Phyllosphere of Submerged Plants in Bathing Lakes as a Reservoir of Fungi—Potential Human Pathogens

The experiment was conducted in three summer seasons (2006–2011–2016) near the bathing sites in three lakes in Olsztyn: Kortowskie, Tyrsko and Skanda, which are the most popular as recreational sites. Microfungi isolated from the phyllosphere of 13 plant species of the littoral zone from dropped leaves of coast plants with no disease symptoms were the study material. Submerged parts of the same plants from each lake were used as follows: Phragmites australis L., Hydrocharis morsus-ranae L., Veronica beccabunga L., Nuphar lutea L., Stratioites aloides L., Persicaria amphibia L., Potamogeton natans L., Lemna minor L., Ceratophyllum demersum L., Urticularia vulgaris L., Myriophyllum sp. and dropped leaves of Alnus glutinosa L. and Acer platanoides L. Each season, the material was collected in late spring, summer, and early autumn three times, giving a total of 27 samples of vegetation. The material was taken along the shoreline in the immediate vicinity of places intended for recreation—bathing areas. On Skanda and Tyrsko lakes, vegetation was collected on the northwestern shore of the lake, while on Kortowskie lake—on the south-eastern shore (Fig. 1).

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Fragments of plants, with an area of ca. 1 cm², were placed in Sabouraud agar and incubated at 37 °C for 48–72 h. The culture was watched for clouding, deposits or a membrane on the broth surface, which were symptoms of yeast presence. Five drops of 20 μl were taken from each culture with visible microfungi growth and inoculated on solid Sabouraud agar with chloramphenicol (0.05%) by the “one drop” method. The samples were incubated for 48–72 h at 37 °C. The colony growth was passaged onto slants with Sabouraud agar and subjected to a standard diagnostic analysis commonly applied in mycological laboratories. The following were evaluated: macroscopic features (colour, sheen, surface structure, growing into agar), microscopic features in microculture on Nickerson agar (presence and position of blastospores, chlamydospores, formation of pseudomycelium) and biochemical features (zymograms, carbohydrate auxanograms) [4]. Due to the adopted method of pure fungi culture, the number of CFU/dm³ was not determined, but rather the observations were focused on differentiation and comparison of the species composition of the isolates with the results of studies conducted earlier in the same waters [1, 2, 3] during which a total of 212 water samples were taken for mycological analyses. The biosafety level (BSL) was determined for microfungi species [22, 23].

The species were identified with identification keys and studies: Kurtzman and Fell [24], Kurtzman et al. [25] and de Hoog et al. [23]. Photographic documentation was performed and the identified species were catalogued.

The significance of differences in the number of species between individual lakes was tested by means of the Kruskal–Wallis test followed by Dunn’s test of multiple comparisons (p < 0.05). In turn, repeated measures ANOVA (Friedmann) and then t test (Wilcoxon, p < 0.05) were employed to check the significance of differences in the number of species between individual research seasons. The correlation between the prevalance of microfungi in water and in the phyllosphere was established by computing the Spearman’s rank correlation coefficient. All statistical analyses were performed using STATISTICA 13 package (StatSoft).

A total of 36 yeast species of 16 genera were identified. The largest number of microfungi species were found on Phragmites australis—29, and on Ceratophyllum demersum—15. The following dominated among yeasts: Candida krusei—found on seven plant species (Fig. 2), and Candida glabrata—on five plant species (Fig. 3). For the other plants, the number of fungi species observed varied between three and six or only individual observations were recorded (Table 1).

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Table 1

Species differentiation of microfungi on the phyllosphere in Lakes Skanda (S), Kortowskie (K) and Tyrsko (T), taking into account the biosafety level (BSL)

No.	Species of microfungi	Phyllosphere
		(Phragmites australis (Cav.) Trin. ex Steud.	(Ceratophyllum demersum L.)	(Lemna minor L.)	(Hydrocharis morsus-ranae L.)	(Veronica beccabunga L.)	(Nuphar lutea L.)	(Alnus glutinosa Gaertn.)	(Acer platanoides L.)	(Stratiotes aloides L.)	(Persicaria amphibia L.)	(Potamogeton perfoliatus L.)	(Myriophyllum L.)	(Utricularia vulgaris L.)	BSL*
1	Brettanomyces bruxellensis	SK													1
2	Candida albicans	SKT													2
3	Candida albicans var. stellatoidea	K						SK							2
4	Candida glabrata	SK		S				S		SK					2
5	Candida glaebosa								S						un
6	Candida guilliermondii	SKT													2
7	Candida krusei	SKT		S	S	S	S				S				2
8	Candida lipolytica	K	SK	S											2
9	Candida melibiosica	SK		S											un
10	Candida tropicalis	SKT													2
11	Debaryomyces hansenii	K	T										S		1
12	Debaryomyces kloeckeri	S	K												un
13	Debaryomyces polymorphus							S							un
14	Endomycopsis bispora	SK				S		S							un
15	Geotrichum capitatum	SK			S		K								2
16	Hanseniaspora occidentalis	T					S	S							un
17	Kluyveromyces lactis var. lactis	SK					S						S		1
18	Lipomyces lipofer									SK					1
19	Metschnikowia pulcherrima	ST					ST								un
20	Pichia anomala	K								K				S	un
21	Pichia farinosa	K	S					S			S	K			un
22	Pichia jadinii									S					un
23	Pichia membranifaciens	SK	S										S		un
24	Pichia polymorpha	K	S							S		S			un
25	Rhodosporidium malvinellum	S	T					S							un
26	Saccharomyces castellanii	K		S										S	un
27	Saccharomyces cerevisiae	SKT	S				S								2
28	Saccharomyces delbruecki		S		S										un
29	Saccharomyces fragilis	K	S												un
30	Saccharomycodes ludwigii	K	S												un
31	Saccharomycopsis capsularis	SK	S								S				un
32	Saccharomycopsis fibuligera	S	T							S					un
33	Saccharomycopsis guttulata		S												un
34	Saccharomycopsis javanensis								S						un
35	Trichosporonoides spathulata	SK													un
36	Zygosaccharomyces mellis	S	T						S						un

un, unclassified

*According de Hoog [22] and de Hoog et al. [23]

Of the microfungi species found, nine were classified as BSL-2 (i.e. potential pathogens) and four species as BSL-1 (i.e. saprotrophs). The other 23 species were not classified into any biosafety group (Table 1).

Rush vegetation plays an important role in the process of inhibiting nutrient inflow, depending on the effectiveness of surface effluents from the drainage basin. It is a natural ecotonic zone [26], with buffer properties against inflowing nutrients. Recreational use of lakes contributes to fragmentation and, in consequence, to the lack of continuity of the zone. This results in easier nutrient inflow to a water body and in water pollution [27]. The most common plants in this zone include Phragmites communis, Ceratophyllum demersum and Polygonum amphibium. Aquatic vegetation in the lacustrine littoral zone is often inhabited by different microorganisms, including microfungi [12, 28]. Yeast present in the phyllosphere often compete with phytopathogens for nutrients, and they also inhibit their growth and development by taking in organic matter from the leaf surface. This is of special importance when the fungi isolated from the phyllosphere can pose a hazard to human health and when the submerged vegetation zone is in close vicinity to the places of recreation [1, 2, 3]. About 200 of the many yeast species occurring in the aquatic environment are pathogens [23, 25].

Rush vegetation has been used for years in bioindication of aquatic ecosystems. Its characteristic structure with an extensive absorption system enables absorption of various microelements from the environment, both harmful and necessary for the plant [29]. Therefore, species of rush plants in assemblies of plants, as well as aesthetic values, also play an important ecological role. The health condition of coastal and aquatic vegetation has an indirect impact on the life of the aquatic fauna, for which it is a shelter and breeding place [30].

As a reservoir of fungi potentially hazardous to humans, the phyllosphere serves as a filter in a water body which provides the first significant barrier for microfungi from the soil environment. When water ripples and washes the leaf surface, fungi cells may be washed down into surface water, posing a serious sanitary and epidemiological hazard. Korniłłowicz et al. [31] and Simi et al. [32] report that various species of fungi in the aquatic environment may derive from birds nesting nearby. However, no bird nests were found near the sampling sites in our study. The lakes which the surveys were conducted in are located within the administrative boundaries of the city, which results in reduced nesting in the coastal area. Fragments of the phyllosphere were collected during a period of intense exploitation of recreational and bathing sites located in the immediate vicinity of designated research areas.

Yeast and bacteria and/or moulds occur on leaf surface, forming aggregates or multicellular biofilms [33]. However, they are not the first group of microorganisms which settle the phyllosphere. This place is first settled by bacteria, which change the structure of cuticle and the lamina of the leaf. They are followed by yeast, with the filamentous fungi, which include numerous pathogens, being the last [28]. The moment of depositing and adhering of yeast on the lamina of the leaf usually occurs in summer, when the lamina surface is well developed. Fungi development is sometimes initiated by an increase in organic matter amount between spring and autumn, which is correlated with the plant growing season. The process of macrophyte decomposition indirectly increases the amount of biogenic elements, including nitrogen and phosphorus, thereby accelerating water eutrophication. Yeast floating in the pelagic zone in water under anthropopressure have higher enzymatic activity, increasing with the degree of eutrophication, which is directly associated with strain pathogenicity [3]. The presence of yeast, including potentially pathogenic species, has been confirmed by the findings of this study. Obtaining as many as 36 fungi species from the phyllosphere of Lake Skanda—which is highly eutrophicated—is worrying because up to eight of them belong to the genus Candida, which are important agents in aetiology of mycosis caused by Saccharomycetes. This can be demonstrated by the fact that the same species, such as Candida albicans, C. krusei and C. tropicalis, were found in clinical materials [1, 34] and at bathing sites in urban lakes [1, 2, 3, 35].

Selected water reservoirs differ from each other in the nature of the shoreline and the catchment area, or in the development of adjacent areas. Tyrsko lake, located in the north-west of Olsztyn, is characterised by the greatest landscape values. It is an area slightly transformed by man. Its shores are high and steep in places [36], and the state of water transparency is included in the second class of cleanliness, making it a great place for diving enthusiasts. An additional advantage of the lake is the lack of surface inflows and outflows. The second lake, Kortowskie, is located in the southwestern part of the city. It is a flow-through reservoir fed by five watercourses: the Starodworski, Parkowy and Leśny Stream, drainage line, and the Kortówka river, which is also its surface outflow [37]. It is a lake with an advanced level of eutrophication, intensively used for tourism and recreation. Its catchment has a forest-agricultural character. From the south-eastern side, it is surrounded by the gardens of the University of Warmia and Mazury and the infrastructure of the academic town with its recreation part (park, marina, water equipment rental, guarded swimming pool) [38]. The established Kortowski experiment has been in operation since 1959, consisting in removing pollutants to the Kortówka river with the help of hypolimnion waters [39]. According to research conducted in 2014 by Smoter et al. [40], the ecological status of the lake was assessed as poor (class IV water quality). The third lake—Skanda, is located on the south-eastern outskirts of Olsztyn [40]. Like Kortowskie lake, it belongs to reservoirs with an advanced degree of eutrophication, intensively used for recreational and tourist purposes [38]. Its shoreline is well developed, and its shores are quite varied, from flat through gently raised to steep [40], with visible traces of anthropopressure [38]. An important role is played by the runoff from agricultural areas, as well as pollution from two non-canalised farms located in the close proximity to the lake. The ecological state of the lake is classified as poor (class V of water quality) [40].

Considering the position of the isolated microfungi in the biosafety classification, it should be noted that the dominant group includes species of the BSL-2 group, i.e. potential human and other vertebrate pathogens [22, 23]. On the other hand, finding such a large number of yeast species inhabiting the littoral zone phyllosphere confirms that the zone acts as a filter for the lake catchment area [41].

Finding many potentially pathogenic yeast species in the lake littoral zone shows the huge human impact on the hydrosphere microbiological quality. Therefore, lake shores at bathing sites should be cleared of vegetation to prevent anthropogenic pollution from accumulating there. This study provides valuable complementary information on the natural composition of the littoral microbiota in water bodies.