First Report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis

In 2018, Navarro et al. (2018a, b) identified two new negative sense coguviruses in citrus, citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA). Since then, the members of the genus Coguvirus have been also detected in other plant species (Xin et al. 2017; Wright et al. 2018; Svanella-Duma et al. 2019). In 2016, leaf flecking with oak leaf patterns were observed in five plants among embryo-rescued navel orange (NO) (Citrus sinensis (L.) Osbeck) trees grafted on C22 (C. sunki x Poncirus trifoliata) rootstock, maintained in a shade house. Madam Vinous (MV) sweet orange trees graft-inoculated with blind buds from the symptomatic NO plants developed the same symptoms in the new growth. These symptoms were similar to those on the citrus concave gum (CG) source tree of the California isolate CG301, one of the standard citrus disease isolates used as a positive control for biological indexing (Roistacher et al. 2000). None of the trees with oak leaf symptoms tested positive with reverse transcription (RT)-PCR for a panel of viruses and viroids commonly infecting citrus (Table S1) . In this study, CG301 leaf RNA-Seq data was used as a platform to identify any viral agent(s) associated with the oak leaf symptoms observed in the symptomatic NO trees (Fig. S1). Of ~162.8 million paired-end CG301 RNA-Seq reads (150 bp), de novo assembly of ~9.6 million reads, not mapped to C. sinensis genome (v.1.1), yielded 5,375 contigs. BLASTn using NCBI virus database (txid 10239) identified two contigs, #49 (6,715 nt) and #20 (2,764 nt), which exhibited ~96% sequence identity, respectively, to RNA1 and 2 of CiVA isolate W4 (MG764565; MG764566) and 71-73% identity to that of CCGaV isolate CGW2 (KX960112; KX960111). 5'-Nuclease assay developed based on contig #20 detected coguviral sequences in the five symptomatic NO and graft-inoculated MV trees as well as in CG301 but not in 44 asymptomatic field trees located near the shade house where the symptomatic NO trees had been kept. A full genomic sequence of the coguviruses present in CG301 and a symptomatic NO tree was reconstructed by RT-PCR. Both CG301 and NO isolate have a 6689 nt long negative sense RNA1 (MT922052; MK689372) encoding RNA-dependent RNA polymerase (RdRp) and a 2739 nt long ambisense RNA2 (MT922053; MK689373) encoding movement protein (MP) and nucleocapsid protein (NP). The isolate CG301 and NO share ~96% nucleotide sequence identity. The genome of both CG301 and NO isolate share 95.4-97.8 % sequence identity to that of CiVA isolate W4 and 70-72.9 % sequence identity to CCGaV isolate CGW2. BLASTp showed that RdRp of CG301 and NO isolate have 96.3-97.7 % sequence identity to CiVA W4 RdRp and ~77 % to CCGaV CGW2 RdRp. These data indicated the presence of CiVA in the symptomatic NO trees and in the concave gum source tree CG301. Recent reports of CiVA in South Africa and Greece indicated a potential wider distribution of CiVA in various citrus growing regions that may be associated with two graft-transmissible citrus diseases, citrus concave gum and impietratura disease (Roistacher et al. 2000; Velázquez et al. 2019; Beris et al. 2021; Bester et al. 2021). Although the source of CiVA in the symptomatic NO trees and the degree of CiVA prevalence in Texas had not been determined yet, a possible involvement of vector(s) or other means of spread (e.g. seed transmission) cannot be ruled out (Timmer et al. 2017). The current study demonstrated the need for further studies to determine the level of threat of coguviruses for citrus production in Texas.