【作者】 司春灿；

【导师】 杜道林；

【作者基本信息】 江苏大学 ， 环境工程， 2014， 博士

【摘要】 生物入侵不仅对生态环境造成了严重的危害,而且会对社会经济和人类健康造成不可估量的损失,已成为21世纪五大全球性环境问题之一。全球科学家投入大量的精力对生物入侵的机制和防治方法进行研究,近年来研究者们发现植物的入侵和土壤微生物之间存在着紧密的联系。丛植菌根真菌(Arbuscular mycorrhizal fungi, AMF)是在农田、草地、森林等陆地生态系统中广泛分布一类功能型微生物,能够和90%以上的植物共生形成菌根。研究者发现,大部分入侵植物都能够与AMF形成共生关系,入侵植物与AMF的相互作用成为当今的研究热点之一。关于入侵植物和AMF相互作用的文章虽然很多,但单一进行野外调查或室内模拟实验往往具有孤立性和片面性。本文构建野外宏观实验-室内盆栽实验-培养皿微观实验体系,克服了过去单一进行野外调查或室内模拟实验的弊端,既避免了室内模拟实验的片面性又保证了实验的可控性,同时兼顾宏观和微观研究,是目前研究AMF与植物的相互关系比较系统和全面的研究方法本文选取中国南部造成严重生态危害的入侵植物南美蟛蜞菊(Wedelia trilobata),从野外宏观实验,室内盆栽实验和培养皿微观实验三个层次全面的研究了南美蟛蜞菊不同程度入侵对土壤中细菌和真菌群落的影响,AMF对南美蟛蜞菊表型可塑性和异速生长的影响,南美蟛蜞菊的菌根依赖性以及南美蟛蜞菊提取物和根系分泌物物对AMF孢子萌发以及菌丝发育的影响,旨在为探讨植物入侵机制,寻找防治植物入侵提供理论依据。本研究主要得到以下结论：1.在中国海南省的五个不同地点分别选取了不同南美蟛蜞菊入侵程度(未入侵、低度入侵和高度入侵)下的根际土壤,系统分析了土壤的理化性质和土壤微生物结构。研究结果显示：(1)低程度的南美蟛蜞菊入侵显著的增加了土壤的pH值,但是高度入侵并没有显著的改变土壤的pH值。(2)南美蟛蜞菊的入侵程度还显著的影响了土壤中K和Ca的含量,但是没有影响其他的理化指标。(3)南美蟛蜞菊低度入侵和高度入侵显著的增加了根际真菌群落的丰度,但是对根际细菌群落没有显著的影响。(4)南美蟛蜞菊的入侵还影响了参与土壤N循环的土壤微生物。这些由南美蟛蜞菊入侵导致的土壤理化性质和微生物群落的变化可能会进一步促进植物的入侵过程。2.为验证土壤微生物对入侵植物的作用,我们在海南岛设计实验来验证密度和AMF对典型克隆植物南美蟛蜞菊表型可塑性与异速生长的影响。通过是否在土壤中添加真菌抑制剂苯菌灵获得高AMF和低AMF两种AMF处理。植物的种植密度设置为25棵/m2和50棵/m2。在种植后的30,60,90和120d进行取样。结果显示：(1)高密度处理和高AMF处理会提高南美蟛蜞菊的菌根侵染率。(2)密度处理和AMF处理都影响了南美蟛蜞菊的表型可塑性和异速生长,并且AMF对其表型可塑性和异速生长的影响在不同的密度下是不同的。(3)低密度和高AMF处理增加了植物的总生物量,但是高AMF处理减少了地下生物量的分配。据我们所了解,本次研究是第一次对AMF和密度处理对入侵克隆植物表型可塑性与异速生长的研究。3.为了更好的研究AMF与南美蟛蜞菊的关系,必须尽可能排除杀菌剂和土壤中其他微生物的影响。我们在江苏大学温室内设计了土壤营养和AMF处理双因素的盆栽实验,验证南美蟛蜞菊在灭菌后的,不同营养条件的土壤中的生长状况和菌根依赖性。在2种土壤营养处理和5种AMF处理下,种植南美蟛蜞菊90d后发现：(1)南美蟛蜞菊可以被多种AMF侵染,并且在不同的营养情况下普遍具有较高的菌根侵染率。(2)分别单独接种幼套球囊霉(Glomus etunicatum)、摩西球囊霉(G. mosseae)、根内球囊霉(G. intraradices)以及同时接种三种AMF均增加了南美蟛蜞菊的茎节数目和地上部分的干重,同时显著的改变了南美蟛蜞菊地下生物量和地上生物量的比值。(3)植物的全根长受到土壤养分和AMF处理的影响。在低营养处理下的南美蟛蜞菊相对于高营养处理下的南美蟛蜞菊具有更长的全根长；未接种AMF的南美蟛蜞菊相对于接种了AMF的南美蟛蜞菊而言具有更长的全根长。(4)通过对接种AMF以及没有接种AMF的南美蟛蜞菊生物量的干重进行计算,我们发现南美蟛蜞菊的菌根依赖性较低,普遍在120%-170%之间。(5)相对于低营养处理而言,高营养处理下的南美蟛蜞菊根部具有更多的AMF孢子,尤其是对于G.i.菌和Mix处理,两种营养状况下的孢子数目差别尤其显著。4.为了更深入的从机制上解释南美蟛蜞菊与AMF之间的作用,我们设计了严格无菌的体外孢子萌发验证实验。首先利用AMFRi-T发根共培养体系繁殖获得了大量无菌的根内球囊霉(G. intraradices G.i.)孢子,然后通过培养皿中的体外孢子萌发实验研究南美蟛蜞菊的浸提物和根系分泌物的几种化学成份对G.i.孢子萌发率和菌丝生长的影响,进一步探讨南美蟛蜞菊对AMF的作用原理。这6种化学成份分别是：南美蟛蜞菊的根系分泌物的水相提取液和有机相提取液,从南美蟛蜞菊根系分泌物中通过GC-MS测定分离鉴定出的邻苯二甲酸二异丁酯、邻苯二甲酸二异辛酯,以及从南美蟛蜞菊全株浸提液中获得的3#纯化物、10#纯化物。实验结果发现：(1)南美蟛蜞菊根系分泌物的水相提取物对G.i.孢子萌发和菌丝生长没有显著的抑制作用和促进作用,平均萌发率都维持在70%左右；根系分泌物的有机相提取物在高浓度下抑制了G.i.孢子的萌发和菌丝生长。(2)邻苯二甲酸二异丁酯抑制了G.i.孢子萌发和菌丝生长；邻苯二甲酸二异辛酯对对G.i.孢子萌发和菌丝生长没有显著的影响。(3)3#纯化物在低浓度时对孢子萌发和菌丝生长没有显著影响,在高浓度时抑制了G.i.孢子萌发和菌丝生长。10#纯化物对G.i.孢子萌发和菌丝生长没有显著的影响。5.本课题从野外的取样调查入手,发现南美蟛蜞菊入侵显著的增加了根际真菌群落的丰度,揭示了土壤真菌群落在入侵植物的入侵过程中可能与土壤真菌有很强的相互联系。在接下来的试验田实验中发现使用苯菌灵抑制AMF和其他真菌会降低南美蟛蜞菊的生物量,说明AMF或其他真菌与南美蟛蜞菊的生长有密切的联系。然后设计菌根依赖性实验,验证南美蟛蜞菊生长过程中对AMF的依赖程度,研究结果显示虽然AMF可以在一定程度上促进南美蟛蜞菊的生长,但南美蟛蜞菊是一种低菌根依赖性的植物。为了进一步解释南美蟛蜞菊与AMF的作用机制,我们研究了南美蟛蜞菊根系分泌物和组织提取物对AMF孢子萌发和菌丝生长的影响,结果显示南美蟛蜞菊根系分泌物和组织提取物中某些成份会抑制纯培养的AMF孢子萌发与菌丝发育。综合以上研究结果,南美蟛蜞菊入侵会增加土壤真菌群落的丰度,但是南美蟛蜞菊的某些根系分泌物和组织提取物成份会抑制AMF的生长,并且AMF在南美蟛蜞菊生长中具有一定的促进作用,我们认为南美蟛蜞菊是不仅仅依赖与AMF共生而入侵新环境的,其入侵过程可能依赖于与其他真菌和环境的相互作用。更多还原

【Abstract】 Biological invasion has become one of the top five global environmental issues of the21st century. It not only does cause serious harm to the environment, but also caused incalculable damage to human health and economy. Scientists have put a lot of effort to the study of biological invasion mechanisms and prevention methods and found that there is a close link between microbial and plant invasion. Arbuscular mycorrhizal fungi (AMF) is a widely distributed fungi in farmland, grassland, forests and other terrestrial ecosystems, which play an important role in the regulation of nutrient, maintaining ecosystem stability and improving plant resistance. As AMF could forming a symbiotic relationship with more than90%plants, which including most of the exotic invasive plants, thus the relationship between invasive plants and AMF has become a hot research. Several studies have been conducted with common garden experiments or field experiments, however it is one-side and incomplete just to use one of the two experiments. We developed an experimental system which including field experiment, common garden experiment and in vitro experiment to avoid these problems. According to current reports, this experiment is a comprehensive and systematic research methods to study the relationship of AMF and invasive plants.Wedelia trilobata, an typical invasive plant caused serious ecological harm in south China, was studied in field experiment, common garden experiment and in vitro experiment in this paper. The the effects of W. trilobata invasions on rhizosphere microbial communities, the effects of AMF on W. trilobata phenotypic plasticity and allometry, the mycorrhizal dependency, the effect of extracts and root exudates of W. trilobata on AMF spore germination and mycelial growth was invested in this study, with the aim to provide a complete relationship between AMF and plant invasions. The main results were as follows:1. Rhizospheric soils of Wedelia trilobata with different degrees of invasion (uninvaded, low-degree, and high-degree using its coverage in the invaded ecosystems) were collected from five discrete areas in Hainan Province. Soil physicochemical properties and the community structure of the soil microorganisms were assessed. Low degrees of W. trilobata invasion significantly increased soil pH values whereas high degrees of invasion did not significantly affected soil pH values. Moreover, the degree of W. trilobata invasion exerted significant effects on soil Ca concentration but did not significantly change other indices of soil physicochemical properties. Low and high degrees of W. trilobata invasion increased the richness of the soil fungal community but did not pose obvious effects on the soil bacterial community. W. trilobata invasion also exerted obvious effects on the community structure of soil microorganisms that take part in soil nitrogen cycling. These changes in soil physicochemical properties and community structure of soil microbial communities mediated by different degrees of W. trilobata invasion may present significant functions in further facilitating the invasion process.2. An experiment was conducted in Hainan Island, to verify the effects of density and AMF on the typical invasive clone plant W. trilobata. Two AMF levels were obtained by applying a fungicide that suppresses the AMF naturally present in soil. Plant sowing density was set to25or50seedlings m-2. Samples were collected at30,60,90, and120d after planting. The colonization rate of W. trilobata was promoted by high-density and high-AMF treatments. Both density and AMF treatments affected the phenotypic plasticity and allometry of W. trilobata, and the effect of AMF on plant phenotypic plasticity and allometry varied at different densities. Low-density and high-AMF treatments increased plant total biomass, whereas high AMF treatment reduced belowground biomass allocation. To the best of our knowledge, this study is the first to test the effects of AMF and density on the phenotypic plasticity and allometry of invasive clonal plants. The results of this study could serve as reference to elucidate the mechanism of clonal plant invasion.3. A common garden experiment, which with two soil nutrition treatments and five AMF treatments, was conducted in Jiangsu University to study the mycorrhizal dependency of W. trilobata. Samples were harvested at120d after planting. W. trilobata could symbiosis with many AMF, and high infection rate was found with high and low soil nutrition treatments. Inoculated independently or together with Glomus etunicatum, Glomus mosseae, Glomus intraradices all increased the shoot biomass and significant changed the proportion of rate of root/shoot. Low nutrient and AMF inoculation reduced the length of plant roots. W. trilobata showed a low mycorrhizal dependency of120%-170%in this study. Relative to the low nutrient treatments, AMF grows more spore in the high nutrient treatments.4. An in vitro system using Ri T-DNA transformed roots and AMF was used to obtain the spore of G.i. first. Then the effects of root exudates and plant extractives of W. trilobata on spore germination and hyphae growth of G.i. were examined. Root exudates aqueous extracts did not affect the spore germination and hyphae growth, while the root exudates organic phase extracts inhibited germination rate and hyphae growth. Isobutyl phthalate inhibited spore germination and hyphal growth. Iso-octyl phthalate did not affect the spore germination and hyphae growth. Spore germination and hyphae growth were not affected by the10#purified composition of plant extractives, while were inhibited by3#purified composition of plant extractives.5. In the field experiment, we found that W. trilobata invasion increased the richness of the soil fungal community. The study results suggest that W. trilobata invasion may have close contact with soil fungi.Ini the common garden experiment, the use of fungal inhibitors affected the growth of W. trilobata. The following mycorrhizal dependency experiment, W. trilobata showed a low mycorrhizal dependency. And some root exudates and plant extractives of W. trilobata inhibited pure cultured AMF spore germination and hyphae growth.In summary, W. trilobata invade new environments may be not only dependent on AMF symbiosis but also associated with other soil fungi and environment factors.更多还原