【作者】 龚明贵；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2012， 博士

【摘要】 沙棘（Hippophae rhamnoides）和狼牙刺（Sophora davidii）是黄土高原地区常见的造林树种。本研究采用孢子密度、侵染率检测和变性凝胶梯度电泳（DGGE）技术相结合的方法，研究了陕西黄土高原不同纬度生态地区、不同季节、不同地貌区沙棘根际丛枝菌根（AM）真菌定殖状况和群落多样性，并分析了沙棘根际AM真菌侵染状况、群落多样性与气候、土壤因子之间的关系。基于DGGE条带基因序列系统发育树所形成的序列群，对风沙草原区、丘陵沟壑区和渭北旱塬区三个地貌区的沙棘根际AM真菌群落结构差异性进行分析。利用盆栽试验探讨了在正常水分和水分胁迫条件下，接种AM真菌对狼牙刺生长、光合作用、根叶形态、营养状况、次生代谢物、渗透调节物质和抗氧化酶的影响，以及接菌对狼牙刺根际土壤中土壤酶、球囊霉素和水稳性团聚体的影响。获得以下主要结论：1.不同纬度生态区沙棘根际AM真菌群落多样性及影响因素五个不同纬度生态区（由南向北依次为：永寿、富县、志丹、横山、榆林）年均温和年均降雨量总体变化趋势是由北向南依次增加，而海拔、土壤pH、速效磷、速效氮和有机碳变化无显著规律。沙棘根际AM真菌总侵染率和孢子密度总体趋势为由北向南依次递增，相关性分析表明，孢子密度与速效磷、总侵染率与中性-微碱性土壤pH呈显著负相关。不同纬度地区AM真菌分子多样性指数差异显著，物种丰富度、Simpsom’s和Shannon-Weiner指数富县最高，均匀度指数志丹最高，永寿物种丰富度、Shannon-Weiner和Simpsom’s指数最低，榆林均匀度最低。不同纬度地区沙棘根际AM真菌群落和环境因子间典范对应分析（CCA）可将AM真菌群落分为4个不同AM真菌种群，各种群对上述气候和土壤因子的响应各不相同。通过Monte Carlo检验表明，土壤中有效磷（P=0.025，F=2.62）和孢子密度（P=0.006，F=1.76）对AM真菌群落结构影响最为显著。2.沙棘根际AM真菌群落季节变化动态及影响因素沙棘根际土壤中土壤酶、球囊霉素、微生物量碳和氮随季节变化均具有一定规律性。不同季节沙棘根际AM真菌定殖状况差异显著，侵染率和孢子密度春季（4月）最低，秋季（10月）最高。相关性分析表明，AM真菌总侵染率与碱性磷酸酶、总球囊霉素、易提取球囊霉素、微生物量碳和量氮呈显著正相关，与过氧化氢酶呈显著负相关；孢子密度与总球囊霉素、易提取球囊霉素、微生物量碳和量氮成显著正相关，与过氧化氢酶呈显著负相关。不同季节AM真菌分子多样性指数除均匀度无显著差异外，物种丰富度、Shannon-Weiner和Simpsom’s指数随季节变化明显，以上三个多样性指数变化趋势均为从春季（4月）至秋季（10月）逐渐上升。主成分分析表明，不同季节间的AM真菌群落多样性存在极显著差异，10月AM群落典型变量值的变异（离散）最小，表明沙棘根际AM真菌在10月最为稳定。不同季节AM真菌群落和土壤酶、球囊霉素、微生物量碳和氮含量变化的CCA分析可将AM真菌群落分为3个不同AM真菌种群。通过Monte Carlo检验表明，微生物量碳（P=0.002，F=2.676）对AM真菌群落结构的季节变化影响最为显著。3.不同地貌区沙棘根际AM真菌群落结构分析黄土高原三个地貌区的沙棘根际AM真菌孢子密度和总侵染率变化趋势为：渭北旱塬区>丘陵沟壑区>风沙草原区。三个地貌区沙棘根际AM真菌DGGE图谱中含有34个条带类型，共计180个条带，其中风沙草原区条带数最少（53个），渭北旱塬区和丘陵沟壑区条带数基本相同（分别为63和64个）。三个地貌区沙棘根际AM真菌DGGE图谱聚类分析表明，渭北旱塬区AM真菌群落结构与风沙草原区、丘陵沟壑区差异显著，风沙草原区和丘陵沟壑区之间AM真菌群落结构差异不显著。从三个地貌区AM真菌DGGE条带得到25个AM真菌基因序列，这些序列通过系统发育树分析，可归纳到Glomus属的12个序列群，其中序列群Glo-C、Glo-D和Glo-G分别与G. indicum、G. constrictum和G.iranicum具有高度相似性，其他九个序列群均与Glomus属的未鉴定种具有高度相似性。12个序列群在三个地貌区分布频率具有一定差异性，Glo-A、C、D、E、G和K为三个地貌区的常见种，Glo-B、F、H、J和L为三个地貌区的偶见种，Glo-I为稀有种。4.干旱胁迫条件下AM真菌对狼牙刺生长及光合特性的影响干旱胁迫条件下，接种G. constrictum和G. mosseae狼牙刺芽干重提高27.5%和15.0%、根干重提高37.3%和17.3%、株高提高36.1%和23.4%、地径提高25.5%和10.2%；狼牙刺叶片净光合速率、最大光化学效率变化趋势为：G. constrictum> G. mosseae>未接种，三处理间差异显著；接种G. constrictum和G. mosseae的狼牙刺相对水分含量提高9.3%和6.1%、水分利用效率提高39.6%和16.2%、实际光化学量子产量提高25.0%和29.2%、光化学淬灭系数提高12.8%和12.8%，而胞间二氧化碳浓度降低29.1%和22.6%、水分饱和亏缺降低29.5%和19.1%，两接种处理间差异不显著；未接种、接种G.constrictum和G. mosseae三处理间狼牙刺叶片叶绿素相对含量、蒸腾速率无显著差异。5.干旱胁迫条件下AM真菌对狼牙刺营养、形态及根际土壤状况的影响水分胁迫条件下，狼牙刺叶片和根系中全N含量以及根系中全P含量变化趋势为：接种G. constrictum>接种G. mosseae>未接种，且三处理间差异显著；接种G.constrictum和G. mosseae叶片中全P含量提高15.4%和14.6%、叶长提高10.6%和7.7%、叶宽提高4.1%和10.0%、根尖数提高11.5%和10.4%，两接种处理间均无显著差异；狼牙刺叶面积、总根长和根表面积的变化趋势为：接种G. constrictum>接种G. mosseae>未接种，碱性磷酸酶活性和易提取球囊霉素含量变化趋势为：接种G. constrictum>接种G. mosseae>未接种，三处理间差异显著；接种G. constrictum和G. mosseae的狼牙刺根际总球囊霉素含量提高34.1%和42.6%、蔗糖酶活性提高40.5%和40.5%、脲酶活性提高61.6%和72.8%，两接种处理间均无显著差异；三处理间的狼牙刺根际过氧化氢酶活性无显著差异；接种G. constrictum和G. mosseae均显著提高了水稳性团聚体比例，其中>2mm土壤提高149.1%和13.5%、0.5-1mm提高23.8%和21.2%、0.25-0.5mm提高14.5%和8.2%、>0.25mm提高20.0%和12.5%。6.干旱胁迫条件下AM真菌对狼牙刺渗透调节物质和抗氧化能力的影响干旱胁迫条件下，根系中淀粉、叶片中游离氨基酸总量以及叶片和根系中黄酮、生物碱、多酚含量变化趋势均为：接种G. constrictum>接种G. mosseae>未接种，根系中游离氨基酸总量变化趋势为接种G. mosseae>接种G. constrictum>未接种，三处理间差异显著；狼牙刺叶片和根系中还原糖、可溶性糖、脯氨酸含量、叶片中淀粉含量以及SOD、CAT和POD活性均为接种AM真菌处理显著高于未接种处理，接种处理之间无显著差异；狼牙刺叶片中MDA、H2O2和O2-含量变化趋势是：接种G. constrictum <接种G. mosseae <未接种，三处理间差异显著；接种G. constrictum和G. mosseae的狼牙刺根系中MDA含量降低15.1%和18.6%、H2O2含量降低29.8%和24.9%、O2-含量降低20.4%和18.9%，两接种处理间无显著差异。更多还原

【Abstract】 Hippophae rhamnoides and Sophora davidii are both common afforestation species inthe Loess Plateau, China. Fine roots and rhizosphere soil of H. rhamnoides were collectedfrom five ecological areas, three geomorphologic regions and in different seasons in the LoessPlateau. Arbuscular mycorrhizal (AM) spore density, colonization rate and communitydiversity in the rhizosphere of H. rhamnoides were analysed by using microscopy andpolymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) respectively.The relationships between climatic, edaphic factors and colonization status, communitydiversities of AM fungi were analysed by using Correlation Analysis (CA) and CanonicalCorrespondence Analysis (CCA). Based on phylogenetic tree formed by AM18S rRNA genesequences of DGGE bands, the differences of AM community structure from threegeomorphologic regions (plain-gully, hilly-gully and desert-grassland) were also analysed andcompared. The effects of AM fungi on the growth and physiological performance of S. davidiiseedlings and on the soil status of S. davidii rhizosphere under well-watered and water-stressedconditions were also investigated.The results are as follows:1. AM community diversity in the rhizosphere of H. rhamnoides from five ecologicalareas and their related affecting factorsThe overall trends of average annual rainfall and mean annual air temperature wereincreasing from north to south in five different latitudes areas (from south to north in order as:Yongshou, Fuxian, Zhidan, Hengshan, Yulin), however changes of elevation, soil pH, Olsen-P,available N and organic carbon. had no significant regularities. The overall trends of totalmycorrhizal colonization and spore density in five latitudes areas were increasingsuccessively from north to south. Spore density had an obviously negative relationship withOlsen-P, and total colonization presented obviously negative correlation with soil pH. Thedifference of diversity indices in five different latitudes areas were obviously different.Species richness, Simpsom’s and Shannon-Weiner indices were highest in Fuxian and lowestin Yongshou, evenness index was highest in Zhidan and lowest in Yulin. AM fungalcommunities in five latitudes areas were clustered into four groups in the canonical correspondence analysis (CCA) biplot, and different groups had their various responses toenvironmental factors. Olsen-P (P=0.025，F=2.62) and spore density (P=0.006，F=1.76)showed significant correlations with AM fungal communities on the basis of Monte Carlo testof CCA.2. Seasonal dynamic of AM community diversity in the rhizosphere of H.rhamnoides and their related affecting factorsThe seasonal dynamic of soil enzyme, glomalin, microbial biomass C and N in therhizosphere of H. rhamnoides had certain regularities. The difference of AM colonizationstatus along easonal changes in the rhizosphere of H. rhamnoides was obvious, totalcolonization and spore density were highest in autumn (October) and lowest in spring (April).AM total colonization presented obviously positive correlation with alkaline phosphatase,total and easily extractable glomalin, microbial biomass C and N, and negative correlationwith catalase. Spore density had obviously negative relationships with total and easilyextractable glomalin, microbial biomass C and N, and negative relationships with catalase.Except evenness index, species richness, Simpsom’s and Shannon-Weiner indices wereobviously different along seasonal changes, and thses three indices were increasing graduallyfrom spring to autumn. AM community diversity in different seasons were obvious differentby using the method of principal component analysis (PCA), the typical variable value of AMcommunity in autumn was lowest, and it indicated that AM community in autumn was moststable in the three seasons. AM fungal communities in the three seasons were clustered intothree groups in CCA biplot, and different groups had various responses to environmentalfactors. Microbial biomass C (P=0.002，F=2.676) showed a significant correlation with AMcommunities on the basis of Monte Carlo test of CCA.3. Structural analysis of AM community in the rhizosphere of H. rhamnoides fromthree geomorphologic regionsThe trends of total colonization and spore density in different geomorphologic regions wereas follows: plain-gully> hilly-gully> desert-grassland.34band-types and total180bandswere found in the DGGE pattern of AM community in the rhizosphere of H. rhamnoides fromthree geomorphologic regions. Desert-grassland area had lowest band number (51),plain-gully and hilly-gully areas had almost same band number (63and64respectively). AMcommunity structure in plain-gully area had obvious diference with those in hilly-gully anddesert-grassland areas, AM community structure between hilly-gully and desert-grasslandareas was no obvious different. A total25gene sequences were found in the AM fungalDGGE gel from three geomorphologic regions. Based on the phylogenesis analysis, thesesequences were classified in12different sequence groups of Glomus genus. Glo-C, Glo-D and Glo-G havd a high degree of similarity with G. indicum, G. constrictum and G.iranicumrespectively. Glo-A, C, D, E, G and K were common species, Glo-B, F, H, J and L wereaccidental species, and Glo-I was rare species.4.The effects of AM fungi on growth and physiological performance of S. davidiiunder water-stressed conditionsUnder water-stressed conditions, G. constrictum-inoculated seedlings had higher dryweight of shoot and root, root length, plant height and stem diameter than non-mycorrhizalseedlings, G. mosseae-inoculated seedlings had higher dry weight of shoot and root, plantheight and stem diameter than non-mycorrhizal seedlings; The total trends of net photosyntheticrate and maximum quantum yield were as follows: G. constrictum> G. mosseae>non-inoculation, and there were significantly different in above parameters among the threetreatments; mycorrhizal seedlings had greater relative water content, water use efficiency,actual quantum yield and photochemical quenching coefficient, and lower intercellular CO2concentration and water saturation deficit than no-mycorrhizal seedlings, there were nosignificantly different in above parameters between G. mosseae and G. constrictum-inoculatedseedlings; there were no significantly different in relative chlorophyll content andtranspiration rate among three treatments.5. The effects of AM fungi on nutrition and morphology of S. davidii andrhizosphere soil status under water-stressed conditionsUnder water-stressed conditions, the total trends of total N in leaves and roots and of total P inroots were as follows: G. constrictum> G. mosseae> non-inoculation, and there weresignificantly different in above parameters among the three treatments; mycorrhizal seedlingshad greater total P than no-mycorrhizal seedlings, there were no significantly different in bothparameters between G. mosseae and G. constrictum-inoculated seedlings; the total trends of leafarea, root total length, root area, alkaline phosphatase activity and easily extractable glomalin content wereas follows: G. constrictum> G. mosseae> non-inoculation, and there were significantlydifferent in above parameters among the three treatments; mycorrhizal seedlings had greatertotal glomalin content, invertase and urease enzyme activities than no-mycorrhizal seedlings,there were no significantly different in above parameters between G. mosseae and G.constrictum-inoculated seedlings; there were no significantly different in hydrogen peroxideactivity among three treatments. Inoculation with G. constrictum and G. mosseae obviouslyimproved the proportion of water stable aggregates at>2mm,0.5-1mm,0.25-0.5mm and>0.25mm.6. The effects of AM fungi on osmotic adjustment substances and antioxidantcapacities of S. davidii under water-stressed conditions Under water-stressed conditions, the total content trends of starch in roots, free aminoacids in leaves, flavonoids, alkaloids, polyphenol in leaves and roots were as follows: G.constrictum> G. mosseae> non-inoculation, the total content trends of free amino acids were asfollows: G. mosseae> G. constrictum> non-inoculation, and there were significantly differentin above parameters among the three treatments. mycorrhizal seedlings had greater contentsof reducing sugar, soluble sugar and proline, activities of SOD、CAT and POD in root andleaves and content of starch in leaves than no-mycorrhizal seedlings, there were nosignificantly different in above parameters between G. mosseae and G. constrictum-inoculatedseedlings; the total content trends of the content of MDA、H2O2and O2-in were G. constrictum <G. mosseae <non-inoculation, there were significantly different among the three treatments;mycorrhizal seedling leaves had lower content of MDA、H2O2and O2-than no-mycorrhizalseedlings, there were no significantly different in three parameters between G. mosseae and G.constrictum-inoculated seedling roots.更多还原