连作和轮作棉田土壤微生物多样性分析及PGPR菌株筛选
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摘要
通过培养法和免培养法相结合研究棉花连作、轮作栽培体系下土壤微生物多样性的演替特点,揭示棉田连作障碍的生物因子,了解棉花连作、轮作体系中土壤微生物性状的现状和趋向。并通过筛选具防病促生功能的PGPR有益微生物菌群调节改善棉田生态环境,为探讨综合治理棉田连作障碍的有效措施,推动棉花种植结构的优化调整,保障棉花产业的健康、持续、高效发展提供科学基础和技术支持。
研究结果表明,棉花多年连作造成土壤中可培养微生物数量减少,连作6-8a、9-12a、>13a的棉田与连作<5a的棉田相比,土壤微生物总量分别下降了40.2%、46.7%、52.4%。连作超过5a后,土壤微生物菌群结构逐渐从高肥的“细菌型”土壤向低肥的“真菌型”土壤转化,细菌/真菌(B/F)比值降低,拮抗菌减少,病原菌积累。氮素生理群氨化细菌、硝化细菌、自生固氮菌数量下降,反硝化细菌数量增加。连作还导致土壤呼吸强度、纤维素分解强度下降,微生物活性降低。经倒茬轮作后,土壤微生物数量明显增长,有益于调节菌群平衡,细菌/真菌(B/F)和放线菌/真菌(A/F)比值增加,固氮菌数量呈现显著增长,微生物活性提高。
建立了一种提取棉田土壤微生物总DNA的方法-改良CTAB-SDS法。提取获得的DNA完整性好,得率为24.20μg/g干土。纯化后A260/A280和A260/A230分别为1.80和1.70,纯化回收率为70.1%,适用于后续的PCR分析。
通过ARDRA和DGGE技术分析比较了连作、轮作棉田土壤微生物多样性的变化,结果显示棉花多年连作使土壤细菌种群数量、结构、优势菌群发生变化,多样性指数、丰富度和均匀度指数下降,连作年限越长此趋势越突出。连作>13a与<5a的相似性仅为64%,菌群结构发生复杂的改变。轮作的种群结构与连作有较大差异,群落多样性增加。番茄和草木樨对细菌群落多样性改善的效果更为明显。
连作土壤中的一些优势菌群数量发生改变,1种未培养细菌减弱或消失,2种未培养细菌得到富集。而轮作则使富集菌群密度下降,同时还使土壤中一些非优势的菌群得到加强,增加了一些特征性菌群。
从分离的棉花根际细菌中筛选出对棉花枯萎病菌、棉花黄萎病菌、棉花立枯病菌具有较高拮抗活性的CRB115菌株。盆栽试验结果显示该菌株接种后表现出一定的防病作用,同时还有促进发芽、增加生物量等促生效果。形态鉴定结合16S rDNA序列分析确定为枯草芽孢杆菌(Bacillus subtilis)。
Research on soil microbial diversity in cotton under continuous and rotation cropping systems have been done through the biotechnology method and culture method, the results show that the continuous cropping obstacles of the bio-factors, continuous and rotation cropping present and trend. The PGPR strains which have prevent growth-promoting effects have been used to improve the ecological condition in the cotton fields, to study the how to deal with continuous cropping obstacles, and improve the cotton growing structure of cotton strains, provide a scientific basis and technical to support cotton industry developing healthy, sustainably and effectively.
The results showed: the continuously cropping of cotton will cause the decrease of the cultivable microorganism number, compare to cropping less than 5a continuously, 6-8a, 9-12a and more than 13a of continuously cropping will cause the decrease of the soil microbe by 40.2%, 46.7%, 52.4%, the structure of the soil microbe translated from a rich nutrition bacteria type to the low nutrition fungi type gradually after 5a, the rate of B/F have decreased, pathogenic bacteria have accumulated.With the increasing of the years of continuously cropping, the number of ammonification bacteria, nitrifying bacteria, free-living nitrogen fixing bacteria in the soil have decreased, meanwhile the number of denitrifying bacteria have increased. Respiratory intensity, cellulolytic activity have decreased in continuously cropping field. Through the rotational cropping,the number of the soil microbe has increased obviously, the rate of B/F and A/F, the number of nitrogen-fixing bacteria and microbial activity have increased.
A simple and efficient DNA extraction of the total microbial cotton method has been establishment-modified CTAB-SDS method. This method of extract DNA are integrity and also have higher rate, 24.20μg/g dry soil. The A260/A280 and A260/A230 after the purifying are 1.80 and 1.70, purification Recovery rate are 70.1%, it is totally fit for the next pcr analyzing, It provides the foundation for the study of the soil microbial diversity without culturing.
The changes of bio-diversity in continuously and rotational cropping soil have been compared through the ARDRA and DGGE, it shows that the continuously cropping lower the number of the bacteria, diversity index, richness index and uniform index, the structure of the bacteria begin to have complicate changes after continuously cropping. 13a of continuously cropping was similar to cropping less than 5a continuously only 45 percent, There have obvious differences in the bio-structure of continuously cropping and rotational cropping, the bio-diversity has increased, and especially the tomato and melilotoides have great effects on improve the bacteria diversity in the continuously cropping field.
The number of superiority soil bacteria have changed. One kinds of uncultured bacteria disappeared or weaked. Two kinds of uncultured bacteria be enriched. A number of nonsuperi- ority soil bacteria has been strengthened to rotational cropping fields.
Screen out activity strains of CRB115 from rhizosphere with high antagonistic to Fusarium oxysporium f.sp. vasinfectum(Atk) Snyder et Hansen, Verticillium dahliae Kleb, Rhizoctonia solani kuhn. A pot experiment results showed it have played roles in preventing the disease, accelerating germination and increasing living weight after inoculation of the strains. Morphological identification combination with 16S rDNA sequence analysis identified as bacillus subtilis.
研究结果表明,棉花多年连作造成土壤中可培养微生物数量减少,连作6-8a、9-12a、>13a的棉田与连作<5a的棉田相比,土壤微生物总量分别下降了40.2%、46.7%、52.4%。连作超过5a后,土壤微生物菌群结构逐渐从高肥的“细菌型”土壤向低肥的“真菌型”土壤转化,细菌/真菌(B/F)比值降低,拮抗菌减少,病原菌积累。氮素生理群氨化细菌、硝化细菌、自生固氮菌数量下降,反硝化细菌数量增加。连作还导致土壤呼吸强度、纤维素分解强度下降,微生物活性降低。经倒茬轮作后,土壤微生物数量明显增长,有益于调节菌群平衡,细菌/真菌(B/F)和放线菌/真菌(A/F)比值增加,固氮菌数量呈现显著增长,微生物活性提高。
建立了一种提取棉田土壤微生物总DNA的方法-改良CTAB-SDS法。提取获得的DNA完整性好,得率为24.20μg/g干土。纯化后A260/A280和A260/A230分别为1.80和1.70,纯化回收率为70.1%,适用于后续的PCR分析。
通过ARDRA和DGGE技术分析比较了连作、轮作棉田土壤微生物多样性的变化,结果显示棉花多年连作使土壤细菌种群数量、结构、优势菌群发生变化,多样性指数、丰富度和均匀度指数下降,连作年限越长此趋势越突出。连作>13a与<5a的相似性仅为64%,菌群结构发生复杂的改变。轮作的种群结构与连作有较大差异,群落多样性增加。番茄和草木樨对细菌群落多样性改善的效果更为明显。
连作土壤中的一些优势菌群数量发生改变,1种未培养细菌减弱或消失,2种未培养细菌得到富集。而轮作则使富集菌群密度下降,同时还使土壤中一些非优势的菌群得到加强,增加了一些特征性菌群。
从分离的棉花根际细菌中筛选出对棉花枯萎病菌、棉花黄萎病菌、棉花立枯病菌具有较高拮抗活性的CRB115菌株。盆栽试验结果显示该菌株接种后表现出一定的防病作用,同时还有促进发芽、增加生物量等促生效果。形态鉴定结合16S rDNA序列分析确定为枯草芽孢杆菌(Bacillus subtilis)。
Research on soil microbial diversity in cotton under continuous and rotation cropping systems have been done through the biotechnology method and culture method, the results show that the continuous cropping obstacles of the bio-factors, continuous and rotation cropping present and trend. The PGPR strains which have prevent growth-promoting effects have been used to improve the ecological condition in the cotton fields, to study the how to deal with continuous cropping obstacles, and improve the cotton growing structure of cotton strains, provide a scientific basis and technical to support cotton industry developing healthy, sustainably and effectively.
The results showed: the continuously cropping of cotton will cause the decrease of the cultivable microorganism number, compare to cropping less than 5a continuously, 6-8a, 9-12a and more than 13a of continuously cropping will cause the decrease of the soil microbe by 40.2%, 46.7%, 52.4%, the structure of the soil microbe translated from a rich nutrition bacteria type to the low nutrition fungi type gradually after 5a, the rate of B/F have decreased, pathogenic bacteria have accumulated.With the increasing of the years of continuously cropping, the number of ammonification bacteria, nitrifying bacteria, free-living nitrogen fixing bacteria in the soil have decreased, meanwhile the number of denitrifying bacteria have increased. Respiratory intensity, cellulolytic activity have decreased in continuously cropping field. Through the rotational cropping,the number of the soil microbe has increased obviously, the rate of B/F and A/F, the number of nitrogen-fixing bacteria and microbial activity have increased.
A simple and efficient DNA extraction of the total microbial cotton method has been establishment-modified CTAB-SDS method. This method of extract DNA are integrity and also have higher rate, 24.20μg/g dry soil. The A260/A280 and A260/A230 after the purifying are 1.80 and 1.70, purification Recovery rate are 70.1%, it is totally fit for the next pcr analyzing, It provides the foundation for the study of the soil microbial diversity without culturing.
The changes of bio-diversity in continuously and rotational cropping soil have been compared through the ARDRA and DGGE, it shows that the continuously cropping lower the number of the bacteria, diversity index, richness index and uniform index, the structure of the bacteria begin to have complicate changes after continuously cropping. 13a of continuously cropping was similar to cropping less than 5a continuously only 45 percent, There have obvious differences in the bio-structure of continuously cropping and rotational cropping, the bio-diversity has increased, and especially the tomato and melilotoides have great effects on improve the bacteria diversity in the continuously cropping field.
The number of superiority soil bacteria have changed. One kinds of uncultured bacteria disappeared or weaked. Two kinds of uncultured bacteria be enriched. A number of nonsuperi- ority soil bacteria has been strengthened to rotational cropping fields.
Screen out activity strains of CRB115 from rhizosphere with high antagonistic to Fusarium oxysporium f.sp. vasinfectum(Atk) Snyder et Hansen, Verticillium dahliae Kleb, Rhizoctonia solani kuhn. A pot experiment results showed it have played roles in preventing the disease, accelerating germination and increasing living weight after inoculation of the strains. Morphological identification combination with 16S rDNA sequence analysis identified as bacillus subtilis.
引文
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