Bacillus pumilus G15 and application thereof in prevention and treatment of potato soil-borne diseases

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus pumilus G15 and application thereof in prevention and treatment of potato soil-borne diseases. The invention provides bacillus pumilus G15, and the preservation number of the bacillus pumilus G15 is CGMCC (China General Microbiological Culture Collection Center) No.32839. The bacillus pumilus G15 has the advantages that the preservation number is CGMCC No.32839; the bacillus pumilus G15 can generate various volatile organic compounds (VOCs) and can destroy cell wall integrity and cell membrane permeability of pathogenic bacteria, so that the effect of preventing soil-borne diseases of potatoes is achieved. Results of the embodiment show that by utilizing the bacillus pumilus G15 disclosed by the invention, hyphae of potato phyllachora solani can be bent, vesicles and other deformity occur, and the activity of the phyllachora solani is influenced, so that the action effect of inhibiting the phyllachora solani is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus pumilus G15 and its application in preventing and controlling potato soil-borne diseases. Background Art

[0002] The pathogen of potato black scurf is Rhizoctonia Solani, which mainly harms young buds, the base of the stem and tubers. The symptoms on the tubers are black sclerotia and scab-like symptoms, seriously affecting the emergence, quality and yield of potatoes. In recent years, the occurrence of potato black scurf has been increasing year by year, hindering the development of the potato industry. The occurrence of potato soil-borne diseases is closely related to the imbalance of the microbial ecosystem in the rhizosphere soil of plants, mainly manifested in the transformation of the soil microbial flora from "bacterial type" to "fungal type", an increase in the number of pathogenic bacteria in the soil, and a change in the diversity of the soil microbial community. Therefore, searching for high-quality biocontrol bacteria resources and exploring biological control methods for controlling soil-borne diseases are of great significance for the sustainable development of agriculture.

[0003] By secreting a large number of secondary metabolites with antibacterial effects, Bacillus can effectively inhibit the growth of various pathogenic bacteria. The research on the secondary metabolites of Bacillus mainly includes lipopeptide compounds, antibacterial proteins and volatile substances, etc. Among them, using microbial volatile organic compounds (mVOCs) for biological fumigation to control diseases has broad research value and application prospects. Volatile organic compounds (VOCs) usually refer to odor compounds with carbon as the basic element, low molecular weight (<300Da), high vapor pressure, low boiling point and lipophilicity (<C15), which can quickly volatilize under normal temperature and pressure conditions and enter the gas phase state, with the advantages of fast moving speed, etc. Using volatile organic compounds (VOCs) to control plant pathogens in the soil is a promising biological control strategy. At present, there is no relevant report on the application of Bacillus that can secrete VOCs in the prevention and control of potato pathogens. Summary of the Invention

[0004] The purpose of the present invention is to provide a Bacillus pumilus G15 and its application in preventing and controlling potato soil-borne diseases. The Bacillus pumilus G15 described in the present invention can secrete volatile organic compounds, affect the integrity of the cell wall and the permeability of the cell membrane of pathogenic bacteria, thereby achieving the prevention and control of potato soil-borne diseases.

[0005] The present invention provides a Bacillus pumilus G15, and the preservation number of the Bacillus pumilus G15 is CGMCC No. 32839.

[0006] The present invention also provides a microbial agent, the active ingredient of which includes the Bacillus pumilus G15 described in the above scheme.

[0007] As a preferred embodiment, the number of viable bacteria of Bacillus pumilus G15 in the microbial agent is ≥ 1×10 6 CFU / mL or ≥1×10 6 CFU / g.

[0008] The present invention also provides the use of the Bacillus pumilus G15 or the microbial agent described in the above scheme in preventing and controlling soil-borne diseases of potatoes.

[0009] As a preferred embodiment, the soil-borne diseases include black mole disease.

[0010] The present invention also provides the use of the Bacillus pumilus G15 or the microbial agent described in the above scheme in improving potato yield.

[0011] The present invention also provides a method for preventing and controlling soil-borne diseases of potatoes, comprising treating potatoes with the Bacillus pumilus G15 or the microbial agent described in the above scheme.

[0012] As a preferred embodiment, the soil-borne diseases include black mole disease.

[0013] As a preferred solution, the treatment period includes the sowing period and / or the seedling period; the treatment method includes furrow application and / or spraying.

[0014] As a preferred solution, the application amount of the microbial agent is 5-10 L / mu or 5-10 kg / mu.

[0015] Beneficial effects: The present invention provides a strain of Bacillus pumilus G15, the deposit number of which is CGMCC No. 32839. The Bacillus pumilus G15 of the present invention can produce a variety of volatile organic compounds (VOCs), which can destroy the cell wall integrity and cell membrane permeability of pathogens, thereby preventing and controlling soil-borne potato diseases. The results of the examples show that the use of the Bacillus pumilus G15 of the present invention can cause the hyphae of the potato black mole fungus (Rhizoetonia solani) to bend and form deformities such as vesicles, affecting the activity of the black mole fungus, thereby exerting an inhibitory effect on the black mole fungus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0017] Figure 1This is the distribution diagram of the antibacterial activity of Bacillus strains against black mole bacteria;

[0018] Figure 2 This is a diagram showing the effect of G15 volatile gas on the pathogenicity of potato black mole pathogen observed by inoculating potato slices in vitro;

[0019] Figure 3 The area of ​​lesions caused by black mole bacteria on potato chips; *** indicates that the data are significantly different, p < 0.001;

[0020] Figure 4 Figure 1 shows the biocontrol effect of G15 volatile gas on potato plants; A shows the incidence of black mole at the base of potato stems; B shows the disease index of potato plants with different treatments in the pot experiment; *** indicates significant differences in the data, p < 0.001;

[0021] Figure 5 This figure shows the effect of G15 volatile organic compounds on the hyphae morphology of black mole pathogens. The left figure represents the hyphae morphology observed under a 10x microscope, and the right figure represents the hyphae morphology observed under a 40x microscope. The triangles in the figure indicate cysts or hyphae bends.

[0022] Figure 6 Scanning electron microscopy images of black mole pathogen hyphae after treatment with VOCs released by G15; a to c are control groups; d to f are treatment groups; white arrows indicate wrinkles; red arrows indicate cysts; green arrows indicate swelling; from left to right, the scale bar in the first column is 100 μm; the scale bar in the first column is 50 μm; the scale bar in the first column is 10 μm;

[0023] Figure 7 Transmission electron microscopy images of black mole pathogen hyphae after treatment with VOCs released by G15; A to C are control groups; D to I are treatment groups; in the figure, 1 indicates loosening of the cell wall; 2 indicates swelling of the vacuole; 3 indicates displacement of the multivesicular body; and 4 indicates displacement of the cytoplasmic contents.

[0024] Figure 8 This is the effect of VOCs released by G15 on the chitin content of potato black mole pathogen;

[0025] Figure 9 This is the effect of G15 volatile organic compounds on the extracellular conductivity of potato black mole pathogen;

[0026] Figure 10 This is the effect of G15 volatile organic compound treatment on the mycelial permeability of potato black mole pathogen;

[0027] Figure 11 This is the effect of VOCs released by G15 on ROS of potato black mole pathogen hyphae;

[0028] Figure 12Figure 1 is a graph showing the relative content of volatile organic compounds released by strain G15 based on the peak area of ​​GC×GC-MS data; A is a graph showing the proportion of different types of volatile organic compounds released by G15; B is a graph showing the peak area proportion of volatile organic compounds released by G15; and C is a SPME (GC-MS) total ion current graph of volatile organic compounds (VOCs) produced by the strain.

[0029] Figure 13 This is the antibacterial effect diagram of a single component of G15 volatile organic compounds;

[0030] Figure 14 This is the fitting curve diagram of the inhibitory effects of the five compounds on melanoma pathogens;

[0031] Figure 15 This is the antibacterial effect diagram of G15 volatile organic compound mixed gas;

[0032] Figure 16 The effect of five compounds on the hyphae morphology of black mole pathogens; the scale bar in the figure is 20 μm; the yellow arrow indicates the cyst; the green arrow indicates the hyphae bending.

[0033] Biological Deposit Certificate

[0034] Bacillus pumilus G15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on November 28, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 2, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 32839. DETAILED DESCRIPTION

[0035] The present invention provides a Bacillus pumilus G15. The deposit number of the Bacillus pumilus G15 is CGMCC No.32839.

[0036] The Bacillus pumilus G15 strain described in the present invention was screened from 211 Bacillus strains using the double-dish method and demonstrated the best antibacterial activity against the black mole pathogen, achieving an inhibition rate of 82.47%. The strain releases various volatile organic compounds, which deform the hyphae of the black mole pathogen and increase chitin content. It also alters the permeability of the cell membrane, causing cytoplasm leakage. Furthermore, the volatile organic compounds increase the level of reactive oxygen species (ROS), disrupting mycelial activity.

[0037] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent includes the Bacillus pumilus G15 described in the above scheme. As a preferred embodiment, the number of viable bacteria of Bacillus pumilus G15 in the microbial agent is ≥1×10 6CFU / mL or ≥1×10 6 CFU / g.

[0038] The present invention also provides the use of the Bacillus pumilus G15 or the microbial agent described in the above-mentioned embodiment for controlling soil-borne potato diseases. In one embodiment, the soil-borne disease includes black spot disease; in another embodiment, the pathogen of black spot disease includes Rhizoctonia solani. The volatile organic compounds produced by the Bacillus pumilus G15 of the present invention can cause mycelial deformities of the pathogen and increase the level of reactive oxygen species, thereby playing a multi-faceted role in controlling soil-borne potato diseases. Taking the black mole pathogen as an example, in terms of hyphae morphology, the volatile organic compounds of Bacillus pumilus G15 cause the hyphae of the black mole pathogen to show deformities such as tip shortening, cysts, bending, wrinkling and twisting; in terms of hyphae structure, the volatile organic compounds of Bacillus pumilus G15 cause the hyphae of the black mole pathogen to leak material, the boundaries of organelles are unclear, the chromatin is condensed, the vacuoles are swollen, and some cells have apoptotic bodies; in terms of energy metabolism, the volatile organic compounds of Bacillus pumilus G15 can increase the level of reactive oxygen species in the black mole pathogen. As the treatment time increases, the level of reactive oxygen species continues to increase, exacerbating the apoptosis of hyphal cells.

[0039] The present invention also provides the use of the Bacillus pumilus G15 or the microbial agent described in the above solution in increasing potato yield. The Bacillus pumilus described in the present invention can effectively prevent and treat potato black spot disease, thereby increasing potato yield.

[0040] The present invention also provides a method for preventing and controlling soil-borne potato diseases, comprising treating potatoes with the Bacillus pumilus G15 or the microbial agent described in the above scheme. In one embodiment, the soil-borne disease includes black mole.

[0041] In one embodiment, the treatment period includes the sowing period and / or the seedling stage; and the treatment method includes furrow application and / or spraying. The treatment period and method of the present invention allow Bacillus pumilus G15 to effectively colonize the soil and plant rhizosphere, facilitating disease prevention and control.

[0042] As an embodiment, the application rate of the microbial agent is 5 to 10 L / mu or 5 to 10 kg / mu. In specific embodiments of the present invention, the application rate of the microbial agent can be any value of 5 to 10 L / mu, such as 5 L / mu, 6 L / mu, 6.5 L / mu, 7 L / mu, 8 L / mu, 9 L / mu, or 10 L / mu. The application rate of the microbial agent can also be any value of 5 to 10 kg / mu, such as 5 kg / mu, 6 kg / mu, 7 kg / mu, 8 kg / mu, 9 kg / mu, or 10 kg / mu. The application rate of the present invention can effectively control pathogens and play a role in preventing and controlling soil-borne diseases.

[0043] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 Screening and identification of biocontrol Bacillus

[0045] (1) The antibacterial activity of volatile organic compounds (VOCs) produced by 211 strains of Bacillus from the Potato Disease and Plant Pathogens Laboratory of Hebei Agricultural University was detected using the double-plate confrontation method.

[0046] Pour LB solid medium into a culture dish and add 200 μL of 1×10 8 CFU / mL of Bacillus fermentation liquid was evenly spread, and PDA was poured into another culture dish. The bacterial cake of black mole pathogen (Rhizoctonia solani, published in the literature [Zhu Mingming, Zhang Dai, Zhao Dongmei, et al. Screening and identification of biocontrol Bacillus for potato black mole disease [J]. Jiangsu Agricultural Science, 2018, 46(14):97-101.]) was placed in the center of the PDA culture medium, and the two plates were buckled and sealed. 200μLLB liquid culture medium was buckled as a control, and co-cultured in a 25℃ incubator for 4 days. When the control was full of the dish, the diameter of the black mole pathogen colony was measured according to the cross-cross method, and the inhibition rate was calculated according to the following formula: mycelium growth inhibition rate = (control colony growth diameter - treatment colony growth diameter) / control colony growth diameter × 100%. The statistical results of the inhibition rate are as follows: Figure 1 shown.

[0047] according to Figure 1 It can be seen that 210 strains had an inhibition rate against melanoma bacteria of over 10%. This indicates that most Bacillus strains can release volatile organic compounds, and the volatile organic compounds they release have a certain antibacterial effect on melanoma bacteria. However, the antibacterial effect of the volatile organic compounds released by different strains on melanoma bacteria varies. 38 strains had an inhibition rate of over 70%, indicating excellent inhibition; 73 strains had an inhibition rate between 50% and 70%, indicating moderate inhibition; 81 strains had an inhibition rate between 30% and 50%, indicating poor inhibition; and 19 strains had an inhibition rate below 30%, indicating poor inhibition.

[0048] The double-dish method was used to screen the antibacterial effects of Bacillus strains. The volatile organic compounds produced by Bacillus pumilus G15 showed the best inhibition rate against melanoma bacteria, at 82.47%. Therefore, the G15 strain was selected for subsequent experiments.

[0049] (2) Biocontrol effect of volatile organic compounds of strain G15 on potato black spot disease

[0050] A. In vitro antibacterial effect of volatile gases from the screened strains on black mole pathogens

[0051] ① Select potato tubers of equal size, wash and dry them, then disinfect them with 75% alcohol for 1 minute. Rinse with sterile water and let them dry. Cut the center of the tubers into slices approximately 8 mm thick to serve as the inoculum. Place the slices on a sterilized iron tray lined with sterilized moist filter paper and seed the center with a 5 mm thick cake of potato black mole bacteria. Set aside.

[0052] ② Group treatment: The potato chips with the mushroom cakes placed in step ① were randomly divided into two groups, with 3 repetitions in each group. Treatment group (G15): The bacterial solution of strain G15 was fumigated separately, and the bacterial solution concentration was set to 10 4 , 10 6 , 10 8 Three gradients of CFU / mL were used for the control group (Control): LB solid culture medium was placed as the control, and 3 replicates were set for each group; after the two groups were placed with G15 bacterial solution or LB solid culture medium, they were sealed tightly with plastic wrap, and the connected potato chips were placed in a constant temperature incubator at 25℃ for 5 days. The results were observed and the diameter of the lethal lesions was measured using the cross method and photographed and recorded. Figure 2 As shown, G15 is 1×10 8 CFU / mL treatment group lesion map. The lesion area of ​​the treatment group and the control group were counted separately, and the results are as follows Figure 3 As shown, the treatment group (G15) is the mean of three bacterial solution concentrations.

[0053] according to Figures 2 and 3 As shown in the figure, after the potato black mole pathogen was inoculated with potato black mole pathogen cakes on the potato chips, the mycelium of potato black mole pathogen expanded and grew on the potato chips. The mycelium expansion area of ​​potato chips treated with volatile organic compounds of strain G15 was smaller, indicating that the volatile organic compounds produced by strain G15 significantly inhibited the growth of potato black mole pathogen and reduced its infection rate on potato tubers. The average lesion area of ​​the control group was 157.2 mm 2 The average lesion area of ​​the treatment group was 79.1 mm 2 In the control group, mycelium grew normally on the potato tubers, while in the treated group, mycelium barely grew at all, and the potato cells surrounding the tubers oxidized and necrotic. This suggests that the volatile organic compounds from strain G15 can significantly inhibit the growth and reproduction of potato black mole pathogens, reducing their pathogenicity.

[0054] (3) Quantitative determination of R. solani in potato roots

[0055] Sample collection: Select tubers of uniform growth potential and size and plant them in pots. Tuber management measures are the same as for ordinary potatoes. Root samples were collected 27 days and 32 days after potato sowing, cut into small segments with scissors and frozen at -80℃. Each treatment was repeated three times. Plant genomic DNA was extracted using the CTAB method, and pathogens were quantitatively determined. The quantitative method can be found in [Li Ruiqin, Liu Xing, Qiu Huizhen, et al. Rapid detection of Rhizoctonia solani in soil with potato damping-off disease by fluorescence quantitative PCR [J]. Acta Prataculturae Sinica, 2013, 22(5): 136-144]. Root DNA from different sampling days was used as template and amplified using specific primers Rs3F (SEQ ID NO.1: 5′-TTGGTTGTAGCTGGTCTATTT-3′) and Rs3R (SEQ ID NO.2: 5′-TATCACGCTGAGTGGAACCA-3′). During the annealing phase of each cycle, fluorescence was collected, the reaction was monitored in real time, and changes in the fluorescence signal were recorded. Finally, the measured values ​​were substituted into the standard curve equation to calculate the gene copy number of R. solani in the test sample. The results are shown in Table 1.

[0056] Table 1 Quantitative determination results of R.solani in potato roots

[0057]

[0058]

[0059] According to Table 1, as time goes by, the number of black mole bacteria in the treatment group decreases and is lower than that in the control group. The higher the concentration of the treatment group, the more obvious the reduction in the number of black mole bacteria. 8 When fumigation was performed with the bacterial solution of 100 CFU / mL, the number of black mole disease on potatoes in the 27-day treatment group was 6.3×10 2 copeis / g, and the number of mole bacteria in the control group was 2.6×10 4 copeis / g; on day 32, the number of black mole bacteria in the treatment group was reduced to 8.7×10 1 copeis / g, while the number of black mole bacteria in the control group was 3.6×10 3 copeis / g. This showed that strain 15 could significantly inhibit the number of black pathogenic bacteria.

[0060] Strain G15 was identified by molecular biology and was found to be Bacillus pumilus. It was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 28, 2024, with the deposit number: CGMCC No. 32839.

[0061] Example 2

[0062] (1) Biocontrol effect of volatile gases from Bacillus pumilus G15 on R. solani

[0063] Preparation of volatile organic compounds of Bacillus pumilus G15: Bacillus pumilus G15 was cultured at 37°C and 200 rpm / min to a concentration of 1×10 8 cfu / mL for future use.

[0064] Preparation of R. solani bacterial suspension: inoculate R. solani into PDA solid culture medium and culture at 25°C until the plate is full. Then, take a 5 mm bacterial cake from the edge of the colony. Pick up the bacterial cake and place it in PD culture medium. Shake the culture at 25°C rpm / min to obtain mycelium and weigh it. Resuspend the mycelium in PD culture medium at a ratio of 1 g / 10 mL for later use.

[0065] Soil pretreatment: The test soil was nutrient soil, which was sterilized in a high pressure sterilizer at 121°C for 20 minutes and then placed in a ventilated place to dry for later use.

[0066] Potato planting: Use 75% ethanol to disinfect the potato seed potatoes. After disinfection, place the potatoes at 18℃ for 15-20 days to germinate. Cut the seed potatoes into small pieces of 20-30g and mix them with talcum powder. After mixing, air them at room temperature for 1 day before sowing. Plant them in flower pots (diameter × height = 20cm × 18cm). Keep the room temperature at 25℃ and the average sunshine time at 16h. Water them every 3-4 days to keep the soil moist.

[0067] Inoculation of bacterial suspension: When the potato plants emerge from the soil, 10 mL of R.solani bacterial suspension is inoculated into each pot and watered. Treatment group: 10 mL of Bacillus pumilus G15 fermentation liquid is used for fumigation treatment, while control group: 10 mL of liquid LB is used for fumigation treatment of mycelium. The disease status is counted 30 days after inoculation. The results are as follows Figure 4 shown.

[0068] according to Figure 4 It can be seen that the disease index of potato plants treated with volatile organic compounds of Bacillus pumilus G15 was 16, while the disease index of the control group was 44. The calculated relative control efficiency was 63.6%, indicating that the volatile substances produced by Bacillus pumilus G15 have the effect of preventing and controlling potato black mole disease.

[0069] (2) Effects of volatile organic compounds from Bacillus pumilus G15 on the mycelial morphology of black mole pathogens

[0070] A. Observe the hyphae morphology of the black mole pathogen using an optical microscope: Use the double-plate method with 1×10 8CFU / mL of Bacillus pumilus G15 culture medium was used to treat potato black mole pathogens. A coverslip was placed 5 mm away from the bacterial cake and the edge of the plate was sealed with multi-layer sealing film. The control group was not treated with Bacillus culture medium. The two groups of culture plates were placed in a 25℃ incubator in the dark for 3 days. The mycelial morphology was observed and photographed using an optical microscope. The results are as follows Figure 5 shown.

[0071] according to Figure 5 It can be seen that the hyphae in the control group had obvious right-angle branches, smooth hyphae, and uniform thickness; while the hyphae in the treated group had shortened tips, cysts, and obvious hyphae bending. Therefore, the volatile organic compounds produced by Bacillus pumilus G15 can cause the hyphae of black mole pathogens to become deformed.

[0072] B. Scanning electron microscopy observation of the hyphae morphology of the black mole pathogen: In a clean bench, a 5 mm sterile punch was used to punch holes at the edge of the activated potato black mole pathogen fungus. A sterile inoculation needle was used to pick up a complete colony, which was placed upside down in the center of a potato solid culture medium plate. Then, 200 μL of 1×10 8 The LB solid medium containing 100 CFU / mL of Bacillus pumilus G15 fermentation broth was placed in a blank culture dish. A culture dish without fermentation broth was set as a blank control. After 3 days, the fungal blocks of the treatment group and the control group were scraped and sliced ​​according to the method of Pan Jialiang [Pan Jialiang. Study on the antibacterial mechanism of matrine on the pathogenic fungus (Botryosphaeria dothidea) of pecan dry rot [D]. Heilongjiang: Northeast Forestry University, 2018]. The ultrastructural changes on the surface of the fungal hyphae were observed using a scanning electron microscope. The results are as follows: Figure 6 shown.

[0073] Scanning electron microscopy revealed that the mycelium of the black mole pathogen in the control group grew normally, with straight mycelium and thicker mycelial cell walls. After treatment with volatile organic compounds from Bacillus pumilus G15, the mycelium became wrinkled, shriveled, swollen and twisted, and the mycelial cell walls became transparent. Figure 6 In (a-c), the mycelia of the control group grew densely, with uniform thickness and surface cell wall, while the mycelia of the treated group ( Figure 6 Middle d) The hyphae are sparse, thin, and more curved. The hyphae are collapsed and wrinkled, and there may be cytoplasm exudation ( Figure 6 Middle e), the mycelial surface is uneven ( Figure 6 (f) The hyphae also swelled and twisted, losing their cylindrical shape. Scanning electron microscopy results showed that the volatile organic compounds released by Bacillus pumilus G15 altered the morphology of the black mole pathogen's hyphae, causing some hyphae to swell, while others shrank and twisted, with cytoplasm leaking, leading to hyphae shrinkage.

[0074] C. Observation of the hyphae morphology of black mole pathogen by transmission electron microscopy: According to the method in step B, potato black mole pathogen was treated with Bacillus sp. to obtain samples of the treatment group and the control group. The ultrastructure changes of fungal hyphae were observed by transmission electron microscopy. The results are as follows: Figure 7 shown.

[0075] like Figure 7 As shown in A to C, the electron microscope images of the internal structure of the hyphae in the control group show complete and clear nuclei, vacuoles, mitochondria, Golgi bodies, cell membranes and cell walls. The edges of the hyphae cell membranes and cell walls are clear and the structure is complete. The intercellular matrix structure is uniform and the structure of the cytoplasm is complete. The hyphae in the treated group have material leakage at the edges, are not clear and smooth, the inner color of the hyphae is darker, and the organelles are not clearly visible. The cells in the treated group have concentrated chromatin and have cladding contents wrapped in a membrane structure; as shown in Figure 2. Figure 7 As shown in middle H, after treatment, the hyphal cell wall became loose; Figure 7 As shown in D, G, and F, the hyphal vacuoles are swollen; Figure 7 As shown in E and F, the hyphal multivesicular bodies were displaced, and the contents of hyphal cells were displaced; some cells formed apoptotic bodies.

[0076] (3) Effects of volatile organic compounds of Bacillus pumilus G15 on the main components of the cell wall of black mole pathogens

[0077] Chitin is the main component of the hyphal cell wall of potato black mole pathogen. Increased chitin content indicates an increase in the number and thickness of the hyphal cell wall. The chitin content of potato black mole pathogen was determined to characterize the effect of volatile organic compounds (VOCs) from Bacillus pumilus G15 on the main cell wall components of potato black mole pathogen.

[0078] First, the potato black mole pathogen cake was inoculated on PDA medium and cultured in the dark at 25℃ for 3 days. Then, another medium was coated with 200μL 1×10 8Solid LB culture plates containing 100 CFU / mL of Bacillus fermentation broth were placed side by side and sealed tightly with parafilm. Treatment times were 0, 12, 24, 36, and 48 h, with blank controls set at each time point. Mycelia scraped after fumigation with volatile organic compounds from Bacillus were used as the treatment group. The chitin content in the mycelia was measured using the KOH digestion method (see [Ou Yang Q, Duan X, Li L, et al. Cinnamaldehyde exerts its antifungal activity by disrupting the cell wall integrity of geotrichum citri-aurantii [J]. Frontiers in cellular and infection microbiology, 2019, 10:55]. The mycelia were dry-ground into a powder and weighed, designated as W1. The powder (W1) was then treated with a saturated KOH solution at 140°C for at least 60 min. The product was poured onto filter paper and slowly rinsed with distilled water. The extract was dehydrated with 95% and 100% ethanol in turn and weighed again, recorded as W2. The chitin content was calculated by the following formula: Chitin content (%) = W2 / W1×1.26×100%, where 1.26 is the conversion factor. The result is as follows Figure 8 shown.

[0079] according to Figure 8 It can be seen that with the extension of treatment time and the growth of mycelium, the chitin content in the mycelium showed an upward trend; at 0h, the chitin content of the control group and the treatment group was 11.34%, and after 12h, the chitin content in the mycelium of the control group changed to 13.06%, while the chitin content in the mycelium of the treatment group changed to 27.28%, and the increase was much greater than that of the control group; after 24h, the chitin content of the control group was 16.13%, and the chitin content of the treatment group was 33.00%; after 36h, the chitin content of the control group was 13.91%, and the chitin content of the mycelium of the treatment group was 33.33%; after 48h, the chitin content of the mycelium of the control group was 19.77%, and the chitin content of the mycelium of the treatment group was 36.24%. The chitin content of the mycelium treated group was greater than that of the mycelium of the control group at each time point, and the chitin content showed an upward trend with the increase of treatment time. This shows that with the increase of treatment time, the chitin content of the mycelium of the treated group is higher in the mycelium of the same weight, which affects the integrity of the cell wall.

[0080] (4) Effects of volatile organic compounds from Bacillus pumilus G15 on the cell membrane permeability of black mole pathogens

[0081] A. To further verify the extent of damage to the cell membrane of potato black mole pathogen by volatile organic compounds released by Bacillus pumilus G15, the permeability of the cell membrane was characterized by measuring the electrical conductivity of the hyphae of potato black mole pathogen after exposure to G15 volatile organic compounds for 0, 12, 24, 36, 48, 60 and 72 hours.

[0082] Determination of extracellular conductivity: The black mole bacteria were cultured in pairs according to the method in 3.1.4. The treatment time was 0, 12, 24, 36, 48, 60 and 72 hours. A blank control was set at each time point. When processing the sample, the black mole bacteria were first rinsed with deionized water, and then soaked in deionized water for 1 hour. The conductivity of the control group and the treatment group was measured using a conductivity meter. Three replicates were set for each sample. The results are shown in the figure below. Figure 9 and as shown in Table 2.

[0083] Table 2 Conductivity data of black pathogenic bacteria under different treatments

[0084] Processing time (h) Control group (μs / cm) Treatment group (μs / cm) 0 219.33 219.33 12 239.67 251.67 24 225.43 266.33 36 209.37 236.33 48 224.67 242.67 60 221.33 250.33 72 212.67 268.00

[0085] according to Figure 9 As shown in Table 2, the conductivity values ​​of the treated groups were all higher than those of the control group, indicating that the volatile organic compounds of Bacillus pumilus G15 can increase the membrane permeability of the potato black mole pathogen mycelium, leading to the outflow of electrolytes from the black mole pathogen. With the increase in treatment time, the conductivity of the control group briefly fluctuated, but the change was generally stable. The conductivity of the treated group first increased, then decreased, and then increased again. This may be due to the initial growth of the treated mycelium and the leakage of intracellular contents, which caused the change in conductivity. This indicates that the permeability of the membrane of the black mole pathogen caused by the volatile organic compounds of Bacillus pumilus G15 does not decrease with time. The change in conductivity is caused by the leakage of intracellular components and the outflow of electrolytes. Electrolytes are related to maintaining ion balance, regulating cellular energy status, transporting substances, and controlling metabolism.

[0086] B. Lactophenol cotton blue staining: Use the double-button method and use 200 μL of 1×10 8 CFU / mL of Bacillus pumilus G15 bacterial solution was spread on solid LB culture medium, and the other side was PDA solid culture medium inoculated with potato black mole bacteria. A cover glass was inserted 1 cm around the bacterial cake and sealed with a sealing film. An LB culture dish without bacterial solution was used as a control and cultured in the dark at 25°C. When the black mole bacteria grew to two-thirds of the cover glass, the cover glass was removed, the culture medium on the back of the cover glass was wiped off with sterile filter paper, and it was stained with lactophenol cotton blue dye. Then the mycelium was placed downward on a slide, and the excess dye was absorbed with lens paper. The mycelium was observed under an optical microscope. The results are as follows: Figure 10 shown.

[0087] according to Figure 10It can be seen that the mycelium of the control group was evenly stained, with right-angled branches and even staining; while the mycelium of the treated group was stained very light blue, unevenly stained, with unstained parts and discontinuous cavities in the mycelium, which indicated changes in the permeability of the mycelial membrane.

[0088] (5) Effects of volatile organic compounds of Bacillus pumilus G15 on energy metabolism of black mole pathogens

[0089] Cellular reactive oxygen species detection was performed using a reactive oxygen species detection kit to observe the effect of volatile gases from Bacillus pumilus G15 on mitochondrial ROS in potato black mole pathogens. The black mole pathogens were treated using a double-dish method, and a coverslip was placed 1 cm away from the black mole pathogen cake. When the hyphae grew to the coverslip, 200 μL / dish of Bacillus pumilus G15 fermentation liquid (1×10 8 CFU / mL) were treated and sealed with sealing film. The treatment time was 6, 10, 12, 24 and 48 hours. After treatment, the coverslip was removed and the culture medium on the coverslip was wiped off with sterile filter paper. After the mycelium was stained according to the kit instructions, it was placed upside down on a slide and DCF was detected using the FITC parameter setting of the laser confocal microscope. The results are shown in the figure. Figure 11 shown.

[0090] according to Figure 11 It can be seen that after 6 hours of treatment with VOCs released by Bacillus pumilus G15, the active oxygen content of the mycelium of potato black mole pathogen in the treatment group did not increase significantly. However, after 10 hours of treatment with volatile organic compounds of Bacillus pumilus G15, some mycelium showed a higher fluorescence intensity. The subsequent 12, 24 and 48 hours of treatment can significantly increase the active oxygen level of the mycelium of potato black mole pathogen, and the active oxygen content of the mycelium increases with the extension of treatment time.

[0091] Example 3

[0092] Volatile organic compounds and their antibacterial activity of Bacillus pumilus G15

[0093] (1) Collection of volatile substances of Bacillus pumilus G15: First, single colonies of volatile substances of Bacillus pumilus G15 were picked and placed in liquid LB. The volatile substances were shaken at 37°C and 200 rpm / min for 1 day as seed solution. The seed solution was added to LB at a volume ratio of 1:100 and the solution was shaken at 25°C and 200 rpm / min until the concentration of the bacterial solution reached 1×10 8 CFU / mL. Sample pretreatment: Before the test, age the chromatographic column until there are no impurity peaks. Then age the DVB / CAR / PDMS extraction head at the gas chromatograph inlet until there are no impurity peaks. Accurately measure 5.0 mL of bacterial solution (concentration of 1×10 8CFU / mL) in a 20 mL headspace vial, equilibrated in a water bath with magnetic stirring at 50°C for 30 min, and then inserted into a conditioned extraction tip for 40 min. The tip was then removed and immediately inserted into the injection port for 5 min of desorption. Gas chromatography conditions included a first-dimensional column, HP-innowax (30 m × 0.25 mm × 0.25 μm); a second-dimensional column, DB17-MS (1.2 m × 0.18 mm × 0.18 μm); and helium as the carrier gas at a flow rate of 1.0 mL / min. The temperature program was as follows: initial temperature at 50°C, increasing at 4°C / min to 240°C, and holding for 10 min. The injection port temperature was 250°C, in splitless mode, with a split ratio of 40.0 mL / min. Mass spectrometry conditions included an EI source, electron energy of 70 eV, a transfer line temperature of 250°C, an ion source temperature of 230°C, and a quadrupole temperature of 150°C. The solvent delay was 3 min, and the proton scanning range (m / z) was 40 to 550. The mass spectra obtained by GC-MS were searched and analyzed by NIST mass spectrum database, and components with relative peak area greater than 1% and retention index greater than 800 were selected for dynamic component analysis.

[0094] Comprehensive two-dimensional gas chromatography-mass spectrometry (GC-MS) was used to identify the volatile organic compounds produced by Bacillus pumilus G15. The total ion current is shown in the figure. Figure 12 After comparing with the database and reading relevant literature, we selected VOCs with peak area%>1.0% and retention index RI>800, and finally determined that Bacillus pumilus G15 produced 20 volatile organic compounds, see Table 3 and Figure 12 A and B. The 20 volatile organic compounds included 6 esters (3.63%), 2 alkanes (1.73%), 2 ketones (4.91%), 7 aromatic compounds (84.21%), 2 alcohols (4.15%), and 1 pyrazine (1.38%). 1,3-Dimethylbenzene accounted for the highest proportion, at 48.35%, followed by 1-ethyl-3-methylbenzene and benzaldehyde, at 16.02% and 10.11% respectively.

[0095] Table 3 Volatile organic compounds released by Bacillus pumilus G15

[0096]

[0097]

[0098] (2) Antibacterial effect of single components of volatile organic compounds released by Bacillus pumilus G15: The inhibitory effect of volatile organic compounds on black mole pathogens was determined by the mycelial growth rate method and the double-plate method. The methods are as follows [Chen Yipeng, Yang Yang, Shi Tao, et al. Analysis of volatile components of endophytic fungus HND5 and determination of its antibacterial effect [J]. Journal of Tropical Crops, 2017, 38(04): 689-694]. Because 3-hydroxy-2-butanone, styrene and 4-methyldecane are hazardous chemicals and their purchase is restricted, only the remaining 17 single products were screened for their antibacterial effects. First, two concentration gradients were set to conduct a preliminary screening of the antibacterial effects of the 17 purchased single products. The dosage of each compound per plate was set at 20 μL and 40 μL, respectively. Sterile water was used as a blank control and the results were repeated three times. After 3 to 5 days, the growth of the black mole pathogen colonies was observed and the colony diameter was measured by the cross-cross method. The antibacterial rate was calculated according to the following formula. Bacterial inhibition rate = (colony diameter of control group - colony diameter of treated group) / colony diameter of control group × 100%. Figure 13 shown.

[0099] according to Figure 13 It can be seen that when 40 μL of compounds were used to fumigate the mycelium of black mole fungus, 8 compounds had an inhibition rate of more than 90%, namely benzaldehyde, 2-decanone, ethyl heptanoate, acetophenone, 3-ethyl-3,5-dimethylpyrazine, 1-nonanol, n-propylbenzene, and 3-ethyltoluene, with inhibition rates of 100.00%, 100.00%, 100.00%, 100.00%, 98.07%, 94.13%, 93.94%, and 90.67%, respectively. When 20 μL of compounds were used to fumigate the mycelium of the black mole fungus, the inhibition rates of five compounds were above 90%, namely benzaldehyde, 2-decanone, ethyl heptanoate, acetophenone, and 1-nonanol, with inhibition rates of 100.00%, 100.00%, 100.00%, 99.73%, and 92.27%, respectively. Therefore, the five compounds of benzaldehyde, 2-decanone, ethyl heptanoate, acetophenone, and 1-nonanol were selected to measure their EC values. 50 .

[0100] (3) Determination of the antibacterial effect of volatile organic compounds with good antibacterial effect on black mole bacteria: The antibacterial rates of the 17 compounds measured in (2) were ranked, and the top 5 compounds with the best antibacterial effect were selected for testing. According to the dosage and antibacterial rate of the compounds, 5 concentration gradients were selected for testing, with 2, 4, 6, 8, and 10 μL per dish, respectively, and the test was carried out according to the method in (2); when calculating the concentration, the used concentration (μL / dish) was converted into the actual concentration (μL / mL) according to the following formula: actual concentration = dosage / culture dish volume. The radius of the double-dish buckle system is 4.00 cm, the height (excluding the gas space of the culture medium thickness) is 1.50 cm, and the calculated volume is 75.39 cm3 The drug concentrations were calculated based on the dosage and culture dish volume to be 0.03, 0.05, 0.08, 0.11, and 0.13 μL / mL, respectively. The colony diameters were measured and the toxicity regression equation and EC were calculated using Spass software. 50 The logarithm of the concentration (x) and the probability value of the percentage of inhibition of colony growth (y) were calculated, and the regression equation y = a + bx and EC50 of the toxicity of each compound to the black mole fungus were obtained using the least squares method using IBM SPASS statistics. The results are shown in Table 4 and Figure 14 shown.

[0101] Table 4 EC50 and toxicity regression equation of the five compounds against potato black mole pathogen

[0102]

[0103] According to Table 4 and Figure 14 It can be seen that benzaldehyde has the best antibacterial effect, with an EC50 of 0.068 μL / mL, followed by 2-decanone, ethyl heptanoate, acetophenone, and n-nonanol, with an EC50 of 0.068 μL / mL. 50 The concentrations of benzaldehyde, 2-decanone, and ethyl heptanoate were 0.083, 0.113, 0.117, and 0.120 μL / mL, respectively. Therefore, benzaldehyde, 2-decanone, and ethyl heptanoate were the main volatile antibacterial components of strain G15.

[0104] (4) Determination of the antibacterial effect of three volatile organic compounds with good antibacterial effect on melanoma bacteria: According to the EC values ​​of the compounds measured in (3), 50 Data results, three compounds with good antibacterial effects, benzaldehyde, 2-decanone, and ethyl heptanoate, were selected and mixed according to the peak area ratio of their gas chromatography-mass spectrometry results of strain G15 (Table 5). 5 μL was set as the dosage and the double-dish method was used to measure the inhibitory effect of the compound on potato black mole pathogen according to the mycelial growth rate. The results are as follows Figure 15 and as shown in Table 6.

[0105] Table 5G15 Volatile Organic Compound Mixing Ratio

[0106] sample Pharmaceutical Ratio Processing 1 2-Decanone Process 2 Ethyl heptanoate Processing Three Benzaldehyde Processing Four 2-Decanone: Ethyl heptanoate = 0.38:0.29 Processing Five Benzaldehyde:2-decanone=10.11:0.38 Processing six Benzaldehyde:ethyl heptanoate=10.11:0.29 Processing Seven Benzaldehyde:2-decanone:ethyl heptanoate=10.11:0.38:0.29

[0107] Table 6 Determination of the antibacterial effect of mixed gas on black mole bacteria

[0108]

[0109] according to Figure 15As shown in Table 6, the antibacterial effects of benzaldehyde, 2-decanone, and ethyl heptanoate alone are consistent with the results measured in 3.7.3. The combination of benzaldehyde and ethyl heptanoate showed the greatest antibacterial effect, with an inhibition rate of 81.50%, followed by the combination of benzaldehyde, 2-decanone, and ethyl heptanoate, with an inhibition rate of 81.20%. The inhibition rates of both combinations were greater than the 80.30% inhibition rate of benzaldehyde alone. This indicates that mixing compounds can enhance the inhibition rate of a single compound, and the enhancement of the inhibition rate by mixing with ethyl heptanoate was greater than that of 2-decanone. However, the inhibition rate of the mixture of 2-decanone and benzaldehyde was lower than that of benzaldehyde, and the inhibition rate of the mixture of 2-decanone and ethyl heptanoate was also lower than that of ethyl heptanoate alone. Therefore, mixing with 2-decanone reduced the inhibition rates of benzaldehyde and ethyl heptanoate. Moreover, the mixture of benzaldehyde and 2-decanone, or the mixture of benzaldehyde and ethyl heptanoate can enhance the antibacterial effect of 2-decanone and ethyl heptanoate. GC×GC-MS experiments showed that benzaldehyde also accounts for a large proportion of the peak area released by Bacillus pumilus G15. Therefore, benzaldehyde is the main volatile antibacterial substance of strain G15, and plays a major antibacterial role in the prevention and treatment of melanoma by Bacillus pumilus G15.

[0110] At the same time, the inhibitory effect on potato black mole was measured using the double-dish method based on the mycelial growth rate, with a dosage of 10 μL. The results are shown in Table 6. This also shows that benzaldehyde is the main volatile antibacterial substance of strain G15 and plays a major antibacterial role in the prevention and treatment of potato black mole by Bacillus pumilus G15.

[0111] (5) Effects of five volatile organic compounds with good antibacterial effects on the mycelial morphology of black mole pathogen: The top five compounds with the best inhibitory effects on black mole pathogen were selected from the compounds produced by Bacillus pumilus G15 for testing. The double-dish method was used to treat black mole pathogen with the compounds at the EC50 concentrations measured in (3). Sterile water was used as a blank control. The insert method was used to observe the effects of the mycelial morphology of the control group and the treatment group using an optical microscope and photographed and recorded the results. Figure 16 shown.

[0112] according to Figure 16 It can be seen that different compounds showed varying degrees of deformity after treatment. After treatment with benzaldehyde, 2-decanone, and ethyl heptanoate, vesicles appeared in the hyphae of the black mole fungus, and the hyphae were slightly curved. After treatment with n-nonanol, no vesicles appeared in the hyphae of the black mole fungus, but the mycelia showed severe curvature. After treatment with acetophenone, the mycelia of the black mole fungus showed curvature, but the curvature was slightly less severe than that after treatment with n-nonanol. The morphology of these hyphae was basically consistent with the previous treatment with volatile organic compounds released by Bacillus pumilus G15.

[0113] Example 4

[0114] Effect of Bacillus pumilus G15 on the control of potato black mole in the field

[0115] On May 6, 2023, the experimental field was sown in Houjingbu Village, Zhangbei County, Zhangjiakou City, Hebei Province. The test variety was Chapoti. The experiment was designed according to a randomized block design with a row spacing of 0.65m and a plant spacing of 0.2m. There were 15 bud blocks per row and 6 rows per plot. There were 4 treatments in total, 4 replicates per treatment, 1 plot per replicate, and a total of 16 plots. Each plot was 20m 2 (5m long, 4m wide). Total area: 320m 2 Treatments included water as a control, azoxystrobin (chemical control), Bacillus amyloliquefaciens-Zhuorun (biocontrol control, purchased from Bayer), and Bacillus pumilus G15. Detailed information on the agents is shown in Table 7. Applications were made by furrow application at potato sowing and by spraying at the base of the stems during the seedling stage.

[0116] Table 7 Pesticides used in potato experimental fields

[0117]

[0118] Survey on potato black mole disease: 50 potato tubers were taken from the two middle ridges of each plot for grading survey. The grading standards are shown in Table 8. The disease conditions of the potato tubers in each plot were recorded according to the table. The incidence rate, disease index and prevention effect were calculated according to the following formulas. The results are shown in Table 9.

[0119]

[0120] Table 8 Disease classification standards for potato black mole

[0121] Disease level Grading standards Level 0 No spots on the tuber surface Level 1 There are sporadic sclerotia on the surface of the tuber, no more than 5 Level 3 The disease is mild, the lesions are small, and the number of lesions does not exceed 15 Level 5 The disease is moderate, the lesions are large, and the area of ​​lesions occupies less than 25% of the whole tuber Level 7 The disease is severe, with the lesion area accounting for 26%-50% of the entire tuber Level 9 The disease is severe, with the lesion area accounting for more than 51% of the entire tuber

[0122] Table 9 Effects of different treatments on field control of potato black mole at harvest time

[0123] deal with Incidence (%) Disease index Prevention effect (%) Azoxystrobin (chemical control) 5.00 0.55±0.53a 98.48 Zhuo Run (biocontrol control) 25.50 9.17±6.51a 74.96 G15 30.50 8.06±8.81a 78.00 Clear water control 68.50 36.61±5.44b -

[0124] Table 9 shows that the chemical agent azoxystrobin is most effective against potato black mole, with a disease index of only 0.55±0.53 and a control efficacy of 98.48%. The biocontrol agent Zhuorun has a control efficacy of 74.96% against potato black mole, with a disease index of 9.17±6.51. Bacillus pumilus G15 has a control efficacy of 78.00% against black mole, with a disease index of 8.06±8.81. Although biocontrol agents are generally less effective than chemical agents, they have less impact on the environment and human health. Therefore, the combined application of chemical and biocontrol agents can be used to achieve green control measures that reduce the use of pesticides.

[0125] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus pumilus G15, characterized in that: The deposit number of the Bacillus pumilus G15 is CGMCC No.32839.

2. A microbial agent, characterized in that: The active ingredient of the microbial agent includes the Bacillus pumilus G15 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The number of viable bacteria of Bacillus pumilus G15 in the microbial agent is ≥1×10 6 CFU / mL or ≥1×10 6 CFU / g.

4. Use of the Bacillus pumilus G15 according to claim 1 or the microbial agent according to claim 2 or 3 in preventing and controlling soil-borne diseases of potato.

5. The use according to claim 4, characterized in that The soil-borne diseases include black mole disease.

6. Use of the Bacillus pumilus G15 according to claim 1 or the microbial agent according to claim 2 or 3 in increasing potato yield.

7. A method for preventing and controlling soil-borne diseases of potatoes, characterized in that: The method comprises treating potatoes with the Bacillus pumilus G15 according to claim 1 or the microbial agent according to claim 2 or 3.

8. The method according to claim 7, characterized in that The soil-borne diseases include black mole disease.

9. The method according to claim 7, characterized in that The treatment period includes sowing period and / or seedling period; the treatment method includes furrow application and / or spraying.

10. The method according to claim 7, characterized in that The application amount of the microbial agent is 5 to 10 L / mu or 5 to 10 kg / mu.

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