Toxoflavin is an Essential Factor for Virulence of Burkholderia glumae Causing Rice Seedling Rot Disease.

Abstract

Mutants of Burkholderia glumae, the causal agent of rice seedling rot and grain rot diseases, were isolated by transposon (Tn) mutagenesis with Tn4431 harboring a tetracycline (Tc) resistance gene, which was carried on the suicide vector pUCD623. The resulting Tc-resistant colonies were screened for a loss of toxin production as well as for a lack of pathogenicity to rice seedlings. One of the screened mutants, the mutant No.19, was shown to have a single copy of Tn insertion on the genomic DNA by Southern hybridization test. Restriction enzyme fragments containing Tn4431-flanking sequences from the genomic DNA of mutant No.19 were cloned and used as probes for colony hybridization of the genomic DNA cosmid library from a wild strain of B. glumae. Six recombinant clones showing homology to the probes were isolated and conjugated into mutant No.19 by triparental mating. The recombinant cosmid pNP147, one of six clones, was able to complement the mutant No.19 for toxin production and pathogenicity. The toxin, purified from a culture of mutant No.19 with pNP147, was identified as toxoflavin by NMR and Mass spectra analyses. These results suggest that toxoflavin is a critical factor in disease development of B. glumae, since the nontoxigenic mutants were unable to induce seedling rot disease to rice plants.