Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.
The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, ‘Candidatus Liberibacter asiaticus’, is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of ‘Ca. L. asiaticus’ in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was ‘Ca L. asiaticus’. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the ‘Ca. L. asiaticus’ contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of ‘Ca L. asiaticus’ in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one ‘Ca. Liberibacter’ cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 ‘Ca. Liberibacter’ cells per phloem cell.