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Allelopathic inhibition of epiphytes by submerged macrophytes
Aquatic Botany 85 (2006) 252–256 www.elsevier.com/locate/aquabot
Short communication
Allelopathic inhibition of epiphytes by submerged macrophytes Sabine Hilt (nee Ko¨rner) * Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mu¨ggelseedamm 301, 12587 Berlin, Germany Received 30 January 2006; received in revised form 19 April 2006; accepted 11 May 2006
Abstract The hypothesis that epiphytes are more vulnerable to allelochemicals released by submerged macrophytes than phytoplankton was tested by measuring growth and photosystem (PS) II activity of three common epiphytic algae and cyanobacteria in coexistence with Myriophyllum spicatum using dialysis tubes. Results were compared with earlier experiments on planktonic species. Contrary to the planktonic species, the tested epiphytes, the green algae Stigeoclonium tenue, the diatom Gomphonema parvulum and the cyanobacterium Oscillatoria limosa, were not significantly inhibited by M. spicatum. Growth and PS II activity of O. limosa were even significantly enhanced by M. spicatum, but this effect disappeared under phosphorus-deficiency due to the allelopathically induced inhibition of the alkaline phosphatase activity or phosphorus leakage by the macrophytes. My findings of a lower vulnerability of epiphytes against allelopathic substances of submerged macrophytes are supported by results of a literature survey. # 2006 Elsevier B.V. All rights reserved. Keywords: Allelopathy; Epiphyton; Myriophyllum spicatum; PAM-fluorometry; Shallow lake
1. Introduction The release of allelopathically active compounds by submerged macrophytes is thought to be an adaptive trait against competing primary producers in their vicinity (Gross, 2003). It is one of the mechanisms that potentially stabilize the macrophyte-dominated clear water state in shallow eutrophic lakes (Phillips et al., 1978). Shading by epiphytes and phytoplankton is a major limiting factor for submerged macrophytes (Phillips et al., 1978). Epiphytes are potentially the primary target of allelopathically active compounds released by submerged plants as they live adjacent to the donor and their contribution to the reduction of light that reaches the plant often exceeds the impact of phytoplankton. Wium-Andersen (1987) recognized a number of almost epiphyte-free macrophytes and found allelopathic effects of their extracts on an epiphytic diatom. On the other hand, common epiphytic species might have developed resistance against allelochemicals from submerged plants in the same habitat by co-evolution (Reigosa et al., 1999). Resistant species co-occurring with the donor plant could even benefit from the production of allelochemicals by the plant. Wium-Andersen
* Tel.: +49 30 64181677; fax: +49 30 64181682. E-mail address: [email protected]. 0304-3770/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2006.05.004
et al. (1982) found a lower sensitivity of an epiphytic diatom as compared to phytoplankton to extracts of Chara and Erhard and Gross (in press) suggested adaptation of epiphytes to plant metabolites as potential reason for differential inhibitory effects of Elodea extracts. The question, whether epiphytes are less or more vulnerable to allelopathic compounds of submerged macrophytes, however, is still unsolved. In the following study, growth and photosystem (PS) II activity of common epiphytic species were measured in coexistence experiments with the Eurasian watermilfoil Myriophyllum spicatum L., one of the best investigated species among allelopathically active submerged macrophytes (Gross, 2003). Results were compared with studies on planktonic species using the same method (Ko¨rner and Nicklisch, 2002) and a metaanalysis on published case studies. Additional experiments were performed to test the influence of phosphorus (P)-deficiency on the reaction of the target organism. 2. Materials and methods Experiments were performed using suspended unialgal cultures of the cyanobacterium Oscillatoria limosa Agardh SAG 42.87, the green alga Stigeoclonium tenue (Agardh) Ku¨tzing SAG 477-9 and the diatom Gomphonema parvulum Ku¨tzing SAG 1032-1, all typically occurring in the epiphyton of eutrophic waters. Cultures were obtained from the Culture
S. Hilt (nee Ko¨rner) / Aquatic Botany 85 (2006) 252–256
Collection of Algae (SAG) at the University of Go¨ttingen and kept in a modified M III nutrient solution (Nicklisch, 1992). Sterile dialysis membrane tubes (three replicates) containing 50 mL algal culture were placed into aquaria containing 4 L of modified M III solution and M. spicatum (20 g fresh weight from plants collected in Lake Flakensee and kept in aquaria containing tap water) or plastic plants as a control for 3 days. Aquarium aerators were used for circulation of the water to prevent gradients. The chlorophyll fluorescence F0 at an irradiance of 1 mmol photons m2 s1 after dark adaptation and the variable fluorescence F0 v as measures of chlorophyll a (chl a) and photosystem (PS) II activity were recorded daily from subsamples (3 mL) using the Phyto-PAM1 Fluorometer (Walz, Germany). A detailed description of the method is given in Ko¨rner and Nicklisch (2002). Means of control and M. spicatum treatment on day 3 were compared using Student’s ttest. The influence of P-deficiency was tested by repeating experiments with O. limosa in M III nutrient solution (normally 50 mM P) without P. Next to chl a and PS II activity, the alkaline phosphatase activity (APA) of O. limosa was measured by fluorescence spectrometry with methylumbelliferyl-phosphate as substrate (see Gross et al., 1996). Water samples were taken from the aquaria at the end of the experiment and soluble reactive phosphorus (SRP) concentrations were determined photometrically by the molybdenum-blue method (Murphy and Riley, 1962). Means of each of the 4 days and two treatments were compared using a repeated measures ANOVA with time as within subjects factor and treatment as between subjects factor in the statistical package SPSS. Additionally, a metaanalysis using a x2 test (four-fold table) was performed to compare the results of all published studies on allelopathic effects of different submerged macrophyte species on epiphytic and planktonic algae and cyanobacteria. Only donor macrophyte species that showed allelopathic activity in any case were included. The distinction between planktonic and epiphytic species was based on the cited literature.
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Fig. 1. Difference (%) in the end of the experiment between control and treatment in chl a content and PS II activity (S.E.) of common epiphytic (e) and planktonic (p) species of chlorophyta (Stigeoclonium tenue, Scenedesmus armatus), diatoms (Gomphonema parvulum, Stephanodiscus minutulus) and cyanobacteria (Oscillatoria limosa, Limnothrix redekei, Planktothrix agardhii, Aphanizomenon flos-aquae, Microcystis aeruginosa) grown in dialysis tubes for 3 days in coexistence with Myriophyllum spicatum (treatment) or plastic plants (control). Data of planktonic species from Ko¨rner and Nicklisch (2002). Significant differences are *p < 0.05 or **p < 0.01.
M. spicatum treatment were not significantly different from the control (Fig. 2, chl a: F 1,4 = 2.4, p = 0.194, PS II activity: F 1,4 = 2.6, p = 0.184). APA was detected and it increased significantly more strongly in the controls as compared to the
3. Results Chl a and PS II activity of the epiphytic cyanobacterium O. limosa were significantly higher than the controls when grown in coexistence with M. spicatum for 3 days (Figs. 1 and 2; chl a: F 1,4 = 18.8, p = 0.012, PS II activity: F 1,4 = 9.04, p = 0.04), whereas no significant differences were found for the tested epiphytic green alga and the diatom (Fig. 1). The results are shown in comparison with data obtained in earlier experiments using the same method, but planktonic species (Scenedesmus armatus Chodat (green alga), Stephanodiscus minutulus (Ku¨tz) Cleve et Mo¨ller (diatom), Limnothrix redekei (Van Goor) Meffert, Planktothrix agardhii (Gomont) Anag. et Kom., Aphanizomenon flos-aquae Ralfs ex Born. et Flah., Microcystis aeruginosa Ku¨tz. emend. Elenkin (cyanobacteria)) (Ko¨rner and Nicklisch, 2002). These species all had a significantly lower chl a and PS II activity when grown in coexistence with a comparable biomass of M. spicatum (except PS II activity of P. agardhii) (Fig. 1). When grown under P-deficiency, chl a and PS II activity of O. limosa in the
Fig. 2. Chl a content, PS II activity and APA (S.E.) of the cyanobacterium O. limosa grown in dialysis membrane tubes in nutrient solution with 50 mM phosphorus (P) (left column) and with P-deficiency (right column) under the influence of M. spicatum compared to controls with plastic plants. PS II activity is given in relative units (RU), APA in nmol methylumbelliferone (MUF)/ mg chlorophyll a h.
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Table 1 Literature review on allelopathic effects of different submerged macrophyte species on epiphytic and planktonic algae and cyanobacteria (+: inhibition, : no effect or enhancement) Target cyanobacteria/algae species Epiphytic Cyanobacteria Oscillatoria limosa Phormidium tenue
Synechococcus sp. a S. nidulansa Pseudanabaena cf. catenataa Chlorophyta Stigeoclonium tenue Scenedesmus brevispinaa Chlorella cf. vulgarisa Diatoms Gomphonema parvulum
Nitzschia palea
Mixture of genera of Navicula spp., Gomphonema spp. and Diploneis spp. Planktonic Cyanobacteria Limnothrix redekei Planktothrix agardhii Aphanizomenon flos-aquae Microcystis aeruginosa M. aeruginosa
Anabaena flos-aquae
Donor macrophyte species
Effect
Reference
Myriophyllum spicatum Cabomba caroliniana Ceratophyllum demersum Eleocharis acicularis Egeria densa Limnophila sessiliflora M. spicatum Potamogeton oxyphyllus Vallisneria denseserrulata Elodea canadensis E. nuttallii E. canadensis E. nuttallii E. canadensis E. nuttallii
+ + + + + + + + +
This study Nakai et al. (1999)
M. spicatum M. spicatum E. canadensis E. nuttallii E. canadensis E. nuttallii
+
This study Gross (1995) Erhard and Gross (2006)
M. spicatum M. spicatum M. verticillatum M. alterniflorum C. demersum C. submersum C. demersum C. demersum C. submersum M. spicatum M. verticillatum M. alterniflorum Chara globularis E. canadensis Stratiotes aloides Nitella sp. Zostera marina
+ + + + + + + + + + + + + + +
This study Gross (1995)
M. spicatum
+ + + + + + + + + + + + + + + +
C. caroliniana C. demersum E. acicularis E. densa L. sessiliflora M. spicatum P. oxyphyllus V. denseserrulata C. caroliniana C. demersum E. acicularis E. densa
Erhard and Gross (2006)
Wium-Andersen et al. (1983) Gross (1995)
Wium-Andersen et al. (1982) Wium-Andersen (1987)
Harrison and Durance (1985)
Ko¨rner and Nicklisch (2002)
Nakai et al. (1999)
Nakai et al. (1999)
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Table 1 (Continued ) Target cyanobacteria/algae species
Synechococcus elongatus Synechococcus leopoliensis Anacystis nidulans Anabaena variabilis P-9
Anabaena sp. PCC 7120 Synechococcus sp. PCC 6911 Synechocystis sp. Chlorophyta Scenedesmus armatus Scenedesmus obliquus Chlorella minutissima Selenastrum capricornutum Scenedesmus communis Ankistrodesmus bibraianus Chlamydomonas globosa Scenedesmus quadricauda Selenastrum capricornutum Scenedesmus acutus Nannochloris sp. Scenedesmus falcatus
Donor macrophyte species
Effect
L. sessiliflora M. spicatum P. oxyphyllus V. denseserrulata E. canadensis E. nuttallii M. verticillatum M. spicatum M. spicatum Fontinalis antipyretica C. demersum C. submersum Chara spp. E. canadensis Hippuris vulgaris Hottonia palustris M. alterniflorum M. verticillatum P. pectinatus P. perfoliatus Ranunculus aquatilis Stratiotes aloides M. spicatum
+ + + + + + + + + + + + + + + + + + + + + + + + +
M. spicatum S. aloides C. globularis/C. contraria C. globularis/C. contraria C. globularis/C. contraria C. globularis
+ + + + + + + + +
Ko¨rner and Nicklisch (2002) Mulderij et al. (2005) Mulderij et al. (2003)
M. spicatum
C. aspera M. spicatum
Reference
Erhard and Gross (2006) Aliotta et al. (1992) Planas et al. (1981) Gross et al. (1996) Gross (1995)
Hootsmans and Blindow (1994) Planas et al. (1981)
van Donk and van de Bund (2002) Gross (1995)
Diatoms Stephanodiscus minutulus
M. spicatum
+
Ko¨rner and Nicklisch (2002)
Mixed Summerplankton from lake Esrom and the pond Ødam
Chara globularis
+
Wium-Andersen et al. (1982)
Only donor macrophyte species that showed allelopathic activity in any case were included. a No typical epiphytic species, but isolated from the epiphyton of submerged plants.
M. spicatum treatment (Fig. 2, F 1,4 = 28.5, p = 0.006). SRP concentrations were 6 mg L1 at day 0 and below the detection limit (3 mg L1) at days 2 and 3 in both control and M. spicatum treatment. Results of the metaanalysis revealed that a significant inhibition by macrophyte allelochemicals was found in a significantly (x2 = 6.37, p = 0.01) lower number of literature case studies (Table 1) using epiphytic species (68% of 38 case studies) as compared to planktonic species (89% of 56 case studies). 4. Discussion My results suggest that epiphytic algae and cyanobacteria might be less vulnerable than planktonic against the allelo-
pathic effect of M. spicatum, at least against hydrophilic compounds that are exuded into the water by the biomass used for the experiments. The chosen biomass density (5 g fresh weight/L) is comparable to those occurring in lakes (Duarte and Kalff, 1990), but M. spicatum plants used in the experiments on planktonic species may have differed in their content of allelopathically active substances as well as their release rates although they originated from the same laboratory culture. The sensitivity of epiphytic species may increase when grown attached to the plant surface instead of suspended due to the additional effect of the more lipophilic compounds and the lack of photolytic and bacterial metabolization during transport. The biofilm, however, may also contain bacteria that metabolize polyphenolic compounds (Gross et al., 1996).
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Additional stress (P-deficiency) changed the reaction of the cyanobacterium O. limosa to M. spicatum. Potential explanations are a higher sensitivity to allelopathy under stress as suggested by Reigosa et al. (1999) or the inhibition of the APA, a known mode of action of M. spicatum allelochemicals (Gross et al., 1996). The APA seemed significantly inhibited in the M. spicatum treatment (Fig. 2), but this difference may also have resulted from P leakage of M. spicatum and direct uptake by O. limosa as suggested by Hilt et al. (in press). P leakage might be more advantageous for epiphytes growing in the biofilm than phytoplankton in case of nutrient interferences on allelopathic effects of submerged macrophytes. In the present study, only three epiphytic target species were tested, but the results of the metaanalysis of other case studies seem to support the findings of a lower vulnerability of epiphytes. More chlorophytes seem to be not inhibited as compared to other groups, but more data are needed for a generalization. Drawbacks of the literature survey are, that (a) the target species used range from recently isolated from the epiphyton of macrophytes to cultures collected decades ago, which potentially results in a different sensitivity, (b) the ratio of inhibitory effects might be overestimated as often negative results (no inhibition) are not reported and (c) the methods used vary considerably. A few investigators, however, tested the influence of macrophytes or allelopathic substances on epiphytic and planktonic algae using the same method (Wium-Andersen et al., 1982; Gross, 1995; Nakai et al., 1999; Erhard and Gross, in press). In these studies, epiphytic algae always proved to be less inhibited than the planktonic species or were not inhibited at all (Table 1). This is in accordance with the hypothesized role of epiphytes in the early stages of the eutrophication process. Phillips et al. (1978) gave evidence that loss of macrophytes is often due to increased growth of, and shading by epiphytes, and that phytoplankton development is subsequent rather than causative. Experiments and the literature review suggest that epiphytic algae and cyanobacteria might indeed have developed resistance against allelopathic substances of macrophytes by co-evolution as proposed by Reigosa et al. (1999). For a final proof of this hypothesis, however, further studies, e.g. using species originating from different habitats or geographical regions are needed. Acknowledgements Special thanks to Lina Wischnewsky for helping with the PAM measurements and maintenance of algae cultures, Christiane Herzog, Antje Lu¨der, Elke Zwirnmann, HansJu¨rgen Exner and Thomas Rossoll for determination of nutrient concentrations, Andreas Nicklisch for scientific discussions and Jan Vermaat and two anonymous reviewers for their comments on the manuscript. The project was financially supported by the ‘‘Berliner Programm zur Fo¨rderung der Chancengleichheit von Frauen in Forschung und Lehre’’.
References Aliotta, G., Molinaro, A., Monaco, P., Pinto, G., Previtera, L., 1992. Three biologically active phenylpropanoid glucosides from Myriophyllum verticillatum. Phytochemistry 31, 109–111. Duarte, C.M., Kalff, J., 1990. Biomass density and the relationship between submerged macrophyte biomass and plant growth form. Hydrobiologia 196, 17–23. Erhard, D., Gross, E.M., 2006. Allelopathic activity of Elodea canadensis and E. nuttallii against epiphytes and phytoplankton. Aquat. Bot. 85, 203– 211. Gross, E.M., 2003. Allelopathy of aquatic autotrophs. Crit. Rev. Plant Sci. 22, 313–339. Gross, E.M., 1995. Allelopathische Interaktionen submerser Makrophyten mit Epiphyten und Phytoplankton: Algizide hydrolisierbare Polyphenole aus Myriophyllum spicatum L. PhD Thesis. Christian-Albrechts-Universita¨t Kiel, Cuvillier, Go¨ttingen. Gross, E.M., Meyer, H., Schilling, G., 1996. Release and ecological impact of algicidal hydrolysable polyphenols in Myriophyllum spicatum. Phytochemistry 41, 133–138. Harrison, P.G., Durance, C.D., 1985. Reductions in photosynthetic carbon uptake in epiphytic diatoms by water-soluble extracts of leaves of Zostera marina. Mar. Biol. 90, 117–119. Hilt (nee Ko¨rner), S., Ghobrial, M., Gross, E.M., in press. In situ allelopathic potential of Myriophyllum verticillatum (Haloragaceae) against selected phytoplankton species. J. Phycol. Hootsmans, M.J.M., Blindow, I., 1994. Allelopathic limitation of algal growth by macrophytes. In: Van Vierssen, W., Hootsmans, M.J.M., Vermaat, J.E. (Eds.), Lake Veluwe, A Macrophyte-dominated System under Eutrophication Stress. Kluwer, Dordrecht, pp. 175–192. Ko¨rner, S., Nicklisch, A., 2002. Allelopathic growth inhibition of selected phytoplankton species by submerged macrophytes. J. Phycol. 38, 862– 871. Mulderij, G., van Donk, E., Roelofs, J.G.M., 2003. Differential sensitivity of green algae to allelopathic substances from Chara. Hydrobiologia 491, 261–271. Mulderij, G., Mooij, W.M., Van Donk, E., 2005. Allelopathic growth inhibition and colony formation of the green alga Scenedesmus obliquus by the aquatic macrophyte Stratiotes aloides. Aquat. Ecol. 39, 11–21. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31–36. Nakai, S., Inoue, Y., Hosomi, M., Murakami, A., 1999. Growth inhibition of blue-green algae by allelopathic effects of macrophytes. Water Sci. Technol. 39, 47–53. Nicklisch, A., 1992. The interaction of irradiance and temperature on the growth rate of Limnothrix redekei and its mathematical description. Arch. Hydrobiol. 91 (Suppl.), 1–18 (Algological Studies 63). Phillips, G.L., Eminson, D., Moss, B., 1978. A mechanism to account for macrophyte decline in progressively eutrophicated freshwaters. Aquat. Bot. 4, 103–126. Planas, D., Sarhan, F., Dube, L., Godmaire, H., Cadieux, C., 1981. Ecological significance of phenolic compounds of Myriophyllum spicatum. Verh. Int. Ver. Limnol. 21, 1492–1496. Reigosa, M.J., Sanchez-Moreiras, A., Gonzalez, L., 1999. Ecophysiological approach in allelopathy. Crit. Rev. Plant Sci. 18, 577–608. van Donk, E., van de Bund, W., 2002. Impact of submerged macrophytes including charophytes on phyto- and zooplankton communities: allelopathy versus other mechanisms. Aquat. Bot. 72, 261–274. Wium-Andersen, S., 1987. Allelopathy among aquatic plants. Arch. Hydrobiol., Beih. Ergeb. Limnol. 27, 167–172. Wium-Andersen, S., Anthoni, U., Christophersen, C., Houen, G., 1982. Allelopathic effects on phytoplankton by substances isolated from aquatic macrophytes (Charales). Oikos 39, 187–190. Wium-Andersen, S., Anthoni, U., Houen, G., 1983. Elemental sulphur, a possible allelopathic compound from Ceratophyllum demersum. Phytochemistry 22, 2613.
Short communication
Allelopathic inhibition of epiphytes by submerged macrophytes Sabine Hilt (nee Ko¨rner) * Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mu¨ggelseedamm 301, 12587 Berlin, Germany Received 30 January 2006; received in revised form 19 April 2006; accepted 11 May 2006
Abstract The hypothesis that epiphytes are more vulnerable to allelochemicals released by submerged macrophytes than phytoplankton was tested by measuring growth and photosystem (PS) II activity of three common epiphytic algae and cyanobacteria in coexistence with Myriophyllum spicatum using dialysis tubes. Results were compared with earlier experiments on planktonic species. Contrary to the planktonic species, the tested epiphytes, the green algae Stigeoclonium tenue, the diatom Gomphonema parvulum and the cyanobacterium Oscillatoria limosa, were not significantly inhibited by M. spicatum. Growth and PS II activity of O. limosa were even significantly enhanced by M. spicatum, but this effect disappeared under phosphorus-deficiency due to the allelopathically induced inhibition of the alkaline phosphatase activity or phosphorus leakage by the macrophytes. My findings of a lower vulnerability of epiphytes against allelopathic substances of submerged macrophytes are supported by results of a literature survey. # 2006 Elsevier B.V. All rights reserved. Keywords: Allelopathy; Epiphyton; Myriophyllum spicatum; PAM-fluorometry; Shallow lake
1. Introduction The release of allelopathically active compounds by submerged macrophytes is thought to be an adaptive trait against competing primary producers in their vicinity (Gross, 2003). It is one of the mechanisms that potentially stabilize the macrophyte-dominated clear water state in shallow eutrophic lakes (Phillips et al., 1978). Shading by epiphytes and phytoplankton is a major limiting factor for submerged macrophytes (Phillips et al., 1978). Epiphytes are potentially the primary target of allelopathically active compounds released by submerged plants as they live adjacent to the donor and their contribution to the reduction of light that reaches the plant often exceeds the impact of phytoplankton. Wium-Andersen (1987) recognized a number of almost epiphyte-free macrophytes and found allelopathic effects of their extracts on an epiphytic diatom. On the other hand, common epiphytic species might have developed resistance against allelochemicals from submerged plants in the same habitat by co-evolution (Reigosa et al., 1999). Resistant species co-occurring with the donor plant could even benefit from the production of allelochemicals by the plant. Wium-Andersen
* Tel.: +49 30 64181677; fax: +49 30 64181682. E-mail address: [email protected]. 0304-3770/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2006.05.004
et al. (1982) found a lower sensitivity of an epiphytic diatom as compared to phytoplankton to extracts of Chara and Erhard and Gross (in press) suggested adaptation of epiphytes to plant metabolites as potential reason for differential inhibitory effects of Elodea extracts. The question, whether epiphytes are less or more vulnerable to allelopathic compounds of submerged macrophytes, however, is still unsolved. In the following study, growth and photosystem (PS) II activity of common epiphytic species were measured in coexistence experiments with the Eurasian watermilfoil Myriophyllum spicatum L., one of the best investigated species among allelopathically active submerged macrophytes (Gross, 2003). Results were compared with studies on planktonic species using the same method (Ko¨rner and Nicklisch, 2002) and a metaanalysis on published case studies. Additional experiments were performed to test the influence of phosphorus (P)-deficiency on the reaction of the target organism. 2. Materials and methods Experiments were performed using suspended unialgal cultures of the cyanobacterium Oscillatoria limosa Agardh SAG 42.87, the green alga Stigeoclonium tenue (Agardh) Ku¨tzing SAG 477-9 and the diatom Gomphonema parvulum Ku¨tzing SAG 1032-1, all typically occurring in the epiphyton of eutrophic waters. Cultures were obtained from the Culture
S. Hilt (nee Ko¨rner) / Aquatic Botany 85 (2006) 252–256
Collection of Algae (SAG) at the University of Go¨ttingen and kept in a modified M III nutrient solution (Nicklisch, 1992). Sterile dialysis membrane tubes (three replicates) containing 50 mL algal culture were placed into aquaria containing 4 L of modified M III solution and M. spicatum (20 g fresh weight from plants collected in Lake Flakensee and kept in aquaria containing tap water) or plastic plants as a control for 3 days. Aquarium aerators were used for circulation of the water to prevent gradients. The chlorophyll fluorescence F0 at an irradiance of 1 mmol photons m2 s1 after dark adaptation and the variable fluorescence F0 v as measures of chlorophyll a (chl a) and photosystem (PS) II activity were recorded daily from subsamples (3 mL) using the Phyto-PAM1 Fluorometer (Walz, Germany). A detailed description of the method is given in Ko¨rner and Nicklisch (2002). Means of control and M. spicatum treatment on day 3 were compared using Student’s ttest. The influence of P-deficiency was tested by repeating experiments with O. limosa in M III nutrient solution (normally 50 mM P) without P. Next to chl a and PS II activity, the alkaline phosphatase activity (APA) of O. limosa was measured by fluorescence spectrometry with methylumbelliferyl-phosphate as substrate (see Gross et al., 1996). Water samples were taken from the aquaria at the end of the experiment and soluble reactive phosphorus (SRP) concentrations were determined photometrically by the molybdenum-blue method (Murphy and Riley, 1962). Means of each of the 4 days and two treatments were compared using a repeated measures ANOVA with time as within subjects factor and treatment as between subjects factor in the statistical package SPSS. Additionally, a metaanalysis using a x2 test (four-fold table) was performed to compare the results of all published studies on allelopathic effects of different submerged macrophyte species on epiphytic and planktonic algae and cyanobacteria. Only donor macrophyte species that showed allelopathic activity in any case were included. The distinction between planktonic and epiphytic species was based on the cited literature.
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Fig. 1. Difference (%) in the end of the experiment between control and treatment in chl a content and PS II activity (S.E.) of common epiphytic (e) and planktonic (p) species of chlorophyta (Stigeoclonium tenue, Scenedesmus armatus), diatoms (Gomphonema parvulum, Stephanodiscus minutulus) and cyanobacteria (Oscillatoria limosa, Limnothrix redekei, Planktothrix agardhii, Aphanizomenon flos-aquae, Microcystis aeruginosa) grown in dialysis tubes for 3 days in coexistence with Myriophyllum spicatum (treatment) or plastic plants (control). Data of planktonic species from Ko¨rner and Nicklisch (2002). Significant differences are *p < 0.05 or **p < 0.01.
M. spicatum treatment were not significantly different from the control (Fig. 2, chl a: F 1,4 = 2.4, p = 0.194, PS II activity: F 1,4 = 2.6, p = 0.184). APA was detected and it increased significantly more strongly in the controls as compared to the
3. Results Chl a and PS II activity of the epiphytic cyanobacterium O. limosa were significantly higher than the controls when grown in coexistence with M. spicatum for 3 days (Figs. 1 and 2; chl a: F 1,4 = 18.8, p = 0.012, PS II activity: F 1,4 = 9.04, p = 0.04), whereas no significant differences were found for the tested epiphytic green alga and the diatom (Fig. 1). The results are shown in comparison with data obtained in earlier experiments using the same method, but planktonic species (Scenedesmus armatus Chodat (green alga), Stephanodiscus minutulus (Ku¨tz) Cleve et Mo¨ller (diatom), Limnothrix redekei (Van Goor) Meffert, Planktothrix agardhii (Gomont) Anag. et Kom., Aphanizomenon flos-aquae Ralfs ex Born. et Flah., Microcystis aeruginosa Ku¨tz. emend. Elenkin (cyanobacteria)) (Ko¨rner and Nicklisch, 2002). These species all had a significantly lower chl a and PS II activity when grown in coexistence with a comparable biomass of M. spicatum (except PS II activity of P. agardhii) (Fig. 1). When grown under P-deficiency, chl a and PS II activity of O. limosa in the
Fig. 2. Chl a content, PS II activity and APA (S.E.) of the cyanobacterium O. limosa grown in dialysis membrane tubes in nutrient solution with 50 mM phosphorus (P) (left column) and with P-deficiency (right column) under the influence of M. spicatum compared to controls with plastic plants. PS II activity is given in relative units (RU), APA in nmol methylumbelliferone (MUF)/ mg chlorophyll a h.
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Table 1 Literature review on allelopathic effects of different submerged macrophyte species on epiphytic and planktonic algae and cyanobacteria (+: inhibition, : no effect or enhancement) Target cyanobacteria/algae species Epiphytic Cyanobacteria Oscillatoria limosa Phormidium tenue
Synechococcus sp. a S. nidulansa Pseudanabaena cf. catenataa Chlorophyta Stigeoclonium tenue Scenedesmus brevispinaa Chlorella cf. vulgarisa Diatoms Gomphonema parvulum
Nitzschia palea
Mixture of genera of Navicula spp., Gomphonema spp. and Diploneis spp. Planktonic Cyanobacteria Limnothrix redekei Planktothrix agardhii Aphanizomenon flos-aquae Microcystis aeruginosa M. aeruginosa
Anabaena flos-aquae
Donor macrophyte species
Effect
Reference
Myriophyllum spicatum Cabomba caroliniana Ceratophyllum demersum Eleocharis acicularis Egeria densa Limnophila sessiliflora M. spicatum Potamogeton oxyphyllus Vallisneria denseserrulata Elodea canadensis E. nuttallii E. canadensis E. nuttallii E. canadensis E. nuttallii
+ + + + + + + + +
This study Nakai et al. (1999)
M. spicatum M. spicatum E. canadensis E. nuttallii E. canadensis E. nuttallii
+
This study Gross (1995) Erhard and Gross (2006)
M. spicatum M. spicatum M. verticillatum M. alterniflorum C. demersum C. submersum C. demersum C. demersum C. submersum M. spicatum M. verticillatum M. alterniflorum Chara globularis E. canadensis Stratiotes aloides Nitella sp. Zostera marina
+ + + + + + + + + + + + + + +
This study Gross (1995)
M. spicatum
+ + + + + + + + + + + + + + + +
C. caroliniana C. demersum E. acicularis E. densa L. sessiliflora M. spicatum P. oxyphyllus V. denseserrulata C. caroliniana C. demersum E. acicularis E. densa
Erhard and Gross (2006)
Wium-Andersen et al. (1983) Gross (1995)
Wium-Andersen et al. (1982) Wium-Andersen (1987)
Harrison and Durance (1985)
Ko¨rner and Nicklisch (2002)
Nakai et al. (1999)
Nakai et al. (1999)
S. Hilt (nee Ko¨rner) / Aquatic Botany 85 (2006) 252–256
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Table 1 (Continued ) Target cyanobacteria/algae species
Synechococcus elongatus Synechococcus leopoliensis Anacystis nidulans Anabaena variabilis P-9
Anabaena sp. PCC 7120 Synechococcus sp. PCC 6911 Synechocystis sp. Chlorophyta Scenedesmus armatus Scenedesmus obliquus Chlorella minutissima Selenastrum capricornutum Scenedesmus communis Ankistrodesmus bibraianus Chlamydomonas globosa Scenedesmus quadricauda Selenastrum capricornutum Scenedesmus acutus Nannochloris sp. Scenedesmus falcatus
Donor macrophyte species
Effect
L. sessiliflora M. spicatum P. oxyphyllus V. denseserrulata E. canadensis E. nuttallii M. verticillatum M. spicatum M. spicatum Fontinalis antipyretica C. demersum C. submersum Chara spp. E. canadensis Hippuris vulgaris Hottonia palustris M. alterniflorum M. verticillatum P. pectinatus P. perfoliatus Ranunculus aquatilis Stratiotes aloides M. spicatum
+ + + + + + + + + + + + + + + + + + + + + + + + +
M. spicatum S. aloides C. globularis/C. contraria C. globularis/C. contraria C. globularis/C. contraria C. globularis
+ + + + + + + + +
Ko¨rner and Nicklisch (2002) Mulderij et al. (2005) Mulderij et al. (2003)
M. spicatum
C. aspera M. spicatum
Reference
Erhard and Gross (2006) Aliotta et al. (1992) Planas et al. (1981) Gross et al. (1996) Gross (1995)
Hootsmans and Blindow (1994) Planas et al. (1981)
van Donk and van de Bund (2002) Gross (1995)
Diatoms Stephanodiscus minutulus
M. spicatum
+
Ko¨rner and Nicklisch (2002)
Mixed Summerplankton from lake Esrom and the pond Ødam
Chara globularis
+
Wium-Andersen et al. (1982)
Only donor macrophyte species that showed allelopathic activity in any case were included. a No typical epiphytic species, but isolated from the epiphyton of submerged plants.
M. spicatum treatment (Fig. 2, F 1,4 = 28.5, p = 0.006). SRP concentrations were 6 mg L1 at day 0 and below the detection limit (3 mg L1) at days 2 and 3 in both control and M. spicatum treatment. Results of the metaanalysis revealed that a significant inhibition by macrophyte allelochemicals was found in a significantly (x2 = 6.37, p = 0.01) lower number of literature case studies (Table 1) using epiphytic species (68% of 38 case studies) as compared to planktonic species (89% of 56 case studies). 4. Discussion My results suggest that epiphytic algae and cyanobacteria might be less vulnerable than planktonic against the allelo-
pathic effect of M. spicatum, at least against hydrophilic compounds that are exuded into the water by the biomass used for the experiments. The chosen biomass density (5 g fresh weight/L) is comparable to those occurring in lakes (Duarte and Kalff, 1990), but M. spicatum plants used in the experiments on planktonic species may have differed in their content of allelopathically active substances as well as their release rates although they originated from the same laboratory culture. The sensitivity of epiphytic species may increase when grown attached to the plant surface instead of suspended due to the additional effect of the more lipophilic compounds and the lack of photolytic and bacterial metabolization during transport. The biofilm, however, may also contain bacteria that metabolize polyphenolic compounds (Gross et al., 1996).
256
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Additional stress (P-deficiency) changed the reaction of the cyanobacterium O. limosa to M. spicatum. Potential explanations are a higher sensitivity to allelopathy under stress as suggested by Reigosa et al. (1999) or the inhibition of the APA, a known mode of action of M. spicatum allelochemicals (Gross et al., 1996). The APA seemed significantly inhibited in the M. spicatum treatment (Fig. 2), but this difference may also have resulted from P leakage of M. spicatum and direct uptake by O. limosa as suggested by Hilt et al. (in press). P leakage might be more advantageous for epiphytes growing in the biofilm than phytoplankton in case of nutrient interferences on allelopathic effects of submerged macrophytes. In the present study, only three epiphytic target species were tested, but the results of the metaanalysis of other case studies seem to support the findings of a lower vulnerability of epiphytes. More chlorophytes seem to be not inhibited as compared to other groups, but more data are needed for a generalization. Drawbacks of the literature survey are, that (a) the target species used range from recently isolated from the epiphyton of macrophytes to cultures collected decades ago, which potentially results in a different sensitivity, (b) the ratio of inhibitory effects might be overestimated as often negative results (no inhibition) are not reported and (c) the methods used vary considerably. A few investigators, however, tested the influence of macrophytes or allelopathic substances on epiphytic and planktonic algae using the same method (Wium-Andersen et al., 1982; Gross, 1995; Nakai et al., 1999; Erhard and Gross, in press). In these studies, epiphytic algae always proved to be less inhibited than the planktonic species or were not inhibited at all (Table 1). This is in accordance with the hypothesized role of epiphytes in the early stages of the eutrophication process. Phillips et al. (1978) gave evidence that loss of macrophytes is often due to increased growth of, and shading by epiphytes, and that phytoplankton development is subsequent rather than causative. Experiments and the literature review suggest that epiphytic algae and cyanobacteria might indeed have developed resistance against allelopathic substances of macrophytes by co-evolution as proposed by Reigosa et al. (1999). For a final proof of this hypothesis, however, further studies, e.g. using species originating from different habitats or geographical regions are needed. Acknowledgements Special thanks to Lina Wischnewsky for helping with the PAM measurements and maintenance of algae cultures, Christiane Herzog, Antje Lu¨der, Elke Zwirnmann, HansJu¨rgen Exner and Thomas Rossoll for determination of nutrient concentrations, Andreas Nicklisch for scientific discussions and Jan Vermaat and two anonymous reviewers for their comments on the manuscript. The project was financially supported by the ‘‘Berliner Programm zur Fo¨rderung der Chancengleichheit von Frauen in Forschung und Lehre’’.
References Aliotta, G., Molinaro, A., Monaco, P., Pinto, G., Previtera, L., 1992. Three biologically active phenylpropanoid glucosides from Myriophyllum verticillatum. Phytochemistry 31, 109–111. Duarte, C.M., Kalff, J., 1990. Biomass density and the relationship between submerged macrophyte biomass and plant growth form. Hydrobiologia 196, 17–23. Erhard, D., Gross, E.M., 2006. Allelopathic activity of Elodea canadensis and E. nuttallii against epiphytes and phytoplankton. Aquat. Bot. 85, 203– 211. Gross, E.M., 2003. Allelopathy of aquatic autotrophs. Crit. Rev. Plant Sci. 22, 313–339. Gross, E.M., 1995. Allelopathische Interaktionen submerser Makrophyten mit Epiphyten und Phytoplankton: Algizide hydrolisierbare Polyphenole aus Myriophyllum spicatum L. PhD Thesis. Christian-Albrechts-Universita¨t Kiel, Cuvillier, Go¨ttingen. Gross, E.M., Meyer, H., Schilling, G., 1996. Release and ecological impact of algicidal hydrolysable polyphenols in Myriophyllum spicatum. Phytochemistry 41, 133–138. Harrison, P.G., Durance, C.D., 1985. Reductions in photosynthetic carbon uptake in epiphytic diatoms by water-soluble extracts of leaves of Zostera marina. Mar. Biol. 90, 117–119. Hilt (nee Ko¨rner), S., Ghobrial, M., Gross, E.M., in press. In situ allelopathic potential of Myriophyllum verticillatum (Haloragaceae) against selected phytoplankton species. J. Phycol. Hootsmans, M.J.M., Blindow, I., 1994. Allelopathic limitation of algal growth by macrophytes. In: Van Vierssen, W., Hootsmans, M.J.M., Vermaat, J.E. (Eds.), Lake Veluwe, A Macrophyte-dominated System under Eutrophication Stress. Kluwer, Dordrecht, pp. 175–192. Ko¨rner, S., Nicklisch, A., 2002. Allelopathic growth inhibition of selected phytoplankton species by submerged macrophytes. J. Phycol. 38, 862– 871. Mulderij, G., van Donk, E., Roelofs, J.G.M., 2003. Differential sensitivity of green algae to allelopathic substances from Chara. Hydrobiologia 491, 261–271. Mulderij, G., Mooij, W.M., Van Donk, E., 2005. Allelopathic growth inhibition and colony formation of the green alga Scenedesmus obliquus by the aquatic macrophyte Stratiotes aloides. Aquat. Ecol. 39, 11–21. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31–36. Nakai, S., Inoue, Y., Hosomi, M., Murakami, A., 1999. Growth inhibition of blue-green algae by allelopathic effects of macrophytes. Water Sci. Technol. 39, 47–53. Nicklisch, A., 1992. The interaction of irradiance and temperature on the growth rate of Limnothrix redekei and its mathematical description. Arch. Hydrobiol. 91 (Suppl.), 1–18 (Algological Studies 63). Phillips, G.L., Eminson, D., Moss, B., 1978. A mechanism to account for macrophyte decline in progressively eutrophicated freshwaters. Aquat. Bot. 4, 103–126. Planas, D., Sarhan, F., Dube, L., Godmaire, H., Cadieux, C., 1981. Ecological significance of phenolic compounds of Myriophyllum spicatum. Verh. Int. Ver. Limnol. 21, 1492–1496. Reigosa, M.J., Sanchez-Moreiras, A., Gonzalez, L., 1999. Ecophysiological approach in allelopathy. Crit. Rev. Plant Sci. 18, 577–608. van Donk, E., van de Bund, W., 2002. Impact of submerged macrophytes including charophytes on phyto- and zooplankton communities: allelopathy versus other mechanisms. Aquat. Bot. 72, 261–274. Wium-Andersen, S., 1987. Allelopathy among aquatic plants. Arch. Hydrobiol., Beih. Ergeb. Limnol. 27, 167–172. Wium-Andersen, S., Anthoni, U., Christophersen, C., Houen, G., 1982. Allelopathic effects on phytoplankton by substances isolated from aquatic macrophytes (Charales). Oikos 39, 187–190. Wium-Andersen, S., Anthoni, U., Houen, G., 1983. Elemental sulphur, a possible allelopathic compound from Ceratophyllum demersum. Phytochemistry 22, 2613.