Compound microbial agent and application thereof in crop disease control

Abstract

The invention belongs to the technical field of microorganisms, and discloses a compound microbial agent and application thereof in crop disease control. According to the invention, two plant endophytes, namely trichoderma asperellum and trichoderma atroviride, are separated, the trichoderma asperellum is Trichoderma asperellum M11, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.40752; the trichoderma atroviride is Trichoderma atroviride T5, and the preservation number of the trichoderma atroviride is CGMCC (China General Microbiological Culture Collection Center) No. 22432. Fermentation liquor of two trichoderma strains and auxiliaries are prepared into the compound microbial agent, the compound microbial agent has excellent prevention and treatment effects on pepper phytophthora blight, cucumber fusarium wilt, watermelon fusarium wilt, cucumber damping off, tomato sesame spot, tomato early blight and tomato leaf mold, in addition, the compound microbial agent can promote growth and increase yield, the disease resistance of plants is improved, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to biological control, and in particular to a composite microbial agent and its application in crop disease control. Background Art

[0002] At present, the prevention and control of vegetable diseases still relies on chemical control. The long-term and large-scale use of chemical agents has caused many problems such as increased soil and water pollution, pesticide residues, and increased disease resistance, which has seriously affected the sustainable development of the vegetable industry. Therefore, it is particularly important to seek alternative methods of chemical control.

[0003] Biological control has become a promising method for crop pest control due to its unique advantages. Compared with chemical pesticide control, it has the advantages of high efficiency, low toxicity, no environmental pollution and low resistance to pesticides, and has broad application prospects. In the research and application of biological control bacteria for the prevention and control of vegetable diseases, Trichoderma is the most common. Trichoderma Pers.) Fungi are widely distributed and can be isolated from soil, plant stems, leaves, seeds and bulb surfaces, and even from plant residues such as dead branches and fallen leaves, and from the fruiting bodies of other fungi. Trichoderma has a wide range of application value in agriculture and environmental protection.

[0004] As an important green control method, biological control aims to discover and screen beneficial microorganisms and make microbial agents to prevent the occurrence of plant diseases and insect pests. This is a new idea and method for future disease and insect pest control. However, most of the current research on microbial agents is in the laboratory research and development stage. Many biocontrol strains that performed well in preliminary tests often face complex and changeable temperature and humidity environments in actual application, and crops often face more than one disease, which increases the difficulty of practical application and promotion of agents. Therefore, it is very necessary to develop composite agents that can play an excellent prevention and control role in the field. Summary of the invention

[0005] In order to enrich the types of existing biocontrol bacteria and achieve efficient prevention and control of diseases of melons and vegetables during field planting, the present invention provides a composite microbial agent and its application in crop disease prevention and control.

[0006] First, the present invention provides a composite microbial agent, wherein the active ingredients of the composite microbial agent are Trichoderma aculeatus and Trichoderma aureum. Trichoderma asperellum M11, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.40752. Trichoderma atrovirideT5, deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No. 22432. The total spore count of Trichoderma spinulosum and Trichoderma aureum in the composite microbial inoculant is not less than 10 10 Pieces / g.

[0007] Furthermore, the components of the composite microbial agent include: by mass, 73 parts of microorganisms and their fermentation products, 3-10 parts of white carbon black, 10-20 parts of wheat bran, 1-3 parts of chitosan oligosaccharides, and 1-5 parts of biochemical fulvic acid. Trichoderma asperellum Solid fermentation product of M11 and Trichoderma aureum Trichoderma atroviride The solid fermentation product of T5 is mixed in a mass ratio of 1:1. The solid fermentation product is mixed and fermented by seed fermentation liquid and solid fermentation medium in a mass ratio of 1:7.5. The solid fermentation medium is mixed and sterilized by wheat bran, corn flour, bran and water in a mass ratio of 9:6:15:70.

[0008] Furthermore, the components of the composite microbial agent include: 73 parts of the microorganisms and their fermentation products, 8 parts of white carbon black, 12 parts of wheat bran, 3 parts of chitosan oligosaccharides, and 4 parts of biochemical fulvic acid.

[0009] Furthermore, the composite microbial agent prevents and controls diseases caused by fungi.

[0010] Specifically, the diseases include: pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight, and tomato leaf mold.

[0011] On the other hand, the present invention also provides the application of the composite microbial agent in crop disease prevention and control, specifically, the composite microbial agent is used to prevent and control pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight, and tomato leaf mold.

[0012] Secondly, the present invention also requests protection for the use of the composite microbial agent in improving the disease resistance of crops, the composite microbial agent has the effect of increasing the content of catalase, superoxide dismutase, and chitinase in plants; the composite microbial agent has the effect of increasing the content of urease, alkaline phosphatase, and sucrase in the rhizosphere soil of plants.

[0013] Finally, the present invention also provides a method for preventing and controlling crop diseases, which comprises: diluting the composite microbial agent by 50 times with water, dipping the agent into the roots of transplanted crop seedlings, and irrigating the roots three times during the crop growth period, using a 100-fold diluted solution, and the interval between medications is 7 to 20 days.

[0014] In the above prevention and control method, the crop diseases include: pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight, and tomato leaf mold.

[0015] Compared with the prior art, the present invention "a composite microbial agent and its application in crop disease prevention and control" has the following beneficial effects: 1) Compatibility: Trichoderma aspergillus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 has good compatibility and can be used as an alternative strain for composite microbial agents.

[0016] 2) Broad spectrum of antibacterial activity: In the plate confrontation test and plate punch test, Trichoderma spinulosa Trichoderma asperellum M11, Trichoderma cyanobacteria Trichoderma atroviride T5 and the combination of the two have significant antagonistic effects on seven pathogens including pepper blight and cucumber wilt, and have a broad spectrum of antibacterial properties.

[0017] 3) Promoting growth and increasing yield: The application of different bacterial solutions and composite microbial solutions has different degrees of growth-promoting effects on pepper, tomato, cucumber and watermelon plants, and the growth-promoting effect of the composite microbial solution is better than that of the solution prepared by a single strain.

[0018] 4) Improving soil microorganisms: Using Trichoderma aculeatus Trichoderma asperellum M11, Trichoderma cyanobacteria Trichoderma atroviride T5 or a combination of the two can help enrich the beneficial bacteria in the soil, improve the soil environment, and enhance the plant's disease resistance.

[0019] 5) Improving plant disease resistance: The composite microbial agent can increase the activities of urease (UE), alkaline phosphatase (ALP), sucrase (SC) in the rhizosphere soil of pepper and catalase (CAT), superoxide dismutase (SOD), and chitinase in pepper plants to varying degrees, indicating that the application of the composite microbial agent has an inducing defense response effect on the plants and can induce the crops themselves to produce disease resistance.

[0020] 6) Adaptability: The composite microbial agent has strong environmental adaptability, excellent field application effect, and has shown excellent disease prevention and control effects in different plots, and has a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Trichoderma acanthosporum Trichoderma asperellum Identification map of M11.

[0022] Figure 2 Trichoderma aureum Trichoderma atroviride Identification map of T5. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 This example describes Trichoderma Trichoderma asperellum M11, Trichoderma cyanobacteria Trichoderma atroviride Isolation and identification of T5.

[0025] 1) Obtaining strains: First, wash the collected tomato and pepper plant samples with tap water and dry them. Then, cut appropriate fragments from different parts of the tissues, soak them in 75% alcohol for 60 seconds, then treat them with 10% sodium hypochlorite solution for 3-5 minutes, and then soak them in 75% alcohol for 30 seconds. The tissue blocks that have been surface disinfected are washed three times with sterile water. The washed different tissues are placed in a sterilized mortar for grinding, and 0.2 mL of juice is taken on the Trichoderma selective culture medium, and evenly spread with a smear stick. After repeating the above steps three times, the culture dish is inverted and cultured in a 25°C constant temperature box. When Trichoderma colonies appear on the plate, they are picked out, further purified, and stored at ultra-low temperatures.

[0026] 2) Morphological identification of Trichoderma: Different Trichoderma strains with obvious differences in colony morphology were selected from each isolated sample for single spore isolation, and then inoculated onto PDA medium. They were cultured at 25°C in the dark for 72 h until mature colonies grew out. The external morphology and color of the colonies were observed, and samples were selected for preparation. The morphological characteristics of the conidiophores of Trichoderma and the size, shape and color of the conidia were observed under an optical microscope. The Trichoderma population classification system revised by Rifai and Bissett (1984), the Manual of Identification of Fungi by Wei Jingchao, and the descriptions and retrieval table classification standards of Wen Chengjing (1993), Zhang Chulong (2005), Sun Jun (2006), Lu Suyun, Dai Fanglan, etc. were used to conduct morphological identification of each Trichoderma strain to clarify its taxonomic status.

[0027] After morphological identification, two Trichoderma strains with obviously different colony morphologies were obtained, one of which had the following colony morphology: the mycelium was initially white on PDA medium, and dark green when the colony was mature, cotton-like or felt-like, thick, colorless or cream-colored on the back, and composed of dense conidia; the phialides on the conidiophores were symmetrically distributed, with two or more phialides at the top, the primary branches generated below the top of the main axis were often opposite, and the angle between the main axis and the phialides was nearly right angles, the phialides were typically generated at the top of the primary, secondary, and tertiary branches, 2 to 3 phialides were arranged in a spiral shape, the phialides were straight and ampoule-shaped, and only slightly thickened in the middle; the conidia were spherical or oval, with a size of 3.7~6.0μm×3.0~5.0μm, smooth, and light green; no diffusible pigment was produced, and there was no odor.

[0028] The colony morphology of the other strain is as follows: the colony grows fast on PDA medium, white at the beginning, dark green and cotton-like when mature, and light yellow or yellow-green on the reverse side; the middle of the colony is a dense spore-producing area (dense disc-like structure), which is the main production area of ​​conidia, and the hyphae gradually become sparse toward the outer edge, and the edge of the colony is dense and felt-like; the main branches of the conidiophores are slightly wavy, and the secondary branches are born at unequal intervals, extending at right angles, often solitary or intercalary, rarely in pairs or branched again; most of the phialides are solitary on the conidiophores, often bending toward the top; the conidia are dark green, subspherical or oval, with a smooth surface, and a size of 2.8~3.5μm×2.5~3.8μm; they produce a coconut smell and no exudate. According to the above identification manual, the two strains of Trichoderma selected are preliminarily judged to be Trichoderma acanthosporum and Trichoderma aureum.

[0029] 3) Molecular biological identification: A molecular biological method combining ITS-rDNA with Tef1-α and rpb2 was used to identify morphologically similar Trichoderma. The internal transcribed spacer (ITS) sequence of rDNA was amplified using PCR technology, and sequence similarity search was performed using the oligonucleotide barcode program TrichOKey 2.0 and the Trichoderma database TrichoBlast. Strains with sequence similarity ≥ 99% were identified as the same species. The two strains of Trichoderma selected were Trichoderma acanthosporum and Trichoderma aureum, and their molecular identification maps were as follows: Figure 1 , Figure 2 Trichoderma acanthosporum Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5.

[0030] Trichoderma acanthosporum Trichoderma asperellum The M11 collection information is as follows: Species name: Trichoderma aculeatus.

[0031] Latin name: Trichoderma asperellum .

[0032] Strain number: M11.

[0033] Depository: General Microbiology Center, China Culture Collection Administration.

[0034] The abbreviation of the collection agency is: CGMCC.

[0035] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] Date of preservation: August 9, 2023.

[0037] Deposit number: CGMCC No.40752.

[0038] Trichoderma cyanobacteria Trichoderma atroviride T5 deposit information is as follows: Species name: dark green Trichoderma.

[0039] Latin name: Trichoderma atroviride .

[0040] Strain number: T5.

[0041] Depository: General Microbiology Center, China Culture Collection Administration.

[0042] The abbreviation of the collection agency is: CGMCC.

[0043] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Date of preservation: May 26, 2021.

[0045] Deposit number: CGMCC No.22432.

[0046] Example 2 This embodiment provides the preparation of a composite microbial agent.

[0047] 1) The Trichoderma acanthoides isolated in Example 1 Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 were inoculated with PDA culture medium, placed in a 28°C biochemical incubator for activation, and cultured for 3 days to produce a large number of conidia. Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride Expansion culture of T5 strain.

[0048] 2) Mix 200g peeled potatoes with 20g glucose, add 1000mL water, adjust the pH to 6, prepare the fermentation culture solution, and put them into 300mL triangular flasks, with each bottle containing 150mL of liquid. After sterilization at 121℃ for 30min, break the expanded culture into 5mm blocks and inoculate into 150mL culture solution, and shake and culture at 28℃ and 180r / min for about 5d to obtain Trichoderma aculeatus. Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 seed fermentation liquid.

[0049] 3) Mix wheat bran, corn flour, bran and water in a mass ratio of 9:6:15:70, sterilize at 121°C for 40 minutes to obtain a solid fermentation medium, and then mix the two seed fermentation liquids obtained in step 2) with the solid fermentation medium in a mass ratio of 1:7.5. After stirring evenly, cover with sterilization 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. Stir 1 to 2 times during the period so that the green bacteria are evenly distributed on the solid fermentation medium. After fermentation, dry it in the air or dry it below 32°C to obtain Trichoderma aspergillus. Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride The solid fermentation product of T5 was stored in a cool place for future use.

[0050] 4) Trichoderma aculeatus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride The solid fermentation product of T5 was mixed in a ratio of 1:1. The mixed solid fermentation product was dry-mixed with white carbon black, wheat bran, chitosan oligosaccharide, and biochemical fulvic acid in a weight ratio of 73:8:12:3:4. The wheat bran was crushed in advance with an ultra-fine crusher and passed through a 100-mesh sieve. After being fully stirred, a green powder was obtained. The conidia content was determined to be 10 10 / g or more to obtain the composite microbial agent.

[0051] Example 3 This example provides Trichoderma aspergillus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 flat plate standoff test.

[0052] The pathogens of pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato leaf mold, tomato early blight, and tomato sesame spot were used as the pathogens to be tested. The experiment was divided into a confrontation treatment group and a control group (CK). The treatment method of the confrontation treatment group was as follows: draw a straight line at the center point of the PDA flat medium, inoculate the pathogen and Trichoderma at equal distances on both sides of the center, and culture in a constant temperature incubator at 28℃ for 5 days after inoculation, and observe the growth of the colony. The treatment method of the CK group was as follows: draw a straight line at the center point of the PDA flat medium, inoculate the pathogen only at the center, and culture in a constant temperature incubator at 28℃ for 5 days after inoculation, and observe the growth of the colony. The colony radius of the confrontation treatment group and the CK group was measured respectively, and the inhibition rate was calculated. The results are shown in Table 1.

[0053] Inhibition rate (%) = (Rd) / R×100%; Among them, R is the average growth radius of the colony of CK (cm), and d is the distance between the pathogen blocks in the confrontation treatment group and the contact points of different Trichoderma species (cm).

[0054] Table 1. Antibacterial rate of each confrontation treatment group

[0055] The results of the test in Table 1 show that Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 has significant antagonistic effect on 7 pathogens and has a broad-spectrum antibacterial activity. Trichoderma asperellum The antibacterial rate of M11 was 89.0~94.27%; Trichoderma atroviride The antibacterial rate of T5 was 84.0~92.4%.

[0056] Example 4 This example provides Trichoderma aspergillus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 flat plate punching test.

[0057] The pathogens of the above 7 diseases were determined by the plate punching method. The experiment was divided into a punching treatment group and a control group (CK). The treatment method of the plate punching treatment group was as follows: a sterilized puncher was used to punch a hole on the PDA culture medium plate, the pathogen was inoculated in the center of the culture medium plate, and 3 holes were punched at equal distances from the center of the pathogen, the culture medium in the hole was taken out, and the fermentation liquid of Trichoderma (preparation method see Example 2) was quantitatively injected into the hole at 40 μL, and then placed in a 28°C incubator for 1 day and then inverted for culturing. After 5 days, the distance between the opposite sides was measured and the inhibition rate was calculated. The treatment method of the control group (CK) was as follows: only the pathogen was inoculated in the center of the culture dish as a control, and the rest of the steps were the same.

[0058] Table 2. Antibacterial rate of each plate punching treatment group (%)

[0059] Note: In Table 2, Echinococcus+dark green means treated with composite fermentation liquid.

[0060] The test results in Table 2 show that different strains and their combinations showed obvious antibacterial effects on pathogens of 7 diseases such as pepper blight, with an inhibition rate of 52.2~93.8%. Among them, the antibacterial effect of the composite fermentation liquid of Echinococcus aculeatus + dark green Trichoderma was the best, which was higher than the antibacterial effect of a single strain, with an inhibition rate of 75.6~93.8%, indicating that the two strains of Trichoderma have a broad-spectrum antibacterial effect on a variety of vegetable diseases.

[0061] Example 5 This example provides Trichoderma aspergillus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride Compatibility determination of T5.

[0062] Using the plate confrontation culture method, a straight line was drawn at the center of the PDA plate culture medium, and Trichoderma aculeatus was inoculated at equal distances on both sides of the center. Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5, repeated 3 times, after inoculation, placed in a 28℃ mold incubator for 3-5 days, observed the growth of colonies, and observed whether there were antibacterial bands at the contact point of the two Trichoderma strains. No bands indicated complete compatibility, while bands indicated incompatibility. Table 3 The test results showed that: Trichoderma Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 has good compatibility and can be used as an alternative strain for composite microbial agents.

[0063] Table 3. Compatibility test results

[0064] Example 6 This example provides Trichoderma aspergillus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride The disease prevention and growth-promoting effects of T5 and its combination (compound bacterial solution) on potted crops and their effects on microorganisms.

[0065] 1. Disease prevention and growth promotion Long flower pots (50cm×20cm) were used to fill the pots with normal field soil, and 10 pepper seedlings were transplanted into each pot. The experiment set up three treatment groups with fermented bacterial liquids of different strains and compound bacterial liquids, and set clear water as a control.

[0066] The pepper seedlings were transplanted after being treated with fermented bacterial liquid at the roots. Two days after transplanting, the roots were irrigated with the pathogen of pepper blight. Under normal management, the peppers were irrigated with fermented bacterial liquid three times during their growth period, once every 7 days, and the growth of the peppers was observed. The control group used water instead of fermented bacterial liquid, and the rest of the steps were the same. The incidence of each group was investigated, and the control effect was calculated. The results are shown in Table 4.

[0067] The pathogen is isolated and purified from pepper plants with pepper blight. The obtained Phytophthora is first inoculated on a carrot solid culture medium for activation, then punched into 5 bacterial cakes with an inner diameter of 5 mm using a sterile puncher, and then inoculated into a 300 mL triangular flask filled with sterilized tomato liquid culture medium, with each bottle containing 200 mL of liquid, and cultured under shaking conditions of 27° C. and 180 r / min for about 7 days to obtain a pepper Phytophthora fermentation liquid (1×10 5 The root irrigation inoculation volume was 20 mL / plant.

[0068] Table 4. The control effect of different treatment groups on pepper blight

[0069] Note: In Table 4, Echinococcus+dark green means treatment with compound bacterial solution, the same below.

[0070] As shown in Table 4, the control efficiency of the composite bacterial solution against pepper blight reached 80.0%, which was higher than that of the single strain bacterial solution and had a synergistic effect.

[0071] Pepper and tomato plants were planted in pots in normal soil, and the growth-promoting effects of different treatments and the control plants were determined at 30 days. The experimental treatments, root dipping and root irrigation methods were the same, but no pathogens were inoculated. The growth-promoting effect determination is shown in Table 5.

[0072] Table 5. Growth-promoting effects of different treatment groups

[0073] Note: In Table 5, “-” means not determined, the same below.

[0074] As shown in Table 5, the application of different bacterial solutions and composite bacterial solutions has different degrees of growth-promoting effects on pepper and tomato plants, and the growth-promoting effect of the composite bacterial solution is better than that of the single strain bacterial solution. Taking pepper as an example, after the composite bacterial solution treatment, the pepper plant height increased by 8.0 cm compared with CK, the root length increased by 2.7 cm, the stem thickness increased by 0.2 cm, the fresh weight increased by 4.5 g / plant, and the dry weight increased by 1.9 g / plant. The prevention effect of Trichoderma aculeatus on pepper blight reached 80.0%, which was higher than the effect of the single strain bacterial solution. Therefore, Trichoderma aculeatus Trichoderma asperellum M11 and Trichoderma aureum Trichoderma atroviride T5 has excellent disease prevention and control capabilities and promotes growth.

[0075] In order to explore whether the composite bacterial liquid has a growth-promoting effect on other crops, further growth-promoting tests were conducted on cucumber and watermelon plants. The results are shown in Table 6.

[0076] Table 6. Growth promotion of cucumber and watermelon plants by compound bacterial solution

[0077] As shown in Table 6, the composite bacterial solution can also significantly promote the growth of cucumber and watermelon plants, which shows that the composite bacterial solution has a broad-spectrum effect in promoting the growth of melon and vegetable plants and has good application prospects in the cultivation of various fruits and vegetables.

[0078] 2. Impact on microorganisms Long flowerpots (50cm×20cm) were filled with normal field soil, and 10 tomato seedlings were transplanted into each pot. The experiment set up three treatment groups with different bacterial solutions and compound bacterial solutions, and clear water was set as a control.

[0079] The tomato seedlings were transplanted after their roots were dipped in fermented bacterial solution and managed normally after transplanting. The roots were irrigated three times during the growth period, once every 7 days. The plants were dug out at 30 days, and the rhizosphere soil and different tissues of the tomato seedlings were taken. Soil microorganisms (fungi, bacteria, actinomycetes) were determined using selective culture media, and the distribution of Trichoderma in the soil and plants was determined. The results are shown in Table 7.

[0080] Table 7. Distribution of rhizosphere soil microorganisms and Trichoderma in each group

[0081] The results in Table 7 show that after treatment with different bacterial solutions and composite bacterial solutions, bacteria and actinomycetes in the rhizosphere soil of tomatoes proliferated significantly, with the bacteria increasing by 3.1-9.8×10 6 CFU / g, actinomycetes increased by 3.0~10.0×10 5 CFU / g, while fungi showed a downward trend. The number of Trichoderma in the soil and plants also increased significantly, with the increase of soil Trichoderma by 5.2~21.4×10 5 CFU / g, the number of Trichoderma in plants increased by 3.0~4.8×10 5 CFU / g, and the number of soil bacteria, actinomycetes and Trichoderma after the composite bacterial solution treatment was higher than that of the single bacterial solution. Trichoderma asperellum M11, Trichoderma cyanobacteria Trichoderma atroviride T5 or a composite bacterial solution combining the two can help enrich the beneficial bacteria in the soil, improve the soil microecological environment, and enhance the plant's disease resistance.

[0082] Example 7 This example provides the disease prevention, yield increase and microbial changes of the composite microbial agent (preparation method see Example 2) after application in the field.

[0083] 1. For chili peppers The compound microbial agent was used to conduct the experiment by combining root dipping and root irrigation. The compound microbial agent was used as the experimental treatment group, and the biological agent Bacillus polymyxa wettable powder (WP), the chemical agent 99% oxadipate WP and no agent (water) were used as the control. Three replicates were set in each group, and the experimental plots of each group were randomly arranged. After the pepper seedlings were transplanted by root dipping, they were managed normally. The roots were irrigated after the plant growth slowing period, and the roots were irrigated 3 times during the growth period, with an interval of 7 to 20 days. The results are shown in Table 8.

[0084] Table 8. Yield increase and microbial changes in peppers

[0085] Note: 50 times diluted + 100 times diluted solution means: the concentration for root dipping is 50 times diluted, the concentration for root irrigation is 100 times diluted, the same below.

[0086] The results in Table 8 show that after root dipping and root irrigation with composite microbial agents, pepper plants showed obvious disease resistance and growth-promoting and yield-increasing effects, with a field control effect of 73.2% on pepper blight and a yield-increasing rate of 48.0%. Both the control effect and yield-increasing rate were higher than those of Paenibacillus polymyxa WP and oxadipazole WP. The number of soil bacteria and actinomycetes increased by 4.67×10 6 CFU / g, 2.66×10 5 CFU / g, and was also higher than that of Paenibacillus polymyxa WP and oxadiazine WP, indicating that the soil environment was significantly improved.

[0087] 2. For watermelon The experimental treatment method is the same as that of pepper, and the results are shown in Table 9.

[0088] Table 9. Yield increase and microbial changes in watermelon

[0089] The results in Table 9 show that after root dipping and root irrigation with composite microbial agents, watermelon plants showed obvious disease resistance and growth-promoting and yield-increasing effects. The field control effect on watermelon wilt reached 74.6%, the yield increase rate was 15.20%, and the longest vine length of watermelon increased by 39.5 cm and the number of leaves increased by 4 compared with the clear water control. The number of soil bacteria increased by an average of 8.5×10 6 CFU / g, the number of soil actinomycetes increased by 1.66×10 5 CFU / g, and was also higher than that of Paenibacillus polymyxa WP and oxazolidinone WP, indicating that the number of beneficial bacteria in the soil increased significantly and the soil environment was significantly improved.

[0090] 3) For tomatoes A combination of root dipping and root irrigation was used to apply compound microbial agents as the test treatment, and clear water was set as the control. 90 tomato seedlings were planted in each plot, and 3 plots were set up for replication, with a total of 270 plants as the test treatment group. When transplanting and planting tomato seedlings, the roots were dipped with a 50-fold dilution of the compound microbial agent. After the seedlings have grown, the roots were irrigated once every 7 to 10 days, and the roots were irrigated 3 times in a row. The root irrigation used a 100-fold dilution, and 200 to 300 mL / plant was irrigated each time. The control group used clear water instead of the compound microbial agent, and the other conditions were the same. The occurrence of wilt disease was regularly observed during the growth period of tomatoes, and the various growth indicators of tomatoes were measured in the early and middle stages of growth. The cumulative yield of tomatoes in the experimental group and the control group was measured in the middle stage of growth, and the incidence of wilt disease, prevention effect and yield increase effect were calculated. The results are shown in Table 10.

[0091] Table 10. Yield increase and microbial changes in tomatoes

[0092] The test results in Table 10 show that after applying the composite microbial agent, the tomato plants showed obvious disease resistance and growth-promoting and yield-increasing effects, among which the height of the tomato plants increased by 9.2 cm, the stem diameter increased by 0.1 cm, the field control effect on tomato wilt reached 76.4%, the yield increase rate was 14.6%, and the incidence of other leaf diseases such as tomato leaf mold and early blight was also relatively mild, indicating that the disease resistance of tomato plants was enhanced. The above field test results also show that the composite microbial agent is a broad-spectrum biological fungicide.

[0093] Example 8 This example provides a field demonstration effect measurement of a composite microbial agent.

[0094] 1) For chili peppers Experimental location: Litong District, Wuzhong, Ningxia; Application method: Combination of root dipping and root irrigation; Test method: The demonstration area was treated with composite microbial agents. The demonstration area was 440m 2 The pepper seedlings were dipped in 50 times diluted liquid and managed normally after transplanting. The roots were irrigated 3 times during the growth period, with an interval of 7 to 20 days between applications. 100 times diluted liquid was used for root irrigation. Clean water was used as a control. The experimental results are shown in Table 11.

[0095] Table 11. Field demonstration effect of composite microbial agents on pepper

[0096] The test results in Table 11 show that after applying the composite microbial agent, the height of pepper plants in the demonstration area increased by 11.1 cm, the stem diameter increased by 0.04 cm, the length of peppers increased by 4.8 cm, and the width of peppers increased by 1.1 cm, showing an obvious growth-promoting effect. The prevention effect against pepper blight reached 78.6%, the yield increase rate was 13.4%, and the effect was significant.

[0097] 2) For watermelon Experimental location: Shapotou District, Zhongwei, Ningxia; Application method: Combination of root dipping and root irrigation; Test method: The demonstration area was treated with composite microbial agents. The demonstration area was 667m 2 The watermelon seedlings were dipped in 50 times diluted solution and managed normally after transplanting. The roots were irrigated 3 times during the growth period, with an interval of 7 to 20 days between applications. 100 times diluted solution was used for root irrigation. Clean water was used as the control. The experimental results are shown in Table 12.

[0098] Table 12. Field demonstration effect of composite microbial agents on watermelon

[0099] The measurement results in Table 12 show that after applying the composite microbial agent, the longest vine length of watermelon in the demonstration area increased by 33.0 cm, the number of leaves increased by 3 on average, and the number of branches increased by 1, showing an obvious growth-promoting effect. The prevention effect against watermelon wilt disease reached 77.4%, the yield increase rate was 26.9%, and the results were good.

[0100] Example 9 This example describes the induced defense response of the composite microbial agent on pepper plants and rhizosphere soil.

[0101] Induced resistance is a potential mechanism for biocontrol microorganisms to control plant diseases. Superoxide dismutase (SOD), catalase (CAT), urease (S-UE), sucrase (SUC), alkaline phosphatase (ALP) and other defense enzymes were used as evaluation indicators to measure the induced defense response of pepper plants and rhizosphere soil after the application of the composite microbial agent. The method was the same as the pepper growth promotion test in Example 6. The test results are shown in Table 13.

[0102] Table 13. Defense enzyme activity after application of composite microbial agents

[0103] The results in Table 13 show that compared with the control group, after the application of the composite microbial agent, the activities of urease (S-UE), alkaline phosphatase (ALP), sucrase (SUC) in the rhizosphere soil of pepper and catalase (CAT), superoxide dismutase (SOD), and chitinase in pepper plants were all increased to varying degrees. This result shows that the application of the composite microbial agent has an inducing defense response effect on the plant and can promote the crop to produce disease resistance.

[0104] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A composite microbial agent, characterized in that: The active ingredients of the composite microbial agent are Trichoderma acanthosporum and Trichoderma aureum; The Trichoderma spinulosa is Trichoderma asperellum M11, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.40752; The dark green Trichoderma is Trichoderma atroviride T5, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.22432; The total spore count of Trichoderma aculeatus and Trichoderma aureum in the composite microbial inoculant is not less than 10 10 Pieces / g.

2. The composite microbial agent according to claim 1, characterized in that: The components of the composite microbial inoculant include: by weight, 73 parts of microorganisms and fermentation products thereof, 3-10 parts of white carbon black, 10-20 parts of wheat bran, 1-3 parts of chitosan oligosaccharide, and 1-5 parts of biochemical fulvic acid; The microorganism and its fermentation product are from Trichoderma aspergillus Trichoderma asperellum Solid fermentation product of M11 and Trichoderma aureum Trichoderma atroviride The solid fermentation products of T5 were mixed in a mass ratio of 1:1; The solid fermentation product is prepared by mixing and fermenting the seed fermentation liquid and the solid fermentation medium in a mass ratio of 1:7.5; The solid fermentation medium is prepared by mixing wheat bran, corn flour, coarse bran and water in a mass ratio of 9:6:15:70 and sterilizing the mixture.

3. The composite microbial agent according to claim 2, characterized in that: The components of the composite microbial inoculant include: by weight, 73 parts of the microorganisms and their fermentation products, 8 parts of white carbon black, 12 parts of wheat bran, 3 parts of chitosan oligosaccharides, and 4 parts of biochemical fulvic acid.

4. The composite microbial agent according to claim 1, characterized in that: The composite microbial agent prevents and controls diseases caused by fungi.

5. The composite microbial agent according to claim 4, characterized in that: The diseases include: pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight, and tomato leaf mold.

6. Use of the composite microbial agent according to claim 1 in crop disease prevention and control, characterized in that: The composite microbial agent is used for preventing and controlling pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight and tomato leaf mold.

7. The use of the composite microbial agent according to claim 1 in improving the disease resistance of crops, characterized in that: The composite microbial agent has the function of increasing the content of catalase, superoxide dismutase and chitinase in plants; The composite microbial agent has the function of increasing the contents of urease, alkaline phosphatase and sucrase in the rhizosphere soil of plants.

8. A method for preventing and controlling crop diseases, characterized in that: The control method comprises: diluting the composite microbial agent of claim 1 by 50 times with water, dipping the agent into the roots of transplanted crop seedlings, and irrigating the roots three times during the crop growth period, using a 100-fold diluted solution, and the interval between applications is 7 to 20 days.

9. The control method according to claim 8, characterized in that: The crop diseases include: pepper blight, cucumber wilt, watermelon wilt, cucumber damping-off, tomato sesame spot, tomato early blight, and tomato leaf mold.

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