Novel artificial inoculation protocols in micro propagated and hardened tissue culture ginger plants (Zingiber...

Soft rot caused by Pythium spp. and bacterial wilt caused by Ralstonia pseudosolanacearum are major constraints in ginger cultivation. Reliable and reproducible inoculation methods are essential for studying host pathogen interactions and screening for resistance. This study aimed to standardize simple and consistent artificial inoculation protocols for Pythium myriotylum and Ralstonia pseudosolanacearum in ginger plant. Inoculation procedures were optimized for rhizome-grown plants, in vitro regenerated plants, and hardened tissue-cultured (TC) plants. For P. myriotylum, inoculum levels were standardized using a sand-maize medium and sporangial suspensions. For R. pseudosolanacearum, a combined stem injection and soil drenching method was evaluated. For P. myriotylum, effective infection was achieved with 0.5 g inoculum in tissue-cultured plants, 1.0 g in rhizome-grown plants, and 1.25 × 10³ sporangia/mL in in vitro plants. For R. pseudosolanacearum, consistent symptom development was obtained by injecting 100 μL of bacterial suspension into the lowermost leaf sheath followed by basal drenching. Pathogen re-isolation from infected plants confirmed the reliability of the protocols. The standardized inoculation methods provide a reliable and reproducible system for year-round disease screening across different growth stages of ginger. These protocols will facilitate resistance evaluation and support the development of disease resistant varieties for sustainable ginger cultivation.