Research Article |
Corresponding author: Samantha C. Karunarathna ( samanthakarunarathna@gmail.com ) Academic editor: Thorsten Lumbsch
© 2018 Saowaluck Tibpromma, Kevin D. Hyde, Jayarama D. Bhat, Peter E. Mortimer, Jianchu Xu, Itthayakorn Promputtha, Mingkwan Doilom, Jun-Bo Yang, Alvin M. C. Tang, Samantha C. Karunarathna.
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Citation:
Tibpromma S, Hyde K, Bhat J, Mortimer P, Xu J, Promputtha I, Doilom M, Yang J, Tang A, Karunarathna S (2018) Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand. MycoKeys 33: 25-67. https://doi.org/10.3897/mycokeys.33.23670
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The authors established the taxonomic status of endophytic fungi associated with leaves of Pandanaceae collected from southern Thailand. Morphotypes were initially identified based on their characteristics in culture and species level identification was done based on both morphological characteristics and phylogenetic analyses of DNA sequence data. Twenty-two isolates from healthy leaves were categorised into eight morphotypes. Appropriate universal primers were used to amplify specific gene regions and phylogenetic analyses were performed to identify these endophytes and established relationships with extant fungi. The authors identified both ascomycete and basidiomycete species, including one new genus, seven new species and nine known species. Morphological descriptions, colour plates and phylogenies are given for each taxon.
Ascomycetes , Basidiomycota , biodiversity, phylogenetic analysis
Endophytic fungi are beneficial to their host plants and have the ability to produce bioactive compounds that have applied uses (
Endophytic fungal strains have been isolated from many different plants including trees, vegetables, fruits, cereal grains and other crops (
The authors have been investigating saprobic and endophytic fungi associated with Pandanaceae (
The objectives of the present study were to establish the endophytic fungal community on selected Pandanaceae collected in southern Thailand. The authors isolated 22 endophytic isolates and sorted them in morphotypes and identified the taxa based on DNA sequence analyses. Both ascomycete and basidiomycete genera were identified, including one new genus, seven new species and nine known species. The recommendations of
Healthy mature leaves of Pandanus and Freycinetia species (Pandanaceae, Figure
The above methods resulted in 22 isolates which were separated into morphotypes based on visual assessment of the similarity of the cultures (
Gene/Loci | PCR primers (Forward/Reverse) | References |
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LSU | LROR/LR5 |
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ITS | ITS5/ITS4 |
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SSU | NS1/NS4 |
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TEF1 | 983F/2218 |
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728F/986R |
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RPB2 | fRPB2-5f/fRPB2–7cR |
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β-tubulin | BT2a/BT2b |
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T1/T2 |
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Actin | 512F/783R |
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CHS-1 | 79F/354R |
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GADPH | Gpd1/Gpd2 |
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GDF/GDR |
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Genomic DNA was extracted from pure fungal cultures using Biospin Fungal Genomic DNA extraction Kit–BSC14S1 (BioFlux, P.R. China). Polymerase chain reaction (PCR) was used to amplify partial gene regions of Internal Transcribed Spacers (ITS), 28S ribosomal RNA (LSU), 18S ribosomal RNA (SSU), RNA polymerase II second largest subunit (RPB2), β-tubulin (Tub2), Actin (ACT), Glyceraldehyde-3- Phosphate Dehydrogenase (GADPH), Chitin synthase 1 (CHS-1) and Translation Elongation Factor 1-alpha (TEF1) using primers as shown in Table
The sequence data generated during this study were the subject of BLAST searches in the nucleotide database of GenBank (www http://blast.ncbi.nlm.nih.gov/) to determine their most probable closely related taxa. Sequence data were retrieved from GenBank based on recent publications. Raw forward and reverse sequences were assembled using Geneious Pro.v4.8.5. Sequence alignments were carried out with MAFFT v.6.864b (
Twenty-two fungal isolates from Pandanus and Freycinetia species were recovered and these mycelia sterilia were separated into eight morphotypes based on the similarity of their culture characteristics, as summarised in Table
Culture characteristics of the 22 strains (8 morphotypes) of mycelia sterilia on PDA.
Morpho-types | Isolate code | Host | Size (cm) of colony | Shape | Colour | Mycelium | Edge | ||
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3 days | 7 days | Above | Reverse | ||||||
1 | PE05 | Pandanus sp. | 4.6 | >A | Circular | 4A1 | 4A2 | Aerial | Undulate |
PE09 | Pandanus sp. | 4.6 | >A | Circular | 6D3 | 6B3 | Aerial | Entire | |
PE15 | Pandanus sp. | >A | >A | Circular | 5B2 | 5B3 | Flat | Entire | |
2 | PE10 | Pandanus sp. | 1.1 | 3.2 | Irregular | 4C1 | 4A3 | Aerial | Undulate |
PE60 | Pandanus sp. | 1.6 | 3.8 | Irregular | 4B1 | 4A3 | Aerial | Undulate | |
FE46 | Freycinetia sp. | 2.1 | 5.6 | Irregular | 5B2 | 5A2 | Aerial | Undulate | |
FE42 | Freycinetia sp. | 1.5 | 5 | Irregular | 4A1 | 4A3 | Aerial | Undulate | |
FE43 | Freycinetia sp. | 1.4 | 4.2 | Irregular | 5D4 | 5C4 | Flat | Undulate | |
PE75 | Pandanus sp. | 1.6 | 5 | Circular | 6A1/6D3 | 6A1/6F5 | Aerial | Undulate | |
PE84 | Pandanus sp. | 1.5 | 3.8 | Circular | 5F4 | 5F7 | Aerial | Curled | |
FE98 | Freycinetia sp. | 1.3 | 3.1 | Irregular | 5B2 | 5D5 | Flat | Filamentous | |
3 | PE25 | Pandanus sp. | >A | >A | Circular | 5E1 | 5F2 | Aerial | Entire |
4 | PE26 | Pandanus sp. | 3.1 | 7.2 | Irregular | 5B3 | 5B5 | Aerial | Undulate |
PE52 | Pandanus sp. | 1.2 | 2.9 | Circular | 5A2 | 5A3 | Aerial | Undulate | |
5 | PE35 | Pandanus sp. | 1.1 | 2.7 | Filamentous | 8E2 | 8F2 | Aerial | Filamentous |
6 | PE92 | Pandanus sp. | 5.1 | >A | Irregular | 4B1 | 4A6 | Aerial | Curled |
PE37 | Pandanus sp. | 2.3 | 7.9 | Circular | 4A1 | 4B3 | Aerial | Curled | |
FE88 | Freycinetia sp. | 2.9 | 6.2 | Circular | 5D3 | 5B2 | Flat | Undulate | |
PE77 | Pandanus sp. | 4.2 | 7.1 | Irregular | 6B1/6E1 | 6B2 | Aerial | Undulate | |
FE41 | Freycinetia sp. | >A | >A | Irregular | 4D2 | 4F6 | Flat | Filamentous | |
7 | PE58 | Pandanus sp. | <B | 1.7 | Circular | 4F2 | 4F8 | Aerial | Entire |
8 | FE101 | Freycinetia sp. | <B | 2 | Circular | 4B2 | 4A3 | Aerial | Entire |
Based on phylogenetic analysis, 22 fungal isolates were identified for 16 species. These include one new genus, seven new species and nine known species. All sequences obtained from this study are summarised in Table
No. | Original code | Species name | Culture collection no. |
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1 | PE26 | Alternaria burnsii |
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2 | PE58 | Cladosporium endophyticum |
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3 | PE09 | Colletotrichum pandanicola |
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4 | FE88 | Colletotrichum fructicola |
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PE84 |
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5 | PE77 | Diaporthe pandanicola |
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6 | PE37 | Diaporthe siamensis |
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7 | FE41 | Endomelanconiopsis freycinetiae |
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8 | FE42 | Endopandanicola thailandica |
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FE43 |
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FE46 |
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PE10 |
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PE60 |
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9 | PE25 | Lasiodiplodia theobromae |
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10 | PE52 | Massarina pandanicola |
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11 | FE98 | Meyerozyma caribbica |
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PE75 |
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12 | FE101 | Mycoleptodiscus endophytica |
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13 | PE05 | Pestalotiopsis jiangxiensis |
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14 | PE92 | Pestalotiopsis microspora |
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15 | PE15 | Phanerochaete chrysosporium |
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16 | PE35 | Phyllosticta capitalensis |
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The family Polyporaceae was introduced by Fr. ex Corda (1839) and includes 92 genera and 636 species (
Named after its habitat as an endophyte of Pandanus.
Endopandanicola thailandica Tibpromma & K.D. Hyde
Colonies on PDA (PE60), superficial, initially white, later becoming yellow-white, smooth at the surface, irregular, with undulate margin, flossy to velvety; reverse white to yellow-white. Generative hyphae simple-septate, branched, sub-hyaline, thin-walled.
Endopandanicola formed a single, well-supported clade (100 % in ML, 100 % in MP), which is distinct as compared to other genera in Polyporaceae (Figure
named after Thailand, the country where the fungus was first discovered.
Colonies on PDA (Figure
THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE60 (
Endopandanicola is introduced and typified by En. thailandica which is represented by six isolates and is described as a novel species based on its asexual morph. The phylogenetic analysis of ITS sequence data showed that this species clustered together with Panus, but there is a high level of statistical support for its separation (100% in ML, 100% in MP) (Figure
Colonies on PDA (Figure
ITS=MG646957.
Burdsall and Eslyn (1974) introduced Phanerochaete chrysosporium which was collected on dead wood of Platanus wrightii in the USA. Phylogenetic analysis of ITS sequence data shows this taxon groups with Phanerochaete chrysosporium (sequences obtained from GenBank) that had been collected from different hosts. The phylogenetic placement of this species is shown in Figure
The order Botryosphaeriales was introduced by
name referring to the host genus on which the fungus was found (Freycinetia).
Colonies on PDA (Figure
THAILAND, Ranong, Muang, on healthy leaves of Freycinetia sp. (Pandanaceae), 3 December 2016, S. Tibpromma FE41 (
Endomelanconiopsis freycinetiae is closely related to the endophytic fungus En. endophytica. Therefore, the culture characteristics of these two taxa were compared and it was found that, in En. endophytica, at first the hyphae are colourless, immersed, later becoming olivaceous in the centre with irregular concentric rings; aerial mycelia are dark olivaceous or grey when dense; shiny black when the aerial mycelia are loose (
Colonies on PDA (Figure
Phyllosticta capitalensis (
Colonies on PDA (Figure
Morphological and phylogenetic data supported placement of this isolate as Lasiodiplodia theobromae. The phylogenetic analysis showed the isolate groups with Lasiodiplodia theobromae. Nucleotide base pairs of published sequences of Lasiodiplodia theobromae (strain EucN188, CBS 111530, PHLO9, CDFA145) were also compared with the sequence and found that the nucleotide base pairs of the ITS gene are 100% similar.
The genus Cladosporium (Cladosporiaceae, Capnodiales) is a large genus of the Ascomycota (
named after its status as an endophytic fungus.
Colonies on PDA attaining 9 cm diam. in six weeks at room temperature, slow growing, dark olivaceous. Mycelium superficial and immersed composed of septate, branched, 2.3–4.5 µm wide, sub-hyaline, with smooth and thick-walled hyphae. Sexual morph Undetermined. Asexual morph Conidiophores 6–10 µm high, 3–4 µm diam. (x̄ = 8.24 × 3.52 µm, n = 10), terminal and intercalary, cylindrical or sub-cylindrical, darkened conidiogenous loci. Conidia 3–6 × 2–4 µm (x̄ = 3.64 × 2.75 µm, n = 30), forming long branched chains, hyaline to pale-olivaceous, smooth and thin-walled, aseptate, globose to ovoid with rounded ends.
Colonies on PDA (Figure
THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE58 (
Cladosporium endophyticum was isolated as an endophyte from Pandanus sp. in Thailand. In the phylogenetic analysis of combined gene sequence data of ITS, LSU, SSU and TEF1, the new taxon Cladosporium endophyticum is sister to C. halotolerans (Figure
The family Massarinaceae was introduced by
name referring to the host genus of the plant on which the fungus was first discovered (Pandanus).
Colonies on PDA attaining 9 cm diam. in 4 weeks at room temperature, slow growing, white to yellow-white. Mycelium superficial and immersed composed of septate, branched, 2.5–7 µm wide, sub-hyaline, with smooth and thick-walled hyphae. Sexual morph Undetermined. Asexual morph Conidiophores 12–25 µm high, 8–14 µm diam. (x̄ = 15.12 × 10.45 µm, n = 10), enteroblastic, phialidic, cylindrical or sub-cylindrical, sub-hyaline. Conidia 3–5 × 1–3 µm (x̄ = 4.34 × 1.75 µm, n = 30), cylindrical, hyaline, smooth and thin-walled, aseptate, rounded ends, guttulate, without sheet or appendages.
Colonies on PDA (Figure
THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE52 (
The genus Massarina has been known as a phylogenetically diverse group in the order Pleosporales based on molecular data (
The family Pleosporaceae was introduced by
Colonies on PDA (Figure
Alternaria burnsii was introduced by
The family Diaporthaceae was introduced by von Höhnel (1917) and was placed in the order Diaporthales. This family comprised two Diaporthe genera (Phomopsis and Mazzantia) (
Name referring to the host genus on which the fungus was first discovered (Pandanus).
Colonies on PDA (Figure
Phylogram generated from maximum likelihood analysis based on ITS, TEF1 and β-tubulin sequenced data. Maximum likelihood (left) and Bayesian inference (right) bootstrap values are given above/below the nodes. The newly generated sequences are in red bold. The tree is rooted with Diaporthe ambigua.
THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE77 (
Diaporthe species are plant pathogens, endophytes or saprobes (
Colonies on PDA (Figure
In the phylogenetic analysis, the authors’ collection grouped with Diaporthe siamensis
The family Glomerellaceae was introduced by Locquin (1984), but was invalidly published. To date, most Glomerellaceae have been recorded to be pathogens (
Colonies on PDA (Figure
The gloeosporioides species complex is mainly plant pathogens (
name referring to the host genus (Freycinetia).
Colonies on PDA attaining 9 cm diam. in 7 days at room temperature, dark-grey. Sexual morph Undetermined. Asexual morph Conidiophores hyaline, smooth-walled, cylindrical to slightly inflated. Conidia 9–18 µm high, 4–8 µm diam. (= 13.39 × 5.35 µm, n = 20), hyaline, cylindrical with rounded ends tapering slightly towards the base, smooth, septate, guttulate.
Colonies on PDA (Figure
THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE09 (
Colletotrichum pandanicola is introduced here as a new species in the gloeosporioides species complex based on morphological and phylogenetic data. The phylogenetic analysis shows that this new taxon is well-separated from other known Colletotrichum species (Figure
Phylogram generated from maximum likelihood analysis based on combined ITS, Actin, β-tubulin, GADPH and CHS-1 sequenced data. Maximum likelihood (left) and Bayesian inference (right) bootstrap values are given above/below the nodes. The newly generated sequences are in red text. The tree is rooted with Colletotrichum truncatum.
The family Magnaporthaceae was introduced by
Named after its original habitat as an endophytic fungus.
Colonies on PDA (Figure
THAILAND, Ranong, Muang, on healthy leaves of Freycinetia sp. (Pandanaceae), 3 December 2016, S. Tibpromma FE101 (
Mycoleptodiscus Ostaz. (1968) belongs to Magnaporthaceae, Magnaporthales. Since 1968, there have been 17 records of Mycoleptodiscus in Index Fungorum. Most of these species were described without molecular data. In this study, a new species Mycoleptodiscus endophyticus is introduced, based on culture characteristics and phylogenetic analysis (100 % in ML). Mycoleptodiscus endophyticus was found as an endophytic fungus on leaves of Freycinetia sp; Mycoleptodiscus freycinetiae Whitton, K.D. Hyde & McKenzie was found as a saprobic fungus on the same host but there was no molecular data available to confirm this identification. The authors were unable to compare the morphological differences between the new taxon and Mycoleptodiscus freycinetiae, because only culture characteristics are presented here for this new taxon (Fig.
Sporocadaceae was introduced by Corda (1842) with Pestalotiopsis-like asexual morphs and confirmed by
Colonies on PDA (Figure
The authors’ collection from Pandanaceae host in Thailand was identified as Pestalotiopsis jiangxiensis. This taxon grouped with Pestalotiopsis jiangxiensis LC4399 which is collected from Eurya sp., with high bootstrap support of 100% in ML.
Colonies on PDA (Figure
Pestalotiopsis microspora was isolated from a Pandanaceae host in Thailand. This strain clusters with Pestalotiopsis microspora DPX3-1 with a strong bootstrap support.
Debaryomycetaceae was introduced by Kurtzman and Suzuki in 2010 and was typified by Debaryomyces Klöcker. Meyerozyma belongs to family Debaryomycetaceae and was detailed in
Colonies on PDA (Figure
Meyerozyma caribbica collected in this study is represented by two endophytic isolates from Pandanaceae. Phylogenetic analysis also supported the identification of this sample as Meyerozyma caribbica.
In this study on fungal endophytes found on leaves of Pandanaceae, it was found that the taxa belonged to both Ascomycota and Basidiomycota. The majority of the taxa were Ascomycota, as found in most previous endophytic studies (
The 22 endophytic fungal strains found in this study were chiefly identified using their microscopic characteristics and DNA sequence data and holotype materials in the form of dried cultures. Future studies are however needed to recollect the taxa which are sporulating to describe sexual and asexual characteristics (sensu
The species composition of endophytic microorganisms is likely to depend on the plant age, genotype, sampled tissue, host type and season of isolation (
We would like to thank Molecular Biology Experimental Centre at Kunming Institute of Botany for their help with sequencing work. Saowaluck Tibpromma thanks the Mushroom Research Foundation (MRF), Chiang Rai, Thailand for financial support. Kevin D. Hyde thanks Mae Fah Luang University for the grant “Biodiversity, phylogeny and role of fungal endophytes of Pandanaceae” (Grant number: 592010200112) and the Chinese Academy of Sciences (project number 2013T2S0030), for the award of Visiting Professorship for Senior International Scientists, at Kunming Institute of Botany and Chiang Mai University. Andrew Stevenson, Fiona Worthy, Sajeewa Maharachchikumbura, Danushka Sandaruwan, Chada Norphanphoun, Asha Dissanayake, Ruvishika Jayawardena and Kasun Thambugala are thanked for their help and valuable suggestions. Samantha C. Karunarathna thanks Yunnan Provincial Department of Human Resources and Social Security funded postdoctoral project (number 179122) and National Science Foundation of China (NSFC) project code 31750110478. Peter E. Mortimer thanks the National Science Foundation of China (NSFC) project codes 41761144055 and 41771063. Fiona Worthy in the World Agroforestry Centre (ICRAF), Kunming Institute of Botany, China is thanked for English editing.