A "vaccine-type" bacteriocin and uses thereof

Abstract

This invention discloses a "vaccine-type" bacteriocin, Yi-VD14, with the amino acid sequence VDFGPHGLLPIRPI, which can induce plant immune responses and enhance plant disease resistance. Taking chili peppers as an example, this bacteriocin significantly improves the storage quality of chili peppers by delaying vitamin C decomposition and chlorophyll degradation and reducing malondialdehyde accumulation. Furthermore, it induces plant immune responses and enhances plant disease resistance by regulating fruit fat metabolism, inhibiting cell membrane degradation, and improving the biosynthesis of plant disease-resistant metabolites. It also increases the activity of disease-resistant enzymes in chili pepper fruits and reshapes the microbial community structure on the surface of the fruits. Ultimately, it enhances the plant's resistance to pathogenic microorganisms from multiple dimensions, reducing the incidence of chili pepper soft rot and minimizing post-harvest losses. This bacteriocin can be used not only to prepare products for controlling chili pepper soft rot but also for controlling other fruit and vegetable diseases, showing promising application prospects in the fruit and vegetable planting and post-harvest distribution industry chain.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a bacteriocin and its application in the prevention and control of fruit and vegetable diseases. Background Technology

[0002] Bacteriocins are a class of polypeptides with antibacterial or bactericidal activity synthesized by certain bacteria through ribosomes during metabolism. Bacteriocins effectively inhibit the growth and reproduction of Gram-positive bacteria, and some also have some inhibitory effect on Gram-negative bacteria. Due to their natural, non-toxic, and residue-free characteristics, bacteriocins are widely used in food preservation, such as in dairy products, meat products, and canned foods. The application of bacteriocins in fruits and vegetables is concentrated in preservation and antiseptic applications, especially showing significant effects in postharvest preservation. For example, nisin, through surface spraying or combined with aerobic packaging and modified atmosphere packaging technologies, can effectively inhibit the growth of spoilage bacteria on the surface of fruits and vegetables; bacteriocin Bac11029 slows down respiration in the preservation of carrots and purple cabbage, reducing the loss of carotenoids, chlorophyll, and phenolic substances. Currently, the application of bacteriocins in preharvest fruits and vegetables is relatively limited.

[0003] Chili peppers are an important economic crop, favored by consumers for their rich nutritional value and unique taste. However, various post-harvest diseases cause significant losses, with soft rot being the most devastating bacterial disease. The main pathogen of soft rot is *Pectobacterium carotovorum*, formerly known as *Erwinia carotovora*. It infects the fruit tissue either latently before harvest or directly after harvest through natural openings in the peel or mechanical damage. Diseased fruits initially show water-soaked dark green spots, which later turn brown and soft rot. The peel changes from green to white, and the internal flesh rots with a foul odor, severely impacting the yield, quality, and storage life of chili peppers. Currently, the control of chili pepper soft rot mainly relies on chemical fungicides, but long-term use of chemical agents can easily lead to increased pathogen resistance, excessive pesticide residues, and environmental pollution. Summary of the Invention

[0004] The purpose of this invention is to conduct research on the screening and application of "vaccine-type" bacteriocins, using chili peppers as an example, in order to provide a new solution for the prevention and control of fruit and vegetable diseases.

[0005] Based on research, the present invention provides the following technical solution:

[0006] 1. Bacteriocin Yi-VD14, amino acid sequence VDFGPHGLLPIRPI (SEQ ID No. 1).

[0007] 2. Application of bacteriocin Yi-VD14 in the preparation of products for the prevention and control of fruit and vegetable diseases.

[0008] Furthermore, the product mentioned in the text is a product for preventing and controlling fruit and vegetable diseases, specifically for preventing and controlling soft rot in peppers.

[0009] 3. The method of using bacteriocin Yi-VD14 to control fruit and vegetable diseases is to apply bacteriocin Yi-VD14 before and / or after harvesting.

[0010] Preferably, the method of using bacteriocin Yi-VD14 to control fruit and vegetable diseases involves applying bacteriocin Yi-VD14 both before and after harvesting the fruits and vegetables.

[0011] Preferably, the pre-harvest application involves soaking fruit and vegetable seeds in an aqueous solution of bacteriocin Yi-VD14, followed by cultivation according to conventional seedling procedures. During the entire growth period, the fruit and vegetables are sprayed with an aqueous solution of bacteriocin Yi-VD14 5-10 times to ensure even spraying on the front and back of the leaves and stems. The post-harvest application involves spraying the surface of the fruit and vegetables with an aqueous solution of bacteriocin Yi-VD14 or soaking the fruit and vegetables in an aqueous solution of bacteriocin Yi-VD14.

[0012] The beneficial effects of this invention are as follows: This invention, for the first time, expands the function of bacteriocins from the traditional "antibacterial function" to "inducing host immune response function," providing a "vaccine-type" bacteriocin, Yi-VD14, that can induce plant immune responses and improve plant disease resistance. This bacteriocin, produced by *Lactobacillus plantarum*, was screened from 30 candidate bacteriocins. Taking pepper as an example, this bacteriocin can significantly improve the storage quality of peppers by delaying vitamin C decomposition and chlorophyll degradation and reducing malondialdehyde accumulation. Furthermore, it can induce plant immune responses and improve plant disease resistance by regulating fruit fat metabolism, inhibiting cell membrane degradation, and improving the biosynthesis of plant disease-resistant metabolites. Simultaneously, it can increase the activity of disease-resistant enzymes in pepper fruits and reshape the microbial community structure on the surface of pepper fruits, increasing beneficial microbial flora and reducing the abundance of pathogenic microorganisms. Ultimately, it improves the plant's resistance to pathogenic microorganism invasion from multiple dimensions, reduces the incidence of pepper soft rot, and minimizes post-harvest rot losses. This bacteriocin can be applied pre-harvest or post-harvest, with the best results achieved when applied simultaneously, as it more comprehensively activates the plant's redox defense system. This bacteriocin can not only be used to prepare products for controlling soft rot in peppers, improving the storage quality of pepper fruits, extending their shelf life, and reducing spoilage losses at various stages of the supply chain, but it can also be used to prepare products for controlling other fruit and vegetable diseases, showing promising application prospects in the fruit and vegetable planting and post-harvest distribution chain. Attached Figure Description

[0013] Figure 1 Mass spectra of bacteriocin Yi-VD14 identified by LC-MS / MS.

[0014] Figure 2 The bar chart shows the change in the diameter of soft rot lesions in peppers treated with different bacteriocins over time during the initial screening of 30 bacteriocins. A represents the initial screening of 5 bacteriocins (numbered 69, 72, 73, 83, and 92) at three concentrations (1, 10, and 100 μM); B represents the initial screening of 9 bacteriocins (numbered 1, 2, 5, 8, 9, 12, 13, 89, and 93) at three concentrations (1, 10, and 100 μM); C represents the initial screening of 8 bacteriocins (numbered 2, 3, 4, 9, 10, 11, 69②, and 73) at three concentrations (1, 10, and 100 μM); D represents the initial screening of 6 bacteriocins (numbered 71, 79, 81, 82, 87, and 88) at three concentrations (1, 10, and 100 μM); and E represents the initial screening of 5 bacteriocins (numbered 74, 75, 80, 85, and 86) at three concentrations (1, 10, and 100 μM).

[0015] Figure 3 Phenotypic diagram of pepper soft rot controlled by 10 μM bacteriocin Yi-VD14 during rescreening.

[0016] Figure 4 The graph shows the determination and analysis of the storage quality of pepper fruits after treatment with bacteriocin Yi-VD14. In the graph, A is malondialdehyde content, B is respiration intensity, C is chlorophyll content, D is soluble solids content, and E is ascorbic acid content.

[0017] Figure 5 Phenotypic diagram (A) of different application times of bacteriocin Yi-VD14 for controlling pepper soft rot and its effects on incidence (B) and lesion diameter (C).

[0018] Figure 6 This is a transcriptomic analysis diagram of pepper fruits treated with bacteriocin Yi-VD14. A is the sample distribution diagram of principal component analysis (PCA), B1-B3 are volcano diagrams of differentially expressed genes (DEGs), C is the GO annotation diagram, and D1-D3 are KEGG enrichment bubble diagrams.

[0019] Figure 7 The diagram shows the activity analysis of disease resistance-related enzymes in pepper fruits after treatment with bacteriocin Yi-VD14. In the diagram, A is catalase (CAT), B is phenylalanine ammonia-lyase (PAL), C is peroxidase (POD), D is polyphenol oxidase (PPO), and E is superoxide dismutase (SOD).

[0020] Figure 8 The diagram shows the bacterial diversity analysis of pepper fruits after treatment with bacteriocin Yi-VD14. A is the Venn diagram, B is the PCA diagram, C is the Alpha diversity index diagram, D is the clustering heatmap, E is the species composition bar chart, and F is the functional prediction analysis diagram.

[0021] Figure 9 The diagram shows the fungal diversity analysis of pepper fruits after treatment with bacteriocin Yi-VD14. A is the Venn diagram, B is the PCA diagram, C is the Alpha diversity index diagram, D is the clustering heatmap, E is the species composition bar chart, and F is the functional prediction analysis diagram. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] Example 1: Identification of bacteriocin Yi-VD14

[0024] Four different *Lactobacillus plantarum* strains, previously demonstrated to have strong antibacterial activity against *Pectinobacillus* (the pathogen of pepper soft rot), were cultured statically in MRS medium at 30°C for 72 h. The fermentation supernatant was collected by centrifugation, filtered through a 0.22 μM membrane, and peptides were extracted using ethyl acetate extraction. The peptides were identified by LC-MS / MS, and the obtained raw mass spectrometry files were used to retrieve the complete genome sequences of the corresponding *Lactobacillus plantarum* strains using MaxQuant software. A total of 30 bacteriocins were identified from the metabolites of the four *Lactobacillus plantarum* strains. The mass spectrum of bacteriocin Yi-VD14 is shown below. Figure 1 As shown, its amino acid sequence is VDFGPHGLLPIRPI (SEQ ID No. 1).

[0025] Example 2: Screening of "vaccine-type" bacteriocins

[0026] Initial screening was conducted using an in vitro fruit inoculation method: ① Fresh, intact pepper fruits were first rinsed with water to remove surface dirt, microorganisms, and other residues. The fruits were then immersed in a 2% sodium hypochlorite solution for 3 minutes for sterilization, rinsed with water, and air-dried. ② Once the fruit surface was free of residual moisture, two holes were made at the top and bottom thirds of the fruit using a 1 mL pipette tip, ensuring the holes were deep but not completely punctured. ③ Each hole was first inoculated with *Bacillus pectinifera*, and after absorption, a bacteriocin was inoculated. ④ The fruit rot rate and rot diameter were recorded daily. Following this method, the inhibitory effects of 30 bacteriocins at concentrations of 1, 10, and 100 μM on the incidence and diameter of pepper soft rot were evaluated. Bacteriocins showing significant effects compared to the control group (blank) (P < 0.05) were included in the secondary screening. Results are as follows: Figure 2As shown, after initial screening, three bacteriocins with activity in controlling pepper soft rot were selected: Syn-1062-15 (number 2), Yi-VD14 (number 13), and YN-27 (number 89). At a concentration of 100 μM, the diameter of the rotten pepper was reduced, and the rot symptoms were significantly lower than those in the control group (P<0.05).

[0027] A secondary screening was conducted using a separate inoculation method (separate treatment of pathogens and bacteriocins): ① Fresh, intact pepper fruits were first rinsed with water to remove surface dirt, microorganisms, and other residues. The fruits were then immersed in a 2% sodium hypochlorite solution for 3 minutes for sterilization, rinsed with water, and air-dried. ② After the fruit surface was free of residual moisture, holes were made at the top and bottom thirds of the fruit using a 1 mL pipette tip, followed by additional holes approximately 5 mm from the top and bottom, for a total of four holes. The holes should be deep but not completely through. ③ Bacteriocins were inoculated into the holes at the top and bottom thirds of the fruit. After 24 hours, *Bacterium tumefaciens* was inoculated into the remaining two holes. ④ The fruit rot rate and rot diameter were recorded daily. Following this method, the three bacteriocins initially selected were rescreened to verify the bacteriocin-induced systemic resistance. Results are as follows: Figure 3 As shown, the bacteriocin Yi-VD14 showed the best effect against pepper soft rot. At a concentration of 10 μM, the rot rate of peppers was significantly reduced, and the rot symptoms were significantly lower than those in the control group.

[0028] Example 3: Determination and analysis of the storage quality of pepper fruits after treatment with bacteriocin Yi-VD14

[0029] 1. Sample Preparation

[0030] Fresh, intact chili peppers were first rinsed with water to remove surface dirt, microorganisms, and other residues. The peppers were then soaked in a 2% sodium hypochlorite solution for 3 minutes for sterilization. After rinsing and air-drying, once the surface of the peppers was free of residual moisture, two holes were made on each pepper using a 1mL pipette tip, positioned perpendicular to the surface of the pepper at the top and bottom third of the fruit. The holes were to be deep but not completely pierced. The perforated peppers were randomly divided into two groups (control group and Yi-VD14 treatment group), with three replicates per group, each containing 10 peppers (30 peppers per group). 30 μL of sterile water was added to each well in the control group, while 30 μL of 10 μM bacteriocin (Yi-VD14) was added to each well in the Yi-VD14 treatment group. After addition, the peppers were left at room temperature for 24 hours. Finally, 15 μL of bacterial culture at a density of 102 was added to each well in both groups. 4 CFU / mL of Bacillus pectinophilus suspension was prepared. The two groups of peppers were stored at 25℃ and 85% relative humidity. Samples were taken daily (cylindrical pulp tissue with a diameter of 1 cm was cut from the inoculation well of the pepper), flash-frozen in liquid nitrogen, and stored at -80℃ for testing.

[0031] 2. Determination of physicochemical properties

[0032] (1) Determination of malondialdehyde content: Weigh 0.5 g of pepper pulp tissue sample, add 8 mL of 5% trichloroacetic acid solution, vortex to mix, centrifuge at 12000 rpm for 10 min, take 2 mL of supernatant and mix with 2 mL of 0.6% thiobarbituric acid solution, react in boiling water bath for 30 min, cool rapidly after the reaction, centrifuge at 12000 rpm for 10 min, take the supernatant and measure the absorbance at wavelengths of 450 nm, 532 nm and 600 nm.

[0033] (2) Respiration intensity measurement: Ten pepper fruits were selected and their accurate weight was recorded. They were then placed in a sealed container, and the initial carbon dioxide content in the container was measured using a gas analyzer. After sealing for 1 hour, the carbon dioxide content in the container was measured again. The respiration intensity was calculated based on the change in carbon dioxide content during the sealing time.

[0034] (3) Determination of chlorophyll content: Weigh 0.5 g of pepper pulp tissue sample, add 8 mL of anhydrous ethanol-acetone (volume ratio 1:1) mixed solvent, extract at room temperature in the dark for 5 h, filter, and measure the absorbance of the filtrate at wavelengths of 645 nm and 663 nm.

[0035] (4) Determination of soluble solids (TSS) content: Take a sample of pepper pulp tissue, squeeze out the juice, add an appropriate amount of juice to a digital refractometer, and determine its TSS value.

[0036] (5) Determination of ascorbic acid (Vc) content: The 2,6-dichlorophenolindophenol titration method was used. Weigh 0.2 g of pepper pulp tissue sample, add 8 mL of pre-cooled 2% oxalic acid solution, mix well, and filter with filter paper (if the filtrate is colored, add 0.4 g of kaolin per gram of sample for decolorization and filter again). Take 4 mL of the filtrate and titrate with standardized 2,6-dichlorophenolindophenol standard solution until the solution turns pink and does not fade within 15 s. A blank control was set up during the experiment.

[0037] The results are as follows Figure 4 As shown, the malondialdehyde (MDA) content in both the control group and the Yi-VD14 treatment group increased with prolonged storage time, but the MDA content in the Yi-VD14 treatment group remained lower than that in the control group. Figure 4 A); In the control group, the respiratory peak occurred on day 3 and was higher, while in the Yi-VD14 treatment group, the respiratory peak was delayed until day 5 and was lower. Figure 4B); The chlorophyll content in both the control group and the Yi-VD14 treatment group decreased over time, but the chlorophyll degradation rate in the Yi-VD14 treatment group was significantly slower than that in the control group ( Figure 4 C); The soluble solids content in both the control group and the Yi-VD14 treatment group initially increased and then decreased, but the peak value in the Yi-VD14 treatment group was delayed, and the rate of decrease was slower in the later stages. Figure 4 D); The vitamin C content in both the control group and the Yi-VD14 treatment group showed a decreasing trend, but the vitamin C retention rate in the Yi-VD14 treatment group was significantly higher than that in the control group ( Figure 4 E). The results above show that bacteriocin Yi-VD14 can delay chlorophyll decomposition, maintain the green appearance of fruits, reduce the oxidative loss of vitamin C, reduce malondialdehyde accumulation, effectively inhibit lipid peroxidation, delay cell aging, slow down the consumption and degradation of nutrients, and maintain the flavor and nutritional value of fruits.

[0038] Example 4: Comparison of the effects of bacteriocin Yi-VD14 at different application times

[0039] Select plump and uniformly sized chili seeds and randomly divide them into four groups: control group, pre-harvest Yi-VD14 treatment group, post-harvest Yi-VD14 treatment group, and pre-harvest + post-harvest Yi-VD14 treatment group. (1) Pre-harvest treatment: Chili seeds in the pre-harvest Yi-VD14 treatment group and the pre-harvest + post-harvest Yi-VD14 treatment group were soaked in 10 μM bacteriocin Yi-VD14 aqueous solution for 12 h; chili seeds in the control group and the post-harvest Yi-VD14 treatment group were soaked in the same amount of ultrapure water simultaneously. After soaking, they were cultivated according to the conventional seedling raising procedure. Pepper plants in the pre-harvest Yi-VD14 treatment group and the pre-harvest + post-harvest Yi-VD14 treatment group were sprayed with 10 μM bacteriocin Yi-VD14 aqueous solution using a pressure sprayer to ensure that the front and back of the leaves and the stems were sprayed evenly. A total of 7 sprays were applied throughout the entire growth period (including seed, transplanting, flowering and fruiting). Pepper plants in the control group and the post-harvest Yi-VD14 treatment group were sprayed with the same amount of ultrapure water at the same time. (2) Post-harvest treatment: Mature pepper fruits of the same size, without mechanical damage and without pests and diseases were harvested from the above four groups of plants. Each group was divided into 3 parallel groups, each containing 10 pepper fruits (a total of 30 pepper fruits per group). After disinfecting, washing, and drying the chili peppers, holes were punched in each fruit (two holes per fruit, with a spacing of at least 1 cm). Ultrapure water was added to each hole in the control group and the pre-harvest Yi-VD14 treatment group. A 10 μM bacteriocin Yi-VD14 aqueous solution was added to each hole in the post-harvest Yi-VD14 treatment group and the pre-harvest + post-harvest Yi-VD14 treatment group. After standing at room temperature for 24 h, a bacterial cell density of 10⁻⁶ cells was added to each hole in all four groups. 4CFU / mL of Bacillus pectinophilus suspension was prepared and then stored at room temperature (25℃) and relative humidity of 85%. The incidence rate of soft rot in pepper fruits of each group was counted on the 1st, 2nd and 3rd days of storage (incidence rate = number of infected fruits / total number of fruits × 100%), and the diameter of lesions on infected fruits was measured. Data are expressed as mean ± standard deviation.

[0040] The results are as follows Figure 5 As shown, by the third day of storage, the incidence of soft rot in the control group was close to 90%, with corresponding lesion diameters of approximately 70 mm. In the post-harvest Yi-VD14 treatment group, the incidence rate dropped to approximately 80%, and the lesion diameter simultaneously shrank to approximately 60 mm. The control effect of the pre-harvest Yi-VD14 treatment group was further improved, with an incidence rate of only about 75% and lesion diameters also shrinking to approximately 50 mm. The combined pre-harvest and post-harvest Yi-VD14 treatment group showed the best control effect, with an incidence rate of only about 50% and lesion diameters of only about 40 mm. Therefore, it can be concluded that the bacteriocin Yi-VD14 can significantly reduce the incidence and lesion diameter of pepper soft rot; among these, the control effect of pre-harvest treatment alone is stronger than that of post-harvest treatment alone, while the combined pre-harvest and post-harvest treatment shows the best control effect.

[0041] Example 5: Transcriptomic analysis of pepper fruits after treatment with bacteriocin Yi-VD14

[0042] Four groups of chili pepper fruits from Example 4 were used: control group (C), preharvest Yi-VD14 treatment group (Q), postharvest Yi-VD14 treatment group (H), and preharvest + postharvest Yi-VD14 treatment group (T). The surface of the chili pepper fruits was perforated according to the perforation procedure in Example 4. Bacteriocin Yi-VD-14 was added to each well of the postharvest Yi-VD14 treatment group (H) and the preharvest + postharvest Yi-VD14 treatment group (T). Ultrapure water was added to each well of the control group (C) and the preharvest Yi-VD14 treatment group (H). After 24 hours of treatment, samples were taken (circular tissue around the well with a diameter of 1 cm) and quickly frozen in liquid nitrogen to extract RNA. Total RNA was then extracted using Trizol or a kit. After passing electrophoresis, Nanodrop, and Agilent 2100 quality control, mRNA was enriched using Oligo(dT) magnetic beads and randomly fragmented. Double-stranded cDNA was synthesized using fragmented mRNA as a template. After purification, end repair, A-tailing, and ligation with sequencing adapters, a sequencing library was constructed by PCR amplification. After the library passed fragment size testing and Q-PCR quantification, it was sequenced using a high-throughput sequencing platform. The obtained raw data were filtered and then subjected to bioinformatics analysis.

[0043] The results are as follows Figure 6 As shown in the figure, the PCA plot reveals a clear spatial separation between the control group and each Yi-VD14 treatment group. Figure 6A). Volcano diagram showing the control group and the postharvest Yi-VD14 treatment group (C-vs-H). Figure 6 B1), pre-harvest Yi-VD14 treatment group (C-vs-Q), Figure 6 B2), pre-harvest + post-harvest Yi-VD14 treatment group (C-vs-T, Figure 6 A certain number of significantly differentially expressed genes were found among B3 and B4, indicating that pre- or post-harvest treatment with bacteriocin Yi-VD14 could induce significant changes in gene expression in peppers. (Annotated by GO) Figure 6 C) and KEGG enrichment analysis ( Figure 6 D) indicates that pre-harvest treatment with bacteriocin Yi-VD14 (C-vs-Q) Figure 6 D2) exerts its effect by altering the lipid physiology of the fruit membrane; postharvest treatment (C-vs-H, Figure 6 D1) showed the most significant enrichment of terpene synthase activity, indicating the induced synthesis of disease-resistant secondary metabolites; while the combined pre-harvest and post-harvest treatment (C-vs-T, Figure 6 D3) showed an extremely high level of enrichment of oxidoreductase activity, indicating that combined pre-harvest and post-harvest application can more comprehensively activate the redox defense system in peppers. In summary, pre-harvest treatment with bacteriocin Yi-VD14 downregulates lipid metabolism to enhance cell membrane structural stability and inhibit cell membrane degradation; while post-harvest treatment significantly activates the biosynthetic pathways of disease-resistant substances such as terpenes, flavonoids, and gingerol, thereby improving the fruit's chemical defense level.

[0044] Example 6: Analysis of disease resistance-related enzyme activities in pepper fruits after treatment with bacteriocin Yi-VD14

[0045] Four groups of chili pepper fruits from Example 4 were used: a control group, a pre-harvest Yi-VD14 treatment group, a post-harvest Yi-VD14 treatment group, and a pre-harvest + post-harvest Yi-VD14 treatment group. The surface of the chili pepper fruits was perforated according to the perforation procedure in Example 4. Bacteriocin Yi-VD-14 was added to each well in the post-harvest Yi-VD14 treatment group and the pre-harvest + post-harvest Yi-VD14 treatment group, while ultrapure water was added to each well in the control group and the pre-harvest Yi-VD14 treatment group. After 24 hours of treatment, samples were taken (circular tissue around the 1 cm diameter pore) and immediately frozen in liquid nitrogen to preserve enzyme activity. The samples were then thoroughly ground into powder using a grinder, and the activities of five disease-resistant enzymes (CAT, PAL, POD, PPO, and SOD) were measured using the corresponding reagent kit (Nanjing Jiancheng Bioengineering Institute).

[0046] The results are as follows Figure 7 As shown, compared with the control group, the pre-harvest + post-harvest Yi-VD14 treatment group significantly improved PAL ( Figure 7 B) and PPO ( Figure 7The activity of α-D was enhanced, promoting the synthesis of lignin and disease-resistant secondary metabolites; at the same time, all three Yi-VD14 treatment groups significantly enhanced the activity of the antioxidant enzyme system CAT ( Figure 7 A), POD ( Figure 7 C) and SOD ( Figure 7 The activity of E) and this synergistic effect effectively reduced cell damage caused by the accumulation of reactive oxygen species and significantly reduced malondialdehyde content. Comparing the Yi-VD14 treatment groups at three different application periods, the "pre-harvest + post-harvest" combined treatment showed the most significant improvement in the activity of five key disease-resistant enzymes (POD, PPO, PAL, SOD, CAT). This is highly consistent with the results of the lowest incidence rate and smallest lesion diameter observed in Example 4, proving that the bacteriocin Yi-VD14 successfully induced a systemic immune response of pepper against soft rot by improving the level of enzymatic defense in multiple dimensions.

[0047] Example 7: Analysis of Microbial Diversity in Pepper Fruits After Treatment with Bacteriocin Yi-VD14

[0048] Mature pepper fruits from the control group (CK) and the pre-harvest Yi-VD14 treatment group (VD14) in Example 4 were harvested in the field and immediately transported back to the laboratory. Each group had three replicates, with each replicate containing 6-7 pepper fruits. Microorganisms on the fruit surface were thoroughly washed into physiological saline, and the bacterial cells were collected by centrifugation. Total DNA was extracted using magnetic beads. PCR amplification was performed targeting the V4 region of bacterial 16S rDNA (primers 515F and 806R) and the ITS1 region of fungi (primers ITS5-1737F and ITS2-2043R). The PCR products were purified by magnetic beads, mixed in equal volumes, and sequenced using the NovaSeq 6000 platform. The obtained sequences were then subjected to bioinformatics analysis.

[0049] Bacterial diversity analysis results as follows Figure 8 As shown, preharvest Yi-VD14 treatment significantly remodeled the bacterial community structure, reducing the relative abundance of the phloem pathogen *Corticimicrobacter* from 47.9% in the control group to near 0, while enriching beneficial bacteria with antagonistic or growth-promoting effects, such as *Pseudomonas*, *Sphingobium*, *Massilia*, and *Methylobacterium*. According to... Figure 8 A, Figure 8 B Figure 8 C can reveal the overall trend of bacterial community diversity. Figure 8 The Venn diagram of A shows that the preharvest Yi-VD14 treatment group has a large number of unique OTUs, indicating that bacteriocin treatment alters the microbial composition on the surface of pepper fruits. Figure 8PCA plot analysis of B showed that the control group and the pre-harvest Yi-VD14 treatment group samples clustered in different regions, indicating that bacteriocin treatment significantly altered the bacterial community structure on the surface of the peppers; from Figure 8 The Alpha diversity index plot of C shows that the Shannon index of the Yi-VD14 treatment group before harvest was significantly higher than that of the control group, indicating that bacteriocin treatment altered the species and uniformity of bacteria on the surface of peppers; combined with Figure 8 The D-species abundance clustering heatmap lists the genera whose abundance was significantly upregulated (red area) and significantly downregulated (blue area) after pre-harvest Yi-VD14 treatment; induced probiotic genera increased after Yi-VD14 treatment, including Stenotrophomonas and Sphingomonas; some harmful genera decreased or even almost disappeared after Yi-VD14 treatment, including Enterococcus, Sphingobacterium, and Providencia; through Figure 8 The bar chart of species composition at the E genus level can be analyzed to reveal changes in dominant bacterial genera before and after Yi-VD14 treatment, especially the near disappearance of the phloem pathogen Corticimicrobacter; analysis Figure 8 The changes in the proportion of functional types between the control group and the pre-harvest Yi-VD14 treatment group in F indicate that the metabolic pathways altered after Yi-VD14 treatment include enhanced functions related to "chemoheterotrophy" and "nitrogen fixation / reduction," which suggests that the microbial environment has increased resistance to pathogen invasion.

[0050] Results of fungal diversity analysis, such as Figure 9 As shown, pre-harvest Yi-VD14 treatment almost completely eliminated *Verticillium*, the pathogen of Verticillium wilt, and significantly reduced the abundance of potential pathogens such as *Nigrospora* and *Pseudopithomyces*, effectively optimizing the microbial community structure on the fruit surface and enhancing disease resistance potential under natural field conditions. Figure 9 A indicates that the control group and the pre-harvest Yi-VD14 treatment group shared 309 fungal OTUs, while the control group had 406 fungal OTUs specific to it; Figure 9 In B, the control group and the pre-harvest Yi-VD14 treatment group samples were completely separated on the first principal component (PC1) axis, indicating that bacteriocin treatment significantly altered the fungal composition of the pepper surface; from Figure 9C shows that the median Shannon index of the Yi-VD14 treatment group before harvest was significantly lower than that of the control group, indicating that Yi-VD14 has an inhibitory effect on certain fungi on the surface of peppers, leading to a decrease in community diversity; Figure 9 In D, there are fungal genera that are extremely low in the pre-harvest Yi-VD14 treatment group (blue area) but high in the control group (red area). These fungal genera are the targets or affected symbiotic fungi of bacteriocins, including Pichia, Nigrospora, Verticillium, Golubevia, Anthracocystis, and Montagnula. Figure 9 E indicates that the proportion of certain fungal genera decreased significantly after Yi-VD14 treatment, including genera such as Verticillium. The decrease in the proportion of some of the above-mentioned molds indicates that Yi-VD14 treatment has a significant inhibitory effect on them. Figure 9 F showed that treatment with bacteriocin Yi-VD14 altered the niche distribution of fungi on the surface of peppers. The most significant change was that the relative abundance of plant pathogens in the pre-harvest Yi-VD14-treated group was significantly lower than that in the control group. This result is consistent with the findings of the genus-level clustering heatmap, which confirms at the functional level that Yi-VD14 can inhibit the colonization of harmful fungi.

[0051] The microbial community results show that bacteriocin Yi-VD14 can reshape the microbial community structure on plant surfaces, increasing beneficial microorganisms and reducing the types and abundance of harmful microorganisms. This is true for peppers, and also for other fruits and vegetables. Furthermore, the broad-based effects of bacteriocin Yi-VD14 on microorganisms indicate that it can control not only pepper soft rot but also other fruit and vegetable diseases.

[0052] Based on the above experimental results, bacteriocin Yi-VD14 can significantly improve the storage quality of chili peppers by delaying the decomposition of vitamin C and chlorophyll degradation and reducing malondialdehyde accumulation. Furthermore, it can induce plant immune responses and enhance plant disease resistance by regulating fruit fat metabolism, inhibiting cell membrane degradation, and improving the biosynthesis of plant disease-resistant metabolites. Simultaneously, it can increase the activity of disease-resistant enzymes in chili pepper fruits and reshape the microbial community structure on the surface of chili pepper fruits, increasing beneficial microbial flora and reducing the abundance of pathogenic microorganisms. Ultimately, it enhances the plant's resistance to pathogenic microorganism invasion from multiple dimensions, reduces the incidence of chili pepper soft rot, and minimizes post-harvest rot losses.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Bacteriocin Yi-VD14, characterized in that, The amino acid sequence is VDFGPHGLLPIRPI.

2. The use of the bacteriocin Yi-VD14 according to claim 1 in the preparation of products for the prevention and control of fruit and vegetable diseases.

3. The application of bacteriocin Yi-VD14 as described in claim 2, characterized in that, The product mentioned is for the prevention and control of soft rot in peppers.

4. A method for controlling fruit and vegetable diseases using the bacteriocin Yi-VD14 as described in claim 1, characterized in that, Apply bacteriocin Yi-VD14 before and / or after harvesting fruits and vegetables.

5. The method for controlling fruit and vegetable diseases using bacteriocin Yi-VD14 as described in claim 4, characterized in that, Apply bacteriocin Yi-VD14 before and after harvesting fruits and vegetables.

6. The method for controlling fruit and vegetable diseases using bacteriocin Yi-VD14 as described in claim 4 or 5, characterized in that, The pre-harvest application involves soaking fruit and vegetable seeds in an aqueous solution of bacteriocin Yi-VD14, followed by cultivation according to conventional seedling procedures. Throughout the entire growth period, the fruit and vegetables are sprayed with an aqueous solution of bacteriocin Yi-VD14 5-10 times, ensuring even spraying on the front and back of the leaves and stems. The post-harvest application involves spraying the surface of the fruit and vegetables with an aqueous solution of bacteriocin Yi-VD14 or soaking the fruit and vegetables in an aqueous solution of bacteriocin Yi-VD14.

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