Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated CandidatusLiberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorinacitri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.
Four DNA citrus plant tissue extraction protocols and three methods of DNA extraction from vector psyllid Diaphorina citriKuwayama (Hemiptera: Psyllidae) were compared as part of the validation process and standardization for detection of huanglongbing (HLB). The comparison was done using several criterias such as integrity, purity and concentration. The best quality parameters presented in terms of extraction of DNA from plant midribs tissue of citrus, were cited by Murray and Thompson (1980) and Rodríguez et al. (2010), while for the DNA extraction from psyllid vectors of HLB, the best extraction method was suggested by Manjunath et al.(2008).
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