犍为县生姜连作土壤微生物特性研究
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摘要
从四川省犍为县榨鼓乡采集了生姜连作土壤,包括患病生姜根际土壤和非根际土壤,正常生姜根际土壤和非根际土壤,采用稀释平板法测定了土壤微生物类群的数量,分别测定了土壤过氧化氢酶、脲酶、中性磷酸酶和微生物量C、N。采用稀释平板法分离获得38株细菌,应用16S rDNA PCR-RFLP、16S rRNA基因全序列分析,研究了这些细菌的遗传多样性,确定了其系统发育及分类地位；采用PCR-DGGE研究了土壤微生物的遗传多样性。其结果如下：
1.患病生姜根际土壤细菌明显增多,放线菌和真菌显著下降,而患病生姜的非根际土壤放线菌有明显增加,真菌和细菌变化不明显。
2.患病生姜根际土壤的中性磷酸酶,脲酶活性降低,过氧化氢酶增加,表明根系分泌物对脲酶、中性磷酸酶活性具有抑制作用,而对过氧化氢酶活性有促进作用。
3.16S rDNA PCR-RFLP分析结果表明,在75%相似性水平处,38株细菌分为6个遗传群。供试菌株表现出一定的差异性,呈现出丰富的多样性。
4.在上述实验基础上,选取了SJ1-4、SJ1-5、SJ1-6、SJ1-9、SJ1-1O、SJ1-11、SJ2-1、SJ2-4、SJ2-8、SJ3-5、SJ4-2、SJ4-3、SJ4-5共13株代表菌株测定了16S rDNA序列,进而构建了系统发育树关系。结果表明,13个代表菌株分布于6个系统发育分支,分别为假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、鞘氨醇杆菌属(Sphingobacterium)、溶杆菌属(Lysobacter)。
5.对土壤DNA的PCR-DGGE分析结果表明,患病生姜根际土壤和正常生姜根际土壤细菌聚类在一起,且细菌种群相似性较高,达到70%左右,显示它们之间具有比较相似的种群结构。
在真菌种群聚类中,患病生姜根际土壤与正常生姜根际土壤聚在一起,患病生姜非根际土壤和正常生姜非根际土壤聚类在一起,各自的相似性不高,说明生姜连作导致了土壤真菌种类改变。
In this study, we collected four kinds of ginger continuous cropping soil sample, including sick ginger rhizosphere soil (SGRS) and sick ginger non-rhizospheric soil (SGNRS), normal ginger rhizospheric soil (NGRS) and normal ginger non-rhizophere soil(NGNRS) from Zhagu town in Qianwei county, Sichuan Province, and determined the microbial flora quantity by pour plate method, and then measeured the soil catalase activity, urease activity, neutral phosphatase activity, and soil microbial biomass C, N. By using pour plated method, 38 bacterial strains were isolated, and 16S rDNA PCR-RFLP,16S rRNA gene sequence analysis was used to do their genetic diversity and phylogeny. Finally, PCR-DGGE was performed to reveal the soil microbial diversity. The results were as follows:
1. The quantity of soil bacteria in SGR increased, but the number of actinomycetes and fungi decreased significantly; however, the quantity of actinomyces in SGNR increased markedly, the fungus and bacteria number did not change obviously.
2. In SGRS, the soil neutral phosphatase activity and urease activity decreased, but catalase activity increased, which suggested that, the plant root exudates inhibited urease activity, neutral phosphatase activity, whereas catalase was then promoted because of continuous cropping.
3. Analysis of 16S rDNA PCR-RFLP revealed that, at the level of 75% similarity,38 bacteria were divided into 6 genetic groups. The results also showed that the difference and diversity was existed among these strains.
4. Based on the results above,13 representative bacterial strains were selected to do 16S rDNA sequence analysis, and the phylogenetic tree was constructed. The results showed that 13 representative strains distributed into six phylogenic branches, including genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Sphingobacterium and Lysobacter.
5. The results of soil fungus PCR-DGGE analysis suggested that, though the fungus flora between SGNRS and NGNRS was closer than that in SGRS and NGRS, the similarity was not high, which revealed that ginger continuous cropping led to the variation of soil microbial population.
1.患病生姜根际土壤细菌明显增多,放线菌和真菌显著下降,而患病生姜的非根际土壤放线菌有明显增加,真菌和细菌变化不明显。
2.患病生姜根际土壤的中性磷酸酶,脲酶活性降低,过氧化氢酶增加,表明根系分泌物对脲酶、中性磷酸酶活性具有抑制作用,而对过氧化氢酶活性有促进作用。
3.16S rDNA PCR-RFLP分析结果表明,在75%相似性水平处,38株细菌分为6个遗传群。供试菌株表现出一定的差异性,呈现出丰富的多样性。
4.在上述实验基础上,选取了SJ1-4、SJ1-5、SJ1-6、SJ1-9、SJ1-1O、SJ1-11、SJ2-1、SJ2-4、SJ2-8、SJ3-5、SJ4-2、SJ4-3、SJ4-5共13株代表菌株测定了16S rDNA序列,进而构建了系统发育树关系。结果表明,13个代表菌株分布于6个系统发育分支,分别为假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、鞘氨醇杆菌属(Sphingobacterium)、溶杆菌属(Lysobacter)。
5.对土壤DNA的PCR-DGGE分析结果表明,患病生姜根际土壤和正常生姜根际土壤细菌聚类在一起,且细菌种群相似性较高,达到70%左右,显示它们之间具有比较相似的种群结构。
在真菌种群聚类中,患病生姜根际土壤与正常生姜根际土壤聚在一起,患病生姜非根际土壤和正常生姜非根际土壤聚类在一起,各自的相似性不高,说明生姜连作导致了土壤真菌种类改变。
In this study, we collected four kinds of ginger continuous cropping soil sample, including sick ginger rhizosphere soil (SGRS) and sick ginger non-rhizospheric soil (SGNRS), normal ginger rhizospheric soil (NGRS) and normal ginger non-rhizophere soil(NGNRS) from Zhagu town in Qianwei county, Sichuan Province, and determined the microbial flora quantity by pour plate method, and then measeured the soil catalase activity, urease activity, neutral phosphatase activity, and soil microbial biomass C, N. By using pour plated method, 38 bacterial strains were isolated, and 16S rDNA PCR-RFLP,16S rRNA gene sequence analysis was used to do their genetic diversity and phylogeny. Finally, PCR-DGGE was performed to reveal the soil microbial diversity. The results were as follows:
1. The quantity of soil bacteria in SGR increased, but the number of actinomycetes and fungi decreased significantly; however, the quantity of actinomyces in SGNR increased markedly, the fungus and bacteria number did not change obviously.
2. In SGRS, the soil neutral phosphatase activity and urease activity decreased, but catalase activity increased, which suggested that, the plant root exudates inhibited urease activity, neutral phosphatase activity, whereas catalase was then promoted because of continuous cropping.
3. Analysis of 16S rDNA PCR-RFLP revealed that, at the level of 75% similarity,38 bacteria were divided into 6 genetic groups. The results also showed that the difference and diversity was existed among these strains.
4. Based on the results above,13 representative bacterial strains were selected to do 16S rDNA sequence analysis, and the phylogenetic tree was constructed. The results showed that 13 representative strains distributed into six phylogenic branches, including genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Sphingobacterium and Lysobacter.
5. The results of soil fungus PCR-DGGE analysis suggested that, though the fungus flora between SGNRS and NGNRS was closer than that in SGRS and NGRS, the similarity was not high, which revealed that ginger continuous cropping led to the variation of soil microbial population.
引文
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