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Diagnosis of huanglongbing-associated Candidatus Liberibacter species in citrus roots by real-time PCR using primers targeting 16s rDNA and nrdB genes

Madhurababu Kunta: Texas A&M University Kingsville Citrus Center

<div>Huanglongbing (HLB) is a destructive disease of citrus which has been associated with three '<em>Candidatus</em> Liberibacter' species, '<em>Ca.</em> Liberibacter' asiaticus (CLas), <em>Ca</em>. L. americanus and <em>Ca</em>. L. africanus. Since HLB has no known cure or resistant cultivars, early detection of its associated bacteria is critical for disease management. Slow symptom development and uneven distribution of associated bacterium in a tree canopy can lead to misdiagnosis when leaves are sampled. Citrus roots have more uniform distribution of CLas compared to the aerial parts of a tree and CLas can be detected in fibrous roots of pre-symptomatic trees. Since the ribonucleotide reductase (RNR) detection assay targets <em>nrdB</em>, a five copy (1.7 X the number of copies of 16S) CLas gene, it is assumed that it will likely be a more sensitive method compared to other CLas detection methods. We compared the incidence of CLas detection of 85 fibrous root samples collected from citrus trees with different rootstock and scion combinations growing in the field using newly designed TXCChlb primers and probe developed based on 16s rDNA sequence with the RNR assay. Sixty-five and 67 of the 85 root samples were CLas positive with the TXCChlb and RNR assays, respectively based on a threshold cycle (Ct) value of ≤ 37. Evaluation of comparative sensitivities of both assays for CLas detection in citrus fibrous root tissues is under progress.</div>