Production of Surfactin and Iturin by Bacillus licheniformis N1 Responsible for Plant Disease Control Activity

공현기,김진철,최경자,이광렬,황은철,문병주,이선우,김현주 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.2

Bacillus licheniformis N1, previously developed as a biofungicide formulation N1E to control gray mold disease of plants, was investigated to study the bacterial traits that may be involved in its biological control activity. Two N1E based formulations, bacterial cell based formulation PN1E and culture supernatant based formulation SN1E, were evaluated for disease control activity against gray mold disease of tomato and strawberry plants. Neither PN1E nor SN1E was as effective as the original formulation N1E. Fractionation of antifungal compounds from the bacterial culture supernatant of B. licheniformis N1 indicated that two different cyclic lipopeptides were responsible for the antimicrobial activity of the N1 strain. These two purified compounds were identified as iturin A and surfactin by HPLC and LCMS. The purified lipopeptides were evaluated for plant disease control activity against seven plant diseases. Crude extracts and purified compounds applied at 500μg/ml concentration controlled tomato gray mold, tomato late blight and pepper anthracnose effectively with over 70% disease control value. While iturin showed broad spectrum activity against all tested plant diseases, the control activity by surfactin was limited to tomato gray mold, tomato late blight, and pepper anthracnose. Although antifungal compounds from B. licheniformis N1 exhibited disease control activity, our results suggested that bacterial cells present in the N1E formulation also contribute to the disease control activity together with the antifungal compounds.

길항세균 Bacillus amyloliquefaciens A-2를 이용한 토마토 잎곰팡이병 방제용 미생물 제제

공현기,전옥주,최기혁,이광렬,백정우,이선우,문병주,센틸쿠마무루가이얀,김현주 한국식물병리학회 2010 식물병연구 Vol.16 No.1

토마토 시설재배에서 증가하는 토마토 잎곰팡이병을 방제할 미생물 제제를 병원균 Fulvia fulva에 길항력이 강한 미생물 Bacillus amyloliquefaciens A-2 균주를 이용하여 개발하기 위해 본 연구를 수행하였다. 미생물 A-2 균주를 현미유가 첨가된 배지에서 대량 발효배양하고 각종 전달매체와 첨가제를 혼합하고 건조하여 제조된 미생물 제제들의 효과를 검정하였다. 제조된 제제 A-2H가 방제효과가 가장 우수하였으나 처리에서 단점으로 인해 이를보완하며 효과가 동일한 제형인 A-2MP를 선발하였다. 선발된 A-2MP는 100배, 500배 희석처리 후 생육상에서 토마토에 처리한 경우 화학농약과 대등한 방제효과를 나타내었다. 더욱이 1,000배 희석처리에서도 화학농약보다 효과가 감소하였으나 72% 정도의 방제효과를 보였다. 토마토를 토경재배한 온실에서 A-2MP의 방제효과를 검정한 결과 100배 희석 처리구의 경우 79.4%의 방제효과를 나타내 화학농약 트리후미졸 처리구의 79.6%의 방제효과와 차이가 없었다. A-2MP 제제를 62.2%의 자연발병한 농가의 온실에서 방제효과 검정한 결과 100배 희석액을 일주일 간격으로 3회 처리한 경우 60%의 방제가로 화학농약 방제가 81.6%보다 낮았으나 화학농약 효과의 73% 수준으로 나타났다. 한편, A-2MP제제를 4℃나 25℃ 보관한 경우 모두 10개월동안 생균수 및 방제효과가 안정적으로 유지되었다. 본 연구결과는 저렴한 원료와 길항균 B.amyloliquefaciens A-2 균주를 활용한 A2-MP제제가 토마토 잎곰팡이병 방제용으로 쓰일 수 있음을 보여준다. This study was performed to develop a formulation using an antagonistic bacterium Bacillus amyloliquefaciens A-2 to control tomato leaf mold caused by Fulvia fulva. B. amyloliquefaciens A-2 was grown in a medium with rice oil and mixed with various carrier and additives. One of the formulations, A2-MP, showed the best disease control value among the tested formulations. The disease control value of A2-MP at 100-fold and 500-fold diluted treatment was not significantly different from that of chemical fungicide triflumizole in a growth chamber. Although disease control effect was decreased by serial diluted treatment of the prepared A2-MP,1,000-fold diluted treatment of A2-MP still showed high disease control value of 72.0%. For the green house experiments, the disease control values of A2-MP was indicated as 79.4% which is similar to that of chemical fungicide, triflumizole showing 79.6%. When the disease control activity of the formulation A2-MP was compared in tomato production conditions, disease control values of 100-fold diluted A2-MP and 3,000 fold diluted triflumizole exhibited 60%, 81.6%, respectively. The disease control efficiency by A-2MP was 73% of the disease control value of chemical fungicide. The formulation A-2MP maintained the stable bacterial viability and disease control activity when stored at 4℃. This result suggested that A-2MP develped from B. amyloliquefaciens A-2 could be used to control tomato leaf mold.

Impact of a Recombinant Biocontrol Bacterium, Pseudomonas fluorescens pc78, on Microbial Community in Tomato Rhizosphere

공현기,김남희,이승엽,이선우 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.2

Pseudomonas fluorescens pc78 is an effective biocontrolagent for soil-borne fungal diseases. We previously constructeda P43-gfp tagged biocontrol bacteria P. fluorescenspc78-48 to investigate bacterial traits in naturalecosystem and the environmental risk of geneticallymodified biocontrol bacteria in tomato rhizosphere. Fluctuation of culturable bacteria profile, microbialcommunity structure, and potential horizontal genetransfer was investigated over time after the bacteriatreatment to the tomato rhizosphere. Tagged genetransfer to other organisms such as tomato plants andbacteria cultured on various media was examined bypolymerase chain reaction, using gene specific primers. Transfer of chromosomally integrated P43-gfp frompc78 to other organisms was not apparent. Populationand colony types of culturable bacteria were not significantlyaffected by the introduction of P. fluorescenspc78 or pc78-48 into tomato rhizosphere. Additionally,terminal restriction fragment length polymorphismprofiles were investigated to estimate the influence onthe microbial community structure in tomato rhizospherebetween non-treated and pc78-48-treated samples. Interestingly, rhizosphere soil treated with strainpc78-48 exhibited a significantly different bacterialcommunity structure compared to that of non-treatedrhizosphere soil. Our results suggest that biocontrolbacteria treatment influences microbial community intomato rhizosphere, while the chromosomally modifiedbiocontrol bacteria may not pose any specific environmentalrisk in terms of gene transfer.

Generation of a Constitutive Green Fluorescent Protein Expression Construct to Mark Biocontrol Bacteria Using P43 Promoter from Bacillus subtilis

공현기,최기혁,허광렬,이광열,이형주,문병주,이선우 한국식물병리학회 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 biocontrol Bacillus species.

Spatial and Temporal Distribution of a Biocontrol Bacterium Bacillus licheniformis N1 on the Strawberry Plants

공현기,이형주,배주영,김남희,문병주,이선우 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.3

Spatial and temporal distribution of Bacillus licheniformis N1 was investigated over time on the leaves,petioles and crowns of the strawberry plants. Bacterial population on the strawberry plants was quantified over time by selective plating. Bacterial population of N1 containing a plasmid pWH43G carrying green fluorescent protein (GFP) declined relatively faster on the plant surface as compared to the Strain N1 itself. However, this result was found to be enough to utilize the strain to visualize bacterial colonization on the plant surface. When B. licheniformis N1 was treated together with Silwet L-77 at 0.03%, the bacterial population on plant surface persisted for up to 7 days. B. licheniformis N1 (pWH43G) containing Silwet L-77 was applied on the strawberry plants and the GFP expressing bacteria were visualized by confocal laser scanning microscopy. Bacterial persistence was also investigated in a growth chamber and in a plastic house after N1 bioformulation treatment on the strawberry plant. The Strain N1colonized three different tissues well and persisted over 3 to 5 days on the strawberry plants. They formed bacterial aggregates on plant surfaces for at least 3 days,resulting in a biofilm to resist fluctuating plant surface environment. However, the bacterial persistence dramatically declined after 7 days in all tested tissues in a plastic house. This study suggest that B. licheniformis N1 colonizes the strawberry plant surface and persists for a long time in a controlled growth chamber, while it can not persist over 7 days on the plant surface in a plastic house.

Identification of virulence-associated genes of Erwinia amylovora by transposon mutagenesis

이승엽,공현기,강인정,오현석,우희종,노은정 충남대학교 농업과학연구소 2023 Korean Journal of Agricultural Science Vol.50 No.2

Erwinia amylovora , which causes fire blight disease on apple and pear trees, is one of the most important phytopathogens because of its devastating impact. Currently, the only way to effectively control fire blight disease is through the use of antibiotics such as streptomycin, kasugamycin, or oxytetracycline. However, problems with the occurrence of resistant strains due to the overuse of antibiotics are constantly being raised. It is therefore necessary to develop novel disease control methods through an advanced understanding of the pathogenesis mechanism of E. amylovora . To better understand the pathogenesis of E. amylovora , we investigated unknown virulence factors by random mutagenesis and screening. Random mutants were generated by Tn5 transposon insertion, and the pathogenicity of the mutants was assessed by inoculation of the mutants on apple fruitlets. A total of 17 avirulent mutants were found through screening of 960 random mutants. Among them, 14 mutants were already reported as non-pathogenic strains, while three mutants, TS3128_M2899 (ΔSUFU ), TS3128_M2939 (ΔwcaG ), and TS3128_M3747 (ΔrecB ), were not reported. Further study of the association between E. amylovora pathogenicity and these 3 novel genes may provide new insight into the development of control methods for fire blight disease.

The Relationship between the Sugar Preference of Bacterial Pathogens and Virulence on Plants

Ismaila Yakubu,공현기 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.6

Plant pathogenic bacteria colonize plant surfaces and inner tissues to acquire essential nutrients. Nonstructural sugars hold paramount significance among these nutrients, as they serve as pivotal carbon sources for bacterial sustenance. They obtain sugar from their host by diverting nonstructural carbohydrates en route to the sink or enzymatic breakdown of structural carbohydrates within plant tissues. Despite the prevalence of research in this domain, the area of sugar selectivity and preferences exhibited by plant pathogenic bacteria remains inadequately explored. Within this expository framework, our present review endeavors to elucidate the intricate variations characterizing the distribution of simple sugars within diverse plant tissues, thus influencing the virulence dynamics of plant pathogenic bacteria. Subsequently, we illustrate the apparent significance of comprehending the bacterial preference for specific sugars and sugar alcohols, postulating this insight as a promising avenue to deepen our comprehension of bacterial pathogenicity. This enriched understanding, in turn, stands to catalyze the development of more efficacious strategies for the mitigation of plant diseases instigated by bacterial pathogens.

Characterization of a Chitinase Gene Exhibiting Antifungal Activity from a Biocontrol Bacterium Bacillus licheniformis N1

이광열,허광률,공현기,남재성,이영병,박성환,이선우,문병주 한국식물병리학회 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 Stransferase 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- β-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl- β-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.

사과 내생 세균 자원에서 화상병원균에 대한 길항미생물 선발 및 분류

김수현,이수인,공현기,조경준,곽연식 한국농약과학회 2022 농약과학회지 Vol.26 No.1

Economic losses by Erwinia amylovora, a causal agent of fire blight disease, effected in various Rosaceae plants. In Korea, the fire blight disease was the first reported in 2015 and rapidly spread to apples and pears orchard nationwide. According to the spread of the fire blight disease, in the early stage of the outbreak, the whole plants within the radius of the diseased orchard were removed or chemical control was performed, but complete eradication of the disease is not possible. Therefore, in this study we conducted isolation and construction of apple endophytic bacterial library and screened the anti-E. amylovora strains. As result, we selected total 11 bacteria, which were classified as Streptomyces sp. and Paenibacillus sp. based on molecular characteristics. The selected bacteria have great potential to be developed as biological control agents in apple cultivation. Erwinia amylovora에 의해 발생하는 화상병은 다양한 장미과 식물에서 경제적 손실을 야기하고 있다 . 국내 에서는 2015년 처음으로 화상병이 발생해 전국 사과와 배 과수원으로 급속도로 확산되고 있다 . 화상병 확산에 따라 발병 초기에는 병든 과수원 반경 내 숙주식물을 모두 제거하고 , 화학물질 방제를 실시했지만 완전한 퇴치는 불가능 하다 . 따라서 본 연구에서는 생물적방제 후보 미생물을 선발하기 위하여 , 사과 내성 세균 미생물 자원을 확보하고 , E. amylovora에 대한 항세균력 균주 선발을 수행하였다 . 그 결과 총 11개 화상병원균의 생육을 억제하는 사과 내생 미 생물을 선발하였고 , 분자생물학적 특성에 따라 Streptomyces sp. 또는 Paenibacillus sp.로 분류되었다 . 선발된 내생미 생물은 사과 잎에 접종하여 화상병에 대한 억제 능력을 검정하였으며 이는 사과 재배에서 화상병의 생물학적 방제제 로 개발될 가능성이 높은 것으로 사료된다 .

Altered Gene Expression and Intracellular Changes of the Viable But Nonculturable State in Ralstonia solanacearum by Copper Treatment

엄해영,이선우,공현기,이형주,최혜경,박은진,김선태,정은숙,강규영,Senthilkumar Murugiyan 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.4

Environmental stresses induce several plant pathogenic bacteria into a viable but nonculturable (VBNC) state,but the basis for VBNC is largely uncharacterized. We investigated the physiology and morphology of the copperinduced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of 200 μM copper sulfate to the liquid microcosm completely suppressed bacterial colony formation on culture media;however, LIVE/DEAD BacLight bacterial viability staining showed that the bacterial cells maintained viability,and that the viable cells contain higher level of DNA. Based on electron microscopic observations, the bacterial cells in the VBNC state were unchanged in size, but heavily aggregated and surrounded by an unknown extracellular material. Cellular ribosome contents, however,were less, resulting in a reduction of the total RNA in VBNC cells. Proteome comparison and reverse transcription PCR analysis showed that the Dps protein production was up-regulated at the transcriptional level and that 2 catalases/peroxidases were present at lower level in VBNC cells. Cell aggregation and elevated levels of Dps protein are typical oxidative stress responses. H2O2 levels also increased in VBNC cells, which could result if catalase/peroxidase levels are reduced. Some of phenotypic changes in VBNC cells of R. solanacearum could be an oxidative stress response due to H2O2 accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.