正红菇菇位土壤可培养细菌多样性与网络结构分析

王英浩; 满家银; 张桃香; 练春兰

doi:10.19303/j.issn.1008-0384.2022.004.014

正红菇菇位土壤可培养细菌多样性与网络结构分析

doi: 10.19303/j.issn.1008-0384.2022.004.014

王英浩^{1, 2,},
满家银^{1, 2},
张桃香^{1, 2, ,},
练春兰³

1.
福建农林大学林学院，福建　福州　350002
2.
福建农林大学森林共生生物学国际联合实验室，福建　福州　350002
3.
东京大学亚洲自然环境科学中心，日本　东京　188-0002

基金项目: 福建农林大学林学高峰学科建设项目（71201800701）

详细信息

作者简介:
王英浩（1997−），男，硕士研究生，研究方向：森林微生物（wangyh705@foxmail.com）

通讯作者:
张桃香（1985−），女，博士，讲师，研究方向：森林培育（ning527@fafu.edu.cn）

中图分类号: S 154
计量
- 文章访问数: 476
- HTML全文浏览量: 150
- PDF下载量: 28
- 被引次数: 0
出版历程
- 收稿日期: 2022-03-08
- 修回日期: 2022-03-28
- 网络出版日期: 2022-04-24
- 刊出日期: 2022-04-28

Diversity and Network Structure Analysis of Culturable Bacteria in Sporophore Site Soil of Russula griseocarnosa

WANG Yinghao^{1, 2
,},
MAN Jiayin^{1, 2},
ZHANG Taoxiang^{1, 2
, ,},
LIAN Chunlan³

1.
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
International Joint Laboratory of Forest Symbiology, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
3.
Asian Natural Environmental Science Center, The University of Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan

摘要

摘要: 目的通过对正红菇菇位土壤细菌的分离培养，探究其可培养细菌的多样性及其种间互作关系，为后续筛选促进正红菇菌丝体生长和子实体形成分化的菌根促生菌奠定基础。方法利用LB和ISP₂两种培养基通过传统分离培养的方法对正红菇菇位土壤中的细菌进行分离纯化，并利用16S rRNA基因序列分析初步确定分离菌株的分类地位，使用R语言中的Bipartite包分析可培养细菌属间的相互作用。结果 6份正红菇菇位土壤中共分离出128株细菌，隶属于3门16属34个OTUs，丰度最高的3个属分别是芽孢杆菌属、伯克霍尔德氏菌属和链霉菌属。属水平-采样品网络分析表明，可培养细菌分布具有随机的嵌套性。Bacillus属和Burkholderia属在细菌群落中存在较多的有效合作值（Effective partners）和亲密度（Closeness），被其他细菌所依赖程度（Species Strength）较高，是该群落中的重要组成类群。结论正红菇菇位土壤中具有丰富的细菌资源，细菌的群落组成和网络结构相结合分析可以更清晰阐明细菌间的相互作用关系。
- 正红菇 /
- 根际土壤 /
- 可培养细菌 /
- 网络结构
Abstract: Objective The diversity of culturable bacteria and their inter-species interactions were explored by isolating and culturing sporophore site soil bacteria of R. griseocarnosa. It provides insights into the mycorrhizal helper bacteria which may promote the growth of the mycelium and the formation and differentiation of sporophore of R. griseocarnosa. Methods The bacteria in the sporophore site soil of R. griseocarnosa were isolated and purified by traditional methods of separation and culture using LB and ISP₂ media, and the species of the isolated strains was preliminarily identified by 16S rRNA gene sequence analysis. Interactions between culturable bacterial genera were analyzed using the Bipartite package in R. Results A total of 128 strains of bacteria were isolated from 6 sporophore site soils of R. griseocarnosa, which belonged to 34 OTUs (16 genera of 3 phyla). The three genera with the highest abundance were Bacillus , Burkholderia and Streptomyces. Genus-level-Sample network analysis showed that the distribution of culturable bacteria was randomly nested. Bacillus and Burkholderia are important groups in the community, which have more Effective Partners, Closeness and high Species Strength in the bacterial community. Conclusion There are abundant bacterial resources in the sporophore site soil of R. griseocarnosa. Combining the analysis of bacterial abundance and network structure can predict the interaction between bacteria more clearly.
- Russula griseocarnosa /
- rhizosphere soil /
- culturable bacteria /
- network structure

HTML全文

图 1 基于正红菇菇位土壤中34个OUTs细菌的16S rRNA序列及其参考序列的系统进化树

Figure 1. Phylogenetic tree based on 16S rRNA sequences of 34 OUTs isolated from rhizosphere soils of R. griseocarnosa and reference

下载: 全尺寸图片幻灯片

图 2 不同正红菇菇位土壤样品中属水平可培养细菌丰度组成

Figure 2. Abundant culturable microbes at genus level in different rhizosphere soils

下载: 全尺寸图片幻灯片

图 3 正红菇菇位土壤可培养细菌(属水平)在不同采样品的分布

Figure 3. Distribution of culturable microbes at genus level in rhizosphere soil sampled at R. griseocarnosa sites

下载: 全尺寸图片幻灯片

表 1 正红菇菇位土壤中分离出的34个OUTs细菌的16S rRNA基因序列比对结果

Table 1. 16S rRNA gene sequences of 34 OUTs microbes isolated from R. griseocarnosa rhizosphere soil

代表菌株 Strain	最大相似菌株及GenBank登录号 Similarity strain and GenBank accession No.	相似度 Similarity/%	菌株数 Number of strains
F422	卤代拟青霉 Amycolatopsis halotolerans(NR_043452)	99	1
L145	蜡样芽孢杆菌 Bacillus cereus(MN999986)	99	15
F124	堀越氏芽孢杆菌 Bacillus horikoshii(MT883498)	99	21
F202	巨大芽孢杆菌 Bacillus megaterium(MW391757)	99	1
L501	蕈状芽孢杆菌 Bacillus mycoides (KU877669)	99	4
L311	稻壳芽孢杆菌 Bacillus oryzaecorticis (MN330145)	99	7
L421	东京芽孢杆菌 Bacillus toyonensis(MN543844)	99	1
L215	热带芽孢杆菌 Bacillus tropicus (MW478751)	99	1
L405	苏云金芽孢杆菌 Bacillus thuringiensis (MN330087)	99	9
L127	越南芽孢杆菌 Bacillus wiedmannii (MH041257)	99	3
L617	芽孢杆菌属 Bacillus sp. (MG309547)	99	6
F301	洋葱伯克霍尔德氏菌 Burkholderia cepacia (LC462133)	99	19
F308	污染伯克霍尔德氏菌 Burkholderia contaminans (MN826151)	100	6
F410	伯克霍尔德氏菌属 Burkholderia sp. (JQ864385)	99	1
L213	柠檬酸杆菌属 Citrobacter sp. (AB673462)	99	1
F414	紫色杆菌属 Janthinobacterium sp. (EU098005)	99	4
L228	细长赖氨酸芽孢杆菌 Lysinibacillus macroides (MN263206)	99	4
F418	Massilia sp. (AB545620)	99	2
F421	马来小四孢菌 Microtetraspora malaysiensis (NR_024780)	99	1
F431	千叶类芽孢杆菌 Paenibacillus chibensis (MN826593)	99	1
F111	Paenibacillus chinjuensis (KP980606)	99	1
F508	松树土壤类芽孢杆菌 Paenibacillus pinihumi (NR_117367)	99	1
F326	类芽孢杆菌属 Paenibacillus sp. (MK215829)	98	1
F321	副伯克霍尔德氏菌属 Paraburkholderia sp. (OK445519)	99	2
L222	植生拉乌尔菌 Raoultella planticola (NR_119279)	99	2
F331	Rhodococcus soli (NR_134799)	99	1
F512	Rummeliibacillus sp. (MN589588)	99	1
F602	黏质沙雷氏菌 Serratia marcescens (GU220796)	99	2
F114	丙氨菌素链霉菌 Streptomyces alanosinicus (KJ571026)	99	2
F129	孔雀石褐链霉菌 Streptomyces malachitofuscus (MN428156)	99	4
F501	生米卡链霉菌 Streptomyces mycarofaciens (EF063483)	99	1
F604	结节链霉菌 Streptomyces nodosus (MN421090)	99	1
F119	链霉菌属 Streptomyces sp. (MF455322)	99	2
F613	Streptoverticillium reticulum (MF062672)	99	2
注：F和L开头指分别用ISP₂培养基和LB培养基分离的菌株，字母后第一个数字为样品编号，后两位数字为菌株编号。 Note: F and L indicate the strains isolated from ISP₂ media and LB media, respectively. The first number after the letter is the sample number, and the last two numbers are the strain number.

下载: 导出CSV

表 2 6个正红菇菇位土壤样品可培养细菌多样性指数

Table 2. Diversity index of culturable microbes in 6 rhizosphere soil samples from sites of R. griseocarnosa

样品 Samples	香农指数 Shannon-Weiner index	优势度指数 Simpson index	丰富度指数 Margalef index	均匀度指数 Pielou index
样品1 Sample 1	2.94	0.81	3.19	1.22
样品2 Sample 2	2.97	0.82	3.24	1.24
样品3 Sample 3	3.55	0.91	4.01	1.38
样品4 Sample 4	3.72	0.91	4.41	1.37
样品5 Sample 5	3.23	0.87	3.61	1.30
样品6 Sample 6	3.40	0.89	3.61	1.37

下载: 导出CSV

表 3 正红菇菇位土壤中可培养细菌属间的网络相互作用

Table 3. Network interactions among culturable microbial genera in R. griseocarnosa rhizosphere soil

属名 Genus	物种依赖程度 Species strength	有效合作值 Effective partners	亲密度 Closeness
拟无枝酸菌属 Amycolatopsis	0.04	1.00	0.06
芽孢杆菌属 Bacillus	3.10	5.79	0.08
伯克霍尔德菌氏属 Burkholderia	1.21	4.68	0.08
柠檬酸杆菌属 Citrobacter	0.05	1.00	0.05
紫色杆菌属 Janthinobacterium	0.17	1.00	0.06
赖氨酸芽孢杆菌属 Lysinibacillus	0.19	4.00	0.07
Massilia	0.09	2.00	0.07
小四孢菌属 Microtetraspora	0.04	1.00	0.06
类芽孢杆菌属 Paenibacillus	0.19	4.00	0.07
副伯克霍尔德氏菌属 Paraburkholderia	0.10	1.00	0.06
拉乌尔菌属 Raoultella	0.09	1.00	0.05
红球菌属 Rhodococcus	0.05	1.00	0.06
Rummeliibacillus	0.05	1.00	0.06
沙雷氏菌属 Serratia	0.10	2.00	0.06
链霉菌属 Streptomyces	0.46	3.60	0.07
轮枝链霉菌属 Streptoverticillium	0.10	1.00	0.05

下载: 导出CSV

参考文献(37)

[1]	钱建新, 陈仁毅, 张惠兰. 正红菇的生长环境研究 [J]. 福建林业科技, 2003, 30(4):52−54. doi: 10.3969/j.issn.1002-7351.2003.04.015 QIAN J X, CHEN R Y, ZHANG H L. The study on growth environment of Russula vinosa [J]. Journal of Fujian Forestry Science and Technology, 2003, 30(4): 52−54.(in Chinese) doi: 10.3969/j.issn.1002-7351.2003.04.015
[2]	涂育合, 陈永聪, 郑肇快. 正红菇依存森林的群落学特征 [J]. 植物资源与环境学报, 2001, 10(2):26−30. doi: 10.3969/j.issn.1674-7895.2001.02.007 TU Y H, CHEN Y C, ZHENG Z K. Feature of forest community with Russula vinosa lindbl Fr. depending on for existance in Dingban Village of Datian County [J]. Journal of Plant Resources and Environment, 2001, 10(2): 26−30.(in Chinese) doi: 10.3969/j.issn.1674-7895.2001.02.007
[3]	MUNSCH P, ALATOSSAVA T, MARTTINEN N, et al. Pseudomonas costantinii sp. nov., another causal agent of brown blotch disease, isolated from cultivated mushroom sporophores in Finland [J]. International Journal of Systematic and Evolutionary Microbiology, 2002, 52: 1973−1983.
[4]	RALPH N, ANDREJA D P, HOBBS P J, et al. Volatile C8 compounds and pseudomonads influence primordium formation of Agaricus bisporus [J]. Mycologia, 2009, 101(5): 583−591. doi: 10.3852/07-194
[5]	YUN Y B, PARK S W, CHA J S, et al. Biological characterization of various strains of Pseudomonas tolaasii that causes brown blotch disease [J]. Journal of the Korean Society for Applied Biological Chemistry, 2013, 56(1): 41−45. doi: 10.1007/s13765-012-2242-y
[6]	盛江梅, 吴小芹. 菌根真菌与植物根际微生物互作关系研究 [J]. 西北林学院学报, 2007, 22(5):104−108,135. doi: 10.3969/j.issn.1001-7461.2007.05.026 SHENG J M, WU X Q. Interaction between mycorrhizal fungi and rhizosphere microorganisms [J]. Journal of Northwest Forestry University, 2007, 22(5): 104−108,135.(in Chinese) doi: 10.3969/j.issn.1001-7461.2007.05.026
[7]	GARBAYE J. Helper Bacteria - a New Dimension to the Mycorrhizal Symbiosis [J]. New Phytologist, 1994, 128(2): 197−210. doi: 10.1111/j.1469-8137.1994.tb04003.x
[8]	ASPRAY T J, JONES E E, DAVIES M W, et al. Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus by the helper bacterium Paenibacillus sp [J]. Mycorrhiza, 2013, 23(5): 403−410. doi: 10.1007/s00572-013-0483-1
[9]	FOUNOUNE H, DUPONNOIS R, BÂ A M, et al. Mycorrhiza helper bacteria stimulate ectomycorrhizal symbiosis of Acacia holosericea with Pisolithus alba [J]. New Phytologist, 2002, 153(1): 81−89. doi: 10.1046/j.0028-646X.2001.00284.x
[10]	FOUNOUNE H, DUPONNOIS R, MEYER J M, et al. Interactions between ectomycorrhizal symbiosis and fluorescent pseudomonads on Acacia holosericea: Isolation of mycorrhiza helper bacteria (MHB) from a Soudano-Sahelian soil [J]. FEMS Microbiology Ecology, 2002, 41(1): 37−46. doi: 10.1111/j.1574-6941.2002.tb00964.x
[11]	陈克华. 野生正红菇生态增产技术示范 [J]. 福建农业, 2007(12):15. CHEN K H. Demonstration of ecological yield increasing technology of wild Pleurotus ostreatus [J]. Fujian Agriculture, 2007(12): 15.(in Chinese)
[12]	郭永红, 罗孝坤, 弓明钦, 等. 大红菇出菇特性研究 [J]. 中国食用菌, 2011, 30(5):30−33. doi: 10.3969/j.issn.1003-8310.2011.05.012 GUO Y H, LUO X K, GONG M Q, et al. Characteristics research of pruducing mushroom about Russula sanguinea [J]. Edible Fungi of China, 2011, 30(5): 30−33.(in Chinese) doi: 10.3969/j.issn.1003-8310.2011.05.012
[13]	李忠, 钟莹莹, 陈逸湘. 广东梅州野生灰肉红菇资源调查 [J]. 食用菌, 2012, 34(4):8−9. LI Z, ZHONG Y Y, CHEN Y X. Investigation on wild red mushroom resources in Meizhou, Guangdong Province [J]. Edible Fungi, 2012, 34(4): 8−9.(in Chinese)
[14]	肖冬来, 陈丽华, 陈宇航, 等. 正红菇菌根际土壤细菌多样性 [J]. 福建食用菌, 2014, 2(4):66−72. XIAO D L, CHEN L H, CHEN Y H, et al. Bacterial Diversity of Russula griseocarnosa Mycorrhizosphere Soil [J]. Fujian Mushroom Journal, 2014, 2(4): 66−72.(in Chinese)
[15]	肖冬来, 陈丽华, 陈宇航, 等. 利用变性梯度凝胶电泳分析正红菇菌根围土壤真菌群落多样性 [J]. 热带作物学报, 2013, 34(12):2508−2512. XIAO D L, CHEN L H, CHEN Y H, et al. Analysis of fungal diversity of Russula griseocarnosa mycorrhizosphere soil with denaturing gradient gel electrophoresis [J]. Chinese Journal of Tropical Crops, 2013, 34(12): 2508−2512.(in Chinese)
[16]	YU F, LIANG J F, SONG J, et al. Bacterial community selection of Russula griseocarnosa mycosphere soil [J]. Frontiers in Microbiology, 2020, 11: 347. doi: 10.3389/fmicb.2020.00347
[17]	STEFANI F, BELL T H, MARCHAND C, et al. Culture-Dependant and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils [J]. PLoS ONE, 2017, 10(6): e0128272.
[18]	SHIRLING E B, GOTTLIEB D. Methods for characterization of Streptomyces species [J]. International Journal of Systematic Bacteriology, 1966, 16(3): 313−340. doi: 10.1099/00207713-16-3-313
[19]	WEISBURG W G, BARNS S M, PELLETIER D A, et al. 16S ribosomal DNA amplification for phylogenetic study [J]. Journal of Bacteriology, 1991, 173(2): 697−703. doi: 10.1128/jb.173.2.697-703.1991
[20]	EDWARDS U, ROGALL T, BLÖCKER H, et al. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA [J]. Nucleic Acids Research, 1989, 17(19): 7843−7853. doi: 10.1093/nar/17.19.7843
[21]	姜华, 何承刚, 于富强, 等. 松口蘑(Tricholoma matsutake)菌塘土壤可培养细菌多样性 [J]. 生态学杂志, 2015, 34(1):150−156. JIANG H, HE C G, YU F Q, et al. Bacterial diversity cultured from shiros of Tricholoma matsutake [J]. Chinese Journal of Ecology, 2015, 34(1): 150−156.(in Chinese)
[22]	余仲东, 唐光辉, 曹支敏. 陕西小叶杨叶内生真菌群体多样性和结构特征 [J]. 林业科学, 2016, 52(6):86−92. YU Z D, TANG G H, CAO Z M. Diversity and community structure of endophytic fungi in the leaves of Populus simonii in Shaanxi Province [J]. Scientia Silvae Sinicae, 2016, 52(6): 86−92.(in Chinese)
[23]	郑梅霞, 朱育菁, 刘波, 等. 云南苍山芽胞杆菌多样性研究 [J]. 福建农业学报, 2019, 34(1):104−116. ZHENG M X, ZHU Y J, LIU B, et al. Microbial diversity of Bacillus community in soils at Cangshan, Yunnan [J]. Fujian Journal of Agricultural Sciences, 2019, 34(1): 104−116.(in Chinese)
[24]	DORMANN C F, FRÜND J, BLÜTHGEN N, et al. Indices, Graphs and Null Models: Analyzing Bipartite Ecological Networks [J]. The Open Ecology Journal, 2009, 2(1): 7−24. doi: 10.2174/1874213000902010007
[25]	CORE R, RDCT R, TEAM R, et al. A Language and Environment for Statistical Computing [J]. Computing, 2015, 1: 12−21.
[26]	BECKER D M, BAGLEY S T, PODILA G K. Effects of mycorrhizal-associated streptomycetes on growth of Laccaria bicolor, Cenococcum geophilum, and Armillaria species and on gene expression in Laccaria bicolor [J]. Mycologia, 1999, 91(1): 33−40. doi: 10.2307/3761191
[27]	FREY-KLETT P, CHAVATTE M, CLAUSSE M L, et al. Ectomycorrhizal symbiosis affects functional diversity of rhizosphere fluorescent pseudomonads [J]. The New Phytologist, 2005, 165(1): 317−328. doi: 10.1111/j.1469-8137.2004.01212.x
[28]	POOLE E J, BENDING G D, WHIPPS J M, et al. Bacteria associated with Pinus sylvestris-Lactarius rufus ectomycorrhizas and their effects on mycorrhiza formation in vitro [J]. New Phytologist, 2001, 151(3): 743−751. doi: 10.1046/j.0028-646x.2001.00219.x
[29]	安忠琦, 詹伟, 吴庆珊, 等. 金钗石斛根际可培养细菌多样性及抑菌活性研究 [J]. 云南大学学报(自然科学版), 2018, 40(3):586−602. AN Z Q, ZHAN W, WU Q S, et al. Diversity and antimicrobial activities of the cultivable rhizosphric bacteria fom Dendrobium nobile [J]. Journal of Yunnan University (Natural Sciences Edition), 2018, 40(3): 586−602.(in Chinese)
[30]	万山平, 郑毅, 汤利, 等. 攀枝花块菌-华山松菌根根际土壤可培养细菌的多样性研究 [J]. 植物分类与资源学报, 2015, 37(6):861−870. WAN S P, ZHENG Y, TANG L, et al. Diversity of culturable bacteria associated with tuber panzhihuanense-Pinus armandii ectomycorrhizosphere soil [J]. Plant Diversity and Resources, 2015, 37(6): 861−870.(in Chinese)
[31]	BARBIERI E, POTENZA L, ROSSI I, et al. Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad. Ectomycorrhizal mycelium [J]. Applied and environmental microbiology, 2000, 66(11): 5035−5042. doi: 10.1128/AEM.66.11.5035-5042.2000
[32]	GARBAYE J, DUPONNOIS R, WAHL J L, et al. The bacteria associated with Laccaria laccata ectomycorrhizas or sporocarps: Effect on symbiosis establishment on Douglas fir [J]. Symbiosis, 1990, 9: 267−273.
[33]	GARBAYE J, DUPONNOIS R. Specificity and function of mycorrhization helper bacteria (MHB) associated with the Pseudotsuga menziesii-Laccaria laccata symbiosis [J]. Symbiosis, 1992, 14(1-3): 335−344.
[34]	OH S Y, LIM Y W. Root-associated bacteria influencing mycelial growth of Tricholoma matsutake (pine mushroom) [J]. Journal of Microbiology, 2018, 56(6): 399−407. doi: 10.1007/s12275-018-7491-y
[35]	盛江梅, 吴小芹, 侯亮亮, 等. 一株黑松-美味牛肝菌菌根辅助细菌的筛选及鉴定 [J]. 应用与环境生物学报, 2010, 16(5):701−704. SHENG J M, WU X Q, HOU L L, et al. Isolation and identification of a MHB strain from the rhizosphere soil of Pinus thunbergi inoculated with Boletus edulis [J]. Chinese Journal of Applied & Environmental Biology, 2010, 16(5): 701−704.(in Chinese)
[36]	RIVETT D W, BELL T. Abundance determines the functional role of bacterial phylotypes in complex communities [J]. Nature Microbiology, 2018, 3(7): 767−772. doi: 10.1038/s41564-018-0180-0
[37]	禹飞. 灰肉红菇多组学特征分析及其与根际微生物的互作[D]. 北京: 中国林业科学研究院, 2020. YU F. Multi-omics analysis of Russula griseocarnosa and its interaction with mycosphere microorganisms[D]. Beijing: Chinese Academy of Forestry, 2020. (in Chinese)