Microbial Diversity of Bacillus Community in Soils at Cangshan, Yunnan
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摘要:
目的 了解芽胞杆菌资源多样性，为芽胞杆菌功能资源挖掘和菌剂开发奠定基础。 方法 从云南苍山采集土壤10份，涂布法分离芽胞杆菌，采用16S rRNA基因同源性进行鉴定。 结果 分离获得了75株芽胞杆菌，5个属、19个种，分别属于芽胞杆菌属Bacillus的14个种、类芽胞杆菌属Paenibacillus的2个种、赖氨酸芽胞杆菌属Lysinibacillus的1个种、嗜冷芽胞杆菌属Psychrobacillus的1个种和短芽胞杆菌属Brevibacillus的1个种。根据分离频度分析得知，云南苍山土壤中的芽胞杆菌优势菌群为韦氏芽胞杆菌B.weihenstephanensis、图瓦永芽胞杆菌B.toyonensis、假蕈状芽胞杆菌B.pseudomycoides和解木糖赖氨酸芽胞杆菌Lysinibacillusxylanilyticus，其他种类分布极其不均匀。苍山路边土壤中的芽胞杆菌的Margalef、Simpson、Shannon、Pielou、Brillouin和Macintosh指数较高，可能与其不受植物影响相关。韦氏芽胞杆菌、图瓦永芽胞杆菌和解木糖赖氨酸芽胞杆菌等3个种在3个生境的土壤中均有分离到，Margalef、Simpson、Shannon、Pielou、Brillouin和Macintosh指数较高，与生境相关性小。根据数量和分离频度对芽胞杆菌种类进行聚类，19个种的芽胞杆菌种类可分为4类。 结论 苍山土壤中蕴藏着丰富芽胞杆菌种类和数量，多样性高，具有很大的开发潜力。 Abstract:Objective To search for new resources as well as information gathering, a survey on the distribution and diversity and a sample collection of Bacillus-like species was conducted in Cangshan, Yunnan. Method Bacillus-like bacteria were isolated from the soil samples and preliminarily identified based on their 16S rRNA gene sequences. Result A total of 75 isolates were obtained from 10 soil samples and identified as 19 species belonging to 5 genera within family Bacillaceae. They included 14 Bacillus species, two Paenibacillus species, one Lysinibacillus species, one Psychrobacillus species and one Brevibacillus species. According to an isolation frequency analysis, the dominant bacteria in the region were B. weihenstephanensis, B. toyonensis, B. pseudomycoides and L. xylanilyticus. The remainders were extremely unevenly distributed among the samples. The high Margalef, Simpson, Shannon, Pielou's, Brillouin and Macintosh indices found in the Bacillus from the roadside soils might indicate that the effect by the plants was not significant. B. weihenstephanensis, B. toyonensis and L. xylanilyticus were isolated from all three sites and had high Margalef, Simpson, Shannon, Pielou, Brillouin and Macintosh indices, but no significant correlation with the sites. Based on the average quantification and frequency cluster analysis, these 19 Bacillus-like species could be divided into 4 groups. Conclusion Cangshan area appeared to have Bacillus-like bacteria in large number with great diversity for further exploitation. -
Key words:
- Bacillus /
- Cangshan, Yunnan /
- 16S rRNA /
- diversity /
- isolating frequency
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图 2 基于16S rRNA基因序列的云南苍山芽胞杆菌系统发育树
注：数字表示各节点的自展支持率数值(> 50%)，圈内的菌株为疑似新种菌株。
Figure 2. 16S rRNA-based phylogenetic tree of Bacillus-like bacteria isolated from soil at Cangshan using neighbor-joining method
Note:Bootstrap values above 50% are shown at the branching points, the strains in the circle suspected as new species resource.
图 3 不同生境中芽胞杆菌与数量的关系
Figure 3. Correlation between species and averaged microbial counts (103 cfu·g-1) of Bacillus-like bacteria isolated from various sites
图 4 芽胞杆菌在不同生境中的数量
Figure 4. Averaged microbial counts (103 cfu·g-1) of Bacillus-like bacteria at various sites
图 5 芽胞杆菌种类分布的聚类分析
Figure 5. Clustering analysis on distribution of Bacillus-like bacteria
表 1 从云南苍山分离得到的芽胞杆菌的种类概况
Table 1. Bacillus-like bacteria isolated from soil at Cangshan, Yunnan
分离来源
Detached source类群
Group代表菌株
Strains登录号
GenBank accession no.最相近菌种
Closest match相似性
Sequence identity/%路边土
The roadside soil芽胞杆菌属Bacillus FJAT-29962 MG905834 大猩猩芽胞杆菌Bacillus massiliogorillae(*) 99.58 FJAT-29971 MG905843 图瓦永芽胞杆菌B.toyonensis 99.93 FJAT-29972 MG905844 土壤芽胞杆菌B.soli(#) 97.82 FJAT-29957 MG910490 蜡样芽胞杆菌B.cereus 99.93 FJAT-29963 MG905835 简单芽胞杆菌B.simplex 99.50 FJAT-29965 MG905837 枯草芽胞杆菌B.subtilis 99.93 FJAT-29960 MG905832 韦氏芽胞杆菌B.weihenstephanensis 99.93 FJAT-29961 MG905833 韦氏芽胞杆菌B.weihenstephanensis 99.93 FJAT-29966 MG905838 韦氏芽胞杆菌B.weihenstephanensis 99.79 FJAT-29967 MG905839 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29969 MG905841 韦氏芽胞杆菌B.weihenstephanensis 99.79 FJAT-29970 MG905842 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29973 MG905845 韦氏芽胞杆菌B.weihenstephanensis 100.00 短芽胞杆菌属Brevibacillus FJAT-29958 MG905752 波茨坦短芽胞杆菌Brevibacillusborstelensis 99.85 赖氨酸芽胞杆菌属Lysinibacillus FJAT-29959 MG905754 解木糖赖氨酸芽胞杆菌Lysinibacillusxylanilyticus 99.79 FJAT-29968 MG905840 解木糖赖氨酸芽胞杆菌L.xylanilyticus 99.93 嗜冷芽胞杆菌属Psychrobacillus FJAT-29964 MG905836 忍冷嗜冷芽胞杆菌Psychrobacilluspsychrodurans 99.73 类芽胞杆菌属Paenibacillus FJAT-29974 MG905846 异味类芽胞杆菌Paenibacillusodorifer 98.56 荨麻根际土
The root soil of nettle芽胞杆菌属Bacillus FJAT-29976 MG905848 维德曼芽胞杆菌B.wiedmannii 98.90 FJAT-40013 MG905885 路西法芽胞杆菌B.luciferensis 98.73 FJAT-29975 MG905847 图瓦永芽胞杆菌B.toyonensis 99.79 FJAT-29989 MG905861 图瓦永芽胞杆菌B.toyonensis 99.93 FJAT-29993 MG905865 图瓦永芽胞杆菌B.toyonensis 99.93 FJAT-29995 MG905867 图瓦永芽胞杆菌B.toyonensis 98.60 FJAT-30000 MG905872 图瓦永芽胞杆菌B.toyonensis 99.86 FJAT-40017 MG905889 图瓦永芽胞杆菌B.toyonensis 99.86 FJAT-40020 MG905892 图瓦永芽胞杆菌B.toyonensis 100.00 FJAT-29990 MG905862 蜡样芽胞杆菌B.cereus 99.93 FJAT-29998 MG905870 苏云金芽胞杆菌B.thuringiensis 99.93 FJAT-29977 MG905849 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29978 MG905850 韦氏芽胞杆菌B.weihenstephanensis 99.79 FJAT-29980 MG905852 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29981 MG905853 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29988 MG905860 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29991 MG905863 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29994 MG905866 韦氏芽胞杆菌B.weihenstephanensis 99.86 FJAT-40016 MG905888 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-40019 MG905891 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29999 MG905871 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-40001 MG905873 韦氏芽胞杆菌B.weihenstephanensis 99.86 FJAT-29996 MG905868 假蕈状芽胞杆菌B.pseudomycoides 100.00 FJAT-29997 MG905869 假蕈状芽胞杆菌B.pseudomycoides 100.00 FJAT-40012 MG905884 假蕈状芽胞杆菌B.pseudomycoides 99.93 FJAT-40014 MG905886 特基拉芽胞杆菌B.tequilensis 99.93 FJAT-40021 MG905893 特基拉芽胞杆菌B.tequilensis 99.86 FJAT-40022 MG905894 特基拉芽胞杆菌B.tequilensis 99.93 FJAT-40015 MG905887 暹罗芽胞杆菌B.siamensis 99.93 赖氨酸芽胞杆菌属Lysinibacillus FJAT-29979 MG905851 解木糖赖氨酸芽胞杆菌L.xylanilyticus 100 FJAT-29992 MG905892 解木糖赖氨酸芽胞杆菌L.xylanilyticus 99.85 FJAT-40018 MG905890 解木糖赖氨酸芽胞杆菌L.xylanilyticus 99.93 松树根际土
The root soil of pine芽胞杆菌属Bacillus FJAT-29986 MG905858 路西法芽胞杆菌B.luciferensis 98.66 FJAT-29985 MG905857 图瓦永芽胞杆菌B.toyonensis 99.93 FJAT-40007 MG905879 图瓦永芽胞杆菌B.toyonensis 99.93 FJAT-40008 MG905880 维德曼芽胞杆菌B.wiedmannii 99.93 FJAT-40030 MG905902 维德曼芽胞杆菌B.wiedmannii 99.65 FJAT-40004 MG905876 阿氏芽胞杆菌B.aryabhattai 99.93 FJAT-40005 MG905877 阿氏芽胞杆菌B.aryabhattai 99.93 FJAT-40010 MG905882 阿氏芽胞杆菌B.aryabhattai 99.93 FJAT-40011 MG905883 阿氏芽胞杆菌B.aryabhattai 100.00 FJAT-40024 MG905896 阿氏芽胞杆菌B.aryabhattai 99.86 FJAT-40026 MG905898 阿氏芽胞杆菌B.aryabhattai 99.93 FJAT-40027 MG905899 阿氏芽胞杆菌B.aryabhattai 99.86 FJAT-40029 MG905901 阿氏芽胞杆菌B.aryabhattai 100.00 FJAT-40023 MG905895 特基拉芽胞杆菌B.tequilensis 99.93 FJAT-40002 MG905874 假蕈状芽胞杆菌B.pseudomycoides 100.00 FJAT-40028 MG905900 假蕈状芽胞杆菌B.pseudomycoides 100.00 FJAT-29982 MG905854 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29983 MG905855 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29984 MG905856 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-29987 MG905859 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-40003 MG905875 韦氏芽胞杆菌B.weihenstephanensis 99.72 FJAT-40025 MG905897 韦氏芽胞杆菌B.weihenstephanensis 100.00 FJAT-40031 MG905903 韦氏芽胞杆菌B.weihenstephanensis 100.00 赖氨酸芽胞杆菌属Lysinibacillus FJAT-40006 MG905878 解木糖赖氨酸芽胞杆菌L.xylanilyticus 99.85 类芽胞杆菌属Paenibacillus FJAT-40009 MG905881 解几丁质类芽胞杆菌P.chitinolyticus 99.44 注：*中国新纪录种；#新种资源。
Note:*New chinese record; # New species resource.
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表 2 云南苍山芽胞杆菌的分离频度和平均数量
Table 2. Frequencies and averaged quantities of Bacillus-like bacteria isolated from sites at Cangshan
芽胞杆菌种类
Bacillus-like species路边土
The roadside soil荨麻根际土
The root soil of nettle松树根际土
The root soil of pine总体
Total分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)Bacillus cereus 33.33 0.70 25.00 310.00 0.00 0.00 20.00 310.70 B.weihenstephanensis 100.00 1.20 100.00 546.00 100.00 403.80 100.00 951.00 B.massiliogorillae 33.33 0.30 0.00 0.00 0.00 0.00 10.00 0.30 B.simplex 33.33 0.10 0.00 0.00 0.00 0.00 10.00 0.10 B.subtilis 33.33 0.10 0.00 0.00 0.00 0.00 10.00 0.10 B.toyonensis 33.33 0.20 100.00 165.00 66.66 90.30 70.00 255.50 B.soli 33.33 0.20 0.00 0.00 0.00 0.00 10.00 0.20 B.tequilensis 0.00 0.00 25.00 60.00 33.33 0.10 20.00 60.10 B.pseudomycoides 0.00 0.00 75.00 225.00 66.66 7.10 50.00 232.10 B.wiedmannii 0.00 0.00 25.00 15.00 66.66 2.10 30.00 17.10 B.thuringiensis 0.00 0.00 25.00 38.00 0.00 0.00 10.00 38.00 B.luciferensis 0.00 0.00 25.00 30.00 33.33 60.00 20.00 90.00 B.siamensis 0.00 0.00 25.00 10.00 0.00 0.00 10.00 10.00 B.aryabhattai 0.00 0.00 0.00 0.00 66.66 4.40 20.00 4.40 Paenibacillus chitinolyticus 0.00 0.00 0.00 0.00 33.33 0.10 10.00 0.10 P.odorifer 33.33 0.10 0.00 0.00 0.00 0.00 10.00 0.10 Psychrobacillus psychrodurans 33.33 0.10 0.00 0.00 0.00 0.00 10.00 0.10 Lysinibacillus xylanilyticus 66.66 0.40 75.00 23.00 33.33 0.10 60.00 23.50 Brevibacillus borstelensis 33.33 0.50 0.00 0.00 0.00 0.00 10.00 0.50 总计Total 24.56 3.90 26.31 1 422 26.31 568 26.32 1993.9
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表 3 基于芽胞杆菌数量的生境分布多样性
Table 3. Diversity of Bacillus habitat by microbial counts
参数
Parameters路边土
The roadside soil荨麻根际土
The root soil of nettle松树根际土
The root soil of pine芽胞杆菌种类数
Number of Bacilli species11 10 9 芽胞杆菌数量
No.of Bacillus/(103cfu·g-1)3.9 1422 568 Margalef 7.348 1.240 1.261 Simpson 1.119 0.769 0.459 Shannon 2.046 1.752 0.890 Pielou 0.853 0.761 0.405 Brillouin 1.160 2.502 1.254 Macintosh 1.196 0.533 0.275
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表 4 基于生境的芽胞杆菌数量分布多样性
Table 4. Diversity of Bacillus count by varied habitats
芽胞杆菌种类
Bacillus-like species含有芽胞杆菌的土壤个数
No.of soil samples数量No./
(103cfu·g-1)Margalef Simpson Shannon Pielou Brillouin Mcintosh L.ylanilyticus 3 23.500 0.634 0.044 0.114 0.103 0.062 0.027 B.weihenstephanensis 3 951.000 0.292 0.491 0.691 0.629 0.989 0.296 B.toyonensis 3 195.500 0.379 0.501 0.698 0.635 0.977 0.314 B.tequilensis 2 60.100 0.244 0.003 0.012 0.018 0.011 0.002 B.pseudomycoides 2 232.100 0.184 0.060 0.137 0.197 0.186 0.032 B.wiedmannii 2 77.100 0.230 0.054 0.125 0.180 0.156 0.030 B.cereus 2 310.700 0.174 0.005 0.016 0.023 0.019 0.002 B.luciferensis 2 90.000 0.222 0.449 0.637 0.918 0.880 0.285
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表 5 芽胞杆菌种类分布
Table 5. Distribution of Bacillus-like species
组别
Group芽胞杆菌种类
Bacillus-like species路边土
The roadside soil荨麻根际土
The root soil of nettle松树根际土
The root soil of pine分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)分离频度
Frequency
/%数量
Quantification/
(103cfu·g-1)第1组The first group
3个样本Three samplesBacillus cereus 33.33 0.70 25.00 310.00 0.00 0.00 B.weihenstephanensis 100.00 1.20 100.00 546.00 100.00 403.80 B.massiliogorillae 33.33 0.30 0.00 0.00 0.00 0.00 平均值Mean value 55.55 0.73 41.67 285.33 33.33 134.60 第2组The second group
5个样本Five samplesB.simplex 33.33 0.10 0.00 0.00 0.00 0.00 B.subtilis 33.33 0.10 0.00 0.00 0.00 0.00 B.soli 33.33 0.20 0.00 0.00 0.00 0.00 Paenibacillus odorifer 33.33 0.10 0.00 0.00 0.00 0.00 Psychrobacillus psychrodurans 33.33 0.10 0.00 0.00 0.00 0.00 平均值Mean value 33.33 0.12 0.00 0.00 0.00 0.00 第3组The third group
3个样本Three samplesB.toyonensis 33.33 0.20 100.00 105.00 66.66 90.30 B.tequilensis 0.00 0.00 25.00 60.00 33.33 0.10 B.pseudomycoides 0.00 0.00 75.00 225.00 66.66 7.10 B.thuringiensis 0.00 0.00 25.00 38.00 0.00 0.00 B.luciferensis 0.00 0.00 25.00 30.00 33.33 60.00 B.siamensis 0.00 0.00 25.00 10.00 0.00 0.00 B.aryabhattai 0.00 0.00 0.00 0.00 66.66 4.40 平均值Mean value 4.76 0.03 39.29 66.86 38.09 23.13 第4组The fourth group
4个样本Four samplesB.wiedmannii 0.00 0.00 50.00 75.00 66.66 2.10 P.chitinolyticus 0.00 0.00 0.00 0.00 33.33 0.10 Lysinibacillus xylanilyticus 66.66 0.40 75.00 23.00 33.33 0.10 Brevibacillus borstelensis 33.33 0.50 0.00 0.00 0.00 0.00 平均值Mean value 25.00 0.22 31.25 24.50 33.33 0.57
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