Selection gains for essential oil traits using micropropagation of Eucalyptus polybractea

The value of eucalyptus oil for medicinal purposes is based largely on its cineole content, and the prime commercial species for pharmaceutical grade eucalyptus oil is Eucalyptus polybractea (blue mallee). This study applies a recently developed micropropagation protocol to selected elite oil yielding blue mallee seedlings and investigates the potential of a trial plantation based on micropropagated clones to increase cineole yields. The study aims to assess two micropropagated clones of blue mallee for growth and key essential oil traits in comparison to two related half-sibling families, and to assess consistency for oil traits within clones. After 23 months growth in a plantation, all ramets from the micropropagated clones appeared healthy and showed similar growth forms to the half-sibling saplings at the time of harvest. Total above ground biomass was significantly greater in the half-sibling families compared to their relative clones. Nevertheless, the foliar essential oil concentration and the proportion of the total oil as cineole was greatest in the clones compared to their related half-sibling families, reflecting the oil traits of the ortets. The oil composition profiles were very consistent within clones. This was particularly evident for cineole which had the remarkably narrow ranges of 90–92% in one clone and 92–94% in the other, suggesting strong genetic control of essential oil composition. Overall, the results suggest that micropropagation has great potential for the establishment of blue mallee plantations using genotypes selected for key oil traits. The application of the micropropagation protocol to older plants of blue mallee (for which both key oil and biomass traits are known) is likely to produce greater selection gains for cineole yield.     

The influence of ontogeny on essential oil traits when micropropagating Eucalyptus polybractea

Eucalyptus polybractea (blue mallee) is the key species used in the commercial production of pharmaceutical-grade (>70% 1,8-cineole) eucalyptus oil in Australia. The establishment of plantations based on clones of selected, elite oil-producing plants has been recognized as the best means by which to increase oil and cineole yields. Blue mallee can be micropropagated using axillary bud proliferation of explants collected from the coppicing lignotubers of decapitated plants. To date, this has been successful for ortets from three different ontogenetic stages: 8-month-old ‘seedlings’, 4-year-old ‘saplings’ and ‘mature’, biannually harvested plants of indeterminate age. The issue of ontogenetic control of oil quality and quantity is an important consideration when selecting ortets for cloning and also when examining the success of a cloning programme. Our aim is to examine if clones derived from ortets of the three different ages reflect their respective ortets, in terms of oil quantity and quality (cineole proportion), and to appraise at what age the clones best reflect the ortets for these traits. Our results suggest that the age of ortets prior to explant collection does not influence micropropagation success or clonal consistency, and that clones generally exhibit the leaf oil traits of their respective ortets. Leaf oil quantity accumulation in ramets followed the ontogenetic trajectory of a relatively rapid increase to a maximum between 3 and 9 months, followed by a period of relative constancy for at least 12 months before beginning to gradually decrease after 21 months. Although oil quality of ramets reflected their corresponding ortets relatively well after 9 months, apparent ontogenetic constraints to cineole accumulation suggest that harvesting at a time between 9 and 21 months may better reflect the ortets for both traits. It appears likely that the proliferation of selected blue mallee genotypes using micropropagation will result in selection gains for key oil traits, thus potentially increasing cineole yields in future plantations.     

不同种源巨桉生长与材性变异*

不同种源巨桉生长与材性变异王豁然，柴修武，郑勇奇，臧道群，方玉霖，王维辉关键词巨桉，种源，材性变异在我国南方，巨按（ＥｕｃａｌｙｐｔｕｓｇｒａｎｄｉｓＷ．ＨｉｌｌｅｘＭａｉｄｅｎ）是生长最快的桉树之一，现已被选作世界银行贷款国家造林项目的主要树种。中...     

Photosynthetic response to green crown pruning in young plantation-grown Eucalyptus pilularis and E. cloeziana

The loss of foliage through pruning of live branches may reduce tree growth or it may be compensated by photosynthetic up-regulation of the remaining crown. Here, the changes in light-saturated photosynthesis following pruning to remove 50% of green crown length were examined in 4-year-old Eucalyptus pilularis Sm. and Eucalyptus cloeziana F. Muell. trees. The objectives of the study were to: (1) compare leaf-level physiological (light-saturated photosynthesis (A_max), stomatal conductance (g), transpiration (T), dark respiration (R_d), quantum yield (Φ), light compensation point (Γ), water-use efficiency (WUE), nitrogen-use efficiency (NUE)) traits in species with contrasting crown dynamics and structure, (2) examine the effect of crown position on these traits, and (3) examine the effect of pruning on A_max, g, T, WUE, NUE, leaf N and P concentrations and specific leaf area (SLA). Prior to pruning there were no differences in R_d, Γ and Φ between E. pilularis and E. cloeziana but differences in A_max, T, g, leaf N, leaf P, WUE, NUE and SLA. Whereas the rate of physiological processes (A_max, T, and g) and leaf N and P concentrations increased with crown height, R_d, Γ, Φ and SLA declined along this vertical gradient, except in the upper crown of E. cloeziana where A_max, T and g were not different to the lower crown. No up-regulation of photosynthesis or changes in leaf physiology occurred between 6 and 13 months after pruning in either species. The results provide an important basis for modelling pruning effects in process-based tree growth models.     

广西丘陵台地巨尾桉、尾叶桉(实生)地位指数表的编制*

从广西南部桉树主要栽培区丘陵台地立地类型现有林人中收集的９５株解析木及９１株固定标准地内平均优势木调查材料，选用ＬｎＹ＝Ａ＋Ｂ×Ｘ＾ｃ公式模拟出巨尾桉和尾叶桉的导向曲线。最后确定了广西丘陵台地两种桉树的立地指数表。经检验，达到了较高的估测精度。     

Effects of light quality on gas exchange and dry matter partitioning in Eucalyptus grandis W. Hill ex Maiden

Stockplants of Eucalyptus grandis were pruned to a height of 7–10 cm and after 3 weeks were placed in growth cabinets set at a photon flux density (PFD) of 200 μmol m⁻² s⁻¹ and red to far-red ratios of 0.4, 0.7, 1.3, 3.5 or 6.5. Experiments tested the effects of light quality on growth and gas exchange of stockplants. Light quality did not affect the total shoot dry weight (DW), root DW or shoot to root ratio of stockplants or their total leaf area. However, there were significant effects of light quality on: (i) plant height, which was greatest at red:far-red (R:FR) ratios of 0.4 and 0.7; (ii) partitioning of DW between leaves and stems, with greater stem DW and less leaf DW at low R:FR ratios (0.4 and 0.7); (iii) partitioning of DW and leaf area between the most dominant shoot and all other (non-dominant) shoots; (iv) specific leaf area, which was greatest at low R:FR ratios. In the above characters, the dominance ratio (ratio of most dominant shoot to sum of all other shoots) was greatest at low R:FR ratios and least at ratios of 3.5 and 6.5. Photosynthetic rate per unit leaf area and leaf chlorophyll concentration significantly increased with increasing R:FR ratio. However, photosynthesis per unit chlorophyll concentration was significantly greater at low R:FR ratios. Generally, light quality had no significant effect on photosynthetic rate per leaf or per unit dry weight, but rates of transpiration, stomatal conductance and water use efficiency increased with an increase in R:FR ratio. These data indicate that compensatory changes in plant morphology and gas exchange caused equality in total dry weight per plant between treatments. The above effects of light quality on dry matter partitioning and gas exchange had important effects on the size, number, morphology and physiology of subsequently collected cuttings for vegetative propagation.     

施肥对蓝桉幼林生长的影响

经５ａ研究结果表明,在省保山市石灰岩红壤区施肥能明显促进蓝桉幼林生长,肥效在１．５ａ时最大,９个施肥处理的树高、胸径平均增长３３．６９％、４８．３９％,造林后５ａ时,生长反应已经呈下降趋势,９个施肥处理的树高、胸径平均增长１６．４５％、１０．７１％。Ｐ 促进蓝桉增产的主要因素,每株施４３０ｇ钙镁磷肥的几个处理产好,Ｎ、Ｋ肥增产效果不明显,有机肥对蓝桉生长有促进作用。在９个施肥处理中,处理６（钙镁磷     

ECM和VAM菌混合接种对尾叶桉生长效应的研究

本文报道混合接种外生菌根真菌彩色豆马勃和ＶＡ菌根真菌苏格兰球囊霉对尾叶桉苗斯有接种效应。根系样口检查表明接种的两个菌朱均能在尾叶桉上成功合成的相应的菌根。接种后５个月试验的苗生长量和生长量的分析结果表明,接种与未接种对照差异极显著,其中,两种混合接种方式苗木干重分别比对照增加了２４０．３％和１９４．３％;苗高生长增加了７３．８％和４１．７％;地径增加了１７．２％和１９．３％。试验苗木对菌根的依赖性     

澳大利亚ECM菌在尾叶桉上的筛选及其接种效应

报道４３种澳大利亚及２种国产外生菌根菌株在广东尾叶桉人工林上的筛选试验的初期研究结果。分别对试验林６、１２和２４个月的生长情况进行了调查,数据分析结果表明,与未接种对照林木相比,接种试验林在树高和地径（或胸径）生长量上均表现出显著的接种优势（Ｐ〈０．０１）;接种林３次调查平均树高生长量最大增幅分别为２６．１％、３３．７％和２７．４％,地径（或胸径）最大增幅分别为１９．２％、２１．５９％和１９．３２％;２４个月时每公顷材积最大幅为５６．８％。Ｅ０７１０（Ｌａｃｃａｒｉａ）、Ｈ４６７０（Ｌａｂｙｒｉｎｔｈｏｍｙｃｅｓ）、Ｅ４５０１（Ａｍａｎｉｔａ）、Ｅ２０１３（Ｌａｃｃａｒｉａ）、Ｃ９３０１（Ｐｉｓｏｌｉｔｈｕｓ）、Ｈ１２７２（Ｓｃｌｅｒｏｄｅｒｍａ）、Ｈ１１９４（Ｔｈａｘｔｅｒｏｇａｓｔｅｒ）、Ｈ０６０３（Ｓｃｌ     

Evaluation of non-destructive methods of measuring growth stress in Eucalyptus globulus: relationships between strain, wood properties and stress

High levels of growth stress are implicated in causing end splitting of logs, deflection during sawing and deformation of boards as stresses are released during sawing operations. Level of stress is a function of strain and the elastic modulus of the wood (MOE). Levels of peripheral strain can be measured on standing trees and, if the MOE is known, stresses can be estimated. The validity of using peripheral strain measurements relies on underlying theoretical models that relate strain to expected patterns of stress distribution and levels of board deflection. This study evaluates these theoretical relationships by determining relationships of stress and strain with board deflection, end splitting and a range of wood properties.

Peripheral strain levels were extremely variable within the bottom log and little evidence was found for consistent patterns of variation, although measurements generally increased with increasing height above ground. Sampling on two sides of the standing tree at breast height appeared to be a suitable strategy, with the mean for these strain readings having a correlation (r) of 0.86 with the average strain in the bottom log.

Growth strain was not a reliable predictor of board deflection and cannot be recommended as a non-destructive sampling method. Overall there was a poor relationship between growth strain and board deflection. No consistent relationships were found between a range of wood properties and growth strain or board deflection across both sites. Stress levels were calculated for each tree as the product of growth strain and modulus of elasticity and the relationship between calculated stress and mean board deflection determined. No relationship was found at either site with correlations being very close to zero.

The underlying theoretical relationships between stress and strain were examined and several questions raised about the validity of such models.     

Allelopathy assessments for the environmental biosafety of the salt-tolerant transgenic Eucalyptus camaldulensis, genotypes codA12-5B, codA 12-5C, and codA 20C

Allelopathy tests were conducted on salt-tolerant transgenic eucalyptus trees conferring bacterial codA gene in the designated net-house conditions under Type II use (contained use) of the Japanese law on environmental biosafety aiming for Type I (field use) application. Three transgenic and corresponding nontransgenic genotypes were employed for four different tests: (1) sandwich bioassay; (2) soil germination method; (3) gas chromatography (GC) for volatile substances from the plants; and (4) high-performance liquid chromatography (HPLC) on phenolic compounds from fresh leaves, which are the primary allelopathic substances on the species. The simple approaches, the bioassays, indicated no significant difference between the transgenic and nongenetically modified genotypes. There was no qualitative difference between the transgenic and nontransgenic lines by GC or HPLC. Absence of any quantitative difference was suggested by repetitive examination and subsequent analysis of variance assessments with the chromatographic methods and bioassays. Moreover, it was also indicated that bioassays should be the primary assessment method for allelopathy in considering the simplicity, speed, low cost, and reproducibility of these methods. Overall, substantial equivalence was considered on the three transgenic genotypes with codA gene when compared with the nontransgenic Eucalyptus camaldulensis lines. The experiments supported the application to isolated field testing of the transgenic Eucalyptus camaldulensis genotypes as the first case and experience in Japanese regulatory approval processes Type I Use for the deliberate release to the environment.     

尾叶桉×细叶桉木材密度与生长的联合选择

[目的]评估Resistograph钻刺法间接测定尾叶桉×细叶桉木材密度的可靠性,检测杂交亲本对子代表型的效应以及生长与木材密度的相关,评选速生、优质的尾细桉杂种。[方法]基于10株尾叶桉与10株细叶桉不完全析因交配产生的56个杂交组合的7.5年生试验林,利用79株分析容积法与Resistograph钻刺法测定的木材密度的相关,通过方差分析检测亲本对杂种生长和木材密度的效应,结合多重比较和独立淘汰法进行材积和木材密度的联合选择。[结果]容积法与Resistograph钻刺法测定的木材密度的表型相关系数为0.52(P0.001),遗传相关系数为0.55(P0.05);树高、胸径及材积的母本间和父本间均呈极显著差异(P0.001或0.01),但母本×父本互作的效应不显著;对钻刺木材密度,父本间呈极显著差异(P0.001),母本×父本互作显著(P0.05),但母本间差异不显著;树高、胸径和材积间的表型相关和遗传相关均极显著(P0.001),其与钻刺木材密度的表型相关极显著(P0.001),但遗传相关不显著;评选出速生、木材密度较高的杂交组合14个、单株17株。[结论]Resistograph钻刺法是一种间接测定尾细桉木材密度的简便、经济和可靠的方法;母本和父本选择以及母本与父本的组配对培育速生、材质优良的尾细桉杂种均较重要;尾细桉生长与木材密度的遗传相关不显著,需要对这两类性状分别进行选择;评选的尾细桉杂交组合和单株为培育速生、优质的桉树良种提供了有用的材料。     

西南桦和尾巨桉凋落叶分解及其与土壤性质的相关性

[目的]探讨南亚热带西南桦和尾巨桉人工纯林的凋落叶分解动态及其与土壤化学性质之间的相关关系.[方法]采用原位分解袋法研究凋落叶的分解过程.[结果]表明:西南桦、尾巨桉人工林凋落叶分解系数分别为0.96 a^-1和0.88 a^-1.在为期12个月的分解试验中,2种凋落叶有机C含量在整个分解过程中呈逐渐下降趋势;全K含量和C/N比在分解前期迅速下降,之后趋于平缓;全N含量和全P含量在整个分解过程中呈逐渐上升趋势;2种凋落叶N/P比则呈先升高后下降的趋势.无论是分解前期还是分解后期,凋落叶质量损失与N含量均呈显著正相关(前期R=0.877;后期R=0.855),与C/N均呈显著负相关(前期R=-0.735;后期R=-0.697).与尾巨桉林地土壤性质相比,西南桦凋落叶分解提高了林地0~10、10~20 cm土壤的有机C、全N、全P、全K、N/P,对2030 cm土壤有机C、全K、pH值、C/N、N/P则未产生显著影响.相关分析表明:凋落叶初始有机C含量与土壤有机C、全N、全P、全K、N/P显著相关;凋落叶初始全N含量与土壤全N、pH值、C/N显著相关.[结论]凋落叶的养分含量与土壤养分的关系紧密;与尾巨桉相比,西南桦凋落叶的养分含量明显较高,分解速率更快,释放到土壤中的养分也更多.     

Provenance and family variation in growth performance of Eucalyptus tereticornis (Sm.) in a provenance cum progeny trial in Midnapore, India

Eucalyptus tereticornis Sm. is an important species for agroforestry and commercial plantations in India. Results of a combined provenance–progeny trial of E. tereticornis laid out in 2002 at Midnapore (West Bengal) located in eastern tropical region of India are reported and discussed. Twelve provenances representing 70 families from Australia and Papua New Guinea were evaluated up to the age of 3 years. As a local seed source open-pollinated seed collected from a land race, i.e., Mysore gum (commonly known as Eucalyptus hybrid) was used to serve as check material (control). Significant variation in plant height, clean stem height, girth at breast height (GBH) and field survival was observed due to provenances, as well as families within provenances. This offers an ample scope to a breeder for increasing the growth and productivity in E. tereticornis through selection of provenances and families having potential for higher productivity. Heritability (narrow sense) values were appreciable for growth traits. Within provenance individual tree heritability estimates for height, clean stem length, GBH and number of branches at age 3 years were 0.318, 0.215, 0.269 and 0.231, respectively (assuming a coefficient of relationship of 0.4 for open-pollinated families of E. tereticornis). Provenance of Walsh River, Queensland Australia performed best for plant height, clean stem height, GBH, number of branches and field survival. Apart from this, the provenance from Oro bay to Emo, Papua New Guinea and Burdekin river, Queensland also showed good performance. Provenance from Yurammie, SF from New South Wales performed poorest for growth and survival. Correlations on growth traits were high and statistically significant, indicating that substantial gains could be achieved through indirect selection for one trait based on the direct selection for another. Age–age genetic correlations between age 1 and 3 years for the growth traits were highly significant and positive. Geographic clinal variation pattern was observed as latitude was negatively correlated with height and GBH (P < 0.05); longitude with number of branches (negative, P < 0.05). Fair differences were observed between phenotypic and genotypic coefficient of variability.     

不同种植年限尾巨桉人工林叶片-凋落物-土壤碳氮磷化学计量特征

[目的]为了解雷州半岛尾巨桉速生人工林生态系统的C、N、P分配格局及化学计量特征。[方法]采用空间换时间的方法,选取雷州半岛4种不同林龄(1、3、5、7 a)的尾巨桉人工林为研究对象,对尾巨桉叶片、凋落物及土壤的C、N、P含量及化学计量特征进行测定分析。[结果]表明:C、N含量表现为叶片凋落物土壤,P含量表现为叶片土壤凋落物,且3个库间差异显著;土壤的C含量随林龄增加而增加,N、P含量差异不显著,土壤C∶N随林龄的增加而增加,说明土壤有机质分解速率逐渐下降;凋落物的C∶N为54. 07 92. 18 ( 25),表明尾巨桉林下凋落物分解速率较慢,N元素成为主要限制凋落物分解的元素,凋落物的C∶N随林龄的增加先增加后下降,凋落物分解速度先降低后升高;叶片的N∶P为10. 80 12. 98,说明中幼林龄尾巨桉受N限制较明显。相关性分析表明:凋落物养分元素含量受叶片限制,土壤养分含量受凋落物限制,表明生态系统内部C、N、P元素在植物、凋落物与土壤之间实现了运输和转换。[结论]雷州半岛尾巨桉中、幼林龄时期土壤有机质及凋落物分解速率较慢;随林龄的增加,土壤有机质、凋落物分解速率下降,N元素成为其主要分解限制性元素,林分生长受N限制明显。     

桉树液流径向变化及其对整树蒸腾估算的影响

目的 分析液流密度径向差异及其对整树蒸腾量估算的影响,以期为中大径级桉树人工林蒸腾的准确估算和尺度扩展提供可靠依据。 方法 采用Granier热扩散探针法(TDP)测定了10年生中等径级尾巨桉(Eucalyptus urophylla × Eucalyptus grandis)3个边材径向深度0～2、2～4、4～6 cm的液流密度。 结果 尾巨桉液流密度径向分布格局为递减型,日变化格局均为单峰型。用单点径向深度0～2、2～4、4～6 cm液流密度代表整个边材液流密度估算的整树日蒸腾量Tr2、Tr4和Tr6较基于多点估算值Tr246分别高估92.9%、低估28.2%和低估74.0%。由于0～2 cm 边材深度液流密度对环境变化较为敏感,并且 Tr2与Tr246之间的拟合精度较高(R2=0.964),因此,基于Tr2构建的整树日蒸腾量估算校正函数Tr246=0.522Tr2有效的提高了整树蒸腾估算的便捷性和准确度。 结论 中等径级尾巨桉液流密度具有明显的径向变化特征,不考虑这种径向变化将会导致整树蒸腾估算的较大误差。基于边材最外侧（0～2 cm）液流密度估算的整树蒸腾经过校正后可以方便准确地估算中等径级桉树人工林蒸腾耗水。     

油桐尺蛾肠道菌群与桉树叶片内生菌差异分析

目的 植食性昆虫肠道微生物的多样性与其食物密切相关,本研究通过探讨油桐尺蛾（Buzura suppressaria）幼虫肠道菌群及其取食的桉树叶内生菌群的多样性,分析两者之间的关系。 方法 利用Illumina Miseq技术对油桐尺蛾5龄幼虫肠道和桉树叶部内生的细菌16s rDNA和真菌ITS rDNA进行测序,分析油桐尺蛾肠道和桉树叶部内生的细菌和真菌的多样性和丰度。 结果 油桐尺蛾肠道细菌共注释到6门、11纲、19目、38科、43属,真菌共注释到8门、29纲、67目、159科、257属;桉树叶部内生细菌共注释到10门、19纲、26目、54科、74属,真菌共注释到8门,31纲,79目,171科,291属。桉树叶内和油桐尺蛾肠道中均发现桉树主要病害病原菌所属菌属,分别为炭疽菌属（Colletotrichum）、假单胞菌属（Pseudomonas）、假尾孢属（Pseudocercospora）、短梗霉属（Aureobasidium）、壳针孢属（Septoria）、毛色二孢属（Lasiodiplodia）、新壳梭孢属（Neofusicoccum）。 结论 油桐尺蛾肠道的细菌、真菌的多样性和丰度与桉树叶之间存在差异,桉树叶对油桐尺蛾肠道微生物的群落结构有重要影响,而携带某些病原菌的油桐尺蛾啃食或其排泄物接触可能是导致该病原菌侵染健康桉树叶的原因。     

Nutrient store and export rates of Eucalyptus urograndis plantations in eastern Amazonia (Jari)

Nutrient loss due to export of wood and bark of 4.5-year-old Eucalyptus urograndis was studied in the Jari project, eastern Amazonia, Brazil. Results are based on estimates of biomass, above-ground nutrient store, and nutrient stores in the soil. Thirteen stands varying in site conditions and the number of previous rotations (up to three), were studied. Average nutrient losses due to export of wood and bark, as a percentage of the element store in the above-ground tree biomass, were: N, 65%, P, 54%; Ca, 76%; K, 57%; Mg, 61%. A comparison of export losses with the present soil stores of Ca, K and Mg cations shows that export is equal to or surpasses the remaining soil store to a depth of 100 cm. As a result of the last narvest, about 200–250 kg Ca and about 100 kg K ha⁻¹ were removed. In spite of uncertainties about other unknown inputs and outputs, one has to conclude that the next rotation may be endangered by acute deficiency of mineral nutrients, especially of Ca.     

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.