Application of volatile substances produced by Bacillus velez K01 in plant disease control

Abstract

The present invention relates to the field of biological control technology, and provides the application of volatile substances produced by Bacillus Velez K01 in plant disease control. The present invention provides the synthesis time law and type identification of volatile compounds of Bacillus Velez K01, the ratio optimization of differential antibacterial compound combinations and their control effects on tomato gray mold. The present invention explores the synthesis time law of antibacterial VOCs produced by strain K01, and explores the number and type of VOCs within a critical time by combining a solid phase microextraction-gas chromatography-mass spectrometry method; focuses on optimizing the ratio of the antibacterial compound combination and further clarifies the control effects of these volatile antibacterial substances on tomato gray mold on living plants.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, in particular to application of volatile substances produced by Bacillus Velez K01 in plant disease control. Background Art

[0002] Plant Growth Promoting Rhizobacteria (PGPR) refers to the general term for bacteria that live freely in the soil, root surface and rhizosphere and are beneficial to plant growth under certain conditions (Sun et al., 2023). In agricultural ecosystems, PGPR strains can directly or indirectly affect pathogens or host plants by secreting secondary metabolites. These secondary metabolites, such as volatiles, play an important role in rhizosphere biological communication and interaction (Sharifi et al., 2022). Volatile organic compounds (VOCs) are generally defined as small molecular compounds (<300Da) with low boiling points and high vapor pressures, and contain lipophilic parts (Lemfacket et al., 2014). These volatile substances can act on a wide range of target organisms, including plants and their pathogens, and mediate different intra- and interspecific interactions above and below ground (Vlot and Rosenkranz, 2022). Since the first study on bacterial volatile compounds promoting plant growth was published in 2003, bacteria have been found to produce more than 2,000 VOCs and non-organic compounds (Lemfack et al., 2018, Lemfack et al., 2014). The chemical structures of microbial VOCs are very diverse, including relatively small molecular weight aliphatic and aromatic compounds and larger molecular weight ketones, alkanes and olefins (Almeida et al., 2023). The low molecular weight and polar properties of volatile organic compounds produced by bacteria can easily diffuse through soil pores over long atmospheric distances, greatly promoting the application potential of bacterial VOCs (Zhao et al., 2022).

[0003] ZL202110114861.2 discloses that Bacillus Velez K01 can produce volatile substances to antagonize a variety of pathogenic fungi, but the synthesis rules and species identification of these volatile antibacterial substances have not been explored. In addition, the ability of volatile substances produced by K01 to prevent and control gray mold on tomato leaves also needs further exploration. At present, the preventive and therapeutic effects of microbial volatile metabolites on plant diseases are usually experimented with two-grid culture dishes. The disadvantage of this culture method is that the space of the two-grid culture dish is limited, and only the preventive and therapeutic effects of microbial volatile metabolites on detached leaves can be studied, and the role of volatile substances in the face of pathogens infecting living plants cannot be fully demonstrated. At present, there is no special experimental device in the field of studying the effects of microbial volatile metabolites on plants, and a potted experimental detection device is required for practical application. Therefore, designing a device for the direct effect of microbial volatile substances on plants has become an urgent problem to be solved. In addition, most of the research on the prevention and control of gray mold by VOCs of Bacillus Velezii has focused on the more superficial aspects such as the ability of a single compound to reduce the incidence or inhibit the growth of Botrytis cinerea, while there has been less research on the interactions between volatile compounds. The research on the regulatory mechanism of these volatile substances as signal substances between pathogens and host plants is also not in-depth enough. Therefore, it is necessary to conduct detailed and in-depth research on the above issues. Summary of the invention

[0004] The purpose of the present invention is to provide application of volatile substances produced by Bacillus Velez K01 in plant disease prevention and treatment.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides any of the following uses of volatile substances produced by Bacillus velezensis K01:

[0006] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0007] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0008] The Bacillus velezensis K01 of the present invention is classified and named Bacillus velezensis K01, and has been deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC NO: M2020871, deposit date December 8, 2020. The volatile substances produced by Bacillus velezensis K01 include 2-heptanone, 2-nonanol, 2-decanone and 2-undecanol.

[0009] In a second aspect, the present invention provides any of the following applications of 2-heptanone:

[0010] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0011] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0012] In a third aspect, the present invention provides any of the following applications of 2-nonanol:

[0013] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0014] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0015] In a fourth aspect, the present invention provides any of the following applications of 2-decanone:

[0016] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0017] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0018] In a fifth aspect, the present invention provides any of the following applications of 2-undecanol:

[0019] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0020] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0021] Furthermore, the pathogen includes but is not limited to Botrytis cinerea, and the plant disease is but not limited to tomato gray mold.

[0022] In a sixth aspect, the present invention provides a composition consisting of two or more of 2-heptanone, 2-nonanol, 2-decanone and 2-undecanol.

[0023] In a seventh aspect, the present invention provides a composition consisting of two or three of 2-heptanone, 2-nonanol and 2-decanone.

[0024] Further, the composition is composed of 2-heptanone and 2-nonanol. The volume ratio of 2-heptanone to 2-nonanol is 1:7 to 7:1, preferably 1:7, 1:3, 3:5, 1:1, 5:3, 3:1 and 7:1, and more preferably 3:5.

[0025] In an eighth aspect, the present invention provides any of the following applications of the composition:

[0026] a) Used to resist pathogens and related diseases or plant diseases caused by pathogens;

[0027] b) Used for the preparation of biocontrol agents, antimicrobial agents, preservatives or disinfectants.

[0028] Furthermore, the pathogen includes but is not limited to Botrytis cinerea, and the plant disease is but not limited to tomato gray mold.

[0029] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0030] The present invention has found out the synthesis time rule of volatile organic compounds (VOCs) of Bacillus velez K01: in the first 48h of culture, the VOCs synthesized by the K01 strain have no antibacterial ability, but the antibacterial ability is enhanced with the extension of culture time. After culture to 72h, the VOCs produced every 12h can not only completely inhibit the growth of Botrytis cinerea, but also effectively prevent and treat gray mold of tomato leaves. The VOCs produced by the K01 strain in the two time periods of 48-60h and 84-96h are identified by SPME-GC-MS method, and a total of 9 VOCs are identified, and the types of these VOCs are mainly ketones, alcohols and alkanes. By comparing the two time points, it is found that there are 5 VOCs with significant differences in VOCs synthesis, and their antibacterial ability is 2-nonanol, 2-decanone, 2-heptanone, 2-undecanol and acetoin in order.

[0031] The invention discloses an optimal antibacterial ratio of a combination of 2-heptanone and 2-nonanol: 2-heptanone and 2-nonanol are prepared according to the formula of 2-heptanone (EC 50 ) concentration was 211.1 μL / mL and 2-nonanol (EC 50 ) concentration was 84.3 μL / mL for compounding, and the compounded mixture EC 50 The concentrations of 2-heptanone and 2-nonanol in the samples were compared with the EC values ​​of single 2-heptanone and 2-nonanol. 50 The concentrations were reduced by 78.8% and 78.1% respectively.

[0032] The present invention also provides a device for detecting the control effect of microbial volatile substances on plant seedling diseases, and uses the device to clarify the control effect of volatile substances produced by Bacillus Velez K01 strain on tomato gray mold. The experiment separates the plant pot and the microbial culture dish by a breathable partition, which solves the problem of ensuring pure culture of microorganisms and facilitating observation of the effects of their volatile metabolites on plants, and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The inhibition ability of VOCs on Botrytis cinerea after 12 hours of culture in the preferred embodiment of the present invention. The upper part shows the expansion of Botrytis cinerea colonies; the lower part shows the inhibition rate of VOCs produced by strain K01 every 12 hours on Botrytis cinerea; different lowercase letters on the column represent significant differences between treatments (p<0.05).

[0034] Figure 2 The inhibition ability of VOCs cultured for 12 hours on gray mold of tomato detached leaves in the preferred embodiment of the present invention. Among them, (a) the incidence of gray mold of tomato detached leaves, (b) the incidence results of detached leaves, and (c) the control effect of VOCs produced by strain K01 every 12 hours on gray mold of tomato. Different lowercase letters on the column represent significant differences between treatments (p<0.05).

[0035] Figure 3 This is a design diagram of a plant-microorganism separation and coexistence system incubator in a preferred embodiment of the present invention.

[0036] Figure 4 This is a demonstration of the use of a plant-microorganism separation coexistence system incubator in a preferred embodiment of the present invention.

[0037] Figure 5 The inhibition ability of VOCs cultured for 12 hours on gray mold of tomato seedling leaves in the preferred embodiment of the present invention. Among them, (a) the incidence of gray mold on tomato seedling leaves, (b) the incidence results of seedling leaves, and (c) the control effect of VOCs produced by strain K01 every 12 hours on tomato gray mold. Different lowercase letters on the column represent significant differences between treatments (p<0.05).

[0038] Figure 6 The chromatographic results of VOCs of K01 strain at key time points in the preferred embodiment of the present invention. Among them, (a) 48-60h VOCs chromatogram, (a) 84-96h VOCs chromatogram, (a) peak area of ​​the compound at two time points, the difference in the peak area of ​​the compound at two time points was analyzed by t-test, and different lowercase letters on the column represent significant differences between the treatments (p<0.05), (d) chromatogram of the compound standard.

[0039] Figure 7 The results of the antibacterial ability verification of the differential compounds in the preferred embodiment of the present invention are shown in Figure 1. Different lowercase letters on the column represent significant differences between the treatments (p<0.05).

[0040] Figure 8The antibacterial concentration gradient of a single compound in a preferred embodiment of the present invention. Among them, (a) the antibacterial concentration gradient of 2-heptanone, (b) the curve fitting result of the logistic equation of 2-heptanone, (c) the antibacterial concentration gradient of 2-decanone, (d) the curve fitting result of the logistic equation of 2-decanone, (e) the antibacterial concentration gradient of 2-nonanol, (f) the curve fitting result of the logistic equation of 2-nonanol. Different lowercase letters on the column represent significant differences between the treatments (p<0.05).

[0041] Fig. 9 The results of screening for the best combination of differential compounds in the preferred embodiments of the present invention are shown in Figure 2. (a) The inhibition rate of five compound combinations on Botrytis cinerea, (b) The inhibition rate of four compound combinations on Botrytis cinerea, (c) The inhibition rate of three compound combinations on Botrytis cinerea, and (d) The inhibition rate of two compound combinations on Botrytis cinerea. Different lowercase letters on the columns represent significant differences between treatments (p<0.05).

[0042] Fig.10 The antibacterial concentration gradient of the H+N combination in the preferred embodiment of the present invention, wherein (a) the antibacterial concentration gradient of H+N, (b) the H+N logistic equation curve fitting result.

[0043] Fig.11 The optimal volume ratio of H+N combination in the preferred embodiment of the present invention. Different lowercase letters on the column represent significant differences between the treatments (p<0.05).

[0044] Fig.12 The antibacterial concentration gradient of the H+N combination (volume ratio 3:5) in the preferred embodiment of the present invention. Among them, (a) the antibacterial concentration gradient of H+N (3:5), (b) the logistic equation curve fitting result of H+N (3:5), (c) the EC of 2-heptanone and 2-nonanol 50 Different lowercase letters above the column indicate significant differences among treatments (p<0.05).

[0045] Fig.13 The control effect of H+N on tomato gray mold on detached leaves in the preferred embodiment of the present invention. Among them, (a) the incidence of tomato gray mold on detached leaves, (b) the incidence results of in vitro experiments, and (c) the control effect of H+N combination on tomato gray mold in in vitro experiments. Different lowercase letters on the column represent significant differences between the treatments (p<0.05).

[0046] Fig.14The control effect of H+N combination on gray mold on tomato seedling leaves in the preferred embodiment of the present invention. Among them, (a) the incidence of gray mold on tomato seedling leaves, (b) the incidence of seedling leaves in the in vivo experiment, and (c) the control effect on gray mold on seedling leaves in the in vivo experiment. Different lowercase letters on the column represent significant differences between the treatments (p<0.05).

[0047] Fig.15 The hyphae morphology of Botrytis cinerea observed under an optical microscope in a preferred embodiment of the present invention. Among them, (a) and (d) are the hyphae morphology of Botrytis cinerea after being treated with anhydrous ethanol (CK), (b) and (e) are the hyphae morphology of Botrytis cinerea after being treated with K01, and (c) and (f) are the hyphae morphology of Botrytis cinerea after being treated with H+N combination. Magnification: ×40 (ac); ×200 (df).

[0048] Fig.16 The hyphae morphology of Botrytis cinerea observed under a scanning electron microscope in a preferred embodiment of the present invention. Among them, (a) and (d) are the hyphae morphology of Botrytis cinerea after being treated with anhydrous ethanol (CK), (b) and (e) are the hyphae morphology of Botrytis cinerea after being treated with K01, and (c) and (f) are the hyphae morphology of Botrytis cinerea after being treated with H+N combination. Magnification: ×1000 (ac); ×5000 (df).

[0049] Fig.17 The cell structure of Botrytis cinerea observed under a transmission electron microscope in a preferred embodiment of the present invention. (a) and (b) are the cell structures of Botrytis cinerea after being treated with anhydrous ethanol (CK), (c) and (d) are the cell structures of Botrytis cinerea after being treated with K01, and (e) and (f) are the cell structures of Botrytis cinerea after being treated with H+N combination. Magnification: ×2000 (a, c, e); ×5000 (b, d, f). DETAILED DESCRIPTION

[0050] The present invention provides the synthesis time law and type identification of volatile compounds of Bacillus velez K01, the ratio optimization of differential antibacterial compound combinations and their control effect on tomato gray mold. The present invention explores the synthesis time law of antibacterial VOCs produced by strain K01, and explores the number and type of VOCs in the critical time by combining solid phase microextraction-gas chromatography-mass spectrometry; focuses on optimizing the ratio of the antibacterial compound combination and further clarifies the control effect of these volatile antibacterial substances on tomato gray mold on living plants.

[0051] The present invention also provides an incubator device for detecting the effects of volatile substances on the prevention and control of plant seedling diseases. The device is a sealing device for the direct impact of microbial volatile substances on plants. The plant and microbial culture are partitioned by partitions, which not only meets the requirement of maintaining humidity during plant cultivation, but also maintains the sterile environment required for microbial culture, and can ensure that the microbial volatile metabolites directly contact the plant leaves, making it easy to detect the effects of the microbial volatile substances on the plants.

[0052] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0053] The Bacillus Velez K01 used in the following examples was provided by Genliduo Biotechnology Co., Ltd.

[0054] The plant pathogen involved in the following examples is Botrytis cinerea B02, that is, Botrytis cinerea strain B02 provided by China Agricultural Culture Collection Center, with the collection number: ACCC 35467. This strain is available to the public and does not need to be preserved.

[0055] The tomato variety used in the following examples is 'Fenteli'.

[0056] Example 1 Antibacterial activity of VOCs produced by strain K01 after 12 h of culture

[0057] To investigate the antibacterial ability of VOCs produced by K01 during each 12-hour culture period, the culture period of K01 was divided into eight time periods: 0-12, 12-24, 24-36, 36-48, 48-60, 60-72, 72-84 and 84-96 h. 8 cfu / mL of bacterial solution was evenly applied to LB plates) and cultured separately for 0, 12, 24, 36, 48, 60, 72 and 84 hours. Then K01 and Botrytis cinerea (5 mm bacterial cake was inoculated in the center of PDA solid plates) were cultured side by side. Each treatment was co-cultured for only 12 hours before the K01 plate was removed, leaving the Botrytis cinerea plate alone for 72 hours. Five replicates were set for each time treatment, and a blank control (LB without bacteria and PDA inoculated with gray mold) was set. The experiments were repeated three times. The diameter of the pathogen in each treatment was measured by the cross method, and the antibacterial effect was calculated according to formulas (1) and (2).

[0058] Colony growth diameter (mm) = colony diameter - cake diameter formula (1)

[0059] Mycelium growth inhibition rate (%) = (Cd-Td) / (Cd×100%) Formula (2)

[0060] Wherein, d represents the colony growth diameter, Cd represents the colony growth diameter of the control group, Td represents the colony growth diameter of the treatment group, and the data are expressed as mean ± standard deviation (SD).

[0061] After dividing the culture time of K01 into 12h units, it was found that the antibacterial ability of VOCs produced by K01 strain was different within each 12h culture time ( Figure 1 ). In the first 48 hours, the VOCs produced by the K01 strain had a weak or even no inhibitory effect on Botrytis cinerea; after 48-60 hours of cultivation, the VOCs produced by the K01 strain began to have a significant inhibitory effect; after K01 was cultivated for 72 hours, the inhibition rate of VOCs produced every 12 hours on Botrytis cinerea could reach 100%.

[0062] Example 2 In vitro control experiment of VOCs produced by 12h culture on gray mold

[0063] According to the plate test method, K01 was cultured alone for 0, 12, 24, 36, 48, 60, 72 and 84 hours; tomato leaves grown to the six-leaf stage were selected, cleaned with sterile water and placed on a plate covered with moist filter paper. After the leaves were dried, Place the bacterial cake upside down on the leaf surface. Place the K01 plate and the tomato leaf plate together and seal them with a sealing film. After incubating at 22°C for 12 hours, remove the K01 plate and continue incubating the leaves for 36 hours. Spread sterile water on LB as a blank control. Set up 5 replicates for each treatment, with 3 leaves in each replicate. After the control is fully diseased, measure the lesion area. Repeat the experiment 3 times. Count the number of diseased leaves in each treatment, and take photos under bright field and ultraviolet light (365nm; Analytik Jena US, Upland, CA, USA), and use the software APS Assess (APS Press, USA) to measure the size of the lesion area. The incidence rate and control effect are calculated according to formula (3) and formula (4), respectively:

[0064] Incidence rate (%) = (Cr-Tr) / (Cr×100%) Formula (3)

[0065] Control effect (%) = (Ca-Ta) / (Ca×100%) Formula (4)

[0066] Among them, Cr represents the number of diseases in the control group, Tr represents the number of diseases in the treatment group, a represents the lesion area, Ca represents the lesion area in the control group, and Ta represents the lesion area in the treatment group; the data are expressed as mean ± standard deviation.

[0067] The results of the in vitro leaf control experiment further verified the results of the plate experiment ( Figure 2 ), the results showed that the VOCs produced by the K01 strain after 72 hours of culture reduced the incidence of gray mold on tomato leaves; in terms of lesion area and control effect, after 60 hours of culture, the VOCs produced by the K01 strain every 12 hours had a control effect of more than 90% on gray mold on tomato detached leaves. Different from the plate experiment results, compared with the control, the VOCs produced by the K01 strain every 12 hours in the first 60 hours did not reduce the incidence of tomato leaves ( Figure 2 b), but the lesion area decreases over time ( Figure 2 a), the control capacity increased (22.1-75.4%, Figure 2 c).

[0068] Example 3 In vivo control of gray mold by VOCs produced at critical time points

[0069] 3.1 Schematic diagram of the design of the incubator for the separation and coexistence of organisms and microorganisms

[0070] According to the experimental requirements, the design Figure 3 Sealable transparent incubators in the dimensions shown:

[0071] Net size inside the box: S1 = 37 (length: cm) × 35 (width: cm) × 40 (height: cm)

[0072] Net size of partition: S2 = 33 (width: cm) × 40 (height: cm)

[0073] Requirements for punching holes in the partition: hole diameter 0.5-0.7cm, 10 holes per row, 8 rows evenly distributed

[0074] Net size of compartment bottom: S3 = 33 (length: cm) × 12 (width: cm)

[0075] Net internal dimensions of the hemmed side cover: S4 = 37 (width: cm) × 40 (length: cm)

[0076] 3.2 Instructions for use of plant-microorganism separation coexistence system incubator

[0077] When using, the incubator should be sterilized as a whole. The perforated partition divides the inside of the culture device into two independent sterile areas. The sterile area with a transparent partition on one side is the microbial culture area, where microbial culture dishes (without lids) can be placed; the sterile area without a partition on the other side can be used as a plant culture area. After placing the experimental samples, the incubator can be strictly sealed (the sealing can be done by applying glue, vaseline, or multi-layer sealing). The specific usage method is as follows: Figure 4 shown.

[0078] 3.3 In vivo experiments

[0079] According to the experimental results of detached leaves, we selected the VOCs produced by K01 at two time points of 60-72h and 72-84h for the prevention and control experiment of gray mold on tomato seedling leaves. According to the method of Chen et al. (Chen et al., 2022), the control effect of VOCs produced by K01 on gray mold on tomato seedling leaves was determined. The specific operation was as follows: K01 was cultured for 60h and 72h in advance for use; tomato seedlings grown to six compound leaves were selected, and the tomato leaves were washed three times with sterile water, and then 3mm Botrytis cinerea cakes were inoculated on the leaves. Three seedlings were treated as biological replicates, and 10 leaflets were inoculated for each plant. The tomato plants inoculated with gray mold were placed in a sealed incubator, and 18 uncovered K01 plates were placed on the other side of the partition. After strict sealing (keeping humidity>90%), the incubator was placed in a 22°C cultivation room for 12 hours, the sealed box was opened, all K01 plates were removed, and ventilation was maintained until no odor remained (about 30 minutes), and the box was resealed and continued to be cultivated for 48 hours. The number of diseased leaves of each tomato plant was counted, and the incidence rate was calculated according to Formula 3; the diseased area of ​​each tomato leaf was counted, and the protective effect of volatile substances produced by K01 on gray mold of tomato seedling leaves in different time periods was calculated according to Formula 4, and the data were expressed as mean ± standard deviation.

[0080] The results of in vivo experiments are as follows Figure 5 As shown: After fumigating seedlings with K01 cultured for 60 and 70 hours for 12 hours, the incidence of gray mold on tomato seedling leaves was significantly reduced (42.2%-53.3%), but there was no significant difference between the two treatments; the lesion area of ​​tomato leaves in the 72-84h treatment group was smaller than that in the 60-72h treatment group, and the control rate reached 76.4%, which was significantly higher than that in the 60-72h treatment group (62.8%). The control experiment of gray mold on tomato leaves further confirmed that the VOCs with strong inhibitory effects produced by K01 were produced after 60 hours of cultivation.

[0081] Example 4 Determination of volatile substances produced by K01 strain

[0082] According to the results of previous experiments, the antibacterial rate and control ability of VOCs of K01 strain in the two time periods of 48-60h, 60-72h and 72-84h, 84-96h showed significant differences. Therefore, it is speculated that the difference in the type or content of VOCs in these two time periods caused the difference in antibacterial activity. In order to avoid the experimental error caused by the close culture time affecting the test results, we selected the volatile components of K01 strain cultured for 48-60h and 84-96h for mass spectrometry identification. Volatile gases were identified by combining solid phase microextraction (SPME) with gas chromatography and mass spectrometry (GC-MS) (Kong et al., 2020).

[0083] 4.1 Collection of volatile substances

[0084] Take 3.5 mL of LB medium at about 60°C and add it to a 20 mL headspace bottle. Seal it with a cotton plug and sterilize it to prepare the LB slant. Soak the cap of the vial in alcohol for disinfection and dry it. Take 20 μL of K01 bacterial solution (concentration 10 8 cfu / mL) were evenly spread on the slant, and then incubated at 22°C for 48h and 84h after drying. The bottle was opened and kept for 20min, and then resealed and incubated for 12h before the VOCs captured in the headspace bottle were analyzed. The blank control was set as the headspace bottle with only the LB slant without bacteria, and three replicates were set for each treatment.

[0085] 4.2 Extraction of volatile substances

[0086] VOCs were enriched using PDMS / DVB (65 μm) solid phase microextraction fiber (Supelco, PA, USA). Before injection, the SPME extraction head was aged at 250°C for 20 min, the sample was balanced at 40°C for 15 min, the fiber head was inserted into the middle of the headspace extraction bottle for adsorption for 30 min, the fiber head was retracted, and the extraction head was immediately inserted into the injection port of the GC chromatograph. After desorption at 250°C for 5 min (without split mode), GC-MS analysis was performed. Adsorption conditions: puncture depth 30 mm, puncture speed 20 mm / s; injection conditions: puncture depth 30 mm, puncture speed 20 mm / s.

[0087] 4.3 Detection of volatile substances

[0088] The gas chromatography-mass spectrometer model was Agilent 7890A / 5975C, and the capillary column HP-5MS (30.0m×0.25mm; 0.25μm) was used to separate VOCs. The injection port temperature was 250℃, the carrier gas was helium, the column flow rate was 1.2mL / min, the injection volume was 1μL, and the injection was splitless. The heating program was: 40℃ for 3min, then heated to 200℃ at a rate of 3℃ / min and held for 2min, then heated to 220℃ at a rate of 8℃ / min and held for 3min.

[0089] 4.4 Mass spectrometry conditions

[0090] The ion source temperature was 230°C, the quadrupole temperature was 150°C, and the transfer line temperature was 230°C; the electron impact EI source, the electron energy was 70 eV, and the full scan mass spectrum range (m / z) was: 30-500u (atomic mass unit). After deducting the peaks detected in the control group, the compounds with significant differences in peak area in the chromatograms at the two time points were found, and the mass spectra of these unknown compounds were automatically searched with the sample mass spectra in the NIST 17.0 spectral library (National Institute of Standards and technology, Gaithersburg, MD, USA), and then the antibacterial ability of the individual compounds was verified.

[0091] 4.5 Verification of compound characteristic peaks

[0092] Standards of 9 compounds, including acetoin, 2-heptanone, 2-nonanone, 2-nonanol, 2-decanone, 2-undecanone, 2-undecanol, 2-dodecanone and 2-tridecanone, were purchased from Sigma. Four treatments were set up: ① 1 μL single standard, ② 1 μL single standard + K01 cultured for 84 h, ③ 1 μL of all standards, ④ 1 μL of all standards + K01 cultured for 84 h. The characteristic peaks of the compounds were verified according to the methods described in 4.2-4.4, and the concentrations of the compounds were quantitatively calculated based on the peak areas. The data were expressed as mean ± standard deviation.

[0093] The results of SPME-GC-MS analysis showed that ( Figure 6 ), we detected and identified a total of 9 volatile organic compounds in two time periods: acetoin, 2-heptanone, 2-nonanone, 2-nonanol, 2-decanone, 2-undecanone, 2-undecanol, 2-dodecanone, and 2-tridecanone; these compounds can be mainly divided into 7 ketones and 2 alcohols ( Figure 6 , a and b); except for acetoin which was only detected in 84-96h, the other 8 compounds were detected in both time periods ( Figure 6The positions of the characteristic peaks of the standard samples of compounds a and b) in the chromatograms further confirmed the existence of these compounds ( Figure 6 d). By comparing the peak areas of 9 compounds, it was found that the peak areas of 5 compounds, including acetoin, 2-heptanone, 2-nonanol, 2-decanone and 2-undecanol, had significant differences ( Figure 6 c).

[0094] Example 5 Detection of antibacterial ability of a single VOC

[0095] The antibacterial ability test of volatile substances was carried out according to the method of Che et al. (Che et al., 2017). The specific operation is as follows: Botrytis cinerea was inoculated in the center of the PDA plate, a sterilized filter paper with a side diameter of 1 cm was placed in the center of the LB plate, 20 μL of standard product (standard product concentration) was added to the filter paper, and a blank control (LB plate) and a positive control (20 μL of anhydrous ethanol) were set up at the same time. After culturing at a constant temperature of 22°C for 12 hours, the LB plate was removed, and the Botrytis cinerea plate was left alone for 72 hours. Five replicates were set for each time treatment, and the experiment was repeated three times. The diameter of the pathogen in each treatment was measured by the cross method, and the antibacterial effect was calculated according to formulas 1 and 2. The data are expressed as mean ± standard deviation.

[0096] The results showed that, except for acetoin, 2-nonanol, 2-decanone, 2-heptanone and 2-undecanol could inhibit the growth of Botrytis cinerea, but the strength of the inhibition ability was significantly different ( Figure 7 a). 2-nonanol and 2-decanone have the highest antibacterial activity, followed by 2-heptanone; while 2-undecanol and acetoin have the weakest antibacterial activity ( Figure 7 b). Compared with the control, anhydrous ethanol had no inhibitory effect on Botrytis cinerea ( Figure 7 a), Anhydrous ethanol was used as solvent to dilute the compounds in subsequent experiments.

[0097] Example 6 Antibacterial concentration gradient of a single compound

[0098] After testing the antibacterial ability of five differential compound standards, we found that only the three pure products of 2-heptanone, 2-nonanol and 2-decanone had an antibacterial ability of more than 50% after only 12 hours of treatment with the bacteria. Therefore, the three compounds were diluted in a gradient to test the antibacterial rate of different concentrations. The 2-heptanone, 2-nonanol and 2-decanone standards were diluted with anhydrous ethanol to the following concentrations: 25, 50, 100, 250, 500, 650, 750, 850 and 1000 μL / mL. Subsequent experimental operations were carried out according to the method described for the antibacterial ability test of a single VOC. In addition, a logistic equation was established to fit the relationship between the inhibition rate (y) and the concentration (x) of a single compound, and the data were expressed as mean ± standard deviation (SD).

[0099] The results are as follows Figure 8 As shown: The logistic equation curve was fitted according to the inhibition rate (y) and concentration (x) of the three compounds at different concentrations ( Figure 8 df), and calculated the EC of 2-heptanone, 2-nonanol, and 2-decanone 50 The EC values ​​of 2-heptanone, 2-nonanol and 2-decanone were 952.8, 385.0 and 715.7 μL / mL respectively. 20 They are 536.1, 128.5 and 181.8 μL / mL respectively.

[0100] Example 7 Screening of the optimal combination of differential antibacterial compounds

[0101] In the subsequent compound screening, the antibacterial ability of the mixture of five compounds must be tested first. In order to avoid the antibacterial ability of the mixture exceeding 100% interfering with the experimental results, we chose to use EC 20 The concentration of 2-heptanone (EC 20 )=536.1μL / mL, 2-nonanol (EC 20 )=128.5μL / mL, 2-decanone (EC 20 )=181.8μL / mL.

[0102] Antibacterial experiment of five different compound mixtures: 20 μL of acetoin and 2-undecanol were taken, and 20 μL of each was taken in EC 202-Heptanone (536.1 μL / mL), 2-nonanol (128.5 μL / mL), and 2-decanone (181.8 μL / mL) at the concentrations were mixed, and 100 μL of the mixture was added to the sterile filter paper in the center of the LB plate. The mixture plate was tightly sealed with the gray mold plate, and cultured at 22°C for 12 hours. The mixture plate was removed and ventilated in the clean bench for 30 minutes. The gray mold plate was covered and continued to be cultured under the same conditions for 72 hours. The experiment was set up with only 100 μL of anhydrous ethanol as a blank control (CK), and 20 μL of a single compound + 80 μL of anhydrous ethanol was set as a single compound reference group. The determination method and calculation formula are the same as those in Experiment 5, and the data are expressed as mean ± standard deviation (SD).

[0103] Five-choice-four screening experiment: According to the antibacterial experimental results of five different compound mixtures, the concentrations of the control and single compounds were kept unchanged, and the five target compounds were randomly combined as shown in Table 1; the five groups of mixtures were subjected to double-plate buckle experiments with Botrytis cinerea, and the group with the strongest antibacterial ability was selected as the screening basis for subsequent experiments.

[0104] Table 1 Random combination scheme of four compounds (unit: μL)

[0105]

[0106] Four-choice-three screening experiment: According to the results of the five-choice-four screening experiment, the four screened compounds were randomly combined as shown in Table 2; the above method was repeated to perform a double-plate buckle experiment, and the group with the strongest antibacterial ability was selected as the screening basis for subsequent experiments.

[0107] Table 2 Random combination scheme of three compounds (unit: μL)

[0108]

[0109] Three-choice-two screening experiment: According to the results of the four-choice-three screening experiment, the three screened compounds were randomly combined as shown in Table 3: The double-plate buckle experiment was repeated, and the group with the strongest antibacterial ability was selected as the basis for subsequent experiments.

[0110] Table 3 Random combination scheme of two compounds (unit: μL)

[0111]

[0112] The results of compound combination screening experiments showed that compared with CK (anhydrous ethanol), the antibacterial rate of the mixture of 2-heptanone (H), 2-nonanol (N), 2-decanone (D), acetoin (A) and 2-undecanol (U) was only about 55% ( Fig. 9a) It is 44% lower than the expected value (Excepted) of the five compounds. The five compounds interact with each other, but the expected synergistic effect is not observed. We speculate that one or several compounds may interfere with the action of the main antibacterial substances.

[0113] The five compounds were randomly combined into four combinations, and we found that no combination of 2-undecanol had an antibacterial rate of 100%, and the antibacterial rate was higher than the expected value of a mixture of 2-heptanone, 2-nonanol, 2-decanone and acetoin ( Fig. 9 b), the synergistic effect was 25.7%, which indicated that the interaction of this group of compounds showed an additive effect; the actual inhibition rates of the other four combinations with 2-undecanol were all lower than the expected values, and the interactions of these four groups showed a negative effect, with a negative effect of 22.6%-44.6%, indicating that 2-undecanol was an interfering substance.

[0114] After randomly combining the four compounds that showed synergistic effects in three combinations, we found that the combination without acetoin had an inhibition rate of 100%, which was significantly higher than the other combinations containing acetoin, and was 26.4% higher than the expected inhibition rate of the mixture of 2-heptanone, 2-nonanol and 2-decanone ( Fig. 9 c), which indicates that there is an additive effect between 2-heptanone, 2-nonanol and 2-decanone. In addition, in the combination of acetoin, 2-nonanol and 2-decanone, the inhibition rate also showed an enhancement (20.6%), but the enhancement of this group was lower than that of the combination of 2-heptanone, 2-nonanol and 2-decanone, and the actual inhibition rate was also significantly lower than that of the combination of 2-heptanone, 2-nonanol and 2-decanone. Negative effects (6.4%-14.3%) appeared between the other two groups of compounds, and acetoin was an interfering substance in these two combinations.

[0115] According to the results of the three-three combination, 2-heptanone, 2-nonanol and 2-decanone were combined in pairs. The results showed that only the inhibition rate of 2-heptanone and 2-nonanol against Botrytis cinerea reached 100%, which was significantly higher than the inhibition rates of the other two groups and also much higher than the expected inhibition rate. This shows that there is a stronger synergistic effect between 2-heptanone and 2-nonanol than before, with an enhancement rate of up to 49.8% ( Fig. 9 d). At the same time, the inhibition rate of the combination of 2-nonanol and 2-decanone increased by 13.5% compared with the predicted value, but the inhibition rate of the combination of 2-heptanone and 2-decanone decreased by 9.1% compared with the predicted value. According to the results of the compound combination screening test, we concluded that there is a strong synergistic antibacterial effect between 2-heptanone and 2-nonanol. When containing 20 μL of 2-heptanone with a concentration of 536.1 μL / mL and 20 μL of 2-nonanol with a concentration of 128.5 μL / mL, the inhibition rate of this group of mixtures against Botrytis cinerea reached 100%.

[0116] Example 8 Antibacterial concentration gradient of 2-heptanone + 2-nonanol combination

[0117] A mixture containing 20 μL 2-heptanone (536.1 μL / mL) and 20 μL 2-nonanol (128.5 μL / mL) was used as the mother solution, and was diluted in a gradient of 10% decreasing in turn, from 100% to 10% to obtain mixed solutions of different concentrations for use; 40 μL of the mixed solution + 60 μL of anhydrous ethanol was added to each LB plate, and the rest of the operations remained the same. The inhibition rate was determined and a logistic equation was established to fit the relationship between the inhibition rate (y) and the mixture concentration (x), and the data were expressed as mean ± standard deviation (SD).

[0118] We diluted H+N in a gradient of 10% decrease in concentration and tested the inhibitory rate of H+N against Botrytis cinerea at different concentrations ( Fig.10 a). The logistic equation curve was fitted based on the antibacterial rate (y) and the concentration percentage of H+N (x). Fig.10 b) and calculated the EC50 of H+N to be 59.6%. (EC50) and 2-nonanol (EC50) The concentrations were 319.5 and 76.6 μL / mL, respectively.

[0119] Example 9 Screening of the Optimal Ratio of 2-Heptanone + 2-Nonanol Combination

[0120] The method described in Example 8 was adjusted to carry out subsequent optimal ratio screening experiments. The specific modified operation was as follows: According to the experimental results of Example 8, the fitted H+N (EC50) is 59.6%, that is, H+N (EC50) The total volume of the H+N mixture in the solution is 23.8 μL, which contains 11.8 μL of 2-heptanone and 11.8 μL of 2-nonanol. H :V N The H+N mixed solutions with different volume ratios were reconfigured to 1:7, 1:3, 3:5, 1:1, 5:3, 3:1 and 7:1; 23.8 μL of H+N mixed solution and 76.2 μL of anhydrous ethanol were added to each LB plate, and the other operations remained the same. The data are expressed as mean ± standard deviation (SD).

[0121] The experimental results of the optimal volume ratio show that when V (2-庚酮) :V (2-壬醇) When the ratio was adjusted from 1:1 to 3:5, the inhibition rate of H+N combination on Botrytis cinerea increased by 7% ( Fig.11 ), it can be concluded that the optimal volume ratio of H+N should be V (2-庚酮) :V (2-壬醇)=3:5. Converted to 2-heptanone (EC50) and 2-nonanol (EC50) The concentrations were 239.6 and 95.8 μL / mL, respectively.

[0122] Example 10 Antibacterial concentration gradient of the optimal ratio of 2-heptanone and 2-nonanol

[0123] According to the results of Example 9, the optimal volume ratio of the 2-heptanone + 2-nonanol combination is H:N = 3:5. After adjusting the concentration of the mixed solution of 2-heptanone (536.1 μL / mL) + 2-nonanol (128.5 μL / mL) according to the above ratio, a mixed solution with concentrations of 2-heptanone (402.1 μL / mL) and 2-nonanol (160.6 μL / mL) was obtained as a mother solution. The method described in Experiment 8 was repeated, and the latest logistic equation of the H+N combination was fitted, and the data were expressed as mean ± standard deviation (SD).

[0124] The latest H+N combination was diluted in a gradient of 10% decrease in concentration, and the inhibition rate of H+N against Botrytis cinerea at different concentrations was tested. Fig.12 a). The new logistic equation curve was fitted based on the antibacterial rate (y) and the concentration percentage of H+N (x). Fig.12 b) and calculate the EC of the new H+N 50 is 52.5%. Converted to 2-heptanone (EC 50 ) and 2-nonanol (EC 50 ) were 211.1 and 84.3 μL / mL, respectively. Based on all the experimental results, we calculated the optimal combination of 2-heptanone (EC 50 ) and 2-nonanol (EC 50 ) concentrations decreased by 78.8% and 78.1% respectively compared with the initial concentrations ( Fig.12 c).

[0125] Example 11 Experiment on the control of gray mold on tomato leaves by the optimal ratio combination of 2-heptanone + 2-nonanol

[0126] Referring to the concentration setting of the plate experiment, the latest H+N combination was diluted in a gradient of 10% decreasing in concentration, and the control effect of H+N of different concentrations on gray mold of tomato detached leaves was tested. H+N mixed solutions of different concentrations were prepared according to the method described in Example 10, and LB plates containing mixed solutions of different concentrations were placed on tomato detached leaf plates for culture according to the method described in Example 2. The incidence rate and control effect were calculated according to formulas (3) and (4), and the data were expressed as mean ± standard deviation (SD).

[0127] The experimental results show that the protective effect of H+N mixture on tomato leaf gray mold is enhanced with the increase of concentration within a certain dilution concentration range ( Fig.13 ), specifically: the incidence rate decreased with increasing concentration, while the expansion area of ​​water-soaked lesions in diseased leaves showed the opposite trend. When the content of H+N mixed solution was greater than 50%, the detached tomato leaves no longer became diseased, and it was speculated that the dosage of the drug may have reached a saturation value when the content was 50%.

[0128] Example 12 Experiment on the control of gray mold on tomato seedling leaves by the optimal ratio combination of 2-heptanone + 2-nonanol

[0129] According to the calculation, the volume of the sealed box is about 350 times that of the double-layer plate. The H+N mixed solution is diluted according to the following gradient: 0.05%, 0.1%, 0.625%, 1.25%, 2.5%, 5% and 10%; the amount of H+N mixed solution is converted to: 3.9, 7.7, 48.2, 96.3, 192.5, 385 and 770 μL. According to the method described in Example 3, the H+N mixed solution plate is used to replace the K01 plate to carry out the control experiment of the optimal ratio combination of 2-heptanone + 2-nonanol on the gray mold of tomato seedling leaves. The incidence rate and control effect are calculated according to formulas (3) and (4), and the data are expressed as mean ± standard deviation.

[0130] The experiment of controlling gray mold of tomato seedlings with H+N mixed solution was carried out in a plant-microorganism separation coexistence system incubator. According to the experimental results ( Fig.14 a): Compared with the CK group, when the dosage of H+N mixture was between 96.3 and 385 μL (i.e. when the concentration of H+N mixture was diluted to 1.25%-5%), the control effect showed a positive correlation with the concentration, while the incidence rate showed a negative correlation with the concentration ( Fig.14 , b and c). When the amount of H+N mixed solution was less than 96.5 μL, the incidence rate could not be reduced, and the inhibition of the expansion area of ​​lesions was much lower than that of the treatment with higher concentration ( Fig.14 , b and c).

[0131] Effects of volatile substances on hyphae morphology and cell structure of Botrytis cinerea:

[0132] The results of observation under optical microscope showed that CK, K01 and EC 80 The morphology of Botrytis cinerea hyphae after 12 hours of treatment with 40% NH4+H2O2 was investigated. The results showed that compared with the control group, the morphology of hyphae in the K01 and H+N treatment groups changed significantly, with the hyphae tips swelling, increased bifurcation, and overflow of intracellular substances ( Fig.15 ).

[0133] Scanning electron microscopy confirmed our results under optical microscopy. The mycelium surface of the control group was smooth and grew evenly ( Fig.16 , a and d); although the hyphae surface of the K01 treatment group was smooth, the thickness was uneven, the hyphae tips had more bifurcations, and were accompanied by swelling ( Fig.16 , b and e); the mycelium abnormalities in the H+N treatment group were more obvious, with irregular bends, wrinkles, and even ruptures ( Fig.16 , c and f).

[0134] By observing the cell structure of Botrytis cinerea treated with volatile antimicrobial substances using transmission electron microscopy, we found that the cell structure of the K01 and H+N treatment groups changed significantly, with an increase in vacuoles and mitochondria ( Fig.17 ); in the control group, the cytoplasm of hyphae cells was uniform, the vacuoles were normal, the endoplasmic reticulum had a regular shape, the cytoplasmic membrane was in close contact with the cell wall, and the nucleus was intact ( Fig.17 , a and b); in the VOCs-treated group K01, the mycelial cells of Botrytis cinerea showed plasmolysis, an increase in the number of mitochondria, rupture of the endoplasmic reticulum, and an increase in vacuoles ( Fig.17 , c and d); the abnormalities of the cell structure of Botrytis cinerea in the H+N treatment group were more serious, manifested by a large number of mitochondrial deformities or even ruptures, degradation of nuclear substances, severe vacuolization, severe plasmolysis, and collapse of intracellular substances ( Fig.17 , e and f).

[0135] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0136] References:

[0137] 1. ALMEIDA OAC, DE ARAUJO NO, DIAS BHS, et al. 2023. The power of the smallest: The inhibitory activity of microbial volatile organic compounds against phytopathogens. Frontiers in Microbiology[J], 13.

[0138] 2、CHE J,LIU B,LIU G,et al.2017.Volatile organic compounds produced byLysinibacillus sp.FJAT-4748 possess antifungal activity againstColletotrichum acutatum.Biocontrol Science and Technology[J],27:1349-1362.

[0139] 3、CHEN Z,SUN H,HU T,et al.2022.Sunflower resistance againstSclerotinia sclerotiorum is potentiated by selenium through regulation ofredox homeostasis and hormones signaling pathways.Environmental Science andPollution Research[J],29:38097-38109.

[0140] 4、KONG W-L,RUI L,NI H,et al.2020.Antifungal Efiects of VolatileOrganic Compounds Produced by Rahnella aquatilis JZ-Gx1 AgainstColletotrichum gloeosporioides in Liriodendron chinense×tulipifera.Frontiersin Microbiology[J],11.

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Claims

1. Any of the following applications of the composition of 2-heptanone and 2-nonanol: a) for the prevention of pathogens and plant diseases caused by pathogens; b) For use in the preparation of antimicrobial agents, preservatives or disinfectants; The composition is obtained by mixing 2-heptanone and 2-nonanol according to the following ratio: 20 μL of 2-heptanone at a concentration of 536.1 μL / mL and 20 μL of 2-nonanol at a concentration of 128.5 μL / mL; or, 2-Heptanone at a concentration of 536.1 μL / mL and 2-nonanol at a concentration of 128.5 μL / mL were mixed in a volume ratio of 3:

5.

2. The use according to claim 1, characterized in that: The pathogen is Botrytis cinerea ( Botrytis cinerea ).

3. The use according to claim 2, characterized in that: The plant disease is tomato gray mold.

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