Virulence, ligninolytic enzymes and metabolic profile of Cryphonectria parasitica virulent and hypovirulent strains Converted by CHV1 hypovirus

  • Omar Abdelaziz Ouni
  • Lurdes Jorge Instituto Politécnico de Bragança, Bragança, Portugal
  • Luísa Moura Instituto Politécnico de Viana do Castelo, Escola Superior Agrária, CISAS, Viana do Castelo, Portugal
  • Valentim Coelho Instituto Politécnico de Bragança, Bragança, Portugal
  • Eugénia Gouveia Instituto Politécnico de Bragança, Bragança, Portugal
Keywords: Cryphonectria parasitica, virulence, metabolic profile, Biolog FF MicroPlates, ligninolytic enzymes, Cryphonectria hypovirus 1 (CHV1)


Introduction: Cryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics, the strains with hypovirus CHV1 are used in Europe as a biological control agent of chestnut blight.

Objectives: The aim of this work is to understand the effect of hypovirus on fungi virulence by comparing the production of some lignin-degrading enzymes and the metabolic profiles of some isogenic virulent and hypovirulent (converted and original) strains.

Methods: The virulence of each strain was evaluated by mycelial inoculation on apple fruits (cv. Golden Delicious) and on detached, one year old, chestnut branches. To detect the activity of ligninolytic enzymes (laccases, peroxidases and cellulases), various substrates and indicator compounds were used. The metabolic profile of C. parasitica was evaluated by the Biolog FF system using 95 different carbon sources.

Results: Virulent strains were found to cause more significant necrotic lesions in chestnut branches (p<0.05) and to produce larger lignin-degrading enzymes. The use of Biolog FF MicroPlates indicated that the use of 95 carbon sources five isolates of C. parasitica were significantly different (p <0.001), when the substrates were grouped into six types of chemical compounds. The highest AWCD values were obtained for carbohydrates, carboxylic acids and polymers, and the lowest values for amines/amides, amino acids and miscellaneous.

Conclusions: Virulence evaluation of C. parasitica strains is important to study the hypovirulence processes mediated by the hypovirus CHV1. The detached branches of chestnut were, in our study, more suitable than apple fruits test in discriminating hypovirulent from virulent strains of C. parasitica. Virulent strains showed higher activity of acid-tannic inducible laccase (Lac3) and other lignin-degrading enzymes (LiP, MnP, and cellulase) when compared with hypovirulent ones. The results of the metabolic profiles studies may lead to new perspectives for understanding the biological process used by the hypovirus therefore, this may suggest a method for discriminating hypovirulent strains and study ecology and field fitness in this fungal strains.


Abreu, C. (1992). A hipovirulência como forma de luta natural contra o cancro do castanheiro. Revista de Ciências Agrárias, 15(1-2), 167–169.

Bragança, H., Simões, S., Santos, N., Marcelino, J., Tenreiro, R. & Rigling, D. (2005). Chestnut Blight in Portugal - Monitoring and vc Types of Cryphonectria parasitica. Acta Horticulturae, 693, 627–34. https://doi: 10.17660/ActaHortic.2005.693.84.

Chen, C., Sun, Q., Narayanan, B., Nuss, D. L. & Herzberg, O. (2010). Structure of Oxaloacetate Acethylhydrolase, a Virulence Factor of the Chestnut Blight Fungus. Journal of Biological Chemistry, 285(34), 26685–96. https://doi:10.1074/jbc.M110.117804.

Chung, H-J., Kwon, B-R., Kim, J-M., Park, S-M., Park, J-K., Cha, B-J., Yang, M-S. & Kim, D-H. (2008). A Tannic Acid-Inducible and Hypoviral-Regulated Laccase3 Contributes to the Virulence of the Chestnut Blight Fungus Cryphonectria parasitica. Molecular Plant-Microbe Interactions: MPMI, 21(12), 1582–90. https://doi:10.1094/MPMI-21-12-1582.

Dawe, A.L., Van Voorhies, W.A., Lau, T.A., Ulanov, A.V. & Li, Z. (2009). Major Impacts on the Primary Metabolism of the Plant Pathogen Cryphonectria parasitica by the Virulence-Attenuating Virus CHV1-EP713. Microbiology Society, 155, 3913–21. https://doi: 10.1099/mic.0.029033-0.

Elliston, J.E. (1985). Characteristics of dsRNA-free and dsRNA containing strains of Endothia parasitica in relation to hypovirulence. Phytopathology, 75, 151-158. https://DOI: 10.1094/phyto-75-151.

Faruk, M.I., Izumimoto, M. & Suzuki, N. (2008). Characterization of mutants of the chestnut blight fungus (Cryphonectria parasitica) with unusual hypovirus symptoms. Journal of General Plant Pathology, 74(6), 425-433.

Fulbright, D.W. (1984). Effect of eliminating dsRNA in hypovirulent Endothia parasitica. Phytopathology, 74, 722-724. https://DOI: 10.1094/phyto-74-722.

Garland, J.L. & Mills, A.L. (1991). Classification and characterization of heterotrophic microbial communities on the basis of patterns of community level sole-carbon-source utilization Applied and Environmental Microbiology, 57, 2351-2359.

Gouveia, E., Cardoso, P. & Monteiro, M.L. (2001). Incidence of chestnut blight and diversity of vegetative compatible types of Cryphonectria parasitica in Trás-os-Montes. Forest Snow and Landscape Research, 76 (3): 387-390.

Gouveia, E., Pereira, E., Araújo, A., Coelho, V., Castro, J., Bragança, H. & Martins, L. (2016). Cancro do Castanheiro em Trás-os-Montes (Portugal): Incidência atual e estudo da estrutura populacional de Cryphonectria parasitica para a introdução da luta biológica por hipovirulência. Gaia Scientia, 10(2), 75-83. ISSN 1981-1268.

Heiniger, U. & Rigling, D. (1994). Biological control of Chestnut blight in Europe. Annual Review of Phytopathology, 32, 581-599. https://doi: 10.1146/

Lee, J.K., Tattar, A.T., Berman, P.M. & Mount, M.S. (1992). A rapid method for testing the virulence of Cryphonectria parasitica using excised bark and wood of American chestnut. Phytopathology, 82, 1454-1456.

Milgroom, M. & Cortesi, P. (1999). Analysis of population structure of the chestnut blight fungus based on vegetative incompatibility. Proceedings of the National Academy of Sciences of the United States of America, 96, 10518-10523.

Rice, A.V. & Currah, R.S. (2005). Profiles from Biolog FF plates and morphological characteristics support the recognition of Oidiodendrum fimicola sp. Nov. Studies in Mycology, 53, 75–82.

Rigling, D. & Prospero, S. (2017). Cryphonectria parasitica, the Causal Agent of Chestnut Blight: Invasion History, Population Biology and Disease Control. Molecular Plant Pathology, 19(1), 7–20. https://doi: 10.1111/mpp.12542.

Rigling, D., Heiniger, U. & Hohl, H.R. (1989). Reduction of Laccase Activity in DsRNA-Containing Hypovirulent Strains of Cryphonectria (Endothia) parasitica. Phytopathology, 79, 219–23. https://doi:10.1094/Phyto-79-219.

Robin, C. & Heiniger, U. (2001). Chestnut Blight in Europe : Diversity of Cryphonectria parasitica, Hypovirulence and Biocontrol. Forest Snow and Landscape Research, 76(3), 361–67.

Zhai, L., Xiang, J., Zhang, M., Fu, M., Yang, Z. & Hong, N. (2016). Characterization of a Novel Double-Stranded RNA Mycovirus Conferring Hypovirulence from the Phytopathogenic Fungus Botryosphaeria dothidea. Virology, 493, 75–85. https://doi:10.1016/j.virol.2016.03.012.

Zhang, T.Y., Wu, Y.H., Zhuang, L.L., Wang, X.X. & Hu, H.Y. (2014). Screening heterotrophic microalgal strains by using the Biolog method for biofuel production from organic wastewater. Algal Research, 6, 175–179. https://doi:10.1016/j.algal.2014.10.003.

Agriculture, Food and Veterinary Sciences