CN113773981B - Biocontrol bacterium for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage - Google Patents
Biocontrol bacterium for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
Biocontrol bacteria for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage, biocontrol bacteriaPan. vagansBWL1 was preserved in China Center for Type Culture Collection (CCTCC) for type 1 and 4 days 2021, and the preservation number is CCTCC NO: m2021001. The invention screens out a strain of biocontrol bacteriaPan. vagansBWL1 can antagonize penicillium and gray mold with high efficiency.
Description
Technical Field
The invention relates to a biocontrol strain for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage, belonging to the technical field of agricultural product storage.
Background
The original production of the kiwi fruits is called as the vitamin C with extremely high content and the fruit is called as the vitamin C, and the kiwi fruits are fragrant in pulp, rich in water and sweet and sour in taste, and are popular with people. However, kiwi fruits are extremely susceptible to pathogen infection during postharvest storage, and the loss of the kiwi fruits caused by postharvest rot is up to 20-30% in the world according to statistics, so that huge economic loss is brought to the kiwi fruit industry, and the rapid development of the industry is greatly limited.
The fungus diseases causing the rot of the picked kiwi fruits mainly comprise gray mold, blue mold and the like. Gray mold (Botrytis cinerea) is the most important postharvest disease on kiwi fruits, the invasion modes of the Botrytis cinerea are various, mycelium, spores or sclerotium can survive for a long time in unfavorable environments, and the kiwi fruits grow rapidly when meeting the proper environments. The penicillium expansum (Penicillium expansum) infection causes the rot of kiwi fruits, and patulin toxic to human bodies is produced, so that the food safety is endangered.
At present, the traditional method for storing and preserving the picked kiwi fruits mainly comprises low-temperature storage, and the respiration of the fruits is reduced at low temperature, so that the kiwi fruits are in a dormant state, the rotting of the kiwi fruits is reduced, and the preservation period is prolonged. However, penicillium and botrytis spores can survive for a long time at a temperature above the freezing point, so that the problem of penicillium and botrytis pollution after kiwi fruit picking cannot be solved by low-temperature storage. Chemical bacteriostats such as 1-methylcyclopropene (1-MCP) and the like are widely applied to the storage and preservation of kiwi fruits, and the storage time and shelf life of the kiwi fruits are remarkably prolonged; however, after the kiwi fruits treated by 1-MCP and stored at low temperature for a long time are taken out of the warehouse, the fruits often cannot be normally ripe and softened after the shelf life, the inherent flavor is light, the eating quality is seriously reduced, and even the phenomenon of 'zombie fruits' in which the fruits are rotten and not soft is also caused, so that the sales and the commodity of the kiwi fruits are greatly influenced. Meanwhile, the chemical bacteriostatic agent not only pollutes the environment, but also is easy to remain, has potential threat to human health, and can cause the appearance of drug-resistant strains after long-term use.
Biocontrol bacteria (English name biocontrol bacteria) refer to beneficial microorganisms, mainly including bacteria, fungi, actinomycetes and the like, and killing or suppressing the number of pathogenic organisms to control the occurrence and development of plant diseases; the essence is that the survival and activity of some pathogens are inhibited by utilizing the antibiotic, competitive, heavy parasitism and bacteriolytic action of microorganism species or species, or inducing plant disease resistance through microorganism metabolites, etc. The biocontrol bacteria are nontoxic to people and animals, have good compatibility to the environment, and the safe and efficient antagonistic bacteria bactericide is a green antiseptic technology by utilizing the biocontrol effect, so that the biocontrol bacteria have important significance and commercial value when applied to the storage and preservation of kiwi fruits.
Disclosure of Invention
The invention aims to provide a biocontrol bacterium for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage.
To achieve the above and other related objects, the present invention provides the following technical solutions: biocontrol bacteria Pan. Vagans BWL1 for antagonizing penicillium and gray mold are preserved in China Center for Type Culture Collection (CCTCC) for 1 month 4 of 2021, and the preservation number is CCTCC NO: m2021001.
To achieve the above and other related objects, the present invention provides the following technical solutions:a microbial preparation of biocontrol bacteria pan.vagans BWL1, characterized in that: the viable count of the biocontrol bacteria Pan. Vagans BWL1 of the microbial preparation of the biocontrol bacteria Pan. Vagans BWL1 is more than or equal to 10 8 CFU/mL。
The preferable technical scheme is as follows: the preparation method comprises the following steps:
step 1: inoculating biocontrol bacteria Pan.vagans BWL1 into an LB culture medium, and culturing in a shaking table at 23-27 ℃ for 12-18h;
step 2: inoculating the bacterial liquid obtained in the step 1 into a new LB culture medium according to the volume percentage of 5-10% for expansion culture;
step 3: centrifuging the culture in the step 2 at 5000-7000 Xg for 4-6min, discarding the supernatant, and then adding 1/5 volume of phosphate buffer solution for re-suspension;
step 4: adding sodium carboxymethylcellulose, dextrin and sodium alginate, stirring, vacuum drying at low temperature, pulverizing, and sieving with 300 mesh sieve to obtain microbial preparation of biocontrol strain Pan. Vagans BWL 1.
To achieve the above and other related objects, the present invention provides the following technical solutions: a method for storing kiwi fruits, wherein bacterial liquid obtained by diluting a microbial preparation of biocontrol bacteria Pan. Vagans BWL1 prepared in claim 2 acts on the outer surface of kiwi fruits.
The preferable technical scheme is as follows: the mass ratio of the microbial preparation of the biocontrol bacteria Pan. Vagans BWL1 to the kiwi fruit is 10:40000-60000.
The preferable technical scheme is as follows: adding 20-30 times of water to the microbial preparation of biocontrol strain Pan. Vagans BWL1, dissolving, and spraying on fructus Actinidiae chinensis.
The preferable technical scheme is as follows: adding 20-30 times of water to the microbial preparation of biocontrol strain Pan. Vagans BWL1, immersing fructus Actinidiae chinensis, and taking out.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the biocontrol strain Pan. Vagans BWL1 obtained by screening can efficiently antagonize penicillium and gray mold.
2. The biological agent prepared from biocontrol strain Pan. Vagans BWL1 can prolong the storage time of kiwi fruits.
Drawings
FIG. 1 is a morphological photograph of Pan. VagansBWL 1.
FIG. 2 shows the bacteriostatic effect of Pan. VagansBWL1 on Penicillium and Gray mold.
FIG. 3 shows the inhibition of spore germination and shoot elongation of Penicillium sp.
FIG. 4 shows the inhibition of spore germination and shoot elongation of Botrytis cinerea by Pan. VagansBWL 1.
FIG. 5 shows the inhibitory effect of Pan. VagansBWL1 on kiwi fruit penicilliosis.
FIG. 6 shows the inhibitory effect of Pan. VagansBWL1 on kiwi fruit gray mold.
FIG. 7 shows the inhibitory effect of Pan. VagansBWL1 on the natural onset of penicilliosis in kiwi fruit.
FIG. 8 shows the inhibitory effect of Pan. VagansBWL1 on the natural onset of kiwi fruit gray mold.
Detailed Description
Further advantages and effects of the present invention will be readily apparent to those skilled in the art from the following disclosure of the present invention by reference to the specific embodiments.
Example 1: biocontrol bacterium for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage
Biocontrol bacteria Pan. Vagans BWL1 capable of efficiently antagonizing penicillium and gray mold are preserved in China center for type culture collection (China center for type culture collection) at 1 month 4 of 2021, and the preservation number is CCTCC NO: m2021001. The biocontrol strain Pan. Vagans BWL1 is separated from the surface of healthy citrus fruits and is fruit habit bacteria, and a flat plate counter method is adopted, so that the biocontrol strain Pan. Vagans BWL1 has an inhibition effect on penicillium and gray mold. The isolation, screening and identification methods of the biocontrol strain Pan.vagans BWL1 are as follows.
(1) Isolation of antagonistic bacteria
The commercial healthy citrus is taken and placed in a sterilized 500mL beaker, 200mL of sterilized water is added, and 150r/min is shaken for 1h. Diluting the suspension culture solution to 10 -1 、10 -2 、10 -3 After that, 100. Mu.L was applied toAnd (3) on an LB solid plate, carrying out inverted static culture at 25 ℃ for 24 hours. And (3) picking the single colony to a new LB solid plate for streak culture, and performing second streak culture on the obtained monoclonal strain to obtain a pure strain.
The obtained strain was preserved in 30% glycerol and placed in a low temperature environment at-80 ℃.
(2) Screening for antagonistic bacteria
And (3) preparing a suspension of pathogenic bacteria penicillium and botrytis cinerea spores after the kiwi fruits are picked. Respectively activating and culturing Penicillium and Botrytis cinerea strain with PDA culture medium (plate or inclined plane), culturing Penicillium for 7 days at appropriate temperature, culturing Botrytis cinerea for 14 days, scraping mycelium, adding into a triangular flask containing 20mL sterile water, shaking for 1min, filtering with double-layer sterile gauze to remove mycelium, measuring spore concentration with a hemocytometer, and adjusting spore concentration to 10 5 And (3) obtaining the penicillium spore suspension and the botrytis cinerea spore suspension by per mL.
PDA medium is commonly used potato dextrose agar medium.
The antibacterial effect test of the strain adopts a plate counter method. Respectively inoculating penicillium and gray mold mycelium blocks on a PDA flat plate, culturing at 25 ℃ for 7 days, wherein the penicillium grows to 2/3-3/4 of the tray, the gray mold grows to 3/4 of the tray to the full tray, taking penicillium and gray mold fungus cakes with diameters of 0.5cm from the position, close to the edge of the mycelium, on the flat plate by using a puncher, inoculating to a new PDA flat plate, and culturing at 25 ℃ for 3-5 days; the purified bacteria were inoculated in a liquid LB medium, cultured overnight at 25℃and streaked on a PDA plate in which penicillium and gray mold were cultured with an inoculating loop dip, and the antibacterial effect was observed after 3 days of culture at 25 ℃.
The preparation method of the LB culture medium comprises the following steps: 5g of yeast extract, 10g of tryptone and 10g of NaCl are added into 950mL of deionized water, the pH is regulated to 7.0 by 5M NaOH after dissolution, the volume is fixed to 1L, and the yeast extract is sterilized by high-pressure steam at 121 ℃ for 20min for later use.
(3) Identification of antagonistic bacteria
Strains with inhibitory effect were selected, identified as pan.vagans by amplifying and sequencing the 16s rDNA, ATP synthase (atpD) and recombinase (recA) genes, aligned in the NCBI (national center for biotechnology information) database, and strain numbered BWL 1. The 16s rDNA, atpD and recA gene amplified fragments were submitted to NCBI database with sequence numbers MW133037, MW221267 and MW221265.
The primers used, the PCR reaction conditions and the amplified fragment sequences were identified as follows:
sequencing of the 16SrDNA Gene amplified fragment:
atpD gene amplified fragment sequencing results:
sequencing results of recA gene amplified fragment:
PCR amplification system:
listed in the table are PCR systems used to amplify the 16s rDNA, ATP synthase (atpD) and recombinase (recA) gene sequences of the biocontrol strain Pan. Vagans BWL1 strain.
Gene primer and PCR conditions:
primers used for amplifying the gene sequences of 16s rDNA, ATP synthase (atpD) and recombinase (recA) of the biocontrol strain Pan. Vagans BWL1 strain are listed in the table, and the annealing temperature and extension time at the time of PCR amplification. The specific conditions of PCR are as follows: the pre-denaturation is performed at 95 ℃ for 4min, [95 ℃ for 20s, annealing temperature for 20s, 72 ℃ for 1 extension time ] for 35 cycles, 72 ℃ for 10min. After the PCR is finished, gel electrophoresis verifies that the fragment size is correct, and the PCR product is sent to a sequencing company for sequencing.
A biocontrol bacterium for antagonizing penicillium and gray mold and its application in storing kiwi fruits comprise the following steps.
(1) Microbial preparation for preparing biocontrol bacteria Pan. Vagans BWL1
Microbial preparation of biocontrol strain Pan. Vagans BWL1, and biocontrol strain Pan. Vagans BWL1 viable count is not less than 10 8 CFU/mL. The bacterial preparation is prepared by the following method:
(1.1) adopting LB culture medium, dipping biocontrol bacteria Pan.vagans BWL1 in an inoculating loop, inoculating in the LB culture medium, and culturing in a shaking table at 25 ℃ for 12-18h;
(1.2) inoculating the bacterial liquid in the step (1) into a fresh LB culture medium according to the proportion of 5-10% (v/v) for expansion culture;
(1.3) centrifugation at 6000 Xg for 5min, discarding the supernatant, and adding 1/5 volume of phosphate buffer (0.5M, pH 7.0) for re-suspension;
(1.4) adding 10% (w/v) sodium carboxymethylcellulose, 0.5% (w/v) dextrin and 0.2% (w/v) sodium alginate, stirring, vacuum drying at low temperature (vacuum gauge pressure-0.05-0.100 MPa, temperature 20-30deg.C), pulverizing, and sieving with 300 mesh sieve. The prepared microbial inoculum is preserved at 2-20deg.C.
(2) Application method of kiwi fruit postharvest disease antagonistic bacteria preparation
The biological antibacterial agent prepared in the step (2.1) is added with 20-30 times (v/w) of water, and after being fully dissolved, the biological antibacterial agent is sprayed on kiwi fruits, and 10g of the biological antibacterial agent can treat 40-60kg of kiwi fruits;
(2.2) storing the kiwi fruits at a low temperature after airing.
The kiwi fruits stored by the method are obviously inhibited from penicilliosis and gray mold; after 30-45d of low-temperature storage, the rot rate is reduced by more than 60% after the materials are taken out of the warehouse and stored for 7d at normal temperature.
Effects of antagonistic bacteria on spore germination and bud tube elongation of Penicillium and Botrytis cinerea
Biocontrol bacteria pan.vagans BWL1 were inoculated into LB medium (tryptone 10g/L, yeast extract 5g/L, naCl5g/L, pH 7.0), cultured overnight at 25℃and centrifuged at 10000 Xg for 10min, and the supernatant was added to PDA medium (available from Baisi Biotechnology Co., hangzhou) at 10% and poured into plates. The spores of Penicillium and Botrytis cinerea are coated on the plate respectively, and after culturing for 12 hours at 25 ℃, the germination rate of the spores and the length of the bud tube are detected. Each replicate contained 9 replicates, each replicate contained 100 spores. PDA without any treatment and PDA control with 10% LB medium added were also set.
As shown in FIG. 3, the biocontrol strain Pan. Vagans BWL1 fermentation broth has a remarkable inhibiting effect on the germination rate of penicillium spores and the length of bud tubes. After addition of the broth, the spore germination rate was reduced by 32.4% (p < 0.05), and the bud tube length was reduced by 73.2% (p < 0.05). As can be seen from FIG. 4, pan. Vagans BWL1 broth had no significant effect on the germination rate of Botrytis cinerea spores, however, the length of the shoot tubes in the treatment with added broth was reduced by 58.4% (p < 0.05). These results indicate that pan.vagans BWL1 treatment significantly inhibited the germination of penicillium and botrytis spores.
Inhibition of antagonistic bacteria on kiwi fruit green mold and gray mold
1. Puncture inoculation
Inoculating Pan. VagansBWL1 into LB medium, culturing at 25deg.C overnight, centrifuging at 6000 Xg for 10min, removing supernatant, adding 50mM phosphate buffer solution (pH 7.0), and resuspending to obtain strain concentration of about 10 8 CFU/mL, selecting damage-free and disease-free kiwi fruits, cleaning with clear water, sterilizing with 1% sodium hypochlorite for 3min, cleaning with sterile water, and air drying in an ultra-clean bench. The middle part of the fruit was perforated, 10. Mu.L of the BWL1 bacterial suspension was added to the holes, and 10. Mu.L of the phosphate buffer solution was used in place of the BWL1 bacterial suspension in the control group. After complete absorption of the liquid, 10. Mu.L of a spore suspension of the pathogenic bacteria (10 5 personal/mL), to be liquidAfter complete absorption, the fruit is placed in a plastic basket, placed in a dish with clean water to maintain humidity, covered with a plastic bag, and placed at 21 ℃ for 7d. After peeling the fruits, the incidence rate is counted, and the diameter of the lesion is measured. Each repetition contained 6 replicates, each repetition contained 3 replicates, each replicate contained 20 kiwi fruits.
As is clear from FIG. 5, the treatment group (inoculated with penicillium spores and Pan. VagansBWL1 strain) showed a smaller incidence of kiwi fruits than the control group (inoculated with penicillium spores only), and a reduction in the lesion diameter by 64.3% (p < 0.05). As can be seen from FIG. 6, the diameter of the lesions of the kiwi fruits in the treatment group (inoculated with the Botrytis cinerea spores and Pan. VagansBWL1 bacterial liquid) was reduced by 63.8% (p < 0.05) compared with that in the control group (inoculated with only the Botrytis cinerea spores). These results indicate that pan.vagansbwl1 treatment significantly inhibited post harvest penicilliosis and gray mold in kiwi fruits.
2. Natural onset of disease
To simulate the effect of Pan. VagansBWL1 on natural disease of kiwi fruits after storage, a spray inoculation method is adopted, pan. VagansBWL1 is inoculated in LB medium, cultured overnight at 25 ℃, centrifuged for 10min at 6000 Xg, the supernatant is removed, and 50mM phosphate buffer (pH 7.0) is added for resuspension, so that the bacterial concentration is approximately 10 8 CFU/mL, selecting damage-free and disease-free kiwi fruits, cleaning with clear water, sterilizing with 1% sodium hypochlorite for 3min, cleaning with sterile water, and air drying in an ultra-clean bench. The pan.vagansbwl1 bacterial suspension was evenly sprayed and the BWL1 bacterial suspension was replaced with 10 μl of phosphate buffer in the control group. After the surface of the kiwi fruit is completely dried, uniformly spraying a spore suspension of pathogenic bacteria (penicillium or gray mold) on the kiwi fruit, after the surface of the kiwi fruit is completely dried, placing the kiwi fruit into a plastic basket, placing a plate filled with clear water to keep humidity, coating a plastic bag on the outer cover of the frame, placing for 15 days at 21 ℃, and counting the morbidity. Each repetition contained 6 replicates, each repetition contained 3 replicates, each replicate contained 20 kiwi fruits.
As can be seen from FIG. 7, the treatment group (spraying penicillium spores and Pan. VagansBWL1 bacteria liquid) showed a lower incidence of kiwi fruits by 72.1% (p < 0.05) than the control group (spraying penicillium spores only). As can be seen from FIG. 8, the incidence of kiwi fruits in the treatment group (spraying the Botrytis cinerea spores and Pan. VagansBWL1 bacteria liquid) was reduced by 74.4% (p < 0.05) compared with the control group (spraying only the Botrytis cinerea spores). These results indicate that pan.vagansbwl1 treatment significantly inhibited post harvest penicilliosis and gray mold in kiwi fruits.
FIG. 1 shows the pattern of Pan. VagansBWL 1. Scale = 50 μm. After Pan. VagansBWL1 was grown on LB medium at 25℃for 1 day, yellow, convex, smooth-edged colonies were formed, and the color gradually changed from the original pale yellow to the bright yellow. As can be seen from a microscope, the cells were short rods, approximately elliptical, and had a length of 10 to 12. Mu.m.
FIG. 2 shows the bacteriostatic effect of Pan. VagansBWL1 on Penicillium and Gray mold. A is a photograph of Pan. VagansBWL1 facing the plate of Penicillium; b is a photograph of Pan. VagansBWL1 as opposed to a plate of Botrytis cinerea. In the direction of pan.vagansbwl1 growth, mycelial growth of penicillium and botrytis cinerea was significantly inhibited.
FIG. 3 shows the inhibition of spore germination and shoot elongation of Penicillium sp. Scale = 10 μm. A is a photograph of germination of penicillium spores; b is the spore germination rate and bud tube length data of the penicillium. As can be seen from the figure, on PDA plates containing Pan. VagansBWL1 fermentation broth (BWL 1), the germination rate of penicillium spores and the length of the sprout tubes were significantly lower than in the control group.
FIG. 4 shows the inhibition of spore germination and shoot elongation of Botrytis cinerea by Pan. VagansBWL 1. Scale = 100 μm. A is a photo of germination of the spores of the Botrytis cinerea; b is the germination rate of the griseofulvin spores and the length data of the bud tubes. As can be seen, there was no significant difference between the germination rate of the Botrytis cinerea spores and the control group on the PDA plate (BWL 1) containing Pan. VagansBWL1 fermentation broth, however, the shoot length was significantly lower than that of the control group.
FIG. 5 shows the inhibitory effect of Pan. VagansBWL1 on kiwi fruit penicilliosis. After puncture inoculation, pan.vagansbwl1 treated group (BWL 1) lesions were significantly smaller than control group.
FIG. 6 shows the inhibitory effect of Pan. VagansBWL1 on kiwi fruit gray mold. After puncture inoculation, pan.vagansbwl1 treated group (BWL 1) lesions were significantly smaller than control group.
FIG. 7 shows the inhibitory effect of Pan. VagansBWL1 on the natural onset of penicilliosis in kiwi fruit. After spray inoculation, pan.vagansbwl1 treatment (BWL 1) had significantly lower incidence than the control.
FIG. 8 shows the inhibitory effect of Pan. VagansBWL1 on the natural onset of kiwi fruit gray mold. After spray inoculation, pan.vagansbwl1 treatment (BWL 1) had significantly lower incidence than the control.
Example 2: biocontrol bacterium for antagonizing penicillium and gray mold and application thereof in kiwi fruit storage
Biocontrol bacteria Pan. Vagans BWL1 for antagonizing penicillium and gray mold are preserved in China Center for Type Culture Collection (CCTCC) for 1 month 4 of 2021, and the preservation number is CCTCC NO: m2021001.
A microbial preparation of biocontrol bacteria pan.vagans BWL1, characterized in that: the viable count of the biocontrol bacteria Pan. Vagans BWL1 of the microbial preparation of the biocontrol bacteria Pan. Vagans BWL1 is more than or equal to 10 8 CFU/mL。
The preferable technical scheme is as follows: the preparation method comprises the following steps:
step 1: inoculating biocontrol bacteria Pan.vagans BWL1 into an LB culture medium, and culturing for 18h in a shaking table at 23 ℃;
step 2: inoculating the bacterial liquid obtained in the step 1 into a new LB culture medium according to the volume percentage of 5%, and performing expansion culture;
step 3: centrifuging the culture in the step 2 at 5000 Xg for 4min, discarding the supernatant, and then adding 1/5 volume of phosphate buffer solution for re-suspension;
step 4: adding sodium carboxymethylcellulose, dextrin and sodium alginate, stirring, vacuum drying at low temperature, pulverizing, and sieving with 300 mesh sieve to obtain microbial preparation of biocontrol strain Pan. Vagans BWL 1.
A method for storing kiwi fruits, wherein bacterial liquid obtained by diluting a microbial preparation of biocontrol bacteria Pan. Vagans BWL1 prepared in claim 2 acts on the outer surface of kiwi fruits.
The preferred embodiments are: the mass ratio of the microbial preparation of the biocontrol bacteria Pan. Vagans BWL1 to the kiwi fruit is 10:40000.
the preferred embodiments are: adding 20-30 times of water to the microbial preparation of biocontrol strain Pan. Vagans BWL1, dissolving, and spraying on fructus Actinidiae chinensis.
The preferred embodiments are: adding 20-30 times of water to the microbial preparation of biocontrol strain Pan. Vagans BWL1, immersing fructus Actinidiae chinensis, and taking out.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.
Sequence listing
<110> university of synthetic fertilizer industry
<120> biocontrol strain for high-efficiency antagonizing penicillium and botrytis cinerea and application thereof in kiwi fruit storage
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 1401
<212> DNA
<213> 16 sequencing of SrDNA Gene amplified fragments
<400> 1
gcagtcggac ggtagcacag agagcttgct cttgggtgac gagtggcgga cgggtgagta 60
atgtctgggg atctgcccga tagaggggga taaccactgg aaacggtggc taataccgca 120
taacgtcgca agaccaaaga gggggacctt cgggcctctc actatcggat gaacccagat 180
gggattagct agtaggcggg gtaatggccc acctaggcga cgatccctag ctggtctgag 240
aggatgacca gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300
gggaatattg cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc 360
ttcgggttgt aaagtacttt cagcggggag gaaggcgatg cggttaataa ccgcgtcgat 420
tgacgttacc cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga 480
gggtgcaagc gttaatcgga attactgggc gtaaagcgca cgcaggcggt ctgttaagtc 540
agatgtgaaa tccccgggct taacctggga actgcatttg aaactggcag gcttgagtct 600
tgtagagggg ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac 660
cggtggcgaa ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcgacttg gaggttgttc 780
ccttgaggag tggcttccgg agctaacgcg ttaagtcgac cgcctgggga gtacggccgc 840
aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgatgcaa cgcgaagaac cttacctact cttgacatcc acggaatttg gcagagatgc 960
cttagtgcct tcgggaaccg tgagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg 1020
aaatgttggg ttaagtcccg caacgagcgc aacccttatc ctttgttgcc agcgattcgg 1080
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catcatggcc cttacgagta gggctacaca cgtgctacaa tggcgcatac aaagagaagc 1200
aacctcgcga gagcaagcgg acctcacaaa gtgcgtcgta gtccggatcg gagtctgcaa 1260
ctcgactccg tgaagtcgga atcgctagta atcgtggatc agaatgccac ggtgaatacg 1320
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<213> result of sequencing amplified fragment of atpD Gene
<400> 2
agtatgaatg gtgatgcgcg tctggtgctg gaagttcagc aacagctcgg cggcggcgta 60
gtacgtacca tcgcaatggg tacgtctgac ggcctgaagc gtggtctgag cgtcaacgac 120
ctgcagaaac cgattcaggt acccgtcggt aaagcgaccc tgggccgtat catgaacgtt 180
ctcggcgagc caatcgatat gaaaggcgag ctgaaagaag aagatggcag cgcagtagag 240
atcgcctcta ttcaccgcgc agccccttct tatgaagatc agtctaactc gcaggaactg 300
ctggaaaccg gcatcaaggt tatcgacctg atgtgtccgt ttgctaaagg cggtaaagtc 360
ggtctgttcg gtggtgcggg tgtaggtaaa accgtcaaca tgatggaact gatccgtaac 420
atcgcggctg aacactcagg ttactcagtg tttgccggtg tgggtgagcg tactcgtgag 480
ggtaacgact tctaccacga aatgactgac tctaacgtta tcgataaagt tgcactggtc 540
tatggccaga tgaacgagcc gccgggtaac cgtctgcgcg tagcactgac cggtctgacc 600
atggcggaaa aattccgtga tgaaggccgc gacgttctgc tgttcatcga taacatctac 660
cgttataccc tggccggtac agaagtttct gcactgctgg gtcgtatgcc atctgcggta 720
ggttaccagc caacgctggc agaagagatg ggtgtgttgc aggagcgtat tacctccacc 780
aagaccggtt caatcacctc cgtacaggcc gtttacgtcc ctgcggatga cctgactgac 840
ccatcaccag caactacctt tgcgcactta gactcaacgg taacgctgag ccgtcagatc 900
gcctctctgg gtatctaccc ggccgttgac ccgctggact ctaccagccg tcagctggat 960
ccgctggttg tcggtcagga gcactatgat gttgcacgtg gcgttcagtc actgctgcag 1020
cgttatcagg aactgaaaga catcatcgcc atcctcggta tggatgagct gtctgaagaa 1080
gacaaactgc tggtggcacg tgcgcgtaag attcagcgct tcctgtctca gccgttcttc 1140
gttgcagaag tattcaccgg ttcaccgggc aaatacgtga cgctgaaaga cactatccgt 1200
ggctttaaag gcatcatgga aggtgagttt gaccacctgc caagagc 1247
<210> 3
<211> 638
<212> DNA
<213> sequencing results of recA Gene amplified fragments
<400> 3
gcgtaaggta aacctgtgcc tttatcgatg ccgagcatgc gcttgatccg gtttacgcca 60
agaaactcgg cgtagacatt gataacctgc tctgctctca gccagacact ggtgagcagg 120
cgctggaaat ttgtgatgcg ctggcgcgct ctggtgccgt tgacgtgatc atcgtcgact 180
ccgtcgcggc gctgacgcct aaagcggaaa tcgaaggtga aatcggtgac tcacacatgg 240
gcctcgcggc acgtatgatg agccaggcga tgcgtaaact ggccggtaac ctgaagcagt 300
ccaataccct gctgattttc atcaaccaga tccgtatgaa aattggtgtg atgtttggta 360
acccggaaac caccactggt ggtaacgcgc tgaagttcta cgcctctgtc cgtcttgata 420
tccgccgtat cggtgcaatc aaagagggcg ataacgtggt cggcagtgag acccgcgtta 480
aagtggtgaa aaacaaaatc gccgcgccat tcaaacaggc tgagttccag atcatgtacg 540
gcgaaggtat caataccttt ggtgagctgg tcgacctggg cgtgaagcac aagctgattg 600
aaaaagcggg tgcatggtac agctacaatg gcgacaag 638
<210> 4
<211> 18
<212> DNA
<213> 16s rDNAF
<400> 4
gagtttgatc ctggctca 18
<210> 5
<211> 19
<212> DNA
<213> 16s rDNAR
<400> 5
ggttaccttg ttacgactt 19
<210> 6
<211> 20
<212> DNA
<213> atpDF
<400> 6
<210> 7
<211> 20
<212> DNA
<213> atpDR
<400> 7
tsgctttttc cacagcttct 20
<210> 8
<211> 20
<212> DNA
<213> recAF
<400> 8
<210> 9
<211> 21
<212> DNA
<213> recAR
<400> 9
ttcgcyttrc cctgrccrat c 21
Claims (7)
1. Biocontrol bacteria agglomerans pantoea strain for antagonizing penicillium and botrytis cinereaPantoea vagans) BWL1 was preserved in China Center for Type Culture Collection (CCTCC) for type 1 and 4 days 2021, and the preservation number is CCTCC NO: m2021001.
2. Biological control fungus agglomeration pantoeaPantoea vagans) A microbial preparation of BWL1, characterized in that: the biocontrol bacteria are formed into pantoea agglomeransPantoea vagans) Biological control bacteria of BWL1 microbial preparation and pantoea agglomeransPantoea vagans) The number of living bacteria of BWL1 is greater than or equal to 10 8 CFU/mL, biocontrol bacterium and pantoea agglomerans @ and the preparation method thereofPantoea vagans) BWL1 was preserved in China Center for Type Culture Collection (CCTCC) for type 1 and 4 days 2021, and the preservation number is CCTCC NO: m2021001.
3. The biocontrol bacterium pantoea agglomerans according to claim 2Pantoea vagans) A microbial preparation of BWL1, characterized in that: the preparation method comprises the following steps:
step 1: the biocontrol bacteria are agglomerated into pantoeaPantoea vagans) Inoculating BWL1 into LB culture medium, and culturing in shake table at 23-27deg.C for 12-18 hr;
step 2: inoculating the bacterial liquid obtained in the step 1 into a new LB culture medium according to the volume percentage of 5-10% for expansion culture;
step 3: centrifuging the culture in the step 2 at 5000-7000 Xg for 4-6min, discarding the supernatant, and then adding 1/5 volume of phosphate buffer solution for re-suspension;
step 4: adding sodium carboxymethyl cellulose, dextrin and sodium alginate, stirring, vacuum drying at low temperature, pulverizing, and sieving with 300 mesh sieve to obtain biological control fungus-forming pantoeaPantoea vagans) A microbial preparation of BWL 1.
4. A method for storing kiwi fruits is characterized by comprising the following steps: the biocontrol bacterium pantoea agglomerans prepared by the method of claim 2 or 3Pantoea vagans) The bacterial liquid obtained by diluting the BWL1 microbial preparation acts on the outer surface of the kiwi fruit.
5. The method for storing kiwi fruits according to claim 4, wherein: biological control fungus agglomeration pantoeaPantoea vagans) The mass ratio of the microbial preparation of BWL1 to the kiwi fruit is 10:40000-60000.
6. The method for storing kiwi fruits according to claim 4, wherein: the bio-control bacteria are clustered into pantoeaPantoea vagans) The BWL1 microbial preparation is added with water of 20-30 times of its volume, and sprayed on fructus Actinidiae chinensis after dissolution.
7. The method for storing kiwi fruits according to claim 4, wherein: the bio-control bacteria are clustered into pantoeaPantoea vagans) The BWL1 microbial preparation is added with water of 20-30 times of its volume, and the kiwi fruit is immersed and then taken out.
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| CN110699275A (en) * | 2019-09-03 | 2020-01-17 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof |
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| CN109234210A (en) * | 2018-11-19 | 2019-01-18 | 江苏科技大学 | For the bacillus subtilis of antagonism grey mould fruit rot of strawberry and its preparation of biocontrol agent and application |
| CN110699275A (en) * | 2019-09-03 | 2020-01-17 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof |
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