Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex

Abstract

Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and culture characteristics. DNA sequence analysis of the partial β-tubulin, histone H3, translation elongation factor 1-α and nuclear ribosomal RNA-Internal Transcribed Spacer (nrRNA-ITS) genes were employed to provide further support for the morphological species resolved among 68 isolates associated with root rot disease symptoms. Of the various loci screened, nrRNA-ITS sequences were the least informative, while histone H3 sequences were the most informative, resolving the same number of species as the combined dataset across the four genes. Within the Ilyonectria radicicola species complex, 12 new taxa are delineated occurring on a diverse range of hosts, the most common being Cyclamen, Lilium, Panax, Pseudotsuga, Quercus and Vitis.

Introduction

The genus Cylindrocarpon was introduced in 1913 by Wollenweber, with C. cylindroides as type. Cylindrocarpon and Cylindrocarpon-like species have since been commonly associated with root and decay of woody and herbaceous plants (Domsch et al. 2007). Cylindrocarpon root rot causes losses up to 30% on ginseng (Panax quinquefolium) (Seifert et al. 2003), and plays an important role in black foot rot of grapevines (Halleen et al. 2004, 2006), apple replant disease (Tewoldemedhin et al. 2010), and beech cankers (Castlebury et al. 2006), to name but a few hosts of economic importance.

In his taxonomic revision of Cylindrocarpon, Booth (1966) divided this genus into four groups based on the presence or absence of microconidia or chlamydospores. Booth’s group 4 represents Neonectria s. str., as it accommodates the type species N. ramulariae (anamorph: C. obtusiusculum). Most of the teleomorphs of Cylindrocarpon species have since this date been classified in Neonectria (Brayford et al. 2004; Halleen et al. 2004, 2006; Mantiri et al. 2001; Rossman et al. 1999). Several phylogenetic studies have, however, revealed that Neonectria/Cylindrocarpon is paraphyletic (Castlebury et al. 2006; Halleen et al. 2004, 2006; Hirooka et al. 2005; Mantiri et al. 2001). The first step in resolving this issue was taken by Halleen et al. (2004), who proposed Campylocarpon for species resembling Cylindrocarpon with 3–5-septate, curved macroconidia, and lacking microconidia. A further phylogenetic study (Chaverri et al. 2011) divided the Neonectria complex into four genera based on a combination of characters linked to perithecial anatomy and conidial septation: Ilyonectria, Neonectria/Cylindrocarpon s. str., Rugonectria and Thelonectria. In this study, a single generic name was proposed for each clade in an attempt to move towards a single nomenclature for pleomorphic fungi, meaning that the Cylindrocarpon-like anamorphs of Ilyonectria, Rugonectria and Thelonectria were placed in teleomorph genera, as recently done with other groups of pleomorphic fungi (Crous et al. 2006, 2007, 2009a; Gräfenhan et al. 2011; Lombard et al. 2010; Schroers et al. 2011).

Cylindrocarpon root rot is commonly associated with “Cylindrocarpon” destructans in the literature (Halleen et al. 2004; Samuels and Brayford 1990). This fungus was originally described as Ramularia destructans from roots of ginseng (Panax quinquefolium) collected in the USA (Zinssmeister 1918). Furthermore, it has been linked to the teleomorph Ilyonectria radicicola (Booth 1966; Chaverri et al. 2011; Samuels and Brayford 1990), which Gerlach and Nilsson (1963) described from rotting bulbs of Cyclamen persicum collected in Sweden. Samuels and Brayford (1990) commented on the morphological variation in collections of I. radicicola and its anamorph “C.” destructans. Seifert et al. (2003) showed that there was more than one “C.” destructans-like species occurring on Panax, and that none of the resolved clades correlated to the ex-type strain of I. radicicola, leading Halleen et al. (2006) to question the purported anamorph/teleomorph link between I. radicicola (from Cyclamen, Sweden) and “C.” destructans (from Panax, USA). Based on a phylogenetic analysis of ITS nrRNA gene sequences, Schroers et al. (2008) concluded that the I. radicicola complex includes “C.” destructans, “C.” destructans var. crassum, I. coprosmae, I. liriodendri, N. austroradicicola and N. macroconidialis.

The aim of the present study was to elucidate the morphological variation present within the I. radicicola complex, and to link fresh collections to older names introduced for species in this complex. This was addressed by combining morphological and culture characteristics with DNA sequence data derived from the Internal Transcribed Spacers (ITS) of the nrRNA gene operon, and partial β-tubulin (TUB), histone H3 (HIS), and translation elongation factor 1-α (TEF) genes.

Materials and methods

Isolates

This study (Table 1) included 42 “C.” destructans s. lat. isolates [including the ex-type strains of I. radicicola (CBS 264.65) and “C.” destructans f.sp. panacis (CBS 124662), “C.” destructans var. destructans and “C.” destructans var. crassum], six “C.” didymum isolates, six I. liriodendri isolates, one N. macroconidialis isolate and one I. coprosmae isolate, all deposited at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (CBS). Also included are two isolates that were previously identified as Ramularia mors-panacis (CBS 306.35) and R. panacicola (CBS 307.35) by Hildebrand (1935).

Table 1 Details pertaining to isolates investigated during this study

Besides those, 10 “Cylindrocarpon” spp. isolates were obtained in Portugal from grapevine plants showing decline symptoms, either 1- to 6-year-old plants in vineyards (Cy22, Cy155, Cy158, Cy190, CBS 129078, CBS 129080, CBS 129081, CBS 129082) or from rootstock nurseries (Cy23), and from a 25-year-old grapevine plant with esca symptoms (CBS 129084). Furthermore, isolates were obtained from a young Malus domestica (Cy164) and from the stem of a young Quercus suber (Cy232) plant, both showing decline symptoms, and from Thymus sp. (Cy231) and Ficus sp. (Cy228). One isolate (Cy131) was made available by P. Lecomte (Institut National de la Recherche Agronomique, Bordeaux-Aquitaine, France) and was obtained from an internal lesion of a stem of Actinidia chinensis ‘Hayward’. Another isolate (Cy122) was made available by W.D. Gubler (University of California, Davis, USA) and was obtained from Vitis sp. All of these isolates are stored in a culture collection at the Laboratório de Patologia Vegetal “Veríssimo de Almeida” (LPVVA-ISA, Lisbon, Portugal).

An additional 25 “C.” destructans isolates used during this study were made available by K.A. Seifert (Agriculture and Agri-Food, Canada), and were isolated from commercial Panax quinquefolium gardens (CBS 120359–120369, CBS 129079, CBS 129083, CD1666, CPC 13535, CPC 13537, NSAC-SH2, NSAC-SH2.5), Picea glauca (94–1628, CPC 13539), Poa pratensis (CPC 13534), Pseudotsuga menziesii (CBS 120370–120372, CPC 13536) and Prunus cerasus (CPC 13532) (Seifert et al. 2003).

Another 109 isolates were also included in the analysis to add phylogenetic support to this study and represent strains of the following taxa: C. cylindroides, C. obtusisporum, C. pauciseptatum, species 1 to 6 (Mostert et al., in preparation; Cabral et al., in preparation), I. macrodidyma, N. ditissima, N. major, N. neomacrospora and N. ramulariae.

DNA isolation, sequencing and phylogenetic analysis

For each isolate, genomic DNA was isolated from mycelium following the protocol of Möller et al. (1992), adapted by Crous et al. (2009b). Sequencing of the ITS and part of the β-tubulin (TUB), histone H3 (HIS) and translation elongation factor 1-α (TEF) genes was performed after PCR amplification using 1× PCR buffer (Bioline, London, UK), 1.5 mM MgCl₂, 32 μM of each dNTPs, 0.24 μM of each primer, 0.5 units Taq DNA Polymerase (Bioline), and 1 μl of diluted gDNA in a final volume of 12.5 μl. The cycle conditions in a iCycler thermocycler (BioRad, Hercules, USA) were 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 80 s, and a final elongation at 72°C for 10 min. Primers were V9G (de Hoog and Gerrits van den Ende 1998) and ITS4 (White et al. 1990) for ITS, T1 (O’Donnell and Cigelnik 1997) and Bt-2b (Glass and Donaldson 1995) for TUB, CYLH3F and CYLH3R (Crous et al. 2004b) for HIS, and EF1 and EF2 (O’Donnell et al. 1998) or CylEF-1 (5’- ATG GGT AAG GAV GAV AAG AC-3’; J.Z. Groenewald, unpublished) and CylEF-R2 (Crous et al. 2004b) for TEF. For TEF, the following modifications were made to the amplification protocol: 2.0 mM of MgCl₂, 40 μM of each dNTPs and addition of 5% of Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Zwijndrecht, Netherlands).

After confirmation by agarose gel electrophoresis, amplicons were sequenced in both directions with the corresponding PCR primers and a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Diegem, Belgium) according to the manufacturer’s recommendations. The products were analysed on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Waltham, USA).

Sequences were assembled and edited to resolve ambiguities, using the EditSeq and SeqMan modules of the Lasergene software package (DNAStar, Madison, USA). Consensus sequences for all isolates were compiled into a single file (Fasta format) and aligned using CLUSTAL X v. 2.0.11 (Larkin et al. 2007). Following manual adjustment of the alignment by eye where necessary, the alignment was subjected to phylogenetic analyses as described by Crous et al. (2004b). Novel sequences were lodged in GenBank (Table 1), taxonomic novelties in MycoBank (Crous et al. 2004a), and the alignments and phylogenetic trees in TreeBASE (http://www.treebase.org).

Morphology

Isolates were grown for up to 5 weeks at 20°C on synthetic nutrient poor agar (SNA; Nirenberg 1976) with and without two 1-cm² filter paper pieces, carnation leaf agar (CLA; Crous et al. 2009b), potato-dextrose agar (PDA; Difco, Detroit, USA) and oatmeal agar (OA; Crous et al. 2009b) under continuous n-UV light (NUV, 400–315 nm; Blacklight-Blue; Sylvania, Capelle a/d Ijssel, Netherlands).

Measurements were done on a 1-cm² agar plug removed from the colony margin, placed on a microscope slide, to which a drop of water and coverslip were added. For each isolate, 30 measurements were obtained for each structure. Measurements were done at ×1,000 magnification using a Nikon Eclipse 80i microscope, or a Leica DM2500. Images were captured using a Nikon DS-Fi1 digital camera with NIS-Elements Software, or a Leica DFC295 digital camera with the Leica Application Suite. Measurements for length and width of conidia and ascospores are given as (Minimum) Lower Limit of a 95% Confidence Interval – Upper Limit of a 95% Confidence Interval (Maximum). For other measurements, only the extreme values are given.

Culture characteristics (texture, density, colour, growth front, transparency and zonation) were described on PDA after incubation at 20°C in the dark for 14 days. Colour (surface and reverse) was described using the colour chart of Rayner (1970). Cardinal temperatures for growth were assessed by inoculating 90-mm-diam PDA dishes with a 3-mm-diam plug cut from the edge of an actively growing colony. Growth was determined after 7 days in two orthogonal directions. Trials were conducted at various temperatures (4, 10, 15, 18, 20, 22, 25, 30 and 35°C) with three replicate plates per strain at each temperature.

To induce the formation of perithecia, isolates were crossed in 60-mm-diam Petri dishes containing a minimal salts medium supplemented with two sterile birch toothpicks (Guerber and Correll 2001). The plates were incubated at 20°C under n-UV light for 8–20 weeks. Two strains were considered sexually compatible if perithecia were formed that exuded masses of viable ascospores. The colour reaction of the perithecia was checked in 3% KOH and in lactic acid. For sectioning, perithecia were mounted in Jung Tissue Freezing Medium (Leica) or in Arabian Gum, and cut in 10- to 15-μm-thick sections using a Leica cryostat CM3050 S or CM1850 at −20°C.

Results

Phylogeny

Amplification products of approximately 700 bases (ITS), 650 bases (TUB), 500 bases (HIS) and 600–800 bases (TEF) were obtained for the isolates listed in Table 1. The manually adjusted combined alignment contains 189 sequences (including the two outgroup sequences) and the statistical parameters for the combined and individual analyses are presented in Table 2. For the combined analysis, only a maximum of 1,000 equally most parsimonious trees were saved, the first of which is presented as Fig. 1. Phylogenetic trees derived from the individual loci are available in TreeBASE. The combined analysis of the four genes enabled the identification of 37 species. However, the analysis of HIS data alone was enough to resolve these taxa. Sequences of TEF could not distinguish species 6, I. robusta, I. europaea, I. lusitanica, I. rufa and N. ditissima; whereas sequences of TUB could not separate I. robusta, species 4, and 6, while “I.” macrodidyma, species 5, I. liliigena and I. pseudodesctructans were supported by low bootstrap values, and CBS 120370 clustered apart from the remaining isolates of I. crassa. Of all loci screened, ITS proved to be the least informative, being unable to resolve 22 of the species in this study. Neighbour-Joining (NJ) analyses using the three substitution models, as well as the parsimony analysis, yielded trees with similar topology and bootstrap support values for the individual and combined gene analyses. The trees obtained supported the same clades, sometimes with rearrangements in the order of these clades between the different analyses (data not shown). The results of the phylogenetic analyses are highlighted below under the taxonomic notes or in the Discussion, where applicable.

Table 2 Statistical information on the individual datasets and number of equally most parsimonious trees for each locus [Internal Transcribed Spacers (ITS) of the nuclear ribosomal RNA gene operon, and partial β-tubulin (TUB), histone H3 (HIS) and translation elongation factor 1-α (TEF) genes]

Fig. 1

The first of 1,000 equally most parsimonious trees obtained from the combined ITS, TUB, HIS and TEF sequence alignment of Cylindrocarpon isolates and relatives with a heuristic search using PAUP v. 4.0b10. The tree was rooted using Campylocarpon isolates as outgroup sequences and bootstrap support values are indicated near the nodes, where”ns” designates not supported. Ex-type strains are indicated in bold. Newly described species are indicated by blue boxes. Scale bar shows 10 changes

Taxonomy

The present study treats isolates that have been freshly collected, or previously identified and maintained in culture collections as “Cylindrocarpon destructans”, meaning cylindrical, rarely curved, 3-septate macroconidia with obtuse apices, abundant microconidia and chlamydospores (Samuels and Brayford 1990). The latter species has in the past been acknowledged as anamorph of I. radicicola (Booth 1966; Chaverri et al. 2011; Samuels and Brayford 1990). However, an examination of the neotype of “C.” destructans in this study [CUP-011985, conidia (18.0)23.0–30.0(35.0) × (6.0)6.5(7.0) μm], found conidia to be considerably smaller than those of I. radicicola (24.0)33.1(47.0) × (4.9)6.4(7.8) μm (Gerlach and Nilsson 1963) (also confirmed in the present study by examination of CBS 264.65, ex-type), revealing them to represent two distinct species. Furthermore, based on the phylogenetic and morphological data obtained in the present study, several novel species could be distinguished that are phylogenetically distinct from I. radicicola, and morphologically distinct based on a range of characters linked to culture characteristics, conidiophores, macro- and microconidium morphology. Some of these could be linked to older names, or taxa long regarded as potential syonyms of “destructans”, which could now be resurrected. These taxa are treated below:

Ilyonectria anthuriicola A. Cabral & Crous, sp. nov. (Fig. 2)

MycoBank 560108.

Fig. 2

Ilyonectria anthuriicola (CBS 564.95). a–c Simple conidiophores on aerial mycelium. d–g Conidiophores giving rise to microconidia, formed on mycelium at agar surface, penicillately mono- or bi-verticillate. h–l Micro- and macroconidia. m Chlamydospores in mycelium. Bars 10 μm

Etymology: Named after its host, Anthurium.

Cylindrocarpi destructantis morphologice simile, sed longitudine media conidiorum longiore, 29.5–32.2 μm, distinguitur.

Conidiophores simple or complex to sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched bearing up to three phialides, 1–3-septate, 40–95 μm long; phialides monophialidic, more or less cylindrical but slightly tapering towards the tip, 10.5–20.5 μm long, 2.5–3.5 μm wide at the base, 3.0–4.5 μm at widest point, 1.5–2.5 μm near the aperture. Conidiophores giving rise to microconidia, formed on mycelium at agar surface, penicillately mono- or bi-verticillate; phialides monophialidic, narrowly flask-shaped, typically with widest point near the middle, 8–15 μm long, 2.0–3.0 μm wide at the base, 2.5–4.5 μm at widest point, 1.0–2.0 μm near the apex. Sporodochial conidiophores irregularly branched; phialides cylindrical, mostly widest near the middle. Macroconidia formed in flat domes of slimy masses, (1–)3-septate, straight or minutely curved, cylindrical with both ends more or less obtusely rounded, mostly without a visible hilum; 1-septate, (20.0)23.5–26.7(29.0) × (5.5)5.9–6.8(7.0) μm (average = 25.1 × 6.4 μm), with a length:width ratio of 3.6–4.8; 2-septate, (25.0)26.6–29.3(32.0) × (6.5)6.8–7.8(8.5) μm (av. = 27.9 × 7.3 μm), with a length:width ratio of 3.2–4.8; 3-septate, (25.0)29.5–32.2(38.0) × (6.0)7.5–8.1(9.0) μm (av. = 30.8 × 7.8 μm) with a length:width ratio of 3.1–5.2. Microconidia 0(−1)-septate, subglobose to ovoid, rarely ellipsoid, mostly with a visible centrally located or slightly laterally displaced hilum; aseptate microconidia, (4.9)5.0–8.1(12.0) × (4.0)4.3–5.5(6.5) μm (av. = 6.5 × 4.9 μm), with a length:width ratio of 1.0–1.8; 1-septate, (11.0)11.6–16.7(18.0) × (5.0)5.4–6.1(6.0) μm (av. = 14.1 × 5.8 μm), with a length:width ratio 1.8–3.0. Chlamydospores globose to subglobose to ellipsoid, 8–14 × 7–12 μm, smooth, but often appearing rough due to deposits, thick-walled, formed intercalary in chains or in clumps and also in the cells of macroconidia, hyaline, becoming golden-brown.

Holotype: Netherlands, Bleiswijk, root rot of Anthurium sp., 1995, coll./isol. R. Pieters, holotype CBS H-20555, culture ex-type CBS 564.95.

Culture characteristics: Mycelium felty with average density. Surface on OA chestnut, with aerial mycelium sparse, saffron; margin pure yellow to orange. Surface on PDA, chestnut with saffron aerial mycelium, growth at margin luteous; zonation absent, transparency homogeneous, margin even; reverse similar to surface, but chestnut to cinnamon on OA, and chestnut on PDA. Colonies on PDA do not grow at 4°C after 7 days. Optimum temperature 20°C when colonies reach 25–27 mm, after 7 days. Colony diam was 20–22 mm at 25°C, after 7 days. Hardly grows at 30°C (2 mm colony diam after 7 days).

Isolate studied: CBS 564.95 (Table 1).

Host and distribution: Roots of Anthurium sp. (Netherlands).

Ilyonectria crassa (Wollenw.) A. Cabral & Crous, comb. et stat. nov. (Fig. 3)

MycoBank 560109.

Fig. 3

Ilyonectria crassa (CBS 129083). a–c Simple conidiophores on aerial mycelium. d–g Micro- and macroconidia. h–i Chlamydospores and macroconidia. Bars 10 μm

Basionym: Cylindrocarpon radicicola var. crassum Wollenw., Z. Parasitenkunde 3: 495. 1931.

≡ Cylindrocarpon destructans var. crassum (Wollenw.) C. Booth, Mycol. Pap. 104: 37. 1966.

Conidiophores simple or complex, to sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched bearing up to two phialides, rarely consisting only of phialides, 1–4-septate, 40–180 μm long; phialides monophialidic, cylindrical to subulate, 20–55 μm long, 2.5–4.0 μm wide at the base, 1.5–2.0 μm near the apex. Complex conidiophores aggregated in small sporodochia (on carnation leaf), repeatedly and irregularly branched; phialides more or less cylindrical, but tapering slightly in the upper part towards the apex, or narrowly flask-shaped, mostly with widest point near the middle, 17–24 μm long, 2.0–3.0 μm wide at the base, 2.5–3.5 μm at the widest point, and 1.5–2.5 μm wide near the apex. Macroconidia predominating, formed on both type of conidiophores, on SNA formed in flat domes of slimy masses, 1–3-septate, straight, cylindrical, but may narrow towards the tip, more or less broadly rounded, and the base appearing somewhat acute due to the presence of the hilum, mostly centrally located; 1-septate, (21.0)25.7–27.3(34.0) × (4.5)5.0–5.3(6.5) μm (av. = 26.5 × 5.1 μm), with a length:width ratio of 3.8–6.7; 2-septate, (23.0)28.5–30.3(37.0) × (4.5)5.3–5.6(6.5) μm (av. = 29.4 × 5.4 μm)with a length:width ratio of 4.2–6.7; 3-septate, (29.0)34.1–36.0(49.0) × (5.0)5.6–5.8(7.0) μm (av. = 35.1 × 5.7 μm), with a length:width ratio of 4.8–8.9. Microconidia 0–1-septate, ellipsoid to subcylindrical, more or less straight, with a visible, truncate hilum; aseptate microconidia, (7.0)9.7–10.9(15.0) × (3.0)3.3–3.6(4.5) μm (av. = 10.3 × 3.5 μm), with a length:width ratio of 1.8–4.3; 1-septate, (12.0)14.2–15.2(19.0) × (3.0)3.8–4.2(5.0) μm (av. = 14.7 × 4.0 μm), with a length:width ratio 2.7–5.0. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose to cylindrical, 7–15 × 6–10 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short lateral branches, rarely intercalary, single, in chains or in clumps, and also in the cells of the macroconidia, hyaline, becoming pale brown.

Lectotype: The Netherlands, on Lilium bulbs, Dec. 1930, coll./isol. W.F. van Hell, lectotype designated here CBS H-20556, culture ex-lectotype CBS 139.30.

Culture characteristics: Mycelium cottony to felty with average to strong density. Surface on OA cinnamon, with aerial mycelium sparse, buff. Surface on PDA saffron with aerial mycelium sparse buff to saffron to pale luteous. No zonation was observed, transparency was homogeneous and growth at margin even. Reverse similar to surface, except in colour, saffron to cinnamon on OA, and chestnut to sienna on PDA. Colonies on PDA grow 5–8 mm diam at 4°C after 7 days. Optimum temperature at 20°C, when colonies reach 31–46 mm diam, after 7 days. Colony diam was 19–34 mm at 25°C, after 7 days. No growth was observed at 30°C.

Isolates studied: CBS 139.30; CBS 158.31; CBS 129083; NSAC-SH-2; NSAC-SH-2.5 (Table 1).

Hosts and distribution: Lilium sp. (bulbs), Narcissus sp. (roots) (Netherlands), Panax quinquefolium (roots) (Canada).

Notes: In the original description, Wollenweber (1931) cites Cylindrocarpon radicicola var. crassum as occurring on roots of Ulmus, Taxus and Lilium in Europe (Germany and the Netherlands). He did not designate any type specimen. However, he specifically refers to a culture sent to him by Prof. J. Westerdijk on Lilium from the CBS in the Netherlands in 1930, which was regarded as authentic for the species. This culture is represented by CBS 139.30 (accessioned in 1930, from Lilium, the Netherlands), and thus we designate a dried, sporulating culture as lectotype for the species.

Ilyonectria cyclaminicola A. Cabral & Crous, sp. nov. (Fig. 4)

MycoBank 560110.

Fig. 4

Ilyonectria cyclaminicola (CBS302.93). a, b Simple conidiophores on aerial mycelium. c Pennicilate conidiophores with aseptate microconidia. d Sporodochial conidiophore on carnation leaf agar. e Isolated chlamydospores formed in lateral branches. f–j Micro- and macroconidia. Bars 10 μm

Etymology: Named after the host from which it was isolated, Cyclamen sp.

Cylindrocarpi destructantis morphologice simile, sed longitudine media conidiorum longiore, 26.9–31.9 μm, distinguitur.

Conidiophores simple or complex to sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–3-septate, 60–120 μm long; phialides monophialidic, more or less cylindrical but slightly tapering towards the tip, 20–60 μm long, 2.0–4.0 μm wide at the base, 3.0–4.5 μm at widest point, 1.5–2.5 μm near the aperture. Conidiophores giving rise to microconidia formed by mycelium at agar surface, penicillate to mono-verticillate; phialides monophialidic, more or less cylindrical, but with slight taper towards the tip, 19–34 μm long, 1.5–2.5 μm wide at the base, 2.0–3.0 μm at widest point, 1.0–2.0 μm near the apex. Sporodochial conidiophores irregularly branched; phialides more or less cylindrical, but slightly tapering towards the tip, or narrowly flask-shaped, with widest point near the base, 14–26 μm long, 2.5–3.5 μm wide at the base 3.0–4.0 μm at widest point, 1.0–2.0 μm near the apex. Macroconidia formed in flat domes of slimy masses, 1(−3)-septate, straight or minutely curved, cylindrical with both ends more or less broadly rounded, sometimes with a constriction at the septa, mostly without a visible hilum; 1-septate, (19.2)21.3–23.6(29.8) × (4.4)5.4–6.0(7.3) μm (av. = 22.5 × 5.7 μm), with a length:width ratio of 3.4–5.5; 2-septate, (23.8)24.0–28.4(29.8) × (5.0)5.5–7.3(8.0) μm (av. = 26.2 × 6.4 μm), with a length:width ratio of 3.1–5.1; 3-septate, (25.3)26.9–31.9(33.6) × (5.8)5.9–6.5(6.9) μm (av. = 29.4 × 6.2 μm), with a length:width ratio of 3.7–5.6. Microconidia formed in heads or on the agar surface, 0–1-septate, subglobose to ovoid to subcylindrical, mostly with a visible, centrally located or slightly laterally displaced hilum; aseptate microconidia, (3.9)7.6–8.9(12.9) × (2.2)3.6–3.9(5.4) μm (av. = 8.2 × 3.7 μm), with a length:width ratio of 1.2– 3.4; 1-septate, (11.5)13.8–15.2(17.5) × (3.7)4.6–4.9(5.5) μm (av. = 14.5 × 4.7 μm), with a length:width ratio of 2.3–3.9. Chlamydospores globose to subglobose, 10–18 × 10–16 μm, smooth, but often appearing rough due to deposits, thick-walled, formed in lateral branches, rarely intercalary, mostly isolated, hyaline, becoming medium brown.

Holotype: Netherlands, Roelofarendsveen, NAKS laboratory, Cyclamen bulb, May 1993, coll./isol. M. Hooftman, iden. E.J. Hermanides-Nijhof, holotype CBS H-20557, culture ex-type CBS 302.93.

Culture characteristics: Mycelium felty with average density. Surface on OA sepia to chestnut. Surface on PDA sepia to chestnut, with sparse, rust, aerial mycelium; no zonation was observed, and transparency was homogeneous; margins predominantly even. Reverse similar to surface, except in colour, sepia to dark brick on OA and chestnut on PDA. Colonies on PDA do not grow at 4°C after 7 days. Optimum temperature at 22°C, when colonies reach 68–70 mm diam, after 7 days. Colony diam was 63–64 mm at 25°C, after 7 days. No growth was observed at 30°C.

Isolate studied: CBS 302.93 (Table 1).

Host and distribution: Bulb of Cyclamen sp. (Netherlands).

Ilyonectria europaea A. Cabral, Rego & Crous, sp. nov. (Fig. 5)

MycoBank 560103.

Fig. 5

Ilyonectria europaea (CBS 129078). a–c Simple conidiophores on aerial mycelium. d Sporodochial conidiophore on carnation leaf agar. e Chlamydospores in aerial mycelium. f–i Micro- and macroconidia. Bars 10 μm

Etymology: Named after the European continent, where this fungus appears to be widely distributed.

Ilyonectriae robustae morphologice similis, sed longitudine media macroconidiorum breviore, 29.7–31.5 μm, distinguitur.

Conidiophores simple or complex to sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to three phialides, 1–3-septate, 50–120 μm long; phialides monophialidic, cylindrical to subulate, 26–60 μm long, 2.5–3.5 μm wide at the base, 1.5–2.5 μm near the apex. Complex conidiophores aggregated in small sporodochia (on carnation leaf), repeatedly and irregularly branched. Macroconidia predominating, formed on both type of conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight or minutely curved, cylindrical with both ends more or less broadly rounded, but may narrow towards the tip, mostly without a visible hilum; 1-septate, (16.4)21.9–23.4(34.0) × (4.0)5.2–5.6(7.8) μm (av. = 22.7 × 5.4 μm), with a length:width ratio of 3.2–5.4; 2-septate, (22.0)26.4–28.1(34.0) × (4.4)5.9–6.4(8.0) μm (av. = 27.2 × 6.1 μm), with a length:width ratio of 3.4–6.4; 3-septate, (22.0)29.7–31.5(40.0) × (5.0)6.5–6.9(8.6) μm (av. = 30.6 × 6.7 μm), with a length:width ratio of 3.5–6.0. Microconidia 0–1-septate, ellipsoid to ovoid, more or less straight, without a visible hilum; aseptate microconidia sometimes curved towards one end, (3.0)8.5–9.8(17.0) × (1.7)3.3–3.5(5.0) μm (av. = 9.1 × 3.4 μm), with a length:width ratio of 1.5–3.4; 1-septate, (9.2)13.4–14.6(18.9) × (3.0)4.0–4.4(5.9) μm (av. = 14.0 × 4.2 μm), with a length:width ratio 2.6–4.0. Conidia formed in heads or on simple conidiophores as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose, 9–14 × 7–14 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short or long lateral branches or intercalary, single, in chains or in clumps, golden-brown.

Holotype: Portugal, Vidigueira, at basal end of a 2-year-old Vitis vinifera plant; scion Petit Verdot, rootstock 110R, 2008, coll./isol. C. Rego, holotype CBS H-20558, culture ex-type CBS 129078 = Cy241 = CPC 19165.

Culture characteristics: Mycelium felty with average density. Surface on OA chestnut, with saffron aerial mycelium. Sienna to saffron on PDA, with luteous aerial mycelium. Concentric zonation, with homogeneous transparency, margins predominantly even. Reverse similar to surface, except in the colour; sepia on OA, and chestnut to umber on PDA. Colonies on PDA grow poorly, 1–5 mm diam at 4°C after 7 days. Optimum temperature for growth is 22°C, when colonies reach 43–57 mm diam, after 7 days. Colony diam was 37–47 mm at 25°C, after 7 days. No growth was observed at 30°C.

Isolates studied: Cy131; Cy155; CBS 537.92; CBS 102892; CBS 129078 (Table 1).

Hosts and distribution: Actinidia chinensis ‘Hayward’ (internal lesion of stem) (France), Aesculus hippocastanum (wood) (Belgium), Phragmites australis (stem) (Germany), Vitis vinifera (Portugal).

Ilyonectria gamsii A. Cabral & Crous, sp. nov. (Fig. 6)

MycoBank 560112.

Fig. 6

Ilyonectria gamsii (CBS 940.97). a–c Simple conidiophores on aerial mycelium. d–h Micro- and macroconidia. i Chlamydospores on mycelium. Bars 10 μm

Etymology: Named after Prof. dr. Walter Gams, who has made a major contribution to our knowledge of Hypocrealean soil fungi.

Ilyonectriae panacis morphologice similis, sed longitudine media macroconidiorum breviore, 34.3–38.5 μm, distinguitur.

Conidiophores simple or complex to sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–3-septate, 50–150 μm long; phialides monophialidic, cylindrical to subulate, 30–60 μm long, 2.5–3.5 μm wide at the base, 1.5–2.0 μm near the aperture. Sporodochial conidiophores irregularly branched; phialides cylindrical, mostly widest near the base. Macroconidia predominating, formed on simple conidiophores, on SNA formed in flat domes of slimy masses, 1–3-septate, straight, cylindrical with both ends broadly rounded, with mostly visible, centrally located hilum; 1-septate, (22.0)25.7–27.9(33.0) × (4.0)5.1–5.5(6.0) μm (av. = 26.8 × 5.3 μm), with a length:width ratio of 4.3–6.2; 2-septate, (25.0)28.2–31.7(39.0) × (5.0)5.5–5.9(6.5) μm (av. = 29.9 × 5.7 μm), with a length:width ratio of 4.2–7.1; 3-septate, (24.0)34.3–38.5(44.0) × (5.0)5.9–6.3(7.0) μm (av. = 36.4 × 6.1 μm), with a length:width ratio of 4.3–7.3. Microconidia 0–1-septate, ellipsoid to subcylindrical, more or less straight, mostly with a visible hilum; aseptate microconidia (4.0)6.9–8.0(10.0) × (3.0)4.0–4.5(5.0) μm (av. = 7.4 × 4.3 μm), with a length:width ratio of 1.3–2.9; 1-septate, (8.0)12.9–15.7(18.0) × (4.0)4.2–4.7(5.5) μm (av. = 14.3 × 4.4 μm), with a length:width ratio 1.8–4.0. Chlamydospores globose to subglobose to ellipsoidal, 8–14 × 7–12 μm, smooth, but often appearing rough due to deposits, thick-walled, mostly intercalary, rarely terminal on short lateral branches, single, in chains or in clumps, hyaline, becoming medium brown.

Holotype: Netherlands, Lelystad, soil, June 1997, coll./isol. J.T. Poll, iden. W. Gams, holotype CBS H-20559, culture ex-type CBS 940.97.

Culture characteristics: Mycelium cottony, dense. Surface on OA cinnamon, with sparse, buff aerial mycelium, on PDA umber to chestnut, with buff to saffron aerial mycelium; zonation absent, transparency homogeneous, margin even; reverse similar to surface, but chestnut on PDA. Colonies on PDA grow 6–7 mm diam at 4°C after 7 days. Optimum temperature at 22°C when colonies reach 44–45 mm diam, after 7 days. Colony diam is 22–24 mm at 25°C, after 7 days. No growth observed at 30°C.

Isolate studied: CBS 940.97 (Table 1).

Habitat and distribution: Soil (Netherlands).

Ilyonectria liliigena A. Cabral & Crous, sp. nov. (Fig. 7)

MycoBank 560114.

Fig. 7

Ilyonectria liliigena (CBS 189.49). a–d Simple conidiophores on aerial mycelium. e Chlamydospores on mycelium. f–i Micro- and macroconidia. Bars 10 μm

Etymology: Named after its host, Lilium regale.

Ilyonectriae panacis morphologice similis, sed longitudine media macroconidiorum 3-septatorum breviore, 27.9–29.8 μm, distinguitur.

Conidiophores simple or complex or sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–4-septate, 50–170 μm long; phialides monophialidic, cylindrical to subulate, 30–65 μm long, 2.0–3.5 μm wide at the base, 1.5–2.0 μm near the apex. Sporodochial conidiophores irregularly branched; phialides cylindrical, mostly widest near the base. Macroconidia predominating, formed on simple conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight or frequently minutely curved, cylindrical or sometimes typically minutely widening towards the tip, therefore appearing somewhat clavate, mostly without a visible hilum; 1-septate, (19.0)22.9–24.6(30.0) × (3.3)4.2–4.5(5.2) μm (av. = 23.8 × 4.3 μm), with a length:width ratio of 4.0–7.0; 2-septate, (21.0)26.1–27.7(32.1) × (4.0)4.7–5(5.7) μm (av. = 26.9 × 4.9 μm)with a length:width ratio of 3.8–7.0; 3-septate, (23.9)27.9–29.8(35.0) × (3.9)4.7–5.1(6.0) μm (av. = 28.9 × 4.9 μm), with a length:width ratio of 4.0–8.3. Microconidia 0–1-septate, ellipsoidal to subcylindrical, more or less straight, mostly with a visible hilum; aseptate, microconidia (5.9)8.9–10.3(17.0) × (2.5)3.0–3.2(4.4) μm (av. = 9.6 × 3.1 μm), with a length:width ratio of 2.0–4.6; 1-septate, (10.0)12.9–14.3(18.0) × (2.5)3.3–3.6(4.5) μm (av. = 13.6 × 3.4 μm), with a length:width ratio 2.8–5.6. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose, 6–14 × 5–12 μm, smooth but often appearing rough due to deposits, thick-walled, mostly in terminal on short lateral branches or rarely intercalary, single, in chains or in clumps, hyaline, becoming slightly brown at margins.

Holotype: Netherlands, Hoorn, bulb rot of Lilium regale, 1949, coll./isol. M.A.A. Schipper, holotype CBS H-20560, culture ex-type CBS 189.49.

Culture characteristics: Mycelium felty, with an average to strong density. Surface on OA sienna, with sparse, saffron, aerial mycelium. Surface on PDA sepia to cinnamon, with saffron to buff aerial mycelium. Zonation absent or concentric, with homogeneous transparency. Margins were even, or sometimes slightly uneven. Reverse similar to surface, except in colour; on OA pale vinaceous to cinnamon; on PDA buff to saffron to chestnut. Colonies on PDA grew poorly (1–4 mm diam) at 4°C after 7 days. Optimum temperature at 22°C, when colonies reach 34–45 mm diam, after 7 days. Colony diam was 16–29 mm at 25°C, after 7 days. No growth was observed at 30°C.

Isolates studied: CBS 189.49; CBS 732.74; CBS 304.85; CBS 305.85 (Table 1).

Host and distribution: Lilium regale bulbs (Netherlands).

Ilyonectria lusitanica A. Cabral, Rego & Crous, sp. nov. (Fig. 8)

MycoBank 560105.

Fig. 8

Ilyonectria lusitanica (CBS 129080). a–c Simple conidiophores of the aerial mycelium. d Chlamydospores on mycelium. e–g Micro- and macroconidia. Bars (a)  20 μm, (b–g) 10 μm

Etymology: Named after the Latin name for the country from where it was collected, Portugal.

Ilyonectriae europaeae morphologice similis, sed longitudine media macroconidiorum breviore, 25–28.4 μm, distinguitur.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–4-septate, 60–220 μm long; phialides monophialidic, cylindrical to subulate, 20–70 μm long, 2.5–3.5 μm wide at the base, 1.5–2.5 μm near the aperture. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed by both type of conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight or minutely curved, cylindrical with both ends more or less broadly rounded, but may narrow towards the tip, without a visible hilum, and may have a constriction on the septa in older cultures; 1-septate, (14.0)17.3–18.8(21.0) × (4.0)4.6–5(5.5) μm (av. = 18.1 × 4.8 μm), with a length:width ratio of 2.8–4.8; 2-septate, (18.0)20.5–22.1(27.0) × (4.0)4.9–5.2(6.0) μm (av. = 21.3 × 5.1 μm), with a length:width ratio of 3.5–5.4; 3-septate, (18.0)25.0–28.4(38.0) × (4.5)5.2–5.5(6.0) μm (av. = 26.7 × 5.4 μm), with a length:width ratio of 3.6–6.8. Microconidia 0–1-septate, ellipsoid to ovoid, more or less straight, without a visible hilum, and may have a constriction at the septum; aseptate, (5.0)6.9–8.2(10.0) × (2.5)3.0–3.3(4.0) μm (av. = 7.6 × 3.2 μm), with a length:width ratio of 1.7–3.3; 1-septate, (8.0)10.0–11.0(14.0) × (3.0)3.4–3.7(4.0) μm (av. = 10.5 × 3.6 μm), with a length:width ratio 2.0–3.7. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores rarely observed, globose to subglobose to cylindrical, 9–13 × 7–11 μm, smooth, but often appearing rough due to deposits, thick-walled, intercalary, hyaline, becoming slightly brown at the margin.

Holotype: Portugal, Melgaço, Alvaredo, on Vitis vinifera, below grafting zone, 6–year-old plant; scion Alvarinho on rootstock 196–17, 2005, coll./isol. N. Cruz, holotype CBS H-20563, culture ex-type CBS 129080 = Cy197 = CPC 19166.

Culture characteristics: Mycelium felty with average density. Surface on OA cinnamon, with aerial mycelium sparse, buff. Surface on PDA, cinnamon, with sparse, ochreous to buff aerial mycelium. Zonation absent, transparency homogeneous, margin even. Reverse similar to surface but buff to cinnamon on OA, and chestnut to cinnamon on PDA. Colonies on PDA grow 5–6 mm at 4°C after 7 days. Optimum temperature between 20 and 22°C, with colonies reaching 42–46 mm and 43–46 mm, respectively, after 7 days. Colony diam was 31–32 mm at 25°C, after 7 days. No growth observed at 30°C.

Isolate studied: CBS 129080 (Table 1).

Host and distribution: Vitis vinifera (Portugal).

Ilyonectria mors-panacis (A.A. Hildebr.) A. Cabral & Crous, comb. nov. (Fig. 9)

MycoBank 560115.

Fig. 9

Ilyonectria mors-panacis (CBS120363). a, b Simple conidiophores on aerial mycelium. c–g Micro- and macroconidia. h–j Chlamydospores on mycelium. Bars 10 μm

Basionym: Ramularia mors-panacis A.A. Hildebr., Can. J. Res. 12: 101. 1935.

= Cylindrocarpon panacis Matuo & Miyaz., Trans. Mycol. Soc. Japan 9: 111. 1969.

≡ Cylindrocarpon destructans f.sp. panacis Matuo & Miyaz., Ann. Phytopath. Soc. Japan 50: 390. 1984.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, rarely consisting only of phialides, 1–3-septate, 45–170 μm long; phialides monophialidic, cylindrical to subulate, 23–55 μm long, 2.0–3.0 μm wide at the base, 1.5–3.0 μm near the apex. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed on simple conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight, cylindrical with both ends more or less broadly rounded, mostly without a hilum; 1-septate, (21.0)28.2–31.6(40.0) × (5.0)5.8–6.3(7.5) μm (av. = 29.9 × 6.1 μm), with a length:width ratio of 3.3–7.0; 2-septate, (28.0)30.5–38.4(42.0) × (5.0)5.9–6.4–7.0(7.1) μm (av. = 34.4 × 6.4 μm), with a length:width ratio of 4.0–6.0; 3-septate, (37.8)39.0–44.2(45.0) × (6.9)7.0–7.5(7.5) μm (av. = 41.0 × 7.2 μm), with a length:width ratio of 5.3–6.0. Microconidia 0–1-septate, ellipsoid to subcylindrical, more or less straight, without a visible hilum; aseptate, (5.0)8.9–10.4(17.0) × (2.5)3.6–3.9(5.0) μm (av. = 9.6 × 3.8 μm), with a length:width ratio of 1.3–3.4; 1-septate, (9.0)12.5–14.1(19.0) × (3.5)4.4–4.8(5.5) μm (av. = 13.3 × 4.6 μm), with a length:width ratio 2.0–4.0. Conidia formed in heads on simple conidiophores or as white, creamy (OA) or hyaline (SNA) masses. Chlamydospores globose to subglobose, 8–16 × 7–15 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short lateral branches or intercalary, single, in chains or in clumps, hyaline, becoming medium brown.

Lectotype: Canada, Ontario, on living roots of Panax quinquefolium, June 1935, A.A. Hildebrand, lectotype designated here CBS H-20561, culture ex-lectotype CBS 306.35.

Culture characteristics: Mycelium felty with an average density. Surface on OA and PDA chestnut, with sparse, buff to rosy-buff to cinnamon or saffron aerial mycelium. Concentric zonation, with homogeneous transparency, and even margins. Reverse similar to surface, ochreous to fulvous, or sepia to dark vinaceous on OA, and chestnut to sienna on PDA. Colonies on PDA grow 3–9 mm diam at 4°C after 7 days. Optimum temperature for growth is 18°C, when colonies reach 22–40 mm diam, after 7 days. Colony diam was 31–40 mm at 25°C after 7 days. No growth was observed at 30°C.

Isolates studied: CBS 306.35; CBS 307.35; CBS 120359; CBS 120360; CBS 120361; CBS 120362; CBS 120363; CBS 120364; CBS 120365; CBS 120366; CBS 120367; CBS 120368; CBS 120369; CBS 124662; CPC 13535; CPC 13537 (Table 1).

Hosts and distribution: Panax ginseng (Japan), P. quinquefolium (Canada).

Notes: Ilyonectria mors-panacis is distinct from “C.” destructans (anamorph: “C”. destructans, neotype CUP-011985, conidia (18.0)23.0–30.0(35.0) × (6.0)6.5(7.0) μm) in having larger conidia, and indistinct hila (being prominent, flat, 2 μm diam in I. radicicola; see also Samuels and Brayford 1990, Fig. 1). “Ramularia” panacicola is distinct by also having shorter conidia than I. mors-panacis, 5.5–34.2 × 2.5–7.2 μm (Zinssmeister 1918), and appears to be another potential synonym of “C.” destructans. However, no authentic material could be located of “R.” panacicola, and the only isolate deposited under this name was a Canadian strain collected by Hildebrand (1935), which in fact represented I. mors-panacis (Fig. 1). The oldest name for the species on Panax treated here, therefore, is R. mors-panacis (CBS 306.35), with the Japanese collections (“C.” panacis ≡ “C.” destructans f.sp. panacis, CBS 124662 = NBRC 31881) being later synonyms (see Fig. 1).

Ilyonectria panacis A. Cabral & Crous, sp. nov. (Fig. 10)

MycoBank 560104.

Fig. 10

Ilyonectria panacis (CBS 129079). a–c Simple, unbranched or sparsely branched conidiophores on aerial mycelium. d, e Chlamydospores on mycelium. f–i Micro- and macroconidia. Bars 10 μm

Etymology: Named after its host, Panax quinquefolium.

Ilyonectriae liliigenae morphologice similis, sed longitudine media macroconidiorum longiore, 31–35 μm, distinguitur.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched bearing up to three phialides, 1–5-septate, 60–220 μm long; phialides monophialidic, cylindrical to subulate, 20–65 μm long, 2.5–3.0 μm wide at the base, 1.5–2.0 μm near the aperture. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed on both type of conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight, cylindrical with both ends more or less broadly rounded, mostly with a visible centrally located hilum; 1-septate, (20.0)23.7–25.9(32.0) × (4.0)4.7–5.0(5.5) μm (av. = 24.8 × 4.8 μm), with a length:width ratio of 4.0–6.0; 2-septate, (23.0)27.0–30.3(37.0) × (4.8)5.0–5.4(6.0) μm (av. = 28.7 × 5.2 μm), with a length:width ratio of 4.6–6.7; 3-septate, (27.0)31.2–35.0(49.0) × (5.0)5.4–5.8(6.0) μm (av. = 33.1 × 5.6 μm), with a length:width ratio of 4.9–8.2. Microconidia 0–1-septate, ellipsoid to ovoid to subcylindrical, more or less straight, mostly with a visible hilum; aseptate, (6.0)8.0–9.8(13.0) × (3.5)3.7–3.9(4.0) μm (av. = 8.9 × 3.8 μm), with a length:width ratio of 1.7–3.3; 1-septate, (8.0)11.3–13.7(16.0) × (3.5)3.8–4.2(4.5) μm (av. = 12.5 × 4.0 μm), with a length:width ratio 1.8–4.3. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose to ellipsoidal, 8–14 × 6–10 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short lateral branches or intercalary, single, in chains or in clumps, hyaline, becoming medium brown.

Holotype: Canada, Alberta, Panax quinquefolium, 1998, coll./isol. K. F. Chang, holotype CBS H-20562, culture ex-type CBS 129079 = CDC-N-9A = CPC 19167.

Culture characteristics: Mycelium felty with strong density. Surface on OA chestnut to sienna, with aerial mycelium sparse, vinaceous-buff. Surface on PDA chestnut to cinnamon, with aerial mycelium sparse, buff to saffron. No zonation was observed, and transparency was homogeneous; margins predominantly even. Reverse similar to surface, except in the colour, fawn to cinnamon on OA, and chestnut on PDA. Colonies on PDA grow 5 mm diam at 4°C after 7 days. Optimum temperature at 20°C, with colonies reaching 40–42 mm diam, after 7 days. Colony diam was 15 mm at 25°C after 7 days. No growth observed at 30°C.

Isolate studied: CBS 129079 (Table 1).

Host and distribution: Panax quinquefolium (Canada).

Notes: Several species have in the past been described on Panax in the genera Ramularia and Cylindrocarpon. The only unresolved species is “C.” destructans (and its potential synonym, “Ramularia” panacicola, see above). “Cylindrocarpon” destructans is clearly different from I. panacis, which has larger conidia, (27.0)31.2–33.1–35.0(49.0) × (5.0)5.4–5.6–5.8(6.0) μm.

Ilyonectria pseudodestructans A. Cabral, Rego & Crous, sp. nov. (Fig. 11)

MycoBank 560106.

Fig. 11

Ilyonectria pseudodestructans (all from CBS 129081, except g and e from CBS117824). a–d Simple, unbranched or sparsely branched conidiophores on aerial mycelium. e–g Chlamydospores on mycelium and macroconidia. h–l Micro- and macroconidia. Bars 10 μm

Etymology: Named after its morphological similarity to “Cylindrocarpon” destructans.

Ilyonectriae crassae morphologice similis, sed macroconidiis clavatis distinguitur.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–3-septate, 50–180 μm long; phialides monophialidic, cylindrical to subulate, 30–58 μm long, 2.5–3.5 μm wide at the base, 1.5–2.0 μm near the aperture. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed by simple conidiophores, on SNA formed in flat domes of slimy masses, 1–3(−4)-septate, straight, typically clavate, mostly centrally located hilum; 1-septate, (19.0)25.8–27.5(35.0) × (4.0)5.0–5.3(6.5) μm (av. = 26.7 × 5.2 μm), with a length:width ratio of 3.8–6.6; 2-septate, (23.0)30.0–31.7(38.0) × (5.0)5.3–5.5(6.0) μm (av. = 30.9 × 5.4 μm), with a length:width ratio of 4.3–7.4; 3-septate, (28.0)34.2–36.2(48.0) × (5.0)5.9–6.2(7.0) μm (av. = 35.2 × 6.0 μm), with a length:width ratio of 4.6–7.4. Microconidia 0–1-septate, ellipsoid to ovoid to subcylindrical, more or less straight, with a visible, centrally located hilum; aseptate (6.0)10.5–11.8(15.0) × (3.0)3.6–3.8(4.5) μm (av. = 11.2 × 3.7 μm), with a length:width ratio of 1.5–4.3; 1-septate, (10.0)14.6–15.6(18.0) × (3.0)4.1–4.4(5.0) μm (av. = 15.1 × 4.2 μm), with a length:width ratio of 2.4–5.0. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose to ellipsoid, 9–18 × 8–14 μm, smooth but often appearing rough due to deposits, thick-walled, terminal on short lateral branches or intercalary, in chains or in clumps, and also in the cells of macroconidia, hyaline, becoming medium brown.

Holotype: Portugal, São Paio, Gouveia, Vitis vinifera, 4-year-old, showing decline symptoms, scion Malvasia fina; rootstock 1103P, 1996, coll./isol. C. Rego, holotype CBS H-20564, culture ex-type CBS 129081 = Cy20 = CPC 19164.

Culture characteristics: Mycelium felty, with average to strong density. Surface on OA cinnamon, with sparse, buff to saffron or chestnut to sienna aerial mycelium. Surface on PDA cinnamon to vinaceous, with sparse, saffron to buff or chestnut to sienna aerial mycelium. Zonation absent, with homogeneous transparency; margins even. Reverse similar to surface, except in colour, sepia to cinnamon on OA and chestnut to cinnamon on PDA. Colonies on PDA grow poorly (4–6 mm diam), at 4°C after 7 days. Optimum temperature between 20–22°C, when colonies reach 32–44 mm and 37–41 mm diam, respectively, after 7 days. Colony diam was 22–29 mm at 25°C after 7 days. No growth was observed at 30°C.

Isolates studied: CPC 13534; CBS 117812; CBS 117824; CBS 129081; Cy22 (Table 1).

Hosts and distribution: Poa pratensis (Canada), Quercus sp. (Austria), Vitis vinifera (Portugal).

Notes: Ilyonectria pseudodestructans is reminiscent of “Cylindrocarpon” destructans, in having a similar conidial morphology (3-septate, with central, truncate hilum). However, conidia of I. pseudodestructans are somewhat longer than those of I. radicicola.

Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous, comb. nov. (Figs. 12 and 13)

MycoBank 560113.

Fig. 12

Ilyonectria robusta (a, b from CPC 13532 × CBS 308.35; c–k from CPC 13532 × CBS 117813). a, b Development of perithecia on the surface of a birch toothpick or agar. c–e Perithecium mounted in lactic acid. d Ostiolar area. e Surface view of perithecium wall region. f–h Longitudinal sections of perithecia showing details of ostiole and wall. i–k Asci and ascospores. Bars (a–c) 50 μm; (d, f) 20 μm; (e, g–k) 10 μm

Fig. 13

Ilyonectria robusta (All from CBS 129084, except f from CBS 605.92). a–c Simple conidiophores on aerial mycelium. d Sporodochial conidiophore on carnation leaf agar. e Chlamydospores on mycelium f–i Micro- and macroconidia. Bars 10 μm

Basionym: Ramularia robusta A.A. Hildebr. Can. J. Res. 12: 102. 1935.

Perithecia formed heterothallically in vitro, disposed solitarily or in groups, developing directly on the agar surface or on sterile pieces of birch wood, ovoid to obpyriform, with a flattened apex, up to 70 μm wide, orange to red, becoming purple-red in 3 % KOH (positive colour reaction), smooth to warted, up to 250 μm diam and high; perithecial wall consisting of two regions; outer region 11–36 μm thick, composed of 1–3 layers of angular to subglobose cells, 10–30 × 6–24 μm; cell walls up to 1 μm thick; inner region 8–14 μm thick, composed of cells that are flat in transverse optical section and angular to oval in subsurface optical face view, 5–11 × 2.5–5 μm; Asci narrowly clavate to cylindrical, 40–50 × 4.5–6 μm, 8-spored; apex subtruncate, with a minutely visible ring. Ascospores medianly 1-septate, ellipsoid to oblong-ellipsoid, somewhat tapering towards both ends, smooth to finely warted, frequently guttulate, hyaline, (8.2)9.4–9.7–10.0(11.5) × (2.5)2.9–3.0–3.1(3.7) μm. Conidiophores simple or complex or sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to three phialides, 1–4-septate, 55–160 μm long; phialides monophialidic, cylindrical to subulate, 20–60 μm long, 2.0–3.0 μm wide at the base, 1.5–2.0 μm near the apex. Complex conidiophores aggregated in small sporodochia (on carnation leaf agar; Crous et al. 2009b), repeatedly and irregularly branched; phialides more or less cylindrical, but tapering slightly in the upper part towards the apex, or narrowly flask-shaped, mostly with widest point near the middle, 15–20 μm long, 2.5–3.5 μm wide at the base, 3.0–4.0 μm at the widest point, and 1.0–2.0 μm wide near the apex. Macroconidia predominating, formed on simple conidiophores, on SNA formed in flat domes of slimy masses, 1–3-septate, straight, minutely curved or sometimes distorted, cylindrical with both ends more or less broadly rounded, but may narrow towards the tip, mostly without a visible hilum; 1-septate, (15.0)22.8–24.6(35.0) × (4.5)6.3–6.7(8.0) μm (av. = 23.7 × 6.5 μm), with a length:width ratio of 2.7–5.2; 2-septate, (20.0)26.2–28.1(38.0) × (5.0)6.9–7.2(8.0) μm (av. = 27.2 × 7.0 μm), with a length:width ratio of 2.9–5.2; 3-septate, (24)32.3–34.7(58) × (6.0)7.2–7.5(9.0) μm (av. = 33.5 × 7.4 μm), with a length:width ratio of 3.1–7.3. Microconidia 0–1-septate, ellipsoid to ovoid to subcylindrical, more or less straight, without a visible hilum; aseptate, (4.0)8.0–9.3(14.0) × (2.5)3.6–4.0(5.5) μm (av. = 8.7 × 3.8 μm), with a length:width ratio of 1.3–4.0; 1-septate, (9.0)13.5–14.7(18.0) × (3.5)4.7–5.1(6.0) μm (av. = 14.1 × 4.9 μm), with a length:width ratio 1.5–4.5. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose, 7–14 × 6–13 μm, smooth, but often appearing rough due to deposits, thick-walled, mostly occurring intercalary in chains, hyaline, becoming golden-brown.

Lecto- and teleotype: Canada, Ontario, on living roots of Panax quinquefolium, 1935, A.A. Hildebrand, lectotype designated here CBS H-20565, as dried culture of CBS 308.35; teleotype designated here CBS H-20566, including fertile perithecia of the teleomorph (CPC 13532 × CBS 308.35), culture ex-lectotype CBS 308.35.

Fertile matings: Perithecia observed after 4 wk in crossings of strains: CPC 13532 × CBS 308.35, CPC 13532 × CBS 773.83, CPC 13532 × CBS 605.92, CPC 13532 × CBS 117813, CBS 129084 × CBS 308.35, CBS 129084 × CBS 605.92, CBS 129084 × CBS 117813.

Culture characteristics: Mycelium felty with an average density. Surface on OA sienna to sepia with aerial mycelium sparse, buff. Surface on PDA cinnamon, with aerial mycelium buff to cinnamon, or rosy buff on PDA. Zonation absent to concentric, with homogeneous transparency; margins predominantly even, but sometimes uneven. Reverse similar to surface, except in the colour, sienna on OA and chestnut at the centre, and sienna to orange towards the margin on PDA. Colonies on PDA grow 4–7 mm at 4°C after 7 days. Optimum temperature at 22°C when colonies reach 40–52 mm diam, after 7 days. Colony diam was 35–48 mm at 25°C after 7 days. No growth to slight growth (0–2 mm) was observed at 30°C.

Isolates studied: CBS 321.34; CBS 308.35; CBS 773.83; CBS 605.92; CBS 117813; CBS 117814; CBS 117815; CBS 117817; CBS 117818; CBS 117819; CBS 117820; CBS 117821; CBS 117822; CBS 117823; CBS 129084; CD1666; CPC 13532; Cy23; Cy158; Cy231 (Table 1).

Hosts and distribution: Loroglossum hircinum (root) (Tunisia), Panax quinquefolium (root) (Canada), Prunus cerasus, Thymus sp., Vitis vinifera (basal end of rootstock) (Portugal), Quercus robur (root), Quercus sp. (root) (Austria), Tilia petiolaris (rootstock) (Germany), water (in aquarium with Anodonta sp.) (Netherlands).

Notes: When Hildebrand (1935) described Ramularia robusta from living roots of Panax quinquefolium in Ontario, Canada, he did not indicate a type specimen. However, he deposited an original culture in the CBS. A sporulating, dried-down culture is thus herewith designated as lectotype, and a new name proposed in Ilyonectria, with a teleotype represented by a fertile mating between CPC 13532 × CBS 308.35.

Ilyonectria rufa A. Cabral & Crous, sp. nov. (Fig. 14)

MycoBank 560116.

Fig. 14

Ilyonectria rufa (All from CBS 156.47, except c from CBS 120372). a–c Simple, sparsely branched conidiophores on aerial mycelium. d–f Chlamydospores in mycelium and in macroconidia. g–k Micro- and macroconidia. Bars 10 μm

Etymology: The epithet “rufa” referring to “Coleomyces rufus”, a provisional name proposed for this species by Moreau and Moreau (1937).

Ilyonectriae crassae morphologice similis, sed macroconidiis brevioribus, 28–31.2 μm longis, distinguitur.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to two phialides, 1–5-septate, 55–210 μm long; phialides monophialidic, cylindrical to subulate, 20–57 μm long, 2.5–3.5 μm wide at the base, 1.5–2.0 μm near the aperture. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed on both types of conidiophores, on SNA formed in flat domes of slimy masses, 1(−3)-septate, straight, cylindrical with both ends broadly round, mostly centrally located hilum; 1-septate, (17.0)22.3–23.8(29.0) × (4.0)5.1–5.4(6.0) μm (av. = 23.1 × 5.3 μm), with a length:width ratio of 3.1–5.6; 2-septate, (19.0)24.5–26.6(32.0) × (4.0)5.2–5.5(6.5) μm (av. = 25.5 × 5.4 μm), with a length:width ratio of 3.4–6.0; 3-septate, (23.0)28.6–31.2(37.0) × (5.0)5.5––5.9(7.0) μm (av. = 29.9 × 5.7 μm), with a length:width ratio of 3.4–7.2. Microconidia 0–1-septate, ellipsoid to subcylindrical, more or less straight, with a visible, centrally located hilum; aseptate, (4.0)8.4–9.8(15.0) × (3.0)3.5–3.8(5.0) μm (av. = 9.1 × 3.6 μm), with a length:width ratio of 1.3–4.0; 1-septate, (9.0)12.1–13.3(17.0) × (3.0)4.2–4.6(5.5) μm (av. = 12.7 × 4.4 μm), with a length:width ratio 2.2–3.8. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores globose to subglobose to cylindrical, 7–12 × 6–9 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short, lateral branches, or intercalary, single, in chains or in clumps, and also in the cells of the macroconidia, hyaline, becoming slightly brown in the outer wall.

Holotype: France, dune sand, Feb. 1937, coll./isol. F. Moreau, holotype CBS H-20567, culture ex-type CBS 153.37.

Culture characteristics: For CBS 153.37, CBS 156.47, CPC 13536 and 94–1628: Mycelium felty with average to strong density. Surface on OA buff to saffron, aerial mycelium sparse, buff. On PDA rosy-buff to cinnamon, with aerial mycelium sparse, buff to rosy-buff or pale luteus in the centre. For CBS 640.77, CBS 120371 and CBS 120372: Mycelium felty, with low to average density. Surface on OA cinnamon to sienna, aerial mycelium sparse, saffron to cinnamon. On PDA saffron to cinnamon, with aerial mycelium cinnamon to rust. Zonation absent or concentric, with homogeneous transparency; margins even or sometimes uneven. Reverse similar, except in colour, saffron on OA, and cinnamon to rosy-buff on PDA, or sienna with pigments, pale vinaceous in OA and umber to chestnut on PDA. Colonies on PDA grow poorly, (2–7 mm diam) at 4°C, after 7 days. Optimum temperature between 20–22°C, when colonies reach 28–42 mm, 31–46 mm diam, respectively, after 7 days. Colony diam was 19–24 mm at 25°C after 7 days. No growth observed at 30°C.

Isolates studied: CBS 153.37; CBS 156.47; CBS 640.77; CBS 120371; CBS 120372; CPC 13536; 94–1628 (Table 1).

Hosts and distribution: Azalea indica (Belgium), dune sand (France), Picea glauca, Pseudotsuga menziesii (Canada).

Notes: The genus Coleomyces represents a later synonym of Cylindrocarpon (Booth 1966). However, Coleomyces, which is based on C. rufus (Moreau and Moreau 1937), was published as “ad interim”, suggesting that Moreau and Moreau were planning to validate the name later, which was not the case. Based on the International Code of Botanical Nomenclature (Art. 34.1, Ex. 6), Chaverri et al. (2011) correctly chose to ignore the name. However, an original strain of C. rufus was deposited in the CBS (CBS 153.37), and the species epithet is herewith validated for the species.

Ilyonectria venezuelensis A. Cabral & Crous, sp. nov. (Fig. 15)

MycoBank 560117.

Fig. 15

Ilyonectria venezuelensis (CBS 102032). a, b Simple conidiophores on aerial mycelium. c–e Conidiophores bearing terminal, penicillate phialides. f–j Micro- and macroconidia. Bars 10 μm

Etymology: Named after the country from where it was collected, Venezuela.

Ilyonectriae robustae morphologice similis, sed conidiophoris cum verticillo terminali phialidum distinguitur.

Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium or from agar surface, solitary to loosely aggregated, unbranched, or bearing terminal, penicillate phialides, 1–4-septate, 35–200 μm long; phialides monophialidic, cylindrical to subulate, 40–60 μm long, 2.5–3.5 μm wide at the base, 1.5–2.5 μm near the apex, or narrowly flask-shaped, 16–40 μm long, 2.0–3.0 μm wide at the base, 3.0–4.0 μm at the widest point, 1.5–2.5 μm near the apex. Complex conidiophores aggregated in small sporodochia, repeatedly and irregularly branched. Macroconidia predominating, formed by both types of conidiophores, on SNA formed in flat domes of slimy masses, 1–3-septate, straight or minutely curved, cylindrical with both ends more or less broadly rounded, but may narrow towards the tip, mostly without a visible hilum; 1-septate, (22.0)24.6–27.3(35.0) × (5.0)5.3–5.7(6.5) μm (av. = 26.0 × 5.5 μm), with a length:width ratio of 3.8–7.0; 2-septate, (25.0)26.3–37.4(44.0) × (5.9)6.0–6.6(7.0) μm (av. = 31.9 × 6.3 μm), with a length:width ratio of 4.2–6.8; 3-septate, (28.0)36.5–41.7(50.0) × (6.0)6.9–7.5(8.0) μm (av. = 39.1 × 7.2 μm), with a length:width ratio of 4.0–6.7. Microconidia 0–1-septate, ellipsoid to ovoid, more or less straight, without a visible hilum; aseptate, (5.0)8.4–10.5(13.0) × (3.0)3.3–3.7(4.0) μm (av. = 9.5 × 3.5 μm), with a length:width ratio of 1.7–3.4; 1-septate, (11.0)14.5–16.3(19.0) × (3.5)3.9–4.3(5.0) μm (av. = 15.4 × 4.1 μm), with a length:width ratio 2.8–4.8. Conidia formed in heads on simple conidiophores or as white (OA) or unpigmented (SNA) masses. Chlamydospores ovoid to ellipsoidal, 6–13 × 5–7 μm, smooth, but often appearing rough due to deposits, thick-walled, terminal on short lateral branches or intercalary, single, in chains or in clumps, hyaline, becoming slightly brown at the margin.

Holotype: Venezuela, Amazonas, Cerro de la Neblina, tree bark, 1985, coll./isol. A. Rossman, holotype CBS H-20568, culture ex-type CBS 102032.

Culture characteristics: Mycelium cottony with average to strong density. Surface on OA saffron, with aerial mycelium sparse, buff, on PDA buff to saffron, with aerial mycelium saffron to pale luteous; zonation absent, transparency homogeneous, margin even; reverse similar to surface, but saffron to cinnamon on PDA. Colonies on PDA grow poorly (2–3 mm) at 4°C, after 7 days. Optimum temperature at 20°C, with colonies reaching 49 mm diam, after 7 days. Colony diam was 35–36 mm at 25°C after 7 days. No growth was observed at 30°C.

Isolate studied: CBS 102032 (Table 1).

Host and distribution: Tree bark (Venezuela).

Ilyonectria vitis A. Cabral, Rego & Crous, sp. nov. (Fig. 16)

MycoBank 560107.

Fig. 16

Ilyonectria vitis (CBS 129082). a–c Simple conidiophores on aerial mycelium. d–g Conidiophores forming microconidia arising from mycelium at agar surface, reduced to a stipe with a terminal arrangement of phialides, ranging from 2 to a dense cluster; sparsely branched or penicillate. i–l Micro- and macroconidia. m Chlamydospores on mycelium. Bars 10 μm

Etymology: Named after the host from which it was collected, Vitis vinifera.

Ilyonectriae anthuriicolae morphologice similis, sed longitudine media macroconidiorum longiore, 41.6–43.5 μm, distinguitur.

Conidiophores simple or complex or sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely branched, bearing up to three phialides, 1–3 septate, 30–70 μm long; monophialides more or less cylindrical, but tapering slightly towards the tip, 11–21 μm long, 2.0–3.0 μm wide at the base, 3.0–4.5 μm at widest point, 1.5–2.5 μm near the apex. Conidiophores forming microconidia arising from mycelium at agar surface, reduced to monophialides, or a stipe with a terminal arrangement of phialides, ranging from 2 to a dense cluster; sparsely branched or penicillate; monophialides narrowly flask-shaped, typically with widest point near the middle, 10–17 μm long, 1.5–3.0 μm wide at the base, 2.5–4.0 μm at widest point, 1.0–2.0 μm near the apex. Sporodochial conidiophores irregularly branched; phialides more or less cylindrical but slightly tapering towards the tip, or narrowly flask-shaped, with widest point near the middle, 14–20 μm long, 2.5–3.5 μm wide at the base, 3.0–4.5 μm at widest point, 1.5–2.5 μm near the apex. Macroconidia formed in flat domes of slimy masses, predominantly 3-septate, rarely 1–2- or 4-septate, straight or minutely curved, cylindrical with both ends more or less broadly rounded, mostly without a visible hilum; 3-septate conidia (34.9)41.6–43.5(51.6) × (6.2)7.9–8.2(9.5) μm (av. =42.5 × 8.0 μm), with a length:width ratio of 3.9–6.7. Microconidia on SNA formed in heads, aseptate, subglobose to ovoid, rarely ellipsoid, mostly with a visible, centrally located or slightly laterally displaced hilum, (3.7)4.9–5.4(6.7) × (3.2)3.7–4.0(4.6) μm (av. = 5.1 × 3.9 μm), with a length:width ratio of 1.1–1.7. Chlamydospores globose to subglobose to ellipsoid, 9–18 × 6–13 μm, smooth, but often appearing rough due to deposits, thick-walled, formed intercalary in chains or in clumps, and also in the cells of macroconidia, hyaline, becoming golden-brown.

Holotype: Portugal, Vidigueira, Vitis vinifera, basal end of a 2-year-old plant; scion Touriga Nacional; rootstock 110R, 2008, coll./isol. C. Rego, holotype CBS H-20569, culture ex-type CBS 129082 = Cy233 = CPC 19168.

Culture characteristics: Mycelium felty with density low to average. Surface on OA sienna, with sparse, saffron aerial mycelium, and luteous growth at margin. Surface on PDA chestnut, with sienna aerial mycelium, with luteous margin. Zonation was absent (OA) or concentric (PDA), transparency was homogeneous (PDA) or not (OA). Growth at margin even to uneven. Reverse similar to surface, except in colour, sienna to saffron on OA, and chestnut to umber on PDA. Colonies on PDA do not grow at 4°C after 7 days. Optimum temperature at 20°C, when colonies reach 29–30 mm diam, after 7 days. Colony diam was 39–40 mm at 25°C and 8–9 mm at 30°C after 7 days. No growth was observed at 35°C.

Isolate studied: CBS 129082 (Table 1).

Host and distribution: Vitis vinifera (Portugal).

Key to species treated

• 1 Growth at margin on OA after 14 days at 20°C, lacking yellow pigmentation

• 2 Colony diameter on PDA after 7 days at 25°C < 30 mm

• 3 Macroconidia forming chlamydospores

• 4 Macroconidia 1–3-septate, 3-septate macroconidia mean range 34.1–36.2 μm long

• 5 Macroconidia cylindrical, with the base appearing somewhat acute I. crassa

• 5* Macroconidia clavate I. pseudodestructans

• 4* Macroconidia predominantly 1-septate; 3-septate macroconidia smaller, mean range 28–31.2 μm long I. rufa

• 3* Macroconidia lacking chlamydospores

• 6 Macroconidia predominantly curved I. liliigena

• 6* Macroconidia straight

• 7 Macroconidia lacking visible hilum I. mors-panacis

• 7* Macroconidia with a visible, centrally located hilum

• 8 Three-septate macroconidia mean range 31.0–35.0 μm long I. panacis

• 8* Three-septate macroconidia mean range 34.3–38.5 μm long I. gamsii

• 2* Colony diameter after 7 days at 25°C >30 mm

• 9 Colony diameter after 7 days at 25°C, >50 mm I. cyclaminicola

• 9* Colony diameter after 7 days at 25°C, 30–50 mm

• 10 Conidiophores bearing a terminal whorl of phialides I. venezuelensis

• 10* Conidiophores unbranched, or different from above

• 11 Teleomorph known, and can be induced in culture

• 12 Three-septate macroconidia mean range 32.3–34.7 μm long; ascospores mean range 9.4–10.0 μm long I. robusta

• 12* Three-septate macroconidia mean range 30.0–36.0 μm long; ascospores mean range longer, 10–13 μm long I. radicicola ^a

• 11* Teleomorph unknown

• 13 Mean range of 3-septate macroconidia, 29.7–31.5 × 6.5–6.9 μm I. europaea

• 13* Mean range of 3-septate macroconidia smaller, 25.0–28.4 × 5.2–5.5 μm I. lusitanica

• 1* Yellow pigmentation present at margin

• 14 Macroconidia 3-septate, mean range 29.5–32.2 μm long I. anthuriicola

• 14* Macroconidia 3-septate, mean range 41.6–43.5 μm long I. vitis

^aNo authentic cultures of “C.” destructans, conidia (18.0–)23.0–30.0(−35.0) × (6.0–)6.5(−7.0) μm, are presently available.

Discussion

“Cylindrocarpon” destructans is a cosmopolitan soil-borne pathogen causing disease on a wide number of herbaceous and woody plant species (Samuels and Brayford 1990). The wide morphological and pathogenic amplitude of “C.” destructans makes it a commonly identified species, with many diseases from the Cylindrocarpon-complex being attributed to it, and ranking at the top of all “Cylindrocarpon” spp. deposited in the NCBI nucleotide database.

In this study, “C.” destructans isolates from the CBS culture collection (deposited under the wider concept of the species) were analysed using a multigene approach in order to clarify taxonomic aspects of this species complex. Molecular analyses show that these isolates cluster in various clades supported by high bootstrap support values. A previous study (Seifert et al. 2003) included a subset of the strains used here, and already highlighted the existence of unexpected divergence in “C.” destructans, as opposed to a large homogeneity in e.g. Neonectria ditissima. Several species have in recent years been separated from the “C.” destructans complex, including “C.” macroconidialis, “C.” coprosmae and “C.” austroradicicola based on morphological (Samuels and Brayford 1990) and molecular characters (Schroers et al. 2008; Seifert et al. 2003). Furthermore, several isolates causing black foot disease of grapevine, previously considered as “C.” destructans, were recently identified as I. liriodendri (Chaverri et al. 2011; Halleen et al. 2006), along with the ex-type strain from Liriodendron tulipifera (CBS 110.81) and a strain from Cyclamen (CBS 301.93). In this study, two further strains isolated from young Malus domestica and Quercus suber trees showing decline symptoms were also identified as I. liriodendri.

Altogether, we analysed 68 strains putatively belonging to “C.” destructans, but none of them clustered together with the ex-type culture of I. radicicola (CBS 264.65), suggesting that this species may not be as common as previously accepted. Halleen et al. (2006) identified a single strain (IMI 313237, isolated from arecoid palm) clustering with CBS 264.65. This also raises questions relating to the correlation between the anamorph, “C.” destructans, and its purported teleomorph, I. radicicola.

“Nectria” radicicola was described by Gerlach and Nilson (1963) from decayed leaves, flowers stalks and corms of Cyclamen persicum collected in Sweden, with a “Cylindrocarpon” anamorph they identified as “C.” radicicola.

In 1924, Wollenweber introduced “C.” radicicola (McAlpine) Wollenw. as a new combination, based on Septocylindrium radicicola McAlpine (1899), described from Citrus trees in Australia. Later, Wollenweber (1928) noted that Septocylindrium radicicola, with catenulate conidia, was different from “C.” radicicola, and the name was therefore based on Wollenweber’s (1928) description. Because of this confusion in names, Booth (1966) suggested that “C.” radicicola should be dropped, and that the name to be used as anamorph for I. radicicola should be “C.” destructans [originally described by Zinssmeister (1918) on Panax quinquefolia from Wisconsin, USA]. Furthermore, Booth (1966) designated a neotype for “C.” destructans, obtained from P. quinquefolia in USA, KY, Washington Co., Springfield; collected by W.B. Edwards in 1922, available as herbarium material in Cornell Plant Pathology Herbarium, CUP-011985. This specimen was re-examined in the present study [conidia (18.0)23.0–30.0(35.0) × (6.0)6.5(7.0) μm] [original description by Zinssmeister (1918), 9.0–32.4 × 3.2–8.1 μm], thus revealing conidia to be smaller than those formed by I. radicicola, which are (24.0)33.1(47.0) × (4.9)6.4(7.8) μm. From these observations, we conclude that “C.” destructans, which occurs on P. quinquefolia in the USA, represents yet another species distinct from I. radicicola, which is not yet represented in our phylogenetic tree (Fig. 1).

A strain deposited in CBS culture collection by Hildebrand in 1935, as “Ramularia” mors-panacis, was found to represent original material (ex-type CBS 306.35), collected from living roots of Panax quinquefolium in Ontario, Canada. The epithet “mors-panacis” is therefore resurrected for this clade, while the Japanese collection identified as “Cylindrocarpon destructans” f. sp. panacis (ex-type CBS 124662 = NBRC 31881), isolated from Panax ginseng in Japan, is treated as synonym.

The ex-type strain of “Ramularia” robusta (CBS 308.35), isolated from living roots of Panax quinquefolium in Ontario, Canada, can be resurrected for a large clade representing isolates from a range of hosts and continents. Similarly, an authentic strain of “C.” destructans var. crassum (Booth 1966; Wollenweber 1931) is available for a species occurring on Panax and Pseudotsuga in Canada, Lilium and Narcissus in the Netherlands, and can thus be resurrected as I. crassa. Although strain CBS 120370 clustered together with other strains of I. crassa for most genes (no nucleotide differences in ITS and TEF, two nucleotide differences in HIS), this strain was not included in that species because of an 8-bp difference in TUB, a slower growth rate (e.g., 21 mm colony diam at 20°C for 7 days, as opposed to 31–46 mm for other isolates), a lower conidial length:width ratio [e.g. for 3-septate conidia 4.0–6.5, as opposed to 4.8–8.9 and smaller conidia (e.g. 3-septate conidia ranging from (26.0)31.2–34.0(40.0) × (6.0)6.6–7.1(8.0) μm (av. 32.6 × 6.9 μm) , as opposed to (29.0)34.1–36(49.0) × (5.0)5.6–5.8(7.0) μm (av. 35.1 × 5.7 μm)]. Further studies should thus be conducted in order to clarify the taxonomy of this strain.

Ilyonectria anthuriicola and I. vitis are very similar in morphology to “C.” pauciseptatum. These species all have predominantly 3-septate macroconidia after 10 days in culture. Ilyonectria anthuriicola is easier to distinguish than “C.” pauciseptatum as the 3-septate conidia are smaller and narrower, (25.0)29.5–32.2(38.0) × (6.0)7.5–8.1(9.0) μm, while in “C.” pauciseptatum they are (37.0)42.0–47.0(54.0) × (7.0)8.5–9.5(10.0) (Schroers et al. 2008). For I. vitis 3-septate macroconidia are of similar size to those of “C.” pauciseptatum, (34.9)41.6–43.5(51.6) × (6.2)7.9–8.2(9.5), making it difficult to distinguish them based on this character. Growth rate at 20°C is slower in I. vitis than in “C.” pauciseptatum, and they also differ regarding colony characteristics and colours. Morphologically, however, they remain difficult to distinguish.

In this study, the genetic structure of the I. radicicola complex was analysed using a multi-locus approach along with morphological and culture characters. Three major groups were identified based on this approach, each group containing several species. Although we have been able to clarify several aspects related to the host range and distribution of taxa in the I. radicicola species complex, further collections, especially from Panax in the USA, will be essential to elucidate the status of “C.” destructans.