CN109880778B - Composite microbial inoculum with growth promoting and yield increasing effects on capsicum and application thereof - Google Patents
Composite microbial inoculum with growth promoting and yield increasing effects on capsicum and application thereof Download PDFInfo
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Abstract
The invention discloses a compound microbial inoculum with growth promoting and yield increasing effects on hot pepper and application thereof. The composite microbial inoculum comprises fermentation liquor of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains; the Bacillus flexus HGD12 is Bacillus flexus, is preserved in China center for type culture Collection in 2019, 4 months and 1 days, and has the preservation number of CCTCC NO: M2019224; the Pseudomonas putida HGD3 is Pseudomonas putida, is preserved in China Center for Type Culture Collection (CCTCC) at 1 month 4 in 2019, and has a preservation number of CCTCC NO: M2019223; the Bacillus velezensis HP9 is Bacillus belgii, is preserved in China center for type culture Collection in 2019, 4 and 8 months, and has the preservation number of CCTCC NO: M2019243. The invention has the advantages of promoting the growth of the pepper, improving the pepper yield, reducing the application of chemical fertilizer, and achieving the effects of reducing the amount of the chemical fertilizer and increasing the pepper yield.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a microbial compound inoculant for promoting growth and yield increase of capsicum and application thereof.
Background
China is the first major pepper producing and consuming countries in the world, the annual sowing area is about 133.3 hectares, but blind fertilization and over-fertilization phenomena commonly exist in pepper planting, soil hardening and fertility reduction are caused by application of a large amount of chemical fertilizers, and finally the yield and quality of peppers are reduced, so that the sustainable development of agriculture is influenced.
The bacterial strain in the microbial agent is derived from beneficial microorganisms in soil or plant materials, is non-toxic and harmless to the environment, can improve the soil and increase the organic matter content of the soil after being applied, and can also promote the growth and development of crops, thereby achieving the effects of increasing the yield and improving the quality. When the fertilizer is matched with the chemical fertilizer, the excessive use of the chemical fertilizer can be reduced, and the ecological environment can be better protected.
At present, few reports are reported on the research and application of the special compound bactericide for the pepper. Patent CN108130289A discloses the preparation of a special compound fungicide for hot pepper consisting of 7 bacillus strains and 1 pseudomonas strain and the effect of the special compound fungicide in field application of big pepper. Patent CN107418906A discloses the use of a complex microbial inoculum consisting of 3 lactobacillus strains for improving the yield of color peppers and resisting root rot. But the yield increase effect is performed under the conventional fertilizer application amount, and only the field test of the compound microbial inoculum on 1 pepper variety is performed.
Disclosure of Invention
The invention aims to provide a compound microbial inoculum with growth promoting and yield increasing effects on capsicum and application thereof. The invention has the advantages of promoting the growth of the pepper, improving the yield of the pepper, reducing the application of chemical fertilizer and achieving the purposes of reducing the amount of the chemical fertilizer and increasing the yield of the pepper.
The technical scheme of the invention is as follows: a complex microbial inoculum with growth promoting and production increasing effects on capsicum and application thereof, comprises fermentation liquor of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains; the Bacillus flexus HGD12 is Bacillus flexus, is preserved in China center for type culture Collection in 2019, 4 and 1 months, and has the preservation number of CCTCC NO: M2019224; the Pseudomonas putida HGD3 is Pseudomonas putida, is preserved in China Center for Type Culture Collection (CCTCC) at 1 month 4 in 2019, and has a preservation number of CCTCC NO: M2019223; the Bacillus velezensis HP9 is Bacillus belgii, is preserved in China center for type culture Collection in 2019, 4 and 8 months, and has the preservation number of CCTCC NO: M2019243.
A method for preparing a compound microbial inoculum with growth promoting and yield increasing effects on Capsici fructus is provided, which is prepared according to the following method;
a. inclined plane activation: respectively inoculating Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains to a solid culture medium, and activating the strains overnight;
b. liquid activation: respectively picking the bacterial lawn of the activated strain in the step a into a liquid culture medium, and carrying out shaking culture on a shaking table at 150rpm to logarithmic phase at the temperature of 28-30 ℃;
c. obtaining fermentation liquor of each strain: respectively inoculating the culture solution of each strain in the logarithmic growth phase in the step b into a liquid culture medium according to the volume ratio of 5%, and performing shaking culture on a shaking table at 150rpm for 18-36h at the temperature of 28-30 ℃ to respectively obtain fermentation liquids of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains;
d. preparing a complex microbial inoculum: and c, uniformly mixing fermentation liquor of the Bacillus flexus HGD12, the Pseudomonas putida HGD3 and the Bacillus velezensis HP9 strains in the step c in equal proportion to obtain the composite microbial inoculum.
In the preparation method of the composite microbial inoculum having growth promoting and yield increasing effects on pepper, in the step a, the solid culture medium is an NA solid culture medium.
In the preparation method of the composite microbial inoculum with growth promoting and yield increasing effects on the capsicum, the NA solid culture medium contains 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 1000mL of distilled water and 15-20g of agar according to the following proportion; the NA solid medium has a pH of 7.0-7.2, and is sterilized at 121 deg.C for 20min before use.
In the preparation method of the composite microbial inoculum with growth promoting and yield increasing effects on the capsicum, in the steps b and c, the liquid culture medium is an NA liquid culture medium.
In the preparation method of the composite microbial inoculum having growth promoting and yield increasing effects on pepper, each liter of the NA liquid culture medium contains: 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride and 1000mL of distilled water; the NA liquid medium has a pH of 7.0-7.2, and is sterilized before use at 121 deg.C for 20 min.
In the preparation method of the compound microbial inoculum with growth promoting and yield increasing effects on the capsicum, in the step d, the compound microbial inoculum is addedThe total number of viable bacteria contained in the synthetic bacterium agent is 3 multiplied by 10 8 -3×10 9 CFU/mL.
A capsicum growth promoting microbial inoculum product comprises the composite microbial inoculum as an active ingredient of the product.
A capsicum growth promoting microbial inoculum product comprises the compound microbial inoculum as an active ingredient.
The capsicum growth promoting microbial inoculum product also comprises conventional components used for growth promoting microbial inoculum.
The invention also provides application of the compound microbial inoculum in the aspects of promoting pepper growth, improving pepper yield and reducing chemical fertilizer application, and the specific method is that the compound microbial inoculum is respectively applied in a pepper seedling stage (10 days after transplantation), a pepper flowering stage (30-40 days after colonization) and a pepper fruit setting stage (60-70 days after colonization), the dosage of each time is 2-4 liters per mu, the total number of viable bacteria is 3 multiplied by 10 8 -3×10 9 And (5) diluting the mixture by adding water between CFU/mL, and irrigating the mixture to roots of the peppers for 3 times in total.
Compared with the prior art, the composite microbial inoculum strain is derived from rhizosphere growth-promoting bacteria of tea garden rhizosphere soil, different strains have the growth-promoting characteristics of nitrogen fixation, phosphorus and potassium dissolution or ACC deaminase generation, and the strains have no antagonism, so that the growth of pepper can be promoted, the yield of pepper can be improved, and the purpose of reducing fertilizer can be achieved by mixed application.
In conclusion, the invention has the beneficial effects of promoting the growth of the pepper, improving the yield of the pepper, reducing the application of chemical fertilizer, and achieving the reduction of the chemical fertilizer and the yield increase of the pepper.
Drawings
FIG. 1 shows the effect of complex microbial inoculum on the height of a brilliant peppery 425 plant;
FIG. 2 shows the effect of the complex microbial inoculum on the stem thickness of the cayenne pepper 425;
FIG. 3 shows the effect of the complex microbial inoculum on the height of No. 10 Linzhu plants;
FIG. 4 shows the effect of the complex microbial inoculum on the thickness of spicy No. 10 stems;
FIG. 5 shows that the compound microbial inoculum promotes growth of root systems conforming to peppery No. 10 in field application.
Detailed Description
The invention is further described with reference to the following figures and examples, which are not to be construed as limiting the invention.
Example 1: molecular identification of 3 strains constituting complex microbial inoculum
Molecular identification of 3 strains: bacterial DNA is extracted from bacterial strains HGD12, HGD3 and HP9, the DNA is respectively used as a template, a bacterial 16S rDNA universal primer is adopted for PCR amplification, an amplification product is sequenced, the sequencing result of 3 bacterial strains is shown in a sequence table, and the amplification lengths of 16S rDNA sequences are 1417, 1442 and 1454bp respectively. Through homology comparison, the 16S rDNA sequence of the HGD12 strain has 99.93 percent of homology with the Bacillus flexus NBRC 1571516S rDNA sequence (NCBI accession number: BCVD01000224) in a GenBank database; the homology of the 16S rDNA sequence of the HGD3 strain and the Pseudomonas putida AA 716S rDNA sequence (NCBI accession number: CP018846) in a GenBank database reaches 99.86 percent; the HP9 strain 16S rDNA sequence has 99.86% homology with Bacillus velezensis CR502(Genbank accession number: AY603658) in GenBank database.
The sequence of the universal primer for amplifying the 16SrDNA is as follows:
27f:5'-AGAGTTTGATCMTGGCTCAG-3'，
1492r：5'-TACGGYTACCTTGTTACGACTT-3'
the PCR system is as follows: 1 × premix rTaq, 0.4. mu.M primer 27f, 0.4. mu.M primer 1492r, 50ng DNA.
The PCR program is as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 45sec, annealing at 55 ℃ for 45sec, extension at 72 ℃ for 90sec, 35 cycles; after 72 ℃ extension for 7 min.
According to the molecular identification results, the HGD12 strain is identified as Bacillus flexus, the HGD3 strain is identified as Pseudomonas putida, and the HP9 strain is identified as Bacillus velezensis.
Example 2: determination of growth promoting characteristics of 3 strains forming composite microbial inoculum
And (3) dissolved phosphorus quantitative determination: transferring the activated bacterial liquid into 100mL of NBRIP liquid culture medium, performing shaking culture at 30 ℃ for 7d, centrifuging the culture solution at 4 ℃ and 10000rpm for 10min, measuring the pH value, measuring the soluble phosphorus content in the supernatant by a molybdenum blue colorimetric method, and deducting the soluble phosphorus content in a blank control, namely the phosphorus dissolving amount of each strain.
Quantitative determination of potassium decomposition: transferring the activated bacterium liquid into 50mL Alexander doff liquid culture medium, shaking and culturing for 5d at 30 ℃, treating the culture liquid by adopting a hydrogen peroxide ashing method, measuring the content of water-soluble potassium in the supernatant by using an atomic absorption method, and deducting the content of soluble potassium in blank control, namely the potassium dissolving amount of each strain.
And (3) ACC-producing deaminase activity determination: activating and culturing the strain for 24h by 60mL of TSB liquid culture medium, centrifuging the culture solution at 8000rpm for 10min, washing the strain for 2 times by using DF culture solution, and then suspending the strain in 24mL of ADF culture medium for 24 h; centrifugally collecting the mycelia, washing with 0.1mol/L Tris-HCl buffer solution (pH7.6), and then suspending in 600. mu.L of 0.1mol/L Tris-HCl buffer solution (pH8.5); then 30 mul of toluene is added and the oscillation is carried out for 30sec to break the cells to obtain a crude enzyme solution; the method for measuring the mycoprotein concentration by a Bradford method and the alpha-ketobutyric acid content in the crude enzyme solution by a 2, 4-dinitrophenylhydrazine method comprises the following steps: under the reaction condition, the micromoles of ACC can be catalyzed to deaminate to form alpha-ketobutyric acid per milligram of mycoprotein per hour, and the unit is mu mol of alpha-ketobutyric acid/h.mg of protein.
And (3) measuring the content of the siderophore: the strain is inoculated in MKB culture medium, shaking culture is carried out for 2d, 3500rpm is carried out for 15min, supernatant fluid is taken, and the content of the siderophore is determined by adopting CAS method.
The formula of the culture medium is as follows:
NBRIP medium: 10g of glucose, 5g of tricalcium phosphate and MgCl 2 ·6H 2 O 5g，MgSO 4 ·7H 2 O 0.25g，KCl 0.2g，(NH 4 ) 2 SO 4 0.1g, 1000mL of distilled water, pH7.0, and sterilized at 115 ℃ for 30 min.
Alexander macroroff medium: sucrose 5g, CaCO 3 0.1g，Na 2 HPO 4 5g，MgSO 4 ·7H 2 0.5g of O, 1g of soil mineral, FeCl 3 0.005g, 1000mL of distilled water, pH7.0-7.5, and sterilizing at 121 ℃ for 20 min.
TSB medium: soybean peptone 3g, tryptone 17g, NaCl 5g, glucose 2.5g, K 2 HPO 4 2.5g, 1000mL of distilled water, pH7.1-7.5, and sterilizing at 115 ℃ for 30 min.
DF Medium: KH (Perkin Elmer) 2 PO 4 4g，Na 2 HPO 4 6g，MgSO 4 ·7H 2 0.2g of O, 2g of glucose, 2g of sodium gluconate, 2g of citric acid, (NH) 4 ) 2 SO 4 2g, 0.1mL of each of the first component solution and the second component solution, 1000mL of distilled water, pH7.2, and sterilization at 115 ℃ for 30 min. Wherein the component is H 3 BO 3 10mg，MnSO 4 ·H 2 O 11.19mg，ZnSO 4 ·7H 2 O 124.6mg，CuSO 4 ·5H 2 O 78.22mg，MoO 3 10mg, dissolved in 100mL of sterilized distilled water; the second component is FeSO 4 ·7H 2 O100 mg was dissolved in 10mL of sterilized distilled water.
ADF culture medium: ACC (1-aminocyclopropane-1-carboxylic acid) was dissolved in ultrapure water, filter-sterilized, and added to the solution containing No (NH) 4 ) 2 SO 4 The final concentration of the medium was 3.0 mmol/L.
MKB medium: casein amino acid 5g, Glycerol 15mL, K 2 HPO 4 2.5g,MgSO 4 ·7H 2 O2.5g, 1000mL of double distilled water, pH7.2, sterilization at 115 ℃ for 20 min.
The result of the growth promoting characteristic test shows that the phosphorus dissolving amount of the HGD12 strain is 218.88mg/L, the potassium dissolving amount is 2.63mg/L, and the secreted ACC deaminase activity is 0.27 mu mol alpha-ketobutyric acid/mg.h; the phosphorus dissolving amount of the HGD3 strain is 334.90mg/L, the potassium dissolving amount is 2.7mg/L, and the relative content of the generated siderophore is 21.20 percent; the phosphorus-dissolving amount of the HP9 strain is 149.30mg/L, the activity of ACC deaminase is 1.58 mu mol alpha-ketobutyric acid/mg.h, and the content of siderophore is 5.70%; all 3 strains had multiple growth promoting properties.
Example 3: preparation of composite microbial inoculum for promoting growth and increasing yield of pepper
The composite microbial inoculum for promoting growth and increasing yield of pepper comprises the following components: fermentation broth of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains.
The preparation method of the compound microbial inoculum for promoting growth and increasing yield of the pepper comprises the following steps:
a. bevel activation: respectively inoculating the strain preserved Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 to a solid culture medium, and activating the strain overnight;
b. liquid activation: respectively picking the bacterial lawn of the activated strain in the step a into a liquid culture medium, and carrying out shaking culture on a shaking table at 150rpm to logarithmic phase at the temperature of 28-30 ℃;
c. obtaining fermentation liquor of each strain: respectively inoculating the culture solution of each strain in the logarithmic growth phase in the step b into a liquid culture medium according to the volume ratio of 5%, shaking and culturing for 18-36h at the temperature of 28-30 ℃ by 150rpm of a shaking table to respectively obtain fermentation liquids of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains;
d. preparing a complex microbial inoculum: c, uniformly mixing the fermentation liquor of the Bacillus flexus HGD12, the Pseudomonas putida HGD3 and the Bacillus velezensis HP9 strains in the step c in equal proportion to obtain the composite microbial inoculum, wherein the total number of viable bacteria contained in the composite microbial inoculum is 3 multiplied by 10 8 -3×10 9 CFU/mL.
In the preparation method, the solid culture medium used in the slant culture in the step a is an NA solid culture medium; in the steps b and c, the liquid culture medium used is NA liquid culture medium.
In the preparation method, the NA solid culture medium contains 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 1000mL of distilled water and 15-20g of agar according to the following proportion; the NA solid medium has a pH of 7.0-7.2, and is sterilized at 121 deg.C for 20min before use.
In the above preparation method, the NA liquid medium contains per liter: 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride and 1000mL of distilled water; the NA liquid medium has a pH of 7.0-7.2, and is sterilized before use at 121 deg.C for 20 min.
Example 4: the field application effect of the compound microbial inoculum for promoting growth and increasing yield of the pepper
Test site: liquidambar formosana township village (106 degrees in east longitude 34 '23 degrees, 27 degrees in north latitude 35' 39 degrees, and 938m altitude) in Caucang city, Guizhou province, Zunyi city.
Test time: 2018.02-2018.10
Test crops: chili pepper
The test varieties are: yan jiao 425 Zun spicy No. 10
Test soil: cultivation soil
The test design adopts a random block design, 2 blocks are provided, each block is provided with 3 repeated cells, and the cell distance is 60 cm; 2 ridges are arranged in each cell, the width of the ridge surface of each ridge is 60cm, the interval between two ridges is 40cm, one ridge and two rows are adopted during planting, the planting distance is 30-40cm, and the area of each cell is 64m 2 . The pepper adopts a floating seedling raising mode, and is transplanted when 6-8 main leaves grow, the cultivation density is 3800-4000 pits/667 m 2 . The water management, the pest control and the like of each district are the same, and the management is carried out according to the local pepper cultivation mode.
This experiment was set up with two treatments: (1) the conventional fertilizer is applied as a control group, and the application amount is compound fertilizer (N-P) 2 O 5 -K 2 O, total nutrient is more than or equal to 45 percent) 60kg, organic fertilizer (total nutrient N + P) 2 O 5 +K 2 More than or equal to 5 percent of O, more than or equal to 45 percent of organic matter) and 100kg of diammonium phosphate, and is applied as a base fertilizer. (2) The compound microbial inoculum and 80% of chemical fertilizer are applied as a treatment group, the application amount of 80% of chemical fertilizer is 48 kg/mu of compound fertilizer, 16 kg/mu of diammonium phosphate, 80 kg/mu of organic fertilizer, and 80% of chemical fertilizer is applied as base fertilizer; the compound microbial inoculum is applied in the growth period of the pepper.
The application method of the compound microbial inoculum comprises the following steps: respectively applying the active ingredients in seedling stage (10 days after transplanting), initial flowering stage (30-40 days after colonization), and fruit setting stage (60-70 days after colonization) of Capsici fructus, wherein the dosage is 2-4L/mu, and the total number of viable bacteria is 3 × 10 8 -3×10 9 Between CFU/mL, diluted 25-fold with tap water and irrigated to the root a total of 3 times.
And (3) test results:
influence on plant height and stem thickness of cayenne pepper 425
As shown in figure 1, the plant heights of the cayenne pepper 425 are measured in the seedling stage, the flowering beginning stage and the fruit setting stage of the growing process respectively, and the plant heights of the compound microbial inoculum treatment group are respectively increased by 7.33%, 11.08% and 12.63% compared with the plant heights of the control group, and the plant heights of the cayenne pepper 425 are obviously increased after the root irrigation of the compound microbial inoculum (P is less than 0.05).
As shown in FIG. 2, the stem thickness of the cayenne pepper 425 at the seedling stage, the flowering beginning stage and the fruit setting stage is respectively measured, and the stem thickness of the cayenne pepper 425 is found to be increased by 11.86%, 22.14% and 22.14% respectively compared with the stem thickness of the control group in the composite microbial inoculum treatment group, and the stem thickness of the cayenne pepper 425 is obviously increased after the root irrigation of the composite microbial inoculum (P < 0.05).
As shown in Table 1, on the basis of reducing the fertilizer dosage by 20%, the effects on the yield, fruit bearing number and per mu yield of the capsicum annuum 425 per plant are respectively improved by 38.76%, 52.89% and 38.76% compared with a control group by inoculating the complex microbial inoculum, and the inoculation of the complex microbial inoculum remarkably promotes the yield, fruit bearing number and per mu yield of the capsicum annuum 425 per plant (P is less than 0.05).
TABLE 1 influence of the Complex microbial inoculum on the yield of cayenne pepper 425
Note: values were taken as the mean ± sd, with different lower case letters in the same column representing significant differences (P < 0.05).
Influence on plant height, stem thickness and root system development of Zunla No. 10
As shown in figure 3, the plant heights of the seedling stage, the flowering phase and the fruit setting stage of the growth of the No. 10 Zunoppon fungicide are respectively measured, the plant heights of the compound fungicide treatment group are respectively improved by 9.83%, 17.00% and 16.34% compared with the control group, and the plant height of the No. 10 Zunoppon fungicide is obviously improved after root irrigation (P is less than 0.05).
As shown in FIG. 4, the plant heights of the seedlings, the flowering phase and the fruit setting phase of the Zuno No. 10 following growth are respectively measured, and the stem thicknesses of the composite microbial inoculum treated group are respectively increased by 16.07%, 18.46% and 18.32% compared with those of the control group, so that the stem thickness of the Zanthoxylum piperitum 425 is remarkably increased after root irrigation by the composite microbial inoculum (P < 0.05).
As shown in fig. 5, after the harvest period is finished, the spicy 10 plants in different treatment groups are taken out, and the development conditions of the root systems are observed, so that the development of the root systems of the plants in the composite microbial inoculum treatment group is more vigorous, the roots are thicker, and the root hairs are more.
As shown in Table 2, on the basis of reducing the fertilizer dosage by 20% for the influence of the yield of the spicy No. 10 single plant, the fruit bearing number of the single plant and the yield per mu, the inoculation of the compound microbial inoculum groups is respectively improved by 22.10%, 7.24% and 22.10% compared with the control group, and the inoculation of the compound microbial inoculum remarkably promotes the yield of the spicy No. 10 single plant and the yield per mu (P < 0.05).
TABLE 2 Effect of Complex microbial inoculum on yield of Zunla No. 10
Note: values were taken as the mean ± sd, with different lower case letters in the same column representing significant differences (P < 0.05).
And (4) conclusion: the field application of the two capsicum varieties of gorgeous pepper 425 and Zunla No. 10 shows that the plant height and stem thickness of capsicum plants are obviously increased by applying the composite microbial inoculum on the premise of reducing the fertilizer dosage by 20 percent, the single plant yield, the single plant fruit bearing number and the per mu yield are improved, and the effects of reducing the fertilizer and increasing the pepper yield are achieved.
Sequence listing
<110> Guizhou university
<120> composite microbial inoculum with growth promoting and yield increasing effects on capsicum and application thereof
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agaactttcc agagatggat tggtgccttc gggaactttg acacaggtgc tgcatggctg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta acgagcgcaa cccttgtcct 1080
tagttaccag cacgtaatgg tgggcactct aaggagactg ccggtgacaa accggaggaa 1140
ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac gtgctacaat 1200
ggtcggtaca gagggttgcc aagccgcgag gtggagctaa tctcacaaaa ccgatcgtag 1260
tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca 1320
gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380
gggttgcacc agaagtagct agtctaacct tcgggaggac ggtaccacgg ttatactgcc 1440
gc 1442
<210> 3
<211> 1454
<212> DNA
<213> Bacillus beilaisi HP9(Bacillus velezensis)
<400> 3
aaatttgtca cttcggcggc tggctcctaa aggttacctc accgacttcg ggtgttacaa 60
actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 120
tgatccgcga ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa 180
ctgagaacag atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgccc 240
attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc 300
ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag 360
atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420
accatgcacc acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga 480
ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg 600
gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc 660
atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc 720
ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta 780
cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt 840
tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc 900
gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc 960
tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtgccgc cctatttgaa 1020
cggcacttgt tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc 1080
ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag 1140
gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat 1200
cgtcgccttg gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa 1260
gtggtagccg aagccacctt ttatgtctga accatgcggt tcaaacaacc atccggtatt 1320
agccccggtt tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc 1380
cgtccgccgc taacatcagg gagcaagctc ccatctgtcc gctcgacttg catgtatagc 1440
tgccccaatt ccac 1454
Claims (10)
1. A compound microbial inoculum with growth promoting and yield increasing effects on pepper is characterized in that: comprises thatBacillus flexusHGD12、Pseudomonas putidaHGD3 andBacillus velezensisfermentation broth of HP9 strain; the above-mentionedBacillus flexusHGD12 is Bacillus flexus, and is preserved in China center for type culture Collection in 2019, 4/1, with the preservation number of CCTCC NO: M2019224; the above-mentionedPseudomonas putidaHGD3 is Pseudomonas putida, which is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4/1, with the preservation number of CCTCC NO: M2019223; the describedBacillus velezensisHP9 is Bacillus belvesii, and is preserved in China center for type culture Collection in 2019, 4-8.M, with the preservation number of CCTCC NO: M2019243.
2. A method for preparing a compound microbial inoculum having growth promoting and yield increasing effects on capsicum annuum according to claim 1, which comprises the following steps: the preparation method comprises the following steps;
a. inclined plane activation: will be provided withBacillus flexus HGD12、Pseudomonas putidaHGD3 andBacillus velezensisrespectively inoculating HP9 strain to solid culture medium, activating strain overnight;
b. liquid activation: respectively picking the bacterial lawn of the activated strain in the step a into a liquid culture medium, and carrying out shaking culture on a shaking table at 150rpm to logarithmic phase at the temperature of 28-30 ℃;
c. obtaining fermentation liquor of each strain: respectively inoculating the culture solution of each strain in logarithmic growth phase in the step b into a liquid culture medium according to the volume ratio of 5%, and performing shaking culture on a shaking table at 150rpm for 18-36h at the temperature of 28-30 ℃ to obtainBacillus flexusHGD12、Pseudomonas putidaHGD3 andBacillus velezensisfermentation broth of HP9 strain;
d. preparing a complex microbial inoculum: in step cBacillus flexus HGD12、Pseudomonas putidaHGD3 andBacillus velezensisand uniformly mixing the fermentation liquor of the HP9 strain in equal proportion to obtain the composite microbial inoculum.
3. The method for preparing a compound microbial inoculum with growth promoting and production increasing effects on pepper as claimed in claim 2, wherein: in the step a, the solid culture medium is an NA solid culture medium.
4. The method for preparing a compound microbial inoculum with growth promoting and production increasing effects on pepper as claimed in claim 3, wherein: according to the following proportion, the NA solid medium contains 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 1000mL of distilled water and 15-20g of agar; the NA solid medium has a pH of 7.0-7.2 and is sterilized at 121 deg.C for 20min before use.
5. The method for preparing a compound microbial inoculum with growth promoting and production increasing effects on pepper as claimed in claim 2, wherein: in the steps b and c, the liquid culture medium is an NA liquid culture medium.
6. The method for preparing a compound microbial inoculum with growth promoting and production increasing effects on pepper as claimed in claim 5, wherein: the NA liquid culture medium contains per liter: 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride and 1000mL of distilled water; the NA liquid culture medium has pH of 7.0-7.2, and is sterilized at 121 deg.C for 20min before use.
7. The method for preparing the compound microbial inoculum with growth promoting and yield increasing effects on the capsicum according to claim 2, which is characterized in that: in the step d, the total number of viable bacteria contained in the composite microbial inoculum is 3 multiplied by 10 8 -3×10 9 CFU/mL.
8. A capsicum growth promoting microbial inoculum product is characterized in that: the active ingredient of the preparation comprises the complex microbial inoculum of claim 1.
9. A capsicum growth promoting microbial inoculum product is characterized in that: the active ingredient of the preparation is the complex microbial inoculum according to claim 1.
10. The pepper growth promoting microbial inoculum preparation as set forth in claim 8 or 9, wherein: also comprises conventional components for promoting the growth of bacteria.
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