Newly isolated phages preying on Pseudomonas syringae pv. garcae: In vitro and ex vivo inactivation studies in coffee plant leafs

Abstract

Coffee canker, or bacterial halo blight (BHB) of coffee, is a disease caused by the phytopathogenic bacterium Pseudomonas syringae pv. garcae (Psg), having been found for the first time in 1955, in the Garça region (State of São Paulo), and which has stood out in the Brazilian coffee plantations in recent years, leading to severe economic losses that seriously affect coffee trade. The treatments available are still scarce, involving frequent spraying of coffee plantations with either copper derivatives or the antibiotic kasugamycin. However, these compounds should be avoided due to environmental toxicity and the development of bacterial resistances. Herein we report the isolation and physical/biological characterisation of two novel lytic phages and their efficacy in the control of Psg. Phages ph002F and ph004F were isolated from coffee plant leaves in Brazil (Sorocaba/SP and Itu/SP cities), using Psg IBSBF-158 as the host. According to the transmission electron microscopy analyses, both phages belong to the class Caudoviricetes and present myovirus-like morphotypes. Phages ph002F and ph004F showed eclipse times of 5 min and 20 min, respectively, and a burst size of 123 PFU/host cell and 12 PFU/host cell, respectively, allowing to conclude they replicate well in Psg IBSBF-158 with latency periods of 50 min. Phage ph002F (reduction of 4.59 log CFU/mL, compared to uninfected culture) was more effective in inactivating Psg than phage ph004F (reduction of 3.85 log CFU/mL) after 10 h of incubation at a MOI of 10. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.26 log CFU/mL at a MOI of 0.1 or reduction of 5.03 log CFU/mL at a MOI of 10, relative to untreated culture), after 12 h of treatment. This study provides evidence that the isolated phages are promising candidates against the causative agent of BHB in coffee plants.