多花黄精叶点霉叶斑病病原菌鉴定及防治研究

【作者】 崔馨燕；

【导师】 尹福强； 张兴端；

【作者基本信息】 重庆三峡学院 ， 农艺与种业， 2023， 硕士

【摘要】 多花黄精（Polygonatum cyrtonema）,属天门冬科（Asparagaceae）多年生草本植物,被2015年版《中国药典》收录,是一味药食同源的中药材,具有健脾润肺、补气养阴、抗病毒、抗肿瘤等功效,在医疗保健、日常饮食及经济增收等领域发挥着重要作用。随着多花黄精的药用和食用价值的不断开发,其栽培面积增长迅速,2020-2021年,在重庆市万州区一管理粗放的多花黄精种植基地发现一种叶部病害,主要危害老叶,发病初期为近圆形或圆形黄褐色病斑,随着病斑扩大,后期多个小斑扩大成不规则的大斑,严重时多个病斑连成一片甚至造成整株枯死,对多花黄精产业发展带来重大影响。为此,对具有典型症状的叶片进行分离鉴定,明确引起该病害的病原菌种类,并在此基础上研究病原菌生物学特性,筛选化学农药和生物农药,进行多花黄精生防菌的分离与鉴定及其防病效果与促生作用研究,具体研究结果如下:1.对多花黄精叶斑病具有典型症状的植株进行病原菌的分离与纯化,依据致病性测定证明其病原菌具有致病性,结合形态学特征和ITS、ACT和TEF多基因联合分析,确定其病原菌为首都叶点霉（Phyllosticta capitalensis）,这是首都叶点霉（P.capitalensis）侵染多花黄精的首次报道。2.在生物学的研究中,病原菌J2在PDA培养基、25℃、p H 7.0、光暗交替有利于菌株J2菌丝生长,最适碳源、氮源分别为蔗糖和胰蛋白胨。产孢最适条件为:PDA培养基,25℃,p H 7.0,全光照,碳源为葡萄糖,氮源为胰蛋白胨。3.在本文研究选取的9种化学农药中,对菌株J2菌丝抑制效果最强为25%三唑酮,EC₅₀为0.292 mg·L^-1;在5种生物农药中,菌株J2菌丝抑制效果最强为10%多抗霉素,EC₅₀为0.205 mg·L^-1。4.从健康的多花黄精根际土中,采用平板对峙法筛选出对多花黄精叶点霉菌防治效果较强的2株菌株,通过形态学特征、生理生化试验和分子生物学（16S r DNA和gyr B）鉴定,确定菌株ZU-6为荧光假单胞菌（Pseudomonas fluorescens）和ZU-17为毕节假单胞菌（Pseudomonas bijieensis）。菌株ZU-6、ZU-17最佳碳源为葡萄糖和可溶性淀粉;最佳氮源为酵母;最佳无机盐分别为Fe So₄、Mg Cl₂利用率最高;菌株ZU-6、ZU-17最佳培养基分别为NYBD和LB。5.通过单因素和正交试验分析得到菌株ZU-6的最佳发酵条件:发酵时间18 h、温度35℃、p H为7.0-8.0、接菌量3%、接种菌龄20 h、装液量75 m L和摇床转速160 r/min;菌株ZU-17的最佳发酵条件:发酵时间20 h、温度32℃、p H为7.0、接菌量3%、接种菌龄20 h、装液量75 m L和摇床转速160 r/min。6.盆栽试验测定菌株ZU-6和ZU-17发酵液生防效果,发现喷洒菌液ZU-6和ZU-17的感病植株未继续扩大病斑,与对照组相比表现出良好的生防效果。7.2株生防菌在不同温度、p H值和贮藏天数中,无菌滤液抗菌活性较高,具有良好的稳定性;无菌发酵液对种子促生作用研究中,2种菌株的发酵液均可以对4种种子表现出显著的促生效果。更多还原

【Abstract】 Polygonatum cyrtonema,also known as King Solomon’s seal,is a perennial herbaceous plant belonging to the Asparagaceae family.It was included in the 2015 edition of the Chinese Pharmacopoeia and is a medicinal and edible herb with the effects of tonifying the spleen and lungs,nourishing qi and yin,and possessing antiviral and anticancer properties.It plays an important role in healthcare,daily diet,and economic development.With the continuous development of its medicinal and culinary values,the cultivation area of Polygonatum cyrtonema has grown rapidly.However,in 2020–2021,a leaf disease was found in a poorly managed extensive Polygonatum cyrtonema planting base in Wanzhou District,Chongqing City.The disease mainly affects the old leaves,initially appearing as nearly circular or circular yellow-brown spots,which then enlarge and become irregularly-shaped large spots with multiple small spots.In severe cases,multiple spots can connect and cause the entire plant to wither and die.Therefore,the identification and separation of leaves with typical symptoms were carried out to determine the type of pathogenic causing the disease.Based on this,the biological characteristics of the pathogenic were studied,and chemical and biological pesticides were screened.The specific research results of the screening of biocontrol bacteria are as follows:1.The pathogenic bacteria were isolated and purified from plants with typical symptoms of leaf spot disease in Polygonatum cyrtonema.Based on pathogenicity determination,the pathogenic were found to be pathogenic.Combined with morphological characteristics and multi-gene analysis of ITS,ACT and TEF,the pathogenic were determined to be Phyllosticta capitalensis.This is the first report of P.capitalensis infecting Polygonatum cyrtonema.2.In the biological study,the J2 strain of pathogenic showed optimal growth on a PDA medium at 25°C and p H 7.0 with alternating light and dark cycles.The optimal carbon and nitrogen sources were sucrose and peptone,respectively.The optimal conditions for sporulation were PDA medium at 25°C,p H 7.0,and full light,with glucose as the carbon source,and peptone as the nitrogen source.3.Among the 9 chemical pesticides selected in this study,25%triazole had the strongest inhibitory effect on the J2 strain of pathogenic,with an EC₅₀of 0.292 mg·L^-1.Among the 5 biopesticides,10%polyoxin had the strongest inhibitory effect on the J2strain of pathogenic,with an EC₅₀of 0.205 mg·L^-1.4.Two strains of bacteria with stronger control effects on P.capitalensis were screened using the plate confrontation method from the healthy soil of P.multiflorum.Through morphological characteristics,physiological and biochemical tests,and molecular biology（16S r DNA and gyr A）identification,strain ZU-6 was identified as Pseudomonas fluorescens,and strain ZU-17 was identified as Pseudomonas bijieensis.The optimal carbon sources for strains ZU-6 and ZU-17 were glucose and soluble starch,respectively.The optimal nitrogen source was yeast.The optimal inorganic salts were Fe SO₄and Mg Cl₂,respectively.The optimal culture media for strains ZU-6 and ZU-17 were NYBD and LB,respectively.5.Through single-factor and orthogonal experiments analysis,the optimal fermentation conditions for strain ZU-6 are a fermentation time of 18 hours,a temperature of 35°C,p H of 7.0-8.0,an inoculum size of 3%,an inoculum age of 20 hours,a liquid volume of 75 m L,and shaking speed of 160 r/min;the optimal fermentation conditions for strain ZU-17 are fermentation time of 20 hours,the temperature of 32℃,p H of 7.0,inoculum size of 3%,inoculum age of 20 hours,the liquid volume of 75 m L,and shaking speed of 160 r/min.6.A pot experiment was conducted to determine the biocontrol effect of the fermentation broth of strains ZU-6 and ZU-17.It was found that the sprayed broth of ZU-6and ZU-17 on diseased plants showed good biocontrol effects compared to the control group,and the disease spots did not continue to expand.7.Two biocontrol strains were tested for their antibacterial activity in sterile filtrates at different temperatures,p H values,and storage times.The results showed that the antibacterial activity of the sterile filtrate was high and had good stability.In the study of the seed-promoting effect of sterile fermentation broth,the fermentation broth of both strains showed significant promotion effects on four types of seeds.更多还原