A soil fumigant for fusarium wilt of banana and a preparation method thereof

Abstract

The application belongs to the field of biology and particularly relates to a soil fumigant for Fusarium oxysporum f. sp. cubense and a preparation method thereof. The application identifies active ingredients of volatile organic compounds generated by Nori spores, finally screens out 2-ethyl-1-hexanol (2EH) which has a significant inhibitory effect on Fusarium oxysporum f. sp. cubense, and the soil fumigant is prepared by taking the component as the main active ingredient, has a significant killing effect on Fusarium oxysporum f. sp. cubense in the soil, has the advantages of good stability and high safety, is not easy to remain in the soil, and is an easily biodegradable substance according to the OECD 301C test, the TOC removal rate reaches 100% within 28 days, the BOD degradation rate is greater than 60%, is used for soil disinfection, can effectively reduce the number of Fusarium oxysporum f. sp. cubense pathogens in the soil, improve the spore inhibition rate, and provide a new method and agent for field prevention and control of banana fusarium wilt.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a soil fumigant for banana wilt pathogen and its preparation method. Background Technology

[0002] Banana wilt is a devastating soil-borne disease caused by a fungus, posing a serious threat to the banana industry. This disease is caused by *Fusarium oxysporum* var. *cubicans*. Fusarium oxysporum Caused by *Fusarium wilt* sp. *cubense*, this disease primarily invades banana plants through the roots, occupying their vascular system and hindering the flow of water and nutrients to the fruit, leading to wilting and eventual death. The pathogen can persist in the soil for over 30 years, making control extremely difficult once an outbreak occurs. Currently, prevention and integrated management of banana wilt are paramount, focusing on blocking transmission routes through agricultural measures such as selecting resistant varieties, using healthy seedlings, crop rotation, and soil management, supplemented by biological and chemical methods to control disease development. Soil is a fundamental condition for the spread of banana wilt; therefore, developing effective soil control methods and pesticides is crucial for the control of this disease. Summary of the Invention

[0003] This invention provides a soil fumigant for Fusarium wilt of banana and its preparation method. Using 2-ethyl-1-hexanol, the active ingredient produced by strain XZF, as the main component, the prepared soil fumigant exhibits significant inhibitory effects against Fusarium wilt of banana in soil. Strain XZF is *Synthia spp.*, classified as... Kitasatospora sp. The strain has been registered and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 64567), with a deposit date of April 26, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Information about this strain has been disclosed in the invention patent application number "CN202410828975.7" entitled "A type of *Synapsium nitidum* and its application".

[0004] The technical solution of this invention is implemented as follows:

[0005] Kitarispora Kitasatospora sp. The application of the generated volatile organic compounds in the preparation of formulations that inhibit and / or control banana wilt pathogens, wherein the preservation number of the *Nyctaginus niger* is GDMCC NO: 64567.

[0006] Furthermore, the volatile organic compound contains 2-ethyl-1-hexanol.

[0007] The method for preparing the volatile organic compounds includes the following steps:

[0008] The *Tetracentron sinense* was inoculated onto YE solid culture medium, sealed, and cultured. Volatile organic matter was collected to obtain the final product.

[0009] Application of volatile substances produced by *Sphaerocera kimosporioides* in any of the following:

[0010] (1) Application in the preparation of formulations that inhibit banana wilt fungus, banana black spot fungus, cucumber wilt fungus, mango anthracnose fungus, banana leaf spot fungus and / or banana anthracnose fungus;

[0011] (2) Application in the preparation of formulations for the prevention and control of diseases caused by Fusarium wilt of banana, Fusarium wilt of banana, Fusarium wilt of cucumber, Fusarium anthracnose of mango, Fusarium leaf spot of banana and / or Fusarium anthracnose of banana;

[0012] (3) Application in the preparation of formulations that inhibit the germination of banana wilt fungus spores.

[0013] (4) Application in the preparation of soil fumigation agents;

[0014] The preservation number of the aforementioned *Cyperus niger* is GDMCC NO: 64567.

[0015] Furthermore, the volatile substance is 2-ethyl-1-hexanol.

[0016] A soil fumigant containing the aforementioned volatile substances.

[0017] Furthermore, the volatile substance accounts for 20-30% of the volume of the soil fumigation agent, and the volatile substance is 2-ethyl-1-hexanol.

[0018] Furthermore, based on the volume of the soil fumigation agent, the volume percentage of the components contained in the soil fumigation agent is as follows: volatile substances 20-30%, cosolvent 10-15%, stabilizer 2-5%, synergist 0.3-2%, emulsifier 2-3%, and deionized water balance.

[0019] The co-solvent is at least one of propylene glycol, glycerin, or polyethylene glycol 400.

[0020] The stabilizer is at least one of ethylene glycol monobutyl ether, glyceryl monostearate, and propylene glycol methyl ether.

[0021] The synergist is at least one of menthol, carvacrol, and dodecyl dimethyl benzyl ammonium chloride.

[0022] The emulsifier is at least one of polyoxyethylene, castor oil, and Tween-80.

[0023] The preparation method of the above-mentioned soil fumigation agent includes the following steps:

[0024] (1) Add a co-solvent to the reactor, turn on low-speed stirring at 300-500 rpm, slowly add volatile substances, stir for 10 minutes to form a homogeneous oil phase system; add stabilizer and continue stirring for 5 minutes.

[0025] (2) Stir at 500 rpm, add deionized water to the vessel in three portions using a metering pump, emulsify at 12000 rpm for 15-20 minutes, then reduce the stirring speed to 300 rpm, add synergist and emulsifier, stir for 10 minutes, and the product is ready.

[0026] The above-mentioned soil fumigation agents may be used in any of the following applications:

[0027] (1) Application in the preparation of agents that inhibit banana wilt pathogens;

[0028] (2) Application in the preparation of formulations for the prevention and control of diseases caused by Fusarium wilt of banana;

[0029] (3) Application in soil disinfection.

[0030] A soil disinfection method includes the following steps:

[0031] Mix the above-mentioned volatile substances and / or soil fumigation agents with the soil and then seal it.

[0032] Furthermore, the volatile substance is 2-ethyl-1-hexanol, and the sealing method is a membrane seal, with the sealing time lasting at least 18 days.

[0033] Beneficial effects:

[0034] This invention identifies the active ingredients in the volatile organic compounds produced by *Fusarium oxysporum*, ultimately screening out 2-ethyl-1-hexanol (2EH), which exhibits a significant inhibitory effect on *Fusarium oxysporum*, the causal agent of banana wilt. A soil fumigant was prepared using this component as the main active ingredient, achieving a disinfect efficiency of over 95% against *Fusarium oxysporum* in the soil. It possesses advantages such as good stability and high safety, and is not prone to residue in the soil. OECD 301C testing shows that 2EH is a readily biodegradable substance, achieving a 100% TOC removal rate and a BOD degradation rate >60% within 28 days. When used for soil disinfection, it can effectively reduce the number of *Fusarium oxysporum* pathogens in the soil and improve spore inhibition rate, providing a new method and agent for the field control of *Fusarium oxysporum*. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0036] Figure 1 Comparison of antibacterial activities of different strains.

[0037] Figure 2 Gas chromatogram of gaseous metabolites of *Kitarechis*.

[0038] Figure 3 The filter paper disc method is used to detect the activity of volatile compounds.

[0039] Figure 4 EC of banana wilt pathogen 50 Measurement results.

[0040] Figure 5 Inhibitory effect of different concentrations of 2EH on FocTR4 spore germination.

[0041] Figure 6 The inhibitory effect of derivatives on banana wilt disease.

[0042] Figure 7 Compound 2EH-1 exhibits antifungal activity against six pathogenic fungi.

[0043] Figure 8 Dynamic trend of banana wilt fungus spore growth and decline in soil.

[0044] Figure 9 The inhibitory effects of different treatments on banana wilt pathogens. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0046] 1. Antagonistic effect of actinomycete VOCs S Antibacterial activity of the components and identification of their active ingredients

[0047] Streptomyces strains that produce VOCs active substances were screened using the plate-to-plate method, and strains with significant inhibitory effects were identified for their active ingredients.

[0048] The plate-on-plate method is performed as follows: Streptomyces strains are streaked onto YE medium and incubated at 28°C for 1-2 days. A 5mm diameter banana wilt pathogen is inoculated onto PDA medium. The plate inoculated with actinomycetes is then inverted onto the plate inoculated with the pathogen, with the bottoms of the two plates facing each other. The gaps between the two plates are sealed with sealing film, creating a closed space for volatile organic compounds (VOCs). The control group's actinomycete plates contain only sterile medium or no inoculation and are incubated upside down at 28°C for 7 days. Smaller colony diameters indicate stronger VOCs-inhibiting activity. The experiment is repeated in triplicate. The colony diameter is measured using the cross-sectional method, and the inhibition rate is calculated.

[0049] Three strains with significant VOC activity were screened, among which strain XZF showed an antibacterial activity of 61.11% ( Figure 1 Therefore, the identification of the active components of the gaseous metabolites of this strain was further determined.

[0050] After identification, strain XZF was determined to be *Hokusei*, and was classified as *Hokusei*. Kitasatospora sp. The strain has been registered and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 64567), with a deposit date of April 26, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Information about this strain has been disclosed in the invention patent application number "CN202410828975.7" entitled "A type of *Synapsium nitidum* and its application".

[0051] The method for identifying active ingredients is as follows: Prepare 100 μL of LYE liquid medium (YE medium without agar) and sterilize it at high temperature for later use. Pick single colonies of Streptomyces from a well-grown bacterial plate and inoculate them into the YE liquid medium. Place the plates on a shaker and incubate at 28°C and 180 rpm for 3 days. Afterward, mix thoroughly to use as the seed culture. Add 100 μL of the seed culture (10... 6The CFU / mL sample was inoculated into a 50 mL sterile flask containing 15 mL of YE solid medium. The flask was then sealed with sterile foil and a sealing film. Distilled water was used as a control instead of the strain. After incubation at 28°C for 7 days, volatile organic compounds (VOCs) were collected and analyzed by solid-phase microextraction (SPME) and gas chromatography-tandem mass spectrometry (GC-MS). The SPME fiber tip was inserted into the upper gas space of the headspace vial through the cap. The fiber tip was then extended to expose the headspace for adsorption. The adsorption time was typically 30 minutes. During this process, the headspace vial could be placed on a mild heated plate (40°C) or a magnetic stirrer to enhance VOC release. After adsorption was complete, the SPME fiber tip was retracted and quickly removed from the headspace vial. The SPME needle was immediately inserted into the GC-MS instrument inlet, the fiber tip was extended, and thermal desorption was performed for 5 minutes (the inlet temperature was typically set to 250-280°C), injecting the VOCs into the column. The chromatographic program was set as follows: Column gradient temperature program: initial temperature 50℃, hold for 5 min; increase to 150℃ at a rate of 5℃ / min and hold for 1 min; then increase to 300℃ at a rate of 20℃ / min and hold for 3 min; GC / MS interface temperature 280℃. Mass spectrometry detection used an EI ion source with a temperature of 230℃, a quadrupole temperature of 150℃, an electron energy of 70 eV, full scan mode (m / z 45~450), and a solvent delay of 3 min. Compound identification was performed by comparing retention times and mass spectra with the NIST05 standard library. Peak areas were quantified as relative abundance. Results are shown below. Figure 2 And Table 1. The activity of the identified volatile compounds was detected using the filter paper disc method. The specific procedure was as follows: Pour PDA medium into a petri dish. After the medium solidified, under aseptic conditions, divide the petri dish into two parts. Inoculate one part with a piece of anthrax fungus cake (5 mm in diameter). Remove the medium from the other part and add sterile filter paper containing 10 μL of the compound, placing it in the opposite position to the fungus cake. Figure 3 As shown, the control was an equal volume of purified water. All petri dishes were incubated at 28°C with the pathogen. Regular observations were made, and when the control group colonies were about to completely cover the plates, the colony diameter of the pathogen was measured using the cross-sectional method, and the inhibition rate of each compound was calculated.

[0052] like Figure 3 As shown in Table 1, among the above compounds, 2-ethyl-1-hexanol (1-Hexanol, 2-ethy-, hereinafter referred to as 2EH) exhibits the best antibacterial activity, achieving a 100% inhibition rate against the mycelial growth of Fusarium wilt of banana. It also possesses advantages such as good stability and high safety, is easily volatilized, and does not easily remain in the soil. OECD 301C testing shows that 2EH is a readily biodegradable substance, achieving a 100% TOC removal rate and a BOD degradation rate > 60% within 28 days.

[0053] Table 1. Main substances identified (first 13)

[0054] Serial Number Compound Name Molecular formula Relative retention time (min) molecular weight Area percentage (%) Antibacterial activity 1 Benzene, 1,4-dichloro- C6H4Cl2 12.215 147 16.10 Inactive 2 2-Methyl-2-bornene C11H18 12.4 150.26 10.53 Inactive 3 1-Hexanol, 2-ethy- C8H18O 13.305 130.22 2.95 It has inhibitory activity 4 2-Heptanone, 6-methyl-5-methylene- C9H16O 13.507 140.22 1.78 Inactive 5 Undecane, 4,7-dimethyl- C13H28 14.448 184.35 3.56 Inactive 6 Benzene, 1-ethenyl-4-methoxy- C9H10O 19.23 134.18 3.01 Inactive 7 2-Methylisoborneol C11H20O 20.882 168.28 2.67 Inactive 8 Benzene, 1-(1,1-dimethylethyl)-4-methoxy- C11H16O 23.403 164.25 2.54 Inactive 9 Dodecane, 4,6-dimethyl- C14H30 25.046 198.39 2.52 Inactive 10 4-tert-Butylcatechol, dimethyl ether C12H18O2 31.435 194.27 0.90 Inactive 11 Sulfurous acid, 2-ethylhexyl isohexyl ester C14H30O3S 33.03 294.45 0.78 Inactive 12 Undecane, 3,8-dimethyl- C13H28 34.122 184.35 0.98 Inactive 13 Pentadecane C15H32 34.649 212.42 2.72 It has inhibitory activity

[0055] Further determination of EC2 of 2EH 50 The specific procedure is as follows: Pour PDA medium into a petri dish. Inoculate the center with a 5mm diameter mushroom cake of Fusarium wilt pathogen. Place a sterile filter paper disc on the inside of the petri dish lid. Based on the total volume of the petri dish, add different amounts of the pure liquid compound to the filter paper disc to create different concentration gradient treatment groups. The total volume of the petri dish is 120cm³. 3 Different amounts of the pure liquid compound 2EH were added to filter paper, and the concentration gradient is shown in Table 2.

[0056] Table 2 Concentration gradient of pure liquid compounds with 2EH

[0057] Concentration (uL / L) Reagent volume (uL) 0.002 0.24 0.004 0.48 0.009 1.08 0.018 2.16 0.0375 4.50 0.075 9 0.15 18 CK 0.0000

[0058] Immediately after adding the solution, cover the petri dish and seal it completely with sealing film to ensure no leakage. Add an equal volume of sterile water or solvent to the control group. Incubate all petri dishes at 28°C with the pathogen. Observe regularly. When the control group colonies are about to cover the entire plate, measure the colony diameter of the pathogen using the cross-sectional method and calculate the inhibition rate at different concentrations. Figure 4 Inhibition rate curves at different concentrations were established, and statistical analysis was performed using SPSS statistical software. The EC50 of the compound *Fusarium wilt* was calculated according to the method of Vanewijk and Hoekstra (1993). 50 Value, EC 50 The smaller the value, the stronger the compound's activity.

[0059] The results showed that the virulence regression equation was: y = 3.363x + 4.448, where x is the logarithm of the agent concentration and y is the percentage of inhibition rate. The calculated EC50 of 2EH against *Fusarium wilt* var. *bacterium* was... 50 The concentration was 0.048 µL / mL. Based on the results, the inhibitory activity of different concentrations of 2EH against Fusarium wilt pathogens of banana was determined, such as... Figure 5 As shown.

[0060] 2. Inhibitory effect of derivatives of compound 2EH on Fusarium wilt pathogens of banana.

[0061] Different derivatives of compound 2EH were determined respectively, such as Figure 6 The nine derivatives shown have different EC values. 50 The method is the same as above. Regarding the inhibitory activity against banana wilt disease, two derivatives were found to have strong antibacterial activity, among which 2-ethylhexanoic acid and hexanal showed strong inhibitory activity against the EC50 of *Fusarium wilt*. 50The concentrations were 0.0058 µL / mL and 0.391 µL / mL, respectively. However, considering the corrosive nature of 2-ethylhexanoic acid and the inconvenience of its handling, which hinders its widespread use in the field, the focus will remain on developing soil disinfectants specifically for 2EH.

[0062] 3. Broad-spectrum antibacterial activity of compound 2EH

[0063] Test pathogen: Physiological race 4 of Fusarium wilt of banana Fusarium oxysporum f. sp. cubenseRace 4 (ATCC 76255) (Foc TR4), banana black spot fungus Curvularia fallax ATCC 34598, Fusarium wilt of cucumber Fusarium oxysporum f. sp. cucumerinum (ATCC 204378), mango anthracnose fungus Colletotrichum gloeosporioides ATCC 58222, Banana leaf spot pathogen Alternaria musae (ATCC66981), Banana anthracnose fungus Colletotrichum musae (ATCC 96726).

[0064] Experimental Method: Pour PDA medium into a petri dish. Inoculate the center with a 5mm diameter cake of Fusarium wilt fungus. Place a sterile filter paper sheet on the inside of the petri dish lid. Adjust the volume according to 1 EC based on the total volume of the petri dish. 50 The pure liquid compound was added dropwise onto filter paper. Immediately after addition, the plate was capped and sealed completely with sealing film to prevent leakage. An equal volume of sterile water was added to the control plate. All plates were incubated at 28°C with the pathogen. Regular observation was performed. When the control group colonies were about to cover the entire plate, the colony diameter was measured using the cross-sectional method, and the inhibition rate at different concentrations was calculated.

[0065] like Figure 7 As shown, compound 2EH exhibits broad-spectrum antifungal activity against six pathogenic fungi, with an efficacy of 1x EC50. 50 Under the specified conditions, it exhibits the strongest inhibitory effect against Fusarium wilt of banana and Fusarium wilt of cucumber.

[0066] 4. Preparation of a soil fumigant for combating Fusarium wilt with 2EH as the main component

[0067] Based on the design logic of "2EH as the core functional component + solubilizing agent + stabilizer to control volatility + synergist to enhance bactericidal effect", a water-based homogeneous liquid agent was prepared, taking into account both fumigation and diffusion properties, and adapting to the needs of banana plantation soil for the control of Fusarium wilt. The screening process followed two principles:

[0068] (1) 2EH characteristics: It is an oily liquid (25℃), insoluble in water, and has a certain volatility (boiling point 183-186℃). It is necessary to break the oil-water interface with a co-solvent to achieve water-based dispersion, while controlling the evaporation rate (to avoid too fast loss or too slow residue).

[0069] (2) Soil fumigation requirements: The agent must be able to quickly penetrate the soil pores to form a fumigation atmosphere, kill wilt pathogens, and be easily degradable with no long-term residue. It should also have low volatility (LD50) and low toxicity (2EH). 50 (3200 mg / kg) meets the basic requirements.

[0070] Based on the above principles, six formulations were designed (calculated according to the volume percentage of the fumigation agent).

[0071] Formula 1: 2-Methylhexanol 20%-30%, propylene glycol (food grade) 10%-15% cosolvent, ethylene glycol monobutyl ether 3%-5% stabilizer, menthol (natural plant extract) 1%-2% synergist, polyoxyethylene castor oil (EL-40) 2%-3% emulsifier, deionized water to 100%.

[0072] Formula 2: 2-Methylhexanol 20%-30%, solubilizer glycerin (glycerol) 12%-18%, stabilizer glyceryl monostearate 2%-4%, synergist carvacrol (oregano extract) 0.8%-1.50%, emulsifier Tween-80 (polyoxyethylene sorbitan monooleate) 2%-3%, deionized water to 100%.

[0073] Formula 3: 2-Methylhexanol 20%-30%, cosolvent polyethylene glycol 400 (PEG-400) 10%-15%, stabilizer propylene glycol methyl ether 3%-5%, synergist dodecyl dimethyl benzyl ammonium chloride (1227) 0.3%-0.50%, emulsifier, Tween-80 (2%-3%), deionized water to 100%.

[0074] Formula 4: 20%-30% 2-methylhexanol, 10%-15% propylene glycol (food grade) as a cosolvent, 2%-3% polyoxyethylene castor oil (EL-40) as an emulsifier, and deionized water to bring the total to 100%.

[0075] Formula 5: 2-Methylhexanol 20%-30%, propylene glycol (food grade) 10%-15% cosolvent, ethylene glycol monobutyl ether 3%-5% stabilizer, polyoxyethylene castor oil (EL-40) 2%-3% emulsifier, and deionized water to bring the total to 100%.

[0076] Formula 6: 2-Methylhexanol 20%-30%, propylene glycol (food grade) 10%-15% cosolvent, menthol (natural plant extract) 1%-2% synergist, polyoxyethylene castor oil (EL-40) 2%-3% emulsifier, deionized water to 100%.

[0077] Taking Formula 1 as an example, the preparation method is as follows: The following ingredients are calculated based on the preparation of 100L of reagent.

[0078] First, add 10-15 L of propylene glycol to the stirred tank and start stirring at a low speed (300-500 rpm). Slowly add 20-30 L of 2EH and stir for 10 minutes to form a homogeneous oil phase system (without obvious stratification or turbidity). Add 3-5 L of ethylene glycol monobutyl ether and continue stirring for 5 minutes to ensure that the stabilizer is fully integrated with the oil phase and to initially inhibit the volatilization of 2EH. While maintaining stirring (500 rpm), slowly add 50-60 L of deionized water to the tank using a metering pump (in 3 portions, 5 minutes apart each time) to avoid rapid addition of the aqueous phase causing stratification. Turn on the high-speed shear emulsifier and adjust the speed to 12,000 rpm. Emulsify for 15-20 minutes and observe the system state: a transparent or semi-transparent homogeneous liquid (without oil droplets or sediment) is formed, indicating that the oil and water have been fully emulsified and dispersed. Reduce the stirring speed to 300 rpm, add 1-2 L of menthol and 2-3 L of polyoxyethylene castor oil, and stir for 10 minutes to allow the synergist and emulsifier to be evenly dispersed in the system. The preparation methods for Formula 2 and Formula 3 are the same as those for Formula 1.

[0079] The efficacy, stability, and safety of the three formulations for Fusarium wilt control in soil were tested to select a suitable formulation for soil disinfection against banana wilt disease. The test results are shown in Table 3.

[0080] Table 3. Test results of different soil fumigation agent formulations

[0081] Comparison indicators stability Antibacterial rate Security Formula 1 No layering or oil separation occurred after 6 months of storage at room temperature. It remained a homogeneous system even after dilution 50-100 times and was minimally affected by temperature fluctuations (5-35℃). The inhibition rate of Fusarium wilt pathogen was 99.50%. All ingredients are low-toxicity substances; menthol is a natural extract with good biodegradability and poses no significant harm to crops. Formula 2 It does not separate into layers after 6 months of storage at room temperature, although there may be trace amounts of oil separation. It remains a homogeneous system after dilution of 50-100 times and is minimally affected by temperature fluctuations (5-35℃). The inhibition rate of Fusarium wilt pathogen was 98.70%. Glycerin, Tween-80, and carvacrol are all food-grade or natural extracts with extremely low toxicity and minimal impact on soil microbial communities; they also cause no significant harm to crops. Formula 3 Slight stratification and trace oil separation may occur after 6 months of storage at room temperature; however, it is minimally affected by temperature fluctuations (5-35℃). The wilt pathogen was inhibited at a rate of 95%. The ingredients have a certain degree of irritation, and their degradation rate in the soil is slower than that of natural synergists. Excessive use may affect beneficial soil microorganisms, making it unsuitable for use in organic agricultural soils. It is suitable for routine farmland disease control. Formula 4 After being stored at room temperature for 2 months, the material showed stratification and poor stability. Inhibition rate 94.5% All ingredients are low-toxicity substances and do not cause significant harm to crops. Formula 5 After 6 months of storage at room temperature, there was no stratification, only a trace amount of oil separation. It remained a homogeneous system after dilution 50-100 times, demonstrating good stability. Inhibition rate 95.5% All ingredients are low-toxicity substances and do not cause significant harm to crops. Formula Six After being stored at room temperature for 2 months, the material showed stratification and poor stability. Inhibition rate 97.5% All ingredients are low-toxicity substances and do not cause significant harm to crops.

[0082] Table 3 shows that different formulations significantly affect the stability, antibacterial rate, and safety of the fumigation agent. Formulation 1 exhibits the best stability, antibacterial rate, and safety. Formulations 2 through 6, with their adjusted components, show varying degrees of decrease in stability and antibacterial rate, thus impacting the effectiveness of the soil fumigation agent. Therefore, subsequent experiments will use the soil fumigation agent prepared with Formulation 1.

[0083] 5. Verify the disinfection effect of the agent on Fusarium wilt of banana (the effect on the dynamics of Fusarium wilt growth).

[0084] Experimental plan:

[0085] 1) For Foc TR4, the same concentration was used for soil inoculation. Two treatments were set up: 2EH (2EH stock solution) and 2EH preparation (i.e., the soil fumigation agent prepared by Formula 1), which were diluted by three different multiples.

[0086] 2) Prepare the spore suspension and calculate the application rate based on the final concentration in 1 kg of soil. Accurately weigh 1 kg of soil and add it to a container, taking a sample once. Then add the spore suspension, mix thoroughly, and let it stand for 5 days, taking another sample. Next, pour in the above-mentioned 2EH and 2EH formulations at different ratios, and quickly seal with plastic wrap. Take a sample of the original soil once, pour in the FOC4 spore suspension, let it stand for 5 days, take another sample, and begin the treatment. Take a sample 24 hours after treatment, and then every 4 days thereafter, for a total of 8 consecutive samplings.

[0087] 3) Sampling method: Five replicates of the original soil sample were randomly selected and mixed into one sample. Five potted soil samples were randomly selected from each treatment as five replicates. The sampling depth was 1-5 cm of the topsoil layer.

[0088] 3) Soil selection: Select non-sterilized soil from banana plantations.

[0089] 4) Foc TR4: Common FOC4 strain

[0090] Experimental Design:

[0091] Foc TR4 is set to a final concentration of 10. 4 The following 9 treatments were set up for CFU / g.soil, with 10 pots in each treatment.

[0092] 1. Control group (CK): Only FOC4 was administered.

[0093] 2. Control group (thiophanate-methyl): FOC4 + thiophanate-methyl

[0094] 3. Dilute the 2EH stock solution (the stock solution needs to be mixed with alcohol at a 1:1 ratio) 5000 times + FOC4

[0095] 4. Dilute the 2EH stock solution (the stock solution needs to be mixed with alcohol at a 1:1 ratio) 2500 times + FOC4

[0096] 5. Dilute the 2EH stock solution (the stock solution needs to be mixed with alcohol at a 1:1 ratio) 1250 times + FOC4

[0097] 6. Dilute 2EH formulation 1000 times + FOC4

[0098] 7. 2EH formulation diluted 500 times + FOC4

[0099] 8. 2EH formulation diluted 250 times + FOC4

[0100] Experimental results showed that, by tracking the number of Fusarium spores in the soil, the number of spores in different treatments exhibited a wave-like trend with increasing treatment time, and the trends were basically consistent, reaching their lowest values ​​at 18 and 34 days, respectively. The dynamic changes in the number of pathogenic spores in the control group, the drug control group, the 2EH stock solution diluted 1250 times, and the 2EH preparation diluted 250 times are shown below. Figure 8 As shown. The number of spores in each treatment at two time points, day 18 and day 34, is as follows. Figure 9 As shown, the 2EH formulation can effectively reduce the number of pathogens in the soil when diluted 250-1000 times. The antibacterial effect is best when diluted 250 times, with a spore inhibition rate of 79.68% compared with the control, while the spore inhibition rate of the control group is 45.31%. Compared with the control group, the spore inhibition rate is increased by more than 75%.

[0101] Based on the above experimental results, the recommended application method for this 2EH formulation is as follows: mix the high-efficiency soil fumigation agent with the soil and then seal it. The dosage of the high-efficiency soil fumigation agent is 10-20 L / mu, and the dilution ratio is 1:250-1000. The sealing method can be achieved by covering the soil with a film. After adding the soil fumigation agent to the soil to be disinfected, cover it with a film, and then bury the film around the edges in the soil to maintain a sealed environment for at least 18 days. To ensure the best antibacterial effect, it can be continued for 18 or 34 days.

[0102] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Kitasatospora sp. producing volatile organic compounds Kitasatospora the use of volatile organic compounds produced by Kitasatospora sp. for the preparation of a preparation for inhibiting Fusarium oxysporum f. sp. cubense and / or for the control of Panama disease, characterized in that, The preservation number of the aforementioned *Cyperus terrestris* is GDMCC NO: 64567; The volatile organic compound contains 2-ethyl-1-hexanol; the method for preparing the volatile organic compound includes the following steps: The *Tetracentron sinense* was inoculated onto YE solid culture medium, sealed, and cultured. Volatile organic matter was collected to obtain the final product.

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