解淀粉芽孢杆菌Y19微生物菌肥的研制及其生物效益研究

发布时间：2018-05-25 15:24

本文选题：内生菌 + 促生长防病机能　；参考：《云南农业大学》2015年硕士论文

【摘要】：目前,农业生产上提高作物单产及防控病虫害的主要方式为增施化肥及使用化学农药。然而它们在保障粮食增产的同时,也引发了环境污染,生态平衡破坏及食品安全等问题。微生物菌肥已被证实具有提高作物产量且能减轻病害的危害程度,因此,开展有益微生物筛选并开发成微生物菌肥的研究工作具有重要的意义。本研究围绕筛选促生长防病的生防菌株、生防菌微生物菌肥的开发及其生物效益等方面开展研究,获得了如下结果:1.通过本实验室先前分离并保存的几株玉米内生细菌的IAA合成、固氮解磷活性及盆栽玉米叶片叶绿素含量的测定,筛选出具有良好应用潜力的内生细菌。结果发现解淀粉芽孢杆菌Y19菌株合成IAA的能力和固氮解磷活性均优于Y13和Y10菌株,盆栽玉米的叶绿素含量也更高。因此,将菌株Y19作为后续研究的供试菌株。2.通过自然转化方式,将含有绿色荧光蛋白基因gfpmut3a的质粒p HAPII导入Y19,获得具有良好荧光表型的标记菌株Y19-GFPmut3a。室内平板对峙、玉米盆栽促生定殖试验的结果表明,质粒的引入对Y19的拮抗病原及促生效果并无明显的影响。在温室玉米盆栽试验中,激光共聚焦显微镜观察结果显示,接菌后的第9天,可观察到其在玉米根、茎及叶部组织的定殖;接种后的第100 d,仍能在玉米、茄子、白菜的根际土壤、根、茎及叶组织中检测到标记菌株。这些结果表明Y19在不同植物体内具有良好的定殖及运输性能。3.使用含有终浓度为0.03%黄原胶的Y19-GFPmut3a(106CFU/m L)发酵液喷施烟草叶面,和(或)直接使用Y19-GFPmut3a(106CFU/m L)发酵液进行灌根处理,结果表明灌根和叶面喷施的双重处理组中烟草的株高、鲜重和干重明显增加。接种后的第30天,仍能在烟草中检测到标记菌株,这表明Y19在烟草体内具有良好的定殖能力,且Y19的定殖并不影响烟草叶内及叶表的细菌种类和数量。4.菌株Y19分别与实验室保存的促生防病效果显著的解淀粉芽孢杆菌B9601-Y2、具有溶磷活性的洋葱伯克氏菌1-2、YN201305分别两两混合培养的结果表明彼此之间无拮抗作用。在此基础上以单独培养的发酵液为对照,将上述的两两混合培养后的发酵液分别与基质复配(体积质量比为1:2)后,种植玉米、萝卜、白菜和油菜。试验结果表明,单一的Y19发酵液与基质复配促生长效果最好。5.将Y19发酵液(106CFU/m L)与不同含量的氮肥、磷肥或钾肥的基质均匀混合后,用于漂浮育苗水稻和盆栽玉米的促生试验。结果发现Y19发酵液与含有3~4%的磷肥的基质复配的促生长效果最好。盆栽30 d后,与对照相比,玉米鲜重可增加448.05%-487.60%。这表明Y19可与含有化学肥料的基质复配且制成的复合基质能显著地促进玉米幼苗生长发育。6.玉米根际土壤微生物群落功能样性及土壤微生物量的变化检测结果发现施入Y19微生物菌肥后,玉米根系细菌、放线菌的数量增加,而真菌的数量却下降,特别是镰刀菌的数量。此外,Y19微生物菌肥也提高了根际微生物群落的碳源利用率(AWCD),主要提高了对碳水化合物类、氨基酸和羧酸类化合物的利用。这些数据说明Y19微生物菌肥可通过改善根际土壤微环境,从而发挥促生和防病的双重功能。7.Y19微生物菌肥菌株货架期检测结果发现Y19在纯基质及含3%磷肥的基质中存活率最高。在温度为28℃、湿度为50%的环境条件下保存1年,其活菌量维持在107CFU/g以上。
[Abstract]:At present, the main way to increase crop yield and prevent and control diseases and pests in agricultural production is to increase chemical fertilizer and use chemical pesticides. However, while ensuring the increase of grain yield, they also cause environmental pollution, ecological balance damage and food safety. Microbial fertilizer has been proved to increase crop yield and reduce the damage of disease. Therefore, it is of great significance to carry out beneficial microorganism screening and development of microbial microbial fertilizer. This study focuses on the screening of biocontrol strains for growth prevention and control of growth prevention, the development of microbial fertilizer for biocontrol bacteria and its biological benefits. The following results are obtained: 1. through several previous isolation and preservation in our laboratory The IAA synthesis of endophytic bacteria, the activity of nitrogen fixing and the determination of the content of chlorophyll in the leaves of the potted maize were measured, and the endophytic bacteria with good application potential were screened out. The results showed that the ability to synthesize IAA and the phosphorus fixing activity of the Bacillus amylobacter Y19 strain were superior to those of the Y13 and Y10 strains, so the content of chlorophyll in the potted maize was also higher. The strain Y19 was used as a follow-up study strain.2. to transfer the plasmid P HAPII containing the green fluorescent protein gene gfpmut3a into Y19 through natural transformation, and to obtain the labelled strain Y19-GFPmut3a. with good fluorescent phenotype, Y19-GFPmut3a. indoor flat confrontation, and the results of maize plant growth promoting colonization test showed that the plasmid was introduced to the antagonism of Y19. In a greenhouse maize pot experiment, the results of laser confocal microscopy showed that the colonization of the roots, stems and leaves could be observed in the ninth days after the inoculation, and the 100th d after inoculation could still be detected in the rhizosphere soil, root, stem and leaf tissues of maize, eggplant and cabbage. These results show that Y19 has good colonization and transport properties in different plants,.3. using Y19-GFPmut3a (106CFU/m L) fermented solution containing 0.03% yellowish original glue to spray tobacco leaf surface and (or) direct use of Y19-GFPmut3a (106CFU/m L) fermentation broth for root irrigation. The results show the double treatment group of root irrigation and foliar spraying. The plant height, fresh weight and dry weight of the tobacco increased obviously. Thirtieth days after inoculation, the labelled strains could still be detected in tobacco, which showed that Y19 had good colonization in tobacco, and the colonization of Y19 did not affect the species of bacteria in the leaf and leaf surface of tobacco and the number of.4. strain Y19, respectively. Bacillus amyloid B9601-Y2, the results of mixed culture of burkderia onions 1-2 and YN201305 with phosphorus soluble activity, showed no antagonism between 22 mixed cultures. On this basis, the fermentation broth of the 22 mixed culture was mixed with the substrate (volume mass ratio 1:2), respectively. Planting corn, radish, cabbage and rapeseed. The results showed that the best combination of Y19 fermentation liquid and matrix was the best.5., and the Y19 fermenting solution (106CFU/m L) was mixed with different content of nitrogen fertilizer, phosphate fertilizer or potash fertilizer. The results were used to promote the growth of floating seedling rice and potted jade rice. The results showed that the Y19 fermentation liquid and 3~4% contained 3~4%. The substrate compound of phosphate fertilizer has the best growth promoting effect. After 30 d potted plants, the fresh weight of corn can be increased by 448.05%-487.60%. compared with the control, which indicates that Y19 can be combined with the matrix containing chemical fertilizer and can significantly promote the microbial community function and soil microbial biomass in the maize seedling growth and development of maize rhizosphere soil. The results showed that the number of bacteria and actinomycetes increased, but the number of fungi decreased, especially the number of Fusarium. In addition, Y19 microbial fertilizer also improved the carbon source utilization (AWCD) of the rhizosphere microbial community (AWCD), which mainly improved carbohydrates, amino acids and carboxylic acids. Using these data, these data show that Y19 microbial fertilizer can improve the microenvironment of Rhizosphere Soil by improving the shelf life detection results of the dual function.7.Y19 microbial fertilizer strain for promoting and preventing disease. It is found that Y19 has the highest survival rate in the pure matrix and the matrix containing 3% phosphate fertilizer. At the temperature of 28, and the humidity of 50%, 1 is preserved. In the year, the amount of living bacteria was more than 107CFU/g.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S144

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