Endophytic Colletotrichum species from Dendrobium spp. in China and Northern Thailand

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, Colletotrichumboninense, 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 Dendrobiumcariniferum and D.harveyanum. Colletotrichumcamelliae-japonicae, C.jiangxiense, and C.orchidophilum are new host records for Thailand.


Introduction
Colletotrichum is the sole genus in family Glomerellaceae (Glomerellales) (Maharachchikumbura et al. 2015(Maharachchikumbura et al. , 2016Jayawardena 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 ), 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;. 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 . 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 et al. 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 mor-phological 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.

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) H 2 O 2 and 70% (v/v) ethanol for 5 minutes, and then rinsed with sterile distilled water for three times. Sterilized materials were cut into 2 mm 2 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

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  (Nylander 2004) were used to estimate the best fitting models according to the Bayesian information criterion (BIC). The GTR model with inverse gamma dis- tribution 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.

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 . The recommendations of Jeewon and Hyde (2016) were followed in establishing new species.

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).

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.

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.

Fungal Name Number: FN570511
Etymology. In reference to the host epithet cariniferum.
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.
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.
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 , 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 ) 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 (Gen-Bank 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. Etymology. In reference to the host sample site Doi tung, Chiang Rai, Thailand.
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.
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 (Gen-Bank 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 . 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 fructicola Prihastuti, L. Cai & K.D. Hyde
Sexual morph forming on CMA. Ascomata globose, pale brown to dark brown. Peridium ( 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. 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 , 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. Figure 9 Description. Sexual morph not observed.
Cultures on PDA flat with entire margin. Growth rate: 0.4cm/day, with 18days 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.
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 . 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 ; 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. Figure 10 Description. Sexual morph not observed.
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.
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 exholotype culture of C. orchidophilum strain CBS 632.80 isolated from Dendrobium sp. in USA ). 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.

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. 2008Tao et al. , 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 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 . 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).
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.