Application of volatile substances produced by a strain of Streptomyces NK27 in the control of plant diseases

Abstract

This invention discloses the application of volatile substances produced by a strain of *Streptomyces northernsis* NK27 in the control of plant diseases, belonging to the field of plant disease control technology. The preservation number of *Streptomyces northernsis* NK27 is CGMCC No. 28268. The volatile substances include β-pinene, 4-methoxystyrene, cyclopentanol, and p-phenylenediamine. This invention found that 4-methoxystyrene, cyclopentanol, β-pinene, and p-phenylenediamine in the volatile substances of *Streptomyces northernsis* NK27 have significant inhibitory effects on *Botrytis cinerea*. Biocontrol experiments showed that the volatile substances produced by *Streptomyces northernsis* NK27 inoculated with wheat grain culture medium can effectively control gray mold. This invention provides a safe and effective formulation for the control of gray mold in fruit and vegetable cultivation and post-harvest storage.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of volatile substances produced by a strain of Streptomyces NK27 of Norilsk in the control of plant diseases. Background Technology

[0002] Grapes (Vitis vinifera L.) are widely cultivated in my country. Their fruit is sweet, refreshing, and nutritious, rich in various minerals. They can be eaten fresh or processed, making them a fruit tree with significant cultivation value. Grapes are berries with thin skin, are juicy, and have soft flesh. They have high water and sugar content, making them highly susceptible to mechanical damage and pathogen invasion during harvesting, storage, transportation, and sales, leading to rot and spoilage and loss of marketability. The annual loss rate due to postharvest diseases is generally 20%–30%, and in some cases even as high as 50%, severely reducing the flavor and quality of the grapes and causing significant economic losses. Postharvest gray mold is a major disease causing a decline in grape quality and yield. Once grapes are infected with gray mold during transportation and storage, the disease becomes severe due to factors such as grape variety, cultivation techniques, and storage conditions. Problems include browning, berry detachment, poor flavor, rapid nutrient loss, and a short shelf life. Currently, the control of postharvest diseases in grapes mainly relies on chemical agents. However, the long-term use of chemical agents has led to problems such as drug residues, food safety, and environmental pollution, and has even resulted in increased drug resistance in diseases. Therefore, seeking safer and more environmentally friendly biological control measures to control postharvest diseases has become a new research trend.

[0003] Microorganisms can produce a large variety of small-molecule volatile organic compounds (VOCs) through metabolism, resulting in a rich diversity of functions. Microbial VOCs are complex in composition and diverse in function, while also exhibiting low toxicity and environmental friendliness. Studies have found that microbial VOCs have a strong antibacterial effect, working synergistically to inhibit or even kill postharvest pathogens in fruits and vegetables. Some can even promote plant growth and increase crop yield. Furthermore, they possess highly efficient diffusion capabilities in air and soil pores, rapidly expanding their effective range. In fruit disease control, volatile substances do not directly contact the fruit or leave residues on the fruit surface, thus reducing the use of chemical fertilizers and pesticides and helping to ensure the quality and safety of agricultural products during planting and postharvest storage. Streptomyces, as a biocontrol strain, produces abundant secondary metabolites and VOCs, which can inhibit the growth of pathogens and effectively control diseases during storage. Therefore, developing Streptomyces volatile substances as a biofumigant has broad prospects in the agricultural field and has great potential to replace traditional chemical fumigants. However, there are few reports of volatile secondary metabolites produced by Streptomyces Norröss. Summary of the Invention

[0004] The purpose of this invention is to provide the application of volatile substances produced by a strain of Streptomyces NK27 of Norilsk in the prevention and control of plant diseases, so as to solve the problems existing in the prior art. This invention finds that the volatile substances produced by fermentation of Streptomyces NK27 of Norilsk can effectively prevent and control gray mold, and can provide a safe and effective preparation for the prevention and control of gray mold in fruit and vegetable planting and post-harvest storage.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of volatile substances produced by a strain of Streptomyces NK27 from Norilsk in the control of plant diseases. The preservation number of Streptomyces NK27 from Norilsk is CGMCC No. 28268. The volatile substances include β-pinene, 4-methoxystyrene, cyclopentanol and p-phenylenediamine.

[0007] Furthermore, the plant disease includes gray mold.

[0008] The present invention also provides a method for producing volatile substances by Streptomyces NK27 from Knowles, wherein the Streptomyces NK27 is inoculated into a grain culture medium and cultured.

[0009] Furthermore, the culture conditions are: cultured at 28℃ for 10-15 days.

[0010] Furthermore, the wheat grain culture medium is prepared by soaking wheat grains and boiling them until the skin cracks, filtering and drying them, and then adding water to the dried wheat grains.

[0011] The present invention also provides the application of the volatile substance described above in the preparation of a formulation for controlling gray mold in plants.

[0012] The present invention also provides an agent for preventing and controlling gray mold in plants, the agent comprising the aforementioned volatile substances.

[0013] The present invention discloses the following technical effects:

[0014] This invention reveals that inoculating *Streptomyces northernsis* NK27 onto wheat grain medium and culturing for 10 days resulted in volatile substances with significantly higher antibacterial activity than those cultured for 8 days. Analysis of the volatile components and their inhibitory effects confirmed that 4-methoxystyrene, cyclopentanol, β-pinene, and diphenyl ether exhibit significant antibacterial effects against *Botrytis cinerea*. Biocontrol experiments demonstrate that the volatile substances produced by *Streptomyces northernsis* NK27 inoculated onto wheat grain medium can effectively control gray mold. This invention provides a safe and effective formulation for the control of gray mold in fruit and vegetable cultivation and post-harvest storage. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a phylogenetic tree based on the 16S rDNA sequences of strain NK27 and related Streptomyces, constructed using the neighbor-joining method.

[0017] Figure 2 The complete genome map of strain NK27;

[0018] Figure 3 The inhibitory effect of volatile substances on gray mold was measured; a-b is the two-plate confrontation method, where a: CK; b: strain NK27; c-d is method two, where c: CK; d: strain NK27;

[0019] Figure 4 To determine the inhibitory effect of volatile products produced by *Streptomyces NK27* cultured for different number of days on *Botrytis cinerea*.

[0020] Figure 5 The effect of volatile substances produced by Streptomyces NK27 of Knowles on the mycelial morphology of Botrytis cinerea;

[0021] Figure 6 The effect of volatile substances produced by *Streptomyces NK27* on the germination of *Botrytis cinerea* spores;

[0022] Figure 7 The effect of volatile substances produced by *Streptomyces NK27* on the ultrastructure of *Botrytis cinerea*; the top image is an electron micrograph of the hyphal morphology of *Botrytis cinerea*, scale bar = 10 μm; the bottom image is an electron micrograph of the protoplasm inside the hyphal cells of *Botrytis cinerea*, scale bar = 0.5 μm.

[0023] Figure 8 The effect of volatile substances produced by Streptomyces NK27 on the control of gray mold on grape leaves;

[0024] Figure 9 The effect of volatile substances produced by Streptomyces NK27 on the control of gray mold in grape fruits. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Collection, isolation and identification of Streptomyces NK27 from Knowles

[0031] (I) Collection and isolation of Streptomyces NK27 from Knowles:

[0032] Streptomyces NK27 from the soil was isolated. The soil samples were collected from the rhizosphere soil of healthy wheat plants in the disease-affected area. The soil sampling method was as follows: the top layer was removed with a sampling shovel, and about 100g of soil from a depth of 20-100mm was placed in a sterile kraft paper bag, which was numbered and the collection location and time were recorded. The soil was then brought back to the laboratory, dried, and used for the isolation of the strain.

[0033] The traditional dilution separation method was used. 0.1 g of sample was taken with a sterile spatula and poured into a sterile 1.5 mL centrifuge tube containing 0.9 mL of LB liquid medium. The sample was incubated at 30°C and 100 rpm for 30 min, followed by a 20 min incubation period. The sample was then diluted 10-fold using a 10-fold serial dilution method to a final volume of 10. -3For gradient culture, take 200 μL of the gradient dilution and spread it on Gao's No. 1 solid medium containing naridinolone acid. Incubate at 30°C for 14 days. Select single colonies of Streptomyces according to their colony characteristics and streak them on Gao's No. 1 solid medium until they are confirmed as pure cultures. Store them frozen at -80°C with 25% glycerol.

[0034] (II) Identification of Streptomyces NK27 from Knowles:

[0035] Species identification of strain NK27 was performed using 16S rDNA pairing technology. Straintestinal DNA was extracted using a Streptomyces whole genome DNA extraction kit. Universal 16S rDNA primers (27F, 1492R) were synthesized by BGI Genomics; primer sequences are shown in Table 1. The PCR reaction system and conditions are as follows.

[0036] Table 1 Primer sequences

[0037]

[0038] PCR reaction system:

[0039] 2×Taq PCR Master mix 25μL, 27F 2μL, 1492R 2μL, template DNA 1μL, ddH2O 20μL.

[0040] PCR reaction conditions:

[0041] Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 45℃ for 15 s, extension at 72℃ for 30 s, 35 cycles; final extension at 72℃ for 10 min.

[0042] After PCR amplification, the PCR products were detected by electrophoresis on a 1% agarose gel. The PCR-obtained gene products were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to NCBI for sequence alignment. Strains with high homology were selected, and multiple sequence alignment was performed using MEGA 7.0 software to construct a phylogenetic tree. Figure 1 ).

[0043] We commissioned Aovison Technology Co., Ltd. to perform second-generation + third-generation (i.e., Illumina Hiseq + PacBio) sequencing and related assembly work on strain NK27. SMRTportal software was used to assemble and align reads, and the sequencing depth distribution to the longest mapped sequence was statistically analyzed. The preliminary assembly results were compared and analyzed to separate chromosome and plasmid sequences. The chromosome sequences were then assembled into a linear genome sequence, resulting in a 0-gap complete genome map of strain NK27. Figure 2 ).

[0044] The 16S rRNA gene fragment of strain NK27, with a sequence length of 1465 bp, was obtained by PCR amplification. This sequence was submitted to the NCBI database for BLAST analysis and comparison. The results showed that strain NK27 had a 97% similarity to *Streptomyces noursei*. Therefore, strain NK27 was identified as *Streptomyces noursei*.

[0045] Genome analysis and sequencing assembly results showed that the total genome length was 9,628,020 bp, with a GC content of 72.24%. Gene function annotation of the whole genome was performed using the NR, KEGG, COG, and GO databases. The results showed that 8,456 open reading frames were predicted, accounting for 84.87% of the total genome length. The composition of the sequenced genome was obtained by predicting coding genes, repetitive sequences, and non-coding RNAs. The genome encodes 69 TRNAs with a total length of 5,239 bp, 7 5S sRNAs with a total length of 812 bp, 7 16S sRNAs with a total length of 10,603 bp, and 7 28S rRNAs with a total length of 21,790 bp.

[0046] Streptomyces noursei NK27 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28268, on August 28, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] Example 2: Plate confrontation test (plate antagonism test) of volatile substances against gray mold.

[0048] (a) Test strains:

[0049] Botrytis cinerea, the causal agent of gray mold, is preserved in the Agricultural Antibiotics Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences at -80℃. Streptomyces noursei NK27 was isolated and purified from soil samples and is deposited at the China General Microbiological Culture Collection Center, with the culture accession number CGMCC No. 28268.

[0050] (II) Determination of the inhibitory effect of volatile substances produced by Streptomyces Norrschneri on Botrytis cinerea.

[0051] Preparation of wheat grain culture medium: Soak wheat grains in water for 24 hours, then boil them in boiling water until the wheat grain skin cracks. After filtering and drying, dispense 20g into 250mL Erlenmeyer flasks, add distilled water at a mass ratio of 1:1, and sterilize at 121℃ for 90min.

[0052] Preparation of Streptomyces NK27 grain culture medium: Strawberry strain NK27 was streaked onto MS medium plates and incubated at 28°C for 7 days. The cultured strain was then cut into 1.0 cm pieces. 2 After reaching a certain size, the cells were inoculated into seed culture medium and cultured at 28℃ with shaking at 220 rpm for 24 h. The OD value was then measured using a UV spectrophotometer. 600 A 20% inoculum was added to wheat grain agar, mixed thoroughly, and then placed in an incubator at 28°C for static cultivation. The flask was shaken twice daily to ensure uniform growth of *Streptomyces* on the wheat grain agar. After 10 days of cultivation, when dense grayish-white colonies covered the surface of the wheat grains, the *Streptomyces* wheat grain agar was obtained for later use. Studies have shown that the antibacterial activity of volatile substances produced is related to the spatial distribution of action. Two assay methods were used to determine the inhibitory effect of the volatile substances produced by the strain on *Botrytis cinerea*.

[0053] Method 1 employs the double-plate confrontation method: A 5mm diameter fungal block is punched from the cultured pathogenic fungus. Two 90mm petri dishes are prepared. One dish is filled with PDA medium, and after cooling, the 5mm diameter gray mold fungal block is placed in the center of the medium. A prepared Streptomyces grain culture is then placed at the bottom of the other dish. The gray mold fungal block is inoculated onto the grain culture medium. A control is prepared by adding an equal volume of uninoculated Streptomyces seed culture to the grain culture medium. The dishes are then sealed with sealing film.

[0054] Method 2: Use a punch to cut 5mm diameter fungal blocks from the cultured pathogenic fungus. Place two 90mm petri dishes and one 70mm petri dish in a 180mm diameter petri dish. Pour PDA medium into the 90mm petri dish and let it cool. Place the 5mm diameter gray mold fungal block in the center of the medium. Place the prepared Streptomyces grain medium in the 70mm petri dish. Use an equal amount of uninoculated Streptomyces seed culture medium as a control. Then seal the 180mm petri dish.

[0055] After the above treatment, the sealed petri dishes were placed in a 25℃ constant temperature incubator and cultured for 5 days. The colony diameters of the treatment and control groups were measured using the cross-sectional method. Each treatment was repeated three times. The inhibition rate of volatile substances was calculated using the formula: Inhibition rate = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0056] Table 2. Determination of the inhibitory effect of volatile substances on gray mold.

[0057]

[0058] Experimental results: According to Table 2, Figure 3 It can be seen that after culturing in a 25℃ constant temperature incubator for 5 days, the control group of gray mold almost completely covered the plate. In the two-plate confrontation, gray mold did not grow under the action of the volatile substances of strain NK27, with an inhibition rate of 100%. Under the same amount of volatile substances, after increasing the interaction area, the inhibition rate of strain NK27 against gray mold was still 100%, which shows that the volatile substances produced by strain NK27 have a good inhibitory effect on gray mold.

[0059] (III) Determination of the inhibitory effect of different incubation days on gray mold

[0060] The inhibitory effect of volatile substances produced by strain NK27 after culturing on wheat grain medium for 8 and 10 days on *Botrytis cinerea* was determined using a two-plate confrontation method. Five-mm diameter fungal blocks were punched from the cultured pathogen and placed in two 90mm petri dishes. One dish was filled with PDA medium, cooled, and then the five-mm diameter *Botrytis cinerea* blocks were placed in the center. The prepared wheat grain culture of strain NK27 was then placed at the bottom of the other dish, and the *Botrytis cinerea* blocks were inoculated onto the wheat grain medium. A wheat grain medium containing an equal volume of uninoculated *Streptomyces* seed culture was used as a control. The dishes were sealed with sealing film. The inhibition rate of the volatile substances was calculated using the formula.

[0061] Antibacterial rate = (colon diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%.

[0062] Table 3. Inhibition of Botrytis cinerea by volatile products produced by Streptomyces Norr's NK27 culture for different number of days on Botrytis cinerea.

[0063]

[0064] Experimental results: According to Table 3, Figure 4 It was found that the inhibition rate of *Streptomyces NK27* on *Botrytis cinerea* was 73.6% after 8 days of culture on wheat grain medium, and the inhibition rate was 100% after 10 days of culture. The antibacterial activity of the volatile substances produced by the *Streptomyces NK27* wheat grain culture after 10 days of culture was significantly enhanced compared with that after 8 days of culture, confirming that the antibacterial effect of the volatile substances produced after 10 days of culture was stable.

[0065] Example 3: Detection and Analysis of Volatile Components Produced by Streptomyces Norilsk

[0066] (I) Detection and analysis of volatile components produced by Streptomyces Norilsk:

[0067] Volatile metabolites of the strain were analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). A 50 / 30µm DVB / CAR / PDMS Stableflex (1cm) manual injection solid-phase microextraction fiber (purchased from Merck Chemical Technology Shanghai Co., Ltd.) was used for collection. The extraction fiber was aged by headspace adsorption at 250℃ for 10 min at the GC-MS inlet. Streptomyces grain culture medium cultured for 8 and 10 days in 250mL Erlenmeyer flasks was placed in a 45℃ water bath for 30 min, and the aged DVB / CAR / PDMS solid-phase microextraction fiber was inserted. Headspace adsorption was performed for 30 min to enrich the volatile substances on the extraction fiber, which was then directly injected into the sample. Gas chromatography-mass spectrometry (GC-MS) was used. After resolution at 250℃ for 5 min, GC-MS analysis was performed. GC-MS operating conditions: Gas chromatography conditions: Column type: VF-5MS (30m×0.25mm×0.25um); Injector temperature: 250℃; Carrier gas: He, purity 99.999%; Split ratio: 10:1; Flow rate: 1mL / min; Temperature program: Initial temperature 40℃, hold for 3 min, increase to 150℃ at 4℃ / min, hold for 1 min, then increase to 250℃ at 8℃ / min, hold for 6 min. Mass spectrometry conditions: Mass separator: ion trap; Ion source: EI; Ionization energy: 70 eV; Ion trap temperature: 220℃; Transfer line temperature: 280℃; Solvent delay time: 1.5 min; Full scan mode; Scan range: 43-500 m / z; The computer automatically compared the obtained gas component mass spectra with data from the international standard database (NISTO17 Mass Spectrometry Library Database) to identify the volatile components. The total peak area measured was taken as 100%, and the relative content of each component was determined by normalization. An identical mass of sterile wheat grain culture medium was used as a control. Substances coexisting in the control and strain wheat grain culture medium were removed. The experiment was repeated three times.

[0068] Table 4. Gas chromatography-mass spectrometry determination of volatile organic compounds produced by *Streptomyces northerneri* NK27 cultured for 8 days.

[0069]

[0070] Table 5. Gas chromatography-mass spectrometry determination of volatile organic compounds produced by Streptomyces Norr's NK27 after 10 days of culture.

[0071]

[0072] Experimental Results: According to Tables 4 and 5, the volatile substances produced after 8 days and 10 days of cultivation mostly belong to alkenes, alcohols, esters, and alkanes. However, the following differences exist: myrcene and 4-methoxystyrene were only present in the 10-day cultured wheat grains but not detected in the 8-day cultured grains. Furthermore, the relative contents of ethyl decanoate, ferrous alcohol, 2-pinene, cyclopentanol, dimethyl terephthalate, and β-pinene in the 10-day cultured volatile substances were higher than those in the 8-day cultured grains. Since previous experiments showed that the antibacterial activity of the volatile substances produced by *Streptomyces* wheat grain cultures after 10 days of cultivation was higher than that after 8 days, it was speculated that these compounds might possess antibacterial activity. Therefore, these compounds were artificially synthesized for the determination of their antibacterial activity.

[0073] (II) Determination of the inhibitory effect of volatile monomers on the mycelial growth of Botrytis cinerea

[0074] Chemical reagents: 2-methylisoborneol, cyclopentanol, ethyl decantereate, 2-pinene, β-pinene, fentanyl alcohol, myrcene, 4-methoxystyrene, p-phenylenediamine.

[0075] The solid monomer was prepared into a solution. 2g of the solution was diluted to 25mL with anhydrous ethanol. 100μL of the solution was then placed into a 0.08L petri dish. At this point, the total concentration of the volatile gas in the petri dish was 100mg / L. The volatile gas was then serially diluted to 50.0, 10.0, 5.0, and 2.5mg / L.

[0076] Liquid monomers: 100.0, 50.0, 10.0, 5.0, and 2.5 μL were directly placed in 0.08 L petri dishes, corresponding to dosages of 1250, 625, 125, 62.5, and 31.25 μL / L, respectively. A slightly modified inverted plate method was used on filter paper: 5 mm diameter mycelial blocks of cultured gray mold were punched and inoculated into the center of a PDA plate. A sterilized filter paper disc with a diameter of approximately 20 mm was placed on the plate lid. 100 μL of volatile monomer was pipetted onto the plate. For the liquid monomer group, the filter paper disc without volatile monomer served as a control; for the solid monomer group, the filter paper disc with 100 μL of anhydrous ethanol served as a blank control. The plates were quickly capped and the edges sealed with sealing film. After incubation in an inverted manner at 25℃ for 3 days, the colony diameter of the treatment groups and the blank control group was measured using the cross-sectional method. Three plates were used for each treatment. The entire experiment was repeated three times. Calculate the inhibition rate of volatile monomers on the mycelial growth of Botrytis cinerea using the formula:

[0077] Antibacterial rate = (colon diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%.

[0078] Table 6. Inhibition Determination of Synthetic Volatile Substances on Gray Mold

[0079]

[0080] * indicates a solid.

[0081] Experimental Results: As shown in Table 6, the inhibition rate of volatile monomers against *Botrytis cinerea* gradually increased with increasing concentration. Among them, 4-methoxystyrene, cyclopentanol, and β-pinene exhibited good inhibitory effects on *Botrytis cinerea*. 4-methoxystyrene, at a concentration of 625 μL / L, completely inhibited the mycelial growth of *Botrytis cinerea*; cyclopentanol and diphenyl ether, at a concentration of 1250 μL / L, completely inhibited the mycelial growth of *Botrytis cinerea*; while β-pinene, at a concentration of 1250 μL / L, showed an inhibition rate of 71.4% against *Botrytis cinerea*.

[0082] Example 4: Evaluation of the effect of Streptomyces Norilsk on the production of volatile substances

[0083] (I) Effects on the mycelial morphology of gray mold

[0084] Using the double-plate confrontation method, 6g of wheat grain medium containing *Streptomyces northerneri* NK27 cultured for 10 days was weighed and placed at the bottom of one dish. PDA medium was poured into the bottom of another dish. After cooling, sterilized cellophane was spread evenly on the PDA medium. 200μL of *Botrytis cinerea* spore suspension (10...) was then added. 6 Spores (per mL) were evenly spread on cellophane, with an appropriate amount of sterile wheat grain culture medium added as a control. The apparatus was sealed with sealing film. The apparatus was incubated at 25°C for 3 days, and the hyphal morphology was observed using an optical microscope.

[0085] Experimental results: According to Figure 5 It can be seen that the control group has a large amount of new mycelium, and the normal gray mold has even and straight segments, while the gray mold mycelium that has been affected by volatile substances has an abnormally distorted shape and short segments.

[0086] (II) Effects on the germination of gray mold spores

[0087] Using the double-plate confrontation method, 6g of wheat grain medium containing *Streptomyces northerneri* NK27 cultured for 10 days was weighed and placed at the bottom of one dish. PDA medium was poured into the bottom of another dish. After cooling, sterilized cellophane was spread evenly on the PDA medium. 200μL of *Botrytis cinerea* spore suspension (10...) was then added. 6 Spores (per mL) were evenly spread on cellophane, with an appropriate amount of sterile wheat grain culture medium added as a control. The apparatus was sealed with sealing film. After incubation at 22°C for 12 hours, spore germination was observed using an optical microscope.

[0088] Experimental results: Figure 6It can be seen that in the control group, all spores germinated after 12 hours, and slender new buds and hyphae could be clearly observed. However, the spores treated with volatile substances had a small amount of new fungi and their germination was inhibited.

[0089] (III) Effects on the ultrastructure of gray mold hyphae

[0090] Sample preparation: The double-plate confrontation method was used. The lower plate contained 6g of Streptomyces NK27 grain culture that had been cultured for 10 days, and the upper plate contained a 5mm mycelial cake of Botrytis cinerea in the center. A grain culture medium with an equal amount of uninoculated Streptomyces seed liquid was used as a blank control. The plates were sealed with sealing film and placed in a constant temperature incubator at 25℃ for 3 days of confrontation culture.

[0091] Scanning electron microscopy (SEM): Edge hyphae were picked and immersed in 4% glutaraldehyde (pH 7.2) and fixed at 4°C in the dark for 16 h. They were then washed three times with PBS buffer (pH 6.8), each time for 20 min. After washing, they were dehydrated sequentially with 30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol, each for 30 min, with the 100% ethanol solution being used three times. After dehydration, the samples were immersed in hexamethyldisilazane (HMDS) in a fume hood for a displacement reaction, repeated twice for 20 min each time. After the displacement reaction, the samples were dried in a vacuum freeze dryer, and finally gold-coated in a vacuum sputtering system. The samples were then observed using a Hitachi SU8000 SEM.

[0092] Transmission electron microscopy: After dehydration, the samples were replaced with alcohol and acetone, and the replacement reaction was repeated twice, each time for 20 min. After replacement, gradient permeation with embedding agent was performed, and the samples were treated with a resin (812#) acetone mixture (1:1, 1:2) for 1 h and 3 h respectively, followed by overnight embedding with pure resin (812#). After permeation, the samples were heated to 70°C overnight in a heating polymerizer. Ultrathin sections were prepared using a Leica M80 stereomicroscope. The sections were first stained with uranyl acetate for 30 min, rinsed 5 times with ddH2O, then stained with lead citrate for 10 min, rinsed 5 times with ddH2O, and finally observed using a Hitachi H-7500 transmission electron microscope.

[0093] Experimental results: According to Figure 7Scanning electron microscopy revealed that the normal gray mold mycelia in the control group exhibited intact morphology and structure, showing good growth, with plump and rounded mycelia. In contrast, the gray mold mycelia treated with volatile substances showed inhibited growth, with twisted and deformed mycelia. Transmission electron microscopy showed that the protoplasm inside the control group mycelial cells was dense and uniform, with consistent cell wall thickness and no extracellular exudate. In contrast, the mycelial cells treated with volatile substances showed endoplasmic reticulum separation, disordered organelles, irregular cell morphology, irregular thickening of the cell wall, and a significant increase in cavities, with some cells being almost entirely cavitary. Deeply stained substances were observed outside the mycelial cells, possibly due to disruption of cell membrane permeability, leading to the deposition of a large amount of unidentified exudate around the cell wall.

[0094] (iv) Determination of the control effect of volatile substances on detached leaves of grape gray mold

[0095] Select a petri dish with a diameter of 15cm, a height of 3cm, and a volume of 0.5L. Line the dish with filter paper and add a small amount of sterile water to keep it moist.

[0096] Grape leaf treatment: Select grape leaves of uniform size, rinse them with sterile water, place them in a petri dish and cover the petiole with cotton to keep it moist. Use a sterile syringe needle to make a wound on the detached grape leaf and inoculate a 5mm diameter mass of gray mold fungus on the wound.

[0097] 20g, 40g, and 60g of Streptomyces NK27 culture medium, which had been cultured for 10 days, were weighed and placed in 70mm petri dishes. Grape leaves and petri dishes containing Streptomyces grain culture were placed in 15cm diameter petri dishes, moistened with a small amount of sterile water using filter paper, and sealed with sealing film. 20g of sterile grain culture medium was added as a control. Each treatment was repeated 3 times. The large petri dishes were placed in a 25℃ incubator. After 4 days, the diameter of the lesions was measured using the cross-sectional method.

[0098] Control efficacy = (diameter of control lesions - diameter of treated lesions) / diameter of control lesions × 100%

[0099] Experimental results: According to Figure 8 It can be seen that the volatile substances produced by Streptomyces NK27 of Knowles have a good control effect on gray mold on detached leaves, and the control effect of the volatile substances on gray mold on detached grape leaves is positively correlated with the amount of wheat grain culture of strain NK27. After 4 days, the diameter of lesions in the control group was 31.0 mm. As the amount of wheat grain culture of strain NK27 increased, the diameter of lesions gradually decreased. When the amount of wheat grain culture of strain NK27 was 20 g, the diameter of lesions was 11.1 mm; when the amount of wheat grain culture of Streptomyces NK27 was 40 g and 60 g, the diameter of lesions was only 6.8 mm and 5.8 mm, respectively.

[0100] (V) Determination of the control effect of volatile substances on detached grape fruits against gray mold

[0101] Choose a food storage container that is 15cm long, 9cm wide, and 8cm high, with a volume of 1.08L. Place a shelf that is 12cm long and 6cm high inside the food storage container. Moisturize the bottom of the food storage container with a small amount of sterile water and degreased cotton beforehand to maintain the humidity inside the container.

[0102] Grape Fruit Treatment: The "Fujiminori" grape variety was selected for the experiment. Grapes of uniform size, good shape, and free from any wounds or lesions were chosen. After soaking in 2% sodium hypochlorite for 5 minutes, the grapes were rinsed three times with sterile water. Using a sterile syringe needle, three evenly spaced wounds (approximately 3 mm deep) were made at the bottom of each grape fruit. 10 μL of a suspension of botrytis cinerea spores was then inoculated into each wound. 6 (Spores / mL), place on a shelf. Immediately take 20g of wheat grain medium of Streptomyces NK27 cultured for 10 days and place it in a 70mm Petri dish. Place the Petri dish at the bottom of the shelf, cover with the lid of the food storage box, and seal with sealing film to allow the volatile substances to act on the grape berries inoculated with gray mold. Add an appropriate amount of uninoculated wheat grain medium as a control. Each treatment is repeated 3 times. Place the food storage box in a 25℃ incubator. Observe the disease situation after 7 days, calculate the disease index according to the disease grading standard, and calculate the control effect.

[0103] Disease index = Σ(number of fruits with disease at each level × corresponding level) / highest disease level × total number of surveys × 100;

[0104] Prevention and control effect (%) = Disease index of control group - Disease index of treatment group / Disease index of control group × 100.

[0105] Table 7. Determination of the control efficacy of volatile substances produced by Streptomyces Norr's NK27 against gray mold on grapes.

[0106]

[0107] Experimental results: According to Table 7, Figure 9 It can be seen that in the blank control group, mycelium grew vigorously around the pores, filled the pores and spread outwards, and the surface of the grapes around the colonies began to rot and a distinct fermentation smell could be detected; while in the volatile matter treatment group, only a small amount of mycelium grew, and there was no rot on the surface of the fruit or a fermentation smell.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of volatile substances produced by a strain of *Streptomyces NK27* in the control of plant diseases, characterized in that... The accession number of the Streptomyces NK27 from Norilsk is CGMCC No. 28268; the volatile substances include β-pinene, 4-methoxystyrene, cyclopentanol and p-phenylenediamine; The plant disease mentioned is gray mold. The method for producing volatile substances by the aforementioned Streptomyces NK27 is to inoculate the Streptomyces NK27 in wheat grain medium and culture it for 10 days.

2. The application according to claim 1, characterized in that, The culture conditions were as follows: cultured at 28 ℃.

3. The application according to claim 1, characterized in that, The wheat grain culture medium is prepared by soaking wheat grains and boiling them until the skin cracks, filtering and drying them, and then adding water to the dried wheat grains.

4. The use of the volatile substance as described in claim 1 in the preparation of an agent for controlling gray mold in plants.

5. A preparation for controlling gray mold in plants, characterized in that, The formulation comprises the volatile substance described in claim 1.

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