Prevention and treatment method for apple alternaria leaf spot

Abstract

The invention provides a control method for apple alternaria leaf spot, and belongs to the technical field of crop disease control. The prevention and treatment method comprises the following steps: spraying a foliar biological pesticide fertilizer to apple trees; the foliar biological medical fertilizer comprises a bacillus fermentation product and a trichoderma fermentation product, the bacillus is at least one of bacillus amyloliquefaciens TS-1203, bacillus subtilis B1 and bacillus subtilis BS-1208, and the bacillus is at least one of bacillus amyloliquefaciens TS-1203, bacillus subtilis B1 and bacillus subtilis BS-1208; the trichoderma is at least one of T6 and Ym. The combined action of bacillus and trichoderma is utilized, the morbidity of apple alternaria leaf spot can be effectively reduced, the growth of apple trees can be promoted, the photosynthetic capacity is improved, the combination of disease prevention and yield increase is realized, and the method is environment-friendly and suitable for green agricultural development.

Description

Technical Field

[0001] The invention belongs to the technical field of crop disease prevention and control, and in particular relates to a method for preventing and controlling apple leaf spot disease. Background Art

[0002] Apple leaf spot, caused by the pathogen Alternaria mali, primarily harms apple tree leaves, leading to premature leaf drop and weakened trees. In severe cases, fruit yield can drop by over 30%, causing significant economic losses to the apple industry. Currently, the disease is primarily controlled with chemical pesticides. However, the long-term and extensive use of chemical agents can lead to increased resistance in pathogens, excessive pesticide residues in fruits, and ecological damage, which is inconsistent with the needs of green agricultural development.

[0003] Therefore, the development of a bio-pesticide fertilizer that is highly effective in inhibiting bacteria, stable and long-lasting, promotes growth and increases yield, and is environmentally friendly is of great significance for solving the problem of preventing and controlling apple leaf spot disease and promoting the green and sustainable development of the apple industry. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for preventing and controlling apple leaf spot disease, which inhibits apple leaf spot disease by combining Bacillus and Trichoderma, reduces the incidence rate, and promotes the growth and yield of apple trees.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for preventing and controlling apple leaf spot disease comprises spraying foliar biopesticide fertilizer on apple trees; the foliar biopesticide fertilizer comprises a fermentation product of Bacillus and a fermentation product of Trichoderma; the Bacillus comprises at least one of Bacillus amyloliquefaciens TS-1203, Bacillus subtilis B1, and Bacillus subtilis BS-1208; and the Trichoderma comprises at least one of T6 and Ym.

[0007] Preferably, the fermentation product is a fermentation broth obtained by activating the Bacillus and Trichoderma species, preparing the seed solution, and fermenting and culturing the bacteria, or a fermentation filtrate obtained by centrifuging and filtering the fermentation broth.

[0008] Preferably, the Bacillus fermentation product comprises 1-2 parts by volume of Bacillus amyloliquefaciens TS-1203 fermentation product and / or 1-2 parts by volume of Bacillus subtilis B1 fermentation product and / or 1-2 parts by volume of Bacillus subtilis BS-1208 fermentation product.

[0009] Preferably, the Trichoderma fermentation product comprises 1-2 parts of Trichoderma T6 fermentation product and / or 1-2 parts of Trichoderma Ym fermentation product by volume.

[0010] Preferably, the Bacillus fermentation product and the Trichoderma fermentation product are compounded in a volume ratio of (1-2):(1-2).

[0011] Preferably, the foliar biopesticide fertilizer further comprises one or more of a surfactant, a stabilizer and a preservative.

[0012] More preferably, the surfactant is added in an amount of 0.1% to 1.0% of the total volume of the foliar biofertilizer, and is selected from one or more of Tween-20, Tween-80, agricultural organosilicon adjuvant and sodium dodecylbenzene sulfonate.

[0013] More preferably, the stabilizer is added in an amount of 0.5% to 1.5% of the total volume of the foliar biofertilizer, and is selected from one or more of sodium alginate, dipotassium hydrogen phosphate, and calcium carbonate.

[0014] More preferably, the amount of the preservative added is 0.05% to 0.15% of the total volume of the foliar biofertilizer, and the preservative is selected from one or more of sodium benzoate, streptomycin sulfate and sorbitol.

[0015] Preferably, the foliar biofertilizer is sprayed after apple blossoms fall and before the fruit expansion period, with an application rate of ≥10 mL / plant.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention utilizes the synergistic effect between microorganisms by compounding the fermentation products of specific Bacillus (Bacillus amyloliquefaciens TS-1203, Bacillus subtilis B1, Bacillus subtilis BS-1208) and Trichoderma (T6, Ym), significantly inhibiting the growth and spread of apple leaf spot pathogens, effectively reducing the incidence of the disease, reducing the problems of premature leaf fall and tree weakness, thereby reducing the risk of yield loss, and the foliar biofertilizer can increase the chlorophyll content of apple leaves while preventing disease, optimize photosynthesis-related physiological parameters, and enhance the photosynthetic capacity of fruit trees. Experiments show that it can promote the improvement of growth indicators such as plant height, stem thickness, fresh weight and root length of apple rootstock seedlings, achieve the dual effect of "disease prevention and growth promotion", and provide strong support for increasing production.

[0018] Compared with traditional chemical pesticides, the present invention uses microbial fermentation products as core ingredients, avoiding the problems of increased pathogen resistance, excessive pesticide residues in fruits, and ecological pollution caused by long-term use of chemical agents. The added surfactants, stabilizers, and preservatives are all low-toxic or biocompatible ingredients, and the dosage is controlled within a safe range. The whole process is environmentally friendly and in line with the concept of sustainable development of green agriculture. By optimizing the ratio of adjuvants, the foliar biofertilizer has good stability, uniform formulation properties, no precipitation and stratification, simple spraying operation, and is easy to promote and apply in large-scale orchards, reducing the difficulty of use in actual production. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Inhibition effect of four Bacillus fermentation broth on Venturia pyrina, in which A-D represent the growth of Venturia pyrina treated by Bacillus TS-1203, B1, BS-1208 and FS-1206 fermentation broth respectively, and E represents the control;

[0020] Figure 2 Inhibition effect of four Bacillus fermentation broth on Venturia pyrina;

[0021] Figure 3 Inhibition effect of four Bacillus fermentation broth on Venturia pyrina, in which A-D represent the growth of Venturia pyrina treated by Bacillus TS-1203, B1, BS-1208 and FS-1206 fermentation broth respectively, and E represents the control;

[0022] Figure 4 Inhibition effect of four Bacillus fermentation broth on Venturia pyrina;

[0023] Figure 5 Inhibition effect of 16 Trichoderma strains on Venturia pyrina, in which A-P represent the growth of Venturia pyrina treated by Trichoderma T6, Ym, S2, S1, S5, D5, T2, An1, S3, S1b, T1, An2, P6, B3, J1 and Lu3 respectively;

[0024] Figure 6 Inhibition effect of 16 Trichoderma strains on Venturia pyrina;

[0025] Figure 7 Inhibition effect of 16 Trichoderma strains on Venturia pyrina, in which A-P represent the growth of Venturia pyrina treated by Trichoderma T6, Ym, S2, S1, S5, D5, T2, An1, S3, S1b, T1, An2, P6, B3, J1 and Lu3 respectively;

[0026] Figure 8 Inhibition effect of 16 Trichoderma strains on Venturia pyrina;

[0027] Figure 9The control effect of different compound ratios of Bacillus fermentation filtrates on apple leaf spot disease is shown in the figure. In the figure, A1 to A18 are treatments with different compound ratios of Bacillus fermentation broth, among which A1 to A13 are treatments with different compound ratios of Bacillus fermentation filtrates in Table 4; A14, A15, and A16 represent treatments with TS-1203, B1, and BS-1208 fermentation filtrates, respectively; A17 is a control sprayed with sterile water; A18 is a blank control;

[0028] Figure 10 The figure shows the effect of different compound ratios of Bacillus fermentation filtrates on the area of ​​apple leaf spot disease lesions. In the figure, A1 to A18 are the front of leaves treated with different compound ratios of Bacillus fermentation filtrates, among which A1 to A13 are leaves treated with different compound ratios of Bacillus fermentation filtrates in Table 4; A14, A15, and A16 are leaves treated with TS-1203, B1, and BS-1208 fermentation filtrates, respectively; A17 is the control sprayed with sterile water; A18 is the blank control;

[0029] Figure 11 The effect of different compound ratios of Bacillus fermentation filtrate on the chlorophyll content of apple leaves;

[0030] Figure 12 The effect of different compound ratios of Trichoderma fermentation filtrates on the area of ​​apple leaf spot disease lesions. In the figure, B1 to B18 are the front of leaves treated with different compound ratios of Trichoderma fermentation filtrates, among which B1 to B13 are leaves treated with different compound ratios of Trichoderma fermentation filtrates in Table 5; B14, B15, and B16 are photos of leaves treated with T6, Ym, and S2 fermentation filtrates, respectively; B17 is a control sprayed with sterile water; B18 is a blank control, the same below;

[0031] Figure 13 The control effect of Trichoderma fermentation filtrate with different compound ratios on apple leaf spot disease;

[0032] Figure 14 The effect of different compound ratios of Trichoderma fermentation filtrate on the chlorophyll content of apple leaves;

[0033] Figure 15 The study is about the effects of different compound ratios of Bacillus fermentation filtrate and Trichoderma fermentation filtrate on the lesion area of ​​apple leaf spot disease.

[0034] Figure 16 The control effect of different compound ratios of Bacillus fermentation filtrate and Trichoderma fermentation filtrate on apple leaf spot disease;

[0035] Figure 17 The study is about the effects of different mixed ratios of Bacillus fermentation filtrate and Trichoderma fermentation filtrate on the chlorophyll content of apple leaves;

[0036] Figure 18The effects of different surfactants and their addition amounts on the antibacterial activity of TB;

[0037] Figure 19 The effects of different stabilizers and their addition amounts on the antibacterial activity of TB;

[0038] Figure 20 The effects of different addition amounts and types of preservatives on TB antibacterial activity;

[0039] Figure 21 Contour map (left) and three-dimensional surface map (right) of the interaction between organosilicon (A) and calcium carbonate (B);

[0040] Figure 22 The contour map (left) and three-dimensional surface map (right) of the interaction between organosilicone (A) and streptomycin sulfate (C);

[0041] Figure 23 The contour map (left) and three-dimensional surface map (right) of the interaction between calcium carbonate (B) and streptomycin sulfate (C);

[0042] Figure 24 To verify the optimal conditions for the inhibition of foliar biofertilizer on apple leaf spot pathogen;

[0043] Figure 25 It is the appearance characteristics of foliar biofertilizer;

[0044] Figure 26 Effects of foliar biofertilizer on the growth of apple rootstock seedlings. DETAILED DESCRIPTION

[0045] The invention provides a method for preventing and treating apple leaf spot disease, comprising spraying a foliar biopesticide fertilizer on apple trees; the foliar biopesticide fertilizer comprises a fermentation product of Bacillus and a fermentation product of Trichoderma; the Bacillus comprises at least one of Bacillus amyloliquefaciens TS-1203, Bacillus subtilis B1, and Bacillus subtilis BS-1208; and the Trichoderma comprises at least one of T6 and Ym.

[0046] The bacillus amyloliquefaciens TS-1203 of the present application is disclosed in "Antifungal Activity of Bacillus amyloliquefaciens TS-1203 against Valsa mali" (Hou B H, Xu B L, Hou T, et al. Antifungal Activity of Bacillus amyloliquefaciens TS-1203 against Valsa mali [J]. Journal of Gansu Agricultural University, 2017, 52(01): 80-86. DOI: 10.13432 / j.cnki.jgsau.2017.01.014.). The bacillus subtilis B1 is disclosed in "Affinity determination of three biocontrol bacteria and their inhibition effect on Valsa mali" (Li E C, Zhang S W, Xu B L, et al. Affinity determination of three biocontrol bacteria and their inhibition effect on Valsa mali [J]. Journal of Gansu Agricultural University, 2020, 55(05): 94-100. DOI: 10.13432 / j.cnki.jgsau.2020.05.011.). The bacillus subtilis BS-1208 is disclosed in "Screening of biocontrol bacteria against Moniliayunnanensis and analysis of its antagonistic mechanism" (Han K D, Liu J, Zhang S W, et al. Screening of biocontrol bacteria against Moniliayunnanensis and analysis of its antagonistic mechanism [J]. China Fruits, 2023, (10): 89-94. DOI: 10.16626 / j.cnki.issn1000-8047.2023.10.014.). The trichoderma T6 and Ym are disclosed in "Efficacy of Trichoderma longibrachiatum SC5 Fermentation Filtrate in Inhibiting the Sclerotinia sclerotiorum Growth and Development in Sunflower" (Li E, Zhu N, Zhang S, et al. Efficacy of Trichoderma longibrachiatum SC5 Fermentation Filtrate in Inhibiting the Sclerotinia sclerotiorum Growth and Development in Sunflower [J]. International Journal of Molecular Sciences, 2024, 26(1): 201.).

[0047] In the present application, the fermentation product is preferably a fermentation broth of bacillus and trichoderma obtained by strain activation, seed liquid preparation and fermentation culture, or a fermentation filtrate obtained by centrifugation and filtration of the fermentation broth.

[0048] Further preferably, the steps of the preparation method are as follows:

[0049] (1) Strain activation: Bacillus and Trichoderma are respectively activated and cultured; more preferably, Bacillus is inoculated in NA culture medium and cultured in a 28°C dark incubator for 12h for activation, and Trichoderma spore suspension is inoculated in PDA culture medium and cultured in a 25°C constant temperature incubator for 4d for activation;

[0050] (2) Seed liquid preparation: the activated Bacillus is inoculated in NB culture medium and cultured at 25-30°C and 150-200rpm for 10-14h to obtain Bacillus seed liquid; the activated Trichoderma is inoculated in PDB culture medium and cultured at 25-30°C and 150-200rpm for 1-2d to obtain Trichoderma seed liquid;

[0051] (3) Fermentation culture: the Bacillus seed liquid is inoculated in NYBD culture medium at an inoculation amount of 5%-10% and fermented and cultured at 25-30°C and 150-200rpm for 40-50h to obtain Bacillus fermentation broth; the Trichoderma seed liquid is inoculated in PDB culture medium at an inoculation amount of 1%-2% and fermented and cultured at 25-30°C and 150-200rpm for 6-8d to obtain Trichoderma fermentation broth;

[0052] More preferably, the viable cell count of the Bacillus fermentation broth is ≥10 8 cfu / mL, and the viable cell count (in spores) of the Trichoderma fermentation broth is ≥10 8 cfu / mL;

[0053] (4) or further centrifuging and filtering the fermentation broth to obtain fermentation filtrate;

[0054] (4.1) Centrifugation: the two kinds of fermentation broth are respectively subjected to centrifugation treatment, the centrifugation conditions being 4°C, 10000-14000rpm, and centrifugation for 20-40min, and the supernatant is taken;

[0055] (4.2) Filtration: the supernatant is filtered using a 0.22μm sterile microporous filter to obtain Bacillus fermentation broth filtrate and Trichoderma fermentation broth filtrate;

[0056] (5) Compounding;

[0057] (5.1) Bacillus fermentation product complex: preferably including Bacillus amyloliquefaciens TS-1203 fermentation product 1-2 parts by volume and / or Bacillus subtilis B1 fermentation product 1-2 parts by volume and / or Bacillus subtilis BS-1208 fermentation product 1-2 parts by volume; further preferably Bacillus amyloliquefaciens TS-1203 fermentation product 2 parts, Bacillus subtilis B1 fermentation product 1 part and Bacillus subtilis BS-1208 fermentation product 1 part;

[0058] (5.2) Trichoderma fermentation product complex: preferably including Trichoderma T6 fermentation product 1-2 parts by volume and / or Trichoderma Ym fermentation product 1-2 parts by volume; preferably Trichoderma T6 fermentation product 2 parts and Trichoderma Ym fermentation product 1 part;

[0059] (5.3) Bacillus fermentation product and Trichoderma fermentation product complex: preferably Bacillus fermentation product and Trichoderma fermentation product are complexed in a volume ratio of (1-2):(1-2); further preferably 2:1.

[0060] In the present application, the above-mentioned foliar bio-pesticide further includes one or more of surfactants, stabilizers and preservatives.

[0061] In the present application, the surfactant is preferably added in an amount of 0.1%-1.0% of the total volume of the foliar bio-pesticide, further preferably the surfactant is selected from one or more of Tween-20, Tween-80, agricultural organic silicon additive and sodium dodecyl benzene sulfonate, more preferably 0.5% agricultural organic silicon additive.

[0062] In the present application, the stabilizer is preferably added in an amount of 0.5%-1.5% of the total volume of the foliar bio-pesticide, further preferably the stabilizer is selected from one or more of sodium alginate, di-potassium hydrogen phosphate and calcium carbonate, more preferably 1.0% calcium carbonate.

[0063] In the present application, the preservative is preferably added in an amount of 0.05%-0.15% of the total volume of the foliar bio-pesticide, further preferably the preservative is selected from one or more of sodium benzoate, streptomycin sulfate and sorbitol, more preferably 0.1% streptomycin sulfate.

[0064] In the present application, one or more of the surfactant, stabilizer and preservative is directly mixed with the fermentation broth or filtrate of the foliar bio-pesticide and stirred uniformly.

[0065] In the present application, the foliar bio-pesticide is preferably sprayed at a period after flower falling and before fruit swelling; further preferably the spraying amount is ≥10 mL / plant, and the spraying amount of the foliar bio-pesticide is gradually increased with the increase of the age of the apple tree; more preferably the foliar bio-pesticide is diluted and sprayed to the apple tree leaves in the form of water droplets but not flowing down.

[0066] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] A method for preventing and treating apple leaf spot disease comprises the following steps:

[0069] Preparation of foliar biofertilizer:

[0070] (1) Bacteria activation:

[0071] Bacillus TS-1203, B1, and BS-1208: Inoculate into NA medium (3 g beef extract, 2.5 g glucose, 5 g peptone, 1 g agar, 1000 mL water), culture in a dark incubator at 28°C for 12 h, and then re-inoculate for later use.

[0072] Trichoderma T6 and Ym: Inoculate the spore suspension into PDA medium (200 g potato, 20 g glucose, 16 g agar, 1000 mL distilled water), culture in a constant temperature incubator at 25°C for 4 days, and then re-inoculate and set aside.

[0073] (2) Seed solution preparation:

[0074] Bacillus seed solution: Take the activated colony and inoculate it into NB culture medium (3g beef extract, 2.5g glucose, 5g peptone, 1000mL water), culture at 28℃ and 180rpm for 12h, and the number of viable cells should be ≥10 8 cfu / mL.

[0075] Trichoderma seed solution: Take the activated mycelium and inoculate it into PDB culture medium (200g potato, 20g glucose, 1000mL distilled water), culture it at 28℃ and 180rpm for 1.5 days, and the spore count should be ≥10 8 cfu / mL.

[0076] (3) Fermentation culture:

[0077] Bacillus fermentation liquid: inoculate 5% of the seed liquid into NYBD culture medium (8g beef extract, 10g glucose, 5g yeast extract, 1000mL water), and culture at 28°C and 180rpm for 48h. The viable cell count should be ≥10 8 cfu / mL.

[0078] Trichoderma fermentation liquid: add 1% seed liquid to PDB culture medium, culture at 28℃ and 180rpm for 7 days, and the spore count should be ≥10 8 cfu / mL.

[0079] (4) Preparation of fermentation filtrate:

[0080] The obtained fermentation broth was centrifuged at 4°C and 12000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm sterile microporous filter to obtain the fermentation broth filtrate;

[0081] (5) Compounding and additive addition:

[0082] Mix the Bacillus fermentation filtrate (TS-1203:B1:BS-1208 = 2:1:1) and the Trichoderma fermentation filtrate (T6:Ym = 2:1) in a volume ratio of 2:1 to obtain a base bacterial solution. Add the following additives (v / v): 0.5% agricultural organosilicon additive, 1.0% calcium carbonate, and 0.1% streptomycin sulfate to the base bacterial solution and stir evenly to obtain a foliar biofertilizer.

[0083] Application of foliar biological fertilizer: dilute and spray the leaves of apple trees after flower fall and before fruit expansion (before bagging) until water droplets appear but do not flow down. The spraying amount for seedlings is 10mL / plant. Gradually increase the spraying amount as the tree age increases.

[0084] Example 2

[0085] A method for preventing and treating apple leaf spot disease comprises the following steps:

[0086] Preparation of foliar biofertilizer:

[0087] (1) The activation of bacterial strains, preparation of seed solution, fermentation culture and preparation of filtrate were the same as those in Example 1.

[0088] (2) Compounding and additive addition:

[0089] The Bacillus fermentation filtrate (TS-1203:B1:BS-1208 = 1:1:1) and the Trichoderma fermentation filtrate (T6:Ym = 1:1) were mixed in a volume ratio of 1:1 to obtain a base filtrate. The following additives (V / V) were added to the base filtrate: 0.1% Tween-20, 0.5% sodium alginate, and 0.05% sodium benzoate. The mixture was stirred evenly to obtain a foliar biofertilizer.

[0090] Application of foliar biological fertilizer: dilute and spray the leaves of apple trees after the flowers fall and before the fruit expansion period (before bagging) until water droplets appear on the leaves but do not flow down.

[0091] Example 3

[0092] A method for preventing and treating apple leaf spot disease comprises the following steps:

[0093] Preparation of foliar biofertilizer:

[0094] (1) Bacteria activation, seed solution preparation, and fermentation culture were the same as in Example 1, but without filtration.

[0095] (2) Compounding and additive addition:

[0096] Mix a Bacillus fermentation solution (TS-1203:B1:BS-1208 = 1:2:1) and a Trichoderma fermentation solution (T6:Ym = 1:2) in a volume ratio of 2:1 to obtain a base solution. Add the following additives (v / v): 0.5% agricultural silicone additive, 0.5% Tween-80, 0.5% dipotassium hydrogen phosphate, 1% calcium carbonate, 0.1% streptomycin sulfate, and 0.05% sorbitol to the base solution and stir evenly to obtain a foliar biofertilizer.

[0097] Application of foliar biological fertilizer: dilute and spray the leaves of apple trees after the flowers fall and before the fruit expansion period (before bagging) until water droplets appear on the leaves but do not flow down.

[0098] Test Example 1

[0099] Screening of Bacillus strains antagonistic to apple leaf spot pathogen

[0100] 1. Test materials

[0101] 1.1 Test strains

[0102] Test pathogen: Apple leaf spot pathogen (Alternaria mali).

[0103] The tested Bacillus strains included Bacillus amyloliquefaciens TS-1203, Bacillus subtilis B1, Bacillus subtilis BS-1208, and Paenibacillus polymyxa FS-1206. All the above strains were provided by the Laboratory of Molecular Plant Virology, College of Plant Protection, Gansu Agricultural University.

[0104] 1.2. Types and components of test culture media

[0105] The main test culture media are shown in Table 1.

[0106] Table 1 Types and components of test culture media

[0107]

[0108]

[0109] 2. Test methods

[0110] 2.1. Activation of test strains

[0111] The apple leaf spot pathogen stored on a low-temperature slant was inoculated onto PDA medium, cultured in a constant temperature incubator at 25°C for 4 days, and then re-inoculated onto PDA medium for use; the test Bacillus was inoculated onto NA medium and cultured in a dark incubator at 35°C for 2 days, and then re-inoculated onto NA medium for use.

[0112] 2.2 Preparation of spore suspension of apple leaf spot pathogen

[0113] Apple leaf spot pathogen was inoculated onto PDA medium and cultured until spores were produced. 10 mL of sterile water containing 0.01% Tween-80 was added to the plate to wash the conidia of the apple leaf spot pathogen. Sterile water was added to 50 mL and the mycelium was filtered out using sterile filter paper. The spore suspension was counted using a hemocytometer and diluted to 1 × 10 with 0.5% sucrose solution. 6 cfu / mL.

[0114] 2.3 Preparation of Antagonistic Bacillus Fermentation Broth and Filtrate

[0115] The activated Bacillus was inoculated into 100 mL of NB culture medium, placed in a constant temperature shaker, and cultured in the dark at 28°C and 180 rpm for 12 hours to obtain a Bacillus seed solution. 3 mL of the seed solution was added to 60 mL of NYBD culture medium sterilized by high temperature using a pipette. The culture was shaken in the dark for 48 hours under the same conditions to obtain a Bacillus fermentation solution and stored at 4°C for later use. The fermentation solution was centrifuged at 4°C and 12,000 rpm for 30 minutes, and the supernatant was taken and filtered with a sterile microporous filter (pore size 0.22 μm) to obtain a Bacillus fermentation filtrate.

[0116] 2.4 Determination of antibacterial activity of antagonistic Bacillus fermentation broth

[0117] Sterile circular paper discs (d = 5 mm) were placed in a Bacillus fermentation broth and soaked for 5 minutes. Four discs were then placed at either end of a cross, 2 cm from the center of the PDA culture medium. Sterile circular paper discs soaked in sterile water served as controls. Apple leaf spot pathogen was inoculated in the center of the PDA culture medium and incubated in a 28°C incubator for 7 days. The cross-hatch method was used to measure the diameter of the pathogen colonies and calculate the inhibition rate. Each treatment and control were replicated three times.

[0118]

[0119] 2.5 Determination of antibacterial activity of antagonistic Bacillus fermentation filtrate

[0120] 6 mL of the Bacillus fermentation filtrate was pipetted into 60 mL of PDA medium to prepare the drug-containing medium. PDA medium supplemented with an equal amount of sterile water served as a control. Apple leaf spot pathogen (d = 5 mm) was inoculated in the center of the PDA medium and incubated in a 28°C incubator. The diameter of the pathogen colony was measured on the 7th day, and the inhibition rate was calculated. Each treatment and control were replicated three times.

[0121] 2.6 Effects of Antagonistic Bacillus Fermentation Filtrate on Spore Germination Activity

[0122] A hydrated culture medium containing 1% agar was prepared, sterilized at high temperature, and poured into a 90 mm diameter sterile Petri dish to create a water agar plate approximately 1-2 mm thick. A 100 μL mixture of a 1:1 ratio of a spore suspension of Pseudomonas aeruginosa and a Bacillus fermentation filtrate was pipetted and dropped onto the center of the water agar plate. The mixture was evenly spread using a sterilized glass spreader. The plate was covered and incubated in a 28°C incubator. A control group was treated with an equal volume of a 1:1 ratio of a spore suspension of Pseudomonas aeruginosa. 100-200 spores were counted for each treatment and control, and this was repeated three times. The germination of pathogen spores in each treatment was observed using a microscope. When the spore germination rate in the control group reached 90% or higher, the total number of spores and the number of germinated spores in each treatment were counted, and the spore germination rate and spore germination inhibition rate were calculated.

[0123]

[0124] 3. Data processing

[0125] The experimental data were statistically analyzed using Origin Pro 2021 software and Excel, and variance analysis and significance test were performed using IBM SPSS 20.0 software.

[0126] 4. Results and Analysis

[0127] 4.1 Determination of antibacterial activity of antagonistic Bacillus fermentation broth

[0128] After 7 days of inoculation and cultivation, the apple leaf spot pathogen colonies treated with the fermentation liquid of Bacillus TS-1203, B1, and BS-1208 grew close to the filter paper, and the hyphae stopped growing toward the filter paper, causing the apple leaf spot pathogen colonies to become irregular in shape. In particular, compared with the size of the control colonies, the diameter of the treated colonies was significantly smaller than that of the control ( Figure 1 A, B, C), the control colony grew normally and almost covered the entire culture dish ( Figure 1 E). However, Paenibacillus polymyxa FS-1206 had no significant effect on the growth of apple leaf spot pathogen colonies, and even the mycelium on the surface of the pathogen colonies covered the FS-1206 colonies ( Figure 1D). Compared with the control, different Bacillus fermentation broths had different growth inhibition effects on apple leaf spot pathogen, and the inhibition rates of different Bacillus fermentation broths were different. The inhibitory effect of Bacillus amyloliquefaciens TS-1203 fermentation broth on apple leaf spot pathogen was higher than that of other Bacillus, with an inhibition rate of 63.25% against the pathogen, followed by Bacillus subtilis B1 and BS-1208, with inhibition rates of 60.68% and 58.03%, respectively. However, the growth inhibition rate of Paenibacillus polymyxa FS-1206 on apple leaf spot pathogen was only 12.43%, which was significantly lower than the inhibition effect of other Bacillus ( Figure 2 ).

[0129] 4.2 Determination of antibacterial activity of antagonistic Bacillus fermentation filtrate

[0130] Different Bacillus fermentation filtrates have different inhibitory effects on apple leaf spot pathogen, and there are certain differences between different Bacillus ( Figure 3 The fermentation filtrate of Bacillus amyloliquefaciens TS-1203 had the best antibacterial effect on apple leaf spot pathogen, with an inhibition rate of 78.52%. The fermentation filtrates of Bacillus subtilis B1 and BS-1208 were second, with inhibition rates of 76.05% and 74.68%, respectively. The fermentation filtrate of Paenibacillus polymyxa FS-1206 had the lowest inhibitory effect on apple leaf spot pathogen, with an inhibition rate of only 12.43%. Figure 4 ).

[0131] 4.3 Effects of Antagonistic Bacillus Fermentation Filtrate on Spore Germination Activity

[0132] Different Bacillus fermentation filtrates exhibited varying degrees of inhibitory activity against the spores of the pathogen causing apple leaf spot. The results showed that treatment with the fermentation filtrates of Bacillus TS-1203, B1, and BS-1208 all achieved inhibition rates exceeding 70% (78.12%, 70.86%, and 70.36%, respectively). Treatment with the fermentation filtrate of Paenibacillus polymyxa FS-1206 exhibited a lower inhibition rate than that of the other three Bacillus strains, at only 26.78%, as shown in Table 2.

[0133] Table 2 Effects of 4 Bacillus fermentation filtrates on spore germination of apple leaf spot pathogen

[0134] strain Germination rate (%) Inhibition rate (%) Bacillus amyloliquefaciens TS-1203 19.70±2.12c 78.12±2.36a Bacillus subtilis B1 26.22±3.21c 70.86±3.56a Bacillus subtilis BS-1208 26.67±2.71c 70.36±3.01a Paenibacillus polymyxa FS-1206 65.89±0.39b 26.78±0.43b CK 90.19±1.67a -

[0135] Test Example 2

[0136] Screening of Trichoderma fungi antagonistic to apple leaf spot pathogen

[0137] 1. Test materials

[0138] Tested Trichoderma fungi: T6, Ym, S2, S1, S5, D5, T2, An1, S3, S1b, T1, An2, P6, B3, J1, Lu3.

[0139] Other details are the same as those in Experiment 1.

[0140] 2. Test methods

[0141] 2.1. Activation of test strains

[0142] Sixteen strains of Trichoderma preserved in low-temperature glycerol and apple leaf spot pathogen preserved in low-temperature slant were inoculated on PDA culture medium respectively, cultured in a constant temperature incubator at 25℃ for 4 days and then re-inoculated on PDA culture medium for use.

[0143] 2.2 Preparation of spore suspension of apple leaf spot pathogen

[0144] Same as Test Example 1.

[0145] 2.3 Determination of antibacterial activity against Trichoderma

[0146] Sixteen strains of Trichoderma were selected as test strains. Activated Trichoderma and the pathogen (5 mm diameter bacterial cake) were inoculated at two points on the same PDA medium, at least 20 mm apart. A PDA medium inoculated with only the pathogen cake served as a control. The cultures were incubated in a 28°C incubator. After 7 days of incubation, the diameters of the colonies were measured using the "cross method" to calculate the inhibition rate of Trichoderma against the pathogen. Each treatment and control were replicated three times.

[0147] 2.4. Preparation of Antagonistic Trichoderma Spore Suspension

[0148] After the Trichoderma strain was cultured on a PDA plate for 5 days, sterile water was added to the center of the plate, and the Trichoderma spores were washed off with a sterile small brush. The spores were transferred to a 10 mL sterile centrifuge tube and mixed. The spores were counted with a hemocytometer to prepare a concentration of 1 × 10 8 cfu / mL of Trichoderma spore suspension.

[0149] 2.5 Preparation of Antagonistic Trichoderma Fermentation Filtrate

[0150] The prepared Trichoderma spore suspension (concentration of 1×10 8 cfu / mL) was added to 100 mL PDB medium at an inoculum size of 1%. After culturing in a shaker at 28°C and 180 rpm for 7 days, the mycelium in the fermentation broth was removed by coarse filtration using sterile filter paper. The broth was centrifuged at 4°C and 12,000 rpm for 15 min, the precipitate was discarded, and the supernatant was filtered using a 0.22 μm sterile microporous filter to obtain the Trichoderma fermentation filtrate, which was stored at 4°C for later use.

[0151] 2.6 Determination of antibacterial activity of Trichoderma fermentation filtrate

[0152] The antibacterial activity of Trichoderma fermentation filtrate against apple leaf spot pathogen was determined using the medicated medium method and growth rate method. Prepared PDA medium was sterilized in an autoclave. Using a pipette, 6 mL of Trichoderma fermentation filtrate was added to 60 mL of PDA medium to create the medicated medium. PDA medium supplemented with an equal amount of sterile water served as a control. Apple leaf spot pathogen (d = 5 mm) was inoculated in the center of the PDA medium. Each treatment and control were replicated three times. After 7 days of incubation at 28°C, colony diameters were measured and the inhibition rate was calculated.

[0153] 2.7 Antagonistic Effects of Trichoderma Fermentation Filtrate on Spore Germination Activity

[0154] The effects of different Trichoderma fermentation filtrates on the spore germination activity of apple leaf spot pathogen were determined.

[0155] 3. Data processing

[0156] The experimental data were statistically analyzed using Origin Pro 2021 software and Excel, and variance analysis and significance test were performed using IBM SPSS 20.0 software.

[0157] 4. Results and Analysis

[0158] 4.1 Determination of antibacterial activity against Trichoderma

[0159] The results of the plate confrontation test showed that 16 strains of Trichoderma (T6, Ym, S2, S1, S5, D5, T2, An1, S3, S1b, T1, An2, P6, B3, J1, and Lu3) showed varying degrees of inhibitory effect on apple leaf spot pathogen. During the culture process, it was found that the tested Trichoderma had a fast growth rate and could quickly occupy most of the living space. After 7 days of culture, the apple leaf spot pathogen in the control group had already filled the culture dish, while the apple leaf spot pathogen in the treatment group was surrounded by Trichoderma and almost stopped growing. The colony diameter was significantly smaller than that of the control group ( Figure 5 A~P). Among them, the T6 strain of Trichoderma had the best inhibitory effect on apple leaf spot pathogen, with a mycelial growth inhibition rate of 67.27%. Ym and S2 had the second best inhibitory effect, with mycelial growth inhibition rates of 63.01% and 62.19% respectively. The three strains of Trichoderma had higher control effects on apple leaf spot pathogen than other Trichoderma strains. The mycelial growth inhibition rates of the other Trichoderma strains ranged from 46.15% to 59.90%. Figure 6 ).

[0160] 4.2 Determination of antibacterial activity of Trichoderma fermentation filtrate

[0161] The fermentation filtrates of 16 Trichoderma strains (T6, Ym, S2, S1, S5, D5, T2, An1, S3, S1b, T1, An2, P6, B3, J1, and Lu3) all had a certain inhibitory effect on the mycelial growth of apple leaf spot pathogen, and the inhibitory effects of different Trichoderma fermentation filtrates on the pathogen varied. On the 7th day, the growth of apple leaf spot pathogen on the medium containing Trichoderma fermentation filtrates was less than that of the control ( Figure 7 A~P). Among them, the fermentation filtrates of Trichoderma strains T6, Ym, and S2 had the best inhibitory effect on apple leaf spot pathogen, with inhibition rates exceeding 50%, namely 69.01%, 57.51%, and 50.05%, respectively. The inhibition rates of the fermentation filtrates of other Trichoderma strains on apple leaf spot pathogen ranged from 8.99% to 48.71%. Figure 8 ).

[0162] 4.3 Antagonistic Effects of Trichoderma Fermentation Filtrate on Spore Germination Activity

[0163] Compared to the control, the fermentation filtrates of different Trichoderma strains all inhibited the germination of pathogen spores, and the inhibition rates of the different Trichoderma fermentation filtrates on pathogen spore germination varied. The results showed that the fermentation filtrates of Trichoderma strains Ym, T6, and S2 were particularly effective in inhibiting the germination of spores of the apple leaf spot pathogen. The T6 fermentation filtrate had the highest inhibition rate against the apple leaf spot pathogen, at 68.09%, followed by Ym and S2, with inhibition rates of 64.01% and 62.91%, respectively. With the exception of the three Trichoderma strains mentioned above, the inhibition rates of the fermentation filtrates of the remaining Trichoderma strains against the apple leaf spot pathogen were all below 60%, as shown in Table 3.

[0164] Table 3 Effects of 16 strains of Trichoderma fermentation filtrate on spore germination of apple leaf spot pathogen

[0165]

[0166]

[0167] Test Example 3

[0168] 1. Test materials

[0169] Test pathogen: Apple leaf spot pathogen.

[0170] Apple leaves for testing were collected from the apple plantation of Gansu Agricultural University, and the variety was 'Fuji'.

[0171] Bacillus fermentation filtrate: same as Test Example 12.3.

[0172] Trichoderma fermentation filtrate: same as Test Example 22.5.

[0173] 2. Test method

[0174] 2.1, Antagonistic bacteria fermentation filtrate complex ratio design

[0175] Bacillus fermentation filtrate complex ratio: the prepared fermentation filtrate of 3 strains of Bacillus (TS-1203, B1, BS-1208) was mixed according to the complex ratio of Table 4, placed in a 10 mL sterile centrifuge tube, and stored at 4°C for standby use.

[0176] Trichoderma fermentation filtrate complex ratio: the prepared fermentation filtrate of 3 strains of Trichoderma (T6, Ym, S2) was mixed according to the complex ratio of Table 5, placed in a 10 mL sterile centrifuge tube, and stored at 4°C for standby use.

[0177] Table 4 Different Bacillus fermentation filtrate complex ratio

[0178]

[0179] Table 5 Different Trichoderma fermentation filtrate complex ratio

[0180]

[0181]

[0182] 2.2, In vitro control effect of different antagonistic bacteria fermentation filtrate complex on apple spot and leaf fall disease

[0183] Select fresh and healthy apple leaves, first rinse with clean water, then disinfect with 75% alcohol for 15s, then rinse with sterile water and dry, use a inoculation needle to make a wound on the apple leaf, inoculate the apple spot and leaf fall disease bacteria cake (d=5mm) cultured for 5-7d on the wound, wrap the stem with cotton, spray sterile water on the cotton for moisturizing treatment, 2 inoculation points per leaf, 3 leaves per treatment, place in a 25°C constant temperature incubator, cultivate for 24h, then spray different Bacillus fermentation filtrate complex liquid and different Trichoderma fermentation filtrate complex liquid in Table 4 and 5, and spray equal amount of TS-1203, B1, BS-1208 and T6, Ym, S2 fermentation filtrate as single strain control, spray equal amount of sterile water as negative control, and only inoculate PDA as blank control. Continue constant temperature and moisturizing cultivation, measure the lesion size after 10d, calculate the lesion area and control effect.

[0184]

[0185] 2.3, Effect of different antagonistic bacteria complex fermentation filtrate on chlorophyll content of apple in vitro leaves

[0186] Apple leaves (0.2 g) treated with the various Bacillus fermentation filtrate mixtures and Trichoderma fermentation filtrate mixtures listed in Tables 4 and 5 were placed in a 10 mL test tube containing an ethanol / acetone solution (v:v = 1:1) and incubated in the dark. The chlorophyll content was calculated using a spectrophotometer using absorbance values ​​at 649 nm and 665 nm. The specific calculation formula is as follows:

[0187] Chlorophyll a (Chla) = 13.95 × D665 - 6.88 × D649;

[0188] Chlorophyll b (Chlb) = 24.96×D649-7.32×D665.

[0189] 2.4. Screening of the optimal compound ratio of antagonistic bacteria fermentation filtrate

[0190] Based on the screened out different Trichoderma fermentation filtrate composite liquids (T) and different Bacillus fermentation filtrate composite liquids (B) with the best control effect on apple leaf spot disease and the most significant effect on chlorophyll content, they were compounded in ratios of 1:1, 1:2 and 2:1, respectively, and the control effect of the composite liquids in different ratios on apple leaf spot disease and the chlorophyll content were measured. The mixing ratio with the best control effect on apple leaf spot disease was screened out, which is the main active ingredient of this biological foliar fertilizer.

[0191] 3. Data processing

[0192] The experimental data were statistically analyzed using Origin Pro 2021 software and Excel, and variance analysis and significance test were performed using IBM SPSS 20.0 software.

[0193] 4. Test results

[0194] 4.1. In vitro efficacy of Bacillus fermentation filtrate complex against apple leaf spot

[0195] The results showed that the control effects of different Bacillus fermentation filtrates on detached leaves inoculated with apple leaf spot pathogen were different ( Figure 9 Among the different compound ratios of Bacillus fermentation filtrates, when the TS-1203:B1:BS-1208 compound ratio was 2:1:1 (B), the control effect on the detached leaves inoculated with pathogens was stronger than that of other compound ratios and higher than that of a single strain, and the area of ​​lesion expansion was the smallest ( Figure 10 ), with a control efficacy of 84.16%, which was significantly different from the control. Furthermore, compared with the fermentation filtrates of single Bacillus TS-1203, B1, and BS-1208, the control efficacy was increased by 16.95%, 22.00%, and 25.62%, respectively.

[0196] 4.2 Effects of Bacillus fermentation filtrate complex on chlorophyll content in apple leaves

[0197] The chlorophyll content in apple leaves increased under the treatment of different Bacillus fermentation filtrates. Figure 11 As shown in the figure, the chlorophyll content of apple leaves treated with different Bacillus fermentation filtrates was significantly higher than that of apple leaves treated with a single Bacillus fermentation filtrate, and there was a certain difference in chlorophyll content compared with the control. When the ratio of TS-1203, B1 and BS-1208 fermentation filtrates was 2:1:1 (B), the chlorophyll a content of the treated apple leaves was 26.38 mg / g ( Figure 11 A); chlorophyll b content is 15.85 mg / g ( Figure 11 B), compared with the control, the chlorophyll a+b content increased by 21.02 mg / g ( Figure 11 C). Furthermore, compared to single Bacillus TS-1203, B1, and BS-1208 fermentation filtrates, chlorophyll content in apple leaves increased by 16.42 g / mg, 17.72 g / mg, and 20.61 g / mg, respectively. This suggests that compounding Bacillus fermentation filtrates increases chlorophyll content in plants.

[0198] 4.3 In vitro control of Trichoderma fermentation filtrate against apple leaf spot

[0199] The results showed that among the different mixture ratios of Trichoderma fermentation filtrates, when the mixture ratio of Trichoderma strain T6 and Ym fermentation filtrates was 2:1 (T), the control effect on detached leaves inoculated with pathogens was stronger than that of other mixture ratios of Trichoderma fermentation filtrates and higher than that of single strain fermentation filtrates, and the area of ​​lesion expansion was the smallest ( Figure 12 B13), the control effect was 78.38%, and the difference was significant compared with the control. At the same time, compared with the fermentation filtrate of single Trichoderma T6 and Ym, the control effect was increased by 15.79% and 18.64% respectively ( Figure 13 ).

[0200] 4.4 Effects of Trichoderma fermentation filtrate on chlorophyll content in apple leaves

[0201] The chlorophyll content of apple leaves increased under the treatment of different Trichoderma fermentation filtrates. Figure 14 As shown in the results, the chlorophyll content of apple leaves treated with a mixture of different Trichoderma fermentation filtrates was significantly higher than that of apple leaves treated with a single Trichoderma fermentation filtrate, and there was a certain difference in chlorophyll content compared with the control. When the mixture ratio of T6 and Ym fermentation filtrate was 2:1 (T), the chlorophyll a content of the treated apple leaves was 26.19 mg / g ( Figure 14 A); chlorophyll b content is 23.67 mg / g ( Figure 14 B), compared with the control, the chlorophyll a+b content increased by 19.90 mg / g ( Figure 14 C). Furthermore, compared to the effects of single Trichoderma T6 and Ym fermentation filtrates on apple leaf chlorophyll content, chlorophyll content increased by 17.39 g / mg and 17.84 g / mg, respectively. This suggests that the combination of Trichoderma fermentation filtrates increases chlorophyll content in plants.

[0202] 4.5. Screening of the optimal compound ratio of antagonistic bacteria fermentation filtrate

[0203] Mixing different Trichoderma fermentation filtrates (T) and different Bacillus fermentation filtrates (B) in the ratio of 1:1, 1:2 and 2:1 respectively can inhibit the expansion of the lesion area of ​​apple leaf spot disease. When T:B is 1:2, the control effect on the detached leaves inoculated with the pathogen is stronger than that of other compound ratios, and the area of ​​lesion expansion is the smallest ( Figure 15 ), the control effect was 88.37% ( Figure 16 The chlorophyll content of apple leaves under the three compound ratio treatments increased. When T:B was 1:2, the chlorophyll a+b content increased by 27.85g / mg compared with the control ( Figure 17 ).

[0204] Test Example 4

[0205] Development of foliar biopesticide fertilizer

[0206] 1. Materials and Methods

[0207] 1.1 Test Materials

[0208] Test pathogen: Apple leaf spot pathogen.

[0209] Test reagent

[0210] Surfactants: Tween-20, Tween-80, agricultural silicone adjuvants and sodium dodecylbenzenesulfonate.

[0211] Stabilizers: sodium alginate, dipotassium hydrogen phosphate and calcium carbonate.

[0212] Preservatives: Sodium benzoate, streptomycin sulfate and sorbitol.

[0213] 2. Test methods

[0214] 2.1. Preparation of foliar biofertilizer stock solution (TB) is the same as that in Experimental Example 32.4.

[0215] 2.2. Additive screening

[0216] 2.2.1. Screening of surfactants

[0217] Four surfactants (Tween-20, Tween-80, an agricultural silicone adjuvant, and sodium dodecylbenzene sulfonate) were added to foliar biofertilizer (TB) at 0.1%, 0.5%, and 1.0% concentrations, respectively. The mixture was then incubated at 25°C. After 48 hours, 6 mL of the surfactant-added TB was pipetted into 60 mL of sterilized PDA medium. The medium was shaken slowly while hot to avoid bubbles. This yielded the drug-containing medium. TB without surfactant served as the negative control (CK), and PDA medium supplemented with an equal volume of sterile water served as the blank control. Apple leaf spot pathogen (d = 5 mm) was inoculated in the center of the PDA plate. After 7 days of incubation at 28°C, colony diameters were measured, and the inhibition rate was calculated. Each treatment and control were replicated three times.

[0218] 2.2.2. Screening of stabilizers

[0219] Three stabilizers (sodium alginate, dipotassium hydrogen phosphate, and calcium carbonate) were added to the foliar fertilizer TB at 0.5%, 1.0%, and 1.5% respectively, and the mixture was thoroughly shaken and evenly mixed. Then, the mixture was placed at 25°C room temperature. After 48 hours, 6 mL of the foliar bio-fertilizer TB with stabilizers added was pipetted and added to 60 mL of PDA culture medium and mixed evenly to prepare a drug-containing culture medium. The foliar bio-fertilizer without stabilizers was used as the negative control (CK), and the PDA culture medium with an equal volume of sterile water was used as the blank control. The apple leaf spot pathogen cake (d = 5 mm) was inoculated in the center of the plate. After culturing in a 28°C incubator for 7 days, the colony diameter was measured, and the antibacterial rate was calculated. The stabilizer with better biocontrol effect on apple leaf spot was screened out based on the antibacterial rate. Each treatment and control in the experiment were repeated 3 times.

[0220] 2.2.3. Screening of preservatives

[0221] Three preservatives (sodium citrate, streptomycin sulfate, and sorbitol) were added to foliar biofertilizer (TB) at 0.05%, 0.10%, and 0.15% respectively. The mixture was then thoroughly mixed and placed at 25°C. After 48 hours, 6 mL of the preservative-added TB was pipetted into 60 mL of PDA medium to obtain the drug-containing medium. The TB without preservative served as the negative control (CK), and an equal volume of sterile water was added to the PDA medium as the blank control. The apple leaf spot pathogen was inoculated in the center of the PDA medium and incubated in a 28°C incubator for 7 days. The pathogen colony diameter was then measured with a vernier caliper, and the inhibition rate was calculated. The preservatives with the best inhibitory effect against the apple leaf spot pathogen were screened based on the inhibition rate. Each treatment and control were replicated three times.

[0222] 2.3, the response surface optimization test of the adjuvant

[0223] According to the results of single factor test, the surfactant organic silicon, stabilizer calcium carbonate and preservative streptomycin sulfate were selected as the best adjuvant of the preparation, which were set as variables A, B and C respectively. The inhibition rate of the leaf biological fertilizer TB was used as the response value, and the Box-Behnken response surface analysis method was used to optimize the addition amount of the adjuvant of the leaf biological fertilizer, so as to obtain the optimal addition amount of the adjuvant, and verify the feasibility of the response surface analysis.

[0224] 2.4, preparation of the leaf biological fertilizer

[0225] The fermentation filtrate compound liquid of trichoderma and bacillus was mixed with the selected surfactant organic silicon, stabilizer calcium carbonate and preservative streptomycin sulfate according to the optimal addition amount to prepare the leaf biological fertilizer, and the characteristics were observed and saved for standby.

[0226] 3, data processing

[0227] The experimental data was statistically analyzed by Origin Pro 2021 software and Excel, and the variance analysis and significant difference test were carried out by IBM SPSS 20.0 software.

[0228] 4, results and analysis

[0229] 4.1, adjuvant screening

[0230] 4.1.1 screening of surfactant

[0231] The screening results of surfactant show that Figure 18 ), the leaf biological fertilizer TB with different surfactants has different inhibition effects on the apple spot and leaf disease fungus, which shows that these surfactants will affect the inhibition activity of the leaf biological fertilizer TB on the apple spot and leaf disease fungus to some extent. Except for the agricultural organic silicon, with the increase of the concentration of surfactant, the inhibition activity of the leaf biological fertilizer is continuously reduced. When the addition amount of Tween-20, Tween-80 and sodium dodecyl benzene sulfonate is 0.1%, the inhibition rate of the leaf biological fertilizer TB on the apple spot and leaf disease fungus is 75.17%, 77.69% and 70.71% respectively. After adding agricultural organic silicon, with the increase of its concentration, the inhibition effect first increases and then decreases, and when the addition amount is 0.5%, the inhibition rate is the highest, which is 84.22%. In summary, 0.5% of agricultural organic silicon adjuvant is selected as the surfactant of the leaf biological fertilizer TB.

[0232] 4.1.2, screening of stabilizer

[0233] From the results of the screening of the stabilizer Figure 19As shown, compared with the control, the addition of different amounts of the three stabilizers reduced the antibacterial activity of TB foliar fertilizer against apple leaf spot pathogen. Different additions of sodium alginate and dipotassium hydrogen phosphate reduced the antibacterial activity of TB foliar fertilizer to varying degrees. When calcium carbonate was added at a 1.0% level, the inhibition rate of TB foliar fertilizer against apple leaf spot pathogen reached 87.83%. While this reduced the antibacterial activity of TB foliar fertilizer compared to the control, the difference was not significant. Therefore, adding 1.0% calcium carbonate was selected as the stabilizer for TB foliar fertilizer.

[0234] 4.1.3 Screening of preservatives

[0235] The different addition amounts of the three preservatives all had different degrees of inhibitory effects on the antibacterial activity of foliar biofertilizer TB ( Figure 20 ), sodium citrate and sorbitol showed the most significant inhibitory effects, achieving inhibition rates of 77.73% and 77.45%, respectively, at a 0.1% addition level. At a 0.1% addition level, streptomycin sulfate (TB), a foliar biofertilizer, exhibited higher antibacterial activity than the other two preservatives, achieving an inhibition rate of 88.51%. Therefore, streptomycin sulfate is a suitable preservative for foliar biofertilizer TB at a 0.1% addition level.

[0236] 4.2. Response surface optimization experiment of additives

[0237] 4.2.1 Response Surface Optimization Design

[0238] Based on the results of the single-factor experiments with the three adjuvants, a Box-Benhnken Design experiment was used to optimize the addition levels of the surfactant agricultural organosilicon, the stabilizer calcium carbonate, and the preservative streptomycin sulfate. The optimal dosage of each factor was determined using the designed factor levels shown in Table 6, with three levels for each factor. A response surface analysis was conducted using Design Expert 8.0. The response value was the inhibition rate against apple leaf spot pathogen. The surfactant agricultural organosilicon, the stabilizer calcium carbonate, and the preservative streptomycin sulfate were selected as independent variables. A total of 17 central combinations (3 factors, 3 levels) were designed, as shown in Table 7.

[0239] Table 6 Box-Behnken factor levels and coding values

[0240]

[0241] Table 7 Box-Behnken test design and results

[0242]

[0243] 4.2.2 Regression Model and Analysis

[0244] The results of multiple regression fitting showed that the regression equation between the influencing factors of organosilicon (A), calcium carbonate (B) and streptomycin sulfate (C) and the inhibition rate of foliar biofertilizer TB against apple leaf spot pathogen was: Y = 79.09-1.11A+2.65B+3.91C-2.06AB-3.97AC+0.485BC-4.69A 2 -6.71B 2 -4.79C 2 In the equation, Y is the predicted value of the inhibition rate of the compound preparation TB against apple leaf spot pathogen.

[0245] Table 8 Analysis of variance of regression model

[0246] source sum of squares degrees of freedom mean square F-number P-value Model 681.61 9 75.73 97.12 <0.0001** A-Silicone 9.86 1 9.86 12.64 0.0093* B-Calcium Carbonate 56.13 1 56.13 71.98 <0.0001** C-Streptomycin sulfate 122.23 1 122.23 156.70 <0.0001** AB 17.02 1 17.02 21.82 0.0023* AC 62.96 1 62.96 80.74 <0.0001** BC 0.9409 1 0.9409 1.21 0.3083 A2 88.63 1 88.63 113.66 <0.0001** B2 189.60 1 189.60 243.14 <0.0001** C2 92.63 1 92.63 118.79 <0.0001** residual 5.46 7 0.7798 - - Lack of Fit 2.63 3 0.8778 1.24 0.4048 Pure error 2.83 4 0.7063 - - sum 687.07 16 - - -

[0247] Note: * indicates significant, ** indicates extremely significant.

[0248] The variance analysis results in Table 8 show that the regression level of the model is significant (p < 0.0001). The three additives and the response values ​​are not simply linearly correlated, and there is an interaction between the additives. R 2 =0.9921, indicating a good fit; the F values ​​of the three factors A, B, and C show that streptomycin sulfate and calcium carbonate foliar biofertilizers have the greatest impact on the antibacterial activity of TB, followed by agricultural organosilicon.

[0249] 4.2.3 Analysis of the interaction effect of additive dosage

[0250] The 3D surface plot and contour plot of the response surface were drawn using Design-Expert software. The results are shown in the figure. Figure 21 、 22 From the 23 two-dimensional contour map, we can see that Figure 21 and Figure 23 The contour map in the figure is close to a circle, indicating that the interaction between the two factors A (organic silicon)-B (calcium carbonate) and B (calcium carbonate)-C (streptomycin sulfate) is not significant; Figure 22 The contour lines in the contour map are close to an ellipse, indicating that the interaction between the two factors A (silicone) and C (streptomycin sulfate) is significant; from the surface graphs 21, 22 and 23, it can be seen that the three-dimensional graph of B (calcium carbonate) and C (streptomycin sulfate) has a larger slope and a strong interaction, indicating that the antibacterial activity sensitivity of foliar biofertilizer TB is greatly affected by these two factors.

[0251] 4.2.4 Optimization and verification of optimal fermentation conditions using response surface analysis

[0252] Based on the above test results, the optimal additive amount formula was simulated: surfactant organosilicon 0.5%, stabilizer calcium carbonate 1.0%, preservative streptomycin sulfate 0.1%, i.e. the optimal additive amount of leaf biological pesticide fertilizer TB is 0.0363 mL of surfactant organosilicon, 0.066 g of stabilizer calcium carbonate, and 0.0066 mL of preservative streptomycin sulfate in 6 mL of leaf biological pesticide fertilizer. In order to test the actual effect of the optimization test, the bacteriostatic test was carried out with the optimized additive formula. As shown in Table 4, the bacteriostatic rate of leaf biological pesticide fertilizer under the optimized additive formula was 82.98%, which was close to the predicted value of 80.09%, proving that the regression model was reliable. Figure 24

[0253] 4.3 Appearance of leaf biological pesticide fertilizer

[0254] Based on the optimal additive amount of surfactant organosilicon, stabilizer calcium carbonate, and preservative streptomycin sulfate, a leaf biological pesticide fertilizer was prepared with Trichoderma and Bacillus fermentation filtrate complex liquid as the main component. The prepared leaf biological pesticide fertilizer was a transparent liquid, easily soluble in water, with strong fluidity; no precipitate was generated after standing, and no stratification phenomenon occurred; fine foam was generated after shaking, and did not dissipate after standing for 30 min; the color was yellow, with a faint fermentation odor. As shown in Table 5. Figure 25

[0255] Test Example 5

[0256] Effect of leaf biological pesticide fertilizer on apple spot and leaf fall disease control and seedling growth

[0257] 1. Materials and methods

[0258] 1.1 Materials

[0259] Test material: apple rootstock seedlings (G935, semi-dwarfing rootstock), purchased from Jingning Zhongguohui Feng Agricultural Technology Co., Ltd.

[0260] Test agent: leaf biological pesticide fertilizer (4.3 leaf biological pesticide fertilizer in Test Example 3).

[0261] Test pathogen: apple spot and leaf fall disease fungus.

[0262] Soil: purchased from Yuzhong County Huimin Flower Industry Development Co., Ltd.

[0263] 1.2 Methods

[0264] 1.2.1 Transplanting of apple rootstock seedlings

[0265] ​​The apple rootstock seedlings were placed in a greenhouse, watered every 3 days, and after 5 days of seedling, the apple rootstock seedlings with uniform height, straight stem and healthy growth were transplanted into plastic pots with the same size. After transplanting, water was sprayed every 3 days in the morning, and the seedlings were cultured in the greenhouse for use.

[0266] 1.2.2, Preparation of the spore suspension of V. mali.

[0267] The same as in Test Example 1.

[0268] 1.3, In vitro control effect of the foliar bio-pesticide on V. mali

[0269] The apple branch with uniform growth was selected, the surface of the leaves was washed with sterilized distilled water, and then naturally dried. The following treatments were carried out:

[0270] (1) Preventive effect: the foliar bio-pesticide was sprayed on the surface of the leaves, and then placed in a greenhouse for 24 hours, and then the spore suspension of V. mali was sprayed;

[0271] (2) Therapeutic effect: the spore suspension of V. mali was sprayed first, and then the foliar bio-pesticide was sprayed after 24 hours of incubation in a greenhouse, and then incubated in a greenhouse;

[0272] (3) Control treatment: the spore suspension of V. mali was sprayed on the leaves, and the treatment with sterile water was used as a control.

[0273] After 10 days of incubation, all the leaves were investigated, the total number of leaves and the number of leaves at each level were recorded according to the classification standard, and the disease index, the control effect and the preventive effect were calculated.

[0274] Classification standard: 0 level: no disease spot; 1 level: disease spot area accounts for less than 10% of the whole leaf area; 3 level: disease spot area accounts for 11% to 25% of the whole leaf area; 5 level: disease spot area accounts for 26% to 40% of the whole leaf area; 7 level: disease spot area accounts for 41% to 65% of the whole leaf area; 9 level: disease spot area accounts for more than 65% of the whole leaf area.

[0275] The calculation formula is as follows:

[0276]

[0277] 2, Test design

[0278] Six apple rootstock seedlings were divided into two groups, three in each group, and the following treatments were carried out simultaneously. Treatment group: 10 mL of leaf bio-pesticide was sprayed on each plant. Control group: 10 mL of sterile water was sprayed. On the 5th day, the spraying treatment was repeated. After 10 days of apple rootstock seedling growth, the relevant indicators were measured, and each treatment and control was repeated three times.

[0279] 3. Determination of indicators and methods

[0280] 3.1. Biomass determination

[0281] After spraying leaf bio-pesticide and sterile water on apple rootstock seedlings, all test seedlings were pulled out and rinsed with sterile water, then the surface water was absorbed with absorbent paper, and the growth indicators (including plant height, stem diameter, aboveground fresh weight, underground fresh weight, and root length) were measured.

[0282] 3.2. Photosynthetic parameter determination

[0283] In the greenhouse of the Plant Protection College, the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) of leaves treated with leaf bio-pesticide and sterile water were measured using a Ciras-3 photosynthetic instrument. Good ventilation was maintained in the greenhouse during testing.

[0284] 3.3. Chlorophyll content determination

[0285] The ethanol / acetone extraction method was used to determine the chlorophyll content. Leaf samples (0.2 g) from apple rootstock seedlings treated with leaf bio-pesticide and sterile water were placed in 10 mL test tubes containing ethanol / acetone solution (v:v = 1:1) and incubated in the dark. The absorbance values at 649 nm and 665 nm were measured using a spectrophotometer, and the chlorophyll content was calculated. The specific calculation formula is as follows:

[0286] Chlorophyll a (Chla) = 13.95 x D665 - 6.88 x D649;

[0287] Chlorophyll b (Chlb) = 24.96 x D649 - 7.32 x D665.

[0288] 4. Data analysis

[0289] The experimental data was statistically analyzed using Origin Pro 2021 software and Excel, and IBM SPSS 20.0 software was used for variance analysis and significant difference test.

[0290] 5. Results and analysis

[0291] 5.1. Treatment and prevention effect of leaf bio-pesticide on apple scab

[0292] Table 9 shows that, in vitro, foliar biofertilizers have a good control effect on leaf spot disease, exceeding 55%. When the biofertilizer was sprayed first and then the leaf spot disease spore suspension was applied, the preventive effect was 64.05%. When the apple leaves were first treated with the leaf spot disease spore suspension and then the biofertilizer was applied, the treatment effect was 56.39%.

[0293] Table 9 Preventive and therapeutic effects of foliar biopesticide fertilizers

[0294]

[0295] 5.2 Effects of foliar biofertilizers on the growth of apple rootstock seedlings

[0296] The potted plant experiment showed that the apple rootstock seedlings in the treatment group (sprayed with foliar biopesticide fertilizer) grew better, with thicker stems and more leaves, while the control group (sprayed with sterile water) appeared shorter than the treatment group, indicating that foliar biopesticide fertilizer had a significant growth-promoting effect on apple rootstock seedlings. Figure 26 shown.

[0297] After the apple rootstock seedlings were pulled out and rinsed, plant height, stem diameter, aboveground fresh weight, belowground fresh weight, and root length were measured for the control and treatment groups. The results in Table 10 show that the plant height, stem diameter, aboveground fresh weight, belowground fresh weight, and root length of the control group were 14.13 cm, 2.09 mm, 2.16 g, 0.55 g, and 91.71 mm, respectively; while the plant height, stem diameter, aboveground fresh weight, belowground fresh weight, and root length of the treatment group were 20.41 cm, 2.33 mm, 4.09 g, 1.31 g, and 104.81 mm, respectively. Compared with the control group, the plant height, stem diameter, aboveground fresh weight, belowground fresh weight, and root length of the treatment group increased by 6.28 cm, 0.24 mm, 1.93 g, 0.76 g, and 13.7 mm, respectively. Therefore, the foliar biofertilizer not only has a good preventive and control effect on apple leaf spot disease, but also can increase the biomass of apple rootstock seedlings and has a good growth-promoting effect on the growth of apple rootstock seedlings.

[0298] Table 10 Effects of foliar biofertilizers on the biomass of apple rootstock seedlings

[0299] deal with Plant height (cm) Stem diameter (mm) Fresh weight above ground (g) Underground fresh weight (g) Root length (mm) Foliar biofertilizer 20.41±0.48a 2.33±0.05a 4.09±0.14a 1.31±0.12a 104.81±4.02a comparison 14.13±0.29b 2.09±0.16b 2.16±0.18b 0.55±0.05b 91.71±2.01b

[0300] 5.3 Effects of foliar biofertilizers on chlorophyll content in apple rootstock seedlings

[0301] The chlorophyll content of the leaf after the foliar fertilizer treatment is increased. As shown in Table 11, after the foliar bio-fertilizer treatment, the chlorophyll content of the leaf is obviously increased compared with the control group. According to the specific analysis, after the foliar bio-fertilizer treatment, the chlorophyll a content of the treatment group is 1.28 times of the control group, and the chlorophyll b content is 1.42 times of the control group.

[0302] Table 11 Influence of foliar bio-fertilizer on chlorophyll content of apple rootstock seedlings

[0303] deal with Chlorophyll a (mg / g) Chlorophyll b (mg / g) Chlorophyllin a+b (mg / g) Foliar biofertilizer 19.69±0.31a 8.41±0.19a 28.10±0.50a comparison 15.28±0.21b 5.94±0.08b 21.22±0.29b

[0304] 5.4, Influence of foliar bio-fertilizer on photosynthetic capacity of apple rootstock seedlings

[0305] As shown in Table 12, after the foliar bio-fertilizer treatment, the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs) are all increased compared with the control, and are increased by 1.7 μmol / m 2 ·s, 0.63 mmol / m 2 ·s, 0.66 μmol / mol, and 8.95 mmol / m 2 ·s, respectively.

[0306] Table 12 Influence of foliar bio-fertilizer on photosynthetic capacity of apple rootstock seedlings

[0307]

[0308] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preventing and treating apple leaf spot disease, characterized in that: Spraying foliar biopesticide fertilizer on apple trees; the foliar biopesticide fertilizer includes a fermentation product of Bacillus and a fermentation product of Trichoderma; the Bacillus includes at least one of Bacillus amyloliquefaciens TS-1203, Bacillus subtilis B1, and Bacillus subtilis BS-1208; the Trichoderma includes at least one of T6 and Ym.

2. The method for prevention and treatment according to claim 1, wherein: The fermentation product is a fermentation liquid obtained by activating the strains of Bacillus and Trichoderma, preparing the seed liquid and fermenting and culturing the bacteria, or a fermentation filtrate obtained by centrifuging and filtering the fermentation liquid.

3. The control method according to claim 1, characterized in that: The Bacillus fermentation product comprises 1-2 parts of Bacillus amyloliquefaciens TS-1203 fermentation product and / or 1-2 parts of Bacillus subtilis B1 fermentation product and / or 1-2 parts of Bacillus subtilis BS-1208 fermentation product by volume.

4. The method for prevention and treatment according to claim 1, wherein: The Trichoderma fermentation product includes 1-2 parts of Trichoderma T6 fermentation product and / or 1-2 parts of Trichoderma Ym fermentation product by volume.

5. The control method according to claim 1, characterized in that: The Bacillus fermentation product and the Trichoderma fermentation product are compounded in a volume ratio of (1-2):(1-2).

6. The control method according to claim 1, characterized in that: The foliar biological fertilizer further comprises one or more of a surfactant, a stabilizer and a preservative.

7. The control method according to claim 6, characterized in that: The added amount of the surfactant is 0.1% to 1.0% of the total volume of the foliar biological fertilizer, and is selected from one or more of Tween-20, Tween-80, agricultural organic silicon adjuvant and sodium dodecylbenzene sulfonate.

8. The control method according to claim 6, characterized in that: The stabilizer is added in an amount of 0.5% to 1.5% of the total volume of the foliar biological fertilizer and is selected from one or more of sodium alginate, dipotassium hydrogen phosphate and calcium carbonate.

9. The control method according to claim 6, characterized in that: The added amount of the preservative is 0.05% to 0.15% of the total volume of the foliar biological fertilizer, and is selected from one or more of sodium benzoate, streptomycin sulfate and sorbitol.

10. The control method according to claim 1, characterized in that: The foliar biofertilizer is sprayed after apple flowers fall and before the fruit expansion period, with an application amount of ≥10 mL / plant.

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