Abstract
BackgroundCitrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use.MethodsDouble-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3).ResultsFrom the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs.ConclusionPhylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).
Highlights
Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV)
Identification of CTV contigs by RNA‐seq The sequencing output of the Double-stranded RNA (dsRNA) library consisted of 39,704,476 reads with a mean quality score of 3355 and the 74.72% of bases called with QC ≥ 30
After the BLASTn search for homologies, the classification of viralderived sequences resulted in a number of 176 contigs belonging to CTV with an average depth of 42,083 X
Summary
Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is relevant in those growing regions where no dedicated certification programs are currently in use. In a citrus industry not ruled by the certification of propagative material, it could be common that budwood grafting spreads the infection. This inadvertent spread of CTV potentially severe strains can irreversibly compromise the nationwide production of citrus for fresh fruit, transformation and export trade
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