稻瘟病生防菌Af1、Af4的初步研究
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摘要
从水稻叶片上分离得到两株生防细菌,将其分别命名为：Af1、Af4。本实验研究内容主要包括：1.通过培养特征、形态学特征、生理生化特征以及16S rRNA序列同源性分析对拮抗菌Af1、Af4进行鉴定。2.通过对稻瘟病菌丝生长的抑制实验、对孢子萌发的抑制实验、广谱抑菌实验以及不同培养时间、不同浓度、不同pH值的发酵液对稻瘟病菌的拮抗性测定等实验对Af1、Af4进行抑菌活性鉴定。3.按照蛋白和脂肽类抗生素分离纯化方法定向地对Af1、Af4发酵液活性物质进行提取,获得粗提物质,并对粗提物质的生化特性进行研究,最后对脂肽类抗生素合成相关基因进行检测。4.水稻叶片离体接种实验,对Al1、Af4进行生物防效测定。
结果表明：1.通过鉴定表明Af1、Af4两个生防菌株同为枯草芽孢杆菌,两者的理化特征大致相似,但是Afl能够产生色素,而Af4不能够产生色素。2.Af1对稻瘟病菌的抑菌半径达到22 mm,Af4对稻瘟病菌的抑菌半径达到23 mm；Af1和Af4均能抑制稻瘟病孢子的萌发,当发酵液浓度为1：1(50%)时,抑制率最高分别可达到88%和92%,当浓度降低到1：124(0.8%)时,抑制率则为16.4%和46%；Af1、Af4具有广谱抑菌作用,除Af1对水稻纹枯病无作用外,Af1、Af4对稻瘟病菌、稻曲病菌等十种供试病原菌均具有抑制作用；Af1、Af4的发酵液在不同浓度梯度检测,均具有抑菌活性；Af1在发酵液培养5 d时抑菌活性最高,而Af4在3 d-4d抑菌活性最高,且两者都耐高温；两者均不能耐酸性环境,适宜在中偏碱性的环境下生存,Afl在发酵液pH 8时,抑菌活性最高,Af4在pH 7时有较高抑菌活性。3.Af1产生多种类型的抗真菌物质,其中包括蛋白质和抗生素,抗生素粗提物对温度、蛋白酶K不敏感,而蛋白质粗提物对蛋白酶K敏感,对温度不敏感,并能够抑制稻瘟病菌黑色素的形成；Af4发酵液粗提物与脂肽类抗生素一致,对温度、对蛋白酶K不敏感；从Af1中检测到fenB、sfp、ituA三个脂肽抗生素合成相关基因,而从Af4中检测到mycB、fenB、sfp、ituA四个脂肽抗生素合成相关基因,表明两个菌株都能够合成抗生素类物质。4.生物防效测定结果表明,菌株Af1,Af4发酵液在不同浓度梯度下均具有生防效果,且Af1的生防效果要略好于Af4的生防效果,二者在1：249低浓度下仍具有防病效果,且效果持续稳定。
From rice leaves which were infected by Magnaporthe oryzae, two bacterial strains were isolated and named as Afl and Af4, respectively. This study mainly consists of four parts:identification of bacterial strains Afl and Af4 based on culture characteristics, physiological and biochemical characteristics and 16S rRNA sequence homology analysis; determination of antifungal activity by growth inhibition to fungal mycelium, inhibition of M.oryzae spore germination, and antifungal spectrum, by using fermentation liquid of Afl and Af4 from different incubation days, concentrations, and pH values; according to separation and purification methods of lipopeptides and proteins, crude substances were obtained from strains Afl and Af4, and the anti-fungal activity was assessed. At the same time, PCR detection of lipopetides synthesis related genes in strains Afl and Af4 was carried out by using primers from the known lipopeptides genes; determination of bio-control efficacy of strains Afl and Af4 by in vivo inoculation experiments.
Two bio-control strains were identified as Bacillus subtilis,both strains had similar physical and chemical characteristics, but strain Afl could produce melanin while Af4 could not. Antifungal radius of strain Afl reached to 22 mm and Af4 antifungal radius reached to 23 mm against M. oryzae. Both of strains could inhibit the germination of M.oryzae spores, when concentration of fermentation liquid was 1:1, the highest inhibition rates could be achieved (88% and 92% for Afl and Af4), and the lower inhibition rates were still observed even the concentration was reduced to 1:124 (16.4% and 46% for Af1 and Af4, respectively). Af1 and Af4 had broad antifungal spectrum, except that Af1 had no effect on rice sheath blight fungus, both strains had obvious antifungal efficacy against 10 kinds of phytophathogenic fungi such as rice blast and rice false smut. Fermentation liquid of Afl showed the highest antifungal efficacy at the fifth day, while Af4 showed highest antifungal efficacy at three to four days after incubated. Both of the fermentation liquids were stable at high temperature, suitable in neutral and alkaline environments. Strain Afl produced different kinds of anti-fungal substances including antifungal proteins and lipopeptides. Crude extract of lipopeptides was insensitive to temperature and proteinase K, crude extract of antifungal proteins could inhibit the formation of melanin in M.oryzae. The properties of crude extract from strain Af4 were consistent with lipopeptides which was not sensitive to temperature and proteinase K.Three genes sfp, fenB and ituA were detected in genome of strain Afl and four genes mycB, sfp, fenB and ituA were detected in genome of strain Af4. Experiment of bio-control efficacy indicated that fermentation liquid of two strains have obviously bio-control effect under different concentrations, and bio-control efficacy of Afl was slightly higher than efficacy of Af4, both strains still had stable efficacy at concentration as low as 1:249.
结果表明：1.通过鉴定表明Af1、Af4两个生防菌株同为枯草芽孢杆菌,两者的理化特征大致相似,但是Afl能够产生色素,而Af4不能够产生色素。2.Af1对稻瘟病菌的抑菌半径达到22 mm,Af4对稻瘟病菌的抑菌半径达到23 mm；Af1和Af4均能抑制稻瘟病孢子的萌发,当发酵液浓度为1：1(50%)时,抑制率最高分别可达到88%和92%,当浓度降低到1：124(0.8%)时,抑制率则为16.4%和46%；Af1、Af4具有广谱抑菌作用,除Af1对水稻纹枯病无作用外,Af1、Af4对稻瘟病菌、稻曲病菌等十种供试病原菌均具有抑制作用；Af1、Af4的发酵液在不同浓度梯度检测,均具有抑菌活性；Af1在发酵液培养5 d时抑菌活性最高,而Af4在3 d-4d抑菌活性最高,且两者都耐高温；两者均不能耐酸性环境,适宜在中偏碱性的环境下生存,Afl在发酵液pH 8时,抑菌活性最高,Af4在pH 7时有较高抑菌活性。3.Af1产生多种类型的抗真菌物质,其中包括蛋白质和抗生素,抗生素粗提物对温度、蛋白酶K不敏感,而蛋白质粗提物对蛋白酶K敏感,对温度不敏感,并能够抑制稻瘟病菌黑色素的形成；Af4发酵液粗提物与脂肽类抗生素一致,对温度、对蛋白酶K不敏感；从Af1中检测到fenB、sfp、ituA三个脂肽抗生素合成相关基因,而从Af4中检测到mycB、fenB、sfp、ituA四个脂肽抗生素合成相关基因,表明两个菌株都能够合成抗生素类物质。4.生物防效测定结果表明,菌株Af1,Af4发酵液在不同浓度梯度下均具有生防效果,且Af1的生防效果要略好于Af4的生防效果,二者在1：249低浓度下仍具有防病效果,且效果持续稳定。
From rice leaves which were infected by Magnaporthe oryzae, two bacterial strains were isolated and named as Afl and Af4, respectively. This study mainly consists of four parts:identification of bacterial strains Afl and Af4 based on culture characteristics, physiological and biochemical characteristics and 16S rRNA sequence homology analysis; determination of antifungal activity by growth inhibition to fungal mycelium, inhibition of M.oryzae spore germination, and antifungal spectrum, by using fermentation liquid of Afl and Af4 from different incubation days, concentrations, and pH values; according to separation and purification methods of lipopeptides and proteins, crude substances were obtained from strains Afl and Af4, and the anti-fungal activity was assessed. At the same time, PCR detection of lipopetides synthesis related genes in strains Afl and Af4 was carried out by using primers from the known lipopeptides genes; determination of bio-control efficacy of strains Afl and Af4 by in vivo inoculation experiments.
Two bio-control strains were identified as Bacillus subtilis,both strains had similar physical and chemical characteristics, but strain Afl could produce melanin while Af4 could not. Antifungal radius of strain Afl reached to 22 mm and Af4 antifungal radius reached to 23 mm against M. oryzae. Both of strains could inhibit the germination of M.oryzae spores, when concentration of fermentation liquid was 1:1, the highest inhibition rates could be achieved (88% and 92% for Afl and Af4), and the lower inhibition rates were still observed even the concentration was reduced to 1:124 (16.4% and 46% for Af1 and Af4, respectively). Af1 and Af4 had broad antifungal spectrum, except that Af1 had no effect on rice sheath blight fungus, both strains had obvious antifungal efficacy against 10 kinds of phytophathogenic fungi such as rice blast and rice false smut. Fermentation liquid of Afl showed the highest antifungal efficacy at the fifth day, while Af4 showed highest antifungal efficacy at three to four days after incubated. Both of the fermentation liquids were stable at high temperature, suitable in neutral and alkaline environments. Strain Afl produced different kinds of anti-fungal substances including antifungal proteins and lipopeptides. Crude extract of lipopeptides was insensitive to temperature and proteinase K, crude extract of antifungal proteins could inhibit the formation of melanin in M.oryzae. The properties of crude extract from strain Af4 were consistent with lipopeptides which was not sensitive to temperature and proteinase K.Three genes sfp, fenB and ituA were detected in genome of strain Afl and four genes mycB, sfp, fenB and ituA were detected in genome of strain Af4. Experiment of bio-control efficacy indicated that fermentation liquid of two strains have obviously bio-control effect under different concentrations, and bio-control efficacy of Afl was slightly higher than efficacy of Af4, both strains still had stable efficacy at concentration as low as 1:249.
引文
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