CN115806907A - Preparation and application of a deep-sea streptomyces and its antifeedant and antifungal active substances - Google Patents

Abstract

The invention relates to the technical field of microbial pesticides, in particular to a deep-sea streptomyces with antifeedant function and antibacterial effect and application thereof. The Streptomyces sp.NA13 derived from the deep sea in the South China Sea was deposited in the China Center for Type Culture Collection on August 29, 2022, with the preservation number: CCTCC M 20221343. The strain has the ability to prepare Antimycin, Candicidin, Naringenin, Surugamide, Fredericamycin, SAL-2242, Albaflavenone, Hopene, SGR PTMs and other antibiotics, especially the use of fermentation to prepare various compounds of antimycin or cyclic octapeptide surugamide, including novel The antimycin compound antimycin Q(1). The prepared antimycin substance is used as an antifeedant for insect pests or a lead compound of an antifungal drug, and has the application prospect of being developed into a natural source of antibacterial and anti-insect pesticides.

Description

Preparation and application of a deep-sea streptomyces and its antifeedant and antifungal active substances

technical field

The invention relates to the technical field of microbial pesticides, in particular to a deep-sea streptomyces with antifeeding function and antibacterial effect and application thereof.

Background technique

Actinomycetes are widely distributed in nature, and they can produce metabolites with various biological functions. It is worth noting that about 76% of commercially available antibiotics’ original molecular structure frameworks are all derived from Actinomycetes, among which the highly developed Streptomyces is the most prominent (Journal of Industrial Microbiology Biotechnology, 2014, 41(2): 425-431). In 2019, according to the report of Professor Li Dehai's team, Streptomyces contributed 54% of the total number of new active secondary metabolites produced by actinomycetes (Current Medicinal Chemistry, 2020, 27(36):6244-6273). What's more interesting is that Professor Zhu Weiming's team found that 67.3% of the natural products of deep-sea Streptomyces showed biological activities, such as cytotoxicity, antibacterial, antimalarial, etc. (Marine Science Journal, 2016, 51:86-124).

The long-term and extensive use of organic synthetic insecticides has led to the development of insecticide resistance in pests, causing serious environmental pollution and agricultural product safety issues (Science, 2002, 297: 2222-2223). With the concept of green prevention and control deeply rooted in the hearts of the people, people are constantly exploring safer and more environmentally friendly pest control methods. The purpose of pest control is to effectively control the damage, not the only means of killing pests. Effective protection can also be achieved by preventing insects from feeding (or repelling). Therefore, insect repellents have become an important alternative to organic insecticides. Cotton bollworm (Helicoverpa Armigera) is a worldwide widespread pest, which seriously harms cotton, African rice, corn and other food crops. It feeds on the young growth points and reproductive organs of crops, and its larvae drill and eat flower buds (Northern Horticulture, 2020 , 20:27-33). At present, chemical pesticides are mainly used to prevent and control cotton bollworms, but cotton bollworms have developed resistance to chemical pesticides, and chemical pesticides degrade slowly and remain. Therefore, the development and application of green, efficient and environment-friendly natural source food repellents has attracted the attention of many scholars.

Plant fungal diseases have a high incidence and a wide range, and their occurrence causes a decline in the yield and quality of agricultural products, seriously affecting the development and safety of the agricultural industry. For example, apple rot is a disease caused by the stubborn weak host parasitic fungus Valsa ceratosperma, also known as rot (Modern Agricultural Science and Technology, 2008, 23: 147-147). Apple rot pathogens have the characteristics of latent infection, that is, when the fruit trees are healthy, the pathogens can maintain a latent state and are not easy to expand and cause diseases to the host; when the tissues around the infection point die and the host's vitality is weak, the pathogens can expand and cause diseases. The disease mainly occurs in mature orchards, resulting in reduced apple production and economic losses. At present, my country has generally entered a period of high incidence of apple rot disease, and about 80% of mature orchards have rot disease (Wang Baojun, 2017, master's thesis of Anhui Agricultural University). Apple rot disease has become one of the important diseases on apple trees in my country. It has plagued fruit farmers for a long time, seriously restricted the yield of apples, and caused serious economic losses. It has become an important disease that restricts the production and export of apples in my country.

At present, there are many varieties of commercialized fungicides. With the long-term use of traditional fungicides, problems such as drug resistance and drug resistance of plant pathogens have appeared, and problems such as pesticide residues and environmental pollution have yet to be resolved (Environmental Science & Technology, 2019, 53 (7 ):3347-3365). Therefore, the development of fungicides with novel mechanism of action, broad bactericidal spectrum, and low risk of resistance is the current focus of fungicide creation.

Contents of the invention

The invention provides a deep-sea streptomyces with anti-feeding function and anti-multiple pathogenic fungi and application thereof.

In order to achieve the above object, the technical solution adopted by the present invention is:

A deep-sea streptomyces, the Streptomyces sp.NA13 derived from the deep sea of the South China Sea, was preserved in the China Center for Type Culture Collection on August 29, 2022, and the preservation number is CCTCC M 20221343.

The deep-sea Streptomyces contains gene clusters that synthesize Antimycin, Candicidin, Naringenin, Isorenieratene, indigoidine-like, Ectoine, Desferrioxamine B, Surugamide A, Fredericamycin A, SAL-2242, Albaflavenone, Hopene, SGR PTMs, Diisonitrileantibiotic SF2768 skeleton compound ( 7.10Mb genome).

An application of Streptomyces deep-sea, the application of the NA13 metabolite of Streptomyces deep-sea in the preparation of antifeedant or antifungal candidate drug.

An application of Streptomyces deep-sea, the use of the NA13 metabolite of Streptomyces deep-sea in the preparation of any type of compound in the antimycin compound antimycin or the cyclic octapeptide compound surugamide.

The preparation of the Streptomyces deep-sea NA13 metabolite:

1) Inoculate the deep-sea Streptomyces NA13 described in claim 1 on the ISP3 solid medium and cultivate it for 2-5d activation, then inoculate it in the ISP3 liquid medium, and cultivate it at 28°C for 2-4d as the seed liquid; then ferment the seed liquid according to the volume Percentage 2-10% inoculated in ISP3 liquid medium, cultured with shaking at 28°C for 5-10d;

2) Centrifuge the fermentation broth to obtain the bacteria, extract the active components in the bacteria with methyl ethyl ketone ultrasonically for 30-50 minutes, and recover the butanone solvent to obtain the crude extract A, which is the metabolite of Streptomyces deep-sea NA13.

The crude extract A was separated by flash silica gel column chromatography, and the dichloromethane:methanol (v/v) ratio was 100:0-0:100 gradient elution; the 100:0-100:1 eluted stream was Part F _B was decontaminated by gel LH20, and was eluted with 80-90% methanol aqueous solution by semi-preparative reverse-phase HPLC at a flow rate of 2.0-4.0mL/min to obtain compounds antimycin Q(1), antimycinA1a(2), antimycin A2a(3), antimycin A3a(4), antimycin A4a(5), antimycin A7a(6); the fraction F _C eluted at 100:2-100:5 was removed by gel LH20, and semi-preparative reaction Phase HPLC, eluted with 30-40% methanol aqueous solution, the flow rate is 2.0-3.0mL/min, to obtain the compound N-formylantimycic acid methyl ester (7); the fraction eluted at 100:18-100:22 F _F Remove impurities by gel LH20, use semi-preparative reverse-phase HPLC, and elute with 40-50% acetonitrile aqueous solution at a flow rate of 2.0-3.0mL/min to obtain compounds surugamide A (8), surugamide B (9), and surugamide D (10), surugamide E (11).

A kind of new antimycin (antimycin) compound produced from deep-sea streptomyces, antimycin (antimycinQ) compound is shown in (I),

An application of an antimycin compound, said compound antimycin Q(1), antimycin A1a(2), antimycin A2a(3), antimycin A3a(4), antimycin A4a(5), antimycinA7a(6) It can be used as antifeedant or antifungal drug lead compound.

The fungus is

Apple tree rot fungus Valsa mali F68-1

Fusarium oxysporum f.sp. Cucurmerimum S19

The advantages that the present invention has:

1. The bacterial strain NA13 obtained in the present invention has uniqueness. Its living environment is special, and it comes from the deep-sea sediments of the South China Sea, and through whole genome sequencing and bioinformatics analysis, it can be seen that its genome contains genes that synthesize and produce 25 kinds of skeleton type compounds such as Antimycin Clusters have the potential to produce diverse active substances.

2. bacterial strain NA13 of the present invention can prepare antimycin compound antimycin or cyclic octapeptide surugamide compound simultaneously; Wherein antimycin antimycin A1a (2), antimycin A2a (3), antimycin A3a (4), antimycin A4a (5 ), the antifeedant function and anti-phytopathogenic activity of antimycin A7a (6) are the first inventions.

3. The present invention obtains antimycin Q through the metabolites of bacterial strain NA13. This compound is a new compound discovered for the first time. The prepared antimycin substance is used as a lead compound of insect pest antifeedant or antifungal drug, and has the ability to be developed into a natural source Antibacterial, anti-insect pesticides and other application prospects.

Description of drawings

Figure 1 shows the taxonomic status of NA13 based on the phylogenetic tree based on the 16S rDNA sequence provided by the present invention.

Fig. 2 is the secondary metabolites of antimycin compounds produced by the strain NA13 provided by the present invention in the ISP3 fermentation medium.

Figure 3 shows the chemical structures of compounds 1-11 obtained by using the strain NA13 provided by the present invention.

Fig. 4 is the HRESIMS spectrum of antimycin Q(1) obtained by using the strain NA13 provided by the present invention.

Fig. 5 is an important correlation between ¹ H- ¹ HCOSY and HMBC of antimycin Q(1) obtained by strain NA13 provided by the present invention.

Fig. 6 is the experimental ECD spectrum and calculated ECD spectrum of antimycin Q(1) in CH ₃ OH obtained by using the strain NA13 provided by the present invention.

Figure 7 shows the antifeedant activity of antimycin compounds 1-6 against cotton bollworm.

Detailed ways

In order to better understand the content of the present invention, the following will be further described in conjunction with specific examples, but the protection content of this patent is not limited thereto.

Identification and characteristics of embodiment 1 deep-sea Streptomyces NA13

Strain NA13 was isolated from deep-sea sediments in the South China Sea (E 113°2.353′, N 13°58.498′), and was deposited in the China Center for Type Culture Collection on August 29, 2022, with the preservation number: CCTCC M20221343. The bacterium grows vigorously on ISP3 and ISP4 medium, and grows generally on ISP2 and Gaoshi No. 1 medium. Round colonies formed without pigmentation (Table 1).

Table 1 Growth characteristics of deep-sea Streptomyces NA13

culture medium growing situation aerial hyphae base hyphae Soluble pigment ISP2 average growth Milky yellow flesh-colored none ISP3 Vigorous growth light yellow flesh-colored none ISP4 Vigorous growth milky flesh-colored none Gao Shi No. 1 average growth light yellow flesh-colored none

Streptomyces NA13 was inoculated in ISP3 liquid medium, cultured at 28°C for 36 hours, and total DNA was extracted according to conventional methods. Then adopt the Whole Genome Shotgun (WGS) strategy, use the second generation sequencing technology (Next Generation Sequencing, NGS) based on the Illumina NovaSeq sequencing platform, and use the third generation single molecule sequencing technology based on the Oxford Nanopore ONT sequencing The platform performs sequencing and obtains the complete genome map of strain NA13. The size of the linear chromosome genome is about 6.91Mb, and the GC content is 73.44%. It contains a linear plasmid with a size of about 186.13kb and a GC content of 68.83%. BLAST analysis of its 16S rRNA gene sequence showed that it had 100% sequence homology with three different standard strains S. daghestanicus NRRL B-5418 ^T , S. albidoflavus DSM 40455 ^T and S. violascens ISP 5183 ^T . A further phylogenetic tree based on the 16S rDNA sequence showed that these strains were clustered in the same branch (Figure 1), showing that they had the closest genetic evolutionary relationship. Finally, calculating the average nucleotide identity (Average Nucleotide Identity, ANI) value of the genome showed that the ANI of Streptomyces NA13 and S.albidoflavus DSM 40455 ^T reached 98.7%, indicating that they are one species. Strain NA13 was therefore identified as Streptomyces albidoflavus.

Wherein, the 16S rRNA gene sequence 16S rDNA of Streptomyces alboflavinus NA13 is:

>Streptomyces albidoflavus NA13 16S rDNA

ACGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCGCTTTCGGGCGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACTGTCCATCGCATGGTGGATGGTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCTGGAGACAGGCGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTT

Using antiSMASH analysis and manual correction, it was found that strain NA13 contained 25 secondary metabolite gene clusters, which were encoded to produce Antimycin, Candicidin, Naringenin, Isorenieratene, indigoidine-like, Ectoine, Desferrioxamine B, Surugamide A, Fredericamycin A, SAL-2242, The potential of Albaflavenone, Hopene, SGR PTMs, Diisonitrile antibiotic SF2768 backbone compounds, and the potential to synthesize a variety of novel PKS, RiPP-like, NRPS, Terpene, and Siderophore compounds (Table 2). It shows that NA13 has the ability to prepare the types of compounds mentioned in the table.

Table 2 Potential of strain NA13 to synthesize various secondary metabolites

Embodiment 2 Deep-sea Streptomyces NA13 produces various types of biologically active substances and preparation methods

Streptomyces NA13 was fermented with ISP3 medium to obtain a series of secondary metabolic active products such as antimycins and cyclic octapeptides (Figure 2); specifically:

Streptomyces deep-sea NA13 was inoculated on ISP3 solid medium and cultured for 72 hours to activate, then inoculated in ISP3 liquid medium, and cultured at 28°C for 72 hours as the seed liquid; then the fermented seed liquid was inoculated in the same fermentation medium at a volume ratio of 1:18 , cultured with shaking at 28°C for 7 days. The fermentation broth was centrifuged to obtain the bacteria, the active ingredients in the bacteria were extracted by ultrasound for 30-50 minutes with butanone, and the crude extract A obtained after recovering the butanone solvent was the metabolite of Streptomyces deep-sea NA13. The crude extract A was separated by flash silica gel column chromatography and eluted according to the dichloromethane:methanol (v/v) ratio of 100:0-0:100 gradient to obtain 7 fractions (F _A -F _G ) . The fraction F _B eluted at 100:1 was removed by gel LH20, and semi-preparative reversed-phase HPLC was used to elute with 85% aqueous methanol at a flow rate of 3.0 mL/min to obtain compound antimycin Q (compound 1) ( 5.7mg, t _R :18.5min), antimycin A1a(2)(6.4mg, t _R :20.2min), antimycin A2a(3)(8.0mg, t _R :15.8min), antimycin A3a(4)(4.8mg ,t _R :12.5min), antimycin A4a(5)(5.3mg,t _R :10.1min), antimycin A7a(6)(4.0mg,t _R :11.5min). The fraction F _C eluted at 100:2-100:5 was removed by gel LH20, and semi-preparative reverse-phase HPLC was used to elute with 40% methanol aqueous solution at a flow rate of 2.5mL/min to obtain the compound N-formylantimycic acid methylester (7) (2.8 mg, t _R : 17.3 min). The 100:20 eluted fraction F _F was removed by gel LH20, and semi-preparative reversed-phase HPLC was used to elute through 45% acetonitrile aqueous solution, and the flow rate was 2.5mL/min to obtain compound surugamide A (8) (52.3 mg,t _R :22.1min), surugamide B(9)(2.5mg,t _R :16.5min), surugamide D(10)(3.8mg,t _R :18.3min), surugamide E(11)(3.6mg, t _R : 19.4min). The structures of the above compounds are shown in Figure 3.

Analyze the structure of the compound obtained above, specifically compound 1 is a light yellow powder, and the HRESIMS data shows a [M+H] ⁺ ion peak of m/z 549.2809 (Figure 4), corresponding to the molecular formula C ₂₈ H ₄₀ N ₂ O ₉ , the degree of unsaturation is 10. The ultraviolet absorption at 228nm and 317nm in the ultraviolet spectrum is the characteristic absorption of antimycin compounds. Combining 1D NMR (DMSO-d ₆ ) and HSQC spectra, it can be seen that the 28 carbons are assigned to 5 carbonyl carbon signals (δ _C 179.4, 173.9, 169.7, 169.4 and 160.4), and 6 aromatic carbon signals (δ _C 150.6, 126.9, 125.0 , 123.1, 118.2 and 114.3), seven methylene carbon signals (δ _C 75.3, 73.2, 72.0, 54.0, 49.7, 39.5 and 32.8), four methylene carbon signals (δ _C 33.5, 28.6, 25.9 and 25.5 ), six methyl carbon signals (δ _C 18.7, 17.8, 16.6, 15.2, 11.4 and 11.1), these NMR signals indicated the presence of antimycin backbone structure. ^{The 1} H- ¹ H COZY correlation signals of NH-3' (δ _H 9.81) and H-8' (δ _H 8.31) can be speculated that there is a fragment of NH-CHO. According to a group of aromatic hydrocarbon signals (δ _C 114.3, δ _C 150.6, δ _C 126.9, δ _H 8.23/δ _C 125.0, δ _H 7.85/δ _C 123.1 and δ _H 6.91/δ _C 118.2) (see Table 3) and NH-3' (δ _H 9.81) and C-4' (δ _C 125.0) HMBC-related signals, inferring the presence of N-formyl-aminosalicylic acid fragments. ¹ H- ¹ H COZY spectrum H-7/H-1″/H-2″/H-3″/H-4″/H-5″/H-6″ correlation signal and HMBC spectrum H- 6″ and the correlation signals of C-2″ and C-4″, it can be speculated that there is a 3-methylhexane group (Figure 5). H-2″′/H-3 in ¹ H- ¹ H COZY spectrum The correlation signals of "'/H-4"'/H-5"' and the correlation signals of H-8, H-3"' and H-5"' in the HMBC spectrum and C-1"' respectively can be speculated to exist A 2-methylbutyryl group (Figure 5). Since this compound has an unsaturation of 10, removing the five carbonyl signals and one benzene ring, the final unsaturation should be a single ring. Combining ^the NH- ³ /H-3/H-4/H-11 and H-10/H-9/H-8/H-7/H-1" related signals in the 1 H- 1 H COZY spectrum and the HMBC spectrum Among them, H-3, H-4 and H-9 are respectively related to C-2, and H-4 and H-7 are respectively related to C-6. It can be inferred that there is a nine-membered dilactone ring fragment (Figure 5) Therefore, the planar structure of compound 1 is shown in Figure 5. The relative configuration of compound 1 is determined according to the coupling constant and NOESY spectrum. Visible H-3/H-4, H-7/H-9/H in the NOESY spectrum -11, the connection of H-8/H-10, and the large coupling constant (9-10Hz) between H-7/H-8 and H-8/H-9, it means that H-3, H-4, H- 8 and H-10 are on one plane, and H-7 and H-9 are on another plane (Fig. 5). The absolute configuration of the parent nucleus of compound 1 obtained by calculating ECD is 3S, 4R, 7R, 8R and 9S (Fig. 6).Therefore compound 1 was named antimycin Q as a new compound (Fig. 3).

Table 3 ¹ H NMR and ¹³ C NMR data of Compound 1 (600MHz, DMSO-d ₆ , δin ppm, J in Hz)

position ¹H NMR ¹³C NMR 2 169.4,C 3 5.32,t,(7.9) 54.0,CH 4 5.58, p, (6.5) 72.0,CH 6 173.9,C 7 2.54,m 49.7,CH 8 4.88,m 75.3,CH 9 4.93,m 73.2,CH 10 1.19,m 17.8, CH₃ 11 1.30, d. (6.7) 15.2, CH₃ 1' 114.3,C 2' 150.6,C 3' 126.9,C 4′ 8.23, d, (7.9) 125.0,CH 5′ 6.91,t,(8.0) 118.3, CH 6' 7.85, d, (8.1) 123.1,CH 7' 169.7,C 8' 8.31, d, (1.8) 160.4,C 1" 1.46,m; 1.30,m 25.5,CH₂ 2" 1.22,m 33.5,CH₂ 3″ 1.18,m 32.8,CH 4″ 1.18,m 28.6, CH₂ 5″ 0.78,m 11.1, CH₃ 6″ 0.78,m 18.7, CH₃ 1"' 174.9,C 2"' 2.45,m 39.5,CH 3"' 1.63,m; 1.46,m 25.9, CH₂ 4"' 0.86,t,(7.4) 11.4, CH₃ 5"' 1.12, d, (7.2) 16.6, CH₃ 2′-OH 12.74,br s 3-NH 9.33,br s 3′-NH 9.81,s

Other known compounds antimycin A1a(2), antimycin A2a(3), antimycin A3a(4), antimycin A4a(5), antimycin A7a(6), N-formylantimycic acid methyl ester(7), surugamide A(8) , surugamide B(9), surugamide D(10), and surugamide E(11) (Fig. 3) were all determined by comparing and reporting relevant literature on 1DNMR spectra and optical rotation data.

Example 3 Determination of Antifeedant Functional Activity of Antimycin Compounds

Determination method: Cotton bollworm (Helicoverpa Armigera) was selected to evaluate the antifeeding function. Measuring method: first punch uniform thickness, clean and fresh Chinese cabbage (Brassica chinensis) leaves into a circular leaflet disc with a diameter of 1 cm, and the treatment group takes the test compound solution (monomer compound (compound 1-6) 50 μL of three different concentrations (1, 0.5, 0.25 mg/ml) were evenly applied to the back of the leaves, and the same volume of methanol solution was applied to the blank control group. After the leaf discs were naturally dried, they were placed in a Petri dish pre-matched with filter paper for moisture retention. (9 cm in diameter), put 2 treatment leaf discs and 2 control leaf discs in each petri dish, and place them crosswise. Insert 2 third-instar cotton bollworms starved for 12 hours into each petri dish, put the petri dish into a light incubator at 25°C, take the high-concentration medium control as the cut-off time when about 80% of the food is eaten, and use coordinate graph paper The ingested area of each leaf disc was measured. The experiment was repeated at least 5 times. Calculation of antifeeding rate: antifeeding rate (%)=(AC-AT)/(AC+AT)×100%, wherein, AC and AT respectively represent the area of the blank control group and the sample treatment group leaf discs being eaten.

Experimental results: The crude extract of NA13 exhibited significant antifeedant activity at 5 mg/mL (AI=83.33%). It can be seen from FIG. 7 that the antimycin compounds 1-6 mentioned in the present invention all have different degrees of antifeedant activity against cotton bollworms. Especially the antifeedant activity of compound 1-4 at 1 mg/mL (AI=87.83%, 88.46%, 89.86%, 90.79%); it can be preliminarily inferred that the nine-membered dilactone ring structure of the antimycin core is necessary for this activity group.

Embodiment 4 Determination of Antimycin Compound Antifungal Activity

The apple tree rot pathogen Valsa mali F68-1 and Fusarium oxysporum f.sp. Cucurmerimum S19 were selected as the strains for antifungal activity evaluation.

Determination method: inoculate the pathogenic fungi into the center of the PSA plate by digging, place in a constant temperature incubator at 28°C, and cultivate for 2-3 days. When the diameter of the colony is about 4cm, place symmetrically on the plate and add 10 μL of the test solution (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg/mL) of filter paper. The negative control was methanol solution, and the positive control was nystatin. The plate was placed in a constant temperature incubator at 28°C for 2 days. The lowest concentration of the test substance at which the sterile growth could be observed with the naked eye was taken as the minimum inhibitory concentration (minimal inhibitory concentration, MIC), and each test was repeated three times.

Experimental results: The test results showed that the isolated antimycin compounds 1-6 showed different degrees of inhibitory activity against the two pathogenic fungi, but the activity was weak. Compound 7 had no inhibitory activity against two pathogenic fungi (Table 4).

Table 4 compound 1-7 antifungal activity

Claims (8)

1. A deep sea streptomycete is characterized in that: streptomyces sp.NA13 is obtained from deep sea in south China, and is preserved in China center for type culture Collection at 29.8.2022 with the preservation number: CCTCC M20221343.

2. The Streptomyces abyssal as claimed in claim 1 wherein: the Streptomyces abyssocyanus contains a gene cluster (7.10 Mb genome) which produces synthetic compounds of the skeleton of Antimycin, candicidin, naringin, isoprenatalene, inditoidine-like, ectoine, desferrioxamine B, surugamid A, fredenicamycin A, SAL-2242, albaflavone, hopen, SGR PTMs, diasonitrile antibody SF 2768.

3. The use of the Streptomyces abyssochlii of claim 1, which comprises: the deep sea streptomyces NA13 metabolite is applied to preparation of antifeedants or antifungal candidate drugs.

4. The use of the Streptomyces abyssochlii of claim 1, which comprises: the deep sea streptomyces NA13 metabolite is applied to preparation of any compound of antimycin compounds or cycloocta peptide compounds surugamide.

5. Use of Streptomyces deep sea according to claim 3 or 4, characterized in that: the preparation of the Streptomyces abyssochlii NA13 metabolite comprises the following steps:

1) Inoculating the Streptomyces abyssochlaini NA13 of claim 1 on an ISP3 solid culture medium for 2-5d activation, then inoculating on an ISP3 liquid culture medium, and culturing at 28 ℃ for 2-4d to serve as a seed solution; inoculating the fermented seed liquid into ISP3 liquid culture medium according to the volume percentage of 2-10%, and performing shaking culture at 28 ℃ for 5-10 days;

2) Centrifuging the fermentation liquor to obtain thalli, extracting active ingredients in the thalli by butanone ultrasonic for 30-50min, and recovering butanone solvent to obtain a crude extract A, namely the Streptomyces abyssocensis NA13 metabolite.

6. Use of a Streptomyces abyssal as claimed in claim 4 in which: separating the crude extract A by adopting a flash silica gel column chromatography, and performing separation according to a dichloromethane ratio: gradient elution with methanol (v/v) ratio of 100; fraction F eluted from 100 _B Removing impurities by gel LH20, adopting semi-preparative reverse phase HPLC, eluting by 80-90% methanol water solution with the flow rate of 2.0-4.0mL/min to obtain compounds of antimycin Q (1), antimycin A1a (2), antimycin A2a (3), antimycin A3a (4), antimycin A4a (5) and antimycin A7a (6); fraction F eluted from 100 _C Removing impurities by gel LH20, eluting with 30-40% methanol water solution at flow rate of 2.0-3.0mL/min by semi-preparative reverse phase HPLC to obtain compound N-formylacetic acid methyl ester (7); fractions F eluted from 100 _F Removing impurities by gel LH20, and adopting semi-prepared reversed phase HPLC eluted with 40-50% acetonitrile in water at a flow rate of 2.0-3.0mL/min to obtain surugamide A (8), surugamide B (9), surugamide D (10), and surugamide E (11).

7. A method for producing novel antimycin compounds from Streptomyces abyssalis, characterized in that: the antimycin (antimycin Q) compound is shown as (I),

8. use of an antimycin (antimycin) compound according to claim 4 or 7, characterized in that: the compounds, namely the antimycin Q (1), the antimycin A1a (2), the antimycin A2a (3), the antimycin A3a (4), the antimycin A4a (5) and the antimycin A7a (6), can be used as antifeedants or antifungal medicine lead compounds.

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