松材线虫(Bursaphelenchus xylophilus)耐久型幼虫(LIV)携带的香茅醇假单胞杆菌(Pseudomonas citronellolis)分离与鉴定

[目的]目前有关松材线虫与伴生细菌的关系及伴生细菌的病原作用是松树枯萎病研究的重点。为了揭示松材线虫与伴生细菌之间存在的密切关系,作者对松材线虫L_IV幼虫携带的细菌进行了分离鉴定。[方法]根据培养性状和16S rDNA序列同源性以及系统发育学等方面进行分析鉴定。[结果]确定L_IV幼虫携带的是香茅醇假单胞杆菌（Pseudomonas citronellolis）,携带率为100%;每条L_IV幼虫携带量在1.4×10⁵~4.5×10⁵。L_IV幼虫生活在松褐天牛体内,是引起松材线虫病侵染流行的唯一虫态;新发现的香茅醇假单胞杆菌能分解纤维素及降解或合成萜烯和酚类化合物。[结论]L_IV幼虫携带香茅醇假单胞杆菌的发现,揭示了松树、松褐天牛、松材线虫、细菌同为一体的紧密关系,并为揭示松树枯萎病机制提供了一种新病原和重要的研究思路。     

Characterization and role of glucose-6-phosphate dehydrogenase of Populus suaveolens in induction of freezing resistance

Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) was purified from the leaves of 8-week-old Populus suaveolens cuttings. The enzyme activity in the absence and presence of reduced dithiothreitol (DTT_red) was determined. The results show that the G6PDH activity is not inactivated by pre-incubation with DTT_red, indicating that the purified enzyme probably presented in cytosol of P. suaveolens. The catalytic characteristics and kinetic parameters of cytosolic G6PDH purified from P. suaveolens cuttings were also studied. The results show that G6PDH is characterized by K _m value of 360 μmol·L⁻¹ for G6P and 16 μmol·L⁻¹ for NADP, a pH range of 7.3-8.9, and the maximum activity around pH 8.2. The enzyme activity is inhibited by various metabolites such as NADPH, NADH, GTP, UTP, ATP, AMP, ADP, CoA, acetyl CoA, fructose-6-phosphate (F6P), erythrose-4-phosphate (E4P), ribose-5-phosphate (R5P) and 3-phosphoglycerate (3-PG) (all at 1 mmol·L⁻¹ except for NADPH and NADH) to different extents. NADPH is the most effective inhibitor of enzyme activity, with an inhibition of 72.0%. The addition of metal ions such as MgCl₂, CaCl₂ and KCl (all 1.0 mmol·L⁻¹) to the standard reaction mixture has no remarkable influence on the cytosolic G6PDH activity. However, CdCl₂ (1.0 mmol·L⁻¹) causes high inhibitory effect on the enzyme activity. To explore the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation, the changes in the cytosolic G6PDH activity and freezing resistance (expressed as LT₅₀) of P. suaveolens cuttings during freezing acclimation at −20 °C were investigated. The results reveal that freezing acclimation decreases LT₅₀ of cuttings, and increases the activity of cytosolic G6PDH compared with control ones, while 2 d of de-acclimation at 25 °C result in a decrease in cytosolic G6PDH activity, and caused an increase in LT₅₀. Furthermore, the change in cytosolic G6PDH activity is found to be closely correlated to the degree of freezing resistance of cuttings during freezing acclimation. It is suggested that cytosolic G6PDH may be involved in the induction of freezing resistance of cuttings. [Supported by the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

Comparative study on antioxidative system in normal and vitrified shoots of Populus suaveolens in tissue culture

To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H₂O₂, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P. suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period. While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H₂O₂, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H₂O₂. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the large accumulation of H₂O₂ and the enhancement of membrane lipid peroxidation, which might be the main cause leading to the occurrence of vitrifying shoots of P. suaveolens in tissue culture. [Supported by National Natural Science Foundation of China (Grant No. 30271093) and the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

山核桃花芽总RNA提取方法研究

以山核桃的花芽为材料,利用改良CTAB法、异硫氰酸胍法和改良热酚法提取其总RNA,并对RNA产率、纯度、电泳图谱和RT-PCR进行分析,结果表明:改良CTAB法明显优于其它两种提取方法,用它提取的28S rRNA、18S rRNA条带清晰、稳定,无降解,OD260/OD280值保持在1.9～2.0,产率相对较高;经RT-PCR检测发现,改良CTAB提取的RNA能被成花基因特异引物扩增出清晰的条带,说明该法完全适合于山核桃花芽总RNA的提取,并可用于Northern杂交、cDNA文库构建等分子生物学操作.     

松材线虫Bxpel2基因的克隆及RNA干扰载体构建

[目的]为了构建松材线虫(Bursaphelenchus xylophilus)果胶酶Bxpel2基因干扰载体.[方法]通过Trizol 法提取松材线虫总RNA,反转录合成cDNA,设计带T₇启动子的果胶酶Bxpel2基因引物,以cDNA为模板扩增出果胶酶Bxpel2基因片段,连接到RNA干扰载体,再以干扰载体为模板,PCR扩增出目的片段后进行测序鉴定,合成果胶酶Bxpel2基因双链RNA(dsRNA),采用RT-PCR检测松材线虫Bxpel2基因干扰后的表达情况.[结果]表明:1)提取的松材线虫总RNA完整性好,无降解;2)成功克隆出松材线虫果胶酶Bxpel2基因片段(790 bp)并将其连接至pMD19-T载体;3)以RNA干扰载体为模板合成dsRNA,浓度分别为1.313 mg·mL^-1和1.152 mg·mL^-1;4)RT-PCR结果显示,松材线虫经过dsRNA干扰后,Bxpel2基因表达基本受到抑制.[结论]成功构建松材线虫果胶酶Bxpel2基因干扰载体,为进一步研究Bxpel2基因在松材线虫致病过程中的作用和功能奠定了良好的基础.     

Potential of the entomopathogenic nematodes Steinernema feltiae , S. weiseri and Heterorhabditis bacteriophora for the biological control of the sugar beet weevil Bothynoderes punctiventris (Coleoptera: Curculionidae)

The sugar beet weevil, Bothynoderes punctiventris Germ. (Coleoptera: Curculionidae), is one of the most damaging pests of sugar beets in Turkey and has been traditionally controlled by application of large amounts of insecticides. The current study used laboratory microcosms to evaluate the possibility of using entomopathogenic nematodes as an alternative control method. The nematodes Steinernema feltiae (strain TUR-S3), Steinernema weiseri (BEY) and Heterorhabditis bacteriophora (TUR-H2) had previously been isolated from Turkey. Nematode-induced mortality generally increased as soil temperature increased from 15 to 25°C but decreased when larvae were located deeper in soil. Heterorhabditis bacteriophora caused the highest larval mortality at 25°C at all depths (5–20 cm). Steinernema feltiae and S. weiseri were more effective than H. bacteriophora at 15°C at all depths. Increasing the application rate of infective nematode dauer juveniles (DJs) affected the number of DJs that penetrated each insect larva and the number of DJs produced per insect. The highest production of DJs per larva occurred at application rates of 50 DJs/larva for S. feltiae and S. weiseri and 100 DJs/larva for H. bacteriophora. Reproduction decreased again at higher application rates. Heterorhabditis bacteriophora had the highest reproductive capability. The results indicate that S. feltiae and S. weiseri might be more effective against this pest early in the growing season when soil temperature is low and that H. bacteriophora might be more effective later in the season when temperature increases.     

Microbial population in rotten living body ofSalix matsudana

The microbial population in rotten living body ofSalix matsudana caused byTrametes suaveolens (L.) was researched. 11 bacteria species (1 species ofBacillus, 2 species ofClostridium and 8 species of non-brood-cell bacteria), 1 species ofActinomyces that belongs to Lavendulac, 8 species of fungi and 6 species ofTrichoderma were isolated from rotten trunk. The hyphae ofTrametes suaveolens mainly existed between rotten sections and discoloration sections. In over-rotten section and healthy section the fungi (Trametes suaveolens) were not isolated. The microbes that lived in the discoloration section were the most in kinds and number and they were the pioneer microbes of wood rotting. Only after they dwelled in wood and eliminated its rot-resistance, could wood-rotting fungi invade wood and caused wood-rotting. Responsible editor: Zhu Hong     

木麻黄青枯病菌的分离及强致病菌株的筛选

[目的]筛选出强致病菌株用于木麻黄抗病育种研究工作。[方法]在广东沿海木麻黄青枯病发病区采集病根,开展病原菌两种不同分离方法的比较研究,对分离出的31个病株进行16s rRNA测序鉴定及致病性测定。[结果]采用稀释分离法及根系溢出法在TTC培养基上共分离出了31个病原菌株,根系溢出法操作简便,杂菌含量低,分离率在60%左右,可作为常规稀释分离法的补充。31个菌株进行分子鉴定,只有22个菌株扩增出了特异性条带,经测序比对确定这22个菌株为青枯菌。青枯菌株致病性测定结果显示菌株致病性在无性系间、菌株间及菌株与无性系间的交互作用均具有极显著差异(P0.01),不同接种方法间菌株致病性相关系数值较小,介于0.496 6~0.731 0之间,即室内水培接种与小苗盆栽接种不存在密切的直线相关关系。[结论]综合选择在不同无性系及不同接种方法中均具有较强致病性的GL-2、H、M、TC-1、F、Q菌株作为下一步木麻黄种质资源抗性鉴定及抗病育种研究试验菌株。     

A new norlignan from Taxodium ascendens

A new norlignan, (2R,3R,4S,5S)-2,4-bis(4-hydroxyphenyl)-3,5-dihydroxy-tetrahydropyran (1), together with 9 known compounds were isolated from the branches and leaves of Taxodium ascendens. Their structures were mainly determined on the basis of MS, IR, 1D and 2D NMR spectral evidences. Methanol extract showed inhibitory activity on carbonic anhydrase II with an IC₅₀ value of 4.27 µg/ml, acetone extract and methanol extract inhibited activity of cathepsin B with IC₅₀ values of 2.12 and 3.71 µg/ml, respectively.     

O_3浓度升高对桢楠和闽楠幼苗光合作用、抗氧化能力及生物量的影响

[目的]探讨O_3浓度升高对两种亚热带树木幼苗的影响,并分析其敏感性差异及原因。[方法]本试验以1年生桢楠和闽楠幼苗为材料,采用开顶式气室(OTCs),研究未过滤大气、100 nmol·mol-1(E1)、150 nmol·mol-1(E2)O_3处理对光合作用、抗氧化能力和生物量的影响。[结果]研究表明:(1) O_3熏蒸主要通过非气孔限制因素降低了两树种的净光合速率(Pn)。其中对于桢楠,E2对Pn的不利影响大于E1。而对于闽楠,8月份E2的Pn大于E1,而9月份和10月份小于E1。(2) O_3熏蒸提高了两树种的抗氧化能力,表现为总酚含量和总抗氧化能力随着O_3浓度的升高逐渐升高,而E2的还原型抗坏血酸含量低于E1。(3) O_3熏蒸降低了两树种根、茎、叶及总干质量,及降低了闽楠的根茎比。[结论]O_3熏蒸降低了两树种的光合作用,提高了抗氧化能力,最终减少了生物量。根据Pn和生物量,确定闽楠较桢楠对O_3浓度升高更加敏感。两树种间的O_3敏感性差异与气孔导度和抗氧化物质的背景水平及其对O_3浓度升高的响应有关。     

Cytotoxicity of withanolides isolated from Acnistus arborescens

A new withanolide identified by spectroscopic analysis as 12β-acetoxy-4-deoxy-5,6-deoxy-Δ⁵–withanolide D and Withanolide D, were isolated from the leaves of Acnistus arborescens. Cytotoxic activity of these two compounds against human tumor cell lines HT-29, MCF-7, MKN-45, HEp-2, HeLa, U-937 and two human normal fibroblast cultures, Fib04 and Fib05, were assessed. Withanolide D presented in vitro cytotoxic activity against tumor cell lines at the low micromolar range (LC₅₀:1.0 to 1.69 µM) and showed a slightly lower activity against Fib04, suggesting moderated selectivity among tumoral and normal cells. No cytotoxic effect was observed for 12β-acetoxy-4-deoxy-5,6-deoxy-Δ⁵–withanolide D.     

林地土壤及其真菌对毛红椿种子发芽及幼苗保存的影响

[目的]通过不同群落毛红椿天然林林地土壤及其真菌对种子萌发和幼苗存活影响的研究,探讨影响毛红椿天然更新的障碍因素。[方法]在江西九连山国家级自然保护区内的3个毛红椿天然优势群落中,分别取距离毛红椿母树3个不同距离(2.5、5.0、7.5 m)处的根区土壤和远离毛红椿母树(25 m以外)的非根区土壤,以非林地土为对照开展室内模拟播种和以根区土壤真菌的水或根系分泌物悬液对毛红椿幼苗进行灌根接种,观察种子萌发、幼苗存活和幼苗感病情况。[结果]各群落土壤中种子发芽均呈先上升后下降规律,且基本在第8 10天达到高峰;林地土与非林地土种子发芽率差异不显著,但林地根区土幼苗的存活率极显著低于非林地土和林地非根区土;距母树3个不同距离根区土的幼苗存活率差异不显著。灌根接种不同处理间幼苗存活率差异极显著,具体表现为接种RS2、RS5根区真菌的幼苗感病率显著高于根系分泌物及无菌水空白对照,且RS2真菌的根系分泌物悬浮液幼苗感病率显著高于其无菌水悬浮液。[结论]毛红椿种子萌发不受土壤环境的影响,但幼苗建成受根区范围内土壤的影响,根系分泌物和致病菌的互作显著降低幼苗保存率。由此推断,毛红椿根区土壤内存在幼苗的潜在致病菌及根系分泌物可增强其致病性。     

山鸡椒1-脱氧木酮糖-5-磷酸还原异构酶 DXR基因的克隆和SNP分析

在转录组测序结果分析基础上,以山鸡椒cDNA 为模板,克隆得到山鸡椒1-脱氧木酮糖-5-磷酸还原异构酶DXR 基因cDNA 全长,以山鸡椒基因组DNA 为模板,设计引物、扩增拼接后获得山鸡椒DXR 基因全长,命名为LcDXR。序列分析表明,LcDXR cDNA 全长为1 501 bp,5'非编码区长34 bp,3'非编码区长53 bp,开放阅读框长1 413 bp,预测编码含有470个氨基酸残基的蛋白质,等电点为6.62,分子量为51.12 kD。LcDXR 基因全长为12 601 bp,其中外显子12 个,内含子11 个。对来自10个种源的LcDXR 基因编码区单核苷酸变异位点进行分析表明:在cDNA 区间内共发现10 个SNP(single nucleotide polymorphism)位点,其中有4 个单核苷酸变异导致了所编码的氨基酸的改变,为了分析氨基酸突变导致的蛋白质精细结构的变化,利用Swiss-PDB Viewer 模拟4 个突变位点氨基酸残基的替换。其中江西安远(AY)的突变Lys119Thr 引起了氢键的变化,推测可能对酶的活性产生影响。研究结果为深入研究山鸡椒脱氧木酮糖5-磷酸还原异构酶的活性和功能奠定了基础,同时为山鸡椒遗传育种提供理论依据。     

杨树PtRRI基因的组织特异性表达模式分析

[目的]模式植物在木本植物中鉴定的许多重要调控因子家族在木本植物中出现了基因家族成员扩张,但ARRs家族作为细胞分裂素响应调节因子在杨树基因组中成员数量反而减少,其在木本植物中如何行使功能需要进一步研究。[方法]本研究通过生物信息学构建PtRRI启动子与GUS融合表达载体,检测植物激素处理后PtRRI表达量和检测PPtRRI::GUS转基因植株在生根过程中GUS信号等方法,对杨树PtRRI基因的组织特异性表达模式进行分析。[结果]表明:PtRRI在杨树根部、形成层、木质部表达量相对较高,PtRRI转录受6-BA激素诱导,与其在不定根发育过程中激素调控下的表达相一致。[结论]PtRRI基因可能参与杨树的次级生长。     

黑木相思与尾叶桉苗期氮素传递的研究

以尾叶桉、黑木相思苗木混栽为研究对象,采用¹⁵N土壤和叶片标记法,在温室盆栽下研究2种植物间在根系完全隔离、部分隔离及无隔离处理下的生长变化及N素传递。结果表明:黑木相思的苗高、地径及生物量等生长指标随着根系隔离的减少而逐渐降低;相反,尾叶桉在根系无隔离处理下,其生长指标都要显著高于根系完全隔离处理,结果表明尾叶桉具有强大的养分吸收能力,混栽条件下会抑制黑木相思的生长。在¹⁵N土壤或叶片标记下,尾叶桉在根系部分隔离、无隔离下的总¹⁵N含量显著高于完全隔离处理(5.0和59.6倍);在土壤标记下,根系无隔离处理的黑木相思总¹⁵N含量显著低于完全隔离、部分隔离处理,而在叶片标记下的完全隔离黑木相思,其总¹⁵N含量显著低于其余2种处理。无论土壤或是叶片标记,黑木相思都能将N素传递至相邻尾叶桉,其N素传递率、传递量随着根系隔离的减少而提高。研究结果表明尾叶桉、黑木相思间能通过土壤渗透等方式传递N素,为桉树、相思的混交模式提供可靠的理论依据。     

Cloning and RNAi construction of a LEAFY homologous gene from Populus tomentosa and preliminary study in tobacco

PtLFY, a LEAFY (LFY) gene, was cloned from Populus tomentosa (LM50) by PCR. Sequencing analysis indicated that PtLFY was 2 629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa (LM50 and 5082). The pBI121-Ptalfy (reverse)-intron-Ptlfy-GUS-nos was constructed using RNA interference (RNAi) technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed. [Supported by the National Natural Science Foundation of China (Grant No. 30371175) and Postdoctoral Foundation of China (Grant No. 2002032041)]     

基于cpDNA rps16序列分析兰考泡桐与白花泡桐和毛泡桐的遗传关系

【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。     

Comparison of terpenes in extracts from the resin and the bark of the resinous stem canker of Chamaecyparis obtusa and Thujopsis dolabrata var. hondae

A monoterpene and 15 diterpenes were isolated from the ethyl acetate extracts of the bark-glued resin from the resinous stem canker ofThujopsis dolabrata var.hondae Makino. A monoterpene (nezukone20) and 4 diterpenes (acetyl torulosol5, acetyl isocupressic acid8, acetyl abietinol11, and 7-methoxytotarol18) were characteristic constituents of the ethyl acetate extracts but were absent in then-hexane extracts from the resinous stem canker ofT. dolabrata var.hondae. These terpenes were first isolated fromT. dolabrata var.hondae andT. dolabrata. The available literature suggests that diterpene18 is a new compound. The resinous stem canker ofChamaecyparis obtusa Endlicher contained larger amounts of manool1,trans-communic acid6, and ferruginol12 and smaller amounts of isocupressic acid7 and abietinol10 than the resinous stem canker ofT. dolabrata var.hondae. The concentration of18 was less than 2% in the extracts, and the resinous stem canker ofC. obtusa lacked this compound. The resinous stem cankers ofC. obtusa andT. dolabrata var.hondae provided extracts 15.6 and 4.96 times, respectively, heavier than the healthy ones. Large differences in the ratios and compositions of terpenes were also observed between the resinous stem canker and the healthy trees. Terpenes isolated from the extracts contained many kinds of diterpene, especially the labdane-type diterpenes, in these diseased trees. These results suggest that the presence of labdane-type diterpenes is closely associated with the resinous stem canker or the causal fungi of this disease.Part of this report was presented at the 50th annual meeting of the Japan Wood Research Society, Kyoto, April 2000     

Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: Nitrogen accumulation in the stands and biological N2 fixation

The sustainability of plantation forests is closely dependent on soil nitrogen availability in short-rotation forests established on low-fertility soils. Planting an understorey of nitrogen-fixing trees might be an attractive option for maintaining the N fertility of soils. The development of mono-specific stands of Acacia mangium (100A:0E) and Eucalyptus grandis (0A:100E) was compared with mixed-species plantations, where A. mangium was planted in a mixture at a density of 50% of that of E. grandis (50A:100E). N₂ fixation by A. mangium was quantified in 100A:0E and 50A:100E at age 18 and 30 months by the ¹⁵N natural abundance method and in 50A:100E at age 30 months by the ¹⁵N dilution method. The consistency of results obtained by isotopic methods was checked against observations of nodulation, Specific Acetylene Reduction Activity (SARA), as well as the dynamics of N accumulation within both species. The different tree components (leaves, branches, stems, stumps, coarse roots, medium-sized roots and fine roots) were sampled on 5–10 trees per species for each age. Litter fall was assessed up to 30 months after planting and used to estimate fine root mortality. Higher N concentrations in A. mangium tree components than in E. grandis might be a result of N₂ fixation. However, no evidence of N transfer from A. mangium to E. grandis was found. SARA values were not significantly different in 100A:0E and 50A:100E but the biomass of nodules was 20–30 times higher in 100A:0E than in 50A:100E. At age 18 months, higher δ¹⁵N values found in A. mangium tree components than in E. grandis components prevented reliable estimations of the percentage of N derived from atmospheric fixation (%Ndfa). At age 30 months, %Ndfa estimated by natural abundance and by ¹⁵N dilution amounted to 10–20 and 60%, respectively. The amount of N derived from N₂ fixation in the standing biomass was estimated at 62 kg N ha⁻¹ in 100A:0E and 3 kg N ha⁻¹ in 50A:100E by the ¹⁵N natural abundance method, and 16 kg N ha⁻¹ in 50A:100E by the ¹⁵N dilution method. The total amount of atmospheric N₂ fixed since planting (including fine root mortality and litter fall) was estimated at 66 kg N ha⁻¹ in 100A:0E and 7 kg N ha⁻¹ in 50A:100E by the ¹⁵N natural abundance method, and 31 kg N ha⁻¹ in 50A:100E by the ¹⁵N dilution method. The most reliable estimation of N₂ fixation was likely to be achieved using the ¹⁵N dilution method and sampling the whole plant.