Pectobacterium araliae sp. nov., a pathogen causing bacterial soft rot of Japanese angelica tree in Japan.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA-DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95-96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

Pseudomonas solani sp. nov. isolated from the rhizosphere of eggplant in Japan.

The search for bacteria that can be used as biocontrol agents to control crop diseases yielded a promising candidate, Sm006T, which was isolated from the rhizosphere of eggplant (Solanum melongena) growing in a field in Aichi Prefecture, Japan, in 2006. The cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with one polar flagellum. The results of homology searches and phylogenetic analyses based on the 16S rRNA gene sequence indicated that Sm006T represents a member of the genus Pseudomonas. The genomic DNA G+C content was 66.3 mol% and the major cellular fatty acids (more than 5 % of the total fatty acids) were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C12 : 0. Phylogenetic analyses using the rpoD gene sequence and phylogenomic analysis of the whole genome sequence revealed that Sm006T represents a member of the Pseudomonas resinovorans group; however, its phylogenetic position does not match that of any known species of the genus Pseudomonas. The average nucleotide identity and digital DNA-DNA hybridisation values between the strain and closely related species were lower than the thresholds for prokaryotic species delineation (95-96 and 70 %, respectively), with the highest values observed for Pseudomonas tohonis TUM18999T (92.05 and 46.3 %, respectively). Phenotypic characteristics, cellular fatty acid composition and possession of 2,4-diacetylphloroglucinol biosynthetic gene cluster could be used to differentiate the strain from its closest relatives. The phenotypic, chemotaxonomic and genotypic data obtained during this study indicated that Sm006T represents a novel species of the genus Pseudomonas, for which we propose the name Pseudomonas solani sp. nov., with Sm006T (= MAFF 212523T = ICMP 24689T) as the type strain.

Comparative genome analysis reveals the presence of multiple quorum-sensing systems in plant pathogenic bacterium, Erwinia rhapontici.

We present the complete genome sequences of 3 Erwinia rhapontici strains, MAFF 311153, 311154, and 311155. These chromosome sequences contained variety types of luxI/luxR gene pair involved in acylhomoserine lactone biosynthesis and reception. Large-scale insertion sequence was observed in the indigenous plasmid of MAFF 311154 and contained eraI3/eraR3 gene pair that make possible to produce acylhomoserine lactone.

A survey of metabolic changes in potato leaves by NMR-based metabolic profiling in relation to resistance to late blight disease under field conditions.

Non-targeted nuclear magnetic resonance (NMR)-based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. 1 H NMR spectra of the foliar metabolites soluble in deuterium oxide- or methanol-d4 -based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l-malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field-grown plants in combination with targeted methods for quantifying marker levels, moving towards marker-assisted screening of new cultivars with durable late blight resistance. Copyright © 2016 John Wiley & Sons, Ltd.

Rhizoxin analogs contribute to the biocontrol activity of a newly isolated pseudomonas strain.

Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol producers. These strains were found to be the same species and were the closest to but different from Pseudomonas protegens among the sequenced pseudomonads, based on 16S ribosomal RNA gene and whole-genome analyses. Strain Os17 was as effective a biocontrol agent as reported for P. protegens Cab57, whereas strain St29 was less effective. The whole-genome sequences of these strains were obtained: the genomes are organized into a single circular chromosome with 6,885,464 bp, 63.5% G+C content, and 6,195 coding sequences for strain Os17; and with 6,833,117 bp, 63.3% G+C content, and 6,217 coding sequences for strain St29. Comparative genome analysis of these strains revealed that the complete rhizoxin analog biosynthesis gene cluster (approximately 79 kb) found in the Os17 genome was absent from the St29 genome. In an rzxB mutant, which lacks the polyketide synthase essential for the production of rhizoxin analogs, the growth inhibition activity against fungal and oomycete pathogens and the plant protection efficacy were attenuated compared with those of wild-type Os17. These findings suggest that rhizoxin analogs are important biocontrol factors of this strain.

The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility, pectate lyase activity, and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209.

Pseudomonas sp. StFLB209 was isolated from potato leaf as an N-acylhomoserine lactone (AHL)-producing bacterium and showed a close phylogenetic relationship with P. cichorii, a known plant pathogen. Although there are no reports of potato disease caused by pseudomonads in Japan, StFLB209 was pathogenic to potato leaf. In this study, we reveal the complete genome sequence of StFLB209, and show that the strain possesses a ppuI-rsaL-ppuR quorum-sensing system, the sequence of which shares a high similarity with that of Pseudomonas putida. Disruption of ppuI results in a loss of AHL production as well as remarkable reduction in motility. StFLB209 possesses strong pectate lyase activity and causes maceration on potato tuber and leaf, which was slightly reduced in the ppuI mutant. These results suggest that the quorum-sensing system is well conserved between StFLB209 and P. putida and that the system is essential for motility, full pectate lyase activity, and virulence in StFLB209.

Identification and characterization of novel N-acylhomoserine lactonase from nonpathogenic Allorhizobium vitis, a candidate for biocontrol agent.

Some strains of nonpathogenic Allorhizobium vitis can control crown gall disease in grapevines caused by pathogenic A. vitis and are considered candidates for biocontrol agents. Many plant pathogenic bacteria regulate the expression of their virulence genes via quorum sensing using N-acylhomoserine lactone (AHL) as a signaling compound. The eight nonpathogenic A. vitis strains used in this study showed AHL-degrading activity. The complete genome sequence of A. vitis MAFF 212306 contained three AHL lactonase gene homologs. When these genes were cloned and transformed into Escherichia coli DH5α, E. coli harboring the aiiV gene (RvVAR031_27660) showed AHL-degrading activity. The aiiV coding region was successfully amplified by polymerase chain reaction from the genomes of all eight strains of nonpathogenic A. vitis. Purified His-tagged AiiV exhibited AHL lactonase activity by hydrolyzing the lactone ring of AHL. AiiV had an optimal temperature of approximately 30 °C; however, its thermostability decreased above 40 °C. When the AiiV-expressing plasmid was transformed into Pectobacterium carotovorum subsp. carotovorum NBRC 3830, AHL production by NBRC 3830 decreased below the detection limit, and its maceration activity, which was controlled by quorum sensing, almost disappeared. These results suggest the potential use of AHL-degrading nonpathogenic A. vitis for the inhibition of crown gall disease in grapevines and other plant diseases controlled by quorum sensing.

Genomic Reclassification and Phenotypic Characterization of Pseudomonas putida Strains Deposited in Japanese Culture Collections.

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 13063T 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 13063T 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 14164T (=JCM 13063T) 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.

N -acylhomoserine lactone-degrading activity of Trichoderma species and its application in the inhibition of bacterial quorum sensing.

Many gram-negative pathogens can activate virulence factors under the control of N-acylhomoserine lactone (AHL）-mediated quorum sensing. AHL-degrading enzymes have been investigated for their application in disease control. Trichoderma is a genus of fungi inhabiting various types of soil and are widely used as biocontrol agents for plant pathogens. When the AHL-degrading activity of 33 strains belonging to Trichoderma species was investigated, most strains can degrade AHL. AHL lactonase catalyzes AHL ring opening by hydrolyzing lactone. Two model strains, Trichoderma atroviride MAFF 242473 and MAFF 242475, degrade AHL using their AHL lactonase activity and rapidly metabolize ring-opening AHL. Moreover, co-inoculation with MAFF 242473 and MAFF 242475 effectively inhibited AHL production by the plant pathogens, Pantoea ananatis and Pectobacterium carotovorum subsp. carotovorum. Our study suggested that Trichoderma might be an effective biocontrol agent to inhibit the expression of virulence factors via AHL-mediated quorum sensing.

AidC, a novel N-acylhomoserine lactonase from the potato root-associated cytophaga-flavobacteria-bacteroides (CFB) group bacterium Chryseobacterium sp. strain StRB126.

N-acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many gram-negative bacteria. We have reported that Chryseobacterium sp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized the aidC gene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-ß-lactamase proteins from Bacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn(2+) is important to AidC activity both in vitro and in vivo. In addition, the aidC gene could also be PCR amplified from several other Chryseobacterium strains. In conclusion, this study indicated that the aidC gene, encoding a novel AHL lactonase, may be widespread throughout the genus Chryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.

Regulation of phenazine-1-carboxamide production by quorum sensing in type strains of Pseudomonas chlororaphis subsp. chlororaphis and Pseudomonas chlororaphis subsp. piscium.

Quorum sensing is a population density-dependent gene regulation mechanism. N-Acyl-l-homoserine lactone (AHL) has been identified as a signal compound in quorum sensing in gram-negative bacteria. Phenazine derivatives are bacterial secondary metabolites known for their broad-spectrum antifungal activity. Pseudomonas chlororaphis has been demonstrated to be a biocontrol strain, and most of its species can produce phenazine derivatives under AHL-mediated quorum sensing. Although P. chlororaphis is divided into four subspecies, the relationship between phenazine production and quorum sensing has not been investigated in two of the subspecies, P. chlororaphis subsp. chlororaphis and piscium. Two luxI/luxR homolog gene sets, phzI and phzR and csaI and csaR, were found in the complete genome sequences of the type strains of P. chlororaphis subsp. chlororaphis JCM 2778T and P. chlororaphis subsp. piscium DSM 21509T. Two major AHLs, N-(3-hydroxyhexanoyl)-l-homoserine lactone and N-(3-hydroxyoctanoyl)-l-homoserine lactone, were detected in JCM 2778 and DSM 21509 samples. PhzI synthesized all AHLs; however, CsaI could not perform AHL biosynthesis in JCM 2778 and DSM 21509. In both strains, disruption of the phzI caused complete disappearance of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production; however, disruption of csaI did not induce significant changes in PCA and PCN production. Phenazine derivatives produced by JCM 2778 and DSM 21509 under quorum sensing are crucial for the control of the plant pathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Fusarium nirenbergiae. These results demonstrated that PhzI/PhzR quorum-sensing system play an important role in production of phenazine derivatives and biocontrol activity.

Genome sequence of Microbacterium testaceum StLB037, an N-acylhomoserine lactone-degrading bacterium isolated from potato leaves.

Microbacterium testaceum is an endophytic Gram-positive bacterium that resides within plant hosts. M. testaceum StLB037 was isolated from potato leaves and shows N-acylhomoserine lactone-degrading activity. Here, we present the 3.98-Mb complete genome sequence of StLB037, with an average GC content of 70.28%.

Genetic and functional diversity of PsyI/PsyR quorum-sensing system in the Pseudomonas syringae complex.

Strains belonging to the Pseudomonas syringae complex often possess quorum-sensing systems that comprise N-acyl-l-homoserine lactone (AHL) synthases (PsyI) and AHL receptors (PsyR). Here, we investigated the diversity of PsyI/PsyR quorum-sensing systems in 630 strains of the P. syringae complex. AHL production was observed in most strains of Pseudomonas amygdali and Pseudomonas meliae, and a few strains of Pseudomonas coronafaciens and P. syringae. The DNA sequences of psyIR and their upstream and downstream regions were categorized into eight types. P. amygdali pv. myricae, Pseudomonas savastanoi, and P. syringae pv. solidagae, maculicola, broussonetiae, and tomato encoded psyI, but did not produce detectable amounts of AHL. In P. savastanoi, an amino acid substitution (R27S) in PsyI caused defective AHL production. The psyI gene of P. syringae pv. tomato was converted to pseudogenes by frameshift mutations. Escherichia coli harboring psyI genes from P. amygdali pv. myricae, P. syringae pv. solidagae and broussonetiae showed high levels of AHL production. Forced expression of functional psyR restored AHL production in P. amygdali pv. myricae and P. syringae pv. solidagae. In conclusion, our study indicates that the PsyI/PsyR quorum-sensing systems in P. syringae strains are genetically and functionally diverse, with diversity being linked to phylogenetic and pathovar classifications.

Detection of Culturable Bacteria from Tomato Seeds on Media Semi-selective for the Canker Pathogen.

Culturable bacteria were isolated from tomato seeds using media selective for the canker pathogen Clavibacter michiganensis subsp. michiganensis. Clustering analysis (>99% identity) revealed the presence of 16 operational taxonomic units (OTUs) among isolates detected on semi-selective media. Three OTUs belonged to the phylum Actinobacteria, including those of Micrococcus and Dermacoccus, and 13 OTUs belonged to the phylum Firmicutes, including Bacillus and related genera. These Gram-positive endophytic bacteria have the potential to provide false-positive results in seed health tests using media considered semi-selective for the cancer pathogen.

Comparative Genome Analysis Reveals Differences in Biocontrol Efficacy According to Each Individual Isolate Belonging to Rhizospheric Fluorescent Pseudomonads.

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.

AiiM, a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many Gram-negative bacteria. We have reported that Microbacterium testaceum StLB037, which was isolated from the leaf surface of potato, has AHL-degrading activity. In this study, we cloned the aiiM gene from the genomic library of StLB037, which has AHL-degrading activity and shows high homology with the alpha/beta hydrolase fold family from Actinobacteria. Purified AiiM as a maltose binding fusion protein showed high degrading activity of AHLs with both short- and long-chain AHLs with or without substitution at carbon 3. High-performance liquid chromatography analysis revealed that AiiM works as an AHL lactonase that catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of AiiM in the plant pathogen Pectobacterium carotovorum subsp. carotovorum reduced pectinase activity markedly and attenuated soft rot symptoms on potato slices. In conclusion, this study indicated that AiiM might be effective in quenching quorum sensing of P. carotovorum subsp. carotovorum.

Identification and Characterization of Quorum-Quenching Activity of N-Acylhomoserine Lactonase from Coagulase-Negative Staphylococci.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signals in Gram-negative bacteria. Many genes encoding AHL-degrading enzymes have been cloned and characterized in various microorganisms. Coagulase-negative staphylococci (CNS) are present on the skin of animals and are considered low-virulent species. The AHL-lactonase gene homologue, ahlS, was present in the genomes of the CNS strains Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri. We cloned the candidate ahlS homologue from six CNS strains into the pBBR1MCS5 vector. AhlS from the CNS strains showed a higher degrading activity against AHLs with short acyl chains compared to those with long acyl chains. AhlS from S. sciuri was expressed and purified as a maltose-binding protein (MBP) fusion. Pseudomonas aeruginosa is an opportunistic pathogen that regulates several virulence factors such as elastase and pyocyanin by quorum-sensing systems. When MBP-AhlS was added to the culture of P. aeruginosa PAO1, pyocyanin production and elastase activity were substantially reduced compared to those in untreated PAO1. These results demonstrate that the AHL-degrading activity of AhlS from the CNS strains can inhibit quorum sensing in P. aeruginosa PAO1.

Discovery of an Antibiotic-Related Small Protein of Biocontrol Strain Pseudomonas sp. Os17 by a Genome-Mining Strategy.

Many root-colonizing Pseudomonas spp. exhibiting biocontrol activities produce a wide range of secondary metabolites that exert antibiotic effects against other microbes, nematodes, and insects in the rhizosphere. The expression of these secondary metabolites depends on the Gac/Rsm signal transduction pathway. Based on the findings of a previous genomic study on newly isolated biocontrol pseudomonad strains, we herein investigated the novel gene cluster OS3, which consists of four genes (Os1348-Os1351) that are located upstream of putative efflux transporter genes (Os1352-Os1355). Os1348 was predicted to encode an 85-aa small precursor protein, the expression of which was under the control of GacA, and an X-ray structural analysis suggested that the Os1348 protein formed a dimer. The mutational loss of the Os1348 gene decreased the antibiotic activity of Pseudomonas sp. Os17 without changing its growth rate. The Os1349-1351 genes were predicted to be involved in post-translational modifications. Intracellular levels of the Os1348 protein in the deficient mutant of each gene differed from that in wild-type cells. These results suggest that Os1348 is involved in antibiotic activity and that the structure or expression of this protein is under the control of downstream gene products.

Diversity of Antibiotic Biosynthesis Gene-possessing Rhizospheric Fluorescent Pseudomonads in Japan and Their Biocontrol Efficacy.

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.

Indigenous bacteria may interfere with the biocontrol of plant diseases.

Prodigiosin is a reddish antibiotic pigment that plays an important role in the biocontrol of plant diseases by the bacterium Serratia marcescens. However, its activity is unstable under agricultural conditions; further, it can be degraded by various environmental factors. To examine the effect of epiphytic microbes on the stability of prodigiosin used for biological control processes, we collected a total of 1,280 bacterial isolates from the phylloplane of cyclamen and tomato plants. Approximately 72% of the bacterial strains isolated from the cyclamen plants and 66% of those isolated from the tomato plants grew on minimal agar medium containing 100 microg ml(-1) prodigiosin. Certain isolates obtained from both plant species exhibited prodigiosin-degrading activity. We compared the 16S rRNA gene sequences derived from the isolates with sequences in a database. The comparison revealed that the sequences determined for the prodigiosin-degrading isolates were homologous to those of the genera Pseudomonas, Caulobacter, Rhizobium, Sphingomonas, Janthinobacterium, Novosphingobium, and Rathayibacter. These results indicate that indigenous epiphytic microorganisms may interfere with the interaction between plant pathogens and biocontrol agents by degrading the antibiotics produced by the agents.