Enhancing identification accuracy for powdery mildews using previously underexploited DNA loci

Abstract

The ITS DNA marker is routinely used for fungal identification but gives a clear result for only three out of four powdery mildew samples. A search for new markers indicates that some genes offer enhanced identification in comparison with ITS. Others fail due to amplification and sequencing difficulties and lack of informative variability. Powdery mildews (Ascomycota, Erysiphales) are biotrophic, fungal plant pathogens, which commonly occur worldwide on a wide range of host plants. They are unsightly and greatly reduce the vigour of their hosts and have major impacts on crops and other cultivated plants. Species within this order are straightforward to spot, but difficult to identify. A citizen science scheme was run 2013-2016 in the UK to gather a wide array of samples on which identification methods could be developed. Current techniques for identification and phylogenetic reconstruction show scope for improvement. In this paper we review genes used in other fungal groups for discrimination at species level. Working protocols for amplification and sequencing of seven genes (Actin, β-tubulin, Calmodulin, Chs, EF1-α, Mcm7, and Tsr1) are developed with varying success; Mcm7 proves to be the most useful at differentiation between closely related, phylogenetically young powdery mildew species for phylogenetic reconstruction when used separately and in tandem with ITS. We therefore propose this as the most appropriate candidate gene to be used commonly in powdery mildew diagnostics alongside the ITS, furthermore this could be transferred to similarly troublesome fungal clades.

Australia: A Continent Without Native Powdery Mildews? The First Comprehensive Catalog Indicates Recent Introductions and Multiple Host Range Expansion Events, and Leads to the Re-discovery of Salmonomyces as a New Lineage of the Erysiphales

“…The use of ITS as the single DNA barcode for species identifications is clearly a bias in powdery mildew research (Kovács et al, 2011 ; Ellingham et al, 2019 ), and the limits of the routine fungal identification procedures based on ITS sequences are well-known in mycology (Nilsson et al, 2006 ; Schoch et al, 2012; Kiss, 2012 ; Nilsson et al, 2014 ; Irinyi et al, 2015 ; Hibbett et al, 2016 ; Selosse et al, 2016 ). ITS sequences are currently still the only reliable molecular tool for powdery mildew species identifications, despite recent advancements toward new DNA barcodes (Desprez-Loustau et al, 2017 ; Ellingham et al, 2019 ). These could be boosted by the recently determined genomes of distinct lineages (Bindschedler et al, 2016 ; Frantzeskakis et al, 2019b ; Bradshaw and Tobin, 2020 ).…”

“…These could be boosted by the recently determined genomes of distinct lineages (Bindschedler et al, 2016 ; Frantzeskakis et al, 2019b ; Bradshaw and Tobin, 2020 ). However, closely related powdery mildew species cannot be reliably distinguished based on their ITS sequences because these are sometimes identical (Takamatsu et al, 2015a ), or the intra-specific, and intra-sample variation is sometimes higher than the differences between the ITS sequences reported for closely related taxa (Kovács et al, 2011 ; Ellingham et al, 2019 ).…”

“…DNA barcodes, particularly sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA), have been most useful for the identification of powdery mildews at the species level, while sequences of the nrDNA 18S and 28S region have supported the identification and classification of the genera (Braun et al, 2002 ; Takamatsu, 2004 , 2013a ; Takamatsu et al, 2015a , b ; Cabrera et al, 2018 ; Marmolejo et al, 2018 ). Other DNA regions have also been tested as molecular barcodes for some groups of the powdery mildews (Inuma et al, 2007 ; Desprez-Loustau et al, 2017 ; Ellingham et al, 2019 ; Qiu et al, 2020 ), but these studies have not yet resulted in the development of new DNA barcodes that are useful to distinguish species across the Erysiphales. To date, ITS sequences is still the sole species-level DNA barcode available for this fungal group, despite its limitations.…”

First records of powdery mildew fungi (Erysiphales) on medicinal plants in Taiwan

“…Erysiphe heraclei is an example for illustrating the con ict between the ITS phylogeny and traditional taxonomy. Most recent new attempts to obtain DNA from herbarium specimens of Erysiphales and to develop alternative barcodes for this group of fungi are very promising for resolving these problems (Ellingham et al 2019;Bradshaw and Tobin 2020).…”

Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales)

“…The application of PCR-based approaches for phylogenetic studies using aDNA is problematic; aDNA can be highly fragmented and there are few small loci (less than 500 bp) which are phylogenetically informative that can be used [36,42]. This study tested nine published primer sets for potential gene regions, that could be utilised as barcodes for powdery mildew molecular species identification [29,30,31,32,33,34,35]. These primer sets proved to be unsuitable in most cases as the target gene regions are too long in length (greater than 550 bp) for the amplification of the fragmented aDNA.…”