A strain of Streptomyces jakartaensis and its application in the control of sweet potato black spot disease

Abstract

The invention discloses a strain of Streptomyces jakartaensis and its application in the prevention and treatment of sweet potato black spot, belonging to the technical field of plant pathogen prevention and treatment. Streptomyces djakartensis ) MEPS155, the deposit number is CGMCC No.27199, the deposit date is April 25, 2023, and the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The Streptomyces jakarta MEPS155 of the present invention can significantly inhibit the growth of sweet potato black spot pathogens, and significantly reduce the diameter and depth of sweet potato black spot lesions; the Streptomyces jakarta MEPS155 of the present invention can be used for the prevention and treatment of sweet potato black spot, and will not affect the quality of sweet potatoes, avoiding environmental pollution caused by pesticide abuse, pesticide residues threatening human health, pathogen resistance and other problems, and has broad application prospects in the prevention and control of sweet potato diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pathogenic bacteria prevention and control, and specifically relates to a strain of Streptomyces jakartaensis and application thereof in the prevention and control of sweet potato black spot disease. Background Art

[0002] Sweet potato black spot is one of the more serious fungal diseases of sweet potatoes during the growth and storage period. The pathogen of this disease is Ceratocystis fimbriata Ellis & Halsted, which belongs to the Ascomycota, Pyromycetes, Coccidioides, Ceratocystis, and Ceratocystis. Sweet potato black spot occurs in all potato regions in the world. At the same time, furan terpenoid toxic substances such as fusarone produced in the diseased potatoes can cause poisoning or even death to humans and livestock. The relevant prevention and control of this disease is urgent.

[0003] Chemical pesticides have a wide spectrum of fungicides, low cost, and rapid effects, making great contributions to the prevention and control of crop diseases and insect pests. However, the problems of drug residues, food safety, and environmental pollution caused by the irrational use of chemical pesticides are becoming increasingly apparent, and even lead to the increase of insect pest resistance. It is urgent to develop new green and efficient prevention and control measures in production. In recent years, biological control has become one of the important measures for green prevention and control of plant diseases and insect pests. The analysis of biological control mechanisms is of great significance for the efficient use of biological control factors. The interaction between plant roots and soil microorganisms plays an important role in plant disease resistance, stress tolerance, nutrient absorption and growth. Most studies have shown that plant rhizosphere soil microorganisms can resist the invasion of pathogens through antagonism, competition, heavy parasitism, and promotion of plant growth and development. Among actinomycetes, Streptomyces is a beneficial microorganism that exists in large quantities in the soil and plant rhizosphere. In terms of plant disease prevention and control, rhizosphere Streptomyces inhibits the growth of pathogens by producing active substances such as antibiotics, antimicrobial peptides, and enzymes. Microbial metabolites are key mediators of the interaction between the environment and organisms, and contain a large number of natural products with novel structures and good functions. Metabolites of Streptomyces play an important role in drug development, environmental ecology, etc. So far, 75% of the antibiotics discovered are produced by Streptomyces, and more than 120 are actually used in production. In terms of plant fungal disease prevention and control, the antimicrobial metabolites produced by rhizosphere Streptomyces have a series of advantages such as broad spectrum, high efficiency, and no cross-resistance, and play an important role in plant growth and agricultural ecological development.

[0004] At present, the research on the pathogenic mechanism of sweet potato black spot and the mechanism of sweet potato resistance to this pathogen is not perfect. Bacteria with potential antagonism to this pathogen provide an alternative method to improve soil and plant health. Streptomyces in rhizosphere soil actinomycetes are high-quality candidates. According to existing literature reports, they produce a wide range of antimicrobial compounds. Therefore, isolating sweet potato rhizosphere soil actinomycetes and exploring the potential mechanism of interaction between actinomycetes and sweet potato black spot will enhance our understanding of plant microbiota, and also provide theoretical support for the subsequent research and development of transformation and formation of bacterial preparations to effectively improve the occurrence of sweet potato black spot. Summary of the invention

[0005] The present invention aims to address the deficiencies of the prior art and provides a strain of Streptomyces djakartensis MEPS155 and application thereof in the prevention and treatment of postharvest black spot disease of sweet potatoes.

[0006] One of the purposes of the present invention is to provide a strain of Streptomyces djakartensis

[0007] MEPS155, the deposit number is CGMCC No.27199, the deposit date is April 25, 2023, the depositor is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] The second object of the present invention is to provide the use of the above-mentioned Streptomyces djakartensis MEPS155 in preventing and controlling black spot disease pathogens, and the use of Streptomyces djakartensis MEPS155 in preventing and controlling black spot disease pathogens.

[0009] In one embodiment of the present invention, the Streptomyces djakartensis MEPS155 can be applied in the form of bacterial suspension, cell-free fermentation filtrate or volatile gas.

[0010] Preferably, the preparation method of the Streptomyces jakarta MEPS155 bacterial suspension is:

[0011] Take the strain of Streptomyces jakarta MEPS155, pick up a small amount of bacteria with a bamboo stick under sterile conditions, inoculate it into the sterilized ISP2 liquid culture medium, and culture it in the dark at 28°C and 180rpm for 4 days; after centrifugation at 12000r / min for 10min, take the bacterial precipitate, add sterile water to adjust the concentration of the bacterial suspension to OD 600 =0.8~1.0, then we get;

[0012] Preferably, the preparation method of the cell-free fermentation filtrate of Streptomyces jakartaensis MEPS155 is:

[0013] Take the strain of Streptomyces jakarta MEPS155, pick up a small amount of bacteria with a bamboo stick under sterile conditions, inoculate it into the sterilized ISP2 liquid culture medium, and culture it in the dark at 28°C and 180rpm for 4 days; after centrifugation at 12000r / min for 10min, take the supernatant, and filter and sterilize the supernatant through a 0.22μm filter membrane under sterile conditions to prepare a cell-free fermentation filtrate.

[0014] The third object of the present invention is to provide the use of the above-mentioned Streptomyces djakartensis MEPS155 in the preparation of a product for preventing and controlling sweet potato black spot disease.

[0015] The fourth object of the present invention is to provide a microbial preparation, the active ingredient of which is the above-mentioned Streptomyces djakartensis MEPS 155. The biological control preparation is sprayed on the harvested sweet potatoes.

[0016] In the early stage, the inventors isolated multiple rhizospheric bacteria from the rhizospheric soil of healthy sweet potatoes in Nanning, Guangxi, screened and verified the antagonistic strains by the plate confrontation method, and found that one of the rhizospheric bacteria had a significant inhibitory effect on the black spot pathogen (Long-beaked Conchocystis). Subsequently, the obtained rhizospheric bacteria MEPS155 were identified mainly from the perspectives of morphology and molecular biology, thereby obtaining Jakarta Streptomyces MEPS155.

[0017] Beneficial effects: The Streptomyces jakarta MEPS155 provided by the present invention can effectively inhibit the pathogen of sweet potato black spot disease, can be used for biological control of sweet potatoes, and reduce the occurrence of sweet potato black spot disease after harvest; in addition, the method provided by the present invention belongs to the field of biological control, and the plant rhizosphere bacteria will not affect the quality of sweet potatoes because they are attached to the plant rhizosphere, and can avoid the problems of excessive pesticide residues, environmental pollution, pathogen resistance, etc. caused by chemical control methods. Therefore, it is more green and environmentally friendly, can ensure the safety of sweet potato consumption, and has broad application prospects in the prevention and control of sweet potato post-harvest diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the morphological characteristics of strain MEPS155 in Example 1. A is the colony morphology after culturing on an ISP2 plate for 4 days; B is the hyphae morphology under a scanning electron microscope.

[0019] Figure 2 In Example 1, the strain MEPS155 has an antagonistic effect on the black spot pathogen on a plate, and A and B are the control group and the experimental group of the plate confrontation experiment, respectively.

[0020] Figure 3 Phylogenetic tree of strain MEPS155 in Example 1 constructed based on 16S rRNA gene.

[0021] Figure 4 Example 2 Inhibitory effect of cell-free fermentation filtrates of strain MEPS155 at different concentration gradients on sweet potato black spot.

[0022] Figure 5 The figure shows the inhibitory effect of the volatile gas produced by the strain MEPS155 in Example 3 on the growth of the black spot pathogen. A and B are the control group and the experimental group, respectively.

[0023] Figure 6 The biological control effect of strain MEPS155 on sweet potato black spot in Example 4 is shown in order from top to bottom: sterile water, pathogen, pathogen plus strain MEPS155 suspension, A is the display of sweet potato lesion diameter, and B is the display of sweet potato lesion depth. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1

[0028] Isolation, Screening and Identification of Streptomyces djakartensis MEPS155

[0029] 1. Isolation of rhizospheric soil bacteria from sweet potato of Guangxi

[0030] The rhizosphere bacteria MEPS155 of the present invention is a strain isolated from the rhizosphere soil of sweet potatoes from Nanning, Guangxi. The specific steps include: (1) air-drying the rhizosphere soil, placing it in a sterile plate, treating it in an oven at 100°C for 60 minutes, cooling it to room temperature, weighing 2g of the soil sample, dissolving it in 18mL of sterile water, and shaking it at 180rpm for 30 minutes at 28°C to make 10 -1 Soil suspension, then diluted to a concentration of 10 -2 Use a pipette to draw 150 μL of 10 -2The concentration dilution was added to the ISP2 plate and evenly spread with a coating rod. After standing for 15 minutes, the culture dish was inverted and placed in a 28°C incubator for 28 days. A single colony grown on the ISP2 plate was picked and inoculated into the same solid culture medium (ISP2) for four-zone line purification. After repeated purification and cultivation, the rhizosphere strain MEPS155 of the present invention was finally obtained. Figure 1 -A, the colony was white, round, opaque, with a raised and wrinkled surface. The strain was preserved in a -80°C refrigerator using the glycerol preservation method.

[0031] ISP2 solid culture medium: malt extract powder 10g / L, yeast extract powder 4g / L, glucose 4g / L, agar 20g / L.

[0032] 2. Plate confrontation test between sweet potato rhizosphere soil bacteria and black spot pathogen

[0033] The test pathogen was B. longibacterium spp.: provided by the Sweet Potato Research Institute of the Chinese Academy of Agricultural Sciences, isolated from diseased potatoes grown in Xuzhou, and later preserved in the Provincial Key Laboratory of Medicinal Plant Biotechnology of Jiangsu Normal University. The strain number is BMPZ13.

[0034] First, the 2mm bacterial cake of the pathogen was transferred to the center of the PDA culture medium, and then the MEPS155 strain was inoculated at a position 2.5cm away from both sides of the pathogen cake, with a length of 4cm, and repeated 3 dishes; in the control group, only the sweet potato black spot pathogen was inoculated without rhizospheric bacteria, and repeated 3 dishes. After dark culture at 28℃ for 5 days, the diameters of the pathogen colonies in the control group and the experimental group were measured using the cross method. Finally, the inhibition rate was calculated according to the following formula: Inhibition rate (%) = [(colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group] × 100. The results are shown in the attached figure. Figure 2 As shown, Figure 2 -A is the control group inoculated with pathogens alone; Figure 2 -B is the experimental group inoculated with strain MEPS155 and pathogens. The results showed that strain MEPS155 had a significant inhibitory effect on the growth of black spot pathogens on the plate, and its inhibition rate on sweet potato black spot pathogens was 41.46%.

[0035] PDA solid culture medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L.

[0036] 3. Identification of strain MEPS155

[0037] (1) Morphological characteristics:

[0038] The purified strain MEPS155 grew well on ISP2 solid medium. The colonies were white, round, opaque, and had raised and wrinkled surfaces (such as Figure 1-A), Gram staining positive. The scanning electron microscopic morphology of strain MEPS155 is as follows Figure 1 -B.

[0039] (2) Physiological and biochemical characteristics

[0040] The strain MEPS155 can produce acid by fermenting glucose. Its xylose fermentation reaction is positive, while its mannitol, urease, gelatin hydrolysis and VP reactions are all negative, as shown in Table 1.

[0041] Table 1 Some physiological and biochemical characteristics of strain MEPS155

[0042]

[0043] (3) Phylogenetic analysis of strain MEPS155 based on 16S rRNA gene

[0044] The total DNA of strain MEPF0303 obtained by separation and screening was extracted using the Novozyme bacterial extraction kit. 16S rDNA PCR amplification was performed using universal primers PA (5'-CAGAGTTTGATCCTGGCT-3') and PB (5'-AGGAGGTGATCCAGCCGCA-3'), and the amplification system was Premix Taq 12.5μL, PA 0.5μL, PB 0.5μL, DNA 0.5μL, ddH2O 12.5μL. PCR amplification conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 1min 45s, 30 cycles, 72℃ total extension for 10min. The amplified product was detected with 1% agarose gel, and the electrophoresis voltage was 135V for 25min. The PCR products were sent to Nanjing Branch of Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the NCBI database. The reference strains were selected according to the comparison results, and the phylogenetic tree was constructed using MEGA 11.0 software ( Figure 3 ), clarifying its taxonomic status. The 16S rRNA gene sequence of strain MEPS155 is shown in SEQ ID NO: 1. The phylogenetic tree results showed that strain MEPS155 and Streptomyces djakartensis were clustered into one branch with a support of 95%. Combining morphological characteristics and molecular methods, strain MEPS155 was identified as Streptomyces djakartensis.

[0045] Example 2

[0046] Effects of the cell-free fermentation filtrate of strain MEPS155 on the growth of black spot pathogen

[0047] The activated strain MEPS155 was inoculated into a triangular flask containing 200mL ISP2 liquid culture medium and shaken at 28℃ and 180r / min. After 4 days of shaking culture, the fermentation liquid of strain MEPS155 was centrifuged and the supernatant was filtered with a 0.22μm microporous filter membrane. The obtained filtrate was the cell-free fermentation filtrate. The fermentation filtrate was added to the PDA culture medium in proportion to make the final volume 200mL to make a poisonous plate, and then the pathogenic fungus cake with a diameter of 2mm was inoculated in the center of the plate. Ordinary PDA culture medium with only pathogens was used as a control. Three replicates were set for each treatment and cultured in a constant temperature incubator at 28℃. After 5 days of culture, the colony diameter was measured by the cross method, and the inhibition rate of 10%, 20%, 30%, 40% and 50% of the cell-free fermentation filtrate of Streptomyces jakarta MEPS155 against sweet potato black spot was calculated. Inhibition rate (%) = [(colony diameter of control group - colony diameter of treatment group) / colony diameter of control group] × 100, see Table 2.

[0048] Table 2 Inhibitory effect of cell-free fermentation filtrate of strain MEPS155 on black spot pathogens

[0049]

[0050] The results showed that (Table 2 and Figure 4 ), the cell-free fermentation filtrate of strain MEPS155 had a significant inhibitory effect on the growth of sweet potato black spot pathogen.

[0051] Example 3 Effect of volatile gases produced by strain MEPS155 on the growth of black spot pathogen

[0052] The partition plate method was used to investigate the effect of volatile gases of strain MEPS155 on the growth of black spot pathogens. The MEPS155 separated and screened in Example 1 was inoculated into the ISP2 solid culture medium on one side of the partition plate by streaking the plate, and then a 2 mm pathogen cake was inoculated into the center of the PDA solid culture medium on the other side, and sealed with Parafilm sealing film. The control group was not inoculated with MEPS155. Three replicates were set for each treatment and cultured in a constant temperature incubator at 28°C. When the pathogens in the control group grew all over one side of the plate, the colony diameter was measured by the cross method, and the inhibition rate (%) = [(colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group] × 100.

[0053] like Figure 5 As shown, the colony diameter of the pathogen treated with strain MEPS155 was 21.17±0.6009 mm, the colony diameter of the pathogen in the control group was 31.50±0.2887 mm, and the inhibition rate (%) was 32.81±1.908, indicating that the volatile gas produced by MEPS155 has a certain inhibitory effect on the growth of black spot disease pathogens.

[0054] Example 4

[0055] Biological Control of Sweet Potato Black Spot by Strain MEPS155

[0056] After strain MEPS155 was selected and fermented in ISP2 liquid medium for 4 days, it was centrifuged at 12000r / min for 10min, the supernatant was discarded, and the precipitated bacteria were diluted with sterile water to OD 600 = 0.8-1.0 and then prepare a bacterial suspension for use. Prepare a spore suspension (10 6 spores / ml) for standby use. The 25 tubers of tobacco yam purchased from a local supermarket were uniform in size, free of lesions and mechanical damage for the experiment. After the tubers were disinfected with 75% ethanol, three cavities (0.5 cm in diameter and 2 cm in depth) were punched out with a sterilized cork puncher. The first hole was a blank control, with 50 μL of sterile water added; the second hole was a CK group, with 50 μL of sterile water added; the third hole was an experimental group, with 50 μL of a suspension of strain MEPS155 added. After drying for one hour, 2 μL of a spore suspension of the long-beaked shell fungus was added to the second and third holes, respectively, and then cultured in the dark at 27°C for 15 days, with 3 replicates set for each group. The diameter and depth of the lesions were measured by the cross method, and the antibacterial rate (%) = [(the colony diameter (or depth) of the control group - the colony diameter (depth) of the treatment group) / the colony diameter (or depth) of the control group] × 100, see Table 3.

[0057] Table 3 Inhibition rate of strain MEPS155 on the diameter and depth of lesions caused by sweet potato black spot

[0058]

[0059] The results are shown in Table 3 and Figure 6 As shown: the lesion diameter of the control group was 8.5±0.5774mm, the lesion diameter of the bacterial suspension treatment group was 4.67±0.333mm, and the inhibition rate was 45.10±3.92%; the lesion depth of the control group was 13.67±0.8819mm, the lesion diameter of the bacterial suspension treatment group was 2.67±0.3333mm, and the inhibition rate was 80.49±2.44%, indicating that the strain MEPS155 has a good biological control effect on sweet potato black spot.

[0060] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A strain of Streptomyces jakartaensis ( Streptomyces djakartensis ) MEPS155, the deposit number is CGMCCNo.27199, the deposit date is April 25, 2023, and the depository is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. The Streptomyces jakartaensis of claim 1 ( Streptomyces djakartensis ) Application of MEPS155 in preventing and controlling black spot pathogens, wherein the plant black spot disease is sweet potato black spot disease.

3. The Streptomyces jakartaensis of claim 1 ( Streptomyces djakartensis ) Application of MEPS155 in the preparation of a product for preventing and controlling plant black spot disease, wherein the plant black spot disease is sweet potato black spot disease.

4. A microbial preparation, characterized in that: The active ingredient is the Streptomyces jakartaensis described in claim 1 ( Streptomyces djakartensis )MEPS155.

5. The microbial preparation according to claim 4, characterized in that The microbial agent is Streptomyces jakartaensis ( Streptomyces djakartensis ) The bacterial suspension of MEPS155 is prepared by: Under sterile conditions, use a bamboo stick to pick up the Streptomyces jakarta MEPS155 cells and inoculate them into the sterilized ISP2 liquid culture medium; culture conditions: temperature 28 ℃, dark culture under shaking condition of 180 rpm for 4 days; after centrifugation at 12000 r / min for 10 min, take the cell pellet, add sterile water to adjust the concentration of the bacterial suspension to OD600 = 0.8 ~ 1.

0.

6. The microbial preparation according to claim 4, characterized in that The microbial agent is Streptomyces jakartaensis ( Streptomyces djakartensis ) The cell-free fermentation filtrate of MEPS155 is prepared by: Under sterile conditions, the cells of Streptomyces jakarta MEPS155 were picked up with a bamboo stick and inoculated into the sterilized ISP2 liquid culture medium. The culture conditions were as follows: dark culture at 28 °C and shaking at 180 rpm for 4 days; after centrifugation at 12000 r / min for 10 min, the supernatant was taken and filtered through a 0.22 μm filter membrane under sterile conditions to prepare a cell-free fermentation filtrate.

7. The microbial preparation according to claim 4, characterized in that The microbial agent is Streptomyces jakartaensis ( Streptomyces djakartensis ) Volatile gases of MEPS155.

8. An application for preventing and controlling sweet potato black spot disease, comprising spraying the microbial preparation according to claims 5-7 on post-harvest sweet potatoes.

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