Effects of the biocontrol agent Bacillus amyloliquefaciens SN16‐1 on the rhizosphere bacterial community and growth of tomato

Fusarium oxysporum is a common soil‐borne pathogen that causes serious economic losses in tomato crops worldwide. The purpose of this study was to evaluate the influence of the bio‐control agents Bacillus amyloliquefaciens SN16‐1 and Pseudomonas fluorescens SN15‐2 and the pathogen Fusarium oxysporum f.sp. lycopersici (FOL) inoculation on tomato rhizosphere bacterial communities and growth, as measured by terminal restriction fragment length polymorphism (T‐RFLP). Treatment with SN16‐1 and SN15‐2 had a transient influence on indigenous bacterial communities, withSN16‐1 showing great potential for controlling FOL. The corresponding genera of terminal restriction fragments (T‐RFs) that were significantly altered after 10 days were obtained using Ribosomal Database Project (RDP) database comparison. Genera that produce antibiotics and promote plant growth were activated by SN16‐1 and FOL treatments, indicating that SN16‐1 responds quickly to FOL invasion. Moreover, the bioremediation activity characteristic of certain genera and the levels of enzymes that degrade pathogen cell walls were decreased while bacterial nutrient cycling and plant growth promotion were enhanced with FOL treatment. In conclusion, we found that SN16‐1 possesses the capacity to control tomato wilt, acts synergistically with soil microbes and does not have a persistent effect on the rhizosphere bacterial communities of tomato.     

内生型解淀粉芽孢杆菌YTB1407对甘薯根系的促生作用及其调控机理

本实验室从西洋参根中分离筛选获得兼具生防和促生效果的内生型解淀粉芽孢杆菌YTB1407.为调查其应用潜力,以甘薯为材料,以无菌水灌根处理(CK)为对照,采用不同浓度YTB1407菌悬液灌根盆栽甘薯植株,通过对甘薯生长前期3个主要生育时间点YTB1407在植株内的内生定殖检测和根系表型效应比较,筛选菌悬液灌根的最适处理浓度.对根系内源生长素吲哚乙酸(IAA)、细胞分裂素玉米素核苷(ZR)、反式玉米素核糖核苷(t-ZR)的含量和吲哚乙酸氧化酶类中的吲哚乙酸氧化酶(IAAO)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性进行分析.结果表明: YTB1407促进了甘薯生长前期根系统的定殖、不定根幼根的伸长生长及侧根的发生,提高了根系活力;在甘薯生长后期形成了更大的吸收根系生物量和更低的地上部和总根系生物量比.其中OD₆₀₀值为0.50的菌悬液处理(T_0.50)促生效应显著,在甘薯茎叶封垄期的单株块根鲜质量和有效薯块数量最高.YTB1407通过提高ZR、t-ZR含量,降低IAAO活性、提高PPO活性和IAA含量以及(t-ZR+ZR)/IAA,促进块根分化建成初期甘薯不定根幼根向块根的分化;通过降低IAAO、PPO活性和提高IAA含量,降低(t-ZR+ZR)/IAA,促进块根分化建成后期甘薯分化根向块根的建成.     

解淀粉芽孢杆菌中温α-淀粉酶基因的克隆、表达与酶学性质分析

以中温α-淀粉酶生产菌株Bacillus amyloliquefaciens M23基因组DNA为模板。PCR扩增得到了2．0kb α-淀粉酶基因全长序列。该基因由上游启动子220bp,结构基因1544bp和终止序列320bp构成。将无信号肽的α-淀粉酶结构基因amyQ,克隆入表达载体pET28a,转化E．coli BL21（DE3）,经诱导,测定α-淀粉酶活性。结果表明：α-淀粉酶基因amyQ获得了活性表达,酶活力为2．297U／mL,SDS-PAGE电泳结果显示出分子量约为58kDa特异性蛋白质条带。酶学性质分析表明,重组α-淀粉酶的最适反应温度为60℃,最适反应pH为6．5,在60℃保温15min保持85％以上活性,超过15min,酶迅速失活,在pH5．5～10．0环境下稳定。水解产物分析表明：淀粉水解终产物主要为麦芽寡糖和糊精和少量葡萄糖。     

Optimization of a protective medium for enhancing the viability of freeze-dried Bacillus amyloliquefaciens B1408 based on response surface methodology

Response surface methodology (RSM) is a commonly used system to optimize cryoprotectants of biocontrol strains when they are subjected to preparations. Various kinds of cryoprotectants and centrifugal conditions were tested to improve the survival of biocontrol agents after freeze-drying. To determine the optimum levels of incorporation of three cryoprotectants (glucose, trehalose and xylitol) in the freeze-drying process of strain Bacillus amyloliquefaciens B1408, a range of experiments based on Box-Behnken Design (BBD) were conducted. The results indicated that the suitable centrifugation conditions were 5000 r/min,10 min and the optimum concentrations of cryoprotectants were glucose 1.00%, trehalose 4.74% and xylitol 1.45%. The proven survival rate of cells after freeze-drying was 91.24%. These results convincingly demonstrated that freeze-drying could be used to preparation of biocontrol strain B1408. This study provides a theoretical basis for commercial possibilities and formulation development.     

Biocontrol potential of two novel grapevine associated Bacillus strains for management of anthracnose disease caused by Colletotrichum gloeosporioides

A study was conducted to identify bacterial antagonists for biological control of anthracnose which is one of the economically important diseases of grapes. In India, it is caused by Colletotrichum gloeosporioides. Two hundred and ninety-three bacteria were isolated from the grape ecosystem of 43 spatially distant vineyards in peninsular India. Of these, 25 bacteria substantially inhibited the radial growth of C. gloeosporioides in in vitro studies and 18 bacteria significantly reduced infections in vivo. Of these 18 bacteria, 5 and 3 bacteria also significantly reduced percent disease index (PDI) of downy and powdery mildew diseases, respectively. These bacteria were labelled as strains, DR-38, DR-39, TL-171, DRo-198, TS-204, TS-205, and DR-219, and were identified as Bacillus species based on morphological and molecular characterisation. Aqueous suspensions of all these strains applied as foliar sprays at 1×10⁸ cfu/ml on field grown vines significantly lowered the PDI and the AUDPC (area under disease progress curve) of anthracnose when compared with the untreated control, except DRo-198. Strains TS-204 and TL-171 recorded lower PDIs and AUDPC when compared with the other five strains, and TS-204 could effectively suppress ripe rot of berries, too, in vivo. Strains TS-204 and TL-171 are identified for biocontrol of anthracnose in grapes.     

解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用

对一株解淀粉芽孢杆菌TF28产生的抗菌脂肽进行分离鉴定及抑菌活性研究,采用酸沉淀、乙酸乙酯和甲醇萃取技术制备了抗菌脂肽粗提物,经过2次HPLC分离纯化,在保留时间32～42min内获得8个抗菌脂肽纯品,经MALDI-TOF-MS鉴定为芬芥素(fengycins),对尖孢镰刀菌和禾谷镰刀菌显示出较强的抑菌活性,该研究为提高菌株TF28抗菌脂肽产量的定向遗传改造奠定基础。     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

牡丹根腐病原菌拮抗细菌抑菌活性物质分析

前期研究发现解淀粉芽胞杆菌(Bacillus amyloliquefaciens)md8和md9对牡丹根腐病原菌具有较好的抑制作用,但其抑菌物质的组成尚不清楚。本文首先明确了2个菌株3种脂肽类物质合成的基因片段,利用酸沉淀和葡聚糖凝胶层析法进行抑菌物质的分离纯化,牛津杯对峙法检测脂肽类粗提物和凝胶层析分离组分的抑菌活性;进一步利用实时荧光定量PCR(RT-qPCR)检测菌株在拮抗根腐病原菌过程中脂肽类物质合成基因相对表达量的变化,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析抑菌物质的种类。结果表明,2个菌株的脂肽类粗提物和凝胶层析分离组分对根腐病原菌均具有良好的平板抑菌效果,RT-qPCR结果表明2个菌株合成伊枯草菌素(iturin)的基因ituD在对峙培养过程中相对表达量显著增加,芬荠素(fengycin)合成基因fenA也呈现上调表达。MALDI-TOF-MS分析表明2个菌株中具有抑菌作用的分离组分主要为伊枯草菌素类物质Iturin B和Bacillomycins D,结合实时荧光定量PCR结果,推测菌株md8和md9在拮抗根腐病原菌过程中发挥主要生防作用的物质为伊...     

一株西瓜枯萎病生防菌的鉴定与田间防效

[背景]西瓜枯萎病是由尖孢镰孢菌西瓜专化型(Fusarium oxysproum f.sp.niveum)引起的土传病害,是西瓜最为严重的病害之一.利用拮抗微生物抑制病原菌繁殖,降低其危害程度,已成为西瓜枯萎病防控领域的研究热点.[目的]筛选对西瓜枯萎病病原菌具有拮抗作用的菌株,并探究其抑菌作用,为有效防控西瓜枯萎病提...