New streptomyces for antagonizing broad-spectrum plant pathogenic fungi and application of new streptomyces

Abstract

The invention provides a streptomyces, which is named as Streptomyces sp. And is registered and preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 65917. The invention also provides a fermentation broth of the streptomyces, and an ethanol extract and a crude extract of the fermentation broth. The novel streptomyces, the crude extract thereof and the like have broad-spectrum antibacterial activity, have a good antagonistic effect on various pathogenic bacteria such as banana long leaf spot germs and banana colletotrichum gloeosporioides, can enable hyphae of the banana colletotrichum gloeosporioides to shrink, deform and break, inhibit spore germination of the banana colletotrichum gloeosporioides, and have a good anti-bacterial effect on the banana colletotrichum gloeosporioides. According to the present invention, with the application of the bacterial strain, the colletotrichum gloeosporioides spores are shrunk and broken, the growth of the colletotrichum gloeosporioides is inhibited, the peroxidase activity, the polyperoxidase activity, the CAT activity and the like in the banana peel can be improved, and the bacterial strain is the potential biological agent for the prevention and control of the colletotrichum gloeosporioides diseases, and has wide development space and good development and application prospects.

Description

Technical Field

[0001] The present invention relates to a strain, and particularly to a novel Streptomyces species antagonistic to a broad spectrum of phytopathogenic fungi and its application. Background Art

[0002] Banana (Musa spp.) is an important crop for both fruits and grains in tropical and subtropical regions. However, banana fruits are extremely vulnerable to anthracnose after harvest, which leads to a decline in the quality of banana fruits and fruit damage. Currently, the methods for preventing the occurrence of banana anthracnose can be roughly divided into three categories: physical preservation, chemical control, and biological control. The commonly used physical preservation methods are mainly low-temperature storage and transportation and controlled atmosphere preservation. The optimal growth temperature of the pathogen is 25 - 28°C. Under low-temperature conditions, the pathogen cannot grow normally and produces spore forms to adapt to the adverse environment. Mechanistically, low temperature inhibits the spore germination and mycelial growth of the pathogen. Appropriate ultraviolet treatment of the fruits can inhibit the number of anthracnose bacteria on the peel and kill the spores of the pathogen without changing the quality of the fruits; Chemical control has the characteristics of high efficiency and broad spectrum. There are many commonly used chemical agents for preventing anthracnose. For example, exogenous application of salicylic acid (SA) and jasmonic acid substances (JA). SA is a phenolic compound that widely exists in various plants. There have been many studies on the prevention and control of post-harvest fruit diseases by spraying SA. A large number of studies have shown that exogenous SA can activate the defense response of plants to help plants resist diseases. However, long-term chemical treatment is likely to make the pathogen develop drug resistance, and at the same time, the leakage of chemical pesticides and other agents will also cause irreversible damage to the environment and affect the balance of the soil microecology.

[0003] Due to the advantages of environmental friendliness and high efficiency, biological control is considered an effective tool for the prevention and control of post-harvest anthracnose. More and more studies focus on the application of antagonistic microorganisms. Various microorganisms have strong application potential in controlling post-harvest anthracnose of fruits. For example, Bacillus velezensis shows a strong antagonistic effect on controlling the rot disease of jujube caused by Colletotrichum gloeosporioides. Streptomyces also has potential application value in the prevention and control of post-harvest diseases of strawberries. In the prevention and control of anthracnose of loquat fruits, the application of the biocontrol yeast Saturnis pora diversa is effective. At the same time, natural Trichoderma has a strong inhibitory ability against anthracnose of sorghum. Therefore, it is safe and sustainable to use biological control to prevent and control post-harvest banana fruit diseases, and it is particularly important to find a better method to prevent and control banana anthracnose. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies in the prior art and provide a novel Streptomyces species antagonistic to a broad spectrum of phytopathogenic fungi and its application.

[0005] The first aspect of the present invention is to provide a Streptomyces sp. named Streptomyces sp. (test number: SX-6), which is registered and preserved in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCC No: 65917, and the preservation date: February 19, 2025.

[0006] The second aspect of the present invention is to provide the fermentation broth of the Streptomyces sp. described in the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide an ethanol extract of the fermentation broth of the Streptomyces sp. described in the first aspect of the present invention.

[0008] The fourth aspect of the present invention is to provide a crude extract of the Streptomyces sp. described in the first aspect of the present invention. The crude extract is the substance adsorbed on the column after the ethanol extract described in the third aspect of the present invention is filtered through macroporous resin, and is obtained by eluting with methanol of different concentrations and concentrating and drying the eluate.

[0009] The fifth aspect of the present invention is to provide a preparation containing the Streptomyces sp. described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention is to provide the Streptomyces sp. described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation for antagonizing Mycosphaerella musicola, and / or Colletotrichum musae, and / or Colletotrichum acutatum, and / or Fusarium oxysporum f. sp. niveum, and / or Colletotrichum gloeosporioides Penz. f. sp. mangiferae, and / or Phyllosticta capitalensis, and / or Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4.

[0011] The seventh aspect of the present invention is to provide the Streptomyces sp. described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation for preventing and treating diseases caused by Mycosphaerella musicola, and / or Colletotrichum musae, and / or Colletotrichum acutatum, and / or Fusarium oxysporum f. sp. niveum, and / or Colletotrichum gloeosporioides Penz. f. sp. mangiferae, and / or Phyllosticta capitalensis, and / or Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4.

[0012] The eighth aspect of the present invention is to provide the application of the Streptomyces described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation that causes the mycelia of Colletotrichum musae to shrink, deform, and break.

[0013] The ninth aspect of the present invention is to provide the application of the Streptomyces described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation that causes the spores of Colletotrichum musae to shrink and rupture.

[0014] The tenth aspect of the present invention is to provide the application of the Streptomyces described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation that increases the activities of peroxidase (POD), and / or polyphenol oxidase (PPO), and / or catalase (CAT) in banana peels.

[0015] Among them, the preparation increases the activities of peroxidase, and / or polyphenol oxidase, and / or catalase in banana peels but does not affect the activities of triacontanol (TA), and / or vitamin C (VC).

[0016] The eleventh aspect of the present invention is to provide the application of the Streptomyces described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the ethanol extract described in the third aspect of the present invention, or the crude extract described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention in the preparation of a preparation that inhibits the growth of Colletotrichum musae.

[0017] The novel Streptomyces SX-6 and its crude extract of the present invention have broad-spectrum antibacterial activity and have good antagonistic effects against pathogens such as Pseudocercospora musae, Colletotrichum musae, Colletotrichum fragariae, Rhizoctonia solani, Colletotrichum gloeosporioides Penz. f. sp. mangiferae, Phyllosticta capitalensis, Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4. Moreover, it can cause the mycelia of Colletotrichum musae to shrink, deform, and break, inhibit the germination of Colletotrichum musae spores, cause the spores of Colletotrichum musae to shrink and rupture, inhibit the growth of Colletotrichum musae, and can also increase the activities of peroxidase, polyphenol oxidase, and CAT in banana peels. It is a potential biological agent for the control of anthracnose diseases and fusarium wilt diseases, etc., with broad development space and good development and application prospects. Description of the Drawings

[0018] Figure 1 Results of primary screening (upper figure) and secondary screening (lower figure) of actinomycetes.

[0019] Figure 2 Results of morphological observation of strain SX-6 under scanning electron microscope (SEM).

[0020] Figure 3 The upper middle figure is the 16S phylogenetic tree of strain SX-6, and the lower figure is the whole genome alignment of strain SX-6 with multiple strains.

[0021] Figure 4 Results of the broad-spectrum experiment on the plate of strain SX-6.

[0022] Figure 5 Results of the broad-spectrum experiment on the plate of the crude extract of strain SX-6.

[0023] Figure 6 Effects of the crude extract of Streptomyces sp. SX-6 on the mycelial growth and its morphology of Colletotrichum musae.

[0024] Figure 7 Experiment on the effect of the crude extract of Streptomyces sp. SX-6 on the spore germination of Colletotrichum musae.

[0025] Figure 8 Experiment on the effect of the crude extract of Streptomyces sp. SX-6 on the spore morphology of Colletotrichum musae.

[0026] Figure 9 Effects of the crude extract of Streptomyces sp. SX-6 on the infection of banana fruits by Colletotrichum musae. A shows the changes in the disease spots of banana fruits, and B shows the comparison of the disease spot diameters of the pathogen of Colletotrichum musae on banana fruits.

[0027] Figure 10 Effects of the crude extract of Streptomyces sp. SX-6 on the enzyme activity of banana peel.

[0028] Figure 11 Effects of the crude extract of Streptomyces sp. SX-6 on the process of C. musae invading banana peel. Detailed implementation manners

[0029] The present invention will be further described below with reference to the accompanying drawings in combination with specific embodiments for better understanding. For those technical or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0030] 1 Isolation of actinomycetes from soil

[0031] Collect soil samples from the rhizosphere of tropical rainforest plants in Bawangling, Changjiang Li Autonomous County, Hainan Province, China. Manually remove large impurities and hard lumps from the collected soil, and then place it in a fume hood to dry at room temperature for 3 days. Subsequently, grind the soil with a mortar and pass it through three filter sieves (10 - 20 mesh). Weigh 1 g of soil on an electronic balance into a 10 ml centrifuge tube, and add 9 ml of sterile water to dilute it to a 10 ml suspension. Dilute it to 10 -2 and 10 -3 suspensions in the same way. Subsequently, respectively pipette 100 μl of the suspensions with three dilution multiples onto SCA, PDA, and Gao's No. 1 medium, and spread them with a spreading rod on the surface of the culture dish without water stains. Place them in an inverted position in a biochemical incubator at 28 °C for 1 - 4 weeks. Select different strains according to color, growth time, mycelial morphology, and colony size onto a new YE medium for strain isolation and purification, and store the pure bacteria in 30% glycerol and place them in a -80 °C ultra-low temperature refrigerator.

[0032] 2 Screening of actinomycetes (primary screening, secondary screening)

[0033] Primary screening: Screen the antagonistic activity of the isolated strains by the four-point confrontation method. First, place the isolated actinomycetes at four points 2.5 cm from the center of the PDA medium. Culture at 28 °C for 3 days. After the actinomycetes grow, inoculate a 5 mm diameter mycelial cake of the banana anthracnose pathogen (Colletotichum musae) at the center point of the medium. Incubate in a biochemical incubator at a constant temperature of 28 °C for 7 days, and repeat the experiment three times. The results show that 84 strains exhibit antibacterial activity (the antibacterial results of some strains are shown in Figure 1 the figure above).

[0034] Secondary screening: Ferment the active antagonistic bacteria obtained from the primary screening in soybean powder medium for 7 days, and then filter the fermentation broth through a 0.22 μm sterile filter pore to obtain a sterile fermentation broth. Use a puncher to make four holes 2.5 cm from the center point on the PDA plate. The four holes are radially symmetric around the center point, and add 200 μl of the filtered sterile fermentation broth to each hole. Inoculate the banana anthracnose pathogen at the center point of the PDA and place it in an incubator for cultivation at a temperature of 28 °C and a humidity of 85%. Select the Streptomyces sp. SX-6 with strong activity and suspected to be a new species (the antibacterial results of some strains are shown in Figure 1 the figure below).

[0035] 3 Identification of actinomycetes

[0036] 3.1 Morphological characteristics

[0037] The morphological characteristics of the antagonistic bacteria were observed on various culture media, including the color of aerial and basal hyphae, as well as the color and size of colonies. The results showed that on ISP2 medium, the aerial hyphae of the antagonistic bacteria were milky yellow, the basal hyphae were brownish yellow, and they did not produce soluble pigments, indicating excellent growth. On ISP4 medium, the aerial hyphae were milky white, the basal hyphae were light yellow, and they did not produce soluble pigments, indicating good growth. On ISP5 medium, the aerial hyphae were pinkish white, the basal hyphae were light yellow, and they did not produce soluble pigments, indicating good growth. On ISP6 medium, the aerial hyphae were yellowish white, the basal hyphae were brownish yellow, and they did not produce soluble pigments, indicating excellent growth. On ISP7 medium, the aerial hyphae of the antagonistic bacteria were white, the basal hyphae were brownish yellow, and they did not produce soluble pigments, indicating good growth.

[0038] 3.2 Scanning electron microscopy (SEM) morphology observation

[0039] The selected antagonistic bacteria were cultured on PDA medium for 10-14 days. The colonies were carefully separated from the medium using a sterile syringe. The flattest and thinnest colonies were selected, and a 0.5 cm square block was cut. After processing, it was fixed with isoamyl acetate and air-dried in a sterile clean bench. Finally, the morphology of hyphae and spores was observed under a scanning electron microscope (Zeiss Sigma 500 / VP, Germany) after gold plating. The results are shown in Figure 2. Figure 2 The antagonistic strain has intricately intertwined hyphae with spores hanging from them, which appear as short rods.

[0040] 3.3 Determination of physiological and biochemical characteristics

[0041] The physiological and biochemical characteristics of actinomycetes were identified with reference to the Manual of Identification of Common Bacterial Systems and Actinomycete Systematics.

[0042] (1) Culture characteristics of strain SX-6

[0043] Strain SX-6 was inoculated into different culture media and cultured at 28°C for 7-14 days before being observed for growth. The results are shown in Table 2. Strain SX-6 grew on all five culture media, with rapid and excellent growth on ISP2, ISP6, and ISP7. SX-6 did not produce soluble pigments on any of the five media. Colonies were mostly powdery and wrinkled, with aerial hyphae mostly white and basal hyphae mostly yellow.

[0044] Table 2

[0045]

[0046] Note: “++” means good growth, and “+” means able to grow on the culture medium.

[0047] (2) Carbon and nitrogen source utilization experiments

[0048] The test strains were inoculated into the culture media containing different carbon sources and nitrogen sources, and their growth was observed after culturing at 28 °C for 7 - 14 d, with a negative control set. The results are shown in Table 3.

[0049] Table 3

[0050]

[0051]

[0052] (3) Antibiotic experiment

[0053] Well - grown single colonies of actinomycetes were picked and inoculated into YE liquid medium, cultured at 28 °C with shaking at 180 r / min for 3 days. A small amount of the bacterial liquid was taken for counting under an optical microscope, and then it was diluted to 1x10 6 CFU / mL by aseptic technique. 100 μL of the spore liquid was evenly spread on the YE solid medium plate, and four antibiotic filter papers were respectively inoculated at four points 2.5 cm away from the plate. The experiment was repeated three times. It was cultured at a constant temperature of 28 °C for 7 days, and whether antibacterial zones were produced around the filter papers was observed. If there were, it indicated that the actinomycetes were sensitive to the antibiotic; otherwise, it meant there was no resistance. The results are shown in Table 4.

[0054] Table 4

[0055]

[0056]

[0057] Note: Sensitivity to antibiotics is recorded as "+", and insensitivity is recorded as "-".

[0058] Being sensitive to an antibiotic means that the antibiotic has an inhibitory effect on it, and vice versa.

[0059] (4) Other physiological and biochemical indicators

[0060] pH experiment: The pH values of the liquid medium were adjusted to 4, 5, 6, 7, 8, 9, and 10 respectively. The test strains were respectively inoculated into the media with different pH values, ensuring that other culture conditions were the same. It was cultured with shaking at 28 °C, observed once a week, cultured for four weeks, and finally the growth of the strains on each medium was determined. The upper and lower limits of the pH values at which the strains could grow and the optimal pH value were determined.

[0061] Sodium chloride experiment: Prepare culture media with different NaCl concentrations (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%). The other components of the culture media are the same. Inoculate the strain to be identified on these culture media and culture at 28°C. Observe once a week for four weeks, and finally record whether the strain grows on the culture media to determine the upper and lower limit concentrations of the NaCl tolerance of the strain to be identified.

[0062] Esterase (Tween 40, Tween 60) experiment: After preparing the esterase culture medium, sterilize the separately packaged Tween 40 and 60 together, and mix the Tween with the culture medium to make plates. Inoculate the strain on the plate and culture for one to two weeks. Observe the plate. If a blurred halo appears around the colony, it is positive; otherwise, it is negative.

[0063] Urease test: Inoculate the strain on the urease culture medium and culture at 28°C for 4 days, then observe whether the culture medium changes color. Test the ability of the test strain to produce urease. If the culture medium turns pink, it is positive; if it does not change color, it is negative.

[0064] Nitrate reduction: Inoculate the strain to be tested in the nitrate reduction culture medium and culture at 28°C for 7 and 14 days, with the culture medium without inoculation as a control. Add a little of the culture solution cultured for 7 days and 14 days respectively to the test tubes, and drop one drop of Solution A and Solution B. Drop the same for the control. When the solution turns pink, rose red, orange or brown, etc., it is positive for nitrate reduction; when no red appears, add 1 or 2 drops of diphenylamine reagent. If it turns blue, the reduction is negative; if it does not turn blue, it is still regarded as positive.

[0065] Gelatin experiment: Inoculate the strain to be identified in a test tube containing gelatin culture medium, and then culture at 28°C. Observe the liquefaction of gelatin on the 5th, 10th, 20th, and 30th days. If there is liquefaction, it is positive, indicating that the strain has the ability to liquefy gelatin; otherwise, it is negative.

[0066] Phosphate solubilization test: Prepare phosphate-solubilizing bacteria culture medium, sterilize and pour plates. Inoculate actinomycetes on the culture plates, and the control is the culture plate without inoculation. After culturing at 28°C for 1 week, observe the culture plates of the culture medium. If there is a clear zone, it indicates that it has the ability to solubilize phosphate; otherwise, it does not.

[0067] Nitrogen fixation test: Prepare Ashby's medium for nitrogen-fixing bacteria, sterilize and pour plates. Inoculate actinomycetes on the culture plates, and the control is the culture plate without inoculation. After culturing at 28°C for 1 week, observe the culture plates of the culture medium. If there is a clear zone, it indicates that it has the ability to fix nitrogen; otherwise, it does not.

[0068] Cellulose degradation: Prepare a sodium carboxymethyl cellulose medium, sterilize it, and pour it into plates. Inoculate actinomycetes on the plates and incubate them at 28 °C for 7 days. Take them out, pour a 1 g / L congo red solution into the plates to submerge the bacterial liquid, and then pour a 1 mol / L NaCl solution into the plates for decolorization for 30 minutes. Observe whether there is a clear zone on the bacterial plates. If a clear zone appears, it is positive, indicating the ability to produce cellulase; otherwise, it is negative.

[0069] Starch hydrolysis: Test whether the strain has amylase activity. Inoculate actinomycetes on a starch hydrolysis medium and incubate them at 28 °C for 5 - 7 days. Then, add iodine solution that covers the hyphae of the strain around the colonies for detection. If a clear zone is produced, it is positive; if there is no change in the clear zone, it is negative.

[0070] The results are shown in Table 5.

[0071] Table 5

[0072]

[0073]

[0074] Note: "+" indicates positive and "-" indicates negative.

[0075] Combined with its cultural characteristics, physiological and biochemical properties, and hyphal morphology, etc., strain SX-6 presents typical morphological characteristics of Streptomyces.

[0076] 3.4 Establishment of Streptomyces 16S phylogenetic tree

[0077] Use a Bioteke bacterial genomic DNA rapid extraction kit to extract the total DNA of actinomycetes. Using the genomic DNA of the actinomycetes to be tested as a template, perform PCR amplification. Universal primers for actinomycetes 16S rRNA, upstream: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream: 5'-TACGGCTACCTTGTTACGACTT-3'. A 5 μl PCR reaction system. PCR amplification program: pre-denaturation at 95 °C (5 min), denaturation at 94 °C (1 min), annealing at 55 °C (1 min), extension at 72 °C (2 min), final extension at 72 °C (10 min), storage at 4 °C. Among them, there are 32 cycles from denaturation to annealing and then to extension, and then there are subsequent processes after the cycles.

[0078] Prepare 1% agarose gel and wait for 15 minutes to solidify. Then, load 5 μL of the PCR product and 5 μL of the Marker. Place the gel in an electrophoresis tank containing 1×TAE electrophoresis buffer and run it at 150 V for 25 minutes. After electrophoresis, observe the results under a UV-visible spectrophotometer at 254 nm. If there is a PCR band at 1400 bp, send the PCR product to Shanghai Sangon Biotech Co., Ltd. for 16S rRNA sequencing.

[0079] After the sequencing company returns the data, upload the obtained sequence to the EzBioCloud and GenBank databases for homologous alignment and search for and download 16S rRNA gene sequences with relatively high similarity. Select the strains for multiple comparison analysis, and use MEGA 11.0 software to construct a phylogenetic tree by the Neighbor-Joining method, with the Bootstrap self-check value set to 1000.

[0080] Based on the results of 16S sequencing, upload the sequence to Ezbiocloud for alignment and find that the closest relative species is Streptomyces orinoci NRRL B-3379. However, when comparing the whole-genome sequences, it is found that its ANi value is 80.78% and its DDH value is 29.70%. The similarity between the two is relatively low. Therefore, upload the sequence to ncbi to establish a phylogenetic tree, download the whole-genome sequences of multiple strains with relatively close genetic relationships for comparison, and finally the comparison result shows a relatively low similarity. The results are as Figure 3 shown. It is determined to be a new species, named Streptomyces sp., and sent to the Guangdong Provincial Microbial Culture Collection Center for preservation. The preservation number is GDMCC No: 65917, the preservation date is February 19, 2025, and the preservation address is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0081] 4 Plate broad-spectrum experiment of actinomycetes

[0082] Pathogenic bacteria: Ciuvularia fallax (ATCC 12017), the pathogenic bacterium of banana long-shaped spot disease; Colletotichum musae (ACCC 31244), the pathogenic bacterium of banana anthracnose; Colletotichim fragariae (ICMP18596), the pathogenic bacterium of strawberry anthracnose; Rhizoctonia solani Kühn (CCTCC NO: M20242043), the pathogenic bacterium of corn root rot; Pestalogiopsis sp. Colletotichum (CGMCC 3.15418), the pathogenic bacterium of mango leaf blight; Colletotichum gloeosporioides (ATCC 16330), the pathogenic bacterium of mango anthracnose; Fusarium oxysporum f. sp. cubense Race 4 (NRRL 36102), the 4th physiological race of banana wilt. All pathogenic fungi were legally obtained from the Banana and Plantain Cultivation Physiology and Ecology Experiment of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. This experiment was conducted with the permission of the other party.

[0083] The target bacteria screened were used to conduct a plate broad-spectrum experiment by the four-point confrontation method. First, the isolated actinomycetes were placed at four points 2.5 cm from the center of the PDA medium. After culturing at 28 °C for 3 days, when the actinomycetes had grown, a 5-mm-diameter fungal cake of different pathogenic bacteria was inoculated at the center point of the medium. It was placed in a biochemical incubator for 7 days, and the experiment was repeated three times.

[0084] The results are as Figure 4 shown. For the 7 selected pathogenic bacteria, compared with 7 untreated pathogenic bacteria, it was found that the antibacterial rate of Streptomyces SX-6 against Ciuvularia fallax was 46.43%, against Colletotichum musae was 50%, against Colletotichim fragariae was 40.54%, against Rhizoctonia solani Kühn was 53.15%, against Pestalogiopsis sp. Colletotichum was 30.3%, against Colletotichum gloeosporioides was 28.79%, and against Fusarium oxysporum f. sp. cubense Race 4 was 57.86%. The results showed that strain SX-6 had broad-spectrum antibacterial activity.

[0085] 5 Properties of the crude extract of Streptomyces SX-6

[0086] 5.1 Fermentation of Streptomyces SX-6 and preparation of the crude extract

[0087] The strain was fermented in large quantities in a soybean powder medium under optimized conditions to obtain 20 L of fermentation broth, which was mixed with an equal volume of absolute ethanol and continued to be placed in a shaker at 28 °C and 180 rpm for extraction for 3 days. After extraction, the mycelium was filtered with filter paper, and the filtrate was distilled under reduced pressure to 500 ml at 40 °C using a rotary evaporator (N-1300, EYELA, Shanghai, China) to obtain the crude fermentation extract.

[0088] The fermented crude extract was extracted with 95% ethanol, and the filtrate was obtained by filtering with filter paper. The filtrate was rotary evaporated and concentrated to 500 ml, and macroporous resin adsorption was carried out to obtain the substances adsorbed on the column. After elution with methanol of different concentrations (the volume concentrations of methanol were 50%-70%-80%-90%-100% in sequence), the eluate with a methanol concentration of 90% was collected and combined. The eluate was rotary evaporated and then freeze-dried to obtain the crude extract for standby.

[0089] 5.2 Plate broad-spectrum experiment of crude extract

[0090] Pathogenic bacteria: Ciuvularia fallax (ATCC 12017), the pathogenic bacterium of banana long-shaped spot disease; Colletotichum musae (ACCC 31244), the pathogenic bacterium of banana anthracnose; Colletotichim fagariae (ICMP18596), the pathogenic bacterium of strawberry anthracnose; Rhizoctonia solani Kühn (CCTCC NO: M20242043), the pathogenic bacterium of corn root rot; Pestalogiopsis sp. Colletotichum (CGMCC3.15418), the pathogenic bacterium of mango leaf blight; Colletotichum gloeosporioides (ATCC 16330), the pathogenic bacterium of mango anthracnose; Fusarium oxysporum f. sp. cubense Race4 (NRRL 36102), the 4th physiological race of banana wilt; Colletotichum acutatim (ATCC56836), the first type of mango anthracnose. All pathogenic fungi were legally obtained from the Banana Cultivation Physiology and Ecology Experiment of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. This experiment was carried out with the permission of the other party.

[0091] The extracted crude extract was freeze-dried and dissolved in dimethyl sulfoxide solution to prepare a stock solution with a crude extract concentration of 20 mg / ml. The PDA medium was mixed with the crude extract solution to pour plates (50 ml of PDA plus 500 μl of the stock solution of the crude extract), and different pathogenic bacteria cakes with a diameter of 5 mm were inoculated at the center point of the medium. It was placed in a biochemical incubator for 7 days (cultured at a constant temperature of 28 °C), and the experiment was repeated three times.

[0092] The results are as Figure 5As shown in the figure, the antibacterial rate of the crude extract against Mycosphaerella musicola was 72.9%, against Colletotrichum musae was 45.49%, against Colletotrichum fragariae was 72.63%, against Fusarium graminearum was 51.88%, against Xanthomonas campestris pv. mangiferaeindicae was 70.93%, against Colletotrichum gloeosporioides was 54.31%, against Fusarium oxysporum f. sp. cubense race 4 was 40.35%, and against Colletotrichum gloeosporioides strain 1 was 80.51%. The results showed that the secreted metabolites of Streptomyces SX-6 had antagonistic effects against multiple phytopathogenic fungi.

[0093] 5.3 Effects of the crude extract on the mycelial growth and morphology of Colletotrichum musae

[0094] The toxicity of the crude extract of the test strain against C. musae was determined by the mycelial growth rate method. The crude extract was thoroughly mixed with autoclaved PDA solid medium to obtain PDA plates with final concentrations of the crude extract of 200, 100, 50.00, 25.00, 12.50, 6.25, and 3.125 μg / mL. A C. musae fungal disc (5 mm in diameter) was inoculated in the center of the PDA plate, and the control was a PDA plate supplemented with an equal volume of aqueous solution. The plates were incubated in a constant temperature incubator at 28 °C for 7 days, and the experiment was set with three biological replicates. The colony diameter was measured by the cross method, and a linear regression was established by the least squares method to establish a toxicity regression equation to calculate the median lethal concentration EC50 of the crude extract, with a value of 42.048 μg / mL.

[0095] Prepare a PDA plate with a concentration of 4 × EC 50 (4 times of EC 50 , the same below) (the crude extract solution was mixed with PDA by the mixing method). A 5-mm-diameter Colletotrichum gloeosporioides fungal disc was inoculated at the center point of the PDA plate, and dimethyl sulfoxide of the same concentration was used as the control. After culturing at 28 °C for 5 days, the mycelial edges of the treated and control groups were cut with a sterile scalpel, and the effects of the crude extract treatment on the structure and morphology of the mycelium were observed under SEM (Zeiss, Germany) and an inverted microscope (cellcutplus, Germany).

[0096] The results are as Figure 6 shown. Under SEM, the mycelial morphology of the control group was round, and plump spores were scattered around the mycelium. However, the mycelium treated at a concentration of 4 × EC50 was shrunk, deformed, and even broken, and the spores scattered around it also showed shrinkage and even rupture. The same results were also observed under the inverted microscope. Robust and uniformly structured substrate mycelium was observed in the control group, while in the treated group, the substrate mycelium showed deformation and swelling, and a part of the mycelium even dissolved, indicating that the extract could significantly inhibit the growth of C. musae and destroy the structure and function of its substrate mycelium.

[0097] 5.4 Effects of the crude extract on the spore germination and spore morphology of Colletotrichum musae

[0098] (1) Spore germination experiment

[0099] Preparation of spore suspension: 5 ml of 0.05% Tween solution was dropped onto the Colletotrichum plate that had been growing for 7 - 10 days. The mycelium was mashed and scraped with a sterilized iron spoon to make it well - mixed. The filtrate was aspirated with a pipette tip with a cut end and filtered through a funnel lined with four layers of wiped paper. 100 μl of the filtrate was taken for counting under an optical microscope. Dilution was carried out according to the situation, and finally a spore suspension of 10 6 CFU / ml was obtained. 100 μl of the spore suspension was mixed with 2 ml of sterile water, and the crude extract dissolved in dimethyl sulfoxide was added to a final concentration of 1×, 2×, 4×, 8×, and 16×EC 50 . The control group was treated with dimethyl sulfoxide. All the liquids were transferred to 5 - ml centrifuge tubes and incubated at 28 °C for 24 h. Each treatment was repeated three times. 30 μl of the liquid was taken onto a cover glass, and the spore germination was observed under an optical microscope. Randomly counting 100 spores as one field, repeating five fields, and calculating the spore germination rate. The spore germination rate of the control group was required to be greater than 90%.

[0100] The results are as Figure 7 shown. Compared with the control group, when the spores of Colletotrichum musae were at a crude extract concentration of 4×EC 50 , they were greatly inhibited. At a concentration of 8×EC 50 , they hardly germinated, and at 16×EC 50 , there was no germination. The crude extract could inhibit the spore germination of Colletotrichum musae at other concentrations.

[0101] (2) Experiment on the effect of the crude extract on spore morphology

[0102] Prepare a spore suspension of 10 6 CFU / ml. Take 100 μl of the spore suspension and 1 ml of sterile water into a 2 - ml centrifuge tube, add the crude extract to a concentration of 4×EC 50 . Use the dimethyl sulfoxide solution as a control and incubate in a biochemical incubator at 28 °C for 6 hours. Take 40 μl of the mixture onto a slide, air - dry it in a laminar flow hood, and observe the effect of the crude extract on the spore structure under SEM.

[0103] The results are as Figure 8 shown. The spores treated in the control group (Control) were round and plump, while the spores in the treatment groups Treatment (b) and (c) were shrunken and ruptured. This indicates that the extract can damage the spore structure of C. musae and make it lose its function.

[0104] 6 Fruit experiment

[0105] Select banana fruits with uniform maturity, consistent size and no disease, plates containing Colletotrichum musae (20 - 25), 5 plastic storage boxes, 75% ethanol, several amounts of sterile water, filter paper, and several amounts of toilet paper. Use a sterile 2 - millimeter punch to make 5 holes with a depth of 2 millimeters on the banana fruits. Inject 30 μl of extracts with different concentrations (1×, 2×, 4×, and 8× EC 50 ) into each wound. Use an equal - volume 10% dimethyl sulfoxide solution (DMSO) as a control. Group the banana fruits into groups of six and place them in a laminar flow hood for disinfection (put these banana fruits into 75% ethanol for 2 minutes, wash them with sterile water for 1 minute, and then air - dry). Air - dry them in the laminar flow hood, and stick 2 - mm Colletotrichum musae fungal discs on the punched holes. Select six banana fruits for each treatment and store them in an artificial climate chamber (Yongjie Technology Instrument Co., Ltd., Shanghai, China) at 28 °C and 85% relative humidity.

[0106] (1) Resistance to Colletotrichum musae

[0107] After culturing for 7 days, take photos of the banana fruits and calculate the lesion area. Use SEM to detect the fruit wounds. The results are as Figure 9 shown. For the banana fruits in the 4xEC 50 treatment group, the lesion diameter changed from 0.47 ± 0.05 cm to 0.78 ± 0.19 cm, and for the control group, the lesion diameter changed from 0.91 ± 0.25 cm to 2.09 ± 0.41 cm. The lesions of the Colletotrichum musae pathogen on the banana fruits in the treatment group were significantly smaller than those in the control group. Phenotypically, it can be seen that the treated banana fruits were more resistant to the infection of Colletotrichum musae than the untreated ones.

[0108] (2) Enzyme activity assay

[0109] To analyze the possible mechanism by which the extract maintains fruit quality, using the DMSO - treated banana peel as a control, the activities of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), triacontanol (TA), and vitamin C (VC) at the punched holes of the banana peel were measured after treating the banana fruits with extracts at four concentrations (1×, 2×, 4×, and 8× EC 50 ) for 7 days. The results are as Figure 10 shown. As the concentration of the extract increased, the activities of the three enzymes POD, PPO, and CAT all showed an upward trend. Among them, it increased successively in POD, and in the enzyme activities of PPO and CAT, the enzyme activities in the treatment group were higher than those in the control group. This also better illustrates that the extract of Streptomyces SX - 6 increased the defensive enzyme activities of the banana peel. However, it had little effect on the two active components TA and VC.

[0110] (3) Effect of the extract on the invasion process of C. musae into the banana peel

[0111] To further study whether the extract inhibits pathogenic growth, dimethyl sulfoxide was used as a control, and the pericarp tissues of the control group (CK) and the 4×EC50 treatment group were sampled. The invasion of C. musae hyphae at the inoculated fruit site was observed using a scanning electron microscope, as Figure 11 shown. In the 4×EC50 group, the extract significantly inhibited the growth of the pathogen after treatment. The germinated spores were scattered around the pores of the treated fruit, and the pathogen was in a defensive state under the treatment of the extract, which was in the early stage of banana anthracnose at this time. In the control group, the pathogen hyphae penetrated the banana pericarp and further invaded the fruit, and it was already in the incidence stage of banana anthracnose.

[0112] In the experiment of treating banana fruits with the extract of Streptomyces sp. SX-6, the lesion diameter of the treatment group was significantly smaller than that of the control group, and the defensive enzyme activity of the banana pericarp was also significantly higher than that of the control group. Further electron microscopy observations found that a large number of C. musae hyphae had adhered to the vicinity of the control group, and a large number of spores also existed on the surface of the banana pericarp under the electron microscopy field of view. However, the hyphae and spores of the C. musae pathogen in the treatment group were relatively scarce under the electron microscopy field of view. In summary, Streptomyces sp. SX-6 can produce a potent antibacterial substance to combat C. musae. At the same time, it can also stimulate the self-defense response of bananas and enhance the ability of banana fruits to resist external biological stresses.

[0113] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to this utility are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A Streptomyces, characterized in that, Named Streptomyces sp., it is registered and preserved in the Guangdong Microbial Culture Collection Center with the preservation number GDMCC No: 65917.

2. The fermentation broth of the Streptomyces as claimed in claim 1.

3. The ethanol extract of the fermentation broth of the Streptomyces as claimed in claim 1.

4. The crude extract of the Streptomyces as claimed in claim 1, wherein the crude extract is the substance adsorbed on the column after the ethanol extract of claim 3 is filtered through macroporous resin and eluted with methanol of different concentrations, and the eluate is concentrated and dried.

5. A preparation, characterized in that, Containing the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4.

6. Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for antagonizing Mycosphaerella musicola, and / or Colletotrichum musae, and / or Colletotrichum acutatum, and / or Fusarium oxysporum f. sp. niveum, and / or Colletotrichum gloeosporioides Penz. f. sp. mangiferae (1), and / or Phyllosticta capitalensis, and / or Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4.

7. Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for controlling diseases caused by Mycosphaerella musicola, and / or Colletotrichum musae, and / or Colletotrichum acutatum, and / or Fusarium oxysporum f. sp. niveum, and / or Colletotrichum gloeosporioides Penz. f. sp. mangiferae (1), and / or Phyllosticta capitalensis, and / or Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4.

8. Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for causing the hyphae of Colletotrichum musae to shrink, deform and break.

9. Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for inhibiting the spore germination of Colletotrichum musae; or Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for causing the spores of Colletotrichum musae to shrink and rupture.

10. Use of the Streptomyces as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the ethanol extract as claimed in claim 3, or the crude extract as claimed in claim 4, or the preparation as claimed in claim 5 in the preparation of a preparation for increasing the activities of peroxidase, and / or polyphenol oxidase, and / or catalase in banana peel; or Use of the Streptomyces according to claim 1, or the fermentation broth according to claim 2, or the ethanol extract according to claim 3, or the crude extract according to claim 4, or the preparation according to claim 5 in the preparation of a preparation for inhibiting the growth of Colletotrichum musae.

11. The application according to claim 10, wherein The preparation enhances the activities of peroxidase, and / or polyphenol oxidase, and / or catalase in banana peel but does not affect the activities of triacontanol, and / or vitamin C.

Images