本文關(guān)鍵詞：蘋果輪紋病生防解淀粉芽胞桿菌（Bacillus amyloliquefaciens）PG12主要生防機(jī)制研究　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 生物防治 解淀粉芽胞桿菌 蘋果輪紋病 脂肽類抗生素 多細(xì)胞行為

【摘要】：芽胞桿菌以其在植物體內(nèi)、根際、土壤等自然環(huán)境中廣泛存在,能夠產(chǎn)生抗逆的芽胞、多樣的抗菌代謝產(chǎn)物,能夠有效地定殖在植物體內(nèi)并保護(hù)植物免受病原菌侵染,被認(rèn)為是一種環(huán)境友好型、應(yīng)用前景廣的生防細(xì)菌。解淀粉芽胞桿菌是其中一類具有生防潛力的細(xì)菌,已經(jīng)成功應(yīng)用于多種植物采后病害的防治。蘋果輪紋病(Botryosphaeria dothidea)是蘋果采后重要病害之一,在中國蘋果園區(qū)普遍發(fā)生。本研究首次以蘋果輪紋病為靶標(biāo),以生防解淀粉芽胞桿菌PG12(B.amyloliquefaciens PG12)為研究對(duì)象,為了揭示PG12防治蘋果輪紋病的主要生防機(jī)制,利用分析化學(xué)和分子生物學(xué)手段探究其主要抑菌活性物質(zhì),以及該活性物質(zhì)對(duì)定殖相關(guān)的多細(xì)胞行為的影響,為蘋果輪紋病生物農(nóng)藥的開發(fā)提供重要的理論指導(dǎo)。1)5. amyloliquefaciens PG12是一株對(duì)蘋果輪紋病有良好生防效果的菌株。B. amyloliquefaciens PG12在蘋果果實(shí)上對(duì)蘋果輪紋病菌絲和孢子的抑制效果顯著。無論在采前還是采后施用,B. amyloliquefaciens PG12在一定的儲(chǔ)藏期內(nèi)能夠減小蘋果采后病害的發(fā)生。在平板拮抗試驗(yàn)中,B. amyloliquefaciens PG12對(duì)蘋果輪紋病菌絲生長有明顯抑制作用,產(chǎn)生顯著的抑菌圈。通過掃描電子顯微鏡(scanning electronic micrographs, SEM)觀察發(fā)現(xiàn),靠近抑菌圈的真菌菌絲畸形,初步推測PG12可能通過產(chǎn)生一些抗菌代謝產(chǎn)物抑制真菌生長。2)脂肽類抗生素是B. amyloliquefaciensPG12抗蘋果輪紋病主要的抑菌活性物質(zhì)。為進(jìn)一步闡明B. amyloliquefaciens PG12對(duì)蘋果輪紋病的主要生防機(jī)制,以抗菌代謝產(chǎn)物為切入點(diǎn),深入探究代謝產(chǎn)物的抑菌活性。PG12無菌上清液中抑菌活性物質(zhì)在紫外線、蛋白酶K、高溫、強(qiáng)酸堿條件下較穩(wěn)定。進(jìn)一步研究發(fā)現(xiàn),理化性質(zhì)穩(wěn)定的脂肽類抗生素是B.amyloliquefaciens PG12抑制蘋果輪紋病菌菌絲和孢子的主要抗菌活性物質(zhì)。經(jīng)PCR檢測,在B.amyloliquefaciens PG12基因組上含有脂肽家族伊枯草素、豐原素和表面活性素的關(guān)鍵合成基因。采用薄層層析(thin layer chromatography, TLC)-生物自顯影技術(shù)分離脂肽粗提物,發(fā)現(xiàn)有兩種脂肽類抗生素家族的化合物對(duì)蘋果輪紋病有顯著地抑制作用,分別為伊枯草素和豐原素。利用反相高效液相色潛(reversed-phase high-performance liquid chromatography, RP-HPLC)和電噴射離子化四級(jí)飛行時(shí)間質(zhì)譜/質(zhì)譜(ESI-Q-TOF MS/MS)進(jìn)一步純化和分析得到其中伊枯草素家族的活性物質(zhì)為伊枯草素A,分子量為1043.55 m/z。為了從分子生物學(xué)角度進(jìn)一步確證脂肽類抗生素的抑菌作用,在已有文獻(xiàn)的基礎(chǔ)上找到了一種適于B. amyloliquefaciens PG12的遺傳操作方法,并適用于其他解淀粉芽胞桿菌野生型菌株。通過同源重組構(gòu)建了脂肽類抗生素合成相關(guān)基因的單、雙和三基因敲除突變體,它們分別喪失一種、兩種和三種脂肽類抗生素的合成能力,無法合成表面活性素、伊枯草素A和豐原素的突變體顯著降低B. amyloliquefaciens PG12對(duì)蘋果輪紋病的抑制作用。伊枯草素A和豐原素的抑菌作用強(qiáng)于表面活性素,且伊枯草素A和豐原素兩種脂肽類抗生素具有協(xié)同增效作用。3)脂肽類抗生素不同程度地促進(jìn)B. amyloliquefaciens PG12定殖相關(guān)的多細(xì)胞行為。近年來一些研究發(fā)現(xiàn)脂肽類抗生素能夠影響枯草芽胞桿菌除抑菌活性外的其他一些生物學(xué)特性,如生物膜形成、運(yùn)動(dòng)性等與定殖相關(guān)的多細(xì)胞行為。為了探究解淀粉芽胞桿菌所產(chǎn)生的脂肽類抗生素是否也有上述的生物學(xué)特性,本研究進(jìn)一步分析了B. amyloliquefaciens PG12脂肽類抗生素對(duì)其多細(xì)胞行為的影響。表面活性素、伊枯草素A和豐原素均不同程度的促進(jìn)了B.amyloliquefaciens PG12薄皮型和菌落生物膜的形成。表面活性素對(duì)整個(gè)生物膜形成都有顯著影響,同時(shí)首次發(fā)現(xiàn)伊枯草素A和豐原素對(duì)解淀粉芽胞桿菌生物膜形成初期的影響較后期顯著。表面活性素、伊枯草素A和豐原素均促進(jìn)B. amyloliquefaciens PG12群集和游動(dòng)的運(yùn)動(dòng)性能,其中伊枯草素A和豐原素在游動(dòng)性能中的貢獻(xiàn)不同于表面活性素,首次證明了伊枯草素家族的伊枯草素A顯著影響解淀粉芽胞桿菌的運(yùn)動(dòng)性。最后,采后果實(shí)生測試驗(yàn)進(jìn)一步證明脂肽類抗生素合成能力的喪失顯著影響B(tài). amyloliquefaciens PG12在一定儲(chǔ)藏期內(nèi)對(duì)蘋果輪紋病發(fā)揮生防作用�？偠灾�,脂肽類抗生素是B. amyloliquefaciens PG12具有良好生防效果的重要因素,該生防效果是脂肽類抗生素對(duì)真菌的拮抗作用、對(duì)芽胞桿菌多細(xì)胞行為等定殖相關(guān)性狀的促進(jìn)作用等生物學(xué)功能綜合作用的結(jié)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.611;S476.1

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