玉米内生细菌群落动态调查及对玉米生长发育影响的研究
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摘要
植物内生细菌数量多，种类繁杂，分布广泛，通过长期协同进化，在植物体内占据有利生态位。合理应用有益内生细菌可减少施用化肥对环境和作物造成的污染，因此将植物内生细菌作为一种生物肥料资源进行开发和利用已势在必行。本研究以黑龙江地区主栽品种玉米为材料，分离其内生细菌进行群落动态调查，从中筛选出具有促进玉米生长发育作用的内生细菌菌株，结合活性验证，对其进行发酵条件优化，以使其达到最佳促生效果。
将分离自供试玉米的内生细菌进行形态及生理生化鉴定，确定其分属芽孢杆菌属、微球菌属、鞘氨醇单胞菌属、棒状杆菌属、肠杆菌属以及假单胞菌属6个菌属。其中芽孢杆菌为绝对优势菌属，将其鉴定为7个不同种，分别为：枯草芽孢杆菌、巨大芽孢杆菌、蜡状芽孢杆菌、蕈状芽孢杆菌、地衣芽孢杆菌、短小芽孢杆菌和环状芽孢杆菌。
筛选出一株具有促进玉米生长发育的内生细菌菌株SYL4，进行16S rDNA核苷酸序列测定及分析，结果显示其与多株枯草芽孢杆菌的16S rDNA基因序列相似性均达99%，结合形态及生理生化鉴定结果，将SYL4鉴定为枯草芽孢杆菌（Bacillussubtilis）。并利用Rif抗性标记法完成SYL4的内生性验证。
对SYL4进行促生发酵优化，优化后培养基成分配比为：葡萄糖0.5%，蛋白胨1.5%，CaCO3_0.25%。优化后培养条件为：在250mL三角瓶中，加入50mL pH7.0的发酵培养基，接入5%的种子液，31℃，200r/min发酵培养48h。发酵优化后，进行发酵液对玉米生长促进的活性验证，结果显示，经优化后发酵液处理与优化前相比苗期叶长及根长、叶重及根重均有提高，且差异极显著，同时玉米苗期POD活性也有显著提高。说明达到了很好的发酵及促生效果。
Numerous, diverse and widespread, endophytic bacteria have coevolved with theirhost plants through long processes and gradually occupied ecological niches with greatadvantages inside their host plants. Making good use of these beneficial endophyticbacteria can reduce the impact of using chemicals on environment and crop. Therefore,the exploration and application of endophytic bacteria as a kind of biological fertilizerresource will be an inevitable trend. In our research, we used maize from Heilongjiangas our experimental materials. Endophytic bacteria were isolated from these maize,these bacteria’s community dynamics were investigated. Among them the endophyticbacteria that can promote the growth of maize were selected. Combined with the growthactivity test, fermentation of these bacteria was optimized and finally achieved theoptimal effect of promoting their growth.
The morphological and physiological-biochemical methods were used to identifythe endophytic bacteria isolated from the tested maize. The results indicated that thesebacteria belong to six different generas including Bacillus sp., Micrococcus sp.,Sphingomonas sp., Corynebacterium sp., Enterobacter sp. and Pseudomonas sp..Among them, Bacillus sp. were the most predominant genera, and were classified asseven different species, they were B. subtilis, B. megaterium, B. cereus, B. mycoides, B.Licheniformis, B. pumilus and B. circulans.
A strain of enophytic bacteria SYL4that can promote the growth of maize wasselected, and16S rDNA gene sequence analysis was done. As a result, it and manyother B. subtilis strains showed the similarities of16S rDNA gene sequence99%.Together with the result of morphological and physiological-biochemical identification,SYL4was preliminarily identified as B. subtilis, and Rif resistance marker method was also used to complete SYL4endogenous test.
Fermentation of SYL4optimizaiton was conducted. The optimal component ratioof culture medium were as follows: glucose0.5%, peptone1.5%, CaCO30.25%. Theoptimal culture conditions were250ml Erlenmeyer flask containing50ml medium,medium pH7.0,5%amount of inoculation,31℃incubating temperature and200r/minculture for48h. Then the growth-promoting effect of fermentation broth that had beenoptimized on maize was verified, the results showed that the length and weight of leavesand roots of maize seedlings treated with optimized fermentation broth were better thanthose untreated, and the differences were significant. Moreover, POD activity of maizeseedlings promoted a lot also. This result successfully proved that the endophyticbactera achieved a great fermentation and growth-promoting effect.
将分离自供试玉米的内生细菌进行形态及生理生化鉴定，确定其分属芽孢杆菌属、微球菌属、鞘氨醇单胞菌属、棒状杆菌属、肠杆菌属以及假单胞菌属6个菌属。其中芽孢杆菌为绝对优势菌属，将其鉴定为7个不同种，分别为：枯草芽孢杆菌、巨大芽孢杆菌、蜡状芽孢杆菌、蕈状芽孢杆菌、地衣芽孢杆菌、短小芽孢杆菌和环状芽孢杆菌。
筛选出一株具有促进玉米生长发育的内生细菌菌株SYL4，进行16S rDNA核苷酸序列测定及分析，结果显示其与多株枯草芽孢杆菌的16S rDNA基因序列相似性均达99%，结合形态及生理生化鉴定结果，将SYL4鉴定为枯草芽孢杆菌（Bacillussubtilis）。并利用Rif抗性标记法完成SYL4的内生性验证。
对SYL4进行促生发酵优化，优化后培养基成分配比为：葡萄糖0.5%，蛋白胨1.5%，CaCO3_0.25%。优化后培养条件为：在250mL三角瓶中，加入50mL pH7.0的发酵培养基，接入5%的种子液，31℃，200r/min发酵培养48h。发酵优化后，进行发酵液对玉米生长促进的活性验证，结果显示，经优化后发酵液处理与优化前相比苗期叶长及根长、叶重及根重均有提高，且差异极显著，同时玉米苗期POD活性也有显著提高。说明达到了很好的发酵及促生效果。
Numerous, diverse and widespread, endophytic bacteria have coevolved with theirhost plants through long processes and gradually occupied ecological niches with greatadvantages inside their host plants. Making good use of these beneficial endophyticbacteria can reduce the impact of using chemicals on environment and crop. Therefore,the exploration and application of endophytic bacteria as a kind of biological fertilizerresource will be an inevitable trend. In our research, we used maize from Heilongjiangas our experimental materials. Endophytic bacteria were isolated from these maize,these bacteria’s community dynamics were investigated. Among them the endophyticbacteria that can promote the growth of maize were selected. Combined with the growthactivity test, fermentation of these bacteria was optimized and finally achieved theoptimal effect of promoting their growth.
The morphological and physiological-biochemical methods were used to identifythe endophytic bacteria isolated from the tested maize. The results indicated that thesebacteria belong to six different generas including Bacillus sp., Micrococcus sp.,Sphingomonas sp., Corynebacterium sp., Enterobacter sp. and Pseudomonas sp..Among them, Bacillus sp. were the most predominant genera, and were classified asseven different species, they were B. subtilis, B. megaterium, B. cereus, B. mycoides, B.Licheniformis, B. pumilus and B. circulans.
A strain of enophytic bacteria SYL4that can promote the growth of maize wasselected, and16S rDNA gene sequence analysis was done. As a result, it and manyother B. subtilis strains showed the similarities of16S rDNA gene sequence99%.Together with the result of morphological and physiological-biochemical identification,SYL4was preliminarily identified as B. subtilis, and Rif resistance marker method was also used to complete SYL4endogenous test.
Fermentation of SYL4optimizaiton was conducted. The optimal component ratioof culture medium were as follows: glucose0.5%, peptone1.5%, CaCO30.25%. Theoptimal culture conditions were250ml Erlenmeyer flask containing50ml medium,medium pH7.0,5%amount of inoculation,31℃incubating temperature and200r/minculture for48h. Then the growth-promoting effect of fermentation broth that had beenoptimized on maize was verified, the results showed that the length and weight of leavesand roots of maize seedlings treated with optimized fermentation broth were better thanthose untreated, and the differences were significant. Moreover, POD activity of maizeseedlings promoted a lot also. This result successfully proved that the endophyticbactera achieved a great fermentation and growth-promoting effect.
引文
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