# Sixty years from the first disease description, a novel badnavirus associated with chestnut mosaic disease

Armelle Marais, Sergio Murolo, Chantal Faure, Yoann Brans, Clement Larue, François Maclot, Sébastien Massart, Michela Chiumenti, Angelantonio Minafra, Gianfranco Romanazzi, et al.

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## To cite this version:

Armelle Marais, Sergio Murolo, Chantal Faure, Yoann Brans, Clement Larue, et al. Sixty years from the first disease description, a novel badnavirus associated with chestnut mosaic disease. Phytopathology, 2021, 111 (6), pp.1051-1058. 10.1094/PHYTO-09-20-0420-R. hal-03036205

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Submitted on 6 Sep 2021

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1 Sixty years from the first disease description, a novel badnavirus associated with

2 chestnut mosaic disease

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4 Armelle Marais*†¹, Sergio Murolot², Chantal Faure¹, Yoann Brans³, Clément Larue⁴,⁵, François

5 Maclot⁶, Sébastien Massart⁶, Michela Chiumenti⁷, Angelantonio Minafra⁷, Gianfranco Romanazzi²,

6 Marie Lefebvre¹, Teresa Barreneche¹, Cécile Robin⁴, Rémy J. Petit⁴, Thierry Candresse¹

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8 ¹Univ. Bordeaux, INRAE, UMR BFP, Villenave d'Ornon, France

9 ²Department Agricultural, Food and Environmental Sciences, UNIVPM, Ancona, Italy

10 ³Laboratoire de Virologie et de Biologie moléculaire, CTIFL, Prigonrieux, France

11 ⁴Univ. Bordeaux, INRAE, UMR Biogeco, Cestas, France

12 ⁵INVENIO, Maison Jeannette, Douville, France

13 ⁶Plant Pathology Laboratory, TERRA-Gembloux Agro-BioTech, University of Liège, Belgium

14 ⁷CNR Institute for Sustainable Plant Protection —Bari, Italy

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16 * Corresponding author: A. Marais; Email: armelle.marais-colombel@inrae.fr

17 †AM and SM contributed equally to this work

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19 Keywords: High throughput sequencing, Castanea sp., etiology, viral disease, Badnavirus,

20 *Caulimoviridae*

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22 The nucleotide sequences reported here have been deposited in GenBank under the accession

23 numbers MT261366, MT269853, MT270664-MT270682, and MT339503-MT339590.

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ABSTRACT

Although the chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not elucidated. Here, using classical approaches in combination with high throughput sequencing (HTS) techniques, we identify a novel *Badnavirus* that is a strong etiological candidate for ChMD.

Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted and sequenced on an Illumina NextSeq500 (2x150 or 2x 75 nt). In each source, we identified a single contig of about 7.2 kilobases that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5% with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons show that this virus is a novel species for which we propose the name *Chestnut mosaic virus* (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak), as well in chestnut trees presenting typical ChMD symptoms in the field (100% and 87% in France and Italy surveys, respectively).

Datamining of publicly available chestnut SRA transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two *Castanea mollissima* sources from the USA, as well as ChMV detection in *C. dentata* from the USA. Preliminary epidemiological studies, performed in France and in Central Eastern Italy, showed that ChMV has a high incidence in some commercial orchards, with a low within-orchard genetic diversity.

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European chestnut (Castanea sativa Mill.) has a long-standing tradition of cultivation in many European countries. It is an important species, both economically as a source of timber and fruit and ecologically through the multiple ecosystemic services it provides. In Europe, chestnut covers about 2.5 million hectares, mainly concentrated in France, Italy, Spain, Portugal, Switzerland, the Balkan regions, and Southern England (Conedera et al. 2016). Chestnut (Castanea spp.) can be heavily affected by various pathogens. The most detrimental are caused by fungal-like organisms (Oomycetes) and fungi such as Phytophthora cambivora Petri and P. cinnamomic Rands., the agents of ink disease, or Cryphonectria parasitica, the causal agent of chestnut blight, which all provoke disorders that can lead to tree mortality (Prospero et al. 2012; Rigling and Prospero, 2018). In Italy, Gualaccini (1958) described a chestnut disease associated with viral symptoms (mosaic, shoots with asymmetric leaf blade deformation) which was again reported in Campania during the 80s, (Ragozzino and Lahoz 1986), and in the Marche region (central eastern Italy) in 2000 (Antonaroli and Perna; 2000). In France, the disease was first identified circa 1987 on cultivars of C. sativa x C. crenata hybrids from commercial orchards located in the south-west of the country. Desvignes (1999b) made a more detailed description of the symptoms, which are characterized by necrotic lesions in the bark and wood that turns into cankers, chlorotic lesions and yellow stripes on leaf veins and partial limb atrophy, and called this disease Chestnut Mosaic Disease (ChMD). This disease can heavily affect the production of both young and secular trees (Antonaroli and Perna, 2000). It has also been reported in Japan and Hungary (Shimada, 1962; Horvath et al. 1975). Even if its etiology has long remained unknown, researchers hypothesized that the causal agent of ChMD could be a virus, introduced in Europe between 1940 and 1960 when a number of C. crenata cultivars were imported from Japan for breeding purposes. Investigations in France and Italy established that the causal agent can be eliminated by thermotherapy, is aphid-transmissible, and is graft-transmissible to Castanea and Quercus species in which it may elicit symptoms (Desvignes
and Lecocq, 1995; Desvignes, 1999b; Vettraino et al. 2005). The susceptibility to the ChMD agent of *Castanea* species/cultivars has been evaluated in several studies (Desvignes, 1992; 1999b; Desvignes and Lecoq, 1995). Three categories of cultivars could thus be defined from tolerant to moderately and fully susceptible. Graft incompatibility was also observed when cultivars of different susceptibilities are assembled by grafting. Most of the *C. sativa* cultivars and hybrids are tolerant to ChMD, although some well-known French hybrids like ‘Maraval’ (Ca 74) are fully susceptible, and used for indexing purposes to detect the ChMD agent in tolerant cultivars (Desvignes and Lecoq, 1995).

In the last decade, a number of studies have highlighted the potential of non-targeted molecular diagnostics based on high-throughput sequencing (HTS) to elucidate the etiology of viral plant diseases and to provide viral sequence data from which rapid diagnostic molecular assays can be developed (Martin et al. 2016; Villamor et al. 2019). Since 2009, HTS combined with bioinformatics have been used for the discovery, characterization, and *de novo* assembly of the genome of known and novel plant viruses and viroids (Rott et al. 2017; Kreuze et al. 2009). This has accelerated the application of HTS technologies in the field diagnostic of diseases (Massart et al. 2014), and in quarantine regulations (Martin et al. 2016; Massart et al. 2017).

Badnaviruses are plant pararetroviruses belonging to the family *Caulimoviridae* that have emerged as serious pathogens causing severe yield losses in a wide range of economically important crops all over the world (Bhat et al. 2016). The genome of badnaviruses is composed of a non-covalently closed, circular double-stranded DNA (ranging from 7.2 to 9.2 kbp) and is encapsidated in bacilliform virions. This genome typically harbors three open reading frames (ORFs) encoding, respectively, a protein of unknown function, the virion-associated protein (VAP), and a polyprotein containing functional and structural domains [movement protein (MP), coat protein (CP), aspartic protease (AP), reverse-transcriptase (RT) and RNase H]] (Hohn and Rothnie, 2013; Bhat et al. 2016).
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Badnaviruses can also be present as integrated sequences in some host plant genomes (endogenous badnaviruses) (Staginnus et al. 2009; Bhat et al. 2016). The contribution of these integrated sequences to host and virus evolution is still poorly understood (Geering et al. 2014).

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## MATERIALS AND METHODS

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**Plant samples and virus isolates.** Virus isolates included in this study are listed in Supplementary Table S1. Isolate LC1224H is originated from a red oak (*Quercus rubra*) artificially inoculated in 1992 with a chestnut mosaic source from a hybrid *Castanea sativa* x *Castanea crenata* included in a French breeding program. Leaves of grafted oaks displayed typical symptoms characterized by chlorotic mottle, yellow veins, and mosaic (Desvignes and Lecoq 1995) (Figure 1A). Isolate FRlc1224A was derived from the same source, and is the result of a back-inoculation by grafting of LC1224H to the natural chestnut hybrid Maraval (Ca 74; *C. crenata* x *C. sativa*) indicator (Desvignes et al. 1992). Isolate LC1224F originated from a Maraval indicator inoculated by aphid transmission from an initial ChMD source in a *C. crenata* x *C. sativa* French hybrid (Desvignes and Cornaggia, 1996). The LCA552 and LCA584 isolates were collected from *C. sativa* trees in France in 2009 and
118 2018, while the T32018 disease source was isolated from a French hybrid *C. crenata* x *C. sativa* in 2018. All of these isolates have been held and propagated on 'Maraval' indicator plants at the CTIFL virology laboratory (Lanxade, France).

121 In the framework of a survey carried out in Italian chestnut orchards to monitor chestnut blight (Acquasanta Terme (AP), locality Umito, Italy) (Murolo et al. 2018), typical leaf symptoms of ChMD were recorded in 2016. Six symptomatic plants were collected, pooled (10 -15 symptomatic shoots) and included in the HTS analysis (ITumito39 source).

125 In order to evaluate the incidence of ChMV, chestnut trees from INRAE chestnut biological resource center (https://www6.bordeaux-aquitaine.inrae.fr/biogeco/Ressources) were sampled. This orchard is located on the Villenave d'Ornon INRAE center (France) with trees distributed in three plots (A, E, or Port, Table S1). A total of 43 *C. sativa*, 14 *C. mollissima*, six *C. crenata* and 32 hybrid chestnut trees were sampled, corresponding to a total of 38 symptomatic trees with typical ChMD symptoms, 47 asymptomatic trees, and 10 trees with dubious or atypical symptoms. In addition, in the Central eastern Italy Marche region, leaves from 60 symptomatic and from 10 asymptomatic grafted *C. sativa* cv. Marrone trees of different ages were collected in a commercial chestnut orchard (Plot I, Table S1).

134 Isolates FRlc1224A and ITumito39 were used for the HTS analysis, whereas all other samples were included either in the incidence analysis or in the causal relationship analysis (Table S1).

136 **Total RNA extraction and RNA-Seq analysis.** Symptomatic leaves from a 'Maraval' indicator (FRlc1224A) were collected and used to extract total RNAs according to the protocol described by Reid et al. (2006). For the Italian material, total RNAs were extracted from symptomatic leaves according to the protocol described by Gambino et al. (2008). Total RNAs were then submitted to a DNAse treatment following the manufacturer's recommendations (Fisher Scientific, Illkirch, France). Ribosomal RNAs were removed using a Ribominus Plant Kit for RNA-Seq (Invitrogen, Fisher
142 Scientific, Illkirch, France) before cDNA library synthesis with the Illumina TruSeq Stranded RNA library Prep kit (Illumina, Inc., San Diego, CA) and sequenced on an Illumina NextSeq500 (2x150 nt or 2x75 nt) in a multiplexed format (GIGA-Genomics facility, Université de Liège, Belgium).

145 **Bioinformatic analysis.** Primary quality analyses were performed using Geneious Prime 2019.2.1 Software (https://www.geneious.com). *De novo* assemblies of quality filtered reads were performed using Velvet (Zerbino and Birney, 2008), Geneious R 11 (https://www.geneious.com), and Spades (Bankevich et al. 2012), or using the CLC genomics workbench 8.0 (http://www.clcbio.com). Contigs were annotated by BlastN and BlastX comparisons with nucleotide and non-redundant protein GenBank databases, respectively. Blast results were screened using e-value thresholds of 10⁻⁶ and 10⁻⁴ for BlastN and BlastX, respectively. Publicly available chestnut RNA-Seq transcriptomic data were retrieved from the NCBI Sequence Read Archive (SRA) and downloaded reads were mapped against the sequence of the FRlc1224A isolate using CLC Genomics Workbench 11.0. When needed, *de novo* assembly and contig annotations were also performed as described above.

156 **Total DNA extraction and PCR confirmation of genome completeness and circularity.** In order to verify both the completeness of the assembled genome sequences and genome circularity, pairs of specific outward-facing primers were designed for each isolate (Ch-Bad-6976F/ Ch-Bad-252R for the isolate FRlc1224A and Bad-Ch-6481F/Bad-Ch-325R for the isolate ITumito39, Table S2). Leaf tissues (0.5 g) were pulverized in liquid nitrogen and total DNA was extracted in CTAB buffer (2% cetyl trimethylammonium bromide, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA), adding 3% polyvinyl pyrrolidone 40, and 0.5% sodium metabisulfite (Doyle and Doyle, 1990). Finally, the DNA pellets were resuspended in 50 µl sterile water. PCRs were performed in a 50 µl reaction volume containing 10 mM Tris-HCl (pH 8.5), 2 mM MgCl₂, 50 mM KCl, 0.2 mM dNTPs, forward and reverse primers at 1µM each, and 1.25U of Dream Taq (ThermoFisher) using 50 ng of the template. After an
166 initial denaturation step at 95°C for 4 min, 40 or 35 cycles, respectively, were set at 94°C for 30 sec,
167 60°C (Ch-Bad-6976F/ Ch-Bad-252R) or 55°C (Bad-Ch-6481F/325R) for 30 sec, and 72°C for 90 sec,
168 followed by a final extension step of 10 min at 72°C. PCR amplification products were sequenced on
169 both strands (GATC, Eurofins, Ebersberg, Germany).

170 **ChMV molecular detection and variant analysis by PCR.** For the molecular detection of ChMV,
171 two sets of primers were designed in conserved regions of ORF3 designed using the sequences of
172 isolates FRlc1224A and ITumito39. One primer pair (Ch-Bad-1466F/Ch-Bad-1800R, Table S2) allows
173 the amplification of a genomic region (335 nt) in the MP domain (Figure 2), whereas the second pair
174 (Ch-Bad-5860F/Ch-Bad-6109R, Table S2) amplifies a 232-nt fragment in the RH domain (Figure 2).
175 An aliquot of 25 ng of total DNA was used for the PCR assays in a 50 µl volume containing 10 mM
176 Tris-HCl (pH 8.5), 2 mM MgCl₂, 50 mM KCl, 0.2 mM dNTPs, forward and reverse primers at 1 µM
177 each, and either 1.25U of DreamTaq or 1U of GoTaq. After an initial denaturation step at 95°C for 4
178 min, 35 cycles were set at 94°C for 30 sec, 56°C for 30 sec, and 72°C for 90 sec, followed by a final
179 extension step of 10 min at 72°C. Amplicons were analyzed by electrophoresis on 1.5% agarose gel
180 and were directly sequenced on both strands (GATC).

181 Possible phytoplasma infection was evaluated using primer pair P1/P7 (Deng and Hiruki. 1991;
182 Smart et al. 1996) and, in nested PCR, primers R16F2n/R2 (Gundersen and Lee 1996).

183 **Sequence and phylogenetic analyses.** The full-length genomes were analyzed by ORF Finder
184 (http://www.ncbi.nlm.nih.gov/projects/gorf/) to identify putative ORFs in the viral genome.
185 Deduced amino acid (aa) sequences were analyzed for conserved protein domains gathered in
186 Conserved Domains Database (CDD) (http://www.ncbi.nlm.nih.gov/structure/cdd.shtml) and
187 theoretical molecular weights were calculated using ExPASy (http://web.expasy.org/compute_pi/).
188 Multiple alignments of nucleotide (nt) or amino acid (aa) sequences were performed using the
189 ClustalW program (Thompson et al. 1994) implemented in MEGA version 7.0 (Kumar et al. 2016).
190 Genetic distances (p-distances using strict nt or aa identity) were calculated using MEGA 7.0.

191 Phylogenetic trees were reconstructed using the neighbor-joining method implemented in MEGA

192 7.0 and robustness of nodes was assessed from 1,000 bootstrap resamplings.

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RESULTS

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Determination of the complete genome sequence of a novel badnavirus from two chestnut
disease sources. Two ChMD sources were included in the HTS analysis. The French source
(FRIc1224A) showed typical ChMD symptoms, with leaf deformation, yellow veins and chlorotic
diffuse mottling (Figure 1B) and the Italian source (ITumito39) is a mixture of six plants showing
intensive vein banding and leaf blade deformation (Figure 1C). High-throughput sequencing of
ribodepleted RNAs extracted from the sources FRlc1224A and ITumito39 yielded a total of
10,737,052 reads and 4,135,330 reads, respectively. De novo assembly and Blast annotation allowed
for the identification of a single long contig with significant homology with badnaviruses. These
contigs were respectively 7,264 and 7,214 bp long and showed short terminal redundancies,
consistent with the structure of the long RNA transcript involved in the replication of badnaviruses
(Teycheney et al. 2020) and suggesting they represented the full coverage of a circular badnaviral
genome. A total of 39,657 reads were integrated in the FRlc1224A contig, representing 0.37% of
total reads, with a mean coverage depth of 795X, whereas 611 reads (0.015% of total reads) were
integrated in the ITumito39 contig, with a mean coverage depth of 14.4X. The circularity and
completion of the DNA genome sequence of each isolate were validated by a PCR on purified DNA
extracted from the host plants and using specific outward-facing primers designed from the contig
sequences. The respective 436 nt- and 1,007 nt fragments were amplified and sequenced,
confirming DNA genome completeness and circularity (data not shown). The assembled sequences

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212 have been deposited in GenBank under accession numbers MT269853 and MT261366, respectively.

213 No other plant virus was detected in the two datasets during the Blast annotation of contigs.

214 **Genome organization of chestnut mosaic virus and determination of its phylogenetic relationships.** The badnaviral genomes characterized independently from the French and Italian ChMD sources are respectively 7,160 bp and 7,161 bp long, within the range of badnavirus genome sizes (Teycheney et al. 2020). The genomic organization is the same for both isolates, comprising three open reading frames (ORFs) encoded on the positive strand (Figure 2), and is typical for badnaviruses (Teycheney et al. 2020). The ORF1 (nt 245-751, numbering according to the isolate FRIc1224A sequence) encodes a protein of 169 aa (19.8 kDa), the ORF2 (nt 751-1161) encodes a 137-aa protein (15 kDa), and the third ORF (nt 1,163-6,721) encodes a polyprotein of 1,853 aa (211.7 kDa) with five conserved protein domains (Figure 2): a viral movement protein (MP, cl03100), a zinc-binding motif (ZnF, pfam00098), a retroviral aspartyl protease domain (RVP, pfam00077), a reverse transcriptase domain (RT, cd01647) and a ribonuclease H domain (RH, cl14782). The two "Cys" motives (C-X₂-C-X₄-H-X₄-C, and C-X₂-C-X₁₁-C-X₂-C-X₄-C-X₂-C) usually found in the coat protein of badnaviruses (Bath et al. 2016) were also detected in the ORF3-deduced protein, between aa positions 777-790 and 902-928.

228 Both isolates are closely related, with an overall 90.5% nt identity. The three indels observed between the two sequences are located in the intergenic region, the isolate ITumito39 ended up being one nucleotide longer. The three ORFs have the same sizes, are strictly colinear and the encoded proteins share respectively 95.2% (ORF1), 95.5% (ORF2) and 94.8% (ORF3) aa identity.

232 To characterize the phylogenetic relationships and taxonomic position of the chestnut badnavirus, a phylogenetic tree was reconstructed using an alignment of full genome nucleotide sequences of *Badnavirus* genus members, with the rice tungro bacilliform virus used as an outgroup (Figure 3). Both isolates cluster in group 3 defined by Wang et al. (2014), together with gooseberry
236 vein banding virus (GVBV), rubus yellow net virus (RYNV), grapevine vein clearing virus (GVCV), birch
237 leafroll-associated virus (BLRaV), wisteria badnavirus 1 (WBV1), and pagoda yellow mosaic-
238 associated virus (PYMaV) (Figure 3). Nevertheless, they are clearly distant from all of these species,
239 defining a novel branch, supported by a 99% bootstrap value (Figure 3). Tree topology was similar
240 when using an alignment of representative badnaviral ORF3 protein sequences (Figure S1). To
241 confirm these analyses, pairwise comparisons of genome sequences showed that the isolate
242 FRlc1224A has only weak identity levels with representative members of the genus *Badnavirus*,
243 comprised between 42.1% nt identity (sugarcane bacilliform IM virus; 42.5% for the isolate
244 ITumito39) and 50.9% (WBV1; 50.8% for the isolate ITumito39). The same tendency is observed
245 when considering the genome proteins. The ORF1-encoded protein shows only weak homology with
246 the corresponding proteins of WBV1 (27.8% aa identity) and PYMAV (26.1% aa identity), and the
247 ORF2-encoded protein shares only 33.1% aa identity with the corresponding protein of the most
248 closely related virus, WBV1. The polyprotein encoded by ORF3 shares 49.5% aa identity with the
249 corresponding protein of the closest relative, PYMAV. Using the ORF3 region (RT and RH domain)
250 used for taxonomical discrimination in the family *Caulimoviridae* (Teycheney et al. 2020), the
251 FRlc1224A isolate shows between 64% (with GVBV) and 68.4% (with BLRaV) nt identity (Table 1),
252 which is below the 80% nt identity value used as the species demarcation threshold in the family.
253 Therefore, this virus represents a novel species in the family *Caulimoviridae*, for which we propose
254 the name *Chestnut mosaic virus* (ChMV). In the same taxonomically informative region, the isolates
255 FRlc1224A and ITumito39 share 91.9% nt identity (97.8% aa identity), indicating that they belong to
256 the same viral species (Table 1).

257 Identification of ChMV in publicly available chestnut HTS data. The datamining of chestnut
258 HTS data from various chestnut sources publicly available at GenBank [EST sequences, whole
259 genome assembly, RNA-Seq and Genotyping-by-sequencing (GBS) reads available as Sequence Read
Archives (SRA)] allowed the identification of ChMV in several of those datasets (Table S3). In particular, two complete genomes were obtained from datasets involving *C. mollissima* cv. Vanuxem in the USA, one from the whole genome assembly (JRKL01079565) and the other by *de novo* assembly of RNA-Seq data (SRX4015368) with 99.2% and 97.4% nt identity, respectively, with the FRIc1224A isolate over the whole genome (Figure 4). In addition, partial ChMV genome assemblies of >3 kbp could be obtained from a range of other datasets generated in the USA or in China from *C. mollissima* (Table S3), all of which showed significant relatedness with the FRIc1224A sequence as shown by a phylogenetic tree reconstructed using nucleotide alignments of concatenated ChMV sequences retrieved from the various datasets (Figure 4). In addition, partial ChMV genomes could also be reconstructed from two datasets obtained from *C. dentata* in the USA. Interestingly, one of these two *C. dentata* isolate sequences shows closest relationship with ITumito39 sequence (Figure 4) with only 89.2% nt identity with the isolate FRIc1224A as compared to 93.9% nt identity with ITumito39. The second isolate of *C. dentata* appears to be equally related to the FRIc1224A and ITumito39 isolates, with 90.9% and 90.6% nt identity, respectively.

**Incidence and genetic variability of ChMV in France and Italy.** The incidence and genetic variability of ChMV were investigated by analyzing two genomic regions of ORF3, one 335-nt long located in the MP domain amplified using primer pair Ch-Bad-1466F/Ch-Bad-1800R and the other 232-nt long in the RNase H domain and amplified with primer pair Ch-Bad-5860F/Ch-Bad-6109R (Table S2, and Figure 2). The two primer pairs were designed to be able to detect both isolates FRIc1224A and ITumito39. In Italy, a total of 70 *C. sativa* cv Marrone samples were collected in the same location, while in France, 95 chestnut accessions belonging to three different *Castanea* species or hybrids were sampled in three plots (A, Port, E). Both symptomatic and asymptomatic samples were collected, as well as some samples with atypical or dubious symptoms. Globally, ChMV was frequent in the surveyed plots, with 57/70 (81.5%) infected *C. sativa* samples in Italy and 65/95 trees
(68%) in France (Table 2). In the Italian orchard, half of the asymptomatic trees were found to be infected by ChMV, compared to 87% of the symptomatic ones (Table 2). None of the analyzed samples were found positive using a phytoplasma-specific PCR assay. In the French collection, the virus was detected in 100% (38/38) of the trees showing typical ChMD symptoms, and in 49% (23/47) of the asymptomatic trees, including two trees that were symptomless but showed strong symptoms on rootstock off-shoots (Figure S2). ChMV was also detected in four out of the 10 trees showing atypical/doubtful symptoms.

The genetic variability of ChMV was evaluated by analyzing the sequences of the two PCR amplicons generated for the incidence survey. Considering the relative homogeneity of the origin of the Italian samples, the number of samples included in this analysis was limited to 13 (four from asymptomatic trees, and nine symptomatic ones) (Table S1). In total, the final dataset consisted in 53 isolates for which the sequence of the two genomic regions were available (49 from the incidence survey and four from independent ChMD sources held in collection at CTIFL, see below). As illustrated by the unrooted neighbor-joining tree reconstructed from the alignments of RT-RnaseH domain nucleotide sequences (Figure S3A), ChMV diversity is structured into two clusters, defined by the geographical origin of the samples (Italy and France). The sequences determined from the four independent French disease sources (FRlc1224A, T30218, LCA552, LCA584) belong to the same «French» cluster. Overall, the level of genetic diversity is very low in this genomic region, with an average pairwise nt divergence (diversity) of 2.2% +/- 0.5%. This value is even lower when considering the intra-group diversity, as 0.2% +/- 0.1% within the French cluster and 0.1% +/- 0.1% within the Italian ones. In contrast, the inter-group diversity reaches 6.3% +/- 1.5%, confirming the existence of two geographical clusters. The same trends are observed when analyzing the genomic region located in the MP domain (Figure S3B). The same geographical clustering could be observed, with the exceptions of three French isolates that seem to be more closely related to the Italian
308 cluster (Figure S3B). Another French isolate, 20971-E remains isolated and does not fit in either
309 group. The average nt divergence in this region is slightly higher than in the RT-RNaseH region, (5.8%
310 +/- 0.7%), and the inter-group diversity reaches a value of 13.4% +/- 1.8%, as compared to the 6.3%
311 value for the other region.

312

DISCUSSION

313 Since the seminal work of Desvignes and collaborators in the 1990s, it has been acknowledged
314 that the agent responsible for ChMD is most likely a thermosensitive, graft-transmissible virus that
315 can be transmitted experimentally and probably naturally by the aphid *Myzocallis castanicola*
316 Desvignes, 1992; 1999a; 1999b; Desvignes and Lecoq, 1995; Desvignes and Cornaggia, 1996).
317 Depending on the chestnut genotype, this infection can either be asymptomatic or can result in the
318 expression of severe and conspicuous ChMD symptoms. In chestnut orchards in the Marche region
319 (Italy), both young and mature plants were shown to be affected, significantly decreasing chestnut
320 production. Symptoms have also been observed in some *Quercus* species following experimental
321 graft inoculation. To date, however, the causal agent remains to be identified.

322 Here, by using HTS-based viral indexing, we were able to identify and characterize, in two
323 independent ChMD sources, two isolates of the same novel virus. Phylogenetic and sequence
324 analyses showed that this virus belongs to the genus *Badnavirus*, in the family *Caulimoviridae*, and
325 could be considered as a new species, for which the name *Chestnut mosaic virus* (ChMV) is
326 proposed. Interestingly, this new virus clusters with a group of badnaviruses that includes RYNV,
327 GVBaV, and GVCV.

328 There is unambiguous evidence that ChMV as reported here is an episomal virus. It was detected
329 in graft-inoculated indicators, and not in non-inoculated control plants of the same variety,
330 demonstrating its graft-transmissibility, a property of episomal viruses. This line of evidence is
331 further reinforced by the detection of ChMV in symptomatic, graft-inoculated indicator *Quercus*
plants and, again, not in the corresponding control plants. In parallel, the HTS detection of ChMV from DNAse-treated RNAs, the failure to detect ChMV in a range of the surveyed chestnut trees and the sequence diversity identified in ChMV all rule out a scenario in which an endogenous ChMV genome, integrated in the chestnut genome could be responsible for the HTS and PCR results reported here. There was in fact no indication of ChMV in the chestnut genome assembly (JRKL01079565) since no integration borders could be identified and a single contig, representing a complete unintegrated viral genome transcript, was identified. Integration of ChMV as an endogenous viral element (EVE, Bhat et al. 2016) therefore does not appear to be a general genomic feature of chestnut.

According to the simplified hierarchical approach proposed by Fox (2020) for assessing causal relationships in plant virology, ChMV appears as a good candidate, if not as the causative agent of ChMD. There are several arguments and experimental evidence supporting this idea. Following HTS analyses, ChMV was the sole virus detected in the French source FRlc1224A, coming from a ChMD source initially involving a *C. sativa* × *C. crenata* hybrid. It was also the sole virus detected in the Italian ChMD source analyzed by HTS. Using molecular detection tests developed in this work, the virus was consistently found in other symptomatic accessions derived from the same diseased source (LC1224H, a *Q. rubra* artificially inoculated and LC1224F, an indicator plant inoculated by aphid transmission; Figure 5). In addition, three other independent chestnut sources shown by biological indexing on the 'Maraval' indicator to be affected by ChMD were found to be infected by ChMV (LCA552, LCA584 and T32018 in Figure 5). There is therefore a correlation between the appearance of ChMD symptoms and the presence of ChMV in the graft-inoculated indicators, supporting the hypothesis of a causal relationship between ChMV infection and ChMD. In total, five independent ChMD sources collected between 1990 and 2018 in two countries (Italy and France)
355 were ChMV positive, satisfying the Bradford-Hill's experimental and consistency criteria (Bradford 356 Hill, 1965; Fox, 2020).

357 Preliminary studies indicate that ChMV is highly prevalent in the analyzed orchards in France 358 and Italy, confirming the earlier results of Desvignes (1999a). In parallel, the identification of ChMV 359 sequences in publicly available HTS data provides a strong indication of the presence of ChMV in *C. mollissima* in the USA and in China and in *C. dentata* in the USA. In the surveys, ChMV was not 360 systematically associated with symptomatic infections, although its frequency was systematically 361 higher in symptomatic plants. This result was expected since previous grafting experiments had 362 demonstrated that not all chestnut varieties/species are susceptible to ChMD and develop 363 symptomatic infections (Desvignes and Lecoq, 1995; Desvignes, 1992; 1999b). Biological indexing 364 on the susceptible 'Maraval' indicator has in particular identified latent ChMV infections in many 365 symptomless *C. sativa* varieties or *C. sativa* × *C. crenata* hybrids (Desvignes and Lecoq, 1995; 366 Desvignes, 1992; 1999b). On the other hand, all surveyed symptomatic plants in France were found 367 to harbor the virus, while it was detected in 52/60 (87%) of tested symptomatic Italian trees. The 368 failure to detect ChMV in eight symptomatic Italian trees might reflect sequence variability and an 369 incomplete inclusiveness of the PCR primers used or low or uneven virus accumulation. Indeed, 370 using biological indexing, Desvignes et al. have previously found an uneven distribution of the ChMD 371 agent in infected trees leading to a failure to detect it in parts of some infected trees (Desvignes and 372 Lecoq, 1995; Desvignes et al. 1999b).

373 Taken together, and even though Koch's postulates were not fully verified, the experiments 374 reported here make a very strong case for a role of ChMV as the causal agent of the chestnut mosaic 375 disease. The low ChMV diversity observed in France and Italy are consistent with the scenario of its 376 recent introduction in Europe (Desvignes and Lecoq, 1995), while the genetic separation of the 377 Italian and French clusters is suggestive of separate introduction events. These results and the 378
379 associated development of molecular tools for the detection of ChMV will help speed up the selection of virus-free mother plants and mitigate the virus spread in new chestnut orchards and layerings. However, many questions remain regarding the variability of symptom intensity in relationship to cultivar susceptibility, ChMV-induced graft incompatibility, the impact of pedoclimatic conditions and of synergic and competitive interferences with other chestnut pathogens, and silvicultural management.

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ACKNOWLEDGMENTS

387 The authors thank the INRAE Vine Experimental Unit of Bordeaux (UEVB) and X. Capdevielle for taking care of chestnut orchards. We would like to thank Ascenzio Santini (Azienda Agricola Laga Nord, Umito, Aquasanta Terme, Italy) for the essential support during the phytosanitary monitoring in chestnut orchards. Thanks to D. Cornaggia and P. Gentit for the information provided on the past work of CTIFL, for the characterization of the symptoms of the disease on biological indicators and the conservation of the isolates. Thanks to Eden Darnige for proofreading the manuscript. Part of this work was based upon COST Action FA1407 (DIVAS), supported by COST (European Cooperation in Science and Technology).

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LITERATURE CITED

397 Antonaroli, R., and Perna, M.R. 2000. Una fitopatia ad eziologia ancora incerta: il giallume dl castagno in Emilia Romagna e nelle Marche. Sherwood 6:43-46.

399 Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G., Alekseyev, M. A., and Pevzner, P. A. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 9:455-477.
403 Bhat, A. I., Hohn, T., and Selvarajan, R. 2016. Badnaviruses: the current global scenario. Viruses 8:177; doi:10.3390/v8060177.

405 Bradford Hill, A. 1965. The environment and disease: association or causation? Proc. R. Soc. Med. 58:295-300.

407 Conedera, M., Tinner, W., Krebs, P., de Rigo, D., and Caudullo, G. 2016. *Castanea sativa* in Europe: distribution, habitat, usage and threats. Pages 78-79. in: European Atlas of Forest Tree Species.
408
409 J. San-Miguel-Ayanz, D. de Rigo, G. Caudullo, T. Houston Durrant, A. Mauri eds.

410 Deng, S. and Hiruki, C. 1991. Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. J. Microbiol. Meth. 14:53-61.

412 Desvignes, J. C. 1992. Characterization of the chestnut mosaic. Acta Hortic. 309:353-358.

413 Desvignes, J. C., and Lecocq, G. 1995. New knowledges on the chestnut mosaic virus disease. Acta Hortic. 386:578-584.

415 Desvignes, J. C., and Cornaggia, D. 1996. Mosaïque du chataignier: Transmission par le puceron *Myzocallis castanicola*. Phytoma 481:39-41.

417 Desvignes, J. C. 1999a. Mosaïque du chataignier. Pages 179-181. in: Maladies à virus des arbres fruitiers. Ctifl Ed. France

419 Desvignes, J. C. 1999b. Sweet chestnut incompatibility and mosaics caused by the chestnut mosaic virus (ChMV). Acta Hortic. 494:451-454.

421 Doyle, J. J., and Doyle, J. L. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13-15.

422 Fox, A. 2020. Reconsidering causal association in plant virology. Plant Pathol. doi: 10.1111/ppa.13199

424 Gambino, G., Perrone, I., and Gribaudo, I. 2008. A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem. Anal. 19:520-525.
426 Geering, A. D. W., Maumus, F., Copetti, D., Choisne, N., Zwickl, D. J., Zytnicki, M., McTaggart, A. R., Scalabrin, S., Vezzulli, S., Wing, R. A., Quesneville, H. and Teycheney, P. Y 2014. Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution. Nat. Comm. 5:5269

430 Gualaccini, G. 1958. Una virosi nuova del castagno. Boll. Staz Patol. Veg. Roma, 16:67-75.

431 Gundersen, D. E. and Lee, I. M. 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol. Mediterr. 35:144-151.

433 Hohn, T., and Rothnie, H. 2013. Plant pararetroviruses: replication and expression. Curr. Opin. Virol. 3:621-628.

435 Horvath, J., Ecke, I., Gal, T., and Dezcery, M. 1975. Demonstration of virus-like particles in sweet chestnut and oak with leaf deformations in Hungary. Z. Pflkrankh. Pfsdrntz. 82:498-502.

437 Kreuze, J. F., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., and Barker, I. 2009. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1-7.

440 Kumar, S., Stecher, G., and Tamura, K. 2016. MEGA 7: Molecular Evolutionary Genetics Analysis version 7 for bigger datasets. Mol. Biol. Evol. 33:1870-1874.

442 Martin, R. R., Constable, F., and Tzanetakis, I. E. 2016. Quarantine regulations and the impact of modern detection methods. Annu. Rev. Phytopathol. 54:189-205.

444 Massart, S., Olmos, A., Jijakli, H., and Candresse, T. 2014. Current impact and future directions of high throughput sequencing in plant virus diagnostics. Virus Res. 188:90-96.

446 Massart, S., Candresse, T. Gil, J., Lacomme, C., Predajna, L., Ravnikar, M., Reynard, J. S., Rumbou, A., Saldarelli, P., Škorić, D., Vainio, E. J., Valkonen, J. P. T., Vanderschuren, H., Varveri, C., and Wetzel, T. 2017. A framework for the evaluation of biosecurity, commercial, regulatory, and scientific
449 impacts of plant viruses and viroids identified by NGS technologies. Front. Microbiol. 8:45. doi: 10.3389/fmicb.2017.00045

451 Murolo, S., De Miccolis Angelini, R. M., Faretra F., and Romanazzi, G. 2018. Phenotypic and molecular investigations on hypovirulent *Cryphonectria parasitica* in Italy. Plant Dis. 102:540-545.

454 Prospero, S., Vannini, A., and Vettraino, A. M. 2012. Phytophthora on *Castanea sativa* Mill. (sweet chestnut). JKI Data Sheets 6, DOI 10.5073/jkidspdd.2012.006

456 Ragozzino, A., and Lahoz, E. 1986. Una malattia virus-simile del castagno in provincia di Avellino. Giornate di studio sul castagno. Soc. Orti. Italiana 307-311.

458 Reid, K. E, Olsson, N., Schlosser, J., Peng, F., and Lund, S. T. 2006. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol. 6:26-37.

461 Rigling, D., and Prospero, S. 2018. *Cryphonectria parasitica*, the causal agent of chestnut blight: invasion history, population biology and disease control. Mol. Plant Pathol. 19:7-20.

463 Rott, M., Xiang, Y., Boyes, I., Belton, M., Saeed, H., Kesanakurti, P., Hayes, S., Lawrence, T., Birch, C., Bhagwat, B., and Rast, H. 2017. Application of Next Generation Sequencing for diagnostic testing of tree fruit viruses and viroids. Plant Dis. 101:14891499.

466 Shimada, S. 1962. Chestnut Yellows. Pl. Prot. Tokyo, 16:253-254.

467 Smart, C. D., Schneider, B., Blomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., Lorenz, K. H., Seemuller, E., and Kirkpatrick, B. C. 1996. Phytoplasma-specific PCR primers based on sequences of 16s-23s rRNA spacer region. Appl. Environ. Microbiol. 62:2988-2993.

470 Staginnus, C., Iskra-Caruana, M. L., Lockhart, B., Hohn, T., and Richert-Pöggeler, K. R. 2009. Suggestions for a nomenclature of endogenous pararetroviral sequences in plants. Arch. Virol. 154:1189-1193.
473 Teycheney, P. Y., Geering, A. D. W., Dasgupta, I., Hull, R., Kreuze, J.F., Lockhart, B., Muller, E., Olszewski, N., Pappu, H., Pooggin, M. M., Richert-Pöggeler, K. R., Schoelz, J. E., Seal, S., Stavolone, L., Umber, M. and ICTV Report Consortium. 2020. ICTV Virus Taxonomy Profile: *Caulimoviridae*. J. Gen. Virol. DOI 10.1099/jgv.0.001497

477 Thompson, J. D., Higgins, D. G., and Gibson, T. J. 1994. CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap, penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.

480 Vettraino, M., Vannini, A., Flamini, L., Lagnese, R., Pizzichini, L., Talevi, S., and Fulbright, D. W. 2005. A new transmissible symptomology on sweet chestnut in Italy. Acta Hortic. 693:547-550.

482 Villamor, D. E. V., Ho, T., Al Rwahnih, M., Martin, R. R., and Tzanetakis, I. E. 2019. High Throughput Sequencing for plant virus detection and discovery. Phytopathology 109:716-725.

484 Wang, Y., Cheng, X., Wu, X., Wang, A., and Wu, X. 2014. Characterization of complete genome and small RNA profile of pagoda yellow mosaic virus, a novel badnavirus in China. Virus Res. 188:103-108.

487 Zerbino, D. R., and Birney, E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18:821-829.
<table><caption>TABLE 1. Percentage of identity between the ORF3 region encoding the Reverse Transcriptase - Rnase Hª of chestnut mosaic virus (ChMV) isolate FRlc1224A and the corresponding genomic regions of the isolate ITumito39 and of the most closely related members of the genus *Badnavirus*</caption><thead><tr><th>Virus<sup>b</sup></th><th>Nucleotide identity (%)</th><th>Amino acid identity (%)</th></tr></thead><tbody><tr><td>ChMV ITumito39</td><td>91.9%</td><td>97.8%</td></tr><tr><td>RYNV</td><td>65.1%</td><td>71.6%</td></tr><tr><td>GVBV</td><td>64%</td><td>68.9%</td></tr><tr><td>GVCV</td><td>66.8%</td><td>72.6%</td></tr><tr><td>BLRaV</td><td>68.4%</td><td>72.6%</td></tr><tr><td>WBV1</td><td>68.2%</td><td>71.6%</td></tr><tr><td>PYMaV</td><td>67.7%</td><td>71.9%</td></tr></tbody></table>

a This region is the one typical used for taxonomic discrimination in the family *Caulimoviridae* (Teycheney et al. 2020)

b Acronyms used: RYNV, rubus yellow net virus; GVBV, gooseberry vein banding virus; GVCV, grapevine vein-clearing virus; BLRaV, birch leafroll-associated virus; WBV1, wisteria badnavirus 1; PYMaV, pagoda yellow mosaic-associated virus.

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<table><caption>**TABLE 2.** Number and percentage of chestnut mosaic virus-infected plants regarding the plot, the *Castanea* species sampled, and the symptomatology</caption><thead><tr><th>Origin of the sampled plants</th><th>Infected/total plants (%)</th><th>Infected/asymptomatic plants (%)</th><th>Infected/symptomatic plants (%)</th><th>Infected/plants with atypical symptoms (%)</th></tr></thead><tbody><tr><td>France (overall)</td><td>65/95 (68.4%)</td><td>23/47 (48.9%)</td><td>38/38 (100%)</td><td>4/10 (40%)</td></tr><tr><td>Plot A</td><td>13/23 (56.5%)</td><td>6/15 (40%)</td><td>6/6 (100%)</td><td>1/2 (50%)</td></tr><tr><td>Plot E</td><td>48/66 (72.7%)</td><td>15/28 (53.6%)</td><td>30/30 (100%)</td><td>3/8 (37.5%)</td></tr><tr><td>Plot Port</td><td>4/6 (66.6%)</td><td>2/4 (50%)</td><td>2/2 (100%)</td><td>na<sup>b</sup></td></tr><tr><td><i>C. crenata</i></td><td>5/6 (83%)</td><td>1/1 (100%)</td><td>4/4 (100%)</td><td>0/1</td></tr><tr><td><i>C. sativa</i></td><td>30/43 (70%)</td><td>11/20 (55%)</td><td>18/18 (100%)</td><td>1/5 (20%)</td></tr><tr><td><i>C. mollissima</i></td><td>13/14 (93%)</td><td>1/2 (50%)</td><td>10/10 (100%)</td><td>2/2 (100%)</td></tr><tr><td>Hybrid<sup>a</sup></td><td>17/32 (53.1%)</td><td>10/24 (41.6%)</td><td>6/6 (100%)</td><td>1/2 (50%)</td></tr><tr><td>Italy <i>Castanea sativa</i></td><td>57/70 (81.5%)</td><td>5/10 (50%)</td><td>52/60 (87%)</td><td>na<sup>b</sup></td></tr></tbody></table>

## CAPTIONS FOR FIGURES

**Fig. 1.** Symptoms of chestnut mosaic disease on various hosts. (A) Isolate LC1224H: Red oak (Quercus rubra) graft-inoculated with a diseased source; (B) Isolate FRlc1224A: 'Maraval' Ca 74 graft-inoculated with LC1224H; (C) Isolate ITumito39: symptomatic leaves from cv Marrone grafted onto *Castanea sativa*; (D) non-inoculated *Q. rubra*; (E) non-inoculated 'Maraval' Ca 74; (F) Asymptomatic leaves from cv Marrone grafted onto *C. sativa*.

<footnote><sup>a</sup> Interspecific hybrids between *C. crenata*, *C. mollissima* and *C. sativa*</footnote>

<footnote><sup>b</sup> not applicable</footnote>
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**Fig. 2: Schematic representation of the genomic organization of the chestnut mosaic virus.** The tRNA binding site is indicated and defines the position 1 on the genome. The three open reading frames (ORFs) are shown as grey arrows, as well as their position in parentheses. Five conserved motifs are identified in the ORF3 polyprotein: MP, Viral movement protein (pfam01107); ZnF, Zinc finger (pfam00098); RVP, Retroviral aspartyl protease (pfam00077); RT, Reverse transcriptase (cd01647); RH, Ribonuclease H (cd09274)

**Fig. 3. Phylogenetic tree reconstructed using the complete genome sequences of badnavirus members.** Virus names as well as GenBank accession numbers are indicated. The tree was reconstructed using the neighbor-joining method, and randomized bootstrapping was performed to evaluate the statistical significance of branches (1,000 replicates). Bootstrap values above 70% are shown. The scale bar represents 5% nucleotide divergence between sequences. The groups as defined by Wang et al. (2014) are indicated. Chestnut mosaic virus isolates determined in this work are indicated by black triangles. Rice tungro bacilliform virus was used as outgroup.

**Fig. 4. Unrooted neighbor-joining phylogenetic tree reconstructed from the alignment of concatenated nucleotide sequences related to chestnut mosaic virus detected by datamining of publicly available transcriptomic chestnut data.** Randomized bootstrapping was performed to evaluate the statistical significance of branches (1,000 replicates). Bootstrap values above 70% are shown. The scale bar represents 10% nucleotide divergence between sequences.

**Fig. 5. Detection of chestnut mosaic virus in various samples by PCR using primers pairs Ch-Bad1466F/1800R (A) and Ch-Bad5860F/6109R (B).** Lane 1: LC1224F; Lane 2: LC1224H; Lane 3: FRlc1224A; Lane 4: T32018; Lane 5: LCA552; Lane 6: LCA584; Lane 7: 'Maraval' Ca 74 non-inoculated plant; Lane 8: *Quercus rubra* non-inoculated plant; Lane 9: no template; L: molecular weight marker. Horizontal bars on the left of the figure indicate the size of the amplification products. The isolates are listed in Table S1.

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e-EXTRA FIGURE CAPTIONS AND TABLE TITLES
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**Supplementary Fig. S1. Phylogenetic tree reconstructed using the ORF3-deduced amino acid sequences of badnavirus members.** Virus names as well as GenBank accession numbers are indicated. The tree was reconstructed using the neighbor-joining method, and randomized bootstrapping was performed to evaluate the statistical significance of branches (1,000 replicates).

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Bootstrap values above 70% are shown. The scale bar represents 10% amino acid divergence between sequences. Chestnut mosaic virus isolates determined in this work are indicated by black triangles. Rice tungro bacilliform virus was used as outgroup.

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**Supplementary Fig. S2. Leaves of the sample 20893. (A) rootstock leaves (Castanea sativa); (B) cultivar leaves**

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**Supplemental Fig. S3. Unrooted neighbor-joining phylogenetic trees reconstructed from the alignment of nucleotide sequences of the PCR fragments targeted partial RT-RnaseH domain (A) and partial MP domain (B) obtained from a range of chestnut mosaic virus isolates (listed in Table S1).** Randomized bootstrapping was performed to evaluate the statistical significance of branches (1,000 replicates). Bootstrap values above 70% are shown. The scale bar represents 5% (A) or 10% (B) nucleotide divergence between sequences.

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**Supplemental Table S1. List of chestnut samples used in the present study together with relevant ChMV accession numbers**

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**Supplemental Table S2. Primers used for genome circularity confirmation and for molecular detection of chestnut mosaic virus by PCR**

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565 Supplemental Table S3. Datamining of publicly available chestnut HTS data for chestnut mosaic

566 virus sequences
<figure><img src="image_3.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /></figure>

Fig. 2: Schematic representation of the genomic organization of the chestnut mosaic virus. The tRNA binding site is indicated and defines the position 1 on the genome. The three open reading frames (ORFs) are shown as grey arrows, as well as their position in parentheses. Five conserved motifs are identified in the ORF3 polyprotein: MP, Viral movement protein (pfam01107); ZnF, Zinc finger (pfam00098); RVP, Retroviral aspartyl protease (pfam00077); RT, Reverse transcriptase (cd01647); RH, Ribonuclease H (cd09274)

338x190mm (96 x 96 DPI)
<figure><img src="image_10.png" /><figcaption>Fig. 3. Phylogenetic tree reconstructed using the complete genome sequences of badnavirus members. Virus names as well as GenBank accession numbers are indicated. The tree was reconstructed using the neighbor-joining method, and randomized bootstrapping was performed to evaluate the statistical significance of branches (1,000 replicates). Bootstrap values above 70% are shown. The scale bar represents 5% nucleotide divergence between sequences. The groups as defined by Wang et al. (2014) are indicated. Chestnut mosaic virus isolates determined in this work are indicated by black triangles. Rice tungro bacilliform virus was used as outgroup.</figcaption></figure>

190x338mm (96 x 96 DPI)
<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

Fig. 5. Detection of chestnut mosaic virus in various samples by PCR using primers pairs Ch-Bad1466F/1800R (A) and Ch-Bad5860F/6109R (B). Lane 1: LC1224F; Lane 2: LC1224H; Lane 3: FRlc1224A; Lane 4: T32018; Lane 5: LCA552; Lane 6: LCA584; Lane 7: 'Maraval' Ca 74 non-inoculated plant; Lane 8: Quercus rubra non-inoculated plant; Lane 9: no template; L: molecular weight marker.
Horizontal bars on the left of the figure indicate the size of the amplification products. The isolates are listed in Table S1.

338x190mm (96 x 96 DPI)
<figure><img src="image_14.png" /><figcaption>Fig. S1</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>Fig. S2</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Fig. S3A</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Fig. S3B</figcaption></figure>
1 Supplementary Table S1. List of chestnut samples used in the present study together with relevant ChMV

2 accession numbers

<table><thead><tr><th>Isolate ID <sup>a</sup></th><th>Chestnut Accession ID</th><th>Chestnut Accession name</th><th>Species <sup>b</sup></th><th>Rootstock</th><th>Country</th><th>Plot <sup>c</sup></th><th>Accession numbers <sup>d</sup></th><th>Use <sup>e</sup></th></tr></thead><tbody><tr><td>20885</td><td>Ca04x<br/>Ca03</td><td>-</td><td>C. crenata</td><td>Not graft</td><td>France</td><td>A</td><td>MT339547;<br/>MT339503</td><td>Incidence</td></tr><tr><td><strong>20887</strong></td><td>Ca 715</td><td>'Merle'</td><td>C. sativa</td><td>G1. Ca15</td><td>France</td><td>A</td><td>MT339548;<br/>MT339504</td><td>Incidence</td></tr><tr><td>20890</td><td>Ca 663</td><td>'Trigueira'</td><td>C. sativa</td><td>unknown</td><td>France</td><td>Port</td><td>MT339549;<br/>MT339505</td><td>Incidence</td></tr><tr><td>20891</td><td>Ca 664</td><td>'Longal<br/>Special'</td><td>C. sativa</td><td>Ca 74</td><td>France</td><td>Port</td><td>MT339550;<br/>MT339506</td><td>Incidence</td></tr><tr><td><u>20895</u></td><td>Ca 43</td><td>'Vignols'</td><td>C. sativa x C. crenata</td><td>Ca 116</td><td>France</td><td>A</td><td>MT339551;<br/>MT339507</td><td>Incidence</td></tr><tr><td>20899</td><td>Ca 599</td><td>'Ibuki'</td><td>C. crenata</td><td>G1.Ca 02</td><td>France</td><td>A</td><td>MT339552;<br/>MT339508</td><td>Incidence</td></tr><tr><td><strong>20901</strong></td><td>Ca 598</td><td>'Rihei'</td><td>C. crenata x C. mollissima</td><td>G1. Ca102</td><td>France</td><td>A</td><td>MT339553;<br/>MT339509</td><td>Incidence</td></tr><tr><td><strong>20902</strong></td><td>Ca 75</td><td>'Fertil'</td><td>C. mollissima</td><td>Ca 07</td><td>France</td><td>A</td><td>MT339554;<br/>MT339510</td><td>Incidence</td></tr><tr><td>20904</td><td>Tree-<br/>A68Ks <sup>i</sup></td><td>-</td><td>C. mollissima</td><td>Not graft</td><td>France</td><td>A</td><td>MT339555;<br/>MT339511</td><td>Incidence</td></tr><tr><td><strong>20907</strong></td><td>Ca 118</td><td>'Marlhac'</td><td>C. sativa x C. crenata</td><td>Not graft</td><td>France</td><td>A</td><td>MT339556;<br/>MT339512</td><td>Incidence</td></tr><tr><td><strong>20908</strong></td><td>Ca 124</td><td>'Maridonne'</td><td>C. sativa x C. crenata</td><td>G1.Ca116</td><td>France</td><td>A</td><td>MT339557;<br/>MT339513</td><td>Incidence</td></tr><tr><td><strong>20909</strong></td><td>Ca 125</td><td>'Bouche de<br/>Bétizac'</td><td>C. sativa x C. crenata</td><td>Ca 74</td><td>France</td><td>A</td><td>MT339558;<br/>MT339514</td><td>Incidence</td></tr><tr><td>20910</td><td>Ca 860</td><td>-</td><td>hybrid SC</td><td>unknown</td><td>France</td><td>E</td><td>MT339559;<br/>MT339515</td><td>Incidence</td></tr><tr><td>20911</td><td>Ca 860</td><td>-</td><td>hybrid SC</td><td>unknown</td><td>France</td><td>E</td><td>MT339560;<br/>MT339516</td><td>Incidence</td></tr><tr><td>20913</td><td>Ca 844 <sup>i</sup></td><td>-</td><td>C. mollissima</td><td>Not graft</td><td>France</td><td>E</td><td>MT339561;<br/>MT339517</td><td>Incidence</td></tr><tr><td>20917</td><td>Ca 837 <sup>h</sup></td><td>-</td><td>C. mollissima</td><td>Not graft</td><td>France</td><td>E</td><td>MT339562;<br/>MT339518</td><td>Incidence</td></tr><tr><td>20923</td><td>Ca 846 <sup>h</sup></td><td>-</td><td>C. mollissima</td><td>Not graft</td><td>France</td><td>E</td><td>MT339563;<br/>MT339519</td><td>Incidence</td></tr><tr><td>20925</td><td>Ca 741</td><td>'Dauphine'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339564;<br/>MT339520</td><td>Incidence</td></tr><tr><td><strong>20926</strong></td><td>Ca 665</td><td>'Longal'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339565;<br/>MT339521</td><td>Incidence</td></tr><tr><td><strong>20930</strong></td><td>Ca 860</td><td>-</td><td>hybrid SC</td><td>unknown</td><td>France</td><td>E</td><td>MT339566;<br/>MT339522</td><td>Incidence</td></tr><tr><td><strong>20932</strong></td><td>Ca 564</td><td>'Ipharra 16'</td><td>C. crenata</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339567;<br/>MT339523</td><td>Incidence</td></tr><tr><td><u>20935</u></td><td>Ca 576</td><td>'Sardonne'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339568;<br/>MT339524</td><td>Incidence</td></tr><tr><td><strong>20937</strong></td><td>Ca 138</td><td>'Marron de<br/>Redon'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339569;<br/>MT339525</td><td>Incidence</td></tr><tr><td><strong>20943</strong></td><td>unknown</td><td>-</td><td>C. sativa</td><td>Ca 74</td><td>France</td><td>E</td><td>MT339570;<br/>MT339526</td><td>Incidence</td></tr><tr><td><strong>20944</strong></td><td>Ca 520</td><td>'Montagne'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339571;<br/>MT339527</td><td>Incidence</td></tr><tr><td><strong>20945</strong></td><td>Ca 106</td><td>'Marron<br/>Comballe'</td><td>C. sativa</td><td>Ca 07</td><td>France</td><td>E</td><td>MT339572;<br/>MT339528</td><td>Incidence</td></tr></tbody></table>
<table>
  <tr>
    <td>20947</td>
    <td>Ca 111</td>
    <td>'Marron de Lyon'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339573;<br>MT339529</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20951</td>
    <td>Ca 126</td>
    <td>-</td>
    <td>C. sativa x C. crenata</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339574;<br>MT339530</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20956</td>
    <td>Ca 105</td>
    <td>'Sardonne'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339575;<br>MT339531</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20959</td>
    <td>Ca 03</td>
    <td>-</td>
    <td>C. crenata</td>
    <td>Ca 74</td>
    <td>France</td>
    <td>E</td>
    <td>MT339576;<br>MT339532</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20960</td>
    <td>Ca 127</td>
    <td>-</td>
    <td>(C. crenata x C. sativa) x C. mollisima</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339577;<br>MT339533</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20961</td>
    <td>Ca 127</td>
    <td>-</td>
    <td>(C. crenata x C. sativa) x C. mollisima</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339578;<br>MT339534</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20964</td>
    <td>Ca 112</td>
    <td>'Bournette'</td>
    <td>C. crenata x C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339579;<br>MT339535</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20965</td>
    <td>Ca 501</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339580;<br>MT339536</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20966</td>
    <td>Ca 501</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339581;<br>MT339537</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20967</td>
    <td>Ca 665</td>
    <td>'Longal'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339582;<br>MT339538</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20968</td>
    <td>Ca 48</td>
    <td>'Précoce Migoule'</td>
    <td>(C. crenata x C. sativa)</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339583;<br>MT339539</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20971</td>
    <td>Ca 151</td>
    <td>'Bouche Rouge'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339584;<br>MT339540</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20975</td>
    <td>unknown</td>
    <td>-</td>
    <td>C. sativa x C. crenata</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>MT339585;<br>MT339541</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20889</td>
    <td>unknown</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>unknown</td>
    <td>France</td>
    <td>Port</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20893</td>
    <td>Ca 106</td>
    <td>'Marron Comballe'</td>
    <td>C. sativa</td>
    <td>unknown</td>
    <td>France</td>
    <td>Port</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20898</td>
    <td>Ca 105</td>
    <td>'Sardonne'</td>
    <td>C. sativa</td>
    <td>G1. Ca394</td>
    <td>France</td>
    <td>A</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20900</td>
    <td>Ca 501</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>G1.Ca486</td>
    <td>France</td>
    <td>A</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20903</td>
    <td>Ca744</td>
    <td>'Quing Zha'</td>
    <td>C. mollissima</td>
    <td>G1.Moll</td>
    <td>France</td>
    <td>A</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20912</td>
    <td>Ca 845<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20914</td>
    <td>Ca 843<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20915</td>
    <td>Ca 842<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20918</td>
    <td>Ca 838<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20919</td>
    <td>Ca 839<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20920</td>
    <td>Ca 840<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20921</td>
    <td>Ca 841<sup>h</sup></td>
    <td>-</td>
    <td>C. mollissima</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20922</td>
    <td>Ca 74</td>
    <td>'Maraval'</td>
    <td>C. crenata x C. sativa</td>
    <td>Not graft</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20924</td>
    <td>Ca 825</td>
    <td>'Précoce de Besse'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20940</td>
    <td>Ca 393</td>
    <td>'Marron de Chevanceaux'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20941</td>
    <td>Ca 460</td>
    <td>-</td>
    <td>(C. crenata x C. sativa) x (C. crenata x C. sativa)</td>
    <td>Ca 74</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>29942</td>
    <td>Ca 511</td>
    <td>'Marrone di Chiusa Pesio'</td>
    <td>C. sativa</td>
    <td>Ca 74</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20948</td>
    <td>Ca 116</td>
    <td>-</td>
    <td>C. sativa x C. crenata</td>
    <td>Ca 74</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20949</td>
    <td>Ca 135</td>
    <td>'Précoce des Vans'</td>
    <td>C. sativa</td>
    <td>Ca 74</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20952</td>
    <td>Ca 521</td>
    <td>-</td>
    <td>C. crenata</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
</table>
<table>
  <tr>
    <td>20955</td>
    <td>Ca 399</td>
    <td>'Comballe'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20962</td>
    <td>Ca 639</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20963</td>
    <td>Ca 730</td>
    <td>'Sauvage Marron'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20969</td>
    <td>Ca 381</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20970</td>
    <td>Ca 381</td>
    <td>-</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>20972</td>
    <td>Ca 151</td>
    <td>'Bouche Rouge'</td>
    <td>C. sativa</td>
    <td>Ca 07</td>
    <td>France</td>
    <td>E</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>FRIC1224A</td>
    <td>Ca 74</td>
    <td>'Maraval'</td>
    <td>C. crenata x C. sativa</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT269853<br>(complete genome)</td>
    <td>HTS</td>
  </tr>
  <tr>
    <td>LC1224H</td>
    <td>na</td>
    <td>na</td>
    <td>Q. rubra</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT339586;<br>MT339542</td>
    <td>CRA</td>
  </tr>
  <tr>
    <td>T32018</td>
    <td>Ca 74</td>
    <td>Maraval</td>
    <td>C. crenata x C. sativa</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT339587;<br>MT339543</td>
    <td>CRA</td>
  </tr>
  <tr>
    <td>LCA552</td>
    <td>Ca 74</td>
    <td>Maraval</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT339588;<br>MT339544</td>
    <td>CRA</td>
  </tr>
  <tr>
    <td>LCA584</td>
    <td>Ca 74</td>
    <td>Maraval</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT339589;<br>MT339545</td>
    <td>CRA</td>
  </tr>
  <tr>
    <td>LC1224F</td>
    <td>Ca 74</td>
    <td>Maraval</td>
    <td>C. crenata x C. sativa</td>
    <td>na</td>
    <td>France</td>
    <td>Ctifl</td>
    <td>MT339590;<br>MT339546</td>
    <td>CRA</td>
  </tr>
  <tr>
    <td>ITUmito39</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT261366<br>(complete genome)</td>
    <td>HTS</td>
  </tr>
  <tr>
    <td>ITCh2</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270674;<br>MT270683</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh3</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270667<sup>f</sup></td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh4</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270668;<br>MT270684</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh8</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270685<sup>g</sup></td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh10</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270669;<br>MT270678</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh11</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270664;<br>MT270679</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh12</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270665;<br>MT270680</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh14</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270666;<br>MT270681</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh20</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270670;<br>MT270675</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITChAs3</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270671;<br>MT270676</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITChAs4</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270672<sup>f</sup></td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITChAs5</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270677<sup>g</sup></td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITChAs8</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>MT270673;<br>MT270682</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITChAs9</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh1</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh5</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh6</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh7</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh9</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh13</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh14</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh15</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh20</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
  <tr>
    <td>ITCh21</td>
    <td>unknown</td>
    <td>Marrone</td>
    <td>C. sativa</td>
    <td>na</td>
    <td>Italy</td>
    <td>I</td>
    <td>na</td>
    <td>Incidence</td>
  </tr>
</table>
<table><tr><td>ITCh22</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh23</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh24</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh25</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh27</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh28</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh29</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh30</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh31</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh32</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh33</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh35</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh36</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh37</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh38</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh39</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh41</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh42</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh43</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh44</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh46</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh47</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh48</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh49</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh50</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh51</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh52</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh54</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh55</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh56</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh58</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh59</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr><tr><td>ITCh60</td><td>unknown Marrone</td><td>C. sativa</td><td>na</td><td>Italy</td><td>I</td><td>na</td><td>Incidence</td></tr></table>

a: Isolates from asymptomatic trees are indicated in bold; isolates with doubtful symptoms are underlined; isolates from asymptomatic cultivars with typical symptoms visible on rootstock regrowths are indicated in bold italic.

3

4

5

b: hybrid SC : hybrid between *C. sativa* and *C. crenata*, regardless the knowledge about which are the female and male parents.

6

c: Five plots have been sampled (A, Port, E, I, Ctifl)

7

d: Accession numbers are relative to the sequences obtained with both PCR detection assays (Ch-Bad-1466F/Ch-Bad-1800R and Ch-Bad-5860F/Ch-Bad-6109R)

8

9

e: Isolates were included either in the HTS analysis, or in the incidence analysis, or in the causal relationship analysis (CRA)

10

f: Only the fragment amplified with Ch-Bad-1466F/Ch-Bad-1800R was sequenced

11

g: Only the fragment amplified with Ch-Bad-5860F/Ch-Bad-6109R was sequenced

12

h: 'Mengshankui' (C. mollissima cultivar) seedling

13

i: C. mollissima seedling

14

15

16

17
<table><caption>**Supplemental TABLE S2.** Primers used for genome circularity confirmation and for molecular detection of chestnut mosaic virus by PCR</caption><thead><tr><th>Primer</th><th>Nucleotide sequence (5' to 3')</th><th>Genome coordinates</th><th>Amplicon size</th><th>Isolates detected</th></tr></thead><tbody><tr><td>Ch-Bad-6976F</td><td>CCCGAGCCATTTAC<br>ACTTCACAAC</td><td>6,976-6,999</td><td>436 nt</td><td>FRIc1224A</td></tr><tr><td>Ch-Bad-252R</td><td>TCACTCATCTACCTC<br>ACACGCTC</td><td>252-230</td><td></td><td></td></tr><tr><td>Ch-Bad-6481F</td><td>GAAATTGAATTGGA<br>AGGAAGA</td><td>6,481-6,501</td><td>1,007 nt</td><td>ITumito39</td></tr><tr><td>Ch-Bad-325R</td><td>TCAGATCAGCAAAC<br>TCGAAC</td><td>344-325-</td><td></td><td></td></tr><tr><td>Ch-Bad-1466F</td><td>TATCAGCACTACAG<br>TGAACAACC</td><td>1,466-1,488</td><td>335 nt</td><td></td></tr><tr><td>Ch-Bad-1800R</td><td>GTCATGACGCAAAC<br>TTGGAA</td><td>1,800-1,781</td><td></td><td>polyvalent</td></tr><tr><td>Ch-Bad-5860F</td><td>AGTATGTAAATGG<br>GCACCGTTC</td><td>5,857-5,878</td><td>232 nt</td><td></td></tr><tr><td>Ch-Bad-6109R</td><td>GTTGATCCATCGCA<br>CTCTTG</td><td>6,109-6,090</td><td></td><td>polyvalent</td></tr></tbody></table>
1 **Supplemental TABLE S3.** Datamining of publicly available chestnut HTS data for chestnut mosaic

2 virus sequences

<table><thead><tr><th>Type of data<sup>a</sup></th><th>Dataset</th><th>Castanea species/cultivar</th><th>Country</th><th>Length of assembled sequence (nt)</th><th>% mapped reads<sup>b</sup></th><th>% nt identity with FRIc1224A</th></tr></thead><tbody><tr><td>EST</td><td>GO917001</td><td>C. mollissima, BX3, clone Vanuxem</td><td>USA</td><td>436</td><td>na</td><td>99%</td></tr><tr><td>WGA</td><td>JRKL01079565</td><td>C. mollissima, cv Vanuxem</td><td>USA</td><td>7,164 (full length)</td><td>na</td><td>99%</td></tr><tr><td>RNA-Seq</td><td>SRX4015368</td><td>C. mollissima</td><td>USA</td><td>7,161 (full length)</td><td>0.004%</td><td>97.4%</td></tr><tr><td>RNA-Seq</td><td>SRX001805</td><td>C. mollissima, cv Vanuxem</td><td>USA</td><td>5,889 (scaffold)</td><td>0.04%</td><td>95-100%</td></tr><tr><td>GBS</td><td>SRX5144434</td><td>C. mollissima</td><td>China</td><td>5,933 (scaffold)</td><td>0.06%</td><td>98.7% (average)</td></tr><tr><td>GSB</td><td>SRX5144449</td><td>C. mollissima, clone Vanuxem</td><td>China</td><td>6,396 (scaffold)</td><td>0.04%</td><td>97.5% (average)</td></tr><tr><td>GBS</td><td>SRX5145051</td><td>C. mollissima, cv Vanuxem</td><td>China</td><td>5,873 (scaffold)</td><td>0.02%</td><td>99% (average)</td></tr><tr><td>GBS</td><td>SRX5145050</td><td>C. mollissima</td><td>China</td><td>6,680 (scaffold)</td><td>0.01%</td><td>99% (average)</td></tr><tr><td>GBS</td><td>SRX51445044</td><td>C. mollissima</td><td>China</td><td>6,183 (scaffold)</td><td>0.01%</td><td>97.4% (average)</td></tr><tr><td>GBS</td><td>SRX51444621</td><td>C. mollissima</td><td>China</td><td>5,875 (scaffold)</td><td>0.02%</td><td>95.6% (average)</td></tr><tr><td>GBS</td><td>SRX51444448</td><td>C. mollissima</td><td>China</td><td>2,948 (scaffold)</td><td>0.04%</td><td>99% (average)</td></tr><tr><td>GBS</td><td>SRX51444446</td><td>C. mollissima</td><td>China</td><td>5,873 (scaffold)</td><td>0.04%</td><td>99% (average)</td></tr></tbody></table>
<table><tr><td>GBS</td><td>SRX5825095</td><td>C. dentata</td><td>USA</td><td>1,857 (scaffold)</td><td>0.0036%</td><td>89.2% (average)</td></tr><tr><td>GBS</td><td>SRX5145077</td><td>C. dentata</td><td>USA</td><td>6,068 (scaffold)</td><td>0.01%</td><td>90.9% (average)</td></tr></table>

3 a EST: expressed sequence tags; WGA: whole genome assembly; GBS: genotyping by sequencing

4 b Reads mapped to the genomic sequence of chestnut mosaic virus (French isolate)