Biocontrol bacterium strain and application thereof in controlling postharvest diseases of fruits and vegetables

Abstract

The invention relates to a fungus strain with a broad-spectrum antibacterial effect and application of the fungus strain in controlling postharvest diseases of fruits and vegetables. The classification name of the fungus is Pichia kluyveri, the Latin literature name is Pichia kluyveri, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing, the preservation number is CGMCC No.32124, and the preservation date is September 30, 2024. The strain has an inhibition effect on a plurality of pathogenic bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a fungal strain with a broad-spectrum antibacterial effect and an application thereof in controlling post-harvest diseases of fruits and vegetables. Background Art

[0002] Fruits and vegetables are rich in nutrients and are an important part of the human diet. However, fruits and vegetables are seriously damaged after harvest, causing huge economic losses every year. Rot caused by pathogens is the main reason for the decline in quality and loss of fruits and vegetables after harvest. At the same time, some post-harvest pathogens can also produce fungal toxins that are harmful to human health, posing a major safety hazard.

[0003] At present, the prevention and control of postharvest diseases of fruits and vegetables mainly include physical control, chemical control and biological control. Due to the limitation of cost and scope of use, the application of physical control methods in agricultural production is subject to certain restrictions. The use of chemical fungicides is currently the most important method for the prevention and control of postharvest diseases, but excessive use of chemical agents will cause environmental pollution, threaten food security and human health, and lead to drug resistance in pathogens. Biological control is the use of antagonistic microorganisms to inhibit the growth and reproduction of pathogens through interactions between species, thereby controlling postharvest diseases of fruits and vegetables and inhibiting the production of fungal toxins. Due to its safety and environmental friendliness, it has gradually become a hot spot in the research of controlling postharvest diseases of fruits and vegetables, and is expected to replace chemical fungicides in the future. So far, hundreds of antagonistic microorganisms have been isolated and identified at home and abroad, including bacteria, molds and yeasts. Among them, antagonistic yeasts have become a commonly used 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 yeast has great potential for improving the floral and fruity aroma characteristics of wine. As one of the most popular species, Pichia kluyveri has attracted much attention in the fermentation process of various beverages. However, there is no research on the biological control effect of P. kluyveri yeast on various pathogenic fungi in postharvest fruits and vegetables. Summary of the invention

[0005] The purpose of the present invention is to provide a fungal strain with a broad-spectrum antibacterial effect, and to apply the fungal strain in the biological control of pathogens of post-harvest fruits and vegetables.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides a fungal strain with a broad-spectrum antibacterial effect, characterized in that the strain is classified and named Pichia kluyveri, and its Latin name is Pichia kluyveri. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No 32124, and the deposit date is September 30, 2024.

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

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

[0010] Preferably, the inhibiting the growth of pathogens refers to inhibiting the growth of plant pathogens.

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

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

[0013] Preferably, the plant is strawberry, apple, yellow peach, blueberry or winter jujube.

[0014] Preferably, the plant pathogen is a pathogen that can parasitize on plant fruits.

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

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

[0017] On the other hand, the present invention provides the use of the aforementioned strain in inhibiting the synthesis of pathogenic bacteria toxins.

[0018] Preferably, the inhibition of pathogen toxin synthesis refers to the inhibition of toxin synthesis of plant pathogens.

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

[0020] Preferably, the plant is a plant of the genus Fragaria, Malus, Peach, Vaccinium or Ziziphus.

[0021] Preferably, the plant is strawberry, apple, yellow peach, blueberry or winter jujube.

[0022] Preferably, the plant pathogen is a pathogen that can parasitize on plant fruits.

[0023] Preferably, the pathogenic bacteria is at least one of Alternaria sp., Trichothecium sp., Monilini sp., Penicillium sp. and Botrytis sp.

[0024] Preferably, the pathogenic bacteria is at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

[0025] Preferably, the inhibition of pathogen toxin synthesis refers to the inhibition of toxin synthesis of patulin (PAT) or alternating spore ol (AOH).

[0026] In another aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria, comprising the steps of:

[0027] 1) Cultivating the aforementioned strain;

[0028] 2) Using the culture product of step 1) to inhibit the growth of pathogenic bacteria.

[0029] Further, step 2) includes:

[0030] 2.1) contacting the culture containing the strain with the pathogen;

[0031] 2.2) Inhibit the growth of pathogens.

[0032] Optionally, the step 2.1) of contacting the culture containing the strain with the pathogens can be carried out by any method that can achieve the effect of contacting the culture with the pathogens, including soaking, spraying, flushing, and any one or more methods that can contact the culture containing the strain with the pathogens.

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

[0034] 2.1) contacting the volatile substances produced by the strain with pathogenic bacteria;

[0035] 2.2) Inhibit the growth of pathogens.

[0036] Optionally, in step 2.1), any method that can achieve the effect of contacting the volatile substances produced by the strain with the pathogens can be used, including bringing the culture and the pathogens into close proximity, placing the culture and the pathogens in a relatively closed space, spraying or atomizing the collected volatile substances on the pathogens, placing the collected volatile substances and the pathogens in a relatively closed space, and any one or more methods that can contact the volatile substances with the pathogens.

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

[0038] 1) Cultivating the aforementioned strain;

[0039] 2) Using the culture product of step 1) to inhibit the synthesis of pathogenic bacteria toxins.

[0040] Further, step 2) includes:

[0041] 2.1) contacting the culture containing the strain with the pathogen;

[0042] 2.2) Inhibit the synthesis of pathogenic toxins.

[0043] Optionally, the step 2.1) of contacting the culture containing the strain with the pathogens can be carried out by any method that can achieve the effect of contacting the culture with the pathogens, including soaking, spraying, flushing, and any one or more methods that can contact the culture containing the strain with the pathogens.

[0044] Optionally, another implementation of step 2) includes:

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

[0046] 2.2) Inhibit the growth of pathogens.

[0047] Optionally, in step 2.1), any method that can achieve the contact effect between the volatile substances produced by the strain and the pathogens can be used to contact the volatile substances produced by the strain and the pathogens, including bringing the culture and the pathogens into close proximity, placing the culture and the pathogens in a relatively closed space, fumigating, spraying, atomizing, or contacting the pathogens with the collected volatile substances by aromatherapy, placing the collected volatile substances and the pathogens in a relatively closed space, and any one or more methods that can contact the volatile substances with the pathogens.

[0048] Preferably, in any of the above schemes, the pathogenic bacteria is at least one of Alternaria alternata, Trichotheciumroseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

[0049] Preferably, in any of the above schemes, inhibiting the synthesis of pathogenic bacteria toxins refers to inhibiting the synthesis of toxins of patulin (PAT) or alpinia spore (AOH).

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

[0051] 1. The Pichia kluyveri yeast strain screened by the present invention has a broad-spectrum antibacterial effect and exhibits antagonistic effects on a variety of postharvest pathogens.

[0052] 2. The volatile substances produced by the Pichia kluyveri yeast strain screened by the present invention can significantly inhibit the growth of various pathogens such as P. expansum and the synthesis of fungal toxins.

[0053] 3. The main volatile organic compounds produced by the Pichia kluyveri yeast strain screened in the present invention can not only inhibit the growth of various pathogens, but also control the biosynthesis of fungal toxins PAT and AOH from the source, showing good application prospects.

[0054] 4. The volatile organic compounds produced by the Pichia kluyveri yeast strain screened by the present invention are used for biological fumigation of post-harvest fruits and vegetables to prevent and control diseases. This is not only applicable to different sales stages of various products, but also to fruits and vegetables such as strawberries, blueberries and grapes that are too fragile to be treated with liquid fungicides. In addition, the fumigation of microbial volatile organic compounds will not come into direct contact with fruits and vegetables, is easy to volatilize and degrade at room temperature, and is not easy to remain on the surface of fruits and vegetables, eliminating consumers' concerns about their safety. Therefore, volatile organic compounds have broad research value and application prospects in preventing and controlling post-harvest diseases of fruits and vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0056] Figure 1 It is a colony morphology.

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

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

[0059] Figure 4 A: The inhibitory effect of volatile organic compounds produced by P. kluyveri on different post-harvest pathogens. B: The growth status of pathogens on PDA medium after treatment with VOCs released at different time periods (3d). C: Statistics of pathogen colony diameter. Data are expressed as mean ± standard deviation, and different letters indicate significant differences among treatments (P<0.05).

[0060] Figure 5 Effects 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 days of culture in CY medium. B: HPLC detection of PAT and AOH production. Data are expressed as mean ± SD, and asterisks represent significant differences (*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 invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The pathogenic fungi used in this experiment, including Alternaria alternata, Trichothecium roseum, Monilinia fructicola and Penicillium expansum, were isolated from naturally diseased fruits and stored in a -80°C refrigerator in the form of a spore suspension (containing 16% glycerol) after single spore separation and purification. The Botrytis cinerea strain is a haploid strain B05.10, which was obtained from Germany Provided by Professor Paul Tudzynski of Wilhelms University. The fruit materials used in this experiment were purchased from a local fruit supermarket. Apples, yellow peaches, strawberries, winter dates, blueberries and other fruits with uniform size and color, uniform maturity and no mechanical damage were selected for the experiment. Before the inoculation test, except for strawberry fruits, other fruits need to be soaked in 2% (v / v) sodium hypochlorite solution for about 2 minutes for surface disinfection, and then rinse the fruits with tap water 2-3 times to remove residual sodium hypochlorite. Dry naturally and set aside.

[0063] Example 1 Strain Isolation and Identification:

[0064] Yeast isolation: harvest ripe nectarines from Pinggu District, Beijing, transport them back to the laboratory, pack them into sterile fresh-keeping bags, add sterile water, and place them in an ultrasonic cleaner to clean the fruit. Collect the washing liquid and perform gradient dilution and plate coating. Pick single colonies of yeast with different morphologies for multiple streaking purification. Finally, pick a single colony and culture it in yeast peptone glucose agar medium at 28°C and 200rpm for 20h. Add glycerol to a final concentration of 16% to the bacterial solution, mix well and store in a -80°C refrigerator.

[0065] Strain identification: Take a yeast strain stored at -80℃, use an inoculation needle to dip a small amount of bacterial liquid and streak it on the YPDA medium for activation, and place the plate in a 28℃ incubator for 2-3 days. Pick a single colony grown after activation and add it to a 10mL centrifuge tube containing 3mL YPD medium, culture it in a shaker at 28℃ and 200rpm for 2-3 days, collect the bacteria by centrifugation at 8000rpm for 10min, and use the Solarbio Yeast Genomic DNA Extraction Kit (Solarbio, D1900) to extract yeast DNA. Using the extracted yeast DNA as a template, sequence amplification was performed using the yeast 26S rDNA D1 / D2 segment universal primers NL-1 and NL-4. The amplification conditions were all 98℃ pre-denaturation for 5min; 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 30s, 35 cycles; 72℃ final extension for 5min. The amplification system is: 1μL genomic template DNA, 25μL 2×Phanta Max Master Mix (Vazyme, P525), 2μL each of primer NL-1 and primer NL-4, and 20μL ddH2O. Take the PCR amplification product and perform electrophoresis detection on 1% agarose gel. The PCR product with detected bands was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequenced 26S rDNA gene sequence (FASTA format) was uploaded to the GenBank database of NCBI for BLAST. In the comparison results, the sequence homology is generally greater than 99%, which can be classified into one category. The strain sequence with higher homology is retrieved, and Clustal X multiple alignment is performed using MEGA 6.0. The bootstrap value (Bootstrap) is 1000, and the phylogenetic evolutionary tree of the strain is constructed using the Neighbor-Joining method.

[0066] Depend on Figure 1 It can be seen that the isolated yeast colonies are white, with a rough and dry surface and jagged edges. Using the genomic DNA of the yeast as a template, the yeast DNA was amplified using the universal primers NL-1 and NL-4 of the yeast 26S rDNA D1 / D2 segment to obtain a fragment of about 500-600 bp ( Figure 2 A). The PCR amplification product was sequenced to obtain a 572 bp sequence. After homology comparison on NCBI, it was found that the homology between the test strain and the strain of Pichia sp. was 99.83%. Other strains with high homology to the test yeast strain were selected to construct a phylogenetic tree. 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 has the highest homology with that of Pichia kluyveri. Therefore, the yeast strain tested was 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 invention is as follows SEQ ID NO.3:

[0070]

[0071] The sequencing results were compared for homology using BLAST in NCBI, and the molecular identification of the bacterial species was performed with a similarity of 99% or more as the standard. The identification result was Pichia kluyveri.

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

[0073] Fruit disease prevention test of embodiment 2 strain:

[0074] The process includes:

[0075] Preparation of different concentrations of yeast solution: Take yeast stored at -80℃, streak and activate, pick a single colony and inoculate it into 3mL of YPD medium, culture it at 28℃, 200rpm for 24h, transfer it to new YPD medium at a ratio of 1:100, continue to culture for 24h, centrifuge at 8000rpm for 10min to collect the cells, count them with a cell counter and adjust the concentration of the yeast suspension to 1×10 7 cell / mL and 1×10 8 cell / mL for later use.

[0076] Preparation of pathogen spore suspension: Take a 1.5mL centrifuge tube, add 1mL sterile water or potato dextrose broth (PDB), cut the pathogenic bacteria blocks that have been cultured for 7-10 days into the above sterile water or PDB, place the centrifuge tube on an oscillator and shake it thoroughly, use a gun to suck the spore suspension in the centrifuge tube, and filter it with a magic filter cloth (Merck Millipore, 475855-1R). Take 20μL of the filtrate, measure the spore concentration with a cell counter, and then dilute it to the working concentration with sterile water or PDB medium for standby use.

[0077] Fruit inoculation test: Select apples, yellow peaches, winter dates, strawberries and other fruits of uniform size, uniform maturity and no mechanical damage, and soak them in a sodium hypochlorite solution with a final concentration of 2% (v / v) for 2 minutes to disinfect the surface of the fruit, then rinse with clean water and dry for later use. Use a sterile inoculation needle to pierce the equator of the fruit (0.2cm (diameter) × 0.5cm (depth)), and after drying, add 10μL of sterile water to the wells of the control group, and add 10μL of different concentrations of yeast suspension to the wells of the treatment group. Naturally dry for 2h, and then add 10μL of 1×10 5 spores / mL of pathogen spore suspension. The inoculated fruits were placed in a plastic basket disinfected with sodium chlorate, wet paper balls were added to keep moisture, and sealed with a fresh-keeping bag. The fruit was placed at 22°C, and the diameter of the lesions was counted and photographed at different times using the cross method.

[0078] like Figure 3 As shown in the figure, the screened P.kluyveri yeast strain can significantly reduce the pathogenicity of P.expansum, T.roseum and M.fructicola on apples and yellow peaches, and its inhibitory effect increases with the increase of the yeast concentration used. The statistical results show that after the winter jujube fruit is treated with P.kluyveri yeast and then inoculated with A.alternata, the fruit is basically not diseased. On the 8th day after inoculation, the diameter of the lesion on the fruit of the control group reached 10.67mm, and the diameter of the lesion on the fruit of the treatment group was 0mm. On the 7th day after inoculation, the diameter of the lesion on the apple of the control group inoculated with P.expansum reached 38.58mm, and 1×10 7 cell / mL and 1×10 8 The diameters of the lesions on the apple fruits treated with cell / mL yeast were 31.50 mm and 24.67 mm, respectively, which were about 4 / 5 and 3 / 5 of those in the control group. The fruits inoculated with M. fructicola developed disease more quickly. On the 4th day after inoculation, the diameter of the lesions on the yellow peaches in the control group reached 57.75 mm, and the diameter of the lesions on the peaches in the 1×10 7 cell / mL and 1×10 8The diameters of the lesions on apple fruits treated with yeast were 52.72 mm and 40.71 mm respectively. After strawberry fruits treated with yeast were inoculated with B. cinerea, the fruits were almost free of disease in the early stage, but they began to become diseased on the third day after treatment, which may be related to the prolonged storage time and the decline in fruit resistance and quality.

[0079] Example 3 Effect of Volatile Organic Compounds on the Growth of Pathogenic Bacteria Colonies

[0080] Activate yeast and adjust the concentration to 1×10 8 cell / mL, 100 μL of yeast suspension was applied to a YPDA plate and incubated in an incubator at 28°C for 12 h and 24 h. Spores were collected according to the method of Example 2, and spore suspensions of different pathogens were prepared. The spore concentration was measured with a cell counter and adjusted to 1×10 with sterile water. 6 spores / mL. 5 μL of spore suspension was added to the center of the PDA medium. After drying naturally, the yeast medium plates incubated for 12 h and 24 h were sealed with the plates inoculated with pathogen spores by the method of two plates buckled. The YPDA medium without yeast suspension was buckled with the PDA plate inoculated with the same pathogen spore suspension as a control. The sealed plates were placed in a fungal incubator at 25 ° C for 3 days. The colony diameter was measured every 1 day by the cross method, and the data were statistically analyzed using SPSS software. Three replicates were set for each treatment, and the entire experiment was repeated 3 times.

[0081] During the cultivation process of the screened P. kluyveri yeast strain, we found that the yeast produced a fruity aroma that was strong and pleasant. In order to explore whether the broad-spectrum antibacterial effect of the yeast was related to the volatile organic compounds (VOCs) it produced, we used a plate-pairing test to detect the effects of volatile organic compounds produced by yeast at different time periods on the growth of various postharvest pathogens. Figure 4It can be seen that the volatile organic compounds produced by P.kluyveri yeast at different time periods have a significant inhibitory effect on the growth of T.roseum, B.cinerea, P.expansum, M.fructicola and A.alternata on PDA plates. In addition, we also found that the volatile organic compounds produced by 12h of culture have a better inhibitory effect on pathogens than those produced by 24h of culture. For T.roseum, the colony diameter of T.roseum that was not treated with volatile organic compounds reached 36.83mm after 3d of inoculation. When treated with volatile organic compounds produced by yeast culture for 12h, T.roseum could not grow on the PDA plate 3d after inoculation. However, when treated with volatile organic compounds produced by yeast culture for 24h, the colony diameter of T.roseum on the PDA plate reached 33.17mm after 3d of inoculation. This result 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 inhibition rates of volatile organic compounds produced after 12 hours of cultivation on B. cinerea, P. expansum, M. fructicola and A. alternata reached 71.2%, 26.4%, 71.9% and 69.3% respectively 3 days after inoculation. The above results show that the volatile organic compounds produced by the screened P. kluyveri yeast strain during the cultivation process can significantly inhibit the expansion of hyphae of various postharvest pathogens.

[0082] Example 4 Effect of Volatile Organic Compounds on Toxin Production by Pathogens

[0083] P. expansum and A. alternata spores were collected according to the method of Example 2, and spore suspension was prepared. The spore concentration was measured by cell counter and adjusted to 1×10 6 spores / mL. Prepare 1×1cm cellophane, sterilize it and spread it on the PDA medium. Pipette 1μL of spore suspension and spread it evenly on the above cellophane. Culture the test yeast in advance, clasp the yeast culture medium plate that has been cultured for 12 hours and the PDA plate coated with pathogens to seal, and culture it in a 25℃ constant temperature incubator for 1.5 days. Then transfer the grown hyphae together with the cellophane to a 24-well plate that has been pre-added with 1mL Czapek Yeast Extract medium (Czapek Yeast Exatract, CY). Add 1mL of melted solid LB medium on the cover of the 24-well plate. After solidification, use a pipette to pipette 20μL of 1×10 8cell / mL of the test yeast culture solution to evenly cover the entire LB medium surface. After drying, cover the 24-well plate with a lid and seal it with a sealing film. After culturing in a 25°C constant temperature incubator for 2 days, collect the mycelium and freeze it with liquid nitrogen for later use. Collect the culture solution and filter it with a 0.22μm water filter membrane before HPLC detection.

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

[0085] Table 1 PAT isocratic elution conditions

[0086]

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

[0088] Table 2 AOH gradient elution conditions

[0089]

[0090] Patulin (PAT) produced by P. expansum and alkalinomycin (AOH) produced by A. alternata are two serious mycotoxins that contaminate fruits and vegetables. Long-term exposure has the risk of teratogenicity, carcinogenicity, and mutagenicity. Figure 5 As shown in A and C, after VOCs treatment, hyphae growth was inhibited and the amount of hyphae was significantly reduced. After removing the hyphae, the CY culture medium inoculated with P. expansum in the VOCs-treated and untreated groups showed obvious color differences. The culture medium of the untreated group was orange-yellow, while the culture medium of the treated group was light yellow. The color of the CY culture medium inoculated with A. alternata did not change significantly. The above results indicate that VOCs treatment may affect the pigment synthesis of P. expansum. The culture medium was collected and the synthesis of PAT and AOH was detected by HPLC. Figure 5 As shown in B and D, the PAT content produced by P. expansum was significantly reduced after VOCs treatment, 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 fungi hyphae, and VOCs treatment is an effective method to achieve source control of fungal toxins.

[0091] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A fungal strain with broad-spectrum antibacterial effect, characterized in that: The strain is classified and named Pichia kluyveri, and its Latin name is Pichia kluyveri. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No 32124, and the deposit date is September 30, 2024.

2. A 26S rDNA sequence for identifying the strain according to claim 1, characterized in that: The sequence is shown as SEQ ID NO.

3.

3. Use of the strain according to claim 1 in inhibiting the growth of pathogenic bacteria.

4. The use according to claim 3, characterized in that The inhibiting the growth of pathogens refers to inhibiting the growth of plant pathogens.

5. The use according to claim 3, characterized in that: The pathogenic bacteria is at least one of Alternariaalternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

6. Use of the strain according to claim 1 in inhibiting the synthesis of pathogenic bacteria toxins.

7. The use according to claim 6, characterized in that The inhibition of pathogen toxin synthesis refers to the inhibition of toxin synthesis of plant pathogens.

8. The use according to claim 6, characterized in that The pathogenic bacteria include at least one of Alternaria sp., Trichothecium sp., Monilinia sp., Penicillium sp. and Botrytis sp.

9. A method for inhibiting the growth of pathogenic bacteria, comprising the following steps: 1) culturing the strain according to claim 1; 2) using the culture product of step 1) to inhibit the growth of pathogenic bacteria.

10. A method for inhibiting the synthesis of pathogenic bacteria toxins, comprising the following steps: 1) Cultivating the strain according to claim 1; 2) Using the culture product of step 1) to inhibit the synthesis of pathogenic bacteria toxins.

Images