A fumigant for controlling postharvest gray mold in tomatoes and its application

Abstract

This invention discloses a fumigant for controlling postharvest gray mold in tomatoes and its application, belonging to the field of biocidal technology. The fumigant is obtained by mixing a first solution and a second solution in a 1:1 volume ratio; the first solution is a 2-ethyl-3-hydroxy-4H-pyran-4-one solution with a concentration of 45-55 μg / mL; the second solution is a heptanoic acid solution with a concentration of 130-150 μL / mL. The fumigant inhibits the infection and spread of gray mold on postharvest tomato fruits through non-contact gas-phase fumigation. This invention utilizes an equal volume combination of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid at a preferred inhibitory concentration for gray mold control during postharvest tomato storage and transportation, replacing or reducing the use of traditional spray-type chemical fungicides. The fumigant not only inhibits gray mold infection but also maintains the postharvest quality of tomato fruits.

Description

Technical Field

[0001] This invention relates to the field of biocides technology, specifically to a fumigant for controlling postharvest gray mold in tomatoes and its application. Background Technology

[0002] tomato( Solanum lycopersicum L . Tomatoes are an important global vegetable with significant nutritional and economic value. However, due to their high water and nutrient content, tomatoes are highly susceptible to pathogenic microorganisms during storage and transportation. This susceptibility can lead to various postharvest diseases, such as those caused by Botrytis cinerea (gray mold). Botrytis cinerea This disease is caused by gray mold. Currently, the main method for controlling this disease is chemical spray fungicides, but long-term and excessive use can easily disrupt the ecological balance, produce pesticide residues, and may lead to drug resistance in pathogens. Therefore, developing safe and environmentally friendly alternative control strategies is crucial.

[0003] Currently, the main methods for controlling gray mold in fruits and vegetables include chemical fungicide treatment, low-temperature storage, modified atmosphere packaging, physical sterilization, and biological control. While chemical fungicides have some efficacy, they face challenges such as resistance, residues, and consumer acceptance. Low temperatures and modified atmosphere packaging primarily delay disease development but are insufficient for stable control when pathogens have already attached or fruits and vegetables have micro-wounds. Direct spraying of some plant-derived antibacterial agents increases free water on the surface of fruits and vegetables, potentially creating favorable humidity conditions for Botrytis cinerea germination. Fumigation is one of the mainstream technologies for post-harvest disease control in fresh agricultural products. Its core advantages lie in three aspects: First, it has strong penetrating power; the fumigant gas can evenly diffuse into the gaps between stacked fruits and vegetables, inside the packaging, and even into the interstitial spaces of the surface tissues, reaching hidden locations that traditional spraying methods struggle to cover, achieving comprehensive sterilization and inhibition, and is particularly suitable for large-scale batch processing needs. Secondly, it boasts high processing efficiency. Modern fumigation processes require only a few hours per treatment, and with specialized equipment, multiple batches can be processed daily, far faster than passive control methods such as low-temperature storage for sterilization. This allows for rapid post-harvest pretreatment to keep pace with market turnover. Simultaneously, it aligns with green requirements. New physical fumigation and low-dose chemical fumigation technologies significantly reduce pesticide usage, not only minimizing the risk of chemical residues but also allowing some inert gas fumigation to naturally dissipate without residue. This meets current consumer demands for fresh food safety and also caters to the industry's need for ready-to-eat and directly processed fruits and vegetables after harvest. Evidence suggests that volatile organic compounds (VOCs) exert antifungal effects both in vitro and in vivo by inhibiting mycelial growth or directly killing fungal cells. The efficacy of these VOCs stems from their dual modes of action: directly disrupting the cell walls of pathogens or inducing systemic resistance in host plants, enhancing the post-harvest disease-related defense capabilities of tomato fruits. In recent years, VOC fumigation, as a non-contact control method, has shown great potential in the field of post-harvest preservation of fruits and vegetables, representing a new approach to developing novel green control agents. Therefore, it is necessary to develop a fumigant for postharvest gray mold in tomatoes, and to clarify its effective components, compounding methods, non-contact fumigation application path, and comprehensive effects on pathogens and fruit quality. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a fumigant for the prevention and control of postharvest gray mold in tomatoes and its application. This invention utilizes an equal volume compound of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid for the prevention and control of gray mold during postharvest tomato storage and transportation, thereby replacing or reducing the use of traditional spray-type chemical fungicides. Simultaneously, the fumigant should not only inhibit gray mold infection but also maintain the postharvest quality of tomato fruits.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fumigant for controlling postharvest gray mold in tomatoes, wherein the fumigant is obtained by mixing a first solution and a second solution in a volume ratio of 1:1; the first solution is a 2-ethyl-3-hydroxy-4H-pyran-4-one solution with a concentration of 45-55 μg / mL; the second solution is a heptanoic acid solution with a concentration of 130-150 μL / mL; the fumigant inhibits gray mold through a non-contact gas-phase fumigation method. Botrytis cinerea Infection and spread on postharvest tomato fruits.

[0006] Preferably, the concentration of the 2-ethyl-3-hydroxy-4H-pyranone solution is 50 μg / mL; and the concentration of the heptanoic acid solution is 140 μL / mL.

[0007] Preferably, both the 2-ethyl-3-hydroxy-4H-pyranone solution and the heptanoic acid solution are prepared from ethanol solutions; the volume fraction of the ethanol solution is 75%.

[0008] In a second aspect, the present invention provides the application of the above-mentioned fumigant in the prevention and control of postharvest gray mold in tomatoes.

[0009] Preferably, the prevention and control of postharvest gray mold in tomatoes includes reducing the incidence and weight loss rate of postharvest gray mold in tomatoes, inhibiting the mycelial growth and spore germination of gray mold, and maintaining the firmness, ascorbic acid content, titratable acidity, and soluble sugar content of tomato fruits.

[0010] Preferably, the method for preventing and controlling postharvest gray mold in tomatoes is as follows: placing the postharvest tomato fruit and the above-mentioned fumigant in a sealed space, and ensuring that the fumigant does not come into direct contact with the tomato fruit, and performing sealed fumigation treatment.

[0011] Preferably, the sealed fumigation treatment is a natural evaporation process; the temperature of the sealed fumigation treatment is 20-28 ℃, and the time is 1-7 days.

[0012] Preferably, the fumigant is used at a dosage of per 1000 cm³. 3 Add 100-1000 μL of fumigant to the enclosed space.

[0013] That is, the amount of fumigant used is 100-1000 μL / L in a closed space.

[0014] Preferably, the fumigant is loaded onto a volatile carrier, which is selected from filter paper sheets, non-woven fabric sheets, absorbent paper sheets, cellulose sheets, silica gel sheets, gel sheets, or open containers.

[0015] Preferably, the sealed space is selected from food storage boxes, food storage bags, polyethylene bags, turnover boxes, storage boxes, cold storage, or transport packaging space.

[0016] The beneficial effects of this invention are: (1) Safety and environmental protection: The present invention uses 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid as the core of prevention and control. Through non-contact fumigation, it avoids the problems of chemical pesticide residues, toxicity and environmental pollution, and meets the requirements of green production.

[0017] (2) Highly effective prevention and control: Through non-contact fumigation, the fumigant and its main components can significantly inhibit Botrytis cinerea in both the external and internal environments. Botrytis cinerea The growth and spore germination of the pathogen were inhibited (with an inhibition rate of nearly 100%). Even direct inoculation of the pathogen onto the fruit could control the disease incidence and index to levels far lower than those in the untreated group.

[0018] (3) The mechanism of action is clear: the compound destroys the integrity of the pathogen cell membrane (leading to increased conductivity, leakage of nucleic acids and proteins), and causes serious damage to hyphal morphology and cell ultrastructure (reduction of mitochondria, etc.), leading to its death.

[0019] (4) Synergistic preservation: The prevention and control scheme of the present invention can not only effectively prevent disease, but also significantly maintain the post-harvest storage quality of tomato fruits, including delaying the decline in fruit firmness, titratable acidity, ascorbic acid (vitamin C) and soluble sugar content, which is better than the treatment of disease.

[0020] (5) Dual prevention and control mode: In addition to directly killing pathogens, the drug provided by this invention can also induce host plants to produce systemic resistance, significantly increase the activity of endogenous antioxidant enzymes in tomato fruits (increase SOD, POD, CAT) and reduce membrane lipid peroxidation products (decrease MDA content), thereby enhancing the fruit's own antioxidant capacity. Attached Figure Description

[0021] Figure 1 The in vivo control effects of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid on Botrytis cinerea, including (a) representative images of the in vivo control of the compounds on Botrytis cinerea; (b) the effect of 2-ethyl-3-hydroxy-4H-pyranone fumigation on the Botrytis cinerea Disease Index (DI); and (c) the effect of heptanoic acid fumigation on the Botrytis cinerea Disease Index (DI). Figure 2 The effects of 2-ethyl-3-hydroxy-4H-pyranone fumigation on the appearance and quality of healthy tomatoes, including (a) the effect of 2-ethyl-3-hydroxy-4H-pyranone fumigation on the appearance of healthy tomatoes; (b) hardness; (c) titratable acidity (TA); (d) soluble sugar content; and (e) ascorbic acid (ASA) content. Figure 3The effects of heptanoic acid fumigation on the appearance and quality of healthy tomatoes, including (a) the effect of heptanoic acid fumigation on the appearance of healthy tomatoes; (b) hardness; (c) titratable acidity (TA); (d) soluble sugar content; and (e) ascorbic acid (ASA) content. Figure 4 The antifungal effects of compound drug fumigation, including (a) the inhibitory effect of compound drugs on the mycelial growth of Botrytis cinerea; (b) quantitative analysis of Botrytis cinerea spore germination; and (c) the effect of compound drug treatment on Botrytis cinerea spore germination. Figure 5 The effects of compound drugs on cell membrane permeability, including (a) conductivity; (b) nucleic acid leakage; and (c) protein leakage. Figure 6 The effects of the compound drug on the ultrastructural changes of Botrytis cinerea hyphae, including (a) scanning electron microscope (SEM) images of Botrytis cinerea hyphae after exposure to the compound drug; and (b) transmission electron microscope (TEM) images of Botrytis cinerea hyphae after exposure to the compound drug (M, mitochondria; N, nucleus; CW, cell wall; C, cytoplasm). Figure 7 The effects of compound drugs on postharvest botrytis cinerea infection in tomato fruits, including (a) the inhibitory effect of compound drugs on botrytis cinerea growth in vivo; (b) morbidity rate; and (c) weight loss rate. Figure 8 The effects of compound drugs on the antioxidant capacity of tomato fruit, including (a) SOD; (b) POD; (c) CAT; and (d) MDA content. Figure 9 The effects of compound drugs on the quality of tomato fruit, including (a) firmness; (b) ascorbic acid content; (c) titratable acidity; and (d) soluble sugar content. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] As described in the background section, while 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid possess some antibacterial activity, they are not broad-spectrum fungicides. Furthermore, fumigation significantly reduces the concentration of the active ingredients, making it unpredictable whether they retain antibacterial activity at extremely low concentrations. In addition, there are no reports of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid being used to control postharvest gray mold in tomatoes.

[0024] Therefore, the purpose of this invention is to provide a fumigant for preventing and controlling postharvest gray mold in tomatoes. This invention aims to address the problem of gray mold (a fungus caused by botrytis cinerea) during postharvest storage and transportation by combining 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid in equal volumes at a preferred inhibitory concentration. Botrytis cinerea This invention addresses the problem of gray mold caused by tomato fungicides, and the technical challenges of insufficient safety, environmental friendliness, and sustainability in existing control methods. Currently, controlling tomato gray mold mainly relies on spray-on fungicides, but excessive use can disrupt the ecological balance and may lead to drug resistance in pathogens, while also posing food safety risks such as pesticide residues. This invention employs fumigation, a safer and more reliable environmentally friendly alternative to spraying, to effectively reduce post-harvest fruit decay while meeting consumers' growing demands for food safety and environmental protection. This fumigation method not only needs to effectively control the disease but should also maintain or improve the storage quality of the fruit (such as firmness, nutritional components, and flavor).

[0025] This invention first involves co-culturing 2-ethyl-3-hydroxy-4H-pyranone or heptanoic acid with *Botrytis cinerea* using a double-plate covering method. This inhibits the growth of *Botrytis cinerea* cells and spores, increases the conductivity of the *Botrytis cinerea* cell suspension, and causes leakage of nucleic acids and proteins. The morphological characteristics of the hyphae are observed using scanning electron microscopy, and the optimal inhibitory concentrations are then determined. An equivalent volume of fumigant is then prepared, and the fumigant is co-cultured with tomato fruits inoculated with *Botrytis cinerea* using a fumigation method. The tomato fruits inoculated with *Botrytis cinerea* and the petri dishes coated with the fumigant are sealed in a transparent polyethylene bag. This reduces the incidence of disease, disease index, and fruit weight loss in the tomatoes; enhances the antioxidant capacity of the tomatoes, specifically by increasing the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) and reducing malondialdehyde (MDA) content; and maintains the quality of the tomatoes by ensuring that the fumigant maintains the firmness, ascorbic acid content, acidity, and soluble sugar content of the tomatoes.

[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0027] Note: The 2-ethyl-3-hydroxy-4H-pyranone used in this invention is also known as 2-ethyl-3-hydroxy-4H-pyran-4-one, ethyl maltol, abbreviated as EM, CAS number: 4940-11-8; heptanoic acid CAS number: 111-14-8.

[0028] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Effects of 2-ethyl-3-hydroxy-4H-pyranone or heptanoic acid on the in vitro and in vivo control of Botrytis cinerea and on fruit quality. A 8 mm diameter block of gray mold mycelium was inoculated at the center of a PDA plate. (The pathogen was provided by the College of Plant Protection, Northeast Agricultural University, and the Key Laboratory of Agricultural Microbiology, Heilongjiang Province; see Biological control of gray mold of tomato by...) Bacillus altitudinis B1-15[J]. (Song J, Ling L, Xu X, et al. Biological Control, 2023, 183(000): 9.)), 2-ethyl-3-hydroxy-4H-pyranone was prepared into solutions of 10, 20, 30, 40, 50, 60 and 70 μg / mL with 75% (v / v) ethanol, and heptanoic acid was prepared into solutions of 50, 100, 120, 140, 160, 180 and 200 μL / mL. 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid were used as volatile sources for double-plate culture. 40 μL of each compound solution was dropped into the bottom of a sealed culture dish (sealed space volume approximately 127.23 mL) as a volatile source, equivalent to approximately 300 μL of fumigant per liter of sealed space. An equal volume of sterile distilled water was used as a blank control. Using an equal volume of ethanol (75% (v / v)) as the solvent control, each treatment was performed in triplicate. Different letters after the data indicate significant differences between groups. P <0.05). After incubation at 25 ℃ for 7 days, the colony diameter was recorded and the inhibition rate was calculated. The results are shown in Table 1.

[0030] Table 1. Inhibition rate of gray mold Table 1 shows that both 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid exhibit significant inhibitory effects on Botrytis cinerea, and the inhibitory activity is concentration-dependent. The preferred inhibitory concentration for 2-ethyl-3-hydroxy-4H-pyranone is 50 μg / mL, and the preferred inhibitory concentration for heptanoic acid is 140 μL / mL.

[0031] Example 2: Postharvest test of 2-ethyl-3-hydroxy-4H-pyranone or heptanoic acid for the control of gray mold in tomatoes Select uniformly sized, undamaged tomatoes (tomato variety: Zhongshu No. 4, maturity: 8-9 ripe). Using a sterile punch, create a 3 mm wide and 2 mm deep incision at the equator of each fruit and inoculate with a block of *Botrytis cinerea* mycelium. Place three tomato fruits in a 3000 mL polyethylene bag. Apply 900 μL of 50 μg / mL 2-ethyl-3-hydroxy-4H-pyranone or 140 μL / mL heptanoic acid to the bottom of the container for fumigation. CK represents untreated tomatoes; BO represents tomatoes inoculated with *Botrytis cinerea* without compound fumigation; EM represents tomatoes inoculated with *Botrytis cinerea* after fumigation with 2-ethyl-3-hydroxy-4H-pyranone; HE represents tomatoes inoculated with *Botrytis cinerea* after fumigation with heptanoic acid. Each treatment has three replicates. Fruit condition and disease index are recorded at 0, 3, and 7 days post-inoculation. The results are shown below. Figure 1 As shown in the figure. The firmness, titratable acidity, soluble sugar content, and ascorbic acid content of tomatoes were also tested, and the results are shown in the figure. Figures 2-3 As shown.

[0032] Hardness testing method: Using a sterile knife, make a 1 cm × 1 cm × 1 cm incision at the equator of the tomato fruit. Insert the zeroed hardness tester (GY-1, China) vertically to the scale line. Read and record the value immediately after the probe is fully inserted into the pulp. Repeat the test on 3 fruits for each sample, and measure 3 times for each fruit at different positions at the equator. Titratable acid (TA) was detected by acid-base titration according to the method described in the literature "2-Phenylethanol biocontrol postharvest tomato gray mold and its effect on tomato quality" ([J]. Wu F, Lin Y, Zheng B, et al. Scientia Horticulturae, 2024, 337: 113550.). The soluble sugar content was determined using the anthrone-sulfuric acid method, referring to the method described in the literature "Aroma enhancement of blueberry wine by postharvest partial dehydration of blueberries" ([J]. Wang Y, Zhang Q, Cui MY, et al. Food Chemistry, 2023, 426: 136593.). The ascorbic acid (AsA) content was determined by titration with 2,6-dichlorophenolindophenol according to the method described in the literature "Effects of variety on the quality of tomato stored under ambient conditions" ([J]. Tigist M, Workneh TS, Woldetsadik K. Journal of food science and technology, 2013, 50(3): 477-486.). Disease severity is assessed using a 4-point scale: Grade 0, no lesion area; Grade 1, 0 < lesion area ≤ 5%; Grade 2, 5% < lesion area ≤ 10%; Grade 3, lesion area ≤ 20% (10% < 20%); Grade 4, lesion area >20%; The formula for calculating the disease index is as follows: .

[0033] like Figure 1 As shown, on day 7, the disease index of B0 tomatoes reached 100%, while the disease index of EM tomatoes was 45.32%, indicating a significant decrease in the disease index. P <0.05%. Furthermore, fumigation with 2-ethyl-3-hydroxy-4H-pyranone partially softened the tomato tissue. Therefore, 2-ethyl-3-hydroxy-4H-pyranone significantly inhibited gray mold in vivo. Similarly, compared to group B0, fumigation with heptanoic acid significantly reduced the severity of disease (…). P <0.05), with a disease index of 47.22%. Meanwhile, heptanoic acid fumigation effectively slowed down the softening of tomato tissue.

[0034] like Figure 2 (a) ~ Figure 2 (b) and Figure 3 (a) Figure 3 As shown in (b), the tomato fruits treated with both compounds did not rot throughout the storage period, effectively inhibiting the decrease in firmness. Furthermore... Figure 2 (c)~ Figure 2 (e) Figure 3 (c)~ Figure 3As shown in (e), compared with the control (CK), neither 2-ethyl-3-hydroxy-4H-pyranone nor heptanoic acid treatment significantly altered ASA, soluble sugar, and TA levels. Overall, fumigation with the tested concentrations of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid effectively controlled tomato fruit rot without affecting key quality attributes such as firmness and nutritional components, thus maintaining post-harvest fruit quality.

[0035] Example 3: Evaluation of the combined toxicity of compound drugs 50 μg / mL of 2-ethyl-3-hydroxy-4H-pyranone was designated as A, and 140 μL / mL of heptanoic acid was designated as B. Compounds were prepared at volume ratios of A:B of 4:1, 2:1, 1:1, 1:2, and 1:4. The optimal compound ratio was determined according to the method in Example 1, and its antibacterial activity was compared with that of a single compound. The antibacterial method was the same as in Example 1, with approximately 300 μL of fumigant added per liter of sealed space. Each treatment was performed in triplicate. Different letters after the data indicate significant differences between groups. P <0.05). The antibacterial rate is shown in Table 2.

[0036] Table 2 Antibacterial rates of different components As shown in Table 2, the combination of 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid in a 1:1 volume ratio exhibited the highest antibacterial activity, which was significantly higher than that of either the single compound 2-ethyl-3-hydroxy-4H-pyranone or heptanoic acid. P <0.05).

[0037] The Wadley method was used to evaluate its combined toxicity, and the calculation formula is as follows: EC 50 (th) = (a+b) / [a / EC 50 (A) + b / EC 50 (B)]; Efficiency coefficient SR = EC 50 (th) / EC 50 (ob); In the above formula, A represents 2-ethyl-3-hydroxy-4H-pyranone, B represents heptanoic acid, a represents the ratio of 2-ethyl-3-hydroxy-4H-pyranone, b represents the ratio of heptanoic acid (a and b are the μg / mL percentages of A and B in the compound system, respectively), and ob represents the actual observed value (i.e., EC 50(ob) represents the measured half-inhibitory concentration obtained by fitting the antibacterial rate-concentration regression equation of the compound system, and th represents the theoretical value. The virulence regression equation for A is: Y = 1.79 + 2.31 log C R 2 = 0.992; the toxicity regression equation for B is Y =-4.91 + 2.05 log C R 2 = 0.989; where C This represents the drug concentration (μg / mL). Heptanoic acid... EC 50 The unit according to ρ =0.92 g / mL conversion, that is, A's EC 50 (ob) = 24.62 μg / mL, B's EC 50 (ob) = 68449.52 μg / mL). When SR When the value is >1.5, it indicates that the compounding effect is synergistic; when 0.5 ≤ SR When ≤ 1.5, it indicates that the compounding result is additive; SR A SR < 0.5 indicates an antagonistic effect in the combination. To highlight the level of significant synergistic effect, SR > 1.5 is defined as significant synergistic effect. The results are shown in Table 3.

[0038] Table 3. Combined toxicity evaluation based on the Wadley method. Note: In the joint toxicity evaluation, to ensure consistency of calculation units, the concentration of heptanoic acid was converted to mass concentration based on density 0.92 g / mL; the EC50 of ethyl maltol and heptanoic acid were calculated by the toxicity regression equation.

[0039] As shown in Table 3, after 2-ethyl-3-hydroxy-4H-pyranone and heptanoic acid are combined in a 1:1 volume ratio, SR The result is 1.55 > 1.5, indicating that the compounding effect is significant.

[0040] Example 4: Fumigant inhibits the growth of Botrytis cinerea in vitro A fumigant was prepared by mixing 50 μg / mL of 2-ethyl-3-hydroxy-4H-pyranone and 140 μL / mL of heptanoic acid in a 1:1 volume ratio. 100 μL of the fumigant was placed on a petri dish (referred to as the treatment), with a dish without the fumigant serving as the blank control (CK). In another clean petri dish, a 2 cm diameter, 0.8 cm high Oxford cup was placed in the center, and 20 μL of a Botrytis cinerea spore suspension (1.0 × 10⁻⁶) was inoculated. 6CFU / mL). The petri dishes coated with fumigant were inverted onto the Botrytis cinerea petri dishes, and the entire assembly was sealed with paraffin film (equivalent to approximately 300 μL of fumigant per liter of sealed space). Each treatment was performed in triplicate, and spore germination was assessed at 4, 6, and 8 h post-treatment. One hundred conidia were randomly selected from each treatment for observation; germination was defined as when the hypocotyl length reached at least half the diameter of the conidia. The results are shown in […]. Figure 4 .

[0041] Botrytis cinerea was cultured on PDA plates at 25 °C for 2 days, and then the plates were fumigated with fumigant for 1, 2, 3, and 4 days. After each fumigation cycle, the hyphae were scraped from the PDA surface, suspended in sterile distilled water, and centrifuged at 3200 g for 5 min. The supernatant was collected, the conductivity was measured, and the absorbance at 260 nm and 280 nm was measured using a UV-Vis spectrophotometer to characterize the leakage of nucleic acid and protein-like substances. The results are shown in [Figure number missing]. Figure 5 .

[0042] Scanning electron microscopy and transmission electron microscopy revealed the ultrastructural changes of Botrytis cinerea hyphae caused by the fumigant. The results are shown in [Figure number missing]. Figure 6 .

[0043] like Figure 4 As shown, compared with the control group (CK), the fumigant treatment significantly inhibited the growth of *Botrytis cinerea* mycelia. After 7 days of cultivation, the colony diameter in the CK group reached 8.89 cm ± 0.05 cm, while no significant mycelial growth was detected in the treatment group, with an inhibition rate of 100%. These results indicate that the fumigant has strong antifungal activity. To further investigate the antifungal activity of the strain's volatile organic compounds (VOCs), spore germination was assessed. Compared with the CK group, spore germination was significantly inhibited at all assessment time points after exposure to VOCs. After 8 hours of cultivation, the spore germination rate in the CK group reached 98.45%, while the spore germination rate in the treatment group was only 3.88%, a relative decrease of approximately 96.06%. These results indicate that the fumigant can inhibit the growth of *Botrytis cinerea* by significantly reducing spore germination.

[0044] like Figure 5 As shown, the control (CK) maintained a low conductivity throughout the experiment. In contrast, the conductivity of *Botrytis cinerea* hyphae gradually increased over time after exposure to the fumigant, with all treatment groups exhibiting higher conductivity than the CK. Cell membrane damage is typically reflected through the efflux of intracellular macromolecules such as proteins and nucleic acids. In the CK, the levels of these substances showed minimal change, while fumigant treatment significantly enhanced extracellular leakage, and this effect gradually increased with prolonged exposure time. These results indicate that fumigation disrupts the cell membrane integrity of *Botrytis cinerea* hyphae.

[0045] like Figure 6 As shown in (a), scanning electron microscopy results indicate that the hyphae in group CK are uniform in thickness, smooth in surface, and exhibit regular branching. The hyphae fumigated with the fumigant show rough surfaces, deformation, and increased branching. Figure 6 In group (b), the fumigant-treated cells exhibited irregular shapes, reduced cytoplasmic matrix, and almost no mitochondria in the cytoplasm. In group CK, *Botrytis cinerea* cells showed normal morphology, with intact cell walls, plasma membranes, and septa, and abundant, structurally intact mitochondria in the cytoplasm. This indicates that the fumigant not only strongly inhibits the growth of *Botrytis cinerea* but also causes severe damage to hyphal structure and significantly reduces the number of mitochondria.

[0046] Example 5: In vivo control of postharvest gray mold in tomatoes using fumigants (1) Prepare a fumigant by mixing 50 μg / mL of 2-ethyl-3-hydroxy-4H-pyranone and 140 μL / mL of heptanoic acid in a 1:1 volume ratio. Place 100 μL of the fumigant in an uncovered Petri dish. Select uniformly sized, undamaged tomatoes (tomato variety: Zhongshu No. 4, maturity: 8-9 ripeness), and use a sterile punch to create a 3 mm wide and 2 mm deep wound at the equator of each fruit, and inoculate with an 8 mm block of Botrytis cinerea mycelium. Place 3 tomatoes and 3 uncovered Petri dishes of fumigant in each transparent polyethylene bag (approximately 3 L in volume), which is equivalent to 100 μL / L of treatment space.

[0047] Four treatments were set up: CK, untreated tomatoes; Y0, tomatoes not inoculated with gray mold that were fumigated with fumigant; B0, tomatoes inoculated with gray mold that were not fumigated with fumigant; and BY, tomatoes inoculated with gray mold that were fumigated with fumigant. Each treatment had 3 replicates.

[0048] like Figure 7 As shown in (a), no rot or disease symptoms were observed at the wound site in groups CK and Y0. In contrast, group B0 showed the most severe disease symptoms 7 days after inoculation with gray mold, characterized by extensive mycelial growth around the wound and fungal penetration into the fruit. In contrast, the fruit in group BY remained in good condition, with only mild gray mold symptoms at the wound site, indicating that the fumigant effectively controlled the disease. Figure 7 (b) and Figure 7 As shown in (c), the incidence of disease and the rate of weight loss in group B0 gradually increased over time. By day 7, the incidence rate in group B0 was 100%, and the rate of weight loss was 3.92%. In contrast, the incidence rate and the rate of weight loss in group BY were significantly lower.

[0049] (2) At 0 (2 h), 1, 3, 5 and 7 days after the treatment in the above experiment (1), 1 g of tissue around the tomato wound was collected and the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined. Group Y0 was not tested because it was not inoculated with gray mold.

[0050] like Figure 8 As shown, the antioxidant enzyme activity in the CK group remained consistent during tomato storage. Figure 8 (a) Figure 8 In the middle (c) group, after inoculation with *Botrytis cinerea*, the activities of SOD, POD, and CAT in both the B0 and BY groups showed a trend of first increasing and then decreasing. In the B0 group, the activities of POD and CAT reached their peak on day 3, increasing by 11% and 79% respectively compared to the CK group. The induction effect was more pronounced in the BY group, with POD and CAT activities reaching 1.86 and 2.24 times the CK level, respectively, both significantly higher than other treatments. P <0.05). Furthermore, the SOD activity in the BY group reached its maximum on day 5, significantly higher than that in the B0 and CK groups. Influenced by the pathogen, the MDA content in the B0 group was 3.126 μmol / g FW, while... Figure 8 Fumigation with fumigant in group (d) BY significantly reduced the MDA content to 1.742 μmol / g FW.

[0051] (3) Seven days after the treatment in the above experiment (1), fruit pulp samples were collected from the same location of each tomato, the fruit hardness was measured using a handheld hardness tester, the ascorbic acid and soluble sugar content were determined using a test kit, and the titratable acidity was determined by titration.

[0052] like Figure 9 As shown in (a), pathogen infection significantly reduced fruit firmness by 26.60%, while fruit fumigated with the fumigant (BY) maintained a firmness level close to that of the control (CK, healthy fruit). Similarly, in the pathogen-inoculated control group (B0), ascorbic acid (ASA) and soluble sugar content were reduced by 42.86% and 40.98%, respectively, compared to the CK. Figure 9 (b) and Figure 9 The fumigation treatment in the (d) BY group effectively mitigated this phenomenon, restoring ASA and soluble sugar levels to levels comparable to healthy fruit. Furthermore, Figure 9 The (c) BY group significantly slowed the decline in titratable acidity (TA) over 7 days, resulting in a significant difference in TA content between the BY and B0 groups. This further demonstrates that fumigation with fumigants helps maintain the quality of tomato fruit during storage.

[0053] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A fumigant for controlling postharvest gray mold in tomatoes, characterized in that, The fumigant is obtained by mixing a first solution and a second solution in a volume ratio of 1:1; the first solution is a 2-ethyl-3-hydroxy-4H-pyran-4-one solution with a concentration of 45-55 μg / mL; the second solution is a heptanoic acid solution with a concentration of 130-150 μL / mL; the fumigant inhibits Botrytis cinerea by a non-contact gas phase fumigation method Botrytis cinerea on postharvest tomato fruits.

2. The fumigant of claim 1, wherein, The concentration of the 2-ethyl-3-hydroxy-4H-pyranone solution is 50 μg / mL; the concentration of the heptanoic acid solution is 140 μL / mL.

3. The fumigant of claim 2, wherein, The 2-ethyl-3-hydroxy-4H-pyranone solution and the heptanoic acid solution were both prepared from ethanol solution; the volume fraction of the ethanol solution was 75%.

4. The use of the fumigant according to any one of claims 1 to 3 in the prevention and control of postharvest gray mold in tomatoes.

5. Use according to claim 4, characterized in that, The control of postharvest gray mold in tomatoes includes reducing the incidence and weight loss of postharvest gray mold, inhibiting the mycelial growth and spore germination of gray mold, and maintaining the firmness, ascorbic acid content, titratable acidity, and soluble sugar content of tomato fruits.

6. Use according to claim 4, characterized in that, The method for preventing and controlling postharvest gray mold of tomatoes is as follows: placing postharvest tomato fruits and the fumigant described in any one of claims 1-3 in a sealed space, and ensuring that the fumigant does not come into direct contact with the tomato fruits, and performing sealed fumigation treatment.

7. Use according to claim 6, characterized in that, The sealed fumigation treatment is a natural volatilization process; the temperature of the sealed fumigation treatment is 20-28 ℃, and the time is 1-7 days.

8. Use according to claim 6, characterized in that, The amount of fumigant used is 100-1000 μL per 1000 cm 3 100-1000 μL of fumigant is added to the closed space.

9. The application according to claim 6, characterized in that, The fumigant is loaded onto a volatile carrier, which is selected from filter paper sheets, non-woven fabric sheets, absorbent paper sheets, cellulose sheets, silica gel sheets, gel sheets, or open containers.

10. The application according to claim 6, characterized in that, The enclosed space is selected from food storage boxes, food storage bags, polyethylene bags, turnover boxes, storage boxes, cold storage, or transport packaging space.

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