Informative value of canker morphology on the presence or absence of virus infection in chestnut blight cankers By S. F. Bryner 1 , K. Sotirovski 2 , S. Akilli 3 , M. Risteski 2 , C. Perlerou 4 and D. Rigling 1,5 1 WSL Swiss Federal Research Institute, CH-8903, Birmensdorf, Switzerland; 2 Faculty of Forestry, University Sv.Kiril i Metodij, Skopje, Republic of Macedonia; 3 Science Faculty, C ßankırı Karatekin University, C ßankırı, Turkey; 4 Forest Research Institute, Vassilika, Thessaloniki, Greece; 5 E-mail: daniel.rigling@wsl.ch (for correspondence) Summary Chestnut blight destroyed the native chestnut forests in North America and also severely affected the European chestnut trees after its introduction in the 20th century. The ascomycete fungus Cryphonectria parasitica is responsible for this serious disease and causes lethal bark cankers on susceptible chestnut trees. In Europe, however, an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV-1) causes hypovirulence in C. parasitica and reduces the severity of the disease. Hypovirulence biologically controls chestnut blight in many regions to date. In this study, our goal was to determine morphological canker characteristics that are indicative of virus presence or absence in C. parasitica. We investigated 677 chestnut blight cankers from seven different geographic locations across Europe. For each canker, we assessed canker length, stem encircling, canker depth, presence of sporulation, canker activity and virus infection. We statistically analysed the informative value of these morphological characteristics for the presence or absence of CHV-1. However, we did not find reliable indica- tors. Our logistic regression analysis revealed that virus infection of C. parasitica is not clearly related to canker morphology. This implies that fungal isolations from chestnut blight cankers and assessments in the laboratory are required to determine infection with CHV-1 unequivocally. 1 Introduction Cryphonectria parasitica is a tree pathogen, which colonizes the bark and cambium of chestnut trees (Castanea spp.) through wounds. The fungus destroys the function of these tissues in susceptible chestnut species and causes lethal bark cankers (Heiniger and Rigling 1994; Milgroom and Cortesi 2004). Such cankers are sunken in appearance, typically expand and girdle the stem and eventually cause death of all tree parts distal to the canker. Cryphonectria parasitica is native to East Asia where chestnut blight is only a minor problem because the Asian chestnuts (primarily Castanea mollissima and Castanea crenata) are resistant to the disease (Milgroom and Cortesi 2004). However, C. parasitica was accidentally introduced to North America around 1904 and to Europe around 1938, where the local chest- nut species had not coevolved with C. parasitica (Anagnostakis 1982). In a devastating epidemic, chestnut blight destroyed the North American chestnut forests and nearly eradicated the American chestnut (Castanea dentata). It became a classical example of an introduced pathogen that had encountered a highly susceptible host population (Anagnostakis 1987). In Europe, initially, a similar story unfolded on European chestnut (Castanea sativa). Cryphonectria parasitica was first recorded in 1938 near Genoa (Italy) from where it spread rapidly throughout the chestnut growing regions, evoking a dra- matic epidemic. However, a few years later, the appearance of superficial cankers was observed, which did not lead to die- back of the infected trees (Heiniger and Rigling 1994). It was discovered that healing of these cankers was due to an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV-1), an unencapsidated RNA virus of the genus Hypovirus (Choi and Nuss 1992). Virus-infected strains of C. parasitica lead to superficial, swollen cankers and do not kill the cells of the cambium. Such cankers are colloquially known as ‘healing or healed cankers’ because they do little or no harm to the tree (Heiniger and Rigling 1994; Milgroom and Cortesi 2004). The formation of such superficial, healing cankers has been attributed to the reaction of the tree to the fungal infection combined with a slow growth of the fungus. It was observed that the chestnut bark reacts to C. parasitica invasion by lignifications and formation of wound periderm (Bramble 1936; Hebard et al. 1984). These tree reactions aim at restricting the growth of the fungus to the outer layers of the bark. How- ever, virulent C. parasitica strains grow fast and may be able to infect the healthy, underlying bark before the protective barrier is fully formed (Bramble 1936). Infection of C. parasitica with CHV-1 does not kill C. parasitica but strongly reduces the parasitic growth of C. parasitica and attenuates asexual sporulation and pigmentation and inhibits sexual reproduction (Milgroom and Cortesi 2004). The pathogenic potential of these so-called hypovirulent strains is greatly reduced (Van Alfen et al. 1975). Virus infection associated with hypovirulence therefore seems to favour the successful formation of wound periderm, which restricts the further advance of the fungus and leads to healed, superficial cankers (Hebard et al. 1984). Cryphonectria hypovirus 1 spread independently throughout the C. parasitica populations in Europe by transmission from one fungal individual to another. Natural dissemination of CHV-1 has been aided by active biocontrol efforts, leading to a high prevalence of virus infection in Europe (Heiniger and Rigling 1994). To date, chestnut blight incidence in Europe is very high, but the disease is maintained at low severity in many regions due to hypovirulence. Four genetically distin- guished subtypes of CHV-1 have been described in Europe: subtype I, F1, F2 and D/E (Allemann et al. 1999; Gobbin et al. Received: 7.3.2013; accepted: 21.5.2013; editor: V. Andrea For. Path. 43 (2013) 496–504 doi: 10.1111/efp.12063 © 2013 Blackwell Verlag GmbH http://wileyonlinelibrary.com/