P-608 - Development of an efficient agroinoculation protocol for cucurbit leaf crumple virus in yellow squash (Cucurbita pepo)

Cucurbit leaf crumple virus (CuLCrV) is a significant pathogen affecting cucurbit crops in the United States. Efficient virus inoculation methods are essential for screening host resistance. However, mechanical (non-vector-mediated) inoculation of CuLCrV is inefficient, necessitating labor-intensive and time-consuming screening via whiteflies. To address this limitation, we developed an infectious partial tandem repeat construct of a CuLCrV isolate from Georgia, USA, using the plant expression vector pCAMBIA2300 and introduced it into Agrobacterium tumefaciens strain EHA105. Agroinfiltration of the construct into the abaxial leaf surface of common bean (Phaseolus vulgaris L.) resulted in systemic CuLCrV infection, but this method was ineffective in yellow squash. To overcome this, we established a highly efficient and reproducible inoculation protocol for squash by inducing micro-wounds with a microneedle followed by rubbing the leaves with a suspension of A. tumefaciens carrying the infectious viral construct. This novel approach facilitates rapid and consistent CuLCrV infection in squash, providing a valuable tool for resistance screening.