苹果轮纹病生防解淀粉芽胞杆菌（Bacillus amyloliquefaciens）PG12主要生防机制研究

发布时间：2017-12-28 17:18

本文关键词：苹果轮纹病生防解淀粉芽胞杆菌（Bacillus amyloliquefaciens）PG12主要生防机制研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：芽胞杆菌以其在植物体内、根际、土壤等自然环境中广泛存在,能够产生抗逆的芽胞、多样的抗菌代谢产物,能够有效地定殖在植物体内并保护植物免受病原菌侵染,被认为是一种环境友好型、应用前景广的生防细菌。解淀粉芽胞杆菌是其中一类具有生防潜力的细菌,已经成功应用于多种植物采后病害的防治。苹果轮纹病(Botryosphaeria dothidea)是苹果采后重要病害之一,在中国苹果园区普遍发生。本研究首次以苹果轮纹病为靶标,以生防解淀粉芽胞杆菌PG12(B.amyloliquefaciens PG12)为研究对象,为了揭示PG12防治苹果轮纹病的主要生防机制,利用分析化学和分子生物学手段探究其主要抑菌活性物质,以及该活性物质对定殖相关的多细胞行为的影响,为苹果轮纹病生物农药的开发提供重要的理论指导。1)5. amyloliquefaciens PG12是一株对苹果轮纹病有良好生防效果的菌株。B. amyloliquefaciens PG12在苹果果实上对苹果轮纹病菌丝和孢子的抑制效果显著。无论在采前还是采后施用,B. amyloliquefaciens PG12在一定的储藏期内能够减小苹果采后病害的发生。在平板拮抗试验中,B. amyloliquefaciens PG12对苹果轮纹病菌丝生长有明显抑制作用,产生显著的抑菌圈。通过扫描电子显微镜(scanning electronic micrographs, SEM)观察发现,靠近抑菌圈的真菌菌丝畸形,初步推测PG12可能通过产生一些抗菌代谢产物抑制真菌生长。2)脂肽类抗生素是B. amyloliquefaciensPG12抗苹果轮纹病主要的抑菌活性物质。为进一步阐明B. amyloliquefaciens PG12对苹果轮纹病的主要生防机制,以抗菌代谢产物为切入点,深入探究代谢产物的抑菌活性。PG12无菌上清液中抑菌活性物质在紫外线、蛋白酶K、高温、强酸碱条件下较稳定。进一步研究发现,理化性质稳定的脂肽类抗生素是B.amyloliquefaciens PG12抑制苹果轮纹病菌菌丝和孢子的主要抗菌活性物质。经PCR检测,在B.amyloliquefaciens PG12基因组上含有脂肽家族伊枯草素、丰原素和表面活性素的关键合成基因。采用薄层层析(thin layer chromatography, TLC)-生物自显影技术分离脂肽粗提物,发现有两种脂肽类抗生素家族的化合物对苹果轮纹病有显著地抑制作用,分别为伊枯草素和丰原素。利用反相高效液相色潜(reversed-phase high-performance liquid chromatography, RP-HPLC)和电喷射离子化四级飞行时间质谱/质谱(ESI-Q-TOF MS/MS)进一步纯化和分析得到其中伊枯草素家族的活性物质为伊枯草素A,分子量为1043.55 m/z。为了从分子生物学角度进一步确证脂肽类抗生素的抑菌作用,在已有文献的基础上找到了一种适于B. amyloliquefaciens PG12的遗传操作方法,并适用于其他解淀粉芽胞杆菌野生型菌株。通过同源重组构建了脂肽类抗生素合成相关基因的单、双和三基因敲除突变体,它们分别丧失一种、两种和三种脂肽类抗生素的合成能力,无法合成表面活性素、伊枯草素A和丰原素的突变体显著降低B. amyloliquefaciens PG12对苹果轮纹病的抑制作用。伊枯草素A和丰原素的抑菌作用强于表面活性素,且伊枯草素A和丰原素两种脂肽类抗生素具有协同增效作用。3)脂肽类抗生素不同程度地促进B. amyloliquefaciens PG12定殖相关的多细胞行为。近年来一些研究发现脂肽类抗生素能够影响枯草芽胞杆菌除抑菌活性外的其他一些生物学特性,如生物膜形成、运动性等与定殖相关的多细胞行为。为了探究解淀粉芽胞杆菌所产生的脂肽类抗生素是否也有上述的生物学特性,本研究进一步分析了B. amyloliquefaciens PG12脂肽类抗生素对其多细胞行为的影响。表面活性素、伊枯草素A和丰原素均不同程度的促进了B.amyloliquefaciens PG12薄皮型和菌落生物膜的形成。表面活性素对整个生物膜形成都有显著影响,同时首次发现伊枯草素A和丰原素对解淀粉芽胞杆菌生物膜形成初期的影响较后期显著。表面活性素、伊枯草素A和丰原素均促进B. amyloliquefaciens PG12群集和游动的运动性能,其中伊枯草素A和丰原素在游动性能中的贡献不同于表面活性素,首次证明了伊枯草素家族的伊枯草素A显著影响解淀粉芽胞杆菌的运动性。最后,采后果实生测试验进一步证明脂肽类抗生素合成能力的丧失显著影响B. amyloliquefaciens PG12在一定储藏期内对苹果轮纹病发挥生防作用。总而言之,脂肽类抗生素是B. amyloliquefaciens PG12具有良好生防效果的重要因素,该生防效果是脂肽类抗生素对真菌的拮抗作用、对芽胞杆菌多细胞行为等定殖相关性状的促进作用等生物学功能综合作用的结果。
[Abstract]:Bacillus with its widely exist in plants, rhizosphere, soil and other natural environment, can produce resistance of Bacillus and various antimicrobial metabolites, can effectively colonize plants and protect plants against pathogen infection, is considered to be a kind of environment friendly and wide application prospect of biocontrol bacteria. Bacillus amyloid is one of the bacteria with potential of biocontrol, which has been successfully applied to the prevention and control of postharvest diseases of various plants. Botryosphaeria dothidea is one of the most important diseases of apple after harvest, and it is widely used in Apple Park in China. This is the first study to physalosporapiricolanose target by Biocontrol Bacillus amyloliquefaciens PG12 (B.amyloliquefaciens PG12) as the research object, in order to reveal the prevention and treatment of PG12 Apple rot mainly biocontrol mechanism, the use of chemical analysis and molecular biology methods to explore the main antibacterial active substances, and the effects of active substances related to multicellular behavior the colonization, provide important theoretical guidance for the development of physalosporapiricolanose biological pesticide. 1) 5. amyloliquefaciens PG12 is a strain of good biocontrol effect on Apple wheel grain disease. The inhibitory effect of B. amyloliquefaciens PG12 on the filaments and spores of Apple RIS fungus on apple fruit was significant. B. amyloliquefaciens PG12 can reduce the occurrence of Apple postharvest diseases in a certain storage period, either before or after harvest. In the plate antagonistic test, B. amyloliquefaciens PG12 had obvious inhibitory effect on the growth of the filaments of Apple rhyme germs, and produced significant bacteriostasis. By observing the scanning electronic micrographs (SEM), it was found that the mycelium deformity near the inhibition zone suggested that PG12 might inhibit the growth of fungi by producing some antibacterial metabolites. 2) lipopeptide antibiotics are the main antibacterial active substances of B. amyloliquefaciensPG12 against apple ring disease. In order to further clarify the main biocontrol mechanisms of B. amyloliquefaciens PG12 on apple ring rot, we take the antibacterial metabolites as the breakthrough point, and further explore the antibacterial activity of the metabolites. The bacteriostasis in PG12 aseptic supernatant is stable under the conditions of ultraviolet, protease K, high temperature and strong acid and alkali. Further studies showed that lipopeptide antibiotics with stable physicochemical properties were the main antibacterial substances inhibiting B.amyloliquefaciens PG12 from mycelia and spores of apple ring rot. By PCR detection, containing lipid peptide family, Iran subtilis surfactin and fengycin key synthetic gene in B.amyloliquefaciens genome PG12. By using thin layer chromatography (thin layer chromatography, TLC) - bioautography separated lipopeptide extracts, found two compounds family lipopeptide antibiotics have a significant inhibitory effect on the apple rot, were Bacillus subtilis and fengycin iran. By reversed-phase high performance liquid chromatography (reversed-phase high-performance liquid chromatography, RP-HPLC) and electrospray ionization time-of-flight mass spectrometry / mass spectrometry grade four (ESI-Q-TOF MS/MS) further purification and analysis of the active substance of Bacillus family for Iraq Iran Bacillus subtilis A, molecular weight of 1043.55 m/z. In order to further confirm the bacteriostatic action of lipopeptide antibiotics from the perspective of molecular biology, a genetic manipulation method suitable for B. amyloliquefaciens PG12 has been found on the basis of existing literature, and is suitable for other wild strains of Bacillus amylus. Constructed by homologous recombination lipopeptide biosynthetic genes and three single and double knockout mutants, their ability to synthesize a loss of two, respectively, and three kinds of lipopeptide antibiotics, cannot synthesize surfactin and Iran Bacillus subtilis A and fengycin mutants significantly decreased B. amyloliquefaciens PG12 of apple the inhibitory effect of rot. Inhibitory effect of Bacillus subtilis A Iran and fengycin is stronger than surfactin, Iran and Bacillus subtilis A and fengycin two kinds of lipopeptide antibiotics have synergistic effects. 3) lipopeptide antibiotics in varying degrees promote the multicellular behavior related to the colonization of B. amyloliquefaciens PG12. In recent years, some studies have found that lipopeptide antibiotics can affect other biological characteristics except Bacillus subtilis, such as biofilm formation, motility and other related cell behaviors. In order to investigate whether these lipopeptide antibiotics produced by Bacillus amylosum have biological characteristics, we further analyzed the effects of B. amyloliquefaciens PG12 lipopeptide antibiotics on their multicellular behavior. Surfactin, Bacillus subtilis A and Iran fengycin had different degree of promoting the formation of B.amyloliquefaciens PG12 thin type and colony biofilm. Surfactin Chengdu has obvious influence on the whole biofilm formation, and for the first time found that Iraq A and Bacillus subtilis fengycin of Bacillus amyloliquefaciens biofilm formation at the early stage of the late effect is significant. Surfactin, Bacillus subtilis A and Iran fengycin could promote the motion performance of B. amyloliquefaciens PG12 cluster and swimming, the contribution of Bacillus subtilis A and Iran in fengycin swimming performance different from the surfactin, for the first time that the Iraqi family Iran in Bacillus subtilis A significantly affects the movement of Bacillus amyloliquefaciens the. Finally, the postharvest fruit growth test further proved that the loss of synthetic ability of lipopeptide antibiotics significantly affected the biocontrol effect of B. amyloliquefaciens PG12 on apple ring rot in a certain storage period. In short, lipopeptide antibiotics is an important factor in B. amyloliquefaciens PG12 has a good biocontrol effect, the biocontrol effect is the combined action of lipopeptide antibiotics, antagonism against fungi of Bacillus colonization and multicellular behavior traits related to promote the role of biological function.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.611;S476.1

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