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Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063 T and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063 T and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164 T (=JCM 13063 T ) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.

Biocontrol fluorescent pseudomonads produce a number of antibiotic organic compounds, including 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, and phenazine. We previously classified rhizospheric fluorescent pseudomonads harboring antibiotic biosynthetic gene clusters into 10 operational taxonomic units (OTUs). In the present study, we report the complete genome sequences of selected strains from these OTUs. The genetic diversity of antibiotic biosynthetic gene clusters and their surrounding sequences correlated with the OTU classification. In comparisons of the biocontrol activity and distribution of antibiotic biosynthetic gene clusters, we found that the pyrrolnitrin biosynthetic gene cluster more effectively controlled the growth of Rhizoctonia solani.

More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In total, 927 hydrogen cyanide (HCN)-, 47 2,4-diacetylphloroglucinol (PHL)-, 6 pyoluteorin (PLT)-, 14 pyrrolnitrin (PRN)-, and 8 phenazine (PHZ)-producing isolates have been detected. A cluster analysis (≥99% identity) identified 10 operational taxonomic units (OTUs) in antibiotic biosynthesis gene-possessing pseudomonads. OTU HLR (PHL, PLT, and PRN) contained four antibiotics: HCN, PHL, PLT, and PRN, while OTU RZ (PRN and PHZ) contained three: HCN, PRN, and PHZ. OTU H1, H2, H3, H4, H5, H6, and H7 (PHL1-7) contained two antibiotics: HCN and PHL, while OTU H8 (PHL8) contained one: PHL. Isolates belonging to OTU HLR and RZ suppressed damping-off disease in cabbage seedlings caused by Rhizoctonia solani. Effective strains belonging to OTU HLR and RZ were related to Pseudomonas protegens and Pseudomonas chlororaphis, respectively. Antibiotic biosynthesis gene-possessing fluorescent pseudomonads are distributed among different geographical sites in Japan and plant species.

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) regulates the expression of virulence factors by N-acylhomoserine lactone (AHL)-mediated quorum sensing. The LuxI family protein, ExpI, catalyzes AHL biosynthesis in Pcc. The structure of the predominant AHL produced by ExpI differs among Pcc strains, which may be divided into two quorum-sensing classes (QS classes) based on the AHL produced. In the present study, AHL produced by 282 Pcc strains were extracted and identified by LC-MS/MS. Seventy Pcc strains produced N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) as the predominant AHL and were categorized into QS class I. Two hundred Pcc strains produced N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) as the predominant AHL, and were categorized into QS class II-1. Twelve Pcc strains produced only small amounts of 3-oxo-C6-HSL, and were categorized into QS class II-2. The phylogenetic analysis revealed that the amino acid sequences of ExpI may be divided into two major clades (I and II). The Pcc strains categorized into ExpI clades I and II entirely matched QS classes I and II, respectively. A multiple alignment analysis demonstrated that only 6 amino acid substitutions were observed among ExpI from QS classes II-1 and II-2. Furthermore, many amino acid substitutions between QS classes I and II were concentrated at the C-terminal region. These amino acid substitutions are assumed to cause significant reductions in 3-oxo-C6-HSL in QS class II-2 or affect the substrate specificity of ExpI between QS classes I and II.

Pseudomonas chlororaphis subsp. aurantiaca StFRB508 regulates phenazine production through N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing. Two sets of AHL-synthase and AHL-receptor genes, phzI/phzR and aurI/aurR, have been identified from the incomplete draft genome of StFRB508. In the present study, the complete genome of StFRB508, comprising a single chromosome of 6,997,933 bp, was sequenced. The complete genome sequence revealed the presence of a third quorum-sensing gene set, designated as csaI/csaR. An LC-MS/MS analysis revealed that StFRB508 produced six types of AHLs, with the most important AHL being N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-OH-C6-HSL). PhzI mainly catalyzed the biosynthesis of 3-OH-C6-HSL, while AurI and CsaI catalyzed that of N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, respectively. A mutation in phzI decreased phenazine production, whereas that in aurI or csaI did not. A phzI aurI csaI triple mutant (508ΔPACI) did not produce phenazine. Phenazine production by 508ΔPACI was stimulated by exogenous AHLs and 3-OH-C6-HSL exerted the strongest effects on phenazine production at the lowest concentration tested (0.1 μM). The plant protection efficacy of 508ΔPACI against an oomycete pathogen was lower than that of wild-type StFRB508. These results demonstrate that the triplicate quorum-sensing system plays an important role in phenazine production by and the biocontrol activity of StFRB508.

Eight genotypes of potato plants with different resistance levels against common scab were grown in a field infested with Streptomyces turgidiscabies. DNA was extracted from the roots, tubers, and rhizosphere soils of each of the eight genotypes at the flowering stage, and the quantity of S. turgidiscabies genomic DNA was assessed by real-time PCR using a TaqMan probe. The results obtained showed that the different potato genotypes had significant impacts on the population levels of S. turgidiscabies between resistant and susceptible genotypes in the tubers, but not in the roots or rhizosphere soils. Clone analyses of 16S rRNA gene libraries from the eight potato genotypes identified three phyla (Proteobacteria, Firmicutes, and Actinobacteria) as dominant taxa in root and tuber clone libraries, while a clustering analysis identified 391 operational taxonomic units (OTUs) at the species level. Eleven OTUs closely related to Aquicella siphonis, Arthrobacter nicotinovorans, Streptomyces rishiriensis, Rhodococcus baikonurensis, Rhizobium radiobacter, Rhizobium etli, Phyllobacterium myrsinacearum, Paenibacillus pabuli, Paenibacillus alginolyticus, and Bacillus halmapalus were detected in the root or tuber libraries of all the potato genotypes examined. Furthermore, an abundance of OTUs related to Aquicella and Rhodococcus was observed in the rhizospheres of resistant and susceptible potato genotypes, respectively. Based on this ecological information, an efficient survey may be conducted for biological agents from the potato rhizosphere.

The bacterial community of a potato phytosphere at the flowering stage was examined using both culture-dependent and -independent methods. Tissues (leaves, stems, roots and tubers) were sampled from field-grown potato plants (cultivar Matilda), and the clone libraries of 16S rRNA genes and the isolate collections using R2A medium were constructed. By analyzing the combined data set of 16S rRNA gene sequences from both clone libraries and isolate collections, 82 genera from 8 phyla were found and 237 OTUs (≥97% identity) at species level were identified across the potato phytosphere. The statistical analyses of clone libraries suggested that stems harbor the lowest diversity among the tissues examined. The phylogenetic analyses revealed that the most dominant phylum was shown to be Proteobacteria for all tissues (62.0%-89.7% and 57.7%-72.9%, respectively), followed by Actinobacteria (5.0%-10.7% and 14.6%-39.4%, respectively). The results of principal coordinates analyses of both clone libraries and isolate collections indicated that distinct differences were observed between above- and below-ground tissues for bacterial community structures. The results also revealed that leaves harbored highly similar community structures to stems, while the tuber community was shown to be distinctly different from the stem and root communities.

N-Acylhomoserine lactone (AHL)-degrading enzyme, AiiM, was identified from the potato leaf-associated Microbacterium testaceum StLB037. In this study, we cloned eight aiiM gene homologues from other AHL-degrading Microbacterium strains. The similarity of the chromosomal locus of the aiiM gene is associated with the phylogenetic classification based on 16S rRNA. Degenerate PCR revealed that the aiiM gene was only conserved in AHL-degrading Microbacterium strains, but not in fifteen Microbacterium type strains or two Microbacterium isolates from other plants. These results suggested that the high level of AHL-degrading activity in Microbacterium strains was caused by the aiiM gene encoded on their chromosome.

A total of 828 isolates of fluorescent pseudomonads (FPs) were obtained from the leaves (305 isolates) and roots (523 isolates) of potato plants grown in different geographical locations in Japan, and 16S rRNA gene sequences of 776 isolates were successfully determined by direct PCR sequencing. Clustering analysis (≥99% identity) identified 13 and 26 operational taxonomic units (OTUs) for leaf- and root-associated FPs, respectively, and 29 OTUs were identified in the phytosphere of potato plants. Among them, 7 and 9 OTUs showed a significantly biased distribution to the leaves and roots, respectively. Phylogenetic analysis revealed that 3 dominant OTUs for leaf-associated FPs were grouped in a cluster of leaf-associated pathogens, such as Pseudomonas cichorii and Pseudomonas viridiflava. In contrast, 4 OTUs were located in a cluster of saprophytic pseudomonads. Among them, 3 OTUs showed high similarity to Pseudomonas koreensis and Pseudomonas vancouverensis, both of which have been reported to be beneficial for biological control or plant growth promotion. These data provide key information for efficient surveying and utilization of beneficial FPs in agricultural practices.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signaling molecules by many Gram-negative bacteria. Here, 413 bacterial strains were obtained from the roots of potato plants and screened for AHL-degrading bacteria using Chromobacterium violaceum reporter strains. Sixty one isolates degraded N-hexanoyl-L-homoserine lactone (C6-HSL) within 24 h. Based on their 16S rRNA gene sequences, most of these isolates were assigned to the genus Chryseobacterium and divided into eight groups. Most of the strains degraded N-decanoyl-L-homoserine lactone (C10-HSL) and were active against 3-oxo-substituted AHLs. Groups V and VI were more effective at inactivating AHLs. One group V strain, StRB402, showed putative AHL-lactonase activity. This is the first report of AHL-degrading Chryseobacterium strains.

A total of 100 isolates of chitinolytic bacteria were obtained from the rhizospheres of various agronomic plants, and the 16S rRNA gene sequences of these isolates were determined. Phylogenetic analyses revealed that 81 isolates belonged to the classes Betaproteobacteria (39 isolates) and Gammaproteobacteria (42 isolates). Of the remaining 19 isolates, 16 belonged to the phylum Firmicutes. Clustering analysis identified 6 and 3 operational taxonomic units (OTUs) in Gammaproteobacteria and Betaproteobacteria, respectively, at the genus level. The majority of chitinolytic bacteria in Gammaproteobacteria belonged to the genera Serratia, Stenotrophomonas, and Lysobacter (14, 15, and 7 isolates, respectively) while those in Betaproteobacteria belonged to the genus Mitsuaria (37 isolates). The 16 isolates placed in Firmicutes belonged to 2 genera, Paenibacillus and Bacillus (8 isolates each). The isolates in the remaining OTUs belonged to the genera Erwinia, Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Microbacterium, in less abundance. These results showed a wide distribution of culturable chitinolytic bacteria in the rhizospheres of various agronomic plants. Considering the potential antagonistic activity of chitinolytic enzymes against phytopathogenic fungi, which is exhibited by fungal cell wall degradation, the above-mentioned native chitinolytic bacteria in rhizospheres could potentially be utilized for the biological control of soil-borne phytopathogenic fungi.

Four hundred and fifty nine isolates of fluorescent pseudomonads were obtained from the leaves and roots of potato plants. Of these, 20 leaf isolates and 28 root isolates induced violacein production in two N-acylhomoserine lactone (AHL)-reporter strains-Chromobacterium violaceum CV026 and VIR24. VIR24 is a new reporter strain for long N-acyl-chain-homoserine lactones, which can not be detected by CV026. Thin-layer chromatography revealed that the isolates produced multiple AHL molecules. We compared the 16S rRNA gene sequences of these isolates with sequences from a known database, and examined phylogenetic relationships. The AHL-producing isolates generally separated into three groups. Group I was mostly composed of leaf isolates, and group III, root isolates. Group II comprised both leaf and root isolates. There was a correlation between the phylogenetic cluster and the AHL molecules produced and some phenotypic characteristics. Our study confirmed that AHL-producing fluorescent pseudomonads could be distinguished in the phyllosphere and rhizosphere of potato plants.