Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine
Graphical abstract
Thirteen phytotoxins were identified from Neofusicoccum parvum. Analysis of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.
Introduction
Grapevine trunk diseases cause decline and premature dieback of vineyards and have become a growing threat to grapevine production worldwide. Losses up to 50% of the normal yield have been reported (Bertsch et al., 2013, Hofstetter et al., 2012, Luque et al., 2009, Úrbez-Torres et al., 2006, Mugnai et al., 1999). Esca and botryosphaeria dieback are complex diseases in which both the symptoms and the time course of their expression are highly variable. Many fungi have been reported to be involved in these diseases, mainly Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Fomitiporia mediterranea, Eutypa lata, and Botryosphaeria spp. (Bertsch et al., 2013). For many years, Botryosphaeriaceous species have been mostly considered saprophytes or secondary colonisers in grapevine (Phillips, 2002). Thus, their pathogenicity has been underestimated, and their epidemiology remains not fully investigated. To date, 30 botryosphaeriaceous species, including Neofusicoccum parvum, have been reported to be involved in dieback disease (Spagnolo et al., 2014a, Luque et al., 2009, Taylor et al., 2005, Úrbez-Torres, 2011). The wood symptoms of botryosphaeria dieback are associated with brown stripes located in the outer xylem. The discoloration appears as an orange/brown colour in the longitudinal direction just beneath the bark and may extend from the trunk to the rootstock and annual stems. The brown stripes are always associated with foliar symptoms (Larignon et al., 2001). Because the pathogens cannot be detected in the leaves of infected plants (Larignon and Dubos, 1997, Mugnai et al., 1999), it was hypothesised that foliar symptoms are caused by phytotoxic compounds produced by the fungi in wood tissue, which are either translocated to the leaves or induce a chain reaction leading to the expression of the symptoms in the leaves. Several secondary metabolites have been found in Botryosphaeriaceae spp., but only three lipophilic compounds has been identified from N. parvum (Evidente et al., 2010), the predominant species recovered from grapevines displaying symptoms of dieback and decline. The present study aimed (i) to further characterise the phytotoxins of N. parvum, (ii) to evaluate their phytotoxicity, (iii) to investigate the expression of plant defence-related genes in response to (−)-terremutin (1), the most abundant phytotoxic compound identified from the culture, and finally, (iv) to determine the presence of the phytotoxins in grapevine wood showing botryosphaeria dieback symptoms.
Section snippets
Extraction, purification and identification of phytotoxins
Thirteen N. parvum fungi isolated from grapevine plants showing decline collected from vineyards or nurseries (Table 1) were each grown on ten potato dextrose plates and extracted with ethyl acetate. The extracts were screened for the production of secondary metabolites by HPLC-DAD analysis and for phytotoxic activity through a leaf disc assay of the host plant Vitis vinifera cv. Chardonnay. The secondary metabolite patterns of all 13 strains were identical to differences in the concentrations
Conclusion
In conclusion, this study showed (i) that N. parvum is able to produce a diverse variety of phytotoxins that confer high flexibility to the fungus that allows it to adapt to several environmental conditions, (ii) the evidence that genes for secondary metabolites are highly conserved in N. parvum of grapevine, (iii) the ability of the plants to respond to fungal toxins, and (iv) the presence of two of the toxins in grapevine wood from plants showing botryosphaeria dieback symptoms.
The role of
General experimental procedure
The 1H and 13C NMR spectra were recorded either on a Bruker 360 MHz or Bruker Avance 500 spectrometer (500 MHz) (Fällanden, Switzerland). The chemical shifts were referenced to TMS. The circular dichroism spectra were recorded on a JASCO PFD 350L spectropolarimeter. The optical rotations were determined using a Perkin-Elmer 241 digital polarimeter with a 1-dm cell. The IR spectra were obtained with a Perkin Elmer 1720× spectrometer. Electrospray mass spectrometry (ESIMS) was performed on a Bruker
Acknowledgements
This work was supported by Grants from the Swiss Secrétariat d'Etat à la Formation, à la Recherche et à l'Innovation (SEFRI-N° C08.0140), works related to COST 858 action-Viticulture – Biotic and abiotic stress – Grapevine defence mechanism and grape development, and The French Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt (CASDAR – Compte d’Affectation Spéciale au Développement Agricole et Rural AAP N°7124 – V902). We thank Dr. Antony Buchala for English revision.
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