CN109456900B

CN109456900B - Composite biological preparation and application thereof

Info

Publication number: CN109456900B
Application number: CN201811307006.8A
Authority: CN
Inventors: 杨文慧; 陈靠山; 赵明松; 辛奕
Original assignee: Shandong Pufang Biotechnology Co ltd
Current assignee: Shandong Pufang Biotechnology Co ltd
Priority date: 2017-03-17
Filing date: 2017-03-17
Publication date: 2022-09-20
Anticipated expiration: 2037-03-17
Also published as: CN106957870B; CN106957870A; CN109456900A

Abstract

The invention discloses a composite biological preparation for preventing and treating soil-borne diseases, which comprises mixed fermentation liquor obtained by submerged fermentation of rhizopus stolonifer and trichoderma viride. Furthermore, the use thereof for controlling soil-borne diseases, preferably blight and verticillium wilt, is disclosed. The invention utilizes the liquid submerged fermentation technology, the two microorganisms have fast propagation speed and extremely high extracellular secretion activity; can realize the technical effect which can not be achieved by simple compounding, and can quickly obtain a large amount of thalli and spores. The composite biological preparation has stronger bactericidal activity on Alternaria fungi, so that the composite biological preparation can effectively prevent and treat soil-borne diseases such as blight or verticillium wilt and the like. When used with fertilizer components, the soil stability and porosity can also be significantly improved.

Description

Compound biological agent and application thereof

The application is a divisional application of an invention patent application with the application date of 2017, 03 and 17, the application number of 201710161435.8 and the name of 'a compound biological agent and application thereof'.

Technical Field

The invention belongs to the field of biotechnology; relates to a compound biological preparation and application thereof, in particular to a compound biological preparation for preventing and treating soil-borne diseases and application thereof.

Background

The plant soil-borne disease refers to a disease in which pathogenic bacteria living in soil infect plants from roots or stems of the plants under appropriate conditions, thereby causing wilting and withering of the plants. The following are the main common soil-borne diseases: common soil-borne diseases in the seedling stage comprise damping-off caused by pythium and rhizoctonia solani and the like; the common outstanding disease types in the adult stage comprise fusarium wilt of melons, verticillium wilt of eggplants, bacterial soft rot of Chinese cabbage, pythium root rot of melons and fruits, plasmodiophora root rot, phytophthora capsici, bacterial wilt of tomatoes, root knot nematode and the like. Among these disease categories, blight and verticillium wilt are two representative soil-borne diseases most widely affected and can damage over 100 kinds of plants such as melons, leguminous plants, flowers and the like.

There are many ways in which soil-borne pathogens can spread, often damaging the roots and stems of plants and causing systemic disease in plants. The degree of the soil-borne diseases is mainly determined by the number of infected pathogenic bacteria. The higher the base number of pathogenic bacteria in one area, the more serious the disease. Diseases occur in the early growth stage of crops, so that roots of seedlings are rotten, or stems are rotten and fall suddenly; the seedlings die quickly, and the crop production is seriously influenced. The crop diseases in the later growth stage, the yield is reduced by 20-30% in general years and 50-60% in severe years, and even the crop is not harvested. After the soil-borne diseases occur, the prevention and the treatment are difficult. Because the germs are hidden in the soil for overwintering, the germs are difficult to kill; the crop continues to be attacked the next year and accumulates as the planting years increase. The occurrence of soil-borne diseases causes a series of adverse phenomena such as deterioration of soil environment, reduction of crop yield and quality reduction. In addition, because organic fertilizers are ignored and a large amount of fertilizers are applied in China for a long time, the disorder of soil microflora is accelerated. Therefore, the soil-borne diseases are aggravated year by year, which becomes an important limiting factor influencing the sustainable development of agriculture, and how to solve the soil-borne diseases is more and more important.

The prevention and control methods of soil-borne diseases, particularly blight and verticillium wilt are more, and are mainly divided into physical prevention and control, chemical prevention and control and biological prevention and control. The chemical prevention and cure is the most main prevention and cure method at present, and has the advantages of quick effect, low cost, simple method and wide insecticidal and antibacterial spectrum. However, long-term application of chemical pesticides not only pollutes the environment and reduces the soil remediation capability, but also causes pathogenic bacteria to generate resistance, and causes great changes in physical, chemical, biological and biochemical properties of the soil. The physical control comprises methods such as soil exposure, water vapor disinfection, flooding and the like, and has certain effect on a few crop varieties. However, physical control is labor intensive and is mainly applied to facility agriculture. The biological control is to control the occurrence of plant diseases and insect pests by using organisms or metabolites thereof, and mainly comprises the following steps: the method for controlling pests by using pests, controlling pests by using bacteria, utilizing antagonism, cross protection and information chemical substances and the like. It has the characteristics of no pollution, ecological balance protection, better control effect and the like, and is called as a green control method. The prevention and treatment of blight and verticillium wilt by biological prevention and treatment methods has become the research focus of scholars at home and abroad. The fact proves that the biological control is the development direction of controlling soil-borne diseases at present and in the future, and meets the requirement of agricultural sustainable development.

For biological control, researchers in various countries have separated many biocontrol bacteria (mainly including bacteria biocontrol bacteria, fungi biocontrol bacteria and actinomycetes), and developed biological control methods with good control effects on blight, such as biological microbial agents, biological organic fertilizers and the like. Among these, a biological control method using fungal biocontrol bacteria is particularly important. Among fungal biocontrol fungi, Alternaria sp (Alternaria sp.) fungi are the main fungi causing the development of blight.

Alternaria fungi have strong adaptability to environment and hosts and are widely distributed in nature. At present, more than 500 species of alternaria alternate are reported all over the world, wherein more than 90 percent of species are facultative parasitic on plants of different families, particularly crops, can cause diseases of dozens of crops such as corn, wheat, tobacco, potato, tomato, apple, pear and the like, and cause field and postpartum losses. According to reports, alternaria fungus causes crop diseases such as tomato blight, apple brown spot, melon leaf spot, pumpkin leaf spot, cabbage black spot, tobacco brown spot, ginseng alternaria, wheat leaf spot, Chinese cabbage black spot, potato early blight, tomato early blight, pepper early blight, eggplant early blight, black nightshade early blight, pear black spot, grape cob brown rot, apricot black spot, cherry black wheel spot, cotton verticillium wilt, eggplant verticillium wilt and the like.

Previously, Alternaria fungi have relied primarily on chemical agents for control. Studies have shown that copper formulations are effective agents for controlling alternaria fungi, but the use of copper formulations affects fruit surface, reduces quality and may lead to systemic resistance by pathogenic bacteria. With the concern of environmental protection and green foods, research on bacteriostatic components using plant extracts is becoming more and more popular. The results of experiments of 13 plants acetone extract on inhibiting alternaria alternata show that only extracts of sophora flavescens, liquorice, medlar and longhairy antenoron have obvious inhibiting effect on alternaria alternata, and other plants have no inhibiting effect. In addition, certain results have been achieved in the application of biological control of Alternaria. Agricultural antibiotics for preventing and treating Alternaria fungal diseases are firstly pushed to polyoxin in Japan and polyoxin in China. The polyoxin is a cytosine nucleoside antibiotic, the producing bacterium is streptomyces theobromae, the main components are polyoxin A and polyoxin B, the action site is the cell wall of fungus, and the polyoxin antibiotic has special effects on alternaria alternate, alternaria leaf spot of apple, pear black spot and the like. The Bacillus polymyxa is separated from the cauliflower seeds by Picard and the like, the bacteria can produce antibiotics to play a role in controlling cauliflower black spot, and the occurrence of diseases can be reduced by using sterile supernatant liquid to perform seed treatment. Trichoderma (Trichoderma spp.) is one of the most used microbial populations in plant disease control worldwide. The Wangcheng and the like have obvious antagonism to the alternaria alternate.

At present, most beneficial or biocontrol microorganisms are screened from soil, and better control effect is achieved on the research and application of single biocontrol bacteria. However, the effect of the single biocontrol bacterium in the actual production application is unstable, and the commercialization process of the biocontrol bacterium is limited.

In recent years, a great deal of research shows that the microbial agent prepared by two or even more biocontrol microorganisms can overcome the defect of single biocontrol bacteria. The inventor has utilized the mixed bacteria of the bacillus subtilis and the trichoderma pseudokoningii to control plant diseases, and develops a series of products on the basis of the plant diseases. Because of remarkable prevention effect and wide prevention spectrum, the pesticide is called as 'fool' pesticide by users. For example, chinese patent ZL201110292116.3 and chinese patent ZL201110292342.1 disclose a crop disease control compound biological agent and a compound biological soil conditioner, respectively, each of which comprises bacillus subtilis fermentation broth, trichoderma pseudokoningii fermentation broth, water-soluble chitin, and potassium fulvate. The product has good prevention and treatment effects on cucumber verticillium wilt and watermelon wilt.

However, in the above-mentioned biological agent or soil improvement agent, the Bacillus subtilis fermentation broth and the Trichoderma pseudokoningii fermentation broth must be obtained by separate fermentation. If mixed fermentation is adopted, the growth and reproduction behaviors of the two fungi interfere with each other, so that the density of hyphae is not as good as the sum of single fermentation, and the spore yield is difficult to satisfy. This will greatly affect the control effect of plant diseases.

Therefore, in order to control the above plant diseases, it is urgently required to find a complex biological agent for controlling soil-borne diseases.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a compound biological agent for preventing and controlling soil-borne diseases. The inventors have surprisingly found that when two specific microorganisms are mixed and fermented, the chemical composition is changed and the antibacterial activity is enhanced. The composite biological agent has the characteristics of stability and strong colonization ability, and the hypha density and sporulation quantity of the fermentation liquor are improved by utilizing the synergistic effect among microorganisms, so that the control effect of plant diseases is improved.

In order to achieve the purpose, the invention adopts the following technical scheme: a composite biological preparation for preventing and treating soil-borne diseases comprises mixed fermentation broth of Rhizopus stolonifer and Trichoderma viride.

The compound biological agent of the invention is characterized in that the rhizopus stolonifer is deposited as follows: CGMCC No. 8435; the preservation name is: rhizopus stolonifer PF 102; the preservation date is as follows: 11/2013, 5/month; the preservation unit is as follows: china general microbiological culture Collection center. Rhizopus stolonifer has extremely strong stress resistance, and can generate a plurality of active substances for decomposing soil organic matters and soil active ingredients in the rapid propagation process, such as: vitamins, amino acids, saccharifying enzymes, proteases, amylases, and the like. Rhizopus stolonifer can produce large amounts of extracellular active substances such as: amino acids, nucleic acids, vitamins, sphingolipids and other compounds, which have the functions of decomposing organic matters, inhibiting harmful microorganisms, promoting plant root growth, etc.; the rhizopus stolonifer can also secrete a plurality of antibacterial substances, has inhibitory activity on a plurality of pathogenic bacteria, has spectral property and strong stress resistance, can prevent diseases and promote the growth of plants.

The composite biological agent is characterized in that the Trichoderma viride is deposited as follows: CGMCC No. 8434; the preservation name is: trichoderma viride PF 1; the preservation date is as follows: 11/2013 and 5/11/month; the preservation unit is as follows: china general microbiological culture Collection center. The trichoderma viride is widely distributed, and the secretion of the trichoderma viride has the effects of promoting plant growth and sterilizing, and has a great soil improvement potential. The trichoderma viride has rich hyphae, the hyphae can wind soil particles, so that the stability and the permeability of soil are improved, soil hardening and hardening are broken, the trichoderma viride can secrete rich extracellular high-molecular polysaccharide and glycoprotein and a high-activity special enzyme system, the acid-base balance of the soil is adjusted, the soil is promoted to release nutrients, and the absorption of plant roots to the nutrient components is promoted.

The composite biological preparation is prepared by simultaneously inoculating a spore suspension of rhizopus stolonifer and a spore suspension of trichoderma viride into a fermentation tank according to a certain weight ratio for liquid submerged fermentation.

The compound biological preparation of the invention, wherein the spore suspension of the rhizopus stolonifer is prepared according to the following method: firstly, inoculating refrigerated strains to a flat PDA culture medium by using an inoculating loop, and transferring for 3 generations; then picking edge hypha with inoculating loop and transferring to PDB culture medium at 20-35 deg.c for 5-15 days, filtering the fermented liquid to obtain rhizopus stolonifer spore suspension with live spore concentration of (5-9) x 10 ⁸ Each/ml.

The compound biological agent of the invention, wherein the PDA culture medium is: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water.

The composite biological agent of the invention, wherein the PDB culture medium is: 200g of potatoes, 20g of cane sugar and 1000mL of distilled water.

The composite biological agent of the invention, wherein the culture temperature is preferably 22 ℃ to 32 ℃, more preferably 25 ℃ to 30 ℃, and most preferably 28 ℃.

The composite biological agent is preferably cultured for 6-14 days, more preferably 7-13 days, even more preferably 8-12 days, and most preferably 10 days.

The composite biological agent of the invention, wherein the filtration is 100 μm sieve filtration.

The composite biological agent is prepared by the following steps of: firstly, inoculating refrigerated strains to a flat PDA culture medium by using an inoculating loop, and transferring for 3 generations; then picking edge hypha by using an inoculating loop, transferring the edge hypha into a PDB culture medium, culturing at the temperature of 20-35 ℃ for 5-15 days, filtering the fermentation liquor to obtain a spore suspension of the trichoderma viride, wherein the concentration of viable spores is (5-9) multiplied by 10 ⁸ One per ml.

The compound biological agent of the invention, wherein the PDA culture medium is: 200g of potato, 20g of cane sugar, 18g of agar and 1000mL of distilled water.

The composite biological preparation is characterized in that the fermentation tank is filled with a PDB culture medium or a bran culture medium; the temperature of the liquid submerged fermentation is 25-35 ℃, preferably 28-32 ℃ and most preferably 30 ℃; the time for the liquid submerged fermentation is 24h to 20 days, preferably 36h to 15 days, more preferably 48h to 12 days, and most preferably 48h to 10 days.

The composite biological agent comprises the following components in a weight ratio of 1:1 to 1: 10. preferably, the weight ratio of the rhizopus stolonifer spore suspension to the trichoderma viride spore suspension is 1: 2 to 1: 8; more preferably, the weight ratio of the rhizopus stolonifer spore suspension to the trichoderma viride spore suspension is 1: 3 to 1: 6; and, most preferably, the ratio by weight of the rhizopus stolonifer spore suspension to the trichoderma viride spore suspension is 1: 4.

the compound biological preparation is applied in the form of a soil conditioner, a biological fertilizer, a foliar spray or a root spray. In the above products, it is preferable to further contain crop nutrients.

Complex organisms according to the inventionFormulations wherein the crop nutrients include, but are not limited to, any one or more of water soluble chitin, potassium fulvate and decomposed organic matter. Wherein, the water-soluble chitin has the title of 'plant vaccine', can induce crops to generate resistance factors to diseases by continuous use, reduces the incidence of diseases and obviously reduces the using amount of pesticides. In addition, the fertilizer has strong leaf surface adhesion performance, and can promote roots, strengthen seedlings and promote fruit ripening. The molecular weight M of the water-soluble chitin is (1-5) x 10 ⁵ The viscosity is 100-200 mPas, the ethyl degree DAC is more than or equal to 90%, and the purity is more than or equal to 90%. The fulvic acid potassium has high loading capacity and physiological activity, and the chelated macronutrients and the micronutrients of the fulvic acid potassium can be better utilized by plants, stimulate the microscopic biological activity of the plants, slowly release fertilizers, improve the absorption of the plants on nutrients, promote the germination and growth of the plants, accelerate the precipitation and decomposition and enhance the waterlogging resistance. The chitin and the potassium fulvate are chelated in different structural forms, so that the mutual promotion effect is achieved, and the soil structure is improved. The specification of the potassium fulvate is as follows: fineness is less than 120 meshes, water soluble substance is more than 99.7 wt%, water-soluble fulvic acid is more than 50 wt%, potassium oxide is 9.5-10 wt%, nitrogen is 2.5-3.0 wt%, phosphorus is 0.5-0.8 wt%, and organic matter is 50-60 wt%. Decomposed organic matter is also a well known crop nutrient in the art. In order to improve the germination rate of the preparation in the initial application stage, common decomposed organic matters can be properly added.

The composite biological preparation comprises the crop nutritional ingredients, and the crop nutritional ingredients are favorably 2-20 wt% of the weight of the composite biological preparation. In one embodiment, the mixed fermentation liquid of rhizopus stolonifer and trichoderma viride is 70-98 wt%, the water-soluble chitin is 0.5-2 wt%, and the potassium fulvate is 0.5-2 wt%, based on the total weight of the composite biological preparation. The dosage of the compound biological preparation is generally 100-5000 ml/mu, preferably 200-4500 ml/mu, more preferably 300-4000 ml/mu, even more preferably 400-3000 ml/mu, and most preferably 500-2500 ml/mu according to the soil fertility.

On the other hand, the invention also provides the application of the compound biological agent in preventing and treating soil-borne diseases, preferably blight or verticillium wilt.

The use according to the invention, wherein the soil-borne disease is preferably a soil-borne disease caused by a fungus of the genus Alternaria.

The application of the invention is characterized in that the soil-borne diseases are selected from tomato blight, apple brown spot, melon leaf spot, pumpkin leaf spot, cabbage black spot, tobacco brown spot, ginseng alternaria alternate, wheat leaf spot, Chinese cabbage black spot, potato early blight, tomato early blight, pepper early blight, eggplant early blight, black nightshade early blight, pear black spot, grape cob brown rot, apricot kernel black spot, cherry black wheel spot, cotton verticillium wilt and eggplant verticillium wilt.

According to the application of the invention, when in use, the compound biological preparation is diluted to a proper concentration, sprayed on the soil surface before planting and then ploughed, or irrigated to roots, or sprayed on the leaf surfaces of crops.

The application of the invention is characterized in that the dilution multiple is 100-500 times. Preferably, the dilution multiple is 100-400 times; more preferably, the dilution multiple is 100-300 times; and, most preferably, the dilution factor is 100 to 200.

Surprisingly, it was found that after submerged fermentation of the liquid, a small piece of mycelium from the mixed fermentation broth was inoculated on a PDA plate and cultured at 28 ℃ for 4 days, and it was found that both white hyphae and black spores of Rhizopus stolonifer and hyphae and green spores of Trichoderma viride were present. This demonstrates that both fungi survive in the fermentation broth after mixed fermentation.

The inventors have also found that after submerged fermentation of the liquid, a small piece of mycelium in the mixed fermentation broth was observed under a 400-fold microscope and compared with the hyphae in the individual fermentation broths of rhizopus stolonifer and trichoderma viride. The results show that the hyphae in the mixed fermentation liquor are more compact and the spore yield is more. This phenomenon is probably due to the addition of Rhizopus stolonifer which results in the growth and propagation of Trichoderma viride being accelerated and secondary metabolites being produced. High Performance Liquid Chromatography (HPLC) confirmed the above results.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) the composite biological preparation has the characteristics of stability and strong colonization ability, and utilizes the synergistic effect among microorganisms, so that the mixed fermentation liquor is generated relative to the single fermentation liquor or the simple mixing of the single fermentation liquorQualitative change(New or more antagonistic substances are produced), andquantitative change(hypha is more compact, and spore yield is more), so that the control effect of plant diseases is improved;

(2) the compound biological agent has high propagation speed of two microorganisms and extremely high extracellular secretion activity in the process of liquid deep fermentation, not only realizes the technical effect which cannot be achieved by simple compounding, but also realizes large-scale production, greatly reduces labor input, can quickly obtain a large amount of thalli and spores, reduces the production cost and is convenient to use;

(3) when the compound biological agent is used together with fertilizer components, the compound biological agent can improve the structure of soil microbial flora, promote the growth of plant root systems, enhance the absorption capacity of the root systems to nutrient components, and can be firmly attached to and wound around soil particles, thereby improving the soil stability and the porosity;

(4) the composite biological preparation has stronger bactericidal activity on Alternaria fungi, so that the composite biological preparation can effectively prevent and treat soil-borne diseases such as blight or verticillium wilt and the like.

The invention also comprises the following:

item 1. a complex biological agent for controlling soil-borne diseases, characterized by comprising a mixed fermentation broth of Rhizopus stolonifer and Trichoderma viride (Trichoderma viride).

Item 2. the complex biological preparation according to item 1, wherein the mixed fermentation is a liquid submerged fermentation in which a spore suspension of Rhizopus stolonifer and a spore suspension of Trichoderma viride are simultaneously inoculated into a fermentation tank at a certain weight ratio.

Item 3. the complex biological preparation according to

item

1 or 2, wherein the spore suspension of Rhizopus stolonifer (or Trichoderma viride) is prepared as follows: first, cool with an inoculating loopInoculating the preserved strain into a flat PDA culture medium, and transferring for 3 generations; then picking edge hypha by using an inoculating loop, transferring the edge hypha into a PDB culture medium, culturing at the temperature of 20-35 ℃ for 5-15 days, filtering the fermentation liquor to obtain a spore suspension of rhizopus stolonifer (or trichoderma viride), wherein the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

Item 4. the composite biological agent according to item 2, wherein the fermenter contains a PDB medium or a bran medium; the temperature of the liquid submerged fermentation is 25-35 ℃; the liquid submerged fermentation time is 24 h-20 days.

Item 5. the complex biological preparation according to item 2, wherein the ratio by weight of the spore suspension of rhizopus stolonifer and the spore suspension of trichoderma viride is 1:1 to 1: 10.

item 6. the composite biological agent according to

item

1 or 2, which is applied in the form of a soil conditioner, a biofertilizer, a foliar spray or a root spray.

The complex biological agent of item 6, wherein the complex biological agent further comprises a crop nutrient; the crop nutrient components comprise one or more of water-soluble chitin, potassium fulvate and decomposed organic matters.

Item 8 the composite biological agent of item 7, wherein the mixed fermentation broth of rhizopus stolonifer and trichoderma viride is 70 to 98 wt%, the water-soluble chitin is 0.5 to 2 wt%, and the potassium fulvate is 0.5 to 2 wt%, based on the total weight of the composite biological agent.

Item 9. use of the complex biological agent according to any one of items 1 to 8 for controlling soil-borne diseases, preferably blight and verticillium wilt.

Item 10 the use of item 9, wherein the soil-borne disease is selected from the group consisting of tomato wilt, apple brown spot, melon leaf spot, pumpkin leaf spot, cabbage black spot, tobacco brown spot, alternaria ginseng, wheat leaf spot, chinese cabbage black spot, potato early blight, tomato early blight, pepper early blight, eggplant early blight, black nightshade early blight, pear black spot, grape cob brown rot, apricot kernel black spot, cherry black wheel spot, cotton verticillium wilt, and eggplant verticillium wilt.

Drawings

FIG. 1 is an HPLC chromatogram of fermentation liquids obtained in example 1 of the present invention and comparative examples 1-2 (from top to bottom: example 1, comparative example 2 and comparative example 1).

FIG. 2 is a graph showing the inhibitory effect on Alternaria mali of the samples obtained in examples 1-3 and comparative examples 1-2 (FIG. 2-1: control; FIG. 2-2: comparative example 1; FIG. 2-3: comparative example 2; FIG. 2-4: example 2; FIG. 2-5: example 1; FIG. 2-6: example 3).

FIG. 3 is a 400-fold microscope image of mycelia in fermentation liquids of example 1 and comparative examples 1-2 of the present invention (one pair on the left and right, and example 1, comparative example 1 and comparative example 2 in this order from bottom to top).

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications can be made by those skilled in the art after reading the contents of the present invention, and those equivalents also fall within the scope of the invention defined by the appended claims.

The following embodiments will help to understand the present invention, but do not limit the scope of the present invention.

Example 1:

the spore suspension of rhizopus stolonifer is prepared according to the following method: firstly, inoculating a strain preserved in a refrigerated way to a flat PDA culture medium by using an inoculating loop, wherein the PDA culture medium comprises the following components: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water, and transferring for 3 generations; then, picking edge hyphae by using an inoculating loop, and transferring the edge hyphae into a PDB culture medium, wherein the PDB culture medium comprises the following components: 200g of potato, 20g of cane sugar and 1000mL of distilled water, the culture temperature is 28 ℃, the culture time is 10 days, the fermentation liquor is filtered to obtain the spore suspension of rhizopus stolonifer, and the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

The spore suspension of trichoderma viride was prepared as follows: firstly, inoculating the strain preserved by refrigeration to a flat PDA culture medium by using an inoculating loop, wherein the PDA culture medium isComprises the following steps: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water, and transferring for 3 generations; then picking edge hyphae by using an inoculating loop, and transferring the edge hyphae into a PDB culture medium, wherein the PDB culture medium comprises the following components: 200g of potatoes, 20g of cane sugar and 1000mL of distilled water, wherein the culture temperature is 28 ℃, the culture time is 10 days, the fermentation liquor is filtered to obtain a spore suspension of trichoderma viride, and the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

Simultaneously inoculating the spore suspension of the rhizopus stolonifer and the spore suspension of the trichoderma viride into a fermentation tank according to the weight ratio of 1: 4 for liquid submerged fermentation; the fermentation tank is filled with a PDB culture medium; the PDB culture medium is as follows: 200g of potatoes, 20g of cane sugar and 1000mL of distilled water; the temperature of the liquid submerged fermentation is 30 ℃; the liquid submerged fermentation time is 48 h. Finally, the composite biological preparation of the invention in the example 1 is obtained. A small piece of mycelium in the fermentation broth was observed under a 400-fold microscope, and the results are shown in FIG. 1.

The antibacterial activity is measured by adopting a confrontation experiment, and the specific method is as follows: alternaria mali was inoculated on one side of a PDA medium with a sterilized inoculator (Φ 5mm) on a clean bench while the composite biological preparation of example 1 was inoculated on the other side of the PDA medium, incubated at a constant temperature of 28 ℃, three to four replicates per treatment were set, the inhibitory effect was observed and recorded, the colony radius of alternaria mali was measured by a cross method, and a plate inoculated with only alternaria mali was used as a control. After culturing at the constant temperature of 28 ℃ for 5-6 days, calculating the bacteriostatic rate of the biocontrol microorganism on the alternaria mali, wherein the calculation method comprises the following steps: the bacteriostatic ratio (%) [ (control group colony radius-experimental group colony radius)/control group colony radius ] × 100. The results are shown in FIG. 2.

The composite biological agent of the invention example 1 is filtered by filter paper, centrifuged at 5000r/min and the supernatant is collected. Filtering with filter (filter membrane phi 0.22um), mixing the obtained fermentation liquid and solid PDA culture medium at a ratio of 1:1, and pouring into a culture dish. Inoculating Alternaria mali fungus cake with sterilized hole puncher to the center of culture medium containing fermentation liquid (one fungus cake per dish, mycelium facing downwards), setting 3-4 parallel treatment groups, and setting control group. After culturing for 5-6 days at constant temperature of 28 ℃, measuring the colony diameter by a cross method, calculating the relative inhibition rate of the growth of fungal hyphae, and comparing the inhibition results. The calculation formula is as follows: relative inhibition ratio (%) (average control group colony diameter-average experimental group colony diameter)/(average control group colony diameter-cake diameter) × 100. See table 1 for results.

A part of the composite biological agent of example 1 was subjected to centrifugal filtration to remove solid impurities, and then concentrated and purified to obtain a sample for chromatography. The samples were analyzed by high performance liquid chromatography. The results are shown in FIG. 3.

Example 2:

mixing the spore suspension of the rhizopus stolonifer and the spore suspension of the trichoderma viride according to the weight ratio of 1:1 simultaneously inoculating the mixture into a fermentation tank for liquid submerged fermentation; the rest of the steps and process parameters were the same as in example 1. Finally, the composite biological preparation of the invention in the embodiment 2 is obtained.

The inhibitory rate against Alternaria mali and the relative inhibitory rate (%) were determined in the same manner as in example 1. See figure 2 and table 1 for results.

Example 3:

mixing a rhizopus stolonifer spore suspension and a trichoderma viride spore suspension according to the weight ratio of 1: 9 inoculating into a fermentation tank for liquid submerged fermentation; the rest of the steps and process parameters were the same as in example 1. Finally, the composite biological preparation of the invention in the embodiment 3 is obtained.

The inhibitory rate and relative inhibition (%) against Alternaria mali were determined in the same manner as in example 1. See figure 2 and table 1 for results.

Comparative example 1:

the spore suspension of rhizopus stolonifer is prepared according to the following method: firstly, inoculating the strain which is refrigerated and preserved to a flat PDA culture medium by using an inoculating loop, wherein the PDA culture medium comprises: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water, and transferring for 3 generations; then picking edge hyphae by using an inoculating loop, and transferring the edge hyphae into a PDB culture medium, wherein the PDB culture medium comprises the following components: 200g of potato, 20g of cane sugar and 1000mL of distilled water, the culture temperature is 28 ℃, the culture time is 10 days, the fermentation liquor is filtered to obtain the spore suspension of rhizopus stolonifer, and the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

Inoculating the spore suspension of rhizopus stolonifer into a fermentation tank for liquid submerged fermentation; the fermentation tank is filled with PDB culture medium; the PDB culture medium is as follows: 200g of potatoes, 20g of cane sugar and 1000mL of distilled water; the temperature of the liquid submerged fermentation is 30 ℃; the liquid submerged fermentation time is 48 h. Finally, the composite biological preparation of comparative example 1 was obtained.

The inhibition rate and relative inhibition rate (%) of alternaria mali were measured in the same manner as in example 1; and the sample was analyzed by high performance liquid chromatography in the same manner as in example 1. See figures 1-3 and table 1 for results.

Comparative example 2:

the spore suspension of trichoderma viride is prepared according to the following method: firstly, inoculating a strain preserved in a refrigerated way to a flat PDA culture medium by using an inoculating loop, wherein the PDA culture medium comprises the following components: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water, and transferring for 3 generations; then picking edge hyphae by using an inoculating loop, and transferring the edge hyphae into a PDB culture medium, wherein the PDB culture medium comprises the following components: 200g of potato, 20g of cane sugar and 1000mL of distilled water, the culture temperature is 28 ℃, the culture time is 10 days, the fermentation liquor is filtered to obtain a spore suspension of trichoderma viride, and the concentration of viable spores is (5-9) multiplied by 10 ⁸ One per ml.

Inoculating the spore suspension of the trichoderma viride into a fermentation tank for liquid submerged fermentation; the fermentation tank is filled with a PDB culture medium; the PDB culture medium comprises: 200g of potatoes, 20g of cane sugar and 1000mL of distilled water; the temperature of the liquid submerged fermentation is 30 ℃; the liquid submerged fermentation time is 48 h. Finally, the composite biological agent of comparative example 2 was obtained.

Comparative example 3:

the spore suspension of rhizopus stolonifer is prepared according to the following method: firstly, inoculating the strain preserved in cold storage into a flat PDA culture medium by using an inoculating loop, and then inoculating the strain into a culture mediumThe PDA culture medium is as follows: 200g of potatoes, 20g of cane sugar, 18g of agar and 1000mL of distilled water, and transferring for 3 generations; then picking edge hyphae by using an inoculating loop, and transferring the edge hyphae into a PDB culture medium, wherein the PDB culture medium comprises the following components: 200g of potato, 20g of cane sugar and 1000mL of distilled water, the culture temperature is 28 ℃, the culture time is 10 days, the fermentation liquor is filtered to obtain the spore suspension of rhizopus stolonifer, the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

Mixing the spore suspension of the rhizopus stolonifer and the spore suspension of the trichoderma viride according to the weight ratio of 1: 4, simple mixing to obtain the composite biological preparation of the comparative example 3.

The inhibitory rate and relative inhibition (%) against Alternaria mali were determined in the same manner as in example 1. See table 1 for results.

FIG. 1 is an HPLC chromatogram of fermentation liquids obtained in example 1 of the present invention and comparative examples 1-2 (from top to bottom: example 1, comparative example 2 and comparative example 1). As can be seen from FIG. 1, the mixed fermentation of example 1 of the present invention is not a simple addition of fermentation components of a single microorganism, but a new substance is produced. This phenomenon is probably due to the addition of Rhizopus stolonifer which results in the growth and propagation of Trichoderma viride being accelerated and secondary metabolites being produced. FIG. 1 is a diagram sufficiently showing the phenomenon of deterioration of the complex biological agent of the present invention. The inventors also determined the HPLC curve of the fermentation broth obtained in comparative example 3, and the peak position was extremely consistent with those of comparative examples 1 and 2, which is substantially equivalent to a simple superposition of the latter.

FIG. 2 is a graph showing the inhibitory effect on Alternaria mali of the samples obtained in examples 1-3 and comparative examples 1-2 (FIG. 2-1: control; FIG. 2-2: comparative example 1; FIG. 2-3: comparative example 2; FIG. 2-4: example 2; FIG. 2-5: example 1; FIG. 2-6: example 3). As can be seen from the graph, although Rhizopus stolonifer and Trichoderma viride both had inhibitory effects on Alternaria inaequalis to different degrees relative to the control group; however, the composite biological agent of examples 1 to 3 of the present invention has significantly better inhibitory effect on alternaria mali.

Table 1 shows the inhibitory effect against Alternaria mali of the sample solutions obtained in examples 1 to 3 and comparative examples 1 to 3 with time.

TABLE 1

Time/day	2	3	4	5	6
						Example 1	57.0％	58.8％	57.2％	56.4％	55.9％
Example 2	63.2％	65.7％	67.1％	65.5％	66.3％
						Example 3	57.4％	57.1％	56.8％	56.6％	56.1％
Comparative example 1	53.9％	51.7％	53.2％	51.4％	51.0％
						Comparative example 2	14.7％	7.6％	13.3％	15.4％	16.8％
Comparative example 3	52.2％	51.5％	50.5％	51.1％	52.0％

As can be seen from Table 1, the mixed fermentation broths of examples 1 to 3 of the present invention have not only higher bacteriostatic ability than the single fermentation broths of comparative examples 1 to 2, but also higher bacteriostatic ability than the single fermentation broths of comparative example 3 when simply mixed. Further, in example 2, the mixing ratio of the spore suspension of rhizopus stolonifer and the spore suspension of trichoderma viride was 1: and 4, the obtained composite biological agent has the highest bacteriostatic rate on alternaria mali.

FIG. 3 is a 400-fold microscope image of mycelia in fermentation liquids of example 1 and comparative examples 1-2 of the present invention (one pair on the left and right, and example 1, comparative example 1 and comparative example 2 in this order from bottom to top). The results show that compared with hyphae in the single fermentation liquor of rhizopus stolonifer and trichoderma viride, the hyphae in the mixed fermentation liquor of the embodiment 1 is more compact and produces more spores. This phenomenon is probably due to the addition of rhizopus stolonifer, which leads to a faster growth and propagation of trichoderma viride and the production of secondary metabolites. This is consistent with the previous HPLC results. FIG. 3 sufficiently reflects the quantitative change phenomenon of the complex biological agent of the present invention.

From the results, the invention can see that the rhizopus stolonifer spore suspension and the trichoderma viride spore suspension are simultaneously inoculated into a fermentation tank for liquid submerged fermentation according to a certain weight proportion, so that the quality change (new or more antagonistic substances are generated) of the mixed fermentation liquid and the quantitative change (more compact hyphae and more spore production) are generated compared with the single fermentation liquid or the simple mixing of the single fermentation liquid. The quality change and the quantity change lead the composite biological preparation obtained by the invention to obviously improve the bacteriostatic effect on alternaria mali.

That is, the composite biological agent not only embodies the synergistic effect of rhizopus stolonifer and trichoderma viride, but also embodies the unique effect of mixed liquid submerged fermentation. This effect is not achievable by a single strain of microorganism and is not simply a superposition of the effects of two strains of microorganism. The two microorganisms in the liquid submerged fermentation process have high propagation speed and extremely high extracellular secretion activity, the liquid submerged fermentation technology is utilized to realize the technical effect which cannot be achieved by simple compounding, large-scale production is realized, the labor input is greatly reduced, a large amount of thalli and spores can be quickly obtained, the production cost is reduced, and the use is convenient.

Further, the present invention studied the effect of the soil conditioner on the improvement of the physicochemical properties of soil and the control effect of blight by preparing 80 parts by weight of the composite biological agent of example 2, 2 parts by weight of water-soluble chitin and 2 parts by weight of potassium fulvate into the soil conditioner 1 for use in crop growth.

Test example 1

The test group 1 is a test group which applies 1000 ml/mu of the soil conditioner 1 (diluted by 100 times) and the test group 2 is a test group which applies 1000 ml/mu of a single trichoderma viride fermentation broth preparation containing the same effective spore amount, the single trichoderma viride fermentation broth preparation is uniformly sprayed on the soil surface before ploughing, the control group is sprayed with the same amount of clear water, the subsequent topdressing treatment is the same, the area of a small area is 100 square meters, and the soil physical and chemical properties are measured and calculated after wheat is harvested. The results are shown in Table 2.

TABLE 2

	Soil volume weight (g/cm) ³ )	pH
			Control group	1.77	5.2
Test group 1	1.42	6.7
			Test group 2	1.49	6.2

The result shows that the volume weight of the soil is reduced relative to that of a control group after the soil conditioner 1 and the green trichoderma preparation are applied, the pH value is close to neutral, the soil acidification condition is reversed, the soil conditioner is suitable for crop growth, and the effect of the soil conditioner 1 is superior to that of a single green trichoderma preparation.

Test example 2

The test group 1 is prepared by spraying 1000 ml/mu of the soil conditioner 1 (diluted by 100 times) on the soil surface before field planting, spraying the same amount of the existing trichoderma viride and bacillus subtilis compound preparation with the same effective thallus quantity on the test group 2, and spraying the same amount of clear water on the control group. Microorganisms in soil not only can regulate plant growth, but also play an important role in maintaining and improving soil fertility, energy conversion, substance circulation and the like, so the microorganisms are an important index of soil quality. The results are shown in Table 3.

TABLE 3

	Actinomycetes (cfu/g)	Bacterium (cfu/g)	Fungus (cfu/g)
				Control group	3.4×10 ⁶	2.3×10 ⁶	3.9×10 ³
Test group 1	4.5×10 ⁶	6.7×10 ⁶	4.2×10 ⁴
				Test group 2	3.7×10 ⁶	4.2×10 ⁶	6.3×10 ³

The results show that compared with the control group treatment, the soil conditioner 1 and the compound preparation of the trichoderma viride and the bacillus subtilis with the same effective thallus quantity improve the quantity of microorganisms in the soil, increase the diversity of the microorganisms, and the quantity of bacteria and fungi improved by the soil conditioner 1 is most obvious and is obviously higher than that of the test group 2.

Test example 3

160 tomato plants with the same size and growth vigor are selected and are divided into four groups to be simultaneously inoculated with blight pathogenic bacteria. The

test groups

1 and 2 are sprayed with the soil conditioner 1 diluted by 100 times and 200 times respectively, and the control group is sprayed with a preparation containing the same effective number of bacillus subtilis single strains. The results are shown in Table 4.

TABLE 4

	Incidence (%)	Disease finger (%)	Control effect (%)
				ControlGroup(s)	2	3	94.6
Test group 1	8	8	85.3
				Test group 2	20	20	66.4

The result shows that compared with the common bacillus subtilis preparation, the biological preparation has obviously improved control effect on the blight, wherein the control effect is the best by 100 times of liquid, and then is 200 times of liquid.

The results of the field test examples show that when the compound biological agent is used together with fertilizer components, the compound biological agent can improve the structure of soil microbial flora, promote the growth of plant root systems, enhance the absorption capacity of the root systems to nutrient components, and can be firmly attached and wound with soil particles, thereby improving the soil stability and the porosity; in addition, the compound biological agent can effectively prevent and treat soil-borne diseases such as blight or verticillium wilt and the like.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A composite biological agent for controlling soil-borne diseases, which comprises Rhizopus stolonifer (A)Rhizopus stolonifer) And Trichoderma viride (Trichoderma viride) The mixed fermentation liquid of (1), wherein the mixed fermentation is prepared by simultaneously inoculating a rhizopus stolonifer spore suspension and a trichoderma viride spore suspension into a fermentation tank according to a certain weight ratio for liquid submerged fermentation, wherein the weight ratio of the rhizopus stolonifer spore suspension to the trichoderma viride spore suspension is 1:1 to 1: 2.

2. the complex biological agent according to claim 1, wherein the spore suspension of rhizopus stolonifer or trichoderma viride is prepared as follows: firstly, inoculating refrigerated strains to a flat PDA culture medium by using an inoculating loop, and transferring for 3 generations; then picking edge hypha by using an inoculating loop, transferring the edge hypha into a PDB culture medium, culturing at the temperature of 20-35 ℃ for 5-15 days, filtering the fermentation liquor to obtain a spore suspension of rhizopus stolonifer or trichoderma viride, wherein the concentration of live spores is (5-9) multiplied by 10 ⁸ One per ml.

3. The composite biological agent of claim 1, wherein the fermentor is filled with PDB medium or bran medium; the temperature of the liquid submerged fermentation is 25-35 ℃; the liquid submerged fermentation time is 24 h-20 days.

4. The complex biological agent of claim 1, further comprising crop nutrients; the crop nutrient components comprise one or more of water-soluble chitin, potassium fulvate and decomposed organic matters.

5. The composite biological agent of claim 4, wherein the mixed fermentation broth of Rhizopus stolonifer and Trichoderma viride is 70-98 wt%, the water-soluble chitin is 0.5-2 wt%, and the potassium fulvate is 0.5-2 wt%, based on the total weight of the composite biological agent.

6. Use of the complex biological agent according to any one of claims 1 to 5 for controlling blight.