Neotypification of Muyocopron dipterocarpi, a new host record on Zanthoxylum fagara (Rutaceae) and the potential for secondary metabolite production in Muyocopronaceae

Muyocopronaceae was validly introduced by Hyde et al.^[1], with a type genus Muyocopron Speg. The family currently comprises nine genera (viz. Arxiella Papendorf, Leptodiscella Papendorf, Mycoleptodiscus Ostaz., Muyocopron Speg., Neocochlearomyces Pinruan, Sommai, Suetrong, J.Z. Groenew. & Crous, Neomycoleptodiscus Hern.-Restr., J.D.P. Bezerra & Crous, Paramycoleptodiscus Crous & M.J. Wingf., Pseudopalawania Mapook & K.D. Hyde, and Setoapiospora Mapook & K.D. Hyde), based on molecular phylogeny and morphology^[2−7]. Interestingly, members of Muyocopronaceae have been reported to have the potential for antimicrobial and cytotoxic activities^[7−10]. Pseudopalawania siamensis polyketide-derived secondary metabolite produced with the potential of antimicrobial and cytotoxic activities. This comprised a new heterodimeric bistetrahydroxanthone, namely pseudopalawanone^[7]. An endophytic fungus, Muyocopron laterale (ECN279), which was isolated from a healthy leaf of Canavalia lineata (Fabaceae), produced two new azaphilones, namely muyocopronones A and B with weak antibacterial activity^[10]. An endophytic species, Mycoleptodiscus indicus, which is currently named Muyocopron sahnii^[3], produced a new triterpenoid^[9] and three new azaphilones, namely mycoleptones A, B, and C with four known polyketides. All compounds were weakly active when tested in antileishmanial and cytotoxicity assays^[8]. However, the study of secondary metabolite production from Muyocopronaceae is still in the initial stages.

Muyocopron is a diverse genus which can be found in tropical and temperate regions, occurring as saprobes and pathogens on various plant parts such as dead aerial twigs, branches, stems and leaves and can also be an opportunistic pathogen on humans and animals^{[3, 11−13]}. A mycoleptodiscus-like asexual morph has been described for the genus with several additional species based on molecular analyses^[3]. The asexual morphs are characterized by irregular sporodochium-like conidiomata, globose or broadly ellipsoidal to ampulliform with enteroblastic, monophialidic conidiogenous cells, and fusiform or fusoid-ellipsoid, curved, hyaline, aseptate or septate conidia with terminal and/or lateral appendages, with or without dark brown appressoria^[3]. Presently, 68 species epithets are listed in Index Fungorum^[14] with 56 probable species listed in Species Fungorum^[15].

In this study, we provide morphological descriptions and illustrations of a new collection of Mu. dipterocarpi from Zanthoxylum fagara, in northern Thailand. The identification was confirmed using both multigene analyses and morphological comparisons. The results of preliminary screening for antimicrobial activity is also provided. The lifestyle and function with the potential for secondary metabolites production are also discussed. In addition, a neotype is designated here for Mu. dipterocarpi due to the original material no longer existing.

Fresh material was collected from Phrae Province (India) in September 2016. The micromorphology was examined following the methodology as described by Mapook et al.^[6]. Single spore isolation and culture morphology were obtained following the methods of Senanayake et al.^[16]. Germinated spores were observed on MEA media (malt extract agar: 33.6 g/l sterile distilled water, Difco malt extract) within 24 h and transferred to new MEA media and incubated at room temperature (25 °C) in the dark. Pure cultures were used for molecular study and pre-screening tests for antimicrobial activity. The specimens with collection details and living cultures are deposited in the Herbarium of Mae Fah Luang University (Herb. MFLU) and Culture collection Mae Fah Luang University (MFLUCC), Chiang Rai, Thailand.

Preliminary screening of antimicrobial activity

Preliminary screening of antimicrobial activity was carried out following Mapook et al.^[6]. Antibacterial discs of penicillin and ciprofloxacin, with antifungal discs of nystatin were used as positive control for screening^[17]. Gram-positive bacteria (Bacillus subtilis, DSM10), Gram-negative bacteria (Escherichia coli, DSM498), and filamentous fungus (Mucor plumbeus, MUCL 49355) were determined by the zone of inhibition using agar plug diffusion method, compared with positive control^[18].

DNA extraction, PCR amplification and sequencing

DNA extraction, PCR amplification and sequencing were carried out following the methodology as described by Mapook et al.^[6]. The partial large subunit nuclear rDNA (LSU) was amplified with primer pairs LROR and LR5^[19]. The partial small subunit nuclear rDNA (SSU) was amplified with primer pairs NS1 and NS4^[20]. The internal transcribed spacer (ITS) was amplified by using primer pairs ITS5 and ITS4^[20]. The translation elongation factor 1-α (TEF1) was amplified by using primers EF1-983F and EF1-2218R^[21]. The PCR products were sent for sequencing at SeqLab GmbH (Microsynth AG), Göttingen, Germany.

Phylogenetic analysis

Phylogenetic analysis was carried out following Mapook et al.^[6] and Dissanayake et al.^[22]. The closest relative strains were selected following Mapook et al.^[6,7]. The combined aligned dataset was analyzed based on maximum likelihood (ML) and Bayesian inference (BI) via the CIPRES Science Gateway platform (V. 3.3, www.phylo.org)^[23]. ML analysis was performed by RAxML-HPC2 on XSEDE (8.2.12) tool^[24,25] using the GTR+I+G model of nucleotide substitution with 1,000 rapid bootstrap replicates. BI analysis was performed by using MrBayes on XSEDE (3.2.7a) tool with the GTR+I+G model. Six simultaneous Markov chains were performed for 5,000,000 generations, and trees were sampled every 1000^th generation. Phylogenetic trees were drawn using FigTree 1.4.0^[26] and edited by Microsoft Office PowerPoint 365 and Adobe Photoshop CS6. The sequences used for analyses with accession numbers are given in Table 1.

Seventy-seven strains are included in the combined dataset of LSU, SSU, ITS, and TEF1 sequence data, including our strain (MFLUCC 17-1464), which comprise 3969 characters with gaps. Tree topology of the ML analysis was similar to the BYPP. A best scoring RAxML tree with a final likelihood value of −25,539.739638 is presented in Fig. 1. The matrix had 1738 distinct alignment patterns, with 49.64% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.237294, C = 0.253374, G = 0.287766, T = 0.221566; substitution rates: AC = 1.187676, AG = 2.326266, AT = 1.446900, CG = 1.082572, CT = 5.256850, GT = 1.000000; gamma distribution shape parameter α = 0.309869. The phylogram generated from ML analysis (Fig. 1) shows that our strain grouped within Muyocopron dipterocarpi clade. In a BLASTn search of NCBI GenBank, the closest match of the LSU, ITS, and TEF1 sequence of MFLUCC 17-1464 is Mu. dipterocarpi with 100% similarity to the strain MFLUCC 14-1103 (ex-holotype), while the closest match of the SSU sequence was identical with 99.90% similarity to Mu. dipterocarpi (strain MFLUCC 14-1103).

Figure 1. 

Phylogram generated from maximum likelihood analysis based on combined dataset of LSU, SSU, ITS, and TEF1 sequence data. Bootstrap support values for maximum likelihood (ML) equal to or greater than 60% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.97 are given above the nodes. Newly generated sequences are in dark red bold and ex-type isolates are in bold. Lophium mytilinum (AFTOL-ID 1609) and Mytilinidion rhenanum (CBS 135.45) are used as outgroup taxa.

Taxonomy

Muyocopron dipterocarpi Mapook, Doilom, Boonmee & K.D. Hyde, Phytotaxa 265(3): 232 (2016)

Index Fungorum number: IF551617, Facesoffungi number: FoF 01889; Fig. 2

Figure 2. 

Muyocopron dipterocarpi (neotype). (a), (b) Superficial ascomata on substrate. (c), (d) Squash mounts showing ascomata walls. (e) Section of ascoma. (f) Peridium. (g) Pseudoparaphyses. (h)−(k). Asci. (l)−(p) Unicellular ascospores. (q) Culture characteristic on MEA. Scale bars: a = 1,000 µm, b = 500 µm, c = 100 µm, e = 50 µm, f, h−k = 20 µm, d, l−p = 10 µm, g = 5 µm.

Saprobic on dead twigs of Zanthoxylum fagara (L.) Sarg. Sexual morph: Ascomata (65–)80–110 µm high × (230–)340–395 µm diam. ($\overline{\chi} $ = 92 × 332 µm, n = 10), superficial, coriaceous, solitary or scattered, appearing as circular, scattered, flattened, brown to dark brown spots, covering the host, without a subiculum, with a poorly developed basal layer and an irregular margin. Ostiole central. Peridium 15–25 µm wide, widest at the sides, outer layer comprising dark brown to black pseudoparenchymatous, occluded cells of textura angularis, inner layer comprising light brown cells of textura angularis. Hamathecium comprising 1–1.5 µm wide, cylindrical to filiform, septate, pseudoparaphyses. Asci 55–75 × 20–30 µm ($\overline {\chi} $ = 64 × 23.5 µm, n = 15), 8-spored, bitunicate, saccate or broadly obpyriform, pedicellate, straight or slightly curved, with a small ocular chamber. Ascospores 14–20 × 8.5–10 µm ($\overline {\chi} $ = 17 × 9.5 µm, n = 25), irregularly arranged, overlapping in the ascus, hyaline, oval or ellipsoid to obovoid with obtuse ends, aseptate, with granular appearance. Asexual morph: Undetermined.

Culture characteristics: Ascospores germinating on MEA within 24 hr at room temperature and germ tubes produced from the ends of the ascospore. Colonies on MEA irregular, initially aerial mycelium white, slightly raised, filiform, becoming light brown from the center, flattened on surface, pale brown to light brown in reverse from the center of the colony with white margin.

Pre-screening for antimicrobial activity: Muyocopron dipterocarpi (MFLUCC 14-1103, ex-holotype) showed antimicrobial activity against Bacillus subtilis and Escherichia coli (11 and 10 mm inhibition zone, respectively), observable as partial inhibition, when compared with the positive control (26 and 9 mm, respectively), but no inhibition of Mucor plumbeus; Mu. dipterocarpi (MFLUCC 17-1464) showed antimicrobial activity against M. plumbeus, B. subtilis and E. coli (11, 8, and 10 mm inhibition zone, respectively), observable as partial inhibition, when compared with the positive control (17, 26 , and 9 mm, respectively).

Known hosts and distribution: On dried twigs of Dipterocarpus tuberculatus (Dipterocarpaceae) in Chiang Rai Province, Thailand^[12]; on dried twig of Hevea brasiliensis (Euphorbiaceae) in Phayao Province, Thailand^[27]; on dead twigs of Mangifera indica (Anacardiaceae) in Sukhothai Province, Thailand^[28]; on decaying pod septum of Delonix regia (Fabaceae) in Phrae Province, Thailand^[29]; on dead leaves and decaying twig of Celtis formosana (Cannabaceae) in Taiwan^{[30, 31]}.

Material examined: THAILAND, Phrae Province, on dead twigs of Zanthoxylum fagara (Rutaceae), 22 September 2016, A. Mapook, (DPKP1, MFLU 23-0072, neotype designated here), ex-neotype culture MFLUCC 17-1464.

Note: We identify our isolate (MFLUCC 17-1464) as Mu. dipterocarpi, based on phylogenetic analyses, together with morphological comparison. However, our isolate was found on different host families as compared with other previously reported strains. Therefore, the isolate is introduced here as a new host record on Zanthoxylum fagara from Thailand.

During this study, we tried to examine the original collection of this species. Unfortunately, the type material of Mu. dipterocarpi could not be located in the MFLU fungarium, where the holotype specimen was deposited with a code number MFLU 15–1132^[12]. A neotype for Mu. dipterocarpi is, therefore, needed to facilitate subsequent taxonomic work in stabilizing the application of name, and to serve as a foundation for further applied research of this species. Although the species have been reported from various hosts and is mostly distributed in Asia, especially in Thailand^{[6,12,27−29]}, there are no collections from the same locality as indicated in the protologue. Neotypes from different locations may also be considered as long as the author are confident that they are conspecific^[32]. Therefore, a neotype specimen is designated here for Mu. dipterocarpi due to the original material no longer existing, and in accordance with Art. 9.16^[33]. This specimen is recent and well-preserved, and has typical morphology suitable to the description given in the protologue, completed with illustrations, molecular data and additional evidence of a potential for antimicrobial activity based on a preliminary screening.

Muyocopron species can be saprobic, endophytic, or pathogenic on various hosts with an ability to produce specialized infection structures such as appressoria from germinating ascospores and therefore probably has an endophytic lifestyle^[3,27,34]. Muyocopron dipterocarpi is mostly reported from northern Thailand and probably not specific to the host, due to the species have been reported on a variety of plant families such as Anacardiaceae, Dipterocarpaceae, Euphorbiaceae, and Fabaceae^{[6,12,27−29]}. The species have not been reported to form any specialized infection structures from the other hosts, as well as our strain in this study except the first isolate from a dried twig of Hevea brasiliensis (Euphorbiaceae), which was assumed to have endophytic lifestyle^{[12,27−31,34,35]}. This suggests that Muyocopron species may have the ability to be endophytic or pathogenic and probably not specific to any hosts due to the genus reported on various hosts. Although fungi have the ability to change their lifestyle which can be exhibited in more than one lifestyle in a different host, the mechanism of appressoria production and the ecological lifestyle of Muyocopron is not well understood. In addition, Mu. dipterocarpi also has potential for antimicrobial activity against the tested organism based on a preliminary screening in this study.