毛乌素沙地土壤生物结皮细菌多样性分析与拮抗菌株5A5-3的筛选鉴定
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摘要
本论文以毛乌素沙地土壤生物结皮为研究对象,采用稀释平板法统计了土壤生物结皮及结皮下土壤层各样品的细菌生物量;根据平板菌落形态差异分离菌株,通过测定分离菌株的16S rDNA序列,进行细菌种属鉴定和种群多样性分析。以分离到的细菌菌株为研究对象,通过对峙试验筛选得到对兰花根腐病病原菌有较强拮抗作用的细菌5A5-3,并对5A5-3进行了生理生化鉴定和16S rDNA序列分析。研究所得的主要结果如下:
可培养细菌分离生物量数量级为105,各取样点结皮层样品细菌生物量略高于土壤层,各方向随距离变化所采集样品分离到的细菌种类数和生物量变化没有相一致的趋势。生物结皮样品分离到的细菌菌落表型丰富,共获得190株菌落表型差异的细菌,结皮下土壤层样品得到表型差异的细菌84株。
获得的274株细菌的16S rDNA有效序列测序后进行BLAST比对分析,除5株与已报道的菌株序列相似性低于97%,其余均大于等于97%。根据序列相似性分析,生物结皮样品中测序的186株细菌划分在60个分类单元,而土壤样品中测序的83株细菌划分在29个分类单元。生物结皮样品的物种丰富度(S),Shannon-Wiener多样性指数(H)和均匀度指数(D)分别为22.25±2.06,2.7016±0.0567和0.9081±0.0189,土壤样品的物种丰富度(S),Shannon-Wiener多样性指数(H)和均匀度指数(E)分别为13.00±2.45,2.3093±0.2037和0.8329±0.0735,其中生物结皮物种丰富度(S),多样性指数(H)和均匀度指数(E)分别都明显优于土壤样品,而优势度指数(D)显示土壤样品高于结皮样品。
细菌种群多样性分析表明,生物结皮样品和土壤样品分离到的细菌类群分布在厚壁菌门(Fimicutes),放线菌门(Actinobacteria),变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。芽孢杆菌属(Bacillus)为所研究样品的优势菌属,在结皮和土壤样品中的分离频率分别为86.84%和56.41%。四个主风向不同距离采集的样品多样性显示各采样点细菌多样性分布差异明显。
从生物结皮分离的菌株,有4株与数据库里已报道的细菌相似性低于97%,土壤样品中只分离出1株相似性低于97%的细菌,证明生物结皮有优于土壤发现新型菌的潜力。并从分离到的274株细菌菌株中筛选到1株对兰花根腐病病原菌尖孢镰刀菌(Fusarium oxysporium)和串珠镰孢菌(Fusarium moniliforme)均有强拮抗作用的菌株5A5-3,通过生理生化试验和16S rDNA序列分析鉴定为Bacillus velezensis。
In order to understand the soil bacterial diversity of biological soil crusts of Mu Us Desert in Inner Mongolia Erdos Wushenqi areas, 54 of biological soil crusts and soil samples were studied. The quantity of bacterium were mesured by plate culture; Isolated strains with different morphological characteristic of colonies; Also, the phylogenetic diversity of generalized bacteria in biological soil crusts and soil was analyzed by 16S rDNA sequences of isolates. A strain named 5A5-3 with a rather higher antagonistic activity against pathogens had inhibition zone diameter than 15 mm was screened via preliminary sereening from bacterias isolated from 54 samples The strain 5A5-3 was further characterized by morphological and culture observation, physiological and biochemical experiments and 16S rDNA sequence analysis. The test results were as follows:
The quantity of culturable bacteria biomass was in the order of 105.Each biomass of bacteria in BSCs sample was higher than in the soil layer. Changes of biomass in different distances of four directions did not have a consistent trend. The diversity of the morphological characteristic of colonies was more relatively rich in BSCs samples than soil samples. 190 of bacterias with different morphological characteristic of colonies were isolated from BSCs samples, and 84 from soil samples.
When 16S rDNA sequences of 274 isolates were aligned the closest relatives in the GenBank database with BLAST, the similarity of 269 isolates were higher than 97%, and 5 isolates were lower than 97%. The strains were clustered into operational taxonomic units (OTUs) at a level of sequence similarity of≥97%, OTUs formed by strains from BSCs samples and soil samples respectively were: 60 and 29. While Species richness (S), Shannon–Wiener index(H) and Evenness index(E) of bacteria in BSCs samples respectively were: 22.25±2.06,2.7016±0.0567和0.9081±0.0189; and which of soil samples respectively were: 13.00±2.45,2.3093±0.2037 and 0.8329±0.0735. Bacteria diversity index of Species richness (S), Diversity index (H) and Evenness index (E) in BSCs samples were significantly higher than the soil samples, while the Dominance index(D) of the soil samples was higher than BSCs samples.
Phylogenetic analysis based on the partial 16S rDNA sequences indicated that isolates were mostly clustered into Fimicutes, Proteobacteria, Actinobacteria and Bacteroidetes. Bacillus was the dominant kind of bacteria, the separation frequency of which in BSCs samples and soil samples were 86.84% and 56.41%. Analysised by genus level, bacterial diversity in different samples showed high distinctive.
The similarity of 4 strains from BSCs samples were lower than 97%,while only 1 isolates from soil samples. BSCs was more potentially to discover new bacterial species. A strain named 5A5-3 with a rather higher antagonistic activity against Fusarium oxysporium and Fusarium moniliforme respectively. Based on the results of physiological and biochemical experiments and 16S rDNA sequence analysis, the strain was initially identified as Bacillus velezensis.
可培养细菌分离生物量数量级为105,各取样点结皮层样品细菌生物量略高于土壤层,各方向随距离变化所采集样品分离到的细菌种类数和生物量变化没有相一致的趋势。生物结皮样品分离到的细菌菌落表型丰富,共获得190株菌落表型差异的细菌,结皮下土壤层样品得到表型差异的细菌84株。
获得的274株细菌的16S rDNA有效序列测序后进行BLAST比对分析,除5株与已报道的菌株序列相似性低于97%,其余均大于等于97%。根据序列相似性分析,生物结皮样品中测序的186株细菌划分在60个分类单元,而土壤样品中测序的83株细菌划分在29个分类单元。生物结皮样品的物种丰富度(S),Shannon-Wiener多样性指数(H)和均匀度指数(D)分别为22.25±2.06,2.7016±0.0567和0.9081±0.0189,土壤样品的物种丰富度(S),Shannon-Wiener多样性指数(H)和均匀度指数(E)分别为13.00±2.45,2.3093±0.2037和0.8329±0.0735,其中生物结皮物种丰富度(S),多样性指数(H)和均匀度指数(E)分别都明显优于土壤样品,而优势度指数(D)显示土壤样品高于结皮样品。
细菌种群多样性分析表明,生物结皮样品和土壤样品分离到的细菌类群分布在厚壁菌门(Fimicutes),放线菌门(Actinobacteria),变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。芽孢杆菌属(Bacillus)为所研究样品的优势菌属,在结皮和土壤样品中的分离频率分别为86.84%和56.41%。四个主风向不同距离采集的样品多样性显示各采样点细菌多样性分布差异明显。
从生物结皮分离的菌株,有4株与数据库里已报道的细菌相似性低于97%,土壤样品中只分离出1株相似性低于97%的细菌,证明生物结皮有优于土壤发现新型菌的潜力。并从分离到的274株细菌菌株中筛选到1株对兰花根腐病病原菌尖孢镰刀菌(Fusarium oxysporium)和串珠镰孢菌(Fusarium moniliforme)均有强拮抗作用的菌株5A5-3,通过生理生化试验和16S rDNA序列分析鉴定为Bacillus velezensis。
In order to understand the soil bacterial diversity of biological soil crusts of Mu Us Desert in Inner Mongolia Erdos Wushenqi areas, 54 of biological soil crusts and soil samples were studied. The quantity of bacterium were mesured by plate culture; Isolated strains with different morphological characteristic of colonies; Also, the phylogenetic diversity of generalized bacteria in biological soil crusts and soil was analyzed by 16S rDNA sequences of isolates. A strain named 5A5-3 with a rather higher antagonistic activity against pathogens had inhibition zone diameter than 15 mm was screened via preliminary sereening from bacterias isolated from 54 samples The strain 5A5-3 was further characterized by morphological and culture observation, physiological and biochemical experiments and 16S rDNA sequence analysis. The test results were as follows:
The quantity of culturable bacteria biomass was in the order of 105.Each biomass of bacteria in BSCs sample was higher than in the soil layer. Changes of biomass in different distances of four directions did not have a consistent trend. The diversity of the morphological characteristic of colonies was more relatively rich in BSCs samples than soil samples. 190 of bacterias with different morphological characteristic of colonies were isolated from BSCs samples, and 84 from soil samples.
When 16S rDNA sequences of 274 isolates were aligned the closest relatives in the GenBank database with BLAST, the similarity of 269 isolates were higher than 97%, and 5 isolates were lower than 97%. The strains were clustered into operational taxonomic units (OTUs) at a level of sequence similarity of≥97%, OTUs formed by strains from BSCs samples and soil samples respectively were: 60 and 29. While Species richness (S), Shannon–Wiener index(H) and Evenness index(E) of bacteria in BSCs samples respectively were: 22.25±2.06,2.7016±0.0567和0.9081±0.0189; and which of soil samples respectively were: 13.00±2.45,2.3093±0.2037 and 0.8329±0.0735. Bacteria diversity index of Species richness (S), Diversity index (H) and Evenness index (E) in BSCs samples were significantly higher than the soil samples, while the Dominance index(D) of the soil samples was higher than BSCs samples.
Phylogenetic analysis based on the partial 16S rDNA sequences indicated that isolates were mostly clustered into Fimicutes, Proteobacteria, Actinobacteria and Bacteroidetes. Bacillus was the dominant kind of bacteria, the separation frequency of which in BSCs samples and soil samples were 86.84% and 56.41%. Analysised by genus level, bacterial diversity in different samples showed high distinctive.
The similarity of 4 strains from BSCs samples were lower than 97%,while only 1 isolates from soil samples. BSCs was more potentially to discover new bacterial species. A strain named 5A5-3 with a rather higher antagonistic activity against Fusarium oxysporium and Fusarium moniliforme respectively. Based on the results of physiological and biochemical experiments and 16S rDNA sequence analysis, the strain was initially identified as Bacillus velezensis.
引文
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