深圳大鹏湾海域锥状斯氏藻赤潮期间细菌群落结构变化研究

目的:近年来,赤潮在我国的发生呈增加的趋势,并造成了极大的经济损失。过去研究赤潮发生的机理主要集中在理化因素的影响,而越来越多的证据表明仅凭借营养盐等环境因素并不能解释大部分赤潮现象,藻际微生物可能发挥着重要作用。本文跟踪观测了2010年7月深圳大鹏湾海域爆发的锥状斯氏藻赤潮生消过程中细菌群落丰度种类的变化,从微生物与赤潮藻相互作用的角度探讨了赤潮的生消过程,讨论了不同时期不同关键菌群的特殊作用,为解释赤潮爆发和消亡提供了新的视角,为赤潮的监控和防治新方法的建立奠定了理论基础。方法:本文按时间顺序共采集该赤潮9次样本,利用末端限制性酶切片段长度多态性分析(T-RFLP)等分子生物学方法,通过主成分分析、克隆文库的构建,研究了微生物群落的变化过程,并探讨了特定种属的微生物在赤潮发生、发展和消亡过程中的作用。结果:从浮游细菌丰度来看,随着锥状斯氏藻细胞数量的波动,浮游细菌总数也随之呈现相应的变化。从浮游细菌的种类来看,它们主要属于变形杆菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)。从浮游细菌的动态变化过程来看,赤潮生消过程中,浮游细菌群落呈现出一定的演替现象,特别是在赤潮后期,群落主成分变化尤为显著。从关键菌群的作用来看,属于γ变形杆菌门的Alteromonas sp.一直占有较高的丰度,赤潮中后期受到菌群相互作用导致比例下降,而赤潮后期其他关键菌群的丰度的增高可能是导致赤潮消亡的重要原因。结论:本文利用T-RFLP这一DNA指纹技术分析微生物群落结构和多样性特征,通过研究赤潮生消过程中藻际浮游细菌群落的动态变化,发现随着赤潮的发展,浮游细菌群落发生着相应的变化。结果说明赤潮藻体丰度数量的改变影响着浮游细菌群落的组成。相对地,细菌群落的适应调整迅速造成赤潮藻体局部生长环境的改变,从而影响赤潮的发展过程。     

锥状斯氏藻藻华期间群体感应信号菌株的动态变化

群体感应信号(quorum sensing, QS)是细菌的一种特殊交流方式, 它具有调节种群密度、生物膜形成、毒素产生以及色素的形成等多种功能。藻菌关系是藻华过程中重要的一环, 为了探求藻华过程中信号微生物的动态变化, 我们以深圳大鹏湾的锥状斯氏藻(Scrippsiella trochoidea)藻华中的QS 菌株为研究对象, 应用报告菌株和环境微生物宏基因组方法, 监测了藻华爆发期间信号微生物的动态变化过程, 并构建了藻类、QS 微生物与其它微生物的相关性关系。结果表明:在筛选的QS 菌株中经去冗余和重复后成功鉴定了7 种不同的细菌, 分别是冷杆菌Psychrobacter cryohalolentis、普罗维登斯菌Providencia sneebia、假单胞菌Pseudomonas stutzeri、微小杆菌Exiguobacterium sp. AT1b、产酸克雷伯菌Klebsiellaoxytoca、球形赖氨酸芽孢杆菌Lysinibacillus sphaericus 和鲍氏不动杆菌Acinetobacter baumannii。相关性分析发现P.sneebia 的丰度与藻类数量变化呈正相关, 而L. sphaericus 和P. stutzeri 的丰度与藻类数量变化呈负相关, 其它QS 微生物中未见显著相关性。综合实验的结果来看, QS 微生物在藻际微生物的群体结构中扮演着一定的生态作用, 它帮助我们从一个新的视角了解藻华过程中QS 微生物的丰度变化和网络关系, 为认识藻菌关系提供了新的思路。     

氯氰菊酯对锥状斯氏藻的毒性效应

为了评估拟除虫菊酯农药对海洋生物以及海洋环境的毒性效应,研究了氯氰菊酯对锥状斯氏藻(Scrippsiellatrochoidea)的毒性效应及其对叶绿素a(Chl.a)、可溶性糖、可溶性蛋白含量以及超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量等生理生化指标的影响.结果表明,低浓度(≤10 μg·L-1)的氯氰菊酯对锥状斯氏藻生长具有明显的促进作用,而高于50μg·L-1则产生较明显的抑制效应.藻细胞所有生理生化指标在暴露初期的6～12 h较为敏感,24 h或48 h后趋于平稳;细胞内含物包括Chl.a、可溶性糖、可溶性蛋白含量以及SOD活性,在低浓度时均出现诱导现象,而高浓度时则呈现先抑制后恢复的现象.而氯氰菊酯对藻体内MDA含量均具有促进作用,浓度越高促进作用越强.研究结果表明,锥状斯氏藻的各种生化指标对氯氰菊酯的响应均较为敏感,但SOD和MDA对剂量反应更为敏感,最低影响剂量为5μg·L-1和1μg·L-1,可作为拟除虫菊酯农药污染早期预警的生物指示指标.     

氮磷限制对锥状斯氏藻孢囊形成的作用

在实验室研究了锥状斯氏藻(Scrippsiellatrochoidea)在N、P单因子营养限制(N:500μg.L-1,P:74～0.74μg.L-1和P:74μg.L-1,N:500～5μg.L-1)条件下的生长和孢囊形成。结果显示N、P限制不利于锥状斯氏藻的快速生长,其中低P对细胞生长的限制作用更显著。其孢囊形成率在15～99%之间,中度N限制能促进孢囊的形成,形成率几乎可达100%。孢囊一般在对数生长期结束、细胞数量达到最大值时开始形成。但由于接种后营养盐浓度的急剧降低,营养极度限制组孢囊可在接种后第1d就开始形成。结果显示稳定生长期孢囊的大量形成大大降低了锥状斯氏藻营养细胞数量,能在一定程度上促进其赤潮的消亡。     

海菖蒲浸提液对锥状斯氏藻的抑制效应研究

海菖蒲(Enhalus acodoides)是典型热带亚热带沿海常见大型海草, 但关于海菖蒲对赤潮藻的抑制作用研究少见报道。研究选取海菖蒲为实验材料, 利用人工海水和两种有机溶剂提取海菖蒲中抑藻活性物质并对锥状斯氏藻进行抑藻试验研究, 结果表明, 三种提取物对锥状斯氏藻均有显著抑制作用, 在试验设定浓度下, 水浸物在 2.5 g·L^–1 时具有最高抑藻率 95.34%, 乙醇提取物在浓度为 12.5 mg·L^–1 时即达到 78.55%, 正己烷提取物在浓度为 50 mg·L^–1 时最高抑制率达到58.28%。研究表明, 海菖蒲水提液抑制效果最好, 具有应用于近海水体富营养化的治理和开发新型抑藻剂的潜力。     

大亚湾海域锥状斯氏藻孢囊形成与萌发的季节变化

锥状斯氏藻(Scrippsiella trochoidea)是南海大亚湾海域优势甲藻。为了解该藻孢囊形成和萌发动态及其对营养细胞种群动态的影响,2001年1月-2002年1月在大亚湾澳头海域用沉积物捕捉器及TFO重力采泥器对其孢囊进行每月一次的周年监测,同时对浮游植物、水温、盐度、溶解氧等也进行了监测。孢囊形成和萌发分别以沉积物捕捉器中的孢囊形成率以及上表层沉积物中空孢囊的百分比来表示。钙质孢囊和非钙质孢囊年平均形成率分别为1.11×104 cysts m-2d-1和2.13×105 cysts m-2d-1。前者在冬季大量形成,而后者在夏季形成较多。孢囊多在春秋季节萌发,夏季萌发较少,而冬季几乎不萌发。在5月份和10月份营养细胞数量峰形成前,孢囊的萌发出现了高峰,而表层沉积物中的孢囊数量及孢囊形成率则在营养细胞数量峰后大幅度上升。由此可见,大亚湾沉积物中该藻孢囊的萌发给水体提供了丰富的营养细胞,反之水体中高密度营养细胞又促使孢囊的大量形成,从而造成了锥状斯氏藻赤潮在大亚湾海域接连发生。     

氮和磷浓度对中肋骨条藻和锥状斯氏藻种间竞争的影响

采用室内单养和混养方法, 设置不同的氮、磷营养条件, 研究了氮、磷对中肋骨条藻(Skeletonema costatum)和锥状斯氏藻(Scrippsiella trochoidea)种间竞争的影响。结果表明: 混养时各氮和磷浓度下均呈现培养初期中肋骨条藻为优势种、培养后期锥状斯氏藻为优势种的变化趋势, 但随着氮、磷浓度的升高, 中肋骨条藻作为优势种的时间延长; 与单养时相比, 混养中两种微藻的最大密度受到不同程度的抑制, 表现出氮、磷浓度越高, 受抑制的程度越大的特征, 且与锥状斯氏藻相比, 中肋骨条藻的最大密度受到抑制的程度更大。混养时两种微藻均是在氮、磷浓度最高时, 抑制起始点出现时间最长, 随着氮、磷浓度的降低, 抑制起始点出现时间缩短; 各氮、磷浓度条件下, 锥状斯氏藻对中肋骨条藻的竞争抑制参数明显高于中肋骨条藻对锥状斯氏藻的竞争抑制参数, 当氮浓度为512 μmol·L^-1、磷浓度为2 μmol·L ^-1时, 竞争结果是锥状斯氏藻获胜; 其余氮、磷浓度条件下为两种微藻不稳定共存。     

广东大亚湾甲藻孢囊及其与锥状斯氏藻赤潮的关系

1999年12月至2001年1月,在大亚湾澳头海域用沉积物捕捉器(Sediment trap)及TFO重力采泥器对甲藻孢囊进行每月一次的周年监测,并同时研究了浮游植物的季节变化.结果显示,晚秋孢囊形成率最高(3.48105 cysts/m2d),冬季形成率较低,年平均为1.28105 cysts/m2d.锥状斯氏藻(Scrippsiella trochoidea)是大亚湾沉积物孢囊中的绝对优势种,除个别季节外,其形成率一般占孢囊总形成率的50%以上.2000年8月至9月,该海域发生了一次较大规模的锥状斯氏藻赤潮,最高细胞密度达4.0104 cells/mL.赤潮中后期,锥状斯氏藻孢囊包括暂时性孢囊和休眠孢囊大量形成,孢囊的形成减少了水体中营养细胞数量,是赤潮消退原因之一.       

海洋卡盾藻与中肋骨条藻和锥状斯氏藻种间竞争研究

研究了不同起始细胞密度与硅酸盐浓度对海洋卡盾藻(Cm)与中肋骨条藻(Sc)及锥状斯氏藻(St)之间种间竞争的影响,利用竞争抑制参数对相互间的竞争关系进行了分析,并引入体积竞争抑制系数的概念。结果表明:海洋卡盾藻的环境容量不受起始细胞密度(100—3600cells/mL)的影响,但随着起始接种密度的增加,达到最大细胞密度和进入稳定生长期的时间均提前。在硅缺乏的条件下,中肋骨条藻仍能保持一定生长,并能在与海洋卡盾藻之间的种间竞争中保持数量优势,但硅酸盐浓度的增加有利于其种间竞争。初始细胞密度和接种比例对3种赤潮藻类之间的种间竞争影响明显,总体来说海洋卡盾藻在竞争中处于劣势。当海洋卡盾藻细胞密度远远高于中肋骨条藻时(Cm:Sc=6:1),海洋卡盾藻才能在与中肋骨条藻的竞争中取胜;而在与海洋卡盾藻共培养条件下,锥状斯氏藻具有一定竞争优势,其对海洋卡盾藻的体积抑制系数(α’)是后者(β’)的27—100倍。但在所有处理中均没有绝对优胜者,竞争结果都会出现不稳定的平衡状态。       

Coupled effects of irradiance and iron on the growth of a harmful algal bloom-causing microalga Scrippsiella trochoidea

Effects of irradiance and iron on the growth of a typical harmful algal blooms (HABs) causative dinoflagellate, Scrippsiella trochoidea, were investigated under various irradiances (high light: 70 μmol m^?2 s^?1 and low light: 4 μmol m^?2 s^?1) and iron concentrations (low iron: 0.063 mg L^?1, medium iron: 0.63 mg L^?1 and high iron: 6.3 mg L^?1), and evaluated by the parameters of algal cell density, specific growth rate, optical density and chlorophyll a content. The results indicated that there was significant difference in the cell density of dinoflagellate S. trochoidea between high light and low light intensity treatments across the entire experiments, 7-fold higher at high irradiance as compared with low irradiance, which was further enhanced by the iron concentration. It was found that the maximum cell density of 25 × 10⁴ cell mL^?1 occurred under the combination of high light intensity and high iron concentration, followed by 23 × 10⁴ cell mL^?1 in the combination of high light and medium iron, and 20 × 10⁴ cell mL^?1 in the combination of high light and low iron. There was no significant effect of iron concentration on the cell density under low light intensity. The cell density maintained about 3 × 10⁴ cell mL^?1 across all combinations of iron concentrations and low light in the end of experiments. Such interactive effects of light intensity and iron level dependent were also observed for the specific growth rate, OD₆₈₀ and chlorophyll a content of S. trochoidea. The maximum values of specific growth rate, OD₆₈₀ and chlorophyll a content peaked at the condition of high irradiance and high iron, which were 0.22 d^?1, 0.282 and 0.673 mg L^?1, respectively. In general, their values increased significantly with the increasing of iron concentration at high irradiance, whereas no significant difference was observed among three iron concentrations at low irradiance, all remaining approximately 0.06 d^?1, 0.03 and 0.050 mg L^?1, respectively. Those results suggest that there may be a strong interactive effect between irradiance and iron on microalgal growth and their physiological characteristics. The combination of high light and high iron concentration may accelerate algal cell growth and pigment biosynthesis, thus leading to massive occurrence of HABs.     

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophyll a liter⁻¹). At this time bacterial abundance abruptly decreased from 2.8 × 10⁶ to 0.75 × 10⁶ ml⁻¹, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Root-Associated Endophytic Bacterial Community Composition of Asparagus officinalis of Three Different Varieties

Asparagus (Asparagus officinalis L) is an economically important crop, rich in nutrients, and is also conducive to solving ecological and environmental problems. Plants may acquire benefits from root-associated endophytic bacteria. However, the composition of the endophytic bacterial community associated with the roots of asparagus is poorly elucidated. In this study, the nine root samples of asparagus from three different varieties including Asparagus officinalis var. Grande (GLD), A. officinalis var. Jinglvlu3 (JL3) and A. officinalis var. Jingzilu2 (JZL) were investigated by high-throughput sequencing technology of the 16S rDNA V5-V7 hypervariable region of endophytic bacteria. A total of 16 phyla, 29 classes, 90 orders, 171 families, and 312 genera were identified. Endophytic bacteria diversity and bacteria structure was different among the three varieties and was influenced by rhizosphere soil properties and varieties. In the GLD variety, the main phyla were Proteobacteria, Actinobacteria, and Firmicutes. The main phylum in JL3 and JZL varieties was Proteobacteria. The observations showed that GLD had the highest diversity of endophytes as indicated by the Shannon index (GLD > JZL > JL3). The order of the endophytes richness was GLD > JL3 > JZL. The PCA and PCoA analysis revealed the microbial communities were different between three different asparagus varieties, and the microbial composition of GLD and JZL was more similar. This report provides an important reference for the study of endophytic microorganisms of asparagus. Supplementary informationThe online version contains supplementary material available at (10.1007/s12088-021-00926-6) contains supplementary material, which is available to authorized users.     

Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom

Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms. The main goal was to compare diversity and succession among free-living (<10 MM) AND PARTICLE-ASSOCIATED (>10 mm) bacteria. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage. Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment. Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied. PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles. Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes. Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria. Bands sequenced from the CF DGGE gel were related to members of the CF cluster. From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.     

Community level physiological study of algicidal bacteria in the phycospheres of Skeletonema costatum and Scrippsiella trochoidea

Bacteria in the phycosphere have a unique ecological relationship with host algae due to their utilization of algal extracellular products as nutrients. Some bacteria control the growth of algal cells and even lyse them. The diversity of bacteria and their community dynamics in the phycosphere of microalgae are still relatively little understood, especially of those associated with red tide-causing algae. In this study, scanning electron microscope (SEM) images of algal cell morphology revealed that the phycosphere bacteria of the red tide-causing algae, Skeletonema costatum and Scrippsiella trochoidea, could lyse them within 72 h. The community level physiology of the algicidal bacteria was studied using Biolog ECO microplates, a common method for the ecological study of microbial communities. The average well color development (AWCD) values of bacteria in the phycospheres of both species were low, indicating that the bacteria had low metabolic activity overall. The diversity indices were both lower than the bacterial diversity from natural environments. However, the bacteria associated with S. trochoidea demonstrated a higher AWCD value and diversity than those in the phycosphere of S. costatum. The utilization of carbon sources significantly changed at different lytic times, reflecting that the bacterial community structure changed during the algae-lysing process. These results revealed that the bacterial communities in phycospheres had a simple structure and low diversity. When the balance between algae and bacteria broke down, the total bacterial density increased while the algicidal bacteria accumulated and became the dominant species, changing the bacterial community structure in this micro-ecosystem.     

上海市内不同水质的河道春季浮游细菌群落结构分析

2008年4月采集上海市城郊结合地区的4条河道午潮港、横港、朝阳河和曹杨环浜的水样, 测定水样的相关理化数据, 计算水环境质量综合指数; 提取水样中浮游细菌的总DNA, 进行变性梯度凝胶电泳(DGGE)分析; 并对水体中的可培养浮游细菌的数量进行测定。结果表明, 污染严重的河道浮游细菌的生物量较高, 而多样性明显偏低, 河道不同采样点的微环境影响浮游细菌的组成, 河道中浮游细菌的组成与生物量的变化与水环境质量密切相关。     

Analysis of Bacterial Community in the Ginseng Soil Using Denaturing Gradient Gel Electrophoresis (DGGE)

The objective of this work was to determine the shifts in the PCR-DGGE profiles of bacterial communities associated with the rhizosphere soil of ginseng at varying age levels. Differences in the dominance of intense DNA bands in the DGGE profile was observed over the age of the plants indicating the fluctuation in the microbial community structure. The bacterial orders of actinomycetales of Actinobacteria and Spingomonadales and Rhizobiales of α-Proteobacteria were predominant in the ginseng soil.     

连作对麦冬根际土壤细菌群落的影响

以中草药麦冬为研究对象,采用变性梯度凝胶电泳(DGGE)方法,考察连作对麦冬根际土壤细菌群落的影响。结果表明,重茬麦冬根际细菌群落的Shannon-Wiener指数从苗期至快速生长期、再到块根膨大期分别为3.36-3.40-3.69,丰富度指数为55.0-61.5-63.5,均匀度指数为0.84-0.82-0.89;而同期正茬根际细菌群落的上述指数值分别为3.66-3.33-3.72、67.5-53.5-63.5和0.87-0.84-0.90。通过对土壤细菌群落的DGGE图谱进行主成分分析,结果显示,上述各个时期重茬与正茬样本均发生显著分离,表明连作改变了麦冬根际细菌群落的多样性变化趋势和结构组成。进一步比较麦冬块根膨大期根际主要功能菌群的数量变化,发现连作后氨化细菌和好气性纤维素分解菌数量增加,而硝化细菌和固氮菌的数量减少。对比发现重茬麦冬的产量仅为正茬的70.6%,表明麦冬连作减产与其根际土壤细菌群落的变化相关。