【作者】 宋会兴；

【导师】 钟章成；

【作者基本信息】 西南大学 ， 生态学， 2007， 博士

【摘要】 喀斯特（Karst）地貌在地球上分布广泛，我国西南地区存在大面积的石灰岩地质。由于石灰岩地区持水力差，土壤贫瘠，石漠化严重，植被恢复异常困难。近来，生理生态学的研究表明，丛枝菌根能够造成水分进入、通过和流出宿主植物的速率发生改变，进而引起组织水分状态和植株生理状态的改变。尽管丛枝菌根影响宿主水分关系的详细机制并不清楚，但有研究者认为，丛枝菌根可能通过促进宿主对营养物质，间接地改变了宿主的水分关系。因此，丛枝菌根真菌在石灰岩地区的植被恢复中具有潜在的应用价值。本文的研究目的就是探讨丛枝菌根真菌对石灰岩地区适生植物种群生理生态学特征的影响，以及石灰岩地区土壤中丛枝菌根真菌的多样性特征。为此，设计如下的5个试验：试验1：土壤水分与丛枝菌根真菌接种对构树根系形态特征和分形特征的影响利用WinRhizo根系分析系统，在石灰土基质上对不同土壤水分条件下接种AM真菌Glomus mosseae对构树幼苗根系的形态特征和分形特征的影响进行了研究。结果表明，土壤水分状况和接种菌根真菌对构树幼苗生物量积累产生了显著的影响；构树幼苗根系干重、总长度、表面积、体积、根节数目、根系平均直径和分形维数表现出随着土壤含水量的降低而降低，接种G.mosseae则使得该参数比同等水分条件下显著升高；未接种植株比根长SRL表现为随着土壤含水量的降低而升高，接种G.mosseae则降低了同等水分条件下构树幼苗的比根长；土壤水分和接种G.mosseae同样也对根节平均长度产生了影响。由此作者认为，接种G.mosseae在不同土壤水分条件下促进了构树幼苗的生物量积累，这种促进作用在土壤干旱条件下尤为明显，表明AM菌根真菌G.mosseae提高了宿主构树的抗旱性；G.mosseae对宿主构树抗旱性的提高与G.mosseae对构树根系形态特征、分形特征的改变密切相关。在接种幼套球囊霉的研究中也得出相同的结论。试验2：接种丛枝菌根真菌对三叶鬼针草保护酶活性的影响本实验研究在石灰土基质上接种丛枝菌根真菌Glomus mosseae对不同土壤水分条件下三叶鬼针草保护酶系统的影响。试验设置正常浇水、中度干旱和重度干旱3个水分处理水平，比较不同水分条件下接种Glomus mosseae的三叶鬼针草植株与未接种植株的丙二醛（MDA）含量、可溶性糖含量、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、抗坏血酸过氧化酶（ASP）、过氧化物酶（POD）和多酚氧化酶（PPO）活性。研究结果表明，干旱胁迫条件下，三叶鬼针草叶片内MDA含量、可溶性糖含量显著升高，SOD、CAT、ASP、POD和PPO的活性都随土壤含水量的降低而升高。在正常浇水条件下，接种Glomus mosseae对三叶鬼针草叶片内MDA含量、可溶性糖含量没有影响，保护酶活性变化不显著；在中度干旱胁迫条件下，接种菌根并没有显著影响ASP与PPO活性，接种植株MDA含量在水分处理28d之前与未接种植株差异不显著，在处理35d时差异显著。可溶性糖含量、SOD和CAT活性则未接种植株显著高于接种植株。POD活性在水分处理前期（7d，14d和21d）接种植株与未接种植株差异不显著，在水分处理后期（28d、35d）未接种植株显著高于接种植株。在重度干旱胁迫条件下，MDA含量、可溶性糖含量、CAT活性未接种植株显著高于接种植株，对POD活性和PPO活性影响不显著，ASP活性则在21d前差异不显著，在28d和35d，未接种植株显著高于接种植株。保护酶系统变化表明，丛枝菌根有效地降低了干旱胁迫对三叶鬼针草的伤害程度，随着土壤含水量的严重亏缺和胁迫时间的延长，菌根对三叶鬼针草的保护作用逐渐减弱。试验3：干旱生境中接种AM真菌对三叶鬼针草光合特征的影响试验设置正常浇水（A）、中度干旱（B）和重度干旱（C）3个水分处理梯度，比较不同水分处理条件下接种丛枝菌根真菌Glomus mosseae和未接种三叶鬼针草之间光合色素含量、净光合速率、气孔导度、蒸腾速率、胞间CO₂浓度、表观量子效率、羧化效率、水分利用效率等特征的差异。结果表明，水分胁迫显著降低三叶鬼针草的净光合速率、气孔导度、蒸腾速率、表观量子效率和羧化效率。光合色素含量因干旱胁迫而升高，胞间CO₂浓度则是在水分处理的前期（7d）因干旱胁迫而降低，在干旱处理的后期随土壤含水量的降低而升高；水分利用效率则是中度胁迫的植株高于正常浇水处理组，而重度胁迫的植株则低于正常浇水处理组。在正常浇水处理条件下接种Glomus mosseae对三叶鬼针草光合参数没有产生显著性影响；在中度胁迫条件下，接种植株较未接种植株在水分处理的28d之前有更高的净光合速率、气孔导度、蒸腾速率、表观量子效率和羧化效率；在重度胁迫条件下，虽然净光合速率、气孔导度、蒸腾速率、表观量子效率和羧化效率接种植株高于未接种植株，但是二者并不显著。研究认为，干旱胁迫对三叶鬼针草光合作用的影响在水分处理的前期表现为气孔因素制约，在后期则主要是非气孔因素的影响；在正常浇水条件下接种丛枝菌根真菌Glomus mosseae对三叶鬼针草的光合作用没有显著性影响，在干旱胁迫条件下，丛枝菌根通过改善三叶鬼针草气孔导度和羧化效率等减弱干旱胁迫对植株的伤害，但是这种保护作用因为土壤水分的严重匮乏以及土壤干旱的时间延长而受到限制。试验4：丛枝菌根真菌对水分胁迫条件下三叶鬼针草（Bidens pilosa L.）开花的影响利用盆栽法研究了接种AM真菌在不同水分胁迫条件下对三叶鬼针草开花时间和单株开花数量的影响。结果表明，水分胁迫对三叶鬼针草的开花时间有提前的趋势，但是不显著；而对单株花序数量影响显著，表现为干旱造成三叶鬼针草单株花序数量升高。接种AM真菌之后，三叶鬼针草植株开花时间明显提前。非胁迫条件下接种摩西球囊霉Glomus mosseae的三叶鬼针草植株和接种幼套球囊霉Glomus etunicatum的植株分别比没有接种的植株始花时间分别提前了23d和13d；在水分胁迫条件下，接种植株较未接种植株开花时间也显著提前。在单株花序数量上，接种AMF的植株不同于未接种植株，在不同水分条件下显示出相同的变化趋势。结果表明，三叶鬼针草在干旱胁迫条件下单株花序数量增加，产生大量种子是其入侵的重要机制；而接种AM菌根使得三叶鬼针草开花期提前、开花数量增加，可能与AM真菌提高了植株对土壤中N、P的利用效率有关。试验5：石灰土上草本植物根系内丛枝菌根真菌多样性特征利用聚丙烯酰胺凝胶电泳（PAGE）和银染显影技术，比较了生长于石灰土和紫色土上的草本植物根系内的丛枝菌根真菌（arbuscular mycorrhizal fungi，AMF）群落的物种多样性特征。分属12个科的17种宿主植物分别种植于石灰土和紫色土上，120d后收获取样，通过CTAB法提取共生菌根内丛枝菌根真菌的DNA，用特异引物U1/U2扩增编码核糖体28S大亚基的rDNA部分序列，并进行聚丙烯酰胺凝胶电泳和银染染色。分析结果表明，生长于石灰土上的17种宿主植物根系内共发现AMF条带29种，平均每种宿主植物感染AMF 8.29种；生长于紫色土上的17种宿主植物根系内发现AMF条带24种，但是平均每种宿主植物感染AMF 9.47种。根系内的AMF包括特异的AMF类群和共有种群。聚类分析的结果表明，AMF对宿主植物的侵染具有科属的专一性，同时，这种专一性又受到土壤环境等外在条件的影响。总之，尽管丛枝菌根对宿主植物的保护作用在严重干旱和长时间干旱的条件下受到限制，但是接种丛枝菌根真菌在一定程度上缓解了由于干旱造成的对植物的伤害，因此，丛枝菌根真菌在石灰岩地区植被恢复过程中具有重要的应用价值。然而在本文中，石灰土中AMF种类并不低于紫色土，同时AMF对宿主抗旱性影响的机制尚不明确，因此丛枝菌根应用于实践还需要作进一步的研究。更多还原

【Abstract】 The distribution of Karst landscape is very general in the world, especially in the southwest of China. In limestone districts, ecological restoration is very difficult due to low water retention capacity, low fertility and growing rocky desertification. Several eco-physiological studies have demonstrated that the arbuscular mycorrhizal （AM） symbiosis often results in altered rates of water movement into, through and out of the host plants, with consequent effects on tissue hydration and plant physiology. Although the real mechanisms were not clear, some researchers conclude that AM symbiosis probably affected the water relations of host plants indirectly improved nutrition, especially P. So AMF might be useful in ecological restoration in limestone districts.The goal of this paper is to investigate the effects of AMF on eco-physiology of dominant plant species in limestone soil. And species diversity of AMF in limestone soil is the other goal of this paper. Following 5 experiments were conducted:Experiment 1:Effects of soil moisture and AM inoculation on root morphology and fractal character in Broussonetia papyrifera （L.） VentEffects of Glomus mosseae and water stress on morphology and fractal characteristic of root system in Broussonetia papyrifera （L.） Vent were studied in this experiment based on scanner-based image analysis. The results showed that drought and AM inoculation had a large, significant effect on plant development. Biomass, total length, surface area, volume, number of root nodes, average diameter and fractal dimension of root system in Broussonetia papyrifera （L.） Vent decreased with the reduction of soil moisture, while they were increased by AM inoculation. Special root length in non-AM plants increased when soil moisture was reducing, but it was decreased by AM inoculation. It was concluded that Glomus mosseae inoculation improved the drought resistance of Broussonetia papyrifera （L.） Vent, especially in moderate stressed conditions. And the contribution of AM symbiosis to plant to avoid the stress or to increase its tolerance might be the result of modification of root morphology and fractal character. The same conclusion was proved by Glomus etunicatum.Experiment 2:Influence of arbuscular mycorrhizae on activities of protective enzyme in Bidens pilosa L. under water stressThe effects of vesicular-arbuscular mycorrhizal fungus （AMF） Glomus mosseae on activities of protective enzyme in Bidens pilosa L. were investigated in pot culture under three water treatments: well watered （A） , middle drought treatment （B） and serious drought treatment （C） .The contents of malondialdehyde （MDA） and soluble sugar, superoxide dismutase （SOD）, catalase （CAT）, peroxide dismutase （POD）, polyphone oxidase （PPO） and ascorbic acid peroxidase （AsP） activities were compared between plants infected with Glomus mosseae （AM plants） and those not infected （Non-AM plants） at different water-treatments. The results showed that water stress significantly increased the activities of SOD, CAT, ASP, POD and PPO; the contents of MDA and soluble sugar were also increased by draught. There were no significant differences between AM plants and non-AM plants when they were well watered. But this was not the case in drought treatment. Soluble sugar content, SOD and CAT activities in non-AM plants were significantly higher than that in AM plants in middle drought treatment. And MDA content was not significantly different before 28d, but it was significant at 35d. POD activity in non-AM plants was markedly higher than in AM plants only at 28d and 35d in middle drought treatment, but AsP and PPO activities in leaves of Bidens pilosa L. were not affected significantly by AM infection. The content of MDA and soluble sugar, CAT activity were higher in non-AM plants than that in AM plants in serious drought treatment, but there were no significant differences in POD and PPO activity between AM and non-AM plants. And AsP activity was higher in non-AM plants than that in AM plants at 28d and 35d. It was concluded that draught stress did affect the protective enzyme system of Bidens pilosa L., and AM weakened the injury caused by draught. But this protection effect was limited by the heavier and longer draught.Experiment 3:Photosynthetic responses of AMF-infection and AMF-free Bidens pilosa L.to drought stress conditionsTo reveal the effects of vesicular-arbuscular mycorrhiza（AM） on the photosynthesis of Bidens pilosa L., three draught treatments were set: well watered （A） , middle drought treatment （B） and serious drought treatment （C） . Total chlorophyll content, net photosynthetic rate（Pn）, Stomatal conductance（Gs）, transpiration rate（Tr）, intercellular CO₂ concentration（Ci）, apparent quantum yield（AQY）, carboxylation efficiency（CE） and water use efficiency （WUE） were compared between plants infected with Glomus mosseae （AM plants） and those not infected （Non-AM plants） at different water-treatments The results showed that the Pn, Gs, Tr, AQY, CE of plants decreased significantly under draught stress, but total chlorophyll content increased under draught stress, while Ci decreased at the early stage of draught stress （7d）, and raised with the soil-water-content decreasing at the late stage of draught stress. The WUE of middle draught stress plants was higher than control, but the WUE of serious stressed plants was lower than control. The photosynthetic parameters of well watered plants were not significantly effected by Glomus mosseae infection. Especially before 28d of treatment, AM plants had higher Pn, Gs, Tr, AQY and CE than non-AM plants under middle draught stress, but they had not significant difference under serious draught stress. It was concluded that draught stress did effect the photosynthesis of Bidens pilosa L. because of Stomatal resistances at the early stage of treatment and nonstomatal limitation at later stage of treatment. The photosynthesis of well watered plants were not effected by Glomus mosseae infection. Under draught stress, AM improved the Gs and CE of plants to weaken the injury caused by draught, but this protection effect was limited by the heavier and longer draught treatment.Experiment 4:Effects of AM fungi on number and flowering time in Bidens pilosa （Compositae） under water stressThe effects of vesicular-arbuscular fungi Glomus etunicatum and Glomus mosseae on number and flowering time in Bidens pilosa L. under water stress were studied in potted culture. The results showed that contents of water in soil played a great role in the processes of flowering in Bidens pilosa L. and water stress brought flowering time forward, although it was not significant, while the number of flowers per plant was increased significantly by water stress. The process of blooming in Bidens pilosa L. was also influenced by vesicular-arbuscular fungi inoculation. The times of first blooming were significantly brought forward by vesicular-arbuscular fungi in different water status and plants infected by vesicular-arbuscular fungi had the same numeric dynamics of flowers. It was concluded that plant growing in water stress habitat had more flowers was its ecological adaptation of the explosive population in Bidens pilosa L.. And plants infected by vesicular-arbuscular fungi growing in different water stress habitats having the same numeric dynamics of flowers was the result of vesicular-arbuscular fungi improving drought tolerance in Bidens pilosa L..Experiment 5:Species diversity of AMF community colonized in roots of herbages in limestone and purple soilThe species diversity of arbuscular mycorrhizal fungi （AMF） in roots of various herbages were studied in this paper. Seventeen common herbages were selected as host plants of AMF colonization in this research and they were grown in limestone soil and purple soil, respectively. The plants were sampled at 120^th day after seedling and DNA of AMF in plant roots was extracted by CTAB method. The partial rDNA sequence encoding ribosomal 28S big unit was amplified with special primers U1/U2 for fungi, and PCR products were analyzed with PAGE silver staining method. Twenty-nine AMF species were found in roots of 17 host plant species in limestone soil and each host plant was colonized by 8.29 AMF species, while only twenty-four AMF species in purple soil and 9.47 AMF species in each host plant in purple soil. All the AMF species in roots were divided into two different groups, unique species group and common species group. Results of cluster analysis showed that colonization of AMF to host plants was family-specific, and the specificity was also affected by soil factors. The possibility of AMF used in ecological restoration of limestone area was also discussed in this paper.In general, AMF may be used in ecological restoration in limestone districts because AM symbiosis weakened the injury caused by draught, though this protection effect was limited by the heavier and longer draught treatment. The species of AMF in limestone soil in this study were not less than that in purple soil, which means more research work should be done in future.更多还原