A neurospora crassa and applications thereof

Abstract

The application belongs to the technical field of microorganisms and particularly relates to a kind of Aspergillus ustus and application thereof.The Aspergillus ustus is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.40820.The Aspergillus ustus can be separated to obtain sesquiterpenes shown in formula I and formula II, and the sesquiterpenes have good bacteriostatic activity on Physarum polycephalum, and the bacteriostatic rates are 79.45% and 76.71% respectively at a final concentration of 100 μg / mL, which is equivalent to the control agent of bercyn in inhibiting mycelial growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Aspergillus pyrolyticus and its applications. Background Technology

[0002] Cucumber, as a major vegetable crop in my country, plays a crucial role in the country's food security. Anthracnose, caused by *Colletotrichum orbiculare*, is one of the main factors leading to significant yield reductions in cucumbers. Cucumber anthracnose seriously threatens cucumber production worldwide. This pathogen can infect more than 40 plant species globally, and its incidence is severe in protected cultivation, causing substantial economic losses.

[0003] Using resistance breeding is the most economical, safe, and effective method for controlling cucumber anthracnose. However, the current severe shortage of resistance resources makes it difficult to achieve fundamental control of the disease. While biological control aligns with environmental protection principles, its inherent limitations hinder its commercialization for anthracnose control at present. Currently, large-scale cucumber production relies heavily on chemical pesticides for anthracnose control. However, the increasing resistance of *Colletotrichum spp.* to traditional chemical pesticides has led to a continuous decline in the effectiveness of local control measures, making long-term effective disease control difficult.

[0004] Therefore, it is imperative to develop new pesticides to control cucumber anthracnose caused by Colletotrichum spp. in order to alleviate the problems of resistance and pollution associated with traditional chemical pesticides currently in use. Summary of the Invention

[0005] This invention discovers a fungus, *Aspergillus ustus* (hereinafter also referred to as *Aspergillus ustus* SN-56147), from which two sesquiterpenoid compounds were isolated. These compounds exhibit good antifungal activity against *Colletotrichum spp.*, with inhibition rates of 79.45% and 76.71% respectively at a final concentration of 100 μg / mL. Their performance in inhibiting mycelial growth is comparable to that of the control fungicide, chlorothalonil. Therefore, the sesquiterpenoid compounds prepared in this invention can be used as pesticides for controlling cucumber anthracnose, effectively mitigating the resistance problems arising from the current use of traditional chemical pesticides for controlling cucumber anthracnose. Furthermore, this invention can reduce environmental pollution and pesticide poisoning in humans and animals, thus promoting safe cucumber production.

[0006] In one aspect, this invention provides a strain of Aspergillus ustus, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40820.

[0007] Another aspect of the present invention provides a composition comprising one or more of the following substances: (a) Aspergillus pyrolyticus of the present invention; (b) pyrolyticus pyrolyticus of the present invention; (c) culture of Aspergillus pyrolyticus of the present invention; (d) fermentation broth of Aspergillus pyrolyticus of the present invention.

[0008] In another aspect, the present invention provides a method for preparing sesquiterpenoid compounds, the structural formula of which is shown in Formula I or Formula II below. The preparation method includes: obtaining them by fermentation with Aspergillus pyrolysis of the present invention.

[0009]

[0010]

[0011] In another aspect, the present invention provides the use of Aspergillus pyroximate of the present invention or the composition thereof as a pesticide for the prevention and treatment of cucumber anthracnose.

[0012] In some specific embodiments, the above applications include: the use of Aspergillus pyrolyticus or the compositions of the present invention as inhibitors of or in the preparation of Colletotrichum spp.

[0013] In another aspect, this invention provides the application of sesquiterpenoid compounds in pesticides for controlling cucumber anthracnose, wherein the structural formula of the sesquiterpenoid compound is shown in Formula I or Formula II below.

[0014]

[0015]

[0016]

[0017] In some specific embodiments, the above applications include: the use of sesquiterpenoid compounds in inhibiting Colchicote sarcodactylis.

[0018] The preservation information of Aspergillus ustus in this invention is as follows: Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: September 12, 2023; Preservation number: CGMCC No. 40820; Classification and name: Aspergillus ustus.

[0019] The beneficial effects of this invention include at least the following: Aspergillus ustus provided by this invention can be used to isolate sesquiterpenoid compounds represented by Formula I and Formula II. These sesquiterpenoid compounds have good antibacterial activity against Colletotrichum spp., with inhibition rates of 79.45% and 76.71% respectively at a final concentration of 100 μg / mL. They are comparable to the control agent chlorothalonil in inhibiting mycelial growth. Attached Figure Description

[0020] Figure 1 Morphological image of Aspergillus pyrolyticus SN-56147;

[0021] Figure 2 The 1H NMR spectrum of sesquiterpene compound 1;

[0022] Figure 3 The carbon NMR spectrum of sesquiterpene compound 1;

[0023] Figure 4 The results are from the single-crystal diffraction of sesquiterpene compound 1.

[0024] Figure 5 The 1H NMR spectrum of sesquiterpene compound 2;

[0025] Figure 6 The carbon NMR spectrum of sesquiterpene compound 2;

[0026] Figure 7 The inhibitory effect of sesquiterpene compound 1 on the mycelial growth of Colchicum spp. under a concentration gradient;

[0027] Figure 8 The inhibitory effect of sesquiterpene compound 2 on the mycelial growth of Colchicum spp. under a concentration gradient;

[0028] Figure 9 The inhibitory effects of sesquiterpenoid compounds 1 and 2 on the mycelial growth of Colletotrichum spp. at a concentration of 100 μg / mL were investigated.

[0029] Figure 10 The effects of sesquiterpenoid compounds 1 and 2 on the control of cucumber anthracnose were studied. Detailed Implementation

[0030] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0031] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0032] This invention provides a strain of Aspergillus ustus, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40820.

[0033] It should be noted that the Aspergillus ustus provided by this invention can be used to isolate sesquiterpenoid compounds represented by Formula I and Formula II. These sesquiterpenoid compounds have good antibacterial activity against Colletotrichum spp., with inhibition rates of 79.45% and 76.71% respectively at a final concentration of 100 μg / mL. They are comparable to the control agent chlorothalonil in inhibiting mycelial growth.

[0034] In some specific embodiments, the 16S rDNA sequence of Aspergillus pyrolysis described above includes the sequence shown in SEQ ID NO.1.

[0035] Another embodiment of the present invention provides a composition comprising one or more of the following substances: (a) Aspergillus pyrolyticus of the present invention; (b) pyrolyticus pyrolyticus of the present invention; (c) culture of Aspergillus pyrolyticus of the present invention; (d) fermentation broth of Aspergillus pyrolyticus of the present invention.

[0036] It should be noted that, as mentioned above, when *Aspergillus pyroximate* is prepared into a composition, the bacteria can be introduced directly as live bacteria to exert its effect, or the bacteria can be inactivated using existing technologies and then introduced into the composition in an inactivated form to exert its effect; alternatively, the lysates of the bacteria can be introduced into the composition to exert its effect; or products such as proteins, peptides, secretions, or metabolites obtained from the culture of the bacteria can be introduced into the composition to exert its effect; or the fermentation broth after the fermentation of the bacteria can be introduced into the composition to exert its effect. In specific use, different forms of the bacteria can be selected to prepare the composition to exert its effect according to specific needs.

[0037] Another embodiment of the present invention provides a method for preparing a sesquiterpene compound, the structural formula of which is shown in Formula I or Formula II below. The preparation method includes: obtaining it by fermentation with Aspergillus pyrolysis of the present invention.

[0038]

[0039]

[0040] In some specific embodiments, the above preparation method includes: (1) incubating PDB culture medium with Aspergillus pyrolysis at a constant temperature; when the colony diameter grows to about 7 cm, punching holes at the edge of the colony with a 5 mm punch and inoculating the bacterial cake into a 25 mL test tube as the primary liquid seed; (2) incubating the fermented primary liquid seed with PDB culture medium, and taking out the secondary liquid seed when the bacterial solution becomes turbid and bacterial blocks are formed; (3) mixing the fermented secondary liquid seed with PDB culture medium and macroporous adsorption resin for fermentation; (4) after the overall fermentation is completed, washing, collecting macroporous adsorption resin, and drying; after drying the resin, eluting with methanol to obtain the eluent; after the eluent is concentrated under reduced pressure, the fermented crude extract is obtained; the fermented crude extract is dissolved with methanol, and then an equal volume of distilled water and dichloromethane are added for extraction, and the dichloromethane phase is collected and concentrated under reduced pressure to obtain the dichloromethane crude extract extract.

[0041] In some specific embodiments, the above preparation method further includes: (5) using a petroleum ether-ethyl acetate system as the eluent for primary silica gel column chromatography to perform gradient elution of the crude dichloromethane extract, using petroleum ether, petroleum ether to ethyl acetate volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16 and 1:1 respectively, and ethyl acetate as the gradient eluent for normal phase silica gel column chromatography, with 0.6 L of mobile phase eluted each time; after primary silica gel column chromatography, the fractions are combined into five components A, B, C, D and E according to their polarity; (6) the mass, TLC and solubility of component A4 are comprehensively analyzed, and finally a 200-300 mesh silica gel column is selected, and dry loading is used. The A4 component was separated and purified by dry column packing using an eluent of V hexane:V dichloromethane = 7:3. The separated components were analyzed by TLC and the target components were combined. (7) The target components were further separated and purified by gel column chromatography with an eluent of V petroleum ether:V methanol:V dichloromethane = 2:1:1. The separated components were analyzed by TLC and the target components were combined according to the detection results to obtain the compound shown in Formula I. Alternatively, the D6 component was subjected to gel column chromatography with V methanol:V dichloromethane = 1:1, silica gel column chromatography with V dichloromethane:V methanol = 100:1, and gel column chromatography with V petroleum ether:V methanol:V dichloromethane = 2:1:1 to obtain the compound shown in Formula II.

[0042] In some specific embodiments, the above preparation method includes:

[0043] (1) Add 5 mL of LPDB culture medium to a 25 mL test tube and sterilize at 121 °C for 30 min; inoculate Aspergillus pyrolyticus SN-56147 stored in a -80 °C refrigerator into PDA medium and culture at 25 °C for 7 days; when the colony diameter grows to about 7 cm, punch holes at the edge of the colony with a 5 mm punch and inoculate the mycelium into a 25 mL test tube as a primary liquid seed. After inoculation, place all the test tubes in a shaker and culture at 180 r / min and 25 °C for 48 h. Remove the tubes when the bacterial solution becomes turbid.

[0044] (2) Transfer all the fermented primary seed to 250mL Erlenmeyer flasks as secondary liquid seed. Add 50mL of LPDB medium to each Erlenmeyer flask, sterilize at 121℃ for 30min, shake at 180r / min and 25℃ for 48h, and remove when the bacterial solution becomes turbid and bacterial clumps form.

[0045] (3) Transfer all the fermented secondary seed to 2L Erlenmeyer flasks. Add 400ml of LPDB medium and 16g of macroporous adsorption resin to each Erlenmeyer flask. Sterilize at 121℃ for 30min, shake at 180r / min and 25℃ for 7d, and ferment a total of 24L.

[0046] (4) After the overall fermentation was completed, a total of 24L of Aspergillus pyrolysis SN-56147 fermentation broth was obtained. The Aspergillus pyrolysis SN-56147 fermentation broth was rinsed multiple times with distilled water to remove all the bacteria and culture medium from the fermentation broth. The macroporous adsorption resin was collected and all the macroporous adsorption resin in the bottle was gathered into a tray and placed in an oven to dry at 30℃. After the resin was dried, it was placed in a 2L separatory funnel and an appropriate amount of methanol was added for elution. The methanol was replaced every 4 hours and the elution was repeated 5 times in total, including one overnight soak. The eluent was collected and combined. The eluent was concentrated under reduced pressure to obtain the crude fermentation extract. The crude fermentation extract was dissolved in methanol and then an equal volume of distilled water and dichloromethane were added for extraction. The extraction was repeated 5 times in total. The dichloromethane phase was collected and concentrated under reduced pressure to obtain the dichloromethane crude extract extract.

[0047] (5) The crude dichloromethane extract was eluted using a petroleum ether-ethyl acetate system as the eluent for primary silica gel column chromatography. Petroleum ether, petroleum ether to ethyl acetate volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16, and 1:1 were selected as the gradient eluents for normal-phase silica gel column chromatography, with 0.6 L of mobile phase eluted each time. After primary silica gel column chromatography, the fractions were combined into five components, A, B, C, D, and E, according to their polarity.

[0048] (6) After comprehensive analysis of the mass, TLC and solubility of component A4, a 25g silica gel column (200-300 mesh) was finally selected. The A4 component was separated and purified by dry loading and dry column packing with an elution buffer of V n-hexane:V dichloromethane = 7:3. The separated components were analyzed by TLC and the target components were weighed and recorded.

[0049] A 25g gel column was used with an eluent of petroleum ether:methanol:dichloromethane = 2:1:1 for further separation and purification of the target component. The separated components were analyzed by TLC, and the target components were combined, weighed, and recorded based on the detection results. This process was repeated until sesquiterpene compound 1 (the compound shown in Formula I) was finally obtained.

[0050] Fraction D6 was subjected to gel column chromatography with a ratio of methanol to dichloromethane of 1:1, silica gel column chromatography with a ratio of dichloromethane to methanol of 100:1, and gel column chromatography with a ratio of petroleum ether to methanol of 2:1:1, ultimately yielding sesquiterpene compound 2 (the compound shown in Formula II).

[0051] Another embodiment of the present invention provides the application of Aspergillus pyroximate of the present invention or the composition of the present invention as a pesticide for controlling or preparing anthracnose in cucumbers.

[0052] In some specific embodiments, the above applications include: the use of Aspergillus pyrolyticus or the compositions of the present invention as inhibitors of or in the preparation of Colletotrichum spp.

[0053] Another embodiment of the present invention provides the application of a sesquiterpene compound in pesticides for controlling cucumber anthracnose. The structural formula of the sesquiterpene compound is shown in Formula I or Formula II below.

[0054]

[0055]

[0056] In some specific embodiments, the above applications include: the use of sesquiterpenoid compounds in inhibiting Colchicote sarcodactylis.

[0057] It should be noted that the Aspergillus ustus provided by this invention can be used to isolate sesquiterpenoid compounds represented by Formula I and Formula II. These sesquiterpenoid compounds have good antibacterial activity against Colletotrichum spp., with inhibition rates of 79.45% and 76.71% respectively at a final concentration of 100 μg / mL. They are comparable to the control agent chlorothalonil in inhibiting mycelial growth.

[0058] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0059] Example 1

[0060] First, the larvae of the ground beetle were cleaned with 95% ethanol and sterile water. Using a sterile scalpel, the mouthparts and tail of the larvae were cut open. The intestines were gently removed from the head using sterile forceps, and the internal organs and appendages were removed. The intestinal contents were collected and diluted with an appropriate amount of phosphate buffer, then evenly spread on PDA medium (components: 200g peeled potato, 20g glucose, 20g agar, 1L distilled water). The medium was incubated at 28℃ for 48 hours. Single colonies of different morphologies were picked using an inoculation needle and repeatedly cultured to obtain a pure strain SN-56147. Based on morphological characteristics and molecular biological analysis, it was preliminarily identified as *Aspergill ustus*. This *Aspergill ustus* SN-56147 has the following characteristics:

[0061] (1) Morphological characteristics: Strain SN56147 colonies grew well, with neat colony edges. Initially white, they gradually turned yellowish-brown, producing a colorless exudate. The reverse side of the colonies was pale yellow. Figure 1 As shown.

[0062] (2) Phylogenetic characteristics: Phylogenetic comparison analysis was performed by amplifying the rDNA-ITS sequence. The results showed that the ITS-rDNA sequence of this strain was 99% similar to that of the standard strain Aspergillus insuetus HE615091.1. The gene sequencing results are as follows (SEQ ID NO.1):

[0063] .

[0064] Example 2

[0065] The Aspergillus pyroximate SN-56147 obtained in Example 1 was subjected to 24L fermentation. A crude extract of Aspergillus pyroximate SN-56147 was obtained using macroporous adsorption resin and solvent extraction techniques. Sesquiterpenoids were then separated from the crude extract using silica gel and gel column chromatography. The specific process is as follows:

[0066] (1) Add 5 mL of PDB culture medium (components: 200 g peeled potato, 20 g glucose, 1 L distilled water) to a 25 mL test tube and sterilize at 121 °C for 30 min; inoculate Aspergillus pyrolyticus SN-56147 stored in a -80 °C refrigerator into PDA medium and culture at 25 °C for 7 days. When the colony diameter grows to about 7 cm, punch holes at the edge of the colony with a 5 mm punch and inoculate the mycelium into a 25 mL test tube as the primary liquid seed. Use 3 uninoculated test tubes as blank control; after inoculation, put all test tubes into a shaker and culture at 180 r / min and 25 °C for 48 h. Take them out when the bacterial solution becomes turbid.

[0067] (2) Transfer all the fermented primary seed to 250mL Erlenmeyer flasks as secondary liquid seed. Add 50mL LDB medium to each Erlenmeyer flask and sterilize at 121℃ for 30min. Use 3 Erlenmeyer flasks without inoculation as blank control. Shake culture at 180r / min and 25℃ for 48h. Remove the flasks when the bacterial solution becomes turbid and bacterial clumps form.

[0068] (3) Transfer all the fermented secondary seeds to 2L Erlenmeyer flasks. Add 400m L PDB medium and 16g macroporous adsorption resin (XAD-16) to each Erlenmeyer flask. Sterilize at 121℃ for 30min. Use 3 Erlenmeyer flasks without inoculation as blank control. Shake culture at 180r / min and 25℃ for 7d. A total of 24L of fermentation was carried out.

[0069] (4) After the overall fermentation was completed, a total of 24L of Aspergillus pyrolysis SN-56147 fermentation broth was obtained. The Aspergillus pyrolysis SN-56147 fermentation broth was rinsed multiple times with distilled water to remove all the bacteria and culture medium from the fermentation broth. The macroporous adsorption resin was collected and all the macroporous adsorption resin in the bottle was gathered into a tray and placed in an oven to dry at 30℃. After the resin was dried, it was placed in a 2L separatory funnel and an appropriate amount of methanol was added for elution. The methanol was changed every 4 hours and a total of 5 elutions were performed, including one overnight soak. The eluents were collected and combined. The eluents were concentrated under reduced pressure to obtain the crude fermentation extract. The crude fermentation extract was dissolved in methanol and then 300ml of distilled water and 300ml of dichloromethane were added for extraction. The extraction was performed 5 times. The dichloromethane phase was collected and concentrated under reduced pressure to obtain 40g of dichloromethane crude extract.

[0070] (5) A petroleum ether-ethyl acetate system was used as the eluent for primary silica gel column chromatography to perform gradient elution of the crude dichloromethane extract. Petroleum ether, petroleum ether to ethyl acetate volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16, and 1:1 were selected as the gradient eluents for normal-phase silica gel columns, with 0.6 L of mobile phase eluted each time. After primary silica gel column chromatography, the fractions were combined into five components A, B, C, D, and E according to their polarity. The mass, TLC, and solubility of component A4 were comprehensively analyzed. Finally, a 25 g silica gel column (200-300 mesh) was selected, and dry loading and dry packing were used to separate and purify component A4 with an eluent system of V hexane:V dichloromethane = 7:3. The separated components were analyzed by TLC, and the target components were weighed and recorded.

[0071] A 25g gel column was used with a petroleum ether:methanol:dichloromethane ratio of 2:1:1 eluent for further separation and purification of the target component. The separated components were analyzed by TLC, and the target components were combined, weighed, and recorded based on the detection results. This process was repeated until a sesquiterpene compound 1 was finally obtained.

[0072] The D6 fraction was subjected to gel column chromatography with a ratio of methanol to dichloromethane of 1:1, silica gel column chromatography with a ratio of dichloromethane to methanol of 100:1, and gel column chromatography with a ratio of petroleum ether to methanol of 2:1:1, ultimately yielding sesquiterpene compound 2.

[0073] Compound 1 was structurally identified as a white crystalline solid, soluble in methanol; single-crystal diffraction data revealed its molecular formula to be C1. 15 H 26 O; 1 H NMR (600MHz, CDCl3) δ: 1.96 (2H, m, H-1, H-2), 1.43 (2H, m, H-3, H-4), 1.17 (2H, m, H-5, H-6), 1.0 7 (1H, m, H-7), 1.87 (2H, m, H-8, H-9), 5.53 (1H, m, H-10), 1.56 (1H, dt, J=13.8Hz, H-11), 3.85 (1 H, dd, J=11.4, 3.6Hz, H-12), 3.73 (1H, dd, J=11.4, 4.8Hz, H-13), 1.87 (3H, s, H-14, H-15, H-16) , 0.86 (3H, s, H-17, H-18, H-19), 0.88 (3H, s, H-20, H-21, H-22), 0.85 (3H, s, H-23, H-24, H-25); 13 C10 NMR (150MHz, CDCl3) δ: 39.89 (C-1), 18.78 (C-2), 42.15 (C-3), 32.93 (C-4), 49.90 (C-5), 23.60 (C-6), 124.09 (C-7), 132.92 (C-8), 57.28 (C-9), 36.07 (C-10), 60.93 (C-11), 21.96 (C-12), 33.27 (C-13), 22.06 (C-14), 14.92 (C-15). These NMR data are consistent with literature reports. Compound 1 is the known compound Drimenol, with the structural formula […]. Its different spectra, such as Figures 2 to 4 As shown.

[0074] Compound 2 was structurally identified as a white crystalline solid, soluble in methanol; [α]22D = -26.1 (c 0.15, CHCl3); according to HR-ESI-MS: m / z 291.1937 [M+Na] + Its molecular formula is C15H26O; 1 H NMR (600MHz, CDCl3) δ: 1.70~1.51 (2H, m, H-1, H-2), 1.49~1.36 (2H, m, H-3, H-4), 1.48~1.25 (2H, m, H -5, H-6), 1.74 (1H, d, J = 10.2Hz, H-7), 3.72 (1H, d, J = 10.2Hz, H-8), 5.63 (1H, s, H-9), 3.76 (1H, d, J = 1 0.8Hz, H-10), 3.55 (1H, d, J = 11.4Hz, H-11), 1.85 (3H, s, H-12, H-12, H-14), 3.33 (3H, s, H-15, H-16, H-17), 1.09 (3H, s, H-18, H-19, H-20), 1.02 (3H, s, H-21, H-22, H-23), 0.86 (3H, s, H-24, H-25, H-26); 13 C10 NMR (150MHz, CDCl3) δ: 32.6 (C-1), 18.6 (C-2), 43.0 (C-3), 33.4 (C-4), 47.0 (C-5), 77.2 (C-6), 127.0 (C-7), 137.5 (C-8), 75.3 (C-9), 42.7 (C-10), 62.1 (C-11), 20.0 (C-12), 54.7 (C-13), 35.8 (C-14), 23.4 (C-15), 17.0 (C-16). These NMR data are consistent with literature reports. Compound 2 is a known compound, O-methylalbrassitriol; its structural formula is [structural formula missing]. Its different spectra, such as 5 to Figure 6 As shown.

[0075] Table 1. Different data for the two compounds

[0076]

[0077] Example 3

[0078] The antifungal activity of sesquiterpenoid compounds 1 and 2 obtained in Example 2 against *Colletotrichum spp.* was determined using the mycelial growth rate method (according to standard GB / T 38480-2020), with chlorothalonil treatment serving as a positive control. Furthermore, the EC50 of compounds 1 and 2 versus chlorothalonil in inhibiting *Colletotrichum spp.* mycelial growth was calculated using a mycelial growth inhibition assay.50 The inhibitory activities of sesquiterpenoid compounds 1 and 2, along with chlorothalonil, at final concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL on the mycelial growth of *Colletotrichum spp.* were investigated. The results are as follows: Figure 7 , Figure 8 , Figure 9 As shown in Table 2.

[0079] Table 2. Inhibitory activities of Compound 1 and Compound 2 on the hyphal growth of Colchicaria stingrosporum.

[0080]

[0081] The results showed that compound 1 and compound 2 had inhibition rates of 79.45% and 76.71% against *Colletotrichum spp.* at a concentration of 100 μg / mL, respectively; and the EC50 of compounds 1, 2, and chlorothalonil against *Colletotrichum spp.* 50 The values ​​were 24.49 μg / mL, 38.41 μg / mL, and 28.67 μg / mL, respectively.

[0082] Example 4

[0083] The control efficacy of fungal secondary metabolites (compound 1 and compound 2) against cucumber anthracnose was determined using the detached leaf method (referring to method guideline GB / T 17980.112-2004). The results are as follows: Figure 10 As shown in Table 4, the specific experimental data are as follows.

[0084] Table 4. Control efficacy of compound 1 and compound 2 against cucumber anthracnose.

[0085] deal with Disease index Prevention and control efficacy % Compound 1 25.6±2.6 63.5±2.1 Compound 2 30.1±2.1 60.3±2.3 Chlorothalonil 40.3±2.1 42.3±2.1 Blank control group 70.6±1.9 /

[0086] The results of the detached leaf method (Table 3) show that when the treatment concentration is 300 mg / L, compound 1 has a control effect of 61.4%–65.6% on cucumber anthracnose, with a disease index of 25.6±2.6. Compound 1 has a control effect of 58.0%–62.6% on cucumber anthracnose, with a disease index of 30.1±2.1. The control effects are better than those of the chlorothalonil positive control.

[0087] In summary, these sesquiterpenoid compounds 1 and 2 demonstrate good antifungal activity against *Colletotrichum spp.*, with inhibition rates of 79.45% and 76.71%, respectively, at a final concentration of 100 μg / mL. Their performance in inhibiting mycelial growth is comparable to that of the control agent, chlorothalonil. Compounds 1 and 2 have the potential to be developed into novel environmentally friendly pesticide lead compounds for the control of cucumber anthracnose.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of sesquiterpenoids in the control of cucumber anthracnose, wherein the structural formula of the sesquiterpenoid is shown in Formula I or Formula II below. I， II.

2. The application according to claim 1, characterized in that, include: The application of the sesquiterpenoid compounds in inhibiting Colchicum spp.

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