Microbial compound bacterium H1 for antagonizing multiple pathogenic fungi of potatoes as well as application and preparation method of microbial compound bacterium H1

Abstract

The invention discloses a microbial compound bacterium H1 for antagonizing multiple pathogenic fungi of potatoes and application and a preparation method thereof, and relates to the field of biological agents, and the microbial compound bacterium H1 is formed by mixing bacillus subtillis, bacillus atrophaeus and bacillus cereus. The microbial compound bacteria H1 disclosed by the invention are used for preparing a microbial agent for antagonizing potato pathogenic fungi. The H1 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 rate is higher than 45%. Wherein the inhibition rate on the grape stem blight germs is the highest and reaches 76.92%. H1 provides a high-quality strain for comprehensively preventing and treating potato soil-borne diseases, and the field application prospect is good.

Description

Technical Field

[0001] The invention belongs to a biological bacterial agent, and specifically relates to a microbial composite bacteria H1 for antagonizing multiple pathogenic fungi of potato, and an application and a preparation method thereof. Background Art

[0002] Potato is the fourth largest food crop in the world and is related to food security. Due to changes in the planting system, the area of ​​long-term continuous potato planting has continued to increase, resulting in soil degradation and serious soil-borne diseases. Potato soil-borne diseases are mainly fungal diseases. The main diseases in my country's main production areas include early blight (also known as brown spot disease), the pathogen of which is Alternaria, which mainly infects stems and leaves; dry rot / wilt disease, the pathogen of which is Fusarium, which mainly infects tubers and is also an important storage disease; leaf / stem spot disease is a common plant disease, and the infected plants produce lesions on the leaves / stems. In severe cases, they are connected into pieces, affecting photosynthesis and causing yield reduction. Among them, grape stem blight (Didymella glomerata) can cause leaf / stem spot disease in nearly 100 plants and is a quarantine plant pathogen in my country. This strain was isolated from potato lesions for the first time in this test area. With the development of biotechnology, more and more antagonistic bacteria products are available. However, due to the complexity of the field environment, the strains have poor stress resistance, and the actual application effect is unstable, it is difficult to achieve the disease suppression effect in the laboratory. Therefore, obtaining antagonistic bacteria with stable field application effects is an urgent problem to be solved in potato soil-borne diseases.

[0003] Bacillus is the most widely used antagonistic bacteria, especially Bacillus subtilis, which has been used in a variety of mature soil-borne disease control agents and bio-organic fertilizers and has been commercialized. Bacillus atrophaeus is a variant of Bacillus subtilis, which 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 control in recent years. Bacillus cereus is mainly used to degrade complex organic compounds and is less used in inhibiting soil-borne diseases. Among them, only Bacillus subtilis is relatively widely used in potato disease control, but it is mainly used to control late blight. Bacillus subtilis, Bacillus atrophaeus or Bacillus cereus that simultaneously inhibit potato early blight, wilt, dry rot and leaf / stem spot pathogens such as Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight have not been reported.

[0004] With the increasing popularity of antagonistic bacteria, people have found that the synergistic antibacterial effect of composite bacteria is often better than that of single bacteria. Therefore, the development and application of beneficial microbial groups have gradually received attention, such as growth-promoting rhizobacteria (PGPR) and synthetic microbial groups (SynCom). However, stable and broad-spectrum composite microbial agents are still being explored, and the combination of Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus 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 H1 for antagonizing multiple pathogenic fungi of potato and its application and preparation method.

[0006] The present invention discloses a microbial composite bacteria H1 for antagonizing multiple pathogenic fungi of potato, wherein the microbial composite bacteria is a mixture of Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus;

[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 cereus is Bacillus cereus Bc19, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date at November 11, 2024, and the deposit number at CGMCC No.32578.

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

[0011] The microbial composite bacteria H1 of the present invention is used for preparing a bacterial agent for antagonizing potato pathogenic fungi.

[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 45-77% against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0015] The preparation method of the microbial composite bacteria H1 for antagonizing multiple pathogenic fungi of potato according to the present invention is as follows:

[0016] Bacillus subtilis Bs3, Bacillus atrophaeus Ba45 and Bacillus cereus Bc19 were inoculated into a liquid LB medium, cultured at a 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 speed of 180 rpm until the OD 600 of the mixed bacterial liquid was 1.0, thereby obtaining a microbial composite bacteria H1.

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

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

[0019] The present invention provides a composite bacterium H1 for inhibiting pathogenic fungi of multiple soil-borne diseases of potatoes. The composite bacterium H1 is composed of three antagonistic bacteria isolated from potato continuous cropping soil in an extreme environment, and has high antibacterial ability. H1 has a strong inhibitory effect on potato early blight pathogenic fungi Alternaria solani and Alternaria solani, wilt / dry rot pathogenic fungi Fusarium solani, Fusarium oxysporum and Fusarium equisetum, and leaf / stem spot pathogenic fungi Grape stem blight, and the inhibition rate is higher than 45%. Among them, the inhibition rate of grape stem blight is the highest, reaching 76.92%. Compared with Ba45 with the highest comprehensive inhibition rate of pathogens, H1 showed a more stable and stronger disease inhibition effect in potted plant experiments, and the prevention effect was 50% higher than that of Ba45. At the same time, in field application experiments, H1 reduced the incidence of early blight and increased potato yield. Therefore, H1 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 1are 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 This is a BLAST comparison of three antagonistic bacteria; from top to bottom, they are Bacillus atrophaeus, Bacillus cereus, 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 cereus, 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 H1;

[0027] Figure 8 These are photos of the potted plant growth promotion experiment of Ba45 and compound bacteria group H1;

[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 triangular flask containing 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 the dilution solution was spread on the NA plate, and each dilution gradient was repeated 3 times. After culturing in a 37℃ constant temperature incubator for 2 days, the culture was purified. A single colony was picked and streaked on a new NA plate 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 cereus.BF15 and Bacillus cereus.MOB-3 in the database was higher than 99.8%, indicating that the strain was Bacillus cereus, named Bacillus cereus.Bc19. The homology with Bacillus subtilis.4ZT, Bacillus subtilis.BEST3102 and Bacillussubtilis.Bs21 in the database reached 100%, indicating that the strain was Bacillus subtilis, named Bacillussubtilis 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 zones of the above antagonistic bacteria were measured to calculate the inhibition rate, and the composite antagonistic bacteria group with antagonistic effects on the above six pathogenic fungi and a high comprehensive inhibition rate was screened out ( Figure 5), Bs3, Bc19 and Ba45 were selected, and strain compatibility experiments were carried out. After confirming that there was no antagonism between the three strains, the compounding ratio was determined according to the growth rate of the strains. The volume ratio of Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus was 1:1:1, and the composite bacterial group H1 of the present invention was obtained. A plate confrontation experiment of the composite bacterial group H1 to inhibit pathogenic bacteria was carried out, and the inhibition rate was calculated (Table 1, Figure 6 ).

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

[0044]

[0045] Example 5 Preparation of composite bacterial inoculation solution

[0046] Bs3, Bc19 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. Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus were mixed at a volume ratio of 1:1:1 and 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, centrifuged, and 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 H1.

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

[0048] 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.

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

[0050] Among the three antagonistic bacteria mentioned above, Ba45 and composite bacteria group H1 with the highest antibacterial rate 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 H1 (NPK+H1). Each treatment was repeated 5 times. NPK fertilizer was applied in equal amounts in each treatment. Experimental procedure: 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 inoculation, Ba45 and H1 were inoculated in the same way, and Ba45 and H1 were inoculated again 10 days later, but NPK was not inoculated. The results showed (Table 2) that compared with NPK, the inoculation of antagonistic bacteria significantly reduced the incidence of plants, especially H1, whose prevention efficiency reached 100%. This shows that compared with Ba45, the disease inhibition effect of the composite bacteria group H1 is more significant, which makes up for the disadvantage that the antagonistic effect of a single strain is easily weakened when antagonizing mixed pathogens in actual crop cultivation.

[0051] Table 2 Disease inhibition of composite bacteria groups H1 and Ba45 potted plants

[0052] 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+H1 5 0 0 100

[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 the pathogenic fungi were not inoculated after the potatoes emerged, and only Ba45 and H1 were inoculated to carry out a comparative test of the growth-promoting effect ( Figure 8 ). The results showed (Table 3) that, when no potato was diseased, the plant height of the plant inoculated with Ba45 increased by 35.4%, and the stem diameter increased by 60% compared with NPK, and the plant height of the plant inoculated with H1 increased by 65.0%, and the stem diameter increased by 100%. It can be seen that the inoculation of antagonistic bacteria has a growth-promoting effect, especially H1, indicating that the Bacillus subtilis Bs3 and Bacillus cereus Bc19 in H1 that inhibit pathogens may synergistically inhibit the reproduction of Ba45 and reduce its plate confrontation inhibition rate. In the case of potted plants growing with plants, the environment is more complex, and the composite bacteria use the synergistic effect between antagonistic bacteria to improve the stability of the disease inhibition system and enhance the antibacterial effect.

[0055] Table 3 Growth promotion effect of composite bacteria group H1 and Ba45 in potted plants

[0056]

[0057]

[0058] Example 9: Field disease suppression effect of composite bacteria group H1

[0059] In order to further verify the stability of the application effect of the composite bacteria group H1 in the field and screen its best application method, the inoculation and compounding experiments with organic materials were carried out in potato continuous cropping fields. Due to continuous cropping, the potato field has been plagued by mixed soil-borne diseases in recent years, including early blight, wilt, stem spot, etc., which is suitable for verifying the disease inhibition effect of the composite bacteria. The treatments included single application of chemical fertilizer (NPK), chemical fertilizer + inoculation of H1 (NPKH1), chemical fertilizer + organic material (NPKM) and chemical fertilizer + organic material + inoculation of H1 (MH1), with 3 replicates per treatment ( Fig. 9 ). The composite bacteria group inoculation solution prepared in Example 5 was inoculated in the seedling stage, the initial flowering stage and the full flowering stage of potatoes, 100 mL was inoculated each time for each plant, 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 although there was no significant difference in the disease index between NPK and NPKH1, NPKH1 significantly reduced the incidence rate, and the potato yield increased by 9.4%. It can be seen that the composite bacteria group H1 has a certain growth-promoting function, which mainly controls the disease by reducing the incidence rate, thereby increasing production. While reducing the disease index, NPKM has a better yield-increasing effect than NPKH1, while the disease index of H1 inoculation increased when organic materials were applied (3.4% higher than NPK), but it greatly reduced the incidence rate, and the yield increased by 10.3% compared with the application of organic materials. This indicates that the composite bacteria group H1 is more suitable for application in combination with organic materials, which may be related to the ability of Bacillus cereus to significantly degrade complex organic compounds. It quickly reduces organic materials and generates nutrients that are beneficial to potato growth, promoting its growth, thereby improving disease resistance and achieving an increased yield effect.

[0060] Table 4 Field application effect of compound bacteria group H1

[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 NPKH1 4.86 40 98.3 32.22 21.0 NPKM — — 100.0 28.89 33.0 MH1 4.86 40 96.7 34.07 36.4

[0062] The present invention obtains a composite bacterial group H1 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 bacterial agents for widely inhibiting pathogenic fungi of potato soil-borne diseases, and laying a foundation.

Claims

1. A microbial composite bacteria H1 for antagonizing multiple pathogenic fungi of potato, characterized in that The microbial composite bacteria is a mixture of Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus; 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 cereus is Bacillus cereus Bc19, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date at November 11, 2024, and the deposit number at CGMCC No.32578.

2. The microbial composite bacteria H1 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 cereus in a volume ratio of (0.5-2):(0.5-2):(0.5-1).

3. The use of a microbial composite bacteria H1 for antagonizing multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The microbial composite bacteria H1 is used for preparing 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 45-77%.

7. A method for preparing the microbial composite bacteria H1 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 cereus Bc19 are inoculated into a liquid LB culture medium, cultured at a speed of 180 rpm until the OD 600 is 0.6, mixed the bacterial liquid in proportion, continued to be inoculated into the liquid LB culture medium, and cultured at a speed of 180 rpm until the OD 600 of the mixed bacterial liquid is 1.0, thereby obtaining the microbial composite bacteria H1.

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

Images