Contributions towards the consistent ID of powdery mildew (Erysiphales, Ascomycota) species Brandon Shaw 2019 Dissertation submitted for the degree of Master of Science in Plant and Fungal Taxonomy, Diversity and Conservation awarded by Queen Mary, University of London. https://doi.org/10.34885/71 © The Author. All rights reserved. Contributions towards the consistent ID of powdery mildew (Erysiphales, Ascomycota) species Brandon Shaw Supervised by Dr Oliver Ellingham and Dr Ester Gaya Abstract Powdery mildews (Erysiphales, Ascomycota) are an order of plant pathogenic fungi that infect 10,000 different angiosperm species, including important crop species. Traditional morphological methods of identifying powdery mildews can be difficult and time consuming, as different character states can be hard to distinguish. Molecular techniques using the ITS region fail to provide a species level identification 1 out of every 4 times and often must be used in conjunction with morphology to provide an identification beyond genus. The Mcm7 gene could potentially be used to provide more accurate identifications and build phylogenies incorporating large number of taxa to help understand the systematics of the order. Here, it is found that incorporating Mcm7 and ITS into a phylogeny greatly increases bootstrap support values, suggesting that Mcm7 may be used in the future to understand the systematics of this previously problematic order. ITS is successfully extracted from more and older specimens than Mcm7 and recommendations on using Mcm7 in the lab are made. Introduction For the first time in decades, the number of undernourished people in the world is rising and is equal to the number seen in 2010-2011 (FAO, 2019), with the current world population of 7.7 billion expected to reach 9.7 billion in 2050 (United Nations, 2019), now more than ever global food security is an issue at the forefront of our minds. An extra 200,000 billion calories a year by 2050 must be produced (Bebber & Gurr, 2015), one way to improve food security will be to improve crop yield. Currently, pests and plant diseases cause a 20-25% decrease in harvest yield and 10% loss post-harvest resulting in a huge economic loss and posing a serious threat to global food security (Bebber & Gurr, 2015; Martinelli et al., 2015). Pathogenic fungi that have the biggest effect, rapidly spread by human trade and transport with species ranges expanding owing to climate change, one such pathogenic group are the powdery mildews(Bebber & Gurr, 2015; Bebber, Holmes, & Gurr, 2014; Ellingham, et al., 2016). Powdery mildews are plant pathogenic fungi in the order Erysiphales in the A B Ascomycota with 872 species currently described (Braun & Cook, 2012) characterised by a talcum powder-like patches growing on host leaves, Figure 1. Powdery mildews infect around 10,000 angiosperm species (Braun, 1987) including popular garden plants such as Asteraceae which are infected by the powdery mildew genus Golovinomyces (Takamatsu, et al., 2006) and the genus Figure 1 Talcum powder-like patches of powdery mildew growing Podosphaera which predominantly infects on Ballota sp. (A) and Lupinus sp. (B). Rosaceae(Takamatsu, et al., 2010). More importantly, powdery mildews infect a wide variety of crop plants, Blumeria graminis which infects important grain crops wheat, rye, and barley and has a worldwide distribution (Braun & Cook, 2012; Zhang et al., 2005), Podosphaera xanthii infects crops belonging to the family Cucurbitaceae and has an almost worldwide distribution (Braun & Cook, 2012; Mcgrath, 2001), and Erysiphe necator which is the most common and damaging pathogen of various grapevine cultivars and is found wherever grape is grown (Braun & Cook, 2012; Gadoury et al., 2001). All 18 genera powdery mildews are obligate biotrophic parasites of angiosperms, 14 genera are epiphytic forming haustoria in the plant epidermal cells, including the most specious genera Erysiphe, and three are endoparasites forming haustoria in plant parenchyma cells (Phyllactinia, Leveillula, and Pleochaeta) (Aghayeva, Abasova, & Takamatsu, 2018). Appendix 1 shows a summary of currently accepted genera within the order Erysiphales. Powdery mildews can be treated in various ways, in the garden, pruning off the infected areas or useing home remedy treatments such as baking soda mixed with soap and water, and diluted milk (Beresford et al., 1996; Bettiol, 1999; Martins et al., 2016) may suffice. On commercial levels however, these treatments are not practical as they may be costly and time consuming. Various fungicides are used, soluble silicone based fungicides are used to help improve host response to infection, increasing the production of antifungal compounds by the plant (Bowen, et al., 1992; Rémus-Borel, Menzies, & Bélanger, 2005). Demethylation inhibitor fungicides are another treatment for powdery mildew however, resistance to these fungicides has been observed, such as in Podosphaera xanthii (Mcgrath, 2001). Powdery mildews have proven to be a taxonomically challenging group, A B with species level identification using morphological and current molecular techniques being problematic and inaccurate at times, morphological techniques especially requiring an in-depth knowledge of the group and a time using light microscopy to reach a potential ID. In order to develop more targeted fungicides and treatment plans for different Figure 2 Chasmothecia (A) and conidiophore (B) viewed using light powdery mildew species, we must microscopy. first be able to accurately identify them and understand their phylogeny in order to predict the response of more closely related taxa to certain treatments. Morphological characters for identification come from the asexual and sexual life stages (Figure 2), the asexual life stage (anamorph) conidiophore is common and can be found in virtually all powdery mildew samples but provide fewer characters for identification whereas the sexual life stage (teleomorph) chasmothecia are rarer and provide many more characters for identification. Chasmothecia can be rare as they are produced in times of stress such as over wintering, chasmothecia have never been observed in some species such as in the genera Oidium, the powdery mildew that infects tomato Oidium neolycopersici has never had chasmothecia observed (Jones, Whipps, & Gurr, 2001). Identification can be further complicated as in the past anamorphs and teleomorphs have been described as different species (Braun, 2012). The advent of molecular techniques has improved the ease of identifying powdery mildews. The ITS region has traditionally been used in fungal barcoding including the powdery mildews (Schoch et al., 2012). However, the ITS region has is less effective for powdery mildews for barcoding than other fungal groups, highlighted by the fact that it fails to provide a species level identification 1 out of every 4 times (Ellingham, 2017). As a result of the inaccuracy of ITS, few if any large-scale order wide phylogenies with a large number of taxa have been published, instead phylogenies covering the different genera of the powdery mildews are available, or phylogenies across the order but with few taxa (Braun et al., 2006). Powdery mildews are often thought of as infecting a small number of hosts, typically within the same plant genus or family, however a few species seem to infect a wide variety of hosts from multiple host families or even orders (JCunnington, Lawrie, & Pascoe, 2010). It is suspected that at least some of these could be species complexes and are multiple different species. Current methods are not strong enough to investigate whether this is true, examples of cases like this which require more molecular analysis include Podosphaera tridactyla a suspected species complex infecting the genus Prunus (Takamatsu et al., 2010), Golovinomyces cichoracearum is a well-known species complex, infecting different plant families including Asteraceae and Solanaceae (Cunnington et al., 2010). The inaccuracy of ITS and need for more accurate molecular tools and methods for identifying powdery mildews has been stated multiple times (Attanayake, et al., 2009; Shin, et al., 2019) Mcm7 is a potential gene that could be used to identify powdery mildews more easily, as well as build better phylogenies and barcoding databases and potentially explore species complexes (Ellingham, 2017). Mcm7 is a gene which codes for the mini-chromosome maintenance complex component 7, mini-chromosome maintenance proteins are essential for DNA replication and is therefore well conserved (Kearsey & Labib, 1998) making it a good potential candidate for use in phylogenetic analysis. Mcm7 has been used in other groups within the Ascomycota to investigate phylogenetic relationships (Raja, et al., 2011) including Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, and Sordariomycetes (Schmitt et al., 2009). This study will make use of the 2215 powdery mildew specimens stored in the RBG Kew fungarium and extractions made during Ellingham, 2017’s citizen science “powdery mildew survey” to test the hypotheses that DNA will be successfully extracted from RBG Kew fungarium using protocols developed by Ivanova et al., (2006) modified for use in the Mycology lab at RBG Kew. Primers used for PCR on fresh powdery mildew samples will be successful for fungarium specimens 10/20/30 years old of both the ITS region and Mcm7.For use in phylogenies it is hypothesised any phylogenies built using Mcm7 will have higher bootstrap support values than those without. It is