A compound microbial agent for preventing and controlling plant diseases and its application

Abstract

The present invention relates to the technical field of biological control, and particularly to a compound microbial agent for preventing and controlling plant diseases and its application. The microbial combination provided by the present invention includes Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3 and Bacillus amyloliquefaciens HC200 which have a synergistic effect; a compound microbial agent is also provided, and the agent includes the microbial combination, a carrier and a synergist provided by the present invention, and also includes a healthy plant leaf extract or a healthy plant rhizosphere soil extract. The agent has a control effect on a variety of major plant diseases (pepper blight, banana wilt, tomato wilt and bacterial wilt, citrus huanglongbing, tea blister blight and tea target spot), and at the same time can change the fungal or bacterial diversity of plant leaves or rhizosphere soil, as well as increase the abundance of beneficial microorganisms or reduce pathogenic microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and in particular to a composite microbial preparation for preventing and controlling plant diseases and its application. Background Art

[0002] Fusarium oxysporum, Phytophthora capsici, Ralstonia solanacearum, Meloidogyne spp.) are devastating soil-borne diseases; Citrus Huanglongbing (HLB) is caused by the Gram-negative bacteria (Candidatus Liberibacter) Asian species (Candidatus Liberibacter asiaticus, CLas) that specifically parasitizes the phloem sieve tube tissue of citrus. Literature reports that after C.Las infects citrus leaves, it mainly colonizes the midrib of the leaves (Shi Yaman, Zeng Jixing, Xue Jinjun, et al. Effect of Huanglongbing on the accumulation of assimilates and mineral nutrient transport in citrus leaves [J]. Journal of Plant Nutrition and Fertilizer, 2023, 29(05):949-960.), which is regarded as the "citrus cancer" and has seriously restricted the healthy development of the citrus industry; Citrus Huanglongbing (HLB) is caused by the Gram-negative bacteria (Candidatus Liberibacter) Asian species (CLas) that specifically parasitizes the phloem sieve tube tissue of citrus. Literature reports that after C.Las infects citrus leaves, it mainly colonizes the midrib of the leaves (Shi Yaman, Zeng Jixing, Xue Jinjun, et al. Effect of Huanglongbing on the accumulation of assimilates and mineral nutrient transport in citrus leaves [J]. Journal of Plant Nutrition and Fertilizer, 2023, 29(05):949-960.). Tea cake disease caused by Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsispiceana, Neopestalotiopsis protearum and Pestalotiopsis theae is one of the most serious diseases that harm tea trees. It mainly harms young leaves and shoots, has a short incubation period, and has a much greater impact on tea yield than other diseases, resulting in serious reduction in tea yield and quality. Tea ring spot disease caused by Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsispiceana, Neopestalotiopsis protearum and Pestalotiopsis theae often starts to infect from the tip or edge of the leaf, followed by the formation of lesions that cause leaf tissue necrosis, resulting in serious reduction in yield. Since the pathogen of citrus Huanglongbing and tea cake disease, Exobasidiomyces necroticus, is difficult to culture in vitro, the research on its prevention and control technology is limited. The above-mentioned major diseases have caused huge losses to my country's agricultural production. Traditional measures that mainly rely on chemical control have not only failed to curb the rising trend of the above-mentioned major diseases, but also inhibited the growth of beneficial microorganisms in the soil and plants, causing pathogens to develop drug resistance and become rampant again. At the same time, they cause harmful substances to remain, leading to a decline in the quality of agricultural products. The use of highly toxic and high-residue pesticides will also cause major safety problems for agricultural products.

[0003] In recent years, microbial control has become an important means of preventing and controlling plant diseases due to its advantages such as pollution-free and easy development. The composite microbial agents containing Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, and Paecilomyces lilacinus have been widely studied and applied, but the diseases they control are single and the control effect is low. Therefore, it is very necessary to find a composite microbial agent with good control effects against multiple diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide a composite microbial agent and its application for preventing and controlling plant diseases to solve the problems existing in the above-mentioned prior art. The present invention selects excellent biocontrol strains with complementary functions to construct a microbial combination, and adds a carrier, a synergist, and an extract of a plant or plant rhizosphere soil as a composite microbial agent, which has good control effects against multiple diseases. The present invention provides a new idea for the research and development of excellent composite microbial agents and scientific field application technologies, and at the same time provides effective technical support for the green and efficient prevention and control of plant diseases.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a microbial combination for preventing and controlling plant diseases, which includes Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200;

[0007] The preservation number of Bacillus amyloliquefaciens HW05 is CGMCC No. 10273; the preservation number of Bacillus subtilis BLG010 is CGMCC No. 5953; the preservation number of Paecilomyces lilacinus E16 is CGMCC No. 5951; the preservation number of Trichoderma viride H06 is CGMCC No. 6229; the preservation number of Bacillus amyloliquefaciens HC200 is CGMCC No. 10371.

[0008] Preferably, the mass ratio of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 in the microbial combination is (5-10):(2-6):(2-4):(2-4):(2-5):(1-5);

[0009] And / or, the plant diseases include one or more of Phytophthora capsici, Fusarium oxysporum f. sp. cubense, Fusarium oxysporum f. sp. lycopersici, Ralstonia solanacearum, Candidatus Liberibacter asiaticus, Exobasidium vexans, and Pestalotiopsis theae.

[0010] Further preferably, the effective viable counts of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 in the microbial combination are not less than 5.7×10 10 CFU / g.

[0011] Further preferably, the mass ratio of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 in the microbial combination is 5:6:2:2:2:1.

[0012] Further preferably, the pathogens causing the plant diseases include: Fusarium oxysporum, Phytophthora capsici, Ralstonia solanacearum, Meloidogyne spp., Candidatus Liberibacter, Xanthomonas axonopodis, Exobasidium vexans, and Pestalotiopsis theae.

[0013] As an additional aspect, the present invention also provides a method for preparing the above microbial combination, comprising the following steps:

[0014] After activating Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200, they are respectively inoculated into a fermentation medium for fermentation culture to obtain fermentation products of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200;

[0015] After drying, pulverizing, and mixing the fermentation products of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 respectively, the microbial combination is obtained.

[0016] Further preferably, the powders of the fermentation products of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 obtained after pulverization are all not less than 5.7×10 10 CFU / g, and the mass ratio is (5-10):(2-6):(2-4):(2-4):(2-5):(1-5).

[0017] Further preferably, the ratio of the effective viable bacteria numbers of the powders of the fermentation products of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 obtained after pulverization is 5:6:2:2:2:1.

[0018] Further preferably, the activation medium used for activation is a liquid activation medium; calculated by 1L of the liquid activation medium: the liquid activation media of Paecilomyces lilacinus E16 and Trichoderma viride H06 include components with the following concentrations, sucrose or glucose 5g / L, tryptone 2.5g / L, yeast powder 2.5g / L, and sodium chloride 5g / L: the liquid activation media of Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 include components with the following concentrations, sucrose or glucose 5g / L, tryptone 2.5g / L, yeast powder 2.5g / L, amino acid chelated calcium, magnesium, boron, and zinc 5g / L, magnesium sulfate 0.1g / L, dipotassium hydrogen phosphate 1g / L, manganese sulfate 0.01g / L, and sodium chloride 5g / L;

[0019] and / or, the activation temperature is 28°C, and the time is 2-4d.

[0020] Further preferably, the fermentation medium includes a solid component and water;

[0021] and / or, the mass ratio of the solid component to the water is 1:1;

[0022] And / or, the solid component includes any 4 or more of corn flour, sucrose, peptone, amino acid chelated calcium, magnesium, boron, zinc, and bioenzyme.

[0023] Further preferably, the temperature of the fermentation culture is 25°C - 37°C, and the time is 3 - 5 days.

[0024] Further preferably, the temperature of the drying is 30 - 40°C, and the time is 48 - 72 hours.

[0025] The present invention provides the application of the above-mentioned microbial combination in the preparation of a compound microbial agent for preventing and controlling plant diseases.

[0026] The present invention provides a compound microbial agent for preventing and controlling plant diseases, and the compound microbial agent includes active ingredient 1; the active ingredient 1 includes the above-mentioned microbial combination.

[0027] Preferably, the compound microbial agent further includes a carrier and a synergist;

[0028] The mass ratio of the active ingredient 1, the carrier, and the synergist is (20 - 50):(10 - 30):(3 - 8).

[0029] Further preferably, the mass ratio of the active ingredient 1, the carrier, and the synergist is 10:10:1.

[0030] Preferably, the carrier includes any 4 or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium, magnesium, boron, zinc, humic acid, and fish protein; when the carrier includes chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium, magnesium, boron, zinc, humic acid, and fish protein, by mass, it includes: 10 - 20 parts of chitosan, 1 - 3 parts of potassium dihydrogen phosphate, 1 - 5 parts of urea, 5 - 10 parts of amino acid chelated calcium, magnesium, boron, zinc, 5 - 10 parts of humic acid, and 5 - 10 parts of fish protein;

[0031] And / or, the synergist includes any 3 or more of silicone, azone, gibberellin, and ascorbic acid; when the synergist includes silicone, azone, gibberellin, and ascorbic acid, by mass, it includes: 50 - 100 parts of silicone, 50 - 100 parts of azone, 5 - 10 parts of gibberellin, and 5 - 10 parts of ascorbic acid.

[0032] Preferably, the compound microbial agent further includes active ingredient 2; the active ingredient 2 includes a healthy plant leaf extract or a healthy plant rhizosphere soil extract;

[0033] And / or, the preparation method of the healthy plant leaf extract includes the step of mixing the leaves of a healthy plant and water in a mass ratio of 1:(10 - 20), and then grinding to obtain the healthy plant leaf extract.

[0034] And / or, the preparation method of the healthy plant rhizosphere soil extract comprises the steps of mixing the rhizosphere soil of healthy plants and water in a mass ratio of 1:(5 - 10), and then filtering to obtain the healthy plant rhizosphere soil extract.

[0035] Preferably, the mass ratio of the mixture of the active ingredient 1, the carrier and the synergist to the active ingredient 2 is (6 - 10):(1 - 2).

[0036] More preferably, the mass ratio of the mixture to the active ingredient 2 is 10:1.

[0037] The compound microbial agent of the present invention combines the microbial combination with the healthy plant leaf extract or the healthy plant rhizosphere soil extract, which can effectively control major plant diseases, such as pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus huanglongbing, tea blister blight and tea target spot disease, etc. It can also improve soil nutrients, change the microbial diversity in the plant or rhizosphere soil, increase the abundance of beneficial microorganisms, and improve the yield and quality.

[0038] As an additional embodiment, when the compound microbial agent contains the active ingredient 1, the carrier and the synergist, the preparation method of the compound microbial agent comprises the steps of mixing the active ingredient 1, the carrier and the synergist to obtain the compound microbial agent; the active ingredient 1 includes the above-mentioned microbial combination.

[0039] More preferably, the mass ratio of the active ingredient 1, the carrier and the synergist is (20 - 50):(10 - 30):(3 - 8).

[0040] More preferably, the mass ratio of the active ingredient 1, the carrier and the synergist is 10:10:1.

[0041] As an additional embodiment, when the compound microbial agent contains the active ingredient 1, the carrier, the synergist and the active ingredient 2, the preparation method of the compound microbial agent comprises the steps of mixing the active ingredient 1, the carrier and the synergist to obtain a mixture; mixing the obtained mixture with the active ingredient 2, and then performing proliferation culture to obtain the compound microbial agent; the active ingredient 2 includes the healthy plant leaf extract or the healthy plant rhizosphere soil extract.

[0042] More preferably, the mass ratio of the active ingredient 1, the carrier and the synergist is (20 - 50):(10 - 30):(3 - 8).

[0043] Further preferably, the mass ratio of the active ingredient 1, the carrier and the synergist is 10:10:1.

[0044] Further preferably, the mass ratio of the mixture and the active ingredient 2 is (6 - 10):(1 - 2).

[0045] Further preferably, the mass ratio of the mixture and the active ingredient 2 is 10:1.

[0046] Further preferably, the environment for the proliferation culture is under natural field conditions, the temperature is 20°C - 40°C, and the time is 24h - 72h.

[0047] As an additional option, when the compound microbial agent of the present invention includes the active ingredient 1, the carrier, the synergist and the active ingredient 2 in separate packages, the specific usage method is as follows:

[0048] Mix the active ingredient 1, the carrier and the synergist to obtain a mixture; mix the obtained mixture with the active ingredient 2 in a mass ratio of (6 - 10):(1 - 2). Before application, dilute it 10 - 20 times with water, and let it stand or stir aerobically at 20°C - 40°C for 24h - 72h to rapidly proliferate the bacterial amount and active substances, then dilute it 50 - 100 times and apply it to the roots and / or leaves of plants;

[0049] Further preferably, the dosage of the compound microbial agent each time is 2.5 - 10 kg / mu, and the number of application times is 3 - 15 times, which can effectively control pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus huanglongbing, tea blister blight and tea target spot, etc., and at the same time can also improve soil nutrients and improve quality.

[0050] The present invention provides the application of the above microbial combination or the above compound microbial agent in preventing and controlling plant diseases.

[0051] The present invention provides a method for preventing and controlling plant diseases, including the following steps:

[0052] After proliferating and culturing the above compound microbial agent, apply it to the surface of plant leaves or rhizosphere.

[0053] The present invention discloses the following technical effects:

[0054] Previously, through microbiome analysis, the inventors found that compared with diseased plants or soil, a large number of microorganisms with different properties, such as fungi and bacteria, survive in healthy plants or rhizosphere soil. The abundance and / or diversity of these microorganisms are significantly different from those in diseased plants or rhizosphere soil. Beneficial microorganisms have the same ecological niche as plant pathogens and coexist with plants for a long time, forming a unique microbial community and a plant-microbe ecological system, which plays an important role in the complex microecological relationship among plants, soil, and microorganisms. Therefore, by appropriately combining the extract of the microbial population in healthy plants or soil with a compound antagonistic microbial agent, the microbial community structure in diseased plants or rhizosphere soil can be improved, including functions such as increasing or decreasing microbial diversity, and / or decreasing the abundance of pathogens in plants or rhizosphere soil, and / or increasing the abundance of beneficial microorganisms, reducing the damage of diseases to plants, and improving yield and quality.

[0055] The microbial combination of the present invention includes Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06, chemotactic antagonist - Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200, which have a synergistic effect. Bacillus velezensis LG14-3 not only has an antagonistic effect but also has a significant chemotactic effect on banana root exudates, glycine, and citric acid, so it is simply called a chemotactic antagonist (public literature: Yang Lihua. Research on the targeted prevention and control of banana wilt by rhizosphere chemotactic bacteria [D]. Guizhou University, 2023.). A compound microbial agent is also provided, which includes the microbial combination, carrier, and synergist provided by the present invention, and also includes an extract of healthy plant leaves or an extract of healthy plant rhizosphere soil. This microbial combination and this agent have a preventive and control effect on a variety of major plant diseases (pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus huanglongbing, tea blister blight, and tea zonate spot blight), and at the same time have a growth-promoting effect. The present invention solves the problem of unstable field application caused by unreasonable synergistic effects of previous compound microbial agents and has the advantages of being green, environmentally friendly, and efficient.

[0056] In this combination, Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06, chemotactic antagonist - Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 are beneficial bacteria in plant rhizosphere soil or plants. Their metabolites contain regulatory substances necessary for crop growth, such as auxin, antibiotics, and cytokinins, which change the microbial diversity in soil or plants. In addition, they also have the effect of inhibiting the reproduction of various plant pathogens.

[0057] The present invention combines a microbial combination composed of Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 beneficial bacteria with a carrier, a synergist, and a healthy plant leaf / healthy plant rhizosphere soil extract, which can produce a synergistic effect. The carrier can supplement the key nutrients required for the proliferation of the microbial inoculant. With the assistance of the synergist, the compound microbial agent is formed on the soil and the plant surface. After the application of the healthy plant leaf / healthy plant rhizosphere soil extract, it can quickly adhere to, grow, spread, and penetrate into the diseased parts of the plant, improve disease resistance, eliminate the pathogens in the diseased parts, enhance the inhibitory effects on citrus huanglongbing, various soil-borne pathogenic fungal diseases (such as banana wilt, tomato wilt, pepper blight, etc.), soil-borne pathogenic bacterial diseases (such as tomato bacterial wilt, etc.), and leaf diseases (such as tea blister blight and tea zonate leaf spot). At the same time, it changes the diversity of microorganisms in the plant or soil, increases the abundance of beneficial microorganisms or reduces the abundance of pathogenic microorganisms, balances the microecology inside the plant or in the plant rhizosphere, thereby enhancing the absorption and utilization efficiency of plant root nutrients, improving root activity, further increasing the yield, and improving the quality.

[0058] The preparation method of the compound microbial agent of the present invention has a simple process and is easy to operate. The prepared compound microbial agent is pollution-free to the environment and has a low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0060] Figure 1 It is the relative abundance diagram of the species at the genus level for each treatment in Example 6;

[0061] Figure 2 It is the relative abundance diagram of the species at the genus level for each treatment in Example 7;

[0062] Figure 3 It is the relative abundance diagram of the species at the genus level for each treatment in Example 8;

[0063] Figure 4 It is the relative abundance diagram of the species at the genus level for each treatment in Example 9;

[0064] Figure 5 It is the relative abundance diagram of the species at the genus level for each treatment in Example 10;

[0065] Figure 6Relative abundance graph of the species at each treatment level in Example 11. Detailed implementation mode

[0066] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation modes of the present invention.

[0067] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0068] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0069] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0070] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0071] Bacillus amyloliquefaciens HW05, with the preservation number of CGMCC No. 10273, was deposited on January 4, 2015, at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is disclosed in the Chinese patent with the application number of "201810578016.9" and the title of "An efficient Bacillus amyloliquefaciens and its bacterium agent and application".

[0072] Bacillus subtilis BLG010, with the preservation number of CGMCC No. 5953, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 6, 2012. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was disclosed in the Chinese patent with the application number of "201210248300.2" and the title of "A Strain of Bacillus subtilis and Its Application".

[0073] Paecilomyces lilacinus E16, with the preservation number of CGMCC No. 5951, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 6, 2012. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was disclosed in the Chinese patent with the application number of "201210358765.3" and the title of "A Strain of Paecilomyces lilacinus and Its Application".

[0074] Trichoderma viride H06, with the preservation number of CGMCC No. 6229, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 15, 2012. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was disclosed in the Chinese patent with the application number of "201210323738.2" and the title of "A Strain of Trichoderma viride and Its Application".

[0075] Bacillus amyloliquefaciens HC200, with the preservation number of CGMCC No. 10371, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2015. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was disclosed in the Chinese patent with the application number of "201510653843.6" and the title of "A Strain of Bacillus amyloliquefaciens, Its Bacterial Agent and Application".

[0076] The chemotactic antagonist - Bacillus velezensis LG14-3 was disclosed in the literature "Research on the Targeted Prevention and Control of Banana Fusarium Wilt by Rhizosphere Chemotactic Bacteria" (Yang Lihua. Research on the Targeted Prevention and Control of Banana Fusarium Wilt by Rhizosphere Chemotactic Bacteria [D]. Guizhou University, 2023.). It is provided by the Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, and a certificate for external distribution for 20 years is promised.

[0077] In the embodiments of the present invention, the selective medium for Fusarium oxysporum was disclosed in the literature "A Simple Selective Medium for Isolating Banana Fusarium Wilt Pathogen" (Jing Xiaohui, Wu Lunying, Ou Xiaoling, Zhu Lilin, Xue Yuxiao, Wu Lin, Huang Junsheng. A Simple Selective Medium for Isolating Banana Fusarium Wilt Pathogen [J]. Chinese Journal of Tropical Crops, 2009, 30(11): 1671-1673.).

[0078] The control strain D34 in the embodiments of the present invention can be found in the published literature (Cao Zhichun. Construction of a Multiple Screening System for Antagonistic Bacillus against Banana Fusarium Wilt and Its Control Effect Research [D]. Hainan University, 2016.).

[0079] In the embodiments of the present invention, the PCR detection method for citrus huanglongbing is carried out with reference to the method in the literature "Comparative Study on PCR Detection Primers for Citrus Huanglongbing" (Comparative Study on PCR Detection Primers for Citrus Huanglongbing. Plant Quarantine. 2022, 36(02): 1-6.).

[0080] In the embodiments of the present invention, the detection method for starch in citrus leaves is carried out with reference to the method in the literature "Effects of Huanglongbing on Assimilation Accumulation and Mineral Nutrient Transport in Citrus Leaves" (Shi Yaman, Zeng Jixing, Xue Jinjun, etc. Effects of Huanglongbing on Assimilation Accumulation and Mineral Nutrient Transport in Citrus Leaves [J]. Journal of Plant Nutrition and Fertilizers, 2023, 29(05): 949-960.).

[0081] In the embodiments of the present invention, the detection method for citrus quality is carried out with reference to the method in the literature "Effects of the Concentration of Citrus Huanglongbing Bacteria on the Fruit Quality of Qiongzhong Green Oranges" (Li Danyang, Huang Linxiao, Yang Yi. Effects of the Concentration of Citrus Huanglongbing Bacteria on the Fruit Quality of Qiongzhong Green Oranges [J]. South China Fruits, 2021, 50(02): 26-29+34. Lu Ying, Qi Yanxiang, Yu Qunfang, etc. Effects of Huanglongbing on the Fruit Quality of Qiongzhong Green Oranges [J]. Journal of Anhui Agricultural Sciences, 2018, 46(11): 122-124.).

[0082] In the embodiments of the present invention, for soil nutrient determination, 200 g of rhizosphere soil with a depth of 10-20 cm is taken from each of 5 plants, mixed evenly, stones and plant residues are removed, air-dried, and passed through a 200-mesh sieve. Then, the organic matter in the soil is measured by the ignition method, and the available nitrogen, available phosphorus, and available potassium are determined using an HM-TYA type soil fertilizer nutrient rapid detector. The salinity is measured by a TR-8D salinity detector, and the pH value is measured by a pH meter.

[0083] In the embodiment of the present invention, the PCR detection method for tomato bacterial wilt refers to the reference "Isolation of Ralstonia solanacearum and Establishment of a Triplex PCR System" (Li Deming, Zhai Zixiang, etc. 2020. Isolation of Ralstonia solanacearum and Establishment of a Triplex PCR System [J]. Molecular Plant Breeding, 18(11): 3655-3661.), and the method for investigating the incidence of bacterial wilt refers to the method in the reference "Field Control Effect of Plant Vaccine Eru Lenglte on Tomato Bacterial Wilt" (Zheng Xuefang, Liu Bo, Zhu Yujing, etc. 2018. Field Control Effect of Plant Vaccine Eru Lenglte on Tomato Bacterial Wilt [J]. Acta Phytophylacica Sinica, 45(5), 1096-1102.).

[0084] In the embodiment of the present invention, for the determination of the growth indexes of tea plants, the growth indexes are determined with reference to the method in the reference (Zhong Shengyun, Chen Guode, Zhang Wei, etc. Influence of Different Shading Treatments on Seasonal Changes in Yield and Quality of Hainan Big-leaf Tea [J]. Modern Agricultural Science and Technology, 2019(17): 5-7, 9.).

[0085] The tested chemical agent in the embodiment of the present invention: 25% pyraclostrobin emulsifiable concentrate is purchased from BASF Plant Protection (Jiangsu) Co., Ltd.

[0086] The microbial bacterial fertilizer used in the embodiment of the present invention is Bacterial fertilizer No. (100 g / L of amino acid, organic matter ≥ 40%, total nutrient N+P2O5+K2O ≥ 12%, total effective viable bacteria count: 50×10 8 cfu / g, among which, the viable bacteria count of Bacillus subtilis BLG010 is 28×10 8 cfu / g, and the viable bacteria count of Bacillus amyloliquefaciens HC200 is 22×10 8 cfu / g, powder.), and this microbial bacterial fertilizer is provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and has been published in the reference (Shao Xuefeng. Research and Application of the Activation Technology between Bacterial Fertilizers No. for Controlling Infectious Diseases [D]. Huazhong Agricultural University, 2020). In the plot test and field test of the embodiment of the present invention, compound microbial preparation 1 and active ingredient 2 are mixed in a mass ratio of 10:1, and after proliferation and cultivation, compound microbial preparation 2 is obtained: If the application time is after plant transplantation, compound microbial preparation 2 is divided into root and leaf applications, and is sprayed onto the roots and leaves at a mass ratio of 5:1 respectively; if the application time of compound microbial preparation 2 is before plant transplantation, it is all applied to the soil. Other comparative / control preparations

[0087] Bacterial fertilizer No. and water control are also applied with reference to the above method.

[0088] ​In the present invention, the specific methods and parameter controls for formulating, culturing, and fermenting using the strain culture medium of the present invention, as well as grinding, shaking, stirring, filtering, etc., can all be accomplished by conventional technical means in the art.

[0089] Example 1 Preparation of Compound Microbial Agent

[0090] 1. Seed liquid preparation:

[0091] Under sterile conditions, pick the mycelia of Paecilomyces lilacinus E16 and Trichoderma viride H06 on the plate medium with an inoculation needle, and inoculate them into the liquid activation medium (calculated based on 1 L of the activation medium, sucrose or glucose 5 g / L, tryptone 2.5 g / L, yeast powder 2.5 g / L, and sodium chloride 5 g / L) respectively, and culture at 28 °C for 4 d to obtain the seed liquids of Paecilomyces lilacinus E16 and Trichoderma viride H06;

[0092] Under sterile conditions, pick Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 on the plate medium with an inoculation needle and inoculate them into the liquid activation medium (calculated based on 1 L of the activation medium, sucrose or glucose 5 g / L, tryptone 2.5 g / L, yeast powder 2.5 g / L, amino acid chelated calcium, magnesium, boron, and zinc 5 g / L, magnesium sulfate 0.1 g / L, dipotassium hydrogen phosphate 1 g / L, manganese sulfate 0.01 g / L, sodium chloride 5 g / L) respectively, and culture at 28 °C for 3 d to obtain the seed liquids of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, chemotactic antagonist - Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 respectively.

[0093] 2. Preparation of Active Ingredient 1:

[0094] Mix corn flour, amino acid chelated calcium, magnesium, boron, and zinc, sucrose, and peptone (at least 4 of corn flour, sucrose, peptone, bioenzyme, and amino acid chelated calcium, magnesium, boron, and zinc can be mixed in any equal proportion) in equal proportion to obtain a mixed material; then mix the obtained mixed material with clear water in a mass ratio of 1:1 and mix evenly, and sterilize by moist heat at 121 °C for 20 min to obtain a fermentation medium;

[0095] Inoculate the seed liquids of Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200 into the fermentation medium for fermentation culture respectively. The inoculation amount is 5% (v / v), the fermentation culture time is 4 d, and the temperature is 30 °C to obtain the fermentation products of each strain;

[0096] The fermentation products of each strain were dried at 30 - 40 °C for 48 h, pulverized, and the fermentation product powders of each strain were obtained; among them, the effective viable count of the fermentation product powder of Bacillus subtilis BLG010 was 54×10 10 CFU / g, the effective viable count of the fermentation product powder of Bacillus amyloliquefaciens HW05 was 23×10 10 CFU / g, the effective viable count of the fermentation product powder of Paecilomyces lilacinus E16 was 6.4×10 10 CFU / g, the effective viable count of the fermentation product powder of Trichoderma viride H06 was 5.7×10 10 CFU / g, the effective viable count of the fermentation product powder of Bacillus velezensis LG14 - 3 was 12×10 10 CFU / g, the effective viable count of the fermentation product powder of Bacillus amyloliquefaciens HC200 was 5.9×10 10 CFU / g;

[0097] The fermentation product powder of Bacillus subtilis BLG010, the fermentation product powder of Bacillus amyloliquefaciens HW05, the fermentation product powder of Paecilomyces lilacinus E16, the fermentation product powder of Trichoderma viride H06, the fermentation product powder of Bacillus velezensis LG14 - 3, and the fermentation product powder of Bacillus amyloliquefaciens HC200 were mixed evenly according to the mass ratio of 5:6:2:2:2:1 to obtain the microbial fermentation product powder.

[0098] 3. Preparation of the compound microbial agent

[0099] (1) Preparation of compound microbial agent 1

[0100] The active ingredient 1, the carrier, and the synergist were mixed according to the mass ratio of 10:10:1 to obtain compound microbial agent 1; among them, the carrier was 4 kinds or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium, magnesium, boron, and zinc, humic acid, and fish protein. In this example, the carrier was obtained by mixing chitosan, potassium dihydrogen phosphate, urea, and humic acid according to the mass ratio of 10:1:5:10; the synergist was 3 kinds or more of silicone, azone, gibberellin, and ascorbic acid. In this example, the synergist was obtained by mixing silicone, azone, and ascorbic acid according to the mass ratio of 100:100:5; the shelf life of compound microbial agent 1 ≥ 18 months, and the total viable count was 12×10 10 CFU / g.

[0101] (2) Preparation of active ingredient 2

[0102] 1) Preparation of healthy plant leaf extract: Mix healthy plant leaves and water in a mass ratio of 1:(10 - 20), grind evenly and filter to obtain it; among them, the plant leaves are those of citrus, tea tree, banana, pepper or tomato, specifically determined according to the prevention and control object to be applied. If the application object is systemic diseases such as citrus huanglongbing in citrus, then healthy citrus leaves are used; if the application object is leaf diseases such as tea blister blight and tea zonate spot, then healthy tea tree leaves are used; in this example, the plant leaves and water are mixed in a mass ratio of 1:10.

[0103] 2) Preparation of healthy plant rhizosphere soil extract: Mix plant rhizosphere soil and water in a mass ratio of 1:(5 - 10), shake or stir for 5 - 10 min, filter and take the supernatant to obtain it; among them, the plant is citrus, tea tree, banana, pepper or tomato, specifically determined according to the prevention and control object to be applied. If the prevention and control object is soil-borne diseases such as pepper blight, banana wilt, tomato bacterial wilt and tomato fusarium wilt, then the rhizosphere soil of pepper, banana and tomato are used respectively; in this example, the plant rhizosphere soil and water are mixed in a mass ratio of 1:5.

[0104] (3) Preparation of compound microbial agent 2

[0105] Under field environmental conditions, mix compound microbial agent 1 and active ingredient 2 in a mass ratio of 10:1 in a container such as a bucket or a pool, then dilute and proliferate culture to obtain compound microbial agent 2; among them, the dilution multiple is 10 times, the diluent is water, the field natural temperature for proliferation culture is 20℃ - 40℃, and the ventilation or stirring time is 48 h; among them, active ingredient 2 is healthy plant leaf extract or healthy plant rhizosphere soil extract.

[0106] The microbial fermentation product powder, active ingredient 1, compound microbial agent 1, active ingredient 2, compound microbial agent 2, carrier and synergist prepared in Example 1 are all used for subsequent experiments.

[0107] Compatibility of the fermentation product powder of a single strain, synergist, carrier and active ingredient 2 prepared in Example 1 of Example 2

[0108] Separate single-fermentation product powders of Bacillus subtilis BLG010 (abbreviation: BLG010), Bacillus amyloliquefaciens HW05 (abbreviation: HW05), Trichoderma viride H06 (abbreviation: H06), Paecilomyces lilacinus E16 (abbreviation: E16), Bacillus velezensis LG14-3 (abbreviation: LG14-3), and Bacillus amyloliquefaciens HC200 (abbreviation: HC200) prepared in Example 1 were diluted 1000 times and evenly spread onto 9-cm petri dishes containing 1 / 4 LB solid medium (0.5% sucrose or glucose, 0.25% tryptone, 0.125% yeast extract, 0.25% NaCl, and 1.5% agar). Then, 0.1 g each of the synergist, carrier, and active ingredient 2 were diluted 500 times, shaken well, and 5 μL was aspirated with a pipette. Taking the center of each petri dish containing different strains as the center, 500-fold dilutions of the synergist, carrier, healthy plant leaf extract, and healthy plant rhizosphere soil extract were inoculated equidistantly at 0.5 cm from both ends of the center along a straight line direction. Using equal-volume clear water as a control, they were cultured in an incubator at 28°C for 2 - 3 d, and the width of the inhibition zone was measured. Each experiment was repeated 3 times, and the experimental results are shown in Table 1.

[0109] Table 1 Results of compatibility investigation

[0110]

[0111]

[0112] Note: - represents no inhibition; + represents weak antagonism, inhibition zone width 1 - 5 mm; ++ represents stronger antagonism, inhibition zone diameter 5 - 10 mm; +++ represents strong antagonism, inhibition zone width > 10 mm.

[0113] The results show that the fermentation product powders of BLG010, HW05, H06, E16, LG14-3, and HC200 containing single strains are compatible with the synergist, carrier, healthy plant leaf extract, and healthy plant rhizosphere soil extract, indicating that this combination has the prospect of preparing a compound microbial agent for preventing and controlling various plant diseases and its application.

[0114] Example 3 Compound microbial agent 1 has a synergistic antagonistic effect against various pathogenic bacteria

[0115] 1. Determination of the antagonistic effect of compound microbial agent 1 against various pathogenic bacteria

[0116] The compound microbial agent 1 prepared in Example 1 and active ingredient 1 were selected, and at the same time, comparative agents (numbered 3 to numbered 14) with indicators exceeding the scope of the strains of the present invention were prepared using the method of Example 1. The bacterial fertilizer, carrier, synergist, and clear water were used as controls to compare the synergistic antagonistic effects of the above preparations against various pathogens. The specific treatments are shown in Table 2.

[0117] Prepare 1 / 4 LB solid medium (0.5% sucrose or glucose, 0.25% tryptone, 0.125% yeast extract, 0.25% NaCl, and 1.5% agar) containing Fusarium oxysporum, Phytophthora capsici, Ralstonia solanacearum, Xanthomonas axonopodis pv. cynodontis, and Escherichia coli DH5α bacterial solutions respectively, pour it into a 9-cm diameter petri dish, and pre-culture it at 28 °C for 12 h. Then, according to the treatments in Table 2, take 0.1 g of Compound Microbial Preparation 1, different combinations of control preparations, synergist, and carrier respectively, dilute them 1000 times with clear water, shake well to obtain a 1000-fold dilution, suck 5 μL with a pipette, and inoculate 4 test bacterial solutions at equal distances with a radius of 2.5 cm centered on the center of each petri dish, using equal-volume clear water as a control. Culture it in an incubator at 28 °C for 3 d, observe the inhibition zone, and repeat each experiment 3 times. Measure the width of the inhibition zone between the edge of the test bacterial colony and the pathogen (the results are shown in Table 2).

[0118] Since the pathogen Exobasidium vexans of tea blister blight is difficult to culture in vitro, pick Exobasidium vexans spores from the tea blister blight lesions on tea tree leaves, and refer to the methods in the literature "Study on the Biocontrol Effect of Bacillus velezensis F85 against Tobacco Anthracnose" (Liu Hanfei, Xu Tingting, Li Xihong, et al. Study on the Biocontrol Effect of Bacillus velezensis F85 against Tobacco Anthracnose [J]. Chinese Tobacco Science, 2023, 44(3): 47-52, 61.) and the literature "Control Effects of Two Strains of Bacillus amyloliquefaciens on Watermelon Fusarium Wilt and Detection of Their Related Biocontrol Factors" (Li Dan, Li Rongmei, Qin Weiying, et al. Control Effects of Two Strains of Bacillus amyloliquefaciens on Watermelon Fusarium Wilt and Detection of Their Related Biocontrol Factors) to prepare a mixed solution containing Compound Microbial Preparation, different combinations of control preparations, synergist, carrier, and No. bacterial fertilizer and Exobasidium vexans conidia, using clear water as a control. The concentration of conidia in this mixed solution is 1×10 6 cfu / mL.

[0119] The mixed solutions of the above treatments were respectively inoculated onto hydrophobic glass slides, using the conidia solution inoculated with sterile water as a control (CK), with 3 replicates for each treatment, and 100 spores were counted for each replicate. Incubate it under moisturized conditions at a constant temperature of 28 °C in the dark for 24 h, observe and calculate the inhibition rate of spore germination under a microscope, [(control spore germination rate - treatment spore germination rate) / control spore germination rate]×100.

[0120] 2. Determination of the inhibition rate of egg hatching of Meloidogyne incognita by Compound Microbial Preparation 1

[0121] Take 1 mL of the root-knot nematode egg suspension with a concentration of 120 eggs / mL, dispense it into 2 mL centrifuge tubes, and then add 200 μL of 1000-fold dilutions of different combinations of control preparations, synergists, and carriers to the centrifuge tubes, using clear water as the control.

[0122] Among them, the banana root-knot nematode egg suspension is composed of Meloidogyne incongnita and Meloidogyne javanica in a ratio of 1:1 by quantity.

[0123] Each experiment is repeated 3 times. On the 3rd day, the egg hatching rate and egg hatching inhibition rate are statistically analyzed. The calculation formula for the hatching rate is: Egg hatching rate (%) = [Number of hatched larvae / (Number of hatched larvae + Number of unhatched eggs)] × 100. The calculation formula for the egg hatching inhibition rate is: Egg hatching inhibition rate (%) = [(Control egg hatching rate - Treated egg hatching rate) / Control egg hatching rate] × 100. The experimental results are shown in Table 2.

[0124] Table 2 Inhibitory effects of different treatments on pathogenic bacteria

[0125]

[0126]

[0127] As can be seen from Table 2, Compound Microbial Preparation 1 has antagonistic activity against various original bacteria in Table 1, is superior to Microbial Active Ingredient 1, and is also superior to other single-strain preparations or preparations of strain combinations. Thus, it can be seen that Compound Microbial Preparation 1 has a broad antibacterial spectrum and has the potential to prepare multifunctional microbial agents.

[0128] Example 4 Antagonistic effects of Compound Microbial Preparation 1 prepared with different combinations of Active Ingredient 1, carrier, and synergist against various pathogens

[0129] According to the method of Example 1, take Active Ingredient 1, carrier, and synergist, and prepare 4 groups of Compound Microbial Preparation 1 with different combinations according to the proportion range [(20 - 50):(10 - 30):(3 - 8)] required by the present invention, numbered B1, B2, B3, B4 in sequence. Use the preparation of 4 groups of Compound Microbial Preparation and clear water outside the proportion range required by the present invention as the control, numbered B5, B6, B7, B8, B9 in sequence. According to the method of Example 3, compare the antagonistic effects of different combinations of Active Ingredient 1, carrier, and synergist against various pathogens.

[0130] Table 3 Inhibitory effects of different treatments on pathogenic bacteria

[0131]

[0132] As can be seen from Table 3, four groups of compound microbial agents 1 (numbered 1 to 4) with different combinations were prepared according to the proportion range protected by the present invention, which are superior to the treatments of other combinations. Thus, it can be seen that the compound microbial agent 1 prepared within the proportion range provided by the present invention has a broad antibacterial spectrum and has the potential to prepare multifunctional microbial agents.

[0133] Field control effect of the compound microbial agent in Example 5 against Phytophthora capsici

[0134] In a pepper planting area in Wenchang, Hainan Province where Phytophthora capsici often occurs, the incidence rate before the experiment was over 90%. The area of each treatment was 1 mu, and the dosage per time was 2.5 kg / mu. It was applied once every 30 days for 6 consecutive times, with a total of 15 kg / mu. It was sprayed by integrating water and fertilizer, and the active ingredient 2 was the extract of healthy pepper rhizosphere soil. For the treatments of applying compound microbial agent 1 alone, control agent bacterial fertilizer No. or active ingredient 2, it was made up with clear water to keep the water consumption of each treatment consistent. Settings: equal amount of clear water (CK1); after compound microbial agent 1 and active ingredient 2 were mixed and then subjected to proliferation culture (diluted 10 times, 25°C - 30°C, aerated culture for 48 h), compound microbial agent 2 was obtained, and then it was diluted 100 times and applied (A1); bacterial fertilizer No. and active ingredient 2 were mixed, subjected to proliferation culture (25°C - 30°C, diluted 10 times, aerated culture for 48 h), and then diluted 100 times and applied (A2); after compound microbial agent 1 was separately subjected to proliferation culture (25°C - 30°C, diluted 10 times, aerated culture for 48 h), it was then diluted 100 times and applied (A3); bacterial fertilizer No. was separately subjected to proliferation culture (25°C - 30°C, diluted 10 times, aerated culture for 48 h), and then diluted 100 times and applied (A4); active ingredient 2 was separately subjected to proliferation culture (25°C - 30°C, diluted 10 times, aerated culture for 48 h), and then diluted 100 times and applied (A5); compound microbial agent 1 was directly diluted 1000 times and applied (A6); bacterial fertilizer No. was directly diluted 1000 times and applied (A7); active ingredient 2 was directly diluted 1000 times and applied (A8). It was repeated 3 times, with a total of 27 plots, and the plots were isolated by trenches. All the test fertilizers for conventional fertilization were used as topdressing, and the base fertilizer, other topdressing and field management were carried out uniformly according to the local normal level.

[0135] The measurement of rhizosphere soil respiration rate refers to the literature (Wang Jun, Zhou You, Yang Layin, Liu Lei, Fu Hongwen, Huang Junsheng. 2019. Effects of applying compound bacterial fertilizer and intercropping on controlling banana wilt disease [J]. China Fruits, (6): 69 - 72.)

[0136] The disease index and control efficacy were statistically analyzed with reference to the literature (Sang Liwei, Liu Aiqin, Gao Shengfeng, et al. Screening of Biopesticides for Controlling Pepper Blast and Their Pot Experiment [J]. Chinese Journal of Tropical Crops, 2017, 38(05): 915-918.).

[0137] Determine soil nutrients, salinity and pH.

[0138] Table 4 Investigation results of rhizosphere soil respiration rate, disease index and control efficacy

[0139] Yield (kg / mu) Soil respiration rate (%) Disease index Control effect (%) CK1 7.52 78.31 / A1 22.48 8.18 89.55 A2 14.14 25.37 62.86 A3 17.21 26.34 61.44 A4 12.24 28.27 58.62 A5 9.25 41.18 39.72 A6 12.14 32.14 52.95 A7 9.27 39.45 42.25 A8 8.24 51.56 24.52

[0140] Table 5 Determination results of soil nutrients, salinity and pH

[0141]

[0142]

[0143] As can be seen from the results recorded in Table 4 and Table 5, under field conditions, Compound Microbial Agent 1 and Compound Microbial Agent 2 obtained by proliferating with the rhizosphere soil extract of healthy pepper plants can significantly improve the disease prevention and growth promotion effects, and are superior to other treatments.

[0144] Example 6 Control Efficacy of Compound Microbial Agent against Banana Fusarium Wilt

[0145] In the banana planting area of Banqiao Town, Dongfang City, Hainan Province, the incidence of banana Fusarium wilt before the experiment was over 90%. Plot experiments and field experiments were carried out. Before cultivation, the land was evenly plowed, and the plowing depth was 0.5 m. The concentration of Fusarium in the soil was detected by a selective medium to reach 4.2×10 4 colony-forming units per gram of soil. The dosage of the compound microbial agent was 5 kg per mu for both.

[0146] 1. Plot control efficacy

[0147] The area of each plot was 0.2 mu, and the dosage per time was 1 kg per plot. It was applied once every 10 days after colonization and 7 days before banana planting, and continuously applied 5 times, with a total of 5 kg per plot, and sprayed by integrated water and fertilizer. The control group and treatment settings were referred to Example 5, where the active ingredient 2 was replaced with the rhizosphere soil extract of healthy bananas. When the control got sick, the incidence of each treatment group was counted, and the control efficacy was calculated. The investigation results are shown in Table 6.

[0148] 2. Field control efficacy

[0149] Control (CK): Treated with clear water;

[0150] T1: After the compound microbial agent 1 and the active ingredient 2 are mixed evenly at a mass ratio of 1:10, they are diluted 10 times with water and then subjected to proliferation culture (aeration culture at 25°C - 30°C for 48 h) to obtain the compound microbial agent 2, which is then diluted 100 times before application;

[0151] T2: The compound microbial agent 1 is directly diluted 1000 times before application;

[0152] T3: The bacterial fertilizer No. is directly diluted 1000 times before application.

[0153] The area of each treatment is 1 mu, randomly arranged, repeated 3 times, with a total of 12 plots, and trenches are dug for isolation between plots. All the tested fertilizers for conventional fertilization are used as topdressing, and the base fertilizer, other topdressings and field management are carried out uniformly according to the local normal level. Then, the yield, incidence, control effect, measurement of rhizosphere soil respiration rate, soil nutrients, salinity, pH and soil bacterial community structure are measured. Among them, the measurement methods of yield, incidence, control effect and rhizosphere soil respiration rate refer to the method in the literature "Influence of Application of Compound Bacterial Fertilizer and Intercropping on the Control of Banana Fusarium Wilt" (Wang Jun, Zhou You, Yang Layin, Liu Lei, Fu Hongwen, Huang Junsheng. 2019. Influence of Application of Compound Bacterial Fertilizer and Intercropping on the Control of Banana Fusarium Wilt [J]. China Fruits, (6): 69 - 72.); the measurement of soil nutrients refers to Example 5; the measurement method of soil bacterial community structure refers to the method in the literature "Research on Targeted Prevention and Control of Banana Fusarium Wilt by Rhizosphere Chemotactic Bacteria" (Literature: Yang Lihua. Research on Targeted Prevention and Control of Banana Fusarium Wilt by Rhizosphere Chemotactic Bacteria [D]. Guizhou University, 2023.). The survey results are shown in Table 7 - Table 9 and Figure 1 。

[0154] Table 6 Investigation Results of Incidence and Control Effect

[0155] Treatment Incidence rate (%) Control effect (%) CK1 82.17 / A1 9.12 88.90 A2 24.18 70.57 A3 28.63 65.16 A4 37.24 54.68 A5 53.56 34.82 A6 36.14 56.02 A7 46.41 43.52 A8 68.69 16.41

[0156] The results show that under plot conditions, the disease prevention and growth promotion effect of the compound microbial agent 2 applied later is better than that of other combined treatments (Table 6).

[0157] Table 7 Detection Results of Yield, Incidence, Control Effect and Measurement of Rhizosphere Soil Respiration Rate

[0158] Treatment Soil respiration rate (%) Yield (kg / mu) Soil respiration rate (%) Disease index Control effect (%) CK 10.17 2549 / 81.22 / T1 19.27 3547 39.15 24.12 93.30 T2 16.42 3120 22.40 34.97 56.94 T3 11.21 2782 9.14 41.21 49.26

[0159] Table 8 Detection Results of Soil Nutrients, Salinity and pH

[0160]

[0161] Table 9 Detection Results of Alpha Diversity Index of Rhizosphere Soil Bacteria

[0162] Treatment Chao1 index goods_coverage observed_otus Shannon index Simpson index CK 861.4055 0.9984 833 3.9199 0.6289 T1 778.1368 0.9981 729 3.6522 0.6097 T2 425.1304 0.9988 392 2.0186 0.4602 T3 447.7711 0.9988 424 2.0946 0.4853

[0163] The results showed that the disease prevention and growth promotion effects of the compound microbial agent 2 under field conditions were better than those of other treatments applied by the same method (Table 7), and the soil nutrient indexes were significantly better than those of other treatments applied by the same method (Table 8).

[0164] Compared with CK, the decline ranges of the richness indexes chao1, observed_otus, the diversity indexes shannon and simpson in the T1 treatment were 3.05%-12.52%, while those in the T2 and T3 treatments reached 22.83%-52.96% (Table 9).

[0165] Figure 1 The results showed the relative abundances of the species at the genus level for each treatment (top 15). Among them, compared with CK, the two Bacillus and Tumebacillus in the T1 treatment increased significantly by 25.12 and 35.58 times, the two microorganisms in the T2 treatment increased by 14.96 and 9.47 times, and the two microorganisms in the T3 treatment increased by 11.86 and 4.22 times.

[0166] Based on the above results, it was shown that the compound microbial agent 2 reduced the bacterial diversity and abundance, and increased the abundance of the beneficial microorganism Bacillus in the rhizosphere soil.

[0167] Example 7 Control Effect of the Compound Microbial Agent on Tomato Bacterial Wilt

[0168] Using the plot where tomato bacterial wilt occurred severely in Qionghai, Hainan in previous years as the test plot, plot control and field experiments were carried out. Before cultivation, the land was evenly plowed, and the plowing depth was 0.5 m, and the application rate was 2.5 kg / mu for all.

[0169] 1. Plot Control Effect

[0170] The area of each treatment was 0.2 mu, and the dosage per time was 0.5 kg / treatment. It was applied once every 10 days after colonization, starting 7 days before tomato colonization, and applied continuously for 5 times, with a total of 2.5 kg / plot, and sprayed by integrated water and fertilizer. There were a total of 8 control groups and treatment settings. Referring to Example 5, the active ingredient 2 was replaced with the extract of healthy tomato rhizosphere soil. When the control got sick, the incidence rates of each treatment group were counted, and the control effect was calculated. The survey results are shown in Table 10.

[0171] 2. Field Experiment

[0172] Four groups of treatments were set up as follows:

[0173] Control (CK): Treated with clear water;

[0174] T1: After the compound microbial agent 1 and the active ingredient 2 are mixed evenly at a mass ratio of 1:10, they are diluted 10 times with water and then subjected to proliferation culture (aerated culture at 25°C - 40°C for 48 h) to obtain the compound microbial agent 2, which is then diluted 100 times before application.

[0175] T2: The compound microbial agent 1 is directly diluted 1000 times before application.

[0176] T3: The bacterial fertilizer No. is directly diluted 1000 times before application.

[0177] The area of each treatment is 1 mu, randomly arranged, repeated 3 times, with a total of 12 plots, and ditches are dug for isolation between plots. On the basis of conventional fertilization, different preparations (CK, T1 - T3) are respectively watered on the roots and leaves of tomatoes during planting, and then watered once every 3 weeks for a total of 5 times.

[0178] After that, growth indexes, control effects, soil nutrients, and soil bacterial microbial diversity are investigated. Among them, the investigation method for growth indexes is to select healthy leaves from the upper and lower parts of each plant under natural light, and use the chlorophyll meter TYS - 4N to measure the relative chlorophyll content (SPAD value) of the leaves; when the fruits are mature, select the first and second clusters of fruits to weigh and measure the single - plant yield; the investigation method for the control effect is that during the tomato harvest period, 15 tomatoes are selected by the five - point sampling method for each treatment to collect their roots, and the root DNA is extracted using the Shengong plant DNA extraction kit. Referring to the method in the literature "Isolation of Ralstonia solanacearum and Establishment of a Triple - PCR System" (Li Deming, Zhai Zixiang, et al. 2020. Isolation of Ralstonia solanacearum and Establishment of a Triple - PCR System [J]. Molecular Plant Breeding, 18(11): 3655 - 3661.), triple - PCR detection of bacterial wilt is carried out. 15 plants are selected for each repetition to detect the bacteria - carrying rate of the roots; referring to the method in the literature "Field Control Effect of Plant Vaccine Eru - Lengte on Tomato Bacterial Wilt" (Zheng Xuefang, Liu Bo, Zhu Yujing, Lin Kangmei, Ge Cibin, Chen Deju. 2018. Field Control Effect of Plant Vaccine Eru - Lengte on Tomato Bacterial Wilt [J]. Acta Phytophylacica Sinica, 45(5), 1096 - 1102.), 100 plants are selected for each repetition to count the incidence rate, and calculate the control effect of different treatments on tomato bacterial wilt. The formula for the incidence rate is: Incidence rate (%) = Number of diseased plants / Total number of investigated plants × 100; The formula for the control effect (%) = (Control incidence rate - Treatment incidence rate) / Control incidence rate × 100; The method for measuring soil bacterial microbial diversity refers to Example 6, which is to take the rhizosphere soil, mix it evenly, remove stones and plant residues, air - dry it, and pass it through a 200 - mesh sieve to obtain the soil sample, extract the total soil DNA, and construct a small - fragment library for sequencing using the paired - end sequencing method based on the Illumina NovaSeq sequencing platform; The investigation results are shown in Table 10 - Table 11 and Figure 2 as shown.

[0179] Table 10 Statistical Results of Incidence and Control Efficiency

[0180] Treatment Incidence rate Control effect (%) CK1 79.82 / A1 6.75 91.54 A2 21.86 72.61 A3 26.27 67.09 A4 31.88 60.06 A5 50.31 36.97 A6 35.79 55.16 A7 42.06 47.31 A8 61.34 23.15

[0181] The results show that, under plot conditions, the disease prevention and growth promotion effects of Compound Microbial Agent 2 are better than those of other combined treatments (Table 10).

[0182] Table 11 Investigation Results of Growth Indexes and Control Efficiency

[0183] Treatment Chlorophyll (SPAD) Yield per plant Bacterial carrying rate of root bacterial wilt pathogen (%) Incidence rate (%) Control effect (%) CK 44.22 2.02 73.33％ 67.00 / T1 53.41 2.58 13.33％ 25.00 62.69％ T2 48.23 2.19 40.00％ 34.00 49.25％ T3 56.87 2.31 46.67％ 44.00 34.33％

[0184] Table 12 Detection Results of Soil Nutrients, Salinity and pH

[0185]

[0186] Table 13 Detection Results of Alpha Diversity Indexes of Rhizosphere Soil Bacteria

[0187] Treatment Characteristic number Feature ACE index Chao1 index Simpson index Shannon index CK 355 358.7403 357.6471 0.9838 6.9025 T1 374 382.2821 384.6875 0.9752 6.3979 T2 359 368.3082 369.5 0.9862 7.0433 T3 373 384.3748 389.6667 0.9823 6.8322

[0188] The results show that, under field conditions, the disease prevention and growth promotion effects of Compound Microbial Agent 2 are better than those of Compound Microbial Agent 1 applied in the same method (Table 11), and the soil nutrient indexes are significantly better than those of other treatments applied in the same method (Table 12).

[0189] As can be seen from Table 13, compared with CK, the characteristic numbers Feature, richness indexes chao1 and ACE of T1 and T3 treatments increased by 5.35% and 5.07%, 6.56% and 7.15%, 7.56% and 8.95% respectively, and the increase of T2 was relatively small; the diversity indexes Simpson and Shannon of each treatment changed insignificantly.

[0190] The relative abundances of each treatment at the genus classification level (the top 20 in relative abundance of OTU numbers) were analyzed. Figure 2 The results show that among each treatment, the relative abundance of Bacillus in the T1 treatment is the highest, ranking the 3rd (6.71%), while the rankings of Bacillus in the CK, T2 and T3 treatments are among the top 10, but the abundances are only 0.79%-2.06%, indicating that Bacillus can colonize well in the rhizosphere soil after applying Compound Microbial Agent 2.

[0191] Based on the above results, it is shown that Compound Microbial Agent 2 has significant disease prevention and growth promotion effects and increases the abundance of the beneficial microorganism Bacillus in the rhizosphere soil.

[0192] Example 8 Control Efficiency of Compound Microbial Agent against Huanglongbing of Citrus

[0193] In the Qiongzhong citrus planting areas with perennial disease incidence, where the incidence of citrus huanglongbing is over 90% (Qiongzhong green oranges), plot experiments and field experiments were conducted. Based on conventional fertilization, it was applied starting from the sprouting period of spring shoots. The active ingredient 2 was replaced with an extract of healthy citrus leaves. The application rate for each treatment was 10 kg / mu each time, and then it was applied once every month thereafter:

[0194] 1. Plot experiment

[0195] The area of each treatment plot was 0.2 mu, with an application rate of 2 kg. It was continuously applied 5 times, totaling 10 kg per treatment. The setting schemes for the treatment group and the control group refer to Example 5. When the control showed symptoms of the disease, PCR was used to detect whether the midribs of citrus leaves in each treatment group were positive for the citrus huanglongbing pathogen. The infection rate was calculated as the number of infected plants / the total number of plants, and the control efficacy was calculated as 100×(control infection rate - treatment infection rate) / control infection rate. This was repeated 3 times, with a total of 12 plots, and trenches were dug for isolation between plots.

[0196] 2. Field experiment

[0197] The area of each treatment was 1 mu, and it was continuously applied 8 times, totaling 80 kg / mu. Four treatment groups were set up, specifically as follows:

[0198] Control (CK): Treated with water;

[0199] T1: Compound microbial agent 1 and active ingredient 2 were mixed evenly at a mass ratio of 1:10, diluted 10 times with water, and then subjected to proliferation culture (aerobically cultured at 20 - 37°C for 48 h) to obtain compound microbial agent 2, which was then diluted 100 times before application;

[0200] T2: Compound microbial agent 1, directly diluted 1000 times before application;

[0201] T3: Bacterial fertilizer No., directly diluted 1000 times before application.

[0202] Each treatment was 1 mu, repeated 3 times, with a total of 12 plots, and trenches were dug for isolation between plots. The carrying situation of huanglongbing pathogen, fruit yield, juice extraction rate, sugar content, starch content in leaves, microbial diversity of endophytic bacteria in leaves, and soil bacterial community structure were investigated. The specific investigation methods were as follows:

[0203] Plant branches and leaves samples were collected regularly. For each treatment, 5 citrus plants were selected at 5 positions in the east, west, south, north, and middle. One leaf sample was randomly collected from each of the 5 positions in the east, west, south, north, and middle of each citrus plant and mixed evenly to measure the starch content in the leaves. After extracting DNA from the collected samples, the PCR method was used to detect the carrying situation of huanglongbing pathogen in the plants; fruits were collected in the same way during the harvest period to measure fruit yield and juice extraction rate;

[0204] After the field experiment, the microbial diversity of endophytic bacteria in leaves was determined by referring to the method in the reference article "Effect of Colletotrichum camelliae Infection on the Community Structure of Endophytic Bacteria in Camellia oleifera Leaves" (Cui Mingqin, Zhang Donghua, Yan Xiaohui, Hong Yingdi, etc. Effect of Colletotrichum camelliae Infection on the Community Structure of Endophytic Bacteria in Camellia oleifera Leaves [J]. Chinese Journal of Biological Control, 2022, 38(4): 911-919.), with slight modifications. Three young leaves were randomly collected, surface disinfected, and DNA was extracted from the midrib of the leaves. Then, based on the Illumina NovaSeq sequencing platform, a small fragment library was constructed using the paired-end sequencing method to sequence the V3-V4 region of 16S rDNA for determining the microbial diversity of endophytic bacteria in leaves.

[0205] The survey results are shown in Table 14 - Table 18 and Figure 3 as follows.

[0206] Table 14 Incidence of Huanglongbing Bacteria and Control Efficacy

[0207] Treatment Bacterial carrying rate (%) Control effect (%) CK1 77.68 / A1 4.66 94.00 A2 19.21 75.27 A3 22.17 71.46 A4 29.32 62.26 A5 47.46 38.90 A6 28.65 63.12 A7 41.71 46.31 A8 64.89 16.46

[0208] The results showed that under plot conditions, the disease prevention and growth promotion effects of Compound Microbial Agent 2 were better than those of other combined treatments (Table 14).

[0209] Table 15 Detection Results of Huanglongbing Bacteria

[0210]

[0211] Note: - represents negative; + represents positive.

[0212] Table 16 Survey Results of Leaf Starch Content

[0213]

[0214] Table 17 Survey Results of Fruit Yield, Juice Extraction Rate, and Brix

[0215]

[0216]

[0217] Table 18 Detection Results of Alpha Diversity Index of Bacteria in the Midrib of Citrus Leaves

[0218] Treatment Characteristic number Feature Ace index Chao1 index Simpson index Shannon index CK 11 11.33 11.33 1.1363 0.4963 T1 23 23.33 23.33 2.0901 0.7927 T2 25 25.33 25.33 2.0694 0.7770 T3 17 17.00 17.00 1.7909 0.7378

[0219] The results showed that compared with the control, Compound Microbial Agent 2 could significantly eliminate the pathogens at the diseased sites (Table 15), reduce the starch content in diseased leaves, relieve the blockage of the ducts by starch in the leaves (Table 16), promote the normal growth of citrus, and thus increase the yield, juice extraction rate, and brix (Table 17);

[0220] Compared with CK, the Ace index and Chao1 index of species abundance indicators processed by T1, T2, and T3 increased by 50.04% - 105.91%; the diversity indicator Simpson index increased by 48.66% - 59.72%, and the Shannon index increased by 57.61% - 83.94% (Table 18). The increases in the chao1 index, ACE index, and Shannon index of T1 and T2 were relatively obvious, reaching more than 80%; the increases in the Simpson index of T1, T2, and T3 treatments reached more than 40%; the increase in the Simpson index of T1 treatment was the most obvious, which was significantly higher than that of T2 and T3 by 3.16 - 11.06 percentage points, and the Shannon index was higher than that of T2 and T3 by 1.82% - 26.33% (Table 18).

[0221] Analysis of the top 1% of the relative abundances at the ASV genus level for each treatment found that ( Figure 3 ), the content of Bacillus in CK was 0, the relative abundance of Bacillus in T1 treatment was 2.43%, ranking 6th; the relative abundances of Bacillus in T1 and T3 treatments were only 0.96% and 0.49, ranking 8th and 11th respectively. The citrus huanglongbing pathogen (Candidatus Liberibacter) in CK, T1, T2, and T3 treatments were 69.22%, 10.96%, 2.88%, and 16.93% respectively).

[0222] Based on the above results, it shows that the control effect and growth promotion of the compound microbial agent 2 are better than other treatments. It significantly improves the Simpson index and Shannon index of the bacterial diversity index of endophytic bacteria in citrus leaves, and at the same time increases the abundances of beneficial microorganisms such as Bacillus.

[0223] Example 9 Control Effect of Compound Microbial Agent on Tomato Fusarium Wilt

[0224] Using the plot with severe occurrence of tomato fusarium wilt in previous years as the experimental plot, the experimental design schemes of the plot experiment and field experiment are the same as those in Example 7.

[0225] The following methods are used for the detection of various indicators:

[0226] Growth indicators: Since the tomato harvest, on sunny days, when measuring, the atmospheric CO2 concentration is kept at (380 ± 6.0) μmol / mol, the air temperature is 26 °C, and the relative humidity is (60 ± 4.0)%. Randomly select 3 plants from each group, and select 3 healthy leaves from the upper and lower parts of each plant respectively. The photosynthesis is measured by an LCPRO-SD portable photosynthesis measuring instrument for 120 s; when the fruits are ripe, select the first and second spike fruits to weigh and measure the single-plant yield;

[0227] Control efficacy investigation: Investigate the incidence of tomato fusarium wilt at the seedling stage, flowering stage, fruiting stage and harvesting stage, calculate the control efficacy of different treatments against tomato fusarium wilt. The formula for calculating the incidence is: Incidence = Number of diseased plants / Total number of investigated plants × 100%; The formula for calculating the control efficacy is: Control efficacy = (Control incidence - Treatment incidence) / Control incidence × 100%;

[0228] Determination of soil fungal microbial diversity: Take the collected soil samples after mixing, stirring evenly, removing stones and plant residues, extract the total soil DNA, and sequence the ITS1 region of fungal ITS1 rDNA based on the Illumina NovaSeq sequencing platform.

[0229] The investigation results are shown in Tables 19 - 21 and Figure 4 as follows.

[0230] Table 19 Statistical results of incidence and control efficacy

[0231] Treatment Incidence rate (%) Control effect (%) CK1 81.24 / A1 8.16 89.96 A2 23.18 69.72 A3 25.64 66.69 A4 33.39 58.17 A5 50.53 36.70 A6 32.21 59.65 A7 44.48 44.27 A8 67.76 15.11

[0232] The results of plot trials show that the disease prevention and growth promotion effects of compound microbial agent 2 are significantly better than those of other combined treatments (Table 19).

[0233] Table 20 Investigation results of growth indicators and control efficacy

[0234] Treatment <![CDATA[Photosynthesis (μmol·m -2 ·s -1 )]]> Yield per plant (kg) Water-soluble sugar (%) Incidence rate (%) Control effect (%) CK 11.78 2.08 4.11 71.00 T1 13.47 2.61 4.66 17.00 87.32 T2 12.58 2.31 4.28 46.00 59.15 T3 12.17 2.27 4.58 48.00 54.93

[0235] Table 21 Detection results of Alpha diversity index of rhizosphere soil fungi

[0236] Treatment Characteristic number Feature ACE index Chao1 index Simpson index Shannon index CK 19 19.0000 19.0000 0.8228 3.1501 T1 17 25.8929 18.0000 0.8148 2.8291 T2 18 18.0000 18.0000 0.6645 2.0998 T3 15 15.0000 15.0000 0.7369 2.2735

[0237] Statistics of field disease prevention and growth promotion results show that treatment T1 significantly increases the yield and quality, with a control efficacy of 87.32%, which is also significantly higher than other treatments (Table 20).

[0238] Analysis of the Alpha diversity index results shows that compared with CK, only for treatment T1, the richness index ACE at the fungal family level increased by 36.28%, and the Chao1, Simpson, and Shannon indices decreased by 0.97% - 10.19%. For treatments T2 and T3, all indicators showed a decrease, with a decrease range of 5.26% - 33.34%. This indicates that after the application of compound microbial agent (T1), the antagonistic microorganisms colonize and become the dominant strains, inhibiting the growth of other microorganisms, thus reducing the microbial diversity index (Table 21).

[0239] Analyze the relative abundances of each treatment at the genus classification level (top 15 in terms of relative abundance of OTU numbers), and the results ( Figure 4)The results showed that the relative abundance of Trichoderma in each treatment was the highest in T1 treatment, ranking second (18.53%), while those in T2 and T3 treatments were 11.52% and 8.56% respectively; the abundances of Fusarium in CK, T1, and T2 treatments were 0.93%, 0.17%, and 2.41% respectively, while the pathogen (Fusarium) in T3 was less than 0.01%. This indicated that the beneficial microorganism Trichoderma could colonize well in the rhizosphere soil after applying Compound Microbial Agent 2.

[0240] Based on the above results, it was shown that Compound Microbial Agent 2 had the effects of preventing diseases and promoting growth, increased the ACE index of the fungal diversity index, decreased the Chao1, Simpson, and Shannon indices, and increased the abundance of the beneficial microorganism Trichoderma in the rhizosphere soil.

[0241] Example 10 Control Effect of Compound Microbial Agent on Tea Blister Blight

[0242] In the plot test and the field, in the Wuzhishan Big-leaf Tea Base where the disease occurred all year round, the incidence of tea blister blight was over 90%. On the basis of conventional fertilization, compound microbial agent was applied starting from the sprouting period of spring shoots. The active ingredient 2 was replaced with the extract of healthy tea tree leaves, and the dosage of each treatment was calculated at 2.5 kg per mu each time.

[0243] 1. Plot Test

[0244] The area of each treatment plot was 0.2 mu, and the dosage was 0.5 kg. It was continuously applied 3 times, with a total of 1.5 kg per treatment. The setting scheme of the treatment group and the control group referred to Example 5. When the control got sick, the incidence of each treatment group was counted, and the control effect was calculated. The investigation results are shown in Table 22.

[0245] 2. Field Test

[0246] The area of each treatment was 1 mu, and it was continuously applied 3 times, once every 7 days, with a total of 7.5 kg per mu. A control (CK) was set. Compound Microbial Agent 1 and the extract of healthy tea tree leaves were mixed and then subjected to proliferation culture (diluted 10 times, at a temperature of 25 - 30 °C, aerated culture for 48 hours) to obtain Compound Microbial Agent 2, which was then diluted 100 times and applied (T1); Compound Microbial Agent 2 was directly diluted 1000 times and applied (T2); 40 g of pyraclostrobin emulsifiable concentrate per mu was diluted 1000 times (T3), with 3 repetitions, a total of 12 plots, and ditches were dug for isolation between plots.

[0247] Determination of control effect: Referring to the method in the literature (Yang Wenbo, Xiang Fen, Liu Hongyan, et al. Field control effect evaluation of different agents against Exobasidium vexans [J]. China Plant Protection, 2022, 42(12): 81-84.), 7 days after the 3rd application of the drug, the disease index was investigated and the control effect was calculated. For each treatment plot, the second leaf below the bud was selected, 50 leaves were investigated, and the grading was recorded. Disease grading standard: Grade 0, no disease spots on the leaves; Grade 1, disease spots accounting for less than 25% of the leaf area; Grade 2, scattered disease spots accounting for 26% - 50% of the leaf area; Grade 3, disease spots accounting for 51% - 75% of the leaf area; Grade 4, the disease spots on the leaves are dense, accounting for 76% and above of the leaf area. The disease index and control effect were calculated according to the following formulas: Disease index = Σ (number of diseased leaves at each level × relative level value) / (total number of investigated leaves × 4) × 100; Control effect = [1 - (disease index before treatment in the control area × disease index after treatment in the treatment area) / (disease index after treatment in the control area × disease index before treatment in the treatment area)] × 100%.

[0248] Determination of main growth indicators of tea plants: Bud length: In each treatment plot, 3 replicates were set, and each replicate was a randomly selected square area of 100 cm × 100 cm. The buds with one bud and two leaves in this area were collected, and the length from the apical bud to the second leaf was measured.

[0249] Bud density: In each treatment plot, 3 replicates were set, and each replicate was a randomly selected healthy bud within a range of 30 cm × 30 cm. The number of buds was recorded.

[0250] Fresh weight: In each treatment plot, 3 replicates were set, and each replicate was a randomly selected square area of 100 cm × 100 cm. All the buds with one bud and two leaves in this area were collected and weighed immediately on the day of picking.

[0251] Relative chlorophyll content: Under natural light, healthy leaves were selected from the upper and lower parts of each plant, and the relative chlorophyll content of the leaves, that is, the SPAD value, was measured with a chlorophyll meter TYS-4N.

[0252] Determination of microbial diversity of endophytic bacteria in leaves. After the field experiment, 3 young leaves of tea plants were randomly collected. After surface disinfection, it was measured according to the method in Example 8. The survey results are shown in Table 22 - Table 24 and Figure 5 as shown.

[0253] Table 22 Survey results of disease index and control effect

[0254] Treatment Disease index Control effect (%) CK1 53.40 / A1 5.35 89.98 A2 11.93 77.66 A3 16.89 68.37 A4 24.04 54.98 A5 33.18 37.87 A6 31.37 41.25 A7 35.43 33.65 A8 45.61 14.59

[0255] The results of the plot experiment showed that the disease prevention and growth promotion effects of the compound microbial agent 2 were significantly better than those of other combined treatments (Table 22).

[0256] Table 23 Survey results of growth indicators, SPAD, disease index and control effect

[0257] Treatment Bud length Bud density Fresh weight SPAD Disease index Control effect (%) CK 1.42 68.24 15.67 32.63 41.52 / T1 2.28 124.22 25.32 43.52 2.41 94.20％ T2 1.91 102.41 22.41 41.51 6.19 85.09％ T3 1.84 87.39 19.24 36.22 3.28 92.10％

[0258] Table 24 Detection results of Alpha diversity index of endophytic bacteria in tea tree leaves

[0259] Treatment Characteristic number Feature ACE index Chao1 index Simpson index Shannon index CK 1057 1060.6050 1058.1957 0.9951 8.9857 T1 2183 2189.9147 2185.2938 0.9940 9.3819 T2 1259 1263.6375 1261.4429 0.9871 8.1245 T3 1212 1217.8239 1214.1000 0.9705 7.3694

[0260] Field trial results: The statistical results of disease prevention and growth promotion showed that the compound microbial agent 2 significantly increased the yield and quality, with a control effect of 94.20%, significantly higher than that of its T2 treatment, and comparable to the chemical agent pyraclostrobin (Table 23).

[0261] Analysis of the Alpha diversity index results showed that compared with CK, the Ace index of the species richness index in the T1, T2, and T3 treatments increased by 106.48%, 19.14%, and 14.82% respectively, and the Chao1 index increased by 106.51%, 19.21%, and 14.73% respectively; the diversity Simpson index decreased by 0.11%, 0.80%, and 2.47%, with no obvious change; the Simpson index in the T2 and T3 treatments decreased by 9.58% and 17.99% respectively, while the Shannon index in the T1 treatment increased by 4.41% (Table 24).

[0262] Analysis was carried out on the top 15 species in terms of relative abundance at the genus level for each treatment, and the results ( Figure 5 ) showed that among the top 5 species in each treatment, Rhizobium was the unique dominant genus in T1 (4.28%), Methylocella was the unique dominant genus in T2 (4.94%), and Stenotrophomonas and Pseudomonas were the unique dominant genera in T3 (13.46% and 7.23%). This indicates that the application of the compound microbial agent changed the proportion of dominant species in tea tree leaves.

[0263] The above results comprehensively showed that the application of the compound microbial agent 2 had an obvious effect on disease prevention and growth promotion, significantly increasing the richness and diversity indexes of endophytic bacteria in tea tree leaves, namely the Ace index, Chao1 index, and Shannon index, and increasing the abundance of beneficial endophytic bacteria such as Rhizobium in tea tree leaves.

[0264] Example 11 Control effect of compound microbial agent on tea zonate spot

[0265] In the Wuzhishan large-leaf tea variety base, tea plants with a tea zonate leaf spot incidence of over 90% were selected. On the basis of conventional fertilization, a compound microbial agent was applied starting from the spring shoot germination period. The treatment setting plan and the efficacy determination were both carried out with reference to the field test plan in Example 10. The sampling of tea tree leaves referred to Example 8, and the determination method of endophytic fungi in the leaves referred to Example 9. The survey results are shown in Table 25 - Table 26 and Figure 6 as follows.

[0266] Table 25 Investigation results of disease index and control efficacy

[0267] Treatment Disease index Control effect (%) CK 64.28 / T1 5.35 91.68 T2 16.46 74.39 T3 5.25 91.83

[0268] The results showed that the control efficacy of treatment T1 reached 91.68%, which was comparable to that of the chemical agent (T3), and was significantly better than that of treatment T2 (Table 25).

[0269] Table 26 Detection results of Alpha diversity index of endophytic fungi in tea trees

[0270] Treatment Characteristic number Feature ACE index Chao1 index Simpson index Shannon index CK 923 1005.8591 970.4802 0.9323 5.3559 T1 1651 1658.7841 1659.2639 0.9946 8.9545 T2 1823 1853.3617 1841.8559 0.9681 7.8788 T3 1508 1552.4282 1536.7972 0.9773 7.4514

[0271] Analysis of the Alpha diversity index results showed that compared with CK, the Ace index of the species richness index of treatments T1, T2, and T3 increased by 64.91%, 84.26%, and 54.34% respectively, and the Chao1 index increased by 70.97%, 89.79%, and 58.35% respectively; the diversity Simpson index increased by 6.68%, 4.83%, and 3.84%, and the Shannon index increased by 67.19%, 47.11%, and 39.13%; the increase in the diversity index of treatment T1 was the most obvious. At the same time, the increase in the Simpson index was 1.86 - 2.84 percentage points higher than that of T2 and T3, and the increase in the Shannon index was 20.08 - 28.06 percentage points higher than that of T2 and T3 (Table 26).

[0272] Analysis was carried out on the top 15 species with relative abundances at the genus level of endophytic fungi in each treatment. The results ( Figure 6 ) showed that the relative abundances of the tea zonate leaf spot pathogens (Neopestalotiopsis, Pseudopestalotiopsis) in CK were as high as 48.99%, while the relative abundances of the tea zonate leaf spot pathogens in T1, T2, and T3 were 0.24%, 0.74%, and 8.34% respectively. Treatment T1 significantly reduced the relative abundance of the tea zonate leaf spot pathogens and changed the proportion of dominant endophytic fungi in tea tree leaves.

[0273] Based on the above results, it was shown that the application of the compound microbial agent 2 had an obvious disease prevention and growth promotion effect, significantly improved the Simpson index and Shannon index of the endophytic fungi diversity index in tea tree leaves, and greatly reduced the abundance of the tea zonate leaf spot pathogens.

[0274] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the design of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of a microbial combination in the preparation of a composite microbial preparation for preventing and controlling plant diseases, characterized in that: The microbial combination consists of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3 and Bacillus amyloliquefaciens HC200; The deposit number of the Bacillus amyloliquefaciens HW05 is CGMCC No.10273; the deposit number of the Bacillus subtilis BLG010 is CGMCC No.5953; the deposit number of the Paecilomyces lilacinus E16 is CGMCC No.5951; the deposit number of the Trichoderma viride H06 is CGMCC No.6229; the deposit number of the Bacillus amyloliquefaciens HC200 is CGMCC No.10371; The plant diseases are one or more of pepper blight, tea cake disease and tea ring spot; The mass ratio of the Bacillus amyloliquefaciens HW05, the Bacillus subtilis BLG010, the Paecilomyces lilacinus E16, the Trichoderma viride H06, the Bacillus velezensii LG14-3 and the Bacillus amyloliquefaciens HC200 in the microbial combination is (5-10): (2-6): (2-4): (2-4): (2-5): (1-5).

2. The use according to claim 1, characterized in that: The composite microbial preparation also includes a carrier and a synergist; The mass ratio of the microbial combination, the carrier and the synergist is (20-50): (10-30): (3-8); The carrier includes any four or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium magnesium boron zinc, humic acid and fish protein; The synergist includes any three or more of organosilicon, azone, gibberellin and ascorbic acid.

3. The application of microbial preparations in preventing and controlling plant diseases, characterized in that: The microbial preparation consists of an active ingredient 1, a mixture of a carrier and a synergist, and an active ingredient 2; the active ingredient 1 is a microbial combination; the microbial combination consists of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200; The carrier includes any four or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium magnesium boron zinc, humic acid and fish protein; The synergist includes any three or more of organosilicon, azone, gibberellin and ascorbic acid; The deposit number of the Bacillus amyloliquefaciens HW05 is CGMCC No.10273; the deposit number of the Bacillus subtilis BLG010 is CGMCC No.5953; the deposit number of the Paecilomyces lilacinus E16 is CGMCC No.5951; the deposit number of the Trichoderma viride H06 is CGMCC No.6229; the deposit number of the Bacillus amyloliquefaciens HC200 is CGMCC No.10371; The plant diseases are one or more of pepper blight, tea cake disease and tea ring spot; The mass ratio of the Bacillus amyloliquefaciens HW05, the Bacillus subtilis BLG010, the Paecilomyces lilacinus E16, the Trichoderma viride H06, the Bacillus velezensii LG14-3 and the Bacillus amyloliquefaciens HC200 in the microbial combination is (5-10): (2-6): (2-4): (2-4): (2-5): (1-5); When the application object is tea cake disease and tea ring spot disease, the active ingredient 2 is an extract of healthy tea leaves; when the application object is pepper blight, the active ingredient 2 is an extract of healthy pepper rhizosphere soil; The preparation method of the healthy tea tree leaf extract comprises the steps of mixing the leaves of the healthy tea tree and water in a mass ratio of 1:(10-20), grinding and filtering, and obtaining the healthy tea tree leaf extract; The preparation method of the healthy pepper rhizosphere soil extract comprises the steps of mixing the healthy pepper rhizosphere soil and water in a mass ratio of 1:(5-10), shaking or stirring for 5-20 minutes, and filtering to obtain the healthy pepper rhizosphere soil extract; The mass ratio of the active ingredient 1, the mixture of the carrier and the synergist, and the active ingredient 2 is (6-10): (1-2); The mass ratio of the microbial combination, the carrier and the enhancer is (20-50): (10-30): (3-8).

4. A method for preventing and controlling plant diseases, characterized in that: The following steps are involved: After the microbial preparation is multiplied and cultured, it is applied to the surface of plant leaves or the rhizosphere; The microbial preparation consists of an active ingredient 1, a mixture of a carrier and a synergist, and an active ingredient 2; the active ingredient 1 is a microbial combination; the microbial combination consists of Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus velezensis LG14-3, and Bacillus amyloliquefaciens HC200; The carrier includes any four or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium magnesium boron zinc, humic acid and fish protein; The synergist includes any three or more of organosilicon, azone, gibberellin and ascorbic acid; The deposit number of the Bacillus amyloliquefaciens HW05 is CGMCC No.10273; the deposit number of the Bacillus subtilis BLG010 is CGMCC No.5953; the deposit number of the Paecilomyces lilacinus E16 is CGMCC No.5951; the deposit number of the Trichoderma viride H06 is CGMCC No.6229; the deposit number of the Bacillus amyloliquefaciens HC200 is CGMCC No.10371; The plant diseases are one or more of pepper blight, tea cake disease and tea ring spot; The mass ratio of the Bacillus amyloliquefaciens HW05, the Bacillus subtilis BLG010, the Paecilomyces lilacinus E16, the Trichoderma viride H06, the Bacillus velezensii LG14-3 and the Bacillus amyloliquefaciens HC200 in the microbial combination is (5-10): (2-6): (2-4): (2-4): (2-5): (1-5); When the application object is tea cake disease and tea ring spot disease, the active ingredient 2 is an extract of healthy tea leaves; when the application object is pepper blight, the active ingredient 2 is an extract of healthy pepper rhizosphere soil; The preparation method of the healthy tea tree leaf extract comprises the steps of mixing the leaves of the healthy tea tree and water in a mass ratio of 1:(10-20), grinding and filtering, and obtaining the healthy tea tree leaf extract; The preparation method of the healthy pepper rhizosphere soil extract comprises the steps of mixing the healthy pepper rhizosphere soil and water in a mass ratio of 1:(5-10), shaking or stirring for 5-20 minutes, and filtering to obtain the healthy pepper rhizosphere soil extract; The mass ratio of the active ingredient 1, the mixture of the carrier and the synergist, and the active ingredient 2 is (6-10): (1-2); The mass ratio of the microbial combination, the carrier and the enhancer is (20-50): (10-30): (3-8).

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