Unraveling Factors Influencing Host Specificity in Xylella fastidiosa

Abstract

Xylella fastidiosa (Xf) is a gram-negative, insect-vectored, plant pathogen that is restricted to the xylem, and impacts various crops such as grapes, blueberry, citrus, peach, pecan, almond, and olives. In Southeastern United States, this pathogen causes bacterial leaf scorch (BLS) on blueberries, but so far this disease has not been reported in other countries. This study broadly explored genetic factors influencing host specificity in Xf strains through genomics, in-vitro, and in-planta experiments. In my first chapter, I explored the relationship between pseudogene makeup and host specificity. By using Pseudofinder, (which accepts genebank format files as an input and blast it against non-redundant protein database) on 151 Xf genomes, I found that Xf subsp. sandyi had the highest pseudogene content followed by subsp. morus and pauca, while subsp. fastidiosa and multiplex had the lowest pseudogene counts. The narrower host range in the first two subspecies compared to the last three supports the hypothesis that more pseudogenes are associated with a restricted host range. In my second chapter, I assessed the potential of a variety of strains to infect blueberry plants. Various Xf strains identified in Europe are known to infect distinct host plants, and exhibit diversity characterized by subspecies and genotypes. From the experiments performed in two different years with different sets of European strains, I found that XYL1966/18 and CFBP8073 might be considered as immediate threat to blueberry production in Europe, as they cause very severe symptoms in plants. As an additional objective of my second chapter, I characterized two Xf genes (Gene_1088 and Gene_2741) proposed to be associated with blueberry hosts. I created mutants by replacing these genes with a kanamycin resistance cassette, and I found that LA-Y3C_1088 mutant showed a different phenotype than its wild-type strain as it produced less biofilm, more planktonic growth, and also showed more twitching motility than its wild type. However, no significant differences were observed between mutants and wild types of the other two strains tested. Similarly, in virulence assessments in planta, LA-Y3C_1088 produced more severe symptoms than the wild-type. These findings suggest that the mutant is still in the exploratory phase, rather than the sticky, vector transmission, clogging xylem phase. The high disease severity of coffee Xf strain CFBP8073 on blueberry plants was noteworthy, since this strain has the two genes studied here as involved in blueberry specificity. This result further supports the hypothesis that these genes are involved in blueberry host specialization. Finally, the research included in my thesis advances the knowledge on host specificity by Xf and will help assess the risk of certain pathogen genotypes for specific hosts.