Enterobacter mori bacteriophage P38 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses enterobacter mori phage P38 and application thereof. Wherein the enterobacter mori bacteriophage P38 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.46285, and the preservation date is December 2, 2024. The enterobacter mori bacteriophage P38 shows a good inhibition effect on enterobacter mori 23LSFQ, can obviously improve yellowing and withering of tomato leaves caused by the enterobacter mori 23LSFQ, is wide in applicable temperature and pH range, and has high application value in the aspect of improving the yield and quality of tomato planting.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Enterobacter mori phage P38 and application thereof. Background Art

[0002] In 2024, researchers first reported that Enterobacter mori Enterobacter mori ) 23LSFQ can cause yellowing and wilting of tomato leaves (Ning X, Liu WT, Wang C, Tian YP. First Report of Enterobacter mori Causing Bacterial Wilt on Tomato in China. Plant Dis. 2024 Apr 30. doi:10.1094 / PDIS-01-24-0174-PDN. Epub ahead of print. PMID: 38687577). Leaves are the key organs for photosynthesis in tomatoes, and their health is directly related to the growth and development of tomatoes. When leaves turn yellow and wilt, the efficiency of photosynthesis will be significantly reduced, which will lead to a decrease in the synthesis of organic matter. This makes the nutrient and energy supply required for the growth of tomato plants insufficient, the growth rate slows down, the plants are short, and in severe cases, they even stop growing. Therefore, preventing and controlling the yellowing and wilting of tomato leaves is crucial to ensuring the yield and quality of tomato cultivation.

[0003] Among the many control methods, bacteriophages, as a natural biological control method, show unique advantages. Compared with traditional chemical pesticides, bacteriophages have high host specificity and can accurately identify and infect specific pathogens. This precision means that bacteriophages will not pollute the environment, nor will they leave residues in plants and soil, which is of great significance to the protection and sustainable development of the ecological environment. However, to date, there have been no reports of bacteriophages targeting Escherichia coli 23LSFQ.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a Bacillus mori phage P38 and its application, aiming to solve the problem of yellowing and wilting of tomato leaves caused by Bacillus mori.

[0006] The technical solution of the present invention is as follows: In the first aspect, a bacteriophage of Enterobacter mulberry is provided ( Enterobacter mori phage) P38, the Enterobacter mulberry phage P38 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNO.46285 and the deposit date of December 2, 2024.

[0007] In a second aspect, a phage composition is provided, wherein the phage composition comprises the Enterobacter mori phage P38 as described in the first aspect.

[0008] According to a preferred technical solution, the bacteriophage composition further comprises an agriculturally acceptable auxiliary material.

[0009] According to a preferred technical solution, the agriculturally acceptable auxiliary material is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

[0010] In a third aspect, there is provided a use of the Enterobacterium mori phage P38 as described in the first aspect or the phage composition as described in the second aspect in preventing and controlling yellowing and wilting of tomato leaves.

[0011] According to a preferred technical solution, when the Enterobacter mulberry phage P38 or the phage composition is used, the temperature of the system is 10-60°C.

[0012] According to a preferred technical solution, when the Enterobacter mulberry phage P38 or the phage composition is used, the pH of the system is 3-11.

[0013] Beneficial effects: The present invention isolates a Bacillus phage P38 having a good inhibitory effect on Escherichia coli from soil, and studies the biological characteristics, morphological observation and control effect evaluation of Bacillus phage P38. The research results show that the Bacillus phage P38 screened by the present invention exhibits a good inhibitory effect on Escherichia coli 23LSFQ, can significantly improve the yellowing and wilting of tomato leaves caused by Escherichia coli 23LSFQ, and has a wide applicable temperature and pH range, and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the plaque image of Enterobacter mori phage P38 in Example 1.

[0015] Figure 2 This is a transmission electron micrograph of the Enterobacter mori phage P38 in Example 2.

[0016] Figure 3 This is a graph showing the results of the thermal stability test of Enterobacter mori phage P38 in Example 4.

[0017] Figure 4 This is a graph showing the results of the pH stability measurement of the Enterobacter mori phage P38 in Example 5.

[0018] Figure 5 This is a graph showing the results of the ultraviolet sensitivity test of Enterobacter mori phage P38 in Example 6.

[0019] Figure 6 This is a graph showing the lysis kinetics curve of the Enterobacter mori phage P38 in Example 7.

[0020] Figure 7 This is a diagram showing the control effect of Enterobacter mulariae phage P38 on tomato wilt in Example 8.

[0021] Figure 8 This is a graph showing the effect of Enterobacter mulberry phage P38 on the incidence of tomato wilt in Example 8.

[0022] Fig. 9 It is a heat map of the genome alignment analysis of Enterobacter mori phage P38 in Example 9. DETAILED DESCRIPTION

[0023] The present invention provides Enterobacter mori phage P38 and applications thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. The embodiment of the present invention provides a bacteriophage of Enterobacter mulberry ( Enterobacter mori phage ) P38, the Enterobacter mulberry phage P38 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNO.46285 and the deposit date of December 2, 2024.

[0024] Specifically, the embodiment of the present invention isolated the Enterobacter mulberry phage P38 from the soil sample of the potato black leg soil sample plot in Zhangjiakou City, Hebei Province. After genome comparison analysis, the maximum similarity between it and the existing Enterobacter mulberry phage is about 80%, which is a new Enterobacter mulberry phage. The study found that the Enterobacter mulberry phage P38 screened by the present invention showed a good inhibitory effect on Enterobacter mulberry 23LSFQ, and can significantly improve the yellowing and wilting of tomato leaves caused by Enterobacter mulberry 23LSFQ, reduce the incidence of wilt disease, and help improve the yield and quality of tomato planting.

[0025] An embodiment of the present invention provides a phage composition, wherein the phage composition includes the Enterobacter mori phage P38 as described above.

[0026] In one embodiment, the bacteriophage composition further comprises an agriculturally acceptable adjuvant.

[0027] In one embodiment, the agriculturally acceptable adjuvant is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

[0028] The embodiments of the present invention provide the use of the Enterobacter mori phage P38 or the phage composition described above in preventing and controlling yellowing and wilting of tomato leaves.

[0029] In one embodiment, the yellowing and wilting of tomato leaves are caused by Enterobacter morifolius 23LSFQ.

[0030] In one embodiment, when the Escherichia coli phage P38 or the phage composition is used, the temperature of the system is 10-60°C, for example, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, but not limited thereto; preferably, the temperature of the system is 10-40°C, for example, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, but not limited thereto; further preferably, the temperature of the system is 20°C or 30°C, but not limited thereto.

[0031] In one embodiment, when the Escherichia coli phage P38 or the phage composition is used, the pH of the system is 3 to 11, for example, it can be 3, 4, 5, 6, 7, 8, 9, 10, 11, but not limited thereto; preferably, the pH of the system is 7 to 11, for example, it can be 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, but not limited thereto; further preferably, the pH of the system is 7, but not limited thereto.

[0032] The present invention will be further described below by means of specific examples.

[0033] In the following embodiments, the materials involved are specifically as follows: The tomato variety is Jinpeng No. 1. Enterobacter mori )23LSFQ was isolated from diseased tomato plants in Longquan Town, Shandong Province in October 2023. Enterobacter mori phage ) P38 was isolated from soil samples from the potato blackleg soil sample plot in Zhangjiakou City, Hebei Province in September 2023. It was deposited in the General Microbiology Center of China Culture Collection on December 2, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.46285.

[0034] Nutrient agar (NA) medium was prepared with water and included: 10 g / L tryptone, 0.5 g / L sodium chloride (NaCl), 3 g / L beef extract, and 20 g / L agar.

[0035] Nutrient broth (NB) medium was prepared with water and included: tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, and beef extract 3 g / L.

[0036] The phage buffer is prepared with water, including: sodium chloride (NaCl) 87.4 g / L, magnesium chloride hexahydrate (MgC12·6H2O) 20.4 g / L, calcium chloride (CaC12) 2.2 g / L, tris(hydroxymethyl)aminomethane (Trisbase) 60.6 g / L, and adjusted to the desired pH with hydrochloric acid (HCl) or sodium hydroxide (NaOH).

[0037] Yeast malt extract (YME) medium was prepared with water and included 5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract, and 10 g / L glucose.

[0038] The preparation method of the suspension of Enterobacter mulberry 23LSFQ includes: taking out Enterobacter mulberry 23LSFQ from a -80°C strain library in a laboratory, placing it in a -20°C refrigerator overnight to slowly thaw. After thawing, diluting the bacterial solution and spreading it on an NA medium, and placing it in a 28°C constant temperature incubator for 2-3 days. Subsequently, picking a single colony for plate streaking purification, repeating 3-4 times to ensure that a pure strain is obtained. Finally, the purified single colony of Enterobacter mulberry 23LSFQ is inoculated into an NB medium, placed on a shaker with a rotation speed of 200 rpm / min, and cultured at 28°C for 2-3 days to obtain a suspension of Enterobacter mulberry 23LSFQ.

[0039] Example 1 In this example, Enterobacter mulberry phage P38 was separated and purified as follows: Soil samples were collected from potato black leg soil sample plots in Zhangjiakou City, Hebei Province. Weigh 3.5 g of soil sample, place in a conical flask, add 500 μL of Enterobacter mulberry 23LSFQ suspension and 6 mL of phage buffer (pH = 7.5), mix thoroughly and place in a shaker at 200 rpm / min, and amplify at 28 ° C for 2-3 days. After amplification, the sample was allowed to stand for 20-30 min, and the supernatant was filtered with a 0.22 μm sterile filter to obtain the sample filtrate. Take 500 μL of sample filtrate and 500 μL of Enterobacter mulberry 23LSFQ suspension, mix well, let stand for 5-10 min, add 7 mL of 0.7% (w / v) water agar (45-50 ° C), mix evenly and pour into NA medium for plating. Take another 500μL of Escherichia coli 23LSFQ suspension, add 7mL of 0.7% (w / v) water agar (45~50℃), mix well, pour into NA medium, let stand for 10min, and after the water agar solidifies, take 10μL of sample filtrate for spotting. Place the plate and spot plate in a 28℃ constant temperature incubator for 1~2 days to observe whether plaques are formed. If plaques appear, it means that there are phages in the sample filtrate that can lyse Escherichia coli 23LSFQ, and further purification can be performed.

[0040] Pick up a single plaque formed by plating, soak it in phage buffer (pH=7.5), crush the agar block with plaques and vortex to ensure that the phage is completely released into the buffer, filter it with a 0.22μm sterile filter, and spread the obtained phage filtrate after gradient dilution. Culture it at 28℃ for 1~2 days. Repeat the above steps 3~4 times to obtain the purified Enterobacter mulberry phage P38. The plaques of the purified Enterobacter mulberry phage P38 are as follows Figure 1 As shown. Figure 1 It can be seen that the plaques of Enterobacter mori phage P38 are transparent and round, uniform in size, and about 3.7 mm in diameter.

[0041] The purified Escherichia coli phage P38 was co-cultured with Escherichia coli 23LSFQ for 3 days by double-layer agar plate method, and the upper agar layer was harvested, crushed, and mixed with an appropriate amount of phage buffer (pH = 7.5). The supernatant was centrifuged (10000g, 15 minutes, 4°C) and filtered through a 0.22μm sterile filter to obtain an Escherichia coli phage P38 suspension, which was stored at 4°C for future use. Another portion of the Escherichia coli phage P38 suspension was mixed with 50% (v / v) glycerol, transferred to a cryovial, and placed in a -80°C refrigerator for long-term storage.

[0042] Example 2 In this example, the morphology of Enterobacter mulberry phage P38 was observed, as follows: The morphology of Enterobacter mulberry phage P38 was observed using a transmission electron microscope (TEM). The sample preparation process was as follows: in a suspension of Enterobacter mulberry phage P38 (~10 8 pfu / mL), PEG8000 (Merck millipore, USA) was added to a final concentration of 10% (w / v) and NaCl to a final concentration of 0.5M, and precipitation was allowed to proceed overnight at 4°C. Subsequently, the mixture was centrifuged at 4°C and 11,000 g for 30 minutes, and the precipitate was resuspended in phage buffer (pH=7.5). After the resuspension was extracted with equal volumes of chloroform three times, the prepared Enterobacter phage P38 was dropped onto a copper mesh carbon support membrane (Beijing Dajikeyi Technology Co., Ltd., China) for adsorption, and then filter paper was used to absorb the unadsorbed Enterobacter phage P38. When performing negative staining, the Enterobacter phage P38 adsorbed on the carbon membrane was treated with 1% (w / v) phosphotungstic acid solution (Sigma, USA) for 1 minute, and then the excess staining solution was absorbed with filter paper. The morphology of Enterobacter phage P38 was observed using TEM at an accelerating voltage of 60 kV, as shown in the following figure. Figure 2 As shown. Figure 2It can be seen that the head of the Enterobacter mulberry phage P38 is hexagonal and has a short rod-shaped tail, and its head diameter is about 56~58nm.

[0043] Example 3 This example determines the host range of Enterobacter mulberry phage P38, as follows: The assay was performed using 34 random strains collected from soil in different regions of China. The process was as follows: First, the target strain was cultured in YME medium for 5 days to obtain the target bacterial solution. Then, 500 μL of the target bacterial solution was mixed with 5 mL of YME medium containing 0.45% (w / v) agar and poured onto a YME medium plate containing 2% (w / v) agar. After that, 10 μL of the Escherichia coli phage P38 suspension (~10 8 PFU / mL) and ensure that the plate contains the target strain. The inoculated plate was cultured at 37°C for 2 days, and then the formation of plaques was observed. The results are shown in Table 1. In Table 1, "+" indicates that the lysis can be achieved, and "-" indicates that the lysis cannot be achieved.

[0044] Table 1 Host spectrum of Enterobacter mulberry phage P38

[0045] As shown in Table 1, the Enterobacterium mori phage P38 can lyse the Enterobacterium mori ZRIMU1424 and the Enterobacter mori 23LSFQ, but has no lyse effect on other strains. This indicates that the Enterobacter mori phage P38 has strong host specificity.

[0046] Example 4 In this example, the thermal stability of Enterobacter mulberry phage P38 was determined as follows: The suspension of Enterobacter mulberry phage P38 (~5×10 9 The titers of PFU / mL were measured after culturing at 10, 20, 30, 40, 50, 60 and 70°C for 12 hours to observe the effect of different temperatures on the stability of Enterobacter mulberry phage P38. Figure 3 As shown. Figure 3 It can be seen that the activity of Escherichia coli phage P38 is relatively stable in the temperature range of 10~40℃, reaches the highest activity at 20 to 30℃, the titer decreases significantly at 50℃, and completely loses activity at 70℃.

[0047] Example 5 In this example, the pH stability of Enterobacter mulberry phage P38 was determined as follows: 100 μL of Enterobacter mulariae phage P38 suspension (~5 × 10 9 PFU / mL) was mixed with 900 μL of phage buffer with a specific pH value (pH = 1, 3, 5, 7, 9, 11, or 13) to obtain a mixture. These mixtures were incubated at 28°C for 12 hours to evaluate the survival of Escherichia coli phage P38 under different pH conditions. The results are shown in Figure 4 As shown. Figure 4 It can be seen that Escherichia coli phage P38 can survive in conditions with a pH value of 3 to 11, and its activity reaches its highest at a pH value of 7; its activity is completely lost under extremely acidic (pH=1) and extremely alkaline (pH=13) conditions. In addition, the activity of Escherichia coli phage P38 in alkaline environments (pH=9 and 11) is significantly higher than its activity in acidic environments (pH=3 and 5).

[0048] Example 6 In this example, the ultraviolet sensitivity of Enterobacter mulberry phage P38 was determined as follows: The suspension of Enterobacter mulberry phage P38 (~5×10 9 The samples were placed 12 cm away from two TUV 15W / G15 T8 lamps with a wavelength of 253.7 nm to simulate the UV irradiation conditions in the natural environment. The experiment was carried out at room temperature and the pH of the Escherichia coli phage P38 suspension was 7.5. The samples were exposed to UV light for 1, 2, 3, 4, 5 and 6 minutes to explore the effect of different irradiation times on the stability of the phage. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that the Escherichia coli phage P38 has a low UV tolerance. Under ultraviolet light, it decreases steadily at the beginning, but after the activity stabilizes during 3 to 4 minutes, it decreases sharply again and finally loses its activity completely at 5 minutes.

[0049] Example 7 In this example, the lysis kinetic curve of Enterobacter mulberry phage P38 was measured, as follows: The concentration of Enterobacter mulberry 23LSFQ suspension was adjusted to 10 6 CFU / mL, and then cultured at 30℃ for 6 hours to promote spore germination, and obtain germinated host bacterial liquid. The treatment group mixed the germinated host bacterial liquid with the suspension of Enterobacter mulberry phage P38 at a multiplicity of infection (MOI) of 0.01 and added it to a 96-well plate, while the control group only added the germinated host bacterial liquid to the 96-well plate. The change in bacterial concentration was monitored by measuring the change in optical density (OD) at 600nm every hour, and the lysis kinetic curve was drawn according to the results. The results are shown in Figure 6 As shown. Figure 6 It can be seen that the bacterial concentration in the treatment group was lower than that in the control group, which indicates that the Escherichia mori phage P38 exhibited an inhibitory effect on Escherichia mori 23LSFQ.

[0050] Example 8 This example evaluates the control effect of Enterobacter mulariae phage P38 on tomato wilt, as follows: Tomato seeds were sown in pots with a diameter of 21 cm. After 30 days of cultivation, the bacteriophage control experiment was carried out. Four groups were set up in the experiment: 23LSFQ group, P38 group, 23LSFQ+P38 group and control group. 5 mL of Escherichia coli 23LSFQ suspension (10 8 cfu / mL) were sprayed evenly on the leaves of tomato plants; 5 mL of Enterobacter mulberry phage P38 suspension (10 6 pfu / mL) were evenly sprayed on the leaves of tomato plants; 23LSFQ+P38 group was first sprayed with 5mL of Escherichia coli 23LSFQ suspension (10 8 cfu / mL), and 5 mL of Enterobacter mulberry phage P38 suspension (10 6 pfu / mL); the control group was sprayed with 5mL of sterile water. After one week of the experiment, the growth of the plants was observed and recorded. Each treatment group contained 3 plants, and the experiment was repeated twice. The experimental results are shown in Figure 7 and Figure 8 As shown. Figure 7 and Figure 8 It can be seen that compared with the 23LSFQ group, the incidence of tomato wilt was significantly reduced in the P38+23LSFQ group after the addition of Enterobacter mulberry phage P38. Among them, the incidence of the 23LSFQ group was about 60%, while the incidence of the P38+23LSFQ group dropped to about 20%, a decrease of about 40%. At the same time, no disease was observed in the P38 group or the control group.

[0051] Example 9 In this example, whole genome sequencing and bioinformatics analysis of Enterobacter mori phage P38 were performed, as follows: The λ phage genomic DNA rapid extraction kit (Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number: ZP317) was used to extract genomic DNA from Enterobacter mulberry phage P38 according to its instructions. The genome was sequenced on the Illumina Novaseq PE150 platform (Shanghai Tanpu Biotechnology Co., Ltd.), and the full genome sequence size of Enterobacter mulberry phage P38 was measured to be 47478bp. In the NCBI database, BLAST was used to compare the similarity of Enterobacter mulberry phage P38 with other phages. The results showed that Enterobacter phage ( Enterobacteria phage )UABPhi20 (GenBank No.: NC_031019.1) and Salmonella phage ( Salmonella phage ) P22 (GenBank No.: NC_002371.2) has the highest similarity with Enterobacter thuringiensis phage P38, both showing 81.53% identity and 6% query coverage. Further, the similarity between Enterobacter thuringiensis phage P38 and other phage genomes was evaluated by DNA-DNA hybridization (DDH) and average nucleotide identity (ANI). In order to display these data more intuitively, the corresponding heat map was generated using R (http: / / www.r-project.org / ), as shown in the following figure. Fig. 9 shown. Fig. 9 In the figure, the DDH and ANI values ​​between the Enterobacter mulberry phage P38 and other phage genomes are displayed in the grid in the form of a percentage system, and the depth of the color reflects the size of the DDH and ANI values. The above results show that the Enterobacter mulberry phage P38 is a new type of phage.

[0052] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Enterobacterium mulberry phage ( Enterobacter mori phage ) P38, characterized in that, The Enterobacter mulberry phage P38 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCCNO.46285 and the deposit date December 2, 2024.

2. A bacteriophage composition, characterized in that: The phage composition comprises the Enterobacter mori phage P38 as claimed in claim 1.

3. The bacteriophage composition according to claim 2, characterized in that The bacteriophage composition further includes an agriculturally acceptable adjuvant.

4. The bacteriophage composition according to claim 3, characterized in that The agriculturally acceptable adjuvant is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

5. Use of the Enterobacterium mori phage P38 according to claim 1 or the phage composition according to any one of claims 2 to 4 in preventing and controlling yellowing and wilting of tomato leaves.

6. The use according to claim 5, characterized in that: When the Enterobacter mulberry phage P38 or the phage composition is used, the temperature of the system is 10-60°C.

7. The use according to claim 5, characterized in that: When the Enterobacter mulberry phage P38 or the phage composition is used, the pH of the system is 3-11.

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