<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.

海洋生境芽孢杆菌(Bacillus spp.)的培养条件及产生的胞外抗菌蛋白

测试了 4株海洋芽孢杆菌的培养条件对其产生抗菌蛋白的影响 ;提取的粗蛋白的性质以及对 6种病原指示菌的抑菌活性 ,结果表明 ,4株海洋芽孢杆菌在2 2 1 6E ,EMB ,CM培养液 ;1 5～ 35℃ ;NaCl浓度 0 %～ 5 % ;pH3～ 9的条件下均能生长 ,适宜生长温度 2 0～ 2 8℃ ;NaCl浓度 1 %～ 3% ;pH5～ 8.菌株分别培养 84～1 0 8h抑菌活性最强 .(NH4) 2 SO4沉淀抗菌粗蛋白的最适饱和度为 5 0 %～ 70 % .粗蛋白对病原菌尤其对病原真菌具有强烈的抑制作用 .粗蛋白性质稳定 ,对高温和蛋白酶不敏感 .

Antagonistic Activities of Volatiles from Four Strains of Bacillus spp. and Paenibacillus spp. Against Soil-Borne Plant Pathogens

The four effective antagonistic Bacillus strains, isolated from the rhizosphere soil of cucumber in a greenhouse, produced antifungal volatiles. These volatiles strongly inhibited the growth of the most tested pathogenic fungi with wide host plants, induced the mycelial morphological abnormalities, and decreased the sclerotoid production of Sclerotinia sclerotiorum in sealed plates. Spores of Botrytis cinerea exposed to these volatiles for 24-48 h in cavity slides cracked and the sporaceous inclusion became brown and effused to the suspension. An interesting phenomenon observed was that all the bacterial volatiles exhibited intense inhibitory activities against the pigment formation of tested pathogenic fungi, including Ascochyta citrullina, Alternaria solani, Alternaria brassicae, and so on. Interactions mediated by microbial volatiles could be widespread in soils, and volatiles may play an important role in reducing disease levels. A phylogenetic analysis based on 16S rDNA sequence placed the four bacteria in three species Paenibacillus polymyxa （BMP-11）, Bacillus subtilis （BL02）, and Bacillus pumilus （BSH-4 and ZB 13）. Through headspace sampling and GC-MS analysis, a rich profile was found from B. subtilis and overlapping volatile patterns could be found among the different species. Studies are under the way to find the possible action mechanisms and to seek the effective application of bacterial volatiles in greenhouse.

芽孢杆菌(Bacillus spp.)防治豇豆根腐病效果

采用生长抑制法,发现芽孢杆菌(Bacillus spp.)对豇豆根腐菌(Fusarium spp.)具有较好的抑制作用,其代谢产物对豇豆根腐菌的抑制中浓度(EC50)为24.4 mg/L。采用拌种法,测定了芽孢杆菌对豇豆根腐病的室内预防效果,其中以药种比100 mL∶1 kg的防效最高,为92.1%,与对照30%噁霉灵水剂防效相当。田间灌根试验结果显示,芽孢杆菌发酵液稀释50倍,对豇豆根腐病的防效达到74.9%,与对照30%噁霉灵水剂防效相当。

Dietary Bacillus spp.enhanced growth and disease resistance of weaned pigs by modulating intestinal microbiota and systemic immunity

Background:Previous research has shown that dietary supplementation of Bacillus spp.probiotics exerts beneficial effects on animals’growth.However,limited studies have evaluated the efficacy of Bacillus spp.on weaned pigs and their effects on host gut health and microbiome,and systemic immunity using a disease challenge model.The objective of this experiment was to investigate the effects of two Bacillus spp.strains(Bacillus subtilis DSM 32540 and Bacillus pumilus DSM 32539)on growth performance,diarrhea,intestinal health,microbiome,and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli(ETEC).Results:Pigs in PRO1(Bacillus subtilis DSM 32540)had greater(P<0.05)body weight on d 7 and 14 PI,greater(P<0.05)ADG from d 0 to 7 and d 7 to 14 PI,compared with pigs in CON(Control).Pigs in PRO1 had milder(P<0.05)diarrhea on d 2 and 3 PI compared with pigs in CON.However,no differences were observed in growth performance and diarrhea score between PRO2(Bacillus pumilus DSM 32539)and CON groups.Supplementation of PRO1 decreased(P<0.05)lymphocyte counts on d 7 and 14 PI,compared with CON.Supplementation of PRO1 and PRO2 both reduced(P<0.05)total coliforms in mesenteric lymph nodes on d 21 PI.Pigs in PRO2 had greater(P<0.05)goblet cell number and sulfomucin percentage in duodenal villi and greater(P<0.05)sialomucin percentage in jejunal villi than pigs in CON.Supplementation of PRO1 up-regulated(P<0.05)MUC2 gene expression in jejunal mucosa and reduced(P<0.05)PTGS-2 and IL1B gene expression in ileal mucosa on d 21 PI,compared with CON.Pigs in PRO1 had reduced(P<0.05)relative abundance of families Lachnospiraceae,Peptostreptococcaceae and Pasteurellaceae in the ileum.Conclusions:Supplementation of Bacillus subtilis DSM 32540 improved growth performance,alleviated diarrhea severity,enhanced gut health,and reduced systemic inflammation of weaned pigs infected with ETEC F18.Although Bacillus pumilus DSM 32539 was able to alleviate systemic inflammation,it had limited impacts on growth performance and severity of diarrhea of ETEC F18 challenged weaned pigs.

Screening of Antagonistic Bacillus spp. Strains against Phytophthora infestans (Mont.) de Bary and Isolation of lipopeptide Substances

[ Objective] The paper was to screen the antagonistic Bacillus spp. strains against Phytophthora infestans （Mont.） de Bary and analyze its lipepeptide substances. [ Methods] Using flat isolation and culture method, the bacteria that could produce Bacillus spp. were isolated from rhizosphere soil, and carried out primary and secondary screening by confrontation culture. The lipopeptide substances of Y-3 strain with better antifungal effect were carried out I-IPLC analysis. [Result] Nine strains of bacteria had certain antagonistic effects against P. infestans in primary screening, Y-1 and Y-3 strains had significant inhibition effect against the growth of P. infestans in secondary screening, with inhibition rate higher than 80% ; the crude extract of lipepeptide produced by Y-3 strain had better antagonistic effect against P. infestans, with inhibition rate higher than 90%. HPLC analysis showed that the crude extract of lipepeptide mainly contained 8 components. [ Conclusion] The lipopeptide substances produced by Y-3 strain had significant inhibition effect, which had potential application value for biological control of potato late blight.

Study on Optimizing Nutrition and Fermentation Conditions for Compound <i>Bacillus spp.</i>

In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L;fermentation temperature is 37&#8451;, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.

改性活性炭构建菌载体及其吸附玉米中黄曲霉毒素B1的工艺研究

旨在为生物处理黄曲霉毒素污染的粮食饲料提供技术支持,以活性炭(SH)为原料进行改性制备磁性活性炭(CSH),选用能高效降解黄曲霉毒素B1(AFB1)的芽孢杆菌属菌株WTX1与CSH载体共培养构建磁性菌载体(FCSH),脱除受污染玉米中的AFB1,对CSH进行BET比表面积、氮吸附-脱附等温线及其对AFB1的等温吸附曲线分析,考察FCSH构建条件和FCSH吸附条件对AFB1脱除效果的影响,并利用傅里叶变换红外光谱(FTIR)和扫描电镜对吸附AFB1前后的FCSH进行表征。结果表明:CSH为大孔结构,其对AFB1的等温吸附符合Freundlich方程;在固定化时间16 h、固定化转速和吸附转速160 r/min、固定化温度和吸附温度35℃、吸附pH 8、振荡时间48 h条件下,FCSH对AFB1脱除率为97.45%;通过FTIR及扫描电镜证实了FCSH对AFB1的吸附作用,且FCSH对AFB1的吸附效果强于CSH。综上,所制备的FCSH能高效脱除玉米中的AFB1。

一株拮抗链格孢(Alternaria spp.)抗生素产生菌研究

从冷冻菌种甘油管中分离到一株生长力极强的污染细菌,该细菌能产生抑制链格孢真菌的活性物质,在研究抑菌活性物质的过程中,对这株细菌进行分类鉴定.通过传统的表型及生理生化方法,将该菌定位到芽孢杆菌属(Bacillus),与枯草芽孢杆菌(B.subtilis)的相似性最大,最终确定这株菌为B.subtilis中的一个株型,即Bacillus subtilis subsp.subtilis str.168.以链格孢属真菌(包括原有的和从空气、小麦病斑分离的)为受试菌,对此株细菌产生抑菌物质的时间进行跟踪,当该菌进入稳定生长期后开始产生抑菌物质,72 h时积累达到最大值.对抑菌物质进行过滤、加热、调节pH值等多种处理,初步确定抑菌物质的特性为外泌型、脂溶性,可透过孔径0.22μm的微孔滤膜,可耐受100℃5 m in,80℃10 m in的高温,微碱性条件不影响其生物活性.

芽胞杆菌抑菌物质作用研究进展

芽胞杆菌是土壤和植物微生态的优势微生物种群,具有很强的抗逆能力和抗菌防病作用,是目前研究和应用较多的一类生防菌。芽胞杆菌可产生醇、酮和酸等多种挥发性物质,能够有效抑制多种病原菌的生长。此外,芽胞杆菌还能合成多种非挥发性抑菌物质如脂肽类化合物、聚酮类化合物和环二肽等抑制某些细菌和真菌的生长。芽胞杆菌的有效抑菌物质主要通过破坏病原菌的细胞壁、细胞膜及影响病原菌的代谢通路发挥其抑菌活性。本文主要总结了芽胞杆菌发酵液中的挥发性、非挥发性抑菌物质及其抑菌机制,为芽胞杆菌作为生防菌的深度开发与应用提供参考。

餐厨堆肥配制基质中添加不同芽孢杆菌对黄瓜育苗效果的影响

为探索不同芽孢杆菌添加至餐厨堆肥配制的基质中对黄瓜育苗效果的影响,试验采用室内穴盘育苗方式,设置该基质中浇灌清水(CK)和枯草芽孢杆菌(T1)、解淀粉芽孢杆菌(T2)和贝莱斯芽孢杆菌(T3)发酵液共4个处理,研究其对黄瓜幼苗生长、育苗后基质的化学性质和镰刀菌属微生物含量的影响。结果表明,三种芽孢杆菌均有较好的促生效果,其中,枯草芽孢杆菌和贝莱斯芽孢杆菌处理在幼苗叶绿素含量、叶面积、地上部鲜重和地上部干重这4项指标上表现最佳,但三种芽孢杆菌对地下部性状尤其是根长并未表现出明显的促进效果。壮苗指数和隶属函数法的综合评价显示,枯草芽孢杆菌和贝莱斯芽孢杆菌处理的壮苗指数相对较高,贝莱斯芽孢杆菌处理的平均隶属度最大,说明贝莱斯芽孢杆菌对黄瓜幼苗的促生效果最佳。三种芽孢杆菌对基质pH值并未产生较大影响,但明显提升基质的EC值,同时没有引起基质中Na^(+)和Cl^(-)含量出现明显下降。在育苗后基质养分含量方面,三种芽孢杆菌处理相对于CK显著增加其碱解氮和速效钾含量;有效磷含量方面,贝莱斯芽孢杆菌处理显著低于其余处理;解淀粉芽孢杆菌处理的有机质含量显著低于其余处理。添加三种芽孢杆菌的餐厨堆肥配制基质栽植黄瓜幼苗具有明显的防病效果,基质中的镰刀菌属微生物含量显著降低,其中贝莱斯芽孢杆菌呈现的效果最佳。建议在本研究结果基础上进一步开展研究以解决餐厨堆肥中盐分离子在基质或土壤中残留的问题。

解淀粉芽胞杆菌Bacillus amyloliquefaciens T-6的分离鉴定及抗病促生潜力

从江苏泗洪桃园发病土壤中筛选出一株对桃树根腐病有拮抗效果且抗菌范围较广泛的芽胞杆菌菌株T-6,经形态学、Biolog生理生化、16S rDNA序列与特异性PCR分析鉴定后,确定该生防菌株为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。抑菌物质酶活、促生性能测定试验表明,该菌株具备产蛋白酶、几丁质酶与溶磷、解磷和解钾的能力。盆栽试验中,T-6处理组番茄幼苗干重、根总长与叶绿素比CK组分别提高31.81%、50.79%、35.11%,防效可达54.69%。菌株T-6在生物防治桃树根腐病等土传病害方面具有较大应用潜力。

猪源芽孢杆菌的分离鉴定及其生物学特性研究

为了分离出潜在的猪源益生芽孢杆菌并探究其益生作用以期获得几株优良的芽孢杆菌菌株用于生产实践。试验采用仔猪粪便为原料,通过菌落形态观察、革兰氏染色、生理生化性质鉴定和16S rDNA序列分析、开展生物学特性分析、进行体外抑菌、药敏和淀粉酶、蛋白酶及纤维素酶活性测定等试验,对用营养琼脂固体培养基分离筛选出的两株菌株进行了研究。经过16S rDNA序列分析,确定培养得到的两株细菌为芽孢杆菌(Bacillus haynesii和Bacillus aerius),Bacillus aerius对大肠杆菌和金黄色葡萄球菌的生长繁殖没有明显的抑制作用;在试验所用到的十种药物中,这两株菌除了对红霉素不敏感外,对其余九种药物均有一定的敏感性;在酶活性试验中,两株菌的蛋白酶和淀粉酶活性比较强,纤维素酶的活性较弱。试验结果表明,这两株芽孢杆菌具有进一步研究并作为益生菌株开发利用的潜能。

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low-and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs

Background： Bacillus spp. seem to be an alternative to antimicrobial growth promoters for improving animals＇ health and performance. However, there is little information on the effect of Bacillus spp. in combination with different dietary crude protein（CP） levels on the ileal digestibility and microbiota composition. Therefore, the objective of this study was to determine the effect of Bacillus spp. supplementation to low-（LP） and high-protein diets（HP） on ileal CP and amino acid（AA） digestibility and intestinal microbiota composition.Methods： Eight ileally cannulated pigs with an initial body weight of 28.5 kg were randomly allocated to a rowcolumn design with 8 pigs and 3 periods of 16 d each. The assay diets were based on wheat-barley-soybean meal with two protein levels： LP（14% CP, as-fed） and HP diet（18% CP, as-fed）. The LP and HP diets were supplemented with or without Bacil us spp. at a level of 0.04%（as-fed）. The apparent ileal digestibility（AID） and standardized ileal digestibility（SID） of CP and AA was determined. Bacterial community composition from ileal digesta was analyzed by Illumina amplicon sequencing and quantitative real-time PCR. Data were analyzed as a 2 × 2 factorial design using the GLIMMIX procedures of SAS.Results： The supplementation with Bacillus spp. did not affect both AID and SID of CP and AA in growing pigs.Moreover, there was no difference in AID of CP and AA between HP and LP diets, but SID of cystine, glutamic acid,glycine, and proline was lower（P 〈 0.05） in pigs fed the HP diets. The HP diets increased abundance of Bifidobacterium spp. and Lactobacillus spp.,（P 〈 0.05） and by amplicon sequencing the latter was identified as predominant genus in microbiota from HP with Bacillus spp., whereas dietary supplementation of Bacillus spp. increased（P 〈 0.05） abundance of Roseburia spp.Conclusions： The HP diet increased abundance of Lactobacillus spp. and Bifidobacterium spp.. The supplementation of Bacil us spp. resulted in a higher abundance of healthy gut associated bacteria without affecting ileal CP and AA digestibility, whereas LP diet may reduce the flow of undigested protein to the large intestine of pigs.

芽孢杆菌嗜铁素研究进展

嗜铁素(Siderophore)是微生物为适应自然界高氧低铁环境,产生的一类能特异性螯合三价铁离子的小分子化合物。绝大多数细菌和真菌都能通过非核糖体肽合成酶途径(NRPS)或非依赖NRPS途径(NIS)合成一种或几种嗜铁素。芽孢杆菌是目前研究与生产上应用最多的一类生防细菌,但芽孢杆菌嗜铁素的生防贡献国内外鲜有报道。本文对芽孢杆菌嗜铁素的种类、合成与调控机制、功能与应用(包括竞争作用、抗生作用、毒力作用、环境污染修复作用以及在医药研发上的应用等)进行了系统概述,为解析自然界中芽孢杆菌具有广谱抗菌活性的生防机制提供理论依据,同时对研发嗜铁素和药物偶联的新型靶向农药具有重要指导意义。

Screening and Identification of an Antagonistic Bacillus Strain against Snap Bean Fusarium Wilt

With Fusarium oxysporum f.sp. phaseoli as the target bacterium,the bacterial strain with strong antagonistic activity was screened from crop rhizosphere soil using plate confrontation method and pot efficiency test. This strain was identified according to morphological observation, physiological and biochemical characteristics and sequence analysis of 16S rRNA gene and gyrB gene. The results showed that LZF-5-3 strain was identified as Bacillus methylotrophicus and the control effect of LZF-5-3 against F.oxysporum in snap bean was 71.42% in pot test.

芽孢杆菌的引入对褐飞虱微生物群和生长发育的影响

【目的】明确褐飞虱Nilaparvata lugens体内可培养共生细菌的种类,探索其中芽孢杆菌类共生菌对褐飞虱微生物群和生长发育的影响。【方法】通过离体培养的方法,从褐飞虱两种不同致害性种群(TN1敏感种群和IR56高致害种群)中分离可培养共生细菌。通过16S rDNA测序对获得的可培养共生菌进行鉴定,在此基础上利用原位杂交研究了共生细菌在褐飞虱体内的分布。利用人工饲料添加抗生素和回补共生细菌的方式,研究了减菌和回补芽孢杆菌的处理对褐飞虱生长发育以及共生菌丰度的影响。比较了通过饲喂和显微注射对芽孢杆菌引入的影响,考察了芽孢杆菌的定殖与褐飞虱TN1种群致害性之间的相关性。【结果】利用离体培养法从褐飞虱中获得了15株不同的共生细菌,其中包括2株源于IR56高致害种群的芽孢杆菌类共生细菌BPH-S36和BPH-S33。原位杂交结果表明共生细菌在褐飞虱成虫唾液腺、肠道、脂肪体和雌虫内生殖器中均有分布,而在雄虫内生殖器中鲜有分布。体内共生细菌对褐飞虱的生长发育至关重要,共生细菌的减少导致褐飞虱存活率在第3和6天时显著下降,而回补共生芽孢杆菌BPH-S36或BPH-S33可使其存活率在第6天时显著上升。褐飞虱通过取食添加共生芽孢杆菌的人工饲料可使芽孢杆菌的相对丰度增加23.2~24.5倍,而通过若虫显微注射并不能够提高芽孢杆菌的相对丰度。随着共生芽孢杆菌的增加,类酵母共生菌(yeast-like symbiont,YLS)的相对丰度也显著增加5~12倍。芽孢杆菌BPH-S36的引入使褐飞虱敏感种群TN1在抗性水稻IR56上的存活率由52.1%±1.5%显著提高至64.2%±3.0%。【结论】共生细菌在褐飞虱唾液腺、肠道和卵巢中广泛分布。抗生素处理能够有效降低其体内共生细菌的丰度,从而导致存活率下降。人工饲喂法可以使外源共生细菌在褐飞虱体内成功定殖。源于致害性强的褐飞虱种群中的芽孢杆菌BPH-S36可提高该种群对抗性水稻品种IR56的致害性。

芽孢杆菌DZY6715生物膜形成对其抗油茶炭疽病的影响

【目的】探究内生芽孢杆菌DZY6715生物膜的形成对其抗油茶炭疽病的影响情况,为油茶炭疽病的绿色防控提供参考依据。【方法】选取分离自油茶健康叶片的内生芽孢杆菌DZY6715为出发菌株,对其进行化学诱变,从中筛选出3株生物膜存在缺陷的突变菌株(其编号分别为-4、15、21)。然后观察DZY6715野生菌株和-4、15、21号突变菌株的生物膜生长特性,测定其群游能力及生物膜形成能力,在此基础上,通过平板对峙实验、扫描电镜观察和油茶离体叶片回接实验,测定不同菌株对油茶炭疽病的抑制能力和生防效果。【结果】DZY6715野生菌株和-4、15、21号突变菌株的生长趋势基本一致,但是,与DZY6715野生菌株相比,3株突变菌株的群游能力均较弱,其生物膜的生长速度均较慢,其生物膜的结构强度均较弱。平板对峙实验结果表明,突变菌株对油茶炭疽病菌的抑制能力随着其生物膜形成能力的降低而降低,其中-4号突变菌株对油茶炭疽病菌的抑制率最低,为57.44%,显著低于DZY6715野生菌株的抑制率(65.70%)。扫描电镜观察中发现,DZY6715野生菌株对油茶炭疽病菌菌丝的破坏力最强,能使菌丝内含物严重渗漏。油茶离体叶片回接实验结果表明,DZY6715野生菌株和3株突变菌株均能不同程度地降低油茶炭疽病的发病率和病情指数,但DZY6715野生菌株的防治效果更好,接种处理7 d后的发病率和病情指数分别为20.22%和5.55,均显著低于突变菌株的。【结论】内生芽孢杆菌DZY6715生物膜的形成,能使其在油茶炭疽病的生物防治中发挥出重要作用。