Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification

Abstract

Guignardia bidwellii is the etiological agent of grape black rot, a disease affecting Vitis and other Vitaceae that can cause heavy crop losses in vineyards. Its identification is based mainly on morphological characters and the symptoms on plants but, due to their variability, they may be difficult to interpret to reliably distinguish the pathogen to species. To date, despite the economic importance of G. bidwellii, no molecular investigations have been carried out on Vitis isolates and few sequence data are available for cultures derived from ornamental host plants. We analyzed samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins by morphological, molecular and proteomic techniques, including ITS1-ITS2 regions and calmodulin gene sequencing, as well as matrix-assisted laser desorption/ionization analysis by time-of-flight mass spectrometry (MALDI-TOF MS). This polyphasic approach allowed assessing the phylogenetic relationships among the different isolates and suggested the existence of two distinct species. The advantages of a polyphasic approach for the identification of G. bidwellii are highlighted.

Key words:

• calmodulin
• grapevine
• ITS
• MALDI-TOF
• molecular analysis
• Parthenocissus
• proteomics
• Vitis

Acknowledgments

We are grateful to Alex Angst (ETHZ, Switzerland), the staff of Mycoscope of Agroscope Changins-Wädenswil ACW (Switzerland) and Andreas Kortekamp (Dienstleistungszen-trum Ländlicher Raum DLR Rheinpfalz, Neustadt a.d. Weinstraβe, Germany) who provided the samples analyzed in this study, as well as Nicole Siebert (Forschungsanstalt Geisenheim, Germany) in isolating conidia from the cultured mycelia. Thanks to all the members of the Plant Pathology Group and especially to Stefano Torriani (ETHZ, Switzerland), to AnnaPaola Caminada, Sophie De Respinis and Cinzia Benagli (Istituto cantonale di microbiologia, Bellinzona, Switzerland) for their technical assistance during the laboratory analyses, as well as to Guido Vogel (Mabritec AG, Riehen, Switzerland) for his advice about MALDI-TOF protocols.

This study was financially supported by the Dipartimento Educazione, Cultura e Sport (Bellinzona, Switzerland).