• Journal of Microbiology and Biotechnology
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• v.23 no.3
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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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• v.30 no.3
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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.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1755-1764
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• 2016

The application of Bacillus sp. in the biological control of plant soilborne diseases has been shown to be an environmentally friendly alternative to the use of chemical fungicides. In this study, the effects of bioorganic fertilizer (BOF) fortified with Bacillus amyloliquefaciens SQY 162 on the suppression of tomato bacterial wilt were investigated in pot experiments. The disease incidence of tomato wilt after the application of BOF was 65.18% and 41.62% lower at 10 and 20 days after transplantation, respectively, than in the control condition. BOF also promoted the plant growth. The SQY 162 populations efficiently colonized the tomato rhizosphere, which directly suppressed the number of Ralstonia solanacearum in the tomato rhizosphere soil. In the presence of BOF, the activities of defense-related enzymes in tomato were lower than in the presence of the control treatment, but the expression levels of the defense-related genes of the plants in the salicylic acid and jasmonic acid pathways were enhanced. It was also found that strain SQY 162 could secrete antibiotic surfactin, but not volatile organic compounds, to suppress Ralstonia. The strain could also produce plant growth promotion compounds such as siderophores and indole-3-acetic acid. Thus, owing to its innate multiple-functional traits and its broad biocontrol activities, we found that this antagonistic strain isolated from the tobacco rhizosphere could establish itself successfully in the tomato rhizosphere to control soilborne diseases.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.748-756
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• 2016

In the present study, a lipopeptide (named AXLP14) antagonistic to Xanthomonas oryzae pv. oryzae (Xoo) was obtained from the culture supernatant of Bacillus amyloliquefaciens B014. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis demonstrated that AXLP14 consisted of surfactin homologs. The minimum inhibition concentration and minimum bactericidal concentration of AXLP14 against Xoo were determined to be 1.25 and 2.50 mg/ml, respectively. At a concentration of 0.613 mg/ml, AXLP14 strongly inhibited the formation of Xoo biofilm. AXLP14 also inhibited the motility of Xoo in a concentration-dependent manner. Applying AXLP14 to rice seedlings significantly reduced the incidence and severity of disease caused by Xoo. In Xoo-infected rice seedlings, AXLP14 strongly and continuously up-regulated the expression of both OsNPR1 and OsWRKY45. In addition, AXLP14 effectively inhibited the Xoo-induced up-regulation of the expression of the abscisic acid biosynthesis gene OsNECD3 and the abscisic acid signalingresponsive gene OsLip9, indicating that AXLP14 may protect rice against Xoo-induced disease by enhancing salicylic acid defense and interfering with the abscisic acid response to virulence.

• Korean Journal of Veterinary Service
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• v.40 no.4
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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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• v.37 no.6
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• pp.580-595
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• 2021

Although late blight is an important disease in ecofriendly potato cultivation in Korea, it is highly dependent on the use of eco-friendly agricultural materials and the development of biological control technology is low. It is a necessary to develop an effective biocontrol agent to inactivate late blight in the field. AFB2-2 strain is a gram-positive with peritrichous flagella. It can utilize 20 types of carbon sources, like L-arabinose, and D-trehalose at 35℃. The optimal growth temperature of the strain is 37℃. It can survive at 20-50℃ in tryptic soy broth. The maximum salt concentration tolerated by AFB2-2 strain is 7.5% NaCl. AFB2-2 strain inhibited the mycelial growth of seven plant pathogens by an average inhibitory zone of 10.2 mm or more. Among the concentrations of AFB2-2, 10⁷ cfu/ml showed the highest control value of 85.7% in the greenhouse. Among the three concentrations of AFB2-2, the disease incidence and severity of potato late blight at 10⁷ cfu/ml was lowest at 0.07 and 6.7, respectively. The nucleotide sequences of AFB2-2 strain were searched in the NCBI GenBank; Bacillus siamensis strain KCTC 13613, Bacillus velezensis strain CR-502, and Bacillus amyloliquefaciens strain DSM7 were found to have a genetic similarity of 99.7%, 99.7%, and 99.5%, respectively. The AFB2-2 strain was found to harbor the biosynthetic genes for bacillomycin D, iturin, and surfactin. Obtained data recommended that the B. velezensis AFB2-2 strain could be considered as a promising biocontrol agent for P. infestans in the field.

• The Plant Pathology Journal
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• v.38 no.2
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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.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.430-448
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• 2023

Recently, strategies for controlling Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of Fusarium wilt of tomato, focus on using effective biocontrol agents. In this study, an analysis of the biocontrol and plant growth promoting (PGP) attributes of 11 isolates of loamy soil Bacillus spp. has been conducted. Among them, the isolates B.PNR1 and B.PNR2 inhibited the mycelial growth of Fol by inducing abnormal fungal cell wall structures and cell wall collapse. Moreover, broad-spectrum activity against four other plant pathogenic fungi, F. oxysporum f. sp. cubense race 1 (Foc), Sclerotium rolfsii, Colletotrichum musae, and C. gloeosporioides were noted for these isolates. These two Bacillus isolates produced indole acetic acid, phosphate solubilization enzymes, and amylolytic and cellulolytic enzymes. In the pot experiment, the culture filtrate from B.PNR1 showed greater inhibition of the fungal pathogens and significantly promoted the growth of tomato plants more than those of the other treatments. Isolate B.PNR1, the best biocontrol and PGP, was identified as Bacillus stercoris by its 16S rRNA gene sequence and whole genome sequencing analysis (WGS). The WGS, through genome mining, confirmed that the B.PNR1 genome contained genes/gene cluster of a nonribosomal peptide synthetase/polyketide synthase, such as fengycin, surfactin, bacillaene, subtilosin A, bacilysin, and bacillibactin, which are involved in antagonistic and PGP activities. Therefore, our finding demonstrates the effectiveness of B. stercoris strain B.PNR1 as an antagonist and for plant growth promotion, highlighting the use of this microorganism as a biocontrol agent against the Fusarium wilt pathogen and PGP abilities in tomatoes.

• The Plant Pathology Journal
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• v.40 no.4
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• pp.346-357
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• 2024

This study was carried out to screen the antifungal activity against Colletotrichum acutatum, Colletotrichum dematium, and Colletotrichum coccodes. Bacterial isolate GP-P8 from pepper soil was found to be effective against the tested pathogens with an average inhibition rate of 70.7% in in vitro dual culture assays. 16S rRNA gene sequencing analysis result showed that the effective bacterial isolate as Bacillus siamensis. Biochemical characterization of GP-P8 was also performed. According to the results, protease and cellulose, siderophore production, phosphate solubilization, starch hydrolysis, and indole-3-acetic acid production were shown by the GP-P8. Using specific primers, genes involved in the production of antibiotics, such as iturin, fengycin, difficidin, bacilysin, bacillibactin, surfactin, macrolactin, and bacillaene were also detected in B. siamensis GP-P8. Identification and analysis of volatile organic compounds through solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) revealed that acetoin and 2,3-butanediol were produced by isolate GP-P8. In vivo tests showed that GP-P8 significantly reduced the anthracnose disease caused by C. acutatum, and enhanced the growth of pepper plant. Reverse transcription polymerase chain reaction analysis of pepper fruits revealed that GP-P8 treated pepper plants showed increased expression of immune genes such as CaPR1, CaPR4, CaNPR1, CaMAPK4, CaJA2, and CaERF53. These results strongly suggest that GP-P8 could be a promising biocontrol agent against pepper anthracnose disease and possibly a pepper plant growth-promoting agent.

• Research in Plant Disease
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• v.28 no.1
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• pp.19-25
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• 2022

Ginseng is an important medicinal plant cultivated in East Asia for thousands of years. It is typically cultivated in the same field for 4 to 6 years and is exposed to a variety of pathogens. Among them, ginseng root rot is the main reason that leads to the most severe losses. In this study, endophytic bacteria were isolated from healthy ginseng, and endophytes with antagonistic effect against ginseng root rot pathogens were screened out. Among the 17 strains, three carried antagonistic effect, and were resistant to radicicol that is a mycotoxin produced by ginseng root rot pathogens. Finally, Bacillus velezensis CH-15 was selected due to excellent antagonistic effect and radicicol resistance. When CH-15 was inoculated on ginseng root, it not only inhibited the mycelial growth of the pathogen, but also inhibited the progression of disease. CH-15 also carried biosynthetic genes for bacillomycin D, iturin A, bacilysin, and surfactin. In addition, CH-15 culture filtrate significantly inhibited the growth and conidial germination of pathogens. This study shows that endophytic bacterium CH-15 had antagonistic effect on ginseng root rot pathogens and inhibited the progression of ginseng root rot. We expected that this strain can be a microbial agent to suppress ginseng root rot.