7株人参病害生防菌株的筛

发布时间：2018-11-24 18:43

【摘要】：人参(Panax ginsengC.A.Meyer)是五加科人参属多年生宿根植物,我国传统名贵中药材。我国人参栽培面积居世界首位,吉林省长白山区是我国人参主产区。人参栽培周期一般5～6年,长时间的单一种植导致农田土壤理化性状恶化、微生物群落结构变化以及病害高发等问题。人参病害问题严重影响人参的产量及品质,化学农药防治常导致农药残留超标等问题,对环境、生态及人畜健康也有较大威胁。生物防治具有环境友好、效果持久、防效显著等优点,且不存在农药残留问题。本研究从人参根围土和商品化微生物菌剂中筛选对人参致病菌具有较好抑菌活性的生防菌株,借助核糖体DNA-ITS序列分析、形态学观察及生理生化指标测定对菌株进行了鉴定;通过田间试验,探究拮抗菌株在田间应用中的防治效果;结合室内研究,对拮抗菌株的抑菌机理进行了初步探究,为生防制剂的开发和拮抗菌株抑菌机理研究奠定了良好基础。具体结果如下:1.从人参栽培土和商品化菌剂产品中分离到菌株83株,其中从人参栽培土中分离到66株,从商品化菌剂中分离到17株。其中拮抗细菌QM、BM2、HZ3、GG和X,拮抗真菌HC和DD对人参根腐菌Fusarium solani、人参锈腐菌Cylindrocarpon destructans、人参黑斑菌Alternariapanax和人参灰霉菌Botrytiscinerea均有较好的抑菌活性。经同源性分析、形态学观察及生理指标测定,初步鉴定菌株QM、BM2和HZ3为甲基营养型芽孢杆菌Bacillus methylorophicus,GG为枯草芽孢杆菌B.subtilis,X为解淀粉芽孢杆菌B.amyloliquefaciens;HC为棘孢木霉Trichoderma asperellum,DD为平展木霉T.effusum。同时,利用BIOLOGOmniLogPlus微生物鉴定仪开展了 5株拮抗细菌的生理生化指标测定。2.田间试验设置了单个拮抗菌株、5株拮抗细菌、2株拮抗真菌、7株拮抗菌等共计12种处理。研究发现,除BM2外,施用菌液的人参出苗率均高于施用多菌灵阳性对照组和无菌水阴性对照组;施用菌液的人参存苗率与多菌灵组持平、均高于阴性对照组。7株菌株对人参苗生长无不良影响。3.人参致病菌与7株拮抗菌株对峙培养,产生明显的抑菌带。通过光学显微镜和电镜观察发现,与对照组相比,与生防菌对峙培养的人参根腐菌、人参锈腐菌、人参黑斑菌和人参灰霉菌菌丝生长畸形,由此可以推测,其与拮抗菌株对人参致病菌生长的抑制作用密切相关。4.拮抗菌株QM、BM2和HZ3的发酵液和发酵上清液对人参致病菌有明显的抑制作用,分别提取上清液中的活性物质,发现脂肽粗提物、蛋白粗提物以及水饱和正丁醇萃取物对人参致病菌均有不同程度的抑菌效果,说明3株拮抗细菌胞外分泌液中含有多种抑菌活性成分,它们可能单一或协同完成对人参致病菌的抑制。
[Abstract]:Ginseng (Panax ginsengC.A.Meyer) is a perennial root plant of Panax ginseng family. The cultivated area of ginseng in China ranks first in the world, and Changbai Mountain area of Jilin Province is the main producing area of Panax ginseng in China. The cultivation period of Panax ginseng is 5 ~ 6 years. The long-term single planting results in the deterioration of soil physical and chemical properties, the change of microbial community structure and the high incidence of diseases. The problem of ginseng disease seriously affects the yield and quality of ginseng, and chemical pesticide control often leads to the problem of pesticide residue exceeding the standard, which is also a great threat to the environment, ecology and human and animal health. Biological control has the advantages of friendly environment, lasting effect and remarkable control effect, and there is no pesticide residue problem. In this study, biocontrol strains with good antibacterial activity against ginseng pathogenic bacteria were screened from rhizosphere and commercial microbial agents. The strains were identified by ribosomal DNA-ITS sequence analysis, morphological observation and physiological and biochemical index determination. Through field experiment, the control effect of antagonistic strain in field application was explored, and the antibacterial mechanism of antagonistic strain was preliminarily explored, which laid a good foundation for the development of biological control agent and the study of bacteriostatic mechanism of antagonistic strain. The results are as follows: 1. 83 strains were isolated from ginseng cultivation soil and commercial bacterial products, among which 66 strains were isolated from ginseng cultivation soil and 17 strains were isolated from commercial fungicides. Among them, antagonistic bacteria QM,BM2,HZ3,GG and X, antagonistic fungi HC and DD had better antibacterial activity against Fusarium solani, Cylindrocarpon destructans, Alternariapanax and Botrytiscinerea. By homology analysis, morphological observation and physiological index determination, the strains QM,BM2 and HZ3 were identified as Bacillus subtilis, B. subtilis X and B. amyloliquefaciens. the results showed that Bacillus subtilis, Bacillus subtilis and Bacillus subtilis X were Bacillus subtilis, B. amyloliquefaciens. HC is Trichoderma spinoides Trichoderma asperellum,DD is T. effusuma. At the same time, the physiological and biochemical indexes of 5 antagonistic bacteria were determined by BIOLOGOmniLogPlus microbiological identification instrument. 2. There were 12 treatments including single antagonistic strain, 5 antagonistic bacteria, 2 antagonistic fungi and 7 antagonistic bacteria. The results showed that, except for BM2, the seedling emergence rate of ginseng treated with bacterial solution was higher than that of carbendazim positive control group and aseptic water negative control group. The survival rate of ginseng seedlings was equal to that of carbendazim group, which was higher than that of negative control group. 7 strains had no adverse effect on the growth of ginseng seedlings. The pathogenic bacteria of ginseng were cultured in confrontation with 7 antagonistic strains to produce obvious bacteriostasis zone. Compared with the control group, the mycelium growth of ginseng root rot, ginseng rust rot, ginseng black spot and ginseng gray fungus were found to be abnormal compared with the control group by optical microscope and electron microscope. Therefore, it can be inferred that the growth of ginseng root rot fungus, ginseng rust rot fungus, ginseng black spot fungus and ginseng gray mold mycelium are abnormal. It is closely related to the inhibitory effect of antagonistic strains on the growth of ginseng pathogenic bacteria. 4. 4. The fermentation broth and fermentation supernatant of antagonistic strains QM,BM2 and HZ3 had obvious inhibitory effects on ginseng pathogenic bacteria. The active substances were extracted from the supernatant respectively and the crude lipopeptide extract was found. The crude protein extract and water-saturated n-butanol extract had different inhibitory effects on ginseng pathogenic bacteria, indicating that the extracellular secretions of three antagonistic bacteria contained a variety of antimicrobial active components. They may be a single or synergistic inhibition of ginseng pathogenic bacteria.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.675;S476.1

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