Actinomycetes capable of producing antibacterial volatile substances and application of actinomycetes in postharvest diseases of grapes

Abstract

The invention discloses actinomycetes for producing bacteriostatic volatile substances and application of the actinomycetes for producing the bacteriostatic volatile substances in postharvest diseases of grapes, and relates to the technical field of microorganism application, the culture name and the indicated identification characteristic of the actinomycetes for producing the bacteriostatic volatile substances are Streptomyces microfavusA89, the actinomycetes are preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M2024842, and the preservation date is May 8, 2024; according to the actinomycete capable of producing the bacteriostatic volatile substance and the application of the actinomycete in the postharvest diseases of the grapes, under the in-vivo condition, the A89 volatile substance and 500 [mu] L / L 2, 5-dimethylpyrazine are best shown in a treatment group after fumigating for 10 days, the scab diameters of the fruits are respectively reduced by 74.11% and 76.70%, and it is worthy of notice that a 125 [mu] L / L acetophenone treatment group can completely inhibit the occurrence of the gray mold of the grapes.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to actinomycetes producing antibacterial volatile substances and application thereof in treating post-harvest diseases of grapes. Background Art

[0002] Grape gray mold is commonly known as "flower rot" and is also called grape gray rot. It is a disease caused by Botrytis cinerea and occurs on ordinary grapes. Grape gray mold is the main disease of grapes. It causes flower rot in spring. When the disease is prevalent, the incidence of flower spikes can reach more than 70%. Ripe fruits are also often rotted during storage, transportation and sales due to this disease. If the diseased fruits of gray mold are accidentally mixed into grape wine, the color of the wine may change and the quality of the wine may deteriorate. Grape gray mold mainly harms inflorescences, young fruits and mature fruits, and sometimes also harms new shoots, leaves and fruit stalks. The flower spikes are usually infected before flowering. In the early stage of infection, the inflorescences are like being scalded by hot water, dark brown, the diseased tissue is soft and rotten, and the surface is densely covered with gray mold. The infected inflorescences wilt and the young fruits fall off easily. After the fruit stalks are infected, they are dark brown, and sometimes black blocky sclerotia appear on the lesions. After the grapes bloom, the fruit spikes are light brown and water-soaked at first, and soon turn dark brown. The whole fruit spike is soft and rotten. If it rains during this period, a layer of light gray mold will grow on the fruit spikes in 2-3 days. When the fruit is infected near maturity, light brown sunken lesions will first appear, which will quickly spread to the whole fruit and cause the fruit to rot. When the disease is serious, the whole or part of the fruit spikes will rot, and a mouse-gray mold layer will grow on it. After the disease, the fruit stalks will turn black, and black blocky sclerotia will grow on the diseased area in the later stage. Finally, after the expansion, the whole spike will be covered with mold and all the fruits will rot. New shoots and leaves can also be susceptible to the disease, producing irregular brown spots, which sometimes have irregular rings. When wet, they have irregular gray mold layers. Generally, each leaf has 2-5 spots. In severe cases, the diseased part can also grow a mouse-gray mold layer and finally dry up. This pathogen can infect grapes, eggplants, tomatoes, cabbages, apples, kidney beans, peppers, cowpeas, cotton bolls, green onions and onions. At present, post-harvest diseases of grapes are mainly controlled by chemical agents, which can achieve relatively good results, such as antibacterial spirit, thiophanate-methyl and thiram. However, chemical agents have the disadvantages of affecting human health, polluting the environment and causing pathogen resistance. Non-chemical control measures are increasingly becoming the focus of preventing and controlling post-harvest gray mold of grapes. Among them, biological control with the characteristics of safety and pollution-free is a very promising control method. Therefore, actinomycetes producing antibacterial volatile substances and their application in post-harvest diseases of grapes are proposed. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides actinomycetes producing antibacterial volatile substances and applications thereof in treating post-harvest diseases of grapes, thereby solving the problems raised in the above-mentioned background technology.

[0004] To achieve the above objectives, the present invention is implemented through the following technical scheme: an actinomycete that produces antibacterial volatile substances, whose culture name and indicated identification characteristics are Streptomyces microfavus A89, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M2024842, and a deposit date of May 8, 2024.

[0005] Application of an actinomycete producing antibacterial volatile substances in treating post-harvest grape diseases comprises the following steps:

[0006] S1: The actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea was obtained by plate-matching method;

[0007] S2: Identify the strains using morphological, physiological, biochemical and molecular biological methods;

[0008] S3: Use headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze its volatile substances, and use pure products to verify and determine the effective antibacterial components;

[0009] S4: Use plastic boxes to analyze the inhibitory effect of volatile substances produced by A89 on postharvest gray mold of Red Globe grapes.

[0010] Optionally, in the step of S1: using the plate-stitching method to screen and obtain the actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea, the plate-stitching method is used, using a sterile cork borer to take the Botrytis cinerea cake that has been cultured for 7 days, inoculate it in the center of the PDA culture medium, streak the activated actinomycete strains A83, A84, A87 and A89 on the ISP2 culture medium, place it at a constant temperature of 30°C for 14 days, remove the culture dish cover, and fasten it with the culture dish containing the Botrytis cinerea cake, and seal it with two layers of sealing film. The control group is the ISP2 culture medium without bacteria. After constant temperature culture at 30°C for 5 days, the colony diameter is measured by the cross method and the result is recorded, and the inhibition rate is calculated according to formula (1).

[0011] Inhibition rate / % = ((colony diameter of control group - colony diameter of treatment group) / colony diameter of control group) × 100 (1)

[0012] Optionally, in the step of S2: identifying the strain using morphological, physiological, biochemical and molecular biological methods, the strain A89 to be observed is inoculated into ISP2 medium and cultured at 30°C for 7 days. When the colonies grow all over the plate, the characteristics of the colonies are observed, recorded and photographed for preservation. The characteristics of mycelium and spores are observed under an optical microscope using the insert method, recorded and photographed for archiving, and the 16SrDNA sequence is sequenced and analyzed. A89 cultured at 30°C for 7 days is collected, and its mycelial DNA is extracted using a bacterial DNA extraction kit, and the 16SrDNA sequence is amplified using this as a template. The primers are 27F and 1492R, and the PCR amplification system (50 μL): 10×TaqDNApolymerasebuffe r5.0μL, dNTP2.0μL, upstream and downstream primers 1.0μL each, TaqDNApolymerase1.2μL, template DNA3.6μL, ddH2O to 50μL; PCR reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 10s, 52.6℃ annealing for 30s, 72℃ extension for 45s, 35 cycles; 72℃ extension for another 5min, 4℃ storage, 1% agarose gel electrophoresis to detect PCR products, Promega gel recovery kit to purify PCR products, and then sequenced, the sequences were blasted on NCBI to obtain sequences with close similarity, and a phylogenetic evolutionary tree was constructed using the MEGA7.0 neighbor-joining method.

[0013] Optionally, the step of S3: using headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze the volatile substances, and verifying and determining the effective antibacterial components with pure products comprises:

[0014] S31: Determination of the composition of volatile substances in strain A89:

[0015] S311: Headspace Solid Phase Microextraction

[0016] Weigh 20g of filtrate sample and put it into a 100mL headspace extraction bottle with a silicone pad cap. After pre-equilibration in a 60℃ water bath for 15min, push out the aged extraction head, adsorb in the headspace for 40min, take out the extraction needle and inject, desorb at 250℃ for 5min;

[0017] S312: GC-MS / MS analysis

[0018] GC conditions: InertCapWAX column (30m×0.25mm, 0.25μm); splitless injection; injection port temperature 250℃; flow rate 1mL / min; programmed temperature rise: initial temperature 35℃ maintained for 5min, increased to 100℃ at 3℃ / min, then increased to 240℃ at 4℃ / min, maintained for 4min, MS conditions: electron bombardment ionization source; carrier gas is helium; ion source temperature 230℃; data acquisition mode Q3 scanning; scanning range m / z45~500, the mass spectrum obtained by analysis was compared and searched with the standard spectrum in the NIST-17 spectral library, and the components with matching degree >80% were determined, and the relative content of each component was calculated by peak area normalization method for quantitative analysis;

[0019] S313: Determination of in vitro antibacterial activity of monomer components against Botrytis cinerea

[0020] The main components of volatile substances, phenylethanol, benzyl alcohol, acetophenone, nonanal, 2,5-dimethylpyrazine, and 2-ethylhexanol, were determined to have antibacterial effects in liquid state at room temperature. The active components were identified. A 7-day cultured Botrytis cinerea cake was taken with a sterile cork puncher and inoculated in the center of the PDA medium. A sterile 2 mL sample cup was placed in another culture dish. 2.187, 4.375, 8.75, 17.5, 35.0, and 70.0 μL of the monomer components were respectively transferred to the centrifuge tube cap with a pipette. The volume of the culture dish was 400 μL. The volume of the culture dish after buckling was 80 mL, minus 20 mL of the volume occupied by the PDA culture medium, and the volume of the culture dish after buckling was 140 mL. The corresponding volatile contents were 15.625, 31.25, 62.5, 125, 250, and 500 μL / L, respectively. In the control group, no volatile pure product was added to the centrifuge tube cover. After removing the culture dish cover, it was quickly buckled together and the edge of the culture dish was sealed with two layers of sealing film. After culturing at 30°C for 7 days, the colony diameters of the treatment group and the blank control group were measured by the cross method. Each group was repeated 3 times, and the antibacterial rate was calculated according to formula (1).

[0021] Optionally, the step of S4: using a plastic box to analyze the inhibitory effect of volatile substances produced by A89 on postharvest gray mold of red globe grapes comprises:

[0022] S41: Determination of the efficacy of volatile substances and active components produced by strain A89 against Botrytis cinerea:

[0023] Select red globe grapes with consistent maturity, uniform size and no diseases and insect pests, soak them in 2% sodium hypochlorite solution for 2 minutes to disinfect the surface of the grapes, dry them for later use, add 30mL of sterile water to the Botrytis cinerea culture medium that has been cultured for 7 days, use an inoculation needle to gently scratch the hyphae, filter the bacterial suspension with 8 layers of sterilized gauze to obtain the Botrytis cinerea spore suspension, use a hemocytometer to adjust the concentration to 1×106 / mL, and set aside, strain A89 and four monomer compounds of nonanal, 2-ethylhexanol, acetophenone and 2,5-dimethylpyrazine were tested for their protective effects on grape gray mold. Wipe the surface of the plastic box with an alcohol cotton ball in the clean bench, and then put an opened PDA plate of strain A89 that has grown for 14 days into it, and use the uninoculated PDA plate as the control CK 1; Take another plastic dish, place a sample cup in the inner corner, and transfer the corresponding volume of volatile monomer compounds into the centrifuge tube cap to reach the set final concentration, among which the added concentrations of nonanal and 2-ethylhexanol are 2×MIC, 8×MIC, and 16×MIC, and the added concentrations of acetophenone and 2,5-dimethylpyrazine are MIC and 2×MIC; Take the empty sample cup as the control CK2, use a sterile syringe to make a hole with a diameter of 0.2 cm and a depth of 0.5 cm at the equator of the grape fruit; Use a pipette to draw 10 μL of spore suspension and inject it into the pre-punched hole, put 15 inoculated grapes in each plastic dish, buckle the lid and seal it, leave it at room temperature for 10 days, and then observe and measure the diameter of the fruit lesions, and set 6 replicates for each treatment;

[0024] S42: Effects of volatile substances produced by strain A89 on the natural incidence of grapevine disease

[0025] Referring to S41, 3 frames of washed, disinfected and dried grapes were placed in the fumigation equipment, with a weight of 5 kg / frame, and then 10 A89 PDA plates with lids opened and grown for 14 days were placed. The control group was an uninoculated PDA culture medium, also with 10 plates. After 30 days, the incidence rate of the fruits was determined and calculated, incidence rate / %=number of diseased fruits / total number of fruits, and each treatment was repeated 6 times.

[0026] Optionally, in the steps S1-S4, the Duncan multiple comparison test of SPSS20.0 software is used to perform a significant difference analysis, P < 0.05, the difference is significant, and a chart is drawn using Excel.

[0027] Optionally, in the step of S41: determining the protective efficacy of volatile substances and active components produced by strain A89 against grape gray mold, red globe grapes with a maturity of eight mature are selected.

[0028] Optionally, in the step S1: using the plate-pairing method to screen and obtain actinomycete A89 antagonistic to grape gray mold fungus Botrytis cinerea and the step S313: determining the in vitro antibacterial activity of monomer components against Botrytis cinerea, the diameter of the Botrytis cinerea cake is 0.8 cm.

[0029] The present invention provides actinomycetes producing antibacterial volatile substances and application thereof in treating post-harvest diseases of grapes, which have the following beneficial effects:

[0030] The actinomycetes producing antibacterial volatile substances and their application in post-harvest diseases of grapes were obtained by screening actinomycetes A89 antagonistic to grape gray mold fungus Botrytis cinerea by using the plate-pairing method, and the strain was identified by morphological, physiological, biochemical and molecular biological methods. The volatile substances were detected and analyzed by headspace solid phase microextraction and gas chromatography-tandem mass spectrometry, and the effective antibacterial components were verified and determined by pure products. The inhibitory effect of volatile substances produced by A89 on post-harvest gray mold of red globe grapes was analyzed using plastic boxes, and it was concluded that the volatile substances of Streptomyces tenuifolius A89 can effectively inhibit the occurrence of post-harvest gray mold of red globe grapes. Under in vivo conditions, the best performing treatment groups were A89 volatile substances and 500μL / L 2,5-dimethylpyrazine after fumigation for 10 days, and the lesion diameters of the fruits were reduced by 74.11% and 76.70%, respectively. It is worth noting that the 125μL / L acetophenone treatment group can completely inhibit the occurrence of grape gray mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the antibacterial effects of different actinomycetes on Botrytis cinerea of ​​the present invention;

[0032] Figure 2 This is a graph showing the inhibition rate of different actinomycetes against Botrytis cinerea in the present invention;

[0033] Figure 3 Figures (A) and (B) show the morphological characteristics of the strain A89 of the present invention;

[0034] Figure 4 This is a phylogenetic tree diagram of the A89 strain based on the 16SrDNA sequence of the present invention;

[0035] Figure 5 This is a diagram showing the antibacterial effect of the volatile substances produced by the monomer compound strain A89 on grape gray mold in the fumigation grape equipment used in the embodiment of the present invention;

[0036] Figure 6 The grape fumigation equipment used in the embodiment of the present invention, the effect of volatile substances produced by strain M6-5 on the natural incidence of grapes (left: treatment group; right: control group);

[0037] Figure 7 This is a diagram of the equipment for large-scale fumigation of grapes according to the present invention.

[0038] In the figure: 1. fermentation tank; 2. first pipeline; 3. second pipeline; 4. gas storage tank; 5. pump; 6. adjustment pipeline; 7. pressure adjustment equipment; 8. temperature adjustment equipment; 9. volume adjustment equipment; 10. fumigation cabinet; 11. drawer board; 12. moving wheel. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0040] See also Figures 1 to 6 The present invention provides a technical solution: an actinomycete that produces antibacterial volatile substances, whose culture name and indicated identification characteristics are Streptomyces microfavus A89, deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCCNO: M2024842, and a deposit date of May 8, 2024. The viability of the culture was tested by China Center for Type Culture Collection on May 15, 2024, and the result was viable.

[0041] Application of an actinomycete producing antibacterial volatile substances in treating post-harvest grape diseases comprises the following steps:

[0042] S1: The actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea was obtained by plate-matching method;

[0043] The plate-stitching method was adopted. A sterile cork hole was used to take a 7-day cultured Botrytis cinerea cake with a diameter of 0.8 cm. It was inoculated in the center of the PDA culture medium. The activated actinomycete strains A83, A84, A87 and A89 were streaked and inoculated on the ISP2 culture medium. The culture dish was placed at 30°C for 14 days. The cover of the culture dish was removed and it was placed on the culture dish containing the Botrytis cinerea cake and sealed with two layers of sealing film. The control group was the ISP2 culture medium without bacteria. After being cultured at 30°C for 5 days, the colony diameter was measured by the cross method and the results were recorded. The inhibition rate was calculated according to formula (1).

[0044] Inhibition rate / % = ((colony diameter of control group - colony diameter of treatment group) / colony diameter of control group) × 100 (1)

[0045] S2: Identify the strains using morphological, physiological, biochemical and molecular biological methods;

[0046] The strain A89 to be observed was inoculated into ISP2 medium and cultured at 30°C for 7 days. When the colonies grew all over the plate, the characteristics of the colonies were observed, recorded, and photographed for preservation. The characteristics of mycelium and spores were observed under an optical microscope using the insert method, and the records were taken for archiving. The 16S rDNA sequence was sequenced and analyzed. A89 cultured at 30°C for 7 days was collected, and its mycelial DNA was extracted using a bacterial DNA extraction kit. The mycelial DNA was used as a template for 16S rDNA sequence amplification. The primers were 27F and 1492R. The PCR amplification system (50 μL) included 10× Taq DNA polymerase buffer 5.0 μL, dNTP 2.0 μL, upstream and downstream primers 1.0 μL each, Taq DNA polymerase 1.2 μL, template DNA 3.6 μL, dd H2O was added to 50 μL; PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 10 s, 52.6℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ extension for another 5 min, 4℃ storage, 1% agarose gel electrophoresis was used to detect PCR products, Promega gel recovery kit was used to purify PCR products, and then sequencing was performed, and the sequences were blasted on NCBI to obtain sequences with close similarity, and the phylogenetic evolutionary tree was constructed using MEGA7.0 neighbor-joining method;

[0047] S3: Use headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze its volatile substances, and use pure products to verify and determine the effective antibacterial components;

[0048] S3: The steps of using headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze the volatile substances and verify and determine the effective antibacterial components with pure products include:

[0049] S31: Determination of the composition of volatile substances in strain A89:

[0050] S311: Headspace Solid Phase Microextraction

[0051] Weigh 20g of filtrate sample and put it into a 100mL headspace extraction bottle with a silicone pad cap. After pre-equilibration in a 60℃ water bath for 15min, push out the aged extraction head, adsorb in the headspace for 40min, take out the extraction needle and inject, desorb at 250℃ for 5min;

[0052] S312: GC-MS / MS analysis

[0053] GC conditions: InertCapWAX column (30m×0.25mm, 0.25μm); splitless injection; injection port temperature 250℃; flow rate 1mL / min; programmed temperature rise: initial temperature 35℃ maintained for 5min, increased to 100℃ at 3℃ / min, then increased to 240℃ at 4℃ / min, maintained for 4min, MS conditions: electron bombardment ionization source; carrier gas is helium; ion source temperature 230℃; data acquisition mode Q3 scanning; scanning range m / z45~500, the mass spectrum obtained by analysis was compared and searched with the standard spectrum in the NIST-17 spectral library, and the components with matching degree >80% were determined, and the relative content of each component was calculated by peak area normalization method for quantitative analysis;

[0054] S313: Determination of in vitro antibacterial activity of monomer components against Botrytis cinerea

[0055] The main components of volatile substances, phenylethanol, benzyl alcohol, acetophenone, nonanal, 2,5-dimethylpyrazine, and 2-ethylhexanol, were determined to have antibacterial effects in liquid state at room temperature. The active components were identified. A sterile cork puncher was used to take a 7-day cultured Botrytis cinerea cake with a diameter of 0.8 cm. It was inoculated in the center of the PDA culture medium. A sterile 2 mL sample cup was placed in another culture dish. 2.187, 4.375, 8.75, 17.5, 35.0, and 70.0 μL of the monomer components were respectively transferred to the centrifuge tube cap using a pipette. The volume of the culture dish was 80 mL, minus the volume of 20 mL occupied by the PDA medium, and the volume of the culture dish after buckling was 140 mL. The corresponding volatile contents were 15.625, 31.25, 62.5, 125, 250, and 500 μL / L, respectively. In the control group, no pure volatiles were added to the centrifuge tube cover. After removing the culture dish cover, it was quickly buckled and the edge of the culture dish was sealed with two layers of sealing film. After culturing at 30°C for 7 days, the colony diameters of the treatment group and the blank control group were measured by the cross method. Each group was repeated 3 times, and the antibacterial rate was calculated according to formula (1);

[0056] S4: Use plastic boxes to analyze the inhibitory effect of volatile substances produced by A89 on postharvest gray mold of Red Globe grapes;

[0057] S41: Determination of the efficacy of volatile substances and active components produced by strain A89 against Botrytis cinerea:

[0058] Select red globe grapes with the same maturity of eight mature, uniform size and free of diseases and insect pests, soak them in 2% sodium hypochlorite solution for 2 minutes to disinfect the surface of the grapes, dry them for later use, add 30mL of sterile water to the Botrytis cinerea culture medium that has been cultured for 7 days, use an inoculation needle to gently scratch the hyphae, filter the bacterial suspension with 8 layers of sterilized gauze to obtain the Botrytis cinerea spore suspension, use a hemocytometer to adjust the concentration to 1×106 / mL, and set aside, strain A89 and four monomer compounds of nonanal, 2-ethylhexanol, acetophenone and 2,5-dimethylpyrazine were tested for their protective efficacy against grape gray mold. Wipe the surface of the plastic box with an alcohol cotton ball in the clean bench, and then put an opened PDA plate of strain A89 that has grown for 14 days into it, and use the uninoculated PDA plate as a control. CK1; Take another plastic dish, place a sample cup in the inner corner, and transfer the corresponding volume of volatile monomer compounds into the centrifuge tube cap to reach the set final concentration, among which the added concentrations of nonanal and 2-ethylhexanol are 2×MIC, 8×MIC, and 16×MIC, and the added concentrations of acetophenone and 2,5-dimethylpyrazine are MIC and 2×MIC; Take the empty sample cup as the control CK2, use a sterile syringe to make a hole with a diameter of 0.2 cm and a depth of 0.5 cm at the equator of the grape fruit; use a pipette to draw 10 μL of spore suspension and inject it into the pre-punched hole, put 15 inoculated grapes in each plastic dish, buckle the lid and seal it, leave it at room temperature for 10 days, and then observe and measure the diameter of the fruit lesions, and set 6 replicates for each treatment;

[0059] S42: Effects of volatile substances produced by strain A89 on the natural incidence of grapevine disease

[0060] Referring to S41, 3 frames of washed, disinfected and dried grapes were placed in the fumigation equipment, with a weight of 5 kg / frame, and then 10 A89 PDA plates with lids opened and grown for 14 days were placed. The control group was an uninoculated PDA culture medium, also with 10 plates. After 30 days, the incidence rate of the fruits was determined and calculated, incidence rate / %=number of diseased fruits / total number of fruits, and each treatment was repeated 6 times.

[0061] S5: Data Analysis:

[0062] S51: Analysis of the antibacterial effects of different actinomycetes on Botrytis cinerea under in vitro conditions:

[0063] like Figure 1 and Figure 2As shown in the results, actinomycete strains A83, A84, A87 and A89 were matched with Botrytis cinerea for 5 days, and different degrees of antibacterial effects were found on Botrytis cinerea. The antibacterial effects were A89, A87, A83 and A84 from strong to weak, among which A89 could completely inhibit the mycelial growth of Botrytis cinerea with an inhibition rate of 100%. The inhibition rates of the other three actinomycete strains A87, A83 and A84 on Botrytis cinerea were 73.91±1.58%, 40.67±2.34% and 30.18±1.69%, respectively. Therefore, A89 was selected as the strain for subsequent experiments.

[0064] S52: Identification of strain A89

[0065] S521: Morphological identification of strain A89:

[0066] After 7 days of growth on ISP2 medium, strain A89 formed circular colonies with a diameter of about 2 to 8 mm, which protruded toward the surface of the medium. The center of the colony was dark yellow, loose and porous ( Figure 3 A); Gram staining is positive; aerial hyphae are white at first, turn light yellow after spore production, grow vigorously, have many branches, are dendrite-like, and have a dry and rough surface ( Figure 3 B); spores are oblong, smooth, and 0.6 to 0.8 × 0.5 to 0.7 μm in size ( Figure 3 C);

[0067] S522: Some physiological and biochemical results of strain A89:

[0068] Actinomycete A89 cultured on ISP2 medium for 7 days was inoculated into six physiological and biochemical identification mediums. According to the culture conditions of the strain in the medium, the physiological and biochemical characteristics of the strain were obtained (Table 1). The experimental results are consistent with the physiological and biochemical characteristics of most Streptomyces;

[0069] Table 1 Some physiological and biochemical results of strain A89

[0070]

[0071]

[0072] S523: Molecular biological identification of strain A89:

[0073] The sequence length of strain A89 was 1047 bp. The sequence was submitted to the GenBank database with the accession number MZ930280.1. BLAST comparison was performed in the EZbiocloud database. The similarity was the highest with Streptomyces s pratensisch24T, reaching 99.85%, followed by Streptomyces durocortorens isRHZ10 (99.57%) and Streptomyces microflavusNBRC13062 (99.37%). Sequences with higher homology were selected to construct a phylogenetic tree ( Figure 4 ), strain A89 and Streptomyces microflavus are in the same branch and have the closest relationship.

[0074] In summary, combining the morphological characteristics, physiological and biochemical and molecular biological results, it can be determined that strain A89 is Streptomyces tenuiflavus.

[0075] S53: Analysis of volatile components of strain A89:

[0076] As shown in Table 2 , strain A89 produced a total of 77 volatile substances detected by GC-MS / MS, of which the main components were 12-methyltridecanoic acid methyl ester, palmitic acid methyl ester, pentadecanoic acid methyl ether, heptadecane, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, etc., accounting for 18.59%, 9.7%, 8.58%, 5.45%, and 4.64% of the total volatile substances, respectively;

[0077] Table 2 GC-MS / MS identification of some volatile components of strain A89

[0078]

[0079]

[0080]

[0081] S54: Analysis of the in vitro antibacterial activity of pure volatile substances produced by strain A89 against Botrytis cinerea:

[0082] Because it is to be used in grapes, 6 spices that can be used in food in the national standard GB2760-201428 were screened out, and the inhibition rate of pure volatile substances with different concentrations on Botrytis cinerea was detected by plate buckling. As shown in Table 3, the 6 volatile substances all had different degrees of antibacterial effect on Botrytis cinerea, and showed a concentration-dependent effect, that is, with the increase of concentration, the inhibition rate also showed an increasing trend. Among them, nonanal and 2-ethylhexanol had the best antibacterial effect, with the minimum inhibitory concentration (MIC) of 7.81μL / L, followed by acetophenone and 2,5-dimethylpyrazine, with MICs of 62.5μL / L and 500μL / L, respectively; phenylethanol and benzyl alcohol had weaker antibacterial effects on Botrytis cinerea, and when the concentration was 500μL / L, the inhibition rates were 70.66% and 49.63%, respectively.

[0083] Table 3 Inhibition rate of pure volatile substances at different concentrations on Botrytis cinerea

[0084]

[0085]

[0086] S55: Antibacterial effect of volatile substances and monomer compounds produced by strain A89 on grape gray mold:

[0087] As shown in Table 4, four volatile substances, nonanal, 2-ethylhexanol, acetophenone and 2,5-dimethylpyrazine, were selected to test the antibacterial effect on gray mold of Red Globe grape under in vivo conditions. Under in vivo conditions, after 10 days of fumigation, there was no significant difference in the diameter of fruit lesions between the control group CK1 and CK2 (P>0.05). The best treatment groups were A89 plate and 500μL / L2,5-dimethylpyrazine treatment groups, with the fruit lesion diameters of 0.51±0.02cm and 0.46±0.03cm, respectively (P>0.05); followed by 62.5μL / L nonanal treatment group, with a fruit lesion diameter of 0.69±0.04cm. It is worth noting that the 62.5μL / L acetophenone treatment group can also significantly reduce the fruit lesion diameter, and no lesions are produced in the 125μL / L acetophenone treatment group.

[0088] Table 4 Effects of different concentrations of monomer compounds and A89 fumigation treatment on the diameter of grape gray mold lesions

[0089]

[0090] Note: Different lowercase letters indicate significant differences (P<0.05). “-” means no antibacterial test was conducted on the volatile substances at that concentration.

[0091] S56: Effects of volatile substances produced by strain A89 on natural incidence of grapevine disease:

[0092] After 30 days of fumigation, the natural disease incidence of grapes was measured to be 10±2.31%, while the disease incidence of grapes in the control group was 35±1.58%. This shows that A89 volatile substances can significantly reduce the natural disease incidence of grapes.

[0093] Example

[0094] Figure 7 As shown, the equipment required for large-scale in vivo grape fumigation experiments includes a fermentation tank 1, a first pipeline 2 is installed on the top of the fermentation tank 1, the other end of the first pipeline 2 is fixedly connected to a gas storage tank 4, a second pipeline 3 is fixedly connected to the top of the gas storage tank 4 and located on the right side of the first pipeline 2, a pump 5 is fixedly connected to one end of the second pipeline 3, an adjustment pipeline 6 is fixedly connected to one end of the pump 5, a pressure adjustment device 7 is installed on the adjustment pipeline 6, a temperature adjustment device 8 is installed on the adjustment pipeline 6 and located on one side of the pressure adjustment device 7, a volume adjustment device 9 is installed on the adjustment pipeline 6 and located on one side of the temperature adjustment device 8, the other end of the adjustment pipeline 6 is fixedly connected to a fumigation cabinet 10, and the fumigation cabinet 10 is fixedly connected with several A dry block drawer board 11, a plurality of moving wheels 12 are arranged at the bottom of the gas storage tank 4, the fermentation tank 1 is connected with the inside of the gas storage tank 4 through the first pipeline 2, the gas storage tank 4 is connected with the inside of the pump 5 through the second pipeline 3, the pump 5 is connected with the inside of the fumigation cabinet 10 through the adjustment pipeline 6, and the fine yellow Streptomyces is fermented in the fermentation tank 1 through the plurality of moving wheels 12 installed at the bottom of the gas storage tank 4, generally for 14 days, and its volatile substances are collected into the gas storage tank 4, and the gas inside the gas storage tank 4 is input into the fumigation cabinet 10 through the pump 5 and the pressure adjustment device 7, the temperature adjustment device 8 and the volume adjustment device 9, and the cubic number of the transported gas is controlled to fumigate the grapes placed on the surface of the drawer board 11 inside the fumigation cabinet 10, such as Figure 6 Grapes are fumigated for this equipment.

[0095] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An actinomycete producing antibacterial volatile substances, characterized in that: The culture name and indicated identification characteristics are Streptomyces microfavusA89. It is deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M2024842 and the deposit date is May 8, 2024.

2. Use of an actinomycete producing antibacterial volatile substances as claimed in claim 1 in treating post-harvest diseases of grapes, characterized in that: The following steps are involved: S1: The actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea was obtained by plate-matching method; S2: Identify the strains using morphological, physiological, biochemical and molecular biological methods; S3: Use headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze its volatile substances, and use pure products to verify and determine the effective antibacterial components; S4: Use plastic boxes to analyze the inhibitory effect of volatile substances produced by A89 on postharvest gray mold of Red Globe grapes.

3. The use of an actinomycete producing antibacterial volatile substances in treating postharvest diseases of grapes according to claim 2, characterized in that: In the step S1: using the plate-stitching method to screen and obtain the actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea, the plate-stitching method is used. A sterile cork borer is used to take a Botrytis cinerea cake that has been cultured for 7 days and inoculate it in the center of a PDA culture medium. The activated actinomycete strains A83, A84, A87 and A89 are streaked and inoculated on an ISP2 culture medium, and the culture dish is placed at a constant temperature of 30° C. for 14 days. The culture dish cover is removed and the culture dish containing the Botrytis cinerea cake is fastened together and sealed with two layers of sealing film. The control group is an ISP2 culture medium without bacteria. After being cultured at a constant temperature of 30° C. for 5 days, the colony diameter is measured by the cross method and the result is recorded. The inhibition rate is calculated according to formula (1). Inhibition rate / %=((colony diameter of control group - colony diameter of treatment group) / colony diameter of control group)×100(1).

4. The use of an actinomycete producing antibacterial volatile substances in treating postharvest diseases of grapes according to claim 2, characterized in that: In the step of identifying the strain using morphological, physiological, biochemical and molecular biological methods, the strain A89 to be observed is inoculated into ISP2 medium and cultured at 30°C for 7 days. When the colony grows all over the plate, the characteristics of the colony are observed, recorded and photographed for preservation. The characteristics of hyphae and spores are observed under an optical microscope using the insert method, recorded and photographed for archiving, and the 16S rDNA sequence is sequenced and analyzed. A89 cultured at 30°C for 7 days is collected, and its mycelial DNA is extracted using a bacterial DNA extraction kit, and the 16S rDNA sequence is amplified using this as a template, with primers 27F and 1492R, and the PCR amplification system (50 μL): 10×TaqDNApolymerasebuffer 5.0 μL, dNTP 2.0 μL, upstream and downstream primers 1.0 μL each, TaqDNApolymerase 1.2 μL, template DNA 3.6μL, ddH2O made up to 50μL; PCR reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 10s, 52.6℃ annealing for 30s, 72℃ extension for 45s, 35 cycles; 72℃ extension for another 5min, 4℃ storage, 1% agarose gel electrophoresis to detect PCR products, Promega gel recovery kit to purify PCR products, and then sequenced, the sequences were blasted on NCBI to obtain sequences with close similarity, and the phylogenetic evolutionary tree was constructed using MEGA7.0 neighbor-joining method.

5. The use of an actinomycete producing antibacterial volatile substances in treating post-harvest diseases of grapes according to claim 2, characterized in that: The step of S3: using headspace solid phase microextraction and gas chromatography-tandem mass spectrometry to detect and analyze the volatile substances, and verifying and determining the effective antibacterial components with pure products comprises: S31: Determination of the composition of volatile substances in strain A89: S311: Headspace Solid Phase Microextraction Weigh 20g of filtrate sample and put it into a 100mL headspace extraction bottle with a silicone pad cap. After pre-equilibration in a 60℃ water bath for 15min, push out the aged extraction head, adsorb in the headspace for 40min, take out the extraction needle and inject, desorb at 250℃ for 5min; S312: GC-MS / MS analysis GC conditions: InertCapWAX column (30m×0.25mm, 0.25μm); splitless injection; injection port temperature 250℃; flow rate 1mL / min; programmed temperature rise: initial temperature 35℃ maintained for 5min, increased to 100℃ at 3℃ / min, then increased to 240℃ at 4℃ / min, maintained for 4min, MS conditions: electron bombardment ionization source; carrier gas is helium; ion source temperature 230℃; data acquisition mode Q3 scanning; scanning range m / z45~500, the mass spectrum obtained by analysis was compared and searched with the standard spectrum in the NIST-17 spectral library, and the components with matching degree >80% were determined, and the relative content of each component was calculated by peak area normalization method for quantitative analysis; S313: Determination of in vitro antibacterial activity of monomer components against Botrytis cinerea The main components of volatile substances, phenylethanol, benzyl alcohol, acetophenone, nonanal, 2,5-dimethylpyrazine, and 2-ethylhexanol, were determined to have antibacterial effects in liquid state at room temperature. The active components were identified. A 7-day cultured Botrytis cinerea cake was taken with a sterile cork puncher and inoculated in the center of the PDA medium. A sterile 2 mL sample cup was placed in another culture dish. 2.187, 4.375, 8.75, 17.5, 35.0, and 70.0 μL of the monomer components were respectively transferred to the centrifuge tube cap with a pipette. The volume of the culture dish was 400 μL. The volume of the culture dish after buckling was 80 mL, minus 20 mL of the volume occupied by the PDA culture medium, and the volume of the culture dish after buckling was 140 mL. The corresponding volatile contents were 15.625, 31.25, 62.5, 125, 250, and 500 μL / L, respectively. In the control group, no volatile pure product was added to the centrifuge tube cover. After removing the culture dish cover, it was quickly buckled together and the edge of the culture dish was sealed with two layers of sealing film. After culturing at 30°C for 7 days, the colony diameters of the treatment group and the blank control group were measured by the cross method. Each group was repeated 3 times, and the antibacterial rate was calculated according to formula (1).

6. The use of an actinomycete producing antibacterial volatile substances in treating postharvest diseases of grapes according to claim 2, characterized in that: The step of using a plastic box to analyze the inhibitory effect of volatile substances produced by A89 on postharvest gray mold of red globe grapes in S4 includes: S41: Determination of the efficacy of volatile substances and active components produced by strain A89 against Botrytis cinerea: Select red globe grapes with consistent maturity, uniform size and no diseases and insect pests, soak them in 2% sodium hypochlorite solution for 2 minutes to disinfect the surface of the grapes, dry them for later use, add 30mL of sterile water to the Botrytis cinerea culture medium that has been cultured for 7 days, use an inoculation needle to gently scratch the hyphae, filter the bacterial suspension with 8 layers of sterilized gauze to obtain the Botrytis cinerea spore suspension, use a hemocytometer to adjust the concentration to 1×106 / mL, and set aside, strain A89 and four monomer compounds of nonanal, 2-ethylhexanol, acetophenone and 2,5-dimethylpyrazine were tested for their protective effects on grape gray mold. Wipe the surface of the plastic box with an alcohol cotton ball in the clean bench, and then put an opened PDA plate of strain A89 that has grown for 14 days into it, and use the uninoculated PDA plate as the control CK 1; Take another plastic dish, place a sample cup in the inner corner, and transfer the corresponding volume of volatile monomer compounds into the centrifuge tube cap to reach the set final concentration, among which the added concentrations of nonanal and 2-ethylhexanol are 2×MIC, 8×MIC, and 16×MIC, and the added concentrations of acetophenone and 2,5-dimethylpyrazine are MIC and 2×MIC; Take the empty sample cup as the control CK2, use a sterile syringe to make a hole with a diameter of 0.2 cm and a depth of 0.5 cm at the equator of the grape fruit; Use a pipette to draw 10 μL of spore suspension and inject it into the pre-punched hole, put 15 inoculated grapes in each plastic dish, buckle the lid and seal it, leave it at room temperature for 10 days, and then observe and measure the diameter of the fruit lesions, and set 6 replicates for each treatment; S42: Effects of volatile substances produced by strain A89 on the natural incidence of grapevine disease Three frames of washed, disinfected and dried grapes were placed in the fumigation equipment, with a weight of 5 kg / frame. Then, 10 A89 PDA plates that had been opened and grown for 14 days were placed in the fumigation equipment. The control group was an uninoculated PDA culture medium, also with 10 plates. After 30 days, the incidence rate of the fruits was determined and calculated. The incidence rate / %=number of diseased fruits / total number of fruits. Each treatment was repeated 6 times.

7. The use of an actinomycete producing antibacterial volatile substances in treating postharvest diseases of grapes according to claim 2, characterized in that: In the steps S1-S4, the Duncan multiple comparison test method of SPSS20.0 software was used to perform a significant difference analysis. When P < 0.05, the difference was significant, and a chart was drawn using Excel.

8. The use of an actinomycete producing antibacterial volatile substances in treating post-harvest diseases of grapes according to claim 2, characterized in that: In the step of S41: determining the protective effect of volatile substances and active components produced by strain A89 on grape gray mold, red globe grapes with a maturity of eight degrees of maturity are selected.

9. The use of an actinomycete producing antibacterial volatile substances in treating postharvest diseases of grapes according to claim 2, characterized in that: In the step S1: using the plate-pairing method to screen and obtain the actinomycete A89 antagonistic to the grape gray mold fungus Botrytis cinerea and the step S313: determining the in vitro antibacterial activity of the monomer components against Botrytis cinerea, the diameter of the Botrytis cinerea cake is 0.8 cm.

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