seedlings (cv. Moneymaker) wilted only when inoculated with soil suspensions exceeding 106 cfu per ml. However, with lower populations down to 1 04 cfu per m], most stems became latently infected. The indirect ELISA detected 104 cfu per ml of non-enriched sample or 103 cfu per ml following enrichment overnight in SMSA broth. R. solanacearum in soil suspensions multiplied 100-fold after enrichment for 20 hrs in SMSA broth. Use of nested-PCR with R. solanacearum specific primers OLI 1 and OLI 2 [1,2] and nested primers JE2 and Y2 [1,2] permitted detection of all inoculated populations of the bacterium from enriched soil suspensions. PCR with primers OLI 1 and Y2 [1] detected down to 101 cfu per ml of soil suspension but only following overnight enrichment and subsequent 10-fold aqueous dilution to reduce PCR inhibitory substances from the soil. The bacterium was not isolated on SMSA agar from noninoculated soil suspensions which also tested negative with the ELISA and PCR protocols.

Culture on modified semi-selective medium SMSA was most effective for quantifying viable populations of R. solanacearum and would therefore be the method of choice for use in epidemiological studies. PCR also has potential for sensitive and specific detection in soil. The simple pathogen-specific PCR method [1] with primers OL11 and Y2 but with an improved preenrichment is preferred since higher specificity of detection is expected than with the nested PCR method.

References
1. Seal S, Jackson LA, Daniels MJ, 1992. Applied and Environmental Microbiology 58, 37513758.
2. Elphinstone JG, Hennessy J, Wilson JK, Stead DE, 1996. EPPO/OEPP Bulletin 26, 663678.