Unravelling unexplored diversity of cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) in tropical Africa

Yalemwork Meswaet; Ralph Mangelsdorff; Nourou S. Yorou; Meike Piepenbring

doi:10.3897/mycokeys.81.67850

Research Article

Unravelling unexplored diversity of cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) in tropical Africa

Yalemwork Meswaet^‡, Ralph Mangelsdorff^‡, Nourou S. Yorou^§, Meike Piepenbring^‡

‡ Goethe University Frankfurt am Main, Frankfurt am Main, Germany

§ University of Parakou, Parakou, Benin

Corresponding author: Meike Piepenbring ( piepenbring@bio.uni-frankfurt.de )

Academic editor: Huzefa Raja

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Meswaet Y, Mangelsdorff R, Yorou NS, Piepenbring M (2021) Unravelling unexplored diversity of cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) in tropical Africa. MycoKeys 81: 69-138. https://doi.org/10.3897/mycokeys.81.67850

Abstract

Cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) are one of the largest and most diverse groups of hyphomycetes causing a wide range of diseases of economically important plants as well as of plants in the wild. Although more than 6000 species are known for this group, the documentation of this fungal group is far from complete. Especially in the tropics, the diversity of cercosporoid fungi is poorly known. The present study aims to identify and characterise cercosporoid fungi collected on host plants belonging to Fabaceae in Benin, West Africa. Information on their morphology, host species and DNA sequence data (18S rDNA, 28S rDNA, ITS and tef1) is provided. DNA sequence data were obtained by a simple and non-culture-based method for DNA isolation which has been applied for cercosporoid fungi for the first time in the context of the present study. Among the loci used for the phylogenetic analysis, tef1 provided the best resolution together with the multigene dataset. Species delimitation in many cases, however, was only possible by combining molecular sequence data with morphological characteristics. Based on forty specimens recently collected in Benin, 18 species are presented with morphological descriptions, illustrations and sequence data. Among these, six species in the genus Cercospora and two species in Pseudocercospora are proposed as species new to science. The newly described species are Cercospora (C.) beninensis on Crotalaria macrocalyx, C. parakouensis on Desmodium tortuosum, C. rhynchophora on Vigna unguiculata, C. vignae-subterraneae on Vigna subterranea, C. tentaculifera on Vigna unguiculata, C. zorniicola on Zornia glochidiata, Pseudocercospora sennicola on Senna occidentalis and Pseudocercospora tabei on Vigna unguiculata. Eight species of cercosporoid fungi are reported for Benin for the first time, three of them, namely C. cf. canscorina, C. cf. fagopyri and C. phaseoli-lunati are new for West Africa. The presence of two species of cercosporoid fungi on Fabaceae previously reported from Benin, namely Nothopassalora personata and Passalora arachidicola, is confirmed.

Keywords

Benin, Cercospora, Fabaceae, Leguminosae, molecular phylogenetic analysis, Nothopassalora, Passalora, Pseudocercospora, West Africa

Introduction

Hyphomycetous anamorphs of Mycosphaerella-like teleomorphs are generally referred to as cercosporoid fungi and are classified in genera with concepts that often changed (Crous and Braun 2003; Braun et al. 2013; Kirschner 2014). Cercosporoid fungi include about 6000 recognized species (Braun et al. 2015), in more than ten genera, with Cercospora Fresen. (C.), Nothopassalora U.Braun, C. Nakash., Videira & Crous (N.), Passalora Fr. (P.) and Pseudocercospora Speg. (Ps.) being the genera relevant for the present publication.

Cercosporoid fungi belonging to Mycosphaerellaceae (Mycosphaerellales, Ascomycota) are one of the largest and most diverse groups of hyphomycetes and cause a wide range of diseases, on numerous economically important plants such as cereals, vegetables and fruits as well as on wild plants. Major diseases include the angular leaf spot of bean caused by Pseudocercospora griseola, black leaf streak of banana caused by Ps. fijiensis (M.Morelet) Deighton, fruit and leaf spot disease of citrus caused by Ps. angolensis (T.Carvalho & O.Mendes) Crous & U.Braun, leaf spot disease of celery (Cercospora apii Fresen.), of sugar beet (C. beticola Sacc.), and foliar diseases of groundnut caused by Nothopassalora personata (Berk. & M.A.Curtis) U.Braun, C. Nakash., Videira & Crous or Passalora arachidicola (Hori) U.Braun (Braun et al. 2013; Videira et al. 2017). Infections by these fungi are mostly evident by leaf spots, but cercosporoid fungi can also cause necrotic lesions on flowers, fruits, seeds and pedicels of numerous hosts in most climatic regions (Agrios 2005). Cercosporoid fungi are known from all parts of the world but they are more abundant and diverse in tropical and subtropical regions (Beilharz et al. 2002; Braun and Freire 2004; Hernández-Gutiérrez and Dianese 2008, 2009).

Cercosporoid fungi are dematiaceous hyphomycetes with conidiophores formed singly or in groups, arranged in sporodochia or in synnemata, with integrated, terminal or intercalary conidiogenous cells (Crous and Braun 2003; Ávila et al. 2005; Braun et al. 2013). Most of the cercosporoid species were previously assigned to a single genus, Cercospora, which was later split into several smaller genera mainly by Deighton (1967, 1973, 1974, 1976, 1979), Braun (1993) and Crous and Braun (2003). Crous and Braun (2003) recognized four genera, namely Cercospora, Passalora, Pseudocercospora and Stenella Syd as important cercosporoid genera. Later, the genus Stenella was assigned to the Teratosphaeriaceae based on the phylogenetic placement of the type species. Stenella-like species remaining in Mycosphaerellaceae were classified in the genus Zasmidium Fr. (Arzanlou et al. 2007; Braun et al. 2013). In the present paper, we follow generic concepts defined by Crous and Braun (2003) and recently updated by Braun et al. (2013), Crous et al. (2013a), Groenewald et al. (2013) and Videira et al. (2017). However, according to recent molecular sequence analyses, most genera of the cercosporoid fungi are not monophyletic (Videira et al. 2017). As many cercosporoid fungi have a strong impact on cultivated plants, a better understanding and stabilisation of the taxonomy of these fungi are urgently needed.

The genus Cercospora was established by Fresenius in 1863 (Fuckel 1863) based on the type species Cercospora apii (Braun and Crous 2016; Videira et al. 2017). It is one of the most species-rich genera of the hyphomycetes and contains numerous important plant pathogenic fungi throughout the world (Crous and Braun 2003). In 1954, the genus was monographed by Chupp (1954), who treated 1419 Cercospora-species using a broad generic concept. Later, several attempts have been made to split Cercospora s. lat. into smaller genera by using characteristics of conidiomatal structure, hyphae, conidiophores, conidiogenous cells, conidiogenous loci and conidia (Ellis 1971, 1976; Deighton 1973, 1979, 1983; Braun 1995a, 1998; Crous and Braun 2003). Currently, Cercospora species are morphologically characterised by pigmented conidiophores, unpigmented conidia, as well as thickened and darkened conidiogenous loci and conidial hila (Crous and Braun 2003; Groenewald et al. 2013). A significant problem in the taxonomy of Cercospora is the host specificity of its species. Most Cercospora species are considered to be distinct based on the host and thus assumed to be specific to a host species or to a host genus (Chupp 1954; Braun 1995a). Some species, such as C. apii and C. beticola, however, were isolated from a high number of host species belonging to several families (Groenewald M et al. 2006). Moreover, phylogenetic approaches based on multi-locus sequences can be problematic for species delimitation in Cercospora due to a high level of conservation in DNA sequences of commonly used loci (i.e., ITS, tef1, actA, cmdA and his3) (Bakhshi et al. 2018).

The genus Pseudocercospora was introduced by Spegazzini (1910) based on the type species Ps. vitis (Lév.) Speg., a foliar pathogen of grapevine. The majority of Pseudocercospora species are known as pathogens occurring on many different plants, mainly in tropical and sub-tropical regions (Chupp 1954; Crous and Braun 2003; Crous et al. 2013). In contrast to Cercospora spp., they are characterised by pigmented conidiophores and conidia, without thickened and darkened conidiogenous loci and conidial hila (Deighton 1976). The monophyly of the genus has not yet been fully resolved (Kirschner 2014). According to molecular sequence data, most species of Pseudocercospora appear to be host specific (Crous et al. 2013).

The genus Passalora Fr. was introduced by Fries (1849) based on the type species Passalora bacilligera (Mont. & Fr.) Mont. & Fr. (≡ Cladosporium bacilligerum Mont. & Fr.) (Videira et al. 2017). Species of Passalora are characterised by pigmented conidiophores and conidia as well as thickened and darkened conidiogenous loci and conidial hila (Crous and Braun 2003).

Several molecular phylogenetic studies are available on species of cercosporoid fungi that are represented by strains in culture collections (Świderska-Burek et al. 2020). These, however, only represent a small fraction of several hundreds of taxa of cercosporoid fungi that are valid species defined by morphological characteristics (Braun et al. 2016; Świderska-Burek et al. 2020). Therefore, the number of cercosporoid species known by detailed morphological characteristics as well as molecular sequence data has to be increased.

Although cercosporoid fungi cause a wide range of diseases on major agricultural crops, the study of cercosporoid fungi in West Africa is still at an early pioneer stage and only very incomplete information is currently available (Piepenbring et al. 2020). To date, approximately 320 species of cercosporoid hyphomycetes are known from 14 West African countries (Piepenbring et al. 2020, Suppl. materials 1, 2). Among these, 12 species of cercosporoid fungi have been reported for Benin (Turner 1971; Marley et al. 2002; Crous and Braun 2003; Houessou et al. 2011; Piątek and Yorou 2018; Soura et al. 2018; Meswaet et al. 2019; Farr and Rossman 2021). Morphological characteristics and molecular sequence data are lacking for most cercosporoid species known for Benin and other West African countries. Although cercosporoid fungi have been investigated for more than 150 years and are important in the agricultural sector, almost no, or only inadequate, studies have been carried out in most West African countries such as Benin. In addition to this lack of species knowledge in tropical regions, many species of cercosporoid fungi are characterised morphologically only. Since many cercosporoid species are known as pathogens on cultivated plants, an accurate diagnosis, identification and documentation of these fungi are a prerequisite and urgent for their control and epidemiological surveys.

As a first step towards a systematic documentation of cercosporoid fungi in tropical Africa, we focus on species infecting hosts belonging to the Fabaceae (Leguminosae) in the present publication. Fabaceae are the third largest family of angiosperms (Gepts et al. 2005). This family includes peas, lentils, beans, peanuts and other plants with pods and/or seeds that are consumed as food (Messina 1999). Several species belonging to Vigna originate from West Africa (Benin, Burkina Faso, Cameroon, Ghana, Niger, Nigeria and Togo) including two important cultivated crops Vigna unguiculata (L.) Walp. and Vigna subterranea (L.) Verdc. (Hepper 1963; Faris 1965; Padulosi and Ng 1990). They provide important nutrients such as proteins, low glycemic index carbohydrates, minerals and vitamins. Legumes are richer in protein than other cultivated plants because of nitrogen-fixing bacteria living in nodules of their roots (Kouris-Blazos and Belski 2016).

We apply an integrative approach that includes sampling in Benin, detailed descriptions and illustrations of collected specimens and herbarium specimens, examination of closely related known species on the same or closely related host species based on herbarium specimens and the isolation, sequencing and analysis of nuclear DNA sequence data. For the isolation of DNA, a new, simple method for DNA isolation has been developed and is presented for the first time for cercosporoid fungi.

Methods

Collections and morphological studies

Samples of leaves infected by cercosporoid fungi were randomly collected in farmlands and fallows in Benin from July–August 2016, July–September 2017 and August–September 2019. Infected leaves were dried in a plant press and deposited in the herbaria Botanische Staatssammlung München (M) and University of Parakou (UNIPAR).

Dried specimens were observed by stereomicroscopy and by light microscopy, using a Zeiss Axioscope 40 microscope. For light microscopy, leaf sections were made with razor blades and mounted in distilled water or 5% KOH without staining. Semi-permanent preparations of sections of the infected leaves were made by a microtome (Leica CM 1510-1) and mounted in lactophenol with cotton blue. For approximately 50 ml lactophenol cotton blue solution we mixed 10 mg phenol, 0.025 mg cotton blue, 10 ml lactic acid, 20 ml glycerin and 10 ml distilled water. Measurements of 30 conidia, conidiophores and other structures have been made for each specimen at a magnification of ×1000. Measurements are presented as mean value ± standard deviation with extreme values in parentheses. Line drawings were made freehand on scaled paper. Images and drawings were edited with Photoshop CS5 (Adobe, San Jose, California). Critical taxa were determined with the help of type specimens and other specimens loaned from the US National Fungus Collections (BPI), the Herbarium of the University of Illinois (ILL) and the New York Botanical Garden (NY).

Host plant identification

Host plants were identified by morphological characteristics and in some cases by molecular methods. Morphological identifications were made by comparison with herbarium specimens, literature (e.g., Akoégninou et al. 2006) and with the help of local botanists. Molecular sequence data for species identifications were obtained by polymerase chain reaction (PCR) for the amplification of the partial region of chloroplast rbcL with the primer pairs rbcLa-F (Levin et al. 2003) and rbcLa-R (Kress et al. 2009). DNA was extracted from approx. 0.05 g of leaf tissue dried with silica gel using the innuPREP Plant DNA Kit (Analytik Jena, Germany) and following the manufacturer’s instructions. Protocols for PCR were carried out as described by Fazekas et al. (2012).

DNA Extraction and PCR amplification of fungal DNA

DNA was isolated from caespituli taken with a needle from dry specimens using the E.Z.N.A Forensic DNA Extraction Kit following the manufacturer’s instructions. Small pieces of leaves containing several clean caespituli, with as little contaminations as possible, were selected under the stereomicroscope. Precautions were taken to avoid picking cells of any other organism (fungi, algae) associated with the leaves. To extract total genomic DNA from caespituli, a small amount of clean hyphae from the leaf surface was transferred into a sterile Eppendorf tube using a sterilized needle or adhesive mini-tapes. The sample was homogenized for 7–10 min. using a Retsch Mixer Mill MM301 with TL buffer and 2.5 mm Zirconia beads. Isolated DNA was re-suspended in elution buffer and stored at -20 °C. DNA concentration was checked by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA).

Four partial nuclear gene regions (three ribosomal loci and one protein-coding gene) were amplified and sequenced: For the large subunit nuclear ribosomal DNA (nrLSU, 28S rDNA) the primers LSU1Fd and LSU3Rd (Crous et al. 2009a), for the small subunit nuclear ribosomal DNA (nrSSU, 18S rDNA) the primers SSU1Fd and SSU1Fd (Crous et al. 2009a), for the internal transcribed spacer region of ribosomal DNA (ITS) the primers V9G (de Hoog and van den Ende 1998) and ITS4 (White et al. 1990) and for the translation elongation factor 1-α (tef1) the primers EF1- 728F and EF1-986R (Carbone and Kohn 1999) were used. PCR amplification and sequencing were conducted following the protocols of Hunter et al. (2006), Crous et al. (2009a, 2012) and Videira et al. (2017). The PCR mixtures consisted of 1 μL genomic DNA, 15× MgCl₂ reaction buffer (Bioline, Luckenwalde, Germany), 25 mM MgCl₂, 25 μM of each dNTP, 10 μM of each primer and 5 U Taq DNA polymerase (VWR) in a total volume of 25 μL. Cycling parameters of the PCR for LSU, SSU and ITS were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of amplification [denaturation at 94 °C for 30 s, primer annealing at 52 °C for 30 s and primer extension at 72 °C for 45 s] and a final extension at 72 °C for 5 min, followed by storage at 8 °C. The PCR mixture for tef1 contained 2 μL of template DNA and the cycling parameters to obtain the partial tef1 were as follows: an initial denaturation at 96 °C for 2 min; followed by 35 cycles of amplification [denaturation at 94 °C for 30 s, primer annealing at 56 °C for 30 s and primer extension at 72 °C for 30 s] and a final extension at 72 °C for 7 min, followed by storage at 8 °C. PCR-products were checked on 1.5% agarose electrophoresis gels containing HDGreenPlus DNA stain. Ampliﬁed PCR products were puriﬁed with the Cycle Pure Kit (VWR-Omega, USA). Sequencing was performed at Seqlab GmbH, Germany.

Molecular phylogeny

Amplification of the SSU, LSU, ITS and tef1 gene regions for all isolates used in this study yielded fragments of approximately 1100 bp, 900 bp, 650 bp and 300 bp, respectively. Consensus sequences of trace files were generated with Geneious 10.2.2 (https://www.geneious.com, Kearse et al. 2012) and searched against GenBank (https://www.ncbi.nlm.nih.gov/, Benson et al. 2014) with MegaBLAST. Sequences with a high similarity (65 sequences of LSU, ITS and tef1 regions) were retrieved (Table 1). A total of 148 sequences for 65 specimens were obtained from GenBank (Table 1) and 92 sequences for 28 specimens from Benin were generated in this study (Table 2). They were aligned with MAFFT v. 7 using the L-INS-i algorithm (Nakamura et al. 2018). The alignments were manually checked by using MEGA v. 7 (Kumar et al. 2016). Gblocks v. 0.91b (Talavera and Castresana 2007) was used to remove poorly aligned positions and divergent regions from the DNA alignment using the parameters for a less stringent selection. Subsequently, a four-locus concatenated alignment (SSU, LSU, ITS and tef1) dataset was assembled for phylogenetic analyses using Geneious 10.2.2. Cladosporium sphaerospermum (G402) served as outgroup taxon, because the genus Cladosporium s. str. was shown to be the sister group of Mycosphaerella s. str. (Braun et al. 2003). PartitionFinder2 v.2.1.1 (Lanfear et al. 2014) on XSEDE (Miller et al. 2010) was used to select the best-fit model of evolution for each gene fragment separately. Data were partitioned by gene and by codon position in the case of protein-coding sequences. The TRNEF+G model was applied to 28S rDNA, K80 model to 18S rDNA, K81+G to ITS and TRN+G model to tef1. The alignment and the tree were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S28032). Phylogenetic analyses of this study were conducted by applying Maximum Likelihood (ML) in RAxML-HPC2 v.8.2.12 (Stamatakis 2014) on XSEDE (Miller et al. 2010) and Bayesian with the program MrBayes 3.2.6 (Ronquist et al. 2012) on XSEDE (Miller et al. 2010) on the CIPRES Science Gateway web portal. (http://www. phylo.org/sub_sections/portal/).

For Maximum Likelihood analyses one thousand nonparametric bootstrap iterations were used with the generalised time-reversible model with a discrete gamma distribution (GTRGAMMA) (Stamatakis et al. 2008). For Bayesian phylogenies, two parallel runs with eight chains of Metropolis-coupled Markov chain Monte Carlo iterations were performed with the heat parameter being set at 0.2. Analyses were run for 100 million generations, with trees sampled every 1000^th generation until the average standard deviation of split frequencies reached 0.01 (stop value). The first 25% of saved trees were discarded as the ‘burn-in’ phase. Posterior probabilities (PP) were determined from the remaining trees. Bayesian posterior probabilities (BPP) ≥ 94% and Bootstrap values (BS) ≥ 70% are considered as significant.

Table 1.

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Data of DNA sequences of cercosporoid fungi downloaded from GenBank and used in this study.

Species	Host	Host family	Country	Source	GenBank Accession Numbers			Reference
Species	Host	Host family	Country	Source	nrLSU	ITS	tef1	Reference
Cercospora cf. apii Fresen.	Cajanus cajan (L.) Millsp.	Fabaceae	S. Africa	CBS 115411	JN941171	JN942278	–	Groenewald JZ et al. (2013)
Cercospora asparagi Sacc.	Asparagus sp.	Asparagaceae	USA	AS16-02	KY549100	KY549098	KY549102	Hay et al. (2017)
Cercospora canescens Ellis & G.Martin	Vigna radiata (L.) R.Wilczek.	Fabaceae	India	Cer70-18	–	MN795675	–	Das et al. 2019
Cercospora capsica Heald & F.A.Wolf	Capsicum annuum L.	Solanaceae	S. Korea	CBS 132622	–	JX143568	JX143323	Groenewald JZ et al. (2013)
Cercospora cf. citrulline Cooke	Citrullus lanatus (Thunb.) Matsum. & Nakai	Cucurbitaceae	Japan	MUCC 576	–	JX143579	JX143337	Groenewald JZ et al. (2013)
Cercospora dubia Speg.	Chenopodium sp.	Amaranthaceae	Mexico	CPC 15600	KX286968	KX287277	–	Videira et al. (2016)
Cercospora kikuchii (Tak. Matsumoto & Tomoy.) M.W.Gardner	Glycine max (L.) Merr.	Fabaceae	USA	DLS5070-3A		AY373573	AY373582	Cai and Schneider (2008)
Cercospora lactucae-sativae Sawada	Lactuca sativa L.	Asteraceae	Japan	MUCC 570	–	JX143623	JX143382	Groenewald JZ et al. (2013)
Cercospora malayensis F.Stevens & Solheim	Abelmoschus esculentus (L.) Moench	Malvaceae	S. Korea	KACC 47769	–	MH129519	MH129517	Ju et al. (2020)
Cercospora cf. maloti Ellis & Everh.	Cucumis melo L.	Cucurbitaceae	Japan	MUCC 575	–	JX143625	JX143384	Groenewald JZ et al. (2013)
Cercospora cf. nicotianae Ellis & Everh.	Nicotiana tabacum L.	Solanaceae	–	CBS 570.69	–	DQ835074	DQ835100	Groenewald JZ et al. (2010)
Cercospora olivascens Sacc.	Aristolochia clematitis L.	Aristolochiaceae	Romania	CBS 253.67	–	JX143632	JX143391	Groenewald JZ et al. (2013)
Cercospora physalidis Ellis	Solanum melongena L.	Solanaceae	India	Cer 69-18	MK027095	MK029358	–	Sinha et al., unpublished
Cercospora rodmanii Conway	Eichhornia sp.	Pontederiaceae	Mexico	15-GTOX	GQ884187	GQ884185	–	Montenegro-Calderón et al. (2011)
Cercospora sojina Hara	Glycine soja Siebold & Zucc.	Fabaceae	S. Korea	CBS 132615	–	JX143659	JX143419	Groenewald JZ et al. (2013)
Cercospora sp. Q JZG-2013	Acacia mangium Willd.	Fabaceae	Thailand	CPC 10550	–	AY752139	AY752172	Groenewald JZ et al. (2013)
Cercospora vignigena C. Nakash., Crous, U.Braun & H.D.Shin	Vigna unguiculata (L.) Walp.	Fabaceae	Japan	MUCC 579	–	JX143736	JX143495	Groenewald JZ et al. (2013)
Cercospora zebrina Pass.	Trifolium subterraneum L.	Fabaceae	Australia	CBS 118790	KF251651	KF251147	–	Quaedvlieg et al. (2013)
Cladosporium sphaerospermum Penz.	–	–	Russia	G402	KJ443113	KJ443245	KJ443201	Grum-Grzhimaylo et al. (2016)
Mycosphaerella keniensis Crous & T.A.Cout.	Eucalyptus grandis W.Hill	Myrtaceae	Kenya	CMW5147	DQ246259	–	DQ235100	Hunter et al. (2006)
Mycosphaerella microsora Syd.	Tilia platyphyllos Scop.	Malvaceae	Romania	CBS 552.71	MH872022	MH860260		Vu et al. (2019)
Mycosphaerella valgourgensis Crous	Yucca sp.	Asparagaceae	France	CPC:18385	JF951175	JF951152	–	Crous et al. (2011)
Nothopassalora personata (Berk. & M.A.Curtis) U.Braun, C. Nakash., Videira & Crous	Arachis hypogaea L.	Fabaceae	Australia	CBS 142236	NG_058496	NR_156379	–	Videira et al. (2017)
Paracercospora egenula (Syd.) Deighton	Solanum melongena L.	Solanaceae	India	CBS 485.81	JQ324940	GU269699	GU384415	Crous et al. (2013a)
Passalora arctostaphyli Moreno-Rico & Crous	Arctostaphylos pungens Kunth	Ericaceae	Mexico	CPC 22067	KJ152785	KJ152782	–	Moreno-Rico et al. (2014)
Neocercosporidium smilacis (Thüm.) U.Braun, C. Nakash., Videira & Crous	Smilax aspera L.	Smilacaceae	Italy	CBS 556.71	KJ633269	KJ633265	–	Collemare et al. (2015)
Pseudocercospora abelmoschi (Ellis & Everh.) Deighton	Hibiscus syriacus L.	Malvaceae	S.Korea	CBS 132103	GU253696	GU269647	GU384365	Crous et al. (2013a)
Pseudocercospora atromarginalis (G.F.Atk.) Deighton	Solanum sp.	Solanaceae	New Zealand	CBS 114640	GU253706	GU269658	GU384376	Crous et al. (2013a)
Pseudocercospora cercidicola Crous, U.Braun & C. Nakash.	Cercis chinensis Bunge	Fabaceae	Japan	MUCC 896	GU253719	GU269671	GU384388	Crous et al. (2013a)
Pseudocercospora chengtuensis (F.L.Tai) Deighton	Lycium chinense Mill.	Solanaceae	S. Korea	CBS 131924	MH877506	MH866053	–	Vu et al. (2019)
Pseudocercospora chiangmaiensis Cheew., K.D.Hyde & Crous	Eucalyptus camaldulensis Dehnh.	Myrtaceae	Thailand	CBS 123244	MH874812	MH863288	–	Vu et al. (2019)
Pseudocercospora cruenta (Sacc.) Deighton	Phaseolus vulgaris L.	Fabaceae	Taiwan	CBS 117232	GU253730	GU269689	GU384405	Crous et al. (2013a)
Pseudocercospora cydoniae (Ellis & Everh.) Y.L.Guo & X.J.Liu	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	S. Korea	CBS 131923	MH877505	MH866052	–	Vu et al. (2019)
Pseudocercospora dingleyae U.Braun & C.F.Hill	Haloragis erecta (Murray) Oken	Haloragaceae	New Zealand	CBS 114645	KX286997	KX287299	–	Videira et al. (2016)
Pseudocercospora dovyalidis (Chupp & Doidge) Deighton	Dovyalis zeyheri (Sond.) Warb.	Salicaceae	S. Africa	CBS 126002	MH875338	MH863877	–	Vu et al. (2019)
Pseudocercospora encephalarti Y.Meswaet, Mangelsdorff, Yorou & M.Piepenbr.	Encephalartos barteri Carruth. ex Miq.	Zamiaceae	Benin	YMMAS78	–	MK397016	–	Meswaet et al. (2019)
Pseudocercospora flavomarginata G.C.Hunter, Crous & M.J.Wingf.	Eucalyptus camaldulensis Dehnh.	Myrtaceae	Thailand	CBS 118824	–	NR_111805	–	Quaedvlieg et al. (2012)
Pseudocercospora fuligena (Roldan) Deighton	Solanum lycopersicum L.	Solanaceae	Japan	MUCC 533	GU253749	GU269712	GU384428	Crous et al. (2013a)
Pseudocercospora griseola f. griseola (Sacc.) Crous & U.Braun	Phaseolus vulgaris L.	Fabaceae	S. Korea	CBS 131929	MH877495	MH866046	–	Vu et al. (2019)
Pseudocercospora hakeae (U.Braun & Crous) U.Braun & Crous	Hakea sp.	Proteaceae	Australia	CBS:144520	MK442553	MK442617	MK442708	Crous et al. (2019)
Pseudocercospora humuli (Hori) Y.L.Guo & X.J.Liu	Humulus lupulus L.	Cannabaceae	Japan	MUCC 742	GU253758	–	GU384439	Crous et al. (2013a)
Pseudocercospora kaki Goh & W.H.Hsieh	Diospyros kaki L.f.	Ebenaceae	Japan	MUCC 900	GU253761	GU269729	GU384442	Crous et al. (2013a)
Pseudocercospora madagascariensis Crous & M.J.Wingf.	Eucalyptus camaldulensis Dehnh.	Myrtaceae	Madagascar	CBS 124155	MH874880	MH863357	–	Vu et al. (2019)
Pseudocercospora metrosideri U.Braun	Metrosideros collina (J.R.Forst. & G.Forst.) A.Gray	Myrtaceae	New Zealand	CBS 118795	GU253774	GU269746	GU384458	Crous et al. (2013a)
Pseudocercospora neriicola Crous, Frisullo & Camele	Nerium oleander L.	Apocynaceae	Italy	CPC 23765	KJ869222	KJ869165	KJ869240	Crous et al. (2014)
Pseudocercospora pallida (Ellis & Everh.) H.D.Shin & U.Braun	Campsis grandiflora (Thunb.) K.Schum.	Bignoniaceae	S. Korea	CBS 131889	–	–	GU384469	Crous et al. (2013a)
Pseudocercospora paraguayensis (Tak. Kobay.) Crous	Eucalyptus nitens (H.Deane & Maiden) Maiden	Myrtaceae	Brazil	CBS:111286	KF901945	KF901619	KF903205	Quaedvlieg et al. (2014)
Pseudocercospora parapseudarthriae Crous & A.R.Wood	Pseudarthria hookeri Wight & Arn.	Fabaceae	S. Africa	CPC 23449	KJ869208	KJ869151	KJ869238	Crous et al. (2014)
Pseudocercospora pittospori (Plakidas) Y.L.Guo & X.J.Liu	Pittosporum sp.	Pittosporaceae	USA	HI-018	MK210475	MK210511	–	Vaghefi et al. 2021
Pseudocercospora proteae Crous	Protea mundii Klotzsch	Proteaceae	S. Africa	CBS 131587	–	–	GU384519	Crous et al. (2013a)
Pseudocercospora prunicola (Ellis & Everh.) U.Braun	Prunus sp.	Rosaceae	China	BJFU ZYP141005.9	KX853057	KX853048	KX853066	Liu et al. (2016)
Pseudocercospora ranjita (S.Chowdhury) Deighton	Gmelina sp.	Lamiaceae	Indonesia	CBS 126005	MH875340	MH863879	GU384500	Crous et al. (2013a)
Pseudocercospora ravenalicola G.C.Hunter & Crous	Ravenala madagascariensis Sonn.	Strelitziaceae	India	CBS 122468	GU253828	–	GU384521	Crous et al. (2013a)
Pseudocercospora schizolobii (M.J.Wingf. & Crous) M.J.Wingf. & Crous	Eucalyptus camaldulensis Dehnh.	Myrtaceae	Thailand	CBS 124990	KF251827	KF251323	KF253270	Verkley et al. (2013)
Pseudocercospora sennae-multijugae Meir. Silva, R.W.Barreto & Crous	Senna multijuga (Rich.) H.S.Irwin & Barneby	(Fabaceae)	Brazil	CPC 25206	KT290169	KT290142	KT290196	Silva et al. (2016)
Pseudocercospora sp.	Citrus grandis (L.) Osbeck	Rutaceae	China	ZJUM 75	KP895896	KP896026	KP896073	Huang et al. (2015)
Pseudocercospora sp.	Eichhornia azurea (Sw.) Kunth	Pontederiaceae	Brazil	CPC 19537	KX287003	KX287304	–	Videira et al. (2016)
Pseudocercospora sp.	Eichhornia azurea (Sw.) Kunth	Pontederiaceae	Brazil	CPC 19535	KX287001	KX287303	–	Videira et al. (2016)
Pseudocercospora sp. A MB-2015	Phaseolus vulgaris L.	Fabaceae	Iran	CCTU 1166	KP717028	KM452864	KM452886	Bakhshi et al. (2014)
Pseudocercospora stizolobii (Syd. & P.Syd.) Deighton	Eucalyptus camaldulensis Dehnh.	Myrtaceae	Thailand	CPC 25217	KT290170	KT290143	KT290197	Silva et al. (2016)
Pseudocercospora tereticornis Crous & Carnegie	Eucalyptus tereticornis Sm.	Myrtaceae	Australia	CBS 125214	MH874960	MH863460	–	Vu et al. (2019)
Pseudocercospora vitis (Lév.) Speg.	Vitis vinifera L.	Vitaceae	S. Korea	CPC 11595	–	–	JX901702	Quaedvlieg et al. (2012)
Pseudocercosporella bakeri (Syd. & P.Syd.) Deighton	Ipomoea indica (Burm.) Merr.	Convolvulaceae	New Zealand	CBS 119488	KX287005	KX287306	KX287862	Videira et al. (2016)
Pseudocercosporella myopori U.Braun & C.F.Hill	Myoporum laetum G.Forst.	Scrophulariaceae	New Zealand	CBS 114644	KX287000	KX287302	JX143491	Groenewald JZ et al. (2013)
Zasmidium daviesiae (Cooke & Massee) U.Braun, C. Nakash., Videira & Crous	Daviesia latifolia R.Br.	Fabaceae	Australia	CBS:116002	KF901928	KF901603	KF903373	Quaedvlieg et al. (2014)

Table 2.

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Data of sequences of cercosporoid fungi from Benin generated during the present study. Names of species proposed as new in this study are written in bold.

Species	Voucher	Host	Host family	GenBank Accession Numbers
Species	Voucher	Host	Host family	nrSSU	nrLSU	ITS	tef1
*Cercospora beninensis*	YMM11	Crotalaria macrocalyx Benth.	Fabaceae	MW834445	MW834433	MW834437	MW848615
Cercospora aff. canescens Ellis & G.Martin	YMM07	Calopogonium sp.	Fabaceae	MW834475	–	MW834492	MW848605
Cercospora aff. canescens Ellis & G.Martin	YMM01	Vigna subterranea (L.) Verdc.	Fabaceae	MW834473	MW834457	MW834490	MW848603
Cercospora cf. canscorina Chidd.	YMM05	Vigna sp.	Fabaceae	MW834474	MW834458	MW834491	MW848604
Cercospora cf. fagopyri K.Nakata & S.Takim.	YMM23A	Lablab sp.	Fabaceae	–	–	MW861543	MW848607
*Cercospora parakouensis*	YMM296A	Desmodium tortuosum (Sw.) DC.	Fabaceae	–	MW834436	MW834442	MW848621
Cercospora phaseoli-lunati U.Braun & Crous	YMM289	Vigna radiata (L.) R.Wilczek	Fabaceae	MW834471	–	MW834483	MW848601
*Cercospora rhynchophora*	YMM03B	Vigna unguiculata (L.) Walp.	Fabaceae	MW834447	MW834431	MW834443	MW848619
Cercospora sp.1	YMM3S	Sorghum bicolor (L.) Moench	Poaceae	MW834466	MW834452	MW834484	MW848600
Cercospora sp.2	YMM48S	Sorghum bicolor (L.) Moench	Poaceae	MW834467	MW834453	MW834485	MW848608
Cercospora sp.3	YMM229	Spigelia sp.	Loganiaceae	–	MW834462	MW834500	MW848599
Cercospora sp.4	YMM297B	Phaseolus lunatus L.	Fabaceae	MW834481	MW834464	MW834501	MW848612
*Cercospora tentaculifera*	YMM75	Vigna unguiculata (L.) Walp.	Fabaceae	MW834448	–	MW834440	MW848614
**Cercospora vignae-subterraneae**	YMM293	Vigna subterranea (L.) Verdc.	Fabaceae	MW834446	–	MW834438	MW848618
*Cercospora zorniicola*	YMM299	Zornia glochidiata DC.	Fabaceae	–	–	–	MW848616
Nothopassalora personata (Berk. & M.A.Curtis) U.Braun, C. Nakash., Videira & Crous	YMM49A	Arachis hypogaea L.	Fabaceae	MW834479	MW844038	MW834497	–
Passalora arachidicola (Hori) U.Braun	YMM49B	Arachis hypogaea L.	Fabaceae	MW845059	MW844039	MW834498	–
Pseudocercospora bradburyae E.Young	YMM275	Centrosema pubescens Benth.	Fabaceae	MW834465	–	–	MW848609
Pseudocercospora cruenta (Sacc.) Deighton	YMM288	Phaseolus sp.	Fabaceae	MW834472	MW834456	MW834489	MW848602
	YMM04	Vigna unguiculata (L.) Walp.	Fabaceae	MW834478	MW834461	MW834496	MW848606
	YMM03A	Vigna unguiculata (L.) Walp.	Fabaceae	MW834482	MW834460	MW834495	MW848613
	YMM294B	Vigna unguiculata (L.) Walp.	Fabaceae	MW834480	MW834463	MW834493	MW848611
	YMM125	Vigna unguiculata (L.) Walp.	Fabaceae	MW834476	MW834451	MW834499	MW848610
Pseudocercospora griseola (Sacc.) Crous & U.Braun	YMM297A	Phaseolus lunatus L.	Fabaceae	MW834477	MW834459	MW834494	–
*Pseudocercospora sennicola*	YMM12	Senna occidentalis (L.) Link	Fabaceae	MW834444	MW834432	MW850550	–
Pseudocercospora sp.3	YMM19	Abelmoschus sp.	Malvaceae	MW834470	–	MW834488	–
Pseudocercospora sp.1	YMM123	Abelmoschus sp.	Malvaceae	MW834468	MW834454	MW834486	–
*Pseudocercospora tabei*	YMM220	Vigna unguiculata (L.) Walp.	Fabaceae	MW834450	MW834434	MW834439

Data availability

The specimen data is available through the Dryad Digital Repository https://datadryad.org/ (https://doi.org/10.5061/dryad.73n5tb2x9).

Results

Phylogeny

We isolated DNA from a total of 28 specimens of cercosporoid fungi recently collected in Benin (Table 2). These specimens represent 18 species found on species of Fabaceae for which 76 sequences are provided: 20 sequences of 18S rDNA, 16 of 28S rDNA, 21 of ITS and 19 of tef1. The separately aligned data sets for each marker consisted of 35 sequences/893 base pairs for 18S rDNA, 60/719 for 28S rDNA, 82/437 for ITS and 74/160 for tef1.

For the four-locus data analysis, DNA sequence data from the 18SrDNA, 28SrDNA, ITS and tef1 gene regions were combined and submitted to Bayesian and Maximum Likelihood (ML) analyses. The final concatenated alignment contained a total of 91 specimens including the out-group (65 specimens from NCBI and 26 specimens from this study) and had an aligned length of 2212 characters including alignment gaps. As the ML analyses produced tree topologies mostly identical to results of Bayesian analyses, bootstrap support values of the ML trees were incorporated into the tree that resulted from Bayesian analyses (Fig. 1). In this tree, the cercosporoid fungi are grouped in three major clades: Cercospora (86/76), Pseudocercospora (87/78) and Passalora together with other species of other genera (100/98) (Fig. 1). Phylogenetic analyses of individual loci are deposited in TreeBASE. Details of results concerning the delimitation of species are mentioned and discussed as part of species notes below.

Figure 1.

The Bayesian phylogenetic tree inferred from DNA sequence data from the multigene alignment (SSU rDNA, LSU rDNA, ITS and tef1) of cercosporoid species. Nodes receiving Bayesian PP ≥ 0.94 or ML BS ≥ 70% are considered as strongly supported and are indicated by thickened branches. Names of newly described species are written in bold and red. Species newly reported for Benin are indicated by green letters. Names of host plants are written with blue letters.

Tef1 sequence data showed differences between closely related species in the genera Cercospora and Pseudocercospora and are more informative than ITS and LSU rDNA sequence data. Therefore, we provide molecular phylogenetic analyses based on new tef1 sequences as well as sequences from GenBank for some newly described species, namely Cercospora rhynchophora, C. parakouensis, C. zorniicola and Pseudocercospora tabei. For Ps. sennicola, we provide an analysis based on ITS sequence data, because we were not able to obtain Tef1 sequence data.

Taxonomy

Based on morphological, molecular phylogenetic and host evidence, the cercosporoid fungi recently collected in Benin are assigned to 18 different taxa belonging to four genera. Among these, eight species are proposed as new to science, six in the genus Cercospora and two in Pseudocercospora. Eight species represent new reports for Benin, three of them are new for the whole of West Africa, namely Cercospora cf. canscorina, C. cf. fagopyri and C. phaseoli-lunati. Two species of cercosporoid fungi were previously reported in Benin and are confirmed.

Cercospora beninensis Y.Meswaet, Mangelsdorff, Yorou & M.Piepenbr., sp. nov.

MycoBank No: 839170

Figs 2A, 3

Etymology

The epithet beninensis refers to the country of origin of the type specimens, Benin.

Diagnosis

Cercospora beninensis differs from four Cercospora spp. known on Crotalaria spp. by having only internal hyphae, darker, shorter and narrower conidiophores [(14.5–)28.5–160(–168) × (3–)3.5–4.5(–5) μm] and mostly smaller and narrower conidia [(19–)23.5–122(–150) × (2.5–)3–4(–4.5) μm] (Table 3).

Table 3.

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Comparison of Cercospora beninensis (YMM11) on Crotalaria macrocalyx with Cercospora spp. known from Crotalaria spp. based on literature ^a–f.

Cercospora species	Leaf spots colour, size	Stromata	Conidiophore size (in μm), branching, septa, colour	Conidium sizes (in μm), septa
Cercospora beninensis (YMM11)	Brown to reddish brown, 0.5(1.5–)–5.5 mm diam.	Small or lacking	(14.5–)28.5–160(–168) × (3–)3.5–4.5(–5), branched, 0–6(–8)-septate, dark brown	(19–)23.5–122(–150) × (2.5–)3.5–4(–4.5), 1–7(–9) septa
C. apii ^ab	Present	Often small or lacking, occasionally developed, (up to 50 μm diam.)	20–300 × 4–6.5, rarely branched, multi-septate, pale brown, uniform in colour and width	25–315 × 3–6, (0–)3–25(–30) septa^b
C. canescens ^a	3–15 mm	Often small	20–200 × 3–6.5, rarely branched, multi-septate, pale to medium dark brown	25–300 × 2.5–5.5, indistinctly multi-septate
C. demetrioniana ^cde	Rusty brown to dark brown, 1–1.5 mm.	Present	40–350 × 4–6 (–7) ^c or up to 1 mm^de, 1–10-septate, unbranched, pale brown	50–210 × 3.5–5.5 /75–230 × 4–7, 7–16, very closely and indistinctly septate
C. demetrioniana f. minor^f	No information	No information	110–130 × 5–6	35–70(–170) × 5–5.5

Type

Benin. Borgou: Parakou, c. 363 m a.s.l., 9°20'29"N, 2°37'28"E, on Crotalaria macrocalyx Benth. (Fabaceae), 21 Sep 2019, Y. Meswaet and R. Dramani, YMM11 (Holotype: M-0312640; Isotype: UNIPAR). Ex holotype sequences. MW834445 (SSU), MW834433 (LSU), MW834437 (ITS), MW848615 (tef1).

Description

Leaf spots amphigenous, subcircular to angular-irregular, (0.5–)1.5–5.5 mm diam., brown to reddish brown, more evident on the adaxial surface of the leaves than on the abaxial side, occasionally with a chlorotic halo, the outermost ring darker than the inner ring, often with indefinite margin. Caespituli amphigenous, mainly epiphyllous, greyish brown to dark brown. Mycelium internal. Internal hyphae conspicuous, branched, 2.5–3.5 μm wide, septate, pale brown. Stromata lacking or formed by few aggregated swollen hyphal cells. Conidiophores in small, loose to moderately dense fascicles of up to approx. 16 conidiophores, occasionally solitary, arising from internal hyphae breaking through the adaxial epidermis of the leaves or penetrating through stomatal openings, erect, straight, subcylindrical, 1–2(–3) times geniculate, sometimes attenuated towards the tips, occasionally branched, (14.5–)28.5–160(–168) × (3–)3.5–4.5(–5) μm, 0–6(–8)-septate, brown to dark brown. Conidiogenous cells monoblastic or proliferating sympodially, sometimes distinctly subdenticulate; loci 1.5–2.5(–3.5) μm wide, thickened and darkened. Conidia solitary, acicular to narrowly obclavate, straight to curved, (19–)23.5–122(–150) × (2.5–)3–4(–4.5) μm, 1–7(–9)-septate, hyaline, smooth, tip acute, base truncate to short obconically truncate, 2.5–3(–4) µm wide, hila thickened and darkened.

Additional specimens examined

Benin. Borgou: Parakou, on the way to Okpara forest, c. 323 m a.s.l., 9°18'11"N, 2°43'50"E, on Crotalaria macrocalyx, 3 Sep 2019, Y. Meswaet and R. Dramani, YMM274 (Paratypes: M-0312641; UNIPAR). Benin. Borgou: N’Dali, c. 380 m a.s.l., 9°52'33"N, 2°41'20"E, same host, 31 Aug 2019, Y. Meswaet and A. Tabé, YMM272 (M-0312642).

Host and distribution

On Crotalaria macrocalyx (Fabaceae) in Benin.

Notes

Currently, three species and one form of Cercospora are known on Crotalaria spp., namely C. apii, C. canescens, C. demetrioniana G.Winter and C. demetrioniana f. minor Gonz. Frag. & Cif. (Farr and Rossman 2021). C. beninensis is morphologically distinct from all of them (Table. 3). C. apii differs by conidiophores that are more abundant on the abaxial surface of the leaves, in large and dense fascicles and longer [20–300 µm versus (14.5–)28.5–160(–168) in C. beninensis] as well as by longer and wider conidia [(25–315 × 3–6 µm versus (19–)23.5–122(–150) × (2.5–)3–4(–4.5) in C. beninensis] with more numerous septa (Chupp 1954; Crous and Braun 2003). C. canescens causes larger leaf spots often along the leaf margin, paler conidiophores that are more abundant on the abaxial leaf surface and longer conidia [(30–300) µm versus (19–)23.5–122(–150) μm in C. beninensis] (Chupp 1954). The distinctness is confirmed by molecular data. C. demetrioniana produces unbranched, paler, longer and wider conidiophores [40–350 × 4–6(–7) µm, in the original description a length of up to 1 mm is mentioned, versus (14.5–)28.5–160(–168) × (3–)3.5–4.5(–5) in C. beninensis] and above all, longer and wider conidia (75–230 × 4–7 µm with 7–16 indistinct septa versus (19–)23.5–122(–150) × (2.5–)3–4(–4.5) μm with 1–7(–9) distinct septa in C. beninensis] (Winter 1884; Saccardo 1886; Chupp 1954). C. demetrioniana f. minor differs from the present species by shorter and wider conidiophores (110–130 × 5–6 µm) and wider conidia (5–5.5 µm) (Ciferri and González-Fragoso 1926).

C. beninensis is distinct from all known species for which DNA sequence data are available based on its position in the multi-gene (Fig. 1) and in the tef1 phylogeny (see Suppl. material 4). In the ITS phylogeny, C. beninensis cannot be distinguished from other Cercospora spp. (see Suppl. material 3).

Cercospora aff. canescens Ellis & G.Martin, Am. Nat. 16(12): 1003 (1882).

MycoBank No: 179841

Figs 2B, C, 4

Type

USA (no further data available), on Phaseolus sp. (Fabaceae), 1882, s.n. (“Type?” NY, n.v.).

For synonyms see Crous and Braun (2003) or MycoBank.

Description

Leaf spots amphigenous, subcircular to irregularly angular, 3–11.5(–13) mm diam., occasionally crossing veins, reddish brown to slightly dark brown, with dark margin. Caespituli amphigenous, greyish brown to dark brown. Mycelium internal and external. Internal hyphae often indistinct. External hyphae branched, 2.5–3.5 μm wide, septate, olivaceous brown to brown, smooth. Stromata lacking or formed by few aggregated swollen hyphal cells, immersed in the mesophyll or in substomatal cavities, dark brown. Conidiophores in small, loose fascicles of up to 8, arising from stromata, breaking through the adaxial epidermis of the leaves or penetrating through stomatal openings, sometimes solitary arising through stomatal openings or erumpent through the cuticle, erect, straight to sinuous or somewhat geniculate, rarely branched, (16.5–)21–152(–165) × (4–)4.5–5.5 μm, 1–6-septate, brown to dark brown. Conidiogenous cells terminal, monoblastic to polyblastic, brown; loci 1.5–2.5 (–3) μm wide, thickened and darkened. Conidia solitary, narrowly obclavate to subacicular, straight to curved, (34–)38–280(–330) × (3–)3.5–4(–4.5) μm, 3–12(–14)-septate, hyaline to subhyaline, smooth, apex subacute or acute, base truncate to short obconically truncate, up to 2.5 μm wide, hila thickened and darkened.

Specimens examined

Benin. Borgou: Parakou, c. 363 m a.s.l., 9°20'29"N, 2°37'28"E, on Calopogonium sp., 21 Sep 2019, Y. Meswaet and A. Tabé, YMM07 (M-0312643, UNIPAR). Benin. Borgou: Parakou, c. 395 m a.s.l., 9°21'27"N, 2°36'44"E, Calopogonium sp., 17 Sep 2019, Y. Meswaet and A. Tabé, YMM08 (M-0312644). Benin. Borgou: Parakou, c. 395 m a.s.l., 9°21'27"N, 2°36'44"E, on Vigna subterranea, 16 Sep 2019, Y. Meswaet and R. Dramani, YMM01 (M-0312645, UNIPAR).

Herbarium specimens examined for comparison

C. canescens. On Vigna unguiculata (as V. sinensis L.): El Salvador. Sacocoyo, 3 Jul 1943, Wellman F. L. 140 (BPI 434127B). On V. unguiculata (as V. sinensis): USA. Illinois: Gallatin County, 8 Sep 1932, G.H. Boewe B331 (ILL23703 Holotype of C. vignicaulis Tehon). On V. unguiculata: USA. Illinois: Pulaski, Olmstead, 17 Sep 1933, G.H. Boewe s.n. (ILL24809 Paratype of C. vignicaulis). On V. unguiculata (as V. sinensis): USA. Illinois: White, Carmi., 10 Sep 1934, G.H. Boewe B588 (ILL 25450 Paratype of C. vignicaulis).

Hosts and distribution

On many species of Fabaceae and of other families (Crous and Braun 2003), known worldwide, from Australia, Bangladesh, Brazil, Bolivia, Brunei, Cambodia, China, Colombia, Costa Rica, Cuba, Dominican Republic, Ecuador, Fiji, Ghana, Guyana, Haiti, Hong Kong, India, Indonesia, Iran, Japan, Kenya, Korea, Malawi, Malaysia, Malawi, Mauritius, Myanmar, Nepal, New Caledonia, New Zealand, Nigeria, Pakistan, Panama, Papua New Guinea, Peru, Philippines, Puerto Rico, Russia, Senegal, Sierra Leone, Solomon Islands, Somalia, South Africa, Saint Vincent and the Grenadines, Sudan, Tadzhikistan, Taiwan, Tanzania, Thailand, Trinidad and Tobago, Togo, Uganda, USA, Uzbekistan, Vanuatu, Venezuela, Zambia, Zimbabwe (Chupp 1954; Ellis 1976; Shin and Kim 2001; Crous and Braun 2003; Farr and Rossman 2021).

Notes

The present Cercospora sp. on Calopogonium sp. also occurs on Vigna subterranea with different leaf spot appearances and caespituli. The lesions on Calopogonium sp. appear to be associated with a species of Pleosporales, whereas the leaf lesions on V. subterranea apparently are not associated with any other fungus and are dark reddish brown to dark brown with a dark margin, which are typical symptoms caused by Cercospora spp. The lesions on V. subterranea are larger and more abundant than those on Calopogonium sp., with abundant, dense caespituli and with dark greyish brown pigmentation (Fig. 2C).

Figure 2.

Leaf spot symptoms associated with Cercospora spp. A Cercospora beninensis on Crotalaria macrocalyx (YMM11) B Cercospora aff. canescens on Calopogonium sp. (YMM07) C Cercospora aff. canescens on Vigna subterranea (YMM01) D Cercospora fagopyri on Lablab sp. (YMM23A) E Cercospora parakouensis on Desmodium tortuosum (YMM296A) F Cercospora phaseoli-lunati on Vigna radiata (YMM289) G Cercospora rhynchophora on Vigna unguiculata (YMM03B) H Cercospora tentaculifera on Vigna unguiculata (YMM75) I Cercospora vignae-subterraneae on Vigna subterranea (YMM293) J Cercospora zorniicola on Zornia glochidiata (YMM299). Scale bars: 10 mm (A, C, F, G); 12 mm (B, D, E, H, J); 6 mm (I).

Figure 3.

Cercospora beninensis on Crotalaria macrocalyx (YMM11) A fascicle of conidiophores B individual conidiophores C conidia. Scale bars: 15 μm (A); 10 μm (B, C).

Cercospora canescens is the only species of Cercospora known for Calopogonium spp. (Farr and Rossman 2021) and has been reported from West Africa (Guinea) on Calopogonium mucunoides (Lenné 1990). Apart from having slightly narrower conidia [(3–)3.5–4(–4.5) μm versus 2.5–5.5(–6) μm in C. canescens] as described by Chupp (1954), Hsieh and Goh (1990) and Mulder and Holliday (1975), the present specimen from Benin is morphologically identical to C. canescens. In the phylogenetic analyses, however, DNA sequences of the two specimens from Benin cluster together but separately from sequences of C. canescens available from India. In the multi-gene tree (Fig. 1), C. canescens is located on a branch in a clade together with sequences of Cercospora spp. YMM3SO and YMM48SO on Sorghum bicolor (Poaceae) from Benin. C. canescens is known to correspond to a species complex that shows diverse morphological characteristics and genetic diversity (Joshi et al. 2006; Groenewald et al. 2013). Although C. canescens is an economically important species, no sequence data from the type or a neotype specimen are available (e.g., Groenewald et al. 2013). These are indispensable to resolve the C. canescens species complex. The specimens collected in Benin are tentatively placed into the species complex of C. canescens until DNA sequence data from the type locality (USA) and from diverse host species are available. C. aff. canescens is cited here for the first time for Benin (Piepenbring et al. 2020).

Figure 4.

Cercospora aff. canescens on Calopogonium sp. (YMM07) A fascicle of conidiophores protruding from a stomatal opening B solitary conidiophores C conidia. Scale bars: 15 μm (A, C); 10 μm (B).

Cercospora cf. canscorina Chidd., Sydowia 13 (1–6): 155. 1959.

MycoBank No: 294326

Fig. 5

Type

India. R. Br. Khandala (Maharashtra), on Canscora diffusa (Vahl) R.Br. ex Roem. & Schult. (Gentianaceae), 9 Nov 1956, Chiddarwar 4 (Holotype: IMI 83165, n.v.; Isotypes: HCIO, BPI, n.v.).

Description

Leaf spots amphigenous, subcircular to irregularly angular, 2.5–8 mm diam., brown to reddish brown, with a dark margin. Caespituli amphigenous, greyish brown to brown. Mycelium internal. Stromata lacking or formed by few substomatal aggregated swollen hyphal cells. Conidiophores in small, loose fascicles to moderately large and dense fascicles of up to approx. 22 conidiophores, arising from internal hyphae breaking through the adaxial epidermis of the leaves or penetrating through stomatal openings, sometimes solitary, erect, straight, subcylindrical, 1–2 times geniculate, unbranched, (12–)20.5–68(–72) × (3–)3.5–4.5 μm, 0–6-septate, brown to dark brown. Conidiogenous cells terminal, usually monoblastic, sometimes polyblastic; loci apical or sometimes located on the shoulders of geniculations, 1.5–2.5(–3) μm wide, thickened and darkened. Conidia solitary, acicular to narrowly obclavate, straight to curved, 22–76(–80) × 2.5–3.5 μm, 1–7-septate, hyaline, smooth, tip acute, base truncate to short obconically truncate, 2–3 µm wide, hila thickened and darkened.

Specimens examined

Benin. Borgou: Parakou, c. 386 m a.s.l., 9°20'35"N, 2°36'37"E, on Vigna sp., 14 Sep 2019, Y. Meswaet and R. Dramani, YMM05 (M-0312646; UNIPAR).

Hosts and distribution

On Canscora diffusa (Gentianaceae) from Khandala, West India (Chiddarwar 1959) and on Vigna unguiculata (as Vigna catjang (Burm.f.) Walp.) from India (Bhat and Pratibha 2010). C. cf. canscorina is reported here for the first time for Benin and for Africa.

Notes

Seven species of Cercospora have previously been recorded on Vigna spp., namely C. apii, C. canescens, C. canscorina, C. caracallae (Speg.) Vassiljevsky & Karak., C. kikuchii, C. longispora Peck and C. vignigena C. Nakash., Crous, U.Braun & H.D. Shin (Farr and Rossman 2021). The present species from Benin is morphologically identical to C. canscorina (Chiddarwar 1959; Bhat and Pratibha 2010) except for narrower conidiophores with (3–)3.5–4.5 μm versus 3–7 μm in C. canscorina as mentioned by Bhat and Pratibha (2010). The original specimen of C. canscorina was not available for morphological examination and no DNA sequence data are currently published for this species. Therefore, a reliable species identification is not possible. The application of the name for the collections from Benin is tentative and must be verified based on sequences derived from the Indian type specimen or similar samples. C. cf. canscorina differs from all species of Cercospora on other members of Fabaceae from Benin by producing unbranched, relatively pale conidiophores and above all, shorter conidiophores [(12–)20.5–68(–72) μm] and conidia [22–76(–80) μm]. Based on the multi-gene tree (Fig. 1) it is not possible to distinguish Cercospora cf. canscorina from many other Cercospora spp.

Figure 5.

Cercospora cf. canscorina on Vigna sp. (YMM05) A fascicle of conidiophores protruding from a stomatal opening B solitary conidiophores C conidia. Scale bars: 15 μm (A); 10 μm (B, C).

Cercospora cf. fagopyri K.Nakata & S.Takim., J. Agric. Exp. Stat. Gov. Gen. Chosen 15: 29. 1928.

MycoBank No: 456931

Figs 2D, 6

Type

South Korea. Suwon, on Fagopyrum esculentum Moench (Polygonaceae), Sep 1934, K. Nakata & S. Takimoto (holotype specimen, not located and not preserved according to Groenewald et al. (2013), neotype: CBS H-21008, n.v).

For synonyms see Groenewald et al. (2013) or MycoBank.

Description

Leaf spots amphigenous, circular to subcircular or rarely irregularly angular, 2–5 mm diam., more or less limited by veins, reddish to pale brown, margin dark brown on the adaxial surface, less conspicuous on the abaxial surface. Caespituli amphigenous, conspicuous, greyish brown to dark brown. Mycelium internal and external. External hyphae branched, often inconspicuous, 1.5–3 μm wide, septate, olivaceous brown to brown, smooth. Stromata lacking to well-developed, 10–45 µm diam., dark brown, substomatal or breaking through the epidermis. Conidiophores in small, loose to moderately dense fascicles of up to approx. 14 conidiophores, arising from stromata breaking through the adaxial epidermis of the leaves or through stomatal openings, sometimes solitary arising from external hyphae, erect, straight, subcylindrical to geniculate, unbranched, (22.5–)36–157(–168) × 3–4(–5) μm, 2–6(–8)-septate, brown to dark brown. Conidiogenous cells terminal, with 1–2 loci; loci mainly apical, sometimes located on the shoulders of geniculations, 1.5–2(–3) μm wide, thickened and darkened. Conidia solitary, acicular to narrowly obclavate, straight to somewhat curved, (24–)27.5–70(–78) × (2–)2.5–3(–4) μm, with 2–5(–6) somewhat indistinct septa, hyaline, smooth, tip acute, base truncate to short obconically truncate, 1.5–2.5 μm wide, hila thickened and darkened.

Specimens examined

Benin. Donga: Taneka-Koko, c. 441 m a.s.l., 9°51'30"N, 1°29'34"E, on Lablab sp., 29 Jul 2017, Y. Meswaet, M. Piepenbring, N. S. Yorou and participants of the summer school 2017, YMM23A ((M-0312647; UNIPAR). Same locality and host, 03 Aug 2016, Y. Meswaet, M. Piepenbring, N. S. Yorou and participants of the summer school 2016, YMM02 (M-0312648).

Hosts and distribution

On Cercis chinensis (Fabaceae), Cosmos bipinnata Cav. (Asteraceae), Fallopia dumetorum (L.) Holub and Fagopyrum esculentum (Polygonaceae), Hibiscus syriacus (Malvaceae), Viola mandshurica W. Becker (Violaceae), from China, Japan, South Korea, Taiwan, Uganda and Venezuela (Hsieh and Goh 1990; Groenewald et al. 2013). C. cf. fagopyri is cited here for the first time on Lablab sp. and the first time for Benin and West Africa.

Notes

Currently there are two species of the genus Cercospora known on hosts belonging to Lablab, namely C. canescens and C. apii. The present Cercospora sp. (YMM23A) differs from C. canescens in leaf spot size, stromata and septation characteristics, as well as unbranched conidiophores. Above all, the sizes of the conidia of the present species are different [(24–)27.5–70(–78) × (2–)2.5–3(–4) μm versus 30–300 × 2.5–5 (–6) µm in C. canescens]. C. apii differs by often small or lacking stromata, dense fascicules of up to 30 conidiophores, branched, longer conidiophores [20–300 μm versus (22.5–)36–157(–168) μm in C. cf. fagopyri] and above all, longer and wider conidia [25–315 × 3–6 μm versus (24–)27.5–70(–78) × (2–)2.5–3(–4) μm in C. cf. fagopyri] (Chupp 1954).

Our sequence of the tef1 region of the specimen YMM23A from Benin is 100% similar to a sequence of Cercospora fagopyri on Fallopia dumetorum (GenBank JX143353) (Identities 233/233, i.e., 100%) and 99% similar to a further sequence of C. fagopyri on Fagopyrum esculentum (GenBank JX143352; Identities; 233/234, i.e., 99%). The identification of the present specimen as C. cf. fagopyri is only based on molecular data. Morphologically, descriptions of specimens of C. fagopyri on diverse host species in the literature differ and are quite confusing (Hsieh and Goh 1990; Groenewald et al. 2013). In order to establish a morphological concept and to know the host range of C. fagopyri, fresh specimens need to be collected once again on Fagopyrum esculentum in Korea, where this species was originally collected and pathogenicity needs to be proven for diverse host species.

Figure 6.

Cercospora cf. fagopyri on Lablab sp. (YMM23A) A fascicle of conidiophores growing out from a slightly developed stroma in the epidermis shown as part of a transverse section of a leaf B solitary conidiophores C conidia. Scale bars: 15 μm (A); 10 μm (B, C).

Cercospora parakouensis Y.Meswaet, Mangelsdorff, Yorou & M.Piepenbr., sp. nov.

MycoBank No: 839171

Figs 2E, 7

Type

Benin. Borgou: Parakou, Tankaro, c. 360 m a.s.l., 9°23'01"N, 2°30'36"E, on Desmodium tortuosum (Sw.) DC. (Fabaceae), 20 Sep 2019, Y. Meswaet and R. Dramani, YMM296A (Holotype: M-0312649; Isotype: UNIPAR). Ex holotype sequences. MW834436 (LSU), MW834442 (ITS), MW848621 (tef1).