Introduction

The Orchidaceae is one of the largest plant families with nearly 25,000 species (Jones 2006). Orchids are fascinating ornamental plants and important research materials for coevolution between plants and fungi because of their special symbiosis with mycorrhizal fungi (Zettler et al. 2004; Stark et al. 2009; Nontachaiyapoom et al. 2010). Recently, the fungal communities in leaves and roots of orchid Bletilla ochracea have been investigated and the results indicated that there is a high diversity of endophytic fungi, including species from the genus Colletotrichum Corda (Tao et al. 2008, 2012).

Endophytic fungi live asymptomatically and internally within different tissues (e.g. leaves, roots) of host plants (Ganley and Newcombe 2006; Promputtha et al. 2007; Hoffman and Arnold 2008). Some endophytes have been demonstrated to be able to enhance the competitive abilities and resistance to herbivores, pathogens, and various abiotic stresses for their hosts (Saikkonen et al. 1998; Newton et al. 2010; Saikkonen et al. 2010). Colletotrichum species are among the most commonly occurring pathogens and foliar endophytes of terrestrial plants and have been recorded from approximately 2,200 plant species (Farr and Rossman 2013). Colletotrichum pathogens are the principal cause of anthracnose, as well as causal agents of pre- and post-harvest fruit rots, damping-off of blossom and seedling blight diseases (Bailey and Jeger 1992). This genus was recently voted the eighth most important group of plant pathogenic fungi in the world, based on perceived scientific and economic importance (Dean et al. 2012). However, it has also been shown that particular Colletotrichum endophytes confer protective benefits to Cacao hosts by reducing disease incidence and damage caused by other plant pathogens (Arnold et al. 2003; Herre et al. 2007). Our understanding on endophytic Colletotrichum species remains very limited.

The nuclear ribosomal internal transcribed spacer (ITS) has been chosen as the universal barcode for the Kingdom of Fungi (Schoch et al. 2012), but it is also widely acknowledged that ITS does not provide sufficient resolution to differentiate species in Colletotrichum (Cai et al. 2009; Crouch et al. 2009a; Prihastuti et al. 2009; Yang et al. 2009; Phoulivong et al. 2010a). ITS sequences have been applied to resolve species of the ‘gloeosporioides’ complex (Sreenivasaprasad et al. 1993; Sreenivasprasad et al. 1996), but the resolution is not satisfactory (Crouch et al. 2009a). Phylogenetic species recognition criterion (Taylor et al. 2000) have been increasingly used in Colletotrichum to recognize and differentiate species. Up to now, significant progress has been achieved in the Colletotrichum acutatum species complex (Damm et al. 2012a), Colletotrichum boninense species complex (Damm et al. 2012b), and the Colletotrichum gloeosporioides species complex (Prihastuti et al. 2009; Phoulivong et al. 2010a; Weir et al. 2012), and some cryptic species in Colletotrichum have been disclosed using multilocus phylogenetics (Phoulivong et al. 2010b; Rojas et al. 2010; Damm et al. 2012a; Damm et al. 2012b; Weir et al. 2012).

The objective of this study was to investigate the endophytic Colletotrichum species associated with Bletilla ochracea (Orchidaceae) in differently geographic sites in Guizhou province, China.

Materials and methods

Sampling sites and treatments

Fifty intact plants of Bletilla ochracea were collected from 5 sites in Guizhou province, China during June–August of 2006 (Table 1). Ten healthy and intact plants with native soil in each site were packed and carefully transported to laboratory within 48 h. Three symptomless leaves of each plant were treated with gently running tap water to remove the surface debris and soil. They were surface-sterilized using 75 % ethanol for 1 min, 0.1 % HgCl2 for 3 min, and washed for five times using sterile distilled water, finally dried on sterile filter paper (Newell 1976). The 5-mm-diam leaf discs treated as above were placed on potato dextrose agar (PDA) plates without antibiotics, which were used as control of microorganisms test.

Table 1 Sampling sites in Guizhou Province, China
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Isolation and spore induction of endophytic Colletotrichum

Six leaf discs treated as above were placed on PDA, malt extract agar (MEA) (Stone et al. 2004) and modified Czapek Dox agar medium (Yamato et al. 2005). Ten repeats were made for each medium type. Streptomycin sulphate and chloramphenicol were added into the media to a final concentration of 100 mg/L and 50 mg/L to inhibit bacterial contamination. Plates were kept in the dark at room temperature (25 °C). When colonies appeared, they were transferred onto new PDA plates and incubated at 25 °C in 12 h light and 12 h dark for morphological examination. All endophytic isolates are deposited in China General Microbiological Culture Collection Center (CGMCC). The specimens of type strains were deposited in the Herbarium of Microbiology, Academia Sinica (HMAS).

Sporulation was induced on pine needle medium (“pine needle” and 1/10-strength PDA, hereafter abbreviated as “PNP”) with exposure to 12 h near-UV light/12 h dark at 25 °C for 7 days, or up to 2 months (Su et al. 2012). Mycelial appressoria were produced and measured using a slide culture technique (Riddell 1950). Five-mm-diam plugs from the margin of actively growing cultures were placed onto PDA plates (Petri dishes diameter: 90 mm) for assessment of growth rates. Colonial diameters were measured at the seventh day (at sixth day for the fast growing cultures). Daily growth rate was calculated (mm/day). Colonial characters were described after 7 days growth on PDA (10 days for slow growing cultures).

DNA extraction, PCR amplification and sequencing

Fungal isolates were incubated on PDA at 25 °C for 7–10 days for DNA extraction. Total genomic DNA of the isolates was extracted using a modified protocol as outlined by Yang and Liu (2005). The ITS1 and ITS4 primers were used to amplify the ITS region following the procedure described by White et al. (1990). The primers of T1 and Bt-2b were used to amplify partial gene of β-tubulin (TUB2) (O’Donnell and Cigelnik 1997; Glass and Donaldson 1995). PCR protocol for TUB2 was performed as follows, an initial step of 3 min at 95 °C, 34 cycles of 1 min at 94 °C , 30 s at 56 °C , and 1 min at 72 °C, followed by 10 min at 72 °C. Primer pairs and PCR amplification conditions of partial genes of actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed the protocols as previously described in Prihastuti et al. (2009) and Crouch et al. (2009b). The PCR amplifications were performed in a 25 μl mixture containing 9.5 μl ddH2O, 12.5 μl 2 × PCR Master Mix (TIANGEN Co. China), 1 μl of DNA template, 1 μl of each primer (10 μM). DNA sequencing was performed at the SinoGenoMax Company Limited, Beijing.

Phylogenetic analysis

Sequences of our isolates, together with reference sequences obtained from GenBank (Table 2), were aligned using Clustal X 1.81 (Thompson et al. 1997) under the default settings. Phylogenetic tree was constructed using combined ITS, TUB2, ACT and GAPDH dataset.

Table 2 Sources of isolates and GenBank accession numbers used in this study
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Phylogenetic analyses were performed using PAUP v. 4.0 b10 (Swofford 2003). 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 1,000 random sequence additions. Branches of zero length were collapsed and all equally most parsimonious trees were saved. Descriptive tree statistics such as tree length [TL], consistency index [CI], retention index [RI], rescaled consistency index [RC], and homoplasy index [HI], were calculated for trees generated. Robustness of clades was estimated by bootstrap analysis (Felsenstein 1985) with 1,000 replications. Trees were visualized in TreeView v. 1.6.6 (Page 1996).

A second phylogenetic analysis using 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 ten millions generations and sampled every 1,000 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.

Results

Phylogeny

The concatenated alignment of four loci from 124 strains comprised 1,710 characters including alignment gaps, of which 902 characters were parsimony informative, 89 variable and parsimony uninformative, and 719 constant. The parsimony analysis resulted in a most parsimonious tree (TL = 4608, CI = 0.4065, RI = 0.8511, RC = 0.3460, HI = 0.6068). The phylogram shows that 36 isolates from Bletilla ochracea belong to 17 distinct clades with high bootstrap support (Fig. 1), thus presumably representing different Colletotrichum species. The Bayesian tree agreed with the topology of the parsimonious tree, Bayesian posterior probability values ≥ 0.95 are shown at the nodes.

Fig. 1
figure 1

Phylogenetic tree generated from a maximum parsimony analysis based on the combined ITS, TUB2, ACT and GAPDH sequence alignment, showing the phylogenetic relationships of endophytes from Bletilla ochracea (strain numbers are in red colour) with other related species. Values at the nodes represent parsimony bootstrap support values (> 50 %) and posterior probability values (≥ 0.95). Novel sequences are printed in bold and the scale bar indicates 10 changes. The tree is rooted with Monilochaetes infuscans. Asterisk (*) indicates the ex-type strains

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Taxonomy

Based on the multi-locus phylogeny and morphological characteristics, 36 endophytic isolates from orchid were identified as 17 species of Colletotrichum (Fig. 1, Table 3), including 7 new species (named as C. bletillum, C. caudasporum, C. duyunensis, C. endophytum, C. excelsum-altitudum, C. guizhouensis and C. ochracea), 8 previously described species (C. boninense, C. cereale, C. destructivum, C. karstii, C. liriopes, C. miscanthi, C. parsonsiae and C. tofieldiae), and 2 sterile mycelia.

Table 3 Morphological synopsis of endophytic Colletotrichum species from Bletilla ochracea
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Colletotrichum bletillum G. Tao, Z. Y. Liu & L. Cai, sp. nov.

MycoBank: MB803643

Figure 2a–o.

Fig. 2
figure 2

Colletotrichum bletillum (from holotype). a–b Colonies on PDA in 7 days, upper (a) and reverse (b); c, e Conidiophores; d, f–h Conidia; i Conidia and appressoria; j–o Appressoria. Scale Bars: c–o = 10 μm

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Etymology: Named after its host plant, Bletilla ochracea.

On PNP: Conidiomata not observed. Setae not observed. Conidiophores rare, formed directly on hyphae, hyaline, septate, sometimes branched. Conidiogenous cells hyaline, cylindrical, apex constricted, 11–26 × 2–2.5 μm, opening 1.5–2 μm diam, collarette distinct, funnel-shaped, 1–1.5 μm long. Conidia hyaline, smooth-walled, lunate, slightly curved, gradually obtuse on both sides, aseptate, 12.5–20.5 × 3–4.5 μm, mean ± SD = 17.5 ± 1.6 × 3.8 ± 0.4 μm, n = 31, L/W ratio = 4.6. Appressoria single, brown, ellipsoidal, ovoid to clavate or irregular with a crenate edge, 7.5–14 (−25) × 5.5–10 μm, mean ± SD = 11.9 ± 3.2 × 7.9 ± 1.2 μm, n = 32, L/W ratio = 1.5.

Culture characteristics: Colonies on PDA reaching 71–84 mm diam in 7 d at 25 °C, white to pale grey aerial mycelia with circular margin, reverse grey to dark grey, sometimes slightly yellowish with age, few visible conidial masses. Mycelial growth rate 8.9–10.4 mm per day, mean ± SD = 9.7 ± 0.7 mm, n = 5.

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao (Holotype HMAS 244278 (dried culture); culture ex-holotype CGMCC 3.15117 = LC2340).

Notes: Colletotrichum bletillum, belonging to the “Spaethianum clade”, is phylogenetically distinct and most closely related to C. liriopes, C. tofieldiae and C. verruculosum (Fig. 1) (Damm et al. 2009; Cannon et al. 2012). Colletotrichum bletillum is different from the three species as it failed to sporulate on SNA media (Damm et al. 2009) despite several attempts. Morphologically, Colletotrichum bletillum (on PNP medium) is different from C. liriopes and C. tofieldiae by producing conidia that are shorter and with lower L/W ratio (12.5–20.5 × 3.0–4.5 μm, L/W ratio = 4.6 in C. bletillum vs. (10.5–)16–23.5(−25.5) × (2.5–)3.5–4.5(−5) μm, L/W ratio = 5.0 in C. liriopes, and (12–)17–21(−23) × 3–3.5(−4) μm, L/W ratio = 5.7 in C. tofieldiae) (Damm et al. 2009). Up to now, the “Spaethianum clade” contains only 7 species including the two new species C. bletillum and C. guizhouensis described in the present study.

Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib., Mycoscience 44(1): 48 (2003)

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15168 = LC2349; Qingzhen, Pianshan mountain, from healthy leaves of B. ochracea, 16 August 2006, Gang Tao, culture CGMCC 3.15125 = LC2327; Guiyang, Yongle mountain, from healthy leaves of B. ochracea, 11 June 2006, Gang Tao, culture CGMCC 3.15124 = LC2314; and Duyun, Xiaba mountain, from healthy leaves of B. ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15165 = LC2346.

Notes: Colletotrichum boninense was isolated as endophyte from leaves of Bletilla ochracea in this study. The conidial shape and dimension are exactly similar to the holotype of Colletotrichum boninense (Moriwaki et al. 2003). The appressoria are, however, slightly shorter than that from ex-holotype and ex-paratype cultures (Moriwaki et al. 2003). In the phylogram, our four endophytic strains confidently clustered together with the type strain of C. boninense (CBS123755) and strain CSSX8, which were reported as pathogens of orchid and Crinum asiaticum (Amaryllidaceae) (Moriwaki et al. 2003; Yang et al. 2009; Damm et al. 2012b) (Fig. 1).

Colletotrichum caudasporum G. Tao, Z. Y. Liu & L. Cai, sp. nov.

MycoBank: MB 803646

Figure 3a–q.

Fig. 3
figure 3

Colletotrichum caudasporum (from holotype). a, b Colonies on PDA in 7 days, upper a and reverse b; c–f Conidiophores cells; g–k Conidia; l–q Appressoria. Scale Bars: c–q = 10 μm

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Etymology: Referring to the falcate conidia with a tail.

On PNP: Conidiomata not observed. Setae not observed. Conidiophores rare, directly formed on hyphae, hyaline, septate, occasionally branched. Conidiogenous cell clavate or cylindrical, apex more or less inflated, collarette not visible. Conidia cymbiform, fusiform, or falcate, apex prolonged into a filiform appendage, 18–30 × 3–5.5 μm, mean ± SD = 25 ± 3.5 × 4.3 ± 0.6 μm, n = 30, L/W ratio = 5.8 (excluding appendage), conidial appendage 2.5–13 long, mean ± SD = 6.3 ± 3.1 μm (n = 30). Appressoria single, brown, ovate to ellipsoidal, sometimes clavate or lobed, 6.5–16 × 5–11.0 μm, mean ± SD = 9.8 ± 1.7 × 7 ± 1.4 μm, n = 50, L/W ratio = 1.4.

Culture characteristics: Colonies on PDA reaching 70–85 mm diam in 5 days at 25 °C, white to grey, and cotton-like aerial mycelia, sometimes with fimbriate margin, reverse grey to dark grey or irregular sectors with slightly purplish pigment with age. Mycelial growth rate 11–16.5 mm per day, mean ± SD = 14.7 ± 2.3 mm, n = 5.

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao (Holotype HMAS 244282 (dried culture); culture ex-holotype CGMCC 3.15106 = LC2311).

Notes: Colletotrichum caudasporum is morphologically similar to Colletotrichum caudatum (Sacc.) Peck, but could be distinguished from the later by the dimension of conidial appendage (2.5–13 μm vs. 10–16 μm) (Sutton 1980). In the phylogram, C. caudasporum appears in a distinct lineage from C. caudatum (Fig. 1). Although there is no type specimen and type-derived sequences of Colletotrichum caudatum, we can confirm that C. caudasporum is a distinct species from C. caudatum based on the comparisons to original descriptions. Colletotrichum caudasporum belongs to the “Graminicola clade” recognised as a distinct assemblage by Crouch et al. (2009a, b) and Cannon et al. (2012). The conidial appendage of Colletotrichum caudasporum is different from all other species in this clade. Several species in the “Graminicola clade” are economically important pathogens, including C. falcatum on sugarcane (Saccharum), C. graminicola on maize (Zea), C. sublineola on Sorghum species, C. cereale and C. eremochloae on cultivated turfgrasses (Wilson 1914; Crouch and Beirn 2009). However, C. caudasporum described here is endophytic.

Colletotrichum cereale Manns, Ohio Agric. Exp. Stn. Bull. 203: 207 (1909)

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15110 = LC2306; Duyun, Xiaba mountain, isolated from healthy leaves of B. ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15111 = LC2308.

Notes: Colletotrichum cereale is the pathogen of grass of the subfamily Pooideae, and was re-described through a designation of epitype by Crouch et al. (2006). Both strains (CGMCC 3.15110 and CGMCC 3.15111) have similar conidia and appressoria to that of C. cereale epitype (Crouch et al. 2006). In the phylogram, they clustered together with the epitype of C. cereale (CBS129663) with highly supported bootstrap and posterior probabilities (100 %/1.00) (Fig. 1).

Colletotrichum destructivum Manandhar, J. B., Hartman, G. L. & Sinclair, J. B., Phytopathology 76 (3): 284 (1986)

Material examined: CHINA, Guizhou Province, Qianxi, Hongshui mountain, isolated from healthy leaves of Bletilla ochracea, 17 July 2006, Gang Tao, culture CGMCC 3.15127 = LC2320; Qianxi, Hongshui mountain, isolated from healthy leaves of B. ochracea, 17 July 2006, Gang Tao, culture CGMCC 3.15128 = LC2323; Qingzhen, Pianshan mountain, isolated from healthy leaves of B. ochracea, 16 August 2006, Gang Tao, culture CGMCC 3.15129 = LC2329.

Notes: Colletotrichum destructivum was reported as pathogen on lucerne (Medicago sativa) and soybean (Glycine max) (Manandhar et al. 1986; Latunde-Dada et al. 1999), and parasitise on a range of plants of Brassicaceae, Cuscutaceae, Lamiaceae and Solanaceae (Hyde et al. 2009). In the present study, three endophytic strains were identified as C. destructivum based on morphology and multi-locus phylogeny. In the phylogram, three strains clustered together with the type strain of C. destructivum (CBS149.34) with bootstrap support/posterior probability values of 100 %/1.00 (Fig. 1).

Colletotrichum duyunensis G. Tao, Z. Y. Liu & L. Cai, sp. nov.

MycoBank: MB 804546

Figure 4a–o.

Fig. 4
figure 4

Colletotrichum duyunensis (from holotype). a, b Colonies on PDA in 7 days, upper a and reverse b; c–e Conidiophores cells; f–i Conidia; j–o Appressoria. Scale Bars: c–o = 10 μm

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Etymology: Referring to the Duyun county in China where this fungus was first collected.

On PNP: Conidiomata not observed. Setae not observed. Conidiophores rare, directly formed on hyphae, hyaline, septate, occasionally branched. Conidiogenous cell clavate or cylindrical, apex more or less inflated, collarette not visible. Conidia cymbiform, falcate, apex prolonged into a filiform appendage, 15.2–28.5 × 3–5.5 μm, mean ± SD = 22 ± 3.1 × 4.3 ± 0.5 μm, n = 42, L/W ratio = 5.1 (excluding appendage), conidial appendage 5.6–20.1 long, mean ± SD = 10.9 ± 5.2 μm (n = 42). Appressoria single, brown, subglobose to ellipsoidal, sometimes clavate or lobed, 7.2–16.3 × 5.2–10.4 μm, mean ± SD = 11.3 ± 2.3 × 7.8 ± 1.3 μm, n = 55, L/W ratio = 1.5.

Culture characteristics: Colonies on PDA reaching 56 mm diam in 7 days at 25 °C, at first white and becoming greyish white, dense, cottony, reverse pale yellowish to brown. Mycelial growth rate 7−9 mm per day, mean ± SD = 8 ± 0.2 mm, n = 5.

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao (Holotype HMAS 244832 (dried culture); culture ex-holotype CGMCC 3.15105 = LC2307).

Notes: Colletotrichum duyunensis is a sister clade of Colletotrichum caudatum (Sacc.) Peck and the new species of C. caudasporum (Fig. 1). C. duyunensis shows highly morphological similarity to C. caudatum and C. caudasporum with a special conidial appendage, but could be distinguished from them by the dimension of conidial appendage (5.6–20.1 μm in C. duyunensis vs. 2.5–13 μm in C. caudasporum) and setae (absence of setae in C. duyunensis vs. abundant setae in C. caudatum) (Sutton 1980). In the phylogram, C. duyunensis appears in a distinct lineage from C. caudasporum and C. caudatum with 100 %/1.00 of bootstrap support and posterior probability values (Fig. 1).

Colletotrichum endophytum G. Tao, Z.Y. Liu & L. Cai, sp. nov.

MycoBank: MB 803647

Figure 5a–n.

Fig. 5
figure 5

Colletotrichum endophytum (holotype). a, b Colonies on PDA in 7 days, upper a and reverse b; c–e Conidiophores; f–h Conidia; i–k Setae, i, j tip and base of a seta; l–n Appressoria. Scale Bars: c–n = 10 μm

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Etymology: Referring to the endophytic life mode of this fungus.

On PNP: Conidiomata not observed. Setae scattered or in small groups under or among hyphae, straight or bent at the base, 2–3-septate, brown, basal cell pale brown, 47.5–113.5 μm long, base more or less inflated, tip usually acute. Conidiophores abundant, formed directly on hyphae, hyaline to pale brown, simple, sometimes branched, septate. Conidiogenous cell enteroblastic, clavate or cylindrical, sometimes elongate ampulliform, apices more or less constricted, 8.5–21.5 × 3–5 μm, opening 1.5–2.5 μm diam, collarette visible, 1–1.5 μm long. Conidia falcate, strongly curved, base obtuse, tapering much more towards apex, 16–27.5 × 3.5–5.5 μm, mean ± SD = 21.4 ± 2.8 × 4.5 ± 0.5 μm, n = 48, L/W ratio = 4.8. Appressoria single, medium to dark brown, globose to subglobose, clavate, the edge entire, sometimes slightly lobed, 8.5–16 × 7–13 μm, mean ± SD = 11.5 ± 1.9 × 9.5 ± 1.3 μm, n = 22, L/W ratio = 1.2.

Culture characteristics: Colonies on PDA reaching 68–70 mm diam in 7 days at 25 °C, white to yellowish green, grey, and felty aerial mycelia, reverse yellowish to grey, and dark grey with age, visible conidial masses. Mycelial growth rate 8.9–9.6 mm per day, mean ± SD = 9.2 ± 0.2 mm, n = 5.

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao (Holotype HMAS 244280 (dried culture); culture ex-holotype CGMCC 3.15108 = LC2338); Guiyang, Yongle mountain, isolated from healthy leaves of B. ochracea, 11 June 2006, Gang Tao, culture CGMCC 3.15107 = LC2318.

Notes: Strains representing Colletotrichum endophytum clustered in a sister clade to C. falcatum (Fig. 1). C. endophytum produces shorter conidia than C. falcatum (8.5–21.5 × 3–5 μm vs. 15.5–26.5 × 4–5 μm). C. endophytum could be further differentiated from C. falcatum by producing relatively abundant seta, and strongly curved or falcate conidia (Sutton 1980; Prihastuti et al. 2010).

Colletotrichum excelsum-altitudum G. Tao, Z.Y. Liu & L. Cai, sp. nov.

MycoBank: MB 803648

Figure 6a–p.

Fig. 6
figure 6

Colletotrichum excelsum-altitudum (from holotype). a, b Colonies on PDA in 7 days, upper a and reverse b; c–f Conidiophores; g Conidia; h–l Setae; m–p Appressoria. Scale Bars: c–p = 10 μm

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Etymology: Referring to the high altitude site where this species was first collected.

On PNP: Conidiomata acervular, conidiophores and setae formed on a basal cushion, hyaline to pale brown, clavate or cylindrical. Setae straight or sometimes slightly bent, 3–5-septate, brown, 70–114 μm long, basal cell pale brown, cylindrical, tip usually acute. Conidiophores abundant, hyaline to pale brown, septate, unbranched. Conidiogenous cell clavate or cylindrical, apex sometimes constricted, 8.5–25 × 4–5 μm, collarette hardly visiable. Conidia cylindrical, straight, sometimes slightly constricted near centre, both ends broadly rounded, 13–16.5 × 5–7 μm, mean ± SD = 14.8 ± 0.8 × 5.8 ± 0.4 μm, n = 61, L/W ratio = 2.6. Appressoria clavate, or irregular, sometimes deeply lobed, medium to dark brown, 7–14.5 × 5–10.5 μm, mean ± SD = 10.9 ± 2.1 × 6.5 ± 1.4 μm, n = 20, L/W ratio = 1.7. Teleomorph not produced in culture after 3 months.

Culture characteristics: Colonies on PDA reaching 41–46 mm diam in 7 days at 25 °C, white to grey, and felty aerial mycelia, sometimes with sectors and circular margin in colonies, reverse yellowish to brown, and dark brown with age. Mycelial growth rate 5.7–6.6 mm per day, mean ± SD = 6 ± 0.3 mm, n = 5.

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao (Holotype HMAS244279 (dried culture); culture ex-holotype CGMCC 3.15130 = LC2344); Shuicheng, Baijipo mountain, isolated from healthy leaves of B. ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15131 = LC2345.

Notes: The cylindrical conidia of C. excelsum-altitudum resemble that of C. boninense and species in C. gloeosporioides complex. However, the phylogram reveals its close affinity to C. tropicicola (Fig. 1). C. excelsum-altitudum can be distinguished from C. tropicicola by the dimensions of conidia and appressoria (conidia 13–16.5 × 5–7, appressoria 7–14.5 × 5–10.5 vs. conidia 15–19 × 6–7 μm, appressoria 13–24 × 7–8 μm) (Noireung et al. 2012).

Colletotrichum guizhouensis G. Tao, Z.Y. Liu & L. Cai, sp. nov.

MycoBank: MB 803649

Figure 7a–s.

Fig. 7
figure 7

Colletotrichum guizhouensis (from holotype). a, b. Colonies on PDA in 7 days, upper a and reverse b; c–f Conidiophores; g–j Conidia; k–m Setae; n–s Appressoria. Scale Bars: c–j, n–s = 10 μm; k–m = 20 μm

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Etymology: Named after Guizhou province where it was first collected.

On PNP: Conidiomata acervular, few developed, setae and few conidiophores formed from a basal cushion of hyaline to pale brown and clavate cells. Setae straight or sometimes slightly bent, 2–4-septate, dark brown, septation hardly visible, 65.5–170 μm long, basal cell brown to dark brown, basel cell cylindrical, tip acute. Conidiophores formed from a cushion of pale brown cells or directly on hyphae, abundant, hyaline to pale brown, usually branched. Conidiogenous cell slightly bent, apex sometimes constricted to acute, clavate, 12–25 × 2–2.5 μm, collarette not observed. Conidia falcate, fusiform, straight to slightly curved, 16–23.5 × 3–4.5 μm, mean ± SD = 19.5 ± 1.9 × 3.6 ± 0.3 μm, n = 52, L/W ratio = 5.4. Appressoria ellipsoidal to clavate, sometimes slightly lobed, or irregular with a crenate edge, dark brown, 6–14.5 × 5–11 μm, mean ± SD = 10.8 ± 1.9 × 8.2 ± 1.6 μm, n = 28, L/W ratio = 1.3.

Culture characteristics: Colonies on PDA reaching 56–74 mm diam in 7 days at 25 °C, white to light grey aerial mycelia, reverse off-white to slightly gray. Mycelial growth rate 8–11 mm per day, mean ± SD = 9.4 ± 1.1 mm, n = 5.

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao (Holotype HMAS244281 (dried culture); culture ex-holotype CGMCC 3.15112 = LC2305); Yongle mountain Guiyan, isolated from healthy leaves of B. ochracea, 11 June 2006, Gang Tao, culture CGMCC 3.15113 = LC2313; Qianxi, Hongshui mountain, isolated from healthy leaves of B. ochracea, 17 July, 2006, Gang Tao, culture CGMCC 3.15167 = LC2348; Qianxi, Hongshui mountain, isolated from healthy leaves of B. ochracea, 17 July, 2006, Gang Tao, culture CGMCC 3.15114 = LC2319; Qianxi, Hongshui mountain, isolated from living leaves of healthy B. ochracea, 17 July, 2006, Gang Tao, culture CGMCC 3.15115 = LC2322.

Notes: Colletotrichum guizhouensis is morphologically similar to C. lilii and C. spaethianum but differs from them in having darker and longer setae (65.5–170 μm in C. guizhouensis vs. 20–70 μm in C. lilii and 30–90 μm in C. spaethianum) (Damm et al. 2009).

Colletotrichum karstii Y.L. Yang, Z.Y. Liu, K.D. Hyde & L. Cai, Cryptogamie Mycologie 32: 241 (2011)

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15119 = LC2342; Shuicheng, Baijipo mountain, isolated from healthy leaves of B. ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15169 = LC2350; Qingzhen, Pianshan mountain, isolated from healthy leaves of B. ochracea, 16 August 2006, Gang Tao, culture CGMCC 3.15120 = LC2328; Qingzhen, Pianshan mountain, isolated from healthy leaves of B. ochracea, 16 August 2006, Gang Tao, culture CGMCC 3.15121 = LC2332; Duyun, Xiaba mountain, isolated from healthy leaves of B. ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15122 = LC2304; Duyun, Xiaba mountain, isolated from leaves of healthy B. ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15123 = LC2312.

Notes: Colletotrichum karstii was described by Yang et al. (2011), and has been known as pathogen and endophyte from the leaf and root of Calanthe argenteo-striata, Eria coronaria and Pleione bulbocodioides (Orchidaceae) in China (Yang et al. 2011). In this study, six strains isolated from leaves of Bletilla ochracea were identified as C. karstii on the basis of morphological and phylogenetic comparison to the holotype (Yang et al. 2011). Recently, many previous records cited as C. boninense have been confirmed to be C. karstii (Damm et al. 2012b), including strains used in Moriwaki et al. (2003), Farr et al. (2006) and Lubbe et al. (2004). Some isolates from Passiflora edulis in Brazil that caused anthracnose fruits (Tozze et al. 2010) were also re-identified to be C. karstii.

Colletotrichum liriopes Damm, P.F. Cannon & Crous, Fungal Diversity 39: 71 (2009)

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15170 = LC2351.

Notes: Colletotrichum liriopes has been known as a pathogen from herbaceous host of Liriope muscari (Damm et al. 2009), and a common endophyte and pathogen from leaves and roots of orchid plants (Yang et al. 2011). In the present study, the strain CGMCC 3.15170 was identified as C. liriopes on the basis of morphology and molecular phylogeny (Fig. 1).

Colletotrichum miscanthi J.A. Crouch, B.B. Clarke, J.F. White & B.I. Hillman, Mycologia 101: 729 (2009)

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao, culture CGMCC 3.15116 = LC2310.

Notes: Colletotrichum miscanthi belongs to the “Graminicola clade” which consists of grass-associated Colletotrichum species (Crouch et al. 2009a). In the original description of C. miscanthi, the morphology of appressoria was not provided (Crouch et al. 2009a). Our strain was identified to be C. miscanthi based on morphology and multi-locus phylogeny (Crouch et al. 2009a, Fig. 1). The hyphal appressoria were successfully induced on the PNP under the 12 h near-UV light and 12 h dark and described and illustrated based on strain CGMCC 3.15116 (Table 3, Fig. 8).

Fig. 8
figure 8

Hyphal appressoria of Colletotrichum miscanthi (from CGMCC 3.15116). a Conidia and Appressoria; b–h Appressoria. Scale Bars: a–h = 10 μm

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Colletotrichum ochracea G. Tao, Z.Y. Liu & L. Cai, sp. nov.

MycoBank: MB804547

Figure 9a–o.

Fig. 9
figure 9

Colletotrichum ochracea (from holotype). a, b Colonies on PDA in 7 days, upper a and reverse b; c–f Conidiogenous cells; g–k Conidia; l–o Appressoria. Scale Bars: c–o = 10 μm

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Etymology: Named after its host plant, Bletilla ochracea.

On PNP: Conidiomata not observed. Setae absent. Conidiophores hyaline, smooth, formed directly on hyphae, septate, branched. Conidiogenous cell straight or slightly curved, enteroblastic, cylindrical, tapering towards the apex, 9−39 × 1.5−3 μm, opening 1−1.5 μm diam, collarette absent. Conidia straight or slightly curved, one-celled, hyaline, guttulate, cylindrical with obtuse ends, 5.5−15 × 1.5−3.5 μm, mean ± SD = 9.4 ± 2.4 × 2.6 ± 0.4 μm, n = 60, L/W ratio = 3.6. Appressoria single, dark brown, subglobose, clavate, the edge entire, 9.3−18.2 × 6.6−9.3 μm, mean ± SD = 12.4 ± 2.3 × 8 ± 0.4 μm, n = 20, L/W ratio = 1.6.

Culture characteristics: Colonies on PDA reaching 48 mm diam in 7 days at 25 °C, white to yellowish, sparse, and with floccose aerial mycelia in centre, reverse slightly yellowish. Mycelial growth rate 6−8 mm per day, mean ± SD = 6.8 ± 0.3 mm, n = 5.

Material examined: CHINA, Guizhou Province, Duyun, Xiaba mountain, isolated from healthy leaves of Bletilla ochracea, 13 July 2006, Gang Tao (Holotype HMAS244831 (dried culture); culture ex-holotype CGMCC 3.15104 = LC2303); Guiyang, Yongle mountain, isolated from healthy leaves of B. ochracea, 11 June 2006, Gang Tao, culture CGMCC 3.15102 = LC2315; Guiyang, Yongle mountain, isolated from healthy leaves of B. ochracea, 11 June 2006, Gang Tao, culture CGMCC 3.15103 = LC2317.

Notes: Colletotrichum ochracea is phylogenetically closely related to C. duyunensis, another new species described in this study (Fig. 1). Although they are similar in producing curved conidia, C. ochracea can be easily distinguished from C. duyunensis by absence of conidial appendage and conidial sizes (5.5−15 × 1.5−3.5 μm in C. ochracea vs. 15.2–28.5 × 3–5.5 μm in C. duyunensis).

Colletotrichum parsonsiae Damm, P.F. Cannon, Crous, P.R. Johnst & B. Weir, Studies in Mycology 73: 27 (2012)

Material examined: CHINA, Guizhou Province, Shuicheng, Baijipo mountain, isolated from healthy leaves of Bletilla ochracea, 28 June 2006, Gang Tao, culture CGMCC 3.15126 = LC2343.

Notes: Colletotrichum parsonsiae is hitherto only known as leaf endophyte from Parsonsia capsularis from New Zealand (Damm et al. 2012b) and B. ochracea from China (this study).

Colletotrichum tofieldiae (Pat.) Damm, P.F. Cannon & Crous, Fungal Diversity 39: 77 (2009)

Material examined: CHINA, Guizhou Province, Qingzhen, Pianshan mountain, isolated from healthy leaves of Bletilla ochracea, 16 August 2006, Gang Tao, culture CGMCC 3.15118 = LC2336.

Notes: Colletotrichum tofieldiae was originally found in China and also known as pathogen of many plants in Europe (Damm et al. 2009). In the present study, the endophytic strain CGMCC 3.15118 was identified as C. tofieldiae based on morphology and multi-locus phylogeny (Fig. 1), and is the first report of C. tofieldiae as endophyte.

Discussion

Diversity and significance of endophytic Colletotrichum species

In the assessment of Hyde et al. (2009), 66 species of the genus Colletotrichum were recognized as “names in current use”. Recently, a further 52 species have been introduced (Damm et al. 2012a; Damm et al. 2012b; Noireung et al. 2012; Weir et al. 2012; Doyle et al. 2013). In our study, 7 new species were described, adding the number of accepted Colletotrichum species to over 120. Among these Colletotrichum species, 30 were known as endophytes (Table 4).

Table 4 Colletotrichum species that have been isolated from healthy plant tissues
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Unlike mycorrhizal fungi that colonize plant roots and grow into the rhizosphere, endophytes inhabit the entire plant tissues and may grow within leaves, roots and stems (Carroll 1988; Stone et al. 2004), and have been disclosed from every major lineage of land plants distributed from the tropics to the tundra (Arnold and Engelbrecht 2007). Over the last few years, the endophytic fungi have been revealed for their important ecological roles and potential applications in the biocontrol of plant diseases (Vasiliauskas et al. 2007; Maciá-Vicente et al. 2008). A good example of mutualism between Colletotrichum species and its host plants is the endophytic C. fioriniae that can be used as natural protectants against insect herbivory (Marcelino et al. 2008). While some strains of C. gloeosporioides sensu lato are also known to be able to protect Theobroma cacao against Phytophthora pathogens (Arnold et al. 2003; Mejía et al. 2008; Rojas et al. 2010).

Colletotrichum species known as endophyts and pathogens from Orchidaceae

Previous studies of fungal communities associated with orchid mainly focused on the mycorrhizal fungi (Taylor and Bruns 1999; Kristiansen et al. 2001; Taylor et al. 2003; Selosse et al. 2009). Recent investigations on non-mycorrhizal fungi of Orchidaceae showed that orchids host a high diversity of endophytes (Tao et al. 2008, 2012; Yang et al. 2011). All the currently known endophytes in the genus Colletotrichum, with their host and distribution information are summarized in Table 4.

The common Colletotrichum pathogens of Orchidaceae include C. boninense, C. crassipes, C. crossandrae, C. gloeosporioides, C. lujae, C. orchidearum, C. stanhopeae and C. vanillae (Allescher et al. 1902; Patel et al. 1953; von Arx 1957; Sutton 1980; Moriwaki et al. 2003; Hyde et al. 2009; Farr and Rossman 2013), which cause brown to black spot on leaves, flowers or stalks (Teoh 2005). Some species appear to be host-specific, e.g. C. graminicola on Zea mays (Sutton 1966; Hyde et al. 2009) but most species infect more than one host (Table 4).

In this paper, 17 endophytic Colletotrichum species from Bletilla ochracea (Orchidaceae) were identified, including 7 new species, 8 previously described species, and 2 mycelia sterilia (Table 3, Fig. 1). These species were isolated as endophytes, but some of them have been known as pathogens on same or different hosts. Species such as C. boninense, C. destructivum, C. karstii, C. miscanthi, C. musae and C. tofieldiae have been known as causal agents of anthracnose of other host plants (Photita et al. 2001; Moriwaki et al. 2003; Damm et al. 2009; Yang et al. 2011). Colletotrichum musae isolated from healthy leaves and roots of Musa acuminata (Pereira et al. 1999; Photita et al. 2001, 2005) can also lead to post-harvest disease of many varieties of banana (Anthony et al. 2004). These examples provide additional support for the hypothesis that endophytes can be latent pathogens (Photita et al. 2001; Romero et al. 2001; Photita et al. 2004).

Induction of sporulation in endophytic Colletotrichum species

Most of the descriptions in this study were based on PNP medium because these fungi failed to sporulate in commonly used media such as PDA and SNA. In endophyte studies, the mycelia sterilia account for 4.5–54 % to the total isolates (Fisher et al. 1994; Guo et al. 1998; Sánchez Márquez et al. 2008; Sun and Guo 2012). Although molecular phylogenetics has been popularized in fungal taxonomy, morphological characters are still essential (Hyde et al. 2010) in species recognition and identification. Spores and fruiting structures are the most important morphological characters to be used for classifying fungi into genera or differentiating closely related species. Su et al. (2012) demonstrated that sporulation was significantly improved for Colletotrichum and Diaporthe species under the special media such as CaCO3 water agar, pine needle agar and 1/10-strength PDA in combination with exposure to near-UV light. In the present study, most of our endophytic fungi failed to sporulate on PDA at 25 °C. Sporulations were induced by using pine needle medium, in combination with the exposure to 12 h’ near-UV light and 12 h’ dark. Most strains have been successfully induced except two that remain sterile (CGMCC 3.15171 and CGMCC 3.15172).