Mycoscience VOL.62 (2021) 260-267 Full paper Revisiting the isolation source after half a century: Emericellopsis mirabilis on a yellow-green alga Yusuke Takashimaa, *, Takeshi Nakayamab, Yousuke Degawaa. a Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Sugadairakogen 1278-294, Nagano 386-2204, Japan b Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan ABSTRACT Fungi-algae interactions, such as lichen-forming fungi and parasitic chytrids on phytoplankton, are common in ecosystems. In contrast, interactions between filamentous fungi and soil algae that can be observed with the naked eye have been given little attention and re- main unexplored. Here, we report a fungus that was associated with a visible symptom of dead algae on a soil surface in Sugadaira-ko- gen, Nagano, central Japan. Acremonium-like conidiophores were growing on vesicles and dead bodies of a yellow-green alga, Botry- dium granulatum. The fungus was identified as Emericellopsis mirabilis based on its morphology by microscopic observation, phylogenetic analysis, and the similarity of the isolation substrate with the first description of the species. Co-culture experiments showed a filamentous cell differentiation of the alga by the fungus, but no harmful or beneficial effects on algal growth. Therefore, we speculate that E. mirabilis is a facultative parasite of B. granulatum under natural conditions. Keywords: cleistothecia, fungi-algae interaction, homothallism, soil algae, Xanthophyceae. Article history: Received 15 February 2021, Revised 30 March 2021, Accepted 30 March 2021, Available online 20 July 2021. 1. Introduction Orpurt, 1961). Species of the genus Emericellopsis, including Acre- monium tubakii W. Gams, which is phylogenetically related to this Fungi-algae interactions are widespread in both aquatic and genus, are often found in aquatic or moist habitats (Tubaki, 1973; terrestrial ecosystems, with lichen-forming fungi and parasitic chy- Zuccaro et al., 2004; Grum-Grzhimaylo, Georgieva, Debets, & Bi- trids on phytoplankton playing important roles in the biogeochem- lanenko, 2013; Phookamsak et al., 2019; Gonçalves et al., 2020). ical cycles in each of these ecosystems, respectively (Frenken et al., The number of isolates and species of this genus has increased fol- 2017; Green, Nash III, & Lange, 2008; Nash III, 2008). However, it lowing the discovery and exploration of such habitats. The yel- is still poorly understood how filamentous fungi and soil algae in- low-green alga, Botrydium granulatum (L.) Greville (Botrydiales, teract with each other. One such group of filamentous fungi is the Xanthophyceae), is regarded as a representative member of soil or genus Emericellopsis (Carreira et al., 2015; Carreira et al., 2020). mud algae and is globally widespread. This species is widely dis- Recently, some isolates phylogenetically related to this genus were tributed around wet and muddy places in Japan, such as paddy obtained from macroalgae, including brown alga (Fucus) and green fields, other agricultural fields, irrigation or drainage ditches, algae (Cladophora and Ulva) (Zuccaro, Summerbell, Gams, Schro- ponds, and wetlands in suburban residential areas (Kamiya, 1960). ers, & Mitchell, 2004; Gonçalves, Vicente, Esteves, & Alves, 2020). In the present study, an isolate of the genus Emericellopsis that oc- van Beyma thoe Kingma (1940) established the genus Emericellop- curred on B. granulatum beside an agricultural field was obtained. sis (Hypocreales, Sordariomycetes) with descriptions of E. terricola The isolate was morphologically, phylogenetically, and ecologically var. terricola J.F.H. Beyma and E. terricola var. glabra J.F.H. Beyma. identified as E. mirabilis (Malan) Stolk. As far as the authors are The genus has been considered somewhat ambiguous and was not aware, this is the first formal report of the isolation of this species reencountered in nature for a decade after the first description. from yellow-green algae. However, a gradual accumulation of isolates of this genus by sever- al researchers, as well as the discovery of antibiotic-producing iso- 2. Materials and methods lates and advances in drug discovery research, has made this genus a familiar one in the field of pharmaceutical research (Backus & 2.1. Algal sample The restrictions on travel brought about during the COVID-19 * Corresponding author: Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Sugadairakogen 1278-294, Nagano 386-2204, Japan. pandemic have encouraged researchers to focus on studying near- E-mail address: [email protected] (Y. Takashima). by places. White circles were formed on a soil surface on a farm This is an open-access paper distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivative 4.0 international license (CC BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/). doi: org/10.47371/mycosci.2021.03.009 ― 260 ― Y. Takashima et al. / Mycoscience VOL.62 (2021) 260-267 road in front of the first author’s house, located in Sugadaira-ko- USA) following the manufacturer’s instructions. For the PCR am- gen, Nagano, central Japan. A small stream is present beside the plification of the ITS1-5.8S-ITS2 region, PCR was conducted fol- farm road, which is flooded during heavy rainfall. After rain, the lowing Takashima et al. (2018) using the ITS5-LR5 primer set (Vil- place becomes muddy but is soon dry again during sunny weather. galys & Hester, 1990; White, Bruns, Lee, & Taylor, 1990) and Ex Taq Around the circles, brownish to whitish colonies of an alga were HS polymerase (Takara Bio, Otsu, Japan). For the PCR amplifica- observed. Following to the monograph of three Botrydium spp. in tion of the partial β-tubulin (tub2) gene, the T10 (O’Donnell & central Japan reported by Kamiya (1960), the alga was distin- Cigelnik, 1997) and Bt2b (Glass & Donaldson, 1995) primer set was guished from B. divisum M.O.P. Iyengar and B. tuberosum M.O.P. used. A PCR mixture containing 1.0 μL of template DNA, 1.5 μL of Iyengar based on unbranched solitary vesicles and no cysts on rhi- each primer solution, 10 μL of 2 mM dNTPs, 1.0 μL of 1.0 U/μL zoids, respectively, and identified as B. granulatum (data not KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan), 25 μL of 2× shown). The algal colony was collected, moved to the laboratory, PCR Buffer for KOD FX Neo DNA polymerase, and 10 μL of steril- and examined under the stereomicroscopes SZ61 (Olympus Corp., ized deionized water was prepared. PCR amplification of the tub2 Tokyo, Japan), SZX16 (Olympus) equipped with a camera DP21 gene was performed as follows: an initial denaturation step of 2 (Olympus), and SZH10 (Olympus) equipped with a camera EOS min at 94 °C, followed by 35 cycles of 98 °C for 10 s, 65 °C for 30 s, kiss X8i (Canon, Tokyo, Japan). Aplanospores of B. granulatum and 68 °C for 30 s. PCR products were purified using polyethylene were isolated onto Bold’s basal medium (BBM) agar using a glycol and ethanol precipitation, and a cycle sequence reaction was flame-sterilized fine needle and incubated under irradiation with then performed with a BigDye Terminator Cycle Sequencing Ready approximately 20 μmol m−2 s−1 and a 16 h light (20 °C) / 8 h dark (20 Reaction Kit (Applied Biosystems) following the manufacturer’s °C) cycle. BBM agar was made according to the following recipe instructions. For the cycle sequencing of the ITS region and the (Bischoff & Bold, 1963): 10 mL of each stock solution of 25 g/L tub2 gene, the ITS5 and ITS4 (White et al., 1990), and the T10 and NaNO3 (Junsei Chemical Co., Ltd., Tokyo, Japan), 2.5 g/L Ca- Bt2b primers were used, respectively. Cycle sequencing products Cl2·2H2O (Iwai Chemicals Company Ltd., Tokyo, Japan), 7.5 g/L were purified by ethanol precipitation, and electrophoresis was MgSO4·7H2O (Junsei), 7.5 g/L K2HPO4·3H2O (Kanto Chemical Co., performed using the Applied Biosystems 3130xl genetic analyzer Inc., Tokyo, Japan), 17.5 g/L KH2PO4 (Wako Pure Chemical Indus- (Applied Biosystems) to determine nucleotide sequences. The se- tries, Osaka, Japan) and 2.5 g/L NaCl (Junsei); as well as 6 mL of quences obtained from the forward and reverse primers of each PIV metal solution containing 750 mg/L Na2EDTA·2H2O (Dojindo gene region were assembled into a single sequence using GeneStu- Laboratories, Kumamoto, Japan), 97 mg/L FeCl3·6H2O (Wako), 41 dio Professional software version 2.2.0.0 (http://www.genestudio. mg/L MnCl2·4H2O (Wako), 5 mg/L ZnCl2 (Kanto Chemical), 4 com/). Sequence data of the isolate was deposited in GenBank mg/L Na2MoO4·2H2O (Wako), and 2 mg/L CoCl2·6H2O (Wako); and [MW595829 (ITS), LC605902 (tub2)]. finally 15 g agar (Wako) in 1 L distilled water. 2.5. Phylogenetic analysis 2.2. Isolation of Acremonium-like asexual stage ITS and tub2 sequences of Emericellopsis spp. and related spe- Conidia of an Acremonium-like asexual stage found on the vesi- cies were obtained from GenBank (Supplementary Table S1). ITS cles of B. granulatum were isolated on LCA (Miura agar) medium and tub2 sequences were aligned independently using MAFFT [0.2 g yeast extract (Difco, Sparks, MD, USA), 1 g glucose (Wako), 2 v7.453 (Katoh & Standley, 2013) and poorly aligned regions were g NaNO3 (Wako), 1 g KH2PO4 (Wako), 0.2 g KCl (Kanto Chemical), trimmed from each multiple sequence alignment with trimal v1.4 0.2 g MgSO4·7H2O (Junsei), and 15 g agar (Wako) in 1 L distilled (Capella-Gutiérrez, Silla-Martínez, & Gabaldón, 2009). Sequence water] (Miura & Kudo, 1970) using a flame-sterilized fine needle alignments were viewed using MEGA X software (Kumar, Stecher, and incubated at room temperature (ca. 20–25 °C). After the estab- Li, Knyaz, & Tamura, 2018) and poorly aligned positions at either lishment of the isolate, single-spore isolation was conducted.