Microbial agent for preventing and treating sclerotinia sclerotiorum and preparation method thereof

Abstract

The application discloses a microbial agent for preventing and treating sclerotinia sclerotiorum of rape and a preparation method thereof, and belongs to the technical field of microorganisms. The microbial agent for preventing and treating sclerotinia sclerotiorum of rape comprises bacillus circulans, microflavus oceanosediminis and paracoagulans brevitalea, and is prepared by compounding the three active strains, has a significant inhibiting effect on sclerotinia sclerotiorum of rape, and has good compatibility and synergistic effect among the three strains through scientific compounding in a specific volume ratio. The field prevention and treatment effect is obviously better than that of each strain used alone, the disease index of sclerotinia sclerotiorum can be effectively reduced, and the prevention and treatment effect of sclerotinia sclerotiorum of rape is improved. The microbial agent is a liquid preparation, is easy to dilute and use, and can be uniformly attached to the surface of rape plants and the rhizosphere through foliar spraying, root irrigation and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial agent for the prevention and control of sclerotinia stem rot in rapeseed and its preparation method. Background Technology

[0002] Sclerotinia sclerotiorum, a global fungal disease of rapeseed, is caused by the fungus *Sclerotinia sclerotiorum*. This pathogen has an extremely wide host range and survives for extended periods in the soil and plant debris as sclerotia, making control extremely difficult. In years with widespread outbreaks, rapeseed yields are often reduced by 20%-30%, with severely affected fields experiencing total crop failure. It also reduces the oil content and quality of the rapeseed. Traditionally, control of this disease has relied on chemical fungicides such as carbendazim and sclerotinia fungicides. However, long-term, large-scale use of single-drug therapy has led to increasing resistance in the pathogen. Furthermore, the impact of chemical pesticide residues on the farmland ecosystem, non-target organisms, and the safety of rapeseed oil is a major concern. Against this backdrop, environmentally friendly microbial agents that are less prone to developing resistance are playing an increasingly important role in the green control system for sclerotinia sclerotiorum.

[0003] Extensive research has been conducted on the use of antagonistic microorganisms to control sclerotinia stem rot in rapeseed. Currently reported biocontrol resources mainly include Trichoderma fungi (such as *Trichoderma harzianum* and *Trichoderma echinococcus*), *Coniothyrium minitans*, and various Bacillus species (such as *Bacillus subtilis*, *Bacillus amyloliquefaciens*, and *Bacillus cereus*). Additionally, *Pseudomonas* and *Streptomyces* have been used to a limited extent. However, single-strain microbial agents still face significant limitations in practical applications. First, most single strains have limited antibacterial spectrums, making it difficult to effectively block sclerotinia stem rot at different infection stages (such as ascospore release, petal infection, and hyphal expansion). Second, biocontrol bacteria are greatly affected by environmental factors such as temperature, humidity, and ultraviolet radiation in the field. Many strains with excellent inhibitory effects indoors often exhibit significant fluctuations in field efficacy, showing significantly lower stability compared to chemical agents. Third, some exogenously introduced biocontrol bacteria have insufficient colonization capacity in the foliar and rhizosphere of rapeseed, resulting in short-lasting effects and difficulty in forming a sustained protective barrier. To compensate for the limitations of single-strain formulations, multi-strain combinations have become an important direction in the research and development of microbial agents in recent years. By scientifically combining strains with different biocontrol mechanisms, it is hoped that complementary functions and synergistic effects can be achieved, thereby improving control efficacy and stability.

[0004] However, in actual research and development, most existing multi-strain compound inoculants are based on empirical mixing, lacking systematic research on the compatibility between strains, optimal ratios, and synergistic mechanisms. Furthermore, existing formulations are mainly powders, which suffer from poor dispersibility and weak adhesion, affecting the colonization and survival of the bacteria on plants. Therefore, screening for new strain combinations with good compatibility, significant synergistic effects, and stable control efficacy against rapeseed sclerotinia stem rot, and developing easy-to-apply, highly effective formulations, remains a pressing issue in current biological control technology. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial agent for the prevention and control of sclerotinia stem rot in rapeseed, which can effectively reduce the disease index of sclerotinia stem rot and improve the quality and yield of rapeseed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A microbial agent for controlling sclerotinia stem rot in rapeseed includes Bacillus circulatoryus, Bacillus microepiplos, and Micrococcus paraagnatus.

[0007] Furthermore, the *Bacillus circulans* was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC1.6486, with an original preservation date of November 1, 2006; *Bacillus microphagainii* was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC1.12406, with an original preservation date of November 7, 2012; and *Bacillus paraaggregatus* was purchased from the China General Microbiological Culture Collection Center (CGMCC1.9068), with an original preservation date of January 19, 2009.

[0008] Furthermore, the microbial agent for controlling sclerotinia stem rot in rapeseed is a liquid preparation.

[0009] A method for preparing a microbial inoculant for controlling sclerotinia stem rot in rapeseed includes the following steps: (1) After thawing Bacillus microepiploicus, activate it in LB solid medium, then pick a single colony and inoculate it into LB liquid medium, and culture it until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10% to cultivate. When the effective viable count reached 1×10 9 When the concentration of CFU / mL is reached, stop the culture to obtain the Bacillus microepiploicus fermentation broth. (2) After thawing Bacillus circularis and Micrococcus paraaggregates, they were activated in nutrient gravy agar solid medium. Single colonies were picked and inoculated into nutrient gravy liquid medium and cultured until OD500. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and fermentation broth of Bacillus circulans and Bacillus paraaggregates was obtained. (3) Mix the above-prepared Bacillus microysoderm fermentation broth, Bacillus circulans fermentation broth and Bacillus paraagnatus fermentation broth to obtain microbial inoculum.

[0010] Furthermore, in step 3, the fermentation broth of Bacillus microglobulus, the fermentation broth of Bacillus circulans, and the fermentation broth of Bacillus paraagnatus are mixed in a volume ratio of (1-2):(1-3):(1-2).

[0011] Furthermore, in step 3, the fermentation broth of Bacillus microglobulus, the fermentation broth of Bacillus circulans, and the fermentation broth of Micrococcus paragallinarum are mixed in a volume ratio of 2:2:1.

[0012] Beneficial effects This invention's microbial inoculant is a compound of three active bacterial strains: Bacillus circinus, Bacillus microepiplos, and Micrococcus paraagnatus. The fermentation broth of these three strains contains antibacterial active substances, which have a significant inhibitory effect on Sclerotinia sclerotinia, the causal agent of rapeseed. Through a scientific compounding process with a specific volume ratio, the three strains exhibit good compatibility and synergistic effects, resulting in significantly better field control than when any of the strains are used alone. This effectively reduces the disease index of Sclerotinia sclerotinia and improves the control effect of Sclerotinia sclerotinia in rapeseed.

[0013] The microbial agent of this invention is a liquid preparation, which is easy to dilute and apply. It can be evenly applied to the surface and rhizosphere of rapeseed plants through foliar spraying, root irrigation, and other methods. The microorganisms have a strong colonization ability on the surface of rapeseed and in the soil, and can occupy ecological niches first, forming a protective barrier and continuously preventing pathogen infection. Attached Figure Description

[0014] Figure 1 Figure A shows the effect of the microbial inoculant of Example 3 of the present invention on the sclerotinia stem rot of rapeseed, while the control group is shown in Figure B. Note: Figure A is the control group figure, and Figure B is the antagonistic figure of the microbial inoculant of Example 3 on the sclerotinia stem rot of rapeseed. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0016] Example 1 A microbial agent for controlling sclerotinia stem rot in rapeseed includes Bacillus circulatoryus, Bacillus microepiplos, and Micrococcus paraagnatus.

[0017] The strain of *Bacillus circulans* is numbered CGMCC1.6486; the strain of *Bacillus microepiplos* is numbered CGMCC1.12406; and the strain of *Bacillus paraaggregatus* is numbered CGMCC1.9068.

[0018] The microbial agent for controlling sclerotinia stem rot in rapeseed is a liquid preparation.

[0019] A method for preparing a microbial inoculant for controlling sclerotinia stem rot in rapeseed includes the following steps: (1) After thawing Bacillus microepiploicus, activate it in LB solid medium, then pick a single colony and inoculate it into LB liquid medium, and culture it until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10% to cultivate. When the effective viable count reached 1×10 9 When the concentration of CFU / mL is reached, stop the culture to obtain the Bacillus microepiploicus fermentation broth. (2) After thawing Bacillus circularis and Micrococcus paraaggregates, they were activated in nutrient gravy agar solid medium. Single colonies were picked and inoculated into nutrient gravy liquid medium and cultured until OD500. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and fermentation broth of Bacillus circulans and Bacillus paraaggregates was obtained. (3) Mix the above-prepared Bacillus microysoderm fermentation broth, Bacillus circulans fermentation broth and Bacillus paraagnatus fermentation broth to obtain microbial inoculum.

[0020] In step 3, the fermentation broth of Bacillus microepiploicus, the fermentation broth of Bacillus circulans, and the fermentation broth of Micrococcus paraagneissus are mixed in a volume ratio of 1:1:1.

[0021] Example 2 A microbial agent for controlling sclerotinia stem rot in rapeseed includes Bacillus circulatoryus, Bacillus microepiplos, and Micrococcus paraagnatus.

[0022] The strain of *Bacillus circulans* is numbered CGMCC1.6486; the strain of *Bacillus microepiplos* is numbered CGMCC1.12406; and the strain of *Bacillus paraaggregatus* is numbered CGMCC1.9068.

[0023] The microbial agent for controlling sclerotinia stem rot in rapeseed is a liquid preparation.

[0024] A method for preparing a microbial inoculant for controlling sclerotinia stem rot in rapeseed includes the following steps: (1) After thawing Bacillus microepiploicus, activate it in LB solid medium, then pick a single colony and inoculate it into LB liquid medium, and culture it until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10% to cultivate. When the effective viable count reached 1×10 9 When the concentration of CFU / mL is reached, stop the culture to obtain the Bacillus microepiploicus fermentation broth. (2) After thawing Bacillus circularis and Micrococcus paraaggregates, they were activated in nutrient gravy agar solid medium. Single colonies were picked and inoculated into nutrient gravy liquid medium and cultured until OD500.600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and fermentation broth of Bacillus circulans and Bacillus paraaggregates was obtained. (3) Mix the above-prepared Bacillus microysoderm fermentation broth, Bacillus circulans fermentation broth and Bacillus paraagnatus fermentation broth to obtain microbial inoculum.

[0025] In step 3, the fermentation broth of Bacillus microepiploicus, the fermentation broth of Bacillus circulans, and the fermentation broth of Micrococcus paraagneissus are mixed in a volume ratio of 2:2:1.

[0026] Example 3 A microbial agent for controlling sclerotinia stem rot in rapeseed includes Bacillus circulatoryus, Bacillus microepiplos, and Micrococcus paraagnatus.

[0027] The strain of *Bacillus circulans* is numbered CGMCC1.6486; the strain of *Bacillus microepiplos* is numbered CGMCC1.12406; and the strain of *Bacillus paraaggregatus* is numbered CGMCC1.9068.

[0028] The microbial agent for controlling sclerotinia stem rot in rapeseed is a liquid preparation.

[0029] A method for preparing a microbial inoculant for controlling sclerotinia stem rot in rapeseed includes the following steps: (1) After thawing Bacillus microepiploicus, activate it in LB solid medium, then pick a single colony and inoculate it into LB liquid medium, and culture it until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10% to cultivate. When the effective viable count reached 1×10 9 When the concentration of CFU / mL is reached, stop the culture to obtain the Bacillus microepiploicus fermentation broth. (2) After thawing Bacillus circularis and Micrococcus paraaggregates, they were activated in nutrient gravy agar solid medium. Single colonies were picked and inoculated into nutrient gravy liquid medium and cultured until OD500. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and fermentation broth of Bacillus circulans and Bacillus paraaggregates was obtained. (3) Mix the above-prepared Bacillus microysoderm fermentation broth, Bacillus circulans fermentation broth and Bacillus paraagnatus fermentation broth to obtain microbial inoculum.

[0030] In step 3, the fermentation broth of Bacillus microepiploicus, the fermentation broth of Bacillus circulans, and the fermentation broth of Micrococcus paraagneissus are mixed in a volume ratio of 2:3:2.

[0031] Comparative Example 1 Compared with Example 2, this comparative example uses only Bacillus microticus fermentation broth as the microbial agent, while the other raw materials and steps are the same as in Example 2.

[0032] Comparative Example 2 Compared with Example 2, this comparative example uses the same raw materials and steps as Example 2, except that only Bacillus circularis is used as the microbial agent.

[0033] Comparative Example 3 Compared with Example 2, this comparative example uses only the fermentation broth of *Bacillus paraaggregatus* as the microbial agent, while the other raw materials and steps are the same as in Example 2.

[0034] Performance testing Test for ability to inhibit pathogens: The antagonistic effect of microbial inoculants on the pathogen of Sclerotinia sclerotinia rot in rapeseed was determined using the plate confrontation culture method. Sclerotinia sclerotinia rot pathogens were cultured on LB agar plates at 30°C for 12 hours. A 5mm diameter Sclerotinia sclerotinia rot fungal disc was inoculated in the center of the petri dish. 10 μL of the microbial inoculant of Example 3 of this invention was inoculated approximately 2.5 cm to both sides of the pathogen center. A plate inoculated with 10 μL of sterile LB nutrient solution served as a control. Each treatment was repeated three times. The plates were inverted and incubated at 28°C for 3-5 days. When the pathogen in the control group had completely colonized the plate, the inhibitory effect of the microbial inoculant of this invention on the pathogen of Sclerotinia sclerotinia rot in rapeseed was observed. (See attached figures). Figure 1 (Figure A is the control group, and Figure B is the antagonistic effect of the microbial agent in Example 3 on sclerotinia rot in rapeseed).

[0035] Depend on Figure 1 As can be seen, compared with the control, the microbial agent prepared in Example 3 of the present invention can significantly inhibit the growth of mycelium of Sclerotinia sclerotinia rot pathogen in rapeseed and has an antagonistic effect on Sclerotinia sclerotinia rot.

[0036] Planting Trial The rapeseed variety tested was March Yellow.

[0037] The experiment consisted of seven treatment groups: treatment groups using the microbial inoculants prepared in Examples 1-3 and Comparative Examples 1-3, and a blank control group using water. Each experimental group was randomly assigned to a block design, and each treatment was repeated three times. 5 kg of sterilized seedling substrate was placed in each seedling tray, and 10 ml of *Sclerotinia sclerotiorum* mycelial suspension was applied to each tray. The mycelial suspension concentration was 5 × 10⁻⁶. 9The concentration of cfu / mL was increased, and 20 mL of the microbial inoculants prepared in Examples 1-3 and Comparative Examples 1-3 were added respectively. Rapeseed seeds were soaked in 5% NaClO for 1 min, then in 75% alcohol for 1 min for disinfection. After rinsing with sterile water, the seeds were sown in seedling trays. After emergence, thinning was carried out, leaving 100 rapeseed plants per tray. Watering was done every 3 days with a fixed amount of 300 mL of water.

[0038] Indicator determination: 30 days after rapeseed emergence, 30 seedlings were selected from each treatment group, i.e., 10 seedlings per replicate. The disease resistance of rapeseed seedlings in each treatment group was determined.

[0039] Grading standards for rapeseed sclerotinia stem rot: Level 0, no disease; Grade 1: The lesion area accounts for less than 10% of the total leaf area; Grade 2, with lesions covering 11-30% of the total leaf area; Grade 3, with lesions covering 31-50% of the total leaf area; Grade 4, with lesions covering more than 51% of the total leaf area.

[0040] Disease index = Σ(disease grade × number of plants at that disease grade) / (highest disease grade × total number of plants) × 100%; Control efficacy = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0041] Table 1. Effects of each treatment group on Sclerotinia rot in rapeseed.

[0042] As shown in Table 1, compared with the blank control, the microbial agents of Examples 1-3 of this invention can effectively control Sclerotinia stem rot in rapeseed. Among them, Example 2 showed the best control effect against Sclerotinia stem rot, reaching over 80%. Compared with Example 2, the control effects of Comparative Examples 1-3, which used only one strain of fermentation broth, on Sclerotinia stem rot were significantly reduced, indicating that the combination of three strains in the microbial agent of this invention is essential. The three strains exerted a synergistic effect, effectively improving the control effect against Sclerotinia stem rot in rapeseed.

[0043] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A microbial inoculant for controlling sclerotinia stem rot in rapeseed, characterized in that, This includes Bacillus circulans, Bacillus microepiploicus, and Paraaggregates.

2. The microbial agent for controlling sclerotinia stem rot in rapeseed according to claim 1, characterized in that, The strain number of the *Bacillus circulans* is CGMCC1.6486; the strain number of *Bacillus microepiplos* is CGMCC1.12406; and the strain number of *Paracillus paraaggregatus* is CGMCC1.9068.

3. The microbial agent for controlling sclerotinia stem rot in rapeseed according to claim 1, characterized in that, The microbial agent for controlling sclerotinia stem rot in rapeseed is a liquid preparation.

4. A method for preparing a microbial inoculant for controlling sclerotinia stem rot in rapeseed as described in claim 1, characterized in that, Preparation includes the following steps: (1) After thawing Bacillus microepiploicus, activate it in LB solid medium, then pick a single colony and inoculate it into LB liquid medium, and culture it until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10% to cultivate. When the effective viable count reached 1×10 9 When the concentration of CFU / mL is reached, stop the culture to obtain the Bacillus microepiploicus fermentation broth. (2) After thawing Bacillus circularis and Micrococcus paraaggregates, they were activated in nutrient gravy agar solid medium. Single colonies were picked and inoculated into nutrient gravy liquid medium and cultured until OD500. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and fermentation broth of Bacillus circulans and Bacillus paraaggregates was obtained. (3) The fermentation broth of Bacillus microysodeoxycholica, Bacillus circulans and Bacillus paraagnatus prepared above are mixed in a volume ratio of (1-2):(1-3):(1-2) to obtain microbial inoculum.

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