Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker

Abstract

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of kiwifruit. In the last years, it has caused severe economic losses to Actinidia spp. cultivations, mainly in Italy and New Zealand. Conventional strategies adopted did not provide adequate control of infection. Phage therapy may be a realistic and safe answer to the urgent need for novel antibacterial agents aiming to control this bacterial pathogen. In this study, we described the isolation and characterization of two bacteriophages able to specifically infect Psa. fPSA1, a member of the Siphoviridae family, is a temperate phage with a narrow host range, a long latency, and a burst size of 178; fPSA2 is a lytic phage of Podoviridae family with a broader host range, a short latency, a burst size of 92 and a higher bactericidal activity as determined by the TOD value. The genomic sequence of fPSA1 has a length of 51,090 bp and a low sequence homology with the other siphophages, whereas fPSA2 has a length of 40 472 bp with a 98% homology with Pseudomonas putida bacteriophage gh-1. Of the two phages examined, fPSA2 may be considered as a candidate for phage therapy of kiwifruit disease, while fPSA1 seems specific toward the recent outbreak's isolates and could be useful for Psa typing.Abbreviations: PSA -Pseudomonas syringae pv. actinidiae; MOI -multiplicity of infection; PFU -plaque forming unit; TE -tris-EDTA; CFU -colony forming unit; TOD -time of death; OD 600 -at 600 nm wavelength Introduction Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is currently damaging both Actinidia deliciosa and A. chinensis worldwide with severe economic losses [1]. On these crops, a pandemic population of the pathogen, most probably originated in China [2,3], incites different kinds of symptoms such as leaf spotting, twig wilting, flower necrosis, reddening of the lenticels, cankers along the leader and trunk as well as exudates oozing out from the canker. This Psa population differs from the one that caused relevant damages to the green-fleshed kiwifruit (i.e., A. deliciosa) in Japan and South Korea in the 1980-1990 period [4-7]. Control measures aiming to reduce the incidence, severity, and spreading of the disease, have been undertaken in all areas of cultivation. A common practice applied everywhere is the cutting and the subsequent destruction of the infected plants or plant parts to reduce the inoculum pressure of the pathogen. Different control strategies have followed in different countries. In New Correspondence: Gustavo Di Lallo, Dipartimento di Biologia, Universita' di Roma "Tor Vergata", I-00133, Rome, Italy E-mail: dilallo@uniroma2.it Phone: þ39 6 72594243 Fax: þ39 6 2023500 Environment Health TechniquesBacteriophages infecting P. syringae pv. actinidiae 1 ß

Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

“…actinidiae [ 27 ], is nearly identical in terms of organisation and sequence (96% nt identity) to a recently sequenced P. syringae pv. actinidiae phage, φPSA2 ( Figure 3 ) [ 59 ]. Both phages were isolated from sewerage, φPsa17 in Dunedin, New Zealand [ 27 ] and φPSA2 from Rome, Italy [ 59 ].…”

φBO1E, a newly discovered lytic bacteriophage targeting carbapenemase-producing Klebsiella pneumoniae of the pandemic Clonal Group 258 clade II lineage

“…Each aliquot was added to 4.5 ml of top agar, mixed, poured onto an LBA plate and incubated O/N at 37 °C. The latency period was defined as the time between infection (excluding the 15 minutes of pretreatment) and the shortest incubation time allowing the production of phages 39 . The burst-size was calculated as the ratio between the number of phage particles released at the plateau level and the initial number of infected bacterial cells.…”

Identification of a newly isolated lytic bacteriophage against K24 capsular type, carbapenem resistant Klebsiella pneumoniae isolates

“…Phage titres were estimated by spotting their dilutions on top agar (0.4%) using the DLA method 39 and incubated overnight (18 h) at 37 °C. The latency period was defined as the time between infection and the shortest incubation time allowing the production of phages 43 . The burst size was calculated as the ratio between the number of phage particles released at the plateau level and the initial number of infected bacterial cells.…”