A biocontrol bacterial strain and application thereof in controlling postharvest diseases of fruits and vegetables

Abstract

The present application relates to a kind of fungal strains with broad-spectrum bacteriostatic effect and its application in the control of postharvest diseases of fruits and vegetables.The classification of the fungus of the present application is named Pichia kluyveri, and the Latin name is Pichia kluyveri.The strain is preserved in China General Microbiological Culture Collection Center (CGMCC), and the address is No.3, Institute of Microbiology, Chinese Academy of Sciences, Beijing Chaoyang District, North Star West Road No.1, and the preservation number is CGMCC No.32124, and the preservation date is September 30, 2024.The strain has inhibitory effect on a variety of pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a fungal strain with broad-spectrum antibacterial effect and its application in controlling postharvest diseases of fruits and vegetables. BACKGROUND

[0002] Fruits and vegetables are rich in nutrients and are an important part of the human dietary structure. However, postharvest rotting of fruits and vegetables is serious, causing huge economic losses every year. Decay caused by pathogenic fungi is the main reason for the decrease in quality and loss of fruits and vegetables after harvest, and some postharvest pathogens can also produce mycotoxins that are harmful to human health, posing a major safety hazard.

[0003] Currently, the prevention and control of postharvest diseases of fruits and vegetables mainly includes physical control, chemical control and biological control. Due to the limitations of cost and its range of use, the physical control method is limited in agricultural production. The use of chemical fungicides is currently the most important method for preventing and controlling postharvest diseases, but the excessive use of chemical agents can cause environmental pollution, threaten food safety and human health, and lead to the development of drug resistance in pathogenic fungi. Biological control is the use of antagonistic microorganisms to inhibit the growth and reproduction of pathogenic fungi through species interactions, thereby controlling postharvest diseases of fruits and vegetables and inhibiting the production of mycotoxins. Due to its safety and environmental friendliness, biological control has gradually become a research hotspot for controlling postharvest diseases of fruits and vegetables and is expected to replace chemical fungicides in the future. To date, hundreds of antagonistic microorganisms have been isolated and identified, including bacteria, molds and yeasts. Among them, antagonistic yeasts have become a common strain for controlling postharvest diseases of fruits and vegetables due to their strong antibacterial ability, high safety, low nutritional requirements, fast growth and reproduction, and strong tolerance to adversity.

[0004] Pichia yeasts have great application potential in improving the floral and fruity characteristics of grape wine. Pichia kluyveri is one of the most concerned strains in the fermentation process of various beverages. However, there is currently no research on the biological control effect of P. kluyveri yeasts on multiple postharvest pathogenic fungi of fruits and vegetables. SUMMARY

[0005] The present application aims to provide a fungal strain with broad-spectrum antibacterial effect and its application in the biological control of postharvest pathogens of fruits and vegetables.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] In one aspect, the present application provides a fungal strain with broad-spectrum antibacterial effect, characterized in that the strain is classified as Pichia kluyveri with Latin name Pichia kluyveri, and the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard 3rd, with the preservation number CGMCC No 32124 and the preservation date September 30, 2024.

[0008] Further, the 26S rDNA sequence of the strain is shown in SEQ ID NO. 3.

[0009] In another aspect, the present application provides the use of the aforementioned strain in inhibiting the growth of pathogenic bacteria.

[0010] Preferably, the inhibition of the growth of pathogenic bacteria refers to the inhibition of the growth of plant pathogenic bacteria.

[0011] Preferably, the plant is a plant of Rosaceae, Ericaceae or Rhamnaceae.

[0012] Preferably, the plant is a plant of Fragaria, Malus, Prunus, Vaccinium or Ziziphus.

[0013] Preferably, the plant is Fragaria, Malus, Prunus, Vaccinium or Ziziphus.

[0014] Preferably, the plant is a plant of Fragaria, Malus, Prunus, Vaccinium or Ziziphus.

[0015] Preferably, the pathogenic bacteria include at least one of Alternaria sp., Trichothecium sp., Moniliniasp., Penicillium sp. and Botrytis sp.

[0016] Preferably, the pathogenic bacteria include at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

[0017] In another aspect, the present application provides the use of the aforementioned strain in inhibiting the synthesis of pathogenic bacterial toxins.

[0018] Preferably, the inhibition of the synthesis of pathogenic bacterial toxins refers to the inhibition of the synthesis of toxins of plant pathogenic bacteria.

[0019] Preferably, the plant is a plant of Rosaceae, Ericaceae or Rhamnaceae.

[0020] Preferably, the plant is a Fragaria, Malus, Prunus, Vaccinium or Ziziphus plant.

[0021] Preferably, the plant is a Fragaria, Malus, Prunus, Vaccinium or Ziziphus plant.

[0022] Preferably, the plant is a Fragaria, Malus, Prunus, Vaccinium or Ziziphus plant.

[0023] Preferably, the pathogenic fungus is a pathogenic fungus that can parasitize on a fruit of a plant.

[0024] Preferably, the pathogenic fungus is a pathogenic fungus that can parasitize on a fruit of a plant.

[0025] Preferably, the pathogenic fungus is at least one of Alternaria sp., Trichothecium sp., Moniliniasp., Penicillium sp. and Botrytis sp.

[0026] Preferably, the pathogenic fungus is at least one of Alternaria sp., Trichothecium sp., Moniliniasp., Penicillium sp. and Botrytis sp.

[0027] 1) cultivating the aforementioned strain;

[0028] 2) inhibiting the growth of the pathogenic fungus using the cultivation product of step 1).

[0029] Further, step 2) comprises:

[0030] 2.1) contacting the cultivation containing the strain with the pathogenic fungus;

[0031] 2.2) inhibiting the growth of the pathogenic fungus.

[0032] Optionally, the contacting the cultivation containing the strain with the pathogenic fungus of step 2.1) can use any method that can achieve the contacting effect, including soaking, spraying, flushing, etc.

[0033] Further, another embodiment of step 2) comprises:

[0034] 2.1) contacting the volatile substance produced by the strain with the pathogenic fungus;

[0035] 2.2) inhibiting the growth of the pathogenic fungus.

[0036] Optionally, step 2.1) said contacting the volatile substances produced by the bacterial strain with the pathogenic bacteria can be achieved by any method that can bring the volatile substances into contact with the pathogenic bacteria, including any one or more of the following methods that can bring the volatile substances into contact with the pathogenic bacteria: placing the culture in close proximity to the pathogenic bacteria; placing the culture in a relatively closed space with the pathogenic bacteria; spraying or misting the collected volatile substances onto the pathogenic bacteria; placing the collected volatile substances in a relatively closed space with the pathogenic bacteria; and the like.

[0037] In another aspect, the present application provides a method for inhibiting the toxin synthesis of pathogenic bacteria, comprising the following steps:

[0038] 1) cultivating the bacterial strain as described above;

[0039] 2) using the cultivation product of step 1) to inhibit the toxin synthesis of pathogenic bacteria.

[0040] Further, step 2) comprises:

[0041] 2.1) contacting the culture containing the bacterial strain with the pathogenic bacteria;

[0042] 2.2) inhibiting the toxin synthesis of the pathogenic bacteria.

[0043] Optionally, step 2.1) said contacting the culture containing the bacterial strain with the pathogenic bacteria can be achieved by any method that can bring the culture into contact with the pathogenic bacteria, including any one or more of the following methods that can bring the culture into contact with the pathogenic bacteria: soaking, spraying, flushing, and the like.

[0044] Optionally, another embodiment of step 2) comprises:

[0045] 2.1) contacting the volatile substances produced by the bacterial strain with the pathogenic bacteria;

[0046] 2.2) inhibiting the growth of the pathogenic bacteria.

[0047] Optionally, step 2.1) said contacting the volatile substances produced by the bacterial strain with the pathogenic bacteria can be achieved by any method that can bring the volatile substances into contact with the pathogenic bacteria, including any one or more of the following methods that can bring the volatile substances into contact with the pathogenic bacteria: placing the culture in close proximity to the pathogenic bacteria; placing the culture in a relatively closed space with the pathogenic bacteria; fumigating, spraying, misting, aromatherapy volatilization, or any other method that can bring the collected volatile substances into contact with the pathogenic bacteria; placing the collected volatile substances in a relatively closed space with the pathogenic bacteria; and the like.

[0048] Preferably, the pathogenic bacteria in any of the preceding aspects is at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

[0049] Preferably, the inhibition of toxin synthesis of the pathogenic bacteria in any of the preceding aspects refers to the inhibition of patulin (PAT) or alternariol (AOH) toxin synthesis.

[0050] Compared with the prior art, the present application has the following advantages:

[0051] 1. The Pichia kluyveri yeast strain screened by the present application has broad-spectrum antibacterial effect and exhibits antagonism to a plurality of postharvest pathogenic bacteria.

[0052] 2. The volatile substances produced by the Pichia kluyveri yeast strain screened by the present application can significantly inhibit the growth of a plurality of pathogenic bacteria such as P. expansum and the synthesis of mycotoxins.

[0053] 3. The main volatile organic compounds produced by the Pichia kluyveri yeast strain screened by the present application can not only inhibit the growth of a plurality of pathogenic bacteria, but also control the biosynthesis of mycotoxins PAT and AOH from the source, showing good application prospect.

[0054] 4. The volatile organic compounds produced by the Pichia kluyveri yeast strain screened by the present application can be used for biological fumigation for disease prevention and control of postharvest fruits and vegetables, which is not only suitable for different sales stages of a plurality of products, but also suitable for fruits and vegetables such as strawberries, blueberries and grapes which are too fragile to be treated with liquid fungicides. In addition, the microbial source volatile organic compound fumigation does not directly contact the fruits and vegetables, is easy to volatilize at room temperature, is easy to degrade, and is not easy to remain on the surface of the fruits and vegetables, thus eliminating the concerns of consumers about its safety, so the volatile organic compounds have wide research value and application prospect in the prevention and control of postharvest diseases of fruits and vegetables. BRIEF DESCRIPTION OF DRAWINGS

[0055] The beneficial effects of the present application will be described in detail below in combination with the drawings and specific embodiments.

[0056] Figure 1 The colony morphology is shown in the following table.

[0057] Figure 2Molecular biological identification of the strains. A: 26S rDNA D1 / D2 segment of the yeasts amplified using universal primers NL-1 and NL-4. B: phylogenetic tree of the tested yeast strains and other Saccharomyces species based on the neighbor-joining analysis of the D1 / D2 26S rDNA sequences.

[0058] Figure 3 Biocontrol effect of P. kluyveri on various postharvest pathogens on different fruits. A: B. cinerea lesion morphology (72 h) and lesion diameter on strawberry fruits. B: A. alternata lesion morphology (8 d) and lesion diameter on winter jujube fruits. C: P. expansum lesion morphology (7 d) and lesion diameter on apple fruits. D: T. roseum lesion morphology (12 d) and lesion diameter on apple fruits. E: M. fructicola lesion morphology (4 d) and lesion diameter on yellow peach fruits. Data are means ± standard deviation, and different letters indicate significant differences (P < 0.05) among treatments.

[0059] Figure 4 Inhibition effect of volatile organic compounds produced by P. kluyveri on different postharvest pathogens. Growth status of pathogens on PDA medium after treatment with VOCs released at different time periods (3 d). B: pathogen colony diameter statistics. Data are means ± standard deviation, and different letters indicate significant differences (P < 0.05) among treatments.

[0060] Figure 5 Effect of volatile organic compounds produced by P. kluyveri on the production of secondary metabolites by P. expansum and A. alternata. A: colony morphology and pigment production of P. expansum and A. alternata after 2 d of culture in CY medium. B: PAT and AOH production detected by HPLC. Data are means ± standard deviation, and the asterisk represents significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test). DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the protection scope of the present application.

[0062] Unless otherwise defined, 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 belongs. The experimental methods described below, unless otherwise indicated, are conventional methods. The experimental materials used in the following examples, unless otherwise indicated, are commercially available from a conventional biochemical reagent store. The pathogenic fungi used in this experiment include Alternaria alternata, Trichothecium roseum, Monilinia fructicola and Penicillium expansum, which are isolated from naturally occurring fruit diseases, and are stored in the form of spore suspension (containing 16% glycerol) at -80°C in the refrigerator after single spore isolation and purification. The Botrytis cinerea strain is a haploid strain B05.10, which is provided by Professor Paul Tudzynski of the University of Wilhelms, Germany. The fruit materials used in this experiment are purchased from a local fruit supermarket. Apples, yellow peaches, strawberries, winter jujubes, blueberries and other fruits of uniform size, color and maturity without mechanical damage are selected for the experiment. Before the inoculation experiment, except for strawberry fruits, other fruits need to be surface sterilized in 2% (v / v) sodium hypochlorite solution for about 2 min, and then washed with tap water for 2-3 times to remove residual sodium hypochlorite. After natural air drying, they are ready for use.

[0063] Strain isolation and identification of Example 1:

[0064] Yeast isolation: mature nectarines were collected in Pinggu District, Beijing, and transported back to the laboratory for packaging into sterile fresh-keeping bags, adding sterile water, and placing the fruit in an ultrasonic cleaning instrument to wash the fruit. The washing liquid was collected and gradient diluted and plated. Different morphological yeast single colonies were picked and purified by multiple streaking. Finally, single colonies were picked on yeast extract peptone glucose agar medium, cultured at 28°C, 200 rpm for 20 h. The final concentration of glycerol in the bacterial solution was 16%, and the mixture was stored in a -80°C refrigerator.

[0065] ​Strain identification: The yeast strain stored at -80°C was used to inoculate a small amount of liquid on YPDA medium, and the plate was incubated at 28°C for 2-3 days. A single colony that grew after activation was picked up and added to a 10 mL centrifuge tube containing 3 mL YPD medium, and incubated at 28°C, 200 rpm for 2-3 days. The cells were collected by centrifugation at 8000 rpm for 10 min, and the yeast DNA was extracted using the Solarbio Yeast Genomic DNA Extraction Kit (Solarbio, D1900). The extracted yeast DNA was used as a template for sequence amplification with yeast 26S rDNA D1 / D2 segment universal primers NL-1 and NL-4. The amplification conditions were as follows: 98°C pre-denaturation for 5 min; 98°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C final extension for 5 min. The amplification system was as follows: genomic template DNA 1 μL, 2×Phanta Max Master Mix 25 μL (Vazyme, P525), primer NL-1 and primer NL-4 each 2 μL, ddH2O 20 μL. The PCR amplification product was detected by 1% agarose gel electrophoresis. The PCR product with a band was sent to Beijing Qikong Biotechnology Co., Ltd. for sequencing. The 26S rDNA gene sequence (FASTA format) obtained by sequencing was uploaded to the GenBank database of NCBI for BLAST. In the comparison results, a sequence homology of more than 99% can be classified into a class. The strain sequences with high homology were retrieved, and MEGA 6.0 was used for Clustal X multiple alignment. The bootstrap value was 1000, and the phylogenetic tree of the strains was constructed by the Neighbor-Joining method.

[0066] By Figure 1 As can be seen, the isolated yeast colony is white, rough and dry on the surface, and the edge is jagged. The genomic DNA of the above yeast was used as a template, and the universal primers NL-1 and NL-4 of the yeast 26S rDNA D1 / D2 segment were used to amplify the yeast DNA, and a fragment of about 500-600 bp (A) was obtained. Figure 2 A) The PCR amplification product was sequenced to obtain a sequence of 572 bp in length. After homology comparison on NCBI, it was found that the test strain had a homology of 99.83% with the Pichia sp. strain. Other strains with high homology with the test yeast strain were selected to construct a phylogenetic tree, and the results are as follows Figure 2As shown in B, it can be seen that the 26S rDNA D1 / D2 region sequence of the yeast strain is most homologous to the sequence of Pichia kluyveri. Therefore, the test yeast strain is finally determined to be Pichia kluyveri.

[0067] The NL-1 primer sequence is SEQ ID NO. 1: 5'-GCATATCAATAAGCGGAGGAAAAG-3';

[0068] The NL-4 primer sequence is SEQ ID NO. 2: 5'-GGTCCGTGTTTCAAGACGG-3';

[0069] The identification sequence of the strain Pichia kluyveri NY 23 of the present application is as follows: SEQ ID NO. 3:

[0070]

[0071] The sequenced results are compared for homology by BLAST in NCBI, and the comparison results are taken as the standard for similarity of 99% or more to carry out molecular identification of the strain, and the identification result is Pichia kluyveri (Pichia kluyveri).

[0072] The strain isolated in this example was preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 30, 2024, the preservation address is No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing Chaoyang District, the preservation number is CGMCC No 32124, and the preservation date is September 30, 2024.

[0073] Fruit disease prevention test of the strain of Example 2:

[0074] The process includes the following:

[0075] Preparation of yeast liquid with different concentrations: take the yeast stored at-80℃, streak for activation, pick single colonies and inoculate into 3 mL of YPD medium, culture at 28℃, 200 rpm for 24 h, transfer into new YPD medium at a ratio of 1:100, continue to culture for 24 h, centrifuge at 8000 rpm for 10 min to collect the bacterial body, and adjust the concentration of the yeast suspension to 1×10 7 cell / mL and 1×10 8 cell / mL for use.

[0076] Preparation of pathogen spore suspension: Take 1.5 mL centrifuge tube, add 1 mL sterile water or potato dextrose broth (PDB), cut the pathogenic bacteria block cultured for 7-10 days into the above-mentioned sterile water or PDB, place the centrifuge tube on the shaker for full shaking, use the gun head to suck the spore suspension in the centrifuge tube, and filter it using the magic filter cloth (Merck Millipore, 475855-1R). Suck 20 μL filtrate, measure the spore concentration using a cell counter, and then dilute it to the working concentration with sterile water or PDB medium, and wait for use.

[0077] Fruit inoculation test: Select fruits such as apples, yellow peaches, winter jujubes, and strawberries that are uniform in size, consistent in maturity, and free of mechanical damage, soak the fruits in a 2% (v / v) sodium hypochlorite solution for 2 min for surface disinfection, then rinse with clean water and dry for standby. Use a sterile inoculation needle to stab (0.2 cm (diameter) x 0.5 cm (depth)) the equatorial part of the fruit, dry it, and then add 10 μL of sterile water to the holes of the control group, and add 10 μL of different concentrations of yeast suspension to the holes of the treatment group. Dry naturally for 2 h, then add 10 μL of pathogenic spore suspension with a concentration of 1 x 10 5 spores / mL to the holes of the control group and the treatment group. Place the inoculated fruits in plastic baskets disinfected with sodium chlorate, add wet paper to keep them moist, seal them with a preservative bag, and place them at 22°C. At different times, use the cross method to count the lesion diameters and take photos.

[0078] As shown in Figure 3 , the screened P. kluyveri yeast strains can significantly reduce the pathogenicity of P. expansum, T. roseum, and M. fructicola on apples and yellow peaches, and the inhibitory effect increases with the increase of the yeast concentration. The statistical results show that after treating winter jujube fruits with P. kluyveri yeast and then inoculating A. alternata, the fruits are basically not diseased. On the 8th day after inoculation, the lesion diameter of the control group fruits reached 10.67 mm, and the lesion diameter of the treatment group fruits was 0 mm. On the 7th day after inoculation, the lesion diameter on the apples inoculated with P. expansum reached 38.58 mm, and the lesion diameters on the apples treated with 1 x 10 7 cell / mL and 1 x 10 8 cell / mL yeast were 31.50 mm and 24.67 mm, respectively, which were about 4 / 5 and 3 / 5 of the control group. The fruits inoculated with M. fructicola became diseased faster, and on the 4th day after inoculation, the lesion diameter on the yellow peaches of the control group reached 57.75 mm, and the lesion diameters on the yellow peaches treated with 1 x 10 7 cell / mL and 1 x 10 8The diameters of lesions on apple fruits treated with cell / mL yeast were 52.72 mm and 40.71 mm, respectively. Strawberry fruits treated with yeast showed almost no disease initially after inoculation with *B. cinerea*, but disease began to appear on the 3rd day after treatment, possibly related to prolonged storage and decreased fruit resistance and quality.

[0079] Example 3: Effects of volatile organic compounds on the growth of pathogenic bacterial colonies

[0080] Activate the yeast and adjust the concentration to 1×10 8 Cells / mL: 100 μL of yeast suspension was spread onto YPDA plates and incubated upside down at 28°C for 12 h and 24 h. Spores were collected according to the method in Example 2, and spore suspensions of different pathogens were prepared. After determining the spore concentration using a cell counter, the concentration was adjusted to 1 × 10⁻⁶ cells / mL with sterile water. 6 spores / mL. 5 μL of spore suspension was added dropwise to the center of a PDA medium. After air-drying, yeast culture plates incubated for 12 h and 24 h were sealed with plates inoculated with pathogen spores using a two-plate sealing method. YPDA medium without yeast suspension was sealed with PDA plates inoculated with the same pathogen spore suspension as a control. The sealed plates were incubated in a fungal incubator at 25°C for 3 days. Colony diameter was measured every day using the cross-hatching method. Data were statistically analyzed using SPSS software. Each treatment was repeated in triplicate, and the entire experiment was repeated three times.

[0081] During the cultivation of the selected *P. kluyveri* yeast strain, we observed that it produced a fruity aroma, which was strong and pleasant. To investigate whether the broad-spectrum antibacterial effect of this yeast was related to the volatile organic compounds (VOCs) it produced, we used a plate-to-plate assay to detect the effects of VOCs produced by the yeast at different time points on the growth of various postharvest pathogens. Figure 4It was found that the volatile organic compounds produced by P. kluyveri at different time periods had significant inhibitory effects on the growth of T. roseum, B. cinerea, P. expansum, M. fructicola and A. alternata on PDA plates. Moreover, we found that the volatile organic compounds produced by the yeast cultured for 12 h had better inhibitory effects on the pathogenic fungi than the volatile organic compounds produced by the yeast cultured for 24 h. For T. roseum, the colony diameter of T. roseum without treatment of volatile organic compounds reached 36.83 mm after 3 d of inoculation, while the volatile organic compounds produced by the yeast cultured for 12 h completely inhibited the growth of T. roseum on PDA plates after 3 d of inoculation, and the colony diameter of T. roseum treated with the volatile organic compounds produced by the yeast cultured for 24 h reached 33.17 mm after 3 d of inoculation. The above results may be related to the differences in the types and abundance of volatile organic compounds produced by the screened P. kluyveri yeast strains at different time periods. The inhibitory rates of the volatile organic compounds produced by the yeast cultured for 12 h on B. cinerea, P. expansum, M. fructicola and A. alternata were 71.2%, 26.4%, 71.9% and 69.3%, respectively, after 3 d of inoculation. The above results showed that the volatile organic compounds produced by the screened P. kluyveri yeast strains during the culture process could significantly inhibit the mycelial growth of multiple postharvest pathogenic fungi.

[0082] Example 4 Effect of volatile organic compounds on the toxin production of pathogenic fungi

[0083] According to the method of Example 2, spores of P. expansum and A. alternata were collected to prepare a spore suspension. After the spore concentration was determined by a cell counter, sterile water was used to adjust the spore concentration to 1 x 10 6 spores / mL. Glass paper with a size of 1 x 1 cm was prepared, sterilized and then laid on PDA medium. 1 μL of the spore suspension was evenly coated on the glass paper. The test yeast was cultured in advance, and the PDA plate coated with the pathogenic fungi was sealed with the yeast culture plate cultured for 12 h, and then placed in a constant temperature incubator at 25 °C for 1.5 d. The mycelium grown was transferred to a 24-well plate containing 1 mL of Czapek yeast extract medium (CY) together with the glass paper. 1 mL of melted solid LB medium was added to the cover of the 24-well plate, and after solidification, 20 μL of the spore suspension with a concentration of 1 x 10 8The test yeast culture solution was diluted to 1.0 x 106cells / mL, and evenly spread on the surface of the LB medium. After drying, the 24-well plate was covered with a lid and sealed with sealing film. The plate was placed in a constant temperature incubator at 25°C for 2 days, and then the mycelium was collected and stored in liquid nitrogen. The culture solution was filtered through a 0.22 μm water filter membrane, and then subjected to HPLC detection.

[0084] The detection conditions for PAT were as follows: mobile phase A: water, mobile phase B: acetonitrile (ACN); detection wavelength: 276 nm, sample injection temperature: 25°C; sample injection volume: 10 μL; flow rate: 1 mL / min; running time: 12 min; isocratic elution, and the elution method is shown in Table 1.

[0085] Table 1: PAT isocratic elution conditions

[0086]

[0087] The detection conditions for AOH were as follows: mobile phase A: water (containing 1 mM oxalic acid), mobile phase B: acetonitrile (containing 1 mM oxalic acid); detection wavelength: 256 nm, sample injection temperature: 35°C; sample injection volume: 10 μL; flow rate: 1 mL / min; running time: 30 min; gradient elution, and the elution method is shown in Table 2.

[0088] Table 2: AOH gradient elution conditions

[0089]

[0090] PAT produced by P. expansum and AOH produced by A. alternata are two kinds of fungal toxins that seriously contaminate fruits and vegetables, and long-term exposure has the risk of causing teratogenicity, carcinogenicity and mutagenicity. For example, PAT can cause liver and kidney damage, and AOH can cause liver and kidney damage, and even death. Figure 5 As shown in A and C, after VOCs treatment, the mycelial growth was inhibited, and the amount of mycelium was significantly reduced. After removing the mycelium, the CY medium inoculated with P. expansum in the VOCs treatment group and the untreated group showed obvious color difference. The medium in the untreated group showed orange yellow, while the medium in the treated group showed light yellow. The CY medium inoculated with A. alternata showed no obvious color change. The above results show that the VOCs treatment may affect the pigment synthesis of P. expansum. Figure 5 As shown in B and D, after VOCs treatment, the PAT content produced by P. expansum was significantly reduced, only 0.9% of the untreated group, and the AOH content produced by A. alternata was only 39% of the control group. The above results show that VOCs treatment can affect the biosynthesis of fungal toxins by affecting the growth of pathogenic fungal mycelium, and VOCs treatment is an effective method for source control of fungal toxins.

[0091] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been described above with reference to preferred embodiments, the present application is not intended to be limited to the above-described embodiments, and any person skilled in the art, without departing from the technical scope of the present application, can make some changes or modifications to the above-described embodiments, or make equivalent embodiments with equivalent changes, as long as the changes or modifications do not depart from the technical scope of the present application. Any modification, change, or equivalent change made to the above-described embodiments, based on the technical essence of the present application, still falls within the scope of the present application.

Claims

1. Use of a fungal strain for inhibiting the growth of a plant pathogenic fungus, characterized in that, The taxonomic name of the strain is Pichia kluyveri (Pichia kluyveri) Pichia kluyveri ) NY23, the strain is preserved in the China General Microbiological Culture Collection Center, the address is No. 1, Yitian West Road, Chaoyang District, Beijing, the Institute of Microbiology of Chinese Academy of Sciences, the preservation number is CGMCC No. 32124, and the preservation date is September 30, 2024, and the plant pathogen is at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea .

2. Use of a fungal strain for inhibiting the synthesis of a pathogenic fungus toxin, characterized in that, The strain was classified as Kluyveromyces oryzae (Kluyveromyces oryzae). Pichia kluyveri NY23, the strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32124 and deposit date of September 30, 2024. The pathogenic toxin is patulin (PAT) and / or albinophenol (AOH).

3. A method of inhibiting the growth of a plant pathogenic fungus, characterized by, comprising the steps of: 1) The cultured strain was classified and named Kluyveromyces kluyveromyces (Kluyveromyces kluyveromyces). Pichia kluyveri NY23, the strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32124 and deposit date of September 30, 2024; 2) using the culture containing the bacterial strain produced in step 1) to inhibit the growth of a plant pathogen, said plant pathogen being at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea .

4. A method of inhibiting the synthesis of pathogenic fungal toxins comprising the steps of: 1) The cultured strain was classified and named Kluyveromyces kluyveromyces (Kluyveromyces kluyveromyces). Pichia kluyveri NY23, the strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32124 and deposit date of September 30, 2024; 2) using the culture containing the strain produced in step 1) to inhibit the synthesis of pathogenic fungal toxins, said pathogenic fungal toxins being patulin (PAT) and / or alternariol (AOH).

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