Citrus Bright Spot Virus: A New Dichorhavirus, Transmitted by Brevipalpus azores, Causing Citrus Leprosis Disease in Brazil

Abstract

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(-)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98-99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis.

Keywords: Mononegavirales; Rhabdoviridae; vector mites.

Figure 1

Chlorotic and necrotic symptoms in leaves and fruits of sweet orange trees (Citrus sinensis) collected in three localities of the southern region of Brazil. SC: State of Santa Catarina; RS: State of Rio Grande do Sul.

Figure 2

Transmission electron microscopy (TEM) and RT-PCR results from sweet orange (Citrus sinensis) tissues, exhibiting symptoms of citrus leprosis disease, collected in small citrus orchards in the Brazilian southern region. (A) Micrograph of a section of a leaf collected in Seara, Santa Catarina (SC) showing a group of particles arranged as spoke wheel inclusions (arrow) in the nucleus (N) of a palisade parenchyma cell, exhibiting an electron-lucent viroplasm (*). (B) Micrograph of a section of a leaf collected in Passo Fundo, Rio Grande do Sul (RS) showing the nucleus (N) with electron-lucent viroplasm (*), with some dispersed particles under formation (arrow) in parenchyma cells. (C) Spoke wheel inclusion (arrow) associated with the endoplasmic reticulum near the nucleus (N) in a section of a leaf collected in Marquês de Souza, RS. (D) The 1% agarose gel electrophoresis of RT-PCR products for the detection of dichorhaviruses. MWM: Molecular weight marker, M1181 Ladder (Sinapse Biotechnology, São Paulo, SP, Brazil). Lane 1: reverse-transcription blank; 2–4: chlorotic lesion from sweet orange leaves collected in: Seara (lane 2); Marquês de Souza (lane 3), Passo Fundo (lane 4); lanes 5–8: control plants infected by CiLV-C (expected amplicon size: 322 bp, lane 5); CiLV-C2 from Colombia (expected amplicon size: 795 bp, lane 6); CiLV-N (expected amplicon size: 362 bp, lane 7); CiCSV (expected amplicon size: 500 bp, lanes 8 and 9).

Figure 3

Nucleotide sequence identity of RNA1 and RNA2 of CiBSV and the genomic sequences of CiLV-N (A) and OFV (B). Data were generated by Simplot version 3.5.1. Window size: 200 nts. Step size: 20 nts. The vertical axis indicates the nucleotide identities, expressed as percentages, and the horizontal axis indicates the nucleotide positions of RNA1 and RNA2 of CiLV-N (A) or OFV (B).

Figure 4

Phylogenetic reconstruction of CiBSV isolates (bold) and members of the genus Dichorhavirus, based on L protein sequences (A) and distribution of citrus leprosis (CL)-associated dichorhaviruses and their Brevipalpus vectors (B). The trees was generated by Bayesian inference using MrBayes with 2,000,000 generations, and the varicosavirus lettuce big-vein associated virus (LBVaV) was used as an outgroup. Figures near the main nodes indicate the posterior probability values. The color of vertical bars, at the right, depicts each phylogenetic subgroup of dichorhaviruses and indicates the identified mite vectors, as described in the table. The phylogenetic tree was edited using iTOL. B. The distribution of CL-associated dichorhaviruses was based on literature data [3,11,14] and the results obtained in this study.

Figure 5

Microphotographs of Brevipalpus azores mites found in leaves and fruits of sweet orange (Citrus sinensis) collected in Marquês de Souza, RS (A) and Seara, SC (B). Evaluated morphological characteristics were as follows: number of dorsolateral setae, number of solenidia on the tarsus of leg II (setae), and reticulation patterns of propodosoma and opisthosoma. Ventral and genital shield reticulations can be observed in the center.

Figure 6

The 1% agarose gel electrophoresis of RT-PCR products using specific pair primers for detection of the N gene of CiBSV. MWM: Molecular weight marker, 100 bp DNA Ladder (Madison, WI, USA); lane 1: reverse-transcription blank; lane 2: healthy citrus plant; lane 3: ClCSV-infected glory-bower plant from Brazil; lane 4: CoRSV-infected coffee from Brazil; lane 5: CiLV-N-infected citrus from Brazil; lane 6: CiCSV-infected citrus from Brazil; lane 7: OFV-infected citrus from Mexico; lane 8: citrus plant infected with CiBSV isolate PFd02; lane 9: citrus plant infected with CiBSV isolate MSo02; lane 10: citrus plant infected with CiBSV isolate Ser01.