Sanitation of carrot seeds infected by Ca. Liberibacter solanacearum through a thermal treatment and assesment of its efficacy by a viability qPCR protocol

Candidatus Liberibacter solanacearum is a non culturable bacterium that may affect several important crop species, including host plants belonging to the Solanaceae and Apiaceae families [1]. In carrots, celery and other Apiaceae this pathogen is seed-borne [2], though its transmissibility is currently a matter of debate [3]. In order to ensure an excellent seed quality, we implemented a heat treatment protocol in order to ensure freedom of viable germs possibly present in carrot seeds. Since a qPCR protocol is not able to discriminate viable from dead bacteria infecting any plant matrix, and cultural methods are not feasible either, we implemented a viability qPCR protocol, where seed extracts are treated with monoazides (PMA or EMA). Through the development of a calibration curve, we were able to assess that the naturally infected seed lots used in the experimets harboured bacterial germs in the order of 10E06 cells/g of seed. Our results showed that a thermal treatment at 50°C for 72 hours was able to sanitise carrot seeds: this was confirmed by the viability qPCR detection method that always gave negative results to the analysis. A further confirmation of seed sanitation was obtained by sowing the sanitised seeds under controlled conditions: such seeds were allowed to germinate and produce seedlings. Analysis of seedlings was negative for the presence of Ca. Liberibacter solanacearum 30, 60 and 90 days after sowing. Finally, seed quality assays done on thermo-treated seeds confirmed that their viability and their ability to develop strong seedlings did not differ from that of untreated seeds. [1] Munyaneza J.E. et al., 2010. Plant Dis. 94, 639–639 [2] Bertolini E. et al., 2014. Plant Pathol. 64, 276–285 [3] Loiseau M., et al., 2017. Plant Dis. 101(12), 2104–2109