• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.296-305
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• 2024

Peach tree gummosis is a botanical anomaly distinguished by the secretion of dark-brown gum from the shoots of peach trees, and Botryosphaeria dothidea has been identified as one of the fungal species responsible for its occurrence. In South Korea, approximately 80% of gummosis cases are linked to infections caused by B. dothidea. In this study, we isolated microbes from the soil surrounding peach trees exhibiting antifungal activity against B. dothidea. Subsequently, we identified several bacterial strains as potential candidates for a biocontrol agent. Among them, Bacillus velezensis KTA01 displayed the most robust antifungal activity and was therefore selected for further analysis. To investigate the antifungal mechanism of B. velezensis KTA01, we performed tests to assess cell wall degradation and siderophore production. Additionally, we conducted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis based on whole-genome sequencing to confirm the presence of genes responsible for the biosynthesis of lipopeptide compounds, a well-known characteristic of Bacillus spp., and to compare gene expression levels. Moreover, we extracted lipopeptide compounds using methanol and subjected them to both antifungal activity testing and high-performance liquid chromatography (HPLC) analysis. The experimental findings presented in this study unequivocally demonstrate the promising potential of B. velezensis KTA01 as a biocontrol agent against B. dothidea KACC45481, the pathogen responsible for causing peach tree gummosis.

• The Plant Pathology Journal
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• 제36권5호
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• pp.491-496
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• 2020

An understanding of the contribution of secondary metabolites (SMs) to the antagonistic and biocontrol activities of bacterial biocontrol agents serves to improve biocontrol potential of the strain. In this study, to evaluate the contribution of each SM produced by Pseudomonas fluorescens NBC275 (Pf275) to its antifungal and biocontrol activity, we combined in silico analysis of the genome with our previous study of transposon (Tn) mutants. Thirteen Tn mutants, which belonged to 6 biosynthetic gene clusters (BGCs) of a total 14 BGCs predicted by the antiSMASH tool were identified by the reduction of antifungal activity. The biocontrol performance of Pf275 was significantly dependent on 2,4-diacetylphloroglucinol and pyoverdine. The clusters that encode for arylpolyene and an unidentified small linear lipopeptide influenced antifungal and biocontrol activities. To our knowledge, our study identified the contribution of SMs, such as a small linear lipopeptide and arylpolyene, to biocontrol efficacy for the first time.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.40-44
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• 2003

Soil-borne infectious disease including Pythium aphanidermatum and Rhizoctonia solani causes severe damage to plants, such as cucumber. This soil-borne infectious disease was not controlled effectively by chemical pesticide. Since these diseases spread through the soil, chemical agents are usually ineffective. Instead, biological control, including antagonistic microbe can be used as a preferred control method. An efficient method was developed to select an antagonistic strain to be used as a biological control agent strain. In this new method, surface tension reduction potential of an isolate was included in the ‘decision factor’ in addition to the other factors, such as growth rate, and pathogen inhibition rate. Considering these 3 decision factors by a statistical method, an isolate from soil was selected and was identified as Bacillus sp. GB16. In the pot test, this strain showed the best performance among the isolated strains. The lowest disease incidence rate and fastest seed growth was observed when Bacillus sp. GB16 was used. Therefore this strain was considered as plant growth promoting rhizobacteria (PGPR). The action of surface tension reducing component was deduced as the enhancement of wetting, spreading, and residing of antagonistic strain in the rhizosphere. This result showed that new selection method was significantly effective in selecting the best antagonistic strain for biological control of soil-borne infectious plant pathogen. The antifungal substances against P. aphanidermatum and R. solani were partially purified from the culture filtrates of Bacillus sp. GB16. In this study, lipopeptide possessing antifungal activity was isolated from Bacillus sp. GB16 cultures by various purification procedures and was identified as a surfactin-like lipopeptide based on the Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), high performance liquid chromatography mass spectroscopy (HPLC-MS), and quadrupole time-of-flight (Q-TOF) ESI-MS/MS data. The lipopeptide, named GB16-BS, completely inhibited the growth of Pythium aphanidermatum, Rhizoctonia solani, Penicillium sp., and Botrytis cineria at concentrations of 10 and 50 mg/L, respectively. A novel method to prevent the foaming and to provide oxygen was developed. During the production of surface active agent, such as lipopeptide (surfactin), large amount of foam was produced by aeration. This resulted in the carryover of cells to the outside of the fermentor, which leads to the significant loss of cells. Instead of using cell-toxic antifoaming agents, low amount of hydrogen peroxide was added. Catalase produced by cells converted hydrogen peroxide into oxygen and water. Also addition of corn oil as an oxygen vector as well as antifoaming agent was attempted. In addition, Ca-stearate, a metal soap, was added to enhance the antifoam activity of com oil. These methods could prevent the foaming significantly and maintained high dissolved oxygen in spite of lower aeration and agitation. Using these methods, high cell density, could be achieved with increased lipopeptide productivity. In conclusion to produce an effective biological control agent for soil-borne infectious disease, following strategies were attempted i) effective screening of antagonist by including surface tension as an important decision factor ii) identification of antifungal compound produced from the isolated strain iii) novel oxygenation by $H_2O_2-catalase$ with vegetable oil for antifungal lipopeptide production.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.528-531
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• 2000

식물의 토양전염병을 유발하는 Pythium sp. 및 Rhizoctonia sp.에 대한 길항력과 생장속도를 고려한 RPI (relative performance indices) 기법을 이용하여 100 여개 후보균 중 #19, #42 등 길항균을 선정하였다. 후보균들은 일반적으로 Pythium sp.보다 Rhizoctonia sp.에 대한 길항력이 우수하였고 Bacillus sp.의 배양 상등액은 항진균활성과 blood agar에서 용혈현상을 보여 상등액으로부터 항균물질인 lipopeptide 계열물질을 분리하였다.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.295-301
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• 2005

한국 전통 젓갈로부터 항균성 lipopeptide를 생산하는 JKK238 균주를 분리하여 16S rDNA및 세포벽의 지방산 조성 분석 등에 따라 Bacillus subtilis로 동정하였다. JKK238 균주를 3일간 배양한 TSB 액체배지의 $25\%$ ammonium sulfate 침전물이 대부분의 lipopeptide를 포함하고 이들이 다양한 식물병원성 곰팡이나 세균의 성장을 강력히 저해함을 알 수 있었다. 또한 FPLC에 의해 분리 및 정제된 각 lipopeptide의 Maldi-tof분석에서, 이 균주가 iturin A, fengycin, surfactin과 같은 세 종류의 lipopeptide의 isomer들을 동시에 생산하는 균주임을 밝혔다.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.784-795
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• 2018

Bacillus amyloliquefaciens Pc3 was isolated from Antarctic seawater with antifungal activity. In order to investigate the metabolic regulation mechanism in the biosynthesis of lipopeptides in B. amyloliquefaciens Pc3, GC/MS-based metabolomics was used when exogenous indole was added. The intracellular metabolite profiles showed decreased asparagine, aspartic acid, glutamine, glutamic acid, threonine, valine, isoleucine, hexadecanoic acid, and octadecanoic acid in the indole-treated groups, which were involved in the biosynthesis of lipopeptides. B. amyloliquefaciens Pc3 exhibited a growth promotion, bacterial total protein increase, and lipopeptide biosynthesis inhibition upon the addition of indole. Besides this, real-time PCR analysis further revealed that the transcription of lipopeptide biosynthesis genes ituD, fenA, and srfA-A were downregulated by indole with 22.4-, 21.98-, and 26.0-fold, respectively. It therefore was speculated that as the metabolic flux of most of the amino acids and fatty acids were transferred to the synthesis of proteins and biomass, lipopeptide biosynthesis was weakened owing to the lack of precursor amino acids and fatty acids.

• 생명과학회지
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• 제14권1호
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• pp.193-197
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• 2004

A continuous enrichment culture procedure was used to isolate bacteria from various soil sources capable of suppressing large patch disease of turfgrass. Six isolates consistently suppressed large patch in turfgrass, and ranged in the spectrum of extracellular enzymes that they expressed. The best disease- suppressing isolate, TBM912, expressed protease, CMCase, and pectinase activity and inhibited the growth of Rhizectonin solani and Betrytis cinerea in vitro. Here we show that this strain also produces an antibiotic that was identified by TLC, SDS-PACE and HPLC analysis as lipopeptide.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1954-1959
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• 2015

Bacillus subtilis HM1 was isolated from the rhizosphere region of halophytes for its antifungal activity against Colletotrichum acutatum, the causative agent of anthracnose. Treatment of postharvest apples with the cell culture or with a cell-free culture supernatant reduced disease severity 80.7% and 69.4%, respectively. Both treatments also exhibited antifungal activity against various phytopathogenic fungi in vitro. The antifungal substances were purified and analyzed by acid precipitation, gel filtration, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Three compounds were identified as fengycin, iturin, and surfactin. The MALDI-TOF/TOF mass spectrum revealed the presence of cyclized fengycin homologs A and B, which were distinguishable on the basis of the presence of either alanine or valine, respectively, at position 6 of the peptide sequence. In addition, the cyclized structure of fengycin was shown to play a critical role in antifungal activity.

• KSBB Journal
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• 제9권2호
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• pp.224-229
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• 1994

Bacillus subtilis에 의한 항진균 물질인 Iturin의 생산에 관한 연구를 수행하였다. 세포성장과 iturin 의 생산은 탄소원인 sucrose, glucose 그리고 fructose에 의해서는 영향을 받지 않았으며, soytone의 농도가 20g/$\ell$일 때가 10g/$\ell$인 경우에 비해 50% 정도 산물생산이 증가하였으나 그 이상의 농도에서는 영향이 없었다. 질소원으로 soytone만을 사용하였을 때 나타나는 diauxic growth는 아미노산과 펩타이드에 대한 세포의 기질 이용의 차이 때문이었다. Iturin의 구성성분의 분포는 배양액의 용존산소 농도에 의해서는 영향을 받지 않았으며, 세포의 비 성장속도에 의해 영향을 받았다.

• 한국미생물·생명공학회지
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• 제21권6호
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• pp.556-563
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• 1993

AF-011A is a novel lipopeptide with potent antifungal activity isolated from Pseudomonas cepacia AF6008 deposited as KFCC 10759. The compound was isolated from the fermentation broth by extraction with 50% isopropyl alcohol. Purification was effected by chromatography on Diaion HP-20, Alumina and C18 followed by HPTLC on silica gel. These techniques affored two closelt related compounds. AF-011A1 ans AF-011A2. The molecular weights of AF-011A1/A2 were determined by fast atom bombardment mass spectrometry(A1 m/z 1,215 : A2 m/z 1,199).