Insect-sand-based myxobacteria bacterial fertilizer and fermentation preparation method thereof

Abstract

The invention discloses a worm sand-based myxobacteria bacterial fertilizer and a fermentation preparation method thereof. The bacterial fertilizer is formed by fermenting myxobacteria sp.KS01 in a solid culture medium, the solid culture medium is a solid basic culture medium prepared from protaetia brevitarsis sand and smashed wheat straw according to the weight ratio of 3: 1, and every 100 g of the solid basic culture medium contains 3-5 g of starch, 1-3 g of wheat bran, 1-3 g of cottonseed meal and 0.5-1 g of yeast powder. The insect-sand-based myxobacteria bacterial fertilizer prepared by the invention not only has a relatively good prevention and control effect in prevention and control of cotton verticillium wilt, but also can remarkably reduce the morbidity and disease index of wheat seedlings in biological prevention and control of wheat stem rot, and has wide application value.

Description

Technical Field

[0001] The invention relates to a biological fertilizer and a preparation method thereof, and in particular to an insect sand-based myxobacterium fertilizer and a preparation method thereof. Background Art

[0002] Today, green agriculture has become the direction of world agricultural development, and environmentally friendly biological preparations are gaining more and more attention and favor. Since many biological preparations are liquid preparations, they will be greatly affected in transportation and the shelf life of their efficacy. In this case, some manufacturers and scientific researchers have begun to develop solid biological preparations with fertilizer and biological control effects to reduce the use of chemical fertilizers and pesticides, so as to save costs, reduce soil pollution, and protect the environment.

[0003] Cotton Verticillium wilt, caused by Verticillium dahliae Kleb., is a soil-borne vascular fungal disease worldwide. It is known as the "cancer" of cotton and seriously affects the yield and fiber quality of cotton. It is one of the main obstacles to high-quality and high-yield cotton and occurs on a large scale all year round. At present, crop rotation, chemical control, and breeding of disease-resistant varieties are the main measures to prevent and control cotton Verticillium wilt. However, due to the wide host range, strong pathogenicity, and rapid mutation of the pathogenic bacteria, disease-resistant cotton varieties are not easy to obtain. Although chemical pesticides play an important role in prevention and control, they seriously affect the safety of humans and animals and ecological sustainable development. In recent years, more and more scholars have realized that the control of cotton Verticillium wilt should adopt a sustainable management system of biological control with the purpose of sustainable agricultural development.

[0004] Wheat crown rot (Fusarium crown rot, FCR) is a worldwide soil-borne fungal disease mainly caused by infection by Fusarium graminearum. In recent years, due to the promotion of tillage cultivation measures such as returning straw to the field, the number of pathogens in the soil has increased year by year, accumulating sufficient fungal sources for the large-scale occurrence of the disease. In addition, affected by climate factors such as global warming, the disease has shown a trend of continuous aggravation and has gradually become an important obstacle to wheat production safety. At present, my country's prevention and control of wheat crown rot mostly adopts a strategy of combining agricultural prevention and control with chemical prevention, and also conducts research on disease-resistant variety screening and biological control. However, most of the disease-resistant germplasm resources screened are medium-resistant varieties, and some varieties are not stable enough in resistance, lacking immune and high-resistant varieties. Studies have shown that dressing wheat seeds with 25% azoxystrobin and other fungicides during sowing can significantly reduce the incidence of wheat crown rot in the seedling stage. Since the seed dressing with chemical seedlings has a significant effect on disease prevention in the wheat seedling stage, the prevention and control effect decreases in the later stage of wheat growth. Biological control has attracted widespread attention due to its advantages such as long-lasting effectiveness and environmental friendliness.

[0005] Myxobacteria are a class of Gram-negative bacteria with complex multicellular group behavior characteristics. They can prey on living microorganisms such as bacteria, fungi and yeast to meet their own nutritional needs. Myxobacteria are widely distributed in different environments including soil, decaying wood, feces and water bodies. Unlike most reported biocontrol microorganisms, myxobacteria can not only actively prey on other microorganisms through "wolf pack" predation behavior and gliding movement, but also differentiate into myxospores with strong stress resistance, have strong environmental adaptability, and can produce rich and diverse secondary metabolites and hydrolases. In addition, myxobacteria are at the top of the soil microbial food web, and their predation on soil-borne pathogens directly affects the soil microecological environment, playing a vital role in maintaining the balance of soil microecology and plant health. These characteristics give myxobacteria unique biocontrol advantages, making them regarded as a new type of biocontrol microorganism. In recent years, some related studies have found that myxobacteria have shown potential application value in the biological control of plant diseases, especially for some soil-borne diseases. At present, some studies have found that the "wolf pack predation" strategy of myxobacteria needs to rely on the population density of predatory bacteria. Compared with directly using myxobacterial fermentation broth, solid fermentation matrix can provide more nutrients and a better living environment for the growth of myxobacteria, enabling myxobacteria to reproduce better and migrate to the roots, thereby achieving better prevention and control effects.

[0006] White-star flower beetle worm sand is mainly made from a mixture of cow dung and cotton straw that is fermented as feed. It is transformed by the larvae of white-star flower beetles through the abdomen and is dry, odorless, black, oblong particles with rich nutrition and stable properties. Its organic matter content is 54.8%, and the total amount of nitrogen, phosphorus and potassium is 9.04%, which is twice the standard for organic fertilizer (NY525-2021 ≥ 4.0%), and it has no phytotoxicity and can be applied to the field as an organic fertilizer. The Chinese invention patent 202211381387.0, which was applied for and disclosed on November 7, 2022, discloses a method for green prevention and control of cotton wilt based on insect-fungus composite technology. The invention mixes the biocontrol agent with the white-star flower beetle worm sand for biological control of cotton wilt, and the best prevention effect reaches 41.03%. The invention simply mixes beneficial biocontrol agents with fertilizers, so the prevention effect is not ideal.

[0007] In the early stage of the present invention, a strain of Myxococcus sp. KS01 with broad-spectrum antibacterial activity was screened out. The Chinese invention patent 202311090290.9, which was applied for and published on August 28, 2023, discloses a Myxococcus sp. KS01 and its microbial fertilizer, preparation method and application, and mentions a Myxococcus sp. KS01 strain (CGMCCNO.27345) and the preparation and application of its microbial fertilizer. The preparation method of the microbial fertilizer is as follows: the strain KS01 is activated and inoculated into LBS culture medium, and cultured at pH 6.5-7.5, 28-32°C, and 180-200r / min for 2-3 days to obtain seed solution; the seed solution is then inoculated into LBS culture medium, and cultured at pH 6.5-7.5, 28-32°C, and 180-200r / min for 3-5 days to obtain fermentation solution; the fermentation solution is further inoculated into a white star flower beetle sand culture medium (white star flower beetle sand and straw crushed material are mixed at a mass ratio of 5-3:1 and fully sterilized), and fermented and cultured at 28-32°C for 5-7 days to obtain microbial fertilizer. The obtained microbial fertilizer can not only effectively prevent and control the occurrence of cotton wilt, but also promote the growth of cotton.

[0008] The above application has no problem in biological control, but the content of myxobacteria in the microbial fertilizer obtained by the above fermentation method can only reach 3.6×10 5 cfu / g, so how to increase the biomass of strain KS01 in microbial fertilizer so that it can achieve better prevention and control effects when used as biological fertilizer has become a technical problem that urgently needs to be solved in this technical field. Summary of the invention

[0009] The technical problem to be solved by the present invention is how to increase the biomass of Myxococcus sp. KS01 in insect sand-based bacterial fertilizer.

[0010] To solve the above technical problems, the present invention is achieved as follows:

[0011] In the first aspect, the present invention provides an insect litter-based myxobacterium fertilizer, which is formed by fermenting Myxococcus sp. KS01 in a solid culture medium; the solid culture medium is a solid basic culture medium prepared by mixing white star flower beetle insect litter with crushed wheat straw in a weight ratio of 3:1, and each 100g of the solid basic culture medium contains 3-5g of starch, 1-3g of wheat bran, 1-3g of cottonseed meal, and 0.5-1g of yeast powder; preferably, each 100g of the solid basic culture medium contains 3.09g of starch, 2.87g of wheat bran, 1.07g of cottonseed meal, and 0.88g of yeast powder.

[0012] In a second aspect, the present invention also provides a method for preparing the insect sand-based slime bacteria fertilizer, and the method steps are as follows:

[0013] (1) Preparation of solid-state fermentation seed solution: After Myxococcus sp. KS01 was activated on a VY / 4 plate, an appropriate amount of biofilm was scraped and transferred to LBS culture medium, and cultured at 30°C and 180 r / min for 48 h to obtain liquid fermentation seed solution, which was inoculated into liquid fermentation medium at a rate of 5% to 7%, and cultured at a temperature of 28-32°C and a rotation speed of 180-220 r / min for 68-80 h to obtain solid-state fermentation seed solution of Myxococcus sp. KS01;

[0014] (2) Preparation of insect-litter-based bacterial fertilizer: A solid basic culture medium for bacterial fertilizer production is prepared with white-star flower beetle insect litter and crushed wheat straw as the main ingredients, starch, wheat bran, cottonseed meal, and yeast powder are added to form a solid culture medium. After sterilization, the liquid bacteria prepared in step (1) are inoculated and cultured and fermented at a constant temperature of 30-34°C for 6-7 days to prepare a solid fermentation product of myxobacteria. After the fermentation is completed, mix well to obtain an insect-litter-based myxobacteria bacterial fertilizer.

[0015] Furthermore, the liquid fermentation medium in step (1) has a ratio of: soluble starch 11.0 g / L, casein peptone 9.9 g / L, MgCl2 1.0 g / L, pH 7.4.

[0016] Furthermore, in step (1), the liquid fermentation is carried out in a container, and the volume ratio of the fermentation medium to the container is (90-120):250, and preferably the volume ratio of the fermentation medium to the container is 125:250. The container can be a container commonly used in the art, such as a conical flask.

[0017] Furthermore, the inoculation amount in step (1) is 6.9-7%.

[0018] Furthermore, in step (1), the culture temperature in the liquid fermentation medium is 30° C., the rotation speed is 200 r / min, and the culture is performed for 72 hours to obtain the solid-state fermentation seed liquid of Myxococcus KS01.

[0019] Furthermore, the preparation steps of the solid culture medium in step (2) are:

[0020] Step A: preparing the sand of the white star beetle and crushed wheat straw in a weight ratio of 3:1 to form a solid basic culture medium;

[0021] Step B. Add starch, wheat bran, cottonseed meal and yeast powder to the solid basic culture medium respectively and stir evenly;

[0022] Step C: Autoclave for 30 min.

[0023] Furthermore, the wheat straw in step A needs to be crushed to a length of 0.5 to 1 cm.

[0024] Furthermore, in step B, the starch required for every 100g of solid basic culture medium is 3-5g, the wheat bran is 1-3g, the cottonseed meal is 1-3g, and the yeast powder is 0.5-1g; preferably, the starch required for every 100g of solid basic culture medium is 3.09g, the wheat bran is 2.87g, the cottonseed meal is 1.07g, and the yeast powder is 0.88g.

[0025] Furthermore, in step (2): the solid culture medium is inoculated with the solid fermentation seed solution of Myxococcus KS01 prepared in step (1) at an inoculation rate of 15%-20%, the water content is adjusted to 65%-70%, and fermented at a constant temperature of 30-34°C for 6-7 days to obtain the solid fermentation product of the myxobacteria. After the fermentation is completed, it is ventilated and dried and mixed evenly to obtain the insect sand-based myxobacteria fertilizer.

[0026] In a third aspect, the present invention also provides the use of the insect litter-based myxobacterial fertilizer in the prevention and treatment of cotton Verticillium wilt.

[0027] In a fourth aspect, the present invention also provides the use of the insect litter-based myxobacteria fertilizer in promoting cotton growth and increasing cotton yield.

[0028] In a fifth aspect, the present invention also provides the use of the insect sand-based myxobacterial fertilizer in the prevention and treatment of wheat stem base rot.

[0029] The white-star flower beetle worm sand of the present invention can be obtained commercially, for example, from Golden Insect Biotechnology Co., Ltd.

[0030] The starch described in the present invention may be starch commonly used in solid fermentation, for example including but not limited to potato starch, bean starch, cereal starch, etc.

[0031] The yeast powder of the present invention may be yeast powder commonly used in solid fermentation.

[0032] The present invention discloses the following technical effects:

[0033] (1) The present invention optimizes the liquid fermentation conditions of the myxobacteria strain KS01, obtains the best culture medium formula and culture conditions, obtains a higher bacterial content, and provides a suitable seed solution for the next solid-state fermentation.

[0034] (2) The present invention provides a solid-state fermentation method for increasing the biomass of insect sand-based slime bacteria fertilizer through optimization. The method is low-cost and easy to operate. Experiments have shown that the solid-state fermentation method obtained after optimization of the present invention increases the biomass of slime bacteria by 128 times compared with the fermentation process before optimization, and can also improve its effect in the biological control of cotton Verticillium wilt and wheat stem base rot. The present invention not only solves the problem of poor biocontrol effect and fertilizer effect caused by separating insect sand from beneficial biocontrol agents or simply mixing beneficial biocontrol agents with fertilizers in the prior art, but also solves the problem of low biomass in existing insect sand-based slime bacteria fertilizers, and provides technical support and theoretical guidance for the industrial production of insect sand-based slime bacteria fertilizers.

[0035] (3) The white-spotted scarab beetle worm sand used for fermentation to prepare microbial fertilizer of the present invention can utilize dung heap or fermented cotton straw waste as feed, and is converted into worm sand rich in organic matter through feeding. The microbial fertilizer of the present invention organically combines biological control and fertilizer use, which can not only improve soil fertility, improve soil structure and physical and chemical quality, and increase the yield and quality of crops, but also promote the resource utilization of agricultural waste such as animal feces and straw, and has economic, social and ecological benefits, and has significant application potential.

[0036] (4) The insect sand-based myxobacterial fertilizer prepared by the present invention not only has a good control effect in the prevention and control of cotton wilt, but also can significantly reduce the incidence and disease index of wheat seedlings in the biological control of wheat stem base rot, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 Effects of different initial fermentation media on the growth of Myxococcus sp. KS01.

[0039] Figure 2 This is the growth curve of Myxococcus sp. KS01 in CYE medium.

[0040] Figure 3 The effects of carbon source and carbon source concentration on the growth of Myxococcus sp.KS01, where a is the effect of different carbon sources on the growth of Myxococcus sp.KS01; b is the effect of different concentrations of soluble starch on the growth of Myxococcus sp.KS01.

[0041] Figure 4 The effects of nitrogen sources and nitrogen source concentrations on the growth of Myxococcus sp. KS01, where a is the effect of different nitrogen sources on the growth of Myxococcus sp. KS01; b is the effect of different concentrations of casein peptone on the growth of Myxococcus sp. KS01.

[0042] Figure 5 The effects of inorganic salts and inorganic salt contents on the growth of Myxococcus sp.KS01, where a is the effect of different inorganic salts on the growth of Myxococcus sp.KS01; b is the effect of different concentrations of MgCl2 on the growth of Myxococcus sp.KS01.

[0043] Figure 6 Figure 3 shows the effects of different culture conditions on the growth of Myxococcus sp.KS01, where a is the effect of the initial pH of the culture medium on the growth of Myxococcus sp.KS01; b is the effect of the culture temperature on the growth of Myxococcus sp.KS01; c is the effect of the culture rotation speed on the growth of Myxococcus sp.KS01; d is the effect of the seed liquid inoculation amount on the growth of Myxococcus sp.KS01; and e is the effect of the liquid volume in the conical flask on the growth of Myxococcus sp.KS01.

[0044] Figure 7 The response surface of the three main effect factors on the growth of Myxococcus sp.KS01, where a is the interaction between casein peptone and seed solution inoculum size; b is the interaction between casein peptone and culture temperature; and c is the interaction between culture temperature and seed solution inoculum size.

[0045] Figure 8 Comparison of the dry weight of Myxococcus sp.KS01 before and after optimization of the solid-state fermentation seed inoculum fermentation method.

[0046] Fig. 9 This is the growth curve of Myxococcus sp.KS01 in different solid-state fermentation basal media.

[0047] Fig.10Figure 3 shows the effects of different culture conditions on the growth of Myxococcus sp.KS01 in the white star flower beetle sand culture medium, where a is the effect of culture temperature on the growth of Myxococcus sp.KS01; b is the effect of initial pH of the culture medium on the growth of Myxococcus sp.KS01; c is the effect of initial water content of the culture medium on the growth of Myxococcus sp.KS01; d is the effect of seed solution inoculation amount on the growth of Myxococcus sp.KS01.

[0048] Fig.11 Figure 1 shows the effect of adding different auxiliary materials to the culture medium of white star beetle sand on the growth of Myxococcus sp.KS01, where Figure a shows the effect of different added amounts of potato starch on the growth of Myxococcus sp.KS01; Figure b shows the effect of different added amounts of wheat bran on the growth of Myxococcus sp.KS01; Figure c shows the effect of different added amounts of yeast powder on the growth of Myxococcus sp.KS01; Figure d shows the effect of different added amounts of cottonseed meal on the growth of Myxococcus sp.KS01;

[0049] Fig.12 This is the response surface of the interaction between different auxiliary materials in solid fermentation medium on the growth of Myxococcus sp.KS01, where a is the interaction between wheat bran and starch; b is the interaction between cottonseed meal and starch; c is the interaction between yeast powder and starch; d is the interaction between cottonseed meal and wheat bran; e is the interaction between yeast powder and wheat bran; and f is the interaction between yeast powder and cottonseed meal.

[0050] Fig.13 Comparison of the Myxococcus sp.KS01 content in the bacterial fertilizer before and after optimization of solid-state fermentation conditions.

[0051] Fig.14 Figure 3 is the control effect of insect sand-based myxobacteria fertilizer on cotton Verticillium wilt, where A is the control; B is the sterile fermentation matrix; C is the insect sand-based myxobacteria fertilizer before optimization; and D is the insect sand-based myxobacteria fertilizer after optimization.

[0052] Fig.15 Figure 3 is the protective effect of insect sand-based myxobacteria fertilizer on wheat stem rot, where A is the control; B is the sterile fermentation matrix; C is the insect sand-based myxobacteria fertilizer before optimization; and D is the insect sand-based myxobacteria fertilizer after optimization. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0057] The culture medium used in the following examples is specifically described as follows:

[0058] VY / 4 medium: Angel Yeast 5.0 g / L, CaCl2·2H2O 1.0 g / L, pH 7.2;

[0059] LBS medium: soluble starch 7.0 g / L, yeast extract powder 5.0 g / L, casein peptone 1.0 g / L, MgSO4·7H2O 1.0 g / L, pH 7.2;

[0060] CYE medium: casein peptone 10.0 g / L, yeast extract 5.0 g / L, MgSO4·7H2O 1.0 g / L, pH 7.6;

[0061] VY / 2 medium: Angel Yeast 5.0 g / L, CaCl2·2H2O 1.0 g / L, pH 7.2 (add VB to a final concentration of 50 μg / mL after sterilization) 12 );

[0062] PDA medium: potato 200 g / L, glucose 20 g / L, agar 15 g / L, natural pH.

[0063] Czapek medium: sodium nitrate 3.0 g / L, potassium dihydrogen phosphate 1.0 g / L, MgSO4·7H2O 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30.0 g / L, natural pH.

[0064] The above culture media were sterilized by high pressure steam at 121°C for 30 min.

[0065] In the following examples, Myxococcus sp. KS01 is Myxococcus sp. KS01 in the literature (Pan Wen, Dou Xinyu, Luo Ming, Ma Deying, Lu Wei, Han Jian. Screening of biocontrol bacteria for cotton Verticillium wilt and its biocontrol potential [J]. Chinese Journal of Biological Control, 2024, 40(05): 1074-1087.), which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the strain collection number: CGMCC No. 27345. The highly pathogenic strain V991 of Verticillium dahliae and the highly pathogenic strain CF14047-2 of Fusarium pseudograminearum are preserved by the Microbiology Laboratory of the College of Agriculture, Xinjiang Agricultural University, where the applicant is located. The public can obtain the biological material from the applicant, which is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0066] Example 1 Optimization of Fermentation Conditions of Myxococcus sp. KS01 Solid-State Fermentation Seed Inoculation Solution

[0067] 1.1 Screening of initial fermentation medium and determination of growth curve of Myxococcus sp. KS01

[0068] Screening of initial fermentation medium: After strain KS01 was activated on VY / 4 plate, appropriate amount of biofilm was scraped and transferred to LBS culture medium, and cultured at 30℃, 180r / min for 48h until OD 600 ≈0.8~1.0 culture solution as seed solution. The seed solution was inoculated into candidate initial fermentation medium LBS, CYE, VY / 4, VY / 2 at an inoculation rate of 5%, and each medium was set up for 3 replicates, 30℃, 180r / min, shaking culture for 72h, 12 000r / min centrifugation for 10min to collect the bacteria, dried in an electric oven until constant weight, and then weighed. By comparing the dry weight of the bacteria in each candidate culture medium, the best initial fermentation medium was screened out.

[0069] Determination of growth curve: Inoculate the seed liquid of strain KS01 into the initial fermentation medium obtained by screening at an inoculum volume ratio of 5%, and culture at 30°C and 180r / min shaking. Take out 3 bottles of fermentation liquid every 4 hours, centrifuge at 12,000r / min for 10 minutes, dry and weigh the bacteria, and finish at 92 hours. Repeat 3 times at each time point. Draw a growth curve with the dry weight of strain KS01 as the ordinate and the culture time as the abscissa.

[0070] The results are as follows Figure 1 As shown, the growth of strain KS01 in the four candidate initial fermentation media was significantly different. The dry weight of strain KS01 in CYE and VY / 4 medium was significantly higher than that in LBS and VY / 2 medium (P<0.05), and the dry weight obtained in CYE medium was the highest, reaching 183.23 mg / 100 mL. Therefore, CYE medium was selected as the initial medium for liquid fermentation of strain KS01.

[0071] The growth curve of strain KS01 was measured using the CYE initial fermentation medium obtained by screening. The results are as follows Figure 2 As shown in the figure, strain KS01 enters the logarithmic phase after about 20 hours of growth in CYE medium, enters the stable phase after 60-76 hours, at which time the cell dry weight reaches the maximum, and enters the decay phase after 76 hours of growth. Therefore, sampling and measuring the cell dry weight at about 72 hours, i.e., the stable phase, is selected as the response value for evaluating the optimization of fermentation conditions.

[0072] 1.2 Single factor screening of liquid fermentation medium components and fermentation conditions of Myxococcus sp. KS01

[0073] 1.2.1 Carbon source screening

[0074] In the CYE initial fermentation medium obtained by screening, different carbon sources (lactose, sucrose, maltose, dextrin, soluble starch, glucose, mannitol, yeast extract) were added at a mass percentage of 1% to replace the carbon source in the initial fermentation medium. The other components in the medium remained unchanged, and each group was repeated 3 times. The seed liquid was inoculated at a volume ratio of 5%, cultured at 30°C, 180r / min for 72h, centrifuged at 12,000r / min for 10min, and the cells were collected and the dry weight was measured to determine the best carbon source. The effects of different carbon source concentrations (0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%) on the biomass of the cells were screened to determine the best carbon source concentration.

[0075] The results are as follows Figure 3a. The strain KS01 can grow in the culture medium with 8 different carbon sources. The dry weight of the strain KS01 is the highest when soluble starch is used as the carbon source, so soluble starch is selected as the best carbon source. The effects of different concentrations of soluble starch on the growth of the strain KS01 are further determined. The results are shown in Figure 3 As shown in Figure b, the dry weight of strain KS01 increased with the increase of soluble starch concentration. When the soluble starch concentration reached 1.1%, the dry weight of strain WCH05 was the highest and basically did not change. Therefore, 1.1% soluble starch was selected as the optimal carbon source concentration for liquid fermentation of strain KS01.

[0076] 1.2.2 Nitrogen source screening

[0077] On the basis of screening for the best carbon source, different nitrogen sources (urea, ammonium sulfate, soy peptone, tryptone, beef extract, peptone, casein peptone) were added at a mass percentage of 0.5% to replace the nitrogen source in the initial fermentation medium. The other components of the medium remained unchanged, and each group was repeated 3 times. The seed liquid was inoculated at a volume ratio of 5%, and cultured at 30°C, 180r / min for 72h, and the cells were collected and the dry weight was measured to determine the best nitrogen source. The effects of different nitrogen source concentrations (0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%) on the biomass of the cells were screened to determine the best nitrogen source concentration.

[0078] The results are as follows Figure 4 As shown in Figure a, strain KS01 can grow in seven different nitrogen source media. When casein peptone is used as the nitrogen source, the dry weight of strain KS01 is significantly higher than that of other nitrogen source treatment groups. Therefore, casein peptone is selected as the best nitrogen source. The effects of different concentrations of casein peptone on the growth of strain KS01 are further determined. The results are shown in Figure 5. Figure 4 As shown in b, the dry weight of strain KS01 increased with the increase of casein peptone concentration, but when the concentration of casein peptone was greater than 0.9%, the dry weight of strain KS01 basically did not change. Therefore, 0.9% casein peptone was selected as the optimal nitrogen source concentration for liquid fermentation culture of strain KS01.

[0079] 1.2.3 Inorganic salt screening

[0080] On the basis of screening out the most suitable carbon source and nitrogen source, different inorganic salts (FeCl3, CaCl2, NaCl, KCl, MgCl2) were added at a mass percentage of 0.1% to replace the inorganic salt MgSO4·7H2O in the initial fermentation medium, and each treatment was repeated 3 times. The seed solution of strain KS01 was inoculated at a volume ratio of 5%, cultured at 30℃, 180r / min for 72h, centrifuged at 12 000r / min for 10min, and the cells were collected and the dry weight was measured to determine the best inorganic salt. The effects of different inorganic salt concentrations (0.01%, 0.04%, 0.07%, 0.1%, 0.2%, 0.3%, 0.5%) on the biomass of the cells were screened to determine the best inorganic salt concentration.

[0081] The results are as follows Figure 5 As shown in a, MgCl2 can significantly promote bacterial growth compared with other inorganic salts, so MgCl2 was selected as the inorganic salt in the culture medium of strain KS01. The effects of different concentrations of MgCl2 on the growth of strain KS01 were measured, and the results are shown in Figure 5 As shown in b, when the MgCl2 concentration was 0.1%, the dry weight of the bacteria was significantly higher than that of other concentrations. Therefore, 0.1% MgCl2 was selected as the optimal inorganic salt concentration for liquid fermentation culture of strain KS01.

[0082] 1.2.4 Optimization of fermentation conditions

[0083] The culture medium was composed of the best carbon source (1.1% soluble starch), nitrogen source (0.9% casein peptone), and inorganic salt (0.1% MgCl2) screened out above, and the initial pH, liquid volume, seed liquid inoculation amount, culture speed, and culture temperature of the strain KS01 were optimized. The initial pH of the culture medium was set at 6.5-8.3, with a gradient of 0.3, and a total of 7 gradients; the liquid volume was set at 25mL / 250mL-175mL / 250mL, with a gradient of 25mL / 250mL, and a total of 7 gradients; the seed liquid inoculation amount was set at 1%-9%, with a gradient of 2%, and a total of 5 gradients; the culture speed was set at 160r / min-240r / min, with a gradient of 20r / min, and a total of 5 gradients; the culture temperature was set at 26°C-32°C, with a gradient of 2°C, and a total of 5 gradients. The basic conditions for shake flask fermentation were liquid volume (100mL / 250mL), inoculation volume 5.0%, 30°C, and shaking culture at 180r / min for 72h. Each treatment was repeated 3 times. When one factor was optimized, the other conditions remained unchanged. After the culture was completed, the cells were collected by centrifugation, dried and weighed, and the optimal liquid fermentation conditions were obtained based on the dry weight of the cells.

[0084] The results are as follows Figure 6As shown, the optimal fermentation initial pH of the screened strain KS01 is 7.4, the liquid volume is 125mL / 250mL, the seed liquid inoculation amount is 5%, the culture speed is 200r / min, and the culture temperature is 30°C.

[0085] 1.3 Optimization of Myxococcus sp. KS01 fermentation medium composition and fermentation conditions by response surface methodology

[0086] 1.3.1 Plackett-Burman (PB) test

[0087] Based on the results of single factor optimization, each factor was selected at two levels, high and low, and the Design-expert 13.0 software was used to design a PB experiment with N=12 times. The dry weight of the bacteria after fermentation was taken as the response value, and the main effect analysis was carried out on 8 factors (carbon source, nitrogen source, inorganic salt, fermentation temperature, initial pH of fermentation, seed liquid inoculation amount, liquid filling amount and culture speed) to screen out the main factors affecting the growth of strain KS01.

[0088] The results of the main effect factor analysis are shown in Table 1. Casein peptone content, MgCl2 content, culture temperature, and seed solution inoculation amount have significant effects on the dry weight of strain KS01 (P<0.05), and casein peptone, seed solution inoculation amount, and culture temperature are the top three factors in terms of their importance in affecting the growth of myxobacterium KS01. Therefore, these three factors were selected as the main factors for constructing the model for response surface method optimization.

[0089] Table 1. Analysis of variance of Plackett-Burman test

[0090] factor F-value p-value Significance importance Soluble starch 0.0175 0.9032 NS 8 Casein Peptone 62.75 0.0042 ** 1 <![CDATA[MgCl2]]> 13.02 0.0365 * 4 Cultivation temperature 23.88 0.0164 * 3 Culture speed 0.6280 0.4860 NS 7 Initial pH 8.17 0.0646 NS 6 Liquid volume 8.52 0.0616 NS 5 Seed liquid inoculation amount 31.55 0.0112 * 2

[0091] Note: *Significant difference (P<0.05); **Extremely significant difference (P<0.01); NSNo significant difference (P>0.05).

[0092] 1.3.2 Box-Behnken test and model prediction

[0093] Based on the three main factors affecting the growth of strain KS01 (casein peptone, seed solution inoculation amount, and culture temperature) screened out in the PB experiment, a three-factor and three-level Box-Behnken experiment was designed using Design-expert 13.0 analysis software with dry weight of bacteria as the response value to determine the optimal combination of each factor.

[0094] According to the test results (Table 2), the quadratic regression equation was solved and variance analysis was performed to obtain the regression equation: Y = 333.44 + 33.35A + 16.63B + 18.35C + 4.70AB + 38.55AC - 22.00BC - 86.67A 2 -36.87B 2 -10.62C 2 .

[0095] From the variance analysis (Table 3), the regression equation P < 0.05, the lack of fit term was not significant (P = 0.3176 > 0.05), R 2 The value of F value is 0.9325, indicating that the model is significant, the model prediction value has a high degree of fit with the actual value, and can better reflect the change of the response value. The analysis results of F value show that the influence of each factor on the dry weight of the strain is: seed liquid inoculation amount > casein peptone > culture temperature. The response surface of the interaction effect on the dry weight of the bacteria obtained by the regression equation shows ( Figure 7 ), the graphs are all three-dimensional space surface graphs with openings downward, indicating that the response surface value has extremely high values, and the optimal value exists in the designed factor level. Solving the regression equation, the optimal concentration of casein peptone is 9.9g / L, the optimal culture temperature is 30℃, and the optimal seed liquid inoculation amount is 6.9%.

[0096] Combining the single factor and response surface optimization results, the optimal liquid fermentation medium formula of strain KS01 was finally determined to be: soluble starch 11.0g / L, casein peptone 9.9g / L, MgCl2 1.0g / L, pH 7.4; the optimal fermentation conditions were: shake bottle liquid volume 125mL / 250mL, seed liquid inoculation 6.9%, rotation speed 200r / min, and culture at 30℃ for 72h. Under these conditions, the model predicted that the maximum dry weight of strain KS01 was 356.16mg / 100mL.

[0097] Table 2 Box-Behnken test design and results

[0098] Serial number Casein peptone (g / L) Temperature(℃) Seed solution inoculation amount (%) Dry weight of bacteria (mg / 100mL) 1 5 28 5 339.2 2 13 28 5 328.2 3 5 32 5 329.6 4 13 32 5 223.6 5 5 30 3 208.7 6 13 30 3 186.7 7 5 30 7 179.2 8 13 30 7 317.3 9 9 28 3 221.2 10 9 32 3 231.2 11 9 28 7 298.6 12 9 32 7 171.6 13 9 30 5 319.2 14 9 30 5 242.5 15 9 30 5 368.7 16 9 30 5 310.5 17 9 30 5 319.2

[0099] Table 3 Analysis of variance in Box-Behnken test

[0100]

[0101]

[0102] Note: *Significant difference (P<0.05); **Very significant difference (P<0.01); NsNo significant difference (P>0.05); R 2 =0.9325

[0103] 1.3.3 Optimization model validation of fermentation medium and culture conditions

[0104] The optimal culture parameters were obtained according to the response surface test optimization, and shake flask fermentation was carried out to obtain the actual dry weight of the bacteria after fermentation. The actual dry weight of the bacteria after fermentation was compared with the predicted value and the dry weight of fermentation before optimization to verify the reliability and effectiveness of the model and obtain the fermentation optimization results.

[0105] The results are as follows Figure 8 As shown in the figure, the actual dry weight of strain KS01 was 357.92 mg / 100 mL, with a relative error of 0.49% from the predicted value. The results confirmed that the model was reliable, indicating that the actual fermentation conditions under the optimized conditions had a good fit with the prediction. Before the optimization, the dry weight of the cells obtained using CYE medium was only 191.6 mg / 100 mL, and the cell content obtained after the optimization fermentation increased by 1.87 times compared with that before the optimization.

[0106] Example 2 Optimization of solid-state fermentation conditions of insect sand-based slime bacteria fertilizer

[0107] 2.1 Preparation of solid-state fermentation seed solution

[0108] The activated strain KS01 was transferred to LBS culture medium and cultured at 30°C, 180r / min shaking for 48h, and then inoculated with an inoculum size of 6.9% into the best liquid fermentation medium (soluble starch 11.0g / L, casein peptone 9.9g / L, MgCl2 1.0g / L, pH 7.4) optimized in Example 1. The solid-state fermentation seed liquid was obtained under the optimal fermentation conditions: shake flask liquid volume 125mL / 250mL, rotation speed 200r / min, and culture at 30°C for 72h.

[0109] 2.2 Screening of solid-state fermentation basal medium

[0110] White-spotted flower beetle sand, yellow mealworm sand, crushed rabbit manure and sheep manure were selected as solid-state fermentation matrices respectively, and crushed wheat straw was added at a mass ratio of 3:1 to prepare a solid-state fermentation basal medium. After high-pressure steam sterilization, the pH was adjusted to 7.0, and the solid-state fermentation seed solution of strain KS01 was added at an inoculation rate of 10%. After uniform mixing, the water content was adjusted to 60%, and the culture was placed in a 30°C incubator for 10 days. 1.0 g of sample was taken every day and inoculated into 9 mL of sterile water. 950 W, 30% output power, ultrasound was applied for 3s with an interval of 10s, and the treatment was carried out for 5 minutes. After gradient dilution with sterile water, 100 μL was taken and coated on the surface of VY / 4 culture medium, and the culture was placed in a 30°C incubator for culturing. The changes in the bacterial content of strain KS01 in different solid-state fermentation basal media were determined.

[0111] The results are as follows Fig. 9As shown in the figure, there are obvious differences in the bacterial content of strain KS01 in different solid-state fermentation basic media. The bacterial content of strain KS01 reached the maximum value on the 6th day when cultured in the medium of white star flower beetle sand and yellow mealworm sand, which were 5.0×10 5 cfu / g and 3.8×10 5 cfu / g; on the 7th day after cultivation in sheep manure medium, the bacterial count of strain KS01 reached a maximum of 5.17×10 5 cfu / g; however, on the 7th day after culture in rabbit feces medium, the bacterial count of strain KS01 reached a maximum value, but it was only 1.67×10 5 Therefore, considering the timeliness and bacterial content of solid-state fermentation, the white star flower beetle sand medium was selected as the basic medium for solid-state fermentation.

[0112] 2.3 Optimization of solid-state fermentation conditions

[0113] The sand of white star flower beetle and crushed wheat straw were mixed in a mass ratio of 3:1, and after being fully sterilized, they were used as the basic medium for solid fermentation. The fermentation temperature, initial pH, water content, and seed liquid inoculation amount were optimized. The fermentation temperature was set at 27℃, 30℃, 34℃, and 38℃, with a total of 4 gradients, the initial pH of the medium was set at 6.7, 7.0, 7.3, 7.6, and 7.9, with a total of 5 gradients; the water content was set at 55%, 60%, 65%, 70%, and 75%, with a total of 5 gradients; the seed liquid inoculation amount was set at 1%, 5%, 10%, 15%, and 20%, with a total of 5 gradients. The seed liquid inoculation amount was 10%, the initial pH of the medium was 7.0, and the fermentation temperature was 30℃, which were the basic conditions for solid fermentation. Each treatment was repeated 3 times. When one factor was optimized, the other conditions remained unchanged. During the 8 days of fermentation, 1 g of sample was taken every 2 days and inoculated into 9 mL of sterile water. The sample was ultrasonically treated for 3 seconds with an interval of 10 seconds at 950 W and 30% output power for 5 minutes. After gradient dilution with sterile water, 100 μL was spread on a VY / 4 culture dish and cultured in a 30°C incubator. The bacterial content was determined and the optimal fermentation conditions were screened.

[0114] The results are as follows Fig.10 As shown, the optimal fermentation temperature range of the screened strain KS01 for solid-state fermentation is 30-34°C, the optimal initial fermentation pH range is 7.0-7.3 (because the pH of the mixed sterilized white star flower beetle sand and crushed wheat straw is in the range of 7.0-7.3, there is no need to adjust the initial pH in subsequent experiments), the optimal fermentation moisture content range is 65%-70%, and the optimal fermentation seed liquid inoculation amount range is 15%-20%.

[0115] 2.4 Screening of auxiliary materials for solid fermentation medium

[0116] 2.4.1 Single factor test of auxiliary material addition in solid-state fermentation medium

[0117] Potato starch, wheat bran, yeast powder and cottonseed meal were selected as auxiliary materials for solid-state fermentation matrix. Each auxiliary material was added to the solid-state fermentation basic medium (mixed with white star flower beetle sand and crushed wheat straw 3:1) at an addition amount of 0%, 0.5%, 1%, 3% and 5%, respectively, and stirred and mixed well. After being fully sterilized, it was adjusted to the optimal culture conditions obtained by screening (seed liquid inoculation amount 15%, initial fermentation pH 7.0, water content 65%, fermentation temperature 30℃) and cultured for 8 days. Samples were taken every 2 days to determine the bacterial content, and each treatment was repeated 3 times.

[0118] The results are as follows Fig.11 As shown, the optimal addition amount of potato starch is 3%, the optimal addition amount of wheat bran is 3%, the optimal addition amount of yeast powder is 1%, and the optimal addition amount of cottonseed meal is 1%.

[0119] 2.4.2 Response surface optimization and model verification of matrix and auxiliary material addition in solid-state fermentation

[0120] According to the results of the single-factor test for screening of solid-state fermentation substrate and auxiliary materials, a response surface experiment with 4 factors and 5 levels was designed using the central composite design (CCD) method to optimize the amount of auxiliary materials added. The corresponding auxiliary material concentrations at each level are shown in Table 4. After the solid-state fermentation substrates for each treatment were configured and fully sterilized, they were adjusted to the optimal culture conditions obtained by screening (seed liquid inoculation amount 15%, initial fermentation pH 7.0, water content 65%, fermentation temperature 30°C), and samples were taken after 6 days of culture to determine the bacterial content.

[0121] Table 4 Design of various levels of CCD test variables

[0122]

[0123] The response surface optimization results of the auxiliary material concentration of solid-state fermentation medium are shown in Table 5. After analysis by Design-expert 13 software, the quadratic polynomial regression equation was obtained: Y = 6.00 + 0.1187A-0.3363B + 0.1421C-0.3438D-0.2856AB-0.1306AC-0.0969AD-0.1406BC-0.4119BD + 0.3356CD-0.8826A 2 -1.20B 2 -0.8176C 2 -1.11D 2 , where: Y represents the bacterial content.

[0124] From the variance analysis (Table 6), the regression equation P < 0.0001, the lack of fit term was not significant (P = 0.3929 > 0.05), R 2The value is 0.9234, indicating that the model is significant, the model prediction value has a high degree of fit with the actual value, and can better reflect the change of the response value. According to the regression equation, the response surface curve of the interaction of various factors on the bacterial content of strain KS01 was obtained ( Fig.12 ), the results showed that the interaction between starch and cottonseed meal and yeast powder was not significant, while the interaction between starch and wheat bran, wheat bran and cottonseed meal, wheat bran and yeast powder, and cottonseed meal and yeast powder was significant. At the same time, the response surface analysis results showed that the bacterial content of strain KS01 increased first and then decreased with the increase of starch, wheat bran, cottonseed meal, and yeast powder, indicating that the response surface has an optimal range. Solving the regression equation, the optimal addition combination of solid-state fermentation medium auxiliary materials was obtained, which was 3.09% starch, 2.87% wheat bran, 1.07% cottonseed meal, and 0.88% yeast powder.

[0125] Based on the results of single factor and response surface optimization experiments of solid-state fermentation, the optimal solid-state fermentation culture conditions of strain KS01 were obtained as follows: after mixing white star flower beetle sand with crushed wheat straw at a ratio of 3:1, 3.09% starch, 2.87% wheat bran, 1.07% cottonseed meal and 0.88% yeast powder were added according to the mass ratio, and the water content was adjusted to 65%-70%, and the pH was adjusted to 7.0-7.3. After sufficient sterilization, the seed liquid was inoculated at an inoculation rate of 15%-20%, and fermented and cultured at 30-34℃ for 6 days after sufficient stirring. Under this condition, the model predicted that the bacterial content of strain KS01 was the highest, with a predicted value of 6.05×10 7 cfu / g.

[0126] Table 5 CCD test design table and test results

[0127] Serial number A: Starch B: Wheat bran C: Cottonseed meal D: Yeast powder <![CDATA[Bacterial content (×10 7 cfu / g)]]> 1 3％ 3％ 0.5％ 0.5％ 1.09 2 4％ 3％ 0.5％ 0.5％ 2.57 3 3％ 4％ 0.5％ 0.5％ 2.43 4 4％ 4％ 0.5％ 0.5％ 3.04 5 3％ 3％ 1.5％ 0.5％ 1.93 6 4％ 3％ 1.5％ 0.5％ 1.69 7 3％ 4％ 1.5％ 0.5％ 1.82 8 4％ 4％ 1.5％ 0.5％ 1.60 9 3％ 3％ 0.5％ 1.5％ 1.87 10 4％ 3％ 0.5％ 1.5％ 1.77 11 3％ 4％ 0.5％ 1.5％ 0.68 12 4％ 4％ 0.5％ 1.5％ 0.59 13 3％ 3％ 1.5％ 1.5％ 1.70 14 4％ 3％ 1.5％ 1.5％ 3.70 15 3％ 4％ 1.5％ 1.5％ 2.26 16 4％ 4％ 1.5％ 1.5％ 0.53 17 2.5％ 3.5％ 1％ 1％ 2.50 18 4.5％ 3.5％ 1％ 1％ 3.07 19 3.5％ 2.5％ 1％ 1％ 2.70 20 3.5％ 4.5％ 1％ 1％ 0.35 21 3.5％ 3.5％ 0 1％ 2.49 22 3.5％ 3.5％ 2％ 1％ 3.60 23 3.5％ 3.5％ 1％ 0 3.17 24 3.5％ 3.5％ 1％ 2％ 0.58 25 3.5％ 3.5％ 1％ 0 5.60 26 3.5％ 3.5％ 1％ 1％ 5.82 27 3.5％ 3.5％ 1％ 1％ 7.40 28 3.5％ 3.5％ 1％ 1％ 5.77 29 3.5％ 3.5％ 1％ 1％ 5.63 30 3.5％ 3.5％ 1％ 1％ 5.76

[0128] Table 6 Analysis of variance of CCD test

[0129]

[0130]

[0131] Note: *Significant difference (P<0.05); **Very significant difference (P<0.01); NSNo significant difference (P>0.05); R 2 =0.9234.

[0132] 2.4.3 Optimization model validation of solid-state fermentation medium and culture conditions

[0133] To verify the accuracy of the model, the fermentation test was carried out with the best solid-state fermentation conditions obtained by the above optimization, and compared with the solid-state fermentation method before optimization. 30℃, 180r / min, shaking culture for 72h

[0134] Preparation of the optimized pre-worm sand-based slime bacteria fertilizer: After activating strain KS01 on a VY / 4 plate, scrape an appropriate amount of bacterial film and transfer it to LBS culture medium. The culture medium was cultured at 30°C and 180r / min for 48h as seed liquid. The seed liquid was inoculated into CYE medium at a 5% inoculation rate, and the solid-state fermentation seed liquid was obtained by shaking and culturing at 30°C and 200r / min for 72h for standby use. The white star flower beetle worm sand and crushed wheat straw were mixed at a mass ratio of 3:1, and the pH was adjusted to 7.0 after high-pressure steam sterilization. The solid-state fermentation seed liquid of strain KS01 was added at a 10% inoculation rate, and the water content was adjusted to 60% after uniform mixing. It was placed in a 30°C incubator for 6d.

[0135] Preparation of optimized insect sand-based myxobacteria fertilizer: After activating strain KS01 on a VY / 4 plate, scrape an appropriate amount of biofilm and transfer it to LBS culture medium, and culture the culture medium at 30°C and 180r / min for 48h as seed liquid. Inoculate the seed liquid into the optimal liquid fermentation medium (soluble starch 11.0g / L, casein peptone 9.9g / L, MgCl2 1.0g / L, pH 7.4) at an inoculum of 6.9%, and culture the solid-state fermentation seed liquid at 30°C and 200r / min for 72h for standby use. The sand of white-starred scarab beetle and crushed straw were mixed in a mass ratio of 3:1, and 3.09% starch, 2.87% wheat bran, 1.07% cottonseed meal and 0.88% yeast powder were added. After thorough mixing and sterilization, the pH was adjusted to 7.0. The solid fermentation seed liquid of strain KS01 was added at an inoculation rate of 15%, the water content was adjusted to 65% after even mixing, and the mixture was placed in a 30°C incubator for 6 days.

[0136] The results are as follows Fig.13 As shown in Figure 2, before the solid-state fermentation conditions were optimized, the bacterial content of strain KS01 in the worm sand-based bacterial fertilizer was only 5.0×10 5 cfu / g, and after the solid-state fermentation conditions were optimized, the bacterial content of strain KS01 in the insect sand-based bacterial fertilizer reached 6.4×10 7 cfu / g, compared with before optimization, the bacterial content increased by 128 times.

[0137] Example 3 Indoor control effect of insect sand-based myxobacteria fertilizer on cotton Verticillium wilt and its effect on cotton growth

[0138] 3.1 Preparation of Verticillium dahliae spore suspension

[0139] Take a small piece of slant-preserved Verticillium dahliae agar, inoculate it on PDA medium, and activate it at 28°C to make a 5mm Verticillium dahliae cake. Inoculate it into a 500mL conical flask containing 200mL Czapek medium. Put 4 Verticillium dahliae cakes in each flask. Incubate it at 28°C and 200r / min for 6 days. Filter the fermentation liquid through 4 layers of sterile gauze to obtain the filtrate as the spore suspension of Verticillium dahliae. After dilution with sterile water, the concentration of 1.0×10 8 / mL of Verticillium dahliae spore suspension for later use.

[0140] 3.2 Preparation of insect sand-based myxobacterial fertilizer

[0141] The preparation of the worm sand-based myxobacterial fertilizer before optimization and the worm sand-based myxobacterial fertilizer after optimization refers to 2.4.3.

[0142] 3.3 Indoor control effect of insect sand-based myxobacterial fertilizer on cotton Verticillium wilt

[0143] The nutrient soil and vermiculite were mixed in a ratio of 4:1 and placed in a plastic box (40cm×20cm×15cm). After soaking the cotton seeds in 2% sodium hypochlorite for 15min, they were rinsed several times with sterile water. The surface-sterilized seeds were placed on moist sterile gauze and germinated at room temperature for later use. There were four treatments in the pot experiment: treatment A: control (only inoculated with spore suspension of Verticillium dahliae); treatment B: sterile fermentation matrix (i.e., solid fermentation medium without myxobacteria, i.e., the white star flower beetle worm sand and crushed straw were mixed in a mass ratio of 3:1, and starch 3.09%, wheat bran 2.87%, cotton meal 1.07%, yeast powder 0.88% were added, and the pH was adjusted to 7.0 after thorough mixing and sterilization); treatment C: worm sand-based myxobacteria fertilizer before optimization; treatment D: worm sand-based myxobacteria fertilizer after optimization. At the time of sowing, 5 g of insect sand-based myxobacterial fertilizer or sterile fermented substrate was applied to the hole below each seed. After covering with a thin layer of soil, the seeds were sown at a density of 15 seeds per box. Every 3 plastic boxes were a treatment, and each treatment was repeated 3 times. The seedlings were transplanted 10 days after sowing, and 10 cotton seedlings were kept in each box. When the two true leaves first appeared, the lateral roots of the cotton seedlings were pierced with sterilized bamboo sticks, and 5 mL of spore suspension of Verticillium dahliae (containing 1.0×10 8 / mL).

[0144] On the 35th day after inoculation with Verticillium dahliae spore suspension, the vascular bundle discoloration of each treatment cotton plant was checked and the incidence rate was calculated. The disease index was investigated according to the 5-level grading standard for cotton Verticillium wilt disease, and the control effect was calculated.

[0145] The classification standards for cotton Verticillium wilt in potted plant experiments are as follows:

[0146] Grade 0: no brown spots on the cross section; Grade 1: the brown area on the cross section accounts for less than 25% of the cross section; Grade 2: the brown area accounts for 25-50% of the cross section; Grade 3: the brown area accounts for 50-75% of the cross section; Grade 4: the brown area accounts for more than 75% of the cross section, and the cross section almost turns brown. The morbidity, disease index and prevention effect are calculated according to the following formulas.

[0147] Incidence rate (%) = number of diseased plants investigated / total number of plants investigated × 100;

[0148] Disease index = ∑[(number of plants at each level × relative level)] / (total number of plants investigated × highest level) × 100;

[0149] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0150] Through the investigation of potted cotton plants, it was found that ( Fig.14 , Table 7), the optimized insect sand-based myxobacteria fertilizer can significantly reduce the disease index of cotton Verticillium wilt, and the prevention effect is significantly higher than that of the insect sand-based myxobacteria fertilizer and sterile fermentation matrix before optimization, and the prevention effect reaches 70.00%, which is 6.99% higher than that before optimization.

[0151] Table 7 The potted control effect of insect sand-based myxobacteria fertilizer on cotton Verticillium wilt

[0152] deal with Incidence (%) Disease index Prevention effect (%) Insect sand-based myxobacteria fertilizer before optimization 26.67±3.33b 12.33±1.67b 63.01±5.00b Optimized insect sand-based myxobacteria fertilizer 16.67±5.77b 10.00±1.44c 70.00±4.33a Sterile fermentation substrate 36.67±2.16ab 21.67±0.83b 34.99±2.50c CK 46.67±3.33a 33.33±3.01a

[0153] Note: Data are expressed as mean ± standard deviation. Different letters after the data in the same column indicate significant differences (P < 0.05), and “-” indicates no such item.

[0154] 3.4 Effect of insect sand-based myxobacterial fertilizer on the growth of potted cotton

[0155] Mix nutrient soil and vermiculite in a ratio of 4:1 and put them into a plastic box (40cm×20cm×15cm). Soak cotton seeds in 2% sodium hypochlorite for 15 minutes, rinse with sterile water several times, place the surface-sterilized seeds on moist sterile gauze, germinate at room temperature, apply 5g of insect sand-based myxobacterial fertilizer or sterile fermentation matrix in the hole below each seed when sowing, cover with a thin layer of soil, and sow at a density of 15 seeds per box. Every 3 plastic boxes are one treatment, and each treatment is repeated 3 times. The seedlings are transplanted 10 days after sowing, and 10 cotton seedlings are retained in each box. There were 4 treatments in the potted experiment, treatment I: optimized worm litter-based myxobacterial fertilizer; treatment II: worm litter-based myxobacterial fertilizer before optimization; treatment III: sterile fermentation matrix (i.e., solid fermentation medium without myxobacteria, i.e., white star flower beetle worm litter and crushed straw in a mass ratio of 3:1, and added with 3.09% starch, 2.87% wheat bran, 1.07% cottonseed meal, and 0.88% yeast powder, mixed thoroughly and sterilized, and then adjusted to pH 7.0); treatment IV: control (no treatment).

[0156] The results of the potted test showed (Table 8) that, compared with the control treatment group and the sterile fermentation matrix treatment group, the application of the optimized insect sand-based myxobacterium fertilizer could significantly (P<0.05) increase the plant height, stem diameter, taproot length, and fresh weight of the aboveground stem and underground part of the cotton plant, indicating that the insect sand-based myxobacterium fertilizer has a certain growth-promoting function for the growth of cotton.

[0157] Table 8 Effects of insect sand-based myxobacteria fertilizer on the growth of potted cotton

[0158]

[0159] Note: Data are expressed as mean ± standard deviation. Different letters after the data in the same column indicate significant differences (P < 0.05).

[0160] 3.5 Field efficacy of insect sand-based myxobacterial fertilizer on cotton Verticillium wilt and its effects on cotton agronomic traits

[0161] The preparation method of the insect sand-based slime bacteria fertilizer is the same as in Example 3. The field test was carried out in the cotton wilt disease nursery (severely diseased field) of the Academy of Agricultural Sciences of Bayinguoleng Mongolian Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Cotton planting adopts drip irrigation under the film, and the row spacing adopts a wide and narrow row configuration. Four rows of cotton are planted per film, and the wide row spacing, narrow row spacing, and film spacing are 86cm, 22cm, and 86cm, respectively, and the spacing between plants is 10cm. The experiment has a total of 5 treatments, and a randomized block design is adopted, with 1 film and 4 rows per treatment, 3 repetitions, that is, 3 films and 12 rows as 1 treatment block group. Myxobacteria fertilizer treatment group: before sowing, 50kg / mu of myxobacteria fertilizer was evenly spread in each row of seed furrows, and then sowed after covering with thin soil; sterile fermentation matrix treatment group: before sowing, 50kg / mu of myxobacteria fertilizer was evenly spread in each row of seed furrows (i.e., solid fermentation medium without myxobacteria, i.e., white star flower beetle sand and crushed straw in a mass ratio of 3:1, and starch 3.09%, wheat bran 2.87%, cottonseed meal 1.07%, yeast powder 0. 88%, fully mixed and sterilized, adjusted to pH 7.0), covered with thin soil and sown; commercial microbial agent treatment group: after sowing, when cotton grows to the three-leaf stage, dilute the microbial agent "Zhongmian Junle Tu" 600 times, and pour it into the cotton roots at 40mL / plant, and repeat the root irrigation once after 14 days; chemical agent treatment group: when cotton grows to the three-leaf stage, dilute the chemical agent "trichloroisocyanuric acid" 6000 times, pour it into the cotton roots at 40mL / plant, and repeat the root irrigation once after 14 days. The plot without agent treatment was used as a blank control. Routine management was performed after cotton sowing, but no other fungicides and other microbial agents or fertilizers were applied during this period. The incidence rate was investigated at the cotton bud stage (July 13) and the flower and boll stage (August 5), and the disease index was calculated according to the field cotton Verticillium wilt grading standard, and the control effect was statistically analyzed. At the same time, agronomic traits such as cotton plant height, stem thickness, number of buds, number of bolls, and number of fruit branches per plant were investigated. During the cotton harvest period, seed cotton was collected from each treatment, weighed and the seed cotton yield was calculated.

[0162] The classification standards for cotton Verticillium wilt in the field are as follows:

[0163] Level 0: The cotton plants are healthy, without diseased leaves, and growing normally; Level 1: 33% or less of the leaves of the cotton plants are diseased, turning yellow and wilting; Level 2: More than 33% and 66% or less of the leaves of the cotton plants are diseased, turning yellow and wilting; Level 3: More than 66% and 99% or less of the leaves of the cotton plants are diseased, turning yellow and wilting; Level 4: All the leaves of the cotton plants fall off and the cotton plants die.

[0164] Incidence rate (%) = number of diseased plants investigated / total number of plants investigated × 100;

[0165] Disease index = ∑[(number of plants at each level × relative level)] / (total number of plants investigated × highest level) × 100;

[0166] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0167] The results of the field survey showed (Table 9) that compared with the control treatment group, the application of the optimized insect sand-based myxobacteria fertilizer could significantly reduce the incidence and disease index of cotton plants. The control effects at the bud stage and boll stage of cotton were 75.91% and 62.44%, respectively, which were significantly (P<0.05) higher than those of the insect sand-based myxobacteria fertilizer, the commercial microbial agent treatment group and the chemical agent treatment group before optimization (Table 9). At the same time, the application of insect sand-based myxobacteria fertilizer could significantly improve the agronomic traits of cotton, such as plant height, stem diameter, bud number, and boll number (Table 10). The yield of the insect sand-based myxobacteria fertilizer before optimization increased by 64.09% compared with the blank control, while the yield of the insect sand-based myxobacteria fertilizer after optimization increased by 81.20% compared with the blank control.

[0168] Table 9 Field efficacy of insect sand-based myxobacterial fertilizer on cotton Verticillium wilt

[0169]

[0170] Note: Data are expressed as mean ± standard deviation. Different letters after the data in the same column indicate significant differences (P < 0.05), and “-” indicates no such item.

[0171] Table 10 Effects of insect sand-based myxobacteria fertilizer on cotton growth

[0172]

[0173]

[0174] Note: Data are expressed as mean ± standard deviation. Different letters after the data in the same column indicate significant differences (P < 0.05).

[0175] Table 11 Yield-increasing effect of insect sand-based slime bacteria fertilizer

[0176]

[0177] Note: Data are expressed as mean ± standard deviation. Different letters after the same column of data indicate significant differences (P < 0.05). "-" indicates no such item. Example 4 Indoor control effect determination of insect sand-based myxobacterial fertilizer on wheat stem base rot

[0178] 4.1 Preparation of wheat stem rot inoculum

[0179] Boil the millet in boiling water for 2 minutes, quickly cool it with cold water and rinse it 3-4 times, dry it, sterilize it with high-pressure steam for 30 minutes, and cool it for later use. Inoculate 5 pieces of Fusarium graminearum cakes (d=5mm) into 100g of millet, and culture it at 25℃ in the dark for 5-7 days. When the mycelium of Fusarium graminearum covers the millet grains, take it out and dry it for later use.

[0180] 4.2 Determination of potted control effect of insect sand-based myxobacterial fertilizer on wheat stem rot

[0181] Mix nutrient soil and vermiculite in a ratio of 4:1 and put them into disposable paper cups (40cm×20cm×15cm). Sprinkle 1g of insect sand-based slime bacteria fertilizer or sterile fermented substrate under the seeds when sowing, cover with a thin layer of soil, sow at a density of 12 seeds per cup, sprinkle 1g of wheat stem rot inoculum, and then cover with 20g of soil. Every 3 paper cups are one treatment, and each treatment is repeated 3 times. After the wheat seedlings emerge, the seedlings are determined, and 8-9 wheat seedlings are retained in each paper cup. A total of four treatments were set up in the pot experiment: Treatment A: control (only inoculated with wheat stem rot pathogen inoculum); Treatment B: sterile fermentation matrix (i.e. solid fermentation medium without myxobacteria, that is, the worm sand and crushed straw are mixed in a mass ratio of 3:1, and 3.09% starch, 2.87% wheat bran, 1.07% cottonseed meal, and 0.88% yeast powder are added, and the pH is adjusted to 7.0 after thorough mixing and sterilization); Treatment C: worm sand-based myxobacterial fertilizer before optimization; Treatment D: worm sand-based myxobacterial fertilizer after optimization.

[0182] The incidence rate was investigated on the 35th day after wheat sowing, and the disease index was investigated according to the 7-level grading standard for wheat stem base rot, and the prevention and control effect was calculated.

[0183] The disease grading standards for wheat base stem rot are as follows: Level 0: no symptoms; Level 1: the embryonic disc node turns brown; Level 2: the embryonic disc node, the underground stem or the leaf sheath turns brown; Level 3: the embryonic disc node, the underground stem or the leaf sheath all turn brown; Level 4: the underground stem, the leaf sheath and the lower stem turn brown; Level 5: the stems show symptoms of drying up and rotting; Level 6: the plant dies due to stem rot; Level 7: seedling withering or seed rot.

[0184] The disease index and control effect were calculated as follows: Incidence rate (%) = number of diseased plants / total number of investigated plants × 100;

[0185] Disease index = [∑(number of diseased plants at each level × level) / (total number of plants surveyed × highest level value)] × 100;

[0186] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0187] Through continuous observation of potted cotton plants, the morbidity rate of the control group reached 93.93% on the 35th day after emergence. At this time, the morbidity rate and disease level were investigated, and the disease index of each treatment and potted control efficacy were calculated. Fig.15 As shown in Table 12, the optimized insect sand-based myxobacteria fertilizer can significantly reduce the incidence and disease index of wheat stem base rot, and the prevention effect is significantly higher than that of the insect sand-based myxobacteria fertilizer and sterile fermentation matrix before optimization, reaching 72.67%, an increase of 7.84% compared with before optimization.

[0188] Table 12 The effect of sand-based myxobacterial fertilizer on the control of wheat stem rot in greenhouse potted plants

[0189] deal with Incidence Disease index Prevention effect CK 93.93±3.61a 45.70±7.64a - Sterile fermentation substrate 55.76±9.48b 32.19±8.20ab 29.55±17.96b Optimization of pre-worm sand-based myxobacteria fertilizer 43.80±10.54b 16.07±2.19b 64.83±4.79a Optimized insect sand-based myxobacteria fertilizer 37.50±5.10b 12.49±2.74b 72.67±5.99a

[0190] Note: The data in the table are mean ± standard deviation, different letters indicate significant differences between groups, P < 0.05

Claims

1. A worm-litter-based myxobacterial fertilizer, wherein the fertilizer is formed by fermenting Myxococcus sp. KS01 in a solid culture medium; the solid culture medium is a solid basic culture medium prepared by mixing white star flower beetle worm-litter with crushed wheat straw in a weight ratio of 3:1, and each 100g of the solid basic culture medium contains 3-5g of starch, 1-3g of wheat bran, 1-3g of cottonseed meal, and 0.5-1g of yeast powder; preferably, each 100g of the solid basic culture medium contains 3.09g of starch, 2.87g of wheat bran, 1.07g of cottonseed meal, and 0.88g of yeast powder.

2. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 1, characterized in that: The method steps are as follows: (1) Preparation of solid-state fermentation seed solution: After Myxococcus sp. KS01 was activated on a VY / 4 plate, an appropriate amount of biofilm was scraped and transferred to LBS culture medium, and cultured at 30°C and 180 r / min for 48 h to obtain liquid fermentation seed solution, which was inoculated into liquid fermentation medium at a rate of 5%-7%, and cultured at a temperature of 28-32°C and a rotation speed of 180-220 r / min for 68-80 h to obtain solid-state fermentation seed solution of Myxococcus sp. KS01; (2) Preparation of insect sand-based bacterial fertilizer: A solid basic culture medium for bacterial fertilizer production is prepared with white star flower beetle insect sand and crushed wheat straw as the main ingredients, and starch, wheat bran, cottonseed meal, and yeast powder are added to form a solid culture medium. After sterilization, the solid fermentation seed liquid prepared in step (1) is inoculated and cultured at a constant temperature of 30-34°C for 6-7 days to prepare a solid fermentation product of myxobacteria. After the fermentation is completed, mix well to obtain an insect sand-based myxobacterial bacterial fertilizer.

3. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 2, characterized in that: The liquid fermentation medium in step (1) has a ratio of: soluble starch 11.0 g / L, casein peptone 9.9 g / L, MgCl2 1.0 g / L, and pH 7.

4.

4. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 2, characterized in that: The liquid fermentation in step (1) is carried out in a container, and the volume ratio of the fermentation medium to the container can be (90-120):250, preferably the volume ratio of the fermentation medium to the container can be 125:250; preferably, the container is a conical flask.

5. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 2, characterized in that: The inoculation amount in step (1) is 6.9-7%; preferably, the culture temperature in the liquid fermentation medium in step (1) is 30°C, the rotation speed is 200r / min, and the solid-state fermentation seed liquid of Myxococcus KS01 is obtained by culturing for 72h.

6. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 2, characterized in that: The steps for preparing the solid culture medium in step (2) are: Step A. Prepare a solid basic culture medium by mixing white star flower beetle sand and crushed wheat straw in a weight ratio of 3:1; preferably, the wheat straw in step A needs to be crushed to a length of 0.5 to 1 cm; Step B. Add starch, wheat bran, cottonseed meal and yeast powder to the solid basic culture medium respectively and stir evenly; preferably, in step B, 3-5 g starch, 1-3 g wheat bran, 1-3 g cottonseed meal and 0.5-1 g yeast powder are required for every 100 g solid basic culture medium; preferably, 3.09 g starch, 2.87 g wheat bran, 1.07 g cottonseed meal and 0.88 g yeast powder are required for every 100 g solid basic culture medium; Step C: Autoclave for 30 min.

7. The method for preparing the insect sand-based slime bacteria fertilizer according to claim 2, characterized in that: In step (2), the solid culture medium is inoculated with the solid fermentation seed solution of Myxococcus KS01 prepared in step (1) at an inoculum amount of 15%-20%, the water content is adjusted to 65%-70%, and the solution is fermented at a constant temperature of 30-34° C. for 6-7 days to obtain the solid fermentation product of the myxobacteria. After the fermentation is completed, the solution is ventilated and dried and then mixed to obtain the insect sand-based myxobacteria fertilizer.

8. Use of the insect sand-based myxobacterial fertilizer according to claim 1 in the prevention and treatment of cotton Verticillium wilt.

9. Use of the insect litter-based myxobacterial fertilizer according to claim 1 in promoting cotton growth and increasing cotton yield.

10. Use of the insect litter-based myxobacterial fertilizer according to claim 1 in the prevention and treatment of wheat stem base rot.

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