Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type.

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

Analysis of 16S rDNA Sequences from Citrus Huanglongbing Bacteria Reveal a Different "Ca. Liberibacter" Strain Associated with Citrus Disease in São Paulo.

Citrus huanglongbing (HLB, ex greening) is one of the most serious and destructive citrus diseases in the world. It is caused by a phloem-limited and nonculturable bacterium, "Candidatus Liberibacter". The disease occurs in some Asian and African countries and recently has been reported in the state of São Paulo, Brazil. Analysis of the 16S ribosomal (r)DNA of the HLB bacteria from orchards in São Paulo revealed the presence of two distinct strains of "Ca. Liberibacter". One of them, named LSg1 (Liberibacter sequence group 1), was 100% identical to strains from Japan (GenBank accessions AB038369 and AB008366), the Asian forms of the bacteria. The other, LSg2, is genetically distant from the Asian (96.1 to 96.3% identity) and African (95.8 to 96.1% identity) strains. Comparison of the 16S rDNA sequences from the LSg2 and the Asian strain revealed the presence of INDELs and point mutations. Specific primers designed for this Brazilian Liberibacter strain revealed that it is more widely distributed throughout the São Paulo orchards compared with the LSg1 strain. The HLB symptoms caused by both strains are almost identical and, interestingly, both strains were found in the same sample, revealing mixed infection in a citrus plant.

First Report of the Causal Agent of Huanglongbing ("Candidatus Liberibacter asiaticus") in Brazil.

Huanglongbing (ex-greening) disease is one of the most serious diseases of citrus. It is caused by the phloem-limited, gram-negative bacterium "Candidatus Liberibacter spp.". This bacterium is not well characterized mainly because it is still uncultured. There are two known strains, Asian ("Candidatus Liberibacter asiaticus") and African ("Candidatus Liberibacter africanus") that cause severe damage to citrus plants including twig dieback, decline, and death. Symptoms first appear as leaf mottling and chlorosis occurring in one shoot or sector of trees. Later, leaf symptoms resemble nutritional deficiencies (Zn, Ca, and N) that vary depending on the strains, with more severe symptoms caused by "Ca. L. asiaticus". The Asian strains are transmitted by the Asian citrus psyllid (AsCP), Diaphorina citri, which is present in Brazil. The bacterium has been detected in citrus plants in many geographic locations including China, Japan, Thailand, India, the Philippines, the Arabian Peninsula, and Africa. In 2004, plants showing Huanglongbing symptoms were observed in the Araraquara County, a central region of the State of Sao Paulo, the largest citrus-producing area in Brazil. To verify the presence of "Ca. L. spp." in these plants, leaf samples of sweet orange cvs. Hamlin and Valencia were used for DNA extraction and polymerase chain reaction amplification using the specific OI1 and Oi2c primers (1). Amplification of the 16S rDNA was positive for 2 (cvs. Hamlin and Valencia) of 10 analyzed plants. The amplified fragments were cloned and sequenced. The amplicons obtained from both plants showed the same sequence, which differed from "Ca. L. africanus", utilized as the positive control in the amplification experiment (27 divergent bases in 1,160). The sequences were used for BLAST searches, and the results showed identities ranging from 94.71 to 100% with "Ca. L. spp." sequences available at the National Center for Biotechnology Information database (on-line publication). The highest scores were obtained with "Ca. L. asiaticus sequences. These analyses confirmed the presence of such agent in the State of Sao Paulo. To our knowledge, this is the first report of "Ca. L. asiaticus" in Brazil as well as elsewhere in the Americas. The significance of this report relates to the potential damage that this pathogen could cause to the citrus industry in the largest citrus-producing country in the world. It remains unclear how and when the pathogen entered Brazil. Reference: (1) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996.