Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.     

DoesAcetobacter diazotrophicusLive and Move in the Xylem of Sugarcane Stems? Anatomical and Physiological Data

We have previously shown that the nitrogen-fixing endophyteof sugarcane,Acetobacter diazotrophicuslives in the sugar solutionin the intercellular-space apoplast of the stem cortex. Variousauthors have claimed that it inhabits the xylem apoplast. Thispossibility has been investigated in the clone Ja 60-5 and shownto be most unlikely for the following reasons: (1) an adequatecarbon source is lacking in the xylem sap, and cannot be suppliedfrom the intercellular-space apoplast of the cortex. Diffusionof solutes into and out of the vascular bundles is preventedby complete lignification and suberization of the bundle sheathcell walls except at the nodes. (2) Longitudinal movement ofparticles as large as bacteria is severely limited at the nodes.Vessel end walls were found in 90% of vessels at each node,and only 1% of open vessels extended through two nodes. Noneextended as far as three nodes. In addition to vessel end walls,vessel continuity at nodes was interrupted by living cells.Dye solution in the transpiration stream in metaxylem vesselsdid not pass through these living cells, but accumulated incrystals (sump formation) in the vessels below the node. Onlyin some protoxylem vessels and cavities did dye solution movethrough many nodes. It is likely that selection of sugarcaneclones such as Ja 60-5 for resistance to bacterial wilt diseaseshave selected for clones that have limited vessel continuity.(3) When culturedA. diazotrophicuswas introduced into the transpirationstream, the xylem parenchyma reacted by secreting a bright redpolymer which killed the bacteria and blocked the movement ofwater. We conclude that the xylem flow-apoplast of this cloneof sugarcane is an unsuitable habitat forA. diazotrophicusandthat additional habitats to those of the intercellular-spaceapoplast should be sought elsewhere. Acetobacter diazotrophicus; endophytic bacteria; nitrogen fixation; sugarcane; vessel end walls; xylem apoplast; xylem bacteria; xylem segmentation     

Partitioning of Nitrogen Derived from N2 Fixation and Reserves in Nodulated Medicago sativa L. During Regrowth

Nodul{macron}ted alfalfa plants were grown hydroponically. Inorder to quantify N₂ fixation and remobilization of N reservesduring regrowth the plants were pulse-chase-labelled with ¹⁵N.Starch and ethanol-soluble sugar contents were analysed to examinechanges associated with those of N compounds. Shoot removalcaused a severe decline in N₂ fixation and starch reserves within6 d after cutting. The tap root was the major storage site formetabolizable carbohydrate compounds used for regrowth; initiallyits starch content decreased and after 14 d started to recoverreaching 50% of the initial value on day 24. Recovery of N₂fixation followed the same pattern as shoot regrowth. Afteran initial decline during the first 10 d following shoot removal,the N₂ fixation, leaf area and shoot dry weight increased sorapidly that their levels on day 24 exceeded initial values.Distribution of ¹⁵N within the plant clearly showed that a significantamount of endogenous nitrogen in the roots was used by regrowingshoots. The greatest use of N reserves (about 80% of N incrementin the regrowing shoot) occurred during the first 10 d and thencompensated for the low N₂ fixation. The distribution of N derivedeither from fixation or from reserves of source organs (taproots and lateral roots) clearly showed that shoots are thestronger sink for nitrogen during regrowth. In non-defoliatedplants, the tap roots and stems were weak sinks for N from reserves.By contrast, relative distribution within the plant of N assimilatedin nodules was unaffected by defoliation treatment. Key words: Medicago sativa L., N₂ fixation, N remobilization, N₂ partitioning, regrowth     

Technical approaches to inoculate micropropagated sugar cane plants were Acetobacter diazotrophicus

Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus. Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol permitted studies of infection and comparison among strains.     

Indentification of Acetobacter diazotrophicus,Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans using biochemical and genetic criteria

Nitrogen-fixing Acetobacter diazotrophicus, Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans colonize sugar cane, and are thought to be capable of supplying high levels of fixed nitrogen to this plant. Eight A. diazotrophicus, two H. seropedicae and four H. rubrisubalbicans isolates were identified and compared by complementary biochemical and genetic methods. Utilization of carbon sources and antibiotic resistance patterns allowed differentiation of A. diazotrophicus from Herbaspirillum species. In order to distinguish strains within A. diazotrophicus species, the polymerase chain reaction was employed, using a Rhizobium meliloti dctA primer under low stringency hybridization conditions.     

Presence of endophytic diazotrophs in sugarcane juice

Summary In this work we investigated the diazotrophs present in the juice of different varieties of sugarcane. Samples of the aerial part of sugarcane, between 3 and 5 months old, were assessed for the presence of diazotrophs. The isolated nitrogen-fixing microorganisms were identified as Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Azospirillum brasilense. These microorganisms use organic acids found in the sugarcane juice as an energy source. They also use sugars, such as free glucose, that are present in all the organs of the aerial part. The distribution of endophytic microorganisms in sugarcane depends on the different organic acid and sugar concentrations present during development and growth. We concluded that in more mature regions of the sugarcane stem Gluconacetobacter diazotrophicus grows more abundantly than Herbaspirillum seropedicae or Azospirillum brasilense.     

Biological Nitrogen Fixation in Sugar Cane: A Key to Energetically Viable Biofuel Production

The advantages of producing biofuels to replace fossil energy sources are derived from the fact that the energy accumulated in the biomass is captured directly from photosynthesis and is thus renewable, and that the cycle of carbon dioxide fixation by the crop, followed by burning of the fuel makes no overall contribution to atmospheric CO₂ or, consequently, to global warming. However, these advantages are negated if large quantities of fossil fuels need to be used to grow or process the biofuel crop. In this regard, the Brazilian bioethanol program, based on the fermentation/distillation of sugar cane juice, is particularly favorable, not only because the crop is principally hand harvested, but also because of the low nitrogen fertilizer use on sugar cane in Brazil. Recent ¹⁵N and N balance studies have shown that in some Brazilian cane varieties, high yields are possible without N fertilization because the plants are able to obtain large contributions of nitrogen from plant-associated biological N₂ fixation (BNF). The N₂-fixing acid-tolerant bacterium Acetobacter diazotrophicus was first found to occur within roots, stems, and leaves of sugar cane. Subsequently, two species of Herbaspirillum also have been found to occur within the interior of all sugar cane tissues. The discovery of these, and other N₂-fixing bacteria that survive poorly in soil but thrive within plant tissue (endophytic bacteria), may account for the high BNF contributions observed in sugar cane. Further study of this system should allow the gradual elimination of N fertilizer use on sugar cane, at least in Brazil, and opens up the possibility of the extension of this efficient N₂-fixing system to cereal and other crops with consequent immense potential benefits to tropical agriculture.     

Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.)

We report studies on the possible effects of fertilisation with high level of N (300 kg of N ha^-1) on the occurrence and numbers of the diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicusin sugar cane plants. In the sugar cane genotype SP79-2312, the N fertilised plants generally showed higher concentrations of this element. These same plants also had lower numbers of A. diazotrophicus, while the population of Herbaspirillum spp. was not affected by N application. These differences in the concentration of N and the numbers of A. diazotrophicus due to N application were not shown in the variety SP70-1143. The numbers of A. diazotrophicus were also shown to be influenced by the harvest time, becoming reduced in the harvests that coincided with dry periods of the year.     

Concurrent Rates of N2 Fixation, Nitrate and Ammonium Uptake by White Clover in Response to Growth at Different Root Temperatures

Nodulated white clover plants (Trifolium repens L. cv. Huia)were grown for 71 d in flowing nutrient solutions containingN as 10 mmol m_–3 NH₄NO₃, under artificial illumination,with shoots at 20/15°C day/night temperatures and root temperaturereduced decrementally from 20 to 5°C. Root temperatureswere then changed to 3, 7, 9, 11, 13, 17 or 25°C, and theacquisition of N by N₂ fixation, NH₄+ and NO₃^– uptakewas measured over 14 d. Shoot specific growth rates (d. wt)doubled with increasing temperature between 7 and 17°C,whilst root specific growth rates showed little response; shoot:root ratios increased with root temperature, and over time at11°C. Net uptake of total N per plant (N₂ fixation + NH₄++ NO₃^–) over 14 d increased three-fold between 3 and 17°C.The proportion contributed by N₂ fixation decreased with increasingtemperature from 51% at 5°C to 18% at 25°C. Uptake ofNH₄+ as a proportion of NH₄+ + NO₃^– uptake over 14 d variedlittle (55–62%) with root temperature between 3 and 25°C,although it increased with time at most temperatures. Mean ratesof total N uptake per unit shoot f. wt over 14 d changed littlebetween 9 and 25°C, but decreased progressively with temperaturebelow 9°C, due to the decline in the rates of NH₄+ and NO₃^–uptake, even though N₂ fixation increased. The results suggestthat N₂ fixation in the presence of sustained low concentrationsof NH₄+ and NO₄^– is less sensitive to low root temperaturethan are either NH₄+ or NO₃^– uptake systems. White clover, Trifolium repens L. cv. Huia, root temperature, nitrogen fixation, ammonium, nitrate     

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10³ cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10⁷/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.The authors are with EMBRAPA-CNPAB, Cx Postal 74.505, Seropédica, Rio de Janeiro, 23851-970, Brazil     

Infection and Root-Nodule Development in Stylosanthes Species by Rhizobium

Root nodules of the tropical forage legume Stylosanthes occurredonly at lateral root junctions and resulted from direct invasionby rhizobia through spaces between epidermal cells. No infectionthreads were present in either the root hairs or nodules. Invasionof the host cortical cells was through structurally alteredcell walls. The bacteria reached the site of nodule initiationin the lateral root cortex by progressive collapse of the initiallyinvaded cells which were compressed by neighbouring cells toform intercellular thread-like infection zones. The bacteriamultiplied in the invaded cells of the nodule initial whichdivided repeatedly to form the nodule. Bacteroids formed onlywhen the host cells ceased to divide. Some abnormal associations occurred in S. capltata and S. hamata40264A. Division of invaded cells was restricted in S. capitataand the bacteria became enlarged and grossly deformed. In S.hamata restricted cell division was immediastely followed bythe brcakdown of the host cells and, although the bacteria multiplied,no bacteroids were formed. Bacteria isolated from these nodulesformed both effective and abnormal nodules when inoculated ontothe same host.     

Site, Scale and Time-course for Adjustments in Lateral Root Initiation in Wheat Following Changes in C and N Supply

The pattern of lateral root initiation in seminal roots of wheat(Triticum aestivumL. cv. Alexandria) and the location, scaleand time-course for adjustments in initiation were studied afterchanges in C and N supply. Macroscopically visible primordiaappeared in a non-acropetal sequence with the frequency (numberper unit length) increasing with distance behind the main rootapex to a maximum at 40–50 mm behind the root tip. Pruningthe root system to a single seminal axis increased the primordiafrequency by 23% within 15 h. After longer periods, the effectof root-pruning was greater. The enhanced primordia frequencywas first observed in tissue located 0–10 mm behind theapex at the start of treatment. Feeding glucose (50 mM) alsoincreased primordia frequency within 15 h, but to a greaterextent, and here additional primordia were initiated in tissuelocated 0–10and10–20 mm behind the apex at the startof treatment. Withdrawing NO₃^-from one part of a split-rootsystem, whilst maintaining the supply to the other, reducedprimordia frequency in the non-fed roots and, in some cases,a compensatory increase in the NO₃^--fed roots was observed.The location and scale of the adjustments were similar to thosefound with root-pruning and glucose-feeding, but were slightlyslower to appear. In spite of some differences in detail, therewas a broad similarity in site, scale and time-course for adjustmentsin lateral root initiation with these treatments, which is consistentwith the operation of a common mechanism. Whenever an increasein primordia frequency was observed, it was associated withan increase in the ethanol-soluble sugar content of the tissue.However, the reduction in frequency in NO₃^--deprived roots wasalso accompanied by an increase in sugar content. There wasno consistent relationship between total N content of the tissueand primordia frequency, but there was between primordia frequencyand the rate of net NO₃^-uptake. The possible mechanisms controllinglateral root initiation are discussed. Compensatory growth; correlative growth; glucose; initiation; lateral root; nitrate; primordium; split-root; Triticum aestivum; wheat     

14C Fixation and Translocation in two Clones of Sugarcane with Contrasting Rates of Sucrose Uptake in vitro

The rates of ¹⁴CO₂ fixation and translocation of ¹⁴C labelledassimilates were measured in field experiments at two timesof the day in two sugar-cane clones known to have differentrates of sucrose uptake in vitro but the same weight of leafper unit weight of cane. The rate of ¹⁴CO₂ fixation and the velocity and rate of translocationwere significantly greater at both times in the clone with thehigher rate of sucrose uptake in vitro. The velocities of translocationwere 2.18 and 2.36 cm/min^–1 for the clone with high sucroseuptake and 1.46 cm min^–1 at both times in the clone withlow uptake. It is suggested that among sugar-cane clones the ability oftheir canes to store sugar may play a part in determining theirrates of photosynthesis and translocation.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

N2 Fixation and Nitrate Uptake by White Clover Swards in Response to Root Temperature in Flowing Solution Culture

Nodulated white clover plants (Trifolium repens L. cv. Huia)were grown as simulated swards for 71 d in flowing nutrientsolutions with roots at 11 C and shoots at 20/15 C, day/night,under natural illumination. Root temperatures were then changedto 3, 5, 7, 11, 13, 17 or 25 C and the total N₂, fixation over21 d was measured in the absence of a supply mineral N. Alltreatments were subsequently supplied with 10 mmol m^–2NO₂ ^– in the flowing solutions for 14 d, and the relativeuptake of N by N₂, fixation and NO₃ ^– uptake was compared.Net uptake of K⁺ was measured on a daily basis. Root temperature had little effect on root d. wt over the 35-dexperimental period, but shoot d. wt increased by a factor of3.5 between 3 and 25 C, with the sharpest increase occurringat 7–11 C. Shoot: root d. wt ratios increased from 25to 68 with increasing temperature at 7–25 C. N₂-fixationper plant (in the absence of NO₂ ^–) increased with roottemperature at 3–13C, but showed little change above13 C. The ratios of N₂ fixation: NO₂ ^– uptake over 14d (mol N: mol N) were 0.47–0.77 at 3–7 C, 092–154at 11–17 C, and 046 at 25 C, reflecting the dominanceof NO₃ ^– uptake over N₂ fixation at extremes of high andlow root temperature. The total uptake of N varied only slightlyat 11–25 –C (095–110 mmol N plant^–1),the decline in N₂ fixation as root temperature increased above11 C was compensated for by the increase in NO^– ₃ uptake.The % N in shoot dry matter declined with decreasing root temperature,from 32% at 13 C to 15% at 3 C. In contrast, concentrationsof N expressed on a shoot water content basis showed a modestdecrease with increasing temperature, from 345 mol m^–3at 3 C to 290 mol m^–3 at 25 C. Trifolium repens L, white clover, root temperature, N₂ fixation, potassium uptake, nitrate uptake, flowing solution culture     

Scanning Electron Microscopy of Plant Roots

A glycol methacrylate infiltration and polymerization techniquewas used to prepare clover roots inoculated with Rhizobium forscanning reflection electron microscopy. Root hairs and epidermalcells were coated with many bacteria; some bacteria seemed tobe embedded in the wall surface. Root hair tips were often smoothbut some older root hair surfaces showed a fibrillar meshworkpattern. Small granules c. 0.18 µm diameter were presenton the root hair and epidermal cell walls. The root cap, someroot hairs, and some epidermal cells were covered by an amorphousfilm thought to be the mucigel.     

Gas and Liquids in Intercellular Spaces of Maize Roots

Oils are spontaneously absorbed by gas-filled intercellularspaces (IS) in maize root cortex. The network of these spacesin living root sections was imaged by confocal laser scanningmicroscopy using a fluorescent solution of Nile red in oil.The gas volume fraction (GVF) of root segments was quantifiedby the increase in weight (differentiated zones) or tissue density(2–3 mm root tips) due to complete vacuum infiltration.Cooling to 6 °C or inhibition of oxidative phosphorylationdiminished the GVF of root tips but did not significantly affectthe GVF of differentiated root zones. The threshold pressuredifference for measurable infiltration of isolated root segmentsis lower (10 to 15  kPa) than the threshold for infiltrationof comparable zones of attached roots or of detached roots withthe cut surface sealed (>60 kPa). In the absence of an opencut, pressure-driven infiltration of the root cortex is acceleratedby microscopic fissures within the epidermal/hypodermal barrier.The GVF of the root cortex was reduced after transferring rootsfrom sugar solutions (0.1 to 0.3M ) to water. This points toefficient water transport from the medium to sugar-containingcortical cell walls through epidermal and hypodermal protoplasts.When 2-cm-long primary roots were vacuum infiltrated in situand then allowed to grow on aerated mineral medium for a further5 d, cortical IS of the originally infiltrated root bases remainedfilled with liquid but the subsequently grown apical root zoneshad a normal GVF. Copyright 1999 Annals of Botany Company Apoplastic and protoplasmic route, maize, infiltration, intercellular spaces, oil absorption, confocal laser scanning microscope, water transport, Zea mays L.     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

Can the 15N Dilution Technique be used to Study N2 Fixation in Tropical Tree Symbioses as Affected by Water Deficit?

Three methods were used to study N₂ fixation and effects ofwater deficit on N₂ fixation: C₂H₂ reduction assay (ARA), ¹⁵Ndilution technique and accumulated N content. In addition, ¹⁵Ndilution was calculated both in a traditional way and in a modifiedway, which takes into consideration N and ¹⁵N content for theplants before the experiment started. The three methods wereapplied on the following Rhizobium-symbioses: Acacia albidaDel (Faidherbia albida (Del) A. Chev.) and Leucaena leucocephala(Lam) de Wit., and the Frankia-symbiosis Casuarina equisetifoliaL. The plants wereabout 4-months-old when they were harvested. Nitrogen derived from N₂ fixation in control plants of Acaciaalbida was 54·2 mg as measured with ARA, while it was28·5 mg as measured with the ¹⁵N dilution technique,compared to 30·7 mg calculated as accumulated N. In comparison,L. leucocephala fixed 41·6 mg N (ARA), 53·5 mgN(15N dilution technique) and 56·3 mg N (accumulatedN). The Frankia-symbiosis had fixed 27·4 mg N as measuredby ARA, 8·1 mg N as measured by ¹⁵N dilution techniqueand 12·3 mg N as accumulated N. There were no differencesbetween the estimates based ontraditional and modified waysof calculating ¹⁵N dilution. The immediate effect of water deficit treatment on N₂ fixationwas continuously measured inall species with ARA, which startedto decrease approximately 10 d after the initiation of the treatment,and declined to less than 5% of the initial level after 21–28d. The decrease in the amount of N derived from N₂ fixation wasstudied in L. leucocephala during the period of treatment. Therewas a 26% decrease in amount of N derived from N₂ fixation asresult of water deficit (as measured with ARA), while the decreasewas 23% when measured withboth the ¹⁵N dilution method and asaccumulated N. The three different methods for measuring N₂ fixation and effectsof water deficit on N₂ fixation are discussed. Key words: Acacia albida, ARA, Casuarina equisetifolia, Leucaena leucocephala, ¹⁵N dilution, N₂N fixation, water deficit     

Responses of N2 Fixation-linked Respiration to Host-plant Energy Status in White Clover Acclimated to a Controlled Environment

Ryle, G. J. A., Powell, C. E. and Gordon, A. J. 1988. Responsesof N₂ fixation-linked respiration to host-plant energy statusin white clover acclimated to a controlled environment.—J.exp. Bot. 39: 879–887. Single plants of white clover, acclimated to a controlled environmentand dependent for nitrogen on N₂ fixation in their root nodules,were darkened, defoliated or exposed to enhanced CO₂ levelsto establish the quantitative relationships between the photosynthesisof the host plant and the N₂ fixation metabolism of root nodules. The nodule respiration associated with N₂ fixation (FLR) declinedrapidly to 10–15% of its normal rate following plant darkeningearly in the photoperiod. Darkening at progressively later intervalsduring the photoperiod demonstrated a positive, apparently linearrelationship between duration of illumination and total FLRduring the photoperiod and the following night period. Completeor partial defoliation reduced FLR according to the leaf arearemoved: again, there was a strong positive correlation betweencurrent rate of photosynthesis, whether of defoliated or undefoliatedplants and the FLR of root nodules. Doubling the current rateof photosynthesis, by enhancing CO₂ levels around the shoots,promoted FLR within 1–2 h when plants were stressed bylack of light. However, enhanced CO₂ levels increased FLR onlyslowly over a period of several hours in plants entrained tothe normal growing conditions. It is concluded that, in these plants acclimated to a uniformand favourable controlled environment, the supply and utilizationof photosynthetic assimilate in N₂ fixation was finely balancedand quantitatively linked during a single diurnal period andthat nodule functioning was not depressed by lack of energysubstrate. Key words: White clover, N₂ fixation, photosynthesis.