Bacterial composite h1 for antagonizing multiple pathogenic fungi in potatoes, use thereof, and preparation method therefor

Abstract

A bacterial composite H1 for antagonizing multiple pathogenic fungi in potatoes, the use thereof, and a preparation method therefor, relating to the field of biological microbial agents. The bacterial composite H1 of the present invention is formed by mixing Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus. The bacterial composite H1 of the present invention is used for preparing a microbial agent that antagonizes pathogenic fungi in potatoes. The H1 of the present invention has potent inhibitory effects on the pathogenic fungi Alternaria solani and Alternaria alternata of potato early blight, the pathogenic fungi Fusarium solani, Fusarium oxysporum and Fusarium equiseti of wilt / dry rot, and the pathogenic fungus Didymella glomerata of leaf spot disease / stem blight, wherein the inhibition rates are all higher than 45%, among which the inhibition rate against Didymella glomerata is the highest, reaching 76.92%. The H1 provides high-quality strains for comprehensive control of soil-borne diseases in potatoes and has a good field application prospect.

Description

A microbial complex H1 antagonizing multiple pathogenic fungi of potato, its application and preparation method Technical Field

[0001] This invention pertains to biological agents, specifically relating to a microbial complex H1 that antagonizes multiple pathogenic fungi of potatoes, its application, and preparation method. Background Technology

[0002] Potatoes are the world's fourth largest food crop, crucial to food security. Due to changes in planting systems, the area under continuous potato cropping has been increasing, leading to soil degradation and serious soil-borne disease problems. Potato soil-borne diseases are mainly fungal diseases. In my country's major producing areas, the main diseases include early blight (also known as brown spot), caused by *Alternaria* fungi, primarily infecting stems and leaves; dry rot / wilt, caused by *Fusarium* fungi, primarily infecting tubers and also an important storage disease; and leaf / stem spot, a common plant disease that causes lesions on the leaves and stems of infected plants, which can merge into large patches, affecting photosynthesis and causing yield reduction. *Didymella glomerata*, the causal agent of grape stem blight, can cause leaf / stem spot in nearly 100 plant species and is a quarantine plant pathogen in my country. This strain was isolated from potato lesions for the first time in this experimental area. With the development of biotechnology, antagonistic fungal agents are becoming increasingly available. However, due to the complexity of the field environment and the poor resistance of bacterial strains, the actual application effects are unstable and difficult to achieve the disease suppression effects observed in the laboratory. Therefore, obtaining antagonistic agents with stable field application effects is an urgent problem to be solved in the treatment of soil-borne diseases of potatoes.

[0003] Bacillus species are the most widely used antagonistic bacteria, especially *Bacillus subtilis*, which has been commercialized and applied in various mature soil-borne disease control agents and bio-organic fertilizers. *Bacillus atrophaeus*, a variant of *Bacillus subtilis*, is widely distributed in the natural environment and has significant biocontrol potential against various soil-borne diseases, and has been widely used in plant disease control in recent years. *Bacillus cereus*, on the other hand, is mainly used for degrading complex organic compounds and has less application in suppressing soil-borne diseases. Among these, only *Bacillus subtilis* has been relatively widely used in potato disease control, primarily for late blight. No reports have been found of *Bacillus subtilis*, *Bacillus atrophaeus*, or *Bacillus cereus* simultaneously inhibiting the pathogens of potato early blight, wilt, dry rot, and leaf / stem spot diseases such as *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium equisetifolium*, and *Botrytis cinerea*.

[0004] With the increasing popularity of antagonistic microbial agents, it has been found that the synergistic antibacterial effect of compound microbial groups is often superior to that of single microorganisms. Therefore, the development and application of beneficial microbial communities are gradually gaining attention, such as rhizosphere growth-promoting bacteria (PGPR) and synthetic microbiome (SynCom). However, stable and broad-spectrum disease-suppressing compound microbial agents are still under investigation, and the combination of Bacillus subtilis, Bacillus atrophicus, and Bacillus cereus in compound microbial agents for inhibiting soil-borne potato diseases has not been reported. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides a microbial complex H1 that antagonizes multiple pathogenic fungi of potatoes, its application and preparation method.

[0006] The present invention provides a microbial complex H1 that antagonizes multiple pathogenic fungi of potatoes, wherein the microbial complex is a mixture of Bacillus subtilis, Bacillus atrophus and Bacillus cereus.

[0007] The Bacillus subtilis described is Bacillus subtilis Bs3, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: November 11, 2024, accession number: CGMCC No. 32579;

[0008] The atrophic bacillus is Bacillus atrophaeus Ba45, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposited on November 11, 2024, with accession number CGMCC No. 32577.

[0009] The Bacillus cereus mentioned is Bacillus cereus Bc19, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposited on November 11, 2024, with accession number CGMCC No. 32578.

[0010] Furthermore, the aforementioned microbial complex is composed of Bacillus subtilis, Bacillus atrophus, and Bacillus cereus in a volume ratio of (0.5–2):(0.5–2):(0.5–1).

[0011] The microbial compound strain H1 of the present invention is used to prepare a microbial agent that antagonizes potato pathogenic fungi.

[0012] Furthermore, the aforementioned potato pathogenic fungus is a soil-borne pathogen.

[0013] Furthermore, the potato pathogenic fungus is a soil-borne pathogen, specifically Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape rot fungus.

[0014] Furthermore, the microbial compound H1 exhibits an inhibition rate of 45-77% against Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stalk blight pathogen.

[0015] The preparation method of the microbial complex H1 antagonizing multiple pathogenic fungi of potatoes according to the present invention is as follows:

[0016] Bacillus subtilis Bs3, Bacillus atrophaeus Ba45, and Bacillus cereus Bc19 were inoculated into liquid LB medium and cultured at 180 rpm until their OD600 reached 0.6. The bacterial solutions were then mixed in a specific ratio and inoculated into liquid LB medium again. The mixture was cultured at 180 rpm until the OD600 of the mixed bacterial solution reached 1.0, thus obtaining the microbial complex H1.

[0017] Furthermore, the bacterial count of the microbial complex H1 is ≥8.0 × 10⁻⁶. 9 cfu / mL.

[0018] The present invention has the following beneficial effects:

[0019] This invention provides a compound bacterium, H1, which inhibits various soil-borne diseases of potato caused by fungi. It consists of three antagonistic strains isolated from potato-grown soil in extreme environments, exhibiting high antibacterial activity. H1 shows strong inhibitory effects against *Alternaria alternata* and *Alternaria alternata*, pathogens of early blight, *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*, and pathogens of leaf / stem spot, *Botrytis cinerea*, with inhibition rates all exceeding 45%. The inhibition rate against *Botrytis cinerea* was the highest, reaching 76.92%. Compared to Ba45, which has the highest overall pathogen inhibition rate, H1 showed a more stable and stronger disease-suppressing effect in pot experiments, with a control efficacy 50% higher than Ba45. Simultaneously, in field application trials, H1 reduced the incidence of early blight and increased potato yield. Therefore, H1 provides a high-quality strain for the integrated control of soil-borne potato diseases and shows promising prospects for field application. Attached Figure Description

[0020] Figure 1 shows the isolation and culture photographs of the pathogenic fungi; where A1 is the culture and isolation photograph, and A2-A4 are photographs of Alternaria alternata, Alternaria solanacea, and Grape rot pathogen, respectively; B1 is the photograph of the diseased plant from which the pathogenic fungi were isolated, and B2-B4 are photographs of Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium, respectively.

[0021] Figure 2 shows the BLAST comparison of pathogenic fungi; from top to bottom, they are Alternaria, Alternaria solanacea, Grape stem blight pathogen, Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium.

[0022] Figure 3 shows the BLAST comparison of three antagonistic bacteria; from top to bottom, they are Bacillus atrophus, Bacillus cereus, and Bacillus subtilis.

[0023] Figure 4 shows the morphological photographs of the three antagonistic bacteria after purification; from left to right, they are Bacillus atrophus, Bacillus cereus, and Bacillus subtilis.

[0024] Figure 5 shows a photograph of the co-culture of three antagonistic bacteria; from left to right, the images show streak co-culture and spread co-culture.

[0025] Figure 6 shows a photograph of plate confrontation inhibition; in the top row from left to right, they are Alternaria, Alternaria solanacearum, and Grape blight pathogen; in the bottom row from left to right, they are Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium.

[0026] Figure 7 shows photos of potted plants that inhibited the disease of Ba45 and compound bacterial group H1.

[0027] Figure 8 shows photos of potted plants promoted by Ba45 and compound bacteria group H1.

[0028] Figure 9 shows a photograph of the intercropping test area of ​​potatoes with compound bacterial group H2 in a continuous cropping field; the left side shows the growth of potatoes without disease during the full bloom stage, and the right side shows the growth when diseased. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0030] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0031] Example 1: Isolation and purification of pathogenic fungi

[0032] From potato plants grown in continuously cropped fields, diseased potato plants were selected and cut into small sections according to their location. Pathogenic fungi were isolated using PDA medium (Figure 1). Colony growth was observed, and the pathogenic fungi were isolated and purified. DNA was extracted from the purified fungi and sequenced. After comparison with the NCBI database (Figure 2), the DNA was stored at -80℃. Common potato pathogenic fungi such as *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium equisetifolium*, and the first isolated pathogen *Botrytis cinerea* were selected for future use.

[0033] The potato fields mentioned above are located in Wuchuan County, Inner Mongolia Autonomous Region, where the soil is chestnut calcareous soil, which is relatively barren, has low organic matter content, and is dry with little rain all year round.

[0034] Example 2: Isolation and purification of bacteria

[0035] Collect soil samples from potato-cropping plants, weigh 5.0 g of the sample, and place it in a sterile Erlenmeyer flask containing 95 mL of LB broth. Cover the flask with a sealing film and incubate at 220 rpm for 15 min on a shaker. Let it stand at room temperature for 10 min, and then dilute the supernatant 10 g to obtain the final product. -3 10 -4 10 -5 Dilute 100 μL of the solution and spread it onto a nanoplate. Perform three replicates for each dilution gradient. Incubate at 37°C for 2 days, then purify. Pile single colonies onto fresh nanoplates to purify the target bacteria, and store at -20°C and -80°C for later use.

[0036] Example 3: Screening and Identification of Antagonistic Bacteria

[0037] Strains exhibiting inhibitory effects against *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium equisetifolium*, and *Botrytis cinerea* causal agent were screened using a plate confrontation culture method. First, colonies of these pathogenic fungi from the culture medium were transferred to new PDA plates using a sterile pipette tip. Then, the isolated bacteria were activated and inoculated onto the plates, with four inoculations per plate and three replicates for each bacterium. The plates were incubated upside down at 30°C for 48 hours, and inhibition zones were observed. Bacteria producing inhibition zones were selected for species identification. DNA was extracted, a PCR reaction system was established, and amplification was performed using universal primers, followed by gel sequencing. The obtained 16S rRNA gene sequence was compared with the NCBI database (Figure 3). The homology with Bacillus atrophaeus.SK3, Bacillus atrophaeus.XJUHX-35, and Bacillus atrophaeus.CNY01 in the database all reached 99.93%, identifying the strain as Bacillus atrophaeus and naming it Ba45 (Bacillus atrophaeus 45). The homology with Bacillus cereus.BF15 and Bacillus cereus.MOB-3 in the database was higher than 99.8%, indicating that the strain is Bacillus cereus and named it Bacillus cereus.Bc19. The homology with Bacillus subtilis.4ZT, Bacillus subtilis.BEST3102, and Bacillus subtilis.Bs21 in the database all reached 100%, indicating that the strain is Bacillus subtilis and named it Bacillus subtilis Bs3.

[0038] The Ba45 (Bacillus atrophaeus 45) screened in this example was milky white in the early stage and milky yellow to dark brown in the later stage on LB solid medium. The colony surface was not smooth, opaque, slightly raised, and the edge was irregularly wavy (Figure 4 left).

[0039] The colony morphology of Bw34 in this embodiment is as follows: After Bw34 was cultured on LB solid medium at 37°C for 2 days, its colonies were observed to be milky white, round, smooth, with neat edges, viscous, and raised in the middle (Figure 4).

[0040] The Bacillus subtilis Bs3 in this embodiment has the following colony morphological characteristics: Gram-positive bacteria, uniformly stained on LB solid medium, with a rough and opaque colony surface, milky white or slightly yellow, and neat edges (Figure 4, right).

[0041] Example 4: Compound formulation and antibacterial rate of the compound bacterial group

[0042] The inhibition zones of the antagonistic bacteria were measured to calculate the inhibition rate. Strains exhibiting antagonistic effects against all six pathogenic fungi and possessing high overall inhibition rates were screened to form a compound antagonistic bacterial group (Figure 5). Bs3, Bc19, and Ba45 were selected, and strain compatibility experiments were conducted. After confirming no antagonism among the three strains, the compound ratio was determined based on their growth rates. The volume ratio of Bacillus subtilis, Bacillus atrophus, and Bacillus cereus was 1:1:1, resulting in the compound bacterial group H1 of this invention. A plate confrontation experiment was conducted to demonstrate the inhibition of pathogenic bacteria by the compound bacterial group H1, and the inhibition rate was calculated (Table 1, Figure 6).

[0043] Table 1. Inhibition rates of antagonistic single bacteria and bacterial complexes against pathogenic fungi.

[0044] Example 5: Preparation of inoculum solution for compound bacterial group

[0045] Bs3, Bc19, and Ba45 were inoculated into liquid LB medium and cultured with shaking at 37°C and 180 rpm until the OD value (OD600) reached 0.6. Then, a mixture of Bacillus subtilis, Bacillus atrophus, and Bacillus cereus was prepared at a volume ratio of 1:1:1 and cultured with shaking at 37°C and 180 rpm until the OD value (OD600) reached 1.0. The supernatant was removed by centrifugation, distilled water was added, vortexed, and centrifuged again. This process was repeated to wash away the LB medium. Water was added to adjust the bacterial count to ≥8.0 × 10⁻⁶. 9 cfu / mL is the bacterial solution to be inoculated in compound bacterial group H1.

[0046] Example 6: Preparation of Mixed Pathogenic Fungal Spore Liquid

[0047] Using an inoculation loop, the mycelium of the above 6 pathogenic fungi on PDA medium was transferred to LB liquid medium and cultured with shaking at 28°C and 200 rpm until the OD value (OD600) reached 1.0. The two were then mixed in equal volumes and shaken for 30 minutes under the same conditions to ensure thorough mixing of the strains, thus obtaining a mixed pathogenic fungal spore solution.

[0048] Example 7: Comparison of antagonistic bacteria and disease-suppressing effects in potted plants

[0049] A comparative experiment was conducted on the antibacterial effects of Ba45 (which had the highest inhibition rate among the three antagonistic bacteria) and the H1 compound bacterial group (which had the highest inhibition rate) on potato pot disease control (Figure 7). Sterilized soil was used to plant potatoes (virus-free seed potatoes). Treatments included NPK (NPK alone), inoculation with Ba45 (NPK+Ba45), and inoculation with H1 (NPK+H1), with each treatment replicated five times. Equal amounts of NPK fertilizer were applied to each treatment. Experimental procedure: After potato seedling emergence, each pot was inoculated with spores of the six mixed pathogenic fungi, 50 mL per pot, with a spore concentration ≥8.0 × 10⁻⁶. 9CFU / mL was administered via root drenching. One day after pathogen inoculation, Ba45 and H1 were inoculated separately in the same manner. Ten days later, Ba45 and H1 were inoculated again, while NPK was not inoculated. The results (Table 2) showed that, compared with NPK, inoculation with antagonistic bacteria significantly reduced the disease incidence of plants, especially H1, which achieved a control efficacy of 100%. This indicates that, compared with Ba45, the H1 compound bacterial group has a more significant disease-suppressing effect, making up for the shortcoming that the antagonistic effect of single strains is easily weakened when antagonizing mixed pathogens in actual crop cultivation.

[0050] Table 2 Disease inhibition of potted plants by compound bacterial groups H1 and Ba45

[0051] Example 8: Comparison of growth-promoting effects of antagonistic bacteria in potted plants

[0052] The potato cultivation method of Example 7 was used, but after potato emergence, no pathogenic fungi were inoculated; only Ba45 and H1 were inoculated, and a comparative experiment on the growth-promoting effect was conducted (Figure 8). The results showed (Table 3) that, under the condition that the potatoes were disease-free, compared with NPK, the plant height of the Ba45 inoculation treatment increased by 35.4% and the stem diameter increased by 60%, while the plant height of the H1 inoculation treatment increased by 65.0% and the stem diameter increased by 100%. It can be seen that inoculation with antagonistic bacteria has a growth-promoting effect, especially H1. This indicates that Bacillus subtilis Bs3 and Bacillus cereus Bc19 in H1, which inhibit pathogenic bacteria, may synergistically inhibit the reproduction of Ba45 and reduce its plate confrontation inhibition rate. In potted plants, with plant growth, the environment is more complex, and the compound bacteria utilize the synergistic effect between antagonistic bacteria to improve the stability of the disease-suppressing system and enhance the antibacterial effect.

[0053] Table 3. Growth-promoting effects of compound microbial groups H1 and Ba45 on potted plants.

[0054] Example 9: Field disease suppression effect of compound bacterial group H1

[0055] To further verify the stability of the field application effect of the compound microbial group H1 and screen its optimal application method, inoculation and compounding experiments with organic materials were conducted in potato fields with continuous cropping. This potato field had experienced a series of mixed soil-borne diseases in recent years due to continuous cropping, including early blight, wilt, and stem spot, making it suitable for verifying the disease-suppressing effect of the compound microbial group. Treatments included single application of chemical fertilizer (NPK), chemical fertilizer + inoculation with H1 (NPKH1), chemical fertilizer + organic material (NPKM), and chemical fertilizer + organic material + inoculation with H1 (MH1), with each treatment replicated three times (Figure 9). Potato seedlings, early flowering, and full bloom were inoculated with the inoculation solution of the compound microbial group prepared according to Example 5, with 100 mL inoculated per plant per time, via root drenching. Due to the favorable climatic conditions of the season for the reproduction of the early blight pathogen, early blight occurred on a large scale. The early blight disease results (Table 4) showed that although there was no significant difference in disease index between NPK and NPKH1, NPKH1 significantly reduced the disease incidence and increased potato yield by 9.4%. This indicates that the compound microbial group H1 has a certain growth-promoting function, mainly by controlling the disease through reducing the disease incidence, thereby increasing yield. NPKM, while reducing the disease index, had a better yield-increasing effect than NPKH1. However, when organic materials were applied, inoculation with H1 actually increased the disease index (3.4% higher than NPK), but it significantly reduced the disease incidence, resulting in a 10.3% increase in yield compared to organic material application. This suggests that the compound microbial group H1 is more suitable for application in conjunction with organic materials. This may be related to the significant ability of Bacillus cereus to degrade complex organic compounds, rapidly reducing organic matter and generating nutrients beneficial to potato growth, thus promoting growth, improving disease resistance, and achieving a yield-increasing effect.

[0056] Table 4 Field application effects of compound microbial group H1

[0057] This invention yielded a compound microbial group H1 that exhibits strong inhibitory effects against six pathogenic fungi of potato. Subsequent research can gradually increase the validation of its inhibitory effects on other pathogenic fungi of potato, and it can also be compounded with other biocontrol bacteria. This provides a high-quality strain and lays the foundation for the development and research of inoculants that can widely inhibit soil-borne pathogenic fungi of potato.

Claims

1. A microbial complex H1 antagonizing multiple pathogenic fungi of potatoes, characterized in that... The aforementioned microbial complex is composed of a mixture of Bacillus subtilis, Bacillus atrophus, and Bacillus cereus. The Bacillus subtilis described is Bacillus subtilis Bs3, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: November 11, 2024, accession number: CGMCC No. 32579; The atrophic bacillus is Bacillus atrophaeus Ba45, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposited on November 11, 2024, with accession number CGMCC No. 32577. The Bacillus cereus mentioned is Bacillus cereus Bc19, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposited on November 11, 2024, with accession number CGMCC No. 32578.

2. The microbial complex H1 antagonizing multiple pathogenic fungi of potatoes according to claim 1, characterized in that... The aforementioned microbial complex is composed of Bacillus subtilis, Bacillus atrophus, and Bacillus cereus in a volume ratio of (0.5–2):(0.5–2):(0.5–1).

3. The application of the microbial complex H1 antagonizing multiple pathogenic fungi of potatoes as described in claim 1, characterized in that... The aforementioned microbial compound H1 is used to prepare an inoculum agent that antagonizes potato pathogenic fungi.

4. The application according to claim 3, characterized in that... The aforementioned potato pathogenic fungus is a soil-borne pathogen.

5. The application according to claim 3 or 4, characterized in that... The aforementioned potato pathogenic fungi are soil-borne pathogens, specifically Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stalk blight pathogen.

6. The application according to claim 5, characterized in that... The microbial compound H1 has an inhibition rate of 45-77% against Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stalk blight pathogen.

7. A method for preparing the microbial complex H1 antagonistic to multiple pathogenic fungi of potatoes as described in claim 1, characterized in that... The preparation method is as follows: Bacillus subtilis Bs3, Bacillus atrophaeus Ba45, and Bacillus cereus Bc19 are inoculated into liquid LB medium and cultured at 180 rpm until their OD 600 is 0.

6. After mixing the bacterial solutions in a certain proportion, they are inoculated into liquid LB medium and cultured at 180 rpm until the OD 600 of the mixed bacterial solution is 1.0, thus obtaining the microbial complex H1.

8. The preparation method according to claim 7, characterized in that... The bacterial count of the microbial complex H1 is ≥8.0×10⁻⁶. 9 cfu / mL.

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