Identification of Endogenous Bacteria in Micropropagated Helleborus ×nigercors

Abstract

During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially contaminated. To determine the identity of contaminants, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S as belonging to the Paenibacillus and Luteibacteri genera, respectively. Strain H7R had highest sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Strains H7R and H7S were unable to grow in the absence of plant tissue and other bacterial species. Paenibacillus sp. H7G was screened using combinations of antibiotics including streptomycin sulfate, gentamicin sulfate, and cefotaxime, and was only eradicated by concentrations of gentamicin sulfate above phytotoxic levels. This is the first documented exploration of bacterial endophytes associated with Helleborus species.