Enterobacteria phage P38 and its applications

Abstract

The present invention relates to the field of microbial technology, and discloses Enterobacter mori phage P38 and its application. Among them, the Enterobacter mori phage P38 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC NO. 46285 and the deposit date of December 2, 2024. The Enterobacter mori phage P38 of the present invention shows a good inhibitory effect on Enterobacter mori 23LSFQ, can significantly improve the yellowing and withering of tomato leaves caused by Enterobacter mori 23LSFQ, and has a wide range of applicable temperatures and pH values, and has high application value in improving the yield and quality of tomato cultivation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to Enterobacter mori phage P38 and its applications. Background Art

[0002] In 2024, researchers first reported that Enterobacter mori Enterobacter mori (23LSFQ) could cause yellowing and withering 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 tomato photosynthesis, and their health is directly related to the growth and development of tomatoes. When the leaves show yellowing and wilting, the photosynthesis efficiency will be significantly reduced, resulting in a decrease in the synthesis of organic substances. This leads to insufficient supply of nutrients and energy required for the growth of tomato plants, slower growth rate, shorter plants, and even cessation of growth in severe cases. Therefore, preventing and controlling the yellowing and withering of tomato leaves is crucial for ensuring the yield and quality of tomato cultivation.

[0003] Among various prevention and control measures, phages, as a natural biological control method, exhibit unique advantages. Compared with traditional chemical pesticides, phages have a high degree of host specificity and can accurately identify and infect specific pathogens. This accuracy means that phages will not pollute the environment and will not leave residues in plants and soil, which is of great significance for the protection of the ecological environment and sustainable development. However, to date, there has been no report on phages against Enterobacter mori 23LSFQ.

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

[0005] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide Enterobacter mori phage P38 and its applications, aiming to solve the problem of yellowing and withering of tomato leaves caused by Enterobacter mori.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect, an Enterobacter mori phage ( Enterobacter mori phage)P38. The Enterobacter sanghuangphage P38 is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC NO. 46285 and the deposit date of December 2, 2024.

[0008] In a second aspect, a phage composition is provided. The phage composition comprises the Enterobacter sanghuangphage P38 as described in the first aspect.

[0009] In a preferred technical solution, the phage composition further comprises agriculturally acceptable excipients.

[0010] In a preferred technical solution, the agriculturally acceptable excipients are selected from one or more of a dispersant, a stabilizer, a filler, and a solvent.

[0011] In a third aspect, an application of the Enterobacter sanghuangphage P38 as described in the first aspect or the phage composition as described in the second aspect in the prevention and control of yellowing and withering of tomato leaves is provided.

[0012] In a preferred technical solution, when the Enterobacter sanghuangphage P38 or the phage composition is used, the temperature of the use system is 10 - 60 °C.

[0013] In a preferred technical solution, when the Enterobacter sanghuangphage P38 or the phage composition is used, the pH of the use system is 3 - 11.

[0014] Beneficial effects: In the present invention, an Enterobacter sanghuangphage P38 with good inhibitory effect on Enterobacter sanghuang is isolated from the soil, and biological characteristics research, morphological observation, and prevention and control effect evaluation of the Enterobacter sanghuangphage P38 are carried out. The research results show that the Enterobacter sanghuangphage P38 screened in the present invention shows good inhibitory effect on Enterobacter sanghuang 23LSFQ, can significantly improve the yellowing and withering of tomato leaves caused by Enterobacter sanghuang 23LSFQ, and has a wide range of applicable temperature and pH, with high application value. Description of the Drawings

[0015] Figure 1 It is the plaque map of Enterobacter sanghuangphage P38 in Example 1.

[0016] Figure 2 It is the transmission electron microscope image of Enterobacter sanghuangphage P38 in Example 2.

[0017] Figure 3 It is the thermal stability measurement result graph of Enterobacter sanghuangphage P38 in Example 4.

[0018] Figure 4 It is the pH stability measurement result graph of Enterobacter sanghuangphage P38 in Example 5.

[0019] Figure 5 It is a graph showing the results of the ultraviolet sensitivity assay of Enterobacter mori phage P38 in Example 6.

[0020] Figure 6 It is a graph showing the results of the lysis kinetics curve assay of Enterobacter mori phage P38 in Example 7.

[0021] Figure 7 It is a graph showing the control effect of Enterobacter mori phage P38 on tomato wilt in Example 8.

[0022] Figure 8 It is a graph showing the results of the effect of Enterobacter mori phage P38 on the incidence of tomato wilt in Example 8.

[0023] Figure 9 It is a heat map of the genomic alignment analysis of Enterobacter mori phage P38 in Example 9. Detailed implementation manners

[0024] The present invention provides Enterobacter mori phage P38 and its applications. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention is further described in detail below.

[0025] An embodiment of the present invention provides Enterobacter mori phage ( Enterobacter mori phage ) P38. The Enterobacter mori phage P38 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO. 46285 and the deposit date December 02, 2024.

[0026] Specifically, Enterobacter mori phage P38 was isolated from the soil sample of the potato blackleg disease plot in Zhangjiakou City, Hebei Province in the embodiment of the present invention. Through genomic alignment analysis, its maximum similarity with the existing Enterobacter mori phages is about 80%, and it is a new Enterobacter mori phage. It was found that the Enterobacter mori phage P38 screened in the present invention shows good inhibitory effects on Enterobacter mori 23LSFQ, can significantly improve the yellowing and withering of tomato leaves caused by Enterobacter mori 23LSFQ, reduce the incidence of wilt disease, and is beneficial to improving the yield and quality of tomato planting.

[0027] An embodiment of the present invention provides a phage composition, which includes the above-mentioned Enterobacter mori phage P38.

[0028] In one embodiment, the phage composition further includes agriculturally acceptable excipients.

[0029] In one embodiment, the agriculturally acceptable excipients are selected from one or more of dispersants, stabilizers, fillers and solvents.

[0030] The embodiments of the present invention provide the application of Enterobacter mori phage P38 or the phage composition as described above in the prevention and control of yellowing and withering of tomato leaves.

[0031] In one embodiment, the yellowing and withering of the tomato leaves are caused by Enterobacter mori 23LSFQ.

[0032] In one embodiment, when the Enterobacter mori phage P38 or the phage composition is used, the temperature of the use system is 10~60°C, for example, it can be 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, but not limited thereto; preferably, the temperature of the use system is 10~40°C, for example, it can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, but not limited thereto; further preferably, the temperature of the use system is 20°C or 30°C, but not limited thereto.

[0033] In one embodiment, when the Enterobacter mori phage P38 or the phage composition is used, the pH of the use system is 3~11, for example, it can be 3, 4, 5, 6, 7, 8, 9, 10, 11, but not limited thereto; preferably, the pH of the use system is 7~11, 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 use system is 7, but not limited thereto.

[0034] The present invention will be further described below through specific examples.

[0035] In the following examples, the materials involved are specifically as follows:

[0036] The tomato variety is Jinpeng No. 1. Enterobacter mori ( Enterobacter mori ) 23LSFQ was isolated from diseased tomato plants in Longquan Town, Shandong Province in October 2023. Enterobacter mori phage ( Enterobacter mori phage ) P38 was isolated from the soil sample of a potato blackleg disease plot in Zhangjiakou City, Hebei Province in September 2023, and was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on December 02, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 46285.

[0037] Nutrient agar (NA) medium is prepared with water and includes: tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, beef extract 3 g / L, agar 20 g / L.

[0038] Nutrient broth (NB) medium is prepared with water and includes: tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, beef extract 3 g / L.

[0039] The phage buffer is prepared with water and includes: sodium chloride (NaCl) 87.4 g / L, magnesium chloride hexahydrate (MgCl₂·6H₂O) 20.4 g / L, calcium chloride (CaCl₂) 2.2 g / L, tris(hydroxymethyl)aminomethane (Trisbase) 60.6 g / L, and is adjusted to the required pH with hydrochloric acid (HCl) or sodium hydroxide (NaOH).

[0040] The yeast malt extract (YME) medium is prepared with water and includes: peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, glucose 10 g / L.

[0041] A method for preparing the suspension of Enterobacter sangaii 23LSFQ includes: taking out Enterobacter sangaii 23LSFQ from the -80 °C strain bank in the laboratory, placing it in a -20 °C refrigerator overnight for slow thawing. After thawing, diluting the bacterial solution and spreading it on NA medium, and culturing it in a 28 °C constant temperature incubator for 2 - 3 days. Subsequently, picking single colonies for streak plate purification, repeating 3 - 4 times to ensure obtaining pure strains. Finally, inoculating the purified single colonies of Enterobacter sangaii 23LSFQ into NB medium, placing it on a shaker with a rotation speed of 200 rpm / min, and culturing it at 28 °C for 2 - 3 days to obtain the suspension of Enterobacter sangaii 23LSFQ.

[0042] Example 1

[0043] In this example, Enterobacter sangaii phage P38 was isolated and purified as follows:

[0044] The soil sample was collected from the potato blackleg disease soil plot in Zhangjiakou City, Hebei Province. Weighed 3.5 g of the soil sample and placed it in a conical flask. Added 500 μL of Enterobacter sanguiensis 23LSFQ suspension and 6 mL of phage buffer (pH = 7.5). After thorough mixing, placed it in a shaker at 200 rpm / min and amplified at 28 °C for 2 - 3 days. After the amplification was completed, the sample was allowed to stand for 20 - 30 min, and the supernatant was filtered through a 0.22 μm sterile filter to obtain the sample filtrate. Took 500 μL of the sample filtrate and mixed it with 500 μL of Enterobacter sanguiensis 23LSFQ suspension, allowed it to stand for 5 - 10 min, added 7 mL of 0.7% (w / v) water agar (45 - 50 °C), mixed evenly and poured it onto the NA medium for plating. Separately, took 500 μL of Enterobacter sanguiensis 23LSFQ suspension, added 7 mL of 0.7% (w / v) water agar (45 - 50 °C), mixed evenly and poured it onto the NA medium, allowed it to stand for 10 min. After the water agar solidified, 10 μL of the sample filtrate was pipetted for spotting. The plated and spotted plates were placed in an incubator at 28 °C for 1 - 2 days to observe whether plaques were formed. If plaques appeared, it indicated that there were phages in the sample filtrate that could lyse Enterobacter sanguiensis 23LSFQ, and further purification could be carried out.

[0045] Picked a single plaque formed on the plate, soaked it in phage buffer (pH = 7.5), mashed the agar block with the plaque and vortexed it to ensure that the phages were completely released into the buffer, filtered it through a 0.22 μm sterile filter, gradient diluted the obtained phage filtrate and plated it, and cultured it at 28 °C for 1 - 2 days. The above steps were repeated 3 - 4 times to obtain the purified Enterobacter sanguiensis phage P38. The plaque of the purified Enterobacter sanguiensis phage P38 is as Figure 1 shown. As Figure 1 can be seen, the plaque of Enterobacter sanguiensis phage P38 is transparent and round, with uniform size, and the diameter is about 3.7 mm.

[0046] Co - cultured the above - purified Enterobacter sanguiensis phage P38 with Enterobacter sanguiensis 23LSFQ by the double - layer agar plate method for 3 days, harvested the upper agar layer, mashed it and added an appropriate amount of phage buffer (pH = 7.5) to mix evenly. The supernatant was centrifuged (10000 g, 15 minutes, 4 °C) and filtered through a 0.22 μm sterile filter to obtain the Enterobacter sanguiensis phage P38 suspension, which was stored at 4 °C for later use. Separately, took part of the Enterobacter sanguiensis phage P38 suspension and mixed it with 50% (v / v) glycerol, transferred it to a cryotube, and placed it in a - 80 °C refrigerator for long - term storage.

[0047] Example 2

[0048] In this example, the morphology of Enterobacter sanguiensis phage P38 was observed as follows:

[0049] The morphology of Enterobacter mori phage P38 was observed using a transmission electron microscope (TEM). The sample preparation process is as follows: In the Enterobacter mori phage P38 suspension (~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 0.5 M, and the mixture was precipitated overnight at 4°C. Subsequently, it 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 resuspended solution was extracted with chloroform three times in equal volumes, the prepared Enterobacter mori phage P38 was dropped onto a copper mesh carbon support film (Beijing Dajikeyi Technology Co., Ltd., China) for adsorption, and then the unadsorbed Enterobacter mori phage P38 was aspirated with filter paper. During negative staining, the Enterobacter mori phage P38 adsorbed on the carbon film was treated with a 1% (w / v) phosphotungstic acid solution (Sigma, USA) for 1 minute, and then the excess staining solution was blotted with filter paper. The morphology of Enterobacter mori phage P38 was observed using TEM at an accelerating voltage of 60 kV, as specifically shown in Figure 2 Figure. As can be seen from Figure 2 this, the head of Enterobacter mori phage P38 is hexagonal and has a short rod-shaped tail, and its head diameter is about 56 - 58 nm.

[0050] Example 3

[0051] In this example, the host range of Enterobacter mori phage P38 was determined as follows:

[0052] Thirty-four random strains collected from soils in different regions of China were used for the determination. The process is as follows: First, the target strains were 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 Enterobacter mori phage P38 suspension (~10 8 PFU / mL) was pipetted onto the surface of the double-layer agar plate, and it was ensured that the plate contained the target strains. 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 lysis can occur, and "-" indicates lysis cannot occur.

[0053] Table 1 Host spectrum of Enterobacter mori phage P38

[0054]

[0055] As can be seen from Table 1, Enterobacter mori phage P38 can lyse Enterobacter mori ZRIMU1424 and Enterobacter mori 23LSFQ, but has no lytic effect on other strains. This indicates that Enterobacter mori phage P38 has strong host specificity.

[0056] Example 4

[0057] In this example, the thermal stability of Enterobacter mori phage P38 was determined as follows:

[0058] The Enterobacter mori phage P38 suspension (~5×10 9 PFU / mL) was incubated at 10, 20, 30, 40, 50, 60, and 70 °C for 12 hours, and then its titer was measured to observe the effect of different temperatures on the stability of Enterobacter mori phage P38. The results are as Figure 3 shown. As Figure 3 can be seen, Enterobacter mori phage P38 is relatively stable in the temperature range of 10 - 40 °C, reaches the highest activity at 20 - 30 °C, the titer decreases significantly at 50 °C, and completely loses its activity at 70 °C.

[0059] Example 5

[0060] In this example, the pH stability of Enterobacter mori phage P38 was determined as follows:

[0061] 100 μL of Enterobacter mori 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 Enterobacter mori phage P38 under different pH conditions. The results are as Figure 4 shown. As Figure 4 can be seen, Enterobacter mori phage P38 can survive at pH values from 3 to 11, and its activity reaches the highest at pH = 7; under extremely acidic (pH = 1) and extremely basic (pH = 13) conditions, its activity will be completely lost. In addition, the activity of Enterobacter mori phage P38 in alkaline environments (pH = 9 and 11) is significantly higher than its activity in acidic environments (pH = 3 and 5).

[0062] Example 6

[0063] In this example, the ultraviolet sensitivity of Enterobacter mori phage P38 was determined as follows:

[0064] The Enterobacter mori phage P38 suspension (~5×10 9The suspension of Enterobacter mori phage P38 (PFU / mL) was placed at a position 12 cm away from two TUV 15W / G15 T8 lamps with a wavelength of 253.7 nm to simulate the ultraviolet irradiation conditions in the natural environment. The experiment was carried out at room temperature, and the pH of the Enterobacter mori phage P38 suspension was 7.5. The samples were exposed to ultraviolet light for 1, 2, 3, 4, 5, and 6 minutes respectively to explore the effect of different irradiation times on the stability of the phage. The results are as Figure 5 shown. It can be seen from Figure 5 that Enterobacter mori phage P38 has low ultraviolet tolerance. Under ultraviolet lamp irradiation, it decreased steadily at first, but after 3 to 4 minutes, its activity tended to be stable and then decreased significantly, and finally completely lost its activity at 5 minutes.

[0065] Example 7

[0066] In this example, the lysis kinetic curve of Enterobacter mori phage P38 was measured as follows:

[0067] The concentration of the suspension of Enterobacter mori 23LSFQ was adjusted to 10 6 CFU / mL with YME medium, and then cultured at 30 °C for 6 hours to promote spore germination to obtain the germinated host bacterial solution. In the treatment group, the germinated host bacterial solution was mixed with the suspension of Enterobacter mori phage P38 at a multiplicity of infection (MOI) of 0.01 and then added to a 96-well plate. In the control group, only the germinated host bacterial solution was added to the 96-well plate. By measuring the change in optical density (OD) at 600 nm every hour to monitor the change in bacterial concentration, the lysis kinetic curve was drawn according to the results. The results are as Figure 6 shown. It can be seen from Figure 6 that the bacterial concentration in the treatment group was lower than that in the control group, indicating that Enterobacter mori phage P38 showed an inhibitory effect on Enterobacter mori 23LSFQ.

[0068] Example 8

[0069] In this example, the control effect of Enterobacter mori phage P38 on tomato wilt was evaluated as follows:

[0070] Tomato seeds were sown in flower pots with a diameter of 21 cm. After 30 days of cultivation, a phage control experiment was carried out. Four groups were set up in the experiment: the 23LSFQ group, the P38 group, the 23LSFQ + P38 group, and the control group. In the 23LSFQ group, 5 mL of the suspension of Enterobacter mori 23LSFQ (10 8 cfu / mL) was evenly sprayed on the leaves of tomato plants; in the P38 group, 5 mL of the suspension of Enterobacter mori phage P38 (10 6Spray evenly on the tomato plant leaves at 10⁸ pfu / mL; for the 23LSFQ + P38 group, first spray 5 mL of the suspension of Enterobacteriaceae bacteriophage 23LSFQ (10⁸ cfu / mL), and then spray 5 mL of the suspension of Enterobacteriaceae bacteriophage P38 (10⁸ pfu / mL) 6 hours later; the control group sprays 5 mL of sterile water. After one week of the experiment, observe the growth of the plants and record. Each treatment group contains 3 plants, and the experiment is repeated twice. The experimental results are as shown in 8 cfu / mL), and then spray 5 mL of the suspension of Enterobacteriaceae bacteriophage P38 (10⁸ 6 pfu / mL) 6 hours later; the control group sprays 5 mL of sterile water. After one week of the experiment, observe the growth of the plants and record. Each treatment group contains 3 plants, and the experiment is repeated twice. The experimental results are as shown in Figure 7 and Figure 8 shown. As can be seen from Figure 7 and Figure 8 compared with the 23LSFQ group, the incidence of tomato wilt disease in the P38 + 23LSFQ group is significantly reduced after adding Enterobacteriaceae bacteriophage P38. Among them, the incidence of the 23LSFQ group is about 60%, while the incidence of the P38 + 23LSFQ group drops to about 20%, a reduction of about 40%. At the same time, no disease occurrence is observed in the P38 group or the control group.

[0071] Example 9

[0072] In this example, the whole genome sequencing and bioinformatics analysis of Enterobacteriaceae bacteriophage P38 are carried out as follows:

[0073] Use the λ phage genomic DNA rapid extraction kit (Beijing Zhuangmeng International Biotechnology Co., Ltd., product number: ZP317), and extract genomic DNA from Enterobacteriaceae bacteriophage P38 following its instructions. Genome sequencing is carried out on the Illumina Novaseq PE150 platform (Shanghai Tanpu Biotechnology Co., Ltd.), and the size of the whole genome sequence of Enterobacteriaceae bacteriophage P38 is measured to be 47478 bp. In the NCBI database, BLAST is used to compare and analyze the similarity between Enterobacteriaceae bacteriophage P38 and other phages. The results show that the similarity between Enterobacteriaceae bacteriophage ( Enterobacteria phage ) UABPhi20 (GenBank accession number: NC_031019.1) and Salmonella phage ( Salmonella phage ) P22 (GenBank accession number: NC_002371.2) and Enterobacteriaceae bacteriophage P38 is the highest, both showing 81.53% identity and 6% query coverage. Further, the similarity between Enterobacteriaceae bacteriophage P38 and other phage genomes is evaluated by DNA-DNA hybridization (DDH) and average nucleotide identity (ANI). To more intuitively display these data, the corresponding heat map is generated using R (http: / / www.r-project.org / ), specifically asFigure 9 as shown Figure 9 Among them, the DDH and ANI values between Enterospora phage P38 and other phage genomes are shown in the grid in percentage form, and the color depth reflects the magnitude of the DDH and ANI values. The above results indicate that Enterospora phage P38 is a novel phage.

[0074] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations shall fall within the protection scope of the appended claims of the present invention.

Claims

1. Enterobacteria phage Enterobacter mori phage ), P38, characterized in that The Enterobacter mori phage P38 is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC NO. 46285 and the deposit date of December 2, 2024.

2. A phage composition, characterized in that, The phage composition includes the Enterobacter mori phage P38 as described in claim 1.

3. The phage composition according to claim 2, wherein The phage composition further includes agriculturally acceptable excipients.

4. The phage composition according to claim 3, wherein The agriculturally acceptable excipients are selected from one or more of dispersants, stabilizers, fillers and solvents.

5. Use of the Enterobacter mori phage P38 as described in claim 1 or the phage composition as described in any one of claims 2 to 4 in the prevention and control of yellowing and withering of tomato leaves; the yellowing and withering of the tomato leaves are caused by Enterobacter mori 23LSFQ.

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

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

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