Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), an economically important disease of cucurbit crop species worldwide. A. citrulli is seedborne and seed transmitted, and seed is the most important source of primary inoculum for BFB outbreaks. During the early stages of watermelon seed germination, A. citrulli colonizes the seed in a saprophytic manner before seed-to-seedling transmission of BFB. To gain a better understanding of this process, we performed transcriptome analysis of A. citrulli cultured in watermelon seed homogenate representing different stages of seed germination. Watermelon seeds were germinated for 24, 48 and 120 h, then crushed to generate seed homogenates (SH). In 24 h SH, the A. citrulli transcriptome showed an overall response to nitrogen starvation. Because glutamine synthetase (GS) plays a central role in bacterial nitrogen starvation response, we studied its role in A. citrulli colonization of germinating watermelon seeds. We observed that in A. citrulli strain AAC00-1, GS was expressed at a constant level in nitrogen rich medium and was up-regulated in response to nitrogen starvation. An A. citrulli mutant (AAC00-13101) that had its uridylytransferase (Aave_3101) gene deleted was unable to up-regulate GS upon nitrogen starvation, but colonized germinating seeds at the wild type (WT) rate. On the other hand, the insertion GS mutant, AAC00-1GSin, displayed reduced seed colonization and reduced BFB seed-to-seedling transmission compared to the WT. These results suggested that the basal GS expression level, but not the induced expression, is required for A. citrulli colonization of germinating watermelon seeds.We also compared the genomes of near clonal pathogenic and non-pathogenic A. citrulli strains to identify novel virulence determinants. Insertion, deletion and point mutations were found between the two genomes. Aave_4140 encoding a glycosyltransferase (gtf) and Aave_4408 encoding flagella motor protein motA were selected for further study. Both genes affected swimming motility of A. citrulli, and mutants with either gene deleted displayed reduced virulence on watermelon seedlings. The motA deletion mutant displayed reduced ability to colonize watermelon cotyledons after inoculation by syringe infiltration, while the gtf deletion mutant colonized seedling tissues at WT levels. Gtf was identified as a novel pathogenicity factor of A. citrulli.