A phage of mulberry bacterial wilt pathogen and its application

Abstract

The present invention relates to the technical field of biological control, and discloses a phage of mulberry bacterial wilt pathogen and its application. The bacterial wilt pathogen phage MuIY4 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC M 20211330. The phage MuIY4 has strong infection activity against bacterial wilt pathogen, strong stability and good safety, and provides an effective solution for the prevention and treatment of bacterial wilt.

Description

Technical Field

[0001] The invention belongs to the field of biological control, and specifically relates to a mulberry bacterial wilt pathogen phage and an application thereof. Background Art

[0002] Mulberry is a deciduous tree belonging to the Urticales, Moraceae, Morus, and Morus alba, which is widely distributed all over the world. In my country, the natural distribution and artificial cultivation of mulberry trees are all over the country, and my country is also the country with the richest mulberry tree variety resources in the world. During the cultivation process of mulberry trees, they are very susceptible to the damage of bacterial wilt, which is serious.

[0003] Plant bacterial wilt is caused by a soil-borne plant pathogenic bacteria, which is mainly distributed in tropical, subtropical and some warm temperate regions, and infects a wide range of plants. It invades plants through wounds, root tips or secondary root germination, colonizes in the root cortex and rapidly reproduces, and enters the vascular bundle to continue spreading to other tissues. At the same time, it secretes pectinase, cellulase and other enzymes to decompose the middle gelatin layer of the plant, causing the plant to wilt and eventually die. Bacterial wilt is one of the most harmful, widely distributed and most damaging plant bacterial diseases. The disease can be called "plant cancer". Once it occurs, it is difficult to control and the economic losses caused are difficult to estimate.

[0004] There have been a lot of explorations on the prevention and control of bacterial wilt at home and abroad, and a variety of prevention and control measures have been proposed, including chemical control, physical control, improved agricultural cultivation methods, and biological control, etc. For example, using chemical fumigants and antibiotics to treat the soil, planting disease-resistant varieties, crop rotation and intercropping, using non-pathogenic bacterial wilt strains, Bacillus, Streptomyces and other biocontrol bacteria, etc. (Yuliar et al., 2015). Although these prevention and control measures have certain effects, they are not stable, and the residues of chemical pesticides seriously pollute the soil and water, endangering the health of humans and animals. Bacteria are also very easy to develop resistance to antibiotics. Biocontrol bacteria cannot be stably colonized in the soil, and their production, storage and use are difficult. Therefore, it is imperative to develop new prevention and control technologies.

[0005] In recent years, the use of phage therapy to control bacterial diseases has attracted widespread attention from researchers. Bacteriophages are a type of virus that can infect bacteria. As a "predator" of bacteria, they can be directly used to control pathogens. Yen et al. confirmed that phage cocktail therapy can inhibit the colonization of Vibrio cholerae in mice and rabbits, and effectively alleviate the diarrhea symptoms caused by the bacteria (Yen et al., 2017). In recent years, there have been more and more reports on the use of phages to control plant pathogenic bacteria. Field test results show that phages have a good control effect on plant pathogens such as Pseudomonas syringae, Streptomyces, Xylella fastidious, Erwinia amylovora and Pseudomonas amylovora (Buttimer et al., 2017). It was also found that the bacterial wilt phages ФRSL1 and PE204 can significantly reduce the incidence of tomato bacterial wilt (Bae et al., 2012; Fujiwara et al., 2011), indicating that phage therapy has great potential in the prevention and control of bacterial wilt.

[0006] Phage therapy has its unique advantages. It is highly specific and has different infection effects on different strains of the same bacteria. This characteristic prevents phages from affecting other surrounding microorganisms and has no adverse effects on the microecological environment. However, during the application of phages, bacteria will gradually develop resistance. Current studies have found that using phages of different types and different host spectra to make mixed phage preparations (phage cocktails) can reduce the emergence of phage resistance and broaden the application range of phage preparations. Summary of the invention

[0007] The purpose of the present invention is to provide a mulberry bacterial wilt pathogen phage, which has strong lytic activity against the bacterial wilt pathogen.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] One object of the present invention is to provide a phage of mulberry bacterial wilt pathogen, wherein the phage is Caudovirales phage MulY4, and the deposit number is CCTCC M 20211330.

[0010] In the present invention, the full length of the bacteriophage MuIY4 genome is 52699 bp, the G+C content is 49.4%, and the end thereof is a 166 bp direct repeat sequence.

[0011] The bacteriophage of the present invention belongs to the order Caudovirales.

[0012] Another object of the present invention is to provide a pharmaceutical preparation comprising the above-mentioned bacteriophage and an agrochemically acceptable carrier.

[0013] Preferably, the pharmaceutical preparation of the present invention further comprises one or more phages of mulberry wilt pathogen that are different from the above phages.

[0014] Preferably, the bacteriophage is a crude extract or a purified product of a lysate of Ralstonia solanacearum.

[0015] More preferably, the crude extract or purified product of the lysate is freeze-dried or dissolved in an appropriate solvent.

[0016] Preferably, the pH of the pharmaceutical preparation of the present invention is 5-9.

[0017] Preferably, the storage temperature of the pharmaceutical preparation of the present invention is below 28°C.

[0018] The present invention also provides the use of the bacteriophage and the pharmaceutical preparation in antagonizing bacterial wilt pathogens.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] The bacteriophage MulY4 of the present invention can infect bacterial wilt pathogens to form clear plaques, and has strong infection activity against bacterial wilt pathogens. The bacteriophage MulY4 has strong lysis activity against bacterial wilt pathogens, and the lysis amount reaches 189 PFU / cell. The bacteriophage MulY4 of the present invention is easy to preserve and can stably exist in a neutral environment below 28°C. There are no virulence-related genes in the genome of bacteriophage MulY4, and the safety is good.

[0021] Collection Instructions

[0022] Bacteriophage Caudovirales phage MulY4 was deposited in the China Center for Type Culture Collection on October 27, 2021, with the deposit number CCTCC M 20211330; address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are plaques formed when bacteriophage MulY4 infects mulberry wilt pathogen.

[0024] Figure 2 is the one-step growth curve of bacteriophage MuIY4;

[0025] Figure 3 is the stability of bacteriophage MuIY4 under different pH conditions;

[0026] Figure 4 is the stability of bacteriophage MuIY4 under different temperature conditions;

[0027] Figure 5This is the inhibitory effect of bacteriophage MulY4 on the bacterial wilt pathogen En5-3. DETAILED DESCRIPTION

[0028] The present invention uses mulberry wilt pathogen En5-3 as an indicator bacteria, uses a plaque purification method to separate phages, and finally separates and purifies MuIY4. Phage MuIY4 can infect En5-3 to form clear plaques ( Figure 1 ), indicating that it has strong infection activity.

[0029] At present, the phage has been preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC M20211330; address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University; preservation date: October 27, 2021, and classification name: Caudovirales phage MulY4.

[0030] The bacteriophage MuIY4 of the present invention has strong lysis activity on host bacteria and can stably exist in a neutral environment below 28°C.

[0031] The genome of bacteriophage MulY4 is 52699 bp in length, with a G+C content of 49.4%, a 166 bp direct repeat sequence at the end, and belongs to the order Caudovirales. The genome of bacteriophage MulY4 has no virulence-related genes and has good application safety.

[0032] According to another aspect of the present invention, there is provided a pharmaceutical preparation comprising the bacteriophage as described above and a pesticide-acceptable carrier thereof.

[0033] The pharmaceutical preparation of the present invention can be produced according to methods well known to those skilled in the art and / or can be commercially available. The pharmaceutical preparation can be in the form of a solution, a dispersant, a suspension, a granule, etc. The use method can be spraying, broadcasting, furrow application or irrigation with water.

[0034] In one embodiment, the pharmaceutical preparation of the present invention further comprises a phage of mulberry bacterial wilt pathogen that is different from the above-mentioned phage of the present invention. The different phages are used in combination to expand the host spectrum of the phages, improve the control effect on bacterial wilt pathogen, and delay the development of drug resistance in bacterial wilt pathogen.

[0035] In one embodiment, the bacteriophage used in the pharmaceutical preparation of the present invention is a crude extract or purified product of a lysate of Ralstonia solanacearum. The extraction or purification method can adopt conventional methods in the art, and the present invention does not limit this. In one embodiment, the crude extract or purified product of the lysate is freeze-dried or dissolved in an appropriate solvent. Freeze drying adopts conventional methods in the art. The solvent can be water, NB culture medium, etc.

[0036] In one embodiment, the pH of the pharmaceutical preparation of the present invention is 5-9. Within this pH range, the bacteriophage MuIY4 can exist stably and maintain its lytic activity.

[0037] In one embodiment, the storage temperature of the pharmaceutical preparation of the present invention is below 28° C., and the phage titer is relatively stable.

[0038] The bacteriophage MuIY4 of the present invention can inhibit the population growth of bacterial wilt pathogens, so the bacteriophage MuIY4 and the pharmaceutical preparation containing the bacteriophage can be used to antagonize bacterial wilt pathogens.

[0039] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0040] Example 1

[0041] Isolation and preparation of bacteriophage

[0042] The bacterial wilt pathogen En5-3 was isolated from the roots of mulberry plants infected with bacterial wilt, and potted root irrigation experiments confirmed that En5-3 is pathogenic to mulberry trees.

[0043] Using En5-3 as the indicator strain, soil was collected from the mulberry garden for phage isolation.

[0044] Weigh 3 g of fresh soil, add it to 10 ml of En5-3 bacterial solution in the logarithmic phase, and culture it in a shaking incubator at 28°C and 220 rpm for 6 h; centrifuge it at 12000 rpm for 10 min, take the supernatant, and filter it with a 0.22 μm sterile filter membrane; take 3 μl of the filtrate and spot it on a double-layer plate (a mixture of the upper semi-solid culture medium and the En5-3 bacterial solution), place the double-layer plate in a constant temperature incubator at 28°C, and culture it for at least 12 h until plaques appear on the plate.

[0045] Take the filtrate that can form plaques, obtain single plaques through gradient dilution method, and pick a single plaque to culture in fresh En5-3 bacterial solution for 6 hours. Centrifuge and filter using the above method to obtain phage suspension. Repeat this step once to obtain pure culture of phage, which is marked as MulY4.

[0046] Example 2

[0047] Identification of the host spectrum of bacteriophage MulY4

[0048] Twelve strains of bacterial wilt (see Table 1) were selected as indicator bacteria, and the double-layer plate dot plating method was used to identify the host spectrum of bacteriophage MulY4.

[0049] Take 1ml of the cultured bacterial wilt pathogen and mix it with 5ml of semi-solid NB medium, pour it into a plate that has been covered with solid NB medium, wait for the upper layer to cool, take 3μl of the prepared phage MulY4 suspension and spot it in the center of the plate, place the plate in a 28℃ constant temperature incubator and culture for 12-24h, if plaques are formed at the location where the phage suspension is spotted, it means that the phage can infect the strain. The results are shown in Table 1.

[0050] The results showed that the bacteriophage MulY4 strain had infection activity against four strains of bacterial wilt, accounting for 33.3% of the tested strains.

[0051] Table 1 Host spectrum of bacteriophage MuIY4

[0052]

[0053]

[0054] Example 3

[0055] One-step growth curve assay of bacteriophage MuIY4

[0056] The one-step growth curve of bacteriophage MulY4 was determined according to the method reported by Hyman et al. (Hyman, 2009).

[0057] Take 0.13 ml of phage MuIY4 suspension (approximately 1.12×10 11 PFU) was added to 100 ml of En5-3 bacterial solution in the logarithmic growth phase (approximately 1.47×10 9 CFU), so that the multiplicity of infection (MOI) is 0.1. Let the mixture of phage and bacterial solution stand for 30 minutes to allow the phage to fully adsorb to the cells. Then centrifuge at 13000g for 1 minute, take out the supernatant (which contains unadsorbed phage, and calculate its titer; the number of bacterial cells infected in this way is the total amount of phage initially added minus the total amount of phage remaining in the supernatant), and use 100ml of fresh NB culture medium to suspend the bacterial cells that adsorb phages, and culture on a shaker at 28°C and 220rpm. After that, take 2ml of bacterial solution every half an hour, centrifuge at 13000g for 1min, and use the double-layer plate method to detect the titer of phage in the supernatant until the phage titer is stable. Draw a one-step growth curve based on the titer of phage at each time point.

[0058] The one-step growth curve shows that after the bacteriophage MulY4 infects the bacteria, the incubation period is 30 minutes, then it enters the exponential growth phase, and reaches the lytic stable phase 5 hours after infection, with the final concentration reaching 10 11 PFU / ml, the phage lysis amount is 189PFU / cell ( Figure 2 ), indicating that MulY4 has strong lytic activity against host bacteria.

[0059] Example 4

[0060] pH stability assay of bacteriophage MuIY4

[0061] 100 μl of the phage MulY4 suspension prepared in Example 2 was added to 900 μl of liquid NB culture medium at pH 3, pH 5, pH 7, and pH 9, mixed, and placed at room temperature for 2 h. The titer was then determined under each pH condition.

[0062] The results showed that the phage MuIY4 could stably exist under the conditions of pH 5, pH 7, and pH 9, but the phage could not be detected under the condition of pH 3 ( Figure 3 ). Therefore, under neutral environmental conditions, bacteriophage MuIY4 can exist stably.

[0063] Example 5

[0064] Temperature stability assay of bacteriophage MulY4

[0065] The fresh phage MuIY4 suspension prepared in Example 2 was taken and its initial titer was determined. The phage MuIY4 suspension was then divided into 4 tubes and incubated in constant temperature incubators at 4°C, 28°C, 37°C, and 50°C, respectively. The incubated phage suspension was taken for titer determination at 1d, 2d, 4d, and 30d.

[0066] The results showed that the titer of phage MuIY4 did not change significantly after being stored at 4℃, 28℃, and 37℃ for 2 days, and it dropped by one order of magnitude on the 4th day. On the 30th day, the titer of phage stored at 4℃ and 28℃ did not change significantly compared with that on the 4th day, but it dropped by 2 orders of magnitude at 37℃ compared with that on the 4th day. At 50℃, the titer of phage dropped by 2 orders of magnitude on the 1st day, and the phage had no activity at all on the 30th day ( Figure 4 ). Therefore, when the storage temperature is below 28°C, the phage is relatively stable.

[0067] Example 6

[0068] Detection of the inhibitory effect of bacteriophage MulY4 on pathogenic bacteria

[0069] Take an appropriate amount of MuIY4 suspension and mix it with 100 ml En5-3 bacterial solution in the logarithmic growth phase (OD600 is about 0.3-0.5) (MOI is about 1), place it in a shaking incubator at 28°C and 220 rpm, and then check the OD600 of the bacterial solution every half an hour until the OD value of the bacterial solution remains stable.

[0070] The results showed that after the addition of bacteriophage, the pathogenic host bacteria began to grow slowly. After 6 hours, the OD value of the bacterial solution remained at a low level, while the OD value of the control group without the addition of bacteriophage continued to increase ( Figure 5 ). This indicates that bacteriophage MulY4 inhibits the population growth of bacterial wilt pathogen and has great application value.

[0071] Example 7

[0072] Extraction, whole genome sequencing and analysis of bacteriophage MulY4

[0073] In order to further explore the characteristics of bacteriophage MulY4, understand its related functional genes and whether it contains some risk genes, we further analyzed the genetic background of MulY4.

[0074] In this example, the zinc chloride (ZnCl2) precipitation method was used to extract a small amount of phage DNA (Santos, 1991). Take 1 mL of the induced phage suspension, add 20 μL of DNaseI (1 mg / mL), 5 μL of RNaseA (10 mg / mL), and incubate at 37°C for 30 to 60 min; add 20 μL of 2 mol / L ZnCl2, mix well and incubate at 37°C for 5 min; centrifuge at 10,000×g for 1 min, discard the supernatant; add 500 μL of TES buffer, break up the precipitate, and incubate at 65°C for 15 min; add 10 μL of proteinase K (20 mg / mL), incubate at 50°C for 1 h; cool after incubation, add 60 μL of pre-cooled 3 mol / L potassium acetate (pH5.2), and place on ice for 10 to 15 min; centrifuge at 13,000×g Centrifuge at 4°C for 10 min; take the supernatant (about 500 μL); add 600 μL phenol / chloroform / isoamyl alcohol (25:24:1, v:v:v) to the supernatant and invert several times; centrifuge at 13,000×g for 5-10 min at room temperature; take the upper liquid, precipitate DNA with 2 / 3 to 1 times the volume of isopropanol, and place at -20°C for no less than 30 min; centrifuge at 13,000×g at 4°C for 10 min, discard the supernatant, wash the precipitate once with 70% ethanol, air-dry at room temperature, and dissolve the DNA in TE (20-50 μl).

[0075] The whole genome sequencing of phages adopts the Whole Genome Shotgun (WGS) strategy to construct libraries with different inserts. The libraries are sequenced by paired-end (PE) sequencing based on the Illumina NovaSeq sequencing platform using the second-generation sequencing technology (NGS), with a sequencing read length of 150bp. The raw sequencing data are processed to remove adapter contamination, filter out reads with a length of less than 50bp, remove reads with an average quality of less than 20, and remove reads with an n number greater than 3, and obtain reads data that can be used for splicing. After that, the software ABySS is used for splicing and assembly, and the terminal sequence of the phage is determined by analyzing the high-frequency sequences in the high-throughput sequencing data. Finally, the complete phage genome sequence is obtained, and the whole genome is annotated using RAST (https: / / rast.nmpdr.org / ).

[0076] Sequencing results showed that the MulY4 genome was 52699 bp long, with a G+C content of 49.4%, and a 166 bp direct repeat sequence at the end, belonging to the Caudovirales order. The full genome of MulY4 was compared with the NCBI NR database, and it was found that it only had 58.49% nucleotide sequence identity with the reported phage Escherichia phage PHB10, indicating that MulY4 is a new type of phage.

[0077] Genomic prediction analysis showed that MulY4 contained 67 coding sequences (CDSs), 32 of which had possible functions, and the rest were proteins of unknown function. These functional protein genes were arranged in clusters to form functional modules, including DNA replication and metabolism, phage structural protein-related modules, DNA packaging-related gene modules, cell lysis modules, and lysogeny control modules. In addition, MulY4 also encodes some proteins related to host functions.

[0078] Whole genome annotation analysis showed that there were no virulence-related genes in the MulY4 genome, which provided a safety basis for the application of this phage.

[0079] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A phage of mulberry bacterial wilt pathogen, characterized in that: The bacteriophage is Caudovirales phage MulY4, the deposit number is CCTCC M 20211330.

2. The bacteriophage according to claim 1, characterized in that The full length of the bacteriophage MulY4 genome is 52699 bp, the G+C content is 49.4%, and the end thereof is a 166 bp direct repeat sequence.

3. A pharmaceutical preparation, characterized in that It comprises the bacteriophage according to any one of claims 1 to 2, and a pesticide-acceptable carrier.

4. The pharmaceutical preparation according to claim 3, characterized in that The pH of the pharmaceutical preparation is 5-9.

5. The pharmaceutical preparation according to claim 3, characterized in that The storage temperature of the pharmaceutical preparation is below 28°C.

6. Use of the bacteriophage according to any one of claims 1 to 2 or the pharmaceutical preparation according to any one of claims 3 to 5 in antagonizing the pathogen of mulberry bacterial wilt, wherein the pathogen of mulberry bacterial wilt is Enterobacter Belong to.

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