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Research Article
The genus Simplicillium
expand article infoDe-Ping Wei§|, Dhanushka N. Wanasinghe|#, Kevin D. Hyde§, Peter E. Mortimer|, Jianchu Xu|#, Yuan-Pin Xiao§§¤, Chitrabhanu S. Bhunjun§§, Chaiwat To-anun
‡ Chiang Mai University, Chiang Mai, Thailand
§ Mae Fah Luang University, Chiang Rai, Thailand
| Kunming Institute of Botany, Chinese Academy of Science, Kunming, China
¶ Mushroom Research Foundation, Chiang Mai, Thailand
# World Agroforestry Centre, East and Central Asia, Kunming, China
¤ Guizhou University, Guiyang, China
Open Access

Abstract

Simplicillium species have a wide host range and an extensive distribution. Some species are associated with rusts, as well as other plant pathogenic fungi and play an important role in biological control. In this study, two specimens of Simplicillium were collected from Chiang Mai Province, Thailand. Simplicillium formicae sp. nov. was isolated from an infected ant and S. lanosoniveum from Ophiocordyceps unilateralis which is a new host record. Species were initially identified using ITS gene sequences and confirmed using morphology coupled with phylogenetic analyses of a combined nrLSU, nrSSU, TEF and RPB1 dataset. Simplicillium formicae differs from other species in the genus by the presence of flask-shaped synnemata and phialides with intercalary nodes. Simplicillium lanosoniveum resembles other collections of the species by its completely solitary, tapering phialides and globose to ellipsoidal conidia which adhere in a slimly head. A key to species of Simplicillium is also provided.

Keywords

new species, Thailand, ant fungi, taxonomy, phylogeny

Introduction

Zare and Gams (2001) introduced Simplicillium to accommodate four taxa including the type species S. lanosoniveum and three other species, S. lamellicola, S. obclavatum and S. wallacei. Simplicillium species were historically placed in Verticillium sect. Prostrata which was described by Gams (1971) for prostrate conidiophore-producing species. Later, most of the species of Verticillium sect. Prostrata were reported as members in Clavicipitaceae, based on molecular data (including SSU, LSU and ITS sequences), whereas Simplicillium species consistently formed a monophyletic group apart from the other described taxa in this family (Zare et al. 2000; Gams and Zare 2001; Sung et al. 2001; Zare and Gams 2001). Recently, Clavicipitaceae was divided into three families, based on multi-gene phylogenetic analyses and Simplicillium was assigned to Cordycipitaceae (Hypocreales, Hypocreomycetidae, Sordariomycetes) (Sung et al. 2007; Maharachchikumbura et al. 2016; Wijayawardene et al. 2018). Zare and Gams (2008) excluded Simplicillium wallacei from Simplicillium and transferred it to Lecanicillium due to the basal position being closer to the latter genus than to the former genus in the cladogram of ITS data. Subsequently, ten species viz. Simplicillium chinense (Liu and Cai 2012), S. aogashimaense, S. cylindrosporum, S. minatense, S. subtropicum, S. sympodiophorum (Nonaka et al. 2013), S. lanosoniveum var. tianjinensis (Dong et al. 2014), S. calcicola (Zhang et al. 2017), S. coffeanum (Gomes et al. 2018) and S. filiforme (Crous et al. 2018) were restricted to Simplicillium, based on the phylogenetic analyses of ITS sequence data and strong morphological evidence. Its sexual-asexual connection has been established with S. lanosoniveum linked to a Torrubiella sp. (Zare and Gams 2001).

Simplicillium species have a wide distribution and are considered as mammal and plant-parasitic, symbiotic, entomopathogenic, fungicolous and nematophagous fungi, as they have a broad spectrum of hosts and substrates, such as insects, plants, rusts, nematodes, human nails, canine tissues and mushrooms, Chroococcus sp., soil, freshwater, marine and terrene environments (Zare and Gams 2001; Guo et al. 2012; Liu and Cai 2012; Dong et al. 2014; Liang et al. 2016; Sun et al. 2019). Several studies have been shown that Simplicillium species have a high ecological and economical value for biocontrol and bioactive compounds (Takata et al. 2013; Yan et al. 2015; Hyde et al. 2019). For example, Simplicillium lanosoniveum can be a phytopathogen, causing brown spots and lesions on flowers (Chen et al. 2008) or a mycoparasite on soybean rust (Ward et al. 2012; Gauthier et al. 2014) or a pathogen on aphids and other phytopathogens (Chen et al. 2017) or an anti-Trichomonas vaginalis agent (Scopel et al. 2013). Simplicillium chinense can be a biological control agent against plant parasitic nematodes (Zhao et al. 2013; Luyen 2017). Simplicillium lamellicola can suppress plant bacterial diseases and grey mould diseases of tomato (Solanum lycopersicum) and ginseng (Panax ginseng) (Dang et al. 2014; Shin et al. 2017). Simplicillium obclavatum has the ability to produce multiple xylanases and endoglucanases that have the potential to be used in biofuels, animal feed and food industry applications (Roy et al. 2013). Bioactive compounds with anti-fungal and anti-bacterial profiles and pharmaceutical exopolysaccharides have been isolated from S. lanosoniveum (Yu et al. 2013; Fukuda et al. 2014; Xing et al. 2016; Dong et al. 2018). Linear and cyclic peptides with anti-fungal and anti-viral properties have also been discovered from the secondary metabolites of S. obclavatum (Liang et al. 2016, 2017).

Recent studies have shown that Thailand supports an amazing fungal diversity with numerous new species that have the potential for biotechnological application (Hyde et al. 2018, 2019). In this study, we introduce a novel species, Simplicillium formicae from northern Thailand and a new record of S. lanosoniveum with evidence from a combination of molecular analyses and morphological characteristics to reserve a natural resource for future studies regarding biocontrol in the forestry, agricultural and pharmaceutical industries.

Material and methods

Sample collection and isolation

The Mushroom Research Centre (MRC) is a disturbed rainforest located in Chiang Mai Province, Thailand (Aung et al. 2008). The forest consists of various tall tree and lower shrubs. The climate of Chiang Mai is controlled by tropical monsoons and the weather is typically hot and humid with temperatures often close to or above 30 °C. Frequent rain and thunder showers usually last from June to late October (Chiang Mai Buddy website: https://chiangmaibuddy.com/welcome-to-chiang-mai/weather-and-climate/, accessed 26.8.2019). Two ant fungi were found anchored to the underside of two different shrubby leaves in the forest at the Mushroom Research Centre. These two fresh specimens; HKAS 102459 and HKAS 102447 were collected and placed in plastic containers and transported to the laboratory for subsequent study. Interestingly, the ant fungus HKAS 102447 was already dead and was colonised by a saprobic fungus. The isolate MFLUCC 18–1385 was separated from this saprobe which occurred on the surface of specimen HKAS 102447 via single spore isolation. The isolate MFLUCC 18–1379 was separated from specimen HKAS 102459 by directly cultivating the hyphae which covered the surface of the ant host. These two isolates were cultured with potato dextrose agar (PDA, 1% w/v peptone) and incubated at room temperature (25 °C).

Morphological studies

For long-term deposit, these two specimens were dried with allochroic silica gel to protect them from contamination of opportunistic fungi and to retain the informative taxonomic characters. The macro-morphological characters were observed with a stereoscope (Olympus SZ61) and the micro-morphological features were examined with a compound microscope (Nikon ECLIPSE Ni). Important characteristics such as mycelium, phialides and conidia were captured with a digital camera (Canon EOS 600D). Measurements of perithecia, synnemata, phialides and conidia were taken using the Tarosoft (R) Image Frame Work programme and the images used were processed with Adobe Photoshop CS3 Extended v. 10.0 (Adobe, San Jose, CA).

DNA extraction, PCR amplification and sequencing

DNA was extracted from fresh mycelium of isolates MFLUCC 18–1379 and MFLUCC 18–1385 and from stromal tissue of ant fungus HKAS 102447 (the host of isolate MFLUCC 18–1385) using a DNA extraction kit (Biospin Fungus Genomic DNA Extraction Kit, BioFlux, China), following the instructions of the manufacturer. Extracted DNA was stored at 4 °C for use in regular work and duplicated at –20 °C for long-term storage. The internal transcribed spacer (ITS1-5.8S-ITS2, ITS) was amplified with primer ITS4 and ITS5 (White et al. 1990) and was used for individual gene phylogenetic analyses. The large subunit (LSU), small subunit rDNA (SSU), translation elongation factor 1-alpha gene (TEF1-α) and RNA polymerase II largest subunit 1 (RPB1) were also amplified as described in Wei et al. (2018) and used for multi-gene phylogenetic analyses. The PCR products were sent to Sangon Company, Kunming City, Yunnan Province, China for sequencing using the above primers. Newly generated sequences, used in the study, were submitted to GenBank to be assigned their accession numbers.

Sequence alignments and phylogenetic analyses

The raw sequences were verified with Finch TV version 1.4.0 (Mccredden 2016) and assembled with BioEdit v. 7.0.9.1 (Hall 1999). Sequence data were downloaded from GenBank based on BLAST searches of ITS sequences and with reference to the recent publications (Table 1). Most Simplicillium species are lacking protein-coding genes, but ITS sequences are available for all the species that are useful in understanding the intraspecific relationships within Simplicillium (Liu and Cai 2012, Nonaka et al. 2013, Dong et al. 2014 and Crous et al. 2018). Therefore, phylogenetic analyses, based on ITS regions, were generated throughout Simplicillium for the primary identification. Multi-gene phylogenetic analysis of the combined SSU, LSU, TEF and RPB1 sequences from representative species in Hypocreales was afterwards performed to confirm the taxonomic placements of our isolates.

Table 1.

Strains and GenBank accession numbers from related references used in multi-gene tree.

Taxon Voucher no. Host/substrate SSU rRNA LSU rRNA tef1 rpb1 Reference
Akanthomyces tuberculata OSC 111002 Lepidoptera DQ522553 DQ518767 DQ522338 DQ522384 Spatafora et al. (2007)
Aschersonia badia BCC 8105 Scale insect DQ522537 DQ518752 DQ522317 DQ522363 Spatafora et al. (2007)
Aschersonia placenta BCC 7957 Scale insect DQ522538 DQ518753 DQ522318 DQ522364 Spatafora et al. (2007)
Balansia henningsiana GAM 16112=AEG96-27a Panicum sp. AY545723 AY545727 AY489610 AY489643 Spatafora et al. (2007)
Balansia pilulaeformis AEG 94-2 Poaceae AF543764 AF543788 DQ522319 DQ522365 Spatafora et al. (2007)
Claviceps fusiformis ATCC 26019 Poaceae DQ522539 U17402 DQ522320 DQ522366 Spatafora et al. (2007)
Claviceps paspali ATCC 13892 Poaceae U32401 U47826 DQ522321 DQ522367 Spatafora et al. (2007)
Claviceps purpurea GAM 12885 Dactylis glomerata AF543765 AF543789 AF543778 AY489648 Spatafora et al. (2007)
Cordyceps farinosa OSC 111005 Lepidoptera pupa DQ522558 DQ518773 DQ522348 DQ522394 Spatafora et al. (2007)
Cordyceps heteropoda OSC 106404 Cicada AY489690 AY489722 AY489617 AY489651 Spatafora et al. (2007)
Cordyceps militaris OSC 93623 Lepidoptera AY184977 AY184966 DQ522332 DQ522377 Spatafora et al. (2007)
Cordyceps ophioglossoides OSC 106405 Elaphomyces sp. AY489691 AY489723 AY489618 AY489652 Spatafora et al. (2007)
Cordyceps pruinosa ARSEF 5413 Iragoides fasciata AY184979 AY184968 DQ522351 DQ522397 Spatafora et al. (2007)
Cordyceps scarabaeicola ARSEF 5689 Scarabaeidae pupa AF339574 AF339524 DQ522335 DQ522380 Spatafora et al. (2007)
Cordyceps tenuipes OSC 111007 Lepidoptera pupa DQ522559 DQ518774 DQ522349 DQ522395 Spatafora et al. (2007)
Drechmeria balanoides CBS 250.82 Nematoda AF339588 AF339539 DQ522342 DQ522388 Spatafora et al. (2007)
Drechmeria gunnii OSC 76404 Lepidoptera larva AF339572 AF339522 AY489616 AY489650 Spatafora et al. (2007)
Drechmeria sinensis CBS 567.95 Nematoda AF339594 AF339545 DQ522343 DQ522389 Spatafora et al. (2007)
Engyodontium aranearum CBS 309.85 Spider AF339576 AF339526 DQ522341 DQ522387 Spatafora et al. (2007)
Epichloë typhina ATCC 56429 Festuca rubra U32405 U17396 AF543777 AY489653 Spatafora et al. (2007)
Hypocrella nectrioides GJS 89-104 Scale insect U32409 U47832 DQ522347 DQ522393 Spatafora et al. (2007)
Hypocrella schizostachyi BCC 14123 Scale insect DQ522557 DQ518771 DQ522346 DQ522392 Spatafora et al. (2007)
Lecanicillium antillanum CBS 350.85 Agaric AF339585 AF339536 DQ522350 DQ522396 Spatafora et al. (2007)
Lecanicillium lecanii CBS 101247=IMI 304807 Coccus viridis AF339604 AF339555 DQ522359 DQ522407 Spatafora et al. (2007)
Lecanicillium wallacei CBS 101237=IMI 331549 Lepidoptera AY184967 EF469073 EF469102 Zare and Gams (2008); Kouvelis et al. (2008)
Metacordyceps chlamydosporia CBS 101244 Mollusca DQ522544 DQ518758 DQ522327 DQ522372 Spatafora et al. (2007)
Metacordyceps taii ARSEF 5714 Lepidoptera AF543763 AF543787 AF543775 DQ522383 Spatafora et al. (2007)
Metapochonia goniodes CBS 891.72 Nematoda AF339599 AF339550 DQ522354 DQ522401 Spatafora et al. (2007)
Metarhizium album ARSEF 2082 Cofana spectra DQ522560 DQ518775 DQ522352 DQ522398 Spatafora et al. (2007)
Metarhizium flavoviride ARSEF 2037 Nilaparvata lugens AF339580 AF339531 DQ522353 DQ522400 Spatafora et al. (2007)
Metarhizium majus ARSEF 3145 Oryctes rhinoceros AF339579 AF339530 AF543774 DQ522399 Spatafora et al. (2007)
Myriogenospora atramentosa AEG 96-32 Andropogon virginicus AY489701 AY489733 AY489628 AY489665 Spatafora et al. (2007)
Ophiocordyceps acicularis OSC 128580 Coleoptera DQ522543 DQ518757 DQ522326 DQ522371 Araújo et al. (2018)
Ophiocordyceps aphodii ARSEF 5498 Coleoptera DQ522541 DQ518755 DQ522323 Araújo et al. (2018)
Ophiocordyceps brunneipunctata OSC 128576 Coleoptera DQ522542 DQ518756 DQ522324 DQ522369 Araújo et al. (2018)
Ophiocordyceps irangiensis OSC 128577 Ant DQ522546 DQ518760 DQ522329 DQ522374 Araújo et al. (2018)
Ophiocordyceps irangiensis OSC 128578 Ant DQ522556 DQ518770 DQ522345 DQ522391 Araújo et al. (2018)
Ophiocordyceps melolonthae OSC 110993 Scarabaeidae larva DQ522548 DQ518762 DQ522331 DQ522376 Araújo et al. (2018)
Ophiocordyceps nutans OSC 110994 Stink bug DQ522549 DQ518763 DQ522333 DQ522378 Araújo et al. (2018)
Ophiocordyceps ravenelii OSC 110995 Phyllophaga sp. DQ522550 DQ518764 DQ522334 DQ522379 Araújo et al. (2018)
Ophiocordyceps sphecocephala OSC 110998 Wasp DQ522551 DQ518765 DQ522336 DQ522381 Araújo et al. (2018)
Ophiocordyceps stylophora OSC 111000 Elateridae grub DQ522552 DQ518766 DQ522337 DQ522382 Araújo et al. (2018)
Ophiocordyceps unilateralis OSC 128574 Ant DQ522554 DQ518768 DQ522339 DQ522385 Araújo et al. (2018)
Ophiocordyceps variabilis ARSEF 5365 Diptera larva DQ522555 DQ518769 DQ522340 DQ522386 Spatafora et al. (2007)
Rotiferophthora angustispora CBS 101437 Rotifera AF339584 AF339535 AF543776 DQ522402 Spatafora et al. (2007)
Simplicillium calcicola LC5586 = CGMCC3.17943 Calcaire KY883301 KU746752 KX855252 Spatafora et al. (2007)
Simplicillium lamellicola CBS 116.25 Agaricus bisporus AF339601 AF339552 DQ522356 DQ522404 Spatafora et al. (2007)
Simplicillium lanosoniveum CBS 704.86 Hemileia vastatrix AF339602 AF339553 DQ522358 DQ522406 Spatafora et al. (2007)
Simplicillium obclavatum CBS 311.74 Air above sugarcane filed AF339567 AF339517 EF468798 Spatafora et al. (2007)
Tolypocladium fractum OSC 110990 Elaphomyces sp. DQ522545 DQ518759 DQ522328 DQ522373 Spatafora et al. (2007)
Tolypocladium japonicum OSC 110991 Elaphomyces sp. DQ522547 DQ518761 DQ522330 DQ522375 Spatafora et al. (2007)
Torrubiella ratticaudata ARSEF 1915 Euophrys sp. DQ522562 DQ518777 DQ522360 DQ522408 Spatafora et al. (2007)
Verticillium epiphytum CBS 384.81 Hemileia vastatrix AF339596 AF339547 DQ522361 DQ522409 Spatafora et al. (2007)

The generated sequences of each gene region were aligned separately with representative sequences using MAFFT v. 7 web server (http://mafft.cbrc.jp/alignment/server) (Kuraku et al. 2013; Katoh et al. 2017). The uninformative gaps and ambiguous regions were manually removed and different gene regions were concatenated using BioEdit v. 7.0.9.1 (Hall 1999). The maximum Likelihood (ML) analyses was performed using RAxML-HPC2 on XSEDE (8.2.10) at CIPRES Science Gateway V. 3.3 (https://www.phylo.org/portal2/home.action), with default setting, except the bootstrap iterations were set to 1,000 and the substitution model set to GTRGAMMA + I (Miller and Blair 2009). Maximum Parsimony analysis (MP) was performed by PAUP v. 4.0b10 (Swofford 2002) with the heuristic search option and Tree-Bisection-Reconnection (TBR) branch-swapping algorithm for 1000 random replicates. Branches that have a minimum branch length of zero were collapsed. Gaps were treated as “missing” and starting tree(s) were generated via stepwise addition (Hillis and Bull 1993). Tree Length [TL], Consistency Index [CI], Retention Index [RI], Rescaled Consistency Index [RC] and Homoplasy Index [HI]) were calculated for all parsimonious trees. For Bayesian analysis, the best models of each gene were selected under Akaike Information Criterion (AIC) employing MrModeltest v. 2.3 (Nylander et al. 2008) and PAUP v. 4.0b10 (Ronquist and Huelsenbeck 2003). Bayesian analysis was performed using MrBayes v. 3.1.2 (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) to evaluate posterior probabilities (BYPP) with the Markov Chain Monte Carlo sampling (MCMC) method. Trees were sampled and printed to output at every 1000 generations. The first 25% of sampled trees were discarded as part of a burn-in procedure, the rest of the trees were used to create the consensus tree and the average standard deviation of split frequencies was set as 0.01.

Phylogenetic trees were visualised with FigTree v1.4.0 (Rambaut 2006) and edited in Microsoft PowerPoint, then saved as a PDF format and finally altered to JPG format using Adobe Illustrator CS6 (Adobe Systems Inc., United States). The finalised alignments and trees were submitted in TreeBASE (http://www.treebase.org/), with submission ID 24238 (ITS) and 24240 (multi-gene).

Results and discussion

Phylogenic analysis

The combined four gene dataset comprised 60 taxa from three families (Cordycipitaceae, Ophiocordycipitaceae and Clavicipitaceae) (Table 1) with Cosmospora coccinea, Nectria cinnabarina, Ophionectria trichospora and Viridispora diparietispora as the outgroup taxa. The RAxML analysis of the combined dataset yielded a best scoring tree (Figure 1) with a final ML optimisation likelihood value of −39792.595439. The alignment comprised 3469 total characters including gaps, of which 2077 were constant, 338 variable characters parsimony-uninformative and 1054 characters parsimony-informative. The Kishino-Hasegawa (KH) test showed CI = 0.281, RI = 0.527, RC = 0.148 and HI = 0.719. The matrix had 1655 distinct alignment patterns, with 6.42% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.241091, C = 0.260362, G = 0.272837, T = 0.225710; substitution rates AC = 0.985172, AG = 2.843760, AT = 0.887714, CG = 0.898140, CT = 6.284116, GT = 1.000000; gamma distribution shape parameter α = 0.585080. MrModeltest v. 2.3 imply that GTR+I+G is the best-fit model for LSU and RPB1, SYM+I+G for SSU and TEF sequences.

Figure 1. 

Phylogram generated from maximum likelihood analysis based on combined SSU, LSU, TEF and RPB1 sequence data. Bootstrap values for maximum likelihood (ML, left) and maximum parsimony (MP, right) equal to or greater than 50% and Bayesian posterior probabilities (BYPP, middle) equal to or greater than 0.90 are placed nearby the note. The newly generated sequences are indicated in red bold.

The ITS dataset comprised 49 taxa from all Simplicillium species that are currently available in GenBank (Figure 2) with Cordyceps militaris (CBS178.59) (Cordycipitaceae, Hypocreales) as the outgroup taxon. The RAxML analysis of the ITS dataset yielded a best scoring tree (Figure 2) with a final ML optimisation likelihood value of −3155.597177. The alignment comprised 570 total characters including gaps, of which 346 were constant, 43 variable characters parsimony-uninformative and 181 characters parsimony-informative. The Kishino-Hasegawa (KH) test showed CI = 0.681, RI = 0.856, RC = 0.583 and HI = 0.319. The matrix had 283 distinct alignment patterns, with 6.45% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.232003, C = 0.283823, G = 0.254774, T = 0.229400; substitution rates AC = 2.623562, AG = 2.645665, AT = 2.248749, CG = 1.653083, CT = 5.842034, GT = 1.000000; gamma distribution shape parameter α = 0.980038. MrModeltest v. 2.3 imply that GTR+I+G is the best-fit model for ITS sequences.

Figure 2. 

Phylogram generated from maximum likelihood analysis based on ITS sequence data. Bootstrap values for maximum likelihood (ML, left) and maximum parsimony (MP, right) equal to or greater than 50% and Bayesian posterior probabilities (BYPP, middle) equal to or greater than 0.90 are placed nearby the branches, respectively. The newly generated sequences are indicated in red bold and the type species are highlighted in black bold.

The multi-gene phylogenetic analyses showed that our isolates MFLUCC 18-1379 and MFLUCC 18–1385 grouped with the remaining Simplicillium species with strong support (100% ML, 1.00 BYPP, 100% MP, Figure 1) in Cordycipitaceae. The host of isolate MFLUCC 18–1385 grouped with Ophiocordyceps unilateralis (OSC 128574) in Ophiocordycipitaceae with a significant statistical support (100% ML, 1.00 BYPP, 100% MP, Figure 1). In the individual ITS-based phylogenetic tree, the isolate MFLUCC 18-1379 constituted a close affiliation to Simplicillium obclavatum with moderate bootstrap support (68% ML, 0.93 BYPP, 87% MP, Figure 2, clade 2). The fungal isolate MFLUCC 18-1385 grouped with the remaining Simplicillium lanosoniveum strains with 85% ML, 0.99 BYPP and 67% MP support (Figure 2, clade 1).

Taxonomy

Simplicillium W. Gams & Zare, Nova Hedwigia 73(1-2): 38 (2001)

Hyperparasitic on rusts or parasitic on nematodes or occurring in soil. Asexual morph: Mycelium thin, hyaline, septate, branched, smooth-walled. Phialides arising from prostrate aerial hyphae or rope-like and flask-shaped synnemata, typically solitary, rarely in whorls of 2–3, gradually tapering towards the apex, elongate, slender, smooth-walled, phialidic. Conidia hyaline, oval, spindle-shaped, cylindrical, subglobose to ellipsoidal, fusoid to filiform, straight to curved, smooth-walled. Conidia commonly form in small globose heads, sometimes in branched, unbranched, zigzag or imbricate chains, occasionally in sympodial proliferation with cylindrical conidium-bearing denticles. Colonies of species in this genus are usually fast growing, reaching 10–38 mm within 10 days on PDA, white, reverse brownish-cream to pale yellow, margin entire, cottony, fluffy or floccose. Some species produce yellow or orange pigment. Crystals are commonly present in the agar. Sexual morph: Torrubiella (Zare and Gams 2001; Liu and Cai 2012; Nonaka et al. 2013; Dong et al. 2014; Gomes et al. 2018; Zhang et al. 2017).

In this study, we introduce a new species and a new host species as described below.

Simplicillium formicae D.P. Wei & K.D. Hyde, sp. nov.

Index Fungorum number: IF556432
Facesoffungi number: FoF 05813
Figure 3, 4

Etymology

the epithet refers to its host–ant.

Holotype

HKAS 102459; living culture: MFLUCC 18–1379.

Description

Parasitic on ant (Formicidae). Asexual morph: Hyphomycetous. Mycelium rarely septate, hyaline, smooth-walled, covering the whole body of the ant host. Synnemata 250–350 × 65–100 (xˉ = 300 × 86, n = 10) µm, forming at the head region of ant host in circular arrangement, flask-shaped, hyaline to yellowish, composed of dense hyphae, somehow curved. Phialides 25–100 × 0.5–1.5 (xˉ = 49 × 1.1, n = 20) µm, arising from procumbent hyphae or synnemata, blastic, enteroblastic, phialidic, monophialidic, discrete, terminal, unbranched, solitary, aseptate, hyaline, smooth-walled, slender, occasionally a swollen node present. Conidia 2–3.5 × 1.5–2.5 (xˉ = 2.6 × 2, n = 30) µm, globose to ellipsoidal, hyaline, one-celled, smooth-walled, round at both ends, adhering in slimy head on the tip of phialides. Sexual morph: Undetermined.

Culture characteristics

The colonies were rapid-growing on PDA medium, reaching a diameter of 2.5–3 (xˉ = 2.6, n = 9) cm, in 13 days at 22 °C, entire margin, circular, velvety and white from above, with radial crack and primrose-yellow on reverse. In vitro, Synnemata absent. Phialides 25–75 × 0.4–0.6 (xˉ = 50 × 0.55, n = 10) µm, arising from procumbent hyphae, blastic, enteroblastic, phialidic, discrete, terminal, unbranched, solitary, aseptate, hyaline, smooth-walled, relatively slender and long. Conidia 1.5–3 × 1.5–2.5 (xˉ = 2.3 × 1.7, n = 100) µm, hyaline, globose to ellipsoidal, aseptate, smooth-walled, slightly guttulate, adhering in slimy head on the tip of phialides.

Material examined

THAILAND, Chiang Mai Province, Mushroom Research Centre, on an adult ant, 1 April 2018, Deping Wei, MRC18040102 (holotype: HKAS 102459; ex-type living culture: MFLUCC 18–1379). Sequences generated from this strain have been deposited in GenBank with accession numbers: SSU = MK765046, LSU = MK766512, ITS = MK766511, TEF = MK926451, RPB1 = MK882623.

Note

Isolate MFLUCC 18–1379 has a close phylogenetic relationship with Simplicillium obclavatum, based on ITS sequence analysis. The new isolate is similar to Simplicillium obclavatum in terms of shape and dimensions of the conidia with slender phialides tapering towards the apex. However, they have a different conidial arrangement, by Simplicillium obclavatum having short-imbricate chains, whereas the new fungus has subglobose to globose head. There are numerous synnemata in a circular arrangement which can be observed in our isolate and those are absent in Simplicillium obclavatum. The comparisons of ITS sequences between our isolate MFLUCC 18–1379 and ex-type strain of Simplicillium obclavatum (CBS 311.74) show 23 bp differences within 550 bp (4.2%). Thereby, we identify our isolates as a new species according to Jeewon and Hyde (2016).

Simplicillium lanosoniveum (J.F.H. Beyma) Zare & W. Gams, Nova Hedwigia 73(1–2): 39 (2001)

Index Fungorum number: 532459 Figure 5

Cephalosporium lanosoniveum J.F.H. Beyma, Antonie van Leeuwenhoek 8: 121 (1942) (Basionym)

Ex-type

Netherlands, on hair of Cibotium schiedei in greenhouse, 1942, F.H. van Beyma, CBS123.42.

Description

Saprophytic on Ophiocordyceps unilateralis. Asexual morph: Hyphomycetous. Mycelium aseptate, hyaline, smooth-walled. Phialides 20–40 × 1.1–2 (xˉ = 30 × 1.6, n = 20) µm, arising from the prostrate mycelium, blastic, enteroblastic, phialidic, monophialidic, discrete, terminal, aseptate, hyaline, smooth-walled, solitary, tapering toward the apex. Conidia 2–4.5 × 1–3 (xˉ = 3 × 1.8, n = 60) µm, hyaline, amerospores, globose to ellipsoidal, smooth-walled, adhering in globose to ellipsoidal head at the apex of phialides. Sexual morph: Undetermined.

Culture characters

The colonies on PDA medium were rapid-growing, reaching a diam. of 5.5 cm in 30 days at 22 °C, white, entire margin, velvety, with radial cracks and primrose-yellow on the reverse.

Host and distribution: Saprophytic on fungi, endophytic or symbiotic or pathogenic on plant, parasitic on rust, nematode and insect, occurring on soil, animal hair or human bronchoalveolar lavage fluid, with a cosmopolitan distribution (see Table 2).

Table 2.

Distribution, host and available sequence data of Simplicillium lanosoniveum strains.

Species Strain no. Host and habitat Origin Available gene region Morphological description Reference
S. lanosoniveum CBS123.42 Hair of Cibotium schiedei (Plant) Netherland ITS, LSU GenBank; Zare and Gams (2001)
Cs0701 Salvinia molesta (Plant) Taiwan ITS Chen et al. (2008)
PSU-ES104 Enhalus acoroides (Plant) Trang Province, Thailand ITS Supaphon et al. (2014)
CBS 531.72 Salvinia rotundifolia (Plant) USA ITS Zare and Gams (2001)
Tr3 Salvia miltiorrhiza (Plant) China ITS GenBank
YLAC-5 Endophytic on Inula aconitum (Plant) China ITS GenBank
Endophytic on seaweed (Plant) India SSU GenBank
E1, E3, E5 Endophytes of Sophora alopecuroides (Plant) Ningxia, China SSU, ITS Yu et al. (2013)
GA-B1 Grewia asiatica (Plant) Shivalik region, Jammu, India SSU GenBank
IMI 303103b Hemileia vastatrix (Rust) Colombia ITS, SSU Zare and Gams (2001), Kouvelis et al. (2008)
AMH 9654 Rust pustules on leaves of Elaeagnus sp. India LSU, ITS Baiswar et al. (2014)
D082307-2A Soybean rust Louisiana ITS Ward et al. (2011)
vecl-02 Rust of Eleagnus latifolia India ITS GenBank
vecl-01 Rust of Eleagnus latifolia India ITS GenBank
CBS 704.86 Hemileia vastatrix (Rust) Venezuela ITS, SSU, LSU, TEF, RPB1, RPB2, ATP GenBank; Zare et al. (2000)
S-599 Coleosporium plumeriae (Rust) Campos dos Goytacazes, GJ, Brazil ITS Berlanga-Padilla et al. (2018)
D082307-2A-GFP15 Phakopsora pachyrhizi (Rust) Florida, USA Gauthier et al. (2014)
HKAS 102447 Ophiocordyceps unilateralis (Fungi) Chiang Mai, Thailand SSU, LSU, ITS, TEF, RPB1 This study
TYL001 Pseudaulacaspis pentagona (Insect) Shanxi Province, China ITS, SSU Wang et al. (2016)
SSBG2 Coccus hesperidum (Insect) The South-Siberian Botanical Garden, Russia ITS Skaptsov et al. (2017)
TAMA 173 Aphidoidea sp. (Insect) Ibaraki, Japan ITS Fukuda et al. (2014)
CHE-CNRCB 373 Diaphorina citri (Insect) Colima, USA ITS Berlanga-Padilla et al. (2018)
ARSEF 8822 Culicid (Insect) Tanzania Hubner-Campos et al. (2013)
ARSEF7550 Coccoidea (Insect) Brazil TEF GenBank
1T9BA Tick (Insect) New York, USA ITS Greengarten et al. (2011)
Btab03 Bemisia tabaci (Insect) South Korea ITS GenBank
113-8 Mosquitoes (Insect) Japan ITS Ishii et al. (2015)
S. lanosoniveum 7S Heterodera schachtii (Nematode) Iran ITS GenBank
Hair of giant panda (Animal) China ITS GenBank
2502 Bronchoalveolar lavage fluid (Human) China ITS GenBank
41559-3 Cave and mine New York State, USA ITS, LSU GenBank
CBS 321.72 Malaysia SSU, LSU, ITS Genbank; Summerbell et al. (2011)
CBS 322.72 Malaysia ITS GenBank

Material examined

THAILAND, Chiang Mai Province, Mushroom Research Centre, on Ophiocordyceps unilateralis, 19 February 2018, Deping Wei, MRC18021901 (HKAS 102447; living culture: MFLUCC 18–1385). Sequences generated from this strain have been deposited in GenBank with accession numbers: SSU = MK752791, LSU = MK752849, ITS = MK752683, TEF = MK926450, RPB1 = MK882622.

Note

Our isolate MFLUCC 18–1385 colonised on a decayed Ophiocordyceps unilateralis with white hyphae. In a thorough examination of the Ophiocordyceps unilateralis host, we found the phialides and conidia of our isolate grown on the surface of the host (Figure 5). Phylogenetically, our isolate grouped with the strains of Simplicillium lanosoniveum with high bootstrap support (85% ML, 0.99 BYPP, 67% MP, Figure 2). The nucleotides comparison between our isolate and the type strain of Simplicillium lanosoniveum (CBS123.42) showed only 5 bp differences out of 539 in the ITS region. This evidence proves that our isolate is a strain of S. lanosoniveum, according to Jeewon and Hyde (2016). Morphologically, it resembles S. lanosoniveum with solitary phialides without verticillate branches and conidia adhering on a slimy head. Most of the previous descriptions of this species were given in hand-drawings and scanning electron microscopy (SEM) patterns (Zare and Gams 2001; Ward et al. 2012; Gauthier et al. 2014). Simplicillium lanosoniveum has been reported from Enhalus acoroides (seagrass) in Trang Province, Thailand. In this study, we introduce our isolate MFLUCC 18–1385 as a new host record of Simplicillium lanosoniveum from Ophiocordyceps unilateralis and provide the updated morphological features for a better understanding of this species. Simplicillium lanosoniveum has been frequently reported as a hyperparasite of rust and plant pathogenic fungi. Therefore, this species has a high potential of being a natural source of microbial agents against microbiological diseases in commercial agriculture (Baiswar et al. 2014; Berlanga-Padilla et al. 2018). At first, we included all available sequences of S. lanosoniveum from GenBank in the individual gene tree. Some strains did not group with other strains but distributed throughout the genus in primary analyses (data not shown), so we excluded those strains from the final phylogenetic analysis. Most of the reported strains of S. lanosoniveum, including the invalid strains, are listed in Table 2 to show their distribution and host range, as well as the sequence data availability.

Figure 3. 

Simplicillium formicae (from HKAS 102459, holotype) a superficial hyphae associated with the ant host b–e flask-shaped synnemata f–k phialides bearing conidia l–p conidia. Scale bars: 1000 µm (a); 500 µm (b); 100 µm (d); 30 µm (e, f); 15 µm (j, k); 10 µm (l–p) (e stained with cotton blue solution).

Figure 4. 

Simplicillium formicae (MFLUCC 18–1379, ex-type living culture) a upper and reverse view of cultures on PDA after 30 days e–g phialides indicated with black arrow c, d, h–j conidial mass on the tip of phialides k–m conidia. Scale bars: 10 µm (c, d, f, g); 20 µm (e); 3 µm (h–j); 1 µm (k–m) (e–j stained with cotton blue solution).

Figure 5. 

Simplicillium lanosoniveum (a–f from HKAS 102447, g–r from MFLUCC 18–1385) a host (Ophiocordyceps unilateralis); b, c hyphae associated with host indicated with black arrows g, h upper and reverse view of cultures on PDA after 40 days incubation i–l conidial mass on the tip of phialides m–o phialides bearing conidia p–r conidia. Scale bars: 15 µm (i–m); 10 µm (d–f, n, o); 3 µm (p–r) (i, l–n stained with cotton blue solution).

Ophiocordyceps unilateralis (Tul. & C. Tul.) Petch, Trans. Br. mycol. Soc. 16(1): 74 (1931)

Figure 6

Description

Parasitic on ants (Formicidae). Sexual morph: Stromata up to 14 mm in length, 0.5 mm wide in the broadest part, cylindrical, brown, slightly tapering towards the apex, single, piercing through the dorsal neck region of the ant host. Ascomatal cushion hemisphere, up to 1.2 mm in diam., laterally attaching to the erect stroma stalk, dark brown, with ostioles protruding from the cushions. Perithecia 200–400 × 50–120 (xˉ = 294 × 81, n = 10) µm, sub-immersed, flask-shaped. Asci and ascospores were too old to observe their features. Asexual morph: Undetermined.

Note

This collection was already decayed and was colonised by other fungi which we introduced as a new host record of Simplicillium lanosoniveum from Thailand. The outline of this specimen was intact, while its asci and ascospores were too old to analyse. We retrieved DNA through direct sequencing from the stromal tissue.

Sequences generated from this specimen have been deposited in GenBank with accession numbers: SSU = MK752759, LSU = MK752812, ITS = MK752874. The herbarium material is deposited at KUN herbarium, Yunnan Province, China. In the multi-gene phylogenetic tree, this collection groups with Ophiocordyceps unilateralis (OSC 128574) with a strongly supported bootstrap value (100% ML, 1.00 BYPP, 100% MP, Figure 1). Therefore, we identify this collection as O. unilateralis, based on its morphologic features and molecular evidence.

Figure 6. 

Ophiocordyceps unilateralis (from HKAS 102447) a stroma emerging from host b ascomata on stroma c host (Formicidae) d, e decayed perithecia. Scale bars: 500 µm (b, c); 300 µm (d); 100µm (e).

Key to accepted species of Simplicillium

1a Conidia formed in sympodia S. sympodiophorum
1b Conidia solitary, borne on the tip of phialides S. calcicole
1c Conidia aggregate in chains 2
1d Conidia aggregate in subglobose to ellipsoidal heads 3
1e Conidia aggregate in globose heads 4
2a Conidia 2.5–3.5 × 1–2 µm, obclavate to ellipsoidal, formed in short imbricate chains S. obclavatum
2b Conidia 3.5–5.0 × 1.0–1.5 μm, oval, ellipsoidal or cylindrical, formed in vertical chains S. chinense
2c Conidia 7.2–12.5 × 1 µm, long, fusiform to short filiform, hyaline, straight to curved, formed in vertical chains S. filiforme
3a Phialides 15–50 × 0.7–1.0 µm, colonies light to dark-brown reverse on PDA, usually with yellow pigment diffusing into the agar S. lamellicola
3b Phialides 11–44 (–70) × 1.0–2.4 µm, colonies cream-coloured reverse on PDA, no diffused pigment S. coffeanum
4a Present flask-shaped synnemata S. formicae
4b Synnemata absent 5
5a Conidia cylindrical 6
5b Conidia globose to subglobose or ellipsoidal 7
6a Phialides 23–53 × 1.2–2.0 µm, long S. cylindrosporum
6b Phialides 17–32 × 1.2–2.0(–2.5) µm, short S. aogashima
7a Phialides 35–75 × 1.2–3.0 µm, conidia 4.5–6.0 × 2.5–3.5 µm, colonies light yellow to deep tawny in reverse view on PDA S. lanosoniveum var. tianjinensis
7b Phialides 15–39 × 0.7–1.9 µm, conidia 1.5–3 × 0.7–1.3 µm, colonies brownish-cream to pale yellow reverse on PDA S. lanosoniveum
7c Phialides 11–31(–47) × 1.0–1.7 µm, conidia 2.0–3.5 × 1.8–2.5(–2.8) µm, colonies brown reverse on PDA S. minatense
7d Phialides (15–)20–42(–50) × 1.0–2.3 µm; conidia 2.3–4.0(–4.5) × 1.5–3.3 µm, colonies brownish-orange to brown reverse on PDA S. subtropicum

Conclusion

A new species Simplicillium formicae and a new host record species Simplicillium lanosoniveum from Ophiocordyceps unilateralis were introduced, based on phylogenetic analyses and morphological evidence. The host and distribution of S. lanosoniveum was summarised and a key to Simplicillium was provided.

Acknowledgements

We are grateful to the Thailand Research Fund (TRF) grant no DBG6080013 entitled ‘the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species’’ for its financial support. Dhanushka Wanasinghe would like to thank CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008) and the 64th batch of China Postdoctoral Science Foundation (grant no.: Y913083271). Peter E. Mortimer and D.N. Wanasinghe thank the National Science Foundation of China and the Chinese Academy of Sciences for financial support under the following grants: 41761144055, 41771063 and Y4ZK111B01. We acknowledge the Kunming Institute of Botany for providing the laboratories and instruments for molecular work. We appreciate the Centre of Excellence in Fungal Research (Mae Fah Luang University) for providing the grant support for collecting trips. We thank Milan Samarakoon and Dr. Sajeewa Maharachchikumbura for their assistance with the phylogenetic analyses and Dr. Shaun Pennycook for his help with the nomenclature of the novel species.

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