Testing the pathogenic potential of Cryphonectria parasitica and related species on three common European Fagaceae

Invasions by non-native pathogens represent a major threat to managed and natural ecosystems worldwide. Although necessary for adopting preventive strategies, the identification of invasive species before they are introduced is particularly difficult. Indeed, most pathogenic species that have become established in the last decades were first described only after they became invasive. To prevent further biological invasions, not only the early identification of potential new invasive plant pathogens is crucial, but also the assessment of their potential host range. In this study, we determined the pathogenicity and the saprotrophic ability of three Cryphonectria species towards three potential hosts in the family Fagaceae. For this, seedlings and dormant stems of European chestnut (Castanea sativa), pedunculate oak (Quercus robur) and European beech (Fagus sylvatica) were inoculated with different genotypes of C. parasitica (Asian species, invasive in Europe), C. naterciae (European species), and C. japonica (Asian species, not present in Europe). Lesion growth was measured and mortality assessed for four months. The highest damage was caused by C. parasitica on European chestnut, while C. japonica and C. naterciae induced significantly smaller lesions on this host species. All three Cryphonectria species did not grow saprophytically on F. sylvatica and Q. robur, but successfully colonized dormant stems of C. sativa. In the context of biological invasions, our study shows that the Asian C. japonica most likely represents a much less severe threat than C. parasitica for the tested European host species. Nonetheless, the ability of C. naterciae and C. japonica to saprotrophically colonize fresh chestnut wood may suggest that they could become established in chestnut forests and eventually infect weakened chestnut trees or other hosts not tested in this study.

but also the assessment of its potential host range. 53 Cryphonectria parasitica, the causal agent of chestnut blight, is one of the most damaging invasive 54 pathogens in forest ecosystems. Native to Asia (China, Japan, Korea), it was introduced to North 55 America at the beginning of the 20 th century and then from there to Europe in the 1930's (Rigling and 56 Prospero, 2018). Additional introductions to Europe as well as to the Caucasus region took place 57 later directly from Asia (Dutech et al., 2012; Prospero and Cleary, 2017). The pathogen spread 58 rapidly in the introduced ranges where it encountered susceptible Castanea species (Anagnostakis, 59 1992) on which it causes lethal bark lesions (so-called cankers). In North America, American 60 chestnut (C. dentata) has virtually become extinct because of chestnut blight (Anagnostakis, 1987). 61 By contrast, in Europe the C. parasitica epidemic on European chestnut (Castanea sativa) showed a milder course due to the appearance and spread in the C. parasitica population of a hyperparasitic 63 mycovirus (CHV-1) that acts as biological control agent, and to higher resistance of C. castanea 64 compared to C. dentata (Rigling and Prospero, 2018). The pathogen can also survive and readily 65 sporulate on fresh dead wood, which is considered to have an important epidemiological role 66 (Prospero et   The study was conducted using three isolates each of C. parasitica, C. naterciae and C. japonica 90 ( Table 1). Isolates of C. parasitica were selected to represent two frequent vegetative compatibility 91 (vc) types (EU-1 and EU-2) and one rare vc type (EU-12) in Switzerland (Prospero and Rigling, 92 2012). Genotyping at 10 microsatellite loci (Prospero and Rigling, 2012) showed that each of them 93 belonged to a different multilocus genotype (M2372 to CpMG15, M2671 to CpMG37 and M4023 to CpMG21). Isolates of C. naterciae and C. japonica originated from Portugal and Japan, respectively, 95 and showed no amplification with the C. parasitica specific microsatellite assay. Cryphonectria species grown on the PDA plates was assessed visually from the culture morphology 119 using reference cultures for each species. 120

Dormant stem experiment 121
Dormant stems of C. sativa, Q. robur and F. sylvatica (3-5 cm diameter) were obtained from Swiss 122 forests. The stems were cut in 50 cm long segments and ends were sealed with paraffin to avoid 123 desiccation. The Cryphonectria spp. isolates were cultured and inoculated on the dormant stems with 124 the same method as described for the seedling experiment. To avoid a stem effect on the experiment, on each stem, one randomly selected isolate of each Cryphonectria species was inoculated. Five 126 replicate inoculations were performed for each isolate on each tree species. The stems were placed in 127 plastic boxes on plastic supports and the boxes filled with 4L demineralized water to avoid 128 desiccation of the stems (Bryner et al., 2012). The boxes were placed at 25°C in the dark for two 129 weeks. Afterwards, the bark around the inoculation point was removed using a scalpel and the length 130 and width of the lesions that had formed was measured. Since the lesions were approximately 131 elliptical, their area was calculated by using the formula for the ellipse area. 132

Data analysis 133
Data were analysed with R version 3.4.1 (R Core Team, 2014). Both in the seedling experiment and 134 in the dormant stem experiment, differences between host-pathogen combinations were analysed 135 with a linear model with tree species and Cryphonectria species as factors. The isolates were nested 136 within the Cryphonectria species. In the greenhouse experiment, one C. naterciae isolate (CO679) 137 had to be excluded from the analyses because it was mixed up with a C. parasitica isolate. In the 138 dormant stem experiment, this error was corrected and the measurements for C. naterciae CO679 139 could be included in the analysis. 140 141 3 Results 142

Seedling experiment 143
Mortality was only observed on chestnut seedlings inoculated with C. parasitica (13 of 15 seedlings 144 died before the end of the experiment). Moreover, this host-pathogen combination resulted in 145 significantly greater lesion areas (average area for single isolates ranged from 26.0 cm 2 to 34.4 cm 2 ) 146 at the end of the experiment than all other combinations (Figs 1 and 2). On oak and beech, no 147 mortality was observed and lesion growth was limited, with the smallest lesions measured on beech 148 for all three Cryphonectria species (Fig 2). Although C. naterciae and C. japonica produced 149 significantly smaller bark lesions on chestnut than C. parasitica (Fig 1), they could establish on the 150 seedlings and at the end of the experiment were successfully re-isolated from 80-100% of the lesions 151 ( Fig. 3). The re-isolations were successful from 20-60% of the lesions on oak for C. parasitica, from 152 40-60% of the lesions for C. naterciae and from 0-20% of the lesions for C. japonica (Fig 3). From 153 beech, the proportion of re-isolations was smaller than those from oak, varying from 0% to 40% of 154 lesions for all three Cryphonectria species.

Dormant stem experiment 157
In the dormant stem experiment, where the three Cryphonectria species were inoculated on chestnut, 158 oak and beech stems to assess saprophytic growth, the results were similar to the seedling experiment 159 for oak and beech. None of the three Cryphonectria species could form lesions on these two tree 160 species (Figs. 4 and 5). For chestnut, however, the results of the stem experiment differed from those 161 of the seedling experiment, with all three Cryphonectria species forming bark lesions on the 162 inoculated stems (Figs. 4 and 5). The lesions caused by C. parasitica and C. naterciae were of 163 similar size (C. parasitica average: 47.1 cm 2 , C. naterciae average: 52.0 cm 2 ), while the lesions 164 caused by C. japonica were significantly smaller (average: 22.2 cm 2 ), but were nonetheless 165 significantly greater than the lesions induced by all three species on oak and beech (Fig 5). its pathogenicity. Other phenotypic traits, such as sporulation and saprotrophic ability may play an 219 important role. Moreover, environmental conditions strongly influence plant diseases, therefore 220 climate changes can also be considered drivers of disease outbreaks (Sturrock et al., 2011). Thus, to 221 obtain a full view it is necessary to experimentally assess as many traits as possible. In our specific 222 case, artificial inoculations showed that an accidental introduction to Europe of C. japonica or the 223 spread within Europe of C. naterciae would most likely have much less devastating consequences 224 than the invasion by C. parasitica. Nonetheless, the ability of both species to saprotrophically 225 colonize fresh chestnut wood may suggest that they could become established in chestnut forests and 226 eventually infect weakened chestnut trees or other hosts not tested in this study. 227

Conflict of Interest 228
The authors declare that the research was conducted in the absence of any commercial or financial 229 relationships that could be construed as a potential conflict of interest.