Method for controlling chestnut blight based on fosetyl-aluminium as immunoactivator mixed with biocontrol bacterium fermentation filtrate

A method for controlling chestnut blight based on fosetyl-aluminium as an immunoactivator mixed with a biocontrol bacterium fermentation filtrate, comprising following steps:

• step 1, culturing Cryphonectria parasitica using a potato dextrose agar (PDA) medium and preparing fungal discs using a puncher;
• step 2, preparing PDA media using fosetyl-aluminium solutions at different concentrations and Bacillus velezensis B268 fermentation filtrates diluted to different multiples, transferring the fungal discs onto the PDA media to allow incubation, and conducting mycelial growth inhibition assay on PDA medium;
• step 3, selecting chestnut seedlings, establishing two control groups and three treatment groups of the chestnut seedlings, comprising:
• a positive control group (spraying with sterile water and inoculating with the Cryphonectria parasitica), a negative control group (spraying with the sterile water and inoculating with sterile PDA), a fosetyl-aluminium treatment group, a B268 fermentation filtrate treatment group, and a fosetyl-aluminium+B268 fermentation filtrate treatment group;
• collecting and preserving leaves from the chestnut seedlings in each of the groups without any chemical spray on day 0 to allow an assay of defense enzyme activities and a malondialdehyde (MDA) content;
• step 4, based on results obtained from the step 2, uniformly spraying the leaves and trunks of the chestnut seedlings in the three treatment groups at concentrations demonstrating a superior indoor antifungal efficacy, wherein the fosetyl-aluminium+B268 fermentation filtrate treatment group requires mixing by preparing a fosetyl-aluminium solution and adding a B268 fermentation filtrate at a specific volume calculated based on a volume of the fosetyl-aluminium solution;
• conducting in vitro inoculation and in vivo inoculation three days later, respectively;
• and step 5:
• collecting and properly preserving leaf samples from each group periodically on days 0, 2, 4, 8, and 16 to allow the assay of the defense enzyme activities and the MDA content, and selecting a part of the samples on day 8 to allow transcriptome sequencing analysis.