葡萄霜霉病生防放线菌PY-1鉴定及抑菌活性物质结构解析

发布时间：2018-04-26 18:35

本文选题：葡萄霜霉病 + 暗黑链霉菌　；参考：《沈阳农业大学》2016年博士论文

【摘要】：葡萄霜霉病是由葡萄生单轴霉Plasmopara viticola (Berk. Curtis) Berl. de Toni引起的真菌性病害,分布广泛且危害严重,是造成葡萄生产损失的主要原因之一。目前,化学药剂仍是防治葡萄霜霉病的主要措施,但大量、持续、单一品种化学药剂的使用导致3R问题日益突出。生物防治因其对环境友好、人畜安全及防治可持续性而受到广泛关注和高度重视。本研究目的是筛选对葡萄霜霉病具有防控效果的生防菌,并探究其生防机制,为葡萄霜霉病生防制剂的研制和应用提供有效菌株和技术指导。1.生防放线菌的分离与鉴定。对采自辽宁、云南和西藏等地葡萄园区的53份土壤样品样进行分离,共获得151株放线菌。采用平板筛选法以辣椒疫霉Plasmopara capsici为指示菌进行初筛,采用离体叶片法以葡萄霜霉病菌Plasmopara viticola为指示菌进行复筛,共获得28株对两种卵菌门真菌均有显著拮抗作用的放线菌,其中菌株PY-1的拮抗作用最强,其发酵液对葡萄霜霉病的抑菌率为91.11%。采用传统分类、化学分类与分子分类相结合的方法,鉴定PY-1菌株为暗黑链霉菌(Streptomyces atratus Higashide et al.), GenBank序列登录号为KJ627770.1。抑菌谱试验表明,菌株PY-1对葡萄灰霉病菌Botrytis cinerea、小麦根腐病菌Bipolaris sorokiniana、番茄晚疫病Phytophthora infestans、辣椒枯萎病菌Fusarium oxysporum、黄瓜炭疽病菌Colletotrichum orbiculare、番茄红粉病菌Trichothecium roseurn、茄子茎基腐病菌Rhizoctonia solani、高粱弯孢菌Curvularia caryopsida、小麦赤霉菌Fusarium graminearum均有不同程度的抑制作用。2.PY-1菌株发酵条件优化。通过单因子试验和正交试验优化生防放线菌PY-1液体发酵培养基的营养成分和发酵条件。确定最适培养基成分为玉米粉5%,葡萄糖0.5%,蛋白胨0.5%,氯化铵0.5%,氯化钠0.05%；最适发酵条件为在250mL三角瓶的装液量为90mL,接种量为5%,摇床转速为180r/min,培养温度28℃,初始培养液的酸碱度为pH7.0,发酵培养时间为5d。对比优化前后的培养基组分和培养条件,优化后的发酵液对葡萄霜霉病的抑菌率提高了8.35%。3.PY-1菌株抑菌活性物质稳定性分析。生防放线菌PY-1抑菌活性物质为胞外次级代谢产物。发酵滤液抑菌活性稳定性试验结果表明：温度低于65℃时抑菌活性稳定;对40W日光灯稳定,在太阳光下照射72h后,抑菌活性开始下降；pH6-10环境下稳定,强酸或强碱均影响其发酵滤液的抑菌活性；4℃下保存6个月抑菌活性无明显变化,常温下保存4个月后,抑菌活性开始下降；蛋白酶溶液对其抑菌活性无明显影响;金属离子导致抑菌活性下降。4.PY-1菌株抑菌机理及田间防效评价。生防放线菌PY-1发酵滤液能够导致葡萄霜霉病菌孢子囊和孢子囊梗出现褶皱、破裂和畸形,进而丧失侵染功能。放线菌PY-1代谢产物中包含几丁质酶、蛋白酶、嗜铁素、ACC脱氨酶、HCN、IAA,不含纤维素酶。田间防效试验表明,生防放线菌PY-1发酵原液对葡萄霜霉病的田间中期防效可达到90%以上,末期防效达86%以上,比52.5%抑快净2000倍液略低,但明显高于58%甲霜锰锌1000倍液；PY-1菌株发酵液稀释700倍液对葡萄霜霉病的末期防效与甲霜锰锌1000倍液防效相当。5.PY-1菌株抑菌活性物质的分离纯化及结构鉴定。PY-1发酵虑液经二氯甲烷萃取、薄层层析、硅胶柱层析、葡聚糖凝胶柱层析和HPLC进行分离纯化,获得两种对葡萄霜霉病菌具有很强抑制活性的化合物纯品PY1-7-1和PY1-7-2。抑菌试验结果显示,不同稀释浓度的PY1-7-1和PY1-7-2 (10-2mg·mL-1、10-4mg·mL-1、10-6mg·mL-1)对葡萄霜霉病菌的抑菌率分别为92.59%、86.30%、64.81%和97.04%、91.85%、84.07%。采用ESI-MS、1HNMR、13CNMR波谱分析技术对活性组分进行结构解析,确定PY1-7-1化合物分子量为339,分子式为C17H25NO6,化学名称为5-Acetoxycycloheximide; PY1-7-2化合物分子量为281,分子式为C15H23NO4,化学名称为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..

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476;S436.631

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