• The Plant Pathology Journal
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• 제37권1호
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• pp.24-35
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• 2021

Blue mold caused by Penicillium expansum is one of the most significant postharvest diseases of apples. Some microorganisms associated with the surface of ripening apples possess the ability to inhibit the growth of P. expansum. However, the existing literature about their colonization in the stages before ripening is not explored in depth. This study aims to characterize the antagonistic capacity of bacterial populations from five fruit development stages of 'Royal Gala' apples. The results have shown that the density of the bacterial populations decreases throughout the ripening stages of fruit (from 1.0 × 105 to 1.1 × 101 cfu/㎠). A total of 25 bacterial morphotypes (corresponding to five genera identified by 16S RNA) were differentiated in which Bacillus stood out as a predominant genus. In the in vitro antagonism tests, 10 Bacillus strains (40%) inhibited the mycelial growth of P. expansum from 30.1% to 60.1%, while in fruit bioassays, the same strains reduced the fruit rot ranging from 12% to 66%. Moreover, the bacterial strains with antagonistic activity increased in the ripening fruit stage. B. subtilis subsp. spiziennii M24 obtained the highest antagonistic activity (66.9% of rot reduction). The matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed that bacteria with antagonistic activity produce anti-fungal lipopeptides from iturin and fengycin families.

• 한국미생물·생명공학회지
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• 제44권1호
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• pp.9-18
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• 2016

청국장에서 강력한 항균력을 지닌 2개의 Bacillus 균주들인 CJW15와 SSD8이 분리되었다. 16S rRNA와 recA 유전자들 염기서열 결정에 의해 두가지 균주 모두 B. amyloliquefaciens로 동정되었다. CJW15는 B. cereus ATCC14579, Listeria monocytogenes ATCC19111, Lactococcus lactis ATCC11454 들의 증식을 강력히 억제하며 SSD8은 B. cereus ATCC14579와 Enterococcus faecium ATCC19953 증식을 억제하였다. 두 균주들의 항균력은 $100^{\circ}C$, 15분 처리에도 감소하지 않았고 산성인 pH 3과 알칼리인 pH 12에서도 안정하였다. 트립신, 펩신, 프로테아제 K, 프로테아제 효소처리에 의해 CJW15 항균력은 변화가 없었지만 SSD8 항균력은 절반으로 감소하였다. 두 균주 공히 surfactin, fengycin, iturin, iturinA와 같은 lipopeptide 생합성 유전자들을 지니고 있고 subtilin과 같은 박테리오신 유전자들도 지니고 있다. 또 두 균주들은 혈전용해능을 지니고 있다.

• 농약과학회지
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• 제20권3호
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• pp.189-196
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• 2016

바실러스는 토양 및 반추동물의 소화기관과 같은 다양한 곳에서 분리되고 있으며, 작물의 생육 촉진과 병방제를 위한 미생물 재제로서 널리 사용되고 있다. 바실러스 GH1-13 균주는 전남 완도의 간척지 논에서 분리되었으며, 16S rRNA 유전자와 gyrB 유전자를 이용하여 계통유전학적으로 분석한 결과 Bacillus velezensis인 것으로 동정되었다. GH1-13 균주의 특성을 분석한 결과 생육촉진에 관련된 IAA를 생성할 뿐 아니라 벼 뿌리의 생육을 촉진하는 것을 확인하였다. 또한 벼의 주요 병원균의 생육을 억제할 뿐 아니라 작물의 병원균인 다양한 곰팡이의 생육을 저해하였다. 더불어 식물병원 진균 및 세균의 생육 억제와 밀접하게 관련된 것으로 판단되는 bacillomycin, bacilycin, fengycin, iturin, surfactin을 생성하는 생합성유전자를 보유한 것으로 확인되었다. 본 연구는 GH1-13균주가 작물의 생육촉진과 병 방제를 동시에 해결할 수 있는 강력한 복합기능성 미생물제로의 가능성이 있음을 보여주었다.

• 식물병연구
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• 제23권2호
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• pp.177-185
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• 2017

장수풍뎅이 유충의 장내세포에서 분리한 Bacillus amyloliquefaciens LM11은 surfactin, iturin, fengycin 같은 biosurfactants lipopeptide를 생산하여 식물병원성 곰팡이의 성장을 강하게 억제하였다. LM11균주 성장단계에 따라 biosurfactant 생산과 surface tension은 상당히 유의한 차이가 있었다. 항균 물질인 surfactin, iturin, fengycin의 생합성 유전자는 정지기에 도달하면서 집중적으로 발현되었고 그 생산량도 높았다. 또한 LM11균주를 제거한 배양 상등액 함량의 농도에 따라 고추 탄저병원균의 포자발아와 높은 부의 상관관계가 있었다(R=0.761, P<0.001). 식물병원성 곰팡이의 균사 생장억제를 위한 최소 surface tension 수준은 38.5 mN/m였다(R=0.951-0.977, P<0.001). 본 연구 결과는 B. amyloliquefaciens LM11의 biosurfactant가 식물병에 대한 생물학적 방제에 중요한 항진균 대사물질로 작용하며, 배양액의 surface tension 측정은 생물학적 방제제의 최적 사용을 위한 기초 지표로 사용될 수 있음을 보여 주었다.

• 생명과학회지
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• 제15권6호
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• pp.1005-1012
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• 2005

식물 내생균 Bacillus sp. CY22는 식물병원균 Rhizoctonia solani, Fusarium oxysporum 및 Phythium ultimum에 대해 강한 항균력을 나타내었다. 일반적으로 많은 Bacillus속 균주들은 iturin, fengycin, mycosubtulin과 같은 항균 물질을 분비한다. 본 연구에서는 식물내생균 Bacillus sp. CY22의 배양액으로부터 항균물질을 분리, 정제하였으며 MALDI-TOF mass로 분자량을 확인하였다. MALDI-TOF mass spectrum분석 결과 분리된 항균물질은 Bacillus 속 균주가 생성하는 항균물질로서 잘 알려져 있는 iturin의 분자량과 거의 일치하였으며, m/z 1043.4, 1057.4, 1071.4에서 molecular ion peak를 나타내었다. 이들은 각각 m/z 14차이를 가진 iturin의 isoform으로 추정되며 이 것은 iturin을 구성하고 있는 지방산의 탄소수 차이로 생각되며 m/z 1065.4, 1079.4 peak는 sodium adduct로서 추정된다. 또한 항균물질 iturin을 생성하는데 관여하는 transacylase 유전자를 크로닝하여 ita22 유전자로 명명하고, 그 특성으로 ita22 유전자는 400 개의 아미노산을 인지하는 1,200 bp의 open reading frame (ORF)을 가지며, 아미노산의 상동성을 조사한 결과 Bacillus subtilis 168의 FenF (BAB69697)와 가장 유사하였다.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.390-396
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• 2013

Biosurfactants have versatile properties and potential industrial applications. A new producer, B. subtilis TU2, was isolated from the underground oil-extraction wastewater of Shengli Oilfield, China. Preliminary flask culture showed that the titer of biosurfactant obtained from the broth of TU2 was ~1.5 g/l at 48 h (718 mg/l after purification), with a reduced surface tension of 32.5 mN/m. The critical micelle concentration was measured as 50 mg/l and the surface tension maintained stability in solution with 50 g/l NaCl and 16 g/l $CaCl_2$ after 5 days of incubation at $70^{\circ}C$. FT-IR spectra exhibited the structure information of both glycolipid and lipopeptide. MALDI-TOF-MS analyses confirmed that the biosurfactant produced by B. subtilis TU2 was a blend of glycolipid and lipopeptide, including rhamnolipid, surfactin, and fengycin. The blended biosurfactant showed 86% of oil-washing efficiency and fine emulsification activity on crude oil, suggesting its potential application in enhanced oil recovery.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.417-426
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• 2020

Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.

• 한국동물위생학회지
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• 제40권4호
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• pp.265-275
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• 2017

The production of enzymes that help digestion, assimilation of essential nutrients, and prevent pathogenic bacteria are important for probiotics used in aquaculture. The objective of this study was to investigate enzyme activities for macromolecular organic matters and antimicrobial properties of the selected potential probiotics isolated from gut of surf clam (Tresus keenae) against well-known shellfish-pathogenic bacteria. Among 65 isolates from guts of 60 surf clams, seven Bacillus strains with outstanding degradation capability of macromolecule organic matter were selected as potential probiotics as follows: TKI01 (B. vietnamensis), TKI02, TKI26 (B. thuringiensis), TKI14, TKI32, TKI42 (B. amyloliquefaciens), and TKI18 (B. stratosphericus). After in vitro antimicrobial activity test was performed against five shellfish-pathogenic bacteria including Listonella anguillarum, Vibrio parahaemolyticus, V. splendidus, V. harveyi, V. tubiashii, PCR assay was performed to detect bacteriocin-producing strain. PCR results revealed that the five Bacillus strains possessed diverse bacteriocin genes including ericinA, coagulin, surfactin, iturin, bacyllomicin, fengycin, bacylisin, subtilin, and lantibiotics. In the present study, the selected seven Bacillus strains showed different enzyme activities according to types of macromolecule organic matters. And their antimicrobial activities varied based on the species of pathogenic bacteria. In addition, at least five Bacillus strains had genetic potential to produce several natural lipopeptide antibiotics that may help biological control of surf clam aquaculture. Therefore, mixed use of probiotics might show co-operative effect and increase the efficiency of probiotics rather than separate use. To the best of our knowledge, it is the first report on antimicrobial properties of Bacillus species isolated from surf clam.

• The Plant Pathology Journal
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• 제34권2호
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• pp.126-138
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• 2018

Rice blast caused by Magnaporthe oryzae is a major disease. In the present study, we aimed to identify and evaluate the novel bacterial isolates from rice rhizosphere for biocontrol of M. oryzae pathogen. Sixty bacterial strains from the rice plant's rhizosphere were tested for their biocontrol activity against M. oryzae under in vitro and in vivo. Among them, B. amyloliquefaciens had significant high activity against the pathogen. The least disease severity and highest germination were recorded in seeds treated with B. amyloliquefaciens UASBR9 (0.96 and 98.00%) compared to untreated control (3.43 and 95.00%, respectively) under in vivo condition. These isolates had high activity of enzymes in relation to growth promoting activity upon challenge inoculation of the pathogen. The potential strains were identified based on 16S rRNA gene sequencing and dominance of these particular genes were associated in Bacillus strains. These strains were also confirmed for the presence of antimicrobial peptide biosynthetic genes viz., srfAA (surfactin), fenD (fengycin), spaS (subtilin), and ituC (iturin) related to secondary metabolite production (e.g., AMPs). Overall, the results suggested that application of potential bacterial strains like B. amyloliquefaciens UASBR9 not only helps in control of the biological suppression of one of the most devastating rice pathogens, M. grisea but also increases plant growth along with a reduction in application of toxic chemical pesticides.

• The Plant Pathology Journal
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• 제38권2호
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• pp.115-130
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• 2022

Though information exists regarding the pathogenesis of the shot-hole disease (SH) in flowering cherry (FC), there has been a lack of research focusing on SH management. Therefore, here, we investigated the inhibitory activities of antagonistic bacteria against SH pathogens both in vitro and in vivo as well as their biochemical characteristics and bioactive compounds. Two biosurfactant-producing bacterial antagonists, identified as Bacillus velezensis strains JCK-1618 and JCK-1696, exhibited the best effects against the growth of both bacterial and fungal SH pathogens in vitro through their cell-free culture filtrates (CFCFs). These two strains also strongly inhibited the growth of the pathogens via the action of their antimicrobial diffusible compounds and antimicrobial volatile organic compounds (VOCs). Crude enzymes, solvent extracts, and biosurfactants of the two strains exhibited antimicrobial activities. Liquid chromatography/electrospray ionization time-of-flight mass spectrometric analysis of the partially purified active fractions revealed that the two antagonists produced three cyclic lipopeptides, including iturin A, fengycin A, and surfactin, and a polyketide, oxydifficidin. In a detached leaf assay, pre-treatment and co-treatment of FC leaves with the CFCFs led to a large reduction in the severity of the leaf spots caused by Epicoccum tobaicum and Bukholderia contaminans, respectively. In addition, the two antagonists produced indole-3-acetic acid, siderophore, and a series of hydrolytic enzymes, along with the formation of a substantial biofilm. To our knowledge, this is the first report of the antimicrobial activities of the diffusible compounds and VOCs of B. velezensis against the SH pathogens and their efficiency in the biocontrol of SH.