GENOMIC ANALYSES AND PATHOGENICITY DETERMINANTS OF XANTHOMONADS ASSOCIATED WITH ARACEAE AND ARALIACEAE

Abstract

Araceae and Araliaceae, two significant plant families, encompass economically important ornamentals, including Anthurium, Dieffenbachia, Philodendron, and Syngonium (Araceae) and Hedera, Schefflera, Fatsia, and Polyscias (Araliaceae). These plants face threats from two bacterial pathogens, Xanthomonas axonopodis pv. dieffenbachiae (Xad) and X. hortorum pv. hederae (Xhh), causing leaf blight and leaf spot diseases globally for several decades. In previous studies, both Xad and Xhh strains were identified as highly heterogeneous groups and were subsequently reclassified into different Xanthomonas species. The Pacific Bacterial Collection at UHM houses over 100 Xad strains and about 20 Xhh strains isolated from various genera of Araceae and Araliaceae, respectively. The taxonomic positions of Xad and Xhh in our culture collection, along with their pathogenicity and virulence factors interacting with Araceae and Araliaceae, await further investigation. This dissertation aims to achieve two primary objectives: (i) To investigate the phylogenetic and taxonomic relationships of Xanthomonas spp. associated with Araceae and Araliaceae; (ii) To explore the molecular mechanisms of pathogenicity and virulence in Xanthomonas spp. associated with Araceae and Araliaceae. In the first study (Chapter 2), we focus on elucidating the phylogenetic and genealogical relationships among Xanthomonas species isolated from Araceae. Multi-locus sequence analysis with five housekeeping genes is employed, analyzing 59 representative strains from various Araceae hosts, temporal and geographical origins, and 100 closely related Xanthomonas and Stenotrophomonas species/pathovars. The results reveal diverse clustering, raising some certainties and some uncertainties about the taxonomic positions of Xad strains. This prompts an exploration of whole genome constituents for genes and gene clusters involved in pathogenicity in the following Chapter 3. In the second study (Chapter 3), we aim to determine the taxonomic positions of selected representative Xad strains and investigate potential pathogenicity factors, including the type III secretion system (T3SS) and type III secretion effectors (T3Es). Genome-scale data unveils accurate taxonomic positions, and the presence or absence of T3SS and T3Es suggests some Araceae strains as potential pathogens, while others are considered commensal strains. In Chapter 4, three new species, X. hawaiiensis sp. nov., S. aracearum sp. nov., and S. oahuensis sp. nov., associated with Araceae, are proposed based on the overall genomic relatedness indices. Pan- and core-genome analyses are conducted for new species along with other type strains of Xanthomonas and Stenotrophomonas species. Lastly, Chapter 5 comprehensively discusses Xhh strains isolated from P. guilfoylei. Based on phylogenetic analysis and genomic relatedness, these representative strains are reclassified as X. euvesicatoria. Comparative genomic analyses reveal that avirulent strain has a big deletion in the flagellar biosynthesis gene cluster 2, resulting in nonmotile and aflagellation. Validation of gene function through reverse mutagenesis and pathogenicity test on P. guilfoylei suggests that two flagellar component genes, fliR and flhB, might be virulence determinants in X. euvesicatoria- P. guilfoylei pathosystem.