A bacteriophage of Ralstonia solanacearum and its application in the control of bacterial wilt of Solanaceae

Abstract

The invention discloses a solanacearum phage and its application in the prevention and treatment of solanaceae bacterial wilt. The solanacearum phage is named PN12‑4, has been deposited in Guangdong Microbial Culture Collection Center, the deposit date is May 14, 2024, the deposit number is GDMCC 64623‑B1, and the classification name is Ralstonia solanacearum phage. The phage has a strong lysis effect on solanacearum GMI1000, and has good thermal stability and acid-base tolerance, which is conducive to the application in agricultural production, has a good prevention and treatment ability for tomato bacterial wilt caused by solanacearum GMI1000, increases the survival rate of tomatoes, and phage PN12‑4 can become a phage source for preventing and treating crop bacterial wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a solanacearum phage and application thereof in the prevention and treatment of solanaceae bacterial wilt. Background Art

[0002] Ralstonia solanacearum is a plant pathogen that mainly causes bacterial soil-borne plant diseases and is known as plant cancer. Ralstonia solanacearum has strong infectivity, a wide range, great harm, complex phylogeny and rapid development of drug resistance, making it one of the most important plant pathogens in global agriculture. Ralstonia solanacearum can infect more than 450 species of plants from 54 families, including important agricultural cash crops such as tomatoes, tobacco, peppers, and bananas. The plant diseases caused by it have brought huge economic losses to the agricultural economy. Ralstonia solanacearum mainly infects the root system of plants, and can infect plants through plant wounds, destroying the middle gelatinous layer and cell walls of plant cells. After infection, the parenchyma tissue of the xylem of the plant will be transformed into an invasive body, thereby blocking the duct, making it impossible for the plant to transport water and nutrients, resulting in withering and death.

[0003] Bacterial wilt is extremely harmful to agricultural cash crops. The prevention and control of bacterial wilt has always been a hot topic in related research fields around the world. In the prevention and control of bacterial wilt, traditional pesticide control and other methods play an important role, but at the same time they also cause many hazards. For example, ecological environment pollution; crop pesticide residues; pathogens are prone to drug resistance and damage soil microbial communities. For these problems, biological control has become a research hotspot. Phage resources are scarce, and collecting phage resources has become a current research focus. Building a phage resource library and enriching phage species are also preliminary preparations for the utilization of phage resources. Phages have strong host specificity and can identify specific pathogens. They have no adverse effects on natural microbial communities, will not cause bacterial imbalance, and will not pollute the environment. In addition, when pathogens are lysed, they provide more niche space and nutrition for other microbial communities, which helps to restore community diversity and reduce the competitiveness of pathogens with other bacterial communities, thereby leading to a further decrease in pathogen abundance. However, the production and application of bacteriophage preparations are currently very rare. Due to the characteristics of bacteriophages, the use of bacteriophages and their derivatives to prevent and control bacterial diseases is a very potential prevention and control method, and the application of bacteriophage preparations is full of development prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a solanacearum phage and its application in the prevention and treatment of solanacearum wilt disease, so as to provide a phage source for the industrial production of phage for biological prevention and treatment of solanacearum wilt disease.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a Ralstonia solanacearum phage, named PN12-4, which has been deposited in Guangdong Microbiological Culture Collection Center on May 14, 2024, with a deposit number of GDMCC 64623-B1, and a classification name of Ralstonia solanacearum phage.

[0007] Furthermore, bacteriophage PN12-4 belongs to the family Autoviridae of the order Caudata, and has a retractable short tail and a hexahedral head.

[0008] Furthermore, the contig genome sequence of bacteriophage PN12-4 is shown in SEQ ID NOs: 1-7.

[0009] Furthermore, phage PN12-4 was treated at 50°C for 60 min, and the phage was still active, with the titer maintained at 10 5 PFU / mL; under the condition of pH 5-11, its titer remains at 10 6 PFU / mL or above.

[0010] The present invention also provides application of the solanacearum phage in preventing and controlling solanaceous bacterial wilt.

[0011] Furthermore, the bacterial wilt disease includes the bacterial wilt disease caused by Ralstonia solanacearum GMI1000.

[0012] Furthermore, the Solanaceae family includes tomatoes.

[0013] Furthermore, bacteriophage PN12-4 can affect the colonization ability of Ralstonia solanacearum in tomato plants and inhibit the reproduction and growth of Ralstonia solanacearum.

[0014] Furthermore, root irrigation is used for prevention and control.

[0015] Compared with the prior art, the beneficial effects of the present invention include:

[0016] (1) The present invention discovered, isolated and purified a strain of Ralstonia solanacearum phage PN12-4, which has good lysis ability and specificity for Ralstonia solanacearum GMI1000, providing a phage source for industrial production of phage for biological control of Ralstonia solanacearum disease.

[0017] (2) The titer of the bacteriophage PN12-4 of the present invention remained at 10 after being treated at 50°C for 60 min. 5 PFU / mL or more; at pH 5-11, the titer remains at 10 after being placed at 28℃ for 1h 6 PFU / mL or above; therefore, the Ralstonia solanacearum bacteriophage PN12-4 has a higher temperature tolerance and a wider acid-base tolerance range, which is conducive to industrial production.

[0018] (3) The present invention directly uses bacteriophage PN12-4 to control soil-borne bacterial wilt by root irrigation, which reduces the number of bacterial wilt colonization in the roots and stems of tomatoes and improves the survival rate of tomato seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a plate culture picture of bacteriophage PN12-4.

[0020] Figure 2 This is the TEM electron microscope image of bacteriophage PN12-4.

[0021] Figure 3 This is the nucleotide sequence evolution tree of the large subunit of DNA helicase of bacteriophage PN12-4.

[0022] Figure 4 This is the one-step growth curve of bacteriophage PN12-4.

[0023] Figure 5 This is the temperature stability diagram of bacteriophage PN12-4.

[0024] Figure 6 This is the pH stability diagram of bacteriophage PN12-4.

[0025] Figure 7 This is the control chart and survival rate of bacteriophage PN12-4 against tomato bacterial wilt.

[0026] Figure 8 It is the colony number of Ralstonia solanacearum strains in the roots and stems of tomatoes. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] In the following examples, the liquid culture medium is a CPG liquid culture medium, and its formula is: 5 g glucose, 10 g peptone, 1 g hydrolyzed casein, and deionized water to a constant volume of 1000 mL, pH 7-8.

[0029] In the following examples, the solid culture medium is a CPG solid culture medium, and its formula is: 5 g glucose, 10 g peptone, 1 g hydrolyzed casein, 3% agar powder, and deionized water to a volume of 1000 mL, pH 7-8.

[0030] In the following examples, the formula of the SM buffer solution is: weigh 2.0 g MgSO 4 7H 2O, 5.8g NaCl, measure 50.0mL, pH 7.5, 1mol / L Tris-HCl, 5.0mL 2% gelatin solution, dissolve with deionized water and make up to 1L, divide and autoclave at 121℃ for 20min, and store at 4℃.

[0031] In the following examples, the specific operation steps of the double-layer plate method are as follows: In a sterile operating table, take an appropriate amount of phage suspension and 1 mL of host bacterial liquid OD 600 =1.0 and mix well. After standing for 15 min, mix the mixture of phage suspension and host bacterial solution with solid culture medium. After mixing well, pour it onto a solidified solid plate and culture it in a 28°C incubator for 24 h.

[0032] The Ralstonia solanacearum phage isolated by the present invention is named PN12-4 and has been deposited in the Guangdong Microbial Culture Collection Center, referred to as GDMCC, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is May 14, 2024, the deposit number is GDMCC 64623-B1, and the classification name is Ralstoniasolanacearum phage.

[0033] Example

[0034] 1. Isolation and purification of bacteriophage PN12-4

[0035] Soil samples were collected, 5 g of soil sample was dissolved in sterile water, and mixed at 220 r / min at 28 °C for 6 h. The soil suspension was centrifuged at 8000 r / min at 4 °C for 10 min. The supernatant was filtered with a 0.22 μm sterile filter membrane, and 5 mL of the filtrate was added to 5 mL of OD 600 =1.0, and placed in a 28°C shaker at 220 r / min for 12 h to obtain a phage enrichment solution. Centrifuge the phage enrichment solution, take the supernatant and filter it again. Take 100 μL of the filtered phage enrichment solution and add 900 μL of the OD 600 = 1.0 host bacterial solution, mix well and let stand at 28℃ for 15min to allow phage to adsorb to the host bacteria, then mix well with 4mL CPG solid medium cooled to about 40℃, immediately pour into the solidified agar solid medium plate, and quickly rotate the culture dish to make the upper medium uniform. After the upper medium solidifies, invert the prepared double-layer plate at 28℃ for 24-48h to observe whether there are plaques. Figure 1When plaques appear in the upper culture medium, pick a single plaque with an inoculation loop and inoculate it into 5 mL of sterile SM suspension, then add it to 5 mL of host bacterial solution (OD value is about 1.0), and culture it overnight at 220 r / min in a 28°C shaker to proliferate the phage. After the proliferation culture, centrifuge the phage-host co-culture solution, collect the supernatant and filter it, repeat the above steps 3 times, and finally mix the phage with the bacterial solution and add 60% glycerol to store it in a -80°C refrigerator, thus completing the purification of the phage and obtaining the single phage PN12-4.

[0036] Take 30 μL of concentrated crude particles of Ralstonia solanacearum phage and stain them with 2% phosphotungstic acid, and let them stand to dry for 30 minutes. Observe the morphology of the phage using a transmission electron microscope (TEM) and take photos to record the morphology. Figure 2 , the phage has a short retractable tail and a hexahedral head.

[0037] 2. Sequencing analysis of bacteriophage PN12-4 genome

[0038] 10 mL of purified phage stock solution was used for phage genome sequencing analysis by Lingen Biotechnology Co., Ltd. The library was constructed with 1 μg of DNA as the starting amount. Covaris M220 ultrasonically sheared the DNA to 300-500 bp, filled in, A was added to the 3' end, and an index adapter was connected (TruSeq TM Nano DNA Sample Prep Kit), library enrichment, PCR amplification for 8 cycles, 2% agarose gel recovery of target bands (Certified Low Range UltraAgarose), TBS380 (Picogreen) quantification, mixed according to data ratio, bridge PCR amplification on cBot solid phase carrier, clusters were generated, and 2x150 bp sequencing was performed on the second-generation sequencing platform. Finally, a contig genome of 40180bp was obtained (SEQ ID NO: 1-7):

[0039]

[0040] The nucleotide sequence of the large subunit of the bacteriophage DNA helicase was analyzed by phylogenetic tree, such as Figure 3 The CG content of phage PN12-4 is 62.34%, which is a dsDNA virus. The genome of phage PN12-4 was compared with the NCBI nucleic acid sequence to determine the closest related phages in evolution and the species to which the comparison belongs. Phage PN12-4 has the highest homology with Serkorvirus ITL1 of Ralstonia solanacearum. The genome size and homology can determine that phage PN12-4 belongs to Caudovirales, Autographiviridae, and is classified as Ralstonia solanacearum phage, which belongs to a new species.

[0041] 3. Determination of the optimal multiplicity of infection (MOI) of bacteriophage PN12-4

[0042] Take the host bacterial suspension in the logarithmic phase and measure the bacterial concentration (OD 600 =0.7), prepare phage liquid at the same time, and measure the phage titer. Add phage liquid to the same amount of host bacterial liquid at different infection multiplicities, mix the mixture and let it stand for 15 minutes, then put it in a 28°C shaker for 6 hours to allow the phage to proliferate. After centrifugation and filtration, measure the titer after culture. The MOI of the test group with the highest titer after culture is the best MOI for this phage for this host bacteria. The experimental results in Table 1 show that the best infection multiplicity (MOI) of phage PN12-4 is 1, and the number of phage progeny is the largest at this time.

[0043] Table 1 Number of progeny of phage PN12-4 at different multiplicity of infection (MOI)

[0044]

[0045] IV. One-step growth curve of bacteriophage PN12-4

[0046] Add phage solution to the logarithmic phase Ralstonia solanacearum solution at the optimal MOI ratio, incubate in a 28°C water bath for 15 minutes, then centrifuge at 5000r / min for 5 minutes, discard the supernatant, and wash the precipitate once with fresh liquid CPG culture medium to remove unsuccessfully adsorbed phage particles. Resuspend the precipitate with 12mL of 28°C preheated CPG liquid culture medium and immediately place in a 28°C water bath. Starting from 0min, take out 1mL every 30min to determine the titer of the culture solution. Draw the one-step growth curve of this phage with the culture time as the horizontal axis and the titer as the vertical axis. Figure 4 It can be seen that the lysis cycle of the bacteriophage PN12-4 is about 1h.

[0047] 5. Thermal stability test of bacteriophage PN12-4

[0048] Take 1mL of the potency of 10 6 The phage suspension with PFU / mL was placed in a water bath at different temperatures (28℃, 40℃, 50℃, 60℃, 70℃ and 80℃) for 1h, and then immediately cooled to room temperature. The titer was then measured using the double-layer plate method. Finally, the phage thermal stability curve was drawn using the titer of the phage obtained from each treatment as the ordinate and the water bath temperature as the abscissa. Figure 5 It can be seen that the phage PN12-4 still maintains a high activity at 50°C, and the phage has good temperature resistance.

[0049] 6. pH stability test of bacteriophage PN12-4

[0050] Take 10 μL of the titer of 10 6 Eight portions of phage suspensions with PFU / mL were mixed with 990 μL of CPG liquid culture medium with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0), respectively, and the titer was measured by double-layer plate method after being incubated in a 28°C water bath for 1 hour. Finally, the pH stability curve of the phage was drawn with the titer of the phage obtained from each treatment as the ordinate and the pH as the abscissa. Figure 6 It can be seen that the titer remains at 10 under pH 5-11 conditions. 6 PFU / mL or above, indicating that bacteriophage PN12-4 has good acid and alkali resistance.

[0051] VII. Biological control of tomato bacterial wilt by bacteriophage PN12-4

[0052] Four treatments were set up, namely, a blank group, a group with Ralstonia solanacearum, a group with Ralstonia solanacearum and phage, and a group with phage. Each treatment was repeated three times, and two tomato plants were planted in each pot. Seven days after planting, the tomato seedlings were in good growth condition before inoculation with strains and phages. The inoculation was carried out by root irrigation, and the strain OD 600 =1.0, phage is 10 9 PFU / mL, 990μL of the strain was treated with 10μL of phage, and all pots were placed in greenhouse conditions (25-28℃ during the day, 20-25℃ at night, and 12h of light). The position of the pots was randomly adjusted every 2 days to reduce environmental errors, and the death time and number of tomato plants in each pot were recorded. Photos were taken after 14 days. The experimental results are as follows Figure 7As shown in a and b, the tomato plants inoculated with Ralstonia solanacearum alone became ill and died on the 3rd day, and all the tomato seedlings died after 9 days. However, the experimental group inoculated with Ralstonia solanacearum and phage did not become ill and die until the 8th and 10th days. During the entire 14-day experimental period, a total of 2 tomato plants died in the experimental group inoculated with Ralstonia solanacearum and phage. This indicates that phage PN12-4 increases the survival rate of tomato plants and has a good effect in preventing and controlling the development of Ralstonia solanacearum, further demonstrating the possibility of using this phage in agricultural biological control.

[0053] 8. Colony count of Ralstonia solanacearum strains in tomato roots and stems

[0054] 14 days after inoculation, three tomato seedlings were selected from each experimental group. The soil around the tomato roots was washed with sterile water. After the surface moisture of the tomato roots was wiped dry with sterile paper towels, 1g of the roots and stems of the tomato plants were weighed with an analytical balance. After being fully ground with a sterile mortar in an ultra-clean workbench, 9mL of sterile water was added and transferred to a 50mL sterile centrifuge tube. The tube was shaken for 20 minutes. After gradient dilution, 100μL was evenly spread on the CPG plate, dried in a sterile operating table and sealed. After inverted culture in a 28°C incubator for 3 days, the plate was taken out and the number of colonies (CFU) on the plate was recorded. The number of colonies of Ralstonia solanacearum in the roots and stems of tomatoes measured represents the colonization ability of Ralstonia solanacearum in the roots and stems of tomato plants. Figure 8 As shown in a and b, the content of Ralstonia solanacearum in the roots and stems of tomato plants inoculated with phages is much lower than that in tomato plants inoculated with Ralstonia solanacearum only, and the difference in the colonization of Ralstonia solanacearum in the stems is more obvious, which further indicates that phage PN12-4 affects the colonization ability of Ralstonia solanacearum in tomato plants and inhibits the reproduction and growth of Ralstonia solanacearum.

[0055] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Ralstonia solanacearum phage, named PN12-4, has been deposited in Guangdong Microbial Culture Collection Center on May 14, 2024, with a deposit number of GDMCC No: 64623-B1, and a classification name Ralstoniasolanacearum phage.

2. Use of the Ralstonia solanacearum phage according to claim 1 in the prevention and treatment of Solanaceae bacterial wilt.

3. The use according to claim 2, characterized in that: The bacterial wilt disease is caused by Ralstonia solanacearum GMI1000.

4. The use according to claim 2, characterized in that: The Solanaceae bacterial wilt disease is tomato bacterial wilt disease.

5. The use according to claim 4, characterized in that: Use root irrigation for prevention and control.

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