Pseudomonas aeruginosa and trichoderma asperellum compound microbial agent as well as preparation method and application thereof

Abstract

The invention relates to a trichoderma asperellum and pseudomonas aeruginosa compound microbial agent as well as a preparation method and application thereof. The compound microbial agent is prepared from a pseudomonas aeruginosa solid fermentation product, a trichoderma asperellum solid fermentation product, an auxiliary agent and a synergist, the number of effective spores in the compound microbial agent is greater than 10 < 10 > CFU / g; the pseudomonas aeruginosa is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2022, and the preservation number of the pseudomonas aeruginosa is CGMCC No.25697; the trichoderma asperellum is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 9, 2023, and the preservation number of the trichoderma asperellum is CGMCC No. 40752. The compound microbial agent can effectively solve the problems of soil-borne diseases such as pepper phytophthora blight and continuous cropping obstacles in the prior art, and has an obvious growth promoting effect on melon and vegetable plants such as pepper.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to a Pseudomonas aeruginosa-Trichoderma aculeatus composite microbial agent and a preparation method and application thereof. Background Art

[0002] Pepper is one of the main vegetable crops grown in greenhouses and open fields. However, with the increase in pepper planting area and the extension of planting years, coupled with the relatively single variety of vegetable planting, changes in climatic conditions, excessive use of fertilizers and pesticides, continuous cropping and other unfavorable factors, soil-borne diseases such as pepper blight and soil continuous cropping obstacles have become more serious, and have become one of the main diseases that seriously affect pepper yield and quality. The pathogen of pepper blight is Phytophthora capsici, which has a wide range of hosts and can infect peppers, tomatoes, cucumbers and other crops. It can survive in the soil for a long time, and when the pathogen accumulates to a certain amount, it can lead to a total crop failure, causing huge economic losses.

[0003] Not only pepper crops, at present, the prevention and control of soil-borne diseases such as pepper blight in production still mainly rely on chemical prevention and control, and the long-term and large-scale use of chemical fungicides has caused many problems such as increased soil and water pollution, excessive residues of agricultural products, and increased disease resistance, resulting in a decline in soil quality and vegetable quality, reduced economic benefits, and a great impact on the soil microecological environment, which seriously affects the sustainable and healthy development of the vegetable industry. Especially in the current era of high-quality agricultural development, the high quality and green development of the vegetable industry are more important. Therefore, microbial pesticides have become the main content of the prevention and control of soil-borne diseases of vegetables such as pepper blight with their unique advantages. Compared with the current use of chemical pesticides for prevention and control, since biocontrol bacteria come from the soil or plant body, they have the advantages of high efficiency, low toxicity, no environmental pollution, and not easy to produce drug resistance, and have broad application prospects.

[0004] Pseudomonas is widely distributed in nature, with a wide variety of species. It is widely distributed in plants and soil. It is the main bacterial group that plays a biological control function around the rhizosphere soil of plants. At present, there have been a large number of reports on the application research of Pseudomonas around the world, involving fields such as agricultural biological control, plant growth regulation, and environmental protection. As one of the widely used biocontrol bacteria, Pseudomonas can inhibit the growth and reproduction of pathogens through parasitism, competition, secretion of secondary metabolites, and induction of plant resistance, thereby reducing the occurrence of diseases, promoting plant growth and development, and the accumulation of products. Its main biological control target is plant pathogenic fungi. At present, the most common and widely used Pseudomonas species are Pseudomonasfluorescens, Pseudomonassyringae, Pseudomonaschlororaphis, Pseudomonasaeeruginosa, etc. In recent years, invention patents related to the application of Pseudomonas aeruginosa in crop biological control are mainly based on single strains (for example, patent number CN202111308578). Moreover, there are few applications in the prevention and control of pepper blight, especially the research and application of composite biocontrol strains with complementary advantages in the prevention and control of pepper blight and other vegetable soil-borne diseases.

[0005] Trichoderma sp. fungi are widely distributed in the world. They are not only one of the important communities of soil microorganisms, but also a kind of endophytic fungi in plants. They can be isolated from soil, plant rhizosphere, stems, leaves, seeds and bulb surfaces, plant residues such as dead branches and fallen leaves, and fruiting bodies of other fungi. Existing research data show that Trichoderma has a wide range of application values ​​in agriculture, industry, environmental protection, etc., and its application in the agricultural field is particularly outstanding. Now, a variety of Trichoderma biocontrol agents, biological fertilizers, inducers, seed coating agents and other products have been registered and commercially produced, and have played an important role in agricultural production. Among them, Trichoderma harzianum is the most common, followed by Trichoderma viride. Compared with soil Trichoderma, the research on endophytic Trichoderma started late, and there are few related reports. However, since endophytic Trichoderma can coexist with host plants, it has obvious advantages in disease resistance, stress resistance, stability, etc., and its own disease resistance and stress resistance are strong, etc. Many advantages have attracted more and more attention from researchers, and the development and application potential is huge. There are relatively many applications of single strains of Trichoderma aculeatus or compounded with different Bacillus to form a microbial agent, but there are few microbial agents compounded with Trichoderma aculeatus and Pseudomonas and their applications (for example, patent No. CN201210365179), and there is a lack of composite microbial agents used in the prevention and treatment of pepper blight. Summary of the invention

[0006] Technical problem to be solved: In view of the shortcomings of existing single biocontrol agents such as low control effect and poor stability against pepper blight, the present invention provides a Pseudomonas aeruginosa-Trichoderma aspergillus composite microbial agent and its preparation method and application. The composite microbial agent can effectively solve the problems of soil-borne diseases such as pepper blight and continuous cropping obstacles existing in the prior art, and has a significant effect on promoting the growth of melon and vegetable plants such as peppers.

[0007] Technical solution: A composite microbial agent of Trichoderma aculeatus and Pseudomonas aeruginosa, comprising the following components by weight:

[0008] Pseudomonas aeruginosa solid fermentation product: 31-42.5 parts,

[0009] Solid fermentation product of Trichoderma aspergillus: 31-42.5 parts,

[0010] Additives: 13-30 parts,

[0011] Synergist: 2-8 parts;

[0012] The effective spore count in the composite microbial agent is > 10 10 CFU / g; the Pseudomonas aeruginosa was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on September 13, 2022, with a deposit number of CGMCC No. 25697; the Trichoderma aspergillus was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on August 9, 2023, with a deposit number of CGMCC No. 40752.

[0013] The auxiliary agent mentioned above is one or both of white carbon black and wheat bran.

[0014] The synergist mentioned above is one or both of chitosan oligosaccharide and biochemical fulvic acid.

[0015] The above-mentioned Pseudomonas aeruginosa and Trichoderma aspergillus composite microbial agent contains the following components by mass: 31 to 42.5 parts of Pseudomonas aeruginosa solid fermentation product, 31 to 42.5 parts of Trichoderma aspergillus solid fermentation product, 3 to 10 parts of white carbon black, 10 to 20 parts of wheat bran, 1 to 3 parts of chitosan oligosaccharide, and 1 to 5 parts of biochemical fulvic acid.

[0016] The above-mentioned Pseudomonas aeruginosa and Trichoderma aspergillus composite microbial agent contains the following components by mass: 36.5 parts of Pseudomonas aeruginosa solid fermentation product, 36.5 parts of Trichoderma aspergillus solid fermentation product, 8 parts of white carbon black, 12 parts of wheat bran, 3 parts of chitosan oligosaccharide, and 4 parts of biochemical fulvic acid.

[0017] The preparation method of the above-mentioned Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent comprises the following steps:

[0018] Step 1: Preparation of solid fermentation products of Pseudomonas aeruginosa and Trichoderma aspergillus:

[0019] 1) taking out the strains of Pseudomonas aeruginosa and Trichoderma aspergillus stored at -80°C, and preparing Pseudomonas aeruginosa seed fermentation liquid and Trichoderma aspergillus seed fermentation liquid respectively,

[0020] 2) wheat bran, corn cob powder, corn stalk powder and water are mixed in a weight ratio of 15:9:6:70, potassium dihydrogen phosphate is added, and sterilized at 121° C. for 40 min to obtain a solid fermentation medium, wherein the weight of potassium dihydrogen phosphate is 1% of the total weight of the wheat bran, corn cob powder, corn stalk powder and water.

[0021] 3) Mix the Pseudomonas aeruginosa seed fermentation liquid and the solid fermentation medium in a weight ratio of 1:7.5, stir evenly, cover with sterilized paper, and culture in an artificial climate box at a temperature of 32° C. and a relative humidity of more than 85% for 3 days, stirring 1 to 2 times during the period. After the fermentation is completed, dry it in the air or dry it below 32° C. to obtain the Pseudomonas aeruginosa solid fermentation product, and store it in a cool place for later use.

[0022] 4) mixing the fermented liquid of Trichoderma aspergillus seeds with the solid fermentation medium at a weight ratio of 1:7.5, stirring evenly, covering with sterilized paper, and culturing in an artificial climate box at a temperature of 28° C. and a relative humidity of more than 85% for 5 days, stirring 1 to 2 times during the period, and drying in the air or at a temperature below 32° C. after the fermentation is completed to obtain a solid fermented material of Trichoderma aspergillus, which is stored in a cool place for later use;

[0023] Step 2: Preparation of composite microbial agent:

[0024] The solid fermentation product of Pseudomonas aeruginosa and the solid fermentation product of Trichoderma aculeatus are mixed, and then dry-mixed with white carbon black, wheat bran, chitosan oligosaccharide, and biochemical fulvic acid, wherein the wheat bran is pre-crushed with an ultra-fine crusher and passed through a 100-mesh sieve, and the solid powder is obtained after being fully stirred. The effective spore number is measured to be >10 10 CFU / g, and you get the composite microbial agent.

[0025] The above-mentioned Pseudomonas aeruginosa and Trichoderma aspergillus composite microbial agent is used in preventing and controlling pepper blight and watermelon wilt.

[0026] Beneficial effects: The Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent provided by the present invention and its preparation method and application have the following beneficial effects:

[0027] 1. The Pseudomonas aeruginosa and Trichoderma aculeatus strains of the present invention have large spore production, especially Pseudomonas aeruginosa S11, which has a spore production of 10 14 cfu / ml or above, and have strong abilities in degrading cellulose, fixing nitrogen and dissolving potassium, enduring high temperature, salt and alkali, and resisting ultraviolet light. Trichoderma can form a symbiotic relationship with the host in the plant body. The two biocontrol bacteria synergize through multiple mechanisms such as competition, heavy parasitism, secretion of secondary metabolites and induction of plant resistance, which can make up for the limitations of the use of a single bacterial agent.

[0028] 2. The composite microbial agent of the present invention has obvious effects on preventing and promoting the growth of pepper blight, and also has good inhibitory effects on pathogens of vegetable wilt, damping-off, early blight, late blight, sclerotinia and other diseases. It is a broad-spectrum biological fungicide with strong stability. It can not only prevent diseases and promote growth, but also cause a large number of beneficial microorganisms in the rhizosphere soil of crops to proliferate, thereby improving the soil microecological environment.

[0029] 3. The auxiliary agents of the composite microbial agent of the present invention are white carbon black and wheat bran, and the synergists are chitosan oligosaccharide and biochemical fulvic acid, wherein the synergists can induce crops to improve disease resistance and improve soil.

[0030] 4. The composite microbial agent of the present invention is pollution-free, harmless, simple to use, and low-priced. After application, it can reduce the use of chemical pesticides and drug resistance, significantly reduce the occurrence of diseases, achieve the purpose of improving quality and efficiency, promote the green development of the vegetable industry, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Phylogenetic tree for molecular species identification of bacterial strain S11.

[0032] Figure 2 Phylogenetic tree for molecular species identification of Trichoderma strain 1.

[0033] Figure 3 This is a bar graph showing the disease prevention and yield-increasing effects of the Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0036] The separation and identification process of the biocontrol bacteria of the present invention is as follows:

[0037] A bacterial strain S11 was isolated from vegetable soil by the dilution plate smear method, and a Trichoderma strain 1 was isolated from tomato plants by the conventional tissue isolation method. They were identified as Pseudomonas aeruginosa and Trichderma asperellum, respectively. The phylogenetic tree analysis of molecular identification is shown in the following table. Figure 1 , Figure 2 shown.

[0038] The two strains were deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on September 13, 2022 and August 9, 2023, respectively, and the deposit numbers were CGMCC No. 25697 and CGMCC No. 40752, respectively. They are Pseudomonas aeruginosa and Trichderma asperellum.

[0039] The preparation method of a Trichoderma aculeatus-Pseudomonas aeruginosa composite microbial agent of the present invention comprises the following steps:

[0040] (1) Preparation of expanded cultures of Pseudomonas aeruginosa and Trichoderma aculeatus

[0041] The Pseudomonas aeruginosa and Trichoderma aspergillus biocontrol strains stored at -80°C were taken out, inoculated into LB and PDA solid culture media respectively, and placed in a 28°C biochemical incubator for activation. Pseudomonas aeruginosa was cultured for 2 to 3 days, and Trichoderma aspergillus was cultured for 3 to 5 days, and then Pseudomonas aeruginosa and Trichoderma aspergillus expanded culture strains could be collected.

[0042] (2) Preparation of Pseudomonas aeruginosa seed fermentation broth

[0043] Mix 10g of peptone, 5g of yeast powder and 10g of sodium chloride, then add 1000mL of water to prepare a fermentation culture medium, and put them into 300mL Erlenmeyer flasks, with each flask containing 150mL of liquid, to make LB solution; after sterilization at 121℃ for 30min, break the expanded culture strains of Pseudomonas aeruginosa into 5mm blocks and inoculate them into 150mL of culture medium, and culture them at 32℃ and 180r / min for about 2 to 3 days to obtain Pseudomonas aeruginosa seed fermentation liquid. (3) Preparation of Trichoderma aeruginosa seed fermentation liquid

[0044] 200 g of peeled potatoes were mixed with 20 g of glucose, and then 1000 mL of water was added, and the pH value was adjusted to 6 to prepare a fermentation culture solution, which was respectively filled into 300 mL triangular flasks, with each bottle having a liquid volume of 150 mL to prepare a PD solution; after sterilization at 121° C. for 30 min, the expanded culture strains of Trichoderma asperellum were respectively beaten into 5 mm blocks and inoculated into 150 mL of culture solution, and shaken and cultured at 28° C. and 180 r / min for about 3 to 5 days to obtain Trichoderma asperellum seed fermentation solution.

[0045] (4) Preparation of solid fermentation medium

[0046] Wheat bran, corn cob powder, corn stalk powder and water are mixed in a weight ratio of 15:9:6:70, potassium dihydrogen phosphate is added, and the mixture is sterilized at 121° C. for 40 minutes to obtain a solid fermentation medium, wherein the weight of the potassium dihydrogen phosphate is 1% of the total weight of the wheat bran, corn cob powder, corn stalk powder and water.

[0047] (5) Preparation of solid fermentation products of Pseudomonas aeruginosa

[0048] The Pseudomonas aeruginosa seed fermentation liquid and the solid fermentation medium are mixed in a weight ratio of 1:7.5, stirred evenly, covered with sterilized paper, and cultured in an artificial climate box with a temperature of 32° C. and a relative humidity of more than 85% for 3 days, stirred 1 to 2 times during the period, and after the fermentation is completed, dried in the air or dried below 32° C. to obtain the Pseudomonas aeruginosa solid fermentation product, which is stored in a cool place for later use.

[0049] (6) Preparation of Trichoderma spinulosa solid fermentation product

[0050] The fermentation liquid of Trichoderma aspergillus seeds and the solid fermentation medium are mixed in a weight ratio of 1:7.5, stirred evenly and covered with sterilized paper, and cultured in an artificial climate box with a temperature of 28°C and a relative humidity of more than 85% for 5 days, stirred 1 to 2 times during the period, and after the fermentation is completed, dried in the air or dried below 32°C to obtain the solid fermentation product of Trichoderma aspergillus, which is stored in a cool place for later use.

[0051] (7) Preparation of composite microbial agents

[0052] The solid fermentation product of Pseudomonas aeruginosa and the solid fermentation product of Trichoderma aculeatus are mixed in a weight ratio of 1:1, and then the mixed solid fermentation product is dry-mixed with white carbon black, wheat bran, chitosan oligosaccharide, and biochemical fulvic acid in a weight ratio of 73:8:12:3:4, wherein the wheat bran is pre-crushed with an ultra-fine crusher and passed through a 100-mesh sieve, and the solid powder is obtained after being fully stirred. The effective spore number of the solid powder is measured to be >10 10 CFU / g (i.e., conidia and spore content is 10 10 / g or more), and a composite microbial agent is obtained.

[0053] The performance of the Pseudomonas aeruginosa / Trichoderma aculeatus composite microbial agent of the present invention was tested in the following experiments, wherein both the indoor experiment and the potted plant experiment were tested with the fermentation liquid of the biocontrol bacteria seeds, and the field experiment was conducted with the Pseudomonas aeruginosa·Trichoderma aculeatus composite microbial agent prepared by the present invention. The process and results are as follows.

[0054] 1. Experimental effect of Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent

[0055] 1.1 Determination of the antibacterial effects of different biocontrol bacteria and their combinations

[0056] The antibacterial effects of different biocontrol bacteria and their combinations on various soil-borne pathogens are shown in Table 1.

[0057] Table 1

[0058]

[0059] The antibacterial effect of different biocontrol strains was determined after fermentation alone or in combination. The results in Table 1 show that different biocontrol strains and their combinations showed significant antibacterial effects on the four soil-borne pathogens of melon and vegetable diseases, with an inhibition rate of 70.7% to 88.0%. Among them, the antibacterial effect of the fermentation liquid of the composite strain was the best, which was higher than the antibacterial effect of a single strain, reaching 77.8% to 88.0%. The inhibition rate of the fungus against pepper blight was as high as 88.0%, and it also showed significant antibacterial effects on other soil-borne pathogens, with a broad spectrum of antibacterial properties.

[0060] 1.2 Determination of the antibacterial spectrum of different biocontrol bacteria

[0061] The broad-spectrum antibacterial activity of different biocontrol bacteria against various leaf diseases of vegetables is shown in Table 2.

[0062] Table 2

[0063]

[0064] Seven leaf disease pathogens, including gray mold of vegetables (cucumber, tomato, pepper), early blight of tomato, and leaf mold, which are currently the most serious in production, were selected as target strains, and the inhibitory effects of biocontrol bacteria on different pathogens were determined by plate confrontation culture method and growth rate method. The results in Table 2 show that different biocontrol bacteria exhibited broad-spectrum antibacterial activity, with significant antagonistic effects, and the antibacterial rate of fermentation liquid reached 73.8%-85.2%, indicating that the composite microbial agent has good broad-spectrum antibacterial activity.

[0065] 1.3 Compatibility determination of different biocontrol strains

[0066] The compatibility between different biocontrol strains is shown in Table 3.

[0067] Table 3

[0068] Different biocontrol strains Pseudomonas aeruginosa Trichoderma acanthosporum Pseudomonas aeruginosa / completely compatible Trichoderma acanthosporum completely compatible /

[0069] The compatibility of different biocontrol strains was determined by the cross-streaking method. The results in Table 3 show that the biocontrol bacteria and Trichoderma strains are easily compatible and have good compatibility, and can be used as alternative strains for composite microbial agents.

[0070] 1.4 Determination of disease prevention and growth promotion effects of potted pepper

[0071] Long flower pots (50 cm × 20 cm) were used to fill the pots with normal field soil. Three treatments were set up with different biocontrol solutions and combinations. Each treatment was repeated 3 times. Ten pepper seedlings were transplanted in each pot, and no biocontrol agent (clear water) was set as a control. The transplanted pepper seedlings were treated by root dipping and root irrigation. 2 days after transplanting, the roots were inoculated with pepper blight pathogens by root irrigation. Normal management was carried out, the growth of peppers was observed regularly, the incidence rate was investigated, and the control effect was calculated. Normal soil pots were used to plant peppers, and the growth-promoting effects of different treatments and the control pepper were measured 30 days later. The treatments were the same as the disease control experiment. The results are shown in Table 4.

[0072] Table 4

[0073]

[0074] The test results in Table 4 show that the control efficacy of different biocontrol liquids and combinations against pepper blight is 66.7% to 80.0%, which has a good biocontrol potential, and has different degrees of growth-promoting effects on pepper seedlings, with the seedling height increased by up to 8.0 cm, the root length increased by up to 2.7 cm, the stem diameter increased by up to 0.2 cm, the fresh weight increased by up to 4.5 g / plant, and the dry weight increased by up to 1.9 g / plant.

[0075] 1.5 Field effect evaluation of composite microbial agents

[0076] The composite microbial agent (S33) of Pseudomonas aeruginosa and Trichoderma aculeatus of the present invention is used to regularly apply the drug by combining root dipping and root irrigation. In a field plot test, the composite microbial agent is used as the test treatment, and the biological agent Paenibacillus polymyxa WP, the chemical agent 99% oxadipate WP and no drug application (clear water) are used as controls. Each treatment is repeated 3 times, and the random block group is arranged. After the pepper seedlings are transplanted by root dipping, they are managed normally. Root irrigation is carried out after the plant growth slowing period, and the root irrigation is carried out 3 times during the growth period, and the interval between drug applications is 7 to 20 days.

[0077] The effects of compound microbial agents on pepper growth, disease prevention and control, and yield - Yinchuan Observatory are shown in Table 5. The effects of compound microbial agents on the microbial flora of pepper rhizosphere soil are shown in Table 6.

[0078] Table 5

[0079]

[0080]

[0081] Table 6

[0082]

[0083] The results of Tables 5 and 6 show that after the composite microbial agent of the present invention was used to dip the roots and regularly irrigate the roots for three times, the pepper plants showed obvious disease prevention, growth promotion and yield increase effects, with a field prevention effect of 72.5% on pepper blight and a yield increase rate of 16.0%. The disease prevention effect and yield increase rate were higher than those of the biological control agent Bacillus polymyxa and the chemical control agent oxadipazole WP, and the plant height increased by 7.33 cm and the stem diameter increased by 0.42 cm compared with the clear water control. After treatment with the composite microbial agent, the number of soil bacteria and actinomycetes increased by 3.33×10 6 cfu / g, 1.66×10 5 cfu / g, indicating that the number of beneficial bacteria in the soil has increased significantly and the soil microecological environment has been significantly improved.

[0084] The effects of compound microbial agents on watermelon growth, wilt disease prevention and control, and yield - Yinchuan Observation Station are shown in Table 7. The effects of compound microbial agents on the microbial flora of watermelon rhizosphere soil are shown in Table 8.

[0085] Table 7

[0086]

[0087] Table 8

[0088]

[0089]

[0090] The results of Tables 7 and 8 show that after the composite microbial agent of the present invention was used to dip the roots and regularly irrigate the roots for three times, the watermelon plants showed obvious disease prevention and growth promotion effects, with a field control effect of 75.4% on watermelon wilt and an increase in yield of 11.48%. The disease prevention effect and increase in yield were higher than those of the biological control agent Bacillus polymyxa and the chemical control agent oxadipazole WP. The longest vine length of the watermelon increased by 39.6 cm and the number of leaves increased by 2 compared with the clear water control. After the composite microbial agent was treated, the number of soil bacteria increased by an average of 44.5×10 6 cfu / g, the number of soil actinomycetes increased by 1.5×10 5cfu / g, indicating that the number of beneficial bacteria in the soil has increased significantly and the soil microecological environment has been significantly improved. The field test results also show that the composite microbial agent of the present invention is a broad-spectrum biofungicide.

[0091] 1.6 Field demonstration effect determination of composite microbial agents

[0092] The technology of the present invention has been maturely developed. The composite microbial agent has been demonstrated in the field on greenhouse peppers and open-field watermelons planted in Litong District, Wuzhong City, Ningxia and Xiangshan Township, Shapotou District, Zhongwei City from 2022 to 2023. The disease prevention and growth promotion effects are obvious, and the soil microbial environment has been significantly improved.

[0093] The effects of composite microbial agents on pepper growth, disease occurrence and yield in Wuzhong Litong District are shown in Table 9. The bar chart of disease prevention and yield increase effect is shown in Figure 3.

[0094] Table 9

[0095]

[0096] The effect of composite microbial agents was verified by demonstration on peppers in the arch shed of Litong District, Wuzhong, Ningxia. The composite agent of Pseudomonas aeruginosa and Trichoderma aculeatus was used as the treatment. The demonstration area was 440m 2 , and set up a conventional control area. The application method is a combination of root dipping and root irrigation. The pepper seedlings are transplanted and managed normally. The roots are irrigated three times during the growth period, and the interval between medications is 7 to 20 days. Table 9 and Figure 3 The results showed that after applying the composite microbial agent, the height of the treated peppers in the demonstration area increased by 11.5 cm, the stem diameter increased by 0.09 cm, the length of the peppers increased by 2.6 cm, and the width of the peppers increased by 1.6 cm, showing a significant growth-promoting effect. The prevention effect on pepper blight reached 78.0%, and the yield increase rate was 15.2%.

[0097] The effects of composite microbial agents on watermelon growth, wilt occurrence and yield in Shapotou District, Zhongwei, are shown in Table 10 below.

[0098] Table 10

[0099]

[0100] The composite microbial agent was used to demonstrate the effect of watermelon in the open field in Shapotou District, Zhongwei City, Ningxia. The composite agent of Pseudomonas aeruginosa and Trichoderma aculeatus was used as the treatment. The demonstration area was 667m 2, and a conventional control area was set up. The method of applying pesticides was a combination of root dipping and root irrigation. The pepper seedlings were transplanted and managed normally. The roots were irrigated three times during the growth period, and the interval between medications was 7 to 20 days. The results of Table 10 showed that after the application of the composite microbial agent, the longest vine length of the watermelon in the demonstration area increased by 44.5 cm, the number of leaves increased by 3 on average, and the number of branches increased by 2, showing a significant growth-promoting effect. The prevention effect on watermelon wilt reached 75.8%, and the yield increase rate was 26.4%.

[0101] 2. Determination of the multifunctionality of different biocontrol strains

[0102] 2.1 Qualitative detection of relevant biocontrol active substances produced by different biocontrol bacteria

[0103] Qualitative detection of relevant biocontrol active substances produced by different biocontrol bacteria is shown in Table 11 below.

[0104] Table 11

[0105]

[0106] Note: “+” means the result was detected, and “-” means it was not detected.

[0107] Through qualitative detection of related biocontrol active substances produced by different biocontrol strains, the results in Table 11 showed that both biocontrol strains produced protease, amylase, cellulase, siderophore and auxin, but did not produce glucanase.

[0108] The effects of the composite microbial agent treatment on the activities of different defense enzymes in the rhizosphere soil and plants of pepper are shown in Table 12 below.

[0109] Table 12

[0110]

[0111]

[0112] Induced systemic resistance is a potential mechanism for biocontrol microorganisms to control plant diseases. PO, PPO and SOD were used as evaluation indicators to determine the induced defense response of pepper plants and rhizosphere soil by applying composite microbial agents. The results in Table 12 showed that compared with the conventional control, the composite microbial agent of Pseudomonas aeruginosa and Trichoderma aculeatus could increase the activities of urease, alkaline phosphatase, sucrase, cellulase in the rhizosphere soil of pepper and phenylalanine ammonia lyase, superoxide dismutase and chitinase in pepper plants to varying degrees, indicating that the composite microbial agent has an induced defense response to pepper and can induce disease resistance in the crop itself.

[0113] 2.2 Determination of nitrogen-fixing, phosphorus-dissolving and potassium-dissolving functions of composite microbial agents

[0114] The effects of composite microbial agent treatment on the available nitrogen, phosphorus and potassium in the soil are shown in Table 13 below.

[0115] Table 13

[0116] deal with Available nitrogen mg / kg Available phosphorus mg / kg Available potassium mg / kg Pseudomonas aeruginosa and Trichoderma aculeatus 199 140 530 Conventional CK 120 105 200

[0117] The determination of nitrogen fixation, phosphorus solubility and potassium solubility of indoor biocontrol bacteria showed that the three biocontrol bacteria not only have disease prevention and growth promotion effects, but also have nitrogen fixation, phosphorus solubility and potassium solubility functions to varying degrees. The results of field effect verification in Table 13 show that compared with the conventional control, the composite microbial agent can increase the available nitrogen in the soil by 79 mg / kg, the available phosphorus by 35 mg / kg, and the available potassium by 330 mg / kg, indicating that the composite microbial agent has good nitrogen fixation and potassium solubility functions and a certain phosphorus solubility effect.

[0118] As can be seen from the above, the Pseudomonas aeruginosa-Trichoderma aspergillus composite microbial agent of the present invention can reproduce rapidly in pepper plants and in the soil after application, effectively inhibits the number of pepper blight pathogens through multiple action mechanisms such as competition, hyperparasitism and antibiotics, and prevents multiple vegetable diseases, has strong stability, and can more effectively prevent and control pepper blight than the single biocontrol strain in the prior art, regulate the soil microbial flora, and make the soil microecological environment develop in a favorable direction; the two added synergists can induce crops to improve disease resistance, improve soil, and promote crop growth and development; the product is pollution-free, pollution-free, simple to use, low-priced, and has significant effects on disease prevention, growth promotion and yield increase, can achieve the purpose of improving quality and efficiency, promote the green development of the vegetable industry, and is suitable for popularization and application.

[0119] The embodiments of the present invention are described in detail above. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A composite microbial agent of Trichoderma aculeatus and Pseudomonas aeruginosa, Features Contains the following ingredients by mass: Pseudomonas aeruginosa solid fermentation product: 31-42.5 parts, Solid fermentation product of Trichoderma aspergillus: 31-42.5 parts, Additives: 13-30 parts, Synergist: 2-8 parts; The effective spore count in the composite microbial agent is > 10 10 CFU / g; the Pseudomonas aeruginosa was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on September 13, 2022, with a deposit number of CGMCC No. 25697; the Trichoderma aspergillus was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on August 9, 2023, with a deposit number of CGMCC No. 40752.

2. The Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent according to claim 1, Features: The auxiliary agent is one or two of white carbon black and wheat bran.

3. The Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent according to claim 2, Features: The synergist is one or two of chitosan oligosaccharide and biochemical fulvic acid.

4. The Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent according to claim 3, Features The invention contains the following ingredients by weight: 31 to 42.5 parts of solid fermentation product of Pseudomonas aeruginosa, 31 to 42.5 parts of solid fermentation product of Trichoderma aspergillus, 3 to 10 parts of white carbon black, 10 to 20 parts of wheat bran, 1 to 3 parts of chitosan oligosaccharide and 1 to 5 parts of biochemical fulvic acid.

5. The Pseudomonas aeruginosa and Trichoderma aculeatus composite microbial agent according to claim 4, Features The composition comprises the following components in parts by mass: 36.5 parts of solid fermentation product of Pseudomonas aeruginosa, 36.5 parts of solid fermentation product of Trichoderma aspergillus, 8 parts of white carbon black, 12 parts of wheat bran, 3 parts of chitosan oligosaccharide and 4 parts of biochemical fulvic acid.

6. A method for preparing a composite microbial agent of Pseudomonas aeruginosa and Trichoderma as claimed in any one of claims 1 to 5, Features The steps of this method are as follows: Step 1: Preparation of solid fermentation products of Pseudomonas aeruginosa and Trichoderma aspergillus: 1) The strains of Pseudomonas aeruginosa and Trichoderma aspergillus stored at -80°C were taken out to prepare Pseudomonas aeruginosa seed fermentation liquid and Trichoderma aspergillus seed fermentation liquid respectively. 2) Mix wheat bran, corn cob powder, corn stalk powder and water in a weight ratio of 15:9:6:70, add potassium dihydrogen phosphate, and sterilize at 121°C for 40 minutes to obtain a solid fermentation medium, wherein the weight of potassium dihydrogen phosphate is 1% of the total weight of wheat bran, corn cob powder, corn stalk powder and water. 3) Mix the Pseudomonas aeruginosa seed fermentation liquid and the solid fermentation medium at a weight ratio of 1:7.5, stir evenly, cover with sterilized paper, and culture in an artificial climate box at a temperature of 32°C and a relative humidity of more than 85% for 3 days, stirring 1 to 2 times during the period. After the fermentation is completed, dry it in the air or dry it below 32°C to obtain the solid fermentation product of Pseudomonas aeruginosa, and store it in a cool place for later use. 4) Mix the fermentation liquid of Trichoderma aspergillus seeds with the solid fermentation medium at a weight ratio of 1:7.5, stir evenly, cover with sterilized paper, and culture in an artificial climate box at a temperature of 28°C and a relative humidity of more than 85% for 5 days, stirring 1 to 2 times during the period. After the fermentation is completed, dry it in the air or dry it below 32°C to obtain the solid fermentation product of Trichoderma aspergillus, and store it in a cool place for later use; Step 2: Preparation of composite microbial agent: The solid fermentation product of Pseudomonas aeruginosa and the solid fermentation product of Trichoderma aculeatus are mixed, and then dry-mixed with white carbon black, wheat bran, chitosan oligosaccharide, and biochemical fulvic acid, wherein the wheat bran is pre-crushed with an ultra-fine crusher and passed through a 100-mesh sieve, and the solid powder is obtained after being fully stirred. The effective spore number is measured to be >10 10 CFU / g, and you get the composite microbial agent.

7. Use of the composite microbial agent of Pseudomonas aeruginosa and Trichoderma as claimed in claim 1 in preventing and controlling pepper blight and watermelon wilt.

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