Young Ryun Chung

Lysobacter capsici sp. nov., with antimicrobial activity, isolated from the rhizosphere of pepper, and emended description of the genus Lysobacter.

The taxonomic position of a novel bacterial strain, YC5194(T), with antimicrobial activity, isolated from the rhizosphere of pepper in Jinju, South Korea, was studied using a polyphasic approach. Cells of the strain were Gram-negative, rod-shaped, facultative anaerobes. It grew at a temperature of 15-37 degrees C (optimum 28 degrees C). Growth of the strain occurred between pH 5.5 and 8.5, with an optimum of pH 7.0-7.5. The strain inhibited mycelial growth of Pythium ultimum, Colletotrichum gloeosporioides, Fusarium oxysporum, Botrytis cinerea, Rhizoctonia solani and Botryosphaeria dothidea and growth of Bacillus subtilis. The G+C content of the total DNA was 65.4 mol%. The 16S rRNA gene sequence of the strain was most closely related to species of the genus Lysobacter (<94.0 to >99.0 % sequence similarity). Chemotaxonomic data (major quinone, Q-8; major polar lipids, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyl-N-methylethanolamine; major fatty acids, iso-C(15 : 0), summed feature 3, C(16 : 0), iso-C(17 : 1)omega9c and C(18 : 1)omega7c) supported the affiliation of strain YC5194(T) to the genus Lysobacter. Phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA hybridization data and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain YC5194(T) from species of Lysobacter with validly published names. Strain YC5194(T) therefore represents a novel species, for which the name Lysobacter capsici sp. nov. is proposed. The type strain is YC5194(T) (=KCTC 22007(T) =DSM 19286(T)).

Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity

Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.

Paenibacillus koreensis sp. nov., a new species that produces an iturin-like antifungal compound.

A bacterial strain, YC300T, that produces an iturin-like antifungal antibiotic was isolated from compost and identified as member of the genus Paenibacillus. Gram reaction of the strain was variable depending upon growth stages and culture media. Three different types of colonies were developed on tryptic soy agar. The organism was facultatively anaerobic and grew at 50 degrees C. The DNA G+C content was 54 mol % and anteiso-C15:0 was the major fatty acid. A 0.9 kb fragment was produced by PCR amplification of strain YC300T DNA using primers PAEN515F and 1377R. Levels of 16S rDNA similarity between strain YC300T and other Paenibacillus species were between 89.8 and 94.8%. Phylogenetically, strain YC300T formed a significant monophyletic clade with Paenibacillus validus. It is clear from polyphasic evidence that the isolate should be classified as Paenibacillus koreensis sp. nov., the type strain of which is YC300T (= KCTC 2393T, KCCM 40903T).

Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli.

ABSTRACT Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using S. cerevisiae as a target fungus.

Changes in fungal population of fly ash and vinasse mixture during vermicomposting by Eudrilus eugeniae and Eisenia fetida: Documentation of cellulase isozymes in vermicompost

Fly ash (FA) and vinasse (VN), two industrial wastes, are generated in huge amounts and cause serious hazards to the environment. In this experiment, different proportions of these two wastes were used as food for two epigeic earthworms (Eisenia fetida and Eudrilus eugeniae) to standardize the recycling technique of these two wastes and to study their effect on fungal especially cellulolytic fungal population, cellulase activity and their isozyme pattern, chitin content and microbial biomass of waste mixture during vermicomposting. Increasing VN proportion from 25% to 50% or even higher, counts of both fungi and cellulolytic fungi in waste mixtures were significantly (P ≤ 0.05) increased during vermicomposting. Higher cellulase activity in treatments having 50% or more vinasse might be attributed to the significantly (P ≤ 0.05) higher concentration of group I isozyme while concentrations of other isozymes (group II and III) of cellulase were statistically at par. Higher chitin content in vinasse-enriched treatments suggested that fungal biomass and fungi-to-microbial biomass ratio in these treatments were also increased due to vermicomposting. Results revealed that Eudrilus eugeniae and Eisenia fetida had comparable effect on FA and VN mixture during vermicomposting. Periodical analysis of above-mentioned biochemical and microbial properties and nutrient content of final vermicompost samples indicated that equal proportion (1:1, w/w) of FA and VN is probably the optimum composition to obtain best quality vermicompost.

Kitasatospora cheerisanensis sp. nov., a new species of the genus Kitasatospora that produces an antifungal agent

An actinomycete, strain YC75T, which produced bafilomycin-like antifungal compounds, was identified as a member of the genus Kitasatospora on the basis of morphological and chemotaxonomic characteristics. The strain produced the aerial and fragmenting vegetative mycelia consisting of straight chains of 20 or more smooth-surfaced spores. Submerged spores were formed in tryptic soy broth. No soluble pigments were formed. Whole-cell hydrolysates contained glucose and mannose, but not galactose. The 16S rDNA sequence of YC75T was compared with those of the other representative kitasatosporae and streptomycetes. Strain YC75T formed a significant monophyletic clade with Kitasatospora phosalacinea. The levels of DNA relatedness between strain YC75T and representatives of the genus Kitasatospora ranged from 16 to 59% including K. phosalacinea (28 and 40%). It is clear from polyphasic evidence that the isolate should be classified as Kitasatospora cheerisanensis sp. nov., whose type strain is YC75T (= KCTC 2395T). The presence of galactose in whole-cell hydrolysates may not be a stable chemical marker for the genus Kitasatospora.

Diversity and Characterization of Endophytic Bacteria Associated with Tidal Flat Plants and their Antagonistic Effects on Oomycetous Plant Pathogens

Endophytic bacterial communities of tidal flat plants antagonistic to oomycete plant pathogens were studied by the isolation of 256 root colonizing endophytic bacteria from surface-disinfected root tissues of six plants (Rosa rugosa, Suaeda maritima, Vitex rotundifolia, Carex scabrifolia, Glehnia littoralis and Elymus mollis) growing in a tidal flat area of Namhae Island, Korea. To understand the antagonistic potential, an in vitro antagonistic assay was performed to characterize and identify strains that were antagonistic to the oomycete plant pathogens Phytophthora capsici and Pythium ultimum from the total population. Nine percent of the total number of isolated bacteria exhibited in vitro inhibitory activity against target plant pathogenic oomycetes. Taxonomic and phylogenetic placement of the antagonistic bacteria was investigated by analysis of the 16S rRNA gene sequences. The sequence analysis classified the antagonistic strains into four major classes of the domain bacteria (Firmicutes, α-Proteobacteria, γ-Proteobacteria and Actinomycetes) and 10 different genera. Further production of secondary metabolites, hydrolytic enzymes and plant growth promoting traits were determined for the putative new species of antagonistic endophytic bacteria. These new strains could not be identified as known species of α-Proteobacteria, and so may represent novel bacterial taxa. The unexpected high antagonistic bacterial diversity associated with the tidal flat plants may be indicative of their importance in tidal flat plants as a promising source of novel antimicrobial compounds and biocontrol agents.

Lysobacter oryzae sp. nov., isolated from the rhizosphere of rice (Oryza sativa L.)

The taxonomic position of a novel bacterial strain, YC6269(T), isolated from the rhizosphere of rice (Oryza sativa L.) managed under no-tillage practice in Jinju, South Korea, was studied using polyphasic approach. Cells of the strain were Gram-negative, rod-shaped and facultatively anaerobic. The novel strain grew at a temperature of 15-42 degrees C (optimum at 28 degrees C). Growth of the strain occurred between pH 5.5 and 11.0, with an optimum at pH 7.0-8.0. The G+C content of the total DNA was 67.4 mol%. The 16S rRNA gene sequence of the strain was most closely related to species of the genus Lysobacter, Lysobacter yangpyeongensis DSM 17635(T) (98.6 %), Lysobacter niabensis GH34-4(T) (97.2 %), Lysobacter enzymogenes DSM 2043(T) (96.9 %), Lysobacter daejeonensis DSM 17634(T) (96.3 %) and Lysobacter niastensis GH41-7(T) (96.2 %). The novel strain showed <96.0 % similarity with other species of the genus Lysobacter. Chemotaxonomic data (major quinone, Q-8; major polar lipids, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyl-N-methylethanolamine, and major fatty acids, C(15 : 0) iso, C(16 : 0) iso, C(17 : 0) iso and C(17 : 1) iso omega9c) supported the affiliation of strain YC6269(T) to the genus Lysobacter. Phylogenetic analysis based on the 16S rRNA gene sequences, DNA-DNA hybridization data and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain YC6269(T) from recognized species of the genus Lysobacter. Strain YC6269(T), therefore, represents a novel member of the genus Lysobacter, for which the name Lysobacter oryzae sp. nov. is proposed. The type strain is YC6269(T) (=KCTC 22249(T)=DSM 21044(T)).

Chitinophaga oryziterrae sp. nov., isolated from the rhizosphere soil of rice (Oryza sativa L.)

A novel bacterial strain, YC7001(T), was isolated from the rhizosphere soil of rice collected at Jinju, Korea, and was characterized using a polyphasic approach. Cells of strain YC7001(T) were Gram-negative-staining, yellow-pigmented, rod-shaped, aerobic, heterotrophic and non-motile. Strain YC7001(T) grew optimally at 25-30 °C, pH 5.0-8.0 and degraded gelatin, cellulose and chitin. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YC7001(T) belonged to the genus Chitinophaga in the family Chitinophagaceae. The most closely related species were Chitinophaga sancti NBRC 15057(T) (96.9%), Chitinophaga ginsengisoli Gsoil 052(T) (95.9%), Chitinophaga pinensis DSM 2588(T) (95.7%), Chitinophaga filiformis IFO 15056(T) (95.7%) and Chitinophaga niabensis JS13-10(T) (95.0%). The DNA-DNA relatedness value of strain YC7001(T) with C. sancti NBRC 15057(T) was 15.2 ± 0.6%. Strain YC7001(T) contained MK-7 as the major menaquinone and homospermidine as the major polyamine. The major fatty acids of strain YC7001(T) were C(15:0) iso, C(16:1)ω5c and C(17:0) iso 3-OH. The polar lipids were phosphatidylethanolamine, unknown aminolipids and unknown lipids. The total genomic DNA G+C content was 41.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain YC7001(T) represents a novel species of the genus Chitinophaga, in the family Chitinophagaceae, for which the name Chitinophaga oryziterrae sp. nov. is proposed. The type strain is YC7001(T) ( = KACC 14533(T) = JCM 16595(T)).

Potential of an Indigenous Fungus, Plectosporium tabacinum, as a Mycoherbicide for Control of Arrowhead (Sagittaria trifolia)

An isolate of the indigenous fungus Plectosporium tabacinum was isolated from arrowhead (Sagittaria trifolia) in Yusung, Korea in 1990 and evaluated in laboratory and growth chamber tests as a potential mycoherbicide. The fungus grew comparatively slowly on potato dextrose agar and corn meal agar, attaining a diameter of 65 mm after 12 days at 25°C. Conidia were mass-produced in shake-cultures or in a fermentor using potato dextrose broth containing yeast extract (0.5%, wt/vol) at 25°C. When arrowhead seedlings at the 2- to 3-leaf stage were inoculated with conidial suspensions (2 × 107 conidia/ml) and incubated in a dew chamber for 18 h at 25°C, the plants developed small, brown spots on the leaves and petioles in 2 days, and were blighted completely within 7 days after inoculation. This effect was consistent on arrowhead plants from the 2- to 5-leaf stage. Another arrowhead species, S. pygmaea, was as susceptible as S. trifolia to the pathogen. Several crops, including rice, barley, and wheat and 34 other common weed species, were immune. In small-scale field tests in paddy fields during the summers of 1992 and 1993, a mean reduction of 71.3% in the number of arrowhead plants was observed following a foliar spray of a conidial suspension (107 conidia/ml). These results indicate that P. tabacinum has potential as a selective mycoherbicide for arrowhead control.