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High-Temperature-Tolerant Fungus and Oomycetes in Korea, Including Saksenaea longicolla sp. nov.

Abstract Global temperatures are steadily increasing, leading to significant changes in microbial diversity and ecology. In the present study, we isolated high-temperature-growing fungi and fungi-like group (Oomycota) strains from freshwater environments of Korea and identified them based on cultural, morphological, and multilocus phylogenetic analyses. As a result, we introduce Saksenaea (Fungi) isolates as a new species, Saksenaea longicolla sp. nov. and record Phytophthora chlamydospora and P. lagoariana (Oomycota) new to Korea. In the growth experiments, they exhibited high-temperature tolerance, which can grow at 35–40 °C but become inactive at 4 °C and below. This study confirms the presence of high-temperature-tolerant fungi and oomycetes in Korea and suggests that the Korean climate conditions are changing in favor of these species. This indicates that climate warming is altering microbial distributions in freshwater environments.


Introduction
Global warming is altering microbial ecology, diversity [1][2][3], physiology [4][5][6], and interaction with abiotic and biotic factors [4,7,8]. For terrestrial Fungi and Oomycota (previously classified under Fungi, but now under Straminipila), climate-changing effects have been established concerning their growth, reproduction, physiology, and distributions [9][10][11][12][13]. However, the impact on the aquatic ecosystem, which is one of the important habitats for fungi and oomycetes, has been poorly studied, although it would result in changes in biotic communities and abiotic environmental factors [14][15][16]. Fungi and oomycetes are abundant in aquatic ecosystems where they play key roles in food web dynamics and carbon cycle [17][18][19]. They will give feedback to changes in climate gradient that may have a significant impact on aquatic environmental changes.
Ecological changes as a consequence of climate change on the Korean Peninsula are inevitable. According to the Intergovernmental Panel on Climate Change (IPCC), Korea can expect extended dry periods with warmer temperatures and frequent extreme precipitation events [20][21][22]. As such, the subtropical zone in Korea is expanding, which will produce significant changes in the ecosystem.
Interestingly, extreme cold events are also becoming more frequent [23], caused by the loss of Arctic sea ice and the change in the Arctic atmosphere [24][25][26]. This unprecedented phenomenon may give rise to considerable changes in Korea's ecosystems. To date, there were relatively few studies focusing on these ecological changes and even fewer on fungal and oomycete communities despite their crucial ecological roles.
Saksenaea, a filamentous fungal genus characterized by large, flask-shaped sporangia, is affiliated with Mucorales [27]. Since its first description from forest soil in India [27], members of Saksenaea have been isolated mainly in soil habitats, but also diverse environments such as on the coast in Costa Rica [28] and Taiwan [29], a banana plantation in Honduras [30], a groundnut field in Israel [31], a pineapple field in Okinawa, Japan [32], tree nurseries in Georgia, USA [33], and forests in Panama [34] and Brazil [35]. S. dorisiae was recently described in freshwater [36]. This species has been found only in the temperate continental region (the Republic of Serbia), while other members of Saksenaea described so far have been isolated in subtropical and tropical regions. Serbia is one of the countries experiencing a warming trend with climate change [37], but it seems to be premature to mention any effect of climatic change on S. dorisiae because this species has been discovered in well water at a depth of 65 m [36]. It should be noted that Saksenaea species are often involved in severe human and animal mucormycosis in tropical and subtropical regions [38][39][40], certainly made possible because of their high-temperature tolerance, growing at 35-40 C [35,41].
The genus Phytophthora belongs to the order Peronosporales of the Oomycota. Phytophthora is currently categorized in ten clades, according to their phylogenetic relationships, morphological and physiological characteristics [42]. Many species of Phytophthora are well known, such as the pathogens which cause sudden oak death [43], alder dieback [44], root rot [45], and late blight of potatoes and tomatoes [46]. Phytophthora species occur abundantly also in riparian ecosystems, irrespective of disease [47][48][49][50][51]. Especially, two phylogenetic clades, 6 and 9, of Phytophthora [42,52,53] include many saprotrophic species that are abundantly distributed in littoral zones of rivers and lakes, or forested streams [48,49,51,54]. They play important roles on food webs in aquatic ecosystems as a decomposer of plant debris or prey for zooplankton by trophic transfer to higher levels [19]. A unique part of these two clades is that they are characterized in terms of containing high-temperature-tolerant species [19,42,48,49,55]. Their optimum temperature ranges mostly from 25 to 30 C, but they are capable of growing at 35-40 C. The high-temperature-tolerant species have been discovered mostly in hot climates, or where the littoral zones can reach high temperatures [48,[55][56][57], although there are some wide temperature-tolerant species within clade 6, e.g., P. chlamydospora [58], P. riparia [59], which have often been recorded even in a cold climate.
Our microbial survey discovered high-temperature-growing fungal and oomycete isolates from freshwater, soil sediment, and decaying plant leaf matter originating from the reservoirs and mountain streams of Korea. These isolates were investigated for cultural and morphological features, and their optimum and minimal/maximal temperatures for growth were determined. Molecular sequencing analyses were carried out to determine their phylogenetic positions using the internal transcribed spacer (ITS) and large subunit (LSU) rDNA, and translation elongation factor 1 (TEF1a) regions for Saksenaea, and cytochrome c oxidase subunit I (cox1) and subunit II (cox2) mtDNA regions for Phytophthora. As a result, a novel species of Saksenaea and two species of Phytophthora, previously unrecorded in Korea, are reported in the present study.

Isolation of fungal and oomycete strains
Fungal and oomycete isolates were collected from freshwater environments in Korea, including water, soil sediment, and decaying plant leaves. The collection details are outlined in Table 1, and climate details for the collection sites are given in Supplementary Table S1. For Saksenaea isolates, a dilution plating method was used for soil samples at a 1:10 dilution. The diluted soil suspension was spread onto solid surfaces of two agar media: potato dextrose agar (PDA; Difco, Detroit, MI, USA) and V8 agar (V8A; 200 mL clarified V8 juice, 10 g CaCO 3 , 15 g agar, 800 mL deionized water). The growth of bacteria and other fungi was suppressed by adding antibiotics to the media of Saksenaea isolates 5 ppm benomyl, 25 ppm nystatin, 100 ppm penicillin G, 10 ppm pimaricin, 100 ppm streptomycin, and 100 ppm chloramphenicol. For Phytophthora isolates, water samples were distributed to both agar plates using a simple plating technique. The decaying plant leaves were cut into 3-5 mm 2 pieces after washing with distilled water and were placed on both types of solid growth media. Bacterial and fungal growth was suppressed by adding 15 ppm rifampicin and 20 ppm nystatin

Determination of temperature range for radial growth
To determine the optimal and minimal/maximal temperatures for growth, three strains (Sak-07, W655, and W694) were inoculated by transferring five replicates for each isolate onto four different media (PDA, V8A, MEA, and CZA) at 4, 10, 15, 20, 25, 30, 35, 40, and 45 C, with additional temperatures of 37 and 42 C for the Saksenaea strain (Sak-07). The colony radial growth was measured every day for 7 days and expressed as mm/day. Relative growth rates and standard deviation were calculated in Microsoft Excel.

Phylogenetic analysis
All sequences were edited using the DNAStar software package 5.05 (DNAStar, Inc., Madison, WI, USA). A BLASTn search was carried out to investigate the sequence similarities with their reference sequences in NCBI GenBank. Multilocus phylogenetic analysis was performed using a combined dataset of TEF1a, ITS, and LSU rDNA for Saksenaea isolates and using a dataset of cox1 and cox2 mtDNA sequences for Phytophthora. In addition, the reference sequences of the type of authentic isolates of Saksenaea and Phytophthora were included in each dataset. The sequences of the individual marker were aligned using MAFFT 7 [66] and then concatenated in SequenceMatrix v1.7.8 [67]. Phylogenetic trees were constructed in MEGA 7.0 [68]. For Saksenaea isolates, minimum evolution (ME) using the Kimura-2 model and maximum likelihood (ML) inferences using the General Time Reversible model were performed. For Phytophthora isolates, the Tamura-Nei model was used for both ME and ML analyses. Bootstrapping analysis was performed with 1000 replicates.

Cultural and morphological analyses
Colonies of five Saksenaea strains (C17, Sak-06, Sak-07, Sak-19, and Sak-21) formed a radiate pattern on five agar media (PDA, V8A, MEA, CMA and CZA) at 25 C in the dark and grew colorless with aerial hyphae ( Figure 1); aerial hyphae were denser on PDA, V8A, and MEA than on CMA and CZA. Radial growth with a circular layer was observed on the reverse side of the colony on PDA. Hyphae were hyaline, sparsely septate, and smooth. Asexual structures, including sporangiophores, sporangia, and sporangiospores, formed only on CZA. The sporangiophores were erect, unbranched, and formed singly. The sporangia were hyaline, flask-shaped, multispored, and developed terminally. The sporangiospores were mostly bacilliform or narrow middle rod-shaped, but rarely trapezoid. The cultural and morphological characteristics were close to those of S. dorisiae, S. oblongispora, and S. trapezispora. However, the sporangiophores of S. trapezispora were significantly longer than those of other species, including the Korean strains. The sporangia of S. oblongispora were smaller than those of S. dorisiae and the Korean ones. Saksenaea dorisiae was distinguished from the present strains by the narrow widths of sporangiophores, neck, and sporangia, and the somewhat smaller size of sporangiospores. The distinguishing morphological features between the Korean isolates and the closely related species of Saksenaea are summarized in Table 2.
All Phytophthora isolates grew colorless on PDA, V8A, CMA, MEA, and CZA at 25 C in the dark, with fewer aerial hyphae. Colonies of W694 formed a radiate pattern on all media. Sporangia were nonpapillate, ovoid to obpyriform, formed on unbranched sporangiophores, and displayed commonly internal proliferation. Chlamydospores formed either terminally or intercalary and were observed abundantly at 25-30 C. These characteristics were identical to those in the original description of Phytophthora chlamydospora [69]. The strains of W655 and W675 were identified as Phytophthora lagoariana based on the colony growth pattern and morphological features described by Wallace [70]. The colonies formed a vague rosaceous pattern on PDA, V8A, and MEA, but a radiate pattern on CMA and CZA. The strains grew submerged on CMA and CZA, while they formed a colorless mat on PDA, V8A, and MEA. These strains produced ovoid to obpyriform, non-papillate, and internally proliferating sporangia. Hyphal swelling was often observed, and chlamydospores were rarely produced. The colony patterns of P. chlamydospora (W694) and P. lagoariana (W655) on PDA, V8A, and CMA, along with morphological characteristics, are shown in Figure 2.

Phylogenetic analysis
Based on a BLASTn search at the NCBI GenBank, the Korean isolates of Saksenaea were determined to be closest to S. trapezispora (UTHSC DI 15-1), with sequence similarities of 90% (229/254 bp) in ITS rDNA, 98% (687/703 bp) in LSU rDNA, and 99% (493/498 bp) in TEF1a. ME and ML analyses of a concatenated alignment of ITS, LSU, and TEF1a sequences were performed to infer the phylogenetic relationships between the Korean isolates and previously published Saksenaea species. As the ME and ML trees were congruent, only the ME tree is shown in Figure 3B. The multilocus tree revealed that the Korean isolates formed a distinct clade with high bootstrapping values in ME (100%) and ML (99%) analyses. They further grouped with S. dorisiae, S.   . As the topologies constructed by both analyses were congruent, only the ME tree is presented. Three Korean isolates were placed in two different clades of Phytophthora, Clade 6 and Clade 9, with high supporting values. The strain W694 fell within Phytophthora Clade 6, which tolerates high temperatures, and further matched P. chlamydospora but was separated from non-chlamydospore-forming species [48,49]. The isolates of P. lagoariana, containing the Korean strains W655 and W675, formed a group with high-temperature-tolerant species of Clade 9. The optimum temperatures for these species range between 25 C and 30 C, but they can also grow at 35-40 C [42,55].

Temperature range for radial growth
The strain Sak-07 of Saksenaea exhibited an optimum growth temperature ranging from 25 C to 37 C, with the lowest temperature at 10 C (reaching 20-30 mm in diameter after a week) and the highest at 40 C ( Figure 5). No growth was observed at 4 C and 42 C. Mean colony radial growth at 25 C was observed to be 13.3 mm/day on PDA, V8A, and CZA, but 10 mm/day on MEA. On V8A and PDA, the colony grew as fast at 30-37 C as at 25 C. On MEA and CZA, the growth rate was lower at 37 C than at 25-35 C. The growth at 15 C was moderately fast (reaching 40-50 mm in 4 days).
For P. lagoariana strain W655, the optimum temperature ranged from 25 C to 35 C ( Figure 6A). At  Compared to the W655, P. chlamydospora stain W694 grew substantially slower on all media ( Figure  6B). The maximum growth temperature was 35 C on MEA and CZA, and 30 C on V8A and PDA. The optimum temperature for W694 ranged from 20 C to 25 C on all culture media. It was capable of growing at 4 C (average 0.5 mm/day) as well as 10 C (average 1.8 mm/day) on all culture media, although the growth rates were significantly lower. The growth rate of W694 was the highest at 25 C on V8A. The colony radial growth on V8A was observed to be 4 mm/day at 25 C. At 10 C (2.6 mm/day) and 30 C (1.4 mm/day); the colonies grew faster on V8A and MEA than on other media. At 30 C on CZA, the strain showed limited growth (0.25 mm/day). Sporangiospores are mostly bacilliform but rarely trapezoid, somewhat narrower at the middle and measuring (5.0)5.6-6.8(7.9) (average 6.2) lm long, (2.5)2.8-3.5(3.9) (average 3.1) lm wide. Zygospores were not observed.

Holotypus.
Korea Notes. Like other members of Saksenaea, S. longicolla is high-temperature tolerant. In terms of being unable to grow above 42 C, S. longicolla is close to its phylogenetically related species, S. trapezispora, S. oblongispora, and S. dorisiae [35,71]. However, the optimum (25-37 C) and the maximum (40 C) growth temperatures of S. longicolla are higher than those of S. trapezispora and S. oblongispora. In addition, S. longicolla grows slowly at 10 C, while the latter two species are inactive [35,72]. Morphologically, Saksenaea longicolla is similar to S. trapezispora [72] and S. oblongispora [35], but distinguishable by the size of the sporangiophores, sporangia, and sporangiospores. Both BLASTn-based comparison and phylogenetic analysis of multilocus sequences supported this morphological identification. Saksenaea stains have been isolated mainly from the soil of diverse environments such as forest, beach, and grain and fruit fields, as well as animal skin infections. Similarly, S. longicolla was isolated from soil sediments of a freshwater reservoir. All species of Saksenaea described so far have a tropical and subtropical distribution in America [28,30,33,73,74], Australia [75,76] and southern Asia [29,32,38,77,78]. A warm-temperate site from which S. dorisiae has been recorded, the Republic of Serbia is experiencing warming trends [37], similar to a Mediterranean climate. Additionally, as mentioned above, S. dorisiae has been discovered in the water at a depth well [36], which is less affected by the air temperature. To date, there has been no record of Saksenaea species from cooler regions. Interestingly, the sporangiospores of S. dorisiae have been reported to be lively at low temperatures in water [36], which could provide a hint that S. longicolla originating from freshwater could survive the cold season of Korea. However, further research is needed to answer this question. Notes. The present study combining morphological and phylogenetic data revealed that the isolate W694 is identical to Phytophthora chlamydospora [69]. The name P. chlamydospora was previously known informally as "P. taxon Pgchlamydo", but Hansen et al. [69] have redesigned this species based on a distinguishing feature of chlamydospore forming at a higher temperature. The Korean isolate also formed chlamydospores abundantly at 25-30 C but not at 20 C and below. In the present study, growth experiments verified that the Korean P. chlamydospora can grow at 35 C but not at 40 C, overlapping with a previous report that this isolate is usually able to survive at 36-37 C [69]. Members of Phytophthora Clade 6, including P. chlamydospora, are high-temperature tolerant and related to freshwater environments [79,80], which is in line with the Korean one obtained from the freshwater of a mountain stream. Phytophthora chlamydospora is often found in irrigation water, rivers, streams, and riparian areas of forest in Australia [81], North America [51], Europe [82], Africa [50], and Asia [83]. Notably, this species is known as a pathogen and a saprophyte; in North America [84] and Turkey [85], it has been reported as a cause of root and crown rot of almonds. Remarkably, these potentially pathogenic species have inhabited the Korean peninsula. Notes. The isolates W655 and W675 were morphologically and phylogenetically identified as P. lagoariana [70]. These isolates fell within Phytophthora Clade 9, of which many species are high-temperature tolerant but also well adapted to aquatic environments, e.g., P. hydrogena [55], P. irrigata [86], and P. aquimorbida [56]. Similarly, the Korean isolates exhibited a high temperature tolerance, with an optimum growth temperature of 10-35 C and were isolated from a freshwater reservoir and mountain stream. This species has been previously isolated from aquatic environments in the Cuyabeno Reserve in Ecuador and Carara National Park in Costa Rica [70]. This is the first record of P. lagoariana in a nontropical region.

Discussion
Here, we report that high-temperature-tolerant fungus (Saksenaea) and oomycetes (Phytophthora) are present in the temperate but cold winter areas of Korea. Saksenaea species and Phytophthora lagoariana has previously been reported only in the tropics and subtropics. It is still unclear how they are able to overcome the cold winter of Korea. In the present growth experiment, S. longicolla did not grow at 4 C and below, and P. lagoariana stopped growing even at 10 C. However, the mean winter temperature of the sampling locations (Gunsan, Yeongi, Namwon, and Imsil) of the present study is below 4 C, with the lowest temperature ranging from À10 to À20 C and frequent frost events. Their presence could be related to climate change as the warming in the Korean peninsula continues to accelerate. According to the Korea Meteorological Administration, Korea's average temperature increased by 1.8 C over the last century. Since 1910, Korea's summer has increased by a month, from 80-110 to 110-140 days. In the sampling locations, the temperature has increased by around 1-2 C for the past 50 years, and extreme heat events have become more frequent. This is an indication that the subtropical zone, restricted to the southern islands and coast of Korea, is expanding to the north.
Another crucial question is whether S. longicolla and two Phytophthora species are indigenous to Korea or exotic species. Given that they still prefer to grow at a much higher optimal temperature (25-35 C) than the yearly mean temperatures (12.0-13.5 C) of Korea, it seems likely that they may have recently immigrated from other (sub-)tropical or temperate regions, and then climate warming of Korea has made it easier for them to settle there. In addition, their ability to grow over a broad spectrum of temperatures (10-40 C for S. longicolla and P. lagoariana and 4-35 C for P. chlamydospora) may have facilitated more rapid adaptation.
It seems certain that the Korean peninsula's climate conditions are changing in favor of high-temperature-tolerant fungus and oomycetes, which could encourage their presence and dominance in the ecosystems. The freshwater environment is an essential habitat for fungus and oomycetes, which are influential as saprophytes and parasites in aquatic food web structure and dynamics. Their diversity and distribution patterns are impacted by environmental settings with climatic factors, e.g., temperature, precipitation [17,87] that can be variable in global climate change. Understanding their diversity and distribution patterns under climate change is essential, and further research focusing on their ecological and functional traits is required.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
Sequence data analyzed during the present study are available in GenBank.