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Abstract
Bacterial fruit blotch (BFB), caused by the bacterium Acidovorax citrulli, is an economically important disease of cucurbitaceous crops worldwide. A. citrulli is seed-borne, and infested seeds are the primary source of inoculum for disease epidemics. Efficacy of current seed management practices varies because the pathogen can ingress through female flowers and become deposited deep within the seed, rendering externally applied seed treatments ineffective. The goal of this research was to inoculate watermelon flowers with biological control agents (BCA) to protect watermelon seeds under field conditions. To select candidate BCAs for field studies, we screened ten BCA bacteria from natural watermelon seed microflora, along with a known BCA, Bacillus mojavensis RRC101 in a series of in vitro assays. We found strains that colonized 24-h watermelon seed exudate and reached higher population levels than A. citrulli therein. These strains also reduced BFB incidence by 41.8-49.4% when applied to watermelon seeds artificially inoculated with A. citrulli. Whole-genome sequence analysis revealed that two of the candidates that provided the best BFB control were Bacillus safensis strains. Furthermore, B. mojavensis RRC101 produced A. citrulli zones of inhibition on culture media, whereas B. safensis strains did not. When A. citrulli was co-inoculated onto watermelon seeds with BCAs, there was a reduction in A. citrulli population growth by 2-3 orders of magnitude; however, the growth of the BCAs was not affected. In the field at anthesis, stigmatic papillae were pollinated and treated with 108 colony-forming units/flower of B. mojavensis RRC101, B. safensis strains, or water as a control. Resulting seeds were inoculated with a cocktail of A. citrulli strains and tested for BFB seed-to-seedling transmission in a grow-out assay. In two experiments, flowers treated with B. mojavensis RRC101 and B. safensis BCA#35 produced seeds with reduced BFB seedling transmission (41.2-47.9%) compared with seeds produced from non-treated flowers (81.2%). Findings from this study will help provide a new approach for protection against A. citrulli.