A microbial agent for preventing and controlling soil-borne diseases of ginger, and its preparation method and application

Abstract

The present invention discloses a microbial agent for preventing and treating soil-borne diseases of ginger, and a preparation method and application thereof, and belongs to the field of microbial technology. The microbial agent of the present invention comprises disease-resistant growth-promoting microorganisms, microbial activity promoters and auxiliary carriers, and the dosage ratio of the three is 10mL:100mL:(30-50)g. The microbial agent of the present invention can effectively prevent and treat ginger blight of ginger, and at the same time can increase the resistance of ginger, promote the growth of ginger, and improve the quality of ginger. At the same time, the strain resources of plant growth-promoting bacteria are enriched. The agent is pollution-free and has no toxic side effects, has strong pertinence, can be stored for a long time after being made into a solid agent, is convenient for field application, and can also effectively improve the soil environment, thereby effectively meeting the nutritional needs of crop growth, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent for preventing and controlling soil-borne diseases of ginger, and a preparation method and application thereof. Background Art

[0002] Ginger contains rich and diverse nutrients, including polysaccharides, fats, proteins, vitamin C and various trace elements. In addition, the rhizomes of ginger are also rich in various functional compounds, such as gingerol, shogaol and ginger essential oil.

[0003] Ginger bacterial wilt, also known as ginger blight, is a vascular soil-borne disease caused by Ralstonia solanacearum. Ralstonia solanacearum is characterized by wide distribution, many species, diverse hosts, and complex infection pathways. The pathogen is easily carried by other hosts in the field, overwinters in the soil, and can also cause disease in other parts of the body. The roots, stems, leaves and other parts of ginger can be infected. After being infected, the ginger pieces initially appear water-soaked and yellow-brown, and later the inside gradually softens and rots. When the diseased part is squeezed, dirty white juice flows out with a foul smell; the stems are infected and dark purple, and the internal tissues turn brown and rot; the leaves are infected, the leaf surface loses its luster, and then gradually turns light yellow, the edges curl, and finally withers and dies.

[0004] Since the disease is soil-borne, it is difficult for conventional chemical agents to play a sustained disease control role in the soil. In the prior art, for example, Chinese patent application CN201410594132.1 relates to a ginger seed treatment agent for preventing ginger blight, which contains active ingredients, inducers and adjuvants, and the active ingredient is a composite composition of pyrimidine nucleoside antibiotics and thiophanate-methyl. It uses a large number of fungicides with chemical components. If used for a long time, crops are very likely to develop resistance and the drug effect is not long-lasting.

[0005] Although crop rotation can reduce the occurrence of ginger blight, a longer rotation cycle is required to achieve better disease prevention effects.

[0006] The use of biological control microorganisms to control ginger wilt is an effective prevention and control method. At present, the biological control of ginger wilt mostly uses a combination of various commercially available microorganisms or fungicides. Although they have broad-spectrum bactericidal and antibacterial effects, they are poorly targeted, have a single function, and are used in large quantities and at high costs. In actual planting, it is difficult to effectively use and promote them, and they are not of practical significance. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention selects a highly effective biocontrol strain-Pseudomonas NTO4, and adds plant extract components, which can effectively inhibit bacteria, promote the growth of ginger, and regulate the soil environment.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0009] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:(30-50) g.

[0010] Furthermore, the disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp.NTO4 ) and Bacillus marineus ( Celeribacter marinus ), the volume ratio of the two is 1:1.

[0011] Furthermore, the Pseudomonas sp. NTO4 ( Pseudomonas sp.NTO4 )’s deposit number is CCTCC NO: M20221479, the deposit date is September 23, 2022, and it is deposited in the China Type Culture Collection in Wuhan, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0012] Furthermore, the Pseudomonas NTO4 of the present invention is isolated from the river sludge of Yihe Road, Linyi City, Shandong Province, and the separation and screening method is: the sludge is dried and placed in a sterilized container, 10g of the sludge soil sample is added to 50mL of sterile water, and after 2h of shaking culture on a shaker at 200r / min, a suspension is filtered using sterile gauze and a 10-fold serial dilution method is used to obtain a 10-fold dilution. 100μL of the dilution is evenly spread on an LB plate, and after culturing in a constant temperature incubator at 25-30℃ for 48h, single colonies with different colors, glossiness, sizes and types are picked, the strains are purified by streaking, and after uniform numbering, they are transferred to LB slant culture medium, preserved with 80% sterilized glycerol, and stored at -70℃ for standby use.

[0013] Plate confrontation method: Select a 2-day cultured ginger blast pathogen - Ralstonia solanacearum bacterial cake (5 mm in diameter) and place it in the center of the PDA plate. Punch holes (5 mm in diameter) at three points 15 mm away from the bacterial cake in a three-point symmetric pattern and inject 100 μL of the fermentation culture medium of the test strain, and add 100 μL NB as a control. After culturing at 28-30℃ for 48 hours, observe and measure the size of the inhibition zone. Repeat the experiment three times and select the strain with the largest inhibition zone, which is Pseudomonas NTO4.

[0014] Biological characteristics: The colonies on the plate culture medium are white, translucent, moist, smooth, non-fluorescent, and Gram-negative. According to the description of the morphological characteristics and physiological and biochemical characteristics of Pseudomonas in the Manual of Systematic Identification of Common Bacteria, the strain Pseudomonas NTO4 meets the characteristics of Pseudomonas. Figure 1 This is the colony image of Pseudomonas NTO4.

[0015] Furthermore, the strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which was purchased from the General Microbiology Center of the China Culture Collection Administration, and the original preservation date is September 20, 2016. The strain can be purchased through the ordering process of the General Microbiology Center of the China Culture Collection Administration, without the need for biological preservation.

[0016] Furthermore, the preparation method of the disease-resistant and growth-promoting microorganisms is:

[0017] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 10-12 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0018] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0019] Furthermore, the composition of the seed culture medium in step (1) is: 10.0 g·L tryptone –1 , yeast extract powder 5.0g•L –1 , NaCl10.0g•L –1 , pH 7.2-7.4, high temperature sterilization at 121℃ for 20min.

[0020] Furthermore, the method for activating Pseudomonas NTO4 and Marine Fast-growing Bacillus in step (1) is as follows: the target strains stored in cryopreservation are inoculated in Erlenmeyer flasks containing 25 mL of LB liquid culture medium, and the culture medium is incubated at 25-30°C and 200 r·min. -1 The cells were cultured under the same conditions for 12 h to complete the activation.

[0021] Furthermore, the composition of the fermentation medium in step (2) is: glucose 20.0 g·L –1 , peptone 10.0 g•L –1 , KH2PO41.0g•L –1 ,MgSO41.0g•L –1 ,MnSO4·H2O 0.2 g·L –1 , sterilize at 115℃ for 20min.

[0022] Furthermore, the preparation method of the microbial activity promoter is as follows: the mushroom solid powder is mixed with distilled water at a mass ratio of 1:30, the pH is adjusted to 9-10 with 1 mol / L sodium hydroxide, and the mixture is placed in a water bath at 45-50°C for 1-2 hours. The solid powder waste is centrifuged at 12000 r / min for 20 minutes to obtain supernatant A, the solid powder waste is recovered and mixed with distilled water at a mass ratio of 1:30, the pH is adjusted to 9-10 with 1 mol / L sodium hydroxide again, the supernatant A and the supernatant B are mixed, and 1% chitosan by mass of the mixed liquid is added after ultrasonic extraction for 1-2 hours and centrifuged at 12000 r / min for 20 minutes to obtain the microbial activity promoter.

[0023] Furthermore, the mushroom powder is obtained by drying, crushing and grinding Pleurotus ostreatus or Flammulina velutipes.

[0024] Furthermore, the auxiliary carrier is diatomaceous earth, humic acid, sucrose, and biochar, with a mass ratio of 5:0.5:1:10.

[0025] A method for preparing a microbial agent for preventing and controlling soil-borne diseases of ginger comprises the following preparation steps:

[0026] (1) preparing disease-resistant and growth-promoting microorganisms;

[0027] (2) preparing a microbial activity promoter;

[0028] (3) Mix the disease-resistant and growth-promoting microorganisms and the microbial activity promoter in proportion to obtain a mixed solution, add an auxiliary carrier and let it stand for 3-5 hours to allow the carrier to fully absorb the mixed solution, and finally granulate and dry to obtain the product.

[0029] Application of a microbial agent for preventing and controlling soil-borne diseases of ginger in ginger cultivation.

[0030] The method for using the microbial agent of the present invention is to apply it in the sowing furrow before sowing ginger, the fertilization depth is 3-5 cm below the ginger seeds, and the application amount is 10 kg / mu.

[0031] Beneficial effects:

[0032] (1) The present invention screened out a Pseudomonas NTO4 from sludge, which has the functions of antagonizing ginger root rot pathogens, dissolving phosphate, secreting proteases, secreting auxins, and significantly promoting the growth of ginger. After being mixed and fermented with marine fast-growing Bacillus in equal proportions, it exhibits obvious synergistic antibacterial and growth-promoting effects, and the inhibitory effect on the pathogen Ralstonia solanacearum is enhanced; at the same time, Pseudomonas NTO4 and marine fast-growing Bacillus are highly effective in inhibiting bacteria. After being used as biocontrol agents to treat ginger, the activity of plant defense enzymes is increased, thereby inducing ginger to produce systemic resistance, thereby fundamentally enhancing the disease resistance of ginger;

[0033] (2) Secondly, the present invention extracts active substances from mushrooms as microbial promoters. On the one hand, the mushroom extract also contains nutrients such as polysaccharides and small molecule proteins. These ingredients may promote the growth of microorganisms and enhance the activity of microorganisms by affecting the metabolic pathways of microorganisms or providing nutritional support. On the other hand, the mushroom extract contains many nutrients required by plants. The residual mycelium can also secrete some enzymes during its growth and development, which can promote biochemical reactions. Therefore, more nutrients that are easily absorbed by plants can be released from complex organic matter, and it can activate the soil.

[0034] (3) Finally, diatomaceous earth, humic acid, sucrose and biochar were used as microbial carriers. Diatomaceous earth, due to its unique porous structure and high porosity, can provide a good growth environment for microorganisms, which is conducive to the reproduction and survival of microorganisms; humic acid can provide carbon and nitrogen sources for soil microorganisms, promoting the number, type and diversity of microorganisms; biochar is rich in organic carbon and contains nutrients such as nitrogen, phosphorus and potassium, has high porosity and water retention, and can provide microorganisms with rich nutrients and a suitable living environment. At the same time, biochar can improve the physical and chemical properties of the soil, such as increasing soil aggregation, improving pH, and increasing cation exchange capacity (CEC) and enzyme activity, thereby providing a better habitat for microorganisms;

[0035] (4) In summary, the microbial agent of the present invention can effectively prevent and treat ginger blight, and at the same time can increase the resistance of ginger, promote the growth of ginger, and improve the quality of ginger. At the same time, it enriches the strain resources of plant growth-promoting bacteria. The agent is pollution-free and has no toxic side effects. It has strong pertinence. After being made into a solid agent, it can be stored for a long time, which is convenient for field application. It can also effectively improve the soil structure, thereby effectively meeting the nutritional needs of crop growth, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a colony diagram of Pseudomonas NTO4 of the present invention;

[0037] Figure 2 The antibacterial properties of disease-resistant and growth-promoting microorganisms in the experimental groups of the examples and comparative examples;

[0038] Figure 3 is the SOD activity of ginger in different treatment groups;

[0039] Figure 4 is the POD activity of ginger in different treatment groups;

[0040] Figure 5 CAT activity of ginger in different treatment groups;

[0041] Figure 6 is the PAL activity of ginger in different treatment groups;

[0042] Figure 7 is the PPO activity of ginger in different treatment groups;

[0043] Figure 8 is the MDA concentration of ginger in different treatment groups. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0045] Example 1

[0046] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:50 g.

[0047] The disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ) and Bacillus marineus ( Celeribacter marinus ), the volume ratio of the two is 1:1.

[0048] The deposit number of the Pseudomonas sp. NTO4 is CCTCC NO: M20221479, and it is deposited in the China Center for Type Culture Collection in Wuhan.

[0049] In this embodiment, Pseudomonas NTO4 was isolated from the river sludge of Yihe Road, Linyi City, Shandong Province. The separation and screening method was as follows: the sludge was dried and placed in a sterilized container, 10 g of the sludge soil sample was added to 50 mL of sterile water, and after 2 hours of shaking culture on a shaker at 200 r / min, a suspension was filtered using sterile gauze and a 10-fold serial dilution method was used to obtain a 10-fold dilution. 100 μL of the dilution was evenly spread on an LB plate, and after culturing in a constant temperature incubator at 25-30°C for 48 hours, single colonies with different colors, glossiness, sizes and types were picked, the strains were purified by streaking, and after being uniformly numbered, they were transferred to LB slant culture medium, preserved with 80% sterilized glycerol, and stored at -70°C for standby use.

[0050] Plate confrontation method: Select ginger blast disease and Ralstonia solanacearum cake (5mm in diameter) cultured for 2 days and place it in the center of the PDA plate. Punch holes (5mm in diameter) at three points 15mm away from the cake and inject 100μL of the fermentation culture medium of the test strain. Add 100μL NB as a control. After culturing at 28-30℃ for 48h, observe and measure the size of the inhibition zone. Repeat the experiment three times and select the strain with the largest inhibition zone, which is Pseudomonas NTO4.

[0051] Biological characteristics: The colonies on the plate culture medium are white, translucent, moist, smooth, non-fluorescent, and Gram-negative. According to the description of the morphological characteristics and physiological and biochemical characteristics of Pseudomonas in the Manual of Systematic Identification of Common Bacteria, the strain Pseudomonas NTO4 meets the characteristics of Pseudomonas.

[0052] The strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which was purchased from the General Microbiology Center of the China Culture Collection Administration. The original preservation date is September 20, 2016. The strain can be purchased through the ordering process of the General Microbiology Center of the China Culture Collection Administration, without the need for biological preservation.

[0053] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0054] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 10 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0055] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0056] Step (1) The composition of the seed culture medium is: 10.0 g·L tryptone –1 , yeast extract powder 5.0 g•L –1 , NaCl10.0g•L –1 , pH 7.2-7.4, high temperature sterilization at 121℃ for 20min.

[0057] Step (1) The method for activating Pseudomonas NTO4 and Marine Fast-growing Bacillus is as follows: the target strains stored in cryopreservation are inoculated into Erlenmeyer flasks containing 25 mL of LB liquid culture medium, and the culture medium is incubated at 25-30°C and 200 r·min. -1 The cells were cultured under the same conditions for 12 h to complete the activation.

[0058] Step (2) The composition of the fermentation medium is: glucose 20.0 g·L –1 , peptone 10.0 g•L –1 , KH2PO41.0g•L –1,MgSO41.0g•L –1 ,MnSO4·H2O 0.2 g·L –1 , sterilize at 115℃ for 20min.

[0059] The preparation method of the microbial activity promoter is as follows: mushroom solid powder is mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide, the mixture is placed in a water bath at 45-50°C for extraction for 1 hour, supernatant A is obtained after centrifugation at 12000 r / min for 20 minutes, solid powder waste is recovered and mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide again, supernatant B is obtained after ultrasonic extraction for 1 hour, supernatant A and supernatant B are mixed, chitosan of 1% by mass of the mixed solution is added after centrifugation at 12000 r / min for 20 minutes, and the mixture is stirred and mixed evenly to obtain the microbial activity promoter.

[0060] Mushroom powder is obtained by drying, crushing and grinding Pleurotus ostreatus.

[0061] The auxiliary carrier is diatomaceous earth, humic acid, sucrose and biochar, and the mass ratio is 5:0.5:1:10.

[0062] A method for preparing a microbial agent for preventing and controlling soil-borne diseases of ginger comprises the following preparation steps:

[0063] (1) preparing disease-resistant and growth-promoting microorganisms;

[0064] (2) preparing a microbial activity promoter;

[0065] (3) The disease-resistant and growth-promoting microorganisms and the microbial activity promoter are mixed uniformly in proportion to obtain a mixed solution, an auxiliary carrier is added and allowed to stand for 3 hours to allow the carrier to fully absorb the mixed solution, and finally granulated and dried to obtain the product.

[0066] Example 2

[0067] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:40 g.

[0068] The disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ) and Bacillus marineus Celeribacter marinus ), the volume ratio of the two is 1:1.

[0069] The deposit number of the Pseudomonas sp. NTO4 is CCTCC NO: M20221479, and it is deposited in the China Center for Type Culture Collection in Wuhan.

[0070] The isolation and screening method of Pseudomonas NTO4 in this example is the same as that in Example 1.

[0071] The strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date is September 20, 2016.

[0072] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0073] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 12 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0074] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0075] Step (1) The composition of the seed culture medium is: 10.0 g·L tryptone –1 , yeast extract powder 5.0 g•L –1 , NaCl10.0g•L –1 , pH 7.2-7.4, high temperature sterilization at 121℃ for 20min.

[0076] Step (1) The method for activating Pseudomonas NTO4 and Marine Fast-growing Bacillus is as follows: the target strains stored in cryopreservation are inoculated into Erlenmeyer flasks containing 25 mL of LB liquid culture medium, and the culture medium is incubated at 25-30°C and 200 r·min. -1 The cells were cultured under the same conditions for 12 h to complete the activation.

[0077] Step (2) The composition of the fermentation medium is: glucose 20.0 g·L –1 , peptone 10.0 g•L –1 , KH2PO41.0g•L –1 ,MgSO41.0g•L –1 ,MnSO4·H2O 0.2 g·L –1 , sterilize at 115℃ for 20min.

[0078] The preparation method of the microbial activity promoter is as follows: mushroom solid powder is mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide, the mixture is placed in a water bath at 45-50°C for leaching for 1.5 hours, centrifuged at 12000 r / min for 20 minutes, and supernatant A is obtained, solid powder waste is recovered and mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide again, supernatant A and supernatant B are obtained after ultrasonic leaching for 1.5 hours, centrifuged at 12000 r / min for 20 minutes, and supernatant A and supernatant B are mixed, chitosan of 1% by mass of the mixed solution is added, and the mixture is stirred and mixed evenly to obtain the microbial activity promoter.

[0079] Mushroom solid powder is obtained by drying, crushing and grinding Flammulina velutipes.

[0080] The auxiliary carrier is diatomaceous earth, humic acid, sucrose and biochar, and the mass ratio is 5:0.5:1:10.

[0081] A method for preparing a microbial agent for preventing and controlling soil-borne diseases of ginger comprises the following preparation steps:

[0082] (1) preparing disease-resistant and growth-promoting microorganisms;

[0083] (2) preparing a microbial activity promoter;

[0084] (3) The disease-resistant and growth-promoting microorganisms and the microbial activity promoter are mixed uniformly in proportion to obtain a mixed solution, an auxiliary carrier is added and allowed to stand for 4 hours to allow the carrier to fully absorb the mixed solution, and finally granulated and dried to obtain the product.

[0085] Example 3

[0086] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:30 g.

[0087] The disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ) and Bacillus marineus ( Celeribacter marinus ), the volume ratio of the two is 1:1.

[0088] The deposit number of the Pseudomonas sp. NTO4 is CCTCC NO: M20221479, and it is deposited in the China Center for Type Culture Collection in Wuhan.

[0089] The isolation and screening method of Pseudomonas NTO4 in this example is the same as that in Example 1.

[0090] The strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date is September 20, 2016.

[0091] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0092] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 12 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0093] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0094] Step (1) The composition of the seed culture medium is: 10.0 g·L tryptone –1 , yeast extract powder 5.0 g•L –1 , NaCl10.0g•L –1 , pH 7.2-7.4, high temperature sterilization at 121℃ for 20min.

[0095] Step (1) The method for activating Pseudomonas NTO4 and Marine Fast-growing Bacillus is as follows: the target strains stored in cryopreservation are inoculated into Erlenmeyer flasks containing 25 mL of LB liquid culture medium, and the culture medium is incubated at 25-30°C and 200 r·min. -1 The cells were cultured under the same conditions for 12 h to complete the activation.

[0096] Step (2) The composition of the fermentation medium is: glucose 20.0 g·L –1 , peptone 10.0 g•L –1 , KH2PO41.0g•L –1 ,MgSO41.0g•L –1 ,MnSO4·H2O 0.2 g·L –1 , sterilize at 115℃ for 20min.

[0097] The preparation method of the microbial activity promoter is as follows: mushroom solid powder is mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide, the mixture is placed in a water bath at 45-50°C for leaching for 2 hours, supernatant A is obtained after centrifugation at 12000 r / min for 20 minutes, solid powder waste is recovered and mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide again, supernatant A and supernatant B are obtained after ultrasonic leaching for 2 hours, chitosan is added at 1% by mass of the mixed solution after centrifugation at 12000 r / min for 20 minutes, and the mixture is stirred and mixed evenly to obtain the microbial activity promoter.

[0098] Mushroom powder is obtained by drying, crushing and grinding Pleurotus ostreatus.

[0099] The auxiliary carrier is diatomaceous earth, humic acid, sucrose and biochar, and the mass ratio is 5:0.5:1:10.

[0100] A method for preparing a microbial agent for preventing and controlling soil-borne diseases of ginger comprises the following preparation steps:

[0101] (1) preparing disease-resistant and growth-promoting microorganisms;

[0102] (2) preparing a microbial activity promoter;

[0103] (3) The disease-resistant and growth-promoting microorganisms and the microbial activity promoter are mixed uniformly in proportion to obtain a mixed solution, an auxiliary carrier is added and allowed to stand for 5 hours to allow the carrier to fully absorb the mixed solution, and finally granulated and dried to obtain the product.

[0104] Comparative Example 1

[0105] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:50 g.

[0106] The disease-resistant and growth-promoting microorganism is Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ).

[0107] The deposit number of the Pseudomonas sp. NTO4 is CCTCC NO: M20221479, and it is deposited in the China Center for Type Culture Collection in Wuhan.

[0108] The isolation and screening method of Pseudomonas NTO4 in this comparative example is the same as that in Example 1.

[0109] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0110] (1) Activating the frozen Pseudomonas NTO4, taking a loop of the activated Pseudomonas NTO4 strain after activation, inoculating it into a 300 mL conical flask filled with seed culture medium, filling it with 25% liquid, and culturing it in a shaking incubator at 25-30° C. and 180-200 r / min for 10 h to obtain a seed solution;

[0111] (2) Inoculate the seed liquid at a 1% inoculum into a 1L conical flask containing fermentation medium, fill it with 50% liquid, and culture it in a shaking incubator at 25-30°C and 200 r / min. Ferment and culture until the effective viable count is greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0112] In this comparative example, except that only Pseudomonas NTO4 was used as the disease-resistant and growth-promoting microorganism, the other raw materials and preparation methods were the same as those in Example 1.

[0113] Comparative Example 2

[0114] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:50 g.

[0115] The disease-resistant and growth-promoting microorganism is marine fast-growing bacillus.

[0116] The strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which was purchased from the General Microbiology Center of the China Culture Collection Administration. The original preservation date is September 20, 2016. The strain can be purchased through the ordering process of the General Microbiology Center of the China Culture Collection Administration, without the need for biological preservation.

[0117] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0118] (1) Activating the frozen marine fast-growing Bacillus strain, taking a ring of the activated marine fast-growing Bacillus strain after activation, inoculating it into a 300 mL conical flask filled with seed culture medium, filling it with 25% liquid, and culturing it in a shaking incubator at 25-30° C. and 180-200 r / min for 10 hours to obtain a seed liquid;

[0119] (2) Inoculate the seed liquid at a 1% inoculum into a 1L conical flask containing fermentation medium, fill it with 50% liquid, and culture it in a shaking incubator at 25-30°C and 200 r / min. Ferment and culture until the effective viable count is greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0120] In this comparative example, except that only marine fast-growing Bacillus was used as the disease-resistant and growth-promoting microorganism, the other raw materials and preparation methods were the same as those in Example 1.

[0121] Comparative Example 3

[0122] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:50 g.

[0123] The disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ) and Bacillus marineus ( Celeribacter marinus ), the volume ratio of the two is 2:1.

[0124] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0125] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 10 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 2:1 to obtain a mixed seed liquid;

[0126] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0127] In this comparative example, except for changing the dosage ratio of Pseudomonas NTO4 and Marine Fast-growing Bacillus in the disease-resistant and growth-promoting microorganisms, the other raw materials and preparation methods are the same as those in Example 1.

[0128] Comparative Example 4

[0129] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant and growth-promoting microorganisms, a microbial activity promoter and an auxiliary carrier, wherein the dosage ratio of the three is 10 mL:100 mL:50 g.

[0130] The disease-resistant and growth-promoting microorganisms include Pseudomonas NTO4 ( Pseudomonas sp. NTO4 ) and Bacillus marineus ( Celeribacter marinus ), the volume ratio of the two is 1:2.

[0131] The preparation method of the disease-resistant and growth-promoting microorganisms is as follows:

[0132] (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 10 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:2 to obtain a mixed seed liquid;

[0133] (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms.

[0134] In this comparative example, except for changing the dosage ratio of Pseudomonas NTO4 and Marine Fast-growing Bacillus in the disease-resistant and growth-promoting microorganisms, the other raw materials and preparation methods are the same as those in Example 1.

[0135] Comparative Example 5

[0136] A microbial agent for preventing and controlling soil-borne diseases of ginger comprises disease-resistant growth-promoting microorganisms, sterile water and an auxiliary carrier, wherein the usage ratio of the three is 10 mL:100 mL:50 g.

[0137] In this comparative example, except that the microbial activity promoter was replaced by an equal amount of sterile water, the other raw materials and preparation method were the same as those in Example 1.

[0138] Performance Testing

[0139] Identification of growth-promoting ability of functional strains:

[0140] Test strains: Pseudomonas sp. NTO4 and T. maritima

[0141] Determination of siderophore production capacity: The CAS detection method was used to record the size of the orange halo. A vernier caliper was used to measure the diameter of the orange siderophore halo (D) and the colony diameter (d) of a single colony. The siderophore production capacity of the strain was determined by the ratio of D / d.

[0142] Phosphorus solubilization capacity determination: 1% of the seed solution of the two strains was pipetted into 100 ml of inorganic phosphorus liquid culture medium, repeated 3 times, and cultured at 180-200 r / min and 25-30°C for 7 days. The pH value of the supernatant after centrifugation of the culture solution was measured every day, and the OD value of the supernatant was measured every day using the molybdenum antimony colorimetric method. 600 The soluble phosphorus content of the strain was determined according to the prepared phosphorus standard curve for 7 days.

[0143] Determination of the ability to produce 3-Indoleacetic acid (IAA): The ability of the strain to produce IAA was determined by the Salkowski colorimetric method. The absorbance was measured at 530 nm, and then the amount of IAA produced was calculated based on the standard curve of IAA.

[0144] Identification of Protease Producers:

[0145] The bacterial strain was inoculated onto a skimmed milk powder medium plate and cultured at a constant temperature of 25-30°C for 24 hours. After repeated verification three times, observe whether a dissolution circle appears and record the diameter.

[0146] Table 1 Results of strain growth-promoting ability test

[0147]

[0148] Antibacterial performance test of disease-resistant and growth-promoting microorganisms:

[0149] The disease-resistant and growth-promoting microbial bacterial liquids obtained in Example 1 (Pseudomonas NTO4 and Marine Fast-growing Bacillus), Comparative Example 1 (Pseudomonas NTO4), Comparative Example 2 (Marine Fast-growing Bacillus), Comparative Example 3 (the volume ratio of Pseudomonas NTO4 and Marine Fast-growing Bacillus is 2:1), and Comparative Example 4 (the volume ratio of Pseudomonas NTO4 and Marine Fast-growing Bacillus is 1:2) are used as the test liquid. An equal amount of sterile water is added dropwise to the control group.

[0150] Prepare a NA medium suspension (4 × 10 7 cfu / mL), take 200μL of pathogen suspension and evenly spread it on the NA plate, add 10μL of the test solution in the middle of the plate, and after the culture medium is fully absorbed, invert it in a 25-30℃ constant temperature incubator for 24h, observe the antibacterial effect and measure the diameter of the inhibition circle. Set it to repeat 3 times and take the average value. Add an equal amount of sterile water to the control group. Plate co-culture inhibition rate (%) = (control group colony area - treatment group colony area) / control group colony area × 100. (Note: The control group has no antibacterial effect, and the recorded diameter is the colony diameter).

[0151] Table 2 Antibacterial test results

[0152]

[0153] From the data in Table 2, we can see that Pseudomonas NTO4 and Marine Fast-growing Bacillus both have good inhibitory effects on pathogenic bacteria, and the inhibition rate of the bacterial solution after fermentation and culture in equal proportion is significantly better than that of a single bacterial solution. It can be proved that after the two strains of the present invention are mixed and cultured in equal proportions, they show significant synergistic antibacterial effects. From the culture effect diagram ( Figure 2 ) It can also be seen that the antibacterial effect of the disease-resistant and growth-promoting microorganisms in Example 1 of the present invention is the best.

[0154] Pot experiment:

[0155] Tested bacterial agents: Examples 1-3, Comparative Examples 1-5, and a blank control, i.e., no bacterial agent was applied.

[0156] That is, the treatment groups are:

[0157] A1: Example 1 + Ralstonia solanacearum;

[0158] A2: Example 2 + Ralstonia solanacearum;

[0159] A3: Example 3 + Ralstonia solanacearum;

[0160] A4: Comparative Example 1 + Ralstonia solanacearum;

[0161] A5: Comparative Example 2 + Ralstonia solanacearum;

[0162] A6: Comparative Example 3 + Ralstonia solanacearum;

[0163] A7: Comparative Example 4 + Ralstonia solanacearum;

[0164] A8: Comparative Example 5 + Ralstonia solanacearum;

[0165] CK: Ralstonia solanacearum;

[0166] There were 10 pots in each treatment, and the results were averaged;

[0167] Inoculation method: Place a round plastic paper of the same size as the bottom of the flowerpot, then put about 1 / 3 of the sterilized substrate in it, then transplant 3 ginger seedlings per pot, put 10g / pot of the test agent near the ginger root system (make sure the ginger root system is in contact with the agent), and continue to pour the sterilized substrate into the flowerpot to 3 / 4 of the flowerpot, making sure that the culture substrate covers the ginger root system. After the plants have grown in the greenhouse for 4 weeks, the above treatments are inoculated with ginger blight pathogens. Root irrigation inoculation, inoculate each pot with 50mL of 10 8 CFU / mL, and the ginger seedlings of each treatment were covered with plastic bags to keep them moist for 24 hours. Physiological and biochemical indicators were measured after harvest, and the disease status of ginger plants was counted after 30 days. Disease classification: Level 0: healthy leaves, no wilting; Level 1: 0%-25% of the leaves wilted; Level 2: 25%-50% of the leaves wilted; Level 3: 50%-75% of the leaves wilted; Level 4: 75%-100% of the leaves wilted.

[0168] Disease index = ∑ (number of diseased plants per treatment × disease level) / (total number of plants × highest disease level);

[0169] Prevention effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100.

[0170] Chlorophyll content and photosynthetic rate determination: Before harvest, choose a clear morning at 9-11 am, randomly select one plant from each pot, and select the mature leaves in the upper middle of the plant, place them in a liquid nitrogen tank and bring them back to the laboratory. The chlorophyll content was determined using the acetone-ethanol mixed solution method; photosynthetic rate and other determinations were determined using a photosynthetic analyzer.

[0171] Table 3 Potted plant experiment results

[0172]

[0173] Determination of defense enzyme activity: The sampling site is the third fully expanded leaf from the top of the ginger plant. The leaves are cleaned and used for enzyme activity determination. The superoxide dismutase (SOD) activity is determined by the nitroblue tetrazolium method, the catalase (CAT) activity is determined by the ultraviolet absorption method, and the peroxidase (POD) and polyphenol oxidase (PPO) activities are determined by the guaiacol method. The malondialdehyde (MDA) content is determined by the thiobarbituric acid colorimetric method. The results of the defense enzyme activity test are as follows: Figure 3-8 shown.

[0174] The above results show that the application of the microbial agent of the present invention can promote the growth of ginger defense enzyme activity to a certain extent, reduce MDA concentration, reduce morbidity, and improve crop resistance, among which the embodiment has the best effect. This shows that the synergistic effect produced by the equal proportion of Pseudomonas NTO4 and Marine Fast-growing Bacillus can effectively improve the prevention and control effect of ginger blight, and has the dual effects of promoting growth and preventing diseases.

[0175] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial agent for preventing and controlling soil-borne diseases of ginger, characterized in that: It includes disease-resistant and growth-promoting microorganisms, microbial activity promoters and auxiliary carriers, and the dosage ratio of the three is 10mL:100mL:(30-50)g; The anti-disease growth-promoting microorganisms include Pseudomonas ( Pseudomonas sp. ) NTO4 and Marine Fast-growing Bacillus ( Celeribacter marinus ), the volume ratio of the two is 1:1; the preservation number of the Pseudomonas NTO4 is CCTCC NO: M20221479, which is preserved in the China Center for Type Culture Collection in Wuhan; the strain number of the marine fast-growing bacillus is CGMCC No.1.15836, which is purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration; the preparation method of the disease-resistant and growth-promoting microorganism is: (1) Activating frozen Pseudomonas NTO4 and marine fast-growing Bacillus, taking a ring of activated Pseudomonas NTO4 and activated strains of marine fast-growing Bacillus after activation, respectively inoculating them into 300 mL conical flasks filled with seed culture medium, filling the flasks with a liquid volume of 25%, placing them in a shaking incubator at 25-30° C. and 180-200 r / min for 10-12 h to obtain seed liquids of the two bacteria, and mixing the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid; (2) The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-30°C and 200 r / min for fermentation until the effective viable bacterial count was greater than 3×10 9 cfu / mL, and the pH was adjusted to 6.5-7.5 to obtain disease-resistant and growth-promoting microorganisms; The preparation method of the microbial activity promoter is as follows: mushroom solid powder is mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide, the mixture is placed in a water bath at 45-50°C for leaching for 1-2 hours, supernatant A is obtained after centrifugation at 12000 r / min for 20 minutes, solid powder waste is recovered and mixed with distilled water at a mass ratio of 1:30, pH is adjusted to 9-10 with 1 mol / L sodium hydroxide again, supernatant B is obtained after ultrasonic leaching for 1-2 hours, supernatant A and supernatant B are mixed, chitosan of 1% by mass of the mixed solution is added after centrifugation at 12000 r / min for 20 minutes, and the mixture is stirred and mixed evenly to obtain the microbial activity promoter.

2. The microbial agent for preventing and controlling soil-borne diseases of ginger according to claim 1, characterized in that: The auxiliary carrier is diatomaceous earth, humic acid, sucrose and biochar, and the mass ratio is 5:0.5:1:

10.

3. A method for preparing the microbial agent for preventing and controlling soil-borne diseases of ginger according to any one of claims 1-2, characterized in that: The method comprises the following preparation steps: (1) preparing disease-resistant and growth-promoting microorganisms; (2) preparing a microbial activity promoter; (3) Mix the disease-resistant and growth-promoting microorganisms and the microbial activity promoter in proportion to obtain a mixed solution, add an auxiliary carrier and let it stand for 3-5 hours to allow the carrier to fully absorb the mixed solution, and finally granulate and dry to obtain the product.

4. Use of the microbial agent for preventing and controlling soil-borne diseases of ginger according to any one of claims 1 to 2 in ginger cultivation.

Images