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Screening Molecules for Control of Citrus Huanglongbing Using an Optimized Regeneration System for ‘Candidatus Liberibacter asiaticus’-Infected Periwinkle (Catharanthus roseus) Cuttings

Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of ‘Candidatus Liberibacter’, of which ‘Ca. L. asiaticus’ is the most widely distributed. An optimized system using ‘Ca. L. asiaticus’-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 μg/ml) and indole-3-butyric acid (4 μg/ml). This system allowed a plant regeneration rate of 60.6% for ‘Ca. L. asiaticus’-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the ‘Ca. L. asiaticus’ bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 μg/ml, all plants regenerated from ‘Ca. L. asiaticus’-infected cuttings were ‘Ca. L. asiaticus’ negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of ‘Ca. L. asiaticus’-positive plants and the titer of the ‘Ca. L. asiaticus’ bacterium at 200 μl/liter.