A Bioassay That Yields Quantifiable Symptoms of Cucurbit Yellow Vine Disease Caused by Serratia marcescens

Cucurbit yellow vine disease (CYVD), which is caused by the gram-negative bacterium Serratia marcescens and transmitted by squash bugs (Anasa tristis DeGeer), is a devastating disease of cucurbit crops that is emerging rapidly in the eastern half of the United States. The lack of a robust pathogenicity assay for CYVD in the laboratory has hampered functional tests using genomic sequences to investigate the biology of this phytopathogen. In this study we developed and validated a bioassay that yielded consistent and quantifiable CYVD symptoms on squash in the laboratory. We compared inoculation by wounding with a multipronged floral pin frog to inoculation by injection in which a needle was moved in and out of the stem multiple times in each of multiple piercings to mimic the feeding behavior of squash bugs. We found that inoculation by needle injection of ≥10⁸ CFU/ml of S. marcescens into the stem of squash (Cucurbita pepo) plants at the cotyledon growth stage reproducibly induced CYVD symptoms, whereas injecting 10⁶ or 10⁷ CFU/ml did not. Additionally, we found that S. marcescens induced symptoms on all of the squash cultivars tested, and induced symptoms that have not been previously reported, including stem elongation and leaf cupping. In short, through our injection approach of mimicking the natural process of S. marcescens transmission by squash bug feeding, we obtained robust and quantifiable CYVD symptoms. This laboratory bioassay provides a crucial tool for investigating the biology and pathology of this emerging pathogen and for plant breeding screens aimed at combatting CYVD.