Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2010
Publication Date: 3/1/2010
Citation: Zhang, M., Duan, Y., Turechek, W., Stover, E.W., Powell, C.A. 2010. Screening molecules for control of citrus Huanglongbing (HLB) using an optimized regeneration system for 'Candidatus Liberibacter asiaticus' infected periwinkle (Catharunthus roseus) cuttings. Phytopathology. 100:239-245. Interpretive Summary: Citrus huanglongbing (HLB) is one of the most destructive bacterial diseases of citrus plants worldwide. The disease is associated with three different species of Candidatus Liberibacter. The occurrence of HLB in both Sao Paulo, Brazil and Florida, United States has caused serious concern since these two countries produce about one third of the world’s citrus. Because perwinkle plants are another good host of ‘Candidatus Liberibacter asiaticus (Las), we used Las-infected periwinkle cuttings as model plants for developing a rapid process for screening chemical compound against the bacterium while simultaneously assessing phytotoxicity. Based on statistics analysis, the optimal conditions for regeneration of the Las-infected perwinkle cuttings occurred in vermiculite treated with half-strength Murashige and Tucker (MT) medium containing 5 mg.L-1 naphthaleneacetic acid and 5 mg.L-1 indole-3-butyric acids. Using the standardized regeneration system, the regeneration rate for HLB-affected cuttings increased from 0.00% to 60.63 %. Two chemical agents, sodium penicillin G (an antibiotic) and DBNPA (a biocide), were effective in eliminating or suppressing the HLB pathogen when used to treat the HLB-affected periwinkle cuttings. All regeneration plants from the HLB-affected periwinkle cuttings treated with 50 mg. L-1 penicillin G were Las-free. The regeneration plants from Las-infected periwinkle cuttings treated with DBNPA also significantly reduced the percentage of Las-positive plants and titer of Las bacterium. These chemicals may useful not only for the control of citrus HLB but also for potato 'zebra chip' disease.
Technical Abstract: Citrus huanglongbing (HLB) ( also known as citrus greening) 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’ (Las) is the most widely-distributed. An improved system using HLB-affected 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 (MT) medium supplemented with both naphthaleneacetic acid (4 µg/ml) and indole-3-butyric acids (4 µg/ml). This system allowed a plant regeneration rate of 60.63% for cuttings severely affected by HLB in contrast to the lower than 1% regeneration rate with water alone. Two chemical agents, Penicillin G sodium\ (an antibiotic) and 2,2-dibromo-3-nitrilopropionamide (DBNPA,a biocide) were found to be effective at eliminating or suppressing the HLB pathogen in this periwinkle regeneration system. When treated with 50 µg/ml penicillin G, all plants propagated from HLB-affected cuttings were Las-negative as determined by both nested PCR and quantitative real-time PCR (qPCR). In addition, 200 µl/ml DBNPA was also able to significantly reduce the percentage of Las-positive plants and the titer of the Las bacteria.