CN116987635A - Streptomyces strain, method for preparing actinomycin and application thereof - Google Patents

Streptomyces strain, method for preparing actinomycin and application thereof Download PDF

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CN116987635A
CN116987635A CN202310877302.6A CN202310877302A CN116987635A CN 116987635 A CN116987635 A CN 116987635A CN 202310877302 A CN202310877302 A CN 202310877302A CN 116987635 A CN116987635 A CN 116987635A
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张军
高良亮
梁燕
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Gannan Normal University
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Abstract

The invention provides a streptomycete strain GLL-9 for high-yield actinomycin, a method for preparing actinomycin with high yield and application of actinomycin in inhibiting growth of agricultural pathogenic microorganisms, and belongs to the fields of agriculture and medicine. The invention separates a streptomycete strain GLL-9 with high actinomycin yield, and the strain can be high in a rice culture medium and a corn culture mediumActinomycin X production 2 Actinomycin D and actinomycin X Oβ . The fermentation product containing actinomycin produced by fermentation and the in-vitro and in-vivo antibacterial activity test result of the purified actinomycin show that the actinomycin has remarkable inhibition effect on canker orange and penicillium citri. Has important application value and good application and development prospect for developing high-efficiency biological pesticides.

Description

Streptomyces strain, method for preparing actinomycin and application thereof
Technical Field
The invention relates to the fields of agriculture and medicine, in particular to a streptomycete strain, a method for producing actinomycin by utilizing the strain through fermentation and application of the actinomycin.
Background
The citrus canker (Xanthomonas citri susp. Citri, xcc, xanthomonas citri subspecies) is a pathogenic bacterium of the citrus canker, and mainly damages citrus spring tips, xia Shao and autumn tips, wherein the summer tips are particularly serious, and the inhibition of the disease is mainly performed by copper preparations such as copper rosinate microemulsion, tannin (copper calcium sulfate) wettable powder, copper (cupric oxychloride) wettable powder and the like. The use of copper formulations in large quantities inevitably leads to pesticide residues and drug resistance problems.
Penicillium citrinum (Penicillium italicum ) is the main pathogenic bacteria of penicillium citrinum, and causes huge loss in the transportation and storage process of citrus, and the economic loss of Gannan navel orange caused by penicillium citrinum is more than 20% each year. The chemical synthetic drugs for preventing and treating the penicilliosis of the citrus are mainly prochloraz, imazalil, and prochloraz, on one hand, the penicillium citrinum can generate drug resistance, and on the other hand, certain food safety hidden trouble exists, so that the search for a safe and novel bactericide is urgent.
To solve the above problems, it is an important direction to seek new antibacterial agents from fermentation products of microorganisms. Actinomycin is an important antibiotic produced by metabolism of streptomycete, and is found in the sixty-seventies of the 20 th century, and later research shows that actinomycin D has medical value for resisting malignant tumor and is widely applied to clinical medical treatment. With the intensive research on actinomycin, the actinomycin has not only anti-tumor activity but also biological activities such as antiviral activity, so that the application of actinomycin in other fields can be further expanded.
Disclosure of Invention
The invention aims to provide streptomyces with high yield of actinomycin, a method for preparing the actinomycin with high yield and application of the actinomycin in inhibiting the growth of agricultural pathogenic microorganisms.
In order to achieve the above object, the present invention provides the following technical solutions:
streptomyces sp GLL-9 is preserved in China Center for Type Culture Collection (CCTCC), and the preservation address is eight-path 299 of Wuhan university in Wuchang district of Wuhan, hubei province, and the preservation number is: cctccc NO: m2023978, date of preservation: 2023, 06, 09.
The invention also provides a preparation method of actinomycin, which comprises the following steps: and fermenting the GLL-9 strain in a culture medium, wherein the obtained fermentation product contains the actinomycin.
The actinomycin is actinomycin X 2 Actinomycin D and actinomycin X Oβ 。
Preferably, the above medium is selected from one or more of PDB, ISP2, MII, TSB, corn medium and rice medium.
More preferably, one or more of yeast extract, beef extract, L-tryptophan and peptone are added to the corn medium.
More preferably, one or more of yeast extract, beef extract, L-tryptophan and peptone are added to the rice culture medium.
Preferably, the fermentation conditions are static fermentation at 25-30 ℃ for 20-60d.
Sequentially extracting the fermentation product with ethanol and ethyl acetate to obtain a fermentation extract containing actinomycin.
Further, subjecting the above fermented extract to silica gel column with different volume fractions of CH 2 Cl 2 Gradient elution with MeOH gives actinomycin.
The invention also provides an application of actinomycin in controlling canker orange and/or penicillium citricum.
The invention has the beneficial effects that:
(1) The invention is from the national navel orange engineering research center germplasm resource nurserySeparating a streptomycete strain GLL-9 with high actinomycin yield from three-year branches, wherein the streptomycete strain can produce actinomycin X in a rice culture medium and a corn culture medium 2 Actinomycin D and actinomycin X Oβ 。
(2) The yield of actinomycin produced by fermentation of streptomycete can be remarkably improved by using the culture method.
(3) The invention discovers that the actinomycin has remarkable inhibition effect on the citrus canker and the citrus penicillium, and discovers that the actinomycin has good control effect on the citrus penicillium caused by the citrus penicillium and the citrus canker caused by the citrus canker through in vivo experimental research, and can be used for developing high-efficiency biopesticide.
(4) The invention further researches the action mechanism of actinomycin antibiosis in detail, and provides a new choice for the research and development of antibacterial pesticides with actinomycin as a main active ingredient.
Drawings
Fig. 1: structural formula of actinomycin, and actinomycin X when R1=R2 is carbonyl 2 The method comprises the steps of carrying out a first treatment on the surface of the R1 and R2 are hydrogen and are actinomycin D; r1 is hydroxy and R2 is hydrogen, actinomycin X Oβ 。
Fig. 2: the retention times on HPLC for the three actinomycins in example 1 were 12.75min, 34.56min and 40.31min, respectively.
Fig. 3: the addition of L-tryptophan (medium in example 1) to the rice grit medium was fermented with time and the yield of actinomycin was varied.
Fig. 4: corn grit medium with beef meal (medium in example 2) added fermented over time, actinomycin yield was varied.
Fig. 5: the organic phase extract has in vitro inhibition effect on the citrus canker (left) and the citrus penicillium (right), and is injected: EE is ethanol extract, EAE is ethyl acetate extract, NBE is n-butanol extract.
Fig. 6: actinomycin has in vitro inhibition effect on canker (left) and penicillium citri (right).
Fig. 7: act-X 2 Influence on the weight of the mycelium of Penicillium citrinum.
Fig. 8: act-X 2 Influence on intracellular protein content of penicillium citrinum.
Fig. 9: act-X 2 Effects on intracellular ROS levels of Penicillium citrinum.
Fig. 10: act-X 2 Effects on DNA replication.
Fig. 11: act-X 2 The control effect on citrus green mold (left) and the lesion diameter (right).
Fig. 12: EAE (ethyl acetate extract mainly containing Act-X) 2 ，Act-D，Act-X Oβ ) The control effect on citrus green mold (left) and the lesion diameter (right).
Fig. 13: EAE (ethyl acetate extract mainly containing Act-X) 2 ，Act-D，Act-X Oβ ) Has effect of preventing and treating citrus ulcer.
Fig. 14: liquid fermentation actinomycin yield in example 7.
Fig. 15: example 8 Rice-based Medium Act-X 2 Influence of yield.
Fig. 16: the effect of rice-based Medium on Act-D production in example 8.
Fig. 17: example 8 Rice-based Medium Act-X Oβ Influence of yield.
Fig. 18: act-X in example 8 corn-based Medium pair 2 Influence of yield.
Fig. 19: effect of corn-based media on Act-D yield in example 8.
Fig. 20: act-X in example 8 corn-based Medium pair Oβ Influence of yield.
Detailed Description
The invention provides streptomyces with high yield of actinomycin, a method for preparing actinomycin with high yield and application of actinomycin in inhibiting growth of agricultural pathogenic microorganisms.
The invention provides a Streptomyces sp GLL-9, which is separated from three annual branches of Neoher navel orange in a national navel orange engineering research center germplasm resource nursery, the effective length of a 16SrRNA gene sequence (GenBank: OQ 726285.1) of the Streptomyces sp is measured, and the effective length of the gene sequence is compared by an NCBI database, so that the Streptomyces sp GLL-9 classified genus is determined and preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation address is eight paths 299 of Wuhan universities in Wuhan region of Hubei province, and the preservation number is: cctccc NO: m2023978, date of preservation: 2023, 06, 09.
The invention also provides a preparation method of actinomycin, which comprises the following steps: and fermenting the GLL-9 strain in a culture medium, wherein the obtained fermentation product contains the actinomycin.
The actinomycin is actinomycin X 2 Actinomycin D and actinomycin X Oβ 。
The structural formula of actinomycin is shown in figure 1, and actinomycin X is obtained when R1=R2 is carbonyl 2 The method comprises the steps of carrying out a first treatment on the surface of the R1 and R2 are hydrogen and are actinomycin D; r1 is hydroxy and R2 is hydrogen, actinomycin X Oβ 。
The culture medium is selected from one or more of PDB, ISP2, MII, TSB, corn culture medium and rice culture medium.
PDB:20% potato, 2% sucrose, ph=7.5;
ISP2:0.4% glucose, 1% malt extract, 0.4% yeast extract, ph=7.5;
MII:2% soybean meal, 0.15% KH 2 PO 4 ，0.05％MgSO 4 ·7H 2 O，PH＝7.5；
TSB:1.7% tryptone, 0.3% soytone, 0.25% KH 2 PO 4 0.25% glucose, ph=7.5;
corn culture medium: 95-110 g of corn residue and 100mL of deionized water;
rice culture medium: 95-110 g of rice and 100mL of deionized water;
preferably, one or more of yeast extract powder, beef extract powder, L-tryptophan and peptone are further added into the corn culture medium; more preferably, 95-110 g rice, 95-110 mL distilled water and 1.0g L-tryptophan are added into a 1000mL conical flask, and the mixture is sterilized by high-pressure steam at 121 ℃ for 30min.
One or more of yeast extract powder, beef extract powder, L-tryptophan and peptone are also added into the rice culture medium; more preferably, 95-110 g of corn residue, 95-110 mL of distilled water and 5.0g of beef extract powder are added into a 1000mL conical flask, and the mixture is sterilized by high-pressure steam at 121 ℃ for 30min.
Preferably, the preparation steps of the streptomyces seed solution are as follows: activating the preserved streptomycete on a potato sucrose agar medium, and then picking a proper amount of hypha in a liquid potato sucrose medium and incubating on a shaking table; preferably, the activation is carried out for 5-7 d, the condition of shaking table incubation is 25 ℃,140rpm, and the incubation is carried out for 24-48 h.
Preferably, the fermentation condition is static fermentation for 20-60d at 25-30 ℃; more preferably, the seed inoculation amount is 5%, and the static fermentation time is 50d.
Further, the fermentation product is sequentially extracted by ethanol and ethyl acetate, and a fermentation extract containing actinomycin is obtained.
Preferably, ethanol is added into the fermentation product to soak overnight, then ultrasonic extraction and filtration are carried out, and the ethanol is removed by a rotary evaporator to obtain ethanol crude extract (EE); suspending the ethanol crude extract in distilled water, extracting with ethyl acetate, removing solvent by rotary evaporator to obtain ethyl acetate phase extract, which is fermented extract EAE (ethyl acetate extract mainly containing Act-X) 2 ，Act-D，Act-X Oβ )。
More preferably, 300-500 mL of industrial ethanol is added to 200g of fermentation product for soaking overnight, 40KHz ultrasonic extraction is carried out for 30-40 min, filter paper filtration is carried out, extraction is repeated for 3 times, and the industrial ethanol is removed by a rotary evaporator, thus obtaining a crude extract (EE). Dissolving the crude extract in distilled water, extracting with ethyl acetate 3 times, and removing solvent by rotary evaporator to obtain ethyl acetate phase (EAE), wherein the ethyl acetate extract is fermented extract EAE (ethyl acetate extract mainly containing Act-X) 2 ，Act-D，Act-X Oβ )。
Still further, subjecting the above fermented extract EAE to silica gel column with different volume fractions of CH 2 Cl 2 Gradient elution with MeOH gives actinomycin.
Preferably, the fermented extract is subjected to silica gel column chromatography with different volume fractions of CH 2 Cl 2 Gradient elution with MeOH to obtain actinomycin; the method comprises the following specific steps of: CH with a volume fraction of 100:0, 50:1,20:1, 10:1,5:1,1:1 2 Cl 2 Gradient elution with MeOH, separating the fermented extract into 6 fractions Fr 1-Fr 6 on a silica gel column; CH with a volume fraction of 100:0,100:1, 50:1,20:1 for Fr2 2 Cl 2 Further gradient elution and separation of MeOH to obtain actinomycin X 2 (1) And actinomycin D (2); CH with a volume fraction of 100:0, 50:1,25:1, 10:1 for Fr3 2 Cl 2 Gradient elution and separation of MeOH to obtain actinomycin X Oβ (3). And, during the elution, each fraction was subjected to a follow-up analysis by thin layer silica gel chromatography (TLC). Dissolving proper amount of compounds 1-3 in 0.5mL deuterated chloroform, and testing 3 compounds by nuclear magnetic resonance apparatus AVNCE 400MHz 1 H spectrum 13 C spectrum, determining the structures of three compounds through comparison of a micro spectrum database; identified actinomycins 1-3 are actinomycin X respectively 2 Actinomycin D and actinomycin X Oβ 。
In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Experimental materials: rice residues and potatoes are purchased from farmers' markets, L-tryptophan is purchased from mikrin Biotechnology Inc., and beef extract and sucrose are purchased from Haibo Biotechnology Inc.; silica gel powder (300-400 mesh) is purchased from Qingdao Spectroscopy separation materials Co., ltd, methanol, ethyl acetate, methylene dichloride, industrial ethanol is purchased from the West Shang Sci Co., ltd; the streptomycete strain is obtained by the inventor by separating from the three-year-old branches of the Newhall navel orange.
1) Preparing seed liquid: activating the separated streptomycete on a potato sucrose agar medium (PDA) for 5-7 d, then picking a proper amount of mycelium into the potato sucrose medium (PDB), and incubating on a shaker for 24-48 h at 25 ℃ and 140rpm;
2) Preparing a culture medium: adding 95-110 g of rice residues, 95-110 mL of distilled water and 1.0g L-tryptophan into a 1000mL conical flask, dissolving the L-tryptophan into the distilled water in the preparation process, adding the conical flask to moisten the rice residues, sterilizing after soaking for 2 hours, and sterilizing for 30 minutes by high-pressure steam at 121 ℃;
3) Inoculating and fermenting: inoculating 10mL of seed suspension into a culture medium, and carrying out static fermentation for 50d at 25-30 ℃;
4) Extracting: adding 300-500 mL of industrial ethanol into each bottle of fermentation product, soaking overnight, performing ultrasonic extraction at 40KHz for 30-40 min, filtering with filter paper, repeating the extraction for 3 times, and removing the industrial ethanol by a rotary evaporator to obtain a crude extract (EE). Dissolving the crude extract in a proper amount of distilled water, extracting 3 times with an equal amount of ethyl acetate, and removing the solvent by a rotary evaporator to obtain an ethyl acetate phase (EAE), wherein the Ethyl Acetate Extract (EAE) mainly contains three actinomycins of the invention;
5) Use of actinomycin isolation and purification: CH with 100:0, 50:1,20:1, 10:1,5:1,1:1 (v/v) 2 Cl 2 MeOH gradient elution, separating the ethyl acetate extract into 6 fractions Fr1 to Fr6 on a silica gel column (silica gel 300-400 mesh). CH with 100:0,100:1, 50:1,20:1 (v/v) for Fr2 2 Cl 2 Further elution with a MeOH gradient afforded compound 1 and compound 2; CH with 100:0, 50:1,25:1, 10:1 (v/v) for Fr3 2 Cl 2 Gradient elution with MeOH afforded compound 3, which was followed by thin layer silica gel chromatography (TLC) for each fraction during the elution. Dissolving appropriate amount in 0.5mL deuterated chloroform, and testing 3 compounds by nuclear magnetic resonance apparatus AVNCE 400MHz 1 H spectrum 13 C spectrum, results are shown in Table 1
Table 1: three actinomycins 13 C NMR data (chemical shift ppm, CDCl) 3 ,400MHz)
The structure of the three compounds was then determined by comparison with a micro-spectral database (see figure 1). Compounds 1 to 3 are actinomycin X respectively 2 Actinomycin D and actinomycin X Oβ 。
6) Determination of actinomycin content: the purified actinomycin is diluted by methanol, the concentration is divided into 0.0625-2 mg/mL, the retention time of the actinomycin and the peak area of different concentrations are detected by a High Performance Liquid Chromatograph (HPLC), and a standard curve equation is calculated by the actinomycin concentration and the peak area. HPLC chromatographic detection conditions:
chromatographic column: XBIdge TM C18 reverse phase column (4.6X100 mm,5 μm);
column pressure and column temperature: 115-125 bar; 30.0-30.2 ℃;
mobile phase: phase A is water and phase B is acetonitrile;
sample injection amount and liquid phase flow rate: 20. Mu.L; 1mL/min;
detection wavelength: 210nm;
elution procedure: 0-45 min,48% acetonitrile; 45-50 min,48% acetonitrile is raised to 90%; the time is 50 to 55 minutes,
90% acetonitrile; 55-60 min,90% acetonitrile is reduced to 48%.
The ethyl acetate extract EAE was dissolved in methanol to prepare a 5mg/mL solution, and the peak areas of 3 actinomycins in the ethyl acetate extract were measured by the above method (see FIG. 2), and the content of 3 actinomycins in the extract was calculated by using a standard curve equation.
Measuring actinomycin X 2 (Act-X 2 ) The yield of (a) was 6.038.+ -. 0.32mg/g (i.e. yield of actinomycin per gram of rice), the yield of actinomycin D (Act-D) was 3.05.+ -. 0.15mg/g and the yield of actinomycin X Oβ (Act-X Oβ ) The yield of (2) is 1.01+/-0.04 mg/g; the unit mg/g refers to milligrams of actinomycin per gram of rice medium.
Example 2
Configuration of the culture medium: 95-110 g of corn residue, 95-110 mL of distilled water and 5.0g of beef extract powder are added into a 1000mL conical flask, the beef extract powder is firstly dissolved into the distilled water in the preparation process, then the conical flask is added to moisten the corn residue, sterilization is carried out after moistening for 2 hours, and high-pressure steam sterilization is carried out for 30 minutes at 121 ℃.
The remaining procedure was as in example 1.
Determination of actinomycin: determination of the content of three actinomycin X in ethyl acetate extract EAE by HPLC 2 The yield of (C) was 5.91.+ -. 0.08mg/g, the yield of actinomycin D was 3.04.+ -. 0.04mg/g and actinomycin X Oβ The yield of (C) was 1.02.+ -. 0.02mg/g.
Example 3
Configuration of the culture medium: as in examples 1 and 2; the remaining operating steps of the respective fermentations 20d,30d,40d,50d,60d were the same as in example 1.
Experimental results: the fermentation media of examples 1 and 2 showed an increasing trend in actinomycin yield (FIGS. 3 and 4) with increasing fermentation time, and the yield was maximized at 50d fermentation, with addition of actinomycin X in L-tryptophan based on rice based media 2 The yield of (a) was 6.038.+ -. 0.32mg/g (i.e. yield of actinomycin per gram of rice residue), the yield of actinomycin D was 3.05.+ -. 0.15mg/g and actinomycin X Oβ The yield of (2) is 1.01+/-0.04 mg/g; actinomycin X in beef extract powder is added into corn residue-based culture medium 2 Yield of 5.91.+ -. 0.0.08mg/g, yield of actinomycin D of 3.04.+ -. 0.04mg/g and actinomycin X Oβ The yield of (C) was 1.02.+ -. 0.02mg/g.
Example 4
Use of crude extract (EE), ethyl acetate phase fermentation extract (EAE) and isolated and purified actinomycin obtained in example 1 for controlling citrus canker and citrus blue mold
1. Evaluation of the controlling Effect of actinomycin and fermented extract on Penicillium citrinum by Oxford cup method
The sample was dissolved in DMSO and then added with an appropriate amount of sterile water, wherein the DMSO content was 10% (v/v), the actinomycin concentration was 2mg/mL, and the fermentation extract was 4mg/mL. 100. Mu.L of spore suspension (10) was inoculated in PDA medium plates (diameter 9 cm) 6 Individual spores/mL), were spread with a spreader, then oxford cups (diameter 6 mm) were placed on the plates, 100 μl of sample was added thereto, 10% dmso was blank control, and 0.01mg/mL prochloraz was positive control. The inhibition was evaluated by measuring the size of the zone of inhibition by calipers by incubating the plates in the dark at 25℃for 2 d.
2. Evaluation of the controlling Effect of actinomycin and fermented extract on canker orange by Oxford cup method
The sample was dissolved in DMSO and then added with an appropriate amount of sterile water, wherein the DMSO content was 10% (v/v), the actinomycin concentration was 1mg/mL, and the fermentation extract was 2mg/mL. Inoculating 100. Mu.L of the bacterial suspension (OD) in nutrient agar plates (diameter 9 cm) 600 =0.2), was spread with a spreader, then an oxford cup (diameter 6 mm) was placed on the plate, 100 μl of actinomycin solution was added thereto, 10% dmso as a blank control, 15 mg/mcuuso 4 Is a positive control. The inhibition was evaluated by measuring the size of the zone of inhibition by calipers by incubating the plates in the dark at 25℃for 2 d.
3. Testing of MIC values
Determination of MIC values against Leuconostoc citruses: MIC values of actinomycin and fermented extract on Leuconostoc using 2,3,5-triphenyltetrazolium chloride (TTC) as a chromogenic agent. The canker citrus is incubated overnight with nutrient broth at 140rpm at 30℃and then diluted with nutrient broth to form a bacterial suspension (OD 600 =0.2). Actinomycin is first dissolved in DMSO, then added with proper amount of nutrient broth, and then added withThe solution was subjected to gradient dilution by the double method. In 96-well plate, 100 μl of bacterial suspension and 100 μl of sample solution are added into each well to make final concentration of sample 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81 μg/mL, and DMSO is used as blank control, cuSO 4 Is a positive control. After incubation of the 96-well plates in the dark for 3.5h, 10. Mu.L TC solution (4 mg/mL) was added to each well. After incubation for 30min, the colour change was observed.
Determination of MIC values against Penicillium citrinum: 100. Mu.L of a sample solution (PDB as solvent in 10% DMSO) of an appropriate concentration was added to a 96-well plate, diluted by a doubling method, and 100. Mu.L of a spore suspension (2X 10) 6 Individual spores/mL), the suspension was PDB to give sample concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63 μg/mL. MIC values were determined by incubating at 25℃for 48h and then observing whether spores germinate and grow.
The results are shown in Table 2:
table 2: inhibition effect of actinomycetes on penicillium citrinum and canker citricola
Wherein the antibacterial activity of EAE is slightly higher than that of other three, and the antibacterial zone and MIC of the EAE on the citrus canker are respectively 30.05+/-1.11 mm and 62.50 mug/mL (shown in figure 5); the inhibition zone and MIC for Penicillium citrinum are 25.69+ -0.96 mm and 31.25 μg/mL, respectively. The EAE is characterized by Thin Layer Chromatography (TLC) to be rich in chemical components and mainly contains three actinomycins described in the invention. Actinomycin has various degrees of inhibition on canker and penicillium citricum, wherein actinomycin X 2 And the inhibition effect of actinomycin D was more remarkable (shown in FIG. 6).
Example 5
Actinomycin X was prepared on the basis of example 4 2 The action mechanism of the penicillium citrinum is explored
1. Actinomycin X 2 Influence on the weight of mycelium
1mL of spore suspension was added to 100mLIn PDB medium, put on shaking table, incubate in dark at 140rpm and 25 ℃ for 24h, then add 100. Mu.L of actinomycin X with proper concentration 2 (DMSO is solvent) to allow actinomycin X to act 2 The concentrations were 0,1/4MIC,1/2MIC and MIC, respectively. Culturing was continued under the same conditions for 24 hours, then the mycelia were filtered off with gauze, washed 3 times with sterile water, and then dried to constant weight at 60℃and each treatment was repeated 3 times.
The experimental results show (FIG. 7), actinomycin X 2 Can effectively inhibit the growth of the penicillium citrinum hyphae, and the inhibiting effect depends on the drug concentration. Hyphae were treated for 24h at MIC concentrations, which reduced the dry weight of hyphae by 66.70% compared to the blank, and at which concentration hyphae growth was significantly inhibited.
2. Actinomycin X 2 Effects on intracellular proteins
The intracellular protein content was determined using coomassie brilliant blue method. Actinomycin X 2 The treatment method for the penicillium citrinum is the same, samples are taken out respectively when medicines are added for culturing for 12 hours and 24 hours, mycelia are filtered out by gauze, and freeze drying is carried out. Taking out 10mg of freeze-dried mycelium, adding 1mLPBS, grinding and homogenizing on ice, centrifuging at 12000rpm for 5min, and collecting supernatant to obtain crude protein extract. 250 μl of coomassie brilliant blue and 10 μl of protein extract were added to each well of a 96-well plate, absorbance at 595nm was measured using a microplate reader, and a standard curve was drawn using bovine serum albumin.
The results are shown in FIG. 8, which shows actinomycin X 2 Can influence the synthesis of intracellular proteins, thereby inhibiting the growth of penicillium citrinum, and has more remarkable influence on the synthesis of the intracellular proteins along with the extension of the action time, and is more remarkable with actinomycin X 2 Is dependent on the concentration of (c). Intracellular proteins are the main contributors to intracellular vital activities, and the inhibition of intracellular protein synthesis affects a series of physiological and biochemical reactions and cellular structures within the cell, thus severely inhibiting hyphal growth.
3. Actinomycin X 2 Effects on intracellular ROS content of Penicillium citrinum
Determination of actinomycin X by fluorescence method using DCFH-DA as ROS indicator 2 Pair of citrusEffects of intracellular ROS content in Penicillium citrinum. 2mL spore suspension and 10. Mu.L of actinomycin X at appropriate concentration 2 (DMSO is solvent) in a glass test tube to allow actinomycin X to be mixed 2 The concentrations were 0,1/8MIC,1/4MIC,1/2MIC and MIC, respectively, and the glass test tubes were placed on a rotary shaker and incubated in the dark at 140rpm and 25℃for 12h. After the incubation was completed, 1mL was removed from each treatment, divided into 2 portions, and washed 3 times with PBS (0.05 mol/L, pH=7), one portion added with 0.5mL of DCFH-DA (10. Mu. Mol/L), the other portion added with 0.5mL of PBS, and incubated in the dark at 25℃for 1 hour, and inverted every 10 minutes. After incubation, the cells were washed 3 times with PBS, centrifuged at 12000rpm for 5min after each wash, spores were collected, and resuspended in PBS. 200 μl of spore suspension was pipetted into a black 96-well plate, the fluorescence value was measured at 485nm for excitation light and 525nm for emission light, and the correction was performed by subtracting the fluorescence value incubated without DCFH-DA from the fluorescence value incubated with DCFH-DA, and each treatment was repeated 3 times.
Experimental results: as shown in fig. 9. Actinomycin X 2 The effect on intracellular ROS of Penicillium citrinum is shown to be progressive, namely, in the concentration range of 1/4MIC, along with actinomycin X 2 Increased concentration of ROS enrichment is exacerbated; after this concentration, ROS accumulation decreased. Presumably, actinomycin X was present at a concentration of 1/4MIC 2 The effect on the germination of the penicillium citrinum spores is small, and as the concentration of the drug increases, the cells are stimulated by the outside, so that the ROS increases sharply. After 1/4MIC concentration, spore germination was greatly affected, with or without a blocked spore biochemical reaction resulting in reduced ROS levels, but with an older increase in intracellular ROS levels compared to the placebo, presumably consistent with reduced intracellular protein levels.
4. In vitro test of actinomycin X 2 Effect on Penicillium citrinum DNA replication
Culturing the penicillium citrinum on a PDA culture medium for 4-5 d, extracting DNA of the penicillium citrinum according to a kit flow, judging the influence of actinomycin X2 on the replication of the DNA of the penicillium citrinum through an in-vitro PCR reaction, and performing a reaction system MIX of 25 mu l: 12.5 μl, ITS1:1.25 μl, ITS4:1.25 μl, DNA:1.5 μl, ddH2O:7.5 μl, actinomycin X2:1 μl of actinomycin X2 is prepared and dissolved by a small amount of ethanol, then diluted to the required concentration by ddH2O, and finally the drug concentration in the reaction system is respectively 10 μg/ml,7.5 μg/ml,5 μg/ml,2.5 μg/ml,1 μg/ml and 0. The PCR reaction conditions were: 95 ℃ C:: 5min;95 ℃ C:: 30s,55 ℃ C: 30s,72 ℃ C: 30s,35 cycles; 72 ℃ C:: 5min. After the PCR reaction is finished, the gel is run on 1% agarose gel for 45min, and then the brightness of the DNA band is checked under an ultraviolet lamp, and the photo is taken.
Previous studies reported that actinomycin D can be inserted into a double helix structure of DNA, form a complex with adjacent bases of guanine and cytosine, affect DNA replication and RNA transcription, and thus affect vital activities of cells. To verify actinomycin X 2 The mechanism of action of Penicillium citrinum was also verified in vitro by PCR. The results are shown in FIG. 10, which shows that following actinomycin X 2 The increase in concentration reduces the brightness of the DNA band, i.e. the DNA content of the in vitro PCR amplification is reduced and the PCR reaction is blocked. After a concentration of 7.5. Mu.g/mL, the DNA band disappeared, i.e.the PCR reaction was completely blocked. From this, it can be seen that actinomycin X 2 The action mechanism of (2) is the same as that of actinomycin D, so that actinomycin X 2 The inhibition mechanism of the penicillium citrinum is to prevent the replication of DNA and influence the transcription of RNA, thereby influencing the synthesis of intracellular proteins and the enrichment of ROS, and further leading to the inhibition of the growth and germination of hyphae or spores.
Example 6
Use of the crude extract (EE), ethyl acetate phase fermentation extract (EAE) and isolated and purified actinomycin obtained in example 1 for controlling citrus canker and citrus blue mold in vivo
1. Penicillium citrinum in-vivo control effect
Evaluation of actinomycin X by fresh fruit experiments 2 And the EAE is used for selecting fresh navel oranges with moderate size and no plant diseases and insect pests for 2min to soak with 2% sodium hypochlorite for surface disinfection, and then washing with distilled water and airing. Cutting wound (length 4mm, width 4mm, depth 3 mm) near fruit equator with scalpel, applying 20 μl of medicine at wound site with concentration of 50, 250, 500 μg/mL, and blankThe control was treated with 40% ethanol. About 2 hours after the drug administration, 20. Mu.L of a suspension of penicillium italicum spores (1X 10) was inoculated on the wound 6 Individual spores/mL, solution is sterile water). Navel orange was placed in a foam box, kept at 25 ℃ and 85% -95% humidity, incubated for 14d, and then lesion diameter was measured with calipers to evaluate disease severity.
The results showed (FIGS. 11 and 12), actinomycin X 2 And EAE can effectively inhibit the occurrence of citrus blue mold, actinomycin can effectively inhibit the growth of citrus blue mold, and can cause the wound of the navel orange to rapidly scab, thereby reducing the occurrence of citrus blue mold, and the navel orange has extremely high potential in the aspects of storage and fresh-keeping of the navel orange without causing phytotoxicity at high concentration.
2. Prevention and treatment effect in citrus canker fungus body
The inhibitory effect of EAE on citrus canker in vivo was tested with fresh neohol navel orange leaves. Collecting healthy navel orange leaf, washing with clear water to remove dirt and residual pesticide on leaf surface, air drying, spraying a layer of EAE solution with concentration of 5, 10, 50 and 100 μg/mL on leaf surface, treating blank control with 5% DMSO, treating positive control with CuSO 4 Solution (1 mg/mL), air-drying the leaf at room temperature, pricking several small holes on both sides of the main vein with inoculating needle, immersing filter paper sheet (diameter 8 mm) into bacterial suspension (OD) 600 =0.2) is overlaid on the leaf aperture. The treated leaf was transferred to a culture dish containing 1% agar, and after culturing at 27℃for 7d, leaf lesions were observed.
The result shows that EAE has good control effect on citrus canker. Can effectively prevent and treat the infection of the citrus canker at 50 mug/mL, and can not have phytotoxicity at 100 mug/mL (shown in figure 13), while the positive control can effectively reduce the occurrence of the citrus canker, but has serious phytotoxicity.
The prevention and treatment effect of EAE on the citrus green mold is tested by a navel orange fresh fruit preservation test, the occurrence of the citrus green mold can be effectively reduced under the condition of 50 mug/mL, the disease can be effectively prevented and treated under the condition of 250 mug/mL, and no phytotoxicity exists under the condition of 500 mug/mL.
Example 7
Liquid medium:
PDB 20% potato, 2% sucrose, ph=7.5;
ISP2, 0.4% glucose, 1% malt extract, 0.4% yeast extract, ph=7.5;
MII 2% soybean meal, 0.15% KH 2 PO 4 ，0.05％MgSO 4 ·7H 2 O，PH＝7.5；
TSB 1.7% tryptone, 0.3% soytone, 0.25% KH 2 PO 4 0.25% glucose, ph=7.5;
solid medium:
wheat bran culture: 75g of wheat bran and 100ml of deionized water;
corn culture medium: 100g of corn grit, 100ml of deionized water;
bean pulp culture medium: 100g of soybean meal and 100ml of deionized water;
orange peel medium: 50g of orange peel and 100ml of deionized water;
when the liquid culture medium is fermented, 10mL of seed liquid is added into each bottle of culture medium (200 mL), shaking culture is carried out for 7d at 25 ℃ and 140rpm, hypha and fermentation liquor are separated during extraction, the fermentation liquor is extracted for 3 times by using equal amount of ethyl acetate, after the hypha is extracted for 3 times by using industrial ethanol in an ultrasonic way, the ethyl acetate is used for extracting for 3 times, and then the two ethyl acetate extracts are combined. The fermentation mode and extraction mode of the solid medium are the same as those described above, and each medium is fermented in 3 bottles. The rest of the procedure is the same as in example 1.
The results showed that although Streptomyces can grow in 4 liquid media, the yield of actinomycin was extremely low, as shown in FIG. 14, and the extraction of fermentation products by liquid fermentation was more cumbersome and more consumed in reagents than solid fermentation, so that the liquid media was not considered. Streptomyces can not grow in wheat bran, bean pulp and orange peel culture mediums, hyphae can be rapidly produced in rice and corn culture mediums, and the yield of actinomycin is high.
Example 8
A further experiment was performed on the basis of example 7, in which rice and corn meal were used as the base medium, and peptone, beef extract, yeast extract, soybean meal and L-tryptophan were added, respectively, with the first 4 additives in the medium at a ratio of 5% and L-tryptophan at a ratio of 1%. The inoculation and fermentation were carried out in the same manner as in example 7, and the production of actinomycin was analyzed by fermentation for 20d,30d,40d,50d and 60d days.
As a result, as shown in FIGS. 15 to 20, the addition of 5% peptone, 5% yeast extract, 5% beef extract and 1% L-tryptophan to the basal medium was improved to various degrees with respect to the yield of actinomycin, but 5% soybean meal in the rice medium prevented the production of actinomycin and also demonstrated that Streptomyces cannot grow in the soybean meal medium. The yield of actinomycin is improved more obviously by adding 5% of beef extract powder and 1% of L-tryptophan into a rice culture medium; the addition of 5% yeast extract, 5% beef extract and 1% L-tryptophan to the corn medium significantly improves the yield of actinomycin, wherein the addition of 5% beef extract improves significantly. And found that addition of 5% peptone to either rice or corn media reduced actinomycin X 2 But the yield of actinomycin D is significantly improved, which is slightly different from other additives.
In the static fermentation process, the yield of actinomycin is increased and then reduced along with the fermentation time, wherein the yield of actinomycin reaches the highest at 40d of fermentation without additives; after the time to peak with the additive was reduced, the fermentation time was 50d. But not absolutely, actinomycin X in the corn-based medium Oβ The yield of (2) always showed an upward trend, but the upward trend became gentle after 50d of fermentation.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The Streptomyces strain GLL-9 is characterized by having a preservation number of CCTCCNO: m2023978.
2. A method for preparing actinomycin, comprising the following steps: fermenting the strain of claim 1 in a medium, wherein the resulting fermentation product contains the actinomycin.
3. The method of claim 2, wherein the actinomycin comprises actinomycin X 2 Actinomycin D and actinomycin X Oβ One or more of the following.
4. The method of claim 3, wherein the medium is selected from one or more of PDB, ISP2, MII, TSB, corn medium, and rice medium.
5. The method of claim 4, wherein one or more of yeast extract, beef extract, L-tryptophan, and peptone are further added to the corn medium.
6. The method according to claim 4, wherein one or more of yeast extract, beef extract, L-tryptophan and peptone are further added to the rice culture medium.
7. The method according to claim 2, wherein the fermentation conditions are static fermentation at 25 to 30 ℃ for 20 to 60 days.
8. The process according to claim 2, wherein the fermentation product is extracted with ethanol and ethyl acetate in sequence to obtain a fermentation extract containing the actinomycin.
9. The use according to claim 8, wherein the fermented extract is applied on silica gelWith different volume fractions of CH on the column 2 Cl 2 Gradient elution with MeOH gives actinomycin.
10. Use of actinomycin obtainable by the method of any one of claims 2 to 9 for controlling canker and/or penicillium citri.
CN202310877302.6A 2023-07-17 2023-07-17 Streptomyces strain, method for preparing actinomycin and application thereof Pending CN116987635A (en)

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