A microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato, and its application and preparation method

Abstract

A microbial composite strain H2, antagonistic to multiple potato pathogens, and its application and preparation method, relates to the field of biological control. The microbial composite strain H2 is a mixture of Bacillus subtilis, Bacillus atrophaeus, and Bacillus Widmannii. It exhibits strong inhibitory effects against potato early blight pathogens Alternaria solani and Alternaria alternata; wilt / dry rot pathogens Fusarium solani, Fusarium oxysporum, and Fusarium equiseti; and leaf / stem spot pathogen Vitis vinifera, with inhibition rates exceeding 46%. The highest inhibition rate against Alternaria alternata reached 75.97%. H2 also exhibits significant growth-promoting and yield-increasing effects, providing a high-quality strain for the comprehensive control of soil-borne potato diseases and promising prospects for field application.

Description

Technical Field

[0001] The invention belongs to the field of biological control, and particularly relates to a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potatoes, and an application and a preparation method thereof. Background Art

[0002] Potatoes are suitable for both food and vegetables, and are the fourth largest food crop in the world. They belong to the Solanaceae family and are prone to soil-borne diseases when grown continuously for a long time. Crop barrier soils usually have multiple pathogens coexisting in the soil. When the water and heat conditions are suitable, a certain pathogen accumulates to a certain number and a corresponding disease occurs. Soil-borne diseases of potatoes are mainly fungal diseases. Early blight, wilt / dry rot and leaf / stem spot are widely distributed in my country's main potato-producing areas. Many potato diseases are caused by mixed pathogens. Among them, the pathogens of early blight mainly include Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternata ); The pathogens of wilt / dry rot mainly include Fusarium solani ( Fusarium solani ), Fusarium equisetum ( Fusarium horsetail ), Fusarium oxysporum ( Fusarium oxysporum ) etc. Leaf / stem spot diseases include early blight, late blight, and anthracnose, all of which cause spots on stems / leaves, affecting photosynthesis and making it easy for large-scale infection to occur, resulting in yield losses. Didymella glomerata ) is a major pathogen causing leaf and stem spot diseases, capable of infecting nearly 100 plant species and a quarantined plant pathogen in my country. This experimental site isolated this strain for the first time from potato stem lesions. Therefore, developing an antagonistic fungus that can comprehensively inhibit multiple pathogens and maintain stable efficacy in the field is an urgent challenge for combating soil-borne potato diseases.

[0003] Bacillus is widely used in agricultural growth promotion and disease resistance. Among them, the most mature commercial product is Bacillus subtilis ( Bacillus subtilis Bacillus atrophaeus ( Bacillus atrophaeus ) is a variant of Bacillus subtilis, which is widely found in the natural environment and has significant biocontrol potential for a variety of soil-borne diseases. In recent years, it has been widely used in plant disease prevention and control. Bacillus wiedmannii ) has the ability to degrade insoluble inorganic phosphorus in soil and has also been reported to inhibit brown root rot, anthracnose, and wilt, but its primary application is crop growth promotion, with limited application for soil-borne disease control. Of the three strains mentioned above, only Bacillus subtilis is relatively widely used for potato disease control, primarily for late blight. There are no reports of Bacillus subtilis, Bacillus atrophaeus, or Bacillus wiedemannii simultaneously inhibiting the fungal pathogens of potato early blight, wilt / dry rot, and leaf / stem spot, Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, and grape stem blight.

[0004] In recent years, advances in biotechnology have improved the efficiency of obtaining antagonistic bacteria. The synergistic antibacterial effects of complex bacterial groups often outperform those of individual bacteria, and the development and application of beneficial microbial groups, such as growth-promoting rhizosphere bacteria (PGPR) and synthetic microbial groups (SynCom), have gradually gained attention. However, the search for stable and broad-spectrum complex microbial agents for the inhibition of soil-borne potato diseases remains ongoing, and the aforementioned combination of Bacillus subtilis, Bacillus atrophaeus, and Bacillus Widmannii has not been reported. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato, and its application and preparation method.

[0006] The present invention provides a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato, wherein the microbial composite bacteria is a mixture of Bacillus subtilis, Bacillus atrophaeus and Bacillus Widmannii;

[0007] The Bacillus subtilis is Bacillus subtilis Bs3, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 11, 2024, deposit number: CGMCC No. 32579;

[0008] The Bacillus atrophaeus is Bacillus atrophaeus Ba45, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 11, 2024, deposit number: CGMCC No. 32577;

[0009] The Widmann's Bacillus Bacillus wiedmannii Bw34 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 11, 2024, and the deposit number is CGMCC No. 32580.

[0010] Furthermore, the microbial composite bacteria is composed of Bacillus subtilis, Bacillus atrophaeus and Bacillus Widmannii in a volume ratio of (0.5~2): (0.5~2): (0.5~1).

[0011] The present invention discloses an application of a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato. The microbial composite bacteria H2 is used for preparing a bacterial agent for antagonizing pathogenic fungi of potato.

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

[0013] Furthermore, the potato pathogenic fungi are pathogens of soil-borne diseases, and the pathogens of soil-borne diseases are Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0014] Furthermore, the microbial composite bacteria H2 has an inhibition rate of 46-76% against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolia and grape stem blight.

[0015] The present invention provides a method for preparing a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potatoes. The preparation method is as follows:

[0016] Bacillus subtilis Bacillus subtilis Bs3, Bacillus atrophaeus Bacillus atrophied Ba45 and Bacillus Widmannii Bacillus wiedmannii Bw34 was inoculated into liquid LB medium and cultured at a speed of 180 rpm until its OD600 reached 0.6. After the bacterial solution was mixed in proportion, it was further inoculated into liquid LB medium and cultured at a speed of 180 rpm until the OD600 of the mixed bacterial solution reached 1.0, thereby obtaining the microbial composite bacteria H2.

[0017] Furthermore, the bacterial count of the microbial composite bacteria H2 is ≥ 8.0×10 9 cfu / mL.

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

[0019] The present invention provides a composite bacterial strain H2 that inhibits multiple soil-borne fungal pathogens of potato diseases and promotes growth. Composed of three antagonistic strains isolated from potato continuous cropping soil in an extreme environment, H2 possesses high antimicrobial activity, is more conducive to potato growth, and exhibits more stable results in potted plants. H2 exhibits strong inhibitory effects against potato early blight pathogens Alternaria solani and Alternaria alternata; wilt / dry rot pathogens Fusarium solani, Fusarium oxysporum, and Fusarium equisetum; and leaf / stem spot pathogen Vitis vinifera, with inhibition rates exceeding 46%. The highest inhibition rate was achieved against Alternaria alternata, reaching 75.97%. Compared to Ba45, which had the highest comprehensive pathogen inhibition rate, H2 demonstrated more stable and robust disease inhibition in potted plant tests, achieving a 50% higher efficacy and 23.5% higher plant height than Ba45. Therefore, H2 provides a high-quality strain for the comprehensive control of potato soil-borne diseases and holds promise for field application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1These are photos of pathogenic fungi isolated and cultured; A1 is a photo of culture and isolation, A2-A4 are photos of Alternaria alternata, Alternaria solani, and grape stem blight, respectively; B1 is a photo of diseased plants from which pathogens were isolated, B2-B4 are photos of Fusarium solani, Fusarium oxysporum, and Fusarium equisetum, respectively;

[0021] Figure 2 This is a BLAST comparison of pathogenic fungi; from top to bottom, they are Alternaria alternata, Alternaria solanacearum, Grape stem blight, Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0022] Figure 3 The BLAST comparison of three antagonistic bacteria strains is shown in Figure 1. From top to bottom, they are Bacillus atrophaeus, Bacillus Widmannii, and Bacillus subtilis.

[0023] Figure 4 The following are photos of the morphology of three antagonistic bacteria after purification; from left to right they are Bacillus atrophaeus, Bacillus Widmannii, and Bacillus subtilis;

[0024] Figure 5 The following are photos of co-culture of three antagonistic bacteria; from left to right, they are streak co-culture and spread co-culture;

[0025] Figure 6 The photos of the plate confrontation and antibacterial activity; the top row from left to right are Alternaria alternata, Alternaria solanacearum, and grape stem blight pathogen; the bottom row from left to right are Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0026] Figure 7 Photos of the disease suppression potted test of Ba45 and compound bacteria group H2;

[0027] Figure 8 Photos of the growth promotion pot experiment of Ba45 and composite bacteria group H2;

[0028] Figure 9 These are photos of the H2 potato continuous cropping field inoculation test area of ​​the composite bacteria group; the left shows the growth of potatoes without disease during the flowering period, and the right shows the growth when the disease occurred. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0030] The exemplary embodiments of the present invention and the description thereof 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 the potato fields with long-term continuous cropping, diseased potato plants were selected and cut into small segments according to their parts. The pathogenic fungi were cultured and isolated using PDA medium ( Figure 1 Observe the growth status of the colony and isolate and purify the pathogenic fungus. Extract DNA from the purified fungus and sequence it. Compare it with the NCBI database ( Figure 2 ) and stored in a -80°C freezer. Common potato pathogens Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, and the first isolated pathogenic fungus, Grape stem blight, were selected for use.

[0033] The potato long-term continuous cropping field is located in Wuchuan County, Inner Mongolia Autonomous Region. The soil is chestnut soil, which is relatively poor, low in organic matter content, and has little rain and drought all year round.

[0034] Example 2 Isolation and purification of bacteria

[0035] Collect potato continuous cropping soil, weigh 5.0 g, and place in a sterile triangular flask filled with 95 mL LB culture medium. Cover with sealing film, place in a shaker at 220 rpm for 15 min, let it stand at room temperature for 10 min, and take the supernatant to dilute 10 -3 , 10 -4 , 10 -5 Pipette 100 μl of the dilution solution onto a NA plate, with three replicates for each dilution gradient. Culture the solution in a 37°C incubator for 2 days before purification. Pick a single colony and streak the target bacteria onto a new NA plate. Store the target bacteria at -20°C and -80°C, respectively, for later use.

[0036] Example 3 Screening and identification of antagonistic bacteria

[0037] The plate confrontation culture method was used to screen strains that have inhibitory effects on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight. First, use a sterile pipette tip to take the colonies on the above pathogenic fungi culture medium and place them on a new PDA plate. Then, activate the isolated bacteria and inoculate them on the plate. Inoculate 4 points on each plate and repeat 3 times for each bacterium. Place the culture dish in a 30°C incubator and invert it for 48 hours to observe the inhibition zone. Select the bacteria that produce the inhibition zone for strain identification. Extract DNA, establish a PCR reaction system, amplify using universal primers, and perform gel excision sequencing. The obtained 16S rRNA gene sequence was compared with the NCBI database ( Figure 3 ): with the database Bacillus atrophaeus. SK3, Bacillus atrophaeus. XJUHX-35 and Bacillus atrophied.The homology of CNY01 reached 99.93%, and the strain was identified as Bacillus atrophaeus and named Ba45 ( Bacillus atrophaeus 45). With the database Bacillus wiedmannii. The homology of KLS4 reached 100%, indicating that the strain was Bacillus Widmannii and was named Bacillus wiedmannii Bw34. With the database Bacillus subtle. 4ZT, Bacillus subtilis. BEST3102 and Bacillus subtilis. The homology of Bs21 reached 100%, indicating that the strain was Bacillus subtilis and was named Bacillus subtilis Bs3.

[0038] The Ba45 ( Bacillus atrophaeus 45) On LB solid medium, the colony is milky white in the early stage, milky yellow in the later stage, and dark brown. The surface of the colony is not smooth, opaque, slightly raised, and the edge is irregularly wavy. Figure 4 Left).

[0039] The colony morphology of Bw34 in this example is as follows: after culturing Bw34 on LB solid medium at 37°C for 2 days, the colonies were milky white, round, smooth in surface, with neat edges, sticky, and convex in the middle ( Figure 4 middle).

[0040] The Bacillus subtilis of this example is Bacillus subtilis Bs3, colony morphology: Gram-positive bacteria, on LB solid medium, the spores are evenly colored, the colony surface is rough and opaque, milky white or slightly yellow, and the edges are neat ( Figure 4 right).

[0041] Example 4 Compounding and Antibacterial Rate of Composite Bacteria Group

[0042] The inhibition zones of the antagonistic bacteria were measured and the inhibition rates were calculated. The composite antagonistic bacteria group with strains that had antagonistic effects on all six pathogenic fungi and had a high comprehensive inhibition rate was screened out. Bs3, Bw34, and Ba45 were selected. After strain compatibility experiments were conducted to confirm that there was no antagonism between the three strains, the composite ratio was determined based on the growth rate of the strains. The volume ratio of Bacillus subtilis, Bacillus atrophaeus, and Bacillus Widmannii was 1:1:1, and the composite bacteria group H2 ( Figure 5 ). Carry out plate confrontation experiment of compound bacteria group H2 to inhibit pathogenic bacteria ( Figure 6 ), the inhibition rate was calculated (Table 1).

[0043] Table 1 Inhibitory rate of antagonistic bacteria and compound bacteria group H2 against pathogenic fungi

[0044]

[0045] Example 5 Preparation of composite bacterial inoculation solution

[0046] Bs3, Bw34, and Ba45 were inoculated into liquid LB medium and cultured at 37°C and 180 rpm with shaking until the OD value (OD600) reached 0.6. After mixing in equal volume ratios, the cells were cultured at 37°C and 180 rpm with shaking until the OD value (OD600) reached 1.0. The supernatant was removed by centrifugation, distilled water was added, vortexed, and centrifuged. The above steps were repeated, the LB medium was washed away, and water was added to adjust the bacterial count to ≥ 8.0 × 10 9 cfu / mL, which is the bacterial solution to be inoculated with the composite bacterial group H2.

[0047] Example 6 Preparation of mixed pathogenic fungal spore solution

[0048] Use an inoculation loop to transfer the mycelia of the above six pathogenic fungi grown on PDA medium to LB liquid medium. Shake and culture at 28°C and 200 rpm until the OD value (OD600) reaches 1.0. Then mix equal volumes and shake under the same conditions for 30 minutes to fully mix the strains to obtain a mixed pathogenic fungal spore solution.

[0049] Example 7 Comparison of the effect of antagonistic bacteria on disease inhibition in potted plants

[0050] Ba45 and H2, which have the highest inhibition rate among the three antagonistic bacteria in Shangshu, were selected to conduct a comparative test on the disease inhibition effect of potato potted plants ( Figure 7 Potatoes (virus-free seed potatoes) were planted in sterilized soil. Treatments included NPK (NPK alone), Ba45 (NPK+Ba45), and H2 (NPK+H2). Each treatment was replicated five times. Equal amounts of NPK fertilizer were applied to each treatment. Experimental Procedure: After emergence, potatoes were inoculated with spores of the six mixed pathogenic fungi (50 mL per pot) at a spore concentration of ≥ 8.0 × 10 9 cfu / mL, inoculated by root irrigation. One day after the pathogen inoculation, Bacillus atrophaeus Ba45 and H2 were inoculated in the same way. After 10 days, Bacillus atrophaeus Ba45 and H2 were inoculated again, and NPK was not inoculated. The results showed (Table 2) that compared with NPK, the inoculation of antagonistic bacteria significantly reduced the incidence of plant disease by 0, and the prevention effect of H2 reached 100%. It can be seen that compared with the single antagonistic bacteria of Ba45, the growth promotion and disease inhibition effects of the composite bacteria group are more significant. This shows that the Bacillus subtilis that inhibits pathogens in H2 is Bacillus subtle Bs3, Bacillus Widmannii Bacillus wiedmannii Bw34 may have synergistically inhibited the reproduction of Ba45 and reduced its plate confrontation inhibition rate. In the presence of plant growth, the potted environment is more complex, and the composite bacteria utilize the synergistic effect between antagonistic bacterial species to improve the stability of the disease inhibition system and enhance the antibacterial effect.

[0051] Table 2 Antagonistic bacteria potted disease inhibition test

[0052]

[0053] Example 8 Comparison of the growth-promoting effect of antagonistic bacteria in potted plants

[0054] The potato cultivation method of Example 7 was selected, but after the potatoes emerged, pathogenic fungi were not inoculated, and only Ba45 and H2 were inoculated to carry out a comparative test on the growth promotion effect ( Figure 8 The results (Table 3) showed that, in the absence of disease in all potatoes, plant height increased by 35.4% and stem diameter increased by 60% in the Ba45-inoculated treatment compared to NPK, while plant height increased by 80.1% and stem diameter increased by 140% in the H2-inoculated treatment. This indicates that inoculation with antagonistic bacteria has a growth-promoting effect, especially in the H2-inoculated treatment.

[0055] Table 3 Growth-promoting effect of composite bacteria groups H2 and Ba45 in potted plants

[0056]

[0057] Example 9: Disease suppression effect of composite bacteria group H2 in the field

[0058] The stability of the field application effect of the composite bacteria group H2 was further verified using a continuous potato cropping test field where mixed soil-borne diseases have occurred continuously in recent years. A field test was also conducted to screen the best application method. The experimental treatments included single chemical fertilizer (NPK), chemical fertilizer + inoculated H2 (NPKH2), chemical fertilizer + organic material (NPKM), and chemical fertilizer + organic material + inoculated H2 (MH2). Each treatment was replicated three times ( Figure 9 ). The potato seedling stage, the beginning of flowering stage and the full flowering stage were inoculated with the composite bacterial group inoculation solution prepared according to Example 5, 100 mL of which was inoculated per plant each time, and the inoculation was carried out by root irrigation. Because the climatic conditions of the season were suitable for the reproduction of early blight pathogens, early blight occurred on a large scale. The results of early blight disease showed (Table 4) that compared with NPK, NPKH2 significantly reduced the incidence rate and disease index, and the potato yield increased by 74.0%, and its disease prevention and yield-increasing effects were similar to those of NPKM. Compared with NPKH2, inoculation of H2 only significantly reduced the incidence rate when organic materials were applied, and there was no significant difference in the effect on the disease index and yield. This shows that the growth-promoting bacteria Bw34 in the composite bacterial group H2 can play a role in promoting growth and increasing yield without applying organic materials, and it is more suitable for use alone.

[0059] Table 4 Field application effects of composite bacteria group H2

[0060]

[0061] The present invention has obtained a composite bacterial group H2 that has a strong inhibitory effect on six potato pathogenic fungi. Subsequent research can gradually increase the inhibition verification of other potato pathogenic fungi, and can also be compounded with other biocontrol bacteria, providing high-quality strains for the development and research of fungal agents that can widely inhibit pathogenic fungi of soil-borne potato diseases, laying a foundation.

Claims

1. A microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato, characterized in that The microbial composite bacteria is a mixture of Bacillus subtilis, Bacillus atrophaeus and Bacillus Widmannii; The Bacillus subtilis is Bacillus subtilis Bs3, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 11, 2024, deposit number: CGMCC No. 32579; The Bacillus atrophaeus is Bacillus atrophaeus Ba45, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 11, 2024, deposit number: CGMCC No. 32577; The Widmann's bacillus is Bacillus wiedmannii Bw34 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 11, 2024, and the deposit number is CGMCC No. 32580.

2. The microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato according to claim 1, characterized in that The microbial composite bacteria is composed of Bacillus subtilis, Bacillus atrophaeus and Bacillus Widmannii in a volume ratio of (0.5-2): (0.5-2): (0.5-1).

3. The use of the microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The microbial composite bacteria H2 is used to prepare a bacterial agent for antagonizing potato pathogenic fungi; the potato pathogenic fungi is Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternata ), Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium equisetum ( Fusarium equiseti ) and grape stem blight ( Didymella glomerata ).

4. A method for preparing the microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato according to claim 1, characterized in that The preparation method is as follows: Bacillus subtilis Bacillus subtilis Bs3, Bacillus atrophaeus Bacillus atrophaeus Ba45 and Bacillus Widmannii Bacillus wiedmannii Bw34 was inoculated into liquid LB medium and cultured until OD600 reached 0.

6. After the bacterial solution was mixed according to the proportion, it was further inoculated into liquid LB medium and cultured until the OD600 of the mixed bacterial solution reached 1.0, thereby obtaining the microbial composite bacteria H2.

5. The preparation method according to claim 4, characterized in that The bacterial count of the microbial composite bacteria H2 is ≥8.0×10 9 cfu / mL.

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