Special antibiological inoculant for rosa chinensis black spot and preparation process of special antibiological inoculant

Abstract

The invention provides a special antibiological inoculant for Chinese rose black spot and a preparation process thereof, and relates to the technical field of agricultural microorganisms. The special biocontrol inoculant comprises compound microorganisms, a filler, a wetting agent, a dispersing agent, an adhesive and an accelerant, the compound microorganism is prepared from trichoderma harzianum, purpureocillium lilacinum and bacillus subtilis. Wherein the trichoderma harzianum can inhibit pathogenic bacteria by competing, parasitizing and secreting antibacterial substances, and simultaneously induce plants to generate systemic resistance; purpureocillium lilacinum can parasitize hyphae and spores of pathogenic bacteria, secrete antibacterial metabolites and directly inhibit the pathogenic bacteria; the bacillus subtilis secretes antibacterial peptides to induce plant resistance and promote plant growth. The three strains have a synergistic effect, multiple defensive lines are formed through different action mechanisms (competition, parasitism, antibacterial substance secretion and resistance induction), and an excellent prevention and treatment effect on the rosa chinensis black spot is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and specifically relates to a special biocontrol agent for rose black spot disease and its preparation process. Background Art

[0002] Roses are ornamental plants widely cultivated worldwide, with extremely high economic value and ornamental value. However, rose black spot disease is one of the most serious diseases in rose cultivation, caused by the fungus Marssonina rosae (synonym: Diplocarpon rosae). This disease occurs widely globally, especially more severely in high-temperature and high-humidity environments. The black spot disease mainly infects rose leaves, causing black spots to appear on the leaves. When severe, the leaves turn yellow and fall off, weakening the photosynthesis ability of the plant, thereby affecting the growth and flowering of the plant, and even causing the death of the plant. The spread of the disease not only reduces the ornamental value of roses but also increases the maintenance cost, bringing huge economic losses to the rose industry.

[0003] Currently, the control of rose black spot disease mainly relies on chemical agents, such as broad-spectrum fungicides of triazoles (such as difenoconazole) and strobilurins (such as azoxystrobin). However, the long-term use of chemical agents not only easily leads to the generation of drug resistance by pathogenic bacteria but also may have a negative impact on the environment and ecosystem. Therefore, developing an efficient, environmentally friendly, and sustainable method for controlling rose black spot disease has become the focus of current research.

[0004] Biological control, as an environmentally friendly disease management strategy, has received extensive attention in recent years. Currently, a variety of microorganisms have been used for the biological control of plant diseases, such as Bacillus subtilis, Trichoderma spp., and Pseudomonas spp. However, there are still some limitations in the existing biocontrol agents for the control of rose black spot disease. First of all, the biocontrol effect of a single strain is often not stable enough and is greatly affected by environmental conditions (such as temperature, humidity, soil pH value). Secondly, some biocontrol agents are difficult to effectively colonize on the plant surface during field application, resulting in poor control efficacy. Therefore, developing a special biocontrol agent for rose black spot disease has important practical significance and application value. Summary of the Invention

[0005] The purpose of the present invention is to provide a special biocontrol agent for rose black spot disease and its preparation process. The special biocontrol agent provided by the present invention can effectively inhibit the growth of pathogenic bacteria and achieve the effect of controlling rose black spot disease.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions: The present invention provides a special biocontrol agent for rose black spot, comprising raw materials in the following weights: 70-80 parts of compound microorganisms, 10-20 parts of filler, 2-5 parts of wetting agent, 2-5 parts of dispersant, 1-2 parts of adhesive, and 1-2 parts of promoter; the compound microorganisms are composed of Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis; the filler is one or both of kaolin and diatomite; the wetting agent is one or both of sodium dodecylbenzenesulfonate and Nekal; the dispersant is one or both of sodium polycarboxylate and sodium carboxymethyl cellulose; the adhesive is one or both of polyvinyl alcohol and gelatin; the promoter is one or both of trehalose and L-alanine.

[0007] Preferably, the viable count of Trichoderma harzianum in the compound microorganisms is 200-500 million CFU / g, the viable count of Purpureocillium lilacinum in the compound microorganisms is 200-400 million CFU / g, and the viable count of Bacillus subtilis in the compound microorganisms is 300-500 million CFU / g.

[0008] The present invention also provides a preparation method of the above special biocontrol agent, comprising: respectively fermenting and culturing Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis to obtain bacterial solutions, centrifuging, collecting precipitates, washing and drying, and then mixing to obtain compound microorganisms; mixing the compound microorganisms, filler, wetting agent, dispersant, adhesive, and promoter, and then performing air flow pulverization and sieving to obtain the special biocontrol agent.

[0009] Preferably, the preparation methods of the Trichoderma harzianum bacterial solution and the Purpureocillium lilacinum bacterial solution include: respectively inoculating Trichoderma harzianum and Purpureocillium lilacinum into PDB liquid medium and culturing at 23-30°C and 150-200 r / min; the preparation method of the Bacillus subtilis bacterial solution includes: inoculating Bacillus subtilis into LB liquid medium and culturing at 35-40°C and 150-200 r / min.

[0010] Preferably, the rotation speed of the centrifugation is 5000-6000 rpm, and the time is 10-15 min.

[0011] Preferably, the drying is spray drying, the inlet air temperature is 140-150°C, and the outlet air temperature is 70-80°C.

[0012] Preferably, the pressure of the air flow pulverization is 0.5-0.8 MPa, and the particle size of the sieving is 150-250 mesh.

[0013] The present invention also provides the application of the above special biocontrol agent in the prevention and treatment of rose black spot.

[0014] Preferably, the application method of the special biocontrol agent is spraying.

[0015] More preferably, the dilution multiple of the special biocontrol agent is 1300 - 1700 times, and the dosage is 100 - 200 g / mu.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a special biocontrol agent for rose black spot, comprising: compound microorganisms, filler, wetting agent, dispersant, adhesive and promoter; the compound microorganisms are composed of Trichoderma harzianum, Purpureocillium lilacinum and Bacillus subtilis. Among them, Trichoderma harzianum can inhibit pathogenic bacteria through competition, parasitism and secretion of antibacterial substances, and at the same time induce plants to produce systemic resistance; Purpureocillium lilacinum can parasitize the hyphae and spores of pathogenic bacteria, secrete antibacterial metabolites, and directly inhibit pathogenic bacteria; Bacillus subtilis secretes antibacterial peptides, induces plant resistance and promotes plant growth. There is a synergistic effect among the three strains, and multiple defense lines are formed through different action mechanisms (competition, parasitism, secretion of antibacterial substances and induction of resistance), which has excellent control effect on rose black spot. Specific embodiments

[0017] The present invention provides a special biocontrol agent for rose black spot, comprising the following raw materials by weight: 70 - 80 parts of compound microorganisms, 10 - 20 parts of filler, 2 - 5 parts of wetting agent, 2 - 5 parts of dispersant, 1 - 2 parts of adhesive and 1 - 2 parts of promoter; the compound microorganisms are composed of Trichoderma harzianum, Purpureocillium lilacinum and Bacillus subtilis.

[0018] The Trichoderma harzianum of the present invention is purchased from the China Center for Agricultural Culture Collection, with the preservation number ACCC32097, the Purpureocillium lilacinum is purchased from the China Center for Agricultural Culture Collection, with the preservation number ACCC 32162, and the Bacillus subtilis is Bacillus subtilis Sjb3, which is isolated and preserved from the Key Laboratory of Plant-Microbe Interactions in Henan Province. The viable count of Trichoderma harzianum in the compound microorganisms is preferably 200 - 500 million CFU / g, the viable count of Purpureocillium lilacinum in the compound microorganisms is preferably 200 - 400 million CFU / g, and the viable count of Bacillus subtilis in the compound microorganisms is preferably 300 - 500 million CFU / g.

[0019] The Trichoderma harzianum in the compound microorganisms of the present invention can inhibit pathogenic bacteria through competition, parasitism and secretion of antibacterial substances, and at the same time induce plants to produce systemic resistance; Purpureocillium lilacinum can parasitize the hyphae and spores of pathogenic bacteria, secrete antibacterial metabolites, and directly inhibit pathogenic bacteria; Bacillus subtilis secretes antibacterial peptides, induces plant resistance and promotes plant growth. There is a synergistic effect among the three strains, and multiple defense lines are formed through different action mechanisms (competition, parasitism, secretion of antibacterial substances and induction of resistance), significantly improving the control effect on rose black spot.

[0020] The filler in the present invention is preferably one or both of kaolin and diatomite. The filler can provide a carrier for the attachment and survival of microorganisms. The microorganisms can adsorb on the surface or pores of the filler and remain in a relatively stable state during storage and use, which helps to maintain the activity and quantity of the microorganisms.

[0021] The wetting agent in the present invention is preferably one or both of sodium dodecylbenzenesulfonate and Nekal. The wetting agent can quickly and evenly disperse various components in the special biocontrol agent, especially the complex microorganisms and other solid components, in water, improve the uniformity of the use effect of the special biocontrol agent, and avoid affecting the control effect due to too high or too low local microorganism concentration; and can make the special biocontrol agent solution quickly spread on the plant surface to form a uniform drug film, reduce the loss of the solution, enable the microorganisms to fully contact the plant surface, and can colonize more effectively inside and outside the plant, playing a role in inhibiting the black spot pathogen of roses.

[0022] The dispersant in the present invention is one or both of sodium polycarboxylate and sodium carboxymethylcellulose. The sodium polycarboxylate is more preferably Geropon T / 36 (copolymer salt of polyacrylic acid and maleic anhydride, Solvay). The dispersant can adsorb on the surface of the complex microorganisms to prevent agglomeration, keep the microorganisms evenly dispersed, prevent caking, and promote the synergistic effect of the complex microorganisms and other components, improving the control effect of the biocontrol agent on rose black spot.

[0023] The adhesive in the present invention is preferably one or both of polyvinyl alcohol (polyvinyl alcohol 1788, molecular weight 75000 - 80000, Macklin) and gelatin. The adhesive in the present invention can enhance the adhesion of the agent on the plant surface, facilitate the colonization of microorganisms, resist external force scouring, extend the action time of the complex microorganisms on rose leaves, and improve the control effect on rose black spot.

[0024] The promoter in the present invention is one or both of trehalose and L-alanine. The promoter can provide an additional nutrient source for Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis, help the growth, reproduction, and metabolism of microorganisms, enable them to proliferate more quickly, help the microorganisms resist adverse environments such as drought and high temperature, maintain the stability and activity of cells, and contribute to the growth of plants, making roses stronger, and improving the overall stress resistance and disease resistance of plants, enabling roses to better resist the invasion of pathogens when facing the threat of black spot disease and reducing the impact of the disease on the growth of roses.

[0025] The present invention also provides a preparation method of the above-mentioned special biocontrol agent, which includes: separately fermenting and culturing Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis to obtain bacterial solutions, centrifuging, collecting precipitates, washing and drying, and then mixing to obtain a composite microorganism; mixing the composite microorganism, filler, wetting agent, dispersant, adhesive, and promoter, followed by air flow pulverization and sieving to obtain the special biocontrol agent.

[0026] The preparation methods of the Trichoderma harzianum bacterial solution and Purpureocillium lilacinum bacterial solution in the present invention preferably include: separately inoculating Trichoderma harzianum and Purpureocillium lilacinum into PDB liquid medium and culturing under the conditions of 23 - 30°C and 150 - 200 r / min; the preparation method of the Bacillus subtilis bacterial solution includes: inoculating Bacillus subtilis into LB liquid medium and culturing under the conditions of 35 - 40°C and 150 - 200 r / min.

[0027] The rotation speed of the centrifugation in the present invention is preferably 5000 - 6000 rpm, more preferably 5500 rpm, and the time is preferably 10 - 15 min, more preferably 13 min. Centrifugation can separate the bacterial cells from the fermentation broth, remove impurities, and concentrate the bacterial cells, facilitating subsequent operations.

[0028] The drying in the present invention is preferably spray drying, the inlet air temperature is preferably 140 - 150°C, more preferably 145°C, and the outlet air temperature is preferably 70 - 80°C, more preferably 75°C. Spray drying can quickly dry the bacterial cells, reduce the loss of activity, and the obtained spore powder has uniform particle size and good solubility.

[0029] The pressure of the air flow pulverization in the present invention is preferably 0.5 - 0.8 MPa, more preferably 0.7 MPa, and the particle size of the sieving is preferably 150 - 250 mesh, more preferably 200 mesh.

[0030] The present invention also provides the application of the above-mentioned special biocontrol agent in the prevention and treatment of rose black spot. The application method of the special biocontrol agent is preferably spraying, and more preferably spraying during the budding and flowering periods of roses. The dilution multiple of the special biocontrol agent is preferably 1300 - 1700 times, more preferably 1500 times, and the dosage is preferably 100 - 200 g / mu, more preferably 150 g / mu. The prevention and treatment rate of the special biocontrol agent of the present invention against rose black spot reaches more than 95%.

[0031] The special biocontrol agent of the present invention not only has excellent prevention and treatment effects on rose black spot, but also has good prevention and treatment effects on diseases on rose leaves (such as leaf spot, downy mildew, gray mold, etc.).

[0032] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0033] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] In the following embodiments, unless otherwise specified, all are conventional methods.

[0035] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0036] In the examples and comparative examples, the polyvinyl alcohol is polyvinyl alcohol 1788, with a molecular weight of 75,000 - 80,000 and the brand is Macklin.

[0037] Example 1 Preparation of a specific biocontrol agent for rose black spot S1. Preparation of compound microorganisms Activate Trichoderma harzianum and prepare a seed solution, inoculate it into a PDB liquid medium, and culture it at 28°C, pH 6.5, and 180 r / min for 10 days to obtain a Trichoderma harzianum fermentation broth; Activate Purpureocillium lilacinum and prepare a seed solution, inoculate it into a PDB liquid medium, and culture it at 25°C, pH 7, and 180 r / min for 24 hours to obtain a Purpureocillium lilacinum fermentation broth; Activate Bacillus subtilis and prepare a seed solution, inoculate it into an LB liquid medium, and culture it at 37°C, pH 7, and 180 r / min for 36 hours to obtain a Bacillus subtilis fermentation broth; Centrifuge the Trichoderma harzianum fermentation broth, Purpureocillium lilacinum fermentation broth, and Bacillus subtilis fermentation broth at 5500 rpm for 13 minutes respectively, collect the precipitate, wash the precipitate, and then spray-dry it at an inlet air temperature of 145°C and an outlet air temperature of 75°C to obtain Trichoderma harzianum spore powder, Purpureocillium lilacinum spore powder, and Bacillus subtilis spore powder. Mix them to obtain compound microorganisms, where the viable count of Trichoderma harzianum spore powder is 200 million CFU / g, the viable count of Purpureocillium lilacinum spore powder is 300 million CFU / g, and the viable count of Bacillus subtilis spore powder is 500 million CFU / g; S2. Preparation of the specific biocontrol agent Weigh 75 parts by weight of compound microorganisms, 8 parts by weight of diatomaceous earth, 7 parts by weight of kaolin, 2 parts by weight of sodium dodecylbenzenesulfonate, 1 part by weight of Nekal BX-78, 2 parts by weight of Geropon T / 36, 2 parts by weight of sodium carboxymethylcellulose, 1 part by weight of polyvinyl alcohol, 0.5 part by weight of gelatin, 1 part by weight of trehalose, and 0.5 part by weight of L-alanine respectively; First, mix the compound microorganism with diatomaceous earth at a rotation speed of 15 rpm for 13 min. Then, successively add sodium dodecylbenzenesulfonate, Nekal BX-78, Geropon T / 36, sodium carboxymethyl cellulose, polyvinyl alcohol, gelatin, trehalose, and L-alanine at a rotation speed of 20 rpm and mix for 15 min. Next, add kaolin at a rotation speed of 25 rpm and mix for 15 min to obtain the special biocontrol agent.

[0038] Example 2 S1. Preparation of compound microorganism Activate Trichoderma harzianum and prepare the seed liquid, then inoculate it into the PDB liquid medium and culture it at 25 °C, pH value of 6, and a rotation speed of 150 r / min for 12 d to obtain the Trichoderma harzianum fermentation broth. Activate Purpureocillium lilacinum and prepare the seed liquid, then inoculate it into the PDB liquid medium and culture it at 23 °C, pH value of 6.5, and a rotation speed of 150 r / min for 30 h to obtain the Purpureocillium lilacinum fermentation broth. Activate Bacillus subtilis and prepare the seed liquid, then inoculate it into the LB liquid medium and culture it at 35 °C, pH value of 6.7, and a rotation speed of 150 r / min for 40 h to obtain the Bacillus subtilis fermentation broth. Centrifuge the Trichoderma harzianum fermentation broth, Purpureocillium lilacinum fermentation broth, and Bacillus subtilis fermentation broth at a rotation speed of 5000 rpm for 15 min respectively, collect the precipitate, wash the precipitate, and then spray-dry it at an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain Trichoderma harzianum spore powder, Purpureocillium lilacinum spore powder, and Bacillus subtilis spore powder. Mix them to obtain the compound microorganism, where the viable count of Trichoderma harzianum spore powder is 500 million CFU / g, the viable count of Purpureocillium lilacinum spore powder is 200 million CFU / g, and the viable count of Bacillus subtilis spore powder is 300 million CFU / g. S2. Preparation of special biocontrol agent Weigh 70 parts by weight of compound microorganism, 10 parts by weight of diatomaceous earth, 10 parts by weight of kaolin, 2 parts by weight of sodium dodecylbenzenesulfonate, 2 parts by weight of Nekal BX-78, 2 parts by weight of Geropon T / 36, 2 parts by weight of sodium carboxymethyl cellulose, 0.5 part by weight of polyvinyl alcohol, 0.5 part by weight of gelatin, 0.5 part by weight of trehalose, and 0.5 part by weight of L-alanine respectively. First, mix the compound microorganism with diatomaceous earth at a rotation speed of 13 rpm for 15 min. Then, successively add sodium dodecylbenzenesulfonate, Nekal BX-78, Geropon T / 36, sodium carboxymethyl cellulose, polyvinyl alcohol, gelatin, trehalose, and L-alanine at a rotation speed of 18 rpm and mix for 17 min. Next, add kaolin at a rotation speed of 23 rpm and mix for 17 min to obtain the special biocontrol agent.

[0039] Example 3 Preparation of Special Biocontrol Bactericide for Rose Black Spot S1. Preparation of Composite Microorganisms Activate Trichoderma harzianum and prepare a seed solution, inoculate it into PDB liquid medium, and culture it at 30 °C, pH 7, and 200 r / min for 8 d to obtain Trichoderma harzianum fermentation broth; Activate Purpureocillium lilacinum and prepare a seed solution, inoculate it into PDB liquid medium, and culture it at 28 °C, pH 7.5, and 200 r / min for 20 h to obtain Purpureocillium lilacinum fermentation broth; Activate Bacillus subtilis and prepare a seed solution, inoculate it into LB liquid medium, and culture it at 40 °C, pH 7.5, and 200 r / min for 30 h to obtain Bacillus subtilis fermentation broth; Centrifuge the Trichoderma harzianum fermentation broth, Purpureocillium lilacinum fermentation broth, and Bacillus subtilis fermentation broth at 6000 rpm for 10 min respectively, collect the precipitate, wash the precipitate, and then spray-dry it at an inlet air temperature of 150 °C and an outlet air temperature of 80 °C to obtain Trichoderma harzianum spore powder, Purpureocillium lilacinum spore powder, and Bacillus subtilis spore powder. Mix them to obtain composite microorganisms, where the viable count of Trichoderma harzianum spore powder is 300 million CFU / g, the viable count of Purpureocillium lilacinum spore powder is 300 million CFU / g, and the viable count of Bacillus subtilis spore powder is 400 million CFU / g; S2. Preparation of Special Biocontrol Bactericide Weigh 80 parts by weight of composite microorganisms, 5 parts by weight of diatomaceous earth, 5 parts by weight of kaolin, 2 parts by weight of sodium dodecylbenzenesulfonate, 1 part by weight of Nekal BX-78, 2 parts by weight of Geropon T / 36, 2 parts by weight of carboxymethylcellulose sodium, 1 part by weight of polyvinyl alcohol, 0.5 part by weight of gelatin, 1 part by weight of trehalose, and 0.5 part by weight of L-alanine respectively; First, mix the composite microorganisms and diatomaceous earth at a rotation speed of 18 rpm for 15 min, and then sequentially add sodium dodecylbenzenesulfonate, Nekal BX-78, Geropon T / 36, carboxymethylcellulose sodium, polyvinyl alcohol, gelatin, trehalose, and L-alanine at a rotation speed of 23 rpm and mix for 13 min. Then add kaolin at a rotation speed of 28 rpm and mix for 13 min to obtain the special biocontrol bactericide.

[0040] Example 4 Preparation of Special Biocontrol Bactericide for Rose Black Spot S1. Preparation of Composite Microorganisms Activate Trichoderma harzianum and prepare a seed solution, inoculate it into PDB liquid medium, and culture it at 27 °C, pH 6.7, and 160 r / min for 9 d to obtain Trichoderma harzianum fermentation broth; Activate and prepare the seed liquid of Purpureocillium lilacinum, inoculate it into the PDB liquid medium, and culture it at 24°C, pH 7.2, and 160 r / min for 26 h to obtain the Purpureocillium lilacinum fermentation broth; Activate and prepare the seed liquid of Bacillus subtilis, inoculate it into the LB liquid medium, and culture it at 38°C, pH 7.6, and 160 r / min for 33 h to obtain the Bacillus subtilis fermentation broth; Centrifuge the Trichoderma harzianum fermentation broth, Purpureocillium lilacinum fermentation broth, and Bacillus subtilis fermentation broth at 5800 rpm for 11 min respectively, collect the precipitate, wash the precipitate, and spray dry it at an inlet air temperature of 148°C and an outlet air temperature of 76°C to obtain Trichoderma harzianum spore powder, Purpureocillium lilacinum spore powder, and Bacillus subtilis spore powder. Mix them to obtain a composite microorganism, where the viable count of Trichoderma harzianum spore powder is 200 million CFU / g, the viable count of Purpureocillium lilacinum spore powder is 300 million CFU / g, and the viable count of Bacillus subtilis spore powder is 500 million CFU / g; S2. Preparation of a specific biocontrol agent Weigh 74 parts by weight of the composite microorganism, 6 parts by weight of diatomaceous earth, 6 parts by weight of kaolin, 3 parts by weight of sodium dodecylbenzene sulfonate, 2 parts by weight of Nekal BX-78, 3 parts by weight of Geropon T / 36, 2 parts by weight of sodium carboxymethyl cellulose, 1 part by weight of polyvinyl alcohol, 1 part by weight of gelatin, 1 part by weight of trehalose, and 1 part by weight of L-alanine respectively; First, mix the composite microorganism and diatomaceous earth at a rotation speed of 12 rpm for 15 min, and then sequentially add sodium dodecylbenzene sulfonate, Nekal BX-78, Geropon T / 36, sodium carboxymethyl cellulose, polyvinyl alcohol, gelatin, trehalose, and L-alanine at a rotation speed of 23 rpm and mix for 12 min. Then add kaolin at a rotation speed of 25 rpm and mix for 15 min to obtain the specific biocontrol agent.

[0041] Example 5 Preparation of a specific biocontrol agent for rose black spot S1. Preparation of the composite microorganism Activate and prepare the seed liquid of Trichoderma harzianum, inoculate it into the PDB liquid medium, and culture it at 29°C, pH 6.2, and 160 r / min for 9 d to obtain the Trichoderma harzianum fermentation broth; Activate and prepare the seed liquid of Purpureocillium lilacinum, inoculate it into the PDB liquid medium, and culture it at 26°C, pH 6.8, and 160 r / min for 23 h to obtain the Purpureocillium lilacinum fermentation broth; Activate and prepare the seed liquid of Bacillus subtilis, inoculate it into the LB liquid medium, and culture it at 36°C, pH 6.7, and 160 r / min for 38 h to obtain the Bacillus subtilis fermentation broth; Centrifuge the fermentation broth of Trichoderma harzianum, the fermentation broth of Purpureocillium lilacinum, and the fermentation broth of Bacillus subtilis at 5200 rpm for 14 minutes respectively, collect the precipitate, wash the precipitate, and then spray-dry it under the conditions of an inlet air temperature of 142 °C and an outlet air temperature of 78 °C to obtain Trichoderma harzianum spore powder, Purpureocillium lilacinum spore powder, and Bacillus subtilis spore powder, and mix them to obtain a composite microorganism. Among them, the viable count of Trichoderma harzianum spore powder is 500 million CFU / g, the viable count of Purpureocillium lilacinum spore powder is 200 million CFU / g, and the viable count of Bacillus subtilis spore powder is 300 million CFU / g; S2. Preparation of special biocontrol agent Weigh 78 parts by weight of the composite microorganism, 6 parts by weight of diatomaceous earth, 6 parts by weight of kaolin, 1 part by weight of sodium dodecylbenzenesulfonate, 1 part by weight of Nekal BX-78, 3 parts by weight of Geropon T / 36, 2 parts by weight of sodium carboxymethylcellulose, 0.5 part by weight of polyvinyl alcohol, 0.5 part by weight of gelatin, 1 part by weight of trehalose, and 1 part by weight of L-alanine respectively; First, mix the composite microorganism and diatomaceous earth at a rotation speed of 15 rpm for 13 minutes, and then sequentially add sodium dodecylbenzenesulfonate, Nekal BX-78, Geropon T / 36, sodium carboxymethylcellulose, polyvinyl alcohol, gelatin, trehalose, and L-alanine at a rotation speed of 20 rpm and mix for 15 minutes. Then add kaolin at a rotation speed of 25 rpm and mix for 15 minutes to obtain the special biocontrol agent.

[0042] Comparative Example 1 The specific implementation method is the same as that of Example 1, except that the composite microorganism is composed of Trichoderma viride, Gliocladium roseum, and Bacillus amyloliquefaciens. Among them, Trichoderma viride is Trichoderma viride CICC ® 13038, purchased from the China Center for Industrial Culture Collection; Gliocladium roseum is Gliocladium roseum MES72701, purchased from the China General Microbiological Culture Collection Center, with the preservation number: CGMCC 3.11519 (disclosed in the patent CN112760230A); Bacillus amyloliquefaciens is Bacillus amyloliquefaciens BA-KA3, purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC No.8342 (disclosed in the patent CN104762223A); The preparation method of the composite microorganism is as follows: Activate Trichoderma viride and prepare a seed solution, inoculate it into a PDB liquid medium, and culture it at 26 °C, pH value 6.5, and 180 r / min for 6 days to obtain the Trichoderma viride fermentation broth; Activate Gliocladium roseum and prepare a seed solution, inoculate it into a PDB liquid medium, and culture it at 25 °C, pH value 7, and 180 r / min for 48 hours to obtain the Gliocladium roseum fermentation broth; The Bacillus amyloliquefaciens was activated and a seed solution was prepared, which was inoculated into an LB liquid medium and cultured at 28 °C, pH 7, and 180 r / min for 36 h to obtain a Bacillus amyloliquefaciens fermentation broth; The fermentation broths of Trichoderma viride, Gliocladium roseum, and Bacillus amyloliquefaciens were centrifuged at 5500 rpm for 13 min respectively. The precipitates were collected, washed, and then spray-dried at an inlet air temperature of 145 °C and an outlet air temperature of 75 °C to obtain Trichoderma viride spore powder, Gliocladium roseum spore powder, and Bacillus amyloliquefaciens spore powder. They were mixed to obtain a composite microorganism; among them, the viable count of Trichoderma viride spore powder was 200 million CFU / g, the viable count of Gliocladium roseum spore powder was 300 million CFU / g, and the viable count of Bacillus amyloliquefaciens spore powder was 500 million CFU / g.

[0043] Comparative Example 2 The specific implementation method was the same as that of Example 1, except that the Trichoderma harzianum was Trichoderma harzianum CICC ® 13010 (purchased from the China Center for Industrial Culture Collection Management), the Purpureocillium lilacinum was Purpureocillium lilacinum pt362 (purchased from the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2016682, disclosed in the patent CN106967613A), and the Bacillus subtilis was the Bacillus subtilis strain NH-6 (purchased from the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC17815); the others remained unchanged.

[0044] Comparative Example 3 The specific implementation method was the same as that of Example 1, except that "2 parts by weight of Geropon T / 36 and 2 parts by weight of sodium carboxymethylcellulose" was replaced with 4 parts by weight of Geropon T / 36.

[0045] Comparative Example 4 The specific implementation method was the same as that of Example 1, except that "2 parts by weight of Geropon T / 36 and 2 parts by weight of sodium carboxymethylcellulose" was replaced with 4 parts by weight of sodium carboxymethylcellulose.

[0046] Test Example 1 Synergistic effect of Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis The antagonistic performance of Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis against the pathogenic fungus Alternaria alternata of rose black spot was determined by the plate confrontation growth method. Among them, Alternaria alternata was Alternaria alternata SQYJAA-1 isolated and purified from "Identification and Biological Characteristics of Rose Black Spot Pathogenic Fungi in Shangqiu, Henan".

[0047] The grouping was as follows: Trichoderma harzianum group: Example 1 Trichoderma harzianum spore powder with a bacterial count of 1 billion CFU; Purpureocillium lilacinum group: Example 1 Purpureocillium lilacinum spore powder with a bacterial count of 1 billion CFU; Bacillus subtilis group: Example 1 Bacillus subtilis spore powder with a bacterial count of 1 billion CFU; Trichoderma harzianum + Purpureocillium lilacinum group: A mixture of Example 1 Trichoderma harzianum spore powder (bacterial count of 400 million CFU) and Example 1 Purpureocillium lilacinum spore powder (bacterial count of 600 million CFU); Trichoderma harzianum + Bacillus subtilis group: A mixture of Example 1 Trichoderma harzianum spore powder (bacterial count of 290 million CFU) and Example 1 Bacillus subtilis spore powder (bacterial count of 710 million CFU); Purpureocillium lilacinum + Bacillus subtilis group: A mixture of Example 1 Purpureocillium lilacinum spore powder (bacterial count of 375 million CFU) and Example 1 Bacillus subtilis spore powder (bacterial count of 625 million CFU); Example 1 group (Trichoderma harzianum + Purpureocillium lilacinum + Bacillus subtilis): A mixture of Example 1 Trichoderma harzianum spore powder (bacterial count of 200 million CFU), Example 1 Purpureocillium lilacinum spore powder (bacterial count of 300 million CFU), and Example 1 Bacillus subtilis spore powder (bacterial count of 500 million CFU).

[0048] Control group 1 (Trichoderma viride + Gliocladium roseum + Bacillus amyloliquefaciens): A mixture of Control 1 Trichoderma viride spore powder (bacterial count of 200 million CFU), Control 1 Gliocladium roseum spore powder (bacterial count of 300 million CFU), and Control 1 Bacillus amyloliquefaciens spore powder (bacterial count of 500 million CFU).

[0049] Control group 2 (Trichoderma harzianum + Purpureocillium lilacinum + Bacillus subtilis): A mixture of Control 2 Trichoderma harzianum spore powder (bacterial count of 200 million CFU), Control 2 Purpureocillium lilacinum spore powder (bacterial count of 300 million CFU), and Control 2 Bacillus subtilis spore powder (bacterial count of 500 million CFU).

[0050] Dilute the above-mentioned bacterial powders 1500 times with water for subsequent determination.

[0051] The measurement method is as follows: Use a 5-mm sterile punch to take Alternaria tenuissima and invert it (with the mycelium facing down) in the center of the PDB solid medium; Use sterile forceps to place three small filter papers at a distance of 3 cm from the center of the petri dish so that the small filter papers form an equilateral triangle; Use a sterile pipette tip to aspirate 5 μL of the well-shaken test bacterial solution and drop it on the small filter papers. Set 3 replicates for each test bacterial solution. Use only Alternaria tenuissima inoculated in the center of the petri dish without inoculating the bacterial solution as the control. Place the treated petri dishes in a constant temperature incubator at 28 °C for 6 days. When the fungal mycelium in the control group covers the entire medium, measure the colony radius of each group and the control group respectively, make records, calculate the inhibition rate, and the inhibition rate results of each test bacterial solution are shown in Table 1.

[0052] Inhibition rate (%) = (colony radius of the control group - colony radius of the test group) / colony radius of the control group × 100%.

[0053] Table 1 Inhibition rate of each test bacterial solution against the pathogenic fungus of rose black spot Group Bacteriostatic rate (%) Trichoderma harzianum group 40.36 Purpureocillium lilacinum group 35.65 Bacillus subtilis group 45.24 Trichoderma harzianum + Purpureocillium lilacinum group 63.48 Trichoderma harzianum + Bacillus subtilis group 70.27 Purpureocillium lilacinum + Bacillus subtilis group 65.41 Example 1 group 96.03 Comparative example 1 group 73.58 Comparative example 2 group 78.72 From the data in Table 1, it can be seen that the group of Example 1 (Trichoderma harzianum + Purpureocillium lilacinum + Bacillus subtilis) can significantly improve the inhibition rate of the pathogenic fungus of rose black spot compared with the Trichoderma harzianum group, Purpureocillium lilacinum group, Bacillus subtilis group, Trichoderma harzianum + Purpureocillium lilacinum group, Trichoderma harzianum + Bacillus subtilis group, and Purpureocillium lilacinum + Bacillus subtilis group, indicating that Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis have a synergistic effect in the prevention and control of rose black spot. From the data comparison between the group of Example 1 and the groups of Comparative Example 1 and Comparative Example 2, it can be seen that the antagonistic effects of different combinations of bacteria and fungi on the pathogenic fungus of rose black spot are different. Therefore, select Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis of the present invention as the composite microorganism for subsequent experiments.

[0054] Test Example 2 Analysis and determination of quality indicators Respectively detect the wetting time and suspension rate of the special biocontrol agents in Examples 1-5 and Comparative Examples 1-4, and the specific results are shown in Table 2.

[0055] The measurement method of the wetting time is GB / T 5451-2001, and the measurement method of the suspension rate is GB / T 14825-2006.

[0056] Table 2 Wetting time and suspension rate of each special biocontrol agent Group Wetting time (s) Suspension rate (%) Example 1 45.35 82.35 Example 2 43.47 83.50 Example 3 45.92 81.93 Example 4 42.13 85.02 Example 5 47.58 82.88 Comparative example 1 44.21 80.92 Comparative example 2 46.74 83.75 Comparative example 3 45.25 72.76 Comparative example 4 47.09 71.04 Analyzing the data in Table 2, the wetting time and suspension rate of the special biocontrol agents of Examples 1-5 and Comparative Examples 1-4 all meet the relevant national standards for pesticides. From the wetting time data, the difference between the examples and the comparative examples is not much; from the suspension rate data, the suspension rates of Comparative Examples 3 and 4 are lower than those of other groups; from the comparison of the suspension data of Example 1 and Comparative Examples 3-4, it can be seen that Geropon T / 36 and sodium carboxymethyl cellulose have a synergistic effect, and the combination of the two can significantly improve the suspension rate.

[0057] Test Example 3 Biocontrol effect of special biocontrol agent on black spot disease of rose leaves in vitro The rose leaves used in the test are all from roses grown naturally in the College of Biology and Food of Shangqiu Normal University. The special biocontrol agents of Examples 1-5 and Comparative Examples 1-4 were diluted 1500 times with water to obtain corresponding special biocontrol agent diluents. Rose leaves with basically the same size, shape, and growth position, good growth, no damage, and darker color were picked, and dust was washed off with clean water. After being naturally dried, they were placed in sterile culture bottles, and alcohol with a volume fraction of 75% was poured in for immersion and disinfection for 30s. The alcohol on the surface of the leaves was washed off with sterile water, and the leaves were placed in sterile culture dishes. A certain amount of sterile water was added to a sterile culture dish covered with a layer of sterile cotton to soak the sterile cotton completely. A leaf was placed on the moist sterile cotton, and 4 small holes were pierced in the non-vein position of the leaf with a sterile toothpick, and the small holes were completely covered with a sterile cotton ball the size of a soybean, and 0.5mL of the special biocontrol agent diluent was inoculated on the sterile cotton ball. The same amount of sterile water was inoculated as a control. Each group was set up with 10 replicates.

[0058] After each group was placed in the light culture room for 48 hours, the cotton balls were removed, and the Alternaria SQYJAA-1 bacterial cakes were taken with a 5mm sterile puncher and inverted on the small holes of the leaves of the control group and the treatment group, respectively. After being placed back in the light culture room for 48 hours, the bacterial cakes were removed. From the day of inoculation of the pathogenic fungus, the radius of the lesion was measured and counted every 4 days (the radius in four directions was measured and the average value was taken). The radius of each group on the 4th and 16th days was counted respectively, and the specific results are shown in Table 3.

[0059] Table 3 Radius of black spot rose leaf lesions in each group Group Radius on the 4th day (mm) Radius on the 16th day (mm) Control 4.42 22.91 Example 1 0.53 6.37 Example 2 0.68 7.45 Example 3 0.70 7.88 Example 4 0.62 7.23 Example 5 0.65 7.36 Comparative example 1 1.31 11.57 Comparative example 2 1.04 10.93 Comparative example 3 1.08 10.19 Comparative example 4 1.03 11.10 From the data in Table 3, it can be seen that, compared with the control group, the use of a special biocontrol agent can significantly reduce the spread of rose black spots. From the data comparison of Example 1 and Comparative Examples 1-2, it can be seen that the use of different composite microorganisms has different control effects on rose black spot. From the data comparison of Example 1 and Comparative Examples 3-4, it can be seen that the use of Geropon T / 36 and sodium carboxymethyl cellulose as dispersants at the same time has an inhibitory effect on the growth of rose leaf black spots.

[0060] Test Example 4 Field control effect on black spot of Rosa chinensis A total of 6 groups were set up in the test, divided into treatment groups (Example 1, Comparative Examples 1-4) and a blank control group.

[0061] For each special biocontrol agent, the plot area was designed to be 30 m 2 , with 3 replicates. The plots were randomly arranged among each other, with no significant differences. At the initial stage of the occurrence of black spot on Rosa chinensis 'Fen Shan', the medicine was applied by spraying method, with a dilution multiple of 1500 times. The medicine was applied continuously 3 times, with an interval of 5 d. The dosage of the special biocontrol agent was 150 g / mu.

[0062] The grading of the disease index of black spot of Rosa chinensis was based on the proportion of the disease spot area on the leaves, and the specific grading criteria were as follows: Grade 0: There are no disease spots on the leaves, the plants are completely healthy and not infected by the black spot pathogen of Rosa chinensis. Grade 1: The disease spot area accounts for less than 5% of the total leaf area. There are only a small number of scattered small black spots on the leaves, and the physiological functions such as photosynthesis of the leaves are less affected. Grade 3: The disease spot area accounts for 6%-10% of the total leaf area. The number of disease spots increases, and it begins to have a certain impact on the normal functions of the leaves, but the whole leaves still basically remain green and functionally intact. Grade 5: The disease spot area accounts for 11%-25% of the total leaf area. The disease spots on the leaves are significantly increased and larger, and some leaves begin to show yellowing, curling and other phenomena, which have a more obvious impact on the growth and ornamental value of the plants. Grade 7: The disease spot area accounts for 26%-50% of the total leaf area. A large number of leaves are damaged by the disease spots, and the phenomena of leaf yellowing and withering are more serious. The photosynthesis ability of the plants is greatly reduced, the growth is significantly inhibited, and the ornamental value is severely damaged. Grade 9: The disease spot area accounts for more than 50% of the total leaf area. Most of the leaves are covered by the disease spots, the leaves are severely withered and yellowed and fallen off, the plants grow weakly, and it may affect the health of the whole plant and even lead to death.

[0063] Disease index = [∑(number of diseased leaf plants at each level × relative level value)] / (total number of plants × 9) × 100; Control effect = (control disease index - treatment group disease index) / control disease index × 100%.

[0064] The incidence base numbers of each group were investigated before treatment, and the incidence situation was investigated 30 d after the last application of the medicine. The incidence situation was recorded according to the grading standard, and the disease index and control effect were calculated. The specific results are shown in Table 4.

[0065] Table 4 Field control effects of each treatment on black spot of Rosa chinensis Group Control effect (%) Example 1 96.32 Comparative example 1 70.13 Comparative example 2 75.85 Comparative example 3 86.58 Comparative example 4 85.76 As can be seen from the data in Table 4, Example 1 has the best control effect on black spot of roses compared with other groups. By comparing Example 1 with Comparative Examples 1-2, it can be seen that the control effects of the special biocontrol agents obtained by different microbial combinations are different, indicating that the use of the composite microorganisms of the present invention has a significant effect on controlling black spot of roses. By comparing Example 1 with Comparative Examples 3-4, it can be seen that the combined use of Geropon T / 36 and sodium carboxymethylcellulose can improve the suspension rate of the special biocontrol agent during use, making it evenly dispersed and improving the control effect of black spot.

[0066] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A special biocontrol agent for black spot of roses, characterized in that, It includes raw materials with the following weights: 70 - 80 parts of compound microorganisms, 10 - 20 parts of filler, 2 - 5 parts of wetting agent, 2 - 5 parts of dispersant, 1 - 2 parts of adhesive, and 1 - 2 parts of accelerator; The compound microorganisms are composed of Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis; The filler is one or both of kaolin and diatomite; The wetting agent is one or both of sodium dodecylbenzenesulfonate and Nekal; The dispersant is one or both of sodium polycarboxylate and sodium carboxymethylcellulose; The adhesive is one or both of polyvinyl alcohol and gelatin; The accelerator is one or both of trehalose and L - alanine.

2. The specific biocontrol agent according to claim 1, wherein The viable count of Trichoderma harzianum in the compound microorganisms is 200 - 500 million CFU / g, the viable count of Purpureocillium lilacinum in the compound microorganisms is 200 - 400 million CFU / g, and the viable count of Bacillus subtilis in the compound microorganisms is 300 - 500 million CFU / g.

3. The preparation method of the specific biocontrol agent according to claim 1 or 2, characterized in that, It includes: Trichoderma harzianum, Purpureocillium lilacinum, and Bacillus subtilis are respectively fermented and cultured to obtain bacterial solutions, centrifuged, the precipitates are collected, washed and dried, and then mixed to obtain compound microorganisms; after mixing the compound microorganisms, filler, wetting agent, dispersant, adhesive, and accelerator, they are air - pulverized and sieved to obtain a special biological control bactericide.

4. The preparation method according to claim 3, characterized in that, The preparation methods of the Trichoderma harzianum bacterial solution and the Purpureocillium lilacinum bacterial solution include: inoculating Trichoderma harzianum and Purpureocillium lilacinum into PDB liquid medium respectively, and culturing under the conditions of 23 - 30°C and 150 - 200 r / min; The preparation method of the Bacillus subtilis bacterial solution includes: inoculating Bacillus subtilis into LB liquid medium and culturing under the conditions of 35 - 40°C and 150 - 200 r / min.

5. The preparation method according to claim 3, characterized in that, The rotation speed of the centrifugation is 5000 - 6000 rpm, and the time is 10 - 15 min.

6. The preparation method according to claim 3, characterized in that, The drying is spray drying, the inlet air temperature is 140 - 150°C, and the outlet air temperature is 70 - 80°C.

7. The preparation method according to claim 3, characterized in that, The pressure of the air - pulverization is 0.5 - 0.8 MPa, and the particle size of the sieving is 150 - 250 mesh.

8. Application of the special biological control bactericide according to claim 1 or 2 in preventing and treating rose black spot.

9. The application according to claim 8, characterized in that, The application method of the special biological control bactericide is spraying.

10. The application according to claim 8 or 9, characterized in that The dilution multiple of the special biological control bactericide is 1300 - 1700 times, and the dosage is 100 - 200 g / mu.