Biocontrol microbial inoculum for preventing and treating watermelon fusarium wilt and use method of biocontrol microbial inoculum

Abstract

The invention relates to a biocontrol inoculant for preventing and treating watermelon fusarium wilt and a use method, and the preparation method of the biocontrol inoculant comprises the following steps: (1) inoculating a bacterial strain activated by bacillus subtilis into a sterilized LB culture medium, and carrying out shake culture in a shaker at 30 DEG C and 150 r / min for 36 hours to prepare a seed solution; (2) inoculating the activated strain of trichoderma asperellum or trichoderma atroviride into a PDA liquid culture medium, and culturing for 72 hours in a shaking table at the constant temperature of 30 DEG C to obtain sufficient bacterial liquid; (3) carrying out co-culture by adopting a co-culture fermentation medium: inoculating bacillus subtilis and trichoderma asperellum or trichoderma atroviride into the co-culture fermentation medium according to a ratio of (3-1): (1-3), and carrying out mixed fermentation culture to obtain a microbial symbiotic culture microbial agent; the biocontrol microbial agent has an obvious growth promoting effect on watermelons, has an excellent prevention and treatment effect on watermelon fusarium wilt, and can be used for field production.

Description

Technical Field

[0001] The invention belongs to the field of biological pesticides, and particularly relates to a biocontrol fungicide for preventing and controlling watermelon wilt disease and a use method thereof. Background Art

[0002] Watermelon wilt, also known as dead seedling disease, wilt disease or vine cutting disease, is a fungal soil-borne disease. The pathogen successfully colonizes and grows in the plant's vascular wall cells by invading the wounds of the plant's roots and the intercellular spaces at the top of the root hairs, and reproduces on a large scale in the vascular bundles, while causing the decomposition and destruction of the cell structure, leading to vascular blockage, thereby hindering the normal water transport of the plant and causing the plant to wilt. The disease occurs throughout the entire growth period of watermelons and is ubiquitous worldwide. When the disease is severe, it leads to a sharp drop in watermelon production, or even a total loss of harvest, causing huge losses to the agricultural economy.

[0003] Biocontrol bacteria refer to beneficial microorganisms that have the function of preventing and controlling plant diseases. These beneficial microorganisms and their metabolites are used to inhibit pathogens to achieve the purpose of controlling plant diseases. Biocontrol bacteria are not only environmentally friendly, but also highly effective, which is of great significance to the sustainable development of modern agriculture. Microorganisms such as actinomycetes, fungi and bacteria are widely used as biocontrol factors in the prevention and control of plant diseases. The research on biocontrol factors of Fusarium oxysporum, the pathogen of watermelon wilt, has been reported both at home and abroad. The biocontrol factors such as Trichoderma, Streptomyces and Bacillus that have been screened have significant control effects on wilt.

[0004] In practical applications, biocontrol agents have the problem of insufficient stability of their protective effects. The method of mixing two or more microorganisms with disease biocontrol functions to form a composite biocontrol agent can achieve a wider colonization on plant roots than single-species preparations. However, there is also mutual antagonism between microorganisms within some mixed agents. Therefore, the study of fermentation technology is very important to solve the problem of low spore production and reproduction capacity of various single agents in biocontrol mixtures. Summary of the invention

[0005] In order to make up for the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a biocontrol agent for preventing and controlling watermelon wilt and a method of use, so as to increase the spore production of the mixture and the control effect on watermelon wilt.

[0006] The object of the present invention is achieved through the following technical scheme, a biocontrol agent for preventing and controlling watermelon wilt and a method of use, the technical points of which are:

[0007] The preparation method of the biocontrol agent comprises the following steps:

[0008] (1) The activated Bacillus subtilis strain was inoculated into a sterile LB medium and cultured in a shaking incubator at 30° C. and 150 rpm for 36 h to prepare a seed solution;

[0009] (2) inoculating the activated strain of Trichoderma spinulosa or Trichoderma aureum into a PDA liquid culture medium and culturing it in a constant temperature shaker at 30° C. for 72 h to obtain a sufficient amount of bacterial liquid;

[0010] (3) Co-cultivation using co-culture fermentation medium:

[0011] Inoculating Bacillus subtilis and Trichoderma aculeatus or Trichoderma aureum in a co-culture fermentation medium at a ratio of 3-1:1-3, performing mixed fermentation culture, and obtaining a microbial symbiotic culture agent;

[0012] The formula of the combined culture fermentation medium is: 5g / L α-lactose, 10g / L wheat bran, 10g / L corn flour, 10g / L peptone, 5g / L yeast extract, 5g / L soybean cake powder, and inorganic salt: 10g / L NaCl;

[0013] The co-culture fermentation conditions are: 32° C., initial pH: 6.5, rotation speed: 180 r / min, inoculation amount: 4%, ventilation volume: 6 L / 10 L, and fermentation culture for 72 h-96 h.

[0014] The method for using the biocontrol agent has the following technical points: the concentration of the biocontrol agent is adjusted to 1×10 7 cfu / ml, and root irrigation was carried out with 50-100ml per plant.

[0015] Beneficial effects of the present invention:

[0016] (1) Through the combined culture formula of the present application, the combined fermentation culture of Bacillus subtilis and Trichoderma acanthosporum and Trichoderma acanthosporum has a good spore production effect, which can increase the individual yields of the two.

[0017] (2) The biocontrol agent has a significant growth-promoting effect on watermelon and an excellent control effect on watermelon wilt, and can be used in field production. DETAILED DESCRIPTION

[0018] Example 1: Co-cultivation of biocontrol Bacillus and biocontrol Trichoderma

[0019] The preparation method of the biocontrol agent comprises the following steps:

[0020] (1) The activated strain of Bacillus subtilis (A) was inoculated into a sterile LB medium and cultured in a shaking incubator at 30° C. and 150 rpm for 36 h to prepare a seed solution;

[0021] (2) inoculating the activated strain of Trichoderma spinulosa (B) or Trichoderma aureum (C) into a PDA liquid culture medium and culturing it in a constant temperature shaker at 30° C. for 72 h to obtain a sufficient amount of bacterial liquid;

[0022] (3) Co-cultivation using co-culture fermentation medium:

[0023] Inoculating Bacillus subtilis and Trichoderma aculeatus or Trichoderma aureum in a co-culture fermentation medium at a ratio of 3-1:1-3, performing mixed fermentation culture, and obtaining a microbial symbiotic culture agent;

[0024] The formula of the combined culture fermentation medium is: 5g / L α-lactose, 10g / L wheat bran, 10g / L corn flour, 10g / L peptone, 5g / L yeast extract, 5g / L soybean cake powder, and inorganic salt: 10g / L NaCl;

[0025] The co-culture fermentation conditions are: 32° C., initial pH: 6.5, rotation speed: 180 r / min, inoculation amount: 4%, ventilation volume: 6 L / 10 L, and fermentation culture for 72 h-96 h.

[0026] Table 1 The number of bacteria in the combined culture of biocontrol Bacillus A and three biocontrol Trichoderma species (×10 9 cfu / ml)

[0027]

[0028] Bacillus subtilis and Trichoderma aculeatus or Trichoderma aurea were inoculated in a co-culture fermentation medium at a ratio of 3:1, 1:1, and 1:3, and mixed fermentation was performed to obtain a microbial symbiotic culture agent. The fermentation was performed for 72 hours and 96 hours, and the number of viable bacteria was determined by plate counting. The results showed that the spore production of Bacillus subtilis and Trichoderma aculeatus and Trichoderma aurea after 96 hours of co-fermentation was significantly greater than that of fermentation for 72 hours; the spore production of co-fermentation with different inoculation ratios also had significant differences, and the 1:1 ratio was greater than the 3:1 and 1:3 ratios, indicating that the biocontrol Bacillus and Trichoderma aculeatus and Trichoderma aurea had the best spore production effect under the condition of equivalent initial concentrations in co-culture.

[0029] Example 2: Effect of biocontrol agents on watermelon wilt pathogen

[0030] The concentration of the biocontrol Bacillus and two kinds of Trichoderma mixture (1:1) was adjusted to 1×10 7 cfu / ml, and the field watermelon growth and wilt control test was carried out. The results of the field watermelon growth test showed that the combination of biological control bacteria and Trichoderma had a good preventive effect on melon wilt in the early stage (10 days). Although the control effect decreased over time, it could still effectively reduce the disease severity within 25 days.

[0031] Table 2 Effects of biocontrol bacteria and Trichoderma mixture on melon wilt

[0032]

Claims

1. A biocontrol agent for preventing and treating watermelon wilt, characterized in that: The preparation method of the biocontrol agent comprises the following steps: (1) The activated strain of Bacillus subtilis was inoculated into a sterile LB medium and cultured in a shaking incubator at 30° C. and 150 rpm for 36 h to prepare a seed solution; (2) inoculating the activated strain of Trichoderma spinulosa or Trichoderma aureum into a PDA liquid culture medium and culturing it in a constant temperature shaker at 30° C. for 72 h to obtain a sufficient amount of bacterial liquid; (3) Co-cultivation using co-culture fermentation medium: Inoculating Bacillus subtilis and Trichoderma aculeatus or Trichoderma aureum in a co-culture fermentation medium at a ratio of 3-1:1-3, performing mixed fermentation culture, and obtaining a microbial symbiotic culture agent; The formula of the combined culture fermentation medium is: 5g / L α-lactose, 10g / L wheat bran, 10g / L corn flour, 10g / L peptone, 5g / L yeast extract, 5g / L soybean cake powder, and inorganic salt: 10g / L NaCl; The co-culture fermentation conditions are: 32° C., initial pH: 6.5, rotation speed: 180 r / min, inoculation amount: 4%, ventilation volume: 6 L / 10 L, and fermentation culture for 72 h-96 h.

2. The method for using the biocontrol agent for preventing and controlling watermelon wilt according to claim 1, characterized in that: The concentration of biocontrol agents was adjusted to 1×10 7 cfu / ml, and root irrigation was carried out with 50-100ml per plant.

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