Compound microbial preparation for preventing and controlling plant diseases and application

Abstract

The invention relates to the technical field of biological control, in particular to a compound microbial preparation for preventing and controlling plant diseases and application. The microbial composition provided by the invention comprises 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. The invention also provides a composite microbial preparation which comprises the microbial composition provided by the invention, a carrier and a synergist, and also comprises a healthy plant leaf extract or a healthy plant rhizosphere soil extract. The preparation has prevention and treatment effects on various major plant diseases (pepper blast, banana fusarium wilt, tomato fusarium wilt and bacterial wilt, citrus huanglongbing, tea cake disease and tea ring spot), can change the diversity of fungi or bacteria in plant leaves or rhizosphere soil, and can improve 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 of being pollution-free and easy to develop. The most widely studied and applied are composite microbial agents containing Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, and Paecilomyces lilacinus, but they only control a single disease and have low control efficacy. Therefore, it is very necessary to find a composite microbial agent that has good control efficacy against multiple diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a composite microbial preparation and 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 carriers and synergists, as well as plant or plant rhizosphere soil extracts as composite microbial preparations, which have good preventive effects on a variety of diseases. The present invention provides new ideas for the research and development of excellent composite microbial preparations and scientific field application technologies, and at the same time provides effective technical support for green and efficient prevention and control of plant diseases.

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

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

[0007] 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; and the deposit number of the Bacillus amyloliquefaciens HC200 is CGMCC No.10371.

[0008] Preferably, 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);

[0009] And / or, the plant diseases include one or more of pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus Huanglongbing, tea cake disease and tea ring spot.

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

[0011] Further preferably, 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 5:6:2:2:2:1.

[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 solution, the present invention also provides a method for preparing the above-mentioned microbial combination, comprising the following steps:

[0014] The Bacillus amyloliquefaciens HW05, the Bacillus subtilis BLG010, the Paecilomyces lilacinus E16, the Trichoderma viride H06, the Bacillus Velez LG14-3 and the Bacillus amyloliquefaciens HC200 are activated and then inoculated into fermentation medium for fermentation culture to obtain a fermentation product of Bacillus amyloliquefaciens HW05, a fermentation product of Bacillus subtilis BLG010, a fermentation product of Paecilomyces lilacinus E16, a fermentation product of Trichoderma viride H06, a fermentation product of Bacillus Velez LG14-3 and a fermentation product of Bacillus amyloliquefaciens HC200;

[0015] The microbial combination is obtained by drying, crushing and mixing the fermentation product of Bacillus amyloliquefaciens HW05, the fermentation product of Bacillus subtilis BLG010, the fermentation product of Paecilomyces lilacinus E16, the fermentation product of Trichoderma viride H06, the fermentation product of Bacillus velezii LG14-3 and the fermentation product of Bacillus amyloliquefaciens HC200.

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

[0017] Further preferably, the ratio of the effective live bacteria count of the fermentation product powder of Bacillus amyloliquefaciens HW05, the fermentation product powder of Bacillus subtilis BLG010, the fermentation product powder of Paecilomyces lilacinus E16, the fermentation product powder of Trichoderma viride H06, the fermentation product powder of Bacillus velezii LG14-3 and the fermentation product powder of Bacillus amyloliquefaciens HC200 obtained after the crushing is 5:6:2:2:2:1.

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

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

[0020] Further preferably, the fermentation medium comprises solid components and water;

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

[0022] And / or, the solid ingredients include any four or more of corn flour, sucrose, peptone, amino acid chelated calcium, magnesium, boron and zinc, and biological enzymes.

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

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

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

[0026] The present invention provides a composite microbial preparation for preventing and controlling plant diseases, wherein the composite microbial preparation comprises an effective ingredient 1; the effective ingredient 1 comprises the above-mentioned microbial combination.

[0027] Preferably, the composite microbial preparation further comprises 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 four 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, the carrier includes, by mass: 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 three or more of organosilicon, azone, gibberellin and ascorbic acid; when the synergist includes organosilicon, azone, gibberellin and ascorbic acid, the synergist includes, by mass, 50-100 parts of organosilicon, 50-100 parts of azone, 5-10 parts of gibberellin and 5-10 parts of ascorbic acid.

[0032] Preferably, the composite microbial preparation further comprises an active ingredient 2; the active ingredient 2 comprises a healthy plant leaf extract or a healthy plant rhizosphere soil extract;

[0033] And / or, the method for preparing the healthy plant leaf extract comprises the steps of mixing healthy plant leaves and water in a mass ratio of 1:(10-20), and then grinding to obtain the healthy plant leaf extract;

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

[0035] Preferably, 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).

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

[0037] The composite microbial preparation of the present invention combines the microbial combination with the extract of healthy plant leaves or the extract of healthy plant rhizosphere soil, which can effectively prevent and treat major plant diseases, such as pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus Huanglongbing, tea cake disease and tea ring spot, and can also improve soil nutrients, change the microbial diversity in plants or rhizosphere soil, increase the abundance of beneficial microorganisms, and improve yield and quality.

[0038] As an additional scheme, when the composite microbial preparation comprises the active ingredient 1, the carrier and the enhancer, the preparation method of the composite microbial preparation comprises the step of mixing the active ingredient 1, the carrier and the enhancer to obtain the composite microbial preparation; the active ingredient 1 comprises the above-mentioned microbial combination.

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

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

[0041] As an additional scheme, when the composite microbial preparation comprises the active ingredient 1, the carrier, the enhancer and the active ingredient 2, the preparation method of the composite microbial preparation comprises the steps of mixing the active ingredient 1, the carrier and the enhancer to obtain a mixture; mixing the obtained mixture with the active ingredient 2, and then carrying out proliferation culture to obtain the composite microbial preparation; the active ingredient 2 comprises an extract from healthy plant leaves or an extract from rhizosphere soil of healthy plants.

[0042] Further 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 to the active ingredient 2 is (6-10):(1-2).

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

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

[0047] As an additional solution, when the composite microbial preparation of the present invention includes an active ingredient 1, a carrier, a synergist and an active ingredient 2 in independent packages, the specific method of use is as follows:

[0048] The active ingredient 1, the carrier and the synergist are mixed to obtain a mixture; the obtained mixture is mixed with the active ingredient 2 in a mass ratio of (6-10):(1-2), and before application, the mixture is diluted 10-20 times with water, and allowed to stand or aerate and stir at 20°C-40°C for 24h-72h to allow the bacterial amount and active substances to proliferate rapidly, and then diluted 50-100 times and applied to the roots and / or leaves of the plant;

[0049] Further preferably, the dosage of the composite microbial preparation is 2.5-10 kg / mu each time, and the number of applications is 3-15 times. It can effectively prevent and control pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus Huanglongbing, tea cake disease and tea ring spot, etc., and at the same time can improve soil nutrients and improve quality.

[0050] The present invention provides the use of the above-mentioned microbial combination or the above-mentioned composite microbial preparation in preventing and controlling plant diseases.

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

[0052] After the above-mentioned composite microbial preparation is proliferated and cultured, it is applied to the surface of plant leaves or the root zone.

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

[0054] The inventors previously found through microbiome analysis that, compared with susceptible plants or soil, a large number of microorganisms of different properties live in healthy plants or plant rhizosphere soils, such as fungi, bacteria, etc. The abundance and / or diversity of microorganisms are significantly different from those of susceptible plants or plant rhizosphere soils. Beneficial microorganisms have the same ecological niche as plant pathogens, coexist with plants for a long time, form a unique microbial community, form a plant microbial ecosystem, and play an important role in the complex microecological relationship of plants, soils, and microorganisms. Therefore, the extracts of microbial populations containing healthy plants or soils are used in combination with composite antagonistic microbial preparations in an appropriate manner, which can improve the microbial community structure in the rhizosphere soil of susceptible plants or plants, including increasing or decreasing microbial diversity, and or decreasing the abundance of pathogens in plants or plant rhizosphere soils, and or increasing the abundance of beneficial microorganisms, etc., reduce the harm of diseases to plants, and improve yield quality.

[0055] The microbial combination of the present invention includes synergistic Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06, chemotactic antagonists-Bacillus velezii LG14-3 and Bacillus amyloliquefaciens HC200. Bacillus velezii LG14-3 not only has an antagonistic effect, but also has a significant chemotactic effect on banana root secretions, glycine and citric acid, so it is referred to as chemotactic antagonists (public literature: Yang Lihua. Research on targeted prevention and control of banana wilt by rhizosphere chemotactic bacteria [D]. Guizhou University, 2023.). A composite microbial preparation is also provided, which includes the microbial combination, carrier and synergist provided by the present invention, and also includes a healthy plant leaf extract or a healthy plant rhizosphere soil extract. The microbial combination and the preparation have a preventive and therapeutic effect on a variety of major plant diseases (pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus Huanglongbing, tea cake disease and tea ring spot), and also have the effect of promoting growth. The invention solves the problem of unstable field application caused by unreasonable synergistic effect of composite microbial agents in the past, 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 antagonistic bacteria - Bacillus Velez 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, cytokinins, etc., which change the microbial diversity in the soil or plants. In addition, they have the effect of inhibiting the reproduction of various plant pathogens.

[0057] The present invention uses a microbial combination consisting of beneficial bacteria such as Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Bacillus velez LG14-3 and Bacillus amyloliquefaciens HC200 in combination with a carrier, a synergist, and a healthy plant leaf / healthy plant rhizosphere soil extract to produce a synergistic effect. The carrier can supplement key nutrients required for the proliferation of the microbial agent. With the assistance of the synergist, the soil and plant surface are composited with the microbial agent. After the healthy plant leaf / healthy plant rhizosphere soil extract is applied, it can quickly attach, grow, diffuse and penetrate. It goes to the diseased parts of the plant, improves disease resistance, removes pathogens in the diseased parts, and enhances the inhibitory effect on citrus Huanglongbing, various soil-borne pathogenic fungal diseases (banana wilt, tomato wilt, pepper blight, etc.) and soil-borne pathogenic bacterial diseases (tomato bacterial wilt, etc.), leaf diseases (tea cake disease and tea ring spot). At the same time, it changes the diversity of microorganisms in plants or soil, increases the abundance of beneficial microorganisms or reduces the abundance of pathogenic microorganisms, and achieves a balance in the microecology inside the plant or in the rhizosphere of the plant, thereby enhancing the absorption and utilization efficiency of nutrients by the plant root system, improving root vitality, further increasing yield and improving quality.

[0058] The preparation method of the composite microbial preparation of the present invention has simple process and is easy to operate. The prepared composite microbial preparation is environmentally friendly and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

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

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

[0065] Figure 6This is a graph of the relative abundance of species at the genus level in each treatment in Example 11. DETAILED DESCRIPTION

[0066] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0067] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0068] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0069] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0070] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0071] Bacillus amyloliquefaciens HW05, whose deposit number is CGMCC No. 10273, was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on January 4, 2015. The deposit address is 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 application number "201810578016.9" and name "A strain of efficient Bacillus amyloliquefaciens, its bacterial agent and application".

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

[0073] Paecilomyces lilacinus E16, whose deposit number is CGMCC No.5951, was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on April 6, 2012, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is disclosed in the Chinese patent with application number "201210358765.3" and name "A strain of Paecilomyces lilacinus and its application".

[0074] Trichoderma viride H06, whose deposit number is CGMCC No.6229, was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on June 15, 2012. The deposit address is 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 application number "201210323738.2" and name "A strain of Trichoderma viride and its application".

[0075] Bacillus amyloliquefaciens HC200, whose deposit number is CGMCC No.10371, was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on January 19, 2015, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is disclosed in the Chinese patent with application number "201510653843.6" and name "A strain of Bacillus amyloliquefaciens, its bacterial agent and application".

[0076] The chemotactic antagonistic bacteria - Bacillus Velez LG14-3 was published in the document "Research on Targeted Prevention and Control of Banana Wilt Disease by Rhizospheric Chemotactic Bacteria" (Yang Lihua. Research on Targeted Prevention and Control of Banana Wilt Disease by Rhizospheric Chemotactic Bacteria [D]. Guizhou University, 2023.), provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and promised to issue a 20-year certificate to the outside world.

[0077] The selective culture medium for Fusarium oxysporum in the embodiment of the present invention is disclosed in the document "A selective culture medium for simply isolating banana wilt pathogen" (Jing Xiaohui, Wu Lunying, Qu Xiaoling, Zhu Lilin, Xue Yuxiao, Wu Lin, Huang Junsheng. A selective culture medium for simply isolating banana wilt pathogen [J]. Journal of Tropical Crops, 2009, 30(11): 1671-1673.).

[0078] The control strain D34 in the examples of the present invention can be found in the public literature (Cao Zhichun. Construction of a multiple screening system for antagonistic Bacillus against banana wilt and research on its prevention effect [D]. Hainan University, 2016.).

[0079] The PCR detection method for citrus Huanglongbing in the embodiment of the present invention is detected by referring to the method in the document "Comparative Study of PCR Detection Primers for Citrus Huanglongbing" (Comparative Study of PCR Detection Primers for Citrus Huanglongbing. Plant Quarantine. 2022, 36(02): 1-6.).

[0080] The citrus leaf starch detection method in the embodiment of the present invention is detected by referring to the method in the document "Effects of Huanglongbing on the Accumulation of Assimilates and the Transport of Mineral Nutrients in Citrus Leaves" (Shi Yaman, Zeng Jixing, Xue Jinjun, et al. Effects of Huanglongbing on the Accumulation of Assimilates and the Transport of Mineral Nutrients in Citrus Leaves [J]. Journal of Plant Nutrition and Fertilizer, 2023, 29(05): 949-960.).

[0081] The citrus quality detection method in the embodiment of the present invention is detected by referring to the method in the document "Effect of Citrus Huanglongbing Bacteria Concentration on the Fruit Quality of Qiongzhong Green Orange" (Li Danyang, Huang Linxiao, Yang Yi. Effect of Citrus Huanglongbing Bacteria Concentration on the Fruit Quality of Qiongzhong Green Orange [J]. China Southern Fruit Trees, 2021, 50(02): 26-29+34. Lu Ying, Qi Yanxiang, Yu Qunfang, et al. Effect of Huanglongbing on the Fruit Quality of Qiongzhong Green Orange [J]. Anhui Agricultural Sciences, 2018, 46(11): 122-124.).

[0082] In the soil nutrient determination of the embodiment of the present invention, 200 g of 10-20 cm deep root soil was taken from each plant, for a total of 5 plants, mixed, stones and plant residues were removed, air-dried, and passed through a 200-mesh sieve. The soil organic matter was measured by the burning method, and the available nitrogen, available phosphorus and available potassium were measured using the HM-TYA soil fertilizer nutrient rapid tester. The salinity was measured by the TR-8D salinity detector, and the acidity was measured by the pH meter.

[0083] The tomato bacterial wilt PCR detection method in the embodiment of the present invention refers to the document "Isolation of tomato bacterial wilt and establishment of a triple PCR system" (Li Deming, Zhai Zixiang, et al. 2020. Isolation of tomato bacterial wilt and establishment of a triple PCR system [J]. Molecular Plant Breeding, 18(11): 3655-3661.), and the bacterial wilt incidence survey method refers to the method in the document "Field control effect of plant vaccine Orulengte on tomato bacterial wilt" (Zheng Xuefang, Liu Bo, Zhu Yujing, et al. 2018. Field control effect of plant vaccine Orulengte on tomato bacterial wilt [J]. Journal of Plant Protection, 45(5), 1096-1102.).

[0084] The tea tree growth index determination in the embodiment of the present invention refers to the method of the literature (Zhong Shengyun, Chen Guode, Zhang Wei, et al. Effects of different shading treatments on seasonal changes in yield and quality of Hainan large-leaf tea [J]. Modern Agricultural Science and Technology, 2019 (17): 5-7, 9.) to determine the growth index.

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

[0086] The microbial fertilizer used in the embodiment of the present invention is No. 41 bacterial fertilizer (amino acid 100g / L, organic matter ≥40%, total nutrients N+P2O5+K2O ≥12%, total effective viable bacteria count: 50×10 8 cfu / g, among which, the viable count of Bacillus subtilis BLG010 was 28×10 8 cfu / g, the viable count of Bacillus amyloliquefaciens HC200 was 22×10 8 cfu / g, powder. ), the microbial fertilizer was provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and has been published in the literature (Shao Xuefeng. Prevention and control of infectious diseases Research and application of inter-activation technology of No. 41 bacterial fertilizer [D]. Huazhong Agricultural University, 2020).

[0087] In the plot test and field test of the embodiment of the present invention, the composite microbial preparation 1 and the active ingredient 2 are mixed at a mass ratio of 10:1, and after proliferation and culture, the composite microbial preparation 2 is obtained: if the application time is after the plant is planted, the composite microbial preparation 2 is applied to the roots and leaves respectively at a mass ratio of 5:1; if the application time of the composite microbial preparation 2 is before the plant is planted, it is all applied to the soil. Other comparative / control preparations No. 41 bacterial fertilizer and clean water control were also applied in the same manner as above.

[0088] In the present invention, the specific methods and parameter control of the strain culture medium preparation, culture, and fermentation, as well as grinding, shaking, stirring, filtering, etc. described in the present invention can all be accomplished by conventional technical means in the art.

[0089] Example 1 Preparation of composite microbial preparation

[0090] 1. Seed solution preparation:

[0091] Under sterile conditions, the cells of Paecilomyces lilacinus E16 and Trichoderma viride H06 on the plate culture medium were picked up with an inoculation needle and inoculated into liquid activation culture medium (1 L of activation culture medium, 5 g / L sucrose or glucose, 2.5 g / L tryptone, 2.5 g / L yeast powder and 5 g / L sodium chloride), respectively, and cultured at 28°C for 4 days to obtain the seed liquid of Paecilomyces lilacinus E16 and Trichoderma viride H06;

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

[0093] 2. Preparation of active ingredient 1:

[0094] Corn flour, amino acid chelated calcium, magnesium, boron and zinc, sucrose and peptone (at least four of corn flour, sucrose, peptone, biological enzyme and amino acid chelated calcium, magnesium, boron and zinc can be mixed in equal proportions) are mixed in equal proportions to obtain a mixed material; then the obtained mixed material is mixed evenly with clean water in a mass ratio of 1:1, and sterilized by wet heat at 121° C. for 20 minutes to obtain a fermentation medium;

[0095] The seed liquid of Paecilomyces lilacinus E16, Trichoderma viride H06, Bacillus amyloliquefaciens HW05, Bacillus subtilis BLG010, Bacillus velez LG14-3 and Bacillus amyloliquefaciens HC200 were inoculated into the fermentation medium for fermentation culture, the inoculation amount was 5% (v / v), the fermentation culture time was 4 days, the temperature was 30°C, and the fermentation products of each strain were obtained;

[0096] The fermentation products of each strain were dried at 30-40°C for 48 h and crushed to obtain the fermentation product powder of each strain. Among them, the effective viable bacteria count of the fermentation product powder of Bacillus subtilis BLG010 was 54×10 10 CFU / g, the effective viable count of Bacillus amyloliquefaciens HW05 fermentation product powder is 23×10 10 CFU / g, the effective viable bacteria count of the powder of the fermentation product of Paecilomyces lilacinus E16 was 6.4×10 10 CFU / g, the effective viable bacteria count of the powder of Trichoderma viride H06 fermentation product was 5.7×10 10 CFU / g, the effective viable count of the powder of Bacillus Velez LG14-3 fermentation product is 12×10 10 CFU / g, the effective viable count of Bacillus amyloliquefaciens HC200 fermentation product powder 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 velezii LG14-3, and the fermentation product powder of Bacillus amyloliquefaciens HC200 were mixed in a mass ratio of 5:6:2:2:2:1 to obtain microbial fermentation product powder.

[0098] 3. Preparation of composite microbial preparations

[0099] (1) Preparation of composite microbial preparation 1

[0100] The active ingredient 1, the carrier and the synergist are mixed in a mass ratio of 10:10:1 to obtain a composite microbial preparation 1; wherein the carrier is 4 or more of chitosan, potassium dihydrogen phosphate, urea, amino acid chelated calcium magnesium boron zinc, humic acid and fish protein, and the carrier of this embodiment is chitosan, potassium dihydrogen phosphate, urea and humic acid mixed in a mass ratio of 10:1:5:10; the synergist is 3 or more of organosilicon, azone, gibberellin and ascorbic acid, and the synergist of this embodiment is organosilicon, azone and ascorbic acid mixed in a mass ratio of 100:100:5; the shelf life of the composite microbial preparation 1 is ≥18 months, and the total viable count is 12×10 10 CFU / g.

[0101] (2) Preparation of Active Ingredient 2

[0102] 1) Preparation of healthy plant leaf extract: healthy plant leaves and water are mixed in a mass ratio of 1: (10-20), and the mixture is ground evenly and filtered to obtain the extract; wherein the plant leaves are leaves of citrus, tea, banana, pepper or tomato, and the specific amount is determined according to the object of prevention and control. If the object of application is a systemic disease such as citrus Huanglongbing, healthy citrus leaves are used; if the object of application is a leaf disease such as tea cake disease and tea ring spot disease, healthy tea leaves are used; in this embodiment, the plant leaves and water are mixed in a mass ratio of 1: 10.

[0103] 2) Preparation of healthy plant rhizosphere soil extract: The plant rhizosphere soil and water are mixed in a mass ratio of 1: (5-10), shaken or stirred for 5-10 minutes, and the supernatant is filtered to obtain the extract; wherein the plant is citrus, tea tree, banana, pepper or tomato, and the specific amount is determined according to the control target. If the control target is soil-borne diseases such as pepper blight, banana wilt, tomato bacterial wilt and tomato wilt, the rhizosphere soil of pepper, banana and tomato is used respectively; in this embodiment, the plant rhizosphere soil and water are mixed in a mass ratio of 1: 5.

[0104] (3) Preparation of composite microbial preparation 2

[0105] Under field environmental conditions, the composite microbial preparation 1 and the active ingredient 2 are mixed in a container such as a bucket or a pool at a mass ratio of 10:1, and then diluted and proliferated to obtain the composite microbial preparation 2; wherein the dilution multiple is 10 times, the diluent is water, the natural field temperature for proliferation and culture is 20°C-40°C, and the ventilation or stirring time is 48h; wherein the active ingredient 2 is a healthy plant leaf extract or a healthy plant rhizosphere soil extract.

[0106] The microbial fermentation product powder, active ingredient 1, composite microbial preparation 1, active ingredient 2, composite microbial preparation 2, carrier and synergist prepared in Example 1 were all used in subsequent experiments.

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

[0108] The single fermentation product powders of Bacillus subtilis BLG010 (abbreviated as BLG010), Bacillus amyloliquefaciens HW05 (abbreviated as HW05), Trichoderma viride H06 (abbreviated as H06), Paecilomyces lilacinus E16 (abbreviated as E16), Bacillus velezii LG14-3 (abbreviated as LG14-3), and Bacillus amyloliquefaciens HC200 (abbreviated as HC200) prepared in Example 1 were respectively diluted 1000 times and evenly coated on a 1 / 4 LB solid medium (sucrose or glucose 0.5%, tryptone 0.25%, yeast extract 0.125%, NaCl 0.25% and agar 1.5%) in a 9 cm culture dish; then take 0.1 g of the synergist, carrier, and active ingredient 2, dilute them 500 times, shake well, and take 5 μL with a pipette. Take the center of each culture dish containing different strains as the center, and insert 500 times dilutions of the synergist, carrier, healthy plant leaf extract, and healthy plant rhizosphere soil extract at equal distances of 0.5 cm from the center of each culture dish along the straight line direction. Take an equal volume of clean water as the control, and culture in a 28°C incubator for 2-3 days, and count the width of the inhibition zone; each experiment was repeated 3 times, and the experimental results are shown in Table 1.

[0109] Table 1 Compatibility survey results

[0110]

[0111]

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

[0113] The results showed that the fermentation product powders containing single strains of BLG010, HW05, H06, E16, LG14-3 and HC200 were compatible with synergists, carriers, healthy plant leaf extracts and healthy plant rhizosphere soil extracts, indicating that the combination has prospects for the preparation and application of composite microbial preparations for the prevention and control of various plant diseases.

[0114] Example 3 Composite microbial preparation 1 has synergistic antagonistic effects on multiple pathogens

[0115] 1. Determination of the antagonistic effect of composite microbial preparation 1 on various pathogens

[0116] The composite microbial preparation 1 and active ingredient 1 prepared in Example 1 were selected, and the method of Example 1 was used to prepare comparative preparations (numbers 3 to 14) whose indicators exceeded the range of the strains of the present invention. No. 41 bacterial fertilizer, carrier, synergist and clean water were used as controls to compare the synergistic antagonistic effects of the above preparations on various pathogens. The specific treatments are shown in Table 2.

[0117] 1 / 4LB solid culture medium (sucrose or glucose 0.5%, tryptone 0.25%, yeast extract 0.125%, NaCl 0.25% and agar 1.5%) containing Fusarium oxysporum, Phytophthora capsici, Ralstonia solanacearum, Xanthomonas fasciatus, and Escherichia coli DH5α bacterial liquid was prepared respectively, poured into a culture dish with a diameter of 9 cm, and pre-cultured at 28°C for 12 h. According to the treatment in Table 2, 0.1 g of composite microbial preparation 1, different combinations of control preparations, enhancers and carriers were taken respectively, diluted 1000 times with clean water, shaken well, and 1000-fold dilutions were obtained. 5 μL was aspirated with a pipette, and 4 test bacterial liquids were inoculated in sequence at equal distances with the center of each culture dish as the center of the circle and 2.5 cm from the center of the circle. An equal volume of clean water was used as a control. The culture was cultured in a 28°C incubator for 3 d, and the inhibition zone was observed. Each experiment was repeated 3 times. The width of the inhibition zone between the edge of the test bacterial colony and the pathogen was measured (the results are shown in Table 2).

[0118] Since the pathogen of tea cake disease, Exobasidiomyces necroticus, is difficult to culture in vitro, spores of Exobasidiomyces necroticus were picked from the tea cake disease spots on tea leaves. According to the method of the literature "Study on the biocontrol effect of Bacillus Velez F85 on tobacco anthracnose" (Liu Hanfei, Xu Tingting, Li Xihong, et al. Study on the biocontrol effect of Bacillus Velez F85 on tobacco anthracnose [J]. Chinese Tobacco Science, 2023, 44(3): 47-52, 61.) and the literature "The control effect of two strains of Bacillus amyloliquefaciens on watermelon wilt and detection of related biocontrol factors" (Li Dan, Li Rongmei, Qin Weiying, et al. The control effect of two strains of Bacillus amyloliquefaciens on watermelon wilt and detection of related biocontrol factors)), composite microbial preparations, control preparations of different combinations, synergists, carriers and The mixture of conidia of the damaged exobasidiomycete in No. 41 bacterial fertilizer was used as a control, and the concentration of conidia in the mixture was 1×10 6 Pieces / mL.

[0119] The mixed solutions of the above treatments were inoculated on hydrophobic glass slides, and the conidia solution with sterile water was inoculated as the control (CK). Each treatment was repeated 3 times, and 100 spores were counted in each repeat. The mixture was kept moisturized for 24 hours at 28°C in the dark, and the spore germination rate inhibition rate was observed and calculated under a microscope, [(control spore germination rate - treatment spore germination rate) / control spore germination rate] × 100.

[0120] 2. Determination of the egg hatching inhibition rate of root-knot nematodes by composite microbial preparation 1

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

[0122] The banana root-knot nematode egg suspension is composed of southern root-knot nematode (Meloidogyne incongnita) and Javan root-knot nematode (Meloidogyne javanica) in a ratio of 1:1.

[0123] Each experiment was repeated 3 times, and the egg hatching rate and egg hatching inhibition rate were calculated on the 3rd day. The calculation formula for the hatching rate was: egg hatching rate (%) = [number of hatched larvae / (number of hatched larvae + number of unhatched eggs)] × 100, and the calculation formula for the hatching inhibition rate was: egg hatching inhibition rate (%) = [(control egg hatching rate - treatment egg hatching rate) / control egg hatching rate] × 100. The experimental results are shown in Table 2.

[0124] Table 2 Inhibition of pathogens by different treatments

[0125]

[0126]

[0127] As shown in Table 2, the composite microbial preparation 1 has antagonistic activity against a variety of bacteria in Table 1, which is better than the effective microbial preparation 1, and is also better than other single strain preparations or strain combination preparations. It can be seen that the composite microbial preparation 1 has a broad antibacterial spectrum and has the potential to prepare a multifunctional microbial agent.

[0128] Example 4 Antagonistic effects of composite microbial preparation 1 prepared from different combinations of active ingredient 1, carrier and synergist against various pathogens

[0129] According to the method of Example 1, the active ingredient 1, the carrier and the synergist were taken, and 4 groups of composite microbial preparations 1 with different combinations were prepared according to the ratio range of [(20-50):(10-30):(3-8)] required by the present invention, and were numbered B1, B2, B3, and B4, respectively. Four groups of composite microbial preparations and clean water were prepared with the ratio exceeding the range required by the present invention as controls, and were numbered B5, B6, B7, B8, and B9, respectively. According to the method of Example 3, the antagonistic effects of different combinations of active ingredients 1, carriers and synergists on various pathogens were compared.

[0130] Table 3 Inhibition of pathogens by different treatments

[0131]

[0132] As shown in Table 3, the four groups of composite microbial preparations 1 (numbered 1 to 4) prepared in different combinations according to the ratio range protected by the present invention are superior to the treatments of other combinations. It can be seen that the composite microbial preparation 1 prepared within the ratio range provided by the present invention has a wide antibacterial spectrum and has the potential to prepare a multifunctional microbial agent.

[0133] Example 5 Field control effect of composite microbial preparation on pepper blast

[0134] In the planting area of ​​Wenchang, Hainan Province, where pepper blast occurs all year round, the incidence rate was more than 90% before the test. The treatment area was 1 mu, and the dosage was 2.5 kg / mu each time. It was applied once every 30 days, and applied 6 times in succession, a total of 15 kg / mu. Water and fertilizer were sprayed in an integrated manner. The active ingredient 2 was the extract from the rhizosphere soil of healthy pepper. The composite microbial preparation 1 and the control preparation were applied alone. For the treatment of No. 41 bacterial fertilizer or active ingredient 2, use clean water to make up for the water consumption of each treatment. Settings: equal amount of clean water (CK1); after the composite microbial preparation 1 + active ingredient 2 are mixed, they are multiplied and cultured (diluted 10 times, 25℃-30℃, aerated culture for 48h), that is, the composite microbial preparation 2 is obtained, and then diluted 100 times before application (A1); No. 41 bacterial fertilizer + active ingredient 2 were mixed, cultured for proliferation (25℃-30℃, diluted 10 times, cultured with ventilation for 48h), and then diluted 100 times before application (A2); composite microbial preparation 1 was cultured alone (25℃-30℃, diluted 10 times, cultured with ventilation for 48h), and then diluted 100 times before application (A3); No. 41 bacterial fertilizer was cultured alone (25℃-30℃, diluted 10 times, cultured with ventilation for 48h), and then diluted 100 times before application (A4); active ingredient 2 was cultured alone (25℃-30℃, diluted 10 times, cultured with ventilation for 48h), and then diluted 100 times before application (A5); composite microbial preparation 1 was directly diluted 1000 times before application (A6); No. 41 bacterial fertilizer was directly diluted 1000 times and applied (A7); active ingredient 2 was directly diluted 1000 times and applied (A8). The experiment was repeated 3 times, with a total of 27 plots, and ditches were opened between the plots to isolate them. All the conventional fertilizers were used as topdressing, and base fertilizer, other topdressing and field management were carried out uniformly according to the local normal level.

[0135] The rhizosphere soil respiration rate was determined by reference to the literature (Wang Jun, Zhou You, Yang Laying, Liu Lei, Fu Hongwen, Huang Junsheng. 2019. Effects of compound bacterial fertilizer and intercropping on the control of banana wilt disease [J]. Chinese Fruit Trees, (6): 69-72.)

[0136] The disease index and control efficacy statistics refer to the literature (Sang Liwei, Liu Aiqin, Gao Shengfeng, et al. Screening of biological pesticides for controlling pepper blast and its potted efficacy test [J]. Journal of Tropical Crops, 2017, 38(05): 915-918.).

[0137] Determine soil nutrients, salinity and pH.

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

[0139] Yield (kg / mu) Soil respiration rate (%) Disease index Prevention 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] deal with Organic matter (%) Ammonium nitrogen (mg / kg) Available phosphorus (mg / kg) Available potassium (mg / kg) salinity(%) pH CK1 1.3 51.2 121.3 174.2 0.19 5.40 A1 2.1 67.5 147.2 255.1 0.11 6.42 A2 1.8 58.7 144.5 240.4 0.14 5.74 A3 1.7 62.6 127.5 228.1 0.12 5.99 A5 1.6 58.2 126.5 210.4 0.16 5.85 A6 1.5 53.4 122.8 208.3 0.18 5.67 A7 1.4 55.5 124.5 201.7 0.17 5.56 A8 1.3 54.1 120.1 179.9 0.19 5.42

[0142] From the results recorded in Tables 4 and 5, it can be seen that under field conditions, the composite microbial preparation 2 obtained by proliferating the composite microbial preparation 1 with the rhizosphere soil extract of healthy pepper rhizosphere plants can significantly improve the effect of disease prevention and growth promotion, and is superior to other treatments.

[0143] Example 6: Effect of the composite microbial preparation on banana wilt

[0144] In the banana planting area of ​​Banqiao Town, Dongfang City, Hainan Province, the incidence of banana wilt was more than 90% before the experiment. The plot experiment and field experiment were carried out. The soil was evenly turned before cultivation, and the turning depth was 0.5m. The concentration of Fusarium in the soil detected by selective culture medium reached 4.2×10 4 The dosage of compound microbial preparation is 5kg / mu.

[0145] 1. Community prevention effect

[0146] Each plot has an area of ​​0.2 mu, and the dosage is 1 kg / plot each time. It is applied once every 10 days before banana planting, and applied 5 times continuously, with a total of 5 kg / plot, and water and fertilizer are sprayed in an integrated manner. The control group and treatment settings refer to Example 5, wherein the active ingredient 2 is replaced with a healthy banana rhizosphere soil extract. When the control group is onset, the incidence rate of each treatment group is counted, and the prevention effect is calculated. The survey results are shown in Table 6.

[0147] 2. Field control effect

[0148] Control (CK): clean water treatment;

[0149] T1: After the composite microbial preparation 1 + active ingredient 2 are mixed at a mass ratio of 1:10, they are diluted 10 times with water and cultured for proliferation (25°C-30°C with aeration for 48 hours) to obtain the composite microbial preparation 2, which is then diluted 100 times before application;

[0150] T2: composite microbial preparation 1, directly diluted 1000 times for application;

[0151] T3: No. 41 bacterial fertilizer should be directly diluted 1000 times before application.

[0152] Each treatment area was 1 mu, randomly arranged, repeated 3 times, with a total of 12 plots, and ditches were opened between the plots. All the test fertilizers for conventional fertilization were used as topdressing, and base fertilizer, other topdressing and field management were carried out uniformly according to the local normal level. Then, the yield, incidence, control effect, rhizosphere soil respiration rate, soil nutrients, salinity, pH and soil bacterial community structure were measured. Among them, the yield, incidence, control effect and rhizosphere soil respiration rate were measured according to the method in the literature "Effects of compound fertilizer and intercropping on the control of banana wilt disease" (Wang Jun, Zhou You, Yang Laying, Liu Lei, Fu Hongwen, Huang Junsheng. 2019. Effects of compound fertilizer and intercropping on the control of banana wilt disease [J]. Chinese Fruit Trees, (6): 69-72.); soil nutrient determination was based on Example 5; soil bacterial community structure was determined according to the method in the literature "Research on targeted prevention and control of banana wilt by rhizospheric chemotactic bacteria" (Literature: Yang Lihua. Research on targeted prevention and control of banana wilt by rhizospheric chemotactic bacteria [D]. Guizhou University, 2023.). The survey results are shown in Tables 7-9 and Figure 1 .

[0153] Table 6 Incidence and prevention efficacy survey results

[0154] deal with Incidence (%) Prevention effect (%) CK 82.17 / T1 9.12 88.90 T2 24.18 70.57 T3 28.63 65.16 T4 37.24 54.68 T5 53.56 34.82 T6 36.14 56.02 T7 46.41 43.52 T8 68.69 16.41

[0155] The results showed that the disease prevention and growth promotion effect of the composite microbial preparation 2 applied after the plot conditions was better than that of other combination treatments (Table 6).

[0156] Table 7 Test results of yield, incidence, control effect and rhizosphere soil respiration rate

[0157] deal with Soil respiration rate (%) Yield (kg / mu) Soil respiration rate (%) Disease index Prevention 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

[0158] Table 8 Test results of soil nutrients, salinity and pH

[0159]

[0160] Table 9 Results of Alpha diversity index test of soil bacteria in rhizosphere soil

[0161] deal with 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

[0162] The results showed that under field conditions, the disease prevention and growth promotion effect of the composite microbial preparation 2 was better than that of other treatments applied in the same way (Table 7), and the soil nutrient indicators were significantly better than those of other treatments applied in the same way (Table 8).

[0163] Compared with CK, the richness index chao1, observed_otus, and diversity index shannon, simpson in T1 treatment decreased by 3.05%-12.52%, while those in T2 and T3 decreased by 22.83%-52.96% (Table 9).

[0164] Figure 1 The results showed the relative abundance of species at the genus level in each treatment (ranked in the top 15). 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.

[0165] Taken together, the above results showed that composite microbial preparation 2 reduced bacterial diversity and abundance and increased the abundance of beneficial microorganisms Bacillus in rhizosphere soil.

[0166] Example 7: Effect of the composite microbial preparation on the prevention of tomato bacterial wilt

[0167] In previous years, plots of land in Qionghai, Hainan Province, where tomato bacterial wilt was severely affected, were used as test sites for plot control and field experiments. The soil was evenly plowed before planting to a depth of 0.5m, and the dosage was 2.5kg / mu.

[0168] 1. Community prevention effect

[0169] Each treatment area is 0.2 mu, and the dosage is 0.5 kg / treatment each time. It is applied 7 days before tomato planting and every 10 days after planting, and applied 5 times continuously, with a total of 2.5 kg / plot, and water and fertilizer are sprayed in an integrated manner. A total of 8 control groups and treatments are set, referring to Example 5, wherein the active ingredient 2 is replaced with a healthy tomato rhizosphere soil extract. When the control group is on the verge of disease, the incidence rate of each treatment group is counted and the prevention effect is calculated. The survey results are shown in Table 10.

[0170] 2. Field experiment

[0171] Set up 4 groups of processing, as follows:

[0172] Control (CK): clean water treatment;

[0173] T1: After the composite microbial preparation 1 + active ingredient 2 are mixed at a mass ratio of 1:10, they are diluted 10 times with water and cultured for proliferation (25°C-40°C with aeration for 48 hours) to obtain the composite microbial preparation 2, which is then diluted 100 times before application;

[0174] T2: composite microbial preparation 1, directly diluted 1000 times for application;

[0175] T3: No. 41 bacterial fertilizer should be directly diluted 1000 times before application.

[0176] Each treatment area was 1 mu, randomly arranged, repeated 3 times, with a total of 12 plots, and ditches were opened between the plots. On the basis of conventional fertilization, different preparations (CK, TI-T3) were irrigated on the roots and leaves of tomatoes at the time of planting, and then irrigated once every 3 weeks, for a total of 5 times.

[0177] Then, growth indicators, prevention efficacy, soil nutrients and soil bacterial microbial diversity were investigated. The growth indicators were investigated by selecting healthy leaves from the upper and lower parts of each plant under natural light, and the relative chlorophyll content (SPAD value) of the leaves was determined using a chlorophyll meter TYS-4N. When the fruit was ripe, the first and second ears of fruit were selected and weighed to determine the yield of each plant. The prevention efficacy was investigated by selecting 15 tomatoes for each treatment using a five-point sampling method during the tomato harvest period, collecting their roots, and using a biotechnology plant DNA extraction kit to extract root DNA. The triple PCR test for bacterial wilt was performed using the method described in the reference "Isolation of Tomato Solanacearum and Establishment of a Triple PCR System" (Li Deming, Zhai Zixiang, et al. 2020. Isolation of Tomato Solanacearum and Establishment of a Triple PCR System [J]. Molecular Plant Breeding, 18(11): 3655-3661.). Fifteen plants were selected for each replicate to detect the root bacterial infection rate. The plant vaccine Orulente was used to detect the bacterial wilt of tomato. The method in "Field Control Effect" (Zheng Xuefang, Liu Bo, Zhu Yujing, Lin Kangmei, Ge Cibin, Chen Deju. 2018. Field Control Effect of Plant Vaccine Orulent on Tomato Bacterial Wilt [J]. Acta Phytophylacica Sinica, 45(5), 1096-1102.) was used. 100 plants were selected for each replicate, and the incidence rate was counted to calculate the control effect of different treatments on tomato bacterial wilt. The calculation formula of the incidence rate was: incidence rate (%) = number of diseased plants / total number of plants investigated × 100; control effect (%) = (control incidence rate - treatment incidence rate) / control incidence rate × 100; the method for determining soil bacterial microbial diversity was based on Example 6, and the rhizosphere soil was mixed, stones and plant residues were removed, air-dried, and sieved through a 200-mesh sieve to extract soil total DNA. Based on the Illumina NovaSeq sequencing platform, a small fragment library was constructed using the paired-end sequencing method for sequencing; the survey results are shown in Tables 10-11 and Figure 2 shown.

[0178] Table 10 Statistical results of incidence and prevention efficacy

[0179] deal with Incidence Prevention 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

[0180] The results showed that the disease prevention and growth promotion effect of the composite microbial preparation 2 under plot conditions was better than that of other combinations (Table 10).

[0181] Table 11 Survey results of growth indexes and control effects

[0182] deal with Chlorophyll (SPAD) Yield / plant Root bacterial wilt pathogen rate (%) Incidence (%) Prevention 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％

[0183] Table 12 Test results of soil nutrients, salinity and pH

[0184]

[0185] Table 13 Alpha diversity index test results of rhizosphere soil bacteria

[0186]

[0187]

[0188] The results showed that under field conditions, the disease prevention and growth promotion effect of composite microbial preparation 2 was better than that of composite microbial preparation 1 applied in the same way (Table 11), and the soil nutrient indicators were significantly better than those of other treatments applied in the same way (Table 12).

[0189] As shown in Table 13, compared with CK, the feature number Feature, richness index 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%, and the increase of T2 was smaller; the diversity index Simpson and Shannon of each treatment did not change significantly.

[0190] The relative abundance of each treatment at the genus classification level (the top 20 in terms of relative abundance of OTUs) was analyzed. Figure 2 The results showed that the relative abundance of Bacillus in the treatments T1 was the highest, ranking third (6.71%), while the Bacillus in the CK, T2 and T3 treatments were ranked in the top 10, but the abundance was only 0.79%-2.06%, indicating that Bacillus could colonize better in the rhizosphere soil after the application of composite microbial preparation 2.

[0191] The above results show that the composite microbial preparation 2 has significant disease prevention and growth promotion effects, and increases the abundance of beneficial microorganism Bacillus in the rhizosphere soil.

[0192] Example 8: Effect of the composite microbial preparation on the prevention of citrus Huanglongbing

[0193] In the Qiongzhong citrus planting area where the disease occurs all year round, the incidence of citrus Huanglongbing is more than 90% (Qiongzhong Green Orange), and plot experiments and field experiments are carried out. On the basis of conventional fertilization, application starts from the spring shoot germination period, and the active ingredient 2 is replaced with healthy citrus leaf extract. The application amount for each treatment is 10kg / mu each time, and then it is applied once every 1 month:

[0194] 1. Community test

[0195] Each treatment plot has an area of ​​0.2 mu, and the dosage is 2 kg. It is applied 5 times in succession, with a total of 10 kg / treatment. The setting scheme of the treatment group and the control group is referred to Example 5. When the control is onset, PCR is used to detect whether the citrus Huanglongbing pathogen in the midrib of the citrus leaves of each treatment group is positive, and the infection rate is calculated as the number of infected plants / total number of plants, and the prevention effect is 100×(control infection rate-treatment infection rate) / control infection rate. Repeat 3 times, with a total of 12 plots, and ditches are opened between the plots for isolation.

[0196] 2. Field experiment

[0197] Each treatment area is 1 mu, and 8 times of continuous application, a total of 80 kg / mu, 8 times of continuous application, 4 groups of treatments are set, as follows:

[0198] Control (CK): clean water treatment;

[0199] T1: After the composite microbial preparation 1 + active ingredient 2 are mixed at a mass ratio of 1:10, they are diluted 10 times with water and cultured for proliferation (20-37°C with aeration for 48 hours) to obtain the composite microbial preparation 2, which is then diluted 100 times before application;

[0200] T2: composite microbial preparation 1, directly diluted 1000 times for application;

[0201] T3: No. 41 bacterial fertilizer should be directly diluted 1000 times before application.

[0202] Each treatment was 1 mu, repeated 3 times, with a total of 12 plots. Ditches were opened between the plots to isolate the Huanglongbing bacteria, fruit yield, juice yield, sugar content, starch content in leaves, microbial diversity of endophytic bacteria in leaves, and soil bacterial community structure. The specific investigation methods were as follows:

[0203] Regularly collect plant branch and leaf samples. For each treatment, select 5 citrus plants in the east, west, south, north and center. Randomly collect 1 leaf sample from each citrus plant in the east, west, south, north and center and mix them evenly. Determine the leaf starch content. After DNA is extracted from the collected samples, the PCR method is used to determine the presence of Huanglongbing bacteria in the plants. During the harvest period, collect fruits in the same way to determine the fruit yield and juice yield.

[0204] After the field experiment, the method in the reference literature "Effects of Colletotrichum oleifera Infection on the Endophytic Bacterial Community Structure of Camellia oleifera Leaves" (Cui Mingqin, Zhang Donghua, Yan Xiaohui, Hong Yingdi, et al. Effects of Colletotrichum oleifera Infection on the Endophytic Bacterial Community Structure of Camellia oleifera Leaves [J]. Chinese Journal of Biological Control, 2022, 38(4): 911-919.) was used to determine the diversity of endophytic bacterial microorganisms in leaves. Three young leaves were randomly collected, the surface was disinfected, and DNA was extracted from the midrib of the leaves. Based on the Illumina NovaSeq sequencing platform, a small fragment library was constructed using the paired-end sequencing method, and the V3-V4 region of 16SrDNA was sequenced to determine the diversity of endophytic bacterial microorganisms in leaves.

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

[0206] Table 14 Huanglong disease bacteria carrying situation and prevention effect

[0207] deal with Bacteria carrier rate (%) Prevention effect (%) CK1 77.68 / A1 4.66 94.00 T2 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 the disease prevention and growth promotion effect of the composite microbial preparation 2 under plot conditions was better than that of other combinations (Table 14).

[0209] Table 15 Huanglong disease bacteria detection results

[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 yield and sugar content

[0215] deal with Yield per plant (kg) Juice yield (%) Sugar content (%) CK 18.5 46.8 9.3 T1 48.2 53.7 14.5 T2 35.4 49.4 12.8 T3 27.1 48.2 11.7

[0216] Table 18 Results of Alpha diversity index test of bacteria in the midrib of citrus leaves

[0217] deal with Feature number Ace Index Chao1 Index Simpson Index Shannon Index CK 11 11.33 11.33 1.1363 0.4963 <![CDATA[T1]]> 23 23.33 23.33 2.0901 0.7927 <![CDATA[T2]]> 25 25.33 25.33 2.0694 0.7770 <![CDATA[T3]]> 17 17.00 17.00 1.7909 0.7378

[0218] The results showed that compared with the control, the composite microbial preparation 2 could significantly remove pathogens in the diseased parts (Table 15), reduce the starch content in the diseased leaves, relieve the blockage of the ducts by starch in the leaves (Table 16), and promote the normal growth of citrus, thereby increasing the yield, juice yield and sugar content (Table 17);

[0219] Compared with CK, the species abundance indexes Ace index and Chao1 index of T1, T2 and T3 increased by 50.04%-105.91%; the diversity index Simpson index increased by 48.66%-59.72%, and the Shannon index increased by 57.61%-83.94% (Table 18). The Chao1 index, ACE index and Shannon index of T1 and T2 increased significantly, reaching more than 80%; the Simpson index of T1, T2 and T3 increased by more than 40%; the Simpson index of T1 treatment increased most significantly, significantly increasing by 3.16-11.06 percentage points compared with T2 and T3, and the Shannon index increased by 1.82%-26.33% compared with T2 and T3 (Table 18).

[0220] Analysis of the top 1% relative abundance of ASV genus level in 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 abundance of Bacillus in T1 and T3 treatments was only 0.96% and 0.49, ranking 8th and 11th. The citrus Huanglongbing pathogen (Candidatus Liberibacter) in CK, T1, T2 and T3 treatments were 69.22%, 10.96%, 2.88% and 16.93%, respectively).

[0221] The above results indicate that the composite microbial preparation 2 is superior to other treatments in terms of prevention and growth promotion. It significantly improves the Simpson index and Shannon index of endophytic bacterial diversity in citrus leaves, and increases the abundance of beneficial microorganisms such as Bacillus.

[0222] Example 9: Effect of the composite microbial preparation on tomato wilt

[0223] The plots where tomato wilt was seriously affected in previous years were used as test plots, and the design schemes of the plot test and field experiment were the same as those in Example 7.

[0224] The following methods are used to test various indicators:

[0225] Growth index: From the time of tomato harvest, in clear weather, the atmospheric CO2 concentration was maintained at (380±6.0)μmol / mol, the temperature was 26℃, and the relative humidity was (60±4.0)%. Three plants were randomly selected from each group, and three healthy leaves were selected from the upper and lower parts of each plant. The photosynthesis was measured using the LCPRO-SD portable photosynthesis meter, and the measurement time was 120s. When the fruit was ripe, the first and second ears of fruit were selected and weighed to measure the yield per plant.

[0226] Prevention effect investigation: investigate the incidence of tomato wilt at the seedling stage, flowering stage, fruiting stage and harvest stage, and calculate the prevention and control effects of different treatments on tomato wilt. The calculation formula of the incidence is: incidence = number of diseased plants / total number of investigated plants × 100%; the calculation formula of the prevention and control effect is: prevention and control effect = (control incidence - treatment incidence) / control incidence × 100%;

[0227] Determination of soil fungal microbial diversity: Soil samples were collected, mixed, and cleaned of stones and plant residues, and total soil DNA was extracted. The ITS1 region of fungal ITS1 rDNA was sequenced based on the IlluminaNovaSeq sequencing platform.

[0228] The survey results are shown in Tables 19-21 and Figure 4 shown.

[0229] Table 19 Statistical results of incidence and prevention efficacy

[0230] deal with Incidence (%) Prevention 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

[0231] The results of the plot test showed that the disease prevention and growth promotion effect of the composite microbial preparation 2 was significantly better than that of other combinations (Table 19).

[0232] Table 20 Growth index and prevention effect survey results

[0233] deal with <![CDATA[Photosynthesis (μmol·m -2 ·s -1 )]]> Yield / plant (kg) Water-soluble sugar (%) Incidence (%) Prevention 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

[0234] Table 21 Alpha diversity index test results of rhizosphere soil fungi

[0235]

[0236]

[0237] The statistics of the results of field disease prevention and growth promotion showed that the T1 treatment significantly improved the yield and quality, with a prevention efficiency of 87.32%, which was also significantly higher than other treatments (Table 20).

[0238] Analysis of the Alpha diversity index showed that compared with CK, only the ACE index of the fungal family level in the T1 treatment increased by 36.28%, and the Chao1, Simpson, and Shannon indexes decreased by 0.97%-10.19%. All the indicators in the T2 and T3 treatments showed a decrease, with a decrease of 5.26%-33.34%. This indicates that after the application of the composite microbial preparation (T1), the antagonistic microorganisms colonized and became the dominant strains, inhibiting the growth of other microorganisms, thereby reducing the microbial diversity indicators (Table 21).

[0239] The relative abundance of each treatment at the genus classification level (the top 15 OTUs in terms of relative abundance) was analyzed. Figure 4 ) showed that the relative abundance of Trichoderma in each treatment was the highest in T1 treatment, ranking second (18.53%), while that in T2 and T3 treatments were 11.52% and 8.56%, respectively; the abundance of Fusarium in CK, T1, and T2 treatments was 0.93%, 0.17%, and 2.41%, respectively, while the pathogenic bacteria (Fusarium) in T3 was less than 0.01%. This indicates that after the application of composite microbial preparation 2, the beneficial microorganism Trichoderma can colonize well in the rhizosphere soil.

[0240] The above results show that the composite microbial preparation 2 has the effect of preventing diseases and promoting growth, improving the ACE index of fungal diversity, reducing the Chao1, Simpson and Shannon indexes, and increasing the abundance of the beneficial microorganism Trichoderma in the rhizosphere soil.

[0241] Example 10: Effect of the composite microbial preparation on tea cake disease

[0242] In the plot test and field test, the incidence of tea cake disease in the Wuzhishan large-leaf tea base, where the disease occurs all year round, is over 90%. On the basis of conventional fertilization, compound microbial preparations were applied from the spring shoot germination period, in which the active ingredient 2 was replaced with healthy tea leaf extract, and the dosage for each treatment was 2.5kg / mu.

[0243] 1. Community test

[0244] Each treatment plot has an area of ​​0.2 mu, and the dosage is 0.5 kg, which is applied three times in succession, with a total of 1.5 kg / treatment. The setting scheme of the treatment group and the control group refers to Example 5. When the control group becomes ill, the incidence rate of each treatment group is counted and the control effect is calculated. The survey results are shown in Table 22.

[0245] 2. Field trials

[0246] Each treatment area is 1 mu, and it is applied continuously for 3 times, once every 7 days, with a total of 7.5 kg / mu. A control (CK) and compound microbial preparation 1 + healthy tea leaf extract are mixed and then cultured for proliferation (diluted 10 times, temperature 25-30°C, aeration culture for 48 hours) to obtain compound microbial preparation 2, which is then diluted 100 times and applied (T1); compound microbial preparation 2 is directly diluted 1000 times and applied (T2); 40 g / mu of pyraclostrobin emulsifiable concentrate is diluted 1000 times (T3), repeated 3 times, with a total of 12 plots, and trenches are opened between the plots for isolation.

[0247] Determination of control effect: Refer to the method in the literature (Yang Wenbo, Xiang Fen, Liu Hongyan, et al. Field control effect evaluation of different agents on tea cake disease [J]. China Plant Protection Guide, 2022, 42 (12): 81-84.), and investigate the disease index and calculate the control effect 7 days after the third application of the pesticide. The second leaf under the bud was selected for each treatment plot, and 50 leaves were investigated and graded. Disease grading standard: Grade 0, no lesions on the leaves; Grade 1, lesions occupy less than 25% of the leaf area; Grade 2, sporadic lesions occupy 26% to 50% of the leaf area; Grade 3, lesions occupy 51% to 75% of the leaf area; Grade 4, lesions on the leaves are dense, accounting for 76% or more of the leaf area. The disease index and control effect were calculated according to the following formula: disease index = Σ(number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × 4) × 100; control effect = [1-(disease index of the control area before drug application × disease index of the treated area after drug application) / (disease index of the control area after drug application × disease index of the treated area before drug application)] × 100%.

[0248] Determination of main growth indicators of tea trees: Bud length: In each treatment plot, three replicates were set up, and each replicate was a randomly selected square area of ​​100 cm × 100 cm. Buds with one bud and two leaves in the area were collected, and the length from the top bud to the second leaf was measured.

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

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

[0251] Relative content of chlorophyll: Select healthy leaves from the upper and lower parts of each plant under natural light, and use a chlorophyll meter TYS-4N to measure the relative content of chlorophyll in the leaves, i.e., the SPAD value.

[0252] Determination of the diversity of endophytic bacterial microorganisms in leaves. After the field test, three young leaves of tea trees were randomly collected, and after surface disinfection, the determination was carried out according to the method of Example 8. The survey results are shown in Tables 22-24 and Figure 5 shown.

[0253] Table 22 Disease index and prevention efficacy survey results

[0254] deal with Disease index Prevention 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 test showed that the disease prevention and growth promotion effect of the composite microbial preparation 2 was significantly better than that of other combinations (Table 22).

[0256] Table 23 Growth index, SPAD, disease index and prevention efficacy survey results

[0257]

[0258]

[0259] Table 24 Results of Alpha diversity index test of endophytic bacteria in tea leaves

[0260] deal with Feature number 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

[0261] Field test results: Statistics of disease prevention and growth promotion results showed that composite microbial preparation 2 significantly improved yield and quality, with a prevention efficiency of 94.20%, significantly higher than its T2 treatment and comparable to the chemical agent pyraclostrobin (Table 23).

[0262] Analysis of the Alpha diversity index showed that compared with CK, the species richness index Ace index of 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%, respectively, and the change was not obvious; the Simpson index of T2 and T3 treatments decreased by 9.58% and 17.99%, respectively, while the Shannon index of T1 treatment increased by 4.41% (Table 24).

[0263] The top 15 species in relative abundance at the genus level in each treatment were analyzed. The results ( Figure 5 ) showed that the top 5 species in each treatment were Rhizobium as the exclusive dominant genus in T1 (4.28%), Methylocella as the exclusive dominant genus in T2 (4.94%), Stenotrophomonas and Pseudomonas as the exclusive dominant genera in T3 (13.46% and 7.23%). This indicates that the application of the composite microbial preparation changed the proportion of dominant species in tea leaves.

[0264] The above results show that the application of composite microbial preparation 2 has obvious disease prevention and growth-promoting effects, significantly improving the richness and diversity indicators of endophytic bacteria in tea leaves, such as Ace index, Chao1 index and Shannon index, and increasing the abundance of endophytic beneficial bacteria in tea leaves, such as Rhizobium.

[0265] Example 11: Effect of the composite microbial preparation on tea ring spot disease

[0266] In the Wuzhishan large-leaf tea base, the incidence of tea ring spot was selected to be more than 90%. On the basis of conventional fertilization, the composite microbial preparation was applied from the spring shoot germination period. The treatment setting scheme and prevention effect determination were all referred to the field test scheme in Example 10. The tea leaf sampling was referred to Example 8, and the leaf endophytic fungus determination method was referred to Example 9. The survey results are shown in Tables 25-26 and Figure 6 shown.

[0267] Table 25 Disease index and prevention efficacy survey results

[0268] deal with Disease index Prevention effect (%) CK 64.28 / T1 5.35 91.68 T2 16.46 74.39 T3 5.25 91.83

[0269] The results showed that the control efficiency of T1 treatment reached 91.68%, which was comparable to that of chemical agent (T3) and significantly better than that of T2 treatment (Table 25).

[0270] Table 26 Alpha diversity index test results of endophytic fungi in tea trees

[0271] deal with Feature number 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

[0272] Analysis of the Alpha diversity index showed that compared with CK, the species richness index Ace index of T1, T2 and T3 treatments 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 diversity index of T1 treatment increased most significantly, and the increase in Simpson index increased by 1.86-2.84 percentage points compared with T2 and T3, and the increase in Shannon index increased by 20.08-28.06 percentage points compared with T2 and T3 (Table 26).

[0273] The top 15 species of endophytic fungi in each treatment at the genus level were analyzed. Figure 6 ) showed that the relative abundance of tea ring spot pathogens (Neopestalotiopsis, Pseudopestalotiopsis) in CK was as high as 48.99%, while the relative abundances of tea ring spot pathogens in T1, T2 and T3 were 0.24%, 0.74% and 8.34%, respectively. T1 treatment significantly reduced the relative abundance of tea ring spot pathogens and changed the proportion of dominant endophytic fungi in tea leaves.

[0274] The above results show that the application of composite microbial preparation 2 has obvious disease prevention and growth-promoting effects, significantly improving the Simpson index and Shannon index of endophytic fungal diversity indicators in tea leaves, and significantly reducing the abundance of tea ring spot pathogens.

[0275] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A microbial combination for preventing and controlling plant diseases, characterized in that: The microbial combination includes 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; and the deposit number of the Bacillus amyloliquefaciens HC200 is CGMCC No.10371.

2. The microbial combination according to claim 1, characterized in that The mass ratio of the Bacillus amyloliquefaciens HW05, the Bacillus subtilis BLG010, the Paecilomyces lilacinus E16, the Trichoderma viride H06, the Bacillus velez LG14-3 and the Bacillus amyloliquefaciens HC200 in the microbial combination is (5-10):(2-6):(2-4):(2-4):(2-5):(1-5); And / or, the plant diseases include one or more of pepper blight, banana wilt, tomato wilt, tomato bacterial wilt, citrus Huanglongbing, tea cake disease and tea ring spot.

3. Use of the microbial combination according to claim 1 or 2 in the preparation of a composite microbial preparation for preventing and controlling plant diseases.

4. A composite microbial preparation for preventing and controlling plant diseases, characterized in that: The composite microbial preparation comprises an active ingredient 1; the active ingredient 1 comprises the microbial combination according to claim 1 or 2.

5. The composite microbial preparation according to claim 4, 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 enhancer is (20-50):(10-30):(3-8).

6. The composite microbial preparation according to claim 5, characterized in that: 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; And / or, the synergist includes any three or more of organosilicon, azone, gibberellin and ascorbic acid.

7. The composite microbial preparation according to claim 5, characterized in that: The composite microbial preparation further comprises an active ingredient 2; the active ingredient 2 comprises a healthy plant leaf extract or a healthy plant rhizosphere soil extract; And / or, the method for preparing the healthy plant leaf extract comprises the steps of mixing healthy plant leaves and water in a mass ratio of 1:(10-20), grinding and filtering, to obtain the healthy plant leaf extract; And / or, the preparation method of the healthy plant rhizosphere soil extract comprises the steps of mixing the rhizosphere soil of the healthy plant and water in a mass ratio of 1:(5-10), shaking or stirring for 5-20 minutes, and filtering to obtain the healthy plant rhizosphere soil extract.

8. The composite microbial preparation according to claim 7, characterized in that: 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).

9. Use of the microbial combination according to claim 1 or 2 or the composite microbial preparation according to any one of claims 4 to 8 in preventing and controlling plant diseases.

10. A method for preventing and controlling plant diseases, characterized in that: The following steps are involved: After the composite microbial preparation according to any one of claims 4 to 8 is proliferated and cultured, it is applied to the surface of plant leaves or the rhizosphere.

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