Microbial agent for preventing and treating cotton verticillium wilt as well as preparation method and application of microbial agent

Abstract

The invention discloses a microbial agent for preventing and treating cotton verticillium wilt and a preparation method and application thereof, the microbial agent comprises a bacterial strain of bacillus velezensis and / or a fermentation broth thereof, and an agriculturally acceptable solid carrier for loading the bacterial strain, and the preservation number of the bacterial strain is CCTCC NO: M 2022954. According to the microbial agent, bacillus velezensis BAC81712 serves as an antagonistic bacterium and is loaded on a solid carrier, and the microbial agent has the advantages of being convenient to use, low in microorganism colonization difficulty, high in bacteriostasis rate, stable in prevention and treatment effect and the like, can be widely used for preventing and treating cotton verticillium wilt in potted plants and fields, can remarkably reduce the morbidity of the cotton verticillium wilt and has a good application prospect. The cotton plant morphology can be obviously improved, the use value is high, and the application prospect is good.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological control technology, specifically relating to a microbial agent for controlling cotton Verticillium wilt, its preparation method, and its application. Background Technology

[0002] Verticillium wilt is one of the major diseases in cotton production, caused by Verticillium dahliae (… Verticillium dahliae ) or Verticillium globosum ( Verticillium albo - atrum Caused by [a disease name - missing in original text], this disease has spread widely in major cotton-producing countries worldwide, severely impacting cotton yield and quality. Typical symptoms of cotton Verticillium wilt include the following types: yellowing, wrinkling, reticulation, and purplish-red.

[0003] Traditional control methods include chemical control and screening for resistant varieties, but these methods suffer from problems such as environmental pollution and increased drug resistance in pathogens. In recent years, biological control has gained attention as a green and environmentally friendly approach, with Bacillus beleales (B. beleales) being a prominent example. Bacillus velezensis Bacillus belyssus is a microorganism with broad-spectrum antibacterial activity, capable of producing various antibacterial substances such as lipopeptides and antibacterial proteins. It has shown promising application prospects in plant disease control and has been successfully applied to the prevention and control of various plant diseases. However, conventional Bacillus belyssus is less effective against Verticillium dahliae (…). Verticillium dahliae ) or Verticillium globosum ( Verticillium albo - atrum The low inhibition rate of Bacillus berleis leads to poor control of cotton Verticillium wilt. At the same time, the existing control methods, which directly apply Bacillus berleis in the form of strains or fermentation broth to cotton seeds, seedlings or fields (soil), also have drawbacks such as high requirements for field conditions, difficulty in microbial colonization, and unstable control effects. These greatly limit the widespread application of Bacillus berleis in the control of cotton Verticillium wilt.

[0004] Therefore, obtaining a microbial agent for controlling cotton Verticillium wilt that is easy to use, has low difficulty in microbial colonization, high antibacterial rate, and stable control effect, as well as its preparation method, plays an important role in effectively controlling cotton Verticillium wilt and improving cotton yield and quality. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a microbial agent for controlling cotton Verticillium wilt that is easy to use, has low difficulty in microbial colonization, high antibacterial rate, and stable control effect, as well as its preparation method and application.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0007] A microbial agent for controlling cotton Verticillium wilt, said microbial agent comprising Bacillus belye ( Bacillus velezensis BAC81712The strain and / or its fermentation broth, and an agriculturally acceptable solid carrier for loading the strain; the strain has the accession number CCTCC NO: M 2022954.

[0008] In a further improvement of the aforementioned microbial inoculant, the solid carrier comprises at least one of mineral materials and biomass materials; the mineral materials comprise at least one of microcrystalline cellulose, diatomaceous earth, bentonite, kaolin, and talc; and the biomass materials comprise at least one of straw, pine shells, rice straw, camellia oleifera shells, peat moss, and animal manure.

[0009] The above-mentioned microbial inoculant is further improved by including plant extracts; the plant extracts are at least one of Phellodendron bark extract, Sophora flavescens extract, and Artemisia argyi extract.

[0010] The above-mentioned microbial inoculant is further improved by including an adjuvant, wherein the adjuvant is at least one of surfactant, binder, stabilizer, and pH adjuster; wherein the surfactant is at least one of Tween 20 and Tween 80; and wherein the stabilizer is an antioxidant.

[0011] As a general technical concept, the present invention also provides a method for preparing the above-mentioned microbial inoculant for controlling cotton Verticillium wilt, comprising the following steps: (1) Mix the Bacillus belye bacterial culture with a solid carrier to obtain a mixture; (2) Remove the liquid components from the mixture to obtain the microbial agent.

[0012] The above preparation method is further improved by adding plant extracts and adjuvants to the mixture in step (1); the amount of plant extracts added is 0.1% to 5% of the total mass of the mixture; the amount of adjuvants added is 0.1% to 10% of the total mass of the mixture.

[0013] In a further improvement to the above preparation method, in step (1), the amount of the solid carrier added is 0.1% to 15% of the total mass of the mixture; the concentration of the Bacillus belyssus culture is 1×10⁻⁶. 9 CFU / mL ~ 1×10 11 CFU / mL.

[0014] In a further improvement to the above preparation method, in step (2), the liquid components in the mixture are removed by centrifugation, and the solid product is collected to obtain the microbial agent; the centrifugation speed is 1000 rpm to 3000 rpm; the centrifugation time is 5 min to 20 min.

[0015] As a general technical concept, the present invention also provides the application of the above-mentioned microbial inoculant or the microbial inoculant prepared by the above-mentioned preparation method in the prevention and control of cotton Verticillium wilt.

[0016] The above application, in a further improvement, includes the following steps: S1. Dilute the microbial inoculant into an agriculturally acceptable formulation; S2. Apply the diluted microbial agent to cotton seeds, seedlings or soil to control cotton Verticillium wilt.

[0017] In a further improvement to the above application, in step S1, the dosage form is an agriculturally acceptable liquid, emulsion, suspension, powder, granule, wettable powder, or water-dispersible granule.

[0018] In a further improvement to the above application, in step S2, the diluted microbial agent is applied by seed coating, soil irrigation, or foliar spraying.

[0019] Compared with the prior art, the advantages of the present invention are as follows: (1) The microbial agent used in this invention for controlling cotton Verticillium wilt employs Bacillus belye (B. belye). Bacillus velezensis BAC81712 This invention exhibits significant inhibitory effects against Verticillium dahliae and Verticillium chrysogenum, effectively antagonizing the pathogen causing cotton Verticillium wilt. Compared to Bacillus belye in nature, the Bacillus belye used in this invention (…) Bacillus velezensis BAC81712 The inhibition rate of *Bacillus belycera* against the pathogen of cotton Verticillium wilt increased by 6.53%, thus effectively reducing the incidence of cotton Verticillium wilt under both potted and field conditions. Meanwhile, *Bacillus belycera* (…) Bacillus velezensis BAC81712 The strain exhibits good stability, with genetically stable antagonistic effects against cotton Verticillium wilt. The inhibitory effects of the fifth and sixth generation strains against cotton Verticillium wilt are not significantly different from the first generation, which is beneficial for improving the control efficacy of microbial agents. Based on this, *Bacillus belye* is loaded onto the interior and surface of a solid carrier. Under the action of the solid carrier, a suitable environment and necessary nutrients are provided for the growth, reproduction, and storage of *Bacillus belye*. On the one hand, this is beneficial for improving the colonization effect of *Bacillus belye*; on the other hand, in practical applications, it can be used directly after simple dilution, making it highly convenient to use. Furthermore, *Bacillus belye* (… Bacillus velezensis BAC81712The fermentation supernatant and cell disruption broth of this invention also have antibacterial effects against the pathogen of cotton Verticillium wilt. The bacterial solution, fermentation broth, and their products can all be subsequently used for the biological control of cotton Verticillium wilt, thus demonstrating good application value in agricultural production. This microbial agent has advantages such as ease of use, low difficulty in microbial colonization, high antibacterial rate, and stable control effect. It can be widely used to control cotton Verticillium wilt, significantly reducing the incidence of the disease and significantly improving cotton plant morphology. It has high application value and promising prospects.

[0020] (2) The present invention also provides a method for preparing a microbial agent for controlling cotton Verticillium wilt. First, the Bacillus berleis bacterial solution is mixed with a solid carrier, which can stably attach Bacillus berleis to the inside and surface of the solid carrier, which is beneficial to improving the adaptability of Bacillus berleis. Then, the liquid component in the mixture is removed, thereby removing the culture medium component in the Bacillus berleis bacterial solution, which can inhibit the growth and reproduction of other strains, and ultimately help to achieve the effective control of cotton Verticillium wilt by the microbial agent of the present invention. Attached Figure Description

[0021] Figure 1 This is a comparison diagram of the growth of cotton under different prevention and control conditions in Example 1 of the present invention.

[0022] Figure 2 The Bacillus belyssus in Example 3 of this invention ( Bacillus velezensis BAC81712 The inhibitory effect of strain 1 and wild-type Bacillus belyssus on the growth of Verticillium dahliae.

[0023] Figure 3 The Bacillus belyssus in Example 5 of this invention ( Bacillus velezensis BAC81712 Genetic stability bar graph of the effect of inhibiting the growth of Verticillium dahliae. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0025] The culture media used in the following examples are as follows: PDA medium: Accurately weigh 5g potato starch, 20g glucose and 15g agar, dissolve in an appropriate amount of distilled water, and then bring the volume up to 1L.

[0026] LB broth medium: Accurately weigh 10g tryptone, 5g yeast powder, 5g sodium chloride, and 1g glucose, dissolve in an appropriate amount of distilled water, and then bring the volume to 1L.

[0027] LB high glucose medium: Accurately weigh 10g tryptone, 5g yeast extract, 5g sodium chloride, 30g sucrose, and 15g glucose, dissolve in an appropriate amount of distilled water, and then bring the volume up to 1L.

[0028] LB agar medium: Accurately weigh 10g tryptone, 5g yeast extract, 5g sodium chloride, 1g glucose, and 15g agar, dissolve in an appropriate amount of distilled water, and then bring the volume to 1L.

[0029] LB agar high sugar medium: Accurately weigh 10g tryptone, 5g yeast powder, 5g sodium chloride, 15g agar, 30g sucrose, and 15g glucose, dissolve in an appropriate amount of distilled water, and then bring the volume to 1L.

[0030] Verticillium dahliae ( Verticillium dahliae The results have been published; see reference 1 (Zhao Weisong, Guo Qinggang, et al., Effects of cotton Verticillium wilt resistant and susceptible varieties on soil bacterial community structure during flowering and boll-forming stage, Chinese Agricultural Science, 2020, 5: 942-954).

[0031] Verticillium blacki Verticillium albo - atrum The information has been published; see reference 2 (Liu Xuetang, Guo Jincheng, et al., RAPD analysis of Verticillium wilt in major cotton-producing areas of China, Journal of North China Agricultural Sciences, 1999, 1: 107-114).

[0032] Example 1 A microbial agent for controlling cotton Verticillium wilt, the microbial agent comprising Bacillus belye ( Bacillus velezensis BAC81712 The strains, and an agriculturally acceptable solid carrier for loading the strains, wherein the solid carrier is a mineral material.

[0033] In this embodiment, Bacillus belye ( Bacillus velezensis BAC81712 The strain with accession number CCTCC NO: M 2022954 was deposited on June 23, 2022, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0034] In this embodiment, the mineral material is microcrystalline cellulose.

[0035] In other embodiments, the mineral material may also be at least one of diatomaceous earth, bentonite, kaolin, talc, and a mixture thereof with microcrystalline cellulose. In other embodiments, the solid carrier may also be a biomass material, wherein the biomass material includes at least one of straw, pine shells, rice straw, camellia shells, peat moss, and animal manure.

[0036] In this embodiment, the microbial inoculant also includes an adjuvant, which is a surfactant, specifically Tween 80. In other embodiments, the adjuvant may also be at least one of an adhesive, a stabilizer, or a pH adjuster, such as an antioxidant.

[0037] In other embodiments, the microbial inoculant also includes plant extracts, wherein the plant extracts are at least one of Phellodendron bark extract, Sophora flavescens extract, and Artemisia argyi extract.

[0038] A method for preparing a microbial inoculant for controlling cotton Verticillium wilt according to the above embodiment includes the following steps: (1) Preparation of Bacillus belesiensis bacterial culture (1.1) Take Bacillus belysinus ( Bacillus velezensis BAC81712 After inoculating the strain with LB high-glucose medium, it was placed in a shaker and cultured at 37°C and 180 rpm for 18 h. The absorbance was measured at 600 nm. When the OD value was higher than 2.5, the bacterial density was adjusted to 1 × 10⁻⁶. 9 CFU / mL was used to obtain Bacillus belye seed culture.

[0039] (1.2) Take 5 mL of Bacillus belye seed culture and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of LB high-glucose medium. Ferment and culture at 37℃ and 180 rpm for 24 h to obtain Bacillus belye bacterial culture. The concentration of this Bacillus belye bacterial culture is 1×10⁻⁶. 10 cfu / mL.

[0040] (2) Preparation of microbial inoculants (2.1) The amount of microcrystalline cellulose added is 12%. The Bacillus vesicle solution of Belize is mixed with microcrystalline cellulose and stirred evenly to obtain mixture A.

[0041] (2.2) Mix the mixture A with Tween 80 at a rate of 1% and stir until homogeneous to obtain mixture B.

[0042] (2.3) Centrifuge the mixture B at 2000 rpm for 15 min to remove the liquid components from the mixture B and collect the solid product to obtain the microbial agent.

[0043] In other embodiments, when the microbial agent also includes plant extracts, the amount of plant extracts added is 0.1% to 5% of the total mass of the mixture.

[0044] The application of the microbial inoculant prepared in this embodiment in the control of cotton Verticillium wilt specifically examines the control effect of the microbial inoculant on cotton Verticillium wilt under pot conditions, including the following steps: S1. Dilute the microbial agent with water to a dilution ratio of 0.5% to prepare a suspension; wherein the concentration of Bacillus belyceae in the suspension is 1×10⁻⁶. 3 CFU / mL ~ 1×10 8 CFU / mL.

[0045] S2. Spray the suspension onto the cotton leaves infected with the pathogen (Verticillium dahliae) in the flowerpot, and continue cultivating the cotton in the flowerpot to complete the control of cotton Verticillium wilt. In this step, the roots of a potted cotton plant with one true leaf stage are pinched off and soaked in the solution, with a pathogen concentration of 1×10⁻⁶. 9 The cotton was obtained by measuring cfu / mL (calculated as spore concentration).

[0046] Control group: Water was used instead of suspending agent, and all other conditions were the same.

[0047] During cotton cultivation, observe the growth of potted plants and calculate the disease incidence rate using the following formula: Incidence rate = (Number of diseased plants / Total number of plants) × 100%.

[0048] Tests showed that the incidence of Verticillium wilt was 86.97% in the control group, while the incidence of Verticillium wilt was 31.54% after spraying with the microbial agent of the present invention.

[0049] In addition, the growth of cotton in the control group and the experimental group was as follows: Figure 1 As shown.

[0050] Figure 1 This is a comparison diagram of the growth of cotton under different prevention and control conditions in Example 1 of the present invention.

[0051] Depend on Figure 1 It can be seen that in the blank group, the cotton leaves showed very serious yellowing due to the lack of spraying of control agents; while in the experimental group of Example 1, the cotton grew well and no yellowing of leaves was observed after spraying with the microbial agent of the present invention.

[0052] Therefore, the microbial agent prepared by this invention can not only significantly reduce the incidence of cotton Verticillium wilt, but also significantly improve the morphology of cotton plants.

[0053] Example 2 The application of a microbial inoculant in the control of cotton Verticillium wilt was investigated, specifically examining the control effect of the microbial inoculant on cotton Verticillium wilt under field conditions, including the following steps: S1. The microbial agent prepared in Example 1 is diluted with water to a dilution ratio of 0.5% to form a suspension; wherein the concentration of Bacillus belyceae in the suspension is 1×10⁻⁶. 3 CFU / mL ~ 1×108 CFU / mL.

[0054] S2. Thirty days after cotton sowing, spray the suspension onto the cotton in the cotton field at a rate of 100 L per acre to continue cultivating the cotton and complete the control of cotton Verticillium wilt.

[0055] Blank group: Water was used instead of the suspending agent in Example 2, and all other conditions were the same.

[0056] CK bacterial agent group: The suspension prepared by wild-type Bacillus belye under natural conditions was used instead of the suspension in Example 2, and other conditions were the same.

[0057] CK bacterial suspension group: CK bacterial suspension prepared from wild-type Bacillus belye under natural conditions was used instead of the suspending agent in Example 2, with other conditions remaining the same.

[0058] In this embodiment, the suspension prepared from wild-type Bacillus belyes under natural conditions is prepared using a method that is basically the same as that used in Example 2, except that wild-type Bacillus belyes under natural conditions is used instead of Bacillus belyes in Example 2. Bacillus velezensis BAC81712 ).

[0059] In this embodiment, the CK bacterial suspension prepared from wild-type Bacillus belyes under natural conditions is prepared using a method that is basically the same as the method for preparing the Bacillus belyes bacterial suspension in Example 1, except that wild-type Bacillus belyes under natural conditions is used instead of Bacillus belyes in Example 1. Bacillus velezensis BAC81712 ).

[0060] During cotton cultivation, the growth of cotton under experimental conditions was observed, and the disease incidence rate was calculated according to the following formula: Incidence rate = (Number of diseased plants / Total number of plants) × 100%.

[0061] Tests showed that the incidence of Verticillium wilt in the blank group was 31.65%, in the CK bacterial inoculum group it was 25.76%, and in the CK bacterial agent group it was 13.29%, while the incidence of Verticillium wilt in Example 2 of this invention was 9.98%. Therefore, it is evident that preparing a microbial agent from Bacillus berberis bacterial inoculum in this invention is more effective in controlling Verticillium wilt in cotton. Furthermore, compared to wild-type Bacillus berberis, the Bacillus berberis used in this invention (…) Bacillus velezensis BAC81712 This is more conducive to improving the control effect of cotton Verticillium wilt.

[0062] Example 3 The inhibitory effect of Bacillus belyss on the pathogen of Verticillium wilt in cotton was investigated. Specifically, an antibacterial experiment was conducted on Bacillus belyss against the pathogen of Verticillium wilt in cotton, including the following steps: (1) The CK strain (wild-type Bacillus belyssus) preserved on slant and the mutagenized Bacillus belyssus ( Bacillus velezensis BAC81712 A single colony (treatment colony) was taken from each strain and inoculated into an Erlenmeyer flask containing 50 mL of LB high-glucose medium. The flasks were incubated at 37 ℃ and 180 rpm for 24 h, and the absorbance was measured to calculate the concentration of *Bacillus belyssiensis*. The concentrations of all strains were then adjusted to be approximately equal using LB high-glucose medium. Finally, the cultures were diluted with sterile physiological saline to adjust the bacterial concentration to an appropriate level.

[0063] (2) Inoculate the sclerotia of the cotton Verticillium wilt pathogen (Verticillium dahliae, Verticillium spp.) in the middle of a plate containing PDA medium.

[0064] (3) Place filter paper discs at both ends 2 cm away from the sclerotium, and take 10 μL of antagonistic bacteria (CK strain and Bacillus belysinus) respectively. BAC81712 The suspension was dropped onto filter paper and incubated at 28 °C for 5 days. The antibacterial effect was measured using calipers, and two replicates were performed for each strain.

[0065] Blank group: Water was used instead of antagonistic bacteria.

[0066] Pathogen inhibition rate = (Coronavirus colony diameter in antagonistic group / Coronavirus colony diameter in blank group) × 100%.

[0067] Antibacterial inhibition rate = (antibacterial inhibition rate of mutant strain - antibacterial inhibition rate of CK strain) / antibacterial inhibition rate of CK strain × 100%.

[0068] Figure 2 The Bacillus belyssus in Example 3 of this invention ( Bacillus velezensis BAC81712 The inhibitory effect of strain 1 and wild-type Bacillus belyssus on the growth of Verticillium dahliae. Figure 3 In the CK strain group, wild-type Bacillus belye was identified. The BAC81712 strain group is Bacillus belesiensis ( Bacillus velezensis BAC81712 ).

[0069] Depend on Figure 2 It can be seen that in the blank group, the colony diameter of Verticillium dahliae was 3.16±0.08 cm; and in the CK strain group, the colony diameter of Verticillium dahliae was 1.01±0.04 cm. BAC81712 The colony diameter of *Verticillium dahliae* in the strain group was 0.90 ± 0.02 cm. It was calculated that *Bacillus belyssioides* (…) Bacillus velezensis BAC81712The inhibition rate of *Verticillium dahliae* was 71.52%, while the inhibition rate of wild-type *Bacillus belye* against *Verticillium dahliae* was 68.04%. Therefore, *Bacillus belye* (…) Bacillus velezensis BAC81712 The *Bacillus belysinus* strain exhibited a 5.11% higher inhibition rate against *Verticillium dahliae* compared to the wild-type strain, indicating that the *Bacillus belysinus* strain of this invention... Bacillus velezensis BAC81712 The strain showed a significant inhibitory effect on the growth of Verticillium dahliae, and the effect was better than that of the CK strain.

[0070] In addition, in this embodiment, the colony diameter of *Verticillium chrysogenum* in the blank group was 3.33 ± 0.07 cm, and the colony diameter of *Verticillium chrysogenum* in the CK strain group was 1.21 ± 0.05 cm. BAC81712 The colony diameter of *Verticillium globosum* in the strain group was 0.96 ± 0.05 cm. It was calculated that *Bacillus belyssioides* (…) Bacillus velezensis BAC81712 The inhibition rate of *Verticillium glomeratum* against *Verticillium glomeratum* was 71.17%, while the inhibition rate of wild-type *Bacillus belyss* against *Verticillium glomeratum* was 63.66%. Therefore, *Bacillus belyss* (…) Bacillus velezensis BAC81712 The inhibitory rate of this strain against Verticillium chrysogenum was 11.80% higher than that of the wild-type strain.

[0071] Example 4 The inhibitory effect of Bacillus belyss fermentation broth on Verticillium wilt pathogen of cotton was investigated. Specifically, an antibacterial experiment was conducted on the pathogen using Bacillus belyss fermentation broth, including the following steps: (1) CK strain (wild-type Bacillus belyssus) preserved on slant and mutant Bacillus belyssus ( Bacillus velezensis BAC81712 Take a single colony (treated colony) from the strain, inoculate it into LB high sugar medium, and place it in a shaker. Incubate at 37°C and 80 rpm for 18 h.

[0072] (2) Shake the bacterial cultures obtained above well and measure their absorbance at 600 nm. When the OD value is higher than 2.5, adjust the bacterial density to 1×10⁻⁶. 9 After obtaining cfu / mL, 5 mL of bacterial culture was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB high-glucose medium and fermented on a shaker at 37℃ and 80 rpm for 24 h.

[0073] (3) The fermentation broth from step (2) was dispensed into 10 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min. The supernatant was then passed through a 0.45 μm organic membrane (Tianjin Jinteng Experimental Equipment Co., Ltd.) and a 0.22 μm cell-specific filter membrane (Millipore, USA) to obtain sterile fermentation supernatant.

[0074] (3) Using a punch, make 6 mm diameter holes in both *Verticillium dahliae* and *Verticillium chrysogenum* plates that have been fully colonized. Place the colonized cells in the center of a PDA-containing medium plate using tweezers. Using the four-point method, place Oxford cups 2.5 cm around the center of each colonized cell. Add 200 μL of sterile fermentation supernatant to each Oxford cup. Add 200 μL of sterile water to the Oxford cup used as a blank control. Perform three replicates for each strain. Incubate the plates at 28°C. After the blank control pathogen has fully colonized the plates, measure the diameter of the pathogen on each plate using calipers. Calculate the inhibition rate.

[0075] Pathogen inhibition rate = (Coronavirus colony diameter in antagonistic group / Coronavirus colony diameter in blank group) × 100%.

[0076] Antibacterial inhibition rate = (antibacterial inhibition rate of mutant strain - antibacterial inhibition rate of CK strain) / antibacterial inhibition rate of CK strain × 100%.

[0077] In this embodiment, the CK strain fermentation broth showed an inhibition rate of 62.11% against *Verticillium dahliae* and 61.59% against *Verticillium chrysogenum*. *Bacillus belyssus* (… Bacillus velezensis BAC81712 The fermentation broth of strain ( ) showed an inhibition rate of 65.31% against *Verticillium dahliae* and 65.09% against *Verticillium spp.*. This indicates that *Bacillus belye* ( ) exhibits strong antibacterial activity against *Verticillium dahliae*. Bacillus velezensis BAC81712 The strain showed a 5.15% increase in inhibition rate against Verticillium dahliae and a 5.68% increase in inhibition rate against Verticillium chrysogenum.

[0078] In addition, in this embodiment, Bacillus belye ( Bacillus velezensis BAC81712 The inoculation density of the strain was 1×10⁻⁶. 9 The cfu / mL was adjusted to 1×10⁻⁶. 7 After CFU / mL, the resulting Bacillus belysinus ( Bacillus velezensis BAC81712 The inhibition rate of the fermentation broth of the strain against Verticillium dahliae became 31.17%, and the inhibition rate against Verticillium chrysogenum became 33.31%.

[0079] In this embodiment, Bacillus belye ( Bacillus velezensis BAC81712 The inoculation density of the strain was 1×10⁻⁶. 9 The cfu / mL was adjusted to 1×10⁻⁶. 8 After CFU / mL, the resulting Bacillus belysinus ( Bacillus velezensis BAC81712 The fermentation broth of the strain showed an inhibition rate of 58.22% against Verticillium dahliae and 57.98% against Verticillium chrysogenum.

[0080] In this embodiment, Bacillus belye ( Bacillus velezensis BAC81712 The inoculation density of the strain was 1×10⁻⁶. 9The cfu / mL was adjusted to 1×10⁻⁶. 10 After CFU / mL, the resulting Bacillus belysinus ( Bacillus velezensis BAC81712 The fermentation broth of the strain showed an inhibition rate of 64.39% against Verticillium dahliae and 63.65% against Verticillium chrysogenum.

[0081] In this embodiment, Bacillus belye ( Bacillus velezensis BAC81712 The inoculation density of the strain was 1×10⁻⁶. 9 The cfu / mL was adjusted to 1×10⁻⁶. 11 After CFU / mL, the resulting Bacillus belysinus ( Bacillus velezensis BAC81712 The fermentation broth of the strain showed an inhibition rate of 48.12% against Verticillium dahliae and 47.98% against Verticillium chrysogenum.

[0082] Example 5 Investigating Bacillus belysinus ( Bacillus velezensis BAC81712 The study of the genetic stability of ) includes the following steps: (1) Subculture: cultured from CK and numbered BAC81712 A loopful of single colonies from the primary generation of Bacillus belye was picked and placed in LB broth. After incubation at 37°C and 180 rpm for 24 h in a shake flask, a loopful was then taken from an Erlenmeyer flask and streaked onto an LB agar plate until single colonies appeared. Then, single colonies were picked and incubated in LB broth at 37°C and 180 rpm for 24 h in a shake flask. This shake flask-plate-shake flask cycle was repeated. Finally, single colonies from the first to sixth generations were picked and streaked onto test tube slants.

[0083] (2) Preparation of Bacillus belysinus bacterial suspension: Strains from the first to sixth generation and the CK strain were selected, along with strains numbered as follows: BAC81712 Bacillus belye was cultured in an Erlenmeyer flask containing 50 mL of LB high-glucose medium at 37 °C and 180 r / min for 16 h. The absorbance was then measured to calculate the concentration of Bacillus belye, and the concentration was adjusted to near the target concentration using LB high-glucose medium.

[0084] (3) Culture of pathogens: Dahlia Verticillium was inoculated into the center of a plate containing PDA medium using the spot inoculation method and cultured at 28°C until the plate was completely covered.

[0085] (4) Genetic stability verification test: Using a punch, create mycelial discs of the same size at the edge of the grown pathogen and inoculate them into the center of a new PDA medium plate. After incubating at 28°C for one day, place filter paper discs at the four ends 2.5 cm from the center of the pathogen mycelial discs using the cross-hatching method. Drip 10 μL of bacterial suspension onto each filter paper disc (each plate contains the same strain). Perform three replicates for each strain (the blank control is a filter paper disc with 10 μL of sterile water). Incubate at 28°C. After the blank control pathogen has fully grown on the plate, measure the antibacterial effect using calipers. Calculate the inhibition rate.

[0086] Pathogen inhibition rate = (Coronavirus colony diameter in antagonistic group / Coronavirus colony diameter in blank group) × 100% Antibacterial inhibition rate = (Antibacterial inhibition rate of mutant strain - Antibacterial inhibition rate of CK strain) / Antibacterial inhibition rate of CK strain × 100% Figure 3 The Bacillus belyssus in Example 5 of this invention ( Bacillus velezensis BAC81712 Genetic stability bar graph of the effect of inhibiting the growth of Verticillium dahliae. Figure 3 In this study, the CK strain was wild-type Bacillus belye. BAC81712 The strain is Bacillus belyssus ( Bacillus velezensis BAC81712 ).

[0087] from Figure 3 It can be known that Bacillus belyssus ( Bacillus velezensis BAC81712 The first to sixth generations showed a consistent effect in inhibiting the growth of Verticillium dahliae, demonstrating good genetic stability.

[0088] The results above show that the microbial agent of the present invention has the advantages of convenient use, low difficulty in microbial colonization, high antibacterial rate, and stable control effect. It can be widely used to control cotton Verticillium wilt, significantly reduce the incidence of cotton Verticillium wilt, and significantly improve the morphology of cotton plants. It has high use value and good application prospects.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A microbial inoculant for controlling Verticillium wilt in cotton, characterized in that, The microbial agent includes Bacillus bellis (B. lesions) Bacillus velezensis BAC81712 The strain and / or its fermentation broth, and an agriculturally acceptable solid carrier for loading the strain; the strain has the accession number CCTCC NO: M 2022954.

2. The microbial agent according to claim 1, characterized in that, The solid carrier includes at least one of mineral materials and biomass materials; the mineral materials include at least one of microcrystalline cellulose, diatomaceous earth, bentonite, kaolin, and talc; the biomass materials include at least one of straw, pine shells, rice straw, camellia shells, peat moss, and animal manure.

3. The microbial agent according to claim 1 or 2, characterized in that, The microbial agent also includes plant extracts; the plant extracts are at least one of Phellodendron bark extract, Sophora flavescens extract, and Artemisia argyi extract.

4. The microbial agent according to claim 1 or 2, characterized in that, The microbial agent also includes adjuvants, which are at least one of surfactants, binders, stabilizers, and pH adjusters; the surfactants are at least one of Tween 20 and Tween 80; and the stabilizers are antioxidants.

5. A method for preparing a microbial inoculant for controlling cotton Verticillium wilt as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Mix the Bacillus belye bacterial culture with a solid carrier to obtain a mixture; (2) Remove the liquid components from the mixture to obtain the microbial agent.

6. The preparation method according to claim 5, characterized in that, Step (1) further includes adding plant extracts and adjuvants to the mixture; the amount of plant extracts added is 0.1% to 5% of the total mass of the mixture; the amount of adjuvants added is 0.1% to 10% of the total mass of the mixture.

7. The preparation method according to claim 6, characterized in that, In step (1), the amount of the solid carrier added is 0.1% to 15% of the total mass of the mixture; the concentration of the Bacillus belyssus culture is 1×10⁻⁶. 9 CFU / mL ~ 1×10 11 CFU / mL; In step (2), the liquid components in the mixture are removed by centrifugation, and the solid product is collected to obtain the microbial agent; the centrifugation speed is 1000 rpm to 3000 rpm; the centrifugation time is 5 min to 20 min.

8. The application of a microbial agent according to any one of claims 1 to 4 or a microbial agent prepared by any one of claims 5 to 7 in the prevention and control of cotton Verticillium wilt.

9. The application according to claim 8, characterized in that, The application includes the following steps: S1. Dilute the microbial inoculant into an agriculturally acceptable formulation; S2. Apply the diluted microbial agent to cotton seeds, seedlings or soil to control cotton Verticillium wilt.

10. The application according to claim 9, characterized in that, In step S1, the dosage form is an agriculturally acceptable liquid, emulsion, suspension, powder, granule, wettable powder, or water-dispersible granule. In step S2, the diluted microbial agent is applied by seed coating, soil irrigation, or foliar spraying.

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