Identification and characterization of Phi1 and Phi3 bacteriophages targeting Xylella fastidiosa subsp. pauca, the causal agent of olive quick decline in Italy.

The xylem-limited bacterium Xylella fastidiosa (Xf) is increasingly threatening global agriculture due to the absence of effective control strategies. Phage biocontrol represents a promising alternative; however, only a limited number of bacteriophages have been identified against Xf. In this study, we report the isolation and characterization of two phages of Xf subsp. pauca (Xfp), namely Xylella phage Phi1 (Phi1) and Xylella phage Phi3 (Phi3) and investigate their potential as safe and suitable biocontrol agents for future agricultural applications. Transmission electron microscopy (TEM) showed that Phi1 and Phi3 exhibited morphological features typical of podovirus and siphovirus morphotypes, respectively. In addition, the interactions between Phi1, Phi3 and Xfp cells were assessed by TEM, indicating efficient phage replication on Xfp cells. The high-throughput sequencing and genomic analysis of Phi1 and Phi3 revealed genome sizes of 44,345 bp and 55,413 bp, respectively. ANI analysis showed that Phi1 sequence shares the highest homology (92.52%) with Xanthomonas phage phi Xc10, suggesting that it is a novel bacteriophage species. In contrast, Phi3 exhibited the highest identity (96.70%) with Xylella phage Salvo, indicating that it is likely the same species. Phylogenetic analysis based on whole-proteome comparisons, allocated Phi1 and Phi3 together with other Xylella phages within the genera Pradovirus and Salvovirus, respectively. The bioinformatics analysis performed on Phi1 and Phi3 genomes predicted that both phages undergo a strictly lytic infection cycle, with no evidence of temperate markers, virulence factors or AMR genes. Furthermore, the off-target effects of Phi1 and Phi3 were evaluated on fourteen bacteria isolated from olive trees in this study and ten beneficial bacteria from our collection, showing no bacteriolytic activity against any of the tested non-target bacteria. These findings make Phi1 and Phi3 suitable candidates for the development of an environmentally friendly, phage-based biocontrol strategy against Xf. However, their efficacy under field conditions remains to be evaluated.