兰州大学机构库 >草地农业科技学院
解淀粉芽孢杆菌对苜蓿炭疽病的生物防治效果及机理研究
Alternative TitleBiological Control Effect and Mechanism of Bacillus amyloliquefaciens against Anthracnose of Alfalfa
胡进玲
Subtype博士
Thesis Advisor李彦忠
2021-05-25
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name农学博士
Degree Discipline草学
Keyword解淀粉芽孢杆菌 平头刺盘孢 炭疽病防治 环状脂肽 抑菌机理
Abstract苜蓿炭疽病是国内外公认的苜蓿上的毁灭性病害,对草产量和草品质的影响高于其他大部分苜蓿病害。为研发出防治该病害的生防制剂,本论文以课题组前期在微生物分离过程中获得的一株对苜蓿病原菌表现出明显抑制作用的细菌为研究材料,通过皿内拮抗、温室和田间接种试验评估了该菌株对平头刺盘孢(Colletotrichum truncatum)引起的苜蓿炭疽病的生防效果,利用基因组学和化学方法对其抑菌活性物质进行鉴定,并从细胞学、生理生化、分子生物学层面揭示了该生防菌对苜蓿炭疽病菌的抑制机理。获得以下主要结果: 1. 解淀粉芽孢杆菌LYZ69对苜蓿病原菌表现出广谱抑菌效果。通过形态学和分子生物学方法将编号为LYZ69的细菌鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。平板拮抗试验表明,LYZ69对供试的9种苜蓿重要的病原真菌中的8种具有皿内拮抗作用,其中对7种抑菌率大于50%。按其抑菌率由大到小排序为:苜蓿小光壳(Leptosphaerulina briosiana;71.11%),苜蓿匍柄霉(Stemphylium botryosum;64.76%),三叶草刺盘孢(Colletotrichum trifolii;60.55%),粉红镰孢(Fusarium roseum;58.90%),腐皮镰刀菌(F. solani;57.71%),尖镰孢(F. oxysporum;54.23%),苜蓿茎点霉(Phoma medicaginis;53.15%)。说明LYZ69对苜蓿病原菌具有广谱抑菌效果。 2. 解淀粉芽孢杆菌LYZ69对苜蓿炭疽病防效显著。LYZ69在平板上对炭疽病菌平头刺盘孢(C. truncatum)的抑制率为84.3%。盆栽条件下,以平头刺盘孢为防治对象检测LYZ69对苜蓿炭疽病的防治效果,结果显示,只接种病原菌时苜蓿植株的发病率为100%,死亡率为85.56%。接种生防菌后再接种病原菌,植株发病率降低了44.68%,无植株死亡,生防菌对苜蓿炭疽病防效高达82.59%。同时,LYZ69使苜蓿株高、茎粗、鲜重和干重分别显著增加了13.38%、 25.00%、28.46%和34.59%。说明该菌株对苜蓿具有促生作用。LYZ69处理还可以使苜蓿植株中粗蛋白含量显著提高6.36%。 3. LYZ69在田间土壤中可以生长繁殖且表现出防病增产的潜力。田间防效测定结果显示,播种后第40 d田间共发生4种地上病害,LYZ69处理使发病最严重的3种病害霜霉病(Peronospora aestivalis Syd.)、褐斑病(P. medicaginis)和茎点霉叶斑病(P. medicaginis)发病率降低了17.66~76.76%,病情指数降低了29.76~69.2%;使苜蓿鲜重、根长、株高和茎粗分别显著增加了76.99%、 8.74%、6.66%和12.99%。播种一年后田间共发生6种地上病害,LYZ69处理使总发病率显著降低了44.68%,使发病最严重的4种病害霜霉病、茎点霉叶斑病、小光壳叶斑病(L. briosiana)、夏季黑茎病(Cercospora medicaginis)的发病率降低了46.42~78.98%,病情指数降低了59.46~76.26%;使炭疽病(Colletotrichum sp.)的发病率和病情指数降低了100%;使根腐病的发病率和中柱病情指数分别显著降低了46%和51.4%。使苜蓿鲜、干草产量分别提高了12.64%和12.7%。通过高通量测序进行宏基因组分析,LYZ69处理的土壤中芽孢杆菌丰度为198.75,显著高于对照组的22.25,表明该生防菌可以在田间土壤中生长繁殖。以上结果表明LYZ69对苜蓿根腐病有显著防效,短期内对苜蓿有显著促生作用,具有防治苜蓿地上病害以及提高苜蓿产量的潜力。 4. LYZ69的抑菌活性物质主要是脂肽类。LYZ69的气体挥发物对平头刺盘孢无抑制作用,而无菌发酵滤液对菌丝生长和孢子萌发具有显著抑制效果,抑制率分别达到59.56%和100%,说明抑菌活性物质为非挥发性物质。LYZ69的全基因组中共有12个基因簇与次级代谢产物合成有关,其中包括3个NRPS合成酶基因簇、2个Ⅰ型PKS合成酶基因簇、1个NRPS/PKS基因簇。其中,3个NRPS基因簇与surfactin、fengycin和bacillibactin合成酶同源性分别高达82%、100%、100%。将LYZ69的抑菌活性物质进行提纯,经LC-MS/MS将其鉴定为脂肽类物质C13 bacillomycin D、C17 fengycin A和C16 fengycin B。说明LYZ69通过产生多种脂肽类抗生素来抑制平头刺盘孢的菌丝生长和孢子萌发。 5. 脂肽通过诱导细胞凋亡抑制平头刺盘孢。脂肽处理后,平头刺盘孢的菌丝细胞膨胀、畸变,菌丝表面粗糙且有明显的凹陷、干瘪和破损迹象。荧光染色结果证明菌丝细胞发生了凋亡,细胞内ROS激增。基于比较转录组分析表明,上调差异基因主要富集在细胞壁、细胞膜和DNA的合成和修复、甘油合成和ROS清除等代谢途径,下调差异基因主要富集在细胞凋亡相关的代谢途径,这说明LYZ69产生的脂肽类物质对平头刺盘孢的抑制机理主要是破坏细胞壁和细胞膜,然后进入细胞内部,诱导甘油合成,造成细胞渗透压改变,从而导致菌丝细胞变形;同时使细胞内活性氧激增,最终造成真菌细胞凋亡式细胞死亡。
Other AbstractAnthracnose is recognized worldwide as a destructive disease of alfalfa, which has a higher effect on grass yield and quality than most other alfalfa diseases. In order to develop biocontrol agents to control the disease, a bacteria strain which showed obvious inhibitory effect on alfalfa pathogens obtained by our research group in the process of microbial isolation was used as the research material. The biocontrol effect of the strain against alfalfa anthracnose caused by Colletotrichum truncatum was evaluated by plate antagonistic test, artificial infection experiment in greenhouse and field, and identified the bacterial metabolites using genomics and chemical methods. In addition, we also revealed the inhibitory mechanism of the biological control bacterium on fungi of alfalfa anthracnose from the aspects of cytology, physiology and biochemistry, and molecular biology. The following results were achieved: 1. Bacillus amyloliticus LYZ69 showed a broad spectrum antibacterial effect against alfalfa pathogens. According to the morphological characteristics and phylogenetic analysis of the bacteria, the strain LYZ69 was identified as B. amyloliquefaciens. A plate antagonistic test showed that LYZ69 had an antagonistic effect on eight of the nine important pathogenic fungi of alfalfa that were tested, and the rate of inhibition of seven fungi was more than 50%. The antifungal rate could be ranked from the highest to the lowest as Leptosphaerulina briosiana (71.11%), Stemphylium botryosum (64.76%), Colletotrichum trifolii (60.55%), Fusarium roseum (58.90%), Fusarium solani (57.71%), Fusarium oxysporum (54.23%), and Phoma medicaginis (53.15%). The results showed that LYZ69 had a broad-spectrum antifungal effect on alfalfa pathogens. 2. LYZ69 has remarkable biological control effect on alfalfa anthracnose. Plate antagonistic results showed that the inhibitory rates of LYZ69 on mycelial growth of Colletotrichum truncatum was 84.3%. Under the potting conditions, the ability of LYZ69 to control alfalfa anthracnose was measured using C. truncatum as the control object. The results showed that the incidence rate of alfalfa plants was 100%, and the mortality rate was 85.56% when inoculated with pathogens only. The rate of disease of plants was reduced by 44.68%, and no plants died when the pathogens were inoculated after the biological control bacteria. The control effect of biological control bacteria on alfalfa Colletotrichum sp. reached as high as 82.59%. Moreover, LYZ69 significantly increased the plant height, stem diameter, fresh weight, and dry weight of alfalfa by 13.38%, 25.00%, 28.46%, and 34.59%, respectively. The results showed that the strain could promote the growth of alfalfa. In addition, the content of crude protein in alfalfa plants increased significantly by 6.36% after the LYZ69 treatment. 3. LYZ69 could grow and reproduce in field soil and showed the potential of disease prevention and yield increase. The results of field control effect determination showed that on day 40 after sowing, four types of aboveground diseases occurred in the field. Compared with the control, the incidence and disease index of 3 most serious diseases, downy mildew, leaf spot diseases caused by Pseudopeziza medicaginis, Phoma medicaginis, decreased by 17.66-76.76% and 29.76-69.2%, respectively after treatment with LYZ69. LYZ69 increased the fresh weight, root length, plant height, and stem diameter by 76.99%, 8.74%, 6.66%, and 12.99%, respectively. One year after sowing, six types of aboveground diseases occurred in the field. The total incidence of the aboveground disease decreased significantly by 44.68% following treatment with LYZ69. The incidence and disease index of four most serious diseases downy mildew, leaf spot diseases caused by Phoma medicaginis, Leptosphaerulina briosiana, black stem diseases caused by Cercospora medicaginis decreased by 46.42-78.98% and 59.46-76.26%. The incidence and disease index of anthracnose decreased by 100%. LYZ69 significantly reduced the rate of incidence of root rot and the stele disease index by 46% and 51.4%, respectively. The yields of fresh and dry alfalfa increased by 12.64% and 12.7% respectively. Based on metagenomic analysis using high-throughput sequencing, the abundance of Bacillus in the control soil was 22.25, and that in the LYZ69 treatment soil was 198.75, which was significantly higher than the control, indicating that the biological control bacteria could reproduce and grow in the field soil. These results indicated that LYZ69 had a significant ability to control alfalfa root rot and a significant growth promotion effect on alfalfa in a short time. It has the potential to control alfalfa aboveground diseases and improve alfalfa yield. 4. The antibacterial active substances of LYZ69 were lipopeptides. The volatile organic compounds of LYZ69 had no inhibitory effect on C. truncatum, while its cell-free culture (CFC) could significantly inhibit the mycelial growth and spore germination of C. truncatum, with inhibition rates of 59.56% and 100%, respectively, which indicated that the antimicrobial active substances were non-volatile substances. There were 12 gene clusters related to secondary metabolites in the whole genome sequencing of LYZ69, including three NRPS synthase gene clusters, two PKS synthase gene clusters of type I, and one NRPS/PKS gene cluster. The three NRPS gene clusters shared 82%, 100% and 100% homology with surfactin, fengycin, and bacillibactin synthase, respectively. The lipopeptides in CFC were purified and identified as C13 bacillomycin D, C17 fengycin A, and C16 fengycin B via LC-MS/MS. The results showed that LYZ69 could inhibit the mycelial growth and spore germination of Colletotrichum truncatum by producing a variety of lipopeptide antibiotics. 5. Lipopeptides inhibit C. truncatum by inducing its apoptotic cell death. Lipopeptide leads to the distortion and expansion of mycelial cells, the surface of mycelium become rough and appear obvious signs of sunken, wizened and damage. The fluorescence staining results showed that apoptosis occurred in the mycelial cells, and reactive oxygen species increased sharply. Based on comparative transcriptome analysis, the up-regulated differential genes were mainly rich in metabolic pathways such as cell wall, cell membrane and DNA synthesis and repair, glycerol synthesis and ROS clearance, while the down-regulated differential genes were rich in apoptotic metabolic pathways. All of these results suggest that the inhibitory mechanism of lipopeptide produced by LYZ69 on C. truncatum was primarily to destroy the cell wall and enter the cell through membrane to induce the synthesis of glycerin, causing a change in cell osmotic pressure, and leading to the deformation of mycelia cells. In addition, the amount of active oxygen in the cell increased, which eventually led to the cell death of fungi by apoptosis.
Pages122
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/462375
Collection草地农业科技学院
Affiliation
草地农业科技学院
First Author AffilicationCollege of Pastoral Agriculture Science and Technology
Recommended Citation
GB/T 7714
胡进玲. 解淀粉芽孢杆菌对苜蓿炭疽病的生物防治效果及机理研究[D]. 兰州. 兰州大学,2021.
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