Allelopathic effect boosts Chrysosporum ovalisporum dominance in summer at the expense of Microcystis panniformis in a shallow coastal water body
The increased occurrence of harmful cyanobacterial species and, with this, higher frequency of cyanobacteria blooms, closely associated with eutrophication and climate change, have attracted increasing attention worldwide. However, competition mechanisms between the different bloom-forming cyanobacteria species remain to be elucidated. In this paper, for the first time, the allelopathic effect of the cyanobacterium Chrysosporum ovalisporum on the cyanobacterium Microcystis panniformis is reported. The results of our study conducted in a Chinese shallow coastal water body demonstrated that the biomass of M. panniformis was relatively low during the C. ovalisporum blooming period. Co-cultivation of a C. ovalisporum strain with a M. panniformis strain showed strong inhibition of the growth of M. panniformis but stimulation of C. ovalisporum. Thus, filtrate of C. ovalisporum culture had a strong inhibitory effect on the performance of M. panniformis by decreasing the maximum optical quantum yield (Fv/Fm), the electron transport rate (ETR) of PS II and the onset of light saturation (Ik) and by increasing the alkaline phosphatase (ALP) activity and superoxide dismutase (SOD) activity of M. panniformis. Our results suggest that the inter-specific allelopathic effect plays an important role in the competition between different cyanobacteria species. We foresee the importance of C. ovalisporum to intensify in a future warmer world, not least in small- to medium-sized, warm and high conductivity coastal water bodies.
KeywordsAllelopathy Chrysosporum ovalisporum Microcystis panniformis Competition Cylindrospermopsin Bloom
For multiple decades, competition between phytoplankton species has been an important topic within aquatic ecology (Sommer 1989; Wacker et al. 2015). Phytoplankton species compete for resources through different physiological adaptations (e.g. photosynthetic capacity, nutrient requirement, luxury uptake, mixotrophy and degree of vertical migration) (Huisman et al. 2004; Legrand et al. 2003; Torres et al. 2015). Also, allelopathy, i.e. the beneficial or harmful effects of one plant on another plant, is known to be an important ecological adaptation mechanism employed by phytoplankton (Legrand et al. 2003). Via secondary metabolites, some phytoplankton species may compete successfully with their opponents (Leflaive and Ten-Hage 2007; Sukenik et al. 2002), and allelopathy is, therefore, considered an important factor in the species succession and formation of algal blooms (Bar-Yosef et al. 2010; Prince et al. 2008).
Although allelopathy is acknowledged for its importance, specific knowledge about its effects remains limited. Several studies have revealed that secondary metabolites may inhibit photosynthesis and enzyme activity and produce oxidative stress (Leflaive and Ten-Hage 2007; Prince et al. 2008; Sukenik et al. 2002). However, a growing number of studies indicate that the inhibition effects vary from species to species; thus, some biochemical substances may inhibit the growth of some species but stimulate that of others (Carey and Rengefors 2010; Pinheiro et al. 2013). To obtain a better understanding of allelopathy and its importance for phytoplankton, more studies are required (Rzymski et al. 2014).
As the frequency of cyanobacteria blooms is increasing worldwide due to eutrophication and global warming (Kosten et al. 2012; Paerl and Huisman 2008), the succession of different cyanobacteria species and their allelopathic influence on other aquatic organisms are topics attracting growing attention (Carey and Rengefors 2010). Sukenik et al. (2002) found that extract filtrate of Microcystis sp. inhibited the growth and photosynthesis of Peridinium gatunense. Moreover, growth and antioxidant enzyme activity have been found to be inhibited by cyanobacterial extracts containing microcystin (MC) and cylindrospermopsin (CYN) (Campos et al. 2013). Allelopathy may also have played a role in the shift to dominance of Cylindrospermopsis raciborskii observed in a tropical reservoir by Mello et al. (2012). However, a recent study has revealed that also non-CYN C. raciborskii may create growth inhibition of Microcystis aeruginosa (Rzymski et al. 2014). Thus, the allelopathic ability of different geographical cyanobacteria populations and the bioactive properties of these species require further study.
Chrysosporum ovalisporum (Zapomělová et al. 2012), previously known as Aphanizomenon ovalisporum, is a nitrogen-fixing toxin-producing cyanobacterium (Banker et al. 1997) that inhabits and forms blooms in warm regions of Europe, especially the Mediterranean area, as well as in reservoirs in Queensland, Australia (Fadel et al. 2014; Pollingher et al. 1998; Rzymski and Poniedziałek 2014; Shaw et al. 1999) and potentially, therefore, may become more important in a future warmer world. Hitherto, only few studies have been conducted to elucidate the allelopathy of C. ovalisporum, including its effects on extracellular alkaline phosphatases of other phytoplankton and the growth inhibition of some green algae (Bar-Yosef et al. 2010; Pinheiro et al. 2013; Viktória et al. 2015).
Blooms of C. ovalisporum, co-existing with Microcystis panniformis, have been observed in a Chinese coastal pond. The aim of this study was to test whether allelopathy was a key factor driving this shift in species dominance. For this purpose, we isolated the two cyanobacteria species and investigated their growth, photosynthesis and enzyme activity in a co-culture and filtrate culture experiment.
Material and methods
Lake description and field sites
Field physical and chemical conditions and phytoplankton dynamics
Average values of physical and chemical parameters in Pond Donghai
TN (mg L−1)
TP (μg L−1)
EC (μs cm−1)
Phytoplankton samples were fixed with a Lugol’s iodine solution (2% final conc.) and settled for 48 h. Cell density was determined microscopically using a Sedgwick-Rafter counting chamber at ×400 magnification. Phytoplankton species were identified and counted according to classical taxonomic references (Hu 2006; Komárek and Kováčik 1989; Zhang et al. 2012). For conversion to biomass, we assumed that 1 mm3 was equivalent to 1 mg fresh weight.
Isolation of strains
One C. ovalisporum strain, CFWA01007, was isolated from Pond Donghai in July 2014. Single filaments of C. ovalisporum were collected using a Pasteur glass micro-pipette under an Olympus BX 53 light microscope (×400 magnification) and cleaned seven to eight times using sterile water, after which it was transferred to a screw cap test tube containing 5-ml sterile CT medium. This procedure was repeated until monocultures of the cyanobacteria were obtained. The isolates were incubated under a constant white light intensity of 40 μmol photons m−2 s−1 on a 12:12 L/D cycle and at a temperature of 25 ± 1 °C. After 3 weeks, a pure algal strain culture was obtained. One M. panniformis strain, CFWA01028, was also isolated from the same pond in Oct. 2014 applying a classical plate method involving incubation with solid agar medium (Waterbury 2006). The culture condition was the same as for the C. ovalisporum strain.
Specific growth rate
where X1 is the algal cell number at the initial incubation time point (T1), and X2 is the cell number at the end time point (T2). M. panniformis cells were counted using a blood cell counting chamber; C. ovalisporum growth was observed under a microscope (Olympus BX53, Japan) in a Bürker chamber.
Laboratory experiment design
Co-culture of C. ovalisporum and M. panniformis
C. ovalisporum and M. panniformis strains were harvested in the late log growth phase, mixed and co-cultured in 250-ml culture flasks (BG11 medium, 80 μmol photons m−2 s−1 in a 12:12 L/D cycle, 25 ± 1 °C) in biovolume proportions (total biovolume 400 mm3 L−1) according to the method of Rzymski et al. (2014): (1) 75C25M: 75% C. ovalisporum–25% M. panniformis; (2) 50C50M: 50% C. ovalisporum–50% M. panniformis; and (3) 25C75M: 25% C. ovalisporum–75% M. panniformis. For each treatment, all cultures were prepared in three duplicates. In the mixed culture samples, the contribution of each species was expressed as the biovolumes of M. panniformis and C. ovalisporum calculated at the beginning of the experiment and after 14 days of incubation.
Allelopathic responses of C. ovalisporum and M. panniformis
In order to clarify which of the co-existing species would induce an allelopathic response, we performed the following experiments: (1) M. panniformis was incubated in filtrate of C. ovalisporum medium (hereafter, Mic in Chr); and (2) C. ovalisporum was incubated in filtrate of M. panniformis medium (hereafter Chr in Mic). For each treatment, all cultures were prepared in three duplicates. All cultures were grown for 14 days at 25 ± 1 °C under 80 μmol m−2 s−1 irradiance using cool white fluorescent light with a photoperiod cycle of 12 h light/12 h dark. Each flask was shaken manually three times a day.
For the experiments, cell-free media were obtained by culturing each species for 15 days in BG-11 medium at 25 ± 1R°C under 80 μmol m−2 s−1, followed by centrifuging at 10000 rpm (Eppendorf, Centrifuge 5804 R ) and filtering through 25 mm Whatman GF/F filters (nominal pore size of 0.7 μm). To ensure that all cells were removed, filtration was performed twice. The filtrates of two strains were then supplemented with nutrients (consistent with the concentrations of N and P in the BG11 medium), stirred evenly and incubated with C. ovalisporum or M. panniformis in a final density of 3.5 × 105 cell mL−1. Simultaneously, two strains were incubated with the same density in standard BG-11 medium as controls.
In order to determine the initial concentration of cylindrospermopsin (CYN) in the filtrate culture experiment, three subsamples (50 mL per sample)—as in the Mic in Chr treatment—were collected. The subsamples were filtered twice (Whatman GF/F filters, pore size 0.7 μm) to remove M. panniformis cells, and the filters were collected to identify CYN by liquid chromatography mass spectrometry using an Agilent 1100 model HPLC linked to an Agilent Trap VL ion-trap mass spectrometer. Chromatographic separation was achieved using a Merck Purospher STAR C18 column (55 mm × 4 mm I.D., 3 mm particle diameter, temperature 40 °C) and a gradient elution of water with 0.05% trifluoroacetic acid (solvent A) and acetonitrile with 0.05% trifluoroacetic acid (solvent B) (gradient 2–70% solvent B over 20 min) at a flow rate of 1 mL min−1 (Antal et al. 2011). The diode array detector scanned within 200–300 nm and the absorption maximum of CYN was determined to 262 nm. Under these conditions, the retention time for CYN is 1.5 min. Quantization was achieved using the m/z 416.1/194.1 transitions for CYN (Antal et al. 2011). In our experiment, the initial concentration of CYN in the Mic in Chr treatment was 3.2 ± 0.3 μg L−1.
Relative growth rate
where Nt is the algal cell number at the t incubation time point, and Nt + 2 is the cell number at t + 2 time point.
Determination of photochemical parameters
Relative ETR (rETR)max is the maximum potential rETR in the absence of photoinhibition and represents the photosynthetic capacity at saturating light, and α is the initial slope of the RLC before the onset of saturation (light-limiting condition efficiency). Ik is the photo adaptive index or minimum point of light saturation.
Enzyme activity assay
The cell cultures were resuspended in 1.5 mL phosphate buffer (0.1 M, pH 7.8) and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatants were used immediately to determine alkaline phosphatase (ALP) activity with detection kits provided by the Jiancheng Bioengineering Institute (Nanjing, China). Subsamples (5 mL) of cultures were resuspended in phosphate buffer, kept on ice and sonicated for 4 min using 60% of the full intensity at 5 s intervals (Sonifier 650–92, Biosafer, China); then, the crude homogenate was centrifuged according to the ALP procedure, and the supernatants used to determine superoxide dismutase (SOD) were tested with detection kits provided by the same institute.
All assays were conducted in three duplicates for each treatment. Data were expressed as the mean ± standard deviation. Differences between groups were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. The tests were performed after analysing the homoscedasticity of variance and the normality of residuals of a linear model fit between the dependent variable and the predicted variable; p < 0.05 was considered statistically significant. Statistical analyses were performed using the SPSS 18.0 statistical package (SPSS Inc., Chicago, IL, USA).
Dominant cyanobacteria dynamics in Pond Donghai
Morphology of isolated strains
The colonies of the M. panniformis strain CFWA01028 were relatively large (∼1.5 cm), spherical–irregular to lobate with irregular indistinct holes, with cells arranged more or less evenly in flat, superficial formations, sometimes indistinct (Fig. 3d). Colonies were “cloudy”, irregular, lobate and disintegrating. The margin of colonies was irregular (Fig. 3c). Mucilage was colourless and did not extend beyond the cells (not forming a slimy margin around colonies). Cells were yellowish blue-green to olive-green, brownish to dark brown, spherical, after division hemispherical, 3–4.5 μm diameter and always with numerous, small aerotopes.
Specific growth rate of the C. ovalisporum strain and the M. panniformis strain
Growth interactions between C. ovalisporum and M. panniformis
Effects of cell-free filtrates on photosynthesis
Effects of cell-free filtrates on ALP and SOD
Our results suggest that C. ovalisporum has a negative allelopathic effect on M. panniformis growth. We found that the biomass of M. panniformis in the field was significantly lower than that of C. ovalisporum during summer (Fig. 2) despite the facts that the two cyanobacteria species are both favoured by higher water temperatures and that M. panniformis has a higher growth rate than its competitor at summer temperatures (Fig. 4). Accordingly, the growth of M. panniformis was strongly inhibited by C. ovalisporum under co-cultivation and filtrate culture conditions; the higher the proportion of competitors, the stronger the observed inhibitory effect (Figs. 5 and 6). The cyanotoxin, CYN, present in the secondary metabolites of C. ovalisporum is likely responsible for its allelopathy as judged from previous studies (Bar-Yosef et al. 2010; Rzymski et al. 2014; Viktória et al. 2015). Despite that the concentration of CYN in our study was relatively low (only 3.2 μg L−1), C. ovalisporum revealed a strong allelopathy, which may indicate that the allelopathic effect is the result of combined effects of CYN and other unknown biochemical compounds, as also suggested in other studies (Campos et al. 2013; Viktória et al. 2015). The allelopathic effects of M. panniformis increased initially followed by a decrease, the latter likely reflecting decreasing biochemical activity with time (Kobayashi 2004). As other toxins/allelochemicals, CYN is an N-rich molecule (five atoms N per molecule), in which production is closely related to the nitrogen (N) resource in the medium (Harada et al. 1994; Stucken et al. 2014). During the 14 days of cultivation without renewal of the medium, the availability of N invariably decreased dramatically as did subsequently the CYN production rates. Contrarily, the growth of C. ovalisporum was not inhibited but rather significantly stimulated by M. panniformis (Figs. 5 and 6). Other studies have revealed a similar, though not as strong, stimulation. Zhang and Song (2006) demonstrated that the cell-free filtrate of M. aeruginosa could accelerate the growth of Aphanizomenon flos-aquae and Anabaena flos-aquae. Duval et al. (2005) found that a lower level of CYN (<12.5 μg L−1) increased the production of Euglena gracilis. Campos et al. (2013) revealed that the growth of Chlorella vulgaris was stimulated by high concentrations of pure CYN (>170 μg L−1) but that growth was inhibited by cell-free filtrates of C. ovalisporum with lower CYN. Thus, the composition of these secondary metabolites and the subsequent allelopathic response of different species are ambiguous and warrant further studies.
Secondly, C. ovalisporum lowered the photosynthetic efficiency of M. panniformis. In our study, the filtrate medium of C. ovalisporum inhibited the photosynthesis of M. panniformis, suggesting a potential allelopathy mechanism (Figs. 7a and 8a). For phytoplankton, the effective quantum yield (Fv/Fm), the relative electron transport rate (rETR) and the onset of light saturation (Ik) in photosystem II (PS II) are restricted by the capacity of the electron transport chain in the photosynthesis process (Huang et al. 2015). Our experiment showed that the Fv/Fm of Mic in Chr was significantly lower in the filtrates than in the controls during the 14-day incubation time. Also, rETR and Ik were significantly lower in the filtrates at the termination of the incubation, suggesting a reduced electron transfer through the electron transport chain and that more excited electrons undergo de-excitation by non-photochemical pathways (Figueredo et al. 2007; Huang et al. 2015); thus, the efficiency of PS II was strongly inhibited. Even though it is difficult to clarify whether reduced efficiency of PS II is a direct target (Prince et al. 2008) or symptom of allelopathy (Sukenik et al. 2002), inhibition of PS II is generally proposed as a main mechanism of allelopathy. For example, Sukenik et al. (2002) found that secondary metabolites produced by Microcystis sp. led to diminished PS II efficiency of P. gatunense. Figueredo et al. (2007) reported that filtrates of C. raciborskii inhibited the photosynthetic efficiency of other phytoplankton species. Shao et al. (2013) showed that the filtrates of Tychonema bourrellyi cultures decreased the rETRmax of M. aeruginosa. Contrarily, in our study, although the Fv/Fm, rETR and Ik of Chr in Mic showed no significant difference from the controls at the end of incubation, Fv/Fm was significantly higher in the filtrates than in the controls during the process of incubation, especially on day 2 (Figs. 7b and 8b). This indicates that the PS II efficiency of C. ovalisporum was stimulated by the secondary metabolites of M. panniformis. Accordingly, it seems that growth inhibition of M. panniformis and stimulation of C. ovalisporum may be caused by photosynthetic efficiency inhibition of M. panniformis and stimulation of C. ovalisporum, respectively.
Thirdly, C. ovalisporum affects the enzyme system of M. panniformis. ALP and SOD of Mic in Chr were higher in the filtrates than in the controls, suggesting that the cells of M. panniformis were under allelopathic stress. The ecological function of ALP is to decompose the phosphate monoesters in the surrounding environment, allowing phytoplankton to utilize organic P when inorganic forms are relatively scarce (Rengefors et al. 2001; Rzymski et al. 2014). Bar-Yosef et al. (2010) revealed that for some green algae (Debarya and Chlamydomonas sp.), CYN-producing C. ovalisporum improved the utilization of organic P by stimulating the production of ALP. Our results suggest that the other cyanobacteria may also be the target of this P utilization mechanism driven by CYN-producing C. ovalisporum. Growing evidence shows that SOD of phytoplankton may play a significant role in the defence against the toxicity of reactive oxygen species (ROS) (Huang et al. 2016; Weckx and Clijsters 1996). SOD of Mic in Chr was significantly higher in the filtrates than in the controls, suggesting that M. panniformis suffered oxidative stress caused by C. ovalisporum. This kind of stress is considered an allelopathic mechanism inhibiting cyanobacterial growth (Huang et al. 2016; Jančula and Maršálek 2011). Most importantly, though, our results suggest that C. ovalisporum (Chr in Mic) does not exert physiological stress on M. panniformis.
In short, our study indicates that bioactive compounds produced by C. ovalisporum can contribute to the inter-specific competition of cyanobacteria by inducing photosynthesis and growth inhibition and physiological metabolic alterations in sympatric Microcystis species during summer. We suggest that, in the future, the allelopathic effects of C. ovalisporum, along with other previously reported eco-physiological adaptations to its invasion (Bar-Yosef et al. 2010; Cirés et al. 2013), may make C. ovalisporum a superior competitor to other phytoplankton species in systems where it is already present. Moreover, it may be expected to colonize and become dominant in many other systems with appropriate lake temperatures, not least in small- to medium-sized, high conductivity coastal water bodies (see Table S1) and perhaps increase in importance with global warming.
This work was supported by the “Shanghai outstanding technical leaders plan” (No. 15XD1522900) and Major Projects on Control and Rectification of Water Body Pollution of China (No. 2012ZX07101-007). EJ was supported by managing aquatic ecosystems and water resources under multiple stress (MARS; Contract No. 603378; http://www.mars-project.eu). We would like to thank Dr. Jianming Deng from Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, for his positive comments. We would also like to express our deep thanks to Anne Mette Poulsen from Aarhus University for her English assistance. The authors are grateful to the two anonymous reviewers for their constructive comments and suggestions.
- Banker R, Carmeli S, Hadas O, Teltsch B, Porat R, Sukenik A (1997) Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. J Phycol 33(4):613–616Google Scholar
- Campos A, Araújo P, Pinheiro C, Azevedo J, Osório H, Vasconcelos V (2013) Effects on growth, antioxidant enzyme activity and levels of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin and cylindrospermopsin. Ecotoxicol Environ Saf 94(5):45–53CrossRefGoogle Scholar
- Duval E, Coffinet S, Bernard C, Briand J (2005) Effects of two cyanotoxins, microcystin-LR and cylindrospermopsin, on Euglena gracilis, Environmental Chemistry. Springer, pp. 659–671Google Scholar
- Hu H (2006) The freshwater algae of China: systematics, taxonomy and ecology. Science Press, BeijingGoogle Scholar
- Mello MM, Soares MCS, Roland F, Lürling M (2012) Growth inhibition and colony formation in the cyanobacterium Microcystis aeruginosa induced by the cyanobacterium Cylindrospermopsis raciborskii. Journal of plankton research: 1–8Google Scholar
- Shaw GR, Sukenik A, Livne A, Chiswell RK, Smith MJ, Seawright AA, Norris RL, Eaglesham GK, Moore MR (1999) Blooms of the cylindrospermopsin containing cyanobacterium, Aphanizomenon ovalisporum (Forti), in newly constructed lakes, Queensland, Australia. Environ Toxicol 14(1):167–177CrossRefGoogle Scholar
- Torres CA, Lürling M, Marinho MM (2015) Assessment of the effects of light availability on growth and competition between strains of Planktothrix agardhii and Microcystis aeruginosa. Microbial ecology: 1–12Google Scholar
- Waterbury JB (2006) The cyanobacteria—isolation, purification and identification, The prokaryotes. Springer, pp. 1053–1073Google Scholar
- Zapomělová E, Skácelová O, Pumann P, Kopp R, Janeček E (2012) Biogeographically interesting planktonic Nostocales (Cyanobacteria) in the Czech Republic and their polyphasic evaluation resulting in taxonomic revisions of Anabaena bergii Ostenfeld 1908 (Chrysosporum gen. nov.) and A. tenericaulis Nygaard 1949 (Dolichospermum tenericaule comb. nova). Hydrobiologia 698(1): 353–365Google Scholar