【作者】 王宇涛；

【导师】 杨中艺；

【作者基本信息】 中山大学 ， 环境科学， 2011， 博士

【摘要】 丛枝菌根真菌（AMF）可能是自然界分布最为广泛的一类共生微生物,能与大部分的陆生植物形成共生关系。由于它们在环境、农林等领域展示出的巨大应用前景,对AMF的研究在国际上一直受到较高的关注。在近年来对海洋环境保护、海洋资源开发日益受到重视的背景下,本研究结合传统的生态、环境领域的研究方法以及最新的分子生物学手段,较为系统地研究了AMF在珠江河口红树林生态系统中的多样性、种类组成、群落结构、生态意义及其对环境因子的响应。主要研究内容和结果如下:1.调查了华南地区珠江河口两处红树林湿地生境中AMF与主要红树植物的共生状况。结果表明,两处红树林生态系统在不同潮间带分布的红树植物根内普遍存在AMF的侵染。根内AMF的形态结构以菌丝为主,泡囊和丛枝结构也均有发现,但是感染强度相对较低。通过诱导培养之后,在两处生境中一共鉴定出属于球囊霉属（Glomus）和无梗囊霉属（Acaulospora）的六个AMF形态种。在两处生境中,分布于高、中潮间带的红树植物根内AMF的感染强度要显著高于低潮间带的红树植物（p < 0.05）。多元线性回归分析表明,水文状况以及土壤P水平是在两处红树林生态系统中影响AMF与红树植物共生强度的主要环境因子（p < 0.05）。2.为了初步探讨AMF在红树林生态系统中潜在的生态功能,通过盆栽试验研究了接种从红树林生境扩繁的AMF菌剂对供试的红树植物无瓣海桑（Sonneratia apetala）的生长发育以及营养元素吸收的影响。结果表明,接种AMF处理显著提高了红树植物的生长:接种AMF的无瓣海桑株高、地径和生物量均显著高于不接种的对照（p < 0.05）。同时,接种AMF处理还显著提高了无瓣海桑对N、P和K的吸收（p < 0.05）。该结果表明,AMF可能在红树林生态系统中具有重要的生态功能。3.调查了珠海红树林处于陆缘的四种半红树植物根内的AMF侵染强度,以及根际土壤中AMF孢子的密度和种类。结果表明,所调查的半红树植物根内存在较高的AMF感染率（总感染强度:33.3%⁶3.7%）。根内AMF的结构以菌丝（27.9%⁵4.5%）为主,但是泡囊（13.8%³8.2%）和丛枝（14.7%²9.8%）结构也较为丰富。根际土壤中AMF的孢子密度相对较低,介于0.1-1.5个g-1干土之间。采用形态学与分子生物学手段相结合的方法在四种植物根际一共分离到属于球囊霉属（Glomus,5个种系型）和无梗囊霉属（Acaulospora,2个种系型）的七个AMF种系型。在每一种宿主植物根际土壤中一共分离到2-4个AMF的种系型,不同宿主植物根际土壤中AMF孢子的多样性和种系型组成均无明显的规律。相关性分析结果表明,半红树植物群落的土壤有机质含量可能是该处生境中影响AMF侵染半红树植物的主要环境因子。4.为了研究淹水强度和宿主植物种类对AMF与宿主植物的共生强度的影响,调查了珠海红树林生境中高、中、低三个潮间带均有分布的三种半红树植物（银叶树、卤蕨和老鼠簕）根内的AMF侵染情况。结果表明,所有调查的半红树植物根内均存在AMF的共生,感染的主要形态是菌丝,囊泡和丛枝结构也很常见。宿主种类对根内AMF的丛枝和泡囊感染强度具有显著的影响（P < 0.05）,而对AMF菌丝感染强度的影响不显著（P > 0.05）。淹水强度对所有类型的感染强度均具有极显著的影响（P < 0.01）:处于中、高潮间带的植物根内的AMF感染强度大多要显著（p < 0.05）高于分布在低潮间带的植物;分布于中潮间带的红树植物根内各种感染强度也要显著高于分布在高潮间带的红树植物（p < 0.05）。另外,宿主种类和淹水强度的交互作用同样对AMF感染强度具有显著的影响（P < 0.05）。5.为了进一步研究红树林生态系统中红树植物根内的AMF多样性及种类组成,并深入探讨淹水强度和宿主种类对AMF多样性和种类组成的影响,采用分子学手段研究了珠海红树林生境中在高、中、低三个潮间带均有分布的三种半红树植物根内的AMF多样性和种类组成。采用“巢式PCR -克隆-测序”的方法,从27个根样中一共获得761条长度约为1500 bp（SSU-ITS-LSU,包含约230 bp的SSU rDNA,480 bp的ITS,约830 bp的LSU rDNA）的AMF序列。1)多样性:以SSU-ITS-LSU序列相似性大于97%作为标准,761条AMF序列归于37个序列分类单元（OTU, operational taxonomic unit）。进一步通过邻接法（neighbor-joining）和贝叶斯（MrBayesian）进化分析,它们分属于23个种系型（phylotype）。在每个单独的根样中检测到2-7个AMF种系型。三种半红树植物根内检测到的AMF多样性要远远高于已报道的其它湿地生态系统中的AMF多样性水平,并且也要高于大部分已报道的陆地生态系统中AMF的多样性。这些结果清楚地表明:红树林生态系统中至少含有与大部分陆地生境相当的AMF多样性,湿地生态系统中也可以具有较高的AMF多样性。2)种类组成:本研究获得的23个AMF种系型中包含了22个（759条序列）属于Glomus属和1个（2条序列）属于Acaulospora属的种系型。其中,属于11个种系型的367条序列（占序列总量的48%）在GenBank中无法找到较高匹配度的序列,可能为新的类型的AMF序列。剩下12个种系型中有四个可以初步界定到AMF种,分别为G. intraradices、‘G. intraradices DAOM197198’、G. mosseae、G. sinuosum。其中,G. intraradices的序列是首次在美国福罗里达州之外的生境中检测到的真正的G. intraradices的DNA序列。剩下的八个种系型分别与一些尚未鉴定到种水平的AMF序列相匹配。3)宿主植物、淹水强度的影响:宿主种类对根内AMF的种系型多度和香浓多样性指数均无显著性影响（P > 0.05）,但是显著影响了根内AMF的群落结构（P < 0.05）,表明在红树林生态系统中存在AMF与宿主植物之间的偏好性。淹水强度对红树植物根内AMF多样性（基于种系型多度、香浓多样性指数）和群落结构具有极显著的影响（P < 0.01）,并且不同的淹水强度对AMF多样性的影响方式不同:相比不淹水条件,高强度的淹水(7^-10 hr day^-1)显著降低了宿主植物根内AMF的多样性,而适宜强度的淹水(2-4 hr day^-1)显著提高了宿主植物根内的AMF多样性水平。淹水强度是红树林生境中影响AMF多样性和群落组成最重要的环境因子。6.为了解红树林生态系统中的重金属污染水平,调查了存在较高污染风险的深圳红树林生境中表层淤泥以及红树植物不同部位（细根、粗根、茎和叶）中六种重金属（Cu、Pb、Cd、Zn、Cr和Ni）的浓度。结果表明,深圳红树林存在中等程度的重金属污染,Cu和Zn是最主要的重金属污染元素。各种调查的重金属在不同潮间带的污泥中具有相似的分布规律,并且均具有较高的DTPA提取态比例,表明该生境中的重金属可能具有相同的来源。各种重金属在六种红树植物中的分布规律相同:细根部位的浓度要极显著的高于其它部位的浓度（p < 0.01）,粗根部位的重金属浓度也大多要高于同一植株的茎和叶部位中的重金属浓度（p < 0.05）,表明所调查的红树植物种类均采取了重金属排除策略来应对重金属污染。不同红树植物种类对同一种重金属的富集能力（富集系数）存在极显著差异（p < 0.05）;相同种类红树植物对不同重金属的富集能力也同样存在极显著差异（p < 0.01）。7.为了评估重金属污染对红树植物的潜在风险,采用盆栽试验的方法研究了不同水平的Cd-Cu复合污染对胚轴和六月龄木榄幼苗的生长和营养元素吸收的影响。结果发现,Cd(c. 1 mg kg^-1, 3 mg kg^-1)和Cu(c. 100 mg kg^-1, 200 mg kg^-1)的复合污染对0³月龄和6⁹月龄木榄幼苗的生长、营养元素吸收均无显著的负面影响（P > 0.05）,表明木榄植株从胚轴阶段就已经具备了较强的重金属耐性。高Cd(约3 mg kg^-1)处理显著降低了0³月龄幼苗叶片的叶绿素a含量和叶绿素a/b的比值（p < 0.05）,但是对6⁹月龄幼苗的叶绿素无显著影响（p < 0.05）。在低Cd(约为1 mg kg^-1)和高Cd(约3 mg kg^-1)处理下,0³月龄木榄植株各个部位（尤其是细根）的重金属浓度要远远高于6⁹月龄木榄幼苗对应部位的重金属浓度（p < 0.01）,表明6⁹月龄的木榄幼苗植株体内排除重金属的机制要比0³月龄的木榄幼苗更为完善,其重金属耐受性更强。该结果表明,在评价重金属污染对红树林生态系统的潜在风险时有必要研究它们对红树植物幼苗的影响。8.为了评估重金属污染对红树林生态系统中AMF与红树植物共生关系的影响,以及AMF在红树植物对重金属污染响应方面的作用,以从红树林湿地扩繁的AMF作为接种菌剂,以菌根敏感型植物玉米（Zea mays L.）作为供试植物,研究了在Cu和Cd复合污染条件下AMF的感染强度、玉米植株体内重金属浓度以及宿主植物的部分生理指标对重金属胁迫的响应。结果表明,Cd处理(c. 1 mg kg^-1, 3 mg kg^-1)和Cu处理(c. 100 mg kg^-1, 200 mg kg^-1)均显著抑制了AMF对宿主植物的感染（P < 0.05）,并显著降低了玉米的株高和生物量（P < 0.05）。AMF侵染显著提高了供试玉米地上部和地下部的Cd和Cu的浓度（p <0.05）,但同时通过提高宿主植物体内脯氨酸浓度增强了宿主植物对重金属的耐受性（p < 0.05）。这些结果显示,AMF能够提高宿主植物的重金属耐性,但是重金属污染会抑制AMF与宿主植物的共生,表明重金属污染很可能会抑制红树林生境中AMF与红树植物的共生,进而对红树林生态系统产生潜在的风险。9.为了进一步探讨红树林生境中影响AMF与红树植物共生的环境因子,在人工构建的秋茄和桐花树湿地中试系统、小试系统中分别模拟生活污水排放,研究生活污水排放对AMF与红树植物共生以及根际孢子密度的影响。结果表明,生活污水排放对AMF的孢子密度无显著的处理效应（P > 0.05）,但是能够明显降低红树植物根内的菌丝、泡囊和丛枝感染强度（p < 0.01）,并且其抑制作用随着污水浓度的增加而增强。AMF泡囊和丛枝结构对生活污水的敏感性要高于AMF菌丝结构的敏感性。由于桐花树根系的放氧能力强于秋茄根系,以及可能存在的其它未知因素的共同作用,在相同的处理条件下,桐花树根内AMF的感染强度总是要显著高于秋茄根内的AMF感染强度（p < 0.05）,充分证明了宿主植物对AMF共生关系的影响。考虑到AMF在红树林生态系统中可能发挥重要的生态功能,本试验的结果清楚的表明,生活污水的排放会通过抑制甚至是破坏AMF与红树植物的共生关系从而对红树林生态系统产生潜在的风险。更多还原

【Abstract】 Arbuscular mycorrhizal fungi （AMF） are probably the most widespread symbiotic microbes in the nature ecosystems, which can form symbiosis with most of the terrestrial plants. They have shown great application perspectives in the areas of environment, agriculture and forestry. Therefore, there has been a great concern in the research of these fungi. During these years, there has been an increasing concern on the exploitaion of marine resources and the protection of the marine environment. The present study systematically investigated the AMF species compostion, community structure, diversity level and their responses to environmental factors in the mangrove swamps near by the estuary of the Pearl River, employing both the traditional methods in ecological and environmental research and the cutting-edge methods in molecular biology. The main contents and results from this research are as follows:1. The symbiosis between AMF and mangrove plant species was investigated in two mangrove swamps near by the Pearl River estuary in south China. AMF were mostly found in the form of hyphae and were commonly associated with all the mangrove species we investigated. The colonization intensities of vesicle and arbuscule were relatively low, though these structures were detected in most of the investigated mangrove species. Six AMF morphological species belonging to the genera Glomus or Acaulospora were identified after a trap culture procedure. The colonization intensity of AMF was significantly higher in the high and middle tide levels than that in the low tide level （p < 0.05） in both swamps. Multiple step-wise linear regression analyses showed that hydrological conditions and soil P levels in the rhizosphere were the main environmental factors affecting the colonization of mangrove species by AMF.2. A greenhouse experiment was conducted to evaluate the effects of AMF inoculation, which is trap-cultured from a manrove swamp, on the growth and nutrient uptake of a true mangrove plant species, Sonneratia apetala B. Ham. The inoculated AMF significantly improved growth, resulting in greater plant height, diameter at ground level and plant biomass （p < 0.05）. The AMF colonization also increased the absorption of N, P and K of the host plants （p < 0.05）. These findings suggest that AMF play important roles in mangrove ecosystems.3. The colonization of AMF to four semi-mangrove species and the AMF spore density and species composition in the rhisosphere soil were investigated in the landward part of the Zhuhai Mangrove forests. A relatively high AMF colonization rate （total colonization rate: 33.3%⁶3.7%） was detected in the investigated plants. Although vesicle （13.8%³8.2%） and arbuscule （14.7%²9.8%） were also commonly detected, hyphae had the highest colonization intensity （27.9%⁵4.5%）. A relatively low AMF spore density was detected in the rhisosphere （0.1 - 1.5 spores g-1 soil）. All the AMF spores were designed to seven phylotypes （five Glomus phylotypes and two Acaulospora phylotypes） according to their morphological features and the phylogenetic analysis based on an 800 bp rDNA sequences. Two to four spore phylotypes were detected from the rhizosphere soil of each semi-mangrove species. No obvious differences were found in the distribution pattern and phylotype diversity of AMF spores from different semi-mangrove species. The results of correlation analysis showed that the soil organic matter content could be the most important environmental factor affecting the symbiosis between AMF and semi-mangrove species.4. To evaluate the effects of host plant and tide level on the AMF colonization to mangrove plants in mangrove ecosystems, the colonization intensity of AMF within the roots of three semi-mangrove species （Heritiera littoralis Ait., Acrostichum aureum L. and Acanthus ilicifolius L.）, which are located in all of the high, middle and low tide levels in Zhuhai Mangrove forest, were investigated. The AMF structures were detected within the roots of all investigated species. AMF hyphae had the highest colonization intensity, but vesicle and arbuscular structures were also commonly detected. The results of Two-way ANOVA showed that the host plants had significant effects on the vesicle and arbuscular colonization intensity （P < 0.05）, but no significant effects on either total or hyphal colonization intensity. The tide level significantly affected all types of AM colonization （P < 0.01）: the AMF colonization intensities in the roots from high and middle tide zones are higher than that from the low tide level, and AMF colonization intensity in the roots of middle tide level is generally higher than that at the high tide level. The interactions between plant species and tide level also significantly affected all types of AM colonization （P < 0.05）.5. To further investigate the diversity and species composition of AMF in mangrove ecosystems, the communities of arbuscular mycorrhizal fungi （AMF） colonizing the roots of three semi-mangrove species were characterized along a tidal gradient in ZH Mangrove forest, using a“Nested PCR– Clone– Sequencing”procedure. A fragment, designated SSU-ITS-LSU, including part of the small subunit （SSU, c. 230 bp）, the entire internal transcribed spacer （ITS, c. 480 bp） and part of the large subunit （LSU, c. 830 bp） of rDNA from samples of AMF-colonized roots was amplified, cloned and sequenced using AMF-specific primers. A total of 761 AMF sequences were obtained.1) AMF diversity within the roots: The results of phylogenetic trees based on the neighbor-joining and MrBayesian analyses indicated that the 761 obtained AMF sequences were grouped into 37 operational taxonomic units （OTUs） based on SSU-ITS-LSU sequence similarities of 97-100%, and finally assigned to 23 AMF phylotypes. Two to seven AMF phylotypes were detected from each individual plant sample. The diversity levels of AMF detected from these three species were much higher than that reported from other wetland ecosystems and most terrestrial ecosystems, indicating that the AMF diversities in mangrove ecosystems could at least be comparable to those in most terrestrial ecosystems, and that the AMF diversity level in wetland ecosystem is not necessarily low.2) AMF species composition: Twenty-two （759 sequences） of the 23 phylotypes detected in the roots belong to the genus Glomus, only one phylotype （2 sequences） belongs to the genus Acaulospora. Eleven （367 sequences） of the 23 phylotypes detected in this study were novel. Four of the other 12 phylotypes were preliminarily identified to G. intraradices,“G. intraradices DAOM197198”, G. mosseae and G. sinuosum. The G. intraradices sequences detected in this study are the first true record of G. intraradices outside Florida. The other phylotypes were related to the AMF sequences that have not yet been identified to species level.3) Effects of host plants and flooding intensity: Plant species had no significant effects on the AMF diversity （based on the phylotype richness and Shannon’s index） （P > 0.05）, but significantly affected the AMF phylotype communities within the roots of three semi-mangrove species （P < 0.05）, indicating the presence of host preference between AMF and host species. The tide level （flooding） has a highly significant effect （P < 0.01） on AMF diversity and phylotype composition, and the effects are dependent on the degree of flooding: intensive flooding (7^-10 hr day^-1) was clearly associated with decreased AMF diversity in the roots of all three mangrove species; while moderate flooding (2-4 hr day^-1) showed a promotion effects on the diversity of AMF in any of the three plant species investigated. The hydrologic conditions should be the most important factors affecting the diversity and community structure of AMF in mangrove ecosystems.6. To investigate the heavy metal contamination level in Shenzhen mangrove swamp, one of the most vulnerable mangrove ecosystems in China, concentrations of six heavy metals （Cu, Ni, Zn, Cd, Cr and Pb） in sediments, and fine roots, thick roots, branches and leaves of six mangrove plant species collected from this site were measured. The results show that the Shenzhen mangrove sediments are moderately contaminated by heavy metals, Zn and Cu being the main contaminants. All investigated metals showed very similar distributions in the sediments, and had relatively high proportions of DTPA extractable heavy metals, implying that they had the same anthropogenic source（s）. High accumulations of the heavy metals were observed in the root tissues, especially the fine roots, and much lower concentrations in the other organs, indicating that the roots strongly immobilize them and （hence） that mangrove plants possess mechanisms that limit the upward transport of heavy metals and exclude them from sensitive tissues. There were significant differences in the accumulation ability of each investigated heavy meatl between different mangrove species （P < 0.05）. The accumulation abilities of the same species to different heavy metals were also significantly different （P < 0.01）. 7. To further evaluate the potential risk of heavy metals posed to mangrove ecosystems, the growth performance of propagules and six-month-old seedlings of Bruguiera gymnorhiza in the presence of contaminating Cd (c. 1 mg kg^-1, 3 mg kg^-1) and Cu (c. 100 mg kg^-1, 200 mg kg^-1) was examined. The results show that the heavy metal contamination set in this study showed no significant effects on the growth and nutrient uptake in both 0³ month seedling and 6⁹ month seedling （P > 0.05）, indicating that this plant is not sufficiently sensitive to heavy metals after its propagule stage for its regeneration and growth to be significantly affected by the heavy metal contamination in the Shenzhen mangrove ecosystem. However, the high Cd (c. 3 mg kg^-1) treatment decreased leaf chlorophyll a contents and chlorophyll a/b ratios in the three-month seedlings （p < 0.05）, but not in the nine-month seedlings. In addition, older mangrove seedlings accumulated much higher Cd level in plants in both the low Cd (c. 1 mg kg^-1) and high Cd (3 mg kg^-1) treatments. These results indicated that the older seedlings are more metal-tolerant than the younger seedlings, due to their more efficient exclusion mechanism. Therefore, it is necessary to assess the effects of metal contamination on young seedlings when evaluating the risks posed by heavy metals in an ecosystem.8. A greenhouse experiment was conducted to evaluate the effects of heavy metal contamination on the symbiosis between AMF and mangrove species, and to study the roles of AMF in the responses of mangrove species to heavy metal contaminants, taking the trap cultures of AMF from mangrove swamp as the fungal materials. Instead of the mangrove plants, maize （Zea mays L.）, a typical sensitive mycorrhizal plant, was used as the test plant. The results show that both the Cd (c. 1 mg kg^-1, 3 mg kg^-1) and Cu (c. 100 mg kg^-1, 200 mg kg^-1) treatments significantly inhibited the symbiosis between AMF and host species （p < 0.05）, and decreased the plant height and biomass of the tested plants （p < 0.05）. AMF colonization significantly increased the concentrations of Cu and Cd in the shoots and roots of the maize plants （p < 0.05）, and improved the heavy metal resistance of plants, partly by increasing the proline levels in host plant （p < 0.05）, resulting in greater plant height and biomass of the host plants （p < 0.05）. These results indicate that the mangrove AMF could decrease the potential risks of heavy metal contaminants through improving the heavy metal tolerance of host plants. In mangrove habitat, heavy metal contamination can inhibite the symbiosis between AMF and host species, posing a risk to mangrove ecosystems.9. A medium-sized mangrove constructed wetland and a small-scale mangrove constructed wetland were set up for the purpose of evaluating the effects of domestic sewage discharge on the symbiosis between AMF and mangrove plants. The colonization intensity in the roots and spore density from the rhisosphere soil of mangrove species locating within varied distances from the sewage inlet in the medium-sized wetlands were tested after approxiamately three years growth of the plants. The colonization intensity and spore density of AMF from the small-scale wetland were also examined after the treatments of sewage and running water （CK） for nine months. The results show that the sewage discharge had no significant effect on the spore density of AMF in both medium-sized and small-scale mangrove constructed wetlands （P > 0.05）. However, it obviously decreased the colonization intensity of the AMF （p < 0.01）, and the inhibition effect of sewage is largely depended on the concentration of the sewage. The vesicle and arbuscular structures were more sensitive than the haphal structure to the sewage dischage. Because the Aegiceras corniculatum had a higher radial oxygen loss ability than the Kandelia candel, combined with some other unknown reasons, AMF colonization intensity in the roots of Aegiceras corniculatum were always significantly higher that that of Kandelia candel （p < 0.05） under the same conditions, providing a solid evidence showing the effects of host plants on the symbiosis between AMF and plants. Given that AMF play important roles in the mangrove ecosystems, the results of the presented research show that sewage discharge to mangrove ecosystems could lead a harmful effect to these habitats through destroying or inhibiting the symbiosis between AMF and mangrove plants.更多还原