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Endophytic Colletotrichum species from Dendrobium spp. in China and Northern Thailand
expand article infoXiaoya Ma§, Sureeporn Nontachaiyapoom, Ruvishika S. Jayawardena, Kevin D. Hyde, Eleni Gentekaki, Sixuan Zhou§|, Yixin Qian§, Tingchi Wen§, Jichuan Kang§
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ Guizhou University, Guiyang, China
| Guizhou Institute of Animal Husbandry and Veterinary, Guiyang, China
Open Access

Abstract

Species of Colletotrichum are commonly found in many plant hosts as pathogens, endophytes and occasionally saprobes. Twenty-two Colletotrichum strains were isolated from three Dendrobium species – D. cariniferum, D. catenatum and D. harveyanum, as well as three unidentified species. The taxa were identified using morphological characterisation and phylogenetic analyses of ITS, GAPDH, ACT and ß–tubulin sequence data. This is the first time to identify endophytic fungi from Dendrobium orchids using the above method. The known species, Colletotrichum boninense, C. camelliae-japonicae, C. fructicola, C. jiangxiense and C. orchidophilum were identified as fungal endophytes of Dendrobium spp., along with the new species, C. cariniferi, C. chiangraiense, C. doitungense, C. parallelophorum and C. watphraense, which are introduced in this paper. One strain is recorded as an unidentified species. Corn meal agar is recommended as a good sporulation medium for Colletotrichum species. This is the first report of fungal endophytes associated with Dendrobium cariniferum and D. harveyanum. Colletotrichum camelliae-japonicae, C. jiangxiense, and C. orchidophilum are new host records for Thailand.

Keywords

Colletotrichum, Dendrobium, Endophytic fungi, multi-loci, new species

Introduction

Colletotrichum is the sole genus in family Glomerellaceae (Glomerellales) (Maharachchikumbura et al. 2015, 2016; Jayawardena et al. 2016b; Hongsanan et al. 2017). Presently, there are 193 accepted Colletotrichum species in eleven species complexes and 23 accepted singleton species (Hyde et al. 2014; Jayawardena et al. 2016a). Colletotrichum species has been listed as one of the top ten fungal pathogenic genera in molecular plant pathology based on scientific/economic importance (Dean et al. 2012). Anthracnose caused by Colletotrichum species can be a devastating disease in many economically important crops, including fruit crops, vegetables, cassava, sorghum, as well as ornamental plant such as orchids (Prusky and Plumbley 1992; Hyde et al. 2009a, b; Cannon et al. 2012; Dean et al. 2012; Jadrane et al. 2012; Jayawardena et al. 2016a; Diao et al. 2017). Many pathogenic Colletotrichum species that adopt biotrophic life strategies are present as symptomless endophytes in living plant tissues (Photita et al. 2004), although a large number of non-pathogenic species also occur as endophytes (e.g. Mendgen and Matthias 2002; Lu et al. 2004; Rojas et al. 2010; Cannon et al. 2012; Kleemann et al. 2012). Interestingly, experiments of Redman et al. (2001) showed that pathogenic Colletotrichum species could express mutualistic lifestyles in plants not known to be hosts and conferred disease resistance, drought tolerance, and/or growth enhancement to the host plants. Even though the diversity of Colletotrichum species associated with cultivated plant hosts have extensively been studied (Yang et al. 2009), a very limited number of studies has been conducted on Colletotrichum species from non-cultivated plants in natural and semi-natural habitats (Cannon et al. 2012).

Dendrobium SW. is the second largest genus in Orchidaceae (The Plant List 2013). Most Dendrobium species/hybrids are important ornamental/floricultural crops, but some species within this genus also possess medicinal values (Xu et al. 1995; Ng et al. 2012). Many Dendrobium orchids have been listed as Chinese medicinal herbs and are used for the treatments of atrophic gastritis, diabetes, faucitis, fever, red tongue, and/or thirsty (Ping et al. 2003; Bulpitt et al. 2007; Xing et al. 2011; Xia et al. 2012; Xu et al. 2014). Moreover, some Dendrobium orchids including D. catenatum Lindl. (widely known as D. officinale Kimura & Migo) have been listed as critically endangered species by the International Union for Conservation of Nature (IUCN) (www.iucnredlist.org). Due to their significance, Dendrobium orchids have been the subject of many studies including the diversity of endophytic fungi (Ma et al. 2015). However, only a limited number of studies on endophytic Colletotrichum in Dendrobium species have been reported and the number of Dendrobium species included in these studies are very few (Yuan et al. 2009; Yang et al. 2011; Mangunwardoyo and Suciatmih 2011; Chen et al. 2012; Noireung et al. 2012; Tao et al. 2013). In the present study, we investigated the diversity of endophytic Colletotrichum in five Dendrobium orchid species collected from a mountain (at an elevation of 1,300–1,400 m) close to the Thailand-Myanmar border and D. catenatum collected from Guizhou Province in China. A total of 22 endophytic Colletotrichum strains were isolated and identified based on both morphological and molecular characteristics. Five Colletotrichum strains, C. cariniferi, C. chiangraiense, C. doitungense, C. parallelophorum and C. watphraense are introduced as new species. The results of this study will contribute to the knowledge on diversity and phylogeny of Colletotrichum.

Material and methods

Sample collection

Healthy roots, stems and leaves of D. cariniferum, D. harveyanum and three unidentified Dendrobium taxa (referred to as Dendrobium sp. 1, 2 and 3) were collected from Wat Phra That Doi Tung (Temple of Doi Tung Pagoda), Mae Fah Luang District, Chiang Rai, Thailand. Healthy roots, stems and leaves of D. catenatum were collected from Guizhou Province in China. Materials were packed in zip-lock bags or tubes containing silica gel on ice. Fungal isolation was carried out within 48 hours following collection.

Fungal isolation and cultivation

Surface sterilization and endophyte isolation were carried out as described by Nontachaiyapoom et al. (2010) with some modifications. First, materials were washed with running water. Roots, stems and leaves were immersed in a solution containing 3% (v/v) H2O2 and 70% (v/v) ethanol for 5 minutes, and then rinsed with sterile distilled water for three times. Sterilized materials were cut into 2 mm2 and placed on potato dextrose agar (PDA) containing 50 μg/ml oxytetracycline, 50 μg/ml penicillin and 50 μg/ml streptomycin (Otero et al. 2002). Samples were incubated at 28 °C under natural light. Single spores were transferred to fresh PDA to obtain pure cultures. The pure cultures were deposited at China General Microbiological Culture Collection Center (CGMCC), International Collection of Micro-organisms from Plants (ICMP) and Mae Fah Luang University Culture Collection (MFLUCC). The dry cultures of new species were deposited in Mae Fah Luang University herbarium (MFLC).

DNA extraction and amplification

DNA samples were prepared from mycelium of pure fungal culture using EZgene Fungal gDNA Kit (GD2416, Biomiga, USA) as described by the manufacturer. Amplification reactions were performed using reagents purchased from BIOMIGA (San Diego, USA). Each 20-μl amplification reaction contained 10 μl of 2*Bench Top Taq Master Mix (0.05 units/μl Taq DNA polymerase, 0.4 mM dNTPs and 4mM MgCl2); 2 μl forward and reverse primers; 1μl of DNA template and 7 μl of water. The primers used in this study were ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), 512F/783R (Carbone and Kohn 1999) and BT2A/BT2B (Glass and Donaldson 1995; Maharachchikumbura et al. 2012). The thermal cycling programs are presented in Table 1. PCR products were sent to Invitrogen (USA), Sangon Biotech and Sino GenoMax (China) for purification and sequencing.

PCR thermal cycling process.

PCR amplification
Region/gene Initial denaturation Cycle number Denaturation Annealing Elongation Final elongation
ITS 95 °C 3 min 30 95 °C 1 min 53 °C 1 min 72 °C 1 min 72 °C 10 min
GAPDH 95 °C 3 min 35 95 °C 1 min 60 °C 30 s 72 °C 45 s
ACT 95 °C 3 min 40 94 °C 45 s 52 °C 30 s 72 °C 90 s
ß-tubulin 95 °C 3 min 35 94 °C 1 min 55 °C 55 s 72 °C 1 min

Sequence analysis

Either single-directional sequencing results (for ITS and GAPDH) or bi -directional sequencing results (for ACT and TUB2) were manually trimmed and/or assembled into contigs using CodonCode aligner software (CodonCode Corporation, Dedham, Massachusetts). Through the latest publications and the observation for ML tree topology, a selected set of ITS, GAPDH, ACT and TUB2 sequences especially those of ex-type/ex-epitype materials used in the phylogenetic analysis were downloaded from GenBank (Table 2). Five datasets of Colletotrichum spp. ITS (134nt), GAPDH (113nt), ACT (119nt), ß–tubulin (125nt) and a concatenated dataset were constructed. Sequences were aligned using MAFFT version 6 (Katoh and Toh 2008; mafft. cbrc. jp/ alignment/server/). Aligned datasets were visually inspected and misaligned regions were manually edited where necessary using Bio-Edit version 7.2.5 (Hall 1999). Ambiguous regions were trimmed using trimAL version 1.3 (Capella–Gutierrez, Silla–Martinez and Gabaldon 2009) available online through Phylemon 2.0 (http://phylemon.bioinfo.cipf.es/). After trimming, the final alignments contained 578 sites for ITS, 298 sites for GAPDH, 290 sites for ACT and 480 sites for ß–tubulin. The concatenated dataset contained a total of 134 taxa and 1646 sites that were used for all subsequent analyses and submitted to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S22431). Gaps were treated as missing data in maximum likelihood (ML), Bayesian inference (BI) and parsimony trees. Parsimony trees were constructed with PAUP (Phylogenetic Analysis Using Parsimony) version 4.0 beta 10 (Swofford 2002). Heuristic searches were conducted as follows: 1000 starting trees were generated using stepwise addition and random addition sequence replicates, followed by branch swapping using the tree–bisection–reconnection (TBR) algorithm. The inferences for MP tree were length = 6732 steps, CI = 0.294, RI = 0.760, RC = 0.223, HI = 0.706. Maximum likelihood analyse was conducted with RAxMLGui 1.31 (Silvestro and Michalak 2012). The general time reversible (GTR) model of nucleotide substitution was used and the inverse gamma distribution option was implemented. The topology of the resulting tree was similar to that of the maximum parsimony tree. Bootstrap support was calculated from 1000 replicates, which were subsequently mapped onto the best-scoring ML tree. Bayesian inference trees were computed using MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). The concatenated dataset was partitioned and the ultrafast bootstrap (Minh et al. 2013) implemented in the IQ-TREE software (Nguyen et al. 2015) as well as Mrmodeltest 2.3 (Nylander 2004) were used to estimate the best fitting models according to the Bayesian information criterion (BIC). The GTR model with inverse gamma distribution and HKY model with gamma distribution were used as the most appropriate for the ITS and GAPDH respectively. The Hasegawa, Kishino & Yano (HKY) model with inverse gamma distribution and GTR model with gamma distribution were selected for the ACT and ß-tubulin datasets. Two sets of four simultaneous independent chains of Markov chains Monte Carlo (MCMC) simulations were run for 6,000,000 generations, 25% of trees were discarded as burn-in and the remaining trees were used to calculate the posterior probabilities. Convergence was assumed when the standard deviation of split sequences was less than 0.01. The fungal isolates and sequences of region/genes used in Colletotrichum phylogenetic analysis are listed in Appendix A.

Colletotrichum strains and species isolated from Dendrobium orchids.

Orchid sample Sample site Tissue Number of fungal strains Colletotrichum species Code
D. cariniferum Chiang Rai, Thailand Root 0 0
Stem 1 C. cariniferi MFLUCC 14-0100
Leaf 0 0
D. harveyanum Chiang Rai, Thailand Root 0 0
Stem 0 0
Leaf 2 C. orchidophilum MFLUCC 14-0161 MFLUCC 14-0162
Dendrobium sp. 1 Chiang Rai, Thailand Root 2 C. parallelophorum MFLUCC 14-0077 MFLUCC 14-0079
Stem 3 C. parallelophorum MFLUCC 14-0082 MFLUCC 14-0083 MFLUCC 14-0085
Leaf 4 C. boninense, C. jiangxiense, C. fructicola MFLUCC 14-0086 MFLUCC 14-0087 MFLUCC 14-0091 MFLUCC 14-0092
Dendrobium sp. 2 Chiang Rai, Thailand Root 2 C. chiangraiense ; C. fructicola MFLUCC 14-0119 MFLUCC 15-0262
Stem 3 C. boninense, C. watphraense, C. sp. indet. MFLUCC 15-0120 MFLUCC 15-0123 MFLUCC 15-0124
Leaf 3 C. citricola, C. doitungense MFLUCC 15-0128 MFLUCC 15-0129 MFLUCC 15-0131
Dendrobium sp. 3 Chiang Rai, Thailand Root 0 0
Stem 0 0
Leaf 1 C. boninense MFLUCC 15-0148
D. catenatum Xing Yi, China Root 0 0
Stem 0 0
Leaf 1 C. boninense MFLUCC 15-0261

Morphological analysis

Sporulation of studied fungi was induced on thin pieces of Corn malt agar medium (CMA). The strains that did not sporulate on CMA were cultured on PDA or Sabouraud dextrose agar (SDA) with sterilized orchid tissues in order to induce sporulation. An autoclaved toothpick was placed on CMA for one strain C. cariniferi to induce sporulation. Cultures were grown in a dark cabinet at room temperature (28 °C) and observed for every seven days or less. The growth rate was evaluated when mycelia nearly covered the whole medium surface. Once an acervuli or ascomata were observed, photos were taken with a stereomicroscope (SteREO Discovery. V8, Carl Zeiss Microscopy GmBH, Germany). Cross-sections and conidiomata crushed in water were observed under a compound microscope (EOS 600D, Nikon, Japan). Ascomata and conidiomata were observed under a Motic SMZ–140 microscope (China). Conidiophore, conidia, appressoria, ascomata, asci, ascospores and other visible structures such as chlamydospore were used for evaluating morphological characteristics in this study (Damm et al. 2014). The recommendations of Jeewon and Hyde (2016) were followed in establishing new species.

Results

Fungal isolation and Identification

Twenty-two endophytic Colletotrichum strains were isolated from six Dendrobium species (Table 2). The highest number of Colletotrichum strains and species were isolated from Dendrobium sp. 1 followed by Dendrobium sp. 2. All three tissue types of the two orchids hosted at least one strains of Colletotrichum. Among the three tissue types, the highest number of Colletotrichum strains and species were isolated from leaves. Colletotrichum boninense and C. fructicola were respectively the most frequently isolated Colletotrichum species. Interestingly, C. boninense was isolated from Dendrobium species collected from both geographical areas studied (i.e., Chiang Rai, Thailand and Guizhou, China).

Figure 1. 

Maximum likelihood (ML) tree of Colletotrichum inferred from 134 taxa and 1646 sites from a concatenated dataset containing ITS, GAPDH, ACT and ß-tubulin sequence data. Values at nodes indicate bootstrap percentages (BP) for ML, Bayesian posterior probabilities (PP) and BP for maximum parsimony (MP) in this order. Only BP over 50%, PP over 0.50 and MP over 50 are shown. Dashes correspond to lower than the above-mentioned values. The isolated fungal endophytes in this study are shown in green bold text. Scale bar corresponds to 0.08 substitutions per site. “*” indicates the new species.

Sporulation results

All Colletotrichum strains could grow on three kinds of media. Colletotrichum citricola, C. doitungense, C. fructicola and C. parallelophorum produced both sexual and asexual morphs in culture. Colletotrichum boninense, C. cariniferi, C. orchidophilum and C. watphraense produced only the asexual morph and C. chiangraiense produced only sexual morph in culture. Measurements of important vegetative and reproductive characteristics of isolated strains are given in Table 3.

Synopsis of size (µm) of structures of Colletotrichum species identified in this study.

Sexual morph Asexual morph
Colletotrichum species N Vegetative hyphae diam (µm) Setae (µm) Ascomata (µm) Size of asci (µm) Size of ascospores (µm) Size of conidiomata (µm) Size of conidiophore (µm) Size of conidia (µm)
C. cariniferi sp. nov. 1 3.5–8.2 50 ×50 (37.5–) 42.3–65 (–71.6) × (3.1–) 3.8–5.9 (–6) (24.1–) 26.8–33.0 (–36.1) × (7.9–) 8.3–9.6 (–10.2), L/W=3.4
C. chiangraiense sp. nov. 1 4.6.± 1.8 (14.4–) 15.3–19.6 (–20.5) × (7.4–) 7.3–7.9 (–8) (30.7–) 33.4–52.7 (–72.2) × (5.7–) 6.5–8.2 (–9.4) (11–) 11.9–15.4 (–16.7) × (2.2–) 2.8–3.8 (–4.4), L/W=4.2
C. citricola 3 3.1 ± 1.1 (51.8–) 54.1–67.8 (–68.5) × (2.3–) 2.4–5.8 (–7.2) (34.5–) 46.4–84.9 (–87.1) × (31.7–) 33.8–46.5 (–50.9) (41.3–) 49.4–65.0 (–71.6) × (8.3–) 9.5–12.9 (–14.3) (14.4–) 14.8–17.5 (–19.3) × (5.4–) 5.7–7.1 (–7.6), L/W = 2.5 (10.8–) 16.7–25.6 (–30.6) × (3.1–) 4–5.3 (–5.6) (12.5–) 13.4–15 (16.5–) × (5–) 5.9–6.9 (–7.2), L/W = 2.2
C. doitungense sp.nov. 2 1.1–3.5 (125.5–) 126.9–133.7 (–135.1) × (101.3–) 101.8–104.3 (–104.8) (51.1–) 53.7–70.6 (–71.6) × (8.5–) 8.8–10.1 (–10.4) (16.1–) 17.5–21.5 (–23.4) × (4.5–) 5.1–7(–7.5), L/W=3.2 (9.1–) 14.3–26.8 (–29.4) × (3–) 3.1–4.5 (–5) (6.6–) 8.6–13.8 (–15) × (2.6–) 3.8–8.9 (–13.8), L/W=1.75
C. fructicola 3 2.6–5 (53–) 57.2–73.1 (–83.3) × (3.4–) 3.5–4 (–4.1) (131.9–) 138.4–163.6 (–171.5) × (120.9–) 123.6–142.1 (–143.2) (57.6–) 61.2–82.6 (–94.3) × (8.7–) 9.3–13.3 (–15.8) (10–) 12.0–20.0 (–20.9) × (3.6–) 4.1–5.2 (–5.3), L/W=3.4 500×400 (12.8–) 13.8–16.6 (–18.6) × (2.7–) 3.5–7.8 (–16), L/W=2.9
C. jiangxiense 2 1.3–2.1 (12.7–) 13.5–21.4 (–23.4) × (1.9–) 2–3 (–3.2) (8.6–) 9–12.4 (–13.2) × (3.5–) 3.6–4.4 (–4.5), L/W=2.6
C. orchidophilum 2 1.9–5.4 200×300 (9.6–) 11.7–14.1 (–14.7) × (2.9–) 3.5–4.4 (–4.8), L/W=3.3
C. parallelophorum sp.nov. 2 2–4.3 (56.7–) 60.2–79.2 (–81.2) × (2.8–) 2.9–3.7 (–3.9) (267–) 261.4–342.3 (–346.2) × (190.4–) 173–272.5 (–280) (43.3–) 44.1–63.3 (–66.5) × (7.6–) 8–9.8 (–10) (13.9–) 14.1–18 (–20.9) × (3.1–) 3.9–5.4 (–5.8), L/W=3.5 200×200 (18.3–) 20.82–34 (–41.2) × (2.6–) 2.8–4.3 (–5.4) (12.1–) 13.8–16.8 (–18.9) × (3.3–) 4.4–7.5 (–7.9), L/W=2.6
C. watphraense sp. nov. 1 1.6–4.3 200×300 (15.8–) 18.5–26.8 (–29.1) × (3.4–) 3.8–5.1 (–5.7) (12.4–) 12.5–14.6 (–15.2) × (4.4–) 4.5–5.8 (–6.1), L/W=2.3

Phylogenetic results

Phylogenetic analyses

All the sequences of ITS, GAPDH, ACT and β-tubulin of 22 strains of Colletotrichum obtained in this study were deposited in GenBank (List in Appendix B). The three selected outgroup species (i.e. Australiasca queenslandica BRIP 24607; Monilochaetes infuscans CBS 869.96 and Monilochaetes guadalcanalensis CBS 346.76) formed a strongly supported cluster (100ML/1.00BI/99MP). The ingroup consisted of all Colletotrichum sequences and was fully supported by all three methods of analysis (100ML/1.00BI/100MP). Five strains grouped within the gloeosporioides complex: MFLUCC 14-0087, MFLCCC 14-0091, MFLUCC 14-0092,

MFLUCC 14-0148 and MFLUCC 14-0262. The sequences of MFLCCC 14-0091 and MFLUCC 14-0092 were nearly identical and close to C. jiangxiense with strong support (99ML/0.95BI/96MP). MFLUCC 14-0087, MFLUCC 14-0148 and MFLUCC 15-0262 clustered with C. fructicola (ICMP 181873) (91ML/0.72BI/83MP).

Nine of the newly sequenced strains clustered within the boninense species complex: MFLUC 14-0086, MFLUCC 14-0119, MFLUCC 14-0120, MFLUCC 14-0123, MFLUCC 14-0124, MFLUCC 14-0128, MFLUCC 14-0129, MFLUCC 14-0131, MFLUCC 15-0261. MFLUCC 14-0086, MFLUCC 14-0124 and MFLUCC 14-0261 shared very similar sequences. MFLUCC 14-0128 grouped as sister to the three above-mentioned strains (66ML/1.00BI/73MP). MFLUCC 14-0123 formed a separated clade from other species by only Bayesian analysis (1.00BI). MFLUCC 14-0120, MFLUCC 14-0129 and MFLUCC 14-0131 formed a cluster with C. camelliae-japonicae and C. fructicola (76ML/1.00BI/62MP). MFLUCC 14-0120 and MFLUCC 14-0129 differed by only three base pairs in trimmed concatenated alignment. MFULCC 14-0119 was placed basally to the boninense species complex with strong support (100ML/0.96BI/90MP).

MFLUCC 14-0161 and MFLUCC 14-0162 grouped outside the currently accepted species complexes. The two had a close relationship and formed a clade with C. orchidophilum, which is a singleton and a sister taxon to the acutatum species complex. They hold the maximum support with all three methods of analysis. Colletotrichum orchidophilum differed 1.5% and 1.3% with MFLUCC 14-0161 and MFLUCC 14-0162 respectively. MFLUCC 14-0077, MFLUCC 14-0079, MFLUCC 14-0082, MFLUCC 14–0083 and MFLUCC 14-0085 formed a novel clade (100ML/1.00BI/100MP), which grouped as sister clade to the C. excelsum-altitudum/C. tropicicola clade and MFLUCC 14-0100 (88ML/1.00BI/59MP). MFLUCC 14-0100 took a solo branch in the basal position among them (99ML/1.0BI/98MP).

Taxonomy

The 22 strains isolated as endophytes were assigned to eleven species, five known species, five new species and one undetermined species. We obtained the sexual and asexual morphs for four strains. The sexual morph only was obtained in the case of C. chiangraiense. The descriptions of the fungal endophytes identified in this study are as follows.

Colletotrichum cariniferi X.Y. Ma, K.D. Hyde & Jayawardena, sp.nov.

Fungal Name Number: FN570511

Etymology

In reference to the host epithet cariniferum.

Holotype

MFLC 17-1199 (ex-holotype culture: MFLUCC 14-0100).

Description

Sexual morph not observed.

Asexual morph on CMA. Vegetative hyphae 3.5–8.2 µm diam (N=20), hyaline to brown, smooth-walled, septate, branched. Appressoria (9.7–) 10.4–17 (–20.5) × (6.5–) 7.1–11.3 (–13.6) µm (N=6), globose to sub-globose, light brown. Conidiomata 50 × 50 µm (N=10), developing with mycelia, globose to irregular, milk orange to orange, in mass brown. Conidiophores (37.5–) 42.3–65 (–71.6) × (3.1–) 3.8–5.9 (–6) µm (N=6), smooth-walled, unbranched, hyaline. The part connected with conidia of conidiogenous cell inflated and some with large guttules. Conidia (24.1–) 26.8–33.0 (–36.1) × (7.9–) 8.3–9.6 (–10.2) µm (N=30), L/W = 3.4, ellipsoidal to cylindrical, with one end inflated when immature state, both ends rounded when mature, with 2 to 3 guttules, hyaline.

Cultures on CMA flat with entire margin. Growth rate: 0.23cm/day, with 50-days for sporulation. Cottony, pale cinnamon to light brown, scattered pale mycelia in spots around the middle inoculum clump, sometimes covered short, floccose-felty, white, aerial mycelium, reverse buff brown.

Figure 2. 

Colletotrichum cariniferi (holotype). A Colony B Conidiomata C, I–J Appressoria D–H Conidiophores K–M Conidia. Scale bars: 100 µm (B), 5 µm (C–D), 10 µm (E–H), 5 µm (I–M).

Material examined

Thailand, Chiang Rai, Wat Phra That Doi Tung (Temple of Doi Tung Pagodas), the host Dendrobium cariniferum was sampled on 19 December 2013, Collector: Sureeporn Nontachaiyapoom, Natdanai Aewsakul, Xiaoya Ma.

Notes

Colletotrichum cariniferi (MFLUCC 14-0100) clusters in a separate branch with a good support (88ML/1.00BI/59MP). The species is most phylogenetically close to Colletotrichum excelsum-altitudum and C. tropicicola, but they are morphologically different. C. cariniferi holds 77 and 91 different base pairs compared with C. tropicicola and C. excelsum-altitudum respectively. Colletotrichum cariniferi has much larger conidia than that of closely related strains in the tree (conidia (24.1–) 26.8–33 (–36.1) × (7.9–) 8.3–9.6 (–10.2) µm (N=30), L/W = 3.4 vs. conidia of C. tropicicola 13–16.5×5–7 μm and C. excelsum-altitudum 14.8 ± 0.8 × 5.8 ± 0.4 μm) (Noireung 2012; Tao et al. 2013). Blastn searches with sequence of MFLUCC 14-0100 resulted in 100% matches with ITS sequence of endophytic Colletotrichum sp. strain S4 isolated from Dendrobuim nobile in China (GenBank FJ042517, Yuan et al. 2009) and 96% identity with TUB2 sequences of C. arxii strain CBS 169.59 isolated from Oncidium excavatum (GenBank KF687868, Liu et al. 2014) in Netherlands and another C. arxii strain CBS 132511 isolated from Paphlopedilum sp. in Germany (GenBank KF687881, Liu et al. 2014) respectively. Colletotrichum cariniferi from stems of D. cariniferum is introduced as a new species.

Colletotrichum chiangraiense X.Y. Ma, K.D. Hyde & Jayawardena, sp.nov.

Fungal Name Number: FN570512
Figure 3

Etymology

In reference to the host sample site Chiang Rai, Thailand.

Holotype

MFLU 17-1201 (ex-holotype culture: MFLUCC 14-0119).

Description

Asexual morph not observed.

Sexual morph on CMA. Vegetative hyphae 4.6.± 1.8 µm diam (N=20), hyaline to pale brown, smooth-walled, septate, branched. Chlamydospore globose, brown. Hyphae fusion and crozier observed. Ascomata rare, covered by mycelia, black. Appressoria (14.4–) 15.3–19.6 (–20.5) × (7.4–) 7.3–7.9 (–8) µm (N=2), single, outline ampulliform or ovate, pale brown. Asci (30.7–) 33.4–52.7 (–72.18) × (5.7–) 6.5–8.2 (–9.4) µm (N=15), cylindrical, straight to curved, unitunicate, 8–spored. Ascospores (11–) 11.9–15.4 (–16.7) × (2.2–) 2.8–3.8 (–4.4) µm (N=20), L/W = 4.2, bi-seriately, smooth-walled, cylindrical or fusiform, one guttule in the middle, hyaline.

Cultures on CMA flat with entire margin. Growth rate: 0.6cm/day, with 20-days for sporulation. Fluffy, dark green in the middle and white margin, reverse black in the middle.

Figure 3. 

Colletotrichum chiangraiense (holotype). A Colony B Spore germination C Conidiophore D Appressoria E Chlamydospore F Mycelia fusion G Crozier H–N Asci O–R Ascospores. Scale bars: 20 µm (D), 20 µm (G), 5 µm (J–N), 10 µm (O–R).

Material examined

Thailand, Chiang Rai, Wat Phra That Doi Tung (Temple of Doi Tung Pagodas), the host Dendrobium sp. 2 was collected on 19 December 2013, Collector: Sureeporn Nontachaiyapoom, Natdanai Aewsakul, Xiaoya Ma.

Notes

Colletotrichum chiangraiense (MFLUCC 14-0119) formed a single branch with the support of 81ML/1.00BI/78MP in boninense species complex. It has 125 different base pairs (mainly in ITS and ACT) from the closest strain C. cymbidiicola. Blastn searches with sequences of MFLUCC 14-0119 resulted in 99% identity with ITS of the endophytic C. crassipes strain DO93 (GenBank KP050648) isolated from Dendrobium officinale in China (Unpublished), 99% identity with ACT of the endophytic Colletotrichum sp. strain COAD 2105 (GenBank KY407893) isolated from Cattleya jongheana in Brazil (Unpublished), 98% identity with TUB2 of the endophytic C. boninense strain CBS 125502 (GenBank KJ955336) isolated from Camellia sinensis in China (Liu et al. 2015) and 98% identity with TUB2 of the endophytic C. boninense strain CGMCC 3.15165 (GenBank KC244156) isolated from Bletilla ochracea in China (Tao et al. 2013). This species was observed antheridium, mycelia fusion and crozier, which indicates that this species may be homothallic. Here we introduce the strain isolated from root of Dendrobium sp. 2 as a new species.

Colletotrichum watphraense X.Y. Ma, K.D. Hyde & Jayawardena, sp. nov.

Fungal Name Number: FN570513
Figure 4

Etymology

In reference to the host sample site – Wat Phra temple in Chiang Rai, Thailand.

Holotype

MFLU 17-1202 (ex-holotype culture: MFLUCC 14-0123).

Description

Sexual morph not observed.

Asexual morph on CMA. Vegetative hyphae 1.6–4.3 µm diam (N=20), smooth-walled, septate, branched, hyaline. Chlamydospores and appressoria not observed. Conidiomata 200 × 300 µm, brown, Conidiophores (15.8–) 18.5–26.8 (–29.1) × (3.4–) 3.8–5.1 (–5.7) µm (N=16), smooth-walled, septate, branched or single, periclinal thickening, hyaline. Conidia (12.4–) 12.5–14.6 (–15.2) × (4.4–) 4.5–5.8 (–6.1) µm (N=5), L/W = 2.3, aseptate, ellipsoidal, single guttules in the middle, the one part inflated, hyaline.

Cultures on CMA flat with entire margin. Growth rate: 0.45cm/day, with 30-days for sporulation. Fluffy, white to light buff orange. Perithecia isolated. Acervuli under white cotton-like mycelia, irregular, asymmetrical surface, light brown to brown.

Figure 4. 

Colletotrichum watphraense (holotype). A Colony B Fruiting body C–J Conidiophores K–N Conidia. Scale bars: 200 µm (B), 5 µm (C–N).

Material examined

Thailand, Chiang Rai, Wat Phra That Doi Tung (Temple of Doi Tung Pagodas), the host Dendrobium sp. 2 was collected on 19 December 2013, Collector: Sureeporn Nontachaiyapoom, Natdanai Aewsakul, Xiaoya Ma.

Note

MFLUCC 14-0123 formed a singular branch with other species and only supported by 1.00BI in boninense species complex. There were 42bp (2.6%) and 85bp (5.2%) differences in GAPDH between Colletotrichum watphraense and its close strains Colletotrichum boninense/C. novae-zelandiae respectively. The closest matches in a blastn search with ITS sequences of the strain MFLUCC 14-0123 are C. cymbidiicola strain FS21 (GenBank KP689224) iaolated from a rare medical plant Huperzia serrata with 99% identity in China (Wang et al. 2016), C. gloeosporioides strain Trtsf02 (GenBank GU479899) isolated from Trillum tschonoskii with 99% identity in China (Unpublished) and pathogenic C. boninense strain CO5016 (GenBank GU935883) isolated from ginseng with 99% identity in Korea (Unpublished). GAPDH and ACT sequences blastn results showed its closest matches are pathogenic C. citricola strain SXC 151 (GenBank KC293736) isolated from Proteaceae with 99% identity in Netherlands (Liu et al. 2012) and C. boninense strain CBS 125502 (GenBank KJ954462) isolated from Camellia sp. with 99% identity in unknown locality (Liu et al. 2015). Blastn search with TUB2 sequence results in 99% identity with two C. boninense strains CBS 125502 (GenBank KJ955336) and the strain CGMCC 3.15165 (GenBank KC244156) as mentioned above. The conidiophores were much longer (40 µm long) in C. boninense. Conidia of the strain CBS 123755 have straight, cylindrical to clavate, conidia with a rounded apex; and base with a prominent hilum, sometimes with two large polar guttules, which is different from Colletotrichum watphraense. Here we assigned the strain isolated from stem of Dendrobium sp. 2 as a new species.

Colletotrichum doitungense X.Y. Ma, K.D. Hyde & Jayawardena, sp.nov.

Fungal Name Number: FN570514
Figure 5

Etymology

In reference to the host sample site Doi tung, Chiang Rai, Thailand.

Holotype

MFLU 17-1200 (ex-holotype culture: MFLUCC 14-0128).

Description

Asexual morph on CMA. Vegetative hyphae 1.1–3.5 µm diam, hyaline, smooth-walled, septate, branched. Setae and chlamydospores not observed. Conidiomata and ascomata cluster together. Conidiophores (9.1–) 14.3–26.8 (–29.4) × (3–) 3.1–4.5 (–5) µm, smooth-walled, unbranched, septate, constricted septum, hyaline. Conidiogenous cell (3.1–) 3.2–5.8 (–7.5) × (2.6–) 3–4 (–4.5) µm (N=14), globose to sub-globose, smooth-walled, hyaline. Conidia (6.6–) 8.6–13.8 (–15) × (2.6–) 3.8–8.9 (–13.8) µm (N=22), L/W = 1.75, globose to ellipsoidal, both ends rounded, smooth-walled, hyaline.

Sexual morph on CMA. Ascomata (125.5–) 126.9–133.7 (–135.1) × (101.3–) 101.8–104.3 (–104.8) µm (N=10), sub-globose, closed, pale brown to brown. Peridium 3–11.5 µm thick, Asci (51.1–) 53.7–70.6 (–71.6) × (8.5–) 8.8–10.1 (–10.4) µm (N=8), cylindrical, slight curved, composed of pale to medium brown flattened angular cells, unitunicate, smooth-walled, 8–spored, hyaline. Ascospores (16.1–) 17.5–21.5 (–23.4) × (4.5–) 5.1–7.0 (–7.5) µm (N=20), L/W = 3.2, fusiform, blunt to somewhat acute or acute both ends, single guttule in the middle, septate, bi-seriately, smooth-walled, hyaline.

Cultures on CMA flat with entire margin. Fluffy, white, reverse same. Growth rate: 0.6cm/day, with 20-days for sporulation. Brown ring in the middle. Perithecia gregarious. Acervuli and ascomata in mass light brown to brown.

Figure 5. 

Colletotrichum doitungense (holotype). A Colony B Fruiting body C–D Ascomata E–J Conidiophores K–L Conidia M Spore germination N–V Asci W–a Ascospores. Scale bars: 100 µm (B), 20 µm (C–D), 5 µm (E–M), 10 µm (N–V), 5 µm (W–a).

Material examined

Thailand, Chiang Rai, Wat Phra That Doi Tung (Temple of Doi Tung Pagodas), the host Dendrobium sp. 2 was collected on 19 December 2013, Collector: Sureeporn Nontachaiyapoom, Natdanai Aewsakul, Xiaoya Ma.

Notes

Colletotrichum doitungense form an independent lineage from other strains with good support (66ML/1.00BI/73MP) in boninense species complex. The ITS sequence of MFLUCC 14-0128 100% matches with unpublished pathogenic C. cymbidiicola strain OORC18 (GenBank JX902424) isolated from orchid in India and C. karstii strain R001 (GenBank JN715846) isolated from blackberry in Colombia (Unpublished). Blastn researches with sequences of MFLUCC 14-0128 results in 98% identity with GAPDH sequence of endophytic C. boninense strain CGMCC 3.15168 (GenBank KC843491) isolated from Bletilla ochracea in China (Tao et al. 2013), 99% identity with ACT sequence of C. boninense strain CBS 125502 (GenBank KJ954462) and 99% identity with TUB2 sequence of C. citricola strain SXC 151 (GenBank KC293656) as mentioned above. Its conidiogenus cell is globose to sub-globose, which differ from cylindrical to ellipsoidal conidiogenus cell in C. boninense (Damm et al. 2012). This strain has 2 and 0 in ITS, 6 and 1 in GAPDH, 3 and 2 in ACT, 17 and 16 base pair differences from its sister taxon C. torulosum and MFLUCC 14-0261 respectively. Here we introduce Colletotrichum doitungense isolated from root of Dendrobium sp. 2 as a new species.

Colletotrichum parallelophorum X.Y. Ma, K.D. Hyde & Jayawardena, sp. nov.

Fungal Name Number: FN570515
Figure 6

Etymology

In reference to the parallel conidiophores.

Holotype

MFLU 17-1198 (ex-holotype culture: MFLUCC 14-0083).

Description

Asexual morph on CMA. Vegetative hyphae 2–4.3 µm diam (N=30), smooth-walled, septate, branched, hyaline to pale brown. Chlamydospores not observed. Conidiomata acervular, orange. Appressoria (56.7–) 60.2–79.2 (–81.2) × (2.8–) 2.9–3.7 (–3.9) µm (N=8), single, sub-globose, brown, rare. Conidiophores and setae formed on a cushion of pale brown cells (1.9–) 2.4–4 (–4.6) µm diam. Setae medium brown, smooth-walled, 2 or 3-septate; base cylindrical, constricted at the base, apex acute. Conidiophores (18.3–) 20.8–34 (–41.2) × (2.6–) 2.8–4.3 (–5.4) µm (N=20), smooth-walled, 2 to 3-septate, branched, hyaline to pale brown. Conidiophores and setae formed on a cushion of pale brown prismatic cells, sometimes with guttules. Conidia (12.1–) 13.8–16.8 (–18.9) × (3.3–) 4.4–7.5 (–7.9) µm (N=50), L/W = 2.6, hyaline, smooth-walled, with 1 to 4 guttules, cylindrical with both ends rounded.

Sexual morph on CMA. Ascomata (267–) 261.4–342.3 (–346.2) × (190.4–) 173.0–272.5 (–280) µm (N=3), globose, glabrous, Ascomata isolated, scattered, irregular and asymmetrical, black. Peridium 13.6–46.4 µm thick, consist of pale to medium brown flattened angular cells. Ascogenous hyphae hyaline, smooth-walled. Asci (43.3–) 44.1–63.3 (–66.5) × (7.6–) 8.0–9.8 (–10) µm (N=7), cylindrical, straight, unitunicate, 8–spored. Ascospores (13.9–) 14.1–18 (–20.9) × (3.1–) 3.9–5.4 (–5.8) µm (N=23), L/W = 3.5, uni-to bi-seriately, aseptate, smooth-walled, ellipsoidal, single guttules in the middle, both ends rounded, hyaline.

Cultures on CMA flat with entire margin. Growth rate: 0.4cm/d, with 20-days for sporulation. With fluffy, light green and white mycelia. Ascomata sometimes growing together with acervuli.

Figure 6. 

Colletotrichum parallelophorum (holotype). A Colony B, C Fruiting body D Setae E–F Ascomata G, J–L Conidiophores I Appressoria M–P Conidia Q–T Asci U–V Ascospore. Scale bars: 50 µm (B), 500 µm (C), 20 µm (D), 100 µm (E), 50 µm (F), 20 µm (G), 10 µm (H–L), 5 µm (M, N), 10 µm (O–V).

Material examined

Thailand, Chiang Rai, Wat Phra That Doi Tung (Temple of Doi Tung Pagodas), the host Dendrobium sp. 1 was collected on 19 December 2013, Collector: Sureeporn Nontachaiyapoom, Natdanai Aewsakul, Xiaoya Ma.

Notes

Strains MFLUCC 14-0077, MFLUCC 14-0079 and MFLUCC 14-0083 had identical sequence data and they formed a single clade with MFLUCC 14-0082 and MFLUCC 14-0085. They are closely related to Colletotrichum excelsum-altitudum and C. tropicicola. MFLUCC 14-0077, MFLUCC 14-0079, MFLUCC 14-0082, MFLUCC 14-0083 and MFLUCC 14-0085 have similar morphological characteristics. Therefore, the five strains are regarded as the same species. There were totally 103bp and 101bp differences between MFLUCC 14-0083 and C. excelsum-altitudum/C. tropicicola respectively (mainly in GAPDH). Blastn researches with four-gene sequences of five strains presented 99% identity with ITS sequence of C. cordylinicola strain LC0886, 80% identity with GAPDH (GenBank JN050229), 90% identity with ACT (GenBank JN050218) and 93% identity with TUB2 (GenBank JN050246) sequences of C. tropicola strain LC0598 respectively as mentioned above. Conidia size and shape were very similar among MFLUCC 14-0083, C. excelsum-altitudum and C. tropicicola. Appressoria were rare and in strain MFLUCC–14–0083 appressoria were not variable like that in C. excelsum-altitudum and C. tropicicola. Here we introduced strains MFLUCC 14-0077, MFLUCC 14-0079, MFLUCC 14-0082 and MFLUCC 14-0083 and MFLUCC–14–0085 isolated from stems and roots of Dendrobium sp. 1 as Colletotrichum parallelophorum sp.nov.

Colletotrichum citricola F. Huang, L. Cai, K.D. Hyde & H.Y. Li

Figure 7

Description

Asexual morph on CMA. Vegetative hyphae 3.1 ± 1.1 µm diam (N=20), smooth-walled, septate, branched, hyaline. Chlamydospores globose, hyaline. Conidiomata ovoid, orange. Setae (51.8–) 54.1–67.8 (–68.5) × (2.3–) 2.4–5.8 (–7.2) µm (N=6), smooth-walled, 1 or 3–septate, contracted to slightly inflated base, tapering to the apex, apex acute, pale brown to brown. Conidiophores (10.8–) 16.7–25.6 (–30.6) × (3.1–) 4–5.3 (–5.6) µm (N=27), smooth-walled, septate, hyaline. Conidia (12.5–) 13.4–15 (16.5–) × (5–) 5.9–6.9 (–7.2) µm (N=40), L/W = 2.2, ellipsoidal, smooth-walled, hyaline.

Sexual morph on CMA. Ascomata (34.5–) 46.4–84.9 (–87.1) × (31.7–) 33.8–46.5 (–50.9) µm (N=5), globose, ostiolate, clustered, pale brown to dark brown. Peridium 1.7–5.8 µm thick, composed of pale to medium brown, flattened, angular cells. Ascogenous hyphae hyaline, smooth-walled. Asci (41.3–) 49.4–65 (–71.6) × (8.3–) 9.5–12.9 (–14.3) µm (N=36), cylindrical, unitunicate, straight or curved, 8–spored. Ascospores (14.4–) 14.8–17.5 (–19.3) × (5.4–) 5.7–7.1 (–7.6) µm (N=25), L/W = 2.5, uni-or bi-seriately, smooth-walled, hyaline, fusiform or one end slightly rounded, with a single guttule in the middle.

Cultures on CMA flat with entire margin. Growth rate: 0.6cm/day, with18-days for sporulation. Fluffy, pale mycelia float on the dark scarlet pigment medium, reverse dark brown. Perithecia gregarious. Orange acervuli and ascomata in mass form thick globules.

Figure 7. 

Colletotrichum citricola. A Colony B Conidiomata C Fruiting bodies D Fruiting body with setae E Setae F Ascomata G–L Conidiophores M, N Conidia O Chlamydospore P–U Young asci V–X Ascospores. Scale bars: 500 µm (B), 200 µm (C), 10 µm (E–F), 5 µm (G), 5 µm (M–N), 10 µm (Q–U), 5 µm (V–X).

Notes

Strains MFLUCC 14-0129 and MFLUCC 14-0131 had similar sequence data, cultures and conidia. There were 5bp and 7bp difference between the strains and Colletotrichum camelliae-japonicae and C. citricola respectively. ITS sequence is 99% identity with unpublished C. boninense strain LD3–8–1 isolated from strawberry in China (Unpublished). Blastn searches sequences results in GAPDH (GenBank KC293736) and TUB2 (GenBank KC293656) sequences of C. citricola strain SXC 151 as mention above. ACT sequence is closest to C. karstii strain 42a (GenBank KT122921) isolated from Coffea arabica in Mexico (Cristobal-Martinez et al. 2016). All morphological characteristics of the two strains were nearly the same as the protologue of C. citricola. Therefore, we name strains MFLUCC 14-0129 and MFLUCC 14-0131 as C. citricola. When compared with C. camelliae-japonicae (conidia: 11–14.5 ×5–6.5μm, mean ±SD = 12.5 ±0.8 ×5.5 ±0.3μm, L/W=1.5; ascospores: 13.5–18.5 ×4–5.5 μm, mean ± SD = 16.5 ±1.1×5±0.4μm, L/W = 3.3), strains MFLUCC 14-0129 and MFLUCC 14-0131 have shorter conidia and wider ascospores.

Colletotrichum fructicola Prihastuti, L. Cai & K.D. Hyde

Figure 8

Description

Asexual morph formed on CMA. Vegetative hyphae 2.6–5 µm diam (N=20), smooth-walled, septate, branched, hyaline. Appressoria and chlamydospores not observed. Conidiomata 500 × 400 µm (N=3), clustered, sub-globose, smooth-walled, orange. Conidiophores rare, septate, hyaline. Conidia (12.8–) 13.8–16.6 (–18.6) × (2.7–) 3.5–7.8 (–16) µm (N=21), L/W = 2.9, ellipsoidal, smooth-walled, septate, hyaline.

Sexual morph forming on CMA. Ascomata globose, pale brown to dark brown. Peridium (131.9–) 138.4–163.6 (–171.5) × (120.9–) 123.6–142.1 (–143.2) µm (N=4), composed of medium brown, flattened, angular cells. Setae (53–) 57.2–73.1 (–83.3) × (3.4–) 3.5–4(–4.1) µm (N=6), grow on the fruiting body, 2-septate, smooth-walled, contracted at the base, apex slightly rounded, brown to dark brown. Asci (57.6–) 61.2–82.6 (–94.3) × (8.7–) 9.3–13.3 (–15.8) µm (N=12), cylindrical, unitunicate, 8–spored. Ascospores (10–) 12–20 (–20.9) × (3.6–) 4.1–5.2 (–5.3) µm (N=10), L/W = 3.4, ellipsoidal to reniform, somewhat fusiform or acute both ends, 1 to 4 guttules, uni-to bi-seriate, smooth-walled, hyaline.

Cultures on CMA flat with slight serrated margin. Growth rate: 0.9cm/day, with 14-days for sporulation. Cottony, light brown to white from middle to the margin, reverse white to light brown with black spots. Ascomata gregarious and/or isolated. Acervuli and ascomata sometimes gregarious.

Figure 8. 

Colletotrichum fructicola. A Colony B Conidiomata and ascomata C, D Conidiomata E, F Ascomata G–J Conidiophores L Setae M–Q Asci R–V Ascospores Scale bars: 500 µm (B–D), 20 µm (E, F), 5 µm (G–J), 10 µm (L), 10 µm (M–Q), 5 µm (R–V).

Notes

Strains MFLUCC 14-0087, MFLUCC 14-0148 and MFLUCC 15-0262 had the identical sequences to Colletotrichum fructicola. The ITS and GAPDH sequences of them 100% match with many different unpublished species. Blastn researches with ACT sequence of them results in 99% identity with the ex-holotype culture of C. fructicola strain ICMP 18581 (GenBank JX009501) isolated from Coffea arabica in Thailand (Weir et al. 2012), which we involved it in phylogenetic analysis. TUB2 sequences of them are 99% identity with C. boninense strain CBS 125502 (GenBank KJ955336) as mentioned above. Their ascomata, conidia, asci and ascospores were also similar. Conidia were the same size as the ex-type strain of the pathogen Colletotrichum fructicola (9.7–14 × 3–4.3µm) found in coffee berries (Prihastuti et al. 2009). However, ascomata were much smaller and asci as well as ascospores were much larger than the ex-type from coffee berries. In the protologue, C. fructicola was introduced with ascomata as 345.67 ± 36.83 × 431.33 ± 69.89 μm, asci as 41.22 ± 7.02 × 7.61 ± 0.58 μm and ascospores as 11.91 ± 1.38 × 3.32 ± 0.35 μm. Here we name strains MFLUCC 14-0087, MFLUCC 14-0148 and MFLUCC 15-0262 isolated from leaves of Dendrobium sp. 1 and Dendrobium sp. 3, root of Dendrobium sp. 2 as Colletotrichum fructicola.

Colletotrichum jiangxiense F. Liu & L. Cai

Figure 9

Description

Sexual morph not observed.

Sexual morph not observed. Asexual morph on PDA. Vegetative hyphae 1.3–2.1 µm diam (N=20), smooth-walled, septate, branched, hyaline. Setae and chlamydospores not observed. Conidiophores (12.7–) 13.5–21.4 (–23.4) × (1.9–) 2–3 (–3.2) µm (N=8), branched, hyaline. Conidia (8.6–) 9–12.4 (–13.2) × (3.5–) 3.6–4.4 (–4.5) µm (N=4), L/W = 2.6, ellipsoidal to cylindrical, smooth-walled, aseptate, one end more blunt than the other end, hyaline.

Cultures on PDA flat with entire margin. Growth rate: 0.4cm/day, with 18-days for sporulation. Aerial mycelia dense, cottony, pale to light brown, with brown outline ring close to the edge, mycelia in the middle dark brown, reverse white to light brown.

Figure 9. 

Colletotrichum jiangxiense. A Colony B Colony from below C–F Conidiophores G–H Conidia. Scale bars: 5 µm (C–F), 2.5 µm (G–H).

Notes

Strains MFLUCC 14-0091 and MFLUCC 14-0092 were the same species as they grouped with high support (98ML/1.0BI/87MP). They formed a very close clade with the pathogen C. jiangxiense isolated from Camellia. However, different media were used in these two studies. Blastn researches with ITS sequences results in 100% identity with C. gloeosporioides strain SS1-MS1 (GenBank KP900279) isolated from Huperzia serrate in China (Wang et al. 2016). GAPDH, ACT and TUB2 sequences of MFLUCC 14-0091 and MFLUCC 14-0092 are closest to C. kahawae subsp. ciggaro strain ICMP 18534 (GenBank JX009904) with 98% identity isolated from Kunzea ericoides in New Zealand, 99% identity with strain ICMP 12952 (GenBank JX009431) isolated from Persea Americana in New Zealand, and 99% identity with strain CO22-1 (GenBank KJ001124) isolated from Rubus glaucus in Colombia respectively (Weir et al. 2012; Afanador-Kafuri et al. 2014). Conidia size reported for C. jiangxiense was 15.2 ± 1 × 5.2 ± 0.4 μm, which was larger and faster growing than the strains isolated in this study. There were 5bp differences between strain MFLUCC 14-0091 and C. jiangxiense. Here we name both of isolates from leaves of Dendrobium sp. 1 as C. jiangxiense.

Colletotrichum orchidophilum Damm, P.F. Cannon & Crous

Figure 10

Description

Sexual morph not observed.

Sexual morph not observed. Asexual morph on SDA. Vegetative hyphae 1.9–5.4 µm diam, smooth-walled, septate, branched, hyaline to pale brown. Chlamydospores not observed. Appressoria brown, smooth-walled. Conidiomata superficial or under mycelia, smooth-walled, 200 × 300 µm, black. Conidiophores smooth-walled, branched or unbranched, hyaline. Conidiophores and appressoria rare. Conidia (9.6–) 11.7–14.1 (–14.7) × (2.9–) 3.5–4.4 (–4.8) µm, L/W = 3.3, cylindrical, straight, with 1 to 4 guttules, one end somewhat acute, hyaline.

Cultures on SDA flat with entire margin. Growth rate: 0.44cm/day, with nearly 20-days for sporulation. White with dark green mycelia around the middle, white edge, reverse white. Cultures on PDA flat with entire margin. Growth rate: 0.45cm/day, with 30-days for sporulation. Fluffy, white, reverse light brown. Acervuli in mass black, irregular, asymmetrical, merging in media.

Figure 10. 

Colletotrichum orchidophilum. A Colony B Fruiting body C–D Conidiophores E Appressoria F–K Conidia. Scale bars: 200 µm (B), 5 µm (F–K).

Notes

Strains MFLUCC–14–0161 and MFLUCC–14–0162 belong to a single species as they have similar conidia, cultures and the nearly identical sequence data. The support values of 100/1.00/100 totally grouped them with C. orchidophilum and their branch lengths are slightly different. Blastn researches sequences of MFLUCC 14-0161 and MFLUCC 14-0162 results in 99% identity with ITS (GenBank NR111729), GAPDH (GenBank JQ948481) and ACT (GenBank JQ949472) sequences of ex-holotype culture of C. orchidophilum strain CBS 632.80 isolated form Dendrobium sp. in USA (Damm et al. 2012). TUB2 sequence is 99% identity with pathogenic C. fructicola strain AV24 (GenBank KX786459) isolated from grapevine shoots in Brazil (Santos et al. 2018) and C. gloeosporioides strain TL-2 (GenBank KC913205) isolated from Camellia sinensis in China (Guo et al. 2014). Because no conidiophores were detected in culture, no measurement for the conidiophores could be given. In this study, strains MFLUCC 14-0161 and MFLUCC 14-0162 of C. orchidophilum were isolated from leaves of D. harveyanum.

Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib.

For an illustrated description please refer Damm et al. (2012a).

Notes

Strains MFLUCC 14-0086, MFLUCC 14-0124 and MFLUCC 15-0261 grouped with C. boninense and MFLUCC 14-0128. All have very similar sequences as those as the ex-type of with C. boninense (only 2bp difference), while there was 11 base pair deviations between these strains and Colletotrichum doitungense sp. nov. Blastn researches with ITS sequences of them result in 100% identity with ITS sequence of endophytic C. boninense strain SL-ML18 (GenBank KP900269) isolated from Huperzia serrate in China (Wang et al. 2016) and strain CGMCC 3.15168 (GenBank KC244158) as mentioned above. GAPDH and ACT sequences of them are 97% identity with C. boninense CGMCC 3.15168 (GenBank KC843491) and 100% identity with C. fructicola strain 1104-7 (GenBank KX885159) isoalted from Malus domestica in China (Liang et al. 2017). TUB2 blastn result are 99% identity with C. fructicola strain AV24 (GenBank KX786459) and C. gloeosporioides strain TL-2 (GenBank KC913205) as mentioned above. Here we identify these three strains isolated from leaves of D. catenatum and Dendrobium sp. 1, stem of D. sp. 2 respectively as Colletotrichum boninense.

Colletotrichum sp. indet

Notes

Strain MFLUCC 14-0120 failed to sporulate and lacks a complete morphological description. It formed a single branch close to C. camelliae-japonicae, MFLUCC 14-0129 / MFLUCC 14-0131 with 67ML/1.00BI/62MP support. There were 15bp and 11bp differences mainly in the ACT gene region among MFLUCC 14-0120 and C. camelliae-japonicae, MFLUCC 14-0129/MFLUCC 14-0131 respectively. ITS sequence blastn of MFLUCC 14-0120 showed many different kinds of species with 99% identity. Blastn searches with GAPDH (GenBank KC293736) and TUB2 (GenBank KC293656) sequences result in 99% identity with C. citricola strain SCX 151 as mentioned above. The ACT of MFLUCC–14–0120 is 98% identity with C. boninense strain CBS 125502 (GenBank KJ954462) as mentioned above. Here we listed it as an unidentified species.

Discussion

Colletotrichum species associated with orchid species

Many Colletotrichum species have been isolated from Orchidaceae plants sampled in China in previous studies (e.g. Yang et al. 2011; Chen et al. 2012; Tao et al. 2008, 2013). Eighteen Colletotrichum species have been reported from these studies. For example, Colletotrichum beeveri isolated from Pleione bulbocodioides; C. bletillum and C. caudasporum isolated from Bletilla ochracea; C. oncidii isolated from Oncidium sp. (Yang et al. 2011; Damm et al. 2012a; Tao et al. 2013). The present study is the first to report endophytic fungi from Dendrobium spp. in Thailand combining both multi-loci sequence data and morphological characteristics. Colletotrichum species in this study were diverse and present in every Dendrobium sample collected from all sites. Therefore, we conclude that Orchidaceae plants are rich source of endophytic Colletotrichum species.

Methods affecting the identification

Hyde and Zhang (2008) and Hyde et al. (2009b) suggested that nucleotide sequence data of holotypes or epitypes is essential for analysing phylogenetic relationships among Colletotrichum species. A polyphasic method combining morphological characteristics and molecular phylogenetics has been applied to define and re-order species in this genus (Cai et al. 2009; Hyde et al. 2009; Damm et al. 2012a, b, c; Jayawardena et al. 2016a, b).

We found some differences in the Colletotrichum gloeosporioides species complex backbone tree as compared to that constructed with more genes in Weir et al (2012), Udayanga et al (2013) and Jayawardena et al. (2016a). Colletotrichum jiangxiense clusters with C. rhexiae rather than C. kahawae. C. fructicola is closer to C. siamense rather than C. nupharicola. The genes CHS-1 and HIS3 were not involved in this study and may be responsible for the differences. Actually CHS-1 and HIS3 could resolve species in sevaral other species complexes of Colletotrichum (Jayawardena et al. 2016a). However, the combination of ApMat and GS turned out to be the most effective genes in species resolution in the Colletotrichum gloeosporioides species complex (Liu et al. 2015). Our study is the first to use multiple gene sequences to analyse fungal endophytes from Dendrobium orchids.

Relationship between Colletotrichum and Dendrobium

Few species identified in this study showed host-specificity. Nevertheless, this study provides evidence that C. orchidophilum colonizes a wide range of hosts in Orchidaceae (Damm et al. 2012b). In addition, we found that leaves contained higher numbers of Colletotrichum species (11 strains from leaves) than other parts (4 strains from roots and 7 strains from stems). All Dendrobium leaves in this study were colonized by Colletotrichum strains. Our results are similar to those of Chen et al. (2011) who isolated more Colletotrichum species from stems and leaves of Dendrobium species than that from roots.

The majority of Colletotrichum species isolated from Dendrobium species in this study were fungal endophytes. This was also reported by Chen et al. (2011) and Yuan et al. (2009). The most common fungal endophytes in leaves of Lepanthes rupestris (Orchidaceae) sampled in a Puerto Rican forest were a Colletotrichum species which showed antagonism against other fungal taxa (Bayman et al. 2002). Most Colletotrichum species have been identified as plant pathogens living a hemibiotrophic life strategy, they adopt a biotrophic phase at an early stage and switch to a necrotrophic phase later (Damm et al. 2010; Cannon et al. 2012).

Here we speculate that most isolates in this study might be latent pathogens (Photita et al. 2004), since in the phylogenies, they were nested with pathogenic strains or have previously been reported to cause plant diseases (Tao et al. 2013, Hou et al 2016). Colletotrichum jiangxiense was isolated as a pathogen from leaf lesions of Camellia sp. (Liu et al. 2015). Colletotrichum boninense was reported as an anthracnose causing agent from Dendrobium kingianum in Japan (Moriwaki et al. 2003).

Acknowledgements

This work was funded by the grants of National Natural Science Foundation of China (Grants Nos. 31670027 & 31460011 & 30870009) and the Agricultural Science and Technology Foundation of Guizhou Province, China (Grant No. NY[2013]3042). We sincerely acknowledge great help from Santi Watthana on identification of orchids collected in Thailand.

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Appendix A

Fungal isolates and sequences of region/genes used in Colletotrichum phylogenetic analysis.

Species Isolatea GenBank accession number
ITS GAPDH ACT ß–tubulin
C. acutatum CBS 128531* JQ005776 JQ948677 JQ005839 JQ005860
C. aeschynomenes CBS 128532* JX010176 JX009930 JX009483 JX010392
C. alcornii CBS 128534* JX076858
C. alienum ICMP 12071* JX010251 JX010028 JX009572 JX010411
C. annellatum CBS 128536* JQ005222 JQ005309 JQ005570 JQ005656
C. anthrisci CBS 125334* GU227845 GU228237 GU227943 GU228139
C. aotearoa CBS 128538* JX010205 JX010005 JX009564 JX010420
C. arxii CBS 132511* NR132055 KF687843 KF687802 KF687881
C. australe CBS 128540* JQ948455 JQ948786 JQ949776 JQ950106
C. beeveri CBS 128541* JQ005171 JQ005258 JQ005519 JQ005605
C. bidentis CBS 128542* KF178481 KF178506 KF178578 KF178602
C. bletillum CBS 128543* JX625178 KC843506 KC843542 JX625207
C. boninense CBS 123755* JQ005153 JQ005240 JQ005501 JQ005588
C. brasiliense CBS 128545* JQ005235 JQ005322 JQ005583 JQ005669
C. brassicola CBS 128546* JQ005172 JQ005259 JQ005520 JQ005606
C. brevisporum CBS 128547* JQ247623 JQ247599 JQ247647 JQ247635
C. camelliae ICMP 10643 JX010224 JX009908 JX009540 JX010436
C. camelliae-japonicae CGMCC3.18117* KX853165 KX893583 KX893575 KX893579
C. caudasporum CGMCC 3.15106* JX625162 KC843512 KC843526 JX625190
C. cereale CBS 129663 JQ005774 JQ005837 JQ005858
C. chlorophyti IMI 103806* GU227894 GU228286 GU227992 GU228188
C. chrysanthemi IMI 364540 JQ948273 JQ948603 JQ949594 JQ949924
C. citricola SXC 151* KC293576 KC293736 KC293616 KC293656
C. clidemiae ICMP 18658* JX010265 JX009989 JX009537 JX010438
C. cliviae CBS 125375* JX519223 GQ856756 JX519240 JX519249
C. coccodes CBS 369.75 JQ005775 HM171673 JQ005838 JQ005859
C. colombiense CBS 129818* JQ005174 JQ005261 JQ005522 JQ005608
C. cordylinicola ICMP 18579* JX010226 JX009975 HM470234 JX010440
C. curcumae IMI 288937* GU227893 GU228285 GU227991 GU228187
C. cymbidiicola IMI 347923* JQ005166 JQ005253 JQ005514 JQ005600
C. dematium CBS 125.25* GU227819 GU228211 GU227917 GU228113
C. dracaenophilum CBS 118199* JX519222 JX519238 JX519247
C. echinochloae MAFF 511473* AB439811
C. eleusines MAFF 511155* JX519218 JX519234 JX519243
C. endophytum CGMCC 3.15108* JX625177 KC843521 KC843533 JX625206
C. eremochloae CBS 129661* CBS 129661 JX519236 JX519245
C. excelsum-altitudum CGMCC 3.15130* HM751815 KC843502 KC843548 JX625211
C. falcatum CBS 147945* JQ005772 JQ005835 JQ005856
C. fioriniae CBS 128517* JQ948292 JQ948622 JQ949613 JQ949943
C. fructi CBS 346.37* GU227844 GU228236 GU227942 GU228138
C. fructicola ICMP 18581* JX010165 JX010033 FJ907426 JX010405
C. fructivorum Coll1414* JX145145 JX145196
C. fusiforme MFLU 130291* NR138010 KT290255 KT290251 KT290256
C. gigasporum MUCL 44947* AM982797 FN557442
C. godetiae CBS 133.44* JQ948402 JQ948733 JQ949723 JQ950053
C. graminicola CBS 130836* JQ005767 JQ005830 JQ005851
C. grevilleae CBS 132879* KC297078 KC297010 KC296941 KC297102
C. guizhouensis CGMCC 3.15112* JX625158 KC843507 KC843536 JX625185
C. hanaui MAFF 305404* JX519217 JX519242
C. henanense LF238* KJ955109 KJ954810 KJ955257
C. hippeastri CBS 125376* JQ005231 JQ005318 JQ005579 JQ005665
C. hemerocallidis CDLG5* JQ400005 JQ400012 JQ399991 JQ400019
C. horii ICMP 10492 GQ329690 GQ329681 JX009438 JX010450
C. incanum ATCC 64682* KC110789 KC110807 KC110825 KC110816
C. jasminigenum MFU 10–0273* HM131513 HM131499 HM131508 HM153770
C. jiangxiense LF 488* KJ955149 KJ954850 KJ954427
C. kahawae ICMP 17816* JX010231 JX010012 JX009452 JX010444
C. kartsii CORCG 6* HM585409 HM585391 HM581995 HM585428
C. laticiphilum CBS 112989* JQ948289 JQ948619 JQ949610 JQ949940
C. lilii CBS 109214 GU227810 GU228202 GU227908 GU228104
C. lindemuthianum CBS 144.31* JQ005779 JX546712 JQ005842 JQ005863
C. linicola CBS 172.51 JQ005765 JQ949476 JQ949806
C. liriopes CBS 119444* GU227804 GU228196 GU227902 GU228098
C. magnisporum CBS 398.84 KF687718 KF687842 KF687803 KF687882
C. malvarum CBS 527.97* KF178480 KF178504 KF178577 KF178601
C. menispermi MFLU 14–0625* KU242357 KU242356 KU242353 KU242354
C. miscanthi MAFF 510857* JX519221 JX519237 JX519246
C. musae ICMP 19119* JX010146 JX010050 JX009433 HQ596280
C. navitas CBS 125086* JQ005769 JQ005832 JQ005853
C. nicholsonii MAFF 511115* JQ005770 JQ005833 JQ005854
C. novae-zelandiae CBS 128505* JQ005228 JQ005315 JQ005576 JQ005662
C. nupharicola ICMP 18187* JX010187 JX009972 JX009437 JX010398
C. ochraceae CGMCC 3.15104* JX625156 KC843513 KC843527 JX625183
C. oncidii CBS 129828* JQ005169 JQ005256 JQ005517 JQ005603
C. orchidophilum CBS 632.80* JQ948151 JQ948481 JQ949472 JQ949802
C. parsonsiae CBS 128525* JQ005233 JQ005320 JQ005581 JQ005667
C. paspali MAFF 305403* JX519219 JX519235 JX519244
C. petchii CBS 378.94* JQ005223 JQ005310 JQ005571 JQ005657
C. phaseolorum CBS 157.36 GU227896 GU228288 GU227994 GU228190
C. phyllanthi CBS 175.67* JQ005221 JQ005308 JQ005569 JQ005655
C. pseudoacutatum CBS 436.77* JQ948480 JQ948811 JQ949801 JQ950131
C. pseudomajus CBS 571.88* NR132059 KF687826 KF687801 KF687883
C. psidii ICMP 19120 JX010219 JX009967 JX009515 JX010443
C. radicis CBS 529.93* NR132057 KF687825 KF687785 KF687869
C. rhexiae Coll 1026* JX145128 JX145179
C. rhombiforme CBS 129953* JQ948457 JQ948788 JQ949778 JQ950108
C. riograndense COAD 928* KM655299 KM655298 KM655295 KM655300
C. rusci CBS 119206* GU227818 GU228210 GU227916 GU228112
C. salsolae ICMP 19051* JX010242 JX009916 JX009562 JX010403
C. siamense ICMP 18578* JX010171 JX009924 FJ907423 JX010404
C. sichuanensis LJTJ3 KP748193 KP823773 KP823738 KP823850
C. spinaciae CBS 128.57 GU227847 GU228239 GU227945 GU228141
C. sublineola CBS 131301* JQ005771 JQ005835 JQ005855
C. syzygicola DNCL 021* KF242094 KF242156 KF157801 KF254880
C. tanaceti CBS 132693* JX218243 JX218238 JX218233
C. tebeestii CBS 522.97* KF178473 KF178505 KF178570 KF178594
C. temperatum Coll 883* JX145159 JX145211
C. theobromicola ICMP 18649 JX010294 JX010006 JX009444 JX010447
C. ti ICMP 4832* JX010269 JX009952 JX009520 JX010442
C. torulosum CBS 128544* JQ005164 JQ005251 JQ005512 JQ005598
C. trichellum CBS 217.64* GU227812 GU228204 GU227910 GU228106
C. trifolii CBS 158.83* KF178478 KF178502 KF178575 KF178599
C. tropicicola BCC 38877* JN050240 JN050229 JN050218 JN050246
C. trucatum CBS 151.35 GU227862 GU228254 GU227960 GU228156
C. verruculosm IMI 45525* GU227806 GU228198 GU227904 GU228100
C. vietnamense CBS 125478* KF687721 KF687832 KF687792 KF687877
C. viniferum GZAAS 5.08601* JN412804 JN412798 JN412795 JN412813
C.$1×anthorrhoeae ICMP 17903* JX010261 JX009927 JX009478 JX010448
C. yunnanense CGMCC AS3.9167* EF369490 JX519239 JX519248
Australiasca queenslandica BRIP 24607 HM237327
Monilochaetes guadalcanalensis CBS 346.76 GU180625
Monilochaetes infuscans CBS 869.96 JQ005780 JX546612 JQ005843 JQ005864

Appendix B

Fungal isolates and sequences of region/genes in this study.

Species Isolate GenBank accession number
ITS GAPDH ACT ß–tubulin
C. boninense MFLU 14-0124 MG792809 MK165700 MH351286
MFLU 14-0086 MG792816 MH673668 MH376390 MH351281
MFLU 14-0261 MG792815 MK165703 MH376400 MH351292
C. cariniferi MFLU 14-0100 MF448521 MH351274
C. chiangraiense MFLU 14-0119 MF448522 MH376383 MH351275
C. citricola MFLU 14-0129 MG792821 MK165697 MH376395 MH351287
MFLU 14-0131 MG792822 MK165696 MH376396 MH351288
C. doitungense MFLU 14-0128 MF448524 MH049480 MH376385 MH351277
C. fructicola MFLU 14-0087 MG792812 MK165691 MH376391 MH351282
MFLU 14-0262 MG792814 MK165698 MH376401 MH351293
MFLU 14-0148 MG792813 MK165701 MH376397 MH351289
C. jiangxiense MFLU 14-0091 MG792806 MH673669 MH376392 MH351283
MFLU 14-0092 MG792807 MH673670 MH376393 MH351284
C. orchidophilum MFLU 14-0161 MG792818 MK165702 MH376398 MH351290
MFLU 14-0162 MG792819 MK165704 MH376399 MH351291
C. parallelophorum MFLU 14-0085 MF448525 MK165695 MH351280
MFLU 14-0077 MG792808 MK165692 MH376387 MH351279
MFLU 14-0079 MG792820 MK165693 MH376388
MFLU 14-0082 MG792810 MK165694 MH376389
MFLU 14-0083 MG792811 MH049478 MH376386 MH351278
C. sp. indet. MFLU 14-0120 MG792817 MK165699 MH376394 MH351285
C. watphraense MFLU 14-0123 MF448523 MH049479 MH376384 MH351276