Ten strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean, which were isolated from various soybean growing regions of Thailand, produced an extracellular diffusible factor (DSF) related to a well-characterized quorum sensing molecule produced by other Xanthomonas spp. Genomic DNA of the 10 strains of X. axonopodis pv. glycines contained rpfF, a gene encoding for the biosynthesis of the DSF in X. campestris pv. campestris. The rpfF gene from one strain of X. axonopodis pv. glycines was fully sequenced, and the 289 aa product is closely related to RpfF of other Xanthomonas spp. (95 to 98% identical). Three independently generated rpfF mutants of X. axonopodis pv. glycines strain No12-2 were defective in the production of a DSF, as expected if rpfF encodes for DSF biosynthesis in X. axonopodis pv. glycines. The rpfF mutants of X. axonopodis pv. glycines exhibited reduced virulence on soybean and produced less than wild-type levels of extracellular polysaccharide and the extracellular enzymes carboxylmethylcellulase, protease, endo-beta-1,4-mannanase, and pectate lyase. Transcripts for three genes that encode for the extracellular enzymes protease, endoglucanase, and pectate lyase were at lower abundance in an rpfF mutant than in the parental strain of X. axonopodis pv. glycines. These results indicate that X. axonopodis pv. glycines produces a diffusible signal related to the DSF of X. campestris pv. campestris, which contributes to virulence and exoenzyme production by this phytopathogenic bacterium.

ABSTRACT Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifHclones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium,Azorhizobium, Herbaspirillum, andThiobacillus and the other, represented by a singlenifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.