Staphylococcus warwick inoculant for inhibiting Pythium spp. in melons and fruits, its preparation method and application

Abstract

The present application relates to the technical field of agricultural microbial agents, and specifically discloses a staphylococcus warneri agent for inhibiting pythium aphanidermatum, a preparation method and application thereof. The staphylococcus warneri agent comprises: staphylococcus warneri bacterial slurry and slow-release adjuvant mixed at a mass ratio of 1:1-3 to form granules; the staphylococcus warneri bacterial slurry comprises staphylococcus warneri bacterial liquid and dry protective agent, the solid content of the staphylococcus warneri bacterial liquid is 60-80%, and the mass fraction of the dry protective agent is 1-10% of the mass fraction of the staphylococcus warneri bacterial liquid; the slow-release adjuvant comprises carrier adjuvant, slow-release adjuvant and trace elements mixed at a mass ratio of 6-8:1.5-3:0.5-1. The staphylococcus warneri agent has a significant effect on inhibiting pythium aphanidermatum, can significantly reduce the harm of pythium aphanidermatum to various crops, and can be used as an efficient and environmentally friendly agricultural disease control method.

Description

Technical Field

[0001] This invention relates to the technical field of agricultural microbial agents, specifically disclosing a Staphylococcus warwick agent for inhibiting Pythium in melons and fruits, its preparation method, and its application. Background Technology

[0002] Pythium spp. is a group of pathogenic fungi with a wide host range and strong infectivity. It is widely distributed throughout the world, found in various habitats including land, saltwater and freshwater, cultivated and fallow soils, and in plants and animals. It is estimated that diseases caused by Pythium spp. in various crops result in billions of dollars in economic losses to global food supplies.

[0003] *Pythium aphanidermatum* is an important plant pathogenic fungus and one of the most invasive pathogens in the *Pythium* genus. It has a wide host range, mainly including common plants in the Cucurbitaceae, Solanaceae, Fabaceae, and Poaceae families, causing serious damage to economic crops such as vegetables and fruits, as well as important food crops. The main symptoms of diseases caused by *Pythium aphanidermatum* are rotting and damping-off in various crops. In recent years, *Pythium* has also been widely reported as an important pathogen causing many seedling diseases in crops, such as damping-off, root rot, stem rot, and fruit rot. Seedling diseases caused by *Pythium aphanidermatum* mainly manifest as water-soaked areas at the base or middle of the hypocotyl, leading to damping-off before the cotyledons wither. *Pythium aphanidermatum* commonly infests cucurbits, beans, peppers, and eggplants, with cucumbers and tomatoes being particularly severely affected, frequently causing spoilage and deterioration of fruits and vegetables, resulting in significant losses to agricultural production and food security. Pythium spp. is a highly adaptable fungus, therefore, tomato wilt caused by Pythium spp. is not limited to the hot season; it can also occur severely in low-temperature, high-humidity environments. The disease cycle of Pythium spp. involves overwintering oospores in the soil. Sporangia and zoospores produced on diseased plant debris can be spread by rainwater to infect the host, or directly germinate into germ tubes to invade the host, eventually forming oospores again in the diseased tissue for overwintering. Furthermore, the severity of diseases caused by Pythium spp. tends to increase with improved soil water and fertilizer conditions.

[0004] Biological control has attracted widespread attention due to its environmental friendliness and non-toxicity to non-target organisms. Currently reported microorganisms with inhibitory effects on Pythium spp. in melons and fruits include Bacillus subtilis, Trichoderma harzianum, and Streptomyces flavus. However, there are no reports on the inhibitory effects of Staphylococcus warwick on Pythium spp. in melons and fruits. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Staphylococcus wartii inoculant for inhibiting Pythium in melons and fruits, its preparation method, and its application.

[0006] In a first aspect, the present invention discloses an agent for inhibiting *Pythium spp.* in melons and fruits, employing the following technical solution:

[0007] A Staphylococcus warwick inoculant for inhibiting Pythium spp. in melons and fruits comprises: Staphylococcus warwick slurry and slow-release excipients mixed and granulated at a mass ratio of 1:1 to 3; the Staphylococcus warwick slurry comprises Staphylococcus warwick liquid and a desiccant, wherein the solid content of the Staphylococcus warwick liquid is 60 to 80%, and the mass of the desiccant is 1 to 10% of the mass fraction of the Staphylococcus warwick liquid; the slow-release excipients comprise carrier excipients, slow-release excipients, and trace elements mixed at a mass ratio of 6 to 8: 1.5 to 3: 0.5 to 1.

[0008] Preferably, the *Staphylococcus wartii* is derived from the China General Microbiological Culture Collection Center (CGMCC), with the culture accession number CGMCC1.2824.

[0009] Preferably, the carrier-type auxiliary material includes diatomaceous earth, attapulgite, and wheat bran mixed in a mass ratio of 1~3:1~3:1.

[0010] Preferably, the slow-release excipient comprises chitosan, humic acid, and amino acid powder mixed in a mass ratio of 1~2:1~2:1.

[0011] Preferably, the trace elements are obtained by mixing zinc sulfate, FeEDTA, manganese sulfate, cobalt chloride, and sodium molybdate in a mass ratio of 2~4:2~4:1~2:1~2:1~2.

[0012] Preferably, the desiccant comprises trehalose, lactose, glycerol, and dimethyl sulfoxide mixed in a mass ratio of 1:1 to 2:1 to 2:2 to 4.

[0013] Secondly, this invention discloses a method for preparing a Staphylococcus warwick inoculant that inhibits Pythium spp. in fruits and vegetables, using the following technical solution:

[0014] A method for preparing a Staphylococcus warwick inoculant that inhibits Pythium spp. in fruits and vegetables includes the following steps:

[0015] Step 1, Primary seed culture: Inoculate Staphylococcus warwick into the seed culture medium and culture it in an incubator at 25-35℃ with shaking for 18-30 hours at a speed of 150-200 rpm to obtain the primary seed culture;

[0016] Step 2, Secondary seed culture: Inoculate the primary seed culture into the primary seed tank at an inoculation rate of 10-20%, and culture under the following conditions: temperature 25-35℃, rotation speed 150-200rpm, pH 6.5-7.2, dissolved oxygen 20-50%, for 18-30 hours to obtain the secondary seed culture;

[0017] Step 3: Fermentation tank cultivation: Inoculate the secondary seed liquid (10-20% by volume) and Pythium spp. (1% by volume) into the fermentation tank. Cultivation conditions are: temperature 25-35℃, rotation speed 150-200 rpm, pH 6.5-7.2, and cultivation for 20-36 hours until Pythium spp. is no longer detected in the fermentation liquid.

[0018] Step 4, bacterial culture treatment: Centrifuge the fermentation broth, adjust the solid content of the Staphylococcus wartii bacterial culture to 60-80%, and add 1-10% by mass of a desiccant to the adjusted Staphylococcus wartii bacterial culture to obtain bacterial slurry;

[0019] Step 5, Granulation and Drying: Mix the bacterial slurry with the excipients and granulate. Vacuum dry the solid particles at a temperature of 30~50℃ for 10~20 hours to obtain the solid product.

[0020] Preferably, the seed culture medium in step one is LB medium.

[0021] Preferably, both the primary seed tank in step two and the fermentation tank in step three are composed of the following components: glucose 2~5g / L, corn steep liquor 2~5g / L, molasses 0.5~1g / L, (NH4)SO4 0.3~0.8g / L, K2HPO4 0.1~0.5g / L, NaH2PO4 0.1~0.5g / L, MnSO4·H2O 0.01~0.05g / L, FeSO4·7H2O 0.01~0.05g / L, Na2MoO4 0.01~0.05g / L, MgSO4·7H2O 0.01~0.05g / L, and CoCl2 0.01~0.05g / L.

[0022] Thirdly, this invention discloses the application of a Staphylococcus warwick inoculant that inhibits Pythium spp. in melons and fruits. It can be used as a base application or top dressing, with an application rate of 1-10 kg / mu.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] The *Staphylococcus warwick* inoculant of this invention has a significant inhibitory effect on *Pythium spp.*, effectively reducing the damage caused by *Pythium spp.* to various crops such as cucumbers, pumpkins, tomatoes, peppers, and eggplants. It can serve as a highly efficient and environmentally friendly method for agricultural disease control. Furthermore, this invention, by mixing *Staphylococcus warwick* slurry with slow-release excipients at a specific mass ratio and granulating the mixture, allows the inoculant to be slowly released into the soil, extending its effective action time and improving its utilization rate and control efficacy. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to specific embodiments.

[0026] Example 1

[0027] This embodiment discloses a Staphylococcus warwick inoculant for inhibiting Pythium spp. in melons and fruits, and its preparation method includes the following steps:

[0028] Step 1, Primary seed culture: Inoculate Staphylococcus warwick into seed culture medium and culture in a 30℃ incubator with shaking for 24 hours at 180 rpm to obtain primary seed culture;

[0029] Step 2, Secondary seed culture: Inoculate the primary seed culture into the primary seed tank at an inoculation rate of 15%, and culture under the following conditions: temperature 30℃, rotation speed 180rpm, pH 7.0, dissolved oxygen 40%, for 24 hours to obtain the secondary seed culture;

[0030] Step 3: Fermentation tank culture: 15% by volume of the secondary seed liquid and 1% by volume of Pythium spp. were inoculated into the fermentation tank. The culture conditions were 30°C, 180 rpm, and pH 7.0. After 30 hours of culture, Pythium spp. was not detected in the fermentation broth.

[0031] Step 4, bacterial culture treatment: Centrifuge the fermentation broth obtained in Step 3, adjust the solid content of the Staphylococcus wartii bacterial culture to 60%, add 5% desiccant to the adjusted bacterial culture to obtain bacterial slurry;

[0032] Step 5, Granulation and Drying: Mix the bacterial slurry and excipients at a mass ratio of 1:1.5 and granulate. Vacuum dry the solid particles at 30°C for 20 hours to obtain the solid product.

[0033] In step one, *Staphylococcus warwick* was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC1.2824. The seed culture medium in step one was LB medium.

[0034] Both the primary seed tank in step two and the fermentation tank in step three are made from the following components: glucose 4 g / L, corn steep liquor 4 g / L, molasses 1 g / L, (NH4)SO4 0.6 g / L, K2HPO4 0.2 g / L, NaH2PO4 0.3 g / L, MnSO4·H2O 0.04 g / L, FeSO4·7H2O 0.04 g / L, Na2MoO4 0.04 g / L, MgSO4·7H2O 0.04 g / L, and CoCl2 0.04 g / L.

[0035] The *Pythium spp.* added in step three is a suspension of zoospores of *Pythium spp.*. The preparation method of the *Pythium spp.* zoospore suspension is as follows:

[0036] (1) The preserved Pythium spp. strain was activated and grown on 10% V8 medium and cultured in the dark in a constant temperature incubator at 25℃. The culture time was generally 1~2 days.

[0037] (2) Cut a 10mm×15mm mycelial block from the above-mentioned Pythium strain cultured for 1-2 days;

[0038] (3) Place 10-20 of the above mycelial blocks in 15 mL of sterile tap water, and change the water on the mycelial surface of the mycelial blocks every 30 minutes.

[0039] (4) Repeat this process 3 times, then add about 8 mL of sterile water, just enough to cover the mycelium surface;

[0040] (5) Place the incubator at 25℃ for 20-24 hours to induce the production of zoospores;

[0041] (6) Adjust the concentration of the zoospore suspension to approximately 1~5×10⁻⁶ with sterile water. 6 The concentration in this example is 1×10⁻⁶ cells / mL. 6 per mL.

[0042] In step four, the drying protectant is obtained by mixing trehalose, lactose, glycerol, and dimethyl sulfoxide in a mass ratio of 1:1.5:2:2.

[0043] In step five, the excipients include a carrier-type excipient, a slow-release excipient, and trace elements mixed in a mass ratio of 6:3:1. Specifically, the carrier-type excipient includes diatomaceous earth, attapulgite, and wheat bran mixed in a mass ratio of 2:2:1. The slow-release excipient includes chitosan, humic acid, and amino acid powder mixed in a mass ratio of 2:2:1. The trace elements include zinc sulfate, FeEDTA, manganese sulfate, cobalt chloride, and sodium molybdate mixed in a mass ratio of 2:2:1:1:1.

[0044] To verify the preservation effect of effective bacteria after solid particle processing, the effective bacterial count and the rate of miscellaneous bacteria in the preservation process of the fermentation broth obtained in step three and the solid product obtained in step five were compared. The test results are shown in Table 1 and Table 2.

[0045] Table 1. Comparison of viable cell counts between fermentation broth and solid products during 0-24 month storage.

[0046]

[0047] Table 2. Comparison of microbial contamination rate (%) between fermentation broth and solid product during 0-24 month storage.

[0048]

[0049] A comparison of the test results in Tables 1 and 2 shows that after bacterial treatment with added desiccant and granulation and drying of mixed carrier excipients, slow-release excipients and trace elements, the solid product still has a high level of effective bacterial count after 24 months of storage, and the rate of miscellaneous bacteria can be maintained at an extremely low level.

[0050] To verify the contribution of the fermentation treatment with added Pythium styracifolium, the bacterial liquid treatment with desiccant, and the granulation and drying treatment with mixed excipients to the product quality of the microbial agent, the following comparative examples 1-3 were conducted for verification.

[0051] Comparative Example 1

[0052] The only difference from Example 1 is that 1% *Pythium spp.* was not added to the fermenter in step three. Everything else was the same as in Example 1. The inhibition rate (%) of the solid product obtained in Comparative Example 1 against *Pythium spp.* was tested. The method for testing the inhibition rate of *Pythium spp.* is as follows:

[0053] Using a sterile punch, mycelial blocks approximately 5 mm in diameter were transferred to the center of a fresh PDA medium. 0.01 g of the inoculum was applied at four equidistant points near the periphery of the culture, and the medium was incubated at 25°C for 7 days. After the pathogen on the control plate had grown to cover the entire surface of the medium, its antifungal activity was estimated by measuring the radial growth diameter of the fungus in the antagonistic experiment. .

[0054] Table 3 shows the comparison of the inhibition rate (%) of solid products against Pythium spp. in melons and fruits.

[0055] Table 3. Comparison of the inhibitory effects of the solid products prepared in Example 1 and Comparative Example 1 on Pythium spp. in melons and fruits.

[0056] Inhibition rate (%) Example 1 83.1 Comparative Example 1 62.5

[0057] As can be seen from the comparison of inhibition effects in Table 3, the inhibition rate of Comparative Example 1, which did not add Pythium spp. during fermentation, was significantly lower than that of Example 1. This proves that adding Pythium spp. during fermentation can improve the inhibitory effect of Staphylococcus warwicki on Pythium spp. This is because adding Pythium spp. during fermentation can significantly improve the inhibitory effect of Staphylococcus warwicki on Pythium spp. through multiple pathways, such as activating the induced resistance mechanism of Staphylococcus warwicki, establishing competitive growth advantage, regulating metabolites, and enhancing the adaptability of the strain.

[0058] Comparative Example 2

[0059] The only difference from Example 1 is that in step four, while maintaining the same amount of desiccant, trehalose, lactose, glycerol, and dimethyl sulfoxide are used as single desiccant to replace the compound desiccant in Example 1. The rest is the same as in Example 1. The microbial count (cfu / g) of the obtained solid product is compared and the test results are shown in Table 4.

[0060] Table 4. Comparison of microbial biomass (cfu / g) of solid products prepared with different desiccants

[0061]

[0062] As can be seen from the microbial biomass test results in Table 4, different types of desiccants have varying degrees of impact on the activity of Staphylococcus warwick. While using trehalose, lactose, glycerol, and dimethyl sulfoxide as single desiccants can provide some desiccant protection for solid products, single desiccants have limitations. For example, although trehalose, lactose, and glycerol have good moisturizing and stability properties and can provide some protection, using trehalose alone at high concentrations can easily lead to excessively high osmotic pressure on the surface of Staphylococcus warwick, affecting the activity of the cells. When used alone, lactose tends to absorb moisture during the drying process, causing clumping on the cell surface and affecting the dispersibility and activity of the cells. When used alone, glycerol's high viscosity can affect the dispersibility of the cells, causing them to aggregate during the drying process and reducing their activity. Dimethyl sulfoxide has good permeability and protective properties, but when used alone, its strong organic solvent properties can easily cause some toxicity to Staphylococcus warwick, affecting the activity of the cells. Only by using a desiccant composed of trehalose, lactose, glycerol, and dimethyl sulfoxide in Example 1 can the synergistic effect of each component be achieved, thereby realizing the ideal level of microbial biomass and ensuring the effectiveness and stability of the solid product in subsequent applications.

[0063] Comparative Example 3

[0064] The only difference from Example 1 is that in step five, while maintaining the same amount of sustained-release excipients, chitosan, humic acid, and amino acid powder are used as single sustained-release excipients to replace the compound sustained-release excipients in Example 1. The rest is the same as in Example 1. The results of the test on the sustained-release effect of the solid product are shown in Table 5.

[0065] Table 5. Comparison of sustained-release effects of solid products prepared with different sustained-release excipients

[0066]

[0067] Note:

[0068] Half-release period: The time required for 50% release of the microbial agent.

[0069] 7d release rate: The percentage of live bacteria released within 7 days out of the total bacterial load.

[0070] 30-day release rate: The percentage of live bacteria released within 30 days out of the total bacterial load.

[0071] As can be seen from the comparison of the sustained-release effect test results in Table 5, different types of sustained-release excipients have varying degrees of impact on the sustained-release effect of Staphylococcus warwick solid products. While using chitosan, humic acid, and amino acid powder as single sustained-release excipients can provide some sustained-release effect on solid products, the release rate is relatively fast and the half-release period is short when using chitosan, humic acid, or amino acid powder alone, which cannot meet the requirements for long-term sustained release. However, by simultaneously adding a combined sustained-release excipient of chitosan, humic acid, and amino acid powder, the synergistic effect of each component can be achieved: chitosan provides good biocompatibility and sustained-release performance, humic acid provides adsorption, and amino acid powder provides biocompatibility and sustained-release performance. This compounding method can comprehensively meet the various needs of the bacteria during the sustained-release process, thereby better achieving the sustained-release effect and ensuring the persistence and effectiveness of the solid product in the soil.

[0072] Example 2

[0073] This embodiment discloses the application of a Staphylococcus warwick inoculant for inhibiting Pythium spp. in melons and fruits, as detailed below:

[0074] Staphylococcus warwick was applied to the roots of various plants at a rate of 4 kg / mu. The control group was not treated with Staphylococcus warwick. After 14 days, the plants were infected with Pythium spp. The disease index and control effect of the plants were calculated after 7 days of treatment. The control results are shown in Table 6. .

[0075] Criteria for judging plant species at each level:

[0076] 0 - Keep the plants green and healthy;

[0077] 1. The leaf sheath ring changes color and the lower leaf blades turn yellow;

[0078] 2- The plant survives, but the leaves turn completely yellow or die;

[0079] 3- The entire plant dies.

[0080] .

[0081] Table 6. Effects of Staphylococcus warwick inoculants on Pythium spp. in melons and fruits.

[0082]

[0083] As can be seen from the comparison of the control effects in Example 2, the solidified Staphylococcus wartii agent of this solution, as a biocontrol agent, can significantly reduce the damage of Pythium spp. to various crops such as cucumber, pumpkin, tomato, pepper, and eggplant, improve the health level and disease resistance of crops, and provide an efficient and environmentally friendly solution for agricultural disease control.

[0084] The technical solution provided by the present invention has been described in detail above. Specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A Staphylococcus warneri bacterial agent for inhibiting Pythium aphanidermatum, characterized by comprising, The application relates to a Staphylococcus warneri granule and a preparation method thereof. The Staphylococcus warneri slurry is prepared by mixing a Staphylococcus warneri slurry and auxiliary materials at a mass ratio of 1:1-3. The Staphylococcus warneri slurry comprises a Staphylococcus warneri liquid and a dry protective agent, the solid content of the Staphylococcus warneri liquid is 60-80%, and the mass fraction of the dry protective agent is 1-10% of the Staphylococcus warneri liquid; the Staphylococcus warneri is fermented by adding Pythium aphanidermatum during fermentation; The auxiliary materials comprise carrier auxiliary materials, slow-release auxiliary materials and trace elements, which are mixed at a mass ratio of 6-8:1.5-3:0.5-1. The carrier auxiliary materials comprise diatomite, attapulgite and bran, which are mixed at a mass ratio of 1-3:1-3:

1. The slow-release auxiliary materials comprise chitosan, humic acid and amino acid powder, which are mixed at a mass ratio of 1-2:1-2:

1. The trace elements comprise zinc sulfate, FeEDTA, manganese sulfate, cobalt chloride and sodium molybdate, which are mixed at a mass ratio of 2-4:2-4:1-2:1-2:1-2. The dry protective agent comprises trehalose, lactose, glycerol and dimethyl sulfoxide, which are mixed at a mass ratio of 1:1-2:1-2:2-4.

2. The S. warneri agent for inhibiting P. carotovorum according to claim 1, characterized by, The Staphylococcus warneri is from the CGMCC China General Microbiological Culture Collection Center, and the strain preservation number is CGMCC1.2824.

3. A method of preparing a S. vitis bacterial agent for inhibiting P. carotovorum according to any one of claims 1-2, characterized in that, The application further discloses a preparation method of the Staphylococcus warneri granule. Step one: primary seed liquid culture: the Staphylococcus warneri is inoculated into a seed liquid culture medium, and is cultured in a 25-35 DEG C incubator at a rotating speed of 150-200 rpm for 18-30 h to obtain a primary seed liquid; Step two: secondary seed liquid culture: the primary seed liquid is inoculated into a primary seed tank at a 10-20% inoculation amount, and the culture conditions are as follows: temperature 25-35 DEG C, rotating speed 150-200 rpm, pH 6.5-7.2, and dissolved oxygen 20-50%, and the secondary seed liquid is obtained after 18-30 h of culture; Step three: fermentation tank culture: the secondary seed liquid with a volume fraction of 10-20% and Pythium aphanidermatum with a volume fraction of 1% are inoculated into a fermentation tank, and the culture conditions are as follows: temperature 25-35 DEG C, rotating speed 150-200 rpm, and pH 6.5-7.2, and the fermentation tank is cultured for 20-36 h until no Pythium aphanidermatum is detected in the fermentation liquid; Step four: liquid treatment: the fermentation liquid is centrifuged, the solid content of the Staphylococcus warneri liquid is adjusted to 60-80%, and the dry protective agent with a mass fraction of 1-10% is added into the adjusted Staphylococcus warneri liquid to obtain a slurry; Step five: granulation and drying: the slurry and the auxiliary materials are mixed to form granules, and the solid granules are vacuum dried at a temperature of 30-50 DEG C for 10-20 h to obtain a solid product.

4. The method of claim 3, wherein the preparation of the S. warneri bacterial agent for inhibiting P. digitatum is characterized by, The seed liquid culture medium in step one is an LB culture medium.

5. The method of claim 3, wherein the preparation of the S. warneri bacterial agent for inhibiting P. digitatum is characterized by, The step two primary seed tank and the step three fermentation tank are made of the following components: glucose 2-5 g / L, corn syrup 2-5 g / L, molasses 0.5-1 g / L, (NH4)2SO4 0.3-0.8 g / L, K2HPO4 0.1-0.5 g / L, NaH2PO4 0.1-0.5 g / L, MnSO4·H2O 0.01-0.05 g / L, FeSO4·7H2O 0.01-0.05 g / L, Na2MoO4 0.01-0.05 g / L, MgSO4·7H2O 0.01-0.05 g / L, CoCl2 0.01-0.05 g / L.

6. Use of a bacterial agent of Staphylococcus woolsii that inhibits Pythium aphanidermatum according to any one of claims 1 to 2, characterized in that, As base application and topdressing, the application amount is 1-10 kg / mu.

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