CN116240126A - Multifunctional bacillus belgium SB10 and application thereof - Google Patents
Multifunctional bacillus belgium SB10 and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to the technical field of soil biocontrol microorganism application, in particular to a multifunctional bacillus beijerinckii SB10 and application thereof. The invention provides bacillus beijerinus (Bacillus velezensis SB) SB10 with the preservation number of: GDMCC No.62370. The bacillus belicus (Bacillus velezensis) SB10 has the function of promoting growth, can remarkably promote the growth of tomato plants, effectively prevent and control pathogenic bacteria of tomato bacterial wilt, has strong growth adaptability in soil, has good antibacterial effect on common pathogenic bacteria in gardening crop planting, and has great application potential in gardening crop planting.
Description
Technical Field
The invention relates to the technical field of soil biocontrol microorganism application, in particular to a multifunctional bacillus beijerinckii SB10 and application thereof.
Background
China is a large agricultural country, and disease control in agricultural planting is one of key factors affecting crop yield and quality. With the rapid development of agricultural economy, chemical medicines such as antibiotics, disinfectants and the like are used for a long time and even abused, so that plant pathogenic bacteria have drug resistance to the medicines and the disease control difficulty is increased, and meanwhile, the soil and the water body are polluted by pesticide residues, so that the microecological balance is destroyed, and the health of human beings is influenced. Therefore, the biological method with low cost, high efficiency, environmental protection and no medicine is adopted to prevent and control the plant diseases, and the method gradually becomes a hot spot at home and abroad. In recent years, the regulatory strength of environmental protection is strengthened in various countries, higher requirements are put on sustainable development of agriculture, and biological control is becoming a popular research for plant disease control with its unique advantages.
Bacterial wilt of solanaceae crops is a typical bacterial vascular bundle soil-borne disease caused by Ralstonia (Ralstonia solanacearum), and pathogenic bacteria of the bacterial vascular bundle soil-borne disease can survive in soil for a long time and have extremely strong harm to plants. The pathogenic bacteria are widely distributed in the tropical, subtropical and temperate regions of the world, have extremely wide host range, and can infect 50 crops such as peanut, banana and the like, as well as various solanaceous crops such as tomatoes, peppers, eggplants and the like. After the crop planting area is ill, a large amount of crop is reduced in yield, even absolute yield is achieved, and huge economic loss is caused for agricultural production.
Bacillus spp is a gram positive bacterium, a most beneficial microorganism. In recent years, the bacillus has increasingly remarkable effects in the application of microorganisms, and not only has a broad-spectrum antibacterial activity, but also can generate various important enzymes, so that the bacillus is widely applied to the production of surfactants, antibiotics, pesticides, biological agents and the like. Therefore, the screening and utilization of biocontrol bacteria have important significance for the research and development of biological control bacterial agents for common diseases of horticultural crops and the sustainable development of agriculture.
Disclosure of Invention
In order to solve the problems in the prior art, the first aim of the invention is to provide bacillus bailii (Bacillus velezensis) SB10 with the capability of efficiently inhibiting pathogenic bacteria of common horticultural crops, and provide a good biological material for preventing and treating the pathogenic bacteria in the cultivation of the common horticultural crops.
The bacillus beleiensis provided by the invention is separated from the root tissues of ginger fever patients in ginger plantations in river north by the inventor in 2021 month 6, and is named as bacillus beleiensis (Bacillus velezensis) SB10.
Further, the common horticultural crop pathogens mainly include rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), banana anthrax (Colletotrichum musae), ralstonia solanacearum (Ralstonia solanacearum), verticillium dahliae (Verticilium dahliae), acidovorax avenae subspecies citrulli (Acidovorax avenae subsp. Citrulli), xanthomonas citri (Xanthomonas citri subsp. Citri, xcc).
The second object of the invention is to provide a SB10 biological microbial agent, which contains the bacillus belicus SB10 as an active ingredient, has obvious effect of promoting tomato growth, and can be applied to promoting tomato growth and preventing and controlling tomato bacterial wilt pathogenic bacteria.
Further, the SB10 biological agent comprises fermentation broth of the Bacillus bailii strain SB10 or supernatant of the fermentation broth or thallus from which the fermentation broth is removed and resuspended in sterile physiological saline.
Further, the preparation method of the SB10 biological agent comprises the following steps: single colony of bacillus bailii SB10 is selected and inoculated in 10ml NB liquid culture medium, shake culture is carried out for 24 hours at 30 ℃ in a shaking table, and fermentation seed liquid is prepared; and inoculating the fermentation seed liquid into a fermentation culture liquid according to the inoculum size of 2%, and shake culturing for 48 hours at the temperature of 30 ℃ under the condition of pH7.2, wherein the fermentation liquid obtained by culturing or supernatant of the fermentation liquid or thallus obtained by removing the fermentation liquid and re-suspending with sterile physiological saline is the biological microbial inoculum.
The invention provides a bacillus bailii (Bacillus velezensis) SB10 which has the physiological and biochemical characteristics that: gram positive bacteria, the shape of the cell is bar-shaped, and the cell has spores with the size of 0.5-1.0X1.5-2.5 μm (see figure 2); the colonies were white, opaque, rough and wrinkled, nearly circular in shape, and thick (see FIG. 1).
Extracting genome DNA of the bacillus SB10, amplifying a 16SrDNA gene by using a 27F/1492R primer, and sequencing to obtain a gene sequence shown in SEQ ID NO.1. The sequence was subjected to homology alignment analysis on NCBI and EzBioCloud websites to construct a phylogenetic tree, and morphological observation was combined, which revealed that the strain was Bacillus belicus (Bacillus velezensis).
Compared with the prior art, the invention has the following beneficial effects:
(1) The bacillus belicus (Bacillus velezensis) SB10 of the invention is used for separating and screening tuber tissues of ginger, and has strong adaptability to plant rhizosphere growth.
(2) The bacillus bailii (Bacillus velezensis) SB10 has broad-spectrum antibacterial effect, and has obvious antibacterial effect on common pathogenic bacteria such as rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), banana anthracis (Colletotrichum musae), lawsonia solanacearum (Ralstonia solanacearum), verticillium dahliae (Verticilium dahliae), acidovorax avenae subspecies (Acidovorax avenae subsp.citrulli) and xanthomonas citri (Xanthomonas citri subsp.citri, xcc) in gardening crop planting.
(3) The bacillus belicus (Bacillus velezensis) SB10 has the function of promoting growth, can obviously promote the growth of tomato plants, and can effectively prevent and treat bacterial wilt pathogenic bacteria of tomatoes.
In conclusion, bacillus beijerinus (Bacillus velezensis) SB10 has strong growth adaptability in soil, good antibacterial effect on common pathogenic bacteria in gardening crop planting, and great application potential in gardening crop planting.
Preservation description:
bacillus bailii (Bacillus velezensis) SB10 of the invention was deposited at the microorganism culture Collection of Guangdong province (GDMCC) on 10 th month 11 of 2021, and was deposited at No. 59 building of Migo 100, va. Jiuzuo, guangdong province under accession number GDMCC No.62370 and taxonomic designation Bacillus velezensis.
Drawings
FIG. 1 is a photograph of Bacillus bailii SB10 grown on NA (nutrient broth) medium.
FIG. 2 is a photograph of Bacillus belicus SB10 stained with crystal violet under an optical microscope (100X).
FIG. 3 is a phylogenetic tree of Bacillus beleiensis SB10 and similar strains.
FIG. 4 is a photograph of a fermentation broth of Bacillus belicus SB10 against pathogenic bacteria Rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), banana anthrax (Colletotrichum musae), laurella multocida (Ralstonia solanacearum), verticillium dahliae (Verticilium dahliae), citrullus natto subspecies (Acidovorax avenae subsp. Citrulli), and Xanthomonas citri (Xanthomonas citri subsp. Citr., xcc) in a zone of inhibition.
FIG. 5 is a photograph of a potting test of Bacillus belicus SB10 for tomato bacterial wilt control.
FIG. 6 is a photograph of B.beijerinus SB10 promoting growth of tomato plants.
Detailed Description
The following examples are provided to illustrate the present invention, but are not intended to limit the scope of the invention, as the parameters, proportions, etc. of the examples may be selected according to the circumstances without materially affecting the results. Unless otherwise indicated, all methods described in the examples are conventional and all reagents used are conventional or formulated in conventional fashion.
EXAMPLE 1 screening separation purification and preservation of Bacillus bailii SB10
In this embodiment, in order to separate more biocontrol strains, three separation media and two different ginger tissue treatment methods are respectively adopted:
the separation culture mediums are respectively nutrient broth culture medium, potato dextrose culture medium and R2A culture medium:
(1) The preparation method of the separation medium (nutrient broth medium) is as follows: taking 3g of beef extract powder, 10g of peptone, 5g of sodium chloride and 15g of agar, adding water to a volume of 1000mL, adjusting the pH to 7.2, and subpackaging into 250mL triangular bottles with 100mL each bottle; sterilizing at 121deg.C under high temperature and high pressure for 20min, and pouring into flat plate.
(2) The preparation method of the separation medium (potato dextrose agar medium) comprises the following steps: taking 300g of potato extract powder, 20.0g of glucose, 15.0g of agar and 0.1g of chloramphenicol, adding water to a volume of 1000mL, adjusting the pH to 7.2, and subpackaging into 250mL triangular bottles with 100mL each bottle; sterilizing at 121deg.C under high temperature and high pressure for 20min, and pouring into flat plate.
(3) The preparation method of the separation medium (R2A agar medium) comprises the following steps: taking 0.25g of tryptone, 0.5g of acid hydrolyzed casein, 0.5g of yeast extract powder, 0.5g of soluble starch, 0.3g of dipotassium hydrogen phosphate, 0.1g of magnesium sulfate, 0.3g of sodium pyruvate, 0.25g of peptone, 0.5g of glucose, 15g of agar, adding water to a volume of 1000mL, adjusting pH to 7.2, and subpackaging into 250mL triangular bottles with 100mL each; sterilizing at 121deg.C under high temperature and high pressure for 20min, and pouring into flat plate.
The screening, separating, purifying and preserving method of bacillus bailii SB10 comprises the following steps:
(1) Fresh ginger root tissues refrigerated at the temperature of 4 ℃ after collection are taken, and a tissue separation method is adopted to separate strains. The specific operation is as follows: the diseased tissue is taken and washed for 5 to 10 minutes by running water, a blade disease-health combined part is cut off by using a sterile blade, the length and the width are about 2cm, the diseased tissue is placed in 75% alcohol for sterilization for 30 seconds and 1% sodium hypochlorite for sterilization for 2 minutes, the diseased tissue is washed for 3 to 5 times by using sterile water after each sterilization, and then tissue separation suspension and ginger tissue treatment are respectively carried out.
The processing method of the ginger tissue comprises the following two steps:
(1) The resulting ginger tissue was crushed, 5mL of sterile water was added thereto, and the mixture was vortexed and homogenized to obtain a suspension. Diluting the suspension liquid for 10 times, then streaking on the three separation culture mediums, and culturing in a constant temperature incubator at 30 ℃ for 3-5 d;
(2) Repeatedly washing the obtained ginger tissue with sterile water for 5 times, placing the ginger tissue on three separation culture mediums, and culturing the ginger tissue in a constant temperature incubator at 30 ℃ for 3-5 d.
The experimental results are shown in fig. 1-2:
the photo of the growth of bacillus belgium SB10 on NA (nutrient broth agar) culture medium is shown in figure 1, the colony morphology is observed visually, and the colony is round, white, opaque, rough in surface and irregular in edge and protrudes; meanwhile, the shape of the cells is rod-shaped, spores are formed, the size of the spores is 0.5-1.0X1.5-2.5 mu m, and the cells are gram-positive bacteria by crystal violet staining under a 100-time oil lens, and the gram-positive bacteria are shown in figure 2.
Further streaking purification was performed, and the purified strain was subjected to expansion culture and the strain was stored in a glycerol tube (-80 ℃) and a lyophilization tube (4 ℃) to thereby obtain Bacillus bailii SB10.
EXAMPLE 2 16S rDNA identification of Bacillus bailii SB10
The genomic DNA of strain SB10 was extracted using a bacterial DNA extraction kit (Meiji Biotechnology Co., ltd.) and amplified using the bacterial 16S rDNA gene amplification universal primer 27F/1492R (5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TACGACTTAACCCCAATCGC-3'), and the PCR reaction system was as follows: accurate Taq Master Mix 12.5.5. Mu.L of 10. Mu.M upstream and downstream primers each 0.5. Mu.L, 1. Mu.L of DNA template was mixed and sterile deionized water was added to 25. Mu.L. The PCR amplification procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min (30 cycles); extending at 72℃for 5min. The obtained PCR product is sent to Jin Weizhi biotechnology limited company for sequence sequencing, and the length of the 16S rDNA sequence obtained after sequencing is 1550bp, and the sequence is shown in SEQ ID NO.1. The sequencing results were subjected to homology alignment analysis with 16S rDNA sequences in NCBI and EzBioCloud databases, then, strain SB10 and similar strains were selected, and phylogenetic tree was built (bootstrap was repeated 1000 times) by using MEGA 6.0, kimura2-parameter model and NJ algorithm, and the obtained evolutionary tree results are shown in FIG. 3, wherein the tree was built by using NJ method, and only > 50% bootstrap coefficients were shown (repeated 1000 times).
The 16S rDNA gene sequence SEQ ID NO.1 is as follows:
TTATCGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT
in the EzBioCloud database, the 16S rDNA gene sequence of Bacillus sp SB10 (i.e.strain SB 10) has the highest similarity (99.93%) to Bacillus velezensis CR-502, but only 95.4% coverage. According to the result of the evolutionary tree, the strain SB10 and Bacillus helencensis CR-502 (99.93%) and Bacillus amyloliquefaciens Bacillus amyloliquefaciens DSM _7 (99.66%) are gathered on one branch, and the strain SB10 is circular, white, opaque, rough in surface and micro-protruding on the edge when being observed on an NA plate in a combined morphology, wherein the colony diameter is 1-3 mm, and is similar to the Bacillus helencensis in shape. Based on the above analysis, it was preliminarily identified that the strain SB10 isolated in example 1 of the present invention is Bacillus bailii. It was named: bacillus bailii (Bacillus velezensis) SB10. The strain was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) on day 10 and 11 of 2021, address: the No. 59 building of the 100 th university of Mitsui, guangzhou, guangdong, has a deposit number of GDMCC No.62370.
EXAMPLE 3 determination of bacteriostatic Effect of Bacillus bailii SB10
(1) Preparing a test bacterial liquid: and (3) picking single colony of the activated strain SB10, inoculating the single colony into 10mL of nutrient broth culture medium, placing the culture medium in a shaking table at 30 ℃ and 180r/min for culturing for 24 hours to prepare seed liquid, inoculating the fermentation seed liquid into the fermentation culture liquid according to the inoculum size of 2%, and performing shaking culture for 48 hours at the temperature of 30 ℃ under the condition of pH7.2 to prepare the bacteria liquid to be tested.
(2) Bacterial pathogen plate preparation: activating the cultured test strain (Table 1 below), and adjusting OD with ultraviolet spectrophotometer 600 The value was 1.0, and then diluted 1000-fold with sterile water, 100. Mu.L of the bacterial liquid was sucked and uniformly spread into the nutrient broth medium, and four holes (aperture 8 mm) were uniformly punched 3cm away from the center, respectively, with a puncher.
(3) Fungal pathogen plate preparation: the pathogenic fungi of the test strain (shown in Table 1 below) were activated with potato agar medium, mycelia with fresh edges were selected with a punch (8 mm) to prepare a cake, and placed in the middle of the plate, and four holes (aperture 8 mm) were uniformly punched with the punch 3cm away from the center, respectively.
(4) Bacteriostasis test
50. Mu.L of the bacterial liquid of the suction strain SB10 was added to each of the pathogen plates containing the test strain, and after culturing in an incubator at 28℃or 30℃for about 48 hours, the bacteriostatic effect was observed (see FIG. 4 for the results). The observation shows that the strain SB10 has remarkable antibacterial effect on seven pathogenic bacteria, and the radius of the antibacterial circle can reach about 1 cm.
Table 1 test strains tested for antibacterial Effect of Bacillus bailii SB10
EXAMPLE 4 potted plant test of Bacillus bailii SB10 for tomato bacterial wilt control
(1) Test plants: the tomato variety is Xinjinfeng No. (susceptible variety of tomato bacterial wilt), and the seedling is transplanted into a seedling soil pot when 3-5 true leaves are grown after seedling cultivation.
(2) Test soil: grinding the natural air-dried soil and sand, sieving with 2mm (10 mesh) sieve, mixing, sterilizing with high pressure steam at 121deg.C for 1 hr for three times, and air drying every one day. Mixing soil and sand uniformly according to a ratio of 3:1 for later use.
(3) Preparation of SB10 biological bacteria: the activated strain SB10 is picked up and inoculated into 10mL nutrient broth culture medium, placed in a shaking table at 30 ℃ and 180r/min for culturing for 24h, and an ultraviolet spectrophotometer is used for adjusting OD 600 The value reaches 1.0, and the SB10 biological microbial inoculum is prepared. And (3) centrifuging part of biocontrol bacteria liquid at 6000r/min for 10min, and taking supernatant, namely preparing the supernatant of the SB10 biological bacteria.
(4) Preparing a pathogenic bacteria liquid: the activated strain HB23 (L.lauchi isolated from ginger diseased root tissue, which was deposited at the microorganism strain collection in Guangdong province at 10 and 11 days 2021, address: no. 59 building of 100. Institute of Hirship, guangzhou City, guangdong, accession number GDMCC 1.3036.) was picked up as a single colony and placed in a nutrient broth, and cultured in a shaker at 30℃and 180rpm for 24 hours to prepare a pathogenic bacterial liquid.
(5) Potting test
Tomato seedlings with the same size are selected and divided into 7 groups, 5 plants are transplanted in each group, and test treatment is carried out after one week of seedling recovery. The tomato seedlings are grouped and treated as shown in Table 2:
TABLE 2
A total of 7 treatments, each treatment being repeated at least 5 times. After the tomato seedlings are treated, the tomato seedlings are placed in an illumination culture room, the temperature is 28-30 ℃, the humidity is 65-80%, all potted plants are placed randomly, quantitative watering is carried out every day, and the water content is controlled at 20%.
(6) Measurement index
The growth condition of tomato seedlings is observed every day, after the tomato plants are in bacterial wilt (the bacterial wilt is inoculated for 5 days in a pre-experiment), the disease time and the disease grade of the tomato plants are observed and recorded, and the disease rate and the disease index of each treatment are counted after one week of disease.
The incidence of bacterial wilt is divided into five stages (Kempe et al, 1983):
0 grade, healthy plants, and no wilting phenomenon;
stage 1, wilting of 1/4 leaves;
stage 3, wilting of 2/3 leaves;
and 4, withering and dying the whole plant.
Plant incidence = (number of plants developed/total number of plants) x 100%
Disease Index (DI) = [ Σ (number of individual stages of disease x number of corresponding grades)/(total number of investigation x highest grade value) ]100
Relative control (BE) = [ (disease index of CK-disease index of test treatment group)/disease index of CK ] ×100%
(7) The test results are shown in Table 3 and FIG. 5.
TABLE 3 Effect of the inoculation of SB10 biological inoculants on Laurencia valica disease
Note that: the values in the tables are mean.+ -. Standard error (n.gtoreq.5), and those with different lower case letters in the same column of data show significant differences (Duncan method, P < 0.05).
The biological control effect of SB10 biological microbial inoculum on bacterial wilt in the potting test is subjected to data statistical analysis, and the results are shown in Table 3. The results show that: the incidence rate of tomato bacterial wilt can be obviously reduced by inoculating SB10 biological bacterial agents in advance. Compared with a group inoculated with bacterial wilt only, the incidence rate of the biocontrol group inoculated with the physiological saline bacterial suspension of the SB10 biological agent is reduced to 60 percent, the disease index is reduced to 15.0 percent from 100 percent, and the relative control effect reaches 85 percent; compared with the bacterial wilt only inoculated treatment group, the incidence rate of the biocontrol group inoculated with the SB10 biological bacterial agent bacterial liquid is reduced to 60 percent, the disease index is reduced to 20.0 percent from 100 percent, and the relative control effect reaches 80 percent.
And for the therapeutic effect of SB10 biological bacteria, the results show that: the SB10 biological bacterial agent can obviously reduce the incidence rate of tomato bacterial wilt. Compared with the group CK1 inoculated with the bacterial wilt only, the incidence rate of the biocontrol treatment group C1 inoculated with the physiological saline bacterial suspension of the SB10 biological agent is reduced to 50 percent, the disease index is reduced to 50.0 percent from 100 percent, and the relative prevention effect reaches 50 percent; compared with the group CK2 inoculated with the bacterial wilt only, the incidence rate of the biocontrol treatment group C2 inoculated with the SB10 biological bacterial agent bacterial liquid is reduced to 50%, the disease index is reduced to 58.0% from 100%, and the relative control effect reaches 42%.
The results show that: the SB10 biological bacterial agent has prevention and treatment effects on the Laurencia laughensis, the prevention effect (relative prevention effect 80%) is obviously higher than the treatment effect (relative prevention effect 42%), and the prevention and treatment effects of the physiological saline bacterial suspension of the SB10 biological bacterial agent are better than those of bacterial liquid.
EXAMPLE 5 test of Bacillus bailii SB10 on tomato growth-promoting pot culture
(1) Test plants: the tomato variety is Xinjinfeng No. one, and after seedling growing, seedling transplanting is carried out when 3-5 true leaves are grown.
(2) Test soil: grinding the natural air-dried soil and sand, sieving with 2mm (10 mesh) sieve, mixing, sterilizing with high pressure steam at 121deg.C for 1 hr for three times, and air drying every one day. Mixing soil and sand uniformly according to a ratio of 3:1 for later use.
(3) Preparation of SB10 biological bacteria: picking single colony of activated strain SB10, inoculating into 10ml nutrient broth culture medium, culturing at 30deg.C in 180r/min shaking table for 24 hr to obtain seed solution, inoculating the seed solution into NB liquid culture medium according to 2% inoculum size, culturing in 30 deg.C in 180r/min shaking table for 48 hr to obtain bacterial solution, centrifuging at 6000r/min for 10min, collecting supernatant, and regulating OD600 to 1.0 (10) 8 CFU/g) to prepare the SB10 biological microbial agent.
(4) Potting test
Selecting tomato seedlings with the same size, setting two treatments, and repeating 3 treatments each for seedling transplanting. The prepared microbial inoculum is 1×10 per pot 7 CFU/g inoculum size was inoculated with SB10 biological inoculum alone, and the blank (CK) was inoculated with an equal amount of sterile deionized water, 3 replicates per treatment. The whole test process is inoculated with the microbial inoculum once every 10 days, and the total microbial inoculum is inoculated for three times. Potted seedlings are placed in a roof greenhouse, watered quantitatively 1 time a day, kept at 20% water content, and sampled after one month of test treatment.
(5) The test results are shown in table 4 and fig. 6:
compared with the CK group, the application of the SB10 biological microbial inoculum increases the fresh weight, the dry weight, the fresh weight, the dry weight and the plant height of the aerial part of the tomato plant; but only increases the fresh weight of the overground part of the tomato very significantly; the SB10 biological bacteria can promote the growth of tomato plants.
TABLE 4 Effect of SB10 biological inoculants on tomato plant growth
| Group of | CK | SB10 biological bacterial agent |
| Fresh weight of overground part (g) | 47.018±0.841 | 52.62±0.335(**) |
| Dry weight of ground part (g) | 6.944±0.326 | 7.54±0.351 |
| Fresh weight of underground part (g) | 13.182±0.230 | 13.41±0.357 |
| Dry weight of underground part (g) | 1.358±0.043 | 1.38±0.031 |
| Height of plant (cm) | 55.5±0.156 | 59.00±0.5 |
| Stem thickness (mm) | 7.322±0.235 | 7.23±0.142 |
Note that: CK is a blank without inoculant. The table shows the significance results of independent sample T-test for each treatment versus control CK, which shows that the treatment and CK have significant differences of 0.01< p.ltoreq.0.05; * Indicating that the treatment was very significantly different from CK, n=3.
The invention discloses a growth-promoting multifunctional biocontrol bacillus beijerinus and application thereof in preventing and controlling tomato bacterial wilt pathogenic bacteria in gardening crop planting. The bacillus is named: bacillus belicus (Bacillus velezensis) SB10 with accession number GDMCC No.62370. The bacillus belicus SB10 has good antibacterial capability: has high-efficiency broad-spectrum antibacterial capability on common pathogenic bacteria planted on horticultural crops. The bacillus belicus SB10 has 80% of relative prevention effect on tomato bacterial wilt and 42% of relative prevention effect on biological prevention and treatment effect in tomato planting. In addition, bacillus beleiensis SB10 is strong in adaptability, can colonize soil, has obvious growth promoting effect on tomato plants, so that comprehensively, bacillus beleiensis SB10 has great application potential on common pathogenic bacteria prevention and treatment in horticultural crop (especially tomatoes) planting.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (9)
1. Bacillus bailii SB10 with accession number: GDMCC No.62370, deposited with the collection of microorganism strains in Guangdong province under the taxonomic designation Bacillus velezensis.
2. Bacillus beleimeris SB10 of claim 1, wherein the bacillus beleimeris SB10 is isolated from root tissue of a ginger fever patient.
3. Use of bacillus beijerinus SB10 according to claim 1 for controlling common horticultural crop pathogens.
4. Use according to claim 3, wherein the common horticultural crop pathogens comprise rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), banana anthrax (Colletotrichum musae), verticillium dahliae (Verticilium dahliae), ralstonia solanacearum (Ralstonia solanacearum), acidovorax avenae subsp (Acidovorax avenae subsp. Citrulli) and xanthomonas citri (Xanthomonas citri subsp. Citr., xcc) pathogens.
5. Use of bacillus beijerinus SB10 according to claim 1 for promoting the growth of tomato plants.
6. An SB10 biological bacterial agent, characterized in that the SB10 biological bacterial agent contains the bacillus belicus SB10 of claim 1 as an active ingredient.
7. A SB10 biological agent according to claim 6 which comprises a fermentation broth of Bacillus belicus strain SB10 or a supernatant of the fermentation broth or a cell from which the fermentation broth is removed and resuspended in sterile physiological saline.
8. A SB10 biological agent according to claim 7, wherein the SB10 biological agent is prepared by the method comprising: inoculating bacillus belicus SB10 into NB liquid culture medium, shake culturing at 30deg.C for 24 hr to obtain fermentation seed liquid; and inoculating the fermentation seed liquid into a fermentation culture liquid, and shake culturing for 48 hours at the temperature of 30 ℃ under the condition of pH7.2, wherein the fermentation liquid obtained by culturing or supernatant of the fermentation liquid or thallus obtained by removing the fermentation liquid and re-suspending with sterile physiological saline is the biological microbial inoculum.
9. Use of a SB10 biological agent according to any one of claims 6 to 8 for promoting tomato growth and controlling bacterial wilt pathogens.
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| CN117701476B (en) * | 2024-02-05 | 2024-04-16 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Bacillus bailii with antagonism to pathogenic fungi and application thereof |
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