CN111172080A - Bacillus belgii and application thereof - Google Patents
Bacillus belgii and application thereof Download PDFInfo
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- CN111172080A CN111172080A CN202010110986.3A CN202010110986A CN111172080A CN 111172080 A CN111172080 A CN 111172080A CN 202010110986 A CN202010110986 A CN 202010110986A CN 111172080 A CN111172080 A CN 111172080A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a Bacillus belgii strainBacillus velezensis) The preservation number is CGMCC No. 19215. The Bacillus belgii and its fermentation liquid and metabolite can be used for inhibiting plant pathogenic bacteria and promoting plant growth. The Bacillus beleisi has biocontrol and growth promoting effects, can effectively reduce the occurrence of soil-borne diseases of crops such as tomatoes, strawberries, corns and the like, and reduces the use amount of chemical agents. The preparation prepared by the method has stable storage performance and good control effect, can directly contact with crop roots, has no adverse effect on plant growth and development, is convenient to process, and has higher production and transportation costsLow.
Description
Technical Field
The invention belongs to the field of biological pesticides, particularly relates to a functional microorganism, and particularly relates to a bacillus beiLeisi capable of antagonizing various pathogenic fungi and bacteria.
Background
Bacillus belgii (B.), (Bacillus velezensis) Is a new species of bacillus, can generate various substances with bacteriostatic effects, such as lipopeptide substances, antibacterial proteins, cyclic dipeptides and the like, has the effects of promoting plant growth and resisting pathogenic microorganisms in agricultural production, has broad-spectrum antibacterial activity, and is a microorganism with great potential in developing biological medicaments.
The tomato soil-borne diseases become more serious and become important factors for restricting the production of tomatoes, wherein the neck rot and root rot, brown root rot and tomato bacterial wilt of tomatoes are the most common diseases in facility tomatoes in recent years. The neck rot and root rot of the tomato are serious in the winter-heating greenhouse and are attacked at low temperature. The neck rot and root rot disease of tomato is characterized by that it is made up by using fusarium oxysporum and special type of neck rot and root rot disease of tomato (Fusarium oxysporumf. sp, radces-lycopersici, FORL) infection, and the two diseases are caused by fusarium oxysporum as well as tomato blight, but due to the difference of specialization types, the two diseases have great difference in the disease period and disease symptoms. Due to the long infection period of the disease, the difficulty in controlling the disease and no registered medicament, great economic loss is caused to the production of tomatoes. The pathogen of tomato brown root rot is Echinospora lycopersici ()Pyrenochaeta lycopersiciSchneider et Ger lach), the tomato main roots are browned and rotted after infection, the overground parts of the plants are poor in growth, the lower leaves are yellow and dry, and the plants die when the disease is serious. The tomato bacterial wilt is made of bacterial wiltRalstonia solanacearum) The bacterial disease caused by the tomato leaf surface disease causes the tomato to wither.
At present, chemical agents are mainly used for preventing and treating tomato soil-borne diseases in China, and frequent and unreasonable use of bactericides can easily cause outbreak of drug resistance of pathogenic bacteria and also seriously threaten the ecological environment and human health. The rhizosphere antagonistic bacteria has strong adaptability to soil environment, and the development and utilization of beneficial microorganisms as biological antagonistic preparations provide abundant resources and methods for biological control of soil-borne diseases. However, the selectable biological agents in the current market often cannot completely meet the requirements of biological control due to poor colonization and competition capability in soil.
Disclosure of Invention
Aiming at the problems of insufficient antagonistic bacteria colonization capacity, poor prevention and control efficiency, wide prevention and control range and the like in the existing biocontrol technology, the invention provides a bacillus belief with biocontrol growth promoting effect and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme.
Bacillus belgiiBacillus velezensis) The preservation number is CGMCC No. 19215.
The fermentation broth and metabolite of Bacillus belgii. The fermentation broth may or may not contain bacteria.
The Bacillus belgii and the application of the fermentation liquor and the metabolite thereof in inhibiting phytopathogens and promoting plant growth.
The phytopathogens include but are not limited to Fusarium oxysporum (F.) (Fusarium oxysporum) L. Ralstonia solanacearum (L.), (L.solanacearum)Ralstonia solanacearum) And Spinospora lycopersici (A), (B), (C)Pyrenochaeta lycopersici) And Fusarium graminearum (F.), (Fusarium. graminearum）。
Such plants include, but are not limited to, tomato, eggplant, strawberry, and corn.
A preparation comprising the above Bacillus belgii, its fermentation broth or metabolite.
Preferably, the formulation is a microbial agent; more preferably, the content of the Bacillus belgii in the microbial agent is 5-10 hundred million cfu/g.
The invention has the following advantages:
the Bacillus beleisi CGMCC No.19215 provided by the invention has the biological control and growth promotion effects, can effectively reduce the occurrence of soil-borne diseases of crops such as tomatoes, strawberries, corns and the like, and reduces the use amount of chemical agents; in the microbial agent prepared from the strain fermentation liquor, thalli are in a semi-dormant state, spores have strong stress resistance, the storage performance is stable, the control effect is good, the spores can directly contact with crop roots, no adverse effect is caused on the growth and development of plants, the processing is convenient, and the production and transportation costs are low.
Biological preservation information
Bacillus belgii (B.), (Bacillus velezensis) And is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 12 months and 16 days, wherein the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 19215.
Drawings
FIG. 1 shows a drawing (a) of streaked plate culture and a drawing (b) of transmission electron microscopy of Bacillus belgii;
FIG. 2 shows antagonistic effect of Bacillus belgii on target pathogenic bacteria, wherein A is antagonistic effect with a plate of tomato neck rot root rot, B is antagonistic effect with a plate of tomato wilt, C is antagonistic effect with a plate of strawberry wilt, D is antagonistic effect with a plate of maize stem rot bacteria, and E is antagonistic effect with a plate of tomato green rot bacteria.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 isolation, purification and characterization of Bacillus beilis
In 2017, in 10 months, collecting soil of a facility greenhouse with good sitting disorder solving effect in villages and towns in Dayue district of Taian city, Shandong, separating bacterial strains from the soil, wherein the specific separation and purification processes are as follows: weighing 10 g of sieved soil sample, placing into a 250 mL sterilized triangular flask filled with 90 mL of sterile water and a small amount of glass beads, placing the triangular flask on a shaker at 37 ℃ and 180 rpm for oscillation for 30min to fully suspend, then performing water bath treatment at 80 ℃ for 10 min, and diluting the bacterial suspension with sterile water to 10-4、10-5、10-6And (4) grading. Respectively sucking 100 mu L of each gradient solution, uniformly coating the gradient solution on an NA culture medium flat plate, repeating each gradient for 3 times, placing the gradient solution in a constant temperature incubator at 28 ℃ for culturing for 1-2 d, and then picking different single colonies for streak purification by the flat plate. The purified strains are inoculated with the tomato rhizoctonia solani serving as a target strainAnd (5) screening to obtain a target strain with the largest inhibition zone, wherein the number of the target strain is SDTB 038. The bacteria are subjected to morphological identification and physiological and biochemical characteristic tests, the culture morphology and transmission electron micrographs of the bacteria are respectively shown in figure 1-a and figure 1-b, and the colony morphology and physiological and biochemical characteristics are shown in table 1. The 16s rDNA sequence determination is carried out on the strain to obtain a gene sequence fragment with the length of 1539 bp, and the sequence is shown as SEQ ID NO. 1.
TABLE 1 colony morphology and physiological and biochemical characteristics
As can be seen from FIG. 1 and Table 1, after the target strain is cultured on the NA solid medium at 37 ℃ for 24h, the colony is milky, opaque, irregular and round, and gram-positive. The rod-shaped bacterial cells are observed under a transmission electron microscope, and the length of the rod-shaped bacterial cells is about 1.2-2.5 mu m.
Combining the morphological, physiological and biochemical characteristics and the 16s rDNA sequence homology comparison result of the target strain, identifying the strain as Bacillus beleisi (B. (R.))Bacillus velezensis). And is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 16 days in 2019, wherein the preservation number is CGMCC No. 19215.
Example 2 antagonistic Effect of Bacillus belgii on target pathogenic bacteria
The method comprises the steps of mixing the rhizoctonia solani (fusarium oxysporum tomato rhizoctonia solani) with a carrier,Fusarium oxysporumf.sp.radicis-lycopersici) And ralstonia solanacearum (ralstonia solanacearum,Ralstonia solanacearum) Tomato fusarium oxysporum (fusarium oxysporum tomato specialization type,Fusarium oxysporumf. sp.Lycopersici Snyder et Hans) And tomato brown root rot (acanthosporium lycopersicum,Pyrenochaeta lycopersicischneider et Gerlach), strawberry blight bacterium (Fusarium oxysporumf.sp.fragriae) And maize stalk rot (fusarium graminearum,F. graminearum) Activating for standby.
On a clean bench, pre-activated and vigorously growing tomato neck rot, tomato brown root rot, tomato wilt, strawberry wilt and corn stalk rot fungus cakes with the diameter of 7 mm are respectively inoculated in the center of a PDA solid culture medium flat plate, after 24 hours of dark culture at 25 ℃, activated single colony of Bacillus belgii described in example 1 is picked up by a sterilization toothpick, inoculated at a position 2 cm away from the center of the fungus cake, sealed by a preservative film, cultured in the dark at 20-25 ℃ for 3-7 days, and the result is observed.
On a clean bench, bacterial solution (bacterial solution concentration 10) of tomato ralstonia solanacearum with vigorous growth is coated on a clean bench7~108cfu/mL) is fully coated on an LB solid culture medium flat plate, a sterilized Oxford cup or a filter paper sheet is placed in the center of the culture medium, 20 mu L of activated Bacillus belezii bacterial liquid is added to the Oxford cup to be sealed by a preservative film, the mixture is cultured in the dark for 1-2 d at the temperature of 28 ℃, and the result is observed.
The antagonistic effect of Bacillus belgii CGMCC No.19215 and target pathogenic bacteria is shown in figure 2, and the inhibition zone result is shown in table 2.
TABLE 2 zone of inhibition of target pathogenic bacteria by B.belgii
As can be seen from fig. 2 and table 2, the inhibitory effect of bacillus belgii on various pathogenic bacteria is very obvious, and the inhibition zone is clearly visible. Thus, the Bacillus belgii has effects on various soil-borne fungi and bacterial diseases.
Example 3 potted plant control Effect on tomato neck rot and root rot
The activated Bacillus belgii CGMCC No.19215 single colony of example 1 is selected, inoculated into 500 mL triangular flask containing 300 mL sterilized LB liquid culture medium, and shake-cultured at 37 deg.C and 150rpm for 24h to obtain 10-degree thallus7~109cfu/mL of Bacillus belgii suspension.
Soil is taken from a land where the tomato neck rot and root rot occur seriously, the land is filled in a plastic flowerpot with the diameter of 15 cm, and the cultured tomato neck rot and root rot are inoculated manually. Transplanting tomato seedlingsSeedling, 1 plant/pot, treating group, each pot of tomato root-irrigation treatment 30 mL of thallus concentration 107~109cfu/mL Bacillus belgii CGMCC No.19215 suspension, and clear water is used to replace bacterial liquid in the control group. Each treatment was set up to 4 replicates, each 10 pots. The morbidity of each treatment group was investigated every 1 time every 30d by adopting conventional cultivation management, and the relative control effect (relative control effect = (control group morbidity-treatment group morbidity)/control group morbidity x 100%) was calculated and continuously investigated for 3 times; at 30d, 10 plants in each of the treated group and the control group were taken for root length measurement. The results are shown in Table 3.
TABLE 3 potted plant control effect of Bacillus belgii bacterial liquid on tomato neck rot root rot
Compared with the control group, the relative prevention effect of the Bacillus belgii CGMCC No.19215 on the tomato neck rot root rot is 46.28%, 75.65% and 80.54% respectively at 30, 60 and 90 days after transplanting, and the long lasting period is achieved. The tomato root length is measured 30 days after treatment, the treatment group root length is 13.08cm which is obviously higher than the treatment group root length, and the Bacillus beleisi CGMCC No.19215 has obvious growth promoting effect on tomato seedlings.
Example 4 preparation of Bacillus belgii microbial inoculum
(1) Preparing a bacterial liquid: a single colony of CGMCC No.19215 purified in example 1, which was previously activated on a solid plate of LB medium, was inoculated into a 500 mL Erlenmeyer flask containing about 300 mL of sterilized LB liquid medium and shake-cultured at 37 ℃ and 150rpm for 24 hours to prepare a strain seed solution. Transferring the seed solution into 100L fermentation tank containing 50L sterilized LB liquid culture medium, culturing at 37 deg.C and 300 rpm for 16 hr to obtain thallus concentration of 108~109cfu/mL of fermentation broth;
(2) and (2) centrifuging the fermentation liquor prepared in the step (1) for 15 min at 4000 rpm, and removing the supernatant to leave a precipitate. Mixing 1kg of precipitate, 0.5 kg of potassium fulvate, 0.5 kg of diatomite and 1kg of humic acid according to a proportion, fully and uniformly mixing, drying at 60 ℃ until the water content is less than 15%, crushing and sieving to obtain the powdery microbial agent with the viable bacteria content of 5-10 hundred million cfu/g.
Example 5 growth promoting action of Bacillus belgii on tomato
The nutrient soil is filled in a plastic flowerpot with the diameter of 15 cm, the tomato seedlings are transplanted in 1 plant/pot, 10 g and 20 g of the Bacillus beiLeisi microbial inoculum prepared in the example 4 are respectively scattered on the tomato rhizosphere of each pot of the treatment group, and the control group is not treated. Each treatment was set up to 4 replicates, each 10 pots. Using conventional cultivation management, the tomato plant heights were measured after treatment, 10d, 20d and 30d, respectively, and the results are shown in Table 4.
TABLE 4 growth promoting effect of Bacillus belgii preparation on tomato plant height
The pot culture experiment shows that compared with a control group treated by clear water, 10, 15 and 20 days after transplanting, the growth promotion effect of the bacillus belief strain microbial inoculum of 20 g/strain on tomato plants is the most obvious, the growth promotion effect is respectively 6.88, 10.08 and 14.37 cm, and the plant height is obviously higher than that of the control group at each period (P is less than 0.05). The growth promoting effect of the applied 10 g/strain of microbial inoculum is slightly lower than that of the high-dose group, but is obviously higher than that of the treated group (P < 0.05), which shows that the high-dose Bacillus belius microbial inoculum treatment has obvious growth promoting effect on the tomato plant height.
Example 6 field control of Bacillus belgii microbial inoculum for tomato neck rot root rot
Planting normal tomato seedlings in a facility greenhouse with serious neck rot and root rot of tomatoes planted for many years continuously, and respectively broadcasting 10 g and 20 g of the bacillus belief microbial inoculum prepared in the example 4 to the rhizosphere of each tomato in a treatment group, wherein a control group is not treated. Each treatment is provided with 3 times of repetition, each repetition is 1 cell, the length of the cell is 15m, the width of the cell is 2m, and the area of the cell is 30m2About 30 tomatoes are planted. The blocks are arranged randomly, and protective rows are arranged between the treatment rooms. The incidence of the neck rot and root rot of each treated tomato was investigated every 30 days for 1 time by conventional cultivation management, the investigation was continued for 3 times, and the relative control effect (relative control effect = (incidence of control group-incidence of treatment group)Rate)/control incidence x 100%) and the results are shown in table 5.
TABLE 5 field control of tomato neck rot root rot with Bacillus belgii preparations
Compared with a control group, at 30 days, 60 days and 90 days after transplanting, the relative control effects of 10 g/strain of the Bacillus belgii microbial inoculum on tomato soil-borne diseases are respectively 76.50%, 68.19%, 62.50% and 61.56%, the relative control effects of 20 g/strain on tomato soil-borne diseases are respectively 82.36%, 77.30%, 75.00% and 73.10%, and the control effects of high-dose Bacillus belgii CGMCC No.19215 microbial inoculum treatment on tomato neck root rot are higher than low dose and the lasting period is longer.
Sequence listing
<110> Shandong university of agriculture
<120> Bacillus belgii and application thereof
<130>20200220
<160>1
<170>PatentIn version 3.5
<210>1
<211>1538
<212>DNA
<213>Bacillus velezensis
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agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 120
cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtttga 180
accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 240
cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc gacctgagag 300
ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360
gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 420
cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 480
gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540
gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600
gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 660
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720
agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840
tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900
aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 1020
gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140
tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1260
aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1320
tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1440
gaggtaacct tttaggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta 1500
acaaggtagc cgtatcggaa ggtgcggctg gatcacct 1538
Claims (8)
1. Bacillus belgiiBacillus velezensis) The preservation number is CGMCC No. 19215.
2. A fermentation broth and metabolite of Bacillus belgii according to claim 1.
3. Use of the Bacillus belgii, fermentation broth and metabolites of claim 2 or 3 for inhibiting phytopathogens and promoting plant growth.
4. Use according to claim 3, wherein the phytopathogen is selected from Fusarium oxysporum (F.) (II.) (III.)Fusarium oxysporum) L. Ralstonia solanacearum (L.), (L.solanacearum)Ralstonia solanacearum) And Spinospora lycopersici (A), (B), (C)Pyrenochaeta lycopersici) And Fusarium graminearum (F.), (Fusarium. graminearum）。
5. Use according to claim 3, wherein the plant is selected from the group consisting of tomato, eggplant, strawberry and corn.
6. A formulation comprising the bacillus belgii, fermentation broth, or metabolite of claim 2 or 3.
7. The formulation of claim 6, wherein the formulation is a microbial agent.
8. The formulation of claim 7, wherein the microbial agent comprises between 5 and 10 hundred million cfu/g of Bacillus beiensis.
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| CN112899201A (en) * | 2021-03-16 | 2021-06-04 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN113502246A (en) * | 2021-07-08 | 2021-10-15 | 广西科学院 | Compound microbial agent and preparation method and application thereof |
| CN113980836A (en) * | 2021-09-23 | 2022-01-28 | 中化化肥有限公司临沂农业研发中心 | Bacillus belgii and application thereof in prevention and treatment of strawberry root rot |
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| CN113969254A (en) * | 2021-12-07 | 2022-01-25 | 山东农业大学 | Sesbania endophytic bacillus ZH60, composite microbial inoculum and corn seed culture method |
| CN114015618A (en) * | 2021-12-09 | 2022-02-08 | 河北农业大学 | Bacillus belgii and application thereof in plant disease control |
| WO2023138678A1 (en) * | 2022-01-21 | 2023-07-27 | 慕恩（广州）生物科技有限公司 | Bacillus velezensis and use thereof |
| CN114437972A (en) * | 2022-01-26 | 2022-05-06 | 云南农业大学 | Application of Bacillus beleisi YC89 or metabolite thereof in promoting plant growth |
| CN116042481A (en) * | 2023-02-16 | 2023-05-02 | 寿光奥康生物制品股份有限公司 | Bacillus bailii compound chitosan oligosaccharide fermentation inoculant and application thereof |
| CN116195600A (en) * | 2023-03-15 | 2023-06-02 | 西安展鹏生物科技有限公司 | Microbial agent for promoting plant growth and development and application thereof |
| CN117165492A (en) * | 2023-10-09 | 2023-12-05 | 山东省果树研究所 | Bacillus bailii and application thereof |
| CN117165492B (en) * | 2023-10-09 | 2024-04-30 | 山东省果树研究所 | Bacillus bailii and application thereof |
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