<i>In Vitro</i>Introduction of Hardy Alcohol Resistant Bacillus spp. through Aseptically Grown Watermelon Seedlings

The present study was taken up with a view to ascertain the possibility of introduction of alcohol resistant bacteria in vitro through the aseptically raised watermelon (Citrullus lanatus) seedlings in the backdrop of isolating such organisms from micropropagated watermelon stocks. Watermelon cv. Arka Manik seedlings grown in vitro from surface-sterilized seeds with the intact seed coat on MS medium appeared visibly clean largely, but upon subjecting them to tissue-indexing, the segments from the collar or root tissue showed bacterial colony growth on Nutrient Agar (NA) from 72% of such healthy seedlings and the cotyledon and hypocotyl tissue of 44% seedlings. The pooled colony growth from NA upon challenge with 90% alcohol yielded 10 distinct colony types, identified as B. pumilus (4×), B. subtilis (4×), B. cereus (1×) or B. safensis (1×) based on partial 16S rRNA sequence analysis. The shoot-tip tissue from the healthy index-negative seedlings cultured on watermelon proliferation medium partly turned index-positive within 2 - 4 sub-culture cycles while being apparently clean. On the other hand, those from the previously index-positive cultures tended to show obvious bacterial growth during subsequent in vitro culturing. The observations suggested the possibility of introduction of spore-forming Bacillus spp. through surface-sterilized seeds, their gradual emergence in vitro in visibly clean seedlings, possible transmittal of spores to the alcohol through tissue-culturing tools and the survival therein with the chances of unsuspected lateral spread. Seed coat removal followed by surface sterilization with sodium hypochlorite facilitated the raising of clean seedlings with no detectable bacterial association.

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera(Hübner)

The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible（96S） and Cry1Ac-resistant H. armigera strain（Cry1Ac-R）. The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance（SPR） showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A＋Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.

Detection and sequencing of plasmid encoded tetracycline resistance determinants(tetA and tetB) from food-borne Bacillus cereus isolates

Objective:To investigate the detection and sequencing of plasmid encoded tetracycline resistance genes(tet A and tet B) from food-borne and standard strains of Bacillus cereus(B. cereus).Methods:PCR was carried out to detect the tetracycline resistance genes(tet A and tet B) in food-borne B.cereus strains and the amplified products were sequenced.Results:The phenotypic resistance against tetracycline was observed in 39 of the 118 food-borne isolates and two reference strains(MTCC 430 and MTCC 1307) of B.cereus.Among the phenotypically resistant isolates,tet A was detected in 36 food-borne isolates and two reference strains(MTCC 430 and MTCC 1307).whereas,tel B was detected in 12 food-bome isolates and MTCC 1307 strain. Conclusions:A close association was therefore found between phenotypic resistance against tetracycline and presence of tetracycline resistance genes.The tet A and tet B gene fragments were amplified,purified and sequenced.The gene sequences of the isolates studied herein were found similar to tetA and tetB gene sequences of other bacteria available in NCBI.The occurrence of tetA and tetB genes in B.cereus indicate the horizontal transfer of antibiotic resistance determinants from other bacteria into B.cereus.The transfer of these resistant determinants to other potentially pathogenic bacteria may be a matter of great concern.

Bacillus velezensis JK-XZ8 prevents and controls crown gall disease on Prunus subhirtella by colonizing and inducing resistance

Crown gall of Prunus subhirtella Miq., flowering cherry, is a soil-borne bacterial disease caused by Agrobacterium tumefaciens Smith & Townsend and has caused serious damage to the species in recent years in China. In this study, the colonization of sapling roots, the biocontrol efficacy, the expression of defense-related genes and the enzyme activity in roots of P. subhirtella inoculated with the Bacillus velezensis JK-XZ8 against A. tumefaciens were determined under greenhouse conditions. The results showed that the JK-XZ8 strain colonized in the rhizosphere and root surfaces of the saplings. Sole application of the strain increased activities of polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) without affecting superoxide dismutase (SOD). SOD and PPO activities increased in the prevention group, and the activity of PAL increased in both the prevention and cure groups. The expression of the defense-related genes PAL and PR-1 of salicylic acid pathway in roots increased in both the prevention and cure groups. Simultaneously on day 3, the expression of both COI and MYC2 genes of the jasmonic acid pathway significantly increased in the two groups. The incidence of crown gall in the prevention and the cure groups were 48.1% and 66.7%, respectively, significantly lower compared to the pathogens alone (87.5%). This study showed that the application of the JK-XZ8 strain reduced the incidence of crown gall in P. subhirtella saplings in the two groups, and the prevention group had better control efficacy. In addition, the JK-XZ8 strain protects against crown gall by developing induced systemic resistance and systemic acquired resistance in the roots of the cherry saplings.

Antibiotics Resistance Profile of Bacillus cereus Strains Isolated from Soil and Pepper in Brazzaville

Introduction: Bacillus cereus and spores produced in various ecological niches are responsible for toxic infections in humans. This study is conducted to determine the antibiotics resistance profile of B. cereus strains isolated from soil and pepper consummated in Brazzaville. Methodology: An antimicrobial susceptibility test of 16 B. cereus strains from soil and peppers was performed using 11 antibiotics by the Kirby-Bauer’s diffusion on disc method. Results: Results revealed 100% (16/16) of resistance in penicillin G, amoxicillin, ceftazidime, rifampicin, and colistin, also 18.75% (3/16), 11.76% (2/16), and 18.75% (3/16) of resistance in doripenem, vancomycin and chloramphenicol respectively. In addition, we have observed 100% (16/16), 81.25% (13/16), 76.47% (13/16), 35.29% (5/16), 35.50% (6/16), and 12.5% (2/16) of sensitivity to line-zolid, tigecycline, ciprofloxacin, vancomycin, doripenem and chloram-phenicol respectively. However, all strains have been multidrug resistant (MDR) to betalactams, polypeptides, and ansamycins. Moreover, 7 strains (43.75%) have been variably multiresistant. One strain, Ri10 has been resistant to beta-lactams, polypeptides, ansamycins, cyclins and glycopeptides. No strain was ultraresistant (XDR) or largely insensitive (PDR) to different antibiotics. Conclusion: This study reveals that 51% of strains have been resistant to antibiotics, 32% are sensitive, and 17% have intermediate resistance. These results partly explain the high rate of gastroenteritis observed in Brazzaville due to food poisoning.

生防解淀粉芽胞杆菌(Bacillus amyloliquefaciens)最新研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)是与枯草芽胞杆菌相似,可产芽胞、革兰阳性(G+)、具有生防活性的益生细菌。其代谢过程中产生的抗菌物质种类多、抗菌谱广,其抗菌制剂或抗菌提取物具有无毒、无害、无残留、抑菌时效长等优点,可广泛应用于果蔬保鲜、果蔬生长期及采后微生物病害的生物防治,是极具开发价值的生防菌。本文从抗菌机理、抗性基因组成、基因工程菌构建、抗性产品开发及对微生物病害防治等几个方面,对解淀粉芽胞杆菌的研究及应用现状进行了归纳和总结,为其在农业生物工程中的规模化安全应用提供参考。

六价铬还原细菌Bacillus cereus S5.4的筛选鉴定及还原特性研究

六价铬生物毒性极大，是造成环境污染的主要重金属之一，其生物治理策略已引起了广泛关注。已经发现许多微生物具有六价铬抗性和还原性，但能工业应用的还十分有限。从宝钢电镀污泥中分离得到一系列高六价铬抗性菌株，其中一株S5．4显示出高六价铬还原性，经形态和生理生化特征及16s rDNA序列比对，鉴定为Bacillus cereus。该菌株好氧生长，在固体LB培养基上培养48h能耐受40mmol／L Cr^6＋，并对Mn^2＋、Ba^2＋和Mo^6＋也显出高抗性；在液体LB培养基中培养72h完全还原2mmol／L Cr^6＋，并能在补充培养基和六价铬的条件下连续还原。该菌株还原六价铬时，最适浓度为2mmol／L Cr^6＋，最适温度范围30～37℃，最适pH7～9。

Bacillus thuringiensis营养期杀虫蛋白Vip3与其转基因植物研究进展

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)是目前应用最多的生物杀虫剂。它能够产生多种杀虫因子,其中,最主要的是杀虫晶体蛋白(Insecticidal Crystal Proteins,ICPs)和营养期杀虫蛋白(Vegetative insectici-dal protein,Vip)。当前,大部分商业化利用的转基因作物均为杀虫晶体蛋白类,随着这些转基因作物种植面积的扩大,害虫对这些较为单一的杀虫蛋白产生抗性已成为一个严峻的问题。Vip3是Vip杀虫蛋白中的一类,不形成蛋白晶体,和ICPs在进化上没有同源性;其对鳞翅目、鞘翅目和同翅目等害虫具有毒杀作用,抗虫谱较广。目前,已经把Vip3基因导入了水稻、玉米和棉花等多种作物中,为作物抗虫育种、延缓害虫产生抗性和减少作物产量损失等带来新的前景。

镉胁迫对节杆菌(Arthrobacter sp．)和芽孢杆菌(Bacillussp．)生理生化特性的影响

将节杆菌和芽孢杆菌分别暴露于不同质量比的镉溶液中,进行不同暴露时间的急性毒性试验。结果表明,20 mg/kg的Cd(II)处理会使节杆菌中还原型谷胱甘肽(GSH)质量浓度显著降低,细胞膜脂质过氧化产物——硫代巴比妥酸活性物质(TBARS)浓度显著升高;0.2mg/kg的Cd(II)处理会使芽孢杆菌中GSH质量浓度、过氧化氢酶(CAT)活性显著降低。节杆菌的可溶性蛋白、可溶性糖质量浓度随暴露时间延长而减少,GSH质量浓度、CAT和超氧化物歧化酶(SOD)活性随暴露时间延长而增加,TBARS浓度呈先减后增的变化趋势;芽孢杆菌的可溶性蛋白质量浓度、CAT活性和TBARS浓度呈先增后减的变化趋势,可溶性糖、GSH质量浓度和SOD活性呈先减后增的变化趋势。镉处理对节杆菌和芽孢杆菌具有一定的胁迫作用,两种菌通过启动不同的抗性系统来抵抗外界胁迫。

铅胁迫对节杆菌(Arthrobacter sp.)和芽孢杆菌(Bacillus sp.)生理生化特性的影响

将节杆菌和芽孢杆菌分别暴露于不同浓度的铅溶液中,并进行不同暴露时间的急性毒性试验。结果表明,500 mg/kg的Pb(II)处理会使节杆菌中的细胞膜脂质过氧化产物——硫代巴比妥酸活性物质(TBARS)浓度显著降低(p<0.05),而Pb(II)质量比变化对其他指标无显著影响(p<0.05);铅处理会使芽孢杆菌中可溶性蛋白质量浓度、GSH质量浓度和超氧化物歧化酶(SOD)活性显著升高,而过氧化氢酶(CAT)活性显著降低(p<0.05)。节杆菌的可溶性糖质量浓度、GSH质量浓度、SOD活性和TBARS浓度随暴露时间延长而显著增加;芽孢杆菌的可溶性糖质量浓度、GSH质量浓度和SOD活性随暴露时间延长而显著增加,TBARS浓度随暴露时间延长呈先增后减的变化趋势,CAT活性也表现出先增后降的变化趋势。铅处理对节杆菌和芽孢杆菌具有一定的胁迫作用,两种菌通过启动不同的抗性系统来抵抗外界胁迫。

Detection,isolation,and identification of cadmium-resistant bacteria based on PCR-DGGE

This study focused on the screening of cadmium-resistant bacterial strains from Pb-Zn tailing. We investigated the diversity of microbial community inhabiting Dong-san-cha Pb-Zn tailing in Beijing, China, by polymerase chain reaction-denaturing gradient gel electrophoresis （DGGE） of 16S rRNA gene of bacterial strain, and found two dominant strains in the DGGE profile. Using special culture media, we isolated two strong cadmium-resistant bacterial strains. On the basis of morphological, physiological, and biochemical characteristics, BIOLOG, and 16S rDNA sequencing, the two strains were identified as Bacillus cereus and Enterobacter cloacae. Minimal inhibitory concentrations （MICs） of heavy metals for the bacteria were determined. E. cloacae showed higher MIC values for heavy metals and a larger range of antibiotic resistance than B. cereus.

Enhanced removal of antibiotic resistance genes during chicken manure composting after combined inoculation of Bacillus subtilis with biochar

This study explored the combined effects of Bacillus subtilis inoculation with biochar on the evolution of bacterial communities,antibiotic resistance genes(ARGs),and mobile genetic elements(MGEs)during the composting of chicken manure.The results showed that B.subtilis inoculation combined with biochar increased bacterial abundance and diversity as well as prolonged the compost thermophilic period.Promoted organic matter biodegradation and facilitated the organic waste compost humification process,reduced the proliferation of ARGs by altering the bacterial composition.Firmicutes and Actinobacteriota were the main resistant bacteria related to ARGs and MGEs.The decrease in ARGs and MGEs was associated with the reduction in the abundance of related host bacteria.Compost inoculation with B.subtilis and the addition of biochar could promote nutrient transformation,reduce the increase in ARGs and MGEs,and increase the abundance of beneficial soil taxa.

Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt

Background:In our previous study,a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt.However,its mechanism for controlling Verticillium wilt remains unclear.The objective of this study was to further clarify its con-trolling effect and mechanism against cotton Verticillium wilt.Results:The results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03%and 59.00%,respectively;the inhibitory effects of sporulation and microsclerotia germination were 47.16%and 70.06%,respec-tively.In the greenhouse test,the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11%on cotton Verticillium wilt,and significantly promoted the growth of cotton seedlings.In the field experi-ment,the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54%at 60 days after cotton sowing,and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking,root irrigation,and spraying treatments significantly increased by 19.48%and 7.42%,30.90%and 2.62%,15.99%and 9.20%,respec-tively.Furthermore,EBS03 improved the resistance of cotton leaves against the infection of V.dahliae,and induced the outbreak of reactive oxygen species and accumulation of callose.In addition,the results of real time fluorescent quantitative polymerase chain reaction(RT-qPCR)detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL,POD,PPO,and PR10 in cotton leaves,enhanced cotton plant resistance to V.dahliae,and inhibited colonization level of this fungal pathogen in cotton.Conclusion:Bacillus subtilis EBS03 has a good biological defense capability,which can inhibit the growth and coloni-zation level of V.dahliae,and activate the resistance of cotton to Verticillium wilt,thus increase cotton yield.

基于全基因组测序的黄水芽胞杆菌Bacillus aquiflavi 3H-10安全性评价

黄水芽胞杆菌Bacillus aquiflavi 3H-10分离自我国四川省宜宾地区传统发酵浓香型白酒黄水样品,具有产生蛋白酶和多种风味物质的能力。对黄水芽胞杆菌3H-10进行高通量基因组测序,并开展耐药基因、毒力基因和致病性分析,以及药物敏感性检测、小鼠毒力实验,以综合评价黄水芽胞杆菌3H-10的安全性。结果显示,黄水芽胞杆菌3H-10基因组为一条环状的DNA,大小3806094 bp,G+C含量为35.66%(摩尔百分含量),基因组中预测到4031个基因、81个tRNA基因、27个rRNA基因、16个sRNA基因;3H-10基因组中未检测到潜在耐药基因,且对常见的9大类、10种抗生素表现为敏感,判断菌株无横向转移耐药性的风险;3H-10基因组中未检测到潜在毒力相关基因,受试小鼠不产生毒性反应或死亡,判断菌株无毒力或人体致病性。黄水芽胞杆菌3H-10在耐药性、毒力与致病性方面无安全性风险。

DNA fingerprinting of Bacillus cereus from diverse sources by restriction fragment length polymorphism analysis

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndrome. It is closely related to animal and human pathogens Bacillus anthracis and the insect pathogen Bacillus thuringiensis. In the present study, antibiotic resistance, heavy metal tolerance &amp;molecular typing of Bacillus cereus from diverse sources such as soil, sewage water, air, fresh water, sea water and milk were studied. Bacillus cereus resistant to Penicillin (10 units/ml) and Amoxycillin (10 &#181;g/ml);sensitive to Cobalt and Mercury even at 1 mM concentration were determined. All the isolate had plasmid ranging in number from 3-5 and their molecular weight ranged from 3.0 kb-2.0 kb. Plasmid DNA band specific to 6,557 bp has been found in Bacillus cereus isolated from sewage water, air, fresh water, sea water and milk.

CRISPR/Cas9-based functional characterization of Px ABCB1 reveals its roles in the resistance of Plutella xylostella(L.)to Cry1Ac,abamectin and emamectin benzoate

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth,Plutella xylostella(L.).To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P.xylostella,we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene.The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain.Intriguingly,the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate.No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed.The knockout strain exhibited no significant fitness costs.Overall,our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand,while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin.The results of this study will help to inform integrated pest management approaches against this destructive pest.

Comparative Study of Disease Suppression on Various Host Plants by <i>Bacillus</i>Cyclic Lipopeptides

In biological controls using Bacillus spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between cyclic lipopeptides and host plants to induce disease resistance. In this study, we aimed to clarify the specificity to induce disease resistance among cyclic lipopeptides on various host plants. Our data clearly showed both cyclic lipopeptides conferred disease suppression on most of host plants, but at different range of cyclic lipopeptide concentration. Our findings contribute to understanding the complex on the specificity of cyclic lipopeptide derived induced disease resistance.

微生物分解硅酸盐矿物增黏对砂土强度的影响

砂土因其无黏聚力而易导致地震液化和渗流侵蚀等工程病害问题,微生物矿化与分解技术的发展为提高砂土黏聚力提供了新的可能。利用胶质芽孢杆菌能够分解硅酸盐矿物的特性,采取人工营养液激励加速含钾长石、黑云母等硅酸盐矿物砂土的生物风化,分析经微生物处理前后砂土的强度参数、黏粒含量、矿物组分和粒间微结构的变化规律。结果表明:胶质芽孢杆菌处理后的砂土中黏粒含量增加了1.6%;黏聚力与处理时长呈线性正相关,120 d内有效黏聚力增加了23 kPa,有效内摩擦角则有轻微的减小;砂土中的钾长石、黑云母等硅酸盐矿物均产生了不同程度的分解破坏,X射线衍射谱图显示,微生物处理120 d后砂土中出现了新的高岭石衍射峰,表明胶质芽孢杆菌分解了黑云母和钾长石等硅酸盐矿物,产生次生黏土矿物高岭石,使砂土中黏粒含量增加。微生物分解硅酸盐矿物产生黏土矿物和胞外聚合物是砂土黏聚力提升的主要原因。营养物激励下矿物分解菌对硅酸盐矿物的风化作用对于改善砂土抗液化和抗渗流性能具有潜在的价值。

Bt对棉铃虫天蚕素抗菌肽基因(HacD)转录的影响

抗菌肽是昆虫体液免疫的重要组成部分,对抵御外源致病微生物的侵染具有重要作用。明确苏云金芽胞杆菌Bacillus thuringiensis(Bt)诱导下棉铃虫天蚕素抗菌肽基因(HacD)转录的空间特征和时序特征,有利于进一步了解昆虫体液免疫与棉铃虫抗Bt发展的关联性。本研究测定了HacD cDNA序列,对序列进行了蛋白质翻译,并通过RT-qPCR技术研究了棉铃虫HacD基因在Bt诱导下的转录规律。结果表明,棉铃虫HacD cDNA全长189 bp,其中GC含量为48.15%,肽链由63个氨基酸组成。Bt诱导后棉铃虫幼虫HacD相对转录水平整体呈显著上调趋势,其中脂肪体中HacD的相对转录水平上调最高,其次为血淋巴。Bt对棉铃虫幼虫进行短期诱导后,脂肪体和血淋巴中的HacD基因的相对转录水平均呈持续上调趋势,分别在诱导24 h和18 h时达到最高,随后均持续下调。对幼虫进行持续诱导后,F 0代初孵幼虫、5龄幼虫、蛹和成虫的HacD基因均可以保持上调的转录特性;传代培养获得的F 1代初孵幼虫、5龄幼虫、蛹和成虫的HacD基因转录较F 0代下调,但均高于对照组。综上所述,Bt能够诱导棉铃虫HacD基因转录水平上调。