Application of metarhizium anisopliae and beauveria bassiana in preparation of microbial agent for preventing and treating clubroot of cruciferae

Abstract

The invention relates to the technical field of biological prevention and treatment, and discloses application of metarhizium anisopliae and beauveria bassiana in preparation of a microbial agent for preventing and treating clubroot of cruciferae, the microbial agent is a complex microbial agent, and the complex microbial agent comprises metarhizium anisopliae and beauveria bassiana. According to the invention, the clubroot of cruciferae is prevented and controlled by utilizing the bacteriostatic activity and composite synergistic effect of metarhizium anisopliae and beauveria bassiana. The fermentation liquor of the metarhizium anisopliae and the beauveria bassiana can directly act on the plasmodiophora brassicae spores, the inactivation rate of the plasmodiophora brassicae spores is remarkably increased, and disease infection is blocked from the source. The problems that disease-resistant varieties lose efficacy due to diversity of physiological races of germs, agricultural management measures have a limited effect on long-term survival spores, and chemical pesticides pollute the environment and induce drug resistance are solved; and the preparation in the technical scheme is environment-friendly, has no residue and is not easy to induce drug resistance of germs.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to the application of Metarhizium anisopliae and Beauveria bassiana in the preparation of microbial agents for controlling clubroot disease in cruciferous plants. Background Technology

[0002] Clubroot is a highly destructive and contagious root disease of cruciferous plants, caused by *Platycodon brassicola* (…). Plasmodiophora brassicae Woron. This pathogen, clubroot, causes disease. Its spores typically remain dormant in the soil or seeds, surviving for up to 20 years. Short-distance spread relies primarily on field irrigation, soil protozoa and insect activity, and human agricultural practices, while long-distance spread occurs through the transport of soil or seedlings. Clubroot can infect various host crops, exhibiting different symptoms in both the above-ground and underground parts of the plant at different growth stages. In the seedling stage, cruciferous crops infected with clubroot generally show no obvious symptoms in the above-ground parts, but symptoms gradually appear after transplanting to the field. In the early stages of infection, crop growth is stunted, leading to dwarfism. Later, the plant suffers severe dehydration, wilting, and even death. The most obvious symptoms appear in the roots. After infecting the host, clubroot alters root hormone secretion, causing excessive proliferation of root parenchyma cells, forming tumors. Currently, clubroot has spread to more than 60 countries worldwide. Its host range is wide, mainly infesting cruciferous plants, and in severe cases it can cause the death of the entire plant, resulting in a 10% to 15% decrease in global cruciferous vegetable production.

[0003] Research on the control of clubroot disease mainly focuses on breeding resistant varieties, implementing agricultural management measures, and chemical and biological control techniques. Breeding clubroot-resistant crop varieties is currently the most direct and effective control method. However, variations in the pathogenicity of the clubroot pathogen or the diversity of its physiological races may cause resistant varieties to lose their resistance. Furthermore, when breeding resistant varieties, usually only a single physiological race of the clubroot pathogen is used as the source of infection, while clubroot pathogens often exist in complex forms in the field, which limits the application scope and effectiveness of the selected varieties. Agricultural management measures can control the occurrence and spread of the disease by reducing the number of dormant spores of the clubroot pathogen in the soil, combined with cultivation management methods. However, due to the long survival time and easy spread of dormant spores, the effectiveness of this method is limited. In agricultural production, farmers often use chemical pesticides to control clubroot disease. However, reports from abroad indicate that although methods such as irrigating the soil with chemical pesticides and adjusting the soil pH with lime can control the spread of clubroot to some extent, these practices can cause significant damage to soil health and lead to environmental pollution problems. Meanwhile, the use of chemical pesticides can lead to pesticide resistance in pathogens and pesticide residues that harm human and animal health. Summary of the Invention

[0004] The application intends to provide applications of Metarhizium anisopliae and Beauveria bassiana in preparing microbial agents for preventing and treating cruciferous root tumour diseases, so as to solve the problems of soil pollution and drug resistance in the prior art.

[0005] To achieve the above-mentioned purposes, the application adopts the following technical scheme: applications of Metarhizium anisopliae and Beauveria bassiana in preparing microbial agents for preventing and treating cruciferous root tumour diseases, wherein the microbial agent comprises Metarhizium anisopliae agent and Beauveria bassiana agent. Preferably, as an improvement, the content of Metarhizium anisopliae in the Metarhizium anisopliae agent is ≥ 80 billion spores / mL, the content of Beauveria bassiana in the Beauveria bassiana agent is ≥ 100 billion spores / mL, and the spore quantity ratio of the Beauveria bassiana agent to the Metarhizium anisopliae agent is 1:9.

[0006] Preferably, as an improvement, the root tumour disease is a cruciferous crop disease caused by Plamodiophora brassicae.

[0007] Preferably, as an improvement, the cruciferous crops include Chinese cabbage, stem tumour mustard and rape.

[0008] Preferably, as an improvement, the microbial agent is a cruciferous crop root tumour disease prevention agent.

[0009] Preferably, as an improvement, the microbial agent is a cruciferous crop development promoting agent.

[0010] Preferably, as an improvement, the microbial agent is applied by irrigation.

[0011] Preferably, as an improvement, the concentration of the microbial agent is 5×10 7 spores / mL, and the application amount of the microbial agent is 10 mL / plant.

[0012] The principle and advantages of the scheme are as follows: in actual application, the technical scheme uses the bacteriostatic activity and complex synergistic effect of Metarhizium anisopliae and Beauveria bassiana to prevent and control cruciferous root tumour diseases, aiming at the problems of soil pollution and drug resistance in the prior art. The fermentation liquor of Metarhizium anisopliae and Beauveria bassiana can directly act on Plamodiophora brassicae spores, significantly improving the inactivation rate and blocking the disease infection from the source. The technical scheme solves the problems of failure of disease-resistant varieties due to the diversity of pathogen physiological races, limited effect of agricultural management measures on long-term surviving spores, environmental pollution of chemical pesticides and induction of drug resistance. Moreover, the preparation of the technical scheme is environment-friendly, has no residue and is not easy to induce drug resistance of the pathogen; has a long stable storage period, and meets the storage and use requirements of agricultural production. In addition, it is found that the microbial agent of the technical scheme can not only effectively reduce the occurrence degree of stem tumour mustard root tumour disease in the field, but also effectively promote the development of stem tumours and improve economic benefits, having unexpected technical effects. Attached Figure Description

[0013] Figure 1 This is a statistical chart showing the root length and stem base diameter of different groups of plants treated with different strains after transplanting *Strombyx mori* in Experiment Example 1 of this invention.

[0014] Figure 2 This shows the disease incidence of different treatment groups treated with different strains after transplanting *Strombyx mori* in Experiment Example 1 of this invention.

[0015] Figure 3 This is a statistical chart of the root length of plants treated with fungal agents 1 and 2 with different spore ratios after transplanting in Experimental Example 2 of the present invention.

[0016] Figure 4 This is an example of the disease incidence in each treatment group treated with fungal agents 1 and 2 with different spore ratios after transplanting *Mucor sibirica* in Experimental Example 2 of this invention.

[0017] Figure 5 This is a statistical chart showing the diameter of the stem base of *Mustela stenoptera* plants treated with compound microbial agent or microbial agent 1 and microbial agent 2 alone after transplanting in Experimental Example 3 of this invention.

[0018] Figure 6 This is an example of the disease incidence in each treatment group after transplanting stem mustard (Mustela stenoptera) in Experimental Example 3 of the present invention, treated with a compound microbial agent or a combination of microbial agents 1 and 2.

[0019] Figure 7 This is a statistical chart showing the root length and stem base diameter of each group of plants treated with compound microbial agents during the seedling cultivation of stem mustard in Experimental Example 4 of this invention.

[0020] Figure 8 This is an example of the disease incidence in each treatment group treated with compound microbial agents during the seedling cultivation of stem mustard in Experimental Example 4 of this invention.

[0021] Figure 9 This is a statistical chart showing the diameter of the stem base of each group treated with compound microbial agent after transplanting rapeseed and Chinese cabbage in Experimental Example 5 of the present invention.

[0022] Figure 10 This is the disease incidence of each group treated with compound microbial agent after rapeseed transplanting in Experimental Example 5 of this invention.

[0023] Figure 11 This is the disease incidence of each group of Chinese cabbage treated with compound microbial agents after transplanting in Experimental Example 5 of this invention.

[0024] Figure 12 The figure shows the results of the experiment on the inhibition of spore activity of different strains of *Plasmodiophora* in Experiment Example 6 of this invention.

[0025] Figure 13 This is a graph showing the statistical analysis results of agronomic indicators of stem mustard plants under different treatments in Experimental Example 7 of this invention. Detailed Implementation

[0026] The following detailed description provides further details on specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials and reagents used are all commercially available.

[0027] Overview of the plan: Application of *Metarhizium anisopliae* and *Beauveria bassiana* in the preparation of microbial agents for the control of clubroot disease in cruciferous plants, using a combination of the two. The *Metarhizium anisopliae* agent contains ≥8 billion spores / mL, while the *Beauveria bassiana* agent contains ≥10 billion spores / mL.

[0028] Clubroot disease specifically refers to disease caused by *Plasmodiophora brassicae* (… Plasmodiophora brassicae Woron. Diseases of cruciferous crops caused by infection.

[0029] Cruciferous plants include Chinese cabbage, stem mustard, and rapeseed.

[0030] The preservation number for *Metarhizium anisopliae* is CGMCC No. 4609. The preservation number for *Beauveria bassiana* is CGMCC NO. 13566. The accession number for *Trichoderma harzianum* is: XZN213-1: CGMCC3.20239 All of the above strains were obtained through purchase. Experimental Example 1 In the pot experiment, transplanted stem mustard was treated with fungal inoculum 1, fungal inoculum 2, and Trichoderma harzianum, respectively. Inoculant 1 is a single inoculant with Beauveria bassiana as the main component. Inoculant 1 is a dispersible oil suspension with the content of its active ingredient Beauveria bassiana being 10 billion spores / mL. Inoculant 2 is a single inoculant with Metarhizium anisopliae as the main component; Inoculant 2 is a dispersible oil suspension with the active ingredient Metarhizium anisopliae containing 8 billion spores / mL. Trichoderma harzianum was cultured on laboratory plate solid culture medium.

[0031] The specific experimental steps are as follows: 1. Seedling of T. caerulecens: After soaking the seeds of T. caerulecens in water for 3 hours, evenly spread them on 90 mm bacterial culture dishes with filter paper, add water to fully wet the filter paper, and then place them in an incubator for 24 hours to germinate. After the seeds germinate, plant them in 32-hole cultivation trays filled with healthy soil, plant two seedlings in each hole, and cultivate them in an environment at 25°C for one week. After one week, thin out the plants, remove the ones that are not growing well, and keep the healthiest ones based on height, leaf number, and size.

[0032] 2. Preparation of Plasmodiophora brassicae resting spore suspension: Take the stored diseased roots from the -20°C refrigerator, wash them, and then rot them in a 25°C dark environment for 5 days. Crush the roots and mix them with sterile water, filter them through 4 layers of gauze, and then place the filtrate in a 50 mL centrifuge tube. Centrifuge at 3100 rpm for 15 minutes, discard the supernatant, and resuspend in sterile water. Repeat the centrifugal washing 2-3 times. Finally, discard the supernatant, resuspend in 15 mL of 50% sucrose solution, centrifuge, transfer the supernatant to a new centrifuge tube, dilute with sterile water, and repeat the centrifugation twice. Determine the concentration of the spore concentrate, label it, and store it at 4°C for future use.

[0033] 3. Preparation of diseased soil: Mix the prepared Plasmodiophora brassicae resting spore suspension with the sterilized nutrient medium at a final concentration of 5 x 10 6 spores per gram, and prepare the diseased soil.

[0034] 4. Transplanting of T. caerulecens seedlings: Transplant after 15 days of sowing, set up an experimental scheme with 3 treatment groups, each containing 10 plants, including: The negative control group is transplanted into disease-free soil and irrigated with water; the positive control group is transplanted into diseased soil and irrigated with water; treatment group 1, treatment group 2, and treatment group 3 are transplanted into diseased soil and irrigated with fungicide 1, fungicide 2, and Trichoderma harzianum spore suspension, respectively.

[0035] 5. After 50 days of transplanting, collect the plants, wash the roots, and record the disease conditions, root length, and stem base diameter data.

[0036] The specific irrigation parameters are: 3 treatment groups are irrigated with 10 mL of spore suspension at a concentration of 5 x 10 7 spores / mL, and the other two control groups are irrigated with an equal amount of water. Irrigation is performed three times on the day of transplanting, 20 days after transplanting, and 40 days after transplanting. After 50 days of transplanting, remove the T. caerulecens seedlings from the soil, wash their roots, and record the disease conditions, root length, and stem base diameter data simultaneously.

[0037] The specific disease classification of the pot experiment T. caerulecens disease is as follows: 0: No tumors on the roots; 1: Main roots slightly enlarged, or small tumors on the root hairs; Grade 3: the taproot is swollen, and its diameter is about 1.5 times of the diameter of the stem base; Grade 5: the taproot is swollen, and its diameter is about 2-3 times of the diameter of the stem base, and no rot appears; Grade 7: the taproot is swollen, and its diameter is about 2-3 times of the diameter of the stem base, and rot appears in the tumor part.

[0038] The disease index is calculated according to the following formula: The relative control effect is compared with the positive control, and the calculation formula is as follows: The disease statistics results are shown in Tables 1, 2 and Figure 2 The disease incidence of treatment group 1 and treatment group 2 is significantly reduced, and the reduction is 60% and 70% respectively, while treatment group 3 fails to inhibit the occurrence of root rot disease. In terms of disease index, the disease index of treatment group 1 and treatment group 2 is 20 and 12.86 respectively, and the relative control effect is 76.67% and 85% respectively. The results show that fungicide 1 and fungicide 2 can effectively inhibit the occurrence of root rot disease.

[0039] Table 1 Disease incidence of root rot of stem tumor mustard in different treatment groups

[0040] Table 2 Disease index and relative control effect of root rot of stem tumor mustard in different treatment groups

[0041] The root length and stem base diameter data statistics are shown in Figure 1 The root length of each treatment group and the positive control group does not appear statistically significant difference, but the stem base diameter of treatment group 1 and treatment group 2 is significantly smaller than that of the positive control and treatment group 3, which is consistent with the disease incidence statistics in the table.

[0042] In summary, the stem tumor mustard treated with fungicide 1 (Beauveria bassiana) and fungicide 2 (Metarrhizium anisopliae) after transplanting can effectively inhibit the occurrence of root rot disease, while the Trichoderma strain cannot effectively prevent and control the root rot disease.

[0043] Experimental Example Two In the pot experiment, the stem tumor mustard seedlings after transplanting were treated with different spore quantity ratios of fungicide 1 and fungicide 2 by root irrigation, and the specific operation steps were as follows: The steps of sowing and seedling raising, preparing the resting spore suspension of Plasmodiophora brassicae and diseased soil were the same as those in experimental example 1, and no treatment was made during seedling raising. When the stem tumor mustard grew to 15 days, transplanting was carried out, and 6 plants were transplanted in each group.

[0044] Three treatments were set: the negative control group was transplanted to the non-disease soil and irrigated with water, the positive control group was transplanted to the disease soil and irrigated with water, and the treatment 1, the treatment 2 and the treatment 3 were all transplanted to the disease soil, wherein: The treatment 1: the spore quantity ratio of the fungicide 1 to the fungicide 2 was 9:1; The treatment 2: the spore quantity ratio of the fungicide 1 to the fungicide 2 was 5:5; The treatment 3: the spore quantity ratio of the fungicide 1 to the fungicide 2 was 1:9.

[0045] When irrigating the roots, 10 mL of the fungicide solution was applied to each plant in the three treatments, and the final spore concentration was 5x10 7 spores / mL, and the two control groups were both irrigated with the same amount of water. The roots were irrigated on the day of transplanting, and the roots were irrigated again 20 days after the transplanting. After 40 days of transplanting, the roots of the stem tumor chenopodium were taken out and washed, and the disease incidence and root length data were recorded.

[0046] The results are shown in Figure 3 , Figure 4 The plant root length of the treatment 1-3 was significantly higher than that of the positive control group, and the plant growth state was obviously better, which indicated that the mixed irrigation of the fungicide 1 and the fungicide 2 in different proportions could also well inhibit the occurrence of the root tumor disease, and when the ratio of the fungicide 1 to the fungicide 2 was 1:9, the effect was better than that of other ratios.

[0047] Experimental example three In the pot experiment, the stem tumor chenopodium seedlings treated by the compound fungicide of the treatment 3 in the experimental example two and the fungicide 1 and the fungicide 2 were transplanted, and the specific operation steps were as follows: The steps of sowing and seedling raising, preparing the root tumor fungus dormant spore suspension and the disease soil were the same as those in the experimental example one, and no treatment was made during the seedling raising. When the stem tumor chenopodium grew to 15 days, 6 plants were transplanted in each group.

[0048] Three treatments were set: the negative control group was transplanted to the non-disease soil and irrigated with water, the positive control group was transplanted to the disease soil and irrigated with water, and the treatment 1, the treatment 2 and the treatment 3 were all transplanted to the disease soil, wherein: The treatment 1 was irrigated with the fungicide 1; The treatment 2 was irrigated with the fungicide 2; The treatment 3 was irrigated with the compound fungicide.

[0049] When irrigating the roots, 10 mL of the fungicide solution was applied to each plant in the three treatments, and the final spore concentration was 5x10 7 spores / mL, and the two control groups were both irrigated with the same amount of water. The roots were irrigated on the day of transplanting, and the roots were irrigated again 20 days after the transplanting. After 40 days of transplanting, the roots of the stem tumor chenopodium were taken out and washed, and the disease incidence and root length data were recorded.

[0050] Table 3 Incidence of root rot of stem canola in different treatment groups

[0051] The results are shown in Table 3, Figure 5 and Figure 6 As shown in Table 3, both treatment group 1 and treatment group 2 showed some degree of disease symptoms, while treatment group 3 showed no disease, indicating that when the fungicide 1 and the fungicide 2 are mixed at a ratio of 1:9, they can exhibit better control effect than the fungicides alone.

[0052] Experimental Example Four In the pot experiment, the stem canola treated with the complex fungicide of treatment 3 in experimental example two during the seedling stage was treated according to the following specific operation steps: The procedures of sowing and seedling, preparation of Agrobacterium tumefaciens spore suspension and preparation of soil with disease were completed according to the same method of experimental example 1. When the stem canola seedlings grew to the two-leaf-one-heart stage, part of the seedlings were irrigated with the complex fungicide (final concentration of 5x10 7 spores / mL), with 10 mL of irrigation per plant. The rest of the seedlings were irrigated with an equal amount of water, and the transplanted after 15 days of sowing.

[0053] Six seedlings treated with the complex fungicide were selected as the treatment group, and the negative control group and the positive control group were set according to the method above, with 6 seedlings in each group. After transplanting, no treatment was done, and after 40 days of growth of the stem canola, the incidence and plant traits of each treatment group were recorded and analyzed.

[0054] Table 4 Incidence of each group after treating stem canola during the seedling stage with the complex fungicide

[0055] The results show that the complex fungicide treatment of stem canola during the seedling stage still has a significant effect on the prevention and control of root rot, as shown in Table 4, after irrigation with the complex fungicide, the incidence of the treatment group is only 16.7%, while the incidence of the positive control group is 100%, and the negative control group shows no disease. As shown in Table 5, the root length of the negative control group is significantly better than that of the other two groups, but there is no significant statistical difference in root length between the treatment group and the positive control group. It is worth noting that in the stem base diameter measurement, the stem base diameter of the negative control group and the treatment group is significantly smaller than that of the positive control group, which is consistent with the disease situation. Figures 7-8

[0056] Experimental Example Five Pot Experiment of Complex Fungicide for Prevention and Control of Other Brassica Crops In the pot experiment, the stem canola seedlings (variety is Brassica napus) and cabbage seedlings (variety is Qijilv) were treated with the complex fungicide of treatment 3 in experimental example two after transplanting, according to the following specific operation steps: ​The steps of sowing and seedling raising, preparing the suspension of Plasmodiophora brassicae resting spores, and soil with the pathogen were the same as in Experimental Example 1, and no treatment was performed during the seedling raising. When the rape seedlings grew to 15 days and the Chinese cabbage seedlings grew to 20 days, they were transplanted, with 10 seedlings transplanted in each group.

[0057] The rape seedlings and the Chinese cabbage seedlings were set as the negative control group and the positive control group, respectively, and the treatment group was the compound microbial agent root irrigation, with 10 mL of the microbial agent solution applied to each seedling, and the final spore concentration was 5 x 10 7 spores / mL.

[0058] The rape seedlings were irrigated once every 15 days after transplanting, and the seedlings were taken out and the root system was cleaned 45 days after transplanting, and the incidence rate and plant trait data were recorded.

[0059] The Chinese cabbage seedlings were irrigated once every 10 days after transplanting, and the seedlings were taken out and the root system was cleaned 30 days after transplanting, and the incidence rate and stem base diameter data were recorded.

[0060] Table 5 Incidence rates of each group after treating the rape seedlings and the Chinese cabbage seedlings with the compound microbial agent

[0061] The incidence rate statistics of the two crops are shown in Table 5, and the data show that the rape seedlings and the Chinese cabbage seedlings treated with the compound microbial agent did not have disease. The stem base diameter statistics of the two plants Figure 9 showed that the stem base diameter of the plants in the positive control group was significantly larger than that in the negative control group and the treatment group, which was consistent with the observed disease conditions Figure 10 , Figure 11 . In summary, the compound microbial agent also has a significant prevention and control effect on the clubroot disease of cruciferous crops such as rape and Chinese cabbage.

[0062] Experimental Example Six: Fermentation liquid of the strains inhibiting the activity of Plasmodiophora brassicae spores The specific operation steps are as follows: First, the suspension of Plasmodiophora brassicae resting spores was prepared according to the method of Experimental Example 1, and the concentration was adjusted to 1 x 10 9 spores / mL for use.

[0063] The preparation process of Metarhizium anisopliae and Beauveria bassiana fermentation liquid is as follows: first, the spore suspension was prepared and inoculated into a conical flask containing 50 mL of 1 / 4 SDAY liquid medium, so that the final spore concentration reached 5 x 10 6 spores / mL, and then the conical flask was placed in a 28℃ constant temperature shaker at 200 revolutions per minute for 3 days. After the culture was completed, the sterile supernatant was obtained by suction filtration as the fermentation liquid.

[0064] The fermentation broth was treated separately with *Plasmodiophora stylosa* spores: Treatment group 1 consisted of 100 μL of *Plasmodiophora stylosa* spore suspension and an equal volume of *Beauveria bassiana* fermentation broth added to a 1.5 mL centrifuge tube, followed by 200 μL of 20 mg / mL Evans blue solution; Treatment group 2 consisted of *Beauveria bassiana* fermentation broth, with all other procedures remaining unchanged; the control group was treated with an equal volume of 1 / 4 SDAY liquid medium instead of the fermentation broth. After mixing, the mixture was allowed to stand in the dark for 1 hour. A portion of the mixture was diluted four times with distilled water, and samples were taken for observation and counting of inactivated spores using an optical microscope.

[0065] like Figure 12 As shown, the spore inactivation rate of both strains increased significantly after treatment with the fermentation broth of *Plasmodiophora*, indicating that the fermentation broth of both strains can further control the occurrence of the disease by inhibiting the spore activity of *Plasmodiophora*.

[0066] Experiment Example 7: Field Experiment on Control of Clubroot Disease in Stem Mustard with Compound Microbial Agent Experimental location: Southeast Chongqing Academy of Agricultural Sciences; Test crop: Stem mustard (variety: Yong'an small leaf); Field management: Except for specific treatments, all other operations are carried out according to conventional production standards.

[0067] Experimental setup: Two treatments were set up, with treatment group 1 and treatment group 2 using the same treatment scheme, and the control group receiving no additional treatment. Specifically, the compound microbial agent (i.e., the compound microbial agent in Experiment Example 3) was mixed with base fertilizer, Bacillus subtilis, and fungal culture medium and applied simultaneously. The dosage of the compound microbial agent was 400 mL / mu, the dosage of Bacillus subtilis was 500 g / mu, and the dosage of fungal culture medium was 1000 g / mu. This mixture was applied simultaneously to both the base fertilizer and the post-transplant topdressing.

[0068] Data collection: At harvest, a five-point sampling method was used to investigate stem-knot mustard, measure its agronomic traits, and observe root tumors to assess disease condition. Disease severity was quantified based on a six-level standard: Grade 0: Root growth, no tumors; Grade 1: Only a small number of tumors are present at the tips of the fibrous roots; Grade 3: Small tumors on the lateral root; Grade 5: Lateral root tumors are enlarged, and there are small tumors at the tip of the main root; the plant has no obvious deformities. Level 7: The main root is completely swollen, the surface of the swollen root is cracked, and the plant's leaves are wilted and yellow. Level 9: The main root tumor begins to crack and rot, and the plant withers and dies.

[0069] The disease index is calculated using the following formula: The relative prevention and control effect was compared with the control group, and the calculation formula was the same as in Experiment 2.

[0070] The experimental results show that the composite microbial agent treatment has a significant effect on the prevention and control of root rot of stem tumor mustard and growth and development. By comparing the incidence, disease index, control effect, yield and plant traits (including plant height, development length and width, stem tumor length and width, etc.) data (see Tables 6-8 and Figure 13 ), it is found that the incidence of the treatment group is 20% and 30% lower than that of the control group, the disease index decreases significantly, and the corresponding relative control effect reaches 28.58% and 37.50%. In terms of yield, the unit yield of the two treatment groups is better than that of the control group. Plant trait analysis shows that the average length of stem tumor in the treatment group is significantly increased compared with the control group, and the development length of treatment 2 and the stem tumor width of treatment 1 are particularly obvious, and the remaining trait parameters do not show significant differences.

[0071] In summary, the composite microbial agent can not only effectively reduce the occurrence of root rot of stem tumor mustard in the field, but also effectively promote the development of stem tumor and improve economic benefits.

[0072] Table 6 Incidence of root rot of stem tumor mustard in different treatments

[0073] Table 7 Disease index and relative control effect of root rot of stem tumor mustard in different treatments

[0074] Table 8 Yield statistics of stem tumor mustard in each treatment

[0075] The above is only an embodiment of the present application, and the specific technical solutions and / or characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. Use of Metarhizium anisopliae and Beauveria bassiana in the preparation of microbial agents for the control of clubroot of crucifers, characterized in that: The microbial agent comprises Metarhizium anisopliae and Beauveria bassiana.

2. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 1 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The content of Metarhizium anisopliae in the Metarhizium anisopliae agent is greater than or equal to 8 billion spores per milliliter, and the content of Beauveria bassiana in the Beauveria bassiana agent is greater than or equal to 10 billion spores per milliliter.

3. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 2 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The root rot is a cruciferous crop disease caused by Plasmodiophora brassicae.

4. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 3 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The cruciferous crops include Chinese cabbage, Brassica napus and rapeseed.

5. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 4 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The microbial agent is a cruciferous crop root rot prevention agent.

6. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 5 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The microbial agent is a cruciferous crop growth promoter.

7. The use of Metarrhizium anisopliae and Beauveria bassiana according to claim 6 for the preparation of a microbial agent for the control of clubroot of crucifers, characterized in that: The microbial agent is applied by irrigation.

8. The use of Metarhizium anisopliae and Beauveria bassiana according to claim 7 for the preparation of a microbial agent for the control of clubroot of cruciferous plants, characterized in that: The concentration of the microbial agent was 5 x 10 7 spores / mL, and the application amount was 10 mL / plant.

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