AJBM Anales del Jardín Botánico de Madrid Anal. Jard. Bot. Madr. 0211-1322 1988-3196 Consejo Superior de Investigaciones Científicas ajbm.584 10.3989/ajbm.584 Articles New records of Anthracoidea pseudofoetidae (Anthracoideaceae) from Russia, and Microbotryum (Microbotryaceae) from Greece and Morocco Nuevos registros de Anthracoidea pseudofoetidae (Anthracoideaceae) de Rusia y Microbotryum (Microbotryaceae) de Grecia y Marruecos https://orcid.org/0000-0002-7242-3307 Denchev Teodor T. Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin St., 1113 Sofia, Bulgaria IUCN SSC Rusts and Smuts Specialist Group https://orcid.org/0000-0001-6301-1629 Denchev Cvetomir M. cmdenchev@yahoo.co.uk Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin St., 1113 Sofia, Bulgaria IUCN SSC Rusts and Smuts Specialist Group https://orcid.org/0000-0002-8286-1597 Begerow Dominik Universität Hamburg, Institute of Plant Science and Microbiology, Organismic Botany and Mycology, Ohnhorststr. 18, 22609 Hamburg, Germany https://orcid.org/0000-0002-0738-4233 Kemler Martin IUCN SSC Rusts and Smuts Specialist Group Universität Hamburg, Institute of Plant Science and Microbiology, Organismic Botany and Mycology, Ohnhorststr. 18, 22609 Hamburg, Germany Dueñas Margarita 01 12 2023 12 2023 80 2 e140 26 01 2023 03 09 2023 10 11 2023 © 2023 CSIC 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. Abstract

A rare smut fungus, Anthracoidea pseudofoetidae, is reported for the first time from Russia, based on a specimen from the Altai Republic, and Carex enervis is reported as a new host. Two new records for Greece, Microbotryum duriaeanum and M. moenchiae-manticae, and one for Morocco, Microbotryum moehringiae, are also presented. Microbotryum moehringiae is recorded for the first time from Africa. Cerastium brachypetalum subsp. roeseri is a new host record for Microbotryum duriaeanum. New molecular data are provided for these smut fungi. Updated phylogenetic trees for Anthracoidea and Microbotryum are also presented.

 Resumen

Anthracoidea pseudofoetidae, un raro hongo tizón, se cita por primera vez para Rusia, a partir de un espécimen de la República de Altai, and Carex enervis se reporta como un nuevo hospedador. Se presentan dos nuevos registros para Grecia, Microbotryum duriaeanum y M. moenchiae-manticae, y uno para Marruecos, Microbotryum moehringiae. Microbotryum moehringiae se registra por primera vez en África. Cerastium brachypetalum subsp. roeseri es un nuevo hospedador de Microbotryum duriaeanum. Se aportan nuevos datos moleculares sobre estos hongos. También se presentan árboles filogenéticos actualizados de Anthracoidea y Microbotryum.

 Phylogenetic analyses smut fungi Análisis filogenéticos hongos tizón Bulgarian National Science Fund KP-06-N51/10/16.11.2021 H2020 Research Infrastructures Programme DE-TAF-8193 This study was supported by the Bulgarian National Science Fund (Grant no. KP-06-N51/10/16.11.2021). The visit of T.T.D. & C.M.D. to the herbarium at the BGBM Berlin received support from the SYNTHESYS Plus Project http://www.synthesys.info, which is financed by the H2020 Research Infrastructures Programme (Grant no. DE-TAF-8193). Introduction

Smut fungi are plant parasites. They are characterized by a specific life cycle that alternates between a dikaryotic, plant-parasitic stage and a haploid, saprobic yeast phase, and by the presence of thick-walled teliospores. Smut fungi represent a phylogenetically heterogeneous group that has evolved three times independently in the Ustilaginomycotina, Microbotryales (Pucciniomycotina), and Entorrhizomycota (Begerow & al. 2014; He & al. 2022). They are mostly found on herbaceous plants, very rarely on woody hosts. More than 1900 species of smut fungi are currently known. The highest species diversity is reported from the northern hemisphere, with most species found in Europe and Asia (He & al. 2022).

The species of Anthracoidea Bref. (Anthracoideaceae, Ustilaginomycotina) have as hosts plants the genera Carex L., Carpha R.Br., Fuirena Rottb., Schoenus L., and Trichophorum Pers. (Cyperaceae). Anthracoidea form sori in female flowers around aborted nuts as ovoid, ellipsoidal or broadly ellipsoidal hard bodies (Denchev & al. 2021). The species of Microbotryum Lév. (Microbotryaceae, Pucciniomycotina) are parasites on hosts in ten dicot families. They form sori in various organs of the infected plants (flowers, anthers, ovules, filaments of stamina, branches of inflorescences, capitula, stems or leaves), filing them with single, subhyaline to dark reddish brown or dark purple, variously ornamented spores (Vánky 2011; Denchev & al. 2020; Kemler & al. 2020).

The aim of this study was to increase the knowledge about the geographical distribution and host specialization of Anthracoidea pseudofoetidae L. Guo and three seed-destroying species of Microbotryum (M. duriaeanum (Tul. & C. Tul.) Vánky, M. moehringiae (Togashi & Y.Maki) Vánky, and M. moenchiae-manticae) (Lindtner) Vánky, as well as to provide new molecular data for these smut fungi.

 Material and methods

The new records are based on collections that were discovered during a visit of two of the authors (T.T.D. & C.M.D.) to the herbarium at the Botanic Garden and Botanical Museum Berlin (B) in March 2022. Dried specimens from B were examined with a light microscope (LM) and scanning electron microscope (SEM). For LM observations and measurements, spores were mounted in lactoglycerol solution (w : la : gl = 1 : 1 : 2) on glass slides, gently heated to boiling point for rehydration, and then cooled. The measurements of spores are given in the form: min-max (extreme values) (mean ± 1 standard deviation). For SEM, spores were attached to specimen holders by double-sided adhesive tape and coated with gold in an ion sputter. SEM images were taken with a Hitachi FE SEM 8010. Information on shapes of spores is arranged in descending order of frequency. The descriptions of spore length range and spore ornamentation of Anthracoidea pseudofoetidae are in accordance with Denchev & al. (2020: 11) and Denchev & al. (2013), respectively. The descriptions given below are based entirely on the specimens examined.

Genomic DNA of the herbarium specimens was isolated using the myBudget Plant DNA KitTM (Bio-Budget Technologies GmbH, Germany) using the SLS protocol according to the manufacturer’s instructions. PCR of the ITS and/or the large subunit (LSU) region of the rDNA was performed using the primer pairs ITS1F/ITS4 and LR0R/LR6, respectively. Amplicons were purified using a modified ExoSAP (1:5 diluted in ddH2O; New England Biolabs, USA) protocol and subsequently sequenced using the respective forward and reverse primers with the Big-DyeTM Terminator Cycle Sequencing Kit V3.1 (Applied Biosystems) on an ABI3130xl Genetic Analyser at the Faculty of Biochemistry of the Ruhr-Universität Bochum, Germany. Forward and reverse reads were quality checked and assembled in Geneious 10.2.6 (Biomatters Ltd, New Zealand).

Multiple alignments were inferred using the online version of MAFFT 7 (Katoh & Standley 2013) with either the E-INS-i (Microbotryum ITS and Anthracoidea LSU) or L-INS-i option (Microbotryum LSU). Leading and trailing gaps, as well as ambiguous sites were removed using GBlocks (Castresana 2000) implemented in SeaView (Gouy & al. 2010), whereby smaller final blocks, gap positions, and less strict flanking positions were allowed. For Microbotryum the ITS and LSU regions were concatenated before phylogenetic analysis. RAxML-NG (Kozlov & al. 2019) implemented in raxmlGUI 2.0 (Edler & al. 2021) was used for phylogenetic inference and bootstrapping (1000 replicates). Before phylogenetic analyses, ModelTest-NG (Darriba & al. 2020) was used to select the most appropriate nucleotide substitution model (Anthracoidea: GTR+I+G; Microbotryum: GTR+I+G). Bootstrap values ≥ 50 are shown above branches.

 Results and discussion Taxonomic treatment

Anthracoidea pseudofoetidae L. Guo, Fungal Diversity 21: 84, 2006. Type: China, Xizang, Gégyai Xian, Alingshan, alt. 5200 m, in ovaries of Carex pseudofoetida Kük., 15 August 1976, Qinghai-Xizang expedition 13486 (HMAS 130321, holotype; isotype HUV 20091). Fig. 1.

<italic>Anthracoidea pseudofoetidae</italic> on <italic>Carex enervis</italic> (B 10 0240205): <bold>a,</bold> habit; <bold>b,</bold> spores in LM; <bold>c, d,</bold> spores in SEM. Scale bars: a = 1 mm, b = 10 μm, c, d = 5 μm.

Infection local. Sori in some female flowers, around aborted nuts as broadly ellipsoidal, subglobose or ovoid hard bodies, 1.0-1.7 mm long, initially covered by a thick, blackish brown peridium that later flakes away exposing a blackish brown, powdery on the surface spore mass. Spores very small-sized, irregularly rounded, subglobose, broadly ellipsoidal, ovoid or ellipsoidal, (8.5-)9-11.5(-12.5) ´ (8-)8.5-10.5(-11.5) (10.2 ± 0.7 ´ 9.3 ± 0.6) μm (n = 100), medium reddish brown; wall unevenly thickened, 0.9-1.5 μm thick, with a few paler, rounded areas with thinner wall (0.5-0.9 μm thick); internal swellings, light refractive areas, and protuberances absent; spore surface minutely verruculose, spore profile not affected. In SEM, spore wall depressed on 3-6 places, ornaments up to 0.15 μm high, usually solitary and sparsely spaced, occasionally partly confluent, forming short rows or small groups.

Specimen examined.-Russia, Altai Republic, Kosh-Agachskiy District, 20 km ENE Kosh-Agach, valley of the Kokorya River, 50°05′30″N, 88°53′57″E, alt. 2080 m, on Carex enervis C.A.Mey., 9 Aug 2008, leg. L. Martins 2476, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 0240205; GenBank LSU OQ067238).

Hosts and Distribution.-On Cyperaceae: on Carex enervis, C. maritima Gunnerus, and C. pseudofoetida. Known from Asia (China and Russia) and North America (Greenland).

Comments.-Anthracoidea pseudofoetidae was recovered as a sister species to a group of A. aspera, A. arenaria, and A. cf. karii, however with low bootstrap support (Fig. 2, Table 1).

<italic>Anthracoidea</italic> specimens, plant hosts, vouchers, and NCBI Genbank accession numbers of the large subunit (LSU) of nuclear ribosomal DNA sequences used in the phylogenetic analysis. The single newly generated sequence is shown in bold.
Species Host Voucher Accession
Anthracoidea arenaria Carex arenaria PUL F916 AY563606
A. aspera C. chordorrhiza HMH 2774 AY563607
A. baldensis C. baldensis HMH 2861 AY563599
A. bigelowii C. bigelowii HMH 2733 AY563566
A. bigelowii C. bigelowii HMH 927 AY563567
A. bigelowii C. bigelowii HMH 2736 AY563568
A. buxbaumii C. buxbaumii HMH 2744 AY563582
A. capillaris C. capillaris HMH 2769 AY563596
A. caricis C. pilulifera HMH 3364 AY563589
A. caricis-albae C. alba HMH 2869 AY563594
A. caricis-albae C. alba HMH 2873 AY563595
A. caricis-meadii C. meadii ISC 428408 JN863083
A. carphae Carpha alpina M-40218 AY563614
A. curvulae Carex curvula HMH 3912 AY563611
A. curvulae C. curvula HMH 2380 AY563612
A. elynae C. myosuroides HMH 3958 AY563609
A. elynae C. myosuroides M 6794 AY563610
A. globularis C. globularis HMH 2422 AY563593
A. hallerianae C. halleriana SOMF 30199 MT628660
A. hallerianae C. halleriana SOMF 30201 MT628657
A. heterospora C. elata HMH 2438 AY563600
A. heterospora C. elata HMH 921 AY563601
A. hostianae C. hostiana HeRB 4706 AY563581
A. inclusa C. rostrata HMH 2883 AY563605
A. irregularis C. ornithopoda HMH 3480 AY563590
A. irregularis C. ornithopoda HMH 3520 AY563591
A. irregularis C. digitata HMH 933 AY563592
A. karii C. brunnescens HMH 2777 AY563575
A. karii C. echinata HMH 3892 AY563576
A. karii C. echinata HMH 3676 AY563577
A. karii C. echinata HMH 3414 AY563578
A. karii C. lachenalii HMH 2644 AY563579
A. karii C. paniculata HMH 3890 AY563574
A. cf. karii C. davalliana HMH 3898 AY563608
A. lasiocarpae C. lasiocarpa HMH 972 AY563583
A. limosa C. limosa HMH 2428 AY563572
A. limosa C. limosa HMH 2790 AY563573
A. misandrae C. atrofusca HMH 2653 AY563584
A. pamiroalaica C. koshewnikowii KRA F-2012-146 KT006854
A. paniceae C. panicea HMH 2818 AY563580
A. pratensis C. flacca HMH 3599 AY563563
A. pratensis C. flacca HMH 1164 AY563564
A. pratensis C. flacca HMH 3870 AY563565
A. pseudofoetidae C. enervis B 10 0240205 OQ067238
A. rupestris C. rupestris HMH 3948 AY563598
A. cf. rupestris C. glacialis HMH 3692 AY563588
A. sclerotiformis C. punicea M 4946 AY563613
A. sempervirentis C. firma HMH 3612 AY563585
A. sempervirentis C. ferruginea HMH 3616 AY563587
A. sempervirentis C. sempervirens HMH 3950 AY563586
A. subinclusa C. hirta HMH 3700 AY563604
A. subinclusa C. riparia PUL F915 AY563603
A. subinclusa C. vesicaria HMH 2809 AY563602
A. turfosa C. dioica HMH 2797 AY563571
A. turfosa C. heleonastes HMH 2662 AY563569
A. turfosa C. parallela HMH 2523 AY563570
A. vankyi C. muricata HMH 1305 AY563597
Maximum Likelihood phylogeny inferred using RAxML-NG based on LSU sequence data representing the species of Anthracoideaceae. The phylogeny was rooted using <italic>Anthracoidea carphae</italic> and <italic>A. sclerotiformis</italic> according to <xref ref-type="bibr" rid="B18">Hendrichs & al. (2005)</xref>. Values above branches indicate bootstrap values inferred by 1000 replicates; only values ≥ 50% are shown.

Anthracoidea pseudofoetidae is recorded for the first time from Russia, on a new host plant species, Carex enervis. This smut fungus is an Arctic-alpine species with a restricted distribution (Denchev & al. 2020). It has been previously known only on Carex pseudofoetida from the type locality in China (Xizang) (Guo 2006), and on C. maritima from two localities in the High Arctic of Greenland (Denchev & al. 2020). The species of Anthracoidea on Carex are host-specific smut fungi restricted to sedges belonging to the same or closely related sections (Denchev & al. 2021). All three hosts of A. pseudofoetidae were traditionally placed in Carex sect. Foetidae (L.H. Bailey) Kük. (Egorova 1999; Reznicek 2002). Currently, these sedges are considered belonging to the Disticha clade that includes 27 species (Roalson & al. 2021). Most species in this clade are distributed in North America and temperate Eurasia. Carex pseudofoetida and C. enervis are Central Asiatic species while C. maritima has a bipolar distribution (Egorova 1999; Reznicek 2002). Anthracoidea pseudofoetidae can be easily distinguished from other Anthracoidea species by a suite of distinctive features that includes: (i) sori covered by a thick, dark brown peridium; (ii) very small-sized spores; and (iii) a characteristic spore wall, depressed on 3-6 places where the wall is paler and thinner (Denchev & al. 2020).

Microbotryum duriaeanum (Tul. & C. Tul.) Vánky, Mycotaxon 67: 43, 1998; Ustilago duriaeana Tul. & C. Tul., Ann. Sci. Nat., Bot., Sér. 3, 7: 105, 1847. Type: ALGERIA, Tlemcen, on Cerastium glomeratum Thuill., 30 May 1842, M.C. Durieu de Maisonneuve (PC s.n., holotype). Fig. 3a-c.

<italic>Microbotryum duriaeanum</italic> on <italic>Cerastium brachypetalum</italic> subsp. <italic>roeseri</italic> (B 10 1224088): <bold>a,</bold> capsule filled with spores; <bold>b, c,</bold> spores in LM (in median and surface view, respectively). <italic>Microbotryum moehringiae</italic> on <italic>Moehringia trinervia</italic> (B 10 0298453): <bold>d,</bold> capsule filled with spores; <bold>e,</bold> spores in LM; <bold>f,</bold> spores in SEM. <italic>Microbotryum moenchiae-manticae</italic> on <italic>Moenchia mantica</italic> (B 10 0255208): <bold>g,</bold> capsule filled with spores; <bold>h,</bold> spores in LM; <bold>i,</bold> spores in SEM. Scale bars: a, d, g = 1 mm, b, c, e, h = 10 μm, f, i = 5 μm.

Infection systemic. Sori destroying the seeds, filling the capsules initially with a semi-agglutinated, later powdery, dark sepia (based on Rayner 1970) or dark purplish date (based on the Color identification chart of Anonymous 1969) spore mass. Spores subglobose, globose or broadly ellipsoidal, sometimes ovoid, (12-)13-16(-17) × (11.5-)12.5-14.5(-15.5) (14.4 ± 0.9 × 13.3 ± 0.8) μm (n = 100), light to medium reddish brown; wall reticulate, 1.9-2.5 μm thick (including reticulum), meshes (5-)6-8(-9) per spore diameter, polyhedral or irregular, 0.6-2.7(-3.5) μm wide, muri (0.8-)1.0-1.4(-1.7) μm high. In SEM, the meshes smooth, often with a hemispherical protuberance at the bottom. The description is based on the infected specimen of Cerastium brachypetalum subsp. roeseri (Boiss. & Heldr.) Nyman.

Specimens examined.-GREECE. Western Macedonia Region: Grevena, W of Kallithea, 39°51′01″N, 21°19′14″E, alt. 1050 m, on Cerastium brachypetalum subsp. roeseri, 16 May 2012, leg. R. Willing & E. Willing 219.396, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 1224088; GenBank ITS OQ096632, LSU OQ067234). Western Macedonia Region: Grevena, Milea, 40°11′00″N, 21°28′22″E, alt. 670 m, on Cerastium semidecandrum L., 24 May 2012, leg. R. Willing & E. Willing 221.910, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 0499209; GenBank ITS OQ096633, LSU OQ067235).

Hosts and Distribution.-On Cerastium spp. (Caryophyllaceae). Known from Europe, North Africa, Asia, and North America.

Comments.-Both specimens of Microbotryum duriaeanum from this study fell into a clade with all other M. duriaeanum specimens sequenced up to date. The specimen on Cerastium brachypetalum subsp. roeseri emerged sister to all other M. duriaeanum specimens, whereas the specimen on C. semidecandrum formed a polytomy with the other specimens (Fig. 4, Table 2).

Maximum Likelihood phylogeny inferred using RAxML-NG based on combined LSU and ITS sequence data representing the species of Microbotryaceae. The tree is rooted with <italic>Microbotryozyma collariae</italic> and <italic>Bauerago abstrusa</italic>. Values above branches indicate bootstrap support inferred by 1000 replicates; only values ≥ 50% are shown. Smut fungi specimens, plant hosts, vouchers, and NCBI accession numbers of the sequences used in the phylogenetic analysis of <italic>Microbotryum</italic>. Newly generated sequences are shown in bold. LSU: large subunit of the nuclear ribosomal DNA.
Species Host Voucher ITS LSU
Bauerago abstrusa Juncus sp. HUV18526 DQ238719 EF621955
Microbotryozyma collariae n/a ATCC:MYA-4666 JN849458 JN849460
Microbotryum adenopetalae Silene adenopetala KRAM F 55201 DQ366848 DQ366876
M. afromontanum Cerastium afromontanum BRIP: HUV 20888 MN657185 MN657208
M. alpinum Pinguicula alpina TUB 015871 EF621944 EF621995
M. anomalum Fallopia convolvulus GLM 59392 EF621921 EF621960
M. arcticum Silene uralensis subsp. arctica SOMF 29999 MK474659 MK474658
M. bardanense S. moorcroftiana KRAM F 54962 DQ366856 DQ366877
M. betonicae Stachys alopecuros GZU 86-98, Scheuer 4983 EF621927 EF621967
M. bistortarum Bistorta vivipara M-0066101 DQ238709 EF621969
M. bosniacum Koenigia alpina M-0066097 DQ238740 EF621977
M. cardui Carduus acanthoides SOMF 30191 MN657187 MN657210
M. cardui C. crispus SOMF 30190 MN657188 MN657211
M. cephalariae Cephalaria humilis BRIP: HUV 10980 MN657203 MN657212
M. chloranthae-verrucosum Silene chlorantha B 70 0007571 AY877404 DQ366878
M. cichorii Cichorium intybus LE 231009 MN657189 MN657213
M. cordae Persicaria hydropiper B 70 0006023 DQ238726 EF621978
M. coronariae Silene flos-cuculi KR 23797 KC684887 KC684886
M. dianthorum Dianthus monspessulanus TUB 011802 AY588080 DQ366871
M. ducellieri Arenaria leptoclados MA-Fungi 37800 MN657190 MN657214
M. duriaeanum Cerastium brachypetalum BRIP: HUV 3638 MN657192 MN657216
M. duriaeanum C. brachypetalum TUB 019596 MN657191 MN657215
M. duriaeanum C. brachypetalum MA 461701 MN657194 -
M. duriaeanum C. brachypetalum subsp. roeseri B 10 1224088 OQ096632 OQ067234
M. duriaeanum C. gracile SOMF 30188 MN657193 MN657217
M. duriaeanum C. semidecandrum B 10 0499209 OQ096633 OQ067235
M. emodense Persicaria chinensis FO17516/DB1037 DQ238743 AY512858
M. flosculorum Knautia arvensis BRIP: HUV 20230 MN657195 MN657218
M. heliospermatis Heliosperma pusillum TUB 019570 HQ832086 HQ832087
M. holostei Holosteum umbellatum B 70 0006032 DQ238722 EF621981
M. intermedium Scabiosa lucida M-0066090 DQ238723 EF621982
M. jehudanum Silene colorata BRIP: HUV 18306 MN657196 MN657219
M. lagerheimii Atocion rupestre TUB 011817 AY588100 DQ366874
M. liroi Pinguicula villosa KRAM 296281 KY421500 KY421502
M. lychnidis-dioicae Silene latifolia TUB 011795 AY588096 DQ366886
M. majus S. otites B 70 0006042 AY877419 DQ366858
M. marginale Bistorta officinalis TUB 015881 EF621940 EF621989
M. minuartiae Minuartia recurva TUB 012519 DQ366853 DQ366862
M. moehringiae Moehringia trinervia BRIP: HUV 19024 MN657197 MN657220
M. moehringiae M. trinervia B 10 0298453 OQ096635 OQ067237
M. moenchiae-manticae Moenchia erecta K(M) 106303 MN657198 MN657221
M. moenchiae-manticae M. mantica BRIP: HUV 4126 MN657199 MN657222
M. moenchiae-manticae M. mantica B 10 0255208 OQ096634 OQ067236
M. onopordi Onopordum bracteatum M-0066075 DQ238735 EF621990
M. parlatorei Rumex maritimus B 70 0007574 DQ238736 EF621991
M. pinguiculae Pinguicula vulgaris STU 10004567401 KY421498 KY421501
M. polycnemoides Polygonum polycnemoides SOMF 30200 MN989380 MN989381
M. pustulatum Bistorta officinalis TUB 015872 EF621947 EF621998
M. reticulatum Persicaria lapathifolia M-0066067 DQ238730 EF621999
M. salviae Salvia pratensis TUB 015858 EF621922 EF621962
M. saponariae Saponaria officinalis TUB 011809 AY588089 DQ366887
M. scabiosae Knautia arvensis TUB 011789 AY588083 DQ366861
M. scabiosae K. longifolia TUB 015875 EF621950 EF622003
M. scolymi n/a n/a AY800113 -
M. scorzonerae Scorzonera humilis M-0066054 DQ238731 EF622006
M. scorzonerae S. humilis TUB 015878 EF621953 EF622007
M. shastense Polygonum shastense M-0066053 DQ238739 EF622008
M. shykoffianum Dianthus sylvestris TUB 011800 AY588082 DQ366857
M. silenes-acaulis Silene acaulis TUB 019585 JN223408 JN223413
M. silenes-dioicae S. dioica TUB 012114 AY877416 DQ366868
M. silenes-inflatae S. vulgaris TUB 011793 AY588105 DQ366884
M. silenes-saxifragae S. saxifraga KR 23889 JN000073 JN000079
M. silybum Silybum marianum SOMF 30193 MN657200 MN657224
M. stellariae Stellaria graminea TUB 011807 AY588109 DQ366872
M. stygium Rumex acetosa M-0066047 DQ238737 EF622009
M. succisae Succisa pratensis M-0066045 MN657204 MN657225
M. succisae S. pratensis B 700007625 MN657201 MN657226
M. superbum Dianthus superbus TUB 011799 AY588081 DQ366867
M. tenuisporum Persicaria glabra M-0066041 DQ238727 EF622011
M. tragopogonis-pratensis Tragopogon pratensis TUB 015879 EF621954 EF622014
M. tragopogonis-pratensis T. pratensis TUB 012509 DQ238733 EF622012
M. tuberculiforme Polygonum runcinatum M-0066035 DQ238744 EF622015
M. violaceoirregulare Silene vulgaris TUB 011816 AY588104 DQ366875
M. violaceoverrucosum S. viscosa TUB 011815 AY588103 DQ366882
M. violaceum S. nutans TUB 011818 AY588099 DQ366880
Sphacelotheca cf. koordersiana n/a JAG 55 AFTOL-ID 1917 DQ832221 DQ832219

Microbotryum duriaeanum is reported here as a new record for Greece, on two host plants, among which Cerastium brachypetalum subsp. roeseri is recorded for the first time.

Microbotryum moehringiae (Togashi & Y.Maki) Vánky, Mycotaxon 67: 46, 1998; Ustilago moehringiae Togashi & Y.Maki, Ann. Phytopathol. Soc. Japan 10: 139, 1940. Type: Japan, Fukuoka Pref., Yoshikawa-mura, on Moehringia trinervia var. platysperma (Maxim.) Makino, 7 May 1938, Y. Maki (TNS s.n., holotype). Fig. 3d-f.

Infection systemic. Sori destroying the seeds, filling the capsules initially with a semi-agglutinated, later powdery, dark bay (based on Rayner 1970) or bay (based on the Color identification chart of Anonymous 1969) spore mass. Spores subglobose, globose or broadly ellipsoidal, sometimes ovoid, (10.5-)11.5-13.5(-14.5) × (10-)11-12.5(-13.5) (12.8 ± 0.6 × 11.8 ± 0.6) μm (n = 100), light to medium reddish brown; wall reticulate, 1.4-2.2 μm thick (including reticulum), meshes (4-)5-6(-7) per spore diameter, polyhedral or irregular, (0.7-)1.0-2.7(-3.3) μm wide, muri 0.6-1.1(-1.3) μm high. In SEM, meshes smooth, often with a hemispherical protuberance at the bottom.

Specimen examined.-MOROCCO. Fès-Meknès Region: Foret de Jaba, ca 15 km from El Hajeb, road to Ifrane, 33°36′N, 5°17′W, alt. 1400 m, on Moehringia trinervia (L.) Clairv., 9 Jun. 1992, leg. B. Valdés & al., 5th Iter Mediterraneum of OPTIMA, Morocco, Jun. 1992, 04-0237, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 0298453; GenBank ITS OQ096635, LSU OQ067237).

Hosts and Distribution.-On Moehringia spp. (Caryophyllaceae). Known from Europe, North Africa, and Asia.

Comments.-The specimen of M. moehringiae from this study formed a well-supported clade with the only other specimen of M. moehringia (on Moehringia trinervia) sequenced so far, indicating that M. moehringiae is indeed a distinct species (Fig. 4, Table 2).

Microbotryum moehringiae is recorded for the first time from Africa. It has been previously reported for Asia (Japan, on Moehringia trinervia var. platysperma; Denchev & al. 2006) and Europe (Spain, on M. pentandra J.Gay; Almaraz 1999, as ‘Ustilago duriaeana’, and France, on M. trinervia; Kemler & al. 2020).

Microbotryum moenchiae-manticae (Lindtner) Vánky, Mycotaxon 67: 46, 1998; Ustilago moenchiae-manticae Lindtner, Bull. Mus. Hist. Nat. Serbe, Ser. B 3-4: 33, 1950. Type: Serbia, Rudnik near Milanovac, on Moenchia mantica Bartl., 15 Jun. 1946, V. Lindtner (HUV 4123, lectotype; Ustilag. Jugosl., no. 3, isolectotypes). Fig. 3g-i.

Infection systemic. Sori destroying the seeds, filling the capsules initially with a semi-agglutinated, later powdery, sepia (based on Rayner 1970) or date brown (based on the Color identification chart of Anonymous 1969) spore mass. Spores subglobose, globose, broadly ellipsoidal or ovoid, (11-)12-15.5(-16.5) × (10-)11-14(-15) (13.8 ± 1.1 × 12.6 ± 1.1) μm (n = 100), medium reddish brown; wall reticulate, 1.4-2.2 μm thick (including reticulum), meshes (5-)6-9 per spore diameter, polyhedral or irregular, 0.7-2.5(-3.0) μm wide, muri 0.7-1.2(-1.5) μm high. In SEM, meshes smooth or rugulose, often with a hemispherical protuberance at the bottom.

Specimens examined.-GREECE. Thessaly: Karditsa, near Kryoneri, 39°19′39″N, 21°42′00″E, alt. 785 m, on Moenchia mantica, 29 May 2005, leg. R. Willing & E. Willing 140.865, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 0255208; GenBank ITS OQ096634, LSU OQ067236). Western Greece Region: Aetolia-Acarnania, Ep. Nafpaktias, 1 km SE of Ano Hora, 38°35′N, 21°55′30″E, alt. 1020 m, on M. mantica, 15 Jun. 1991, leg. R. Willing 15.741, fungus comm. & det. T.T. Denchev & C.M. Denchev (B 10 1224076).

Hosts and Distribution.-On Moenchia spp. (Caryophyllaceae). Known from Europe and North Africa.

Comments.-The sequence of the specimen B 10 0255208 formed a well-supported clade with two sequences from this species (Fig. 4, Table 2).

Microbotryum moenchiae-manticae is recorded for the first time from Greece. It has been previously reported from Europe - Bulgaria, Spain, and UK, on Moenchia erecta G.Gaertn., B.Mey. & Scherb. subsp. erecta (Denchev 1997; Denchev & al. 2010; Denchev & Denchev 2017), and Romania and Serbia, on M. mantica subsp. mantica (Lindtner 1950; Vánky 1985), and Africa-Algeria and Morocco, on M. erecta subsp. octandra (Ziz ex Mert. & W.D.J.Koch) Gürke ex Cout. (Denchev & Denchev 2017).

 Acknowledgements

This study was supported by the Bulgarian National Science Fund (Grant no. KP-06-N51/10/16.11.2021). The visit of T.T.D. & C.M.D. to the herbarium at the BGBM Berlin received support from the SYNTHESYS Plus Project http://www.synthesys.info, which is financed by the H2020 Research Infrastructures Programme (Grant no. DE-TAF-8193).

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