First report of genotypes of citrus tristeza virus breaking resistance in sweet orange and clementine in Spain.

Abstract

Citrus tristeza virus (CTV) classified as Closterovirus tristezae within the genus Closterovirus, family Closteroviridae is broadly distributed in the major citrus-growing regions. CTV has eight main strains (genotypes or phylogenetic groups): T36, T30, T3, T68, VT, RB, HA16-5, and S1, that can induce different symptoms (Sun et al., 2024) with RB isolates being the only variants able to infect and spread within Poncirus trifoliata (Yokomi et al., 2017). In Spain, most isolates belong to the biologically mild T30 strain. Citriculture relies on CTV-resistant P. trifoliata rootstock and its hybrids to prevent virus-induced graft incompatibility. Only rarely, severe VT isolates inducing stem pitting on sweet orange have been reported (Moreno et al., 2008). From February to June 2023, citrus orchards in 4 areas in the Valencian Community were surveyed for monitoring circulanting strains. Five plants (3 sweet orange and 2 clementine trees) showing symptoms of tristeza disease and decline, grafted onto the resistant rootstock Carrizo citrange (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) were individually analyzed by high-throughput sequencing (HTS): sweet orange trees 137.4 in Moncada, 141.4 and 141.5 in Sagunto and clementine trees 142.13 in Nules and 152.7 in Burriana. Five leaves showing vein chlorosis and leaf cupping were pooled and analyzed per tree. Total RNA purified from leaf tissue was sequenced in a Novaseq 6000 platform using TrueSeq Illumina methodology with ribo-depletion. Bioinformatic analysis by CLC Genomics Workbench 10.1.1, according to Ruiz-García et al. (2019), allowed the recovery of 13 near full-length CTV genomes from different strains. Interestingly, 3 isolates belonged to the RB strain, 5 to the VT strain and 5 to the T30 strain. Sweet orange trees exhibited co-infections with CTV isolates from different strains: VT and T30 (OQ714508 and PQ576736) in 137.4; RB, VT and T30 (PQ538530, PQ576743 and PQ576742) in 141.4; and RB, VT and T30 (OQ848758, PQ576738 and PQ576737) in 141.5. In clementine trees CTV co-infections were also observed: RB, VT and T30 (PQ538529, PQ576740 and PQ576739) in 142.13; and VT and T30 (PQ576741 and PQ576744) in 152.7. BLASTn analysis showed that the RB isolates PQ538530 and OQ848758 belong to RB2 group, with the highest nucleotide identity of 99.63% (19198/19269 nt) and 99.62% (19197/19270 nt), respectively, to the isolate B390-5 (KU883265); whereas RB isolate PQ538529 belongs to RB1 group, with the highest nucleotide identity of 99.1% (19082/19255 nt) to the isolate B301 (JF957196). Supplementary Table 1 shows data from the HTS analysis, including additional viruses and viroids detected for which no association with the symptoms was established. The presence of the RB isolates found by HTS was confirmed by RT-PCR analysis and Sanger sequencing, using CTV-RB specific primers previously reported (Cook et al., 2016). In a survey of 147 citrus trees with tristeza symptoms across 7 orchards in Burriana, Nules, and Sagunto, the presence of RB isolates was evaluated. The results identified 46 CTV-RB-infected plants: 35 belonging to RB1 group and 11 to RB2 group, indicating that RB isolates are widespread. To our knowledge, this is the first report of CTV-RB isolates in Spain. The co-infection of biologically mild symptomless RB isolates with Spanish severe VT isolates poses a potential risk for the emergence of recombinant strains, threatening the Spanish citrus industry.