CN110699275A

CN110699275A - Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof

Info

Publication number: CN110699275A
Application number: CN201910829402.5A
Authority: CN
Inventors: 施俊凤; 杜静婷; 孙常青; 冯志宏; 焦旋; 白宇皓; 杨志国; 陈嘉; 高振峰; 陈会燕
Original assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Current assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Priority date: 2019-09-03
Filing date: 2019-09-03
Publication date: 2020-01-17
Anticipated expiration: 2039-09-03
Also published as: CN110699275B

Abstract

The antagonistic bacterium is Bacillus velezensis H-1(Bacillus velezensis H1), is preserved in China center for type culture collection, has the preservation number of CCTCC NO: M2019274, is separated from the surface of pear fruit, has very obvious effect on inhibiting the botrytis cinerea after fruit and vegetable picking, has very strong inhibiting effect on botrytis cinerea, can obviously inhibit the germination and the growth of botrytis cinerea spores, has very good inhibiting effect on the botrytis cinerea after pear picking, the natural rot of winter jujube and the natural rot of grapes after fruit and vegetable picking, and provides an environment-friendly, simple and effective way for fruit and vegetable fresh-keeping after fruit and vegetable picking.

Description

Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacterial strain for antagonizing botrytis cinerea pathogenic bacteria of fruits and vegetables after picking and application thereof.

Background

The fruit industry is the third major industry behind the ranking of grains and vegetables in the planting industry of China and plays a very important role in national economy. However, during the storage and transportation of fruits and vegetables after picking, the fruits and vegetables are easy to rot and deteriorate due to pathogen infection, respiration, aging and the like, and the loss caused by the fruits and vegetables is very large. It is estimated that 20-25% of fruits and vegetables in developed countries are rotten due to pathogenic bacteria infection during the harvesting process every year, and the loss is more serious in developing countries. According to statistics, the loss caused by the decay of picked fruits in China accounts for about 30 percent of the total yield every year, and the economic loss reaches hundreds of billions of yuan RMB. With the post-harvest physiological aging process of fruits and vegetables being effectively controlled, more and more researchers have come to appreciate that controlling the ultimate loss of fruits and vegetables must also begin with controlling the post-harvest disease caused by pathogenic microorganisms. Therefore, grasping the types and infection ways of pathogenic bacteria causing postharvest diseases of fruits and vegetables and predicting the postharvest pathogenic rules of the pathogenic bacteria are the precondition for establishing control measures.

At present, the main measure for controlling postharvest diseases of fruits and vegetables is to use chemical bactericides. Although the chemical bactericide has obvious effect of preventing and treating postharvest diseases of fruits and vegetables, the residue of chemical agents and adverse effects on human health are gradually known by people. Many bactericides, such as benlate, carbendazim, double-muscle salt, etc., cannot be used for postharvest storage of fruits and vegetables. Therefore, new, non-toxic and efficient preservation technologies are urgently needed to be searched for to gradually replace the use of chemical bactericides on the picked fruits and vegetables.

Microbial control technology has evolved from laboratory to commercial production applications over two decades of research, and many studies have demonstrated that the use of antagonistic microorganisms to control disease is an emerging technology with great potential. The relatively stable storage environment condition of the harvested fruits and the greatly reduced volume of the harvested fruits compared with the field biomass are beneficial to economically and effectively implementing the biological control method. At present, the separated biocontrol bacteria mainly comprise small filamentous fungi, bacteria, yeasts and the like. To date, dozens of antagonistic microorganisms have been screened from more than 10 fruits such as citrus, apple, peach, pear, kiwi, fresh jujube, etc.

Commercial products of microbial preservatives for postharvest disease control currently on the market are products (us) based on the antagonistic bacterium Pseudomonas syringae (for controlling sweet potato and potato diseases) and Shemer (israel) based on the antagonistic yeast Metschnikowia fructicola (for controlling sweet potato and carrot diseases). Progress in the field of microbial preservation has also been actively pursued, for example, Bionext corporation, Belgium, and Leasafffreintenal corporation, France, both develop antagonistic antimicrobial Candida oleophila products, and Neova technologies, Canada, also develop Candida saitoina-based products. In addition, recently, in spain, a product Gandifruit based on Candida lake is registered, which is mainly used for preserving pome fruits, and at present, the number of microorganisms having the functions of preserving freshness and preserving fruits is small.

Disclosure of Invention

The invention aims to solve the technical problems that pathogenic bacteria cause fruit and vegetable rot and mildew after fruit and vegetable picking, and provides a bacterial strain Bacillus belius H-1(Bacillus velezensis H1) capable of antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking, which is preserved in China Center for Type Culture Collection (CCTCC) NO: M2019274 with the preservation number of 2019, 4 and 19 days.

Furthermore, the botrytis cinerea pathogenic bacteria of the picked fruits and vegetables are fusarium moniliforme, alternaria tenuissima and botrytis cinerea.

The invention also aims to provide the fermentation liquor of the bacterial strain.

Further, the preparation method of the fermentation liquor of the bacterial strain comprises the following steps:

selecting a single colony of Bacillus belgii H-1, culturing at 28 ℃ for 24H at 200r/min in an LB liquid culture medium, and diluting by 1-10 times to obtain the bacillus belgii H-1.

The invention also aims to provide a gray mold fungus protobacteria biological antibacterial agent taking the bacterial strain as an active ingredient.

The fourth purpose of the invention is to provide the application of the bacterial strain in resisting gray mold of picked fruits and vegetables.

Furthermore, the fruits and vegetables are pears.

The fifth purpose of the invention is to provide the application of the bacterial strain in inhibiting the natural decay of the picked winter jujubes or grapes.

The invention has the beneficial effects that:

the bacillus beiLeisi H-1 of the bacterial strain for antagonizing the post-harvest gray mold of fruits and vegetables has extremely obvious effect on inhibiting the post-harvest gray mold of fruits and vegetables, has very strong inhibiting effect on botrytis cinerea, can obviously inhibit the spore germination and the growth of a sprout canal of the botrytis cinerea, has very good inhibiting effect on the post-harvest gray mold of pears, the natural rot of winter jujubes and the natural rot of grapes, and provides an environment-friendly, simple and effective way for the post-harvest fresh keeping of fruits and vegetables.

Biological material preservation information description

Bacillus velezensis H1, also called Bacillus belezensis H-1 in the application, has been deposited in China center for type culture Collection in 19.4.2019 with the deposition number of CCTCC NO: M2019274, the deposition unit address is Wuhan university in Wuhan, China, the postal code is 430072, and the classification name is Bacillus velezensis H1.

Drawings

FIG. 1 is a colony morphology of Bacillus belgii H-1 in LB medium.

FIG. 2 is a microscopic morphology (1000-fold) of Bacillus belgii H-1.

FIG. 3 is a phylogenetic tree of Bacillus belgii H-1 based on the 16S rDNA sequence; the numbering of the corresponding strain in Genbank follows the name Latin in the figure.

FIG. 4 is a phylogenetic tree of Bacillus beleisi H-1 based on the gyrB gene sequence; the numbering of the corresponding strain in Genbank follows the name Latin in the figure.

FIG. 5 shows the inhibitory effect of different concentrations of Bacillus belgii H-1 suspensions on 3 pathogenic bacteria.

FIG. 6 shows the inhibition effect of Bacillus belgii H-1 on gray mold after pear harvest.

FIG. 7 shows the inhibitory effect of Bacillus belgii H-1 on the natural decay of winter jujube.

FIG. 8 shows the effect of different treatment solutions of Bacillus belgii H-1 on the inhibition of the natural rot of grapes at 26 ℃.

FIG. 9 shows the effect of different treatment solutions of Bacillus belgii H-1 on the inhibition of the natural rot of grapes at 16 ℃.

FIG. 10 shows the effect of different treatment solutions of Bacillus belgii H-1 on the inhibition of the natural rot of grapes at 0 ℃.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The methods in the following examples are conventional methods, unless otherwise specified. The percentages in the following examples, unless otherwise specified, are by weight.

Example 1

Isolation and selection of Bacillus beilis H-1 Strain

The tomato Botrytis cinerea (Botrytis cinerea) pathogen is derived from naturally-occurring tomato fruitsSeparating surface, purifying, inoculating to PDA culture medium, culturing at 26 deg.C for 7 days, and preparing into 1 × 10 with sterile water by hemacytometer⁶Spore mL^-1The spore suspension of (1). The pear fruits used for separating the antagonistic bacteria are Yulu fragrant pear gardens which are taken from original plain cities of Shanxi province; cherry tomatoes for inoculation were purchased from small areas in taiyuan.

Isolation of Bacillus belgii H-1 Strain: respectively putting the pomes in 3 storage periods into 0.2M phosphate buffer solution, performing rotary oscillation in a shaking table at a speed of 100r/min for 10min, discarding the washing solution, adding the phosphate buffer solution, washing in an ultrasonic cleaner for 30s, and performing secondary washing. The liquid obtained by the second washing is diluted by gradient of 10 times, 100 times and 1000 times, 100 mu L of each liquid is taken out and coated on PDA culture medium, and the liquid is cultured for 2-3d at 26 ℃. And (4) picking each different single colony growing in the culture dish, performing streak culture on the PDA plate, and further purifying.

Screening: single colonies were picked from PDA medium and shake-cultured in Lb medium at 28 ℃ for 24 h. Picking single colony, shake culturing at 28 deg.C for 24h at 200r to obtain 1 xl 0¹⁰cfu/mL of the suspension is ready for use. Sterilizing the surfaces of the cherry tomatoes by using 2% NaClO solution for 2min, washing the cherry tomatoes by using tap water, and drying the cherry tomatoes by using sterile wind. Pricking 3mm × 3mm wound at waist of cherry tomato fruit with sterile inoculating needle, inoculating 50 μ L of separated antagonistic bacteria suspension at wound site, inoculating 15 μ L of 1 × 10 suspension after 2 hr⁶And (3) taking the spore/mL pathogenic bacteria spore suspension as a reference by using sterile water, drying the suspension by using sterile air, putting the fruits into a plastic box, and carrying out constant-temperature moisturizing culture at 26 ℃. After 7d, the incidence rate and lesion diameter of the fruits are measured. Each treatment of 10 fruits, 3 replicates.

The method comprises the steps of totally separating 48 strains on the surface of pear, inoculating the 48 strains to the wound of the cherry tomato after fermentation culture, and after 5-7 days of moisture-preserving culture, finding that the strains with biocontrol effect are 21 strains compared with a control, wherein the tomato incidence rate of the H-1 strain treatment is 0%, and the tomato incidence rate of the control is 100%.

Example 2

Identification of Bacillus beilesiensis H-1 Strain

2.1 cultivation of the cells

After the strain preserved by freeze-drying is subjected to replica culture, streaking separation is carried out on a flat plate, a single colony is picked and inoculated into 20ml of liquid culture medium after 20 hours of culture, and the culture is carried out for 15 hours at 30 ℃ and 200 rpm.

2.2 genomic DNA extraction

(1) A single colony was inoculated into 5ml of the corresponding medium and cultured at an appropriate temperature. The incubation time may vary from hours to days depending on the growth rate of the bacteria.

(2) 1.0ml of the bacterial solution was put into a 1.5ml centrifuge tube, centrifuged at 13000rpm for 2min, and the supernatant was discarded.

(3) The pellet was resuspended in 1.0ml 0.85% NaCl.

(4) Centrifuge at 13,000rpm for 2min at room temperature and discard the supernatant.

(5) The pellet was resuspended in 550. mu.l of 1 XTE.

(6) Add 17. mu.l lysozyme (35mg/ml) and incubate at 37 ℃ for 30 min.

(7) Add 3. mu.l proteinase K (20mg/ml) and incubate at 37 ℃ for 30 min.

(8) Add 30. mu.l 10% SDS and incubate at 37 ℃ for 30 min.

(9) Add 100. mu.l of 5M NaCl and mix well.

(10) Adding 80 μ l CTAB/NaCl solution, mixing, and water bath at 65 deg.C for 10 min.

(11) Adding chloroform/isoamyl alcohol (24:1) with the same volume (0.7-0.8 ml), and lightly shaking and uniformly mixing.

(12) Centrifuge at 13,000rpm for 10min at room temperature.

(13) The supernatant was transferred to a new 1.5ml centrifuge tube and mixed by gentle shaking with an equal volume of phenol/chloroform/isoamyl alcohol (25:24: 1).

(14) And (4) repeating the step (12).

(15) The supernatant was transferred to a new 1.5ml centrifuge tube and mixed by gentle shaking with an equal volume of chloroform/isoamyl alcohol (24: 1).

(16) And (4) repeating the step (12).

(17) The supernatant was transferred to a new 1.5ml centrifuge tube, 0.6 volume isopropanol was added, mixed well and allowed to stand at 4 ℃ for 30 min.

(18) Centrifuge at 13,000rpm for 7min at 20 ℃.

(19) The supernatant was discarded, 500. mu.l of 70% ethanol was added, and the mixture was gently inverted several times (salt washing).

(20) Centrifuge at 15,000rpm for 7min at 4 ℃.

(21) The salt washing was repeated twice.

(22) The centrifuge tube was inverted and the DNA pellet was dried for 7 min.

(23) The DNA pellet was dissolved in 100. mu.l of 1 XTE buffer and stored at-20 ℃ as a template DNA for future use.

3. Cloning of 16S rDNA of Bacillus belgii H-1

The primers are as follows:

27F: 5'-AGA GTT TGA TCC TGG CTC AG-3' (shown in SEQ ID NO. 1);

1492R: 5'-ACG GTT ACC TTG TTA CGA CTT-3' (shown in SEQ ID NO. 2).

The 16S rDNA reaction system was 100. mu.l: taq (5U/ml) 0.8. mu.l; 10 × PCR Buffer (Mg)²⁺Plus) 10. mu.l; dNTP mix (2.5mM/each) 8. mu.l; 2.5ng of template DNA; 2. mu.l of primer F1(10mmol/L), 2. mu.l of primer R1(10 mmol/L); ddH₂O make up to 100. mu.l.

The PCR reaction conditions are as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) annealing at 95 deg.C for 1min, annealing at 57 deg.C for 1min, and extending at 72 deg.C for 1min for 20s for 30 cycles; (3) stop at 72 ℃ for 5 min.

The PCR product is analyzed by an ultraviolet gel imaging system after 1 percent agarose gel electrophoresis and EB staining, a target fragment is recovered and purified, and the sequence is determined by Shanghai Yingjun Gene Co., Ltd, and the sequencing result is shown as a sequence table SEQ ID NO. 3.

4. Cloning of the gyrB Gene of Bacillus beilis H-1

The primers are as follows:

gyrB-34F: 5'-ggTgTWRgKgCNgTCgTAAACg-3' (shown in SEQ ID NO. 4);

gyrB-977R: 5 '-CCSgCAgARTCACCTCCTACg-3' (shown in SEQ ID NO. 5);

the reaction system is 100 ul: taq (5U/. mu.l) 0.8. mu.l; 10 × PCR Buffer (Mg)²⁺Plus) 10. mu.l; dNTPmix (2.5mM/each) 8. mu.l; 2.5ng of template DNA; 2 μ L of gyrB-F (10mmol/L), 2 μ L of gyrB-R (10 mmol/L); ddH₂O make up to 100. mu.l.

The reaction procedure is as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) denaturation at 95 deg.C for 1min, annealing at 55 deg.C for 2min 15s, and extension at 72 deg.C for 2min 15s for 3 cycles; (3) denaturation at 95 deg.C for 35s, annealing at 55 deg.C for 1min, and extension at 72 deg.C for 1min for 15s for 30 cycles; (4) terminate at 72 ℃ for 7 min.

The PCR product is analyzed by an ultraviolet gel imaging system after 1 percent agarose gel electrophoresis and EB staining, a target fragment is recovered and purified, and the sequence is determined by Shanghai Yingjun Gene Co., Ltd, and the sequencing result is shown as a sequence table SEQ ID NO. 6.

5. Homology comparison and construction of phylogenetic trees

5.1 16S rDNA homology analysis and phylogenetic Tree construction of Bacillus beilesiensis H-1

The full length of the 16S rDNA amplification product sequence is 1437 bp. After homology comparison of the measured sequences by Blast, using Mycobacterium geneva (Mycobacterium genavense) as an exocolony, constructing a 16S rDNA phylogenetic tree (figure 2) of the strain by adopting an orthotopic ligation method by MEGA4.1 software, and carrying out similarity repeated calculation for 1000 times, wherein the developmental tree node only shows a Bootstrap value of more than 50%. The clustering result shows that the strain H-1 is clustered with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Bacillus siamensis (Bacillus siamensis) into a class, and the relative relationship is relatively close.

5.2 phylogenetic evolution Tree of gyrB Gene sequences of Bacillus belgii H-1 and related species

By further verifying the housekeeping gene gyrB, the sequence length of the amplified fragment obtained by sequencing is 892 bp. To further characterize the strain H-1, the sequences determined were compared for homology by Blast and then treated with Bacillus safensis FO-036b^TFor the outbreak, the MeGA4.1 software was used to construct the gyrB gene phylogenetic tree of the strain H-1 by the orthotopic ligation method (FIG. 3), the strain H-1 and Bacillus velezensis BCRC17467^T) The strains are grouped into one group, the bootstrap support rate is 100 percent, so the strain H-1 is determined to belong to the Bacillus belgii.

6. Morphological identification of Bacillus belgii H-1

Bacillus belgii H-1 is rod-shaped and has spores. The colonies on the bacterial culture plate were round, irregular in edges, creamy and opaque, as shown in FIG. 1.

7. Biochemical and physiological assays for Bacillus beilis H-1

According to the 'common bacteria identification manual', the physiological and biochemical reactions of the Bacillus beiLeisi H-1 are further detected, and the form and the basic physiological and biochemical characteristics are determined. The morphology and the basic physiological and biochemical characteristics of the Bacillus belgii H-1 of the invention are shown in Table 1:

TABLE 1 physiological and biochemical characteristics of Bacillus belgii H-1

Note: "+" indicates positive, "-" indicates negative, and "w" indicates weak positive

Example 3

Research on bacteriostatic effect of Bacillus beleisi H-1

1. In vitro inhibition of pathogenic bacteria by Bacillus belgii H-1

Purifying Bacillus belgii H-1 by bacterial disc method, selecting single colony, inoculating into 100mL LB liquid culture medium, performing shake culture at 30 deg.C for 24 hr, determining concentration by dilution plate method, and preparing into 1 × 10 concentration with sterile water⁵、1×10⁶、1×10⁷、1×10⁸、1×10⁹The suspension of antagonist bacteria of (1). In a culture dish (diameter is 9cm) with PDA, after the culture medium is solidified, 100 microliter of antagonistic bacteria H-1 suspension with different concentrations are respectively added, after the uniform coating, the suspension is dried by aseptic wind, then a botrytis cinerea mould cake with the diameter of 5mm is inoculated at the central point, and aseptic water is added in contrast. Culturing at 28 deg.C, measuring colony diameter after 5-7d, and calculating its inhibition rate, wherein each treatment is repeated for 3 times, and the experiment is repeated for 3 times.

Inhibition ratio (%) - (dC-dT)/(dC-5). times.100

Wherein: dC is the control colony diameter (mm), dT is the colony diameter (mm) under different treatment conditions;

as shown in FIG. 5, different concentrations of Bacillus belgii H-1 can effectively inhibit the normal growth of Fusarium moniliforme (Fusarium verticillioides), Alternaria tenuissima (Alternaria tenuissima) and Botrytis cinerea (Botrytis cinerea) hyphae, and the higher the concentration of the antagonistic bacteria liquid is, the stronger the bacteriostatic action is.

2. Effect of Bacillus beleisi H-1 on Botrytis cinerea spore germination and germ tube elongation

And (3) spreading multiple layers of sterilized filter paper in the sterilized culture dish, wetting the filter paper by sterile water, and placing a concave glass slide on the filter paper. The spore number is 3 × 10 by using 2% sucrose solution⁵Taking a/mL botrytis cinerea spore suspension, sucking 20 mu L of the suspension on a concave glass slide, respectively adding 15 mu L of antagonistic bacteria H-1 fermentation stock solution for 24H of culture and fermentation liquor diluted by 5, 10, 20 and 40 times, adding sterile water in a contrast, adding a cover glass, after covering a culture dish, preserving moisture and culturing for 72H at 25 ℃, randomly selecting 200 spores, observing the spore germination condition, measuring the length of a germ tube, repeating the treatment for 3 times, and repeating the test for 3 times.

As a result: after the Bacillus beiLeisi H-1 fermentation broth with different concentrations and pathogenic bacterium Botrytis cinerea are co-cultured for 72H, the result shows that the higher the concentration of the fermentation broth is, the stronger the inhibition effect is. The germination rate of the control spores reaches 92.34 percent, the average length is 212.17 mu m, the spores are obviously inhibited after being treated by the fermentation liquor, the spores cannot germinate completely after being treated by the fermentation stock solution and the fermentation liquor diluted by 5 times, only few spores slightly germinate after being diluted by 10 times, most of the tube lengths of the fermentation liquor treated by 20 times are equal to or slightly longer than the diameter of the spores, the spores of the fermentation liquor diluted by 40 times are longer than 20 times, the spores are germinated more, but the spore germination rate is far smaller than the control, and the spore germination rate is only 32.76 percent (Table 2).

TABLE 2 Effect of different concentrations of Bacillus belgii H-1 culture on Botrytis cinerea spore germination and sprout tube length

3. Inhibition effect of Bacillus beleisi H-1 on gray mold after pear harvest

Taking healthy pome without plant diseases and insect pests, sterilizing the surface of the pome by using 2% NaClO, and pricking wounds with the diameter of 5mm and the depth of 3mm at the waist part of the pome, wherein 2 wounds are formed on each pome. Adding 1 × 10 to the wound⁸、1×10⁹、 1×10¹⁰、1×10¹¹cfu/ml suspension of Bacillus belgii H-1, Control (CK) plus sterile water. After 2h, the wound is connected with 1X 10⁵20 μ l of spore/ml Botrytis cinerea (B. cinerea), culturing at room temperature under moisture, measuring lesion diameter after 5d, and calculating the inhibition rate. 10 fruits per treatment. The experiment was repeated 3 times.

As a result: after inoculating Bacillus belgii H-1 suspension with different concentrations, the incidence rate statistics result is shown in figure 6, and the incidence rate of pear gray mold is reduced along with the increase of the concentration. The incidence rate of CK gray mold is 100 percent; the concentration of antagonistic bacteria is 1 × 10⁸The incidence rate is 56.41% at cfu/ml, 1X 10⁹cfu/ml 47.63%, 1X 10¹⁰cfu/ml is only 5.24%, at the highest concentration 1X 10¹¹The occurrence of diseases can be completely inhibited when cfu/ml is adopted; and the larger the concentration of the antagonistic bacteria is, the smaller the lesion diameter is.

4. Inhibition effect of Bacillus beleisi H-1 on natural rot of winter jujube

Taking out Bacillus beleisi H-1 from-80 ℃, performing streak culture, performing shake flask culture in LB liquid medium (10 g of tryptone, 5g of yeast extract and 10g of NaCl, adding water to a constant volume of 1L) at 28 ℃ for 200r/min, soaking the jujube fruits for 2min by using fermentation stock solution, diluted 5 times of fermentation solution and diluted 10 times of fermentation solution respectively, taking out and draining, placing in a 0 ℃ refrigeration house, keeping moisture of the surfaces of the fruits dry, placing at 0 ℃ by using a PE bag (contrast without soaking treatment), and counting the rotting rate of the jujube fruits after 90 days. Each time 5kg of jujube fruits are treated, 3 times of experiments are repeated.

As shown in FIG. 7, the Bacillus belgii H-1 treatment solutions of different concentrations were effective in inhibiting the postharvest natural decay of winter jujube fruits after storage at 0 ℃ for 90 days. Compared with the common jujube fruit, the rot rate of the common jujube fruit is 94.56%, while the lowest rot rate of the fermentation liquid diluted by 5 times is 46.23%, the biocontrol efficiency reaches 51.11%, and the biocontrol efficiency is 39.36% because the fermentation stock solution is 57.34%.

5. Inhibitory effect of Bacillus beleisi H-1 on postharvest natural rot of grapes

Selecting a single colony of Bacillus belgii H-1 in 100/250mL LB liquid culture medium at 28 ℃ and 200r/min, culturing for 24H, and then carrying out the following treatment: a. fermenting the stock solution; b. diluting the fermentation liquor by 5 times; c. diluting the fermentation liquor by 10 times; soaking the healthy rose-scented grapes in the mature period in the 5 solutions for 2min, taking out, drying by sterile wind, checking the rotting degree of fruits and fruit stalks when the fruits and the fruit stalks are stored for 15 days, 25 days and 70 days at 26 ℃, 16 ℃ and 0 ℃, classifying the fruits according to the rotting degree, weighing, and calculating the rotting rate of the fruit stalks, the rotting index of the fruits and the control effect. The weight of the grape fruits per treatment was 5kg, each treatment was repeated 3 times, and the experiment was repeated 2 times.

Wherein the fruit stalks are normal without mildew phenomenon, otherwise, the fruit stalks are rotten. The fruits are classified into 5 grades according to the rotting degree, and the grading standard is as follows: level 0: the fruits are intact without any rot; level 1: the surface of the fruit is slightly speckled, and the rotten area accounts for below 1/3 of the surface of the fruit; and 2, stage: the surface of the fruit is obviously rotten, and the rotten area accounts for 1/3-1/2 of the surface of the fruit; and 3, level: the fruit has rot exceeding 1/2 on the surface of the fruit, and the fruit has certain hardness; 4, level: the fruit is totally ulcerated. The fruits were graded and weighed separately. Pulp decay index calculation formula:

as a result: the adopted different treatment liquids of the antagonistic bacteria can inhibit the postharvest rot of the grape fruits respectively at 26 ℃, 16 ℃ and 0 ℃ corresponding to the results shown in fig. 8, 9 and 10, and the different treatment liquids of the antagonistic bacteria can effectively inhibit the postharvest rot of the grape fruits, wherein the fermentation liquid and the bacterial suspension have better rot-preventing effect. In the fermentation liquor, the inhibition effect of the fermentation liquor diluted by 5 times is better than that of the fermentation stock solution and the fermentation liquor diluted by 10 times. When the fermented liquid is stored for 15 days, 25 days and 70 days under 3 temperature conditions, the rot rates of the control fruit stalks are respectively 88.89%, 100.00% and 100.00%, while the rot rates of the fruit stalks diluted by 5 times under the same conditions are respectively 30.42%, 25.96% and 16.67%, and the rot rates of the fruit stalks of the bacterial suspension are respectively only 34.02%, 28.33% and 17.20%. When stored at 0 ℃ for 70 days, the pulp decay index was 67.14%, while the 5-fold dilution of the fermentation broth and bacterial suspension was 3.21% and 6.77%. The effect of the fermentation liquor diluted by 5 times is the best for inhibiting the fruit stalk rot and the fruit pulp rot. Compared with the control effect at 3 temperatures, the control effect is best at 0 ℃ at 26, 16 ℃ and 0 ℃ regardless of fruits or fruit stalks. Therefore, the control effect of the fermentation liquor diluted by 5 times can reach 95.22 percent at the temperature of 0 ℃ (Table 3).

TABLE 3 prevention and treatment of grape fruit stalks and flesh with different treatment solutions of Bacillus beilesiensis H-1

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> institute for storing and preserving agricultural products of academy of agricultural sciences of Shanxi province

<120> bacterial strain for antagonizing botrytis cinerea pathogenic bacteria of picked fruits and vegetables and application thereof

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial sequence

<400>1

agagtttgat cctggctcag 20

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<400>2

acggttacct tgttacgact t 21

<210>3

<211>1437

<212>DNA

<213> Artificial sequence

<400>3

tgcctataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60

cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120

ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180

acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240

cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300

tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360

ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420

cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480

cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540

tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600

gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660

cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720

aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780

atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840

tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900

agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960

cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020

gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080

atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140

aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200

atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260

agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320

cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380

gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aaggtgg 1437

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

ggtgtwrgkg cngtcgtaaa cg 22

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<400>5

ccsgcagart caccctctac g 21

<210>6

<211>892

<212>DNA

<213> Artificial sequence

<400>6

tcgtaaacgc cttgtcgacc actcttgacg ttacggttca tcgtgacgga aaaatccact 60

atcaggcgta cgagcgcggt gtacctgtgg ccgatcttga agtgatcggt gatactgata 120

agaccggaac gattacgcac ttcgttccgg atccggaaat tttcaaagaa acaaccgaat 180

acgactatga cctgctttca aaccgtgtcc gggaattggc cttcctgaca aaaggtgtaa 240

acatcacgat tgaagacaaa cgtgaaggac aagaacggaa aaacgagtac cactacgaag 300

gcggaatcaa aagctatgtt gagtacttaa accgttccaa agaagtcgtt catgaagagc 360

cgatttatat cgaaggcgag aaagacggca taacggttga agttgcattg caatacaacg 420

acagctatac aagcaatatt tattctttca caaataatat caacacatac gaaggcggca 480

cgcacgaagc cggatttaaa accggtctga cccgtgttat aaacgactat gcaagaagaa 540

aagggatttt caaagaaaat gatccgaatt taagcgggga tgatgtgagg gaagggctga 600

ctgccattat ttcaattaag caccctgatc cgcaattcga agggcagacg aaaacgaagc 660

tcggcaactc cgaagcgaga acgatcactg atacgctgtt ttcttctgcg ctggaaacat 720

tccttcttga aaatccggac tcagcccgca aaatcgttga aaaaggttta atggccgcaa 780

gagcgcggat ggcagcgaaa aaagcgcggg aattgacccg ccgcaaaagt gcgcttgaga 840

tttccaatct gccgggcaaa ctggcggact gttcttctaa agatccgagc at 892

Claims

1. A bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking is characterized in that the bacterial strain is Bacillus velezensis H1 and is preserved in China center for type culture collection with the preservation number of M2019274 and the preservation date of 19 months 4 in 2019.

2. The bacterial strain of claim 1, wherein the post-harvest botrytis cinerea is fusarium moniliforme, alternaria tenuis, or botrytis cinerea.

3. A fermentation broth of the bacterial strain of claim 1.

4. A method of preparing a fermentation broth of a bacterial strain according to claim 3, comprising the steps of:

5. A biological antibacterial agent against Botrytis cinerea comprising the bacterial strain of claim 1 as an active ingredient.

6. The use of the bacterial strain of claim 1 for antagonizing post-harvest gray mold of fruits and vegetables.

7. The use of claim 6, wherein the fruit or vegetable is pear.

8. Use of a bacterial strain according to claim 1 for inhibiting the natural decay of winter jujube or grape after harvest.