一种马尾松菌根辅助细菌——短芽孢杆菌的筛选及鉴定

doi:10.11707/j.1001-7488.20150519

摘要/Abstract

摘要：

【目的】菌根辅助细菌与菌根真菌是林木根际重要的微生物菌群,为探讨二者互作关系,采集马尾松人工林中有外生菌根真菌彩色豆马勃子实体的菌根根际土壤,对分离获得的74株细菌进行菌根辅助细菌的筛选。【方法】通过干皿对抗法筛选获得1株能高效促进外生菌根真菌彩色豆马勃(Pt2)和红绒盖牛肝菌(Xc)生长的菌根辅助细菌MPt17。【结果】 MPt17菌株对Pt2和Xc菌丝生长的平均增长率分别为48.6%和25.0%,其胞外代谢产物对Pt2和Xc菌丝生长也有明显的促进作用,平均增长率分别为129.6%和29.5%,该菌株的胞外代谢产物对Pt2和Xc的生物量也有相一致的影响,生物量增长率分别为124.2%和34.2%。盆栽试验结果表明,双接种Pt2-MPt17处理、双接种Xc-MPt17处理、单接种Pt2、单接种Xc和单接种MPt17较未接种处理的马尾松苗高分别提高78.0%,68.2%,32.7%,39.2%和53.4%,地径分别提高46.3%,57.5%,17.5%,23.8%和25.0%。由此可以确定MPt17既是菌根辅助细菌又是根际促生细菌。通过对MPt17菌株的菌落形态观察、BIOLOG细菌自动鉴定仪鉴定及16SrDNA序列测定,初步鉴定该菌株为短芽孢杆菌(Brevibacillus reuszeri),在GenBank数据库中的登录号为KF891383。【结论】 MPt17菌株与外生菌根真菌Pt2和Xc双接种马尾松幼苗对苗木的生长都具有明显的促进作用, 而且MPt17菌株单接种马尾松幼苗也能在一定程度上促进苗木的生长。通过外生菌根真菌(EMF)-菌根辅助细菌(MHB)-植物三者互作机制进行植物生长研究的方法,对类似研究具有重要意义。

关键词: 马尾松, 外生菌根真菌, 菌根辅助细菌, 短芽孢杆菌

Abstract:

【Objective】 To investigate the interaction between ectomycorrhizal fungi and mycorrhiza helper bacteria, we screened 74 bacterial strains isolated from the rhizosphere soil of Pinus massoniana that bore the fruiting body of Pisolithus tinctorius.【Method】The dry-plate confrontation assay was used to screen the bacterial strains, and one strain of MHB was obtained and defined as MPt17 which can efficiently promote the growth of Pisolithus tinctorius (Pt2) and Xerocamus chrysenteron (Xc). 【Result】The result showed that obtained MPt17 significantly promoted in vitro hyphal growth of Pt2 and Xc with an increasing rate of 48.6% and 25.0%. The extracellular metabolites of Mpt17 were able to also obviously promote the increment rate of Pt2 and Xc hypha by 129.6% and 29.5%, and consistently influence on Pt2 and Xc biomass increment with an increasing rate of 124.2% and 34.2%. Compared with the blank control, the pine seedlings co-inoculated with Pt2 and MPt17 strain, co-inoculated with Xc and MPt17 strain, Pt2 alone, Xc alone, and MPt17 alone all significantly increased their mean height with increasing rates of 78.0%, 68.2%, 32.7%, 39.2% and 53.4%, and their diameter with increasing rates of 46.3%, 57.5%, 17.5%, 23.8% and 25.0%. According to our results, Mpt17 was not only a Mycorrhiza Helper Bacterium but also a plant growth-promoting rhizobacterium. The MHB stain was identified based on an analysis of morphologiacal characters, the BIOLOG system and 16SrRNA gene sequences homology. MPt17 was identified as Brevibacillus reuszeri, and the 16SrRNA sequences have been registered at GenBank database under the accession number of KF891383.【Conclusion】In this experiment the pine seedlings co-inoculated with Pt2 and MPt17 strain, and co-inoculated with Xc and MPt17 strain significantly improved the growth of pine seedlings, and MPt17 alone also improved the growth of seedlings to a certain extent. This study method with the interactions of ectomycorrhizal fungi, Mycorrhiza Helper Bacteriun and plant has important reference implications for similar studies.

Key words: Pinus massoniana, ectomycorrhizal fungi, mycorrhiza helper bacteria, Brevibacillus reuszeri

中图分类号:

S718.8

李倩, 吴小芹, 叶建仁. 一种马尾松菌根辅助细菌——短芽孢杆菌的筛选及鉴定[J]. 林业科学, 2015, 51(5): 159-164.

Li Qian, Wu Xiaoqin, Ye Jianren. Isolation and Identification of a Mycorrhiza Helper Bacteria(MHB)—Brevibacillus reuszeri on Pinus massoniana Roots[J]. Scientia Silvae Sinicae, 2015, 51(5): 159-164.

参考文献

程丽娟.1993.微生物实验技术.西安:天则出版社.(Chen L J.1993.Laboratory Manual of Microbiology. Xi'an:Day,Press.[in Chinese])
黄鹏飞.2012.马尾松复合微生物菌剂的研制及应用.长沙:中南林业科技大学硕士学位论文.(Huang P F.2012.Development and application of compound fungal and bacterial fertilizers of Pinus massoniana.Changsha:MS thesis of Central South University of Forestry&Technology.[in Chinese])
蒋萍,叶建仁.2008.马尾松叶面拮抗细菌NA的分子鉴定.新疆农业大学学报,31(3):39-42.(Jiang P,Ye J R.2008.Identification on molecular of an antagonistic bacteria NA isolated from leaf surface of Pinus massoniana.Journal of Xinjiang Agricultural University,31(3):39-42.[in Chinese])
刘辉,吴小芹,任嘉红,等.2012.荧光假单胞菌及红绒盖牛肝菌接种对NL-895杨生长和光合特征的影响.西部林业科学,41(1):53-59.(Liu H,Wu X Q,Ren J H,et al.2012.Effect of Inoculating Pesudomonas fluorenscens and Xerocomus chrysenteron on growth and photosynthetic characteristics of NL-895 poplar.Journal of West China Forestry Science,41(1):53-59.[in Chinese])
盛江梅.2014.黑松—黄色须腹菌共生体根际菌根辅助细菌的筛选及鉴定.东北林业大学学报,42(5):110-114.(Sheng J M.2014.Isolating and identifying mycorrhiza helper bacteria from the rhizosphere soil of Pinus thunbergii inoculated with Rhizipogen luteous.Journal of Northeast Forestry University,42(5):110-114.[in Chinese])
盛江梅.2008.黑松菌根根际菌根辅助细菌(MHB)筛选研究.南京:南京林业大学硕士学位论文.(Sheng J M.2008.Isolating and Identifying mycorrhiza helper bacteria from the Pinus thunbergii.Nanjing: MS thesis of Nanjing Forestry University.[in Chinese])
孙民琴,吴小芹,叶建仁.2007.外生菌根真菌对不同松树出苗和生长的影响.南京林业大学学报:自然科学版,31(5):39-43.(Sun M Q,Wu X Q.Ye J R.2007.Effects of ectomycorrhizal fungi on germination and growth of pines.Journal of Nanjing Forestry University:Natural Sciences Edition,31(5):39-43.[in Chinese])
吴小芹,孙民琴.2006.七株外生菌根真菌与三种松苗菌根的形成能力.生态学报,26(12):4186-4191.(Wu X Q, Sun M Q.2006.Mycorrhizal formation between seven ectomycorrhizal fungi and seedlings of three pines species.Acta Ecologica Sinica,26(12):4186-4191.[in Chinese])
张海涵,唐明,陈辉,等.2007.不同生态条件下油松(Pinus tabulaeformis)菌根根际土壤微生物群落.生态学报,27(12):5463-5470.(Zhang H H,Tang M,Chen H.2007.Microbial communities in Pinus tabulaeformis mycorrhizosphere under different ecological conditions.Acta Ecologica Sinica,27(12):5463-5470.[in Chinese])
Aspray T J,Frey-Klett P,Jones J E.2006.Mycorrhization helper bacteria:a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation.Mycorrhiza,16(8):533-541.
Ausubel F M, Brent R, Kingston R E, et al.1999.Short Protocols in Molecular Biology.4th ed.Greene Pub Associates,1-1319.
Bianciotto V,Minerdi D, Perotto S.1996.Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria.Protoplasma,193:123-131.
Dunstan W A,Malajczuk N,Dell B.1998.Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F Muell seedlings.Plant Soil,201:241-249.
Duponnois R,Kisa M.2006.The possible role of trehalose in the mycorrhiza helper effect.Can J Bot,84:1005-1008.
Garbaye J.1994.Helper bacteria:a new dimension to the mycorrhizal symbiosis.New Phytologist,128(2):197-210.
Garbaye J,Bowen G D.1989.Stimulation of mycorrhizal infection of Pinus radiata by some micro-organisms associated with the mantle of ectomycorrhizas.New Phytol,112:383-388.
Riedlinger J.2006.Die stofflichen Grundlagen der Modulation des Myzelwachstums von sym-biotischen und pathogenen Pilzen durch Streptomyzeten. PhD thesis of University of Tubingen,Tubingen, Germany.
Mosse B.1962.The establishment of vesicular-arbuscular mycorrhiza under aseptic conditions. J Gen Microbiol,27:509-520.
Xavier L J C,Germida J J.2003.Bacteria associated with Glomus clarum spores influence mycorrhizal activity.Soil Biol Biochem,35:471-478.

[1]	邓秀秀, 肖文发, 曾立雄, 雷蕾, 施征. 马尾松幼苗光合产物的运输与分配特征[J]. 林业科学, 2019, 55(7): 27-34.
[2]	金国庆, 张振, 余启新, 冯随起, 丰忠平, 赵世荣, 周志春. 马尾松2个世代种子园6年生家系生长的遗传变异与增益比较[J]. 林业科学, 2019, 55(7): 57-67.
[3]	李亚藏, 冯仲科. 气候敏感的马尾松生物量相容性方程系统研建[J]. 林业科学, 2019, 55(5): 65-73.
[4]	王好运, 吴峰, 朱小坤, 谢维斌. 叶型对马尾松幼苗生长及叶绿素荧光特征的影响[J]. 林业科学, 2019, 55(3): 183-192.
[5]	陈金磊, 方晰, 辜翔, 李雷达, 刘兆丹, 王留芳, 张仕吉. 中亚热带2种森林群落组成、结构及区系特征[J]. 林业科学, 2019, 55(2): 159-172.
[6]	陈坦, 张振, 楚秀丽, 金国庆, 周志春, 丰忠平. 马尾松二代无性系种子园的花期同步性[J]. 林业科学, 2019, 55(1): 146-156.
[7]	罗晓蔓, 丁贵杰, 王艺. 菌根化马尾松苗木根际土壤浸提物有效成份及其对种子萌发的影响[J]. 林业科学, 2018, 54(8): 32-38.
[8]	姚虹宇, 刘亚敏, 张盛楠, 刘玉民, 周文颖, 王针针. 外源柠檬酸对铝胁迫下马尾松生理特性的影响[J]. 林业科学, 2018, 54(7): 155-164.
[9]	魏永成, 刘青华, 周志春, 徐六一, 陈雪莲, 郝焰平. 不同抗性马尾松接种松材线虫后针叶内化学信号物质的变化[J]. 林业科学, 2018, 54(2): 119-125.
[10]	辜夕容, 倪亚兰, 江亚男, 贾豪. 接种双色蜡蘑对酸性铝胁迫下马尾松幼苗生长、养分和铝吸收与分布的影响[J]. 林业科学, 2018, 54(2): 170-178.
[11]	尹焕焕, 刘青华, 周志春, 余启新, 丰忠平. 马尾松产脂性状与生长性状的无性系变异及相关性[J]. 林业科学, 2018, 54(12): 82-91.
[12]	高尚坤, 肖文发, 曾立雄, 雷蕾, 黄志霖, 王松. 马尾松人工林干扰对土壤微生物群落结构的短期影响[J]. 林业科学, 2018, 54(12): 92-101.
[13]	张振, 金国庆, 余启新, 刘青华, 丰忠平, 董虹妤, 周志春. 马尾松三代测定林抽梢性状的遗传效应及与针叶氮磷钾养分的遗传相关[J]. 林业科学, 2017, 53(8): 26-34.
[14]	杜明凤, 丁贵杰, 赵熙州. 不同家系马尾松对持续干旱的响应及抗旱性[J]. 林业科学, 2017, 53(6): 21-29.
[15]	贺同鑫, 孙建飞, 李艳鹏, 俞有志, 胡宝清, 王清奎. 环割对杉木和马尾松人工林土壤微生物群落结构的影响[J]. 林业科学, 2017, 53(6): 77-84.