CN114517172B

CN114517172B - Staphylococcus equi and application thereof in prevention and control of gray mold of fruits and vegetables

Info

Publication number: CN114517172B
Application number: CN202210337953.1A
Authority: CN
Inventors: 王彩霞; 郝柏慧; 赵润欣; 孙子豪; 李保华
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2023-04-28
Anticipated expiration: 2042-04-01
Also published as: CN114517172A

Abstract

The invention discloses a staphylococcus equi strain and application thereof, the strain is named as staphylococcus equi (Staphylococcus equorum) F1, and is preserved in China general microbiological culture collection center (CGMCC) No.21658 in the 1 st month 18 of 2021, and the preservation address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. The staphylococcus equi F1 has a high-efficiency antibacterial effect on fruit and vegetable botrytis cinerea, can obviously inhibit the growth of botrytis cinerea hyphae and the germination of spores, can be applied to the preparation of biocontrol bactericides and fruit and vegetable fresh-keeping agents, has a wide antibacterial spectrum, has the characteristics of stable control effect, environmental friendliness and the like, is suitable for large-scale production, and has low production cost.

Description

Staphylococcus equi and application thereof in prevention and control of gray mold of fruits and vegetables

Technical Field

The invention relates to the technical field of microorganisms, in particular to a staphylococcus equi and application thereof in preventing and controlling gray mold of fruits and vegetables.

Background

Gray mold caused by Botrytis cinerea is one of important diseases during postharvest storage of fruits and vegetables, and the disease can occur in various links of fruit and vegetable transportation, storage and the like, and causes serious economic loss each year. After the fruit and vegetable are infected with the gray mold, round or nearly round water stain-shaped soft rot spots are formed at the disease-affected parts, and the gray mold and conidia of the mice are produced on the later stage spots. Because the pathogenic bacteria have strong low temperature resistance, the pathogenic bacteria can cause fruit and vegetable rot even under refrigeration condition, and contact infection exists. The effective control of gray mold is significant for improving the storage and fresh-keeping of the picked fruits and vegetables.

At present, the traditional method for preventing and treating the gray mold of the picked fruits and vegetables mainly relies on chemical bactericides, but the problems of chemical bactericide residue, easy resistance of pathogenic bacteria to the chemical bactericide, serious environmental pollution and the like are increasingly serious, and various chemical bactericides are limited or forbidden to be used. Therefore, the search for safe and effective control measures to replace chemical bactericides has become a problem to be solved in postharvest storage of apples. A large number of researches show that biological control is an effective way for solving the sustainable development of agriculture due to the high efficiency and no pollution of the biological control, and some biological control preparations are applied to production.

The biological control microbial inoculum is used for controlling the gray mold of fruits and vegetables, is environment-friendly, and can avoid a series of problems caused by chemical control. The biocontrol microbial inoculum can reduce or replace the use of chemical agents, thereby achieving the purpose of preventing and controlling the gray mold of fruits and vegetables.

However, the existing biological strains for preparing the biocontrol microbial agents for preventing and treating the gray mold of fruits and vegetables are few, and the corresponding effective biocontrol microbial agents are lacking, so that the technical problems are solved, and the technical problems which need to be solved in the technical field of microorganisms at present.

Disclosure of Invention

In order to solve the problems, the invention provides the staphylococcus equi (Staphylococcus equorum) F1 and the application thereof in preventing and controlling the fruit and vegetable gray mold, and the staphylococcus equi F1 has high-efficiency antibacterial effect on the fruit and vegetable gray mold, has a wide antibacterial spectrum, can be widely applied to preventing and controlling the fruit and vegetable gray mold, and has the advantages of stable preventing and controlling effect and environmental friendliness.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a strain of staphylococcus equi designated staphylococcus equi (Staphylococcus equorum) F1. The strain is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) No.21658 at 18 days of 1 month 2021, and the preservation address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

In a second aspect, the invention also provides a biocontrol microbial inoculum prepared from the staphylococcus equine, wherein the biocontrol microbial inoculum is thalli, bacterial suspension, fermentation filtrate or active extract of the staphylococcus equine F1.

In a third aspect, the invention provides an application of the staphylococcus equi or the biocontrol microbial inoculum in preventing and controlling fruit and vegetable diseases; preferably, the fruit and vegetable diseases comprise fruit and vegetable gray mold. Fruits and vegetables include various fruits and vegetables that can be infested with fruits and vegetables. In the following specific examples, tomato, grape and apple are taken as examples for experiments.

Preferably, the staphylococcus equi is used for inhibiting the growth of botrytis cinerea hyphae and spore germination.

In a fourth aspect, the invention also provides a preparation method of the biocontrol microbial agent, which comprises the following steps:

s1, streaking and inoculating the staphylococcus equi F1 in a solid culture medium, and performing activation culture to obtain activated single colonies;

s2, inoculating the activated staphylococcus equi F1 single colony into a liquid culture medium, and shake culturing to obtain fermentation seed liquid;

s3, inoculating the fermentation seed liquid into a liquid culture medium for expansion culture to obtain a cell culture liquid of staphylococcus equi F1;

s4, performing centrifugal separation on the cell culture solution to obtain bacterial precipitate, and re-suspending the bacterial precipitate by using sterile water to obtain bacterial suspension of staphylococcus equine F1, namely the biocontrol microbial inoculum.

Preferably, the solid medium is PDA medium and the liquid medium is PDB medium.

Preferably, the culture condition of the step S1 is 28 ℃ constant temperature activation culture for 24 hours.

Preferably, the culture conditions in step S2 are that the activated strain F1 single colony is inoculated into PDB culture medium with the bottling quantity of 80mL/250mL and 180 r.min ^-1 Shake culturing at 28 deg.C for 48 hr.

Preferably, the culture condition of the step S3 is that the fermentation seed liquid is inoculated into a PDB culture medium for expansion culture according to the inoculum size of 1:100, and the constant temperature shake culture is carried out for 48 hours at 28 ℃ to obtain 10 ⁹ cfu·mL ^-1 Cell culture broth of F1 strain of (C).

Preferably, the culture conditions for step S4 are 10 ⁹ cfu·mL ^-1 Cell culture solution c of F1 strain 12000 r.min at 4deg.C ^-1 Centrifuging for 15min, collecting F1 thallus precipitate, and re-suspending with sterile water to obtain 10 ⁹ cfu·mL ^-1 F1 bacterial suspension of (a).

In a fifth aspect, the invention also provides a fruit and vegetable preservative, the active ingredient of which is the staphylococcus equi. Preferably, the fruit and vegetable preservative directly adopts the bacterial liquid or bacterial heavy suspension of the staphylococcus equi F1 or bacterial fermentation supernatant, or takes the bacterial liquid or bacterial heavy suspension as an active ingredient, and auxiliary materials are added to prepare different formulations.

Experiments prove that after the staphylococcus equi F1 bacterial liquid is soaked, the disease spots caused by the gray mold of fruits and vegetables can be effectively inhibited. Therefore, the strain can be used for preparing fruit and vegetable preservative, and the preservative containing the bacterial liquid of the strain is used for carrying out soaking treatment or spraying treatment on fruits and vegetables to be treated and then storing or transporting the fruits and vegetables, so that the preservation time of the fruits and vegetables can be effectively prolonged, and the fruits and vegetables can be prevented from being rapidly rotten due to gray mold infection.

In a sixth aspect, the invention also provides a fruit and vegetable fresh-keeping method, which comprises the following steps: soaking fruits and vegetables in the fruit and vegetable preservative, or spraying the fruit and vegetable preservative on the surfaces of the fruits and vegetables.

Preferably, in the fruit and vegetable fresh-keeping method, the fruits and vegetables are tomatoes, grapes and apples.

Advantageous effects

1. According to the invention, a staphylococcus equi (Staphylococcus equorum) strain F1 is firstly separated, and has remarkable control effect on gray mold of fruits and vegetables, and researches in the embodiment show that the strain F1 can remarkably inhibit the growth of gray mold hyphae and the germination of spores; in addition, the bacterial strain F1 has a wide bacteriostasis spectrum, has excellent antagonism on various plant pathogenic bacteria such as apple tree rot bacteria, apple ring rot bacteria, apple anthracnose and the like besides fruit and vegetable gray mold bacteria, and can be used for preventing and treating various plant diseases at the same time.

2. The bacterial strain F1 bacterial suspension has excellent control effect on the gray mold of fruits and vegetables, which is more than 81%, wherein the control effect on the gray mold of apples is as high as 93.51%; the strain F1 can induce the activity of relevant enzymes in plant tissues to be increased, and the expression level of disease resistance genes is improved; the biocontrol microbial inoculum has the characteristics of stable control effect, environmental friendliness and the like, is suitable for large-scale production, has simple fermentation process and low production cost, and has good market prospect.

3. Based on the excellent control effect of the strain F1 on the botrytis cinerea, the strain can be used for preparing fruit and vegetable preservative, and the preservative containing the bacterial liquid of the strain is used for carrying out soaking treatment or spraying treatment on fruits and vegetables to be treated and then storing or transporting the fruits and vegetables, so that the preservation time of the fruits and vegetables can be effectively prolonged, and the fruits and vegetables can be prevented from being rapidly rotten due to gray mold infection.

Drawings

FIG. 1 shows colony morphology of strain F1 on LB medium;

FIG. 2 is a schematic diagram of PCR amplification of 16S rDNA sequences of extracted DNA of strain F1;

wherein M in the figure is nucleic acid Marker DL2000; FIG. 1 shows the result of amplification of the 16SrDNA sequence of the genomic DNA of strain F1;

FIG. 3 is the effect of strain F1 on fruit and vegetable Botrytis cinerea mycelium growth (a) and spore germination (b);

wherein, A is the influence of the strain F1 on the growth of the botrytis cinerea hyphae; in the graph, B is the influence of the strain F1 on the germination of botrytis cinerea spores;

FIG. 4 shows the control effect of strain F1 on tomato and grape gray mold;

wherein, graph A shows the incidence of gray mold of tomato and grape after treatment with strain F1; panel B is the diameter of the lesions of tomato and grape gray mold after treatment with strain F1;

FIG. 5 shows the control effect of strain F1 on apple gray mold;

wherein in the figure, A, B and C are controls of inoculating the apple fruits with a PDB culture medium and then inoculating the apple fruits with botrytis cinerea; in the graph, D, E and F are the control effects of the strain F1 on the gray mold of apples;

FIG. 6 is the effect of strain F1 treatment on defense-related enzyme activity in apple fruit tissue;

FIG. 7 shows the effect of strain F1 suspension treatment on the expression of disease course related protein genes in apple fruit tissue.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified. The reagents used, unless otherwise specified, are commercially available.

Culture medium for experiment and formula thereof

1. LB medium: 10g of tryptone, 5g of yeast extract, 5g of NaCl and adjusting the pH to 7.0.

2. PDA medium: weighing 200g of potato after peeling, cutting into small pieces, boiling in water for 15-20 min, filtering with four layers of gauze, adding 20g of glucose and 15g of agar powder, fixing volume to 1000mL, sterilizing with high pressure steam at 121 ℃ for 20min

3. PDB medium: weighing 200g of potato after peeling, cutting into small pieces, boiling in water for 15-20 min, filtering with four layers of gauze, adding 20g of glucose, fixing volume to 1000mL, sterilizing with natural pH value at 121 ℃ under high pressure steam for 20min.

EXAMPLE 1 isolation screening and Strain identification of Staphylococcus equi F1

1. Isolation and screening of strains

Mature healthy fruits are collected from a Shandong commercial Fuji apple garden, surface disinfection is carried out, fruit tissues are taken and ground, sterile water is added, a conventional gradient dilution coating separation method is adopted, PDA culture media are used for respectively culturing at 28 ℃, colonies with obvious colony morphology difference are selected, purified and stored on the PDA culture media, primary screening and multiple secondary screening of antagonistic bacteria are carried out by taking apple tree rot germs as target pathogenic bacteria, and finally a bacterial strain with bacteriostasis and remarkable prevention and treatment effects is obtained, and the bacterial strain is named as F1.

2. Identification of Strain F1

(1) Morphological features

As shown in the results of FIG. 1, strain F1 was identified as gram-positive bacteria by having small colonies, off-white, rounded (slightly irregular) protrusions, opaque, and wet and smooth surfaces on LB medium.

(2) Physiological and biochemical characteristics

Through physiological and biochemical tests, the strain F1 can use glucose, sucrose, mannitol and the like as carbon sources, and galactose, lactose and the like cannot be used as carbon sources; the catalase, the V-P, the methyl red and the urease react positively, can liquefy the legendary, hydrolyze starch and do not form a bacterial film, and the characteristics are consistent with that of the staphylococcus equi.

(3) Gene identification

A. The experimental method comprises the following steps: extracting genome DNA of the strain F1 by using the kit, taking the genome DNA as a DNA template, and adopting a universal Primer F:5'-AGAGTTTGATCCTGGCTCAG-3' Primer R:5'-AAGGAGGTGATCCAGCCGCA-3' PCR amplification was performed on the DNA template.

PCR amplification reaction System (50. Mu.L): DNA template 2. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, taq enzyme (TaKaRa) 1. Mu.L, dNTP 1. Mu.L, 2×Taq buffer 25. Mu.L, ddH ₂ O17. Mu.L. PCR amplification procedure: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 2min, and extension at 72℃for 10min were performed for 35 cycles. The amplified product (5. Mu.L) was detected by 1% agarose gel electrophoresis. The PCR amplification result is shown in FIG. 2, and the PCR amplification product was recovered and sent to Shanghai Bioengineering Co., ltd.

B. Results and analysis: the determination result shows that the length of the 16S rDNA sequence amplified fragment is 1431bp, and the sequence is shown as SEQ ID NO. 1 of the sequence table. The resulting sequences were aligned with the nucleic acid sequences in GenBank using BLAST software from NCBI database (http:// www.ncbi.nlm.nih.gov), and were 99.9% homologous to the 16S rDNA sequences of the C4044 strain and the C2060 strain of Staphylococcus equi (Staphylococcus equorum) (Accession No: KF439736 and KF 439734).

Based on the results of the morphological, physiological and biochemical characteristics and sequence analysis described above, strain F1 was identified as staphylococcus equi (Staphylococcus equorum).

Example 2 Effect of Staphylococcus equi F1 on the hyphal growth and spore germination of Botrytis cinerea on fruits and vegetables

(1) Effect of Strain F1 on Botrytis cinerea mycelium growth

A. The experimental method comprises the following steps: inoculating a seed fermentation broth of staphylococcus equi (Staphylococcus equorum) F1 into a PDB culture medium, performing shake culture at a constant temperature of 28 ℃ for 48 hours to obtain a cell culture broth of the strain F1, centrifuging, and collecting bacterial cells of the strain F1. PDA culture medium was prepared, and cells of strain F1 were added thereto so as to have a final concentration of 10 ⁵ 、10 ⁷ And 10 ⁹ cfu·mL ^-1 PDA without strain F1 was used as a control. Inoculating activated fruit and vegetable botrytis cinerea in the PDA culture medium. The colonies were cultivated in dark at a constant temperature of 25℃for 3d, and the diameters of the colonies were observed and measured.

B. Results and analysis:

the result shows that the addition of F1 bacteria with different concentrations has a certain inhibition effect on the botrytis cinerea of fruits and vegetables, and the inhibition effect is obviously improved along with the improvement of the concentration of the strain F1. When the strain F1 is not added, the bacterial colony diameter of the gray mold is 7.46cm; the bacterial concentration of the strain F1 is 10 ⁵ cfu·mL ^-1 When the bacterial colony diameter of the gray mold is 5.01cm; f1 cell concentration of 10 ⁷ cfu·mL ^-1 When the bacterial colony diameter of the gray mold is only 0.89cm; the concentration of the bacterial F1 is 10 ⁹ cfu·mL ^-1 When the colony diameter was less than 0.6cm (FIG. 3A).

(2) Effect of Strain F1 on Botrytis cinerea spore germination

A. The experimental method comprises the following steps: preparing spore suspension of Botrytis cinerea, dark culturing pathogenic bacteria on PDA plate, washing conidium on mycelium with 0.05% Tween 20 after 7dFiltering and collecting Botrytis cinerea spores by using four layers of mirror wiping paper, and preparing a conidium suspension to adjust the concentration to 10 ⁶ Individual mL ^-1 。

Inoculating seed fermentation broth of strain F1 into PDB culture medium, shake culturing at 28deg.C for 48 hr to obtain cell culture broth of strain F1, centrifuging, collecting F1 thallus, re-suspending with sterile water to obtain bacterial suspension 10 with different concentrations ⁹ cfu·mL ^-1 ，10 ⁷ cfu·mL ^-1 And 10 ⁵ cfu·mL ^-1 . The F1 strain bacterial suspension with each concentration is respectively mixed with the equal volume of the conidium suspension 10 of the botrytis cinerea ⁶ Individual mL ^-1 Mixing, and standing in dark at 25deg.C for 24 hr to observe the germination rate of Botrytis cinerea spores.

B. Results and analysis:

as shown in the results of FIG. 3B, the germination rate of the conidia of the Botrytis cinerea in the control group (CK) is 84.50%, and the F1 strain has remarkable inhibition effect on the germination of the conidia of the Botrytis cinerea at different concentrations, wherein the concentration is 10 ⁹ cfu·mL ^-1 The inhibition effect of F1 thalli is most obvious, the spore germination rate is only 2.38%, and the inhibition rate is as high as 97.19%.

EXAMPLE 3 inhibition of Staphylococcus equi F1 against various plant pathogens

A. The experimental method comprises the following steps: preparation of a Staphylococcus equi (Staphylococcus equorum) F1 suspension to a final concentration of 10 ⁹ cfu·mL ^-1 PDA without strain F1 was used as a control, and each pathogen was inoculated with an activating culture.

Antibacterial ratio= (control colony diameter-treatment colony diameter)/control colony diameter x 100%.

B. Results and analysis: as shown in the results of Table 1, the strain F1 has remarkable inhibition effect on main plant pathogenic bacteria such as fruit and vegetable botrytis cinerea, apple tree rot pathogen, apple ring spot pathogen, apple anthracnose pathogen and the like, and has good broad-spectrum antibacterial property, and the antibacterial rate of the strain F1 in 4d cultivation is shown in Table 1.

TABLE 1 inhibition of major plant pathogens by Staphylococcus equi (Staphylococcus equorum) Strain F1

Example 4 preparation of biocontrol microbial agent

The embodiment provides a biocontrol microbial agent prepared by adopting a strain F1, and the preparation method specifically comprises the following steps:

(1) Streaking and inoculating the strain F1 in a PDA culture medium, and performing constant-temperature activation culture for 24 hours at the temperature of 28 ℃ to obtain an activated strain F1 single colony a;

(2) Inoculating the activated strain F1 single colony a into PDB culture medium with a bottling amount of 80mL/250mL, and 180 r.min ^-1 Shake culturing at 28 ℃ for 12h to obtain a strain F1 fermentation seed liquid b;

(3) Inoculating the fermentation seed liquid b into a PDB culture medium according to an inoculum size of 1:100 for expansion culture,

shake culturing at 28deg.C for 48 hr to obtain 10 ⁹ cfu·mL ^-1 Cell culture broth c of F1 strain;

(4) Will 10 ⁹ cfu·mL ^-1 Cell culture solution c of F1 strain 12000 r.min at 4deg.C ^-1 Centrifuging for 15min, and re-suspending thallus precipitate with sterile water to obtain 10 ⁹ cfu·mL ^-1 The bacterial suspension of (2) is the biocontrol bacterial agent.

The above-mentioned 10 ⁹ cfu·mL ^-1 When in use, the bacterial suspension is diluted by 0-10000 times.

EXAMPLE 5 control Effect of Staphylococcus equi F1 on tomato and grape gray mold

1. The experimental method comprises the following steps:

preparation of Staphylococcus equi (Staphylococcus equorum) Strain F1 suspension 10 ⁹ cfu·mL ^-1 After the gray mold bacteria of the fruits and vegetables are activated and cultured on PDA, the gray mold bacteria of the fruits and vegetables are induced to produce spores at the constant temperature of 25 ℃ in a dark place; 7d later, the conidium on the mycelium is washed down by 0.05% Tween 20, and the concentration of the conidium is adjusted to 10 ⁶ Individual mL ^-1 。

Selecting cherry tomato and grape fruits with uniform and healthy size, soaking in bacterial suspensions of different concentrations of the strain F1 for 30min, naturally airing at room temperature, and storing in dark under the condition of 4 ℃ and relative humidity of 95%. After 30d of storage, the fruit and vegetable botrytis cinerea spore suspension is inoculated by stabbing, the morbidity is counted 2d after inoculation, the diameter of the disease spots is measured, and the technical control effect is achieved.

Control effect= (control group lesion diameter-treatment group lesion diameter)/control group lesion diameter x 100%.

2. Experimental results and analysis

As shown in the results of fig. 4, the different bacterial suspensions of the strain F1 have remarkable control effects on tomato and grape gray mold; the incidence rate of the control group is 100%, and the diameters of the lesions of the grape control group and the tomato control group are respectively 1.55cm and 1.50cm; after the grape and the tomato are treated by the strain F1 with different concentrations, gray mold bacteria are inoculated, the incidence rate of the tomato is reduced to about 75%, and the incidence rate of the grape is reduced to 66.67%; the diameter of the disease spots on the grapes is only 0.28cm, and the control effect is as high as 81.97%; after tomato is treated by the strain F1, the diameter of the disease spots is 0.28cm, and the control effect is up to 81.07%.

EXAMPLE 6 control Effect of Staphylococcus equi F1 on apple gray mold

1. The experimental method comprises the following steps:

preparation of Strain F1 suspension 10 ⁹ cfu·mL ^-1 The Botrytis cinerea is activated and cultured on PDA for 3d, and bacterial cakes with the diameter of 5mm are picked up at the edge of bacterial colony for standby.

Selecting healthy Fuji apples with uniform size, sterilizing the surfaces of the Fuji apples with 75% alcohol, airing at room temperature, and making 3 wounds at the equatorial position of the apples by using a sterile puncher, wherein the diameter of each wound is 5mm, and the depth of each wound is 3mm. Respectively inoculating 30 mu L F bacteria suspension to fruit wounds, airing at room temperature, inoculating the activated fruit and vegetable botrytis cinerea bacterial cake, and taking the treatment of inoculating PDB culture medium and then inoculating botrytis cinerea as a control group. And calculating the control effect according to the diameter of the disease spots.

2. Experimental results and analysis:

as shown in FIG. 5, the diameter of the lesion in the control group after 3d inoculation is1.85cm ² The diameter of the disease spot is obviously reduced after F1 bacterial suspension is treated and is only 0.12cm, and the prevention effect is as high as 93.51%.

EXAMPLE 7 Effect of Staphylococcus equi F1 bacterial suspension on defense-related enzyme Activity in apple tissue

1. The experimental method comprises the following steps:

healthy Fuji apple fruit was selected and uniformly sprayed 10 according to the method of example 4 ⁹ cfu·mL ^-1 F1 bacterial suspension of (2) with sterile water treated fruits as control. And respectively taking apple peel tissues at 0, 12, 24, 48, 72 and 120 hours after treatment, and measuring the change trend of the activity of the defense-related enzymes in the apple tissues. Wherein, the activity measurement of POD (peroxidase) and SOD (superoxide dismutase) adopts guaiacol method and xanthine oxidase method respectively; PAL (phenylalanine ammonia lyase) and PPO (polyphenol oxidase) activities were measured by phenylalanine method and catechol method, respectively.

2. Experimental results and analysis

The results are shown in FIG. 6, 10 ⁹ cfu·mL ^-1 After apple fruit is treated by the F1 bacterial suspension of (2), peroxidase (POD) and Phenylalanine Ammonia Lyase (PAL) activities in branch tissues are increased to different degrees compared with a control, and the activities reach enzyme activity peaks respectively at 48h and 72h after treatment, which shows that the staphylococcus equine F1 can induce the resistance of a host to diseases by improving the activities of relevant enzymes in the fruit.

Example 8 Effect of bacterial suspension of Staphylococcus equi F1 on the expression of disease-related protein genes in apple tissue

1. The experimental method comprises the following steps:

healthy Fuji apple shoots were selected and uniformly sprayed 10 according to the method of example 4 ⁹ cfu·mL ^-1 F1 bacterial suspension of (2) with sterile water treated shoots as control. And respectively taking

apple peel tissues

0, 24 and 48 hours after treatment, extracting total RNA of a sample, performing reverse transcription, and determining the expression condition of protein genes related to the disease course in the apple tissues by using an RT-qPCR technology.

2. Experimental results and analysis

As a result, as shown in FIG. 7, the concentration was 10 ⁹ cfu·mL ^-1 F1 bacterium of (2)After the apple fruits are treated by the suspension, the expression level of the disease course related protein genes MdPR1 and MdPR5 in the tissues is obviously increased compared with the control; 24 hours after treatment, the expression quantity of MdPR1 reaches the highest, and is respectively improved by 21.63 times compared with the control; when 48h is treated, the expression level of MdPR5 reaches the highest, and is improved by 18.51 times compared with a control, which shows that the staphylococcus equine F1 can induce the up-regulated expression of the protein genes related to the disease course in apple tissues, thereby improving the resistance of hosts to diseases.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Qingdao university of agriculture

<120> a strain of staphylococcus equi and application thereof in prevention and treatment of gray mold of fruits and vegetables

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<213> 16S rDNA of Staphylococcus equi F1 (16S rDNA of Staphylococcus equorum F1)

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tatacatgca agtcgagcga acggataagg agcttgctcc tttgaagtta gcggcggacg 60

ggtgagtaac acgtgggtaa cctacctata agactggaat aacttcggga aaccggagct 120

aatgccggat aacatttgga accgcatggt tctaaagtaa aagatggttt tgctatcact 180

tatagatgga cccgcgccgt attagctagt tggtaaggta acggcttacc aaggcaacga 240

tacgtagccg acctgagagg gtgatcggcc acactggaac tgagacacgg tccagactcc 300

tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg 360

cgtgagtgat gaaggttttc ggatcgtaaa actctgttat tagggaagaa caaatgtgta 420

agtaactgtg cacatcttga cggtacctaa tcagaaagcc acggctaact acgtgccagc 480

agccgcggta atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagcgcgcgt 540

aggcggtttc ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg tcattggaaa 600

ctgggaaact tgagtacaga agaggaaagt ggaattccat gtgtagcggt gaaatgcgca 660

gagatatgga ggaacaccag tggcgaaggc gactttctgg tctgtaactg acgctgatgt 720

gcgaaagcgt ggggatcaaa caggattaga taccctggta gtccacgccg taaacgatga 780

gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840

cctggggagt acgaccgcaa ggttgaaact caaaggaatt gacggggacc cgcacaagcg 900

gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaaatct tgacatcctt 960

tgaaaactct agagatagag ccttcccctt cgggggacaa agtgacaggt ggtgcatggt 1020

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttaaa 1080

cttagttgcc agcatttagt tgggcactct aggttgactg ccggtgacaa accggaggaa 1140

ggtggggatg acgtcaaatc atcatgcccc ttatgatttg ggctacacac gtgctacaat 1200

ggacaataca aagggcagct aaaccgcgag gtcatgcaaa tcccataaag ttgttctcag 1260

ttcggattgt agtctgcaac tcgactacat gaagctggaa tcgctagtaa tcgtagatca 1320

gcatgctacg gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca ccacgagagt 1380

ttgtaacacc cgaagccggt ggagtaacca tttatggagc tagccgtcga a 1431

Claims

1. A strain of staphylococcus equi is characterized by being named as staphylococcus equi (Staphylococcus equorum) F1, and the preservation number of the staphylococcus equi is CGMCC No.21658.

2. A biocontrol agent prepared by utilizing the staphylococcus equine of claim 1, wherein: the biocontrol microbial inoculum is thallus, bacterial suspension, fermentation filtrate or active extract of staphylococcus equi F1.

3. Use of the staphylococcus equi of claim 1 or the biocontrol agent of claim 2 for controlling gray mold of fruits and vegetables.

4. The use according to claim 3, wherein said staphylococcus equi is used for inhibiting the growth of botrytis cinerea hyphae and the germination of botrytis cinerea spores.

5. A method for preparing the biocontrol microbial agent as claimed in claim 2, comprising the steps of:

s1, streaking and inoculating the staphylococcus equi F1 of claim 1 in a solid culture medium, and performing activation culture to obtain activated single colonies;

6. The method for producing a biocontrol agent according to claim 5, wherein the solid medium is PDA medium and the liquid medium is PDB medium.

7. A fruit and vegetable preservative, characterized in that the effective component is staphylococcus equi according to claim 1.

8. The fruit and vegetable fresh-keeping method is characterized by comprising the following steps: the fruit and vegetable fresh-keeping agent according to claim 7 is soaked in the fruit and vegetable fresh-keeping agent or the fruit and vegetable fresh-keeping agent according to claim 7 is sprayed on the surface of the fruit and vegetable.