Compounds metabolized by actinomyces 769 and their use in inhibiting plant pathogenic fungi

Abstract

The application provides a compound metabolized by actinomyces 769 and use of the compound in inhibiting plant pathogenic fungi, and belongs to the technical field of pesticides. The compound has the following structure: the application separates and purifies a compound from metabolized products of actinomyces 769, the compound is named 2,6-piperidinedione, 4-[2-hydroxy-2-(2-hydroxy-3,5-dimethylcyclohexyl)ethyl]-2,6-piperidinedione in English, and it is found through experiments that the compound has strong anti-plant pathogenic fungal activity and can be used for development of pesticides.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticides, in particular to a compound metabolized by actinomycete 769 and the use thereof in inhibiting plant pathogenic fungi. BACKGROUND

[0002] Streptomyces ahygroscopicus n. var. gongzhulingensis Ruan et Zhang, also known as actinomycete 769, belongs to the genus Actinomyces and was isolated from soil in Gongzhuling by experts from Jilin Academy of Agricultural Sciences in 1976. The antibiotic produced by the actinomycete is called gongzhulingmycin. Gongzhulingmycin is a broad-spectrum agricultural fungicide and has been used for the prevention and treatment of smut of cereal crops. Gongzhulingmycin has the unique advantage of low toxicity to humans and animals and has a good application prospect.

[0003] However, there are few reports on the chemical components produced by actinomycete 769, but it has good fungicidal effect. Therefore, increasing the research on the effective chemical components of actinomycete 769 can provide a reference for the development and application of pesticides. SUMMARY

[0004] The purpose of the present application is to provide a compound metabolized by actinomycete 769 and the use thereof in inhibiting plant pathogenic fungi. It is found through experiments that the compound has strong anti-plant pathogenic fungal activity and can be used for the development of pesticides.

[0005] The technical solution of the present application is as follows:

[0006] The present application provides a compound isolated from the fermentation product of actinomycete 769, which has the structure shown in formula I:

[0007]

[0008] The present application further protects a preparation method of the above-mentioned compound isolated from the fermentation product of actinomycete 769, which comprises the following steps:

[0009] S1. Culturing actinomycete 769 on modified Gao's culture medium for a first time period, transferring to corn cob culture medium, and culturing for a second time period. After the surface of the corn cob culture medium produces gray powdery substances, active detection is performed. When the number of target bacteria reaches 1×10 6 CFU / mL, it is considered to be sufficient cultivation. The qualified culture is naturally aired, dried, and crushed to obtain a solid fermentation product dry powder of agricultural resistance “769”;

[0010] S2. The dry powder is extracted with ethanol, and the extract is concentrated to obtain a crude extract infusion;

[0011] S3. The crude extract was dispersed in water and extracted with petroleum ether, ethyl acetate and n-butanol, respectively. After concentration, an extract paste was obtained.

[0012] S4. Separate the extract using a silica gel column;

[0013] S5. Collect the eluent obtained by elution, remove the solvent under reduced pressure, and continue purification using a gel column and preparative high performance liquid chromatography. The chromatographic conditions are a C18 column, methanol and water as the mobile phase, and the target compound is obtained by elution.

[0014] As a further improvement of the present invention, the conditions for the first time period of cultivation are 25-30℃, relative humidity of 55-65%, and dark cultivation for 10-13 days.

[0015] As a further improvement of the present invention, the conditions for the second cultivation period are 25-30℃ and 5-10 days.

[0016] As a further improvement of the present invention, the corn grits culture medium is prepared by soaking corn grits thoroughly to absorb water, draining off the water, and then sterilizing at high temperature.

[0017] As a further improvement of the present invention, the silica gel column separation method is to elute sequentially with dichloromethane:methanol = 50:1, 10:1, 5:1, 3:1.

[0018] As a further improvement of the present invention, the volume ratio of methanol to water in the mobile phase is 70:30, 80:20, and 90:10 for elution.

[0019] As a further improvement of the present invention, the eluent obtained by collection and elution is an eluent with a dichloromethane:methanol ratio of 10:1.

[0020] As a further improvement of the present invention, the formulation of the modified Gao's culture medium is as follows:

[0021] The present invention further protects the use of the above-mentioned compound isolated from the fermentation product of Actinomycetes 769 in pesticides.

[0022] The present invention has the following beneficial effects: The present invention isolates and purifies a compound from the metabolic products of Actinomycete 769, named 2,6-Piperidinedione,4-[2-Hydroxy-2-(2-hydroxy-3,5-dimethylcyclohexyl)ethyl]-2,6-piperidinedione. Experiments have shown that this compound has strong anti-plant pathogenic fungal activity and can be used for pesticide development. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The 1H-NMR spectrum of the compound described in this invention;

[0025] Figure 2 The 13C-NMR spectrum of the compound described in this invention;

[0026] Figure 3 This is a high-resolution ion mass spectrum of the compound described in this invention;

[0027] Figure 4 The 1H-1H COSY spectrum of the compound described in this invention;

[0028] Figure 5 The HSQC spectrum of the compound described in this invention;

[0029] Figure 6 This is the HMBC spectrum of the compound described in this invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Preparation of the compound described in this invention

[0032] S1. Actinomycetes 769 was cultured in the dark on modified Gao's medium at 28°C and 60% relative humidity for 12 days, then transferred to corn grits medium (corn grits were thoroughly soaked to absorb water, drained, and sterilized at high temperature). After culturing at 28°C for 7 days, activity was tested when a gray powdery substance appeared on the surface of the corn grits medium. The target bacterial count reached 1×10⁻⁶. 6 CFU / mL or higher is considered sufficient culture; qualified cultures are naturally sun-dried, dried and pulverized to obtain the solid fermentation product dry powder of Nongkang "769".

[0033] S2. Extract the dry powder with ethanol and concentrate it to obtain a crude extract.

[0034] S3. The crude extract was dispersed in water and extracted with petroleum ether, ethyl acetate and n-butanol respectively. After concentration, an ethyl acetate extract was obtained.

[0035] S4. Separate the ethyl acetate extract using a silica gel column, eluting sequentially with dichloromethane:methanol = 50:1, 10:1, 5:1, and 3:1.

[0036] The extract obtained by elution with S5.10:1 was further purified by gel column and preparative high performance liquid chromatography. The chromatographic conditions were C18 column, methanol and water (volume ratio: 70:30, 80:20, 90:10) as mobile phase, and the target compound was obtained by elution.

[0037] Example 2: Structural identification of the compound described in this invention

[0038] The powder obtained in Example 1 of this invention is a white powder, such as... Figure 3 High-resolution ion mass spectrometry yielded a quasi-molecular ion peak at m / z 282.17086 [MH]. - , combined 1 H-NMR and 13 C1-NMR determined the molecular formula to be C10. 15 H 24 NO4, the calculated degree of unsaturation is 4. For example... Figures 1-2 ,according to 1 H-NMR and 13 C1-NMR analysis, the results are shown in Table 1. This compound contains two carbonyl groups (δ¹⁴NMR). C 175.7 and δ C 175.58), two methyl groups (δ) C 23.23 and δ C 17.20), two oxygen-bound carbons (δ C 71.81 and δ C 71.77), five methylene groups, four methine groups, these data indicate that the compound contains two cyclic structures, such as Figure 4 ,exist 1 H- 1 In the H COSY spectrum, with C-3 / C-5 / C-4 / C-7 / C-8 /

[0039] The CH2-CH2-CH-CH2-CH(OH)-CH-CH(OH)-CH(OH)-CH(CH3)-CH2-CH(CH3)-CH2 related to C-9 / C-10 / C-11(C-15) / C-12 / C-13(C16) / C-14 systems are in the same spin system. For example... Figure 6In the HMBC spectrum, H-3 is correlated with C-2 and C-4; H-5 is correlated with C-6 and C-4; H-10 is correlated with C-12, C-14, and C-15; H-15 is correlated with C-10, C-11, and C-12; and H-16 is correlated with C-2 and C-4.

[0040] C-12, C-13, and C-14 correlations determined the positions of the methyl and hydroxyl groups. In the HSQC spectrum, C-3 (δ) C

[0041] 37.96) / H-3(δ H 2.77m, 2.32m), C-4 (δ C 28.92) / H-4(δ H 2.40m), C-5(δ C 39.53) / H-5(δ H 2.68m, 2.40m), C-7 (δ C 41.11) / H-7(δ H 1.56m), C-8(δ) C 71.81) / H-8(δ H 3.66ddd), C-9(δ) C 42.78) / H-9(δ H 1.52m), C-10(δ C 71.77) / H-10(δ H 3.94t), C-11(δ C 36.78) / H-11(δ H 1.95dtp), C-12(δ) C 33.67) / H-12(δ H 1.31m, 1.22m), C-13 (δ C 27.35) / H-13(δ H 1.66m), C-14 (δ C 35.73) / H-14(δ H 1.52m, 1.56m), C-15 (δ C 17.20) / H-15(δ H 0.97d), C-16(δ) C 23.23) / H-16(δ H 0.91d) directly linked. These data determined the structure of the compound and named it 2,

[0042] 6-Piperidinedione, 4-[2-Hydroxy-2-(2-hydroxy-3,5-dimethylcyclohexyl)ethyl]-2,6-piperidinedione. Table 1 lists the compounds mentioned above. 1 H-NMR and 13 C-NMR data, chemical structure as follows:

[0043]

[0044] Table 1: Compounds of the present invention 1 H-NMR (400MHz) and 13 C-NMR (101MHz) data

[0045]

[0046] Example 3: Test of the antifungal activity of the compound described in this invention against plant pathogenic fungi.

[0047] The compound obtained in Example 1 of this invention was dissolved in dimethyl sulfoxide (DMSO) solution and then added to PDA medium at a concentration gradient of 1.25 μg·mL. -1 2.5 μg·mL -1 5.0 μg·mL -1 and 10.0 μg·mL -1 An equal volume of DMSO was used as the control group. Plant pathogenic fungi were inoculated into the center of PDA culture medium containing the compound and cultured at 25°C for 7 days. All experiments were repeated three times, and the inhibition rate was calculated using the following formula:

[0048] Inhibition rate (%) = [(d 对照 -d 处理 ) / (d 对照 -5mm)]×100

[0049] The experimental results are shown in Table 2.

[0050] Table 2: Half-inhibitory concentrations (IC50) of compounds in this invention against plant pathogenic fungi 50 )

[0051] Plant pathogenic fungi IC 50 (μg·mL -1 )]]> Magnaporthe grisea 16.45 Fusarium graminearum 22.18 Rhizoctonia solani >100 Botrytis cinerea 37.71 Exserohilum turcicum 28.65

[0052] As shown in the table above, the compounds of this invention have significant inhibitory effects on many plant pathogenic fungi. These compounds exhibit significant inhibitory activity against *Magnaporum oryzae*, *Fusarium graminearum*, *Botrytis cinerea*, and *Cordyceps militaris*, with the most significant inhibitory activity against *Magnaporum oryzae*. The compounds of this invention possess strong antifungal activity against plant pathogenic fungi and can be used in pesticide development.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a compound isolated from the fermentation product of Actinomycetes 769, characterized in that, The compound isolated from the fermentation product of Actinomycete 769 has the structure shown in Formula I: The preparation method includes the following steps: S1. Actinomycete 769 was cultured on modified Gao's medium for the first time period, then transferred to corn grits medium for the second time period. Activity was tested after a gray powdery substance appeared on the surface of the corn grits medium. The activity was determined when the target bacterial count reached 1×10⁻⁶. 6 CFU / mL or higher is considered sufficient culture; qualified cultures are naturally air-dried, dried and pulverized to obtain the solid fermentation product dry powder of agricultural antibiotic "769"; the conditions for the first time period of culture are 25-30℃, relative humidity of 55-65%, and dark culture for 10-13 days; the conditions for the second time period of culture are 25-30℃ and culture for 5-10 days. S2. Extract the dry powder with ethanol and concentrate it to obtain a crude extract. S3. The crude extract was dispersed in water and extracted with petroleum ether, ethyl acetate and n-butanol, respectively. After concentration, an extract paste was obtained. S4. Separate the extract using a silica gel column; the silica gel column separation method is to elute sequentially with dichloromethane:methanol = 50:1, 10:1, 5:1, 3:1; S5. Collect the eluent obtained by elution, remove the solvent under reduced pressure, and continue purification using gel column and preparative high performance liquid chromatography. The chromatographic conditions are C18 column, methanol and water as mobile phase, and elution to obtain the target compound. The volume ratio of methanol to water in the mobile phase is 70:30, 80:20, and 90:10, respectively. The eluent obtained by elution is a dichloromethane:methanol = 10:1 eluent.

2. The method for preparing the compound isolated from the fermentation product of Actinomycetes 769 according to claim 1, characterized in that, The corn grits culture medium is prepared by soaking corn grits thoroughly to absorb water, draining off the water, and then sterilizing at high temperature.

3. The method for preparing the compound isolated from the fermentation product of Actinomycetes 769 according to claim 1, characterized in that, The modified Gao's medium formula is as follows: NaCl 0.5g, K2HPO4 0.5g, FeSO4·7H2O 0.01g, KNO3 1g, MgSO4·7H2O 0.5g, soluble starch 20g, agar 20g, distilled water 1L, sterilized at 121℃ for 20 minutes.

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