Identification and control of endophytic bacteria during in vitro cultures of Staphylea pinnata L.

Abstract

This study focused on the identification and elimination of endophytic bacterial contaminations during in vitro propagation of European bladdernut (Staphylea pinnata). Axillary shoots were propagated on Murashige and Skoog medium with 20 mg ∙ dm−3 FeEDDHA, 5 μM BA and 0.5 μM NAA at 20/18°C (day/night) and a 16-h photoperiod. Clouding by endophytic bacterial colonies was observed where shoots contacted the media. Bacteria were isolated and separated by repeated streaking as two strains. Gram staining revealed that both strains were Gram-negative. The colonies were very precisely identified as Acinetobacter johnsonii, strain ATCC 17909, and Methylobacterium rhodesianum, strain DSM 5687, using VITEK®2—a rapid bacterial identification system—and the 16S rRNA gene sequencing method. The agar disc-diffusion test proved that both bacterial strains were susceptible to 13 antibiotics (out of 25 tested), derived from the groups of fluoroquinolones, aminoglycosides and tetracyclines. Doxycycline or gentamicin (100–300 mg ∙ dm−3) was added to the S. pinnata shoot propagation medium to eliminate bacteria. Gentamicin 100 mg ∙ dm−3 showed the best effect, inhibiting the growth of endogenous bacteria (63%) when applied in the medium for 4 weeks. After the following transfer to media without antibiotics, shoots developed axillary buds and bacterial colonies were not observed.

Keywords

• acinetobacter
• antibiotics
• decontamination
• gene sequencing
• methylobacterium
• vitek®2 system