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Article

Morphology and Multi-Gene Phylogeny Reveal Pestalotiopsis pinicola sp. nov. and a New Host Record of Cladosporium anthropophilum from Edible Pine (Pinus armandii) Seeds in Yunnan Province, China

1
College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, Yunnan, China
2
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China
3
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
4
Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Department of Biology, Faculty of Science, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand
*
Authors to whom correspondence should be addressed.
Pathogens 2019, 8(4), 285; https://doi.org/10.3390/pathogens8040285
Received: 17 October 2019 / Revised: 29 November 2019 / Accepted: 30 November 2019 / Published: 4 December 2019
(This article belongs to the Section Plant Pathogens)

Abstract

This study contributes new knowledge on the diversity of conidial fungi in edible pine (Pinus armandii) seeds found in Yunnan Province, China and emphasizes the importance of edible seed products to ensure food safety standards. We isolated two fungal species, one on the pine seed coat and the other on the endosperm of the pine seed. The two fungal species were identified as Pestalotiopsis pinicola sp. nov. and a new host record Cladosporium anthropophilum. Characteristic morphological features of Pestalotiopsis pinicola were used alongside results from multi-gene phylogenetic analysis to distinguish it from currently known species within the genus. Cladosporium anthropophilum was identified as a new host record based on morphological features and phylogenetic analysis. In addition, detailed descriptions, scanned electron microscopy morphology, illustrations, and phylogenetic trees are provided to show the placement of these species.
Keywords: ascomycota; endophytic fungi; new taxon; saprobic fungi; taxonomy; weak pathogen ascomycota; endophytic fungi; new taxon; saprobic fungi; taxonomy; weak pathogen

1. Introduction

Chinese white pine (Pinus armandii), one of the endemic conifer species of East Asia, is known throughout China, and particularly Yunnan Province, for its substantial ecological and economic value [1,2]. Pinus armandii seeds are suitable for use as a culinary ingredient after roasting, because the fatty acid profile of the seeds has a higher level of taxoleic acid and lower levels of octadecenoic acids compared to other species in Pinus [3,4].
Seeds are colonized by various types of fungi including fungal pathogens [5]. Several fungal species exist in seeds in the forms of spores and mycelium and can subsist for long periods of time on the seed coat and in the inner areas [6]. In general, fungi that are present within seeds are more harmful than those that merely contaminate the outer seed coat [6]. Common fungi genera that have been reported as associated with various seeds are Aspergillus, Mucor, Penicillium, Pestalotiopsis, Rhizopus, and Trichoderma [7]. Some studies have confirmed that fungi that are usually thought to be saprobes act as pathogens under certain circumstances, while endophytes can also switch to a saprobic lifestyle [8,9]. Fungal invasions happen after injury to the seed or seed coat as well as when moisture levels and temperatures are favorable for fungal growth [10]. Many seed fungi are also important sources of bioactive compounds [11,12]. In this study, we were able to isolate and identify two micro-fungi belonging to the genera Cladosporium and Pestalotiopsis from the seeds of Pinus armandii.
The genus Cladosporium (Cladosporiaceae, Capnodiales) was introduced by Link [13] with C. herbarum (Pers.) Link as the type species. The members of this genus can be endophytes, pathogens, and saprobes with worldwide distribution across a wide range of disparate substrates [14,15,16,17,18]. Cladosporium species are also known as the most abundant fungi in indoor and outdoor environments and are also important as spoilage organisms and discoloration which have been screened from cereal grains, fruits, peanuts, and chilled meat [19,20,21,22]. While Cladosporium species have not been reported as mycotoxin producers, they may nonetheless represent a health threat. Furthermore, some species have been reported causing fungal allergies, especially in patients with severe asthma [23,24,25,26,27,28,29,30,31]. Recently, several Cladosporium species have been reported in China, Thailand, and the United Kingdom on the decaying seed pods of Delonix regia, Entada phaseoloides, Laburnum anagyroides, and Magnolia grandiflora [32]. Only two species, Cladosporium nigrellum and C. psoraleae, have been reported from Pinus armandii in China [33,34].
The genus Pestalotiopsis (Sporocadaceae, Amphisphaeriales) was introduced by Steyaert [35] with P. guepinii (Desm.) Steyaert as the type species. The members of this genus can be found worldwide as endophytes, saprobes or opportunistic pathogens [18,36,37,38,39,40,41,42,43,44]. Some of them are confirmed to cause human and animal diseases [42,45,46]. For example, Pestalotiopsis spp. have been isolated from a bronchial biopsy, corneal abrasions, eyes, feet, fingernails, scalp, and sinuses from the human body [45]. In addition, this genus is known as one of the common fungi genera reported on various seeds [7]. Pestalotiopsis algeriensis, P. carveri, P. caudata, P. cocculi, P. disseminate, P. heterocornis, P. lespedezae, P. neglecta, P. olivacea, and P. vismiae have been reported from Pinus armandii in China [33,34,38,47].
In the present study, we used multi-gene sequence analysis, morphological examinations, and culture characteristics for the identification and delimitation of fungi isolates belonging to the genera Cladosporium and Pestalotiopsis from seeds of Pinus armandii collected in Yunnan Province, China.

2. Materials and Methods

2.1. Sample Collection and Specimen Examination

Fresh fungal structures (mycelia and spore masses) were directly isolated in potato dextrose agar (PDA) from seed coats and endosperms of Pinus armandii seeds using aseptic techniques, and the PDA plates were incubated at room temperature. Pine seeds were obtained outside Kunming, Yunnan Province, China (Figure 1). The seeds were then carefully analyzed. Morphological structures of the fungi were examined under a stereo microscope. Scanning electron microscopy (SEM) micrographs were obtained under a ZEISS GeminiSEM and ZEISS Sigma 300 apparatus, following the methods described by Figueras and Guarro [48]. To observe the fungal structures, sporulated cultures were mounted on water. Microscopic fungal structures were observed under a compound microscope and photographs were captured with a digital camera fitted on to the microscope. All microscopic structures of fungi were measured by the Tarosoft Image Framework program v.0.9.0.7., and Adobe Photoshop CS3 Extended version 10.0 (Adobe Systems, USA) was used to process and edit the images used in the figures.

Isolation

The PDA medium was used for culturing the isolated fungi. Spore masses from the seed coat and mycelia from the endosperm were aseptically transferred to PDA plates (two isolates of each species). The pure culture plates were incubated at room temperature (20–25 °C) for 14–21 days, and the fungal colonies were carefully observed and described. The herbarium specimens of the fungi were dehydrated using silica gel and deposited in the Mae Fah Luang University Herbarium. The pure cultures were deposited in the Kunming Institute of Botany Culture Collection (KMUCC). Index Fungorum (IF) and Facesoffungi (FoF) numbers were obtained as described by Index Fungorum [49] and Jayasiri et al. [50].

2.2. DNA Extraction, PCR Amplification, and DNA Sequencing

The mycelia of the cultures grown on PDA at room temperature for 4 weeks were used for DNA extraction. The fungal mycelia were scraped off with a sterile scalpel and transferred to 1.5 mL micro-centrifuge tubes under aseptic conditions and kept at −20 °C to avoid contaminations until use. The Biospin Fungal Genomic DNA Extraction Kit (BioFlux, China) was used to perform DNA extraction from the fungal cultures, following the manufacturer’s protocols. To amplify partial gene regions of the 5.8S rRNA gene in the internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1), actin gene (ACT), and beta-tubulin gene (TUB2), polymerase chain reaction (PCR) was used. The PCR conditions and primers were set under standard conditions as shown in Table 1. The total volume of PCR mixtures for amplifications was set as described in Tibpromma et al. [18]. Purification and sequencing of PCR products were done by Sangon Biotech Co., Shanghai, China.

2.3. Phylogenetic Analyses

The ITS and TEF1 sequence data produced in this study were used in BLAST searches in the GenBank database (www.http://blast.ncbi.nlm.nih.gov/) to determine their most probable closely related taxa. The sequence data generated in this study were analyzed with closely related taxa retrieved from GenBank based on BLAST searches and recent publications [9,16,18,55,56]. Single gene sequence datasets were aligned using the MAFFT v.7.215 website [57] and manually edited in BioEdit v.7.0 when necessary [58]. Single sequence alignment datasets were combined using BioEdit v.7.2.5 [58]. The alignment of combined datasets in FASTA format was converted to PHYLIP and NEXUS formats using the Alignment Transformation Environment (ALTER) website [59]. Phylogenetic trees were run in randomized accelerated maximum likelihood (RAxML) and Bayesian posterior probabilities (BYPPs). The maximum likelihood (ML) analysis was performed via the CIPRES Science Gateway [60] using the RAxML-HPC BlackBox (8.2.4) section [61,62] with the general time reversible model (GTR) using a discrete gamma distribution as the evolutionary model. To carry out Bayesian analysis, the model of evolution was estimated using MrModeltest 2.2 [63] with HKY+I+G (for the Pestalotiopsis dataset) and GTR+I+G (for the Cladosporium dataset) as nucleotide substitution models selected for combined datasets. Posterior probabilities (PPs) [64] were determined by Markov chain Monte Carlo sampling (MCMC) in MrBayes v.3.0b4 [65]. The parameters were set as six simultaneous Markov chains ran for 5,000,000 generations and sampling every 100th generation for a total of 50,000 trees [66]. The first trees representing the burn-in phase of the analysis (20%) were discarded and the remaining (post-burn) trees were used for calculating PPs in the majority rule consensus tree (the critical value for the topological convergence diagnostic values reached 0.01) [67,68].
The phylograms were figured in FigTree v.1.4 [69] and reorganized using Microsoft Office PowerPoint 2007 and Adobe illustrator CS3 (Adobe Systems Inc., USA). The sequences generated in this study were submitted to GenBank (Table 2 and Table 3).

3. Results

3.1. Phylogenetic Analysis of Combined Sequence Data

The combined dataset of genera Cladosporium and Pestalotiopsis were analyzed using maximum likelihood and Bayesian analyses (Figure 2; Figure 4). Both the ML and BYPP trees showed similar results in topology and no significant differences were seen (data not presented).
In the Cladosporium tree (Figure 2), the final alignments contained 104 strains with 1484 characters, including 594 characters for TEF1, 306 characters for ACT, and 584 characters for ITS. Cercospora beticola (CBS 116456) was used as an outgroup taxon. The tree topology of the ML analysis was similar to the BYPP. The best scoring RAxML tree with a final likelihood value of −14,457.527098 is presented. The matrix had 681 distinct alignment patterns with 30.20% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.228336, C = 0.290122, G = 0.251877, T = 0.229664; substitution rates AC = 1.724785, AG = 2.866615, AT = 1.692026, CG = 1.001444, CT = 5.300862, GT = 1.000000; gamma distribution shape parameter a = 0.312597. The phylogram of the genus Cladosporium based on a combined dataset showed that our strains grouped together with Cladosporium anthropophilum clade with relatively high bootstrap supports (Figure 2).
In the Pestalotiopsis tree (Figure 4), the final alignments contained 81 strains with 1562 characters, including 549 characters for TEF1, 440 characters for TUB2, and 573 characters for ITS. Neopestalotiopsis formicarum (CBS 362.72) and N. clavispora (CBS 447.73) were used as outgroup taxa. The tree topology of the ML analysis was similar to the BYPP. The best scoring RAxML tree with a final likelihood value of −11413.131729 is presented. The matrix had 696 distinct alignment patterns, with 12.40% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.235816, C = 0.293897, G = 0.211788, T = 0.258500; substitution rates AC = 1.049115, AG = 3.327441, AT = 1.067008, CG = 0.861291, CT = 3.485808, GT = 1.000000; gamma distribution shape parameter a = 0.276615. The Pestalotiopsis phylogram, based on a combined dataset, showed that our new species, Pestalotiopsis pinicola, was well separated from P. rosea with relatively high bootstrap supports (100% ML/ 1 BYPP, Figure 4). Therefore, we propose Pestalotiopsis pinicola as a distinct new species and Cladosporium anthropophilum as a previously known species.

3.2. Taxonomy

Cladosporium anthropophilum Sand.-Den., Gené and Wiederhold, Persoonia 36: 290 (2016) [16].
Index Fungorum number: IF815334, Facesoffungi number: FoF 06275, Figure 3.
Saprobic or weak pathogen on seed coat of Pinus armandii. Sexual morph: Undetermined. Asexual morph: Mycelium sparsely formed, superficial, overgrowing entire pod, thin to dense, later often forming colonies on the surface, hyphae straight to strongly flexuous-sinuous, branched, subhyaline to olivaceous-brown. Conidiophores erect, stipes, slightly attenuated towards the apex, yellow-brown to dark-brown, smooth and thick-walled, branched, septate. Conidiogenous cells 5–15 × 2.5–5.5 μm ( x ¯ = 8.7 × 4 μm; n = 20), cylindrical, sometimes geniculate-sinuous, proliferation sympodia with distinctive scar. Secondary ramoconidia 5.9–9.1 × 2–3.5 μm ( x ¯ = 7.7 × 2.9 μm; n = 40), olivaceous-brown, ellipsoid-ovoid, obovoid, fusiform, subcylindrical, aseptate, smooth to rough-walled, granulate and scars. Conidia 2.7–5.6 × 2–3.2 μm ( x ¯ = 4.1 × 2.7 μm; n = 40), in simple or branched chains, subhyaline to olivaceous, ellipsoid-ovoid, aseptate, a scar at base, rough-walled with granulate.
Culture characters: Colonies on PDA reaching 9 cm in diameter after 3 weeks at room temperature. Colonies olivaceous-grey to olivaceous, pale-olivaceous to black at the margin and circular with slightly regular colony, powdery, radially furrowed, aerial mycelium sparse with raised elevation, numerous small prominent exudates formed, sporulation profuse.
Material examined: CHINA, Yunnan Province, on seed coat of Pinus armandii Franch., May 2019, Kai Yan, Seed01 (MFLU19-2362); living culture KUMCC 19-0182 = KUMCC 19-0202.
Note that Cladosporium anthropophilum was established by Sandoval-Denis et al. [16] which belongs to the C. cladosporioides species complex. Cladosporium anthropophilum is probably known as a common saprobic fungus and also represents a clinically relevant fungus [16,70]. In this study, we found a strain of C. anthropophilum from a seed coat of Pinus armandii which was confirmed based on morphology and multi-gene analysis (Figure 2 and Figure 3). The morphology of our strain was similar to the C. anthropophilum described by Sandoval-Denis et al. [16]. In addition, this is the first report of C. anthropophilum from P. armandii (Figure 4).
Pestalotiopsis pinicola Tibpromma, Karunaratha and Mortimer, sp. nov.
  • Index Fungorum number: IF556765, Facesoffungi number: FoF 06276, Figure 5.
  • Etymology: named after the host genus, Pinus.
  • Holotype: MFLU19-2363.
Saprobic or endophytic on seed endosperm of Pinus armandii. Sexual morph: Undetermined. Asexual morph: Conidiophores short, unbranched, reduced to conidiogenous cells. Conidiogenous cells discrete, holoblastic, simple, filiform, smooth and thin-walled, hyaline. Conidia fusoid to ellipsoid, straight to slightly curved, 3–4 septate (mostly 4 septate), 18–23 × 5–7 μm ( x ¯ = 21 × 6 μm, n = 40), basal cell conic to obconic with obtuse end, subhyaline, thin-walled, verruculose, 3.5–5 μm long ( x ¯ = 4 μm); three median cells, doliiform, yellow-brown and becoming brown with age, septa and periclinal walls darker than rest of the cell, together 11–16 μm long ( x ¯ = 13 μm); second cell from base 3–6 μm long ( x ¯ = 4.5 μm); third cell 3–5.5 μm long ( x ¯ = 4.6 μm); fourth cell 3–5 μm long ( x ¯ = 3.9 μm); apical cell hyaline, conic 3–5 μm long ( x ¯ = 3.9 μm), with 2(–3) tubular apical appendages; appendages arising from the apex of the apical cell, unbranched, 5–17 μm long ( x ¯ = 10.3 μm); single basal appendage usually present, 2–7 μm long ( x ¯ = 4.7 μm), tubular, unbranched, centric.
Culture characteristics: Colonies on PDA reaching 9 cm in diameter after 2 weeks at room temperature, edge undulate with curled, whitish, aerial mycelium on surface, spore masses form after 1 month, black spore masses; reverse of culture yellow-white to yellow-orange with black dots.
Material examined: China, Yunnan Province, on endosperm of pine seed of Pinus armandii Franch., May 2019, Kai Yan, Seed02 (MFLU19-2363, holotype); ex-type living culture KUMCC 19-0183 = KUMCC 19-0203.
Note that Pestalotiopsis pinicola is introduced based on morphological and phylogenetic data. In the phylogenetic analysis, our new species cluster with P. rosea Maharachch. and K.D. Hyde [40] but are well separated with high support (100% ML/1 BYPP, Figure 4). In addition, base pair differences of our new taxa with closest taxa were checked based on the recommendations of Jeewon and Hyde [71]; our isolate differs from P. rosea (MFLUCC12-0258 and CL0441) with five ITS base pairs (2.65%), four TUB base pairs (1.64%), and ten RPB2 base pairs (4.65%). In addition, the culture of P. rosea was seen as a reddish colony [40], while our new species produces a whitish colony.
In a BLASTn search on the NCBI GenBank, the closest ITS sequence match of KUMCC 19-0183 is Pestalotiopsis sp. with a 99.31% identity to the strain JSM 06261592 (KY086253), KUMCC 19-0203 is P. neglecta with 99.82% identity to the strain CBS 357.71 (MH860161.1), the closest TEF1 sequence matches of KUMCC 19-0183 and KUMCC 19-0203 were with the P. rosea strain MFLUCC12-0258 with 98.72% (JX399069), while the closest matches with the TUB2 sequence were with the 99.53% identical P. olivacea strain PSHI2002 (DQ787834) by KUMCC 19-0183 and 99.53% identical P. vismiae strain Q15DY (EF055259) by KUMCC 19-0203.

4. Discussion

In this paper, we describe a novel taxon belonging to Pestalotiopsis and a new host record of Cladosporium isolated from seeds of Pinus armandii obtained from Yunnan Province, China. Mature agar colonies sporulated in cultures with masses of conidia.
We isolated a new Pestalotiopsis species from mycelia-covered endosperms of pine seeds. Past research has yielded new species from Pestalotiopsis with similar origins; for example, several endophytic Pestalotiopsis species were isolated from the bark and needles of Pinus armandii in China [38]. Furthermore, Pestalotiopsis brassicae and P. oryzae were isolated from seeds from Oryza sativa and Brassica napus [42]. Several have often been isolated as endophytes and many pathogens or endophytes may persist as saprobes, which mean Pestalotiopsis species are able to switch life-modes [42]. The present study illustrates a novel species of Pestalotiopsis as Pestalotiopsis pinicola, taking both morphology and phylogeny into consideration (Figure 4 and Figure 5). The phylogenetic tree construction of the DNA sequences of single and combined genes provides confirmation with high bootstrap support that P. pinicola is a characteristic new species separate from other known species of the genus (Figure 4). Moreover, this genus is known as one of the fungal groups that can produce a wide range of chemically novel secondary metabolites and mycotoxins; for example, pestaloside exhibiting significant antifungal properties was produced by P. microspora, obtained from Torreya taxifolia [42,72,73,74]. There is, consequently, a potential health threat in the sale of these seeds as an edible foodstuff. Follow-up research investigating the potential toxins produced by P. pinicola should be conducted to clarify this issue. We conclude that fungi live inside seeds as endophytes and then switch life-modes to saprobes or weak pathogens when conditions become unfavorable. In the future, knowledge about pestalotioid fungi associated with seeds will help provide a basis for developing proper management of these pathogens.
We found another species, Cladosporium anthropophilum, growing on pine seed coats. The etymology of this species comes from Greek which refers to the sample’s source which was isolated from a human clinical sample [16]. This species can be found in human clinical samples, indoor air, food and plant materials, such as seeds or leaves, and it is also a common saprobic fungus [56]. In addition, this species is known as the second-most prevalent species from clinical environments from the US after C. halotolerans, and it also has been isolated quite frequently from indoor environments [16,69]. However, we continue to lack information about the chemistry or secondary metabolites of this species along with the potential serious health effects associated with long-term exposure to a large amount of Cladosporium anthropophilum.
The present study illustrates two species of Pestalotiopsis and Cladosporium based on both morphology and phylogeny. These two species of fungi were isolated from pine seeds from Yunnan Province, China. The fungal mycelia in the seeds were observed after the seeds were broken open to eat, and these seeds can be found in many food markets around Yunnan Province. We recommend that consumers should carefully check seed products before purchase and consumption, as these fungi may cause adverse health effects in the long term. Therefore, to address this health concern, in the future we will focus our research on the secondary metabolites and mycotoxins of Cladosporium anthropophilum and Pestalotiopsis pinicola.

Author Contributions

S.T., P.E.M., S.C.K. and K.Y. designed the experiments. S.T. and S.C.K. conducted the experiments. S.T. performed the morphological study and phylogenetic data. S.T., P.E.M., K.Y., and S.C.K. analyzed the data. S.T., P.E.M., I.P., F.Z., K.Y., and S.C.K. provided funding and financial support for this study. S.T. wrote the manuscript and P.E.M., F.Z., J.X., S.C.K., I.P., and K.Y. gave comments, suggestions, and edited the manuscript. All authors reviewed and approved the final manuscript.

Funding

This research was funded by National Science Foundation of China (NSFC) [project codes 41761144055, 31750110478, 41807524 and 41771063], the Southeast Asian Biodiversity Research Institute [project code Y4ZK111B01] and Chiang Mai University.

Acknowledgments

S.T. would like to thank the International Postdoctoral Exchange Fellowship Program (number Y9180822S1), CAS President’s International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation, and the Yunnan Human Resources and Social Security Department Foundation for funding her postdoctoral research. Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming, China and Gu Zhi-Jia are thanked for the scanning electron microscopy morphology. P.E.M. thanks the National Science Foundation of China (NSFC), project codes 41761144055 and 41771063, and the Southeast Asian Biodiversity Research Institute (Y4ZK111B01) for support. S.C.K. thanks CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2018PC0006) and the National Science Foundation of China (NSFC) for funding this research work under project code 31750110478. This research was partially supported by the Chinese National Science Foundation (41807524) and Chiang Mai University. Austin Smith at World Agroforestry (ICRAF), Kunming Institute of Botany, China, is thanked for English editing.

Conflicts of Interest

There is no conflict of interest.

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Figure 1. (a) Seeds of Pinus armandii from Yunnan Province, China. (b) Fungal mass on a seed. (c) Endosperm covered with mycelia. (d) Mycelia on endosperm under a SEM micrograph. (eh) Rows of rounded cells present on a seed coat skin under SEM micrographs that form aerial hyphae, conidia, and conidiophores. Scale bars: d = 1 mm, e = 100 µm, f = 10 µm, g = 2 µm, h = 1 µm.
Figure 1. (a) Seeds of Pinus armandii from Yunnan Province, China. (b) Fungal mass on a seed. (c) Endosperm covered with mycelia. (d) Mycelia on endosperm under a SEM micrograph. (eh) Rows of rounded cells present on a seed coat skin under SEM micrographs that form aerial hyphae, conidia, and conidiophores. Scale bars: d = 1 mm, e = 100 µm, f = 10 µm, g = 2 µm, h = 1 µm.
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Figure 2. Phylogram generated from RAxML analysis based on combined TEF1, ACT and ITS sequence data of the genus Cladosporium. Related sequences were obtained from Sandoval-Denis et al. [16] and Bensch et al. [56]. Bootstrap support values for ML equal to or greater than 60% and BYPP from MCMC analyses equal to or greater than 0.95 are given above/below the nodes. The ex-type strains are indicated in bold type. Newly generated sequences are indicated in red with two asterisks.
Figure 2. Phylogram generated from RAxML analysis based on combined TEF1, ACT and ITS sequence data of the genus Cladosporium. Related sequences were obtained from Sandoval-Denis et al. [16] and Bensch et al. [56]. Bootstrap support values for ML equal to or greater than 60% and BYPP from MCMC analyses equal to or greater than 0.95 are given above/below the nodes. The ex-type strains are indicated in bold type. Newly generated sequences are indicated in red with two asterisks.
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Figure 3. Cladosporium anthropophilum (KUMCC 19-0182). (a,b) Colony on PDA media. (c) Mycelium masses. (d,e) Conidiophores and conidiogenous cells and conidia. (fi) Conidiogenous cells with secondary ramoconidia and coidia. (j) Conidia. Scale bars: (d,e) = 10 µm, (fi) = 5 µm, (j) = 2 µm.
Figure 3. Cladosporium anthropophilum (KUMCC 19-0182). (a,b) Colony on PDA media. (c) Mycelium masses. (d,e) Conidiophores and conidiogenous cells and conidia. (fi) Conidiogenous cells with secondary ramoconidia and coidia. (j) Conidia. Scale bars: (d,e) = 10 µm, (fi) = 5 µm, (j) = 2 µm.
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Figure 4. Phylogram generated from RAxML analysis based on combined TEF1, TUB2, and ITS sequence data of the genus Pestalotiopsis. Related sequences were obtained from Ariyawansa et al. [55] and Tibpromma et al. [9,18]. Bootstrap support values for ML equal to or greater than 60% and BYPP from MCMC analyses equal to or greater than 0.95 are given above/below the nodes. The ex-type strains are indicated in bold type. Newly generated sequences are indicated in red with two asterisks (**).
Figure 4. Phylogram generated from RAxML analysis based on combined TEF1, TUB2, and ITS sequence data of the genus Pestalotiopsis. Related sequences were obtained from Ariyawansa et al. [55] and Tibpromma et al. [9,18]. Bootstrap support values for ML equal to or greater than 60% and BYPP from MCMC analyses equal to or greater than 0.95 are given above/below the nodes. The ex-type strains are indicated in bold type. Newly generated sequences are indicated in red with two asterisks (**).
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Figure 5. Pestalotiopsis pinicola (KUMCC 19-0183, ex-type). (a,b) Colony on PDA media. (c) Fruiting body on PDA media. (dg) Conidia, conidiogenous cells and conidia. (hj) Conidia. Scale bars: (d) = 5 µm, (e) = 10 µm, (f,g) = 5 µm, (h) = 20 µm, (ij) = 5 µm.
Figure 5. Pestalotiopsis pinicola (KUMCC 19-0183, ex-type). (a,b) Colony on PDA media. (c) Fruiting body on PDA media. (dg) Conidia, conidiogenous cells and conidia. (hj) Conidia. Scale bars: (d) = 5 µm, (e) = 10 µm, (f,g) = 5 µm, (h) = 20 µm, (ij) = 5 µm.
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Table 1. Gene regions and primers used in this study.
Table 1. Gene regions and primers used in this study.
GenesPrimers (Forward/Reverse)References
Cladosporium
ACT512F/783R[51]
ITSITS5/ITS4[52]
TEF1728F/986R [51]
Pestalotiopsis
ITSITS5/ITS4[52]
TEF1526F/1567R[53]
TUB2T1/T2[54]
Table 2. GenBank accession numbers and culture collection numbers of the nucleotide sequences of Cladosporium taxa used in this study. The new sequences generated in this study are in bold type.
Table 2. GenBank accession numbers and culture collection numbers of the nucleotide sequences of Cladosporium taxa used in this study. The new sequences generated in this study are in bold type.
SpeciesCulture Collection NumberGenBank Accession Numbers
ITSTEF1ACT
Cercospora beticolaCBS 116456NR_121315AY840494AY840458
Cladosporium acalyphaeCBS 125982HM147994HM148235HM148481
C. alboflavescensCBS 140690LN834420LN834516LN834604
C. angustisporumCBS 125983HM147995HM148236HM148482
C. angustisporumUTHSC-DI-13-240LN834356LN834452LN834540
C. anthropophilumCBS 117483HM148007HM148248HM148494
C. anthropophilumCBS 140685LN834437LN834533LN834621
C. anthropophilumKUMCC 19-0182MN412638MN417513MN417511
C. anthropophilumKUMCC 19-0202MN412639MN417514MN417512
C. anthropophilumUTHSC-DI-13-168LN834407LN834503LN834591
C. anthropophilumUTHSC-DI-13-169LN834408LN834504LN834592
C. anthropophilumUTHSC-DI-13-178LN834410LN834506LN834594
C. anthropophilumUTHSC-DI-13-179LN834411LN834507LN834595
C. anthropophilumUTHSC-DI-13-207LN834413LN834509LN834597
C. anthropophilumUTHSC-DI-13-226LN834421LN834517LN834605
C. anthropophilumUTHSC-DI-13-228LN834423LN834519LN834607
C. anthropophilumUTHSC-DI-13-244LN834428LN834524LN834612
C. anthropophilumUTHSC-DI-13-246LN834430LN834526LN834614
C. anthropophilumUTHSC-DI-13-269LN834437LN834533LN834621
C. anthropophilumUTHSC-DI-13-271LN834439LN834535LN834623
C. asperulatumCBS 126339HM147997HM148238HM148484
C. asperulatumCBS 126340HM147998HM148239HM148485
C. australienseCBS 125984HM147999HM148240HM148486
C. austroafricanumCPC 16763KT600381KT600478KT600577
C. chalastosporoidesCBS 125985HM148001HM148242HM148488
C. chubutenseCBS 124457FJ936158FJ936161FJ936165
C. cladosporioidesCBS 112388HM148003HM148244HM148490
C. cladosporioidesCBS 113738HM148004HM148245HM148491
C. cladosporioidesCPC 14292HM148046HM148287HM148533
C. cladosporioidesUTHSC-DI-13-215LN834360LN834456LN834544
C. colocasiaeCBS 119542HM148066HM148309HM148554
C. colocasiaeCBS 386.64HM148067HM148310HM148555
C. colombiaeCBS 274.80BFJ936159FJ936163FJ936166
C. crousiiUTHSC-DI-13-247LN834431LN834527LN834615
C. cucumerinumCBS 171.52HM148072HM148316HM148561
C. cucumerinumCBS 173.54HM148074HM148318HM148563
C. delicatulumCBS 126342HM148079HM148323HM148568
C. delicatulumCBS 126344HM148081HM148325HM148570
C. exileCBS 125987HM148091HM148335HM148580
C. flabelliformeCBS 126345HM148092HM148336HM148581
C. flabelliformeUTHSC-DI-13-267LN834361LN834457LN834545
C. flavovirensUTHSC-DI-13-273LN834440LN834536LN834624
C. funiculosumCBS 122128HM148093HM148337HM148582
C. funiculosumCBS 122129HM148094HM148338HM148583
C. gamsianumCBS 125989HM148095HM148339HM148584
C. globisporumCBS 812.96HM148096HM148340HM148585
C. grevilleaeCBS 114271JF770450JF770472JF770473
C. hillianumCBS 125988HM148097HM148341HM148586
C. inversicolorCBS 143.65HM148100HM148344HM148589
C. ipereniaeCBS 140483KT600394KT600491KT600589
C. ipereniaeCPC 16855KT600395KT600492KT600590
C. iranicumCBS 126346HM148110HM148354HM148599
C. limoniformeCBS 113737KT600396KT600493KT600591
C. longicatenatumCPC 17189KT600403KT600500KT600598
C. lycoperdinumCBS 126347HM148112HM148356HM148601
C. lycoperdinumCBS 574.78CHM148115HM148359HM148604
C. montecillanumCPC 15605KT600407KT600505KT600603
C. montecillanumCPC 17953KT600406KT600504KT600602
C. myrtacearumCBS 126350HM148117HM148361HM148606
C. oxysporumCBS 125991HM148118HM148362HM148607
C. oxysporumCBS 126351HM148119HM148363HM148608
C. paracladosporioidesCBS 171.54HM148120HM148364HM148609
C. parapenidielloidesCPC 17193KT600410KT600508KT600606
C. phaenocomaeCBS 128769JF499837JF499875JF499881
C. phyllactiniicolaCBS 126353HM148151HM148395HM148640
C. phyllactiniicolaCBS 126355HM148153HM148397HM148642
C. phyllophilumCBS 125992HM148154HM148398HM148643
C. phyllophilumCBS 125990HM148111HM148355HM148600
C. pini-ponderosaeCBS 124456FJ936160FJ936164FJ936167
C. pseudochalastosporoidesCPC 17823KT600415KT600513KT600611
C. pseudocladosporioidesCBS 125993HM148158HM148402HM148647
C. pseudocladosporioidesCBS 667.80HM148165HM148409HM148654
C. pseudocladosporioidesCPC 13683HM148173HM148417HM148662
C. pseudocladosporioidesCPC 14020HM148185HM148429HM148674
C. pseudocladosporioidesCPC 14295HM148188HM148432HM148677
C. pseudocladosporioidesUTHSC-DI-13-165LN834406LN834502LN834590
C. pseudocladosporioidesUTHSC-DI-13-190LN834412LN834508LN834596
C. pseudocladosporioidesUTHSC-DI-13-210LN834414LN834510LN834598
C. pseudocladosporioidesUTHSC-DI-13-218LN834418LN834514LN834602
C. pseudocladosporioidesUTHSC-DI-13-227LN834422LN834518LN834606
C. pseudocladosporioidesUTHSC-DI-13-234LN834424LN834520LN834608
C. pseudocladosporioidesUTHSC-DI-13-238LN834426LN834522LN834610
C. pseudocladosporioidesUTHSC-DI-13-241LN834427LN834523LN834611
C. pseudocladosporioidesUTHSC-DI-13-245LN834429LN834525LN834613
C. pseudocladosporioidesUTHSC-DI-13-251LN834432LN834528LN834616
C. pseudocladosporioidesUTHSC-DI-13-261LN834384LN834480LN834568
C. pseudocladosporioidesUTHSC-DI-13-265LN834435LN834531LN834619
C. pseudocladosporioidesUTHSC-DI-13-268LN834436LN834532LN834620
C. pseudocladosporioidesUTHSC-DI-13-270LN834438LN834534LN834622
C. rectoidesCBS 125994HM148193HM148438HM148683
C. ruguloflabelliformeCPC 19707KT600458KT600557KT600655
C. scabrellumCBS 126358HM148195HM148440HM148685
C. silenesCBS 109082EF679354EF679429EF679506
C. subinflatumCBS 121630EF679389EF679467EF679543
C. subinflatumCBS 121630EF679389EF679467EF679543
C. subuliformeCBS 126500HM148196HM148441HM148686
C. tenuissimumCPC 13222HM148210HM148455HM148700
C. tenuissimumCPC 14250HM148211HM148456HM148701
C. tenuissimumUTHSC-DI-13-258LN834404LN834500LN834588
C. variansCBS 126362HM148224HM148470HM148715
C. verrucocladosporioidesCBS 126363HM148226HM148472HM148717
C. versiformeCPC 19053KT600417KT600515KT600613
C. xantochromaticumCBS 140691LN834415LN834511LN834599
C. xylophilumCBS 125997HM148230HM148476HM148721
Table 3. GenBank accession numbers and culture collection numbers of the nucleotide sequences of the Pestalotiopsis taxa used in this study. The new sequences generated in this study are in black bold type.
Table 3. GenBank accession numbers and culture collection numbers of the nucleotide sequences of the Pestalotiopsis taxa used in this study. The new sequences generated in this study are in black bold type.
SpeciesCulture Collection NumberGenBank Accession Numbers
ITSTUB2TEF1
Neopestalotiopsis clavisporaCBS 447.73KM199374KM199443KM199539
N. formicarumCBS 362.72KM199358KM199455KM199517
Pestalotiopsis adustaMFLUCC 10-0146JX399006JX399037JX399070
P. aggestorumLC8186KY464140KY464160KY464150
P. anacardiacearumIFRDCC 2397KC247154KC247155KC247156
P. arceuthobiiCBS 434.65KM199341KM199427KM199516
P. arengaeCBS 331.92KM199340KM199426KM199515
P. australasiaeCBS 114126KM199297KM199409KM199499
P. australisCBS 114193KM199332KM199383KM199475
P. biciliataCBS 124463KM199308KM199399KM199505
P. biciliataCBS 790.68KM199305KM199400KM199507
P. biciliataMFLUCC 12-0598KX816920KX816948KX816890
P. brachiataLC2988KX894933KX895265KX895150
P. brassicaeCBS 170.26KM199379-KM199558
P. camelliaeMFLUCC 12-0277JX399010JX399041JX399074
P. chamaeropisCBS 186.71KM199326KM199391KM199473
P. clavataMFLUCC 12-0268JX398990JX399025JX399056
P. colombiensisCBS 118553KM199307KM199421KM199488
P. digitalisICMP 5434KP781879KP781883-
P. dilucidaLC3232KX894961KX895293KX895178
P. diploclisiaeCBS 115587KM199320KM199419KM199486
P. distinctaLC8185KY464139KY464159KY464149
P. diversisetaMFLUCC 12-0287JX399009JX399040JX399073
P. dracontomelonMFUCC 10-0149KP781877-KP781880
P. ericacearumIFRDCC 2439KC537807KC537821KC537814
P. ericacearumOP023KC537807KC537821KC537814
P. formosanaNTUCC 17-010MH809382MH809386MH809390
P. furcataMFLUCC 12-0054JQ683724JQ683708JQ683740
P. gaultheriaIFRD 411-014KC537805KC537819KC537812
P. gaultheriaOP137KC537805KC537819KC537812
P. grevilleaeCBS 114127KM199300KM199407KM199504
P. hawaiiensisCBS 114491KM199339KM199428KM199514
P. hispanicaCBS 115391MH553981MH554640MH554399
P. hollandicaCBS 265.33KM199328KM199388KM199481
P. humusCBS 336.97KM199317KM199420KM199484
P. inflexaMFLUCC 12-0270JX399008JX399039JX399072
P. intermediaMFLUCC 12-0259JX398993JX399028JX399059
P. italianaMFLUCC 12-0657KP781878KP781882KP781881
P. jesteriCBS109350KM199380KM199468KM199554
P. jiangxiensisLC4399KX895009KX895341KX895227
P. jinchanghensisLC8191KY464145KY464165KY464155
P. kenyanaCBS 442.67KM199302KM199395KM199502
P. knightiaeCBS 114138KM199310KM199408KM199497
P. krabiensisMFLUCC 16-0260MH388360MH412722MH388395
P. leucadendriCBS 121417MH553987MH554654MH554412
P. licualacolaHGUP 4057KC492509KC481683KC481684
P. linearisMFLUCC 12-0271JX398992JX399027JX399058
P. longiappendiculataLC3013KX894939KX895271KX895156
P. lushanensisLC8183KY464137KY464157KY464147
P. lushanensisOP086KC537804KC537818KC537811
P. macadamiaeBRIP 63738KX186588KX186680KX186621
P. malayanaCBS 102220KM199306KM199411KM199482
P. monochaetaCBS 144.97KM199327KM199386KM199479
P. montelicaMFLUCC 12-0279JX399012JX399043JX399076
P. neolitseaeNTUCC 17-012MH809384MH809388MH809392
P. novae-hollandiaeCBS 130973KM199337KM199425KM199511
P. oryzaeCBS 353.69KM199299KM199398KM199496
P. pandanicolaMFLUCC 16-0255MH388361MH412723MH388396
P. papuanaCBS 331.96KM199321KM199413KM199491
P. parvaCBS 265.37KM199312KM199404KM199508
P. pinicolaKUMCC 19-0183MN412636MN417507MN417509
P. pinicolaKUMCC 19-0203MN412637MN417508MN417510
P. portugalicaCBS 393.48KM199335KM199422KM199510
P. rhododendriIFRDCC 2399KC537804KC537818KC537811
P. rhodomyrtusHGUP 4230KF412648KC537818KF412645
P. roseaCL0441KY228790--
P. roseaMFLUCC 12-0258JX399005JX399036JX399069
P. scopariaCBS 176.25KM199330KM199393KM199478
P. sequoiaMFLUCC 13-0399KX572339--
P. shoreaMFLUCC 12-0314KJ503811KJ503814KJ503817
P. spathulataCBS 356.86KM199338KM199423KM199513
P. spathuliappendiculataCBS 144035MH554172MH554845MH554607
P. telopeaeCBS 114161KM199296KM199403KM199500
P. terricolaCBS 141.69MH554004MH554680MH554438
P. trachicarpicolaIFRDCC 2240NR_120109--
P. trachicarpicolaOP143JQ845947JQ845945JQ845946
P. unicolorMFLUCC 12-0276JX398999JX399030JX399063
P. verruculosaMFLUCC 12-0274JX398996-JX399061
P. yanglingensisLC3375KX894975KX895307KX895192
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