First report of citrus-associated rhabdovirus in Australia in citrus.

Abstract

Citrus-associated rhabdovirus (CiaRV), putatively classified in the Cytorhabdovirus genus as a strain of Cytorhabdovirus caricae, was first reported in China in 2021. Budwood from an asymptomatic kumquat (Citrus japonica Thunb.) collected in the Northern Territory, Australia in November 2021 a daughter tree was propagated from this budwood onto Carrizo citrange (Citrus ×insitorum Mabb.) rootstock and grown in a controlled environment greenhouse at the Elizabeth Macarthur Agricultural Institute (EMAI). Green bark tissue (100 mg) was sampled from the daughter plant and total RNA was extracted using Invitrogen Trizol TM Reagent (ThermoFisher Scientific, Australia), with DNA removed using DNaseI (Bioline, Australia). The Ramaciotti Centre for Genomics (Sydney, Australia) completed library preparation using a Truseq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, California, United States) and high throughput sequencing using an Illumina NovaSeq6000 sequencing system resulting in 128,234,594 raw reads (2x150bp). The data was trimmed using BBduk (Bushnell 2017) and de novo assembled using rnaSPAdes (v3.15.5) (Bushmanova et al. 2019). The resulting contigs were analysed using a local NCBI ref_viruses_rep_genomes BLAST database (downloaded 15 December 2023). There were eight contigs showing 95-99% identity to citrus leaf blotch virus (CLBV), family Betaflexiviridae (NC_003877), and three contigs (length - 6646, 6044, 1144 nucleotides; coverage - 2043, 174, 214x, respectively) generated showed 80.6, 78.6 and 83% identity to C. caricae (NC_055504). These contigs were analysed using the NCBI BLASTn database online and were 94.5% (MT302546), 90.7% (MT302546) and 94.7% (MT302545) identical to CiaRV isolates from China. Mapping of reads to MT302546 using Geneious Prime (Biomatters, New Zealand) resulted in a consensus sequence (PP998055) of a CiaRV genome with 92.8% identity to both MT302545 and MT302546 isolates. To confirm the presence of CiaRV in the sample, two primer sets, LF117/LR120 (Zhang et al. 2021) and CiaRV-F/CiaRV-R (Huang et al. 2023), were engaged to amplify regions of the L gene or nucleocapsid protein gene respectively. Bidirectional amplicon sequencing was followed by alignment using Geneious Prime (Biomatters) to the consensus sequence obtained by HTS, resulting in 100% identity. A further 176 citrus samples from Australia were tested by RT-PCR using the CiaRV-F/CiaRV-R primers that partially amplify the nucleoprotein (N) gene. Four trees (3 × kumquat, 1 × lemon (Citrus ×limon (L.) Osbeck)) were found to contain CiaRV including a kumquat which was held at EMAI as a living control for CLBV for over 20 years. Bidirectional Sanger sequencing was completed for each of the four positive samples with >99% similarity of each to the original sample. CiaRV was first detected in citrus (Citrus spp.), passionfruit (Passiflora edulis Sims) and paper bush (Edgeworthia chrysantha Lindl.) trees (Zhang et al. 2021) with no symptoms in infected citrus and foliar symptoms observed in the latter two hosts. The papaya virus E strain of C. caricae causes mild leaf vein clearing in Macroptilium lathyroides. When co-infected with cowpea severe mosaic virus (CPSMV), severe leaf mosaic is observed (Cornejo-Franco et al. 2022), noting that CPSMV causes mild leaf mottling in single infection of the host. The likely transmission of CiaRV by whitefly (Zhang et al. 2021), may result in the transmission of CiaRV to other hosts and possible mixed infections, increasing symptom severity. To our knowledge, this is the first report of CiaRV outside of China and it likely has been present in Australia for decades, possibly imported in budwood.