浑源黄芪内生细菌的菌群组成及其功能

Composition and function of endophytic bacteria residing the root tissue of Astragalus mongholicus in Hunyuan

摘要:

[目的] 分离、鉴定浑源黄芪内生细菌，筛选潜在促生菌，并研究绿叶挥发物对其生长的影响。[方法] 以山西浑源7年生传统采收期黄芪为材料，采用平板培养法分离内生菌，16S rRNA基因序列进行菌株鉴定；通过培养基中添加1-氨基环丙烷-1-羧酸（1-aminocyclopropane-1-carboxylate，ACC）、色氨酸及缺氮素培养的方式进行含ACC脱氨酶、吲哚乙酸产生及固氮菌初筛；通过培养基中添加Ca₃（PO₄）₂、钾长石的方式进行解磷、解钾菌初筛；电感耦合等离子体质谱等方法进行定量；通过液体培养基中添加绿叶挥发物的方式，研究其对含ACC脱氨酶菌株的影响；利用顶空气相色谱-质谱法测定黄芪绿叶挥发物含量。[结果] 从浑源黄芪根中分离得85株代表性内生菌株，分别属于变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）和拟杆菌门（Bacteroidetes）的13个属，其中假单胞菌属（Pseudomonas）、泛菌属（Pantoea）和葡萄球菌属（Staphylococcus）菌株数量较高，占分离菌株总数的80.00%。筛选获得的促生菌中，具有吲哚乙酸合成能力的菌株所占比例最高（69.41%），其次为含ACC脱氨酶和具有固氮活性的菌株，分别占分离菌株的40.00%和31.76%，而解磷、解钾菌株所占比例较低（分别为14.12%、7.06%）；双重促生效应菌株中，兼具吲哚乙酸合成与含ACC脱氨酶菌株所占比例最高（37.65%），其次为兼具吲哚乙酸合成和固氮活性、兼具含ACC脱氨酶和固氮活性的菌株，分别占分离菌株的28.24%和24.71%，兼具含ACC脱氨酶和解钾活性的菌株占比最低（1.18%）。2-50 μmol/L正己醛和Z-3-己烯醛、5-125 μmol/L正己醇具有促进部分含ACC脱氨酶内生菌株生长的作用。[结论] 吲哚乙酸产生和含ACC脱氨酶的内生菌株占比高、绿叶挥发物促生菌的存在很可能是浑源黄芪内生菌群适应栖息地独特生境的产物，绿叶挥发物很可能是影响浑源黄芪内生菌群组成与功能的重要因素之一。

Abstract:

[Objective] This study aimed to isolate and screen bacterial endophytes with potential plant growth-promoting activities from Astragali Radix, and to explore effects of green leaf volatiles (GLVs) on the endophytes.[Methods] Plate culture method was used to isolate bacterial endophytes from the root tissue of 7-year old plants of A. mongholicus in Hunyuan, Shanxi, China and 16S rRNA gene sequencing was used to identify the isolates. The screening for plant growth-promoting endophytes was performed by adding exogenous 1-aminocycline-1-carboxylic acid (ACC), tryptophan, calcium phosphate and feldspar into the medium and by incubation in the medium lacking nitrogen source, further quantification was carried out by inductively coupled plasma mass spectrometry (ICP-MS) and spectrophotometry. Effect of the main GLVs in Astragali Radix on the isolates was investigated by adding synthetic chemicals into the liquid medium and the ten isolates containing ACC deaminase were used as representative strains. Headspace GC-MS was performed to determine the contents of the main GLVs in Astragali Radix.[Results] A total of 85 bacterial strains were obtained and classified into the follow phyla:Proteobacteria, Firmicutes, actinobacteria and Bacteroides. At the genus level, they were categorized into 13 genera and the stains of Pseudomonas, Pantoea and Staphylococcus were more abundant (80.00%) than the ones of the others. Among candidate plant growth-promoting isolates, these that can synthesize indoleacetic acid (IAA) shared the biggest proportion (69.41%), followed by these with ACC deaminase (40.00%) and nitrogen fixation activity (31.76%), and these that can solubilize phosphorus and potassium shared relatively lower proportions (i.e., 14.12% and 7.06%). Among the isolates with double plant growth-promoting activities, the IAA-producing strains containing ACC deaminase shared the biggest proportion (37.65%), followed by the IAA-producing strains with nitrogen fixation activity and the strains with ACC deaminase and nitrogen fixation activity, accounting for 28.24% and 24.71%, respectively, while these that not only contain ACC deaminase but can solubilize potassium bearing minerals shared the smallest part (1.18%). Besides, low concentrations of GLVs (i.e., 2-50 μmol/L of hexanal and Z-3-hexenal, and 5-125 μmol/L hexanol) exerted positive effects on the growth of a few strains with ACC deaminase.[Conclusion] There were bigger proportions of the isolates that can produce IAA and/or contain ACC deaminase and GLV growth-promoting strains in the bacterial community residing the root tissue of A. mongholicus in Hunyuan, which might be resulted from the local adaptation. GLVs might be involved in the shaping of the composition and function of endophytic communities in Astragali Radix in Hunyuan.