定殖植物根内和根围放线菌的分离鉴定及其体外抑菌促生效应

Isolation and identification of antagonizing and growth-promoting actinobacteria colonized in plant roots and rhizosphere

摘要:

[目的]旨在分离、筛选并鉴定体外具抑菌促生作用的定殖于植物根内和根围的放线菌，以期丰富放线菌种质资源，为研制植物病害生防菌剂提供技术依据。[方法]采用稀释涂布平板法分离盐碱地、湿地、工业污染土壤中优势植物根内及其根围中的放线菌；通过平板对峙试验筛选具有抑菌效应的菌株，进而采用Salkowski比色法、CAS平板检测法和无氮源培养法进一步检测抑菌菌株的促生作用；通过形态观测、生理生化特性检测及16S rRNA基因序列分析鉴定菌种。[结果]共分离到链霉菌属（Streptomyces）、诺卡氏菌属（Nocardia）和小单孢菌属（Micromonospora）3属共283株定殖于植物根内和根围的放线菌，3个采样地中湿地数量最多，均为根围土>根内；其中链霉菌属占总数的77%，可分为10个类群。经筛选获得7株抑菌活性和促生效应较强的菌株，其中菌株H6-1抑菌效应最大，其无菌发酵液对尖孢镰刀菌（Fusarium oxysporum）、禾谷镰刀菌（Fusarium graminearum）、灰葡萄孢菌（Botrytis cinerea）、立枯丝核菌（Rhizoctonia solani）、轮纹大茎点霉（Macrophoma kawatsukai）和瓜类炭疽菌（Colletotrichum orbiculare）的抑制率分别为32.3%、42.6%、48%、72.2%、58.1%和60.5%；而D11-4菌株的促生作用最强，能产吲哚乙酸（22.3 mg/L）、产铁载体（晕圈直径25.2 mm）和固氮。经鉴定这7株放线菌是吸水链霉菌变种（Streptomyces angustmyceticus）H4-6、娄彻氏链霉菌（Streptomyces rochei）S2-2、浑圆链霉菌（Streptomyces globosus）H6-1、（Streptomyces iakyrus）GD8-4、波卓链霉菌（Streptomyces bottropensis）GH8-6、（Streptomyces paradoxus）H8-2和（Streptomyces coralus）D11-4。[结论]三个生境中定殖于植物根内和根围的放线菌类群丰富且所筛选获得的7株放线菌具有生防潜力，值得进一步研发。

Abstract:

[Objective] The aim of this study was to enrich species resources of actinobacteria colonized in plant roots and rhizosphere through isolating, screening and identifying and to provide resources for the development of biocontrol agents via examining the effects of these actinobacteria on antagonizing plant pathogens and promoting plant growth. [Methods] Actinobacteria were isolated from roots and rhizosphere of dominant herbaceous plants grown in three habitats (saline-sodic, wet, and polluted land) using the dilution plate method. The antibacterial strains were screened by plate confrontation test. Then Salkowski colorimetry, Chrome Azurol S plate assay and nitrogen free culture were used to further detect their effect on promoting plant growth. The isolated strains were examined by the combined morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis for taxonomical identification. [Results] A total of 283 isolates of actinobacteria colonized in plant roots and rhizosphere, belonging to genera Streptomyces, Nocardia and Micromonospora, were obtained. Among them, 77% belonged to 10 groups of the genus Streptomyces. Seven strains showed strong antagonizing activity and plant growth-promoting effects. Strain H6-1 gave the most significant antagonizing effect among the seven strains, and its fermentation broths inhibition ratio to pathogens Fusarium oxysporum, Fusarium, Botrytis cinerea, Rhizoctonia solani, Macrophoma kawatsukai, and Colletotrichum orbiculare were 32.3%, 42.6%, 48%, 72.2%, 58.1% and 60.5%, respectively. Strain D11-4 showed the strongest plant growth-promoting effect, which was found to be capable of producing IAA (22.3 mg/L), iron carrier (halo diameter 25.2 mm) and fixing nitrogen from air. The seven species of the actinobacteria were identified as Streptomyces angustmyceticus H4-6, Streptomyces rochei S2-2, Streptomyces globosus H6-1, Streptomyces iakyrus GD8-4, Streptomyces bottropensis GH8-6, Streptomyces paradoxus H8-2, and Streptomyces coralus D11-4. [Conclusion] The three habitats had abundant species of actinobacteria colonized in plant roots and rhizosphere, and the seven selected actinobacteria isolates were proven to possess biocontrol potential, which is worth of further research and development.