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1 1 2 3 2 Viromes in fungi infecting avocado in Spain 4 5 1+ 2 1 3 Leonardo Velasco , Isabel Arjona-Girona , Enrico Cretazzo and Carlos López- 6 7 2 8 4 Herrera 9 10 5 1 Instituto Andaluz de Investigación y Formación Agraria (IFAPA), 29140 Churriana, 11 12 13 6 Málaga, Spain 14 15 7 2 Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible, C.S.I.C., 16 17 18 8 Córdoba, Spain. 19 20 9 21 22 23 10 + Correspondence may be sent to L. Velasco 24 25 11 Instituto Andaluz de Investigación y Formación Agraria, Pesquera, Alimentaria y de la 26 27 12 Producción Ecológica (IFAPA) 28 29 30 13 Cortijo de la Cruz s/n, 29140 Churriana, Málaga (Spain) 31 32 14 Telephone: + 34 671 532821 33 34 35 15 Tel.: + 34 671 532822 (lab) 36 37 16 Fax: + 34 951 036227 38 39 40 17 E-mail: [email protected] 41 42 18 43 44 45 19 46 47 20 48 49 50 21 51 52 22 53 54 55 56 57 58 59 60 61 62 1 63 64 65 23 Abstract 1 2 3 24 Four isolates of Entoleuca sp., family Xylariaceae, , recovered from 4 5 6 25 avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the 7 8 26 dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that 9 10 27 revealed the presence of eleven viruses putatively belonging to families Partitiviridae, 11 12 13 28 Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition 14 15 29 to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence 16 17 18 30 with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris 19 20 31 alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the 21 22 23 32 virome composition, the four isolates only differed in the presence of the bunyavirus 24 25 33 and the ourmia-like virus, while all other viruses showed common patterns. Specific 26 27 28 34 primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca 29 30 35 sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results 31 32 36 indicate that intra- and interspecies horizontal virus transmission occur frequently in this 33 34 35 37 pathosystem. 36 37 38 38 Keywords: mycoviruses, negative single-stranded RNA virus, gammaflexivirus, 39 40 41 39 ourmia-like virus, endornavirus, multiple virus infections, Entoleuca, Rosellinia 42 43 40 necatrix, horizontal virus transmission 44 45 46 47 41 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 2 63 64 65 42 Introduction 1 2 3 43 In the last years, widespread analysis of plant-pathogenic fungi metatranscriptomes has 4 5 6 44 revealed the presence of complex infections by multiple viruses belonging to different 7 8 45 taxonomic groups including families Alphaflexiviridae, Chrysoviridae, Endornaviridae, 9 10 46 Gammaflexiviridae, Hypoviridae, Megabirnaviridae, Narnaviridae, Deltaflexiviridae, 11 12 13 47 Partitiviridae, “Ourmiaviridae”, Totiviridae, Virgaviridae and order Bunyavirales, 14 15 48 whilst many others mycoviruses do not belong to known virus families (Marzano et al., 16 17 18 49 2016a; Marzano et al., 2016b; Osaki et al., 2016; Deakin et al., 2017; Xin et al., 2017; 19 20 50 Rott et al., 2018; Arjona-López et al., 2018; Mu et al., 2018). Among the viruses 21 22 23 51 identified, most of them have double-stranded (ds) RNA or positive single-stranded (ss) 24 25 52 RNA genomes, being scarce those with negative ssRNA genomes. Mycoviruses are 26 27 28 53 often associated with families containing plant-infecting and/or invertebrate-infecting 29 30 54 members as evidenced by recent metagenomics analysis (reviewed in: Dolja and 31 32 55 Koonin, 2018), that is also an extremely helpful tool in clarifying taxonomic 33 34 35 56 relationships (Shi et al., 2018). 36 37 57 Mycovirus research aims to control fungal pathogens by identifying mycoviruses 38 39 40 58 capable of weakening or limiting fungal damages (Ghabrial and Suzuki, 2009; Xie and 41 42 59 Jiang, 2014; Ghabrial et al., 2015). On the other hand, there is growing evidence for 43 44 45 60 horizontal virus transmission (HVT) among species from different taxa as a major 46 47 61 driver of virus evolution (Dolja and Koonin, 2018). Direct confirmation of plant-fungi 48 49 62 HVT was provided recently when a plant virus, cucumber mosaic virus, was detected in 50 51 52 63 natural infection in the plant-pathogenic Rhizoctonia solani and HVT could be 53 54 64 experimentally demonstrated (Andika et al., 2017). 55 56 57 65 Avocado cultivation was introduced in the southern coast of Spain in the '70s as an 58 59 66 alternative to traditional Mediterranean crops such as olive, almond or grapevine 60 61 62 3 63 64 65 67 (López-Herrera and Zea-Bonilla, 2007). Since then, avocado white root rot, caused by 1 2 68 R. necatrix, has become one of the most serious threats to the cultivation of this crop in 3 4 5 69 the area (López-Herrera, 1998). Chemical treatments have been ineffective in 6 7 70 controlling the disease, especially when the harmful effects on the environment and 8 9 10 71 human health of their application are considered. Thus, alternative methods are being 11 12 72 considered, such as grafting on resistant rootstocks or the use of biological control 13 14 73 agents. Among those, and in parallel with similar approaches used in other trees, the 15 16 17 74 identification of mycoviruses capable of limiting the growth of fungal pathogens 18 19 75 appeared to us as a potential control strategy. Mycoviruses infecting Rosellinia necatrix, 20 21 22 76 a fungal species that severely infects many crops have been extensively studied in 23 24 77 different plant-fungus pathosystems, including pears, apple (reviewed in: Kondo et al., 25 26 27 78 2013) and, more recently, avocado (Persea americana Mill.) (Arjona-López et al., 28 29 79 2018; Velasco et al., 2018). 30 31 32 80 With this goal in mind, we investigated the presence of mycoviruses in avirulent fungal 33 34 81 isolates supposed to be R. necatrix by high-throughput sequencing (HTS) on dsRNA 35 36 37 82 extracts of four different isolates. Then, based on molecular tools, these avirulent 38 39 83 isolates were shown to be another fungus species morphologically indistinguishable 40 41 42 84 from R. necatrix, namely Entoleuca sp., a closely related member of family Xylariaceae 43 44 85 (Arjona-Girona and López-Herrera, 2018). Xylariaceae comprise one of the largest and 45 46 47 86 most diverse families of filamentous Ascomycota that includes saprotrophs occurring in 48 49 87 wood, litter, soil, and dung, and a few plant pathogens that cause canker diseases, root 50 51 88 rots, and needle blight in agricultural and natural systems (Whalley, 1996). Despite a 52 53 54 89 member of the genus Entoleuca (E. mammata) is pathogenic for forest trees (Ostry and 55 56 90 Anderson, 2009), the Entoleuca sp. isolated from avocado has not shown any 57 58 59 91 pathogenicity on this crop yet but seems to compete with R. necatrix for root 60 61 62 4 63 64 65 92 colonization (Arjona-Girona and López-Herrera, 2018). Therefore, and because of the 1 2 93 multiple virus infection observed in the four isolates of Entoleuca sp. as revealed by 3 4 5 94 HTS analysis, we extended the determination of the identified viruses to a collection 6 7 95 composed of 19 isolates of Entoleuca sp. and 18 of R. necatrix. 8 9 10 11 96 Materials and Methods 12 13 14 97 Virus sources and culture media. Four isolates of Entoleuca sp. (E97-14, E112-4, 15 16 98 E115-15 and E117-4) were originally obtained from the rhizosphere of different 17 18 19 99 avocado escape trees located in orchards and the mycelia grown on PDA medium 20 21 100 (Difco) was used later for subsequent analyses. These isolates belong to a collection of 22 23 24 101 37 fungal isolates from avocado trees that were used for mycovirus detection afterward. 25 26 102 The escape trees were normally vegetating in diseased patches located in orchards in the 27 28 29 103 Málaga province (Spain) (Arjona-Girona and López-Herrera, 2018) and in the same 30 31 104 orchard, other threes showed symptoms of white root rot due to R. necatrix infection. 32 33 34 105 dsRNA extraction for sequence analyses. For each Entoleuca sp. isolate, 1 g of fungal 35 36 106 mycelium was grown for 10 days on cellophane membrane-covered PDA Petri plates. 37 38 39 107 Then, dsRNAs were extracted using CF-11 cellulose as described by Morris and Dodds 40 41 108 (1979). Quality and yield were determined using the NanoDrop ND-1000 42 43 44 109 spectrophotometer (NanoDrop Technologies, USA). DsRNA bands sizes were analyzed 45 46 110 in 1% agarose gels after RedSafeTM staining under UV light. After extraction, dsRNAs 47 48 49 111 were eluted in 50 µl RNase-free water and stored at -70°C until further use. 50 51 52 112 Sequence determination from high-throughput sequencing (HTS). For HTS, 5-10 53 54 113 g of dsRNA were used, denatured at 95ºC for 10 min and immediately cooled on ice. 55 56 57 114 Next, RNA fragmentation and cDNA synthesis were carried out. Libraries were 58 59 115 prepared following procedures developed at the CRG Genomic Unit (Barcelona, Spain). 60 61 62 5 63 64 65 116 Illumina sequencing was performed using the Hiseq2000 for 50 nt reads. Reads were 1 2 117 used to generate contigs by de novo assembly using the algorithm Velvet v.12.08 (k-mer 3 4 5 118 = 31) at the SCBI Picasso supercomputing server (Malaga, Spain). Contig data sets 6 7 119 were subjected to BLASTN and BLASTX in the NCBI GenBank through the software 8 9 10 120 Geneious 7.1.9 (Biomatters). 11 12 5’- and 3’-ends determination of the mycoviral genomes. For completion of several 13 121 14 15 122 mycoviral genomic sequences we analysed the 5'- and 3'- ends using a modification of 16 17 123 the protocol developed by Potgieter et al., (2009) as described elsewhere (Velasco et al., 18 19 20 124 2018). Briefly, the T4 RNA ligase (Ambion, USA) ligates the PC3-T7 loop primer to 21 22 125 the 3’ end of each dsRNA strand allowing the cDNA synthesis by reverse transcription. 23 24 25 126 Amplification of cDNA ends is performed with the PC2 primer, that is internal to the 26 27 127 PC3-T7 loop primer, and template specific primers. 28 29 30 128 Phylogenetic analyses. The genomic sequences obtained from scaffolds were translated 31 32 33 129 accordingly to amino acids in the correct frame and aligned to sets of sequences 34 35 130 retrieved from the GenBank employing the MAFFT algorithm (Katoh & Standley, 36 37 131 2013) available in Geneious R11.1 (Biomatters, New Zealand). Maximum likelihood 38 39 40 132 (ML) phylogenetic trees were inferred for the different amino acid sequences using the 41 42 133 IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) that also calculated the most 43 44 45 134 appropriate substitution model for each case (Trifinopoulos et al., 2016). Estimation of 46 47 135 branch support was done using the aBayes test as implemented in IQ-TREE. 48 49 50 136 RT-PCR detection of mycoviruses in Entoleuca sp. and Rosellinia necatrix isolates. 51 52 53 137 The detection of the different hypothetical mycoviruses revealed by HTS was done 54 55 138 using RT-PCR after the availability of specific primers based on the sequences for the 56 57 139 different viral genomes. For that, dsRNA was extracted from Entoleuca sp. and R. 58 59 60 140 necatrix collection (IAS, Córdoba, Spain) using the viral dsRNA extraction kit (Intron, 61 62 6 63 64 65 141 South Korea). The synthesis of cDNA by reverse transcription was performed with 200 1 2 142 ng of dsRNA with the High-Capacity cDNA reverse transcription kit (Applied 3 4 5 143 Biosystems, USA). PCR reactions were carried out with the AmpliTools master mix 6 7 144 (Biotools, Spain) using 50 pmol each of forward and reverse primers (Table S1) in the 8 9 10 145 following conditions: an initial denaturing cycle of 2 min at 94 ºC, then 40 cycles of 30 11 12 146 s at 95 ºC, 30 s at 55-60ºC and 40 s at 72 ºC, and a final extension step of 5 min at 72 13 14 147 ºC. The amplicons were cloned and subjected to Sanger sequencing and compared with 15 16 17 148 the HTS derived sequences. 18 19 149 Phenotypic characterization of Entoleuca sp. isolates. Three parameters of mycelia 20 21 22 150 were evaluated: colony aspect, radial growth, and fresh weight. Phenoloxidase activity 23 24 151 was tested by using Bavendamm's medium (Bavendamm, 1928). Each treatment 25 26 27 152 consisted of four replicates per isolate, and each experiment was repeated once. To 28 29 153 compare fungal radial growth, the values for the Standardized Area Under the Growth 30 31 154 Curve (AUGCs) was calculated and statistical tests were performed. 32 33 34 35 155 Results 36 37 38 39 156 A collection of 37 isolates of Xylariaceae fungi isolated from avocado roots were used 40 41 157 for dsRNA extraction. The dsRNA profiles showed a range of sizes, indicative of the 42 43 44 158 probable occurrence of virus(es) in the fungal tissues (some representative dsRNA 45 46 159 profiles are displayed in Fig. 1). Four fungal isolates were selected for HTS on the basis 47 48 49 160 their dsRNA profiles, lack on virulence to avocado and differential characteristics 50 51 161 regarding both hyphae phenotype, phenoloxidase activity, and colony morphology (see 52 53 162 supplementary materials, Tables S7-S8 and Figs. S2-S5). Also, incompatibility barriers 54 55 56 163 (IB) were observed for all isolate combinations (Fig. S6), indicating that the four 57 58 164 isolates considered here belong to different vegetative compatibility groups (VCG). 59 60 61 62 7 63 64 65 165 Taxonomic adscription of the mycoviral sequences. Genomic sequences (complete or 1 2 166 partial) were obtained by alignment of contigs from the different viruses, completion 3 4 5 167 with Sanger sequencing of gaps or undetermined regions and, when necessary, 6 7 168 determination of RNA ends. Phylogenetic analysis allowed their adscription to different 8 9 10 169 virus families and genera. Eleven mycoviruses putatively belonging to different 11 12 170 taxonomic groups resulted from the analysis (Table 1). 13 14 15 171 Two viral sequences related to positive single-strand RNA viruses, order 16 17 172 Tymovirales. An RNA virus resembling positive ssRNA viruses was first determined in 18 19 20 173 Entoleuca isolate E97-14 and then detected in other fungal isolates (Table 1 and Table 21 22 174 2). This new species was named Entoleuca gammaflexivirus 1 (EnGFV1). The complete 23 24 25 175 genomic sequence was 9,221 nucleotides (nt) length, presenting three ORFs and no 26 27 176 tRNA-like structure at both 5’and 3’ ends. ORF1 consists of 5,790 nt starting at position 28 29 30 177 278 from the 5’ end. It encodes for a putative protein of 1,929 amino acids (aa) with a 31 32 178 molecular mass of 213.38 kDa. NCBI’s CDD search identified three putative domains: 33 34 179 methyltransferase (pfam01660, E-value = 1.10e-37), helicase (pfam01443, E-value = 35 36 37 180 7.78e-14) and RNA dependent RNA polymerase (pfam00978, E-value = 2.37e-11). 38 39 181 Compared to other members of order Tymovirales, the EnGFV1 replicase showed the 40 41 42 182 highest similarity to Botrytis virus F (BVF), the only known member of the family 43 44 183 Gammaflexiviridae in Tymovirales (Howitt et al., 2001), being the values of identity and 45 46 47 184 similarity 18.8% and 33.6%, respectively. However, when the specific domains in the 48 49 185 replicase (Met, Hel, RdRp) are considered, the homology scores varied greatly (Table 50 51 186 S2). Phylogenetic analysis using the aa sequence of the core RdRp clustered this virus 52 53 54 187 species in the family Gammaflexiviridae (Fig. 3A) Next, the ORF2 extends from 55 56 188 positions 6,074 to 8,086. It encodes for a protein of 670 aa and a molecular mass of 57 58 59 189 17.41 kDa and presents a domain homologous to DEAD-like helicases (smart00487, E- 60 61 62 8 63 64 65 190 value = 3.47e-11). This protein appears to be related to the putative movement proteins 1 2 191 encoded by two recently described mycoviruses provisionally ascribed to family 3 4 5 192 Virgaviridae, Macrophomina phaseolina tobamo-like virus 1 (MpTLV1; Marzano et al., 6 7 193 2016) and Podosphaera prunicola tobamo-like virus 1 (PPrTLV1; Pandei et al., 2018) 8 9 10 194 (Fig. 3B). The ORF3 of EnGFV1 consists of 984 nts, and encodes for a putative coat 11 12 195 protein of 327 aa and a molecular mass of 35.54 kDa. The aa sequence of the ORF3 13 14 196 showed the highest similarities with MpTLV1 and PPrTLV1 (41.0% and 40.5%, 15 16 17 197 respectively) (Table S4) and in the phylogenetic tree resulted grouped as the ORF2 with 18 19 198 these two same viruses (Fig. 3C). 20 21 22 199 Another mycovirus, originally identified in E97-14 and apparently belonging to a new 23 24 25 200 species, was named Entoleuca gammaflexivirus 2 (EnGFV2). The complete genome 26 27 201 consists of 7,321 nt and three possible ORFs were predicted. The ORF1, extending from 28 29 30 202 position 121 to 5,820, encodes for a putative replicase of 1,899 aa and molecular mass 31 32 203 of 209.85 kDa. Three domains were identified in this replicase: methyltransferase 33 34 204 (pfam01660, E-value = 1.12e-44), helicase (pfam01443, E-value =-6.02e-21) and RNA 35 36 37 205 dependent RNA polymerase (pfam00978, E-value = 7.76e-15). Phylogenetic analysis of 38 39 206 the aa sequence of the core RdRp allocated EnGFV2 in the Gammaflexiviridae (Fig. 40 41 42 207 3A; Table S3). On the contrary to BVF, the type species of family Gammaflexiviridae, 43 44 208 the replicase-encoding ORFs of EnGFV1 and EnGFV2 do not present a leaky stop 45 46 47 209 codon between the RdRp core and the helicase domains (Howitt et al., 2001). Next, the 48 49 210 ORF2 extends from position 5,872 to 6,447. It encodes for a protein of 191 aa and a 50 51 211 predicted molecular mass of 21.25 kDa. No domains or significant similarities with 52 53 54 212 known proteins could be detected. Phylogenetic analysis clustered the putative protein 55 56 213 encoded by ORF2 with Tymovirales, in the same branch of the betaflexivirus apple 57 58 59 214 chlorotic leaf spot virus (ACLSV) (Fig. 3B) but showing the highest similarities with 60 61 62 9 63 64 65 215 the alphaflexivirus white clover mosaic virus (WCMV) (15.6% and 37.2%, identity and 1 2 216 similarity, respectively) (Table S4). Finally, ORF3 is comprised between positions 3 4 5 217 6,548 and 7,159 and encodes for a protein of 203 aa with a predicted molecular mass of 6 7 218 22.08 kDa. NCBI’s CDD search allowed the detection of a domain with similarity to the 8 9 10 219 superfamily of closterovirus’ coat proteins (pfam01785, E-value = 4.09e-08). However, 11 12 220 phylogenetic analysis grouped this protein with members of Tymovirales, in the same 13 14 221 clade of the betaflexivirus apple chlorotic leaf spot virus (ACLSV) (Fig. 3C). The 15 16 17 222 highest similarities were observed when compared with the betaflexivirus banana mild 18 19 223 mosaic virus (BaMMV) and the alphaflexivirus potato virus X (PVX) (Table S4). 20 21 22 224 Two novel partitiviruses. The two partitiviruses, named EnPV1 and EnPV2, are shared 23 24 25 225 by the four Entoleuca sp. isolates analyzed by HTS and resulted very common in the 26 27 226 Entoleuca and R. necatrix isolate collection (Table 2). They belong to genus Alpha- and 28 29 30 227 Betapartitivirus, respectively. Deduced aa sequences from the RNA1 segments of both 31 32 228 viruses showed an RT-like superfamily domain (Acc. cl02808) (E-values = 2.62e-8 and 33 34 229 8.41e-6, for EnPV1 and EnPV2, respectively). These two partitiviruses are described in 35 36 37 230 detail in a separate article and shall not be discussed here (Velasco et al., in 38 39 231 preparation). 40 41 42 232 Entoleuca megabirnavirus 1. A sequence related to dsRNA1 segment of 43 44 45 233 megabirnaviruses was identified by HTS in all four Entoleuca sp. isolates and named 46 47 234 Entoleuca megabirnavirus 1 (EnMBV1). RT-PCR using consensus primers (Table S1) 48 49 50 235 showed that this virus was common in the Xylariaceae fungi in our work collection 51 52 236 (Table 2). The complete genome sequence of EnMBV1 consists of 8,927 nt and present 53 54 237 two ORFs, putatively encoding the CP and the RdRp proteins. The sequenced strain 55 56 57 238 from isolate E97-14 showed 87.1% of nucleotide identity compared with Rosellinia 58 59 239 necatrix megabirnavirus 3 (RnMBV3), previously described in R. necatrix isolate 60 61 62 10 63 64 65 240 Rn454 of the same fungal collection (Arjona-López et al., 2018). The coat protein 1 2 241 sequence is 78.8% identical among these two viruses while for the RdRp the nucleotide 3 4 5 242 identity is 81.2%. Coat protein derived amplicons of 529 bp were sequenced for ten 6 7 243 EnMBV1 strains isolated from Entoleuca sp. and R. necatrix and compared with the 8 9 10 244 corresponding sequence of RnMBV3. The nucleotide distances ranged between 77.2% 11 12 245 and 99.8% (Table S5). Interestingly, the partial sequence of the CP region of EnMBV1 13 14 246 Rn114-15R strain from R. necatrix and EnMBV1 E94-14 from Entoleuca sp. are 100% 15 16 17 247 identical (Table S5). Hence, RnMBV3 and EnMBV1 are strains of the same virus 18 19 248 species. No contigs with homology to the dsRNA2 segment of megabirnaviruses could 20 21 22 249 be identified after BLASTX and BLASTN searches. 23 24 25 250 Entoleuca hypovirus 1. A sequence resembling members of family Hypoviridae was 26 27 251 identified as shared by the four Entoleuca sp. isolates and named Entoleuca hypovirus 1 28 29 30 252 (EnHV1). It has been described previously (Velasco et al., 2018) and is no longer 31 32 253 considered here. 33 34 35 254 A sequence related to ourmia-like viruses. A viral sequence with similarity to ourmia- 36 37 255 like viruses was identified in Entoleuca sp. isolates E112-4 and E117-4. This virus was 38 39 40 256 named Entoleuca ourmia-like virus 1 (EnOLV1) and its genome has 3,412 nt, longer 41 42 257 than any other fungal ourmia-like virus described so far (Fig. 4A). In this work, we 43 44 45 258 characterized the EnOLV1 strain from E112-4. A single ORF of 2,556 nts was 46 47 259 identified starting at position 323 from the 5’ end. It encodes for a putative protein of 48 49 50 260 851 aa and a predicted molecular mass of 97.5 kDa that showed no significant domains 51 52 261 in NCBI’s CDD. It probably encodes for the replicase. After the ORF a long 3’UTR of 53 54 262 534 nt was present. The highest similarity of the putative protein of EnOLV1 ORF was 55 56 57 263 observed with the aa sequence of Botrytis ourmia-like virus (BOLV; Donaire et al., 58 59 264 2016a) (23.5% of identity, 40.2% of similarity) followed by Sclerotinia sclerotiorum 60 61 62 11 63 64 65 265 ourmia-like virus 2 (SScOLV2; Marzano et al., 2016a) (21.4%, 34.5%) and Sclerotinia 1 2 266 sclerotiorum ourmia-like virus 3 (SScOLV3; Mu et al., 2018) (20.4%; 33.8%). It is 3 4 5 267 remarkable that the protein encoded by the ORF in EnOLV1 is 12% larger to that from 6 7 268 BOLV (722 aa) but comparable to the one of OuMV (860 aa) a plant virus more distant 8 9 10 269 phylogenetically. Given that in the sequence available for SScOLV2 the 5’ end is 11 12 270 lacking we cannot compare with EnOLV1. On the other hand, the ORF of the 13 14 271 SScOLV3 replicase encodes for a protein of only 645 aa. A highly conserved region 15 16 17 272 among ourmiaviruses was present in the middle part of the protein (Fig. S1). EnOLV1 18 19 273 aa sequence features conserved residues in the RdRp shared by positive ssRNA viruses 20 21 22 274 (Fig. S1). A phylogenetic tree grouped EnOLV1 in the same clade (87% bootstrap 23 24 275 support) of BOLV and SScOLV2 and separated from other ourmiaviruses (Fig. 4B). 25 26 27 276 The 5’ end of EnOLV1 RNA presents neither a secondary structure nor the 5’-CCC 28 29 277 terminal sequence, both typical in other ourmiaviruses, so the genomic sequence 30 31 278 obtained for EnOLV1 may be incomplete. 32 33 34 279 A virus related to negative single-stranded RNA viruses, order Bunyavirales. In 35 36 37 280 Entoleuca sp. isolate E115-5 two viral sequences were identified and attributed to a 38 39 281 novel mycovirus species, namely Entoleuca phenui-like virus 1 (EnPLV1). Its genome 40 41 42 282 consists of two RNA sequences. RNA1 has a size of 7,256 nt and presents one single 43 44 283 ORF of 7,062 nt that starts at position 96 from the 5’end. NCBI’s CDD search identified 45 46 47 284 a domain from nt position 2,066 (aa position 652) to position 4,106 (aa 1,333) with 48 49 285 homology to Bunya-RNA dependent RNA polymerases superfamily (pfam04196, E- 50 51 286 value=2.6e-32). The alignment with other viral sequences showed the six conserved 52 53 54 287 motifs (premotif A and motifs A–E) typical of bunyaviruses (Fig. 5A): motif A 55 56 288 (DATKWC), motif B (QGILHDASC), motif C (VQGSDDSA; including the SDD 57 58 59 289 amino acids), motif D (GIWCSEAKSS), and finally motif E (EYNSEWYMNG) that 60 61 62 12 63 64 65 290 includes the tetrapeptide E(F/Y)xS, which is specific in polymerases of segmented 1 2 291 negative-sense RNA viruses (Kormelink et al., 2011). Besides, we identified three basic 3 4 5 292 residues (K, R, and R/K) in premotif A and a glutamic acid (E) residue downstream of 6 7 293 premotif A (Fig. 5A), which are commonly conserved in RdRps of bunyaviruses 8 9 10 294 (Elbeaino et al., 2009). A phylogenetic analysis of the aa sequences of RdRp allocated 11 12 295 EnPLV1 to the group IV of negative ssRNA viruses, order Bunyavirales within the 13 14 296 family Phenuiviridae (Fig. 5B), that includes members consisting of either two or three 15 16 17 297 genomic segments (L, M, S) (Fig. 6A). 18 19 298 EnPLV1 RNA2 is related to the M and S genomic segments of bunyaviruses and 20 21 22 299 consists of 2,816 nt, presenting two ORFs in opposite directions (Fig. 6A). ORF1, from 23 24 300 nt 120-1,178 encodes a putative protein of 352 aa and a predicted molecular mass of 25 26 27 301 38.6 kDa, with similarity to nucleocapsid proteins of bunyaviruses. A domain typical of 28 29 302 tenuivirus-phlebovirus nucleocapsid proteins (pfam05733, E-value: 1.13e-07) was 30 31 303 predicted between the aa positions 92-240. ORF2 is homologous to MPs. It ends 285 nt 32 33 34 304 downstream ORF1 and consists of 1,221 nt, encoding for a putative protein of 406 aa 35 36 305 and a molecular mass of 45.27 kDa. The MP protein of EnPLV1 (403 aa) is comparable 37 38 39 306 in size to the corresponding of watermelon crinkle leaf-associated virus 2 (WCLaV2). 40 41 307 The highest and lowest similarities were observed in comparison to WCLaV2 and Rift 42 43 44 308 Valley fever virus (RVFV), respectively (Table S6). The intergenic region (IR) in 45 46 309 ambisense RNA segments are highly rich in A- and U- stretches and predicted to fold 47 48 49 310 into a stable hairpin structure (Kiening et al., 2017). In the case of the RNA2 of 50 51 311 EnPLV1, the IR is rich in A-U, and shows strong secondary hairpin structure (Fig. 6B). 52 53 312 Interestingly, the IR of RVFV, which is proximal phylogenetically to EnPLV1, has no 54 55 56 313 A-U predominance; on the contrary, it has a high C-content (56%) and is considerably 57 58 314 shorter (82 nt) than that of EnPLV1. The terminal sequences of the RNA1 and RNA2 of 59 60 61 62 13 63 64 65 315 EnPLV1 at the 5’ and 3’ ends resemble those of phleboviruses and tenuiviruses (Fig. 1 2 316 6C). Like other segmented negative-sense RNA viruses (Pettersson and Bonsdorff, 3 4 5 317 1975), the inverted repeats of their terminal sequences conform a panhandle structure 6 7 318 (Fig. 6C). 8 9 10 319 Other positive ssRNA and dsRNA viruses. A mycoviral sequence was identified in 11 12 the Entoleuca isolates resembling the recently described Rosellinia necatrix 13 320 14 15 321 fusagravirus 1 (RnFSV1; LC333734). RnFSV1 was identified in R. necatrix Rn459 16 17 322 isolated in the same avocado orchards of this study (Arjona-López et al., 2018). The 18 19 20 323 mycoviral sequence identified in Entoleuca sp. presented a genome of 9,327 nt in length 21 22 324 and includes two putative ORFs. ORF1 encodes for a protein of 1,422 aa and does not 23 24 25 325 present similarity with known sequences or predicted domains. ORF2 encodes for a 26 27 326 1,311 aa protein and includes a RT-like domain (cl02808; E-value = 4.04e-11). The 28 29 30 327 genomic sequence of this mycovirus showed high nucleotide identity (89.0%) with 31 32 328 RnFSV1. Considering that this identity is 92% along the coding regions (not shown), 33 34 329 this mycovirus can be considered as a strain of RnFSV1, ascribed to the newly proposed 35 36 37 330 family “Fusagraviridae” (Wang et al., 2016). 38 39 40 331 Another mycovirus sequence could be identified in Entoleuca consisting of 5,860 nt in 41 42 332 length. The only predicted ORF presented a RT-like domain (cl02808; E-value = 1.00e- 43 44 45 333 19). This ORF showed similarities with the so-called Yado-kari viruses (YkV1, -2, -3 46 47 334 and -4), recently identified in R. necatrix isolates from the same pathosystem (Arjona- 48 49 50 335 López et al., 2018). The predicted aa sequence of this ORF showed 84.8% and 39.8% of 51 52 336 identity compared to YkV2 and YkV4, respectively. In the case of the 5'UTR the 53 54 337 nucleotide identity between this mycovirus and YkV2 was 85.6% and in the case of the 55 56 57 338 3'UTR it was 91.3%. Thus, the new mycovirus detected originally in Entoleuca 58 59 339 probably represents a different strain of YkV2. The published sequences for the three 60 61 62 14 63 64 65 340 Yado-kari viruses showed a C/U indetermination at the 5’ terminal nucleotide of the 1 2 341 RNA sequences that could not be defined either in the genomic sequence of this 3 4 5 342 mycovirus after specific sequencing. 6 7 8 343 Phaseolus vulgaris alphaendornavirus 1, strain E97-14. A sequence showing 98.1% 9 10 344 of nucleotide identity (98.6% identity for the deduced aa sequence of the polyprotein) to 11 12 Phaseolus vulgaris alphaendornavirus 1 (PvEV1; AB719397; Okada et al., 2013) was 13 345 14 15 346 identified in the four Entoleuca sp. isolates (Table 1). Therefore, this endornavirus 16 17 347 represents a new strain of PvEV1. The nucleotide sequence of PvEV1 E97-14 was 18 19 20 348 identical in the four Entoleuca sp. isolates. RT-PCR with specific primers (Table S1) 21 22 349 allowed the detection of this virus from dsRNA extracts of Entoleuca sp. isolates but 23 24 25 350 not in R. necatrix. Moreover, PCR with the same primer set using extracts of Entoleuca 26 27 351 sp. E97-14 total DNA showed that the virus was not integrated into the fungal genome 28 29 30 352 (not shown). Up to our knowledge, this endornavirus species has not been detected 31 32 353 previously in any other fungal host. 33 34 35 354 Multiple virus infections in Entoleuca sp. isolates. An in-depth analysis of the contigs 36 37 355 and scaffolds obtained from HTS revealed that only single virus strains of the families 38 39 40 356 Hypoviridae, Megabirna-, Partiti-, “Ourmia-” and order Bunyavirales were present in 41 42 357 the Entoleuca sp. isolates (not shown). For each sample, the total number of Illumina 43 44 45 358 reads matching to the mycoviral genomes detected in this work is shown in Table 1. 46 47 359 Differential virome composition among the four isolates arises from these results (Table 48 49 50 360 1). There are nine virus species shared by the four Entoleuca sp. isolates and two other 51 52 361 viruses that appear differentially. Specifically, the ourmia-like virus is present in 53 54 362 isolates E112-4 and E117-4. In contrast, the bunyavirus-like virus is present only in 55 56 57 363 Entoleuca E115-15, whiles both viruses are absent in E97-14. Also, there are huge 58 59 364 differences in the number of reads, ranging from 83.1% (5.95 x 106 reads) in the case of 60 61 62 15 63 64 65 365 the RnFSV1 from isolate 97-14 to below 0.01% (16 reads) for RnFSV1 in isolate E117- 1 2 366 4, regarding the total of mycovirus-related reads. RT-PCR confirmed the presence of 3 4 5 367 viruses or viral genomic segments displaying a low number of reads in some fungal 6 7 368 isolates (e.g. EnGFV1, EnGFV2, EnMBV1, EnPV1 or EnPV2) from dsRNA and total 8 9 10 369 RNA extracts (not shown). The presence of circular DNA viruses was discarded after 11 12 370 the RCA analysis of DNA extractions from the fungal isolates tested negative (not 13 14 371 shown). 15 16 17 372 Screening mycoviruses in the Entoleuca sp. and R. necatrix collection. The 18 19 20 373 availability of partial sequences for the viruses described in this work allowed the 21 22 374 design of specific primers for RT-PCR detection. For that, consensus primers were 23 24 25 375 obtained based on the genomic sequences obtained for each virus in the four Entoleuca 26 27 376 sp. isolates (Table S1). A collection of Entoleuca sp. and R. necatrix isolates recovered 28 29 30 377 from avocado trees in orchards in Spain was investigated for the presence of the 31 32 378 mycoviruses described in this work (Table 2). The result of this survey showed that the 33 34 379 mycoviruses are well spread in these fungi, showing recurrent multiple virus infections. 35 36 37 380 Moreover, several viruses are present simultaneously in Entoleuca sp. and R. necatrix, 38 39 381 being the most frequent EnHV1, and EnMBV1, RnFSV1, YkV2 and the two 40 41 42 382 partitiviruses. 43 44 45 383 Discussion 46 47 48 49 384 Novel mycoviruses detected in Xylariaceae species isolated from avocado 50 51 52 385 In the four Entoleuca sp. isolates that are the main object of this work we could identify 53 54 386 14 sequence segments apparently belonging to eleven different mycovirus species. The 55 56 387 number of reads obtained after HT sequencing matching to the reconstructed genomes 57 58 59 388 varied greatly depending not only on the specific virus species but also on the fungal 60 61 62 16 63 64 65 389 isolate from which they were isolated. These differences must be related to virome 1 2 390 composition, the replication strategy for each virus species as well as the characteristics 3 4 5 391 of the fungal isolate. Given the variability observed in the virus genomic sequences, the 6 7 392 RT-PCR primers designed in this work could be not suitable to detect all the variants, as 8 9 10 393 some unspecific amplification bands have been observed in some cases (results not 11 12 394 shown). Strains of four of the pool of virus species have been already identified in R. 13 14 395 necatrix (Arjona-López et al., 2018), including EnHV1, RnFSV1 and YkV2. Another 15 16 17 396 megabirnavirus identified, EnMBV1, appears to be the same species as RnMBV3 18 19 397 according to genome structure comparison and genetic analysis. The two flexiviruses 20 21 22 398 reported here showed striking genome structure and features. EnGFV1 replicase 23 24 399 corresponds to gammaflexiviruses but the MP and the CP shares homologies with 25 26 27 400 virgaviruses. The polyphyletic origin of the CPs and MPs of alpha-like viruses, such as 28 29 401 the two novel gammaflexiviruses described here, complicates the elaboration of a 30 31 402 coherent evolutionary history for this group of viruses (Dolja and Koonin, 2018). 32 33 34 403 Recently, a novel virus with similarity to gammaflexiviruses (FbLFV1) has been 35 36 404 reported presenting a single ORF encoding for the replicase (Mizutani et al., 2018). 37 38 39 405 Clearly, more genomic sequences from different phyla are necessary to elucidate the 40 41 406 evolution of this important group of viruses. The ourmia-like virus identified in this 42 43 44 407 study (EnOLV1) was phylogenetically grouped in a clade with other two ourmia-like 45 46 408 viruses whose hosts are fungi, separated from mitoviruses and fungal narnaviruses and 47 48 49 409 allocated proximal to other plant and fungal ourmia-like viruses that have a genome 50 51 410 structure consisting of a single ORF. Another viral species, EnPLV1, has been ascribed 52 53 411 to negative ssRNA viruses order Bunyavirales, according to genetic and phylogenetic 54 55 56 412 analysis. The structure of its genome is bipartite, like that of CCGaV, recently described 57 58 413 in orange trees (Navarro et al., 2018), although the genetic sequence is more similar to 59 60 61 62 17 63 64 65 414 phlebo-like viruses with tripartite genomes, reflecting in-parallel genome arrangements 1 2 415 in plant and fungal phlebo-like viruses. Ambisense RNA segments are relatively scarce 3 4 5 416 in viruses and up to now found only in members of the family Arenaviridae, in order 6 7 417 Bunyavirales (genus Tenuivirus and Phlebovirus) and tospoviruses (Nguyen & Haenni, 8 9 10 418 2003). The detection of an endornavirus species in the fungal isolates that it is 98% 11 12 419 homologous to the endornavirus PvEV1 originally identified in common bean (Okada et 13 14 420 al., 2013), suggests that in some cases the plant host may act as a bridge between 15 16 17 421 incompatible fungal species or isolates for inter-specific virus transmission. In their 18 19 422 interaction, plant cells and colonizing fungi exchange macromolecules that may include 20 21 22 423 virion particles or large dsRNA molecules (Roossinck, 2019). Whereas endornaviruses 23 24 424 are found in plants, fungi or oomycetes, up to our knowledge, there are no reports of a 25 26 27 425 same endornavirus species present across kingdoms, even though it has been suggested 28 29 426 that endornaviruses have originated in fungi (Fermin et al., 2018). Phylogenetic 30 31 427 analyses of endornaviruses point out to extensive horizontal transfer between plants and 32 33 34 428 fungi (Roossinck, 2019). An endornavirus-like dsRNA (Persea americana endornavirus 35 36 429 1, PaEV1) different from the one described here, has been isolated from leaves of an 37 38 39 430 avocado tree belonging to a germplasm collection in Spain (Estación Experimental “La 40 41 431 Mayora”, Algarrobo-Costa, Málaga, Spain) (Villanueva et al., 2012). However, 42 43 44 432 preliminary RT-PCR of Spanish avocado trees using PvEV1-derived primers tested 45 46 433 negative (results not shown). 47 48 49 50 434 Virome composition and host phenotype 51 52 435 Mycoviruses infecting R. necatrix have been extensively investigated (Kondo et al., 53 54 436 2013; Zhang et al., 2014; Yaegashi and Kanematsu, 2015; Chiba et al., 2016) especially 55 56 57 437 in Japanese fruit tree pathosystems. The presence of simple and multiple mycovirus 58 59 438 infections has been related with hypovirulence phenomena in R. necatrix (Chiba et al., 60 61 62 18 63 64 65 439 2009; Sasaki et al., 2015). Virus effects on their hosts can include limitation of hyphae 1 2 440 development and/or a decrease of the fungus virulence, but in most cases, there is a 3 4 5 441 negligible impact in the fungus-plant host interaction (Kondo et al., 2013). The 6 7 442 biological characterization of all the viruses identified in this work is far for being 8 9 10 443 completed. Curing these viruses in the fungal isolates has been unsuccessful after 11 12 444 hyphal tip cultivation combined with antiviral compounds (Velasco et al., 2018). From 13 14 445 the complete virome analysis of the four Entoleuca isolates studied in the present work, 15 16 17 446 it became evident that three different sets of viruses (viromes) appeared. On the other 18 19 447 hand, variation in the biological characteristics of the four Entoleuca sp. isolates 20 21 22 448 resulted high, however, we cannot definitively attribute those differences to virome 23 24 449 composition or intrinsic biological characteristics of each fungal isolate (or a 25 26 27 450 combination of both). 28 29 30 451 Horizontal virus transmission in Xylariaceae fungi from avocado 31 32 33 452 We found vegetative incompatibility among the four Entoleuca sp. isolates object of 34 35 453 this work. In R. necatrix, the isolates collected from avocado roots in Spain present 36 37 454 different VCGs (Pérez-Jiménez et al., 2002). Despite that, a same set of mycoviruses is 38 39 40 455 shared by isolates of Entoleuca sp. and R. necatrix that are colonizing the same avocado 41 42 456 trees, evidencing the probable horizontal virus transmission intra- and interspecies. 43 44 45 457 Moreover, a population analysis in one of the mycovirus species (EnHV1) revealed 46 47 458 polymorphism among strains not linked to host origin (Velasco et al., 2018), that has 48 49 50 459 also been observed in the other virus species investigated in this work (EnMBV1). 51 52 460 Transmission of mycoviruses intra- and interspecies of the same genera of fungi in 53 54 461 natural environments has been reported previously (Liu et al., 2003; Ikeda et al., 2004; 55 56 57 462 Vainio et al., 2011; Liu et al; 2012). Artificial transmission of the mycoviruses between 58 59 463 different fungal species has been achieved (Kanematsu et al., 2010). Moreover, it has 60 61 62 19 63 64 65 464 been experimentally demonstrated the transmission of a virus between vegetatively 1 2 465 incompatible R. necatrix isolates using zinc ions in the growth medium (Ikeda et al., 3 4 5 466 2014). Interestingly, Yaegashi and col. (2013) showed a virus infection of a traceable R. 6 7 467 necatrix isolate from an unknown vector source after the inoculation during a three-year 8 9 10 468 period in the soil of an apple orchard. Thus, it is not surprising that we could detect the 11 12 469 partitiviruses EnPV1 and EnPV2 in a Fusarium sp. isolate collected from the same 13 14 470 avocado orchards (results not shown). Avocado, the perennial plant that harbors the two 15 16 17 471 species of fungi studied here, conforms a particular ecological niche in the rhizosphere 18 19 472 that might include mycophagous species (insects, Trichoderma, etc.) as possible vectors 20 21 22 473 for mycoviruses. We foresee that these same viruses or some others will be detected in 23 24 474 future surveys in other fungal species or oomycetes collected in this pathosystem. 25 26 27 475 Finally, this work provides supporting evidence for HVT among two sympatric fungal 28 29 476 species in natural conditions involving several mycovirus species belonging to different 30 31 477 taxa. 32 33 34 35 478 Although the R. necatrix isolates identified so far in Spain are virulent to avocado, the 36 37 479 eventual finding of a virus species capable of conferring hypovirulence and the probable 38 39 40 480 horizontal transmission of the mycoviruses observed in this work suggests that 41 42 481 virocontrol of R. necatrix in avocado is plausible. Finally, high-throughput sequencing 43 44 45 482 technologies that enable the determination of complete viral infection status in fungal 46 47 483 hosts, help to clarify the role of viruses in the mycovirus-fungal-plant interaction, to 48 49 50 484 explore virocontrol strategies for fungal diseases, and to better understand virus 51 52 485 evolution. 53 54 55 56 486 Accessions Numbers 57 58 59 60 61 62 20 63 64 65 487 The sequences described in this work were deposited in GenBank under the following 1 2 488 accession numbers: EnPLV1 (MF375882; MK140653), EnGFV1 (MF375883), 3 4 5 489 EnGFV2 (MF375884), EnHV1 (MF375885), EnMBV1 (MF375886), RnFSV1 6 7 490 (MF375887), YkV2 (MF375888), EnOLV1 (MF375889), EnPV1 (MF375890), EnPV2 8 9 10 491 (MF375891) and PvEV1 (MF375892). 11 12 13 492 Acknowledgments 14 15 16 17 493 This research was funded by the Spanish Ministry of Science and Innovation project 18 19 494 AGL2014-52518-C2-2-R and the IFAPA projects AVA201301.13 and AVA201601.14, 20 21 22 495 co-financed by FEDER. 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MM: -HindIII DNA marker, Rn95-16 corresponds to R. necatrix, while 16 17 645 the rest of the dsRNAs belong to Entoleuca sp. isolates. Samples selected for HTS 18 19 646 analysis are indicated in bold. 20 21 647 Fig. 2. Comparison of the genome organization and size in selected members of order 22 23 648 Tymovirales with Entoleuca gammaflexivirus 1 (EnGFV1) and Entoleuca 24 25 649 gammaflexivirus 2 (EnGFV2). CYVCV: citrus yellow vein clearing virus (KP313240), 26 27 650 PVX: potato virus X (D00344), WCMV: white clover mosaic virus (X06728), 28 651 BanMMV: banana mild mosaic virus (AF314662), GVA: grapevine virus A (X75433), 29 30 652 BVF: Botrytis virus F (AF238884), FbLFV1: Fusarium boothii large flexivirus 1 31 32 653 (LC425115), MpTLV1: Macrophomina phaseolina tobamo-like virus 1 (KF537660). 33 34 654 Color interpretation of protein homologies: blank: RdRp, orange: movement protein; 35 36 655 green: coat protein, grey: unknown or other functions. 37 38 656 Fig. 3. ML phylogenetic tree of the aa sequences of the RdRp domain in the replicase 39 40 657 (A), movement protein (B) and coat protein (C) of representative members of order 41 42 658 Tymovirales, family Virgaviridae and other clades as generated with IQ-TREE. The 43 44 659 best-fit model of substitution according to BIC for the replicases was the VT+F+R5. 45 660 EnGFV1 and EnGFV2 resulted allocated in the same clade as BFV, the only known 46 47 661 member of the family Gammaflexiviridae that consists of a single ORF. The best-fit 48 49 662 model of substitution for MPs, according to BIC scores was the Blosum62+F+I+G4. In 50 51 663 this case, EnGFV1 MP protein resulted grouped with other tobamo-like mycoviruses 52 53 664 ascribed to the Virgaviridae and close to MPs of tobamoviruses. In the case of 54 55 665 EnGFV2, the CP clustered with members of order Tymovirales. For both the MP and 56 666 the CPs, we introduced in the analysis members of family Closteroviridae as reference. 57 58 667 In this last case, the best-fit model of substitution was the VT+G4. EnGFV1 CP 59 60 668 grouped with mycoviruses ascribed to the Virgaviridae, while EnGFV2 CP resulted 61 62 27 63 64 65 669 allocated with Tymovirales. Bootstrap support values (SH-aLRT) written on the 1 2 670 branches. 3 4 671 Fig. 4. A. Comparisons of the genome sizes and organization for Botrytis ourmia-like 5 6 672 virus (BOLV), Entoleuca ourmia-like virus 1 (EnOLV1), Ourmia melon virus (OuMV), 7 8 673 rhizoctonia solani ourmia-like virus 1 RNA 1 (RsOLV1), Sclerotinia sclerotiorum 9 674 ourmia-like virus 1 (SScOLV1), Sclerotinia sclerotiorum ourmia-like virus 2 10 11 675 (SScOLV2), Sclerotinia sclerotiorum ourmia-like virus 3 (SScOLV3), and soybean leaf- 12 13 676 associated ourmiavirus 1 (SLOV1). B. ML phylogenetic tree of the amino acid 14 15 677 sequences of RNA-dependent RNA polymerase (RdRp) for selected members of family 16 17 678 Narnaviridae (genera Narnavirus and Mitovirus), and genus Ourmiavirus as generated 18 19 679 with IQ-TREE. The best-fit model of substitution according to BIC was the 20 680 Blosum62+F+R4. EnOLV1 was grouped with other ourmiaviruses. Tomato bushy stunt 21 22 681 virus, a Tombusvirus was used as out-group. Bootstrap support values written on the 23 24 682 branches. 25 26 683 Fig. 5. A. Amino acid alignment between RdRp domains showing the premotif A and 27 28 684 motifs A–E of EnPLV1 and selected negative single stranded RNA viruses. EMARaV, 29 30 685 European mountain ash ringspot-associated virus (AY563040); RRV, rose rosette virus 31 32 686 (HQ871942); RLBV, raspberry leaf blotch virus (FR823299); WSMoV, watermelon 33 34 687 silver mottle virus (AF133128); RSV, rice stripe virus (D31879); RVFV, Rift Valley 35 36 688 fever virus (DQ375403); SFTSV, severe fever with thrombocytopenia syndrome 37 689 (SFTS) virus (HM745930); RGSV, rice grassy stunt virus (AB009656), GOLV, 38 39 690 Gouleako virus (HQ541738), SFTSV, severe fever with thrombocytopenia syndrome 40 41 691 virus (HM745930), WCLaV-1 and -2, watermelon crinkle leaf-associated viruses 1 and 42 43 692 2 (KY781184, KY781187). B. ML phylogenetic tree of the amino acid sequences of 44 45 693 RNA-dependent RNA polymerase (RdRp) for a selection of bunyaviruses. The best-fit 46 47 694 model of substitution according to BIC was the rtREV+F+R6. Botrytis virus F (BVF) 48 695 was predicted as out-group by IQ-TREE. Bootstrap support values written on the 49 50 696 branches. 51 52 53 697 Fig. 6. A. Comparisons of the genome organization and sizes of Entoleuca bunyavirus 1 54 698 (EnPLV1), citrus concave gum-associated virus (CCGaV), apple rubbery wood virus 2 55 56 699 (ARVV2) and watermelon crinkle leaf-associated virus 2 (WCLaV1). Blank boxes 57 58 700 represent the RdRp ORF, green boxes refer to the MP ORF and orange boxes represent 59 60 701 the CP ORF. The double arrows in the ORF boxes represent the sense of the RNA 61 62 28 63 64 65 702 transcription. B. Secondary structure and analysis of the intergenic regions of the RNA2 1 2 703 of (a) EnPLV1 and (b) Citrus concave gum-associated virus (CCGaV) (Navarro et al., 3 704 2018), a phlebo-like virus isolated from orange trees. C. Structural characteristics of 4 5 705 EnPLV1 RNA1 terminal sequences. (a) Panhandle structure formed by the 3' and 5' 6 7 706 termini. (b) Comparison of the terminal sequences of the L segments for representative 8 9 707 bunyaviruses (see text for virus acronyms). Matching base pairing is indicated in bold. 10 11 708 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 29 63 64 65 1 2 3 4 5 6 7 TABLE 1. 8 9 Entoleuca sp. isolate 10 11 Virus Species acronym* Genomic segment 97-14 112-4 115-15 117-4 12 Gammaflexiviridae EnGFV1 RdRp 185,784 40ǂ 3,915 16ǂ 13 EnGFV2 RdRp 74,350 23ǂ 1,751 16ǂ 14 Endornaviridae PvEV1 RdRp 13,219 3,717 14,075 3,048 15 16 Partitiviridae EnPV1 RdRp 3,351 3,873 764 1,267 17 EnPV2 RdRp 215ǂ 268ǂ 52 ǂ 71ǂ 18 Hypoviridae EnHV1 RdRp 201,521 272,696 241,467 988,555 19 Megabirnaviridae EnMBV1 RdRp 145,724 216ǂ 4,255 700,821 20 21 “Fusagraviridae” RnFSV1 RdRp 12,304,829 10,533 12,197,523 5,613 22 Bunyavirales EnPLV1 RdRp 0 0 5,589 0 23 EnPLV1 CP/MP 0 0 12,767 0 24 25 “Ourmiaviridae” EnOLV1 RdRp 0 2,224 0 1,441 26 “Yadokariviridae” YkV2 RdRp 136,635 156,788 151,046 3,024,844 27 Mycoviral reads 13,593,056 4,009,386 14,771,674 4,727,082 28 Total reads 25,405,202 28,056,398 26,562,997 28,337,624 29 30 % Mycoviral dsRNA reads 53.51% 14.29% 55.61% 16.68% 31 ǂ Confirmed by RT-PCR: most of the reads matched to core RdRp 32 *Acronym definition in the text 33 34 35 36 37 38 39 40 41 42 43 44 45 46 30 47 48 49 1 2 3 4 5 6 7 8 9 TABLE 2. 10 11 12 Host Isolate EnPLV1 EnGFV1 EnGFV2 EnHV1 EnMBV1 EnPLV1 EnPV2 RnFSV1 YkV2 EnOLV1 13 Entoleuca sp. 97-09 + + + + + + 14 Entoleuca sp. 97-14 + + + + + + + + 15 16 Entoleuca sp. 102-4 + + + + + + 17 Entoleuca sp. 104-10 + + + + + + 18 Entoleuca sp. 106-14 + + + + + + 19 20 Entoleuca sp. 106-4 nt nt + + nt + 21 Entoleuca sp. 107-12 + + + + + + + + 22 Entoleuca sp. 107-13 + + + + + + 23 24 Entoleuca sp. 108-8 + + + + + + 25 Entoleuca sp. 110-1 + + + + + + 26 Entoleuca sp. 110-15R + + + + 27 28 Entoleuca sp. 112-4 + + + + + + + + + 29 Entoleuca sp. 112-8 + + + + + + + 30 Entoleuca sp. 114-15CH + + + + + + 31 32 Entoleuca sp. 115-14 + + + 33 Entoleuca sp. 115-15 + + + + + + + + + 34 Entoleuca sp. 117-4 + + + + + + + + + 35 36 Entoleuca sp. 117-7 + + + + 37 Entoleuca sp. 118-7 + + + 38 R. necatrix 95-12 + + + + + 39 40 R. necatrix 95-16 + + + + + 41 R. necatrix 97-11 nt nt + + nt + 42 R. necatrix 103-7 + + + + + + 43 44 45 46 31 47 48 49 1 2 3 4 5 6 7 R. necatrix 106-11 + + + + 8 R. necatrix 108-10 + + + 9 10 R. necatrix 108-15 + + ? + + + 11 R. necatrix 110-15N + + + + + 12 R. necatrix 114-15R + + + + + 13 14 R. necatrix 114-16 + + + + + 15 R. necatrix 114-4 + + + + 16 R. necatrix 114-5 + + + + + + 17 18 R. necatrix 118-8 + + + + 19 nt: not tested 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 32 47 48 49 FIG. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 FIG. 2 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 33 63 64 65 FIG. 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 34 63 64 65 FIG. 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 35 63 64 65 FIG. 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 36 63 64 65 FIG. 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 37 63 64 65 Supporting information 1 2 Table S1. Primers used for the detection of the different mycoviruses identified in 3 4 Entoleuca sp. and R. necatrix isolated from avocado. 5 6 7 Tables S2 and S3. Percentages of identity and similarity of the three domains in the 8 9 replicase protein of EnGFV1 and EnGFV2 compared with representative tymoviruses. 10 11 Table S4. Percentages of identity and similarity in the protein sequences of ORF2 (MP) 12 13 and ORF3 (CP) of EnGFV1 and EnGFV2 compared with representative members of 14 15 positive single-stranded RNA viruses. For taxonomic adscription see Fig. 3. 16 17 Table S5. Genetic distance matrix (%) for the nucleotide sequences of a 529 nt length 18 19 fragment within the CP gene of Entoleuca megabirnavirus 1 (EnMBV1) and Rosellinia 20 21 necatrix megabirnavirus 3 (RnMBV3) strains isolated from Entoleuca sp. and R. 22 necatrix. 23 24 25 Table S6 Comparison of the amino acid sequences for the three putative proteins 26 27 encoded in the genome of Entoleuca phenui-like virus 1 (EnPLV1) and apple rubbery 28 wood virus 1 and 2 (ARVV1, -2), citrus concave gum-associated virus (CCGaV), laurel 29 30 lake virus (LLV), watermelon crinkle leaf-associated virus 1 and 2 (WCLaV1, -2) and 31 32 Rift Valley fever virus (RVFV). 33 34 Table S7. Hyphae diameter of Entoleuca sp. isolates. Standard deviation is shown on 35 36 hyphae diameter column. In each column, numbers followed by the same letter are not 37 38 significantly different according to the LSD test. The hyphae diameters were 39 40 significantly different (F=5.76; df=3.53; P<0.05), owing isolate E117-4 the thickest 41 42 hyphae. Hyphae diameter of isolate E97-14 is homogeneous, while the biggest 43 44 differences are observed on E112-4, E115-15 and E117-4 isolates. 45 46 Table S8. Radial growth (ABCCAS) of Entoleuca sp. isolates on PDA and PDA-TA. 47 48 For each isolate and in each column, numbers followed by the same letter are not 49 50 significantly different according to the LSD test (P < 0.05). Higher significant 51 differences in radial growth (F=29.81; df=1.52; P<0.05) of the four Entoleuca sp. 52 53 isolates were observed in PDA versus PDA-TA. Besides, significant differences in fresh 54 55 weight from the four Entoleuca isolates in PDA and PDA-TA were detected (F=7.77; 56 57 df=1,46; P<0.05). 58 59 60 61 62 38 63 64 65 Fig. S1. Alignment of the conserved region in the replicase protein of representative 1 2 ourmia-like viruses. Invariable residues in the protein aa sequences of RdRps of positive 3 single-stranded RNA viruses are indicated. Botrytis ourmia-like virus (BOLV), cassava 4 5 virus C (CaVC), Epirus cherry virus (ECheV), Entoleuca ourmia-like virus 1 6 7 (EnOLV1), Magnaporthe oryzae ourmia-like virus (MgOLV), Ourmia melon virus 8 9 (OuMV), Rhizoctonia solani ourmia-like virus 1 (RsOLV1), Sclerotinia sclerotiorum 10 11 ourmia-like virus 1 (SScOLV1), Sclerotinia sclerotiorum ourmia-like virus 2 12 13 (SScOLV2) and soybean leaf-associated ourmiavirus 1 (SLaOV1). 14 15 Fig. S2. Microscopical image of Entoleuca sp. isolates. 400x. Oblate swellings of 16 17 hyphae before the septa are frequent on E97-14 and E115-15 but are not present on 18 19 E112-4 and E117-4. Young hyphae are thinner than old ones and when young hyphal 20 contact with an older one the first growth parallel to the second one (isolate E115-15). 21 22 There is a narrowing of the diameter of the hyphae before the septum when lateral 23 24 hyphae arise or bifurcation appears. 25 26 Fig. S3. Colony morphology of the four Entoleuca sp. isolates grown on PDA or PDA- 27 28 TA. 29 30 31 Fig. S4. Radial growth of Entoleuca sp. isolates on PDA or PDA-TA. 32 33 Fig. S5. Fresh weight of Entoleuca sp. isolates grown on PDA and PDA-TA. In each 34 35 column, numbers followed by the same letter are not significantly different according to 36 37 the LSD test. 38 39 Fig. S6. Study of formation of incompatibility barriers between Entoleuca sp. isolates. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 39 63 64 65 FIG. S1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 FIG. S2 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 40 63 64 65

1 2 3 4 FIG. S3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 FIG. S4 31 32 33 34 35 E97‐14 E112‐4 36 PDA PDA‐TA PDA PDA‐TA

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18 e r 19 F b 20 500 21 22 0 23 E97‐14 E112‐4 E115‐15 E117‐4 24 Entoleuca sp. isolates 25 26 27 28 29 30 31 32 33 FIG. S6 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 42 63 64 65 TABLE S1 1 Amplicon size Virus Primer Sequence 5'-3' Acc. No. 2 (bp) 3 EnHV1 HYP10879F GCGTACCAGGAACCAGAGTATAG 684 MF375885 4 5 HYP11562R AGGGGCGAGTATTATAACACGTC 6 EnPV1 CP269F TCAATGGATATCGCCGCTCC 335 MF375890 7 CP603R AGATTCGTGCGCTTCAGTGA 8 9 EnPV2 CP41F AACAAGCCTCAAAACTTTGCCA 353 MF375891 10 CP394R GAGCAGGAAGAAACCTTGGAGA 11 EnMBV1 MEGA-F1 AGTGTACAATATGAACATGCGCAA 395 MF375886 12 MEGA-R2 TCCGAGCTCACAAAACCCCA 13 14 EnGFV1 EnGFV1-RDP-2450F CCGTGAACCGAAACCCTAAGA 639 MF375883 15 EnGFV1-RDP-2958R GGTCGATCTAGGGCTCCAATG 16 EnGFV2 EnGFV2-RDP-2262F CGCCATCTCCGAGGTGCCCT 509 MF375884 17 EnGFV2-RDP- TTCTTAGCCTGTCGGAAAGC 18 2900BiR 19 EnPLV1 EnPLV1-RDP-2918F AAGTAGCACTGTGGGTCATGG 530 MF375882 20 21 EnPLV1-RDP-3447R ACCTCGCGGTTCTAAGAAAGG 22 RnFSV1 MyV1 -RDP-1208F GTGGTTCAACAGACGCATGTT 485 MF375887 23 MyV1 - RDP-1672R CGTAGTACAGTAACTGCATTT 24 YkV2 MyV2-RDP-2573F GCGTTTCTCGAGAGGGCCCCG 486 MF375888 25 26 MyV2-RDP-3038R CGGGCGAATTACCTCGTCCGA 27 EnOMV1 EnOLV1-RDP-2279R TCAAACCCAACACACTCCTTCA 609 MF375889 28 EnOLV1-RDP-1671F CAAAAGGAATTGGGATGGCGTT 29 PvEV1 Ev12806F GTACGATAACACCAGCACCGA 648 MF375892 30 31 Ev13411R CCGTTAATCGCATGGCTTGG 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 43 63 64 65 TABLES S2-S3 1 2 3 TABLE S4 4 5 MP

6 EnGFV1 EnGFV2

7 Virus species %Identity %Similarity %Identity %Similarity 8 Apple chlorotic leaf spot virus 6.0 26.9 12.2 37.8 9 Banana mild mosaic virus 9.1 31.6 11.8 36.3 10 Potato virus X 7.3 31.5 12.9 34.8 11 White clover mosaic virus 7.3 24.8 15.6 37.2 12 Clover yellow mosaic virus 8.4 35.3 11.0 37.4 13 Red clover vein mosaic virus 9.1 34.3 7.7 30.2 14 Entoleuca gammaflexivirus 2 8.3 34.8

15 Bean yellow disorder virus 11.0 37.0 12.0 35.3 16 Lettuce chlorosis virus 10.7 38.6 11.6 38.0 17 Tomato chlorosis virus 10.8 39.5 10.1 35.0 18 Tomato infectious chlorosis virus 12.2 38.9 10.7 37.0 19 Blackberry vein banding-associated virus 9.8 31.1 8.7 30.9 20 Grapevine leafroll-associated virus 3 8.2 30.0 9.8 28.8 21 Grapevine leafroll-associated virus 4 12.2 39.4 11.3 34.2 22 Pineapple mealybug wilt-associated virus 1 10.3 37.2 11.6 36.3 23 Citrus tristeza virus 9.2 33.2 10.6 34.3 24 Grapevine leafroll-associated virus 2 7.0 28.1 13.6 36.7 25 Raspberry leaf mottle virus 8.7 29.5 11.3 35.7 26 Cucumber green mottle mosaic virus 7.1 22.8 5.7 31.6 27 Zucchini green mottle mosaic virus 6.0 25.4 9.1 32.5 28 Pepper mild mottle virus 6.4 25.8 8.8 34.6 29 Tobacco mild green mosaic virus 8.3 25.6 10.6 32.4 30 Tobacco mosaic virus 5.3 24.4 10.1 33.3 31 Entoleuca gammaflexivirus 1 8.3 34.8

32 Macrophomina phaseolina tobamo-like virus 1 15.0 41.0 8.6 30.0 33 Podosphaera prunicola tobamo-like virus 1 14.7 40.5 8.5 29.6 34 Bee Macula-like virus 8.4 29.3 6.9 27.9 35 36 37 CP

38 EnGFV1 EnGFV2

39 Virus species %Identity %Similarity %Identity %Similarity 40 Apple chlorotic leaf spot virus 6.4 21.6 5.2 15.9 41 Cucumber green mottle mosaic virus 4.1 19.3 2.7 15.2 42 Zucchini green mottle mosaic virus 3.8 18.1 4.3 14.3 43 Pepper mild mottle virus 2.6 16.4 3.8 15.7 44 Tobacco mosaic virus 3.5 18.7 4.0 17.2 45 Tobacco mild green mosaic virus 3.3 17.8 3.4 16.0 46 Soil-borne wheat mosaic virus 3.6 16.9 5.4 15.0 47 Banana mild mosaic virus 5.7 19.4 5.9 20.4 48 Cherry rusty mottle associated virus 6.2 20.6 4.5 19.9 49 Potato virus X 5.6 19.9 5.7 21.5 50 Red clover vein mosaic virus 6.2 19.1 7.3 17.8 51 Strawberry mild yellow edge virus 7.0 18.7 6.6 18.8 52 White clover mosaic virus 6.4 19.5 7.3 17.9 53 Cymbidium mosaic virus 5.7 17.5 5.9 17.7 54 Pepino mosaic virus 5.8 19.4 6.9 19.3 55 Barley stripe mosaic virus 4.8 15.8 2.9 11.6 56 Lychnis ringspot virus 6.9 20.8 4.2 14.6 57 Peanut clump virus 5.7 17.0 5.3 15.3 58 Bean yellow disorder virus 6.4 21.6 3.9 12.4 59 Lettuce chlorosis virus 6.8 21.9 3.5 11.9 60 Tomato chlorosis virus 6.6 22.3 3.7 12.8 61 62 44 63 64 65 Tomato infectious chlorosis virus 6.7 22.1 3.7 12.1 1 Citrus tristeza virus 6.8 25.1 4.4 13.1 2 Raspberry leaf mottle virus 6.0 23.2 4.2 14.1 3 Grapevine leafroll-associated virus 2 6.6 22.2 3.8 12.9 4 Grapevine leafroll-associated virus 3 4.5 20.1 4.5 12.2 5 Blackberry vein banding-associated virus 5.6 20.4 4.6 12.6 6 Grapevine leafroll-associated virus 4 5.3 18.8 4.8 14.4 Entoleuca gammaflexivirus 1 3.8 12.1 7 8 Macrophomina phaseolina tobamo-like virus 1 17.0 31.6 5.0 12.2 9 Podosphaera prunicola tobamo-like virus 1 15.2 32.5 2.8 7.5 10 Entoleuca gammaflexivirus 2 3.8 12.1 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 45 63 64 65 1 2 3 4 5 6 7 TABLE S5 8 9 E117-4b E108-8 Rn114-15R E97-14 E102-4 E115-15 E114-15-CH E117-4a RnMBV3 Rn103-7 10 E108-8 89.2 11 Rn114-15R 89.8 99.4 12 13 E97-14 89.8 99.4 100.0 14 E102-4 90.3 97.7 98.3 98.3 15 E115-15 90.3 97.7 98.3 98.3 98.9 16 17 E114-15-CH 89.8 97.2 97.7 97.7 98.3 98.3 18 E117-4a 92.0 95.5 96.0 96.0 96.6 96.6 96.0 19 RnMBV3 92.6 95.5 96.0 96.0 96.6 96.6 96.0 97.7 20 21 Rn103-7 71.6 78.4 78.4 78.4 76.7 77.3 76.1 76.1 77.3 22 Rn95-12 72.7 80.1 80.1 80.1 78.4 79.0 77.8 76.7 77.8 85.8 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 46 47 48 49

1 2 3 TABLE S6 4 RDRP MP CP 5 6 %Identity %Similarity %Identity %Similarity %Identity %Similarity 7 ARVV 1 42.9 19.9 31.8 13.7 35.2 19.7 8 ARVV 2 42.2 20.5 34.2 17.0 34.1 19.3 9 10 CCGaV 46.8 26.5 43.0 20.7 39.8 19.3 11 LLV 47.4 25.2 27.2 15.2 35.8 18.5 12 WCLaV1 47.4 27.0 38.9 17.4 42.6 22.3 13 WCLaV2 46.1 25.1 42.9 19.8 42.9 22.2 14 15 RVFV 37.7 19.3 26.4 17.4 32.1 15.7 16 17 18 19 TABLE S7 20 21 Entoleuca sp. isolate Hyphae diameter (μm) 22 23 E97-14 4.46±0.24 b 24 E112-4 5.31±1.37 b 25 26 E115-15 4.95±1.03 b 27 E117-4 6.21±0.82 a 28 29 30 31 TABLE S8 32 33 34 Entoleuca sp. isolate PDA-TA PDA 35 E97-14 1.1491 a 0.8259 b 36 37 E112-4 2.6500 a 1.7411 b 38 E115-15 2.3259 a 1.3161 b 39 E117-4 2.7104 a 1.5813 b 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 65 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure S1 Click here to download Supplementary Material (To be Published): FIGURE S1.tif

Figure S2 Click here to download Supplementary Material (To be Published): FIGURE S2.tif

Figure S3 Click here to download Supplementary Material (To be Published): FIGURE S3.tif

Figure S4 Click here to download Supplementary Material (To be Published): FIGURE S4.tif

Figure S5 Click here to download Supplementary Material (To be Published): FIGURE S5.tif

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Tables S2 and S3 Click here to download Supplementary Material (To be Published): Tables S2 & S3.docx