Antibiotics and thermotherapy have limited effectiveness in eliminating Candidatus Liberibacter asiaticus from citrus budwood

Epigenetic markers related to resistance require regenerating healthy trees from previously infected plant tissues. Thus, sanitizing and regenerating healthy citrus plants from previously Candidatus Liberibacter asiaticus (CLas)-infected tissues constitute a valuable need for breeding programs. Immersion in ampicillin + streptomycin solution and thermotherapy were evaluated separately for eliminating CLas from infected citrus propagules. Valencia sweet orange (Citrus × sinensis) and trifoliate orange (Citrus trifoliata) were chosen as genotypes that were highly and moderately susceptible to CLas, respectively. Infected budwood was used to graft nucellar material with or without CLas. Thirty-two months after inoculation, 2.5-cm grafts were collected and treated by 12 h immersion in an antibiotic solution (1 g/L ampicillin + 0.1 g/L streptomycin) or in autoclaved Milli-Q water, and subsequently grafted onto Rangpur lime (C. × limonia) rootstocks. In a second experiment, thermotherapy was evaluated for treating stem cuttings kept for 0, 1.5 and 10 min at 55 °C in a water bath. The antibiotics did not influence plant regeneration, but CLas was effectively eliminated from only 22.2% of the infected propagules of trifoliate orange up to 15 months after treatment. Thermotherapy at 55 °C for 1.5 min was ineffective in completely suppressing CLas from trifoliate orange plants eight months after treatment, and was harmful to citrus propagation, notably sweet orange. Antibiotic treatment did not eliminate CLas in sweet orange propagules, but a decrease in titer was recorded for both genotypes.