红灰链霉菌HDZ-9-47生产技术及对土壤生物群落的影响研究

发布时间：2017-12-28 00:02

本文关键词：红灰链霉菌HDZ-9-47生产技术及对土壤生物群落的影响研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：根结线虫Meloidogyne spp.是一类固着型植物内寄生线虫,分布广泛且危害严重,是威胁农业生产的重要病原物。生物防治作为一种安全有效手段在根结线虫防治中得到了广泛的研究和应用。红灰链霉菌Streptomyces rubrogriseus HDZ-9-47分离自根结线虫的卵,可寄生南方根结线虫M. incongnita的卵,产生具有杀线活性的代谢产物；在温室及田间试验中对南方根结线虫都有良好防治效果,开发前景较好。本文初步研究了HDZ-9-47防治南方根结线虫的机制及其发酵滤液中杀线化合物的理化性质,重点研究HDZ-9-47的产业化生产、储存、田间应用技术及其对土壤微生物群落的影响,从而为其产业化奠定基础。1、首先在体外及盆栽试验中研究了红灰链霉菌HDZ-9-47防治南方根结线虫的机制。体外实验结果表明HDZ-9-47具有几丁质酶及蛋白酶活性,透射电镜观察发现HDZ-9-47可寄生南方根结线虫卵。盆栽实验结果表明红灰链霉菌HDZ-9-47的代谢产物和孢子在防治南方根结线虫上都发挥了重要的作用；此外施用红灰链霉菌HDZ-9-47可以促进番茄根部防御酶活性增加,进而诱导番茄产生抗性,并且可以提高番茄产量及果实中可溶性糖的含量。2、研究了红灰链霉菌HDZ-9-47发酵液中杀线化合物的特点：分子量大于3 KDa、对蛋白酶敏感、对酸碱稳定,耐高温和紫外线照射；对不同属线虫的致死率不同；可有效地抑制小麦全蚀病菌的菌丝生长。在盆栽试验中,对小麦全蚀病的防效与化学农药立克秀的防效相当。3、用单因素和正交法优化了红灰链霉菌HDZ-9-47工业培养基组成,筛选出了适合其大规模生产,活性高,价格低廉的工业培养基SPR-2。将HDZ-9-47接种到SPR-2中在160 rpm,28。C培养3天后,发酵滤液稀释50倍后对南方根结线虫二龄幼虫的致死率达90%以上,发酵液中的孢子量达108/ml。在50 L发酵罐上小试发酵技术,并优化发酵参数。工作体积为30 L,接种量为3%,温度为28。C,罐压0.2 MPa,通气量2 L/min,起始转速300 rpm,溶氧35%,发酵周期72 h。红灰链霉菌发酵滤液在稀释40倍时对南方根结线虫二龄幼虫的致死率达90%以上,孢子量可达1010/ml。4、红灰链霉菌HDZ-9-47孢子在以草炭灰为载体,常温、含水量5%的条件下储存12个月后,孢子量较初始菌量增加一个数量级,达108个/g。但是发酵滤液储存4个月后活性显著下降。5、田间试验表明,SPR-2培养的红灰链霉菌HDZ-9-47发酵液可有效地防治番茄上的南方根结线虫病,防效达48%以上；HDZ-9-47发酵液和福气多结合施用可在作物生长前期提高HDZ-9-47防效,减少福气多用量,但是在作物生长后期,其防治效果和单独施用HDZ-9-47相当；虾壳可将HDZ-9-47的防效提高10%左右；HDZ-9-47与土壤生物熏蒸联合应用在防治南方根结线虫病上具有协同增效作用,两者联合应用的防效达67%以上,较单独施用提高19%,且其防效高于化学农药福气多：不同生物熏蒸材料和HDZ-9-47结合的防效相近。6、用平板培养法和变性梯度凝胶电泳PCR-DGGE法研究了红灰链霉菌HDZ-9-47发酵液与土壤生物熏蒸联合施用对土壤微生物群落的影响。结果表明生物熏蒸后施用HDZ-9-47发酵液增加了土壤中有益细菌、真菌和放线菌的数量和种类,降低土壤中病原真菌的数量和种类,提高了土壤真菌多样性,从而有助于红灰链霉菌HDZ-9-47和生物熏蒸对南方根结线虫的防治。
[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.
【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】：S476;S154.3

【相似文献】