Armelle Marais
email: armelle.marais-colombel@inrae.fr

Sergio Murolo

Chantal Faure

Yoann Brans

Clement Larue

François Maclot

Sébastien Massart

Michela Chiumenti

Angelantonio Minafra

Gianfranco Romanazzi

Sergio Muroloꝉ

Clément Larue

Marie Lefebvre

Teresa Barreneche

Cécile Robin

Rémy J Petit

Thierry Candresse

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

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

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Armelle Marais

Phytopathology 3

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 [START_REF] Conedera | Amplification of 16S rRNA genes from culturable and non-culturable mollicutes[END_REF]. 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 [START_REF] Prospero | Phytophthora on Castanea sativa Mill. (sweet chestnut)[END_REF][START_REF] Rigling | Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control[END_REF]. In Italy, [START_REF] Gualaccini | Una virosi nuova del castagno[END_REF] described a chestnut disease associated with viral symptoms (mosaic, shoots with asymmetric leaf blade deformation) which was again reported in Campania during the 80s, [START_REF] Ragozzino | Una malattia virus-simile del castagno in provincia di Avellino. Giornate di studio sul castagno[END_REF], 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 [START_REF] Shimada | Chestnut Yellows[END_REF][START_REF] Horvath | Demonstration of virus-like particles in sweet chestnut and oak with leaf deformations in Hungary[END_REF]. 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 Armelle Marais Phytopathology 4 and [START_REF] Desvignes | New knowledges on the chestnut mosaic virus disease[END_REF]Desvignes, 1999b;[START_REF] Vettraino | A new transmissible symptomology on sweet chestnut in Italy[END_REF]. The susceptibility to the ChMD agent of Castanea species/cultivars has been evaluated in several studies [START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]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 [START_REF] Martin | Quarantine regulations and the impact of modern detection methods[END_REF][START_REF] Villamor | High Throughput Sequencing for plant virus detection and discovery[END_REF]. 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 [START_REF] Rott | Application of Next Generation Sequencing for diagnostic testing of tree fruit viruses and viroids[END_REF][START_REF] Kreuze | 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[END_REF]). This has accelerated the application of HTS technologies in the field diagnostic of diseases [START_REF] Massart | Current impact and future directions of high throughput sequencing in plant virus diagnostics[END_REF], and in quarantine regulations [START_REF] Martin | Quarantine regulations and the impact of modern detection methods[END_REF][START_REF] Massart | A framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by NGS technologies[END_REF].

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 [START_REF] Bhat | Badnaviruses: the current global scenario[END_REF]. The genome of badnaviruses is composed of a noncovalently 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)] [START_REF] Hohn | Plant pararetroviruses: replication and expression[END_REF][START_REF] Bhat | Badnaviruses: the current global scenario[END_REF].

Armelle Marais Phytopathology 5

Badnaviruses can also be present as integrated sequences in some host plant genomes (endogenous badnaviruses) [START_REF] Staginnus | Suggestions for a nomenclature of endogenous pararetroviral sequences in plants[END_REF][START_REF] Bhat | Badnaviruses: the current global scenario[END_REF]. The contribution of these integrated sequences to host and virus evolution is still poorly understood [START_REF] Geering | Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution[END_REF].

Given the very limited knowledge on the etiology of ChMD, and based on previously published studies [START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]1999a, 1999b;Desvignes and Lecoq, 1995;[START_REF] Desvignes | Mosaïque du chataignier: Transmission par le puceron Myzocallis castanicola[END_REF], we investigated the hypothesis that a virus might be involved in this disease. Combining HTSbased viral indexing and classical approaches, we report here the complete genome sequence of a novel badnavirus species for which the name Chestnut mosaic virus (ChMV) is proposed. We further show that there is a strict correlation between the presence of the virus and the appearance of typical ChMD symptoms in various graft-inoculated indicator plants. Preliminary epidemiological studies carried out in Italy and in France reveal that the virus can have high incidence in some orchards, and, as expected, can be associated with symptomatic or asymptomatic infections.

MATERIALS AND METHODS

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 [START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]. 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 [START_REF] Desvignes | Mosaïque du chataignier: Transmission par le puceron Myzocallis castanicola[END_REF]. The LCA552 and LCA584 isolates were collected from C. sativa trees in France in 2009 and 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).

In the framework of a survey carried out in Italian chestnut orchards to monitor chestnut blight (Acquasanta Terme (AP), locality Umito, Italy) [START_REF] Murolo | Phenotypic and molecular investigations on hypovirulent Cryphonectria parasitica in Italy[END_REF], 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).

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).

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).

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 [START_REF] Reid | An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[END_REF]. For the Italian material, total RNAs were extracted from symptomatic leaves according to the protocol described by [START_REF] Gambino | A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants[END_REF]. 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 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). 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 [START_REF] Zerbino | Velvet: algorithms for de novo short read assembly using de Bruijn graphs[END_REF], 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 evalue 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.

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 [START_REF] Doyle | Isolation of plant DNA from fresh tissue[END_REF]. 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 Armelle Marais Phytopathology 8 initial denaturation step at 95°C for 4 min, 40 or 35 cycles, respectively, were set at 94˚C for 30 sec, 60°C (Ch-Bad-6976F/ Ch-Bad-252R) or 55˚C (Bad-Ch-6481F/325R) for 30 sec, and 72˚C for 90 sec, followed by a final extension step of 10 min at 72˚C. PCR amplification products were sequenced on both strands (GATC, Eurofins, Ebersberg, Germany).

ChMV molecular detection and variant analysis by PCR. For the molecular detection of ChMV, two sets of primers were designed in conserved regions of ORF3 designed using the sequences of isolates FRlc1224A and ITumito39. One primer pair (Ch-Bad-1466F/Ch-Bad-1800R, Table S2) allows the amplification of a genomic region (335 nt) in the MP domain (Figure 2), whereas the second pair (Ch-Bad-5860F/Ch-Bad-6109R, Table S2) amplifies a 232-nt fragment in the RH domain (Figure 2).

An aliquot of 25 ng of total DNA was used for the PCR assays in a 50 μl volume containing 10 mM Tris-HCl (pH 8.5), 2 mM MgCl2, 50 mM KCl, 0.2 mM dNTPs, forward and reverse primers at 1 μM each, and either 1.25U of DreamTaq or 1U of GoTaq. After an initial denaturation step at 95°C for 4 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 extension step of 10 min at 72˚C. Amplicons were analyzed by electrophoresis on 1.5% agarose gel and were directly sequenced on both strands (GATC).

Possible phytoplasma infection was evaluated using primer pair P1/P7 (Deng and Hiruki. 1991;[START_REF] Smart | Phytoplasma-specific PCR primers based on sequences of 16s-23s rRNA spacer region[END_REF] and, in nested PCR, primers R16F2n/R2 [START_REF] Gundersen | Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs[END_REF].

Sequence and phylogenetic analyses. The full-length genomes were analyzed by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) to identify putative ORFs in the viral genome.

Deduced amino acid (aa) sequences were analyzed for conserved protein domains gathered in Conserved Domains Database (CDD) (http://www.ncbi.nlm.nih.gov/structure/cdd.shtml) and theoretical molecular weights were calculated using ExPASy (http://web.expasy.org/compute_pi/).

Multiple alignments of nucleotide (nt) or amino acid (aa) sequences were performed using the ClustalW program [START_REF] Thompson | CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap, penalties and weight matrix choice[END_REF] implemented in MEGA version 7.0 [START_REF] Kumar | MEGA 7: Molecular Evolutionary Genetics Analysis version 7 for bigger datasets[END_REF].

Genetic distances (p-distances using strict nt or aa identity) were calculated using MEGA 7.0. Phylogenetic trees were reconstructed using the neighbor-joining method implemented in MEGA 7.0 and robustness of nodes was assessed from 1,000 bootstrap resamplings.

RESULTS

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 (FRlc1224A) 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 [START_REF] Teycheney | ICTV Virus Taxonomy Profile: Caulimoviridae[END_REF]) 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 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.

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 [START_REF] Wang | Characterization of complete genome and small RNA profile of pagoda yellow mosaic virus, a novel badnavirus in China[END_REF], together with gooseberry vein banding virus (GVBV), rubus yellow net virus (RYNV), grapevine vein clearing virus (GVCV), birch leafroll-associated virus (BLRaV), wisteria badnavirus 1 (WBV1), and pagoda yellow mosaicassociated virus (PYMaV) (Figure 3). Nevertheless, they are clearly distant from all of these species, defining a novel branch, supported by a 99% bootstrap value (Figure 3). Tree topology was similar when using an alignment of representative badnaviral ORF3 protein sequences (Figure S1). To confirm these analyses, pairwise comparisons of genome sequences showed that the isolate FRlc1224A isolate shows between 64% (with GVBV) and 68.4% (with BLRaV) nt identity (Table 1), which is below the 80% nt identity value used as the species demarcation threshold in the family.

Therefore, this virus represents a novel species in the family Caulimoviridae, for which we propose the name Chestnut mosaic virus (ChMV). In the same taxonomically informative region, the isolates FRlc1224A and ITumito39 share 91.9% nt identity (97.8% aa identity), indicating that they belong to the same viral species (Table 1). 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 FRlc1224A 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 FRlc1224A as compared to 93.9% nt identity with ITumito39. The second isolate of C. dentata appears to be equally related to the FRlc1224A 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 FRlc1224A 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 Armelle Marais Phytopathology 13 (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 Armelle Marais Phytopathology 14 cluster (Figure S3B). Another French isolate, 20971-E remains isolated and does not fit in either group. The average nt divergence in this region is slightly higher than in the RT-RNaseH region, (5.8% +/-0.7%), and the inter-group diversity reaches a value of 13.4% +/-1.8%, as compared to the 6.3% value for the other region.

DISCUSSION

Since the seminal work of Desvignes and collaborators in the 1990s, it has been acknowledged that the agent responsible for ChMD is most likely a thermosensitive, graft-transmissible virus that can be transmitted experimentally and probably naturally by the aphid Myzocallis castanicola [START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]1999a;1999b;Desvignes and Lecoq, 1995;[START_REF] Desvignes | Mosaïque du chataignier: Transmission par le puceron Myzocallis castanicola[END_REF].

Depending on the chestnut genotype, this infection can either be asymptomatic or can result in the expression of severe and conspicuous ChMD symptoms. In chestnut orchards in the Marche region (Italy), both young and mature plants were shown to be affected, significantly decreasing chestnut production. Symptoms have also been observed in some Quercus species following experimental graft inoculation. To date, however, the causal agent remains to be identified.

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

There is unambiguous evidence that ChMV as reported here is an episomal virus. It was detected in graft-inoculated indicators, and not in non-inoculated control plants of the same variety, demonstrating its graft-transmissibility, a property of episomal viruses. This line of evidence is further reinforced by the detection of ChMV in symptomatic, graft-inoculated indicator Quercus Armelle Marais Phytopathology 15 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, [START_REF] Bhat | Badnaviruses: the current global scenario[END_REF]) 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 x 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)

were ChMV positive, satisfying the Bradford-Hill's experimental and consistency criteria [START_REF] Hill | The environment and disease: association or causation?[END_REF][START_REF] Fox | Reconsidering causal association in plant virology[END_REF].

Preliminary studies indicate that ChMV is highly prevalent in the analyzed orchards in France and Italy, confirming the earlier results of Desvignes (1999a). In parallel, the identification of ChMV 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 systematically associated with symptomatic infections, although its frequency was systematically higher in symptomatic plants. This result was expected since previous grafting experiments had demonstrated that not all chestnut varieties/species are susceptible to ChMD and develop symptomatic infections (Desvignes and Lecoq, 1995;[START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]1999b). Biological indexing on the susceptible 'Maraval' indicator has in particular identified latent ChMV infections in many symptomless C. sativa varieties or C. sativa x C crenata hybrids (Desvignes and Lecoq, 1995;[START_REF] Desvignes | Characterization of the chestnut mosaic[END_REF]1999b). On the other hand, all surveyed symptomatic plants in France were found to harbor the virus, while it was detected in 52/60 (87%) of tested symptomatic Italian trees. The failure to detect ChMV in eight symptomatic Italian trees might reflect sequence variability and an incomplete inclusiveness of the PCR primers used or low or uneven virus accumulation. Indeed, using biological indexing, Desvignes et al. have previously found an uneven distribution of the ChMD agent in infected trees leading to a failure to detect it in parts of some infected trees (Desvignes and Lecoq, 1995;Desvignes et al. 1999b).

Taken together, and even though Koch's postulates were not fully verified, the experiments reported here make a very strong case for a role of ChMV as the causal agent of the chestnut mosaic disease. The low ChMV diversity observed in France and Italy are consistent with the scenario of its recent introduction in Europe (Desvignes and Lecoq, 1995), while the genetic separation of the Italian and French clusters is suggestive of separate introduction events. These results and the

Armelle Marais

Phytopathology 17 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. S1.

e-EXTRA FIGURE CAPTIONS AND TABLE TITLES

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

  in GenBank under accession numbers MT269853 and MT261366, respectively.No other plant virus was detected in the two datasets during the Blast annotation of contigs.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[START_REF] Teycheney | ICTV Virus Taxonomy Profile: Caulimoviridae[END_REF]. The genomic organization is the same for both isolates, comprising three open reading frames (ORFs) encoded on the positive strand (Figure2), and is typical for badnaviruses[START_REF] Teycheney | ICTV Virus Taxonomy Profile: Caulimoviridae[END_REF]. The ORF1 (nt 245-751, numbering according to the isolate FRlc1224A 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 (Figure2): a viral movement protein (MP, cl03100), a zincbinding 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.

FRlc1224A

  has only weak identity levels with representative members of the genus Badnavirus, comprised between 42.1% nt identity (sugarcane bacilliform IM virus; 42.5% for the isolate ITumito39) and 50.9% (WBV1; 50.8% for the isolate ITumito39). The same tendency is observed when considering the genome proteins. The ORF1-encoded protein shows only weak homology with the corresponding proteins of WBV1 (27.8% aa identity) and PYMAV (26.1% aa identity), and the ORF2-encoded protein shares only 33.1% aa identity with the corresponding protein of the most closely related virus, WBV1. The polyprotein encoded by ORF3 shares 49.5% aa identity with the corresponding protein of the closest relative, PYMAV. Using the ORF3 region (RT and RH domain) used for taxonomical discrimination in the family Caulimoviridae (Teycheney et al. 2020), the

Fig. 2 :

 2 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. 4 .

 4 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 .

 5 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 TableS1.

Fig. 2 :

 2 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)

Fig. S2

  Fig. S2

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

  c : Five plots have been sampled (A, Port, E, I, Ctifl) 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) e: Isolates were included either in the HTS analysis, or in the incidence analysis, or in the causal relationship analysis (CRA) f : Only the fragment amplified with Ch-Bad-1466F/Ch-Bad-1800R was sequenced g : Only the fragment amplified with Ch-Bad-5860F/Ch-Bad-6109R was sequenced h 'Mengshankui' (C. mollissima cultivar)

  Identification of ChMV in publicly available chestnut HTS data. The datamining of chestnut HTS data from various chestnut sources publicly available at GenBank [EST sequences, whole genome assembly, RNA-Seq and Genotyping-by-sequencing (GBS) reads available as Sequence Read

	Armelle Marais
	Phytopathology 12

TABLE 1 .

 1 Percentage of identity between the ORF3 region encoding the Reverse Transcriptase -Rnase H a 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

	Armelle Marais
	Phytopathology 22

a This region is the one typical used for taxonomic discrimination in the family Caulimoviridae

[START_REF] Teycheney | ICTV Virus Taxonomy Profile: Caulimoviridae[END_REF]

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.

TABLE 2 .

 2 Number and percentage of chestnut mosaic virus-infected plants regarding the plot, the Castanea species sampled, and the symptomatology

	Origin of the	Infected/total	Infected/asympto	Infected/sympto	Infected/plants
	sampled plants	plants (%)	matic plants (%)	matic plants (%)	with atypical
					symptoms (%)
	France (overall)	65/95 (68.4%)	23/47 (48.9%)	38/38 (100%)	4/10 (40%)
	Plot A	13/23 (56.5%)	6/15 (40%)	6/6 (100%)	1/2 (50%)
	Plot E	48/66 (72.7%)	15/28(53.6%)	30/30 (100%)	3/8 (37.5%)
	Plot Port	4/6 (66.6%)	2/4 (50%)	2/2 (100%)	na b
	C. crenata	5/6 (83%)	1/1 (100%)	4/4 (100%)	0/1
	C. sativa	30/43 (70%)	11/20 (55%)	18/18 (100%)	1/5 (20%)
	C. mollissima	13/14 (93%)	1/2 (50%)	10/10 (100%)	2/2 (100%)
	Hybrid a	17/32 (53.1%)	10/24 (41.6%)	6/6 (100%)	1/2 (50%)
	Italy Castanea	57/70 (81.5%)	5/10 (50%)	52/60 (87%)	na b
	sativa
	b not applicable
	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

a Interspecific hybrids between C. crenata, C. mollisima and C. sativa 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.

  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.

	a	Ca 844 i Ca 837 h --Ca 846 h -Ca 741 'Dauphine' Ca 665 'Longal' Ca 860 -Ca 564 'Ipharra 16' Ca 576 'Sardonne' Ca 138 'Marron de Redon' unknown -Ca 520 'Montagne' Ca 106 'Marron Comballe' Ca 511 Ca 116 -Ca 135 Vans' Ca 521 -unknown Marrone unknown Marrone unknown Marrone 'Précoce des unknown Marrone unknown Marrone Chiusa Pesio' unknown Marrone 'Marrone di unknown Marrone Ca 460 -unknown Marrone unknown Marrone Ca 74 'Maraval' Ca 825 Besse' Ca 393 'Marron de unknown Marrone Chevanceaux' unknown Marrone unknown Marrone 'Précoce de unknown Marrone unknown Marrone Ca 111 'Marron de Lyon' Ca 399 'Comballe' unknown Marrone Ca 126 -Ca 639 -unknown Marrone Ca 105 'Sardonne' Ca 03 -Ca 127 -Ca 127 -Ca 112 'Bournette' Ca 501 -Ca 501 -Ca 665 'Longal' Ca 48 'Précoce Migoule' Ca 151 'Bouche Rouge' unknown -unknown -Ca 106 'Marron Comballe' Ca 105 'Sardonne' Ca 501 -Ca744 'Quing Zha' Ca 845 h -Ca 843 h -Ca 842 h -Ca 838 h -Ca 839 h -Ca 840 h -Ca 841 h -Ca 730 'Sauvage Marron' unknown Marrone Ca 381 -unknown Marrone Ca 381 -unknown Marrone Ca 151 unknown Marrone 'Bouche unknown Marrone Rouge' FRlC1224A Ca 74 ITCh21 ITCh20 ITCh15 ITCh14 ITCh13 ITCh9 ITCh7 ITCh6 ITCh5 ITCh1 ITChAs9 ITChAs8 ITChAs5 ITChAs4 20955 ITCh22 20962 ITCh23 20963 ITCh24 20969 ITCh25 20970 ITCh27 20972 ITCh28 ITCh29 'Maraval' ITCh30 unknown Marrone ITCh31 unknown Marrone LC1224H na na ITCh32 unknown Marrone ITCh33 unknown Marrone T32018 Ca 74 Maraval ITCh35 unknown Marrone ITCh36 unknown Marrone LCA552 Ca 74 Maraval ITCh37 unknown Marrone LCA584 Ca 74 Maraval ITCh38 unknown Marrone ITCh39 unknown Marrone LC1224F Ca 74 Maraval ITCh41 unknown Marrone ITCh42 unknown Marrone ITUmito39 unknown Marrone ITCh43 unknown Marrone ITCh44 unknown Marrone ITCh2 unknown Marrone ITCh46 unknown Marrone ITCh47 unknown Marrone ITCh3 unknown Marrone ITCh48 unknown Marrone ITCh4 unknown Marrone ITCh49 unknown Marrone ITCh50 unknown Marrone ITCh8 unknown Marrone ITCh51 unknown Marrone ITCh10 unknown Marrone ITCh52 unknown Marrone ITCh54 unknown Marrone ITCh11 unknown Marrone ITCh55 unknown Marrone ITCh12 unknown Marrone ITCh56 unknown Marrone ITCh58 unknown Marrone ITCh14 unknown Marrone ITCh59 unknown Marrone ITCh60 unknown Marrone ITCh20 unknown Marrone ITChAs3 unknown Marrone	C. mollissima C. mollissima C. mollissima C. sativa C. sativa hybrid SC C. crenata C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa x C. crenata C. sativa C. crenata C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa (C. crenata x C. sativa) x (C. crenata x C. sativa) C. sativa C. sativa C. crenata x C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa x C. crenata C. sativa C. sativa C. sativa C. crenata (C. crenata x C. sativa) x C. mollisima (C. crenata x C. sativa) x C. mollisima C. crenata x C. sativa C. sativa C. sativa C. sativa (C. crenata x C. sativa) C. sativa C. sativa x C. crenata C. sativa C. sativa C. sativa C. sativa C. mollissima C. mollissima C. mollissima C. mollissima C. mollissima C. mollissima C. mollissima C. mollissima C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. crenata x C. sativa C. sativa C. sativa Q. rubra C. sativa C. sativa C. crenata x C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. crenata x C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa C. sativa	Not graft France E Not graft France E Not graft France E Ca 07 France E Ca 07 France E unknown France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 74 France E Ca 07 France E Ca 07 France E Ca 74 France E Ca 74 France E Ca 07 France E na Italy I na Italy I na Italy I na Italy I na Italy I Ca 74 France E na Italy I na Italy I Ca 74 France E na Italy I na Italy I Not graft France E Ca 07 France E na Italy I Ca 07 France E na Italy I na Italy I na Italy I na Italy I Ca 07 France E Ca 07 France E na Italy I Ca 07 France E Ca 07 France E na Italy I Ca 07 France E Ca 74 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E Ca 07 France E unknown France Port unknown France Port G1. Ca394 France A G1.Ca486 France A G1.Moll France A Not graft France E Not graft France E Not graft France E Not graft France E Not graft France E Not graft France E Not graft France E Ca 07 France E na Italy I Ca 07 France E na Italy I Ca 07 France E na Italy I na Italy I Ca 07 France E na Italy I na France Ctifl na Italy I na Italy I na France Ctifl na Italy I na Italy I na France Ctifl na Italy I na Italy I na France Ctifl na Italy I na France Ctifl na Italy I na Italy I na France Ctifl na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I na Italy I	MT339561; MT339517 MT339562; MT339518 MT339563; MT339519 MT339564; MT339520 MT339565; MT339521 MT339566; MT339522 MT339567; MT339524 MT339525 MT339570; MT339526 MT339571; MT339527 MT339572; MT339528 na na na na na na na na na na na MT339569; na na na MT339568; MT339523 na na na na na MT270673; MT270682 MT270677 g MT270672 f MT339573; MT339529 na na na na MT339574; MT339530 MT339575; MT339531 MT339576; MT339532 MT339577; MT339533 MT339578; MT339534 MT339579; MT339535 MT339580; MT339536 MT339581; MT339537 MT339582; MT339538 MT339583; MT339539 MT339584; MT339540 MT339585; MT339541 na na na na na na na na na na na na na na na na na na na na na na MT269853 (complete na genome) na MT339586; MT339542 na MT339587; na na MT339543 MT339588; MT339544 na na MT339589; MT339545 na na MT339590; na MT339546 na MT261366 (complete na genome) na MT270674; MT270683 na MT270667 f na na MT270668; na MT270684 MT270685 g na MT270669; na na MT270678 MT270664; MT270679 na na MT270665; na MT270680 MT270666; na na MT270681 MT270670; MT270676 MT270671; MT270675	Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence HTS Incidence Incidence Incidence CRA Incidence CRA Incidence Incidence CRA Incidence Incidence CRA Incidence CRA Incidence Incidence Incidence HTS Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence Incidence

ACKNOWLEDGMENTS

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). LITERATURE CITED 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. 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.

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). 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. S1

Supplemental Table


Hugues Clivot

Fabien Ferchaud

Florent Levavasseur

Sabine Houot

Anne-Isabelle Graux

Alice Cadéro

Françoise Vertès

Alain Mollier

Annie Duparque

Jean-Christophe Mouny

Olivier Therond

Bruno Mary

Simulating soil organic carbon dynamics in long-term bare fallow and arable experiments with STICS model

Keywords: soil organic carbon, mineralization, carbon inputs, cropped soils, bare fallow soils

Introduction

Accurate modelling of soil organic carbon (SOC) dynamics on the long-term is required to better predict the environmental impacts of cropping systems and notably their potential to sequester atmospheric CO2 into SOC that can play an important role in greenhouse gas mitigation and soil fertility. To date, a limited number of studies have been conducted to evaluate the ability of STICS to simulate soil organic nitrogen dynamics (e.g. [START_REF] Constantin | Long-term nitrogen dynamics in various catch crop scenarios: Test and simulations with STICS model in a temperate climate[END_REF][START_REF] Autret | Long-term modelling of crop yield, nitrogen losses and GHG balance in organic cropping systems[END_REF][START_REF] Yin | Long-term modelling of soil N mineralization and N fate using STICS in a 34-year crop rotation experiment[END_REF]. There is therefore a need to assess the ability of STICS to simulate SOC dynamics and to compare its modelling performances with models validated for various situations.

Methods

A research version of STICS [START_REF] Autret | Long-term modelling of crop yield, nitrogen losses and GHG balance in organic cropping systems[END_REF] was used to simulate SOC dynamics (0 to 20-30 cm) in long-term field experiments that were either under bare fallow [START_REF] Farina | Multi-metric evaluation of an ensemble of biogeochemical models for the estimation of organic carbon content in long-term bare fallow soils[END_REF] or cultivated conditions (Table 1). The model performances were compared with those of the annual time-step carbon model AMG v2, which had been previously validated for various pedoclimatic conditions and cropping systems [START_REF] Clivot | Modeling soil organic carbon evolution in long-term arable experiments with AMG model[END_REF].

Results and discussion

Results show that STICS could predict satisfactorily final C stocks after a long-term monitoring of SOC in bare fallow and cultivated systems (Fig. 1). The diversity of experiments suggests that STICS was able to simulate well both decomposition and mineralization derived from native soil organic matter and from fresh organic residues that are incorporated into humified organic matter. The performances of STICS in simulating SOC are comparable to those of AMG model (mean RRMSE of 6.3% and 4.3% for final SOC stocks simulated with STICS and AMG, respectively), for which C inputs into soil require measured crop yields to be calculated, while STICS provides the advantage of simulating crop growth and residues returned to the soil.

Further studies will be needed to assess the model ability to simulate SOC dynamics in other systems such as grasslands or cropping systems including perennial species.

Figure 1 .

 1 Figure 1. Differences between final and initial observed SOC stocks vs simulated with STICS and AMG models for bare fallow (BF) and cultivated soils in long-term experiments. RRMSE = relative root mean square error and MD = mean difference for final SOC stocks.

Table 1 .

 1 Description of the selected long-term experiments.

	Experiment	Code	Rotation	Duration (years)	Initial SOC stock (t C ha -1 )	Final SOC stock (t C ha -1 )
	Askov-B3/B4	Ask3/4	Bare fallow	29	52.1/47.7	36.4/33.0
	Grignon	Grign	Bare fallow	48	41.7	25.4
	Kursk	Kursk	Bare fallow	36	100.3	79.4
	Rothamsted	Roth	Bare fallow	49	71.7	28.6
	Ultuna	Ult	Bare fallow	51	42.5	26.9
	Versailles	Vers	Bare fallow	79	65.5	22.7
	La Cage-Co-B1/B2	Cage-Co1/2	Pea-Wheat-Rapeseed	16	43.4/37.5	44.7/38.8
	La Cage-Li-B1/B2	Cage-Li1/2	Pea-Wheat-Rapeseed	16	49.5/37.6	48.3/40.0
	Feucherolles-Min	Feu-Min	Wheat-Grain Maize	15	43.4	43.3
	Feucherolles-T0	Feu-T0	Wheat-Grain Maize	15	42.7	39.0
	Kerbernez-A	Kerb-A	Silage Maize	26	81.7	57.8