【作者】 班宜辉；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 本研究在调查陕西凤县铅硐山铅锌矿区不同程度铅锌污染样地植物丛枝菌根真菌（Arbuscular Mycorrhizal Fungi，AMF）和深色有隔内生真菌（Dark Septate Endophytes，DSE）分布的基础上，从DSE侵染率较高的沙打旺（Astragalus adsurgens）根中分离DSE，经过形态特征和分子鉴定、回接宿主和重金属耐性测定，筛选出具有较强Pb耐受性的菌株——柱孢顶囊壳（Gaeumannomyces cylindrosporus）。研究了该菌株对Pb胁迫的响应和不同pH、温度、Pb浓度、培养时间对该菌株Pb吸附特性的影响以及Pb胁迫下接种该菌株对玉米幼苗生长特性、光合特征和Pb吸收和转移的作用。获得以下主要结论：1.铅锌污染区AMF和DSE侵染特征对陕西凤县铅锌矿区4个不同程度铅锌污染样地植物根系的AMF和DSE资源调查发现，在调查的20科36种常见植物的55个根样中，49个（占总样品的89.1%）观察到AM典型结构，且Arum型菌根占优势；孢子密度与AMF侵染率之间没有显著相关性（P>0.05）；所调查根样中有46个被DSE侵染，占样品总数的83.6%，AMF和DSE同时侵染的根样有40个，占调查样品总数的72.7%，且DSE侵染率与土壤Zn含量显著正相关（P<0.05）。结果表明，AMF和DSE能够广泛存在于铅硐山铅锌矿区，尤其是DSE表现出较强的耐受性和适应性。2. AMF、DSE与土壤理化性质的关系对铅硐山尾矿区沙打旺（A. adsurgens）、山蒿（Artemisia brachyloba）、鹅绒委陵菜（Potentilla anserina）、芦苇（Phragmites australis）、博落回（Macleaya cordata）和狭叶米口袋（Gueldenstaedtia stenophylla）6种优势植物根系AMF和DSE侵染率、根际土中球囊霉素含量与土壤理化性质（Pb、Zn含量、pH、速效N、速效P、速效K、有机质）的关系进行研究。结果表明，AMF侵染率与土壤有机质含量和Zn在植物体的转运系数显著负相关，与速效P含量极显著负相关；DSE侵染率与Pb在植物体的转运系数呈显著负相关。冗余分析表明，土壤Pb含量是影响AMF群落组成的决定性因素；典范对应分析发现，土壤有机质是影响根内AMF群落组成的关键因子。3. DSE的鉴定和重金属耐性菌株的筛选从尾矿优势植物沙打旺（A. adsurgens）根内分离得到5株DSE，回接宿主后在根内形成有隔菌丝和典型微菌核结构，经形态特征和分子鉴定，5株DSE为柱孢顶囊壳（Gaeumannomyces cylindrosporus）、菊异茎点霉（Paraphoma chrysanthemicola）、甘瓶霉（Phialophora mustea）、沙门外瓶柄霉（Exophiala salmonis）和枝状枝孢菌（Cladosporiumcladosporioides）。对5株DSE的最低抑制浓度（MIC）和半致死浓度（EC50）等指标的测定结果表明，柱孢顶囊壳（G. cylindrosporus）对Pb有较强的耐受性，其最佳培养条件是25°C、120rpm和pH5.0，最佳碳源是甘露醇，最佳氮源是蛋白胨。4. G. cylindrosporus对Pb胁迫的响应不同浓度Pb胁迫对G. cylindrosporus菌落特征有影响，在0～1.0mg/mL的Pb²⁺浓度范围内，菌落颜色随着Pb浓度的升高先变黑后变黄；Pb胁迫引起菌丝卷曲，形成典型的菌丝圈，菌丝圈数量和卷曲程度与Pb浓度没有显著相关性。液体培养条件下，低浓度Pb胁迫下菌丝发生缠绕和粘连，胁迫加剧后，菌丝变形膨胀。在0～0.3mg/mL的Pb²⁺浓度范围内，G. cylindrosporus菌丝黑色素含量随着Pb浓度增加而逐渐升高，在0.4和0.5mg/mL的胁迫浓度下，菌丝黑色素含量下降，但仍分别为对照的4.1和3.5倍。低浓度Pb胁迫下G. cylindrosporus菌丝内可溶性蛋白和还原型谷胱甘肽含量、SOD和CAT活性均明显提高，高浓度Pb胁迫则引起下降。SOD活性与Pb浓度呈显著正相关，说明SOD在抵抗Pb胁迫的过程中发挥重要作用。5. G. cylindrosporus对Pb的吸附特性G. cylindrosporus对Pb有较强的吸附能力，菌丝在50⁵00mg/L的Pb²⁺浓度范围内，G. cylindrosporus菌丝吸附量随着Pb²⁺初始浓度的增加而提高；非活性菌丝的吸附量和吸附率均高于活性菌丝。当温度为30°C和pH5.0时，活性和非活性菌丝均达到最大吸附量，在80～140rpm的转速范围内，非活性菌丝对Pb的吸附量随着转速的提高而逐渐增加，活性菌丝在120rpm时达到最大吸附量。傅立叶红外光谱分析发现，G.cylindrosporus菌丝吸附Pb后C-O键、C=O键和N-H键的伸缩振动峰以及N-H键的面内弯曲振动峰的锋面积增大，表明菌丝糖类和蛋白质成分发生改变。6. G. cylindrosporus对植物生长和Pb吸收和转移的影响不同浓度Pb胁迫下接种G. cylindrosporus于玉米，苗高、地茎、生物量和根系活力都明显高于对照，高浓度Pb胁迫效果更为明显。接种的幼苗在1000mg/kg的Pb²⁺浓度下，相对含水量提高19.3%，水分饱和亏降低了8.5%，提高了叶片水分利用效率。在1000mg/kg的Pb²⁺浓度下，接种的幼苗叶绿素a、叶绿素b和总叶绿素含量分别为比对照提高66.9%、36.8%和59.5%；在500和1000mg/kg的Pb²⁺浓度下，接种的幼苗净光合速率分别为对照的2.14和4.11倍，蒸腾速率分别比对照提高76.2%和114.3%，胞间CO2浓度分别为对照的45.3%和39.0%；在1000mg/kg的Pb²⁺浓度下，接种幼苗的Fv/Fm值是对照的11.3倍。接种G. cylindrosporus后玉米对Pb的吸收量明显增加，500和1000mg/kg的Pb²⁺浓度下分别是对照吸收量的1.17和1.36倍。接种G. cylindrosporus使更多的Pb积累在植物根部，阻止了Pb向地上部的转移，降低了地上部的Pb含量，从而缓解了Pb对植物的毒害作用。盆栽条件下，G. cylindrosporus侵染率随着Pb²⁺浓度的增加而提高，与野外调查结果一致。更多还原

【Abstract】 In this study, we investigated the distributions of arbuscular mycorrhizal fungi （AMF）and dark septate endophytes （DSE） in different degree Pb-Zn polluted areas at QiandongshanPb-Zn mine, located in Feng county, Shaanxi province, and isolated several DSE strains fromthe roots of Astragalus adsurgens according to the DSE colonization. Based on morphologicalcharacteristics and molecular identification, the resynthesis experiment and the screening andselection of heavy metal-resistant DSE fungi, we finally chose the most resistantGaeumannomyces cylindrosporus as the object for further study. We studied the responses ofG. cylindrosporus to Pb stress, the effects of pH, temperature, Pb concentration and culturetime on the biosorption characteristics of Pb. In addition, we researched the effects ofinoculation with G. cylindrosporus on the growth characteristics, photosynthetic properties,and the uptake and translocation of Pb in maize seedings under Pb stress. The main results asfollows:1. Colonization characteristics of AMF and DSE in Pb-Zn polluted areasThe resources and distribution of AMF and DSE in four sampling areas in Pb-Zn mine,Feng county, Shaanxi province, were investigated.55root samples of36different plantsspecies in20families were analyzed, and the results showed that49samples （about89.1%ofthe total） were typically colonized by AMF, and Arum-type was more dominant than othertypes. No correlations were observed between the spore denstity and the AMF colonizationrate （P>0.05）.46root samples （about83.6%of the total） were found colonized by DSE.40samples were simultaneously colonized by AMF and DSE and accounted for72.7%of thetotal samples. DSE colonization rate was significantly positively correlated with the total Zncontent of soil （P<0.05）. These results indicated that AMF and DSE commonly occur inQiandongshan Pb-Zn mine, especially DSE has excellent heavy metal tolerance andadaptability.2. The relationships of AMF and DSE with soil chemistry propertiesWe explored the relationships of root colonization by AMF and DSE and GRSP contentin rhizosphere soils of six dominant plant species, including A. adsurgens, Artemisiabrachyloba, Potentilla anserina, Phragmites australis, Macleaya cordata, andGueldenstaedtia stenophylla, grown on Qiandongshan Pb-Zn mine tailings with soilchemistry properties, such as Pb and Zn content in soil, pH, available N, available P, available K, and organic matter. The results showed that AMF colonization was negatively correlatedwith organic matter content and the translocation of Zn in plant, and had extreme significantnegative correlation with available P content. DSE colonization was negatively correlatedwith the translocation of Pb in plant. Redundancy analysis revealed a significant effect of Pbcontent in soil on AMF community structure in soil samples, and canonical correspondenceanalysis of DGGE profiles showed that soil organic matter had significant relationship withAMF community structure in the roots.3. Identification and screening of heavy metal-resistant DSE fungiFive DSE isolates have been obtained from the roots of A. adsurgens, and all of themproduced melanized septate hyphae and microsclerotia in host plant roots after resynthesis.Based on morphological characteristics and DNA sequence analyses, the isolates wereidentified as G. cylindrosporus, Paraphoma chrysanthemicola, Phialophora mustea,Exophiala salmonis, and Cladosporium cladosporioides, respectively. The minimuminhibitory concentration （MIC） and the50%effective concentration （EC50） indicated that G.cylindrosporus was specifically resistant to Pb. Meanwhile, we found that the best culturingconditions for G. cylindrosporus were25°C,120rpm, and pH5.0, and the best carbon andnitrogen source were mannitol and peptone, respectively.4. The responses of G. cylindrosporus to Pb stressThere were remarkable changes in colony morphology in the presence of different Pbconcentrations. In the concentration range of0¹.0mg/mL of Pb²⁺, the colony color turnedblack firstly and then yellow with the increasing Pb concentration. There was twisting andlooping of individual hyphae and formation of intertwined hyphal strands under Pb stress,however, there was no obvious relationship between the number of hyphal coils or the extentof hyphal twisting and the concentrations of Pb. Under liquid culture condition, hyphae curledand twisted when Pb concentration was low, and obviously became thick and swollen hyphaewith the increase of Pb concentration.In the concentration range of0⁰.3mg/mL of Pb²⁺, melanin content of the hyphaegradually increased with the increase of Pb concentration. Melanin content declined when theconcentration of Pb²⁺was up to0.4and0.5mg/mL, however, still was4.1and3.5times thatof the control, respectively. The contents of soluble protein and GSH and the activities ofSOD and CAT in G. cylindrosporus were significantly higher than controls under low Pbconcentration, but higher Pb concentrations decreased these parameters. SOD activity wassignificantly positively correlated with Pb concentrations, thus, SOD may play an importantrole in abating the hazards of heavy metals.5. Adsorption characteristics of G. cylindrosporus for Pb We found G. cylindrosporus had strong ability to absorb Pb ions. When the Pbconcentrations increased from50to500mg/L, the Pb content of the hyphae absorbedincreased gradually, and the absorption content and rate of dead hyphae were higher than livehyphae. Under the condition of30°C and pH5.0, the live and dead hyphae reached themaximum absorption capacity. In a range of agitation speeds from80to140rpm, theabsorption capacity of Pb in dead hyphae gradually increased with the increase of agitationspeed, and the live hyphae reached the maximum absorption capacity at120rpm. Fouriertransform infrared spectroscopy showed that the stretch vibration absorption peaks of C-O,C=O and N-H and the bending vibration absorption peak of N-H were broadened, whichindicated that the sugar and protein content significantly changed after the adsorption of Pb.6. The effects of G. cylindrosporus inoculation on plant growth and the uptake andtranslocation of Pb in maizeG. cylindrosporus inoculation significantly enhanced the seedling height, basal diameter,biomass and the root activity of maize seedlings, the enhancements were more obvious underhigh Pb stress. Relative water content of leaves was increased by19.3%while watersaturation deficient was declined by8.5%after inoculated with G. cylindrosporus under Pb²⁺concentration of1000mg/kg, indicating that the water use efficiency of maize leaves hadbeen significantly improved. When the concentration of Pb²⁺was1000mg/kg, the content ofchlorophyll a, chlorophyll b and total chlorophyll under inoculation treatment were increasedby66.9%,36.8%and59.5%, respectively. Under the Pb²⁺concentration of500and1000mg/kg, Pn under inoculation treatment were2.14and4.11times that of the control, Tr wereimproved by45.3%and39.0%, and Ci under inoculation treatment were45.3%and39.0%ofthe control, respectively; Fv/Fmof leaves after inoculation was11.3times that of the control atthe concentration of1000mg/kg.Pb content of maize seedlings inoculated with G. cylindrosporus were1.17and1.36times that of the control under the Pb²⁺concentration of500and1000mg/kg, respectively.More Pb content were accumulated in the roots under inoculation treatment, indicating that G.cylindrosporus inoculation decreased Pb translocation from roots to shoots, and decreased Pbcontent of shoots, thus it alleviated the Pb toxicity to maize. In pot experiment, the maize rootcolonization by G. cylindrosporus increased gradually with the increasing Pb stress, whichwas consistent with the field investigation results.更多还原