A biological control agent for winter jujube spot disease, a preparation method and application thereof

Abstract

The application discloses a biological control agent for winter jujube spot disease, and a preparation method and application thereof, and the raw materials of the biological control agent comprise biochemical fulvic acid, chelated biochemical calcium fulvate, Trichoderma spore suspension, Penicillium spore suspension, Bacillus subtilis spore suspension, vitamin C, sophorose lipid and sodium carboxymethyl cellulose; the biological control agent prepared by the application realizes and improves the disease prevention ability by utilizing the synergistic effect of multiple bacteria; on the other hand, the beneficial microorganism and the biochemical fulvic acid have the effects of activating soil nutrients, enhancing water retention, resisting drought, improving fertilizer utilization rate, enhancing the growth of young leaves of fruit trees, and inducing and improving the disease resistance of fruit trees, and can further strengthen the antagonistic control effect of the biocontrol bacteria on the winter jujube spot pathogen.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for fruit tree diseases, specifically relating to a biological control agent for jujube spot disease, its preparation method, and its application. Background Technology

[0002] Because of its rich nutrition and high economic value, winter jujube is widely planted in Shandong, Hebei, Shanxi, Shaanxi and other provinces in my country. Winter jujube has low requirements for soil, strong adaptability, and is tolerant of saline-alkali soil, poor soil, drought and flood. It can grow well in moderate to severe saline-alkali soil with a pH of 5.0 to 8.5 and a salt content of 0.3 to 0.5%, making it an important industry for farmers in saline-alkali areas to increase their income and become rich.

[0003] Jujube spot disease, commonly known as black spot disease or spot disease, is a major disease that damages jujube fruit. It has a great impact on the yield and quality of jujubes. A severe outbreak of jujube spot disease can reduce jujube production by 20-40%, and the resulting defective fruit accounts for more than 60%, causing huge economic losses to the jujube industry.

[0004] Jujube leaf spot is a weakly parasitic disease caused by a mixed infection of bacteria and fungi. The bacteria include *Pseudomonas* sp., *Xanthomonas* sp., and *Erwinia* sp.; the fungi mainly include *Alternaria* sp. and *Alternaria* sp. If jujube leaf spot is not controlled in a timely manner, the sores formed on the infected fruit become infection points for other fungal diseases such as anthracnose and ring rot, often leading to cross-infection and causing widespread outbreaks of fruit rot and drop, resulting in significant losses. Therefore, strengthening the prevention and control of jujube leaf spot is one of the important measures to increase jujube yield and improve jujube quality.

[0005] The prevention and control of jujube leaf spot disease mainly involves a combination of agricultural control measures and chemical control. Chemical control agents include carbendazim, DuPont's cymoxanil, tebuconazole, agricultural streptomycin, and chlorpyrifos. However, the long-term and frequent use of chemical agents can lead to the development of drug resistance in pathogens, resulting not only in ineffective control and pesticide residues, but also serious social problems such as environmental pollution and ecological imbalance. Summary of the Invention

[0006] To address the above problems, this invention provides a biological control agent for jujube spot disease, its preparation method, and its application.

[0007] The technical solution of this invention is: a biological control agent for jujube spot disease, its preparation method, and its application.

[0008] A biological control agent for jujube spot disease, wherein the raw materials of the biological control agent, by mass percentage, include: 20-30% chelated biochemical fulvic acid calcium, 10-15% Trichoderma mycelium suspension, 10-15% Penicillium mycelium suspension, 5-10% Bacillus subtilis mycelium suspension, 2-5% vitamin C, 0.1-2.0% sophorolipid, 0.1-1.0% sodium carboxymethyl cellulose, and the balance being biochemical fulvic acid.

[0009] Explanation: By selecting and proportioning the above-mentioned raw materials, the control effect and stability of jujube spot disease can be improved. It can also increase soil nutrient content, improve the soil microecological environment, and induce fruit trees to improve disease resistance, without causing pesticide residues or environmental pollution. The biological control of jujube spot disease using a complex of biochemical fulvic acid, chelated biochemical fulvic acid calcium, biocontrol microorganisms, vitamin C, sophorolipids, and sodium carboxymethyl cellulose utilizes biocontrol microorganisms (Trichoderma, Penicillium, Bacillus subtilis) and their metabolites to antagonize pathogens, achieving synergistic effects and enhancing disease resistance. Furthermore, the beneficial microorganisms and biochemical fulvic acid activate soil nutrients, enhance water retention, improve drought resistance, and increase fertilizer utilization. The chelated biochemical fulvic acid calcium, vitamin C, and sophorolipids promote the synthesis and increase the strength of fruit cell walls, enhance the growth of young leaves, and induce improved disease resistance in fruit trees, further strengthening the antagonistic control effect of biocontrol bacteria against the pathogens causing jujube spot disease.

[0010] Furthermore, the viable count of the Trichoderma suspension is ≥10. 8 cfu / mL, the viable count of the Penicillium suspension is ≥10 8 cfu / mL, the viable count of the Bacillus subtilis suspension is ≥10 9 cfu / mL.

[0011] Note: By further setting the viable bacteria count of the bacterial suspension as described above, the number of bacteria in the raw material components can reach the required level, thereby improving the control effect of the obtained biocontrol agent.

[0012] A method for preparing a biocontrol agent for jujube leaf spot disease includes the following steps:

[0013] S1. Preparation of chelated biochemical calcium humate:

[0014] Soluble calcium salts are added to biochemical fulvic acid until the calcium ion concentration in the biochemical fulvic acid reaches 0.05–0.2 mol / L. Then, a chelation reaction is carried out at 40–50 °C and pH 3–4 for 2–3 hours to obtain chelated biochemical fulvic acid calcium.

[0015] S2. Preparation of high-concentration bacterial suspension:

[0016] Trichoderma, Penicillium, and Bacillus subtilis cells were sequentially cultured in test tubes, shake flasks, and seed tanks, respectively. Then, the resulting seed tank culture was inoculated into the fermentation tank liquid at an inoculation rate of 10–15% (w / w). The culture temperature for Trichoderma and Penicillium was 25–28°C, and the culture temperature for Bacillus subtilis was 30–37°C. The culture time was 48–72 hours. After culture, the Trichoderma, Penicillium, and Bacillus subtilis suspensions were obtained.

[0017] S3. Mix and prepare biological control agents:

[0018] The chelated biochemical calcium fulvic acid obtained in step S1, the Trichoderma suspension, Penicillium suspension, and Bacillus subtilis suspension prepared in step S2, along with vitamin C, sophorolipid, and sodium carboxymethyl cellulose, are added to the biochemical fulvic acid and stirred to obtain the biocontrol agent.

[0019] Explanation: The chelated biochemical calcium humate prepared through the above method has high active calcium potency and rapid absorption, which can effectively alleviate fruit diseases caused by calcium deficiency-related physiological disorders in fruit trees. All three microbial strains have highly effective antagonistic effects against plant fungal pathogens, and there is a good synergistic effect among the three strains, ensuring the viable count and stability of the compound microbial agent, improving disease resistance and prevention, and effectively controlling leaf spot disease. The selected microbial strains have strong soil and environmental adaptability; after application, the strains can quickly colonize on leaves, stems, and soil, effectively improving the quantity and structure of the microbial community. Through the mixing and formulation of various raw materials, the resulting biocontrol agent can significantly improve the antagonistic control effect against the leaf spot pathogen of jujube compared to existing technologies. Simultaneously, it can increase fruit tree yield by more than 12% and chlorophyll content by more than 20%.

[0020] Further, in step S1, the soluble calcium salt is one or more of calcium nitrate, calcium chloride, calcium bicarbonate, and calcium dihydrogen phosphate.

[0021] Note: By selecting the above examples of soluble calcium salts, a good calcium source can be provided for the chelation of calcium ions by biochemical fulvic acid, and it is not easy to cause pollution from excess elemental components.

[0022] The application of a biological control agent for jujube leaf spot disease, and its use in the prevention and control of plant leaf spot disease.

[0023] Furthermore, it is used for the prevention and control of leaf spot disease in fruit trees, including jujube, apple, pear, and hawthorn.

[0024] Note: Biological control agents are most effective against jujube trees.

[0025] Furthermore, a combination of spraying and / or root drenching can be used for prevention and control.

[0026] A method for applying a biological control agent for jujube spot disease includes the following steps:

[0027] S1. Pre-disease prevention:

[0028] Before the onset of plant disease, determine whether prevention is necessary. If not, do not carry out prevention. If so, start spraying prevention from the flowering / young fruit stage / latent stage. Take 2-4 L / acre of biological control agent and dilute it with water 20-60 times. Then, spray the diluted biological control agent evenly onto the plant stems, leaves, leaf backs, and fruits. Spray 1-2 times, with an interval of 5-8 days between each spray.

[0029] S2. Disease prevention and control:

[0030] When plant spot disease occurs, a combination of spraying and root drenching should be used for prevention and control. For spraying, take 2-4 L / acre of biological control agent, dilute it with water 20-60 times, and then spray the diluted biological control agent evenly onto the plant stems, leaves, leaf undersides, and fruits. Spray 1-3 times, with an interval of 5-8 days between each spraying. For root drenching, take 5-10 L / acre of biological control agent, dilute it with water 50-100 times, and apply it in holes or furrows. Drench the diluted biological control agent onto the plant roots. Drench 1-3 times, with an interval of 10-15 days between each drench. Orchards with severe disease should have more spraying and root drenching times than orchards with mild disease. The severity of the disease is judged by the number, extent, and density of leaf spots, the darker the color of the spots, and the more and larger the spots on the fruit surface. Conversely, fewer spots indicate a milder disease.

[0031] Explanation: By setting the above application methods, the control effect of biological control agents can be maximized, thereby achieving a superior level of control. Through pre-disease prevention, spraying agents can kill pathogens before they multiply rapidly, greatly reducing the probability of disease in fruit trees and other plants, and controlling the spread of disease to a certain extent. By combining root drenching and spraying during disease onset, the control effect can be further strengthened. Spraying directly kills pathogens on the fruit tree plants, while root drenching kills pathogens in the soil around the roots. At the same time, the active ingredients in biological control agents can improve the soil and tree microbial environment, enhance the fruit tree's own disease resistance and defense capabilities, thus providing comprehensive protection for the fruit tree and promoting its growth and fruiting.

[0032] Furthermore, in step S1, the method for determining whether prevention is needed is as follows: if the orchard has experienced spot disease in previous years, then pre-disease prevention of spot disease is required, and pre-disease prevention should be carried out according to the method in step S1; if the orchard has never experienced spot disease, then pre-disease prevention of spot disease is not required.

[0033] Note: By using the above judgment method, corresponding prevention and control measures can be taken based on the actual condition of the fruit trees.

[0034] The beneficial effects of this invention are:

[0035] (1) The biological control agent and application method of the present invention can not only improve the control effect and control stability of jujube spot disease, but also increase the soil nutrient content, improve the soil micro-ecological environment, induce fruit trees to improve disease resistance, and will not cause pesticide residues and environmental pollution. It is a green and environmentally friendly control technology; it is pollution-free, residue-free, and ecotoxicological, can enhance tree vigor, and can increase fruit tree yield by more than 12% and increase chlorophyll content by more than 20%.

[0036] (2) The biocontrol agent prepared in this invention uses a complex of biochemical fulvic acid, chelated biochemical fulvic acid calcium, biocontrol microorganisms, vitamin C, sophorolipids and sodium carboxymethyl cellulose for the biocontrol of jujube spot disease. On the one hand, it utilizes biocontrol microorganisms (Trichoderma, Penicillium, Bacillus subtilis) and their metabolites to antagonize pathogens, and the synergistic effect of multiple microorganisms can achieve and improve disease control ability. On the other hand, the beneficial microorganisms and biochemical fulvic acid have the effects of activating soil nutrients, enhancing water retention, drought resistance and improving fertilizer utilization. The chelated biochemical fulvic acid calcium, vitamin C and sophorolipids can promote the synthesis and increase the strength of fruit cell walls, enhance the growth of young leaves of fruit trees and induce and improve the disease resistance of fruit trees, which can further enhance the antagonistic control effect of biocontrol bacteria on the pathogens of jujube spot disease.

[0037] (3) The present invention uses a multi-species compound antagonistic biocontrol agent, which can effectively solve the problem of narrow spectrum of antagonistic pathogens of single species; the selected microbial species all have a high-efficiency antagonistic effect against plant fungal pathogens, and there is a good synergistic effect among the three species, which can ensure the viable number and stability of the compound microbial agent, improve the disease resistance and prevention ability, and effectively prevent and control leaf spot disease; the selected microbial species have a strong soil and environmental adaptability. After application, the strains can quickly colonize the leaves, stems and soil, effectively improving the quantity and structure of microbial community.

[0038] (4) The chelated biochemical fulvic acid calcium selected in this invention has high active calcium potency and fast absorption, which can effectively reduce fruit diseases caused by calcium deficiency-type physiological disorders in fruit trees; biochemical fulvic acid (BFA) has the characteristics of small molecular weight and high active groups, which makes the biological control agent of this invention have the effects of enhancing fertilizer nutrients, promoting plant root development, effectively regulating soil pH, solving soil compaction, and improving soil; foliar spraying can reduce the opening of plant leaf stomata and enhance plant drought resistance in dry climate conditions; vitamin C is a highly active antioxidant that participates in a variety of plant metabolic processes, can help plants resist drought, ozone and ultraviolet damage, and is one of the protective factors that eliminate free radical damage in plants. Meanwhile, Vitamin C can activate plant cells, increase the extensibility of plant cell walls and the interaction between cells, and greatly improve the toughness of fruit peel, thus reducing fruit cracking and enhancing the fruit's defense against pathogenic microorganisms. The selected sophorolipid is a highly efficient biosurfactant with three main characteristics: 1. High performance, wide tolerance to temperature and acid / alkali conditions, high salt tolerance, and wide range of applications; 2. Sophorolipid possesses antibacterial, antitumor, and anti-inflammatory biological activities; 3. Low toxicity, biodegradable, environmentally friendly, and will not cause residual pollution. Therefore, adding sophorolipid can significantly improve the dispersibility and stability of microbial cells in the system, enhance the wetting and adsorption capacity of the control agent on the plant surface, and synergistically enhance the antagonistic disease control capabilities of biocontrol bacteria. Detailed Implementation

[0039] The present invention will now be described in more detail with reference to specific embodiments, so as to better demonstrate the advantages of the present invention.

[0040] Example 1:

[0041] A biological control agent for jujube spot disease, wherein the raw materials of the biological control agent comprise, by weight percentage: 25% chelated biochemical calcium fulvic acid, 15% Trichoderma suspension, 10% Penicillium suspension, 5% Bacillus subtilis suspension, 2% vitamin C, 0.1% sophorolipid, 1.0% sodium carboxymethyl cellulose, and the balance being biochemical fulvic acid;

[0042] The microbial strains used were purchased from the China Agricultural Microbial Culture Collection Center (ACCC), with the following strain numbers: *Trichoderma afroharzianum* (ACCC-33109), *Penicillium* sp. (ACCC-30287), and *Bacillus subtilis* (ACCC-60383). The microbial suspensions for each strain were prepared according to the methods described in the protocol. The viable count of the *Trichoderma* suspension was 5.6 × 10⁻⁶. 8 CFU / mL, viable count of Penicillium suspension: 6.2*10⁻⁶8 CFU / mL, viable count of Bacillus subtilis suspension: 3.4*10⁻⁶ 9 cfu / mL;

[0043] A method for preparing a biocontrol agent for jujube leaf spot disease includes the following steps:

[0044] S1. Preparation of chelated biochemical calcium humate:

[0045] Calcium nitrate was added to biochemical fulvic acid until the calcium ion concentration in the biochemical fulvic acid reached 0.1 mol / L. Then, a chelation reaction was carried out at 43℃ and pH 3.5 for 2.5 h to obtain chelated biochemical fulvic acid calcium; at this time, the calcium ion chelation rate was 95%.

[0046] The biochemical fulvic acid solution was prepared using the method described in patent 200610162041.6. The prepared liquid biochemical fulvic acid had a pH of 3.0 and a viable bacteria count of 2.0 x 10⁻⁶. 8 cfu / ml;

[0047] S2. Preparation of high-concentration bacterial suspension:

[0048] Trichoderma, Penicillium, and Bacillus subtilis cells were sequentially cultured in test tubes, shake flasks, and seed tanks. Then, the seed tank culture solution was inoculated into the fermentation tank liquid at an inoculation rate of 12% (w / w). The culture temperature for Trichoderma and Penicillium was 26℃, and the culture temperature for Bacillus subtilis was 35℃. The culture time was 50h. After culture, the Trichoderma, Penicillium, and Bacillus subtilis suspensions were obtained.

[0049] S3. Mix and prepare biological control agents:

[0050] The chelated biochemical calcium fulvic acid obtained in step S1, the Trichoderma suspension, Penicillium suspension, and Bacillus subtilis suspension prepared in step S2, along with vitamin C, sophorolipid, and sodium carboxymethyl cellulose, are added to the biochemical fulvic acid and stirred to obtain the biocontrol agent.

[0051] It can be used to control leaf spot disease in jujube trees. Take 10 jujube trees.

[0052] S1. Pre-disease prevention:

[0053] Before the onset of plant disease, it was determined that prevention was necessary. Since this orchard has been affected by leaf spot disease for many years, preventive spraying was carried out starting from the young fruit stage. The biological control agent was diluted with water at a rate of 3L / acre and then evenly sprayed onto the plant stems, leaves, undersides of leaves, and fruits. The spraying was done twice, with an interval of 7 days between each spraying.

[0054] S2. Disease prevention and control:

[0055] When plant spot disease occurs, control it by combining spraying and root drenching. For spraying, take 3L / acre of biological control agent and dilute it 40 times with water. Then, spray the diluted biological control agent evenly onto the plant stems, leaves, leaf undersides, and fruits. Spray 3 times, with an interval of 7 days between each spraying. For root drenching, take 7L / acre of biological control agent and dilute it 70 times with water. Apply the diluted biological control agent to the plant roots by hole application or trench application. Apply root drenching twice, with an interval of 12 days between each root drenching.

[0056] Example 2

[0057] The difference between this embodiment and Embodiment 1 is that the proportions of the raw material components are different. The raw materials of the biocontrol agent include, by mass percentage: 20% chelated biochemical calcium fulvic acid, 10% Trichoderma suspension, 15% Penicillium suspension, 10% Bacillus subtilis suspension, 3.5% vitamin C, 1% sophorolipid, 0.5% sodium carboxymethyl cellulose; the balance is biochemical fulvic acid.

[0058] Example 3

[0059] The difference between this embodiment and Embodiment 1 is that the proportions of the raw material components are different. The raw materials of the biocontrol agent include, by mass percentage: 30% chelated biochemical calcium humate, 13% Trichoderma suspension, 12% Penicillium suspension, 8% Bacillus subtilis suspension, 5% vitamin C, 2% sophorolipid, 0.1% sodium carboxymethyl cellulose; the balance is biochemical humic acid.

[0060] Example 4

[0061] The difference between this embodiment and Embodiment 1 lies in the production parameters of the bacterial suspension. In step S2, *Trichoderma*, *Penicillium*, and *Bacillus subtilis* cells are sequentially cultured in test tubes, shake flasks, and seed tanks, respectively. Then, at an inoculation rate of 10% (w / w), the resulting seed tank culture is inoculated into the fermentation tank liquid for further cultivation. The culture temperature for *Trichoderma* and *Penicillium* is 28°C, and the culture temperature for *Bacillus subtilis* is 37°C. The cultivation time is 48 hours. After cultivation, the *Trichoderma*, *Penicillium*, and *Bacillus subtilis* bacterial suspensions are obtained. The viable count of the *Trichoderma* bacterial suspension is 3.4 * 10-1. 8 CFU / mL, viable count of Penicillium suspension 5.5*10 8 CFU / mL, viable count of Bacillus subtilis suspension: 2.8 x 10⁻⁶ 9 cfu / mL.

[0062] Example 5

[0063] The difference between this embodiment and Embodiment 1 lies in the production parameters of the bacterial suspension. In step S2, *Trichoderma*, *Penicillium*, and *Bacillus subtilis* cells are sequentially cultured in test tubes, shake flasks, and seed tanks, respectively. Then, the resulting seed tank culture solution is inoculated into the fermentation tank liquid at an inoculation rate of 15% (w / w). The culture temperature for *Trichoderma* and *Penicillium* is 25°C, and the culture temperature for *Bacillus subtilis* is 30°C. The culture time is 72 hours. After culture, the *Trichoderma*, *Penicillium*, and *Bacillus subtilis* bacterial suspensions are obtained. The viable count of the *Trichoderma* bacterial suspension is 4.9*10^9. 8 CFU / mL, viable count of Penicillium suspension: 3.7*10⁻⁶ 8 CFU / mL, viable count of Bacillus subtilis suspension: 5.1*10⁻⁶ 9 cfu / mL.

[0064] Example 6

[0065] The difference between this embodiment and Example 1 is that the preparation parameters for chelated biochemical calcium fulvic acid are different. The calcium ion concentration in the biochemical fulvic acid reaches 0.2 mol / L, and then the chelation reaction is carried out at 45℃ and pH 4 for 3 hours to obtain chelated biochemical calcium fulvic acid. At this time, the calcium ion chelation rate is 91%.

[0066] Example 7

[0067] The difference between this embodiment and Example 1 is that the preparation parameters for chelated biochemical calcium fulvic acid are different. The calcium ion concentration in the biochemical fulvic acid reaches 0.05 mol / L, and then the chelation reaction is carried out at 40℃ and pH 3 for 2 hours to obtain chelated biochemical calcium fulvic acid. At this time, the calcium ion chelation rate is 93%.

[0068] Example 8

[0069] The difference between this embodiment and Example 1 is that the calcium source for preparing chelated biochemical fulvic acid calcium is different; calcium bicarbonate is added to biochemical fulvic acid.

[0070] Example 9

[0071] The difference between this embodiment and Example 1 is that the calcium source for preparing chelated biochemical fulvic acid calcium is different; calcium chloride is added to biochemical fulvic acid.

[0072] Example 10

[0073] The difference between this embodiment and Example 1 is that the calcium source for preparing chelated biochemical fulvic acid calcium is different; calcium dihydrogen phosphate is added to biochemical fulvic acid.

[0074] Example 11

[0075] The difference between this embodiment and Example 1 is that the calcium source for preparing chelated biochemical fulvic acid calcium is different; calcium dihydrogen phosphate and calcium chloride in a mass ratio of 1:1 are added to biochemical fulvic acid.

[0076] Example 12

[0077] The difference between this embodiment and Embodiment 1 is that the prevention and control parameters are different. S1, Pre-disease prevention:

[0078] Before the onset of plant disease, it was determined that prevention was necessary. Since leaf spot disease occasionally occurred in this orchard in previous years, preventive spraying was carried out from the incubation period. The biological control agent was diluted with water 60 times at a dosage of 2L / acre. The diluted biological control agent was then evenly sprayed onto the plant stems, leaves, undersides of leaves, and fruits once.

[0079] S2. Disease prevention and control:

[0080] When plant spot disease occurs, control it by combining spraying and root drenching. For spraying, take 2L / acre of biological control agent, dilute it 60 times with water, and then spray the diluted biological control agent evenly onto the plant stems, leaves, leaf undersides, and fruits, spraying once. For root drenching, take 5L / acre of biological control agent, dilute it 100 times with water, and apply it in holes or furrows, irrigating the roots of the plant once.

[0081] Example 13

[0082] The difference between this embodiment and Embodiment 1 is that the prevention and control parameters are different. S1, Pre-disease prevention:

[0083] Before the onset of plant disease, it was determined that prevention was necessary. Since the leaf spot disease in this orchard was severe in previous years, preventive spraying was carried out starting from the flowering period. The biological control agent was diluted 20 times with water at a dosage of 4L / acre. The diluted biological control agent was then evenly sprayed onto the plant stems, leaves, undersides of leaves, and fruits. The spraying was done twice, with an interval of 8 days between each spraying.

[0084] S2. Disease prevention and control:

[0085] When plant spot disease occurs, control it by combining spraying and root drenching. For spraying, take 4L / acre of biological control agent and dilute it 20 times with water. Then, spray the diluted biological control agent evenly onto the plant stems, leaves, leaf undersides, and fruits. Spray 3 times, with an interval of 5 days between each spraying. For root drenching, take 10L / acre of biological control agent and dilute it 50 times with water. Apply the diluted biological control agent to the plant roots by hole application or trench application. Apply the root drenching 3 times, with an interval of 10 days between each root drenching.

[0086] Experimental Example

[0087] I. The results obtained from Comparative Examples 1-5 and Examples 1-13 are compared respectively, and the results are as follows:

[0088] 1. To investigate the effects of different parameters on the control of jujube leaf spot disease;

[0089] Comparative Example 1: The biological control agent was replaced with water, and the rest of the treatment was the same as in Example 1;

[0090] Comparative Example 2: The biological control agent was replaced with 90% carbendazim. The application method was to dilute it 1200 times and spray it once every 7 days, starting from the young fruit stage of winter jujube, for a total of 3 sprays.

[0091] Comparative Example 3: No chelated biochemical calcium fulvic acid was added; biochemical fulvic acid was added instead of the chelated biochemical calcium fulvic acid component, and the rest of the treatment was the same as in Example 1.

[0092] Comparative Example 4: Only spraying was used for prevention and control, and all other treatments were the same as in Example 1;

[0093] Comparative Example 5: Only root irrigation was used for prevention and control, and all other treatments were the same as in Example 1;

[0094] 1.1 Incidence Statistics:

[0095] In the prevention and control experiment, 20 jujube fruits were marked on each jujube tree in the east, west, south, and north of the canopy with waterproof small tags. A total of 200 jujube fruits were marked in each treatment. Before harvesting, the disease status of the jujube fruits was investigated uniformly.

[0096] Calculation method:

[0097] Disease incidence rate (%) = (Total number of diseased fruits at each level / Total number of fruits surveyed) × 100;

[0098] Control efficacy (%) = [(Disease rate of Comparative Example 1 - Disease rate of fruit) / Disease rate of Comparative Example 1] × 100;

[0099] 1.2 Statistics on chlorophyll content, yield, and vitamin C content of jujube leaves and fruits

[0100] The chlorophyll content of jujube leaves was measured 15 days after all control measures were completed using a chloroplast method. Twenty jujube leaves were measured for each treatment, and the average value was taken. After harvest, the yield was counted and the vitamin C content of jujube fruits was measured. The vitamin C content was measured using the 2,4-dinitrophenol method. Twenty jujube fruits were measured for each treatment, and the average value was taken.

[0101] The results are shown in Table 1 below:

[0102] Table 1. Experimental results for controlling jujube leaf spot disease.

[0103]

[0104] As shown in Table 1, the incidence of jujube leaf spot disease in Comparative Example 1 was 26.8%, the chlorophyll content was 23.45 SPAD, and the V... C The content was 229.3 mg / 100g, and the yield was 1656 kg / mu; in Comparative Example 2, chemical control with 90% carbendazim resulted in a 12.3% incidence of jujube leaf spot disease and a control effect of 54.1%; the chlorophyll content was 31.74 SPAD, V C The content was 247.5 mg / 100g, and the yield was 1742 kg / mu, which were 15.63%, 7.94%, and 5.19% higher than those of control example 1, respectively; the incidence rate in example 1 was 4.4%, and the control effect was 83.6%; the chlorophyll content was 37.15 SPAD, and the V C The content was 275.0 mg / 100g, and the yield was 1923 kg / mu, which were 36.65%, 19.93%, and 16.12% higher than those of control group 1, respectively.

[0105] 2. Investigate the impact of raw material components on the prevention and control effect;

[0106] As shown in Table 1, comparative examples 1-3 and examples 1-5 were used for comparison:

[0107] Comparing Example 1 with Comparative Example 1, it can be seen that, compared with water, the biological control agent used in Example 1 has outstanding effects in all indicators.

[0108] Comparing Example 1 and Comparative Example 2, it can be concluded that compared with the chemical control agents in the prior art, the control effect of the present invention is obvious. Using Example 1, the disease rate of fruit is low and the yield is high. At the same time, due to the addition of raw material components, the chlorophyll and vitamin C content are increased.

[0109] Comparing Example 1 and Comparative Example 3, it can be concluded that the active calcium in the chelated biochemical calcium fulvic acid added in Example 1 can effectively alleviate fruit diseases caused by calcium deficiency-type physiological disorders in fruit trees, and has a strengthening effect on prevention and control.

[0110] Comparing Examples 1 to 5, it can be seen that the proportion of each raw material group in Example 1 has the best prevention and control effect;

[0111] 3. To investigate the effects of the conditions and parameters for preparing chelated biochemical calcium humate on the control effect;

[0112] As shown in Table 1, Examples 1 and 6-11 are compared:

[0113] Comparing Example 1 with Examples 6-7, it can be seen that the calcium ion concentration, temperature, pH, and chelation time in Example 1 are more optimized.

[0114] Comparing Example 1 with Examples 8-11, it can be seen that the selection of different calcium sources has a small impact on the control effect of biocontrol agents, and the calcium source in Example 1 is preferred.

[0115] 4. Investigate the impact of control parameters on control effectiveness;

[0116] As shown in Table 1, Examples 1, 12, and 13, and Comparative Example 4 and Comparative Example 5 are compared:

[0117] Comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the prevention and control method of Example 1 is more preferred.

[0118] Comparing Examples 1, 12, and 13, it can be seen that the orchards in the three cases had different degrees of disease damage in previous years, but all of them achieved better control effects through corresponding prevention and control measures.

[0119] The experimental data above show that the biological control agent for jujube spot disease proposed in this invention can effectively reduce the incidence of jujube disease, increase yield, enhance tree vigor, and improve fruit quality.

Claims

1. A biological control agent for jujube leaf spot disease, characterized in that, The raw materials of the biological control agent, by mass percentage, include: 20-30% chelated biochemical calcium fulvic acid, 10-15% Trichoderma suspension, 10-15% Penicillium suspension, 5-10% Bacillus subtilis suspension, 2-5% vitamin C, 0.1-2.0% sophorolipid, 0.1-1.0% sodium carboxymethyl cellulose, and the balance being biochemical fulvic acid; The viable count of the Trichoderma suspension is ≥10. 8 cfu / mL, the viable count of the Penicillium suspension is ≥10 8 The cfu / mL concentration of the Bacillus subtilis suspension is ≥10⁻⁶. 9 cfu / mL; The preparation method of the biological control agent for jujube spot disease includes the following steps: S1. Preparation of chelated biochemical calcium humate: Add soluble calcium salt to biochemical fulvic acid until the calcium ion concentration in the biochemical fulvic acid reaches 0.05~0.2 mol / L, and then carry out a chelation reaction at 40~50℃ and pH 3~4 for 2~3 hours to obtain chelated biochemical fulvic acid calcium. S2. Preparation of high-concentration bacterial suspension: Trichoderma, Penicillium, and Bacillus subtilis cells were sequentially cultured in test tubes, shake flasks, and seed tanks, respectively. Then, the resulting seed tank culture was inoculated into the fermentation tank liquid at an inoculation rate of 10-15% W / W. The culture temperature for Trichoderma and Penicillium was 25-28℃, and the culture temperature for Bacillus subtilis was 30-37℃. The culture time was 48-72 hours. After culture, the Trichoderma, Penicillium, and Bacillus subtilis suspensions were obtained. S3. Mix and prepare biological control agents: The chelated biochemical calcium fulvic acid obtained in step S1, the Trichoderma suspension, Penicillium suspension, and Bacillus subtilis suspension prepared in step S2, along with vitamin C, sophorolipid, and sodium carboxymethyl cellulose, are added to the biochemical fulvic acid and stirred to obtain the biocontrol agent. The *Trichoderma* species is *Trichoderma* ACCC-33109, the *Penicillium* species is *Penicillium* ACCC-30287, and the *Bacillus subtilis* species is *Bacillus subtilis* ACCC-60383. ， The viable count of the Trichoderma suspension was 5.6 x 10⁻⁶. 8 CFU / mL, viable count of Penicillium suspension: 6.2*10⁻⁶ 8 CFU / mL, viable count of Bacillus subtilis suspension: 3.4*10⁻⁶ 9 cfu / mL.

2. The method for preparing a biological control agent for jujube spot disease as described in claim 1, characterized in that, In step S1, the soluble calcium salt is one or more of calcium nitrate, calcium chloride, calcium bicarbonate, and calcium dihydrogen phosphate.

3. The application of the biological control agent for jujube spot disease as described in claim 1, characterized in that, It is used for the prevention and control of plant spot disease.

4. The application of the biological control agent for jujube spot disease as described in claim 3, characterized in that, It is used for the prevention and control of leaf spot disease in fruit trees, including jujube, apple, pear, and hawthorn.

5. The application method of the biological control agent for jujube spot disease as described in claim 3, characterized in that, Control measures can be implemented by combining spraying and / or root drenching.

6. The application method of the biological control agent for jujube spot disease as described in claim 5, characterized in that, Includes the following steps: S1. Pre-disease prevention: Before the onset of plant disease, determine whether prevention is necessary. If not, do not take preventive measures. If so, start spraying prevention from the flowering / young fruit stage / latent stage. Take 2-4 L / acre of biological control agent and dilute it with water 20-60 times. Then, spray the diluted biological control agent evenly onto the plant stems, leaves, leaf backs, and fruits. Spray 1-2 times, with an interval of 5-8 days between each spray. S2. Disease prevention and control: When plant spot disease occurs, control it by combining spraying and root drenching. Spraying: Take 2-4 L / acre of biological control agent and dilute it with water 20-60 times. Then, spray the diluted biological control agent evenly onto the plant stems, leaves, leaf undersides, and fruits. Spray 1-3 times, with an interval of 5-8 days between each spraying. Root drenching: Take 5-10 L / acre of biological control agent and dilute it with water 50-100 times. Apply it in holes or furrows, and drench the diluted biological control agent onto the plant roots. Drench 1-3 times, with an interval of 10-15 days between each drench.

7. The application method of the biological control agent for jujube spot disease as described in claim 6, characterized in that, In step S1, the method for determining whether prevention is needed is as follows: if the orchard has experienced spot disease in previous years, then pre-disease prevention of spot disease is needed, and pre-disease prevention should be carried out according to the method in step S1; if the orchard has never experienced spot disease, then pre-disease prevention of spot disease is not needed.

Images