https://doi.org/10.4081/jbr.2024.12459
Molecular characterization of Cryphonectria parasitica isolates from Basilicata region (Southern Italy) and mycovirus identification
Submitted: March 12, 2024
Accepted: October 7, 2024
Published: December 20, 2024
Accepted: October 7, 2024
Abstract Views: 25
PDF: 7
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
Chestnut is a valuable species that grows widely throughout the Italian peninsula. It is susceptible to different diseases and among them chestnut blight caused by Cryphonectria parasitica is the most devastating one. This study aimed to molecularly characterize and distinguish virulent and hypovirulent C. parasitica isolates from the Basilicata region. Pure fungal cultures were obtained from symptomatic plant material, their genomic DNA was extracted and amplified using rDNA Internal Transcribed Spacer (ITS), M13 minisatellite and (GACA)4 microsatellite primers. ITS sequencing allowed the identification of the fungal species while minisatellite and microsatellite Polymerase Chain Reactions (PCRs) differentiate between virulent and hypovirulent isolates placing them in distinct clusters. Additionally, sequence analysis of hypovirus partial genome showed that all identified hypoviruses, belonged to Cryphonectria hypovirus 1 (CHV-1) subtype, sharing a 99% sequence identity with the Italian isolate Marche of CHV-1. To our knowledge, this is the first study to differentiate virulent and hypovirulent isolates of C. parasitica at molecular level and identify the hypovirus subtype associated with the hypovirulent isolates in the Basilicata region.
Rigling D, Prospero S. Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control. Molec Plant Pathol 2018;19:7-20.
DOI: https://doi.org/10.1111/mpp.12542
Milgroom MG, Cortesi P. Biological control of chestnut blight with hypovirulence: a critical analysis. Annu Rev Phytopathol 2004;42:311-38.
DOI: https://doi.org/10.1146/annurev.phyto.42.040803.140325
Turina M, Rostagno L. Virus-induced hypovirulence in Cryphonectria parasitica: an unresolved conundrum. J Plant Pathol 2007;89:165-78.
Peters FS, Busskamp J, Prospero S, et al. Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany. Fungal Biol 2014;118:193-210.
DOI: https://doi.org/10.1016/j.funbio.2013.11.009
Forgia M, Isgandarli E, Aghayeva DN, et al. Virome characterization of Cryphonectria parasitica isolates from Azerbaijan unveiled a new mymonavirus and a putative new RNA virus unrelated to described viral sequences. Virology 2021;563:51-61.
DOI: https://doi.org/10.1016/j.virol.2020.10.008
Nuskern L, Stojanović M, Milanović-Litre M, Šibenik T. Filling the gap in Southern Europe-diversity of Cryphonectria parasitica and associated mycovirus (Cryphonectria hypovirus 1) in Montenegro. J Fungi 2022;8:552.
DOI: https://doi.org/10.3390/jof8060552
Alleman C, Hoegger P, Heiniger U, Rigling D. Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment polymorphism (RFLP) markers. Mol Ecol 1999;8:843-54.
DOI: https://doi.org/10.1046/j.1365-294X.1999.00639.x
Gobbin D, Hoegger PJ, Heiniger U, Rigling D. Sequence variation and evolution of Cryphonectria hypovirus 1 (CHV-1) in Europe. Virus Res 2003;97:39-46.
DOI: https://doi.org/10.1016/S0168-1702(03)00220-X
Hillman BI, Suzuki N. Viruses of the chestnut blight fungus Cryphonectria parasitica. Adv Virus Res 2004;63:423-72.
DOI: https://doi.org/10.1016/S0065-3527(04)63007-7
EFSA PLH Panel (EFSA Panel on Plant Health). Scientific Opinion on the pest categorization of Cryphonectria parasitica (Murrill) Barr. EFSA Journal 2014;12:3859.
DOI: https://doi.org/10.2903/j.efsa.2014.3859
Li R, Zhou S, Li Y, et al. Comparative methylome analysis reveals perturbation of host epigenome in chestnut blight fungus by a hypovirus. Front Microbiol 2018;9:1026.
DOI: https://doi.org/10.3389/fmicb.2018.01026
Suzuki N, Cornejo C, Aulia A, et al. In-tree behavior of diverse viruses harbored in the chestnut blight fungus, Cryphonectria parasitica. J Virol 2021;95:e01962-20.
DOI: https://doi.org/10.1128/JVI.01962-20
Ramon-Ochoa P, Foster J, Chitty R, et al. Canker development and biocontrol potential of CHV-1 infected English isolates of Cryphonectria parasitica is dependent on the virus concentration and the compatibility of the fungal inoculums. Viruses 2022;14:2678.
DOI: https://doi.org/10.3390/v14122678
Robin C, Heiniger U. Chestnut blight in Europe: Diversity of Cryphonectria parasitica, hypovirulence and biocontrol. For Snow Landsc Res 2001;76:361-7.
Akilli S, Serce CU, Katircioglu YZ, et al. Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey. Eur J Plant Pathol 2013;135: 323-34.
DOI: https://doi.org/10.1007/s10658-012-0089-z
Mang SM, Racioppi R, Camele I, Rana GL. Use of volatile metabolite profiles to distinguish three Monilinia species. J Plant Pathol 2015;97:55-9.
Mang SM, Scrano L, Camele I. Preliminary studies on fungal contamination of two rupestrian churches from Matera (Southern Italy). Sustainability 2020;12:6988.
DOI: https://doi.org/10.3390/su12176988
Mentana A, Camele I, Mang SM, et al. Volatolomics approach by HS-SPME-GC-MS and multivariate analysis to discriminate olive tree varieties infected by Xylella fastidiosa. Phytochem Anal 2019;30:623-34.
DOI: https://doi.org/10.1002/pca.2835
White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M.A., Gelfand D.H., Sninsky J.J., White T.J. (eds.). 1990. PCR Protocols: A Guide to methods and Applications, pp. 315-322. Academic Press, San Diego, California.
DOI: https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Frisullo S, ElShafie H, Mang SM. First report of two Phomospsis species on olive trees in Italy. J Plant Pathol 2015;97:401.
Mang SM, Marcone C, Maxim A, Camele I. Investigations on Fungi isolated from apple trees with die-back symptoms from Basilicata Region (Southern Italy). Plants 2022;11:1374.
DOI: https://doi.org/10.3390/plants11101374
Mang SM, Figliuolo G. Species delimination in Pleurotus eryngii species-complex inferred from ITS and EF-1α gene sequences. Mycology 2010;1:269-80.
DOI: https://doi.org/10.1080/21501203.2010.536171
Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol 1990;215:403-10.
DOI: https://doi.org/10.1016/S0022-2836(05)80360-2
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389-402.
DOI: https://doi.org/10.1093/nar/25.17.3389
Latouche GN, Daniel HM, Lee OC, et al. Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species. J Clin Microbiol 1997;35(12):3171-80.
DOI: https://doi.org/10.1128/jcm.35.12.3171-3180.1997
Meyer W, Mitchell T, Freedman E,Vilgalys R. Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans. J Clin Microbiol 1993;31:2274-80.
DOI: https://doi.org/10.1128/jcm.31.9.2274-2280.1993
Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Molec Biol Evol 2021;38:3022-7.
DOI: https://doi.org/10.1093/molbev/msab120
Larkin M, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8.
DOI: https://doi.org/10.1093/bioinformatics/btm404
Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985;39:783-91.
DOI: https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molec Biol Evol 1993;10:512-26.
Anagnostakis SL. Chestnut breeding in the United States for disease and insect resistance. Plant Dis 2012;96:1392-403.
DOI: https://doi.org/10.1094/PDIS-04-12-0350-FE
Sabbatini Peverieri G, Alma A, Manzo A, et al. Linee guida per la gestione delle problematiche fitosanitarie del castagno.[Guidelines for the management of chestnut phytosanitary problems] Consiglio per la Ricerca e la sperimentazione in Agricoltura (CRA), 2014; Roma, Italia.
Trapiello E, Rigling D, González AJ. Occurrence of hypovirus-infected Cryphonectria parasitica isolates in northern Spain: an encouraging situation for biological control of chestnut blight in Asturian forests. Eur J Plant Pathol 2017;149:503-14.
DOI: https://doi.org/10.1007/s10658-017-1199-4
Nieddu S. Studi sulla struttura delle popolazioni di Cryphonectria parasitica dei castagneti del centro Sardegna finalizzati alla selezione di ceppi ipovirulenti da utilizzare nella lotta biologica. Tesi di dottorato, 2009.[Studies on the structure of Cryphonectria parasitica populations in chestnut groves in Sardinia aimed at the selection of hypovirulent strains to be used in biological control. PhD thesis, 2009]. Università degli Studi di Sassari, Sassari, Italia.
Cooke DEL, Drenth A, Duncan JM, et al. A molecular phylogeny of Phytophthora and related Oomycetes. Fungal Genet Biol 2000;30:17-32.
DOI: https://doi.org/10.1006/fgbi.2000.1202
Görcsös G, Irinyi L, Radócz L, et al. Diversity of Cryphonectria parasitica populations from the Carpathian Basin. Acta Microbiol Immunol Hung 2015;62:247-66.
DOI: https://doi.org/10.1556/030.62.2015.3.3
Murolo S, De Miccolis Angelini RM, Faretra F, Romanazzi G. Phenotypic and molecular investigations on hypovirulent Cryphonectria parasitica in Italy. Plant Dis 2018;102:540-5.
DOI: https://doi.org/10.1094/PDIS-04-17-0517-RE
Breullin F, Dutech C, Robin C. Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations by microsatellite markers. Mycol Res 2006;110:288-96.
DOI: https://doi.org/10.1016/j.mycres.2005.09.016
Kubsiak TL, Dutech C, Milgroom G. Fifty-three polymorphic loci in the chestnut blight fungus, Cryphonectria parasitica. Mol Ecol Notes 2007;7:428-32.
DOI: https://doi.org/10.1111/j.1471-8286.2006.01608.x
Çelik A, Çakar D, Derviş S, et al. New detection methods for Cryphonectria Hypovirus 1 (CHV1) through SYBR Green-Based Real-Time PCR and Loop-Mediated Isothermal Amplification (LAMP). Viruses 2024;16:1203.
DOI: https://doi.org/10.3390/v16081203
Çakar D, Özer G, Şimşek SA, et al. Characterisation of Cryphonectria hypovirus 1 strains in Turkey and their transmission to various vegetative compatibility types of Cryphonectria parasitica. J Plant Pathol 2024. https://doi.org/10.1007/s42161-024-01691-3
DOI: https://doi.org/10.1007/s42161-024-01691-3
Popović M, Nuskern L, Peranić K, Vuković R, et al. Physiological variations in hypovirus-infected wild and model long-term laboratory strains of Cryphonectria parasitica. Front Microbiol 2023;14:1192996.
DOI: https://doi.org/10.3389/fmicb.2023.1192996
Supporting Agencies
This research was funded by the Basilicata Region-Phytosanitary Office (Matera, Italy), under the “Agreement for collaboration between the Basilicata Region and the University of Basilicata (SAFE) for carrying out technical-scientific activities in phytosanitary field” (REP. nr. 600 of 21.12.2020) within the project: “Epidemiological studies regarding the presence and spread in Basilicata of agricultural and forestry phytopathogens with particular focus on quarantine ones. Molecular characterization of phytopathogens and their possible control”.How to Cite
Mang, S. M., Marcone, C., Marcone, M., Malvasi, G., Chira, D., Chira, F., Maxim, A., & Camele, I. (2024). Molecular characterization of <i>Cryphonectria parasitica</i> isolates from Basilicata region (Southern Italy) and mycovirus identification. Journal of Biological Research - Bollettino Della Società Italiana Di Biologia Sperimentale. https://doi.org/10.4081/jbr.2024.12459
Copyright (c) 2024 the Author(s)
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
PAGEPress has chosen to apply the Creative Commons Attribution NonCommercial 4.0 International License (CC BY-NC 4.0) to all manuscripts to be published.