CN113773981A

CN113773981A - Biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application of biocontrol bacterium in kiwi fruit storage

Info

Publication number: CN113773981A
Application number: CN202110878838.0A
Authority: CN
Inventors: 张丹凤; 毕婉玲; 马志桃; 叶应旺
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-12-10
Anticipated expiration: 2041-08-02
Also published as: CN113773981B

Abstract

Biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea, application of biocontrol bacterium in kiwi fruit storage, and biocontrol bacteriumPan.vagansBWL1, which has been preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 4 days 2021, with the preservation number of CCTCC NO: m2021001. The biocontrol bacterium is obtained by screeningPan.vagansBWL1 can antagonize Penicillium and Botrytis cinerea with high efficiency.

Description

Biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application of biocontrol bacterium in kiwi fruit storage

Technical Field

The invention relates to a biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application thereof in kiwi fruit storage, belonging to the technical field of agricultural product storage.

Background

The kiwi fruits are originally produced in China and are called as the king of VC and the king of fruits due to the extremely high content of vitamin C, and the kiwi fruits have fragrant pulp, rich water and delicious sour and sweet taste, and are deeply loved by people. However, the kiwi fruits are very easily infected by pathogenic bacteria during the storage period after the kiwi fruits are picked, and according to statistics, the worldwide loss of the kiwi fruits caused by rotting after the kiwi fruits are picked is up to 20-30%, so that huge economic loss is brought to the kiwi fruit industry, and the rapid development of the industry is greatly limited.

The fungal diseases causing the rot of the picked kiwi fruits mainly comprise gray mold, penicilliosis and the like. Botrytis cinerea (Botrytis cinerea) is the most important postharvest disease of kiwi fruits, the invasion mode of the Botrytis cinerea is various, mycelium, spores or sclerotia can survive for a long time in adverse environments, and the mycelia, the spores or the sclerotia can grow rapidly when meeting appropriate environments. Penicillium disease is caused by Penicillium expansum (Penicillium expansum) infection, which not only can cause the fruit of the kiwi fruit to rot, but also can generate patulin which is toxic to human bodies, thereby endangering the food safety.

At present, the traditional method for storing and preserving the picked kiwi fruits mainly adopts low-temperature storage, and reduces the respiration of the fruits at low temperature so as to reach a dormant state, reduce the rot of the kiwi fruits and prolong the preservation period. However, the penicillium and botrytis cinerea spores can survive for a long time at a temperature above the freezing point, so that the problem of penicillium and botrytis cinerea pollution after kiwi fruit picking cannot be solved by low-temperature storage. Chemical bacteriostatic agents, such as 1-methylcyclopropene (1-MCP) and the like, are widely applied to the storage and preservation of the kiwi fruits, and the storage time and shelf life of the kiwi fruits are remarkably prolonged; however, after the kiwi fruits treated by the 1-MCP and stored at low temperature for a long time are taken out of a warehouse, the fruits often have the problems of abnormal after-ripening and softening, light flavor and serious reduction of edible quality in the shelf life, and even the phenomenon of rotten and not soft dead bodies of the fruits is caused, which greatly influences the sales and marketability of the kiwi fruits. Meanwhile, the chemical bacteriostatic agent not only pollutes the environment, but also is easy to remain, has potential threat to human health, and may cause the appearance of drug-resistant strains after long-term use.

Biocontrol bacteria (the english name biocontrol bacteria) refer to beneficial microorganisms, mainly including bacteria, fungi, actinomycetes, etc., and kill or suppress the number of pathogenic organisms to control the occurrence and development of plant diseases; the essence is to inhibit the survival and activity of some pathogens by using the antibiotic, competitive, parasitism and bacteriolysis between or in the microbial species, or inducing plant disease resistance by microbial metabolites. The biocontrol bacteria are nontoxic to human and livestock, have good compatibility to the environment, utilize biological control function, adopt safe and efficient antagonistic bacteria bactericide as a 'green anticorrosion' technology, and have important significance and commercial value when being applied to the storage and fresh keeping of the kiwi fruits.

Disclosure of Invention

The invention aims to provide a biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application thereof in kiwi fruit storage.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a biocontrol bacterium Pan.vagans BWL1 with high-efficiency antagonism to penicillium and botrytis cinerea, which is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 4 days in 2021, and the preservation number is CCTCC NO: m2021001.

In order to achieve the above objects 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 bacterium Pan.vagans BWL1 of the microbial preparation of the biocontrol bacterium Pan.vagans BWL1 is more than or equal to 10⁸CFU/mL。

The preferable technical scheme is as follows: the preparation method comprises the following steps:

step 1: inoculating biocontrol bacteria Pan.vagans BWL1 in LB culture medium, and culturing in a shaking table at 23-27 ℃ for 12-18 h;

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 amplification culture;

and step 3: centrifuging the culture obtained in the step 2 for 4-6min at 7000 Xg of 5000-;

and 4, 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 bacteria Pan.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for storing kiwi fruits, comprising applying a bacterial solution obtained by diluting the microbial preparation of biocontrol bacteria Pan. vagans BWL1 prepared in claim 2 on the outer surface of kiwi fruits.

The preferable technical scheme is as follows: the mass ratio of the microbial preparation of the biocontrol bacterium Pan.vagans BWL1 to the kiwi fruit is 10: 40000-60000.

The preferable technical scheme is as follows: adding water 20-30 times of the microbial preparation of biocontrol bacteria Pan. vagans BWL1, dissolving, and spraying on fructus Actinidiae chinensis.

The preferable technical scheme is as follows: adding water 20-30 times of the microbial preparation of biocontrol bacteria 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 bacterium Pan.vagans BWL1 obtained by screening can efficiently antagonize penicillium and botrytis cinerea.

2. The biological agent prepared from biocontrol bacterium Pan. vagans BWL1 can prolong the storage time of fructus Actinidiae chinensis.

Drawings

Fig. 1 is a photograph of a morphology of pan.

Fig. 2 shows the bacteriostatic effect of pan. vagans bwl1 on penicillium and botrytis cinerea.

Fig. 3 shows the inhibitory effect of pan. vagans bwl1 on spore germination and germ tube elongation of penicillium.

Fig. 4 shows the inhibitory effect of pan. vagans bwl1 on spore germination and germ tube elongation of botrytis cinerea.

Fig. 5 shows the inhibitory effect of pan. vagans bwl1 on penicilliosis of kiwifruit.

Fig. 6 shows the inhibition effect of pan. vagans bwl1 on grey mould of kiwi fruit.

Fig. 7 shows the inhibitory effect of pan. vagans bwl1 on the natural onset of penicilliosis in kiwifruit.

Fig. 8 shows the inhibition effect of pan. vagans bwl1 on the natural onset of grey mould of kiwi fruits.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be apparent to those skilled in the art from the present disclosure.

Example 1: biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application of biocontrol bacterium in kiwi fruit storage

A biocontrol bacterium Pan. vagans BWL1 with high-efficiency antagonism to penicillium and botrytis cinerea, which is preserved in China center for type culture Collection at 1 month and 4 days 2021, with the address: in Wuhan university school of eight-channel No. 299 in Wuhan district of Wuhan city, Hubei China, the preservation number is CCTCC NO: m2021001. The biocontrol bacterium Pan.vagans BWL1 is separated from the surface of healthy citrus fruits and is a fruit inhabitation bacterium, and the biocontrol bacterium Pan.vagans BWL1 is found to have an inhibiting effect on penicillium and botrytis cinerea by adopting a plate confrontation method. The method for separating, screening and identifying biocontrol bacteria Pan. vagans BWL1 is as follows.

(1) Isolation of antagonistic bacteria

Taking commercially available healthy citrus, putting into a sterilized 500mL beaker, adding 200mL of sterilized water, and oscillating at 150r/min for 1 h. Diluting suspension culture solution to 10^-1、10^-2、10^-3Then, 100. mu.L of the suspension was spread on an LB solid plate, and the plate was subjected to static culture at 25 ℃ in an inverted state for 24 hours. And (3) selecting a single bacterial colony to a new LB solid plate for streak culture, and carrying out second streak culture on the obtained monoclonal bacterial strain to obtain a pure bacterial strain.

The obtained strain is preserved in 30% glycerol and placed in a low-temperature environment of-80 ℃.

(2) Screening of antagonistic bacteria

And (3) preparing a suspension of pathogenic bacteria penicillium and botrytis cinerea spores after the kiwi fruits are picked. Respectively performing activation culture on Penicillium and Botrytis cinerea strains by using PDA culture medium (flat plate or inclined plane), culturing Penicillium and Botrytis cinerea at appropriate temperature for 7 days, culturing Botrytis cinerea for 14 days, scraping off mycelia, adding into a triangular flask containing 20mL of sterile water, shaking for 1min, filtering with double-layer sterilizing gauze to remove mycelia, measuring spore concentration by using a blood count plate, and adjusting the spore concentration to 10⁵Obtaining penicillium spore suspension and botrytis cinerea spore suspension.

PDA medium is the usual potato dextrose agar medium.

The bacterial strain bacteriostasis effect test adopts a plate confrontation method. Respectively inoculating penicillium and botrytis cinerea mycelium blocks on a PDA (personal digital assistant) plate, culturing at 25 ℃ for 7 days, wherein the penicillium grows to 2/3-3/4 discs and the botrytis cinerea grows to 3/4 discs to full discs, taking penicillium and botrytis cinerea fungus cakes with the diameter of 0.5cm from the positions, close to the edges of hypha, on the plate by using a puncher, inoculating the penicillium and botrytis cinerea fungus cakes on a new PDA plate, and culturing at 25 ℃ for 3-5 days; inoculating the purified bacteria into liquid LB culture medium, culturing overnight at 25 deg.C, taking bacterial liquid by inoculating loop, streaking on PDA plate with penicillium and botrytis, culturing at 25 deg.C for 3 days, and observing antibacterial effect.

The preparation method of the LB culture medium comprises the following steps: adding yeast extract 5g, tryptone 10g and NaCl 10g into 950mL deionized water, dissolving, adjusting pH to 7.0 with 5M NaOH, diluting to 1L, and sterilizing with high pressure steam at 121 deg.C for 20 min.

(3) Identification of antagonistic bacteria

Selecting a strain with an inhibitory effect, amplifying and sequencing 16s rDNA, ATP synthsase (atpD) and recombinase (recA) genes, comparing in an NCBI (national center for Biotechnology information) database, identifying the strain as Pan.vagans, and numbering the strain as BWL 1. The 16s rDNA, atpD and recA gene amplified fragments have been submitted to the NCBI database with sequence numbers MW133037, MW221267 and MW221265 in that order.

The primers used, PCR reaction conditions and amplified fragment sequences were identified as follows:

sequencing result of 16SrDNA gene amplification fragment:

5’-GCAGTCGGACGGTAGCACAGAGAGCTTGCTCTTGGGTGACGAGTGGCGGACGGGTGAGTAAT GTCTGGGGATCTGCCCGATAGAGGGGGATAACCACTGGAAACGGTGGCTAATACCGCATAACGTCGC AAGACCAAAGAGGGGGACCTTCGGGCCTCTCACTATCGGATGAACCCAGATGGGATTAGCTAGTAGG CGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAAC TGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGA TGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCG ATGCGGTTAATAACCGCGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCA GCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTC TGTTAAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTC TTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGG CGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGG AGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTG ACATCCACGGAATTTGGCAGAGATGCCTTAGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCT GTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGC CAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGT CAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCAA CCTCGCGAGAGCAAGCGGACCTCACAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCC GTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACA CACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTCGGAGGCGCT-3’。

sequencing of amplified fragments of atpD Gene:

5’-AGTATGAATGGTGATGCGCGTCTGGTGCTGGAAGTTCAGCAACAGCTCGGCGGCGGCGTAGTA CGTACCATCGCAATGGGTACGTCTGACGGCCTGAAGCGTGGTCTGAGCGTCAACGACCTGCAGAAAC CGATTCAGGTACCCGTCGGTAAAGCGACCCTGGGCCGTATCATGAACGTTCTCGGCGAGCCAATCGAT ATGAAAGGCGAGCTGAAAGAAGAAGATGGCAGCGCAGTAGAGATCGCCTCTATTCACCGCGCAGCCC CTTCTTATGAAGATCAGTCTAACTCGCAGGAACTGCTGGAAACCGGCATCAAGGTTATCGACCTGATG TGTCCGTTTGCTAAAGGCGGTAAAGTCGGTCTGTTCGGTGGTGCGGGTGTAGGTAAAACCGTCAACA TGATGGAACTGATCCGTAACATCGCGGCTGAACACTCAGGTTACTCAGTGTTTGCCGGTGTGGGTGAG CGTACTCGTGAGGGTAACGACTTCTACCACGAAATGACTGACTCTAACGTTATCGATAAAGTTGCACT GGTCTATGGCCAGATGAACGAGCCGCCGGGTAACCGTCTGCGCGTAGCACTGACCGGTCTGACCATG GCGGAAAAATTCCGTGATGAAGGCCGCGACGTTCTGCTGTTCATCGATAACATCTACCGTTATACCCTG GCCGGTACAGAAGTTTCTGCACTGCTGGGTCGTATGCCATCTGCGGTAGGTTACCAGCCAACGCTGGC AGAAGAGATGGGTGTGTTGCAGGAGCGTATTACCTCCACCAAGACCGGTTCAATCACCTCCGTACAG GCCGTTTACGTCCCTGCGGATGACCTGACTGACCCATCACCAGCAACTACCTTTGCGCACTTAGACTC AACGGTAACGCTGAGCCGTCAGATCGCCTCTCTGGGTATCTACCCGGCCGTTGACCCGCTGGACTCTA CCAGCCGTCAGCTGGATCCGCTGGTTGTCGGTCAGGAGCACTATGATGTTGCACGTGGCGTTCAGTCA CTGCTGCAGCGTTATCAGGAACTGAAAGACATCATCGCCATCCTCGGTATGGATGAGCTGTCTGAAGA AGACAAACTGCTGGTGGCACGTGCGCGTAAGATTCAGCGCTTCCTGTCTCAGCCGTTCTTCGTTGCAG AAGTATTCACCGGTTCACCGGGCAAATACGTGACGCTGAAAGACACTATCCGTGGCTTTAAAGGCATC ATGGAAGGTGAGTTTGACCACCTGCCAAGAGC-3’。

sequencing results of the recA gene amplification fragment:

5’-GCGTAAGGTAAACCTGTGCCTTTATCGATGCCGAGCATGCGCTTGATCCGGTTTACGCCAAGA AACTCGGCGTAGACATTGATAACCTGCTCTGCTCTCAGCCAGACACTGGTGAGCAGGCGCTGGAAAT TTGTGATGCGCTGGCGCGCTCTGGTGCCGTTGACGTGATCATCGTCGACTCCGTCGCGGCGCTGACGC CTAAAGCGGAAATCGAAGGTGAAATCGGTGACTCACACATGGGCCTCGCGGCACGTATGATGAGCCA GGCGATGCGTAAACTGGCCGGTAACCTGAAGCAGTCCAATACCCTGCTGATTTTCATCAACCAGATCC GTATGAAAATTGGTGTGATGTTTGGTAACCCGGAAACCACCACTGGTGGTAACGCGCTGAAGTTCTAC GCCTCTGTCCGTCTTGATATCCGCCGTATCGGTGCAATCAAAGAGGGCGATAACGTGGTCGGCAGTGA GACCCGCGTTAAAGTGGTGAAAAACAAAATCGCCGCGCCATTCAAACAGGCTGAGTTCCAGATCATG TACGGCGAAGGTATCAATACCTTTGGTGAGCTGGTCGACCTGGGCGTGAAGCACAAGCTGATTGAAA AAGCGGGTGCATGGTACAGCTACAATGGCGACAAG-3’。

PCR amplification System:

the PCR system for amplifying gene sequences of biocontrol bacteria Pan. vagans BWL1 strain 16s rDNA, ATP synthsase (atpD) and recombiase (recA) is listed in the table.

Gene primers and PCR conditions:

the primers used for amplifying the 16s rDNA, ATP synthsase (atpD) and recombinase (recA) gene sequences of the biocontrol bacterium Pan. vagans BWL1 strain, and the annealing temperature and extension time during PCR amplification are listed in the table. The specific PCR conditions are as follows: pre-denaturation at 95 ℃ for 4min, [95 ℃ for 20s, [ annealing temperature for 20s, [ 72 ℃ for 1 extension time ]. times.35 cycles, and (72 ℃ for 10 min). After the PCR is finished, the size of the fragment is verified to be correct by gel electrophoresis, and then the PCR product is sent to a sequencing company for sequencing.

A biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and an application thereof in kiwi fruit storage comprise the following technical steps.

(1) Preparation of microbial preparation of biocontrol bacterium Pan. vagans BWL1

A microbial preparation containing biocontrol bacteria Pan. vagans BWL1 with viable count of not less than 10⁸CFU/mL. The bacterium preparation is prepared by the following method:

(1.1) adopting an LB culture medium, dipping biocontrol bacteria Pan. vagans BWL1 by using an inoculating loop, inoculating the biocontrol bacteria Pan. vagans BWL1 into the LB culture medium, and then culturing for 12-18h in a shaking table at 25 ℃;

(1.2) inoculating the bacterial liquid in the step (1) to a fresh LB culture medium according to the ratio of 5-10% (v/v) for amplification culture;

(1.3) centrifuging at 6000 Xg for 5min, discarding the supernatant, adding 1/5 volume of phosphate buffer (0.5M, pH7.0) and resuspending;

(1.4) adding 10% (w/v) sodium carboxymethylcellulose, 0.5% (w/v) dextrin and 0.2% (w/v) sodium alginate, uniformly stirring, drying at low temperature in vacuum (vacuum gauge pressure of-0.05 to-0.100 MPa, temperature of 20-30 ℃), crushing and sieving with a 300-mesh sieve. The prepared microbial inoculum is preserved at the temperature of 2-20 ℃.

(2) Method for using disease antagonistic bacteria preparation after picking kiwi fruit

(2.1) adding 20-30 times (v/w) of water into the prepared biological antibacterial agent, fully dissolving, and spraying the obtained solution on the kiwi fruit, wherein 40-60kg of kiwi fruit can be treated by 10g of microbial inoculum;

and (2.2) airing the kiwi fruits, and storing at low temperature.

When the kiwi fruits are stored by the method, the penicilliosis and the gray mold are obviously inhibited; after the low-temperature storage for 30-45 days, the materials are taken out of the warehouse and stored for 7 days at normal temperature, and the rotting rate is reduced by more than 60%.

Effect of antagonistic bacteria on spore germination and shoot elongation of Penicillium and Botrytis

Inoculating biocontrol bacteria Pan. vagans BWL1 into LB culture medium (tryptone 10g/L, yeast extract 5g/L, NaCl 5g/L, pH7.0), culturing overnight at 25 deg.C, centrifuging at 10000 Xg for 10min, collecting supernatant, adding 10% into PDA culture medium (purchased from Hangzhou Baisi Biotech Co., Ltd.), and pouring into plate. The penicillium and botrytis cinerea spores are respectively coated on the flat plate, and after the cultivation at 25 ℃ for 12 hours, the spore germination rate and the length of a germ tube are detected. Each replicate contained 9 replicates, each replicate containing 100 spores. PDA without any treatment and PDA control supplemented with 10% LB medium were also set.

As can be seen from fig. 3, the biocontrol bacterium pan. vagans BWL1 fermentation broth has significant inhibitory effect on the germination rate of penicillium spores and the length of germ tubes. After the fermentation liquor is added, the spore germination rate is reduced by 32.4 percent (p is less than 0.05), and the germ tube length is reduced by 73.2 percent (p is less than 0.05). As can be seen from fig. 4, pan. vagans BWL1 broth had no significant effect on botrytis cinerea spore germination rate, however, the germ tube length in the treatment with the addition of broth decreased by 58.4% (p < 0.05). These results indicate that pan. vagans BWL1 treatment significantly inhibited the germination of penicillium and botrytis cinerea spores.

Inhibitory action of antagonistic bacteria on penicilliosis and gray mold of kiwi fruit

1. Puncture inoculation

Inoculating Pan. vagans BWL1 into LB culture medium, culturing at 25 deg.C overnight, centrifuging at 6000 Xg for 10min, removing supernatant, adding 50mM phosphate buffer (pH 7.0) and re-suspending to make bacterial concentration approximately equal to 10⁸CFU/mL, selecting non-damaged and disease-free kiwi fruits, cleaning with clear water, sterilizing with 1% sodium hypochlorite surface for 3min, washing with sterile water, and air drying in a super clean bench. A hole is punched in the middle of the fruit, 10 mu L of LPAn. vagans BWL1 bacterial suspension is added into the hole, and BWL1 bacterial suspension is replaced by 10 mu L of phosphate buffer solution in a control group. After complete absorption of the liquid, the wells were inoculated with 10. mu.L of a pathogen spore suspension (10)⁵one/mL), after the liquid is completely absorbed, the fruits are placed in a plastic basket, a dish filled with clear water is placed to keep humidity, a plastic bag is sleeved outside the frame, and the fruits are placed for 7 days at 21 ℃. The incidence of disease is counted after peeling the fruit, and the diameter of the disease spot is measured. Each one of which isThe replicates contained 6 replicates, each replicate contained 3 replicates, each replicate contained 20 kiwi fruits.

As can be seen from fig. 5, compared to the control group (inoculated with only penicillium spores), the treated group (inoculated with penicillium spores and pan. vagans bwl1 bacterial solution) showed a lower incidence of kiwifruit, and the lesion size was reduced by 64.3% (p < 0.05). As can be seen from fig. 6, the lesion diameter of kiwifruit in the treated group (inoculated with grifola frondosa spores and pan. vagans bwl1 bacterial liquid) was reduced by 63.8% (p <0.05) compared to the control group (inoculated with grifola frondosa spores only). These results indicate that pan. vagans bwl1 treatment can significantly inhibit post-harvest penicilliosis and gray mold in kiwifruits.

2. Natural disease onset

To simulate the effect of Pan. vagans BWL1 on the natural onset of kiwi fruits after storage, we used a spray inoculation method, and inoculated Pan. vagans BWL1 into LB medium, cultured overnight at 25 ℃, centrifuged at 6000 Xg for 10min, removed the supernatant, added 50mM phosphate buffer (pH 7.0) for resuspension, and the bacterial concentration was about 10⁸CFU/mL, selecting non-damaged and disease-free kiwi fruits, cleaning with clear water, sterilizing with 1% sodium hypochlorite surface for 3min, washing with sterile water, and air drying in a super clean bench. The liquid suspension of Pan. vagans BWL1 was sprayed evenly, and BWL1 liquid suspension was replaced with 10. mu.L of phosphate buffer solution in the control group. After the surface of the kiwi fruit is completely dried, uniformly spraying a pathogenic bacteria (penicillium or botrytis cinerea) spore suspension to the kiwi fruit, after the surface of the kiwi fruit is completely dried, putting the kiwi fruit into a plastic basket, putting a flat dish filled with clear water to keep humidity, covering a plastic bag outside the frame, standing at 21 ℃ for 15 days, and counting the morbidity. Each replicate contained 6 replicates, each replicate contained 3 replicates, each replicate contained 20 kiwi fruits.

As can be seen from fig. 7, compared to the control group (only penicillium spores were sprayed), the treated kiwifruit (penicillium spores and pan. vagans bwl1 bacterial liquid were sprayed) had a lower incidence of disease and the incidence of disease was reduced by 72.1% (p < 0.05). As can be seen from fig. 8, the incidence of kiwifruit in the treated group (sprayed with grifola frondosa spores and pan. vagans bwl1 bacteria) was reduced by 74.4% (p <0.05) compared to the control group (sprayed with grifola spores only). These results indicate that pan. vagans bwl1 treatment can significantly inhibit post-harvest penicilliosis and gray mold in kiwifruits.

Fig. 1 is a pan. vagans bwl1 morphology. Scale bar 50 μm. Van bwl1 forms yellow, convex, smooth-edged colonies after 1 day of growth on LB medium at 25 ℃, gradually changing from an initial pale yellow color to a bright yellow color. As can be seen by microscopic observation, the cells were short rods, nearly elliptical, and 10-12 μm in length.

Fig. 2 shows the bacteriostatic effect of pan. vagans bwl1 on penicillium and botrytis cinerea. A is a photo of plate confrontation of Pan.vagasBWL1 and penicillium; b is a photograph of pan. vagasbwl 1 in opposition to a plate of botrytis cinerea. In the direction of pan. vagans bwl1 growth, penicillium and botrytis hyphae growth was significantly inhibited.

Fig. 3 shows the inhibitory effect of pan. vagans bwl1 on spore germination and germ tube elongation of penicillium. Scale bar 10 μm. A is a picture of spore germination of penicillium; and B is the germination rate and the length data of the tube of the penicillium spores. It can be seen from the figure that on PDA plates containing pan. vagans BWL1 fermentation broth (BWL1), the germination rate and germ tube length of penicillium spores were significantly lower than those of the control group.

Fig. 4 shows the inhibitory effect of pan. vagans bwl1 on spore germination and germ tube elongation of botrytis cinerea. Scale bar 100 μm. A is a botrytis cinerea spore germination photo; and B is the germination rate of the botrytis cinerea spores and the length data of the germination pipes. As can be seen, on PDA plates containing pan. vagans BWL1 broth (BWL1), the germination rate of botrytis cinerea spores was not significantly different from that of the control group, however, the germ tube length was significantly lower than that of the control group.

Fig. 5 shows the inhibitory effect of pan. vagans bwl1 on penicilliosis of kiwifruit. After puncture inoculation, pan. vagans BWL1 treated group (BWL1) had significantly smaller lesions than control group.

Fig. 6 shows the inhibition effect of pan. vagans bwl1 on grey mould of kiwi fruit. After puncture inoculation, pan. vagans BWL1 treated group (BWL1) had significantly smaller lesions than control group.

Fig. 7 shows the inhibitory effect of pan. vagans bwl1 on the natural onset of penicilliosis in kiwifruit. After spray vaccination, the pan. vagans BWL1 treated group (BWL1) had significantly lower incidence than the control group.

Fig. 8 shows the inhibition effect of pan. vagans bwl1 on the natural onset of grey mould of kiwi fruits. After spray vaccination, the pan. vagans BWL1 treated group (BWL1) had significantly lower incidence than the control group.

Example 2: biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application of biocontrol bacterium in kiwi fruit storage

A biocontrol bacterium Pan.vagans BWL1 with high-efficiency antagonism to penicillium and botrytis cinerea, which is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 4 days in 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 bacterium Pan.vagans BWL1 of the microbial preparation of the biocontrol bacterium Pan.vagans BWL1 is more than or equal to 10⁸CFU/mL。

step 1: inoculating biocontrol bacteria Pan.vagans BWL1 in an LB culture medium, and culturing in a shaking table at 23 ℃ for 18 h;

step 2: inoculating the bacterial liquid obtained in the step 1 into a new LB culture medium according to the volume percentage of 5% for amplification culture;

and step 3: centrifuging the culture obtained in the step 2 at 5000 Xg for 4min, discarding the supernatant, and adding 1/5 volumes of phosphate buffer solution for resuspension;

A method for storing kiwi fruits, comprising applying a bacterial solution obtained by diluting the microbial preparation of biocontrol bacteria Pan. vagans BWL1 prepared in claim 2 on the outer surface of kiwi fruits.

The preferred embodiment is: the mass ratio of the microbial preparation of the biocontrol bacterium Pan.vagans BWL1 to the kiwi fruit is 10: 40000.

The preferred embodiment is: adding water 20-30 times of the microbial preparation of biocontrol bacteria Pan. vagans BWL1, dissolving, and spraying on fructus Actinidiae chinensis.

The preferred embodiment is: adding water 20-30 times of the microbial preparation of biocontrol bacteria Pan. vagans BWL1, immersing fructus Actinidiae chinensis, and taking out.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Sequence listing

<110> university of fertilizer industry

<120> biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinerea and application of biocontrol bacterium in kiwi fruit storage

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 1401

<212> DNA

<213> sequencing results of amplified fragment of 16SrDNA Gene

<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

tcgggaactc aaaggagact gccggtgata aaccggagga aggtggggat gacgtcaagt 1140

catcatggcc cttacgagta gggctacaca cgtgctacaa tggcgcatac aaagagaagc 1200

aacctcgcga gagcaagcgg acctcacaaa gtgcgtcgta gtccggatcg gagtctgcaa 1260

ctcgactccg tgaagtcgga atcgctagta atcgtggatc agaatgccac ggtgaatacg 1320

ttcccgggcc ttgtacacac cgcccgtcac accatgggag tgggttgcaa aagaagtagg 1380

tagcttaacc tcggaggcgc t 1401

<210> 2

<211> 1247

<212> DNA

<213> sequencing result of 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 result of amplified fragment of recA Gene

<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

gacgtcgaat tccctcarga 20

<210> 7

<211> 20

<212> DNA

<213> atpDR

<400> 7

tsgctttttc cacagcttct 20

<210> 8

<211> 20

<212> DNA

<213> recAF

<400> 8

garkcbtcng gtaaaacvac 20

<210> 9

<211> 21

<212> DNA

<213> recAR

<400> 9

ttcgcyttrc cctgrccrat c 21

Claims

1. Biocontrol bacterium for efficiently antagonizing penicillium and botrytis cinereaPan. vagansBWL1, which has been preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 4 days 2021, with the preservation number of CCTCC NO: m2021001.

2. Biocontrol bacteriumPan. vagansMicrobial preparation of BWL1, characterized in that: the biocontrol bacteriumPan. vagansBiocontrol bacterium of microbial preparation of BWL1Pan. vagansThe viable count of BWL1 is greater than or equal to 10⁸CFU/mL。

3. The biocontrol bacterium of claim 2Pan. vagansMicrobial preparation of BWL1, characterized in that: the preparation method comprises the following steps:

step 1: biological control bacteriaPan. vagansBWL1 is inoculated in LB culture medium and cultured for 12-18h in a shaking table at 23-27 ℃;

and 4, step 4: adding sodium carboxymethylcellulose, dextrin and sodium alginate, stirring, vacuum drying at low temperature, pulverizing, and sieving with 300 mesh sieve to obtain biocontrol bacteriaPan. vagansA microbial preparation of BWL 1.

4. A kiwi fruit storage method is characterized by comprising the following steps: biocontrol bacteria prepared by claim 2 or 3Pan. vagansBacterial liquid obtained by diluting the microbial preparation of BWL1 acts on the outer surface of kiwi fruit.

5. A method of kiwi storage according to claim 4, wherein: biocontrol bacteriumPan. vagansThe mass ratio of the BWL1 microbial preparation to the kiwi fruit is 10: 40000-60000.

6. A method of kiwi storage according to claim 4, wherein: biocontrol bacteriaPan. vagansThe microbial preparation BWL1 is added with water 20-30 times of its volume, dissolved and sprayed on the kiwi fruit.

7. A method of kiwi storage according to claim 4, wherein: biocontrol bacteriaPan. vagansAdding water 20-30 times the volume of BWL1 into the microbial preparation, immersing fructus Actinidiae chinensis, and taking out.