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Lee, Kwang-Youll,Heo, Kwang-Ryool,Choi, Ki-Hyuck,Kong, Hyun-Gi,Nam, Jae-Sung,Yi, Young-Byung,Park, Seung-Hwan,Lee, Seon-Woo,Moon, Byung-Ju The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.4
A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.
Lee, Kwang Youll,Kong, Hyun-Gi,Lee, Seon-Woo The Korean Society of Plant Pathology 2012 Plant Pathology Journal Vol.28 No.4
Burkholderia pyrrocinia CH-67 is a biocontrol bacterium with strong antifungal activity against several plant pathogenic fungi. Transposon mutagenesis was performed to identify the genes responsible for the antifungal activity of B. pyrrocinia CH-67. Of the 2,500 mutants tested using the Fulvia fulva spore screening method, a mutant deficient in antifungal activity, M208, was selected. DNA sequence analysis of the transposon-inserted region revealed that a gene encoding an adenylate kinase-related kinase was disrupted in M208. Antifungal activity was restored in M208 when a full-length adenylate kinase gene with its promoter was introduced in trans. The deduced amino acid sequence of adenylate kinase from CH-67 was 80% identical to that of B. cenocepacia MCO-3. Adenosine diphosphate supplementation or high levels of adenosine triphosphate and adenosine monophosphate together restored antifungal activity in M208, suggesting that adenylate kinase of B. pyrrocinia CH-67 is involved in antifungal activity expression.
Kwang Youll Lee,공현기,이선우 한국식물병리학회 2012 Plant Pathology Journal Vol.28 No.4
Burkholderia pyrrocinia CH-67 is a biocontrol bacterium with strong antifungal activity against several plant pathogenic fungi. Transposon mutagenesis was performed to identify the genes responsible for the antifungal activity of B. pyrrocinia CH-67. Of the 2,500 mutants tested using the Fulvia fulva spore screening method, a mutant deficient in antifungal activity, M208, was selected. DNA sequence analysis of the transposon-inserted region revealed that a gene encoding an adenylate kinase-related kinase was disrupted in M208. Antifungal activity was restored in M208 when a full-length adenylate kinase gene with its promoter was introduced in trans. The deduced amino acid sequence of adenylate kinase from CH-67 was 80% identical to that of B. cenocepacia MCO-3. Adenosine diphosphate supplementation or high levels of adenosine triphosphate and adenosine monophosphate together restored antifungal activity in M208, suggesting that adenylate kinase of B. pyrrocinia CH-67 is involved in antifungal activity expression.
Kwang Ryool Heo,Kwang Youll Lee,Sang Hyun Lee,Soon Je Jung,이선우,Byung Ju Moon 한국식물병리학회 2008 Plant Pathology Journal Vol.24 No.1
Seedling damping-off and bottom rot caused by Rhizoctonia solani are yield limiting diseases of crisphead lettuce. To provide biocontrol measure in the management of the diseases, biocontrol strain Pseudomonas aeruginosa LY-11 was isolated from lettuce rhizosphere and introduced into crisphead lettuce rhizosphere by the seed coating delivery method. Alginate was used as a coating material to generate beads containing 106-106.5 colony-forming units (CFUs) of viable bacterial cells of LY-11. When seeds germinated from the alginate beads containing the strain LY-11, the bacteria established mostly in plant rhizosphere to maintain at least 104 CFU per gram of plant tissues. Crisphead lettuce seedlings germinated from the entrapped seeds were less affected from damping-off and bottom rot with disease control values of 70.4% and 85.4% respectively. Although P. aeruginosa LY-11 colonized plant rhizosphere and not phyllosphere, the result indicated that bottom rot caused by the foliar inoculation of R. solani was effectively reduced by the rhizobacteria. All data suggested that immobilized rhizobacterial application in seeds by alginate coating could control damping-off and induce induced systemic resistance of crisphead lettuce to reduce bottom rot.
Lee, Kwang-Youll,Kong, Hyun-Gi,Choi, Ki-Hyuck,Lee, Seon-Woo,Moon, Byung-Ju The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.1
A bacterial strain CH-67 which exhibits antagonism towards several plant pathogenic fungi such as Botrytis cinerea, Fulvia fulva, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum sp. and Phytophthora sp. was isolated from forest soil by a chitin-baiting method. This strain was identified as Burkholderia cepacia complex (Bcc) and belonging to genomovar IX (Burkholderia pyrrocinia) by colony morphology, biochemical traits and molecular method like 16S rRNA and recA gene analysis. This strain was used to develop a bio-fungicide for the control of tomato leaf mold caused by Fulvia fulva. Various formulations of B. pyrrocinia CH-67 were prepared using fermentation cultures of the bacterium in rice oil medium. The result of pot experiments led to selection of the wettable powder formulation CH67-C containing modified starch as the best formulation for the control of tomato leaf mold. CH67-C, at 100-fold dilution, showed a control value of 85% against tomato leaf mold. Its disease control efficacy was not significantly different from that of the chemical fungicide triflumidazole. B. pyrrocinia CH-67 was also effective in controlling damping-off caused by Rhizoctonia solani PY-1 in crisphead lettuce and tomato plants. CH67-C formulation was recognized as a cell-free formulation since B. pyrrocinia CH-67 was all lethal during formulation process. This study provides an effective biocontrol formulation of biofungicide using B. pyrrocinia CH-67 to control tomato leaf mold and damping-off crisphead lettuce and tomato.
Heo, Kwang-Ryool,Lee, Kwang-Youll,Lee, Sang-Hyun,Jung, Soon-Je,Lee, Seon-Woo,Moon, Byung-Ju The Korean Society of Plant Pathology 2008 Plant Pathology Journal Vol.24 No.1
Seedling damping-off and bottom rot caused by Rhizoctonia solani are yield limiting diseases of crisphead lettuce. To provide biocontrol measure in the management of the diseases, biocontrol strain Pseudomonas aeruginosa LY-11 was isolated from lettuce rhizosphere and introduced into crisphead lettuce rhizosphere by the seed coating delivery method. Alginate was used as a coating material to generate beads containing $10^6-10^{6.5}$ colony-forming units (CFUs) of viable bacterial cells of LY-11. When seeds germinated from the alginate beads containing the strain LY-11, the bacteria established mostly in plant rhizosphere to maintain at least $10^4$ CFU per gram of plant tissues. Crisphead lettuce seedlings germinated from the entrapped seeds were less affected from damping-off and bottom rot with disease control values of 70.4% and 85.4% respectively. Although P. aeruginosa LY-11 colonized plant rhizosphere and not phyllosphere, the result indicated that bottom rot caused by the foliar inoculation of R. solani was effectively reduced by the rhizobacteria. All data suggested that immobilized rhizobacterial application in seeds by alginate coating could control damping-off and induce induced systemic resistance of crisphead lettuce to reduce bottom rot.
Kong, Hyun-Gi,Choi, Ki-Hyuck,Heo, Kwang-Ryool,Lee, Kwang-Youll,Lee, Hyoung-Ju,Moon, Byung-Ju,Lee, Seon-Woo The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.2
Marking biocontrol bacteria is an essential step to monitor bacterial behavior in natural environments before application in agricultural ecosystem. In this study, we presented the simple green fluorescent protein (GFP) reporter system driven by the promoter active in Bacillus species for tagging of the biocontrol bacteria. A constitutive promoter P43 from Bacillus subtilis was fused to an enhanced promoterless gfp gene by overlap extension PCR. The GFP expression was demonstrated by the high fluorescence intensity detected in B. subtilis and Escherichia coli transformed with the P43-gfp fusion construct, respectively. The GFP reporter system was further investigated in two bacterial biocontrol strains B. licheniformis and Pseudomonas fluorescens. When the reconstructed plasmid pWH34G was introduced into B. licheniformis, GFP level measured with the fluorescence intensity in B. licheniformis was almost equivalent to that in B. subtilis. However, GFP expression level was extremely low in other biocontrol bacteria P. fluorescens by transposon based stable insertion of the P43-gfp construct into the bacterial chromosome. This study provides information regarding to the efficient biomarker P43-gfp fusion construct for bio-control Bacillus species.