Scanning electron microscopy and in vitro cultivation of endophytic bacteria from potato tubers afflicted with zebra chip disease

Abstract

Zebra chip disease (ZCD) of potato (Solanum tuberosum L.) has caused significant economic losses to the potato industry in the USA. ‘Candidatus Liberibacter solanacearum’ Liefting et al. has been associated with ZCD. This non-culturable bacterium dwells within the phloem of plants where other endophytic bacteria may also live. Knowledge concerning phloem colonization by ‘Ca. L. solanacearum’ and other endophytic bacteria may provide a better understanding about disease biology and could facilitate the development of novel disease management strategies. In this study, endophytic bacteria in potato tubers afflicted with ZCD were evaluated by scanning electron microscopy (SEM), in vitro cultivation, and PCR analyses. Bacillus, coccus, and filamentous bacterial cells were observed by SEM in phloem tissue of ZCD-diseased tubers, but were absent in healthy potato tubers. Fifty-one bacterial isolates were obtained in vitro from ZCD-affected tubers and 34 isolates were from non-ZCD tubers. Comparison of 16S rDNA sequences identified bacteria belonging to 16 genera. Seven (Brachybacterium, Enterobacter, Microbacterium, Paenibacillus, Staphylococcus, Stenotrophomonas and Variovorax) were only isolated from ZCD-affected potato tubers, whereas four (Bosea, Nocardia, Sphingomonas and Sphingopyxis) were only isolated from non-ZCD potato tubers. However, a more sensitive PCR analysis diminished the specific association of the bacteria to either ZCD or non-ZCD tubers, suggesting that infection by ‘Ca. L. solanacearum’ influenced the titres of other endophytic bacteria in potato tubers.