|Anthracnose 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.