Taxonomy and phylogeny of Smaragdinisetamusae sp. nov. and Albifimbriaverrucaria (Hypocreales, Stachybotryaceae) on Musa from Thailand

Abstract Background Smaragdinisetamusae 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 Albifimbriaverrucaria for the first time from Thailand, based on morpho-molecular evidence.

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 andKirk 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 myrotheciumlike 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.

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 . 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 (gmsmicrofun gi.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.

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)

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). . 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 100 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.

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. Conidiophores arising from a thin stromatic layer, hyaline, smooth, 30-48 × 1-2 μm (x= 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).

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

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 verrucaria has been reported as a plant pathogen that causes stem necrosis and leaf spots on various crops, such as  ). 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.