CN108676778B - Bacteriophage for preventing and treating soil-borne bacterial wilt and application thereof - Google Patents
Bacteriophage for preventing and treating soil-borne bacterial wilt and application thereof Download PDFInfo
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Abstract
The invention discloses a bacteriophage for preventing and treating soil-borne bacterial wilt and application thereof, wherein the strain of lawsonia specialized bacteriophage NN-P42 of solanaceae is preserved in China center for type culture collection in 2018, 3 and 6 days, and the preservation number is as follows: CCTCC NO: M2018102, and is classified and named as Podoviridae phase. The pathogenic bacteria specific phage is used for biological control, can efficiently crack bacteria to die, and has no toxicity to the environment; strong specificity, can not damage other normal flora, and can keep soil microbial balance. The bacteriophage of the invention can specifically crack bacterial wilt pathogenic bacteria, and can be used for preventing and treating soil-borne bacterial wilt of tomato.
Description
Technical Field
The invention belongs to an environment-friendly biocontrol preparation, and relates to a brachyury bacteriophage NN-P42 and application thereof, which are specially used for preventing and treating soil-borne bacterial wilt.
Background
Tomato bacterial wilt is a bacterial vascular bundle disease caused by Laurella species (Ralstonia solanacearum) and has a devastating disaster on solanaceous crops. The ralstonia solanacearum has wide host range, strong environment adaptability, large variation and complex intraspecific inheritance. At present, bacterial wilt occurs in most areas of China, but the disease is serious in southern areas. The traditional prevention and control measures such as resistant varieties, grafting rotation, chemical pesticides and the like have the problems of difficult research and development, unstable effect, soil ecology damage and food safety, and the development of environment-friendly prevention and control measures is urgently needed.
The microbial community of the plant rhizosphere has the capacity of 'instinct' resisting soil-borne diseases, and a first defense line for pathogenic bacteria to invade plants is constructed. The soil microorganisms are mainly used for preventing and controlling diseases through resource competition, antagonistic competition, induction of plant resistance, predation, parasitism and other ways. But the biocontrol potential of bacterial viruses has been neglected as a more minute but abundant group in the rhizosphere. Bacterial viruses, also known as bacteriophages, are widely present in the environment in quantities more than 10 times greater than bacteria. Wherein the lytic bacteriophage depends on a host to replicate and lyse host bacteria, and is a main biocontrol 'reserve force'.
The phage has host specificity and the capability of efficient cracking and rapid proliferation, and compared with the unidirectional evolution of bacterial antibiotic resistance, the synergistic evolution of the phage and host bacteria has the advantage of biocontrol application. With the research on the biological characteristics of the bacteriophage, the interaction relationship between the bacteriophage and host bacteria, the mastering on the safety and controllability of the bacteriophage, the deep research on the genome of the bacteriophage, the modification research on the bacteriophage through biotechnology, and the great application potential of the bacteriophage in the aspect of biological prevention and control. Aiming at the technical problems, a strain of obligate bacteriophage of lytic ralstonia solanacearum needs to be screened.
Disclosure of Invention
Aiming at the current biological control of the tomato bacterial wilt, a new thought and means are provided, and the occurrence of diseases is reduced. The pathogenic bacteria specific phage is used for biological control, bacteria can be efficiently cracked to die, and the bacterial vaccine has no toxicity to the environment; strong specificity, does not destroy other normal flora, and can keep the soil microbial balance. The applicant separates and screens a lytic bacterial wilt specific phage from rhizosphere soil with serious bacterial wilt, and researches the biological characteristics and the lytic performance of the lytic bacterial wilt specific phage.
Another object of the present invention is to provide a method for rapidly and effectively inhibiting Ralstonia solanacearum using the bacteriophage.
The purpose of the invention can be realized by the following technical scheme:
a Laurella sp.solanacearum specific phage NN-P42 is preserved in the China center for type culture Collection in 2018, 3 and 6 months, with the preservation number: CCTCC NO: M2018102, and is classified and named as Podoviridae phase.
The Laurella solanacearum (Ralstonia solanacearum) obligate phage NN-P42 is isolated from rhizosphere soil with serious bacterial wilt, is a tailed phage order brachyucatenae, and has a genome size of about 42.28 kb. The phage culture solution can be stored for more than 2 months at 4 ℃ after being filtered and sterilized, and can be stored for more than 2 years in a glycerin pipe at-70 ℃; the stock solution concentration of the phage culture solution is 10 11 PFU/mL. The high activity can be kept within 7 days at 25 ℃; higher activity was maintained at 37 ℃ by addition of 30% glycerol or SM buffer. The phage has stable lytic capacity at low resource levels.
The Laurella solanacearum specific bacteriophage NN-P42 has obvious effect in the application of preventing and treating soil-borne bacterial wilt or preparing a preparation for preventing and treating the soil-borne bacterial wilt phage.
A phage preparation for preventing and treating soil-borne bacterial wilt comprises the Laurella speciality phage NN-P42 of the family Solanaceae. As a preferred technical scheme, the content of the phage in the phage preparation is more than or equal to 10 6 PFU/mL, and more preferably, the phage content in the phage preparation is 10 6 ～10 11 PFU/mL. The phage preparation can be phage culture solution, and SM buffer solution or 30% glycerol is also added into the phage preparation.
The phage preparation can keep higher activity for a long time and has obvious effect in the application of preventing and treating soil-borne bacterial wilt.
A method for preventing and treating the soil-borne bacterial wilt of crops features that the culture liquid or phage preparation is directly inoculated to the rhizosphere of plant in root irrigation mode, and the additive amount is greater than or equal to 10 6 PFU/g dry soil, the disease prevention rate reaches 60 percent.
The invention has the beneficial effects that:
the bacteriophage of the invention can specifically crack bacterial wilt pathogenic bacteria, and can be used for preventing and treating tomato soil-borne bacterial wilt.
Drawings
FIG. 1 plaques on double agar plates.
FIG. 2 morphology of phage NN-P42 under electron microscope.
FIG. 3 SDS-PAGE analysis of the phage NN-P42 protein.
FIG. 4 phylogenetic tree of the phage NN-P42 gene.
FIG. 5 effect of different temperatures on phage NN-P42 titer.
FIG. 6 the titer of the phage NN-P42 in different solvents at different temperatures.
FIG. 7 bacteriostatic ability of bacteriophage under different nutritional conditions.
FIG. 8 incidence of bacterial wilt (A) and number of rhizosphere pathogenic bacteria (B) after inoculation with bacteriophage NN-P42.
Detailed Description
Isolation and characterization of phages
1. Separation and purification of phage
Host bacteria: ralstonia solanacearum QL-Rs1115(Wei et al.2011), which is called RS for short and has strong pathogenicity and is separated from tomato plants of kylin town of Nanjing in the laboratory.
The phage NN-P42 is obtained by screening rhizosphere soil of a vegetable greenhouse with serious bacterial wilt disease after long-term tomato planting in Guangxi Nanning (108 degrees 21 'E and 22 degrees 49' N). The separation and purification process comprises the following steps:
culture medium:
NA liquid medium: 10g of glucose, 5g of peptone, 3g of yeast extract, 0.5g of yeast extract and 1000mL of deionized water, adjusting the pH value to 7.2-7.4, and carrying out autoclaving at 115 ℃ for 30 min.
NA solid medium: agar powder (1.8% (g/100 ml)) was added to the NA liquid medium.
NA semisolid medium: 0.8% (g/100ml) agar powder was added to the NA liquid medium.
SM buffer solution: taking MgSO 4 ·7H 2 O2.0 g, NaCl 5.8g, 1mol/L Tris-HCl (pH7.5)50mL, 2% gelatin 5mL, deionized water, constant volume to 1000mL, subpackaging, autoclaving at 121 ℃ for 20min, and storing at 4 ℃, wherein SM buffer solution is used for diluting and storing bacteriophage.
Adding the collected soil sample into a triangular flask according to the proportion of adding 10g of soil sample into every 90mL of SM buffer solution, oscillating overnight, taking out and standing. Filtering the soil suspension with a 0.22 μm filter membrane, adding 5mL of the soil suspension into logarithmic phase R.solanacearum bacterial liquid, performing shake culture for 12h, and filtering the co-culture solution with a 0.22 μm filter membrane to obtain phage stock solution. Mixing 0.5mL of logarithmic phase R.solanacearum bacterial liquid with 6mL of NA semisolid culture medium cooled to about 50 ℃, immediately pouring the mixture onto the solidified NA solid culture medium plate to prepare a double-layer plate, partitioning the plate after the upper layer culture medium is solidified, and respectively dotting 2 μ L of phage stock solution (10, 10) diluted in gradient 2 ，10 3 ，10 4 ，10 5 ，10 6 ，10 7 ，10 8 ) Inverted culture is carried out for 24-48 h at 30 ℃, and the existence of plaques is observed (figure 1).
And when the plaques appear, picking the single largest plaque, inoculating the single largest plaque into the R.solanacearum bacterial liquid in the logarithmic growth phase, shaking uniformly, standing for 15min, putting the mixture into a shaking table, performing shaking culture at the temperature of 30 ℃ at 120r/min for 12h to proliferate the phage, filtering the co-culture solution through a 0.22 mu m filter membrane after proliferation culture, and obtaining the plaques by the double-layer agar culture method. Repeating for 3-5 times to obtain the pure phage.
The obtained phage solution without host bacteria is directly stored at 4 ℃, or is mixed with host bacteria and 30% glycerol (v/v) and then stored at-70 ℃.
The phage NN-P42 is classified and named as Podoviridae phage, is preserved in the China center for type culture Collection in 2018, 3 and 6 months, and has the preservation number: CCTCC NO: M2018102.
2. Morphological Observation of bacteriophages
Taking 20 mu L of fresh phage suspension liquid, dripping the fresh phage suspension liquid on a copper net, standing for natural precipitation for 15min, then sucking excessive liquid on the side surface by using filter paper, then dripping a drop of 2% PTA (phosphotungstic acid) on the copper net to dye phage particles, standing for 10 min, then sucking excessive dyeing liquid on the side surface by using filter paper, standing for about 5min continuously to dry a sample, and finally observing the form of the phage by using an electron microscope and taking a picture for recording.
The phage NN-P42 was observed to be tadpole-shaped by transmission electron microscopy, with an icosahedron-shaped head, a diameter of about 100nm, and a short tail, as shown in FIG. 2. The phage NN-P42 belongs to the order caudada (Caudovirales), the family Brevibacterium (Podoviridae), by alignment with the classification standards of the International Committee for viral classification.
3. SDS-PAGE analysis of phage proteins
Taking 15 mu L of concentrated phage NN-P42 particle liquid, uniformly mixing with 3 mu L of 6 multiplied sample buffer solution, boiling for 5min at 100 ℃ to achieve the purpose of protein denaturation, adding a gel sample adding hole (two pore canals can not be adjacent) into a sample and a protein Marker which are subjected to high-temperature treatment respectively, switching on a power supply after marking, carrying out electrophoresis on the sample to be detected to the junction of concentrated gel and separation gel after about 120min under the voltage of 90V, increasing the voltage to 150V at the moment until the sample is electrophoresed to the lower edge of the separation gel, closing an electrophoresis apparatus, taking out the gel, cutting off the concentrated gel, putting the separation gel in a glass plate, dyeing according to a conventional protein silver dyeing method, and taking out for scanning after protein strips are clearly developed.
As shown in FIG. 3, at least 6 protein bands were observed after the protein silver, indicating that the phage particles contained at least 6 structural proteins with relative molecular masses between 25kDa and 100 kDa.
4. Genomic information of bacteriophages
Phage genome DNA is extracted by using a kit (lambda phage DNA extraction kit, Abigen) according to the operation steps, the purity and the concentration of the genome DNA are detected by using a NanoDrop 2000 ultramicro spectrophotometer (NanoDrop company in America), and the obtained product is sent to Meiji biology company in Shanghai for sequencing.
The sequencing result showed that the genome size of phage NN-P42 was approximately 42.28kb, and the GC content was approximately 62.10%. The genomic sequence of phage NN-P42 was BLAST analyzed against selected ones of the standard phages and phylogenetic trees were constructed. As shown in FIG. 4, the NN-P42, the phage isolated by us, has the highest homology with Pelagibacter _ phase _ HTVC010P, so that it can be determined that the phage NN-P4 belongs to the order of the tailed bacteriophages (Caudovirales), the family of short-tailed bacteriophages (Podoviridae).
Shelf life evaluation of (II) phages
Since the development of phage products for practical production is possible in the middle of long-distance transportation, we must consider the shelf life of phage, i.e., the effective conditions for phage preservation.
1. Preservation temperature
Suspension of activated phage NN-P42 (approximately 10) 11 PFU/mL) were kept in multi-tube format, incubated at 4, 25, 37 ℃ for one week, and changes in phage titer were recorded at different times (1, 2, 4, 7 days) with 3 replicates per treatment. As a result, it was found that the titer of the phage was greatly affected by the high temperature of 37 ℃ while the phage remained higher in order of magnitude and slightly decreased in 7 days at the low temperature (4 ℃) and the normal temperature (25 ℃) (FIG. 5).
2. Preserving solvent
Suspension of activated phage NN-P42 (approximately 10) 11 PFU/mL) are respectively mixed with 30 percent of glycerol,Sterile water, 0.85% physiological saline, and SM buffer solution in a ratio of 1: 1, standing at 4, 25, and 37 ℃ for one week, recording the change in phage titer at different times (1, 2, 4, and 7 days), with 3 replicates per treatment. Under the same temperature, different solvents have different effects on the titer of the phage, wherein the treatment effect by using physiological saline as a solvent is the worst, and the number of the phage is reduced most rapidly along with time. 30% Glycerol, SM buffer and ddH at 4 ℃ 2 The differences between the O treatments were not significant (fig. 6-a); ddH at 25 deg.C 2 O and SM buffer treatment is better (FIG. 6-B); SM buffer and 30% glycerol performed better at 37 deg.C (FIG. 6-C). It is demonstrated that we can extend the time and activity of phage preservation by these two solvents even under high temperature conditions.
(III) bacterial wilt prevention and control effect of bacteriophage
1. Inhibitory effect of bacteriophage on Ralstonia solanacearum QL-RS1115 under different resource levels
5 resource levels were prepared: NA, 1/2NA, 1/10NA, 1/20NA, 1/100NA culture medium, utilize the micropore board system, this system comprises 4 links: culture medium, bacterium inoculation, shake culture and enzyme labeling instrument OD determination 600 . The inoculation amount of ralstonia solanacearum is 10 7 CFU/mL, the inoculation amount of the phage is 10 6 PFU/mL. Shake culturing at 30 deg.C for 96h, and determining OD at different times 600 。
As shown in FIG. 7, different nutrient conditions had an effect on the growth of Ralstonia solanacearum and the bacteriostatic effect of the phage. Under low nutritional level (1/20NA), the phage can stably inhibit the growth of ralstonia solanacearum; under high nutrition level, the growth of ralstonia solanacearum can be inhibited about 35 hours after the phage is inoculated, and the inhibition effect of the phage is weakened along with the prolonging of the culture time. The reason is probably that the resistance of the pseudomonas solanacearum to the phage is generated in the later period, but the pseudomonas solanacearum is difficult to generate the resistance under the low resource level, so that the method has certain guiding significance on how to regulate and control plant rhizosphere resources in actual production so as to improve the prevention and control efficiency of the phage.
2. Greenhouse experiment in pots
The effect of the bacteriophage NN-P42 on preventing and treating tomato soil-borne bacterial wilt is investigated in a greenhouse of the Yixing organic solid waste resource synergistic innovation center.
The test soil was rice soil without ralstonia solanacearum. The tomato variety is Micro-Tom (dwarf variety).
The experiment set up 2 treatments, as follows:
control, CK: inoculating only pathogenic bacteria;
treatment, NN-P42: the pathogens and the phage NN-P42 were inoculated.
After surface disinfection of the Micro-Tom tomato seeds: soaking in 70% ethanol for 1min, washing with sterile water for 1 time, soaking in 3% NaClO solution for 5min, and rinsing with sterile water for 6 times. The sterilized seeds were put on a sterile plate padded with sterile double distilled water soaked filter paper and were germinated for two days at 30 ℃. The germinated seeds were transplanted into 9-well nursery trays (filled with soil). In each treatment of 3 disks (9 replicates, total 27 tomato seedlings), tomato seedlings at 3 leaf stage were inoculated with ralstonia solanacearum at a concentration of 5.0X 10 7 CFU/g dry soil; inoculating bacterial suspension 5 days after inoculating Ralstonia solanacearum, the concentration is 5.0 multiplied by 10 6 PFU/g dry soil. After inoculation is completed, the morbidity is counted every day, and finally 3 healthy tomato seedlings are randomly collected for each treatment at the end of the test, and rhizosphere soil is collected.
And (3) measuring the total amount of the tomato rhizosphere ralstonia solanacearum by adopting quantitative PCR. After rhizosphere soil was collected, soil DNA was extracted with a kit. The ralstonia solanacearum specific primer is fliC F: 5'-GAA CGC CAA CGG TGC GAA CT-3', fliC R: 5'-GGC GGC CTT CAG GGA GGT C-3' (Schonfeld et al 2003) (synthesized by Kinshire Bio Inc., Nanjing). Fluorescent quantitative PCR method for pathogenic bacteria
Premix Ex Taq TM (Takara, Dalian) kits according to the methods described in ABI (applied BIO systems)
Amplification on 7500Real-time PCR System amplimer. And calculating the copy number of the key gene according to the drawn standard curve.
Mixing 1g of rhizosphere soil with 9mL of sterile deionized water, shaking at 170rpm for 30min, and filtering the prepared soil suspension with a 0.22-micron filter membrane to obtain phage stock solution. Uniformly mixing 0.5mL of RS bacterial liquid in logarithmic phase with 6mL of NA semisolid culture medium cooled to about 50 ℃, immediately pouring the mixture onto a solidified NA solid culture medium flat plate to prepare a double-layer flat plate, partitioning the flat plate after the upper layer culture medium is solidified, respectively dotting 50 mu L of phage stock solution diluted in a gradient manner, culturing for 24-48 h at 30 ℃, and counting the number of plaques to obtain the number of phage.
The control effect of the phage NN-P42 is shown in FIG. 8, and the inoculated phage NN-P42 shows good control effect. The incidence of the control group was 83.33%, while the incidence of the NN-P42 treatment was 33.33%, which was significantly lower than the control group. The number of rhizosphere pathogens treated group was also lower than the control group (as shown in fig. 8B). The test results show that the phage NN-P42 can significantly reduce the incidence rate of bacterial wilt. The invention analyzes the biological characteristics, evaluates the shelf life and detects the disease prevention effect of the bacterial wilt bacterial specificity phage obtained by screening.
Schonfeld,J.,Heuer,H.,van Elsas,J.D.&Smalla,K.(2003).Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments.Appl. Environ.Microbiol.,69,7248-7256.
Wei,Z.,Yang,X.M.,Yin,S.X.,Shen,Q.R.,Ran,W.&Xu,Y.C.(2011).Efficacy of Bacillus- fortified organic fertiliser in controlling bacterial wilt of tomato in the field.Appl.Soil Ecol., 48,152-159。
Claims (8)
1. A strain of Laurella of Solanaceae: (Ralstonia solanacearum) The obligate phage NN-P42, deposited in the China center for type culture Collection at 3.6.2018 with the deposit number: CCTCC NO of M2018102, classified and namedPodoviridae phage。
2. The use of the lawsonia specialized phage of claim 1 for the control of soil-borne bacterial wilt.
3. The use of the lawsonia specialized bacteriophage of claim 1 in preparing a bacteriophage preparation for controlling soil-borne bacterial wilt.
4. A phage preparation for controlling soil-borne bacterial wilt, characterized in that the phage preparation comprises the Laurella speciality phage NN-P42 of claim 1.
5. The phage preparation for preventing and treating soil-borne bacterial wilt according to claim 4, wherein the content of phage in the phage preparation is not less than 10 6 PFU/mL。
6. The phage preparation for controlling soil-borne bacterial wilt according to claim 4 or 5, wherein SM buffer or 30% glycerol is further added to the phage preparation.
7. The use of the phage preparation of claim 4 or 5 for the control of soil-borne bacterial wilt.
8. A method for preventing and controlling soil-borne bacterial wilt of crops, which is characterized in that the phage preparation of claim 4 or 5 is inoculated to the rhizosphere of a plant of a crop, and the addition amount of the phage preparation is more than or equal to 10 6 PFU/g dry soil.
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