Application of compound of paenibacillus polymyxa and chemical bactericide in preparation of medicament for preventing and treating wheat stem rot

Abstract

The invention discloses application of a compound of paenibacillus polymyxa and a chemical bactericide in preparation of a medicament for preventing and treating wheat stem rot. Belongs to the technical field of plant disease control. The invention studies the control effect of compound seed dressing treatment of paenibacillus polymyxa NPDY05-8 and seven common bactericides on the growth condition of wheat in the seedling stage and the control effect of wheat stem rot in the seedling stage, and the result shows that the compound seed dressing treatment of paenibacillus polymyxa NPDY05-8 and the bactericides can effectively improve the control effect of the bactericides on wheat stem rot; and compared with wheat treated by a single bactericide, wheat treated by mixed seed dressing of the bactericide and the paenibacillus polymyxa NPDY05-8 is thicker in leaf color and thicker in stalk, has the effects of promoting rooting and growth, and has huge application potential.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and more specifically to the application of Paenibacillus polymyxa and a chemical fungicide in preparing a medicine for preventing and controlling wheat stem base rot. Background Art

[0002] Wheat stem rot is mainly a soil-borne disease, which can not only cause yield reduction, but also lead to the accumulation of toxins in the ear, seriously affecting the health of humans and livestock, and causing unpredictable losses. The pathogenic composition of wheat stem rot is complex, and the pathogenic mechanism is not very clear. According to the research of many scholars at home and abroad, Fusarium gramiuearum, F. pseudogramiuearum and F. culmorum are the main pathogens of wheat stem rot, among which F. pseudogramiuearum is the main pathogen of the disease in most wheat-growing areas.

[0003] The prevention and control measures for wheat stem rot are mainly divided into four categories: agricultural prevention, planting disease-resistant varieties, biological prevention and chemical prevention. However, most of the wheat varieties currently promoted in production are susceptible varieties. The application of chemical agents has a poor control effect on soil-borne diseases and is prone to cause a series of problems such as drug resistance, pesticide residues, and environmental pollution. In recent years, microbial agents have been widely used in soil microbial environments to promote crop growth and prevent soil-borne diseases. However, microbial agents have the disadvantages of slow effectiveness and lower control effects than chemical agents.

[0004] In summary, how to provide a method for controlling wheat stem base rot with fast effect and good control effect is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides the use of a compound of Paenibacillus polymyxa and a chemical fungicide in the preparation of an agent for preventing and treating wheat stem base rot.

[0006] Paenibacillus polymyxa NPDY05-8 was isolated from the soil of the Fusarium spore resistance identification nursery of the Nanping Agricultural Science Research Institute experimental base, Tongyou Street, Jianyang District, Nanping City, Fujian Province. At present, there are few reports on whether Paenibacillus polymyxa can be mixed with chemical agents for application. The present invention uses Paenibacillus polymyxa NPDY05-8 to prevent and treat wheat stem base rot by reasonably matching and optimizing the application method with chemical agents, thereby achieving the purpose of complementing the defects of chemical agents and biological agents and superimposing the prevention and control effects. It is expected to provide a theoretical basis for the prevention and control of wheat stem base rot.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The application of a compound of Bacillus polymyxa and a chemical fungicide in the preparation of an agent for preventing and treating wheat stem base rot, wherein the Bacillus polymyxa is Bacillus polymyxa NPDY05-8, and its classification name is Paenibacillus polymyxa. It was deposited in the China Center for Type Culture Collection on May 12, 2023, with a deposit number of CCTCC NO: M 2023740, and the deposit address is Wuhan University, Wuhan, China.

[0009] Furthermore, the chemical fungicide is one or more of prochloraz, azoxystrobin, tebuconazole, fludioxonil, difenoconazole, thiophanate-methyl, and metalaxyl.

[0010] Furthermore, the chemical fungicide is prochloraz or fludioxonil.

[0011] Furthermore, 1×10 7 cfu·mL -1 The bacterial solution was mixed with 6% prochloraz.

[0012] Furthermore, 1×10 7 cfu·mL -1 The bacterial solution was mixed with 3% fludioxonil.

[0013] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0014] The combination of biocontrol bacteria and fungicides demonstrates the potential compatibility between biocontrol bacteria and chemical agents. The use of low-dose fungicides can inhibit pathogens to a certain extent and help biocontrol bacteria to colonize stably, becoming dominant strains. The present invention studies the effects of seed dressing treatment with Paenibacillus polymyxa NPDY05-8 and 7 commonly used fungicides on the growth of wheat seedlings and the control effect of wheat stem base rot at the seedling stage. The results show that seed dressing with Paenibacillus polymyxa NPDY05-8 and fungicides can effectively improve the control effect of fungicides on wheat stem base rot, and the fungicide and Paenibacillus polymyxa NPDY05-8 bacterial solution mixed with seeds have darker green leaves and thicker stems than those treated with fungicides alone, and have the effect of promoting rooting and growth, and have great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 The colony morphology (A) and bacterial morphology (×1600) (B) of Paenibacillus polymyxa NPDY05-8 on PDA medium in Example 1 of the present invention;

[0017] Figure 2 This is a graph showing the inhibition of Paenibacillus polymyxa NPDY05-8 against Fusarium graminearum in Example 2 of the present invention;

[0018] Figure 3 The control effect of Paenibacillus polymyxa NPDY05-8 and 7 common fungicides on wheat stem base rot at the seedling stage in Example 5 of the present invention is shown, wherein M represents prochloraz; MN represents prochloraz+NPDY05-8; J represents azoxystrobin; JN represents azoxystrobin+NPDY05-8; W represents tebuconazole; WN represents tebuconazole+NPDY05-8; S represents thiophanate-methyl; SN represents thiophanate-methyl+NPDY05-8; B represents difenoconazole; BN represents difenoconazole+NPDY05-8; L represents fludioxonil; LN represents fludioxonil+NPDY05-8; JS represents metalaxyl; JSN represents metalaxyl+NPDY05-8; N represents NPDY05-8; CK represents clear water control. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The materials used in the following examples are as follows:

[0021] Wheat variety: Le Tu 808, highly susceptible to stem base rot.

[0022] Source of biocontrol strain: Paenibacillus polymyxa NPDY05-8 was isolated from the soil of the Fusarium spore resistance identification nursery of the Nanping Agricultural Science Research Institute Experimental Base, Tongyou Street, Jianyang District, Nanping City, Fujian Province. Paenibacillus polymyxa, named NPDY05-8, is classified as Paenibacillus polymyxa and was deposited in the China Center for Type Culture Collection on May 12, 2023. The deposit number is CCTCC NO: M 2023740. The deposit address is Wuhan University, Wuhan, China.

[0023] The pathogenic strain of wheat stem rot: F. pseudogramiuearum, was isolated from the experimental site in Luohe, Henan.

[0024] Chemical agents for testing:

[0025] 97% prochloraz technical, Shandong Huayang Pesticide Chemical Group Co., Ltd.;

[0026] 94.8% azoxystrobin, Syngenta China Investment Co., Ltd.;

[0027] 98% tebuconazole suspension, Guangxi Tianyuan Biochemical Co., Ltd.;

[0028] 97.9% Fludioxonil SC, Syngenta China Investment Co., Ltd.;

[0029] 10% difenoconazole water dispersible granules, Zhejiang Yifan Biological Company;

[0030] Thifluzamide 24% SC, Syngenta Nantong Crop Protection Co., Ltd.;

[0031] Metalaxyl 35% powder, Zhejiang Heben Technology Co., Ltd.

[0032] Preparation of culture medium:

[0033] PDA medium (fungi or bacteria): 250 g potato, 20 g glucose, 20 g agar, dilute to 1000 mL.

[0034] PDB medium: 200 g potato, 20 g glucose, dilute to 1000 mL.

[0035] The drugs required for the present invention are conventional experimental drugs, purchased from commercial channels;

[0036] The experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0037] Example 1

[0038] Morphological Observation of Paenibacillus polymyxa NPDY05-8

[0039] Paenibacillus polymyxa NPDY05-8 appears as a transparent light yellow moist state on PDA solid medium, with irregular, smooth, and raised ( Figure 1 A); Gram staining is positive, and the bacteria are rod-shaped under a 1600x microscope, with a size of (2.0-5.0) μm × (0.5-1.0) μm ( Figure 1 B).

[0040] Example 2

[0041] Antibacterial rate of Paenibacillus polymyxa NPDY05-8 against Fusarium graminearum

[0042] The plate confrontation method was used to determine the inhibition rate of Bacillus polymyxa NPDY05-8 against Fusarium graminearum. Fresh fungus blocks of the target pathogenic fungus Fusarium graminearum were selected and inoculated into the center of the PDA culture medium; then Bacillus polymyxa NPDY05-8 was inoculated 2.5 cm away from the center of the mycelial block of the fungal strain to be tested; only the target pathogenic fungus was inoculated as a control; the culture was carried out at 28°C for 5 to 7 days, the growth was observed, the width of the fungal mycelial growth was measured, and the inhibition rate I was calculated.

[0043] I=(D 1 -D 2 ) / (D 1 -5)×100%;

[0044] D 1 is the diameter of the fungal colony of CK (without Paenibacillus polymyxa NPDY05-8) (mm), D 2 is the diameter (mm) of the fungal colony containing Paenibacillus polymyxa NPDY05-8.

[0045] Figure 2 The results showed that Paenibacillus polymyxa NPDY05-8 had a good inhibition rate against Fusarium graminearum, reaching 62.5%.

[0046] Example 3

[0047] Toxicity determination of test agents

[0048] The mycelial growth rate method was used to determine the toxicity of seven chemical agents to pathogens. PDA culture medium containing different mass concentrations of the test agents was prepared, inoculated with 5 mm diameter Fusarium graminearum cakes, and inverted cultured at 28°C for 5 to 7 days. The colony diameter was measured and the inhibition rate (I) was calculated.

[0049] I=(D 1 -D 2 ) / (D 1 -5)×100%;

[0050] D 1 is the diameter of CK (without drug) colony (mm), D 2 is the diameter of the colony containing the test agent (mm).

[0051] SPSS software was used to calculate the toxicity regression equation and median effective concentration.

[0052] Table 1 Results of indoor toxicity tests of seven fungicides against Fusarium graminearum

[0053]

[0054] The results of indoor toxicity test of 7 fungicides on Fusarium graminearum showed (Table 1) that the 7 fungicides, prochloraz, azoxystrobin, tebuconazole, fludioxonil, difenoconazole, thiophanate-methyl and metalaxyl, all had a certain inhibitory effect on Fusarium graminearum. Among them, difenoconazole had the strongest toxicity, with EC50 of 0.2175 μg / mL, followed by prochloraz and azoxystrobin, which were 0.3259 μg / mL and 0.5526 μg / mL, respectively. The toxicity of thiophanate-methyl and metalaxyl was relatively weak, which were 1.2556 μg / mL and 1.2388 μg / mL, respectively.

[0055] Example 4

[0056] Tolerance of Paenibacillus polymyxa NPDY05-8 to common fungicides

[0057] If Paenibacillus polymyxa NPDY05-8 is to be made into a compound seed dressing agent with chemical fungicides or used together, it is necessary to study the toxicity of different concentrations of commonly used seed dressing fungicides to Paenibacillus polymyxa NPDY05-8, that is, the proportion of strains that survive high concentrations of fungicides.

[0058] Paenibacillus polymyxa NPDY05-8 was activated on PDA medium, and the activated strain was added to seven commonly used fungicides with high concentrations to prepare a Paenibacillus polymyxa NPDY05-8 strain with a content of 1×10 7 cfu·mL -1 A mixture of microorganisms and chemical agents was prepared, with clean water as the control. After 10 days, the survival rate relative to the control was observed using a cell counting plate under a microscope using the dilution method to understand the tolerance of Bacillus polymyxa NPDY05-8 to fungicides.

[0059] Table 2 Tolerance of Paenibacillus polymyxa NPDY05-8 to 7 commonly used seed dressing agents

[0060]

[0061] According to the results of indoor toxicity test and the dosage of seed dressing agent, the tolerance of Paenibacillus polymyxa NPDY05-8 to 7 different doses of fungicides was set (Table 2). Paenibacillus polymyxa NPDY05-85 had high tolerance to fludioxonil and thiophanate-methyl, and could still maintain a high relative survival rate after 10 days. The survival rates in 6% and 3% fludioxonil suspension concentrates were 76.23% and 83.74%, and the survival rates in 8% and 4% thiophanate-methyl suspension concentrates were 66.64% and 73.25%, respectively. The survival rates in 6% prochloraz, 8% azoxystrobin, 6% tebuconazole, 3% difenoconazole, and 6% metalaxyl fungicides were all over 50%, and it can be used as a seed dressing agent in combination.

[0062] Example 5

[0063] Effects of chemical agents and Paenibacillus polymyxa NPDY05-8 seed dressing on wheat growth and control of wheat stem rot

[0064] Soak the wheat seeds in 1% NaClO solution for 10 minutes to disinfect the surface, then wash with sterile water 2-3 times to wash away the residual NaClO. Treatments 1, 3, 5, 7, 9, 11, 13: Dress the seeds with the above seven agents, 6% prochloraz, 8% azoxystrobin, 6% tebuconazole, 3% fludioxonil, 3% difenoconazole, 4% thiofuran, and 6% metalaxyl, at a dosage of 1:400. Treatments 2, 4, 6, 8, 10, 12, 14: 1×10 7 cfu·mL -1 The bacterial suspension of Paenibacillus polymyxa NPDY05-8 was mixed with 6% prochloraz, 8% azoxystrobin, 6% tebuconazole, 3% fludioxonil, 3% difenoconazole, 4% thiofuran, and 6% metalaxyl in a volume ratio of 1:1 and then applied to the seeds at a dosage of 1:400; Treatment 15: 1×10 7 cfu·mL -1 The polymyxa bacillus NPDY05-8 solution was mixed with seeds at a dilution of 1:400; treatment 16: water control mixed with seeds. After drying for 3 hours, the treated seeds were placed in a germination tray with a layer of gauze at 28°C for germination.

[0065] The control effect of different seed dressing methods on wheat stem rot was determined by the fungus plate method. 200g of soil was sterilized in a 9cm wide tissue culture bottle, and the pseudograminearum Fusarium was activated and cultured on a PDA plate. After the mycelium grew all over the culture dish, the mycelium and culture medium in the culture dish were dug out as a whole, with the mycelium facing up, and placed above the soil layer of the tissue culture bottle. The seeds of the above 16 treatments were sown in tissue culture bottles, 20 grains of wheat were sown in each tissue culture bottle, and three replicates were used for each treatment. The germination rate was calculated after germination, and the plant height and root length of different treatments were measured after 20 days of culture, and the disease was observed.

[0066] The leaf sheath symptom score is based on the 0-4 standard for investigating the 1st to 3rd leaf sheaths, and then adding up the scores of the 3 leaves (the maximum total score is 12 points). The standard is:

[0067] Level 0, leaf sheath is completely healthy;

[0068] Grade 1, leaf sheath browning <25%;

[0069] Level 2, the leaf sheath browning is 25% to 50%;

[0070] Level 3, the leaf sheath browning is 51% to 75%;

[0071] Level 4, leaf sheath browning >75%.

[0072] Disease index = Σ (number of diseased plants at each level × level number) / (total number of plants surveyed × 4) × 100%;

[0073] Control effect (%) = (average disease index of control group - disease index of treatment group) / average disease index of control group × 100%.

[0074] Calculation method of synergistic coefficient CTC:

[0075] The method of equal toxicity of binary mixtures by Mansour et al. was used to determine the synergistic effect. Assume that the mixed agents are A and B respectively, and the actual control effects of single use are expressed as percentages C a and C b , the actual control effect of the mixture is CS, the theoretical control effect C 1 of the mixture of A and B and the calculation formula of the synergistic coefficient CTC are as follows:

[0076] C 1 = C a +(1 - C a )×C b ;

[0077] CTC = (CS - C 1 )×100;

[0078] In the formula, CTC ≥ 20 indicates a synergistic effect, CTC ≤ -20 indicates an antagonistic effect, and -20 < CTC < 20 indicates an additive effect.

[0079] Table 3 Growth situation of wheat seedlings treated with the mixture of Paenibacillus polymyxa NPDY05 - 8 and 7 common fungicides for seed dressing

[0080]

[0081] As can be seen from Table 3 and Figure 3 , in terms of germination rate, seed dressing with 8% azoxystrobin has a certain impact on the germination rate, and the germination rate is only 62.78%, but 8% azoxystrobin and 1×10 7 cfu·mL -1The combination of Paenibacillus polymyxa NPDY05-8 with seed dressing can increase the germination rate to 73.43%. The combination of Paenibacillus polymyxa NPDY05-8 with the other 6 fungicides also increased the germination rate to varying degrees. In terms of root length, the root length of Paenibacillus polymyxa NPDY05-8 and water ck after seed dressing alone was only 3.11cm and 3.69cm, and the root length of Paenibacillus polymyxa NPDY05-8 after 3% fludioxonil and 4% thiophanate-methyl with Paenibacillus polymyxa NPDY05-8 was not significantly improved compared with the original agent alone. The root length of the other 5 fungicides after being combined with Paenibacillus polymyxa NPDY05-8 was improved compared with the original agent alone. In terms of plant height, the compounding of 4% thiofuran and 6% metalaxyl with Paenibacillus polymyxa NPDY05-8 did not significantly improve plant height compared with the original agent alone, while the compounding of the other five fungicides with Paenibacillus polymyxa NPDY05-8 increased plant height compared with the original agent alone. Therefore, it is inferred that Paenibacillus polymyxa NPDY05-8 has the function of promoting wheat seed germination and growth.

[0082] Table 4 The control effect of Paenibacillus polymyxa NPDY05-8 and 7 common fungicides on wheat stem rot at the seedling stage

[0083]

[0084] As shown in Table 4, the control effect of Paenibacillus polymyxa NPDY05-8 on wheat stem rot can reach 25.25% when used alone, which is higher than 6% tebuconazole and 3% fludioxonil. Among them, 8% azoxystrobin and 3% difenoconazole have the best control effects, which are 53.56% and 64.24% respectively. Paenibacillus polymyxa NPDY05-8 significantly improved the control effect after compounding with 7 fungicides, especially the compounding with 6% prochloraz, 8% azoxystrobin and 3% difenoconazole, which had the best control effect, reaching 73.34%, 80.49% and 78.96% respectively. The highest synergistic coefficient was compounded with 6% prochloraz and 3% fludioxonil, with synergistic coefficients of 24.37 and 26.16 respectively. It also achieved an additive effect with the other 5 fungicides, and no antagonism occurred. This shows that the combination of Bacillus polymyxa NPDY05-8 and fungicides can effectively improve the control effect of wheat stem rot. Figure 3 It can be seen that the wheat leaves treated with a mixture of fungicide and Paenibacillus polymyxa NPDY05-8 solution are darker green and the stems are thicker than those treated with fungicide alone.

[0085] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0086] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Paenibacillus polymyxa and chemical fungicides in the preparation of a medicine for preventing and treating wheat stem rot, characterized in that: The polymyxa Paenibacillus is polymyxa Paenibacillus NPDY05-8, which is classified and named as Paenibacillus polymyxa. It was deposited in the China Center for Type Culture Collection on May 12, 2023, with a deposit number of CCTCC NO: M 2023740, and the deposit address is Wuhan University, Wuhan, China.

2. The use according to claim 1, characterized in that The chemical fungicide is one or more of prochloraz, azoxystrobin, tebuconazole, fludioxonil, difenoconazole, thiophanate-methyl, and metalaxyl.

3. The use according to claim 1, characterized in that The chemical fungicide is prochloraz or fludioxonil.

4. The use according to claim 3, characterized in that 1×10 of Paenibacillus polymyxa NPDY05-8 7 cfu·mL -1 The bacterial solution was mixed with 6% prochloraz.

5. The use according to claim 3, characterized in that 1×10 of Paenibacillus polymyxa NPDY05-8 7 cfu·mL -1 The bacterial solution was compounded with 3% fludioxonil.

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