Environmental Science and Pollution Research

, Volume 24, Issue 5, pp 4666–4675 | Cite as

Allelopathic effect boosts Chrysosporum ovalisporum dominance in summer at the expense of Microcystis panniformis in a shallow coastal water body

  • Wei Zhang
  • Erik Jeppesen
  • Mengmeng Wang
  • Xiaoying Xu
  • Liqing Wang
Research Article

Abstract

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.

Keywords

Allelopathy Chrysosporum ovalisporum Microcystis panniformis Competition Cylindrospermopsin Bloom 

Introduction

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

Lake Dishui is located in southeastern Shanghai, found in the southeastern part of the Yangtze River Delta, China (31° 53′ N; 121° 55′ E). It is the largest, shallow (mean depth ∼6.1 m) man-made coastal lake in China, with a total surface area of 5.56 km2, a diameter of 2.5 km and a catchment area of 36,500 km2. The lake was created in October 2003 by covering the bottom of a beach with clay and filling it with river water. Since its creation, precipitation has become the main water source to the lake. It is a brackish lake with a salinity ranging between 0.9 and 3.2‰ (Zhu et al. 2013). The nearby Pond Donghai (31° 52′ 16″ N; 121° 54′ 23″ E) is an artificial water body connected with Lake Dishui and is managed by the Shanghai branch of the China Oceanic Administration. The pond has an area of 0.012 km2 and an average depth of 2.7 m (Fig. 1). In summer 2013, the bloom-forming cyanobacteria C. ovalisporum was, for the first time, observed in Lake Dishui but had a low biomass (about 0.03 mg L−1). At the same time, a cyanobacteria bloom occurred in Pond Donghai, first dominated by C. ovalisporum in summer followed by Microcystis spp. in the autumn. Phytoplankton samples from Pond Donghai were analysed in 2014.
Fig. 1

Location of Lake Dishui and Pond Donghai

Field physical and chemical conditions and phytoplankton dynamics

In 2014, water samples were taken at three sampling sites in Pond Donghai from the surface to 0.5 m depth using a vertical sampler. During the bloom period (Jun. 2014 to Nov. 2014), sampling was conducted weekly, otherwise monthly. In situ water temperature (WT), pH, electrical conductivity (EC) and salinity (SAL) were measured using a Yellow Springs Instruments (YSI) Pro-plus multisensor sonde. Total nitrogen (TN) and total phosphorus (TP) were analysed using combined persulphate digestion (Ebina et al. 1983). Average values of physical and chemical parameters in Pond Donghai are shown in Table 1.
Table 1

Average values of physical and chemical parameters in Pond Donghai

 

TN (mg L−1)

TP (μg L−1)

WT (°C)

EC (μs cm−1)

pH

SAL (‰)

Range

0.96–1.87

45–92

4–35

1992–2740

8.4–10.3

1.1–2.0

Average

1.32

56

20.3

2307

9.4

1.3

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

The strains were grown in 250-ml Erlenmeyer flasks with 150-ml BG11 medium under a constant white light intensity of 80 μmol photons m−2 s−1, in a 12:12 L/D cycle. The incubation lasted 14 days, and all cultures were prepared in triplicate. The specific growth rate (μ) under various temperatures (14, 17, 20, 23, 26, 29, 32 °C) was determined by cell counts of collected subsamples according to the following equation:
$$ \upmu = \ln \left({\mathrm{X}}_2/{\mathrm{X}}_1\right)/{\mathrm{T}}_2-{\mathrm{T}}_1 $$

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.

CYN analysis

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

The relative growth rate (RGR) of the cultures was determined by cell counts of the collected subsamples and calculated according to the following equation:
$$ RGR=\left({\mathrm{N}}_{t+ 2}-{\mathrm{N}}_t\right)/{\mathrm{N}}_t\times 100\% $$

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

Maximum optical quantum yield (Fv/Fm), electron transport rate (ETR) of PS II and onset of light saturation (Ik) of the M. panniformis strain incubated in spent C. ovalisporum medium, of the C. ovalisporum strain incubated in spent M. panniformis medium and of the control strains incubated in BG11 medium were measured with a pulse–amplitude-modulated fluorescence monitoring system (PAM, Walz, Effeltrich, Germany). Cells were dark-acclimated for 5 min before analysis. F0 was determined as the fluorescence of dark-adapted cells stimulated by a weak probe light immediately following 5 min of darkness. Fm was the maximum fluorescence signal following the closure of all reaction canters by a 600-ms pulse of saturating irradiance. Simultaneously, Fm′ was the maximum fluorescence signal in the light-adapted state. Fluorescence parameters were calculated according to the following equations after subtraction of the blank fluorescence value obtained by measuring the fluorescence of a 0.22-μm filtered sample:
$$ {F}_{\mathrm{v}}/{F}_{\mathrm{m}}=\left({F}_{\mathrm{m}}-{F}_0\right)/{F}_{\mathrm{m}} $$
Fv/Fm is the maximum optical quantum yield (Juneau and Harrison 2005) measured once every second day. Light curves were obtained by running a rapid light curve (RLC) protocol in Phyto-Win software (Walz). The ETR of PS II (McMinn and Hegseth 2004) was calculated as
$$ \mathrm{relative}\kern.3em ETR=\mathrm{\triangle}F/{F}_{\mathrm{m}}^{\prime}\times PAR,\mathrm{where}\mathrm{\triangle }F={F}_{\mathrm{m}}^{\prime }-{F}_{\mathrm{t}} $$
Ft is the transient fluorescence level at a given time. The data were fitted to the following equation (Zonneveld 1998) using a Levenberg–Marquardt regression algorithm:
$$ rETR=I/\left({aI}^2+ bI+c\right) $$
I is irradiance, and a, b and c are regression coefficients that indicate the fit of the curve (Zhang et al. 2011). The photosynthetic parameters were calculated as
$$ \begin{array}{c}\kern1em \alpha =1/\mathrm{c}\kern1em \\ {}\kern1em {rETR}_{\max }={\left[b+2{(ac)}^{0.5}\right]}^{-1}\kern1em \\ {}\kern1em {I}_{\mathrm{k}}={rETR}_{\max /\alpha}\kern1em \end{array} $$

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.

Data analysis

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).

Results

Dominant cyanobacteria dynamics in Pond Donghai

From 15 Jan. 2014 to 15 Dec. 2014, the water temperature in Pond Donghai ranged from ∼4 to 35 °C, reaching annual maxima in July and August and minima in December and January (Fig. 2). C. ovalisporum occurred from the middle of June and rose to a peak of 13.9 mg L−1 in early August, after which it was relatively constant for 1 month, rerising to a peak on 18 Sep. (42.6 mg L−1). Thereafter, when the water temperature decreased to <25 °C, a rapid decline in biomass occurred. The other dominant algae group, Microcystis spp., appeared in the pond almost simultaneously with C. ovalisporum. Its biomass was relatively low during the C. ovalisporum bloom, reaching a maximum of 2.2 mg L−1 in August. Coinciding with the decrease in C. ovalisporum, the biomass of Microcystis spp. exhibited a rapid increase, reaching a peak (11.9 mg L−1) on 30 Oct. 2014. The Microcystis spp. group was dominated by M. panniformis with a small contribution of M. aeruginosa and M. wesenbergii.
Fig. 2

Changes in Chrysosporum ovalisporum (Chr) and Microcystis spp. (Mic) biomass dynamics and water temperature

Morphology of isolated strains

The trichomes of the C. ovalisporum strain CFWA01007 were solitary (Fig. 3a) (l = 60–500 μm) with low occurrence of terminal hyaline cells (Fig. 3b); they were slightly narrowed towards the ends and had gas vesicle-containing cells (l = 3.5–12.3 μm, w = 3.4–6.0 μm). Heterocysts (Fig. 3c) were ellipsoidal (l = 4.3–7.5 μm, w = 4.1–5.5 μm), sometimes with obvious slimes. Akinetes (l = 8.9–16.5 μm, w = 8.5–15.4 μm) were oviform to spherical and located in the middle of the trichomes (Fig. 3a, b).
Fig. 3

The morphology of the Chrysosporum ovalisporum strain CFWA01007 and the Microcystis panniformis strain CFWA01028 isolated from Pond Donghai (acC. ovalisporum, A akinete, H heterocyst; d, eM. panniformis)

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

At the onset of the lab experiment from March 2015, the specific growth rates of both the C. ovalisporum and the M. panniformis strain, incubated at gradient temperatures (14, 17, 20, 23, 26, 29 and 32 °C), showed a trend towards an initial increase followed by a slight decrease (Fig. 4). Thus, the growth of the two species benefitted from the higher water temperature, exhibiting a relatively high growth rate when the temperature was >26 °C. Growth rates differed significantly, however, at low (<23 °C) and high (26, 29 °C) temperatures (p < 0.05). At 20 °C, the growth rate of C. ovalisporum was 0.12 ± 0.07 day−1, whereas the growth rate of M. panniformis was 0.26 ± 0.08 day−1. At 29 °C, the growth rates of C. ovalisporum and M. panniformis were 0.59 ± 0.06 and 0.84 ± 0.07 day−1, respectively. Although the growth rate of M. panniformis decreased at 32 °C (0.55 ± 0.13 day−1), it was still higher than that of C. ovalisporum (0.49 ± 0.10 day−1).
Fig. 4

Specific growth rates of Chrysosporum ovalisporum and Microcystis panniformis in BG11 medium at different temperatures. Both species have a relatively high growth rate at >26 °C. However, M. panniformis seems to be more adaptable to low temperatures than C. ovalisporum. Asterisks indicate statistically significant differences from the controls; *p < 0.05

Growth interactions between C. ovalisporum and M. panniformis

During a 14-day co-cultivation experiment, an average increase in C. ovalisporum biomass of 11.2 ± 2.3% occurred at the expense of M. panniformis, regardless of its initial contribution. The higher the initial biomass of C. ovalisporum, the higher the increase percentage was observed (Fig. 5). The greatest change was recorded in the 75C25M sample—13.8 ± 2.9%. In all the analysed samples, the increase of C. ovalisporum and the decrease of M. panniformis were statistically significant (p < 0.05).
Fig. 5

Variation in biomass contribution of Microcystis panniformis and Chrysosporum ovalisporum at the start and at the end of co-culture experiment (grey bar, M. panniformis; white bar, C. ovalisporum). Asterisks indicate statistically significant differences from the controls; *p < 0.05; ***p < 0.001

Incubation of M. panniformis in spent C. ovalisporum medium resulted in significantly lower growth rates than in the controls during the 14-day incubation (p < 0.05). The greatest RGR was observed on day 4 (RGR = 107.5 ± 32%), but it was significantly lower than in the controls (RGR = 238.4 ± 22%) (p < 0.01). At the end of the incubation, RGR (4.69 ± 3.8%) was also significantly lower than in the controls (34.8 ± 7%). Chr in Mic always had a significantly higher RGR than in the controls (p < 0.05). The greatest RGR was observed on day 4 (RGR = 256.1 ± 21%), being 1.4 times higher than in the controls. The greatest change was observed on day 6 (RGR = 157.8 ± 19%) (p < 0.01). At the end of incubation, RGR of Chr in Mic remained 1.4 times higher than in the controls (p < 0.05) (Fig. 6).
Fig. 6

Daily relative growth rate (RGR) of Microcystis panniformis incubated in spent Chrysosporum ovalisporum medium (Mic in Chr) and C. ovalisporum incubated in spent M. panniformis medium (Chr in Mic). Asterisks indicate statistically significant differences from the controls; *p < 0.05; ***p < 0.001

Effects of cell-free filtrates on photosynthesis

Fv/Fm of M. panniformis incubated in the cell-free filtrates of C. ovalisporum (Mic in Chr) ranged from 0.30 ± 0.01 to 0.43 ± 0.02, showing an initial increase followed by a decrease (Fig. 7a). The maximum value was observed on day 6 and the minimum value on day 14. As from day 6, the Fv/Fm of the Mic in Chr was significantly lower than when M. panniformis was incubated in BG11 medium (p < 0.05). At the end of incubation, the Fv/Fm of the Mic in Chr amounted to 77.7% of the control. During the whole incubation, the Fv/Fm of C. ovalisporum incubated in the cell-free filtrates of M. panniformis (Chr in Mic) ranged from 0.38 ± 0.01 to 0.52 ± 0.01 and was always higher than when C. ovalisporum was incubated in the BG11 medium (Fig. 7b). From day 0 to day 4, the Fv/Fm of the Mic in Chr showed a dramatic increase, and from day 8, a decrease. On day 2, 8 and 12, the Fv/Fm of Chr in Mic was statistically significantly higher than in the controls (p < 0.05 or p < 0.01).
Fig. 7

The maximum optimal quantum yield of photosystem II (Fv/Fm) of Microcystis panniformis in spent Chrysosporum ovalisporum medium (Mic in Chr) and C. ovalisporum in spent M. panniformis medium (Chr in Mic). Asterisks indicate statistically significant differences from the controls; *p < 0.05; ***p < 0.001

The value of the ETR of PS II and the onset of light saturation were determined at the end of incubation (Fig. 8). The ETR of Mic in Chr was 77.6 ± 5.7 μmol photons m−2 s−1, which was significantly lower than in the control (122.0 ± 8.4 μmol photons m−2 s−1) (p < 0.05) (Fig. 8a). However, the ETR of Chr in Mic (180.6 ± 9.40 μmol photons m−2 s−1) showed no significant difference from the C. ovalisporum (192.2 ± 9.7 μmol photons m−2 s−1) incubated in the BG11 medium. Ik of Mic in Chr was 563 ± 48.4 μmol photons m−2 s−1, again significantly lower than in the control (656.1 ± 19.6 μmol photons m−2 s−1) (p < 0.05). The Ik of Chr in Mic was 786.0 ± 14.2 μmol photons m−2 s−1, which was lower than in the control (822.3 ± 20.2 μmol photons m−2 s−1), but not significantly so (p > 0.05) (Fig. 8b).
Fig. 8

Electron transport rate (ETR) and photo adaptive index (Ik) of Microcystis panniformis in spent Chrysosporum ovalisporum medium (Mic in Chr) and C. ovalisporum in spent M. panniformis medium (Chr in Mic) after 14 days of incubation. Asterisks indicate statistically significant differences from the controls; *p < 0.05; ***p < 0.001

Effects of cell-free filtrates on ALP and SOD

At the end of incubation, the ALP activity of Mic in Chr had apparently increased (by 16.2 ± 3.3%) compared with the controls (p < 0.05). In contrast, the ALP activity of Chr in Mic tended to be slightly lower than in the controls (5.1 ± 2.0%) (p > 0.05) (Fig. 9). SOD activity of Mic in Chr had increased by 151.4 ± 25.6% compared with the level in the controls (p < 0.05). It differed significantly from the SOD activity of Chr in Mic, which was only 5.8 ± 3.0% higher (but not significantly so, p > 0.05) than in the controls (Fig. 9).
Fig. 9

Mean changes in alkaline phosphatase (ALP) and superoxide dismutase (SOD) of Microcystis panniformis in spent Chrysosporum ovalisporum medium (Mic in Chr) and C. ovalisporum in spent M. panniformis medium (Chr in Mic) after 14 days of incubation. Asterisks indicate statistically significant differences from the controls; *p < 0.05

Discussion

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.

Notes

Acknowledgements

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.

Supplementary material

11356_2016_8149_MOESM1_ESM.doc (68 kb)
ESM 1(DOC 67 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Wei Zhang
    • 1
  • Erik Jeppesen
    • 2
    • 3
  • Mengmeng Wang
    • 1
  • Xiaoying Xu
    • 1
  • Liqing Wang
    • 1
  1. 1.College of Fisheries and Life ScienceShanghai Ocean UniversityShanghaiChina
  2. 2.Department of BioscienceAarhus UniversitySilkeborgDenmark
  3. 3.Sino-Danish Centre for Education and Research (SDC)BeijingChina

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