CN113846126B

CN113846126B - Preparation method and application of banana vascular wilt resistant small molecular compound

Info

Publication number: CN113846126B
Application number: CN202111205934.5A
Authority: CN
Inventors: 王尉; 周登博; 陈宇丰; 谢江辉; 张璐; 李凯; 赵炎坤; 井涛; 起登凤
Original assignee: Hainan Normal University; Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences; Haikou Experimental Station of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Hainan Normal University; Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences; Haikou Experimental Station of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2023-08-04
Anticipated expiration: 2041-10-15
Also published as: CN113846126A

Abstract

The fourth aspect of the invention provides a preparation method of the aspergillus terreus ketone, which is separated from Streptomyces samsonii XJC-2-6 fermentation broth. The invention adopts actinomycetes for fermentation and separation to obtain the aflatone for the first time, and further researches show that the compound can effectively antagonize No. 4 physiological race of banana fusarium wilt, destroy mycelium structure, inhibit mycelium growth and EC ₅₀ 、EC ₇₅ And EC (EC) ₉₅ The total sugar content, the total protein content, the fat content, the mitochondrial respiratory chain complex enzyme I-IV activity and the like in the banana fusarium wilt 4 physiological race can be obviously reduced by 25.76 mug/mL, 86.30 mug/mL and 491.37 mug/mL respectively. The invention provides a new thought for preparing the aspergillum terrestris, expands a new field for preventing and treating plant diseases such as fusarium wilt and the like, and has wide development space and good development and application prospects.

Description

Preparation method and application of banana vascular wilt resistant small molecular compound

Technical Field

The invention relates to a preparation method and application of a small molecular compound, in particular to a preparation method and application of a small molecular compound for resisting banana vascular wilt.

Background

Bananas (Musa spp.) belong to the genus Musa of the family Musaceae (Musaceae), one of the most valuable primary agricultural products in the world, the highest fruit in the world (135 countries in 2016 total yield 1.48 million tons), the main food for about 4 million people worldwide, the fourth largest food crop located next to rice, wheat, corn by the world's grain and agriculture organization (ducaneli, 2017; li et al, 2018). Bananas are also the eighth world food crop, and have very important roles in the world (Dong et al, 2015) because of their rapid growth, rich nutrients and high economic value. However, the occurrence and spread of banana vascular wilt also presents the most serious threat and challenge, while the global banana industry is rapidly developing. Banana wilt, also known as banana panama disease, yellow mosaic disease, is a destructive soil-borne disease caused by fusarium oxysporum cubeba specialization bacteria (Fusarium oxysporum f, sp. Cube, foc), which damages banana vascular bundles and causes death of plants, is one of the most serious fungal diseases worldwide, and is also a main limiting factor for banana production (Zhang et al, 2014).

Banana wilt first appears in australia and was found and reported in panama in 1896 (Ploetz et al, 2015). 1935-1939 it has exploded in large areas in south america and spread rapidly throughout the world. At present, the disease is commonly generated in tropical and subtropical banana planting areas such as Latin America, africa and Asia, and the development of banana industry worldwide is seriously affected (Hwang et al, 2004).

The perennial nature and polycyclic nature of banana vascular wilt complicates and limits long term control measures (Ploetz, 2015). In recent years, due to the great investment of pesticides, concerns on economy, environment and food safety are raised, biological control is widely focused on plant pathology control, a biological control technology is one of important means for controlling fusarium wilt, the method has the characteristics of high efficiency, safety and the like, and the acquisition of high-efficiency antagonistic microorganisms is the basis of research on biological control (Fu et al, 2017). The use of biocontrol agents has proven to be an environmentally friendly control strategy (Xue et al 2015, deltaur et al 2017; fu et al 2017). Some studies have shown that biological control agents have an inhibitory effect on the growth of Foc both under in vitro conditions and in potting experiments (Thangavelu et al, 2004; mohammed et al, 2011; gnanasekaran et al, 2015; ho et al, 2015; sekhar et al, 2015). Chen et al (2018) reported that treatment of banana plants inoculated with F.oxysporum No. 4 race with Streptomyces in potting experiments did not show symptoms of banana wilt such as leaf wilt and internal discoloration of the plant. In field trials, trichoderma harzianum applied in soil effectively controlled banana wilt, with an effect comparable to carbendazim (Thangavelu et al, 2004). Xue et al (2015) screened bacillus for a potential biocontrol agent that plays an important role in banana vascular wilt control. The research of Cao et al (2005) shows that Streptomyces griseus has control effect on fusarium wilt and can be developed into biological control agent for controlling banana fusarium wilt. The success of biocontrol depends not only on the production method but also on the costs involved and on the effective biocontrol agents, which furthermore have to be able to be stored as dry preparations for a long period of time (Jackson, 1997).

Disclosure of Invention

The invention aims to overcome the defects in the prior art, takes banana fusarium wilt No. 4 physiological race (Foc TR 4) as target pathogen, adopts the culture medium with the optimal proportion and fermentation conditions to ferment a strain of marine streptomycete, extracts to obtain active crude extract, and adopts the modern chromatographic separation technologies such as forward and reverse phase silica gel column chromatography, sephadex LH-20 gel column chromatography, preparative HPLC and the like to research active secondary metabolites. The co-separation is carried out to obtain 1 banana wilt resistant active micromolecular monomer compound, and the structure of the compound is identified by adopting modern spectrum technologies such as 1D-NMR,2D-NMR, HR-MS and the like and combining literature report data information.

A first aspect of the present invention provides the use of Streptomyces samsonii XJC-2-6 in the preparation of aspergilli.

The second aspect of the invention provides the application of Streptomyces samsonii XJC-2-6 fermentation broth in the preparation of the aspergillone.

In a third aspect, the invention provides an application of Streptomyces samsonii XJC-2-6 fermentation broth ethyl acetate extract in preparation of aspergillone.

Wherein the ethyl acetate extract of the fermentation liquor of the streptomyces samsonii XJC-2-6 is obtained by adding ethyl acetate into supernatant obtained by adding ethanol into the fermentation liquor of the streptomyces samsonii XJC-2-6 for extraction and filtration, and concentrating an ethyl acetate phase.

The amount of ethanol to be added is not particularly limited, and may be added empirically by those skilled in the art.

The amount of ethyl acetate to be added is not particularly limited, and may be added empirically by those skilled in the art.

The fourth aspect of the invention provides a preparation method of the aspergillus terreus ketone, which is separated from Streptomyces samsonii XJC-2-6 fermentation broth.

Preferably, the preparation method comprises the following steps: (1) Inoculating Streptomyces samsoni XJC-2-6 into a fermentation culture solution for fermentation culture to obtain a fermentation solution; (2)Adding proper amount of ethanol into the fermentation broth for extraction, filtering, taking supernatant, adding proper amount of ethyl acetate for extraction, taking ethyl acetate phase, and concentrating to obtain ethyl acetate extract; (3) Separating ethyl acetate extract by silica gel column chromatography, and separating with CH ₂ Cl ₂ Gradient elution with MeOH System (v/v: 100% CH ₂ Cl ₂ Performing antibacterial tests on the obtained components by taking Foc TR4 as target pathogenic bacteria and performing TLC-direct bioautography antibacterial activity detection on the components to obtain a main active component Fr.A, wherein the antibacterial tests comprise 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1,5:1,2:1 and 100% MeOH; (4) Subjecting the active component Fr.A to Sephadex LH-20 gel column chromatography, eluting with CH2Cl2/MeOH (v/v: 2:1) eluent, performing antibacterial test on each obtained component, and detecting the antibacterial activity of TLC-direct bioautography to obtain higher active component Fr.A-4; (5) Repeatedly separating and purifying the active component Fr.A-4 by RP-HPLC to obtain the aspergillone.

Further preferably, in the step (1), the fermentation broth is M6 liquid medium, the inoculation amount is 5%, and the shaking culture is carried out at 28 ℃ for 8d at 180 r/min.

The fifth aspect of the invention is to provide the application of the aflatoxin in antagonizing the No. 4 physiological race of banana fusarium wilt.

The sixth aspect of the invention provides an application of aspergillosis in preparing a pesticide for preventing and treating diseases caused by banana fusarium wilt 4 physiological race.

The seventh aspect of the invention is to provide the application of the aflatoxin in reducing the content of soluble total sugar and/or the content of soluble total protein and/or the content of fat and/or the activity of mitochondrial respiratory chain complex enzymes I-IV in the physiological race 4 of banana fusarium wilt.

The invention takes banana fusarium wilt No. 4 physiological race (Foc TR 4) as target pathogen, and takes samson chain as the target pathogenThe method comprises the steps of fermenting and extracting mould XJC-2-6 to obtain active crude extract, researching active secondary metabolites by adopting modern chromatographic separation technologies such as forward and reverse phase silica gel column chromatography, sephadex LH-20 gel column chromatography, preparative HPLC and the like, finally separating to obtain 1 banana wilt resistant active micromolecular monomer compound, identifying the structure of the compound by adopting modern spectroscopic technologies such as 1D-NMR,2D-NMR, HR-MS and the like and combining literature report data information, namely aspergillus terreus (terrain), namely the aspergillus terreus is obtained by fermenting and separating actinomyces for the first time, further researching and finding that the compound can effectively antagonize banana wilt bacteria No. 4 physiological seeds, destroy mycelium structures, inhibit mycelium growth and realize the EC ₅₀ 、EC ₇₅ And EC (EC) ₉₅ The total sugar content, the total protein content, the fat content, the mitochondrial respiratory chain complex enzyme I-IV activity and the like in the banana fusarium wilt 4 physiological race thallus can be obviously reduced by 25.76 mug/mL, 86.30 mug/mL and 491.37 mug/mL respectively. The invention provides a new thought for preparing the aspergillum terrestris, expands a new field for preventing and treating plant diseases such as fusarium wilt and the like, and has wide development space and good development and application prospects.

Drawings

FIG. 1 is a flow chart for separating and purifying chemical components of Streptomyces 2-6 ethyl acetate extract.

FIG. 2 shows the structure of Compound A7.

FIG. 3 shows the inhibition of Foc TR4 hyphal growth by Compound A7.

FIG. 4 shows the effect of compound A7 on the morphological changes of Foc TR4 hyphae under scanning electron microscope, with CK on the left and compound A7 on the right.

FIG. 5 shows the effect of compound A7 on the change in conidium morphology of Foc TR4 under transmission electron microscopy, with CK on the left and compound A7 on the right.

FIG. 6 shows the effect of compound A7 on the ultrastructural change of Foc TR4 under transmission electron microscopy, A being the control, B and C being the treatment with compound A7.

FIG. 7 shows the effect of active compound A7 on the N-acetylglucosamine content in Foc TR4 cells.

FIG. 8 shows the effect of active compound A7 on total sugar in banana vascular wilt No. 4 micro-organisms.

FIG. 9 shows the effect of active compound A7 on the soluble protein content in banana vascular wilt No. 4 micro-organisms.

FIG. 10 shows the effect of active compound A7 on the fat content in banana vascular wilt No. 4 micro-organisms.

FIG. 11 shows the effect of active compound A7 on mitochondrial complex enzymes in banana vascular wilt No. 4 race.

Detailed Description

The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1 test materials

1.1 Test strain

Streptomyces samsonii XJC-2-6%Streptomyces samsunensisXJC-2-6) (hereinafter abbreviated as "Streptomyces 2-6") was isolated and screened from samples collected from Corallium japonicum Kishinouye of south China, and was deposited at China center for type culture Collection (CCTCC NO) at 10/23 of 2017: m2017620. The morphological characteristics, culture characteristics, physiological and biochemical characteristics, 16S rRNA molecular biology characteristics and the like are identified by' patent numbers: 201711435432.5, patent name: the present invention is not described in detail herein.

1.2 Test pathogenic bacteria

Physiological Race No. 4 of fusarium oxysporum f.sp.cube Race 4 (ATCC 76255) (Foc TR 4).

1.3 Test medium

The main culture media used in this chapter of study are shown in table 1.

TABLE 1 fermentation Medium and formulation

Name of the name	Formulation of
		FA7	Soluble starch 10, g, yeast extract 4, g, peptone 2 g, glucose 20 g, K ₂ HPO ₄ 0.5 g，CaCO ₃ 2 g，MgSO ₄ 0.5 g，(NH ₄ ) ₂ SO ₄ 0.5 g，ddH ₂ O1000 mL, natural pH.
FM2	Soluble starch 20 g, soybean powder 15 g, yeast extract Extract 5 g, peptone 2 g, caCO ₃ 4 g，NaCl 4 g，ddH ₂ O 1000 mL，pH 7.2-7.4。
		FM3	Soya flour 3 g, glucose 2 g, K ₂ HPO ₄ 0.5 g， NaCl 0.5 g，MgSO ₄ 0.5 g, beef extract 3, g, yeast extract 10 g, soluble starch 10 g, CaCO ₃ 2 g，ddH ₂ O 1000ml, pH 7.0。
FM4	corn flour 30 g, soybean flour 15 g, soluble starch 15 g, yeast extract 0.9 g, NH ₄ SO ₄ 0.5 g， KH ₂ PO ₄ 3 g，Na ₂ HPO ₄ 12 g，CaCl ₂ 0.12 g，ddH ₂ O 1000ml，pH 7.5。
		FM5	Corn flour 30 g, soybean flour 20 g and MgSO ₄ 0.2 g， NaH ₂ PO 0.5 g，CaCl ₂ .2H ₂ O 0.1 g， K ₂ HPO ₄ 0.5 g，ddH ₂ O 1000ml，pH 7.0。

1.4 major reagents

The main reagents used in this study are shown in table 2.

TABLE 2 Main Biochemical reagents and sources

Biochemical reagent	Source
		The common organic reagent is a domestic AR-grade reagent	Miou, tianjin, fuchen, tianjin and Ke Ji
Tetramethyl azoazole blue (MTT)	Sigma Co
		RMPI1640	Sigma Co
Silica gel for column chromatography (60-80 mesh, 200-300 mesh)	Qingdao Ocean Chemical Group Co.,Ltd.
		Thin layer chromatography silica gel H	Qingdao Ocean Chemical Group Co.,Ltd.
Thin layer chromatography silica gel plate (GF) ₂₅₄ )	Qingdao Ocean Chemical Factory Branch
		Glass sample application capillary	Instrument factory of university of Huaxi medical science

1.5 major instruments

The main instruments used in this study are shown in Table 3.

TABLE 3 Main instruments

Name of the name	Model number	Manufacturer (S)
			Microscopic melting point tester	X-5 constant temperature type	Beijing Tech Instrument Co.,Ltd.
Mass spectrometer	ESI-MS、EI-4000	Micromass Autospec-Uitima-TOF
			Nuclear magnetic resonance apparatus	Bruker AV-400，AV-500	Bruker
Infrared spectrometer	Nicolet 380	Thermo, USA

2. Test methods and results

All experiments were performed in triplicate, three replicates were set, and data results were expressed as mean ± Standard Deviation (SD). Differences between the means obtained in each treatment were assessed by analysis of variance (ANOVA; SAS 9.2), p <0.05 representing statistically significant differences.

2.1 Streptomyces fermentation and metabolite extraction

Streptomyces 2-6 is inoculated into ISP2 liquid culture medium, and shake culture is carried out at 28 ℃ and 180 r/min for 4 d. The fresh bacterial liquid was inoculated into a 5L flask containing 1L M6 liquid medium at an inoculum size of 5%, and cultured with shaking at 28℃for 8d at 180 r/min to obtain 120L of fermentation broth. Mixing with absolute ethanol at a ratio of 1:1 (v/v), ultrasonic extracting for 1h, filtering and collecting supernatant, and concentrating under reduced pressure at 45deg.C for 10: 10L. Ultrasonic extraction with ethyl acetate 5 times (2L times, 30 min) at 1:1 (v/v) volume, combining ethyl acetate phases, concentrating under reduced pressure at 45deg.C, and removing ethyl acetate to give ethyl acetate extract 26.33 g. Through activity test, the ethyl acetate extract has obvious antibacterial activity on Foc TR4 pathogenic bacteria.

2.2 TLC detection

The TLC analysis method is to suck the sample by a capillary suction tube of 0.3. 0.3 mm, sample the sample at a distance of l cm from the bottom of the GF254 silica gel plate, and spread the sample after the solvent volatilizes. Spreading the sample by adopting an upright upward spreading method, adding a spreading agent in advance in a chromatography cylinder, placing a thin layer chromatography plate after 20min, carrying out chromatography, taking out the silica gel layer chromatography plate when the front edge of the spreading agent is 1.0-cm from the edge of the silica gel plate, marking the position of the front edge of the solvent, observing the result at an ultraviolet analyzer 254-nm, and calculating the Rf value.

2.3 TLC-bioAutography biological Activity assay

The bacterial inhibition activity of the metabolite is detected by TLC-biological autoradiography (TLC-biological autoradiography) by taking banana wilt No. 4 physiological race (Foc TR 4) as target pathogen. Preparing PDA culture medium, inoculating Foc TR4, culturing at 24deg.C for 7-10d, adding 10 mL sterile water to elute spores, preparing conidium suspension, adding appropriate amount of PDB liquid culture medium, and preparing into liquid culture medium with concentration of 3.0X10 ⁵ spores/mLSpore suspension mixture. The crude extract was dissolved in methanol to a concentration of 20mg/mL and samples of 4. Mu.L and 8. Mu.L were spotted on TLC plates using calibrated capillaries, and a spore suspension (Foc TR 4) was sprayed evenly on the TLC plates (3.0X10) ⁵ spores/mL) were incubated three times in a humidified box at 25 ℃ in an incubator for 12h light, 12h dark, and 4d day-night exchange incubation, when blank areas appear on the TLC plate, indicating inhibition of fungal growth, the crude extract contained antifungal components, and the zone diameter was recorded.

2.4 Ethyl acetate extract silica gel column chromatography

Dissolving ethyl acetate extract with methanol, adding equal volume of n-hexane, extracting for three times, removing small polar components such as oil in the extract, completely drying the residual methanol layer sample, adding small amount of CH ₂ Cl ₂ (DCM) dissolving (a little MeOH is required to be added dropwise in incomplete dissolving), weighing equal amount of 100-200 meshes of silica gel, dripping sample liquid, uniformly stirring, and completely drying the silica gel sample for later use. Taking a small amount of sample, performing silica gel Thin Layer Chromatography (TLC) detection, and selecting CH ₂ Cl ₂ MeOH system as eluent, subjecting the extract to normal phase silica gel column chromatography (200-300 mesh). By CH ₂ Cl ₂ Gradient elution with MeOH System (v/v: 100% CH ₂ Cl ₂ Fractions were collected, 10 mL per tube, and 7 fractions (Fr. A-Fr. G) were pooled by TLC detection, 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1,5:1,2:1, 100% MeOH). And (3) performing antibacterial activity detection by adopting a TLC-bioAutograph method, wherein the target pathogenic bacteria is Foc TR4, and obtaining an active component Fr. A. The methanol dissolution of the active component is repeatedly purified by silica gel column chromatography.

Detecting Streptomyces 2-6 ethyl acetate extract by silica gel Thin Layer Chromatography (TLC) sample application, selecting CH ₂ Cl ₂ The MeOH system eluted as the mobile phase. Subjecting 26.33. 26.33 g crude extract to silica gel column chromatography (200-300 mesh), CH ₂ Cl ₂ MeOH System gradient elution (v/v: 100% CH ₂ Cl ₂ The 65 tube fractions were collected together and combined to 7 fractions fr.a (2.0782 g), fr.b (3.0783 g), fr.c (5) after TLC detection, 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1,5:1,2:1, 100% MeOH5961 g), fr.d (0.7065 g), fr.e (1.3725 g), fr.f (0.9760 g), fr.g (1.5719 g), foc TR4 as target pathogen, and obtaining the main active component fr.a (2.0782 g) by TLC-direct bioautography antibacterial activity detection.

2.5 Active component Sephadex LH-20 gel column chromatography

The active components obtained after silica gel column chromatography, TLC detection and biological activity measurement are dissolved by a small amount of methanol, are loaded by a wet method and are separated by Sephadex LH-20 gel column chromatography (3 cm multiplied by 150 cm). By CH ₂ Cl ₂ The mobile phase of MeOH (v/v: 2:1) was eluted, fractions were collected, 8 mL per tube and pooled by TLC. And (3) performing antibacterial activity detection by adopting a TLC-bioAutograph method, wherein the target pathogenic bacteria is Foc TR4, and obtaining an active component Fr.A-4.

Subjecting the active component Fr.A to Sephadex LH-20 gel column chromatography, and subjecting to CH ₂ Cl ₂ The elution is carried out by eluting with MeOH (2:1), 51 pipe fractions are collected altogether, after TLC detection, 5 fractions Fr.A-1 (0.9233 g), fr. A-2 (0.1685 g), fr. A-3 (0.2759 g), fr. A-4 (0.3396 g), fr. A-5 (0.1922 g) are obtained after TLC detection, and higher active fractions Fr.A-4 (0.3396 g) are obtained after TLC-direct bioautography antibacterial activity detection.

2.6 RP-HPLC separation and purification of active components

The semi-preparation conditions of the high performance liquid chromatography are as follows: agilent 1100, uv/RID detector, column temperature 30 ℃; the chromatographic column is YMC Pack ODS-A (250' -10 mm,5 μm); the mobile phase is MeOH/H2O; the detection wavelengths are 210, 230, 254 and 305, nm respectively, and the manual sample injection amount is 50 mu L; the flow rate was 0.2 mL/min.

The active component Fr.A-4 obtained by Sephadex LH-20 gel column chromatography is repeatedly separated and purified by RP-HPLC to obtain high purity monomer compound A1 (5.1 mg, meOH: H) ₂ O =60 : 40, 2 mL/min, tR = 59 min）、A5（9.1mg, MeOH : H ₂ O =60 : 40, 2 mL/min, tR = 59 min）、A6（3.2mg, MeOH : H ₂ O =60 : 40, 2 mL/min, tR = 59 min）、A7（15.3mg, MeOH : H ₂ O=60:40, 2 mL/min, tr=30.5 min). Through antibacterial activity detection, A7 is determined to be a high-activity compound, and structural identification is carried outAnd (5) setting.

Compound A7 is a dark brown solid with the molecular formula C ₈ H ₁₀ O ₃ Melting point 133-135, mass spectrum (EI, 70 eV)), M/z:154 [ M] ⁺ , 139, 121, 109, 95, 79。 ¹ H-NMR (600 MHz, CD ₃ OD) δ _H 4.68 (1H, d, J = 2.5 Hz, H-3), 4.08 (1H, d, J = 2.5 Hz, H-2), 6.00 (1H, s, H-5), 6.44 (1H, m, J = 16.0 Hz, 1.2 Hz, H-6), 6.83 (1H, dq, J = 16.0 Hz, 1.2Hz, H-7), 1.94 (3H, dd, J = 6.5 Hz, 1.2 Hz, CH ₃ -8); ¹³ C-NMR (150 MHz, CD ₃ OD) δ _C 205.60 (C-1), 82.41(C-2), 78.11 (C-3), 170.83 (C-4), 125.92 (C-5), 126.42 (C-6), 141.83 (C-7), 19.49 (C-8)。 ¹ H-NMR spectra showed 3 double bond hydrogen signals in the low field: delta _H 6.83 (dq, j=16.0 Hz, 1.2Hz, 1H), 6.44 (m, j=16.0 Hz, 1.2Hz, 1H), 6.00 (s, 1H); 2 oxyhydrogen signals: delta _H 4.08 (d, j=2.5 Hz, 1H), 4.68 (d, j=2.5 Hz, 1H); the high field region has 1 methyl hydrogen signal: delta _H 1.94 (dd, J = 6.5 Hz, 1.2 Hz, 3H)； ³ C-NMR and DEPT spectra show that the compound contains 8 carbon atoms in total, and the low field region has a1 carbonyl carbon signal: delta _C 205.60,4 double bond carbon signals: delta _C 170.83 141.83, 125.92, 126.42, it was determined that the compound contained two double bonds; 2 oxygen-carbon signals: delta _C 82.41 78.11, from the determination of chemical shift as typical ring-on hydroxy substitution structure, it can be inferred that the compound contains two hydroxy substitutions; the high field region has 1 methyl carbon signal: delta _C 19.49. According to the compound ¹ H-NMR、 ¹³ C-NMR and DEPT spectra, looking at literature comparison, compound A7 spectra data were found to be consistent with the reports in the literature, and compound A7 was identified as terrein (Raistrick et al, 1935; kim et al, 2005; asfour et al, 2019). The planar structure of the compound A7 was analyzed by 2D-NMR spectrum, and the nuclear magnetic data of the compound is shown in Table 4 as shown in FIG. 2.

TABLE 4 NMR data for Compound A7 (J units: hz)

Position of	δ _H (mult, J in Hz)	δ _C	Position of	δ _H (mult, J in Hz)	δ _C
						1		205.60, C	5	6.00 (s)	125.92, CH
2	4.08 (d)	82.41, CH	6	6.44, (m)	126.42, CH
						3	4.68 (d)	78.11, CH	7	6.83, (dq)	141.83, CH
4		170.83, C	8	1.94, (dd)	19.49, CH ₃

¹ H-NMR represents 600 MHz; ¹³ C-NMR represents 150 MHz.

2.7 Antibacterial effect of active ingredient A7 on Foc TR4 hypha growth

The inhibitory activity of the Streptomyces metabolite against the growth of banana fusarium wilt filaments was evaluated by a growth rate method (Shalma et al, 2016; shalma et al, 2017). Active ingredient A7 dissolved in sterile water (10.0 mg/mL) is added into PDA culture medium at 45-50 ℃, PDA plates with different active ingredient concentrations are prepared by adopting a 2-fold serial dilution method, and the final concentrations of the plates are respectively 100.0, 50.0, 25.0, 12.5, 6.25 and 3.125 mug/mL, so that equal amounts of sterile water are added as a control. A fungal cake of Foc TR4 (Φ=5 mm) was inoculated into the center of each plate and incubated at 28±2 ℃ until the control mycelia reached the edges of the plates. The average value of the vertical diameters of the respective colonies was measured. Each treatment was repeated 3 times. The hypha growth inhibition was calculated as follows (nimichand et al 2015):

wherein: c is the average diameter of the colonies of the control group, and T is the average diameter of the colonies of the treatment group.

The effect of active ingredient A7 on the growth of the mycelium of the target pathogen Foc TR4 at concentrations of 100.0, 50.0, 25.0, 12.5, 6.25 and 3.125 μg/mL was determined using a growth rate method, as shown in figure 3. By measuring colony diameter and model calculation, a virulence regression equation is calculated, as shown in Table 5, and EC of Compound A7 is calculated ₅₀ 、 EC ₇₅ And EC (EC) ₉₅ 25.76 mug/mL, 86 respectively30 mug/mL and 491.37 mug/mL. The concentration of the active ingredient is positively correlated with the inhibition of hypha growth, and the greater the concentration, the stronger the inhibition.

TABLE 4 bacteriostatic action of Compound A7 on Foc TR4

Compounds of formula (I)	Toxicity regression equation	R ²	EC ₅₀	EC ₇₅	EC ₉₅
						A7	y=3.1874+1.2846x	0.9933	25.76µg/mL	86.30µg/mL	491.37µg/mL

2.8 Effect of active ingredient A7 on Foc TR4 pathogen morphology and intracellular ultrastructure

And respectively adopting a scanning electron microscope and a transmission electron microscope observation method to research the teratogenicity of the streptomycete active ingredient A7 on Foc TR4 pathogenic fungus hyphae, pathogenic fungus conidia and pathogenic fungus intracellular ultrastructures.

(1) Scanning electron microscope for observing Foc TR4 pathogenic bacteria mycelium change

0.5 cm bacterial cake is taken out from the edge of target pathogenic bacteria Foc TR4 colony by a puncher and is connected to the center of a plate containing active ingredient A7. After 5d incubation at 28℃the mycelium tips were cut with a blade and the medium was cut off as much as possible at the colony edges, fixed overnight at 4℃with 2.5% (w/v) glutaraldehyde solution, rinsed three times with phosphate buffer, then dehydrated stepwise with 30%, 50%, 70%, 90% ethanol once, 100% ethanol twice, 20min each time, and finally eluted ethanol twice with isoamyl acetate, 30min each time, and then gold sprayed after vacuum drying.

As can be seen from FIG. 4, the mycelium surface of the control group was dense and smooth, uniform and full in shape, good in integrity, and a large number of microspores were produced around the mycelium. After the active ingredient A7 is treated, the surface of the pathogenic bacteria mycelium is rough and uneven, the mycelium is atrophic and thinned, the integrity is destroyed, the phenomena of fracture and rupture occur, and the generation of conidium is inhibited. Through the experiment, the compound A7 can damage the mycelium structure of pathogenic bacteria, thereby inhibiting the growth of the pathogenic bacteria and the generation of conidium.

(2) Scanning electron microscope observation of Foc TR4 pathogen conidium change

Preparation of Foc TR4 spore suspension (1X 10) ⁶ CFU/mL), 5 μl spore suspension was placed on the slide, with 5 μl EC ₅₀ Active ingredient A7 at the concentration was treated with sterile water as a control and incubated for 24h. The slide glass is fixed overnight at 4 ℃ by 2.5% glutaraldehyde, is rinsed three times by phosphoric acid buffer solution, is dehydrated step by 30%, 50%, 70% and 90% ethanol once, is dehydrated by 100% ethanol twice for 20min each time, is eluted by isoamyl acetate twice for 30min each time, and is observed by metal spraying after vacuum drying.

The effect of active ingredient A7 of Streptomyces 2-6 on the conidia of Foc TR4 pathogenic bacteria was observed by scanning electron microscopy as shown in FIG. 5. SEM images show the active ingredient treated pathogenic bacteria, spore deformation, shrinkage, collapse, bending and head swelling, and the integrity is destroyed, and fracture and rupture occurs. And the spores of the control group are full, the surfaces of the spores are smooth, and the spore forms of the spores are complete. Through the experiment, the compound A7 can damage the conidium structure of pathogenic bacteria, thereby inhibiting the growth of the pathogenic bacteria.

(3) The change of ultrastructure of Foc TR4 pathogenic bacteria cells is observed by a transmission electron microscope

And (3) taking 0.5 cm bacterial cake from the edge of the target bacterial colony by using a puncher, and inoculating the bacterial cake into the exact center of the plate containing the active ingredient A7. After 5d incubation at 28℃the mycelium tips were cut with a blade and the medium was cut off as much as possible at the colony edges, fixed overnight at 4℃with 2.5% (w/v) glutaraldehyde solution, rinsed three times with phosphate buffer, then dehydrated stepwise with 30%, 50%, 70%, 90% ethanol once, 100% ethanol twice for 20min each time, and the samples were immersed in propylene oxide for 2 exchanges for 20min each time. The sample was prepared on propylene oxide: after embedding in epoxy (1:1) solution for 1h, the embedded material was cut into 70 nm ultra-thin sections with a diamond knife. Sections were stained with uranyl acetate and lead citrate, respectively, for 30min and observed with a transmission electron microscope (Phillips et al, 2003).

The effect of active ingredient A7 of Streptomyces 2-6 on the ultrastructural structure of Foc TR4 pathogenic bacteria cells is shown in FIG. 6. The control group pathogenic bacteria cells were observed by transmission electron microscopy to be plump in morphology, complete in structure, complete in organelle type, complete in cell wall, uniform in cytoplasm, regular in mitochondrial morphology and uniform in body type (fig. 6A). After treatment with the active compound, the Foc TR4 pathogenic bacteria cell wall is obviously thinned, the organelles are dissolved and disappeared, the cell tissue disintegrates, the cell vacuoles are formed, and the vesicles appear (fig. 6B); the cytoplasmic electron density increased, the number of mitochondria increased significantly, the mitochondrial morphology was abnormal, exhibiting surface roughness collapse, lack of matrix, clear visualization of the inner and outer membranes, cristae swelling and structural disorder (fig. 6C).

2.9 Effect of active ingredient A7 on physiological metabolism of Foc TR4 pathogenic bacteria

(1) Effect of active ingredient A7 on Foc TR4 pathogenic cell wall chitinase

N-acetylglucosamine standard curve: preparing 100 mug/mL of N-acetylglucosamine standard solution, and diluting the standard solution into 20, 40, 60, 80 and 100 mug/mL of gradient standard solution. Adding 1mL of gradient standard solution into a test tube, adding 0.5 mL potassium borate (0.8 mol/L and 0.5 mL potassium borate solution (0.8 mol/L)) into the test tube, carrying out boiling water bath for 3 min, cooling, adding 3 mL DMAB (p-dimethylaminobenzaldehyde) with the mass fraction of 1%, carrying out heat preservation at 36 ℃ for 20min, cooling, measuring absorbance by an ultraviolet spectrophotometer at the wavelength of 544 nm, and drawing a standard curve by taking the N-acetylglucosamine content as an abscissa and the absorbance as an ordinate.

100 mL of PDB culture medium is added into a 250 mL triangular flask, foc TR4 pathogenic bacteria are inoculated, active ingredients A7 with different concentration gradients (the concentration is 3.125 mug/mL, 6.25 mug/mL, 12.5 mug/mL, 25.0 mug/mL and 50.0 mug/mL) are added, the mycelia are collected by shaking culture at 180 r/min and 28 ℃ for 5d, centrifugation at 5000 r/min is carried out for 15 min, and the mycelia are washed by sterile water and washed 3 times, and then the mycelia are to be detected. 1.0. 1.0 g mycelium is weighed, 5mL of Tris-HCl is added, grinding is carried out in an ice bath, 10000 r/min is carried out, centrifugation is carried out at 4 ℃, and supernatant fluid is taken and stored at-20 ℃ for standby. Adding 1.0 mL of the cell supernatant liquid with different treatments into a clean test tube, adding 0.5 mL potassium borate solution (0.8 mol/L), carrying out boiling water bath for 3 min, cooling, adding 3 mL mass percent of 1% DMAB (p-dimethylaminobenzaldehyde), carrying out heat preservation at 36 ℃ for 20min, cooling, and measuring absorbance at a wavelength of 544 nm by using an ultraviolet spectrophotometer. The N-acetylglucosamine content of the chitin hydrolysate was calculated by a standard curve.

The effect of the active ingredient on the change in intracellular N-acetylglucosamine content of Foc TR4 pathogenic bacteria is shown in FIG. 7. As the concentration of active compound A7 increases, the N-acetylglucosamine content shows a gradual upward trend. The content of N-acetylglucosamine is rapidly increased in the concentration range of 0-25 mug/mL in the compound A7 treatment group, and the increasing slope is larger. And in the concentration range of 25-50 mug/mL, the rising trend of the N-acetylglucosamine content is gentle, and the rising slope is small. Thus, 25 μg/mL is the concentration node for compound A7 treatment, which has an N-acetylglucosamine content of 56.48 μg/g. Chitin is the main component of the cell wall of pathogenic bacteria, N-acetylglucosamine is the final product of chitin hydrolysis, and the change of N-acetylglucosamine can reflect the change of the cell wall of pathogenic bacteria. The increase in N-acetylglucosamine content with increasing treatment concentration indicates that the increase in concentration of active ingredient A7 leads to an increase in the degree of chitin hydrolysis, and the cell wall is destroyed and the degree of destruction is continuously increased. The active compound of streptomycete 2-6 can decompose the cell wall of pathogenic bacteria to reduce the viability of pathogenic bacteria and inhibit the growth of pathogenic bacteria.

(2) Effect of active Compound A7 on total sugar, protein and fat content of Foc TR4 pathogenic bacteria

(1) Determination of total sugar (DNS method) content: the final concentration of 2 mL was set to 0, 40, 80, 120, 160, 200. Mu.g/mL glucose solution and 1.5mL 3,5-dinitrosalicylic acid (3, 5-Dinitrosalicylic acid, DNS) reagent was added. Mixing, incubating in a 100deg.C constant temperature water bath for 5min, cooling to room temperature, diluting with distilled water to 20mL, mixing, and measuring absorbance at 540 nm wavelength. And drawing a standard curve by taking the glucose content as an abscissa and the absorbance as an ordinate. 100 mL of PDB culture medium is added into a 250 mL triangular flask, foc TR4 pathogenic bacteria are inoculated, active ingredients A7 with different concentration gradients (the concentration is 3.125 mug/mL, 6.25 mug/mL, 12.5 mug/mL, 25.0 mug/mL and 50.0 mug/mL) are added, mycelia are collected by shaking culture at 180 r/min and 28 ℃ for 5d, centrifugation at 5000 r/min is carried out for 15 min, and the mycelia are washed by sterile water and dried. Weighing the prepared mycelium 1.0 g, placing in a precooled mortar, adding 6 mLTris-HCl leaching solution, grinding in ice bath to homogenate, and centrifuging at 4deg.C and 10000 r/min for 10 min. Adding 2 mL of HCl (6 mol/L) into 1mL supernatant, boiling for 30min, cooling by flowing, neutralizing with NaOH (6 mol/L) solution to neutrality, adding 1.5mL of DNS, boiling for 5min, adding 1.5mL of DNS into 2 mL supernatant, and boiling for 5 min. Taking out, cooling to room temperature, adding distilled water, diluting to 20mL, and mixing. Absorbance was measured at wavelength 540, nm and total sugar content was calculated by standard curve (river et al, 1984).

As can be seen from fig. 8, the total soluble sugar content in the Foc TR4 pathogenic bacteria cells was gradually reduced with increasing concentration of the active ingredient A7 by the treatment with the active compound A7, and the treatment with the low concentration was not significantly different from the control, and the treatment with the high concentration was significantly different. After treatment with Compound A7 at concentrations of 3.125 [ mu ] g/mL, 6.25 [ mu ] g/mL, 12.5 [ mu ] g/mL, 25.0 [ mu ] g/mL and 50.0 [ mu ] g/mL, the total sugar content of the Foc TR4 pathogenic bacteria was 1.59.+ -. 0.04mg/g, 1.53.+ -. 0.0. 0.0 mg/g, 1.47.+ -. 0.07 mg/g, 1.28.+ -. 0.02 mg/g, 1.12.+ -. 0.04mg/g, respectively, whereas the control was 1.62.+ -. 0. 0.04mg/g, 3.125 [ mu ] g/mLNo significant difference in the irradiation; the soluble total sugar content of the treated group was reduced by 2.06%,5.56%,9.47%,20.78% and 30.66% respectively, compared with the control. Sugar is a main carbon source and energy reserve substance for microbial metabolism, when the concentration of the active ingredient A7 is increased, the growth and metabolism rate of banana fusarium wilt bacteria is reduced, the rate of synthetic energy substance is also reduced, the energy consumption rate is increased, and the total sugar content is reduced in a same ratio. The results show that after treatment with active compound A7, the total sugar content of the pathogenic bacteria Foc TR4 showed a sharp decrease at 25. Mu.g/mL, which is the EC of this compound A7 ₅₀ Regional ranges of values.

(2) Determination of protein content: 100 mL of PDB culture medium is added into a 250 mL triangular flask, foc TR4 pathogenic bacteria are inoculated, active ingredients A7 with different concentration gradients (the concentration is 3.125 mug/mL, 6.25 mug/mL, 12.5 mug/mL, 25.0 mug/mL and 50.0 mug/mL) are added, mycelia are collected by shaking culture at 180 r/min and 28 ℃ for 5d, centrifugation at 5000 r/min is carried out for 15 min, and the mycelia are washed by sterile water and dried. Weighing the prepared mycelium 1.0 g, placing into precooled grinding body, adding 6 mLTris-HCl leaching solution, grinding in ice bath to homogenate, centrifuging at 4deg.C and 10000 r/min for 10 min. Taking 0.1. 0.1 mL supernatant, adding 0.9. 0.9 mL distilled water, adding 5.5 mL Coomassie brilliant blue G-250 solution, mixing thoroughly, standing for 5min, measuring absorbance at wavelength 595. 595 nm, comparing with standard curve, and calculating protein content (Bradford, 1976).

As is clear from FIG. 9, the content of soluble protein in the Foc TR4 pathogenic bacteria after the treatment with the active compound A7 gradually decreases with the increase of the concentration of the active ingredient A7. After treatment with compound A7 at concentrations of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25.0 μg/mL and 50.0 μg/mL, the Foc TR4 pathogenic bacteria soluble protein content was 2.70+ -0.06 mg/g, 2.37+ -0.11 mg/g, 2.20+ -0.12 mg/g, 2.06+ -0.12 mg/g, 1.74+ -0.06 mg/g, and the control group was 2.81+ -0.10 mg/g, respectively, all concentration treatments differing significantly from the control; the soluble total protein content of the treated group was reduced by 3.78%,15.54%, 21.86%, 26.67% and 37.91%, respectively, compared to the control.

(3) Determination of fat content: the method of measuring fat content in ginseng literature (Chenquan, 2002) is carried out: 100 mL of PDB culture medium is added into a 250 mL triangular flask, foc TR4 pathogenic bacteria are inoculated, active ingredients A7 with different concentration gradients (the concentration is 3.125 mug/mL, 6.25 mug/mL, 12.5 mug/mL, 25.0 mug/mL and 50.0 mug/mL) are added, the mycelia are collected by shaking culture at 180 r/min and 28 ℃ for 5d, centrifugation at 5000 r/min is carried out for 15 min, and the mycelia are washed by sterile water. Accurately weighing mycelium 0.5. 0.5 g, placing in a filter paper cylinder, sealing two ends of the paper cylinder, and placing in a Soxhlet fat extractor. The constant weight liposuction bottle was connected, and anhydrous diethyl ether (boiling range: 30 to 60 ℃ C.) was added from the upper end of the condenser. Heating and extracting 12-16 h in water bath at 50-60 deg.c. After the extraction is finished, residual diethyl ether is distilled off on a water bath kettle, and then the mixture is dried in an oven at 100-105 ℃ to constant weight by 8 h. The calculation formula is as follows:

the effect of active compound A7 on fat content in Foc TR4 pathogenic bacteria is shown in FIG. 10. After treatment with compound A7 at concentrations of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25.0 μg/mL and 50.0 μg/mL, the Foc TR4 pathogen fat content was 5.37%,5.24%,5.14%,4.79% and 3.25%, respectively, whereas the control group was 5.43%, with low concentrations of compound A7 (3.125 μg/mL and 6.25 μg/mL), the fat content was not significantly different from the control group; and the fat content in pathogenic bacteria is obviously reduced by 11.72 percent and 40.09 percent respectively when the pathogenic bacteria are treated at higher concentrations of 25 mug/mL and 50 mug/mL. Thus, an active compound A7 concentration of 25 μg/mL can have a significant effect on target pathogen biosynthesis.

(3) Effect of active ingredients on mitochondrial respiratory chain (ETC) Complex enzyme I-IV Activity

(1) Mitochondrial extraction and pretreatment

And centrifuging to collect mycelia treated with active ingredients with different concentrations. Mycelium mitochondria were extracted by sucrose differential centrifugation (Mizutani et al 1995), 5 g washed three times with pre-chilled HEPES-Trisbuffer (20 mM, pH 7.2), the mycelium was resuspended in 3 volumes of pre-chilled mitochondrial extraction buffer (250 mM sucrose, 10 mM KCl,5 mM EDTA,20 mM HEPES-Tris, pH 7.2,1.5 mg/mL BSA), the mycelium placed in a 50 mL centrifuge tube and homogenized and broken under a glass bead vortex shaker (Fishbein et al 1993). Centrifuging at 4000 r/min at 4deg.C for 15 min, collecting supernatant, and discarding precipitate. Taking supernatant, continuing to centrifuge, centrifuging at 12000 r/min and 4 ℃, discarding supernatant, flushing precipitate with mitochondrial extraction buffer without BSA, and centrifuging at 10000 r/min for 20min at 4 ℃. The precipitate was a crude extract of mitochondrial particles (barrens, 2002).

The mitochondrial particles were resuspended in 400. Mu.L of pre-chilled mitochondrial preservation buffer (2 mM HEPES,0.1 mM EGTA,250 mM sucrose, pH 7.4) leaving 60-70. Mu.L and the complex enzyme III activity assayed. Taking 10 mu L of mitochondrial solution, taking BSA as a standard protein, measuring the protein concentration by using a Bradford method (Bradford M, 1976), regulating the protein concentration to 1mg/mL by using a mitochondrial preservation buffer, placing on ice for standby, or quick freezing by liquid nitrogen after split charging, and preserving at-20 ℃. Before measuring enzyme activity, the mitochondrial solution was repeatedly frozen and thawed 3 times in liquid nitrogen/room temperature.

The remaining 330-340. Mu.L of solution was centrifuged at 10000 r/min for 20min at 4℃and the pellet was resuspended in 1mL hypotonic buffer, and after centrifugation at 10000 r/min for 20min at 4℃the pellet was resuspended in 300. Mu.L hypotonic buffer. Is used for the complex enzyme I, the complex enzyme II and the complex enzyme IV. Taking 10 mu L of mitochondrial solution, taking BSA as a standard protein, measuring the protein concentration by using a Bradford method, regulating the protein concentration to 1mg/mL by using a hypotonic buffer solution, placing on ice for standby, or quick freezing by liquid nitrogen after split charging, and preserving at-20 ℃. Before mitochondrial complex enzyme detection, the solution is placed in liquid nitrogen/room temperature for 3 times of repeated freezing and thawing, and then subsequent detection is carried out. The measurement of the complex enzyme I-IV is carried out at 37 ℃, the total volume of the reaction system is 1mL, and the addition amount of mitochondrial protein is 20-40 mug. Mitochondrial complex enzyme I-IV activity assays were performed with reference to known literature methods (Spinazzi, et al 2012).

(2) Effects on mitochondrial respiratory chain complex enzyme I Activity

Complex enzyme I (rotenone-sensitive NADH-decyl ubiquinone oxidoreductase): by measuring decyl ubiquinone as electron acceptor and NADH as electron donorThe decrease in the absorption of 340 nm by oxidation of NADH. 700. Mu.L of purified water was added to a 1mL cuvette, 8. Mu.L of the pretreated mitochondrial solution (1 mg/mL) was added and incubated at 37℃for 2 min. mu.L of potassium phosphate buffer (0.5M, pH 7.5), 60. Mu.L of fatty acid-free BSA (50 mg/mL), 30. Mu.L of KCN (10 mM) and 10. Mu.L of LNADH (10 mM) were added, respectively. Another cuvette for the parallel test was added with 10. Mu.L of rotenone (1 mM), and the activity of the rotenone-insensitive complex enzyme was determined, and the activity of rotenone-sensitive complex enzyme I was quantified after subtraction. The volume of the reaction mixture was adjusted to 994. Mu.L with pure water. The reaction mixture was mixed by inverting the Parafilm-capped cuvette and monitored for baseline at 340 nm for 2 min. The reaction was started by adding 6 μl of decylubiquinone (10 mM), and the decrease in absorbance value of 340 nm (NADH molar extinction coefficient epsilon=6.2 mM) was measured over 2 min ^-1 ·cm ^-1 ). The calculation formula is as follows:

l: optical path (cm); v = sample volume (mL); [ prot ] = sample protein concentration (mg/mL)

(3) Effects on mitochondrial respiratory chain complex enzyme II Activity

Complex enzyme II (decyl ubiquinone succinate DCPIP reductase, succinic acid-ubiquinone oxidoreductase): DCPIP was used as an electron acceptor, and succinic acid was used as an electron donor, and the decrease in absorption value of 600 nm was measured by reduction of DCPIP. 600 μl of purified water was added to a 1mL cuvette, 50 μl of potassium phosphate buffer (0.5M, pH 7.5), 20 μl of fatty acid-free BSA (50 mg/mL), 30 μl of KCN (10 mM), 50 μl of succinic acid (400 mM), 8 μl of mitochondrial protein (1 mg/mL) and 145 μl of DCPIP (0.15 mg/mL) were added, respectively, and the reaction mixture volume was adjusted to 995 μl with purified water. The reaction mixture was incubated at 37 ℃ for 10 min after tumbling the cuvette sealed with a Parafilm, and the baseline was monitored at 600 nm for the last 2 min. The reaction was started by adding 5. Mu.L of decylubiquinone (10 mM), and the decrease in absorbance at 600 nm (DCPIP extinction coefficient ε=19.1 mM-1 cm-1) was measured over 3 minutes. The calculation formula is as follows:

(4) effects on mitochondrial respiratory chain complex enzyme III Activity

Complex enzyme III (ubiquinol Cyt c reductase, decyl ubiquinol Cyt c oxidoreductase): the increase in 550nm absorption value by reduction of oxidized Cyt c was measured using oxidized Cyt c as an electron acceptor and decyl panthenol as an electron donor. 730 μl of pure water was added to a 1mL cuvette, and 50 μl of potassium phosphate buffer (0.5M, pH 7.5), 75 μl of oxidized Cyt c (1 mM), 50 μl of KCN (10 mM), 20 μl of LETTA (5 mM, pH 7.5), 10 μl of Tween 20 (2.5%) and 8 μl of mitochondrial protein (1 mg/mL) were added, respectively. Parallel test cuvette except for the above reagent, 10. Mu.L of antimycin A (1 mg/mL) was added to determine the activity of antimycin A insensitive complex enzyme III, and the activity of antimycin A sensitive complex enzyme III was quantified after subtraction. The volume of the reaction mixture was adjusted to 990. Mu.L with pure water. The reaction mixture was mixed by flipping a cuvette sealed with a Parafilm, and the baseline was monitored at 550nm for 2 min. The reaction was started by adding 10. Mu.L of decyl panthenol (10 mM), and immediately after mixing the reaction mixture, the increase in absorbance at 550nm (reduced Cyt c extinction coefficient ε=18.5 mM-1 cm-1) was measured within 2 min. The calculation formula is as follows:

(5) effects on mitochondrial respiratory chain complex enzyme I-IV Activity

Complex enzyme IV (Cyt c oxidase): the reduction type Cyt c was used as an electron donor, and the decrease in the absorption value of 550nm due to oxidation of the reduction type Cyt c was measured. 400. Mu.L of pure water was added to a 1mL cuvette, and 250. Mu.L of potassium phosphate buffer (100 mM, pH 7.0) and 50. Mu.L of reduced Cyt c (1 mM) were added, respectively. The reaction mixture was mixed by inverting the Parafilm-capped cuvette and monitored for baseline at 550nm for 2 min. The volume of the reaction mixture was adjusted to 992. Mu.L with pure water. The reaction was started by adding 8 μl of mitochondrial protein (1 mg/mL) and the decrease in absorbance value of 550nm (reduced Cyt c extinction coefficient epsilon=18.5 mM-1 cm-1) was measured over 3 min. The calculation formula is as follows:

to investigate the effect of active compounds on mitochondrial electron transport chain complex enzyme activity, foc TR4 was treated with compound A7 at concentrations of 3.125,6.25, 12.5, 25 and 50 μg/mL, respectively, and the activity of ETC complex enzymes I-IV was examined (fig. 11). Compared with the control group, the activity of ETC complex enzymes I-IV is increased under the low-concentration treatment; however, as the treatment concentration increases, the activity or content thereof gradually decreases without exception. Under the treatment of Compound A7, the activities of the complex enzymes III to III increased to a maximum at a concentration of 6.25. Mu.g/mL and began to decrease, while the activity of the complex enzyme IV increased to a maximum at a concentration of 3.125. Mu.g/mL and began to decrease. The activities of Complex enzymes I-IV were reduced by 31.10%,47.87%,51.27% and 59.34%, respectively, at 50. Mu.g/mL compared to the control.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims

1. A preparation method of the aspergillus terreus ketone is characterized by separating the aspergillus terreus ketone from fermentation liquor of streptomyces samsonii XJC-2-6, wherein the preservation number of the streptomyces samsonii XJC-2-6 is CCTCC NO: m2017620, comprising the steps of:

(1) Inoculating Streptomyces samsoni XJC-2-6 into a fermentation culture solution for fermentation culture to obtain a fermentation solution;

(2) Adding proper amount of ethanol into the fermentation broth for extraction, filtering, taking supernatant, adding proper amount of ethyl acetate for extraction, taking ethyl acetate phase, and concentrating to obtain ethyl acetate extract;

(3) Separating ethyl acetate extract by silica gel column chromatography, and separating with CH ₂ Cl ₂ Gradient elution with MeOH System, CH ₂ Cl ₂ The MeOH system was: 100% CH ₂ Cl ₂ CH in a volume ratio of 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1,5:1 and 2:1, respectively ₂ Cl ₂ And MeOH, and 100% MeOH; performing antibacterial tests on the obtained components, taking Foc TR4 as target pathogenic bacteria, and performing TLC-direct bioautography antibacterial activity detection to obtain a main active component Fr.A;

(4) Subjecting the active component Fr.A to Sephadex LH-20 gel column chromatography, and subjecting to CH ₂ Cl ₂ MeOH eluent elution, CH ₂ Cl ₂ And MeOH in a volume ratio of 2:1, performing antibacterial test on each obtained component, and detecting the antibacterial activity of TLC-direct bioautography to obtain a higher-activity component Fr.A-4;

(5) Repeatedly separating and purifying the active component Fr.A-4 by RP-HPLC to obtain the aspergillone.

2. The method according to claim 1, wherein in the step (1), the fermentation broth is M6 liquid medium, the inoculation amount is 5%, and the shaking culture is carried out at 28 ℃ 180 r/min for 8d.

3. The application of the aspergillone prepared by the preparation method according to claim 1 in antagonizing No. 4 physiological race of banana fusarium wilt.

4. The application of the aspergillone prepared by the preparation method according to claim 1 in preventing and controlling diseases caused by banana fusarium wilt bacteria No. 4 physiological race.

5. The use of the aflatoxin prepared by the preparation method according to claim 1 for reducing the soluble total sugar content, and/or the soluble total protein content, and/or the fat content, and/or the mitochondrial respiratory chain complex enzyme I-IV activity in the physiological race 4 of banana fusarium wilt.