Bacillus velezensis tcs001 volatile gas composition and application thereof

Abstract

The application provides a Bacillus velezensis TCS001 volatile gas composition, which comprises 2-ethylhexanol, methyl nonyl methyl ketone, 2-decanol and 2-dodecanone, and the mass ratio of 2-ethylhexanol:methyl nonyl methyl ketone:2-decanol:2-dodecanone is 2-7:5-13:2-6:2-23. The Bacillus velezensis TCS001 volatile gas composition can be used as a safe and efficient gas-phase bacteriostatic material for the research and development of a novel fumigation preparation, is suitable for the prevention and treatment of diseases in a closed environment such as a greenhouse, a warehouse and the like, promotes crop growth, and has wide application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of crop disease prevention and crop growth regulation, in particular to the application of Bacillus velezensis TCS001 volatile gas composition in preventing plant diseases and regulating plant growth, and to the identification of each component in the Bacillus velezensis TCS001 volatile gas composition and the determination of its activity. BACKGROUND

[0002] Bacillus velezensis is a new type of biocontrol bacteria. It was first isolated by Spanish scholar Ruiz-García et al. in 2005, and two strains CR-14b and CR-502T were obtained. They can synthesize a large amount of lipopeptide substances and have strong antibacterial activity. Bacillus velezensis is widely distributed and can survive in adversity such as drought and salinity. There are few related products, and the development prospect is broad.

[0003] Bacillus velezensis TCS001 is isolated from the sludge of Bohai Sea. The original strain is Bacillus marinus CT2628, which is identified and named as Bacillus velezensis TCS001 after mutagenesis and stabilization. It has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.8921, and was first published in the patent ZL201410168402.2. After growth on NA medium, the single colony of TCS001 is nearly round, light yellow and opaque. The colony surface is smooth at the early stage of culture, with neat edges. The surface has wrinkles at the later stage, with slightly uneven edges, a convex middle and a cloud-like diffusion around. Gram staining shows that the Bacillus velezensis TCS001 strain is gram-positive and rod-shaped. It has certain inhibitory effect on cucumber gray mold, cucumber blight, cucumber sclerotium blight, cucumber brown spot, cotton wilt and banana leaf spot, among which the inhibition rate on cucumber gray mold is the highest, reaching 87.66%.

[0004] Previous studies have shown the presence of volatile metabolites in B. velezensis C2 by GC-MS analysis, which mainly contain volatile metabolites known for their antifungal activity, namely tetradecane, phenylacetic acid, benzaldehyde, 1-decene and phenylethanol (protease, chitosanase and beta-glucanase), siderophores and indole-3-acetic acid, and the ability to solubilize inorganic phosphate. In addition, GC-MS analysis of B. velezensis C2 showed the presence of bacteriostatic active VOCs, which reduced the incidence of tomato plants by 70.43 ± 7.08% compared to the untreated control group. After treatment with B. velezensis C2, it was also found that the plant growth of tomato plants could be significantly promoted by stem elongation and leaf number. The results of this study indicate that B. velezensis C2 has great potential for commercialization as a bio- agent and can be effectively treated as a biocontrol agent for tomato wilt (Dhouib et al., 2019).

[0005] Manel et al. (2019) showed that the B. velezensis OEE1 strain has a large number of secreted and volatile secondary metabolites. Gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOCs) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of secondary metabolites of the OEE1 strain determined a number of PGP-capable molecules, which are known to interfere with the development of pathogens. GC-MS analysis of the main volatile organic compounds of the B. velezensis strain OEE1 showed the presence of phenylacetic acid, and ethylbenzene, phenylethanol, E-sesquiterpene and cyclo(Leu-Pro) were also detected in the volatile organic compounds of the B. velezensis strain OEE1, which have insecticidal, antibacterial, acaricidal and antifungal activity, respectively. In addition, in greenhouse experiments, B. velezensis OEE1 was shown to be effective in protecting olive trees from Fusarium solani and was able to colonize the roots of olive trees (Manel et al., 2019).

[0006] Peng et al. (2019) demonstrated that acetoin (3-hydroxybutanone) is the main component of VOCs produced by the B. velezensis GJ11 strain, which can induce the priming state of plants (e.g., H2O2 burst and callose deposition) (Peng et al., 2019).

[0007] In the prior art, it has been disclosed that volatile gases of Bacillus velezensis have antibacterial effects, for example, patent CN201710538989.5 discloses the inhibitory effect of volatile gases of Bacillus velezensis JS25R on Fusarium graminearum; CN201910519896.7 discloses the antibacterial effect of volatile substances of Bacillus velezensis strain BPC6 on Sclerotium and soft rot bacteria; CN202010747157.6 discloses the inhibitory effect of fermentation broth and volatile gases of Bacillus velezensis WH-P2-20 on Monilinia fructicola; CN202011286285.1 discloses that the volatile gases of Bacillus velezensis ZHX-7 make Aspergillus flavus color fade, etc. However, there is no research on the volatile gases of Bacillus velezensis TCS001.

[0008] Bacillus velezensis TCS001 is first isolated and found by the applicant of the present application. The applicant found that the crops in the adjacent control area of the treatment area applied with Bacillus velezensis TCS001 showed a disease prevention effect, which was speculated to be caused by the volatile substances of Bacillus velezensis TCS001, and after research, the technical scheme of the present application was obtained. SUMMARY

[0009] One of the main purposes of the present application is to provide the application of the volatile gas composition of Bacillus velezensis TCS001 in inhibiting plant pathogenic fungi.

[0010] The plant pathogenic fungi include Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum, Curvularia inaequalis, Magnaporthe grisea, Gaeumannomyces graminis, Colletotrichum gloeosporioides and Cucurbita leaf spot bacteria.

[0011] Preferably, the plant pathogenic fungi are Sclerotinia sclerotiorum and Botrytis cinerea.

[0012] Another purpose of the present application is to provide the application of the volatile gas composition of Bacillus velezensis TCS001 in promoting plant growth.

[0013] The plants are cucumber and rape;

[0014] The plant growth indicators include plant height, root length, fresh weight, etc.

[0015] Another purpose of the present application is to provide the volatile gas composition of Bacillus velezensis TCS001, which comprises 2-ethylhexanol, methyl nonyl ketone, 2-decanol and 2-dodecanone.

[0016] Preferably, the mass ratio of 2-ethylhexanol, methyl nonyl ketone, 2-decanol and 2-dodecanone is 2-7:5-13:2-6:2-21.

[0017] Preferably, the mass ratio of 2-ethylhexanol: methyl nonyl ketone: 2-decanol: 2-dodecanone is 7:6:4:2; or 4:5:6:3; or 2:12:2:16; or 3:13:6:21.

[0018] Another object of the present application is to provide the use of 2-decanol in the preparation of a medicine for preventing and treating Botrytis cinerea of cucumber.

[0019] Another object of the present application is to provide the use of 2-dodecanone in the preparation of a medicine for preventing and treating Botrytis cinerea of cucumber.

[0020] Another object of the present application is to provide the use of 2-ethylhexanol in the preparation of a medicine for preventing and treating Botrytis cinerea of cucumber.

[0021] Another object of the present application is to provide the use of any two or three combinations selected from 2-decanol, 2-dodecanone and 2-ethylhexanol in the preparation of a medicine for preventing and treating Botrytis cinerea of cucumber.

[0022] Another object of the present application is to provide a method for preparing the volatile gas composition of Bacillus velezensis TCS001, wherein the volatile gas composition of Bacillus velezensis TCS001 is obtained by culturing Bacillus velezensis TCS001 in a culture medium; or 2-ethylhexanol, 2-dodecanone, 2-decanol and methyl nonyl ketone are mixed.

[0023] The beneficial technical effects of the present application

[0024] The present application first discovers that the volatile gas composition of Bacillus velezensis TCS001 has the use of inhibiting plant pathogenic fungi, especially the inhibition effect on Sclerotinia sclerotiorum is the strongest, reaching 98.50%; the inhibition effect on Botrytis cinerea is the second, reaching 66.96%.

[0025] The present application first discovers that the volatile gas composition of Bacillus velezensis TCS001 has the use of promoting plant growth, which can significantly improve the plant height, root length and fresh weight of cucumber and rape.

[0026] The present application first detects and analyzes that the volatile gas composition of Bacillus velezensis TCS001 includes 2-ethylhexanol, methyl nonyl ketone, 2-decanol and 2-dodecanone.

[0027] The present application first discovers that 2-decanol, 2-dodecanone and 2-ethylhexanol have the effect of preventing and treating Botrytis cinerea of cucumber.

[0028] The present application researches and finds that the volatile gas composition of Bacillus velezensis TCS001 has the characteristics of low residual amount, high safety and good prevention effect.

[0029] The volatile gas composition of Bacillus velezensis TCS001 provided by the application can be used as a safe and efficient gas-phase bacteriostatic material for the research and development of new fumigation preparations, and is suitable for the prevention and treatment of diseases in closed environments such as greenhouses and warehouses, and has wide application value.

[0030] The accompanying drawings

[0031] Figure 1 The inhibition effect of the volatile substances of Bacillus velezensis TCS001 on various plant pathogenic fungi;

[0032] Figure 2 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 The effect diagram of the “double dish system” of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0033] Figure 3 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 The single plant result (5d) of the “double dish system” of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0034] Figure 4 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 Statistical data analysis result of the growth index of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0035] Figure 5 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 The effect diagram of the “double dish system” of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0036] Figure 6 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 The single plant result (4d) of the “double dish system” of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0037] Figure 7 The fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10 9 Statistical data analysis result of the growth index of the fermentation liquid of Bacillus velezensis TCS001 with a concentration of 10

[0038] Figure 8 The total ion flow chromatogram of the volatile substances of Bacillus velezensis TCS001 in the LB solid culture medium sample;

[0039] Figure 9 The total ion flow chromatogram of the volatile substances of the 5d fermentation liquid of Bacillus velezensis TCS001;

[0040] Figure 10Inhibition of Botrytis cinerea mycelium growth by volatile organic compounds from Bacillus velezensis TCS001 after 4 days of treatment

[0041] Figure 11 Botrytis cinerea not co-cultured with Bacillus velezensis TCS001

[0042] Figure 12 Botrytis cinerea co-cultured with Bacillus velezensis TCS001

[0043] Note: A: Botrytis cinerea; B: Colletotrichum gloeosporioides; C: Sclerotinia sclerotiorum; D: Fusarium oxysporum; E: Rhizoctonia cerealis; F: Pseudomonas lachrymans; G: Magnaporthe grisea; H: Curvularia inaequalis; I: Pythium porphyricum; A-I: blank control group; A'-I': treatment group; J: blank control; K: 20 μL of ethyl hexanol; N: 40 μL of isooctanol; L: 20 μL of 2-decanol; O: 40 μL of 2-decanol; M: 20 μL of 2-dodecanone; P: 40 mL of 2-dodecanone.

[0044] Example 1

[0045] Test of volatile substances from Bacillus velezensis TCS001 to inhibit plant pathogenic fungi

[0046] Test strains:

[0047] Bacillus velezensis TCS001, preserved by the Green Pesticide Collaboration Center of Zhejiang Agricultural University.

[0048] Pathogenic fungi:

[0049] Sclerotinia sclerotiorum, Botrytis cinerea, Magnaporthe grisea, Fusarium oxysporum, Curvularia inaequalis, Pythium porphyricum, Colletotrichum gloeosporioides, Pseudomonas lachrymans.

[0050] Test method

[0051] Culture medium preparation:

[0052] Potato dextrose agar medium (PDA): 200.0 g of potato, 20.0 g of glucose, 18.0 g of agar powder, 1000 mL of distilled water. Used for culturing plant pathogenic fungi.

[0053] LB solid medium: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 18 g of agar powder, 1000 mL of water, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 single colonies.

[0054] LB liquid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, water 1000 mL, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 fermentation broth.

[0055] Antibacterial spectrum test

[0056] In one compartment of a 9 cm diameter two-compartment culture dish, 8 mL of PDA medium was added, and in the other compartment, 8 mL of LB solid medium was added. A single colony of Bacillus velezensis was picked up with a inoculating loop and inoculated into 100 mL of LB liquid medium, which was cultured at 28°C, 180 rpm for 24 h. 40 μL of Bacillus velezensis TCS001 fermentation broth (10 9 CFU / mL) was applied to one side of the LB solid medium, and a pathogenic fungus colony (Φ = 5 mm) taken from the edge of the colony was inoculated in the center of the other side of the PDA, which was sealed with Parafilm film and cultured at 28°C in the dark for 72 h.

[0057] The growth of the pathogenic fungus was observed, and the colony diameter of the pathogenic fungus was measured to calculate the inhibition rate.

[0058] The plate without Bacillus velezensis TCS001 fermentation broth was used as a blank control.

[0059] Each treatment was set up in triplicate.

[0060] Results investigation

[0061] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - colony diameter) x 100%.

[0062] Table 1 Inhibition of plant pathogenic fungi by volatile substances of Bacillus velezensis TCS001

[0063] Plant pathogenic fungi % Volatile substance inhibition rate Sclerotinia sclerotiorum 98.50±0.52a Botrytis cinerea 66.96±0.41b Rhizoctonia solani 31.27±0.13d Fusarium oxysporum 3.39±0.38i Cercospora beticola 19.05±0.35g Phytophthora cactorum 4.85±0.33h Rhizoctonia cerealis 44.19±0.33c Gloeosporium lognatum 28.29±0.03e Pseudoperonospora cubensis 20.20±0.05f

[0064] The inhibition effect of volatile substances of Bacillus velezensis TCS001 on plant pathogenic fungi was as shown in Figure 1 As shown in Table 1, the results showed that the volatile substances of Bacillus velezensis TCS001 had broad-spectrum antibacterial selective activity, among which the inhibition effect on Sclerotinia sclerotiorum and Botrytis cinerea was the strongest, reaching 98.50% and 66.96%, respectively.

[0065] Example 2

[0066] Test of plant growth promotion by volatile substances of Bacillus velezensis TCS001

[0067] Test strain:

[0068] Bacillus velezensis TCS001, preserved by Zhejiang A & F University Green Pesticide Collaboration Center.

[0069] Tested plants:

[0070] Cucumber, rape.

[0071] Test method

[0072] Culture medium preparation:

[0073] (1) LB medium: tryptone 10 g, yeast powder 5 g, NaCl 10 g, agar powder 18 g, water 1000 mL, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 single colony.

[0074] (2) LB liquid medium: tryptone 10 g, yeast powder 5 g, NaCl 10 g, water 1000 mL, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 fermentation broth.

[0075] (3) Wheat medium: sterilized wheat grains.

[0076] Select uniform and full seeds of rape or cucumber. Place the seeds in 2 mL EP tubes. First, sterilize the rape or cucumber seeds with 75% (V / V) medical alcohol for 5 min. Then, use a pipette to suck out the alcohol solution and wash the seeds with sterile water for 5-6 times, 30 s each time. Then, sterilize the seeds with 2.5% sodium hypochlorite solution (V / V) for 5 min. Then, use a pipette to suck out the sodium hypochlorite solution and wash the seeds with sterile water for 5-6 times, 30 s each time.

[0077] Place the sterilized seeds in a large sterile nutrient soil dish (diameter 12 cm, weigh 70 g of sterile soil). Place a small dish (diameter 6 cm) in the center of the large dish. Add 10 sterilized wheat grains to the small dish to make a wheat medium. Place 10 9 Add 1 mL of Bacillus velezensis TCS001 fermentation broth with a CFU / mL of 10 to the wheat medium. Place the double dishes (large dish and small dish) in a constant temperature light incubator at 20°C with light and dark alternation (16 h / 8 h, light / dark). Add 2 mL of sterile water to keep the soil moist every day. Measure the fresh weight, root length, and plant height of the rape after 4 days of culture. Measure the fresh weight, root length, and plant height of the cucumber after 5 days of culture. Use 1 mL of LB liquid medium as a blank control. Each dish contains 4 seeds, and the experiment is repeated 3 times.

[0078] Results investigation

[0079] The experimental results are shown in Figures 2-7 .

[0080] Measure the fresh weight, root length, and plant height of the cucumber and rape using a ruler and analyze the data.

[0081] As shown in Figure 4 , the fresh weight and plant height of cucumber seedlings treated with Bacillus velezensis TCS001 were significantly different from those of cucumber seedlings treated with CK.

[0082] According to Figure 2 , 3 , after co-culturing Bacillus velezensis TCS001 with cucumber seeds for 5 days, the fresh weight of cucumber seedlings was significantly higher than that of CK treatment by about 81.75%, and the plant height was significantly higher than that of CK treatment by about 62.16%.

[0083] As shown in Figure 7 , the fresh weight and plant height of rape seedlings treated with Bacillus velezensis TCS001 were significantly different from those of rape seedlings treated with CK.

[0084] According to Figure 5 , 6 , after co-culturing Bacillus velezensis TCS001 with rape seeds for 4 days, the fresh weight of rape seedlings was significantly higher than that of CK treatment by about 50.33%, and the plant height was significantly higher than that of CK treatment by about 34.56%.

[0085] Investigation conclusion: The volatile substances produced by Bacillus velezensis TCS001 have significant growth-promoting effect on cucumber and rape.

[0086] Example 3

[0087] Analysis of volatile substance components of Bacillus velezensis TCS001

[0088] Test strain

[0089] Bacillus velezensis TCS001 was preserved by the Green Pesticide Collaboration Center of Zhejiang Agricultural University.

[0090] Test method

[0091] Culture medium preparation

[0092] LB medium: tryptone 10g, yeast powder 5g, NaCl 10g, agar powder 18g, water 1000mL, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 single colony.

[0093] LB liquid medium: tryptone 10g, yeast powder 5g, NaCl 10g, water 1000mL, pH 7.0-7.2. Used for culturing Bacillus velezensis TCS001 fermentation broth.

[0094] Analysis of volatile substance components

[0095] The method of Headspace Solid Phase Micro-extraction (HS-SPME) and Gas Chromatography-Mass Spectrometer (GC-MS) was used for collecting and analyzing the volatile substances of Bacillus velezensis TCS001.

[0096] In this experiment, the volatile substances produced by the fermentation broth of Bacillus velezensis TCS001 cultured for 1d, 2d, 3d, 4d, and 5d were adsorbed by Headspace Solid Phase Micro-extraction (HS-SPME). Then the collected volatile substances were detected by GC-MC technology. Full scan mode was used for detection, and the detection range was 50-600amu. After detection, automatic database (National Institute of Standards and technology NIST) matching was performed, and components with relative peak area greater than 1.0% and RSI (Reverse similarity) and SI (Similarity) greater than 80 were selected for dynamic analysis of components.

[0097] Specific operation steps:

[0098] A single colony of Bacillus velezensis TCS001 was picked up with an inoculation loop and inoculated into 100mL of LB liquid medium, which was cultured at 28℃ and 180rpm for 24h. The volatile substances were collected using static headspace.

[0099] Without cooling (about 55℃), 3mL of LB solid medium was taken and poured into a 20mL sterile headspace bottle, and the bottle was inclined at 15°. After cooling and solidification, 100μL of pre-prepared TCS001 was evenly coated on the inclined surface of the medium, and the bottle was quickly sealed with a bottle cap with an internal polytetrafluoroethylene coating and a glass bottle. The headspace was placed in a 28℃ incubator and kept and cultured in the dark.

[0100] The treatment of Bacillus velezensis TCS001 fermentation broth was blank control, and each sample was repeated 3 times.

[0101] After equilibrating the overhead flask at room temperature for 20min, 50 / 30μm DVB / CAR / PDMS extraction head was used for sampling. Before using the extraction head, the SPME fiber was aged at 250℃ for 1h. The SPME needle was inserted into the headspace bottle, and then the extraction head was pushed into the top 1 / 3 of the headspace bottle to collect the volatile organic compounds produced by TCS001. The sampling time was 30min. After the collection was completed, the extraction head was removed, the needle was removed from the sample bottle, and the sample collection was completed.

[0102] LB medium without Bacillus velezensis TCS001 inoculation was blank control.

[0103] Solid phase microextraction fiber head (SPME): Divinylbenzene / carboxen / polydimethylsiloxane (DVB / PDMS / CAR);

[0104] Chromatographic column: Agilent HP-5MS capillary column (30 m x 0.25 mm x 0.25 μm);

[0105] Acquisition mode: full scan;

[0106] GC:

[0107] Program name Program setting Column oven temperature 40.0℃ Injection port temperature 250.0℃ Injection mode Split Injection time 2.0 min Flow control mode Pressure Pressure 63.9 Kpa Total flow rate 76.2 mL / min Column flow rate 1.20 mL / min Linear velocity 39.5 cm / sec Purge flow rate 3.0 mL / min Split ratio 60.0 Total program time 60.0 min

[0108] Program: column oven temperature

[0109] Serial number Rate Final temperature (°C) Hold time (min) 0 - 40.0 3.00 1 3.00 160.0 5.00 2 8.00 220.0 5.00 4 0.00 0.00 0.00

[0110] MS:

[0111] Ion source temperature 230℃ Interface temperature 200℃ Solvent delay time 2 min GC degree time 60.50 min

[0112] The total ion current chromatogram of volatile substances in the LB solid medium sample of Bacillus velezensis TCS001 is shown in Figure 8 .

[0113] The total ion current chromatogram of volatile substances in the fermentation broth of Bacillus velezensis TCS001 is shown in Figure 9 .

[0114] Table 2. Analysis of volatile gas components and relative content of Bacillus velezensis TCS001

[0115]

[0116] The structures of the compounds are shown as follows:

[0117]

[0118] Example 4

[0119] Inhibition test of 2-dodecanol, 2-ethyl ethanol and 2-dodecanol in volatile substances of Bacillus velezensis TCS001 on plant pathogenic fungi

[0120] Test pathogenic fungi: Botrytis cinerea

[0121] Test method

[0122] Medium preparation:

[0123] Potato dextrose agar medium (PDA): Potato 200.0 g, glucose 20.0 g, agar powder 18.0 g, distilled water 1000 mL. Used for culturing plant pathogenic fungi.

[0124] Filter paper disc test

[0125] Control group: without adding any compound

[0126] Treatment group: sterile filter paper discs with a diameter of 8 mm were placed on one side of the plate, and the corresponding volume (20, 40 μL) of 2-hexanol, 2-ethyl ethanol, 2-dodecanol was added dropwise (the upper limit of liquid added to each filter paper disc was 10 μL). After sealing with Parafilm, the plates were incubated at 28°C for 4 days, and the colony diameter was measured once a day.

[0127] The other side of all plates was inoculated with a 5 mm diameter P. cucumerina plug on PDA.

[0128] Result investigation

[0129] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - plug diameter) x 100%.

[0130] Inhibition of mycelial growth of P. cucumerina by Bacillus velezensis TCS001 volatile organic compound monomers after 4 days of treatment, as shown in Table 3 and Figure 10 .

[0131] Test results: After 4 days of incubation, it was found that 2-ethylhexanol, 2-decanol, and 2-dodecanol with a volume of 20 μL and 40 μL added to filter paper discs could all inhibit the growth of P. cucumerina mycelium to varying degrees. The compound monomer with the strongest inhibitory effect on P. cucumerina mycelium among the volatile organic compound monomers produced by Bacillus velezensis TCS001 was 2-ethylhexanol.

[0132] Table 3 Inhibition of mycelial growth of P. cucumerina by three organic synthetic compounds produced by Bacillus velezensis TCS001

[0133]

[0134] Example 5

[0135] Effect of volatile substances produced by Bacillus velezensis TCS001 on the morphology of P. cucumerina mycelium

[0136] Pathogenic fungus: P. cucumerina

[0137] Test method

[0138] One compartment of a 9 cm diameter two-compartment Petri dish was filled with 8 mL PDA medium and the other compartment was filled with 8 mL LB medium. A single colony of B. velezensis was picked up with a loop and inoculated into 100 mL LB liquid medium, which was incubated at 28°C, 180 rpm for 24 h. 40 μL of B. velezensis TCS001 fermentation broth with a CFU / mL of 10 9 CFU / mL of B. velezensis TCS001 fermentation broth was inoculated on one side of the PDA medium, and a fungal cake (Φ = 5 mm) of cucumber gray mold taken from the edge of the colony was inoculated in the center of the other side. The Petri dish was sealed with Parafilm and incubated at 28°C in the dark for 72 h. A control test was set up in which cucumber gray mold was inoculated alone without B. velezensis TCS001.

[0139] Cucumber gray mold mycelial morphology scanning

[0140] Scanning electron microscopy (sample preparation):

[0141] (1) The cucumber gray mold was divided into small pieces with a size of 0.5 x 0.3 cm.

[0142] (2) The small pieces were placed in 2 mL centrifuge tubes and 2.5% glutaraldehyde was added, and incubated at 4°C overnight.

[0143] (3) The glutaraldehyde was removed with a pipette, and the sample was immersed in 0.1M PBS buffer with pH 7.0 for 15 min, and the immersion was repeated 3 times. (PBS buffer: 600 μL KH2PO4 + 400 μL 0.1M Na2HPO4)

[0144] (4) The sample was sequentially immersed in ethanol solutions with concentrations of 30%, 50%, 70%, 80%, 90%, and 95%, respectively, for 15 min each time.

[0145] (5) The ethanol solution was removed with a pipette, and the sample was eluted with 100% ethanol for 20 min, and the elution was repeated 2 times

[0146] (6) The ethanol was poured off, and the sample was immersed in a mixed solution of ethanol and isopropyl acetate (V / V = 1 / 1) for 30 min.

[0147] (7) The sample was immersed in 100% isopropyl acetate for 2 h.

[0148] (8) Conductive glue was applied to the sample stage, and the sample edge was lightly clamped with tweezers and applied to the conductive glue.

[0149] (9) The sample was dried in a clean bench and gold plated at the critical point, and finally observed with a scanning electron microscope.

[0150] Results investigation

[0151] The sample was observed under a scanning electron microscope at 30 μm.

[0152] Test results: from Figure 11 It can be seen that the mycelium surface of cucumber botrytis cinerea is relatively smooth without co-culturing with bacillus velezensis TCS 001. From Figure 12 It can be seen that the mycelium surface of cucumber botrytis cinerea appears shrinkage phenomenon after co-culturing with bacillus velezensis TCS 001. It shows that the growth and development of botrytis cinerea mycelium is inhibited after being treated by volatile substances produced by bacillus velezensis TCS 001.

Claims

1. A volatile gas composition of Bacillus belye TCS001, characterized in that, It includes 2-ethylhexanol, methyl nonyl ketone, 2-decyl alcohol and 2-dodecane; the mass ratio of 2-ethylhexanol: methyl nonyl ketone: 2-decyl alcohol: 2-dodecane is 2-7: 5-13: 2-6: 2-21.

2. The Bacillus belyssus TCS001 volatile gas composition according to claim 1, characterized in that, The mass ratio of 2-ethylhexanol: methyl nonyl ketone: 2-decyl alcohol: 2-dodecane ketone is 7:6:4:2; or 4:5:6:3; or 2:12:2:16; or 3:13:6:

21.

3. A method for preparing the Bacillus belye TCS001 volatile gas composition according to any one of claims 1-2, characterized in that, The volatile gas composition of Bacillus belye TCS001 is obtained by volatilization of Bacillus belye TCS001.

4. A method for preparing the Bacillus belye TCS001 volatile gas composition according to any one of claims 1-2, characterized in that, The volatile gas composition of Bacillus belye TCS001 is obtained by mixing 2-ethylhexanol, methyl nonyl ketone, 2-decyl alcohol, and 2-dodecane.

5. Use of the Bacillus vesiculosus TCS001 volatile gas composition according to any one of claims 1-2 for inhibiting plant pathogenic fungi.

6. The use according to claim 5, characterized in that, The plant pathogenic fungi include *Sclerotinia sclerotiorum*, *Gray mold*, *Rhizoctonia solani*, *Corn rot*, *Stem rot*, *Colletotrichum gloeosporioides*, and *Cyclocarya paliurus*.

7. Use of the Bacillus vesiculosus TCS001 volatile gas composition according to any one of claims 1-2 for promoting plant growth.

8. The use according to claim 7, characterized in that, The plants mentioned are cucumber and rapeseed.

9. The use according to claim 7, characterized in that, The promotion of plant growth includes plant height, root length, and fresh weight.

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