Isolation, molecular identification, and in vitro control of Campomanesia rufa (O. Berg) Nied. endogenous bacteria

Abstract

ABSTRACT Campomanesia rufa (O. Berg) Nied. is a Myrtaceae species native to the Brazilian Cerrado. As C. rufa is considered endangered, in vitro propagation is an alternative for its conservation. However, the lack of effective disinfection protocols for endophytic microorganisms naturally present in plant tissues hinders the success of micropropagation. In this context, the objective of this study was to isolate, molecular identify, and control endogenous occurring bacteria of C. rufa propagated in vitro. Purified PCR products of bacterial isolates were sequenced by the Sanger method and aligned with homologous sequences using the Basic Local Alignment Search Tool (BLAST) available in National Centre for Biotechnology Information (NCBI) database. MEGA7 software was used to align all sequences and to draw phylogenetic trees. Survival, shoot height, and efficiency of antibiotics (streptomycin, ampicillin, and chloramphenicol) at different concentrations in the culture medium were evaluated. Different Bacillus sp. strains were observed in the plant tissues. When testing the control of Bacillus sp. with antibiotics, 32 mg L-1 ampicillin caused a significant reduction in bacterial contamination with no effect on explant survival. On the other hand, 256 mg L-1 streptomycin caused the greatest reduction in contamination but was lethal to over 90% of the explants. This study is the first report on the occurrence of endogenous bacteria and their control in the context of in vitro native species conservation.

Keywords

• ‘Casaqueira’
• phylogenetic tree
• Bacillus sp.
• antibiotics