Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

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Citrus tristeza virus (CTV) exists in field isolates as a complex of virus isolates. This complex may contain both mild and severe CTV. Using single and multiple aphid transmissions, subiso-lates of the various field isolates were separated. Some CTV isolates that tested negative with the monoclonal antibody MCA13 consisted of MCA13-positive subisolates. Using primers to specific and variable regions of the CTV genome, molecular profiles of the isolates and subisolates were generated and compared. The profiles of the subisolates sometimes were very different from the parent field isolates from which they were transmitted.

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The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different ‘Candidatus Liberibacter species’, ‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’, and ‘Ca. Liberibacter africanus’, which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of ‘Ca. Liberibacter’ at the genus level, providing increased convenience. Recent molecular analyses of ‘Ca. Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus ‘Ca. Liberibacter species’, enabling detection of ‘Ca. Liberibacter’ at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected ‘Ca. Liberibacter solanacearum’, a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

The complete genome of citrus leprosis virus nuclear type (CiLV-N) was identified by small RNA sequencing utilizing leprosis-affected citrus samples collected from the state of Querétaro, Mexico. The nucleotide identity and phylogenetic analysis indicate that CiLV-N is very closely related to orchid fleck virus, which typically infects Cymbidium species.