鸡腿菇菌柄腐烂病病原鉴定与防治技术初探

发布时间：2018-06-24 15:36

本文选题：鸡腿菇 + 氧化木糖无色杆菌　；参考：《华中农业大学》2017年硕士论文

【摘要】：鸡腿菇(Coprinus comatus)是一种风味和口感十分独特的美味食用菌,营养和药用价值高,深受消费者的喜爱。鸡腿菇从菌丝生长到子实体形成耗时较短,同时生物转化效率非常高,栽培过程简单容易掌握。但栽培的一系列过程中病虫害发生严重,常造成惨重的经济损失。特别是细菌性病害发生快、蔓延快和传播范围广,防治相当困难。本课题研究了鸡腿菇菌柄腐烂病的发生规律,后根据柯赫氏法则对病原分离物的致病性进行验证,同时鉴定病原物所属属名和种名,并将分离的病原物和对应的临床标准菌株进行了动物致病性验证。采集不同地点的土壤样品,分析病原物的来源。筛选植物提取物和生防细菌,进行了鸡腿菇菌柄腐烂病防治初探。为保障鸡腿菇产业良好发展,进一步对细菌性病害进行防治,食品安全研究工作奠定一定的基础。2015年7月在山东省平阴县鸡腿菇栽培场内发现一种鸡腿菇菌柄腐烂病。从发病部位分离纯化到了3种细菌菌株,分别命名为JTG-A,JTG-B1,JTG-B2。根据柯赫氏法则,验证3种分离物的致病性,采用磨伤接种的方式,分为四组进行接种,每组25个健康的鸡腿菇子实体,重复4次,在接种16 h、36 h和52 h后连续观察发病情况。结果显示,JTG-B1在16 h后可使50%以上的子实体菌柄发病,形成明显的病斑,并有菌脓出现,后期整个子实体腐败,直至死亡。JTG-A和JTG-B2接种后仅少数子实体出现症状,致病力明显低于JTG-B1。对JTG-B1采用菌落形态观察、生理生化反应和16s r DNA序列分析进行鉴定,确定其为人体病原菌,氧化木糖无色杆菌(Achromobacter xylosoxidans)。从华中科技大学医学院附属同济医院得到对应的临床标准菌株TJ-ade,采用腹腔注射小白鼠验证JTG-B1和TJ-ade的动物致病性。结果显示80%JTG-B1悬浮液和20%生理盐水混合物腹腔注射小白鼠,在21 d后小白鼠进食率为33%,TJ-ade在相同处理下进食率为29%。小鼠在各处理组中均表现出明显的不活跃性,眼紧闭,有抱团现象,体毛有脱落现象,但未出现死亡现象。对两个菌株最适p H和温度进行测定,JTG-B1和TJ-ade在16-34℃时均可生长,28℃为最适生长温度,在p H4-11之间两者均可生长,p H7为最适生长p H值。对JTG-B1和TJ-ade的7个管家基因nus A、rpoB、eno、glt B、lepA、nuoL、和nrd A进行扩增,测序后输入MLST数据库,将等位基因编号进行一对一的比对,从而确定对应的ST型。PCR扩增后对产物进行琼脂糖凝胶电泳,可知管家基因扩增片段大小为300-400 bp,JTG-B1和TJ-ade分别为ST14和ST29。采用上述相同方法进行鸡腿菇子实体致病性实验,TJ-ade在16h时磨伤接种也能使50%以上的健康鸡腿菇子实体菌柄发病,症状与JTG-B1接种后相同。生理生化反应观察两者对不同糖类分解情况,CIT(柠檬酸盐利用试验),VP(丙酮酸盐试验),GLU(葡萄糖发酵试验),SAC(蔗糖发酵试验),AMY(苦杏仁苷发酵试验)和ARA(阿拉伯糖试验)的反应均为阳性,但JTG-B1所有阳性反应小管的颜色反应均深于TJ-ade,可知其代谢反应速率和相关酶类表达量高于TJ-ade,结合动物致病性实验,可综合分析两者种间差异情况。从山东省平阴县鸡腿菇栽培基地周边的7处位置采集土壤样品,提取土壤总DNA,扩增目的病原菌JTG-B1的bla OXA-114基因。洞穴外地面土壤PCR反应产物在电泳时出现了大小500 bp左右的条带,表明病原物可能在栽培场地周边存在,通过人为携带、覆土材料或栽培料等进入栽培场地中。制备大蒜、梧桐树叶、银杏、蒲空、龙葵、茶叶、肉桂油、石榴皮和辣椒等植物的提取物。结果显示,仅肉桂油在浓度100 mg/m L和120 mg/m L时,可以对病原菌产生直径1.0 mm和9.0 mm的抑菌圈。荧光假单胞菌(Pseudomonas fluorescens)和恶臭假单胞菌(Pseudomonas putida)发酵液对病原菌没有抑制作用。120 mg/m L肉桂油对鸡腿菇菌丝生长无影响,且在12 h和24 h时肉桂油在鸡腿菇子实体上可以预防病原菌感染。
[Abstract]:Coprinus comatus is a delicious edible fungus with unique flavor and taste. It has high nutritional and medicinal value. It is deeply loved by consumers. The growth of the coprinus comatus from mycelia to the fruiting body is short, the efficiency of biological transformation is very high, and the cultivation process is easy to be grasped. Serious, often causing heavy economic loss. Especially, bacterial diseases occur quickly, spread quickly and spread widely. The prevention and control of the bacterial stalk rot of Coprinus comatus is very difficult. The pathogenesis of the stalk rot of Coprinus comatus is studied, and the pathogenicity of the pathogen is verified by the Coch's rule, and the name and name of the genus are identified, and the separation will be separated. The pathogenic bacteria and the corresponding clinical standard strains were tested for animal pathogenicity. The soil samples in different locations were collected, the source of the pathogens were analyzed, the plant extracts and biocontrol bacteria were screened. The prevention and control of the stalk rot disease of Coprinus comatus was carried out to protect the development of the coprinus comatus industry and the prevention and control of the bacterial diseases. The safety research work laid a foundation for the discovery of a kind of Coprinus comatus stalk rot disease in the Pingyin County of Shandong Province in July. 3 strains of bacterial strains were isolated and purified from the site of the disease, named JTG-A, JTG-B1, and JTG-B2., according to Kirsch's rule, to verify the pathogenicity of the 3 species, and the inoculation method was adopted. There were four groups of inoculation in four groups, 25 healthy Coprinus Comus fruiting bodies in each group, repeated 4 times. After inoculation of 16 h, 36 h and 52 h, the incidence of the disease was observed continuously. The results showed that after 16 h, more than 50% of the subsolid stalk could cause the pathogenesis of the subfruiting body, formed a distinct spot, and had the occurrence of bacterial pus, and later the whole body was corrupt, until the death.JTG-A and JTG-B2 inoculation. Only a few of the offspring had symptoms, and the pathogenicity was obviously lower than that of JTG-B1.. JTG-B1 was observed by colony morphology, physiological and biochemical reactions and 16S R DNA sequence analysis to identify it as human pathogens and Achromobacter xylosoxidans (Achromobacter xylosoxidans). From the affiliated Tongji Hospital of the Medical College of Huazhong University of Science and Technology, the corresponding presence was obtained. The standard strain TJ-ade of the bed was injected into the mice by intraperitoneal injection to verify the pathogenicity of JTG-B1 and TJ-ade. The results showed that the mixture of 80%JTG-B1 suspension and 20% physiological saline was intraperitoneally injected with mice, the feeding rate of mice was 33% after 21 d, and the eating rate of TJ-ade under the same treatment was obviously inactive in the treatment groups. Sex, eye tight, hairs and body hair fall off, but there is no death. The optimum P H and temperature for two strains are measured, JTG-B1 and TJ-ade can grow at 16-34 C, 28 C is the optimum growth temperature, both between P and H4-11 can grow, P H7 is the optimum P H value. JTG-B1 and TJ-ade 7 housekeeping genes B, Eno, GLT B, lepA, nuoL, and NRD A were amplified and sequenced, and then the allele number was compared to the MLST database, and then the corresponding ST type.PCR amplification was determined by agarose gel electrophoresis. The size of the gene amplification fragment of the housekeeper was 300-400 BP. Methods the pathogenicity experiment of the coprinus comatus fruiting body was carried out. The inoculation of TJ-ade at 16h could also cause more than 50% of the healthy Coprinus comatus. The symptoms were the same as that after the JTG-B1 inoculation. The physiological and biochemical reactions observed the decomposition of different carbohydrates, CIT (citrate test), VP (pyruvate test), GLU (glucose fermentation test). The reaction of SAC (sucrose fermentation test), AMY (bitter amygdalin fermentation test) and ARA (Arabia sugar test) were all positive, but the color reaction of all JTG-B1 positive tubules was deeper than that of TJ-ade. The metabolic reaction rate and the expression of related enzymes were higher than TJ-ade, and the pathogenicity experiment of nodal animals could be used to analyze the difference of the two species. Soil samples were collected from 7 locations around the culture base of Coprinus comatus in Pingyin County, Shandong province. The total DNA of the soil was extracted and the BLA OXA-114 gene of the target pathogen JTG-B1 was amplified. The PCR reaction product of the cave field surface soil appeared in the strip of about 500 BP at the time of electrophoresis, indicating that the disease originals may exist around the cultivation site and are carried by human beings. The extracts of garlic, Chinese parasol leaves, Ginkgo biloba, Pubei, dragon anflower, tea, cinnamon oil, pomegranate skin and capsicum were prepared. The results showed that only cinnamon oil could produce 1 mm and 9 mm diameters of 1 mm and 9 mm when the concentration of Cinnamomum cinnamomi oil was 100 mg/m L and 120 mg/m L. Onas fluorescens) and malodus Pseudomonas (Pseudomonas putida) fermentation broth did not inhibit the pathogenic bacteria..120 mg/m L cinnamon oil had no effect on the growth of the mycelium of Coprinus Comus, and cinnamon oil could prevent the pathogen infection at 12 h and 24 h in the coprinus comatus fruiting body.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.46

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