施用甘蔗糖厂酒精废液对蔗田微生物多样性的影响
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摘要
为了探明施用甘蔗糖厂酒精废液对土壤生物群落的影响,为科学施用甘蔗糖厂酒精废液提供科学依据,本研究利用传统的分离培养方法及PCR-DGGE分子标记技术,对在不同甘蔗糖厂酒精废液处理条件下甘蔗各个生育时期的蔗田微生物数量及成熟期微生物群落DNA序列进行了分析,得到不同处理条件下的土壤微生物数量和DNA序列的多样性之间的差异,并对影响土壤微生物群落的因素进行了探讨。
本研究获得的主要结论如下:
1.在甘蔗生育期内,施用酒精废液的处理土壤中细菌、真菌、放线菌及微生物总数均比不施酒精废液的处理有明显增加。
2.比较了两种不同的土壤DNA提取方法,发现直接法的提取效率较高,且产量也高,为后期试验提供良好的DNA模板。
3.对目的基因的PCR扩增进行了优化,由于采用特殊的带有“GC夹板”结构的引物,所以分别采用常规PCR及降落式PCR对16S-rDNA V3区进行扩增,结果显示,降落式PCR方法在扩增中具有明显的优势,不但提高了对目的产物扩增的特异性,同时也使产量得以提高。
4.土壤微生物物种丰富度次序由高到低的顺序为:75t/ha酒精废液＞105t/ha酒精废液＞4St/ha酒精废液＞CK2＞CK1＞75t/ha酒精废液+不种处理,各处理土壤间微生物群落相似性指数以45t/ha与75t/ha酒精废液处理的最高,达41%;75t/ha酒精废液+不种与CK2、CK1与105t/ha酒精废液处理之间的最低,为19%。UPGMA聚类也显示了类似的结果,75t/ha和45t/ha酒精废液的处理相似性达75%。
5.施用酒精废液处理对蔗田土壤微生物的影响总体上是有利的,可使蔗田土壤细菌多样性更丰富,数量更多,有利于改良土壤结果,提高土壤肥力。
In order to investigate the effects of vinasse application on the microbial diversity in soil and provide with references for rational application of vinasse in sugarcane field, the number of microbes at different growth stages and the DNA sequence of microbial Community in the soil treated with different rates of vinasse were studied with traditional isolating and culturing methods and PCR-DGGE Molecular marker technique. The changes and differences of the number of soil microbe and the genetic diversity were analyzed, and the factors that may influence the soil microbial community were discussed.
The main results were summarized as follows:
1. During sugarcane growth, the quantities of bacterium, fungi and actinomyces, and the total number of microbes in the soil treated with vinasse increased obviously as compared with the controls without vinasse application.
2. The effectiveness of two soil DNA extraction methods was compared. The results showed that the direct method gave better DNA and was more efficient, and provided ideal template DNA for further molecular study.
3. The PCR method was optimized. Both traditional and touchdown strateges were experimented comparatively for PCR because the primer used in the present study has "GC clamp" structures. The result showed that better aim gene fragment could be amplified with the touchdown strategy, suggesting that this method is more suitable for the present study.
4. Micobial richness in soil was in the order of 75t/ha vinasse >105t/ha vinasse > 45t/ha vinasse > CK2 > CK1 > 75t/ha vinasse + no planting treatments. The soil microbial similarity index was the highest (41%) in 45t/ha vinasse and 75t/ha vinasse treatments, and the lowest in 75t/ha vinasse + no planting and CK2 treatments, CK1 and 7t/ha vinasse (both 19%). UPGMA cluster analysis showed that microorganisms in the soils of 75t/ha vinasse and 45t/ha vinasse treatments were in the same cluster, and the genetic similarity reached 75 %.
5. In general, application of vinasse is good for microorganisms in the soil of sugarcane field, which resulted in higher microbial richness and higher populations as compared with the controls without vinasse application, which is beneficial to amelioration of soil structure and improvement of soil fertility.
本研究获得的主要结论如下:
1.在甘蔗生育期内,施用酒精废液的处理土壤中细菌、真菌、放线菌及微生物总数均比不施酒精废液的处理有明显增加。
2.比较了两种不同的土壤DNA提取方法,发现直接法的提取效率较高,且产量也高,为后期试验提供良好的DNA模板。
3.对目的基因的PCR扩增进行了优化,由于采用特殊的带有“GC夹板”结构的引物,所以分别采用常规PCR及降落式PCR对16S-rDNA V3区进行扩增,结果显示,降落式PCR方法在扩增中具有明显的优势,不但提高了对目的产物扩增的特异性,同时也使产量得以提高。
4.土壤微生物物种丰富度次序由高到低的顺序为:75t/ha酒精废液＞105t/ha酒精废液＞4St/ha酒精废液＞CK2＞CK1＞75t/ha酒精废液+不种处理,各处理土壤间微生物群落相似性指数以45t/ha与75t/ha酒精废液处理的最高,达41%;75t/ha酒精废液+不种与CK2、CK1与105t/ha酒精废液处理之间的最低,为19%。UPGMA聚类也显示了类似的结果,75t/ha和45t/ha酒精废液的处理相似性达75%。
5.施用酒精废液处理对蔗田土壤微生物的影响总体上是有利的,可使蔗田土壤细菌多样性更丰富,数量更多,有利于改良土壤结果,提高土壤肥力。
In order to investigate the effects of vinasse application on the microbial diversity in soil and provide with references for rational application of vinasse in sugarcane field, the number of microbes at different growth stages and the DNA sequence of microbial Community in the soil treated with different rates of vinasse were studied with traditional isolating and culturing methods and PCR-DGGE Molecular marker technique. The changes and differences of the number of soil microbe and the genetic diversity were analyzed, and the factors that may influence the soil microbial community were discussed.
The main results were summarized as follows:
1. During sugarcane growth, the quantities of bacterium, fungi and actinomyces, and the total number of microbes in the soil treated with vinasse increased obviously as compared with the controls without vinasse application.
2. The effectiveness of two soil DNA extraction methods was compared. The results showed that the direct method gave better DNA and was more efficient, and provided ideal template DNA for further molecular study.
3. The PCR method was optimized. Both traditional and touchdown strateges were experimented comparatively for PCR because the primer used in the present study has "GC clamp" structures. The result showed that better aim gene fragment could be amplified with the touchdown strategy, suggesting that this method is more suitable for the present study.
4. Micobial richness in soil was in the order of 75t/ha vinasse >105t/ha vinasse > 45t/ha vinasse > CK2 > CK1 > 75t/ha vinasse + no planting treatments. The soil microbial similarity index was the highest (41%) in 45t/ha vinasse and 75t/ha vinasse treatments, and the lowest in 75t/ha vinasse + no planting and CK2 treatments, CK1 and 7t/ha vinasse (both 19%). UPGMA cluster analysis showed that microorganisms in the soils of 75t/ha vinasse and 45t/ha vinasse treatments were in the same cluster, and the genetic similarity reached 75 %.
5. In general, application of vinasse is good for microorganisms in the soil of sugarcane field, which resulted in higher microbial richness and higher populations as compared with the controls without vinasse application, which is beneficial to amelioration of soil structure and improvement of soil fertility.
引文
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