Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host-endophyte interaction in vitro and in vivo

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (approximately 1 cm) of papaya (Carica papaya L. 'Surya') planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2-4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea (P. ananatis), Enterobacter (E. cloacae), Brevundimonas (B. aurantiaca), Sphingomonas, Methylobacterium (M. rhodesianum), and Agrobacterium (A. tumefaciens) or two Gram-positive genera, Microbacterium (M. esteraromaticum) and Bacillus (B. benzoevorans) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550=0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.