A new strain of streptomyces antagonizing a broad spectrum of plant pathogenic fungi and its use

Abstract

The present application provides a kind of streptomyces, named Streptomyces sp., is registered and preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No:65917.The present application also provides fermentation broth of the streptomyces and ethanol extract and crude extract of fermentation broth thereof.The new species of streptomyces and its crude extract and the like of the present application have broad-spectrum antibacterial activity, have good antagonism to many pathogenic bacteria such as banana long-shaped spot fungus and banana anthracnose fungus, and can make banana anthracnose fungus hyphae wrinkle, deform, break, inhibit banana anthracnose fungus spore germination, make banana anthracnose fungus spore germination wrinkle, break, inhibit banana anthracnose fungus growth, and can also improve peroxidase, polyperoxidase, CAT activity and the like in banana peel, is a potential biological agent for preventing and treating anthracnose disease, stem rot disease and the like, has broad development space, and has good development and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain, in particular to a new species of Streptomyces antagonizing a broad spectrum of plant pathogenic fungi and application thereof. BACKGROUND

[0002] Banana (Musa spp.) is an important fruit and food crop in tropical and subtropical regions. However, banana fruits are extremely susceptible to anthracnose disease after harvest, which leads to a decrease in fruit quality and damage to the fruits. Currently, there are three methods to prevent the occurrence of banana anthracnose disease: physical preservation, chemical control and biological control. The commonly used physical preservation method is low-temperature storage and transportation and controlled atmosphere preservation. The optimal growth temperature of the pathogen is 25-28℃. Under low-temperature conditions, the pathogen cannot grow normally, and the spore morphology adapts to the adverse environment. From the mechanism, low temperature inhibits the spore germination and mycelial growth of the pathogen. Appropriate ultraviolet treatment of the fruits can inhibit the number of anthracnose on the peel and kill the spores of the pathogen, while not changing the quality of the fruits. Chemical control has the characteristics of high efficiency and broad spectrum. There are many chemical agents commonly used to control anthracnose disease. For example, exogenous application of salicylic acid (SA) and jasmonic acid (JA). SA is a phenolic compound widely present in various plants. Spraying SA has been studied for disease control on postharvest fruits. 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 can easily make the pathogen resistant, and the leakage of chemical pesticides and other agents can cause irreversible damage to the environment and affect the balance of soil microecology.

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

[0004] The present application aims to overcome the deficiencies in the prior art, and provides a new species of Streptomyces antagonizing a broad spectrum of plant pathogenic fungi and application thereof.

[0005] The first aspect of the present application provides a Streptomyces, named Streptomyces sp. (test number: SX-6), which is deposited in the Guangdong Microbial Culture Collection Center and has a deposit number of GDMCC No: 65917 and a deposit date of February 19, 2025.

[0006] The second aspect of the present application provides a fermentation liquor of the Streptomyces according to the first aspect of the present application.

[0007] The third aspect of the present application provides an ethanol extract of the fermentation liquor of the Streptomyces according to the first aspect of the present application.

[0008] The fourth aspect of the present application provides a crude extract of the Streptomyces according to the first aspect of the present application, which is obtained by filtering the ethanol extract according to the third aspect of the present application through a macroporous resin column, adsorbing the substance on the column, and eluting the substance with methanol of different concentrations, and then drying the eluate.

[0009] The fifth aspect of the present application provides a preparation containing the Streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application.

[0010] The sixth aspect of the present application provides use of the Streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for antagonizing Mycosphaerella musicola, and / or Mycosphaerella musicola, and / or Mycosphaerella fragariae, and / or Fusarium verticillioides, and / or Colletotrichum gloeosporioides, and / or Mycosphaerella musicola, and / or Fusarium oxysporum f. sp. cubense race 4.

[0011] The seventh aspect of the present application provides use of the Streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for preventing and treating diseases caused by Mycosphaerella musicola, and / or Mycosphaerella musicola, and / or Mycosphaerella fragariae, and / or Fusarium verticillioides, and / or Colletotrichum gloeosporioides, and / or Mycosphaerella musicola, and / or Fusarium oxysporum f. sp. cubense race 4.

[0012] The eighth aspect of the present application provides use of the streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for causing the mycelium of Colletotrichum musae to wrinkle, deform and break.

[0013] The ninth aspect of the present application provides use of the streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for causing the spores of Colletotrichum musae to wrinkle and break.

[0014] The tenth aspect of the present application provides use of the streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for improving the activities of peroxidase (POD), and / or polyphenol oxidase (PPO), and / or catalase (CAT) in the banana peel.

[0015] The preparation improves the activities of peroxidase, and / or polyphenol oxidase, and / or catalase in the banana peel but does not affect the activities of tricosanol (TA) and / or vitamin C (VC).

[0016] The eleventh aspect of the present application provides use of the streptomyces according to the first aspect of the present application, or the fermentation liquor according to the second aspect of the present application, or the ethanol extract according to the third aspect of the present application, or the crude extract according to the fourth aspect of the present application, or the preparation according to the fifth aspect of the present application in the preparation of a preparation for inhibiting the growth of Colletotrichum musae.

[0017] The novel streptomyces SX-6 and the crude extract thereof and the like of the present application have broad-spectrum antibacterial activity, have good antagonistic effect on Mycosphaerella musicola, Colletotrichum musae, Colletotrichum acutatum, Fusarium decemcellulare, Colletotrichum gloeosporioides, Mycosphaerella musicola, Colletotrichum gloeosporioides, and / or Fusarium oxysporum f. sp. cubense race 4, can cause the mycelium of Colletotrichum musae to wrinkle, deform and break, inhibit the spore germination of Colletotrichum musae, cause the spores of Colletotrichum musae to wrinkle and break, inhibit the growth of Colletotrichum musae, and can improve the activities of peroxidase, polyphenol oxidase and CAT in the banana peel, and is a potential biological preparation for preventing and treating anthracnose disease, fusarium wilt disease and the like, has broad development space and good development and application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

[0023] Figure 6 Effects of crude extract of Streptomyces SX-6 on hyphal growth and morphology of C. musae.

[0024] Figure 7 Effects of crude extract of Streptomyces SX-6 on spore germination of C. musae.

[0025] Figure 8 Effects of crude extract of Streptomyces SX-6 on spore morphology of C. musae.

[0026] Figure 9 Effects of crude extract of Streptomyces SX-6 on banana fruit infection of C. musae, A is banana fruit lesion change, and B is comparison of lesion diameter of C. musae pathogen.

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

[0028] Figure 11 Effects of crude extract of Streptomyces SX-6 on C. musae invasion process of banana peel. DETAILED DESCRIPTION

[0029] The present application will be further described below with reference to the drawings and specific examples, so as to better understand the present application. In the examples, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not mentioned are all conventional products that can be commercially available.

[0030] 1 Isolation of actinomycetes from soil

[0031] Soil samples were collected from the rhizosphere of plants in the tropical rain forest of Bawangling, Changjiang Li Autonomous County, Hainan Province, China. The collected soil was manually cleaned of large debris and hard clumps, then dried at room temperature in a fume hood for 3 days. Subsequently, the soil was ground in a mortar and passed through a three-pass filter sieve (10-20 mesh). 1 g of soil was weighed on an electronic balance into a 10 ml centrifuge tube, diluted to 10 ml with sterile water to make a suspension. The same dilution was made to 10 -2 and 10 -3 ml suspensions. Subsequently, 100 μl of each dilution was pipetted onto SCA, PDA and Gause No. 1, respectively, and spread onto the surface of the Petri dish with a spreader bar to avoid water spots. The plates were placed in an inverted position in a 28°C incubator for 1-4 weeks. Different strains were selected according to color, growth time, mycelium morphology and colony size onto new YE medium for strain isolation and purification. The pure strains were preserved in 30% glycerol and stored in a -80°C ultra-low temperature freezer.

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

[0033] Primary screening: The isolated strains were screened for antagonistic activity using the four-point confrontation method. First, 2.5 cm from the center of the PDA medium, four points were each placed with an isolated actinomycete. After 3 days of incubation at 28°C, a 5 mm diameter mycelial disc of the banana anthracnose pathogen (Colletotrichum musae) was placed in the center of the medium. The plates were incubated at 28°C for 7 days in a biochemical incubator. The test was repeated three times. The results showed that 84 strains exhibited antibacterial activity (see Figure 1 above).

[0034] Secondary screening: The active antagonistic bacteria obtained from the primary screening were fermented in a soybean flour medium for 7 days, then the fermentation broth was filtered through a sterile 0.22 μm filter to obtain sterile fermentation broth. A puncher was used to punch four holes in the PDA plate at a distance of 2.5 cm from the center point, the four holes were radially symmetric around the center point, and 200 μl of the filtered sterile fermentation broth was added to each hole. The banana anthracnose pathogen was placed in the center of the PDA, and the plates were incubated in a biochemical incubator at 28°C and 85% humidity. A Streptomyces strain SX-6 with strong activity and suspected to be a new species was selected (see Figure 1 below).

[0035] 3 Identification of actinomycetes

[0036] 3.1 Morphological characteristics

[0037] The morphological characteristics of the antagonistic bacteria on various media were observed, including aerial hyphae, color of suberaneous hyphae, color and size of colonies. The results showed that the aerial hyphae of the antagonistic bacteria on ISP2 medium was cream, the suberaneous hyphae was brownish yellow, no soluble pigment was produced, and the growth condition was excellent; on ISP4 medium, the aerial hyphae was cream, the suberaneous hyphae was light yellow, no soluble pigment was produced, and the growth condition was good; on ISP5 medium, the aerial hyphae was powder white, the suberaneous hyphae was light yellow, no soluble pigment was produced, and the growth condition was good; on ISP6 medium, the aerial hyphae was yellowish white, the suberaneous hyphae was brownish yellow, no soluble pigment was produced, and the growth condition was excellent; on ISP7 medium, the aerial hyphae of the antagonistic bacteria was white, the suberaneous hyphae was brownish yellow, no soluble pigment was produced, and the growth condition was good.

[0038] 3.2 Scanning electron microscope (SEM) morphological observation

[0039] The selected antagonistic bacteria were cultured on PDA medium for 10-14 days, and the colonies were carefully separated from the medium with a sterile syringe. Flat and thin colonies were selected as much as possible, and a 0.5 cm square piece of the colony was cut off. After treatment, it was fixed with isoamyl acetate, and after completion, it was dried in a sterile clean bench. Finally, the morphology of the mycelium and spores was observed by scanning electron microscope (Zeiss Sigma 500 / VP, Germany). The results are shown in Figure 2 The mycelium of the antagonistic strain was interlaced, and the spores were suspended thereon, and the spores were short rod-shaped.

[0040] 3.3 Determination of physiological and biochemical characteristics

[0041] The physiological and biochemical characteristics of the antagonistic bacteria were identified according to the Handbook of Systematic Identification of Common Bacteria and Systematics of Actinomycetes.

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

[0043] Strain SX-6 was inoculated into different media, and the growth condition of the strain was observed after 7-14 days of culture at 28°C. The results are shown in Table 2. Strain SX-6 grew on all five media, grew rapidly and well on ISP2, ISP6 and ISP7 media, and did not produce soluble pigment on the five media. The colonies were mostly powder and wrinkled, the aerial hyphae was mostly white, and the suberaneous hyphae was mostly yellow.

[0044] Table 2

[0045]

[0046] Note: “++” represents good growth, and “+” represents growth on the medium.

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

[0048] The test strains were inoculated into culture medium containing different carbon sources and nitrogen sources, and were cultured at 28°C for 7-14 days, and the growth was observed. The negative control was set. The results are shown in Table 3.

[0049] Table 3

[0050]

[0051]

[0052] (3) Antibiotic experiment

[0053] A single colony of well-grown actinomycetes was inoculated into YE liquid medium, and was cultured at 28°C with 180 r / min shaking for 3 days. A small amount of bacterial liquid was counted under an optical microscope, and was then diluted to 1x10 6 CFU / mL by aseptic technique. 100 ul of spore liquid was uniformly coated on a YE solid culture medium plate, and four antibiotic filter paper pieces were inoculated at four points 2.5 cm away from the plate, and the experiment was repeated three times. The plates were incubated at 28°C for 7 days, and whether an antibacterial circle was produced around the filter paper pieces was observed. If yes, it indicated that the actinomycetes were sensitive to the antibiotic, and vice versa. The results are shown in Table 4.

[0054] Table 4

[0055]

[0056]

[0057] Note: "+" means sensitive to the antibiotic, and "-" means not sensitive;

[0058] Sensitivity to the antibiotic means that the antibiotic has inhibitory effect on the actinomycetes, and vice versa.

[0059] (4) Other physiological and biochemical indexes

[0060] pH experiment: The pH values of the liquid culture medium were adjusted to 4, 5, 6, 7, 8, 9 and 10, respectively. The test strains were inoculated into the culture medium with different pH values, and other culture conditions were ensured to be consistent. The strains were cultured at 28°C with shaking, and were observed once a week. After four weeks of culture, the growth of the strains in each culture medium was determined. The upper limit and lower limit of the pH value at which the strains could grow and the optimal pH value were determined.

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

[0062] Esterase (Tween 40, Tween 60) experiment: After preparing the esterase culture medium, sterilize the separately packaged Tween 40 and 60 together, mix the Tween with the culture medium to plate. Inoculate the strains on the plate, and incubate for one to two weeks. Observe the plate. If a fuzzy halo is produced around the colonies, it is positive, otherwise, it is negative.

[0063] Urease test: Inoculate the strains on the urease culture medium, and incubate at 28°C for 4 days. Observe whether the culture medium changes color. Test the ability of the test strains to produce urease. The culture medium changes to pink for positive, and no color change is negative.

[0064] Nitrate reduction: Inoculate the test strains in the nitrate reduction culture medium, and incubate at 28°C for 7 and 14 days. Use the un-inoculated culture medium as a control. Add a little of the 7-day and 14-day culture liquid to the test tube, and add one drop of A liquid and B liquid. The control is also added. When the solution turns pink, rose red, orange, or brown, it is positive for nitrate reduction. If no red color appears, add one or two drops of diphenylamine reagent. If it is blue, the reduction is negative. If it is not blue, it is still treated as positive.

[0065] Gelatin experiment: Inoculate the strains to be identified in test tubes containing gelatin culture medium, and then incubate at 28°C. Observe the liquefaction of the 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] Phosphorus dissolving test: Prepare phosphorus dissolving bacteria culture medium, sterilize and pour the plate. Inoculate the actinomycetes in the plate, and use the un-inoculated plate as a control. After incubating at 28°C for one week, observe the culture medium plate. If there is a transparent circle, it indicates that it has the ability to dissolve phosphorus, otherwise, it does not.

[0067] Nitrogen fixation test: Prepare Azotobacter culture medium for nitrogen fixation bacteria, sterilize and pour the plate. Inoculate the actinomycetes in the plate, and use the un-inoculated plate as a control. After incubating at 28°C for one week, observe the culture medium plate. If there is a transparent circle, it indicates that it has the ability to fix nitrogen, otherwise, it does not.

[0068] Cellulose-decomposing: prepare sodium carboxymethyl cellulose culture medium, sterilize and pour into plates. Inoculate actinomycetes into the plates, and incubate at 28℃ for 7 days. Take out the plates, pour 1g / L Congo red solution into the plates to immerse the bacterial liquid, and then pour 1 mol / L NaCl solution into the plates to decolorize for 30 min. Observe whether transparent circles are generated on the plates. If transparent circles are generated, it is positive, indicating that the strain has cellulase-producing ability, otherwise, it is negative.

[0069] Starch hydrolysis: test whether the strain has amylase activity. Inoculate actinomycetes into starch hydrolysis culture medium, and incubate at 28℃ for 5-7 days. Then, add iodine solution around the colonies to immerse the bacterial hyphae, and detect. If transparent circles are generated, it is positive, otherwise, it is negative.

[0070] The results are shown in Table 5.

[0071] Table 5

[0072]

[0073]

[0074] Note: "+" is positive, "-" is negative

[0075] Combined with its culture characteristics, physiological and biochemical characteristics and mycelial morphology, the strain SX-6 shows typical Streptomyces morphological characteristics.

[0076] 3.4 Establishment of Streptomyces 16S phylogenetic tree

[0077] The total DNA of actinomycetes was extracted by Bioteke bacterial genome DNA rapid extraction kit. The genome of the tested actinomycetes was used as a template for PCR amplification. The universal primer of actinomycetes 16S rRNA was used, the upstream primer was 5'-AGAGTTTGATCCTGGCTCAG-3', and the downstream primer was 5'-TACGGCTACCTTGTTACGACTT-3'. The PCR reaction system was 5ul. The PCR amplification program was pre-denaturation at 95℃ for 5min, denaturation at 94C for 1min, annealing at 55℃ for 1min, extension at 72℃ for 2min, post-extension at 72℃ for 10min, storage at 4℃, and 32 cycles of denaturation, annealing and extension, followed by subsequent processes.

[0078] Agarose gel (1%) was configured, and after waiting for 15 min for solidification, the PCR product (5 μl) and Marker (5 μl) were spotted. The gel was placed in an electrophoresis tank containing 1x TAE electrophoresis buffer, and electrophoresis was performed at 150 V for 25 min. After electrophoresis, the results were observed under ultraviolet-visible spectrophotometer at 254 nm. If there was a PCR band at 1400 bp, the PCR product was sent to Shanghai Biotechnology Co., Ltd. for 16S rRNA sequencing.

[0079] After the sequencing company returned the data, the obtained sequence was uploaded to EzBioCloud and GenBank database for homologous comparison and download of 16S rRNA gene sequences with higher similarity. The strains were selected for multiple comparison analysis, and MEGA11.0 software was used to construct a phylogenetic tree by Neighbor-Joining method, with Bootstrap self-check value set to 1000.

[0080] After the 16S sequencing returned results, the sequence was uploaded to Ezbiocloud for comparison, and it was found that the closest relative species was Streptomyces orinoci NRRL B-3379, but when the whole genome sequence was compared, the ANi value was 80.78%, and the DDH value was 29.70%. The similarity between the two was low, so the sequence was uploaded to ncbi to establish a phylogenetic tree, and the whole genome sequences of multiple strains with close genetic relationship were downloaded and compared. The final comparison result was low similarity. The results are shown in Figure 3 . It was determined to be a new species, named Streptomyces sp., and sent to Guangdong Microbial Culture Collection Center for preservation, with the preservation number GDMCC No: 65917, the preservation time February 19, 2025, and the preservation address 5th Floor, No. 59, Building, 100, Martyrs' Road, Guangzhou.

[0081] 4 Plate broad-spectrum experiment of actinomycetes

[0082] Pathogenic bacteria: banana long-shaped spot pathogen Ciuvularia fallax (ATCC 12017); banana anthracnose pathogen Colletotichum musae (ACCC 31244); strawberry anthracnose pathogen Colletotichim fagariae (ICMP 18596); corn root rot pathogen Rhizoctonia solani Kühn (CCTCC NO: M20242043); mango leaf blight pathogen Pestalogiopsis sp. Colletotichum (CGMCC 3.15418); mango anthracnose pathogen gloeosporioides (ATCC 16330); banana wilt 4th physiological race pathogen Fusarium oxysporum f. sp. cubense Race 4 (NRRL 36102). All pathogenic fungi were obtained legally from the Banana Cultivation Physiological Ecology Experiment of the Institute of Tropical Biosciences, Chinese Academy of Tropical Agricultural Sciences. This experiment was used with the permission of the other party.

[0083] The target bacteria selected were used to perform plate broad-spectrum experiments using the four-point confrontation method. First, 2.5 cm apart at the center of the PDA medium were placed into the isolated actinomycetes. After 3 days of culture at 28°C, the actinomycetes were grown, and a 5 mm diameter bacterial cake of different pathogenic bacteria was inoculated at the center of the medium. The biochemical incubator was placed for 7 days, and the experiment was repeated three times.

[0084] The results are shown in Table 1. Figure 4 According to the control of the 7 strains of pathogenic bacteria without treatment, it was found that the inhibition rate of Streptomyces SX-6 against banana long-shaped spot pathogen was 46.43%, against banana anthracnose pathogen was 50%, against strawberry anthracnose pathogen was 40.54%, against corn root rot pathogen was 53.15%, against mango leaf blight pathogen was 30.3%, against mango anthracnose pathogen was 28.79%, and against banana wilt 4th physiological race pathogen was 57.86%. The results showed that the strain SX-6 had broad-spectrum antibacterial activity.

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

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

[0087] The strain was fermented in soybean powder medium under the optimized conditions to obtain 20 L of fermentation broth. An equal volume of anhydrous ethanol was added, and the mixture was further placed on a shaking bed at 28°C and 180 rpm for extraction for 3 days. After extraction, the bacterial cells were filtered with filter paper, and the filtrate was distilled to 500 ml under reduced pressure at 40°C using a rotary evaporator (N-1300, EYELA, Shanghai, China). The fermentation crude extract was obtained.

[0088] The fermentation crude extract was extracted with 95% ethanol, filtered with filter paper to obtain filtrate, the filtrate was concentrated to 500 ml by rotary evaporation, and subjected to macroporous resin adsorption to obtain the material adsorbed on the column. After elution with different concentrations of methanol (methanol concentrations were 50%-70%-80%-90%-100% by volume), the eluate with 90% methanol was combined. The eluate was freeze-dried after rotary evaporation to obtain the crude extract, which was ready for use.

[0089] 5.2 Broad-spectrum experiment of crude extract

[0090] Pathogenic fungi: banana long-shaped spot pathogen Ciuvularia fallax (ATCC 12017); banana anthracnose pathogen Colletotrichum musae (ACCC 31244); strawberry anthracnose pathogen Colletotrichum mfagariae (ICMP18596); corn root rot pathogen Rhizoctonia solani Kühn (CCTCC NO: M20242043); mango leaf blight pathogen Pestalogiopsis sp. Colletotrichum (CGMCC3.15418); mango anthracnose pathogen gloeosporioides (ATCC 16330); banana wilt 4 physiological race Fusarium oxysporum f. sp. cubense Race 4 (NRRL 36102); mango anthracnose pathogen Colletotrichum acutatim (ATCC 56836). All pathogenic fungi were obtained legally from the Institute of Tropical Biological Technology, Chinese Academy of Tropical Agricultural Sciences. This experiment was used with the permission of the other party.

[0091] After freeze-drying the extracted crude extract, it was dissolved in dimethyl sulfoxide solution to prepare a mother liquor with a crude extract concentration of 20 mg / ml. PDA medium was mixed with the crude extract extract to pour plates (50 ml PDA plus 500 ul crude extract mother liquor), and different pathogenic fungus cakes with a diameter of 5 mm were inoculated in the center of the medium. The biochemical incubator was placed for 7 days (28°C constant temperature culture), and the test was repeated three times.

[0092] The results are as follows Figure 5As shown, the crude extract exhibited an inhibition rate of 72.9% against *Streptomyces simonii*, 45.49% against *Hydrocotyle spp.*, 72.63% against *Hydrocotyle spp.*, 51.88% against *Hydrocotyle spp.*, 70.93% against *Hydrocotyle spp.*, 54.31% against *Hydrocotyle spp.*, 40.35% against *Fusarium wilt* race 4 of banana, and 80.51% against *Hydrocotyle spp.* race 1 of mango. These results indicate that the secreted metabolites of *Streptomyces SX-6* have antagonistic effects against multiple plant pathogenic fungi.

[0093] 5.3 Effects of crude extract on the growth and morphology of banana anthracnose mycelium

[0094] The mycelial growth rate method was used to determine the toxicity of C. musae in the crude extract of the test strain. The crude extract was thoroughly mixed with hyperthermically sterilized PDA solid medium to obtain PDA plates with final extract concentrations of 200, 100, 50.00, 25.00, 12.50, 6.25, and 3.125 μg / mL. C. musae mycelial discs (5 mm in diameter) were inoculated into the center of the PDA plates. A control was a PDA plate supplemented with an equal volume of aqueous solution. The plates were incubated at 28°C for 7 days, with three biological replicates. Colony diameter was measured using the cross-multiplication method. A linear regression was established using the least squares method to calculate the toxicity regression equation, which yielded a median lethal concentration (EC50) of 42.048 μg / mL.

[0095] The prepared concentration is 4×EC 50 (EC 50 The crude extract was mixed with PDA plates (4 times the concentration of the control group) using a cross-linking method. Anthrax mycelial cakes with a diameter of 5 mm were inoculated at the center of the PDA plates, with a control group using the same concentration of dimethyl sulfoxide. After 5 days of incubation at 28°C, the edges of the hyphae from both the treated and control groups were cut off with a sterile scalpel. The effects of the crude extract treatment on the structure and morphology of the hyphae were observed using SEM (Zeiss, Germany) and an inverted microscope (Cellcutplus, Germany).

[0096] The results are as follows Figure 6 As shown in the figure, under SEM, the hyphae in the control group were rounded with plump spores scattered around them, while the hyphae treated with 4×EC50 concentration were shrunken, deformed, and even broken, and the scattered spores around them also shrunken or even ruptured. The same results were observed under an inverted microscope; the control group showed robust and uniformly structured hyphae, while the treated group showed deformed and swollen hyphae, with some hyphae even dissolving. This indicates that the extract can significantly inhibit the growth of *C. musae* and disrupt the structure and function of its hyphae.

[0097] 5.4 Effect of crude extract on spore germination and spore morphology of C. musae

[0098] (1) Spore germination experiment

[0099] Preparation of spore suspension: 5 ml of 0.05% Tween solution was dropped on the 7-10 day old C. musae culture plate, and the mycelium was crushed and scraped with a sterile iron spoon to make it fully mixed. The filtrate was filtered with a funnel with four layers of clean paper, and 100 μl of the filtrate was counted under an optical microscope. It was diluted 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 1x, 2x, 4x, 8x and 16xEC 50 The control group was treated with dimethyl sulfoxide, and all the liquids were moved to 5 ml centrifuge tubes and treated at 28°C for 24 h, with three repeats for each treatment. 30 μl of the liquid was taken on a cover glass, and spore germination was observed under an optical microscope. 100 spores were randomly counted as one field, and five fields were repeated. The spore germination rate was calculated, and the spore germination rate of the control group was required to be greater than 90%.

[0100] The results are shown in Table 1. Figure 7 Compared with the control group, the spores of the banana anthracnose pathogen were greatly inhibited at a crude extract concentration of 4xEC 50 , could hardly germinate at a concentration of 8xEC 50 , and had no germination at a concentration of 16xEC 50 . The crude extract could inhibit the germination of the spores of the banana anthracnose pathogen at other concentrations.

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

[0102] Preparation of a spore suspension of 10 6 CFU / ml. 100 μl of the spore suspension and 1 ml of sterile water were taken into a 2 ml centrifuge tube, and the crude extract was added to a concentration of 4xEC 50 . The dimethyl sulfoxide solution was used as a control, and the mixture was cultured at 28°C in a biochemical incubator for 6 h. 40 ul of the mixture was taken onto a glass slide, which was dried in a super-clean bench and then observed under SEM to observe the effect of the crude extract on the spore structure.

[0103] The results are shown in Table 2. Figure 8 The spores in the control group (Control) were round and full, while the spores in the treatment groups Treatment (b) and (c) were shriveled and broken. This shows that the extract can destroy the spore structure of C. musae, making it lose its function.

[0104] 6 Fruit experiment

[0105] Select maturity uniform, size consistent and no disease of banana fruit, containing banana anthracnose plate (20-25), plastic preservation box 5, 75% ethanol, sterile water, filter paper, several pieces of toilet paper. On the banana fruit with sterile 2 mm puncher punch 5 holes with a depth of 2 mm. Each wound injection of 30 μl different concentrations of extract (1 x, 2 x, 4 x and 8 x EC 50 ) control. Using equal volume of 10% dimethyl sulfoxide solution (DMSO) as a control. The banana fruit with every 6 as a group, and put into the clean bench for disinfection (put these banana fruit in 75% ethanol for 2 minutes, then washed with sterile water for 1 minute, then air dry) in the clean bench air dry, 2 mm anthracnose fungus cake paste in the punch. Each treatment selected six banana fruit, respectively, at 28 ℃ and 85% relative humidity stored in artificial climate chamber (Yongjie Technology Instrument Co., Ltd., Shanghai, China).

[0106] (1) against banana anthracnose fungus

[0107] After 7 days of culture, take pictures of banana fruit, and calculate the lesion area. SEM was used to detect fruit wounds. The results are shown in Figure 9 . The lesion diameter of banana fruit treated with 4 x EC 50 changed from 0.47 ± 0.05 cm to 0.78 ± 0.19 cm, and the lesion diameter of the control group changed from 0.91 ± 0.25 cm to 2.09 ± 0.41 cm. The banana anthracnose lesion of the treatment group was significantly smaller than that of the control group. Phenotypically, the treated banana fruit was more resistant to banana anthracnose than the untreated banana fruit.

[0108] (2) enzyme activity determination

[0109] To analyze the possible mechanism of extract maintaining fruit quality, dimethyl sulfoxide treated banana peel was used as a control, and the activities of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), triacontanol (TA), and vitamin C (VC) in the banana peel after 7 days of treatment with four concentrations (1 x, 2 x, 4 x and 8 x EC 50 ) extract were determined. The results are shown in Figure 10 . With the increase of extract concentration, the activities of POD, PPO and CAT enzymes showed an upward trend. Among them, the POD activity increased successively, and the enzyme activities of PPO and CAT were higher in the treatment group than in the control group. This better explained that the extract of Streptomyces SX-6 improved the defense enzyme activity of banana peel. However, the activities of TA and VC were not significantly affected.

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

[0111] In order to further study whether the extract inhibits pathogenic growth, the peel tissue of the control group (CK) and the 4xEC50 treatment group was sampled, and the invasion of C. musae hyphae at the inoculated fruit site was observed by scanning electron microscopy, as shown in Figure 11 In the 4xEC50 group, the growth of the pathogenic bacteria was significantly inhibited after the extract treatment, the spores germinated and spread around the pores of the treated fruit, and the pathogenic bacteria were in a defensive state under the treatment of the extract, which was in the early stage of banana anthracnose. In the control group, the hyphae of the pathogenic bacteria penetrated the banana peel and further infected the fruit, which was in the onset stage of banana anthracnose.

[0112] In the experiment of banana fruit treated with the extract of Streptomyces SX-6, the lesion diameter of the treatment group was significantly smaller than that of the control group, and the defense enzyme activity of the banana peel was also significantly higher than that of the control group. Further electron microscope observation found that the control group was attached by a large number of banana anthracnose hyphae, and a large number of spores existed on the surface of the banana peel under the electron microscope observation field, while the hyphae and spores of the banana anthracnose pathogen in the treatment group were relatively rare under the electron microscope observation field. In summary, Streptomyces SX-6 can produce potent antibacterial substances to resist C. musae, and at the same time, it can stimulate the defense response of banana and enhance the ability of banana fruit to resist external biological stress.

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

Claims

1. A Streptomyces sp. deposited at Guangdong Microbial Culture Collection Center and assigned accession number GDMCC No: 65917.

2. A fermentation broth of the Streptomyces sp. of claim 1.

3. A formulation characterized in that, A composition comprising the Streptomyces sp. of claim 1 or the fermentation broth of claim 2.

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