Isolation of bacteriophage against Pseudomonas syringae pv. syringae in North-West of Iran
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sevda ahadi,1,*
1. University of Maragheh
- Introduction: Plant diseases caused by bacteria are a major economic liability to agricultural production. Disease control has been a major challenge for many bacterial diseases. There is a well-recognized need to develop new environmentally friendly control strategies to combat bacterial crop disease.
Bacteriophages(phages), the viruses that infected bacterial cells, have received increased research interest in recent years as a realistic environmentally friendly means of controlling bacterial diseases.
Their use presents a viable control measure for a number of destructive bacterial crop diseases, with some phage-based products already becoming available on the market.
Phage biocontrol possesses advantages over chemical controls in that phage cocktails can be adapted to target specific disease-causing bacteria.
Unlike chemical control measures, phage mixtures can be easily adapted for bacterial resistance which may develop over time.
In addition to phage cocktails, proteins produced by and isolated from bacteriophage have been investigated as alternatives to using complete and intact phage to treat crop infections.
Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees.
- Methods: Media :
Nutrient broth medium (peptone 5 g; beef extract 3 g; NaCl 5 g per liter) was used as a broth or solidified with 1.8% agar (NA) to grow bacterial hosts. Soft agar for phage plaque-assays contained 0.9% agar. SM buffer (10 mM Tris–HCl, pH 7.5; 100 mM NaCl; 10 mM MgSO4) was used for suspending and titrating the bacteriophages.
Bacterial Strain:
Bacterial strain used in this study is standard strain of Pseudomonas syringae pv. Syringae
Bacteriophage Isolation:
Phages were isolated from soil samples taken from Maragheh apricot orchard that had symptoms of the disease.
The samples were treated as follows: 5 g of soil samples were mixed with 45 mL Sterile distilled water and centrifuged (3000× g for 15 min at 4 ◦C). The supernatant filtered through a 0.45 mm filter and mixed with 5 ml of nutrient broth and 5 ml of 4-hour culture of bacteria in nutrient broth and incubated for 18-24 hour in 37◦C. After incubation 3000 ml volume of chloroform was added to the mixture and incubated at 4 °C for 20 minutes then centrifuged (3000× g for 15 min at 4 ◦C). After filtration supernatant through a 0.45 mm filter, supernatant plated with the double layer agar technique.
The phage titer (PFU/mL) was determined using a standard double layer agar.
- Results: Phage isolated from soil sample and formed clear plaques on the host strain with a diameter of 0.3–1 mm. Phages were purified by successive single plaque isolation.
- Conclusion: Food supplies from agriculture will need to intensify as a result of the anticipated growth in the human population. Phages and phage-based technologies are promising approaches for the treatment and biocontrol of bacterial diseases, including resistant pathogens.
To fully implement phage-based applications, research into their efficacy in the real-world control of diseases in agriculture are needed
- Keywords: Bacteriophages, biocontrol, Pseudomonas syringae pv. syringae
