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Multiple virus and viroid infection of Citrus wilsonii

Citrus wilsonii Tanaka (Citrus ichangensis Swingle x C. grandis L.) is a chance hybrid of a hardy citrus known as ichang papeda and pommelo. It was evaluated in Croatia as a citrus rootstock and an indicator as it displayed severe reactions to Citrus tristeza virus (CTV). Cultivated citrus harbors different types of pathogens including viruses and viroids. The appearance of symptoms in C. wilsonii grown from seeds and bud inoculated with different CTV-strains was monitored over the years. Severe symptoms appeared in some of the trees and we hypothesized that they were caused by additional viruses and/or viroids in the inoculum. The hypothesis was tested by subjecting a representative sample of C. wilsonii showing severe symptoms to high throughput sequencing (HTS). The preliminary results are reported here. C. wilsonii seedlings inoculated with Madam Vinous sweet orange CTV-positive buds showing stunting, severe stem pitting and leaf yellowing symptoms over at least five years were kept in an insect proof greenhouse and monitored for symptoms. Total nucleic acids were isolated using TRI Reagent Solution (Sigma) from leaf vein and bark phloem and DNase treated. The HTS was performed from total RNA ribodepleted on Illumina Nextseq 500 platform yielding a total number of 4.2 million reads (2×150 nt). Contigs resulting from de novo assembly performed by Geneious R10 (https://www.geneious.com) were used for BLAST searches against NCBI viral database. The full genome sequences of CTV, Citrus vein enation virus (CVEV), and Citrus dwarfing viroid (CDVd) were obtained in the C. wilsonii sample. Results suggest the presence of Citrus endogenous pararetrovirus (CEPRV) but due to the low number of reads (˂100) covering 75% of the genome, this needs to be confirmed. CTV strain detected showed 99.8% identities with Italian severe VT (KJ790175.1) and SY genotypes (KC748392.1). CVEV had 98.5% identity with Korean isolate (LC433634.1) and CDVd was 100% identical to a Japanese isolate (AB054623.1). CEPRV was most similar (90% identity) to a Brazilian orange source isolate (KY609920.1). Although CBVd was already known to be present in Croatia, this constitutes the first report of CVEV and CEPRV (if confirmed) in Croatia. The significance of this finding regarding the plant–pathogen complex interactions needs to be investigated further especially in light of the citrus as a host of multiple pathogens and their potential synergistic effects. Judging by the intensity of the symptoms, C. wilsonii has a promising potential to be a good indicator plant.

Citrus wilsonii, high throughput sequencing, Citrus tristeza virus, Citrus dwarfing viroid, Citrus vein enation virus, symptoms

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