粉绿狐尾藻（Myriophyllum aquaticum）对铜绿微囊藻（Microcystis aeruginosa）的化感抑制效应及其生理机制

由于生物法,尤其是水生植物化感控藻方法在治理水体富营养化及水华现象中具有多方面的优势,近年来该领域的理论和技术研究备受世界各国关注.采用初始添加种植水和连续添加种植水的方法,研究挺水型粉绿狐尾藻对铜绿微囊藻的化感抑藻效应,并从光合作用的角度探讨其对铜绿微囊藻化感抑制的生理生态机制及作用靶点.研究结果表明:粉绿狐尾藻能够分泌某些化感物质有效抑制铜绿微囊藻的正常生长,其抑藻效应是通过连续释放某些化感物质作用于铜绿微囊藻而实现的,且具有累积性;粉绿狐尾藻分泌化感物质对铜绿微囊藻的Chl a、PC及APC的损伤程度存在差异(如处理5 d后,Chl a、PC、APC的相对含量分别降至52.7%、15.3%、7.6%),其中藻胆蛋白(尤其是APC)比Chl a 更为敏感,说明藻胆蛋白是粉绿狐尾藻化感物质抑制铜绿微囊藻的关键靶点.研究为水生植物化感控藻技术的发展提供了新的材料,并有助于深入了解水体生态系统的化学生态作用及其机制.     

绿藻栅藻对微囊藻的抑制效应及评价

为筛选具有控制微囊藻水华潜力的绿藻, 研究基于藻类之间的化感作用, 对34株绿藻开展筛选及评价工作。结果发现, 栅藻FACHB-1229的抑制率最高, 其滤液对微囊藻FACHB-3550和FACHB-905的抑制率分别为53.95%和48.39%; 通过对滤液中的物质进行GC-MS检测, 推测该藻株的效应物质可能为邻苯二甲酸二(2-甲氧基乙基)酯。进一步对FACHB-1229生长和光合放氧、氨氮耐受等指标进行评价, 结果发现: 该藻株在孔板中的生长速率(0.38±0.06)/d高于微囊藻生长速率(0.13±0.03)/d; 栅藻FACHB-1229的光合放氧速率高于同期测定的其他绿藻光合放氧速率, 可达(229.91±10.49) μmol O₂/(mg Chl.a·h); 同时该藻株对氨氮的耐受能力最强, 在氨氮浓度为1888.60 mg/L时其生长速率可达(0.30±0.08)/d。此外, 在与微囊藻FACHB-3550进行共培养时, 栅藻FACHB-1229所占比例持续升高。综上, 栅藻FACHB-1229具备与微囊藻竞争的优势, 有望成为微囊藻水华生物控制的优选材料。     

微囊藻碳酸酐酶活性在不同环境因素下的调节与适应

测定了3种微囊藻水华中的优势种类,即铜锈微囊藻（Microcystis aetlzginosa Kutz．）,绿色微囊藻（Microcystis viridis（A．Br．）Lemm）,惠氏微囊藻（Microcystis wesenbergii（Kom．）Kom．）,以及微囊藻573（Microcystis sp．573）的碳酸酐酶活性;研究了无机碳、pH、温度、光强、NIP比等环境因素和外源葡萄糖对铜锈微囊藻碳酸酐酶活性的影响,发现微囊藻碳酸酐酶活性受环境中碳酸氢根浓度的调节,故推断碳酸氢根是铜锈微囊藻利用的主要无机碳形式;相比添加葡萄糖进行混合营养培养的细胞,无外源葡萄糖和暗饥饿培养的微囊藻细胞会产生高约6倍的碳酸酐酶活性;光强的改变也会影响碳酸酐酶的活性。     

Modulation and adaptation of carbonic anhydrase activity in Microcystis spp. under different environmental factors

The carbonic anhydrase (CA) activities were determined in three cyanobacterial species, namely Microcystis aeruginosa Kütz., Microcystis viridis (A.Br.) Lemm, and Microcystis wesenbergii (Kom.) Kom, which were dominant in a lake (Dianchi Lake) subject to major blooms. In more detailed experiments on M. aeruginosa, the effects of inorganic carbon, pH, temperature, nitrogen/phosphorus ratio, glucose, and light intensity on CA activity were also investigated. Because of the relatively alkaline pH value of the culture media for the optimum growth of algal cells, bicarbonate ions were the main form of exogenous inorganic carbon. The results showed that the CA activity of M. aeruginosa was influenced dramatically by the concentration of bicarbonate. Consequently, it was suggested that bicarbonate ions were the main form of exogenous inorganic carbon that M. aeruginosa could utilize. Cultures grown in the dark exhibited CA activity six times higher than that of cells cultured mixotrophically with the addition of glucose. Features of eutrophic water bodies promoted an increase in CA activity, and the resulting higher CA activity would accelerate the utilization of inorganic carbon and favor the growth and blooming of Microcystis spp. in eutrophic lakes. Although the experiments were carried out under controlled experimental conditions, they could provide some basic data that would prove useful for the control of cyanobacterial blooms in nature.     

Allelopathic effects of the Baltic cyanobacteria Nodularia spumdigena, Aphanizomenon flos-aquae and Anabaena lemmermannii on algal monocultures

Allelopathy, the release of extracellular compounds that inhibit the growth of other microorganisms, may be one factor contributing to the formation and/or maintenance of cyanobacterial blooms. We investigated the allelopathic effects of three cyanobacterial species (Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena lemmermannii) that frequently form mass-occurrences in the Baltic Sea. We exposed monocultures of three phytoplankton species (Thalassiosira weissflogii, Rhodomonas sp. and Prymnesium parvum) to cell-free filtrates of the three cyanobacteria, and quantified allelopathic effects with cell counts. We also investigated the role of the growth phase of cyanobacteria in their allelopathy, by comparing the effects of an exponential and a stationary phase culture of N. spumigena. All tested cyanobacteria inhibited the growth of Rhodomonas sp., but none of them affected P. parvum. The effects on T. weissflogii were more variable, and they were amplified by repeated filtrate additions compared to a single filtrate addition. N. spumigena was more allelopathic in exponential than in stationary growth phase, whereas the culture filtrate was more hepatotoxic in stationary phase. Hepatotoxins were thus probably not involved in the allelopathic effects, which is also indicated by the allelopathic properties of the non-toxic A. flos-aquae and A. lemmermannii. The results demonstrate that the common Baltic cyanobacteria affect some coexisting phytoplankton species negatively. Allelopathy may therefore play a role in interspecific competition and contribute to cyanobacterial bloom maintenance.     

铜绿微囊藻(Microcystis aeruginosa)对惠氏微囊藻(Microcystis wesenbergii)的化感作用

通过混合培养和添加过滤液两种方式观察铜绿微囊藻和惠氏微囊藻的生长曲线,探讨两种微囊藻之间的化感作用。结果表明:在混合培养条件下,两者能够形成相互抑制作用;当两者起始藻密度高于0.5×106cells.mL-1、混合比为1:1时,惠氏微囊藻的生长因化感作用而受到显著抑制(P<0.05),同时惠氏微囊藻也会对铜绿微囊藻产生一定的胁迫作用;处于对数生长期的铜绿微囊藻过滤液能抑制惠氏微囊藻的生长,且惠氏微囊藻起始藻密度低于0.5×106cells.mL-1,连续滴加该过滤液后,其生长受到极显著抑制(P<0.01)。     

东海原甲藻与中肋骨条藻的种间竞争特征

对东海原甲藻和中肋骨条藻按照起始Chl-a比1∶5、1∶1和5∶1进行了f/2条件下的共培养实验,以探讨这两种藻的种间竞争特征。实验结果表明在共培养体系中,中肋骨条藻完全占优势,而东海原甲藻的生长受到明显的抑制。应用Lotka-Volterra种间竞争模型对共培养实验进行模拟的结果表明,东海原甲藻与中肋骨条藻的种间竞争结果与初始密度配比无关,中肋骨条藻总会竞争胜过东海原甲藻。为了探讨他感作用对东海原甲藻和中肋骨条藻种间竞争的影响,采用了中肋骨条藻的无藻细胞滤液来进行培养实验。实验结果显示,中肋骨条藻滤液对东海原甲藻及其本身的生长均无明显影响,这表明他感作用并非中肋骨条藻获得优势的主要竞争方式。     

铜绿微囊藻的分子研究进展

中国淡水湖泊、水库众多,富营养化问题严重。铜绿微囊藻是中国湖泊、水库及其他水域生态系统发生、形成富营养化危害的主要藻类。目前对铜绿微囊藻的研究主要集中在水华成因、生长的特点、作用机理等,对其生命活动相关的分子机制研究不多。该文主要从生物节律、毒素合成、藻胆蛋白合成及其调控机制和ATP合成酶等四个方面综述了铜绿微囊藻的分子研究进展,为今后进一步研究铜绿微囊藻的分子作用机理及其防治具有重要意义。     

Allelopathic activity of Elodea canadensis and Elodea nuttallii against epiphytes and phytoplankton

Elodea nuttallii and Elodea canadensis have both been introduced from North America to Europe. They are now common in many water bodies where they often form dominating stands. It was suggested that negative relationships between Elodea and phytoplankton or epiphytic covers exist, probably due to the release of growth inhibiting allelochemicals. This would be an effective strategy to avoid light limitation caused by algae and cyanobacteria. We investigated the allelopathic potential of both E. nuttallii and E. canadensis against different photoautotrophs, focussing on epiphytic algae and cyanobacteria isolated from different submersed macrophytes and culture strains. Methanolic extracts of both species inhibited the growth of most of these organisms. Only a culture strain of Scenedesmus brevispina was stimulated. Further separation of extracts yielded several active fractions, indicating that hydrophilic and slightly lipophilic compounds were responsible for growth reduction. At least some of the activity seems to be related to phenolic substances, but flavonoids in these species are inactive. Since growth declined also in a moderately lipophilic fraction of culture filtrate of E. nuttallii, we assume that active compounds were exuded in the water. Allelopathy might thus be relevant in situ and suppress cyanobacteria and algae. We furthermore found differences in the susceptibility of target organisms, which could (1) at least partly be a result of adaptation to the respective host plants and (2) indicate that allelopathic interference might reduce the abundance of some species, especially cyanobacteria, in epiphytic biofilms.     

Isolation and characterization of a novel antialgal allelochemical from Phragmites communis

Antialgal allelochemicals were isolated from Phragmites communis Tris. The isolated allelopathic fraction showed strong inhibition activity on the growth of Chlorella pyrenoidosa and Microcystis aeruginosa but had no inhibition on Chlorella vulgaris. The 50% effective concentrations (EC50) of the allelopathic fractions on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.79 mg/liter, respectively. The allelopathic activity of the fraction was species-specific. The isolated allelopathic fraction caused metal ion leakage from algal cells. The fraction decreased the activities of antioxidant enzymes, such as superoxide dismutase and peroxidase. The addition of the isolated fraction increased the concentration of unsaturated lipid fatty acids in cell membrane of C. pyrenoidosa and M. aeruginosa. This caused a change in plasma membrane integrity and the leakage of ions in the protoplast. The allelopathic compound was identified by nuclear magnetic resonance and gas chromatography-mass spectrometry as ethyl 2-methylacetoacetate. Synthesized ethyl 2-methylacetoacetate also showed allelopathic activity on C. pyrenoidosa and M. aeruginosa. The EC50 of synthesized ethyl 2-methylacetoacetate on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.65 mg/liter, respectively.     

铜绿微囊藻对紫外辐射的生理代谢响应

采用紫外(UV)滤膜过滤日光UV以及紫外灯添加UV的方法,研究了UV辐射对铜绿微囊藻Microcystis aeruginosa单细胞藻株PCC7806和群体藻XW01生长及生理代谢的影响。结果显示,在室内条件下低剂量UV辐射可促进群体微囊藻XW01生长;室外条件下与滤除了UV的光照相比,含有UV的完全日光更有利于微囊藻生长;而相同的UV辐射强度均导致单细胞株死亡,群体株显示了较强的UV抗性;日光中的UV可促进XW01合成抗氧化相关的超氧化物歧化酶(SOD)和过氧化氢酶(CAT)、促进胞外多糖的产生并形成较大的群体、促进UV屏障物质类菌孢素氨基酸(MAAs)和伪枝藻素(Scy)积累。这些生理代谢的改变,消除了阳光辐射中UV对微囊藻的伤害。研究的结果提示,自然条件下阳光中的UV有助于群体微囊藻生长。     

Toxic and non-toxic strains of Microcystis aeruginosa induce temperature dependent allelopathy toward growth and photosynthesis of Chlorella vulgaris

Global warming was believed to accelerate the expansion of cyanobacterial blooms. However, the impact of changes due to the allelopathic effects of cyanobacterial blooms with or without algal toxin production on the ecophysiology of its coexisting phytoplankton species arising from global warming were unknown until recently. In this study, the allelopathic effects of toxic and non-toxic Microcystis aeruginosa strains on the growth of green alga Chlorella vulgaris and photosynthesis of the co-cultivations of C. vulgaris and toxic M. aeruginosa FACHB-905 or non-toxic M. aeruginosa FACHB-469 were investigated at different temperatures. The growth of C. vulgaris, co-cultured with the toxic or non-toxic M. aeruginosa strains, was promoted at 20 °C but inhibited at temperatures ≥25 °C. The inhibitory effects of the toxic and non-toxic M. aeruginosa strains on of the co-cultivations (C. vulgaris and non-toxic M. aeruginosa FACHB-469 or toxic M. aeruginosa FACHB-905) also linearly increased with elevated temperatures. Furthermore, toxic M. aeruginosa FACHB-905 induced more inhibition toward growth of C. vulgaris or P_max and R_d of the mixtures than non-toxic M. aeruginosa FACHB-469. C. vulgaris dominated over non-toxic M. aeruginosa FACHB-469 but toxic M. aeruginosa FACHB-905 overcame C. vulgaris when they were co-cultured in mesocosms in water temperatures from 20 to 25 °C. The results indicate that allelopathic effects of M. aeruginosa strains on C. vulgaris are both temperature- and species-dependent: it was stimulative for C. vulgaris at low temperatures such as 20 °C, but inhibitory at high temperatures (≥25 °C); the toxic strain was determined to be more harmful to C. vulgaris than the non-toxic one. This suggests that global warming may aggravate the ecological risk of cyanobacteria blooms, especially those with toxic species as the main contributors.     

Simultaneous cultivation of Spirulina platensis and the toxigenic cyanobacteria Microcystis aeruginosa

Mangueira Lagoon, located in the extreme south of Brazil, has water with physicochemical characteristics such as alkaline pH and carbonate levels propitious for the growth of the cyanobacterium Spirulina platensis. Previously published studies have shown that Mangueira Lagoon water supplemented with small quantities of carbon and nitrogen is suitable for S. platensis cultivation and can significantly reduce production costs. We studied mixed cultures of Spirulina platensis and the toxic cyanobacterium Microcystis aeruginosa using a 2(3) factorial design in which the three factors were the initial biomass concentration of S. platensis and M. aeruginosa and the type of culture medium (100% Zarrouk's medium or 80% Mangueira Lagoon water plus 20% Zarrouk's medium). The highest S. platensis maximum specific growth rate (mu(max)) occurred in the culture with the highest M. aeruginosa biomass concentration and when undiluted culture medium was used (micro(max) = 0.283 d(-1)). The highest M. aeruginosa specific death rate (k) was obtained in the presence of S. platensis (k = 0.555 d(-1)) and was independent of the initial M. aeruginosa biomass concentration and culture medium, demonstrating that S. platensis cultures are not susceptible to contamination by M. aeruginosa. The culture medium had no significant influence (p > 0.05) on S. platensis micro(max) values, indicating that production costs could be reduced by using a medium consisting of 80% Mangueira Lagoon water plus 20% Zarrouk's medium.     

Comparative studies on the allelopathic effects of two different strains of Ulva pertusa on Heterosigma akashiwo and Alexandrium tamarense

Allelopathic effects of fresh tissue and dry powder of a nonsexual and a sexual strain of the macroalga Ulva pertusa on the growth of the microalgae Heterosigma akashiwo and Alexandium tamarense were evaluated using long-term coexistence culture systems in which several concentrations of macroalga fresh tissue and dry powder were used. The effects of macroalga culture medium filtrate on the two HAB algae were also investigated. Moreover, isolation co-culture systems were built to confirm the existence of allelochemicals and preclude the growth inhibition by direct contact. Short-term algicidal effect assays of macroalgae on H. akashiwo were carried out to measure the rate of algal cell lysis. The results of the coexistence assays showed that the growth of H. akashiwo and A. tamarense was strongly inhibited by fresh tissue and by dry powder of both strains of U. pertusa. The allelochemicals were lethal to H. akashiwo at relatively higher concentrations. The macroalga culture medium filtrate exhibited no apparent growth inhibitory effect on the two HAB algae under initial or semicontinuous filtrate addition, which suggested that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of both strains of U. pertusa was essential to effectively inhibit the growth of H. akashiwo and A. tamarense.     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

Potassium Salts Inhibit Growth of the Cyanobacteria Microcystis spp. in Pond Water and Defined Media: Implications for Control of Microcystin-Producing Aquatic Blooms

Ten metals were assayed in 21 Indian ponds which comprised three groups: (i) eutrophic alkaline ponds containing <2.5 mM potassium and thick growths of Microcystis aeruginosa or Microcystis flos-aquae during most of the year, (ii) equally eutrophic alkaline ponds containing >2.8 mM potassium and no detectable Microcystis growth, and (iii) oligo- or mesotrophic ponds with various potassium and hydrogen ion concentrations and no persistent Microcystis blooms. The effects of potassium on Microcystis growth were examined in filter-sterilized pond water and in defined culture media. A 50% reduction in the 10-day yield of cultured M. aeruginosa was observed in DP medium and pond water supplemented with 1 and 3 mM KCl, respectively. In contrast, the addition of 2 to 30 mM NaCl did not suppress the growth of M. aeruginosa in either DP medium or pond water. Both 5 mM KCl and 20 mM KHCO(inf3) in J medium strongly inhibited the growth of M. flos-aquae C3-9, whereas 5 to 30 mM NaCl had no effect and 20 mM NaHCO(inf3) was stimulatory. For pond water cultured with a mixture of M. aeruginosa and the duckweed Wolffia arrhiza, M. aeruginosa dominated in unsupplemented water and W. arrhiza dominated in water supplemented with 4.8 mM KCl. Implications for the ecology and control of Microcystis blooms are discussed.     

Isolation of a Poterioochromonas capable of feeding on Microcystis aeruginosa and degrading microcystin-LR

Algal blooms have become a worldwide issue recently, especially those comprised of toxic cyanobacteria. Grazers' predation of bloom-forming algae plays an important role in water clearing. In this study, a species of golden alga (strain ZX1), capable of feeding on the toxic cyanobacteria Microcystis aeruginosa, was isolated and identified as Poterioochromonas sp. (GenBank accession: EU586184) on the basis of morphological characteristics and 18s rRNA gene sequencing. Feeding experiments showed that ZX1 could clear high densities of M. aeruginosa (7.3 x 10(5)-4.3 x 10(6) cells mL(-1)) in a short time (40 h), with inhibition ratios higher than 99.9%. ZX1 grew during the feeding processes and achieved a maximum density of 10-20% of the initial M. aeruginosa density. Furthermore, this study is the first to report that ZX1 was able to degrade microcystin-LR (MC-LR) in cells of M. aeruginosa while digesting the whole cells, and that the degradation process was determined to be carried out inside the ZX1 cell. For a total MC-LR (intra- and extracellular) concentration of up to 114 microg L(-1), 82.7% was removed in 40 h. This study sheds light on the importance of golden alga in aquatic microbial ecosystems and in the natural transportation/transformation of MC-LR.     

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 μmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.     

青草沙水库中一株铜绿微囊藻抑藻细菌的作用研究

【目的】为了研究青草沙水库中土著微生物对藻类生长的抑制作用,从水库水体中筛选出对藻类有抑制作用的细菌并研究其对铜绿微囊藻的抑制效果。【方法】通过对水库水体中的细菌进行划线分离和筛选,挑选出一株对铜绿微囊藻生长有较好抑制作用的菌株CL。考察其对铜绿微囊藻的抑制效果及不同培养时间和菌液浓度对抑藻效果的影响,并对菌株进行16S rDNA序列分析。【结果】实验菌液浓度为4.5×108CFU/mL 8.4×108CFU/mL时,细菌对铜绿微囊藻的抑制率可达45.4%。抑藻效果随培养时间先增后降,在静置培养第6天抑藻效果达到最大。该菌经过16S rDNA序列分析,属于黄单胞菌科的寡养单胞菌。【结论】从青草沙水库中筛选出了对铜绿微囊藻有抑制作用的土著细菌寡养单胞菌,对青草沙水体铜绿微囊藻的控制具有一定的潜在应用价值。     

渗透与非渗透性抗冻剂联用技术对铜绿微囊藻的超低温保藏研究

铜绿微囊藻(Microcystis aeruginosa Kütz)是一种在全世界分部很广的淡水水华蓝藻。在实验室长期的研究工作中, 为了使铜绿微囊藻能维持稳定的生理学特征, 通常使用超低温保藏技术长期冻存藻细胞。研究发现, 同时使用渗透性和非渗透性的抗冻剂比只使用传统的渗透性保护剂能显著提高Microcystis aeruginosa超低温保藏的存活率。以三株铜绿微囊藻为材料进行二步法超低温保藏, 对4 种抗冻剂(甲醇、二甲亚砜、丙三醇、聚乙烯吡咯烷酮), 两种降温速率(-1 /℃min、-0.5℃/min), 第一步温度设置(-30℃、-40℃、-80℃)进行筛选; 用流式细胞仪和细胞计数检测存活率, 并监测冻后相关生理参数、PSII、细胞色素、生长曲线等以确保该方法可保持藻株的活性和生理状态。结果表明, 5%的二甲亚砜和30%的聚乙烯吡咯烷酮(PVP)同时使用时能达到最好效果, 并能保持生理活性与冻前一致。