CN108424860B - Bacillus subtilis and screening method and application thereof - Google Patents
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Abstract
The invention provides a Bacillus subtilis strain and a screening method and application thereof, wherein the Bacillus subtilis strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2017729 and is classified and named as Bacillus subtilis L501, and is mainly applied to degradation of an autotoxicity barrier substance benzoic acid in strawberry continuous cropping; the highest degradation rate of the benzaldehyde reaches 87.5 percent. The method effectively degrades benzoic acid in strawberry rhizosphere, overcomes main obstacles of strawberry continuous cropping, provides a technical method for effectively utilizing microbial inoculum to treat the obstacles of strawberry continuous cropping in the subsequent strawberry planting production, thereby realizing the problem of large-scale production of strawberries, increasing the yield and income of strawberries, providing technical support for effectively utilizing the microbial inoculum to treat the obstacles of strawberry continuous cropping in the strawberry planting production, and having wider market prospect.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, particularly relates to application of bacillus subtilis in degradation of benzoic acid serving as an autotoxicity obstacle substance in strawberry continuous cropping, and particularly relates to bacillus subtilis and a screening method and application thereof.
Background art:
strawberry (English name: Strawberry, scientific name: Fragaria ananassa Duch), plant of Strawberry of Rosaceae, praised as fruit queen, have the advantages of short cultivation period, quick effect, high economic benefit, suitability for facility cultivation and the like, and become hot spots of agricultural cultivation of various countries. At present, the strawberry is produced in 62 countries all over the world, the annual production total amount of the strawberry in the world is over 300 ten thousand tons, and the cultivation area is over 20 ten thousand hm2. Strawberry cultivation in China becomes the underpinning industry for rural economic development in many areas. The strawberry planting benefit is larger than that of other planting industries, the situation that the market demand is larger than the production capacity is still unchanged, in order to meet the supply and demand of the market, greenhouse strawberries are increasingly popular, and huge economic benefits are brought to farmers. However, as the continuous cropping time increases, the successive cropping obstacles of strawberries become more and more obvious. The obstacle of facility strawberry continuous cropping mainly shows that: the plant height is reduced, the leaf number is reduced, the biomass is reduced, the growth period is delayed, adverse climatic conditions are met, and the seedling death rate is high.
One of the main reasons for restricting the large-scale production of strawberries is continuous cropping obstacle, and the main reason for the continuous cropping obstacle is the self-toxicity of strawberries. Strawberry autotoxicity causes soil nutrient imbalance and soil microflora disorder, and a large amount of toxic substances are accumulated in root exudates. The research on the influence of root autotoxicants on the yield of cultivated crops is a hot point of current research. Some allelochemicals in strawberry root secretion have self-toxicity, and benzoic acid has high content in the allelochemicals and strong toxicity. Accelerated degradation or activity reduction of the autotoxic substances of the strawberry root system can help to control the occurrence of successive cropping obstacles of the strawberry. Screening and developing microbial repairing agents are hot spots for solving the continuous cropping obstacles of crops. Strains which have been reported to degrade bacteria are Erwinea herbicola, Microbacterium hydrocarbonoxydans, Eubacterium oxidans, Ochrobacterium sp.
The degrading bacteria with the strawberry continuous cropping self-toxicity function are mostly actinomycetes in the prior report, such as Streptomyces lavonoensis and Streptomyces sp reported by P-hydroxybenzoic acid degrading bacteria strain B3512 and Ninin reported by the Jilingjun and the like, and research reports show that the actinomycetes preparation prepared from the degrading bacteria can effectively reduce the self-toxicity function of allelochemicals on the strawberry continuous cropping. Researches on Sunjiao and the like show that actinomycetes can colonize on the root surface of the strawberry and play a role in preventing diseases and promoting growth in the repair of strawberry continuous cropping obstacles. Therefore, the microorganism can carry out bioremediation on the strawberry continuous cropping obstacle. The bacteria reported by Fagafene et al to have a good effect on the degradation of benzoic acid include Pseudomonas, Acinetobacter and Alcaligenes. However, the bacillus subtilis is not reported as a degrading bacterium with the strawberry continuous cropping autotoxicity.
The main reason of strawberry continuous cropping obstacle is autotoxicity generated by root system, and benzoic acid is one of the main substances of strawberry continuous cropping autotoxicity obstacle. In order to reduce the harm of benzoic acid to strawberries, how to screen out bacteria for degrading benzoic acid in the rhizosphere of strawberries is realized, so that the continuous cropping obstacle of strawberries is overcome, the problem of large-scale production of strawberries is realized, and the yield and income of strawberries are increased. Provides technical support for effectively utilizing the microbial inoculum to treat the strawberry continuous cropping obstacles in the strawberry planting production. Has wide market prospect.
Disclosure of Invention
The invention provides a bacillus subtilis strain and a screening method and application thereof, which are mainly applied to the degradation of an autotoxicity obstacle substance benzoic acid in strawberry continuous cropping; the highest degradation rate of the benzaldehyde reaches 87.5 percent. The method effectively degrades benzoic acid in strawberry rhizosphere, overcomes main obstacles of strawberry continuous cropping, and provides a technical method for effectively utilizing microbial inoculum to treat the obstacles of strawberry continuous cropping in the subsequent strawberry planting production, thereby realizing the problem of large-scale production of strawberries and realizing the yield and income increase of strawberries.
The Bacillus subtilis is characterized in that the Bacillus subtilis is preserved in China center for type culture Collection, has a preservation number of CCTCC No. M2017729, is classified and named as Bacillus subtilis L501, and has a preservation date: 27 th month in 2017, address, eight-way Lodokya in Wuchang district, Wuhan university, Hubei province, post code: 430072.
the invention discloses application of bacillus subtilis in degradation of strawberry continuous cropping autotoxicity obstacle substances;
the application of the bacillus subtilis is applied to degradation of the autotoxicity obstacle substance benzoic acid in strawberry continuous cropping.
Preferably, the bacillus subtilis is applied to antagonizing strawberry root rot.
The bacillus subtilis strain is characterized in that a culture medium of the bacillus subtilis is an improved inorganic salt culture medium and comprises (NH)4)2SO4 1.5-2.5g.L-1，Na2HPO4 1.1-1.5g.L-1，KH2PO4 1.5-2.5g.L-1Benzoic acid 100-1000mg-1，NaCl 4-7g.L-1The balance of distilled water; the pH of the medium was controlled to 7.2.
The invention discloses a screening method of bacillus subtilis, which adopts an enrichment culture method and comprises the following specific steps: 1) preparing an inoculation improved inorganic salt culture medium, numbering successive cropping eight-to ten-year strawberry planting fields as L1, L2, L3, L4, L5 and successive cropping three-to five-year strawberry planting fields as R1, R2, R3, R4 and R5, randomly sampling 10-20g of the soil of the strawberry planting fields at different times, inoculating the soil into the prepared improved inorganic salt culture medium as an inoculation amount to inoculate the improved inorganic salt culture medium, 2) preparing a liquid inoculation culture medium, preparing the inoculation improved inorganic salt culture medium into a liquid inoculation culture medium, controlling benzoic acid in a culture solution of the liquid inoculation culture medium as a unique carbon source, 3) controlling the concentration, and controlling the concentration gradient of the benzoic acid in the culture solution in the step 2) to be as follows: l1 and R1 were 100mg.L-1L2 and R2 were 200mg.L-1L3 and R3 were 300mg.L-1L4 and R4 were 400mg.L-1L5 and R5 were 500mg.L-1The contrast group is not inoculated with a soil sample, 4) shaking table shaking culture is carried out, the culture solution obtained in the step 2) is subjected to shaking table shaking culture, and the shaking table shaking culture condition is controlled to be 140r.min-1At the temperature of 35-40 ℃ for 6-8d, namely shaking table shaking culture solution, 5) preparing the bacillus subtilis, and coating 4) shaking table shaking culture solution on a flat plateImproving inorganic salt solid culture medium, culturing at 35-40 deg.C for 60-80h to obtain solid culture medium colony, selecting single colony, and purifying to obtain Bacillus subtilis.
The screening method of the invention also comprises primary screening and secondary screening:
primary screening:
performing single colony selection experiment on a solid culture medium colony which grows well after the plate is coated, repeating each colony for 2-4 times, taking the strain obtained by screening, purifying and culturing as a degradation strain, and numbering L101, L102, L103, L201, L202, L203, L301, L302, L303, L401, L402, L403, L501, L502 and L503 in sequence; r101, R102, R103, R201, R202, R203, R301, R302, R303, R401, R402, R403, R501, R502, R503;
re-screening:
inoculating the degrading strain obtained by primary screening into the liquid inoculation culture medium, and controlling the concentration of benzoic acid in the liquid inoculation culture medium to be 1000mg.L-1Controlling the inoculation amount of the degrading strain to be 1mL per 200mL of liquid inoculation culture medium degrading strain, inoculating 1mL of sterile water to a control group, performing shake culture on a shaking table, and controlling the shake culture condition of the shaking table to be 140r.min-1And shake culturing for 6-8 days at 37 ℃ in a shaking table, observing the growth condition, and detecting the residual amount of the benzoic acid after the culture is finished.
The invention relates to a bacillus subtilis, which is characterized in that a 16S rDNA sequence of the bacillus subtilis constructs a phylogenetic tree as follows:
the 16S rDNA sequence of the bacillus subtilis L501 is as follows:
or the 16S rDNA sequence of the Bacillus subtilis strain L501, and the meanings are the same.
The bacillus subtilis is screened, and has a good degradation effect on the main autotoxic substance benzoic acid generated by the root system of the strawberry continuous cropping obstacle; the harm of benzoic acid to strawberries is reduced, and the degradation rate of the benzoic acid is up to 87.5 percent. Meanwhile, the compound bactericide has a good inhibition effect on strawberry root rot and can effectively antagonize strawberry root rot. The strain Bacillus subtilis L501 has dual functions of detoxification and antibiosis, and provides theoretical basis for effectively utilizing microbial inoculum to treat strawberry continuous cropping obstacle in strawberry planting production
Description of the drawings:
FIG. 1 is a 1000-fold microscopic characteristic diagram of a Bacillus subtilis strain L501 screened by the invention;
FIG. 2 is a phylogenetic tree constructed from the 16S rDNA sequence of the L501Bacillus subtilis screened by the present invention;
FIG. 3 is a 16S rDNA sequence diagram of Bacillus subtilis L501.
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to specific embodiments, and the degrading bacteria in the present specification are all Bacillus subtilis of the present invention. The L501Bacillus subtilis has the same meaning as the Bacillus subtilis L501Bacillus subtilis; 7d, Table 7 days, h represents hours.
The culture medium of the bacillus subtilis is preferably an improved inorganic salt culture medium which comprises the following components (NH)4)2SO4 2.0 g.L-1，Na2HPO4 1.3g.L-1，KH2PO4 2.0g.L-1Benzoic acid 100-1000mg-1，NaCl 5g.L-1The balance of distilled water; the pH of the medium was controlled to 7.2.
The invention relates to a screening method of bacillus subtilis, which adopts an enrichment culture method and comprises the following specific steps: 1) preparing an inoculation improved inorganic salt culture medium, numbering successive cropping eight-to ten-year strawberry planting fields as L1, L2, L3, L4, L5 and successive cropping three-to five-year strawberry planting fields as R1, R2, R3, R4 and R5, randomly sampling 10-20g of the soil of the strawberry planting fields at different times, inoculating the soil into the prepared improved inorganic salt culture medium as an inoculation amount to inoculate the improved inorganic salt culture medium, 2) preparing a liquid inoculation culture medium, preparing the inoculation improved inorganic salt culture medium into a liquid inoculation culture medium, controlling benzoic acid in a culture solution of the liquid inoculation culture medium as a unique carbon source, 3) controlling the concentration, and controlling the concentration gradient of the benzoic acid in the culture solution in the step 2) to be as follows: l1 and R1 were 100mg.L-1L2 and R2 were 200mg.L-1L3 and R3 were 300mg.L-1L4 and R4 were 400mg.L-1L5 and R5 were 500mg.L-1The contrast group is not inoculated with a soil sample, 4) shaking table shaking culture is carried out, the culture solution obtained in the step 2) is subjected to shaking table shaking culture, and the shaking table shaking culture condition is controlled to be 140r.min-1And the temperature is 37 ℃, the shaking table shaking culture time is 6-8d, and the shaking table shaking culture solution is obtained, 5) the bacillus subtilis is prepared, 4) the shaking table shaking culture solution is coated on an improved inorganic salt solid culture medium in a flat plate mode, the obtained product is cultured for 60-80h at the temperature of 37 ℃ to obtain a solid culture medium bacterial colony, and then a single bacterial colony is picked up and subjected to purification culture, so that the bacillus subtilis is obtained.
The screening method of the bacillus subtilis also comprises primary screening and secondary screening;
primary screening:
performing single colony selection experiment on a solid culture medium colony which grows well after the plate is coated, repeating each colony for 2-4 times, taking the strain obtained by screening, purifying and culturing as a degradation strain, and numbering L101, L102, L103, L201, L202, L203, L301, L302, L303, L401, L402, L403, L501, L502 and L503 in sequence; r101, R102, R103, R201, R202, R203, R301, R302, R303, R401, R402, R403, R501, R502, R503.
Re-screening:
inoculating the degrading strain obtained by primary screening into the liquid inoculation culture medium, and controlling the concentration of benzoic acid in the liquid inoculation culture medium to be 1000mg.L-1Controlling the inoculation amount of the degrading strain to be 1mL per 200mL of liquid inoculation culture medium degrading strain, inoculating 1mL of sterile water to a control group, performing shake culture on a shaking table, and controlling the shake culture condition of the shaking table to be 140r.min-1And shake culturing for 6-8 days at 37 ℃ in a shaking table, observing the growth condition, and detecting the residual amount of the benzoic acid after the culture is finished.
The following are specific screening method steps and performance detection and identification of the bacillus subtilis strain.
Materials and methods
Test material
Variety of test strawberries: "Weizhou" strawberry; soil samples for screening degrading strains: two strawberry cultivation greenhouses are extracted from a certain place in Yuan State of Yichun city of Jiangxi province at a certain day in 2016, wherein the strawberry cultivation greenhouses in the continuous cropping decade randomly take 10-15cm soil layers of 5 types of soil, and the strawberry cultivation greenhouses in the continuous cropping three years randomly take 10-15cm soil layers of 5 types of soil.
The pathogenic fungi to be tested, F405Fusarium solani, F403Fusarium oxysporum, and F407Fusarium yellow cumorum, are all strawberry root rot pathogens and are strains stored in a microbiological laboratory of the academy of China.
Primary reagents and instrumentation
The analytical purity of the benzoic acid to be tested is produced by Tianjin certain chemical reagent company Limited. Ezup column type bacteria genome DNA extraction kit, 2 xEasyTaq PCR Supermix, Marker 2000ladder, 16S rRNA amplification primers (27F:5'-attccggttgatcctgc-3',1541R:5', -aggaggtgatccagccgca-3') were purchased from a bioengineering company, Inc.
PH100-DB00U-IPL digital microscope, optical group of Jiangxi, Inc., TGL-16G desk-top high speed centrifuge (Shanghai, scientific Instrument factory, 752N ultraviolet visible light spectrophotometer, Instrument Co., Ltd., T100 gradient PCR instrument Bio-Rad, etc.).
Culture medium
The improved inorganic salt culture medium comprises the following components (NH)4)2SO4 2g.L-1，Na2HPO41.3g.L-1，KH2PO4 2 g.L-1100-100 mg.L of benzoic acid-1，NaCl 5g.L-1The balance of distilled water; the modified inorganic salt liquid culture medium is prepared, and the pH value of the modified inorganic salt liquid culture medium is controlled to be 7.2.
Enrichment and separation of bacillus subtilis degrading strain
An enrichment culture domestication method is adopted, specifically, ten-year-old continuous cropping strawberry planting places are numbered as L1, L2, L3, L4, L5 and three-year-old continuous cropping strawberry planting places are numbered as R1, R2, R3, R4 and R5, 10g of samples are randomly sampled from each strawberry planting place and are inoculated into a prepared basic inorganic salt liquid culture medium as an inoculation amount, the culture medium is inoculated into a liquid, or is called as an inorganic salt liquid inoculation culture medium, benzoic acid in a culture solution of the liquid inoculation culture medium is controlled as a unique carbon source, and the concentration gradient of the benzoic acid in the culture solution is controlled as follows: l1 and R1 were 100mg.L-1L2 and R2 were 200mg.L-1L3 and R3 were 300mg.L-1L4 and R4 were 400mg.L-1L5 and R5 were 500mg.L-1The two control groups are not inoculated to soil and set at 140r.min-1Shaking the culture plate at 37 ℃ for 7d by shaking, then plating the culture plate on a culture dish of a solid culture medium, wherein the solid culture medium is a solid modified inorganic salt culture medium, placing the culture dish of the solid culture medium at 37 ℃ for culture for 72h, then picking a single colony, and performing purification culture.
Screening and identification of Bacillus subtilis degrading strains
Primary screening: observing the growth of the obtained solid culture medium colonies after the plate is coated, wherein the solid culture medium is the solid form of an inorganic salt culture medium, performing a single colony selection experiment on the solid culture medium colonies with good growth vigor, repeating each strain for 3 times, and performing screening, purification and culture on the degraded strains respectively according to the numbers L101, L102, L103, L201, L202, L203, L301, L302, L303, L401, L402, L403, L501, L502 and L503; r101, R102, R103, R201, R202, R203, R301, R302, R303, R401, R402, R403, R501, R502, R503. Primarily screening degradation strains, namely primarily screening bacillus subtilis strains.
Re-screening:
transferring the primary-screened degrading strain obtained by primary screening into a triangular flask liquid culture medium, and controlling the benzoic acid concentration to be 1000mg.L-1Controlling the inoculation amount of the degrading strain to be 1mL per 200mL of liquid inoculation culture medium, inoculating 1mL of sterile water to a control group, setting the inoculation amount to be 1mL per bottle and the control group to be 1mL of sterile water, and setting the inoculation amount to be 140r.min-1And shake culturing for 7d at 37 ℃ by a shaking table, observing the growth condition of the mixture, and detecting the residual quantity of the benzoic acid.
Degradation assay for benzoic acid by degrading bacterial strains
Determination of benzoic acid Standard Curve
Taking 0.00mL, 1.00 mL, 2.00 mL, 4.00 mL, 6.00 mL, 8.00 mL and 10.00mL of benzoic acid standard solution, respectively adding into a 100mL volumetric flask, adding a small amount of distilled water, shaking up, then fixing the volume with the distilled water, taking a zero tube as reference comparison, respectively measuring the absorbance (OD230) of the solution by using a quartz cuvette at the wavelength of 230nm, and drawing a standard curve by taking the absorbance (OD230) as an ordinate and taking the abscissa as the concentration of the benzoic acid standard solution, namely the existing standard curve of the benzoic acid.
Determination of residual amount of benzoic acid in sample
In the process of shake culture for 7d in a liquid culture medium (liquid inoculation culture medium) which is inoculated with the bacillus subtilis, 5mL of culture solution is taken out and added into a 10mL sterile centrifuge tube at an interval of 24h for 8000r.min-1Centrifuging at 25 deg.C for 8min, filtering (0.22 μm), and removing thallus to obtain solution to be detected; and (3) determining the absorbance (OD230) of the solution to be detected in the quartz cuvette under the wavelength of 230nm by using an ultraviolet-visible spectrophotometry, and calculating the residual content of the benzoic acid in the sample according to the absorbance (OD230) and a benzoic acid standard curve.
The degradation rate DR (%) of benzoic acid is represented by the following formula:
wherein Cck is a control CK without inoculation in liquid culture, and Ct is the concentration of benzoic acid remained in the culture solution after respective inoculation treatment; the inoculation is to inoculate the bacillus subtilis cultured by the invention; hereinafter referred to as "degrading bacterium L501" or "degrading strain L501".
Identification of degrading bacterium L501
The best strain L501 of the degrading bacteria, namely the bacillus subtilis strain is identified by microscopic morphological observation through morphology and gram staining; determining 16rDNA sequence of Bacillus subtilis strain; selecting universal primers (27F:5'-attccggttgatcctgc-3',1541R:5', -aggaggtgatccagccgca-3') to carry out PCR amplification, wherein a PCR reaction system is 2xTaq PCR Master12.5 mu l, upstream and downstream primers are 0.5 mu l respectively, a DNA template is 1.5 mu l, and water is added to 25 mu l; the amplification conditions were: 5min at 94 ℃, 40s at 94 ℃, 45s at 55 ℃, 60s at 72 ℃ and 30 cycles; extension at 72 ℃ for 10 min. And sequencing analysis was performed by Biotech. Submitting the sequencing result to GenBank of an NCBI website, logging in a BLAST program for comparison, constructing a strain phylogenetic tree by MEGA6.0 software, and setting the self-development times to be 1000 by using an NJ algorithm; identified as the strain L501 by combining with the manual of identifying common bacteria.
The in-dish antagonistic action of the degrading bacteria on the strawberry root rot disease:
the degrading bacteria with the best effect obtained by re-screening are shake-cultured in LB culture solution under the culture condition of 140r.min-148h later at 10000 r.min-1Centrifuging at speed for 5min, and filtering with 0.2 μm filter membrane to obtain supernatant.
Inoculating 3 strains of strawberry root rot pathogenic bacteria, F405Fusarium solani (Fusarium solani), F403Fusarium oxysporum (Fusarium oxysporum) and F407Fusarium flavum (Fusarium culmorum) on PDA slant, culturing at 28 deg.C for 5 days, adding sterile water, scraping off spores, mixing to obtain spore suspension, and mixing to obtain 1 × 10 spore suspension7cfu.ml-1; adding the suspension into a 100m L melted P DA culture medium cooled to about 45 ℃, shaking uniformly and preparing into a PDA plate containing root rot pathogenic bacteria.
After solidification, a sterile hole puncher is used for punching the middle of a PDA plate containing pathogenic bacteria, filtered degrading bacteria L501100, mu L are taken and cultured for 5D at 28 ℃, and the diameter (D) of a bacteriostatic circle is measured by a cross method.
Example 1 screening of degrading Strain L501 and detection of degradation Rate of benzoic acid
The standard curve of benzoic acid can be plotted based on the data determined from the standard curve of benzoic acid, which is shown in table 1 below. The standard curve is the standard curve of the existing benzoic acid.
TABLE 1 benzoic acid Standard Curve survey data Table
Table 1Table of standard curve for benzoic acid determination
According to experimental analysis, as shown in the following table 2, 9 of the 67 isolated and purified strains, namely L203, L302, L403, L501, L502, R401, R402, R403 and R503, have degradation capability on benzoic acid. The degradation rates are respectively 13.3%, 17.1%, 43.2%, 87.5%, 40.8%, 49.5%, 46.1%, 57.3% and 42.7% in 168h, wherein the L501 reaches the highest value in 168h and is 87.5%. The degradation effect of the bacillus subtilis on benzoic acid is obvious.
The degradation rate of 9 degrading bacteria to the benzoic acid is 13.3-87.5% in the culture process of 7 d. The bacterial strain separated and screened from the strawberry root soil has effective degradation capability on benzoic acid. The rescreening result shows that the degrading strain L501bacillus subtilis has the strongest capacity of degrading benzoic acid, so that the L501 is used for antagonistic experiment and identification.
TABLE 2 degradation Rate DR of benzoic acid and residual amount C of benzoic acid by 9 strains of degrading bacteria of the present invention
Table 2Degradation rate of benzoic acid by 9strains of degrading bacteria and Residual benzoic acid
Example 2 identification of the Strain of the invention Bacillus subtilis L501
The degradation rate of the bacillus subtilis L501 to the benzoic acid reaches the highest at 168h, and the degradation rate is 87.5%. Thus authenticating it. Morphological observation shows that the bacterial colony of the degrading bacteria is milky white and nearly beige, round, smooth and moist in edge, slightly provided with small teeth, wrinkled in surface, bulged in middle and opaque in texture. Under an optical microscope, the strain is gram-positive bacteria, is Brevibacterium, and has the size of 0.7-0.9 Mumx (2.0-3.0) Mum; the spores were growing and slightly enlarged (FIG. 1).
The length of the 16S rDNA sequence of the molecular identification strain L501 is 1456bp, GenBank is submitted to obtain a sequence number MG519283, the sequence of the L501 and the sequence with extremely high similarity are compared and analyzed by using a BLAST program in an NCBI website, hundreds of strains with homology of more than 99 percent with the 16S rDNA sequence of the degrading strain L501 are Bacillus subtilis, the strain is preliminarily classified into the genus Bacillus, 5 strains with higher similarity to the L501 strain in the Genbank are selected to construct a phylogenetic tree, and the result is shown in figure 2. In conclusion, according to 16S rDNA sequence similarity analysis, the strain L501 is identified as Bacillus subtilis by combining morphological structure observation of the strain L501 and contrasting common bacteria identification manual.
The 16S rDNA sequence of the bacillus subtilis strain L501 is as follows:
example 3 in-dish antagonism of the degrading bacteria of the present invention on strawberry root rot
The fermentation broth of degrading strain L501 showed antagonistic activity, and the results are shown in Table 3. Wherein the strain has strong inhibiting effect on the growth of F405Fusarium solani strain, the diameter of an antagonistic circle reaches 15.0mm, and the diameter of antagonism on F403Fusarium oxysporum is 14.5 mm; the antagonistic diameter of the degrading strain L501 to strawberry root rot pathogen F407Fusarium culmorum is 11.0 mm; and the transparency is good. In conclusion, the degrading strain L501 has a better inhibiting effect on the root rot of strawberries.
TABLE 3 antagonistic ring diameter of L501 against pathogenic bacteria of root rot of 3 strawberries
Tab.3Diameters of the inhibitory zones of strain L501against three strawberry root rot
The research shows that the bacillus subtilis L501 is a biocontrol bacterium, and is primarily developed into a related biocontrol agent to be put into a market test. The L501bacillus subtilis can be used as a biocontrol bacterium to inhibit fusarium oxysporum and fusarium oxysporum of strawberry root rot.
The technology of the invention shows that a bacterial strain L501Bacillus subtilis is obtained by screening, has stronger degradation effect on benzoic acid which is a main harmful allelopathy substance in strawberry root exudates, and can effectively antagonize strawberry root rot bacteria. The L501bacillus subtilis is a degrading bacterium secreted by strawberry roots from toxic substance benzoic acid, has the biocontrol bacterium potential of becoming strawberry root rot, and can be used for repairing strawberry continuous cropping. In addition, the actual repairing effect can be explored through subsequent field experiments, and the microbial agent is expected to be prepared.
The 16S rDNA sequence of the strain L501Bacillus subtilis is used for constructing a phylogenetic tree:
<110> college of Yichun
<120> bacillus subtilis strain and screening method and application thereof
<210> 1
<211> 1441
<212> DNA
<213> Artificial sequence (Bacillus subtilis sp.)
<400> 1
1 gtgggggggg tgcttataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt
61 agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg
121 aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata aaaggtggct
181 tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca
241 ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac
301 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac
361 ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag
421 aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta
481 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc
541 gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga
601 gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc
661 ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa
721 ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg
781 ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc
841 attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg
901 gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg
961 tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg
1021 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg
1081 caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca
1141 aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca
1201 cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa
1261 tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta
1321 atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac
1381 accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttgtaggagc cagccgccga
1441 aaggtggaca caattg
Claims (3)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) The bacillus subtilis is characterized in that the bacillus subtilis is preserved in China center for type culture Collection, the preservation number is CCTCC No. M2017729, the bacillus subtilis is named as bacillus subtilis L501, and the preservation date is as follows: 27 th month in 2017, address, eight-way Lodokya in Wuchang district, Wuhan university, Hubei province, post code: 430072.
2. the use of the bacillus subtilis according to claim 1 for degrading benzoic acid which is a strawberry continuous cropping autotoxicity barrier substance.
3. The bacillus subtilis strain of claim 1 for antagonizing fusarium solani of rhizoctonia solani of strawberry (f)Fusarium solani) Fusarium oxysporum (F.), (Fusarium oxysporum) Or Fusarium yellow (Fusarium culmorum) The use of (1).
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