Differential characterization of two novel antinematodal biological control agents : Streptomyces costaricus sp. nov. and a strain of Bacillus thuringiensis.
Two novel antinematodal strains of bacteria, Streptomyces sp. (CR-43) and Bacillus thuringiensis is (Bt) (CR-371) originating in Costa Rica were differentiated from other closely related strains and compared to rifampicin-resistant derivatives in greenhouse and field studies. The name Streptomyces costaricus is proposed for strain CR-43. The generic attribution was based on its typical morphology, production of LL-diaminopimelic acid and fatty acid composition. To clarify its taxonomic position, the isolate was compared with type strains of similar Streptomyces spp. The results of biochemical tests and profile analysis of hydrolyzable fatty acids indicated that CR-43 differed from previously described species and represents a new species. CR-43 (= ATCC 55274 = NRRL B-16897) is the type strain. A genotaxonomic approach to the differentiation of the streptomycetes was also taken. A modified RAPD analysis, conserved mini-sequence primed PCR (CMSP-PCR), was designed to target short conserved sequences dispersed throughout the streptomycete genome. All species had distinct DNA profiles when DNA was amplified with either 15-mer primer 5SSU3 (5\sp{\prime}-TGCGGCCGTACTCCC) or SSU5 (5\sp{\prime}-CGGCAGGCCTAACAC). The DNA profile of CR-43 resembled most that of S. h. decoyicus using primer SSU5. Species delineation was easily achieved with CMSP-PCR DNA amplification without further enzymatic processing. The differentiation of the novel Bt strain from five patented antinematodal Bt strains was required for patent prosecution. This was achieved by analysis of hydrolyzable fatty acid compositions of the strains by GC-MS and by DNA profile analysis. Thirty compounds in the total ion chromatograms were identified and analyzed using non-parametric statistics, the major fatty acids being i-15:0 (13.7-23.2%), i-13:0 (6.8-10.7%), i-14:0 (5.0-7.7%), 14:0 (3.0-4.1%), a-15:0 (3.9-9.9%), i-16:0 (3.6-7.2%), 16:0 (3.2-11.6%), i-17:0 (3.2-9.8%), 18:0 (tr-13.5%), a monounsaturated C16 (4.5-9.9%), a monounsaturated branched C17 (2.8-5.8%), a diunsaturated C18 (0-5.8%), and a monounsaturated C18 acid (0.3-4.8%). Molecular amplification of the DNA using a random nanomeric primer 5\sp{\prime}-CCGAGTCCA (that could discriminate serovars) showed that RAPD profiles of the Bt\u27s were different. Spontaneous rifampicin-resistance (rif\sp+) was selected in CR-371 and CR-43. Rif\sp+ had a positively pleiotropic effect on the biocontrol activity of the Bt strain and a negatively pleiotropic effect on the novel streptomycete. Rif\sp+ was a useful marker for monitoring survival of CR-371 and CR-43 in soil
