J Appl Phycol (2013) 25:1567–1573 DOI 10.1007/s10811-012-9969-z
First report on the allelopathic effect of Tychonema bourrellyi (Cyanobacteria) against Microcystis aeruginosa (Cyanobacteria) Jihai Shao & Liang Peng & Si Luo & Gongliang Yu & Ji-dong Gu & Shen Lin & Renhui Li
Received: 17 August 2012 / Revised and accepted: 17 December 2012 / Published online: 6 January 2013 # Springer Science+Business Media Dordrecht 2013
Abstract The allelopathic effect of the cyanobacterium Tychonema bourrellyi against the cyanobacterium Microcystis aeruginosa is reported for the first time in this paper. The filtrate of T. bourrellyi CHAB663 culture showed strong inhibitory effect on M. aeruginosa NIES-843, but the inhibitory effect was weakened by shaking culture, and such results implied that the allelopathic effect was probably mediated by the volatile substances secreted by T. bourrellyi. β-Ionone was identified as a major ingredient in the volatile substances in the cultures of T. bourrellyi, and it may act as an important allelochemical responsible for this allelopathic activity. The filtrates of T. bourrellyi culture were shown to decrease the maximum electron transport rate (ETRmax) and elevate the reactive oxygen species (ROS) levels in the cells of M. aeruginosa NIES-843. J. Shao : L. Peng : S. Luo College of Resources and Environment, Hunan Agricultural University, Changsha 410128, People’s Republic of China G. Yu : S. Lin : R. Li (*) Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Republic of China e-mail:
[email protected] J. Shao : J.-d. Gu Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources Use, Hunan Agricultural University, Changsha 410128, People’s Republic of China J.-d. Gu Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, Hong Kong Special Administrative Region, People’s Republic of China
Keywords Tychonema bourrellyi . Microcystis aeruginosa . Allelopathy . Volatile substances . β-Ionone . Cyanobacteria
Introduction The word “allelopathy” was coined by Molisch (1937) to describe the phenomenon of direct or indirect effects of one plant on another by releasing chemical compounds into the environment. Rice (1984) extended the definition of allelopathy and microorganisms were included in it. Although allelopathy originally included both advantageous and deleterious effects, this word usually refers to adverse effects now (Gross et al. 2007). Allelopathic activity is a widespread phenomenon amongst freshwater primary producers (Inderjit and Dakshini 1994). Cyanobacteria are an important group of prokaryotes in the aquatic community, and they also produce wide variety of secondary metabolites demonstrating powerful biological activities such as antiviral, antibacterial, antifungal, and antimalarial effects (Sharma et al. 2011). Some secondary metabolites are allelochemicals responsible for the interactions between cyanobacteria and other kinds of algae as well as among cyanobacteria themselves. For example, two alkaloids, 12-epi-hapalindole E and calothrixin A, produced by the filamentous cyanobacteria Fischerella sp. and Calothrix sp., respectively, exhibited inhibitory activities against the RNA polymerase of green algae (Doan et al. 2000). The violet pigment nostocine A, from Nostoc spongiaeforme, was found to be highly cytotoxic for several green algae (Hirata et al. 2003). The chemical compound 4,4′-dihydroxybiphenyl, produced by Nostoc insulare, showed a strong allelopathic activity against the unicellular
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cyanobacterium Synechocystis aquatilis (Volk and Furkert 2006). Allelopathy has been regarded as a major driving force for algal succession and the formation and ending of blooms (Takamo et al. 2003). The cyanobacterium Tychonema bourrellyi (J. W. G.Lund) Anagnostidis & Komárek (Oscillatoriales) was recently found as a main component of phytoplankton community in some areas in Lake Erhai, a lightly eutrophicated lake in Yunnan province, China. It was also found that Microcystis species were rarely detected in the Tychonema-dominated areas of the lake. Tychonema has received less attention since it was just recently recorded in China, and the information about the ecology and physiology of Tychonema is therefore very limited. In order to explain the above phenomenon for the inhibitory potential of Tychonema against Microcystis, we isolated a typical T. bourrellyi strain from the lake and studied the interaction between T. bourrellyi and Microcystis aeruginosa under coculture condition. To further prove that the inhibitory effect of T. bourrellyi against M. aeruginosa was caused by the allelochemicals released by T. bourrellyi, the filtrates were used to detect the influence on the growth and physiological characteristics of M. aeruginosa, and then gas chromatography coupled with mass spectrometry (GC–MS) was used to identify the possible allelochemicals responsible for the inhibitory effect. The findings presented in this paper provided a new candidate microorganism against the typical bloomforming cyanobacterium M. aeruginosa. Like other reported algicidal microorganisms, Tychonema may be useful in the control of Microcystis-dominated blooms. The allelochemical identified in this paper may be used as a biologically originated algicide in the control of the harmful cyanobacterium Microcystis.
Materials and methods Tychonema bourrellyi CHAB663 was isolated from Lake Erhai, China by Dr S Lin. Microcystis aeruginosa NIES-843 was kindly provided by National Institute of Environmental Science, Japan. They were all grown in CT liquid medium (pH 8.2) (Ichimura 1979), under a 12:12 LD cycle with an intensity of 20 μmol photons m−2s−1 provided by cool white fluorescent tubes at 25±1 °C.
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T. bourrellyi and M. aeruginosa at the same inoculating cell densities were set as controls. Each treatment was replicated three times. All treatments were cultured under the same conditions mentioned above. The cells of T. bourrellyi and M. aeruginosa were counted at an interval of 3 days using a plankton counting chamber according to the method described by LeGresley and McDermott (2010). Effect of the filtrate of T. bourrellyi culture on the growth of M. aeruginosa Cultures of T. bourrellyi in stationary phase were centrifuged at 10,000×g for 10 min, and the supernatants were filtered through the 0.22-μm cellulose acetate membrane (Millipore, USA). Fifty milliliter filtrate was added into 250 mL conical flask containing 45 mL of CT medium, and 5 mL exponential phase of M. aeruginosa was added to give a final volume of 100 mL. The initial cell density of M. aeruginosa NIES-843 was about 1.1×105 cells mL−1. The filtrate was substituted with ddH2O in control treatments. Each treatment received two kinds of culture: static culture and shaking culture (80 rpm) under the conditions mentioned above. In order to maintain the homogeneity of the static treatments, we shook the conical flask by hand every 12 h. Identification of volatile compounds in T. bourrellyi culture Volatile compounds in the cultures of T. bourrellyi were extracted using headspace solid phase micro-extraction according to Shao et al. (2011). GC−MS (HP6890GC5973MSD, Hewlett-Packard, USA) was used to separate and identify volatile compounds. The separation of extracted compounds was conducted on a capillary column (TC series, WondaCap 5, 0.25 mm × 30 m × 0.25 μm, Shimadzu, Japan). The injector temperature was set as 250 °C. The oven temperature program was as follows: initial temperature was set at 60 °C for 2 min, increased to 200 °C at a rate of 5 °C min−1 and then maintained at this temperature for 2 min, followed by 250 °C at 20 °C min−1 which was held for 2 min. High purity N2 (≥99.99 %) was used as the carrier gas under a pressure of 150 Kpa. Mass analyses were performed in the electron ionization mode at 70 eV; the mass range was m/z 50–500. The temperatures of quadrupole and ion source were set at 150 and 230 °C, respectively.
Co-culture of T. bourrellyi and M. aeruginosa Exponential phase of T. bourrellyi and M. aeruginosa was inoculated into the same 250-mL conical flasks containing CT medium. The cell densities of T. bourrellyi and M. aeruginosa were initially set about 1.4×103 cells mL−1 and 1.1×105 cells mL−1 respectively. The monocultures of
Effect of the filtrate of T. bourrellyi culture on the ETRmax in M. aeruginosa As described above, exponential phase M. aeruginosa was inoculated into CT medium containing 50 % (v/v) filtrate of T. bourrellyi culture. The filtrate was substituted with
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ddH2O in control treatments. The maximum relative electron transport rate (ETRmax) of static cultured M. aeruginosa was measured using a pulse–amplitude-modulated fluorescence monitoring system (PAM, Walz, Germany). The numerical values of chlorophyll fluorescence of samples exposed to 12 intensities of actinic light increasing from 0 to 1,265 μmol photons m−2s−1 photosynthetically active radiation (PAR) was recorded during a 3-min time series. Effect of the filtrate of T. bourrellyi culture on the reactive oxygen species levels in the cells of M. aeruginosa As described in above, M. aeruginosa was cultured using CT medium containing 50 % (v/v) filtrate of T. bourrellyi culture. The intracellular reactive oxygen species (ROS) levels of static cultured M. aeruginosa were detected using 2,7-dichlorofluorescein diacetate (Sigma Chemicals, USA) based on the method described by Hong et al. (2008). The fluorescence intensity of 2,7-dichlorofluorescein was obtained using a microplate reader (Molecular Device M2, USA). Excitation and emission wavelengths were 485 and 530 nm, respectively. Statistics Significant differences were determined by one-way ANOVA followed by least significant difference post-hoc test using analysis software (SPSS, version 13.0, SPSS Inc., USA). Differences were considered to be significant at P
