Biodiversity Data Journal : Taxonomy & Inventories
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Taxonomy & Inventories
Taxonomy and phylogeny of Smaragdiniseta musae sp. nov. and Albifimbria verrucaria (Hypocreales, Stachybotryaceae) on Musa from Thailand
expand article infoBinu C. Samarakoon‡,§, Dhanushka N. Wanasinghe|,, Jayarama Bhat#, Putarak Chomnunti¤
‡ Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand
§ School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand
| CIFOR-ICRAF China Program, World Agroforestry (ICRAF), Kunming 650201, China
¶ Centre for Mountain Futures (CMF), Kunming Institute of Botany, Chinese Academy of Sciences, Honghe 654400, Yunnan, China
# Department of Botany, Goa University, Goa, India
¤ School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
Open Access

Abstract

Background

Smaragdiniseta musae is introduced as a leaf-based novel saprobic species from Musa. Multi-gene phylogenetic analyses of internal transcribed spacer (ITS), RNA polymerase II second largest subunit (rpb2) and β-tubulin (tub2) data support the taxonomic placement of the new collection in Smaragdiniseta (Hypocreales, Stachybotryaceae). The novel species is characterised by cup-shaped sporodochia covered by numerous peripheral setae and simple hyaline, guttulate conidia produced by the ultimate branches (phialides) of conidiophores.

New information

This is the first report of Smaragdiniseta from Thailand and on Musaceae. In addition, we report Albifimbria verrucaria for the first time from Thailand, based on morpho-molecular evidence.

Keywords

one new species, banana, fungi, host, hyaline conidia, Musaceae, myrothecium-like, saprobes

Introduction

Crous et al. (2014) established Stachybotryaceae and Lombard et al. (2016) further revised the family, based on phenotypic characteristics and molecular analyses of LSU, ITS, rpb2, cmdA, tef1 and tub2 sequences. These arguments were accepted in the recent classifications of Sordariomycetes by Hyde et al. (2020) and Wijayawardene et al. (2020). The polyphyletic nature of Myrothecium (Chen et al. 2016) was further justified by Lombard et al. (2016) who established 13 new genera in Stachybotryaceae with myrothecium-like morphology viz. Albifimbria, Capitofimbria, Dimorphiseta, Gregatothecium, Inaequalispora, Myxospora, Neomyrothecium, Paramyrothecium, Parvothecium, Smaragdiniseta, Striaticonidium, Tangerinosporium and Xenomyrothecium. Smaragdiniseta was established to accommodate S. bisetosa (=Myrothecium bisetosum), which is characterised by cup-shaped sporodochia with straight, hyaline, smooth-walled peripheral setae (Rao and De Hoog 1983, Lombard et al. 2016). The setae grow rapidly from sporodochia and are soon covered by a weft of emerald green, echinulate marginal hyphae (Rao and De Hoog 1983). The sexual morph of Smaragdiniseta is yet to be determined.

The taxonomic placement of M. bisetosum was doubted by Rao and De Hoog (1983) as it is morphologically resembling Sarcopodium by the formation of setae (Ehrenberg 1818). However, Rao and De Hoog (1983) distinguished M. bisetosum from Sarcopodium by the macroscopic colour of the conidiomata. They interpreted the new collection that was similar in morphology to Sarcopodium and Myrothecium. In the phylogenetic analyses of Lombard et al. (2016), M. bisetosum formed a monophyletic lineage sister to Albifimbria that was well separated from Myrothecium. Smaragdiniseta remains monospecific (Index Fungorum 2022, Cooper and Kirk 2022) and no additional taxonomic work has been conducted since Lombard et al. (2016).

In Lombard et al. (2016), the ex-neotype strain of Myrothecium verrucaria formed another highly supported monophyletic clade in Stachybotryaceae. Albifimbria was introduced to classify this lineage and M. verrucaria was synonymised and typified under Albifimbria. In addition, A. lateralis, A. terrestris and A. viridis were introduced as novel taxa (Lombard et al. 2016). Albifimbria is characterised by the production of verrucose setae around the sporodochia (Lombard et al. 2016). In addition, some Albifimbria species bear funnel-shaped mucoid appendages in conidia (Lombard et al. 2016). Currently, four species of Albifimbria are listed in Index Fungorum (Cooper and Kirk 2022).

We are studying the saprobic fungi associated with Musa spp. from Thailand with the intention of providing a better understanding of their taxonomy, based on both morphology and phylogeny (Samarakoon et al. 2020a, Samarakoon et al. 2020b, Samarakoon et al. 2021a, Samarakoon et al. 2021b). This study is aimed at documenting two myrothecium-like taxa in Stachybotryaceae isolated from the dead leaves of Musa. Based on morphological illustrations, descriptions and phylogenetic analyses, we introduce one of our collections as Smaragdiniseta musae sp. nov. from Mae Sai, Chiang Rai, Thailand. This is the second species in Smaragdiniseta which further validates the taxonomic establishment of Lombard et al. (2016) and breaks the monotypic nature of the genus. In addition, we report Albifimbria verrucaria on Musa sp. as a new country record to Thailand.

Materials and methods

Sample collection, morphological studies and isolation

Dead leaves of Musa with characteristic sporodochia were collected from Thailand from January to October 2019. Specimens were transferred to the laboratory in small cardboard boxes. Fungi were observed using a Motic SMZ 168 series microscope (Motic Asia, Kowloon, Hong Kong). Conidiomata were mounted on glass slides in tap water and lactoglycerol for examination and photomicrography. The specimens were further observed using a Nikon ECLIPSE 80i compound microscope (Nikon Instruments Inc., Melville, NY, USA) and photographed using a Canon 550D digital camera (Canon Inc., Ota, Tokyo, Japan). Measurements were taken with the aid of Tarosoft (R) Image Frame Work programme. More than 10 measurements were made for the structures. The images were further arranged using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, USA).

Single spore isolations for the samples were conducted according to Senanayake et al. (2020). Germinated conidia were individually transferred to potato dextrose agar (PDA) plates and incubated at 25°C. Colony characters were examined after two weeks. Dried herbarium specimens were deposited in the Mae Fah Luang University Herbarium (Herb. MFLU), Chiang Rai, Thailand. Living cultures in PDA were deposited in the Culture Collection of Mae Fah Luang University (MFLUCC). Facesoffungi numbers (Jayasiri et al. 2015) and MycoBank numbers (http://www.MycoBank.org) were received for the isolates. The illustrations and descriptions were submitted to the GMS MICROFUNGI (gmsmicrofungi.org) database (Chaiwan et al. 2021). The finalised alignment and tree were submitted to Zenodo (https://zenodo.org/record/6867700#.Ytghz3ZBzIU).

DNA extraction, PCR amplification and sequencing

DNA was extracted from the mycelium of 14 days-old cultures. The mycelium was crushed using a plastic pestle and DNA was extracted using Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) following the manufacturer's guidelines. Three gene regions; viz. internal transcribed spacer (ITS), partial β-tubulin (tub2) and partial second largest subunit of the DNA-directed RNA polymerase II (rpb2), were amplified using ITS5/ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995) and fRPB2-5f/fRPB2-7cR (Liu et al. 1999), respectively.

Polymerase chain reaction (PCR) was conducted using the following protocol. The total volume of the PCR reaction was 25 μl and comprised 12.5 μl of 2 × Power Taq PCR MasterMix (a premix and ready-to-use solution, including 0.1 Units/μl Taq DNA Polymerase, 500 μm dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris-HCL pH 8.3, 100 mM KCl, 3 mM MgCl2, stabiliser and enhancer), 1 μl of each primer (10 pM), 2 μl genomic DNA and 8.5 μl of deionised water. The total reaction comprised 35 cycles. The annealing temperatures were according to Lombard et al. (2016) and Samarakoon et al. (2021b). The amplified PCR fragments were sent to TsingKe Biological Technology (Beijing) Co., China for sequencing. DNA sequence data obtained were deposited in GenBank (https://www.ncbi.nlm.nih.gov/).

Sequence alignment

Newly-generated sequence data of different gene regions were subjected to BLAST searches using BLASTn in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to retrieve similar sequences. The results and initial morphology indicated that our strains belong to Stachybotryaceae (Hypocreales). The collection numbers for these similar sequences (Table 1) were then downloaded from GenBank, based on BLASTn results and Lombard et al. (2016). Single gene alignments were made by MAFFT v. 7.036 (http://mafft.cbrc.jp/alignment/server/large.html, Katoh et al. 2019) using the default settings and later refined as necessary using BioEdit v. 7.0.5.2 (Hall 1999).

Table 1.

Names, culture collection numbers and their respective GenBank accession numbers of the Stachybotryaceae taxa that have been subjected to phylogenetic analyses. Type strains are superscripted with T and new collections are indicated in bold black.

Species

Strain

GenBank Accession Numbers

ITS

rpb2

tub2

Albifimbria lateralis

CBS 117712T

KU845881

KU845919

KU845957

A. terrestris

CBS 109378

KU845882

KU845920

KU845958

A. terrestris

CBS 126186

KU845883

KU845921

KU845959

A. terrestris

CBS 127838

KU845884

KU845922

KU845960

A. verrucaria

CPC 30056

KU845885

KU845923

KU845961

A. verrucaria

CBS 328.52T

KU845886

KU845924

KU845962

A. verrucaria

CBS 188.46

KU845888

KU845926

KU845964

A. verrucaria

MFLUCC 22-0017

ON563487

NA

ON586153

A. viridis

CBS 449.71T

KU845898

KU845936

KU845974

A. viridis

CBS 127346

KU845899

KU845937

KU845975

Alfaria caricicola

CBS 113567T

KU845983

KU846001

KU846014

Alf. terrestris

CBS 168.97

KU845987

KU846005

KU846018

Capitofimbria compacta

CBS 111739T

KU846287

KU846349

KU846404

Dimorphiseta terrestris

CBS 127345T

KU846314

KU846375

KU846431

Fusarium sambucinum

CBS 146.95

KM231813

KM232381

KM232078

Gregatothecium humicola

CBS 205.96T

KU846315

KU846376

KU846432

Inaequalispora prestonii

CBS 175.73T

KU846316

KU846377

KU846433

Myrothecium inundatum

CBS 196.74T

KU846451

NA

KU846532

M. simplex

CBS 582.93T

KU846456

NA

KU846537

Myxospora aptrootii

CBS 101263T

KU846458

KU846496

KU846539

Neomyrothecium humicola

CBS 310.96T

KU846467

KU846505

NA

Paramyrothecium acadiense

CBS 123.96

KU846288

KU846350

KU846405

Pa. breviseta

CBS 544.75T

KU846289

KU846351

KU846406

Pa. cupuliforme

CBS 126167T

KU846290

KU846352

KU846407

Pa. foeniculicola

CBS 331.51T

KU846292

KU846354

KU846409

Pa. foliicola

CBS 419.93

KU846293

KU846355

KU846410

Pa. humicola

CBS 127295T

KU846295

KU846356

KU846412

Pa. nigrum

CBS 116537

KU846296

KU846357

KU846413

Pa. parvum

CBS 142.42

KU846297

KU846358

KU846414

Peethambara sundara

CBS 521.96

KU846470

KU846508

KU846550

Septomyrothecium maraitiense

MUCL 47202T

NA

KU846510

NA

Smaragdiniseta bisetosa

CBS 459.82T

KU847229

KU847281

KU847319

S. musae

MFLUCC 22-0015T

ON563485

ON586151

ON572191

S. musae

MFLUCC 22-0016

ON563486

ON586152

ON572192

Striaticonidium brachysporum

CBS 131.71

KU847230

KU847282

KU847320

Tangerinosporium thalitricola

CBS 317.61T

KU847243

NA

KU847333

Virgatospora echinofibrosa

CBS 110115

KU847244

KU847293

KU847334

Xenomyrothecium tongaense

CBS 598.80T

KU847246

KU847295

KU847336

Xepicula crassiseta

CBS 392.71T

KU847247

KU847296

KU847337

Abbreviations of culture collections; CBS: CentraalbureauvoorSchimmelcultures, Utrecht, The Netherlands, CPC: Working collection of Pedro Crous housed at CBS, MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand, MUCL: Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada, NA: Sequence data are not available in GenBank.

Phylogenetic analyses

Maximum Likelihood (ML) trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008, Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) with GTR+I+G model of evolution for single and multi-gene alignments. Bootstrap supports were gained by running 1000 pseudo-replicates. Maximum Likelihood (ML) bootstrap values equal to or greater than 60% are given above each node of the phylogenetic tree (Fig. 3).

Figure 1.  

Smaragdiniseta musae (MFLU 22-0047, holotype) a, b sporodochia on the host; c, d cupulate sporodochia; e-i, q setae and marginal hyphae; j-l attachments of conidiophores and phialides; m-t conidia; u colony on PDA after 8 weeks. Scale bars: 400 μm (a, b), 100 μm (c, d, i, q), 50 μm (e-h), 20 μm (j), 10 μm (k-t).

Figure 2.  

Albifimbria verrucaria (MFLU 22-0048) a, b sporodochia on the host; c, e marginal hyphae; d, f, g conidiophores and phialides; h-l conidia. Scale bars: 400 μm (a, b), 100 μm (i-j), 50 μm (c), 10 μm (d-h), 5 μm (i-l).

Figure 3.  

Maximum Likelihood tree revealed by RAxML analyses of ITS, rpb2 and tub2 sequence data of selected genera of Stachybotryaceae, showing the phylogenetic position of Albifimbria verrucaria (MFLUCC 22-0017) and Smaragdiniseta musae (MFLUCC 22-0015, MFLUCC 22-0016). ML bootstrap supports (≥ 60%) and Bayesian posterior probabilities (≥ 0.95 BYPP) are given above the nodes, respectively. The tree is rooted with Fusarium sambucinum (CBS146.95) (Nectriaceae). Strains generated in this study are indicated in bold red. Ex-type strains are indicated in bold black. The scale bar represents the expected number of nucleotide substitutions per site.

A Bayesian analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate posterior probabilities (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (MCMC). Two parallel runs were conducted, using the default settings and with the adjustments as follows; four simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation and 20000 trees were obtained. The first 4,000 trees of the burn-in phase were discarded. The remaining 16,000 trees were taken for calculating PP in the majority rule consensus tree. Branches with Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are written above each node of the multigene tree (Fig. 3). The trees were displayed with FigTree v.1.4.0 (Rambaut 2011) and re-arranged in Microsoft PowerPoint.

Taxon treatments

Smaragdiniseta musae Samarakoon & Chomnunti, sp. nov.

Material    Download as CSV 
Holotype:
  1. scientificName:
    Smaragdiniseta musae Samarakoon & Chomnunti
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Sordariomycetes
    ; order:
    Hypocreales
    ; family:
    Stachybotryaceae
    ; taxonRank:
    species
    ; genus:
    Smaragdiniseta
    ; specificEpithet:
    musae
    ; scientificNameAuthorship:
    Samarakoon & Chomnunti
    ; continent:
    Asia
    ; country:
    Thailand
    ; stateProvince:
    Chiang Rai
    ; locality:
    Mae Sai
    ; year:
    2019
    ; month:
    June
    ; day:
    16
    ; habitat:
    Terrestrial
    ; occurrenceRemarks:
    Found on a dead leaf of Musa sp.
    ; recordNumber:
    BNS264
    ; recordedBy:
    Binu C. Samarakoon
    ; disposition:
    Living cultures: MFLUCC 22-0015 and MFLUCC 22-0016
    ; associatedSequences:
    GenBank MFLUCC 22-0015: ON563485 (ITS), ON586151 (rpb2), ON572191 (tub2); MFLUCC 22-0016:ON563486 (ITS), ON58615(rpb2), ON572192 (tub2)
    ; identifiedBy:
    Binu C. Samarakoon
    ; institutionID:
    MFLU
    ; collectionID:
    MFLU 22-0047

Description

Saprobic on dead leaves of Musa sp. Sexual morph: Undetermined. Asexual morph: Sporodochia 0.2–0.35 mm diam., cup-shaped, scattered, solitary, circular with an entire margin, initially emerald green, later becoming black, composed of hyaline, erect conidiophores, surrounded by filamentous white marginal hyphae and setae at periphery. Fungal hyphae arranged in parallel like a palisade layer, compacted, verrucose, olivaceous-brown or hyaline, sometimes warticulate, septate, branched, irregularly thick-walled, with ultimate hyphal cell rounded at apex and flat at base; hyphal cells: 10–14 × 0.8–2.5 μm (x̄ = 13.3 × 1.6 μm, n = 30). Aggregated hyphae hyaline or emerald green when immature, tan brown at maturity. Setae fast growing from marginal hyphae, numerous, aggregating as a pale grey brush, elongated, straight or slightly curved, tapering towards apex, hyaline, septate, unbranched, apiculate at apex, sometimes caudate, truncate or rounded, swollen to globose to ovate or rounded at base, setae wall 0.4–1.5 μm (x̄ = 0.8 μm, n = 30) thick, often smooth, sometimes rough with hyaline acellular coatings, (60–)90–160(–250) × 0.8–2.8 μm (x̄ = 126.4 × 1.6 μm, n = 30); septa 4–8 μm apart. Marginal hyphae often coiling or growing around the setae or projecting out, forming a wefty cover around setae, 95–125 μm (x̄ = 106 μm, n = 30) wide. Conidiophores 4–14 × 1.3–2.5 μm (x̄ = 12.9 × 1.7 μm, n = 30), macronematous, hyaline, smooth, thin-walled, arising from sub hyaline or pale brown, with slightly thickened and swollen basal cells, often with a narrow truncate base, wider in the middle, tapering to rounded at apex, 3–4 × 5–6 μm (x̄ = 3.5 × 5.5 μm, n = 10), septate, rarely unbranched, mostly branched and with 2–3 conidiogenous sub-branches at each node. Conidiogenous cells phialidic, rough or thin-walled, rod-shaped, elongated or ovate, 4–9 × 1–3 μm (x̄ = 6.8 × 1.9 μm, n = 20), without a collarette, pinching off simple conidia at the apex of each phialide. Conidia simple, hyaline, smooth, thin-walled, elliptic or slightly ovate, rounded at one end and acute at other end, with two distinct guttules at vertical ends, sometimes with 3–5 guttules, 6–10 × 2–3.5 μm (x̄ = 8.3 × 2.7 μm, n = 10).

Culture characteristics. Conidia germinating on PDA after 48 hours, germ tubes being produced from the acute end. Colonies growing on PDA reaching 20 mm diam. after 2 weeks in light conditions at 25°C, mycelium mostly immersed, not slimy, cottony, pinkish-white, dense in the middle and comparatively sparse at the periphery. Radially and unevenly striated, colonies have a slightly wrinkled appearance from the top. The formation of sporodochia was not observed in mature colonies.

Etymology

The species epithet reflects the host genus, Musa.

Notes

Based on BLASTn search results of ITS, tub2 and rpb2 sequence data, Smaragdiniseta musae (Fig. 1) showed a high percentage identity (ITS = 97.23%, tub2 = 89.16% and rpb2 = 91.10%) without gaps to S. bisetosa (CBS 459.82). In the multigene phylogeny, S. musae clustered with S. bisetosa in having strong statistical support (100% ML, 1.00 BYPP) (Fig. 3). The base pair comparison of ITS, tub2 and rpb2 of our new taxon revealed 3.14% (17/540), 12.36% (35/283) and 9.34% (63/674) nucleotide differences with S. bisetosa. Besides, S. musae differs from S. bisetosa by the conidial morphology. The conidia of S. musae have two distinct guttules at the vertical poles. In addition, some conidia bear minute guttules at the centre. However, the taxonomic illustration and description of S. bisetosa did not indicate the guttule formation in the conidia (Rao and De Hoog 1983). Moreover, the conidia of S. bisetosa are obclavate, narrowly ellipsoidal or rod-shaped, whereas our new taxon has elliptic or slightly ovate-shaped conidia with a rounded top and an acute base. Both ends of the conidia of S. bisetosa are rounded or sometimes are found with a truncate base (Rao and De Hoog 1983). We have not observed a truncate base in the conidia of S. musae. Furthermore, the marginal hyphae of S. musae often coil or grow around the setae, but have never overgrown. The marginal hyphae always reach around 95–125 μm of the setae and terminate at a point. According to the description of Rao and De Hoog (1983), in S. bisetosa, the marginal hyphae always covered the entire setae. Our new collection is similar in morphology to the other genera in Stachybotryaceae in having conidiophores where the ultimate branches become phialides (Lombard et al. 2016). This feature phenotypically justifies the placement of our new collection in Stachybotryaceae. Based on distinct morphological characteristics and strong statistical support from our molecular phylogeny, Smaragdiniseta musae is, therefore, herein introduced as a new species on Musa sp. from Chiang Rai Province, Thailand. This is the first report of Smaragdiniseta on Musaceae from Southeast Asia. In addition, S. musae is the second taxon that is being described in this genus.

Albifimbria verrucaria (Alb. & Schwein.) L. Lombard & Crous

Material    Download as CSV 
  1. scientificName:
    Albifimbria verrucaria (Alb. & Schwein.) L. Lombard & Crous
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Sordariomycetes
    ; order:
    Hypocreales
    ; family:
    Stachybotryaceae
    ; taxonRank:
    species
    ; genus:
    Albifimbria
    ; specificEpithet:
    verrucaria
    ; scientificNameAuthorship:
    (Alb. & Schwein.) L. Lombard & Crous
    ; continent:
    Asia
    ; country:
    Thailand
    ; stateProvince:
    Chiang Rai
    ; municipality:
    Mae Sai
    ; year:
    2019
    ; month:
    June
    ; day:
    25
    ; habitat:
    Terrestrial
    ; occurrenceRemarks:
    Found on a dead leaf of Musa sp.
    ; recordNumber:
    BNS292
    ; recordedBy:
    Binu C. Samarakoon
    ; disposition:
    Living culture: MFLUCC 22-0017
    ; associatedSequences:
    GenBank: MFLUCC 22-0017: ON563487(ITS), ON586153(tub2)
    ; identifiedBy:
    Binu C. Samarakoon
    ; institutionID:
    MFLU
    ; collectionID:
    MFLU 22-0048

Description

Saprobic on dead leaves of Musa sp. Sexual morph: Undetermined. Asexual morph: Sporodochia cupulate or discoid, scattered or gregarious, having irregular or rounded outline composed of white marginal hypha, with conidial mass flattened or convex, pale olivaceous-green at an immature stage, black and shiny at maturity, 10–18 × 0.8–3 μm (x̄ = 12.3 × 2.4 μm, n = 20). Stroma rarely well-developed, usually with a thin layer of isodiametric or elongated hyaline cells 15–25 (x̄ = 16.8 μm, n = 10). Setae: not observed. Marginal hyphae hyaline, usually verrucose, septate, curling and coiling, some branched, rounded or blunt at apex, 1.5–4 (x̄ = 3.3, n = 20) in diam. Conidiophores arising from a thin stromatic layer, hyaline, smooth, 30–48 × 1–2 μm (x̄ = 42.2 × 1.7 μm, n = 30) septate, branching repeatedly, forming 2–4 branches at each level, with ultimate branches becoming phialides, which give rise to numerous conidia, conidiophores sometimes also arising from the hyphae. Phialides hyaline, rough-walled 30–48 × 1–2 μm (x̄ = 42.2 × 1.7 μm, n = 30), 3–7 in a whorl, closely packed in a dense parallel layer, cylindrical, hyaline, collate at the base, rounded or acute at apex, sometimes slightly tapering towards apex, 8–16 × 1–3.5 μm (x̄ = 12.2×1.8 μm, n = 30). Conidia broadly fusiform, always pointed at one end, mostly truncate or rounded at the other end, hyaline, sometimes sub hyaline, smooth, 5–9.5 × 2–3.5 μm (x̄ = 7.6 × 3.0 μm, n = 30).

Culture characteristics. Conidia germinated on PDA after 12 hours. Colonies growing on PDA reaching 40 mm diam. after 2 weeks in the light conditions at 25°C, mycelium is mostly immersed, not slimy, cottony, white, dense in the middle and comparatively sparse at the periphery, fast-growing. Sporodochia formed after 12 days at the centre as a black uneven ring.

Notes

Based on BLASTn search results of ITS, tub2 and rpb2 sequence data, our stain (MFLUCC 22-0017) showed a high similarity (ITS = 100% tub2 = 100% and rpb2 = 99%), excluding gaps to Albifimbria verrucaria (CBS 188.46). In the multigene phylogeny, MFLUCC 22-0017 grouped with A. verrucaria strains with strong statistical supports (97% ML, 1.00 BYPP) (Fig. 3). Morphologically, our collection (Fig. 2) is similar to the descriptions of Tulloch (1972) and Lombard et al. (2016). Albifimbria verrucaria has previously been reported from Musa sp. as a saprobe in Venezuela (Dennis 1970). This is the first report of Albifimbria from Thailand. MFLUCC 22-0017 is the first saprobic A. verrucaria strain found in Thailand. In addition, this is the second report of Albifimbria on Musaceae.

Analysis

Phylogenetic analyses

The combined ITS (1–599), rpb2 (604–1281) and tub2 (1286–1596) gene alignment was composed of 40 sequences that represented some of the selected taxa in Stachybotryaceae. The best scoring RAxML tree is presented (Fig. 3) with a final ML optimisation likelihood value of -13217.698. The matrix had 714 distinct alignment patterns with 15.47% of undetermined characters or gaps. Estimated base frequencies were: A = 0.239962, C = 0.278798, G = 0.255871, T = 0.225369; substitution rates AC = 1.609807, AG = 6.303653, AT = 1.525739, CG = 1.563045, CT = 10.838331, GT = 1.0; proportion of invariable sites I = 0.479596; gamma distribution shape parameter α = 0.654983. All trees (ML and BI) resulting from the multi-gene alignment were equal in topology, without notable differences from Lombard et al. (2016). Smaragdiniseta musae (MFLUCC 22-0015, MFLUCC 22-0016) formed an independent lineage sister to S. bisetosa (CBS 459.82) (ML = 100%, BYPP = 1.00) with strong statistical support. In addition, MFLUCC 22-0017 grouped with Albifimbria verrucaria (CBS 188.46) (ML = 97%, BYPP = 1.00), respectively. RAxML trees generated from single gene allignments of ITS, rpb2 and tub2 sequences were attached as supplementray data (Suppl. material 1).

Discussion

Smaragdiniseta has been previously documented as a saprobe only from terrestrial habitats (Rao and De Hoog 1983). There were no reports on pathogenic and endophytic lifestyles or sexual morphs that represent the genus. Smaragdiniseta was only discovered in India, while no other reports have been published on the occurrence worldwide. Albifimbria was subsequently discovered to show all three nutritional modes viz. as endophytes (Li et al. 2020, Wei et al. 2022), as pathogens (Gilardi et al. 2020, Herman et al. 2020, Rehman et al. 2021, Sharma et al. 2021) and as saprobes (Tulloch 1972). However, Albifimbria has mostly been discovered from plant hosts in terrestrial habitats (Tulloch 1972). In addition, the genus has also been reported in human blood (Masetti et al. 2020) and soil (Gurung et al. 2019, Murgia et al. 2019). Several genera of Stachybotryaceae, such as Albifimbria, Memnoniella, Myrothecium and Stachybotrys, are capable of producing bioactive compounds (Pervez et al. 2015, Wang et al. 2015, Li et al. 2020, Sun et al. 2020).

Albifimbria verrucaria has been reported as a plant pathogen that causes stem necrosis and leaf spots on various crops, such as Glycine latifolia (Herman et al. 2020), leafy vegetables (Matić et al. 2019, Rehman et al. 2021), ornamental crops (Matić et al. 2019) and tomato (Gilardi et al. 2020). In addition, A. verrucaria was also reported as an antagonistic agent on grapevine pathogens (Li et al. 2020). Additionally, A. verrucaria has been applied as a bio-pesticide to many weeds and nematodes (Assaf 2020). Albifimbria verrucaria can produce many lytic enzymes (viz. lipase, protease and kinase) which can degrade the cuticles of insects and, thus, can be used as an insecticide (Assaf 2020, Weaver et al. 2021). Chemical screening of Smaragdiniseta isolates has not been conducted so far and still, the profiles remain undiscovered. Hence, apart from the taxonomic treatments, the chemical profiles of these genera also can be investigated as they are excellent sources of secondary metabolites. In addition, A. verrucaria was reported as a human pathogen causing keratomycosis (Moreno-Flores et al. 2020, Liu et al. 2021). Hence, there are opportunities for taxonomists to conduct sampling, isolation and identification of these hidden taxa from various hosts and provide baseline data for future research work.

Acknowledgements

This project is funded by the National Research Council of Thailand (NRCT) grant number N41A640165. Binu C. Samarakoon extends her heartfelt gratitude to Mae Fah Luang University for granting the tuition scholarship for her Ph.D. studies and research and the financial support of the dissertation support grant. Putarak Chomnunti would like to thank Reinventing University 2021 for supporting the research assistant. Binu C. Samarakoon also expresses her sincere gratitude to Dr. Samantha Karunarathna and Digvijayini Bundhun for their valuable support in manuscript writing and editing. Dhanushka Wanasinghe would like to thank CAS President’s International Fellowship Initiative (grant number 2021FYB0005), the National Science Foundation of China (NSFC) under the project code 32150410362 and the Postdoctoral Fund from the Human Resources and Social Security Bureau of Yunnan Province.

References

Supplementary material

Suppl. material 1: Maximum Likelihood trees 
Authors:  Binu C. Samarakoon
Data type:  Phylogenetic
Brief description: 

Maximum Likelihood trees revealed by RAxML analyses ITS, btub and rpb2 single gene regions