CN107881129B - Bacillus amyloliquefaciens and microbial inoculum thereof, and preparation method and application of microbial inoculum - Google Patents
Bacillus amyloliquefaciens and microbial inoculum thereof, and preparation method and application of microbial inoculum Download PDFInfo
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Abstract
The invention relates to a bacillus amyloliquefaciens strain, a microbial inoculum thereof, a preparation method of the microbial inoculum and application of the microbial inoculum. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) AMCC100200 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14170. The microbial inoculum of the bacillus amyloliquefaciens is obtained by spray drying the bacterial liquid of the bacillus amyloliquefaciens AMCC 100200. The bacillus amyloliquefaciens AMCC100200 has strong inhibiting effect on various plant pathogenic bacteria. The field test result shows that after the treatment by the microbial inoculum of the bacillus amyloliquefaciens AMCC100200, the disease index of cucumber anthracnose of the plant is reduced by 74.41 percent, and the disease index of tomato root-knot nematode disease is reduced by 65 percent.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens, a microbial inoculum thereof, a preparation method of the microbial inoculum and application of the microbial inoculum.
Background
Plant diseases are one of the main factors influencing high and stable yield and high quality of agriculture, and pathogens of the plant diseases mainly comprise fungi, bacteria, nematodes, viruses and the like. For example, fusarium oxysporum is a soil-borne fungal disease, can cause blight of hundreds of plants such as melons, solanaceous plants and the like, is common in all parts of the country, generally has the disease rate of 10-30 percent, and can reach more than 50 percent in serious cases, and the blight becomes one of the most main factors for restricting melon production along with the gradual expansion of melon planting areas; root knot nematode disease is a disease which is caused by root knot nematodes and damages the roots of plants, and statistically, the annual loss caused by plant parasitic nematodes reaches $ 1000 billion, and the economic loss caused by the root knot nematodes reaches $ several billion; bacterial wilt is a destructive soil-borne disease caused by Laurella of Solanaceae, is widely distributed in tropical, subtropical and temperate regions, can cause wilting death of plants in a large area, has a disease rate of more than 80 percent in seriously ill fields, and seriously harms the growth of solanaceae crops.
Chemical control is an effective method for controlling plant diseases, has the advantages of quick effect, wide bactericidal spectrum, low cost, simple and convenient use and the like, but the long-term use of a large amount of chemical bactericides can cause environmental pollution such as soil, atmosphere and the like and destroy ecological balance. With the recent concern about food safety and environmental pollution, some chemical fungicides have been severely restricted in many developed countries and regions.
Biological control of plant diseases refers to a technique and method for effectively controlling plant diseases by using beneficial microorganisms or metabolites of microorganisms. The biological control has the characteristics of environmental friendliness, no residue, safety to people and livestock in the using process and the like, is gradually concerned by scholars at home and abroad, and with the continuous and deep research, the biological control method gradually replaces the traditional chemical control means and has wide application prospect. Although biological control is more and more emphasized, the number of microorganisms which can be practically applied to plant disease control is small, and microorganisms which are separated in a laboratory and have good biological control effect have the problems of low survival rate or incapability of survival in soil and the like due to environmental factors or strains in the application process, and the control effect is unstable.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bacillus amyloliquefaciens strain, a microbial inoculum thereof, a preparation method of the microbial inoculum and application of the microbial inoculum.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a broad-spectrum plant disease-resistant Bacillus amyloliquefaciens strain is disclosed, wherein Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) AMCC100200 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14170.
A microbial agent of the bacillus amyloliquefaciens: the microbial inoculum is obtained by spray drying of the bacterial liquid of the bacillus amyloliquefaciens AMCC 100200.
Further, the concentration of the bacterial liquid is 1.0 × 1010～1.2×1010CFU/mL。
Furthermore, the effective viable count of the microbial inoculum is 5.0 multiplied by 1010～1.0×1011CFU/g。
The preparation method of the microbial inoculum comprises the following steps:
s1, strain activation: streaking and inoculating the bacillus amyloliquefaciens AMCC100200 on an LB solid culture medium plate, and culturing for 24h at 37 ℃;
s2, preparing seed liquid: inoculating the activated bacillus amyloliquefaciens AMCC100200 into a triangular flask filled with LB liquid culture medium, and performing shaking culture in a constant temperature oscillator at 37 ℃ and 180rpm for 24 hours to obtain seed liquid;
s3, fermentation culture in a seed tank: inoculating the seed solution obtained in the step S2 into a 50L seed tank by an inoculation amount of 5-8%, wherein the liquid loading volume of a liquid culture medium in the seed tank is 30L, and culturing at 37 ℃ for 8h to obtain a fermentation culture;
s4, fermentation culture: inoculating the fermentation culture obtained in the step S3 into a 500L fermentation tank by an inoculation amount of 5-8%, wherein the liquid filling amount of a liquid culture medium in the fermentation tank is 350L, and culturing at 37 ℃ for 24h to obtain a fermentation liquid of the bacillus amyloliquefaciens AMCC 100200;
s5, concentrating the fermentation liquid obtained in the step S4 to obtain the bacterial liquid, and performing spray drying on the bacterial liquid to obtain the bacterial agent of the bacillus amyloliquefaciens AMCC 100200.
Further, LB solid medium preparation in said step S1The method comprises the following steps: 0.5 wt% of yeast extract, 1 wt% of peptone, 1 wt% of sodium chloride and 1.6 wt% of agar, wherein the LB liquid medium formula in the step S2 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone and 1 wt% of sodium chloride, wherein the pH values of the LB solid medium in the step S1 and the LB liquid medium in the step S2 are 7.0, and the raw materials are autoclaved at 121 ℃ for 20min before use; the liquid medium formulation used in the steps S3 and S4 includes: corn flour 2.88 wt%, bean cake powder 1.68 wt%, CaCO30.7 wt%, glucose 0.5 wt%, KH2PO4 0.5wt％，MgSO4·7H2O 0.02wt％，MnSO4·H20.02 wt% of O, and the pHs of the liquid culture media used in the steps S3 and S4 are 7.0-7.2; the temperature of the spray drying in the step S5 is 200 ℃ of inlet air and 80 ℃ of outlet air.
The application of the bacillus amyloliquefaciens AMCC100200 strain or the microbial inoculum in preventing and treating tomato root knot nematode disease.
The application of the bacillus amyloliquefaciens AMCC100200 strain or the microbial inoculum in preventing and treating cucumber anthracnose.
Further, the microbial inoculum is dissolved in water to form a bacterial solution, and is applied in furrows before the field planting of the tomatoes, wherein the using amount of the microbial inoculum is 10 g/mu.
Further, the microbial inoculum is dissolved in water to form a bacterial liquid, and foliage spraying is carried out on plants, wherein the using amount of the microbial inoculum is 1 kg/mu, and the concentration of the bacterial liquid is 500 hundred million CFU/mL.
The invention has the beneficial effects that:
(1) the bacillus amyloliquefaciens AMCC100200 has strong inhibiting effect on various plant pathogenic bacteria, such as: the inhibition rate of the compound preparation on cucumber anthracnose pathogenic bacteria is 73.78%, the inhibition rate on wheat sharp eyespot pathogenic bacteria is 70.22%, the inhibition rate on asparagus rolling eyespot pathogenic bacteria is 50.02%, the inhibition rate on apple ring rot pathogenic bacteria is 74.20%, the inhibition rate on wheat root rot pathogenic bacteria is 57.34%, the inhibition rate on fusarium oxysporum is 62.80%, the inhibition rate on fusarium oxysporum is 61.31%, the inhibition rate on fusarium moniliforme is 57.87%, and the inhibition rate on fusarium solani is 71.41%. In addition, the strain has a strong killing effect on second-instar larvae of the root-knot nematodes, and the lethality of the strain can reach 90% after the 48-hour fermentation broth is used for treating the second-instar larvae of the root-knot nematodes for 24 hours.
(2) The field test result shows that after the treatment by the microbial inoculum of the bacillus amyloliquefaciens AMCC100200, the disease index of cucumber anthracnose of the plant is reduced by 74.41 percent, and the disease index of tomato root-knot nematode disease is reduced by 65 percent.
Drawings
FIG. 1 is the colony morphology of Bacillus amyloliquefaciens AMCC100200 strain.
FIG. 2 is a microscopic morphology of the Bacillus amyloliquefaciens AMCC100200 strain.
FIG. 3 shows the antagonistic effect of Bacillus amyloliquefaciens AMCC100200 on cucumber anthracnose pathogen.
FIG. 4 shows the antagonistic action of Bacillus amyloliquefaciens AMCC100200 on the pathogenic bacteria of wheat sharp eyespot.
FIG. 5 shows the antagonistic effect of Bacillus amyloliquefaciens AMCC100200 on apple ring rot pathogen.
FIG. 6 shows the antagonistic effect of Bacillus amyloliquefaciens AMCC100200 on wheat root rot pathogens.
FIG. 7 is the antagonism of Bacillus amyloliquefaciens AMCC100200 against Fusarium oxysporum.
FIG. 8 is the antagonism of Bacillus amyloliquefaciens AMCC100200 against Fusarium heterothecioides.
FIG. 9 shows the antagonistic effect of Bacillus amyloliquefaciens AMCC100200 on Fusarium moniliforme.
FIG. 10 is the antagonism of Bacillus amyloliquefaciens AMCC100200 against Fusarium solani.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 isolation, identification and preservation of the AMCC100200 Strain
1.1 isolation of the AMCC100200 Strain
Screening a broad-spectrum plant disease-resistant strain from plant rhizosphere soil, and naming the strain as AMCC100200, wherein the specific separation method comprises the following steps:
10g of plant rhizosphere soil is taken and filled into a 250mL conical flask filled with sterile glass beads and 90mL of sterile water, and the mixture is fully shaken up. The conical flask after shaking is placed in a shaker at 37 ℃ and shaken at 180rpm for 30 min. Taking out, and performing gradient dilution on a superclean bench to obtain 10-3Gradient, 10-4Gradient, 10-5Gradient, 10-6Gradient sum 10-7A gradient of dilutions. The dilutions of the gradients were spread on LB medium plates, 6 dilutions were set in parallel, sealed with sealing films, and cultured in 37 ℃ incubator. And observing the growth condition after 24h, and classifying according to the characteristics of colony size, shape, color and the like. And selecting single colonies with differences by using an inoculating loop, streaking the single colonies on an LB medium flat plate for purification, removing repeated strains according to the characteristics of colonies, selecting the obtained strains, transferring the strains to an LB medium inclined plane, and preserving at 4 ℃.
1.2 identification of the AMCC100200 Strain
The colony morphology of the AMCC100200 strain is shown in fig. 1.
The microscopic morphology of the AMCC100200 strain is shown in fig. 2.
The colonies of the AMCC100200 strain are light yellow, opaque, irregular in edges and rough in surface. And (5) gram staining microscopic observation shows that the cells are positive, rod-shaped and oval in spore shape. The bacillus amyloliquefaciens is positive in nitrate reduction experiments, gelatin hydrolysis and starch hydrolysis, negative in V-P experiments, citrate utilization experiments, oxidase experiments and indole production experiments, and has the same characteristics as bacillus amyloliquefaciens model species.
Through identification, the AMCC100200 strain belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The 16SrDNA sequence of the AMCC100200 strain is shown as a sequence 1 in a sequence table.
1.3 deposition of AMCC100200 Strain
The AMCC100200 strain is preserved in the China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu No.1 Beijing, Chaoyang district) in 5 and 17 months in 2017, and the preservation number is CGMCC No. 14170.
Example 2 preparation of microbial inoculum
Example 2-1
S1, strain activation: streaking and inoculating the bacillus amyloliquefaciens AMCC100200 on an LB solid culture medium plate, and culturing for 24h at 37 ℃;
s2, preparing seed liquid: inoculating the activated bacillus amyloliquefaciens AMCC100200 into a triangular flask filled with LB liquid culture medium in an inoculation amount of 5%, and performing shaking culture in a constant temperature oscillator at 37 ℃ and 180rpm for 24 hours to obtain seed liquid;
s3, fermentation culture in a seed tank: inoculating the seed liquid obtained in the step S2 into a 50L seed tank at an inoculation amount of 5%, wherein the liquid loading capacity of a liquid culture medium in the seed tank is 30L, and culturing at 37 ℃ for 8h to obtain a fermentation culture;
s4, fermentation culture: inoculating the fermentation culture obtained in the step S3 into a 500L fermentation tank by an inoculation amount of 5%, wherein the liquid loading capacity of a liquid culture medium in the fermentation tank is 350L, and culturing at 37 ℃ for 24h to obtain a fermentation liquid of the bacillus amyloliquefaciens AMCC 100200;
s5, concentrating the fermentation liquid obtained in the step S4 to obtain the bacterial liquid, and performing spray drying on the bacterial liquid to obtain the bacterial agent of the bacillus amyloliquefaciens AMCC 100200.
The LB medium formula in the steps S1 and S2 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone, 1 wt% of sodium chloride and 1.6 wt% of agar, wherein the LB culture medium pH7.0 in the steps S1 and S2 is autoclaved at 121 ℃ for 20min before use; the liquid medium formulation used in the steps S3 and S4 includes: corn flour 2.88 wt%, bean cake powder 1.68 wt%, CaCO30.7 wt%, glucose 0.5 wt%, KH2PO4 0.5wt％，MgSO4·7H2O 0.02wt％，MnSO4·H20.02 wt% of O, and the pHs of the liquid culture media used in the steps S3 and S4 are 7.0-7.2; the temperature of the spray drying in the step S5 is 200 ℃ of inlet air and 80 ℃ of outlet air.
Examples 2 to 2
S1, strain activation: streaking and inoculating the bacillus amyloliquefaciens AMCC100200 on an LB solid culture medium plate, and culturing for 24h at 37 ℃;
s2, preparing seed liquid: inoculating the activated bacillus amyloliquefaciens AMCC100200 into a triangular flask filled with LB liquid culture medium, and performing shaking culture in a constant temperature oscillator at 37 ℃ and 180rpm for 24 hours to obtain seed liquid;
s3, fermentation culture in a seed tank: inoculating the seed liquid in the step S2 into a 50L seed tank at an inoculation amount of 8%, wherein the liquid loading volume of a liquid culture medium in the seed tank is 30L, and culturing at 37 ℃ for 8h to obtain a fermentation culture;
s4, fermentation culture: inoculating the fermentation culture obtained in the step S3 into a 500L fermentation tank by 8% of inoculation amount, wherein the liquid loading capacity of a liquid culture medium in the fermentation tank is 350L, and culturing at 37 ℃ for 24h to obtain a fermentation liquid of the bacillus amyloliquefaciens AMCC 100200;
s5, concentrating the fermentation liquid obtained in the step S4 to obtain the bacterial liquid, and performing spray drying on the bacterial liquid to obtain the bacterial agent of the bacillus amyloliquefaciens AMCC 100200.
The LB medium formula in the steps S1 and S2 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone, 1 wt% of sodium chloride and 1.6 wt% of agar, wherein the LB culture medium pH7.0 in the steps S1 and S2 is autoclaved at 121 ℃ for 20min before use; the liquid medium formulation used in the steps S3 and S4 includes: corn flour 2.88 wt%, bean cake powder 1.68 wt%, CaCO30.7 wt%, glucose 0.5 wt%, KH2PO4 0.5wt％，MgSO4·7H2O 0.02wt％，MnSO4·H20.02 wt% of O, and the pHs of the liquid culture media used in the steps S3 and S4 are 7.0-7.2; the temperature of the spray drying in the step S5 is 200 ℃ of inlet air and 80 ℃ of outlet air.
Examples 2 to 3
S1, strain activation: streaking and inoculating the bacillus amyloliquefaciens AMCC100200 on an LB solid culture medium plate, and culturing for 24h at 37 ℃;
s2, preparing seed liquid: inoculating the activated bacillus amyloliquefaciens AMCC100200 into a triangular flask filled with LB liquid culture medium, and performing shaking culture in a constant temperature oscillator at 37 ℃ and 180rpm for 24 hours to obtain seed liquid;
s3, fermentation culture in a seed tank: inoculating the seed liquid in the step S2 into a 50L seed tank at an inoculation amount of 6.5%, wherein the liquid culture medium liquid loading amount in the seed tank is 30L, and culturing at 37 ℃ for 8h to obtain a fermentation culture;
s4, fermentation culture: inoculating the fermentation culture obtained in the step S3 into a 500L fermentation tank by 6.5% of inoculation amount, wherein the liquid filling amount of a liquid culture medium in the fermentation tank is 350L, and culturing at 37 ℃ for 24h to obtain a fermentation liquid of the bacillus amyloliquefaciens AMCC 100200;
s5, concentrating the fermentation liquid obtained in the step S4 to obtain the bacterial liquid, and performing spray drying on the bacterial liquid to obtain the bacterial agent of the bacillus amyloliquefaciens AMCC 100200.
The LB medium formula in the steps S1 and S2 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone, 1 wt% of sodium chloride and 1.6 wt% of agar, wherein the LB culture medium pH7.0 in the steps S1 and S2 is autoclaved at 121 ℃ for 20min before use; the liquid medium formulation used in the steps S3 and S4 includes: corn flour 2.88 wt%, bean cake powder 1.68 wt%, CaCO30.7 wt%, glucose 0.5 wt%, KH2PO4 0.5wt％，MgSO4·7H2O 0.02wt％，MnSO4·H20.02 wt% of O, and the pHs of the liquid culture media used in the steps S3 and S4 are 7.0-7.2; the temperature of the spray drying in the step S5 is 200 ℃ of inlet air and 80 ℃ of outlet air.
Example 3 determination of broad-spectrum plant disease resistance of Bacillus amyloliquefaciens strain AMCC100200
3.1 determination of ability of Bacillus amyloliquefaciens AMCC100200 to antagonize fungal diseases
Activated bacteria and hypha blocks of pathogenic fungi are picked by inoculating loops and inoculated into a culture dish plate (the diameter of the culture dish is 9cm, the distance between the two is 5cm, and the two are repeated for 3 times) at the same time respectively. And then, respectively taking pure culture of the bacteria and the pathogenic fungi on a culture dish plate as a control, culturing at constant temperature, and observing the growth of bacterial colonies and the inhibition of the bacteria day by day. And continuously observing, measuring the diameter of the bacterial colony of the treated pathogenic bacteria when the hypha of the control strain grows over the culture dish, and calculating the bacteriostasis rate. Inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100 (%).
The inhibitory effect of Bacillus amyloliquefaciens AMCC100200 on various pathogenic fungi is shown in FIGS. 2-10, in which the control colonies are present in the left side of the plate and the treatment colonies are present in the right side of the plate.
The calculation result shows that: the bacillus amyloliquefaciens AMCC100200 has strong inhibiting effect on various plant pathogenic bacteria, has the inhibiting rate of 73.78 percent on cucumber anthracnose pathogenic bacteria, 70.22 percent on wheat sheath blight pathogenic bacteria, 50.02 percent on asparagus roll blight pathogenic bacteria, 74.20 percent on apple ring rot pathogenic bacteria, 57.34 percent on wheat root rot pathogenic bacteria, 62.80 percent on fusarium oxysporum, 61.31 percent on fusarium oxysporum, 57.87 percent on fusarium moniliforme and 71.41 percent on fusarium solani.
3.2 determination of nematicidal ability of Bacillus amyloliquefaciens AMCC100200
Activating the strain according to the method, inoculating the activated bacillus amyloliquefaciens AMCC100200 seed liquid into an LB liquid culture medium (the composition and the quality of the culture medium are 10g of peptone, 10g of NaCl, 10g of NaCl, 5g of yeast extract, 1000mL of distilled water, pH7.0, sterilizing at 121 ℃ for 20min), and performing shake culture at 37 ℃ and 180r/min to obtain the fermentation liquid. 0.2mL (about 100) of nematode suspension and 0.8mL of 48-hour fermentation liquid are added into a 1.5mL centrifuge tube, after the mixture is cultured for 24 hours at 28 ℃, 0.8mL of supernatant is carefully absorbed, 0.2mL of NaOH solution with the concentration of 1mol/L is added and mixed evenly, and the mixture is immediately observed under a microscope to calculate the mortality of the nematodes. Judging the dead and alive nematode standard: enabling the nematodes to be active and bent, and adding NaOH solution to the nematodes to bend the nematodes, wherein the nematodes are alive; the nematodes are dead after the nematodes are still stiff and the nematodes are dead after the NaOH solution is added.
The experimental results show that: the strain has a strong killing effect on second-instar larvae of the root-knot nematodes, and the lethality of the strain can reach 90% after the 48-hour fermentation broth is used for treating the second-instar larvae of the root-knot nematodes for 24 hours.
Example 4 control Effect verification experiment
4.1 cucumber anthracnose field plot experiment
In 2015, field experiments were carried out in a cucumber greenhouse from Linyi city of Shandong province to Hezhuangcun city of Town in 5-7 months. Experiment shedSevere cucumber anthracnose has occurred in the past year. The cucumber variety is Zhongnong No. 8, no bactericide is used in the test period, and 3 treatment groups are arranged in the test: (1) dissolving an AMCC100200 microbial inoculum in water, and then spraying the leaves of the leaves by using a manual sprayer, wherein the concentration of the AMCC100200 microbial inoculum after being dissolved in water is 500 hundred million CFU/mL, and the usage amount of the AMCC100200 microbial inoculum is as follows: 1 kg/mu; (2) dissolving 50% prochloraz wettable powder in water, and then spraying the solution on the leaf surface by using a manual sprayer, wherein the 50% prochloraz wettable powder has the dilution multiple: 500 times, using amount: 25 g/mu); (3) and (5) clear water control. Each cell is 30m2The method is characterized in that each cell is provided with 60 cucumbers, each treatment group is provided with 3 repeated groups, the cells are randomly distributed, spraying is started at the early stage of cucumber anthracnose attack, and then spraying is carried out every other week for 3 times. The test and evaluation were carried out according to the relevant GB/T17980.112-2004 standard. Each cell randomly selects 15 plants for investigation, and each plant investigates 10 leaves from bottom to top. Disease base is investigated before application, disease index is investigated 14 days after 3 times of application, and prevention and treatment effects are calculated.
TABLE 1 prevention and treatment effect of AMCC100200 bacterial agent on cucumber anthracnose
From the results in Table 1, it can be seen that: the AMCC100200 microbial inoculum has good control effect on cucumber anthracnose after being dissolved in water, the control effect can reach 74.41 percent, and the control effect of the control medicament 50 percent prochloraz wettable powder on the cucumber anthracnose can reach 85.94 percent.
4.2 tomato root knot nematode disease field plot test
The test is carried out in a plastic vegetable greenhouse in Dingzhuang village and village in Ling county of Texas province, Shandong province. Tomato and fennel crop rotation is carried out for years, and tomato root-knot nematode disease is serious. The experiment had 3 treatment groups: (1) performing bottom application treatment on the AMCC100200 microbial inoculum, ditching and applying the bottom application treatment before planting tomatoes, wherein the usage amount of the AMCC100200 microbial inoculum is as follows: 10 kg/mu; (2) blank group treatment: no treatment is carried out; (3) chemical pesticide treatment: the abamectin 2000-time diluent is used for root irrigation treatment during tomato transplanting, the soil is covered after the tomato is planted, and the using amount of the abamectin is as follows: 2 kg/mu.Each treatment group was provided with 3 repeating subgroups, each subgroup having a treatment area of about 27m2(3.6m multiplied by 7.5m) comprising 3 ridges, 6 rows in total, and 121 tomato plants. And (4) counting indexes such as root knot indexes, insect population density and the like after harvesting the tomatoes.
TABLE 2 influence of Bacillus amyloliquefaciens AMCC100200 on Meloidogyne
The AMCC100200 microbial inoculum has good prevention and treatment effect on the tomato root-knot nematode after being dissolved in water, and compared with a blank group, the root knot number is reduced by 68.06%, the oocyst number is reduced by 92.01%, and the density of insect population is reduced by 62.40%, which shows that the AMCC100200 microbial inoculum has strong inhibition effect on the generation and development of the root-knot nematode.
Sequence listing
<110> Anhui six nations chemical industry, Inc., Shandong university of agriculture
<120> bacillus amyloliquefaciens strain, microbial inoculum thereof, preparation method of microbial inoculum and application of microbial inoculum
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agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840
ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960
gacaatccta gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggttgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080
agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200
cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260
ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380
taacacccga agtcggtgag gtaacctttt aggagccagc cgccga 1426
Claims (10)
1. A strain of Bacillus amyloliquefaciens is characterized in that: the Bacillus amyloliquefaciens (A), (B), (C) and (C)Bacillus amyloliquefaciens) AMCC100200 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 14170.
2. A bacterial preparation of the Bacillus amyloliquefaciens strain of claim 1: the method is characterized in that: the microbial inoculum is obtained by spray drying of the bacterial liquid of the bacillus amyloliquefaciens AMCC 100200.
3. The bacterial agent of bacillus amyloliquefaciens of claim 2: the method is characterized in that: the concentration of the bacterial liquid is 1.0 multiplied by 1010～1.2×1010CFU/mL。
4. The bacterial agent of bacillus amyloliquefaciens of claim 2: the method is characterized in that: the effective viable count of the microbial inoculum is 5.0 multiplied by 1010～1.0×1011CFU/g。
5. A method for preparing the microbial agent according to claim 2, comprising the steps of:
s1, strain activation: streaking and inoculating the bacillus amyloliquefaciens AMCC100200 on an LB solid culture medium plate, and culturing for 24h at 37 ℃;
s2, preparing seed liquid: inoculating the activated bacillus amyloliquefaciens AMCC100200 into a triangular flask filled with LB liquid culture medium, and performing shaking culture in a constant temperature oscillator at 37 ℃ and 180rpm for 24 hours to obtain seed liquid;
s3, fermentation culture in a seed tank: inoculating the seed solution obtained in the step S2 into a 50L seed tank by an inoculation amount of 5-8%, wherein the liquid loading volume of a liquid culture medium in the seed tank is 30L, and culturing at 37 ℃ for 8h to obtain a fermentation culture;
s4, fermentation culture: inoculating the fermentation culture obtained in the step S3 into a 500L fermentation tank by an inoculation amount of 5-8%, wherein the liquid filling amount of a liquid culture medium in the fermentation tank is 350L, and culturing at 37 ℃ for 24h to obtain a fermentation liquid of the bacillus amyloliquefaciens AMCC 100200;
s5, concentrating the fermentation liquid obtained in the step S4 to obtain the bacterial liquid, and performing spray drying on the bacterial liquid to obtain the bacterial agent of the bacillus amyloliquefaciens AMCC 100200.
6. The method for producing the microbial agent according to claim 5, wherein: the LB solid medium formula in the step S1 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone, 1 wt% of sodium chloride and 1.6 wt% of agar, wherein the LB liquid medium formula in the step S2 comprises: 0.5 wt% of yeast extract, 1 wt% of peptone and 1 wt% of sodium chloride, wherein the pH values of the LB solid medium in the step S1 and the LB liquid medium in the step S2 are 7.0, and the raw materials are autoclaved at 121 ℃ for 20min before use; the liquid medium formulation used in the steps S3 and S4 includes: corn flour 2.88 wt%, bean cake powder 1.68 wt%, CaCO30.7 wt%, glucose 0.5 wt%, KH2PO4 0.5wt％，MgSO4·7H2O 0.02wt％，MnSO4·H20.02 wt% of O, and the pHs of the liquid culture media used in the steps S3 and S4 are 7.0-7.2; the temperature of the spray drying in the step S5 is 200 ℃ of inlet air and 80 ℃ of outlet air.
7. The application of the bacillus amyloliquefaciens AMCC100200 strain of claim 1 or the microbial inoculum of claim 2 in controlling tomato root knot nematode disease.
8. The use of the bacillus amyloliquefaciens AMCC100200 strain of claim 1 or the bacterial agent of claim 2 for controlling cucumber anthracnose.
9. The use of the bacterial agent of claim 7 for controlling tomato root knot nematode disease, characterized in that: and (3) dissolving the microbial inoculum into water to form a bacterial solution, and ditching and applying the bacterial solution before the field planting of the tomatoes, wherein the using amount of the microbial inoculum is 10 kg/mu.
10. The use of the bacterial agent of claim 8 for controlling cucumber anthracnose, characterized in that: and dissolving the microbial inoculum into water to form a bacterial liquid, and spraying the bacterial liquid to the leaf surfaces of plants, wherein the using amount of the microbial inoculum is 1 kg/mu, and the concentration of the bacterial liquid is 500 hundred million CFU/mL.
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| CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
| CN110951656A (en) * | 2020-01-02 | 2020-04-03 | 山东农业大学 | Acetobacter and application thereof in preventing and treating plant parasitic nematode disease |
| CN111440743B (en) * | 2020-04-09 | 2022-05-27 | 山东农业大学 | Bacillus amyloliquefaciens PEBA20 capable of preventing diseases, promoting growth and improving soil and application thereof |
| WO2022036551A1 (en) * | 2020-08-18 | 2022-02-24 | 江苏滋百农生态农业股份有限公司 | Bacillus altitudinis strain and use of active substance compound liquid and microbial inoculant thereof in prevention and treatment of root-knot nematode diseases |
| CN112694990B (en) * | 2020-12-25 | 2022-10-04 | 中农绿康（北京）生物技术有限公司 | Bacillus amyloliquefaciens and application thereof in tomato nematode disease control |
| CN113966747B (en) * | 2021-06-30 | 2022-04-29 | 中国热带农业科学院环境与植物保护研究所 | Nano-silver composite bacillus agent for preventing and treating syzygium samarangense fruit rot and anthracnose of syzygium samarangense fruit and preparation method thereof |
| CN115125159B (en) * | 2022-04-24 | 2023-05-23 | 沈阳农业大学 | Bacillus amyloliquefaciens, microbial inoculum thereof, preparation method and application of microbial inoculum |
| CN115197869A (en) * | 2022-05-19 | 2022-10-18 | 广东海洋大学 | Bacillus amyloliquefaciens ZJLMBA1908 with broad-spectrum antibacterial activity and application thereof |
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