云南仓储烟叶霉变及其生物防治研究
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摘要
本文对云南部分地区烟草储藏期霉变菌及其拮抗菌进行了分离鉴定，对霉变菌有强拮抗活性的细菌菌株抗菌物质的种类、部分理化性质进行了初步研究。
从云南弥勒、曲靖、泸西和临沧等地采集霉变烟样，采用稀释平板法共分离到霉变菌株38株，经鉴定分别属于5个属：曲霉属(Aspergillus)、青霉属(Penicillium)、根霉属(Rhizopus)、毛霉属(Mucor)和木霉属(Trichoderma)。其中曲霉属和青霉属真菌是引起烟叶霉变的主要微生物类群。黄曲霉原变种(Aspergillus flavus var．flavus)、烟曲霉椭孢变种(Aspergillus fumigatus var.ellipticus)和黑曲霉(Aspergillus niger)为采集地引起烟叶霉变的优势菌种。
通过混合平板法从云85、云87混合烟叶表面分离到对霉变菌有抑制作用的细菌65株。同时测定了分离自烟草根际的43株细菌菌株的拮抗活性。以黄曲霉原变种为指示菌株，以R值为筛选标准。R=病原菌向拮抗菌生长的距离／对照菌落半径。R值与拮抗作用大小成反比。结果表明，在所测试的菌株中，有较强拮抗作用的菌株有5个：DY11、B011、B022、B054、B055，其R值分别为0.368、0.365、0.37、0.368、0.35。B055经鉴定为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。
对5株拮抗作用强的菌株分别进行烟叶防霉试验，拮抗菌悬液的浓度为1×10~8cfu／ml，在不同温度、湿度条件下，B055表现出很强的拮抗效果，霉变率在40％以下。储藏烟叶的安全水分有显著增加，烟叶的常规化学成分(总糖量、总烟碱、总氮、蛋白质、K~+、Cl~-)没有明显变化。单料烟评吸结果除B054菌株处理的烟样杂气重，刺激性大外，与对照无明显差异。
B055菌株的拮抗机制在于产生抗菌物质。抑菌活性检测跟踪表明B055菌株产生的抗菌物质在胰蛋白胨、葡萄糖、马铃薯培养液中培养72h，抑菌活性达到最大值。该物质可通过硫酸铵盐析沉淀获得，盐析饱和度为30％-60％。该物质经Tricine-SDS-PAGE电泳，只有微弱条带。121℃高温灭菌30min及在常温(23-26℃)保存2月后，对黄曲霉原变种的抑制作用不减。且经紫外照射和不同酸碱(pH2.6-11.8)处理后，除在pH4.0-5.2之间为可逆沉淀外，其余抑菌作用也未有明显变化。经固相萃取后，50％甲醇洗脱下的组分活性最高。综合以上性质，初步断定该物质为小分子的肽类物质。
The fungi caused tobacco mold in the storage stage were investigated in Mile, Qujing, Luxi, Lincang in Yunnan province. Thirty-eight molds were isolated from moldy tobacco, and were identified as species of Aspergillus, Penicillium, Rhizopus, Mucor, Trichoderma. The species of Aspergillus and Penicillium were dominant ones, and Aspergillus flavus var. flavus, Aspergillus fumigatus var. ellipticus, Aspergillus niger were detected in all samples.
Bacteria were isolated from the tobacco leaves with good quality in the storage stage in two samples and plant rhizosphere bacterial isolates were selected to suppress Aspergillus flavus var. flavus according to its R value (R = distance from center to margin of the pathogen colony under suppression by antibiotic bacteria / radium of unsuppressed pathogen colony). An isolate B055 was demonstrated to be highly suppressive to tobacco moldy fungi in dual-culture tests on agar plates with R value 0.35. The bacterium was identified as Bacillus amyloliquefaciens.
Five bacterial isolates were applied to control tobacco mold in the storage stage. Tobacco leaves
were sprayed with bacterial suspension at the concentration of 1x10 cells/ml and placed in different temperature, humility for one month. The B055 was demonstrated to be effective in controlling molds. And there were no significant variation of the general gradients such as concentrations of general sugar, general nitrogen, nicotine, protein, K+, Cl- in tobacco leaves.
The isolate B055 inhibited molds growth by producing antifungal compounds. Antifungal compounds were isolated and purified by fractionation with ammonium sulphate in saturation 30%-60%. There were faint belts obtained by Tricine-SDS-PAGE. The compound was active after autoclaving at 121# for 30min and storing at about 23-26# for two months. It was insensitive to pH from 2.6 to 11.8 and UV radiation. But it produced reversible sediment at pH 4.0 to 5.2. They were further extracted with a Clg solid phase extraction (SPE), and eluted by MeOH. The 50% fraction was active. So we conferred it low molecular weight peptides.
从云南弥勒、曲靖、泸西和临沧等地采集霉变烟样，采用稀释平板法共分离到霉变菌株38株，经鉴定分别属于5个属：曲霉属(Aspergillus)、青霉属(Penicillium)、根霉属(Rhizopus)、毛霉属(Mucor)和木霉属(Trichoderma)。其中曲霉属和青霉属真菌是引起烟叶霉变的主要微生物类群。黄曲霉原变种(Aspergillus flavus var．flavus)、烟曲霉椭孢变种(Aspergillus fumigatus var.ellipticus)和黑曲霉(Aspergillus niger)为采集地引起烟叶霉变的优势菌种。
通过混合平板法从云85、云87混合烟叶表面分离到对霉变菌有抑制作用的细菌65株。同时测定了分离自烟草根际的43株细菌菌株的拮抗活性。以黄曲霉原变种为指示菌株，以R值为筛选标准。R=病原菌向拮抗菌生长的距离／对照菌落半径。R值与拮抗作用大小成反比。结果表明，在所测试的菌株中，有较强拮抗作用的菌株有5个：DY11、B011、B022、B054、B055，其R值分别为0.368、0.365、0.37、0.368、0.35。B055经鉴定为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。
对5株拮抗作用强的菌株分别进行烟叶防霉试验，拮抗菌悬液的浓度为1×10~8cfu／ml，在不同温度、湿度条件下，B055表现出很强的拮抗效果，霉变率在40％以下。储藏烟叶的安全水分有显著增加，烟叶的常规化学成分(总糖量、总烟碱、总氮、蛋白质、K~+、Cl~-)没有明显变化。单料烟评吸结果除B054菌株处理的烟样杂气重，刺激性大外，与对照无明显差异。
B055菌株的拮抗机制在于产生抗菌物质。抑菌活性检测跟踪表明B055菌株产生的抗菌物质在胰蛋白胨、葡萄糖、马铃薯培养液中培养72h，抑菌活性达到最大值。该物质可通过硫酸铵盐析沉淀获得，盐析饱和度为30％-60％。该物质经Tricine-SDS-PAGE电泳，只有微弱条带。121℃高温灭菌30min及在常温(23-26℃)保存2月后，对黄曲霉原变种的抑制作用不减。且经紫外照射和不同酸碱(pH2.6-11.8)处理后，除在pH4.0-5.2之间为可逆沉淀外，其余抑菌作用也未有明显变化。经固相萃取后，50％甲醇洗脱下的组分活性最高。综合以上性质，初步断定该物质为小分子的肽类物质。
The fungi caused tobacco mold in the storage stage were investigated in Mile, Qujing, Luxi, Lincang in Yunnan province. Thirty-eight molds were isolated from moldy tobacco, and were identified as species of Aspergillus, Penicillium, Rhizopus, Mucor, Trichoderma. The species of Aspergillus and Penicillium were dominant ones, and Aspergillus flavus var. flavus, Aspergillus fumigatus var. ellipticus, Aspergillus niger were detected in all samples.
Bacteria were isolated from the tobacco leaves with good quality in the storage stage in two samples and plant rhizosphere bacterial isolates were selected to suppress Aspergillus flavus var. flavus according to its R value (R = distance from center to margin of the pathogen colony under suppression by antibiotic bacteria / radium of unsuppressed pathogen colony). An isolate B055 was demonstrated to be highly suppressive to tobacco moldy fungi in dual-culture tests on agar plates with R value 0.35. The bacterium was identified as Bacillus amyloliquefaciens.
Five bacterial isolates were applied to control tobacco mold in the storage stage. Tobacco leaves
were sprayed with bacterial suspension at the concentration of 1x10 cells/ml and placed in different temperature, humility for one month. The B055 was demonstrated to be effective in controlling molds. And there were no significant variation of the general gradients such as concentrations of general sugar, general nitrogen, nicotine, protein, K+, Cl- in tobacco leaves.
The isolate B055 inhibited molds growth by producing antifungal compounds. Antifungal compounds were isolated and purified by fractionation with ammonium sulphate in saturation 30%-60%. There were faint belts obtained by Tricine-SDS-PAGE. The compound was active after autoclaving at 121# for 30min and storing at about 23-26# for two months. It was insensitive to pH from 2.6 to 11.8 and UV radiation. But it produced reversible sediment at pH 4.0 to 5.2. They were further extracted with a Clg solid phase extraction (SPE), and eluted by MeOH. The 50% fraction was active. So we conferred it low molecular weight peptides.
引文
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