Efficient and Rapid Bioassay for <i>Xanthomonas citri</i> pv <i>. punicae</i> Blight on Pomegranate

It is crucial that experiments dealing with plant-pathogen interactions are robust and reproducible. For evaluating the disease-resistance ability of a host plant against any pathogen, controlled conditions are a prerequisite that cannot be obtained with soil-based systems. Variation in environmental conditions during challenge inoculation of a pathogen, when performed in fields or a polyhouse, may lead to spurious results. This is of particular importance when dealing with disease resistance in a fruit tree like pomegranate, as determined after years of research efforts. Due to lack of uniformity and environment variability, the possibility of pathogen escapes and false positives is large under field or polyhouse conditions. To address these limitations, we have used tissue-cultured plants and developed an easy, reliable, rapid, and efficient in vitro bioassay for bacterial blight on pomegranate. Owing to its performance under a closed system, this system minimizes the risk of pathogen escapes, reduces environmental variability, and provides a robust high-throughput screening platform for disease resistance and tissue sampling for downstream functional genomics studies. The protocols enable reproducible experiments to study plant response to bacterial blight infection. Stepwise protocols and detailed instructions are provided to carry out the screening experiment in pomegranate or other similar plant species. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Preparation of nodal explants and in vitro rooting Basic Protocol 2: In vitro blight bioassay Basic Protocol 3: Pathogen re-isolation and confirmation.