A microbial inoculum for preventing root-knot nematode disease and clubroot disease simultaneously and its preparation method

Abstract

The present invention discloses a microbial inoculant for preventing both root-knot nematode disease and clubroot disease and a preparation method thereof, relating to the technical field of fertilizers, and solving the technical problems in the prior art that the field symptoms of root-knot nematode disease and clubroot disease are similar and difficult to quickly distinguish and accurately select agents, and that root-knot nematode disease and clubroot disease are likely to occur simultaneously and difficult to control in a timely manner. The preparation method of the microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention comprises the following steps: preparing methylotrophic bacillus powder and bacillus subtilis powder; preparing paecilomyces lilacinus powder; and compounding the prepared methylotrophic bacillus powder, paecilomyces lilacinus powder and bacillus subtilis powder in proportion and mixing them evenly to obtain the microbial inoculant for preventing both root-knot nematode disease and clubroot disease. The microbial inoculant for preventing both root-knot nematode disease and clubroot disease prepared by the present invention can conduct unified prevention and control of the two diseases, avoiding the golden period of early prevention and control being delayed due to incorrect agent selection in agricultural production.

Description

Technical Field

[0001] The present invention relates to the technical field of fertilizers, and particularly relates to a microbial inoculant for preventing both root-knot nematode disease and clubroot disease and a preparation method thereof. Background Art

[0002] Clubroot disease is caused by Plasmodiophora brassicae and is a fungal disease that only occurs in cruciferous vegetables such as Chinese cabbage, pakchoi, cabbage, radish, etc. After the occurrence of clubroot disease, the main roots and lateral roots of the plant expand to form tumors or chicken-intestine roots of different sizes, and their shape and size are greatly affected by the attachment position. The tumors on the main roots are mostly near the upper part, spherical or nearly spherical, with uneven, rough surfaces, and sometimes the epidermis cracks in the later stage; the tumors on the lateral roots are mostly cylindrical or finger-shaped. After some plants are diseased, many new fibrous roots grow on the upper part of the main root due to the stimulation of the pathogen. These fibrous roots sometimes extend all the way to the base of the stem, and the tumors on the fibrous roots are numerous and strung together. In the later stage of the disease, it is easily infected by soft rot bacteria, causing tissue decay and emitting a stench. The above-ground part of the diseased plant shows slow growth, the plant is stunted and shrunk, and the leaves have no fresh green luster, which is particularly obvious around noon on sunny days. In severe cases, the whole plant wilts and dies.

[0003] Root-knot nematode disease occurs not only in the roots of cruciferous vegetables such as Chinese cabbage, but also in the roots of carrots, eggplants, tomatoes, lettuce, lettuce, cucumbers, watermelons, melons, coriander, zucchini, flue-cured tobacco, etc., and root nodules (also known as root knots) grow on the roots. The shape and size of the root nodules vary greatly depending on the crop and the type of nematode. Generally, thin and weak fibrous roots grow on the root knots, and secondary infections form root-knot-like nodules. When the plant is pulled out with soil and the soil is washed away in water, many nodular small tumors can be seen on the roots. The small tumors are initially milky white and turn brown in the later stage, and there are few fibrous roots.

[0004] After the occurrence of root-knot nematode disease in the roots of vegetables, the symptoms shown in the above-ground part are very similar to those of clubroot disease, both of which slow down the growth of the plant. The differences between the two are as follows: (1) Clubroot disease only damages cruciferous vegetables and does not damage other vegetables. Root-knot nematode disease damages other vegetables in addition to cruciferous vegetables; (2) The roots of clubroot disease are mainly the root tissues that expand into colon-like shapes, while root-knot nematode disease forms small nodules on the fibrous roots; (3) The two can be distinguished by dissecting the tumors or root knots on the roots. Whether there are milky white pear-shaped female adults of root-knot nematodes in the tumors is the most accurate diagnostic method for distinguishing the two. There is nothing in the tumors of clubroot disease.

[0005] Currently, in the prevention and control of agricultural diseases, a biological control product generally only acts on one disease. When used in the field, if the disease type cannot be accurately identified, the situation of inaccurate disease prevention and control often occurs. Especially for diseases with similar symptoms, if there is no current rapid detection technology in the field, the situation of incorrect selection of pesticides and delay in the prevention and control opportunity often occurs.

[0006] Since the symptoms of root-knot nematode disease and clubroot are very similar, it is often difficult to quickly distinguish and prevent them in agricultural production. At the same time, the occurrence of root-knot nematode disease will reduce plant resistance, often leading to the simultaneous occurrence of clubroot disease. When the two occur at the same time, it is often more difficult to distinguish and prevent them. Because the prevention and control agents for the two diseases are completely different, if only a single disease is controlled, the symptoms of the disease cannot be improved at all. Once the two diseases occur together, it means that the production of vegetables in the season will be lost, causing huge losses to farmers. Therefore, providing a solution for the comprehensive prevention and control of the two diseases is a technical problem that needs to be solved urgently in current agricultural production. Summary of the invention

[0007] The purpose of the present invention is to provide a microbial agent for preventing both root-knot nematode disease and clubroot disease and a preparation method thereof, which solves the technical problems in the prior art that the field symptoms of root-knot nematode disease and clubroot disease are similar and difficult to quickly distinguish and accurately select agents, and that root-knot nematode disease and clubroot disease are prone to occur simultaneously and are difficult to prevent and control in time. The many technical effects that can be produced by the preferred technical scheme of the present invention are described in detail below.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The preparation method of the microbial agent for preventing both root-knot nematode disease and clubroot disease of the present invention comprises the following steps:

[0010] Preparation of methylotrophic Bacillus powder and Bacillus subtilis powder.

[0011] Preparation of Paecilomyces lilacinus powder.

[0012] The prepared methylotrophic Bacillus powder, Paecilomyces lilacinus powder and Bacillus subtilis powder are compounded according to the following weight proportions: 3-8 parts of methylotrophic Bacillus powder, 1 part of Paecilomyces lilacinus powder and 2-5 parts of Bacillus subtilis powder, and mixed evenly to obtain a microbial agent for preventing both root-knot nematode disease and clubroot disease.

[0013] According to a preferred embodiment, the preparation of methylotrophic Bacillus powder and Bacillus subtilis powder comprises the following steps:

[0014] The methylotrophic Bacillus and Bacillus subtilis preserved by glycerol were taken out from -80°C, inoculated into liquid LB medium at a ratio of 3‰ to 5‰, and cultured under shaking at 35°C to 38°C for 24h to 48h to obtain seed liquids of methylotrophic Bacillus and Bacillus subtilis.

[0015] The activated seed liquid is inoculated into the first-stage culture medium at a ratio of 5% - 10% respectively for cultivation to obtain the first-stage seed liquid, and then the first-stage seed liquid is inoculated into the second-stage culture medium at a ratio of 60% - 70% by volume respectively for cultivation to obtain the second-stage seed liquid.

[0016] The second-stage seed liquid is respectively introduced into a disc centrifuge and centrifuged at a speed of 6000 r / min for 15 min. After centrifugation, the upper-layer bacterial suspension is removed to obtain two kinds of bacterial strain concentrates.

[0017] The two kinds of bacterial strain concentrates are respectively poured into a blender and 10% - 13% of auxiliary materials are added. The bacterial strain concentrate is pressed to a rotary nozzle under a pressure of 0.9 Pa, and the nozzle is kept spraying at a speed of 20000 r / min. At the same time, hot air at 150 °C and 4000 m 3 / h is introduced into the drying tower for drying. After spray drying, two kinds of bacillus microbial bacterial powders with an effective viable count of not less than 1×10 11 CFU / g are obtained.

[0018] The methylotrophic bacillus (Bacillus methylotrophicus) of the present invention has the number WSWFJ-29, the preservation registration number is CGMCC No. 18269, the preservation date is July 22, 2019, and the preservation unit is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Morphological characteristics of the methylotrophic bacillus WSWFJ-29: After culturing on an LB medium for 3 days under dark conditions, it shows a milky white round colony, the surface and edge are rough, with wrinkles, the colony is semi-transparent and relatively thick.

[0019] The bacillus subtilis of the present invention has the number WSWFJ44, the preservation registration number is CGMCC No. 20144, the preservation date is June 28, 2020, and the preservation unit is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Morphological characteristics of the bacillus subtilis WSWFJ44: After culturing on an LB medium for 3 days under dark conditions, it shows a slightly yellow colony, the colony shape is irregular, the colony surface is rough with wrinkles, the colony is opaque and relatively thick.

[0020] According to a preferred embodiment, the components of the primary culture medium are: soybean powder 3 - 7 g / L, corn flour 3 - 7 g / L, sucrose 8 - 16 g / L, peptone 5 - 15 g / L, fish meal 5 - 10 g / L, calcium carbonate 2 - 6 g / L, potassium dihydrogen phosphate 0.2 - 1.4 g / L, manganese sulfate 0.5 - 1.5 g / L, ammonium sulfate 1 - 1.5 g / L; the culture conditions of the activated seed liquid in the primary culture medium are: culture temperature 35°C - 37°C, culture time 17 h - 20 h, and the effective viable count is not less than 1×10 8 CFU / mL.

[0021] According to a preferred embodiment, the components of the secondary culture medium are: soybean powder 10 - 18 g / L, corn flour 2 - 6 g / L, sucrose 2 - 4 g / L, peptone 1 - 3 g / L, fish meal 1 - 3 g / L, calcium carbonate 4 - 8 g / L, potassium dihydrogen phosphate 0.1 - 0.5 g / L, magnesium sulfate 0.1 - 0.5 g / L, manganese sulfate 0.1 - 0.3 g / L, dipotassium hydrogen phosphate 0.1 - 0.5 g / L, sodium chloride 0.1 - 0.5 g / L, ammonium sulfate 0.2 - 0.8 g / L, sodium hydroxide 0.1 - 0.4 g / L; the culture conditions of the primary seed liquid in the secondary culture medium are: culture temperature 35°C - 37°C, oxygen supply 15 m 3 / h, culture time 23 h - 25 h, and the effective viable count is not less than 1×10 9 CFU / mL.

[0022] According to a preferred embodiment, the auxiliary materials include the following components in parts by weight: diatomaceous earth 55 - 65 parts, calcium carbonate 15 - 25 parts, starch 15 - 25 parts.

[0023] According to a preferred embodiment, the preparation of Paecilomyces lilacinus powder comprises the following steps:

[0024] Take Paecilomyces lilacinus preserved by the filter paper preservation method from the strain library, inoculate it into the PDA liquid medium, and shake culture for 72 h - 120 h at 20°C - 28°C to obtain the seed liquid.

[0025] Inoculate the activated strain into the primary liquid medium at a ratio of 10% - 20% to obtain the primary seed liquid, and then inoculate the primary seed liquid into the secondary liquid medium at a ratio of 60% - 70% to obtain the secondary seed culture solution.

[0026] Inoculate the secondary seed culture solution into the solid medium for culture, collect the Paecilomyces lilacinus spore powder, and obtain the Paecilomyces lilacinus powder.

[0027] The Paecilomyces lilacinus of the present invention has the number WSWFJ-28, the preservation registration number is CGMCC No. 18274, the preservation date is July 22, 2019, the preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, the preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The morphological characteristics of Paecilomyces lilacinus WSWFJ-28: Under dark conditions, after culturing on a PDA plate for 3 days, white circular colonies grow out, and gradually turn light pink after spore production. After culturing for 7 days, the colonies are purple-red, circular, raised, powdery, with no secretions on the surface. The more spores are produced, the darker the colony color is.

[0028] According to a preferred embodiment, the components of the first-stage liquid medium are: soluble starch 10-20 g / L, potassium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.2-0.8 g / L, magnesium sulfate heptahydrate 0.2-0.8 g / L, sodium chloride 0.2-0.8 g / L, ferric sulfate 0.01-0.03 g / L; the culture conditions of the activated strain in the first-stage liquid medium are: the inoculation amount of the activated strain is 0.5-1.5% of the total volume of the first-stage liquid medium, the culture time is 35-50 h, the culture temperature is 25-30 °C, and the initial pH value of the culture is 6.0-7.0; ventilation is carried out during the culture process. Within 24 h of culture, the ventilation volume is 10-20 m 3 / h. After 24 h of culture, the ventilation volume is 15-24 m 3 / h; the culture is carried out in the dark.

[0029] According to a preferred embodiment, the components of the second-stage liquid medium are: sucrose 10-80 g / L, soybean powder 1-30 g / L, sodium nitrate 0.1-20 g / L, dipotassium hydrogen phosphate 0.05-5 g / L, zinc sulfate 0.03-3 g / L, ferric sulfate 0.01-1 g / L; the culture conditions of the first-stage seed liquid in the second-stage liquid medium are: the inoculation amount of the first-stage seed liquid is 5-15% of the total volume of the second-stage liquid medium, the culture time is 55-75 h, the culture temperature is 25-30 °C, and the initial pH value of the culture is 6.0-7.0; ventilation is carried out during the culture process. Within 24 h of culture, the ventilation volume is 130-170 m 3 / h. After 24 h of culture, the ventilation volume is 200-260 m 3 / h; the culture is carried out under stirring, the stirring frequency is 1-2 times / h, and the stirring time each time is 10-20 min; the culture is carried out in the dark.

[0030] According to a preferred embodiment, the solid medium comprises the following components in parts by weight: 30-80 parts of tobacco stem and tobacco dust, 25-60 parts of mineral source humic acid, 1-5 parts of lime, 3-8 parts of fertilizer-grade calcium hydrogen phosphate, and 10-20 parts of secondary seed liquid; the culture conditions of the secondary seed culture solution in the solid medium are as follows: the ratio of solid matter to water in the raw material is 10:4-8, the natural pH of the raw material, the culture temperature is 26-28 °C, the fermentation time is 6-12 d, and the culture is carried out in the dark; at the end of fermentation, the spore count reaches 1×10 10 CFU / g.

[0031] The microbial inoculum for preventing both root-knot nematode disease and clubroot disease of the present invention is prepared by the preparation method described in any one of the technical solutions of the present invention.

[0032] The microbial inoculum for preventing both root-knot nematode disease and clubroot disease and the preparation method thereof provided by the present invention at least have the following beneficial technical effects:

[0033] The preparation method of the microbial inoculum for preventing both root-knot nematode disease and clubroot disease of the present invention comprises the following steps: preparing methylotrophic Bacillus powder and Bacillus subtilis powder; preparing Paecilomyces lilacinus powder; compounding the prepared methylotrophic Bacillus powder, Paecilomyces lilacinus powder and Bacillus subtilis powder according to the following weight parts: 3-8 parts of methylotrophic Bacillus powder, 1 part of Paecilomyces lilacinus powder, and 2-5 parts of Bacillus subtilis powder, and mixing them evenly to obtain the microbial inoculum for preventing both root-knot nematode disease and clubroot disease. The microbial inoculum obtained by this method, after being applied to the soil or substrate and under suitable conditions, the methylotrophic Bacillus with the function of preventing clubroot disease and the Bacillus subtilis with the functions of activating the soil and inhibiting soil-borne diseases can rapidly germinate and reproduce within 1-2 days, form Bacillus spore colonization in the soil, and enter the crop interior through the crop roots to become crop endophytes. The methylotrophic Bacillus can inhibit and kill Plasmodiophora brassicae in the soil and inside the crop, and the Bacillus subtilis can activate the nutrients in the soil, stimulate the growth of crop roots, so as to improve the absorption and utilization of water and fertilizer in the soil by the crop and promote the improvement of crop resistance; while the Paecilomyces lilacinus with the function of preventing root-knot nematode disease can reproduce into vegetative mycelium within 3-5 days and parasitize in the eggs of root-knot nematodes, effectively killing nematodes. At the same time, this bacterium can produce rich derivatives, and one of the products is similar to indole acetic acid. Its most significant physiological effect is to promote the growth of plant roots and plants at low concentrations. Therefore, applying the bacterial strain to the plant roots can not only significantly inhibit nematode infection, but also promote the growth of plant vegetative organs, and at the same time has a promoting effect on the germination and growth of seeds.

[0034] On the other hand, the preparation method of the microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention effectively guarantees the number of viable bacteria in the compound microbial inoculant. Compared with the liquid compound microbial inoculant, the volume of the product is greatly reduced, the transportation cost is effectively reduced, the mixing aging period of the inoculant is extended, and convenience is added to field production.

[0035] The microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention conducts unified prevention and control of these two diseases according to the occurrence characteristics and indistinguishable early characteristics of these two diseases, avoiding the golden period of early prevention and control being delayed due to incorrect pesticide selection in agricultural production. In some crops, these two diseases may occur simultaneously. Using this inoculant for prevention and control can act on both diseases simultaneously, greatly reducing agricultural losses. At the same time, the use of chemical pesticides is reduced to a certain extent, promoting the development of green agriculture. Using this inoculant for prevention and control, whether it is the single occurrence of clubroot disease or root-knot nematode disease, or the simultaneous occurrence of both, can be effectively prevented and controlled. At the same time, the problem of difficult rapid detection and differentiation of the two diseases in field production is avoided. It not only meets the requirements of rapid prevention and control and early prevention and control in agricultural production, but also avoids the pollution and residues caused by the abuse of pesticides. It is a new prevention and control mode that is economical, fast, effective, green and healthy.

[0036] On the other hand, the usage method of the microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention is diverse. It can be applied in holes as a powdery microbial inoculant, can be diluted with water after mixing for dipping roots, irrigating roots and watering, and can also be added as beneficial microorganisms to substrates and fertilizers for prevention and control during the seedling raising stage and fertilization stage, having good application and promotion value. Detailed implementation modes

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0038] The microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention and its preparation method will be described in detail below in combination with Examples 1 to 3.

[0039] Example 1

[0040] This example provides a microbial inoculant for preventing both root-knot nematode disease and clubroot disease. The specific preparation method is as follows:

[0041] The first step: Prepare methylotrophic bacillus powder and bacillus subtilis powder

[0042] Take out the methylotrophic Bacillus and Bacillus subtilis preserved with glycerol under the condition of -80°C, and inoculate them into the liquid LB medium at a ratio of 3‰ respectively. After shaking culture at 38°C for 24 h, the seed solutions of the two strains of bacteria are obtained. Inoculate the activated seed solutions into the primary medium at a ratio of 10% respectively for culture to obtain the primary seed solutions, and then inoculate the primary seed solutions into the secondary medium at a ratio of 60% by volume respectively for culture to obtain the secondary seed solutions. Then, introduce the secondary seed solutions into a disc centrifuge respectively, and centrifuge at a speed of 6000 r / min for 15 min. After centrifugation, remove the upper bacterial suspension to obtain two kinds of bacterial concentrate solutions. Pour the two kinds of bacterial concentrate solutions into a blender respectively, add 13% of auxiliary materials, press the bacterial concentrate solutions to a rotary nozzle under a pressure of 0.9 Pa, and keep the nozzle spraying at a speed of 20000 r / min. At the same time, introduce hot air at 150°C and 4000 m 3 / h into the drying tower for drying. After spray drying, two kinds of Bacillus microbial bacterial powders with an effective viable count of not less than 1×10 11 CFU / g are prepared.

[0043] Among them, the components of the primary medium are: soybean powder 3 g / L, corn flour 7 g / L, sucrose 8 g / L, peptone 15 g / L, fish meal 5 g / L, calcium carbonate 6 g / L, potassium dihydrogen phosphate 0.2 g / L, manganese sulfate 1.5 g / L, ammonium sulfate 1 g / L. The culture conditions of the activated seed solution in the primary medium are: culture temperature 37°C, culture time 17 h, and the effective viable count is not less than 1×10 8 CFU / mL.

[0044] The components of the secondary medium are: soybean powder 10 g / L, corn flour 6 g / L, sucrose 2 g / L, peptone 3 g / L, fish meal 1 g / L, calcium carbonate 8 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 0.1 g / L, ammonium sulfate 0.8 g / L, sodium hydroxide 0.1 g / L. The culture conditions of the primary seed solution in the secondary medium are: culture temperature 37°C, oxygen supply 15 m 3 / h, culture time 23 h, and the effective viable count is not less than 1×10 9 CFU / mL.

[0045] The auxiliary materials include the following components in parts by weight: 55 parts of diatomite, 25 parts of calcium carbonate, and 15 parts of starch.

[0046] Step 2: Prepare Paecilomyces lilacinus bacterial powder

[0047] Take Paecilomyces lilacinus preserved by the filter paper preservation method from the strain library, inoculate it into the PDA liquid medium, and obtain the seed liquid after shaking culture at 20 °C for 120 h. Inoculate the activated strain into the first-stage liquid medium at a ratio of 10%, obtain the first-stage seed liquid, and then inoculate the first-stage seed liquid into the second-stage liquid medium at a ratio of 70% by volume to obtain the second-stage seed culture solution. Inoculate the second-stage seed culture solution into the solid medium for culture, collect the Paecilomyces lilacinus spore powder, and obtain the Paecilomyces lilacinus powder.

[0048] Among them: The components of the first-stage liquid medium are: soluble starch 10 g / L, potassium nitrate 3 g / L, dipotassium hydrogen phosphate 0.2 g / L, magnesium sulfate heptahydrate 0.8 g / L, sodium chloride 0.2 g / L, ferric sulfate 0.03 g / L. The culture conditions of the activated strain in the first-stage liquid medium are: the inoculation amount is 0.5% of the total volume of the liquid medium, the culture time is 50 h, the culture temperature is 25 °C, and the initial pH value of the culture is 7.0; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 10 m 3 / h. After 24 h of culture, the aeration volume is 24 m 3 / h; The culture is carried out in the dark.

[0049] The components of the second-stage liquid medium are: sucrose 10 g / L, soybean powder 30 g / L, sodium nitrate 0.1 g / L, dipotassium hydrogen phosphate 5 g / L, zinc sulfate 0.03 g / L, ferric sulfate 1 g / L. The culture conditions of the first-stage seed liquid in the second-stage liquid medium are: the inoculation amount is 5% of the total volume of the liquid medium, the culture time is 75 h, the culture temperature is 25 °C, and the initial pH value of the culture is 7.0; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 130 m 3 / h. After 24 h of culture, the aeration volume is 260 m 3 / h; The culture is carried out under stirring, the stirring frequency is 1 time / h, and the stirring time for each time is 20 min; The culture is carried out in the dark.

[0050] The solid medium includes the following components in parts by weight: tobacco stem tobacco dust 30 parts, mineral source humic acid 60 parts, lime 1 part, fertilizer-grade calcium hydrogen phosphate 8 parts, and second-stage seed liquid 10 parts. The culture conditions of the second-stage seed culture solution in the solid medium are: the ratio of solid matter to water in the raw material is 10∶4, the natural pH of the raw material, the culture temperature is 28 °C, the fermentation time is 6 d, and the culture is carried out in the dark. At the end of fermentation, the spore amount reaches 1×10 10 CFU / g.

[0051] Step 3: Prepare the compound microbial agent

[0052] Mix the methylotrophic Bacillus powder, Paecilomyces lilacinus spore powder, and Bacillus subtilis powder prepared in the first and second steps in a physical mixing ratio of 3 parts: 1 part: 5 parts by weight. After mixing evenly, it is a microbial inoculant that can prevent both root-knot nematode disease and clubroot disease.

[0053] Furthermore: The three kinds of powder should be packaged separately and mixed during use. After mixing, it should be used within 48 hours to avoid the mutual influence between the strains due to too long mixing time, resulting in a reduction in the number of effective viable bacteria.

[0054] The microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention can be applied to crops by one or more methods of root dipping, root irrigation, watering, and hole application.

[0055] Example 2

[0056] This example provides a microbial inoculant for preventing both root-knot nematode disease and clubroot disease. The specific preparation method is as follows:

[0057] The first step: Prepare methylotrophic Bacillus powder and Bacillus subtilis powder.

[0058] Take out the methylotrophic Bacillus and Bacillus subtilis preserved with glycerol under the condition of -80°C, and inoculate them into the liquid LB medium at a ratio of 5‰ respectively. After shaking culture at 35°C for 48 hours, the seed solutions of the two strains are obtained. The activated seed solutions are inoculated into the first-stage medium for culture at a ratio of 5% respectively to obtain the first-stage seed solutions, and then the first-stage seed solutions are inoculated into the second-stage medium for culture at a ratio of 70% by volume respectively to obtain the second-stage seed solutions. Then, the second-stage seed solutions are respectively introduced into a disc centrifuge and centrifuged at a speed of 6000 r / min for 15 minutes. After centrifugation, the upper layer of the bacterial suspension is removed to obtain two kinds of bacterial strain concentrates. Pour the two kinds of bacterial strain concentrates into a blender respectively, add 10% of auxiliary materials, press the bacterial strain concentrates to a rotary nozzle under a pressure of 0.9 Pa, and keep the nozzle spraying at a speed of 20000 r / min. At the same time, hot air at 150°C and 4000 m 3 / h is introduced into the drying tower for drying. After spray drying, two kinds of Bacillus microbial powders with an effective viable bacteria number of not less than 1×10 11 CFU / g are obtained.

[0059] Among them, the components of the first-stage medium are: soybean powder 7 g / L, corn flour 3 g / L, sucrose 16 g / L, peptone 5 g / L, fish meal 10 g / L, calcium carbonate 2 g / L, potassium dihydrogen phosphate 1.4 g / L, manganese sulfate 0.5 g / L, ammonium sulfate 1.5 g / L. The culture conditions of the activated seed solution in the first-stage medium are: culture temperature 35°C, culture time 20 h, and the effective viable bacteria number is not less than 1×10 8CFU / mL.

[0060] The components of the secondary culture medium are: soybean powder 18 g / L, corn starch 2 g / L, sucrose 4 g / L, peptone 1 g / L, fish meal 3 g / L, calcium carbonate 4 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.3 g / L, dipotassium hydrogen phosphate 0.1 g / L, sodium chloride 0.5 g / L, ammonium sulfate 0.2 g / L, sodium hydroxide 0.4 g / L. The culture conditions of the primary seed liquid in the secondary culture medium are: culture temperature 35°C, oxygen supply 15 m 3 / h, culture time 25 h, and the number of viable bacteria is not less than 1×10 9 CFU / mL.

[0061] The excipients include the following components in parts by weight: diatomaceous earth 65 parts, calcium carbonate 15 parts, and starch 25 parts.

[0062] Step 2: Prepare Paecilomyces lilacinus powder

[0063] Take out Paecilomyces lilacinus preserved by the filter paper preservation method from the strain library and inoculate it into the PDA liquid medium. After shaking culture at 28°C for 72 h, a seed liquid is obtained. The activated strain is inoculated into the primary liquid medium at a ratio of 20% to obtain the primary seed liquid, and then the primary seed liquid is inoculated into the secondary liquid medium at a ratio of 60% by volume to obtain the secondary seed culture solution. The secondary seed culture solution is inoculated into the solid medium for culture, and the Paecilomyces lilacinus spore powder is collected to obtain Paecilomyces lilacinus powder.

[0064] Among them: The components of the primary liquid medium are: soluble starch 20 g / L, potassium nitrate 1 g / L, dipotassium hydrogen phosphate 0.8 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.8 g / L, ferric sulfate 0.01 g / L. The culture conditions of the activated strain in the primary liquid medium are: the inoculation amount is 1.5% of the total volume of the liquid medium, the culture time is 35 h, the culture temperature is 30°C, and the initial culture pH value is 6.0; ventilation is carried out during the culture process. Within 24 h of culture, the ventilation volume is 20 m 3 / h, after 24 h of culture, the ventilation volume is 15 m 3 / h; the culture is carried out in the dark.

[0065] The components of the secondary liquid medium are: sucrose 80 g / L, soybean powder 1 g / L, sodium nitrate 20 g / L, dipotassium hydrogen phosphate 0.05 g / L, zinc sulfate 3 g / L, ferric sulfate 0.01 g / L. The culture conditions of the primary seed liquid in the secondary liquid medium are: the inoculation amount is 15% of the total volume of the liquid medium, the culture time is 55 h, the culture temperature is 30°C, and the initial culture pH value is 6.0; ventilation is carried out during the culture process. Within 24 h of culture, the ventilation volume is 170 m 3 / h, after culturing for 24 h, the ventilation volume is 200 m 3 / h; The culturing is carried out under stirring, the stirring frequency is 2 times / h, and the time for each stirring is 10 min; The culturing is carried out in the dark.

[0066] The solid medium comprises the following components in parts by weight: 80 parts of tobacco stem and tobacco dust, 25 parts of mineral source humic acid, 5 parts of lime, 3 parts of fertilizer-grade calcium hydrogen phosphate, and 20 parts of secondary seed liquid. The culturing conditions of the secondary seed culture solution in the solid medium are: the ratio of solid matter to water in the raw material is 10∶8, the natural pH of the raw material, the culturing temperature is 26 °C, the fermentation time is 12 d, and the culturing is carried out in the dark. At the end of fermentation, the spore amount reaches 1×10 10 CFU / g.

[0067] The third step: Prepare the compound microbial agent

[0068] The methylotrophic bacillus powder, Paecilomyces lilacinus spore powder, and Bacillus subtilis powder prepared in the first step and the second step are physically mixed in a weight ratio of 8 parts∶1 part∶2 parts, and after mixing evenly, it is a microbial agent for jointly preventing root-knot nematode disease and clubroot disease.

[0069] Furthermore: The three kinds of powder should be packaged separately, mixed when in use, and should be used within 48 h after mixing to avoid the mutual influence between the strains due to too long mixing time, resulting in a reduction in the number of effective viable bacteria.

[0070] The microbial agent for jointly preventing root-knot nematode disease and clubroot disease of the present invention can be formulated with a substrate into a functional seedling-raising substrate for use, and the prevention and control of diseases can be carried out at the seedling-raising stage, which can effectively reduce the outbreak of diseases after crop transplantation.

[0071] Example 3

[0072] This example provides a microbial agent for jointly preventing root-knot nematode disease and clubroot disease, and the specific preparation method is as follows:

[0073] The first step: Prepare methylotrophic bacillus powder and Bacillus subtilis powder.

[0074] The methylotrophic Bacillus and Bacillus subtilis preserved with glycerol were taken out under the condition of -80°C and inoculated into liquid LB medium at a ratio of 4‰ respectively. After shaking culture at 37°C for 35 h, the seed solutions of the two strains of bacteria were obtained. The activated seed solutions were inoculated into the first-stage medium for culture at a ratio of 8% respectively to obtain the first-stage seed solutions, and then the first-stage seed solutions were inoculated into the second-stage medium for culture at a ratio of 65% by volume respectively to obtain the second-stage seed solutions. Then the second-stage seed solutions were respectively introduced into a disc centrifuge and centrifuged at a speed of 6000 r / min for 15 min. After centrifugation, the upper-layer bacterial suspension was removed to obtain two kinds of bacterial concentrate solutions. The two kinds of bacterial concentrate solutions were poured into a blender, 12% of auxiliary materials were added, and the bacterial concentrate solutions were pressed to a rotary nozzle under a pressure of 0.9 Pa, and the nozzle was kept spraying at a speed of 20000 r / min. At the same time, hot air at 150°C and 4000 m 3 / h was introduced into the drying tower for drying. After spray drying, two kinds of Bacillus microbial bacterial powders with an effective viable count of not less than 1×10 11 CFU / g were prepared.

[0075] Among them, the components of the first-stage medium are: soybean powder 5 g / L, corn flour 5 g / L, sucrose 12 g / L, peptone 10 g / L, fish meal 7 g / L, calcium carbonate 4 g / L, potassium dihydrogen phosphate 0.8 g / L, manganese sulfate 1 g / L, ammonium sulfate 1.2 g / L. The culture conditions of the activated seed solution in the first-stage medium are: culture temperature 36°C, culture time 18 h, and the effective viable count is not less than 1×10 8 CFU / mL.

[0076] The components of the second-stage medium are: soybean powder 14 g / L, corn flour 4 g / L, sucrose 3 g / L, peptone 2 g / L, fish meal 2 g / L, calcium carbonate 6 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.2 g / L, dipotassium hydrogen phosphate 0.4 g / L, sodium chloride 0.3 g / L, ammonium sulfate 0.6 g / L, sodium hydroxide 0.2 g / L. The culture conditions of the first-stage seed solution in the second-stage medium are: culture temperature 36°C, oxygen supply 15 m 3 / h, culture time 24 h, and the effective viable count is not less than 1×10 9 CFU / mL.

[0077] The auxiliary materials include the following components in parts by weight: 60 parts of diatomite, 20 parts of calcium carbonate, and 22 parts of starch.

[0078] Step 2: Prepare Paecilomyces lilacinus bacterial powder

[0079] Take the Paecilomyces lilacinus preserved by the filter paper preservation method from the strain library, inoculate it into the PDA liquid medium, and obtain the seed liquid after shaking culture at 25 °C for 90 h. Inoculate the activated strain into the first-stage liquid medium at a ratio of 15%, obtain the first-stage seed liquid, and then inoculate the first-stage seed liquid into the second-stage liquid medium at a ratio of 66% by volume to obtain the second-stage seed culture solution. Inoculate the second-stage seed culture solution into the solid medium for culture, collect the Paecilomyces lilacinus spore powder, and obtain the Paecilomyces lilacinus powder.

[0080] Among them, the components of the first-stage liquid medium are: soluble starch 15 g / L, potassium nitrate 2 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium chloride 0.5 g / L, ferric sulfate 0.02 g / L. The culture conditions of the activated strain in the first-stage liquid medium are: the inoculation amount is 1% of the total volume of the liquid medium, the culture time is 40 h, the culture temperature is 28 °C, and the initial pH value of the culture is 6.5; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 15 m 3 / h. After 24 h of culture, the aeration volume is 19 m 3 / h; the culture is carried out in the dark.

[0081] The components of the second-stage liquid medium are: sucrose 50 g / L, soybean powder 10 g / L, sodium nitrate 15 g / L, dipotassium hydrogen phosphate 2 g / L, zinc sulfate 1 g / L, ferric sulfate 0.5 g / L. The culture conditions of the first-stage seed liquid in the second-stage liquid medium are: the inoculation amount is 10% of the total volume of the liquid medium, the culture time is 60 h, the culture temperature is 28 °C, and the initial pH value of the culture is 6.5; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 150 m 3 / h. After 24 h of culture, the aeration volume is 230 m 3 / h; the culture is carried out under stirring, the stirring frequency is 2 times / h, and the stirring time for each time is 15 min; the culture is carried out in the dark.

[0082] The solid medium includes the following components in parts by weight: tobacco stem tobacco dust 50 parts, mineral source humic acid 40 parts, lime 3 parts, fertilizer-grade calcium hydrogen phosphate 5 parts, and second-stage seed liquid 15 parts. The culture conditions of the second-stage seed culture solution in the solid medium are: the ratio of solid matter to water in the raw material is 10∶6, the natural pH of the raw material, the culture temperature is 27 °C, the fermentation time is 10 d, and the culture is carried out in the dark. At the end of fermentation, the spore amount reaches 1×10 10 CFU / g.

[0083] Step 3: Prepare the compound microbial agent

[0084] The methylotrophic Bacillus powder, Paecilomyces lilacinus spore powder, and Bacillus subtilis powder prepared in the first and second steps are physically mixed in a weight ratio of 5 parts: 1 part: 4 parts. After mixing evenly, it is a microbial inoculant for preventing both root-knot nematode disease and clubroot disease.

[0085] Furthermore: The three kinds of powder should be packaged separately and mixed during use. After mixing, it should be used within 48 hours to avoid the mutual influence between the strains due to too long mixing time, resulting in a reduction in the number of effective viable bacteria.

[0086] The microbial inoculant for preventing both root-knot nematode disease and clubroot disease of the present invention can be compatible with fertilizers to make functional bio-fertilizers. It enters the soil when applying the base fertilizer and controls diseases during crop transplantation, which can effectively reduce the outbreak of diseases after crop transplantation.

[0087] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. Preparation method of microbial inoculum for preventing root-knot nematode disease and clubroot disease simultaneously Characterized in that: It includes the following steps: Prepare Bacillus methylotrophicus bacterial powder and Bacillus subtilis bacterial powder; wherein, the Bacillus methylotrophicus has the number WSWFJ-29 and the preservation registration number CGMCC No. 18269; the Bacillus subtilis has the number WSWFJ44 and the preservation registration number CGMCC No. 20144; Prepare Paecilomyces lilacinus bacterial powder, and the Paecilomyces lilacinus has the number WSWFJ-28 and the preservation registration number CGMCC No. 18274; Mix the prepared Bacillus methylotrophicus bacterial powder, Paecilomyces lilacinus bacterial powder and Bacillus subtilis bacterial powder according to the following weight parts: 3-8 parts of Bacillus methylotrophicus bacterial powder, 1 part of Paecilomyces lilacinus bacterial powder, 2-5 parts of Bacillus subtilis bacterial powder. After mixing, a microbial inoculum for preventing root-knot nematode disease and clubroot disease simultaneously is obtained; The preparation of Paecilomyces lilacinus bacterial powder includes the following steps: First, take out the Paecilomyces lilacinus preserved by the filter paper preservation method from the strain library, inoculate it into the PDA liquid medium, and shake and culture it at 20°C - 28°C for 72h - 120h to obtain a seed liquid; Secondly, inoculate the activated strain into the first-stage liquid medium according to the ratio of 10% - 20% to obtain the first-stage seed liquid, and then inoculate the first-stage seed liquid into the second-stage liquid medium according to the ratio of 60% - 70% to obtain the second-stage seed culture solution; Inoculate the second-stage seed culture solution into the solid medium for culture, collect the Paecilomyces lilacinus spore powder, and obtain the Paecilomyces lilacinus bacterial powder.

2. The preparation method of the microbial inoculum for preventing root-knot nematode disease and clubroot disease simultaneously according to claim 1, Characterized in that: The preparation of Bacillus methylotrophicus bacterial powder and Bacillus subtilis bacterial powder includes the following steps: Take out the Bacillus methylotrophicus and Bacillus subtilis preserved by glycerol at -80°C, inoculate them into the liquid LB medium according to the ratio of 3‰ - 5‰ respectively, and shake and culture them at 35°C - 38°C for 24h - 48h to obtain the seed liquids of Bacillus methylotrophicus and Bacillus subtilis; Inoculate the activated seed liquids into the first-stage medium according to the ratio of 5% - 10% respectively for culture to obtain the first-stage seed liquids, and then inoculate the first-stage seed liquids into the second-stage medium according to the volume ratio of 60% - 70% respectively for culture to obtain the second-stage seed liquids; Respectively introduce the second-stage seed liquids into a disc centrifuge, centrifuge at a speed of 6000r / min for 15min, and after centrifugation, remove the upper-layer bacterial suspension to obtain two kinds of concentrated bacterial solutions; Pour the two kinds of concentrated bacterial liquid into a blender respectively and add 10% - 13% of auxiliary materials. Press the concentrated bacterial liquid to a rotary nozzle through a pressure of 0.9 Pa, and keep the nozzle spraying at a rotation speed of 20,000 r / min. At the same time, introduce hot air at 150 °C and 4,000 m 3 / h for drying in a drying tower. After spray drying, two kinds of bacillus microbial bacterial powders with the effective viable count not less than 1×10 11 CFU / g are obtained; Among them, the components of the primary medium are: soybean powder 3 - 7 g / L, corn flour 3 - 7 g / L, sucrose 8 - 16 g / L, peptone 5 - 15 g / L, fish meal 5 - 10 g / L, calcium carbonate 2 - 6 g / L, potassium dihydrogen phosphate 0.2 - 1.4 g / L, manganese sulfate 0.5 - 1.5 g / L, ammonium sulfate 1 - 1.5 g / L; The culture conditions of the activated seed liquid in the first-stage culture medium are as follows: the culture temperature is 35°C to 37°C, the culture time is 17h to 20h, and the number of viable bacteria is not less than 1×10 8 CFU / mL; The components of the secondary medium are: soybean powder 10 - 18 g / L, corn flour 2 - 6 g / L, sucrose 2 - 4 g / L, peptone 1 - 3 g / L, fish meal 1 - 3 g / L, calcium carbonate 4 - 8 g / L, potassium dihydrogen phosphate 0.1 - 0.5 g / L, magnesium sulfate 0.1 - 0.5 g / L, manganese sulfate 0.1 - 0.3 g / L, dipotassium hydrogen phosphate 0.1 - 0.5 g / L, sodium chloride 0.1 - 0.5 g / L, ammonium sulfate 0.2 - 0.8 g / L, sodium hydroxide 0.1 - 0.4 g / L; The culture conditions of the primary seed liquid in the secondary culture medium are as follows: the culture temperature is 35°C to 37°C, the oxygen supply is 15 m 3 / h, the culture time is 23 h to 25 h, and the number of viable bacteria is not less than 1×10 9 CFU / mL.

3. The preparation method of the microbial inoculum for preventing both root - knot nematode disease and clubroot disease according to claim 1, characterized in that, The auxiliary materials include the following components in parts by weight: diatomite 55 - 65 parts, calcium carbonate 15 - 25 parts, starch 15 - 25 parts.

4. The preparation method of the microbial inoculum for preventing both root - knot nematode disease and clubroot disease according to claim 1, characterized in that, The components of the primary liquid medium are: soluble starch 10 - 20 g / L, potassium nitrate 1 - 3 g / L, dipotassium hydrogen phosphate 0.2 - 0.8 g / L, magnesium sulfate heptahydrate 0.2 - 0.8 g / L, sodium chloride 0.2 - 0.8 g / L, ferric sulfate 0.01 - 0.03 g / L; The culture conditions of the activated bacterial strain in the first-stage liquid medium are as follows: the inoculation amount of the activated bacterial strain is 0.5-1.5% of the total volume of the first-stage liquid medium, the culture time is 35-50 h, the culture temperature is 25-30 °C, and the initial pH value of the culture is 6.0-7.0; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 10-20 m 3 / h. After 24 h of culture, the aeration volume is 15-24 m 3 / h; the culture is carried out in the dark.

5. The preparation method of the microbial inoculum for preventing both root - knot nematode disease and clubroot disease according to claim 1, characterized in that, The components of the secondary liquid medium are: sucrose 10 - 80 g / L, soybean powder 1 - 30 g / L, sodium nitrate 0.1 - 20 g / L, dipotassium hydrogen phosphate 0.05 - 5 g / L, zinc sulfate 0.03 - 3 g / L, ferric sulfate 0.01 - 1 g / L; The culture conditions of the primary seed liquid in the secondary liquid medium are as follows: the inoculation amount of the primary seed liquid is 5-15% of the total volume of the secondary liquid medium, the culture time is 55-75 h, the culture temperature is 25-30 °C, and the initial pH value of the culture is 6.0-7.0; aeration is carried out during the culture process. Within 24 h of culture, the aeration volume is 130-170 m 3 / h. After 24 h of culture, the aeration volume is 200-260 m 3 / h; the culture is carried out under stirring, the stirring frequency is 1-2 times / h, and the stirring time for each time is 10-20 min; the culture is carried out in the dark.

6. The preparation method of the microbial inoculum for preventing both root - knot nematode disease and clubroot disease according to claim 1, characterized in that, The solid medium includes the following components in parts by weight: tobacco stem and tobacco dust 30 - 80 parts, mineral - source humic acid 25 - 60 parts, lime 1 - 5 parts, fertilizer - grade calcium hydrogen phosphate 3 - 8 parts, secondary seed liquid 10 - 20 parts; The culture conditions of the secondary seed culture solution in the solid medium are as follows: the ratio of solid substances to water in the raw material is 10∶4 - 8, the natural pH of the raw material, the culture temperature is 26 - 28 °C, the fermentation time is 6 - 12 d, and the culture is carried out in the dark; at the end of fermentation, the spore count reaches 1×10 10 CFU / g.

7. A microbial inoculum for preventing both root - knot nematode disease and clubroot disease, characterized in that, The microbial inoculum is prepared by the preparation method described in any one of claims 1 to 6.