Introduction
Freshwater is a diverse and complex environment for microorganisms and provides several types of habitat for fungi, e.g., plant litter (for example, fallen leaves and dec- aying wood in freshwater), soil, aquatic insects, and aqua- tic plants. Especially, plant litter is an important habitat for many kinds of fungi because it provides surfaces for adhesion and nutrient sources. Freshwater fungi were iden- tified in plant litter, and many of their characteristics are unclear.
Pleosporales Luttr. ex M.E. Barris is the largest order in he fungal class Dothideomycetes including 23 families, 332 genera, and 4,764 species [1]. The majority of species re saprobes on decaying plant litter in freshwater, marine, or terrestrial environments, but several species are also associated with living plants as parasites, epiphytes, or en- dophytes [2-4]. Montagnulaceae is the one of the families in Pleosporales and contains 7 genera and approximately 100 species [1, 5]. A tentative genus of Montagnulaceae is Paraphaeosphaeria, whose anamorph type is found to be Paraconiothyrium. Montagnulaceae fungi are known as sa- probic or parasitic species in terrestrial or aquatic envir- onments [6]. Nevertheless, only 2 species of Montagnula- ceae have been reported in Korea until now [7].
In this study, we found and described 4 species of Mon- tagnulaceae (Paraconiothyrium archidendri, Paraphaeos- phaeria sporulosa, Paraphaeosphaeria michotii, and Para- phaeosphaeria viridescens), which were isolated from plant itter in freshwater for the first time in Korea. Herein, we described phylogenetic and morphological characteristics of these species.
Materials and Methods
Isolation of fungal strains and culture conditions
We collected deposits of plant litter in a freshwater tream and pond from October to December 2015. After washing with distilled water more than twice, we incub- ated plant litter in a pretreatment liquid medium (0.05% 3-morpholinopropane-1-sulfonic acid [w/v], 0.05% KNO3 w/v], 0.025% KH2PO4 [w/v], and 0.025% K2HPO4 [w/v])at 20°C overnight. Next, 100 mL of the pretreatment me- dium was spread on a 1% water agar plate, and incubated at 20°C for 2 days. Hyphal tip and germinated conidia were isolated under a microscope and transferred onto 24-well plate of V8 agar (V8A; 8% V8 juice [v/v] and 1.5% agar [w/v] adjusted to pH 6.0 using 10N NaOH). All strains used in this study were grown on malt extract agar (MEA; 2% malt extract [w/v] and 1.5% agar [w/v]), oatmeal agar (OA; 5% oat meal [w/v] and 2% agar [w/ v]), yeast peptone dextrose agar (YPDA; 0.3% yeast ext- ract [w/v], 1% peptone [w/v], 1% dextrose [w/v], and 2% agar), and V8A at 25°C. Collection information of all strains in this study is listed in Table 1
DNA extraction and phylogenetic analysis
Fungal genomic DNA was isolated by means of the Nu- cleoSpin Plant II DNA extraction Kit (Macherey-Nagel, Düren, Germany). For identification of the fungi, ampli- fication of an internal transcribed spacer (ITS) region using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') was perf- ormed [8]. Homology searches of DNA were carried out by BLAST algorithms available at the National Center for Biotechnology Information (NCBI). For the phylogenetic analysis, MEGA6 software was used [9]. A phylogenetic tree was constructed by the neighbor-joining (NJ) method, maximum-likelihood (ML) method with 1,000 bootstrap replications, and by the minimum evolution (ME) method. Reference sequences of other fungi were obtained from GenBank at NCBI (Table 2).
Results and Discussion
Phylogenetic analysis
We obtained sequences of reference species from NCBI (https://www.ncbi.nlm.nih.gov) for the phylogenetic ana- lysis, which are listed in Table 2. A total of 23 taxa were used to analyze phylogenetic relations among the 4 unre- corded species and other Montagnulaceae species. Phylo- genetic analysis was conducted by the NJ method after ClustalW alignment of ITS sequences for identification because our target species have distinct ITS sequence variance with other closely related species. The phyloge- netic tree constructed by other statistical methods − ML method and ME method − showed a clustering pattern similar to that of NJ methods. Fig. 1 shows a phylogenetic relation between the newly recorded species and refer- ence strains in Montagnulaceae. Three strains − NNIBR FG29, NNIBRFG99, and NNIBRFG116 (isolated in Sam- cheok) − were found to be in a cluster with P. ar chi d e ndr i CBS 168.77 showing 99.38% similarity. Although we isol- ated 3 strains of P. a r chi d e ndr i from plant litter in fresh-water, the holotype of P. ar chid endr i CBS 168.77 was iso- lated from leaf spots of a leguminouse tree in Burma [10]. Three other strains − NNIBRFG403, NNIBRFG404, and NNIBRFG436 − are clustered with P. sporulosa strains CBS 105.76 and CBS 109.72 showing 100% simil- arity. Although our strains of P. sp o r ul o s a were isolated from plant litter in freshwater, CBS 105.76 was isolated from roots of Norway spruce (Picea abies), and another specimen (CBS 218.68) was isolated from wheat field soil [10]. NNIBRFG243 is in a cluster with P. mi ch o t i i strain CBS 340.86 showing 100% similarity. Some of P. mi cho t i i strains were reported as pathogens of water plants, oak, sugarcane, and endophytic fungi of timothy [11, 12]. NN IBRFG469 isolated from Jeju was found to be in a cluster with P. v i r i de s c ens strain CBS 854.73 showing 99.8% sim- ilarity. P. v i r i d e s c e n s living Ex-type culture CBS 854.73 was isolated from freshwater, just as our strain NNIBR FG469 was [10]. This phylogenetic tree showed trends similar to those of other studies on phylogenetics of Mon- tagnulaceae [13]. Above all, these results indicated that all four species (for the first time reported in Korea in this study) were isolated from a new habitat (except for P. viridescens): plant litter in freshwater. These species are worthy of further research focused on diversity of fresh- water fungi.
Phylogenetic analysis
Paraconiothyrium archidendri Verkley, Göker & St ie- low, Persoonia 32: 37 (2014) (Fig. 2)
Shape of conidiomata was pycnidial globose, and dia- meter was 250~350 (~400) μm. Color of conidiomata became black because conidia were produced inside. Co- nidia shapes were variable, most of them ellipsoid, often subglobose, round-ended. Color of conidia was brown- ish. Conidia had no septa, and their size was 3.60~6.29 × 1.98~4.87 μm (average length 5.10 ± 0.65 μm, average width 3.10 ± 0.59 μm; n = 50). Average L/W (length/ width) ratio was 1.69 ± 0.37 (n = 50). Colonies on MEA in 10 days showed growth of mycelia (50~55 mm in dia- meter) and glabrous-colorless margin; an aerial mycelium was absent. Immersed mycelium was reddish brown to cinnamon. Conidiomata developed after 20~25 days. Col- onies on OA in 10 days showed growth of mycelia (55~ 60 mm in diameter), a colorless to buff margin, and fluffy white to gray aerial mycelia. Reverse side of colonies on OA was predominantly white to tawny, in center: olivac- eous black. Conidiomata developed after 20~25 days. Colonies on YPDA in 10 days showed growth of mycelia 48~53 mm in diameter, colorless to buff margin, and highly dense light-yellowish aerial mycelia. Reverse of colonies on YPDA was predominantly lemon-colored to yellow, in center dark yellow with wrinkles. Colonies on V8A in 10 days showed growth of mycelia, 55~60 mm in diameter, colorless margin, highly fluffy aerial mycelium, and light gray aerial mycelia in center.
Specimens examined
Korea, Gangwon-do, Samcheok-si, Geundeok-myeon, Hamaengbang-ri, 37°23'14.4" N, 129°11'53.1" E, isolated from plant litter deposited in the stream Sohancheon, 13 October 2015, J. Goh, NNIBRFG29 (GenBank accession no. KY327411), NNIBRFG99 (GenBank accession no. KY 327412), and NNIBRFG116 (GenBank accession no. KY 327413).
Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Arkiv før Botanik 6 (4–5): 405 (1967) (Fig. 3)
Shape of conidiomata was pycnidial globose, and dia- meter was 250~350 (~400) μm. Color of conidiomata later turned black because of conidia produced inside. Conidia shapes were broadly fusiform to ellipsoid, ends rounded. Color of conidia was brownish. Conidia had no septa, and the size was 4.38~9.00 × 2.82~6.25 μm (aver- age length 6.73 ± 1.05 μm, average width 4.70 ± 0.69 μm;n = 60). Average L/W ratio was 1.45 ± 0.25 (n = 60). Colonies on MEA in 10 days showed growth of mycelia 50~55 mm in diameter, glabrous and colorless margin, and aerial mycelia were absent. Immersed mycelium was reddish brown to cinnamon. Conidiomata developed after 20~25 days. Colonies on OA in 10 days showed growth of mycelia 55~60 mm in diameter, wide colorless to buff margin, and white to light brown fluffy aerial mycelia. Reverse side of colonies on OA was predominantly white to tawny. Conidiomata developed after 20~25 days. Col- onies on YPDA in 10 days showed growth of mycelia 43 ~48 mm in diameter, narrow colorless to white margin, and highly dense goldenrod aerial mycelia. Center of col- ony on YPDA was weakly pinkish lemon-colored with aerial mycelium. Colonies on V8A in 10 days showed growth of mycelia 50~55 mm in diameter, wide colorless margin, and smooth yellowish brown aerial mycelium.
Specimens examined
Korea, Gyeongsangbuk-do, Mungyeong-si, Dongno- myeon, Noeun-ri, 36°46'23.3" N, 128°18'43.7" E, isolated rom plant litter deposited in the stream Geumcheon, 12 October 2015, J. Goh, NNIBRFG243 (GenBank accession no. KY327414).
Paraphaeosphaeria sporulosa (W. Gams & Domsch) Verkley, Göker & Stielow, Persoonia 32: 47 (2014) (Fig. 4)
Shape of conidiomata was pycnidial globose, and dia- meter was 120~250 (~350) μm. Color of conidiomata was initially pale to dark brown and finally black due to mature conidia inside. Conidia shape was ellipsoid, obo- void-pyriform, or subglobose. Color of conidia was initi- ally hyaline to yellowish brown. Conidia had no septa and had one large and often 1~2 additional smaller oil drop- lets. Size of conidia was 2.58~6.20 × 2.30~4.92 μm (ave- rage length 4.94 ± 0.76 μm, average width 3.48 ± 0.53 μm; n = 60). Average L/W ratio was 1.45 ± 0.33 (n = 60) Colonies on MEA after 10 days showed growth of myc- elia 46~50 mm in diameter, glabrous and with colorless margin, and hyaline reddish brown immersed mycelium aerial mycelium was absent. Conidiomata developed after 20~25 days. Colonies on OA in 10 days showed growth of mycelia 55~60 mm in diameter, wide colorless margin and fluffy light gray aerial mycelia. Reverse of colonies on OA appeared yellow in center where numerous pycnidia formed. Conidiomata developed after 7~10 days. Colonies on YPDA in 10 days showed growth of mycelia 48~53 mm in diameter, narrow colorless margin, and highly dense dove gray aerial mycelia. Center of colonies on YPDA in 10 days showed weakly pinkish lemon-colored aerial my- celium. Reverse was predominantly lemon-colored to yel- low, in center: dark yellow with wrinkles.
Specimens examined
Korea, Gyeongsangbuk-do, Andong-si, Dosan-myeon, Togye-ri, 36°43'21.0" N, 128°51'49.6" E, isolated from plant litter deposited in Nakdong river, 29 October 2015, J. Goh, NNIBRFG403 (GenBank accession no. KY327415), NNI BRFG404 (GenBank accession no. KY327416); Korea, Gyeongsangbuk-do, Yecheon-gun, Yonggung-myeon, Dae- eun-ri, 36°35'13.0" N, 128°19'38.5" E, isolated from plant litter deposited in stream Naeseongcheon, 29 October 2015, J. Goh, NNIBRFG436 (GenBank accession no. KY 327417).
Paraphaeosphaeria viridescens Verkley, Göker & Stie- low, Persoonia 32: 49 (2014) (Fig. 5)
Shape of conidiomata was pycnidial globose, and dia- meter was 250~450 μm. Color of conidiomata was init- ially light sea green, and turned black because of mature conidia inside. Shape of conidia was consistently color- less ellipsoid with thin and smooth wall. Conidia had no septa and had 1~2 large oil droplets. Size of conidia was 3.27~5.00 × 1.76~3.16 μm (average length 4.25 ± 0.36 μm, average width 2.40 ± 0.25 μm; n = 73). Average L/ W ratio was 1.80 ± 0.26 (n = 70). Sexual morph is un- known. Colonies on MEA in 10 days showed growth of mycelia 55~60 mm in diameter and colorless to dark green mycelium with smooth surface. Reverse of colonies on MEA in center was dark brown to grayish-green. Col- onies on OA in 10 days showed growth of mycelia 57~ 63 mm in diameter, slightly ruffled margin, and teal aerial mycelia. Immersed mycelium was initially colorless to very light green, later with dark green pigment. Reverse of colonies on OA in center was dark bluish green. Col- onies on YPDA in 10 days showed growth of mycelia 55~60 mm in diameter, colorless glabrous margin, and rin-ged yellow to gray aerial mycelia. Colonies on V8A in 10 days showed growth of mycelia 60~65 mm in diameter, colorless glabrous margin, and ringed olivaceous to gray aerial mycelia. This species is notable for producing green pigment diffusing in agar and for conidia with consist- ently ellipsoid shape and relatively high L/W ratio (1.80 ± 0.26), and relatively light green yellowish wall at maturity.
Specimens examined
Korea, Jeju-do, Jeju-si, Hallim-eup, Geumak-ri, 33°21' 21.9" N, 126°18'21.4" E, isolated from plant litter deposited in Geumorem, 5 December 2015, J. Goh, NNIBRFG469 (GenBank accession no. KY327418).
In this study, we for the first time reported 4 Montag- nulaceae species in Korea. In particular, these fungi were isolated from plant litter deposited in freshwater. This finding indicated that a freshwater environment may be a good habitat for coniothyrium-like fungi. Further studies on a new substrate or habitat for unknown fungi may en- hance domestic fungal biodiversity in Korea.
