III LICEUM OGÓLNOKSZTAŁCĄCE Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 7: XX–XX, 2022 ISSN 2543-8832, e-ISSN 2545-0999 DOI: 10.24917/25438832.7.X Gracjana Budzałek*, Sylwia Śliwińska-Wilczewska Institute of Oceanography, University of Gdansk, Gdynia, Poland, *gbudzalek@gmail.com Allelopathic effect of the green macroalga Ulva intestinalis (Ulvaceae, Chlorophyta) on selected Baltic cyanobacteria Introduction Allelopathy is a unique strategy to deter or eliminate organisms coexisting in the same ecosystem (Molisch, 1937; Śliwińska-Wilczewska et al., 2021). In aquatic ecosystems, allelopathic activity depends on the production and secretion of active allelopathic compounds and their effective dispersal in the environment (Lewis, 1986). The benthic zone is limited compared to the extensive pelagic zone in the sea, so allelopathic interactions in these coastal ecosystems may be stronger. Macroalgae have been found to produce active metabolites that inhibit other organisms that compete with them for light and space (Harlin, 1987; Jeong et al., 2000), but their allelopathic activity on Baltic cyanobacteria is still insufficiently recognised. Previous studies have confirmed that green algae of the genus Ulva L. (Chlorophyta) are capable of allelopathic effects on selected microalgal species (Jin, Dong, 2003; Nan et al., 2004; Jin et al., 2005; Wang et al., 2007; Tang, Gobler, 2011). Jin and Dong (2003) studied the effects of extract, filtrate, and fresh thallus of Ulva pertusa Kjellman on Heterosigma akashiwo (Y. Hada) Y. Hada ex Y. Hara & M. Chihara and Alexandrium tamarense (Lebour) Balech. H. akashiwo and A. tamarense were strongly inhibited by aqueous extract and fresh tissue from U. pertusa. In contrast, the authors showed that the green algae-derived filtrate had no significant effect on the cell counts of the test organisms. A year later, Nan et al. (2004) investigated the effect of fresh tissue and filtrate from U. pertusa on the Tetraselmis subcordiformis (Wille) Butcher (species form phylum Chlorophyta), the Heterosigma akashiwo and Alexandrium tamarense (representative of Miozoa), the Skeletonema costatum (Greville) Cleve, Nitzschia Closterium (Ehrenberg) W. Smith, and Chaetoceros gracile F. Schütt (member of Bacillariophyta), the Chroomonas placoidea Butcher ex G. Novarino & I.A.N. Lucas (species from phylum Cryptophyta), and the Isochrysis galbana Parke (representative of Haptophyta). Growth was significantly inhibited for each tested species. Additionally, the authors’ results suggest that allelopathic compounds from U. pertusa are highly degradable. The inhibitory effect of extract, filtrate, and fresh thallus from U. lactuca was also demonstrated by Tang and Gobler (2011). This green macroalgae adversely affected the cell concentrations of Aureococcus anophagefferens Hargraves & Sieburth in Sieburth, P.W. Johnson & Hargreaves and Chattonella marina (Subrahmanyan) Y. Hara & M. Chihara (member of Ochrophyta), the Cochlodinium polykrikoides Margalef, Karlodinium veneficum (D. Ballantine) J. Larsen in Daugbjerg & al., Karenia brevis (C.C. Davis) Gert Hansen & Moestrup, and Prorocentrum minimum (Pavillard) J. Schiller (species form phylum Miozoa), and Pseudo-nitzschia multiseries (Pavillard) J. Schiller (representative of Bacillariophyta). Wang et al. (2007) investigated the inhibitory effect of extract, filtrate, and fresh thallus of Ulva pertusa on the growth of two dinoflagellates H. akashiwo and Alexandrium tamarense. On the other hand, Jin et al. (2005) investigated the inhibitory effect of fresh thallus and extract of U. pertusa and U. linza L. on the dinoflagellata Prorocentrum micans Ehrenberg. A recent work indicates that Ulva intestinalis L. also has allelopathic properties (Budzałek et al., 2021a). It was shown that the addition of the cell-free filtrate obtained from U. intestinalis significantly inhibited growth and photosynthetic efficiency of filamentous cyanobacteria Nodularia spumigena Mertens ex Bornet & Flahault and Nostoc sp. Suprysingly, the authors found that the addition of different concentrations of aqueous extract as well as filtrate stimulated the Aphanizomenon sp. There is only one report of allelopathic activity of Spirogyra sp. (Charophyta) (Irfanullah, Moss, 2005). In their work, they studied the effects of allelopathic compounds secreted by Spirogyra sp. on phytoplankton communities. Phytoplankton species dynamics and species composition were apparently not influenced by allelopathy of living or decomposing Spirogyra sp. In addition, they investigated the allelopathic effects of filtrate and living thallus of Spirogyra sp. on phytoplankton. There was no change in phytoplankton growth and species composition under the allelopathy of living or decomposing Spirogyra sp. This study showed that filamentous macroalgae from Charophyta phylum probably cannot control phytoplankton biomass in a nutrient-rich environment by secreting allelopathic compounds. Allelopathic activity was also confirmed for macroalgae belonging to the genus Chara (Charophyta) (Donk, Bund, 2002; Berger, Schagerl, 2003; Mulderij et al., 2003; Pakdel et al., 2013; Mähnert et al., 2017; Złoch et al., 2018). Pakdel et al. (2013) studied the effects of extract, filtrate, and live material from the stonewort – Chara australis R. Brown on the cyanobacteria Anabaena variabilis Kützing ex Bornet & Flahault and the green alga Scenedesmus quadricauda (Turpin) Brébisson in Brébisson & Godey. C. australis had a highly inhibitory effect on the growth of A. variabilis. In contrast, the extract had no significant effect on S. quadricauda. Recently, the effect of the extract of Baltic C. aspera Wild., C. baltica Bruzelius, and C. canescens Loiseleur on the cyanobacteria Synechococcus sp. was demonstrated by Złoch et al. (2018). Both inhibition and stimulation of growth and photosynthesis of the tested species were demonstrated. C. aspera had both stimulatory and inhibitory effects on the studied cyanobacteria. The other two Chara sp. inhibited the growth of cyanobacteria cell numbers. Mähnert et al. (2017) showed inhibitory effects of extracts as well as fresh thallus of C. aspera, C. globularis, C. rudis, and C. tomentosa on the green microalgae Chlorella vulgaris L., Acutodesmus acuminatus (Lagerheim) P.M. Tsarenko in Tsarenko & Petlovanny, the cyanobacteria Synechococcus elongatus, S. leopoliensis, and the bacterium Aliivibrio fischeri Beijerinck. Strong inhibitory effects of the extract and fresh thallus on cyanobacteria and bacteria were demonstrated. Berger and Schagerl (2003) demonstrated the inhibitory effect of C. aspera extract on the growth of the filamentous cyanobacteria Anabaena cylindrica Lemmermann. The allelopathic inhibitory effect of C. aspera was also studied on the green algae Scenedesmus acutus Meyen (Donk, Bund, 2002). On the other hand, Mulderij et al. (2003) studied the effect of filtrate obtained from Chara globularis var. globularis (Detharding ex Willdenow) R.D. Wood and C. contraria var. contraria (A. Braun ex Kützing) J.A. Moore on three green algae. They showed growth stimulation of Selenastrum capricornutum Printz and Chlorella minutissima Fott & Nováková. However, they showed no significant differences for Scenedesmus obliquus (Turpin) Kützing. Toxic cyanobacterial blooms pose a serious threat to the environment and human health, and restoration of affected water bodies can be a challenge. Although cyanobacterial blooms are a common phenomenon already, their occurrence and severity are expected to increase in the future due to climate change (Reichwaldt, Ghadouani, 2012). In fresh and brackish water bodies toxic cyanobacterial blooms have been recorded for at least 2 millennia (Zillén, Conley, 2010). Massive cyanobacterial blooms appear in the Baltic Sea almost every year during summer, however it is very difficult to predict their location and intensity of occurrence (Kahru et al., 2020). The genus Aphanizomenon sp. commonly dominates the water column biomass during blooms in the Baltic Sea, along with Nodularia spumigena, which has consistently caused fatalities of both wild and domesticated animals in the Baltic Sea (Wasmund, 1997). A third common species in this basin is Nostoc sp., which also exhibits cytotoxic properties (Surakka et al., 2005). Thus, the aim of this study was to demonstrate the allelopathic effects of Baltic macroalga Ulva intestinalis L. (syn. Enteromorpha intestinalis (L.) Nees) thallus on growth and photosynthetic activity of three bloom-forming cyanobacteria Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. These studies help define the role of U. intestinalis allelopathy as a biological factor in the distribution of bloom-forming cyanobacteria in the coastal Baltic Sea region. Fig. 1. Ulva intestinalis L. thalli from habitat and light micrographs of tubular thallus (young cell arranged in longitudinal rows). Scale bar: 100 mm (A), 60 μm (B), 40 μm (C), 20 μm (D). (Photo. G. Budzałek) Material and methods Place of sampling and material cultivation The material used in the experiments consisted of strains of Baltic cyanobacteria Aphanizomenon sp. (CCBA-69), Nodularia spumigena (CCBA15), and Nostoc sp. (CCBA- 81) (so-called target organisms). Strains of cyanobacteria were isolated from the natural phytoplankton communities of the coastal waters of the Gulf of Gdańsk (southern Baltic Sea) (54°30'53.7"N; 18°54'23.5"E) and are maintained in the Culture Collection of Baltic Algae (CCBA) at the Laboratory of Marine Plant Ecophysiology at the University of Gdańsk (Latała et al., 2006). The macroalga Ulva intestinalis used in the study (donor organism) was collected from the coastal zones of the southern Baltic Sea region (54°30'08.7"N; 18°33'32.3"E). Determination of U. intestinalis (Fig. 1) based on the examination of morphological features (such as number of pyrenoids and shape of cells) using an identification keys (Starmach, 1972; Škaloud et al., 2018). The herbarium sheets (voucher number: BA M50; herbarium website: https://zielnik.ug.edu.pl/en/home/) were prepared in accordance with guidelines in Drobnik (2007) and Rybak (2018) and deposited at the Institute of Oceanography, University of Gdansk (Poland). The studied macroalga and cyanobacteria were cultured on sterile mineral medium f/2 (Guillard, 1975) prepared with Baltic Sea water filtered through glass fiber filters (Whatman GF/C) and autoclaved. The salinity was 8 PSU as measured with a salinometer (inoLab Cond Level 1, Germany). The U. intestinalis and cyanobacterial strains used in the experiments was maintained in 300-mL and 50-mL glass Erlenmeyer flasks, respectively. Donor and target organisms were cultured at a PAR intensity of 10 μmol photons m–2s–1 (16:8 h light: dark cycle) and a temperature of 18°C. Photosynthetically active irradiance (PAR) was measured using a quantum meter (LI-COR, USA). The light sources used in the experiment were lamps (Cool White 40W, USA). The cultures were acclimated to these conditions for 2 days, and these growth conditions were used for the experiments. Determination of the allelopathic effect of Ulva intestinalis thallus The allelopathic effect of Ulva intestinalis thallus was tested according to a method proposed by Wang et al. (2007) with modifications. The cyanobacteria monocultures were exposed to three different concentrations of the presence of live thallus of U. intestinalis. Target cyanobacterial strains were maintained in 25-mL Erlenmeyer flasks. In all experiments, the starting concentration of chlorophyll a in cyanobacterial cultures was 0.4 μg chl a mL–1. The coexistence assays were performed using a mixed culture system of one macroalga and one strain of cyanobacteria. Different initial inoculation concentrations (0.01, 0.05, and 0.1 g wet weight mL–1) of fresh algal thallus were inoculated into 25-mL Erlenmeyer flasks containing 20 mL of the target cyanobacteria strains. Control cultures were prepared analogously, but f/2 medium was added instead of thallus at 0.01, 0.05, and 0.1 mL– 1. The concentration of major nutrients in the control and experimental samples were adjusted to the same level as in the f/2 standard. Therefore, the influence of nutrients, micronutrients and vitamins on the experimental result can be excluded. After 7 days of the exposure, before the measurements, the Ulva sp. thallus was removed and the cyanobacterial cells concentration as well as chlorophyll a fluorescence parameters were determined. All allelopathic tests were conducted in triplicate. Determination of cyanobacterial number of cells The number of cells (N) in Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. cultures was estimated with previously determined linear correlations between cell abundance (N mL– 1) and optical density (OD). N was counted using a Bürker chamber (48 squares per count) and light microscope following a procedure according to Guillard and Sieracki (2005), and the OD was measured spectrophotometrically at 750 nm with a Multiskan GO UV-VIS spectrophotometer (Thermo Scientific, Massachusetts, USA). The linear correlation between N and OD for mentioned cyanobacteria was described by Budzałek et al. (2021). OD measurements were performed on the 7th days of the experiment and control and converted to cyanobacteria cells. In addition, absorption spectra in the wavelength range from 400 nm to 750 nm with a frequency of 1 nm was determined for cyanobacterial abundances in controls and allelopathic treatments. Determination of the chlorophyll a fluorescence In the conducted experiments, the Pulsed Amplitude Modulation (PAM) method was used to measure the chlorophyll a fluorescence (FMS1, Hansatech). This method is widely used to measure chlorophyll a fluorescence in cyanobacteria, both in the laboratory and in the natural environment (Schreiber et al., 1995; Campbell et al., 1998). Samples were taken for chlorophyll fluorescence analysis after the 7th days of the experiment. About 5 mL of target cyanobacteria were filtered through 13-mm glass fiber filters (Whatman GF/C). In the next step, the filters were placed in holders. The samples were kept in the dark for 5 min before measurement. In this study, the maximum PSII quantum efficiency (Fv/Fm) and the effective quantum yield of PSII photochemistry (ΦPSII) were determined (Campbell et al., 1998). Statistical analysis To confirm the allelopathic effect of the extracts obtained from macroalgae on the number of cells and chlorophyll a fluorescence parameters of target cyanobacteria, a one-way ANOVA was performed. Data are reported as the means ± standard deviations (SD), where level of significance was p < 0.05. Statistical analysis and graphs were performed using Statistica® 13.1 software and OriginPro program, Version 2021 (OriginLab Corporation, Northampton, MA, USA). Fig. 2. The number of cells (N 105 mL–1) of Aphanizomenon sp. (A) Nodularia spumigena Mertens ex Bornet & Flahault (B), and Nostoc sp. (C) in controls (C) and cultures to which were added different concentrations (g mL–1) of fresh thallus (FT) obtained from Ulva intestinalis L. after 7 days of the experiment (a) and PAR absorption spectra determined for this strains at thallus concentrations: 0.01 (b), 0.05 (c), and 0.1 (d). The values shown are mean values (n = 3, mean ± SD). Different letters indicate significant differences between the means of the treatments (p < 0.05, one-way ANOVA) Results Allelopathic effect of Ulva intestinalis thallus on cyanobacterial abundances In this study, the number of cells (N 105 mL–1) of Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. in controls and cultures to which were added: 0.01, 0.05, and 0.1 g wet weight mL–1 of fresh thallus obtained from Ulva intestinalis after 7 days of the experiment were determined. It was found that thallus obtained from U. intestinalis had no statistically significant effect on the number of cells of the cyanobacterium Aphanizomenon sp. at higher concentrations (0.05 and 0.1 g mL–1). On the other hand, it was examined a stimulating effect of 0.01 g mL–1 of the fresh thallus on the number of Aphanizomenon sp. cells which constituted 168% (one-way ANOVA, p < 0.05), relative to the control treatment (Fig. 2A). On the other hand, it was shown that the fresh thallus addition resulted in a decrease in the number of N. spumigena cells. For this cyanobacterium, the growth was, relative to the control, 45%, 27%, and 46% (one-way ANOVA, p < 0.05, for all), after addition of 0.01, 0.05, and 0.1 g wet weight mL−1 of fresh thallus, respectively (Fig. 2B). In experiments with Nostoc sp. and U. intestinalis thallus addition, the negative effect on cyanobacterial growth was detected on the 7th day of exposure at 0.1 g wet weight mL−1 and constituted 97% (one- way ANOVA, p < 0.05) of control. In turn, the U. intestinalis thallus addition had no significant effect on the growth of Nostoc sp. at lower concentrations (0.01 and 0.05 g mL−1) (Fig. 2C). Fig. 3. The values of the fluorescence parameter Fv/Fm (a) and ΦPSII (b) for Aphanizomenon sp. (A) Nodularia spumigena Mertens ex Bornet & Flahault (B), and Nostoc sp. (C) in controls (C) and cultures to which were added: 0.01, 0.05, and 0.1 (g mL–1) of fresh thallus (FT) obtained from Ulva intestinalis L. after 7 days of the experiment. The values shown are mean values (n = 3, mean ± SD). Different letters indicate significant differences between the means of the treatments (p < 0.05, one-way ANOVA) Allelopathic effect of Ulva intestinalis thallus on fluorescence parameters The values of the fluorescence parameter Fv/Fm (the maximum PSII quantum efficiency) and ΦPSII (the effective quantum yield of PSII photochemistry) for Aphanizomenon sp., N. spumigena, and Nostoc sp. in control and cultures to which were added a different concentrations (0.01, 0.05, and 0.1 g wet weight mL–1) of fresh thallus obtained from U. intestinalis after 7 days of experiment were examined. A stimulating effect of concentrations of 0.01, 0.05, and 0.1 g wet weight mL–1 of U. intestinalis thallus on the values of ΦPSII of cyanobacteria Aphanizomenon sp. were observed and in these conditions these parameters constituted 176%, 129%, and 148% (one-way ANOVA, p < 0.05, for all), respectively (Fig. 3Ab). Surprisingly, addition of thallus of U. intestinalis did not affect the value of Fv/Fm of Aphanizomenon sp. (Fig. 3Aa). Moreover, the addition of fresh thallus obtained from U. intestinalis had no statistically significant effect on the fluorescence parameters Fv/Fm and ΦPSII of N. spumigena (Fig. 3Ba-b). In turn, the Fv/Fm of Nostoc sp. was positively affected after addition of 0.01, 0.05, and 0.1 g wet weight mL−1 of thallus, being, relative to the control, 142%, 153%, and 124% (one-way ANOVA, p < 0.05, for all), respectively (Fig. 3Ca). Moreover, in the fresh thallus addition experiments, the value of ΦPSII for the mentioned cyanobacterium was significantly different from the control at the concentration of 0.01 g mL−1, when it constituted 202% (Fig. 3Cb). Discussion Effects of allelopathic compounds produced by Baltic macroalga Ulva intestinalis on cyanobacterial growth In the present study, the allelopathic effect of Baltic green macroalga Ulva intestinalis thallus on growth of three bloom-forming cyanobacteria Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. was investigated. The live thallus had an inhibitory effect on the number of cells of N. spumigena, and Nostoc sp. in the first week. Whereas cell growth in the Aphanizomenon sp. sample was stimulated by U. intestinalis thallus at the lowest concentration (0.01 g wet weight mL–1). In several other works, the authors also showed allelopathic activity of fresh thallus of green algae of the genus Ulva on co-occurring phytoplankton species. Wang et al. (2007) noted that Heterosigma akashiwo and Alexandrium tamarense showed different responses when exposed to live U. pertusa thallus. The authors showed that relatively low concentrations had a lethal effect on H. akashiwo, while A. tamarense cells were not completely degraded even at the highest macroalgal concentration. Differences in the cell surface structure of H. akashiwo and A. tamarense may account for their different sensitivity to allelopathic compounds (Wang et al., 2007). Kakisawa et al. (1988) suggested that allelopathic compounds produced by macroalgae may be more active against organisms that lack cell walls. In the present study, three species of Baltic cyanobacteria that possess cell walls were used for testing. In addition, cyanobacteria are covered with a mucous envelope of varying thickness, which may also diminish the effect of allelochemicals. This may partly explain the lack of effect of the filtrate and live U. intestinalis mollusks on the cell abundance of Aphanizomenon sp. It should be noted here that the composition of allelopathic compounds from green macroalge is still not well known. In a review by Budzalek et al. (2021b), known compounds from macroalgae with an active effect on other organisms were listed. Among them, it has been shown that the U. intestinalis can produce Penostatins A–H, Cytochalasans, penochalasins A–H, Chaetoglobosin, and Communesins A–B with cytotoxic activities. However, more research is needed to unequivocally show which compounds are responsible for inhibiting or stimulating cyanobacteria in the Baltic ecosystem. In contrast, Uchida et al. (1995) reported that Heterocapsa circularisquama Horiguchi completely annihilated Gyrodinium instriatum Freudenthal & J.J.Lee by direct cell contact. In the present study, the possibility of inhibition of cyanobacterial growth by direct contact with U. intestinalis was also considered and this possibility was finally ruled out by additionally performing experiments with the addition of the filtrate of this donor green alga alone. Since significant changes in the growth of Baltic cyanobacteria were also observed under the influence of the filtrate, it can be assumed that the secretion of allelopathic compounds by U. intestinalis is the most probable explanation for the observed growth inhibition of the organisms studied. Effects of allelopathic compounds produced by Baltic macroalga Ulva intestinalis on photosynthesis performance In this work, we also investigated the allelopathic effect of fresh thallus obtained from U. intestinalis on the maximum PSII quantum efficiency (Fv/Fm) and the effective quantum yield of PSII photochemistry (ΦPSII) of some Baltic cyanobacteria. It was found that U. intestinalis had no allelopathic effect on fluorescence parameters of tested cyanobacterium N. spumigena. On the other hand, all tested concentrations (0.01, 0.05, and 0.1 g wet weight mL–1) of thallus from mentioned green macroalga stimulated the values of Fv/Fm or ΦPSII of cyanobacteria Aphanizomenon sp. and Nostoc sp. compared to the control. In other works, Budzałek et al. (2018, 2021b) and Złoch et al. (2018) demonstrated that extract and filtrate of U. intestinalis contains water-soluble allelochemical(s) is able to influence the fluorescence parameters of some Baltic cyanobacteria. Budzałek et al. (2018) noted that the highest decrease in Fv/Fm for Nostoc sp. was observed after addition of 100 µL mL–1 U. inestinalis extract. On the other hand, in some cases, the extract and filtrate from U. inestinalis caused the stimulation of fluorescence parameters of Aphanizomenon sp. and Nostoc sp. (Budzałek et al., 2021). Moreover, extract obtained from Chara baltica and C. canescens caused an increase in fluorescence parameters for Synechococcus sp. after the seventh day of the exposure (Złoch et al., 2018). The low level of these parameters may be evidence of certain disturbances in the photosynthesis process due to allelochemicals (Song et al., 2017). On the other hand, stimulating photosynthesis at the low concentration of thallus may indicate the phenomenon of hormesis (Stebbing, 1982). It is worth mentioned here, that this is the first report of the allelopathic effect of U. intestinalis thallus on the maximum PSII quantum efficiency and the effective quantum yield of PSII photochemistry of Baltic cyanobacteria. Therefore, more studies should be done to further investigate the interactions between macroalgae and cyanobacteria in the aquatic reservoirs. Ecological significance of Ulva intestinalis allelochemicals and possibilities of their application Direct competitive interaction between species is one of the major causes of plant extinction worldwide (Jarchow, Cook, 2009). Competition for available resources is often considered the main competitive mechanism that influences the success of some organisms. Studies have shown that the green algae Ulva sp., which have a high surface area to volume ratio, exhibit high rates of nutrient uptake. However, Ulva sp. have limited ability to concentrate and store nitrogen internally (Xu et al., 2012). Furthermore, Tait and Schiel (2011) indicated that light intensity plays an important role in macroalgae productivity. The authors noted that high densities of the brown alga Sargassum muticum (Yendo) Fensholt exclude native species and reduce biodiversity by shading the associated microalgae (White, Shurin, 2011). In contrast, Svirski et al. (1993) found that growth inhibition of Gracilaria sp. cultured in the presence of U. lactuca was not due to shading or nutrient depletion but appeared to be caused by competition for inorganic carbon through the production of allelopathic compounds. Nan et al. (2004) found that it is difficult to distinguish competition for available resources from allelopathic interactions in the natural aquatic habitats. Therefore, the authors in their study focused on performing experiments with the addition of filtrate obtained from U. pertusa to exclude the effect of competition for available nutrients in the culture and the effect of elevated pH. The authors showed that the growth of H. akashiwo and S. costatum was completely inhibited, even with high nutrient availability. Therefore, competition for nutrients cannot explain the growth inhibition of the tested microalgae by U. pertusa. The experiments conducted in the present study were refined to be able to exclude the effect of competition for resources. In the work described above, the experiments were also conducted using mineral medium f/2. Hence, the effect of nutrient depletion as a cause of growth inhibition of the cyanobacteria studied in our work can also be excluded. Macroalgae are known for their good nutrient uptake capacity (Valiela et al., 1997; Neori et al., 2004; Zertuche-González et al., 2009) and are intentionally used in many parts of the world to reduce nutrient levels in coastal waters (Neori et al., 2004; Carmona et al., 2006; Chopin et al., 2001, 2008). Many bloom-forming species are directly or indirectly promoted by nutrients (Heisler et al., 2008; Anderson et al., 2008) thus macroalgae may reduce the occurrence of these species by decreasing the levels of nutrients available in the ecosystem. However, experiments conducted by Tang and Gobler (2011) showed that the observed negative effects of U. lactuca on bloom-forming species were not due to nutrient limitation of microalgae or nutrient competition between micro- and macroalgae. In the present study, the studied cyanobacteria grown on a nutrient-sufficient mineral medium, so it can be concluded that the negative effect of U. intestinalis was the result of the allelopathic compounds released into the medium. Macroalgae belonging to the genus Ulva are widely distributed. Harvesting these macroalgae from shorelines provides an easy and environmentally friendly way to potentially control the species responsible for creating massive blooms (Jeong et al., 2000). Our results have shown that U. intestinalis can produce and secrete some allelopathic compounds that are able to inhibit the growth of the common, bloom-forming cyanobacteria. 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Journal of Applied Phycology, 21(6), 729. https://doi.org/10.1007/s10811-009-9408-y Zillén, L., Conley, D.J. (2010). Hypoxia and cyanobacterial blooms are not natural features of the Baltic Sea. Biogeosciences Discussions, 7(2), 1783–1812. http://dx.doi.org/10.5194/bgd-7-1783-2010 Złoch, I., Śliwińska-Wilczewska, S., Kucharska, M., Kozłowska, W. (2018). Allelopathic effects of Chara species (C. aspera, C. baltica, and C. canescens) on the bloom-forming picocyanobacterium Synechococcus sp. Environmental Science and Pollution Research, 25, 36403–36411. https://doi.org/10.1007/s11356-018- 3579-5 Abstract Macroalgae have been found to produce active allelochemicals that inhibit of growth other organisms that compete with them for light and space. However, their allelopathic activity on Baltic cyanobacteria is still insufficiently recognised. Therefore, this study aimed to demonstrate the allelopathic effects of Baltic macroalga thallus (Ulva intestinalis) on the growth and photosynthetic activity of three bloom-forming cyanobacteria: Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. This study investigated the cell count of the analysed cyanobacteria (N 105 mL–1), the maximum quantum yield of the second photosystem (PSII) in the dark (Fv/Fm), and the real quantum yield of PSII in the light (ΦPSII) (in the control and the experiments). After 7 days of exposure, the following were added: 0.01, 0.05, and 0.1 g mL–1 of U. intestinalis fresh thallus. It was found that thallus obtained from U. intestinalis had no statistically significant effect on the number of cells of the cyanobacterium Aphanizomenon sp. (at 0.05 and 0.1 g mL–1) and Nostoc sp. (at concentrations of 0.01 and 0.05 g mL−1). On the other hand, it was examined a stimulating effect of 0.01 g mL–1 of the fresh thallus on the number of Aphanizomenon sp. cells which constituted 168%, relative to the control. It was shown that the fresh thallus addition resulted in a decrease in the number of N. spumigena cells (45%, 27%, and 46% after addition of 0.01, 0.05, and 0.1 g wet weight mL−1 of fresh thallus, respectively). In experiments with Nostoc sp., the addition of U. intestinalis thallus has been a negative effect on cyanobacterial growth at 0.1 g mL−1 and constituted 97% of control. It was also found, that U. intestinalis had no allelopathic effect on fluorescence parameters of N. spumigena. All tested concentrations of thallus U. intestinalis (0.01, 0.05, and 0.1 g wet weight mL–1) stimulated the values of Fv/Fm or ΦPSII of cyanobacteria Aphanizomenon sp. and Nostoc sp. compared to the control. These studies help define the role of U. intestinalis allelopathy as a biological factor in the distribution of bloom- forming cyanobacteria in the coastal Baltic Sea region. Key words: allelopathy, cyanobacteria, green macroalgae, growth, fluorescence, thallus Received: [2021.11.05] Accepted: [2022.03.22] Wpływ allelopatyczny Ulva intestinalis na wybrane gatunki bałtyckich sinic Streszczenie Makroglony mają zdolność do produkowania aktywnych związków allelopatycznych, które wpływają na inne organizmy, konkurujące z nimi o światło i przestrzeń. Jednakże ich działanie allelopatyczne na bałtyckie sinice jest wciąż niedostatecznie rozpoznane. Dlatego głównym celem niniejszej pracy było wykazanie aktywności allelopatycznej plechy bałtyckiej zielenicy, ulwa (lub taśma czy błonica) kiszkowata – Ulva intestinalis, na wzrost i aktywność fotosyntetyczną i wzrost trzech sinic bałtyckich, tworzących masowe zakwity: Aphanizomenon sp., Nodularia spumigena oraz Nostoc sp. W niniejszej pracy badano liczebność komórek analizowanych sinic (N 105 mL–1), maksymalną wydajność kwantową drugiego fotosystemu (PSII) w ciemności (Fv/Fm) oraz rzeczywistą wydajność kwantową PSII w świetle (ΦPSII) (w kontroli oraz w eksperymentach). Po 7 dniach ekspozycji, dodawano do nich: 0,01, 0,05, i 0,1 g mL–1 świeżej plechy U. intestinalis. Wykazano, że obecność plech U. intestinalis nie miała istotnego wpływu na liczebność komórek Aphanizomenon sp. w ilości (0,05 i 0,1 g mL–1) oraz Nostoc sp. w ilości plechy (0,01 i 0,05 g mL−1). Wykazano także stymulujący wpływ plechy dodawanej w najmniejszej ilości 0,01 g mL–1 na liczebność komórek Aphanizomenon sp., która wynosiła 168%, w stosunku do kontroli. Dodanie 0,01, 0,05 oraz 0,1 g mL−1 świeżej plechy powodowało obniżenie liczebności komórek N. spumigena (o odpowiednio: 45%, 27%, i 46%, w stosunku do kontroli). Dodatkowo, dodanie 0,1 g mL−1 plechy U. intestinalis powodowało zahamowanie wzrostu Nostoc sp. o 97%. W pracy wykazano także, że plecha U. intestinalis nie miała wpływu na wartość parametrów fluorescencji u N. spumigena. Plecha tego gatunku (w każdym testowanym stężeniu: 0,01, 0,05 oraz 0,1 g mL–1) stymulowała wartość parametrów Fv/Fm lub ΦPSII u sinic Aphanizomenon sp. i Nostoc sp. Wyniki uzyskane w niniejszej pracy definiują rolę allelopatii U. intestinalis jako ważnego czynnika biologicznego, wpływającego na występowanie zakwitów sinic w przybrzeżnych rejonach Morza Bałtyckiego. Słowa kluczowe: allelopatia, sinice, zielenice, wzrost, fluorescencja, plecha Information on the authors Gracjana Budzałek The field of her interest is allelopathic interactions between macroalgae and other phytoplankton species. In her research she is focusing mostly on Baltic species from Puck Bay. She is investigating what influence of allelopathic compounds produced by macroalgae have on bloom-forming cyanobacteria and microalgae. Sylwia Śliwińska-Wilczewska https://orcid.org/0000-0002-3147-6605 She is interested in allelopathy of cyanobacteria and microalgae; in particular of picocyanobacteria Synechococcus sp. Allelopathy plays an important role in interspecific competition and contributes to cyanobacterial bloom maintenance. In her study, the influence of allelochemicals on the growth, chlorophyll fluorescence and photosynthesis irradiance curves of different phytoplankton species was investigated. She is also investigating what influences have environmental factors on produced allelopathic compounds on algae and cyanobacteria.