發(fā)布時間：2018-04-26 18:35

  本文選題：葡萄霜霉病 + 暗黑鏈霉菌��； 參考：《沈陽農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：葡萄霜霉病是由葡萄生單軸霉Plasmopara viticola (Berk. Curtis) Berl. de Toni引起的真菌性病害,分布廣泛且危害嚴(yán)重,是造成葡萄生產(chǎn)損失的主要原因之一。目前,化學(xué)藥劑仍是防治葡萄霜霉病的主要措施,但大量、持續(xù)、單一品種化學(xué)藥劑的使用導(dǎo)致3R問題日益突出。生物防治因其對環(huán)境友好、人畜安全及防治可持續(xù)性而受到廣泛關(guān)注和高度重視。本研究目的是篩選對葡萄霜霉病具有防控效果的生防菌,并探究其生防機(jī)制,為葡萄霜霉病生防制劑的研制和應(yīng)用提供有效菌株和技術(shù)指導(dǎo)。1.生防放線菌的分離與鑒定。對采自遼寧、云南和西藏等地葡萄園區(qū)的53份土壤樣品樣進(jìn)行分離,共獲得151株放線菌。采用平板篩選法以辣椒疫霉Plasmopara capsici為指示菌進(jìn)行初篩,采用離體葉片法以葡萄霜霉病菌Plasmopara viticola為指示菌進(jìn)行復(fù)篩,共獲得28株對兩種卵菌門真菌均有顯著拮抗作用的放線菌,其中菌株P(guān)Y-1的拮抗作用最強(qiáng),其發(fā)酵液對葡萄霜霉病的抑菌率為91.11%。采用傳統(tǒng)分類、化學(xué)分類與分子分類相結(jié)合的方法,鑒定PY-1菌株為暗黑鏈霉菌(Streptomyces atratus Higashide et al.), GenBank序列登錄號為KJ627770.1。抑菌譜試驗表明,菌株P(guān)Y-1對葡萄灰霉病菌Botrytis cinerea、小麥根腐病菌Bipolaris sorokiniana、番茄晚疫病Phytophthora infestans、辣椒枯萎病菌Fusarium oxysporum、黃瓜炭疽病菌Colletotrichum orbiculare、番茄紅粉病菌Trichothecium roseurn、茄子莖基腐病菌Rhizoctonia solani、高粱彎孢菌Curvularia caryopsida、小麥赤霉菌Fusarium graminearum均有不同程度的抑制作用。2.PY-1菌株發(fā)酵條件優(yōu)化。通過單因子試驗和正交試驗優(yōu)化生防放線菌PY-1液體發(fā)酵培養(yǎng)基的營養(yǎng)成分和發(fā)酵條件。確定最適培養(yǎng)基成分為玉米粉5%,葡萄糖0.5%,蛋白胨0.5%,氯化銨0.5%,氯化鈉0.05%；最適發(fā)酵條件為在250mL三角瓶的裝液量為90mL,接種量為5%,搖床轉(zhuǎn)速為180r/min,培養(yǎng)溫度28℃,初始培養(yǎng)液的酸堿度為pH7.0,發(fā)酵培養(yǎng)時間為5d。對比優(yōu)化前后的培養(yǎng)基組分和培養(yǎng)條件,優(yōu)化后的發(fā)酵液對葡萄霜霉病的抑菌率提高了8.35%。3.PY-1菌株抑菌活性物質(zhì)穩(wěn)定性分析。生防放線菌PY-1抑菌活性物質(zhì)為胞外次級代謝產(chǎn)物。發(fā)酵濾液抑菌活性穩(wěn)定性試驗結(jié)果表明：溫度低于65℃時抑菌活性穩(wěn)定;對40W日光燈穩(wěn)定,在太陽光下照射72h后,抑菌活性開始下降；pH6-10環(huán)境下穩(wěn)定,強(qiáng)酸或強(qiáng)堿均影響其發(fā)酵濾液的抑菌活性；4℃下保存6個月抑菌活性無明顯變化,常溫下保存4個月后,抑菌活性開始下降；蛋白酶溶液對其抑菌活性無明顯影響;金屬離子導(dǎo)致抑菌活性下降。4.PY-1菌株抑菌機(jī)理及田間防效評價。生防放線菌PY-1發(fā)酵濾液能夠?qū)е缕咸阉�霉病菌孢子囊和孢子囊梗出現(xiàn)褶皺、破裂和畸形,進(jìn)而喪失侵染功能。放線菌PY-1代謝產(chǎn)物中包含幾丁質(zhì)酶、蛋白酶、嗜鐵素、ACC脫氨酶、HCN、IAA,不含纖維素酶。田間防效試驗表明,生防放線菌PY-1發(fā)酵原液對葡萄霜霉病的田間中期防效可達(dá)到90%以上,末期防效達(dá)86%以上,比52.5%抑快凈2000倍液略低,但明顯高于58%甲霜錳鋅1000倍液；PY-1菌株發(fā)酵液稀釋700倍液對葡萄霜霉病的末期防效與甲霜錳鋅1000倍液防效相當(dāng)。5.PY-1菌株抑菌活性物質(zhì)的分離純化及結(jié)構(gòu)鑒定。PY-1發(fā)酵慮液經(jīng)二氯甲烷萃取、薄層層析、硅膠柱層析、葡聚糖凝膠柱層析和HPLC進(jìn)行分離純化,獲得兩種對葡萄霜霉病菌具有很強(qiáng)抑制活性的化合物純品PY1-7-1和PY1-7-2。抑菌試驗結(jié)果顯示,不同稀釋濃度的PY1-7-1和PY1-7-2 (10-2mg·mL-1、10-4mg·mL-1、10-6mg·mL-1)對葡萄霜霉病菌的抑菌率分別為92.59%、86.30%、64.81%和97.04%、91.85%、84.07%。采用ESI-MS、1HNMR、13CNMR波譜分析技術(shù)對活性組分進(jìn)行結(jié)構(gòu)解析,確定PY1-7-1化合物分子量為339,分子式為C17H25NO6,化學(xué)名稱為5-Acetoxycycloheximide; PY1-7-2化合物分子量為281,分子式為C15H23NO4,化學(xué)名稱為Cycloheximide。
[Abstract]:Grape downy mildew is a fungal disease caused by Plasmopara viticola (Berk. Curtis) Berl. de Toni of Vitis Vitis, which is widely distributed and seriously harmful. It is one of the main causes for the loss of grape production. At present, chemical agents are still the main measures to prevent downy mildew of grape, but a large number, continuous, single variety chemical agents are made. The problem of 3R is becoming more and more prominent. Biological control is widely concerned and highly valued for its environmental friendliness, human and animal safety and the sustainability of prevention and control. The purpose of this study is to screen the biocontrol bacteria that have the control effect on the grape downy mildew, and to explore the biological control mechanism to provide effective strains for the development and application of the biocontrol preparations for the grape frosting disease. And technical guidance for isolation and identification of.1. actinomycetes. 151 actinomycetes were isolated from 53 soil sample samples collected from Liaoning, Yunnan and Tibet and other places, and 151 strains of actinomycetes were obtained by the plate screening method, and the isolated leaf blade method was used for the grape downy mildew fungus Plasmopara vitic. Ola was used to resieve the indicative bacteria, and 28 actinomycetes had a significant antagonistic effect on two species of actinomycetes. The antagonistic effect of strain PY-1 was the strongest. The bacteriostasis rate of the fermentation broth on downy mildew was 91.11%. by traditional classification, chemical classification and molecular classification, and PY-1 strain was identified as Streptomyces dark Streptomyces (Strept) Omyces atratus Higashide et al.), the sequence number of GenBank sequence is KJ627770.1. bacteriostasis test, which shows that strain PY-1 pairs Botrytis cinerea, Bipolaris sorokiniana of wheat root rot fungus, tomato late blight, pepper Fusarium wilt pathogen and cucumber anthrax pathogen, Tomato red powder bacteria Trichothecium roseurn, eggplant stem base rot pathogen Rhizoctonia solani, sorghum Curvularia Curvularia caryopsida, wheat scab Fusarium graminearum have different inhibition effect on the fermentation conditions of.2.PY-1 strain. Through single factor test and orthogonal test, the liquid fermentation medium of actinomycetes PY-1 is optimized. The optimum medium is 5% of corn flour, 0.5% of glucose, 0.5% of peptone, 0.5% of ammonium chloride and 0.05% of sodium chloride. The optimum fermentation condition is that the liquid quantity of 250mL triangle bottle is 90mL, the inoculation amount is 5%, the rotational speed of the rocking bed is 180r/min, the culture temperature is 28, the acidity alkalinity of the initial culture is pH7.0. Between the culture medium and the culture conditions of 5d. before and after the optimization, the bacteriostasis rate of the optimized fermentation broth to downy mildew was enhanced by the analysis of the stability of the antibacterial active substance of the 8.35%.3.PY-1 strain. The antifungal activity of the biocontrol actinomycete PY-1 was the extracellular secondary metabolite. The stability test results of the fermentation filtrate showed that the temperature was temperature The bacteriostasis activity was stable at less than 65 C; the bacteriostatic activity of 40W fluorescent lamp was stable and 72h was irradiated under the sun light. The bacteriostasis activity began to decrease in the sun light. The bacteriostatic activity of the filtrate was affected by the pH6-10 environment, strong acid or strong alkali all affected the bacteriostasis activity of the fermentation filtrate; the bacteriostasis activity was not obviously changed at 4 centigrade for 6 months, and the bacteriostasis activity began to decline after 4 months of preservation at normal temperature; protease was reduced. The solution has no obvious effect on its bacteriostasis activity; metal ions lead to bacteriostasis activity to decrease the bacteriostasis mechanism of.4.PY-1 strain and field efficacy evaluation. PY-1 fermentation filtrate of biocontrol actinomycetes can cause folds, rupture and malformation of spores and spore sac of downy mildew strain of grape downy mildew, and then lose the infection function. The PY-1 metabolites of actinomycetes contain several The results of field control test showed that the field control effect of the biocontrol actinomycetes PY-1 fermentation broth could reach more than 90% in the middle stage of Grape Downy Mildew in the field, and the end effect reached more than 86% at the end of the stage, and was slightly lower than that of 52.5%, but was significantly higher than that of 58% methodezine and zinc 1000 times, and PY-1 strain was produced by PY-1 strain. The solution of 700 times dilution of yeast solution to the end stage of grape downy mildew and the antifungal activity of 1000 times liquid of methyl cream and zinc is equivalent to the isolation and purification of bacteriostasis of.5.PY-1 strain and its structure identification..PY-1 was separated and purified by dichloromethane, thin layer chromatography, silica gel column chromatography, dextran gel column chromatography and HPLC, and two kinds of grape cream were obtained. The results of bacteriostasis test of pure products with strong inhibitory activity of bacteria PY1-7-1 and PY1-7-2. showed that the bacteriostasis rates of PY1-7-1 and PY1-7-2 (10-2mg. ML-1,10-4mg. ML-1,10-6mg. ML-1) at different diluted concentrations were 92.59%, 86.30%, 64.81% and 97.04%, 91.85%, 84.07%. adopted ESI-MS, 1HNMR, 13CNMR spectrum analysis technique The molecular weight of the PY1-7-1 compound is 339, the molecular formula is C17H25NO6, the chemical name is 5-Acetoxycycloheximide, the molecular weight of the PY1-7-2 compound is 281, the molecular formula is C15H23NO4, and the chemical name is Cycloheximide..

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S476;S436.631

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