First report of ‘ <i>Candidatus</i> Liberibacter asiaticus’ associated with Huanglongbing in Cuba

Abstract

Citrus greening, also known as Huanglongbing (HLB), is a destructive citrus disease caused by an uncultured phloem-restricted bacterium of alpha subdivision of proteobacteria for which three species, ‘Candidatus Liberibacter asiaticus’ (Las), ‘Ca. L. africanus’ (Laf), and ‘Ca. L. americanus’ (Lam), have been characterized (Batool et al., 2007). Characteristic symptoms of HLB appear both on fruit and on leaves as chlorosis and mottling, the latter resembling zinc deficiency symptoms. In some cases a green colour develops on fruit at the peduncular end. Recently characteristic HLB patterns of mottling and asymmetric chlorosis have been observed in citrus production areas throughout Cuba. Ten leaf samples were collected from five trees with and five trees without symptoms, and DNA was isolated from leaf midribs using the method described by Dellaporta et al. (1983). DNA was used as template in a simple PCR reaction with specific primers A2/J5 (Hocquellet et al., 1999) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon of both ‘Ca. L. africanum’ and ‘Ca. L. asiaticus’, allowing the differentiation of these two strains. PCR amplicons of 703 bp corresponding to ‘Ca. L. asiaticus’ (Hocquellet et al., 1999) were obtained from three plants showing symptoms, but not from symptomless plants. Two amplicons were purified, cloned (pGEM T-Easy vector, Promega) and sequenced. blast and phylogeny analysis revealed that the ribosomal protein sequences (EU649780 and EU649781) shared 100% of identity with each other, and 99% with those of the ribosomal protein and RNA polymerase (rplKAJL-rpoB) of ‘Ca. Liberibacter asiaticus’ identified in Brazil (DQ471904), and Asia, including Japan (AY342001), China (DQ157277, DQ157276), and India (AY266352). The cloned amplicons from ‘Ca. L. asiaticus’ identified in Cuba were further used as probes to detect ‘Ca. L. asiaticus’ in 49/109 plants showing symptoms randomly collected from HLB-affected citrus fields by a dot-blot hybridization assay. These results are the first record of HLB and the molecular identification of the associated bacterium, ‘Ca. L. asiaticus’, in Cuba.