Mycoscience: Advance Publication doi: 10.47371/mycosci.2020.12.003 Full Paper (Received July 3, 2020; Accepted December 9, 2020) J-STAGE Advance Published Date: March 13, 2021 Full paper Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology Miao Liua,d,*, Uwe Braunb,d, Susumu Takamatsuc, Sarah Hambletona, Parivash Shoukouhia, Kassandra R. Bissona, Keith Hubbarda a Biodiversity and Bioresources, Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada. b Martin Luther University, Institute of Biology, Department of Geobotany, Herbarium, Halle (Saale) 06099, Germany. c Faculty of Bioresources, Mie University, Tsu 514-8507, Japan. d First two authors should be considered as havingPublication equal contribution. *Corresponding author email: [email protected] Text: 61 pages; tables: 1; figures: 9 Supplemental materials: 4 Supplementary Figures, 1 Supplementary Appendix text Advance - 1 - Mycoscience: Advance Publication ABSTRACT A taxonomic revision of the hitherto monotypic genus Blumeria was conducted incorporating multi-gene sequence analyses, host preference data and morphological criteria. The sequenced loci included rDNA ITS, partial chitin synthase gene (CHS1), as well as fragments of two unnamed orthologous genes (Bgt-1929, Bgt-4572). The combined evidence led to a reassessment and a new neotypification of B. graminis s. str. (emend.), and the description of seven additional species, viz. B. americana sp. nov. (mainly on hosts of the Triticeae), B. avenae sp. nov. (on Avena spp.), B. bromi-cathartici sp. nov. (on Bromus catharticus), B. bulbigera comb. nov. (on Bromus spp.), B. dactylidis sp. nov. (on Dactylis glomerata as the main host, but also on various other hosts), B. graminicola sp. nov. (on Poa spp. as principal hosts, but also on various other hosts), and B. hordei sp. nov. (on Hordeum spp.). Synonyms were assessed, some were lectotypified, and questionable names previously associated with powdery mildew on monocots were discussed although their identities remained unresolved. Keys to the described species were developed. Keywords: Ascomycota, E3 ubiquitin-protein ligase, Poaceae, typification, unnamed orthologous genes Advance Publication - 2 - Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology 1. Introduction Powdery mildew fungi are responsible for a variety of common and important diseases of cereals and grasses (Poaceae), many have world-wide distributions and some cause significant yield losses and quality reduction in the cereal production (Everts, Leath, & Finney, 2001). The causal pathogen of the powdery mildew disease on wheat (Triticum aestivum L.) was first described as Erysiphe graminis DC. (De Candolle, 1815). Thereafter, the species has been redescribed many times under various names legitimately or illegitimately, as reviewed by Braun and Cook (2012). According to Speer (1975 [1973–1974]), Golovin erected Blumeria to separate wheat powdery mildew from other Erysiphe spp. based on Blumer’s observations (Blumer, 1933; Golovin, 1958). However, the erection of a new genus failed to comply with the rules of nomenclature in force at the time due to the absence of a Latin description, therefore the generic name, Blumeria Golovin 1958, was considered illegitimate. Speer redescribed the genus Blumeria properly, made the combination B. graminis (DC.) Speer and identified seven host species in addition to T. aestivum (Speer, 1975 [1973–1974]). Intra-specific variation was noted very early by Saccardo who distinguished the form E. graminis f. dactylis-glomeratae (Exs: Sacc. Mycoth. Ven. 606, 1876) from others. Jaczewski (1927) introduced 26 formae (host/substrate forms), corresponding to each host genus. Marchal (1902) introduced the first formae speciales (f. spp.) for E. graminis based on inoculation experiments. Several subsequent authors added additional formae and formae specialesAdvance (Marchal, 1902; Jaczewski, 1927; Publication Mains, 1933; Cherewick, 1944; Bunkina, 1967, 1973, 1974). In the latest classification of powdery mildew, B. graminis is the only species in this genus, which is classified in the monotypic tribe Blumerieae within Erysiphaceae (Braun - 3 - Mycoscience: Advance Publication & Cook, 2012). The host species have been recorded in 107 genera of Poaceae (Braun & Cook, 2012, also see Taxonomy section). Some are common hosts with a wide distribution, such as Avena, Bromus, Dactylis, Elymus, Hordeum, Poa, Secale, and Triticum. Some are rarely recorded, suggesting occasional host jumps by the pathogen. Multi-locus sequences analyses (MLSA) conducted by Inuma, Khodaparast and Takamatsu (2007) recovered nine lineages correlated with host specialization, and demonstrated reproductive isolation between the lineages. Among these lineages, some had a restricted host range of a single genus or species, i.e. lineages on Avena, Bromus, Diarrhena and Hordeum, while others infected host species in 2 or 3 genera, i.e. lineages of Dactylis, Poa and Triticum. Bromus hosted three lineages. Validation of these phylogenetic species (lineages) within the B. graminis complex required comprehensive morphological analyses. The purpose of this study was to describe and distinguish eight of them by a combination of molecular characters, morphology and putative host preference, and provide formal taxonomic names. The ninth lineage on Diarrhena (see Inuma et al., 2007) was not included because only one specimen was available. 2. Materials and methods 2.1. Fungal specimens and gDNA extraction For DNAAdvance MLSA and morphological examination, Publication 163 specimens of B. graminis s. lat. on cereal crops and various grasses were borrowed from five international herbaria: Canadian National Mycological Herbarium (DAOM), U.S. National Fungus Collections (BPI), Martin- - 4 - Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology Luther-Universität, Institut für Biologie, Bereich Geobotanik und Botanischer Garten Herbarium (HAL), the National Museum of Nature and Science (Tokyo, TNS), and Conservatoire et Jardin botaniques de la Ville de Genève (G). Previously developed DNA sequences, i.e. 49 rDNA-ITS, 25 CHS1 were downloaded from GenBank as references. An additional 126 specimens from Senckenberg Museum für Naturkunde Görlitz (GLM) were examined for morphology (by UB). For genomic DNA extraction, three or four 2 mm discs of infected leaf tissues were excised from specimens using Disposable Biopsy Punches Integra™ Miltex® (VWR, Mississauga Ontario, Canada). Alternatively, similar amounts of mycelia and/or chasmothecia were removed from leaf surfaces using tweezers. Macherey-Nagel Nucleomag® 96 Trace kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) on a KingFisher Flex magnetic particle processor (Thermo Fisher Scientific Oy, Vantaa, Finland), or an E.Z.N.A. Forensic DNA Kit (Omega Biotek, Inc., Norcross, Georgia, United States) were used for DNA extraction according to the manufacture’s protocols with minor modifications. The modifications were as follows: prior to extraction, samples were frozen using liquid nitrogen and ground using sterile disposable micro-centrifuge tube pestles (PES-15-B-SI Axygen, Corning, New York, USA), and DNA was suspended in 70 µL of elution buffer. Extracted DNA aliquots were stored at -20 °C and the stock DNA was stored at -80 °C. 2.2. PCR, sequencing and analyses The rDNAAdvance-18S (~100 bps)-ITS-28S (~50 bps, Publication abbreviated as ITS region in followed text) region was amplified with two forward and two reverse primers in various combinations, P7 (Mori, Sato, & Takamatsu, 2000), PMITS1, PMITS2 (Cunnington, Takamatsu, Lawrie, & Pascoe, - 5 - Mycoscience: Advance Publication 2003) and ITS4 (White, Bruns, Lee, & Taylor, 1990), and a portion of the chitin synthase gene (CHS1) with primers CHS1-E1f (Seko, Heluta, Grigaliunaite, & Takamatsu, 2011) and CHS1-B3r or CHS1-2r (Inuma et al., 2007). Fragments of two unnamed orthologous genes were amplified using primers designed using the published alignment of 93 phylogenetic informative genes from 31 whole genome sequences of B. graminis (Menardo, Wicker, & Keller, 2017). Geneious R10 primer design module (Biomatters, Aukland, New Zealand) was used to search for candidate oligos with all parameters set as default. The final primer sequences were: locus 1 (Bgt-1929 hypothetic protein exon 1–2 on chromosome 4): FM_27522F 5’- TGTGACGATGGAGATTGTGA-3’ and FM_27868R 5’-CCCATTCGCTGATTGCATAA-3’; and locus 2 (Bgt-4572 exon 3 on chromosome 9, 81% match with E3 ubiquitin-protein ligase in Venustampulla echinocandica Unter., Réblová & Bills): FM_110716F 5’- ATGGAAGGAGTTGATGCAGA-3’ and FM_110946R 5’-GAACTGCTCATCAATTCGCT-3’ (Etymology of primers: FM stands for the initial of Fabrizio Menardo, first author of the 31 B. graminis genome sequences; numbers = location on the concatenated alignment of 93 orthologous loci; F = forward, R = reverse). Polymerase chain reaction (PCR) was performed in 10 μL reactions containing 1 μL of gDNA, 1× Titanium Taq buffer (with 3.5 mM MgCl2), 0.1 mM dNTPs, 0.08 µM each of forward and reverse primer, 0.5× Titanium Taq DNA Polymerase (BD Biosciences, San Jose, California, USA), and 0.01 mg bovine serum albumin (BSA) on a TProfessional thermocycler (Biometra, Göttingen,Advance Germany). Touchdown thermocycling Publication protocols were used for rDNA-ITS and two anonymous loci: initial denaturation at 95 °C for 3 min, followed