一株樟子松-褐环乳牛肝菌菌根辅助细菌的筛选和鉴定

Isolation and Identification of a MHB Strain from the Mycorrhiza of Pinus sylvestris var.mongolica Inoculated with Suillus luteus

菌根辅助细菌(mycorrhiza helper bacteria, MHB)可以促进菌根真菌在宿主植物根部定殖形成菌根共生结构,是外生菌根菌际重要微生物类群。为探讨菌根际有益微生物之间的互作对樟子松(Pinus sylvestris var. mongolica)实生苗生长的影响,对分离自樟子松-褐环乳牛肝菌(Suillus luteus)菌根根际土壤的75株细菌,采用干皿对抗法、细菌胞外代谢产物促生法筛选MHB潜力菌株。以MHB潜力菌株对樟子松1年生苗的苗高、地径、菌根侵染率的影响为指标,筛选出菌根辅助细菌。通过细菌的形态、生理生化指标、16S rRNA基因序列鉴定细菌种类。结果表明:菌株MHBZ039和MHBZ043处理在干皿对抗试验中,同对照相比,褐环乳牛肝菌N94菌丝增长率分别为20.11%和18.63%,胞外代谢产物促生实验N94菌落直径增长率分别为91.89%和85.41%,菌丝生物量的增长率分别达到42.11%和25.32%。盆栽试验中,双接种处理褐环乳牛肝菌N94+MHBZ039和N94+MHBZ043,菌根侵染率分别为57.77%和56.46%,苗高和地径同对照相比分别提高28.42%,40.32%和21.41%,34.86%。确定MHBZ039为樟子松-褐环乳牛肝菌菌根辅助细菌。经生理生化和16S rRNA基因分子鉴定,确定菌株MHBZ039为适冷假单胞杆菌(Pseudomonas extremaustralis),在GenBank数据库中的登录号为MN814017。综上,菌根辅助细菌MHBZ039对菌根菌生长和菌根合成效率具有显著的促进作用,同时,MHBZ039与外生菌根菌双接种可促进樟子松1年生苗的生长,为进一步研究褐环乳牛肝菌-菌根辅助细菌-樟子松三者互作机制提供理论基础。

Mycorrhiza helper bacteria(MHB)can promote the colonization of mycorrhiza fungi in the root of host plant,which is an important microbial group among ectomycorrhizal fungi.To investigate the effect of ectomycorrhiza co-inoculated with MHBon the growth of pine seedlings,the potential strains of MHB were screened by dry-plate confrontation assay and bacterial extracellular metabolite promoting assay from 75 bacterial strains isolated from rhizosphere soil of Pinus sylvestris var.mongolica inoculated with Suillus luteus N94.The MHB strain was screened based on seedling height,ground diameter of the stem,and mycorrhizal infection rate of pine seedlings.The MHB strain was then identified after morphological,biochemical and physiological analyses,in conjunction with 16 S rRNA gene sequencing.The result showed that strains MHBZ039 and MHBZ043 significantly promoted in vitro hyphal growth of N94 with an increasing rate of 20.11%and 18.63%.The extracellular metabolites of MHBZ039 and MHBZ043 were able to obviously promote the increment rate of N94 hypha by 91.89%and 85.41%,and consistently influence on N94 biomass increment with an increasing rate of 42.11%and 25.32%.Compared with the blank control,the pine seedlings co-inoculated treatments include N94+MHBZ039 and N94+MHBZ043,the mycorrhizal infection rates were 57.77%and 56.46%,respectively.The seedling height with increasing rates of 28.42%and 21.41%,the ground diameter with increasing rates of 40.32%and 34.86%.According to our results,MHBZ039 was a Mycorrhiza Helper Bacterium.The MHB strain was identified based on an analysis of morphologiacal charactersand 16 S rRNA gene sequences homology.MHBZ039 was identified as Pseudomonas extremaustralis,and the 16 SrRNA gene sequences have been registered at GenBank database with the accession number of MN814017.In summary,the pine seedlings co-inoculated with MHBZ039 strain can significantly improved the seedling growth and mycorrhizal infection rates,this study laid a good foundation for further study on the interaction mechanism of S.luteus,mycorrhiza helper bacteria and P.sylvestris var.mongolica.