Untargeted and Targeted LC-MS/MS Based Metabolomics Study on In Vitro Culture of Phaeoacremonium Species.

Pierluigi Reveglia,Genoveffa Nuzzo,Angelo Fontana,Marco Masi,Antonia Carlucci,Gaetano Corso,Antonio Evidente,Maria Luisa Raimondo,Alessio Cimmino

doi:10.3390/jof8010055

Abstract

Grapevine (Vitis vinifera L.) can be affected by many different biotic agents, including tracheomycotic fungi such as Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are the main causal agent of Esca and Petri diseases. Both fungi produce phytotoxic naphthalenone polyketides, namely scytalone and isosclerone, that are related to symptom development. The main objective of this study was to investigate the secondary metabolites produced by three Phaeoacremonium species and to assess their phytotoxicity by in vitro bioassay. To this aim, untargeted and targeted LC-MS/MS-based metabolomics were performed. High resolution mass spectrometer UHPLC-Orbitrap was used for the untargeted profiling and dereplication of secondary metabolites. A sensitive multi reaction monitoring (MRM) method for the absolute quantification of scytalone and isosclerone was developed on a UPLC-QTrap. Different isolates of P. italicum, P. alvesii and P. rubrigenum were grown in vitro and the culture filtrates and organic extracts were assayed for phytotoxicity. The toxic effects varied within and among fungal isolates. Isosclerone and scytalone were dereplicated by matching retention times and HRMS and MS/MS data with pure standards. The amount of scytalone and isosclerone differed within and among fungal species. To our best knowledge, this is the first study that applies an approach of LC-MS/MS-based metabolomics to investigate differences in the metabolic composition of organic extracts of Phaeoacremonium species culture filtrates.

Highlights

Vitis vinifera is one of the most economically important crops worldwide, with approximately 71% of the world’s grape production being used for wine production
The highest yield in organic extract was obtained in the range between 7.13 and 28.15 mg per 40 mL filtrate when the culture filtrates were extracted at acid pH (Table 2)
As previous attempts to quantify scytalone (1) and isosclerone (2) from culture filtrates of P. minimum by LC–MS failed [63], this study reports for the first time a valid targeted MS method for their quantification in in vitro culture of Phaeoacremonium species

Summary

Introduction

Vitis vinifera is one of the most economically important crops worldwide, with approximately 71% of the world’s grape production being used for wine production. A variety of fungal diseases threatens viticultural regions all over the world, compromising the yield and quality of wine [1,2]. Grapevine trunk diseases (GTDs), caused by one or several xylem-inhabiting fungi, produce a progressive decline in vines, consisting of a loss in productivity and eventually death of the vines [3]. Over the past few decades, they have been extensively studied [4]. The relationship between pathogenic fungi involved in GTDs and abiotic agents, the expression of symptoms, and the lack of effective management strategies, requires further investigation [5,6].

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