Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Abstract

ABSTRACT Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K 2 TeO 3 was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K 2 TeO 3 concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K 2 TeO 3 -amended medium. The bona fide agrobacterium colonies growing on media amended with K 2 TeO 3 were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species ( Agrobacterium rubi and Agrobacterium fici ) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium -like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10 3 to 10 4 agrobacteria · g of dry soil −1 in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.