Complex microbial inoculant, compound fertilizer, bio-organic fertilizer and application

Abstract

The invention provides a complex microbial inoculant, a compound fertilizer, a bio-organic fertilizer and application, and belongs to the technical field of microbial inoculants, fertilizers and agriculture. The composite microbial agent is prepared from the following components in parts by mass: 20 to 60 parts of bacillus subtilis, 20 to 40 parts of bacillus amyloliquefaciens and 20 to 40 parts of bacillus velezensis, wherein the total mass of the bacillus subtilis, the bacillus amyloliquefaciens and the bacillus velezensis is 100 parts.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, fertilizers and agriculture, and in particular relates to a composite microbial agent, a composite fertilizer, a bio-organic fertilizer and applications. Background Art

[0002] When crops are grown in areas with infertile soil (such as wheat, corn, cabbage, citrus, pomelos, mandarin oranges, and other cash crops), soil fertility and soil-penetrating diseases can alter the soil's microbial environment during the planting process, affecting crop growth and leading to frequent root rot. Root rot is caused by infection with a variety of pathogens, including Pythium, Fusarium, and Phytophthora. The pathogens overwinter in the soil or on diseased plant debris, becoming the main source of initial infection the following year. The pathogens invade through rhizomes or root wounds, spreading and distributing through rainwater or irrigation water. The disease is most severe in fields with low terrain, poor drainage, waterlogging, continuous cropping, dripping and leaking greenhouses, and root injuries. For example: 70% of citrus is planted on hilly slopes with poor soil. During the citrus planting process, chemical fertilizers are used for a long time. Due to the decline in fertility, soil compaction, and the aggravation of soil-borne diseases, the structure and function of the rhizosphere microbial community in the orchard soil are unbalanced, the soil has weak water and fertilizer retention capacity, the citrus root system grows poorly, the tree is weak, and the incidence of root rot increases.

[0003] Canker is a serious disease of citrus. With the exception of varieties such as sugar oranges and pomelo, which are less susceptible, most citrus varieties, such as mandarin oranges and navel oranges, are severely affected. Citrus canker harms leaves and fruits, and once the fruit is infected, it seriously affects its marketability. In the field, canker reduces the marketable fruit rate, generally between 10% and 30%, and in severe cases, up to 50%. The current main measure is the use of copper preparations for prevention and control, but there are problems such as poor preventive effect, lack of systemic therapeutic effect, poor compatibility and safety, killing of natural enemies, and pollution of the environment and soil. This is one of the current difficulties in citrus plant protection. The use of microbial agents for prevention and control is an important and effective way to solve these problems.

[0004] At present, the strains and functions of various microbial agents on the market are relatively simple and cannot achieve the comprehensive effects of root promotion, disease prevention, and yield increase. Summary of the Invention

[0005] In view of this, and in response to the above technical problems, the present invention provides a composite bacterial agent, a composite fertilizer, a bio-organic fertilizer, and applications thereof, in order to at least partially solve the above technical problems. The technical solutions provided by the present invention are as follows.

[0006] As a first aspect of the present invention, a composite bacterial agent is provided, which comprises, based on 100 parts by total mass, 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez.

[0007] As a second aspect of the present invention, there is provided an application of a composite bacterial agent in preventing and treating root rot or canker.

[0008] As a third aspect of the present invention, a compound fertilizer is provided, comprising: a composite bacterial agent and a synergist, wherein the mass ratio of the composite bacterial agent to the synergist is 1-10:1-4; wherein the synergist comprises a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or an auxiliary synergist selected from at least one of L-arginine, seaweed polysaccharide, and chitosan; based on 100 parts of the total mass of the composite bacterial agent, the composite bacterial agent comprises: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez.

[0009] As a fourth aspect of the present invention, a bio-organic fertilizer is provided, which is composed of a composite bacterial agent, a synergist and an organic material, wherein, in parts by mass, the mass ratio of the organic material, the composite bacterial agent and the synergist is 1000:2-10:1; wherein, based on the total mass of the composite bacterial agent as 100 parts, the composite bacterial agent includes: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus Velez; the synergist includes a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or an auxiliary synergist selected from at least one of L-arginine, seaweed polysaccharide and chitosan; the organic material includes at least one of straw, livestock and poultry manure, and plant residues.

[0010] As a fifth aspect of the present invention, there is provided an application of a bio-organic fertilizer in vegetable and fruit planting.

[0011] Based on the above technical solution, the composite bacterial agent, compound fertilizer, bio-organic fertilizer and their application provided by the present invention have at least one of the following beneficial effects:

[0012] (1) The present invention uses three Bacillus species: Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis. Through the mutual cooperation of the various strains, they have a significant antibacterial effect on soil-borne pathogens such as Fusarium and Phytophthora in the planting soil, and also have a significant antibacterial effect on citrus canker. They also have a significant promoting effect on the growth of plant roots and aboveground parts. Furthermore, when the various Bacillus species are compounded in a certain proportion, the obtained composite bacterial agent has a significantly better effect on promoting the growth of plant roots and aboveground parts and inhibiting soil-borne root rot and citrus canker. In addition, the sources of these three strains are not limited, and the same effects as those of the present invention can be achieved by compounding them using the formula provided by the present invention.

[0013] (2) In the present invention, the composite microbial agent and the synergistic adjuvant are mixed in proportion. In addition to the above-mentioned antibacterial effect and the effect of promoting plant root growth, the synergistic adjuvant therein can significantly improve the plant height, stem thickness, above-ground fresh weight, underground fresh weight and other indicators.

[0014] (3) In the present invention, the composite bacterial agent, the synergistic adjuvant and the organic fertilizer are mixed in proportion, and the obtained bio-organic fertilizer can be used as a commercial product. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Graph showing the test results of a plate confrontation test of different strains against soil-borne pathogens in an embodiment of the present invention;

[0016] Figure 2 This is a diagram showing the results of a plate confrontation test of antagonism between Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velez in the present invention;

[0017] Figure 3 This is a graph showing the antibacterial effect of the composite bacterial agent on citrus canker in an embodiment of the present invention;

[0018] Figure 4A This is a diagram showing the effect of applying the bio-organic fertilizer in Example 3 of the present invention on two-year-old saplings of the Ju Jiuyuehong navel orange;

[0019] Figure 4B This is a picture of the effect of a competitor's product applied on two-year-old saplings of the Jiuyuehong navel orange;

[0020] Figure 4C This is the effect diagram of the control group applying the product on two-year-old saplings of Ju Jiuyuehong navel orange;

[0021] Figure 5 This is a diagram showing the effect of applying the bio-organic fertilizer on lettuce in Example 4 of the present invention. DETAILED DESCRIPTION

[0022] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of embodiments of the present invention. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of the concept of the present invention.

[0023] In view of the fact that the current various biological microbial agents have single functions and cannot achieve the comprehensive effects of promoting root growth, preventing diseases, the present invention provides a composite microbial agent, a compound fertilizer, and a biological organic fertilizer. When applied to crop planting, they can significantly improve the promotion of root growth and achieve the purpose of disease prevention at the same time.

[0024] Specifically, as a first aspect of the present invention, a composite bacterial agent is provided. Based on 100 parts by total mass, the composite bacterial agent comprises: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velezensis.

[0025] The present invention selects three kinds of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus veleziaceus. Through the mutual cooperation of each strain, it has a significant antibacterial effect on soil-borne pathogens such as Fusarium and Phytophthora in the planting soil, and also has a significant antibacterial effect on citrus canker, and also has a significant promoting effect on the growth of plant roots and aboveground parts. Furthermore, when various Bacillus are compounded in a certain proportion, the obtained composite bacterial agent has a significantly better effect on promoting the growth of plant roots and aboveground parts and inhibiting soil-borne root rot and citrus canker. In addition, the sources of these three strains are not limited, and the same effect as the present invention can be achieved by compounding using the formula provided by the present invention.

[0026] Further preferably, based on 100 parts by weight, the composite bacterial agent comprises: 30-50 parts of Bacillus subtilis, 20-30 parts of Bacillus amyloliquefaciens, and 20-30 parts of Bacillus velez, wherein the effective viable count of the Bacillus is 10 billion to 100 billion per CFU. More preferably, based on 100 parts by weight, the composite bacterial agent comprises: 40-50 parts of Bacillus subtilis, 25-30 parts of Bacillus amyloliquefaciens, and 20-25 parts of Bacillus velez. The present invention is not particularly limited to the specific Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velez, and the sources thereof can achieve the same effects as in the examples of the present application.

[0027] As a second aspect of the present invention, there is provided an application of a composite bacterial agent in preventing and treating root rot or canker.

[0028] As a third aspect of the present invention, a compound fertilizer is provided, comprising: a composite bacterial agent and a synergist, wherein the mass ratio of the composite bacterial agent to the synergist is 1-10:1-4; wherein the synergist comprises a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or an auxiliary synergist selected from at least one of L-arginine, seaweed polysaccharide, and chitosan; based on 100 parts of the total mass of the composite bacterial agent, the composite bacterial agent comprises: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez.

[0029] In the present invention, the synergist is a substance commonly used in plant cultivation, which has a promoting effect on plant height, stem thickness, above-ground fresh weight, underground fresh weight and other indicators. The present invention has no special limitation on the source of the synergist, and commercially available products can be used.

[0030] Furthermore, the synergistic adjuvants are preferably L-tryptophan, L-phenylalanine, L-arginine, and chitosan oligosaccharides, with L-tryptophan, L-phenylalanine, and chitosan oligosaccharides being more preferred, followed by L-tryptophan and chitosan oligosaccharides, and L-phenylalanine and chitosan oligosaccharides. The compound fertilizer obtained by combining the synergistic adjuvant with the composite microbial agent not only has a significant antibacterial effect on soil-borne pathogens, but also promotes growth and increases yield.

[0031] According to an embodiment of the present invention, the primary synergists are L-tryptophan and L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan, L-phenylalanine, and any one of the co-synergists is 10-30:1-10:1-10:1-3, more preferably 25:4:4:1. Furthermore, the primary synergist is L-tryptophan or L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan or L-phenylalanine and any one of the co-synergists is 25:8:1. Furthermore, the mass ratio of the composite bacterial agent to L-arginine and chitosan oligosaccharide is 25:8:1. Within these ratio ranges, high compounding effects can be achieved.

[0032] L-tryptophan, molecular structure is C 11 H 12 N₂O₂, with a molecular weight of 204.23, is a precursor for the synthesis of the endogenous hormone auxin (indoleacetic acid). It is involved in plant growth and development regulation, photosynthesis, stress resistance, and defense, playing an important role in plant growth and adaptation to the environment. L-tryptophan of the present invention can be obtained commercially.

[0033] L-phenylalanine (3-phenyl-L-alanine), molecular formula is C9H 11 NO2, with a molecular weight of 165.19. The L-phenylalanine in the present invention can be obtained through commercial purchase channels. Its main functions include: 1) Promoting growth and development: L-phenylalanine can be used as a raw material for plant growth and participate in the synthesis of various plant hormones, such as auxin, gibberellins, etc., thereby promoting plant growth and development, and increasing the number of leaves and stem length. 2) Enhancing disease resistance: L-phenylalanine can improve the adaptability of plants to the external environment, and enhance their disease resistance and stress resistance. For example, when resisting viral and bacterial infections, L-phenylalanine can increase the immunity of plants, inhibit the growth of pathogens, and reduce disease losses. 3) Improving yield and quality: L-phenylalanine can promote plant photosynthesis, increase leaf area and photosynthetic rate, thereby increasing plant yield, promoting the synthesis of lignin, and is also a precursor substance for anthocyanin synthesis, so it can effectively promote fruit coloring.

[0034] Chitosan oligosaccharides, also known as chitosan oligosaccharides and oligochitosans, have the functions of promoting root growth, activating rhizosphere conditions, shrinking and strengthening joints, having strong bactericidal ability, improving soil quality, improving crop quality, increasing yield, improving fertilizer efficiency, and enhancing plant immunity. The chitosan oligosaccharides in the present invention can be obtained through commercial purchase channels.

[0035] As a fourth aspect of the present invention, a bio-organic fertilizer is provided, which is composed of a composite bacterial agent, a synergist and an organic material, wherein the mass ratio of the organic material, the composite bacterial agent and the synergist is 1000:2-10:1, based on the total mass of the composite bacterial agent being 100 parts, the composite bacterial agent comprises: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez; the synergist comprises a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or a co-synergist selected from at least one of L-arginine, seaweed polysaccharide and chitosan; and the organic material comprises at least one of straw, livestock and poultry manure and plant residues.

[0036] In the present invention, by mixing according to the above ratio, a bio-organic fertilizer that can be used directly can be obtained. The bio-organic fertilizer has the same effect as the composite microbial agent and compound fertilizer and can be directly sold as a product.

[0037] According to an embodiment of the present invention, the main synergistic adjuvants are L-tryptophan and L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan, L-phenylalanine and any one of the synergistic adjuvants is 10-30:1-10:1-10:1-3; wherein the number of viable bacteria in the composite bacterial agent is 200 million to 1 billion / g, the organic matter content is ≥45%, and the total nutrient content in the biological organic fertilizer is ≥5%.

[0038] As a fifth aspect of the present invention, there is provided application of bio-organic fertilizer in vegetable and fruit planting, wherein the vegetables and fruits include any one of citrus, orange, lettuce, wheat, and corn.

[0039] The following is a detailed description of the composite bacterial agent, compound fertilizer, bio-organic fertilizer and application of the present invention in conjunction with specific examples. The composite bacterial agent, synergistic adjuvant and test results shown are only part of the substances and corresponding test results, and the test methods are all conventional test methods.

[0040] (1) Microbial agent screening test

[0041] Test crops: corn potted plants (artificial climate greenhouse, day:night = 12:12);

[0042] Treatment method: Equal bacterial dose screening test, using 30 billion bacterial agents per plant; weigh equal bacterial doses of the agents and add them quantitatively to each treatment using a pipette, with each treatment repeated for 12 plant pots.

[0043] Investigation Indicators: 15 days after treatment, the effects of different treatments on corn seedling plant height, stem diameter, and fresh weight were measured. The growth-promoting effects of different microbial agents on corn are shown in Table 1. An equal-dose screening experiment was conducted on mycorrhizal crops (corn), using 30 billion microbial agents per plant. In this study, 1, 2, and 3 represent different strains of the same bacterial species (from a commercially available product). The following data were obtained for various growth indicators.

[0044] Table 1

[0045]

[0046]

[0047] As shown in Table 1, for the same strain, Bacillus amyloliquefaciens (such as Bacillus amyloliquefaciens 1) had the best growth-promoting effect on the aboveground dry weight, followed by Bacillus megaterium (such as Bacillus megaterium 1).

[0048] In terms of underground dry weight of the same strain, Bacillus amyloliquefaciens (such as Bacillus amyloliquefaciens 1) has the best growth-promoting effect, followed by Bacillus gelatinosa.

[0049] In terms of chlorophyll content, the best growth-promoting effect of the same strain is Bacillus gelatinosa (such as Bacillus gelatinosa 1), followed by Bacillus amyloliquefaciens (such as Bacillus amyloliquefaciens 1) and Bacillus velez (such as Bacillus velez 1).

[0050] For the same strain, Bacillus amyloliquefaciens (such as Bacillus amyloliquefaciens 1) and Bacillus subtilis (such as Bacillus subtilis 3) had the best growth-promoting effect, followed by Bacillus gelatinosa (such as Bacillus gelatinosa 1) and Bacillus velez (such as Bacillus velez 1).

[0051] For the same strain, Bacillus amyloliquefaciens (such as Bacillus amyloliquefaciens 1) had the best growth-promoting effect, followed by Bacillus Velezii (such as Bacillus Velezii 1) and Bacillus gelatinosa (such as Bacillus gelatinosa 1).

[0052] Therefore, based on comprehensive indicators such as aboveground dry weight, underground dry weight, chlorophyll, stem diameter, and plant height, it was found that Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, and Bacillus gelatinosa had more outstanding comprehensive performance.

[0053] Further, the above-mentioned strains are activated, and the plate confrontation test is carried out by using Pestalotiopsis versicolor, Fusarium oxysporum and Fusarium solani as pathogenic bacteria. After adjusting the concentration of each biocontrol strain, the plate confrontation test is carried out, and the plate antagonism rate is measured. The specific operation is as follows: PDA (potato dextrose agar) medium is prepared, and a 5mm-diameter fungus cake is punched at the edge of the pathogenic bacteria by using a puncher, and then the fungus cake is transplanted to the center of the plate. Different strains are cultured, and then the pathogenic bacteria are inoculated around the plate. The pathogenic bacteria and the different strains are placed in the central part of the position from the center of the pathogenic bacteria to the edge of the culture plate. After inoculation, the culture plate is incubated at 28℃, the inhibition effect of different strains on pathogenic bacteria is observed, and the plate antagonism rate is measured after 3-5 days.

[0054] The plate antagonism rate calculation formula is: antagonism rate (%) = (control colony diameter-treatment colony diameter) / control colony diameter * 100.

[0055] The test results of the plate confrontation test of the strains on the soil-borne disease pathogenic bacteria are shown in Figure 1 , and the inhibition rate is shown in Table 2.

[0056] Table 2

[0057]

[0058] Figure 1 The test results of the plate confrontation test of the strains on the soil-borne disease pathogenic bacteria are shown in

[0059] As shown in Figure 1 and Table 2, Bacillus velezensis and Bacillus amyloliquefaciens have strong inhibition effect on Fusarium oxysporum, Bacillus velezensis and Bacillus subtilis have strong inhibition effect on Fusarium solani, and Bacillus velezensis and Bacillus subtilis have strong inhibition effect on P. erythrophylla. Therefore, it can be determined that the bacterial agents with significant inhibition effect on soil-borne diseases and promoting root growth and growth are Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus velezensis.

[0060] Further, in order to verify whether there is obvious antagonism between Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus velezensis, the plate confrontation test is carried out.

[0061] For example, the three strains with the best growth-promoting effect in Table 1, i.e., Bacillus subtilis 3, Bacillus amyloliquefaciens 1 and Bacillus velezensis 1, are used for the plate confrontation test for antagonism between each other. The test method adopts the plate confrontation method, and the test results are shown in Figure 2 .

[0062] Figure 2 The test results of the plate confrontation test for antagonism between each other of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus velezensis are shown in .

[0063] like Figure 2 As shown, there is no antagonism between the three bacteria and they can be used in combination.

[0064] (2) Strain combination test

[0065] The different bacterial agents with the best performance in terms of aboveground dry weight, underground dry weight, chlorophyll, stem diameter, plant height and other indicators in Table 1 were used, combined in different proportions and used in combination experiments on corn seedlings. The specific experimental combinations and test results are shown in Table 3.

[0066] Table 3

[0067]

[0068]

[0069] The experiment in Table 3 shows that: B12-treated Bacillus subtilis 3: Bacillus amyloliquefaciens 1: Bacillus Velez 1 were combined in a ratio of (40%:30%:30%), totaling 100% (referred to as composite bacterial powder KJB01). The combined system had the best growth-promoting effect in terms of plant height, stem diameter, dry weight, etc., and had the best growth-promoting effect on corn.

[0070] (III) Synergist screening experiment

[0071] Different synergistic adjuvants to be tested were applied to wheat to conduct growth promotion screening tests. The specific experimental plan is as follows.

[0072] Test crops: Wheat potted plants (artificial climate chamber, day:night = 12:12, 28°C, 50% relative humidity).

[0073] Crop status before the test: two-leaf wheat with the same growth, 3 boxes for each treatment, 20 plants / box.

[0074] Treatment: Root irrigation was conducted twice every seven days. Samples were taken eight days after the second irrigation to analyze the aboveground and underground wheat parts.

[0075] Synergistic additives: L-phenylalanine, L-tryptophan, L-methionine, L-arginine, polyglutamic acid, malic acid, mineral fulvic acid, chitosan oligosaccharide, alginic acid, salicylic acid, calcium chloride, manganese sulfate, zinc sulfate, ammonium molybdate, succinic acid (succinic acid), lignin sulfonate, etc., concentrations are shown in Table 4.

[0076] Table 4

[0077]

[0078] Table 5

[0079]

[0080] Table 6

[0081]

[0082] Table 7

[0083]

[0084] Table 8

[0085]

[0086] Table 9

[0087]

[0088] As shown in Table 4-9, chitosan oligosaccharide, L-arginine, chitin, L-tryptophan, polyaspartic acid, melatonin, L-phenylalanine can obviously promote wheat aboveground dry weight, and wherein chitosan oligosaccharide improves the most remarkable aspect the aboveground dry weight, followed by L-arginine and L-tryptophan.Algae polysaccharide, alginic acid, polyglutamic acid, chitin, chitosan oligosaccharide, succinic acid (succinic acid), L-tryptophan, L-arginine, L-phenylalanine can obviously promote wheat belowground dry weight, and wherein, seaweed polysaccharide improves the most remarkable aspect the belowground dry weight, followed by polyglutamic acid and chitosan oligosaccharide.Chitosan oligosaccharide, L-arginine, L-tryptophan, calcium chloride, L-phenylalanine, polyglutamic acid powder can obviously promote wheat aboveground length, and wherein chitosan oligosaccharide improves the most remarkable aspect the aboveground length, followed by L-arginine and L-tryptophan. Alginic acid, melatonin, L-arginine, L-tryptophan, choline chloride and polyglutamic acid can significantly increase the length of the underground part of wheat, among which L-tryptophan has the most significant effect on the length of the underground part, followed by chitosan oligosaccharides and polyglutamic acid.

[0089] Taking into account the stability of the material and the appropriate production cost, in a preferred embodiment of the present invention, the synergist is selected from at least one of L-phenylalanine and L-tryptophan as the main synergist, and / or is selected from at least one of L-arginine, seaweed polysaccharide, and chitosan as a secondary synergist.

[0090] Furthermore, in order to verify the synergistic effect of compounding each synergistic adjuvant with the composite microbial agent, a test of compounding the composite microbial agent with synergistic substances was conducted. The specific experiment is as follows.

[0091] Test crops: maize potted plants (artificial climate chamber, day:night = 12:12, 28°C, 50% relative humidity).

[0092] Crop status before the test: Two-leaf, one-heart corn seedlings with the same growth, 30 plants per treatment.

[0093] Experimental treatment method: The composite bacterial agent (Bacillus subtilis 3: Bacillus amyloliquefaciens 1: Bacillus Velez 1 = 40%:30%:30%) and the synergistic adjuvant were weighed and mixed according to the mass ratio as shown in Table 10. The mixture was diluted 1000 times with clean water at a dosage of 0.3 g / plant and irrigated into the roots of corn seedlings with two leaves and one heart that grew neatly. After 15 days, the plant height, stem diameter, chlorophyll, root length, aboveground dry weight, and single plant root dry weight of each group were measured. The specific test results are shown in Table 11.

[0094] Table 10

[0095]

[0096] Table 11

[0097]

[0098] As shown in Table 11, the T4 treatment (composite microbial agent + L-tryptophan + L-phenylalanine + chitosan oligosaccharide in a ratio of 25:4:4:1) showed the best performance in terms of plant height, stem diameter, chlorophyll content, root length, and dry weight; followed by the T7 treatment (composite microbial agent + L-tryptophan + chitosan oligosaccharide in a ratio of 25:8:1) and the T6 treatment (composite microbial agent + L-phenylalanine + chitosan oligosaccharide in a ratio of 25:8:1). In view of this, the preferred synergistic adjuvants of the present invention are L-tryptophan, L-phenylalanine, and chitosan oligosaccharide, followed by L-tryptophan and chitosan oligosaccharide, and L-phenylalanine and chitosan oligosaccharide.

[0099] Furthermore, the present invention investigated the effects of a composite bacterial agent on citrus canker and found that it exhibited significant antibacterial activity against the disease. The composite agent included 40-50 parts of Bacillus subtilis, 25-30 parts of Bacillus amyloliquefaciens, and 20-25 parts of Bacillus Velez-Bézia (preferably a 40%:30%:30% ratio of Bacillus subtilis:Bacillus amyloliquefaciens:Bacillus Velez-Bézia ratio; more preferably, a 40%:30%:30% ratio of Bacillus subtilis 3:Bacillus amyloliquefaciens 1:Bacillus Velez-Bézia ratio). Specifically, the experimental procedure for the composite bacterial agent against citrus canker is as follows.

[0100] Inoculate a loopful of X. citri inoculum into 40 mL of LB medium and incubate at 28°C at 200°C for 48 hours. Pour into 400 mL of LB solid medium, mix thoroughly, and plate. Dilute the biocontrol agent to 200 million cfu / cfu, and place 1 μL of the agent on the pathogen plate. Observe the size of the inhibition zone. The biocontrol agents include Bacillus licheniformis (labeled "1"), Bacillus subtilis (labeled "2"), Bacillus megaterium (labeled "3"), Bacillus velezensis (labeled "4"), Bacillus amyloliquefaciens (labeled "5"), Bacillus gelatinous (labeled "6"), Bacillus laterosporus (labeled "7"), Bacillus polymyxa (labeled "8"), a composite agent (labeled "9"), and a blank control (labeled "10"). Agents 1-8 are commercially available.

[0101] Figure 3 This is a diagram showing the antibacterial effect of the composite bacterial agent on citrus canker in an embodiment of the present invention.

[0102] like Figure 3 As shown in the figure, under the same test conditions, No. 9 has the largest inhibition zone, indicating that the composite bacterial agent provided by the present invention has the most significant antibacterial effect on ulcer pathogens.

[0103] To verify the effectiveness of the composite inoculant of the present invention in the field, a field trial was conducted in an orchard in Wen'an Town, Zhijiang City, Hubei Province. The orchard was a six-year-old Newhall navel orange orchard, which had previously suffered from severe canker. The composite inoculant contained 40 parts of Bacillus subtilis (3), 30 parts of Bacillus amyloliquefaciens (1), and 30 parts of Bacillus velezensis (1). A 33% copper quinoline solution was used as a control group.

[0104] Test method: The first spray was conducted in spring, at 2 cm bud break. The second spray was conducted 15 days later, and the third spray was conducted 15 days later. After the three sprays, the disease index and control efficacy of each treatment were calculated.

[0105] Survey indicators: disease index, prevention efficacy. The specific survey methods are shown in Table 12, and the specific test results are shown in Table 13.

[0106] Table 12

[0107]

[0108] Table 13

[0109]

[0110] As shown in Table 13, the composite bacterial agent of the present invention has a certain antibacterial effect on citrus canker, and its effect is close to that of the commonly used chemical agent quinoline copper.

[0111] Furthermore, the compound bacterial agent, organic matter and synergistic adjuvant are compounded to prepare bio-organic fertilizer, and its effect in field application is verified.

[0112] The bio-organic fertilizer in Examples 1, 2, 3, and 4 below refers to the bio-organic fertilizer produced from the present invention's microbial composite agent, synergistic agent, and organic materials. It has a viable count of 500 million / g, an organic matter content of 45%, and a nitrogen, phosphorus, and potassium content of 5%. This bio-organic fertilizer was prepared by weighing and mixing the organic materials: composite agent (Bacillus subtilis 3: Bacillus amyloliquefaciens 1: Bacillus velezensis 1 = 40%:30%:30%): synergistic agent in a ratio of 1000:9:1 by weight. The competing product was a commercially available bio-organic fertilizer of the same specification in the field (Biological Organic Fertilizer - Rujin, with a viable count of 500 million / g, an organic matter content of 45%, and a nitrogen, phosphorus, and potassium content of 5%). The application rate for fruit trees was 5 kg / plant, and for vegetables was 200 kg / mu. The control group (CK) received no treatment except water.

[0113] Example 1: Application on Cara Cara navel oranges in Wanzhou, Chongqing. The application effect is shown in Table 14 below.

[0114] Table 14

[0115]

[0116] Example 2: The application effect on 6-year-old Newhall navel orange trees in Zhijiang, Hubei Province is shown in Table 15 below.

[0117] Table 15

[0118]

[0119] Example 3: The application effect on two-year-old seedlings of Hubei Zigui Citrus September Red Navel Orange is shown in Table 16 below.

[0120] Table 16

[0121]

[0122] Figure 4A This is a diagram showing the effect of applying the bio-organic fertilizer in Example 3 of the present invention on two-year-old saplings of the Ju Jiuyuehong navel orange; Figure 4B This is a picture of the effect of a competitor's product applied on two-year-old saplings of the Jiuyuehong navel orange; Figure 4C This is the effect diagram of the control group applying Ju Jiuyuehong navel orange saplings.

[0123] like Figures 4A-4C As shown, the bio-organic fertilizer provided by the present invention has significant rooting and growth-promoting effects.

[0124] Example 4: The application effect on lettuce is shown in Table 17 below.

[0125] Table 17

[0126]

[0127] Figure 5 This is a diagram showing the effect of applying the bio-organic fertilizer on lettuce in Example 4 of the present invention.

[0128] like Figure 5 As shown, the bio-organic fertilizer of the present invention has a good yield-increasing effect on lettuce cultivation.

[0129] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composite bacterial agent, characterized in that: Based on 100 parts by total mass, the composite bacterial agent comprises: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez.

2. Use of the composite bacterial agent according to claim 1 in preventing and treating root rot or canker.

3. A compound fertilizer, characterized in that The compound fertilizer comprises: a compound bacterial agent and a synergistic auxiliary agent, wherein the mass ratio of the compound bacterial agent to the synergistic auxiliary agent is 1-10:1-4; Wherein, the synergist comprises a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or a secondary synergist selected from at least one of L-arginine, seaweed polysaccharide and chitosan; Based on 100 parts of the total mass of the composite bacterial agent, the composite bacterial agent includes: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez.

4. The compound fertilizer according to claim 3, characterized in that The main synergistic adjuvants are L-tryptophan and L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan, L-phenylalanine and any one of the auxiliary synergistic adjuvants is 10-30:1-10:1-10:1-3.

5. The compound fertilizer according to claim 3, characterized in that The main synergist is L-tryptophan or L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan or L-phenylalanine and any one of the auxiliary synergists is 25:8:

1.

6. The compound fertilizer according to claim 3, characterized in that The mass ratio of the composite bacterial agent to L-arginine and chitosan oligosaccharide is 25:8:

1.

7. A bio-organic fertilizer, characterized in that: The bio-organic fertilizer is composed of a composite bacterial agent, a synergistic agent and an organic material, and the mass ratio of the organic material, the composite bacterial agent and the synergistic agent is 1000:2-10:

1. Wherein, based on the total mass of the composite bacterial agent as 100 parts, the composite bacterial agent includes: 20-60 parts of Bacillus subtilis, 20-40 parts of Bacillus amyloliquefaciens, and 20-40 parts of Bacillus velez; The synergist comprises a main synergist selected from at least one of L-tryptophan and L-phenylalanine and / or a secondary synergist selected from at least one of L-arginine, seaweed polysaccharide and chitosan; The organic material includes at least one of straw, livestock and poultry manure, and plant residues.

8. The bio-organic fertilizer according to claim 7, wherein The main synergistic adjuvants are L-tryptophan and L-phenylalanine, and the mass ratio of the composite bacterial agent to L-tryptophan, L-phenylalanine and any one of the auxiliary synergistic adjuvants is 10-30:1-10:1-10:1-3; The number of viable bacteria in the composite bacterial agent is 200 million to 1 billion per gram, the organic matter content is ≥45%, and the total nutrient content in the bio-organic fertilizer is ≥5%.

9. Use of the bio-organic fertilizer according to any one of claims 7 to 8 in vegetable and fruit planting.

10. The use according to claim 9, characterized in that The vegetables and fruits include any one of citrus, orange, lettuce, corn and wheat.

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