不同生长条件下铁皮石斛内生细菌多样性及活性菌株的筛选

Diversity of endophytic bacteria of Dendrobium officinale under different growth conditions and screening of active strains

【目的】比较和分析野生与人工栽培铁皮石斛Dendrobium officinale内生细菌分布特点,筛选有较强定殖能力的活性菌株,为促进野生铁皮石斛驯化、提高药材品质奠定基础。【方法】采用组织块分离法分离铁皮石斛根、茎、叶各部位的内生细菌,利用16S rDNA序列和系统发育树对分离菌株进行分析鉴定,体外筛选具有解磷、解钾、固氮、产铁载体、产IAA、拮抗病原菌的促生活性菌株,回接后再分离,观察内生细菌在组培苗中的定殖动态。【结果】从铁皮石斛中分离到285株内生细菌,其中,217株分离自野生铁皮石斛,归类于3门9属,以芽孢杆菌属Bacillus和不动杆菌属Acinetobacter为优势菌群存在于根、茎、叶中,其菌株数分别占总分离菌株数的79.26%和8.76%;68株内生细菌分离自人工栽培铁皮石斛,归类于1门3属,以伯克霍尔德菌属Burkholderia和埃希菌属Escherichia为优势菌群存在于根、茎中,其菌株数分别占总分离菌株数的54.41%和30.88%。泛菌属Pantoea在野生和人工栽培铁皮石斛中均有分布。野生铁皮石斛内生细菌的物种数(9)和多样性指数(0.85)明显高于人工栽培铁皮石斛(物种数为3,多样性指数为0.61)。活性筛选共获得38株菌株,占筛选菌株的45%,4株野生铁皮石斛的活性内生细菌中有3株在人工组培苗具有较好的定殖性。【结论】野生与人工栽培铁皮石斛内生细菌的类群结构和生物多样性具有明显差异,铁皮石斛内生细菌蕴含丰富的促生潜力。

【Objective】To compare and analyze the distribution characteristics of endophytic bacteria in wild and cultivated Dendrobium officinale,and screen active strains with strong colonization ability.To lay the foundation for improving the domestication of wild D.officinale and the quality of medicinal materials.【Method】The endophytic bacteria were isolated from the root,stem,leaf of D.officinale by tissue block separation method,and 16 S rDNA sequence and phylogenetic tree were used to identify the isolated strains.The probioticactive bacterial strains with the abilities of phosphate-solubilizing,potassium-dissolving,nitrogenfixing,siderophore-synthesizing,IAA-producting and antagonistic activity of pathogen were screened in vitro.The colonization dynamics of endophytic bacteria in tissue culture seedlings were observed after backgrafting and separation.【Result】A total of 285 endophytic bacteria strains were isolated from D.officinale,among which 217 were isolated from wild D.officinale,which were classified into three phylums and nine genera.Bacillus and Acinetobacter strains accounted for 79.26%and 8.76%of the total isolated strains respectively,and were the dominant bacteria.Only 68 endophytic bacteria strains were isolated from the cultivated D.officinale,which were classified into one phylum three genera,Burkholderia and Escherichia accounted for 54.41%and30.88%of the total isolated strains respectively,and were the dominant bacteria.Only Pantoea was distributed in both wild and cultivated D.officinale.The number of endophytic species(9)and diversity index(0.85)of wild D.officinale were obviously higher than those of cultivated D.officinale(3 and 0.61 respectively).A total of 38 strains were obtained by active screening,accounting for 45%of the screened strains and three of four strains of endophytic bacteria from wild D.officinale showed good colonization in artificial tissue culture seedlings.【Conclusion】The biodiversity and community structure of endophytic bacteria of wild and artificially cultivated D.officinale are obviously different and the endophytic bacteria contains abundant potential of growth promotion.