Characterization of four <i>Acidovorax</i> phages and their potential in phage biocontrol for lamb’s lettuce seed decontamination

Bacterial black spot, caused by <i>Acidovorax valerianellae</i>, is responsible for significant yield losses in lamb's lettuce (<i>Valerianella locusta</i>) in many producing countries, especially Europe. Currently, no resistant varieties of <i>V. locusta</i> are available that effectively control the disease under field conditions. Bacteriophage-based biocontrol has been suggested as a sustainable and natural alternative strategy to combat bacterial pathogens. In this study, novel phages infecting <i>A. valerianellae</i> and <i>Acidovorax cattleyae</i>, Alfacinha1, Alfacinha3, Acica, and Aval, were isolated and characterized. Based on comparative genomics, these phages represent three new phage genera. Aval and Acica phages revealed genomic features characteristic of temperate lifestyle, encoding toxins likely associated with lysogenic conversion, which contrasts with Alfacinha1 and Alfacinha3. The latter was selected for application as a biocontrol agent during seed steeping. This phage reaches an 87% reduction in the <i>A. valerianellae</i> concentration on artificially infested seeds. Importantly, this reduction results in an increased germination rate from 58.9% to 93.3%. Moreover, the infected seedlings had a dramatic reduction in vigor index after 22 days of growth, whereas the phage-treated ones had a vigor index similar to the negative control, reinforcing the ability of bacteriophages to effectively reduce disease progression. We further evaluated the impact of lipopolysaccharides in phage suspensions on the development of seedlings. Here, we demonstrated that the presence of lipopolysaccharides do have an impact on seedling development, significantly reducing the number of roots developed. This study shows how genomic analyses and tailored bioassays represent an essential route to ensure safe phage application and demonstrates the potential of a phage-based biocontrol strategy against <i>A. valerianellae</i>.IMPORTANCEBacteria continue to globally cause serious damage to a variety of crops. One example is a bacterial black spot of lamb's lettuce caused by <i>Acidovorax valerianellae</i>. It has spread across Europe, resulting in economic losses of at least 10% in tonnage annually. Faced with the inefficiency of conventional control methods, an alternative and sustainable strategy based on the use of bacteriophages was pursued in this study. We present for the first time the isolation and characterization of <i>A. valerianellae</i>-specific phages. Moreover, we assessed their biocontrol potential in seed decontamination since the disease primarily spreads from seeds to seedlings. Interestingly, seed treatment with one of our phages reaches an 87% reduction in bacterial concentration. More importantly, this reduction results in an increased germination rate from 58.9% to 93.3%. Finally, our study demonstrated for the first time the need for removing endotoxins from phage suspensions as they impact plant development when used as a biocontrol agent.