Microscopic fungi on Schoenoplectus lacustris in Płociczno and Płociowe lakes in Drawa National Park (northwest Poland)

In the period 2009–2011, the micromycetes causing symptoms on the leaves of Schoenoplectus lacustris (SL) from Płociczno and Płociowe lakes in Drawa National Park (DNP) were investigated. A total of 39 taxa of microscopic fungi and chromistan fungi were found. The taxonomic structure of the mycobiota was dominated by anamorphs of Ascomycota. In each year of the study, the SL fungal species richness was higher in Płociowe Lake than in Płociczno Lake. In all the years of study in both lakes, the fungal community on SL was composed of the following six dominant species: Alternaria alternata, Cladosporium herbarum, Fusarium incarnatum, F. sacchari, Gibberella avenacea, and Pleospora scirpicola. The species occurring exclusively in Płociczno Lake were Papulaspora immersa, Puccinia scirpi, and Trichothecium roseum, and those found only in Płociowe Lake were Acremoniella atra, Alternaria atra, Aspergillus niger, A. versicolor, Fusarium oxysporum, Gonatobotrys simplex, Massariosphaeria scirpina, Microascus brevicaulis, Penicillium chrysogenum, and Stagonospora aquatica. Only one confirmation of the occurrence of Puccinia scirpi and the lack of other specialized obligate parasites indicate the good health of SL in DNP. The facultative parasites, inter alia, Boeremia exigua, Chaetomium globosum, Fusarium culmorum, F. incarnatum, F. sacchari, Gibberella avenacea, and Stagonospora aquatica that are present on plants as part of the natural process of apoptosis may influence the health of SL.


Introduction
Plants of the genus Schoenoplectus (Rchb.) Palla (lake bulrush) belong to the family Cyperaceae and are represented by 77 species and subspecies of hydrophytes and helophytes [1]. In Poland, among the five species of Schoenoplectus, the most common and the only one not present on the red list is Schoenoplectus lacustris (L.) Palla (SL) [2]. In rush phytocoenoses, SL is a characteristic species of the Scirpetum lacustris (Allorge 1922) Chouard 1924 [3] community and is the belt of emergent macrophytes that extend the farthest from the shore. Schoenoplectus lacustris phytocoenoses are an important component of lake littoral zones and a cocreator of habitats for other hydrobionts. Schoenoplectus along with Typha and Phragmites, form one of the most productive zones in lake ecosystems [4,5]. This zone increased heterogeneity in water reservoirs, shaping the conditions for fishery exploitation. Many fish species from the families Cyprinidae, Esoxidae, and Siluridae use bulrushes as substrate for spawned eggs and as nurseries for fry that cannot yet swim well. Like most macrophytes, SL Digital signature This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website.
phytocoenoses facilitate the self-purification of waters. Therefore, they are increasingly used, for example, in the Netherlands to prevent the erosion of drained terrain [6]. In addition to its ecological significance, SL is also an important and increasingly popular natural raw material for construction and furniture manufacture. The quality and durability of the raw materials harvested from these plants are determined by the presence of the microorganisms accompanying SL vegetation [6]. In the context of the economic/utilitarian exploitation of SL, the occurrence of these microorganisms can also have significant health consequences because of the allergens, and frequently even toxins, produced by many microscopic fungi species co-occurring with plant vegetation. Among microorganisms that contribute to the health of macrophytes, phytopathogenic and saprotrophic mycobiota play significant roles, as they shorten plant vegetation. They initiate the decomposition of tissues, and their dominance, lasting for 1-4 days, can exceed by at least nine times greater biomass than that of bacteria [4]. Only after a time is the share of mycobiota suppressed by bacteria [7].
Microscopic mycobiota participate in the decomposition of plants and determine the release rate of soluble organic compounds, which modifies the hydrochemical conditions of lakes. However, knowledge about the diversity of these microorganisms remains fragmentary, and such studies of SL, both globally and in Poland, are sporadic; however, the few studies that have been published do provide interesting, often groundbreaking, information.
The aim of the study was to identify the microscopic fungi participating in the natural functioning of SL phytocoenoses in Płociczno and Płociowe lakes in Drawa National Park (DNP) and the frequency of their occurrence.

Description of the study area
Drawa National Park is located in the mesoregion of Drawsko Plain, northwestern Poland. Drawa National Park represents a young landscape of glacial outwash plains. The whole area (11 342 ha) is in the drainage basin of the Drawa River, one of the cleanest rivers in Poland. The Drawa River is also the only lowland Polish river, which in its course has over 20 km of rapid current, with the nature of the mountain river [8].
Drawa National Park is a large abundance of ecosystems and plant communities. The area is dominated by forests (80%), but the distinguishing constituents of nature are mires, meadows, and aquatic ecosystems. In DNP, there are several lakes, differentiated in terms of trophies, physicochemical and morphic characteristics. Morphological differences are also observed in the Płociczno and Płociowe lakes, which are the subject of the study (Tab. 1) [8].
Płociczno Lake is a fluvial water body through which the Płociczna River flows (Tab. 1). The mouth of this river has been blocked for over 20 years by rising lake levels, which have resulted in the creation of a characteristic delta. In the 1980s, the Płociczna River delta was proposed for classification as a natural monument [9]. Among [3,10] (also, Kraska M, Piotrowicz R, 1998, unpublished data). These plant communities are composed of extensive vegetation beds at opposite ends of the lake concentrated in the river inflow and outflow zones, where there is heavy organic and mineral sedimentation, and along the lake shoreline [9,10] (also, Kraska M, Piotrowicz R, 1998, unpublished data).
Płociowe Lake (also called Rakowy Lake or Płocica Lake) is not a fluvial lake (Tab. 1). The vegetation in Płociowe Lake is composed of the phytocoenoses of 20 plant communities. Communities of lake bulrush, Scirpetum lacustris, form more or less dense beds that extend along the entire shoreline of the lake. Among other plant communities  [3,10] (also, Kraska M, Piotrowicz R, 1998, unpublished data).

Field and laboratory study
Field studies were conducted in three subsequent vegetation seasons (2009-2011) in lake bulrush, Schoenoplectus lacustris (SL), that formed bulrush communities in lakes Płociczno and Płociowe in DNP. During each vegetation season, SL was collected twice from each lake, depending on weather conditions, in late June to early July and in the first 10 days of August (12 total field trips in three years). The SL plants with visible disease symptoms were collected from a boat alongside the shoreline (route method).
At each sampling, the collected material was composed of approximately 10 SL specimens exhibiting symptoms (Fig. 1a,b). As much as possible, the entirety of the SL specimen, including its submerged portions, was collected. The nomenclature of the host plant is provided in accordance with Mirek et al. [18].
Fresh parts of SL plants exhibiting disease symptoms were used in the laboratory studies to isolate mycobiota. The samples were rinsed under running freshwater, and then their surfaces were disinfected (70% C 2 H 5 OH). Further procedures were conducted under sterile conditions. Cuttings (3-5 cm) were taken from each plant with disease symptom marks 3 to 15 mm in diameter. Five to 10 sections were obtained from each plant depending on the severity and variety of disease symptoms. The sections were placed on sterile paper in moist Petri dishes (Ø 10 and 15 cm) and incubated for 2 to 21 days at 20 ±2°C. During incubation, the chambers were systematically moistened with sterile water aerosol, and the plant sections were examined daily under a stereo microscope to observe the morphological structure of the mycelium causing etiological symptoms. These structures were used to create microscope preparations in drops of lactic acid with dye (methylene blue), and they were observed under a Nikon Eclipse 200 at magnifications of 12.5×10, 40, and 66.
When fully formed fungi were not present and direct identification was not possible, they were moved to Petri dishes with PDA, CDA, MEA, and Sabouraud culture media (MERCK) with the addition of chloramphenicol. The preparation of fungal cultures and acquisition of single-spore cultures was performed using standard methods according to Waller et al. [19]. The microscopic fungi (fungi and fungus-like organisms) were identified (directly on host plants and in isolates cultured on media) using the following keys: Majewski [20], Sutton [21], Borowska [22], Kwaśna et al. [23], Ellis and Ellis [24], and Rietmüller [25]. The systematic framework used for the mycobiota was that of Kirk et al. [26], and the nomenclature was verified with Index Fungorum [27].
The results were interpreted by calculating biocenotic indexes such as the species richness and diversity of micromycetes according to Krebs [28] based on the taxonomic affiliation, frequency, and spatial structure of the mycobiota species [29,30]. Additionally, the Sørensen (SI) similarity index was calculated for species of microscopic fungi occurring on the SL in lakes Płociczno and Płociowe and in the different years of the study [28].

Morphometric features and water exchange rate
Płociczno Lake Płociowe Lake In each year of the study, microfungi species richness was higher on the SL from Płociowe Lake than that from Płociczno Lake. The highest species richness on the SL from Płociowe Lake was 23 taxa (2010), while in Płociczno Lake it was 15 taxa   Fig. 1c). The species occurring exclusively in Płociczno Lake were Papulaspora immersa, Puccinia scirpi, and Trichothecium roseum, and those found only in Płociowe Lake were Acremoniella atra, Alternaria atra, Aspergillus niger, A. versicolor, Fusarium oxysporum, Gonatobotrys simplex, Massariosphaeria scirpina, Microascus brevicaulis, Penicillium chrysogenum, and Stagonospora aquatic (Fig. 2f, Tab. 2). The frequency index of the micromycete species confirmed in both lakes indicated that in 2009-2011 the majority of them occurred abundantly (54%), while the remaining species occurred commonly (46%) (Tab. 2). No species were confirmed that occurred frequently, occasionally, or rarely.
6.19%, for subdominants 2.06%, 3.09%, and 4.12%, and for recedents 1.03%. Among all the species noted, none was confirmed to belong to the classes of eudominants or subrecedents. In Płociczno Lake, the majority of fungal taxa on SL was represented by the class of subdominants (14 taxa), while the rest was from the class of dominants (six taxa) and recedents (four taxa) (Tab. 2). The most abundant group noted on SL in Płociowe Lake was also that of subdominants (15 taxa), and the least abundant was that of dominants (six taxa), while the remaining 14 taxa belonged to the class of recedents (Tab. 2). The comparison of the mycobiota species richness on SL in lakes Płociczno and Płociowe indicated that throughout the 3-year study, the degree of species similarity was 67.8%, which corresponded to 20 common taxa that were confirmed on this host plant in both lakes (Tab. 3). The microfungi species similarity for SL in both lakes varied depending on the year of the study and fluctuated from 48.5% (eight common taxa) to 75% (12 common taxa) in 2010 and 2011, respectively. However, lesser differences in the coefficient of mycobiota species similarity on SL were confirmed in the two

Discussion
The results of the current study that revealed 39 fungal taxa associated with the SL in lakes Płociczno and Płociowe in DNP are impressive compared with other previous studies conducted in Poland. In the Masurian Lake District, Durska [31] noted one species, Myrioconium scirpi, on SL, which infected 50% of the plants. Rather modest data are also reported for Glinno Lake (Western Pomerania), where the occurrence of Epicoccum nigrum was only noted on SL [32], while two species, Phyllosticta scirpi and Stagonospora schoenoplecti, caused disease symptoms on SL in Czarne Sosnowickie Lake (Łęczna-Włodawa Lake District) [33]. Low species richness of microfungi associated with SL in Poland from studies cited above could arise from what they primarily refer to as pathogenic species, while the study discussed in the current paper focused on all the micromycete species associated with SL vegetation. Species isolated from SL in England also include Coleosperma lacustrae Ingold, Trichobelonium questphalicum Rehm, Hypoderma scirpinum DC. [34], and Physalospora aquatica [35], while in Ukraine, the SL species richness is characterized by the occurrence of other micromycete species such as Niptera melanosphaera Rehm., N. pilosa (Cross & Boudier), Lasiosphaeria sp., Lophiotrema culmifraga Speg., Leptosphaeria clavicarpa Ev. [36], species which seem to be of highly local significance. In contrast, according to Nannfeldt [37], Belonopsis mediella (P. Karst.) Aebi is characterized by a much wider range of occurrence and is confirmed on SL in England, Finland, France, Germany, Ireland, Sweden, and Switzerland. This author also reports the occurrence of Niptera lacustris (Fr.) in Scandinavia. The current study has not verified the occurrence of the species mentioned above in DNP or in Poland. Literature data indicate that the only other location where a similarly wide-ranging study was conducted is Hong Kong, China, where 39 taxa of these microorganisms were identified on SL [38]. Despite the approximate mycobiota species richness noted on SL from Hong Kong and in lakes Płociczno and Płociowe, there are notably distinct qualitative differences in their species diversity. Just six taxa [Alternaria alternata, Cladosporium cladopsorioides, Fusarium incarnatum (= F. semitectum) (Fig. 2c,d), Periconia sp., Pseudocecosporella sp. (Fig. 1d), and Trichotecium roseum] that are associated with SL are common in the areas discussed. Wong et al. [38] report 27 taxa on SL that were not noted in our study. None of these species was confirmed in the present study, and they are also lacking from the report by Ellis and Ellis [24], in which they identified 13 micromycete species that are potentially linked to Schoenoplectus in Europe. Among these, the occurrence of the following seven taxa was confirmed on SL in the present study: Alternaria sp., Puccinia scirpi (Fig. 1e) Massariosphaeria scirpina (= Leptosphaeria scirpina), Pleospora scirpicola (= Pyrenophora scirpicola) (Fig. 1c), Phaeosphaeria sowerbyi (= Leptosphaeria sowerbyi), Pseudocercosporella sp. (Fig. 1d), and Stagonospora aquatica (Fig. 2f). In the research by Wong et al. [38] and in each year of the current study, the species confirmed on SL were dominated by anamorphs of Ascomycota, which occurred in mass and commonly. The constancy of these tendencies is evidenced by the biocoenotic stability of the phytocoenoses studied, which is also a suggested point by Subramanian [39] and Adamska [40]. Teleomorphs rarely occur in Europe. They have been described in Mecklenburg -West Pomerania, where only Coronellaria pulicaris s. l. (P. Karsten) Sacc. [= Peziza palustris (P. Karst.)] was observed [41].
Most of micromycete taxa found on SL are facultative parasites and saprotrophic fungal species. Such trophic groups of fungi on other host plants with similar ecological requirements include Alternaria, Cladosporium, and Fusarium, which were previously identified in Drawa National Park [42] and in Słowiński National Park [40]. Our and other authors' previous studies indicate that microfungi species richness and diversity on host plants are specific to vegetation regions, and their occurrence is shaped by local environmental conditions. These environmental differences result in low mycobiota species similarity coefficient values at different times and from different areas of occurrence of given host plant species. In the present study, these relationships were confirmed by the SI index of less than 60%, which expressed the mycobiota species similarity on SL in each of the lakes (Płociowe and Płociczno) in comparable years of the study (Tab. 4). Mycological diversity in different years of the study could arise from different weather conditions, which, depending on the year of the study, facilitated the development of other microorganisms [43]. However, during each vegetation season, the microscopic fungi diversity coefficient may be affected by specific local abiotic and biotic conditions in addition to weather factors like element accessibility and plant vulnerability [44,45]. The significance of these conditions is confirmed by the wide range of the SI index of mycobiota species similarity (48.5-75%), which indicates the share of common micromycetes on SL in the lakes compared in the same year of the study (Tab. 3). Lower SI index values for the microorganisms occurring on the same host plant determine increases in the diversity of the phytocoenoses created by these microorganisms.
Among the fungi that determine the health of SL, the Pleospora rubelloides, associated with Schoenoplectus tabernaemontani [24], and P. scirpicola (Fig. 1c), which were confirmed in the current study on SL, are noteworthy. In DNP, the significance of P. scirpicola is confirmed by its presence on SL in both lakes in all years of the study and by the fact that it is one of six representatives of the dominants class (Tab. 2) alongside with such abundant polyphagous species as Alternaria alternata, Cladosporium cladosporioides, Fusarium incarnatum, F. saccharii, and Gibberella avenacea. Pleospora scirpicola appears to have a significant impact on the health of SL in Europe, which is confirmed by its wide range of distribution extending from England [35] to Ukraine (the former USSR) [36].
Nevertheless, it is noteworthy that in the current study the occurrence of telial stage (stage III) of Puccinia scirpi was confirmed on SL in Płociczno Lake (Tab. 2, Fig. 1e). This is the first confirmed occurrence of this stage on SL since the nineteenth century, when, according to Majewski [46], this species was described on Wolin Island, in Kołobrzeg, near Legnica and Ścinawa, and in the vicinity of Cracow and Międzyrzec Podlaski. The rare occurrence of this rust may be associated with the equally sporadic, though dispersed, occurrence of Nymphoides peltata, its ecidial host (stage 0; I), in its natural habitats [47]. However, in Europe, the telial stage of P. scripi has also been noted on other species from the family Cyperaceae, including Bolboschoenus maritimus and Blysmus compressus (in the former Czechoslovakia) [46]. Increasing host range is often associated with changes in the epidemiology of pathogenic factors [48]. In the context of the possibility of increasing pathogen host range, information about two new fungus species of Ustilaginales confirmed by Vánky and Shivas [49] on representatives of the genus Schoenoplectus in the subtropical zone of Australia is important. This refers to Dermatosorus schoeoplecti associated with Schoeoplectus mucronatum and Dermatosorus thirumalachari confirmed on Scirpus supinus (= Schoenoplectus supinus). In Taiwan, in addition to the well-known Pestalotia palmarum, two new species of mycobiota, Hyalotia lateripes and Polyschema olivacea, were also described on S. tiquer [50]. Most probably, SL is also a potential host of these and other phytopathogens, which will certainly appear in Polish latitudes, which is just a question of time. Evidence of this fact is found in the Netherlands, where recent studies describe the new species Bloxamia hesterae M. Spooren sp. nov. that was first noted on Schoenoplectus tabernaemontani and SL and then on other representatives of the family Cyperaceae (i.e., Eleocharis palustris) [51].
Climate change plays a significant role in the dynamics of widening the spectrum of hosts, often associated with the widening of the range of microorganisms [52]. Such dependencies, related to, i.a., higher temperatures, are explained by the increased intensity of the powdery mildew (Erysiphe flexuosa) epidemic in urban areas [53]. The dynamics of the emergence of one species and the withdrawal of other species of micromycetes can affect the stability of the ecosystem. This is manifested by the predominant role of saprotrophs and facultative pathogens that guarantees the natural course of seasonal decomposition of host plants.

Conclusions
■ The abundance and species diversity of microscopic fungi on Schoenoplectus lacustris differ depending on the study area. ■ Anamorphic forms dominate between micromycetes associated with S. lacustris. ■ The dominant trophic and ecological group of micromycetes isolated from S. lacustris are saprobionts and facultative parasites. ■ In Poland, the presence of Puccinia scirpi on S. lacustris was observed and confirmed for the first time since the nineteenth century.