Microbial complex bacteria H1 for antagonizing multiple pathogenic fungi of potato and application and preparation method thereof

Abstract

The application relates to a microbial compound bacterium H1 antagonizing multiple potato pathogenic fungi and an application and a preparation method thereof, and relates to the field of biological bacterial agents. The microbial compound bacterium H1 is mixed by bacillus subtilis, bacillus atrophaeus and bacillus cereus. The microbial compound bacterium H1 is used for preparing a bacterium agent antagonizing potato pathogenic fungi. The H1 has a strong inhibiting effect on the early blight pathogenic fungi alternaria solani and alternaria alternata, the dry rot pathogenic fungi fusarium solani, fusarium oxysporum and fusarium equiseti, and the leaf / stem spot pathogenic fungi botryodiplodia theobromae, and the inhibiting rates are all higher than 45%. The inhibiting rate on the botryodiplodia theobromae is the highest, reaching 76.92%. The H1 provides a high-quality strain for comprehensively preventing and treating potato soil-borne diseases, and has a good field application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to biological bacterial agent, and particularly relates to a microorganism compound bacteria H1 antagonizing multiple pathogenic fungi of potato and an application and a preparation method thereof. BACKGROUND

[0002] Potato is the fourth largest food crop in the world, which is related to food security. Due to the change of planting system, the area of long-term continuous cropping of potato is increasing, which leads to soil degradation and serious soil-borne disease problems. The main potato soil-borne disease is fungal disease. The main diseases in the main production areas of China include early blight (also known as brown spot disease), the pathogenic fungus of which is Alternaria, which mainly infects stems and leaves; dry rot / wilt disease, the pathogenic fungus of which is Fusarium, which mainly infects tubers and is also an important storage period disease; leaf / stem spot disease, which is a common plant disease, and the diseased plant produces disease spots on the leaves / stems, which are connected into pieces when serious, affecting photosynthesis and causing yield reduction. Among them, Didymella glomerata can cause leaf / stem spot disease in nearly 100 kinds of plants, and is a quarantine plant pathogenic fungus in China. The strain is isolated from potato disease spots for the first time in the test area. With the development of biotechnology, more and more antagonistic bacterial agents are produced. However, due to the complexity of the field environment, the strain has poor stress resistance, the actual application effect is unstable, and it is difficult to achieve the disease inhibition effect in the laboratory. Therefore, obtaining an antagonistic bacterial agent with stable field application effect is an urgent problem to be solved for potato soil-borne diseases.

[0003] Bacillus is the most widely used antagonistic bacterial species, especially Bacillus subtilis, which has been applied in a variety of mature soil-borne disease control agents and biological organic fertilizers, and commercialized. Bacillus atrophaeus is a variety of Bacillus subtilis, which widely exists in the natural environment and has significant biocontrol potential for a variety of soil-borne diseases, and has been widely used in plant disease control in recent years. Bacillus cereus is mainly used for degrading complex organic compounds, and its application in inhibiting soil-borne diseases is less. Among them, only Bacillus subtilis is relatively more applied in potato disease control, but it mainly prevents late blight. Meanwhile, Bacillus subtilis, Bacillus atrophaeus or Bacillus cereus for inhibiting potato early blight, wilt disease, dry rot disease and leaf / stem spot disease pathogenic fungi Alternaria solani, Alternaria solani, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Didymella glomerata have not been reported.

[0004] With the popularization of antagonistic bacteria, it is found that the synergistic inhibition effect of complex bacteria group is often better than that of single bacteria. Therefore, the development and application of beneficial microorganism group, such as rhizosphere growth promoting bacteria (PGPR) and synthetic microorganism group (SynCom), are gradually valued. However, stable and broad-spectrum disease-inhibiting complex microbial agents are still being explored, and the combination of the above Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus has not been reported in the complex microbial agents for inhibiting soil-borne diseases of potato. SUMMARY

[0005] The present application provides a kind of microbial complex bacteria H1 for antagonizing multiple pathogenic fungi of potato and its application and preparation method.

[0006] The microbial complex bacteria H1 for antagonizing multiple pathogenic fungi of potato is mixed by Bacillus subtilis, Bacillus atrophaeus and Bacillus cereus.

[0007] The Bacillus subtilis is Bacillus subtilis Bs3, preserved in China General Microbiological Culture Collection Center, with the preservation address of No.3, Beichen West Road, Chaoyang District, Beijing, the preservation date of November 11, 2024, and the preservation number of CGMCC No.32579.

[0008] The Bacillus atrophaeus is Bacillus atrophaeus Ba45, preserved in China General Microbiological Culture Collection Center, with the preservation address of No.3, Beichen West Road, Chaoyang District, Beijing, the preservation date of November 11, 2024, and the preservation number of CGMCC No.32577.

[0009] The Bacillus cereus is Bacillus cereus Bc19, preserved in China General Microbiological Culture Collection Center, with the preservation address of No.3, Beichen West Road, Chaoyang District, Beijing, the preservation date of November 11, 2024, and the preservation number of CGMCC No.32578.

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

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

[0012] Further, the pathogenic fungi of potato is the pathogenic bacteria of soil-borne disease.

[0013] Further, the potato pathogenic fungi is a pathogenic fungus of soil-borne disease, and the pathogenic fungus of soil-borne disease is Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Botryodiplodia theobromae.

[0014] Further, the bacterium number of the microorganism complex bacteria H1 is ≥8.0×10

[0015] The preparation method of the microorganism complex bacteria H1 for antagonizing multiple pathogenic fungi of potato is as follows:

[0016] Bacillus subtilis Bs3, Bacillus atrophaeus Ba45 and Bacillus cereus Bc19 are inoculated into liquid LB medium, and when the OD 600 of the bacteria is 0.6, the bacteria liquid is mixed in proportion, and then inoculated into liquid LB medium, and when the OD 600 of the mixed bacteria liquid is 1.0, the microorganism complex bacteria H1 is obtained.

[0017] Further, the bacterium number of the microorganism complex bacteria H1 is ≥8.0×10 9 cfu / mL.

[0018] The present application comprises the following beneficial effects:

[0019] The present application provides a complex bacteria H1 for inhibiting multiple soil-borne disease pathogenic fungi of potato, which is composed of three antagonistic bacteria isolated from potato continuous cropping soil in an extreme environment, and has high antibacterial ability. H1 has strong inhibition on potato early blight pathogenic fungi Alternaria solani and Alternaria alternata, fusarium wilt / dry rot pathogenic fungi Fusarium solani, Fusarium oxysporum and Fusarium equiseti, and leaf / stem spot pathogenic fungi Botryodiplodia theobromae, and the inhibition rate is higher than 45%. Among them, the inhibition rate on Botryodiplodia theobromae is the highest, reaching 76.92%. Compared with Ba45 with the highest comprehensive inhibition rate on pathogenic fungi, H1 shows more stable and stronger disease inhibition effect in potting test, and the control effect is 50% higher than that of Ba45. At the same time, in the field application test, H1 reduces the incidence of early blight and improves the yield of potato. Therefore, H1 provides a high-quality strain for comprehensive prevention and control of potato soil-borne diseases, and has good field application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Isolation culture photos of pathogenic fungi; wherein, A1 is a culture isolation photo, A2-A4 are Alternaria alternata, A. solani, and E. gloeopora photos respectively; B1 is a diseased plant photo for isolating pathogenic fungi, B2-B4 are F. solani, F. oxysporum, and F. equiseti photos respectively;

[0021] Figure 2 BLAST alignment chart of pathogenic fungi; wherein, from top to bottom are Alternaria alternata, A. solani, E. gloeopora, F. solani, F. oxysporum, and F. equiseti respectively;

[0022] Figure 3 BLAST alignment chart of 3 antagonistic bacteria; wherein, from top to bottom are B. atrophaeus, B. cereus, and B. subtilis respectively;

[0023] Figure 4 Morphology photos of 3 antagonistic bacteria after purification; wherein, from left to right are B. atrophaeus, B. cereus, and B. subtilis respectively;

[0024] Figure 5 Co-culture photos of 3 antagonistic bacteria; wherein, from left to right are streak co-culture and spread co-culture respectively;

[0025] Figure 6 Plate confrontation photos of bacteria inhibition; wherein, from left to right in the upper row are Alternaria alternata, A. solani, and E. gloeopora respectively; from left to right in the lower row are F. solani, F. oxysporum, and F. equiseti respectively;

[0026] Figure 7 Photos of Ba45 and complex bacteria group H1 disease inhibition pot experiment;

[0027] Figure 8 Photos of Ba45 and complex bacteria group H1 growth promotion pot experiment;

[0028] Figure 9 Photos of complex bacteria group H2 potato continuous cropping field inoculation experiment area; wherein, the left is potato growth at flowering stage without disease, and the right is growth at disease stage. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear and apparent, the spirit of the present application will be described in detail below, and any person skilled in the art can make changes and modifications to the technology taught by the present application content after understanding the embodiments of the present application content, without departing from the spirit and scope of the present application content.

[0030] The schematic embodiments of the present application and the descriptions thereof are used to explain the present application, but not as a limitation on the present application.

[0031] Isolation and purification of pathogenic fungi in example 1

[0032] The potato disease strains were selected from the long-term continuous cropping field of potatoes, cut into small pieces according to the parts, and the pathogenic fungi were isolated and cultured by using PDA medium. Figure 1 The growth state of the colonies was observed, and the pathogenic fungi were isolated and purified. The purified fungi were extracted and sequenced, and after comparison with the NCBI database, Figure 2 The common pathogenic fungi of potatoes, Alternaria solani, A. alternata, Fusarium solani, F. oxysporum, F. equiseti and the first isolated pathogenic fungus of grape stem blight were selected for standby.

[0033] The long-term continuous cropping field of potatoes is located in Wuchuan County, Inner Mongolia Autonomous Region, and the soil is chestnut soil, which is relatively poor, with low organic matter content and perennial drought.

[0034] Isolation and purification of bacteria in example 2

[0035] Potato continuous cropping soil was collected, 5.0 g was weighed and placed in a sterile triangular flask containing 95 mL of LB culture solution, covered with a sealing film, and placed in a shaking bed at 220 rpm for 15 min, and then left at room temperature for 10 min. The supernatant was diluted 10 -3 , 10 -4 , 10 -5 times, 100 uL of the diluted solution was taken and spread on NA plates, 3 repeats were set for each dilution gradient, and then placed in a 37℃ constant temperature incubator for culture for 2 days, and then purified and cultured. The single colonies were picked and streaked on new NA plates to purify the target bacteria, and stored in -20 and -80℃ refrigerators for standby.

[0036] Screening and identification of antagonistic bacteria in example 3

[0037] The strains with inhibitory effect on Alternaria solani, A. alternata, Fusarium solani, F. oxysporum, F. equiseti and grape stem blight were screened by plate confrontation culture method. First, the colonies on the above-mentioned pathogenic fungus culture medium were taken with a sterile pipette gun head and placed on new PDA plates, and then the isolated bacteria were activated and inoculated on the plates, 4 points were inoculated on each plate, and 3 repeats were made for each bacterium. The culture dishes were placed in a 30℃ incubator and cultured for 48h, and then the inhibition zone was observed. The bacteria producing inhibition zone were selected for strain identification. DNA was extracted, PCR reaction system was established, universal primers were used for amplification, and gel was cut for sequencing. The obtained 16S rRNA gene sequence was compared in NCBI database Figure 3The strain showed 99.93% homology with Bacillus atrophaeus.SK3, Bacillus atrophaeus.XJUHX-35, and Bacillus atrophaeus.CNY01 in the database, identifying it as Bacillus atrophaeus and naming it Ba45 (Bacillus atrophaeus 45). It also showed over 99.8% homology with Bacillus cereus.BF15 and Bacillus cereus.MOB-3 in the database, indicating it was Bacillus cereus and naming it Bacillus cereus.Bc19. Finally, it showed 100% homology with Bacillus subtilis.4ZT, Bacillus subtilis.BEST3102, and Bacillus subtilis.Bs21 in the database, indicating it was Bacillus subtilis and naming it Bacillus subtilis Bs3.

[0038] The Ba45 (Bacillus atrophaeus 45) screened in this example was initially milky white on LB solid medium, later turning milky yellow and dark brown. The colony surface was not smooth, opaque, slightly raised, and had irregular wavy edges. Figure 4 Left).

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

[0040] The Bacillus subtilis strain used in this embodiment is Bacillus subtilis Bs3. Its colony morphology is as follows: Gram-positive bacteria; on LB solid medium, the spores are uniformly stained; the colony surface is rough and opaque, milky white or slightly yellow, with neat edges. Figure 4 right).

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

[0042] The inhibition zones of the above antagonistic bacteria were measured to calculate the inhibition rate. Strains exhibiting antagonistic effects against all six pathogenic fungi and demonstrating high overall inhibition rates were screened and combined with a compound antagonistic bacterial group. Figure 5), Bs3, Bc19 and Ba45 were selected to carry out strain compatibility experiment, after it was determined that there was no antagonism among the three strains, the compound ratio was determined according to the growth rate of the strains, the volume ratio of Bacillus subtilis, Bacillus atrophaie and Bacillus cereus was 1:1:1, and the compound bacteria group H1 of the application was obtained. The plate confrontation experiment of the compound bacteria group H1 for inhibiting pathogenic bacteria was carried out, and the inhibition rate was calculated (Table 1, Figure 6 )。

[0043] Table 1 Inhibition rate of antagonistic bacteria single strain and compound bacteria group on pathogenic fungi

[0044]

[0045] Example 5 Preparation of compound bacteria group inoculation bacterial liquid

[0046] Bs3, Bc19 and Ba45 were inoculated into liquid LB medium, and cultured at 37℃ and 180rpm until the OD value (OD600) was 0.6, then mixed according to the volume ratio of Bacillus subtilis, Bacillus atrophaie and Bacillus cereus 1:1:1, and cultured at 37℃ and 180rpm until the OD value (OD600) was 1.0, then centrifuged to remove the supernatant, added distilled water and vortexed, centrifuged, and the above operation was repeated to wash away the LB medium, and then added water to adjust the bacterial number to be greater than or equal to 8.0x10 9 cfu / mL, which was the compound bacteria group H1 to be inoculated.

[0047] Example 6 Preparation of mixed pathogenic fungus spore liquid

[0048] The mycelium of the above-mentioned six kinds of pathogenic fungi on PDA medium was taken by a bacteria inoculation ring into LB liquid medium, and cultured at 28℃ and 200rpm until the OD value (OD600) was 1.0, then mixed by equal volume, and mixed the strains sufficiently under the same conditions for 30 minutes, and the mixed pathogenic fungus spore liquid was obtained.

[0049] Example 7 Comparison of antagonistic bacteria inhibition effect by potting

[0050] The highest inhibition rate of Ba45 and the compound bacteria group H1 among the above-mentioned three antagonistic bacteria were selected to carry out the comparison test of potato potting disease inhibition effect Figure 7 ). Sterilized soil was used to plant potatoes (detoxification seed potatoes), and the treatments included NPK (single NPK), inoculation of Ba45 (NPK+Ba45) and inoculation of H1 (NPK+H1), with 5 repetitions for each treatment, wherein the NPK fertilizers of each treatment were applied in equal amounts. The test procedure was as follows: after the potatoes were emerged, the above-mentioned six kinds of mixed pathogenic fungus spore liquid was inoculated, 50mL per pot, and the spore concentration was greater than or equal to 8.0x10 9cfu / mL, and were inoculated in a root irrigation manner. One day after inoculation of the pathogenic bacteria, Ba45 and H1 were inoculated in the same manner, and Ba45 and H1 were inoculated again after 10 days. NPK was not inoculated. The results (Table 2) show that, compared with NPK, inoculation of the antagonistic bacteria significantly reduced the plant disease incidence, and H1 reached 100% control effect. Compared with Ba45, the complex bacteria group H1 has more significant disease inhibition effect, which compensates for the disadvantage of single strain in actual crop cultivation that the antagonistic effect is easily weakened when mixed with pathogenic bacteria.

[0051] Table 2 Potting disease inhibition of complex bacteria group H1 and Ba45

[0052] Treatment Number of plants Number of diseased plants Disease incidence (%) Control efficiency (%) NPK 5 2 40.0 — NPK+Ba45 5 1 20.0 50.0 NPK+H1 5 0 0 100

[0053] Example 8 Potting comparison of the growth promotion effect of antagonistic bacteria

[0054] The potato cultivation method of Example 7 was selected, but after the potato seedlings emerged, no pathogenic fungi were inoculated, and only Ba45 and H1 were inoculated to carry out a growth promotion effect comparison test. Figure 8 The results (Table 3) show that, under the condition that the potatoes did not have diseases, compared with NPK, the plant height of the Ba45 inoculation treatment increased by 35.4%, and the stem diameter increased by 60%; 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 of antagonistic bacteria has a growth promotion effect, especially H1, which indicates that the B. subtilis Bs3 and B. cereus Bc19 in H1 may synergistically inhibit the reproduction of Ba45, reducing the plate confrontation inhibition rate. In the potting environment, the environment is more complex with plant growth, and the complex bacteria utilize the synergistic effect between antagonistic bacteria to improve the stability of the disease inhibition system and enhance the inhibition effect.

[0055] Table 3 Growth promotion effect of complex bacteria group H1 and Ba45 in potting

[0056]

[0057]

[0058] Example 9 Field disease inhibition effect of complex bacteria group H1

[0059] In order to further verify the stability of the application effect of the complex bacteria group H1 in the field, the best application mode is selected, and the inoculation and organic material compounding test is carried out in the potato continuous cropping field. The potato field has been continuously suffering from mixed soil-borne diseases in recent years, including early blight, fusarium wilt, stem spot and the like, which is suitable for verifying the disease inhibition effect of the complex bacteria. The treatment includes 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), and each treatment has 3 repetitions Figure 9 ). The complex bacteria group inoculation bacteria liquid prepared according to the preparation method of the complex bacteria group in the embodiment 5 is inoculated at the seedling stage, the initial flowering stage and the full flowering stage of the potato, 100 mL per plant per time, and the inoculation is carried out in the form of root irrigation. Because the climate condition is suitable for the reproduction of the early blight pathogen in the season, the early blight occurs in a large area. The early blight disease index result (table 4) shows that although there is no obvious difference between the disease index of NPK and NPKH1, the disease incidence of NPKH1 is significantly reduced, and the potato yield is increased by 9.4%. It can be seen that the complex bacteria group H1 has a certain growth promoting function, mainly by reducing the disease incidence to control the disease and then increase the yield. NPKM reduces the disease index, and the yield increasing effect is better than that of NPKH1, and the disease index is increased (3.4% higher than that of NPK) when the H1 is inoculated in the case of applying the organic material, but the disease incidence is greatly reduced, and the yield is increased by 10.3% than that of applying the organic material. It is indicated that the complex bacteria group H1 is more suitable for being applied in combination with the organic material, which may be related to the fact that bacillus cereus has a significant ability to degrade complex organic compounds, and the organic material is rapidly reduced to generate nutrients beneficial to the growth of the potato, so that the growth of the potato is promoted, and the disease resistance is improved, and the yield increasing effect is achieved.

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

[0061] Treatment Inoculum area (m 2 )]]> Number of inoculated plants Disease incidence (%) Disease index Yield (t / ha) NPK — — 100.0 32.96 19.2 NPK H1 4.86 40 98.3 32.22 21.0 NPK M — — 100.0 28.89 33.0 M H1 4.86 40 96.7 34.07 36.4

[0062] The complex bacteria group H1 with strong inhibition effect on six pathogenic fungi of potato is obtained, and the subsequent research can gradually increase the inhibition verification of other pathogenic fungi of potato, and the complex bacteria group H1 can also be compounded with other biocontrol bacteria, so that a high-quality strain is provided for the development and research of a broad-spectrum pathogenic fungus agent for inhibiting soil-borne diseases of potato, and a foundation is laid.

Claims

1. A microbial complex H1 antagonizing a variety of pathogenic fungi of potato, characterized in that The microorganism composite bacteria is mixed by bacillus subtilis ( Bacillus subtilis ), bacillus atrophaeus ( Bacillus atrophaeus ) and bacillus cereus ( Bacillus cereus ). The Bacillus subtilis is Bacillus subtilis Bs3, preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation date of November 11, 2024, and a preservation number of CGMCC No. 32579; The atrophied Bacillus is Bacillus atrophaeus Ba45, preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on November 11, 2024, with a preservation number of CGMCC No. 32577; The Bacillus cereus is Bacillus cereus Bc19, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xibaixili, Chaoyang District, Beijing, on November 11, 2024, and has a preservation number of CGMCC No. 32578.

2. The microbial complex H1 antagonizing a plurality of pathogenic fungi of potato according to claim 1, characterized in that The microbial complex bacteria are 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 the microbial complex H1 antagonizing a number of pathogenic fungi of potato according to claim 1, characterized in that The microorganism complex bacteria H1 is used for preparing a bacteria agent for antagonizing potato pathogenic fungi, the potato pathogenic fungi are pathogenic bacteria of soil-borne diseases, and the soil-borne diseases are alternaria solani ( Alternaria solani ), alternaria macrospora ( Alternaria alternata ), fusarium solani ( Fusarium solani ), fusarium oxysporum ( Fusarium oxysporum ), fusarium equiseti ( Fusarium equiseti ) and botryosphaeria dothidea ( Didymella glomerata ).

4. Use according to claim 3, characterized in that The microbial complex bacteria H1 have an inhibition rate of 45-77% on alternaria solani, alternaria alternata, fusarium solani, fusarium oxysporum, fusarium equiseti and didymella glomerata.

5. A method for preparing the antagonistic microbial complex Hl of various pathogenic fungi of potato according to claim 1, characterized in that The preparation method is as follows: Bacillus subtilis ( Bacillus subtilis Bs3, Bacillus atrophus ( Bacillus atrophaeus Ba45 and Bacillus cereus ( Bacillus cereus Bc19 was inoculated into liquid LB medium and cultured at 180 rpm until its OD 600 was 0.

6. After mixing the bacterial solution in proportion, it was inoculated into liquid LB medium again and cultured at 180 rpm until the OD 600 of the mixed bacterial solution was 1.0, thus obtaining the microbial complex H1.

6. The method of claim 5, wherein The number of bacteria of the microbial complex bacteria H1 is ≥8.0×10 9 cfu / mL.

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