Fungicide composition for preventing and treating peanut root rot as well as preparation and application thereof

Abstract

The invention relates to a fungicide composition for preventing and treating peanut root rot as well as preparation and application of the fungicide composition. The invention aims to solve the technical problems of unstable field control effect, high chemical pesticide residue and high drug resistance risk of biological control in peanut root rot control. Bacillus velezensis YJ02 with good biocompatibility is compounded with azoxystrobin (a biological fungicide generates antibacterial substances and induces plant defensive enzymes, and a chemical fungicide is combined to rapidly inhibit respiration of pathogenic bacteria) to prevent and treat peanut root rot; the technical effects that the efficient synergistic control effect (for example, the control effect reaches up to 86.79%) is achieved, the application amount of chemical pesticides is reduced (for example, the dosage of azoxystrobin is only 9.82% of the conventional dosage), the drug resistance risk of pathogenic bacteria is effectively reduced, the morbidity and disease index are reduced, and environment friendliness is achieved are achieved, and high economic benefits and social benefits are achieved.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a fungal-agent composition for controlling peanut root rot and its preparation and application. Background Technology

[0002] Peanut root rot, commonly known as "rat's tail" or "root rot," is caused by Fusarium oxysporum (…). Fusarium oxysporum This fungal disease, caused by [unspecified pathogen], is distributed throughout peanut-producing areas in my country, with particularly severe damage in southern regions. It can infect peanuts throughout their entire growth cycle, primarily affecting the roots, but can also extend to the pegs and pods. In the early stages, the tip of the main root becomes wet and brown, with irregular small spots appearing on the epidermis. In the middle stage, the lesions enlarge to a dark brown, the cortex rots and falls off, and lateral roots decrease. In the later stage, the root system rots severely, leaving only remnants resembling a "rat's tail." Above-ground parts subsequently show yellowing and wilting of leaves, stunted growth, weak stems, and reduced branching. During the pod-forming stage, pods develop poorly, the pericarp turns brown or cracks, and the kernels become shriveled. Under high humidity, a white to pale red mold layer or conidiophores may appear on the diseased parts.

[0003] The prevalence of this disease is closely related to soil, climate, and cultivation practices. Continuous cropping, heavy clay soil, poor drainage, and conditions such as low temperatures, overcast skies with rain, or sudden shifts from drought to sunshine can exacerbate the damage. The pathogen overwinters in the soil as mycelium, conidia, or chlamydospores within diseased plant debris. It spreads through water flow, fertilization, and agricultural operations, directly invading through wounds or the epidermis. In general, disease incidence in affected fields is 10%-20%, resulting in a yield reduction of 5%-8%; in severely affected fields, the incidence reaches 20%-30%, leading to yield reductions exceeding 20%, and in severe cases, complete crop failure.

[0004] Existing control methods have limitations: chemical control, while rapid, easily leaves pesticide residues and induces resistance; biological control, while safe, is slow to take effect and its effectiveness is unstable; agricultural control methods such as crop rotation and variety selection can reduce disease, but are limited by land resources and variety resistance. Therefore, developing new, highly efficient, low-toxicity, and environmentally friendly compound pesticides has become a key need to overcome the current bottlenecks in disease control.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors discovered that Bacillus vesiculosus YJ02 and azoxystrobin have good biocompatibility. The combination of the two can significantly inhibit Fusarium oxysporum and has excellent synergistic effects. It can solve the problem of unstable field control efficacy of biocontrol bacteria, reduce the amount of chemical pesticides used, reduce the incidence and disease index of peanut root rot, and significantly improve the disease resistance of peanuts, providing a new approach for the prevention and control of peanut root rot.

[0007] According to one aspect of the present disclosure, a microbial composition is provided, which contains azoxystrobin 2.45-22.10 μg / mL, Bacillus velezensis (CGMCC NO. 23110) Bacillus velezensis ) YJ02 0.5×10 2 ~0.5×10 8 CFU / mL.

[0008] In some embodiments of the present disclosure, the microbial composition contains azoxystrobin 7.36-22.10 μg / mL, Bacillus velezensis (CGMCC NO. 23110) Bacillus velezensis ) YJ02 0.5×10 5 ~0.5×10 8 CFU / mL.

[0009] According to another aspect of the present disclosure, a preparation method of the microbial composition is provided, which comprises the following steps: (1) Bacillus velezensis YJ02 with a preservation number of CGMCC NO. 23110 is activated and inoculated in NA culture solution, and then cultured in a shaking incubator at 28°C and 160 rpm for 48 h to obtain a microbial agent raw material (the composition of the NA culture solution: yeast extract powder 3 g, peptone 5 g, glucose 10 g, and distilled water 1000 mL); (2) the microbial agent raw material is uniformly mixed with azoxystrobin technical material and corresponding adjuvants in proportion, so that the azoxystrobin content reaches 2.45-22.10 μg / mL, and the number of Bacillus velezensis YJ02 reaches 0.5×10 2 ~ 0.5×10 8 CFU / mL.

[0010] According to a third aspect of the present disclosure, the microbial composition is applied to inhibit Fusarium oxysporum.

[0011] According to a fourth aspect of the present disclosure, the microbial composition is applied to improve the disease resistance of peanuts.

[0012] According to a fifth aspect of the present disclosure, the microbial composition is applied to prevent and treat peanut root rot.

[0013] In some embodiments of the present disclosure, the root irrigation is repeated three times at intervals of 5-8 days before or after the occurrence of root rot, and the amount of irrigation per plant is 5-15 mL each time.

[0014] One or more technical solutions provided in the embodiments of the present disclosure have at least any of the following technical effects or advantages: 1. Synergistic effect, improve the control effect: Bacillus velezensis YJ02 can inhibit the colonization and expansion of Fusarium oxysporum and other pathogens by competing for nutrients and space, producing antagonistic substances (such as antibiotics, enzymes), inducing the expression of peanut root resistance genes (such as PR proteins, chitinase), and azoxystrobin directly inhibits the spore germination and mycelial growth of the pathogen, rapidly reduces the pathogen base in the field, and the two form a "quick-acting + long-acting" prevention and control system, which is significantly better than using biological agents or chemical agents alone.

[0015] 2. Dual improvement of prevention and control effect and resistance management: potting prevention and control effect test proves that the compound composition can reduce the peanut root rot disease rate to 11.67%, and the optimal ratio of the compound composition has a prevention and control effect of up to 86.79%, and the use amount of azoxystrobin in the highest concentration of the compound composition is only 9.82% of the conventional use amount in the field, which indicates that the compound composition can effectively inhibit the pathogen while greatly reducing the use amount of chemical agents, effectively reducing the risk of pathogen resistance, and providing technical support for delaying the development of resistance.

[0016] 3. Innovative disease control strategy: Bacillus velezensis YJ02 with good biocompatibility is first selected to be compounded with azoxystrobin to prevent and control peanut root rot, which breaks through the limitation of single active ingredient and provides an efficient and environmentally friendly new solution for peanut root rot prevention and control. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The YJ02 0.5x10 2 cfu / mL+azoxystrobin 2.46 μg / mL compound composition inhibits peanut root rot fungus effect result graph, wherein A is 1x10 2 cfu / mL Bacillus velezensis YJ02 on Fusarium oxysporum; B is the inhibition effect of 4.91 μg / mL azoxystrobin single agent on Fusarium oxysporum; C is the inhibition effect of YJ02 0.5x10 2 cfu / mL+azoxystrobin 2.46 μg / mL on Fusarium oxysporum; and D is the blank control of Fusarium oxysporum. DETAILED DESCRIPTION

[0018] In order to better understand the technical scheme of the present application, the above technical scheme will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0019] The main test raw materials and reagents involved in the following examples include: Test strain: peanut root rot fungus (Fusarium oxysporum Fusarium oxysporum ), Bacillus velezensis Bacillus velezensisYJ02, the preservation number of which is CGMCC NO. 23110, and the preservation date of which is August 4, 2021; see Chinese patent document CN113817642A.

[0020] PDA medium: potato 200.0 g, glucose 20.0 g, agar 17.0 g, distilled water 1000 mL.

[0021] NA medium: yeast extract powder 3.0 g, peptone 5.0 g, glucose 10.0 g, agar 17.0 g, distilled water 1000 mL.

[0022] Tested peanut varieties: Yuhua 22, commercially available.

[0023] The tested fungicides are shown in Table 1.

[0024] Table 1: Tested fungicides .

[0025] The Bacillus velezensis agent described in the present application is prepared by the following method: After the Bacillus velezensis YJ02 is activated, a single colony is inoculated in NA culture solution, and is cultured in a shaking incubator at 28℃ and 160 rpm for 18-30 h, thereby obtaining the Bacillus velezensis YJ02.

[0026] Example 1: Compatibility determination of Bacillus velezensis YJ02 and fungicides 1. Test method Five chemical fungicides commonly used for peanut root rot, pyraclostrobin, tebuconazole, difenoconazole, azoxystrobin and fludioxonil, were selected for testing. The compatibility of Bacillus velezensis YJ02 and the five fungicides was determined by plate counting method. Each chemical fungicide was added to the NA medium to make the final concentration 50, 100, 150, 200 and 250 μg / mL; 1 mL of 1.0×10 8 CFU / mL Bacillus velezensis YJ02 solution was diluted 10 times in series for 5 times, 100 μL of the diluted bacterial suspension was uniformly coated on the NA plate, sterile water was used as a control, and the plate was cultured at 28℃ in the dark. After 48 h, the number of Bacillus velezensis YJ02 colonies was counted, the bacterial amount of YJ02 per milliliter of bacterial solution was calculated, and each group was repeated 3 times. DPS software was used for statistical analysis of biological compatibility.

[0027] 2. Test results The results of the chemical fungicide compatibility test with Bacillus velezensis YJ02 showed that YJ02 could not grow on pyraclostrobin and fludioxonil plates with a concentration of 50 μg / mL; the number of YJ02 colonies on the 50 μg / mL tebuconazole plate was only 0.20×10 8 CFU / mL, which was significantly lower than that of the control group (P <0.05); but on azoxystrobin plates with concentrations of 50, 100, and 150 μg / mL, the number of YJ02 colonies was 1.09×10 8 , 1.06×10 8 , and 1.05×10 8 CFU / mL, respectively, which showed no significant difference with the control group (Table 2), indicating that Bacillus velezensis YJ02 had good biocompatibility with azoxystrobin at a concentration of less than 150 μg / mL and could be used for mixing.

[0028] Table 2 Effects of five fungicides on the growth of Bacillus velezensis YJ02 .

[0029] Example Two, Bacillus velezensis YJ02 and azoxystrobin indoor virulence test on Fusarium oxysporum 1. Test method The inhibition rate of Bacillus velezensis YJ02 and azoxystrobin on the main pathogen Fusarium oxysporum of peanut root rot was determined by using the toxic medium method. Azoxystrobin and Bacillus velezensis YJ02 were added to PDA medium cooled to about 40℃, respectively, to prepare different concentration gradients of drug (bacteria) containing medium (Table 3), and then poured into plates after mixing evenly. The blank control group had no addition. A 5.0 mm diameter Fusarium oxysporum cake was inoculated in the center of each prepared drug-containing PDA plate at different concentrations, with 3 replicates for each concentration. After 7 days of culture at 25℃ in the dark, the colony diameter of Fusarium oxysporum was measured, and the inhibition rate was calculated.

[0030] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - cake diameter) × 100.

[0031] Table 3 Concentration gradient of Bacillus velezensis YJ02 and azoxystrobin

[0032] Note: The concentration in the table is the final concentration in the medium.

[0033] 2. Test results The indoor virulence of Bacillus velezensis YJ02 and azoxystrobin on Fusarium oxysporum is shown in Table 4. The EC 50 value of Bacillus velezensis YJ02 on Fusarium oxysporum was 1.19×102 CFU / mL, EC50 of azoxystrobin against Fusarium oxysporum 50 The value was 4.91 μg / mL. To facilitate the calculation of the YJ02 concentration, the EC50 of *Bacillus belyi* YJ02 against *Fusarium oxysporum* was measured in subsequent experiments. 50 Rounded to the nearest whole number, the result is 1.0 × 10⁻⁵. 2 cfu / mL.

[0034] Table 4. Results of toxicity assays of Bacillus vesiculosus YJ02 and azoxystrobin against Fusarium oxysporum. .

[0035] Example 3: Screening of the ratio of Bacillus belye YJ02 to azoxystrobin 1. Test Methods The optimal ratio of Bacillus belycetamol YJ02 to azoxystrobin was screened using an interactive assay. Two single-agent EC assays were used. 50 Based on this, 11 different mixing ratios were set up for toxicity testing. Two single-dose formulations were tested using their respective EC50 values. 50 The content is 100%, and in the mixed combination, the amount of component A is respectively its EC 50 The amounts of 10 / 10, 9 / 10, 8 / 10, 7 / 10, 6 / 10, 5 / 10, 4 / 10, 3 / 10, 2 / 10, 1 / 10, and 0, respectively, and the amounts of component B are respectively their EC values. 50 Eleven mixtures with different mixing ratios (0 / 10, 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, and 10 / 10) were prepared and their toxicity was determined. The toxicity ratios of each mixture were calculated based on the toxicity test results.

[0036] Toxicity ratio = Tested inhibition rate / Theoretical inhibition rate; Theoretical antibacterial rate = drug A in its EC 50 The tested inhibition rate at the dose × the proportion of drug A used + the EC50 of drug B 50 The tested inhibition rate at the dosage × the proportion of drug B used; In the formula: A is 1×10 2 cfu / mL Bacillus berberis YJ02, B is 4.91 μg / mL azoxystrobin.

[0037] A toxicity ratio <0.75 indicates an antagonistic effect; a toxicity ratio around 1 indicates an additive effect; and a toxicity-efficacy ratio >1.25 indicates a synergistic effect.

[0038] 2. Test Results The inhibitory effects of different ratios of Bacillus vesiculosus YJ02 and azoxystrobin on Fusarium oxysporum are shown in Table 5. Figure 1 The results showed that the two active ingredients, Bacillus belycetamol YJ02 and azoxystrobin, were present at their respective EC50 values. 50 The measured antibacterial rates of Fusarium oxysporum in the three mixtures of 0.5:0.5, 0.2:0.8, and 0.1:0.9 were significantly higher than the theoretical antibacterial rates, and the toxicity ratios were all greater than 1.25, namely 1.55, 1.47, and 1.41, respectively, showing a synergistic effect. The synergistic effect was most obvious in the 0.5:0.5 ratio.

[0039] Table 5. Inhibition rate and toxicity ratio of Bacillus vesiculosus YJ02 (A) and azoxystrobin (B) against peanut root rot pathogens. .

[0040] Example 4: Verification of Prevention Efficacy Test 1. Test Methods The mycelial cake of *Fusarium oxysporum*, the causal agent of peanut root rot, was inoculated into sterilized wheat grains for large-scale cultivation and cultured in the dark at 25°C for 7-10 days for later use. Peanut seeds were disinfected with 75% alcohol for 1 min, rinsed thoroughly with sterile water, and then sown in 19 cm × 14 cm × 10 cm square pots, each containing 0.2 kg of sterilized soil. All pots were placed in a greenhouse at 22-28°C, with 12 hours of light and approximately 70% relative humidity, and watered regularly. The efficacy test was conducted when the peanut seedlings reached the 4-5 leaf stage.

[0041] Considering that the actual field concentration of the pesticide is often much higher than the plate inhibitory concentration, the two mixed components were separated at their respective EC50 concentrations. 50 For low concentrations, medium and high concentrations were added proportionally. Bacillus belyssus YJ02 was added in 1000-fold gradients (1×10⁻⁶). 2 CFU / mL, 1×10 5 CFU / mL, 1×10 8 The concentrations of azoxystrobin were high, with a 3-fold gradient (4.91 μg / mL, 14.73 μg / mL, 44.19 μg / mL). Low, medium, and high gradient combinations were prepared by mixing the two components at a ratio of 0.5:0.5 for their respective gradients. Preventive and therapeutic treatment groups were established to investigate the mechanism of action of Bacillus belyssus YJ02 combined with azoxystrobin against peanut root rot.

[0042] Preventive measures settings: T1: Single dose of Bacillus belysin YJ02 1×10 2 CFU / mL (single dose); T2: Azoxystrobin 4.91 μg / mL as a single agent; T3: Combination Bacillus velezensis YJ02 0.5 x 10 2 CFU / mL + azoxystrobin 2.46 μg / mL (combination); T4: Bacillus velezensis YJ02 1 x 10 5 CFU / mL (single agent); T5: Azoxystrobin 14.73 μg / mL; T6: Combination Bacillus velezensis YJ02 0.5 x 10 5 CFU / mL + azoxystrobin 7.37 μg / mL (combination); T7: Bacillus velezensis YJ02 1 x 10 8 CFU / mL (single agent); T8: Azoxystrobin 44.19 μg / mL; T9: Combination Bacillus velezensis YJ02 0.5 x 10 8 CFU / mL + azoxystrobin 22.10 μg / mL (combination); T10: Azoxystrobin 250 μg / mL (control agent); T11: Blank control.

[0043] The application method was root drenching, and the amount of each peanut plant was 10 mL. After 48 h of prophylactic application, all treatments were inoculated uniformly. Wheat kernels covered with Fusarium oxysporum mycelium were placed at the base of each peanut seedling stem, with 5 kernels per plant. The first application was repeated 7 days later, and a total of 3 times. The treatment group was treated with the application of the fungicide 48 h after inoculation, and the treatment settings were the same as the prophylactic group. The blank control group was treated with water. Each treatment had 20 peanuts, and there were 3 replicates. After the blank control group was fully diseased, the disease incidence of each treatment was investigated and the control effect was calculated.

[0044] Disease incidence (%) = number of diseased plants / total number of plants x 100; Control effect (%) = (disease incidence of the blank control group - disease incidence of the treatment group) / disease incidence of the blank control group x 100.

[0045] 2. Test results: potting data (Table 6) showed that the control effects of the three complex agents in the preventive treatment group and the treatment group were significantly higher than the control effects of the respective single agents, indicating that Bacillus velezensis YJ02 and azoxystrobin had a significant synergistic effect when they were compounded according to the single agent concentration of 0.5:0.5. The control effects of each treatment in the preventive treatment group were higher than the control effects of the corresponding treatments in the treatment group, indicating that the complex agent and each single agent and the conventional field use amount of azoxystrobin had a preventive effect on peanut root rot that was greater than the treatment effect, and the complex agent was more suitable for application before the disease occurred. Among the three concentrations of the complex agent, the control effect of Bacillus velezensis YJ02 1×10 8 CFU / mL compounded with azoxystrobin 44.19 μg / mL at a ratio of 0.5:0.5 was the highest, reaching 86.79%, which was equivalent to the control effect of the conventional field use amount of azoxystrobin, but the content of azoxystrobin was only 9.82% of the conventional field use amount (250 μg / mL). The control effects of Bacillus velezensis YJ02 1×10 5 CFU / mL compounded with azoxystrobin 14.73 μg / mL at a ratio of 0.5:0.5 and Bacillus velezensis YJ02 1×10 2 CFU / mL compounded with azoxystrobin 4.91 μg / mL at a ratio of 0.5:0.5 were 62.26% and 28.30%, respectively, which were significantly higher than the control effects of the respective single agents, but the control effect of Bacillus velezensis YJ02 1×10 2 CFU / mL compounded with azoxystrobin 4.91 μg / mL at a ratio of 0.5:0.5 had poor potting control effect due to the low concentration, so the medium and high concentrations of the two single agents were selected as the suitable complexing concentrations, and therefore the suitable complexing concentration range of Bacillus velezensis YJ02 was 1×10 5 ~ 1×10 8 CFU / mL, and the suitable complexing concentration range of azoxystrobin was 14.73 ~ 44.19 μg / mL, and the best complexing ratio was 0.5:0.5.

[0046] Table 6 Potting control effects of complex combinations on peanut white thread blight .

[0047] Although some preferred embodiments of the present application have been described, those skilled in the art who understand the basic inventive concept can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0048] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A fungal drug composition, characterized in that, Bacillus belye, containing the active ingredient azoxystrobin at concentrations of 2.45–22.10 μg / mL and with the preservation number CGMCC NO. 23110, is a strain of Bacillus belye. Bacillus velezensis YJ02 0.5×10 2 ~ 0.5×10 8 CFU / mL.

2. The fungal drug composition according to claim 1, characterized in that, Bacillus belyssus YJ02, with a pyraclostrobin content of 7.36 ~ 22.10 μg / mL and preservation number CGMCC NO. 23110, was tested at 0.5 × 10⁻⁶. 5 ~ 0.5×10 8 CFU / mL.

3. A method for preparing the fungal drug composition according to claim 1, characterized in that, Includes the following steps: (1) After activating Bacillus belye YJ02 with preservation number CGMCC NO. 23110, it was inoculated into NA culture medium and cultured in a shaking incubator at 28℃ and 160 rpm for 48 h to obtain the bacterial agent raw material; (2) The bacterial agent raw material is mixed evenly with azoxystrobin technical and corresponding adjuvants in a certain proportion, so that the azoxystrobin content reaches 2.45~22.10 μg / mL and the Bacillus belysin YJ02 colony count reaches 0.5×10 2 ~ 0.5×10 8 CFU / mL.

4. The use of the fungal composition according to claim 1 or 2 in at least one of the following (1) to (3): (1) Inhibits Fusarium oxysporum; (2) Improve peanut disease resistance; (3) Prevention and control of peanut root rot.

5. The application according to claim 4, characterized in that, Before or after the occurrence of root rot, repeat the root irrigation 2-4 times at 5-8 day intervals, with each irrigation dose being 5-15 mL per plant.

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