Microbial compound bacterium H2 for antagonizing multiple pathogenic fungi of potatoes as well as application and preparation method of microbial compound bacterium H2

Abstract

The invention discloses a microbial compound bacterium H2 for antagonizing multiple pathogenic fungi of potatoes and application and a preparation method of the microbial compound bacterium H2, and relates to the field of biological control, and the microbial compound bacterium H2 is formed by mixing bacillus subtillis, bacillus atrophaeus and bacillus vedmannii. The compound has a relatively strong inhibition effect on potato early blight pathogenic fungi alternaria solani and alternaria alternata, fusarium solani, fusarium oxysporum and fusarium equisetum pathogenic fungi of fusarium wilt / dry rot, and leaf / stem leaf spot pathogenic fungi grape stem blight, and the inhibition rates are all higher than 46%. Wherein the inhibition rate on alternaria alternata is the highest and reaches 75.97%. Meanwhile, H2 has good growth-promoting and yield-increasing effects, a high-quality strain is provided for comprehensively preventing and treating potato soil-borne diseases, and the field application prospect is good.

Description

Technical Field

[0001] The invention belongs to the field of biological control, and specifically relates to a microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato, 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 they are continuously planted for a long time. Crop barrier soils usually contain 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. Potato soil-borne diseases 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 and Alternaria alternata; the pathogens of wilt / dry rot mainly include Fusarium solani, Fusarium equiseti, Fusarium oxysporum, etc.; leaf / stem spot diseases include early blight, late blight, and anthracnose, all of which cause lesions on stems / leaves, affect photosynthesis, and are prone to large-scale infection, resulting in yield losses. Didymella glomerata is an important leaf / stem spot pathogen that can infect nearly 100 species of plants and is a quarantine plant pathogen in my country. This experimental area isolated this strain from potato stem lesions for the first time. It can be seen that obtaining antagonistic bacteria that can comprehensively inhibit multiple pathogens and have stable field application effects is an urgent problem to be solved for potato soil-borne diseases.

[0003] Bacillus is widely used in agricultural growth promotion and disease resistance. Among them, the most mature commercial product is Bacillus subtilis. Bacillus atrophaeus is a variant of Bacillus subtilis. It is widely present in the natural environment and has significant biocontrol potential for a variety of soil-borne diseases. It has been widely used in plant disease prevention and control in recent years. Bacillus wiedmannii has the function of degrading insoluble inorganic phosphorus in the soil. It has also been reported that it can inhibit brown root rot, anthracnose, wilt, etc., but it is mainly used for crop growth promotion and is less used in inhibiting soil-borne diseases. Among the above three strains, only Bacillus subtilis is relatively widely used in potato disease prevention and control, but it is mainly used to prevent and control late blight. There are no reports of Bacillus subtilis, Bacillus atrophaeus or Bacillus Widmannii that can simultaneously inhibit the pathogenic fungi of potato early blight, wilt / dry rot and leaf / stem spot, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0004] In recent years, with the development of biotechnology, the efficiency of obtaining antagonistic bacteria has been improved. The synergistic antibacterial effect based on composite bacteria is often better than that of single bacteria, and the development and application of beneficial microbial groups have gradually received attention, such as growth-promoting rhizosphere bacteria (PGPR) and synthetic microbial groups (SynCom). However, stable and broad-spectrum disease-inhibiting composite microbial agents are still being explored, and the combination of the above-mentioned Bacillus subtilis, Bacillus atrophaeus and Bacillus wiedemannii has not been reported in composite microbial agents for inhibiting potato soil-borne diseases. 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 discloses 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, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No. 32579;

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

[0009] The Bacillus wiedmannii Bw34 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 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 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 fungi are pathogens 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 H1 has an inhibition rate of 46-76% against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

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

[0016] Bacillus subtilis Bs3, Bacillusatrophaeus Ba45 and Bacillus wiedmannii Bw34 were inoculated into a liquid LB medium, cultured at a rotation speed of 180 rpm until the OD 600 was 0.6, mixed the bacterial liquids in proportion, and then continued to be inoculated into a liquid LB medium, and cultured at a rotation speed of 180 rpm until the OD 600 of the mixed bacterial liquid was 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 group H2 that inhibits multiple soil-borne pathogenic fungi of potato and promotes growth. The composite bacterial group H2 is composed of three antagonistic bacteria isolated from potato continuous cropping soil in an extreme environment. The composite bacterial group H2 has a high antibacterial ability and is more conducive to potato growth, and has a more stable application effect in potted plants. H2 has a strong inhibitory effect on potato early blight pathogenic fungi Alternaria solani and Alternaria alternata, wilt / dry rot pathogenic fungi Fusarium solani, Fusarium oxysporum and Fusarium equiseti, and leaf / stem spot pathogenic fungi Grape stem blight, and the inhibition rate is higher than 46%. Among them, the inhibition rate of Alternaria alternata is the highest, reaching 75.97%. Compared with Ba45 with the highest comprehensive inhibition rate of pathogens, H2 showed a more stable and stronger disease inhibition effect in potted plant experiments, with a prevention effect 50% higher than Ba45 and a plant height 23.5% higher. Therefore, H2 provides a high-quality strain for the comprehensive prevention and control of potato soil-borne diseases, and has a good prospect for field application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 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 pathogens; B1 is a photo of diseased plants of isolated pathogens, B2-B4 are photos of Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

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

[0022] Figure 3 The BLAST comparison diagram of three antagonistic bacteria, from top to bottom, is Bacillus atrophaeus, Bacillus wiedemannii, and Bacillus subtilis;

[0023] Figure 4 The morphological photos of the three antagonistic bacteria after purification are shown below. From left to right, they are Bacillus atrophaeus, Bacillus Widmannii, and Bacillus subtilis.

[0024] Figure 5 The photo shows the co-culture of three antagonistic bacteria; from left to right, they are streak co-culture and coating co-culture;

[0025] Figure 6 These are photos of the antibacterial confrontation on flat plates; among them, the top row from left to right are Alternaria alternata, Alternaria solani, and grape stem blight pathogen; the bottom row from left to right are Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0026] Figure 7 These are photos of the disease inhibition potted test of Ba45 and compound bacteria group H2;

[0027] Figure 8 These are photos of the potted plant growth promotion experiment of Ba45 and compound bacteria group H2;

[0028] Fig. 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 by 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 them according to 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 pieces according to their parts. Pathogenic fungi were isolated using PDA medium ( Figure 1 ). Observe the growth status of the colony and isolate and purify the pathogenic fungi. Extract DNA from the purified fungi and sequence it. Compare it with the NCBI database ( Figure 2 ) and then stored in a -80°C refrigerator. Common potato pathogenic fungi 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, place in a sterile Erlenmeyer flask filled with 95 mL LB culture medium, cover with sealing film, place in a shaker at 220 rpm for 15 min, let stand at room temperature for 10 min, take the supernatant and dilute 10 -3 , 10 -4 , 10 -5 100ul of dilution was spread on NA plates, and 3 replicates were set for each dilution gradient. After culturing in a 37℃ constant temperature incubator for 2 days, the culture was purified. Single colonies were picked and streaked on new NA plates to purify the target bacteria, and stored in -20 and -80℃ refrigerators 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 alternata, 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, the isolated bacteria are activated and inoculated on the plate. Four points are inoculated on each plate, and each bacterium is repeated three times. The culture dish was placed in a 30°C incubator and inverted for 48 hours, and the inhibition zone was observed. The bacteria that produced the inhibition zone were selected for strain identification. DNA was extracted, a PCR reaction system was established, and universal primers were used for amplification, followed by gel excision and 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 Bacillusatrophaeus.CNY01 in the database reached 99.93%, and the strain was identified as Bacillus atrophaeus, named Ba45 (Bacillus atrophaeus 45). The homology with Bacillus wiedmannii.KLS4 in the database reached 100%, indicating that the strain was Bacillus wiedmannii, named Bacillus wiedmannii Bw34. The homology with Bacillus subtilis.4ZT, Bacillus subtilis.BEST3102 and Bacillus subtilis.Bs21 in the database reached 100%, indicating that the strain was Bacillus subtilis, named Bacillus subtilis Bs3.

[0038] The Ba45 (Bacillus atrophaeus 45) screened in this example was milky white in the early stage, milky yellow in the later stage, and dark brown in color. The surface of the colony was not smooth, opaque, slightly raised, and the edge was 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 are milky white, round, smooth, with neat edges, sticky, and convex in the middle ( Figure 4 middle).

[0040] The Bacillus subtilis in this example is Bacillus subtilis Bs3, and the colony morphology is as follows: Gram-positive bacteria, on LB solid medium, the cells 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 zone of the antagonistic bacteria was measured to calculate the inhibition rate, and the composite antagonistic bacteria group with antagonistic effects on the above 6 pathogenic fungi and a high comprehensive inhibition rate was screened out. Bs3, Bw34 and Ba45 were selected, and strain compatibility experiments were carried out. After confirming that there was no antagonism between the three strains, the composite ratio was determined according to 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), and the inhibition rate was calculated (Table 1).

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

[0044]

[0045]

[0046] Example 5 Preparation of composite bacterial inoculation solution

[0047] 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 ratio, they 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 operation was 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 composite bacterial group H2.

[0048] Example 6 Preparation of mixed pathogenic fungal spore solution

[0049] Use an inoculation loop to take the mycelia of the above six pathogenic fungi on the PDA medium into the LB liquid medium, shake and culture at 28°C and 200 rpm until the OD value (OD600) is 1.0, mix equal volumes, shake for 30 minutes under the same conditions to fully mix the strains, and obtain a mixed pathogenic fungal spore solution.

[0050] Example 7 Comparison of the effect of antagonistic bacteria on disease inhibition in potted plants

[0051] Ba45 and H2, which have the highest antibacterial 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. The treatments included NPK (NPK alone), Ba45 (NPK+Ba45) and H2 (NPK+H2). Each treatment was repeated 5 times. NPK fertilizer was applied in equal amounts in each treatment. Experimental process: After the potatoes emerged, the spores of the above six mixed pathogenic fungi were inoculated, 50 mL per pot, and the spore concentration was ≥8.0×10 9cfu / mL, inoculated by root irrigation. One day after the pathogen was inoculated, Bacillus atrophaeus Ba45 and H2 were inoculated in the same way, and Bacillus atrophaeus Ba45 and H2 were inoculated again 10 days later, and NPK was not inoculated. The results showed (Table 2) that compared with NPK, the inoculation of antagonistic bacteria significantly reduced the incidence of plants 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 were more significant. It shows that the Bacillus subtilis in H2 that inhibits pathogens is Bacillus subtilis Bs3 and Bacillus wiedmannii Bw34, which may synergistically inhibit the reproduction of Ba45 and reduce its plate confrontation inhibition rate. In the case of plant growth, the environment of potted plants is more complicated. The composite bacteria use the synergistic effect between antagonistic bacteria to improve the stability of the disease inhibition system and enhance the antibacterial effect.

[0052] Table 2 Antagonistic bacteria potted disease inhibition test

[0053] deal with Number of potted plants Number of diseased plants Incidence (%) Prevention effect (%) NPK 5 2 40.0 — NPK+Ba45 5 1 20.0 50.0 NPK+H2 5 0 0 100

[0054] Example 8 Comparison of the growth-promoting effect of antagonistic bacteria in potted plants

[0055] The potato cultivation method of Example 7 was selected, but the pathogenic fungi were not inoculated after the potatoes emerged, and only Ba45 and H2 were inoculated to carry out a comparative test of the growth-promoting effect ( Figure 8 The results showed (Table 3) that, when all potatoes were disease-free, the plant height increased by 35.4% and the stem diameter increased by 60% in the Ba45 inoculation treatment, and the plant height increased by 80.1% and the stem diameter increased by 140% in the H2 inoculation treatment compared with NPK. It can be seen that the inoculation of antagonistic bacteria has a growth-promoting effect, especially H2.

[0056] Table 3 Growth-promoting effects of composite bacteria groups H2 and Ba45 in potted plants

[0057] deal with Number of potted plants Average plant height (cm) Average stem diameter (cm) Number of diseased plants NPK 3 15.55 0.5 0 NPK+Ba45 3 21.06 0.8 0 NPK+H2 3 28.00 1.2 0

[0058] Example 9: Disease suppression effect of composite bacteria group H2 in the field

[0059] The stability of the field application effect of the composite bacteria group H2 was further verified by using a potato continuous cropping test field where mixed soil-borne diseases have occurred continuously in recent years, and a field test for screening the best application method was carried out. The test treatments included single application of chemical fertilizer (NPK), chemical fertilizer + inoculation of H2 (NPKH2), chemical fertilizer + organic material (NPKM) and chemical fertilizer + organic material + inoculation of H2 (MH2), with 3 replicates per treatment ( Fig. 9). The composite bacteria group inoculation solution prepared according to Example 5 was inoculated in the seedling stage, the initial flowering stage and the full flowering stage of potatoes, 100 mL was inoculated each 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. It shows that the growth-promoting bacteria Bw34 in the composite bacteria group H2 can play a role in promoting growth and increasing production without the application of organic materials, and it is more suitable for use alone.

[0060] Table 4 Field application effect of compound bacteria group H2

[0061] deal with <![CDATA[Inoculation area (m 2 )]]> Number of inoculated plants Incidence (%) Disease index Yield (t / ha) NPK — — 100.0 32.96 19.2 NPKH2 4.86 40 98.3 28.33 33.4 NPKM — — 100.0 28.89 33.0 MH2 4.86 40 96.7 28.15 33.3

[0062] The present invention obtains a composite bacterial group H2 which has a strong inhibitory effect on 6 kinds of potato pathogenic fungi. Subsequent research can gradually increase the inhibition verification of other potato pathogenic fungi, and it can also be compounded with other biocontrol bacteria, providing high-quality strains for the development and research of fungal agents for widely inhibiting pathogenic fungi of potato soil-borne diseases, and 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 Wiedemannii; The Bacillus subtilis is Bacillus subtilis Bs3, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No. 32579; The atrophaeus bacillus is Bacillus atrophaeus Ba45, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No. 32577; The Bacillus wiedmannii Bw34 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 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 Wiedemannii in a volume ratio of (0.5-2):(0.5-2):(0.5-1).

3. The use of a 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.

4. The use according to claim 3, characterized in that The potato pathogenic fungi are pathogenic fungi of soil-borne diseases.

5. The use according to claim 3 or 4, characterized in that 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.

6. The use according to claim 5, characterized in that The antibacterial rate of the microbial composite bacteria H1 to Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 46-76%.

7. A method for preparing the microbial composite bacteria H2 for antagonizing multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The preparation method is as follows: Bacillus subtilis Bs3, Bacillus atrophaeus Ba45 and Bacillus wiedmannii Bw34 are inoculated into a liquid LB culture medium, cultured until OD600 is 0.6, mixed the bacterial liquid in proportion, and then continued to be inoculated into the liquid LB culture medium, and cultured until the OD600 of the mixed bacterial liquid is 1.0, so as to obtain the microbial composite bacteria H2.

8. The preparation method according to claim 7, characterized in that The bacterial count of the microbial composite bacteria H2 is ≥ 8.0×10 9 cfu / mL.

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