CN116200301B

CN116200301B - A strain of Westernella edodes for controlling gray mold in fruits and vegetables and its application

Info

Publication number: CN116200301B
Application number: CN202310032807.2A
Authority: CN
Inventors: 张丹凤; 胡婷婷; 毕婉玲; 叶应旺; 许雪华; 田心蕊
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2023-01-10
Filing date: 2023-01-10
Publication date: 2025-11-04
Anticipated expiration: 2043-01-10
Also published as: CN116200301A

Abstract

This invention discloses a strain of *Weissella cibaria* for controlling gray mold in fruits and vegetables and its application, belonging to the field of agricultural product storage technology. The *Weissella cibaria* strain, named *Weissella cibaria* BWL4, was deposited at the China Center for Type Culture Collection (CCTCC) on January 19, 2021, with accession number CCTCC NO: M2021109. This invention used the plate confrontation method and the plate overlap method to find that this strain has an inhibitory effect on *Gypsophila*. Experiments verified that this strain has a significant inhibitory effect on gray mold in grapes and kiwifruit. Microbial preparations made from this strain reduce the incidence of gray mold by inhibiting the growth of *Gypsophila* on the surface of harvested fruits and vegetables, improving the storage quality of fruits and vegetables, and extending their storage time, providing a new direction for the prevention and control of gray mold in fruits and vegetables.

Description

Weissella food for controlling gray mold of fruits and vegetables and application thereof

Technical Field

The invention relates to the technical field of agricultural product storage, in particular to a Weissella food for controlling gray mold of fruits and vegetables and application thereof.

Background

The botrytis cinerea (Botrytiscinerea) is widely distributed, can survive in water, air and soil, has extremely high adaptability, and is one of the most destructive fungal plant pathogens in horticultural crops. The hosts of the plant are very wide, and can infect more than 400 plants to cause gray mold on a plurality of organs of the plants, including flowers, fruits, leaves, branches, underground tubers and the like, wherein the leaves and the fruits are most seriously damaged, so that a large number of leaves fall off and the fruits rot, and the yield and quality of agricultural products such as vegetables, fruits and the like are seriously affected. In the plant pathogenic fungi hazard assessment, gray mold bacteria are ranked second to rice blast bacteria. The mold often enters these tissues at an early stage of crop development and remains dormant in the tissues for a long period of time when the environment is unfavorable, and therefore, healthy looking crops at harvest may decay during subsequent storage and transportation, resulting in serious economic losses. In addition, in products such as kiwi fruits, grapes, apples and pears, the gray mold can survive for a long time at the refrigerating temperature higher than the freezing point, and the storage and fresh-keeping of the picked fruits and vegetables are provided with serious challenges.

Grape and kiwi fruit are fruits which are popular with people. However, during postharvest storage they may be infested with a variety of pathogenic fungi, with gray mold caused gray mold being one of the most important postharvest diseases of grapes and kiwi fruits, the course of the disease developing rapidly once the fruit is infested with gray mold, and it is difficult to control, resulting in complete loss of commodity value to the fruit.

For a long time, the control of gray mold is mainly chemical control, but a large amount of pesticides can cause higher pesticide residues, in addition, gray mold can easily generate drug resistance to chemical pesticides, and the currently applied pesticides have found resistant strains at home and abroad, so that the control effect is continuously reduced. Therefore, biological control is becoming an increasingly important way to control gray mold.

Disclosure of Invention

The invention aims to provide a Weissella antrum for controlling gray mold of fruits and vegetables and application thereof, so as to solve the problems in the prior art. The Weissella antrum is named WeissellacibariaBWL to be preserved in China Center for Type Culture Collection (CCTCC) in 2021, 1 and 19 days, and the preservation number is CCTCCNO: M2021109, so that the gray mold of fruits and vegetables can be effectively inhibited.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a antral Weissella (Weissellacibaria) BWL4 for controlling fruit and vegetable gray mold, wherein the antral Weissella BWL4 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2021109 in 1 month 19 of 2021.

The invention also provides a microbial preparation comprising the Weissella antrum BWL4.

The invention also provides a method for preventing and treating gray mold of fruits and vegetables, wherein the bacterial liquid of the antrum Weissella BWL4 or the microbial preparation is sprayed or smeared on the surfaces of the fruits and vegetables.

Further, the concentration of the bacterial liquid of the Weissella antrum BWL4 is more than or equal to 10 ⁸ CFU/mL.

Further, the microbial preparation is diluted according to the mass-volume ratio of the microbial preparation to water of 1kg:50-100L and then used.

Further, the mass ratio of the dosage of the microbial preparation to the fruits and vegetables is 1:1000-10000.

The invention also provides application of the antral Weissella BWL4 or the microbial preparation in preventing and treating gray mold of fruits and vegetables.

The invention also provides application of the Weissella antrum BWL4 or the microbial preparation in storage of picked fruits and vegetables.

Further, by inhibiting the growth of gray mold on the surface of the picked fruits and vegetables, the occurrence rate of gray mold is reduced, and the storage quality of the fruits and vegetables is improved.

Further, inhibiting the growth of the botrytis cinerea on the surface of the picked fruit and vegetable comprises inhibiting spore germination, bud tube elongation and hypha penetration of the botrytis cinerea.

The invention discloses the following technical effects:

The invention separates and screens a strain of Weissella antrum WeissellacibariaBWL from the surface of healthy grape fruits, the strain is fruit-inhabiting bacteria, a flat-plate counter method and a flat-plate buckling method are adopted to find that the strain has an inhibitory effect on gray mold, and experiments prove that the strain has an obvious inhibitory effect on gray mold of grapes and kiwi fruits. The microbial preparation prepared from the strain can reduce the occurrence rate of gray mold on the surface of the picked fruits and vegetables by inhibiting the growth of gray mold on the surface of the picked fruits and vegetables, improve the storage quality of the fruits and vegetables and prolong the storage time of the fruits and vegetables.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 shows the morphology of strain w.cibariabwl4, wherein a is the plate culture result, B is the microscopic observation result, scale = 50 μm;

FIG. 2 shows the growth curve and pH change of strain W.cibariaBWL4, wherein A is OD ₆₀₀ during growth of strain W.cibariaBWL4 and B is pH change during growth of strain W.cibariaBWL 4;

FIG. 3 shows the antibacterial effect of the strain W.cibariaBWL4 on Botrytis cinerea, wherein A is a photograph of the W.cibariaBWL4 against a plate of Botrytis cinerea, and B is a photograph of the W.cibariaBWL4 against a plate of Botrytis cinerea;

fig. 4 is the inhibition of spore germination and shoot elongation of botrytis cinerea by strain w.cibariabwl4, scale = 100 μm, where a is a photograph of spore germination of botrytis cinerea, B is data of spore germination rate and shoot length of botrytis cinerea;

FIG. 5 shows the inhibitory effect of strain W.cibariaBWL4 on the penetration of Botrytis cinerea spores;

FIG. 6 shows the inhibitory effect of strain W.cibariaBWL4 on grape gray mold;

FIG. 7 shows the inhibitory effect of the strain W.cibariaBWL4 on kiwi fruit gray mold.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 isolation, screening and identification of biocontrol strain W.cibariaBWL4

The biocontrol strain W.cibariaBWL4 is separated from the surface of healthy grape fruits and is fruit-bearing growth strain, and a flat plate counter method is adopted to find that the biocontrol strain W.cibariaBWL4 has an inhibitory effect on Botrytis cinerea.

1. Isolation of strains

Commercially available healthy grapes are taken, sheared from pedicles with scissors, placed in a sterilized 500mL beaker, and sterilized water is added 200mL and 150r/min is shaken for 1h. After the suspension culture solutions were diluted to 10 ^-1、10^-2、10^-3 respectively, 100. Mu.L of each was spread on MRS solid plates, and subjected to inverted stationary culture at 25℃for 24 hours. And (3) picking single colonies to a new MRS solid plate for streak culture, and performing second streak culture on the obtained monoclonal strain to obtain a pure strain W.cibariaBWL4 (shown in figure 1). After 1 day of growth of the strain w.cibariabwl4 on 25 ° C, MRS medium, milky white, convex, smooth-edged colonies were formed. As can be seen from a microscope, the cells were short rods and had a length of 10 to 12. Mu.m. The strain obtained was preserved in 30% glycerol and placed in a low temperature environment at-80 ℃.

2. Screening of strains

The preparation of the spore suspension of the pathogenic bacteria, namely the botrytis cinerea after fruit and vegetable harvest comprises the steps of carrying out activation culture on the botrytis cinerea by adopting a V8 culture medium plate, culturing for 7d at 25 ℃, scraping hyphae, adding the obtained product into a triangular flask containing 20mL of sterile water, vibrating for 1min, filtering by double-layer sterile gauze to remove the hyphae, measuring the spore concentration by a blood cell counting plate, and adjusting the spore concentration to 10 ⁵/mL to obtain the spore suspension of the botrytis cinerea.

The V8 medium, commonly used potato dextrose agar medium, was supplemented with 20% V8 vegetable juice (V/V) and 0.3% CaCO ₃ (w/V), wherein the V8 vegetable juice was produced by Campbell' sSoup (Australia).

The antibacterial effect test of the strain adopts two methods of plate opposition and plate buckling.

The plate facing method comprises inoculating Botrytis cinerea mycelium block at the center of PDA plate, culturing at 25deg.C for 7d, growing Botrytis cinerea to 3/4 of the tray till full tray, taking Botrytis cinerea mycelium cake with diameter of 0.5cm from the position near mycelium edge, inoculating to the center of new PDA plate, culturing at 25deg.C for 3-5d, inoculating purified W.cibaimiaBWL 4 to liquid MRS medium, culturing at 25deg.C overnight, inoculating loop, dipping fungus solution, streaking on the PDA plate, culturing at 25deg.C, and observing antibacterial effect.

The plate buckling method comprises the steps of respectively pouring 15mL of MRS culture medium and 15mL of PDA culture medium into two plates with the diameter of 90mm, inoculating a Botrytis cinerea bacterial cake with the diameter of 5mm at the center of the PDA culture medium, and streaking W.cibariaBWL4 bacterial liquid with the OD ₆₀₀ = 0.8 on the MRS culture medium. After the inoculation, the PDA plate inoculated with the gray mold was snapped onto the MRS plate inoculated with W.cibaiaaBWL 4, sealed with a sealing film, and incubated at 25℃for 6d, and the results were observed.

The MRS medium was purchased from Guangdong CycloKai Biotech Co.

As shown in FIG. 3, W.cibariaBWL4 significantly inhibited the growth of Botrytis cinerea mycelium.

3. Identification of strains

Strains with inhibitory effects were selected by PCR amplification and sequencing of the 16srDNA, phenylalanyl-tRNAsynthetasesubunit alpha (Phes), chromosomalreplicationinitiatorprotein (dnaA) and ATPsynthasesubunit alpha (atpA) genes, wherein the PCR amplification system used to amplify the biocontrol strain W.cibariaBWL4 strain 16srDNA, phes, dnaA and the atpA gene sequences is shown in Table 1:

TABLE 1PCR amplification System

Component name	Additive amount
		10×LATaqBufferII(Mg²⁺Plus)	5μL
dNTPs(2.5mM)	9μL
		Primer F (1. Mu.M)	2μL
Primer R (1. Mu.M)	2μL
		DNA template	0.5μL
High-fidelity Taq enzyme	0.5μL
		ddH₂O	31μL

Primers used for amplifying the anti-bacterial strain W.cibariaBWL4 strain 16srDNA, phes, dnaA and the atpA gene sequences and annealing temperatures and extension times at the time of PCR amplification are shown in Table 2. The specific conditions for PCR were pre-denaturation at 95℃X 4min, [95℃X 20s, annealing temperature X20 s,72℃X 1 extension time ]. Times.35 cycles, 72℃X 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.

TABLE 2 PCR primers for each Gene and reaction conditions

Sequencing of the PCR products was as follows:

16SrDNA Gene amplified fragment sequencing results (SEQ ID NO. 1):

5'-GCAGTCGACGCTTTGTGGTTCAACTGATTTGAAGAGCTTGCTCAGATATGA CGATGGACATTGCAAAGAGTGGCGAACGGGTGAGTAACACGTGGGAAACCTACCTCTTAGCAGGGGATAACATTTGGAAACAGATGCTAATACCGTATAACAATAGCAACCGCATGGTTGCTACTTAAAAGATGGTTCTGCTATCACTAAGAGATGGTCCCGCGGTGCATTAGTTAGTTGGTGAGGTAATGGCTCACCAAGACGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGGTTTCGGCTCGTAAAACACTGTTGTAAGAGAAGAATGACATTGAGAGTAACTGTTCAATGTGTGACGGTATCTTACCAGAAAGGAACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTTCCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTATTTAAGTCTGAAGTGAAAGCCCTCAGCTCAACTGAGGAATTGCTTTGGAAACTGGATGACTTGAGTGCAGTAGAGGAAAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGTTGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAGTGCTAGGTGTTTGAGGGTTTCCGCCCTTAAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTTGACAACTCCAGAGATGGAGCGTTCCCTTCGGGGACAAGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTCGGAGC-3'.

phes sequencing of the amplified fragment of the gene (SEQ ID NO. 2):

5'-ATCTTGATGCGTACGCAAACGTCACCCGTTCAAGCCCGCACGTTGGAGTC ACACGACTTTAATGCTGGACCTTTGAAGATGGTATCACCTGGTCGTGTTTACCGTCGCGATACGGATGATGCAACGCACTCACACCAATTCCACCAAATGGAAGGACTTGTGATCGATAAGCACATCACGATGGGTGATTTGAAGGGAACGCTTTTGGCAGTTGCGCGCAACTTGTTTGGTGAAGACCATGATATTCGTTTGCGTCCATCGTACTTCCCATTCACGGAACCTTCTGTTGAAGTCGATGTGTCATGGAACGCGGTAACGCCTGACATGAACCCTGAAGATATCGAATGGATTGAAGTGCTT-3'.

dnaA sequencing of the amplified fragment of the gene (SEQ ID NO. 3):

5'-AACAATCCCAACGCCAAAGTTAAGTTCATTACGACTGAAGATTTCATTAAT GACTTCACGGAAGCTTTGCGTCGTGGTCAAAAAGAGACCGAAGCATTTAAGCGCGAATACCGCTCGACAGATCTCTTGCTAGTTGACGACGTCCAATTCTTGGCTGGTAAAGAAAAGATTCAAGAAGAATTCTTTAATACCTTCAATGCCATTACGCGAGAAAATCACCAAATCGTCCTGACATCAGATAAGTTACCAAAGGAAATTCCTGGCCTAGAAATGCGTTTGGTCACGCGTTTCGGGCAAGGTTATTCAGCAAACATTACGAAGCCTGACTTACCAACACGTGTCGCCATCCTACGTAACAAGTCAGATCAAGAAAACCTCAATATTCCAAATGATGTGATTGATGAAATCGCTGCGGCTGTTGATACGAACGTCCGTGATCTGGAAGGGGTCTTCAATCAAGTTGCTGGTAAGTTACGATTCGCATCACAGCCGGTCACGGTTGATACTGCTCGTGACATTCTCGAGACAATGAACTTCAAGCGGCAACGCGCCATCACGATTCCTATCATTCAAGATACGGTTGCAAAGTTCTTTGACGTGACCGTGCAAGACCTTAATGGTAAGAAGCGTAATAAGGAAATCGTGGTACCACGCCAAATCGCCATGTATTTGGCACGTGAGCTCACTCAAGAC-3'.

atpA gene amplified fragment sequencing results (SEQ ID NO. 4):

5'-TTGGTCGAGTTTGAGAACGGCGTATTTGGTATGGCACAAAACCTTGAGTC TAATGATGTTGGTATCATTATCCTAGGTAAGTACGACGAGATTCGCGAAGGCGATACTGTTAAGCGCACTGGTCGTATCATGGAAGTGCCTGTTGGTGAGGGATTGATCGGACGTGTTGTTAACGCATTGGGTCAACCAATCGACGGAATGGGACCAATTAACTCGACGAGCACTCGTCCAGTTGAAGTAAAGGCCCCAGGAGTTATGGAGCGTAAGTCTGTTTTCGAACCATTGCAAACTGGTTTGAAGGCCGTCGACGCCTTGGTTCCTATTGGTCGTGGACAACGTGAGTTGATCATCGGTGACCGTAAGACGGGTAAGACGTCTGTTGCCATCGACACGATCTTGAACCAAAAGGATCAAGACATGATCGTTATCTACGTGGCTATTGGACAAAAGGACTCAACTGTGCGTACGCAAGTTGAAACTTTGCGTCAAATGGGTGCTTTGGATTACACGATTGTTGTCTCAGCTGGTCCTTCAGAACCAGCCCCAATGTTGTACTTGGCACCTTATGCCGGAGCAGCAATGGGTGAAGAGTTCATGTACAACGGCAAGCACGTCTTGATTGTGTACGATGATTTGTCAAAGCAAGCTACGGCTTACCGTGAGCTGTCATTGATTCTTCGTCGTCCTCCTGGACGTGAAGCTTACCCTGGTGACGTCTTCTACTTGCACTCACGTTTGCTAGAACGTGCGGCTAAGTTGTCAGACGAATTGGGTGGCGGTTCTATGACTGCTTTGCCAGTTATCGAAACGCAAGCGGGTGACGTTTCTGCGTACATCCCAACGAACGTTATCTCAATCACCGACGGACAAATCTTCTTGGATGCCGACCAATTCTACGCCGGCGTACGTCCTGCCATCGAT-3'.

the sequencing results were aligned in the NCBI (national center for Biotechnology information) database, identifying the strain as Weissella antrum Weissellacibaria, and numbering the strain as BWL 4. 16srDNA, phes, dnaA and atpA gene amplified fragments have been submitted to NCBI database, with sequence numbers MW133053, MW364859, MW364860 and MW364861, in order. The strain is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of M2021109 in 2021, 1 and 19 days.

4. Growth curve of strain

Strain w.cibariabwl4 was inoculated in MRS liquid medium at 1% inoculum size, and cultured at 25 ℃ for stationary culture, and OD ₆₀₀ and pH were measured at 2, 4, 6, 8, 12, 16, 24, 36, 48h, respectively. As a result, as shown in FIG. 2, the strain W.cibariaBWL4 was cultured for 2 hours, i.e., it was grown in the logarithmic phase, and it was grown in the stationary phase for about 6 hours. The pH value is reduced from 5.5 to about 4.0 in 12h, and then the pH value is stable.

Example 2 Effect of strains on spore germination and bud tube elongation of Botrytis cinerea

Strain w.cibariabwl4 was inoculated in MRS liquid medium (available from guangdong-cykai biotechnology limited) and cultured overnight at 25 ℃,10000×g was centrifuged for 10min, and the supernatant, i.e., w.cibariabwl4 broth, was added to PDA medium (available from hangzhou baisi biotechnology limited) at 10% and plated (thickness 1-2 mm). The spore suspension of Botrytis cinerea was spread on the plate, and after culturing at 25℃for 12 hours, the spore germination rate and the length of the sprout tube were measured. Each treatment contained 9 replicates, each replicate containing 100 spores. PDA controls supplemented with 10% mrs medium were also set. The results are shown in FIG. 4.

As can be seen from FIG. 4, the W.cibariaBWL4 fermentation broth has a remarkable inhibition effect on the germination rate of the spores of the Botrytis cinerea and the length of the bud tubes, the germination rate of the spores in the fermentation broth treatment is reduced by 10.6% (p < 0.05), and the length of the bud tubes is reduced by 54.6% (p < 0.05). These results indicate that w.cibariabwl4 treatment significantly inhibited the germination of the botrytis cinerea spores.

EXAMPLE 3 inhibition of the penetration of the Gray mold wire by the Strain

Hyphal penetration indicates the pathogenicity of the gray mold, and a decrease in penetration indicates a decrease in gray mold pathogenicity. The Botrytis cinerea was inoculated on a V8 medium containing 10% W.cibariaBWL4 supernatant (W.cibariaBWL 4 fermentation broth in example 2), cultured at 25℃for 7d, and spores were collected to prepare a Botrytis cinerea spore suspension having a concentration of 10 ⁵ pieces/mL. The inner skin of the onion was cut into 1cm square pieces with a razor blade, and then torn off, spread in a petri dish, and water-bath at 68 ℃ for 1 hour to kill the onion cells. 200. Mu.L of the Botrytis cinerea spore suspension was dropped onto onion skin, covered with a petri dish cover, cultured at 25℃for 12 hours, the skin was stained with 0.25% trypan blue for 5 minutes, gently washed 3 times with phosphate buffer (50 mM, pH 7.0) to remove excess trypan blue dye, and whether or not the hyphae could penetrate onion cells was observed using an optical microscope. Hyphae capable of penetrating onion cells are difficult to be dyed by trypan blue inside the cells, and the hyphae are colorless or light blue, while hyphae incapable of penetrating grow on the surface of onion skin and are dyed by trypan blue to be dark blue. The experiment was repeated 3 times with the botrytis spores collected from normal V8 medium as a control. As a result, as shown in FIG. 5, most of the mycelia of the control group were colorless, i.e., penetrated onion cells, while most of the mycelia of the W.cibariaBWL4 supernatant treatment group were stained dark blue.

EXAMPLE 4 inhibition of grape and Kiwi berry Gray mold by Strain

1. Inhibition of grape gray mold by bacterial strain

Inoculating strain W.cibariaBWL4 into LB culture medium, culturing at 25deg.C overnight, centrifuging at 6000 Xg for 10min, removing supernatant, adding 50mM phosphate buffer (pH 7.0) for resuspension to make bacterial concentration about 10 ⁸ CFU/mL, selecting non-damaged and disease-free grape, shearing off fruit from pedicel with scissors, cleaning with clear water, sterilizing with 1% sodium hypochlorite surface for 3min, washing with sterile water, and air drying in ultra clean bench. The middle part of the fruit was perforated, 10. Mu.LW. CibariaBWL4 bacterial suspension was added to the holes, and 10. Mu.L phosphate buffer was used instead of BWL4 bacterial suspension in the control group. After complete absorption of the liquid, 10. Mu.L of pathogenic spore suspension (10 ⁵/mL) was inoculated in the wells, after complete absorption of the liquid, the fruit was placed in a plastic basket, placed in a dish with clear 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 treatment contained 6 replicates, each replicate contained 3 replicates, each replicate contained 30 grape fruits.

As shown in FIG. 6, it is clear from FIG. 6 that the incidence of grape was lighter in the treated group (inoculated with Botrytis cinerea spores and W.cibaimiaBWL 4 strain) than in the control group (inoculated with only Botrytis cinerea spores), the incidence was reduced by 38.4% (p < 0.05), and the disease index was reduced by 57.6% (p < 0.05). These results indicate that w.cibariabwl4 treatment significantly inhibited postharvest gray mold in grapes.

2. Inhibition of bacterial strain on kiwi gray mold

In order to simulate the influence of the strain W.cibariaBWL4 on natural morbidity of the kiwi fruits after storage, a spray inoculation mode is adopted, the strain W.cibariaBWL4 is inoculated in an MRS culture medium, cultured at 25 ℃ overnight, centrifuged for 10min at 6000 Xg, supernatant is removed, 50mM phosphate buffer (pH 7.0) is added for resuspension, the bacterial concentration is approximately equal to 10 ⁸ CFU/mL, the kiwi fruits without damage and diseases are selected, after the kiwi fruits are washed by clear water, the kiwi fruits are washed clean by sterile water after being sterilized for 3min on the surface of 1% sodium hypochlorite, and the kiwi fruits are placed in an ultra-clean bench for airing. The w.cibariabwl4 bacterial suspension was evenly sprayed, and 10 μl of phosphate buffer was used instead of the w.cibariabwl4 bacterial suspension in the control group. After the surface of the kiwi fruit is completely dried, the kiwi fruit is uniformly sprayed with the ash mold spore suspension, after the surface of the kiwi fruit is completely dried, the kiwi fruit is placed into a plastic basket, a plate filled with clear water is placed into the plastic basket to keep humidity, a plastic bag is sleeved outside the frame, and the kiwi fruit is placed for 15 days at 21 ℃, so that the morbidity is counted. Each treatment contained 3 replicates, each replicate contained 2 replicates, each replicate contained 20 kiwi fruits.

As shown in fig. 7, it is clear from fig. 7 that the treatment group (spraying the botrytis cinerea spores and w.cibaia bwl4 bacteria liquid) had a lower incidence of kiwi fruits by 64.1% (p < 0.05) and a lower disease index by 56.4% (p < 0.05) than the control group (spraying only the botrytis cinerea spores). These results indicate that w.cibariabwl4 treatment significantly inhibited post-harvest gray mold in kiwi fruits.

Example 5 application of Strain W.cibariaBWL4 in fruit and vegetable storage

Preparation of microbial preparation with strain w.cibariabwl 4:

step 1, preparing a protective agent, namely 12 percent of skim milk, 1 percent of trehalose, 1 percent of sodium glutamate and 3 percent of glycerol, and sterilizing for later use;

step 2, inoculating biocontrol bacteria W.cibariaBWL4 into a 50mLMRS culture medium, and culturing for 18 hours in a 25 ℃ shaking table;

Step 3, inoculating the bacterial liquid obtained in the step 2 into a new MRS culture medium according to the volume percentage of 5% for expansion culture, wherein the culture conditions are the same as those in the step 2;

Step 4, centrifuging the culture in the step 3 at 5000 Xg for 4min, discarding the supernatant, washing twice with phosphate buffer (50 mM, pH 7.0), centrifuging at 5000 Xg for 4min, discarding the supernatant to obtain fresh thalli, and weighing;

And 5, adding the protective agent prepared in the step 1 into fresh thalli according to the proportion of 1g to 1mL, uniformly stirring, vacuum freeze-drying, crushing and sieving with a 300-mesh sieve to obtain the microbial preparation of the biocontrol strain W.cibariaBWL 4.

Adding the obtained microbial preparation into water with the mass being 100 times of that of the microbial preparation, diluting, and spraying the microbial preparation on the picked kiwi fruits, grapes, apples and pears after dissolving, wherein the mass ratio of the microbial preparation of W.cibaiaaBWL 4 to the kiwi fruits, the grapes, the apples and the pears is 1:10000.

After 3 weeks of storage, the fruits and vegetables have no obvious disease occurrence phenomenon.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A strain of Weissellacibaria BWL4 for controlling gray mold in fruits and vegetables, characterized in that the Weissellacibaria BWL4 was deposited at the China Center for Type Culture Collection on January 19, 2021, with accession number CCTCCNO: M2021109.

2. A microbial preparation, characterized in that it comprises Weissella esculenta BWL4 as described in claim 1.

3. A method for preventing and controlling gray mold in fruits and vegetables, characterized in that the bacterial solution of Weissella tamariscina BWL4 as described in claim 1 or the microbial preparation as described in claim 2 is sprayed or applied to the surface of fruits and vegetables.

4. The method according to claim 3, wherein the bacterial concentration of Weissella esculenta BWL4 is greater than or equal to ^10⁸ CFU/mL.

5. The method according to claim 3, wherein the microbial preparation is diluted at a mass-volume ratio of 1 kg of microbial preparation to 50-100 L of water before use.

6. The method according to claim 3, wherein the ratio of the amount of the microbial agent to the mass of the fruits and vegetables is 1:1000-10000.

7. The application of *Westernella esculenta* BWL4 as described in claim 1 or the microbial preparation as described in claim 2 in the prevention and control of gray mold in fruits and vegetables.

8. The application of *Westernella esculenta* BWL4 as described in claim 1 or the microbial preparation as described in claim 2 in the storage of harvested fruits and vegetables.

9. The application according to claim 8, characterized in that, by inhibiting the growth of gray mold on the surface of harvested fruits and vegetables, the incidence of gray mold is reduced, and the storage quality of fruits and vegetables is improved.

10. The application according to claim 9, wherein the inhibition of the growth of gray mold on the surface of harvested fruits and vegetables includes inhibiting the spore germination, germ tube elongation and hyphal penetration of the gray mold.