Phage Ca1 and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a bacteriophage Ca1 and a preparation method thereof, the bacteriophage is the bacteriophage Ca1 capable of infecting a strain Xcc8004 and is preserved in Guangdong Microbiological Culture Collection Center, and the preservation number is GDMCC66226-B1. According to the phage Ca1 and the preparation method thereof disclosed by the invention, the highest average nucleotide similarity between the genome sequence of the phage Ca1 and the known phage sequence is 94% and is lower than a new species threshold (95%), and the genome does not contain integrase, so that the phage Ca1 is a new lytic phage; the titer is relatively stable in a pH range of 3-11; the titer of the bacteriophage is relatively stably maintained at 109 PFU / mL at 4-60 DEG C; after the bacteriophage Ca1 is treated by 25% chloroform, the titer is not obviously changed, which indicates that the bacteriophage is insensitive to chloroform and free of lipid coating; the results show that the bacteriophage Ca1 is a new bacteriophage resistant to acid, alkali, high temperature and organic solvent, and has strong stability in natural environment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bacteriophage Cal and a preparation method thereof. Background Art

[0002] Bacteriophages have been widely used as fungicides against pathogenic microorganisms. However, phages vary significantly in their adaptability to their environments, with different phages exhibiting varying tolerances to temperature, pH, and organic matter. Xanthomonas campestris pv. campestris (Xcc) is a pathogenic bacterium that causes black rot in nearly all cruciferous plants, including cabbage, kale, rapeseed, and Chinese cabbage. It is widely distributed in diverse environments worldwide and causes black rot in nearly all cruciferous plants, severely impacting vegetable production. While chemical fungicides for combating X. campestris are effective and low-cost, their broad, non-targeted activity not only harms numerous beneficial microorganisms in the soil, but also contributes to water pollution and ultimately harms humans. Bacteriophages, as bacterial viruses, are widely distributed in bacterial habitats. Their host specificity and ability to lyse host bacteria make them promising biocides. Currently, phages are widely used in biocontrol applications in agriculture, forestry, livestock, and aquaculture. Furthermore, phages have been approved for clinical use in the treatment of pulmonary tuberculosis caused by Mycobacterium tuberculosis in some countries. However, the limited tolerance of most phages severely restricts their application. Furthermore, research has shown that using a cocktail biocontrol approach is currently an effective way to mitigate host resistance evolution, as a single phage can accelerate the evolution of host resistance. Therefore, isolating and identifying new phages with broad environmental adaptability not only expands their application as biocontrol agents but also provides a phage resource for cocktail biocontrol approaches. Summary of the Invention

[0003] In response to the above-mentioned deficiencies in the prior art, the present invention provides a bacteriophage Ca1 and a preparation method thereof, the purpose of which is to provide a bacteriophage Ca1 that can infect Xanthomonas campestris and has strong tolerance, which is of great significance for preventing and controlling Xanthomonas campestris in different environments and studying phage tolerance.

[0004] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0005] A bacteriophage Cal, which can infect strain Xcc8004 (Xanthomonas phage), is deposited in Guangdong Provincial Microorganism Collection Center with the deposit number GDMCC66226-B1 and the deposit date of April 27, 2025.

[0006] Furthermore, phage Ca1 has a large polyhedral head with a diameter of 60±5 nm and a retractable long tail with a length of 220±3 nm.

[0007] Furthermore, the optimal multiplicity of infection of phage Ca1 against strain Xcc8004 was 0.01.

[0008] Furthermore, treatment with 25% chloroform revealed no significant change in the potency of phage Ca1. It remained stable within the 4°C-60°C range, began to decline at 70°C, and was completely inactivated at 80°C, indicating that Ca1 is relatively stable at room temperature. Phage Ca1 was completely inactivated at pH ≤ 2, and its potency was relatively stable in the alkaline range, demonstrating that the phage has a wide range of acid and alkaline tolerance. Furthermore, plant experiments confirmed that Ca1 significantly inhibited the cabbage disease caused by Xcc8004.

[0009] A method for preparing bacteriophage Cal comprises the following steps:

[0010] S1: Isolation of phage from strain Xcc8004: Soil from different cultivated cruciferous crops and different regions was incubated overnight in LB liquid medium. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane. First, strain Xcc8004 was used as a host and co-cultured with the filtrate overnight. The culture medium was centrifuged and filtered through a 0.22 μm filter membrane and used as the viral stock. Phage that could infect strain Xcc8004 was isolated and purified using the double-layer plate method.

[0011] S2: Biological characteristics of the phage to be tested: The biological characteristics of the phage are determined by electron microscopy, optimal multiplicity of infection, thermal stability and tolerance;

[0012] S3: First, spray the cabbage leaves with bacteriophage Ca1 until they drip water, and spray with sterile water as a control. Adjust the OD600 of the overnight culture of Xcc8004 to 0.1. 24 hours after spraying the bacteriophage, use sterile scissors to pick up the culture solution and cut off 1-2 cm of the leaf tip perpendicular to the main vein. Observe and photograph 14 days after inoculation.

[0013] S4: Genome analysis: TaKaRa's viral genome extraction kit was used to extract the nucleic acid of the phage to be tested, and second-generation sequencing was performed. Trimmomatic was used to quality control the offline data, and then SPAdes was used to assemble the phage genome to be tested. Glimmer, GeneMarkS, Prodiga, and blastp were used to annotate the genome. CGView was used for genome visualization and analysis. VipTree and blastn were used to classify the phage to be tested.

[0014] The beneficial effects of the present invention are:

[0015] The present invention provides a bacteriophage Ca1 and a preparation method thereof. The average nucleotide similarity between the phage Ca1 genome sequence and the known phage sequences is as high as 94%, which is lower than the new species threshold of 95%, and the genome does not contain integrase, indicating that the phage Ca1 is a new lytic phage with relatively stable potency in a wide acid-base range. The phage has strong acid-base tolerance and is insensitive to organic matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a microscopic observation image of the bacteriophage Ca1 of the present invention;

[0017] Figure 2 is the acid-base tolerance range of the bacteriophage Ca1 of the present invention;

[0018] Figure 3 is the temperature tolerance range of the bacteriophage Ca1 of the present invention;

[0019] Figure 4 is the sensitivity of the bacteriophage Ca1 of the present invention to organic matter;

[0020] Figure 5 is the genome map of the bacteriophage Ca1 of the present invention;

[0021] Figure 6 is a graph showing the average nucleotide similarity of the genome between the bacteriophage Cal of the present invention and the closest known bacteriophage;

[0022] Figure 7 Schematic diagram of the proteomic tree of the bacteriophage Cal and its most related phages of the present invention;

[0023] Figure 8 This is a schematic diagram of the bacteriophage Ca1 plant control method of the present invention;

[0024] Figure 9 is the optimal multiplicity of infection of the bacteriophage Cal1 of the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below with reference to the accompanying drawings.

[0026] Example 1:

[0027] A method for preparing bacteriophage Cal comprises the following steps:

[0028] Isolation of bacteriophage from strain Xcc8004: Soil from different cultivated cruciferous crops and different regions was incubated overnight in LB liquid medium. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane. First, strain Xcc8004 was used as a host and co-cultured with the filtrate overnight. The culture medium was centrifuged and filtered through a 0.22 μm filter membrane and used as the viral stock. Phage capable of infecting strain Xcc8004 was isolated and purified using the double-layer plate method using strain Xcc8004 as a host.

[0029] Biological characteristics of the phage to be tested: The biological characteristics of the phage are determined by electron microscopy, one-step growth curve, optimal multiplicity of infection, thermal stability and tolerance;

[0030] First, spray the cabbage leaves with bacteriophage Ca1 until they drip water, using sterile water as a control. Adjust the OD600 of an overnight culture of Xcc8004 to 0.1. 24 hours after spraying the bacteriophage, use sterile scissors to remove the bacterial solution and cut off 1-2 cm from the leaf tip perpendicular to the main vein. Observe and photograph the leaves 14 days after inoculation.

[0031] Genome analysis: TaKaRa's viral genome extraction kit was used to extract the nucleic acid of the phage to be tested, and second-generation sequencing was performed. Trimmomatic was used to quality control the data. The phage genome to be tested was then assembled using SPAdes. Glimmer, GeneMarkS, Prodiga, and blastp were used to annotate the genome. CGView was used for genome visualization and analysis. VipTree and blastn were used for classification of the phage to be tested.

[0032] like Figure 1 As shown, a bacteriophage Ca1 capable of infecting strain Xcc8004 was isolated.

[0033] Electron microscopy results showed that phage Ca1 had a large polyhedral head with a diameter of 60±3 nm and a retractable long tail with a length of 220±3 nm.

[0034] like Figure 9 As shown in Figure 2, the optimal infection multiplicity (MOI) of phage Ca1 for strain Xcc8004 is 0.01, and the progeny phage yield at this MOI is 7.8×10 7 PFU / mL.

[0035] Example 2:

[0036] Phage stability experiment

[0037] like Figure 2 As shown, the titer of phage Ca1 was relatively stable within the pH range of 3-11 and maintained at 10 8PFU / mL. When pH ≤ 3 or pH ≥ 11, the phage was completely inactivated, indicating that the phage had strong acid-base tolerance.

[0038] like Figure 3 As shown, it is relatively stable in the range of 4℃-60℃, begins to decline at 70℃, and is completely inactivated at 80℃, indicating that Ca1 is highly resistant to high temperatures.

[0039] like Figure 4 As shown, the titer of phage Ca1 did not change significantly after treatment with 25% chloroform, indicating that the phage was insensitive to organic matter.

[0040] like Figure 6 As shown, the average nucleotide similarity between phage Ca1 and known phages is 94%, which is lower than the threshold of 95% for new species; and the genome does not contain integrase ( Figure 5 ), indicating that phage Ca1 is a new lytic phage.

[0041] like Figure 7 As shown, the phylogenetic tree at the protein level constructed using VipTree showed that Xanthomonas phage Ca1 was most closely related to Xylella phage Bacata (NC_052973.1).

[0042] like Figure 8 As shown in the results, spraying bacteriophage Ca1 could significantly alleviate the disease symptoms caused by Xcc8004 on cabbage.

[0043] like Figure 2 As shown, phage Ca1 has a wide range of pH applications. In the pH range of 3-11, the titer is relatively stable at 10 8 PFU / mL, when pH≤3 or pH≥11, the phage is completely inactivated. Figure 3 It can be seen that phage Ca1 has a strong tolerance to temperature, and the phage titer is relatively stable at 10 in the range of 4-60℃. 9 PFU / mL, when the temperature reaches 80℃, the phage is almost completely inactivated. After the phage Ca1 is treated with 25% chloroform, Figure 4 The titers shown did not change significantly, indicating that this phage strain was insensitive to chloroform and had no lipid coating.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A bacteriophage Ca1, characterized in that The phage can infect strain Xcc8004 and is deposited in Guangdong Provincial Microbiological Collection Center with the deposit number GDMCC66226-B1.

2. The method for preparing bacteriophage Cal according to claim 1, characterized in that: The following steps are involved: S1: Isolation of phage strain Xcc8004: Soil from different cultivated cruciferous crops and different regions was incubated overnight in LB liquid medium. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane. The strain Xcc8004 was used as a host and co-cultured with the filtrate overnight. The culture solution was centrifuged and filtered through a 0.22 μm filter membrane and regarded as the virus stock solution. The phage capable of infecting the strain Xcc8004 was isolated and purified using the double-layer plate method using the strain Xcc8004 as a host. S2: Biological characteristics of the phage to be tested: The biological characteristics of the phage are determined by electron microscopy, optimal multiplicity of infection, thermal stability and tolerance; S3: First, spray the cabbage leaves with bacteriophage Ca1 until they drip water, and spray with sterile water as a control. Adjust the OD600 of the overnight culture of Xcc8004 to 0.

1. 24 hours after spraying the bacteriophage, use sterile scissors to pick up the culture solution and cut off 1-2 cm of the leaf tip perpendicular to the main vein. Observe and photograph 14 days after inoculation. S4: Genome analysis: TaKaRa's viral genome extraction kit was used to extract the nucleic acid of the phage to be tested, and second-generation sequencing was performed. Trimmomatic was used to quality control the offline data, and then SPAdes was used to assemble the phage genome to be tested. Glimmer, GeneMarkS, Prodiga, and blastp were used to annotate the genome. CGView was used for genome visualization and analysis. VipTree and blastn were used to classify the phage to be tested.

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