CN114045268A - Bacteriophage for preventing and treating plant protection Ralstonia solanacearum and Pseudomonas solanacearum diseases and application thereof - Google Patents
Bacteriophage for preventing and treating plant protection Ralstonia solanacearum and Pseudomonas solanacearum diseases and application thereof Download PDFInfo
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Abstract
The invention provides a bacteriophage for preventing and treating diseases of plant protection Ralstonia solanacearum and Pseudomonas solanacearum and application thereof, wherein the bacteriophage is preserved in China center for type culture collection (CCTCC M2020944) in 12-21 months in 2020. The phage has a wide bacterial phage spectrum, can simultaneously crack Ralstonia solanacearum and Pseudomonas solanacearum, can more effectively solve the problem of bacterial disease infection in crop planting, and can be used as a microecological preparation for preventing and treating diseases of plant protection Ralstonia solanacearum and Pseudomonas solanacearum.
Description
Technical Field
The invention relates to the technical field of plant protection prevention and treatment, and particularly relates to a bacteriophage for preventing and treating plant protection Ralstonia solanacearum and Pseudomonas solanacearum diseases and application thereof.
Background
Bacterial wilt is a bacterial soil-borne disease with high incidence in crop production, which is mainly composed of Ralstonia solanacearumRalstonia solanacearumRs for short, Pseudomonas solanacearum (C.M.)Pseudomonas solanacearum) The infection is caused. The pathogen is distributed all over the world, has wide host range, can infect more than 450 plants of 54 families, mainly infect solanaceae crops such as tobacco, tomato and pepper, and can infect peanut, pepper, corn,ginger, banana, mulberry and other important crops. Because the host range of the ralstonia solanacearum is wide, the latent infection capability is strong, and the variation capability is extremely strong, the control of the ralstonia solanacearum is always a big problem in the aspect of plant disease control. Once the ralstonia solanacearum infects plants, the plants show the symptoms of rapid wilting and withering, stem leaves still keep green, and the browning parts of diseased stems are squeezed by hands and are discharged by milky bacteria liquid. And after the plant diseases reach the middle stage, the plants cannot be cured by the pesticide, so that great loss is caused to farmers.
Aiming at the bacterial wilt disease, the bacterial wilt disease can be only treated by preventing in advance or treating at the outbreak stage, but the treatment effect of pesticides, antibiotics and chemical reagents on the bacterial wilt disease is not good, and pesticide and antibiotic residues are caused. And the biological control of bacterial wilt is environment-friendly and can not bring potential threat to human health, thereby arousing the attention of vast scholars at home and abroad. The bacteriophage can selectively kill a specific harmful bacterial strain due to high-efficiency specific lysis and specific host property, and cannot cause damage to other beneficial flora in the ecological environment when in use. The phage not only has the self-replicating capacity of exponential type proliferation, but also can kill corresponding pathogenic bacteria only with a very small amount, thereby achieving the purpose of preventing or controlling bacterial infection, and the phage has high environmental abundance, the corresponding phage of bacteria commonly exist in nature, is easy to separate and has short purification period, can be stored at normal temperature, and is convenient for transportation and application. In conclusion, the phage preparation product is a safer food safety biological prevention and control means, has the advantages of safety, high efficiency, strong specificity, high stability, fast self proliferation, short screening period, no influence on the sensory quality of food and the like, and has very wide development prospect. The substitution of phage products for antibiotics is a situational requirement, and therefore, the development of phage products is of great significance.
The project takes the high-density production technology of the phage composite preparation as the core, researches and develops a microecological preparation capable of treating plant bacterial wilt, establishes a whole set of domestication and screening of a series of high-activity phages and a fermentation production process of the high-density phage composite preparation, and realizes large-scale industrial production and trial of the phage composite preparation. Compared with the conventional phage preparation, the developed phage composite preparation is innovative to a series of phage with harmful pathogen specificity, and avoids the defects of narrow spectrum, slow effect and poor stability of the conventional phage product in treating harmful pathogens; compared with the conventional probiotic preparation, the phage composite preparation product has the characteristics of quick treatment effect, safety and harmlessness. The plant protection phage microecological preparation has the advantages of environmental friendliness, safety to non-target organisms, specific bactericidal effect on plant protection diseases and the like, is different from antibiotics and chemical preparations, is a green and environment-friendly product completely derived from nature, is widely applied to the field of plant protection cultivation and the field of food safety, replaces antibiotics in the cultivation industry for preventing and resisting diseases, overcomes the problem of pathogenic bacteria drug resistance caused by excessive use of antibiotics, and becomes a hotspot of current biotechnology research. However, phages are highly specific to the host, and thus phages with narrow phage spectra generally do not solve the problem of infection with a variety of bacterial diseases that occur during plant protection farming.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention provides the phage for preventing and treating the diseases of the plant protection Ralstonia solanacearum and the plant protection Pseudomonas solanacearum, the phage has a wide phage spectrum, and can crack 21 strains of the plant protection Ralstonia solanacearum and 8 strains of the plant protection Pseudomonas solanacearum. Can more effectively solve the problem of bacterial disease infection in crop planting and has good application prospect.
Therefore, in the first aspect of the invention, the invention provides a bacteriophage for preventing and controlling diseases of plant protection Ralstonia solanacearum and Pseudomonas solanacearum, wherein the bacteriophage is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2020944 in 12-21 months of 2020, and is classified and named as:Ralstonia solanacearumphage 10RS306A, with the collection address: wuhan, Wuhan university. The phage 10RS306A obtained by separation and screening according to the invention has a wider phage spectrum, can crack 21 strains of Ralstonia solanacearum and 8 strains of Pseudomonas solanacearum, can effectively solve the problem of bacterial disease infection in crop planting, and has good performanceAnd the application prospect is good.
In a second aspect of the invention, the invention provides a microecological preparation which comprises the phage for controlling diseases of plant protection Ralstonia solanacearum and Pseudomonas solanacearum. According to the embodiment of the invention, the microecological preparation can be used for preventing and treating the ralstonia solanacearum diseases such as ralstonia solanacearum and pseudomonas solanacearum diseases.
Optionally, the bacteriophage has a titer of 1.8 × 1011pfu/mL。
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a plaque map of bacteriophage 10RS306A according to an embodiment of the present invention;
FIG. 2 is a BLAST alignment of bacteriophage 10RS306A at NCBI according to an embodiment of the present invention;
FIG. 3 is an electrophoresis diagram of PCR amplification products of 2 specific genes according to an embodiment of the present invention.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1 screening and purification of phage 10RS306A
1. Separation of tomato bacterial wilt bacterium Ralstonia solanacearum
Taking tomato rhizomes suffering from tomato bacterial wilt in Fujian Xiamen area in an aseptic mortar, taking 50 mL of 75% alcohol to wash the surface of the rhizomes, putting the rhizomes in the aseptic mortar for grinding, dipping a inoculating loop in grinding fluid, scribing on a TC (bacterial wilt syndrome) plate of a bacterial wilt identification culture medium, culturing for 24 h at 30 ℃, picking red colonies, scribing and purifying on the same selective culture medium, repeatedly purifying for 3 times, picking single colonies of strains with consistent shapes by using the inoculating loop, inoculating into an LB (LB) liquid culture medium, culturing for 12 h at 30 ℃, mixing 500 mu L of bacterial liquid with 500 mu L of 40% glycerol, and then putting the mixture in a refrigerator at-80 ℃ for storage.
2. Expanded culture of Ralstonia solanacearum
Carrying out enlarged culture on host bacteria, preparing 1L LB liquid culture medium, carrying out autoclaving at 121 ℃ for 20 min, cooling to room temperature, adopting the following two inoculation methods, wherein the first inoculation method is single colony inoculation, selecting a single colony on a seed-preserving plate under an aseptic condition for inoculation, and carrying out fermentation at 30 ℃, at a rotation speed of 150 rpm for 12 h; the second inoculation method is liquid inoculation, and 10% of culture medium is added at a concentration of 108And (3) fermenting the cfu/mL ralstonia solanacearum bacterial liquid in an LB liquid culture medium at the fermentation temperature of 30 ℃ and the rotation speed of 150 rpm for 12 h.
3. Screening of phage 10RS306A
Collecting soil samples from 45 tomato planting greenhouses of Xiamen, Quanzhou, Nanping, Sanming and the like in Fujian province, respectively mixing 1L of the Ralstonia solanacearum bacterial liquid cultured in the step 2 and 500g of the soil samples together by adopting a bacterial liquid-soil sample mixing enrichment method, adding 1L of fresh LB liquid culture medium for enrichment culture overnight, extracting the enriched liquid, centrifuging at 12000 rpm for 10min, filtering twice by using a 0.22 mu m filter membrane, and screening by using a coating spotting method to obtain the phage. The phage 10RS306A of the present application was obtained by screening tomato soil from Xiamen city, Fujian province.
4. Purification of phage 10RS306A
Individual plaques (the plaque pattern of phage 10RS306A is shown in FIG. 1) were picked, incubated overnight in 1mL of SM buffer (Scientific Phygene), centrifuged at 12000 rpm for 10min, filtered twice with a 0.22 μm filter, and diluted to 10 μm-6First, only 100. mu.L of the solution with the concentration of 4.2X 10 is added8And (3) carrying out control on double-layer plates of cfu/mL host bacteria, taking 100 mu L of host bacteria and phage for each dilution gradient, mixing the double-layer plates, carrying out constant-temperature culture at 30 ℃ overnight, observing the growth condition of plaques, and selecting a single plaque to continuously purify for 3 times.
5. High temperature resistance assay for bacteriophage 10RS306A
Taking 6 containers 200mL of 5.4X 1010The pfu/mL phage liquid 10RS306A triangular flask, respectively placed in 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ water bath, placed for 12 h, through double-layer plate mixing plate calculation processing phage content to determine the phage 10RS306A high temperature resistance. The results are shown in table 1 below: the phage 10RS306A can still survive after being treated at 70 ℃ for 12 h, the content is still high, and the phage generally die in large amount or even all die after being treated at 60 ℃, so the phage 10RS306A has strong high temperature resistance.
Table 1: high temperature resistance assay for bacteriophage 10RS306A
6. Determination of the acid and base resistance of bacteriophage 10RS306A
Taking 7 containers 200mL of which the content is 6.5X 1010The pfu/mL phage solution is prepared by dripping concentrated sulfuric acid solution and hydrogenThe pH value of the phage bacterium liquid is adjusted to 4, 5, 6, 7, 8, 9 and 10 respectively by using a sodium oxide solution, the phage bacterium liquid is placed for 12 hours, and the content of the processed phage is calculated by using a double-layer plate mixing plate to measure the acid and alkali resistance of the phage 10RS 306A. The results are shown in table 2: the phage 10RS306A can still survive and still have high content under the treatment of pH 5 and 10, while the general phage will die in large amount or even all die under the treatment of pH 5 and 8, which indicates that the phage 10RS306A has certain acid resistance and strong alkali resistance.
Table 2: determination of the acid and base resistance of bacteriophage 10RS306A
Example 2
1. Determination of the optimal multiplicity of infection (MOI) of bacteriophage 10RS306A against Ralstonia solanacearum
8 100mL portions of fresh LB liquid medium were taken and treated separately. Treatment 1 while adding 1mL of 106 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 109 pfu/mL phage solution, treatment 2 while adding 1mL 107 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 109 pfu/mL phage solution, treatment 3 while adding 1mL phage solution with 10 content8 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 109 pfu/mL phage solution, treatment 4 while adding 1mL 108cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 108 pfu/mL phage solution, treatment 5 while adding 1mL 108 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 107 pfu/mL phage solution, treatment 6 while adding 1mL 108 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 106 pfu/mL phage solution, treatment 7 while adding 1mL 108 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 105 pfu/mL phage solution, treated 8 while adding 1mL 108 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 104 pfu/mL phage solution, cultured at 30 ℃ and 150 rpm for 12 h, and then assayedPhage titer. The results are shown in Table 3: the phage titer was highest when the optimal multiplicity of infection was 0.01.
Table 3: determination of the optimal multiplicity of infection (MOI) of phage 10RS306A against Ralstonia solanacearum
2. Amplification culture of bacteriophage 10RS306A
Carrying out amplification culture on the separated phage: preparing LB liquid culture medium 1L, autoclaving at 121 deg.C for 20 min, cooling to room temperature, and adding 1mL 109cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 107putting pfu/mL phage liquid in a shaker at 30 ℃ and 150 rpm for 12 h, centrifuging at 8000 rpm for 10min to settle host bacteria at the bottom, taking the upper clear part, and obtaining the liquid which is the phage 10RS306A in enlarged culture.
3. Preparation of phage 10RS306A Microecological preparation
Preparing LB liquid culture medium 1L, autoclaving at 121 deg.C for 20 min, cooling to room temperature, and adding 1mL 109 cfu/mL Ralstonia solanacearum bacterial liquid and 1mL of Ralstonia solanacearum bacterial liquid with the content of 107 pfu/mL phage liquid is placed in a shaking table with the temperature of 30 ℃ and the rotating speed of 150 rpm for 12 hours to serve as seed liquid, the seed liquid is inoculated into a 50L fermentation tank, the seed liquid is inoculated into a 500L fermentation tank after the seed liquid is cultured for 12 hours under the fermentation condition with the temperature of 30 ℃ and the rotating speed of 150 rpm, the obtained fermentation liquid is centrifuged for 10 minutes at 8000 rpm, so that host bacteria are settled at the bottom, the upper clear part is taken, and then the supernatant is subjected to graded filtration by using a ceramic membrane with the wavelength of 500 nm and 200 nm to obtain the phage microecological preparation.
4. Determination of the content of bacteriophage 10RS306A
The phage was diluted to 10-10First, only 100. mu.L of the mixture is added to the mixture with the concentration of 5.8X 108control was performed on double-layer plates of cfu/mL Ralstonia solanacearum from 10-10Starting the concentrations, 100. mu.L each of phage dilutions andpreparing a double-layer plate mixed plate by 100 mu L of bacterial ralstonia solanacearum, culturing overnight at constant temperature of 30 ℃, and calculating the content of phage by observing the number of plaques. The results showed that the phage content was 1.8X 1011pfu/mL, high content and strong infection ability to Ralstonia solanacearum.
Table 4: determination of the content of bacteriophage 10RS306A
Example 3 lytic ability and control Effect of bacteriophage 10RS306A
1. Determination of the lytic Activity of bacteriophage 10RS306A against Ralstonia solanacearum
Phage 10RS306A were counted and diluted to 1X 10 each8、1×107、1×106、1×105、1×104pfu/mL 5 concentrations, with sterile water as a blank, three replicates for each concentration. Taking 6 tubes of cultured Ralstonia solanacearum bacterial liquid, calculating the content of Ralstonia solanacearum by a dilution plate coating method before treatment, respectively adding 1mL of phage liquid with 5 concentrations and sterile water, culturing for 12 h at 30 ℃ at 150 r/min, and calculating the content of Ralstonia solanacearum treated by the dilution plate coating method to determine the cracking capacity of the phage 10RS306A on the Ralstonia solanacearum. The result shows that the bacteriophage 10RS306A has a good sterilization effect on Ralstonia solanacearum, and the effect is better when the concentration is higher.
Table 5: determination of the lytic Activity of bacteriophage 10RS306A against Ralstonia solanacearum
2. Host Spectroscopy assay for bacteriophage 10RS306A
The phage host bacteria 10RS306A and other Ralstonia solanacearum are cultured to obtain 62 strains, wherein the strains comprise Ralstonia solanacearum 42 strain and Pseudomonas solanacearum 20 strain. Culturing at 30 deg.C and 150 r/min for 12 h, and directly spotting to obtain 100 μ L of 3.5 × 108cfu/mL host bacteria liquid is evenly coated on an LB plate, and 20 mu L of the host bacteria liquid with the concentration of 2.3 multiplied by 10 is taken10Dripping 4 spots of pfu/mL phage liquid on the plate, culturing overnight at 30 ℃, observing whether the plate has transparent plaques, and determining whether the phage 10RS306A has the capability of cracking 62 strains of pathogenic bacteria.
The results are shown in the following table: the phage 10RS306A can cleave 29 of 62 strains of ralstonia solanacearum, among which, the bacterial strain ralstonia solanacearum 21 and the bacterial strain pseudomonas solanacearum 8. And compared with other phages, the phage 10RS306A has stronger lytic capacity and wider host spectrum. Zhengxiashu 'screening of vibrio parahaemolyticus broad-spectrum lytic phage and application thereof in marine product safety control' was carried out infection experiment on 42 strains of vibrio parahaemolyticus, and it was found that phage VppYZU68 can only lyse 5 strains thereof. Infection experiments of 37 strains of staphylococcus aureus and 74 strains of other species are carried out in the 'cracking spectrum specificity and molecular classification research of staphylococcus aureus bacteriophage' of Zhang Qihong, and the bacteriophage vB _ SauH _ SAP1 is found to be only capable of cracking 10 strains of staphylococcus aureus. Infection experiments are carried out on 26 strains of vibrio (vibrio parahaemolyticus, vibrio cholerae and vibrio alginolyticus) in screening wide lytic vibrio phage by using vibrio cholerae SWBC-a of the Yang Jixia as a target bacterium, and the result shows that the phage SWBC-a-3 can only lyse 3 strains of vibrio therein. From this, it is clear that the bacteriophage 10RS306A of the present application has a strong lytic ability and a broad host spectrum.
TABLE 6 host spectra of bacteriophage 10RS306A
3. Determination of prevention and treatment effects of wide-spectrum phage 10RS306A and narrow-spectrum phage on ralstonia solanacearum
500 mL of the bacterial solution of Ralstonia solanacearum RS306 and the bacterial solution of Pseudomonas solanacearum P301 were cultured, and the concentration was 2.6X 108cfu/mL supernatant. Phage 10RS301 and 10RS307 which can only crack Ralstonia solanacearum RS306 are cultured, 500 mL of phage with concentration of 6.5X 1010pfu/mL phage liquid.
Taking 15 ridges of tomato seedlings in a field as a test field. The treatment groups were divided into 5 treatment groups of 3 replicates each, each replicate 20 tomato seedlings. The group A is treated as a blank control without treatment, each tomato seedling in the group B is treated, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 and 10 mL of prepared supernatant of Pseudomonas solanacearum P301 are added, each tomato seedling in the group C is treated, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 and 10 mL of 10RS301 phage liquid are added, each tomato seedling in the group D is treated, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 and 10 mL of 10RS307 phage liquid are added, each tomato seedling in the group E is treated, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 and 10 mL of 10RS306A preparation in the example 2 are added, the growth condition of the tomato seedlings is observed every day, and the incidence rate of the tomato seedlings in each group is recorded.
The control effects of the broad-spectrum bacteriophage 10RS306A and the bacteriophages 10RS301 and 10RS307 with narrow bacteriophagic spectrums on ralstonia solanacearum are shown in Table 7, and the tomato seedlings without disease in the group A are treated; when the tomato seedlings are treated in the group B for 3 days, the average incidence rate is 43.33 percent, and after 7 days, the incidence rate of the tomato seedlings is 58 percent, and the average incidence rate is up to 96.67 percent; the tomato seedlings are attacked at 16 days after the treatment of the group C, the average incidence rate is 26.67%, and the incidence rate of 34 tomato seedlings after 7 days is 56.67%; the tomato seedlings are attacked by 19 days after the treatment of the group D, the average incidence rate is 31.67 percent, and the incidence rate of the tomato seedlings after 7 days is 35 plants, and the incidence rate is 58.33 percent; the tomato seedlings in the group E after 3d had 2 diseases, the average incidence rate was 3.33%, and the incidence rate of the tomato seedlings after 7d was 7, the incidence rate was only 11.67%. By comparison, it was found that the tomato seedlings incidence was 85% lower in treatment E than in treatment a, 45% lower in treatment C and 46.66% lower in treatment D. The results show that: in practical application, the broad-spectrum bacteriophage 10RS306A microecological preparation has obvious advantages compared with bacteriophage 10RS301 and 05A021 with narrow bacteriophagia spectrum, can obviously reduce the morbidity of tomato seedlings, and reduce the harm caused by ralstonia solanacearum in crop planting.
TABLE 7 determination of control effect of broad-spectrum 10RS306A and narrow-spectrum phage on Ralstonia solanacearum
| Group number | Treatment of | Mean incidence of 3d | Mean 7d incidence | |
| | Blank control | 0% | 0% | |
| B | Each of 2 ralstonia solanacearum solutions is 100mL | 43.33% | 96.67% | |
| C | 2 bacterial wilt liquid 100mL and 100mL10 RS301 phage liquid | 26.67% | 56.67% | |
| D | 2 bacterial wilt liquid 100mL and 100mL10RS307 phage liquid | 31.67% | 58.33% | |
| E | 2 bacterial wilt liquid 100mL and 10RS306A phage liquid 100mL respectively | 3.33% | 11.67% |
4. Determination of prevention and treatment effects of phage 10RS306A microecological preparation on Ralstonia solanacearum
And taking 12 ridges of tomato seedlings in the field as a test field. The treatment groups were divided into 4 treatment groups of 3 replicates each, each replicate 20 tomato seedlings. The treatment group A is a blank control without treatment, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 is added to each tomato seedling in the treatment group B, 10 mL of prepared supernatant of Ralstonia solanacearum RS306 and 10 mL of 10RS306A phage liquid are added to each tomato seedling in the treatment group C simultaneously, 10 mL of prepared phage 10RS306A in example 2 is added to each tomato seedling in the treatment group D independently, the growth condition of the tomato seedlings is observed every day, and the incidence rate of the tomato seedlings in each treatment group is recorded.
The control effect of the phage 10RS306A preparation on the ralstonia solanacearum disease of the tomato seedlings is shown in Table 8, and the blank group A and the treated group D have no disease tomato seedlings; when the tomato seedlings are treated in the group B for 3 days, 29 tomato seedlings are attacked, the average incidence rate is 48.33%, and after 7 days, the incidence rate of 57 tomato seedlings is up to 95%; the disease-free tomato seedlings in the group C after 3d treatment and 1 tomato seedling after 7d treatment have the disease, the disease incidence rate is only 1.7 percent, and the disease incidence rate is reduced by 93.3 percent. The results show that: the phage 10RS306A microecological preparation has no influence on the growth of tomato seedlings, and has obvious control effect on the wilt disease of the tomato seedlings.
Table 8: control effect of phage 10RS306A microecological preparation on diseases of tomato seedling Ralstonia solanacearum
| Group number | Treatment of | Mean incidence of 3d | Mean 7d incidence | |
| | Blank control | 0% | 0% | |
| B | 100mL bacterial liquid of ralstonia solanacearum | 48.33% | 95% | |
| C | 100mL of bacterial liquid of ralstonia solanacearum and 100mL of |
0% | 1.7% | |
| D | 100mL |
0% | 0% |
Example 4 phage 10RS306A Whole genome determination and analysis
1. Purification of phage 10RS306A
2mL of 10RS306A phage liquid is taken, centrifuged for 10min at 8000 rpm, the supernatant is filtered twice by a 0.22 mu m filter membrane and then stored in a refrigerator at 4 ℃.
2. Extraction of phage 10RS306A genomic DNA
The phage extracted from the separated and purified phage was extracted from phage genome DNA/RNA using phage genome DNA/RNA extraction kit from Tiangen Biotechnology (Beijing) Ltd, and sent to Meiji organism (Shanghai) Ltd for sequencing.
3. Phage 10RS306A Whole genome sequence analysis
Through second-generation genome sequencing of the Pacbio platform, the whole genome sequence of the screened phage 10RS306A is 39742bp in length and is circular double-stranded DNA. BLAST alignment of the genome sequences at the NCBI official website resulted in an alignment coverage of 87% and homology of 86.46% with the closest genomic sequence of a Ralstonia solanacearum phage (GeneBank accession No.: NC 047751.1), as shown in FIG. 3; next, the alignment coverage with the genomic sequence of a Ralstonia solanacearum phage (GeneBank accession AB 597179.1) was 70%, and the homology was 78.55%. Thus, the selected phage is a new phage. Phage 10RS306A has been deposited in the China center for type culture Collection in Wuhan at 21.12.2020 with the deposition number CCTCC M2020944.
Differential fragments of phage 05A034 genome and two phage genome sequences with the highest homology are found out through DNAMAN 7 software to obtain specific genes A1 (SEQ ID NO. 1) and A2 (SEQ ID NO. 2); specific PCR amplification primers were designed by Primer Premier6 software and sent to Bodhands Biotech (Shanghai) Ltd for Primer synthesis, the results are shown in FIG. 3.
A1F:5’-CGGTCCTTCCAGTGTAGA-3’；
A1R: 5’-ACTCCTCGTATCGCTTGA-3’；
A2F: 5’-GAGATGCCAATGTCAACAG-3’；
A2R: 5’-AACGAGCCAGCGAATATC-3’；
In conclusion, according to the embodiment of the invention, the phage 10RS306A is separated and screened from the tomato planting base in the mansion area of Fujian province, has high bactericidal activity on Ralstonia solanacearum, has a wider bacteriophagic spectrum on other Ralstonia solanacearum, can crack 21 strains of Ralstonia solanacearum and 8 strains of Pseudomonas solanacearum, and can effectively solve the problem of bacterial disease infection in crop planting.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (4)
1. The phage for preventing and treating diseases of plant protection Ralstonia solanacearum and Pseudomonas solanacearum is characterized in that the phage is preserved in China center for type culture collection (CCTCC M2020944) in 12-21.2020.
2. The bacteriophage of claim 1, wherein said bacteriophage has 2 specific gene segments having a nucleic acid sequence as set forth in SEQ ID nos. 1-4.
3. A microecological preparation comprising the bacteriophage of claim 1 for controlling diseases of Ralstonia solanacearum and Pseudomonas solanacearum.
4. The probiotic according to claim 3, characterized in that the bacteriophage has a titer of 1.8 x 1011pfu/mL。
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| CN108676778A (en) * | 2018-04-20 | 2018-10-19 | 南京农业大学 | One plant of prevention soil passes bacteriophage and its application of bacterial wilt |
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| CN108676778A (en) * | 2018-04-20 | 2018-10-19 | 南京农业大学 | One plant of prevention soil passes bacteriophage and its application of bacterial wilt |
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