A green biocontrol bacteria-containing sclerotinia rot of rape fungicidal composition and application thereof

Abstract

The present application belongs to the field of agricultural fungicides, and particularly relates to a rapeseed sclerotinia disease fungicide composition containing green biocontrol bacteria and application thereof, which comprises Bacillus amyloliquefaciens WHIG bacterial agent and cantharidin; the mass ratio of the Bacillus amyloliquefaciens WHIG bacterial agent and the cantharidin is 20:1-1:20. The present application adopts a biological control strategy, which can not only reduce the application amount of chemical agents, improve the stability of biological control, but also achieve the effect of effectively controlling diseases, and the composition shows significant synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fungicides, specifically relating to a fungicide composition for controlling sclerotinia stem rot in rapeseed containing green biocontrol bacteria and its application. Background Technology

[0002] Rapeseed is one of my country's most important oilseed crops and one of the world's four major oilseed crops. Its seeds contain 33%-50% oil, giving it significant economic value and wide applications in medicine, food, and industrial raw materials. Rapeseed has a long growing season and is susceptible to numerous diseases. Among these, sclerotinia stem rot is the most serious. Sclerotinia stem rot can occur at any stage of the rapeseed's growth, but is most severe during the flowering and fruiting period. The pathogen can infect all above-ground parts of the rapeseed plant, with the greatest damage caused by infection of the stems. The pathogen mostly infects through withered flowers, initially appearing as water-soaked or pale lesions. Later, the fallen petals cause infection of the stems and leaves. When the stem is infected with sclerotinia stem rot, it initially shows water-soaked lesions, which gradually turn brown, become sunken, and develop white, cottony mycelium and black, rat-dung-like sclerotia. When the air becomes dry, the diseased parts at the base of the stem will dry up, wither, and die. When plant leaves are infected with sclerotinia stem rot, the initial symptoms are mainly dark blue, water-soaked, and irregular lesions with concentric rings. When the air becomes humid, the lesions grow rapidly, causing the leaves to rot quickly. Subsequently, white, cottony mycelium and small, black, rat-dung-like sclerotia will grow on the diseased area. The typical symptoms of sclerotinia stem rot in rapeseed are the growth of white, cottony mold and black, rat-dung-like sclerotia on the diseased area.

[0003] For a long time, the control of rapeseed sclerotinia stem rot has mainly relied on chemical control. While the application of fungicides has a certain control effect on rapeseed sclerotinia stem rot, its effectiveness is affected by various factors such as the timing of application, the plant's developmental stage, and environmental conditions. Furthermore, the large-scale use of chemical agents not only leads to the accumulation of pesticide residues, affecting human health, but also disrupts the ecological balance and causes environmental pollution. Long-term use of chemical agents can lead to drug resistance in pathogens, resulting in decreased or even failed control, forcing people to seek safer and more effective control methods. Rapeseed, as a global oilseed crop, has wide applications in food and medicine. Using biocontrol agents to control the disease can not only reduce the use of chemical pesticides but also mitigate environmental pollution, making it an increasingly popular environmentally friendly control technology.

[0004] *Bacillus amyloliquefaciens* is a Gram-positive spore-forming bacterium highly related to *Bacillus subtilis* and widely distributed in nature. During its growth, *Bacillus amyloliquefaciens* produces a series of metabolites with broad-spectrum inhibitory activity against fungi and bacteria, showing great promise for biocontrol and gradually becoming a microorganism with potential for biopesticide development. *Bacillus amyloliquefaciens* WHIG was isolated by the Rice Disease Control Research Group of the Institute of Plant Protection and Agricultural Product Quality Safety, Anhui Academy of Agricultural Sciences, and deposited on April 15, 2015, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 10722. This strain was named WHIG and its classification is *Bacillus amyloliquefaciens* (…). Bacillus amyloliquefaciens ) (reference CN107136120A).

[0005] Cantharidin, also known as blister beetle, is a sesquiterpene defense substance produced by insects of the blister beetle family (Brassica davidii). It has a unique defensive effect against the natural enemies of blister beetles. For a long time, cantharidin has been extensively researched and applied in medicine, and its pharmacology has been thoroughly studied. In addition to its pharmaceutical applications, it has been found to have strong stomach poisoning, antifeedant, and contact toxicity effects on some agricultural pests. As a novel biological insecticide, cantharidin has seen the registration and application of aqueous formulations in recent years (Chen Lin et al., Field Application Study of 0.1% Cantharidin Aqueous Formulation).

[0006] Research on cantharidin against plant diseases is limited. Yun Yueli et al. (Inhibitory effect of cantharidin on plant pathogens) were the first to demonstrate the inhibitory effect of cantharidin on plant pathogens. They found that a 500 PPM cantharidin solution significantly inhibited six of the eight plant pathogenic fungi tested, including *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, *Anthracnose fungus*, *Sclerotinia sclerotiorum*, and *Fusarium wilt*, but had no significant effect on the nine plant pathogenic bacteria tested. Wang Lin et al. (Inhibitory mechanism of 3-halophenyl aromatic amine norcantharidin derivatives against *Sclerotinia sclerotiorum*) disclosed that cantharidin derivatives inhibit the mycelial growth of a large number of plant pathogenic fungi, such as those causing *Fusarium wilt*, *Rhizoctonia solani*, and *Sclerotinia sclerotiorum*.

[0007] Biological control strategies can reduce the amount of chemical pesticides used, improve the stability of biological control, and effectively control diseases. However, there are no existing reports on the combined use of Bacillus amyloliquefaciens WHIG and cantharidin. The applicant's research has found that the combination of Bacillus amyloliquefaciens WHIG and cantharidin has a good synergistic effect against sclerotinia stem rot in rapeseed. Summary of the Invention

[0008] This invention employs a biological control strategy, which can reduce the amount of chemical agents used and improve the stability of biological control, and the composition exhibits a significant synergistic effect.

[0009] The technical solution of this invention is as follows: A bactericidal composition comprising Bacillus amyloliquefaciens, characterized in that it comprises Bacillus amyloliquefaciens WHIG inoculant and cantharidin; the mass ratio of Bacillus amyloliquefaciens WHIG inoculant to cantharidin is 20:1-1:20; Bacillus amyloliquefaciens WHIG is deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 10722; the concentration of Bacillus amyloliquefaciens WHIG inoculant is 1×10⁻⁶. 7 -1×10 9 cfu / mL.

[0010] Preferably, the mass ratio of the Bacillus amyloliquefaciens WHIG inoculant to cantharidin is 10:1 to 1:20. More preferably, the concentration of the Bacillus amyloliquefaciens WHIG inoculant is 1×10⁻⁶. 8 cfu / mL; the mass ratio of the Bacillus amyloliquefaciens WHIG inoculum and cantharidin is 10:1-1:10.

[0011] The bactericidal composition further comprises pesticide excipients, and is prepared into a pesticide formulation. The pesticide formulation is a suspension concentrate, wettable powder, or water-dispersible granule. The excipients are selected from conventional excipients in the art and formulated using conventional preparation methods.

[0012] The aforementioned bactericidal composition can be used to control crop diseases. The preferred crop disease is sclerotinia stem rot in rapeseed.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects: This invention utilizes a control strategy that combines biological and chemical control, which can reduce the amount of chemical agents used, improve the stability of biological control, and effectively control diseases.

[0014] When the mass ratio of WH1G bacterial suspension to cantharidin is 20:1-1:20, the composition exhibits a synergistic effect against Sclerotinia sclerotiorum var. sclerotiorum, effectively reducing the dosage of single agents and delaying the development of drug resistance in the pathogen. Detailed Implementation

[0015] The present invention will be further explained below with reference to specific embodiments.

[0016] Example 1: Indoor toxicity test of compound agent against sclerotinia stem rot in rapeseed 1.1 Test materials Biocontrol agent: Bacillus amyloliquefaciens WH1G, deposited at Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences. The strain is cultured on modified NA medium (beef extract 1.0g, peptone 5.0g, yeast extract 5.0g, NaCl 5.0g, sucrose 10.0g, agar 20.0g, distilled water 1000mL, pH 6.8-7.0) in a constant temperature incubator at 28°C. Prepare a Bacillus amyloliquefaciens WH1G suspension with a concentration of 1×10 8 cfu / mL.

[0017] Sclerotinia sclerotiorum (the pathogen of rapeseed sclerotinia rot) Sclerotinia sclerotiorum was isolated, screened, identified and preserved by Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences.

[0018] Cantharidin is commercially available.

[0019] 1.2 Test method The mycelial growth rate method was adopted. Based on the preliminary experiment, PDA plates containing different gradient concentrations of the agents were prepared, and a mycelial plug of Sclerotinia sclerotiorum (5 mm in diameter) was placed in the center of the drug-containing plate, followed by culturing in an incubator at 28°C. A control group was set up, with 4 replicates for each treatment. When the control colony covered two-thirds of the culture dish, the colony diameter of each treatment was measured by the cross method, and the average inhibition rate was calculated.

[0020] Average inhibition rate = [(average colony diameter of control group - average colony diameter of treatment group) / (average colony diameter of control group - 5.0)] × 100%.

[0021] Convert the mycelial growth inhibition rate to probit value of inhibition (y), and convert the agent concentration to logarithm of concentration (x). Draw a toxicity regression line with the logarithm of concentration as the abscissa and the probit value as the ordinate, obtain the toxicity regression equation of the single agent and its mixture against the pathogen, and calculate the EC 50 value and the correlation coefficient r value.

[0022] Calculate the toxicity index of each agent and the co-toxicity coefficient (CTC value) of the mixture according to Sun Yunpei's method, where CTC ≤ 80 indicates antagonism, 80 < CTC < 120 indicates addition effect, and CTC ≥ 120 indicates synergism.

[0023] Co-toxicity coefficient (CTC) = actual toxicity index of the mixture (ATI) / theoretical toxicity index of the mixture (TTI).

[0024] Actual toxicity index (ATI) = EC of standard agent 50 / EC of test agent 50 × 100 Theoretical toxicity index (TTI) = toxicity index of agent A × percentage content of A in the mixture + toxicity index of agent B × percentage content of B in the mixture.

[0025] 1.3 Test Results Table 1 shows that the WH1G bacterial suspension has an EC50 effect on Sclerotinia sclerotiorum var. sclerotiorum in rapeseed. 50 The value was 21.62 mg / L; the EC50 of cantharidin against Sclerotinia sclerotiorum was... 50 The value was 5.30 mg / L. When the mass ratio of WH1G bacterial suspension to cantharidin was 20:1-1:20, it showed a synergistic effect against Sclerotinia sclerotiorum causal agent of rapeseed. When the combination showed a synergistic effect, it could effectively reduce the dosage of single agent and slow down the development of drug resistance in the disease.

[0026] Table 1. Indoor toxicity test of compound agents against Sclerotinia sclerotinia in rapeseed. , Note: 1 mL of bacterial suspension is calculated as 1 g.

[0027] Example 2: Pot test of compound agent against sclerotinia stem rot in rapeseed 2.1 Test materials Biocontrol bacteria: *Bacillus amyloliquefaciens* WH1G, Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences. The bacteria were cultured in a modified NA medium (1.0g beef extract, 5.0g peptone, 5.0g yeast extract, 5.0g NaCl, 10.0g sucrose, 20.0g agar, 1000mL distilled water, pH 6.8-7.0) at 28℃. A *Bacillus amyloliquefaciens* WH1G bacterial suspension was prepared with a bacterial count of 1×10⁻⁶. 8 cfu / mL.

[0028] Sclerotinia stem rot pathogen ( S. sclerotiorum The samples were separated, screened, identified, and preserved by the Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences.

[0029] Cantharidin, commercially available.

[0030] 2.2 Test Method Different concentrations of the pesticide were evenly sprayed onto the surface of rapeseed leaves. Twenty-four hours later, sclerotinia rot pathogens were inoculated onto the leaf surface. A control group was included. Each treatment was replicated with 10 plants, and the experiment was repeated three times. The plants were incubated in a humidified incubator. Disease incidence was assessed 7 days after inoculation, disease severity was recorded, and the disease index was calculated.

[0031] Disease severity grading standards: Level 0, no disease; Grade 1: The affected area accounts for less than 5% of the total leaf area; Level 3, with the affected area accounting for 5%-10% of the total leaf area; Level 5, with the affected area accounting for 11%-30% of the total leaf area; Level 7, with the affected area accounting for 31%-50% of the total leaf area; Level 9, with the affected area accounting for more than 50% of the total leaf area.

[0032] Disease index = ∑ [(Number of diseased plants at each level × Relative disease severity value) / (Total number of plants surveyed × Highest disease severity value)] × 100 Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100 2.3 Test Results As shown in Table 1, the combination of WH1G bacterial suspension and cantharidin has excellent control effect on sclerotinia stem rot in rapeseed and can be used in field production.

[0033] Table 2 Pot tests of compound pesticides against Sclerotinia sclerotinia in rapeseed. , Note: 1 mL of bacterial suspension is calculated as 1 g.

[0034] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention are considered to be within the scope of protection of this invention.

Claims

1. A bactericidal composition for sclerotinia stem rot control in rapeseed containing green biocontrol bacteria, characterized in that, It is composed of Bacillus amyloliquefaciens WHIG bacterial agent and cantharidin; the mass ratio of Bacillus amyloliquefaciens WHIG bacterial agent and cantharidin is 20:1-1:20; Bacillus amyloliquefaciens WHIG is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 10722; Bacillus amyloliquefaciens WHIG inoculum concentration 1×10 8 cfu / mL.

2. The bactericidal composition according to claim 1, characterized in that, The mass ratio of Bacillus amyloliquefaciens WHIG inoculum to cantharidin is 10:1 to 1:

20.

3. The bactericidal composition according to claim 2, characterized in that, The mass ratio of the Bacillus amyloliquefaciens WHIG inoculum to cantharidin is 10:1 to 1:

10.

4. The bactericidal composition according to any one of claims 1-3, characterized in that, It also contains pesticide excipients, which are used to prepare pesticide formulations.

5. The bactericidal composition according to claim 4, characterized in that, The pesticide formulation is a suspension concentrate, wettable powder, or water-dispersible granule.

6. The use of the bactericidal composition according to any one of claims 1-5 for the prevention and control of sclerotinia stem rot in rapeseed.