本文選題：雞腿菇 + 氧化木糖無色桿菌　； 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：雞腿菇(Coprinus comatus)是一種風味和口感十分獨特的美味食用菌,營養(yǎng)和藥用價值高,深受消費者的喜愛。雞腿菇從菌絲生長到子實體形成耗時較短,同時生物轉(zhuǎn)化效率非常高,栽培過程簡單容易掌握。但栽培的一系列過程中病蟲害發(fā)生嚴重,常造成慘重的經(jīng)濟損失。特別是細菌性病害發(fā)生快、蔓延快和傳播范圍廣,防治相當困難。本課題研究了雞腿菇菌柄腐爛病的發(fā)生規(guī)律,后根據(jù)柯赫氏法則對病原分離物的致病性進行驗證,同時鑒定病原物所屬屬名和種名,并將分離的病原物和對應(yīng)的臨床標準菌株進行了動物致病性驗證。采集不同地點的土壤樣品,分析病原物的來源。篩選植物提取物和生防細菌,進行了雞腿菇菌柄腐爛病防治初探。為保障雞腿菇產(chǎn)業(yè)良好發(fā)展,進一步對細菌性病害進行防治,食品安全研究工作奠定一定的基礎(chǔ)。2015年7月在山東省平陰縣雞腿菇栽培場內(nèi)發(fā)現(xiàn)一種雞腿菇菌柄腐爛病。從發(fā)病部位分離純化到了3種細菌菌株,分別命名為JTG-A,JTG-B1,JTG-B2。根據(jù)柯赫氏法則,驗證3種分離物的致病性,采用磨傷接種的方式,分為四組進行接種,每組25個健康的雞腿菇子實體,重復(fù)4次,在接種16 h、36 h和52 h后連續(xù)觀察發(fā)病情況。結(jié)果顯示,JTG-B1在16 h后可使50%以上的子實體菌柄發(fā)病,形成明顯的病斑,并有菌膿出現(xiàn),后期整個子實體腐敗,直至死亡。JTG-A和JTG-B2接種后僅少數(shù)子實體出現(xiàn)癥狀,致病力明顯低于JTG-B1。對JTG-B1采用菌落形態(tài)觀察、生理生化反應(yīng)和16s r DNA序列分析進行鑒定,確定其為人體病原菌,氧化木糖無色桿菌(Achromobacter xylosoxidans)。從華中科技大學醫(yī)學院附屬同濟醫(yī)院得到對應(yīng)的臨床標準菌株TJ-ade,采用腹腔注射小白鼠驗證JTG-B1和TJ-ade的動物致病性。結(jié)果顯示80%JTG-B1懸浮液和20%生理鹽水混合物腹腔注射小白鼠,在21 d后小白鼠進食率為33%,TJ-ade在相同處理下進食率為29%。小鼠在各處理組中均表現(xiàn)出明顯的不活躍性,眼緊閉,有抱團現(xiàn)象,體毛有脫落現(xiàn)象,但未出現(xiàn)死亡現(xiàn)象。對兩個菌株最適p H和溫度進行測定,JTG-B1和TJ-ade在16-34℃時均可生長,28℃為最適生長溫度,在p H4-11之間兩者均可生長,p H7為最適生長p H值。對JTG-B1和TJ-ade的7個管家基因nus A、rpoB、eno、glt B、lepA、nuoL、和nrd A進行擴增,測序后輸入MLST數(shù)據(jù)庫,將等位基因編號進行一對一的比對,從而確定對應(yīng)的ST型。PCR擴增后對產(chǎn)物進行瓊脂糖凝膠電泳,可知管家基因擴增片段大小為300-400 bp,JTG-B1和TJ-ade分別為ST14和ST29。采用上述相同方法進行雞腿菇子實體致病性實驗,TJ-ade在16h時磨傷接種也能使50%以上的健康雞腿菇子實體菌柄發(fā)病,癥狀與JTG-B1接種后相同。生理生化反應(yīng)觀察兩者對不同糖類分解情況,CIT(檸檬酸鹽利用試驗),VP(丙酮酸鹽試驗),GLU(葡萄糖發(fā)酵試驗),SAC(蔗糖發(fā)酵試驗),AMY(苦杏仁苷發(fā)酵試驗)和ARA(阿拉伯糖試驗)的反應(yīng)均為陽性,但JTG-B1所有陽性反應(yīng)小管的顏色反應(yīng)均深于TJ-ade,可知其代謝反應(yīng)速率和相關(guān)酶類表達量高于TJ-ade,結(jié)合動物致病性實驗,可綜合分析兩者種間差異情況。從山東省平陰縣雞腿菇栽培基地周邊的7處位置采集土壤樣品,提取土壤總DNA,擴增目的病原菌JTG-B1的bla OXA-114基因。洞穴外地面土壤PCR反應(yīng)產(chǎn)物在電泳時出現(xiàn)了大小500 bp左右的條帶,表明病原物可能在栽培場地周邊存在,通過人為攜帶、覆土材料或栽培料等進入栽培場地中。制備大蒜、梧桐樹葉、銀杏、蒲空、龍葵、茶葉、肉桂油、石榴皮和辣椒等植物的提取物。結(jié)果顯示,僅肉桂油在濃度100 mg/m L和120 mg/m L時,可以對病原菌產(chǎn)生直徑1.0 mm和9.0 mm的抑菌圈。熒光假單胞菌(Pseudomonas fluorescens)和惡臭假單胞菌(Pseudomonas putida)發(fā)酵液對病原菌沒有抑制作用。120 mg/m L肉桂油對雞腿菇菌絲生長無影響,且在12 h和24 h時肉桂油在雞腿菇子實體上可以預(yù)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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S436.46

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