Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpea in India, suppressed Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris, and Pythium damping-off of bean, caused by Pythium splendens. When grown in culture, PNA1 produced the phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine, and inhibited mycelial growth of F. oxysporum f. sp. ciceris, P. splendens, and certain other phytopathogenic fungi. Two mutants (FM29 and FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1. The transposon in the genome of FM29 was localized to phnA, which is thought to encode a subunit of anthranilate synthase II involved in the phenazine biosynthesis. The FM13 mutation was complemented by trpC, which encodes indole glycerol phosphate synthase in the tryptophan biosynthesis pathway; consequently, FM13 could not grow on a minimal medium in the absence of tryptophan. Neither FM29 nor FM13 suppressed Fusarium wilt of chickpea to the level achieved by the wild-type strain, indicating that phenazine production contributed to the biocontrol of this disease by P. aeruginosa PNA1. FM29 was also less effective than the phenazine-producing parental strain in biological control of Pythium damping-off of bean, but FM13 was as effective as the parental strain in suppressing this disease. Anthranilate, an intermediate in the tryptophan biosynthesis pathway, suppressed mycelial growth of Pythium spp. in culture and Pythium damping-off of bean and lettuce. Anthranilate, excreted by FM13 as a consequence of the trpC mutation, may have contributed to the suppression of Pythium damping-off by the mutant.

Microevolution and origins of Bradyrhizobium populations associated with soybeans at two field sites (A and B, 280 km apart in Canada) with contrasting histories of inoculation was investigated using probabilistic analyses of six core (housekeeping) gene sequences. These analyses supported division of 220 isolates in five lineages corresponding either to B. japonicum groups 1 and 1a or to one of three novel lineages within the genus Bradyrhizobium. None of the isolates from site A and about 20% from site B (the only site with a recent inoculation history) were attributed to inoculation sources. The data suggest that most isolates were of indigenous origin based on sequence analysis of 148 isolates of soybean-nodulating bacteria from native legumes (Amphicarpaea bracteata and Desmodium canadense). Isolates from D. canadense clustered with B. japonicum group 1, whereas those from A. bracteata were placed in two novel lineages encountered at soybean field sites. One of these novel lineages predominated at soybean sites and exhibited a significant clonal expansion likely reflecting selection by the plant host. Homologous recombination events detected in the 35 sequence types from soybean sites had an effect on genetic diversification that was approximately equal to mutation. Interlineage transfer of core genes was infrequent and mostly attributable to gyrB that had a history of frequent recombination. Symbiotic gene sequences (nodC and nifH) of isolates from soybean sites and native legumes clustered in two lineages corresponding to B. japonicum and B. elkani with the inheritance of these genes appearing predominantly by vertical transmission. The data suggest that soybean-nodulating bacteria associated with native legumes represent a novel source of ecologically adapted bacteria for soybean inoculation.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA–DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency~68 %) but was recovered infrequently (~1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Aims: The development and evaluation of a sensitive and specific TaqMan Ò real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results: A TaqMan Ò -based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan Ò PCR assay detected 1 pg of purified DNA and 10 4 P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92AE0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan Ò real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.