Compound enzyme biocontrol preparation for preventing and treating tomato diseases as well as preparation method and application of compound enzyme biocontrol preparation

Abstract

The invention provides a compound enzyme biocontrol preparation for preventing and treating tomato diseases. The compound enzyme biocontrol preparation is prepared from beta-glucanase, chitinase and alpha-galactosidase, the enzyme activity ratio of the beta-glucanase to the chitinase to the alpha-galactosidase is (1-4): (2-8): (1-2); the beta-glucanase, the chitinase and the alpha-galactosidase are obtained by carrying out mixed induced fermentation on trichoderma harzianum and trichoderma reesei and carrying out separation and purification; when the mixed induced fermentation is carried out, botrytis cinerea cell walls are added into the enzyme-producing culture medium as induction primers. The preparation method is ingenious in conception, three enzymes generated through mixed induction fermentation of trichoderma are prepared according to the enzyme activity ratio, and a novel compound enzyme biocontrol preparation is obtained; the compound enzyme biocontrol agent has an unexpected control effect on fungal diseases, and the obtained compound enzyme biocontrol agent has an excellent control effect on various fungal diseases of tomatoes, can promote the growth of tomato seedlings, and has an application prospect of replacing or reducing the use of chemical pesticides.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop disease control, and particularly relates to a compound enzyme biocontrol preparation for preventing and treating tomato diseases, a preparation method thereof, and an application thereof. Background Art

[0002] Tomatoes are widely planted across the country due to their high yield, strong adaptability, and rich nutrition. Soil-borne diseases are one of the main problems faced in tomato production. Due to continuous continuous cropping, the soil fertility decreases and the microbial flora is out of balance, resulting in the increasing severity of tomato soil-borne diseases, which has become a bottleneck in the development of the tomato industry. The main soil-borne diseases in tomato production in China include late blight, leaf mold, fusarium wilt, bacterial wilt, scab, etc. Due to their own biological characteristics and the natural barrier effect of the soil, tomato soil-borne diseases are less sensitive to chemical agents and are more difficult to control. Coupled with the concealment of the occurrence of soil-borne diseases, greater economic losses are often caused due to untimely prevention and control. At present, the prevention and control of tomato soil-borne diseases mainly combines agricultural control and chemical control. Although certain effects have been achieved, there are also problems such as a lack of high-quality resistant variety resources and environmental pollution by chemical agents. In recent years, with the public's attention to the quality and safety of agricultural products and the safety of the ecological environment, under the background of the country's strong advocacy of green agriculture, it is particularly important to develop green and environmentally friendly biocontrol preparations to prevent and treat tomato soil-borne diseases.

[0003] In the research of biological control agents for tomato diseases, although certain progress has been made, it usually relies on specific biocontrol bacteria. For example, CN106520595B provides an Arthrobacter and its application in biological control of tomato bacterial wilt, and CN112063554B provides a biocontrol bacterium Pantoea jilinensis D25, which can effectively control tomato gray mold. However, these biocontrol bacteria are limited by the microbial ecology of different soils and are extremely vulnerable to environmental changes, making it difficult for them to exert their best control effects and having poor adaptability to different planting areas. Therefore, developing a specific solution based on biological control technology that is not restricted by soil and environmental factors and can be better applied on a large scale is an important research and development direction in this field. As a biological control method, bioenzymes have the advantages of only targeting pathogenic bacteria without harming other beneficial organisms, not generating drug resistance, and being environmentally friendly. Exploring the role of bioenzymes in plant disease control and developing new green biopesticides based on the specific catalytic functions of bioenzymes are the development trends of modern plant protection technologies. For example, Zuo Yuhu et al. found in the research paper "Relationship between β-1,3-glucanase and chitinase activities and soybean resistance to Phytophthora root rot" that the mixture of β-1,3-glucanase and chitinase has a synergistic effect on the inhibition of Phytophthora sojae; Lin Zhenya et al. optimized the fermentation conditions for the co-expression of natamycin and chitinase in the research paper "Optimization of fermentation conditions for the co-expression of natamycin and chitinase by Streptomyces A01-chit33CT". However, in the prevention and control of tomato diseases, there is still relatively little research in this area. The selection of bioenzyme types, the composition ratio of complex enzyme preparations, and the optimization of the technological conditions for microbial fermentation to express target enzymes are problems that urgently need to be solved by researchers in this field. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a complex enzyme biocontrol preparation for preventing and controlling tomato diseases and its preparation method. The complex enzyme biocontrol preparation has simple components, is easy to produce on a large scale, has a synergistic effect on the prevention and control of common fungal diseases of tomatoes, and has a growth-promoting effect on tomato seedlings.

[0005] The present invention provides the following technical solutions to achieve the foregoing purpose.

[0006] A complex enzyme biocontrol preparation for preventing and controlling tomato diseases, characterized in that the complex enzyme biocontrol preparation contains β-glucanase, chitinase, and α-galactosidase;

[0007] The β-glucanase, chitinase, and α-galactosidase are obtained by mixed induction fermentation of Trichoderma harzianum and Trichoderma reesei and separation and purification; the preservation number of the Trichoderma harzianum strain is ACCC 30371, and the preservation number of the Trichoderma reesei strain is ACCC 30911.

[0008] The enzyme activity ratio of the β-glucanase, chitinase and α-galactosidase is 1-4:2-8:1-2;

[0009] When performing the induced fermentation, the enzyme-producing induction medium is added with the cell wall of Botrytis cinerea.

[0010] Preferably, the formula of the enzyme-producing induction medium is: 6-10 g·L -1 Cell wall of Botrytis cinerea, 2-5 g·L -1 Peptone, 2-5 g·L -1 Beef extract, 0.8 g·L -1 KH2PO4, 0.1 g·L -1 CaCl2·2H2O, 0.05 g·L - 1 MgSO4·7H2O, 0.1 g·L -1 CuSO4·5H2O, 0.06 g·L -1 MnCl2·4H2O, 0.05 g·L -1 ZnSO4·7H2O, 0.01 g·L -1 FeS04·7H2O, 0.02 g·L -1 CoCl2·6H2O, and the rest is distilled water.

[0011] More preferably, the formula of the enzyme-producing induction medium is: 8 g·L -1 Cell wall of Botrytis cinerea, 2 g·L -1 Peptone, 3 g·L -1 Beef extract, 0.8 g·L -1 KH2PO4, 0.1 g·L -1 CaCl2·2H2O, 0.05 g·L -1 MgSO4·7H2O, 0.1 g·L -1 CuSO4·5H2O, 0.06 g·L -1 MnCl2·4H2O, 0.05 g·L -1 ZnSO4·7H2O, 0.01 g·L -1 FeS04·7H2O, 0.02 g·L -1 CoCl2·6H2O, and the rest is distilled water.

[0012] Preferably, the preparation method of the cell wall of Botrytis cinerea is: inoculating Botrytis cinerea on a PDA plate medium for activation culture for 96 h, picking hyphae and inoculating them into a yeast sucrose culture solution, and at 26 °C at 150 r·min -1Oscillate and culture for 120 h, filter with multiple layers of gauze, and wash the obtained mycelia with distilled water 4 times until clear; at 4 °C, centrifuge at 12000 r·min -1 for 10 min, add distilled water to dissolve the precipitate, and repeat centrifugation until the supernatant is clear; homogenize the mycelia with a homogenizer, add distilled water, centrifuge at 12000 r·min -1 for 10 min, dry the precipitate under vacuum and grind it into fine powder, pass through a 20-mesh sieve, add distilled water and stir to dissolve, centrifuge at 12000 r·min -1 for 10 min, repeat several times until it is determined that the supernatant contains no protein, and obtain the cell wall of Botrytis cinerea of tomato.

[0013] Preferably, when inducing fermentation of Trichoderma, inoculate the seed solutions of Trichoderma harzianum and Trichoderma reesei into the enzyme production induction medium simultaneously, with an inoculation amount of 6 mL each, a liquid loading volume of 50 mL in a 250-mL shaking flask, at 28 °C, an initial pH of 6.8, and a shaking speed of 140 r·min -1 Perform mixed fermentation for 84 hours.

[0014] Preferably, when performing the separation and purification, first perform ammonium sulfate salting-out, and then perform chromatographic column chromatography separation.

[0015] Preferably, centrifuge the induced fermentation broth at 4 °C and 5000 r·min -1 for 10 min to obtain the supernatant, which is the crude enzyme solution. Take 100 mL of the crude enzyme solution, filter it through a 0.20-μm microporous filter membrane to remove conidia, slowly add dispersed (NH4)2SO4 to a saturation of 50%, stir evenly and let it stand overnight at room temperature. Centrifuge the salting-out solution at 4 °C and 8000 r·min -1 for 20 min, discard the supernatant, add 10 mL of HAc-NaAc buffer solution to dissolve it fully. If turbidity occurs, centrifuge again for 10 min, repeat several times, and store it at 4 °C for standby.

[0016] Preferably, when performing the chromatographic column chromatography separation, dialyze the enzyme solution obtained by preliminary purification with ammonium sulfate for 4 h until there is no SO4 2- , concentrate it to 4 mL with polyethylene glycol, load the concentrated enzyme solution onto an agarose gel DEAE-Sepharose CL-6B (2.6 cm × 60 cm) chromatographic column for chromatographic separation; the flow rate is 14.1 mL·h -1 ; linearly elute with 0 - 1.0 mol·L -1 NaCl solution. Collect one tube of eluate every 5 min, measure the soluble protein content, β-glucanase activity, chitinase activity, and α-galactosidase activity of each tube of collected solution respectively, draw the chromatographic elution curve, and collect the eluate according to the overlap of the enzyme activity elution peak and the protein elution peak to obtain purified chitinase, β-glucanase, and α-galactosidase.

[0017] The present invention also provides a preparation method for preparing the foregoing compound enzyme biocontrol agent for preventing and treating tomato diseases. The preparation method is to formulate β-glucanase, chitinase, and α-galactosidase in the foregoing compound enzyme biocontrol agent for preventing and treating tomato diseases according to the enzyme activity ratio, and then the product is obtained.

[0018] The present invention also provides the application of the foregoing compound enzyme biocontrol agent for preventing and treating tomato diseases in preventing and treating tomato fungal diseases, and the tomato fungal diseases include at least one of tomato bacterial wilt, tomato late blight, and tomato leaf mold.

[0019] Advantages of the present invention:

[0020] The present invention has a clever concept. Three enzymes produced by mixed induction fermentation of Trichoderma harzianum and Trichoderma reesei are compounded to obtain a new compound enzyme biocontrol agent. The prevention and treatment effect of the present invention on fungal diseases is surprising. The obtained compound enzyme biocontrol agent has excellent prevention and treatment effects on various tomato fungal diseases and has a growth-promoting effect on tomato seedlings, and has an application prospect of replacing or reducing the use of chemical pesticides. Specific embodiments

[0021] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still belong to the protection scope of the present invention.

[0022] Example 1

[0023] 1.1 Preparation of the cell wall of Botrytis cinerea of tomato:

[0024] Inoculate the Botrytis cinerea of tomato on a PDA plate medium for activation culture for 96 h. Pick the hyphae and inoculate them into a yeast sucrose culture solution, and culture them at 26 °C with shaking at 150 r·min -1 for 120 h, and perform filtration treatment with multiple layers of gauze. The obtained hyphae are washed with distilled water until clear for 4 times, and centrifuged at 4 °C and 12,000 r·min -1 for 10 min. The precipitate is dissolved in distilled water, and the centrifugation is repeated until the supernatant is clear. The hyphae are homogenized with a homogenizer, distilled water is added, and centrifuged at 4 °C and 12,000 r·min -1 for 10 min. The precipitate is vacuum dried and ground into fine powder, passed through a 20-mesh sieve, stirred and dissolved in distilled water, and centrifuged at 4 °C and 12,000 r·min -1 for 10 min, and repeated several times until it is determined that the supernatant does not contain protein, and the cell wall of Botrytis cinerea of tomato is obtained.

[0025] 1.2 Preparation of Trichoderma seed liquid:

[0026] The Harzianum strain was activated and cultured until conidia were produced. 10 mL of sterile normal saline was used to rinse the conidia in each petri dish to obtain a conidia suspension. The suspension was transferred to an Erlenmeyer flask and shaken thoroughly to form a single-spore suspension. After filtering with sterilized absorbent cotton, the concentration of Trichoderma spores was adjusted to 1×10 6 cfu·mL -1 to obtain the Harzianum seed liquid. Among them, the original preservation institution of the Harzianum strain is the China Center for Agricultural Culture Collection, and the preservation number is ACCC 30371. This strain was purchased from the China Center for Agricultural Culture Collection, and this patent does not involve the preservation of this strain.

[0027] The Trichoderma reesei strain was activated and cultured until conidia were produced. 10 mL of sterile normal saline was used to rinse the conidia in each petri dish to obtain a conidia suspension. The suspension was transferred to an Erlenmeyer flask and shaken thoroughly to form a single-spore suspension. After filtering with sterilized absorbent cotton, the concentration of Trichoderma spores was adjusted to 1×10 6 cfu·mL -1 to obtain the Trichoderma reesei seed liquid. Among them, the original preservation institution of the Trichoderma reesei strain is the China Center for Agricultural Culture Collection, and the preservation number is ACCC30911. This strain was purchased from the China Center for Agricultural Culture Collection, and this patent does not involve the preservation of this strain.

[0028] 1.3 Enzyme production induction medium:

[0029] Composition of the enzyme production induction medium: 8 g·L -1 cell wall of Botrytis cinerea, 2 g·L -1 peptone, 3 g·L -1 beef extract, 0.8 g·L -1 KH2PO4, 0.1 g·L -1 CaCl2·2H2O, 0.05 g·L -1 MgSO4·7H2O, 0.1 g·L -1 CuSO4·5H2O, 0.06 g·L -1 MnCl2·4H2O, 0.05 g·L -1 ZnSO4·7H2O, 0.01 g·L -1 FeS04·7H2O, 0.02 g·L - 1 CoCl2·6H2O, and the rest is distilled water. The medium was sterilized in an autoclave and cooled for standby.

[0030] 1.4 Preparation of composite enzyme induction fermentation broth:

[0031] Inoculate the Trichoderma harzianum seed liquid and Trichoderma reesei seed liquid into the enzyme production induction medium simultaneously, with an inoculation amount of 6 mL each. The liquid volume in a 250 mL shaking flask is 50 mL, at 28 °C, an initial pH of 6.8, and a shaking speed of 140 r·min -1 Perform shaking flask fermentation for 84 h to obtain the induced fermentation broth of β-glucanase, chitinase, and α-galactosidase.

[0032] 1.5 Separation and purification of enzymes

[0033] (1) Ammonium sulfate salting out

[0034] The induced fermentation broth is centrifuged at 4 °C and 5000 r·min -1 for 10 min. The supernatant is the crude enzyme solution. Take 100 mL of the crude enzyme solution, filter it through a 0.20 μm microporous filter membrane to remove conidia, slowly add dispersed (NH4)2SO4 to a saturation of 50%, stir evenly, and let it stand overnight at room temperature. The salted-out solution is centrifuged at 4 °C and 8000 r·min -1 for 20 min. Discard the supernatant, add 10 mL of HAc-NaAc buffer solution to dissolve it fully. If turbidity occurs, centrifuge it again for 10 min and repeat several times. Store it at 4 °C for standby.

[0035] (2) Chromatographic column chromatography separation

[0036] Dialyze the enzyme solution obtained by preliminary purification with ammonium sulfate for 4 h until there is no SO4 2- , concentrate it to 4 mL with polyethylene glycol, and load the concentrated enzyme solution onto an agarose gel DEAE-Sepharose CL-6B (2.6 cm × 60 cm) chromatographic column for chromatographic separation; flow rate, 14.1 mL·h -1 ; linear elution with 0 - 1.0 mol·L -1 NaCl solution. Collect one tube of eluate every 5 min, and measure the soluble protein content, β-glucanase activity, chitinase activity, and α-galactosidase activity of each tube of collected eluate respectively. Plot the chromatographic elution curve, and collect the eluate according to the overlap of the enzyme activity elution peak and the protein elution peak to obtain purified chitinase, β-glucanase, and α-galactosidase.

[0037] 1.6 Preparation of the complex enzyme:

[0038] Take 3 portions of the purified β-glucanase solution, and dilute them with distilled water respectively until the β-glucanase activity in the enzyme solution is 1.0 U·mL -1 , 2.0 U·mL -1 , 3.0 U·mL -1 ; take 3 portions of the purified chitinase solution, and dilute them with distilled water respectively until the chitinase activity in the enzyme solution is 2.0 U·mL -1, 4.0 U·mL -1 , 6.0 U·mL -1 , Take 3 portions of the purified α-galactosidase solution, and add distilled water for dilution until the α-galactosidase activity in the enzyme solution is 1.0 U·mL -1 , 2.0 U·mL -1 , 3.0 U·mL -1 .

[0039] Take the dilution of β-glucanase solution at 3.0 U·mL -1 , the dilution of chitinase solution at 6.0 U·mL -1 , and the dilution of α-galactosidase solution at 3.0 U·mL -1 and mix one portion of each to make the β-glucanase activity in the mixed solution 1.0 U·mL -1 , the chitinase activity 2.0 U·mL -1 , and the α-galactosidase activity 1.0 U·mL -1 , thus obtaining a compound enzyme preparation with the enzyme activity ratio of β-glucanase, chitinase and α-galactosidase being 1:2:1.

[0040] Example 2

[0041] In this example, except that the content of Botrytis cinerea cell wall in the enzyme-producing induction medium is 6 g·L -1 , the content of peptone is 3 g·L -1 , and the content of beef extract is 2 g·L -1 , the rest is the same as in Example 1.

[0042] Example 3

[0043] In this example, except that the content of Botrytis cinerea cell wall in the enzyme-producing induction medium is 10 g·L -1 , the content of peptone is 5 g·L -1 , and the content of beef extract is 5 g·L -1 , the rest is the same as in Example 1.

[0044] Example 4

[0045] In this example, except that in the compound enzyme, the enzyme activity ratio of β-glucanase, chitinase and α-galactosidase is 1:4:1, the rest is the same as in Example 1.

[0046] Example 5

[0047] In this example, except that in the compound enzyme, the enzyme activity ratio of β-glucanase, chitinase and α-galactosidase is 2:4:1, the rest is the same as in Example 1.

[0048] Comparative Example 1

[0049] In this comparative example, compared with Examples 1 to 3, the fermentation parameters during induced fermentation were adjusted. Specifically, the scheme in Step 1.4 was adjusted, and the rest was the same as in Example 1.

[0050] Preparation of complex enzyme induced fermentation broth:

[0051] Inoculate the Trichoderma harzianum seed liquid and Trichoderma reesei seed liquid into the enzyme production induction medium simultaneously. The inoculation amount is 8 mL each, the liquid volume in a 250 mL shaking flask is 70 mL, at 28 °C, the initial pH is 7.2, and the shaking speed of the shaker is 120 r·min -1 Perform shaking flask fermentation for 72 h to obtain the induced fermentation broth of β-glucanase, chitinase, and α-galactosidase.

[0052] Comparative Example 2

[0053] In this comparative example, except that the 8 g·L -1 tomato gray mold cell wall in the enzyme production induction medium of Example 1 was replaced with 8 g·L -1 yeast extract, the rest was the same as in Example 1.

[0054] Comparative Example 3

[0055] In this comparative example, except that the enzyme activity ratio of β-glucanase, chitinase, and α-galactosidase in the complex enzyme preparation was 1:16:1, the rest was the same as in Example 1.

[0056] Comparative Example 4

[0057] In this comparative example, except that the enzyme activity ratio of β-glucanase, chitinase, and α-galactosidase in the complex enzyme preparation was 5:2:1, the rest was the same as in Example 1.

[0058] Comparative Example 5

[0059] The complex enzyme preparation in this comparative example only added β-glucanase, and the rest was the same as in Example 1.

[0060] Comparative Example 6

[0061] The complex enzyme preparation in this comparative example only added chitinase, and the rest was the same as in Example 1.

[0062] Comparative Example 7

[0063] The complex enzyme preparation in this comparative example only added α-galactosidase, and the rest was the same as in Example 1.

[0064] Comparative Example 8

[0065] In this comparative example, except that α-galactosidase was not added to the complex enzyme preparation, the rest was the same as in Example 1.

[0066] Comparative Example 9

[0067] This comparative example is the same as Example 1 except that chitinase is not added to the complex enzyme preparation.

[0068] Comparative Example 10

[0069] This comparative example is the same as Example 1 except that β-glucanase is not added to the complex enzyme preparation.

[0070] Experimental Example 1

[0071] The DNS method was used to measure the activities of β-glucanase and chitinase in the enzyme solution, and the p-NPG method was used to measure the activity of α-galactosidase in the enzyme solution. The amount of enzyme required to hydrolyze β-glucan to produce 1 μmol of reducing sugar per minute was defined as one unit of β-glucanase activity (U·mL -1 ), the amount of enzyme required to hydrolyze chitin to produce 1 μmol of reducing sugar per minute was defined as one unit of chitinase activity (U·mL -1 ), and the amount of enzyme required to degrade p-nitrophenyl-α-D-galactopyranose to release 1 mmol of p-nitrophenol per minute was defined as one unit of α-galactosidase activity (U·mL -1 ). The enzyme activities of the enzyme solutions prepared in Examples 1-3 and Comparative Examples 1-2 were measured, and the results are shown in Table 1.

[0072] Table 1

[0073]

[0074] Experimental Example 2

[0075] This experimental example investigated the growth promotion of tomato seedlings by the complex enzyme preparation.

[0076] Tomato seeds (variety: Chunli) were soaked in 10% sodium hypochlorite solution for 10 min, rinsed with water, and then placed in a constant temperature incubator at 28 °C for germination; when the radicle was about 5 mm long, they were sown in a plastic seedling tray, with 12 h of light per day. The seedling substrate was vegetable field soil, peat, and high vermiculite (volume ratio 1:2:1), sterilized by high-temperature steam (134 °C, 30 min), and seedlings were grown in a light incubator at 20-25 °C.

[0077] Tomato seedlings were transplanted into nutrient pots at the two-cotyledon fully-expanded stage, with one plant per pot. After 24 h of transplantation, they were treated with the complex enzyme preparations prepared in Example 1, Example 4, and Example 5 by root irrigation, with 30 mL of enzyme solution applied per plant; at the same time, the biocontrol agents obtained in Comparative Examples 3-10 were used as test controls, and distilled water treatment was used as a blank control. The enzyme solution application amounts for the test control and the blank control were also 30 mL. Each treatment had 3 replicates, with 30 plants per replicate. After being cultured in a greenhouse for 30 d, the plants were gently pulled out, rinsed repeatedly with water, and then blotted dry with absorbent paper.

[0078] The following methods were used to measure the growth indexes of tomato seedlings:

[0079] The plant height was measured with a tape measure as the length from the base of the tomato plant stem to the growth point at the stem tip; the stem diameter was measured with a vernier caliper as the diameter of the stem 1 mm above the cotyledon node of the tomato; the leaf area of the tomato plant was measured with a leaf area meter, and the total root volume of the tomato plant was measured with a root scanner; the fresh weight of the aboveground part and the fresh weight of the underground part of the tomato plant were determined with an analytical balance; the fresh samples were blanched at 105 °C for 15 min and dried to a constant weight at 70 °C, and then the dry weights of the aboveground part and the underground part of the plant were measured respectively. Calculate the root-shoot ratio and the strong seedling index: root-shoot ratio = dry weight of underground part / dry weight of aboveground part, strong seedling index = (stem diameter / plant height + dry weight of underground part / dry weight of aboveground part) × dry weight of whole plant. The results are shown in Table 2.

[0080] Table 2

[0081]

[0082]

[0083] Experimental Example 3

[0084] This experimental example investigated the prevention and control effect of the compound enzyme preparation on tomato diseases.

[0085] Tomato seeds (variety: Chunli) were soaked in 10% sodium hypochlorite solution for 10 min, rinsed with clean water, and then placed in a constant temperature incubator for germination at 28 °C; when the radicle was about 5 mm long, they were sown in a plastic seedling tray, with 12 h of light per day. The seedling-raising substrate was vegetable field soil, peat, and high vermiculite (volume ratio 1:2:1), sterilized by high-temperature steam (134 °C, 30 min), and seedlings were raised in a light incubator at 20 - 25 °C. Tomato seedlings were cultivated until the two cotyledons were flattened and then transplanted into nutrient pots, with 1 plant per pot. The tomato seedlings were treated with the compound enzyme preparations prepared in Example 1, Example 4, and Example 5, using the biocontrol preparations obtained in Comparative Examples 3 - 10 as the test control, and distilled water treatment as the blank control. Each treatment had 3 replicates, with 30 plants per replicate. Using a combination of root irrigation and foliar spraying, root irrigation treatment was carried out 24 h after transplantation, with 30 mL of enzyme solution applied per plant, and 7 d after transplantation, the tomato leaves were sprayed with the same enzyme solution. The following methods were used to investigate the prevention and control effects of different biocontrol preparations on the main tomato diseases:

[0086] (1) According to the technical regulations for the identification of disease resistance of main tomato diseases, Part 1: Technical regulations for the identification of tomato resistance to late blight (NYT 1858.1 - 2010), the methods of inoculating Phytophthora infestans, seedling management, and disease investigation were carried out, and the results are shown in Table 3. The disease grades of tomato seedlings and the corresponding symptom descriptions are as follows:

[0087] Grade 0: No symptoms;

[0088] Level 1: The lesions on the leaves are small, and the total area of the lesions accounts for ≤ 5% of the leaf area;

[0089] Level 2: The lesions on the leaves are larger, and 5% < the total area of the lesions accounts for ≤ 15% of the leaf area;

[0090] Level 3: The lesions on the leaves further expand, and 15% < the total area of the lesions accounts for ≤ 30% of the leaf area;

[0091] Level 4: Some of the lesions on the leaves are connected, 30% < the total area of the lesions accounts for ≤ 50% of the leaf area, and there are a small number of lesions on the stem;

[0092] Level 5: The lesions on the leaves are connected to each other, 50% < the total area of the lesions accounts for ≤ 70% of the leaf area; or there are spreading lesions on the stem;

[0093] Level 6: The lesions on the leaves are almost contiguous, 70% < the total area of the lesions accounts for ≤ 100% of the leaf area; or the stem is severely damaged and even the plant dies.

[0094] The disease index and the biocontrol effect are calculated according to the following formulas (1) and (2) respectively:

[0095] Disease index (disease severity) = ∑(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100% (1)

[0096] Biocontrol effect = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100% (2)

[0097] Table 3

[0098]

[0099]

[0100] (2) According to the technical regulations for the identification of disease resistance of main tomato diseases, Part 2: Technical regulations for the identification of tomato resistance to Cladosporium fulvum (NYT 1858.2 - 2010), the inoculation of Cladosporium fulvum, seedling management and disease investigation were carried out. The results are shown in Table 4. The disease levels of tomato seedlings and the corresponding symptom descriptions are as follows:

[0101] Level 0: Asymptomatic;

[0102] Level 1: The inoculated leaves show chlorotic to yellow lesions;

[0103] Level 3: The lesions on the inoculated leaves produce a thin layer of sparse greyish-yellow mold;

[0104] Level 5: The lesions on the inoculated leaves produce obvious greyish-brownish mold;

[0105] Level 7: The inoculated leaves develop a thick layer of dark brown mold, and the upper leaves are also infected.

[0106] Level 9: In addition to the thick dark brown mold layer on the inoculated leaf lesions, the mold layer on the lesions of the upper leaves is also obvious.

[0107] The disease index and the biocontrol effect are calculated according to the following formulas (3) and (4) respectively:

[0108] Disease index (disease severity) = ∑(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100% (3)

[0109] Biocontrol effect = (control group disease index - treatment group disease index) / control group disease index × 100% (4)

[0110] Table 4

[0111]

[0112]

[0113] (3) According to the technical regulations for the identification of disease resistance of main tomato diseases, Part 4: Technical regulations for the identification of tomato resistance to bacterial wilt (NYT 1858.4 - 2010), the inoculation of Ralstonia solanacearum, seedling management and disease investigation were carried out. The results are shown in Table 5. The disease levels of tomato seedlings and the corresponding symptom descriptions are as follows:

[0114] Level 0: No symptoms

[0115] Level 1: One leaf wilts

[0116] Level 2: Two to three leaves wilt

[0117] Level 3: Except for the top two to three leaves, all other leaves wilt

[0118] Level 4: All the leaves of the whole plant wilt

[0119] The disease index and the biocontrol effect are calculated according to the following formulas (5) and (6) respectively:

[0120] Disease index (disease severity) = ∑(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100% (5)

[0121] Biocontrol effect = (control group disease index - treatment group disease index) / control group disease index × 100% (6)

[0122] Table 5

[0123]

[0124]

[0125] It can be seen from the test results of Experimental Example 2 and Experimental Example 3 that different biocontrol agents can all promote the growth of tomato seedlings and significantly improve the disease resistance of tomatoes against late blight, leaf mold and bacterial wilt. The control effects of Comparative Example 8 (β-glucanase + chitinase), Comparative Example 9 (β-glucanase + α-galactosidase) and Comparative Example 10 (chitinase + α-galactosidase) on the three tomato fungal diseases are significantly higher than those of Comparative Example 5 (only containing β-glucanase), Comparative Example 6 (only containing chitinase) and Comparative Example 7 (only containing α-galactosidase), and are significantly lower than those of Example 1 (the enzyme activity ratio of β-glucanase, chitinase and α-galactosidase is 1:2:1), Example 4 (enzyme activity ratio 1:4:1), Example 5 (enzyme activity ratio 2:4:1) and Comparative Example 3 (enzyme activity ratio 1:16:1) and Comparative Example 4 (enzyme activity ratio 5:2:1). Among them, Example 1 has the best control effects on tomato late blight, leaf mold and bacterial wilt, reaching 83.58%, 86.12% and 76.28% respectively. The results show that β-glucanase, chitinase and α-galactosidase have obvious synergistic effects in controlling tomato fungal diseases.

Claims

1. A compound enzyme biocontrol preparation for preventing and controlling tomato diseases, characterized in that, The compound enzyme biocontrol agent contains β-glucanase, chitinase and α-galactosidase; The β-glucanase, chitinase and α-galactosidase are obtained by mixed induction fermentation of Trichoderma harzianum and Trichoderma reesei followed by separation and purification; the preservation number of the Trichoderma harzianum strain is ACCC 30371, and the preservation number of the Trichoderma reesei strain is ACCC 30911; The enzyme activity ratio of the β-glucanase, chitinase and α-galactosidase is 1-4:2-8:1-2; When carrying out the induction fermentation, the cell wall of Botrytis cinerea of tomato is added to the enzyme-producing induction medium.

2. The compound enzyme biocontrol preparation for preventing and treating tomato diseases according to claim 1, characterized in that, The formula of the enzyme-producing induction medium is: 6-10 g·L -1 Botrytis cinerea cell wall, 2-5 g·L -1 Peptone, 2-5 g·L -1 Beef extract, 0.8 g·L -1 KH2PO4, 0.1 g·L -1 CaCl2·2H2O, 0.05 g·L -1 MgSO4·7H2O, 0.1 g·L -1 CuSO4·5H2O, 0.06 g·L -1 MnCl2·4H2O, 0.05 g·L -1 ZnSO4·7H2O, 0.01 g·L -1 FeS04·7H2O, 0.02 g·L - 1 CoCl2·6H2O, and the rest is distilled water.

3. The compound enzyme biocontrol agent for preventing and controlling tomato diseases according to claim 2, characterized in that, The formula of the enzyme-producing induction medium is: 8 g·L -1 cell wall of Botrytis cinerea, 2 g·L -1 peptone, 3 g·L -1 beef extract, 0.8 g·L -1 KH2PO4, 0.1 g·L -1 CaCl2·2H2O, 0.05 g·L -1 MgSO4·7H2O, 0.1 g·L -1 CuSO4·5H2O, 0.06 g·L -1 MnCl2·4H2O, 0.05 g·L -1 ZnSO4·7H2O, 0.01 g·L -1 FeS04·7H2O, 0.02 g·L -1 CoCl2·6H2O, and the rest is distilled water.

4. A composite enzyme biocontrol preparation for preventing and controlling tomato diseases according to claim 2 or 3, characterized in that, The preparation method of the cell wall of Botrytis cinerea on tomatoes is as follows: inoculate Botrytis cinerea on tomatoes on a PDA plate medium for activation culture for 96 h, pick hyphae and inoculate them into a yeast sucrose culture solution, and culture them at 26 °C with shaking at 150 r·min -1 for 120 h, filter with multiple layers of gauze, and wash the obtained hyphae with distilled water until clear for 4 times; at 4 °C, centrifuge at 12000 r·min -1 for 10 min, add distilled water to dissolve the precipitate, and repeat centrifugation until the supernatant is clear; homogenize the hyphae with a homogenizer, add distilled water, and centrifuge at 12000 r·min -1 for 10 min, vacuum-dry the precipitate and grind it into a fine powder, pass it through a 20-mesh sieve, add distilled water and stir to dissolve, and centrifuge at 12000 r·min -1 for 10 min, repeat several times until it is determined that the supernatant does not contain protein, and obtain the cell wall of Botrytis cinerea on tomatoes.

5. A composite enzyme biocontrol agent for preventing and controlling tomato diseases according to claim 1, characterized in that, When inducing the fermentation of Trichoderma, the seed solutions of Trichoderma harzianum and Trichoderma reesei are inoculated into the enzyme production induction medium simultaneously, with an inoculation amount of 6 mL each. The liquid volume in a 250 mL shaking flask is 50 mL, at 28 °C, an initial pH of 6.8, and a shaking speed of 140 r·min -1 for 84 hours of mixed fermentation.

6. The composite enzyme biocontrol agent for preventing and treating tomato diseases according to claim 1, characterized in that, When carrying out the separation and purification, ammonium sulfate salting-out is first used, and then chromatographic column chromatography is used for separation.

7. The compound enzyme biocontrol preparation for preventing and controlling tomato diseases according to claim 6, wherein, When performing the ammonium sulfate salting-out, the induced fermentation broth is centrifuged at 4 °C and 5000 r·min -1 for 10 min to obtain the supernatant, i.e., the crude enzyme solution. Take 100 mL of the crude enzyme solution, filter it through a 0.20-μm microporous membrane to remove conidia, slowly add dispersed (NH4)2SO4 to a saturation of 50%, stir evenly, and leave it to stand overnight at room temperature. Then centrifuge the salting-out solution at 4 °C and 8000 r·min -1 for 20 min, discard the supernatant, add 10 mL of HAc-NaAc buffer solution to dissolve it fully. If turbidity appears, centrifuge it again for 10 min and repeat several times, and store it at 4 °C for standby.

8. The composite enzyme biocontrol preparation for preventing and controlling tomato diseases according to claim 6, wherein, When performing the chromatographic column chromatography separation, the enzyme solution obtained by preliminary purification with ammonium sulfate is dialyzed for 4 h until there is no SO4 2- , concentrated to 4 mL with polyethylene glycol, and the concentrated enzyme solution is loaded onto an agarose gel DEAE-Sepharose CL-6B (2.6 cm × 60 cm) chromatographic column for chromatography separation; the flow rate is 14.1 mL·h -1 ; eluted linearly with 0-1.0 mol·L -1 NaCl solution. An elution fraction is collected every 5 min, and the soluble protein content, β-glucanase activity, chitinase activity, and α-galactosidase activity of each collected fraction are measured respectively. A chromatographic elution curve is plotted, and the elution fractions are collected respectively according to the overlap of the enzyme activity elution peak and the protein elution peak to obtain purified chitinase, β-glucanase, and α-galactosidase.

9. The preparation method of a compound enzyme biocontrol agent for preventing and controlling tomato diseases according to any one of claims 1 to 8, characterized in that, The β-glucanase, chitinase and α-galactosidase are formulated according to the enzyme activity ratio to obtain the compound enzyme preparation.

10. Use of a compound enzyme biocontrol agent for preventing and treating tomato diseases according to any one of claims 1 to 8 and the preparation method according to claim 9 in preventing and treating tomato fungal diseases, characterized in that, The tomato diseases include at least one of tomato bacterial wilt, tomato late blight and tomato leaf mold.

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