Mite killing application of aspergillus dianhydride compound for microbial metabolism

Abstract

The invention relates to the technical field of microorganisms, in particular to acarid killing application of an aspergillus dianhydride compound metabolized by microorganisms, the aspergillus dianhydride compound is extracted from mangrove forest endophytic fungi through fermentation, the mangrove forest endophytic fungi are Aspergillus sp.GXNU-31, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number of the strain is CGMCC NO. The preservation number is GDMCC (China General Microbiological Culture Collection Center) The maximum extraction rate of an aspergillus dianhydride compound in an extract of a fermentation product of the strain can reach 25%, the acaricidal activity of the aspergillus dianhydride compound glaucic acid to citrus red mites is superior to that of a positive control bifenazate suspending agent after 10 days, 15 days, 20 days and 30 days, and a great development way is provided for industrialization of microbial acaricides.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to the application of an aspergillus dianhydride compound metabolized by microorganisms in killing mites. Background Art

[0002] With the development of society and the improvement of people's quality of life, most synthetic pesticide chemicals cause irreversible damage to humans and the environment and can no longer meet the requirements of modern agriculture. Agricultural pest control is also a huge challenge to agricultural economy and ecology worldwide. Pest control has become one of the important areas of concern for agricultural development and has greatly attracted the attention of the scientific community. Compared with other methods, chemical disinfecting is considered to be the most effective pest control method due to its simple operation and fast action. Chemical synthetic pesticides, such as pyrethroids and pyridoxine, have always had a good killing effect on pest control and are highly favored in the market. However, due to their single ingredients, chemical synthetic pesticides not only leave residues on agricultural products, but also produce pest resistance. Among them, arachnid pests, such as red spider mites and tea yellow mites, are one of the important pests that harm a variety of crops. The discovery and development of new chemical insecticides and miticides are very important to minimize the damage caused by pests to agriculture, which will achieve sustainable production of commodities and provide affordable food for the growing world population. Insecticide and acaricide resistance is a major obstacle to effective pest control worldwide and, as noted by the Insecticide Resistance Action Committee, implementation of resistance management strategies based on mode-of-action rotation, including new chemical classes / modes of action, is a prerequisite for modern applied agriculture.

[0003] Natural products are secondary metabolites naturally present in animals, plants and microorganisms in nature, and are a valuable source of bioactive molecules. For example, cyclic dipeptides isolated from Bacillus amyloliquefaciens exhibit significant acaricide activity, indicating that these cyclic dipeptides have the potential to become safe and environmentally friendly acaricides (Li XY, Wang YH, Yang J, et al. J. Agric. Food Chem. 2018, 66, 10163-10168.), and macrolides isolated and identified from Streptomyces avermitilis have significant nematicidal and acaricide activities, and have the potential to be used as insecticides and acaricides (Wang XJ, Wang M, Wang JD, et al. J. Agric. Food Chem. 2010, 58, 2710–2714). Natural products are considered to be an important resource for pest control applications due to their rich structures, excellent activity and environmentally friendly characteristics.

[0004] Glauconic acid is a fungal metabolite, and its structural formula is shown below:

[0005]

[0006] In 2015, Wang Rong et al. isolated glauconic acid from Aspergillus sp. HDf2 (Wang Rong, Lin Guoyao, Chen Fuxiao, Shen Minghui, Zhan Xiafei. Purple sea urchin intestinal fungus Aspergillus sp. HDf2 produces nonadride secondary metabolites [J]. Anhui Agricultural Sciences, 2015, 43(21): 1-2.). However, the extraction rate of glauconic acid by this method is extremely low, only 0.0857%, and there is no relevant literature reporting the biological activity and application of glauconic acid. Therefore, it is necessary to further study the extraction method of glauconic acid with a higher extraction rate, explore its biological activity and clarify its role. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing an Aspergillus dianhydride compound metabolized by microorganisms and the use of the Aspergillus dianhydride compound in killing mites in response to the above-mentioned problems.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a method for preparing a microbially metabolized Aspergillus dianhydride compound, the preparation method comprising fermenting and extracting the Aspergillus dianhydride compound from endophytic fungi in mangroves, wherein the endophytic fungi in mangroves are Aspergillus sp. GXNU-31, which was deposited in the Guangdong Microbiological Culture Collection Center on November 10, 2024, with a deposit number of GDMCC No. 60923.

[0009] The specific preparation method comprises the following steps:

[0010] (1) Inoculate Aspergillus sp. GXNU-31 grown in PDA medium into PDB medium, culture in a constant temperature shaker cabinet at 110-150 rpm and 25-35° C. for 2-6 days, then inoculate into solid culture medium and culture statically for 20-40 days;

[0011] (2) Soaking the fermented strain in methanol for 3-5 times, combining the extracts, concentrating under reduced pressure, extracting with ethyl acetate for 3-5 times, concentrating under reduced pressure to obtain a crude extract, subjecting the extract to silica gel column chromatography, using ethyl acetate-petroleum ether as an eluent, performing gradient elution from 0:100-100:0, subjecting the extract to silica gel column chromatography again, using ethyl acetate-petroleum ether as an eluent, performing gradient elution from 0:100-100:0, and using a recrystallization method for a 20%-30% portion to obtain the aspergillus dianhydride compound.

[0012] In the above preparation method, the composition of the PDB culture medium is as follows: 200 g / L potato and 20-40 g / L glucose.

[0013] In the above preparation method, the composition of the solid culture medium is as follows: 50-90 g of rice and 3-5 g / L of sea salt.

[0014] The present application also provides the use of the Aspergillus dianhydride compound in killing mites, specifically, the Aspergillus dianhydride compound is used to kill crop pests mites.

[0015] The mite described in the above application is citrus red spider.

[0016] Specifically, the present application also provides the use of the Aspergillus dianhydride compound in the preparation of acaricides.

[0017] Furthermore, the pharmaceutical dosage form of the acaricide is a spray, emulsion, granule, suspension, emulsifiable concentrate, powder, aqueous solution, microemulsion or aqueous dispersion.

[0018] Preferably, the pharmaceutical dosage form of the acaricide is a suspension.

[0019] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0020] 1. The present invention is the first to extract glauconic acid from Aspergillus sp. GXNU-31 by fermentation. The extraction rate of glauconic acid in the fermentation extract of the strain is as high as 25%, which is significantly higher than that of the prior art.

[0021] 2. The present invention uses the method described in the existing literature to test the acaricidal activity of the Aspergillus dianhydride compound glauconic acid against citrus red spider mites. The test results show that the prevention effect of the Aspergillus dianhydride compound on citrus red spider mites is above 80%, and is superior to the positive control group 43% bifenazate suspension at 10 days, 15 days, 20 days, and 30 days after the drug is applied. This indicates that the Aspergillus dianhydride compound glauconic acid has significant insecticidal activity against citrus red spider mites, providing a great development path for the industrialization of microbial acaricides.

[0022] 3. The glauconic acid, an aspergillus dianhydride compound produced by fermentation of the mangrove endophytic fungi of the present invention, can be produced on a large scale without being restricted by resources, environment, conditions, equipment, etc. DETAILED DESCRIPTION

[0023] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0024] 1. Compound Preparation

[0025] Example 1

[0026] Isolation and Purification of Aspergillus sp. GXNU-31

[0027] Materials: Leaves of the mangrove plant Acanthus sphaerocephalus collected from Beihai City, Guangxi.

[0028] (1) Plant sample pretreatment: The roots, stems, leaves, and flowers of Acanthus sphaerocephalus were collected from the Beihai mangroves, washed with sterile water, wiped dry, then soaked in 75% alcohol, wiped dry, and finally washed with sterile water and wiped dry. The roots, stems, leaves, and flowers were sliced ​​with a sterile scalpel.

[0029] (2) Isolation of Aspergillus sp. GXNU-31: The prepared plant slices were obliquely inserted into PDA medium and cultured at room temperature, wherein the PDA medium components were as follows: 200 g / L potato, 20-40 g / L glucose, and 15-30 g / L agar;

[0030] (3) Purification of Aspergillus sp. GXNU-31: Culture at room temperature for about 2-3 days, observe whether colonies grow in the PDA medium, transfer the grown colonies to new PDA medium, and purify repeatedly until only a single colony grows to obtain a single strain, which is Aspergillus sp. GXNU-31.

[0031] (4) Preservation of strains: Use an inoculation loop to pick up the mycelium of the strain and place it into a freezing tube. After it has grown well, store it in a refrigerator at low temperature.

[0032] Example 2

[0033] This embodiment provides a method for preparing an aspergillus dianhydride compound metabolized by microorganisms, comprising the following steps.

[0034] (1) In a clean bench, use an inoculation loop to inoculate the strain grown in PDA medium into a 500 mL conical flask containing 200 mL PDB medium, and culture it dynamically in a constant temperature shaker cabinet. After culturing for 6 days at 110 rpm and 25°C, inoculate it into a solid culture medium and culture it statically for 20 days;

[0035] The composition of the PDB culture medium is as follows: 200 g / L potato and 20 g / L glucose, and the composition of the solid culture medium is as follows: 50 g rice and 3 g / L sea salt.

[0036] (2) Soaking the fermented product obtained in step (1) with methanol for 3-5 times, combining the extracts, concentrating under reduced pressure, extracting with ethyl acetate for 3-5 times, and concentrating under reduced pressure to obtain a crude extract (200.3 g), subjecting the extract to silica gel column chromatography with ethyl acetate-petroleum ether as eluent, performing gradient elution from 0:100-100:0, and subjecting the extract to silica gel column chromatography again, using ethyl acetate-petroleum ether as eluent, performing gradient elution from 0:100-100:0, and recrystallizing the 20%-30% portion to obtain a high-purity glauconic acid compound (49.5 g).

[0037] Example 3

[0038] This embodiment provides a method for preparing an aspergillus dianhydride compound metabolized by microorganisms, comprising the following steps.

[0039] (1) In a clean bench, use an inoculation loop to inoculate the strain grown in PDA medium into a 500 mL conical flask containing 200 mL PDB medium, and culture it dynamically in a constant temperature shaker cabinet. After culturing at 130 rpm and 30°C for 4 days, inoculate it into a solid culture medium and culture it statically for 28 days;

[0040] The composition of the PDB culture medium is as follows: 200 g / L potato and 30 g / L glucose, and the composition of the solid culture medium is as follows: 70 g rice and 4 g / L sea salt.

[0041] (2) The fermented product obtained in step (1) was soaked in methanol for 3-5 times, the extracts were combined, concentrated under reduced pressure, extracted with ethyl acetate for 3-5 times, and concentrated under reduced pressure to obtain a crude extract (396.5 g). After silica gel column chromatography, ethyl acetate-petroleum ether was used as an eluent and gradient elution was performed from 0:100 to 100:0, and then silica gel column chromatography was performed again, ethyl acetate-petroleum ether was used as an eluent and gradient elution was performed from 0:100 to 100:0. A 20%-30% portion was recrystallized to obtain a high-purity glauconic acid compound (95.3 g).

[0042] Example 4

[0043] This embodiment provides a method for preparing an aspergillus dianhydride compound metabolized by microorganisms, comprising the following steps.

[0044] (1) In a clean bench, the strain grown in the PDA medium was inoculated into a 500 mL conical flask containing 200 mL PDB medium using an inoculation loop, and dynamically cultured in a constant temperature shaker cabinet. After culturing at 150 rpm and 35° C. for 2 days, it was inoculated into a solid culture medium and statically cultured for 40 days to obtain the fermented strain GXNU-31;

[0045] The composition of the PDB culture medium is as follows: 200 g / L potato and 40 g / L glucose, and the composition of the solid culture medium is as follows: 90 g rice and 5 g / L sea salt.

[0046] (2) The fermented product obtained in step (1) was soaked in methanol for 3-5 times, the extracts were combined, concentrated under reduced pressure, extracted with ethyl acetate for 3-5 times, and concentrated under reduced pressure to obtain a crude extract (2200.6 g). After silica gel column chromatography, ethyl acetate-petroleum ether was used as an eluent and gradient elution was performed from 0:100 to 100:0, and then silica gel column chromatography was performed again, ethyl acetate-petroleum ether was used as an eluent and gradient elution was performed from 0:100 to 100:0. A 20%-30% portion was recrystallized to obtain a high-purity glauconic acid compound (550.5 g).

[0047] The extraction rate of the Aspergillus dianhydride compound prepared in Example 2-4 is shown in Table 1. The strain stably metabolizes 24%-25% of the target product, and the target extraction rate can reach up to 25%.

[0048] Table 1 Extraction rate of aspergillus dianhydride compounds

[0049]

[0050]

[0051] 2. Compound Confirmation

[0052] The aspergillus dianhydride compound glauconic acid obtained above was identified by nuclear magnetic resonance spectroscopy (NMR) to obtain the following test data:

[0053] 1H NMR (400MHz, Chloroform-d)δ H2.47(1H,m,H-1),2.13(1H,m,H-2),5.09(1H,s,H-3-OH),3.78(1H,d,J= 13.8Hz,H-3),3.06(1H,s,H-6a),3.43(1H,d,J=13.8Hz,H-6b),6.87(1H, d,J=12.8Hz,H-9),1.14(3H,t,J=7.3Hz,H-1′),1.14(1H,m,H-2′),1.53 -1.70(3H,m,H-2′b,4′),0.89(3H,t,J=7.4Hz,H-3′),1.71(3H,s,H-5′). 13 C NMR(100MHz,Chloroform-d)δC37.9(C-1),53.1(C-2),65.6(C-3),129.3(C-4),143.4(C-5),31.9(C-6),47.5(C-7),146.4(C-8),15 0.6(C-9),163.5(C-10),164.7(C-11),174.2(C-12),164.7(C-13),13.1(C-1′),19.7(C-2′),12.6(C-3′),27.8(C-4′),26.5(C-5′).

[0054] The above data were compared with the data in the relevant literature, and it was determined that the compounds obtained in Examples 2, 3 and 4 were the aspergillus dianhydride compound glauconic acid.

[0055] 3. Biological Activity Test of Aspergillus dianhydride Compounds

[0056] Evaluation of the acaricidal activity of glauconic acid, an Aspergillus dianhydride compound, against citrus spider mites:

[0057] The test method for acaricidal activity against citrus red spider mites refers to Zhu L, Chen L, Min Y, et al. Acaricidal Activity and Field Efficacy Analysis of the Potential Biocontrol Agent Bacillus vallismortis NBIF-001. Microorganisms 2022, 10, 1750. Using 43% bifenazate suspension as the positive control, the number of citrus red spider mites was recorded before and 10, 15, 20 and 30 days after medication.

[0058] Mite reduction rate = (number before medication - number after medication) / number before medication × 100%;

[0059] Corrected control effect = (mite reduction rate % - mite reduction rate % of blank control group) / (100 - mite reduction rate % of blank control group) × 100%.

[0060] The test results are shown in Table 2 and Table 3:

[0061] Table 2 Reduction rate of citrus red spider mites

[0062]

[0063] Table 3 Control effect of Aspergillus dianhydride compounds on citrus red spider mites

[0064]

[0065] As shown in Table 2, the Aspergillus dianhydride compound can effectively reduce citrus red spider mites, and the mite reduction rate is significantly greater than 43% bifenazate suspension; As shown in Table 3, the Aspergillus dianhydride compound has a control effect on citrus red spider mites of more than 80%, and is superior to the positive control group 43% bifenazate suspension at 10 days, 15 days, 20 days, and 30 days after the drug is applied. This shows that the Aspergillus dianhydride compound glauconic acid has significant insecticidal activity against citrus red spider mites, providing a great development path for the industrialization of microbial acaricides.

[0066] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing a microbial metabolized aspergillus dianhydride compound, characterized in that: The aspergillus dianhydride compound is obtained by fermentation extraction from endophytic fungi in mangroves. The endophytic fungi in mangroves are Aspergillus sp. GXNU-31, which is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC No.60923.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Inoculate Aspergillus sp. GXNU-31 grown in PDA medium into PDB medium, culture in a constant temperature shaker cabinet at 110-150 rpm and 25-35° C. for 2-6 days, then inoculate into solid culture medium and culture statically for 20-40 days; (2) Soaking the fermented product obtained in step (1) with methanol for 3-5 times, combining the extracts, concentrating under reduced pressure, extracting with ethyl acetate for 3-5 times, and concentrating under reduced pressure to obtain a crude extract; and subjecting the extract to silica gel column chromatography, After gradient elution with ethyl acetate-petroleum ether as eluent at a ratio of 0 / 100-100 / 0, the product is subjected to silica gel column chromatography again, with ethyl acetate-petroleum ether as eluent at a ratio of 0 / 100-100 / 0, wherein the fraction with 20%-30% ethyl acetate-petroleum ether is recrystallized to obtain the aspergillus dianhydride compound.

3. The preparation method according to claim 2, characterized in that: The composition of the PDB culture medium is as follows: 200 g / L potato and 20-40 g / L glucose.

4. The preparation method according to claim 2, characterized in that: The composition of the solid culture medium is as follows: 50-90 g of rice and 3-5 g / L of sea salt.

5. Use of the Aspergillus dianhydride compound for killing mites according to any one of claims 1 to 4, characterized in that: The aspergillus dianhydride compound is used for killing crop pests mites.

6. The use according to claim 5, characterized in that: The mite is citrus red spider.

7. The use according to claim 5, characterized in that: Application of the aspergillus dianhydride compound in the preparation of acaricides.

8. The use according to claim 7, characterized in that: The pharmaceutical dosage form of the acaricide is a spray, emulsion, granule, suspension, emulsifiable concentrate, powder, aqueous solution, microemulsion or aqueous dispersion.

9. Use of the aspergillus dianhydride compound according to claim 7 in the preparation of acaricides, characterized in that: The pharmaceutical dosage form of the acaricide is a suspension.

Images