黄芪新品系内生固氮菌的分离鉴定及其对连作小麦的促生作用

Endophytic nitrogen-fixing bacteria from new lines of Astragalus mongholicus var. mongholicus: isolation, identification, and evaluation of growth-promoting effect on wheat in continuous cropping

摘要:

【背景】 蒙古黄芪(Astragalus membranaceus var. mongholicus)是甘肃省定西市陇西县道地药材，多生长在较为干旱和贫瘠的土壤中，其生长环境决定蒙古黄芪内生固氮菌抗逆性强。【目的】 为挖掘蒙古黄芪新品系内生固氮菌资源，筛选与小麦高效联合固氮菌株，减轻连作障碍对小麦生长的抑制，减少化肥用量。【方法】 以甘肃省定西市陇西县育成蒙古黄芪2个品系(‘蒙芪07-22-1’和‘蒙芪07-22-2’)种苗为材料，采用组织研磨法和平板划线法分离纯化获得其内生菌，通过Ashby培养基筛选菌落饱满的固氮菌，通过菌落形态观察，结合16S rRNA基因序列分析进行菌株鉴定。利用多种功能鉴定培养基测定固氮菌的固氮、溶磷、解钾、产吲哚-3-乙酸(indole-3-acetic acid, IAA)和铁载体合成等促生能力。最后通过2次施用有机肥和不施肥连作小麦幼苗促生长盆栽试验，测试固氮菌对连作小麦的促生能力。【结果】 从‘蒙芪07-22-1’中得到2株优良固氮菌株，从‘蒙芪07-22-2’中得到3株优良固氮菌株，经鉴定菌株G2B58为鸡眼草申氏菌(Shinella kummerowiae)；菌株G2B59为放射形农杆菌(Agrobacterium radiobacter)；菌株G2B65为苦马豆根瘤菌(Rhizobium sphaerophysae)；菌株G1B66为维斯氏博斯氏菌(Bosea vestrisii)；菌株G1B89为腓特烈斯堡分枝杆菌(Mycobacterium frederiksbergense)。经筛选得到5株内生固氮菌兼具固氮、溶磷、解钾、产IAA、产铁载体等功能。盆栽试验表明，连作小麦幼苗接种菌株后，菌株G2B58、G2B59和混合处理均能在2次盆栽试验中表现出促生作用，在施肥条件下菌株G2B58处理的株高、茎粗、单株鲜重和单株干重分别显著提高11.65%、9.61%、13.24%、18.92%。【结论】 5株内生固氮菌均有良好促生特性，为研发微生物菌剂提供优质菌种资源。接种菌株G2B58促生效果最好，能显著提高连作小麦幼苗生长指标。连作土壤中施加菌剂时，适量有机肥可以提升黄芪内生菌株的促生活力。

Abstract:

[Background] Astragalus membranaceus var. mongholicus is a genuine medicinal herb in Longxi County, mostly grown in arid and infertile soil. The growth environment determines that the endogenous nitrogen-fixing bacteria of A. membranaceus var. mongholicus have strong stress resistance. [Objective] To explore the endophytic nitrogen-fixing bacterial resources of the new lines of A. membranaceus var. mongholicus, screen strains for efficient joint nitrogen fixation with wheat, mitigate the effects of continuous cropping obstacles on wheat, and reduce the application of chemical fertilizers. [Methods] The seedlings of two A. membranaceus var. mongholicus lines ("Mengqi 07-22-1" and "Mengqi 07-22-2") bred in Longxi, Gansu were used for the isolation of endophytic bacteria by tissue milling and plate streaking. The Ashby solid medium was used for screening of nitrogen-fixing bacteria with full colonies. Strain identification was performed by colony morphology observation combined with 16S rRNA gene sequence analysis. Multifunctional identification media were used to determine the nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, indole-3-acetic acid (IAA)- producing, and siderophore-synthesizing abilities of nitrogen-fixing bacteria. Finally, the ability of nitrogen-fixing bacteria to promote the growth of wheat in continuous cropping was tested by pot experiments with and without the application of organic fertilizers. [Results] Two and three excellent strains of nitrogen-fixing bacteria were obtained from "Mengqi 07-22-1" and "Mengqi 07-22-2", respectively. Strains G2B58, G2B59, G2B65, G1B66, and G1B89 were identified as Shinella kummerowiae, Agrobacterium radiobacter, Rhizobium sphaerophysae, Bosea vestrisii, and Mycobacterium frederiksbergens, respectively. Five strains of endophytic nitrogen-fixing bacteria were screened out for their functions of nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA production, and siderophore production. Pot experiments with wheat seedlings in continuous cropping showed that the inoculation with G2B58, G2B59, and mixed strains promoted wheat growth. The plant height, stem diameter, fresh weight per plant, and dry weight per plant were significantly increased by 11.65%, 9.61%, 13.24%, and 18.92%, respectively, in the G2B58 treatment under fertilization. [Conclusion] All the five strains of endophytic nitrogen-fixing bacteria demonstrate good plant growth-promoting properties, providing high-quality strain resources for the development of microbial agents. Inoculation with G2B58 has the best plant growth-promoting effect and significantly improves the growth indexes of wheat seedlings in continuous cropping. In the case of applying microbial agents in the soil with continuous cropping, moderate application of organic fertilizer can enhance the plant growth-promoting effects of the endophytes of A. membranaceus var. mongholicus.