2022 Volume 63 Issue 5 Pages 222-234
Phylogenetic and morphological analyses have been conducted on powdery mildew specimens on different Berberis and Mahonia spp. from Asia, Europe and North America. The present study showed that collections of Erysiphe berberidis exhibit a high degree of morphological plasticity of the sexual morph, in contrast to their morphologically, rather uniform, asexual morph. In phylogenetic tree, all sequences cluster in a large strongly supported clade, without any indication and support for further differentiation into cryptic species. There are three morphological types within E. berberidis s. lat. that contain consistent differences. Until future multi-locus analyses will be available, we prefer to treat these ‘morphological types’ as varieties. These include Erysiphe berberidis var. berberidis, E. berberidis var. asiatica, and E. berberidis var. dimorpha comb. nov. (≡ Microsphaera berberidis var. dimorpha, M. berberidicola, and M. multappendicis). To fix the application of species name E. berberidis, an appropriate epitype was designated, with an ITS sequences.
The family Berberidaceae comprises approximately 17 genera and 650 species (Flora of China Editorial Committee, 2001). Berberis, a genus of about 500 species of deciduous and evergreen shrubs, is distributed in temperate and subtropical regions of Asia, Europe and the Americas (Sehrawat, Rajasekaran, & Pant, 2008). Berberis species are thought to contain medicinal properties and almost all parts of the plant are used for medical purposes (Mokhber-Dezfuli, Saeidnia, Gohari, & Kurepaz-Mahmoodabadi, 2014). Mahonia, the second largest genus of Berberidaceae, contains about 60 species (Flora of China Editorial Committee, 2001). The status of Mahonia as a genus of its own or as heterotypic synonym of Berberis has been controversially debated for a long time, but the latest phylogenetic studies are clearly in favor of Mahonia as a genus of its own (Yu & Chung, 2017). Mahonia spp. are used in the traditional Chinese medicine as a remedy for multiple ailments including tuberculosis, periodontitis, etc. (He & Mu, 2015).
The history of Erysiphe spp. on species of Berberis and Mahonia is long and intricate. Braun and Cook (2012) recognized seven taxa belonging to Erysiphe sect. Microsphaera, including E. berberidicola (F.L. Tai) U. Braun & S. Takam., E. berberidis var. berberidis DC., E. berberidis var. asiatica (U. Braun) U. Braun & S. Takam., E. dimorpha (Y.N. Yu & Z.Y. Zhao) U. Braun & S. Takam., E. golovinii (Domashova) U. Braun & S. Takam., E. multappendicis (Z.Y. Zhao & Y.N. Yu) U. Braun & S. Takam., E. sichuanica (Y.N. Yu) U. Braun & S. Takam., and E. thaxteri (Havryl. & U. Braun) U. Braun & S. Takam. All taxa, except for E. golovinii [originally described as Trichocladia golovinii Domashova (Domashova, 1959)] and E. thaxteri [originally introduced as Microsphaera thaxteri Havryl. & U. Braun (Havrylenko & Braun, 1998)], pertain to the E. berberidis complex. Tai (1946) introduced Microsphaera berberidicola F.L. Tai as a new powdery mildew on the basis of type material on Berberis sp. collected in Henan Province, China (holotype: HMAS11528, collected in 1939). Yu and Zhao (1981) identified the type host of M. berberidicola as B. dasystachya Maxim. Braun (1983) divided Microsphaera berberidis (DC.) Lév. into two varieties: M. berberidis var. berberidis (DC.) Lév. from Europe on Berberis vulgaris L. and M. berberidis var. asiatica U. Braun. Microsphaera berberidis var. asiatica was introduced for Asian collections on Berberis amurensis Rupr., which were characterized by having uniformly shorter chasmothecial appendages [1-2 (mostly 1-1.5) times as long as the diameter of the chasmothecia], with more regularly branched apices of chasmothecial appendages, vs. longer appendages [1-3 (mostly 1.5-2) times as long as the chasmothecial diameter], with more irregularly branched apices in M. berberidis var. berberidis. Braun and Cook (2012) maintained the former variety as Erysiphe berberidis var. asiatica. Yu and Zhao (1981) described a new species, Microsphaera multappendicis Z.Y. Zhao & Y.N. Yu, based on type material from Xinjiang Autonomous Region on Berberis amurensis. Braun (1983) identified powdery mildew on B. amurensis var. japonica (Regel) Rehder from Japan as M. multappendicis. Yu and Zhao (1981) published a new variety of M. berberidis on Berberis heteropoda Schrenk under the name M. berberidis var. dimorpha Y.N. Yu & Z.Y. Zhao, based on differences between M. berberidis var. dimorpha and M. berberidis var. berberidis in their chasmothecial appendages, and the numbers of asci and ascospores. Braun (1983) regarded these differences as sufficient to recognize M. berberidis var. dimorpha as a species of its own and introduced the combination Microsphaera dimorpha (Y.N. Yu & Z.Y. Zhao) U. Braun. Wei (1942) initially identified a Chinese powdery mildew collection on B. diaphana Maxim. as M. alni (Wallr.) E.S. Salmon, which was later described as a new species, M. sichuanica Y.N. Yu (Yu & Zhao, 1981). All taxa involved in this complex were later treated as species of the genus Erysiphe, based on Braun and Takamatsu (2000). In phylogenetic analysis of species of the Microsphaera lineage within the genus Erysiphe, based on ITS and 28S rDNA sequences, Takamatsu, Ito, Shiroya, Kiss, and Heluta (2015) verified that sequences retrieved from collections identified as E. berberidicola, E. berberidis, and E. multappendicis formed a single clade, without further subdivision. In these analyses, it was demonstrated that E. thaxteri forms an independent cluster with E. myoschili (Neger) U. Braun & S. Takam., distant from the E. berberidis clade (Takamatsu et al., 2015). Sequences obtained from E. sichuanica and E. golovinii are not yet available, so the phylogenetic status of these taxa remains unclear. In regards to E. dimorpha, there is only a sequence obtained from a non-type collection on Berberis carolii C.K. Schneid. In order to elucidate the phylogeny and taxonomy of the E. berberidis complex, comprehensive morphological re-examinations, based on Asian, European and North American collections, including type material, and broad-scale molecular analyses of the collections concerned have been performed with the goal of clarifying the taxonomy within this complex.
To examine the asexual morphs, small pieces of infected leaves were mounted in lactic acid and rehydrated by gently boiling the microscope slide, and then asexual structures were scrapped off from the leaf surface (Abasova et al., 2018a). For examinations of the sexual morphs, chasmothecia were stripped off from the leaf surface with a clean needle, mounted on a microscope slide, and examined in 3% NaOH using optical microscopy and differential interference contrast optical instruments and devices. Thirty measurements for each structure were made. Herbarium specimens were deposited at the Herbarium of Martin-Luther-University, Halle (Saale), Germany (HAL), Harvard University Farlow Herbarium (FH), the Herbarium of Mycology of Jilin Agricultural University (HMJAU), the Mycological Herbarium of Chifeng University (CFSZ), and the Herbarium of Iranian Research Institute of Plant Protection (IRAN).
Whole-cell DNA was extracted from mycelia or chasmothecia using the Chelex 100 as previously described (Walsh, Metzger, & Higuchi, 1991; Hirata & Takamatsu, 1996). The 5′ end of the 28S (large subunit) rDNA gene, was amplified by the polymerase chain reaction (PCR) with the primer pair PMITS3 (Takamatsu & Kano, 2001) and TW14 (Mori, Sato, & Takamatsu, 2000) for the first amplification. The first PCR products were used for the templates of the second PCR using the nested primer set PMITS3 and LSU2 (Scholin, Herzog, Sogin, & Anderson, 1994). Internal transcribed spacer (ITS) regions were amplified by the primers PMITS1 and PMITS2 (Cunnington, Lawrie, & Pascoe, 2003). Then, the PCR product was subjected to the second PCR using primer set PMITS10 (Bradshaw & Tobin, 2020) and PMITS2. The reaction components were 2 μL of total genomic DNA, 2.5 μL 10 × PCR buffer (Mg2+plus), 2 μL dNTP Mixture (10 mM total, 2.5 mM each), 1 μL each primer (20 ng/μL), 0.3 μL rTaq polymerase (5 U/μL) and sterile ddH2O up to a final volume of 25 μL. PCR reactions were conducted under the following thermal cycling conditions: an initial predenaturation of 3 min at 95 °C, followed by 35 cycles of 30 s of denaturation at 94 °C, 30 s at 56 °C for annealing and 30 s at 72 °C for extension, and a final extension for 8 min at 72 °C. A negative control lacking template DNA was included in each set of reactions. PCR products were subjected to electrophoresis in a 1.2% agarose gel in 0.5 × TBE buffer and sequenced by Sangon Biotech (Shanghai) with the same primers used in the PCR.
In Germany, whole-cell DNA was extracted from infected leaves with the DNeasy plant mini kit (Qiagen, Germany), following the manufacturers protocol. For the first PCR reaction the primers AITS (Bradshaw & Tobin, 2020) / TW14 were used followed by PM1 (Cunnington et al., 2003) / NLP2 (Mori et al., 2000). The reaction components were 2 μL of total genomic DNA, 12.5 μL MyFiTM Mix (Meridian Bioscience), 0.5 μL each primer (10 µM) and sterile ddH2O up to a final volume of 25 μL.
PCR reactions included an initial predenaturation of 3 min at 94 °C, followed by 35 cycles of 30 s of denaturation at 94 °C, 45 s at 48 °C for annealing and 60 s at 72 °C for extension, and a final extension for 10 min at 72 °C. Negative controls lacking template DNA were included. Amplicons were purified (ExoSAP-IT, ThermoFisher Scientific) and sequenced in both directions (LGC Genomics GmbH, Germany) using the primers PM1/NLP2. Consensus sequences were generated and edited (CLC Main Workbench version 21.0.1, Qiagen Digital Insights).
A phylogenetic tree was constructed from the ITS+28S sequences of Erysiphe specimens on Berberis spp. and other closely related powdery mildew species (Fig. 1). Erysiphe syringae Schwein. was selected as the outgroup taxon based on the phylogenetic analyses by Bradshaw, Braun, Götz, and Jurick (2021). Sequences were aligned and edited using MUSCLE in MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 (Kumar, Stecher, & Tamura, 2016). Alignments were deposited in TreeBASE (http://www.treebase.org) under the accession number 29257. A GTR+G+I evolutionary model was used for phylogenetic analyses as it is the most inclusive model of evolution and includes all other evolutionary models (Abadi, Azouri, Mayrose, & Pupko, 2019). A fixed parameter-rich model (such as GTR+G+I) can be used in lieu of running a test to select the most suitable evolutionary model (Abadi et al., 2019). The phylogeny was inferred using Bayesian analysis of the combined ITS and 28S rDNA using a Yule tree prior (Gernhard, 2008) and a strict molecular clock, in the program BEAST version 1.10.4 (Suchard et al., 2018). A single MCMC chain of 106 steps was run, with a burn-in of 10%. Posterior probabilities were calculated from the remaining 9,000 sampled trees. A maximum clade credibility tree was produced using Tree Annotator version 1.10.4 (part of the BEAST package). Stationarity was confirmed by running the analysis multiple times, which revealed convergence among runs. The resulting tree was visualized using FigTree version 1.3.1 (Rambaut, 2009). A maximum likelihood analysis was accomplished using raxmlGUI (Silvestro & Michalak, 2012) under the default settings with a GTR+G+I evolutionary model. Bootstrap analyses were conducted using 1000 replications (Felsenstein, 1985).
Amplicon sequences were deposited in GenBank (Table 1). The type specimen of Erysiphe multappendicis (HMAS13608) was too old to get sequences. Sequences from the ITS and 28S rDNA regions were combined for phylogenetic analyses. The phylogenetic analyses contained 64 sequences from powdery mildew specimens throughout the world. Of these 64 sequences, 41 were obtained for the current study. A maximum clade credibility tree was constructed using Bayesian analyses from the combined ITS and 28S sequences. Posterior probabilities > 90 are displayed followed by bootstrap values greater than 70% for the maximum likelihood (ML) analyses conducted (Fig. 1). The representative maximum clade credibility tree is illustrated in Figure 1. Molecular analyses revealed that sequences retrieved fell into one highly supported clade. No support was found for the different Erysiphe berberidis varieties.
| Taxon | Host Species | Vouchera | Collection Locality | GenBank Numberb |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM91999 | China | OL636058 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92000 | China | OL636059 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92001 | China | OL636060 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92002 | China | OL636061 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92003 | China | OL636062 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92004 | China | OL636063 |
| Erysiphe berberidis var. dimorpha (=Erysiphe multappendicis) | Berberis thunbergii | HMJAU-PM92005 | China | OL636064 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92006 | China | OL636065 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92007 | China | OL636066 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92008 | China | OL636067 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92009 | China | OL636068 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92010 | China | OL636069 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92011 | China | OL636070 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92012 | China | OL636071 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92013 | China | OL636072 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92014 | China | OL636073 |
| Erysiphe berberidis | Berberis thunbergii | HMJAU-PM92015 | China | OL636074 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92016 | China | OL636075 |
| Erysiphe berberidis | Mahonia fortunei | HMJAU-PM92017 | China | OL636076 |
| Erysiphe berberidis var. dimorpha (=Erysiphe multappendicis) | Berberis sp. | HMJAU-PM92018 | Russia | OL636077 |
| Erysiphe berberidis var. dimorpha (= Erysiphe berberidicola) | Berberis thunbergii | CFSZ 1433 | China | OL636078 |
| E. berberidis var. dimorpha (= Erysiphe dimorpha) | Berberis carolii | CFSZ 05267 | China | OL636079 |
| E. berberidis var. dimorpha (= Erysiphe multappendicis) | Berberis sp. | GUM1803 | Iran | OL690571 / OL690548 |
| E. berberidis var. dimorpha (= Erysiphe multappendicis) | Berberis thunbergii | GUM 1715 | Iran | OL690572 / OL690549 |
| Erysiphe berberidis var. berberidis | Berberis sp. | IRAN 10545 | Iran | OL690573 / OL690550 |
| Erysiphe berberidis var. asiatica | Berberis sp. | IRAN 11287 | Iran | OL690574 / OL690551 |
| Erysiphe berberidis | Berberis vulgaris | IRAN 10812 | Iran | OL690575 / OL690552 |
| Erysiphe berberidis var. asiatica | Berberis sp. | IRAN 15773 | Iran | OL690576 / OL690553 |
| E. berberidis var. dimorpha (=Erysiphe multappendicis) | Berberis integrimma | IRAN 15771 | Iran | OL690577 / OL690554 |
| E. berberidis var. dimorpha (= Erysiphe multappendicis) | Berberis vulgaris | GUM73 | Iran | AB104520 / AB103076 |
| Erysiphe berberidis var. asiatica | Berberis thunbergii | HAL 003450F | Austria | OL739135 |
| Erysiphe berberidis var. asiatica | Berberis thunbergii | HAL 003449F | Austria | OL739136 |
| Erysiphe berberidis var. berberidis | Berberis vulgaris | GLM-F78691 | Germany | OL739137 |
| Erysiphe berberidis var. berberidis | Berberis koreana | GLM-F99423 | Germany | OL739138 |
| Erysiphe berberidis var. asiatica | Berberis thunbergii | HAL 003460F | Germany | OL739139 |
| Erysiphe berberidis var. asiatica | Berberis thunbergii | HAL 003468 F | Germany | OL739140 |
| Erysiphe verberidis var. asiatica | Berberis thunbergii | HAL 003469 F | Germany | OL739141 |
| Erysiphe berberidis var. berberidis | Berberis × ottawensis | HAL 003470 F | Germany | OL739142 |
| Erysiphe berberidis | Nandina domestica | FH00941192 | USA | OL691787 |
| Erysiphe berberidis | Berberis repens | FH00941193 | USA | OL691788 |
| Erysiphe berberidis | Berberis nervosa | FH00941194 | USA | OL691789 |
a CFSZ=the Mycological Herbarium of Chifeng University; FH=Harvard University Farlow Herbarium; GUM=University of Guilan Mycological Herbarium; HAL=Martin-Luther-Universität Herbarium; HMJAU=the Herbarium of Mycology of Jilin Agricultural University; IRAN=the Herbarium of Iranian Research Institute of Plant Protection
b GenBank number refers to the ITS, 28S or the combined ITS+28S region (if available)
Morphology of collections assigned to Erysiphe berberidis s. lat. (including E. berberidicola, E. berberidis var. asiatica, E. dimorpha, and E. multappendicis)
Collections assigned to Erysiphe berberidis var. berberidis
Illustration: Braun and Cook (2012: 439, fig. 512 A).
Mycelium amphigenous, mostly epiphyllous, sometimes also on stems and fruits, effuse or in patches, thin, white, evanescent to almost persistent; hyphae about 4-7 µm wide; hyphal appressoria nipple-shaped to lobed; conidiophores arising from the upper surface of the mother cell, up to 80 µm long, foot-cells cylindrical, slender, 15-35(-45) × 5.5-10 µm, followed by 1-2 shorter cells or sometimes by a cell about as long as the foot-cell, forming conidia singly; conidia cylindrical or almost so, slender, both ends rounded, 20-50 × 9-18 µm, germ tubes terminal or almost so, short to moderately long 0.25-2.5 (median 0.75) times the conidial width excluding a few (up to 10%) showing longitubus pattern, conidial appressoria often mostly unlobed, up to 5 (median 0-3) lobes, highest lobing on rare 2-sided appressoria, many unlobed tubes with short unlobed extensions. Chasmothecia gregarious or scattered, 80-130 µm diam; peridium cells irregularly polygonal, 8-23 µm diam; appendages 5-22, equatorial, stiff to flexuous, 1-2.5(-3) times as long as the chasmothecial diam, 7-12 µm wide below, 0-1-septate, hyaline except pigmented at the very base, walls smooth to rough, thin but thicker towards base, sometimes moderately thick throughout, apices 3-5 times dichotomously branched, branching mostly loose and diffuse, primary and secondary branches often elongated, occasionally more tight and regular, tips of the ultimate branchlets straight, not recurved, also in fully mature specimens collected late in the season, rarely a single or few tips somewhat recurved; asci 4-10, ellipsoid-obovoid, saccate, 40-60 × 25-40 µm, sessile or short-stalked, 3-5(-6)-spored; ascospores ellipsoid-ovoid, 16-25 × 9-14 µm, colorless.
Additional specimens examined [Note - the description of E. berberidis var. berberidis is based on Braun and Cook (2012), which resulted from examinations of many specimens. The data was supplemented from additionally examined samples]: (1) On Berberis spp., specimens with mature chasmothecia collected late in season: GERMANY, on Berberis × ottawensis C.K. Schneid., Wolfenbüttel, Forstweg/Am Schwedendamm, Niedersachsen, 17 Sep 2021, M. Götz (HAL 3470 F); on Berberis vulgaris, Niesky, Sachsen, Landkreis Görlitz, 8 Nov 2006, H. Boyle & S. Hoeflich (GLM-F78691); on Berberis sp., Quitzdorf, OT Kollm, Landkreis Görlitz, Sachsen, 16 Nov 2006, H. Boyle (GLM-F78752); on Berberis koreana Palib., Klitten, Schäferei, Boxberg, Landkreis Görlitz, Sachsen, 7 Sep 2009, P. Gebauer (GLM-F99423); on Berberis vulgaris, between Betonwerk and Parneaer Grund, Könnern, Salzlandkreis, Sachsen-Anhalt, 19 Oct 2005, H. Zimmermann (GLM-F84402). IRAN, on Berberis sp., Shirvan, North Khorasan province, 12 Sep 1994, Abbasi, Fatehi and Foitzik (IRAN 10545). RUSSIA, on Berberis vulgaris, St. Petersburg, Pulkovsky park, 17 Sep 2007, H. Boyle (GLM-F80986). (2) On Mahonia aquifolium (Pursh) Nutt. with chasmothecia: GERMANY, Lankow, Schwerin, Mecklenburg-Vorpommern, 30 Sep 2015, B. Schurig (GLM-F85294); Rauschwalde, Görlitz, Sachsen, 18 Aug 2004, H. Boyle & S. Hoeflich (GLM-F533687); Görlitz, centre, Sachsen, 30 Nov 2008, S. Hoeflich (GLM-F89749); Teuchern, OT Prittitz, Burgenlandkreis, Sachsen-Anhalt, 22 Oct 2004, H. Jage (GLM-F66291); Kemberg, Landkreis Wittenberg, Sachsen-Anhalt, 24 Oct 2006, H. Jage (GLM-F85806); Leau, cemetery, Bernburg, Salzlandkreis, Sachsen-Anhalt, 13 Aug 2007, H. Zimmermann (GLM-F96380); Friedrichroda, Thüringen, 25 Sep 2004, H. Jage (GLM-F64231). SWEDEN, Uppsala, Uppland, 17 May 2007, H. Boyle (GLM-F79287). (3) Specimens (asexual morphs) from the USA: on Berberis repens Lindl., University of Washington, Main Campus, Seattle, Washington, 30 Jul 2018, M. Bradshaw (FH00941193); on Berberis nervosa Pursh, Oxbow Nursery, Carnation, Washington, 3 Aug 2018, M. Bradshaw (FH00941194); on Nandinia domestica Thunb., University of Washington, Main Campus, Seattle, Washington, 15 May 2018, M. Bradshaw (FH00941192); Specimens (asexual morphs) from the IRAN: on Berberis vulgaris, Amarlou, Guilan province, 13 Aug 1998, Seyed Akber Khodaparast (IRAN 10812); Specimens (asexual morphs) from the China: on Berberis vulgaris, Beijing Institute of Technology, 28 May 2018, Shu-rong Tang, Peng-lei Qiu & Dan-ni Jin (HMJAU-PM91999); Railway Station in Gaoping city, Shanxi Province, 17 Aug 2018, Shu-rong Tang & Li Liu (HMJAU-PM92000); Near hospital in Shennong Town, Gaoping City, Shanxi Province, 18 Aug 2018, Shu-rong Tang & Li Liu (HMJAU-PM92001); Taihang Park, Changzhi City, Shanxi Province, 19 Aug 2018, Shu-rong Tang & Li Liu (HMJAU-PM92002); Zhanqian Park, Jinzhong City, Shanxi Province, 20 Aug 2018, Shu-rong Tang & Li Liu (HMJAU-PM92003); Shanxi Agricultural University, Jinzhong City, Shanxi Province, 20 Aug 2018, Shu-rong Tang & Li Liu (HMJAU-PM92004); Hebei Normal University, Langfang City, Hebei Province, 20 Oct 2018, Shu-rong Tang & Li Liu (HMJAU-PM92006); Fenghuang Mountain, Tangshan city, Hebei Province, 21 Oct 2018, Shu-rong Tang & Li Liu (HMJAU-PM92007); Jinzhou University of Technology, Jinzhou city, Liaoning Province, 23 Oct 2018, Shu-rong Tang & Li Liu (HMJAU-PM92008); Chongqing Technology and Business University, 23 Nov 2018, Shu-rong Tang & Li Liu (HMJAU-PM92012); Gaoping City, Shanxi Province, 6 Jul 2020, Li Liu (HMJAU-PM92013); Shenyang City, Liaoning Province, 26 Sep 2020, Li Liu & Dan-ni Jin (HMJAU-PM92014); Jinjiang Star, Dalian City, Liaoning Province, 28 Sep 2020, Li Liu & Dan-ni Jin (HMJAU-PM92015); on Mahonia fortunei (Lindl.) Fedde, Sichuan Agricultural University, Chengdu, Sichuan Province, 18 Nov 2018, Shu-rong Tang & Li Liu (HMJAU-PM92009); Qingchengshan Railway Station, Chengdu city, Sichuan Province, 19 Nov 2018, Shu-rong Tang & Li Liu (HMJAU-PM92010); Sichuan food and drug school, Emeishan City, Sichuan Province, 22 Nov 2018, Shu-rong Tang & Li Liu (HMJAU-PM92011); Gushan Scenic Spot, Fuzhou City, Fujian Province, 23 Mar 2020, Jing Feng, Ren-jing Jiang & Li Liu (HMJAU-PM92016); Lenin Park, Wuyishan City, Fujian Province, 24 Mar 2020, Jing Feng, Ren-jing Jiang & Li Liu (HMJAU-PM92017).
Collections assigned to Erysiphe berberidicola (≡ Microsphaera berberidicola) Figs. 2, 3
Mycelium amphigenous, persistent, forming circular or irregular patches; hyphal cells usually straight to somewhat sinuous, 4.3-5.9 µm wide; hyphal appressoria solitary or in opposite pairs, nipple-shaped, slightly lobed to multilobed; conidiophores arising ± centrally from upper surface of the mother cell, erect, 30.6-53.8(58.1) × 6.3-8.9 µm, foot-cells straight or slightly curved at the base, cylindrical, about 15.9-28.8(35.5) × 5.2-7.8µm, followed by 1-2 shorter cells, forming conidia singly; conidia oblong-ellipsoid, cylindrical, 22.3-34.7 × 8.5-14.5 µm, length/width ratio 2.0-3.2, germ tubes longitubus pattern, straight or sinuous, produced in perihilar position of conidia. Chasmothecia scattered to almost gregarious, brown, depressed globose, 105-136 µm diam; peridium cells irregularly polygonal, about 12-25 × 10-19 µm diam; appendages 6-27, equatorial, straight, flexuous or curved, 0.6-2 times as long as the chasmothecial diam, 6-9 µm wide near the base, aseptate, hyaline or pigmented at the base, moderately thick throughout or thin above and thick towards the base, smooth to rough, apices 2-5 times loosely and widely dichotomously branched with primary branches sometimes recurved, tips of the ultimate branchlets straight or recurved; asci 3-7, ellipsoid, obovoid, 46-68 × 39-50 µm, sessile or short-stalked, 2-6-spored; ascospores ellipsoid, ovoid, 18-24 × 9-13 µm, colorless.
Specimens examined: CHINA, on Berberis thunbergii DC., Chifeng City, Inner Mongolia Autonomous Region, 3 Sep 2008, Tie-zhi Liu (CFSZ1433).
Collections assigned to Erysiphe berberidis var. asiatica (≡ Microsphaera berberidis var. asiatica) Figs. 4, 5
Mycelium amphigenous on leaves, forming persistent white patches or a complete cover, persistent; hyphae thin-walled, 4.1-6.3µm wide, straight or slightly sinuous; hyphal appressoria solitary or in opposite pairs, nipple-shaped or lobed; conidiophores arising mostly ± centrally from the upper surface of the mother cells, 29.7-59.6 × 6.1-9.8 µm, foot-cells straight, occasionally somewhat flexuous, cylindrical, about 11.4-30.1 × 5.3-8.5 µm, followed by 1-2 cells, forming conidia singly; conidia cylindrical, ellipsoid, 23.5-35.4 × 8.8-15.2 µm, length/width ratio 1.8-3.5, germ tubes short, simple or with lobed terminal appressorium. Chasmothecia scattered to gregarious, 80-125 µm diam; peridium cells irregularly polygonal, 8-17 × 6-15 µm diam; appendages 4-16, equatorial, straight to somewhat curved, (0.5-)0.75-1.5(-2) times as long as the chasmothecial diam, 7-11 µm wide, aseptate, hyaline or pigmented at the base, walls thin, becoming gradually thicker towards the base, smooth to rough, apices 3-5 times tightly and regularly branched, tips straight, not recurved, in some collections recurved with maturity; asci 3-6, ellipsoid, obovoid, 42-61 × 32-43 µm, sessile or short-stalked, 4-5 spored; ascospores ellipsoid, 17-27 × 9-15 µm, colorless.
Specimens examined: AUSTRIA, on Berberis thunbergii, Northern Tyrol, Kufstein, Sparchen, 14 Aug 2021, W.K. Hofbauer (HAL 3447 F); on Berberis thunbergii, Northern Tyrol, Kufstein, Sparchen, Anton Karg Street, 20 Aug 2021, W. Hofbauer (HAL 3449 F); on Berberis thunbergii, Northern Tyrol, Kufstein, Sparchen, Kaiserberg Street, 20 Aug 2021, W. Hofbauer (HAL 3450 F). CHINA, on Berberis amurensis, Chifeng City, Inner Mongolia Autonomous Region, 5 Aug 1997, Tie-zhi Liu (CFSZ97054); on Berberis poiretii C.K. Schneid., Chifeng City, Inner Mongolia Autonomous Region, 30 Aug 1997, Tie-zhi Liu (CFSZ97028); GERMANY, Berberis thunbergii, Rothenburg ob der Taubern, Landkreis Ansbach, Bayern, 22 Jun 2008, H. Boyle (GLM-F88871); on Berberis thunbergii, Halle (Saale), August Bebel Street, Sachsen-Anhalt, 8 Sep 2021, U. Braun (HAL 3460 F); on Berberis thunbergii, Wolfenbüttel, Schweiger Street, spots field, Niedersachsen, 29 Sep 2021, M. Götz (HAL 3468 F); Berberis thunbergii, Wolfenbüttel, Johan Street, church, Niedersachsen, 29 Sep 2021, M. Götz (HAL 3469 F); Löbau, Sachsen, 23 Nov 2006, H. Boyle & S. Hoeflich (GLM-F78784), with chasmothecia; Johnsdorf, Landkreis Görlitz, Sachsen, 19 Oct 2006, H. Boyle & S. Hoeflich (GLM-F 78481); Oberodewitz, Landkreis Görlitz, Sachsen, 19 Jul 2007, H. Boyle (GLM-F79650). IRAN, on Berberis sp., Tehran, Tehran province, 10 Nov 1998, Abbasi (IRAN 15773); on Berberis sp., Arasbaran, East Azerbaijan province, 27. Oct 1999, Gholam-Hasan Tavanaei (IRAN 11287).
Collection assigned to Erysiphe dimorpha Fig. 6
Chasmothecia gregarious or scattered, dark brown, 80-123 µm diam; peridium cells irregularly polygonal, 6-17 × 5-12 µm diam; appendages 5-17, equatorial, straight to curved, 0.7-1.9 times as long as the chasmothecial diam, 5-9 µm wide below, aseptate, hyaline, sometimes brown at the base, walls smooth to slightly rough, walls thin, becoming gradually thicker towards the base, apices 3-6 times regularly dichotomously branched, loose to denser, primary and/or secondary branches sometimes somewhat elongated, tips obtuse or at least not recurved, but tending to be recurved when mature; asci 4-10, ellipsoid, oblong-ellipsoid, obovoid, saccate, doliiform, 40-60 × 25-40 µm, sessile or short-stalked, 2-6-spored; ascospores, ellipsoid, 14-24 × 10-15 µm, colorless.
Specimens examined: CHINA, on Berberis carolii C. K. Schneid., Alxa League of Inner Mongolia Autonomous Region, 17 Sep 2005, Tie-zhi Liu (CFSZ05267).
Collections assigned to Erysiphe multappendicis (≡ Microsphaera multappendicis) Fig. 7
Mycelium amphigenous, mostly epiphyllous; hyphae thin-walled, 3.1-4.7 µm wide, straight or slightly sinuous; hyphal appressoria solitary, nipple-shaped or slightly lobed; conidia cylindrical, ellipsoid-cylindrical, 24-34 × 9-13 µm, length/width ratio 1.9-3.4, germ tubes almost terminal, longitubus pattern. Chasmothecia gregarious or scattered to gregarious, 85-138 µm diam; peridium cells irregularly polygonal, 9-19 × 6-13 µm diam; appendages 8-24(-29), arising equatorially, straight or curved or somewhat flexuous, 0.7-2.4 times as long as the chasmothecial diam, 6-9 µm wide, 0-2-septate, hyaline above, pigmented at the base, walls moderately thick throughout or thick below and thin above, smooth or somewhat rough, apices 2-5 times loosely dichotomously branched, 31-102 × 22-64 µm, primary branches often elongated, occasionally forked in the lower half, tips straight or recurved; asci 4-9(-10), broad ellipsoid, subglobose, saccate, 52-68(-73) × 37-53 µm, sessile or short-stalked, 3-6 spored; ascospores ellipsoid or ovoid, 20-28 × 11-17 µm, colorless.
Specimen examined: CHINA, on Berberis amurensis Rupr., Xinjiang Uygur, Ürümqui Shi, 15 Sep 1977, Zhen-Yu Zhao (HMAS 13608, holotype); on Berberis thunbergii, Wuquanshan Park, Lanzhou City, Gansu Province, 22 Sep 2018, Shu-rong Tang & Li Liu (HMJAU-PM92005);. IRAN, on Berberis vulgaris, Deileman-Kalishom road, km. 13, Guilan province, 13 Aug 1998, Seyed Akbar Khodaparast (GUM73); on Berberis integerrima Bunge, Yazd, Yazd province, 25 Jun 2010, Esmaeelzadeh-Hoseini (IRAN 15771); on Berberis sp., Qazvin, Qazvin province, 19 Jun 2019, Seyed Akbar Khodaparast (GUM1803); on Berberis thunbergii, Zarin Shahr, Isfahan province, 5 Aug 2017, K. Sharifi (GUM1715). RUSSIA, on Berberis thunbergii, Krasnodar region, Sochi, Kurortny Avenue, 9 Oct 2018, Timur S. Bulgakov (HMJAU-PM92018).
Taxonomic conclusion based on phylogenetic analyses and reassessments of the morphological traits of collections assigned to Erysiphe berberidis s. lat.
Erysiphe berberidis DC., Fl. franç. 2: 275, 1805 var. berberidis
MycoBank no.: MBT10004749.
Holotype: on Berberis sp., France, Paris, Mont-Rouge, garden of C. Cels (G 00122113).
Epitype (designated here): Germany, Niesky, Sachsen, Landkreis Görlitz, Berberis vulgaris, 8 Nov 2006, H. Boyle & S. Hoeflich (GLM-F78691);
Gene sequences from epitype: OL739137.
≡ Alphitomorpha penicillata γ berberidis (DC.) Wallr., Verh. Ges. Naturf. Freunde Berlin 1(1): 40, 1819.
≡ Erysibe penicillata var. berberidis (DC.) Link, Sp. pl. 4, 6(1): 114, 1824.
≡ Erysiphe penicillata a. berberidis (DC.) Fr., Syst. mycol. 3: 244, 1829.
≡ Podosphaera berberidis (DC.) Quel., Champ. Jur. Vosg. 3: 106, 1875.
≡ Microsphaera berberidis (DC.) Lév., Ann. Sci. Nat., Bot., Sér. 3, 15: 159, 381, 1851.
= Microsphaera berberidis f. mahoniae Jacz., Karm. Opred. Grib., Vip. 2. Muchn.-rosj. griby: 319, 1927.
= M. penicillata f. ilicis Potebia, Gribnye parazity vysshikh rasteny Kharkovskoy i smezhnykh guberny: 243, 1915.
≡ M. ilicis (Poteb.) Sandu-Ville, Mem. Sect. Şti. Acad. Română, Ser. III, 11(5): 205, 1936.
Morphology, see above under “Collections assigned to Erysiphe berberidis var. berberidis”. Chasmothecial appendages relatively long and stiff, 1-2.5(-3) times as long as the chasmothecial diam, apex mostly loosely branched, tips of the ultimate branchlets straight, also in mature samples, collected late in the season, rarely with a single or few somewhat recurved tips in mature samples.
Erysiphe berberidis var. asiatica (U. Braun) U. Braun & S. Takam., Schlechtendalia 4: 6, 2000
≡ Microsphaera berberidis var. asiatica U. Braun, Mycotaxon 18(1): 114, 1983.
Holotype: on Berberis amurensis var. japonica, Japan, Sapporo, 3 Oct 1925, Y. Homma (TNS-F-214333).
= M. berberidis auct. p.p.
= M. berberidis var. berberidis sensu Yu & Lai (1981).
Morphology, see above under “Collections assigned to Erysiphe berberidis var. asiatica”. Chasmothecial appendages uniformly short, (0.5-)0.75-1.5(-2) times as long as the chasmothecial diam, apex with a tendency to be more regularly and tightly branched, but primary branched often also elongated, tips of the ultimate branchlets straight, rarely a single or few somewhat recurved.
Erysiphe berberidis var. dimorpha (Y.N. Yu & Z.Y. Zhao) L. Liu & U. Braun, comb. nov.
MycoBank no.: MB844902
Basionym: Microsphaera berberidis var. dimorpha Y.N. Yu & Z.Y. Zhao, Acta Microbiol. Sin. 21(2): 145, 1981 [holotype: on Berberis heteropoda [= B. sphaerocarpa Kar. & Kir.], China, Guliu, Xinjiang Uygur, 23 Aug 1973, C.Y. Zhao 73206 (HMAS 33706)].
≡ Microsphaera dimorpha (Y.N. Yu & Z.Y. Zhao) U. Braun, Mycotaxon 18(1): 115, 1983.
≡ Erysiphe dimorpha (Y.N. Yu & Z.Y. Zhao) U. Braun & S. Takam., Schlechtendalia 4: 7, 2000.
= Microsphaera berberidicola F.L. Tai, Bull. Torrey Bot. Club 73(2): 115, 1946 [holotype: on Berberis dasystachya, China, Prov. Henan, Oct 1939, Wang, J.X. 507 (HMAS 11528)].
≡ Erysiphe berberidicola (F.L. Tai) U. Braun & S. Takam., Schlechtendalia 4: 6, 2000.
= Microsphaera multappendicis Z.Y. Zhao & Y.N. Yu, Acta Microbiol. Sin. 21(2): 146, 1981 [holotype: on Berberis amurensis, China, Xinjiang Uygur, Ürümqui Shi, 15 Sep 1977, Zhao (HMAS 13608).].
≡ Erysiphe multappendicis (Z.Y. Zhao & Y.N. Yu) U. Braun & S. Takam., Schlechtendalia 4: 11, 2000.
Morphology, see above under “Collections assigned to Erysiphe multappendicis”. Chasmothecial appendages in number and length largely agreeing with those in E. berberidis var. berberidis, but with a stronger tendency to become more curved, sinuous or flexuous, terminal branched part variform, more regularly and tightly to loosely branched, but tips of the ultimate branchlets always becoming recurved with maturity, at least a certain part of them.
A wide range of ITS and 28S rDNA sequences retrieved from collections assigned to E. berberidis var. berberidis and E. berberidis var. asiatica, E. berberidicola, E. dimorpha, and E. multappendicis, have been used to perform phylogenetic analyses and to construct a phylogenetic tree. Phylogenetic analyses confirmed a strongly supported E. berberidis clade, including sequences obtained from collections previously referred to as E. berberidis var. berberidis and E. berberidis var. asiatica, E. berberidicola, E. dimorpha, and E. multappendicis. All subgroups found in the tree are weakly supported and there is no evidence or support for any cryptic speciation. The weakly supported subgroupings seem to reflect a geographic aspect with the specimens from China grouping on the bottom of the tree. The structure of the E. berberidis clade is standard for clades composed of many sequences from throughout the world on a broad range of hosts. Trees with sequences from different regions of the world tend to show a certain degree of variation likely pertaining to the different species populations. In conclusion, the whole clade is considered to represent a single species, E. berberidis emend. There are three morphological types within E. berberidis s. lat. that contain consistent differences, collected on Berberis and Mahonia spp. However, sequences obtained from specimens belonging to these morphological types are spread over the entire phylogenetic tree, i.e., they do not form uniform well-supported clades or subclades, and cannot be treated as separate species. Genuine species should, at least ideally, form well-supported clades in phylogenetic analyses. Therefore, we prefer to consider these groups as (morphological) varieties until future multi-locus work can be accomplished.
The main difference between Erysiphe berberidis var. berberidis and E. berberidis var. asiatica lies in the length of the appendages: 1-3 times the diameter of the chasmothecia (mostly 1.5-2 times) in E. berberidis var. berberidis, and (0.5-)1-1.5(-2) times in E. berberidis var. asiatica. In this study, additional specimens were examined which revealed that E. berberidis var. berberidis on Berberis vulgaris (the type host), is extremely variable in morphology. In some collections, some chasmothecia have uniformly short appendages (1-1.5 times the chasmothecial diameter), whereas other chasmothecia have longer ones (1.5-2.5 times the chasmothecial diameter), i.e., chasmothecia with uniformly shorter and uniformly longer appendages may even be mixed in a single collection. Such collections on B. vulgaris (and other hosts with similar characteristics of the appendages) cannot be assigned to E. berberidis var. asiatica. On the other hand, genuine E. berberidis var. asiatica may also occur on B. vulgaris (Abasova et al., 2018b). In Europe, Erysiphe berberidis var. asiatica has undoubtedly been introduced. It occurs mainly on cultivated Berberis thunbergii, which is a popular shrub in gardens and often planted as hedges for a long time, but nevertheless first reports of E. berberis on this host in Germany are not older than 20 y (Jage, Klenke, & Kummer, 2010). Now the powdery mildew on B. thunbergii is rather common in Germany. However, sequences retrieved from collections that are morphologically assignable to E. berberidis var. asiatica cluster throughout the E. berberidis clade. There is no genetic support for E. berberidis var. asiatica in the form of a separate clade or subclade. Therefore, we prefer to maintain this taxon as a morphological variety.
Erysiphe berberidis and E. berberidicola (here referred to as E. berberidis var. E. dimorpha) can be distinguished by the chasmothecial appendages. In E. berberidicola, appendages are flexuous with a tendency to form more frequently recurved tips of the ultimate branchlets in contrast to E. berberidis which possesses stiff appendages with uniformly straight tips and only occasionally with recurved tips in fully mature specimens collected late in the season. Stiff or more flexuous appendages are to a certain extent correlated with the length of the appendages, i.e., longer appendages tend to be more flexuous, while shorter ones are stiffer. These gradual differences are the only characteristics that discriminate Asian collections previously assigned to E. berberidicola and E. berberidis var. berberidis. To evaluate the value of recurved tips within the E. berberidis complex, additional specimens, collected late in the season and fully mature, were examined (see above under E. berberidis var. berberidis). However, it could be confirmed that the ultimate tips in the branched part of the appendages of E. berberidis var. berberidis remain straight, also in fully mature samples (except for very few tips that may have become somewhat recurved with age). The reasons and influences for the development of recurved tips are not quite clear. The time of collection in the season seems to play a certain role, but other influences can also be a factor, above all the climate (warmer conditions seem to promote recurved tips).
The main difference between E. berberidicola and E. multappendicis lies in the appendages that tend to be more flexuous in the former and more straight and stiff in the latter species. The microscopic examination of the type specimen (HMAS13608) revealed that the appendages of E. multappendicis were straight to curved, i.e., this trait of the appendages, either more flexuous or stiffer, is only gradual and not diagnostic. On the other hand, E. berberidicola and E. multappendicis are both characterized by having straight to recurved tips of the ultimate branchlets in the terminal branched part of the appendages. Other postulated “differences” between E. berberidicola and E. multappendicis refer to the mode of branching in the apical portion of the appendages. Branched apices of E. berberidicola were described to be denser, with shorter secondary branches, which is in contrast to E. multappendicis which has terminal branchings that were described to be closer to E. berberidis. However, these “differences” are also only gradual and difficult to comprehend. The phylogenetic tree (Fig. 1) contains nine sequences obtained from samples previously identified as E. multappendicis. Seven sequences from Iran cluster in the upper group of the tree, and the two sequences from China in the lower part. Attempts to obtain sequences from type material failed.
The introduction of E. dimorpha (≡ Microsphaera berberidis var. dimorpha) was based on differences in the branched terminal part of the chasmothecial appendages in comparison to E. berberidis, i.e., the former has more tightly branched appendages with some curved tips of the ultimate branchlets (Yu & Zhao, 1981). These characteristics resemble those of E. berberidicola. Unfortunately, the type material of Microsphaera berberidis var. dimorpha is in poor condition, and could not be evaluated for morphological examinations and sequencing. However, another Chinese specimen (CFSZ05267), of E. dimorpha on Berberis carolii from Inner Mongolia Autonomous Region of China was evaluated in place of the type material , and had been tentatively referred to as E. dimorpha by Tie-zhi Liu (Liu, 2010 & Liu, 2022). This sample agrees well with E. dimorpha in terms of the number, length and branching pattern of the appendages. Based on the number and size of asci and ascospores, this specimen seems to be morphologically closer to E. multappendicis. The re-examination of this material showed that the morphological characteristics of this sample are consistent with E. berberidis, except that the appendages have more regularly and compactly branched apices and a higher proportion of recurved tips of the ultimate branchlets in the branched terminal part. The ITS sequence retrieved from this specimen clustered at the base of the E. berberidis clade. Currently, suitable collections of Microsphaera berberidis var. dimorpha on its type host, which could serve as epitype with ex-epitype sequence, are not available. Therefore, for the interim the sequence obtained from the collection on Berberis carolii (CFSZ05267) should be used as reference sequence for the name Microsphaera berberidis var. dimorpha.
It is currently not possible to clarify the taxonomic status of Erysiphe sichuanica (≡ Microsphaera sichuanica) with certainty. This species undoubtedly pertains to the E. berberidis complex, but the type material is limited, i.e., detailed morphological re-examinations are not possible, and attempts to isolate DNA from this material is not permitted. Thus, a clarification of its status requires newly collected specimens on the type host that are suitable for sequencing and epitypification.
Finally, there are Asian collections on some Asian hosts of Berberis and Mahonia spp. that were previously assigned to Microsphaera berberidicola, M. berberidis var. dimorpha and M. multappendicis. Most morphological traits of the chasmothecial appendages are very multiform and variable. Differences in the texture of the chasmothecial appendages, i.e., more stiff or more flexuous, are inconstant and barely applicable as distinguishing feature. This also holds true for the patterns of the branching types of the appendages, which are rather variable, similar to E. berberidis var. berberidis. The only consistent difference lies in the characteristic of the tips of the ultimate branchlets in the branched terminal portion of the appendages, which are often recurved when mature, as is seen with all Asian taxa cited above, which we now refer to as E. berberidis var. dimorpha.
However, this is not the end of the “E. berberidis story”. There are various open problems. Microsphaera berberidicola, M. berberidis var. dimorpha and M. multappendicis need to be epitypified with ex-epitype sequences of suitable new specimens. Furthermore, phylogenetic multi-locus approaches based on similarly broad sampling would be useful to reach a better understanding of speciation processes within the E. berberidis complex.
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
We would like to thank the curation team at FH for helping with the North American specimens. This research was supported by the National Natural Science Foundation of China (No.31970019, No.U21A20177) and partially support from National Science Foundation of Iran (No. 96007836). We are very grateful to the Herbarium Mycologicum Academiae Sinicae for allowing the re-examination of type specimens.
