Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures

The study was undertaken with a view to unravel the source of bacterial colony growth observed in a section of micropropagated triploid watermelon cultures that were supposedly cleansed of the associated endophytic bacteria through antibiotic treatment, and thereafter maintained under stringent sterility checks to prevent lateral intrusion of contaminants. Five different bacteria were retrieved from colony growth-displaying watermelon cultures that were previously treated with gentamycin and five isolates from cefazolin-treated stocks with the organisms showing tolerance to the respective antibiotic. These watermelon cultures were in degeneration phase (over 6 months after the previous sub-culturing), while the actively maintained counterpart stocks appeared healthy with no colony growth on different bacteriological media during tissue-screenings. The latter cultures, however, revealed abundant motile, tetrazolium-stained bacterial cells in microscopy, suggesting tissue colonization by non-culturable endophytes. PCR screening on healthy cultures endorsed tissue colonization by different bacterial phylogenic groups. A few organisms could be activated to cultivation from healthy watermelon stocks through host tissue extract supplementation, which also enhanced the growth of all the organisms. The study indicated that a fraction of antibiotic-tolerant bacteria survived intra-tissue in non-culturable form during the preceding cleansing activity, multiplied to substantial numbers thereafter, and turned cultivable in degenerating cultures contributed by tissue breakdown products. This study brings out the existence of a deep endophyte association in tissue cultures which is not easily dissociable. It also signifies the utility of in vitro system for investigations into plant-endophyte association and to bring normally non-culturable novel organisms to cultivation facilitating their future exploitation.