刘国红, 刘波
, 王阶平, 朱育菁, 陈峥, 车建美, 陈倩倩 福建省农业科学院农业生物资源所, 福建 福州 350003
收稿日期：2018-10-12；修回日期：2018-11-29；网络出版日期：2018-12-14
基金项目：农业部农业微生物资源收集与保藏重点实验室开放基金（MRCP-2016-1）；福建省农科院青年英才计划（YC2015-17）；科技创新团队（STIT2017-1-11）；农业生物基因组项目（A2015-3，A2017-4）
作者简介：刘波，1957年出生，男，博士、研究员，原福建省农科院院长，长期从事微生物系统分类、微生物生物技术与农业生物药物研究。先后主持中德国际合作项目、国家自然科学基金、863项目、国家支撑项目、国家成果转化项目、发改委项目、省科技重大攻关项目等科研课题150余项，建立了“中德生防合作研究实验室”、“国家引进国外智力成果示范推广基地-生物防治技术”、“福建省生物农药工程研究中心”、“福建省科技厅农业生物药物研究平台”、“福建省农业科学院微生物创新团队”，形成了较为完整的农业微生物生物技术与农业生物药物研究的技术体系。在国内外期刊上发表论文200多篇(第一作者或通讯作者).
*通信作者：刘波, Tel/Fax:+86-591-87882571, E-mail:fzliubo@163.com.
摘要：[目的] 为了解云南腾冲小空山火山谷土壤中可培养芽胞杆菌种类分布特征。[方法] 采用可培养手段对小空山火山谷阳坡、谷底和阴坡土壤中的芽胞杆菌进行分离培养，根据16S rRNA基因序列同源性对分离菌株进行鉴定，并分析系统发育地位。利用Canoco 5软件分析采样点芽胞杆菌种类分布特征与土壤样品理化性质的相关性。[结果] 从火山谷土壤样品中共分离获得180株芽胞杆菌，16S rRNA基因测序鉴定结果表明分离菌株隶属于芽胞杆菌纲2个科（芽胞杆菌科和类芽胞杆菌科）、6个属、34个种，其中芽胞杆菌属（Bacillus）11个种，类芽胞杆菌属（Paenibacillus）14个种，短芽胞杆菌属（Brevibacillus）3个种，赖氨酸芽胞杆菌属（Lysinibacillus）4个种，嗜冷芽胞杆菌属（Psychrobacillus）1个种和绿芽胞杆菌属（Viridibacillus）2个种，其中7个菌株与其最近模式菌株16S rRNA相似性低于种的界定阈值（98.65%），为芽胞杆菌潜在新物种。优势属为芽胞杆菌属和类芽胞杆菌属，优势种为蕈状芽胞杆菌（Bacillus mycoides），图瓦永芽胞杆菌（Bacillus toyonensis），蜡状芽胞杆菌（Bacillus cereus），解木糖赖氨酸芽胞杆菌（Lysinibacillus xylanilyticus），蜂房类芽胞杆菌（Paenibacillus alvei）和沙地绿芽胞杆菌（Viridibacillus arenosi）。其中16个种分离自阳坡，29个种分离自阴坡，9个种分离自谷底，三者共同种类为6种。阳坡、谷底和阴坡的芽胞杆菌种群分布Bray-Curtis相似性为62.4%，多样性分析结果表明，Shannon指数（H'）大小次序为阴坡>阳坡>谷底。环境因子分析发现，芽胞杆菌种群分布多样性特征与其土壤的海拔高度、碳氮比和硫含量呈负相关，而和碳源和氮源含量呈正相关。[结论] 从以上结果得出，云南腾冲火山谷有着较为丰富的芽胞杆菌资源，且还存在可分离培养的芽胞杆菌的潜在新物种，为利用火山微生物资源提供了保障。
关键词：可培养芽胞杆菌火山谷物种多样性
Distribution characteristics of Bacillus-like species of Xiaokong Mountain volcanic valley in Tengchong County, Yunnan Province
Guohong Liu, Bo Liu
, Jieping Wang, Yujing Zhu, Zheng Chen, Jianmei Che, Qianqian Chen Agricultural Bio-resources Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, Fujian Province, China
*Corresponding author: Bo Liu, Tel/Fax:+86-591-87882571, E-mail:fzliubo@163.com.
Foundation item: Supported by the Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, China (MRCP-2016-1), by the Youth Science and Technology Talent (YC2015-17), by the Science and Technology Innovation Team Program (STIT2017-1-11) and by the Agrobiogenome Program of Fujian Academy of Agricultural Sciences (A2015-3, A2017-4)
Abstract: [Objective] The study was aimed to determine the distribution of Bacillus-like species in volcanic valley of Xiaokong Montain, Tengchong County, Yunnan Province. [Methods] On the basis of culturable method, we isolated Bacillus-like species from soil samples collected in shady-slope, sunny-slope and valley bottom of Xiaokong Mountain. Then the isolates were identified using the 16S rRNA gene and phylogenetic tree was constructed to explore the relation between isolates and related type species. The correlation between environmental factors and community of Bacillus-like species was analyzed by software Cano 5. [Results] We obtained 180 isolates from the soil samples, and identified them as 2 families, 6 genera, 34 species using 16S rRNA gene sequences, including genus Bacillus (11 species), Paenibacillus (13 species), Brevibacillus (3 species), Lysinibacillus (4 species), Psychrobacillus (1 species) and Viridibacillus (2 species). Among them, 7 strains had the lower 16S rRNA similarities with their closest type strain, below the species threshold valus 98.65%, indicating that they were potential novel Bacillus species. The dominant genera were Bacillus and Paenibaciilus, and dominant species were B. mycoides, B. toyonensis, B. cereus, L. xylanilyticus, P. alvei and V. arenosi. Among the Bacillus-like species, we got 16 species from sunny-slope, 29 species from shady-slope and 10 species from valley bottom. The diversity index was showed that the order of Shannon index was shady-slope > sunny-slope > valley bottom. We also found that the correlation between Bacillus-like species community structure, altitude and carbon source was negative, but with S and nitrogen source was positive. [Conclusion] There were rich Bacillus-like species in volcanic valley, Tengchong County, Yunnan Province, and some novel potential Bacillus-like species.
Keywords: cultural Bacillus-like speciesvolcanic valleydiversity
极端特殊环境微生物具有较重要的研究和开发利用价值，能产生多种特殊的活性产物，如产菊糖酶[1]、植酸酶[2]、抗菌物质[3]，在农业、工业、医学等领域具有良好的研发和应用意义。地球上极端特殊环境主要为沙漠、冰川、海洋、火山等，火山作用被定义为岩浆爆发至地球表面或其他星球之固体表面的现象，在此过程中，熔岩、火山碎屑岩与火山气体通过地面的裂口向外喷发[4-5]。仅少数的文献研究揭示了火山喷气口处土壤中含有丰富的原核生物(细菌和古菌)[6-7]。Benson等报道火山喷气孔是极端微生物的多样性热点，易发现潜在生物新分类单元[8]。Verma等的研究表明安达曼海火山贫瘠岛具有较高的细菌多样性[9]。为了确定火山灰先锋细菌的定殖，Wiit等首次调查了火山灰作为先锋细菌定殖的底物，重点分析了火山灰中细菌的多样性[10]。Kelly等报道冰岛的陆地结晶火山岩中有很多细菌[11]。
芽胞杆菌是一类重要的微生物资源，在自然界分布广泛，极端环境中常有芽胞杆菌的踪迹。芽胞杆菌属(Bacillus)和类芽胞杆菌属(Paenibacillus)种类是常见的石生细菌[12]，具有耐盐、强紫外线和寡营养的特殊能力。中国泥火山中含有高度的细菌多样性，杨娟等利用高通量测序技术分析发现新疆泥火山土壤细菌多样性丰富，含有少量的芽胞杆菌，含量最高的为未分类类群[13]。李智等从新疆独山子泥火山分离获得了2株中度嗜盐的芽胞杆菌[14]。云南省腾冲县位于亚欧板块与印度板块相撞交接地带，是世界上罕见且为典型的火山地热并存区。小空山火山谷是火山喷发形成的山谷，小空山高仅五十来米，但火山口却大得出奇，直径150米有余，且深达70米。植被主要是矮灌木和杂草，阴坡温度低，气候阴凉，长满绿色植被，谷底植被稀疏，主要为杂草，而阳坡阳光充裕，温度高，植被为干枯植物覆盖。这种独特的环境使得小空山火山谷的微生物多样性独特，该环境下的微生物可能会产生多种活性物质，研究火山谷芽胞杆菌多样性为挖掘功能菌株提供了资源保障和基础，也为微生物适应性和群落演化研究提供了宝贵材料和科学理论依据。本研究分别从小空山的阳坡、谷底和阴坡采集了14份土壤样品，分析小空山火山谷土壤中可培养芽胞杆菌多样性，比较不同区域芽胞杆菌种类异同，结合采样点环境参数，研究芽胞杆菌群落结构特征及其与土壤性质的相关性，为丰富芽胞杆菌资源库，促进芽胞杆菌资源的开发利用提供基础。
1 材料和方法 1.1 土壤样品的采集 2015年3月，分别从云南腾冲小空山火山谷小空山的阳坡、谷底和阴坡采集表层0-20 cm土壤样品，置于无菌自封采样袋中。小空山阴坡植被繁茂，阳坡植被干枯，谷底无植被或植被稀疏。采样点信息见表 1。样品采集完后带回实验室立即进行处理。
表 1. 供试土壤样品信息 Table 1. The information of soil samples
| Soil No. | Sampling sites | Latitude and longitude | Habitat | C/(g/kg) | N/% | S/(mg/kg) | Altitude/m |
| TL47 | Sunny slope | 25°13′N, 98°30′E | Dry plant | 80.6 | 0.62 | 3.48 | 1914 |
| TL45 | 25°13′N, 98°30′E | Dry plant | 98.6 | 1.11 | 26.9 | 1905 | |
| TL44 | 25°13′N, 98°30′E | Dry plant | 125.1 | 0.96 | 10.2 | 1891 | |
| TL57 | 25°13′N, 98°30′E | Dry plant | 143.8 | 1.19 | 12.1 | 1876 | |
| TL62 | 25°13′N, 98°30′E | Dry plant | 110.2 | 0.92 | 15.5 | 1833 | |
| TL61 | Bottom | 25°12′N, 98°30′E | No plant | 120.2 | 096 | 5.53 | 1873 |
| TL40 | 25°12′N, 98°30′E | No plant | 102.5 | 0.87 | 16.8 | 1875 | |
| TL43 | Shady slope | 25°12′N, 98°30′E | Green plant | 122 | 0.78 | 6.3 | 1932 |
| TL42 | 25°12′N, 98°30′E | Green plant | 27.8 | 0.23 | 37.3 | 1925 | |
| TL37 | 25°12′N, 98°30′E | Green plant | 22 | 0.17 | 2.82 | 1924 | |
| TL63 | 25°12′N, 98°30′E | Green plant | 82.4 | 0.65 | 3.13 | 1912 | |
| TL59 | 25°12′N, 98°30′E | Green plant | 156.4 | 1.11 | 18.7 | 1906 | |
| TL58 | 25°12′N, 98°30′E | Green plant | 170.4 | 1.33 | 13.8 | 1896 | |
| TL60 | 25°12′N, 98°30′E | Green plant | 156.9 | 1.22 | 21.9 | 1888 | |
| T, the initial of Tengchong; L, the initial of the first name of my supervisor. | |||||||
表选项
1.2 芽胞杆菌的分离、纯化与保存 采用温度梯度法和稀释涂布法进行芽胞杆菌的分离，具体参考刘国红等[15]描述的方法。简单来说，称取10 g土壤样品制备土壤悬浮液，80 ℃水浴10 min，吸取0.2 mL悬浮液涂布于LB培养基平板上，30 ℃培养2 d。观察并拍照记录菌株的菌落大小、形状、颜色、光泽、透明度、湿润度、边缘等特征。根据菌落特征挑取单菌落，采用连续划线法进行纯化，获得的单菌落采用20%甘油冷冻法保藏于-80 ℃超低温冰箱。
1.3 DNA提取、16S rRNA基因PCR扩增 芽胞杆菌基因组DNA提取主要是采用苯酚-氯仿法进行提取，具体参照Cheng和Ning[16]描述的方法。通过细菌16S rRNA通用引物27F (5′-AG AGTTTGATCMTGGCTCAG-3′)和1492R(5′-TAC GGYTACCTTGTTACGACTT-3′)对分离菌株DNA进行扩增。PCR反应条件：95 ℃ 5 min，95 ℃ 30 s，55 ℃ 45 s，72 ℃ 1.5 min，35个循环，72 ℃ 10 min。PCR产物以1.5%琼脂糖凝胶电泳检测，检测有条带的菌株PCR产物送至铂尚生物技术有限公司进行测序。测序的序列提交至NCBI的GenBank数据库并获得序列号。
1.4 基于16S rRNA基因的系统发育树构建 将芽胞杆菌菌株的16S rRNA基因序列上传至EzBioCloud (https://www.ezbiocloud.net/)[17]和NCBI数据库与已知模式菌株的16S rRNA序列进行比对，初步判断分离菌株的分类地位。利用MEGA 7.0软件进行构建系统发育进化树[18]，采用Neighbor- Joining方法推断亲缘关系[19]，进行1000次重复验证[20]，进化距离借助p-distance法进行计算[21]。
1.5 芽胞杆菌菌落结构多样性分析 将两种菌之间的16S rRNA基因序列相似性98.65%作为种的界定阈值[22]。通过PRIMER 5.0软件，采用Shannon多样性指数、丰富度指数、均匀度指数和优势度指数进行芽胞杆菌多样性分析和比较。
1.6 芽胞杆菌分布特征与环境因子相关性分析 采用Canoco5.0分析土壤中芽胞杆菌种群结构与土壤样品性质的相关性。土壤样品的碳、氮及硫含量是由有资质的检测机构福建省农业科学院土壤肥料研究所测试技术中心测定的，检测依据：有效硫按NY/T1121.14-2006方法，全氮按NY/T53-1987方法，有机碳按NY/T1121.6-2006方法测定。
2 结果和分析 2.1 芽胞杆菌的分离与鉴定 从LB培养基平板上，根据菌落形态特征差异，从14份土壤样品中共分离获得芽胞杆菌180株，其中从阳坡分离获得64株，谷底获得24株，从阴坡获得101株，数量最多。分离菌株纯化后采用-80 ℃甘油冷冻法进行保藏。
基于芽胞杆菌的16S rRNA基因鉴定，16S rRNA基因相似性分析结果表明大部分芽胞杆菌菌株与其近缘菌株的相似性为98.7%-100%。181株芽胞杆菌分属于2个科、6个属、35个种；其中芽胞杆菌属(Bacillus) 85株、11个种，类芽胞杆菌属(Paenibacillus) 23株、13个种，短芽胞杆菌属(Brevibacillus) 6株、3个种，赖氨酸芽胞杆菌属(Lysinibacillus) 52株、4个种，嗜冷芽胞杆菌属(Psychrobacillus) 5株、1个种和绿芽胞杆菌属(Viridibacillus) 9株、2个种。根据16S rRNA分析结果，每份土样中去除合并相似性完全相同的菌株，见表 2。
表 2. 基于16S rRNA基因的芽胞杆菌种类鉴定 Table 2. Isolation and identification of Bacillus-like species based on 16S rRNA gene sequence analysis
| Strain No. | Closed match | 16S rRNA similarity/% | Accession No. | Location |
| FJAT-45841 | Bacillus aryabhattai B8W22T | 99.9 | KY038659 | Sunny slope |
| FJAT-45847 | Bacillus cereus ATCC 14579T | 100.0 | KY038660 | Sunny slope |
| FJAT-45974 | Bacillus methylotrophicus KACC 13105T | 100.0 | KY038669 | Sunny slope |
| FJAT-45973 | Bacillus mycoides DSM 2048T | 100.0 | KY038670 | Sunny slope |
| FJAT-45836 | Bacillus simplex NBRC 15720T | 100.0 | KY038656 | Sunny slope |
| FJAT-45827 | Bacillus toyonensis BCT-7112T | 100.0 | KY038657 | Sunny slope |
| FJAT-45977 | Brevibacillus laterosporus DSM 25T | 99.6 | KY038673 | Sunny slope |
| FJAT-45899 | Lysinibacillus contaminans FSt3AT | 99.3 | KY038679 | Sunny slope |
| FJAT-45840 | Lysinibacillus parviboronicapiens BAM-582T | 99.2 | KY038664 | Sunny slope |
| FJAT-45830 | Lysinibacillus xylanilyticus XDB9T | 100.0 | KY038658 | Sunny slope |
| FJAT-45851 | Paenibacillus alvei DSM 29T | 99.1 | KY038666 | Sunny slope |
| FJAT-45898 | Paenibacillus assamensis GPTSA 11T | 98.1 | KY038753 | Sunny slope |
| FJAT-45829 | Paenibacillus odorifer DSM 15391T | 98.3 | KY038752 | Sunny slope |
| FJAT-45846 | Paenibacillus selenitireducens ES3-24T | 98.9 | KY038667 | Sunny slope |
| FJAT-45903 | Paenibacillus vulneris CCUG 5327T | 96.7 | KY038754 | Sunny slope |
| FJAT-45902 | Viridibacillus arenosi LMG 22166T | 100.0 | KY038682 | Sunny slope |
| FJAT-46012 | Bacillus cereus ATCC 14579T | 99.9 | KY038686 | Bottom |
| FJAT-46010 | Bacillus mycoides DSM 2048T | 100 | KY038687 | Bottom |
| FJAT-45917 | Bacillus toyonensis BCT-7112T | 100 | KY038688 | Bottom |
| FJAT-46013 | Brevibacillus laterosporus DSM 25T | 97.5 | KY038755 | Bottom |
| FJAT-45925 | Lysinibacillus xylanilyticus XDB9T | 99.9 | KY038697 | Bottom |
| FJAT-46015 | Paenibacillus alvei DSM 29T | 99.1 | KY038690 | Bottom |
| FJAT-45928 | Paenibacillus uliginis N3-975T | 99.6 | KY038698 | Bottom |
| FJAT-46038 | Psychrobacillus psychrodurans DSM 11713T | 98.7 | KY038692 | Bottom |
| FJAT-46009 | Viridibacillus arenosi LMG 22166T | 100.0 | KY038693 | Bottom |
| FJAT-45987 | Bacillus aryabhattai B8W22T | 100.0 | KY038734 | Shady slope |
| FJAT-45863 | Bacillus cereus ATCC 14579T | 99.9 | KY038701 | Shady slope |
| FJAT-45942 | Bacillus isronensis B3W22T | 100.0 | KY038710 | Shady slope |
| FJAT-45861 | Bacillus mycoides DSM 2048T | 100.0 | KY038716 | Shady slope |
| FJAT-45868 | Bacillus niacini IFO 15566T | 97.6 | KY038756 | Shady slope |
| FJAT-45985 | Bacillus patagoniensis PAT 05T | 99.6 | KY038736 | Shady slope |
| FJAT-45956 | Bacillus pseudomycoides DSM 12442T | 99.9 | KY038745 | Shady slope |
| FJAT-45961 | Bacillus simplex NBRC 15720 | 99.9 | KY038746 | Shady slope |
| FJAT-45939 | Bacillus timonensis MM10403188T | 99.2 | KY038711 | Shady slope |
| FJAT-45938 | Bacillus toyonensis BCT-7112T | 100.0 | KY038712 | Shady slope |
| FJAT-46005 | Brevibacillus brevis NBRC 15304T | 99.7 | KY038702 | Shady slope |
| FJAT-45996 | Brevibacillus laterosporus DSM 25T | 99.8 | KY038729 | Shady slope |
| FJAT-45883 | Lysinibacillus contaminans FSt3AT | 99.2 | KY038721 | Shady slope |
| FJAT-46006 | Lysinibacillus fusiformis NBRC 15717T | 100.0 | KY038703 | Shady slope |
| FJAT-45860 | Lysinibacillus parviboronicapiens BAM-582T | 99.6 | KY038717 | Shady slope |
| FJAT-45945 | Lysinibacillus xylanilyticus XDB9T | 100.0 | KY038714 | Shady slope |
| FJAT-45968 | Paenibacillus alvei DSM 29T | 99.1 | KY038750 | Shady slope |
| FJAT-45941 | Paenibacillus amylolyticus NRRL NRS-290T | 98.0 | KY038757 | Shady slope |
| FJAT-46022 | Paenibacillus assamensis GPTSA 11T | 98.1 | MG022441 | Shady slope |
| FJAT-46002 | Paenibacillus chitinolyticus NBRC 15660T | 99.2 | KY038705 | Shady slope |
| FJAT-45891 | Paenibacillus montaniterrae MXC2-2T | 96.4 | KY038758 | Shady slope |
| FJAT-45867 | Paenibacillus pinisoli NB5T | 99.4 | KY038706 | Shady slope |
| FJAT-45884 | Paenibacillus pinisoli NB5T | 98.5 | KY038724 | Shady slope |
| FJAT-46004 | Paenibacillus taichungensis BCRC 17757T | 99.9 | KY038707 | Shady slope |
| FJAT-45876 | Paenibacillus terrigena A35T | 99.0 | KY038732 | Shady slope |
| FJAT-45885 | Paenibacillus uliginis N3-975T | 100.0 | KY038725 | Shady slope |
| FJAT-46008 | Psychrobacillus psychrodurans DSM 11713T | 99.2 | KY038708 | Shady slope |
| FJAT-45864 | Viridibacillus arenosi LMG 22166T | 100.0 | KY038709 | Shady slope |
| FJAT-45874 | Viridibacillus arvi LMG 22165T | 100.0 | KY038733 | Shady slope |
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2.2 芽胞杆菌的系统发育分析 选取34株代表性芽胞杆菌菌株，构建小孔山火山谷土壤中芽胞杆菌系统发育树(图 1)，其中7株与其近缘种间的亲缘关系较远，与已知模式菌株的16S rRNA基因序列相似性较低。菌株FJAT-45903与其最相近模式种伤口类芽胞杆菌Paenibacillus vulneris CCUG 5327T的16S rRNA基因相似性为96.7%，FJAT-45868与烟酸芽胞杆菌Bacillus niacini IFO 15566T的16S rRNA基因相似性为97.6%，FJAT-46013与侧胞短芽胞杆菌Brevibacillus laterosporus DSM 25T的为97.5%，FJAT-45898与阿萨姆类芽胞杆菌Paenibacillus assamensis GPTSA 11T的为98.1%，FJAT-45891与山土类芽胞杆菌Paenibacillus montaniterrae MXC2-2T的为96.4%，FJAT-45829与载味类芽胞杆菌Paenibacillus odorifer DSM 15391T的为98.3%，FJAT-45884与针叶林土类芽胞杆菌Paenibacillus pinisoli NB5T的为98.5%。
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| 图 1 云南小孔山火山谷可培养芽胞杆菌系统发育分析 Figure 1 The phylogenetic tree of cultural Bacillus-like species of Xiaokong Montain volcanic valley. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 0.01 substitutions per site. The content in the bracket was the accession number of 16S rRNA gene sequence of each strain |
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2.3 火山谷芽胞杆菌种类分布
2.3.1 不同地理位置种类分布: 分析结果见图 2。从火山谷土壤中共分离获得了34种芽胞杆菌，其中16个种分离自阳坡，29个种分离自阴坡，9个种分离自火山谷谷底。火山谷阳坡和阴坡土壤样品中皆有的芽胞杆菌种类有12个种，阳坡和谷底皆有的种类为6种，阴坡和谷底皆有8个种，其中6种芽胞杆菌从阳坡、阴坡和谷底皆分离到，为蕈状芽胞杆菌(Bacillus mycoides)、图瓦永芽胞杆菌(Bacillus toyonensis)、蜡状芽胞杆菌(Bacillus cereus)、解木糖赖氨酸芽胞杆菌(Lysinibacillus xylanilyticus)、蜂房芽胞杆菌(Paenibacillus alvei)和沙地绿芽胞杆菌(Viridibacillus arenosi)。
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| 图 2 火山谷芽胞杆菌种类分布 Figure 2 The Venn diagram of the cultivable aerobic Bacillus-like species in the sunny slope, shady slope and valley bottom. The numbers in the overlapping parts represent the shared number of Bacillus-like species in different site |
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2.3.2 阳坡芽胞杆菌种类分布: 从阳坡分离到了63株芽胞杆菌，鉴定为5个属、16个种，芽胞杆菌属(31株，6个种)，短芽胞杆菌属(2株，1个种)，赖氨酸芽胞杆菌属(20株，3个种)，类芽胞杆菌属(8株，5个种)和Viridibacillus (2株，1个种)。芽胞杆菌属由6个种组成，分别为13株菌属于图瓦永芽胞杆菌，7株属于简单芽胞杆菌(Bacillus simplex)，4株蜡状芽胞杆菌，3株阿氏芽胞杆菌(Bacillus aryabhattai)，3株蕈状芽胞杆菌和1株甲基营养型芽胞杆菌(Bacillusmethylotrophicus)。赖氨酸芽胞杆菌属包含3个种，17株解木糖赖氨酸芽胞杆菌，1株污染赖氨酸芽胞杆菌(Lysinibacillus contaminans)，2株含低硼赖氨酸芽胞杆菌(Lysinibacillus parviboronicapiens)。类芽胞杆菌属包含2个种，分别为4株蜂房芽胞杆菌和1株硒还原类芽胞杆菌(Paenibacillus selenitireducens)。此外，还包含2株侧胞短芽胞杆菌和2株沙地绿芽胞杆菌。3株类芽胞杆菌FJAT-45898、FJAT-45829和FJAT-45903与已知种类的分类地位都不同，可能为潜在新种。
2.3.3 阴坡芽胞杆菌种类分布: 从阴坡土壤中分离到93株芽胞杆菌，鉴定为6个属的29个种。阴坡土壤中优势属为芽胞杆菌属，包含11株蕈状芽胞杆菌、12株图瓦永芽胞杆菌、4株蜡状芽胞杆菌、4株简单芽胞杆菌、3株印空研芽胞杆菌(Bacillus isronensis)、2株阿氏芽胞杆菌、1株假蕈状芽胞杆菌(Bacillus pseudomycoides)、3株提蒙类芽胞杆菌(Bacillus timonensis)和1株巴塔哥尼亚芽胞杆菌(Bacillus patagoniensis)。次优势属为赖氨酸芽胞杆菌属，包含13株解木糖赖氨酸芽胞杆菌属、8株含低硼赖氨酸芽胞杆菌、2株污染赖氨酸芽胞杆菌和2株纺锤形赖氨酸芽胞杆菌(Lysinibacillus fusiformis)。类芽胞杆菌属包含2株蜂房类芽胞杆菌、2株解几丁质类芽胞杆菌(Paenibacillus chitinolyticus)、1株针叶林土类芽胞杆菌，1株台中类芽胞杆菌(Paenibacillus taichungensis)，2株潮湿类芽胞杆菌(Paenibacillus uliginis)和2株土地类芽胞杆菌(Paenibacillus terrigena)。短芽胞杆菌属由2株侧胞短芽胞杆菌和1株短短芽胞杆菌(Brevibacillus brevis)组成。阴坡土壤中种类最少的是3株忍冷嗜冷芽胞杆菌(Psychrobacillus psychrodurans)和7株沙地绿芽胞杆菌。此外，芽胞杆菌属菌株FJAT-45868和3株类芽胞杆菌(FJAT- 45941、FJAT-45884和FJAT-45891)为潜在新种。
2.3.4 谷底芽胞杆菌种类分布: 从火山谷底分离获得了9种芽胞杆菌，由24株菌组成。9种芽胞杆菌归于6个属芽胞杆菌属、短芽胞杆菌属、赖氨酸芽胞杆菌属、类芽胞杆菌属、绿芽胞杆菌属和嗜冷芽胞杆菌属。其优势属为芽胞杆菌属，包含9株图瓦永芽胞杆菌、2株蜡状芽胞杆菌和1株蕈状芽胞杆菌，第二优势属为解木糖赖氨酸芽胞杆菌属(7株)。此外，还包含1株蜂房类芽胞杆菌、1株潮湿类芽胞杆菌、2株忍冷嗜冷芽胞杆菌和1个潜在新种(Brevibacillus sp. FJAT-46013)。
2.4 火山谷芽胞杆菌分布多样性分析 以土壤样本为样本，芽胞杆菌种类含量为指标，进行聚类分析。由图 3可知，阳坡、谷底和阴坡土壤中芽胞杆菌种群分布具有62.4%的Bray-Curtis相似性，阳坡和谷底土壤中芽胞杆菌的种群特征相似性为72%，高于与阴坡的。通过PRIMER 5.0的SIMPER分析发现，图瓦永芽胞杆菌和解木糖赖氨酸芽胞杆菌偏好于阳坡和谷底，表明这两个种可能是火山谷普遍存在的芽胞杆菌种类。
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| 图 3 火山谷芽胞杆菌种类组成及相关性 Figure 3 The composition and relation of Bacillus-like species in volcanic valley. The clustering tree on the left was constructed based on the Bray-Curtis similarity of Bacillus-like species compositions which is on the right half of the figure, in which cluster 1 was combination of bottom and sunny and cluster 2 included shady. A: The cluster analysis of soil samples; B: the relative abundance of Bacillus-like species in soil samples. Ba: Bacillus aryabhattai; Bc:Bacillus cereus; Bi:Bacillus isronensis; Bm1: Bacillus methylotrophicus; Bm2: Bacillusmycoides; Bn: Bacillus niacini FJAT-45868; Bp1: Bacillus patagoniensis; Bp2: Bacillus pseudomycoides; Bs: Bacillus simplex; Bt1: Bacillus timonensis; Bt2: Bacillus toyonensis; Bb: Brevibacillus brevis; Bl: Brevibacillus laterosporus; Bl1:Brevibacillus laterosporusFJAT-46013; Lc: Lysinibacillus contaminans; Lf: Lysinibacillus fusiformis; Lp: Lysinibacillus parviboronicapiens; Lx: Lysinibacillus xylanilyticus; Pa1: Paenibacillus alvei; Pa2: Paenibacillus amylolyticus; Pa3: Paenibacillus assamensis FJAT-45898; Pc: Paenibacillus chitinolyticus; Pm: Paenibacillus montaniterrae FJAT-45891; Po: Paenibacillus odorifer FJAT-45829; Pp1: Paenibacillus pinisoli; Pp2: Paenibacillus pinisoli FJAT-45884; Ps: Paenibacillus selenitireducens; Pt1: Paenibacillus taichungensis; Pt2: Paenibacillus terrigena; Pu: Paenibacillus uliginis; Pv: Paenibacillus vulneris FJAT-45903; Pp3: Psychrobacillus psychrodurans; Va1: Viridibacillus arenosi; Va2: Viridibacillus arvi |
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换言之，火山谷阳坡、谷底和阴坡芽胞杆菌种类具有37.8%的种类差异。阿氏芽胞杆菌、简单芽胞杆菌、污染赖氨酸芽胞杆菌、侧胞短芽胞杆菌和含低硼赖氨酸芽胞杆菌仅分布在阳坡和阴坡，潮湿类芽胞杆菌和忍冷嗜冷芽胞杆菌分布在阴坡和谷底，甲基营养型芽胞杆菌、硒还原类芽胞杆菌和2个类芽胞杆菌潜在新种FJAT-45903和FJAT-45898仅在阳坡土壤中分离获得，潜在新种Brevibacillus sp. FJAT-46013和Paenibacillus sp. FJAT-45920仅分布在谷底，14种芽胞杆菌只分布在阴坡。阳坡和谷底差异贡献最大的是蜡状芽胞杆菌，阳坡和阴坡差异贡献最大的为蕈状芽胞杆菌，阴坡和谷底的为图瓦永芽胞杆菌。
多样性指数分析结果表明，火山谷阴坡、谷底和阳坡多样性指数Shannon (H′)和丰富度指数具有明显的差异，阴坡土壤中芽胞杆菌的Shannon (H′)指数明显高于阳坡和谷底样品中分离到的芽胞杆菌菌株，而均匀度指数(J′, Pielou’s evenness)差异较小(表 3)。
表 3. 火山谷芽胞杆菌多样性指数 Table 3. The diversity indices in volcanic valley
| Sample | S | N | D (Species richness) | J′ (Pielou’s evenness) | H′ | Simpson |
| Sunny | 16 | 263200 | 1.2019 | 0.74748 | 2.0725 | 0.82877 |
| Bottom | 9 | 276000 | 0.71838 | 0.75651 | 1.7419 | 0.77894 |
| Shady | 29 | 256600 | 2.248 | 0.68781 | 2.6161 | 0.85724 |
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2.5 芽胞杆菌种群分布与环境因子相关性 为了分析环境因子对火山谷土壤芽胞杆菌种群分布特征的影响，利用Canoco 5分析得出，芽胞杆菌种群分布特征与其土壤样品的海拔高度、碳氮比和硫含量与呈负相关，而和碳源和氮源含量呈正相关(图 4)。
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| 图 4 环境因子与芽胞杆菌种群分布的相关性 Figure 4 The correlation between environmental factors and Bacillus-like communication structure |
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3 讨论 16S rRNA基因是原核生物分类地位判断的黄金指标之一，当两株菌之间16S rRNA基因序列相似性低于98.65%时，可认为是两种不同的种[22]。基于可培养方法和16S rRNA基因，本文首次调查了云南小空山火山谷的芽胞杆菌资源，结果表明小空山火山谷含有丰富的芽胞杆菌资源，其中芽胞杆菌属为第一优势属，优势种主要为蕈状芽胞杆菌、图瓦永芽胞杆菌、蜡状芽胞杆菌、解木糖赖氨酸芽胞杆菌、蜂房类芽胞杆菌和沙地绿芽胞杆菌。环境因子对小空山火山谷阴坡、阳坡和谷底芽胞杆菌种群分布特征具有一定影响。此外，小空山火山谷还蕴藏着一定的新资源，本研究发现了潜在7个芽胞杆菌新种资源，其准确分类地位需要进一步分类学特征实验的验证。
我国自然资源丰富多样，形成了各种特殊环境，如盐湖、沙漠、海洋、火山谷等，这些特殊环境孕育了丰富的微生物资源，而芽胞杆菌为上述环境条件下普遍存在且占优势的种群。如，Sahay等[23]从印度喜马拉雅地区分离获得了芽胞杆菌属、短芽胞杆菌属和类芽胞杆菌属等种类，芽胞杆菌属是青海茶卡盐湖的第一优势种群[24]，亦是青海省察尔汗盐湖地区的卤水与湖泥样品中的优势属[25]。Mohammad等[26]从Jordanian热泉分离获得了大量的地衣芽胞杆菌，Piubeli等[27]研究发现阿塔卡马沙漠(Atacama Desert)死谷(Death valley)的优势菌群为产芽胞的大洋芽胞杆菌属和芽胞杆菌属，Liu等[28]从Taklamakan沙漠分离获得了1个芽胞杆菌新种，基于焦磷酸测序，海南八门湾沉积泥中的芽胞杆菌类群主要为9个属，分别为芽胞杆菌属(42%)、类芽胞杆菌属(16%)、Halobacillus (13%)、Alicyclobacillus (11%)、Rummeliibacillus (5%)、Cohnella (5%)、Tumebacillus (4%)、Pontibacillus (3%)和Aneurinibacillus (2%)，其中优势属为芽胞杆菌属[29]。上述研究表明，自然界特殊生境中含有较为丰富的芽胞杆菌资源，芽胞杆菌作为一类具有重要应用价值的微生物资源，研究其种群分布特征具有重要的理论依据和科学意义。
据文献统计，关于火山谷微生物资源研究报道的较少，挖掘筛选火山谷生境的芽胞杆菌资源可为开发活性物质提供重要的菌种资源保障。Kolumb海底火山含有丰富的细菌资源，从中分离获得了丰富的功能芽胞杆菌资源[3]。世界最高火山Ojos del Salado区域的高海拔湖沉积物中含有一定量的细菌种类[30]。厄立特里亚Alid火山区域的5大热泉中微生物资源丰富多样，Garbanabra热泉mat样品中芽胞杆菌属具有较高的丰富度(占36.2%)，在其他样品中芽胞杆菌属比例为0.1%至3.0%[31]。454焦磷酸测序分析发现酸性火山土壤中的主要优势类群为类芽胞杆菌属等[32]，从长有牧草的火山土壤中分离获得了可培养的类芽胞杆菌属和芽胞杆菌属种类[2]。本研究发现，小空山火山谷土壤中含有丰富的芽胞杆菌属和类芽胞杆菌属种类，这两个属的种类在土壤环境中广泛分布，本文研究结果与前人报道研究结果具有一致性。Ge等[33]从长白山休眠火山土壤中分离获得了甲基营养型芽胞杆菌，本研究亦从小空山火山谷土壤中分离到了该种。Rodrigues等[1]从S?o Miguel分离获得了4株具有生物催化剂的芽胞杆菌，其中枯草芽胞杆菌的活性最高，但本研究未分离获得枯草芽胞杆菌类群的种类，与文献报道不一致。墨西哥Paricutín火山喷气孔的细菌多样性较低[34]，多样性指数分析结果表明，本研究中芽胞杆菌的多样性较高，阴坡的多样性指数最高，这可能与地形和采样点具有一定的关联，阴坡植被类型丰富，湿度较高，适合芽胞杆菌的生存，而阳坡和谷底植被单一，土壤相对较干燥，从而造成两者的芽胞杆菌种类分布特征相似性更高。
火山生境中存在一定量的微生物新物种资源，如Lee等[35]从火山岩从分离到1个新物种Pseudokineococcus basanitobsidens sp. nov.，Norman等[36]从火山土壤中分离中度嗜热寡营养的Rubrobacter spartanus sp. nov.，火山洞含有丰富的放线菌资源及大量的新未知物种[37]，Liu等[38]从中国著名火山之乡五大连池分离获得了芽胞杆菌新种Bacillus wudalianchiensis sp. nov。根据Logan等[39]和Tindall等[40]描述的产芽胞细菌新种鉴定标准，本研究从火山谷土壤中分离到的7个芽胞杆菌潜在新种资源，其与最相近模式菌株的相似性均低于98.65%，其中5个种为类芽胞杆菌属的潜在新种，类芽胞杆菌能分泌多种水解多糖的酶，本研究发现的类芽胞杆菌新种具有很大的功能挖掘价值。由此说明，火山谷土壤也蕴藏着一定量的芽胞杆菌新资源，这为挖掘开发产新活性物质的功能芽胞杆菌提供了新的菌种来源。
环境因子对微生物种群分布特征具有一定的相关性。土壤营养成分显著影响了阿尔卑斯山森林土壤中可培养细菌的丰富度和多样性[41]，低海拔高度细菌多样性最高[42]，Xu等[43]研究发现长白山低海拔高度的微生物多样性高于高海拔的，但武夷山芽胞杆菌多样性与海拔高度相关性不显著[44]，而Kerfahi等[45]研究表明pH、N和C对火山灰中的细菌多样性影响不显著。本研究发现海拔高度和碳氮比对芽胞杆菌种群分布特征影响呈负相关，与前人的研究结果基本相符合。阴坡、谷底和阳坡的芽胞杆菌种类差异明显，阳光、温度等环境因子对其芽胞杆菌种群结构具有一定的影响。本文研究结果表明，云南腾冲小空山火山谷蕴藏着丰富的芽胞杆菌资源，且阳坡和阴坡植被类型差异显著，是研究微生物物种适应和群落演化的最佳地点。
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