First report of cherry leafroll virus infecting tree of heaven (Ailanthus altissima), detected in Greece

Authors: OLMEDO-VELARDE, ALEJANDRO - Cornell University; TANNIERES, MELANIE - European Biological Control Laboratory (EBCL); KASHEFI, JAVID - European Biological Control Laboratory (EBCL); SHAKHZADYAN, HAYK - Cornell University; BREUGNOT, DAMIEN - European Biological Control Laboratory (EBCL); Heck, Michelle

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Tree of heaven (Ailanthus altissima) is an invasive species in Europe and North America that serves as a primary host of the destructive spotted lanternfly. Because tree of heaven contributes to ecological disruption and agricultural pest spread, identifying pathogens that infect this invasive plant is important for understanding potential virus reservoirs and evaluating biological control options. Using high-throughput RNA sequencing and confirmatory RT-PCR assays, scientists detected and molecularly characterized cherry leafroll virus (CLRV) from a symptomatic tree in Greece, documenting the first report of this virus infecting tree of heaven globally. Genome analysis revealed high similarity to CLRV isolates from other plant species previously identified in Canada and Germany and identified a putative nepovirus satellite sequence that may represent a novel satellite type. Additional testing confirmed the association of CLRV with the symptomatic tree and its absence in asymptomatic trees sampled at the site. This discovery expands the known host range of CLRV and highlights the potential for tree of heaven to serve as a reservoir that could contribute to virus spread into susceptible agronomic crops.

Technical Abstract: Identifying viruses in Ailanthus altissima (tree of heaven), a fast-growing invasive species (Soler & Izquierdo, 2024) in Europe and North American and host of the invasive and damaging spotted lanternfly, Lycorma delicatula (Barringer & Ciafre, 2020), is critical for exploring potential biological control strategies and understanding the role of A. altissima as virus reservoir. A single A. altissima tree was identified in a visual survey of 30 trees in Thessaloniki, Greece showed leaflets with chlorotic ringspots and mild leaf distortion, with symptoms primarily observed on lower and middle canopy, including basal shoots. Three symptomatic leaf samples exhibiting chlorotic spots (Supplementary Figure 1) were first collected from this tree on June 15, 2022. Total RNA was extracted from each sample using the Spectrum Plant Total RNA kit (Sigma-Aldrich) and pooled to make a composite sample. RNA was subjected to ribosomal depletion using the RiboMinus Plant kit for RNA-seq (ThermoFisher Scientific) and converted to cDNA using the TruSeq RNA library prep kit (Illumina). Paired-end (100-bp) sequencing was performed on an Illumina NovaSeq 6000. De novo assembly of ~19.2 M trimmed reads using rnaviralSPAdes (v 4.2.0) produced 33,502 contigs. Blastn analysis against the NCBI viral database identified two contigs (2061x-2476x coverage) corresponding to cherry leafroll virus (CLRV), sharing the highest identities with CLRV isolates from Canada and Germany. The RNA1 contig shared 92% nucleotide identity (nt) with Canadian isolate MT935684 (6145/6694 nt) and German isolate MW848519 (5763/6389 nt), and the RNA2 contig shared 90% nucleotide identity with Canadian isolate MT935685 (5754/6387 nt) and German isolate MW848519 (5763/6389 nt). Translated protein alignments (blastx) revealed a third contig (2646x coverage) with 30.1% (109/362) amino acid identity to Green Sichuan pepper nepovirus satellite (QWY93780), but no match was found for this sequence when using blastn. No other viruses were identified. RNA1 (6708 nt, accession PV769470), RNA2 (6387 nt, accession PV769471), and putative satellite (1210 nt, accession PV769472) sequences cover 85-100% of CLRV reference genomes. Three contig-specific primers (Supplementary Table 1) were designed for specific detection of CLRV in an RT-PCR assay. Total RNA was extracted from six additional samples collected from the symptomatic tree and two samples collected from trees not displaying symptoms during the initial sampling event using the RNeasy plant mini kit (Qiagen). RT-PCR, followed by sequencing of the PCR products, was conducted using the OneStep RT-PCR kit (Qiagen) according to the manufacturer’s instructions confirming the presence of CLRV in the original samples and its absence in the two samples from trees that did not display the symptoms in Supplementary Figure 1. This first identification of CLRV in A. altissima expands the host range of the virus and highlights its potential role as a reservoir driving CLRV spread into agronomic crops. Its satellite may correspond to a new type of satellite for nepoviruses. The potential of CLRV as a biological control agent for A. altissima is low without further research. While host plants primarily structure CLRV populations, Rebenstorf and colleagues (2006) suggest that mechanisms of inter-species transmission cannot be ruled out, thus posing ecological risks. Additionally, the pollen-borne transmission pathway of CLRV poses a second risk to its use as a biological control agent for A. altissima. Engineering the CLRV genome to reduce virus particle production could enhance its potential as a biocontrol tool for A. altissima. Villar-Alvarez and colleagues (2022) showed that virus particles might not be essential for systemic infection, enabling the development of non-transmissible virus variants with reduced encapsidation potential for biologic