Potential of Local Hypovirulent Strains of Cryphonectria parasitica for Biological Control of Chestnut Blight

Cryphonectria parasitica, introduced in the NE of Portugal since 1989, is now an important and widespread pathogen of chestnut (Castanea sativa Mill.) which causes losses in chestnut fruit production and led to decline and death of many chestnut trees. Hypovirulence, a virus mediated attenuation of fungal pathogenesis, has not been frequent until now in Portugal but recently some cases of healing cankers were reported by chestnut growers. We assessed these sites and one of them, where healing cankers were clustered, was intensively surveyed. From all the trees with healing cankers, white and orange phenotypes of C. parasitica were isolated, which are both of the same vc type (EU-11). White isolates of C. parasitica showed reduced pigmentation in colony colour which was not completely stable, suppressed conidiation, reduction of phenol oxidase but no significant differences in lesion length and sporulation on chestnut twigs on dormant chestnut trees. Hypovirulent strains have dsRNA virus in the cytoplasm which is the typical element that reduces virulence in C. parasitica. Bands of dsRNA were identified in all of the white isolates and some variation in band dimension and number of bands was detected. The LdsRNA (12 kbp), which is the diagnostic fragment of C. parasitica infection, was detected. The presence of white phenotypes of infected C. parasitica (CHV) and the fact that spreads occur in natural conditions is promising for its development as a biological control agent of chestnut blight. INTRODUCTION Chestnut blight is an important and widespread disease of chestnut (Castanea sativa Mill.) in Portugal. Cryphonectria parasitica, the causal organism of the disease is classified as a quarantine organism, list A2, by the EPPO (OEPP/EPPO, 1982) and is also of quarantine significance for NAPPO and IAPSC. European chestnut is an important tree species in the ecological region of “Terra Fria” in the northeast of Portugal where more than 80% of the total area (28 200 ha) of chestnut is found. Initial extensive surveys in that region, from 115 different sites in 20 different communities and around 10,000 inspected trees, had revealed 8-10% of blighted trees with varied disease incidence among sites and communities (Gouveia et al., 2001). Recent surveys in areas where the disease was previously recognised have revealed an increase in disease incidence with 40-100% of infected trees (Bragança et al., 2007) which together with the large number of new infected sites greatly increase losses in chestnut fruit production and lead to decline and death of many chestnut trees. The disease kills the infected tree branches and the rapid death of the entire tree takes place which is causing high environmental and economic concerns. Fungicides are not efficient in managing the disease and only canker excision and the removal of diseased branches are available in Portugal to reduce the dispersal of the disease. Despite the importance of these cultural measures to reduce the inoculum of the pathogen, the fragile chestnut ecosystem demands different disease management approaches. Biological control by hypovirulence, a virus mediated attenuation of fungal pathogenesis, is considered an efficient mean to control the disease and improve chestnut recovery (Heiniger and Rigling, 1994; Robin and Heiniger, 2001; Cortesi et al., 1989). Proc. 1 European Congress on Chestnut Castanea 2009 Eds.: G. Bounous and G.L. Beccaro Acta Hort. 866, ISHS 2010 444 Hypovirulent strains of C. parasitica are associated with the presence of an uncapsidated double stranded dsRNA virus in the cytoplasm of C. parasitica (Anagnostakis and Day, 1979; Dawe and Nuss, 2001). Hypovirus has no extracellular phase and virulent strains are converted to hypovirulent strains by the transmission of dsRNA through hyphal anastomoses between C. parasitica strains (Bissenger et al., 1997; Dawe and Nuss, 2001). Hypovirulence alters some morphological characteristics of the host fungi and hypovirulent strains show white colony morphology and reduced or absent conidial formation (Heiniger and Rigling, 1994; Dawe and Nuss, 2001). Hypovirulence is not frequent in initial phases of the disease and ten or more years frequently elapse between blight arrival and hypovirulence appearance (Robin and Heiniger, 2001). In Portugal, after two decades of chestnut blight introduction, hypovirulence is not currently frequent. Despite the fact that some isolates have shown reduced levels of pigmentation and sporulation on PDA and some cankers seemed to be healing cankers, only one isolate of C. parasitica was identified until now in Portugal as CHV infected (Bragança et al., 2007). The population of C. parasitica in Portugal is characterized by general low vc type diversity (Bragança et al., 2007) and in the northeast many sub-populations have only one vc type (Gouveia et al., 2001). The dominant vc type is the EU-11, which is somewhat rare outside of Portugal, and is present only in some sites in Italy (Cortesi et al., 1996) and France (Robin and Heiniger, 2001). The success of hypovirulence depends on population structure related to vc and mating types (Heiniger and Rigling, 1994; Bissenger et al., 1997) however, as a therapeutic treatment the success of the hypovirulent strain depends on the vc type present in each individually treated canker. Healing cankers in blighted chestnut trees are nowadays more frequently reported by chestnut growers in the NE of Portugal. The main objectives of this work were to isolate local hypovirulent strains of C. parasitica, to detect the presence of L-dsRNA, and to compare cultural characteristics, vegetative compatibility, phenol oxidase activity, hypovirulence conversion capacity and growth on dormant chestnut twigs to assess their potential for biological control of chestnut blight. MATERIALS AND METHODS In this study we focused on a chestnut orchard located in Vinhais in the northeastern region of Portugal where healing cankers of C. parasitica were reported. Chestnut trees were heavily blighted with extended and multiple cankers but some of them had visible healing cankers. The sampling was focused on the detection of hypovirulent strains of the pathogen. Individual samples of bark were collected from all the healing cankers, the surface was disinfected in 70% ethanol and promptly flamed. The samples were then placed on potato dextrose agar (PDAmb, 39 g L, Difco) supplemented with 1 mg L of biotine and 100 mg L of methionine. Plates were incubated at 25oC in the dark for seven days and then exposed to daylight for seven days. All the isolates were scored for the white mycelium and for the absence of picnydia and also scored for few and large pycnidia which are all indicators of infection by Cryphonectria hypovirus (CHV-1). One mycelial plug from each isolate was then transferred to fresh PDA. All white C. parasitica isolates were chosen along with some random orange isolates to determine the vc-type using the method of Bissengger et al. (1997). Vegetative compatibility of the strains was assessed according to the barrage/merging response. Conversion capacity was evaluated by pairing agar disks containing mycelia from white isolates and virulent isolates of known vc type on PDAmb, merging and spread of white colour were evaluated after 10 and 30 days. Phenol oxidase activity test was performed by growing the isolates on modified Bavendamn’s medium (Rigling et al., 1989) (0,5g taninic acid, 1,5% Difco malt extract, 2% Difco bacto agar, pH 4,5 adjusted with 1N NaOH). Mycelial growth, in the three replications of each isolate, was measured after five and ten days of incubation at 25oC. Changes in color were also checked.