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. 2023 Dec 27;51(6):401–409. doi: 10.1080/12298093.2023.2283272

Diversity of Nigrospora (Xylariales, Apiosporaceae) Species Identified in Korean Macroalgae Including Five Unrecorded Species

Wonjun Lee 1, Dong-Geon Kim 1, Rekhani H Perera 1, Ji Seon Kim 1, Yoonhee Cho 1, Jun Won Lee 1, Chang Wan Seo 1, Young Woon Lim 1,✉
PMCID: PMC10763912  PMID: 38179117

Abstract

Nigrospora (Xylariales, Apiosporaceae) consists of species of terrestrial plant endophytes and pathogens. Nigrospora has also been reported in marine environments such as mangroves, sea fans, and macroalgae. However, limited research has been conducted on Nigrospora associated with macroalgae. Here, we isolated Nigrospora species from three types of algae (brown, green, and red algae) from Korean islands (Chuja, Jeju, and Ulleung) based on phylogenetic analyses of multigenetic markers: the internal transcribed spacers (ITS), beta-tubulin (BenA), and translation elongation factor 1 (TEF1-α). A total of 17 Nigrospora strains were isolated from macroalgae and identified as nine distinct species. The majority of Nigrospora species (seven) were found on brown algae, followed by red algae (three), and then green algae (two). To our understanding, this study represents the first account of N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, N. osmanthi, N. pyriformis, and N. rubi occurring in marine environments. Additionally, this study provides the first report of the occurrence of N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, and N. osmanthi in South Korea. This study will provide valuable insights for future research exploring the functions of fungi in macroalgal communities.

Keywords: Marine fungi, taxonomy, seaweed, algicolous fungi, Ascomycota

1. Introduction

Nigrospora Zimm. is characterized by large dark conidiospores [1]. Since the first report of the type species, N. panici, from leaves in Indonesia [1], Nigrospora species have been reported globally [2–6]. The phylogenetic analysis based on the internal transcribed spacer (ITS), beta-tubulin (BenA), and translation elongation factor 1-a (TEF1-α) affirmed the placement of Nigrospora in Apiosporaceae of Xylariales [7]. Up to date, 44 Nigrospora species have been recorded in the MycoBank database (https://www.mycobank.org/; accessed on 2023.06.14). Among them, 33 species have DNA sequence data in GenBank.

Nigrospora is usually reported to occur in terrestrial environments such as indoors [8,9], lichens [5,10], and plants [4,11], but it has also been reported to occur in marine environments. Specifically, Nigrospora oryzae and Nigrospora sphaerica have predominantly been isolated from marine organisms such as corals [12], mangroves [13–15], macroalgae [16–19], sea fans [19,20], and sponges [21,22]. Furthermore, Nigrospora camelliae-sinensis has been isolated from a mangrove [23], and Nigrospora aurantiaca has been found in sponges [24]. Most of these studies aimed at discovering bioactive compounds derived from Nigrospora rather than exploring its diversity or ecological interactions.

Macroalgae are integral components of marine ecosystems, providing habitats for diverse organisms and contributing to carbon sequestration [25–27]. Microbial associations with macroalgae have been extensively studied [28,29]. Bacterial communities associated with macroalgae have been found to play roles in nutrient supply to macroalgae, defense against unwanted colonization, and even morphogenesis of macroalgae [28]. However, fungal contributions to macroalgae remain poorly understood. Given the significant value of macroalgae and their microorganisms, it is crucial to investigate the relationship between macroalgae and fungi to gain a comprehensive understanding of their ecological significance and potential application.

In this study, we investigated i) Nigrospora species inhabiting macroalgae collected from three Korean islands (Chuja, Jeju, and Ulleung) and ii) whether they have a specific correlation with algal types. We isolated 17 Nigrospora strains from brown, green, and red algae. Nine Nigrospora species were identified at the species level based on both multigenetic markers (ITS, BenA, and TEF1-α) and morphological analysis. Seven of these species are, for the first time, reported to be associated with macroalgae and marine environments. Brown algae exhibited the highest level of Nigrospora species diversity of the three algal types.

2. Materials and methods

2.1. Sampling and fungal isolation

Fourteen macroalgae samples were collected from three islands (Chuja, Jeju, and Ulleung) in South Korea in August of 2018 and 2021 (Table 1). Macroalgae were morphologically identified according to [30] and Marine Bio-Resource Information System (https://www.mbris.kr/pub/info/encyclopedia/algae.do; accessed on 2023.05.17).

Table 1.

List of strains, collection information, and GenBank accession numbers of sequences used in the phylogenetic analysis. Newly reported strains are indicated in bold and holotypes are indicated by “*”.

Species Strain number Habitat/host Country GenBank accession numbers
ITS BenA TEF1-α
Apiospora sargassi KUC21287 Sargassum fulvellum Jeju, South Korea MF615227 MF615232 MN868934
Nigrospora aurantiaca CGMCC 3.18130* = LC 7302 Nelumbo sp. China KX986064 KY019465 KY019295
  SFC20230324-M01 Polyopes sp. (Rhodophyta) Chuja, South Korea OQ726356 OQ735177 OQ735194
  SFC20230324-M02 Hypnea sp. (Rhodophyta) Chuja, South Korea OQ726355 OQ735178 OQ735195
N. bambusae CGMCC 3.18327* = LC 7114 Bamboo (leaf) China KY385307 KY385319 KY385313
N. cooperae BRIP 72440a* Heteropogon contortus Australia OP035048 OP039540 OP039539
  SFC20230324-M03 Ishige sp. (Phaeophyceae) Chuja, South Korea OQ726361 OQ735179 OQ735196
N. camelliae-sinensis CGMCC 3.18125* = LC 3500 Camellia sinensis China KX985986 KY019460 KY019293
N. covidalis CGMCC 3.20538* Lithocarpus sp. China OK335209 OK431479 OK431485
  SFC20230324-M04 Ishige sp. (Phaeophyceae) Chuja, South Korea OQ726371 OQ735180 OQ735197
N. chinensis CGMCC 3.18127* = LC 4575 Machilus breviflora China KX986023 KY019462 KY019422
N. falsivesicularis CGMCC 3.19678* Saccharum officinarum China MN215778 MN329942 MN264017
N. globosa CGMCC 3.19633* Soil of cave China MK329121 MK336134 –
N. globospora CGMCC 3.20539* Petasites hybridus China OK335211 OK431481 OK431487
N. gorlenkoana CBS 480.73* Vitis vinifera Kazakhstan KX986048 KY019456 KY019420
N. guangdongensis CFCC 53917 Cunninghamia lanceolata (needle) China MT017509 MT024495 MT024493
N. guilinensis CGMCC 3.18124* = LC 3481 Camellia sinensis China KX985983 KY019459 KY019292
  SFC20230324-M05 Sargassum sp. (Phaeophyceae) Ulleung, South Korea OQ726362 OQ735181 OQ735198
N. hainanensis CGMCC 3.18129* = LC 7030 Musa paradisiaca (leaf) China KX986091 KY019464 KY019415
N. lacticolonia CGMCC 3.18123* = LC 3324 Camellia sinensis China KX985978 KY019458 KY019291
  SFC20230324-M06 Sargassum sp. (Phaeophyceae) Chuja, South Korea OQ726357 OQ735182 OQ735199
N. macarangae MFLUCC 19-0141* Macaranga tanarius Taiwan MW114318 – –
  NCYUCC 19-0177 Macaranga tanarius Taiwan MW114319 – –
N. magnoliae MFLUCC 19-0112* Magnolia liliifera China MW285092 MW438334 –
N. musae CBS 319.34* Musa paradisiaca (fruit) Australia KX986076 KY019455 KY019419
N. oryzae LC 7306 Nelumbo sp. (leaf) China KX986068 KY019612 KY019408
  LC 2689 Rhododendron sp. China KX985942 KY019469 KY019423
  LC 4265 Rhododendron sp. China KX985994 KY019518 KY019335
  LC 4338 Camellia sp. China KX986008 KY019532 KY019349
  SFC20230324-M07 Ulva sp. (Chlorophyta) Jeju, South Korea OQ726369 OQ735183 OQ735200
  SFC20230324-M08 Myagropsis sp. (Phaeophyceae) Ulleung, South Korea OQ726367 OQ735184 OQ735201
  SFC20230324-M09 Chondria sp. (Rhodophyta) Ulleung, South Korea OQ726368 OQ735185 OQ735202
  SFC20230324-M10 Grateloupia sp. (Rhodophyta) Chuja, South Korea OQ726366 OQ735186 OQ735203
N. osmanthi CGMCC 3.18126* = LC 4350 Osmanthus sp. China KX986010 KY019461 KY019421
  SFC20230324-M11 Codium sp. (Chlorophyta) Jeju, South Korea OQ726360 OQ735187 OQ735204
  SFC20230324-M12 Ulva sp. (Chlorophyta) Jeju, South Korea OQ726358 OQ735188 OQ735205
  SFC20230324-M13 Codium sp. (Chlorophyta) Jeju, South Korea OQ726359 OQ735189 OQ735206
N. philosophiae-doctoris CGMCC 3.20540* Disporum sessile China OK335213 OK431483 OK431489
N. pyriformis CGMCC 3.18122* = LC 2045 Citrus sinensis China KX985940 KY019457 KY019290
  SFC20230324-M14 Sargassum sp. (Phaeophyceae) Chuja, South Korea OQ726363 OQ735190 OQ735207
  SFC20230324-M15 Polyopes sp. (Rhodophyta) Chuja, South Korea OQ726364 OQ735191 OQ735208
  SFC20230324-M16 Laurencia sp. (Rhodophyta) Chuja, South Korea OQ726365 OQ735192 OQ735209
N. rubi CGMCC 3.18326* = LC 2698 Rubus sp. China KX985948 KY019475 KY019302
  SFC20230324-M17 Sargassum sp. (Phaeophyceae) Chuja, South Korea OQ726370 OQ735193 OQ735210
N. saccharicola CGMCC 3.19362* Saccharum officinarum China MN215788 MN329951 MN264027
N. sacchari-officinarum CGMCC 3.19335* Saccharum officinarum China MN215791 MN329954 MN264030
N. singularis CGMCC 3.19334* Saccharum officinarum China MN215793 MN329956 MN264032
N. sphaerica LC 2840 Harpullia longipetala China KX985965 KY019492 KY019318
  LC 2958 Cleyera japonica China KX985966 KY019493 KY019319
  LC 4372 Rhododendron arboreum China KX986012 KY019535 KY019351
  LC 6969 Musa paradisiaca (leaf) China KX986077 KY019584 KY019386
N. vesicularifera CGMCC 3.19333* Saccharum officinarum China MN215812 MN329975 MN264051
N. vesicularis CGMCC 3.18128* = LC 7010 Musa paradisiaca (leaf) China KX986088 KY019463 KY019294
N. zimmermanii CBS 290.62* Saccharum officinarum (leaf) Ecuador KY385309 KY385317 KY385311
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Macroalgae samples were cut into 0.5 × 0.5 cm2 pieces and placed on dichloran rose bengal chloramphenicol (DRBC) agar (Difco, Sparks, MD, USA) media supplemented with sterilized seawater (SSW). Fungal colonies grown from the samples were isolated and transferred to potato dextrose agar (PDA; Difco, Sparks, MD, USA) media supplemented with SSW. The living cultures of each isolate were stocked in 20% (v/v) glycerol at −80 °C and deposited into the Seoul National University Fungus Collection (SFC).

2.2. Molecular analyses (DNA extraction, PCR amplification, sequencing, and phylogenetic analysis)

The mycelium of each fungal isolate grown on PDA was ground by a Bead Ruptor Elite Homogenizer (OMNI International, Kennesaw, GA, USA). DNA extraction was conducted using an AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea) following the manufacturer’s protocol with a small modification where cetyltrimethylammonium bromide (CTAB) extraction solution (Biosesang, Incheon, South Korea) was used instead of the TL buffer included in the kit.

The ITS region was amplified by PCR using a C1000 thermal cycler (Bio-Rad, Richmond, CA, USA) with the primer sets ITS1F/ITS4 [31,32]. BenA and TEF1-α were subsequently amplified with Bt2a/Bt2b [33] and EF1-728F/EF2 [34,35] primers, respectively. The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 40 s, and extension at 72 °C for 60 s, followed by a final extension at 72 °C for 5 min. Purification was done using an Expin™ PCR SV kit (GeneAll Biotechnology, Seoul, South Korea), following the manufacturer’s protocol. Sanger sequencing was performed in both forward and reverse directions using the PCR primers in an ABI prism 3730xl Genetic Analyzer (Life Technologies, Gaithersburg, MD, USA) at Macrogen (Seoul, South Korea). Obtained sequences were merged using the De novo assemble function in the Geneious Prime software ver. 2023. 1. 1. (Biomatters Ltd., San Diego, CA, USA) and were then proofread and edited manually. The proofread sequences were deposited in GenBank (Table 1).

The generated sequences, reference GenBank Nigrospora sequences, and an outgroup sequence of Apiospora sargassi (KUC 21287) were aligned by each genetic marker (ITS, BenA, and TEF1-α) using MAFFT v7.490 [36] in the Geneious Prime software ver. 2023.1.1. (Biomatters Ltd., San Diego, CA, USA). The alignments were then concatenated. The best model test was investigated in MEGA 7.0.26 [37] to conduct the maximum likelihood analysis. The phylogenetic tree was inferred through RAxML analysis [38] with 1,000 replications using the GTR GAMMA model in the Geneious Prime software.

2.3. Morphological observation

For an effective observation and measurement of microscopic features, Nigrospora strains were initially subcultured on PDA and subsequently transferred to both PDA and synthetic nutrient-poor agar media (SNA; KH2PO4 1 g, KNO3 1 g, MgSO4·7H2O 5 g, KCl 0.5 g, Glucose 0.2 g, Saccharose 0.2 g, and Bacto agar 20 g per 1 L). The colonies on PDA were incubated for 7 d at 25 °C in the dark to observe the culture morphology. The color of the colonies was determined using the Methuen Handbook of Color [39]. Representative strains of each species were cultivated on SNA for conidial structure observation. All observations were done using a Nikon 80i light microscope (Tokyo, Japan). At least 30 measurements were obtained per strain to calculate the mean size of the microscopic structures. The colonies and the conidial structures were measured using ImageJ software [40].

3. Results

Seventeen Nigrospora strains were isolated from macroalgae, and ITS and two protein-coding genes (BenA and TEF1-α) were used to infer a maximum likelihood tree to identify the strains at the species level. The total number of molecular characters was 1,853 (586 in ITS, 402 in BenA, and 865 in TEF1-α). Nine Nigrospora species (N. aurantiaca, N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, N. oryzae, N. osmanthi, N. pyriformis, and N. rubi) were identified through the phylogenetic analysis (Figure 1). Each strain matched its corresponding species with at least 98% bootstrap support.

Figure 1.

Figure 1.

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The maximum likelihood tree of Nigrospora species with outgroup Apiospora sargassi (KUC21287). ITS, BenA, and TEF1-α genetic markers were used in phylogenetic analyses. The newly collected strains are enclosed in colored boxes. Bootstrap values of more than 70% are shown at the nodes. Branches that lead to nodes of bootstrap values of 100 are indicated by a bold line. Colored circles indicate the color of algae (brown, green, and red) where each strain was isolated, followed by the island on which the strain was collected.

Nigrospora species were categorized based on the algal types to which they were associated (Figure 1). The macroalgae were morphologically identified as brown (Sargassum spp., Ishige okamurae, and Myagropsis myagroides), green (Ulva sp. and Codium fragile), and red algae (Hypnea sp., Polyopes sp., Laurencia sp., Grateloupia sp., and Chondia sp.) (Table 1). The largest number of Nigrospora species (seven spp.)—N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, N. oryzae, N. pyriformis, and N. rubi—were isolated from brown algae. Three Nigrospora species (N. aurantiaca, N. oryzae, and N. pyriformis) were isolated from red algae. Nigrospora oryzae and N. osmanthi were isolated from green algae. Nigrospora oryzae appeared on all types of algae, and N. pyriformis was detected in both brown and red algae (Figure 1).

4. Taxonomy

Nigrospora cooperae Y.P. Tan, Bishop-Hurley, Bransgr. & R.G. Shivas (2022) (Figure 2A).

Figure 2.

Figure 2.

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Cultures of five Nigrospora species from this study. On the left, surface (left) and reverse (right) sides of strains on PDA are shown in halves. On the right, conidial structures on SNA media are shown. (A) N. cooperae, (B) N. covidalis, (C) N. guilinensis, (D) N. lacticolonia, and (E) N. osmanthi. Scale bar: 10 μm.

Sexual morph: Undetermined. Asexual morph on SNA: Hyphae branched, guttulate, septate, pale brown, 1.6–5.4 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells monoblastic, discrete, pale brown, doliiform to ampulliform to subglobose, 5.6–13.9 × 4–7 μm (av. = 8.6 ± 2.07 × 5.47 ± 0.83). Conidia solitary, spherical or ellipsoidal, aseptate, black, shiny, smooth-walled, spherical 10.3–14 μm (av. = 12.2 ± 0.84), ellipsoidal 11.4–14.6 × 8.2–11.4 μm (av. = 12.67 ± 0.84 × 9.63 ± 0.79).

Culture characters on PDA: Colonies sparse, velvety, fimbriate, irregular at the margin, surface gray-green (1C4) to bile yellow (30C5), reverse concolorous, not producing pigments in PDA, with prominent exudates, reaching 18–45 mm diameter in 7 d at 25 °C.

Materials examined: South Korea. South Sea, Chuja island, 33°57′11″N, 126°18′07″E, from Ishige sp. (Phaeophyceae), 31 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M03, stored in a metabolically inactive state).

Notes: SFC20230324-M03 produces prominent exudates, and the conidiogenous cells of this isolate displayed a range of length variations. However, the original description of N. cooperae does not produce any prominent exudates, and the length variation (7–10 μm) of conidiogenous cells in the holotype is lower than our isolate [41].

Nigrospora covidalis M. Raza, Qian Chen & L. Cai. (2017) (Figure 2B).

Sexual morph: Undetermined. Asexual morph on SNA: Hyphae branched, septate, guttulate, hyaline to pale brown, 1.8–4.6 μm diam. Conidiophores monoblastic, flexuous or straight, pale brown, and some conidiophores reduced to conidiogenous cells. Conidiogenous cells monoblastic, discrete, pale brown, doliiform to ampulliform, 5.4–12.7 × 3.7–8.8 μm (av. = 8.24 ± 1.81 × 6.32 ± 1.32). Hyaline vesicles delimited the conidia from their conidiogenous cells. Conidia sparse, solitary, spherical or ellipsoidal, aseptate, mostly black, discrete on aerial hyphae, spherical 10.9–15.2 μm diam. (av. = 12.9 ± 1.21), ellipsoidal 11.8–15.8 × 8.8–12.3 μm (av. = 13.86 ± 1.12 × 11.03 ± 0.81).

Culture characters on PDA: Colonies floccose, surface white (1A1) to light grey (1D1), sometimes deep green (1C8), reverse pale grey (1B1) to grayish yellow (1B4) with black patches, mostly producing yellow pigments in PDA, reaching 90 mm diameter in 4–5 d at 25 °C.

Materials examined: South Korea. South Sea, Chuja islands, 33°57′11″N, 126°18′07″E, from Ishige sp. (Phaeophyceae), 31 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M04, stored in a metabolically inactive state).

Notes: Nigrospora covidalis can be morphologically distinguished from N. musae, which is phylogenetically a sister species, by the smaller size of its conidia [7,41]. Even though the absence of vesicles is a taxonomic key to delimiting N. covidalis from N. musae [7], hyaline vesicles are observed in this isolate. Furthermore, SFC20230324-M04 produces yellow pigment and grew faster on PDA media compared to what is reported in the original description [42].

Nigrospora guilinensis Mei Wang & L. Cai (2017) (Figure 2C).

Sexual morph: Undetermined. Asexual morph on SNA: Hyphae branched, smooth, hyaline to pale brown, septate, 1.9–6.1 μm diam. Conidiophores usually reduced to conidiogenous cells, aggregated in clusters on hyphae. Conidiogenous cells monoblastic, determinate, hyaline to pale brown, smooth-walled, doliiform to ampulliform, in clusters on aerial mycelia, 5.0–13.6 × 3.7–12.9 μm (av. = 8.02 ± 2.12 × 6.53 ± 1.93). Conidia solitary, spherical or ellipsoidal, aseptate, black, shiny, smooth-walled, spherical, 10.8–13.4 μm diam. (av. = 12 ± 0.7), ellipsoidal, 11.4–14.2 × 8.7–11.2 μm (av. = 12.77 ± 0.66 × 9.97 ± 0.63).

Culture characters on PDA: Colonies wooly, cottony, margin irregular, undulate, surface and reverse white (1A1) with a few black patches, sometimes producing red pigment, reaching 54–68 mm diameter after 7 d at 25 °C.

Materials examined: South Korea. East Sea, Ulleung island, 37°30′52″N, 130°47′41″E, from Sargassum sp. (Phaeophyceae), 29 August 2018, M. S. Park & Y. W. Lim (SFC20230324-M05, stored in a metabolically inactive state).

Notes: Nigrospora guilinensis can be distinguished from closely related species by morphological characteristics such as the ability to produce diffusible pigment on PDA and the arrangement of conidiogenous cells [7]. Nevertheless, pigment production is not consistently observed in SFC20230324-M05, and the isolate forms wider conidiogenous cells (6–11 × 4–7.5 μm) compared to that reported in the original description [7].

Nigrospora lacticolonia Mei Wang & L. Cai. (2017) (Figure 2D).

Sexual morph: Undetermined. Asexual morph on SNA: Hyphae branched, smooth, hyaline, septate, 1.5–4.6 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells sometimes aggregated in clusters on hyphae, pale brown, smooth-walled, mostly spherical, sometimes doliiform, 5–13.2 × 4–8 μm (av. = 8.63 ± 1.89 × 6.09 ± 1.02). Conidia sparse, solitary, spherical or ellipsoidal, aseptate, black, shiny, smooth-walled, spherical 9.7–15.1 μm diam. (av. = 12.28 ± 1.45), ellipsoidal 10.2–15.2 × 7.9–12.1 μm (av. = 12.52 ± 1.13 × 9.73 ± 1.03).

Culture characters on PDA: Colonies floccose, entire edge, surface, and reverse white (1A1), without any patches, reaching 90 mm diameter in 3–4 d at 25 °C.

Materials examined: South Korea. South Sea, Chuja island, 33°57′11″N, 126°18′07″E, from Sargassum sp. (Phaeophyceae), 31 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M06, stored in a metabolically inactive state).

Notes: Nigrospora lacticolonia derived its name from the creamy white colonies on PDA [7]. Similarly, the isolate SFC20230324-M06, from Sargassum sp., colonized PDA in white. SFC20230324-M06 sparsely produces conidia and does not show a prominent tendency to aggregate conidiogenous cells in clusters. Moreover, narrower ellipsoidal conidia are observed than those in the original description (13.5–17.5 × 10.5–13.5 μm) [7].

Nigrospora osmanthi Mei Wang & L. Cai. (2017) (Figure 2E).

Sexual morph: Undetermined. Asexual morph on SNA: Hyphae branched, guttulate, septate, hyaline to pale brown, 1.8–6.4 μm diam. Conidiophores mostly reduced to conidiogenous cells. Conidiogenous cells monoblastic, discrete, determinate, brown, subspherical, ampulliform to cylindrical, 4.8–15.3 × 4.1–11.9 μm (av. = 8.58 ± 3.28 × 6.34 ± 1.76). Conidia solitary, globose or subglobose, aseptate, initially pale brown, becoming black with age, shiny, smooth-walled, sometimes formed directly from the mycelia, 9.9–16.7 μm diam. (av. = 13.07 ± 1.29).

Culture characters on PDA: Colonies flat, floccose, undulate, surface initially white (1A1), becoming grayish green (1D3), abundant aerial mycelium, reverse concolorous with dark patches, reaching 90 mm diameter in 5 d at 25 °C.

Materials examined: South Korea. South Sea, Jeju island, 33°23′53″N, 126°14′24″E, from Codium sp. (Chlorophyta), 15 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M11, stored in a metabolically inactive state); ibid., from Ulva sp. (Chlorophyta), 15 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M12, stored in a metabolically inactive state) ibid., from Codium sp. (Chlorophyta), 15 August 2021, M. S. Park & Y. W. Lim (SFC20230324-M13, stored in a metabolically inactive state).

Notes: Three Nigrospora osmanthi strains (SFC20230324-M11, SFC20230324-M12, and SFC20230324-M13) have bigger conidiogenous cells (5.5–12 μm) and smaller conidia (13.5–16.5 μm) than those of the strains in the original description [7]. Regarding growth on PDA, our isolates achieve a 90 mm diameter on plates within a span of 5 d at 25 °C, whereas the strains in the original description take 10 days to reach the same diameter [7].

5. Discussion

Nigrospora can be identified at the genus level by large dark conidiospores. However, morphological variation sometimes appears in strains despite them belonging to the same species, and different species can share similar morphological characteristics. Therefore, multigenetic marker analysis is imperative for the detection and identification of Nigrospora, given that the phylogeny of Nigrospora has been well-established using multigenetic markers, including ITS, BenA, and TEF1-α [7]. A total of nine Nigrospora species were identified in this study using multigenetic markers analysis and morphological data. This study provided the first report of five of these species in South Korea (N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, and N. osmanthi), and their morphological characteristics are provided in the taxonomy section.

Two Nigrospora species, N. oryzae and N. sphaerica, are frequently encountered. Nigrospora oryzae has been consistently reported from macroalgae [16,43] and was also commonly detected in this study on three islands and on three types of algae. However, Nigrospora sphaerica was not detected on marine macroalgae in this study, although it has been commonly reported in various marine environments [44,45]. Taritla et al. [18] isolated a strain L18/35 from the macroalga Sargassum muticum and identified it as N. sphaerica, but we confirmed that the ITS sequence (MF457920) of the strain L18/35 did not match that of N. sphaerica provided on NCBI. This creates doubt regarding the viability of its association with macroalga. Nigrospora aurantiaca has been reported on sponges [24] but was only isolated from red algae in this study. Until this study, there were no records of N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, N. osmanthi, N. pyriformis, and N. rubi in marine environments. These findings indicate that some Nigrospora species can adapt and inhabit both terrestrial and marine habitats, but further research is required to elucidate the underlying mechanisms of such environmental adaptation.

Although secondary metabolites produced by Nigrospora isolated from macroalgae have received limited attention in previous studies, numerous valuable secondary metabolites have been isolated from Nigrospora species [19]. One such example is nigrosporone B, which has shown anti-cancer, anti-bacterial, cytotoxic, and anti-malarial activities [46]. Nigrospora aurantiaca produces a red pigment known as bostrycin that can be used as a natural dye [47], and the same color pigment was observed in our N. aurantiaca strains SFC20230324-M01 and SFC20230324-M02 as well. Notably, Nigrospora species isolated from marine environments also produce a diverse range of secondary metabolites, many of which exhibit beneficial properties such as antimicrobial, antitumor, and cytotoxic activities [48–50].

The diversity of Nigrospora species was found to be highest in brown algae, followed by red algae and green algae. With the exception of N. oryzae and N. pyriformis, all species were exclusively isolated from a specific algal type (Figure 1). It is too early to conclude that Nigrospora species have a symbiotic relationship with algae due to the limited number of studied samples. However, considering that Arthrinium spp., a sister genus of Nigrospora, improves the survival of brown algae by providing antioxidants in response to decreased photosynthetic activity [51], it is possible that Nigrospora may also interact with algae. Moreover, pyrenocines isolated from Phaeosphaeria sp. can protect macroalgae against protistan pathogens, such as Olpidiopsis pyropia (Oomycota), through the collapse of the zoosporangia of O. pyropia [52]. Therefore, further investigations are required to elucidate the ecological role of Nigrospora associated with macroalgae and whether it acts as an endophyte or a pathogen. This study provides insights and discusses the possibility of biologically meaningful interactions between Nigrospora and macroalgae. Further studies aimed at comprehending the role of algicolous Nigrospora will greatly contribute to the effective management of macroalgal aquaculture and pathogenicity.

Acknowledgments

We thank Editage (www.editage.co.kr) for English language editing.

Funding Statement

This work was supported by the management of Marine Fishery Bio-resources Center (2023), funded by the National Marine Biodiversity Institute of Korea (MABIK).

Disclosure statement

The authors have declared that no competing interests exist.

Data availability

All the data that support the findings of this study are available within the article.

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Data Availability Statement

All the data that support the findings of this study are available within the article.

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