J Appl Phycol DOI 10.1007/s10811-014-0326-2 Allelopathic interactions between microcystin-producing and non-microcystin-producing cyanobacteria and green microalgae: implications for microcystins production Maria do Carmo Bittencourt-Oliveira & Mathias Ahii Chia & Helton Soriano Bezerra de Oliveira & Micheline Kézia Cordeiro Araújo & Renato José Reis Molica & Carlos Tadeu Santos Dias Received: 10 March 2014 /Revised and accepted: 24 April 2014 # Springer Science+Business Media Dordrecht 2014 Abstract Most mixed culture studies on the allelopathic in- the experiment. On the other hand, the extracts of the teractions between toxic and nontoxic cyanobacteria with cyanobacteria had no significant inhibitory effect on the green phytoplankton species rarely investigate the role of algal strains investigated, while those of the green algae also microcystins (MC) production and regulation in the course had significant inhibitory effect on the growth of of the studies. This study investigated the interactions between M. aeruginosa. In conclusion, both cyanobacterial and green intact cells of toxic (Microcystis aeruginosa (Kützing) algal strains investigated were negatively affected by the Kützing) and nontoxic (Microcystis panniformis Komárek presence of competing species. M. aeruginosa responded to et al.) cyanobacteria with those of green algae the presence of green algae by increasing its MC production. (Monoraphidium convolutum (Corda) Komárková-Legnerová The green algal strains significantly inhibited the growth of and Scenedesmus acuminatus (Largerheim) Chodat) as well M. aeruginosa. as the effects of their respective crude extracts (5 and 10 μg.L−1) on their growth under controlled conditions. Keywords Allelopathy . Species competition . M. aeruginosa and M. panniformis were able to significantly Phytoplankton . Mixed algal cultivation . Cyanotoxins . (p<0.05) inhibit the growth of the green algae with Monoraphidium . Scenedesmus . Microcystis M. convolutum being the most affected. The green alga S. acuminatus in return was able to inhibit the growth of the both cyanobacteria. In response to the presence of a compet- Introduction ing species in the growth medium, M. aeruginosa significant- ly increased its MC production per cell with the progression of In recent decades, algal blooms have become widespread in the experiment, having the highest concentration at the end of many water bodies around the world. These have also been characterized by the increased occurrence of toxic M. C. Bittencourt-Oliveira : M. A. Chia : H. S. B. de Oliveira : cyanobacteria blooms, in addition to those of diatoms, dino- M. K. Cordeiro Araújo flagellates, and green algae (Zhang et al. 2013), which have Department of Biological Sciences, Luiz de Queiroz College of serious social and economic implications due to the degrada- Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, tion of water resources, and as a result, generated a lot of Piracicaba, SP 13418-900, Brazil interest into investigations on the environmental factors and * : : M. C. Bittencourt-Oliveira: ( ) H. S. B. de Oliveira mechanisms that promote these blooms as well as how M. K. Cordeiro Araújo R. J. R. Molica cyanotoxins confer a competitive advantage to the producing Graduate Program in Botany, Rural and Federal University of species (Jonsson et al. 2009). Pernambuco, Rua D. Manoel de Medeiros, S/N, Dois Irmãos, Recife, PE 52171-030, Brazil Different water bodies around the world are characterized e-mail: [email protected] by dominant species of phytoplankton that alternate between cyanobacteria and other microalgae (diatoms, green algae). C. T. S. Dias The dominance of different algal species changes from season Department of Exact Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, to season during bloom formation in most water bodies world- Piracicaba, SP 13418-900, Brazil wide (Chen et al. 2003;Frossardetal.2014). In order to J Appl Phycol understand the succession mechanisms responsible for monitoring changes in their concentrations (Yang et al. cyanobacteria and other phytoplankton species, experiments 2014). Studies of the action of MC on microalgae are mostly involving mixed cultures have been encouraged to be carried restricted to lysates of toxic cyanobacterial extracts or purified out by researchers. This has led to investigations on the MC, and often with much higher concentrations than those interactions of cyanotoxin-producing and nonproducing- commonly found in natural environments (Nagata et al.1997; cyanobacteria with microalgae strains as a means of contrib- Kemp and John 2006; Máthé et al. 2007; B-Beres et al. 2012). uting to the understanding of how successional processes Conclusions on the allelopathy of MC resulting from studies operate in nature. A comprehensive understanding of the that used non-environmentally relevant concentrations can be competition between algae and cyanobacteria can be crucial misleading and not applicable in real life situations (Leao et al. to control strategies for bloom formation and outbreaks in 2009). Therefore, our study aimed to (1) evaluate the possible aquatic ecosystems (Zhang et al. 2013). allelopathic interaction between intact cells of microcystin- Most studies have considered the effect of chemical and producing and -nonproducing cyanobacteria with the green physical factors on the competition between cyanobacteria microalgae Scenedesmus acuminatus and Monoraphidium and other microalgae (Takeya et al. 2004; Shen and Song convolutum under controlled conditions and (2) the effect of 2007;LiandLi2012), and shown that nutrient concentration crude extracts of cyanobacteria and green algae on the growth of the medium does not always determine the phytoplankton of the green algal and cyanobacterial strains, respectively, succession in mixed cultures (Kuwata and Miyazaki 2000; using environmentally relevant concentrations. Zhang et al. 2013). According to You et al. (2007), Dunker et al. (2013), and Zhang et al. (2013), the competition between species and the ecological success of a competitor can be described by resource exploitation and interference models. Materials and methods Whereas most studies have focused on the indirect interaction by exploitation that is based on the competition for limited The microcystin-producing (MC+) Microcystis aeruginosa resources (Zhang et al. 2013), in hypertrophic environments, BCCUSP232 (Bittencourt-Oliveira et al. 2011) and non- interference, for example, the production of microcystins (MCs) microcystins-producing (MC−) Microcystis panniformis and other allelochemicals is a direct form of interspecific inter- BCCUSP200 (Bittencourt-Oliveira 2003) cyanobacterial strains action that is very important (Leflaive and ten-Hage L, 2007; from the Brazilian Cyanobacteria Collection of University of São Bar-Yosef et al. 2010; B-Beres et al. 2012; Magrann et al. 2012). Paulo (BCCUSP). The two strains of green microalgae used in The microcystins belong to a family of cyclic heptapeptide this study were Monoraphidium convolutum (CMEA/UFF0201) known to inhibit protein phosphatases. They cause liver fail- obtained from Elizabeth Aidar Collection of Microalgae ure in wild and domestic animals and humans and have (CMEA/UFF) and Scenedesmus acuminatus (UFSCar036) from heterogenous effects on the physiology of plants (Jochimsen the Federal University of São Carlos (UFSCar). For all exper- et al. 1998; Papadimitriou et al. 2013). Unfortunately, most of iments, the cyanobacteria and green algae were maintained in the mixed culture experiments rarely monitor the production environmentally controlled growth chambers set to 24±1 °C, of microcystins during the experiments (B-Beres et al. 2012; 14 h:10 h (light/dark) photoperiod, and 40 μmol photons − − Zhang et al. 2013), in addition to the fact that most of the m 2 s 1 light intensity. Light intensity was measured using a published results are conflicting. Due to the varied responses LI-COR model 250 light meter equipped with a spherical of different microalgae species to MCs (Sedmak et al. 2008,Li underwater sensor. The cyanobacteria and green microalgae and Li 2012; Bittencourt-Oliveira et al. 2013;Camposetal. were grown in BG-11 (Rippka et al. 1979) at pH 7.4 according 2013), it becomes imperative to investigate the behavior of to the modifications of Bittencourt-Oliveira et al. (2011)that different phytoplankton lineages in the presence of toxic and involved the substitution of iron for ferric ammonium citrate nontoxic cyanobacteria and microalgae. In addition, it is not FeCl3·H2O chloride. The cultures were stirred manually, and clear whether microcystins or other released compounds by their positions changed on a daily basis. cyanobacteria or other microalgae are responsible for allelop- athy (Leao et al. 2009; B-Beres et al. 2012). This makes it Mixed culture experiments difficult for generalizations or conclusions to be made on the relationship between dominant phytoplankton species in- All experiments were carried out in 1,000-mL Erlenmeyer volved in standing water species succession. flasks containing 600 mL final culture volume. In the mixed Investigations on allelopathic effects between cultures, 300 mL of the culture of each strain with an equal cyanobacteria and green algae should not only be based on number of cells (ratio, 1:1) was used. Briefly, intact cells of the mutual interactions in mixed culture experiments but also M. aeruginosa (BCCUSP232) were cocultured with those of