毛竹Mariner-like element自主转座子的鉴定与生物信息学分析

【目的】Mariner-like element(MLE)转座子是一类在真核生物基因组内广泛分布的DNA类转座子，以“剪切-黏贴”模式在基因组中复制扩张，可以作为一类高效稳定的工具应用于转基因、基因功能研究等领域。为了探究MLE转座子在毛竹基因组中的分布规律，揭示MLE转座子在毛竹进化过程中的演化模式，本研究对毛竹MLE转座子进行了全基因组挖掘和分析。【方法】基于水稻、大豆和毛竹目前发表的MLE转座子序列，利用IRF、Blast和Fasta等工具，根据MLE转座子序列的特异性结构末端倒置重复序列(TIR)及其所编码转座酶的保守性，对毛竹基因组中的MLE转座子进行挖掘，从毛竹基因组中筛选具有完整结构的MLE转座子。分析所获得的MLE转座子的结构特征，根据完整MLE转座子的转座调控元件，从毛竹基因组中筛选出所对应非自主转座子，通过序列提取和比对，对自主与非自主转座子的分布特点以及非自主转座子结构缺失特点进行分析。【结果】共鉴定了2个完整结构的自主MLE转座子，PhV2MLE1A和PhV2MLE2A。PhV2MLE1A全长3 950 bp,编码414个氨基酸，TIR长度30 bp;PhV2M...     

一个毛竹典型LTR转座子的克隆、鉴定及进化分析

LTR反转录转座子普遍存在于植物界中。为了进一步了解LTR反转录转座子及探索毛竹中存在的可能具有潜在活性的LTR反转录转座子,研究借助毛竹基因组草图数据库,通过LTR-structure软件分析得到一个典型LTR反转录转座子Ph-LTR1,并对其进行鉴定及插入时间的估算,结果表明PhLTR1属于Ty3-gypsy类反转录转座子,其两端长末端重复序列同源性高达98.78%,插入时间约为47.9万年,是一个自身具备转座条件的年轻反转录转座子,并且极有可能具有潜在活性。采用同源性搜索方法搜索到52条Ph-LTR1家族元件,并对这些元件结构及其所在基因组的位置进行分析,结果表明PhLTR1家族元件中存在大量缺失的元件,且Ph-LTR1家族元件偏向分布于远离基因的区域。研究结果为后续具有潜在活性的LTR反转录转座子转化拟南芥实验奠定了基础,Ph-LTR1家族元件结构及进化分析促进了对于LTR反转录转座子在基因组进化角色的认识与理解。     

毛竹Stowaway-like MITEs转座子的分离与分析

微型颠倒重复序列(miniature inverted repeat transposable elements,MITEs)是一类对基因组进化和基因表达有重要调节作用的转座子。为分析MITEs在毛竹基因组中的分布特性,借鉴Fast Isolation by AFLP of Sequences Containing repeats(FIASCO)方法,首次构建Stowaway-like MITEs富集文库。随机挑取21个克隆,测序发现2个含有Stowaway-like MITEs序列(Stow-Ph1和Stow-Ph2),阳性率为9.52%。Stow-Ph1和Stow-Ph2的长度分别为260和258bp,具有Stowaway-like MITEs典型的末端颠倒重复序列(terminal inverted repeats,TIRs)和靶位点重复序列(target site duplication,TSD)。Stow-Ph1和Stow-Ph2与水稻的Stow-Os8(FJ266024)的同源性分别为46.4%和52.6%。Stow-Ph1和Stow-Ph2与水稻16类Stowaway-like MITEs的TI...     

毛竹NIP基因的分子特征及应答胁迫的表达模式

【目的】膜内在水通道蛋白(NIPs)是细胞膜上运输水分子和一些小分子物质的膜蛋白,在植物生长以及抵御逆境胁迫等方面发挥着重要作用。温度和水分是影响竹子生长发育的重要环境因子,研究温度和干旱胁迫条件下毛竹NIP家族成员的表达模式,以揭示其在毛竹应答胁迫中的功能。【方法】利用生物信息学软件对毛竹基因组中NIP家族成员基因及其启动子序列进行系统分析,基于转录组数据分析基因在毛竹不同组织中的表达模式,并用实时荧光定量PCR(q PCR)技术检测基因在温度和干旱胁迫条件下的表达特征,构建2个NIP基因的酵母表达载体,分析它们的表达对酵母抗干旱和盐胁迫能力的影响。【结果】在毛竹基因组中共获得14个编码完整NIP蛋白的基因(Pe NIP1-1—Pe NIP1-6、Pe NIP2-1—Pe NIP2-4和Pe NIP3-1—Pe NIP3-4),含有3～5个内含子;在Pe NIPs启动子区域中含有多种与胁迫、激素相关的调控元件; Pe NIPs编码蛋白的氨基酸长度为235～297 aa,相对分子量为24.03～31.84 k Da;亚细胞定位预测显示所有PeNIPs均定位于细胞质膜上。共线性分析表明,有12个Pe NIPs与水稻8个NIP基因存在27对片段重复,它们的非同义对同义取代比(Ka/Ks)均小于1,表明毛竹Pe NIPs经复制后主要经历了较强的纯化选择。系统进化分析表明,来自毛竹和其他5个物种的NIP蛋白可分为3类(Ⅰ、Ⅱ、Ⅲ),毛竹在各类中的成员依次为6、4和4个。PeNIPs共包含10个保守基序,其中基序1、2和4为PeNIPs所共有。转录组表达谱热图分析表明,Pe NIPs在不同组织中的表达存在一定的差异,如Ⅰ类的Pe NIP1-3、Pe NIP1-4和Pe NIP1-5在根中表达,而在笋中几乎不表达;Ⅱ类的4个Pe NIP2s以及Ⅲ类的Pe NIP3-1和Pe NIP3-2在根和笋中表达量较高。q PCR结果显示,随着胁迫时间的延长,4℃处理下8个基因上调表达,2个基因下调表达; 42℃处理下,2个基因上调表达,4个基因下调表达;而干旱胁迫下3个基因上调表达。表达Pe NIP1-1和Pe NIP2-2的酵母在添加山梨醇或Na Cl培养基上的生长优于对照。【结论】从毛竹中共鉴定出14个NIP家族基因成员(Pe NIPs),各基因的分子特征、表达模式均存在着一定差异,说明它们在毛竹生长发育的不同阶段和响应环境胁迫中可能发挥着不同的作用;在酵母表达的Pe NIP1-1和Pe NIP2-2能够一定程度上提高酵母的抗干旱和盐胁迫能力,说明它们在毛竹应对逆境胁迫中可能发挥着重要作用。     

毛竹PeIRX10基因在木聚糖合成中的功能

【目的】克隆毛竹木聚糖合成关键酶基因Pe IRX10,并对其进行表达分析和功能初探,为毛竹中木聚糖合成分子机制的探究提供理论依据。【方法】以毛竹为研究对象,根据毛竹基因组数据库中序列信息设计引物,通过RT-PCR克隆得到1个在模式植物拟南芥木聚糖合成中起关键作用的同源基因,命名为Pe IRX10;运用生物信息学的方法对其核苷酸序列及其编码的氨基酸序列进行分析;通过qRT-PCR对毛竹的根、茎、叶、花和笋5个部位进行组织表达特异性分析。利用Gateway克隆技术构建Pe IRX10的植物表达载体,通过蘸花法转化拟南芥irx10l(-/-)irx10(+/-)双突植株,经BASTA筛选及PCR植株表型鉴定获得具有irx10l(-/-)irx10(-/-)双突背景的转基因植株;通过表型观察、茎部切片甲苯胺蓝染色观察、细胞壁单糖成分分析以及木聚糖免疫定位观察探讨该植株中Pe IRX10基因的功能。【结果】从毛竹中克隆得到Pe IRX10(PH01004923G0080,http:∥www.bamboogdb.org/)基因的ORF序列,长度为1 251 bp,编码416个氨基酸,蛋白质的理论分子质量为46 821 Da,等电点为6.26;其氨基酸序列与其他植物的IRX10基因具有很高的相似性(80%),属于GT47家族;qRT-PCR结果表明,Pe IRX10基因在毛竹笋和茎中高度表达。过量表达Pe IRX10基因的转基因拟南芥植株可以互补于拟南芥IRX10双突植株irx10l(-/-)irx10(-/-),表现出正常的株高、茎粗以及叶片大小和数量;转基因拟南芥植株的茎部切片甲苯胺蓝染色观察发现,其维管束间纤维和木质部导管细胞的细胞壁厚度与野生型拟南芥相近;细胞壁单糖分析发现,互补植株木糖含量以及其他单糖(例如阿拉伯糖、半乳糖和葡萄糖等)的含量与野生型相近;木聚糖免疫定位分析表明,Pe IRX10基因的过表达可以使irx10l(-/-)irx10(-/-)双突植株中木质部和束间纤维中LM10信号几乎完全恢复。【结论】在irx10l(-/-)irx10(-/-)双突拟南芥植株中过量表达Pe IRX10基因可以使植株的表型和次生细胞壁缺陷恢复,表明毛竹Pe IRX10基因与拟南芥IRX10基因具有相似的功能,都在木聚糖的合成过程中发挥重要作用。     

毛竹SQUAMOSA启动子结合蛋白SBP家族基因的鉴定及表达模式分析

【目的】SBP家族基因编码的一类转录因子可识别并结合MADS-box基因SQUAMOSA(SQUA)的启动子,参与植物营养生长和生殖生长的调控。系统地鉴定和分析毛竹SBP家族基因,有助于理解SBP家族基因在毛竹营养生长和生殖生长过程中的调控作用,为毛竹SBP家族基因功能分析奠定基础。【方法】利用已公布的毛竹基因组数据,通过生物信息学方法鉴定毛竹SBP家族基因。利用MEGA 6.0、DNAMAN、psRNATarget等生物信息学软件获得毛竹SBP家族基因基本生物学信息。通过实时荧光定量PCR分析SBP家族基因在毛竹根茎叶、花序发育不同时期和实生苗受到高盐胁迫时的表达模式。【结果】在毛竹基因组中共鉴定到18条SBP家族基因,这些基因都有单一且高度保守的SBP结构域,其中有10条SBP家族基因具有miR156靶位点。毛竹SBP结构域有74个氨基酸残基,包括2个锌指结构域和1个NLS结构域。毛竹SBP转录因子进化关系、基序分布和DNA结构结果表明,进化关系相近的毛竹SBP转录因子的Motif和基因结构均具相似性。对不同物种来源的SBP家族基因进行进化分析发现,毛竹SBP家族基因与水稻SBP家族基因进化关系较近。对SBP基因在毛竹根茎叶中的相对表达量进行研究发现,PeSBP11和PeSBP16表达量分别在茎和根中最高。在花序发育P1-P4期(P1:花序形成初期;P2:小穗的分化;P3:颖花的分化;P4:小穗的生长),有6个基因(PeSBP1,PeSBP2,PeSBP3,PeSBP7,PeSBP15和PeSBP17)表达量较低,且无明显的表达量变化;7个基因(PeSBP5,PeSBP9,PeSBP10,PeSBP12,PeSBP14,PeSBP16和PeSBP18)在P1-P2期表达量上升,其中PeSBP10与PeSBP14的高表达延续到P3期;7个基因(PeSBP6,PeSBP8,PeSBP9,PeSBP10,PeSBP14,PeSBP16和PeSBP18)在P3期表达值较高;5个基因(PeSBP4,PeSBP5,PeSBP9,PeSBP13和PeSBP16)在P4期表达值较高。在高盐胁迫环境下,随着高盐胁迫处理时间的延长,PeSBP2,PeSBP3,PeSBP4,PeSBP8,PeSBP10,PeSBP11,PeSBP12与PeSBP14相对表达量逐渐上升。【结论】毛竹SBP家族基因在进化上非常保守,在进化过程中毛竹基因组可能并未出现特异的SBP基因。实时荧光定量PCR分析显示毛竹SBP家族基因参与了毛竹的营养器官生长,而且在毛竹开花过程中也发挥了重要的作用。在逆境盐胁迫条件下,有4个毛竹SBP基因的表达量发生了明显上调,表明毛竹SBP家族基因可能参与了毛竹对高盐的响应。     

模拟氮沉降对毛竹非结构性碳的影响

【目的】分析不同强度氮沉降对毛竹体内非结构性碳水化合物(NSC)含量的影响,为全面认识氮沉降对毛竹林生态系统的影响提供科学依据。【方法】在临安市毛竹林长期试验基地,采用典型选样法,设置12块20 m×20 m样地,包括低氮(30 kg·hm~(-2)a~(-1))、中氮(60 kg·hm~(-2)a~(-1))、高氮(90 kg·hm~(-2)a~(-1))和对照(0 kg·hm~(-2)a~(-1))4种处理,每处理设3个重复样地,处理32个月,探讨模拟氮沉降对1龄和3龄毛竹NSC含量的影响。【结果】可溶性糖和淀粉在NSC中所占比例分别为80%和20%,NSC主要以可溶性糖的形式存在;竹叶NSC含量最高,分别是枝和秆的1.32和1.27倍;3龄竹枝和秆NSC含量显著高于1龄竹(P0.05),竹龄对竹叶NSC含量影响不显著(P0.05);氮沉降显著降低毛竹叶、3龄竹枝和1龄竹秆的NSC含量(P0.05);氮沉降和竹龄的交互作用对毛竹枝、秆的NSC含量影响显著(P0.01),对毛竹叶影响不显著。【结论】氮沉降显著影响毛竹地上器官的NSC含量及其分配,且随竹龄而显著变化,高强度的氮沉降不利于毛竹NSC的积累和固碳。     

毛竹简易基因组文库的快速构建

以毛竹（Phyllostachys edulis）当年所发新叶为材料,用改良的CTAB法提取基因组DNA,通过超声波震断DNA,琼脂糖凝胶电泳回收大小为700bp-2000bp的片段,经T4DNA聚合酶末端补平,与经过牛小肠碱性磷酸酶处理的pUCl9载体连接,通过电转化转染受体菌XL-1 Blue,构建了毛竹基因组文库。文库容量为2．8×10^7,通过PCR验证,插入片段大小为750 bp-2000bp,插入效率为91％,符合基因组文库构建的要求,为毛竹特异功能基因的克隆和分子标记的研究奠定了基础。     

苹果反转录转座子Mdtcr5序列分析研究

该研究利用苹果反转录转座子Mdtcr5反转录序列为材料,在线通过BLASTn、BLASTx、DNAMAN软件、Clustal X1.8、Gene Doc和PLACE数据库进行分析,结果表明:Mdtcr5是典型的Ty1-copia组反转录转座子基因结构,其POL基因编码的蛋白按PR-INT-RT-RNase H顺序排列;Mdtcr5具有Ty1-copia组反转录转座子不同区域的特征基序,包括GAG区域的C-C-H-C基序、PR区域的D(T/S)G位点等;调控元件查找表明,从苹果基因组中分离的这个Ty1-copia组反转录转座子的3′端LTR序列包含多种逆境响应元件和激素应答元件,并且Mdtcr5序列在开放读码框(ORF)的完整性、LTR序列大小及结构上存在异质性。     

基于转录组序列的楠木SSR分子标记开发

【目的】通过对楠木转录组数据的分析,开发楠木SSR分子标记技术,为楠木遗传多样性分析和资源保护提供保障。【方法】将楠木高通量转录组测序获得的67 331条Unigene进行简单重复序列(SSR)位点挖掘和分析,并结合引物验证,初步证明其可行性。【结果】通过软件分析,共获得9 405个SSR位点,出现频率为13.97%,涉及序列数量为6 667条,发生频率为9.90%。SSR序列中包括166种重复基元类型,其中,单核苷酸、二核苷酸和三核苷酸为优势重复类型,SSR位点数分别为3 890(41.36%),2 885(30.68%),2 489(26.46%)。SSR位点重复次数差别较大,其中重复10次比例最高,SSR位点数为1 621(17.24%),其次是5次(1 549,16.47%)和6次(1 495,15.90%)。主导重复基元类型为A/T(40.67%),AG/CT(27.59%),AAG/CTT(11.29%)。运用多态性分析的方式初步验证了SSR位点在楠木标记中的可行性。同时,利用Primer 3.0进行引物设计,随机筛选18对进行验证,9对引物可以扩增出预期大小的条带。【结论】通过对楠木高通量转录组序列的SSR信息的研究,获得6 667条SSR序列,主导重复类型为A/T,AG/CT和AAG/CTT。同时随机挑选18对引物对SSR分子标记方法进行验证。本文对楠木SSR标记的研究将有助于楠木基因挖掘、分子标记育种和资源保护。     

Browsing Damage on Pine (Pinus sylvestris and P. contorta) by a migrating moose (Alces alces) Population in Winter: Relation to Habitat Composition and Road Barriers

In Scandinavia, moose (Alces alces L.) sometimes cause severe browsing damage to economically-important pine. Moose-vehicle accidents have spurred construction of fences along roads, and these may interfere with moose migration between summer and winter ranges, or the road alone may be a barrier. If this happens and moose build up along roads, landowners may suffer economically. Therefore, this study investigated whether roads, fences or other factors influence the use of young pine stands by moose. Eighty stands along roads in northern Sweden were evaluated in which individually-browsed branches were counted on 9972 pines. Moose browsing was not significantly related to birch (Betula pendula Roth, B. pubescens Ehrh.) density, nor did it differ between pines (Pinus contorta Douglas or P. sylvestris L.). However, increased pine density, site productivity and proximity to a highway were associated with increased browsing. Further large-scale studies are needed to understand moose habitat selection and the effects of roads.     

Effects of Root Freezing on the Physiology and Growth of Picea glauca,Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes

The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings were submitted to various frost temperatures during an artificial frost to induce different levels of root damage. Frost-damaged and control seedlings were placed in a greenhouse under high and low soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedling survival was reduced when root damage reached levels of 60-80%. Root systems of all three species showed partial to total recovery by the end of the experiment. In general, root freezing damage caused reductions in seedling growth, with these reductions becoming less significant over time. Root damage had little to no effect on black spruce and jack pine seedling physiology, while white spruce CO 2 uptake decreased with increasing root damage. Shoot nitrogen content of all three species decreased slightly with increasing root damage.     

Stand Structure in Pine,Spruce and Deciduous Forests: A Comparison Between Finnish and Russian Karelia

This study investigated the stand structure in pine, spruce and deciduous forests in the border district of Finland and Russia. A total of 46 mature forest stands was selected as pairs, the members of each pair being as similar as possible with respect to their forest site type, age, moisture and topography. The stands were then compared between the two countries by means of basal areas and number of stems. The proportions of dominating tree species were 2-12% lower, and correspondingly the proportions of secondary tree species higher, in Russian forests. The density of the forest stock was also higher in each forest type in Russia. The forests in the two countries differed most radically in terms of the abundance of dead trees. The amount was two to four times higher in Russian deciduous and spruce forests, and in pine forests the difference was 10-fold. The stand structures indicated that Russian coniferous stands, in particular, were more heterogeneous than intensively managed pine and spruce stands in Finland.     

Growth and Terminal Bud Formation in Picea abies Seedlings Grown with Alternating Diurnal Temperature and Different Light Qualities

The root collar diameter and the height:diameter ratio are of particular importance in container-grown seedlings where a high density in the containers may produce spindly seedlings. Temperature regimes and light quality are known to affect plant growth. The aim of this study was to identify responses in Picea abies (L.) Karst. seedlings grown with light providing different red:far-red ratios and under temperature regimes with alternating day (DT) and night temperature (NT) from negative (DT < NT) to positive (DT > NT) difference (DIF) between DT and NT. Experiments were conducted in controlled environment chambers and in a daylight phytotron. Only limited thermoperiodic responses appeared in P . abies seedlings with respect to seedling height and dry weight accumulation. The formation of terminal buds, however, was clearly delayed in seedlings grown at negative DIF. The results indicate a requirement for day extension light that is high in far-red, to prevent terminal bud formation under natural short-day conditions. An extended study should be conducted to clarify the minimum level of light intensity and the optimal light quality needed to prevent terminal bud formation under natural short-day conditions.     

CAD与建筑设计的关系

本文分析了CAD在设计中引起的正面和负面影响,并进行了系统的阐述,从而使设计者在应用中能够保持客观的态度。     

Natural Regeneration in Clear-cuts: Effects of Scarification,Slash Removal and Clear-cut Age

The effects of soil scarification (mounding), slash removal and clear-cut age on the natural regeneration in clear-cuts was evaluated using data from four sites in southern Sweden. The treatments were carried out during a good seed and establishment year for birch ( Betula pubescens Ehrh. and B. pendula Roth). Scarification had the strongest positive effect on the density of naturally regenerated seedlings, especially in birch, but also in pine ( Pinus sylvestris L.) and spruce [ Picea abies (L.) Karst]. Slash removal had a positive effect on birch density. No statistically significant effect of clear-cut age was found. In addition, no statistically significant interactions between clear-cut age and scarification or slash removal were found. The ingrowth of field vegetation was the fastest in areas that were not scarified, less rapid in areas scarified on old clear-cuts, and the slowest after scarification in fresh clear-cuts. In conclusion, it may be possible to control the density of birch during a good establishment year for birch. If birch is desired, the best combination of treatments is to remove the slash and scarify; otherwise, these treatments should be avoided.     

Risk Perception Among Non-industrial Private Forest Owners

Based on an enquiry, risk perception among non-industrial private forest owners is described in relation to climate change and forestry hazards. Of the respondents, 11% took action to remedy the effects of climate change. Out of a given set, hazards were ranked according to each respondent's experience of recent substantial financial loss to the estate and in relation to his or her willingness to make investments aimed at risk reduction. For each hazard, the respondent assessed the risk in four classes ranging from very high to negligible risk. Six hazards were considered most problematic in all three aspects: browsing damage, falling timber prices, damage by wind, spruce bark beetle, root rot and pine weevil. A majority of the respondents claimed to take action to reduce the risk associated with at least one hazard, while 35% did not know whether they did. Excluding climate change, the need for decision support was the largest in relation to damage by wind owing to a combination of perceived high risk and a high level of ignorance in relation to whether risk-reducing measures were taken.     

Interactions in decomposition and N mineralization between tropical legume residue components

In situ produced plant residues contain a mixture of different plant components of varying quality. To assess synergistic or antagonistic interactions occurring during the decomposition and mineralization of such mixtures, individual plant parts (stems, leaves, leaf litter and roots) or the mixture of stems, leaves and leaf litter of the agroforestry species pigeonpea (Cajanus cajan) or of crop residues of peanut (Arachis hypogaea) or of the weed hairy indigo (Indigofera hirsuta) were incubated in pots for 19 weeks. Periodically, remaining plant residues were sieved out (>2 mm), weighed and N content as well as soil mineral N determined. Above- and below-ground residues of peanut decomposed fastest and showed the largest N release in agreement with their high N concentration and low-acid detergent fibre (ADF) : N ratio. Hairy indigo was hypothesized to be of lower quality than pigeonpea because of its high-polyphenol content. However, it decomposed faster than pigeonpea, largely because of the higher N and lower lignin concentration of its components. Ranking of individual plant components for N mineralization resulted in the following pattern, leaves > leaf litter > roots > stems. In mixtures of the different plant components a similar species order in decomposition was obtained, e.g. peanut > hairy indigo > pigeonpea. The amount of N released from the mixture was dominated by stem material that comprised 46–61% of the mixture. The interactions in mixtures were relatively small for peanut (generally high-quality components) as well as for pigeonpea (low proportion of high-quality components, i.e. N rich leaf material). However, a positive interaction occurred during later stages of N mineralization in the mixture of hairy indigo as it had a significant proportion of N rich components and absence of highly reactive polyphenols. Thus, for plants with low to intermediate chemical quality attributes, manipulating plant composition (e.g. by varying harvest age, affecting stem and leaf proportions) will be important to obtain significant interactions during decomposition when its components are mixed.     

Pasture production and tree growth in a young pine plantation fertilized with inorganic fertilizers and milk sewage in northwestern Spain

The use of organic waste materials such as milk sewage as an organic fertilizer could have the dual advantages of organic-waste disposal and reduced dependence on inorganic fertilizers. The effects of fertilization with (1) conventional mineral fertilization, (2) milk sewage sludge at 40 kg N ha⁻¹ target rate and (3) no fertilization on pasture production and tree growth were examined in an experiment consisting of two pasture mixtures under a one-year-old Pinus radiata plantation with a density of 2500 trees ha⁻¹. The two pasture mixtures were: (1) Dactylis glomerata L. var. saborto (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹); (2) Lolium perenne L. var. Tove (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹). The experiment began in the spring of 1995 using a randomized block design with three replicates in Castro Riberas de Lea (Lugo, Galicia, north-western Spain). Plot size was 12 × 8 m², with a 1 m buffer strip between plots. Two-year data showed that fertilization with either material had a positive effect on pasture production, with no significant difference between the two fertilization treatments. Tree growth in the milk sewage sludge plot was significantly higher than in the control plots. Inorganic fertilization increased pasture production, but affected tree growth negatively. The results show that milk sewage sludge could be used as a fertilizer in silvo-pastoral systems. This revised version was published online in June 2006 with corrections to the Cover Date.