Bacillus safensis K112, culture method thereof, microbial inoculum containing bacillus safensis K112 and application of microbial inoculum

Abstract

The invention belongs to the technical field of microorganisms and application, and particularly relates to bacillus safensis K112, a culture method of the bacillus safensis K112, a fungicide containing the bacillus safensis K112 and application of the fungicide. The invention provides a bacillus safensis K112 with a preservation number of CCTCC NO: M 2025656, the bacillus safensis K112 is preserved in China Center for Type Culture Collection on March 31, 2025, and the preservation address is No. 299 on eight road in Wuchang District, Wuhan City, Hubei Province. The strain K112 not only can promote the mycelial growth of the morchella, but also can inhibit the growth of mortierella alpina, which is one of endophytes of the morchella, and the bacteriostasis rate is 63.53%; meanwhile, the strain can also promote the formation of bacterial cream, and the yield of the morchella esculenta is greatly improved by about 30%. The bacillus safensis K112 provided by the invention is of great significance to the cultivation industry of morchella esculenta.

Description

Technical Field

[0001] This invention belongs to the field of microbial and applied technology, specifically relating to a strain of Bacillus sabolicii K112 and its culture method, bacterial agents containing it, and its applications. Background Technology

[0002] Morel mushrooms are a rare edible fungus with high economic value, and their artificial cultivation industry has rapidly emerged in recent years. Morel mushrooms require soil cultivation to produce fruiting bodies, so soil microorganisms likely have a significant impact on their growth. Current research indicates that *Morchella alpineensis*, one of the endophytic fungi in morels, negatively affects their quality; however, a method has not yet been found that can inhibit *Morchella alpineensis* growth without negatively impacting morel quality.

[0003] *Bacillus safras*, belonging to the genus *Bacillus*, exhibits a variety of biocontrol functions: *Bacillus safras* isolated from tobacco rhizosphere soil showed an average control efficacy of 80.06% against tobacco black shank. *Bacillus safras* isolated from rapeseed rhizosphere soil in Linzhi, Tibet, achieved an antagonistic effect of over 50% against wheat scab; *Bacillus safras* isolated from peanut rhizosphere soil showed strong antagonistic activity against *Xanthomonas oryzaepv. oryzicola*, effectively preventing bacterial leaf streak in rice, and so on. These studies all indicate that *Bacillus safras*, as a potential biocontrol agent, possesses broad-spectrum and high-efficiency potential in the biocontrol of agricultural diseases. However, strains suitable for the biocontrol of alpine *Morchella* have not yet been discovered. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Bacillus salsa K112 and its culture method, a fungal agent containing the strain and its application. The Bacillus salsa K112 not only has an antagonistic effect on the endophytic fungus Moringa alpinea, but also promotes the mycelial growth and mycelial bloom formation of morel mushrooms, significantly increasing the yield of morel mushrooms.

[0005] To solve the above technical problems, the first aspect of the present invention provides a strain of Bacillus safensis K112, which was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCCNO: M 2025656, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The *Bacillus salsa* K112 strain provided by this invention was isolated and screened from saline-alkali soil. Based on morphological, physiological, and biochemical characteristics, and 16S rDNA sequencing results, strain K112 was preliminarily identified as *Bacillus salsa*. The colonies of strain K112 are milky white, smooth, viscous, and opaque, with irregular edges. The strain has a rod-like structure and is Gram-positive. Applying *Bacillus salsa* K112 provided by this invention to the soil before planting morel mushrooms significantly increases the yield per acre of morel mushrooms, which is of great significance for the artificial industrial cultivation of morel mushrooms.

[0007] The second aspect of this invention provides a method for culturing Bacillus safensis K112 with accession number CCTCC NO: M 2025656. Specifically, strain K112 is inoculated into LB medium, NA medium, SDA medium, BHI medium or NYBD medium and cultured in shake flasks at 23-31°C with an initial pH of 6.5-8.5. The volume of the shake flask medium is 30-150 mL / 250 mL, the inoculum size is 0.5%-5%, and the rotation speed is 140-220 r / min.

[0008] Preferably, the cultivation method of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 is as follows: strain K112 is inoculated into LB liquid medium and cultured in a shake flask at 25-27°C with an initial pH of 6.5-7.5; wherein the volume of the shake flask medium is 30-60 mL / 250 mL, the inoculation amount is 2%-3%, and the rotation speed is 200-220 r / min.

[0009] More preferably, the cultivation method of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 is as follows: strain K112 is inoculated into LB liquid medium and cultured in a shake flask at 25°C with an initial pH of 7; wherein the volume of the shake flask medium is 30 mL / 250 mL, the inoculation amount is 2%, and the rotation speed is 200 r / min.

[0010] A third aspect of the present invention provides a microbial agent, the active ingredient of which includes Bacillus safensis K112 with accession number CCTCC NO: M 2025656.

[0011] In conjunction with the third aspect, the dosage form of the microbial agent includes, but is not limited to, fermentation broth, powder, or suspension.

[0012] The fourth aspect of this invention provides the application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in the preparation of plant biocontrol agents and / or bio-organic fertilizers.

[0013] The Bacillus sarcodactylis K112 or its culture medium can be used as an active ingredient and combined with other excipients to prepare biocontrol agents and / or bio-organic fertilizers.

[0014] The fifth aspect of this invention provides the application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in inhibiting the growth of endophytic bacteria in morel mushrooms.

[0015] In conjunction with the fifth aspect, the morel mushrooms include, but are not limited to, morel mushrooms of the sixth genus, morel mushrooms of the seventh genus, and morel mushrooms of the tiered genus, and may also be other types of culturable morel mushrooms; the endophytic fungi include, but are not limited to, *Morchella alpina*.

[0016] The sixth aspect of this invention provides the application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in promoting morel mycelial growth, mycelial bloom formation and / or increasing morel yield.

[0017] In conjunction with the sixth aspect, the morel mushrooms mentioned include, but are not limited to, morel mushrooms of the sixth rank, morel mushrooms of the seventh rank, and morel mushrooms of the tiered ridge, and may also be other types of cultivable morel mushrooms.

[0018] The seventh aspect of the present invention provides a method for increasing morel mushroom yield, specifically: spraying an agent containing Bacillus saffron K112, a biocontrol agent, or a bio-organic fertilizer onto the soil surface, then turning the soil over, planting morel mushrooms, and then covering with a 0.5-1 cm thick layer of the same soil substrate, followed by conventional mushroom management.

[0019] In conjunction with the seventh aspect, inoculants, biocontrol agents, or bio-organic fertilizers containing Bacillus sabolicii K112 should be used at a bacterial concentration of 10... 7 ~10 8 After spraying CFU / mL onto the soil surface, turn the soil over, sow morel spawn, and then cover with 0.5–1 cm of the same soil substrate. Perform routine mushroom cultivation management.

[0020] The bacterial agent, biocontrol agent, or bio-organic fertilizer containing the above-mentioned Bacillus sabensis K112 provided by this invention can be sprayed or irrigated into the soil before planting morel mushrooms, or sprayed on the surface of morel mushroom fruiting bodies at the early stage of disease. It can also be applied in other ways at all times during the morel mushroom planting season, or used for soil improvement.

[0021] This invention provides a strain of *Bacillus safranin* K112, its cultivation method, a fungal agent containing the strain, and its applications. Strain K112 not only inhibits the growth of *Morchella alpineensis*, one of the endophytic fungi of *Morchella esculenta*, with an inhibition rate of 63.53%, but also promotes mycelial growth and the formation of mycelial blooms in *Morchella esculenta*, significantly increasing the yield of *Morchella esculenta* (increasing the yield per mu of *Morchella esculenta* 'Six Sister' and *Morchella esculenta* 'Seven Sister' by 29.43% and 34.8%, respectively). The *Bacillus safranin* K112 provided by this invention is of great significance to the *Morchella esculenta* cultivation industry. Attached Figure Description

[0022] Figure 1 Photographs of plate confrontation experiments between *Morchella alpina* and *Morchella spp.* KS1 and KS5, respectively.

[0023] Figure 2 The images are from the screening experiment of Example 1. Among them, A is a photo of the endophytic fungus *Morchella alpineensis* growing in the culture medium, B is a photo of the confrontation experiment between *Bacillus salsa* K112 and *Morchella alpineensis*, C is a photo of the confrontation experiment between *Bacillus salsa* K112 and *Morchella septemlobus* KS5, and D is a photo of the confrontation experiment between *Bacillus salsa* K112 and *Morchella septemlobus* KS1.

[0024] Figure 3 Morphological characterization of Bacillus sabovella K112, where A is a photograph of colony morphology and B is a microscopic image of the bacterial cells after Gram staining;

[0025] Figure 4 Phylogenetic tree of Bacillus saffron K112;

[0026] Figure 5 The growth curve of Bacillus sabovella K112;

[0027] Figure 6 The effects of different types of culture media on the growth of Bacillus sabovella K112;

[0028] Figure 7 The effect of culture temperature on the growth of Bacillus sabovella K112;

[0029] Figure 8 The effect of initial pH of the culture medium on the growth of Bacillus sabovella K112;

[0030] Figure 9 The effect of rotation speed on the growth of Bacillus sabovellae K112;

[0031] Figure 10 The effect of liquid volume on the growth of Bacillus sabovellae K112;

[0032] Figure 11The effect of inoculum size on the growth of Bacillus sabovella K112;

[0033] Figure 12 Photographs of bacterial blooms during co-culturing of *Bacillus salsa* K112 and *Morchella esculenta* KS1 at different cell or culture concentrations. In the images, A through E represent cell concentrations of 0, 10, and 10, respectively. 6 , 10 7 , 10 8 , 10 9 Photographs of bacterial blooms from co-culture of *Bacillus salsa* K112 and *Morchella esculenta* KS1 at CFU / mL; F-J represent bacterial concentrations of 0, 10, and 10, respectively. 6 , 10 7 , 10 8 , 10 9 Photograph of bacterial frost co-cultured with Bacillus salsa K112 (CFU / mL) and Morel KS1;

[0034] Figure 13 Photographs of bacterial blooms during co-culturing of *Bacillus salsa* K112 and *Morchella esculenta* KS5 at different bacterial cell or culture concentrations. K to O represent bacterial cell concentrations of 0, 10, and 10, respectively. 6 , 10 7 , 10 8 , 10 9 Photographs of bacterial blooms from co-culture of *Bacillus salsa* K112 (CFU / mL) and *Morchella esculenta* KS5; P-T represent bacterial concentrations of 0, 10, and 10, respectively. 6 , 10 7 , 10 8 , 10 9 Photograph of bacterial frost formed by co-culturing CFU / mL Bacillus sarfusae K112 and Morel mushroom KS5. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0036] During morel cultivation, we isolated and screened a strain of *Morchella alpineensis* from diseased morel fruiting bodies. This strain significantly inhibited the growth of morel mycelia. He Peixin et al. have confirmed that *Morchella alpineensis* is one of the endophytic fungi of morels. Wang Bangxiang, through re-inoculation experiments, found that morel fruiting bodies inoculated with *Morchella alpineensis* showed suspected disease symptoms, suggesting that this strain may have formed a nutrient competition relationship with morels. Although the pathogenic mechanism of *Morchella alpineensis* on morels still needs systematic verification, current technology has confirmed that the presence of *Morchella alpineensis* does indeed affect the quality of morels. Therefore, targeted control research on this fungus is of significant practical importance for morel cultivation.

[0037] In view of this, the present invention provides a strain of Bacillus salsa K112 and its culture method, a bacterial agent containing the strain and its application. The Bacillus salsa K112 can not only antagonize the endophytic fungus Moringa alpinea, but also promote the growth of morel mushrooms and the formation of mycelial bloom, thus significantly increasing the yield of morel mushrooms.

[0038] All raw materials used in the following embodiments of the present invention are commercially available products. In the following embodiments, the *Morchella esculenta* strains KS5 and KS1 are commercially available. In practical applications, the morel mushrooms in the embodiments can be replaced with *Morchella tessmannii* or other culturable morel mushrooms.

[0039] The endophytic fungus *Morchella alpineensis* was obtained as follows: a fungus strain was isolated from naturally diseased morel fruiting bodies collected from the morel cultivation area of ​​Yuanshi County, Shijiazhuang City, Hebei Province. This fungus was then subjected to plate confrontation culture with morel strains at 20℃. The results showed that this fungus significantly inhibited the mycelial growth of *Morchella esculenta* strains KS1 and KS5 (e.g., *Morchella esculenta* strain KS5). Figure 1 As shown in the image, the fungus was identified as *Morchella alpineensis*.

[0040] In the following examples, the SDA medium used had the following formulations: 40 g / L glucose and 10 g / L peptone; the NA medium used had the following formulations: 3 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride; the LB medium used had the following formulations: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride; the BHI medium used had the following formulations: 38.5 g / L brain and heart broth; and the NYBD medium used had the following formulations: 8 g / L beef extract, 5 g / L yeast extract, and 20 g / L glucose.

[0041] Example 1

[0042] This embodiment provides a method for isolating, screening, and identifying Bacillus sabovella strain K112.

[0043] 1. Isolation and screening of Bacillus sabovella strain K112

[0044] (1) Separation: 10g of fresh soil was collected from the saline-alkali land of Houxianzhuang Village, Huanghua City, Cangzhou, Hebei Province, and placed in a 250mL Erlenmeyer flask containing 90mL of sterile water. The mixture was shaken for 30min. The bacterial suspension was then diluted 10-fold sequentially to a concentration of 10 using a serial dilution method. -1 ~10 -6 Take 10 -4 ~10 -6 100 μL of the bacterial suspension was evenly spread on LB agar plates and incubated at 28 °C. After single bacterial colonies were formed, single colonies with different morphological characteristics were selected and purified. Finally, 145 bacterial strains were isolated.

[0045] (2) Screening: The flat standoff method (e.g.) is used. Figure 2 As shown in the figure, *Morchella alpina* mycelium cakes were inoculated in the center of PDA medium. At a distance of 2.5–3 cm from the mycelium cake, 145 isolated bacterial strains were inoculated onto the medium in a triangular shape. After culturing at 25℃ for 5 days, the colony growth was observed. A control group was also set up with only *Morchella alpina* mycelium cakes inoculated on the plates. Once the control group plates were fully colonized with mycelia, the inhibition rate of each bacterial strain against the target strain was calculated. Based on the inhibition rate, bacteria exhibiting antagonistic effects against *Morchella alpina* were screened. These antagonistic bacteria were then subjected to confrontation culture with *Morchella spp.* KS1 and *Morchella spp.* KS5, respectively. Finally, strain K112 was selected as significantly inhibiting the growth of *Morchella alpina* with an inhibition rate of 63.53%, and had no inhibitory effect on the mycelial growth of *Morchella spp.* KS1 and *Morchella spp.* KS5 (as shown in Table 1).

[0046] Table 1

[0047]

[0048] 2. Identification of Bacillus sabolicii strain K112

[0049] (1) Morphological characteristics

[0050] Observe the colony morphology and microscopic morphology of the antagonistic strain K112 on LB plates (e.g. Figure 3 As shown in the figure, the colonies of strain K112 are milky white, smooth, viscous, and opaque, with irregular edges. The strain has a rod-shaped structure and is a Gram-positive bacterium.

[0051] (2) Determination of physiological and biochemical characteristics

[0052] The physiological and biochemical tests of the screened antagonistic bacterium K112 were performed using micro-biochemical identification tubes. After culturing K112 on manganese sulfate nutrient agar medium at 28°C for 24 hours, both the medium and K112 turned brown. The results (as shown in Table 2) indicate that antagonistic bacterium K112 can utilize glucose and mannose for sugar fermentation; it cannot perform nitrate reduction, and starch hydrolysis, MR reaction, and VP reaction were negative. Based on the identification results and consultation with identification manuals, strain K112 was preliminarily classified into the genus *Bacillus* spp.

[0053] Table 2

[0054] strain manganese ions Mannitol glucose Starch hydrolysis Nitrate reduction MR VP K112 + + + - - - -

[0055] Note: In the table, "+" indicates a positive result or that the product can be used; "-" indicates a negative result or that the product cannot be used.

[0056] (3) 16S rDNA identification

[0057] Using bacterial K112 genomic DNA as a template, PCR products were amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-GGTTACCTTGTTACGACTT-3'). 16S rDNA sequencing was then performed. The sequencing results (sequence shown in SEQ ID NO.1) were compared with NCBI data using BLAST to construct a phylogenetic tree as shown below. Figure 4 As shown, the results indicate that bacteria K112 is most closely related to Bacillus safensis.

[0058] Based on morphological structure, physiological and biochemical characteristics and 16S rDNA sequencing results, strain K112 was preliminarily identified as Bacillus safensis.

[0059] The Bacillus safensis K112 provided by this invention was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCC NO: M 2025656, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0060] Example 2

[0061] This embodiment provides an experiment to optimize the culture process of Bacillus salsa K112.

[0062] Strawberry strain K112 was streaked onto LB medium and incubated at 28°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and cultured overnight at 25°C with shaking to prepare a seed culture for later use.

[0063] (1) Strain K112 was inoculated into LB medium at a 2% inoculum and cultured at 28℃ and 180 r / min for 44 h. Samples were taken every 2 hours during the first 8 hours of culture, and every 4 hours after that. The optical density (OD value) of the culture medium at 600 nm was measured, and the growth curve of the growth-promoting strain was plotted based on the obtained data. The results are as follows: Figure 5 As shown in the figure, it can be seen that from the 36th hour onwards, strain 112 gradually transitioned into the stationary phase, and its growth rate gradually decreased to a plateau. Therefore, the subsequent experimental culture time was selected as 36 hours.

[0064] (2) Prepare SDA, NA, LB and NYBD culture media, inoculate 2% into the culture media, and incubate at 28℃ and 180r / min for 36h to measure the OD in each culture medium. 600 Value. Result as follows Figure 6 As shown, the optimal culture medium for strain 112 was determined to be LB medium.

[0065] (3) To investigate the suitable culture conditions for the growth of strain K112, the culture temperature, initial pH, volume of the culture medium (250 mL Erlenmeyer flask), inoculum size, and shaking speed were changed sequentially (results are shown in the table below). Figures 7-11 (As shown). Based on the experimental results, the most suitable culture conditions were determined to be: culture temperature: 25℃, initial pH: 7, liquid volume: 30mL / 250mL, inoculum size: 2%, and rotation speed: 200r / min.

[0066] Example 3

[0067] This embodiment provides the application of strain K112 in promoting the mycelial growth rate of morel mushrooms.

[0068] (1) After culturing strain K112 for 36 h under the optimal culture process determined in Example 2, the bacterial cells were collected by centrifugation at 4000 r / min for 10 min, resuspended in sterile water, and the OD was adjusted. 600 The value was 0.8. 0.005 μL, 0.05 μL, 0.5 μL, and 5 μL of resuspension were added to 10 mL of the upper culture medium, respectively, to prepare a concentration of 4 × 10⁻⁶. 2 ~4×10 5 Morel mushroom cakes of strains KS1 and KS5 were inoculated into the center of plates using a double-layer medium containing CFU / mL, with an unweighted suspension medium as a blank control. The plates were incubated at 20℃, and the mycelial growth rate was measured on days 2 and 3. The results are shown in Table 3. It can be seen that when the concentration of strain K112 is 4×10⁻⁶ CFU / mL... 2At a concentration of CFU / mL, the mycelial growth rate of Morel KS1 reached its fastest, significantly higher than that of the control and other groups (P<0.05); when the concentration of strain K112 was 4×10⁻⁶ CFU / mL, the mycelial growth rate was significantly higher than that of the control and other groups (P<0.05); 3 At CFU / mL, the mycelial growth rate of Morel KS5 reached its fastest, significantly higher than that of the control and other groups (P<0.05).

[0069] Table 3

[0070] Concentration (CFU / mL) KS1 mycelial growth rate (mm / d) KS5 mycelial growth rate (mm / d) 0 24.74±1.62ab 22.21±1.20c <![CDATA[4×10 2 ]]> 28.43±0.42a 24.90±0.49b <![CDATA[4×10 3 ]]> 26.81±3.09ab 29.45±2.06a <![CDATA[4×10 4 ]]> 26.05±2.79ab 28.03±0.95b <![CDATA[4×10 5 ]]> 24.16±0.62b 26.43±0.48b

[0071] (2) After culturing strain K112 for 36 hours under the optimal culture process determined in Example 2, the bacterial solution was diluted with sterile water to remove OD. 600 The pH was adjusted to 0.8, and the bacterial suspension was collected after filtration through a 0.22 μm filter membrane. 5 μL, 50 μL, 250 μL, 500 μL, and 750 μL of bacterial suspension were added to 10 mL of the upper culture medium to prepare bilayer media with volume fractions of 0.05%, 0.5%, 2.5%, 5%, and 7.5%, respectively. A blank control without bacterial suspension was used. Morel mushroom cakes of strains KS1 and KS5 were inoculated into the center of the plates. The plates were incubated at 20℃, and the mycelial growth rate was measured on days 2 and 3. The results are shown in Table 4. It can be seen that when the addition amount of strain K112 was 0.5%, the mycelial growth rate of KS1 and KS5 reached its fastest, significantly higher than the control (P<0.05).

[0072] Table 4

[0073]

[0074]

[0075] Example 4

[0076] This embodiment provides a microbial agent in the form of a suspension, the active ingredient of which includes Bacillus safensis K112 with preservation number CCTCC NO: M 2025656.

[0077] Example 5

[0078] This embodiment provides the application of strain K112 in the growth of morel mushrooms.

[0079] Cultivars of *Morchella esculenta* KS1 and *Morchella esculenta* KS5 were prepared separately, and cultivars with a concentration of 10 were also prepared separately. 6 , 10 7 , 10 8 and 10 9CFU / mL of antagonistic bacteria K112 cells and bacterial suspensions were used. Flowerpots with a diameter of 8.5 cm, a height of 7.5 cm, and a volume of 0.43 L were selected. 200 g of a substrate, accurately weighed from each pot, was prepared by mixing soil, potting mix, and vermiculite in a 1:1:1 volume ratio. Using a pipette, 0.4 mL of the above-mentioned K112 cell suspensions and bacterial suspensions at different concentrations were sprayed into the substrate, ensuring even distribution. Subsequently, 3 g of KS1 or KS5 inoculum was evenly sown onto the substrate surface, and immediately covered with a layer of the same mixed substrate approximately 0.5–1 cm thick. The treated flowerpots were incubated at 18℃ for 7 days. A control group was prepared using substrate without any bacterial suspension or bacterial suspension. Photos of the potted experiments are shown below. Figures 12-13 As shown in Table 5, the results are as follows.

[0080] Table 5

[0081]

[0082] Depend on Figure 11 As shown in Table 5, when the cell concentration of strain K112 reaches 10... 7 At CFU / mL, the bacterial bloom of both KS1 and KS5 reached its maximum value (+++); once the bacterial concentration exceeded 10... 7 At CFU / mL, the amount of bacterial bloom in KS1 decreases or even disappears completely as the concentration increases, while the amount of bacterial bloom in KS5 decreases or disappears completely as the bacterial concentration reaches 10. 9 Even at CFU / mL, a certain amount of bacterial bloom (+) can still be maintained. In comparison, KS5 has a wider range of suitable bacterial species for spraying than KS1. In pot experiments, KS1 showed better performance at a bacterial concentration of 10... 7 The optimal concentration for bacterial bloom formation (++) is CFU / mL, while KS5 has a wider optimal concentration range for bloom formation, around 10... 7 CFU / mL and 10 8 The peak bacterial bloom (+++) can be reached at CFU / mL.

[0083] Example 6

[0084] This embodiment provides the application of strain K112 in increasing morel mushroom yield.

[0085] Field experiments were conducted in greenhouses in Yuanshi County, Shijiazhuang City, Hebei Province, from November 2023 to April 2024. K112 cells were cultured in the optimal medium with shaking for 36 hours. After centrifugation at 4000 rpm for 10 minutes, the cells were collected, resuspended in sterile water, and the OD was adjusted. 600 The value is 1.0, and the bacterial cell concentration is configured to be 10. 7 and 10 8CFU / mL bacterial suspension. 10.67 L of bacterial suspension per acre was sprayed onto the surface of the substrate soil in each plot, with unsprayed plots serving as a control (bacterial concentration of 0). Each concentration was replicated in triplicate. Afterward, the soil was turned over, and 84 g each of *Morchella truncatum* KS1 and *Morchella truncatum* KS5 spawn were broadcast into the corresponding plots. Immediately after broadcasting, a layer of soil about 0.5–1 cm thick was covered, and mushroom management was carried out according to standard field operations.

[0086] Table 6

[0087]

[0088] Note: The area of ​​the residential area is 5.6m². 2 Yield per mu (kg / mu) = Plot yield / 5.6m 2 ×667m 2 ×0.7

[0089] Table 6 shows that, compared with the control group, spraying different concentrations of K112 bacterial suspension significantly promoted the increase in morel mushroom yield. At a bacterial concentration of 10... 7 Under the condition of CFU / mL, the yield of morel mushroom KS1 was 1049.337 kg / mu and the yield of morel mushroom KS5 was 868.434 kg / mu, which increased by 29.43% and 34.80% respectively compared with the control.

[0090] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus safensis K112, characterized in that, It was deposited on March 31, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025656, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A method for culturing Bacillus safensis K112 with accession number CCTCC NO: M 2025656, characterized in that, Strain K112 was inoculated into LB medium, NA medium, SDA medium, BHI medium or NYBD medium and cultured in shake flasks at 23-31°C with an initial pH of 6.5-8.

5. The volume of the shake flask medium was 30-150 mL / 250 mL, the inoculum size was 0.5%-5%, and the rotation speed was 140-220 r / min.

3. A microbial inoculant, characterized in that, The active ingredient includes Bacillus safensis K112 with preservation number CCTCCNO: M 2025656.

4. The microbial agent as described in claim 3, characterized in that, The formulations of the microbial agent include fermentation broth, powder, or suspension.

5. The application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in the preparation of plant biocontrol agents and / or bio-organic fertilizers.

6. The application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in inhibiting the growth of endophytic bacteria in morel mushrooms.

7. The application as described in claim 6, characterized in that, The morel mushrooms include *Morchella esculenta*, *Morchella esculenta*, and *Morchella tilapia*; the endophytes include *Morchella alpineensis*.

8. The application of Bacillus safensis K112 with accession number CCTCC NO: M 2025656 in promoting morel mycelial growth, mycelial bloom formation and / or increasing morel yield.

9. The application as described in claim 8, characterized in that, The morel mushrooms include morel mushrooms of the sixth rank, morel mushrooms of the seventh rank, and morel mushrooms of the tiered ridge.

10. A method for increasing morel mushroom yield, characterized in that, After spraying the soil surface with the inoculant, biocontrol agent, or bio-organic fertilizer containing Bacillus sabolicus K112 as described in claim 1, the soil is turned over, morel mushrooms are planted, and then covered with 0.5-1 cm of the same soil substrate for routine mushroom production management.

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