本文關(guān)鍵詞：拮抗油菜菌核病的功能菌篩選與作用機(jī)制研究　出處：《南京農(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： 油菜 菌核病 拮抗菌 促生作用 微生物有機(jī)肥

【摘要】：油菜作為重要的油料作物和經(jīng)濟(jì)作物,對(duì)農(nóng)業(yè)生產(chǎn)具有重要的意義。然而油菜生產(chǎn)過(guò)程中,菌核病的發(fā)生嚴(yán)重制約著菜籽的產(chǎn)量,嚴(yán)重時(shí)大田發(fā)病率達(dá)80%以上。由于菌核病兼具土傳和氣傳兩種傳播方式,一直以來(lái)難以防治。目前,生物防治作為替代傳統(tǒng)化學(xué)防治受到廣泛關(guān)注。在本實(shí)驗(yàn)室利用生物有機(jī)肥防控多種作物土傳病害取得重要成果的基礎(chǔ)上,本文通過(guò)篩選獲得對(duì)油菜菌核病病原菌具有高效抑制作用的兩株拮抗菌,對(duì)其葉面防病能力和抑菌機(jī)理進(jìn)行了研究,并探討了其中一株防控效果較好的拮抗菌制成的生物有機(jī)肥對(duì)油菜生長(zhǎng)的促進(jìn)作用。全文取得如下成果:1、從紫金山森林土壤中分離得到多株對(duì)油菜菌核病病原菌核盤菌有抑制能力的菌株。平板對(duì)峙實(shí)驗(yàn)發(fā)現(xiàn),菌株NJZJSB3和菌株NJZJSA2對(duì)核盤菌的抑制作用最強(qiáng),其發(fā)酵濾液抑菌圈直徑分別達(dá)到27 mm和44 mm。并且兩株菌都具有廣譜的抑菌效果,對(duì)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等多種植物土傳病原菌具有強(qiáng)烈的抑制作用。同時(shí),油菜離體葉片侵染實(shí)驗(yàn)表明兩株菌均能很好地抑制核盤菌的侵染。通過(guò)傳統(tǒng)的形態(tài)、生理生化特性分析結(jié)合分子生物學(xué)的方法對(duì)菌株NJZJSB3和NJZJSA2進(jìn)行了鑒定,確定其分別為解淀粉芽孢桿菌(Bacillus amyloliquefaciens)和小白鏈霉菌(Streptomyces albulus)。2、菌株NJZJSB3在初始pH為7的Landy培養(yǎng)基中,35℃/170r·min-1條件下培養(yǎng)60 h,其發(fā)酵濾液抑菌直徑達(dá)到36.7mm;菌株NJZJSA2在初始pH為6的CGA培養(yǎng)基,30℃/170 r.min-1條件下培養(yǎng)7 d,其發(fā)酵濾液抑菌直徑達(dá)到46.8 mm。菌株NJZJSB3和菌株NJZJSA2發(fā)酵產(chǎn)物分別在pH2～7和pH6～8作用下抑菌活性相對(duì)穩(wěn)定;經(jīng)50～70℃和蛋白酶(蛋白酶K、胰蛋白酶、胃蛋白)處理后發(fā)酵產(chǎn)物活性相對(duì)穩(wěn)定,抑菌圈直徑下降不明顯。盆栽葉面防病試驗(yàn)結(jié)果表明:相對(duì)于對(duì)照處理的發(fā)病率為100%時(shí),噴施菌株NJZJSB3和NJZJSA2菌懸液后菌核病發(fā)病率則分別為13.3%和36.7%;噴施菌株NJZJSB3和NJZJA2發(fā)酵濾液后菌核病發(fā)病率則分別為16.0%和0%;且各處理發(fā)病葉片病斑直徑顯著低于對(duì)照處理葉片病斑直徑。3、以菌株NJZJSB3的總DNA為模板進(jìn)行PCR擴(kuò)增得到8個(gè)脂肽類抗生素合成相關(guān)的基因。通過(guò)酸沉淀-甲醇抽提法對(duì)菌株.NJZJSB3發(fā)酵液中脂肽類抑菌物質(zhì)進(jìn)行提取,粗提物對(duì)核盤菌菌絲生長(zhǎng)的抑制率為71.1 %。通過(guò)HPLC-ESI-MS分析從粗提物中檢測(cè)出質(zhì)荷比分子量為1,030.6,1,044.7,1,058.8, 1,072.8,1,086.8 Da的抑菌物質(zhì)。經(jīng)高效液相色譜(HPLC)分析發(fā)現(xiàn)粗提物出峰的保留時(shí)間與iturin A標(biāo)準(zhǔn)物質(zhì)出峰保留時(shí)間一致,確定菌株NJZJSB3分泌抑菌物質(zhì)為iturinA類脂肽類物質(zhì)。此外,菌株NJZJSB3還能產(chǎn)生揮發(fā)性有機(jī)物,其對(duì)核盤菌菌絲生長(zhǎng)的抑制率為40.3%。通過(guò)GC-MS分析檢測(cè)出其中含有苯并噻唑、十七烷和植酮等物質(zhì)。其中,苯并噻唑的純品對(duì)核盤菌菌絲生長(zhǎng)具有抑制能力。菌株NJZJSB3還能分泌嗜鐵素、葡聚糖酶和蛋白酶等與抑菌相關(guān)的物質(zhì)。4、在基礎(chǔ)培養(yǎng)基中添加滅活核盤菌菌絲作為碳源可以誘導(dǎo)菌株NJZJSA2分泌多種胞外酶,發(fā)酵濾液中幾丁質(zhì)酶、β-1,3-葡聚糖酶和蛋白酶的活性分別比對(duì)照提高了4.4、9.5和2.7倍。通過(guò)減硫酸銨沉淀法提取發(fā)酵濾液中的粗蛋白,對(duì)核盤菌菌絲的生長(zhǎng)具有抑制作用。掃描電鏡(SEM)分析結(jié)果表明,經(jīng)粗蛋白溶液處理的核盤菌菌絲和菌核表皮細(xì)胞壁坍塌、破裂。在油菜離體葉片防病實(shí)驗(yàn)中,經(jīng)粗蛋白酶溶液噴施處理的油菜葉片菌核病病斑平均直徑比對(duì)照小12.2 mm。菌株NJZJSA2利用小麥粒培養(yǎng)基產(chǎn)生的揮發(fā)性有機(jī)物,能顯著抑制核盤菌菌絲生長(zhǎng)及菌核萌發(fā)。通過(guò)GC-MS分析從揮發(fā)性有機(jī)物中檢測(cè)出12種物質(zhì),分別屬于芳烴、烷烴、醚類、脂類和烯烴。其中,五種物質(zhì)對(duì)核盤菌均有抑制作用,其抑菌效果排序?yàn)?4-甲氧基苯乙烯2-戊基呋喃苯甲醚苯乙烯 正十四烷。掃描電鏡(SEM)觀察4-甲氧基苯乙烯處理后的核盤菌菌絲,發(fā)現(xiàn)菌絲末端腫脹和縮短,細(xì)胞壁凹陷和坍塌,但未破損。胎盤藍(lán)染色結(jié)果表明,4-甲氧基苯乙烯破壞了核盤菌菌絲的細(xì)胞膜的活性,使其失去選擇通透性。推測(cè)菌株NJZJSA2產(chǎn)揮發(fā)性氣體通過(guò)抑制核盤菌菌絲細(xì)胞膜活性,達(dá)到抑制菌絲生長(zhǎng)的目的。5、經(jīng)菌株NJZJSA2菌懸液灌根處理,油菜幼苗地上部和地下部生物量分別提高了45.0%和30.4%,且顯著提高了油菜總根長(zhǎng)、根表面積、根體積。對(duì)菌株NJZJSA2促生機(jī)理分析結(jié)果顯示,菌株NJZJSA2具有解磷和分泌鐵載體的能力,并在其發(fā)酵液中檢測(cè)到促生類物質(zhì)IAA。菌株NJZJSA2能利用根系分泌物定殖到油菜根系上生長(zhǎng),油菜幼苗根系分泌物中的草酸、蘋果酸、檸檬酸和琥珀酸對(duì)菌株NJZJSA2菌絲的生長(zhǎng)及孢子萌發(fā)具有促進(jìn)作用。第一次盆栽促生實(shí)驗(yàn)結(jié)果顯示,施用含有NJZJSA2的生物有機(jī)肥(BOF )能顯著增加油菜苗的生物量,地上部和地上部鮮重分別比施用化肥(CF)提高31.8%和25.5%。施用普通有機(jī)肥(OF)的油菜地上部和地下部鮮重與CF處理比較沒(méi)有顯著差異。與CF相比,施用BOF后土壤中的脲酶、過(guò)氧化氫酶、蔗糖酶和酸性磷酸酶的酶活分別提高了 18.4%, 13.5%, 70.6%和45.4%;而施用OF的上述土壤酶活分別提高了6.69%,6.97%,69.8%和33.2%。第二次盆栽促生實(shí)驗(yàn)結(jié)果顯示,施用菌株NJZJSA2菌懸液(A2)比空白對(duì)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%。兩次盆栽結(jié)果表明菌株NJZJSA2制備的生物有機(jī)肥比單純施用NJZJSA2菌體具有更好的促生能力。6、田間試驗(yàn)表明,CF,OF和BOF處理的田塊油菜地上部生物量均顯著高于不施肥處理(CK) 。 BOF處理油菜地上部鮮重、株高和莖粗顯著高于OF與CF處理,OF與CF處理的油菜地上部生物量無(wú)顯著差異。對(duì)不同時(shí)期油菜植株根際土壤和土體土壤微生物計(jì)數(shù)結(jié)果顯示,油菜根際土微生物數(shù)量顯著高于土體土。與CF相比,施用BOF顯著促進(jìn)油菜根際土壤細(xì)菌和放線菌的數(shù)量增加,減少真菌的數(shù)量。施用OF在提高油菜根際土壤細(xì)菌、放線菌的數(shù)量,同時(shí)也促進(jìn)了真菌數(shù)量的增加。油菜籽產(chǎn)量及油菜菌核病統(tǒng)計(jì)結(jié)果顯示BOF、OF、CF和CK處理菌核病發(fā)病率分別是42.7%,53.0%,46.3%和58.0%,沒(méi)有顯著差異。各田塊產(chǎn)量分別是241.9、221.1、215.2和96.9 kg/畝。第二次田間實(shí)驗(yàn)結(jié)果顯示,施用BOF聯(lián)合葉面噴施菌株NJZJSA2發(fā)酵液處理(BOF+噴施A2)田塊的菌核病發(fā)病率為40.0%,比單施BOF低15.3%。CF加葉面噴施菌株NJZJSA2發(fā)酵液處理(CF+噴施A2)田塊的菌核病發(fā)病率為52.0%,比單施CF處理低16.0%。BOF+噴施A2、BOF、CF+噴施A2和CF處理的油菜籽產(chǎn)量分別為284.8、181.9、240.8和 155.8 kg/畝。綜上所述:拮抗菌NJZJSB3和NJZJSA2能分泌多種抑菌物質(zhì),在盆栽實(shí)驗(yàn)中能較好的抑制核盤菌侵染油菜葉片。菌株NJZJSA2能分泌多種促生相關(guān)的物質(zhì),能顯著促進(jìn)油菜的生長(zhǎng)。其與有機(jī)肥二次發(fā)酵制成的生物有機(jī)肥不僅能促進(jìn)油菜的生長(zhǎng)和提高油菜籽產(chǎn)量,對(duì)油菜菌核病也有一定的防效。結(jié)合施用生物有機(jī)肥與葉面噴施菌株NJZJSA2對(duì)油菜菌核病的防治效果更好。本文的研究結(jié)果有助于研發(fā)油菜菌核病綜合防控技術(shù),為油菜菌核病的有效防控提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京農(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)】：S435.654

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