一株分泌型铁载体真菌分离鉴定及生物活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0394

摘要/Abstract

摘要：

从云南省国家自然保护区哀牢山原始森林土壤环境中分离1株分泌型铁载体真菌,并探讨了其对罹烟草青枯病土壤微生物生理生化功能的影响,为菌株的开发利用提供理论依据。稀释涂布法从土壤样品中分离纯化分泌型铁载体真菌菌株,形态学结合rDNA-ITS基因序列鉴定菌株分类学地位。铬天青S（chromeazurol S,CAS）液检测法结合全波段紫外光吸收法,判定铁载体化学结构类型,检测其活性。DNS比色法和Biolog-ECO分析法,分析菌株次生代谢产物对罹烟草青枯病土壤酶活性和微生物代谢的影响。结合形态学观察结果和序列对比分析鉴定该分泌型铁载体真菌为百岁兰曲霉（Aspergillus welwitschiae）;菌株分泌型铁载体的化学结构类型为羧酸盐型（carboxylates）,铁载体活性为78.79%。菌株次生代谢产物可显著提高土壤蔗糖酶和脲酶活性（P<0.05）,分别提高13.7%和14.46%;菌株次生代谢产物亦可显著提高碳源利用能力：平均颜色变化率（average well color development,AWCD）和多样性指数（Shannon指数和Richness指数）,分别提高86.15%、50.43%和66.67%。原始森林土壤环境贮藏铁载体微生物资源,利用菌株分泌型铁载体介导改善土壤微生物群落生理生化功能是实施土壤生态系统的优化管理的重要技术手段。

关键词: 原始森林土壤, 分泌型铁载体真菌, 烟草青枯病, 土壤酶, Biolog-ECO

Abstract:

A secretory siderophore producing fungus was isolated from the soil environment of Ailao Mountain primeval forest in Yunnan National Nature Reserve,and its effects on the physiological and biochemical functions of soil microorganisms infected with tobacco bacterial wilt were studied,which provides a theoretical basis for the development and utilization of the strain. Dilution coating method was used to isolate and purify the fungus producing the secretory siderophore from soil sample,the taxonomic status of the strain was identified by morphology and rDNA-ITS gene sequence. Chromazurol S（CAS）solution detection combined with full band ultraviolet absorption method was to determine the chemical structure type and activity of siderophore. DNS colorimetric method and Biolog-ECO analysis method were used to analyze the effects of strain secondary metabolites on the enzyme activity and microbial metabolism of tobacco bacterial wilt soil. The secretory siderophore-producing fungus was identified as Aspergillus welwitschiae based on morphological observations and sequence alignment analysis;the chemical structure type of the secretory siderophore of the strain was carboxylates,and its siderophore activity was 78.79%. The secondary metabolites of the strain significantly increased the activities of soil invertase and urease（P<0.05）by 13.7% and 14.46% respectively. The secondary metabolites of the strain also significantly improved the carbon source utilization capacity：Average color change rate（AWCD）and diversity index（Shannon index and richness index）increased by 86.15%,50.43% and 66.67% respectively. In conclusion,siderophore microbial resources are stored in primeval forest soil environment. The improvement of physiological and biochemical functions of the soil microbial community mediated by strain-secreted siderophore is an important technical means to optimize the management of the soil ecosystem.

Key words: soil in primeval forest, siderophore, Ralstonia solanaceous, soil enzyme, Biolog-ECO

邹雪峰, 李铭刚, 包玲风, 陈齐斌, 赵江源, 汪林, 濮永瑜, 郝大程, 张庆, 杨佩文. 一株分泌型铁载体真菌分离鉴定及生物活性研究[J]. 生物技术通报, 2022, 38(3): 130-138.

ZOU Xue-feng, LI Ming-gang, BAO Ling-feng, CHEN Qi-bin, ZHAO Jiang-yuan, WANG Lin, PU Yong-yu, HAO Da-cheng, ZHANG Qing, YANG Pei-wen. Isolation and Identification of a Secretory Siderophore Fungus,and Its Biological Activity[J]. Biotechnology Bulletin, 2022, 38(3): 130-138.

图/表 8

图1 菌株平板培养（PDA与CAS平板） A：1-5F1菌株;B：1-5F1菌株接种于双层平板培养（下层为添加铬天青S（chromeazurol S,CAS）的水琼脂培养基,上层为无铁查氏固体培养基）

Fig. 1 Plate culture of the strain（PDA and CAS plate） A：1-5F1 strain. B：1-5F1 strain was inoculated in double plate culture（The lower layer was water agar medium supplemented with chromeazurol S（CAS）,and the upper layer was solid medium without iron）

图2 1-5F1菌株产铁载体在全波段下吸光度值

Fig.2 Absorbance values of strain 1-5F1 siderophores in full band

图3 菌株分生孢子电镜形态 A：10 000倍;B：2 500倍

Fig. 3 Microscopic morphologies of conidia of the strain A：10 000 times. B：2 500 times

图4 菌株1-5F1基于ITS基因序列构建的系统发育进化树系统发育进化树采用邻接法构建,自展数为1 000。每隔节点上的数值表示自展数的贡献值。每个菌株名称后括号中的数字是GenBank中的ITS基因序列登录号

Fig. 4 Phylogenetic tree of strain 1-5F1 based on ITS gene sequence The phylogenetic evolutionary tree is constructed by the adjacent method,and the self-propagating number was 1 000. The values at each of the nodes refer to the contribution values of the self-expanding number. The number in parenthesis after each strain name is ITS gene sequence entry number in GenBank

图5 1-5F1菌株代谢物对烟草青枯病根际土壤蔗糖酶（S-SC）与脲酶（S-UE）活力的影响

Fig.5 Effects of metabolites of 1-5F1 strain on the activities of sucrase（S-SC）and urease（S-UE）in the rhiz-osphere soil of tobacco bacterial wilt

图6 1-5F1菌株代谢物对烟草青枯病根际土壤平均颜色变化率（AWCD）随时间的变化

Fig.6 Mean color change rate（AWCD）of tobacco bacte-rial wilt rhizosphere soil treated by the metabolites of 1-5F1 strain with time

图7 菌株不同浓度代谢物处理下土壤微生物群落对碳源利用能力的差异

Fig.7 Differences in carbon source utilization abilities of soil microbial communities treated with different concentrations of metabolites of bacterial strains

图8 菌株不同浓度代谢物处理下土壤微生物功能多样性指数随时间的变化

Fig.8 Changes in soil microbial functional diversity index with time under different concentrations of metabolites from the strain

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