Fusaricidins in Paenibacillus polymyxa A21 and their antagonistic activity against Botrytis cinerea on tomato

Four kinds of antifungal compounds from an extract of Paenibacillus polymyxa A21 with molecular masses of 883.56, 897.59, 947.55, and 961.58 Da were characterized as the members of fusaricidin-type of antibiotics according to LC-MS analysis. Fusaricidins isolated from culture filtrate displayed high antagonistic activity against several plant fungal pathogens, especially Botrytis cinerea, the causal agent of gray mold. The fusaricidins biosynthetic gene cluster (BGC) from A21 was cloned by PCR and comparative cluster analysis revealed that gene fusTE, the 3′ boundary of the fusaricidin BGC in strain PKB1, was not present in fusaricidin BGC of A21, indicating that fusTE is not necessary for fusaricidin synthesis. Fusaricidin extract from A21 significantly reduced gray mold disease incidence and severity on tomato. The mRNA levels for three patho-genesis-related proteins (PRs) revealed that treatment of tomato leaves with fusaricidin extract induced the expression of PR genes to different levels, suggesting that one reason for the reduction of gray mold infection by fusaricidin is induction of PR proteins, which lead to increased resistance to pathogens. This is the first report of the application of fusaricidins to control tomato gray mold and the comparative cluster analysis provides important molecular basis for research on fusaricidin biosynthesis.     

3％井冈霉素A·(2亿芽胞/毫升)多粘类芽胞杆菌水剂防治水稻纹枯病试验

以3％井冈霉素A·(2亿芽胞/毫升)多粘类芽胞杆菌水剂为供试药剂,通过田间试验研究不同剂量、不同施药时期对水稻纹枯病的防治效果.2年试验结果表明,当用药量在45～90 g.a.i/hm2 时,药后20 d,发病初期(即病株率在5％以下)预先防治的田间防效达80.38％～89.06％,而发病期(即病株率在10％以上)施药的田间防效仅达41.12％～53.26％;在发病初期预先防治效果要明显优于发病期再施药,从而确定该药剂防治水稻纹枯病时期应适当前移.     

奶牛瘤胃液中浸麻类芽孢杆菌的分离鉴定

采用平板划线分离法从健康奶牛瘤胃液中分离得到1株细菌Y-1,体外模拟瘤胃环境增殖Y-1,进行形态学观察、生化反应和16S rDNA基因同源性比较,并对其进行系统发育树的构建等系统鉴定。结果表明,该细菌为浸麻类芽孢杆菌(Paenibacillus macerans),本研究还对瘤胃液中存在该菌的原因做了分析。     

生防菌BMP-11对瓜果腐霉病菌的抑制活性及其鉴定

为明确生防细菌BMP-11对黄瓜猝倒病的控制作用及其分类地位,采用平板对峙法结合显微观察确定其对致病菌瓜果腐霉的抑制活性,进行BMP-11发酵液对黄瓜猝倒病的防病促生试验,并采用形态及培养特征观察、生理生化测定结合16S rDNA序列分析对其进行鉴定.结果表明,BMP-11对瓜果腐霉病菌具有较强的抑制活性,并可使菌丝体细胞壁裂解,原生质聚集、外溢,进而抑制卵孢子形成;发酵液土壤处理对种子发芽率无显著影响,并可提高黄瓜出苗率,促进黄瓜植株生长;盆栽防病试验结果显示,BMP-11对由瓜果腐霉病菌引起的黄瓜猝倒病的防治效果为62.2%,与对照药剂霜霉威无显著差异.根据16S rDNA序列分析的系统发育树,BMP-11与多粘类芽孢杆菌Paenibacillus polymyxa GBR-1基因序列的同源性最高,结合形态及培养特征观察和生理生化鉴定结果,初步确定该菌株为多粘类芽孢杆菌.     

多粘类芽孢杆菌Cp-S316抗真菌活性物质的提取及其部分性质研究

多粘类芽孢杆菌Cp-S316对动植物真菌、细菌病原物具有广谱抗性,为研究抗真菌活性物质的化学结构、理化性质并探讨其工业化生产的可行性,研究了大孔吸附树脂对多粘类孢杆菌Cp-S316产生的抗真菌活性物质的吸附、解吸性能,筛选出其最佳解吸剂,并测定了抗真菌活性物质的部分性质.结果表明,大孔吸附树脂X-5对抗真菌活性物质的吸附及解吸性能最好,其饱和吸附量为5.36×104 μg·g^-1,其最佳解吸剂为0.01 mol·L^-1 HCl-70%乙醇,以解吸剂∶树脂=2∶1（v∶m）进行静态解吸,解吸率达93.28%.该活性物质对热、紫外线稳定,对胰蛋白酶、蛋白酶K有一定的耐受性,对氯仿部分敏感,对酸稳定,对碱敏感.抗真菌活性物质提取方便、理化性质稳定,显示了一定的应用和开发前景.     

灰葡萄孢拮抗细菌的种类鉴定及生长条件研究

经形态和生理生化鉴定,对灰葡萄孢（Botrytis cinerea Pers.）拮抗能力强、防病效果好的3株细菌进行分类,W10为地衣芽孢杆菌[Bacillus licheniformis(Weigmann)Chester],W3和Y2－11－1为多粘类芽孢杆菌[Paenibacillus polymyxa(Prazmowski)Ash et al]。这2种对蔬菜灰霉病的高效拮抗菌为中国首次报道和应用。3株拮抗细菌的生长条件基本一致,最适培养时间为24－72h,最适生长温度是27－30℃,最适培养液pH值为7－8。     

极端嗜热多肽的纯化、性质初探及对苗瘟的防效

本试验用加热法从多粘类芽孢杆菌(Paenibacillus polymyxa)LM-3菌株的发酵液中纯化得到2个极端嗜热多肽。平板拮抗实验表明,5μl纯化多肽对稻瘟病菌(Magnaporthe grisea)抑菌率达89.6%。SDS-PAGE电泳显示纯化多肽分子量介于6 000~7000 Da之间。多肽复性后,其中之一对稻瘟病菌表现出强的拮抗活性,命名为APPLM3(Antagonism Polypeptide fromPaenibacilluspolymyxaLM-3);另一个则无此活性,命名为PPLM3(Polypeptide fromPaenibacillus polymyxaLM-3)。APPLM3经氨基酸测序,获得了其N-末端5个氨基酸序列(H2N-ANDPR);以该序列为靶序列在NCBI上进行相似性检索,发现其可能与3个假设蛋白(或推导蛋白)相关。以APPLM3为主效成分配得5种微生物源生防乳剂,对水稻苗瘟进行了田间防效试验。试验结果表明,这5种乳剂的防效都优于LM-3拮抗液直接喷雾,其中AB4处理的防效最佳,达80.73%,其拮抗成分、吐温80、乳化剂OP、溶剂的质量比为50∶6.5∶1.75∶41.75。APPLM3为首次报道的兼具极端嗜热性和稻瘟病菌拮抗活性的小分子多肽。     

土壤因子对海洋多粘类芽胞杆菌L_1-9在黄瓜根表土壤定殖的影响

本试验考察了土壤温度、有机质含量和pH对海洋生防细菌多粘类芽胞杆菌L_1-9在黄瓜根表土壤中定殖的影响。采用实时荧光定量PCR方法检测了不同时期黄瓜根表土壤中菌株L_1-9的16S r DNA拷贝数。结果表明,土壤有机质含量、土壤温度和pH对菌株L_1-9在黄瓜根表的定殖有显著影响(P0.01)。25℃定殖数量最高,为5.79×108拷贝/g土,是15℃时的3.4倍;添加有机肥20~40 g/kg土壤,可以促进菌株L_1-9在黄瓜根际定殖;pH 7时,菌株L_1-9在黄瓜根表土壤中的定殖数量最高。本研究为海洋生防菌L_1-9防治黄瓜土传病害提供了理论依据。     

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.     

多粘芽孢杆菌中2,3-丁二醇脱氢酶的克隆和表达

将来自于多粘芽孢杆菌(Paenibacillus polymyxa)中的2,3-丁二醇脱氢酶基因pla C在大肠杆菌(Escherichia coli)BL21中进行了诱导表达,并对其酶学性质进行研究。结果表明,该基因的表达产物主要是生成内消旋型2,3-丁二醇,其氧化2,3-丁二醇的最适温度是50℃,最适pH为10,而还原乙偶姻的最适温度为60℃,最适pH为6。