Alternaria alternata and metabolites and applications thereof

Abstract

The application provides an Alternaria alternata and metabolites and applications thereof, and belongs to the technical field of agricultural insecticides and herbicides. The Alternaria alternata (Alternaria alternata) FL7 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2023428. A new Tenuazamine type isomerization isomer compound, Tenuazamine A, is separated from a fermentation extract of the Alternaria alternata. The compound has good insecticidal and herbicidal activity, and can be applied to crop disease, pest and weed control. The application provides a source and a method for producing and preparing the new Tenuazamine type compound.

Description

Technical Field

[0001] This invention belongs to the technical field of agricultural insecticides and herbicides, and particularly relates to a very fine Alternariae bacterium, its metabolites, and their applications. Background Technology

[0002] Alternaria spp. is one of the most potent producers of toxic metabolites among Alternaria fungi. It is widely distributed in various substrates and exhibits parasitic, saprophytic, and pathogenic characteristics (FENG Zhonghong, SUN Guangyu. Advances in the classification of Alternaria and related genera[J]. Journal of Fungal Research, 2020, 18(4):294-303). Alternaria spp. has been reported to cause wheat leaf blight, onion purple spot disease, maize leaf blight, citrus brown spot disease, and cruciferous black spot disease (NIU Junke, LU Baohui, LIULiping, LI Meng, WANG Tian, ​​GAO Jie. Identification of the pathogens causing Tobacco brown spot disease in Jilin and Heilongjiang provinces[J]. Chinese Tobacco Science, 2019-10, 40(5)), Tobacco Red Spot Disease (Jiang Yunyun, Qian Cen, Hao Zhigang, et al. Preliminary screening of biocontrol strains of Alternaria spp. pathogen of Tomato Black Spot Disease in Xinjiang [C]. Chinese Society for Plant Pathology. Technological Innovation and Green Control in Plant Pathology - Proceedings of the 2021 Annual Meeting of the Chinese Society for Plant Pathology. China Agricultural Science and Technology Press, 2021:1. DOI:10.26914 / c.cnkihy.2021.064157.) More than ten diseases, including gray spot and wilt of Notopterygium incisum (Jiang Yunyun, Qian Cen, Hao Zhigang, et al. Preliminary screening of biocontrol strains of Alternaria spp. pathogen of black spot of tomato in Xinjiang [C]. Chinese Society for Plant Pathology. Technological Innovation and Green Control in Plant Pathology - Proceedings of the 2021 Annual Meeting of the Chinese Society for Plant Pathology. China Agricultural Science and Technology Press, 2021:1.DOI:10.26914 / c.cnkihy.2021.064157), early blight of eggplant, black spot of cabbage, etc. Secondary metabolites are mainly toxins. Currently, there are more than 70 substances with identified structures and obvious toxicity. According to different chemical structures, they can be divided into the following categories: Alternaria solaniol (AOH), Alternaria solaniol monomethyl ether (AME), Alternaria solaniol (ALT), Alternaria solaniol ketone acid (TeA), Alternaria solanitoxin (ATx), Tengtoxin (TEN), etc.

[0003] Alternaria alternata causes pollution and yield reduction in economic crops, and the residues of its secondary metabolites in crops can seriously affect the health of livestock and humans (Wang Yan, Chen Xiurong, Du Tao, et al. Investigation and pathogen identification of diseases of Notopterygium incisum in Gansu Province [J]. Chinese Journal of Traditional Chinese Medicine, 2009, 34(15): 1898-1901.). Although the detection of harmful substances in crops is very strict, due to the large number of toxins produced by Alternaria alternata, there is currently no complete detection system for this type of pathogenic fungus.

[0004] According to their source, insecticides can be divided into inorganic and mineral insecticides, chemically synthesized insecticides, and bio-based insecticides. (1) Inorganic and mineral insecticides: such as lead arsenate, calcium, etc. These insecticides have low efficacy, are prone to causing crop damage, and are highly toxic to humans. Most of them have been phased out. (2) Chemically synthesized insecticides: such as DDT, hexachlorocyclohexane, dichlorvos, etc. These are also the most widely used and widely applied methods. However, they are prone to accumulation in organisms and cause great harm. (3) Bio-based insecticides: such as camptothecin, juvenile hormones, and sex pheromones. These substances not only have good insecticidal effects, but also have a complete degradation pathway developed since they exist in nature. They have great potential. Taking the TeA compound produced by Alternaria alternata as an example, Zhou Bing et al. of Nanjing Agricultural University studied the degradation of this type of substance in soil in 2007. Its half-life is 3.22 days, and it can be completely degraded after 20 days, which is an easily degradable substance (Zhou Bing, Qiang Sheng. Study on the degradation of Alternaria alternata toxin Alternaria alternata ketone acid in soil [J]. Journal of Agricultural Environmental Science, 2007, 26(002):572-576.DOI:10.3321 / j.issn:1672-2043.2007.02.032.).

[0005] In terms of weed control, chemical herbicides are currently the mainstay. However, with the widespread use of chemical herbicides such as glyphosate, weed resistance has increased, seriously threatening the ecological environment (Zhou Wenguan, Meng Yongjie, Chen Feng, et al. Current status and prospects of herbicide research and development and its compound use [J]. Grassland Science, 2018, 35(1):13.DOI:10.11829 / j.issn.1001-0629.2017-0046.). Currently, herbicides derived from natural products are receiving much attention due to their diverse target sites and environmental friendliness, and are also one of the important research directions in the field of herbicides (Li Yanpeng, Wang Bing, Shen Zheng, et al. Research progress on biosynthesis of biological herbicide thaxtomin [J]. Chinese Journal of Antibiotics [2023-06-26]:1-9.https: / / doi.org / 10.13461 / j.cnki.cja.007510.).

[0006] Currently, the types of pesticides and herbicides derived from natural products are relatively limited. Developing more pesticides and herbicides derived from natural products is of great significance for agricultural development. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide Alternaria microphylla, its metabolites, and their applications. The metabolites of Alternaria microphylla FL7 have good insecticidal and herbicidal activities and can be applied to the control of agricultural pests.

[0008] This invention provides a strain of Alternaria alternata FL7, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2023428.

[0009] This invention provides the application of Alternaria filamentosa FL7 in the control of crop pests.

[0010] Preferably, the metabolites of Alternaria microphylla FL7 are used as insecticides.

[0011] This invention provides a novel amine base compound with the structure shown in Formula I or Formula II:

[0012]

[0013] Preferably, R is NH2, and the compound is named Tenuazamine A.

[0014] This invention provides a method for preparing the aforementioned novel amine base compound, comprising the following steps:

[0015] 1) The cell cells obtained by fermentation of Alternaria FL7 are mixed with an alcohol solvent to extract an alcohol extract, and the alcohol extract is concentrated to obtain alcohol extract A;

[0016] 2) The alcohol extract A is mixed with water to obtain a suspension. The suspension is extracted three times with petroleum ether. The petroleum ether phase is concentrated to obtain extract B. The remaining aqueous phase is extracted three times with ethyl acetate. The ethyl acetate phase is concentrated to obtain extract C.

[0017] 3) Extract C was subjected to silica gel column chromatography and eluted with four gradients of petroleum ether:ethyl acetate volume ratios of 3:1, 1:1, 1:3, and 0:1 to obtain fractions Fr.1 to Fr.4.

[0018] Fraction Fr.4 was subjected to MCI column chromatography and eluted with eight gradient eluents of methanol:water volume ratios of 5:95, 10:90, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain fractions Fr.4-1 to Fr.4-8.

[0019] Fraction Fr.4-3 was subjected to silica column chromatography and eluted with six gradients of dichloromethane:methanol at volume ratios of 30:1, 20:1, 10:1, 5:1, 1:1, and 1:3 to obtain fractions Fr.4-3-1 to Fr.4-3-6.

[0020] Fraction Fr.4-3-1 was separated by preparative high performance liquid chromatography: the mobile phase was 35% methanol + 65% water, the flow rate was 10 mL / min, and the detector wavelength was 295 nm; tenuazamine A was obtained at a retention time of 23 min.

[0021] Preferably, the alcohol solvent in step 1) is methanol; the volume ratio of the bacterial cells to methanol is 1:(0.8-1.2); the number of extractions is 10-12; and the extraction time for each extraction is 20-30 hours.

[0022] Preferably, in step 2), the volume ratio of extract A to water is 1:(0.8 to 1.2).

[0023] This invention provides the application of the aforementioned novel amine base compounds as insecticides.

[0024] This invention provides the application of the aforementioned novel amine base compounds as herbicides.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a very fine Alternaria FL7, which is isolated from the leaf tissue of Humulus scandens. The very fine Alternaria FL7 is fermented to produce a new type of amine base compound, Tenuazamine A. This type of amine base compound has good insecticidal and herbicidal activity and can be applied to the control of crop diseases, pests and weeds. Attached Figure Description

[0026] Figure 1 The morphology of Alternaria microphylla of the present invention under three culture media are shown in A and a, which are front and back views of FL7 growing on P DA medium; B and b, which are front and back views of FL7 growing on CYA medium; and C and c, which are front and back views of FL7 growing on YES medium.

[0027] Figure 2These are microscopic images of the hyphae (A) and spores (B) of the *Alternaria spp.* of the present invention. The hyphae are grayish-white, septate, and branched. The conidia are brown, obclavate, and have 3-6 septa.

[0028] Figure 3 The present invention relates to the structural formula of Tenuazamine A, a metabolite of Alternaria microphylla.

[0029] Figure 4 This is a schematic diagram of the preparative liquid phase separation of compound Tenuazamine A.

[0030] Figure 5 The stomach poisoning efficacy of different concentrations of the compound Tenuazamine A against the larvae of the beet armyworm was measured. AF represents six experimental groups: 20 mg / g, 1 mg / g, 2 mg / g, 0.5 mg / g, 0.1 mg / g, and 0.05 mg / g, respectively. A1-F1 represent repeated experimental groups.

[0031] Figure 6 The image shows the effect of different concentrations of the compound Tenuazamine A on the germination of barnyard grass seeds. A, G, and A represent the blank group, negative control group, and experimental groups of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL, respectively.

[0032] Figure 7 The figure shows the effect of different concentrations of the compound Tenuazamine A on the radicle growth of barnyard grass seeds. A, G, and A represent the blank group, negative control group, and experimental groups of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL, respectively.

[0033] Biological Preservation Instructions

[0034] The Alternaria alternata FL7 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2023428, deposit date March 28, 2023, and address Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Detailed Implementation

[0035] This invention provides a strain of Alternaria alternata FL7, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2023428.

[0036] In this invention, the extremely fine Alternaria FL7 was isolated from the leaf tissue of Humulus scandens and has the following morphology. Figure 1 As shown, colonies on PDA medium are white with a gray center, a black center on the back, and white edges. The surface is uneven, the hyphae are fluffy, and the texture is relatively thick. On CYA medium, the colonies on the front are entirely white, with an uneven surface, fluffy hyphae, and a relatively thick texture. They develop more slowly than those on PDA medium, and the center of the back is light brown. On YES medium, the front shows the same appearance as the former, but the center of the back is dark brown, and the affected area is larger.

[0037] The present invention also provides the application of Alternaria filamentosa FL7 in the control of crop pests or weeds. In the present invention, the metabolites of Alternaria filamentosa FL7 fermentation have good insecticidal and herbicidal activities and can be applied to the control of crop diseases, pests and weeds.

[0038] In this invention, the Alternaria microphylla FL7 is preferably obtained by culturing using the following method:

[0039] S1) The activated Alternaria microphylla FL7 was inoculated into the seed culture medium for seed culture to obtain seed liquid;

[0040] S2) The seed liquid is inoculated into a fermentation medium for fermentation culture to obtain bacterial cells.

[0041] In this invention, the *Alternaria filamentosa* FL7 is activated on a solid plate medium and then inoculated into a seed culture medium. The solid plate medium is preferably a potato agar (PDA) plate. The seed culture medium uses water as a solvent and comprises 150–250 g / L potato juice and 15–25 g / L glucose; preferably, it comprises 180–220 g / L potato juice and 17–23 g / L glucose, and more preferably, it comprises 200 g / L potato juice and 20 g / L glucose. In this invention, the seed culture temperature is preferably 27–29°C, more preferably 28°C; the seed culture rotation speed is preferably 150–200 rpm, more preferably 180 rpm; and the seed culture time is preferably 2–4 days, more preferably 3 days.

[0042] In this invention, after obtaining the seed liquid, the seed liquid is inoculated into a fermentation medium to obtain bacterial cells. In this invention, the fermentation medium preferably comprises rice and water, and the mass ratio of rice to water is preferably 16:(10-14), more preferably 16:(11-13), and most preferably 16:12. In this invention, the fermentation medium is used after sterilization, and the sterilization is preferably performed by high-temperature moist heat sterilization. In this invention, the inoculation amount of the seed liquid is preferably 2%-3%, more preferably 2.5%. The fermentation culture is preferably static culture, the fermentation culture temperature is preferably 27-29℃, more preferably 28℃, and the fermentation culture time is preferably 25-35 days, more preferably 28-32 days, and most preferably 30 days.

[0043] This invention also provides a novel amine base compound with the structure shown in Formula I or Formula II:

[0044]

[0045] Preferably, R is NH2; the compound is named Tenuazamine A;

[0046] In this invention, the aforementioned novel amine base compound is a metabolite of Alternaria filamentosa FL7, obtained by isolating it from the fermentation cells of Alternaria filamentosa FL7.

[0047] This invention provides a method for preparing the aforementioned novel amine base compound, comprising the following steps:

[0048] 1) The bacterial cells obtained by fermenting Alternaria FL7 are mixed with an alcohol solvent to extract an alcoholic extract, and the alcoholic extract is concentrated to obtain alcoholic extract A;

[0049] 2) Mix the alcohol extract A with water to obtain a suspension, extract the suspension with petroleum ether and concentrate the petroleum ether phase to obtain extract B; extract the remaining aqueous phase with ethyl acetate and concentrate the ethyl acetate phase to obtain extract C;

[0050] 3) Extract C was subjected to silica gel column chromatography and eluted with four gradients of petroleum ether:ethyl acetate volume ratios of 3:1, 1:1, 1:3, and 0:1 to obtain fractions Fr.1 to Fr.4.

[0051] Then, the fractions were eluted with four gradients of ethyl acetate:methanol volume ratios of 3:1, 1:1, 1:3, and 0:1 to obtain fractions Fr.5 to Fr.8.

[0052] Fraction Fr.4 was subjected to MCI column chromatography and eluted with eight gradient eluents of methanol:water volume ratios of 5:95, 10:90, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain fractions Fr.4-1 to Fr.4-8.

[0053] Fraction Fr.4-3 was subjected to silica column chromatography and eluted with six gradients of dichloromethane:methanol at volume ratios of 30:1, 20:1, 10:1, 5:1, 1:1, and 1:3 to obtain fractions Fr.4-3-1 to Fr.4-3-6.

[0054] Fraction Fr.4-3-1 was separated by preparative high performance liquid chromatography: the mobile phase was 35% methanol + 65% water, the flow rate was 10 mL / min, and the detector wavelength was 295 nm; tenuazamine A was obtained at a retention time of 23 min.

[0055] In this invention, the bacterial cells obtained by fermentation culture using the above-described culture method are preferably used as raw materials for the preparation of novel amine base compounds. In this invention, the bacterial cells obtained by fermenting *Alternaria filamentosa* FL7 are mixed with an alcohol solvent for extraction to obtain an alcohol extract, and the alcohol extract is concentrated to obtain alcohol extract A. In this invention, the alcohol solvent is preferably methanol; the volume ratio of bacterial cells to methanol is preferably 1:(0.8-1.2), more preferably 1:1; the number of extractions is preferably 11 times, the number of extractions is preferably such that the methanol extract is nearly colorless, and the extraction time for each extraction is preferably 20-30 hours, more preferably 22-28 hours, and most preferably 24 hours.

[0056] In this invention, the volume ratio of extract A to water is preferably 1:(0.8-1.2), more preferably 1:(0.9-1.1), and most preferably 1:1. In this invention, the mixing of extract A with water is preferably accompanied by ultrasonic treatment to promote dissolution.

[0057] In this invention, the petroleum ether phase obtained by extracting the suspension with petroleum ether is concentrated to obtain extract B. Preferably, the petroleum ether extraction is performed 2 to 4 times, more preferably 3 times. The volume ratio of petroleum ether to suspension in each extraction is preferably 1:(0.8 to 1.2), more preferably 1:(0.9 to 1.1), and most preferably 1:1. Following the petroleum ether extraction, ethyl acetate extraction is performed. Preferably, the ethyl acetate extraction is performed 2 to 4 times, more preferably 3 times. The volume ratio of ethyl acetate to suspension in each extraction is preferably 1:(0.8 to 1.2), more preferably 1:(0.9 to 1.1), and most preferably 1:1. In this invention, the petroleum ether phases obtained from multiple extractions are mixed and concentrated to obtain extract B; the ethyl acetate phases obtained from multiple extractions are mixed and concentrated to obtain extract C.

[0058] In this invention, the preferred method for obtaining extracts A, B and C by concentration is vacuum concentration until no solvent is refluxed.

[0059] This invention also provides the application of the aforementioned novel amine base compounds as insecticides. In this invention, the novel amine base compounds are capable of killing the beet armyworm; the novel amine base compounds kill the beet armyworm through stomach poisoning.

[0060] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Fermentation culture of Alternaria microphylla FL7

[0063] (1) Preparation of culture medium:

[0064] Seed culture medium preparation: 200g / L potato juice, 20g / L glucose, tap water, natural pH.

[0065] Fermentation medium preparation: Each 1L Erlenmeyer flask contains: 160g rice, 120g tap water, sealed with disposable sealing film, and then sterilized in an autoclave at 121℃ for 28 minutes.

[0066] (2) Fermentation:

[0067] The FL7 strain preserved in glycerol tubes was transferred to potato agar (PDA) plates for activation. After culturing at 28°C for 7 days, it was inoculated into a 500 mL shake flask containing 120 mL of seed culture medium. After culturing at 28°C and 180 rpm for 3 days, it was inoculated into the prepared fermentation medium at a volume of 3 mL. Finally, it was fermented at 28°C for 30 days.

[0068] Fermentation culture: The seed culture was inoculated into the fermentation medium at a rate of 3 mL per Erlenmeyer flask. The fermentation medium consisted of 1L Erlenmeyer flasks containing 160g of rice and 120g of water, which were sterilized in an autoclave. After fermentation at 28℃ for 30 days, the entire fermentation medium was removed to obtain the bacterial cells.

[0069] Example 2

[0070] 1. Take out the bacterial cells obtained in the example and put them into a plastic bucket.

[0071] 2. Pour commercially available analytical grade methanol into a plastic bucket containing fermentation cells, ensuring the methanol level is 15 cm above the cells. Stir and let stand for 24 hours. Take the supernatant methanol extract and concentrate it under reduced pressure in a rotary evaporator. Repeat the above process 11 times until the methanol extract is almost colorless. Combine all the methanol extracts and concentrate under reduced pressure to obtain extract A.

[0072] 3. Extraction of extracts B and C

[0073] Extract A (10.25 kg) was dissolved in distilled water (8 L) by sonication to obtain an aqueous suspension. Then, an equal volume of petroleum ether was added for extraction. This process was repeated three times. The petroleum ether phases were combined and concentrated under reduced pressure to obtain extract B. The remaining aqueous phase was then extracted with ethyl acetate. This process was repeated three times. The ethyl acetate phases were combined and concentrated under reduced pressure to obtain extract C (191 g).

[0074] 4. Isolation and purification of compounds

[0075] Extract C was subjected to silica gel column chromatography, using petroleum ether / ethyl acetate and ethyl acetate / methanol as eluents, with gradient elution at volume ratios of 3:1, 1:1, 1:3, 0:1, 3:1, 1:1, 1:3, and 0:1. The fractions were concentrated under reduced pressure to obtain fractions Fr.1-8. Fraction Fr.4 was subjected to MCI column chromatography, using methanol / water as eluent, with gradient elution at methanol / water ratios of 5:95, 10:90, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain fractions Fr.4-1 to Fr.4-8. Fraction Fr.4-3 was subjected to silica column chromatography and eluted with a gradient of dichloromethane / methanol at volume ratios of 30:1, 20:1, 10:1, 5:1, 1:1, and 1:3 to obtain fractions Fr.4-3-1 to Fr.4-3-6.

[0076] Fraction Fr.4-3-1 was subjected to preparative high-performance liquid chromatography (35% methanol + 65% water, flow rate 10 mL / min, 295 nm) to obtain compound A (R). t =23min, 59.5mg).

[0077] 5. Structural identification of isolated compounds

[0078] The compounds obtained through separation and purification were identified using 1D, 2D NMR, and HR-ESI-MS high-resolution mass spectrometry. 1D and 2D NMR were obtained from a 500 MHz NMR spectrometer (AVANCE NEO 500, Bruker, Switzerland), and HR-ESI-MS high-resolution mass spectrometry was obtained from a high-resolution mass spectrometer (model...). 4600, obtained by AB SCIE (Company).

[0079] Test results:

[0080] Compound Tenuazamine A: pale yellow oil droplets, HR-ESI-MS (m / z 197.1297 [M+H]) + ); 1 H NMR (600MHz, DMSO-d6); δ (a): 3.44 (1H, br d, 2.9, H-5), 2.30 (3H, s, H-7), 1.72 (1H, m, H-8), 1.08, 1.23 (each 1H, m, H-9), 0.79 (3H, t, 7.4, H-10), 0.88 (3H, d, 7.0, H-11), 7.53 (1H, brs, 1-NH), 8.64, 9.42 (each 1H, br s, NH2); δ (b): 3.53 (1H, br d, 2.9, H-5), 2.33 (3H, s, H-7), 1.72 (1H, m, H-8), 1.08, 1.23 (each 1H, m, H-9), 0.79 (3H, t, 7.4, H-10), 0.87 (3H, d, 6.9, H-11), 7.31 (1H, br s, 1-NH), 8.78, 9.67 (each 1H, br s, 12-NH2); 13 C NMR(150MHz,DMS O-d6); δ(a): 174.6(s, C-2), 95.4(s, C-3), 195.9(s, C-4), 64.9(d, C-5), 167.2 (s, C-6), 18.5 (q, C-7), 36.5 (d, C-8), 23.0 (t, C-9), 11.9 (q, C-10), 15.8 (q, C-1 1); δ(b): 172.1(s, C-2), 97.0(s, C-3), 198.5(s, C-4), 63.6(d, C-5), 167.3(s, C-6), 17.6(q, C-7), 36.6(d, C-8), 23.3(t, C-9), 11.9(q, C-10), 15.6(q, C-11). All the above data were obtained through... 1 H, 13 Identified by C NMR and HR-ESI-MS.

[0081] 6. Nomenclature of compounds

[0082] Tenuazamine compounds and Tenuazonic acid compounds share the same origin but differ in structure: 1) Both types of compounds have the same basic skeleton, so Tenuaz- (acid skeleton) is used; 2) The group attached to C-6 is -NH2, not -OH, so -amine is used; 3) Because they are non-enol isomers, they are not weakly acidic, so acid is removed. Based on the above combination order, the specific name is Tenuazamine A.

[0083] Example 3

[0084] Stomach poisoning assay to determine the ability of the insect to kill the larvae of the beet armyworm.

[0085] 1. Rearing and selection of Spodoptera litura larvae

[0086] The eggs of the beet armyworm (from Henan Keyun Biological Pesticide Co., Ltd.) were placed in an incubator at 28℃. The larvae hatched in 2-3 days. Artificial feed (from Henan Keyun Biological Pesticide Co., Ltd.) was cut into pieces and placed in a petri dish. The food was kept plentiful and fresh. After 5-6 days of growth, 3rd-4th instar larvae (with black and white stripes on their bodies) were selected as experimental subjects.

[0087] 2. Feed formulation, larval grouping, and drug administration regimen

[0088] Taking the compound Tenuazamine A as an example, the compound was mixed with feed at mass ratios (mg / g) of 20:1, 10:1, 2:1, 0.5:1, 0.1:1, and 0.05:1. The beet armyworms were divided into 3 groups of 10 larvae each and fed the feed containing the drug mixture. The changes in the larvae were recorded every 12 hours for 72 hours, and the number of larvae that died at each stage was counted.

[0089] 3. Experimental Results

[0090] Feed and observe for 72 hours ( Figure 5 The results (Table 1) are as follows: At a material ratio of 20:1, larval feeding was significantly affected, and individual mortality occurred, with a mortality rate of 30%–40%. At a material ratio of 10:1, larval feeding was significantly affected, but no individual mortality occurred. At ratios of 2:1–0.05:1, larval feeding and mortality were not affected. In conclusion, Tenuazamine compounds are effective in killing Spodoptera litura larvae and can be applied in the pesticide field.

[0091] Table 1. Lethal effects of different concentrations of Tenuazamine A on Spodoptera litura larvae.

[0092]

[0093] As can be seen from the above embodiments, the Alternaria filamentosa FL7 provided by the present invention, which produces novel amine base compounds through fermentation, has good insecticidal activity and can be applied to the prevention and control of crop diseases and pests.

[0094] Example 4

[0095] Determination of the ability of Tenuzamine A to inhibit barnyardgrass seed germination and radicle growth

[0096] 1. Preparation of experimental solutions

[0097] The solutions were prepared in three groups: a blank group, a negative control group, and an experimental group. The blank group was prepared with ultrapure water, the negative control group was prepared with 1% methanol solution, and the experimental group was prepared by first dissolving Tenuazamine A in an appropriate amount of methanol and then diluting it with ultrapure water to five concentrations: 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL, ensuring that the methanol content was below 1%.

[0098] 2. Treatment of barnyard grass seeds

[0099] First, soak the seeds in an aqueous solution containing 2% NaClO for 15 minutes, then rinse them repeatedly with distilled water 10 times, and finally drain the water from the surface of the seeds.

[0100] 3. Specific experimental plan and procedure

[0101] The entire process was carried out in a clean bench. Sterile filter paper was cut to an appropriate size and laid flat on the bottom of a disposable three-part culture dish (90mm). 50 barnyard grass seeds were placed in each compartment of the culture dish, for a total of 150 seeds per culture dish. Then, 2mL of the prepared experimental solution was added to each compartment. Finally, the culture dish was covered, sealed with sealing film, and placed in a light incubator at 28℃ for observation. The experimental data were collected after 60 hours.

[0102] 4. Statistical analysis of experimental results

[0103] Each of the three compartments in the petri dish was considered a parallel unit. Germination was defined as barnyard grass seeds with a radicle length exceeding 2 mm. The germination rate and radicle length of the seeds were then statistically analyzed. It was found that at a Tenuazamine A concentration of 1 mg / mL, seeds barely germinated, and radicle growth was significantly inhibited; at a Tenuazamine A concentration of 0.5 mg / mL, almost all seeds germinated, and radicle growth was significantly inhibited; at a Tenuazamine A concentration of 0.25 mg / mL, almost all seeds germinated, and radicle growth was inhibited; at other concentrations, the inhibitory effect on seed germination and radicle growth was not significant. Figure 6 and Figure 7 .

[0104] In summary, these compounds have a significant impact on the germination and radicle growth of barnyard grass seeds and can be applied in the field of crop herbicides.

[0105] 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 novel amine base compound, characterized in that, The structure is shown in Equation I or Equation II: Formula I, Formula II; Where R is NH2, the compound is named Tenuazamine A.

2. The method for preparing the novel amine base compound according to claim 1, characterized in that, Includes the following steps: 1) The cells obtained by fermentation of Alternaria filamentosa FL7 with preservation number CCTCC NO: M 2023428 were mixed with alcohol solvent to extract alcohol extract, and the alcohol extract was concentrated to obtain alcohol extract A. 2) The alcohol extract A is mixed with water to obtain a suspension. The suspension is extracted three times with petroleum ether. The petroleum ether phase is concentrated to obtain extract B. The remaining aqueous phase is extracted three times with ethyl acetate. The ethyl acetate phase is concentrated to obtain extract C. 3) Extract C was subjected to silica gel column chromatography and eluted with four gradients of petroleum ether:ethyl acetate volume ratios of 3:1, 1:1, 1:3, and 0:1 to obtain fractions Fr.1 to Fr.

4. Fraction Fr.4 was subjected to MCI column chromatography and eluted with eight gradient eluents of methanol:water volume ratios of 5:95, 10:90, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain fractions Fr.4-1 to Fr.4-8. Fraction Fr.4-3 was subjected to silica column chromatography and eluted with six gradients of dichloromethane:methanol at volume ratios of 30:1, 20:1, 10:1, 5:1, 1:1, and 1:3 to obtain fractions Fr.4-3-1 to Fr.4-3-6. Fraction Fr.4-3-1 was separated by preparative high performance liquid chromatography: the mobile phase was 35% methanol + 65% water, the flow rate was 10 mL / min, and the detector wavelength was 295 nm; tenuazamine A was obtained at a retention time of 23 min.

3. The preparation method according to claim 2, characterized in that, The alcohol solvent in step 1) is methanol; the volume ratio of the bacterial cells to methanol is 1:(0.8~1.2); the number of extractions is 10~12 times; and the extraction time for each extraction is 20~30 hours.

4. The preparation method according to claim 2, characterized in that, In step 2), the volume ratio of extract A to water is 1:(0.8~1.2).

5. The application of the novel amine base compound as described in claim 1 as an insecticide.

6. The application of the novel amine base compound of claim 1 as a herbicide.

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