拮抗油菜菌核病的功能菌筛选与作用机制研究

发布时间：2017-12-27 01:20

本文关键词：拮抗油菜菌核病的功能菌筛选与作用机制研究　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：油菜作为重要的油料作物和经济作物,对农业生产具有重要的意义。然而油菜生产过程中,菌核病的发生严重制约着菜籽的产量,严重时大田发病率达80%以上。由于菌核病兼具土传和气传两种传播方式,一直以来难以防治。目前,生物防治作为替代传统化学防治受到广泛关注。在本实验室利用生物有机肥防控多种作物土传病害取得重要成果的基础上,本文通过筛选获得对油菜菌核病病原菌具有高效抑制作用的两株拮抗菌,对其叶面防病能力和抑菌机理进行了研究,并探讨了其中一株防控效果较好的拮抗菌制成的生物有机肥对油菜生长的促进作用。全文取得如下成果:1、从紫金山森林土壤中分离得到多株对油菜菌核病病原菌核盘菌有抑制能力的菌株。平板对峙实验发现,菌株NJZJSB3和菌株NJZJSA2对核盘菌的抑制作用最强,其发酵滤液抑菌圈直径分别达到27 mm和44 mm。并且两株菌都具有广谱的抑菌效果,对F.oxysporum f.sp. niveum, F. oxysporum f.sp. cucumuerimu, F. oxysporum f.sp.melonis, Verticillium dahliae Kleb, Rhizoctonia solani, F. oxysporum f.sp. cubense, F.oxysporum Schlecht等多种植物土传病原菌具有强烈的抑制作用。同时,油菜离体叶片侵染实验表明两株菌均能很好地抑制核盘菌的侵染。通过传统的形态、生理生化特性分析结合分子生物学的方法对菌株NJZJSB3和NJZJSA2进行了鉴定,确定其分别为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和小白链霉菌(Streptomyces albulus)。2、菌株NJZJSB3在初始pH为7的Landy培养基中,35℃/170r·min-1条件下培养60 h,其发酵滤液抑菌直径达到36.7mm;菌株NJZJSA2在初始pH为6的CGA培养基,30℃/170 r.min-1条件下培养7 d,其发酵滤液抑菌直径达到46.8 mm。菌株NJZJSB3和菌株NJZJSA2发酵产物分别在pH2～7和pH6～8作用下抑菌活性相对稳定;经50～70℃和蛋白酶(蛋白酶K、胰蛋白酶、胃蛋白)处理后发酵产物活性相对稳定,抑菌圈直径下降不明显。盆栽叶面防病试验结果表明:相对于对照处理的发病率为100%时,喷施菌株NJZJSB3和NJZJSA2菌悬液后菌核病发病率则分别为13.3%和36.7%;喷施菌株NJZJSB3和NJZJA2发酵滤液后菌核病发病率则分别为16.0%和0%;且各处理发病叶片病斑直径显著低于对照处理叶片病斑直径。3、以菌株NJZJSB3的总DNA为模板进行PCR扩增得到8个脂肽类抗生素合成相关的基因。通过酸沉淀-甲醇抽提法对菌株.NJZJSB3发酵液中脂肽类抑菌物质进行提取,粗提物对核盘菌菌丝生长的抑制率为71.1 %。通过HPLC-ESI-MS分析从粗提物中检测出质荷比分子量为1,030.6,1,044.7,1,058.8, 1,072.8,1,086.8 Da的抑菌物质。经高效液相色谱(HPLC)分析发现粗提物出峰的保留时间与iturin A标准物质出峰保留时间一致,确定菌株NJZJSB3分泌抑菌物质为iturinA类脂肽类物质。此外,菌株NJZJSB3还能产生挥发性有机物,其对核盘菌菌丝生长的抑制率为40.3%。通过GC-MS分析检测出其中含有苯并噻唑、十七烷和植酮等物质。其中,苯并噻唑的纯品对核盘菌菌丝生长具有抑制能力。菌株NJZJSB3还能分泌嗜铁素、葡聚糖酶和蛋白酶等与抑菌相关的物质。4、在基础培养基中添加灭活核盘菌菌丝作为碳源可以诱导菌株NJZJSA2分泌多种胞外酶,发酵滤液中几丁质酶、β-1,3-葡聚糖酶和蛋白酶的活性分别比对照提高了4.4、9.5和2.7倍。通过减硫酸铵沉淀法提取发酵滤液中的粗蛋白,对核盘菌菌丝的生长具有抑制作用。扫描电镜(SEM)分析结果表明,经粗蛋白溶液处理的核盘菌菌丝和菌核表皮细胞壁坍塌、破裂。在油菜离体叶片防病实验中,经粗蛋白酶溶液喷施处理的油菜叶片菌核病病斑平均直径比对照小12.2 mm。菌株NJZJSA2利用小麦粒培养基产生的挥发性有机物,能显著抑制核盘菌菌丝生长及菌核萌发。通过GC-MS分析从挥发性有机物中检测出12种物质,分别属于芳烃、烷烃、醚类、脂类和烯烃。其中,五种物质对核盘菌均有抑制作用,其抑菌效果排序为:4-甲氧基苯乙烯2-戊基呋喃苯甲醚苯乙烯正十四烷。扫描电镜(SEM)观察4-甲氧基苯乙烯处理后的核盘菌菌丝,发现菌丝末端肿胀和缩短,细胞壁凹陷和坍塌,但未破损。胎盘蓝染色结果表明,4-甲氧基苯乙烯破坏了核盘菌菌丝的细胞膜的活性,使其失去选择通透性。推测菌株NJZJSA2产挥发性气体通过抑制核盘菌菌丝细胞膜活性,达到抑制菌丝生长的目的。5、经菌株NJZJSA2菌悬液灌根处理,油菜幼苗地上部和地下部生物量分别提高了45.0%和30.4%,且显著提高了油菜总根长、根表面积、根体积。对菌株NJZJSA2促生机理分析结果显示,菌株NJZJSA2具有解磷和分泌铁载体的能力,并在其发酵液中检测到促生类物质IAA。菌株NJZJSA2能利用根系分泌物定殖到油菜根系上生长,油菜幼苗根系分泌物中的草酸、苹果酸、柠檬酸和琥珀酸对菌株NJZJSA2菌丝的生长及孢子萌发具有促进作用。第一次盆栽促生实验结果显示,施用含有NJZJSA2的生物有机肥(BOF )能显著增加油菜苗的生物量,地上部和地上部鲜重分别比施用化肥(CF)提高31.8%和25.5%。施用普通有机肥(OF)的油菜地上部和地下部鲜重与CF处理比较没有显著差异。与CF相比,施用BOF后土壤中的脲酶、过氧化氢酶、蔗糖酶和酸性磷酸酶的酶活分别提高了 18.4%, 13.5%, 70.6%和45.4%;而施用OF的上述土壤酶活分别提高了6.69%,6.97%,69.8%和33.2%。第二次盆栽促生实验结果显示,施用菌株NJZJSA2菌悬液(A2)比空白对照(CK)油菜地上部生物量提高11.7%,施用BOF油菜地上部生物量比OF提高了 25.7%。两次盆栽结果表明菌株NJZJSA2制备的生物有机肥比单纯施用NJZJSA2菌体具有更好的促生能力。6、田间试验表明,CF,OF和BOF处理的田块油菜地上部生物量均显著高于不施肥处理(CK) 。 BOF处理油菜地上部鲜重、株高和茎粗显著高于OF与CF处理,OF与CF处理的油菜地上部生物量无显著差异。对不同时期油菜植株根际土壤和土体土壤微生物计数结果显示,油菜根际土微生物数量显著高于土体土。与CF相比,施用BOF显著促进油菜根际土壤细菌和放线菌的数量增加,减少真菌的数量。施用OF在提高油菜根际土壤细菌、放线菌的数量,同时也促进了真菌数量的增加。油菜籽产量及油菜菌核病统计结果显示BOF、OF、CF和CK处理菌核病发病率分别是42.7%,53.0%,46.3%和58.0%,没有显著差异。各田块产量分别是241.9、221.1、215.2和96.9 kg/亩。第二次田间实验结果显示,施用BOF联合叶面喷施菌株NJZJSA2发酵液处理(BOF+喷施A2)田块的菌核病发病率为40.0%,比单施BOF低15.3%。CF加叶面喷施菌株NJZJSA2发酵液处理(CF+喷施A2)田块的菌核病发病率为52.0%,比单施CF处理低16.0%。BOF+喷施A2、BOF、CF+喷施A2和CF处理的油菜籽产量分别为284.8、181.9、240.8和 155.8 kg/亩。综上所述:拮抗菌NJZJSB3和NJZJSA2能分泌多种抑菌物质,在盆栽实验中能较好的抑制核盘菌侵染油菜叶片。菌株NJZJSA2能分泌多种促生相关的物质,能显著促进油菜的生长。其与有机肥二次发酵制成的生物有机肥不仅能促进油菜的生长和提高油菜籽产量,对油菜菌核病也有一定的防效。结合施用生物有机肥与叶面喷施菌株NJZJSA2对油菜菌核病的防治效果更好。本文的研究结果有助于研发油菜菌核病综合防控技术,为油菜菌核病的有效防控提供理论基础。
[Abstract]:As an important oil crop and economic crop, rapeseed is of great significance to agricultural production. However, in the process of rape production, the occurrence of Sclerotinia seriously restricts the yield of rapeseed, and the incidence of the field is over 80%. It is difficult to prevent and cure Sclerotinia Sclerotinia because it has two modes of transmission with both soil transmission and gas transmission. At present, biological control, as a substitute for traditional chemical control, has attracted wide attention. Based on the laboratory utilization of bio organic fertilizer crop soil borne disease prevention and control of various important achievements, this paper obtain high inhibitory effect of two strains of Antagonistic Bacteria on pathogen Sclerotinia screening, the leaf disease prevention ability and antibacterial mechanism was studied and discussed including a better control effect of antagonistic bacteria made of bio organic fertilizer on rape growth promoting effect. The following achievements were obtained as follows: 1. Isolated strains from the forest soil of Zijin Mountain were isolated and isolated from the pathogen of sclerotia of rape Sclerotinia sclerotiorum. The test of plate confrontation showed that strain NJZJSB3 and strain NJZJSA2 had the strongest inhibition effect on the bacteria, and the bacteriostasis diameter of the fermentation filtrate reached 27 mm and 44 mm respectively. The antibacterial effect and two strains with a broad spectrum of F.oxysporum f.sp. niveum, F. oxysporum f.sp. cucumuerimu, F. oxysporum f.sp.melonis, Verticillium dahliae Kleb, Rhizoctonia solani, F. oxysporum f.sp. cubense, F.oxysporum Schlecht and other plant soil borne pathogens had a strong inhibitory effect. At the same time, the infection experiment of the leaves of rape in vitro showed that two strains of bacteria could well inhibit the infection of sclerotius. The strains NJZJSB3 and NJZJSA2 were identified by traditional morphological, physiological and biochemical characteristics analysis and molecular biology methods. They were identified as Bacillus amyloliquefaciens and Streptomyces albulus respectively. 2, strain NJZJSB3 at initial pH 7 Landy medium, 35 C /170r min-1 cultured under 60 h, the fermentation filtrate of antibacterial diameter reached 36.7mm; strain NJZJSA2 at initial pH 6 CGA medium, 30 C /170 r.min-1 cultured under 7 d, the fermentation filtrate of antibacterial diameter reached 46.8 mm. The fermentation products of strain NJZJSB3 and strain NJZJSA2 were relatively stable under the action of pH2 ~ 7 and pH6 ~ 8 respectively. After 50~70 h treatment and protease (protease K, trypsin and stomach protein) treatment, the activity of fermentation products was relatively stable, and the diameter of inhibition zone was not obvious. Potted foliage disease test results showed that: compared to the control treatment the incidence rate was 100%, with strain NJZJSB3 and NJZJSA2 suspension after the disease rate is respectively 13.3% and 36.7%; spraying strains NJZJSB3 and NJZJA2 fermentation filtrate after the disease rate is respectively 16% and 0%; and the diameter of each treatment onset of leaf lesion significantly lower than CK leaf lesion diameter. 3. The total DNA of strain NJZJSB3 was used as the template for PCR amplification to get 8 genes related to the synthesis of lipopeptide antibiotics. The inhibitory substances of lipopeptide in fermentation broth of strain.NJZJSB3 were extracted by acid precipitation methanol extraction. The inhibition rate of crude extracts against sclerotiorum of Sclerotinia sclerotiorum was 71.1%. Through the detection and analysis of HPLC-ESI-MS from the crude extract of mass molecular weight of 1030.6,1044.7,1058.8 and 1072.8,1086.8 Da antibacterial substances. High performance liquid chromatography (HPLC) analysis showed that the peak retention time of crude extracts was consistent with the peak storage time of iturin A standard substances, indicating that the NJZJSB3 strain secreting bacteriostatic substances was iturinA lipoid peptides. In addition, the strain NJZJSB3 can produce volatile organic compounds, which inhibit the growth of hypha by 40.3%. The GC-MS analysis was used to detect the contents of benzothiazole, seventeen alkanes and ketones. The pure products of benzothiazole inhibited the growth of mycelium of Sclerotinia sclerotiorum. Strain NJZJSB3 can also secrete substances such as ferric, glucan enzyme and protease, which are related to bacteriostasis. 4, based on the cultivation of live Sclerotinia mycelium added out medium as the carbon source of strain NJZJSA2 can induce the secretion of extracellular enzymes, fermentation filtrate -1,3- glucanase and chitinase and protease activity were increased to 4.4, 9.5 and 2.7 times. The crude protein in the filtrate was extracted by ammonium sulfate precipitation, and it could inhibit the growth of mycelium of Sclerotinia sclerotiorum. The results of scanning electron microscopy (SEM) analysis showed that the cell walls of sclerotia and sclerotia were collapsed and ruptured by the treatment of crude protein solution. In the leaves of control experiment, the sclerotium disease spot of rape leaf crude protease solution spraying. The average diameter of 12.2 mm smaller than control. Strain NJZJSA2 can inhibit the growth of hypha and the germination of Sclerotinia sclerotiorum by using the volatile organic compounds produced by wheat grain medium. 12 compounds were detected from volatile organic compounds by GC-MS analysis, which belong to aromatics, alkanes, ethers, lipids and olefin. Among them, five kinds of substances have inhibitory effect on the bacteria, and their bacteriostasis effect is: 4- methoxy styrene 2- amyl furanodimethyl ether styrene positive fourteen alkane. Scanning electron microscopy (SEM) was used to observe the hypha after 4- methoxy styrene treatment. It was found that the end of the mycelium was swollen and shortened, and the cell wall was depressed and collapsed, but it was not damaged. The results of placental blue staining showed that 4- methoxy styrene destroyed the cell membrane activity of the mycelium of Sclerotinia sclerotiorum and lost the selective permeability. It is inferred that the production of volatile gases from strain NJZJSA2 can inhibit the growth of mycelium by inhibiting the membrane activity of the mycelium of Sclerotinia. 5. After root NJZJSA2 treatment, the aboveground and underground biomass of rape seedlings increased by 45% and 30.4% respectively, and the total root length, root surface area and root volume of rape significantly increased. The result of the analysis of the growth mechanism of strain NJZJSA2 showed that the strain NJZJSA2 had the ability to Dissolve Phosphorus and secrete iron carrier, and in its fermentation liquid.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.654

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