The Structure and Fungal Associates of Mycorrhizas in Leucopogon parviflorus (Andr.) Lindl.

Mycorrhizal root systems ofLeucopogon parviflorus (Andr.) Lindl.were collected from wild populations at three sites on the coastof New South Wales, Australia and examined by light and electronmicroscopy. The structure of the hair roots is typical of thefamily, there being an epidermal layer in which ericoid mycorrhizasare formed, two cortical layers (an exodermis and endodermis)and a very small stele. The colour, size and coil structureof the fungal symbionts indicate that there were at least twodifferent fungi that consistently formed ericoid mycorrhizalstructures at these sites. Transmission electron microscopyof the endophytes showed only ascomycete fungi. Plants fromtwo of the populations were used for fungal isolations. Fungiwere isolated by incubating surface sterilized hair-root piecesin a solution of bovine serum albumin with penicillin and streptomycin.Twenty-one different culture types were obtained, four of whichwere common to both sites. Two of the common culture types weredark, sterile, slow-growing cultures similar to the ericoidendophyteHymenoscyphus ericae(Read) Korf & Kernan. Leucopogon parviflorus ; bearded heath; Epacridaceae; Ericaceae; mycorrhiza; ultrastructure; endophytes; callose     

Phytogeography, Biology and Conservation of Western Australian Epacridaceae

The Epacridaceae of Western Australia comprise 181 named speciesdistributed in 17 genera. One hundred and seventy seven species(98% of the total) and ten genera are endemic to the region.within Western Australia, all members are confined to the south,with a bimodal pattern of species diversity centred on the sandplainsand uplands around the Stirling Ranges and Mount Lesueur.Leucopogon, the largest genus, reflects this pattern, however, speciesdiversity is greatest on the south coastal sandplains. All species are woody shrubs, mostly found on neutral or acidicsandy soils. Plants of at least 120 species are killed by fire;Astrolomacontainsthe only significant number of resprouting species. Althoughsome species are in flower every month every year, autumn/winterflowering is a common feature of many members of the family.All species are biotically pollinated. Bird pollination is foundin 18 species in six genera. The remaining species are insectpollinated. Bees are the major pollinators inLeucopogon, ConostephiumandColeanthera.Moths and butterflies are important pollinators inAndersonia,Leucopogon,LysinemaandStyphelia. Flies are minor pollinators of some speciesofAndersonia, LeucopogonandMonotoca. All species studied areprotandrous and outbreeding, with onlyCosmeliabeing capableof self pollination. Fruits are fleshy inAstroloma, Brachyloma,Croninia, in certain species ofLeucopogonandStypheliaand areanimal dispersed. Other genera have dry, dehiscent fruit withgravity or wind dispersed seeds. Many species have relatively narrow distribution ranges, 90species having ranges of over 300km, 54 species have rangesof 100–300km and 37 have ranges of less than 100km (andthere are many undescribed species in this group). The conservationstatus of many species is still poorly known. Dieback diseaseis a major threat to the family, and could cause the extinctionof several species in the wild. Western Australia; Epacridaceae; phytogeography; biology; pollination; dispersal; conservation; disease     

Salicylic Acid Levels in Thermogenic and Non-Thermogenic Plants

The natural trigger for heat production in the thermogenic inflorescencesof Sauromatum guttatum Schott (voodoo lily) was recently identifiedas salicylic acid (SA), which induced heat production at levelsas low as 13 ng g f. wt^–1. Since then the levels of SAwere determined in other thermogenic and non-thermogenic plantspecies. In thermogenic inflorescences of five aroid species,and in male cones of at least four thermogenic cycads SA levelsduring heat production exceeded 1 µg g f. wt^–1.SA was not detected in the thermogenic flowers of a water lily,Victoria regia Lindl. (Nymphaeaceae), and Bactris major Jacq.(Palmae). Levels of salicylic acid varied substantially in thefloral parts of seven non-thermogenic species and in the leavesof 27 non-thermogenic species. Amorphophallus campanulatus Blume ex Decne, Arum italicum Mill., Arum dioscoridis Sibth. & Son., Philodendron selloum Koch, Monstera deliciosa Liebm., Encephalartosferox Bertol. f., Encephalartos hildebrandtii A. Br. & Bouché, Encephalartos gratus Prain, Dioon edule Lindl. cv. edule, Dioon edule Lindl. cv angustifolium, Sauromatum guttatum Schott, voodoo lily, Victoria regia Lindl., Bactris major Jack, salicylic acid, thermogenicity, heat production     

The Pollination Mechanism of Three Sympatric Prescottia(Orchidaceae: Prescottinae) Species in Southeastern Brazil

The pollination biology of three Prescottia(Orchidaceae: Prescottinae)species was studied in Picinguaba, São Paulo State, south-easternBrazil. Plants are self-compatible but pollinator-dependentand offer nectar as the only reward for pollinators. Prescottiaplantaginea Lindl. and Prescottia stachyodes(Swartz) Lindl.are pollinated by pyralid moths (Lepidoptera: Pyralidae). Moth-pollinationis reported for the first time in the genus Prescottia and thesubtribe Prescottinae. Pollination by halictid bees (Halictidae)is confirmed for Prescottia densiflora Lindl. The pollinationmechanism is the same regardless of pollinator-type: pollinariaare fixed on the ventral surface of the insect's proboscis throughthe pad-like viscidium and are removed when the insect leavesthe flower. A pollinarium-carrying insect visiting another flowerwill brush the stigmatic surface and leave clumps of pollen,thus effecting pollination. Prescottia stachyodes is protandrous,a feature that increases the chances of cross-pollination. Asfar as we know, this is the first report of protandry in thegenus Prescottia and also the subtribe Prescottinae. Copyright2001 Annals of Botany Company Prescottia, Orchidaceae, orchids, Prescottinae, moths, Pyralidae, bees, Halictidae, flower morphology, protandry     

Water Relations of Tropical Epiphytes: III. EVIDENCE FOR CRASSULACEAN ACID METABOLISM

Five tropical epiphytes were examined for evidence of CrassulaceanAcid Metabolism (CAM), namely the orchids Eria velutina Lindl.,Dendrobium tortile Lindl. and Dendrobium crumenatum Sw., andthe ferns Pyrrosia adnascens (Forst.) Ching and Pyrrosia angustata(Sw.) Ching, family Polypodiaceae. Diurnal variations in leaftitratable acidity, diffusive conductance and water potentialwere measured at various levels of water stress. The three orchidsshowed typical CAM behaviour, namely large diurnal fluctuationsin leaf acidity, day-time closure and night opening of stomataand a very slow decline in water potential under stress. Theferns showed some evidence of CAM, but this was not as well-developedas had been reported for two other tropical epiphytic membersof the same family. Key words: Crassulacean acid metabolism, Tropical epiphytes, Water stress     

Effects of Fluctuating Temperatures on Germination

Germination responses to alternating daily cycles involvingtransfers between different temperatures over the range 0 toc. 40?C were investigated in four species: Lycopus europaeusL., Clarkia unguiculata Lindl., Apium graveolens L. cv. GiantRed, and Silene dioica (L.) Clairv. Comparisons of the germination of these species revealed distinctivedifferences which could be related to particular features ofthe geographical regions or ecological habitats in which theyoccur naturally. The effects of fluctuating as opposed to constant temperaturesvaried from one species to another. L. europaeus depended almostabsolutely on fluctuating temperatures for germination. Germinationof S. dioica was greatly increased by fluctuating temperatures.Moderate increases occurred over a restricted temperature rangein A. graveolens cv. Giant Red and C. unguiculata was affectedin only minor ways. These results are discussed in relationto previous attempts to explain the mechanisms responsible forthe frequently beneficial effects of fluctuating temperatureson germination.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Water Relations of Tropical Epiphytes: I. RELATIONSHIPS BETWEEN STOMATAL RESISTANCE, RELATIVE WATER CONTENT AND THE COMPONENTS OF WATER POTENTIAL

The water relations of five species of tropical vascular epiphytesnative to Malaysia were studied. The species were ferns: Pyrrosiaadnascens (Forst.) Ching. and Pyrrosia angustata (Sw.) Ching.;orchids: Eria velutina Lindl., Dendrobium tortile Lindl. andDendrobium crumenatum Sw. Leaf resistance as a function of leafwater potential was measured for the two ferns. The criticalwater potential at which stomata closed was found to be highin each case; –0.75 MPa and –0.5 MPa respectively.The components of water potential were estimated with the pressurechamber as functions of relative water content. For each speciescell sap was found to be dilute, pressure potential low at fullturgor, and the change in relative water content between fullturgor and wilting point small. Small values of solute potentialat full turgor were also found for the ferns and E. velutinausing a vapour pressure osmometer. Values of the bulk modulusof elasticity of the leaf tissue for each species lay withinthe range of published data. The significance of these resultsfor the epiphytic way of life is discussed. Key words: Water potential, Epiphytes, Diffusive resistance, Orchid, Fern     

Effects of Environmental Factors on Germination and Seedling Growth in Quercus floribunda and Cupressus torulosa, Tree Species of Central Himalaya

Quercus floribunda Lindl. (tilonj oak), a late successionalspecies, and Cupressus torulosa D. Don. (surai), an early successionalspecies, were compared in terms of seed germination and seedlinggrowth. In Petri dish germination tests in which light quality,temperature and osmotic water stress were varied, the germinationof both species was reduced by continuous dark or far-red lightand by osmotic water stress. These effects were greater on thegermination of surai than on that of tilonj oak. The growthof seedlings in soil was measured over a twelve-month periodduring which different levels of shading and different wateringregimes were applied. Surai was less affected than tilonj oakby reduced soil moisture, but the effect of deep shade treatmentswas greater on surai than with tilonj oak. At higher soil moisturestress seedlings of surai attained greater height then tilonjoak, but the latter achieved higher dry weight and a higherroot: shoot and lower leaf-weight ratio. Quercus floribunda Lindl. (tilonj oak), Cupressus torulosa D. Don. (surai), seed germination, seedling growth, environmental factors, regeneration     

Water Relations of Tropical Epiphytes: II. PERFORMANCE DURING DROUGHTING

Relative water content R, water potential and leaf diffusiveconductance C₅ were monitored while five tropical epiphyteswere subjected to an extended period of drought. The speciesstudied were: ferns Pyrrosia adnascens (Forst.) Ching and Pyrrosiaangustata (Sw.) Ching, family Polypodiaceae; orchids: Eria velutinaLindl., Dendrobium tortile Lindl. and Dendrobium crumenatumSw. The ferns reached zero turgor rapidly and after only small declinesin relative water content (R) and . Beyond this point stomatalmovement seemed strongly suppressed, but the leaves continuedto lose water vapour until very low values of R were reached.Nevertheless, on re-watering water potential (), R and diffusiveconductance (C₅) returned to pre-stress levels within 3 d. The orchids showed a more gradual decline in R and , stomatalactivity was not so strongly suppressed, and night opening ofstomata was observed under stress. The relationship between and R was found for each species,curves being fitted to the data points by non-linear regression.From these analyses it was concluded that these species hadvery dilute cell sap, and consequently the change in for agiven decline in R was smaller than for most other species recordedin the literature. Key words: Orchid, Fern, Drought, Relative water content, Diffusive conductance, Water potential     

EVOLUTION OF ASEXUALITY VIA DIFFERENT MECHANISMS IN GRASS THRIPS (THYSANOPTERA: Aptinothrips)

Asexual lineages can derive from sexual ancestors via different mechanisms and at variable rates, which affects the diversity of the asexual population and thereby its ecological success. We investigated the variation and evolution of reproductive systems in Aptinothrips, a genus of grass thrips comprising four species. Extensive population surveys and breeding experiments indicated sexual reproduction in A. elegans, asexuality in A. stylifer and A. karnyi, and both sexual and asexual lineages in A. rufus. Asexuality in A. stylifer and A. rufus coincides with a worldwide distribution, with sexual A. rufus lineages confined to a limited area. Inference of molecular phylogenies and antibiotic treatment revealed different causes of asexuality in different species. Asexuality in A. stylifer and A. karnyi has most likely genetic causes, while it is induced by endosymbionts in A. rufus. Endosymbiont‐community characterization revealed presence of Wolbachia, and lack of other bacteria known to manipulate host reproduction. However, only 69% asexual A. rufus females are Wolbachia‐infected, indicating that either an undescribed endosymbiont causes asexuality in this species or that Wolbachia was lost in several lineages that remained asexual. These results open new perspectives for studies on the maintenance of mixed sexual and asexual reproduction in natural populations.     

Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus by the helper bacterium Paenibacillus sp

Paenibacillus sp. EJP73 has been previously demonstrated as a mycorrhization helper bacterium (MHB) for the Lactarius rufus–Pinus sylvestris symbiosis in both laboratory and glasshouse experiments. In the present study, the effect of Paenibacillus sp. EJP73 metabolites on L. rufus EO3 pre-symbiotic growth was tested in two agar plate-based systems. Specifically, volatile metabolites were investigated using a dual plate system, in which the presence of strain EJP73 resulted in a significant negative effect on L. rufus EO3 hyphal radial growth but enhanced hyphal branching and reduced internode distance. Soluble metabolites produced by strain EJP73 were tested on L. rufus EO3 growth in single-agar plate assays by incorporating bacterial cell-free whole or molecular weight fraction spent broth into the agar. Whole spent broth had a negative effect on hyphal growth, whereas a low molecular weight fraction (100–1,000) promoted colony radial growth. Headspace and spent broth analysis of strain EJP73 cultures revealed 2,5-diisopropylpyrazine to be the most significant component. Synthesised 2,5-diisopropylpyrazine and elevated CO₂ (2,000 ppm) were tested as specific volatile metabolites in the dual plate system, but neither produced the response shown when strain EJP73 was present. Increased pre-symbiotic hyphal branching leading to increased likelihood of plant infection may be an important MHB mechanism for strain EJP73. Although the precise signal molecules could not be identified, the work suggests a number of metabolites may work synergistically to increase L. rufus root colonisation.     

Comparative account of nectary structure in Hexisea imbricata (Lindl.) Rchb.f. (Orchidaceae)

• Background and Aims Despite the number of orchid speciesthat are thought to be pollinated by hummingbirds, our knowledgeof the nectaries of these orchids is based solely on a singlespecies, Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge.Nevertheless, it is predicted that such nectaries are likelyto be very diverse and the purpose of this paper is to comparethe nectary and the process of nectar secretion in Hexisea imbricata(Lindl.) Rchb.f. with that of Maxillaria coccinea so as to beginto characterize the nectaries of presumed ornithophilous Neotropicalorchids. • Methods Light microscopy, transmission electronmicroscopyand histochemistry were used to examine the histology and chemicalcomposition of nectary tissue and the process of nectar secretionin H. imbricata. • Key Results and Conclusions The nectary of H. imbricatahas a vascular supply, is bound by a single-layered epidermiswith few stomata and comprises two or three layers of subepidermalsecretory cells beneath which lie several layers of palisade-likeparenchymatous cells, some of which contain raphides or mucilage.The secretory cells are collenchymatous and their walls havenumerous pits with associated plasmodesmata. They contain thefull complement of organelles characteristic of secretory cellsas well as intravacuolar protein bodies but some of the secretoryepidermal cells, following secretion, collapse and their anticlinalwalls seem to fold. Nectar secretion is thought to be granulocrineand, following starch depletion, lipid droplets collect withinthe plastids. The nectar accumulates beneath the cuticle whichsubsequently forms swellings. Finally, nectar collects in thesaccate nectary spur formed by the fusion of the margins ofthe labellum and the base of the column-foot. Thus, althoughthe nectary of H. imbricata and M. coccinea have many featuresin common, they nevertheless display a number of important differences.     

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation

Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.     

Eyes and vision in Arion rufus and Deroceras agreste (Mollusca; Gastropoda; Pulmonata): What role does photoreception play in the orientation of these terrestrial slugs?

This paper deals with the orientational behaviour in the two terrestrial slugs Arion rufus and Deroceras agreste. It presents anatomical details of their eyes and provides an appraisal of the eyes’ optical system. In both species the retinae contain two principal types of cell: photoreceptive and pigmented supportive cells. While only the eye of A. rufus apparently contains neurosecretory neurones, that of D. agreste is the only one equipped with a small additional retina with its own separate lens. Lens shapes vary between ovoid (A. rufus) and spherical (D. agreste). Our results demonstrate that the camera‐type eyes in A. rufus and D. agreste have optical systems that do not allow the production of a sharp image on the retina. The slugs demonstrate negative visually mediated phototactic behaviour, but no polarization sensitivity. Only one aspect of the visual environment, namely the overall distribution of light and dark, seems to be important for these slugs. As the main role of the slugs’ photoreceptors is to monitor environmental brightness and to assist the animal in orientating towards dark places, we conclude that these slugs do not need to perceive sharp images.     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

Extreme genetic diversity in asexual grass thrips populations

The continuous generation of genetic variation has been proposed as one of the main factors explaining the maintenance of sexual reproduction in nature. However, populations of asexual individuals may attain high levels of genetic diversity through within‐lineage diversification, replicate transitions to asexuality from sexual ancestors and migration. How these mechanisms affect genetic variation in populations of closely related sexual and asexual taxa can therefore provide insights into the role of genetic diversity for the maintenance of sexual reproduction. Here, we evaluate patterns of intra‐ and interpopulation genetic diversity in sexual and asexual populations of Aptinothrips rufus grass thrips. Asexual A. rufus populations are found throughout the world, whereas sexual populations appear to be confined to few locations in the Mediterranean region. We found that asexual A. rufus populations are characterized by extremely high levels of genetic diversity, both in comparison with their sexual relatives and in comparison with other asexual species. Migration is extensive among asexual populations over large geographic distances, whereas close sexual populations are strongly isolated from each other. The combination of extensive migration with replicate evolution of asexual lineages, and a past demographic expansion in at least one of them, generated high local clone diversities in A. rufus. These high clone diversities in asexual populations may mimic certain benefits conferred by sex via genetic diversity and could help explain the extreme success of asexual A. rufus populations.     

Using Stable Isotopes to Infer the Impacts of Habitat Change on the Diets and Vertical Stratification of Frugivorous Bats in Madagascar

Human-modified habitats are expanding rapidly; many tropical countries have highly fragmented and degraded forests. Preserving biodiversity in these areas involves protecting species–like frugivorous bats–that are important to forest regeneration. Fruit bats provide critical ecosystem services including seed dispersal, but studies of how their diets are affected by habitat change have often been rather localized. This study used stable isotope analyses (δ¹⁵N and δ¹³C measurement) to examine how two fruit bat species in Madagascar, Pteropus rufus (n = 138) and Eidolon dupreanum (n = 52) are impacted by habitat change across a large spatial scale. Limited data for Rousettus madagascariensis are also presented. Our results indicated that the three species had broadly overlapping diets. Differences in diet were nonetheless detectable between P. rufus and E. dupreanum, and these diets shifted when they co-occurred, suggesting resource partitioning across habitats and vertical strata within the canopy to avoid competition. Changes in diet were correlated with a decrease in forest cover, though at a larger spatial scale in P. rufus than in E. dupreanum. These results suggest fruit bat species exhibit differing responses to habitat change, highlight the threats fruit bats face from habitat change, and clarify the spatial scales at which conservation efforts could be implemented.     

Regulation of Organ Formation by Cytokinin and Auxin in Lilium Bulbscales Grown In Vitro

Regulation of organ formation by cytokinin and auxin was investigatedin vitro using Lilium auratum Lindl. (wild species habituatedin Japan) and Lilium speciosum Thunb. cv. "Uchida". The interactionof -naphthylacetic acid (NAA) and kinetin on bulbscale or rootdifferentiation was examined. NAA and kinetin showed mainlyindividual but also some synergistic effects. The effects ofbenzyladenine (BA) and kinetin were compared and the resultindicated that BA has a stronger physiological effect on organformation than kinetin and that their effects on Lilium auratumand Lilium speciosum were BA or kinetin-specific. The actionof kinetin on Lilium was affected by sucrose concentration andthe strength of the Murashige and Skoog medium (MS medium),and thus their high concentrations inhibited the kinetin-inducedbulbscale differentiation. Furthermore, a high sucrose levelnegated the kinetin inhibition of root formation, while highMS medium strength in itself inhibited root formation. Morphologicalobservation of bulbscale differentiation induced under a highkinetin level revealed that the new-formed structures are homologousto normally grown bulbs in soil in spite of their particularfeatures. (Received August 3, 1981; Accepted November 2, 1981)     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning