Isolation and Speciation of a New Bacteriophage for Controlling Pectobacterium atrosepticum

Many trials were performed in this study for isolation and purification of bacteriophage; that has lytic activity against Pectobacterium atrosepticum, previously known as Erwinia carotovora sub sp. atroseptica. This pathogen is a common phytopathogen that causes significant economic losses in Egypt and worldwide. For developing a biocontrol agent for this blackleg or soft rot pathogen of solanaceous plants, a virulent P. atrosepticum phage (ϕPaP2) affiliated to the family Podoviridae was successfully isolated from soil in the Nile Delta of Egypt. Its morphological, epidemiological characteristics, and lytic activity was included in this study. The phage was isolated and purified following a single plaque isolate approach, then propagated in bacterial cell suspension inoculated by the Multiplicity Optimal Infection (MOI) of the phage. The phage plaque size was 2.0–3.0 mm in diameter. Morphological examination by Electron microscope showed that phage has an icosahedral head of 55-60 nm in diameter and a short tail of 15-20 nm in length that might belong to the family Podoviridae order caudovirales. The entire head structural protein was approximately 37 kDa in molecular weight, detected by the usual SDS–PAGE technique. The genomic size and genes function of the phage will be studied in a future study. The optimal MOI value of the phage was 1.0 based on the average count of plaque-forming units (PFU). The single-step growth curve showed that the latent period of infection for ϕPaP2 phage was 50 min, while its complete infection cycle was 150 min. The phage has an average burst size of ∼38 virions per bacterial cell.
The in vitro stability of phage has shown that it has the ability to retain activity at temperature up to 70ºC but lost its activity at 75ºC or higher, while the optimal temperature of phage activity was at 25ºC. The phage showed optimal activity at pH 7, while lytic stability was maintained at pH range 4-9. 
The host range of the ϕPaP2 phage /specificity was tested against 18 isolates; 7 belong to P. atrosepticum, 11 were non-target bacterial isolates, including other bacterial pathogens of potato and some isolated antagonistic bacteria.
The bacteriolytic activity of phage was expressed In Planta (Potato tubers cv. Spunta) that displayed 100% inhibition of soft rotting of tissues (maceration) caused by the pathogen after incubation at 25ºC for 48 h compared to phage- untreated tubers. The longevity of phage ϕPaP2 was studied in different soil types. In general, the longevity of the phage was found higher in clay soil than sandy soil. The presence of the host bacterium in both clay and sandy soil showed a remarkable increase in the longevity of the phage compared to bacteria- untreated soil. In conclusion, the isolated bacteriophage has had the ability to suppress P. atrosepticum in vitro and to protect potato tuber from rotting caused by the pathogenic bacteria in concern.