Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control

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1 bs_bs_banner MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 DOI: /mpp Pathogen profile Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control DANIEL RIGLING* AND SIMONE PROSPERO Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf 8903, Switzerland SUMMARY Chestnut blight, caused by Cryphonectria parasitica, is a devastating disease infecting American and European chestnut trees. The pathogen is native to East Asia and was spread to other continents via infected chestnut plants. This review summarizes the current state of research on this pathogen with a special emphasis on its interaction with a hyperparasitic mycovirus that acts as a biological control agent of chestnut blight. TAXONOMY: Cryphonectria parasitica (Murr.) Barr. is a Sordariomycete (ascomycete) fungus in the family Cryphonectriaceae (Order Diaporthales). Closely related species that can also be found on chestnut include Cryphonectria radicalis, Cryphonectria naterciae and Cryphonectria japonica. HOST RANGE: Major hosts are species in the genus Castanea (Family Fagaceae), particularly the American chestnut (C. dentata), the European chestnut (C. sativa), the Chinese chestnut (C. mollissima) and the Japanese chestnut (C. crenata). Minor incidental hosts include oaks (Quercus spp.), maples (Acer spp.), European hornbeam (Carpinus betulus) and American chinkapin (Castanea pumila). DISEASE SYMPTOMS: Cryphonectria parasitica causes perennial necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees, eventually leading to wilting of the plant part distal to the infection. Chestnut blight cankers are characterized by the presence of mycelial fans and fruiting bodies of the pathogen. Below the canker the tree may react by producing epicormic shoots. Non-lethal, superficial or callusing cankers on susceptible host trees are usually associated with mycovirus-induced hypovirulence. DISEASE CONTROL: After the introduction of C. parasitica into a new area, eradication efforts by cutting and burning the infected plants/trees have mostly failed. In Europe, the mycovirus Cryphonectria hypovirus 1 (CHV-1) acts as a successful biological control agent of chestnut blight by causing so-called hypovirulence. CHV-1 infects C. parasitica and reduces its parasitic growth and sporulation capacity. Individual cankers can be therapeutically treated with hypovirus-infected C. parasitica strains. The hypovirus may subsequently spread to untreated cankers and become established in the C. parasitica population. *Correspondence: daniel.rigling@wsl.ch Hypovirulence is present in many chestnut-growing regions of Europe, either resulting naturally or after biological control treatments. In North America, disease management of chestnut blight is mainly focused on breeding with the goal to backcross the Chinese chestnut s blight resistance into the American chestnut genome. Keywords: chestnut blight, Cryphonectria hypovirus, Cryphonectria parasitica, disease management, hypovirulence, review. INTRODUCTION As with many other plant pathogens, Cryphonectria parasitica was first described on a non-native host outside its natural distribution range. The pathogen was first reported in 1904 on American chestnut [Castanea dentata (Marsh.) Borkh.] in the Zoological Park of New York City and the disease that it caused was named chestnut blight. The fungus was first described as Diaporthe parasitica Murrill. and later transferred to the genus Endothia (Anderson and Anderson, 1912). In 1978, it received its current name, Cryphonectria parasitica (Murr.) Barr. (Barr, 1978). Cryphonectria parasitica has become a textbook example of an introduced pathogen that has caused devastating disease epidemics on native tree species. In North America, it has largely eliminated American chestnut as a dominant overstorey tree species and, in Europe, it has caused widespread decline of European chestnut (Castanea sativa Mill.). Other severe disease epidemics of trees caused by introduced fungal pathogens have followed, with ash dieback (caused by Hymenoscyphus fraxineus) in Europe as the last example (Gross et al., 2014). Chestnut blight has also become famous for hypovirulence, a virus-attenuated reduction of virulence, which provides the basis for biocontrol of the disease, and for a conservation-breeding programme aiming to restore American chestnut as a forest tree species. The main aim of this review is to summarize the current knowledge of the biology and epidemiology of the chestnut blight fungus C. parasitica and to highlight perspectives for disease control with special emphasis on hypovirulence. VC 2017 BSPP AND JOHN WILEY & SONS LTD 7

2 8 D. RIGLING AND S. PROSPERO Fig. 1 Symptoms of chestnut blight on Castanea sativa. (a) Branch wilting caused by a Cryphonectria parasitica infection. The wilted leaves typically remain hanging on the branches even after leaf fall. They produce a so-called flag, which is the most pronounced early symptom of chestnut blight in the crown of adult trees. (b) Extended dieback after several years of infection. (c e) Various virulent chestnut blight cankers. Cankers typically appear as sunken, reddish-brown bark lesions. Below the cankers, trees typically react by producing epicormic shoots. (f) Cryphonectria parasitica forms pale brown mycelial fans, which advance intercellularly in the bark and cambium of the chestnut tree. (g) Grafted chestnut seedling infected by C. parasitica. (h) Blight infection (reddish discoloration) of a chestnut plant in a nursery. (i, j) Passive (healed) chestnut blight canker associated with hypovirulence of C. parasitica. In contrast with virulent cankers, hypovirus-infected cankers typically have a swollen appearance and are superficial or callused. DISEASE SYMPTOMS Cryphonectria parasitica is a bark pathogen, which only infects above-ground tree parts, i.e. stems, branches and, eventually, twigs. The manifestation of symptoms (Fig. 1) induced by the pathogen on susceptible hosts (European and American chestnut) varies depending on both the virulence of the particular C. parasitica strain and the age of the infected tree part (Heiniger and Rigling, 1994; Prospero and Rigling, 2013). Virulent strains of C. parasitica typically produce necrotic lesions (so-called cankers) on the bark, which can kill smaller branches or twigs within a few months (Fig. 1d, e, g, h). Perennial cankers are formed on thicker branches or stems, which may develop over years before causing mortality (Fig. 1c). Bark cankers on smooth-barked young stems/ branches are orange to reddish-brown on the surface. On older stems/branches, canker coloration is generally less pronounced. On stems/branches with thick bark, C. parasitica is difficult to recognize until longitudinal splits appear in the bark (Diller, 1965). In the bark and cambium, C. parasitica develops typical pale brown mycelial fans (see below), which are a clear sign of a chestnut blight infection (Fig. 1f). If the cambium is killed, the bark sinks inwards, giving the canker a characteristic sunken appearance. Bark cankers may enlarge rapidly and girdle the affected tree part, leading to the death of the stem/branch part distal to the canker. The leaves wilt, turn yellow or brown, and typically remain hanging on the infected dead branches, producing a so-called flag (Fig. 1a). On adult trees, flags are the most pronounced early symptom of a C. parasitica MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 VC 2017 BSPP AND JOHN WILEY & SONS LTD

3 Cryphonectria parasitica 9 infection in the crown. Trees react to an infection by producing numerous epicormic shoots below the cankers (Fig. 1e). Cankers induced by hypovirus-infected C. parasitica strains (see below) initially show similar characteristics to those caused by virulent strains. Successively, their expansion may slow down and stop. The tree forms new layers of bark under the affected area, the outer bark cracks and the canker takes on a swollen appearance (Fig. 1j). As the cambium is not colonized and killed by the fungus, the plant part distal to the canker survives. In contrast with cankers caused by virulent fungal strains (commonly called virulent or active cankers), cankers caused by hypovirusinfected strains (commonly called hypovirulent, healed or passive cankers) are usually superficial or callused (Fig. 1i, j). The absence of epicormic shoots below the canker is generally a sign of such non-lethal chestnut blight cankers (Fig. 1j). However, a recent study by Bryner et al. (2013) indicated that canker morphology and hypovirus infection are not always clearly related. The authors concluded that hypovirus infections can be definitively verified only with laboratory analyses. On the surface of the cankers, stromata harbouring the fruiting bodies of C. parasitica may develop (see below). On oaks, symptoms consist of slowly developing, callusing cankers that usually do not kill the affected stems or branches (Tziros et al., 2015). HOST RANGE The main host species of C. parasitica in particular, the American chestnut, the European chestnut, the Chinese chestnut (C. mollissima Blume) and the Japanese chestnut (C. crenata Siebold & Zucc.) belong to the genus Castanea in the family Fagaceae (Roane et al., 1986). The most susceptible species is C. dentata, followed by C. sativa. On the other hand, the two Asian chestnut species, thanks to their co-evolution with the pathogen, are resistant to C. parasitica (Anagnostakis, 1992). Hosts other than Castanea species include mainly oaks (Quercus spp.), such as post oak (Q. stellata Wangenh.), scarlet oak (Q. coccinea M unchh.), live oak (Q. virginiana Mill.) and white oak (Q. alba L.) in the USA, and holm oak (Q. ilex L.), sessile oak [Q. petraea (Mattuschka) Lieblein], downy oak (Q. pubescens Willd.) and Hungarian oak (Q. frainetto Ten.) in Europe (Diller, 1965; Radocz and Tarcali, 2009; Torsello et al., 1994; Tziros et al., 2015). Occasionally, C. parasitica hasalsobeenreportedonmaples(acer spp.), European hornbeam (Carpinus betulus L.) and American chinkapin (Castanea pumila L. var. pumila and C. pumila var. ozarkensis) (Diller, 1965; Dallavalle and Zambonelli, 1999; Gryzenhout et al., 2009; Paillet, 1993). According to Turchetti and Maresi (2008), hosts other than Castanea species may be infected, especially when they are weakened and disease pressure is particularly high. In addition to C. parasitica, the genus Cryphonectria includes several other species. Three, mostly saprotrophic species are often isolated together with C. parasitica from chestnut or oak, namely C. radicalis (Schwein.) M.E. Barr in Europe and North America (Hoegger et al., 2002), C. naterciae M.H. Bragança, E. Diogo & A.J.L. Phillips (syn. C. decipiens Gryzeh. & M.J. Wingf.) in Europe (Bragança et al., 2011) and C. japonica (Tak. Kobay. & Kaz. It^o) Gryzeh. & M.J. Wingf [syn. C. nitschkei (G.H. Otth) M.E. Barr] in Japan (Gryzenhout et al., 2009; Liu et al., 2003). Cryphonectria cubensis (Bruner) Hodges [now Chrysoporthe cubensis (Bruner) Gryzenh. & M.J. Wingf.] is one of the most significant pathogens on eucalyptus (Eucalyptus spp.) worldwide (Gryzenhout, 2013). Symptoms include diffuse, sunken or deforming cankers on roots, trunks and branches. As observed in C. parasitica, cankers may girdle stems and branches, resulting in dieback or tree death. LIFE CYCLE AND REPRODUCTION As a necrotrophic pathogen, C. parasitica requires fresh wounds or growth cracks in the bark to penetrate into the host tissue (Roane et al., 1986). Moribund woody tissue generated by drought, fire, natural competition or cutting of trees might also serve as an entry point for C. parasitica (Prospero et al., 2006). Recently, it has been shown that C. parasitica infects abandoned galls of the chestnut gall wasp (Dryocosmus kuriphilus), which apparently represent a new way for the pathogen to penetrate into host tissue (Meyer et al., 2015). Both sexual and asexual spores of C. parasitica can cause infections. After spore germination, an initial lesion is formed which subsequently develops into a bark canker. Later, C. parasitica may sporulate on the infected bark, as well as on the bark of recently dead chestnut wood (Prospero et al., 2006). Sexual (perithecia) and asexual (pycnidia) fruiting bodies develop in masses of yellow orange to red brown pustules named stromata (Fig. 2). These are mm in diameter and up to 2.5 mm in height and, except for the upper part, are embedded in the bark. Both types of fruiting body can co-exist in close proximity on the same bark portion (Prospero et al., 2006). Pycnidia ( mm in diameter) are formed as irregular cavities within the stromatic tissue. The sticky conidia ( mm) range from ellipsoidal to bacilliform in shape, are occasionally slightly curved and are aseptate and hyaline [European and Mediterranean Plant Protection Organization (EPPO), 2005]. They are released from the pycnidia in long (up to more than 1 cm), twisted, yellow tendrils during moist conditions (Fig. 2c). The production of tendrils can be induced artificially by incubating infected bark in moist chambers (EPPO, 2005). Conidia are mainly splash dispersed by rain over short distances (a few metres), or washed down the stem and branches (Griffin, 1986). However, birds, insects, mites or windborne dust may also transport them over long distances (Heald and Studhalter, 1914; Russin et al., 1984; Wendt et al., 1983). If conidia reach the ground, they may remain viable in the soil for a long time (Heald and Gardner, 1914). VC 2017 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20

4 10 D. RIGLING AND S. PROSPERO Fig. 2 Sporulation of Cryphonectria parasitica. (a) On the infected bark, the fungus produces masses of yellow orange to reddish brown pustules (stromata) harbouring sexual or asexual fruiting bodies. (b) Sexual fruiting bodies (perithecia). (c) Asexual fruiting bodies (pycnidia). The asexual spores (conidia) are extruded from the pycnidia as spore tendrils. The flask-shaped perithecia ( mm) are globose and deeply immersed in the stromatic tissue (Fig. 2b). They are characterized by a long cylindrical neck (up to mm in length and 200 mm in diameter) that ends in an ostiole. The relatively short-lived ascospores ( mm) are ellipsoidal, twocelled, smooth and arranged in the asci in two rows of four (EPPO, 2005). They are actively ejected into the air through a small opening in the ostiole and can be wind dispersed over long distances (up to a few hundred metres). In France, the peak of ascospore discharge is in May, but ascospores are continuously trapped from spring to autumn (Guerin et al., 2001). In North America, ascospores are also discharged during warm rains from spring to autumn, but the peak is in late summer/autumn (Heald and Gardner, 1913). In C. parasitica, mating is controlled by a single mating type (MAT) locus, which contains either the MAT-1 or MAT-2 allele (Marra and Milgroom, 2001). Investigations in natural populations have shown that C. parasitica has a mixed mating system, with outcrossing and self-fertilization occurring at variable frequencies (Marra et al., 2004). Most self-fertile isolates are heterokaryotic for mating type (McGuire et al., 2004). Such heterokaryons form through biparental inbreeding or parasexual recombination involving mitotic crossing over (Milgroom et al., 2009). EPIDEMIOLOGY New populations of C. parasitica are frequently established by one or a few genotypes (e.g. Hoegger et al., 2000; Milgroom et al., 2008; Prospero and Rigling, 2012). These genotypes may reach the new hosts via accidental introduction with infected plant material or by natural spore dispersal. At the beginning of new epidemics, mating opportunities for C. parasitica are generally reduced because of the presence of a single or predominant mating type. Consequently, the pathogen initially reproduces mainly asexually and spreads by conidia. Sexual reproduction may occur when both mating types are present from the beginning, through selfing in self-fertile isolates or after successive introduction/arrival of the opposite mating type (Bragança et al., 2007). Ten to 25 years after the establishment of a new C. parasitica population, natural hypovirulence (see below) has been known to appear and reduce the severity of chestnut blight in Europe (Heiniger and Rigling, 1994). An environmental factor that seems to strongly influence canker development and, potentially, the spread of C. parasitica is air temperature. Anagnostakis and Aylor (1984) showed that, in North America, at an average daily temperature of 20 8C, cankers expand by 1 mm per day. In a growth chamber experiment, canker expansion on European chestnut was fastest at 27 8C and considerably slowed below 20 8C (Bazzigher, 1981). The availability of water from humid air, rain or dew is also important for the survival, development and spread of C. parasitica. For example, spore release is induced by moist conditions (Griffin, 1986). Water stress in bark has been shown to promote canker expansion, but not necessarily canker initiation, on stems of American chestnut (Gao and Shain, 1995). Summer drought has been found to increase the incidence of chestnut blight on Japanese chestnut in Japan (Roane et al., 1986) and the decline and mortality of infected European chestnut trees in northern Italy (Waldboth and Oberhuber, 2009). According to Guerin and Robin (2003), the susceptibility of European chestnut to C. parasitica shows a seasonal pattern, with a maximum in spring and summer and a minimum in autumn and winter. The authors proposed that this variation in susceptibility may be related besides temperature to the nutritional value and water content of the bark. The age of a wound in the bark appears to strongly influence the probability of C. parasitica becoming established. Bazzigher and Schmid (1962) found that, on European chestnut, the largest lesions developed after inoculation of 1-day-old wounds, whereas, in 4-day-old wounds, C. parasitica could no longer become established. INVASION HISTORY Cryphonectria parasitica is native to Eastern Asia, where it has been reported in China, Japan and Korea (Lee et al., 2005; MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 VC 2017 BSPP AND JOHN WILEY & SONS LTD

5 Cryphonectria parasitica 11 Myburg et al., 2004). During the 20th century, the pathogen was accidentally introduced into North America and Europe through infected chestnut plants (Griffin, 1986). Following its first discovery in 1904 in New York City, C. parasitica spread at a rate of more than 30 km per year throughout the native distribution range of the American chestnut (about 3.6 million hectares) in eastern North America (Anagnostakis, 1987; Evans and Finkral, 2010; Roane et al., 1986). Within 50 years, the formerly canopyforming American chestnut was confined to the understorey, with significant ecological and economic consequences (Elliott and Swank, 2008). Historical records, as well as population genetic analyses, have shown that C. parasitica was initially introduced into North America mainly from Japan, in particular from the main island of Honshu (Dutech et al., 2012). Successively, additional introductions from other Japanese regions, China or Korea also occurred (Dutech et al., 2012). In Europe, C. parasitica was officially first detected in 1938 in Italy near Genoa, a main international port. Genetic analyses suggest that North America was the most likely source for this initial introduction into Italy (Dutech et al., 2012). By 1950, chestnut blight was already widespread in the main Italian chestnut-growing regions. From Italy, the disease rapidly spread into the neighbouring regions of France, Switzerland and Slovenia (Heiniger and Rigling, 1994; Krstin et al., 2008; Prospero and Rigling, 2012), and beyond to the chestnut-growing regions in eastern and south-eastern Europe and Turkey (Robin and Heiniger, 2001). Many of these regions were invaded by only one predominant genotype, possibly originating from the founding population in Italy (Milgroom et al., 2008). In south-western Europe (Atlantic and south-western France, Spain and Portugal), divergent genetic lineages of C. parasitica have been observed, suggesting additional introductions into Europe. Dutech et al. (2010, 2012) revealed at least two independent introductions directly from Asia into south-western France. The C. parasitica populations in Spain and Portugal were probably not only founded by genotypes originating from the French, but also from the Italian introduction (Bragança et al., 2007; Montenegro et al., 2008; Robin et al., 2000; Zamora et al., 2012). Multiple imports of chestnut trees into Europe from Asia and the USA between the 1920s and 1950s were most probably responsible for the introduction of C. parasitica (Dutech et al., 2012; Heiniger and Rigling, 1994). Ironically, many of these imports aimed to obtain chestnut trees resistant to ink disease (caused by Phythophthora cinnamomi and P. cambivora), which was the most significant chestnut disease before the arrival of chestnut blight. To date, chestnut blight is present in all main chestnut-growing areas in continental Europe. In 2011, an outbreak of the disease was reported in southern England on planted chestnut trees imported from France (Hunter et al., 2013). The chestnut stands in the most eastern distribution range of European chestnut (Georgia, Azerbaijan and Iran) are also affected by C. parasitica (Aghayeva and Harrington, 2007; Kazempour et al., 2006; Prospero et al., 2013). Based on microsatellite analyses, the Georgian population was not established by genotypes coming from Europe and is probably the result of an independent introduction (Prospero et al., 2013). Historical records and the level of genetic diversity found in Georgian populations suggest that C. parasitica has an old invasion history in this region and was probably introduced already in the 1930s. INFECTION PROCESS AND VIRULENCE FACTORS The infection process during canker development has been studied in detail using histological methods (Hebard et al., 1984). After spore germination, the rate and extent of mycelial fan formation by C. parasitica appears to be a key process in canker enlargement. By building up physical pressure, the mycelial fans split the host cells and advance intercellularly in the bark and cambium of susceptible chestnut species. The host tree reacts against the infection by lignification of cell walls and subsequent wound periderm formation. Mycelial fans, however, are able to penetrate through zones of lignified host cells and developing wound periderm. Only fully developed wound periderm prevents further penetration of mycelial fans. Wound periderm formation is continuously inhibited in susceptible chestnut species because the advancing mycelial fans kill the host cells by means of toxins and cell wall-degrading enzymes (Roane et al., 1986). Oxalic acid is one metabolite which is probably involved in this process. It is secreted by C. parasitica at the advancing edge of the infection, and is assumed to have a toxic effect on host cells and to enhance cell wall degradation (Havir and Anagnostakis, 1983). Knock-out mutation of the gene that encodes for the oxalic acid-producing enzyme (oxalacetate acetylhydolase) confirms the important role of oxalic acid in pathogenesis (Chen et al., 2010). A number of other virulence factors have been identified in C. parasitica using specific gene mutations. These factors include G-protein signalling (Gao and Nuss, 1996), a kexin-like protease involved in protein secretion (Jacob-Wilk et al., 2009), an Ste12 transcription factor homologue (Deng et al., 2007), a tannic acid-inducible laccase (Chung et al., 2008), a cyclophilin (Chen et al., 2011), protein kinase 2 (CK2)-mediated signalling (Salamon et al., 2010) and an inhibitor (CpBir1) of apoptosis proteins (Gao et al., 2013). The availability of the genome sequence of C. parasitica ( genome.jgi.doe.gov/crypa2/crypa2.home.html) will probably benefit research on the mechanisms underlying its pathogenesis. VEGETATIVE INCOMPATIBILITY Vegetative incompatibility is common in fungi and is assumed to function, amongst others, as a defence mechanism against cytoplasmically transmitted diseases by preventing the formation of stable anastomosis (hyphal fusion) and cytoplasmic exchange between individuals (Caten, 1972). The vegetative incompatibility system in C. parasitica has attracted interest because it restricts VC 2017 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20

6 12 D. RIGLING AND S. PROSPERO Fig. 3 Vegetative incompatibility and hypovirus transmission in Cryphonectria parasitica. (a) Vegetative compatibility tests. For vc type determination, the unknown C. parasitica strain is paired with tester strains of known vc types. Compatible strains merge (M) into a single culture, whereas a barrage zone (B) is formed between incompatible strains. (b) Hypovirus transmission between strains of C. parasitica. Pairs of hypovirus-infected (white) and hypovirus-free (orange) strains are cocultured on potato dextrose agar. A colour change from orange to white indicates successful hypovirus transmission (left plate). Note that vegetative incompatibility prevents hypovirus transmission in the right plate, but not in the left. the horizontal transmission of virulence-attenuating mycoviruses between fungal individuals (Anagnostakis, 1977). To date, six unlinked vegetative incompatibility (vic) loci, each with two alleles, have been identified (Cortesi and Milgroom, 1998). These six di-allelic vic loci define vic genotypes, which correspond to 64 different vc types. Cryphonectria parasitica isolates belong to the same vc type, i.e. are vegetatively compatible, if they have the same alleles at all vic loci. When co-culturing on agar plates, compatible isolates merge into a single culture, whereas a barrage line is formed along the contact zone between two incompatible isolates (Fig. 3a). This barrage line is the result of a programmed cell death (apoptosis), which is triggered when incompatible cells anastomose (Biella et al., 2002). The six genetically defined vic loci have also recently been characterized at the molecular level (Choi et al., 2012, Zhang et al., 2014). These studies revealed complex allelic and non-allelic interactions at these vic loci involving polymorphic and idiomorphic genes. Based on the molecular characterization of the six vic loci, a polymerase chain reaction (PCR)-based method has been developed to identify all 64 currently known vic genotypes (Short et al., 2015). Surveys of vc types have been conducted in many countries worldwide to assess the genetic structure of C. parasitica populations. The availability of a defined collection of 64 European vc type tester strains (EU-1 to EU-64) has largely facilitated comparisons amongst different studies (Milgroom and Cortesi, 1999). The highest vc type diversity was detected, as expected, in native populations of C. parasitica in Asia, with most of the isolates being incompatible with all the EU tester strains (Liu and Milgroom, 2007). In the introduced range of the pathogen, vc type diversity in local populations is generally lower in Europe than in the USA (Milgroom and Cortesi, 2004). In both continents, newly founded populations or populations at the margin of the distribution range of C. parasitica typically show a lower vc type diversity than longestablished, central populations (Bryner and Rigling, 2012a; Dutech et al., 2012; Hoegger et al., 2000; Milgroom et al., 2008; Springer et al., 2013). Founder events having introduced only a few polymorphic vic loci and restricted immigration of additional vic genotypes are probably the main reasons for this low vc type diversity in newly founded C. parasitica populations. In addition, limited sexual recombination, as observed in many European C. parasitica populations, contributes to the low diversity of vc types (Milgroom and Cortesi, 1999). The vc types that are incompatible with all 64 EU vc type tester strains have not only been found in Asia, but also in the USA (Milgroom and Cortesi, 1999) and in Europe (Bragança et al., 2007; Montenegro et al., 2008; Robin et al., 2009), including Caucasian Georgia (Prospero et al., 2013). In addition, vegetatively incompatible isolates have been found that have the same allele pattern at all six known vic loci (Short et al., 2015). These findings indicate either that vegetative incompatibility in C. parasitica is controlled by more than six vic loci or that additional alleles exist at the known vic loci. DISEASE MANAGEMENT To prevent the introduction and spread of C. parasitica, quarantine regulations have been adopted worldwide, affecting the movement and trade of chestnut (and sometimes oak) wood and bark, seeds and living plants. In Europe, EPPO still recommends the regulation of C. parasitica as an A2 quarantine organism, i.e. a pathogen/pest locally present in the EPPO region (EPPO, 2005). Chestnut and oak plants for planting can only be moved within Europe if they are accompanied by a plant passport which certifies that: (i) the plants originated from areas free from C. parasitica; or (ii) no observation of C. parasitica has been made at the place of production or its immediate vicinity since the beginning of the last complete cycle of vegetation. Quarantine regulations, however, have proven to be ineffective in completely stopping the spread of the pathogen. In this regard, a major problem is caused by asymptomatic infected plants, which cannot be detected through visual inspections. MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 VC 2017 BSPP AND JOHN WILEY & SONS LTD

7 Cryphonectria parasitica 13 After the introduction of C. parasitica into a new area, eradication efforts, specifically cutting and burning of the infected plants/ trees, have frequently been undertaken. Such efforts have, however, mainly failed, especially in forests, because of the difficulty in finding and eliminating all inoculum sources. Eradication efforts may be successful in chestnut orchards, in particular, if they are well separated from infected chestnut forests (Prospero and Rigling, 2013). At the level of single trees, infections by C. parasitica may be prevented by avoiding wounds in the bark. When this is not possible (e.g. pruning or grafting), wounding should be avoided during the periods of spore production (in the Northern Hemisphere between March and October), disinfected tools should be used and less invasive grafting techniques should be chosen. Therapeutic treatment with chemicals does not seem to be a practicable option for the control of chestnut blight. First, in most countries, the use of chemicals in forests is restricted or prohibited. Second, fungicides may be phytotoxic or induce the development of resistance. Trapiello et al. (2015) have shown that the application of triazole fungicides (specifically epoxiconazole) may be helpful in managed conditions, as in nurseries or for single chestnut trees. BIOLOGICAL CONTROL USING HYPOVIRULENCE Cryphonectria hypoviruses Hypovirulence refers to a viral disease in C. parasitica caused by mycoviruses in the family Hypoviridae (Choi and Nuss, 1992). Hypoviruses are positive-strand RNA viruses, located in the cytoplasm of the fungal host. They do not have a coat protein and are associated with host-derived membrane vesicles (Hansen et al., 1985, Fahima et al., 1993). Hypoviruses were tradionally detected in the fungal mycelia by extracting double-stranded RNA, which represents the replicative form of the virus (Day et al., 1977). The genus Hypovirus currently comprises four well-characterized species, namely Cryphonectria hypovirus 1 (CHV-1), CHV-2, CHV-3 and CHV-4 (Hillman and Suzuki, 2004; Turina and Rostagno, 2007). The best-studied hypovirus is CHV-1, which acts as a biological control agent of chestnut blight in Europe. CHV-1 induces a hypovirulent phenotype in C. parasitica by reducing the parasitic growth and sporulation capacity of the pathogen. The phenomenon of hypovirulence was first observed in Italy in the 1950s in heavily infected chestnut stands that showed signs of recovery from the disease (Heiniger and Rigling, 1994). The biocontrol potential of hypovirulence was subsequently discovered by Grente (1965) and led to many further studies on the biology and application of hypovirulence (reviewed by Anagnostakis, 1987; Dawe and Nuss, 2001; Elliston, 1982; Fulbright et al., 1983; Grente, 1965; Griffin, 1986; MacDonald and Fulbright, 1991; McCabe et al., 1999; Nuss, 1992; Van Alfen, 1982). The success of hypovirulence is determined by the ability of CHV-1 to infect a large proportion of a C. parasitica population. This kind of invasion depends on the combination of horizontal transmission (to other fungal individuals) and vertical transmission (to spores) of the hypovirus. From a hypovirus-infected fungal individual, the hypovirus is only transmitted into asexual spores (conidia), but not into sexual ascospores (Prospero et al., 2006). The hypovirus is dispersed with the conidia and then transmitted from the outgrowing spores to other fungal individuals via hyphal anastomosis (Hoegger et al., 2003; Milgroom and Cortesi, 2004). Hypovirus-infected conidia are most probably disseminated by the same vectors as normal conidia. Mites that feed on fungal conidia or mycelium may be important vectors for the spread of hypovirulence (Bouneb et al., 2016; Nannelli and Turchetti, 1999; Simoni et al., 2014). Grente (1975) was the first to point out that hypovirus transmission is restricted between vegetatively incompatible strains of C. parasitica. The success or failure of hypovirus transmission can be easily assessed in vitro by co-culturing pairs of hypovirusinfected and hypovirus-free strains on potato dextrose agar (PDA) plates (Fig. 3b). The white culture morphology of hypovirusinfected strains compared with the orange morphology of hypovirus-free strains enables the visual assessment of hypovirus transmission. This simple plate assay has been widely used to investigate the influence of vegetative incompatibility on hypovirus transmission in C. parasitica. Pairing tests have demonstrated that hypovirus transmission readily occurs between vegetatively compatible strains, but is largely reduced if strains are heteroallelic at five of the six known vic loci (Cortesi et al., 2001; Papazova-Anakieva et al., 2008). The locus vic 4 has apparently no effect on hypovirus transmission. The restriction of hypovirus transmission is variable among the other vic loci and, in most cases, asymmetric depending on which vic allele is present in the hypovirus donor or recipient strain. The frequency at which the hypovirus is transmitted negatively correlates with the number of heterogenic vic loci between strains (Liu and Milgroom, 1996). Recently, gene disruption studies have formally confirmed the role of the vic loci in restricting hypovirus transmission (Choi et al., 2012; Zhang et al., 2014). Based on in vitro transmission frequencies between vc types, Cortesi et al. (2001) developed a regression model to predict the mean probability of horizontal hypovirus transmission in a C. parasitica population. Field surveys, however, have shown that hypovirus incidence is not clearly correlated with the expected rate of hypovirus transmission (Milgroom and Cortesi, 2004; Robin et al., 2010). Brusini and Robin (2013) concluded that vegetative incompatibility barriers may be less restrictive in the field than estimated from in vitro studies. They found that horizontal transmission of CHV-1 between different vc types is more frequent on chestnut stems than on agar medium. Hypovirus transmission between VC 2017 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20

8 14 D. RIGLING AND S. PROSPERO different vc types in the field has also been demonstrated using genealogy-based methods (Carbone et al., 2004). In Europe, CHV- 1 typically occurs in common and rare vc types (Bissegger et al., 1997; Bryner and Rigling, 2012a; Krstin et al., 2008, 2011), which is additional evidence that hypovirus transmission among vc types is not completely restricted. In Europe, hypovirulence is largely a natural phenomenon (Heiniger and Rigling, 1994; Milgroom and Cortesi, 2004). CHV-1 was probably introduced together with its fungal host and has spread widely throughout the European C. parasitica population (Bryner et al., 2012). In many regions, C. parasitica cankers display a high prevalence of hypovirus infection. In local chestnut stands, the proportion of hypovirus-infected cankers is often more than 50%. Examples of this have been found in Switzerland (Bissegger et al., 1997; Bryner and Rigling, 2012a), France (Robin et al., 2010), Croatia (Krstin et al., 2008), Slovenia (Krstin et al., 2011), Bosnia and Macedonia (Bryner and Rigling, 2012a). Several genetically distinct subtypes of CHV-1 have been identified in Europe (Allemann et al., 1999; Gobbin et al., 2003). The Italian subtype (subtype I) is most widespread and commonly associated with a high natural prevalence of hypovirulence. This subtype is dominant in Italy, Switzerland, south-eastern France, Bosnia-Herzegovina, Croatia, Slovenia, Macedonia, Greece and Turkey (Allemann et al., 1999; Akilli et al., 2013; Krstin et al., 2011; Sotirovski et al., 2006). Other CHV-1 subtypes (i.e. F1, F2, D, E) have been found in France (Feau et al., 2014), Spain (Zamora et al., 2012), Germany (Peters et al., 2014) and eastern Turkey (Akilli et al., 2013). The geographical distribution of the different CHV-1 subtypes in Europe coincides well with the distribution of different gene pools of C. parasitica (see section on Invasion history), which suggests multiple introductions of both the fungus and the hypovirus. Several recombination events appear to have contributed to the evolution of CHV-1 in Europe (Feau et al., 2014). It should be noted that there are still areas in which chestnut blight is present but CHV-1 has not been detected or its prevalence is very low. Such areas include Portugal (Bragança et al., 2007), Spain (Castano et al., 2015; Zamora et al., 2012), Bulgaria (Risteski et al., 2013), Romania (Adamcikova et al., 2015), northern Switzerland (Hoegger et al., 2000) and the Aegean region of Turkey (Erincik et al., 2011). CHV-1 has also been detected in C. parasitica in East Asia (Japan, China, Korea), from where it was probably introduced to Europe (Liu et al., 2007; Park et al., 2004; Peever et al., 1998). CHV-1 does not occur in North America, with the exception of a few locations in which it has been released for the biological control of chestnut blight (Milgroom and Cortesi, 2004). The other Cryphonectria hypoviruses, CHV-2, CHV-3 and CHV-4, are taxonomically related to CHV-1, but have different genome organizations and different effects on C. parasitica (reviewed by Hillman and Suzuki, 2004). Two hypoviruses, CHV-2 and CHV-3, also induce a hypovirulent phenotype in C. parasitica, whereas CHV-4 causes no significant symptoms in its fungal host. Cryphonectria parasitica is a natural host for other fungal viruses, some of which may also be associated with the debilitation and reduced virulence of the infected fungal strains (Hillman and Suzuki, 2004). These additional viruses that attenuate fungal virulence include two mycoreoviruses (family Reoviridae) (Suzuki et al., 2004) and a mitovirus (family Narnaviridae) (Polashock and Hillman, 1994). Application of artificial hypovirulence In areas with low or no natural hypovirulence, the hypovirus (typically CHV-1) can be artificially introduced into a chestnut stand by the treatment of bark cankers with a hypovirus-infected C. parasitica strain (Heiniger and Rigling, 2009; Prospero and Rigling, 2016; Robin et al., 2010). The introduced hypovirus is then horizontally transmitted via hyphal anastomosis to the fungal strain that causes the canker. The infection by the hypovirus weakens the fungus, and the treated canker eventually stops expanding and becomes passive. A canker treatment usually involves the following main steps (Heiniger and Rigling, 2009). First, the vc type(s) occurring in the infected chestnut stand must be determined. Ideally, the hypovirus-infected strain used for canker treatment should have the same vc type as the canker strain. However, if several vc types occur in the chestnut stand, a mixture of hypovirus-infected strains belonging to the different vc types can be used(robin et al., 2000). Second, in the laboratory, the hypovirus is transmitted into local virulent strains of the target vc type(s) using the in vitro virus transmission assay described above (Fig. 3b), and hypovirulent inoculum is produced. Third, holes are made with a cork borer at the margin of the canker to be treated and filled with the hypovirulent inoculum. After at least one growing season, the success of canker treatment may be evaluated by the assessment of canker morphology and by re-isolation of C. parasitica from the treated cankers to verify the presence of the hypovirus. Molecular markers can be used to track the persistence and spread of a hypovirus applied for biological control (Hoegger et al., 2003; Prospero and Rigling, 2016). The likelihood of hypovirus establishment in the treated cankers can be maximized by performing the treatments during the growing season (April to October in central Europe). European experience In Europe, therapeutic canker treatment is generally successful, i.e. the treated cankers usually stop expanding and the tree confines the infection (e.g. Heiniger and Rigling, 2009; Prospero and Rigling, 2016). Treatments are particularly effective in cankers on young chestnut trees with a smooth bark and easily identifiable cankers, as in coppice stands (Hoegger et al., 2003; Robin et al., 2000). Cankers on older trees, however, are more difficult to treat MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 VC 2017 BSPP AND JOHN WILEY & SONS LTD

9 Cryphonectria parasitica 15 because of the thick bark, their unclear margins and sometimes their location in the crown. Despite its high therapeutic efficacy, spontaneous spread of the artificially introduced hypovirus to untreated cankers is not always observed (Milgroom and Cortesi, 2004). Several main factors seem to affect hypovirus dissemination within the local C. parasitica population. The first is the intensity of treatment, which must be as high as possible (Diamandis et al., 2014; Heiniger and Rigling, 2009). Another important factor appears to be the hypovirus used as the biocontrol agent (Mac- Donald and Fulbright, 1991). Different subtypes of CHV-1 show varying degrees of reduction of fungal growth and sporulation (Bryner and Rigling, 2011; Robin et al., 2010). French subtypes (F1 and F2) have been proven to have a strong therapeutic effect, as they significantly suppress both the growth and sporulation of the affected C. parasitica strains (Chen and Nuss, 1999). In contrast, the Italian subtype (I) is better at disseminating and becoming established in a treated chestnut stand because of its milder effect on C. parasitica (Robin et al., 2010). These field observations have been confirmed by a model developed by Morozov et al. (2007), which shows that mild strains of the hypovirus (i.e. strains with low virulence and high transmission rates) have a better chance to become established than virulent strains. The diversity of vc types may also affect the spontaneous dissemination of the artificially introduced hypovirus. Liu et al. (2000) used a stochastic model to calculate a theoretical threshold of vc type diversity for successful hypovirus spread in C. parasitica populations. Remarkably, in European populations, vc type diversity is below this threshold. Finally, the degree of susceptibility of chestnut trees to C. parasitica may also affect the success of hypovirulence. Although European chestnut is considered to be susceptible, canker expansion is sufficiently slow to allow time for a successful hypovirus infection (Griffin, 1986). North American experience Biocontrol of chestnut blight using artificially applied hypovirulence has failed almost completely in eastern North America. High vc type diversity is considered to be one of the main reasons for this failure (Milgroom and Cortesi, 2004). Remarkably, vc type diversity in North American C. parasitica populations is above the theoretical threshold determined by Liu et al. (2000) for successful spread of the hypovirus. The chances of a successful biocontrol of chestnut blight are further limited by the high blight susceptibility of the American chestnut, with trees being killed before the released hypovirus can actually infect the cankers. Naturally occurring hypovirulence in North America is only observed in some chestnut stands in Michigan and Ontario, outside the natural distribution range of the American chestnut (Milgroom and Cortesi, 2004). In Michigan, C. parasitica strains infected by the hypovirus CHV-3 have been isolated from recovering cankers since the early 1980s (Fulbright et al., 1983). By comparing different C. parasitica populations, Springer et al. (2013) showed that vc type diversity in hypovirus-free populations is rather higher than that in hypovirus-infected populations. Moreover, in chestnut stands invaded by the hypovirus, the authors detected a decrease in vc type diversity over time, which they explained by a hypovirusinduced inhibition of sexual reproduction. To improve the establishment and spread of the hypovirus, molecular approaches have been used by Donald Nuss and associates (Dawe and Nuss, 2001). Transgenic C. parasitica strains carrying an infectious cdna copy of CHV-1 integrated into the fungal genome have provided enhanced dissemination potential for the hypovirus (Chen et al., 1993). These strains transmit the hypovirus via the nuclear copy of the viral cdna into sexual spores, which do not carry the hypovirus naturally. As a result, the hypovirus cannot only be disseminated by sexual spores, but can potentially also infect all vc types produced in a sexual cross (Anagnostakis et al., 1998). In addition, every conidium produced by a transgenic strain is hypovirus infected, whereas not all conidia from a naturally hypovirulent strain contain the hypovirus. Field performance studies, however, have failed, so far, to demonstrate the successful establishment of transgenic hypovirulence (Root et al., 2005). This result is in accordance with model predictions suggesting that hypovirus dissemination is only slightly enhanced when applying such transgenic strains (Liu et al., 2000). Most recently, super hypovirus donor strains of C. parasitica have been engineered by disrupting four of the five vegetative incompatibility genes (Zhang and Nuss, 2016). These mutants are capable of transmitting the hypovirus to recipient strains that are heteroallelic at three or more vic loci. Potentially, these strains can be used as carriers to deliver hypoviruses into C. parasitica populations with high vc type diversity, as found in North America. Hypovirus fungus interactions The interaction between C. parasitica and CHV-1 has been studied intensively at the molecular level (reviewed in Dawe and Nuss, 2001; McCabe et al., 1999; Nuss, 2005; Turina and Rostagno, 2007). In addition to reduced virulence, CHV-1-infected strains also display other phenotypic symptoms, including reduced pigmentation and reduced sporulation. It has been demonstrated that CHV-1 induces these phenotypic changes by interfering with fungal signal transduction pathways (Choi et al., 1995; Larson et al., 1992; Park et al., 2004). The hypovirus effect on cellular signalling can be seen when C. parasitica is grown on PDA and exposed to light. As a response to the light signal, hypovirus-free cultures typically produce an orange pigmentation and abundant asexual sporulation, whereas hypovirus-infected cultures largely remain white with no or only little sporulation (Fig. 3b). Nuss (2005) pointed out that signal transduction processes in C. parasitica are also important for the infection process (i.e. fungus plant interaction), and that reduced virulence of hypovirus- VC 2017 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20

10 16 D. RIGLING AND S. PROSPERO infected strains is probably the result of the perturbation of these processes by the hypovirus. Studies on the fungus hypovirus interaction at the cellular level have also discovered RNA silencing to be a novel defence mechanism of fungi against virus infections (Segers et al., 2006, 2007). Thanks to its genetic manipulability and its hosting of viruses from at least six established families, C. parasitica is becoming a model host for the study of virus host and virus virus interactions (Eusebio-Cope et al., 2015). In the future, this system should contribute further to research progress in fungal virology (e.g. virus replication, symptom expression and antiviral defence). Evolution of hypovirulence Hypovirulence is still a recent phenomenon in the introduced range of C. parasitica and the evolutionary dynamics in this tritrophic pathosystem (tree fungus hypovirus) are difficult to predict. The evolution of the hypovirus fungus interaction is crucial for the sustainability of the biological control system, which, in Europe, is largely a natural phenomenon. What makes the hypovirus CHV-1 successful as a biological control agent is the combination of hypovirus virulence and fitness. Virulence, defined as the ability of CHV-1 to debilitate the fungal host and induce hypovirulence, is the primary requirement for disease control. At the same time, CHV-1 fully relies on its host for its spread and establishment in a host population. Thus, a host debilitation that is too severe would largely reduce the ecological fitness of the hypovirus. Considerable variation in hypovirus virulence has been reported for CHV-1 in several studies, even within local populations (Bryner and Rigling, 2012a; Peever et al., 2000). Genomic comparisons of low- and high-virulent CHV-1 strains and mutational analysis have revealed specific regions in the viral genome that are associated with these variations (Chen and Nuss, 1999; Lin et al., 2007). The observed variation in hypovirus virulence is probably linked to the mutation rate of the viral RNA genome, and indicates the potential for hypovirus evolution. The mutation rate of CHV-1 has been estimated to average out at nucleotide substitutions per site and year, which is much higher than the mutation rate (approximately ) reported for ascomycetes (Bryner et al., 2012). Thus, although little is known about the evolutionary trajectory of the fungus hypovirus interaction, CHV-1 is expected to evolve faster than its fungal host. In an experimental study, Bryner and Rigling (2011) suggested that the level of hypovirulence is not only determined by hypovirus host interactions, but potentially also by environmental factors, such as temperature. As a consequence of such genotype-by-genotype-byenvironment interactions, different host and hypovirus genotypes may be selected under different environmental conditions. It has been hypothesized that fungal viruses will evolve towards lower virulence when vegetative incompatibility barriers increasingly restrict horizontal virus transmission (Brusini et al., 2011; Milgroom, 1995). This hypothesis was tested by comparing the virulence of CHV-1 in C. parasitica populations with low and high vc type diversity (Bryner and Rigling, 2012a). However, no such trend towards lower hypovirus virulence in populations with higher vc type diversity was observed. In addition, a further study revealed a positive association between hypovirus virulence and hypovirus transmission across vegetative incompatibility barriers (Bryner and Rigling, 2012b). The reason for this association is not known. Virulent hypoviruses possibly interfere with the vegetative incompatibly reaction, which normally is induced on hyphal fusion of incompatible fungal strains. Nevertheless, the higher transmission potential of virulent hypoviruses may compensate for the otherwise debilitating effect they have on their fungal host, and may explain why virulent hypoviruses persist in diverse C. parasitica populations. Ecological factors might also support the persistence of virulent hypoviruses in C. parasitica populations. An important characteristic of CHV-1 is that it mainly affects the parasitic, but not saprophytic, growth of C. parasitica. This can be observed on agar medium on which the growth of C. parasitica cultures is not or only slightly affected by hypovirus infections (e.g. Rigling et al., 1989; Robin et al., 2010). In the field, hypovirus-infected strains can survive and reproduce (asexually) on the bark of dead chestnut wood, which thereby can serve as a reservoir for hypovirus inoculum (Prospero et al., 2006). In contrast, sporulation on hypovirus-infected cankers on living trees is typically absent or rare. The fact that the saprophytic and parasitic potential of hypovirus-infected C. parasitica are not strongly linked most probably contributes to the observed variation in hypovirus virulence. RESISTANCE BREEDING Breeding for resistance against chestnut blight is mainly based on the genetic resistance found in Asian chestnut species (Anagnostakis, 2012). Of the two main chestnut species present in Asia, Chinese chestnut is considered to be more blight resistant than Japanese chestnut, but a considerable variation in resistance has been reported for both species (Graves, 1950; Huang et al., 1996). Three putative blight-resistant loci have been identified in the genome of Chinese chestnut (Kubisiak et al., 1997). Two of these loci share synteny with two quantitative trait loci (QTL) which are associated with powdery mildew resistance in peach (Kubisiak et al., 2013). By using comparative transcriptome analysis of American and Chinese chestnut, several candidate genes that are possibly involved in the defence response against chestnut blight have been identified (Barakat et al., 2009, 2012). A major ongoing breeding programme was initiated in the 1980s by the American chestnut foundation with the goal to restore American chestnut as an important forest tree species (Burnham, 1981; Hebard, 2005). The approach of this programme is to backcross the blight resistance of Chinese chestnut into the American chestnut genome. Three successive generations of backcrosses have resulted in MOLECULAR PLANT PATHOLOGY (2018) 19(1), 7 20 VC 2017 BSPP AND JOHN WILEY & SONS LTD