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Long term (in)stability of vegetative incompatibility type diversity and hypovirulence in Cryphonectria parasitica populations

Chestnut blight is a dangerous disease of the American (Castanea dentata) and European chestnut (C. sativa) caused by the growth of the fungus Cryphonectria parasitica inside the infected trees’ bark. The spreading of mycelia inside the plant’s tissues causes damage to the bark, cambium and the vascular tissue. If the active growth of the virulent fungus girdles the entire branch or trunk of the tree, distal parts of the plant die off, and the lower dormant lateral buds sprout and grow (Dutech et al 2012). The most effective known way to combat this disease is by means of biological control using a hyperparasite – Cryphonectria hypovirus 1 (CHV-1) (Prospero and Rigling 2013). This virus infects C. parasitica, causing morphological shift of the affected mycelia: from the brightly orange to white, which indicates the attenuation of the virulence of the infected strain. Thus, the white colored, CHV-1-infected C. parasitica strain is said to be hypovirulent, because the infection induced by such a strain causes only slight, superficial, and non-lethal wounds (Nuss 2005). The efficacy of the biocontrol mediated by the presence of the CHV-1 infected strains of C. parasitica is directly associated with vegetative compatibility (vc) type diversity of the C. parastica population, the prevalence of the hypovirulent isolates, and their distribution among different C. parasitica vc types. Vegetative compatibility of C. parasitica in European fungal isolates is controlled by at least six bialleic loci called vegetative incompatibility (vic) loci. The difference in one or more loci obstructs the hyphal fusion of the growing mycelia, which prohibits free transportation of the cytoplasmic elements between the incompatible mycelia, thus preventing the efficient dissemination of the CHV-1 within population (Cortesi et al. 2001). Sexual reproduction is the most efficient way the vc type diversity can increase within a population, thus potentially obstructing CHV-1 transmission and biological control of this pathogen. Cryphonectria parasitica appeared in Croatia in the 1950-ies and rapidly spread over most of the country in the next couple of decades. By the 1980-ies none of the native chestnut populations were free of this disease. The first comprehensive study was carried by Krstin et al. (2008) and it showed pronounced differences between populations of C. parasitica in Croatia. In our recent study we wanted to discern whether the population structure and CHV-1 distribution changed in the last decade i.e. to see whether the populations’ vc diversity increased or decreased and if that affected the prevalence of the hypovirulent C. parasitica isolates. We have revisited three chestnut populations in Croatia – two continental: Hrvatska Kostajnica and Ozalj, and one in Istria, approximately 10 km from the Adriatic coast (Buje). These populations were chosen because of the differences in population structure, hypovirulence prevalence and climate differences between them. In the previous study, sexual structures (perithecia) as well as the presence of two idiomorphs of the MAT gene were found in all those populations, suggesting a potential for sexual reproduction and increase of the vc type diversity (Krstin et al. 2008). In the first study, done in 2006, the coastal population in Istria, Buje, had the lowest number of hypovirulent isolates – only 12, 7%. Continental populations had much higher prevalence of the hypovirulent isolates: 44, 1% in Ozalj, and 50, 8% in Hrvatska Kostajnica. These numbers changed by the 2014 when our study was conducted ; the proportion of the hypovirulent isolates in Buje increased slightly to 19, 0%, while at the same time substantially decreased to 27, 8% in Ozalj and to 30, 9% in Hrvatska Kostajnica. However, general forest health seemed much better in continental populations than in Buje ; while in Buje a lot of completely dead trees were observed, in continental populations signs of healing were observed, despite the reduction of hypovirulent strains’ prevalence in populations. Furthermore, the population structure changed in the last decade as we observed an increase of Shannon’s diversity index in all three populations: in Buje from 1, 69 to 2, 46, in Ozalj from 0, 63 to 1, 78, and in Hrvatska Kostajnica from 1, 59 to 2, 36. This was mainly because of the appearance of the new, and in some cases, previously unobserved, vc types in all populations in Croatia. The most probable reason for that is recombination during the sexual reproduction of the fungus and, at least to some degree, the influx of the new individuals from the surrounding areas. We have also determined that the abundance of different vc types varies between populations. However, no significant difference in vc type distribution between virulent and hypovirulent isolates within a population was observed. This indicates that CHV-1 is fairly well represented in most of the vc types in each population. Still, some changes, or rather a drift, in population structure when considering vc types was observed. For example, in Ozalj in 2006 vc type EU-1 was absolutely dominant with more over 87% of the isolates belonging to that particular vc group. This changed quite drastically and in 2014 we found that EU-2 was the most abundant vc type, represented with about 42% isolates, while only 24% of the isolates had EU-1 vc type. Similar observation was made in Buje, where in 2006 about 38% isolates were EU-1, and 25% were EU-17, while in 2014 EU-13 became the most common vc type, represented with about 21% of isolates. Approximately 15% of all isolates belonged to EU-4 vc type. Other vc types were represented with less than 10%. Such dramatic change in the percentages of vc types was not observed in Hrvatska Kostajnica, although, numerous new and rare vc types were identified. Our results indicate that chestnut-C. parasitica-CHV-1 pathosystem is a highly dynamic one i.e. that a dramatic change in vc type diversity and CHV-1 prevalence in populations can occur in less than a decade. It is worth noting that even though the percentages of hypovirulent isolates were reduced in Hrvatska Kostajnica and Ozalj when compared to 2006, thus far these two chestnut populations remained in a fair overall state, while the Istrian population Buje, showed a severe disease symptoms and significant dieback of the trees. This indicates that when both virulent and hypovirulent isolates of the major vc groups in populations are present, the overall prevalence of CHV-1 infection is the most important factor contributing significantly to the health status of the chestnut populations. The trend of reducing hypovirulence prevalence in two populations is somewhat worrying, because in the future, skewing the ratios of hypovirulent and virulent C. parasitica isolates in populations might hinder the natural dissemination of the viruses within populations, reducing the CHV-1 prevalence even further and possibly increasing the severity of the disease symptoms in populations, resulting with more dieback and significant loss of chestnuts in the forests, as is observed in Buje.

chestnut blight ; hypovirus ; population structure

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