• ISSN 1008-505X
  • CN 11-3996/S
陆海飞, 郑金伟, 余喜初, 周惠民, 郑聚锋, 张旭辉, 刘晓雨, 程琨, 李恋卿, 潘根兴. 长期无机有机肥配施对红壤性水稻土微生物群落多样性及酶活性的影响[J]. 植物营养与肥料学报, 2015, 21(3): 632-643. DOI: 10.11674/zwyf.2015.0310
引用本文: 陆海飞, 郑金伟, 余喜初, 周惠民, 郑聚锋, 张旭辉, 刘晓雨, 程琨, 李恋卿, 潘根兴. 长期无机有机肥配施对红壤性水稻土微生物群落多样性及酶活性的影响[J]. 植物营养与肥料学报, 2015, 21(3): 632-643. DOI: 10.11674/zwyf.2015.0310
LU Hai-fei, ZHENG Jin-wei, YU Xi-chu, ZHOU Hui-min, ZHENG Ju-feng, ZHANG Xu-hui, LIU Xiao-yu, CHENG Kun, LI Lian-qing, PAN Gen-xing. Microbial community diversity and enzyme activity of red paddy soil underlong-term combined inorganic-organic fertilization[J]. Journal of Plant Nutrition and Fertilizers, 2015, 21(3): 632-643. DOI: 10.11674/zwyf.2015.0310
Citation: LU Hai-fei, ZHENG Jin-wei, YU Xi-chu, ZHOU Hui-min, ZHENG Ju-feng, ZHANG Xu-hui, LIU Xiao-yu, CHENG Kun, LI Lian-qing, PAN Gen-xing. Microbial community diversity and enzyme activity of red paddy soil underlong-term combined inorganic-organic fertilization[J]. Journal of Plant Nutrition and Fertilizers, 2015, 21(3): 632-643. DOI: 10.11674/zwyf.2015.0310

长期无机有机肥配施对红壤性水稻土微生物群落多样性及酶活性的影响

Microbial community diversity and enzyme activity of red paddy soil underlong-term combined inorganic-organic fertilization

  • 摘要: 【目的】长期有机与无机肥配合施用是促进农田生产力和土壤有机碳固定的重要技术途径。本文以江西省红壤研究所长期不同施肥试验田的表土(015 cm)为对象,探讨不同施肥措施对土壤微生物群落多样性和酶活性的影响。 【方法】在水稻收获后,采集表土壤样品,提取土壤总DNA。采用聚合酶链反应结合变性梯度凝胶电泳(PCR-DGGE)的方法研究土壤微生物的群落结构多样性,并结合克隆测序研究土壤微生物的群落组成;用实时荧光定量PCR (qPCR)的方法研究土壤微生物的丰度。土壤细菌定量和群落结构分析的分子标靶基因分别为16S rRNA基因V3区和V6区片段,土壤真菌定量和群落结构分析的标靶基因均为18S rRNA基因。DGGE分析采用8%的聚丙烯酰胺凝胶分离细菌和真菌,所用变性梯度分别为35%~65%和20%~40%。同时采用荧光微孔板检测技术测定土壤几丁质酶、-葡萄糖苷酶、-葡萄糖苷酶、纤维素酶、酸性磷酸单脂酶和木聚糖酶活性;用紫外分光光度计法测定土壤过氧化物酶活性。【结果】PCR-DGGE分析表明,与不施肥对照(CK)相比,有机无机肥配施(NPKM),土壤细菌的香农指数和丰富度指数显著增大,而土壤真菌的香农指数和丰富度指数在不同施肥处理间无显著差异。DGGE图谱聚类分析显示,NPKM处理的土壤细菌和真菌的群落结构显著区别于其他3个处理。后续的切胶测序得出,土壤细菌分属于Chloroflexi(绿弯菌门),Proteobacteria(变形菌门)和Firmicutes(厚壁菌门);NPKM处理下隶属于Clostridum(梭菌属)和Anaerolineaceae(厌氧绳菌科)的两类细菌显著增加。土壤真菌主要分属于Basidiomycota(担子菌门)和Ascomycota(子囊菌门),这些真菌条带在DGGE图谱上的分布不同处理间均无明显的规律性,因而不同处理间真菌的群落分布未出现较清晰的变化。qPCR的结果显示,土壤细菌和真菌拷贝数在不同处理间无显著差异。土壤酶的检测结果表明,与CK相比, 单施氮肥(N)处理的土壤几丁质酶活性显著提高,常规氮磷钾处理(NPK)处理的几丁质酶和-葡萄糖苷酶活性显著增强,NPKM处理提高了土壤几丁质酶、纤维素酶和过氧化物酶活性;酸性磷酸单酯酶和木聚糖酶活性在各处理间无显著差异。归一化酶活性值,NPKM处理显著高于CK和其他施肥处理。【结论】 长期有机无机肥配施可显著提高土壤细菌多样性,并改变土壤细菌和真菌的群落结构,提高土壤酶活性,因而提高了农田生态系统的生产力并对生态系统健康有改善作用。

     

    Abstract: 【Objective】 Amendment of soil with organic and inorganic fertilizers has been considered an important source for carbon sequestration. However, it needs further research on influences of carbon sequestration to soil microbial health and reasons of improving soil biological activity. Soil samples were collected from Jiangxi Institute of Red Soils experimental fields. The aim of our study was to discuss the influences to microbial community diversity and enzyme activity of soil causing by different fertilizations. 【Methods】 Soil samples were collected after the harvest of rice, and total soil DNA was extracted. Soil microbial community diversity, composition and abundance were investigated using polymerase chain reaction combined with denaturing gradient gel electrophoresis(PCR-DGGE), cloning combined with sequencing and quantitative real-time PCR(qPCR). And the target genes for qPCR and PCR-DGGE were V3 and V6 partial bacterial 16S rRNA or partial fungal 18S rRNA. The DGGE analysis of using an 8% polyacrylamide gel with a denaturant gradient of 35%-65% for bacteria and 20%-40% for fungi. 96-well microplate fluorimetric was applied to analysis activities of -N-acetylglucosaminidase, -glucosidase, -glucosidase, cellobiohydrolase, acid phosphatase and -xylosidase of soil, and ultraviolet spectrophotometry was used for investigating activity of soil peroxidase. 【Results】 The Shannon index and richness index of soil bacteria are increased significantly under the treatment of organic manure plus chemical fertilization(NPKM) compared to the control without application of fertilizer(CK), while there are no changes in the two indexes of soil fungi among different treatments based on the PCR-DGGE analysis. Moreover the clustering analysis demonstrates that the NPKM treatment alters the community structure of soil bacteria and fungi. The results of sequencing indicate that soil bacteria is affiliated with Chloroflexi, Proteobacteria and Firmicutes, while soil fungi is belonged to Basidiomycota and Ascomycota. The bacterial phyla belonged to Clostridium and Anaerolineaceae is stimulated under the NPKM treatment, and the community of fungi is not changes significantly. The results of qPCR show that the copies of soil bacteria and fungi are not altered under different treatments. The soil enzyme assay indicates that comparing to CK, the treatment of nitrogen(N) significantly improves the activity of soil -N-acetylglucosaminidase, the treatment of nitrogen, phosphorus and potassium(NPK) enhances the activities of soil -N-acetylglucosaminidase and -glucosdiase, and the NPKM treatment increases the activities of -N-acetylglucosaminidase, cellobiohydrolase and peroxidase of soil. The activities of soil acid phosphatase and -xylosidase are not altered among different treatments. Meanwhile, the normalized enzyme activity is increased under the NPKM treatment compared to the CK, N and NPK treatments.【Conclusions】 The findings of this study conclude that the long-term fertilization treatment of NPKM significantly increases soil bacterial diversity, changes soil microbial community structure and improves soil enzyme activity. Therefore, the combined inorganic/organic fertilization can enhance the productivity of agricultural ecosystem and improve the health of soil ecosystem.

     

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