Irpex bacteria, terpenoids and application of terpenoids in resisting tobacco pathogenic bacteria

Abstract

The invention discloses Irpex lacteus YYY-16, and the preservation number of the Irpex lacteus YYY-16 is GDMCC No: 67140. The invention further discloses a terpenoid and a preparation method thereof. The terpenoid has a structure shown in a formula I or a formula II. The invention further discloses application of the terpenoid in preparing tobacco pathogenic bacterium resisting drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to *Pteris vittata* species, terpenoid compounds, and their application in combating tobacco pathogens. Background Technology

[0002] In the long-term co-evolution with plant hosts, plant endophytic fungi have developed unique secondary metabolic pathways, producing numerous novel and diverse natural products, which are of great significance for the discovery of new drug lead compounds. In a systematic study of the secondary metabolites of the tobacco endophytic fungus *Irpex lacteus* YYY-16, two novel terpenoid compounds were isolated for the first time. Both compounds exhibit strong activity against *Aureobasidium aizoni* and *Wildfire*, providing new lead molecules for the development of pesticides against tobacco pathogens. This is of great value in improving the current situation where the selection of anti-tobacco pathogen drugs is limited and their efficacy is unsatisfactory. Summary of the Invention

[0003] This invention provides an endophytic fungus from tobacco. The preservation information of the biological material sample involved in this invention is as follows: the reference microorganism (strain) is YYY-16, classified and named as Irpex lacteus, and was deposited by Guangdong Provincial Microbial Culture Collection Center (GDMCC) on November 6, 2025, with accession number GDMCC No:67140. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0004] The technical solution of the present invention is as follows:

[0005] The first aspect of this invention discloses a species of fungus, Irpex lacteus YYY-16, with accession number GDMCC No:67140.

[0006] A second aspect of this invention discloses a terpene compound having the structure shown in Formula I or Formula II:

[0007]

[0008] A third aspect of this invention discloses a method for preparing the terpenoid compound, comprising the following steps:

[0009] (1) Activate the Irpex lacteus YYY-16 of claim 1;

[0010] (2) The activated strains from step (1) are cultured for two generations to obtain the fermentation product of the genus *Pteris*.

[0011] (3) Extract the *Rhizoctonia* species obtained in step (2) with methanol and ethyl acetate, and take the upper organic layer for vacuum distillation until the solvent evaporates to dryness to obtain crude extract;

[0012] (4) The crude extract obtained in step (3) is eluted by silica gel column chromatography to obtain the first eluent;

[0013] (5) The first eluent obtained in step (4) is purified using a Sephadex LH-20 dextran gel column to obtain the second eluent;

[0014] (6) The second eluent obtained in step (5) is subjected to gradient elution on a reverse-phase silica RP-C18 column, and the terpenoid compound is obtained after drying.

[0015] Preferably, the culture medium used for activating Irpex lacteus YYY-16 in step (1) is potato dextrose agar (PDA) medium, and the culture conditions are to place it in an incubator at 28°C for 2-3 days.

[0016] Preferably, in step (2), the first-generation culture uses activated *Pteris vittata*, with an inoculum amount of 5wt%-10wt% of the culture medium used. The culture medium is potato dextrose (PDB) medium, and the culture conditions are as follows: cultured in a shaker at 200 rpm and 28°C for 2-3 days to form the first-generation seed culture solution; the second-generation culture uses the first-generation seed culture solution, with an inoculum amount of 5wt%-10wt% of the culture medium used. The culture medium is rice medium, and the culture conditions are as follows: cultured in a constant temperature incubator at 25-29°C for 30-40 days; thus obtaining *Pteris vittata* fermentation product.

[0017] Preferably, in step (3), the volume ratio of the fermented product of *Pteris vittata* to the volume of methanol is 1:1, the ultrasonic power is 200-400W, the ultrasonic time is 10-20min, the ultrasonication is performed 3 times, and after each ultrasonication, there is an interval of 10-20min before the next ultrasonication is performed. The standing time is 12h, the pressure of vacuum distillation is 6-12kPa, and the temperature is 45-55℃. The obtained product is then mixed with ethyl acetate in a volume ratio of 1:1, the standing time is 12h, and extraction is performed. The pressure of vacuum distillation is 6-12kPa, and the temperature is 45-55℃.

[0018] Preferably, the specific operation steps of silica gel column chromatography elution in step (4) are as follows: the crude extract is fully dissolved in methanol solution at a ratio of 200 g / L to obtain a crude extract solution; the crude extract solution is mixed with 200 mesh silica gel, wherein the mass of silica gel powder is 0.8-1.4 times the dry weight of the crude extract; the solvent is removed by vacuum distillation at a pressure of 6-12 kPa and a temperature of 45-55℃; the column is packed with silica gel, and gradient elution is performed sequentially using mixed solutions of dichloromethane and methanol with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain eluents with volume ratios of dichloromethane and methanol of 1:0, 50:1, 30:1, 10:1, and 0:1, respectively; the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 is selected as the first eluent.

[0019] Preferably, step (5) specifically involves: removing the solvent in the first eluent by vacuum distillation at a pressure of 6-12 kPa and a temperature of 45-55°C, then dissolving it in methanol solution, purifying it using a Sephadex LH-20 dextran gel column at a flow rate of 0.3-0.6 mL / min, and using the washing solution as the second eluent.

[0020] Preferably, step (6) specifically involves: removing the solvent in the second eluent by vacuum distillation at a pressure of 6-12 kPa and a temperature of 45-55°C; then dissolving it in methanol solution and adding the resulting solution to defatted cotton, allowing it to stand and evaporate to dryness, then loading it into an RP-C18 column, and sequentially eluting with a mixed solution of methanol and water in volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 to obtain eluents with methanol and water in volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 respectively; drying to obtain two terpenoid compounds from the eluents in volume ratios of 2:3 and 1:1 methanol and water respectively.

[0021] The fourth aspect of this invention discloses the application of the terpenoid compounds in the preparation of drugs against tobacco pathogens. The terpenoid compounds exhibit strong inhibitory activity against *Aureobasidium tobaccois* and *Bacillus thuringiensis*.

[0022] The beneficial effects of this invention are:

[0023] 1. The *Irpex lacteus* YYY-16 of the present invention is an endophytic fungus from tobacco and is a new species. Its preservation number is GDMCC No:67140.

[0024] 2. The two terpenoid compounds of this invention are newly discovered compounds, which can enrich the diversity of terpenoid compounds. They have good anti-tobacco star fungus and wildfire fungus activity, and can be used to prepare anti-tobacco pathogen drugs, providing a new approach for the development and research of anti-tobacco pathogen drugs.

[0025] 3. Screening tests of the two terpenoid compounds against *Aureobasidium tobaccois* and *Bacillus thuringiensis* showed that the two terpenoid compounds had strong inhibitory activity against *Aureobasidium tobaccois* and *Bacillus thuringiensis*.

[0026] 4. The present invention uses the fermentation and separation method of Irpex lacteus YYY-16 to prepare two terpenoid compounds. It has the advantages of high efficiency, low cost and mild conditions, and is easy to industrialize. It can also provide a new option for the mass production of terpenoid compounds. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described; unless otherwise specified, the reagents used in the present invention are all conventional commercially available reagents.

[0028] The strain Irpex lacteus YYY16 used in this invention has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:67140. Therefore, this strain can be purchased from the Guangdong Provincial Center for Microbial Culture Collection.

[0029] Preparation of the culture medium used in this invention:

[0030] Rice culture medium: Soak rice for 12 hours, then put it into a 300mL bottle, with the rice occupying one-third of the bottle; place the prepared culture in a 121℃ high-temperature sterilizer for 30 minutes, then remove it, cool it and store it for later use.

[0031] Potato glucose medium (PDB medium): Take 200g of washed and peeled potatoes, cut them into pieces about 1cm in diameter, add water and boil until the potato pieces can be pierced with a glass rod; filter with gauze to obtain the filtrate, add 20g of glucose to the filtrate, and add water to 1000mL; place the prepared culture in a high-temperature sterilizer at 121℃ for 30 minutes, remove it, cool it and store it for later use.

[0032] Potato Dextrose Agar Medium (PDA): Take 200g of washed and peeled potatoes, cut them into pieces about 1cm in diameter, add water and boil until the potato pieces can be pierced with a glass rod; filter the filtrate through gauze, add 20g of glucose and 18g of agar powder to the filtrate, and add water to 1000mL; sterilize the prepared culture in a 121℃ autoclave for 30 minutes, then remove, cool and store for later use.

[0033] Example 1: Preparation of two terpenoid compounds, the specific operation steps are as follows:

[0034] (1) Irpex lacteus YYY-16 was inoculated onto PDA medium and placed in an incubator for 2 days at a temperature of 28℃.

[0035] (2) The activated Irpex lacteus YYY-16 from step (1) was inoculated into PDB medium at a rate of 10% and cultured at 28°C and 200 rpm for 3 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation rice medium at a rate of 10% and cultured at 28°C for 30 days to obtain the Irpex lacteus ferment.

[0036] (3) The fermentation product of *Pteris vittata* obtained in step (2) was thoroughly mixed with methanol. The volume ratio of the solid fermentation product of *Pteris vittata* to methanol was 1:1. The ultrasonic power was 400W, the ultrasonic time was 10min, and the ultrasonication was performed 3 times. After each ultrasonication, there was a 10min interval before the next ultrasonication. The mixture was allowed to stand for 12h. The upper organic phase solution was then subjected to vacuum distillation at a pressure of 9kPa and a temperature of 50℃. The volume ratio of the obtained product to ethyl acetate was 1:1. The mixture was allowed to stand for 12h and then extracted at a pressure of 9kPa and a temperature of 50℃ to obtain the crude extract.

[0037] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (200 mesh). The mass of the silica gel used is 1.2 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 9 kPa and a temperature of 50°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions of volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents of volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution of volume ratio of 30:1 as the first eluent.

[0038] (5) Remove the solvent in the eluent 1 obtained in step (4) by vacuum distillation at a pressure of 9 kPa and a temperature of 50 °C. Dissolve the solute in methanol solution to obtain a solution. Purify the solution using a Sephadex LH-20 dextran gel column at a flow rate of 0.4 mL / min. Use the eluent as the second eluent.

[0039] (6) The solvent of the second eluent obtained in step (5) was removed by vacuum distillation at a pressure of 9 kPa and a temperature of 50 °C. The solute was dissolved in methanol solution to obtain a solution. The solution was added dropwise to cotton in three portions, allowed to stand and evaporate, and then packed into an RP-C18 column. Gradient elution was performed using methanol-water mixed solutions with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 to obtain methanol-water eluents with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0, respectively. The eluents were dried, and two terpenoid compounds were obtained from the methanol and water volume ratios of 2:3 and 1:1, respectively.

[0040] The terpenoid compounds obtained in this embodiment were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry).

[0041] From HSQC spectrum and 13 The chemical shifts δ of H and C of this terpene compound can be obtained from the C-spectrum, as shown in Tables 1 and 2.

[0042] Table 1. Terpenoids of Formula I prepared in Example 1 13 C and 1 HNMR data, CD3OD is the solvent.

[0043] No. <![CDATA[δ C ]]> <![CDATA[δ H (J in Hz)(The space represents C atoms without H)]]> 1 175.9,C 2 130.0,C 3 66.9,CH 4.52,m 4 <![CDATA[37.8,CH2]]> 2.05,ddd(10.88,8.52,13.8); 1.75,ddt(13.8,6.1,1.1) 5 30.3,CH 1.85,m 6 48.7, CH 2.62,m 7 <![CDATA[40.2,CH2]]> 2.11,dd(13.84,8.44); 1.53,dd(13.8,8.8) 8 45.2,C 9 76.9, CH 3.62,s 10 69.1,C 11 158.8,C 12 <![CDATA[71.0,CH2]]> 4.74,dd(18.0,2.4); 4.54,dd(18.0,1.2) 13 <![CDATA[20.3,CH3]]> 1.00,d(7.2) 14 <![CDATA[68.9,CH2]]> 3.57,s 15 <![CDATA[19.9,CH3]]> 1.11,s

[0044] HR-ESI-MS m / z of terpenoid compounds: [M+Na] + The quasi-molecular ion peak is at 303.1205 (C). 15 H 20 O5[M+Na] + The calculated value is 303.1203, indicating that its molecular formula is C. 12 H 16 O3 contains six degrees of unsaturation. Through the analysis of compounds... 1 H, 13 C indicates that the compound contains 15 carbon signals and two methyl groups (δ-C). C 20.3, 19.9, δ H 1.00, 1.11), four methylene groups (δ C 37.8, 40.2, 71.0, 68.9, δ H 2.05, 1.75, 2.11, 1.53, 4.74, 4.54, 3.75), four methines (δ C 66.9, 30.3, 48.7, 76.9, δ H 4.52, 1.85, 2.62, 3.62), and five quaternary carbons (δ).C 175.9, 130.0, 45.2, 69.1, 158.8).

[0045] Analysis of the 2D nuclear magnetic resonance spectra of terpenoids revealed, through the HMBC plot, that H-12 (δ) H 4.74) to C-1(δ C 175.9), C-2(δ) C The correlations between 130.0), C-11 (158.8), and H2-12 (δ) H 4.54) to C-10(δ C The correlation of 69.1), H-9(δ) H 3.62) to C-7(δ) C 40.2), C-8(δ) C

[0046] 45.2), C-10(δ) C 69.1), C-11(δ) C 158.8), C-14 (δ) C 68.9), H-14 (δ) H 3.57) to C-15 (δ) C 19.9), C-7 (δ) C 40.2), C-8(δ) C HMBC correlation of 45.2), H-6 (δ) H

[0047] 2.62) and C-4(δ) C 37.8), C-7 (δ) C 40.2), C-10(δ) C 69.1), C-9 (δ) C 76.9), C-5 (δ) C HMBC correlation of 30.3), H-7 (δ) H 2.11) to C-15 (δ) C 19.9), C-5 (δ) C 30.3), C-8(δ) C 45.2), C-10(δ) C HMBC correlation of 69.1), H-4 (δ) H 2.05) to C-5(δ C 30.3), C-2(δ) C The correlations between 130.0), C-3 (66.9), and H2-4 (δ) H 1.75) to C-13 (δ C The correlation between 20.3), C-1 (175.9), and H-5 (δ)H 1.85) to C-13 (δ) C 20.3), C-3(δ) C The correlation of 66.9), H-15 (δ) H 1.11) to C-8(δ) C 45.2), C-9 (76.9), H-13 (δ) correlation. H

[0048] 1.00) to C-4(δ) C The correlation between C-6 (37.8) and C-6 (48.7) is used. Combined with the molecular formula, the planar structure of the terpenoid compound is thus derived.

[0049] Analysis of the 2D NMR spectra of terpenoid compounds revealed a correlation between H-4 / H-14 / H-9 / H-10, indicating that H-4, H-14, H-9, and H-10 share the same spatial orientation, as did H-5 / H-6, indicating that H-5 and H-6 share the same spatial orientation. Combined with the molecular formula, the relative stereostructures of the terpenoid compounds were thus determined.

[0050] In summary, the structural formula of compound I prepared in Example 1 can be determined as follows:

[0051] Table 2 shows the terpenoid compounds of formula II prepared in Example 1. 13 C and 1 HNMR data, CD3OD is the solvent.

[0052] No. <![CDATA[δ C ]]> <![CDATA[δ H (J in Hz)(The space represents C atoms without H)]]> 1 175.8,C 2 130.1,C 3 66.7,CH 4.56,m 4 <![CDATA[38.5,CH2]]> 1.88,m 5 30.4,CH 1.85,m 6 48.4, CH 2.60,m 7 <![CDATA[40.0,CH2]]> 1.70,dd(8.2,6.1) 8 45.0,C 9 76.4, CH 3.54,s 10 68.7,C 11 159.0,C 12 <![CDATA[70.9,CH2]]> 4.75,dd(18.0,2.3); 4.57,dd(18.08,1.2) 13 <![CDATA[20.0,CH3]]> 1.02,d(7.0) 14 <![CDATA[67.9,CH2]]> 3.37,d(1.1); 3.63,d(10.5) 15 <![CDATA[22.0,CH3]]> 1.28,s

[0053] HR-ESI-MS m / z of terpenoid compounds: [M+Na] + The quasi-molecular ion peak is at 303.1205.

[0054] (C 15 H 20 O5[M+Na] + The calculated value is 303.1203, indicating that its molecular formula is C. 12 H 16 O3 contains six degrees of unsaturation. Through the analysis of compounds... 1 H, 13 C indicates that the compound contains 15 carbon signals and two methyl groups (δ-C). C 20.0, 22.0, δ H 1.02, 1.28), four methylene groups (δ C 38.5, 40.0, 70.9, 67.9, δ H1.88, 1.70, 4.75, 4.57, 3.37, 3.63), four methines (δ C 66.7, 30.4, 48.4, 76.4, δ H 4.56, 1.86, 2.60, 3.54), and five quaternary carbons (δ). C 175.8, 130.1, 45.0, 68.7, 159.0).

[0055] Analysis of the 2D nuclear magnetic resonance spectra of terpenoids revealed, through the HMBC plot, that H-12 (δ) H 4.75) to C-2(δ C The correlation between 130.1), C-11 (159.0), and H2-12 (δ) H 4.57) to C-1(δ C The correlation between 175.8) and H-9 (δ) H 3.54) to C-7 (δ) C 40.0), C-8 (δ) C 45.0), C-11 (δ) C

[0056] 159.0), H-14 (δ) H 3.63) to C-15 (δ) C 22.0), C-7 (δ) C 40.0), C-8 (δ) C 45.0), C-9 (δ) C HMBC correlation of 76.4), H-6 (δ) H 2.60) and C-4(δ) C 38.5), C-10 (δ) C

[0057] 68.7), C-9 (δ) C 76.4), C-5 (δ) C HMBC correlation of 30.4), H-7 (δ) H 1.70) to C-15 (δ) C 22.0), C-5 (δ) C 30.4), C-8 (δ) C 45.0), C-10 (δ) C HMBC correlations of C-6 (68.7) and C-6 (48.4), and H-4 (δ) H 1.88) to C-5 (δ) C 30.4), C-2(δ) C The correlation between 130.1), C-3 (66.7), and H-5 (δ)H 1.86) to C-13 (δ) C 20.0), C-3(δ) C The correlation of 66.7), H-15 (δ) H

[0058] 1.28) to C-8(δ) C The correlation between C-9 (76.4) and H-13 (δ) is 45.0). H 1.02) to C-4(δ C

[0059] The correlation between C-6 (38.5) and C-6 (48.4) was used. Combined with the molecular formula, the planar structure of the terpenoid compound was thus derived.

[0060] Analysis of the 2D NMR spectra of terpenoid compounds revealed a correlation between H-4, H-9, and H-10, indicating a shared spatial orientation, as well as a correlation between H-5, H-6, and H-14, suggesting a shared spatial orientation. Combined with the molecular formula, the relative stereostructures of the terpenoid compounds were thus determined.

[0061] In summary, the structural formula of compound II prepared in Example 1 can be determined as follows:

[0062] Example 2: Preparation of two terpenoid compounds, the specific operation steps are as follows:

[0063] (1) Irpex lacteus YYY-16 was inoculated onto PDA medium and placed in an incubator for 3 days at a temperature of 28℃.

[0064] (2) The activated Irpex lacteus YYY-16 from step (1) was inoculated into PDB medium at a rate of 5% and cultured at 28°C and 200 rpm for 2 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation PDA medium at a rate of 5% and cultured at 28°C for 40 days to obtain the Irpex lacteus ferment.

[0065] (3) The fermentation product of *Pteris vittata* obtained in step (2) is thoroughly mixed with methanol. The volume ratio of fermentation product to methanol is 1:1. The ultrasonic power is 200W, the ultrasonic time is 15min, and the ultrasonication is performed 3 times. After each ultrasonication, there is a 15min interval before the next ultrasonication. After standing for 12h, the upper organic phase solution is taken and subjected to vacuum distillation at a pressure of 6kPa and a temperature of 45℃. The volume ratio of the obtained product to ethyl acetate is 1:1. After standing for 12h, extraction is performed at a pressure of 6kPa and a temperature of 45℃ to obtain the crude extract.

[0066] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (200 mesh). The mass of the silica gel used is 0.8 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 6 kPa and a temperature of 45°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 as the first eluent.

[0067] (5) Remove the solvent in the first eluent obtained in step (4) by vacuum distillation at a pressure of 6 kPa and a temperature of 45 °C. Dissolve the solute in methanol solution to obtain a solution. Add the solution dropwise to cotton in three portions, let it stand to evaporate, and then pack it into an RP-C18 column. Perform gradient elution with methanol-water mixed solutions with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 to obtain methanol-water eluents with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 respectively. Select the eluent with a methanol-water volume ratio of 1:1 as the second eluent.

[0068] (6) The solvent of the second eluent obtained in step (5) was removed by vacuum distillation at a pressure of 6 kPa and a temperature of 45 °C. The solvent was methanol, and the solution was purified using a dextran gel (Sephadex LH-20) column at a flow rate of 0.3 mL / min. After drying, two terpenoid compounds were obtained.

[0069] The structures of the two terpenoid compounds obtained in this example were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). The results were the same as in Example 1.

[0070] Example 3: Preparation of two terpenoid compounds, the specific operation steps are as follows:

[0071] (1) Irpex lacteus YYY-16 was inoculated onto PDA medium and placed in an incubator for 3 days at a temperature of 28℃.

[0072] (2) The activated Irpex lacteus YYY-16 from step (1) was inoculated into PDB medium at a rate of 5% and cultured at 28°C and 200 rpm for 3 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation rice medium at a rate of 10% and cultured at 28°C for 40 days to obtain the Irpex lacteus ferment.

[0073] (3) The fermentation product of the genus *Pteris* obtained in step (2) is thoroughly mixed with ethyl acetate. The volume ratio of the fermentation product to ethyl acetate is 1:1. The mixed solution is placed in an ultrasonic extractor for ultrasonic extraction. The ultrasonic extraction conditions are 300W power for 20 minutes, a total of 3 times, with an interval of 20 minutes between each time. After ultrasonic extraction, the mixture is allowed to stand for 12 hours. The upper organic phase solution is taken and subjected to vacuum distillation until dry. The pressure of vacuum distillation is 12 kPa and the temperature is 55℃ to obtain the crude extract.

[0074] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (300 mesh). The mass of the silica gel used is 1.4 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 12 kPa and a temperature of 55°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 as the first eluent.

[0075] (5) Remove the solvent in the first eluent obtained in step (4) by vacuum distillation at a pressure of 12 kPa and a temperature of 55 °C. Dissolve the solute in methanol solution to obtain a solution. Add the solution dropwise onto cotton in three portions, let it stand to evaporate, and then pack it into an RP-C18 column. Perform gradient elution with methanol-water mixed solutions with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 to obtain methanol-water eluents with methanol and water volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 respectively. Select the eluent with a methanol-water volume ratio of 1:1 as the second eluent.

[0076] (6) The solvent of the eluent 2 obtained in step (5) was removed by vacuum distillation at a pressure of 12 kPa and a temperature of 55 °C. The solvent was methanol, and the solution was purified using a dextran gel (Sephadex LH-20) column at a flow rate of 0.6 mL / min. After drying, two terpenoid compounds were obtained.

[0077] The structures of the two terpenoid compounds obtained in this example were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). The results were the same as in Example 1.

[0078] Example 4: Test of the activity of terpenoid compounds against Tobacco Star Bacterium and Wildfire Bacteria.

[0079] The MICs of two terpenoid compounds against *Erythromyces boulardii* and *Bacillus thuringiensis* were determined using a 96-well plate two-fold dilution method.

[0080] Methods: PDA slant tubes inoculated with *Tobacco Star Fever* and *Wildfire Bacteria* were incubated at 28°C for 96 hours. 1 mL of sterile water was added to each slant tube, and the suspensions were then transferred to PDB medium to prepare bacterial suspensions. A certain amount of the two terpenoid compounds prepared in Example 1 and the positive control nystatin were dissolved in 50% DMSO aqueous solution. The samples were added to 96-well plates using a 2-fold serial dilution method and diluted to 50 μL. Then, 100 μL of the corresponding pathogen suspension was added, bringing the final compound concentration to 512 μg / mL - 0.5 μg / mL, with the final DMSO concentration not exceeding 5%. Simultaneously, 100 μL of indicator bacterial suspension and 7.5 μL of DMSO solution were added to the last well of the 96-well plate and diluted to 150 μL as a negative control. The 96-well plates containing the fungal pathogen indicator bacteria were incubated at 28°C for 18 hours. The growth of pathogen indicator bacteria can be observed with the naked eye. The minimum concentration of the compound at which no pathogen growth occurs is taken as the minimum inhibitory concentration (MIC) of the sample to be tested. Three replicates are performed for each sample.

[0081] The experimental results are shown in Table 3. Table 3 shows that in the experiment of anti-*A. tobacco star spp.* and *Bacillus thuringiensis* (positive control: nystatin), both compounds exhibited strong inhibitory activity against both pathogens (MICs of 1-4 μg / mL). Compared with the positive control nystatin, both terpenoid compounds showed comparable or even superior inhibitory activity against these two pathogens. Therefore, the two terpenoid compounds provided by this invention demonstrate significant research value in the development of pesticides against *A. tobacco star spp.* and *Bacillus thuringiensis*. Thus, the method of obtaining terpenoid compounds with anti-*A. tobacco star spp.* and *Bacillus thuringiensis* through bacterial fermentation not only achieves high efficiency, low cost, and convenient operation, but also provides an option for the further mass production of these two terpenoid compounds.

[0082] Table 3. Minimum inhibitory concentrations (μg / mL) of the compounds.

[0083] sample Tobacco star fungus Wildfire pathogens Compound I 1 2 Compound II 2 4 Nystatin 2 4

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fungus of the genus Irpex, Irpex lacteus YYY-16, characterized in that, It has a preservation number of GDMCC No: 67140.

2. A terpenoid compound, characterized in that, It has a structure shown in formula I or formula II:

3. The method for preparing terpenoid compounds according to claim 2, characterized in that, It comprises the following steps: (1) activating the Irpex lacteus YYY-16 in claim 1; (2) culturing the activated strain in step (1) for two generations to obtain the Irpex lacteus fermentation product; (3) extracting the Irpex lacteus obtained in step (2) with methanol and ethyl acetate, taking the upper organic layer to perform vacuum distillation until the solvent is evaporated to dryness to obtain a crude extract; (4) performing silica gel column chromatography elution on the crude extract obtained in step (3) to obtain a first eluate; (5) purifying the first eluate obtained in step (4) with a dextran gel Sephadex LH-20 column to obtain a second eluate; (6) performing gradient elution on the second eluate obtained in step (5) with a reversed-phase silica gel RP-C18 column, and drying to obtain the terpenoid compound.

4. The method for preparing terpenoid compounds according to claim 3, characterized in that, The culture medium for activating the Irpex lacteus YYY-16 in step (1) is potato dextrose agar (PDA) medium, and the culture conditions are placed in a 28°C incubator and cultured for 2-3 days.

5. The method for preparing terpenoid compounds according to claim 3, characterized in that, The activated Irpex lacteus is used for one-generation culture in step (2), and the inoculation amount is 5wt%-10wt% of the inoculation amount of the used culture medium, the culture medium is potato dextrose (PDB) medium, and the culture conditions are to form a one-generation seed culture in a shaker with a rotation speed of 200 rpm and a temperature of 28°C for 2-3 days; the one-generation seed culture is used for two-generation culture, and the inoculation amount is 5wt%-10wt% of the used culture medium, the culture medium is rice medium, and the culture conditions are to culture in a thermostat with a temperature of 25-29°C for 30-40 days; and the Irpex lacteus fermentation product is obtained.

6. The method for preparing terpenoid compounds according to claim 3, characterized in that, In step (3), the volume ratio of the Irpex lacteus fermentation product to methanol is 1:1, the ultrasonic power is 200-400W, the ultrasonic time is 10-20min, the ultrasonic is performed for 3 times, each time is separated by 10-20min, the standing time is 12h, the pressure for vacuum distillation is 6-12kPa, and the temperature is 45-55°C; the obtained product is mixed with ethyl acetate at a volume ratio of 1:1, the standing time is 12h, the extraction is performed, the pressure for vacuum distillation is 6-12kPa, and the temperature is 45-55°C.

7. The method for preparing terpenoid compounds according to claim 3, characterized in that, The specific operation steps of the silica gel column chromatography elution in step (4) are as follows: the crude extract is dissolved in methanol solution at a ratio of 200 g / L to obtain a crude extract solution, the crude extract solution is mixed with 200 mesh silica gel, and the mass of the silica gel powder is 0.8-1.4 times the dry weight of the crude extract; the solvent is removed by distillation under reduced pressure, the pressure of the distillation under reduced pressure is 6-12 kPa, and the temperature is 45-55°C; the silica gel column is loaded, and gradient elution is performed using mixed solutions of dichloromethane and methanol with volume ratios of 1:0, 50:1, 30:1, 10:1 and 0:1, respectively, to obtain eluents with volume ratios of 1:0, 50:1, 30:1, 10:1 and 0:1, respectively; and the eluent of the mixed solution of dichloromethane and methanol with a volume ratio of 30:1 is selected as the first eluent.

8. The method for preparing the terpenoid compound according to claim 3, characterized in that, Step (5) is specifically: the solvent in the first eluent is removed by distillation under reduced pressure at a pressure of 6-12 kPa and a temperature of 45-55°C, then dissolved in a methanol solution, and purified using a dextran gel column Sephadex LH-20 at a flow rate of 0.3-0.6 mL / min, and the eluent is used as the second eluent.

9. The method for preparing the terpenoid compound according to claim 3, characterized in that, Step (6) is specifically: the solvent in the second eluent is removed by distillation under reduced pressure at a pressure of 6-12 kPa and a temperature of 45-55°C; then dissolved in a methanol solution to obtain a solution, which is added to defatted cotton, and the volatile is allowed to dry, then loaded into an RP-C18 column, and gradient elution is performed using mixed solutions of methanol and water with volume ratios of 1:4, 2:3, 1:1, 3:2 and 1:0, respectively, to obtain eluents with volume ratios of 1:4, 2:3, 1:1, 3:2 and 1:0, respectively; dried, and two terpenoid compounds are obtained from the eluents with volume ratios of 2:3 and 1:1, respectively.

10. The use of the terpenoid compound according to claim 2 for preparing an anti-tobacco pathogenic fungus drug.

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