本文關(guān)鍵詞：紅灰鏈霉菌HDZ-9-47生產(chǎn)技術(shù)及對(duì)土壤生物群落的影響研究　出處：《中國(guó)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： 紅灰鏈霉菌 南方根結(jié)線蟲(chóng) 培養(yǎng)基優(yōu)化 發(fā)酵技術(shù) 生物熏蒸 協(xié)同增效 生物群落

【摘要】：根結(jié)線蟲(chóng)Meloidogyne spp.是一類固著型植物內(nèi)寄生線蟲(chóng),分布廣泛且危害嚴(yán)重,是威脅農(nóng)業(yè)生產(chǎn)的重要病原物。生物防治作為一種安全有效手段在根結(jié)線蟲(chóng)防治中得到了廣泛的研究和應(yīng)用。紅灰鏈霉菌Streptomyces rubrogriseus HDZ-9-47分離自根結(jié)線蟲(chóng)的卵,可寄生南方根結(jié)線蟲(chóng)M. incongnita的卵,產(chǎn)生具有殺線活性的代謝產(chǎn)物；在溫室及田間試驗(yàn)中對(duì)南方根結(jié)線蟲(chóng)都有良好防治效果,開(kāi)發(fā)前景較好。本文初步研究了HDZ-9-47防治南方根結(jié)線蟲(chóng)的機(jī)制及其發(fā)酵濾液中殺線化合物的理化性質(zhì),重點(diǎn)研究HDZ-9-47的產(chǎn)業(yè)化生產(chǎn)、儲(chǔ)存、田間應(yīng)用技術(shù)及其對(duì)土壤微生物群落的影響,從而為其產(chǎn)業(yè)化奠定基礎(chǔ)。1、首先在體外及盆栽試驗(yàn)中研究了紅灰鏈霉菌HDZ-9-47防治南方根結(jié)線蟲(chóng)的機(jī)制。體外實(shí)驗(yàn)結(jié)果表明HDZ-9-47具有幾丁質(zhì)酶及蛋白酶活性,透射電鏡觀察發(fā)現(xiàn)HDZ-9-47可寄生南方根結(jié)線蟲(chóng)卵。盆栽實(shí)驗(yàn)結(jié)果表明紅灰鏈霉菌HDZ-9-47的代謝產(chǎn)物和孢子在防治南方根結(jié)線蟲(chóng)上都發(fā)揮了重要的作用；此外施用紅灰鏈霉菌HDZ-9-47可以促進(jìn)番茄根部防御酶活性增加,進(jìn)而誘導(dǎo)番茄產(chǎn)生抗性,并且可以提高番茄產(chǎn)量及果實(shí)中可溶性糖的含量。2、研究了紅灰鏈霉菌HDZ-9-47發(fā)酵液中殺線化合物的特點(diǎn)：分子量大于3 KDa、對(duì)蛋白酶敏感、對(duì)酸堿穩(wěn)定,耐高溫和紫外線照射；對(duì)不同屬線蟲(chóng)的致死率不同；可有效地抑制小麥全蝕病菌的菌絲生長(zhǎng)。在盆栽試驗(yàn)中,對(duì)小麥全蝕病的防效與化學(xué)農(nóng)藥立克秀的防效相當(dāng)。3、用單因素和正交法優(yōu)化了紅灰鏈霉菌HDZ-9-47工業(yè)培養(yǎng)基組成,篩選出了適合其大規(guī)模生產(chǎn),活性高,價(jià)格低廉的工業(yè)培養(yǎng)基SPR-2。將HDZ-9-47接種到SPR-2中在160 rpm,28。C培養(yǎng)3天后,發(fā)酵濾液稀釋50倍后對(duì)南方根結(jié)線蟲(chóng)二齡幼蟲(chóng)的致死率達(dá)90%以上,發(fā)酵液中的孢子量達(dá)108/ml。在50 L發(fā)酵罐上小試發(fā)酵技術(shù),并優(yōu)化發(fā)酵參數(shù)。工作體積為30 L,接種量為3%,溫度為28。C,罐壓0.2 MPa,通氣量2 L/min,起始轉(zhuǎn)速300 rpm,溶氧35%,發(fā)酵周期72 h。紅灰鏈霉菌發(fā)酵濾液在稀釋40倍時(shí)對(duì)南方根結(jié)線蟲(chóng)二齡幼蟲(chóng)的致死率達(dá)90%以上,孢子量可達(dá)1010/ml。4、紅灰鏈霉菌HDZ-9-47孢子在以草炭灰為載體,常溫、含水量5%的條件下儲(chǔ)存12個(gè)月后,孢子量較初始菌量增加一個(gè)數(shù)量級(jí),達(dá)108個(gè)/g。但是發(fā)酵濾液儲(chǔ)存4個(gè)月后活性顯著下降。5、田間試驗(yàn)表明,SPR-2培養(yǎng)的紅灰鏈霉菌HDZ-9-47發(fā)酵液可有效地防治番茄上的南方根結(jié)線蟲(chóng)病,防效達(dá)48%以上；HDZ-9-47發(fā)酵液和福氣多結(jié)合施用可在作物生長(zhǎng)前期提高HDZ-9-47防效,減少福氣多用量,但是在作物生長(zhǎng)后期,其防治效果和單獨(dú)施用HDZ-9-47相當(dāng)；蝦殼可將HDZ-9-47的防效提高10%左右；HDZ-9-47與土壤生物熏蒸聯(lián)合應(yīng)用在防治南方根結(jié)線蟲(chóng)病上具有協(xié)同增效作用,兩者聯(lián)合應(yīng)用的防效達(dá)67%以上,較單獨(dú)施用提高19%,且其防效高于化學(xué)農(nóng)藥福氣多：不同生物熏蒸材料和HDZ-9-47結(jié)合的防效相近。6、用平板培養(yǎng)法和變性梯度凝膠電泳PCR-DGGE法研究了紅灰鏈霉菌HDZ-9-47發(fā)酵液與土壤生物熏蒸聯(lián)合施用對(duì)土壤微生物群落的影響。結(jié)果表明生物熏蒸后施用HDZ-9-47發(fā)酵液增加了土壤中有益細(xì)菌、真菌和放線菌的數(shù)量和種類,降低土壤中病原真菌的數(shù)量和種類,提高了土壤真菌多樣性,從而有助于紅灰鏈霉菌HDZ-9-47和生物熏蒸對(duì)南方根結(jié)線蟲(chóng)的防治。
[Abstract]:Root knot nematode Meloidogyne spp. is a kind of solid plant parasitic nematode, which is widely distributed and seriously endangers. It is an important disease source that threatens agricultural production. Biological control, as a safe and effective means, has been widely studied and applied in the prevention and control of root knot nematode. Red and grey rubrogriseus HDZ-9-47 of Streptomyces Streptomyces isolated from root knot nematode eggs, can M. incongnita Southern root knot nematode parasitic eggs produce metabolites with nematicidal activity; in greenhouse and field tests have good control effect on the root knot nematode, better prospects for development. This is a preliminary study on the physicochemical properties of the mechanism of HDZ-9-47 control meloidogyneincognita and fermentation filtrate nematicidal compounds, HDZ-9-47 focuses on industrial production, storage, field application and its effect on soil microbial communities, so as to lay the foundation for its industrialization. 1. First, in vitro and potted experiments, the mechanism of Streptomyces soot HDZ-9-47 to control the root knot nematode in the South was studied. In vitro experiments showed that HDZ-9-47 had the activity of chitinase and protease, and the transmission electron microscope showed that HDZ-9-47 could parasitism of the eggs of the southern root knot nematode. The results showed that the red ash pot experiment of Streptomyces HDZ-9-47 metabolites and spores have played an important role in the prevention and control of root knot nematode; moreover applying red gray Streptomyces HDZ-9-47 can promote root defense enzyme activity of tomato increased, thereby inducing tomato resistance, and can increase the content of soluble sugar in the fruit and yield of tomato. 2, to study the characteristics of red ash in fermentation liquid of Streptomyces HDZ-9-47 nematicidal compounds: a molecular weight greater than 3 KDa, the protease sensitive, high temperature resistance and acid-base stability, ultraviolet irradiation; different mortality rate for different genera of nematodes; mycelium can effectively inhibit the growth of Gaeumannomyces graminis. In the pot experiment, the wheat take all disease prevention effect with chemical pesticides like show the control effect is quite. 3, single factor and orthogonal method were used to optimize the composition of industrial medium of Streptomyces sotomyces HDZ-9-47, and SPR-2 suitable for mass production, high activity and low cost was screened out. HDZ-9-47 was inoculated into SPR- 2 in 160 rpm, 28. After 3 days of C culture, 50 times the dilution of the fermentation filtrate, the mortality of the two instar larvae of the southern root knot nematode was more than 90%, and the spores in the fermentation broth were up to 108/ml. The fermentation technology was tested on 50 L fermentor and the fermentation parameters were optimized. The work volume is 30 L, the inoculation amount is 3%, the temperature is 28. C, the pressure of the tank is 0.2 MPa, the ventilation volume is 2 L/min, the starting speed is 300 rpm, the dissolved oxygen is 35%, and the fermentation period is 72 h. When the filtrate of Streptomyces sodii was diluted 40 times, the death rate of the two instar larvae of the southern root knot nematode was over 90%, and the spores amount could reach 1010/ml. 4, the red ash of Streptomyces HDZ-9-47 spores in the peat ash as the carrier, the storage water content of 5% at room temperature and under the condition of 12 months after the amount of spore is the initial bacteria amount increased by an order of magnitude, up to 108 /g. But the activity of the fermentation filtrate decreased significantly after 4 months. That 5, field test, red ash fermentation broth of strain HDZ-9-47 cultured in SPR-2 can effectively control on tomato root knot nematode disease, the control efficiency reached above 48%; HDZ-9-47 fermentation liquid and the good fortune with the application of crop growth in early HDZ-9-47 improve control efficiency, reduce the amount of good fortune, but in the later period of crop growth, the the control effect and the separate application of HDZ-9-47; shrimp shell can be HDZ-9-47 the control effect increased by about 10%; the combined application of HDZ-9-47 and soil biofumigation has a synergistic effect in the prevention and control of root knot nematode disease prevention effect, combined with the above 67%, a single application is increased by 19%, and the control effect is higher than that of chemical pesticides blessing control effect of biofumigation: different materials and HDZ-9-47 combined with similar. 6, plate culture and denaturing gradient gel electrophoresis (PCR-DGGE) were used to study the effect of combined application of Streptomyces Sox HDZ-9-47 and soil fumigation on soil microbial community. The results showed that the number and types of biofumigation application after HDZ-9-47 fermentation increased, beneficial bacteria and fungi actinomycetes in soil, reduce the number and types of soil fungi, improve soil fungal diversity, which contributes to the red ash of Streptomyces HDZ-9-47 fumigation and biological control of root knot nematode.
【學(xué)位授予單位】：中國(guó)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)】：S476;S154.3

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