The use of mycoviruses in the control of forest diseases

1 The use of mycoviruses in the control of forest diseases 1 2 E. Jordán Muñoz-Adalia 1,2* , M. Mercedes Fernández 1,3 & Julio J. Diez 1,2 3 4 1: Sustainable Forest Management Research Institute, University of Valladolid – INIA, 5 Avenida de Madrid 44, 34071 Palencia, Spain. 6 2: Department of Vegetal Production and Forest Resources, University of Valladolid. 7 Avenida de Madrid 44, 34071 Palencia, Spain. 8 3: Department of Agroforestry Sciences, University of Valladolid. Avenida de Madrid 9 44, 34071 Palencia, Spain. 10 11 * Corresponding author: 12 E. Jordán Muñoz-Adalia. 13 Tel.: (34) 979108432. 14 Email: [email protected] / ejordanm[email protected]. 15 Co-authors: 16 M. Mercedes Fernández 17 Tel.: (34) 979108392. 18 Email: [email protected]. 19 Julio J. Diez 20 Tel.: (34) 979108420. 21 Email: [email protected]. 22 23 2 Number of tables: 1. 24 Number of figures: 1 (black and white). 25 3 The use of mycoviruses in the control of forest diseases 26 Abstract 27 Fifteen families of mycoviruses have been described and 80% of these catalogued. 28 However, their evolutionary relationship with fungi is not clear. The mycovirus genome 29 can be formed by single or double-stranded RNA or single-stranded DNA. The effects of 30 mycoviruses range from the induction of a cryptic state (asymptomatic) to promotion of 31 hyper- or hypovirulence in the host. Horizontal transmission of mycoviruses is 32 determined by the presence of different vegetative compatibility types and mating types. 33 Biocontrol of chestnut blight (Cryphonectria parasitica) has been found to be a successful 34 mycovirus-based treatment and is considered a model in forest disease management. 35 Development of this type of biological control tool for use in other forest pathologies 36 requires a sound knowledge of viral symptomatology and transmission. The present 37 review focuses on the application of mycoviruses and the prospects for future use in the 38 biological control of forest diseases as well as on advances in mycovirus-applied research 39 in forestry, landscape and culture of woody plants. 40 Keywords: biological control, forest protection, hypovirulence, vc types, virocontrol, 41 virus transmission. 42 4 1. Introduction 43 Viruses that infect fungi, i.e. mycoviruses, are frequent in the subkingdom Dikarya 44 (phyla Ascomycota and Basidiomycota), phyla Blastocladiomycota and 45 Neocallimastigomycota (formerly Chytridiomycota) and Glomeromycota (formerly 46 Zygomycota) (Herrero, Dueñas, Quesada-Moraga, & Zabalgogeazcoa, 2012; Hibbett et 47 al., 2007). Most fungal genera, ranging from microscopic yeasts to the more evolved 48 edible mushrooms, have been described as hosts of mycoviruses (Hammond, Andrewski, 49 Roossinck, & Keller, 2008; Lim et al., 2005; Magae, 2012; Ro et al., 2007; Schmitt & 50 Breinig, 2006; Stielow, Klenk, Winter, & Menzel, 2011; Strauss, Lakshman, & Tavantzis, 51 2000). This also applied to filamentous fungi that cause plant diseases. 52 Despite the apparent abundance of mycoviruses in nature, research on these infective 53 agents is relatively scarce. Some recent studies have attempted to uncover the biological 54 mechanisms that drive viral infection, replication and transmission in fungi and the 55 ecological and management implications. As a result, agroforestry researchers have 56 discovered the potential use of these viruses in biocontrol, with special attention given to 57 mycoviruses that confer hypovirulence (weakened state) in their pathogenic hosts. 58 In this article, we review studies concerning the use of mycoviruses to control 59 devastating forest diseases. Our main goal is to provide background information about 60 biocontrol based on fungal virus research as well as on the degree to which different 61 protection strategies are being implemented. 62 5 2. General aspects of mycoviruses 63 2.1. Taxonomy, diversity and biology 64 More than 250 fungus-related viral sequences have been identified and sequenced 65 according to National Center for Biotechnology Information (NCBI, 2014; Xie & Jiang, 66 2014), resulting in 22 genera divided among 15 families according to the list published 67 by the International Committee on Taxonomy of Viruses (ICTV, 2014) (Figure 1). 68 Nevertheless, 20% of mycoviruses have not yet been catalogued (Pearson, Beever, Boine, 69 & Arthur, 2009; Van Regenmortel et al., 2010). 70 Mycoviruses usually replicate in the cytoplasm, although some (e.g. Mitovirus sp.) 71 replicate in mitochondria of the host species (Göker, Scheuner, Klenk, Stielow, & 72 Menzel, 2011; Milgroom & Hillman, 2011). Structurally, mycovirus genomes contain 73 one or more open reading frames (ORFs) that encode proteins required for virus 74 replication and sometimes for capsid synthesis. The molecular size of mycovirus genomes 75 varies somewhat, e.g. Rosellinia necatrix quadrivirus 1 (RnQV1) segments range in size 76 from 3.70-4.90 kbp with a single ORF (Chiba et al., 2009), while the maximum size of 77 Chalara elegans RNA Virus 1 (CeRV1) has been reported to be 5.31 kbp in length and 78 contain three ORFs (Park, James, & Punja, 2005). Other mycoviruses may be longer, e.g. 79 Cryphonectria hypovirus 1 (CHV-1) is 12.70 kbp in length and has at least two ORFs 80 (Allemann, Hoegger, Heiniger, & Rigling, 1999; Shapira, Choi, & Nuss, 1991). Overall, 81 the size of genome ranges between the extremes of Partitiviridae viruses (1.4-2.4 kbp and 82 a single ORF) and Hypoviridae viruses (~9-13 kb and two overlapping ORFs); in 83 addition, some families such as Alphaflexiviridae may contain several more or less 84 overlapping ORFs (e.g. Botrytis virus X: ~7.0 kb and five ORFs) (Ghabrial, Castón, 85 Jiang, Nibert, & Suzuki, 2015). In some cases, small RNA molecules may also occur as 86 6 satellite elements associated with the main genome particles (e.g. 0.9-1.4 kb elements 87 associated with 3.7-5.0 kpb mycovirus genome in basydiomicetous yeast 88 Xanthophyllomyces dendrorhous; anamorph: Phaffia rhodozyma (Flores, Alcaíno, 89 Fernandez-Lobato, Cifuentes, & Baeza, 2015)). 90 Mycoviruses can be differentiated on the basis of molecular structure. Thus seven 91 families possess double-stranded RNA (dsRNA) genomes, and six families have single- 92 stranded RNA (ssRNA) genomes. The latter are further divided into two subcategories: 93 five families have ss(+)RNA genomes and one family has a ss(-)RNA genome (Figure 94 1). The mycoviruses belonging to ss(+)RNA families possess viral RNA with the same 95 base sequence as mRNA. The functions of the RNA are similar to mRNA during 96 replication, serving as a template for protein synthesis such as RNA-dependent RNA 97 polymerase (RdRp) or capsid. On the other hand, ss(-)RNA mycoviruses require 98 participation of RNA replicase for their single strain genome to be transcribed into 99 positive sense RNA. Only a few mycoviruses are formed by single circular molecules of 100 DNA (ssDNA) (Ghabrial, Castón, Jiang, Nibert, & Suzuki, 2015; Pearson et al., 2009). 101 <<Insert Figure 1 around here>> 102 The evolutionary relationship between mycoviruses and their hosts remains unclear. 103 Two main hypotheses have been proposed. Briefly, one hypothesis is based on ancient 104 co-evolution of mycoviruses and fungi whereby the speciation of viruses is closely related 105 to vertical transmission (see below), and the asymptomatic presence of mycoviruses may 106 denote a long period of coexistence between viruses and fungi. This would explain the 107 complex relationships between host species and mycoviruses, which range between 108 severe disadvantage to the host (antagonism) and mutualism where the infected host 109 obtains some benefit under certain conditions, as suggested in other viral associations 110 7 (Botella, Vainio, Hantula, Diez, & Jankovsky, 2015; Roossinck, 2015a, 2015b). The other 111 hypothesis suggests the eventual transfer of viruses from plants to saprophytic or 112 pathogenic fungi. In this case, viral transmission may take place during co-existence of 113 fungal endophytes with plants, and small differences detected even within mycovirus 114 families can be explained by a recent change of host (Chiba et al., 2011; Ghabrial, 1998; 115 Liu et al., 2010; Pearson et al., 2009). 116 2.2. Transmission of mycovirus 117 The mechanism of viral transmission is another important aspect of viral biology. 118 Mycoviruses can be transmitted in three ways: by horizontal, vertical or extracellular 119 transfer. Horizontal transmission takes place when a mycovirus colonises a new host 120 through hyphal contact and subsequent mycelia fusion (anastomosis) between individuals 121 during heterokaryon formation (mediated by a self/non-self recognition system). 122 Nevertheless, isolates of the same species are not always compatible, even in the same 123 population. In this type of transfer, different vegetative compatability groups (vc types or 124 VCGs) play a special role, sometimes restricting movement of the virus (Leslie, 1993). 125 Heterokaryon formation is genetically controlled by a specific het or vic loci. 126 Heteroallelism in the het locus is not possible, resulting in reduction in cell lysis or 127 mycelial growth (Saupe, 2000). At the same time, the presence of different mating types 128 (MAT´s) in fungal populations makes transmission more complex (Coppin, Debuchy, 129 Arnaise, & Picard, 1997; Milgroom & Hillman, 2011). 130 In vertical transmission, mycoviruses commonly infect asexual spores. Nevertheless, 131 prevalence rates may vary significantly between species, e.g. in Heterobasidion annosum 132 only 3% of conidia are infected (Ihrmark, Johannesson, Stenström, & Stenlid, 2002) in 133 contrast to 100% infection in Cryphonectria parasitica (Ding et al., 2007). Fungal viruses 134 8 can also colonise sexual spores, infecting a new generation of the host: 8-13% dsRNA 135 infected ascospores of Magnaporthe grisea (Chun & Lee, 2009) whereas 10-84% dsRNA 136 infected basidiospores of H. annosum (Ihrmark, Stenström, & Stenlid, 2004). However, 137 in a more recent study, lower vertical transmission of Heterobasidion parviporum to 138 basidiospores (8.3%) was observed in a spruce forest (Vainio, Müller, Korhonen, Piri, & 139 Hantula, 2014). The authors of the latter study suggested that continuous spore load in 140 stumps may be related to the high rate of infected basidiospores, in contrast to low rates 141 of infection in standing trees, as previously reported. It is now considered that the 142 predominant route of viral transmission is via asexual spores, and vertical transmission 143 has not been reported to occur in many fungal species (Carbone, Liu, Hillman, & 144 Milgroom, 2004; Milgroom & Hillman, 2011). 145 Extracellular transmission, in which purified viral particles of Sclerotinia sclerotiorum 146 hypovirulence-associated DNA virus 1 (SsHADV-1) infected extracellularly virus-free 147 protoplasts, intact hyphae and hyphal fragments of white mould fungus (Sclerotinia 148 sclerotiorum) either in vitro (PDA culture) or in vivo (leaves of infected plants has 149 recently been described (Yu et al., 2013). These authors also mentioned that purified viral 150 DNA did not infect mycelia or fungal protoplasts, suggesting that whole viral particles 151 are needed for extracellular infection. 152 On a larger scale, transmission of mycoviruses between species has also been reported 153 (Lee, Yu, Son, Lee, & Kim, 2011; Liu, Linder-Basso, Hillman, Kaneko, & Milgroom, 154 2003; Vainio et al., 2011a), opening up new research lines focusing on the genetic, 155 evolutionary and ecological factors involved in transmission. 156 2.3. Hypovirulence process 157 9 The effects of mycoviruses infection can range from cryptic symptoms 158 (asymptomatic) to the promotion of hypervirulence, through variations of colonial 159 morphology and inducement of color changes (Ghabrial & Suzuki, 2009). In fact, the 160 same mycovirus can have different effects on their host depending on ecological 161 conditions (Hyder et al., 2013). One phenomenon caused by mycoviruses, especially 162 interesting for agroforestry science, is hypovirulence. Only a few mycoviruses reduce 163 spore production, causing slow mycelial growth or less aggressive invasion in pathogenic 164 hosts, making viruses effective in biocontrol (Milgroom & Hillman, 2011; Nuss, 2005) 165 or virocontrol (Chiba, Kondo, Kanematsu, & Suzuki, 2010). In this sense, hypovirulence 166 have been proved according to Koch´s postulates using infectious cDNA of C. parasitica 167 (Chen & Nuss, 1999) and S. sclerotiorum (Marzano et al., 2015), hyphae infection of 168 Sclerotinia spp. using viral particles (Yu et al., 2013) and protoplast infection using 169 dsRNA (Chiba, Lin, Kondo, Kanematsu, & Suzuki, 2013; Hillman, Supyani, Kondo, & 170 Suzuki, 2004; Lee et al., 2011). Hence, knowledge about mycovirus-mediated 171 hypovirulence is improving biocontrol strategies in many cases of agroforestry health (see 172 next section). 173 Both hyper- and hypovirulence are strongly related to the presence of specific viruses, 174 even in co-infection. Four dsRNA mycoviruses have been detected in Nectria radicicola 175 (anamorph: Cylindrocarpon destructans) (Ahn and Lee, 2001). Removal of one virus, L1 176 (6.0 kbp), caused a reduction in virulence of the fungus, while later reinfection through 177 anastomosis recovered the virulence of the isolate. Detailed laboratory studies 178 complemented with pathogenicity field assays are essential for developing virocontrol 179 techniques. 180 One challenge in plant pathology and the use of mycoviruses is the antiviral response 181 of fungi or RNA silencing. When viruses infect healthy cells, dicer-type nucleases initiate 182 16 Many coniferous tree species (mainly Picea, Pinus, Abies and Larix species) in 313 Northern and Central Europe, North America and Japan host the fungus Gremmeniella 314 abietina (anamorph: Brunchorstia pinea), leading to the appearance of stem cankers and 315 shoot dieback and causing severe damage in woods and plantations when weather 316 conditions are favourable. Three races of this fungus (European, North American and 317 Asian) have been catalogued. The European race is subdivided into three biotypes (A, B 318 and alpine) (Botella et al., 2010; Donaubauer, 1972; Hamelin, Lecours, Hansson, 319 Hellgren, & Laflamme, 1996; Kaitera & Jalkanen, 1992; Romeralo, Botella, Santamaria, 320 & Diez, 2012; Santamaria, Alves-Santos, & Diez, 2005; Senn, 1999), although the 321 taxonomy is currently under revision (Romeralo pers. com.). 322 Three families of dsRNA mycoviruses have been detected in this forest pathogen: 323 Gremmeniella abietina mitocondrial RNA virus S1 (GaMRV-S1, Narnaviridae) 324 (Tuomivirta & Hantula, 2003a); Gremmeniella abietina RNA virus L1 (GaRV-L1, 325 Totiviridae); and Gremmeniella abietina RNA virus MS1 (GaRV-MS1, Partitiviridae) 326 (Tuomivirta & Hantula, 2003b), with a high frequency of occurrence; e.g. the 327 mycoviruses have been detected in 89% of Spanish isolates (Botella, Tuomivirta, 328 Hantula, and Diez, 2012) and in 50% of Turkish isolates (Aday et al., 2012). In addition, 329 three mycoviruses were found together infecting the same isolates of G. abietina var. 330 abietina type A (Tuomivirta and Hantula, 2005). Later, Botella, Tuomivirta, Vervuurt, 331 Diez, and Hantula (2012) reported the absence of mitoviruses in biotype B from Turkey, 332 biotype A from North America and European Alpine biotype. On the contrary, biotype A 333 from Finland and Spain hosted mycoviruses. Specifically, Spanish populations hosted 334 two mycoviruses (GMV1 and GMV2) in high proportion (74% of isolates hosted 335 dsRNA). These authors discussed the possible factors determining presence and 336 transmission of mitoviruses between fungal races and highlighted the role of asexual 337 17 reproduction in virus widespread. In fact, the higher proportion of mitovirus presence was 338 detected in Spain where only asexual reproduction has been reported. Regarding the high 339 presence and the low genetic variability detected in GMV2 in Spanish isolates, the 340 researchers suggested a possible recent host switch and a subsequent adaptation to these 341 new conditions. The findings of recent RdRp sequencing studies support the idea of a low 342 degree of genetic variation in G. abietina mitoviruses in the European population 343 (Botella, Tuomivirta, Hantula, Diez, & Jankovsky, 2014). 344 3.5. Fusarium circinatum 345 Pine pitch canker is a virulent disease caused by Fusarium circinatum (teleomorph: 346 Gibberella circinata) in many pine species and in Douglas fir (Pseudotsuga menziesii) 347 worldwide. Infections have also been observed to cause significant damage in Abies alba, 348 S. giganteum, Larix decidua and Picea abies (Martínez-Álvarez, Pando, and Diez, 349 2014a). The pathogen was first detected in the southeastern USA and Mexico (where it is 350 probably endemic) an then in Haiti, South Africa, Chile, France, Korea, Spain, Italy, 351 Japan, Portugal, Uruguay and Brazil (Aegerter, Gordon, Storer, & Wood, 2003; Enebak 352 & Stanosz, 2003; Gordon, Kirkpatrick, Aegerter, Wood, & Storer, 2006; Martínez- 353 Álvarez, Alves-Santos, & Diez, 2012; Pfenning, Costa, Melo, Costa, & Aires, 2014). This 354 fungus causes dieback in trees due to the formation of bleeding and resinous cankers on 355 trunk and branches. Moreover, F. circinatum frequently causes death and damping-off of 356 seedlings through both pre- and post-emergence infection, making such infections a 357 significant threat to nurseries and afforestations (Aegerter et al., 2003; Hammerbacher, 358 Ganley, Steenkamp, Gordon, & Coutinho, 2008). 359 Three putative Mitovirus spp. (Narnaviridae) were recently identified in F. circinatum 360 isolates from Pinus radiata in northern Spain and named Fusarium circinatum mitovirus 361 18 1, 2-1 and 2-2 (FcMV1, FcMV2-1 and FcMV2-2) (Martínez-Álvarez, Vainio, Botella, 362 Hantula, and Diez, 2014b). The genetic structure of the mycoviruses hosted by F. 363 circinatum isolates from Spain and South Africa has also been studied (Vainio, Martínez- 364 Álvarez, Bezos, Hantula, and Diez, 2015). Only Spanish isolates were found to host 365 mycoviruses, which showed very similar sequence variants (>95% similarity). Indeed, a 366 high rate of asexual spore transmission of mycoviruses (ranging between 70% and 100%) 367 has been preliminary observed (Bezos, Martínez-Álvarez, Romeralo, and Diez, 2015), 368 indicating the potential use of the mycoviruses as biocontrol agents. 369 3.6. Botryosphaeria spp. 370 Botryosphaeria spp. commonly occur as endophytic fungi in healthy hosts, but may 371 become virulent when their host is subjected to environmental stress or physical damage 372 (Burgess, Sakalidis, & Hardy, 2006; Smith, Crous, Wingfield, Coutinho, & Wingfield, 373 2001; Smith, Wingfield, Crous, & Coutinho, 1996). Despite its taxonomic complexity, 374 Botryosphaeria dothidea (anamorph: Fusicoccum aesculi) is cited as the causal agent of 375 stem and branch cankers on apple trees (Malus domestica), ring spot on pear trees (Pyrus 376 communis) and dieback and stem cankers on eucalyptus trees (Eucalyptus spp.) among 377 many other woody species (Brown-Rytlewski & McManus, 2000; Slippers & Wingfield, 378 2007). Eucalyptus sp. is one of the most common trees planted in commercial and clonal 379 forestry at an international level. Eucalyptus dieback and cankers are of special interest 380 in forest science because of the reduced growth, offspring failure and adult tree death 381 caused by the pathogen (Pérez, Wingfield, Slippers, Altier, & Blanchette, 2010). The 382 gummy exudation produced in cankers also makes the wood less valuable, causing 383 significant economic losses in the forest industry (Rodas, Slippers, Gryzenhout, & 384 Wingfield, 2009). 385 19 Two dsRNA mycoviruses were recently detected in non virulent isolates of B. dothidea 386 infecting Pyrus pyrifolia (Wang et al., 2014). These researchers reported Botryosphaeria 387 dothidea chrysovirus 1 (BdCV1) as a new member of Chrysoviridae and also identified 388 Botryosphaeria dothidea partitivirus 1 (BdPV1). Although BdPV1 was included in 389 Partitiviridae, the capsid proteins of the mycovirus do not show significant similarity to 390 any other capsid proteins. Analysis of the RdRp sequence also suggests the inclusion of 391 this mycovirus in a new Partitiviridae clade (with 39% RdRp similarity to the most 392 closely related Chrysovirus sp.). 393 3.7. Hymenoscyphus fraxineus 394 Ash dieback is an invasive disease caused by the fungus Hymenoscyphus fraxineus 395 (synonym: Hymenoscyphus pseudoalbidus; anamorph: Chalara fraxinea). The fungus 396 infects Fraxinus spp. with notable incidence in common ash (Fraxinus excelsior) and 397 narrow-leafed ash (Fraxinus angustifolia). This pathogen has been spreading in Europe 398 since the 1990s and causes severe damage in forests (pure or mixed stands), nurseries and 399 urban green areas (Hietala, Timmermann, Børja, & Solheim, 2013; Kowalski, 2006; 400 Timmermann, Børja, Hietala, Kirisits, & Solheim, 2011). It has also been cited in East 401 Asia and Japan infecting Fraxinus mandshurica and Fraxinus chinensis (Gross, 402 Holdenrieder, Pautasso, Queloz, & Sieber, 2014). The fungus infects ash trees of all ages, 403 causing rapid crown dieback in adult trees, cankers and bark lesions on stem and twigs, 404 and also leaf wilt. The disease frequently causes the death of young trees a few years after 405 infection. However, it may become a chronic disease in older trees, reducing the tree’s 406 defences against other pathogens and pests or environmental factors (Gross et al., 2014; 407 Kowalski & Holdenrieder, 2009; Timmermann et al., 2011). 408 20 A new ssRNA mycovirus that infects this pathogenic fungus was recently discovered 409 (Schoebel, Zoller, and Rigling, 2014). The authors proposed inclusion of the virus in the 410 genus Mitovirus (Narnaviridae) and named it Hymenoscyphus fraxineus mitovirus 1 411 (HfMV1). They noted the possibility of rapid genetic divergence based on their findings 412 of large differences in the strains isolated in Switzerland, Poland, Germany, Lithuania 413 and Japan. They hypothesised that the similarities between Swiss and Japanese strains 414 may denote a European pathogen introduction across infected host material from Asia. 415 Moreover, the prevalence of this mycovirus was high (90% in Swiss isolates according 416 to Schoebel et al., (2014)), supporting the most accepted hypothesis of predominance of 417 vertical transmission via ascospores. 418 3.8. Other fungal pathogens in woody plants 419 Botrytis cinerea (teleomorph Botryotinia fuckeliana) causes grey mould disease in 420 more than 200 crops species over the world, including farmland crops, ornamental species 421 and fruit crops such as grapes (Vitis vinifera), pear trees, raspberries and blackberries 422 (Rubus spp.) (Rodríguez-García, Medina, Alonso, & Ayllón, 2014; Williamson, 423 Tudzynski, Tudzynski, & van Kan, 2007). The presence of different genera of mycovirus 424 in this fungus has been widely reported (Castro, Kramer, Valdivia, Ortiz, & Castillo, 425 2003; Potgieter, Castillo, Castro, Cottet, & Morales, 2013; Rodríguez-García et al., 2014; 426 Wu et al., 2007; Zhang, De Wu, Li, Jiang, & Huang, 2010). These studies highlight the 427 wide diversity of viruses that this fungus is able to host and which provide a wide range 428 of opportunities for research in the field of fungal virology. Another three mycoviruses 429 that infect Botrytis sp. have recently been sequenced: Botrytis virus F (BVF, 430 Gammaflexiviridae), Botrytis virus X (BVX, Alphaflexiviridae) and Botrytis porri RNA 431 virus 1 (BpRV1, dsRNA virus) (Xie & Jiang, 2014). 432 21 Verticillium dahliae and Verticillium albo-atrum are both causal agents of Verticillium 433 wilt disease. They have been cited in a broad range of hosts and more than 200 species, 434 including bushes and trees (Schall & Davis, 2009; Smith, 1965). Specifically, V. dahliae 435 can infect economically important woody crops such as gooseberry (Ribes grossularia), 436 apricot (Prunus armeniana), olive (Olea europea), quince (Cydonia oblonga) and roses 437 (Rosa spp.), as well as other species of ecological interest such as maple (Acer palmatum), 438 sycamore (Acer pseudoplatanus), raspberry, honeysuckle (Lonicera sp.) and broom 439 (Cytisus scoparius). V. albo-atrum causes damage to the tree of heaven (Ailanthus 440 altissima), striped maple (Acer pennsylvanicum), yellow poplar (Liriodendron tulipifera) 441 and other landscape species (Morehart, Donohue, & Melchior, 1980; Schall & Davis, 442 2009; Smith, 1965). Some studies have demonstrated the presence of mycoviruses in 443 these pathogenic fungi. For example, a Chrysovirus sp. named Verticillium dahliae 444 chrysovirus 1 (VdCV1) was identified by Cao et al. (2011). A novel member of the family 445 Partitiviridae was identified in V. albo-atrum: Verticillium albo-atrum partitivirus 1 446 (VaaPV1) (Cañizares, Pérez-Artés, and García-Pedrajas, 2014), although no details were 447 provided about the pathogenic effect of the mycovirus in its fungal host. 448 Some opportunistic fungal pathogens of Pinus spp., such as Diplodia pinea (synonym: 449 Sphaeropsis sapinea) and Diplodia scrobiculata (Smith et al., 1996), also host 450 mycoviruses. Two dsRNA mycoviruses have been identified in D. pinea: Sphaeropsis 451 sapinea RNA virus 1 and 2 (SsRV1, SsRV2 respectively; Totiviridae) (Preisig, Wingfield, 452 & Wingfield, 1998); and one in D. scrobiculata: Diplodia scrobiculata RNA virus 1 453 (DsRV1; related to Chrysoviridae) (De Wet, Bihon, Preisig, Wingfield, & Wingfield, 454 2011; De Wet, Preisig, Wingfield, & Wingfield, 2008). 455 Another pathogenic fungi of interest in agroforestry is the causal agent of root rot 456 disease, Rosellinia necatrix (anamorph: Dematophora necatrix). The interest is due to the 457 22 pathogenicity of the fungus in several woody species e.g. apple, olive, grape and poplar 458 (Populus spp.) (Pérez-Jiménez, 2006). Many families of mycoviruses are known to infect 459 this fungus, e.g. Chrysoviridae, Quadriviridae, Partitiviridae, Reoviridae and Totiviridae 460 (Xie and Jiang, 2014). Two dsRNA mycoviruses have also been associated with 461 hypovirulence: Rosellinia necatrix megabirnavirus 1 (RnMBV1), included in a new 462 family of mycoviruses (Megabirnaviridae) and Rosellinia necatrix partitivirus 2 (RnPV2) 463 (Xie and Jiang, 2014). 464 23 4. Future perspectives for use of mycoviruses in biocontrol 465 As already mentioned, many forest, horticultural and ornamental species harbour 466 mycoviruses to a greater or lesser extent (Table 1). Although many of these have not yet 467 been found to be of use for biocontrol purposes, many of them provide new opportunities 468 for research in forestry science. Despite the promising outlook, the use of mycoviruses in 469 biological control is limited by the need for detailed analysis of (a) the symptoms 470 associated with mycovirus-caused hypovirulence, (b) transmission mechanisms and 471 biological and ecological conditions, (c) treatment effectiveness in the field and (d) 472 subsequent persistence in the host population. 473 4.1. Identification of factors leading to hypovirulence: research in progress and lessons 474 learned 475 The best known example of a disease managed by mycoviruses is chestnut blight. In 476 Europe, CHV-1 has been used to induce hypovirulence (Robin & Heiniger, 2001) with 477 goods results in field inoculation trials (Juhásová, Adamcíková, & Robin, 2005; Robin, 478 Anziani, & Cortesi, 2000; Zamora et al., 2014). CHV-1 and CHV-3 have been used with 479 less success in the USA than in Europe, with natural hypovirulence being reported in 480 Michigan (Milgroom & Cortesi, 2004). For other pandemics such as DED, mycoviruses 481 infecting O. novo-ulmi appear promising for biocontrol, because of the symptoms that 482 they cause in host isolates, such as slow mycelial growth, abnormal or amoeboid colony 483 formation, reduction in asexual spore production, low cytochrome oxidase level and 484 formation of mitochondrial DNA plasmids (Hong et al., 1999). 485 In relation to the application of biocontrol in diseased forests in boreal areas, no clear 486 relationship between viral presence and fungus growth rate was observed in H. annosum 487 s.l. (Vainio et al., 2010). However, significant variations in growth and changes in the 488 24 effects of virus were observed in relation to the culture conditions. The effect of HetPV6 489 infection in relation to multiple variables (geographical, culture conditions and host) has 490 been investigated in four Heterobasidion species (Vainio et al., 2012). No significant 491 differences in growth were found in H. parviporum (in vivo and in vitro) or H. annosum 492 (in vivo). However, significantly increased mycelial growth was observed in infected H. 493 annosum cultures (laboratory assays condition: 6º C and 15ºC culture on MOS agar 494 plates). Consequently, these results do not support a possible use of HetPV6 in 495 virocontrol, although HetPV6 is very frequent in fungal populations and apparently does 496 not interfere in subsequent viral infection (Vainio et al. 2013b). 497 Mycoviruses may eventually be used as tools in the management of invasive diseases, 498 for example in ash dieback. Although Hymenoscyphus fraxineus mitovirus 1 does not 499 show harmful effects in its host, future perspectives for its application in biocontrol are 500 promising because of the phylogenetic position of this mitovirus relative to others that 501 are known to cause hypovirulence (Schoebel et al., 2014). In fact, HfMV1 is closely 502 related to Cryphonectria cubensis, S. sclerotiorum and Helicobasidium mompa 503 mitoviruses. 504 Several Totiviridae, Chrysoviridae and Partiviridae mycoviruses have been identified 505 in Fusarium graminearum (Lee, Son, & Kim, 2011; Yu et al. 2011). More specifically, a 506 mycovirus described in F. graminearum infecting maize in Korea (named Fusarium 507 graminearum virus 1-DK2; FgV1-DK2) is capable of reducing mycelial growth and 508 sporulation, decreasing mycotoxin production and increasing pigmentation (Chu et al., 509 2002). In a later study addressing this topic, a mixed infection of two dsRNA viruses was 510 reported, with no changes in mycelial morphology but with a high rate of transmission in 511 conidia and ascospores (30-100%) (Chu et al., 2004). A recent study identified a new 512 mycovirus associated with hypovirulence in Fusarium virguliforme and closely related to 513 25 F. graminearum mycoviruses (Marvelli et al., 2014). Moreover, two new putative 514 mycoviruses belonging to the Mitovirus genus have been described in Fusarium 515 coeruleum isolates, in addition to one new Alphapartitivirus sp. in Fusarium solani f. sp. 516 pisi (Osaki et al., 2015). Mycoviruses infecting in F. coeruleum are closely related to 517 FcMV1, which opens up a new line of phylogenetic research. Together these results 518 encourage the continued study of hypovirulence induced by mycoviruses in Fusarium 519 spp. (with special focus on F. circinatum) whose use in biocontrol may prove to be a 520 profitable consequence of in-depth studies of this species. 521 Grey mold, caused by B. cinerea, is being investigated by various research groups 522 around the world because of the global importance of this disease. The rare formation of 523 multicellular penetration structures (infection cushions) and decreased mycelial growth 524 are probably caused by hypovirulence induced by mycoviruses (especially Botrytis 525 cinerea mitovirus 1 (BcMV1), main mycovirus implied in hypovirulence process) as 526 suggested by Rodríguez-García et al. (2014); Wang et al. (2014) and Zhang et al. (2010). 527 These advances are very encouraging in agroforestry technology and are leading the way 528 to the development of new treatments in the control of tree diseases, at least for incipient 529 infections, thus possibly reducing economic and ecological damage. 530 4.2. Mycoviruses transmission and biological conditions 531 The existence of vegetative incompatibility is a major limitation in virocontrol, due to 532 the instability of hyphal fusion between fungi that have not the same vc type. In the case 533 of C. parasitica, fungal viruses can be transferred thought anastomosis among different 534 vc types (0.13-0.50 transmission rates between CHV-1 strains differentiated by one or 535 two vegetative incompatibly genes), although slowly and in less proportion (3-4%) 536 (Cortesi, McCulloch, Song, Lin, & Milgroom, 2001; Liu & Milgroom, 1996; Peters, 537 32 biocontrol treatment was regarded as doubtful. Another study reported the disappearance 687 of CHV-1 in European treated plots 24 years after biocontrol application (Robin, Lanz, 688 Soutrenon, & Rigling, 2010). The authors pointed out that the low diversity of vc is not 689 necessarily related to low persistence, because similar results have been reported in other 690 chestnut forests in Europe, and they concluded that differences in CHV-1 subtype fitness 691 may be the most important factor in the persistence of mycoviruses in field. More research 692 is required to establish the long-term effects of the use of mycoviruses in the field. 693 <<Insert Table 1 around here>> 694 33 5. Conclusions 695 1. Mycoviruses represent a relatively unknown group in virology and plant 696 pathology sciences. However, the taxonomy of mycoviruses based on genetic 697 sequences and biological characteristics (including antiviral response by hosts) 698 is being improved greatly. 699 2. Chestnut blight caused by C. parasitica is the best known and most successful 700 mycovirus-based biocontrol method in forest pathology. Moreover, it is the only 701 case in which a mycovirus-based biocontrol technique has been satisfactorily 702 implemented. This disease serves as a study model in forestry protection, with 703 particular relevance in the development of new preventive and therapeutic 704 measures centred on several tree species. 705 3. Mycovirus research focused on diseases caused by the O. novo-ulmi, H. 706 annosum complex, G. abietina, F. circinatum, B. dothidea, H. fraxineus and R. 707 necatrix is currently being developed in the forest context. Further studies 708 involving D. pinea, D. scrobiculata, V. dahliae and V. albo-atrum pathologies 709 are also needed. 710 4. Mycovirus-mediated hypovirulence is a current challenge in biocontrol research 711 because of its potential role in the prevention and/or management of plant 712 diseases. It could become an important tool for maintaining the health of woody 713 species, complementing or totally replacing chemical treatments. 714 5. Inoculation of fungi with mycoviruses may become a new management tool for 715 forest protection, as used in the treatment of chestnut blight disease. 716 34 6. The main targets of study in mycovirus-based biological control are: (i) the 717 mycoviruses that induce hypovirulence in their hosts, (ii) the conditions that 718 affect hypovirulence and the virus silencing process, (iii) the transmission 719 ecology and its biological limitations, (iv) the taxonomical and phylogenetic 720 relationships between mycoviruses and (v) the viability of field biocontrol 721 measures. 722 35 Acknowledgements 723 E. J. Muñoz-Adalia is in receipt of funding from the European Social Fund and the Consejería 724 de Educación de Castilla y León (JCyL). This study was supported by the Ministerio de Economía 725 y Competitividad (Project number: AGL2012-39912). Comments by Pablo Martínez-Álvarez 726 helped to improve an earlier version of the manuscript. 727 36 References 728 Aday, A. G., Lehtijarvi, A., & Doğmuş-Lehtijarvi, H. T. (2012). 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Summary of mycoviruses of agroforestry interest. 1364 Fungus Main Hosts Mycoviruses Family References C. parasitica Castanea spp. CHV-1; CHV-2; CHV-3; CHV-4; MyRV-1; MyRV2; CpMV1 Hypoviridae; Reoviridae Narnaviridae Hillman et al. (1994); Hillman and Suzuki (2004); Linder- Basso et al. (2005); Shapira et al. (1991); Smart et al. (1999); Suzuki et al. (2004) O. novo-ulmi Ulmus spp. OnuMV1a; OnuMV1b; OnuMV1c; OnuMV2; OnuMV3a; OnuMV3b; OnuMV4-Ld; OnuMV5-Ld; OnuMV6-Ld; OnuMV7-Ld; DsRNA01_ORF; DsRNA02_ORF Narnaviridae Hong et al. (1998a,b, 1999); Doherty et al. (2006); Hintz et al. (2013) H. annosum complex Various HaV; HaV-P; HetPV1; HetPV2; HetPV3; HetPV4; HetPV5; Partitiviridae Ihrmark et al. (2001) Vainio et al. (2010, 2011a,b, 2012, 2013c, 2014) 54 HetPV6; HetPV7; HetPV8; HetPV2-pa1; HetPV7-pa1 G. abietina Pinus spp., Picea spp., Abies spp., Larix spp. GaMRV-S1; GaRV-L1; GaRV-MS1 Narnaviridae; Totiviridae; Partitiviridae; Tuomivirta and Hantula (2003a,b) F. circinatum Pinus spp.; Pseudotsuga menziesii FcMV1; FcMV2-1; FcMV2-2 Narnaviridae Martínez-Álvarez et al. (2014b) B. dothidea Pyrus spp., Malus spp., Eucalyptus spp. BdCV1; BdPV1 Chrysoviridae; Partitiviridae Wang et al. (2014) H. fraxineus Fraxinus spp. HfMV1 Narnaviridae Schoebel et al. (2014) B. cinerea Various BcMV1 Narnaviridae Wu et al. (2010) V. dahliae Various VdCV1 Chrysoviridae Cao et al. (2011) V. albo-atrum Various VaaPV1 Partitiviridae Cañizares et al. (2014) D. pinea Pinus spp. SsRV1; SsRV2 Totiviridae Preisig et al. (1998) D. scrobiculata Pinus spp. DsRV1 Chrysoviridaerelated De Wet et al. (2011) R. necatrix Various RnMBV1; RnPV2 Megabirnaviridae; Partitiviridae Chiba et al. (2009, 2013) 1365 55 Figure 1. General taxonomy of mycoviruses according to ICTV classification criteria, 1366 Virus Taxonomy 2014 Release.*Classification under consideration; **Family proposed 1367 by Ghabrial et al. (2015). 1368 1369