Evaluation of an RNA-based PCR assay to detect viable Candidatus Liberibacter solanacearum (Lso) in Lso-contaminated carrot seeds using different disinfection methods

Candidatus Liberibacter solanacearum (Lso) is a plant pathogenic bacterium and the causal agent of potato ‘zebra chip’ and carrot Lso diseases. Because this pathogen is suspected to be transmitted via carrot seeds, seed Lso disinfection is important for seed transportation and distribution worldwide. However, since Lso is unculturable, disinfection efficacy cannot be confirmed using bacterial culture assays. Here, we focused on Lso RNA to detect viable Lso cells in Lso-contaminated carrot seeds. Because bacterial RNA is only transcribed in living bacteria, we reasoned that non-detection of Lso RNA after seed disinfection would signify that Lso had not survived, which could be confirmed using reverse-transcription quantitative PCR. Indeed, when six culturable seed-transmitted bacterial strains were all killed by autoclaving or sodium hypochlorite treatment, neither RNA nor colony formation was detected, confirming that non-detection of RNA indicated successful sterilization. When Lso-contaminated carrot seeds were separately treated with five disinfection methods, four, including hot water treatment, resulted in non-detection of Lso RNA, even though carrot RNA was detected and the seeds germinated normally. We suggest that an RNA-based PCR assay is suitable for identifying the presence of plant pathogenic bacteria, which will be useful for screening disinfection methods for Lso-contaminated seeds.