【作者】 潘欣；

【导师】 张健； 朱天辉；

【作者基本信息】 四川农业大学 ， 森林培育， 2011， 博士

【摘要】 巨桉作为四川短轮伐期纸浆林和退耕还林的主要桉树树种之一,其人工林对立地条件的需求以及所产生的影响(如地力衰退),巨桉与伴生物种的相互作用,巨桉的遗传变异,巨桉对环境的作用,巨桉生态系统的稳定性,巨桉原料林与地方生态要求及经济发展的相关关系等已成为目前研究的重点问题。巨桉是典型的菌根营养型树种,菌根真菌在巨桉人工林地力维护及生态系统稳定性中发挥的作用已日益显现。为进一步开发和利用四川地区巨桉菌根资源,提高巨桉的生长量与抗逆性,合理规划和科学经营巨桉人工林,本研究通过对四川省主要巨桉人工林地大型真菌(外生菌根真菌)生物多样性及其生态特性的研究,深入探讨外生菌根与植物的共生关系,掌握本省巨桉外生菌根真菌生态分布规律,为维持巨桉人工林生态系统的稳定和持续发展提供一定的理论依据。在此基础上,研究巨桉林下马勃系统发育关系、巨桉组培苗外生菌根真菌的接种效应,为巨桉菌根真菌在“退耕还林”中的应用和菌类资源开发利用提供依据。主要研究结果如下：(1)通过野外调查和室内鉴定,在四川省巨桉人工林下共鉴定出大型真菌17种,隶属于7科,10属,其中外生菌根真菌7种,食用真菌11种,药用菌10种,食毒不明4种。硬皮马勃科及马勃科真菌占优势,银灰口蘑为四川省巨桉外生菌根真菌新记录种。(2)生态因子的研究表明,同海拔条件下,阴坡、半阴坡土层厚的地方,子实体发生较多；真菌子实体大量出现在海拔400-600m之间的巨桉林下,在林分郁闭度小于0.7时,马勃状硬皮马勃、橙黄硬皮马勃为林下主要的大型真菌类群,随着郁闭度增加,林下大型真菌的种类逐渐增多,草地横模马勃、多根硬皮马勃形成次优势菌群,林龄对外生菌根真菌分布的影响较小。土壤样品的测定表明,四川省巨桉林下大型真菌喜好酸性环境,其周围土壤pH值在4.2-5.8之间,土壤中的有机质、N、P、K养分含量均明显高于对照土。大型真菌丰富度以乐山井研最高,乐山沙湾、眉山彭山及其丹棱次之；均匀度指数与丰富度不同,其变化情况为宜宾高县>眉山彭山>乐山沙湾>乐山井研>眉山丹棱>泸州泸县>雅安名山；Simpson指数最大的是眉山彭山,大于0.6,其次是眉山丹棱、乐山井研和沙湾；而多样性指数则是眉山地区最高,接近2.0,井研、丹棱、乐山次之,均在1.2以上。(3)对巨桉林下马勃类真菌进行鉴定、组织培养及ITS序列结果鉴定,得到6种马勃纯培养物。使用通用ITS序列引物对马勃总DNA进行扩增,测序后分析其序列特征。将序列在Genbank中注册,序列号如下：大秃马勃C. gigantea (HM237179)、白刺马勃L. wrightii (HM237177)、草地横隔膜马勃V. pretense (HM237178)、橙黄硬皮马勃S. citrinum (HM237176)、金黄硬皮马勃S. aurantium (HM237174)、大孢硬皮马勃S. bovista (HM237175)、多根硬皮马勃S. polyrhizum (HM237173)及三个待定种硬皮马勃Scleroderma sp.11-1 (HM237170)、Scleroderma sp.2-2 (HM23717)和Scleroderma sp.5-2 (HM237172)。ITS区序列特征分析表明,马勃种类多样性及差异主要体现在18S rDNA的5’端与28S rDNA的3’端之间的ITS(ITS1、ITS2)中度保守区域。构建基于ITS1-5.8S rDNA-ITS2区全序列的系统发育树,发现硬皮马勃属Scleroderma、豆包菌Pisolithus亲缘关系较近,秃马勃属Calvatia、马勃属Lycoper don、横膜马勃属Vascellum之间亲缘关系近；三个待定种待定种硬皮马勃Scleroderma sp.11-1、Scleroderma sp.2-2和Scleroderma sp.5-2与金黄硬皮马勃(S. aurantium)在同一分支中。利用ITS1-5.8SrDNA-ITS2标记建立属间发育树支持度较高,ITS2区信息可用于分析属内差异。(4)采用多因素混合水平正交试验设计,对巨桉无性系苗进行人工接种外生菌根真菌,菌根处理组合的侵染率均在50%以上,对巨桉生长的各项指标的测定表明,无论是单接种还是混合接种,苗高、地径、鲜重及地上部分地下部分比值均值均高于复合肥处理或无菌处理,但不同接种处理间存在一定的差异。对菌根化巨桉苗木的叶绿素测定结果表明,接种后120d,不同处理组合对叶绿素a、叶绿素b及叶绿素总量的影响有一定的差异,总体变化趋势为：菌根处理>复合肥处理>无菌处理。不同处理组合在生长季节的不同时期,对巨桉苗木光合作用的影响力具有不一致性,且光合速率与胞间CO2浓度成负相关,与蒸腾作用正相关,气孔导度与蒸腾速率、胞间CO2浓度正相关。菌根化处理的苗木体内的N、P、Ca、Mg元素含量要高于无菌处理,对K元素含量的影响不显著。接种菌根可以提高苗木可溶性糖含量,使游离氨基酸含量保持在相对稳定且较高的水平,但与还原糖及总蛋白含量无明显相关性。(5)菌根化巨桉苗木根际土壤中脲酶保持较高的活性,各处理均具有酸性、碱性及中性磷酸酶活性,其中酸性磷酸酶活性较高,均高于中性磷酸酶和碱性磷酸酶的活性,且酶的活性与时间相关,普遍表现为7月份土壤磷酸酶活性普遍高于5、9月。接种外生菌根后土壤中微生物数量与酶之间有着密切关系,5月份,土壤微生物数量增多,酶的分泌增加,随着时间的变化,7、9月份后,随着微生物数量的变化,酶活性虽高,至7月后达到最高,但已表现出下降趋势。菌根化巨桉苗木根际土壤中发现真菌8种,细菌19种(含固氮菌8种),放线菌6种,接种后60d,真菌、细菌与pH显著负相关,有机质与真菌、细菌、放线菌显著相关；接种后120d,速效N与固氮菌极显著相关,有机质与真菌、细菌、放线菌及固氮菌数量极显著相关;接种后180d,pH与真菌数量间极显著负相关,但有机质、全氮等其它指标与微生物数量的相关性发生一定变化。更多还原

【Abstract】 Eucalyptus grandis is the main kind of Eucalyptus spp. for pulpwood forest and returning the grain plots to forestry. Nowadays research on Eucalyptus grandis in the fields of man-made forest demand for land capability, interaction between Eucalyptus grandis and associated species, genetic variation, stability of ecosystem, correlativity of ecological demands and economic development, etc. have been the key items.Eucalyptus grandis is the typical mycorrhiza nutritional type tree, and it plays an important role in man-made forest’s land capability maintenance and stability of ecological system. To develop and utilize mycorrhiza resource, improve growth and stress resistance of Eucalyptus grandis, our study focused on diversity and ecosystem characterization of macromycetes in the Eucalyptus grandis inforest, inquiring into relations between mycorrhiza and Eucalyptus grandis, grasping ectotrophic mycorrhiza regularities of distribution of Eucalyptus grandis and providing theory evidence of stabilizing man-made forest ecological system and sustainable development.On the base of the study, we can provide evidence in the application of mycorrhiza and utilization of fungi resource. The results were as the follows:(1) The article surveyed the mycorrhizal fungi of Eucalyptus grandis in Sichuan. The results showed that there are 17 species belong to 10 genus under the Eucalyptus grandis inforest, among which seven species were ectotrophic mycorrhiza, eleven edible fungi, ten medicindl fungi and four others. Vascellum pretense and Scleroderma areolatum were the predominant fungus, and Trichlloma argyreum was the new record species in Sichuan provience.(2) The macrofungi resources were surveyed termly from the Eucalyptus grandis inforest to know the diversity of the macrofungi and its ecological distribution. The results showed that there are 17 species belong to 10 genus under the Eucalyptus grandis inforest, Vascellum pretense and Scleroderma areolatum were the predominant fungus, and five species of Scleroderma, Pisolithus tinctorius and Tricholoma argyreum were ectomycorrhizal fungi. The living conditions of macrofungi were as follows:they grown in the areas of schattenseite and semi-nouthward, the altitude was between 400 and 650m, crown density exceeded 0.7, pH was between 4.2 to 5.8, and the content of the organic matter, N, P and K was higher than the contrast. The diversity analysis revealed that the macrofungus in Shawan and Jinyan of Leshan, Pengshan and Danling of Meishan were copious, and they had a good degree of homogeneity, dominance and diversity. The macrofungus from the Eucalyptus grandis inforest contribute to the quality of forestry and stability of the man-made forest ecosystem. Simpson index showed that it was highest in Pengshan (>0.6), then Danling, Jinyan and Shawan. The diversity index also showed that it was highest in Meishan (about 2.0), and the index was above 1.2 in Jinyan, Danling and Leshan.(3) The mycelium was cultivated from the fruiting body of puffballs which collected from the Eucalyptus grandis inforest, and the genomic DNA was extracted as template. Six puffballs pure culture were obtained, and the ITS sequences were also submitted to Genbank. The accession numbers were as belows, Calvatia gigantea (HM237179), Lycoperdon wrightii, Vascellum pretense (HM237178),S. citrinum (HM237176),S. aurantium (HM237174),S. bovista (HM237175),S. polyrhizum (HM237173), Scleroderma sp.11-1 (HM237170), Scleroderma sp.2-2 (HM23717) and Scleroderma sp.5-2 (HM237172), respectively.Subsequently, the internal transcribed spacer (ITS) region in rDNA gene was amplified, cloned and sequenced. The phylogenetic tree of puffballs was first built based on different part of ITS region. The results showed that six species of puffballs was cultivated successfully using PDA medium after comparing the sequences of puffballs fruiting body and mycelium ITS regions. The rDNA-ITS region of different puffballs was 607 bp to 766 bp in length. Analysis of phylogenesis revealed that the genetic relationship between the Scleroderma and Pisolithus, Calvatia, Lycoperdon and Vascellum was close. The three undetermined species, Scleroderma sp.11-1, Scleroderma sp.2-2 and Scleroderma sp.5-2, could probably be S. aurantium. The sequence of ITS1-5.8S rDNA-ITS2 can used as an evolutionary marker between different genus, and ITS2 as an evolutionary marker in genus. Thus, this work laid the foundation of studying the ectotrophic mycorrhiza, the mechanisms of action, the classification of puffballs and its exploitation and utilization.(4) The ectotrophic mycorrhiza fungi were inoculated into culture clone of Eucalyptus grandis using multiple factor orthogonal test. The results showed that infection of treatment using ectotrophic mycorrhiza fungi was above 50%. The value of seedling height, caliper, fresh weight and the overground/underground ratio were higher than compound fertilizer or sterile treatments.The chlorophyll content measurement showed that the content of Chlorophyll a, Chlorophyll b and total Chlorophyll varied in different treatments. The variation trend was as follows:ctotrophic mycorrhiza treatment> compound fertilizer treatment> sterile treatment treatment. However, it was not same in photosynthesis index. It was found that negative correlation between photosynthetic rate and CO2 concentration, positive correlation between photosynthetic rate and transpiration. Besides, the positive correlation between stomatal conductance and transpiration rate, CO2 concentration was observed.The N, P, Ca and Mg contents were higher than sterile treatments in nursery stock, but it was not significant in K content. The soluble sugar in nursery stock can be increased after inoculation, moreover, free amino acid content can also stabilized in high degree, but there was not significant correlations between reducing sugar and total protein content.(5) Soil urease activities of E. grandis rhizosphere was high, there are activities of phosphate enzyme in the each treatments of acidic, medium and alkali, the acid phosphate enzyme activity was higher than activities of acidic, medium phosphate enzyme, and the enzyme activities had correlation with time, the results generally showed that soil phosphate enzyme activity in July were higher than that in May and September. The number of soil microbes had close relation to enzyme after vaccinating Ectomycorrhiza, and the number of soil microbes and secretion of enzyme increased in may, but soil microbes decreased though secretion of enzyme increased after July and September, secretion of enzyme reached its highest level after July, but had the decreased trend.Eight funguses,19 bacterial (eight nitrogen-fixing bacteria) and six actinomycete were found in the soil after vaccinating Ectomycorrhiza of E. grandis seedling. A negative correlation was found between fungus, bacterial and pH, and fungus, bacterial, actinomycete had positive correlation with organic matter after 60 days. Significant correlation was found between Available N and the number of nitrogen-fixing bacteria, organic matter had significant correlation between fungus, bacteria, actinomycete and the number of nitrogen-fixing bacteria respectively after 120 days. A negative correlation was found between pH and the number of fungus, but the relationship of correlation between organic matter, TN indicators and number of microbes changed after 180 days.更多还原