STUDIES ON THE POPULATION DYNAMICS OF RALSTONIA SOLANACEARUM BIOVAR 2 (RACE 3) BY USING METHODS BASED ON MICROBIAL ENRICHMENT, SEROLOGY AND SPECIFIC AMPLIFICATION OF NUCLEIC ACID SEQUENCES
STUDIES ON THE POPULATION DYNAMICS OF RALSTONIA SOLANACEARUM BIOVAR 2 (RACE 3) BY USING METHODS BASED ON MICROBIAL ENRICHMENT, SEROLOGY AND SPECIFIC AMPLIFICATION OF NUCLEIC ACID SEQUENCES JM VAN DER WOLF, JD VAN ELSAS, P KASTELEIN AND GOM LEONE DLO Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands Background and objectives Ralstonia (Pseudomonas) solanacearum, the causal organism of bacterial wilt, is a destructive disease on many economically important crops, in particular in tropical countries. At least 3 biovars are distinguished on the basis of biochemical properties, of which biovar 2 (nearly synonymous to race 3), is adapted to low temperatures and is found in temperate zones. Biovar 2 has a restricted host range and is the causative agent of brown rot in potato. Recently, outbreaks of brown rot have been reported in several EU-countries where R. solanacearum is declared as a quarantine pathogen. To take appropriate measures for confinement and eradication, a basic knowledge of the population dynamics is required. This paper describes the development of detection methods to study the population dynamics of R. solanacearum in the potato cropping ecosystem. Materials and methods lmmunofluorescence colony-staining (IFC) was adapted for specific detection of R. solanacearum in soil . The identity of target bacteria in IFC-positive colonies was confirmed by PCR-amplification using R. solanacearum specific 16S RRNA primers. To determine persistence of R. solanacearum on farm equipment, plating on the semi-selective SMSA-medium and the isothermal NASBA-amplification method , based on RNA-amplification using primers directed to 16S RRNA sequences. Results and conclusions IFC allowed detection of c. 1 000 cfu per gram of artificially inoculated sandy loam and clay soil. Crossreacting bacteria in IFC-preparations, occasionally present in soil samples, could be recognized on the basis of the colony morphology and staining intensity, but identity of bacteria sampled from fluorescent colonies could also be confirmed efficiently by PCR-amplification. In sandy soil, the pathogen survived at least 92 days in microcosms.at constant temperatures of 15 o C and 4 oC. At 20 OC, the pathogen was not detectable after 50 days. Freezing and thawing had a detrimental effect on the pathogen. Using IFC, high population densities of up to 10 5 cfu per gram of dry soil could be detected in two naturally brown rot infected sandy field soils, up to five months after chemical destruction of potato haulms. Population densities will also be determined in the soil after the winter. So far, results indicate that R. solanacearum will not easily survive in regions with a temperate climate for long periods in bulk soil. To assess the risk of dissemination of the pathogen during cultural practices, its persistence on farm equipment and materials was determined using dilution plating methods, and for detection of low densities on iron surfaces also with NASBA. Cell death is accompanied by RNA disappearance, due to the activity of RNases in crude samples. Thus by using NASBA, the viability of cells can be distinguished, also when cells are present in a non-culturable state. A high correlation was found between NASBA and dilution plating results for studies on the persistence of R. solanacearum on iron; the pathogen died within 24 h. According to plate counts, on wood and clothing, survival was less than 1 day, while on rubber the pathogen persisted for 2 days. These results indicate that the pathogen rapidly dies on the surfaces of equipment and materials. References 1. Van der Wolf JM, Van Bekkum PJ, Van Eisas JD, Nijhuis EH, Vriend SGC, Ruissen MA, 1997. Bulletin OEPP/EPPO Bulletin 27. In press. 2. Van der Vliet GME, Schepers P, Schukkink RAF, Van Gemen B and Klatser PR, 1994. Antimicrobial agents and chemotherapy 38, 1959-1965.