A new bacterial strain, <Emphasis Type="Italic">Pseudomonas koreensis</Emphasis> IB-4, as a promising agent for plant pathogen biological control

A bacterial strain IB-4, antagonistic to plant pathogenic fungi of the genera Fusarium, Bipolaris, and Alternaria, was isolated from arable soils of the Mechetlinskii district, Bashkortostan. Physiological, biochemical, and culture morphological properties of strain IB-4 supported its classification within the genus Pseudomonas. In spite of some discrepancies in the results of phenotypic and chemotaxonomic research, analysis of the 16S rRNA gene sequence, DNA–DNA hybridization, GC-content, and MALDI mass spectral data provide considerable evidence supporting its identification as a Pseudomonas koreensis strain. P. koreensis strain IB-4 was shown to possess the valuable features characteristic of PGPR microorganisms: antifungal and nitrogenase activities and ability to synthesize indole-3-acetic acid (IAA) and cytokinin-like compounds. Field test, in which potato plants were treated with the culture liquid of P. koreensis strain IB-4 revealed a positive effect on potato yield and resistance to plant pathogens.     

<Emphasis Type="Italic">Paenibacillus oryzisoli</Emphasis> sp. nov., isolated from the rhizosphere of rice

A novel bacterium, strain 1ZS3-15^T, was isolated from rhizosphere of rice. Its taxonomic position was investigated using a polyphasic approach. The novel strain was observed to be Gram-stain positive, spore-forming, aerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 1ZS3-15^T was recovered within the genus Paenibacillus. It is closely related to Paenibacillus pectinilyticus KCTC 13222^T (97.9 % similarity), Paenibacillus frigoriresistens CCTCC AB 2011150^T (96.8 %), Paenibacillus alginolyticus JCM 9068^T (96.4 %) and Paenibacillus chondroitinus DSM 5051^T (95.5 %). The fatty acid profile of strain 1ZS3-15^T, which showed a predominance of anteiso-C_15:0 and iso-C_16:0, supported the allocation of the strain into the genus Paenibacillus. The predominant menaquinone was found to be MK-7. The polar lipids profile of strain 1ZS3-15^T was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified lipid and two unidentified aminophospholipids. The cell wall peptidoglycan contains meso-diaminopimelic acid. Based on draft genome sequences, the DNA–DNA relatedness between strain 1ZS3-15^T and the closely related species P. pectinilyticus KCTC 13222^T are 24.2 ± 1.0 %, and the Average Nucleotide Identity values between the strains are 78.9 ± 0.1 %, which demonstrated that this isolate represents a new species in the genus Paenibacillus. The DNA G+C content was determined to be 45.3 mol%, which is within the range reported for Paenibacillus species. Characterisation by genotypic, chemotaxonomic and phenotypic analysis indicated that strain 1ZS3-15^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus oryzisoli sp. nov. is proposed. The type strain is 1ZS3-15^T (= ACCC 19783^T = JCM 30487^T).     

<Emphasis Type="Italic">Paenibacillus bryophyllum</Emphasis> sp. nov., a nitrogen-fixing species isolated from <Emphasis Type="Italic">Bryophyllum pinnatum</Emphasis>

A nitrogen-fixing, endospore-forming bacterium, designated strain L201^T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201^T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3^T (97.4%), Paenibacillus odorifer DSM 15391^T (97.3%) and Paenibacillus borealis DSM 13188^T (97.2%). The main fatty acids components was anteiso-C_15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201^T was 43.9%. DNA–DNA relatedness between L201^T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201^T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201^T(=?KCTC 33951 ^T?=?GDMCC 1.1251 ^T).     

Purification and Characterization of Exo-Inulinase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. d9 Strain

This study intended to purify and characterise exo-inulinase of diesel-degrading Paenibacillus sp. D9. The whole genome sequencing of Paenibacillus sp. D9 revealed to possess the sacC gene that is encoded as exo-inulinase/levanase. This isolate was capable of producing a maximum of 50.9 IU/mL of exo-inulinase activity within 3 days at 30?°C, 200 rpm and pH of 7.0 on minimal salt medium agar supplemented with 1% (w/v) inulin. An exo-inulinase of 58.5 kDa was purified using ammonium sulphate precipitation, HiTrap QFF column and MMC column chromatographies with a specific activity of 4333 IU/mg, 7.1% recovery and a 4.3-fold increase in purity. The purified D9 exo-inulinase had temperature and pH optimum at 40?°C and pH 4.0, respectively, with the Michaelis constant of 5.5 mM and a maximal velocity of 476.2 IU/mg, respectively. Catalytic constant, k _cat was calculated to be 42.6 s^?1 with a catalytic efficiency (k _cat /K _m ) of 7.6 s^?1 mM^?1. The presence of Ca²⁺ enhanced the activity of D9 exo-inulinase while Hg²⁺ completely inhibited the activity, other compounds such as Fe³⁺ and Cu²⁺ had an inhibitory effect. The results of amino acid alignment and the complete degradation of inulin into fructose by the purified enzyme confirmed that inulinase from Paenibacillus sp. D9 is an exo-form. The phylogenetic tree based on the protein sequences indicates that bacterial exo-inulinases possess a common ancestry.     

Isolation,characterization and virulence of bacteria from <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.     

Production of macrolide antibiotics from a cytotoxic soil <Emphasis Type="Italic">Streptomyces</Emphasis> sp. strain ZDB

Crude extract from a culture of a soil Streptomyces sp. strain ZDB showed toxicity towards Artemia salina and antimicrobial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Chlorella vulgaris, and Chlorella sorokiniana. Large scale fermentation of the strain led to the isolation of the macrolide antibiotics, bafilomycins A1 (1), B1 (2), and D (3) together with nonactic acid (4) and bostrycoidin-9-methyl ether (5). Structures of the antibiotics were determined based on spectral data analysis. We describe the isolation of the compounds and characterization of the producing strain.     

Growth and wax ester production of an <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> ADP1 mutant deficient in exopolysaccharide capsule synthesis

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA ^? strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA ^? cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA ^? strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA ^? strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

Isolation and identification of cultivated bacteria associated with soybeans and their biocontrol activity against <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

One hundred bacteria, isolated from rhizospheric soil and rhizoplane of healthy soybean plants, were assayed for antifungal activity against six Phytophthora sojae isolates. Nine of the tested bacteria inhibited the hyphal growth of P. sojae in vitro. They were subsequently evaluated for their in vitro traits and identified using the 16S rRNA gene sequences. Four of them (Paenibacillus sp.,—S1; Streptomyces sp.,—S9, S10 and S11) were further selected on the basis of their strongest antagonistic activity in vitro against P. sojae race 4, the predominant race in most growing soybean areas in Canada, and tested for their beneficial effects on soybean plants in the greenhouse. Results showed that application of bacterial strain S11 as seed coating reduced the disease severity by 57.1% and increased the root and shoot weight by and 140 and 108% respectively, in comparison to the diseased control. Overall, a positive correlation was recorded between the in vitro and in planta effects of the selected bacteria. This is promising for further application as select environmentally safe biological control agents in the protection of soybean against root rot diseases.     

Role of bacterial pyrroloquinoline quinone in phosphate solubilizing ability and in plant growth promotion on strain <Emphasis Type="Italic">Serratia</Emphasis> sp. S119

The aim of this study was to analyze if cofactor pyrroquinoline quinone from Serratia sp. S119 is involved in the inorganic phosphate solubilization mechanism and in its ability to promote the plant growth. Site directed mutagenesis was performed to obtain a pqqE- minus mutant of strain Serratia sp. S119. The phosphate solubilization ability, gluconate and PQQ production of the mutant Serratia sp. RSL (pqqE-) was analyzed. Mutant RSL (pqqE-) showed significant decrease in P soluble and gluconic acid levels produced and undetectable levels of PQQ cofactor compared with wild-type strain. Complementation with synthetic PQQ cofactor restored P solubilization and gluconate production reaching the levels produced by wild-type strain. PqqE gene sequence indicated that it is highly conserved within Serratia strains and its product shows conserved motifs found in other PqqE proteins of several bacteria. The effect of the inoculation of the PQQ- mutant on peanut and maize plants was evaluated in pot assays. Plants growth parameters showed no differences among the different treatments indicating that PQQ from Serratia sp. S119 is not involved in the growth promotion of these plants. PQQ cofactor is essential for phosphate solubilization ability of Serratia sp. S119 but is not required for growth promotion of peanut and maize plants.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Root-associated bacteria influencing mycelial growth of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> (pine mushroom)

Tricholoma matsutake is an ectomycorrhizal fungus usually associated with Pinus densiflora in South Korea. Fruiting bodies (mushrooms) of T. matsutake are economically important due to their attractive aroma; yet, T. matsutake is uncultivatable and its habitat is rapidly being eradicated due to global climate change. Root-associated bacteria can influence the growth of ectomycorrhizal fungi that co-exist in the host rhizosphere and distinctive bacterial communities are associated with T. matsutake. In this study, we investigated how these bacterial communities affect T. matsutake growth by isolating bacteria from the roots of P. densiflora colonized by ectomycorrhizae of T. matsutake and co-culturing rootassociated bacteria with T. matsutake isolates. Thirteen species of bacteria (27 isolates) were found in pine roots, all belonging to the orders Bacillales or Burkholderiales. Two species in the genus Paenibacillus promoted the growth of T. matsutake in glucose poor conditions, likely using soluble metabolites. In contrast, other bacteria suppressed the growth of T. matsutake using both soluble and volatile metabolites. Antifungal activity was more frequent in glucose poor conditions. In general, pine rhizospheres harbored many bacteria that had a negative impact on T. matsutake growth and the few Paenibacillus species that promoted T. matsutake growth. Paenibacillus species, therefore, may represent a promising resource toward successful cultivation of T. matsutake.     

<Emphasis Type="Italic">Amycolatopsis vastitatis</Emphasis> sp. nov., an isolate from a high altitude subsurface soil on Cerro Chajnantor,northern Chile

The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5^T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H₄) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5^T (= NCIMB 14970^T = NRRL B-65279^T).     

Biodegradation of sulfonamides by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 and <Emphasis Type="Italic">Shewanella</Emphasis> sp. strain MR-4

Because of extensive sulfonamides application in aquaculture and animal husbandry and the consequent increase in sulfonamides discharged into the environment, strategies to remediate sulfonamide-contaminated environments are essential. In this study, the resistance of Shewanella oneidensis MR-1 and Shewanella sp. strain MR-4 to the sulfonamides sulfapyridine (SPY) and sulfamethoxazole (SMX) were determined, and sulfonamides degradation by these strains was assessed. Shewanella oneidensis MR-1 and Shewanella sp. strain MR-4 were resistant to SPY and SMX concentrations as high as 60 mg/L. After incubation for 5 days, 23.91 ± 1.80 and 23.43 ± 2.98% of SPY and 59.88 ± 1.23 and 63.89 ± 3.09% of SMX contained in the medium were degraded by S. oneidensis MR-1 and Shewanella sp. strain MR-4, respectively. The effects of the initial concentration of the sulfonamides and initial pH of the medium on biodegradation, and the degradation of different sulfonamides were assessed. The products were measured by LC–MS; with SPY as a substrate, 2-AP (2-aminopyridine) was the main stable metabolite, and with SMX as a substrate, 3A5MI (3-amino-5-methyl-isoxazole) was the main stable metabolite. The co-occurrence of 2-AP or 3A5MI and 4-aminobenzenesulfonic acid suggests that the initial step in the biodegradation of the two sulfonamides is S–N bond cleavage. These results suggest that S. oneidensis MR-1 and Shewanella sp. strain MR-4 are potential bacterial resources for biodegrading sulfonamides and therefore bioremediation of sulfonamide-polluted environments.