• The Korean Journal of Mycology
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• v.47 no.3
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• pp.259-269
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• 2019

Brown spot and sheath rot of rice are caused by fungal pathogens such as Curvularia lunata, Cochliobolus miyabeanus, and Sarocladium oryzae, and cause losses such as reduced rice yield and quality, which is an enormous problem with serious long-term effects. To search biological control agents of phytopathogenic fungi, five kinds of useful Bacillus-like isolates which are excellent in extracellular enzyme activity and produce siderophore were selected from paddy soil of Sunchang in Korea. The selected isolates were tested for excellent antifungal activity against three of the phytopathogenic fungi that frequently occur in rice, and JSRB 177 strain had the most excellent antifungal activity. Based on the experimental results, JSRB 177 is finally selected as a candidate for biological control and identified to Bacillus subtilis through 16S rRNA sequence analysis. In addition, physiological characteristics of JSRB 177 confirmed by analysis of carbohydrate fermentation patterns and enzyme production ability. Based on the above results, JSRB 177 is expected to be used as a biological control agent for the rice pathogenic fungi. In the future, further studies related to industrialization such as port test and establishment of mass production process are needed.

• The Korean Journal of Mycology
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• v.20 no.2
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• pp.109-117
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• 1992

Chemotactic responses of five motile saprophytic and one phytopathogenic bacteria e.g. Agrobacterium radiobacter, Bacillus subtilis, B. polymyxa, Pseudomonas aeruginosa, P. fluorescens and Xanthomonas malvacearum towards exudate of Cochliobolus sativus conidia, Fusarium of oxysporum f. sp. ciceri chlamydospores, Macrophomina phaseolina sclerotia and Phytophthora drechsleri f. sp. cajani oospores were determined in vitro at different abiotic conditions. In general, a positive correlation (r=0.76 to 0.89; P=0.05) was observed between concentration of fungal exudates and attraction of bacterial cells. Similarly, a significant (P=0.05; r=+0.82 to 0.95) positive correlation was noticed between chemotactic response and incubation period. The chemotactic response of bacteria was greatly influenced by temperature and pH of the test fungal exudate. The optimum temperature for maximum chemotaxis was $25^{\circ}C$ for A. radiobacter, $30^{\circ}C$ for B. polymyxa, P. aerugionosa, P. fluorescens and X. malvacearum and $35^{\circ}C$ for B. subtilis. Fungal exudates maintained at pH 7 attracted maximum number of bacteria. The response of bacterial cells to exudates at pH 3 and 11 was not significantly (P=0.05) different than that to the buffer (control). Chemotaxis of bacteria was observed towards attractants (fungal propagules and their exudates) when they were kept apart and bridged with the capillaries filled with non-attractant (buffer) or attractant (exudate).

• Journal of Ginseng Research
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• v.40 no.2
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• pp.97-104
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• 2016

Background: Rhizobacteria play an important role in plant defense and could be promising sources of biocontrol agents. This study aimed to screen antagonistic bacteria and develop a biocontrol system for root rot complex of Panax notoginseng. Methods: Pure-culture methods were used to isolate bacteria from the rhizosphere soil of notoginseng plants. The identification of isolates was based on the analysis of 16S ribosomal RNA (rRNA) sequences. Results: A total of 279 bacteria were obtained from rhizosphere soils of healthy and root-rot notoginseng plants, and uncultivated soil. Among all the isolates, 88 showed antagonistic activity to at least one of three phytopathogenic fungi, Fusarium oxysporum, Fusarium solani, and Phoma herbarum mainly causing root rot disease of P. notoginseng. Based on the 16S rRNA sequencing, the antagonistic bacteria were characterized into four clusters, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetesi. The genus Bacillus was the most frequently isolated, and Bacillus siamensis (Hs02), Bacillus atrophaeus (Hs09) showed strong antagonistic activity to the three pathogens. The distribution pattern differed in soil types, genera Achromobacter, Acidovorax, Brevibacterium, Brevundimonas, Flavimonas, and Streptomyces were only found in rhizosphere of healthy plants, while Delftia, Leclercia, Brevibacillus, Microbacterium, Pantoea, Rhizobium, and Stenotrophomonas only exist in soil of diseased plant, and Acinetobacter only exist in uncultivated soil. Conclusion: The results suggest that diverse bacteria exist in the P. notoginseng rhizosphere soil, with differences in community in the same field, and antagonistic isolates may be good potential biological control agent for the notoginseng root-rot diseases caused by F. oxysporum, Fusarium solani, and Panax herbarum.

• The Plant Pathology Journal
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• v.30 no.3
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• pp.245-253
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• 2014

Antimicrobial peptides (AMPs) are small but effective cationic peptides with variable length. In previous study, four hexapeptides were identified that showed antimicrobial activities against various phytopathogenic bacteria. KCM21, the most effective antimicrobial peptide, was selected for further analysis to understand its modes of action by monitoring inhibitory effects of various cations, time-dependent antimicrobial kinetics, and observing cell disruption by electron microscopy. The effects of KCM21 on Gram-negative strain, Pseudomonas syringae pv. tomato DC3000 and Gram-positive strain, Clavibacter michiganensis subsp. michiganensis were compared. Treatment with divalent cations such as $Ca^{2+}$ and $Mg^{2+}$ inhibited the bactericidal activities of KCM21 significantly against P. syringae pv. tomato DC3000. The bactericidal kinetic study showed that KCM21 killed both bacteria rapidly and the process was faster against C. michiganensis subsp. michiganensis. The electron microscopic analysis revealed that KCM21 induced the formation of micelles and blebs on the surface of P. syringae pv. tomato DC3000 cells, while it caused cell rupture against C. michiganensis subsp. michiganensis cells. The outer membrane alteration and higher sensitivity to $Ca^{2+}$ suggest that KCM21 interact with the outer membrane of P. syringae pv. tomato DC3000 cells during the process of killing, but not with C. michiganensis subsp. michiganensis cells that lack outer membrane. Considering that both strains had similar sensitivity to KCM21 in LB medium, outer membrane could not be the main target of KCM21, instead common compartments such as cytoplasmic membrane or internal macromolecules might be a possible target(s) of KCM21.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.151-156
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• 1994

Pseudomonas fluorescens produces the antibiotic, 2,4-diacetylphloroglucinol (Phl), which promotes plant growth by inhibiting bacteria and fungi. Cosmids (genomic library) were mobilized into Phl-nonproducing mutants through the triparental matings with pRK2013 as the helper plasmid at the frequency of 8.37$\times$10-4. Complemented mutants that showed antibiotic activity were selected among about 2,000 transconjugants. The complemented mutants were confirmed by acquired drug resistances (kanamycin and tetracycline). The antibiotic substances of wild type and complemented mutants showed the most excellent anti-bacterial activity. Inhibitory effects of complemented P. fluorescens against phytopathogenic fungi were equal to the parental strain. Complemented mutant and wild type of P. fluorescens were causal microbes of fungal morphological abnormalities. Complemented mutants in potato dextrose agar supplemented with bromothymol blue also showed restoration of glucose utilization as wild type. Plasmids of complemented mutants were isolated from transconjugant sand transformed into competent cells of E. coli DH5$\alpha$. The plamid DNA was reisolated from transformed E. coli DH5$\alpha$.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.184-192
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• 2006

Six-hundred bacterial strains from human milk and milk from Sahiwal cows, Holstein Friesian cows, and buffaloes were screened for their ability to suppress phytopathogenic fungi under in vitro conditions. A consortium of 3 strains, viz., Bacillus lentimorbus B-30486 (B-30486), B. subtilis B-30487 (B-30487), and B. lentimorbus B-30488 (B-30488), isolated from Sahiwal cow milk resulted in better biological control and plant-growth promotion than single-strain treatments. For commercial-scale production of a bioinoculant, the solid-state fermentation of sugarcane agro-industrial residues, i.e., molasses, press mud, and spent wash, using the consortium of B-30486, B-30487, and B-30488, resulted in a value-added product, useful for enhancing plant growth. The application of the consortium to sugarcane fields infested with Fusarium moniliforme and Colletotrichum falcatum resulted in a reduction of mortality and significantly higher (P=0.05) plant height, number of tillers, and cane girth when compared with the control. Furthermore, under field conditions, the treatment of sugarcane with the consortium resulted in significantly (P=0.05) greater plant growth compared with nonbacterized plants. Accordingly, this is the first report on the effective use of bacteria isolated from milk for biological control and enhancing plant growth under field conditions. Furthormore, a solid-state fermentation technology was developed that facilitates the economic utilization of agro-industrial residues for environmental conservation and improving plant and soil health.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.1
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• pp.11-18
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• 1997

This study was to provide the basic data in improving protoplast formation and regeneration of antagonistic bacteria against phytopathogenic fungi and pest. The antagonistic rhizobacterium, BS 101, against Rhizoctonia solrmi and Fusurium oxyspomm was isolated and identified as Bacillus subtilis. Another bacterium for protoplast formation and regeneration was B. thuringiensis subsp. kurstcJtiHD-l (BT 37669) which have insectcidal toxin in the orders Coleopteria, Dipteria etc.. Auxotrophic mutants, BS 1013 and BT 69, were isolated by treating with NTG 300 ug/ml for 40 min. at $37^{\circ}C$, and with NTG 300 ug/ml for 30 min. at $37^{\circ}C$, respectively. The BS 1013 and BT 69 were converted to protoplas by treating with lysozyme 300 ugh1 for 30 min. at 37C, and lysozyme 9 mglml for 60 min. at $37^{\circ}C$, respectively. The fequencies of the protoplast formation of BS 1013 and BT 69 were 90.00 and 92.83% respectively, after 1~2 day at $37^{\circ}C$. The regeneration kequencies of the protoplasts BS 1013 and B T 69 were 0.52 and 0.10%, respectively, after 4~6 days at $37^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.235-241
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• 1991

- A phthalic acid derivative and basic macrolide antibiotics, with antimicrobial activity against Gram positive bacteria and phytopathogenic fungi, respectively, were found to be produced by a strain 88-GT-161 identified as being a variety of Streptomyces melanosporofaciens. This paper describes an isolation procedure of the active compounds produced by this strain, their in vitro and in vivo (pot test) antimicrobial activites, and structure determination of one of the compounds, bis (2-ethylhexyl) phthalate, a phthalic acid derivative antibiotic. This compounds, upon cornparision with authentic bis (2-ethylhexyl) phthalate, dioctyl phthalate, revealed a difference in antimicrobial activity even though physico-chemical properties of these two compounds seemed indentical. This is the first report that dioctyl phthalate is biosynthetically produced by a Streptomyces sp. and shows antimicrobial activity.

• The Korean Journal of Mycology
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• v.44 no.1
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• pp.36-47
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• 2016

This study was conducted to evaluate five different strains of rhizobacterial isolates viz. PA1, PA2, PA4, PA5 and PA12 for biological control against Colletotrichum acutatum, C. coccodes, C. gloeosporioides, C. dematium, Botrytis cinerea, Rhizoctonia solani, Sclerotinia minor and Fusarium sp. In vitro inhibition assay was performed on three different growth mediums, potato dextrose agar (PDA), tryptic soy agar (TSA), and PDA-TSA (1:1 v/v) for the selection of potential antagonistic isolates. According to the result, isolate PA2 showed the highest inhibitory effect with 65.5% against C. coccodes on PDA and with 96.5% against S. minor on TSA. However, the same isolate showed the highest inhibition with 58.5% against C. acutatum on PDA-TSA. In addition, an in vivo experiment was performed to evaluate these bacterial isolates for biological control against fungal pathogens. Plants treated with bacteria were analyzed with phytopathogens and plants inoculated with phytopathogens were treated with isolates to determine the biological control effect against fungi. According to the result, all five isolates tested showed inhibitory effects against phytopathogens at various levels. Mode of action of these rhizobacterial isolates was evaluated with siderophore production, protease assay, chitinase assay and phosphate solubilizing assay. Bacterial isolates were identified by 16S rDNA sequencing, which showed that isolates PA1 and PA2 belong to Bacillus subtilis, whereas, PA4, PA5, and PA12 were identified as Bacilus altitudinis, Paenibacillus polymyxa and Bacillus amyloliquefaciens, respectively. Results of the current study suggest that rhizobacterial isolates can be used for the plant growth promoting rhizobacteria (PGPR) effect as well as for biological control of various phytopathogens.