Black pepper, the "King of Spices", is a globally acclaimed spice, renowned for its distinctive flavour and aroma. To meet the increasing demand for high-quality planting materials, various in vitro propagation techniques have been developed. Despite these efforts, persistent contamination either with fungi or bacteria occurs during the culture process, necessitating a comprehensive investigation into the underlying causes and possible origin of microbial contaminants.

In the present investigation, explants (shoot tips) derived from the black pepper variety IISR Thevam were subjected to various sterilization protocols to minimize contamination and tissue browning. The protocols assessed included combinations of 70% alcohol for 30 seconds to 1 minute, 0.1% mercuric chloridefor 2 to 10 minutes, carbendazim at 0.2% and 0.4% for 30 minutes, propiconazole at 0.1% and 0.2% for 30 minutes, and sodium hypochlorite at 0.1% for 2 minutes. The percentage of contamination before subculturing (14 days after inoculation) was 81%, while after subculturing, the contamination reached 100%. The increased concentration and treatment time led to higher browning rates, while reduced treatment time and concentrations resulted in higher contamination rates. Despite efforts to achieve surface sterilization, fungal growth was observed in treatments with fungicides involving carbendazimand propiconazole at tested concentrations. However, the final rinse samples showed no microbial growth, indicating the effectiveness of surface sterilization and the possible role of endophytic microbial contaminants. Subsequently, the fungal contaminants were collected from the contaminated culture tubes and cultured on potato dextrose agar medium; morphological characteristics were documented and subjected to molecular analysis. The morpho-molecular analyses identified the fungal contaminants as Colletotrichum fructicola, Fusarium proliferatum, and Fusarium equiseti. The present study highlights the persistence of endophytic contaminants in black pepper micropropagation and emphasizes the need to evolve effective sterilization protocols in tissue culture protocols to ensure successful in vitro propagation.