Microbial seed coating as well as preparation method and application thereof

Abstract

The invention discloses a microbial seed coating agent as well as a preparation method and application thereof. The microbial seed coating agent comprises a composite flora bacterial suspension and a film-forming agent solution in a volume ratio of 1: (0.5-2), the composite flora comprises pseudomonas chlororaphis with the login number of CP019399.1, pseudomonas kirschner with the login number of PP218673 and bacillus velezensis with the login number of MF192765.1, and the film forming agent solution comprises sodium alginate with the concentration of 1 wt%-4 wt% and Arabic gum with the concentration of 6 wt%-9 wt%. The microbial seed coating agent has no obvious influence on crop seed germination, has excellent physical and chemical properties such as film forming time, water absorption rate, swelling rate, coating uniformity and coating shedding rate, has good low-temperature storage and thermal storage stability, and can be applied to coating treatment of pepper seeds. The growth promoting effect can be well achieved, and the obvious effect is achieved in the aspect of inhibiting the pepper bacterial wilt.

Description

[0001] This invention claims the priority of a Chinese patent application titled "Microbial Seed Coating Agent and Its Preparation Method and Application" with the application number 2025105315129, which was filed with the Chinese Patent Office on April 25, 2025. The entire content of this application is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of plant disease resistance and drug development, and particularly relates to a microbial seed coating agent, its preparation method, and its application in promoting crop growth and controlling bacterial wilt of crops. Background Art

[0003] A seed coating agent is an innovative pesticide formulation that encapsulates active ingredients using a film-forming agent or adhesive to ensure efficient delivery of the active ingredients. By using a seed coating agent, seeds are made spherical or maintained in their original form to control pests and diseases, enhance stress resistance and disease resistance, promote germination and seedling emergence, and ultimately increase crop yield and quality. This is an efficient and environmentally friendly way of seed treatment.

[0004] There are a wide variety of seed coating agents on the market. Based on differences in composition, they can be mainly classified into two categories: chemical seed coating agents and biological seed coating agents. For a long time, chemical seed coating agents have dominated agricultural production. However, with the in-depth implementation of the concept of sustainable agricultural development, the drawbacks of chemical seed coating agents have gradually emerged. Their safety issues such as high pollution and high residues not only damage the soil ecological environment, leading to soil microbial community imbalance, but also may cause drug resistance in pathogenic bacteria, increasing the difficulty of controlling soil-borne diseases. To address the problems caused by chemical seed coating agents, biological seed coating agents have emerged. Microbial seed coating agents mainly use live microorganisms, secondary metabolites of microorganisms, and bioactive substances as active ingredients, and are processed with some auxiliaries that have no impact on the strains or microbial metabolites. Compared with traditional chemical seed coating agents, microbial seed coating agents have great advantages: (1) During the seed coating process, using a microbial seed coating agent for coating can significantly improve the fusion of seeds and soil; (2) It can effectively solve the "3R" problems caused by long-term use of pesticides, achieving environmental protection; (3) Microbial seed coating agents are harmless to humans and animals, and are safe to use; (4) It constructs a beneficial microbial environment, enhances the stress resistance of plants, and has a wide range of applications.

[0005] However, there are few reports on microbial seed coating agents for promoting the growth of peppers and controlling bacterial wilt. In addition, microbial seed coating agents also face the defect that their physical and chemical properties and storage stability are not ideal enough. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a microbial seed coating agent with excellent physical and chemical properties, good low-temperature stability and thermal stability, which can be used for promoting crop growth and controlling bacterial wilt.

[0007] The technical solutions for achieving the above-mentioned invention objectives are as follows.

[0008] In the first aspect of the present invention, a microbial seed coating agent is provided, which comprises a composite bacterial suspension and a film-forming agent solution with a volume ratio of 1:0.5 - 2; the composite bacteria group is Pseudomonas chlororaphis with accession number CP019399.1, Pseudomonas kribbensis with accession number PP218673, and Bacillus velezensis with accession number MF192765.1, and the film-forming agent solution comprises sodium alginate with a concentration of 1wt% - 4wt% and arabic gum with a concentration of 6wt% - 9wt%.

[0009] In the second aspect of the present invention, a preparation method of a microbial seed coating agent is provided, which comprises the following steps:

[0010] (1) Prepare a composite bacterial suspension from Pseudomonas chlororaphis with accession number CP019399.1, Pseudomonas kribbensis with accession number PP218673, and Bacillus velezensis with accession number MF192765.1;

[0011] (2) Dissolve sodium alginate and arabic gum in water to obtain a film-forming agent solution;

[0012] (3) Mix the composite bacterial suspension and the film-forming agent solution in a volume ratio of 1:0.5 - 2 to obtain the product.

[0013] In the third aspect of the present invention, the application of the above-mentioned microbial seed coating agent in promoting crop growth or preventing and controlling crop bacterial wilt is provided.

[0014] In the fourth aspect of the present invention, a method for promoting crop growth or preventing and controlling crop bacterial wilt is provided, which comprises the following step: using the above-mentioned microbial seed coating agent to coat crop seeds.

[0015] The inventor of the present invention found that: a seed coating agent prepared by mixing a composite strain with the function of preventing and controlling bacterial wilt, which is isolated, cultured and screened from the roots and leaves of healthy pepper samples, as an active ingredient, with a film-forming agent solution comprising sodium alginate and arabic gum in a certain concentration ratio in a certain volume ratio has no obvious influence on the germination of crop seeds, and has excellent physical and chemical properties such as film-forming time, water absorption rate, swelling rate, coating uniformity and coating shedding rate, and at the same time has good low-temperature storage and heat storage stability, and is an ideal seed coating agent.

[0016] After coating pepper seeds with the microbial seed coating agent of the present invention and then raising seedlings, 30 days after transplantation, the plant height, fresh weight, and chlorophyll content of pepper seedlings were all significantly increased, and the total root length, total surface area, and root volume were all significantly enlarged. This indicates that the microbial seed coating agent of the present invention can significantly improve the accumulation of pepper biomass and promote root development, and can better play its growth-promoting role.

[0017] Through pepper pot experiments, it was found that after coating pepper seeds with the microbial seed coating agent of the present invention, the onset time of pepper bacterial wilt can be significantly delayed, and the development speed of the disease condition is also slowed down, indicating that it has a significant effect in inhibiting pepper bacterial wilt. Description of the Drawings

[0018] Figure 1 It is the storage stability of each microbial seed coating agent in Example 6 of the present invention at 37°C. Among them, from left to right, from top to bottom, they represent microbial seed coating agents RHB191, RHB192, RHB193, RHB281, HRB282, RHB283, RHB371, RHB372, RHB373, RHB461, RHB462, and RHB463 in sequence.

[0019] Figure 2 It is a graph showing the change in the viable count of different microbial seed coating agents in Example 6 of the present invention within 56 days.

[0020] Figure 3 It is a picture of coated and uncoated pepper seeds in Example 7 of the present invention; among them, a is without coating treatment; b is coating treatment with microbial seed coating agent RHB463.

[0021] Figure 4 It is the result of the influence of the microbial seed coating agent on the growth of pepper seedlings in Example 7 of the present invention; among them, a is without coating treatment; b is coating treatment with microbial seed coating agent RHB463.

[0022] Figure 5 It is the result of the influence of the microbial seed coating agent on the root growth of pepper seedlings in Example 7 of the present invention; among them, a is without coating treatment; b is coating treatment with microbial seed coating agent RHB463.

[0023] Figure 6 It is the control effect of the microbial seed coating agent on pepper bacterial wilt in Example 8 of the present invention; among them, a is CK + RS (uncoated); b is microbial seed coating agent RHB463 + RS. Detailed Embodiments

[0024] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0026] The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as the conditions described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the embodiments are commercially available products.

[0027] In some embodiments of the present invention, a microbial seed coating agent is disclosed, which comprises a complex microbial community suspension and a film-forming agent solution with a volume ratio of 1:0.5 - 2; the complex microbial community is Pseudomonas chlororaphis with accession number CP019399.1, Pseudomonas kribbensis with accession number PP218673, and Bacillus velezensis with accession number MF192765.1, and the film-forming agent solution comprises sodium alginate with a concentration of 1 wt% - 4 wt% and gum arabic with a concentration of 6 wt% - 9 wt%.

[0028] In some embodiments, the concentration of sodium alginate in the film-forming agent solution is 3 wt% - 4 wt%, and the concentration of gum arabic is 6 wt% - 7 wt%.

[0029] In some embodiments, the concentration of sodium alginate in the film-forming agent solution is 3.8 wt% - 4 wt%, and the concentration of gum arabic is 6 wt% - 6.2 wt%.

[0030] In some embodiments, the concentration of sodium alginate in the film-forming agent solution is 3.9 wt% - 4 wt%, and the concentration of gum arabic is 6 wt% - 6.1 wt%.

[0031] In some embodiments, the concentration of the complex microbial community suspension is 1.0×10 8 cfu - 1.0×10 9 cfu.

[0032] In some of these embodiments, the volume ratio of the complex microbial community bacterial suspension to the film-forming agent solution is 1:1 to 2.

[0033] In some of these embodiments, the volume ratio of the complex microbial community bacterial suspension to the film-forming agent solution is 1:1.8 to 2.

[0034] In some of these embodiments, the 16S rDNA sequence of Pseudomonas chlororaphis is as shown in SEQ ID NO:1, the 16S rDNA sequence of Pseudomonas kribbensis is as shown in SEQ ID NO:2, and the 16S rDNA sequence of Bacillus velezensis is as shown in SEQ ID NO:3.

[0035] In some of these embodiments, the ratio of the colony counts of Pseudomonas chlororaphis, Pseudomonas kribbensis, and Bacillus velezensis is 1:1:0.2 to 5.

[0036] In some of these embodiments, the ratio of the colony counts of Pseudomonas chlororaphis, Pseudomonas kribbensis, and Bacillus velezensis is 1:1:0.5 to 1.5.

[0037] In some of these embodiments, the ratio of the colony counts of Pseudomonas chlororaphis, Pseudomonas kribbensis, and Bacillus velezensis is 1:1:0.8 to 1.2.

[0038] In some of these embodiments, the ratio of the colony counts of Pseudomonas chlororaphis, Pseudomonas kribbensis, and Bacillus velezensis is 1:1:0.9 to 1.1.

[0039] In some of these embodiments, the microbial seed coating agent further includes an adsorbent and an emulsifier. The dosage of the adsorbent accounts for 0.2 wt% to 0.5 wt% of the seed coating agent, and the dosage of the emulsifier accounts for 0.2 wt% to 0.5 wt% of the seed coating agent.

[0040] In some of these embodiments, the adsorbent is bentonite or kaolin.

[0041] In some of these embodiments, the emulsifier is methylcellulose or sodium carboxymethylcellulose.

[0042] In some other embodiments of the present invention, a method for preparing the above-mentioned microbial seed coating agent is disclosed, including the following steps:

[0043] (1) Prepare a complex microbial community bacterial suspension from Pseudomonas chlororaphis with the accession number CP019399.1, Pseudomonas kribbensis with the accession number PP218673, and Bacillus velezensis with the accession number MF192765.1;

[0044] (2) Dissolve sodium alginate and arabic gum in water to prepare a film-forming agent solution;

[0045] (3) Mix the complex microbial community bacterial suspension and the film-forming agent solution in a volume ratio of 1:0.5 - 2 to obtain the product.

[0046] In some other embodiments of the present invention, the application of the above microbial seed coating agent in promoting crop growth or preventing and controlling bacterial wilt of crops is disclosed.

[0047] In some other embodiments of the present invention, a method for promoting crop growth is disclosed, which includes the following steps: coating crop seeds with the above microbial seed coating agent.

[0048] In some other embodiments of the present invention, a method for preventing and controlling bacterial wilt of crops is disclosed, which includes the following steps: coating crop seeds with the above microbial seed coating agent.

[0049] In some of the embodiments, the crops are chili peppers, tomatoes, eggplants or potatoes.

[0050] The chili pepper samples used in the following examples of the present invention are healthy chili pepper samples collected from Dawanzhen, Yingde.

[0051] The present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0052] Example 1

[0053] In this example, endophytes were isolated and cultured from the roots and leaves of the collected healthy chili pepper samples as the tested biocontrol bacteria, and antagonistic bacteria against bacterial wilt were screened. The specific method is as follows:

[0054] 1. Cut the chili pepper roots and leaves, first wash the surface soil with tap water, and then repeatedly wash with sterile water for several times; after washing, dry with sterile filter paper, soak in 75% alcohol for 2 min and 3% sodium hypochlorite for 5 min for surface disinfection respectively, discard the disinfectant solution and then rinse with sterile water for several times, and dry with sterile filter paper; take 100 μL of the sterile water from the last rinse and spread it on the LB medium (tryptone 10 g, yeast extract powder 5 g, sodium chloride 10 g, deionized water 1000 mL, add 1.8% agar powder to obtain the solid medium, autoclave at 121 °C for 20 min) as a control to verify the disinfection effect;

[0055] 2. Grind the chili pepper samples with a sterilized mortar, and dilute the grinding solution with 0.01 mol / L phosphate buffer (PBS) in gradient (10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7After dilution (by a factor of [X]), 100 μL of each was spread on LB medium and cultured at 30 °C for 2 - 3 days, followed by further streak purification for 2 - 3 times. The isolated endophytes were identified by 16S rDNA molecular biology and stored at -80 °C in a refrigerator with a glycerol concentration of 30% for future use.

[0056] 3. Using Ralstonia solanacearum (screened from pepper plants suffering from bacterial wilt by conventional methods) as a biocontrol indicator bacterium, the strains with antagonistic ability against Ralstonia solanacearum were screened by the plate confrontation method. After activating Ralstonia solanacearum, it was inoculated into 250 mL of modified NA liquid medium (10 g of glucose, 5 g of bacteriological peptone, 3 g of beef extract, 0.5 g of yeast powder, 1000 mL of deionized water, autoclaved at 121 °C for 20 min). It was cultured with shaking at 30 °C and 180 rpm in a constant temperature shaker for 48 h, and the bacterial concentration OD 600 was adjusted to 1.0 and added to the modified NA solid medium (10 g of glucose, 5 g of bacteriological peptone, 3 g of beef extract, 0.5 g of yeast powder, 1000 mL of deionized water, plus 1.8% agar powder to make it a solid medium, autoclaved at 121 °C for 20 min) cooled to about 50 °C at an addition amount of 1% to make a plate with the pathogenic bacterium. After the plate solidified, a hole with a diameter of 9 mm was punched in the center of the plate containing the pathogenic bacterium. 100 μL of the overnight-cultured test biocontrol bacterial solution (OD 600 = 1) was added to each hole, and each treatment was repeated 3 times. It was cultured in an incubator at 30 °C for 2 days, and the presence of an inhibition zone was observed.

[0057] A total of 14 strains with inhibitory ability against Ralstonia solanacearum were preliminarily screened from the test strains (Table 1), namely J△, X5, A2, A10, A19, Z50, Z67, Z68, Z81 - 1, Z104, Z108, Z117, C5, C33.

[0058] Table 1 Determination of the antagonistic ability of the test strains

[0059]

[0060]

[0061] Note: In the table, the symbol "+" indicates having antagonistic ability, and the symbol "-" indicates not having antagonistic ability.

[0062] The plate antagonistic activities of the 14 screened strains were compared, and the results are shown in Table 2.

[0063] Table 2 Statistics of the plate antagonistic activities of the antagonistic bacteria

[0064]

[0065] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the numbers in the same column indicate the significance of differences between treatments (Duncan's method, P = 0.05).

[0066] Table 2 results showed that strains C5, C33, A2, Z68, Z117, J△, and Z50 had higher antagonistic activities, and the diameters of the inhibition zones were 25.50 mm, 28.42 mm, 29.50 mm, 28.15 mm, 28.60 mm, 28.29 mm, and 29.14 mm, respectively. Among them, the subsequent activities of strains J△ and Z50 were not good and were not suitable as strains for controlling bacterial wilt. Strains C5, C33, A2, Z68, and Z117 could be used as alternative strains for controlling bacterial wilt.

[0067] Example 2

[0068] Analysis of the ability of the five strains C5, C33, A2, Z68, and Z117 screened in Example 1 to metabolize disease-resistant promoting substances was carried out, and the results are shown in Table 3.

[0069] Table 3 Analysis of the ability to metabolize disease-resistant promoting substances

[0070]

[0071]

[0072] Note: In the table, the symbol "+" indicates the ability to metabolize this substance, and the symbol "-" indicates the inability to metabolize this substance

[0073] It can be seen from Table 3 that all five strains had the ability to produce auxin and form biofilms, and none of them had the ability to fix nitrogen and produce cellulase. Strains C33, A2, and Z68 had the ability to produce protease and siderophore, and strain Z117 did not have the ability to produce siderophore. Therefore, strains C33, A2, and Z68 may have the effect of controlling bacterial wilt and were used for further experiments.

[0074] The method of Example 1 was used to compare the plate antagonistic activities of strains C33, A2, Z68, and their combinations, and the results are shown in Table 4.

[0075] Table 4 Statistics of the plate antagonistic activities of antagonistic bacteria

[0076]

[0077] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the numbers in the same column indicate the significance of differences between treatments (Duncan's method, P = 0.05).

[0078] As can be seen from the results in Table 4, there were no significant differences in the diameters of the inhibition zones among strains C33, Z68, and the combinations of strains A2, C33, and Z68; while for strain A2 and the combination of strains A2, C33, and Z68, the diameters of the inhibition zones were significantly larger than those of other strains or their combinations. This result preliminarily suggests that strain A2 or the complex microbial community composed of strains A2, C33, and Z68 may have a more excellent effect on controlling bacterial wilt.

[0079] The 16S rDNA sequence of C33 is shown in SEQ ID NO:1, and it is Pseudomonas chlororaphis with the accession number CP019399.1; the 16S rDNA sequence of A2 is shown in SEQ ID NO:2, and it is Pseudomonas kribbensis with the accession number PP218673; the 16S rDNA sequence of Z68 is shown in SEQ ID NO:3, and it is Bacillus velezensis with the accession number MF192765.1.

[0080] C33 16S rDNA(SEQ ID NO:1)

[0081]

[0082] A2 16S rDNA (SEQ ID NO:2)

[0083]

[0084] Z68 16S rDNA (SEQ ID NO:3)

[0085]

[0086] Example 3 Influence of Different Auxiliaries on the Composite Microbial Population

[0087] By the plate confrontation method, bentonite, kaolin, sodium dodecyl benzene sulfonate, methyl cellulose, sodium carboxymethyl cellulose, lecithin, gum arabic (GA), and sodium alginate (SA) were respectively subjected to confrontation experiments with the plate of the composite microbial population A2+C33+Z68 screened in Example 2 at the addition amounts of 1%, 2%, and 3%. The results are shown in Table 5.

[0088] Table 5

[0089]

[0090]

[0091] Note: In the table, the symbol "+" indicates an antagonistic effect between the auxiliary and the strain, and the symbol "-" indicates no antagonistic effect between the auxiliary and the strain.

[0092] The results in Table 5 show that sodium dodecyl benzene sulfonate at 1%, 2%, and 3% has an inhibitory effect on the composite microbial population A2+C33+Z68, and other auxiliaries have no inhibitory effect on the composite microbial population. Since lecithin needs to be stored at low temperature, it is not suitable as an auxiliary for seed coating agents. Sodium alginate and gum arabic can be used as film-forming agents for composite microbial seed coating agents, bentonite and kaolin can be used as adsorbents, and sodium carboxymethyl cellulose and methyl cellulose can be used as emulsifiers.

[0093] Example 4 Comparison of the Influence of Two Film-Forming Agents with Different Ratios on the Germination of Pepper Seeds

[0094] In this example, the influence of different weight ratios of sodium alginate and gum arabic in the film-forming agent solution on the germination of pepper seeds (pepper seeds wrapped with the film-forming agent) was compared. The results are shown in Table 6.

[0095] Table 6

[0096]

[0097] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the same-column numbers indicate the significance of differences between treatments (Duncan's method, p < 0.05). Among them, ck is pepper seeds without any treatment.

[0098] Table 6 results show that the higher the concentration of sodium alginate in the film-forming agent solution, the lower the germination rate of pepper seeds. Among them, the film-forming agent solutions with the best germination rates are those with sodium alginate SA: gum arabic GA = 1:9, sodium alginate SA: gum arabic GA = 2:8, sodium alginate SA: gum arabic GA = 3:7, and sodium alginate SA: gum arabic GA = 4:6. When the sodium alginate and gum arabic in the film-forming agent solution are in these ratios, there is no significant effect on seed germination.

[0099] Comparison of Physicochemical Properties of Two Film-Forming Agents with Different Ratios in Example 5

[0100] In this example, the film-forming agent solution was dropped on the surface of a glass slide and observed after air-drying. Among them, the mass ratios of sodium alginate and gum arabic in the film-forming agent solution were 1:9, 2:8, 3:7, and 4:6 respectively ((taking the total amount of the film-forming agent as 4 g, for example, sodium alginate: gum arabic = 1:9, take 0.4 g of sodium alginate and 3.6 g of gum arabic, dissolve in 100 mL of water to obtain). The results showed that there were fewer bubbles in the SA:GA = 1:9 and SA:GA = 2:8 treatments. Through optical microscopy, it was found that its surface was relatively smooth, but there were more cracks and obvious protrusions.

[0101] In addition, the physicochemical properties at different ratios were compared in terms of film-forming time, water absorption rate, swelling rate, coating uniformity, and coating shedding rate. The results are shown in Table 7.

[0102] Table 7

[0103]

[0104] Note: The data in the table represent the mean ± standard error (n = 3), and different letters after the same column of numbers indicate the significance of differences between treatments.

[0105] It can be seen from Table 7 that when the sodium alginate and gum arabic in the film-forming agent solution are in different ratios, the coating uniformity is above 90% and the film-forming time is within 10 min, indicating that it has a fast film-forming speed and is suitable for actual production applications. In terms of the coating shedding rate, the shedding rate of all treatments is lower than 10%. Among them, the shedding rate of SA:GA = 4:6 is the lowest, only 7.70%, showing excellent coating stability. At the same time, SA:GA = 4:6 also shows the best performance in terms of swelling rate and water absorption rate, reaching 31.2% and 19.0% respectively, indicating that it has good water absorption performance and swelling characteristics, can provide a suitable water environment for seeds, and promote seed germination and seedling growth.

[0106] Comparison of Low-Temperature Storage and Thermal Storage Stability of Different Microbial Seed Coatings in Example 6

[0107] High temperatures can accelerate the chemical reactions of microorganisms, leading to the degradation or inactivation of active ingredients. Good heat storage stability ensures that the seed coating agent can maintain long-term stability in high-temperature environments and reduce spoilage. Low temperatures can cause the seed coating agent to solidify, stratify, or crystallize, affecting its uniformity and application effect, and also affecting the release and action of active ingredients. Good low-temperature stability can ensure that it can still achieve the expected effect in low-temperature environments.

[0108] In this example, the stabilities of 12 different formulations of microbial seed coating agents in Table 8 were tested under low-temperature and heat storage conditions.

[0109] Table 8

[0110]

[0111]

[0112] The results showed that within 24 hours, HRB191, HRB192, HRB193, HRB282, HRB283, and HRB372 showed varying degrees of stratification at 4°C, 25°C, and 37°C. After 7 days, HRB281, HRB371, HRB461, and HRB462 showed stratification at 4°C, 25°C, and 37°C, while HRB373 and HRB463 did not show stratification at 4°C, 25°C, and 37°C. Among them, the storage stability results of different formulations of microbial seed coating agents at 37°C are as Figure 1 shown.

[0113] The strain activities of microbial seed coating agents HRB373 and HRB463 were measured at 40°C for 1 day, 7 days, 14 days, 28 days, 42 days, and 56 days. The results are as Figure 2 shown, and the results showed that the initial colony counts of RHB373 and RHB463 were 5.27×10 8 , 5.61×10 8 , respectively. The viable counts of live bacteria decreased slowly from 1 to 14 days. At 56 days, the viable counts of RHB373 and RHB463 were 1.25×10 8 , 1.38×10 8 , respectively. The active ingredients of RHB373 and RHB463 decreased by 76.28% and 75.40% respectively after two months. Although the strain activity decreased, after two months of storage, the effective microorganisms in the seed coating agent still maintained a certain activity and met the requirements of the GB 20287-2006 "Agricultural Microbial Agents" standard. Therefore, a certain proportion of film-forming agent can effectively protect the strain activity for a long time and meet the needs of actual production applications. However, stratification of RHB373 was observed after 56 days of storage.

[0114] Therefore, overall, the microbial seed coating agent RHB463 (4 g of sodium alginate, 6 g of gum arabic, 0.5 g of bentonite, 0.5 g of methyl cellulose, 1 g of warning color (scarlet red pigment), adding 100 mL of water, stirring evenly, and then mixing with the complex microbial suspension (1.0×10 8 cfu) in a volume ratio of 2:1) shows excellent performance in terms of film-forming time, coating uniformity, coating shedding rate, swelling rate, water absorption rate, storage stability, etc., and is an ideal seed coating agent formula.

[0115] Example 7 Effect of Microbial Seed Coating Agent Coated on Pepper Seeds on Their Growth

[0116] The uncoated and microbial seed coating agent RHB463-coated pepper seeds (as Figure 3 shown) were used for seedling raising. After transplanting and continuously observing for 30 days, the biomass of peppers was counted. To explore the effect of the complex microbial seed coating agent on the growth of pepper seeds at the seedling stage.

[0117] The effects of uncoated and microbial seed coating agent coating on the growth promotion effect of peppers are as Figure 4 and Table 9 shown.

[0118] Table 9

[0119] Index CK RHB463 Plant height (cm) 24.90±2.00b 28.94±2.66a Stem girth (mm) 4.29±0.38a 4.51±0.46a Fresh weight (g) 13.06±1.63b 16.18±1.27a Dry weight (g) 1.44±0.33a 1.52±0.20a Chlorophyll (mg / g) 8.15±0.11b 10.27±0.42a

[0120] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the same column of numbers indicate the significance of differences between treatments (t-test, p < 0.05).

[0121] Figure 4 The results in and Table 9 show that the plant height and fresh weight of the microbial seed coating agent RHB463 treatment group are significantly higher than those of the control CK treatment group, increasing by 16.22% and 23.89% respectively compared with the control group; there is no significant difference in the stem girth, above-ground dry weight between the microbial seed coating agent RHB463 treatment group and the CK treatment group. In addition, compared with the uncoated CK treatment group, the chlorophyll content in the microbial seed coating agent coating treatment group increases significantly, increasing by 26.01%.

[0122] The effects of uncoated and microbial seed coating agent coating on the root growth of peppers are as Figure 5 and Table 10 shown.

[0123] Table 10

[0124] Index CK RHB463 Total root length (cm) 1196.35±238.11b 1745.76±204.66a <![CDATA[Surface area (cm 2 )]]> 213.86±47.06b 321.29±29.00a <![CDATA[Root volume (cm 3 )]]> 3.06±0.79b 4.71±0.37a Average diameter (mm) 0.57±0.05a 0.59±0.03a

[0125] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the same column of numbers indicate the significance of differences between treatments (t-test, p < 0.05).

[0126] Figure 5 The results in Table 10 show that after coating with the microbial seed coating agent RHB463, the total root length, total surface area, and root volume are significantly higher than those of the uncoated treatment group. The total root length increased by 45.92%, the total surface area increased by 50.23%, and the root volume increased by 53.92%. There is no significant difference in the average root diameter between the microbial seed coating agent RHB463 and the uncoated treatment group.

[0127] The results of this example show that coating with the microbial seed coating agent can significantly increase the accumulation of pepper biomass and promote root development, and can better play its growth-promoting role.

[0128] Example 8 Control effect of microbial seed coating agent on bacterial wilt of pepper

[0129] This example compared the control effects of uncoated and microbial seed coating agent-coated on bacterial wilt of pepper. Through pot experiments, the disease onset time was continuously observed after inoculating with Ralstonia solanacearum. After 3 days of inoculating with Ralstonia solanacearum, the uncoated treatment (CK) began to show symptoms; after 5 days of inoculation, the disease symptoms began to appear in the treatment group coated with the microbial seed coating agent RHB463+RS. The control effects and disease index of each treatment group were statistically analyzed on the 10th day. The results are shown in Table 11 and Figure 6 as follows.

[0130] Table 11

[0131] Index CK RHB463 + RS Incidence rate (%) 76.67±15.28a 43.33±15.28b Disease index (%) 57.00±13.53a 23.33±3.51b Control effect (%) — 59.65±5.26

[0132] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the same column of numbers indicate the significance of differences between treatments (t-test, p < 0.05).

[0133] The results in Table 11 show that the disease index of the CK treatment reached 57.00%, while the disease index of the treatment group coated with the microbial seed coating agent RHB463+RS was 23.33%, which was 33.67% lower than that of the CK treatment group. It can be seen that compared with the treatment only inoculated with Ralstonia solanacearum, the microbial seed coating agent-coated treatment significantly delayed the onset time of bacterial wilt of pepper, and the disease development rate also slowed down. The control effect of the microbial seed coating agent treatment reached 59.65%, indicating that it has a significant effect in inhibiting bacterial wilt of pepper.

[0134] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0135] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A microbial seed coating agent, characterized in that, It includes a composite bacterial suspension and a film-forming agent solution with a volume ratio of 1:0.5 - 2; the composite bacteria group is Pseudomonas chlororaphis with accession number CP019399.1, Pseudomonas kribbensis with accession number PP218673, and Bacillus velezensis with accession number MF192765.1, and the film-forming agent solution includes sodium alginate with a concentration of 1wt% - 4wt% and arabic gum with a concentration of 6wt% - 9wt%.

2. The microbial seed coating agent according to claim 1, characterized in that, In the film-forming agent solution, the concentration of sodium alginate is 3wt% - 4wt%, and the concentration of arabic gum is 6wt% - 7wt%; preferably, the concentration of sodium alginate in the film-forming agent solution is 3.8wt% - 4wt%, and the concentration of arabic gum is 6wt% - 6.2wt%; more preferably, the concentration of sodium alginate in the film-forming agent solution is 3.9wt% - 4wt%, and the concentration of arabic gum is 6wt% - 6.1wt%.

3. The microbial seed coating agent according to claim 1, characterized in that, The concentration of the complex bacterial community bacterial suspension is 1.0×10 8 cfu to 1.0×10 9 cfu; And / or, the volume ratio of the composite bacterial suspension to the film-forming agent solution is 1:1 - 2, more preferably 1:

2.

4. The microbial seed coating agent according to claim 3, characterized in that, The 16S rDNA sequence of Pseudomonas chlororaphis is as shown in SEQ ID NO:1, the 16S rDNA sequence of Pseudomonas kribbensis is as shown in SEQ ID NO:2, and the 16S rDNA sequence of Bacillus velezensis is as shown in SEQ ID NO:3; And / or, the ratio of the colony numbers of Pseudomonas chlororaphis, Pseudomonas kribbensis, and Bacillus velezensis is 1:1:0.2 - 5, preferably 1:1:0.5 - 1.5, preferably 1:1:0.8 - 1.2, more preferably 1:1:0.9 - 1.

1.

5. The microbial seed coating agent according to any one of claims 1 to 4, characterized in that The seed coating agent further includes an adsorbent and an emulsifier, the dosage of the adsorbent accounts for 0.2wt% - 0.5wt% of the seed coating agent, and the dosage of the emulsifier accounts for 0.2wt% - 0.5wt% of the seed coating agent.

6. The microbial seed coating agent according to claim 5, wherein The adsorbent is bentonite or kaolin; the emulsifier is methylcellulose or sodium carboxymethylcellulose.

7. A method for preparing the microbial seed coating agent according to any one of claims 1 to 6, characterized in that, It includes the following steps: (1) Prepare a composite bacterial suspension from Pseudomonas chlororaphis with accession number CP019399.1, Pseudomonas kribbensis with accession number PP218673, and Bacillus velezensis with accession number MF192765.1; (2) Dissolve sodium alginate and arabic gum in water to obtain a film-forming agent solution; (3) Mix the composite bacterial suspension and the film-forming agent solution in a volume ratio of 1:0.5 - 2 to obtain the product.

8. Use of the microbial seed coating agent according to any one of claims 1 - 6 in promoting crop growth or controlling crop bacterial wilt.

9. A method for promoting crop growth or controlling bacterial wilt of crops, characterized in that, It includes the following steps: Coating crop seeds with the microbial seed coating agent according to any one of claims 1 - 6.

10. The method according to claim 9, wherein The crop is pepper, tomato, eggplant or potato, preferably pepper.

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