Application of tryptophan in improving the efficacy of yeast in controlling postharvest diseases of fruits and vegetables

Abstract

The present invention discloses the use of tryptophan in improving the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables. The invention is characterized in that the yeast is Scheffersomyces spartinae W9, which has a preservation number of CGMCC No. 27149, and the post-harvest diseases of fruits and vegetables are strawberry gray mold and peach brown rot. The invention can further prepare a preparation for improving the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables. The preparation is characterized in that the yeast is a yeast having a concentration of 1×10 8 cells / mL of yeast suspension is added with tryptophan to a final concentration of 25 μM. The preparation is useful for preparing inhibitors of Botrytis cinerea and / or Sclerotinia sclerotiorum, and for preparing inhibitors of strawberry gray mold and / or peach brown rot. The advantage is that after storage at shelf temperature for three days, the incidence of gray mold in strawberries is reduced by 100%, and the incidence of brown rot in peaches is reduced by 56%.

Description

Technical Field

[0001] The present invention relates to the application in the field of biological control, and in particular to the application of tryptophan in improving the efficacy of yeast in controlling post-harvest diseases of fruits and vegetables. Background Art

[0002] Fruits and vegetables often suffer from fungal infections during production, harvesting, and post-harvest storage and transportation, leading to rot, reduced yield and quality, and significant economic losses. In recent years, the use of antagonistic bacteria to control postharvest diseases of fruits and vegetables has become a safe and effective emerging technology and one of the most promising alternatives to chemical fungicides. Yeast is a hot topic in postharvest disease biocontrol research due to its safety, stability, lack of toxin production, and ability to grow and colonize on fruit and vegetable surfaces. However, commercial production and application are limited. Marine yeast, a general term for yeasts that live in the ocean, is exposed to low temperatures, high salt levels, and high pressure. Compared to terrestrial yeasts, marine yeasts possess superior properties such as greater tolerance to cold and high osmotic pressure, making them more advantageous for biocontrol of postharvest diseases of fruits and vegetables. However, the effectiveness of antagonistic yeasts alone in controlling postharvest diseases of fruits and vegetables remains limited, and improving their biocontrol effectiveness has become a key research topic.

[0003] Tryptophol, molecular formula C 10 H 11 NO, with a molecular weight of 161.20, is an indole derivative with multiple activities. Tryptophan can induce apoptosis in normal leukemia U937 cells without affecting normal cells. In addition to its potential biological activity in humans, it also inhibits the growth of pathogens. For example, some endophytic fungi in plants can enhance their resistance to pathogenic fungi by producing tryptophan. Tryptophan also has inhibitory effects on the foodborne pathogen Salmonella. Currently, there are no published reports domestically or internationally on the use of tryptophan to enhance the biocontrol effectiveness of antagonistic yeasts against postharvest diseases of fruits and vegetables through quorum sensing. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of tryptophan in improving the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables, which can effectively prevent and control gray mold of strawberries and brown rot of peaches.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] 1. The application of color alcohol in improving the effectiveness of yeast in controlling post-harvest diseases of fruits and vegetables.

[0007] Furthermore, the tryptophan is used to improve the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables. The yeast is Scheffersomyces spartinae W9, with a deposit number of CGMCC No. 27149.

[0008] Furthermore, the tryptophan is used to improve the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables, wherein the post-harvest diseases of fruits and vegetables are gray mold of strawberry and brown rot of peach.

[0009] 2. A preparation for improving the efficacy of yeast in preventing and controlling post-harvest diseases of fruits and vegetables, wherein the preparation is 8 Tryptophan was added to a yeast suspension of 10 cells / mL to a final concentration of 25 μM.

[0010] Furthermore, the preparation is used in the preparation of Botrytis cinerea inhibitors and / or Sclerotinia sclerotiorum inhibitors.

[0011] Furthermore, the preparation is used in the preparation of an inhibitor of strawberry gray mold and / or peach brown rot.

[0012] Compared with the prior art, the advantages of the present invention are:

[0013] 1. The marine-derived yeast S. spartinae W9 secretes a secondary metabolite, tryptophan. This metabolite regulates the population density of S. spartinae W9, enabling rapid growth and colonization and enhancing its ability to form biofilms. This enhances S. spartinae W9's effectiveness in controlling postharvest fruit diseases, improving the effectiveness of postharvest biocontrol efforts and extending the fruit's storage life.

[0014] 2. Experiments conducted in this paper demonstrate that tryptophan enhances the biocontrol efficacy of marine-derived S. spartinae W9 yeast against postharvest fruit diseases. Experimental results show that adding 25 μM tryptophan to a S. spartinae W9 yeast suspension and applying it to strawberries and peaches, after three days of storage at shelf temperature, reduced the incidence of gray mold by 100% in strawberries and 56% in peaches. This demonstrates superior biocontrol efficacy and economic benefits compared to using S. spartinae W9 yeast alone.

[0015] The above-mentioned Scheffersomyces spartinae is the W9 strain, classified and named Scheffersomyces spartinae, and was deposited in the General Microbiology Center of the China Culture Collection Administration on July 5, 2023, with the deposit number CGMCC No. 27149. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The effects of secondary metabolites of marine yeast S. spartinae W9 on its growth;

[0017] Figure 2 To analyze the concentration of secondary metabolites in the fermentation broth of marine yeast S. spartinae W9 by high performance liquid chromatography;

[0018] Figure 3 To enhance the population density of marine yeast S. spartinae W9 through quorum sensing for tryptophan;

[0019] Figure 4 To enhance the biofilm formation ability of marine yeast S. spartinae W9 through quorum sensing;

[0020] Figure 5 The phenomenon of tryptophan promoting the aggregation of marine yeast S. spartinae W9 cells through quorum sensing;

[0021] Figure 6 To improve the inhibitory effect of marine yeast S. spartinae W9 on the spore germination of Botrytis cinerea and Sclerotinia sclerotiorum;

[0022] Figure 7 To improve the biocontrol effect of marine yeast S. spartinae W9 on strawberry gray mold;

[0023] Figure 8 Scanning electron microscopy observations show that tryptophan enhances the ability of the marine yeast S. spartinae W9 to form biofilms on strawberry wounds. ((a) Sterile water as a control; (b) 25 μM tryptophan; (c) S. spartinae W9; (d) S. spartinae W9 and 25 μM tryptophan. Images were taken at 3000x (a1-d1) and 5000x (a2-d2) after storage at 20°C for 2 days.)

[0024] Figure 9 To improve the biocontrol effect of marine yeast S. spartinae W9 on peach brown rot. DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0026] 1. Test methods

[0027] 1. Yeast culture

[0028] Take 50 μL of the marine yeast Scheffersomyces spartinae W9 glycerol culture stored in a -80°C refrigerator and inoculate it into 100 mL of NYDB medium (formula: 8 g beef extract, 5 g yeast extract, 10 g glucose, 1 L of pure water). Incubate it at 28°C, 180 rpm on a shaker for 24 h to obtain the first-generation yeast. The first-generation yeast solution is inoculated into 100 mL of NYDB medium at a volume ratio of 1% and incubated at 28°C, 180 rpm on a shaker for another 24 h to obtain the second-generation yeast fermentation broth. After that, the yeast cells are collected by centrifugation at 8000 rpm and 4°C for 5 min, washed twice with sterile water to remove the residual culture medium, and then resuspended in sterile water to obtain the first yeast suspension. The concentration of the yeast suspension is counted using a hemocytometer.

[0029] 2. Counting the number of live yeast

[0030] The second generation activated yeast suspension (1) in method 1 was inoculated into NYDB medium at a volume ratio of 1%. After shaking at 28°C and 180 rpm for 5 days, the culture medium was centrifuged and the supernatant was filtered through a 0.22 μm membrane to obtain a conditioned medium (also known as CM medium). The absorbance at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer to characterize the effect of different treatments on yeast cell growth. The number of viable yeast was determined using the plate counting method, and the colony forming unit (CFU) expressed the viable bacterial count.

[0031] 3. Determination of colorant content

[0032] The tryptophan content in standards and samples was determined by high-performance liquid chromatography (HPLC). An HPLC instrument (Agilent 1260 Series; USA) was equipped with an Agilent poroshell 120EC-C18 (4.6 mm × 150 mm) column and a 280 nm UV detector. Quantification was performed using the peak area external standard method. The column temperature was 30°C, the flow rate was 0.75 mL / min, and the injection volume was 10 μL. The mobile phase consisted of 50% (v / v) methanol and 50% pure water.

[0033] 4. Analysis of the ability of yeast to form biofilms on polystyrene plastic using crystal violet (CV) staining. 96-well microplates were washed twice with 200 μL of PBS to remove free cells and dehydrated with methanol for 15 min. 200 μL of 0.3% crystal violet was then added and stained for 20 min. Excess dye was removed with a pipette tip, and the plates were washed with sterile water. The crystal violet was then eluted with 200 μL of 33% glacial acetic acid at room temperature for 30 min. Finally, the absorbance at 590 nm was measured using a microplate reader (Spectra Max 190; Molecular Devices, USA).

[0034] 5. Count the germination of pathogenic fungal spores using an optical microscope

[0035] The number of germinated conidia per 200 conidia was counted under a microscope. Conidia were considered germinated when the length of the germ tube exceeded half of the conidia diameter. The germination rate (%) = number of germinated conidia / total number of conidia × 100.

[0036] 6. Use scanning electron microscopy to analyze and observe the wound status of strawberry fruits

[0037] After storage, fruit samples were taken from wounds using a sterile knife and placed in pre-cooled PBS (0.1 mol / L, pH 6.8) containing 2.5% glutaraldehyde, followed by fixation overnight (16-24 h) in a 4°C refrigerator. The fixed samples were rinsed three times with 0.1 mol / L PBS (pH 6.8), then dehydrated with graded ethanol (dehydrated once with 30%, 50%, 70%, 80%, and 90% ethanol, and twice with 100% ethanol, each dehydration time being 10 min). After dehydration, the samples were replaced with tert-butanol (replaced once with 50%, 70%, 90%, and 95% tert-butanol, and then replaced twice with 100% tert-butanol, each replacement time being 10 min). After replacement, the samples were dried in a vacuum drying oven overnight. Finally, the dried samples were gold-sprayed and then observed and photographed using an S-3400NII scanning electron microscope from HITACHI, Japan. 2. Specific embodiments

[0039] 1. Isolation and identification of strains

[0040] 1.0 g of sea mud was weighed and placed in a conical flask containing 50 mL of sterile distilled water. The mixture was incubated at 180 rpm at 28°C for 30 min. The supernatant was collected and diluted 10-fold. 100 μL of the dilution was spread onto a NYDA plate (8 g beef extract, 5 g yeast extract, 10 g glucose, 20 g agar, 1 L purified water) containing 0.1 g / L streptomycin sulfate. The plate was incubated in a 28°C incubator for 48 h. Colonies with typical yeast characteristics were selected by microscopic examination and streaked multiple times on the NYDA plate to obtain single colonies, thus obtaining the isolated and purified yeast strain. Next, the yeast isolate was molecularly identified. The ITS region of the isolate DNA was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR amplification procedure was as follows: 95°C predenaturation for 5 minutes, 95°C denaturation for 15 seconds, 53°C annealing for 30 seconds, 72°C extension for 60 seconds, 35 cycles, and 72°C extension for 5 minutes. Amplified products were confirmed by agarose gel electrophoresis and then sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. Sequencing results were compared using the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). Phylogenetic trees were constructed for each isolate and verified using the bootstrap method.

[0041] Phylogenetic analysis revealed 18 isolates identified as yeast belonging to three distinct genera: Kluyveromyces lactis (K. lactis), S. spartinae (S. spartinae), and C. pseudoambica (C. pseudoambica), accounting for 40.91%, 31.82%, and 9.09% of the total isolates, respectively. The strain used in this study was named Scheffersomyces spartinae W9 and deposited with the China General Microbiological Culture Collection on July 5, 2023, under the accession number CGMCC No. 27149.

[0042] 2. Analysis of strain metabolites

[0043] CM medium was added to each NYDB culture medium to make the volume concentration of 5%, 25%, 50%, 75%, and 100%, respectively. The second-generation activated marine yeast S. spartinae W9 suspension (1) in method 1 was inoculated into each NYDB culture medium to make the final concentration of S. spartinae W9 cells 1×10 4cells / mL, cultured in a shaker at 28°C and 180 rpm. The yeast fermentation broth was collected at 8, 12, 24, 36, 48, 60, and 72 h post-inoculation, and cell density and colony-forming units (CFU) (viable bacterial count) were measured using a UV spectrophotometer. The control group consisted of S. spartinae W9 cultured in NYDB medium without CM.

[0044] The results are as follows Figure 1 A shows that the yeast fermentation broth with 5% CM culture medium had a weak promoting effect on the OD value at 36h compared with the control group, while the treatment groups with 25%, 50%, 75%, and 100% CM culture medium significantly inhibited the growth of S. spartinaeW9. Figure 1 B shows that adding 5% CM culture medium to the yeast fermentation broth can slightly increase the number of viable yeast after 8 and 36 hours of culture of S. spartinae W9. The viable bacterial count at 8 hours (4.66log10 CFU / mL) was higher than that of the control culture medium without CM culture medium (4.51log10 CFU / mL). This indicates that the yeast S. spartinae W9 can communicate between cells by secreting secondary metabolites and quorum sensing molecules. Secondary metabolites have an impact on its growth. Low concentrations promote its growth, while high concentrations inhibit it.

[0045] 3. Analysis of the ability of yeast S. spartinae W9 to secrete tryptophan

[0046] The yeast fermentation broth obtained in the above specific example 2 was centrifuged at 8000 rpm and 4°C for 5 min. 2 mL of the supernatant was mixed with 2 mL of a 10 wt% trichloroacetic acid solution and vortexed for 1 min. The mixture was allowed to stand at 4°C for 4 h. 1 mL of the supernatant was filtered through a 0.22 μm filter membrane, aliquoted into chromatographic vials, and stored at 4°C for the determination of tryptophan content.

[0047] The results are as follows Figure 2 As shown, S. spartinae W9 yeast cell growth gradually increased with fermentation time, exhibiting an S-shaped growth curve. Growth was relatively slow from 0 to 8 hours, representing the lag phase, and then increased rapidly from 10 to 72 hours, representing the logarithmic phase. After 72 hours, yeast cell growth entered the stationary phase. Furthermore, tryptophan production gradually increased with fermentation time, indicating that S. spartinae W9 can secrete tryptophan as a secondary metabolite during fermentation. The maximum tryptophan production reached 9.21 mg / L (approximately 58.35 μM) at 120 hours of fermentation.

[0048] 4. Effects of tryptophan on the population density of yeast S. spartinae W9

[0049] Tryptophan and the yeast suspension (1) obtained in the above experimental method 1 were added to 50 mL of NYDB medium in sequence to make the final concentration of the yeast suspension 1×10 4 cells / mL, and the final tryptophan concentration was 25 μM. The cells were cultured in a shaking incubator at 28°C and 180 rpm. Samples were taken every 6, 8, 10, 12, 24, 36, and 48 h, and the absorbance at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer to characterize the effects of different tryptophan concentrations on the growth of S. spartinae W9 cells. The control group consisted of 50 μL of the yeast suspension (1) obtained in the above experimental method 1 added to 50 mL of NYDB medium to a final concentration of 1×10 4 cells / mL, and the 25 μM treatment group was prepared by adding 50 μL of the yeast suspension (1) obtained in the above experimental method 1 and 5 mL of tryptophan solution to 45 mL of NYDB medium to make the final concentration of the yeast suspension 1×10 4 cells / mL, and the final tryptophan concentration was 25 μM.

[0050] like Figure 3 As shown, the control group was cultured for 24 and 36 hOD 600 were 11.06 and 13.9 respectively, while the OD of the 25 μM tryptophan treatment group was 600 11.27 and 14.3 respectively, indicating that the addition of tryptophan can significantly promote the growth of marine yeast S. spartinae W9 and increase its population density when cultured for 24 and 36 hours. ( Figure 3 The same letters indicate no significant difference, and different letters indicate significant difference).

[0051] 5. Analysis of the biofilm formation ability of yeast S. spartinae W9

[0052] The first-generation yeast fermentation broth obtained in the above experimental method 1 was added to 50 mL of YNB medium (6.7 g of amino-free yeast nitrogen source and 5 g of glucose were weighed and dissolved in 100 mL of sterile water, sterilized by filtration with a 0.22 μM filter membrane, and then 1 L of sterile water was added to make YNB medium) at a volume ratio of 1%. After incubation at 28°C and 180 rpm for 24 h, the yeast precipitate was centrifuged at 8000 rpm and 4°C for 5 min to obtain the yeast precipitate, which was washed twice with sterile water. The yeast concentration was adjusted to 1 × 10 7cells / mL to obtain a second yeast suspension. 20 μL of this second yeast suspension was added to a 96-well microplate. 160 μL of YNB medium and 20 μL of tryptophan were then added to give a final tryptophan concentration of 25 μM. The cells were incubated at 28°C for 12, 24, and 48 hours. The ability of the yeast to form biofilms on polystyrene plastic was analyzed using crystal violet (CV) staining. A control group consisted of 20 μL of the second yeast suspension, 160 μL of YNB medium, and 20 μL of sterile water added to a 96-well microplate. A treatment group consisted of 20 μL of the second yeast suspension, 160 μL of YNB medium, and 20 μL of tryptophan added to a 96-well microplate.

[0053] The results are as follows Figure 4 As shown in the figure, compared with the control group, the addition of tryptophan can significantly improve the biofilm formation ability of S. spartinae W9 in the early stage of culture (12h). The OD 590 The values ​​were 0.84 and 1.49 respectively; its biofilm formation ability increased by 77%. ( Figure 4 The same letters indicate no significant difference, and different letters indicate significant difference).

[0054] 6. Optical microscope observation of the morphology of marine yeast S. spartinae W9 cells: The treatment group was: 100 μL of the solution obtained in the above experimental method 1 with a final concentration of 1×10 4 cells / mL of the first yeast suspension and 100 μL of tryptophan with a final concentration of 25 μM were added to 100 mL of NYDB medium in sequence.

[0055] The control group was: 100 μL of the solution obtained in the above experimental method 1 was used to obtain a final concentration of 1×10 4 cells / mL of the first yeast suspension and 100 μL of sterile water were added into 100 mL of NYDB medium in sequence.

[0056] The treated group and the control group were cultured at 28°C and 180 rpm for 24 h, and 100 μL of 1×10 3 The cells / mL diluted bacterial solution was spread on the prepared PDA (200 g potato, 20 g agar, 20 g glucose, 1 L pure water) plates and cultured in a 28°C incubator. Every 2, 3, and 5 days, a single colony was taken and dissolved in 1 mL sterile water. 50 μL of the bacterial solution was dropped onto a glass slide and cedar oil was added to the coverslip. The changes in yeast cell morphology were observed under a 100x oil microscope.

[0057] like Figure 5 As shown, the addition of tryptophan can promote the aggregation of marine yeast S. spartinae W9 cells, with obvious adhesion phenomenon.

[0058] 7. Effect of exogenous addition of tryptophan on the in vitro biocontrol effect of yeast S. spartinae W9

[0059] (1) In vitro biocontrol effect on Botrytis cinerea

[0060] Scrape the 12-day-old Botrytis cinerea and add sterile water to make 1×10 5 spores / mL (spores / mL) of Botrytis cinerea spore suspension. Take multiple centrifuge tubes, add 100 μL of Botrytis cinerea spore suspension and 800 μL of sterile PDB medium to each centrifuge tube for later use. Then take one of the centrifuge tubes and add 100 μL of sterile water as the control group; take one of the above centrifuge tubes and add 100 μL of tryptophan with a final concentration of 25 μM as treatment group 1; take one of the above centrifuge tubes and add 100 μL of 1×10 8 cells / mL yeast W9 bacterial suspension (obtained by experimental method 1) was used as treatment group 2; 100 μL of a suspension containing 1×10 8 cells / mL yeast W9 and a mixture of tryptophan at a final concentration of 25 μM was used as treatment group 3. The control group, treatment group 1, treatment group 2, and treatment group 3 were cultured at 25°C and 120 rpm in a shaking incubator for 8-10 h, and the number of spores germinated under these four different treatments was counted.

[0061] like Figure 6 As shown in A, the spore germination rates of Botrytis cinerea in the 25 μM tryptophan treatment group, the S. spartinae W9 treatment group, and the 25 μM tryptophan and S. spartinae W9 treatment groups were respectively reduced by 28.3%, 51.6%, and 66.4% compared with the control group ( Figure 6 A).

[0062] (2) In vitro biocontrol effect on Sclerotinia sclerotiorum

[0063] Scrape the fruit-growing Streptomyces sclerotiorum cultured for 12 days and add sterile water to make 1×10 5 spores / mL (spores / ml) of a spore suspension of Sclerotinia sclerotiorum. Take multiple centrifuge tubes, add 100 μL of a spore suspension of Sclerotinia sclerotiorum and 800 μL of sterile PDB medium to each centrifuge tube for later use. Then, take one of the centrifuge tubes and add 100 μL of sterile water as the control group; take one of the centrifuge tubes and add 100 μL of tryptophan with a final concentration of 25 μM as treatment group 1; take one of the centrifuge tubes and add 100 μL of a 1×10 8 cells / mL yeast W9 bacterial suspension (obtained by experimental method 1) was used as treatment group 2; 100 μL of a suspension containing 1×10 8cells / mL yeast W9 and a mixture of tryptophan at a final concentration of 25 μM was used as treatment group 3. The control group, treatment group 1, treatment group 2, and treatment group 3 were cultured at 25°C and 120 rpm in a shaking incubator for 8-10 h, and the number of spores germinated under these four different treatments was counted.

[0064] like Figure 6 B shows that the spore germination rates of Sclerotinia sclerotiorum in the 25 μM tryptophan treatment group, S. spartinae W9 treatment group, and 25 μM tryptophan and S. spartinae W9 treatment groups were respectively reduced by 9.5%, 49.0%, and 62.1% compared with the control group.

[0065] In summary, exogenous addition of tryptophan can promote the inhibitory effect of S. spartinaeW9 on the spore germination of Botrytis cinerea and Sclerotinia sclerotiorum.

[0066] 8. Effect of exogenous addition of tryptophan on the biocontrol effect of yeast S. spartinae W9 on strawberry Several ripe strawberry fruits were pricked with a sterile nail at the equator for later use; 10 μL of sterile water was added to the wound of the ripe strawberry fruit as the control group; 10 μL of tryptophan with a final concentration of 25 μM was added to the wound of the ripe strawberry fruit as treatment group 1; 10 μL of tryptophan with a final concentration of 1×10 8 cells / mL yeast W9 suspension (obtained by method 1) was used as treatment group 2; 10 μL of yeast suspension containing 1×10 8 cells / mL yeast W9 and a mixture of tryptophan at a final concentration of 25 μM was used as treatment group 3. After standing for 1 h to dry, 10 μL of 1×10 5 spores / mL (spores / mL) of Botrytis cinerea spore suspension. After air-drying, the fruit was stored in an incubator at 20°C and 95% humidity. The incidence of infection was measured after three days of storage. Four treatments were performed, each with 10 strawberries, and each group was replicated three times.

[0067] The results are as follows Figure 7 A. The incidence rate and Figure 7 B shows the symptoms of gray mold on strawberries after storage at 20°C for 3 days in one of the repetitions. After 3 days of storage, adding tryptophan to the suspension of marine yeast S. spartinae W9 reduced the incidence of gray mold on strawberries by 100%. Tryptophan can effectively increase the resistance of marine yeast S. spartinae W9 to gray mold on strawberries and enhance the biocontrol effect of yeast.

[0068] Scanning electron microscopy was used to analyze the effect of tryptophan on the biofilm formation of marine yeast S. spartinae W9 on strawberry fruit wounds. Figure 8As shown in the figure, tryptophan can promote the growth of yeast S. spartinae W9 in the wounds of strawberry fruits, closely adhere to the surface of the fruit wound, increase the population density of S. spartinae W9 on the fruit surface, thereby forming a strong spatial competitive advantage. At the same time, it can also regulate S. spartinae W9 to produce a large number of biofilms (the membrane pointed by the red arrow in the figure is the biofilm) to exhibit quorum sensing phenomenon, thereby improving the biocontrol effect of S. spartinae W9.

[0069] 9. Effect of exogenous addition of tryptophan on the biocontrol effect of yeast S. spartinae W9 on peaches. Use sterile nails to make two wounds at the equator of mature peach fruits.

[0070] 10 μL of sterile water was added to the wound of ripe peach fruit as the control group;

[0071] 10 μL of tryptophan with a final concentration of 25 μM was added to the wound of mature peach fruit as treatment group 1;

[0072] Add 10 μL of 1×10 8 cells / mL yeast W9 bacterial suspension (obtained by method 1) was used as treatment group 2;

[0073] Add 10 μL of 1×10 8 cells / mL yeast W9 and a mixture of tryptophan at a final concentration of 25 μM was used as treatment group 3. After standing for 1 hour to dry, 10 μL of a solution with a concentration of 1×10 5 spores / mL (spores / ml) of a spore suspension of Sclerotinia sclerotiorum. After air-drying, the fruit was stored in an incubator at 20°C and 95% humidity. The incidence of disease was measured after three days of storage. Four treatments were conducted, each with five peaches, and each group was replicated three times.

[0074] The results are as follows Figure 9 A. The incidence rate and Figure 9 B selected one of the repetitions, and the symptoms of brown rot of peaches appeared after storage at 20℃ for 3 days. After storage for 3 days, adding tryptophan to the suspension of marine yeast S.spartinae W9 reduced the incidence of brown rot of peaches by 56%. Tryptophan can effectively increase the susceptibility of marine yeast S.spartinae W9 to the occurrence of brown rot of peaches and enhance the biocontrol effect of yeast.

[0075] The above experiments demonstrate that the tryptophan of the present invention is a secondary metabolite secreted by the marine-derived S. spartinae W9 yeast. It can regulate the population density of the yeast S. spartinae W9, enabling rapid growth and colonization with stronger spatial competitiveness. Furthermore, the addition of tryptophan imparts a strong adhesion ability to the yeast community, allowing it to adhere to the surface of fruit wounds, forming a physical barrier. This enhances the effectiveness of the yeast S. spartinae W9 in controlling postharvest diseases of strawberries and peaches, resulting in a better biological control effect without causing environmental pollution. This addresses the problem of poor control effectiveness of existing biocontrol fungi against postharvest diseases of fruits and vegetables.

[0076] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. The use of tryptophan in improving the efficacy of yeast in controlling postharvest diseases of fruits and vegetables is characterized by: The yeast is Spartania serrata Scheffersomyces spartinae W9, the deposit number is CGMCC No. 27149, and the post-harvest diseases of fruits and vegetables are strawberry gray mold and / or peach brown rot.

2. A preparation for improving the efficacy of yeast in controlling postharvest diseases of fruits and vegetables, characterized by: The preparation is at a concentration of 1×10 8 cells / mL of yeast suspension was added with tryptophan to a final concentration of 25 μM. The yeast was S. spartanica. Scheffersomyces spartinae W9, the deposit number is CGMCC No.27149.

3. A preparation for improving the efficacy of yeast in controlling postharvest diseases of fruits and vegetables according to claim 2, characterized in that The preparation is used for preparing Botrytis cinerea inhibitor and / or Sclerotinia sclerotiorum inhibitor.

4. A preparation for improving the efficacy of yeast in controlling postharvest diseases of fruits and vegetables according to claim 2, characterized in that The preparation is used for preparing a strawberry gray mold inhibitor and / or a peach brown rot inhibitor.

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