Diversity and control of bacterial contamination of plants propagated in temporary immersion bioreactor system

Microbial contamination can cause major loss to micropropagated plants. In this research, we studied the genera of bacteria contaminated in sugarcane plantlets and rice seedlings grown in different temporary immersion bioreactor (TIB) systems. Samples were collected from the contaminated medium of a TIB system, used in the plant tissue culture laboratory of the Faculty of Science, Maejo University (Chiangmai, Thailand). All bacterial isolates were characterized by the 16S rRNA gene base sequencing technique. A total of 24 bacterial isolates was obtained from sugarcane plantlets of the Saccharum sp. 'LK92-11'. These bacterial isolates were characterized into 4 genera. It was found that the contamination in 20 L TIB container was caused by Bacillus amyloliquifaciens, B. pumilus and B. subtilis, whereas in 700 mL TIB container was by Methylobacterium sp., Paenibacillus sp. and Sphingomonas sp.; all belonging to Firmicutes and Proteobacteria groups. These six bacteria species were not endophytic bacteria of sugarcane, indicating that they were from the environment. Meanwhile, in rice seeds germinated in TIB system, 48 bacterial isolates were identified into 8 genera. These bacteria include Bacillus, Curtobacterium, Microbacterium, Achromobacter, Acidovorax, Burkholderia, Enterobacter, Klebsiella and Pantoea, belonging to Actinobacteria, Firmicutes and Proteobacteria groups. Most of them have previously been reported as endophytic bacteria in rice. These results suggest that the diversity of bacterial contamination in TIB system depends on plant species and explant types, and it is also linked to environmental conditions. Furthermore, procedures in preventing bacterial contamination from various sources during sugarcane propagation in TIB system were designed, based on the obtained results. Obviously, the level of bacterial contamination decreased under controlled conditions and good practices in laboratory.