【作者】 王星；

【导师】 柳陈坚； 朱红惠；

【作者基本信息】 昆明理工大学 ， 微生物学， 2024， 硕士

【摘要】 茶叶是我国重要的叶用经济作物之一,茶树的种植面积和茶叶产量均居世界首位。随着茶园种植面积的不断扩大和茶园的不规范管理,导致茶树根际微生物群落多样性改变、茶树的病虫害显著增加,严重影响茶叶的产量和品质。本研究以鹤山地区健康和失管茶园茶树根际土壤微生物为研究对象,探究健康和失管茶园中微生物群落结构的差异以及土壤理化性质对微生物群落结构的影响;同时分离鉴定了对茶叶斑病具有高效拮抗活性的微生物资源,并对贝莱斯芽孢杆菌LS9Y的抑菌机制进行了研究,以期为茶树的病害防治提供理论支持。主要结果如下:(1)通过对鹤山地区健康和失管茶园中土壤微生物群落特征进行分析,研究发现在细菌群落组成中变形菌门、酸杆菌门、绿弯菌门、放线菌门为优势门;热酸菌属、鞘鞍醇单胞菌属、慢生根瘤菌属为优势属;且失管茶园的α多样性显著低于健康茶园。在健康茶园和失管茶园中分别检出157和136个核心OTU;并且分别发现了25个和32差异的细菌类群,其中,8个属可作为潜在的生物标志物;细菌功能预测的结果表明健康茶园中18个功能种群的表达量显著高于失管茶园。(2)在根际土壤真菌群落中担子菌门、被孢霉门和子囊菌门是优势门;Saitozyma属和被孢霉属、木霉属为优势属;失管茶园中的土壤真菌群落α多样性指数显著低于健康茶园;健康和失管茶园中检出共有核心OTU是10个;总氮、总磷、有效磷、有机质和水解性氮是真菌β多样性差异的主要环境驱动因子;p H、总磷、交换性镁、交换性钙、有效磷和有效钾等与真菌类群间存在显著相关性;真菌功能预测的结果表明失管茶园中病原营养型、病原-腐生-共生过渡型和共生营养型真菌的相对丰度明显增加;而腐生-共生过渡型真菌的则显著降低。(3)在鹤山古劳镇茶园中发病的叶片上分离得到4株茶树病原真菌,分别属于炭疽菌属、间座壳菌属、藤仓镰刀菌属和叶点霉菌属;采用培养组学技术在鹤山茶园根际土壤与叶片上共分离得到116株细菌;通过对峙实验筛选出了10株对茶树病原真菌具有拮抗活性的细菌,包括芽孢杆菌属3株、伯克霍尔德菌属3株、链霉菌属3株和节杆菌属1株;最终选择拮抗活性强、生物安全性高的芽孢杆菌Bacillus sp.LS9Y进行后续实验。(4)经鉴定Bacillus sp.LS9Y属于贝莱斯芽孢杆菌,具有产生多种酶、IAA和铁载体及溶磷的能力;贝莱斯芽孢杆菌LS9Y的发酵上清滤液(CFS)对茶树病原真菌具有很强的抑制作用,且对其他的植物病原真菌也表现出广谱抗性;菌株LS9Y可能通过破坏真菌的细胞膜和细胞壁,从而抑制茶树病原真菌的生长;菌株LS9Y的CFS及代谢粗提物对茶树病原真菌的侵染具有较好的防控效果。更多还原

【Abstract】 Tea is one of the most important economic crops.The area of plantation and yield of tea rank first in the world.With the continuous expansion of tea plantation area and nonstandard management of tea plantation,change of the diversity of tea rhizosphere microbial communities and increase of diseases and pests has seriously affected the yield and quality of tea.In this study,we focused on the rhizosphere soil microorganisms of tea trees in healthy and mismanaging tea plantations of Heshan black tea production to investigate the differences in microbial community structure between healthy and mismanaging tea plantation and the impact of soil physicochemical properties on microbial community structure.We further screened the microbial resources with efficient antagonistic activity against the pathogen of tea spot disease and explored the antibacterial mechanism of Bacillus sp.LS9Y.Our results provided a theoretical support for controlling tea diseases.The main results are as follows:（1）Based on the analysis of soil microbial diversity in healthy and mismanaging tea plantation in Heshan area,we found that Proteobacteria,Acidobacteria,Chlorobacteria,and Actinobacteria were dominant phyla in bacterial community composition.The analysis results at the genus level indicate that the dominant genera were Acidothermus,Sphingomonas,Bradyrhizobium.Theαdiversity index were significantly decreased in mismanaging tea plantation compared with healthy tea plantation.Respectively,157 and 136 core OTUs were detected in healthy tea plantation and mismanaging tea plantation.In addition,25 and 32 characterizing bacterial taxa were identified in health and mismanaging plantation.Eight genera in two plantations can be used as potential biomarkers.Besides,bacterial function predicted 18 functional groups with significant differences in health and mismanaging plantations.（2）Basidiomycota,Mortierellomycota and Ascomycota were dominant phyla in the rhizosphere soil fungal community;Saitozyma genus,Mortierella genus,and Trichoderma genus were dominant genera;Theαdiversity index were significantly decreased in mismanaging tea plantation compared with the health tea plantation.The total nitrogen,total phosphorus,available phosphorus,organic matter and hydrolyzed nitrogen were the primary driving factors of soil fungalβdiversity.Ten of OTUs was common between health and mismanaging tea plantation.The p H,total phosphorus,exchangeable Ca²⁺and Mg²⁺,available phosphorus and available potassium were significantly correlated with the fungal species.The relative abundance of pathotroph,pathotroph-saprotroph-symbiotroph and symbiotroph fungi was significantly increased,but the abundance of saprotroph-symbiotroph was decreased in mismanaging tea plantation.（3）We characterized four tea pathogenic fungi from the diseased leaves of the tea plantation in Gulao Town,including Colletotrichum siamense HT-1,Diaporthe phaseolorum HT-3,Fusarium fujikuroi HT-4,Phyllosticta captalensis HT-5.Furthermore,116 bacterial strains were isolated from the ecosystem of Heshan tea production using culturomics techniques.Based on the antifungal activity,ten bacteria were screened using antagonistic assay,including three strains of Bacillus,three strains of Burkholderia,three strains of Streptomyces,and one strain of Arthrobacter.We ultimately chose Bacillus sp.LS9Y for subsequent experiments,which has strong antagonistic activity and high biological safety.（4）The results of phylogenetic analysis suggested that LS9Y belonged to Bacillus velezensis,which could generate multiple enzymes,IAA,iron carrier,and solubilized phosphorus.It was found that the fermentation supernatant filtrate（CFS）of B.velezensis LS9Y exhibited antibacterial activity and broad-spectrum resistance to other pathogenic fungi;In addition,the results of fungal co-culture suggested that LS9Y may inhibit the growth of tea plant pathogenic fungi by disrupting their cell membranes and walls;It was also found that the CFS and metabolic crude extract of LS9Y have good control effects on tea plant pathogenic fungi更多还原