Introduction

The Proteaceae is a family of the Proteales in the Rosidae which developed approximately 96 million years ago, representing one of the most prominent plant families of the Southern Hemisphere, in southern Africa, Asia, Australia, Central and South America, especially in areas with long dry seasons (Crous et al. 2004a). The majority of genera, however, are found in Australia and South Africa (Taylor et al. 2001c). Given their beauty, unique appearance and relatively long shelf life, some members of the Proteaceae have been sought-after for the export market being commercially valuable as cut flowers (Crous and Palm 1999). Many species of South African Proteaceae are cultivated in Australia, the Azores, Chile, France, Israel, New Zealand, Portugal (including Madeira Island), Spain (including Canary Islands), Thailand, USA (California, Hawaii) and Zimbabwe. Some Australian Proteaceae species are also cultivated in countries other than Australia (Crous et al. 2000).

One of the factors limiting commercial production of Proteaceae is damage caused by fungal diseases (Knox-Davies 1981; Wright and Saunderson 1995; Crous et al. 2004a). Some pathogens were even considered as actionable quarantine organisms (Crous et al. 2000). Colletotrichum spp. belong to the most devastating fungal pathogens of Proteaceae, causing seedling damping off, shepherd’s crook, anthracnose, leaf lesions, pruning wound dieback and stem dieback (Knox-Davies 1981; Knox-Davies et al. 1986; Von Broembsen 1989; Crous et al. 2004a).

Colletotrichum gloeosporioides was previously regarded as the only Colletotrichum species to infect species of the Proteaceae (Baxter et al. 1983). Based on morphology, sequence data of the internal transcribed spacer region (ITS) and partial sequences of the Beta-tubulin gene (TUB2), Lubbe et al. (2004) differentiated four species of Colletotrichum (C. acutatum, C. boninense, C. crassipes, C. gloeosporioides) and one forma specialis (C. acutatum f. sp. hakeae) associated with diseased Proteaceae. An additional strain identified as C. gloeosporioides based on ITS and 28S rDNA gene (LSU) sequence data was included in the study of Marincowitz et al. (2008a).

Recently, systematic studies of Colletotrichum species complexes have started to employ a polyphasic approach to species identification, emphasizing multi-locus phylogeny in conjunction with recognisable phenotypic characters (Cai et al. 2009; Damm et al. 2009, 2012a, b; Rojas et al. 2010; Liu et al. 2011; Weir et al. 2012). Using this approach, many cryptic and new species associated with Proteaceae have been revealed, e.g. C. acutatum, C. australe, C. fioriniae, C. nymphaeae and C. simmondsii in the C. acutatum species complex (Damm et al. 2012a) and C. boninense and C. karstii in the C. boninense species complex (Damm et al. 2012b). However, in the recent revision of the C. gloeosporioides species complex (Weir et al. 2012), only one strain from Proteaceae (Banksia, series Dryandra) was included. The aim of the present study is therefore to reassess the identification of strains associated with Proteaceae that belong to the C. gloeosporioides species complex.

Materials and methods

Isolates

Isolates previously identified as C. gloeosporioides and related strains associated with Proteaceae obtained from the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands, were used for morphological and phylogenetic analyses and presented in Table 1. Type specimens of the species newly described here are located in the fungarium of the CBS. Descriptions of new species are based on an examination of ex-type cultures.

Table 1 Strains of Colletotrichum studied in this paper with details about host/substrate and location, and accession numbers of reference sequences from GenBank
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Morphological analysis

Agar plugs (5-mm-diam) were taken from the periphery of actively growing cultures and transferred to the centre of 9-cm-diam Petri dishes containing 2 % potato dextrose agar (PDA; Difco) or synthetic nutrient-poor agar medium (SNA; Nirenberg 1976) amended with double-autoclaved stems of Anthriscus sylvestris placed onto the agar surface. Cultures were incubated at 20 °C under near UV light with a 12 h photoperiod for 10 d. Colony characters and pigment production on PDA and SNA were noted after 10 d. Colony colours were rated according to Rayner (1970). Growth rates were measured after 7 and 10 d.

Conidia were taken from acervuli and mounted in lactic acid. Cultures were examined periodically for the development of perithecia. Ascospores were described from perithecia crushed in lactic acid. Appressoria on hyphae were observed on the reverse side of colonies grown on SNA plates. At least 30 measurements per structure were noted and observed with a Nikon Eclipse 80i microscope using differential interference contrast (DIC) illumination. Range measurements were made according to methods described by Liu et al. (2012).

Phylogenetic analysis

Genomic DNA of the isolates was extracted using the method of Damm et al. (2008). Eight loci including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), chitin synthase 1 (CHS-1), beta-tubulin (TUB2), calmodulin (CAL), histon3 (HIS3) and glutamine synthetase (GS) gene were amplified and sequenced using the primer pairs ITS1F (Gardes and Bruns 1993) + ITS4 (White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), ACT-512F + ACT-783R (Carbone and Kohn 1999), CHS-79F + CHS-354R (Carbone and Kohn 1999), T1 (O’Donnell and Cigelnik 1997) + Bt-2b (Glass and Donaldson 1995), CL1 + CL2A (O’Donnell et al. 2000) or CL1C + CL2C (Weir et al. 2012), CYLH3F + CYLH3R (Crous et al. 2004c) and GSF1 + GSR1 (Stephenson et al. 1997), respectively. The PCR protocols were performed as described by Damm et al. (2009). Some isolates occasionally gave two bands (GS and TUB2), which were then amplified using a touchdown PCR program (Zhou et al. 2006). The DNA sequences generated with forward and reverse primers were used to obtain consensus sequences using MEGA5 (Tamura et al. 2011), and subsequently aligned using MAFFT v.6 (Katoh and Toh 2010), and the alignments edited manually using MEGA5.

A maximum parsimony analysis was performed on the multi-locus alignment (ACT, CAL, CHS-1, GAPDH, GS, ITS, TUB2) using PAUP v.4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded from all analyses. Unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability was assessed in a bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa.

A second phylogenetic analysis using a Markov Chain Monte Carlo (MCMC) algorithm was conducted to generate trees with Bayesian posterior probabilities in MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003). Nucleotide substitution models were determined using MrModeltest v.2.3 (Nylander 2004) for each gene region and included in the analyses. Two analyses of four MCMC chains were run from random trees for 10 million generations and sampled every 1000 generations. The first 25 % of trees were discarded as the burn-in phase of each analysis and posterior probabilities determined from the remaining trees.

Sequences derived in this study were deposited in GenBank (Table 1), the concatenated alignment in TreeBASE (www.treebase.org) (S13708), and taxonomic novelties in MycoBank (Crous et al. 2004b).

Results

Phylogeny

The phylogenetic analysis included 86 strains with Colletotrichum boninense (MAFF 305972) as outgroup. The dataset of seven genes (ACT, CAL, CHS-1, GAPDH, GS, ITS, TUB2) comprised 3745 characters including the alignment gaps, of which 967 characters were parsimony-informative, 505 parsimony-uninformative and 2251 constant. Parsimony analysis resulted in 121 equally parsimonious trees, and one of them (Length = 2394, CI = 0.765, RI = 0.924, RC = 0.706) is shown in Fig. 1. The Bayesian tree confirmed the tree topology of the trees obtained with maximum parsimony.

Fig. 1
figure 1

One of 121 equally parsimonious trees obtained from a heuristic search of combined ACT, CAL, CHS-1, GAPDH, GS, ITS and TUB2 gene sequences of 85 isolates from the Colletotrichum gloeosporioides species complex and one outgroup C. boninense. Bootstrap support values (1000 replicates) above 50 % and Bayesian posterior probability values above 0.95 are shown at the nodes. Ex-type cultures are emphasised in bold, and include the taxonomic name as originally described. Strain number is followed by host and country of origin. Isolates associated with Proteaceae are marked with a red square

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The isolates from Proteaceae studied here (indicated with red squares) belong to six clades (Fig. 1). Nine strains clustered with C. alienum, six strains with C. aotearoa and four strains with C. kahawae subsp. ciggaro. Three isolates from Protea sp. identified as C. proteae form a clade on a long branch (100/1.00), which was basal to the top part of the phylogeny formed by 12 closely related species that correspond to the clade addressed as the Musae clade by Weir et al. (2012). A single strain lineage, representing C. grevilleae, formed a sister lineage to C. theobromicola; the two species form a clade that is basal to all other species in the C. gloeosporioides complex. Two strains from Proteaceae clustered with the ex-type strains of C. siamense (ICMP 18578), C. jasmine-sambac (CBS 130420), C. hymenocallidis (CBS 125378) and strains from Murraya in China (Fig. 1). The ex-type strain of C. populi (HMBFU 191) and other authentic cultures of C. populi grouped with the ex-type strain of C. aenigma (ICMP 18608) and also formed a well-supported monophyletic lineage.

Taxonomy

Based on results of the multigene phylogeny, the 25 Colletotrichum strains from Proteaceae hosts studied belong to six species within the C. gloeosporioides complex, including two species that proved to be new to science. In addition, two synonymies of recently described species were recognised. All species associated with Proteaceae are characterised and illustrated below, except for C. siamense.

Colletotrichum aenigma B. Weir & P.R. Johnst., Stud. Mycol. 73: 135 (2012)

= Colletotrichum populi C.M. Tian & Z. Li, Mycotaxon 120: 283 (2012)

Descriptions of this species are provided by Li et al. (2012) and Weir et al. (2012).

Notes: Colletotrichum aenigma and C. populi were both described recently (Li et al. 2012; Weir et al. 2012). Their ex-holotype cultures however belong to the same terminal clade (Fig. 1). There is only one base pair difference in the ITS and one difference in the GS sequences between C. aenigma and C. populi. Furthermore, the GAPDH, ACT and TUB sequences are identical; CAL and CHS-1 sequences were not generated by Li et al. (2012) and therefore not included in this study. Since C. aenigma was published online on 21 Aug. 2012 prior to C. populi (28 Sep. 2012), C. populi is regarded as a synonym of C. aenigma.

Colletotrichum alienum B. Weir & P.R. Johnst., Stud. Mycol. 73: 139 (2012) Fig. 2

Fig. 2
figure 2

Colletotrichum alienum (from strain CBS 115183). a–b, d. Ascomata; c, g. Conidiophores; e. Paraphyses; f. Conidia; h–j. Asci; k, q. Ascospores; l–o. Appressoria-like structures; p. Outer surface of peridium. a, c–h, k, p. from PDA; b, i–j, q. from Anthriscus stem; l–o. from SNA. a–b. DM; c–q. DIC.—Scale bars: a = 100 μm; f = 10 μm; a applies to a–b; f applies to c–q

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On PDA: Vegetative hyphae hyaline to medium brown, usually smooth-walled, sometimes verrucose, septate, branched. Chlamydospores not observed. Conidiomata not developed, conidiophores formed directly on hyphae of the aerial mycelium. Setae not observed. Conidiophores rarely observed, hyaline to pale brown, simple or septate, sometimes branched. Conidiogenous cells hyaline to pale brown, cylindrical, up to 62.5 μm long, apex 1–1.5 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, 13.5–16.5 × 4–5.5 μm, mean ± SD = 14.7 ± 0.8 × 4.7 ± 0.4 μm, L/W ratio = 3.1.

Sexual morph developed on PDA. Ascomata globose, sometimes obpyriform, brown to black, 150–500 μm diam, usually covered by aerial mycelium, ostiolate, neck brown, outer wall composed of flattened angular cells, 5–13 μm diam. Interascal tissue composed of paraphyses, thin-walled, hyaline, septate, with rounded apex. Asci cylindrical, 50–89.5 × 8–10.5 μm, 8-spored. Ascospores uni- or biseriately arranged, hyaline, aseptate, smooth-walled, allantoid with rounded ends, 9.5–21.5 × 3–5.5 μm, mean ± SD = 16.6 ± 3.0 × 4.2 ± 0.6 μm, L/W ratio = 4.0.

On SNA: Asexual and sexual morph not observed. Appressoria not observed on the undersurface of the medium, but appressoria-like structures that possibly function as chlamydospores were observed within the medium. These are single, aseptate, smooth-walled, brown, globose, obovoid or irregular, 11–21.5 × 5–9 μm.

On Anthriscus stem: Asexual morph not observed. Ascomata globose, brown to black, covered by aerial mycelium, outer wall composed of flattened angular cells, 6–13 μm diam. Interascal tissue composed of paraphyses, thin-walled, hyaline, septate, 2.5–4.5 μm diam, the apex rounded. Asci cylindrical, 50–84.5 × 5–12.5 μm, 8-spored. Ascospores uni- or biseriately arranged, hyaline, aseptate, smooth-walled, allantoid or fusiform with rounded to slightly acute ends, 15–22.5 × 4–5.5 μm, mean ± SD = 19 ± 2 × 4.7 ± 0.4 μm, L/W ratio = 4.0.

Culture characteristics: Colonies on PDA low convex with entire margin, entirely covered with dense, whitish aerial mycelium, surface olivaceous grey with white margin; reverse iron grey to greenish black with white margin; colony diam 76–78 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, filter paper and Anthriscus stem covered with sparse whitish mycelium; colony diam 70–72 mm in 7 d, > 90 mm in 10 d.

Materials examined: AUSTRALIA, New South Wales, on Grevillea sp., 1999, P.W. Crous, culture CBS 111982 = CPC 2925; on Grevillea sp., 1999, P.W. Crous, culture CBS 132880 = CPC 2926. PORTUGAL, on Leucadendron sp., cv. ‘High Gold’, Apr. 2000, S. Denman, culture CBS 115183 = STE-U 5226 (strain described); on Leucadendron sp., cv. ‘High Gold’, Apr. 2001, J.E. Taylor, culture CBS 112991 = STE-U 4450 = JT 1168.1; Madeira Island, Florialis Estate, on Protea cynaroides, 2 Jan. 2002, S. Denman, culture CBS 133930 = CPC 5204. SOUTH AFRICA, Western Cape Province, on Serruria sp., 4 Jan. 2009, K. Bezuidenhout, culture CBS 132883 = CPC 16168; Caledon, on P. cynaroides, 1 Jun. 2001, S. Denman, culture CBS 113192 = STE-U 4455 = B 5712.2; Caledon, on P. cynaroides, Jun. 2001, S. Denman, culture CBS 113001 = STE-U 4454 = B 5678.1; Betty’s Bay, on leaf litter of Leucadendron sp., 26 Jun. 2000, S. Marincowitz, culture CBS 122687 = CPC 13164 = PREM 59587 = CMW 22211 = SL 587.

Notes: Strains CBS 113001, CBS 112991 and CBS 113192 were previously identified as C. gloeoporioides based on ITS and Beta-tubulin sequences (Lubbe et al. 2004). Strain CBS 122687 was also identified as C. gloeosporioides based on ITS and LSU sequences (Marincowitz et al. 2008a). However, the multi-locus phylogenetic analysis in this study showed that these strains clustered together with the ex-type culture of C. alienum (ICMP 12071) (Fig. 1). This species is common on members of Proteaceae in Australia, South Africa and Europe.

Colletotrichum aotearoa B. Weir & P.R. Johnst., Stud. Mycol. 73: 139 (2012) Fig. 3

Fig. 3
figure 3

Colletotrichum aotearoa (from strain CBS 114140). a–b. Acervuli; c, d, m. Setae; e–f. Conidiophores; k, l. Conidia; g–j. Appressoria. a, c–e, k. from PDA; b, f–j, l–m. from SNA. a–b. DM; c–m. DIC.—Scale bars: b = 100 μm; f = 10 μm; b applies to a–b; f applies to c–m

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On PDA: Vegetative hyphae hyaline to pale brown, smooth-walled, septate, branched. Chlamydospores not observed. Conidiomata acervular, conidiophores and setae either directly formed from hyphae or on a cushion of roundish hyaline cells. Setae pale to dark brown, smooth-walled to verruculose, 1–3-septate, 40–110 μm long, base inflated or cylindrical, 3.5–6 μm diam, tip more or less acute. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical to ampulliform, 8.5–16 × 3–5.5 μm, apex 1.5–2.5 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, contents sometimes with guttulae, 11–21.5(−28) × 4–6 μm, mean ± SD = 15.5 ± 3.8 × 5.0 ± 0.5 μm, L/W ratio = 3.1.

On SNA: Chlamydospores not observed. Conidiomata acervular. Setae pale to dark brown, smooth-walled to verruculose, 1–3-septate, 59–66 μm long, base cylindrical or inflated, 3.5–5.5 μm diam, tip ± acute. Conidiophores hyaline to pale brown, septate, branched. Conidiogenous cells hyaline to pale brown, cylindrical or ampulliform, 9–21 μm long, apex 1.5–2.5 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, contents with small guttulae, 12.5–16 × 4–5 μm, mean ± SD = 14.0 ± 0.8 × 4.5 ± 0.3 μm, L/W ratio = 3.1. Appressoria not observed in strain CBS 114140, appressoria of strain CBS 111971 medium to dark brown, solitary, aseptate, circular, ellipsoidal or irregular in outline, crenate or slightly lobed at edge, 6.5–12 × 6–7.5 μm, mean ± SD = 9.2 ± 1.4 × 6.3 ± 0.6 μm, L/W ratio = 1.5.

Culture characteristics: Colonies on PDA low convex with entire margin, olivaceous grey to greenish black, conidial masses salmon; reverse greenish grey to greenish black; colony diam 76–80 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, umber, white to buff pigment, Anthriscus stem and medium covered with salmon conidial masses; colony diam 65–67 mm in 7 d, > 90 mm after 10 d.

Materials examined: AUSTRALIA, Victoria, Victoria Valley road, Dunkeld, on Banksia marginata, 17 Oct. 1999, I. Pascoe, culture CBS 132448 = CPC 17784 = VPRI 41610A. NEW ZEALAND, Buried Village, on Knightia sp., 1999, P.W. Crous, culture CBS 114140 = STE-U 2900 (strain described); Buried Village, on Knightia sp., 1999, P.W. Crous, culture CBS 111971 = STE-U 2898; Buried Village, on Knightia sp., 1999, P.W. Crous, culture CBS 111965 = STE-U 2897; Buried Village, on Knightia sp., 1999, P.W. Crous, culture CBS 114139 = STE-U 2902; Buried Village, on Knightia sp., 1999, P.W. Crous, culture CBS 111962 = STE-U 2901.

Notes: Colletotrichum aotearoa was recently described and reported on a wide host range in New Zealand, and was assumed to be a native species (Weir et al. 2012). However, results of this study report this species in Australia as well and extend its host range to Banksia and Knightia. Based on ITS sequence comparison, Weir et al. (2012) suggested the possible occurrence of this species on Boehmeria in China (GenBank records GQ120479 and GQ120480, Wang et al. 2010), which would need to be confirmed with DNA sequence data from additional gene loci.

Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, sp. nov. Fig. 4

Fig. 4
figure 4

Colletotrichum grevilleae (from ex-holotype strain CBS 132879). a. Acervulus; b–c. Conidia; d–f. Conidiophores. a, c, f. from SNA; b, d–e. from PDA. a. DM; b–f. DIC.—Scale bars: a = 100 μm; b = 10 μm; b applies to b–f

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MycoBank MB 802496

Etymology: Referring to the host genus, Grevillea.

On PDA: Vegetative hyphae hyaline to medium brown, usually smooth-walled, sometimes verrucose, septate, branched. Chlamydospores not observed. Conidiomata not observed, conidiophores formed directly on hyphae of the aerial mycelium. Setae not observed. Conidiophores hyaline to pale brown, simple or septate, sometimes branched. Conidiogenous cells hyaline to pale brown, cylindrical to ampulliform, straight to flexuous, 11–53 × 1.5–4 μm, apex 1–1.5 μm diam, collarette rarely observed, 0.5–1.5 μm long. Conidia hyaline, usually aseptate, sometimes becoming 1–3-septate with age, smooth-walled, cylindrical to clavate, both ends rounded, or one end rounded and one end ± acute, 7–22.5(−37) × 3–6 μm, mean ± SD = 15.4 ± 6.6 × 3.9 ± 0.8 μm, L/W ratio = 3.9.

On SNA: Chlamydospores not observed. Conidiomata acervular. Setae not observed. Conidiophores hyaline to pale brown, septate, branched. Conidiogenous cells hyaline to pale brown, straight or flexuous, cylindrical to ampulliform, 9.5–27.5 × 2.5–4.5 μm, opening 1.5–2.5 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, 12.5–17 × 3.5–5.5 μm, mean ± SD = 14.5 ± 1.1 × 4.4 ± 0.3 μm, L/W ratio = 3.3. Appressoria not observed.

Culture characteristics: Colonies on PDA low convex with entire margin, surface olivaceous black to dark slate blue with white margin, reverse dark slate blue; colony diam 82–84 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, short sparse white aerial mycelium and buff pigment around Anthriscus stem, conidial mass salmon; colony diam 70–74 mm in 7 d, colonial diam > 90 mm in 10 d.

Material examined: ITALY, Catania, from root and collar rot of Grevillea sp., Jan. 2000, G. Polizzi (CBS H-21120 holotype, culture ex-type CBS 132879 = CPC 15481 = DISTEF.GREV.Z).

Notes: Sequence data derived from the ITS region does not separate C. grevilleae from C. theobromicola, but they can be distinguished based on CAL or GAPDH. Although C. grevilleae is only represented by a single isolate, it shows sufficient phylogenetic distance to C. theobromicola (Fig. 1). Apart from C. grevilleae and C. alienum that are treated in this study, there are three Colletotrichum species that were previously reported from Grevillea. Colletotrichum acutatum and C. fioriniae that were found on Grevillea from Australia and Germany, respectively, are both with a broad host spectrum and belong to the C. acutatum species complex (Damm et al. 2012a). Another Colletotrichum species, C. palhinhae, was described by González Fragoso (1924) as parasite of Lamproderma echinulatum (Stemonitida, Amoebozoa) growing on branches and leaves of Grevillea robusta in Portugal. However, the conidia of C. palhinhae (9–12 × 1.5–2 μm) are smaller than that of C. grevilleae, fusoid and green, and the width of conidia ≤ 2 μm, which is unlikely to be a Colletotrichum species in our current understanding.

Colletotrichum kahawae subsp. ciggaro B. Weir & P.R. Johnst., Stud. Mycol. 73: 158 (2012) Fig. 5

Fig. 5
figure 5

Colletotrichum kahawae subsp. ciggaro (from strain CBS 114499). a–b. Acervuli; c–h. Appressoria; i–j, l–m. Conidiophores; k, n. Conidia. a, i, k–l. from PDA; b–h, j, m–n. from SNA. a–b. DM; c–n. DIC.—Scale bars: b = 100 μm; n = 10 μm; b applies to a–b; n applies to c–n

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On PDA: Vegetative hyphae hyaline to pale brown, smooth-walled, septate, branched. Chlamydospores not observed. Conidiomata acervular, conidiophores formed from a cushion of roundish hyaline cells. Setae absent. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical, ampulliform or elongate ampulliform, 10.5–13 × 2–4 μm, apex 1.5–2 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, 10–14 × 4–5.5 μm, mean ± SD = 12.2 ± 0.9 × 4.7 ± 0.3 μm, L/W ratio = 2.6.

On SNA: Chlamydospores not observed. Conidiomata acervular. Setae not observed. Conidiophores hyaline to pale brown, simple or septate, branched or unbranched. Conidiogenous cells hyaline to pale brown, cylindrical or ampulliform, up to 40 μm long, apex 1.5–2.0 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, 9.5–13.5(−22) × 3.5–5.5 μm, mean ± SD = 12.1 ± 2.0 × 4.2 ± 0.4 μm, L/W ratio = 2.9. Appressoria medium to dark brown, aseptate, solitary or in groups, with a circular, ovoid, ellipsoidal to irregular outline, and crenate or lobed margin, 6–12.5 × 4.5–8.5 μm, mean ± SD = 9.0 ± 1.6 × 6.6 ± 0.8 μm, L/W ratio = 1.4.

Culture characteristics: Colonies on PDA low convex with entire margin, surface greenish grey with a whitish margin, conidial masses salmon; reverse greenish grey with a whitish margin; colony diam 76–80 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, filter paper and Anthriscus stem covered with whitish to pale mouse grey aerial mycelium, conidial mass on SNA medium pale mouse grey to mouse grey, buff pigment around Anthriscus stem; colony diam 72–74 mm in 7 d, > 90 mm in 10 d.

Materials examined: PORTUGAL, Madeira Island, on Banksia sp., 1 Apr. 2001, J.E. Taylor, culture CBS 112984 = STE-U 4445 = ICMP 17932. SPAIN, Santa de Serra, on Banksia sp., 1 Apr. 2002, S. Denman, culture CBS 115194 = STE-U 5196. USA, Hawaii, on Leucospermum sp. cv. ‘Safari Sunset’, 26 Jan. 1999, P.W. Crous, culture CBS 114499 = STE-U 2192; Hawaii, on Leucospermum sp. cv. ‘Safari Sunset’, 26 Jan. 1999, P.W. Crous, culture CBS 111861 = STE-U 2191.

Notes: Colletotrichum kahawae sensu Waller et al. (1993) was divided into two subspecies, C. kahawae subsp. kahawae and C. kahawae subsp. ciggaro (Weir et al. 2012). Colletotrichum kahawae subsp. kahawae was distinguished from C. kahawae subsp. ciggaro on the basis of its host range and GS gene sequence to stress the biosecurity importance of the coffee berry pathogen (Weir et al. 2012). Isolates of C. kahawae occurring on Proteaceae were identified as C. kahawae subsp. ciggaro according to Weir et al. (2012) by the presence of a 22 bp insertion in their GS sequences that is missing in those of C. kahawae subsp. kahawae.

Strain CBS 112984 included in this study was regarded as C. crassipes by Lubbe et al. (2004), probably due to the name tag applied to it and a strain from Dryas in Switserland (CBS 112988 = IMI 359911) in the CBS and IMI culture collections. Colletotrichum crassipes was originally described as Gloeosporium crassipes (Spegazzini 1878) from fruits of Vitis vinifera in Italy and forms conidia that are larger than those of C. kahawae, measuring 20–30 × 7–8 μm; the two species are therefore unlikely to be conspecific. However, the taxonomic status of C. crassipes remains uncertain. Isolates from grapes from the original location of Gloeosporium crassipes in Italy are required to serve as epitype to stabilize the application of the name, and to resolve its relationship with other taxa in the genus.

Colletotrichum proteae F. Liu, Damm, L. Cai & Crous, sp. nov. Fig. 6

Fig. 6
figure 6

Colletotrichum proteae (from ex-holotype strain CBS 132882). a, c. Acervuli; b, d. Conidia; e. Seta; f–g. Conidiophores. a–b, e–f. from PDA; c–d, g. from SNA. a, c. DM; b, d–g. DIC.—Scale bars: a = 100 μm; b = 10 μm; a applies to a, c; b applies to b, d–g

Full size image

MycoBank MB 802498

Etymology: Referring to the host genus, Protea.

On PDA: Vegetative hyphae hyaline to pale brown, smooth-walled, septate, branched. Chlamydospores not observed. Conidiomata acervular, conidiophores formed from a cushion of roundish, hyaline or pale brown cells. Setae rare, only one observed, medium brown, smooth-walled, 1- septate, basal cell pale brown, cylindrical, tip round. Conidiophores hyaline to pale brown, septate, branched. Conidiogenous cells hyaline to pale brown, cylindrical, ampulliform to elongate ampulliform, 12.5–19 × 2–3 μm, apex 1–2 μm diam. Conidia hyaline to pale brown, aseptate, smooth-walled, fusiform to ellipsoidal, 15.5–19 × 4–5 μm, mean ± SD = 17.0 ± 0.9 × 4.6 ± 0.3 μm, L/W ratio = 3.7.

On SNA: Chlamydospores not observed. Conidiomata acervular. Setae not observed. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical, ampulliform to elongate ampulliform, 12.5–20 × 2.5–4.5 μm, apex 1.5–2.5 μm diam. Conidia hyaline, aseptate, smooth-walled, fusiform to ellipsoidal, 14.5–18.5 × 3.5–5.5 μm, mean ± SD = 16.9 ± 1.1 × 4.5 ± 0.4 μm, L/W ratio = 3.8. Appressoria not observed.

Culture characteristics: Colonies on PDA raised with entire margin, white, sparse aerial mycelium, conidial mass apricot, sepia or brown-vinaceous; reverse white; colony diam 70–74 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, aerial mycelium lacking, medium buff to honey pigment, conidial mass salmon, apricot, or iron-grey, colony diam 61–65 mm in 7 d, > 90 mm in 10 d.

Materials examined: SOUTH AFRICA, Western Cape Province, Tsitsikamma National Park, Nature’s Valley, on Protea sp., 9 Jan. 2008, P.W. Crous (CBS H-21119 holotype, culture ex-type CBS 132882 = CPC 14859); Western Cape, Tsitsikamma National Park, Nature’s Valley, on Protea sp., 9 Jan. 2008, P.W. Crous, culture CBS 134301 = CPC 14860; Western Cape, Tsitsikamma National Park, Nature’s Valley, on Protea sp., 9 Jan. 2008, P.W. Crous, culture CBS 134302 = CPC 14861.

Notes: Although the fusiform to ellipsoidal conidia of C. proteae are reminiscent of species belonging to the C. acutatum complex (Damm et al. 2012a), DNA sequence data demonstrate that this species belongs to the C. gloeosporioides species complex. To our knowledge, no species in C. gloeosporioides species complex was described on Protea before, except two further Colletotrichum species treated in this study, which are C. alienum and C. siamense from Protea in South Africa and Zimbabwe, respectively. Another species was identified as C. boninense by Lubbe et al. (2004); strains from Protea cynaroides in Zimbabwe and Protea obtusifolia in Portugal (Madeira Island) proved to be C. karstii, belonging to the C. boninense species complex (Damm et al. 2012b).

Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde, Fungal Divers 39: 98 (2009)

A descriptions of this species was provided by Prihastuti et al. (2009).

Materials examined: THAILAND, Chiang Mai, Mae Lod Village, on Coffea arabica berries, 12 Dec. 2007, H. Prihastuti, ex-holotype culture CBS 130417 = ICMP 18578 = CGMCC 3.14174 = MFLU 090230 = BPD-I2. ZIMBABWE, on P. cynaroides, Mar. 1999, S. Denman, culture CBS 112983 = STE-U 2291 = JT814; on P. cynaroides, Mar. 1999, S. Denman, culture CBS 113199 = STE-U 2290 = JT813.

Notes: Colletotrichum jasmini-sambac and C. hymenocallidis were synonymised with C. siamense based on a multi-locus (ACT, CAL, CHS-1, GAPDH and ITS) phylogenetic analysis by Weir et al. (2012), while a recent study based on sequence data of ITS, TUB2, DNA lyase (APN2) and an intergenic spacer between the 3’ end of the DNA lyase and the mating type locus MAT1-2 (apn2mat/IGS) recognised a further species closely related to C. siamense, C. melanocaulon, and two unnamed clades (Doyle et al. 2013). A study by Sharma et al. (2013, this issue) based on ApMat sequences of a large set of strains recognised several clades within C. siamense suggesting C. siamense to be a species complex. In our study, C. siamense shows high sequence variability as well. However, more strains need to be included to support a further splitting of C. siamense, possible resurrecting C. jasmini-sambac and C. hymenocallidis and possible recognising the strains from Protea as a distinct species.

The name Colletotrichum murrayae Gutner was originally applied to isolates associated with Murraya exotica (synonym of M. paniculata) in Russia (Bondartseva-Monteverde et al. 1936). Colletotrichum murrayae Gutner was placed in synonymy with C. gloeosporioides by von Arx (1957). Although the name C. murrayae Gutner has had not been used since its description, it is still a legitimate name. Recently, Peng et al. (2012) described a new species associated with leaf spots of Murraya sp. in China as C. murrayae L.J. Peng & K.D. Hyde, using the same epithet as the species published by Bondartseva-Monteverde et al. in 1936. Therefore, C. murrayae L.J. Peng & K.D. Hyde is an illegitimate name. Our multi-locus phylogeny (Fig. 1) revealed that the ex-holotype strain of C. murrayae L.J. Peng & K.D. Hyde clusters with C. siamense, however a further study is needed to verify the taxonomic status of the strains from Murraya sp. in China. Another species, C. exoticum was described on Murraya exotica in India by Pavgi and Singh (1964). Epitypification of C. murrayae Gutner and C. exoticum is needed and their relationships within the C. gloeosporioides species complex remain to be clarified.

Discussion

The name Colletotrichum gloeosporioides was originally applied to Colletotrichum isolates associated with diseases of Citrus from Italy (Penzig 1882). Since then, many morphologically similar species were described on the basis of host association (Hyde et al. 2009). Based on morphological characteristics, von Arx (1957) synonymised around 600 names under C. gloeosporioides. Colletotrichum gloeosporioides has long been regarded as a species complex comprising many morphologically similar species. It was only after the epitypification of C. gloeosporioides (Cannon et al. 2008) however, that phylogenetically distinct species were defined within this complex. An important contribution to the taxonomy of C. gloeosporioides species complex was made by Weir et al. (2012), who applied multi-locus phylogenetic analyses to a large number of isolates revealing this complex to consist of at least 23 taxa. Some of the synonyms of C. gloeosporioides treated by von Arx (1957) have been found to represent distinct species in other species complexes, e.g. C. dracaenae and C. godetiae (Damm et al. 2012a, b). Thus far, C. gloeosporioides sensu lato isolates from only approximately 100 host plants have been restudied using multi-locus phylogenetic analyses, the majority of the host plants with anthracnose disease symptoms in von Arx’s treatment (von Arx 1957) remain to be recollected and restudied.

Proteaceae cut-flowers are extensively cultivated in South Africa, Zimbabwe, Israel, Australia and New Zealand. However, comprehensive phytosanitary regulations induced by the World Trade Organisation (WTO) restrict the trade of Proteaceae cut-flowers (World Trade Organisation 1994; Crous et al. 2000; Taylor et al. 2001a, b). Thus rapid and accurate identification of plant pathogenic fungi is essential to ensure appropriate phytosanitary measures and suitable control strategies (Crous and Groenewald 2005). A large number of fungal pathogens are known to occur on Proteaceae (Crous et al. 2004a). The taxonomy of some of these species has changed considerably since they were first reported, of which many have been restudied (Crous et al. 2011). While strains from Proteaceae previously identified as C. acutatum and C. boninense were included in the revisions of the respective species complexes (Damm et al. 2012a, b), the present study is the first to revisit the taxonomy of the C. gloeosporioides species complex on Proteaceae since the first phylogenetic study on this topic published by Lubbe et al. (2004).

Based on the multi-locus phylogenetic analysis conducted in this study, isolates belonging to the C. gloeosporioides complex associated with Proteaceae are revealed to belong to six species. Some of the strains included here were previously regarded as C. gloeosporioides or C. crassipes based on ITS, TUB2 or LSU sequence data (Lubbe et al. 2004; Marincowitz et al. 2008a), but are now shown to belong to C. alienum, C. kahawae subsp. ciggaro or C. siamense based on our analysis and recent treatments published on this species complex (Weir et al. 2012).

Together with the seven species in the C. boninense and C. acutatum species complexes (Damm et al. 2012a, b), there are now 13 Colletotrichum species known to be associated with Proteaceae hosts. Most of these taxa have a diverse host range. The species so far only known from Proteaceae are C. grevilleae and C. proteae.

This study also revealed a wider host distribution of several species. For example, although C. aotearoa was originally reported as having a restricted distribution in New Zealand (Weir et al. 2012), it was found here to also occur in Australia. While C. alienum was originally described from New Zealand and Australia, it is shown here to also occur in Africa and Europe.

Various species of Banksia, Grevillea, Leucadendron, Leucospermum and Protea are commercially cultivated for the cut-flower industry (Crous et al. 2000; Marincowitz et al. 2008b). These plants host relatively high species diversities within the C. gloeosporioides complex. Protea can be infected by several Colletotrichum species, e.g. C. alienum, C. siamense or C. proteae, which are reported from Portugal, South Africa, Spain or Zimbabwe, respectively (Table 1). More strains need to be included to evaluate if the strains from Protea identified as C. siamense represent a distinct species. Further studies are also needed to determine the identity of the C. gloeosporioides isolates which were originally regarded as pathogens of Protea in Australia, California, Hawaii and the Madeira Islands (Crous et al. 2004a) and are not included in this study.

Presently C. aotearoa has been recorded from Banksia and Knightia (Australia and New Zealand); C. kahawae subsp. ciggaro was collected on Banksia and Leucospermum (Portugal, Spain and USA/Hawaii), C. proteae on Protea (South Africa), and C. grevilleae on Grevillea (Italy). As these records are based on random, chance collections, it calls for more detailed surveys to determine their host range and distribution, and relative importance.

Among species in the C. gloeosporioides complex, C. alienum seems to be the economically most important species in Proteaceae cultivation because of its wide host range and pathogenicity. Colletotrichum alienum is associated with most of the popular Proteaceae cut flowers in South Africa and Europe, such as Grevillea, Leucadendron, Leucospermum, Protea, and Serruria. Strain CBS 113001 (identified as C. gloeosporioides in Lubbe et al. 2004, here identified as C. alienum) was shown to be highly virulent to leaves of Protea cultivars (Lubbe et al. 2006), thus further collections and pathogenicity tests are necessary to characterise its distribution and importance as a pathogen of other genera in Proteaceae.

According to current data, Colletotrichum species associated with Proteaceae have seldom been isolated from other symptomatic or asymptomatic host plants in South Africa, except Carica papaya and Persea americana (Weir et al. 2012). Although this could be due to the generally limited number of samples investigated, it could also be due to the limited host range of the species studied. Further studies are thus required to resolve the host range, distribution and pathogenicity of the Colletotrichum species reported on Proteaceae.