CN113005048A - Streptomyces nigricans CYS22, metabolite thereof and application thereof - Google Patents

Abstract

The invention belongs to the field of biological control of plant diseases, and discloses preparation and application of Streptomyces melanogenes and metabolites thereof. The Streptomyces nigricans has the strain number of CYS22, and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 18476. The streptomyces nigricans CYS22 has an inhibiting effect on a plurality of plant pathogenic fungi such as bitter gourd blight bacteria, rice blast bacteria, apple ring rot bacteria and the like; the strain CYS22 can effectively prevent and treat the bitter gourd fusarium wilt and the rice blast; the pot experiment result shows that the prevention and treatment effect of the Streptomyces nigricans CYS22 fermentation liquor on the bitter gourd fusarium wilt is 60.61%. The active compound P1 is prepared from streptomycete metabolite, and can effectively inhibit spore germination and germ tube growth of rice blast fungi.

Description

Streptomyces nigricans CYS22, metabolite thereof and application thereof

Technical Field

The invention belongs to the field of biological control of plant diseases, and particularly relates to streptomyces nigricans CYS22, a metabolite thereof and application thereof.

Background

Plant diseases seriously affect agricultural production. At present, the prevention and control of plant diseases in China mainly depend on chemical agents, but the problems of environmental pollution, pesticide residues, drug resistance of pathogenic bacteria and the like can be caused by long-term application of the chemical agents. Therefore, exploring a novel plant disease control technology with low toxicity, high efficiency, broad spectrum and environmental protection is a hotspot of the current agricultural production. Biological control is a control technology which utilizes the actions of antagonism, competition, parasitism, antibiotics and the like among microorganisms or induces plants to generate disease resistance through the microorganisms, has the characteristics of safety and innocuity to the environment and human health, difficult generation of drug resistance by pathogenic bacteria and the like, is widely valued and plays an increasingly important role, and is an effective way for green control of plant diseases.

Soil actinomycetes are important microbial resources and are widely applied to the fields of industry, medicine, health, agriculture and the like. Meanwhile, soil actinomycetes are also important plant disease biocontrol factors, and can play an important role in biological control of plant diseases through action modes and ways such as antagonism, competition, parasitism and induction of plant disease resistance. At present, most antibiotics in the world are produced by actinomycetes, and a plurality of antibiotics derived from actinomycetes are applied to agricultural production practice. More than 20 agricultural antibiotics such as validamycin, ningnanmycin, blasticidin and the like registered in China have been registered, and the application of the agricultural antibiotics in agricultural production has great economic and ecological benefits. Streptomyces (Streptomyces) and its related groups are the major actinomycete groups used for biological control of plant diseases. The streptomyces can generate active metabolites such as various antibiotics, biological enzymes, plant hormones and the like, can inhibit the growth of pathogenic bacteria, enhances the plant resistance, plays an important role in biological control of plant diseases, and has great biopesticide development potential. The active streptomyces resource with antagonistic plant pathogenic fungi is screened, and the development of novel microbial pesticide to replace the traditional chemical pesticide has important significance for preventing and controlling the green color of plant diseases.

Streptomyces melanogenes (Streptomyces melanogenes) was first isolated from a soil sample from Tokyo, Japan. The strain is reported to produce the melanomycins and has antitumor, antibacterial and insecticidal activities. However, the antagonistic activity of Streptomyces melanogenes against plant pathogenic bacteria and the control effect thereof on plant diseases have not been reported.

Through the above analysis, the problems and defects of the prior art are as follows: many actinomycete strains with plant disease biological control potential are reported at present, but the strains and related preparations thereof are few in large-scale industrial production and applied to plant disease control practice, and the main reason is that (1) the biological control effect of the strains is greatly influenced by environmental conditions, so that the control effect is unstable; (2) the action mechanism of the strain is complex, and the application technology is not easy to master; (3) the secondary metabolism active substances of the strains are various, and the main active ingredients are not clear or the separation and purification difficulty is high, so that the development cost is high.

The difficulty in solving the above problems and defects is: in order to solve the problems, the target strain must be systematically researched, the biological characteristics and the genetic background of the target strain are determined, and the biocontrol action mechanism and the active substance structure and the physicochemical characteristics of the secondary metabolite of the target strain are determined. However, these studies can be solved by the cooperation of multiple disciplines and division of labor and joint attack and customs.

The significance of solving the problems and the defects is as follows: the problems and the defects are solved, a theoretical basis can be laid for the fermentation process optimization, the preparation and the application technology of the biocontrol strain, and theoretical guidance is provided for the industrial production, popularization and application of the biocontrol strain.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the streptomyces nigricans CYS22, the metabolite thereof and the application thereof, which can inhibit various plant pathogenic fungi and have good plant disease control effect, thereby having good application prospect.

The technical scheme for solving the problems is as follows:

the Streptomyces nigrogens provided by the invention is separated from a yellow brown soil sample in Nanyang city, Henan province, and is characterized in that the Streptomyces nigrogens (Streptomyces melanogenes) with the strain number of CYS22 has the preservation number of the common microorganism center of China Committee for culture Collection of microorganisms: CGMCC No. 18476. Hereinafter, Streptomyces melanogenesis (Streptomyces melanogenes) CYS22 is referred to as "Streptomyces melanogenesis".

Streptomyces melanogenes CYS22 has white oyster mycelium, black red mycelium, and ochre brown soluble pigment on Gao's I culture medium (FIG. 1A, B, C). Under a scanning electron microscope, it can be observed that the intracellular hyphae of Streptomyces melanogenes (CYS 22 basal have no diaphragm, and the aerial hyphae are directly branched; the spores were cylindrical and split transversely (FIG. 1D, E, F). Cultures of Streptomyces melanogenes (CYS 22) are also within the scope of the invention.

Another purpose of the invention is to provide a secondary metabolite of the Streptomyces nigrocaudus CYS22, wherein the secondary metabolite is a compound P1, a compound P1 is a light yellow solid, and the molecular formula is C according to mass spectrum inference₁₃H₁₆O₆N₂The relative molecular mass was about 296.

Further, of said metabolites¹A total of 8 hydrogen signals were shown in the H-NMR (500MHz, in MeOD) data, including 1 single peak methyl signal δ_H3.78(3H, s), 1 bimodal methyl Signal δ_H1.25(3H, d, J ═ 6.4Hz), 1 bimodal methine signal δ_H4.74(1H, d, J ═ 2.4Hz), 1 multiplet methine signal δ_H4.40(1H, dd, J. 6.1, 3.3Hz), 1 monomodal aldehyde signal δ_H8.36(1H, s), 2 bimodal PHENE signals delta_H 8.31(1H，d，J＝7.6Hz)，δ_H7.65(1H, d, J ═ 7.9Hz), 1 triplet benzene ring hydrogen signal δ_H6.88(1H, t, J ═ 7.9 Hz). FIG. 20 is a drawing ofOf compound P1¹³C-NMR spectrum.

Further, the metabolite is N-formamylastatic acid methyl ester, and the chemical structure of the metabolite is as follows:

by combining all the technical schemes, the invention has the advantages and positive effects that: streptomyces melanogenesis (Streptomyces melanogenes) CYS22 has inhibitory effect on 8 plant pathogenic fungi such as plant pathogenic fungi including Fusarium oxysporum, Magnaporthe grisea and Phyllospora Mali (FIG. 3). The test result of Streptomyces melanogenes (CYS 22) on the prevention and treatment of the bitter gourd fusarium wilt shows that only bitter gourd leaves inoculated with the bitter gourd fusarium wilt SD-1 are attacked after 7 days of inoculation, and the symptoms of large-area yellowing can be seen after the leaves extend upwards from the leaf stalks of the leaves; simultaneously inoculating the strain CYS22 and pathogenic bacteria SD-1 to the base of the bitter gourd leaf, wherein the base is slightly yellow; the bitter gourd leaves inoculated with the strain CYS22 and then the pathogenic bacteria SD-1 and the strain CYS22 have no yellowing symptom (figure 4). The test result of the strain CYS22 on the rice blast control shows that after 7d of inoculation, the rice leaves only inoculated with the rice blast fungus P131 are more severe in disease, namely, the leaves are yellow and serious with the inoculation point as the center, and spread to the two ends of the leaves to cause the whole leaves to yellow, and the disease spots are obvious; meanwhile, the rice leaves inoculated with the CYS22 strain and the pathogenic bacteria P131 have spots at the inoculation point, but do not spread to the two ends of the leaves; no obvious lesion is generated on the leaves of the rice which are inoculated with the CYS22 strain firstly and then inoculated with the pathogenic bacteria P131 and the CYS22 strain firstly and then inoculated with the pathogenic bacteria P131 (figure 5). Pot experiments show that the effect of Streptomyces melanogenes (CYS 22) fermentation liquor on preventing and treating the bitter gourd fusarium wilt is 60.61% (table 2, figure 6), in addition, the balsam pear plants treated by the Streptomyces melanogenes (CYS 22) fermentation liquor have no significant difference in indexes such as plant height, stem thickness, root length, root dry weight and the like with a control group, but can significantly increase the fresh weight of the bitter gourd roots, and the strain CYS22 has a certain growth promoting effect on the plants while preventing and treating the bitter gourd fusarium wilt (table 3).

After an active compound P1 is prepared from the streptomycete metabolite and treated by a compound P1 for 4 hours at the concentrations of 5 mu g/mL, 25 mu g/mL and 50 mu g/mL, the germination rates of rice blast fungus spores are 70.59%, 9.76% and 0%, the tube lengths of germinated spores are 42.7 +/-4.7 mu m and 6.8 +/-2.2 mu m respectively, the germination rate of control group spores is 90%, and the tube length of the germinated spores is 51.5 +/-6.5 mu m; after 8h of treatment, the germination rates of Magnaporthe grisea spores were 100%, 68.42% and 0%, respectively, the germ tube lengths of the germinated spores were 73.8 + -8.8 μm and 47.2 + -12.2 μm, respectively, the germination rate of the control group spores was 100%, and the germ tube length was 107.8 + -8.8 μm (Table 10). Can effectively inhibit the germination of the rice blast fungus spores and the growth of germ tubes, and the inhibition rate of the compound P1 on the germination of the rice blast fungus spores is in direct proportion to the concentration of the compound. Streptomyces melanogenes (CYS 22) can be artificially cultured, has broad-spectrum activity against plant pathogenic fungi, has good secondary metabolite activity, and has great development and application values. Streptomyces nigrogene CYS22 can inhibit plant pathogenic fungi by generating active substances, and the active compound P1 can be used for preventing and treating rice blast, and can also be used as a precursor for synthesizing antifungal derivatives and the like. The streptomyces nigrogens CYS22 has good application prospect in the field of biological control of plant diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without any creative effort.

FIG. 1 is a schematic diagram of the morphological characteristics of the strain CYS22 provided by the examples of the invention; a: culture morphology of the culture medium 7d of Gao's No. I (front); b: culture form of the Gao's No. 7d medium (back side); c: 7d single colony morphology of Gao's medium I; d: aerial hyphae were observed by scanning electron microscopy (3500 ×); e: scanning electron microscopy of the spore chains (6000 ×); f: spores were observed by scanning electron microscopy (6000 ×).

FIG. 2 is a schematic diagram of a Neighbour-Joining phylogenetic tree constructed by the strain CYS22 based on a 16S rRNA gene sequence according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of an antagonism map of Streptomyces melanogenesis CYS22 provided in the example of the present invention.

FIG. 4 is a schematic view showing the controlling effect of the in vitro leaves of bitter gourd fusarium wilt provided by the embodiment of the present invention.

FIG. 5 is a schematic view of the effect of controlling rice blast fungus on isolated leaves of rice provided by an embodiment of the present invention.

FIG. 6 is a schematic diagram of the prevention and treatment effect of the strain CYS22 on the fusarium wilt of potted bitter gourds provided by the embodiment of the invention.

FIG. 7 is a schematic diagram of the determination of bacteriostatic activity by a filter paper sheet method according to an embodiment of the present invention.

FIG. 8 is a schematic diagram of the bacteriostatic activity of a methanol solution of a solid fermentation crude extract of the strain CYS22 provided by the embodiment of the invention.

FIG. 9 is a schematic diagram of the detection result of thin layer chromatography of crude solid fermentation extract of the strain CYS22 provided by the embodiment of the invention; a: dichloromethane: methanol 20: 1 (V/V); b: dichloromethane: methanol 10: 1 (V/V); c: dichloromethane: methanol 5: 1 (V/V); d: petroleum ether: ethyl acetate ═ 1: 1 (V/V).

FIG. 10 is a diagram showing the separation result of the first-stage silica gel column chromatography provided in the embodiment of the present invention.

FIG. 11 is a schematic diagram showing the bacteriostatic activity of components T1-T8 on Magnaporthe grisea provided by the example of the present invention; 1: confronting the flat plates; 2: solid fermentation crude extract methanol solution; 3-10: the components are T1, T2, T3, T4, T5, T6, T7 and T8 in sequence.

FIG. 12 is a diagram showing the separation result of the component T2 by two-stage silica gel column chromatography provided by the embodiment of the present invention.

FIG. 13 is a schematic diagram of the bacteriostatic activity of components T2-1 to T2-5 on Magnaporthe grisea provided by the embodiment of the invention.

FIG. 14 is a schematic diagram of the bacteriostatic activity of components T2-2-1 to T2-2-3 on Magnaporthe grisea provided by the embodiment of the invention.

FIG. 15 is a schematic diagram of the separation result of the circulation preparation of the bacteriostatic active substance of component T2-2-2 provided by the embodiment of the invention.

FIG. 16 is a schematic diagram of the detection result of thin layer chromatography of compound P1 provided by the embodiment of the present invention.

FIG. 17 is a high performance liquid chromatogram of compound P1 provided in the examples of the present invention.

FIG. 18 is a flow chart of the separation and purification of the bacteriostatic activity product of the strain CYS22 provided by the embodiment of the invention;

1: separating by secondary silica gel column chromatography; 2: performing Sephades LH-20 gel column chromatography separation; 3: and (4) circulating preparative HPLC separation.

FIG. 19 shows Compound P1 provided by example of the present invention¹H-NMR spectrum.

FIG. 20 is a drawing of Compound P1 provided in an example of the invention¹³C-NMR spectrum.

FIG. 21 is a schematic diagram showing the effect of different concentrations of compound P1 on the germination of Magnaporthe grisea spores provided by an example of the present invention; mock: sterile water control; treatment 1: the concentration of the compound P1 is 50 mug/mL; and (3) treatment 2: the concentration of the compound P1 is 25 mu g/mL; and (3) treatment: the concentration of the compound P1 is 5 mug/mL; a scale: 20 μm.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides streptomyces nigrogenes CYS22, a metabolite thereof and application thereof, and the invention is described in detail with reference to the accompanying drawings.

The streptomyces nigrogenes CYS22 provided by the invention is preserved in the center of common microorganisms of China Committee for culture Collection of microorganisms in 2019, 9 months and 9 days, and the registration number is CGMCC No. 18476; reference biomaterial CYS22, suggested classification nomenclature: streptomyces melanogenes (Streptomyces melanogenes) is produced, the biological material is received by the depository at 9.2019, is registered into a book, is stored for thirty years from the day according to a request, is stored for five years after receiving a request for a sample of the biological material before the expiration, and the survivability of the biological material is detected by the depository at 9.2019, and is survivable.

Classification and identification of biocontrol strain CYS22

1 morphological feature Observation

Morphological characteristics of the strain CYS22 were observed by the patch method, and after the plated dish was cultured at 28 ℃ for 7 days, the slide was carefully removed, and the morphology of hyphae and aerial hyphae in the substrate was observed by a microscope. The hyphae, spore chain and spore morphology of strain CYS22 were observed by scanning electron microscopy.

2 Observation of culture characteristics

The purified strain CYS22 is inoculated on 6 culture media such as Gao's first, PDA, ISP2, ISP3, ISP4 and ISP5 respectively, and cultured at 28 ℃ for 7-30d, during which the color of intrabasal hyphae and aerial hyphae of the strain CYS22 on different culture media, the colony morphology characteristics and the production of soluble pigment are observed.

3 determination of physiological and biochemical characteristics

The physiological and biochemical indexes of the strain CYS22 are determined according to test methods in Streptomyces appraisal handbook and actinomycete systematics-principle, method and practice.

(1) Carbon source utilization: the carbon source provides energy for the growth of the microorganism for vital activities and provides a carbon skeleton for the synthesis of metabolites. And a certain carbon source is used as a unique carbon source for selective culture, so that a basis can be provided for strain classification and identification. 8 carbon sources such as sucrose, glycerol, glucose, mannitol, xylose, galactose, rhamnose and sorbitol are respectively added into a carbon-source-free basic culture medium in equal amount (the carbon source concentration is 1.0%) to serve as unique carbon sources. Inoculating CYS22 strain, culturing at 28 deg.C for 7-30d, observing the utilization of carbon source, and repeating the test for 3 times with the culture medium without carbon source as control.

(2) Nitrogen source utilization: the nitrogen source is a raw material for synthesizing proteins, nucleic acids and other nitrogen compounds constituting the microorganism, and can provide the microorganism with essential nutrient elements. The different types of microorganisms have different nitrogen source utilization capacities, and therefore, the microorganisms can be used as the basis for the classification and identification of strains. A basic culture medium without nitrogen source is selected, and 7 nitrogen sources such as yeast extract powder, potassium nitrate, ammonium dihydrogen sulfate, peptone, ammonium nitrate, urea, glycine and the like are added into the basic culture medium according to the concentration of 0.5%. The control was a basal medium without nitrogen source. Inoculating strain CYS22, culturing at 28 deg.C for 7-30d, observing growth state of strain, and determining utilization of nitrogen source.

(3) Starch hydrolysis test: inoculating CYS22 strain on starch hydrolysis culture medium plate, and culturing at 28 deg.C for 7-10 days. When the strain grows to the optimal state, adding Lugol iodine solution for dyeing treatment, if the culture medium around the strain can not be dyed, indicating that the starch in the culture medium is decomposed, and the strain can generate amylase to hydrolyze the starch, wherein the reaction is positive; if the culture medium surrounding the strain is stained blue, it is indicated that starch in the culture medium is not decomposed, the strain does not produce amylase, and the reaction is negative. The experiment was repeated 3 times.

(4) Milk clotting and peptonization test: subpackaging skim milk culture medium, sterilizing in test tubes, inoculating CYS22 strain, culturing at 28 deg.C, and observing when culturing for 5d, 10d, 20d, and 30 d. If the milk in the tube is coagulated, coagulation is indicated, indicating that the strain produces rennet. If the milk clot in the tube was further hydrolyzed to a clear liquid, peptonization occurred, indicating that the strain produced protease. The experiment was repeated 3 times with no inoculation as a blank control.

(5) Cellulose decomposition test: the cellulose decomposition test of the strain was carried out by using a fiber filter paper method. The upper end of a sterile filter paper strip is inoculated with the CYS22 strain, the lower end is immersed in a cellulose hydrolysis medium, and the filter paper strip is cultured for 30 days at 28 ℃, and whether the strain grows on the filter paper strip or not is observed in the period. If the strain can decompose the filter paper strips and grows well, the strain produces cellulase, and the result is positive; if the strain is unable to decompose the filter paper strip and is unable to grow, the strain does not produce cellulase, and the result is negative. The experiment was repeated 3 times.

(6) Gelatin liquefaction test: the phenomenon that gelatin is decomposed into liquid by strains is called gelatin liquefaction, and the gelatin liquefaction capacity of different strains is obviously different, so that the gelatin liquefaction capacity is a characteristic for identifying the strains. Gelatin medium was filled into tubes and inoculated with the CYS22 strain. The cultures were incubated at 28 ℃ for 30d and the experimental observations were made at 5, 10, 20 and 30d post inoculation, respectively. Before observation, the test tube is put into a refrigerator at 4 ℃ to be refrigerated for 30min and then observed. If the gelatin in the test tube is in a liquid state, the surface gelatin is hydrolyzed, and the strain can produce protease and is marked as positive; if the gelatin is in the solid state in the tube, the strain does not produce protease and is marked negative. The experiment was repeated 3 times.

(7) Hydrogen sulfide generation test: the CYS22 strain was inoculated on a Ctenons medium plate and cultured at 28 ℃ for 7-10 days. Observing the conditions around the bacterial colony, and if the bacterial colony is blackened, the bacterial strain can generate hydrogen sulfide and is marked as positive; if the colony does not turn black around, the strain does not produce hydrogen sulfide and is marked as negative. The experiment was repeated 3 times with no inoculation as a blank control.

(8) Melanin production test: the CYS22 strain is inoculated on a tyrosine culture medium plate, and the culture is carried out for 10-15d at 28 ℃, during which the generation of melanin around the colony is observed, and the generation of melanin is positive, and the generation of no melanin is negative. The experiment was repeated 3 times with no inoculation as a blank control.

(9) MR (mrthyl red test) assay: most microorganisms are capable of breaking down and utilizing glucose, but the pathways and products for breaking down glucose are not completely the same. The MR test is used for measuring the strength of acid production capability of the strain by utilizing glucose and is a standard for strain identification. The sterilized MR medium was dispensed into test tubes, inoculated with CYS22 strain, cultured at 28 ℃ for 7 days and investigated. Methyl red reagent is prepared before investigation, and one drop of methyl red reagent is respectively added to the treatment group and the blank control group. If the color of the solution in the test tube changes from yellow to red, the test tube is positive, and the test tube is negative if the solution changes from yellow to red. The experiment was repeated 3 times with no inoculation as a blank control.

(10) V-P test: the diacetyl produced by the microorganisms during metabolism can combine with the guanidino group on arginine in the culture medium to form a red compound, which is a standard for strain identification. The sterilized V-P test medium was dispensed into test tubes, inoculated with CYS22 strain, cultured at 28 ℃ for 7 days and investigated. During investigation, 5mL of culture solution and a blank control culture solution are respectively sucked and placed into a clean test tube, an equal amount of 40% NaOH is added, and a small amount of creatine is added after uniform shaking. After reacting for 30min, observing the color of the mixed solution, wherein the red reaction is positive reaction, and the yellow reaction is negative reaction. The experiment was repeated three times with no inoculation as a blank control.

(11) Salt resistance test: the salt tolerance of different microorganisms is different, so that the microorganisms have different salt tolerance. The CYS22 strain was inoculated in the high-number one medium containing NaCl in an amount of 0%, 1.0%, 2.5%, 5.0%, 7.5%, 10.0%, 15.0%, respectively, and cultured at 28 ℃ for 7 days. Whether the strain grew on the plate was observed. The experiment was repeated three times with no inoculation as a blank control.

416S rRNA Gene sequence analysis

CYS22 strain 16S rRNA gene sequence analysis, and bacterial universal primer 27F/1492R is selected for sequence amplification.

Biocontrol test of the (II) Strain CYS22

1 antagonistic Activity assay

Firstly, activating target bacteria on a PDA culture medium, beating a freshly activated target bacteria cake by using a sterile puncher (with the diameter of 6mm), inversely sticking hypha to the middle of the PDA culture medium, respectively inoculating actinomycetes at the position with the same distance from the target bacteria by using an inoculating needle, inversely culturing at 28 ℃ for 7 days, and observing whether a bacteriostatic zone exists.

2 preparation of fermentation broth

6 newly activated CYS22 bacterial blocks are selected and inoculated in a PDB culture medium, and the culture is carried out for 3d under the condition of shaking at the temperature of 28 ℃ and 180r/min, thus obtaining the strain CYS22 seed liquid. Inoculating the seed liquid into a newly configured PDB culture medium according to the volume ratio of 6%, shaking and culturing at 28 ℃ for 7d at 180r/min, and collecting a bacterial liquid, namely a fermentation liquid for later use.

Preparation of 3-bitter gourd fusarium wilt germ SD-1 spore suspension

Selecting 6 blocks of freshly activated SD-1 strain of Momordica charantia Fusarium wilt, inoculating in PDB culture medium, culturing at 28 deg.C for 5d with shaking, filtering with sterile filter cloth to obtain SD-1 spore solution, microscopic examination, and preparing into about 1 × 10 with sterile water⁷Spore suspension per mL for use.

4 in vitro leaf method for determining prevention and treatment effect of strain CYS22 on bitter gourd fusarium wilt

Taking bitter gourd leaves with consistent shape and size, cleaning the bitter gourd leaves with sterile water, and putting the bitter gourd leaves into a moisturizing culture dish. 5 treatments were set, and 3 replicates were inoculated per treatment. Treatment 1: only inoculating bitter gourd fusarium wilt bacteria SD-1; and (3) treatment 2: simultaneously inoculating CYS22 fermentation liquor and bitter gourd fusarium oxysporum SD-1; and (3) treatment: firstly inoculating CYS22 fermentation liquor, and inoculating bitter gourd fusarium oxysporum SD-1 after 2 hours; and (4) treatment: firstly inoculating bitter gourd fusarium wilt bacteria SD-1, and inoculating CYS22 fermentation liquor after 2 h; comparison: only the moisture retention treatment is performed. Placing in a climatic chamber with 30 deg.C and 80% humidity after inoculation for 12h, lighting, dark treating for 12h, and investigating disease after 7 d.

5 in vitro leaf method for determining prevention and treatment effect of strain CYS22 on rice blast

The leaves of the four-leaf stage of the rice are selected and cut into 5cm long leaves, and the leaves are put into a culture dish with moisture preservation after being washed clean by sterile water. 5 treatments were set, and 3 replicates of each treatment were inoculated. Treatment 1: only inoculating rice blast fungi; and (3) treatment 2: inoculating CYS22 fermentation liquor and Magnaporthe grisea at the same time; and (3) treatment: inoculating CYS22 fermentation liquor at first, and inoculating rice blast fungi after 2 h; and (4) treatment: inoculating rice blast fungi firstly, and inoculating CYS22 fermentation liquor after 2 hours; comparison: only the blade is scratched. After inoculation, the cells were cultured in the dark at 28 ℃ and 100% relative humidity for 36 hours, and then cultured under light. The disease was investigated 5d after inoculation.

6 pot test

Sterilizing fructus Momordicae Charantiae seed with 75% ethanol, soaking in 55 deg.C warm water for 15min, soaking in water at room temperature for 8-10 hr, and placing at 30 deg.C for moistening and accelerating germination. Transplanting the germinated seeds into a plastic pot filled with nutrient soil. After 2 true leaves grow on the balsam pear seedlings, pouring biocontrol strain CYS22 fermentation liquor into roots, and inoculating 20mL of seedlings in each pot; after 24h, the final concentration is 10 by adopting a root irrigation method⁷And inoculating 10mL of bitter gourd fusarium wilt pathogen spores per mL of bitter gourd seedlings. 4 treatments were set, 3 replicates of each inoculation: treatment 1: inoculating only SD-1 spore suspension; and (3) treatment 2: inoculating CYS22 fermentation liquor, and inoculating a pathogen spore suspension 24 hours later; and (3) treatment: inoculating only the fermentation broth of strain CYS 22; and (4) treatment: an equal amount of sterile water was inoculated. To be inoculated with pathogenic spores onlyAfter treatment of the suspension (about 30 days), the disease index of different treatments and the physiological index of the bitter gourd plants were investigated.

The disease index ∑ (plant tree × representative value of this level)/(total plant number × representative value of highest level) × 100.

The control effect (%) is [ (disease index of control group-disease index of treatment group)/disease index of control group ] × 100.

(III) separation and purification of metabolically active substance of strain CYS22

1 solid fermentation and preparation of crude extract

The seed liquid of the streptomyces nigrogenicus CYS22 is inoculated into a sterile bag filled with 200g of solid fermentation medium in an inoculation amount of 10 percent and is uniformly mixed, and the solid fermentation medium which is not inoculated is used as a control. Culturing at 28 deg.C in dark for 30 days. Obtaining a solid fermentation product. After completion of the fermentation, the mixture was extracted 3 times with 3 times the volume of the solid fermentation product of an organic solvent (ethyl acetate: methanol: 2: 1(V/V)) and the resulting extract was filtered by suction filtration using a buchner funnel packed with 2 layers of filter paper to remove impurities. The resulting extract was concentrated by rotary evaporator (40 ℃ C., 65 r/min). Obtaining crude extract, taking out a little of crude extract, dissolving with methanol, adjusting the concentration to 10mg/mL for later use, and treating the solid fermentation culture medium by the same way.

2 determination of bacteriostatic Activity of crude extract

Taking methanol solution of solid fermentation crude extract and methanol solution of control culture medium crude extract, and respectively preparing into water solution of crude extract with concentration of 200 μ g/mL with sterile water for standby. And (3) determining the bacteriostatic activity of the crude extract by adopting a filter paper sheet method. And (4) preparing the qualitative filter paper into a circular filter paper sheet with the diameter of 6mm by using a puncher, and sterilizing for later use. A drug-carrying filter paper sheet: soaking the sterilized filter paper sheets in methanol solutions of the crude extracts extracted from different components respectively, taking out and airing; filter paper without drug: and (3) soaking the sterilized filter paper sheet in a methanol solvent, taking out and airing. The activated target bacteria are beaten into bacterial cakes by an aseptic puncher with the diameter of 6mm, hypha downwards inoculates the activated target bacteria to the middle of a PDA plate, a sterilized filter paper sheet is taken, 3 filter paper sheets (2 with medicines and 1 without medicines) are clamped by aseptic forceps and evenly placed in the PDA plate with the bacterial cakes, and the distance between the filter paper sheets and the bacterial cakes is 25mm (figure 7). And (5) observing and recording the bacteriostasis condition after culturing for 7d at 28 ℃.

3 stability assay of crude extracts

(1) And (3) determining the antibacterial activity of the solid fermentation crude extract after acid and alkali treatment: 5mL of the aqueous solution of the crude extract (200. mu.g/mL) was transferred into a sterile tube, and the pH of the sample was adjusted to five gradients of 3, 5, 7, 9 and 11 with 0.1mol/mL of HCl and NaOH, respectively, and after stabilization at room temperature for 2 hours, the pH was adjusted to 7. Inhibition rate of each treatment on rice blast fungus (m.oryzae) P131 was determined by a hyphal growth rate method, and repeated 3 times.

(2) And (3) determining the antibacterial activity of the solid fermentation crude extract after treatment at different temperatures: treating the crude extract aqueous solution (200 μ g/mL) at 20 deg.C, 40 deg.C, 60 deg.C, 80 deg.C, 100 deg.C and 121 deg.C for 30min, cooling to room temperature, measuring the inhibition rate of each treatment on Magnaporthe grisea P131, and repeating for 3 times.

4 thin layer chromatography detection of crude extract

(1) Sample application: cutting GF254 thin layer chromatography silica gel plate (10cm × 10cm) into silica gel plate of 2cm × 10cm specification for use. When sample application is carried out, a base line is drawn by a pencil at the position of 0.5cm from the bottom end of the thin-layer chromatography plate, sample application points are spaced by 0.5cm from each other, the sample application points at the two ends of the thin-layer chromatography plate are 1cm away from the edge, and sample application is carried out on the base line. During the spotting process, a capillary is used for sucking a trace amount of sample to perform spotting, and the diffusion diameter of the sample is controlled as much as possible. And after the thin layer chromatography plate is naturally dried, putting the thin layer chromatography plate into a layer spreading cylinder for chromatography.

(2) Selecting a spreading agent: the spreading agent is selected according to the polarity of a sample, on the premise that the solvent can fully dissolve the sample, generally, a compound with small polarity needs to select petroleum ether and ethyl acetate as the spreading agent, and a compound with large polarity needs to select dichloromethane and methanol as the spreading agent. The layer spreading capability of the same system of the spreading agent is generally proportional to the polarity of the solvent, and the Rf value after layer spreading is most suitable between 0.4 and 0.6. 4 kinds of spreading agents are selected in the test: dichloromethane: methanol 20: 1 (V/V); dichloromethane: methanol 10: 1 (V/V); dichloromethane: methanol 5: 1 (V/V); petroleum ether: ethyl acetate ═ 1: 1 (V/V). And screening out the optimal spreading agent system according to the test result.

(3) Spreading a layer: preparing the spreading agents with different systems by using a pipette, sucking 20mL of the spreading agent, adding the spreading agent into a chromatographic cylinder which is cleaned and dried completely in advance, and covering a cover to vibrate fully to saturate the spreading agent in the chromatographic cylinder. Taking the dried thin-layer chromatography plate, putting the plate into a chromatography cylinder in parallel, taking care that the liquid level does not exceed the sampling point, and covering a cover to spread the layer. And after the spreading is finished, taking out the thin-layer chromatography plate, and detecting after the spreading agent is completely volatilized.

(4) And (3) detecting by using a thin-layer chromatography plate: and (3) placing the dried thin-layer chromatography plate into an ultraviolet analyzer, and detecting and photographing at the wavelengths of 254nm and 365 nm. After the completion of the ultraviolet detection, the thin layer chromatography plate is dyed with phosphomolybdic acid dye solution, developed after heating, observed, photographed and calculated for Rf value.

Relative mobility (Rf value) is the distance of the sample spread point from the baseline/the distance of the solvent front from the baseline

5 first-order silica gel column chromatography of crude extract

(1) Sample pretreatment: completely dissolving 70g of the strain CYS22 solid fermentation crude extract with a proper amount of methanol, weighing equal mass of silica gel, mixing, grinding uniformly, placing in a fume hood, and standing until the methanol is completely volatilized, and the sample is uniform, fine and dark brown for later use.

(2) Silica gel column packing: weighing 100-200 meshes of silica gel with the mass being 10-15 times that of a sample, putting the silica gel into a glass beaker, pouring a proper amount of initial elution liquid to mix with the silica gel, uniformly stirring the mixture by using a glass rod, and removing bubbles in the mixture by ultrasonic treatment for 30 min. And selecting a proper silica gel chromatographic column according to the amount of silica gel filled in the column. Before loading, the silica gel chromatographic column must be cleaned, and no water can be remained in the column. 1/3 column volumes of initial eluent is added to the chromatographic column, and the lower valve is opened, which is not too large to affect the packing. Slowly pouring silica gel homogenate into the chromatographic column to enable the silica gel homogenate to naturally settle, sucking initial eluent by a Pasteur glass dropper, flushing the silica gel remained on the inner wall of the chromatographic column, and tapping the outer wall of the chromatographic column by an aurilave to enable the silica gel to be uniformly distributed into the chromatographic column, wherein the liquid level in the column is higher than the silica gel level. And after the sedimentation is finished, observing whether bubbles and faults exist in the chromatographic column or not to enable the upper surface of the silica gel to be flat. Adding a continuous liquid ball, adding 3-5 initial eluents of column volume to wash the column, opening the lower valve to the maximum, quickly eluting and compacting, and removing impurities doped with silica gel in the column.

(3) Loading: stirring the prepared sample uniformly, opening a valve at the lower part of the silica gel chromatographic column, and closing the valve when the liquid level of the eluent at the middle upper part of the chromatographic column is reduced to 0.5-1cm of the plane of the silica gel. Slowly pouring the sample into the column, sucking initial eluent by a Papanicolaou glass dropper after the sample is completely added, and slowly washing the sample remained on the inner wall of the chromatographic column. And finally, adding silica gel with the length of at least 1cm above the sample to serve as a protective layer, and preventing the sample layer from being washed away by adding eluent. Then the lower valve is opened, and the eluent is added from low polarity to high polarity in sequence, and the elution is carried out in sequence.

(4) Sample elution and collection: the gradient elution method is adopted, the eluent is proportioned according to a certain proportion, and then the eluent with different proportions is added into the silica gel column from low polarity to high polarity. In order to ensure the concentration of the eluent to be accurate, the eluent with the former concentration needs to be put on the upper part of the silica gel protective layer (closest to the silica gel layer as far as possible) when the eluent is replaced every time. The eluent ratios are shown in Table 1.

TABLE 1 crude extract first-stage silica gel column chromatography eluent

Collecting the eluent by using a 500mL round bottom distillation flask, collecting 200mL eluent in each bottle, concentrating the eluent by using a rotary evaporator at 40 ℃ and 65r/min, filling the concentrated components into glass vials, and numbering according to the sequence. After elution is finished, carrying out thin-layer chromatography detection on the concentrated eluent, analyzing the concentrated eluent by an ultraviolet analyzer and carrying out phosphomolybdic acid dyeing treatment, merging the same components, numbering again, airing and weighing for later use.

6 Activity determination of each component of crude extract first-stage silica gel column chromatography

After the first-stage silica gel column chromatography elution, the thin-layer chromatography, the ultraviolet and the dyeing treatment, according to the result of the thin-layer chromatography plate, the similar substances are combined to obtain 8 components (T1-T8). The rice blast fungus and the bitter gourd fusarium wilt fungus are selected as target bacteria, and the bacteriostatic activity of the obtained 8 components on the indicator bacteria is detected by adopting a filter paper method. And judging the bacteriostatic activity of each component according to the width of the bacteriostatic band, and separating and purifying the components with good bacteriostatic activity in the next step.

Separation and purification of 7-component T2

The eluate of fraction T2 was selected by thin layer chromatography using dichloromethane: methanol 100:1, performing secondary silica gel column chromatography separation, and loading the silica gel column according to a method in the primary silica gel column chromatography of the crude extract. Concentrating the collected eluate with rotary evaporator by isocratic elution, performing thin layer chromatography, ultraviolet coloration and phosphomolybdic acid staining, and mixing similar components to obtain 5 secondary components (T2-1-T2-5). And the 5 secondary components are subjected to bacteriostatic activity determination. And judging the bacteriostatic activity of each component according to the bacteriostatic bandwidth, and separating and purifying the components with good bacteriostatic activity in the next step.

8-component T2-2 gel column chromatographic separation

(1) Column packing and chromatographic column balancing: wet packing is selected, a certain amount of sephadex LH-20 gel is weighed according to the specification of a gel chromatographic column, and eluent with the volume of about 10 times that of the sephadex LH-20 gel (dichloromethane: methanol-1: 1(V/V)) is added for overnight swelling. Before loading the column, about 10mL of eluent is added into the column, then the swollen gel is slowly and uniformly added, and the gel is slowly settled after the addition. After the gel is settled, the lower valve of the gel column is opened, and the column is washed for 6-8h at the flow rate of 2mL/min to balance the column.

(2) Sample treatment and loading: the fraction T2-2 was dissolved in a small amount of an eluent (dichloromethane: methanol ═ 1: 1(V/V)), and then passed through a 0.45 μm organic microporous filter. After the eluent level dropped to the gel level, the sample was pipetted using a glass pipette and added slowly to the column along the column wall (taking care not to break the gel level). After the sample is completely added, after the liquid level of the sample is reduced to the gel level, a small amount of eluent is used for slowly flushing the residual sample in the column wall. When the liquid level of the residual sample is reduced to the gel level, the eluent is filled.

(3) Sample elution and collection: after adding the eluent, the mixture is passed through a gel column under normal pressure, and the elution flow rate is adjusted to 1 mL/min. The eluates were collected in 10mL glass vials and numbered sequentially. After completion of the collection, the fractions were examined by thin layer chromatography, and fractions with similar spots and Rf values were pooled by UV absorption and staining results, renumbered, and activity was measured.

9 high performance liquid chromatography for determining component T2-2-2 to prepare liquid phase mobile phase

(1) Sample pretreatment: the component T2-2-2 is dissolved by methanol, the concentration is adjusted to 10mg/mL, and impurities and insoluble substances are removed by filtering through a microporous filter.

(2) Mobile phase selection and treatment: the organic phase and the aqueous phase are selected to be prepared into a mobile phase. In the experiment, chromatographic grade methanol is selected as an organic phase, and purified water is selected as a water phase.

(3) Analysis conditions of high performance liquid chromatography: before sample introduction, ultraviolet scanning is carried out by a UV-3010 type ultraviolet-visible spectrophotometer under the wavelength of 190 nm-600 nm, and the absorption wavelength of a sample is preliminarily determined. Methanol and water in different proportions are used as mobile phases, and the specifications of a chromatographic column are as follows: AgilentTC-C18 (250X 4.6mm, 5 μm), the injection volume is 5 μ L, the flow rate is set to 1mL/min, the ratio of the organic phase and the aqueous phase in the mobile phase is changed, and the optimal peak-producing conditions are screened out.

10 preparation of liquid phase purification component T2-2 by circulation

According to the peak pattern results shown by HPLC, an analytical liquid phase was flowed using a preparative chromatographic column Inertsil PREP-ODS column (20. phi. times.250 mm). Determining the wavelength of 254nm, the flow rate of 3.5mL/min, adjusting the sample concentration to 10mg/mL, the sample amount to 0.5mL, the column temperature of 25 ℃, and performing multiple preparations. Collecting the same single peak, carrying out rotary evaporation concentration on the collected sample, drying to obtain a pure product, weighing and marking. Weighing a trace amount of pure compound, dissolving the compound with chromatographic methanol completely, removing an organic filter membrane, transferring the organic filter membrane into a clean liquid bottle, and carrying out chromatographic column specification: agilent TC-C18(4.6 × 250mm, 5 μm), sample size 5 μ L, mobile phase: 70% methanol water, flow rate 1.0 mL/min.

11 Nuclear Magnetic Resonance (NMR) analysis

The pure compound P1 obtained by separation was placed in a fume hood and air-dried to remove the residual organic solvent and water in the sample. After complete dissolution of the compound with 1mL of deuterated methanol, it was transferred to a clean nuclear magnetic tube. To carry out¹H NMR、¹³C NMR nuclear magnetic resonance detection. The determination of the nuclear magnetic resonance spectrum is completed by Beijing university of traditional Chinese medicine.

12 high resolution Mass Spectrometry (MS)

And (3) taking a trace amount of dried pure compound P1, completely dissolving the sample by using chromatographic grade methanol, transferring the sample into a clean liquid bottle, and performing mass spectrometry to determine the molecular mass. The experiment is completed by Beijing university of traditional Chinese medicine.

(IV) Effect of active Compounds on spore Germination of Pyricularia oryzae

(1) Preparation of a rice blast fungus spore suspension: washing the cultured rice blast fungus hypha with sterile water and a sterile cotton swab, sucking 500 mu L of fungus liquid to a tomato and oat culture medium plate by using a liquid transfer gun, uniformly coating the bacterial liquid with a sterile coating rod, and airing the bacterial liquid in a super clean bench. Culturing at 28 deg.C until mycelia grow out, adding small amount of sterile water, breaking off aerial mycelia with sterilized cotton swab, coating with sterilized gauze, culturing in 28 deg.C light incubator for 1-2d, growing gray spores, washing with sterile water, and filtering with three-layer sterilized mirror paper. Counting the spore suspension with a hemocytometer, diluting the spore suspension with sterile water to 1X 10⁵one/mL for use.

(2) And (3) rice blast fungus spore germination determination: the influence of the compound P1 on the spore germination of Magnaporthe grisea was determined by the gloeosporium spore germination method. The pure compound P1 is prepared into three concentrations of 10 mu g/mL, 50 mu g/mL and 100 mu g/mL by sterile water, the spore suspension is taken and evenly mixed with liquid medicines with different concentrations in equal volume to prepare mixed liquid with final concentrations of 5 mu g/mL, 25 mu g/mL and 50 mu g/mL, and the sterile water and the spore suspension in equal volume are mixed to serve as a control. 60 mu L of the mixed solution is absorbed and dripped on a clean and sterile concave glass slide, and the moisturizing treatment is carried out. Culturing at 28 ℃, observing the spore germination condition under an optical microscope at 4h and 8h, and counting the spore germination rate and the germ tube length. Each treatment was 3 replicates. The spore germination rate is calculated according to the following formula:

R＝(N_g/N_t)×100

I＝[(R₀-R_e)/R₀]×100

wherein, R: spore germination rate (%); n is a radical of_g: the germination number of spores; n is a radical of_t: the total number of spores investigated; i: the spore germination inhibition rate; r₀: blank control spore germination rate; r_e: treatment group spore germination rate.

II, identification result of strain CYS22

1.1 morphological characteristics, colony characteristics observations

Strain CYS22 grew well on the gao's No. one medium plate (fig. 1A-fig. 1C). Comparing with a Roel color comparison card, the aerial hyphae of the strain CYS22 grown in Gao's I plate have a color of oyster white (1013), the basal hyphae have a color of black red (3007), and the soluble pigment is ochre brown (8001); the culture characteristics differed on PDA, ISP2, ISP3, ISP4 and ISP5 media with raw hyphae of agate grey (7038), brown beige (1011), pure white (9010), off-white (9002) and wine red (3005) respectively, and with intrabasal hyphae of black grey (7021), grey beige (1019), beige (1001), pale green (6021) and maroon brown (8015) respectively, with soluble pigments produced on PDA and ISP4 media in the colours of sand yellow (1002) and pink green (6019) respectively (table 2). Through the observation of a scanning electron microscope, the intrabasal hyphae of the strain CYS22 have no diaphragm, and the aerial hyphae are straight and branched; the spores were cylindrical and divided by transverse dissection (FIGS. 1D-1F).

TABLE 2 culture characteristics of the Strain CYS22

1.2 measurement results of physiological and biochemical characteristics

In order to classify and identify the strain CYS22, the physiological and biochemical indexes of the strain CYS22 are measured, and the result shows that the strain CYS22 can utilize cane sugar, glycerol and grapesSugar, mannitol, xylose, galactose and rhamnose grow as single carbon source without sorbitol; yeast extract powder, potassium nitrate, ammonium dihydrogen sulfate, peptone, ammonium nitrate, urea and glycine can be used as a single nitrogen source for growth; the starch can be hydrolyzed; milk coagulation peptonization cannot be achieved; cellulose cannot be decomposed and utilized; can liquefy gelatin; produce H₂S; producing melanin; negative in MR and V-P experiments; in the salt tolerance test, when the salt content is higher than 7.5%, the strain CYS22 cannot grow (Table 3). The physiological and biochemical characteristics of the strain CYS22 are similar to those of Streptomyces melanogenesis (Streptomyces melanogene) in Bergey bacteria identification handbook (eighth edition), but slightly different in rhamnose utilization.

TABLE 3 physiological and biochemical characteristics of Strain CYS22

Note: "+" indicates that the strain grew, "+" indicates that the strain grew well, and "-" indicates that the strain did not grow

1.316S rRNA Gene sequence analysis results

The 16S rRNA gene sequencing length of the strain CYS22 is 1357bp, and the sequence is SEQ ID NO: 1 is as follows:

gggttgggccaccggcttcgggtgttaccgactttcgtgacgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcagcaatgctgatctgcgatt actagcaactccgacttcatggggtcgagttgcagaccccaatccgaactgagaccggctttttgagattcgctccgcctcacggcatcgcagctcattgtaccggcca ttgtagcacgtgtgcagcccaagacataaggggcatgatgacttgacgtcgtccccaccttcctccgagttgaccccggcagtctcctgtgagtccccatcaccccga agggcatgctggcaacacagaacaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacagccatgcaccacctgtataccgacca caagggggcgactatctctggtggtttccggtatatgtcaagccttggtaaggttcttcgcgttgcgtcgaattaagccacatgctccgctgcttgtgcgggcccccgtc aattcctttgagttttagccttgcggccgtactccccaggcggggaacttaatgcgttagctgcggcaccgacgacgtggaatgtcgccaacacctagttcccaacgttt acggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagtaatggcccagagatccgccttcgccaccggtgttcctcctgatat ctgcgcatttcaccgctacaccaggaattccgatctcccctaccacactctagctagcccgtatcgaatgcagacccggggttaagccccgggctttcacatccgacgt gacaagccgcctacgagctctttacgcccaataattccggacaacgcttgcgccctacgtattaccgcggctgctggcacgtagttagccggcgcttcttctgcaggta ccgtcactttcgcttcttccctgctgaaagaggtttacaacccgaaggccgtcatccctcacgcggcgtcgctgcatcaggctttcgcccattgtgcaatattccccactg ctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggccggtcgccctctcaggccggctacccgtcgtcgccttggtaggccattaccccaccaacaag ctgataggccgcgggctcatccttcaccgccggagctttccacccagaagcatgcgtttccgggtcgtatccggtattagaccccgtttccagggcttgtcccagagtg aagggcagattgcccacgtgttactcacccgttcgccactaatccaccccgaaggg

this sequence has been filed at NCBI, accession number MT 740348. As shown in FIG. 2, the phylogenetic tree constructed based on the sequence has the closest genetic relationship between the strain CYS22 and Streptomyces melanogenesis (GenBank accession number: NR 041089.1).

Thirdly, the result of the biocontrol effect test of the strain CYS22

1 spectrum of antagonistic activity

The results of the plate confronting test show that the streptomyces nigromaculatus CYS22 has inhibitory activity on 8 plant pathogenic bacteria and shows broad-spectrum antifungal activity. Wherein, the compound has obvious bacteriostasis zone on rice blast (M.oryzae) and bitter gourd fusarium wilt (F.oxysporum f.sp.modosidase) and strongest antagonistic effect (figure 3).

2 in vitro leaf method

The test result of the prevention and treatment of the bitter gourd fusarium wilt shows that the in vitro leaf biocontrol test result of the bitter gourd fusarium wilt shows that after 7 days of inoculation, the symptoms of large-area yellowing can be seen when only the bitter gourd leaves inoculated with the bitter gourd fusarium wilt SD-1 extend upwards from the leaf stalks of the leaves; the base of the balsam pear leaves inoculated with CYS22 strain and pathogenic bacteria SD-1 is slightly yellow; the bitter gourd leaves inoculated with the CYS22 strain and then the pathogenic bacteria SD-1 and then the CYS22 strain have no yellowing symptom (figure 4). This preliminarily shows that the strain CYS22 has good control effect on the bitter gourd wilt.

The test result of the rice blast control shows that after 7 days of inoculation, the rice leaves only inoculated with the rice blast fungus P131 are seriously attacked, namely, the rice leaves are seriously yellowed by taking an inoculation point as the center, and spread to the two ends of the leaves to cause the whole leaves to be yellowed, so that the disease spots are obvious; meanwhile, the rice leaves inoculated with the CYS22 strain and the pathogenic bacteria P131 have spots at the inoculation point, but do not spread to the two ends of the leaves; no obvious lesion is generated on the leaves of the rice which are inoculated with the CYS22 strain firstly and then inoculated with the pathogenic bacteria P131 and the CYS22 strain firstly and then inoculated with the pathogenic bacteria P131 (figure 5). This preliminarily indicates that the strain CYS22 has good control effect on rice blast.

3 pot culture test results

Pot experiment results show that after 30 days of inoculation, the diseased plants of the balsam pears only inoculated with SD-1 spore liquid show wilting phenomena, and the stems and leaves show typical wilting symptoms; the balsam pear plants inoculated with the SD-1 spore liquid after being inoculated with the CYS22 fermentation liquid for 24 hours have good growth vigor which is equivalent to the growth vigor of the balsam pear plants treated by two groups which are not inoculated with the SD-1 spore liquid (figure 6), and the disease index statistical result shows that the disease index of the treatment group only inoculated with the SD-1 spore liquid is 66; the disease index of a treatment group inoculated with SD-1 spore liquid after being treated by CYS22 fermentation liquid is 26, the prevention and treatment effect of the fermentation liquid of the strain CYS22 on the bitter gourd fusarium wilt is 60.61%, the strain CYS22 can obviously reduce the occurrence of the bitter gourd fusarium wilt, and the strain has better prevention and treatment potential (Table 4). The physiological indexes of the balsam pear plants of different treatment groups show that the balsam pear plants treated by the strain CYS22 fermentation liquor and a control group have no significant difference in indexes such as plant height, stem thickness, root length, root dry weight and the like, but can significantly increase the fresh weight of the balsam pear roots, and the strain CYS22 has a certain growth promoting effect on the balsam pear plants while preventing and treating the blight of the balsam pear (Table 5).

TABLE 4 preventive and therapeutic effects of the strain CYS22 on fusarium wilt of bitter gourd

TABLE 5 physiological indices of different treatment groups of bitter gourd plants

The above researches show that the strain CYS22 has good prevention and treatment effects on the bitter gourd fusarium wilt. Combining the results of in vitro leaf and pot experiments, the strain CYS22 has certain biological control potential on the bitter gourd fusarium wilt and the rice blast.

Fourthly, separating and purifying the metabolic active substance of the strain CYS22

1 Activity of crude solid fermentation extract

The crude solid fermentation extract of the strain CYS22 has certain inhibition effect on different target bacteria, and the methanol solution of the crude solid culture medium extract has no inhibition effect on the target bacteria by taking the methanol solution as a contrast, so that the influence of the culture medium on the activity of the solid fermentation product of the strain CYS22 is eliminated. As shown in FIG. 8, the crude fermentation extracts showed the highest inhibitory activity against Pyricularia oryzae. In the subsequent experiments, Magnaporthe grisea was selected as an indicator bacterium and activity evaluation was performed.

2.1 stability of crude extract

The determination result shows (table 6), the biological activity of the crude extract is strongest after the treatment under the condition of pH 7, and the inhibition rate of the crude extract on the rice blast fungi reaches 74.05 percent; the biological activity can still be kept higher after the treatment under the conditions of pH 3 and pH 5, and the inhibition rates are respectively 50.56% and 49.66%; the biological activity of the crude extract is obviously reduced after the treatment under the conditions of pH 9 and pH 11, and the inhibition rate is 11.63 percent and 11.41 percent respectively. The crude extract of the strain CYS22 is stable at 20 ℃ and 40 ℃, and the inhibition rate is 73.15%; after the treatment at 60 ℃, the biological activity of the crude extract is slightly reduced, and the inhibition rate is 64.88%; after treatment at 80 ℃, 100 ℃ and 121 ℃, the activity of the crude extract is obviously reduced, and the inhibition rates are 40.72%, 36.24% and 14.99% respectively. The results show that the antibacterial substance in the CYS22 crude extract is relatively stable under acidic conditions and is relatively sensitive to the temperature above 60 ℃.

TABLE 6 acid-base and thermal stability test results of crude fermentation of strain CYS22

2.2 thin layer chromatography assay of crude extract

The results of the thin layer chromatography are shown in FIG. 9. The Rf value of the methylene dichloride-methanol system is 0.18-1.00, while the Rf value of the petroleum ether-ethyl acetate system is 0, which can not carry out effective layer development. The layer spreading effect of the dichloromethane-methanol system is better than that of the ethyl acetate-petroleum ether system, and the dichloromethane-methanol system is selected as an elution solvent in subsequent experiments.

2.3 first-order silica gel column chromatography separation result

According to the detection result of thin layer chromatography, a dichloromethane-methanol system is selected as an eluent. Setting 7 concentration gradients, and carrying out gradient elution on the sample according to the sequence from low polarity to high polarity. And combining the collected liquid with the approximate Rf value to obtain 8 components in total, wherein the components are numbered from T1 to T8, and are dried and weighed for later use. As shown in fig. 10, table 7.

TABLE 7 quality and bacteriostatic activity of components T1-T8

Note: "+" indicates bacteriostatic activity; "-" indicates no bacteriostatic activity.

The results of inhibition experiments on Magnaporthe grisea of each component show (figure 11), and the components T2, T3 and T5 have certain bacteriostatic activity on Magnaporthe grisea, wherein the component T2 has the strongest inhibitory activity on Magnaporthe grisea.

According to the above results, component T2 was further separated and purified.

2.4 two-stage silica gel column chromatography separation result

According to the result of thin layer chromatography analysis, the component T2 is separated by two-stage silica gel column chromatography by adopting an isocratic elution method. And combining the collected liquid with the approximate Rf value to obtain 5 components with the serial numbers of T2-1-T2-5 in total, and airing and weighing the components for later use. As shown in fig. 12, table 8.

TABLE 8 quality and bacteriostatic activity of components T2-1-T2-5

Note: "+" indicates bacteriostatic activity; "-" indicates no bacteriostatic activity.

The bioassay results of the components T2-1-T2-5 on Magnaporthe grisea show (figure 13), and the components T2-1, T2-2 and T2-3 all have the inhibiting effect on Magnaporthe grisea, wherein the inhibiting activity of the component T2-2 is strongest.

According to the above results, component T2-2 was selected for further separation and purification.

Separating component T2-2 by gel column chromatography (sephadexLH-20), detecting by thin layer chromatography, and mixing the collected liquid with approximate Rf value to obtain 3 components, numbered as T2-2-1-T2-2-3. Referring to FIG. 14, according to the determination of antibacterial activity, component T2-2-2 was subjected to high performance liquid chromatography, and liquid chromatography cycle preparation was performed after determining the mobile phase ratio.

2.5 high performance liquid chromatography Cyclic preparation of active substances

The results are shown in FIG. 15. The component T2-2-2 is prepared by circulation to obtain a monomer compound P1, the fluorescence detection wavelength is 254nm and 312nm, as shown in figure 16. After concentration by rotary evaporation, a dark yellow powder was obtained, weighing 10 mg. The purity of compound P1 was measured by HPLC, the measurement result at 254nm is shown in FIG. 17, the retention time of compound P1 is 3.093min, and the purity is higher than 95%.

2.6 Process flow for separation and purification of active substances

The separation and purification process of the metabolic active substances of S.melanogenesis CYS22 is defined. Extracting a solid fermentation product of streptomyces nigricans CYS22 with an organic solvent (ethyl acetate: methanol: 2: 1) to obtain a crude extract; separating the crude extract by first-stage silica gel column chromatography, and performing gradient elution with dichloromethane-methanol (100: 0) (V/V) as eluent to obtain active component T2; performing secondary silica gel column chromatography separation on the component T2, and continuously eluting by taking dichloromethane and methanol as eluent at the ratio of 100:1(V/V) to obtain an active component T2-2; and then adding dichloromethane: methanol 1: 1(V/V) is used as eluent, and T2-2 is subjected to gel column chromatography separation; and finally, circularly preparing by high performance liquid chromatography (the mobile phase is 70% methanol water) to obtain the pure active compound P1. The separation and purification process flow is shown in figure 18.

3 structural characterization of the active Compounds

Compound P1 is a pale yellow solid with molecular formula C as deduced from mass spectrometry₁₃H₁₆O₆N₂The relative molecular mass was about 296.

¹A total of 8 hydrogen signals, including 1 single-peak methyl signal delta, are shown in H-NMR (500MHz, in MeOD) data (see FIG. 19)_H3.78(3H, s), 1 bimodal methyl Signal δ_H1.25(3H, d, J ═ 6.4Hz), 1 bimodal methine signal δ_H4.74(1H, d, J ═ 2.4Hz), 1 multiplet methine signal δ_H4.40(1H, dd, J. 6.1, 3.3Hz), 1 monomodal aldehyde signal δ_H8.36(1H, s), 2 bimodal PHENE signals delta_H 8.31(1H，d，J＝7.6Hz)，δ_H7.65(1H, d, J ═ 7.9Hz), 1 Triplex phenylring hydrogen signal δ_H 6.88(1H，t，J＝7.9Hz)。

¹³C-NMR (125MHz, in MeOD) data (see FIG. 20) showed a total of 13 carbon signals, including 1 aldehyde carbon signal (. delta. )_C160.63), 2 methyl signals (. delta.))_C19.03, 51.50), 6 benzene ring carbon signals (δ)_C124.60, 126.99, 151.36, 114.38, 122.22, 117.50), 2 methine signals (δ_C58.01, 67.05), 2 carbonyl carbon signals (. delta.)_C170.33，171.04)。

The nuclear magnetic and mass spectrum data of the compound P1 are shown in Table 9, and each parameter is consistent with the reported data of the known compound and is identified as N-formamylanic acid methyl ester, and the chemical structure of the N-formamylanic acid methyl ester is as follows:

TABLE 9 of Compound P1¹H-NMR and¹³C-NMR data and reference data

Fifth, influence of Compound P1 on spore germination of Pyricularia oryzae

The effect of different concentrations of compound P1 on the germination of Magnaporthe grisea spores is shown in FIG. 21. The inhibition rate of the compound P1 on the germination of the rice blast fungus spores is directly proportional to the concentration of the compound P1. After the compound P1 with the concentrations of 5 mu g/mL, 25 mu g/mL and 50 mu g/mL is treated for 4 hours, the germination rates of the rice blast fungus spores are 70.59%, 9.76% and 0%, the tube lengths of the germinated spores are 42.7 +/-4.7 mu m and 6.8 +/-2.2 mu m respectively, the germination rate of the control group spores is 90%, and the tube lengths of the germinated spores are 51.5 +/-6.5 mu m; after 8h of treatment, the germination rates of the rice blast fungus spores were 100%, 68.42% and 0%, respectively, the germ tube lengths of the germinated spores were 73.8 + -8.8 μm and 47.2 + -12.2 μm, respectively, the germination rate of the control group spores was 100%, and the germ tube length was 107.8 + -8.8 μm (Table 10).

TABLE 10 Effect of different concentrations of Compound P1 on spore germination of Pyricularia oryzae

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Henan university of agriculture

<120> Streptomyces nigrogens CYS22, metabolite thereof and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gggttgggcc accggcttcg ggtgttaccg actttcgtga cgtgacgggc ggtgtgtaca 60

aggcccggga acgtattcac cgcagcaatg ctgatctgcg attactagca actccgactt 120

catggggtcg agttgcagac cccaatccga actgagaccg gctttttgag attcgctccg 180

cctcacggca tcgcagctca ttgtaccggc cattgtagca cgtgtgcagc ccaagacata 240

aggggcatga tgacttgacg tcgtccccac cttcctccga gttgaccccg gcagtctcct 300

gtgagtcccc atcaccccga agggcatgct ggcaacacag aacaagggtt gcgctcgttg 360

cgggacttaa cccaacatct cacgacacga gctgacgaca gccatgcacc acctgtatac 420

cgaccacaag ggggcgacta tctctggtgg tttccggtat atgtcaagcc ttggtaaggt 480

tcttcgcgtt gcgtcgaatt aagccacatg ctccgctgct tgtgcgggcc cccgtcaatt 540

cctttgagtt ttagccttgc ggccgtactc cccaggcggg gaacttaatg cgttagctgc 600

ggcaccgacg acgtggaatg tcgccaacac ctagttccca acgtttacgg cgtggactac 660

cagggtatct aatcctgttc gctccccacg ctttcgctcc tcagcgtcag taatggccca 720

gagatccgcc ttcgccaccg gtgttcctcc tgatatctgc gcatttcacc gctacaccag 780

gaattccgat ctcccctacc acactctagc tagcccgtat cgaatgcaga cccggggtta 840

agccccgggc tttcacatcc gacgtgacaa gccgcctacg agctctttac gcccaataat 900

tccggacaac gcttgcgccc tacgtattac cgcggctgct ggcacgtagt tagccggcgc 960

ttcttctgca ggtaccgtca ctttcgcttc ttccctgctg aaagaggttt acaacccgaa 1020

ggccgtcatc cctcacgcgg cgtcgctgca tcaggctttc gcccattgtg caatattccc 1080

cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc cggtcgccct 1140

ctcaggccgg ctacccgtcg tcgccttggt aggccattac cccaccaaca agctgatagg 1200

ccgcgggctc atccttcacc gccggagctt tccacccaga agcatgcgtt tccgggtcgt 1260

atccggtatt agaccccgtt tccagggctt gtcccagagt gaagggcaga ttgcccacgt 1320

gttactcacc cgttcgccac taatccaccc cgaaggg 1357

Claims (6)

1. The streptomyces nigrogenicus strain is CYS22, and the preservation number of the streptomyces nigrogenicus CYS22 in the China general microbiological culture Collection center is as follows: CGMCC No. 18476.

2. A culture obtained by culturing the Streptomyces melanogenes of claim 1 in a microbial culture medium.

3. A pathogen inhibitor comprising the Streptomyces melanogenesis of claim 1 and/or a metabolite of the Streptomyces melanogenesis of claim 1.

4. A metabolite of Streptomyces melanogenesis as claimed in claim 3, wherein: the metabolite is a compound P1, the compound P1 is a light yellow solid, and the molecular formula is C according to mass spectrum inference₁₃H₁₆O₆N₂The relative molecular mass was about 296.

5. A metabolite of Streptomyces nigrogenes CYS22 as claimed in claim 4, wherein the metabolite is¹A total of 8 hydrogen signals, including 1 unimodal methyl signal delta, were shown in the H-NMR (500MHz, in MeOD) data_H3.78(3H, s), 1 bimodal methyl Signal δ_H1.25(3H, d, J ═ 6.4Hz), 1 bimodal methine signal δ_H4.74(1H，d, J ═ 2.4Hz), methine signal δ of 1 multiplet_H4.40(1H, dd, J. 6.1, 3.3Hz), 1 monomodal aldehyde signal δ_H8.36(1H, s), 2 bimodal PHENE signals delta_H8.31(1H，d，J＝7.6Hz)，δ_H7.65(1H, d, J ═ 7.9Hz), 1 Triplex phenylring hydrogen signal δ_H6.88(1H, t, J ═ 7.9 Hz). FIG. 20 is a drawing of compound P1¹³C-NMR spectrum.

6. The metabolite of Streptomyces nigrogenes CYS22 as claimed in claim 4, wherein the metabolite is N-formimylanic acid methyl ester having the chemical structure:

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