Bacillus pumilus K40, culture method of bacillus pumilus K40, microbial inoculum containing bacillus pumilus K40 and application of bacillus pumilus K40

Abstract

The invention belongs to the technical field of microorganisms and application, and particularly relates to bacillus pumilus K40, a culture method of the bacillus pumilus K40, a microbial agent containing the bacillus pumilus K40 and application of the bacillus pumilus K40. The invention provides a bacillus pumilus K40 with a preservation number of CCTCC NO: M 2025655, the bacillus pumilus K40 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 K40 provided by the invention can inhibit the growth of pathogenic bacteria pseudomonas elongata of white mold of morchella esculenta, and the bacteriostasis rate is 56.06%; meanwhile, the strain can promote formation of bacterial cream, the yield of morchella esculenta is increased by 20% or above, and the occurrence rate of white mold is reduced by 85% or above. The bacillus pumilus K40 provided by the invention is of great significance to the cultivation industry of morchella esculenta.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and their applications, and particularly relates to a strain of Bacillus pumilus K40, a culture method thereof, a bacterial agent containing the same, and applications thereof. Background Art

[0002] As a rare edible fungus with high economic value, the artificial cultivation industry of Morchella has developed rapidly in recent years. However, the frequent occurrence of soil-borne diseases has seriously restricted the sustainable development of the Morchella cultivation industry, causing the quality of Morchella to decline, the yield to decrease, and even the complete extinction of Morchella. Su Wenying et al. isolated and purified the pathogen from the diseased fruiting bodies of Morchella and conducted research on its identification and biological characteristics. They concluded that the pathogen of white mold disease may be Monosporus longisporus ( longispora; Sun Jingzu et al. renamed the fungus as Pseudomonas longispora ( longispora), and through LC-MS / MS analysis combined with phylogenetic relationships and secondary mass spectrometry ion fragmentation, it was identified that the differential substances between infected and uninfected morels and their extracts were peptaibois-type antimicrobial peptides. These substances often lead to the stunted growth of Morel hyphae, necrosis of Morel fruiting body tissue, and the appearance of white velvety spots on the fruiting body surface, which seriously affect the yield and quality of Morel.

[0003] Currently, chemical fungicides remain the primary means of disease control for morels. However, long-term use can easily lead to pathogen resistance, microbial imbalance, and food safety risks. Therefore, developing environmentally friendly, efficient, and stable biological control technologies has become a research priority. Currently, relatively few chemical agents are suitable for disease control and ensure the safety of morels. Furthermore, prolonged and excessive use of the same chemical often leads to pathogen resistance, reducing control effectiveness, increasing pesticide residue levels, and increasing control costs. Compared to chemical control, biological control offers significant advantages such as increased safety and environmental friendliness.

[0004] Antagonistic bacteria show great promise in agricultural disease prevention and control because they can inhibit pathogens by competing for nutrients, secreting antimicrobial substances, or inducing host resistance. Studies have shown that some bacterial genera can inhibit the growth of certain pathogens, thereby alleviating symptoms to a certain extent. Du Yifan et al. conducted experiments and found that Bacillus pumilus had a greenhouse and field efficacy of 76.16% and 54.70% against tobacco black shank, respectively, and could significantly promote tobacco plant growth. Su Jiewen et al. screened Bacillus pumilus using the Oxford cup method, which had a strong antagonistic effect against cucumber-specific Fusarium oxysporum, with an inhibition rate of 69.11%, and had a significant biological control effect against cucumber wilt. However, there is still a lack of systematic research on the antagonistic bacteria resources of Morchella pathogens, and the interaction mechanism between antagonistic bacteria and Morchella is still unclear. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Bacillus pumilus K40, a culture method thereof, a bacterial agent containing the same, and an application thereof. The Bacillus pumilus K40 can not only produce an antagonistic effect on the pathogen Pseudomonas longisporus, effectively reducing the incidence of morels, but also promote the growth of morel hyphae and the formation of fungal frost, significantly increasing the yield of morels.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a strain of Bacillus pumilus K40, which was deposited in the China Center for Type Culture Collection on March 31, 2025, with a deposit number of CCTCCNO: M 2025655, and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The Bacillus pumilus K40 provided by the present invention was isolated and screened from saline-alkali soil. Strain K40 was preliminarily identified as Bacillus pumilus based on morphological, physiological and biochemical characteristics and 16S rDNA sequencing results. The colonies of strain K40 are milky yellow, smooth, moist, opaque, and have regular, short, rod-shaped structures. It is a Gram-positive bacterium. Applying the Bacillus pumilus K40 provided by the present invention to the soil before planting morels can significantly reduce the incidence of morels and significantly increase the per-acre yield of morels, which is of great significance for the artificial industrial cultivation of morels.

[0008] A second aspect of the present invention provides a method for culturing Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655, specifically comprising: inoculating strain K40 into LB liquid medium, NA medium, SDA medium, BHI medium or NYBD medium, and culturing in a shake flask at 20-40°C with an initial pH of 6.0-8.0; wherein the liquid volume of the shake flask medium is 30-150mL / 250mL, the inoculum size is 0.5%-5%, and the rotation speed is 140-220r / min.

[0009] Preferably, the culture method of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 is: inoculating strain K40 into LB liquid culture medium, culturing in a shake flask at 25-35°C, and the initial pH is 7.0-7.5; wherein the liquid volume of the shake flask culture medium is 30-60mL / 250mL, the inoculum size is 1%-3%, and the rotation speed is 180-220r / min.

[0010] Further preferably, the culture method of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 is: inoculating strain K40 into LB liquid culture medium and culturing in a shake flask at 30°C with an initial pH of 7.5; wherein the liquid volume of the shake flask culture medium is 30mL / 250mL, the inoculum size is 2%, and the rotation speed is 200r / min.

[0011] A third aspect of the present invention provides a microbial agent, wherein the active ingredient of the microbial agent comprises: Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655.

[0012] In conjunction with the third aspect, the dosage form of the bacterial agent includes but is not limited to fermentation broth, powder or suspension.

[0013] A fourth aspect of the present invention provides a use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in the preparation of a plant biological control agent and / or a bio-organic fertilizer.

[0014] The Bacillus pumilus K40 or its culture solution can be used as an active ingredient and combined with other auxiliary materials to prepare a biological control agent and / or a biological organic fertilizer.

[0015] In a fifth aspect, the present invention provides a use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in inhibiting the growth of Morchella pathogens and / or reducing the incidence of Morchella.

[0016] In combination with the fifth aspect, the morels include but are not limited to six-sister morels, seven-sister morels and ladder-edge morels, and can also be other types of cultivable morels; the pathogens include but are not limited to Pseudomonas longisporus.

[0017] In a sixth aspect, the present invention provides a use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in promoting the growth of Morchella mycelium, the formation of fungal frost and / or increasing the yield of Morchella.

[0018] In combination with the sixth aspect, the morels include but are not limited to six-sister morels, seven-sister morels and ladder-edge morels, and may also be other types of cultivable morels.

[0019] In a seventh aspect, the present invention provides a use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in inhibiting white mold disease of Morchella fruiting bodies.

[0020] The eighth aspect of the present invention provides a method for increasing the yield of morels and reducing the incidence of diseases, specifically: spraying a bacterial agent containing Bacillus pumilus K40, a biological control agent or a biological organic fertilizer on the soil surface, turning the soil, planting morels, and then covering with the same soil matrix with a thickness of 0.5 to 1 cm, and carrying out conventional mushroom production management.

[0021] In combination with the eighth aspect, the bacterial agent, biological control agent or biological organic fertilizer containing Bacillus pumilus K40 is added at a bacterial concentration of 10 7 ~10 8 After spraying the soil surface with 1000 CFU / mL, the soil was turned over and the Morchella spp. were sown. Then, the soil was covered with 0.5-1 cm thick layer of the same soil matrix and conventional mushroom production management was carried out.

[0022] The bacterial agent, biological control agent or biological organic fertilizer containing Bacillus pumilus K40 provided by the present invention can be sprayed or irrigated into the soil before planting morels, and can also be sprayed on the surface of morel fruiting bodies at the early stage of disease onset of morel fruiting bodies. It can also be applied in other ways at all times during the morel planting season, or used for soil improvement.

[0023] The present invention provides a strain of Bacillus pumilus K40, a cultivation method thereof, a microbial agent containing the same, and applications thereof. The strain K40 can not only inhibit the growth of the Morchella pathogen Pseudomonas longisporus, with an inhibition rate of 56.06%, but also promote the mycelial growth and frost formation of Morchella, greatly increasing the yield of Morchella (increasing the per-acre yield of Morchella pumilus KS1 and Morchella pumilus KS5 by 22.38% and 22.97%, respectively), and reducing the incidence of diseases (reducing the incidence of white mold in Morchella pumilus KS1 and Morchella pumilus KS5 by 85.06% and 89.85%, respectively). The Bacillus pumilus K40 provided by the present invention is of great significance to the Morchella cultivation industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are photos of the pathogen Pseudomonas longisporus confronting Morchella edulis. Picture A shows the pathogen confronting Morchella edulis KS1, and Picture B shows the pathogen confronting Morchella edulis KS5.

[0025] Figure 2 The following are photos of the screening experiment in Example 1, wherein A is a photo of the pathogen Pseudomonas longisporus growing in a culture medium, B is a photo of a confrontation experiment between Bacillus pumilus K40 and the pathogen, C is a photo of a confrontation experiment between Bacillus pumilus K40 and Morchella septempunctata KS5, and D is a photo of a confrontation experiment between Bacillus pumilus K40 and Morchella septempunctata KS1;

[0026] Figure 3 The morphological characterization of Bacillus pumilus K40, where A is a photo of the colony morphology and B is a microscopic image of the bacteria after Gram staining;

[0027] Figure 4 is the phylogenetic tree of Bacillus pumilus K40;

[0028] Figure 5 is the growth curve of Bacillus pumilus K40;

[0029] Figure 6 The effect of different culture media on the growth of Bacillus pumilus K40;

[0030] Figure 7 The effect of culture temperature on the growth of Bacillus pumilus K40;

[0031] Figure 8 is the effect of the initial pH of the culture medium on the growth of Bacillus pumilus K40;

[0032] Figure 9 is the effect of rotation speed on the growth of Bacillus pumilus K40;

[0033] Figure 10The effect of liquid volume on the growth of Bacillus pumilus K40;

[0034] Figure 11 is the effect of inoculum size on the growth of Bacillus pumilus K40;

[0035] Figure 12 Figure 4 is the effect of antagonistic bacteria 40 on the mycelial growth rate of Morchella esculenta. Figure A shows the effect of antagonistic bacteria 40 on the mycelial growth of Morchella esculenta KS1, and Figure B shows the effect of antagonistic bacteria 40 on the mycelial growth of Morchella esculenta KS5. 1 to 5 are 0, 4×10 2 , 4×10 3 , 4×10 4 and 4×10 5 CFU / mL bacterial resuspension concentration;

[0036] Figure 13 Figure 40 shows the effect of antagonistic bacteria 40 on the mycelial growth rate of Morchella esculenta. Figure A shows the effect of antagonistic bacteria 40 on the mycelial growth of Morchella esculenta KS1, and Figure B shows the effect of antagonistic bacteria 40 on the mycelial growth of Morchella esculenta KS5.

[0037] Figure 14 The photos of the fungus frost when Bacillus pumilus K40 and Morchella edulis were co-cultured with different bacterial concentrations or bacterial solution concentrations, among which A to E are the bacterial concentrations of 0, 10, 6 , 10 7 , 10 8 , 10 9 CFU / mL of co-cultured bacterial frost of Bacillus pumilus K40 and Morchella lilii KS1; F to J are bacterial solution concentrations of 0, 10, 6 , 10 7 , 10 8 , 10 9 Photo of the bacterial frost of co-culture of Bacillus pumilus K40 and Morchella siliquae KS1; K~O are bacterial concentrations of 0, 10, and 10 CFU / mL, respectively. 6 , 10 7 , 10 8 , 10 9 Photo of the bacterial frost co-cultured with Bacillus pumilus K40 and Morchella serrata KS5; P~T are the bacterial solution concentrations of 0, 10, 6 , 10 7 , 10 8 , 10 9 Photo of the bacterial frost of co-culture of Bacillus pumilus K40 and Morchella oleracea KS1 at CFU / mL. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] During the cultivation of Morchella, we isolated a strain of Pseudomonas longisporus from the infected fruiting bodies of Morchella. Su Wenying et al. isolated and purified the pathogen from the diseased fruiting bodies of Morchella and conducted research on its identification and biological characteristics, and concluded that the pathogen of white mold disease may be Pseudomonas longisporus ( longispora, Sun Jingzu et al. renamed the fungus as Pseudomonas longispora ( longispora) and identified differentially expressed substances between infected and uninfected Morchella and their extracts through LC-MS / MS analysis combined with phylogenetic relationships and secondary mass spectrometry ion fragmentation as peptaibois-class antimicrobial peptides. These substances often cause mycelial growth stagnation, necrosis of Morchella fruiting body tissue, and the appearance of white, fuzzy lesions, severely impacting the yield and quality of Morchella. Therefore, targeted control research on this fungus is of great practical significance.

[0040] In view of this, the present invention provides a strain of Bacillus pumilus K40 and a culture method thereof, a bacterial agent containing the same and an application thereof. The Bacillus pumilus K40 can not only produce an antagonistic effect on the pathogen Pseudomonas longisporus, but also promote the growth of Morchella mycelium and the formation of fungal frost, significantly increase the yield of Morchella, and effectively reduce the incidence of Morchella.

[0041] The raw materials used in the following examples of the present invention are all common commercially available products. In the following examples, the strains of Morchella serrata KS5 and Morchella serrata KS1 used are both commercially available. In actual applications, the Morchella serrata in the examples can also be replaced with Morchella serrata or other cultivable Morchella.

[0042] The pathogen Pseudomonas longisporus was obtained by isolating a fungus from naturally infected Morchella fruiting bodies collected from the Morchella cultivation area in Yuanshi County, Shijiazhuang City, Hebei Province, and cultured it on plates with Morchella strains at 20°C. It was found that the fungus could significantly inhibit the growth of the hyphae of the sixth sister Morchella strain KS1 and the seventh sister Morchella strain KS5 (e.g. Figure 1 The fungus was identified as Pseudomonas longisporus.

[0043] In the following examples, the formula of the SDA medium used is: 40 g / L glucose, 10 g / L peptone; the formula of the NA medium used is: 3 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride; the formula of the LB medium used is: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride; the formula of the BHI medium used is: 38.5 g / L brain heart infusion broth; and the formula of the NYBD medium used is: 8 g / L beef extract, 5 g / L yeast powder, and 20 g / L glucose.

[0044] Example 1

[0045] This example provides a method for the isolation, screening and identification of Bacillus pumilus strain K40

[0046] 1. Isolation and screening of Bacillus pumilus strain K40

[0047] (1) Isolation: 10 g of fresh soil collected from the saline-alkali land of Houxianzhuang Village, Huanghua City was placed in a 250 mL conical flask containing 90 mL of sterile water and shaken in a shaker for 30 min. The resulting bacterial suspension was diluted to 10 -1 to 10 -6 Then, select 10 -4 , 10 -5 , 10 -6 100 μL of each bacterial suspension was evenly spread onto LB medium plates, with each gradient repeated three times. Cultures were maintained at 28°C, and single colonies with distinct morphological structures were selected and purified, ultimately yielding 145 bacterial strains.

[0048] (2) Screening: Use the plate standoff method (such as Figure 2 As shown), the pathogen (Pseudomonas longisporus) was inoculated in the center of the PDA culture medium, and the 145 isolated bacterial strains were inoculated on the culture medium in a triangular shape at a distance of 2.5 to 3 cm from the pathogen cake. After culturing at a constant temperature of 25°C for 5 days, the growth status of the colony was observed. At the same time, a plate culture medium inoculated only with pathogens was set as a control group. When the plate culture medium of the control group was full of hyphae, the inhibition rate of each bacterial strain on the pathogen was calculated, and the bacteria that could produce antagonistic effects on the pathogen were screened out according to the inhibition rate. The antagonistic bacteria obtained were cultured against the sixth sister morel KS1 and the seventh sister morel KS5, respectively. Finally, the strain K40 was screened out, which could significantly inhibit the growth of Pseudomonas longisporus, with an inhibition rate of 56.06%, and had no inhibitory effect on the hyphae growth of the sixth sister morel and the seventh sister morel (as shown in Table 1).

[0049] Table 1

[0050]

[0051] 2. Identification of Bacillus pumilus strain K40

[0052] (1) Morphological characteristics

[0053] Observe the colony morphology and microscopic morphology of the antagonistic strain K40 on the LB plate (such as Figure 3 As shown, the colonies of antagonistic strain 40 have regular, smooth, moist, creamy-yellow, and opaque edges. They have moderate colony elevation, and cross-sections show a slightly thicker center and tapering edges. The colonies have a uniform, creamy texture. Observed under transmitted light, the edges appear translucent, while the center is denser. These short, rod-shaped bacteria are Gram-positive.

[0054] (2) Determination of physiological and biochemical characteristics

[0055] Physiological and biochemical tests were performed on the selected antagonistic bacterium, K40, using microbiological identification tubes. The K40 strain tested negative for VP, but positive for starch hydrolysis. It fermented glucose but not mannitol, and was capable of nitrate reduction. After 24 hours of incubation on manganese sulfate nutrient agar in a 28°C incubator, the medium and K40 turned brown (Table 2). Based on the identification results and reference to the identification manual, strain K40 was preliminarily assigned to the genus Bacillus.

[0056] Table 2

[0057]

[0058] Note: “+” in the table represents positive or can be used; “-” represents negative or cannot be used.

[0059] (3) 16S rDNA identification

[0060] The genomic DNA of bacterial K40 was used as a template and the PCR product was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rDNA sequence was sequenced and the sequencing result (sequence shown in SEQ ID NO.1) was compared with the NCBI data by BLAST to construct the phylogenetic tree. Figure 4 As shown, the results showed that the bacterium K40 was most closely related to Bacillus pumilus.

[0061] Combining the morphological structure, physiological and biochemical characteristics and 16S rDNA sequencing results, strain K40 was preliminarily identified as Bacillus pumilus.

[0062] The Bacillus pumilus K40 provided by the present invention has been deposited in the China Center for Type Culture Collection on March 31, 2025, with a deposit number of CCTCC NO: M 2025655, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0063] Example 2

[0064] This example provides an experiment to optimize the culture process of Bacillus pumilus K40.

[0065] The strain K40 was streaked and inoculated into LB medium, cultured at 28°C for 24 h, and a single colony was picked and inoculated into LB liquid medium, cultured with shaking at 28°C overnight, and used as seed liquid.

[0066] (1) Strain K40 was inoculated into LB medium at a 2% inoculum size and cultured at 28°C and 180 rpm for 40 h. The sampling frequency was set to: sampling every 2 hours for the first 8 hours, and then adjusted to sampling every 4 hours. The absorbance of the culture solution at 600 nm (OD) was measured using a spectrophotometer. 600 The strain growth curve was drawn based on the test results. Three parallel samples were set at each time point (the average value was taken at the end). Figure 5 As shown in the figure, the bacterial growth entered the stable phase after 32-40 hours of culture. Therefore, the culture time for subsequent experiments was selected to be 32 hours.

[0067] (2) Prepare SDA, NA, LB, BHI, and NYBD culture media respectively, inoculate 2% of the culture medium, and culture at 28°C and 180 rpm for 32 h to measure the OD value of each culture medium. 600 The result is as follows Figure 6 As shown, the optimal culture medium for K40 was determined to be LB medium.

[0068] (3) Based on the optimal culture medium, the effects of different culture parameters on the growth of the strain were explored: culture temperature (20, 25, 30, 35, and 40°C), initial pH value (6.0, 6.5, 7.0, 7.5, and 8.0), shaker speed (140, 160, 180, 200, and 220 r / min), liquid volume (30, 60, 90, 120, and 150 mL / 250 mL), and inoculum size (0.5%, 1%, 2%, 3%, 4%, and 5%). All culture experiments were performed after 32 hours of shaking culture. 600 Each set of conditions is repeated three times (the results are as follows Figures 7 to 11According to the experimental results, the optimal culture conditions were determined to be initial pH 7.5, culture temperature 30°C, liquid volume 30mL / 250mL, rotation speed 200r / min, and inoculum size 2%.

[0069] Example 3

[0070] This example provides the use of strain K40 in promoting the mycelial growth rate of Morchella

[0071] (1) After strain K40 was cultured for 32 h under the optimal culture process determined in Example 2, the cells were collected by centrifugation at 4000 rpm for 10 min and resuspended in sterile water to an OD of 600 0.005, 0.05, 0.5, and 5 μL of the resuspension were added to 10 mL of the upper culture medium to prepare 4×10 2 , 4×10 3 , 4×10 4 and 4×10 5 CFU / mL double-layer culture medium, with no added suspension as blank control. Morchella KS1 and KS5 cakes were inoculated into the center of the plate, cultured at 20℃, and the mycelial growth rate was measured on the 2nd and 3rd day. Figure 12 As shown in the figure, it can be seen that when the concentration of the bacterial resuspension of strain K40 is 4×10 2 and 4×10 3 CFU / mL significantly promoted the growth of KS1 hyphae ( Figure 12 A), which was significantly higher than that of the control and other groups (P<0.05); when the concentration of the resuspension of strain K40 was 4×10 2 CFU / mL significantly promoted the growth of KS5 hyphae ( Figure 12 B), which was significantly higher than that of the control and other groups (P<0.05).

[0072] (2) After culturing strain K40 for 32 h under the optimal culture process determined in Example 2, the bacterial solution was adjusted to a bacterial solution OD 600 =0.8. After filtering through a 0.22 μm filter membrane, 5, 50, 250, 500, and 750 μL of the bacterial solution were added to 10 mL of the upper culture medium, respectively, to prepare a double-layer culture medium with 0.05%, 0.5%, 2.5%, 5%, and 7.5% (v / v). A blank control was used without the addition of bacterial solution. Morchella KS1 and KS5 cakes were inoculated into the center of the plate. Cultured at 20°C, the growth rate of Morchella mycelium was measured on the second and third days. The results are shown in Figure 2. Figure 13 As shown in A and B, it can be seen that under the 0.05% bacterial solution concentration treatment, the mycelial growth rate of KS1 and KS5 was significantly higher than that of other treatments (P<0.05).

[0073] Example 4

[0074] This embodiment provides a microbial agent in the form of a suspension, wherein the active ingredient includes: Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655.

[0075] Example 5

[0076] This example provides the application of strain K40 in the growth of Morchella

[0077] Prepare the cultivation species of Six Sister Morchella KS1 and Seven Sister Morchella KS5 respectively, and prepare the cultivars of 10 6 , 10 7 , 10 8 , 10 9 CFU / mL of antagonistic bacteria K40 bacterial resuspension and bacterial solution. Use 0.43L flower pots (diameter 8.5cm × height 7.5cm), put 200g of mixed matrix (soil: nutrient soil: vermiculite = 1:1:1) in each pot, evenly spray 0.4mL of antagonistic bacteria K40 bacterial suspension and bacterial solution of different concentrations, sow 3g of KS1 or KS5 bacteria on the surface, and then cover with a layer of about 0.5-1cm of the same matrix. At the same time, the matrix without any bacterial suspension or bacterial solution was used as the control group, and all treatments were cultured at a constant temperature of 18℃ for 7 days. The potted experiment photos are as follows Figure 14 The results are shown in Table 3.

[0078] Table 3

[0079]

[0080] Depend on Figure 14 As can be seen from Table 3, 10 6 The bacterial resuspension and bacterial solution treatment groups with CFU / mL had no significant effect on the growth of KS1 bacterial frost. 7 The bacterial resuspension and bacterial solution treatment groups with 10 CFU / mL significantly promoted the occurrence of bacterial frost (++), and the promoting effect weakened with the increase of application concentration; 7 CFU / mL bacterial resuspension and bacterial solution significantly promoted the growth of KS5 bacterial frost (+++). 8 The promotion effect of bacterial frost growth on the CFU / mL bacterial resuspension and bacterial solution was slightly weaker (++). 7 CFU / mL bacterial resuspension and bacterial solution significantly promoted the formation of KS1 and KS5 bacterial frost.

[0081] Example 6

[0082] This example provides the use of strain K40 in increasing Morchella yield and reducing the incidence of Morchella white mold

[0083] Field experiments were conducted in a greenhouse in Yuanshi County, Shijiazhuang City, Hebei Province from December 2023 to April 2024. K40 was placed in the optimal culture medium and shaken for 32 hours. After centrifugation at 4000 rpm for 10 minutes, the cells were collected and resuspended in sterile water to adjust the OD 600 The value is 1.0, and the bacterial solution concentration is 10 7 and 10 8 CFU / mL. 10.67L of bacterial suspension per mu was sprayed on the surface of the substrate soil in each plot. The plot that was not sprayed was used as a control (bacteria concentration was 0). Then the soil was turned over and 84g each of the Six Sister Morel KS1 and Seven Sister Morel KS5 strains were sown in the corresponding plots. After sowing, a layer of soil about 0.5-1cm thick was immediately covered and mushroom production management was carried out according to conventional field operations.

[0084] Table 4

[0085]

[0086] Note: The area of ​​the plot is 5.6m 2 , yield per mu (kg / mu) = plot yield / 5.6m 2 ×667m 2 ×0.7

[0087] It can be seen from Table 4 that compared with the control group, the yield of the groups treated with microbial agents increased, whether it was KS1 or KS5. 7 The CFU / mL heavy suspension treatment group had the best yield-promoting effect, with the yields of Six Sister Morchella KS1 and Seven Sister Morchella KS5 reaching 992.20kg / mu and 792.18kg / mu, respectively, which were 22.38% and 22.97% higher than the control. 7 CFU / mL and 10 8 The incidence rates of the CFU / mL bacterial resuspension treatment groups were lower than those of the control group, among which 10 7 The highest inhibition rate was 85.06% CFU / mL against white mold of KS1 and 89.85% against KS5. These results indicate that the application of a heavy suspension of the antagonistic bacterium K40 can effectively inhibit the occurrence of white mold of Morchella oleracea, demonstrating a significant biocontrol effect.

[0088] The above is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A strain of Bacillus pumilus K40, characterized in that: It was deposited in the China Center for Type Culture Collection on March 31, 2025, with the deposit number CCTCC NO: M2025655, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A method for culturing Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655, characterized in that: The strain K40 is inoculated into LB medium, NA medium, SDA medium, BHI medium or NYBD medium, and cultured in a shake flask at 20-40° C. with an initial pH of 6.0-8.0; wherein the liquid 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.

3. A microbial agent, characterized in that: The active ingredients include: Bacillus pumilus K40 with a deposit number of CCTCCNO: M 2025655.

4. The microbial agent according to claim 3, wherein The dosage form of the bacterial agent includes fermentation broth, powder or suspension.

5. Use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in the preparation of a plant biological control agent and / or a bio-organic fertilizer.

6. Use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in inhibiting the growth of Morchella pathogens and / or reducing the incidence of Morchella.

7. The use according to claim 6, characterized in that The morels include six-sister morels, seven-sister morels and ladder-shaped morels; the pathogenic bacteria include Pseudomonas longisporus.

8. Use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in promoting mycelial growth, frost formation and / or increasing the yield of Morchella esculenta.

9. Use of Bacillus pumilus K40 with a deposit number of CCTCC NO: M 2025655 in inhibiting white mold disease of Morchella fruiting bodies.

10. A method for increasing the yield of Morchella edulis, characterized in that: After spraying a microbial agent containing Bacillus pumilus K40, a biological control agent or a biological organic fertilizer on the soil surface, the soil is turned over and morels are planted. After that, the soil is covered with a 0.5 to 1 cm thick layer of the same soil matrix and conventional mushroom production management is carried out.

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