CN115851480A - Bacillus belgii strain P87 and application thereof in prevention and treatment of pepper diseases - Google Patents
Bacillus belgii strain P87 and application thereof in prevention and treatment of pepper diseases Download PDFInfo
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Abstract
The invention relates to the technical field of biological control of plant diseases, in particular to a Bacillus beili (Bacillus velezensis) strain P87 and application thereof in control of pepper diseases; the invention aims to provide a Bacillus belezii strain P87 for antagonizing xanthoxylum bungeanum, phytophthora parasitica, xeromyces and deciduous germs aiming at the defects of the prior control technology of the pepper diseases, the strain is preserved in China center for type culture Collection of Wuhan university at 6 months and 7 days in 2022, and the preservation number is CCTCCNo: M2022819; the Bacillus beilesiensis strain P87 has obvious antagonistic action on xanthoxylum, xanthoxylum and deciduous pepper germs; meanwhile, the invention provides a biological preparation with the active ingredient of the Bacillus beiLeisi strain P87, the bacterial colony growth bacteriostasis rate of the preparation to the root rot of the pepper is 82.88%, and the control effect to the root rot of the pepper is 81.47%.
Description
Technical Field
The invention relates to the technical field of biological control of plant diseases, in particular to a Bacillus beiLeisi strain P87 and application thereof in control of pepper diseases.
Background
The wild pepper is an important economic crop in China, the wild pepper is the first country of wild pepper cultivation area yield in China, the wild pepper is distributed in most areas except northeast, inner Mongolia and other areas, and the wild pepper is mainly distributed in Shanxi, sichuan, hebei, shandong, gansu and other provinces. In recent years, with the guidance and the drive of the national policy of vigorously developing the rural cash crops, the planting region and the area of the pepper are rapidly increased. With the expansion of the planting area of the pepper, the appearance of the planting mode of large-area concentrated connection of the pepper and the increasing aggravation of the environment and climate, the problem of pepper diseases is increasingly prominent, and huge loss is caused to the development of the pepper industry. The dry rot, root rot, black shank and defoliation of the pepper are serious diseases in pepper producing areas in China, and the yield and the quality of the pepper are seriously influenced. At present, the prevention and treatment research and application of the diseases are mainly focused on a single prevention and treatment method mainly based on chemical prevention and treatment, and the phenomenon of heavy chemical prevention and light prevention often occurs in actual production, so that the prevention and treatment cost is increased due to the large use of chemical pesticides, and the problems of environmental pollution, pesticide residue, pesticide resistance of pests and the like are seriously threatened to human health and ecological environment. Along with the rapid development of national economy and the continuous improvement of the living standard of people, the pollution-free and green food is generally regarded by people, and the acceleration of the pollution-free production of the pepper is imperative.
Biological control is an important research direction for green and pollution-free control of plant diseases, and microorganisms are used as an important resource bank for biological control of plant diseases, and have remarkable effect in the aspect of biological control of plant diseases. The implementation of biological control is premised on the acquisition of biocontrol strains and the development of microbial inoculum thereof. The bacillus widely exists in natural environments such as soil, plant rhizosphere, plant body and the like, can generate stress-resistant spores, generate various antibacterial metabolites, effectively colonize in the plant body and protect the plant from being infected by pathogenic bacteria, and is considered to be an environment-friendly biocontrol factor with wide application prospect. Regarding the successful case of utilizing bacillus to control plant diseases in production, research and development of bacillus and microbial inoculum thereof aiming at pepper disease control are a new expectable way for carrying out pollution-free control on pepper diseases.
Disclosure of Invention
The invention aims to provide a Bacillus beijerinckii strain P87 for antagonizing xanthoxylum bungeanum, phytophthora parasitica, xeromyces and deciduous sporulata aiming at the defects of the prior control technology of the pepper diseases.
In order to achieve the purpose, the invention provides a Bacillus velezensis strain P87, which is preserved in China Center for Type Culture Collection (CCTCC) at 6.7.2022 with the preservation number of M2022819, the preservation address: china center for type culture Collection, wuhan university, wuhan, china.
The invention also provides application of the bacillus beleisi strain P87 in antagonizing xanthoxylum rhizoctonia, phytophthora parasitica, xeromyces and deciduous fungi.
The invention also aims to provide a biological agent for preventing and treating pepper diseases, which comprises the bacillus beilesensis strain P87 as an active ingredient.
Further, the biological agent is wettable powder prepared from fermentation liquor of bacillus beilesensis strain P87.
Further, the fermentation liquor is prepared by the following steps:
activating a Bacillus belgii strain P87, culturing in a fermentation medium containing 18g of glucose, 30g of sucrose, 10g of peptone, 5g of sodium chloride and 1L of water, and stirring and culturing at 25-29 ℃ and 100-150 rpm for 4-6 days to obtain the fermentation liquid.
Further, the formula of the wettable powder is as follows: 25g/L of carrier white carbon black, 8000.0.5 g/L of wetting agent PEG, 0.75g/L of dispersant sodium tripolyphosphate, 0.025g/L of protective agent sodium dodecyl sulfate, and the spore content is more than or equal to 1 multiplied by 10 8 The suspension percentage is 90.52 percent, the wetting time is 43.6s, the pH value is 7.1, the fineness is 98 percent, and the dry weight loss is 0.9 percent.
Furthermore, the invention also provides application of the biological preparation for preventing and treating the pepper diseases in preventing and treating the pepper root rot.
Compared with the prior art for preventing and treating pepper diseases, the invention has the beneficial effects that:
(1) The screened strain P87 of Bacillus beleisi has obvious antagonistic activity on pathogenic bacteria (root rot, phytophthora parasitica, deciduous leaf and xeromyces) of main diseases of pepper;
(2) The fermentation process of the bacillus belgii strain P87 is optimized, the antibacterial activity of the fermentation liquor is used as a detection index, the optimal fermentation condition of the bacillus belgii strain P87 is determined, and the large-scale fermentation culture of the bacillus belgii strain P87 is facilitated;
(3) The invention develops the wettable powder of the Bacillus beiLeisi strain P87, the best formulation of the Bacillus beiLeisi strain P87 wettable powder is definite, and the quality of the wettable powder reaches the standard, the Bacillus beiLeisi strain P87 wettable powder of the best formulation is applied, the colony growth bacteriostasis rate of the Bacillus beiLeisi can reach 82.88%, the control effect to the root rot of the pepper can reach 81.47%, have good application prospect in the pollution-free control of the pepper disease, also lay a foundation for the commercialization, the industrialized production of the Bacillus beiLeisi strain P87 wettable powder later stage;
(4) By applying the biological preparation prepared by the embodiment of the invention, a series of problems caused by the use of chemical pesticides are completely avoided, the use amount of other chemical pesticides for preventing and controlling the root rot of the pepper can be omitted or reduced, the pollution-free production of the pepper is facilitated, the problems of pesticide residue, drug resistance, pest reexplosion and the like caused by the use of the chemical pesticides can be made up, and the sustainable development of a agriculture and forestry ecosystem is facilitated to be maintained;
(5) Because the wild pepper is a biological strain preparation, a series of problems caused by the use of chemical pesticides are completely avoided, so that the wild pepper is beneficial to pollution-free production, farmers can not use or reduce the use amount of other chemical pesticides, the expense of the farmers can be saved, and the wild pepper is beneficial to export of wild pepper products.
Drawings
FIG. 1 is a plate confrontation picture of Bacillus belgii strain P87 and four target pathogenic bacteria; wherein, (A) is a control map of antagonistic activity against xanthorrhizos, and (B) is a control map of antagonistic activity against phytophthora parasitica, and (C) is a control map of antagonistic activity against Laurencia and (D) is a control map of antagonistic activity against Laurencia;
FIG. 2 is a graph of colony morphology, gram stain and spore stain of Bacillus belgii strain P87; wherein (A) is a colony morphology map of Bacillus belgii strain P87, (B) is a gram staining map of Bacillus belgii strain P87, and (C) is a spore staining map of Bacillus belgii strain P87;
FIG. 3 is a graph (A) showing the influence of different fermentation liquids of Bacillus belgii strain P87 on the colony growth of Rhizopus zanthoxyli and the bacteriostasis rate (B);
FIG. 4 shows the bacteriostatic activity of Bacillus belgii strain P87 wettable powder on Rhizopus zanthoxyli;
FIG. 5 is a comparison graph of the control effect of Bacillus belgii strain P87 wettable powder on pepper root rot.
Detailed Description
The present invention is further described with reference to specific embodiments, which are not intended to be limiting, and any variations or alterations may occur to those skilled in the art without departing from the scope of the invention.
Example 1: separation, screening and identification of antagonistic bacillus beilesensis strain P87
The biocontrol Bacillus velezensis strain P87 is preserved in China center for type culture Collection at 6 months and 7 days in 2022, and the preservation number is CCTCC NO: M2022819. The strain is separated from pepper rhizosphere soil microorganisms, and the flat plate confronting culture is adopted to discover that the Bacillus belgii strain P87 shows obvious antagonistic action on tested target bacteria, and the separation, screening and identification methods of the strain are as follows:
(1) Isolation of antagonistic bacteria
Collecting the pricklyash root soil in a pricklyash garden of a Shaanxi Yangling agricultural comprehensive test demonstration station in 8-month middle of 2020, wherein the specific collection process is as follows: setting 3 standard lands of 20m multiplied by 30m in a pepper garden, respectively selecting 3 plants of pepper trees of 3 years, 5 years and 8 years in each standard land, setting 5 sampling points at a position 40cm away from the base of a trunk, using a soil drill with the inner diameter of 10cm to dig and pick 10-30 cm of soil in layers, collecting fine pepper roots with the diameter of 0.2-0.5 cm in a soil layer, shaking off large-particle soil adhered to the pepper roots, then bringing the fine pepper roots back to a laboratory, and shaking off soil on the fine pepper roots with force to obtain rhizosphere soil; mixing rhizosphere soil of the same age, filling the rhizosphere soil into a sterilized sealed plastic bag, putting the plastic bag into an ice box, and taking the plastic bag back to form rhizosphere soil samples of 3 different ages: sieving 3 years (RS 1-Zb), 5 years (RS 2-Zb) and 8 years (RS 3-Zb) with 0.2mm sieve, and freezing at-80 deg.C.
Collecting 5g of pepper rhizosphere soil RS1-Zb, RS2-Zb and RS3-Zb, mixing uniformly, adding into a sterile glass triangular flask containing 135mL of sterile water (containing 30-40 sterile glass beads), and placing on a shaker for shaking for 15min to obtain the final product with the concentration of 10 -1 g/mL soil suspension, then respectively preparing a concentration gradient of 10 -2 、10 -3 g/mL and 10 -4 g/mL soil diluent, absorbing 50 mu L of soil suspension liquid with each concentration, dripping the soil suspension liquid on a PDA culture medium flat plate, uniformly coating the soil suspension liquid with a sterile coater, sealing the culture dish with a sealing film, placing the culture dish in an incubator at 28 ℃, observing and counting single microbial colonies growing on the PDA culture medium every day, picking the newly-appeared single microbial colonies with different colony morphologies from the previous single microbial colonies with a sterile toothpick, and inoculating the single microbial colonies with the sterile toothpickThe strain is purified on a new PDA culture medium to obtain a pure strain.
The obtained strain is preserved in 30% glycerol and placed in a low-temperature environment of-80 ℃.
The preparation method of the PDA culture medium comprises the following steps: weighing 200g of potato without peel, cutting into small pieces, adding a proper amount of deionized water, boiling for 20min, cooling, filtering with gauze, collecting filtrate, adding 20g of glucose and 20g of agar into the filtrate, diluting to 1L with deionized water, and sterilizing with high-pressure steam at 121 ℃ for 20min for later use.
(2) Screening of antagonistic bacteria
The antagonistic activity of all microbial strains separated from the pepper rhizosphere soil on pepper root rot, phytophthora parasitica, deciduous leaf pathogen and dry rot is detected by adopting a plate confronting culture method, and the tested target pathogens are obtained by laboratory prophase separation and Koehz's rule verification and identification.
Primary screening of antagonistic strains: taking out four test target pathogen strains stored in a refrigerator, respectively inoculating the four test target pathogen strains on a PDA (personal digital assistant) flat plate for activated culture, after dark culture at 28 ℃ for 10 days, taking a pathogenic bacteria cake with the diameter of 0.5cm from the position, close to the edge of hypha, on the flat plate by using a sterile puncher, and inoculating the pathogenic bacteria cake to the center of a new PDA flat plate; inoculating the purified pepper rhizosphere soil microbial strains on a PDA culture medium for activated culture for 5 days, then picking thalli on bacterial colonies by using a sterile toothpick, inoculating four different test strains at a position 2cm away from a pathogenic bacteria cake by adopting a cross inoculation method, culturing for 10 days at 28 ℃, observing the bacteriostatic effect of each microbial strain in the pepper rhizosphere soil on a target pathogen, screening out the strains simultaneously showing stronger bacteriostatic activity (the bacteriostatic bandwidth is more than or equal to 10 mm) on four pathogenic bacteria, and primarily screening to obtain 20 antagonistic strains which are bacteria.
Re-screening antagonistic strains: taking the preliminarily screened antagonistic strains and pathogenic bacteria, carrying out a confrontation test on a PDA (personal digital assistant) flat plate, and carrying out re-screening. And (3) preparing a pathogenic bacteria cake with the diameter of 0.5cm from the position, close to the edge of the hypha, on the plate inoculated with the pathogenic bacteria by using an aseptic puncher, and inoculating the pathogenic bacteria cake to the center of a new PDA plate. The antagonistic bacterial strains obtained by primary screening are inoculated on a PDA culture medium for culture for 2D, thalli on bacterial colonies are dipped by a sterile cotton swab, the same antagonistic bacterial strain is inoculated at a position 2cm away from a pathogenic bacterium cake by adopting a cross inoculation method and cultured for 10D at 28 ℃, the bacterial strain P87 which simultaneously shows the most remarkable antagonistic activity to four target pathogens is screened, the antagonistic effect on the target pathogens is shown in figure 1, (A) is an antagonistic activity comparison chart on xanthophyllum rot bacteria, (B) is an antagonistic activity comparison chart on phytophthora parasitica, (C) is an antagonistic activity comparison chart on alternata, and (D) is an antagonistic activity comparison chart on xerophyte rot bacteria, and the antagonistic effect of the bacterial cake treated by the bacterial strain P87 is remarkable compared with that of the control bacterial cake by adopting a plate antagonistic culture method.
(3) Identification of antagonistic Strain P87
A single colony of the antagonistic strain P87 was selected and inoculated on a PDA medium, and the colony morphology is shown in FIG. 2 (A). The gram-stained strain P87 showed purple color, indicating that the gram-positive strain P87 is shown in FIG. 2 (B). Whether the strain P87 produces spores or not is detected by using a Schaeffer-Fulton spore staining kit, and the strain P87 is found to produce a large number of spores as shown in figure 2 (C).
The DNA of the strain P87 is extracted by a CATB method, then the 16S rDNA of the strain P87 is amplified and sequenced, and the primer synthesis and the sequencing are synthesized by Shanghai Biotechnology Limited. According to the comparison result of the 16S rDNA sequence of the strain P87 in the NCBI database, the strain is identified as Bacillus velezensis. The antibacterial lipopeptide synthetic genes of the bacillus beleisis have certain conservative property, on the basis, 7 antibacterial lipopeptide synthetic genes (srfAA, srfAC, ituC, ituD, fenD, bmyB and bacA) of the strain P87 are subjected to PCR amplification and sequencing, the sequencing results are compared in an NCBI database, and the homology of the results and the corresponding genes of the bacillus beleisis up to more than 99%, so that the result accuracy of identifying the strain P87 as the bacillus beleisis proved. The PCR amplification primer synthesis and sequencing of the strain P8716S rDNA and 7 antibacterial lipopeptide synthetic genes are completed by Shanghai biological engineering Co.
The primers for PCR amplification of 16S rDNA and 7 antimicrobial lipopeptide synthetic genes from strain P87 are shown below:
the reaction system for PCR amplification of 16S rDNA and 7 antibacterial lipopeptide synthetic genes of the strain P87 is 30 mu L, and comprises 2 XS Taq MasterMix30 mu L, ddH2O 12.5 mu L, a forward primer 1 mu L, a reverse primer 1 mu L and a DNA template 0.5 mu L; the PCR amplification procedure was: pre-denaturation at 94 ℃ X3 min, [94 ℃ X30 s, 52 ℃ X30 s, 72 ℃ X45 s ]. Times.35 cycles, 72 ℃ X10 min. After the PCR is finished and the size of the fragment is verified to be correct by gel electrophoresis, the PCR product is sent to a sequencing company for sequencing.
Sequencing result of 16S rDNA amplified fragment of strain P87:
GGACTGGCGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCC TGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACAT AAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCAC ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCG TAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACG GTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCT GATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG AACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA GTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCT CTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT TGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGT GCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAG TAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAG CCGCCGAAGTGACAGATGA
sequencing result of srfAA gene amplified fragment of strain P87:
GGGGAAATGGGACTTGCTCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTT GTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGAC CCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG AGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTT CAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGG CTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGT GAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC ACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAAT TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGA ACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA GTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCC CTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGATGCCAC
sequencing of the srfAC gene amplified fragment of strain P87:
GGGTTGGGCAGCTGGAGCGGGAGGGCATTTACCCGCAGGCCGTGATCATCTCAGC CATTCAGCCGCCGCATGTGGAAAGGAAAAAAGTGTCTCATCTTGATGATGAAAAATTTCTCGCCCATATTATCGAGCTTGGCGGAATGCCGCAGGAGTTAGTGGAGAATAAGGAGG TCATGTCATTTTTCCTGCCTTCTTTCAGATCCGACTACCGCGCGCTTGAAAGCTTCCGTCCGTCTGATTCTCACATGATTCAATCACCCGTCCATATTTTTAACGGGCGGAAAGATAA AAAATGTATCAAAGATGCGGACGGATGGAAAAAATGGGCCGACAATCCCGTATTTCAT GAGTTTTCGGACGGCCACATGTTCATATTAAGTGAAACTGAAAAG
sequencing result of the ituC gene amplified fragment of the strain P87:
ACCGTACACTTCTCGTTGACATCAAACTGCGAAAACTGAATATCCGCGGGATAAG ACGGTTTATCAGTTATTCGAAGAACAGATGAAACGAACACCGGATCAAGCAGCCGTTA TTTGCGGAGAAAAGCAATTCACATATCGCCAGCTCAATGAACGTGCCAATCAATTAGCCCGAACGTTAAGGAAAAAGGGGGTAAAGACGGACCGGCTCACTGCGATTATTTGCGA ACATGAGATCGAATTGGTCGTGGGAATACTGGCCGTTTTAAAAGCGGGAGGGGCCTATGTGCCAATTGATCCGGATTACCCGAAGCATCGCATACAGTATATAGTAGAAGACTCCCA AGCTGATATCATCCTGACGCAGAGCCATCTTCAAAAACAGTTGGAACTTGCGGGCACAATGGTTTTCCTCGATCAAGAAAACTCTTACCACGAAGACGGCTCTGACCTGGAACCGA TCAGCAGTACGAAGGATTTGGCTTATGTCATTTATACGTCAGGCTCCACAGGCAAGCCTAAAGGAGTGGCAATTGAGCATCAGGCTTAAACCAAA
sequencing of the ituD gene amplified fragment of strain P87:
TTTCCCCCTGTTTTTTTAGATCGGAGGAATCTCATGAACAATCTTGCCTTTTTATTT CCTGGACAAGGGTCTCAATTTGTAGGAATGGGCAAACAATTTTGGAATGATTTTGTGCTCGCAAAGAGATTGTTTGAAGAAGCGAGCGATGCGATCTCCTTGGATGTAAAAAAACTG TGTTTTAACGGAGATATGAATGAATTGACAAAGACAATGAACGCGCAGCCCGCTATTTTAACGGTCAGTGTCATTGCTTTTCAAGTGTATATGCAGGAAATAGGGGTGAAGCCACGC TTCCTGGCAGGCCATAGCTTAGGCGAATATTCAGCGCTTGTCTGTGCCGGCGCCCTTTCTTTTCAGGATGCCGTTACACTTGTAAGGGAGCGGGGAATTCTTATGCAAAATGCGGATC CTCAGCAGCAGGGGGCGATGGCCGCCGTGACTCACCTCTCTCTTCAAACCTTGCAAGAAATATGTTCGAAAGTGTCGACGGAAGACTTTCCGGCAGGTGTAGCCTGCATGAATTCA GAACAGCAGCATGTGATTTCCGGACACCGGCAAGCTGTGGAACGTGTCATCAAGATG GCGGAGGAAAAGGGTGCGGCATACACTTATTTGAATGTCAGTGCGCCTTTTCACAGTCAGATGGTACGATCAGCATCTGAACAATTCCAGACTGTATTACACCGGTATTCCTTCCGG GATGCCGCATGGCCGATCATTTCAAATGTCACCGCACGCCCTTACAGCAGCGGAAATTCGATCCGCGAACATCTCAAGCAGCACATGACGATGCCGGTTAGATGGCAAAA
sequencing result of fenD gene amplification fragment of strain P87:
GGGTCGGCGGGCAATGGTTCTTCACACAAGACATGAAGGAAGCAAATCATTTTAA CCAGTCCGTCATGCTGACGAGAACAAATTCAATAGATGAAGAGATCCTGAGGAAAACGTTAAAAGCCATTACCGTTCATCATGATGCCTTGCGCCTTGTCTGCAAAAAAGACGAA GAGAAAGGTCTTCTCCTTTTCAACAGACCGGCTGACCTCCCAGATGAGCAGCTTTACAGCCTGACCATTTTAGAAACGGAAGACGATGAGCAA
sequencing of the BmyB gene amplified fragment of the strain P87:
AGATGCGCTTAATGCCGGACATTTTTCTAACGGCAGGTTTTGATTTTTCAGAGAAT CATTAACGGCTTCTTTCAGCTGCCCGAATACGGTTTTAAATGTGCTCTCGTTTGAAAGCGTATTTTTTAAGAGAACAATGTTGTTAACGGCTGACTGACCGGCTTTTTGCTTCGATAC CGTTGGAATGCCAAGAATCAGGCTCGTTCGATCCGTATATTTATACAACAAGCATTCAATACCCGCCAATAAAATCAGATAGGCCGCCATATCGGAATGATTCGCCATTGTCATGATTCTTTGAGATACTTCCGGAGATAAGGAACGGCAGATGCATTTTTCA
sequencing result of bacA gene amplified fragment of the strain P87:
GGGGGAACAGCAAATTATCACTGCATGATACATTTGAATCGTGCATTGAAAAAAC GCTTCAAAGTCCGCTGGAATACACAATTGTGCCTCACGCGTATGACGGCATTAAGCACTTCTACATGAGACCGGATCTGCAGCTTCTGCAAATTTTCAGGTGCGACACCCCGATGTA CGGCCTGGCTGTCCGGCCCGGTTTTGAATATACAGATGACATGCTTGAACAGGCTGTCATCGTATCCCACCCGTCTCCTATCAATTTGATTAAGTATTTTACCCGTAAAGATGTCACGT TTGATCTGGTGAACTCGACGAGCGCAGCCGCGAAAAGAGTGAAGGACGGTTTGTCCGACATCGCGCTGACAAATGAGCTGGCCAGACAAAAGTACGGCCTTCACTTTGTCAAAA CATTTAAAAGCATTCCCATGAGCTGGTCATTATTCGGAAAAGGAGAGATTCATGATGAAAACTGAACAAGATTTGCAGGAACTTTATTTCCCGACGCCTAAATTAATAGAATGGGATA ACGGAGTAAGACAATATTCGTCCGTCAGAGGAGATACGGAAGTTCTGCTTTCTTACGT TCCGCCGCATACAAACGTGGAACCGCATCAGCATAAGGAAGTCCAAATCGGCCTTGTCGTTTCCGGCGAGCTGTCCATGACTGTCGGAGATGTCACAAGAAAGATGACAGCGCTG GAATCAGCCTATATCGCGCCTCCTAACGTGCCTCACGGCGCAAGAAATGAGACGGATCAAGAAGTGATCGCCATTGATATCAAACGCCTGAAAGCCGTAAAA
in conclusion, the antagonistic strain P87 is identified as Bacillus velezensis (Bacillus velezensis), which has been deposited in China center for type culture Collection in 2022, 6, 7 days and has a deposition number of CCTCC NO: M2022819.
Example 2: fermentation process of Bacillus belgii strain P87
(1) 6 different fermentation media were designed. Culture medium A: 5g of glucose, 7.5g of yeast extract, 7.5g of peptone and (NH) 4 ) 2 SO 4 5g, naCl 5g, water 1L, pH 7.0; and (3) a culture medium B: 5g of glucose, 5g of yeast extract, 10g of peptone, 5g of beef extract, 5g of NaCl, 1L of water, pH =70; and (3) a culture medium C: 10g of glucose, 4g of peptone, 1L of water, pH =7.0; and (3) a culture medium D: 10g of soluble starch, 10g of peptone and MgSO 4 0.1g、KH 2 PO 4 0.5g, 1L of water and natural pH; and (3) a culture medium E: 20g of glucose, 10g of yeast powder, 10g of peptone, 5g of NaCl and CaCO 3 1g, water 1l, ph =7.0; and (3) a culture medium F: 18g of glucose, 30g of sucrose, 10g of peptone, 5g of NaCl and 1L of water, and the pH value is natural. After the preparation of the culture medium, the culture medium is sterilized at high temperature and high pressure, and after the culture medium is cooled to room temperature, the culture medium is filled into glass triangular flasks with the volume of 100mL in 30mL per flask.
(2) A small piece of culture medium with bacteria was prepared on a colony of Bacillus beiLeisi strain P87 with a sterile toothpick, and placed in a glass flask containing 30mL of LB liquid medium, 3 cakes were inoculated per bottle, 3 bottles were inoculated in total, and placed at 27 ℃ for 2 days with shaking at 150rpm, and then used as a seed culture medium.
(3) 2mL of the P87 seed culture medium is sucked by a pipette and transferred into a triangular flask containing 30mL of a fermentation medium, the triangular flask is placed in a constant-temperature shaking incubator and subjected to dark culture at 27 ℃ and 125rpm for 5 days, then fermentation liquor is collected and centrifuged at 12000rpm for 10min, thalli are removed, and supernatant is collected.
(4) Filtering the supernatant with a filter (0.22 μm), adding into PDA culture medium which has not been solidified after autoclaving, mixing, and pouring into flat plate to make the content of Bacillus beiLeisi strain P87 fermentation liquid in PDA culture medium 45%.
(5) The formula of the fermentation medium which takes the root rot of the zanthoxylum bungeanum as a target pathogen, detects the inhibiting effect of six different fermentation liquids of A, B, C, D, E and F of the Bacillus belezii strain P87 on the colony growth of the root rot of the zanthoxylum bungeanum, takes the bacteriostasis rate as an index, and screens out the fermentation medium which is most beneficial to the P87 to play the antibacterial activity effect is as follows: and (3) a culture medium F: 18g of glucose, 30g of sucrose, 10g of peptone, 5g of NaCl and 1L of water, and the pH value is natural. Under the condition, the inhibition rate of the growth of the colonies of the pepper root rot fungi reaches 70.86 percent; A. the influence of the Bacillus belgii strain P87 in the six media B, C, D, E and F on the growth of the colonies of the disease pricklyash root rot fungi is shown in FIG. 3 (A), and the results of the detection of the inhibition of the Bacillus belgii strain P87 in the fermentation liquors of the six media on the growth of the colonies of the disease pricklyash root rot fungi are shown in FIG. 3 (B).
Example 3: preparation of bacillus belgii strain P87 wettable powder
(1) Preparation of Bacillus beilesiensis Strain P87 stock
Based on the optimal fermentation process for the B.beiLensis strain P87 determined in example 2, a liquid fermentation stock of B.beiLensis strain P87 was obtained.
(2) Preparation of bacillus belgii strain P87 wettable powder
Uniformly mixing a carrier and an auxiliary agent with a mother strain P87 of the Bacillus belezensis strain according to a ratio, wherein the carrier is 25g/L of white carbon black, the wetting agent is PEG80000.5g/L, the dispersing agent is 0.75g/L of sodium tripolyphosphate, and the protective agent is 0.025g/L of sodium dodecyl sulfate. After all the components are mixed uniformly, the mixture is put into an air freezing dryer to be frozen and dried for 24 hours and then put into a micro crusher to be crushed, and the bacillus beilaisi strain P87 wettable powder can be obtained.
(3) Quality detection of Bacillus belgii strain P87 wettable powder
According to the national quality standard, the quality detection is carried out on the Bacillus belgii strain P87 wettable powder, and the method is characterized by comprising the following steps: the spore content is not less than 1 × 10 8 The suspension percentage is 90.52 percent, the wetting time is 43.6s, the pH value is 7.1, the fineness is 98 percent, and the dry weight loss is 0.9 percent.
Example 4: application of bacillus beilaisi strain P87 wettable powder in antagonizing xanthoxylum nivalens
(1) Determination of bacteriostatic activity of Bacillus beleisi strain P87 wettable powder on xanthoxylum
Preparing fungus cake of xanthoxylum, inoculating in PDA culture medium, punching holes with diameter of 5mm at 2cm positions on left and right sides of the fungus cake with puncher, and diluting wettable powder of Bacillus beleisi strain P87 to 1 × 10 7 After CFU/g, 0.3mL of the solution is aspirated by a pipette and injected into each well. Then placing the mixture in a dark culture at 28 ℃ for 8 days, and finding that the Bacillus belgii strain P87 wettable powder has obvious colony growth inhibition effect on the xanthorrhizoma bungeanum, and the inhibition rate can reach 8288% as shown in figure 4.
(2) Prevention effect of bacillus beilaisi strain P87 wettable powder on pepper root rot
Healthy 2-year-old 'Dahongpao' pepper pot seedlings are selected to verify the prevention and control effect of the Bacillus beilesiensis strain P87 wettable powder on pepper root rot. The potted seedling of pricklyash peel is planted in sterilized soil after high pressure steam sterilization, the soil of pricklyash peel seedling passing through the base part is poked, on the main root of pricklyash peel with the base part about 1cm downward, 15 times of needle punching is carried out by using sterile inoculating needle to make micro wound, then 1mL10 is taken 5 cfu/mL xanthoxylum bungeanum spore suspension is injected into soil along the main root of xanthoxylum bungeanum for root irrigation inoculation of dry rot pathogen, the contrast and treatment groups are subjected to root irrigation inoculation of root rot pathogen, and then the treatment group is inoculated with 1mL of 10mg/mL (the viable count of the Bacillus belgii strain P87 is 10) 6 cfu/mL) of the wettable powder of Bacillus belgii strain P87, and a control group was inoculated with 1mL of the sterile aqueous solution. All the seedlings of Zanthoxylum bungeanum after inoculation were placed in a greenhouse for culture (25 ℃, photoperiod: 12 (L: D) h).
After the pepper seedlings are treated for 40 days, photographing and recording the disease occurrence condition of the pepper roots, averagely dividing the pepper roots into 5 sections, respectively grading the severity, counting the results, and calculating the disease index and the prevention and treatment effect. The severity classification standard of the pepper root rot disease is as follows: level 0: health care; stage I: the cortex of the root is discolored, and the cortex is not peeled off; and II, stage: xylem is browned and cortex is peeled off; grade III: the xylem becomes black and the cortex falls off; IV stage: the wood part is rotten and softened, and the cortex is peeled off.
As shown in fig. 5, the roots of the zanthoxylum bungeanum in the control group are seriously attacked, the cortex of the whole root is rotten and falls off, the xylem is blackened and softened, a bad odor is generated, and the disease index can reach 94.35; the disease part of the treated group skin is mainly positioned at the bottom end of the root or the tip end of the fibrous root, the disease does not occur at the middle upper part, the skin layer slightly falls off, the xylem is slightly browned, the disease index is 17.48, the prevention and treatment effect of the obtained Bacillus beilaisi strain P87 wettable powder on the root rot of the pepper is 81.47 percent, and the prevention and treatment effect is obvious.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Sequence table information:
DTD version V1_3
Filename ST26.Xml
Software name WIPO Sequence
Software version 2.1.1
Production date 2022-07-20
Basic information:
filing name of present application/applicant, northwest A & F University
Name or name of applicant, northwest university of agriculture and forestry
Zh name or name/language of applicant
Applicant's name or name/Latin name Northwest A & F University
The invention discloses a Bacillus beleisi strain P87 and application thereof in pepper disease control (zh)
Total sequence amount 8
The sequence is as follows:
serial number (ID): 1
Length of 1454
Molecular type DNA
Feature location/qualifier:
-source,1..1454
>mol_type,other DNA
(> organism), bacillus velezensis residue:
serial number (ID): 2
Length 1315
Molecular type DNA
Feature location/qualifier:
-source,1..1315
>mol_type,other DNA
>organism,Bacillus velezensis
residue:
serial number (ID): 3
Length of 392
Molecular type DNA
Feature location/qualifier:
-source,1..392
>mol_type,other DNA
>organism,Bacillus velezensis
residue:
serial number (ID): 4
Length of 556%
Molecular type DNA
Feature location/qualifier:
-source,1..556
>mol_type,other DNA
>organism,Bacillus velezensis
residue:
serial number (ID): 5
Length: 809
Molecular type DNA
Feature location/qualifier:
-source,1..809
>mol_type,other DNA
>organism,Bacillus velezensis
residue:
serial number (ID): 6
Length of 263
Molecular type DNA
Feature location/qualifier:
-source,1..263
>mol_type,other DNA
>organism,Bacillus velezensis
residue:
serial number (ID): 7
Length of 337
Molecular type DNA
Feature location/qualifier:
-source,1..337
>mol_type,other DNA
>organism,Bacillus velezensis
residue (b):
serial number (ID): 8
Length of 798
Molecular type DNA
Feature location/qualifier:
-source,1..798
>mol_type,other DNA
>organism,Bacillus velezensis
residue (b):
Claims (7)
1. the Bacillus subtilis P87 is characterized in that the strain is preserved in China center for type culture Collection at 6 months and 7 days in 2022, and the preservation number is CCTCCNO: M2022819.
2. Use of the bacillus beiLeishiensis (Bacillusvelezensis) strain P87 according to claim 1 for antagonizing Rhizopus zanthoxyli, rhizopus nigricans, rhizoctonia solani and Laternaria deciduous.
3. A biological agent for controlling pepper diseases, characterized in that the effective component of the biological agent is the Bacillus belgii strain P87 of claim 1.
4. The biological agent according to claim 3, characterized in that it is a wettable powder prepared from a fermentation broth of Bacillus belgii strain P87.
5. The biological agent according to claim 4, wherein the fermentation broth is prepared by the following steps:
activating a Bacillus belgii strain P87, culturing in a fermentation medium containing 18g of glucose, 30g of sucrose, 10g of peptone, 5g of sodium chloride and 1L of water, and stirring and culturing at 25-29 ℃ and 100-150 rpm for 4-6 days to obtain the fermentation liquid.
6. The biological preparation according to claim 4, wherein the wettable powder has a formulation of: 25g/L of white carbon black as a carrier, 0.75g/L of PEG80000.5g/L of wetting agent, 0.75g/L of sodium tripolyphosphate as a dispersing agent, 0.025g/L of sodium dodecyl sulfate as a protective agent, and the spore content of the carrier is more than or equal to 1 multiplied by 10 8 The suspension percentage is 90.52 percent, the wetting time is 43.6s, the pH value is 7.1, the fineness is 98 percent, and the dry weight loss is 0.9 percent.
7. The use of the biological agent according to any one of claims 3 to 6 for the control of pepper root rot.
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| INÉS MARTÍNEZ-RAUDALES等: "Draft genome sequence of Bacillus velezensis 2A-2B strain: a rhizospheric inhabitant of Sporobolus airoides (Torr.) Torr., with antifungal activity against root rot causing phytopathogens", STANDARDS IN GENOMIC SCIENCES, 5 December 2017 (2017-12-05), pages 1 - 10 * |
| 田凤鸣等: "一株花椒根腐病拮抗菌的分离鉴定及全基因组序列分析", 微生物学通报, 9 May 2022 (2022-05-09), pages 3205 - 3219 * |
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