Studies on the Physiology of Nodule Formation: With two Figures in the Text: A Reappraisal of the Effect of Preplanting

Nutman (Arm. Bot. 21, 321, 1957) found that preplanting agarslopes with Trifolium pratense L. or Medicago sativa L. advancedthe time when second lots of plants of these species grown onthe same slopes initiated nodules, and depressed the total numberof nodules formed. He attributed these effects to root secretionswhich at low concentration hasten initial nodulation but athigher concentrations inhibit nodule formation. Further workhas now shown that initial nodulation is stimulated becausethe preplant removes traces of nitrate from the medium. Theamount of nitrate in the tap water used to prepare the medium(6?5 p.p.m. N) also increases the number of nodules formed onthe control plants, and this effect explains to a considerableextent the depression of nodule numbers by preplanting. Initial nodulation was delayed by small amounts of nitrate andnitrite but not by other forms of combined nitrogen (ammonium,asparagine, and urea). All forms of combined nitrogen testedincreased the number of nodules formed over a period of 8 weekswhen supplied at an initial concentration of 20 p.p.m. N.     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

Biological and Cultural Characteristics of the Effective Frankia Strain HFPCcl3 (Actinomycetales) from Casuarina cunninghamiana (Casuarinaceae)

From homogenates prepared from surface-sterilized nodules ofseedlings of Casuarina cunninghamiana grown aeroponically, astrain of Frankia designated HFPCc13 was isolated and has beengrown in pure filamentous culture in a defined synthetic nutrientmedium. Vesicle and sporangium formation can be induced by removalof combined nitrogen from the medium.Frankia strain HFPCc13nodulates young seedlings of C. cunninghamiana and C. equisetifoliawithin three weeks of inoculation with an optimum root mediumpH of 6–7 for nodulation and optimum temperature of 30–35^°C. The presence of combined nitrogen in the root mediuminhibits nodulation with NH₄⁺ more inhibitory than NO₃^–.Frankia HFPCc13 does not nodulate Allocasuarina species withinthe same family nor several other possible actinorhizal plantstested. Thus this strain is quite precise in its host specificity.The rate of acetylene reduction was greater in C. cunninghamianathan the closely related species C. equisetifolia. In neitherof these host species were vesicles observed to occur withinthe infected root nodules which had been demonstrated to beactively fixing dinitrogen. Root nodules were shown to be activein acetylene reduction over a range of O₂ concentration in thegaseous environment with an optimum at about 20 per cent O₂,the ambient P_O2 of the air. The mechanism(s) for oxygen protectionof nitrogenase within the filamentous form of Frankia withinthese nodules remains to be explained. Casuarina, Frankia, nodulation, nitrogen fixation     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: I. PHYSIOLOGICAL CHARACTERIZATION

Two ineffectively nodulating, allelic mutants of the commonbean (Phaseolus vulgaris L.) lines RIZ30 and RIZ36 were studied.In both mutants the nodulation phenotype was characterized bythe formation of tumour-like swellings when inoculated withbean Rhizobium strains. Late formation of pink nodules was observedin mutant plants grown in pots with soil or in hydroponic conditionswith perlite. In the absence of mineral nitrogen, mutant growthand nodulation were poor; a low peak of acetylene reductionactivity being detected 45 d after inoculation. Mutant growthin the presence of mineral nitrogen was similar to that of thewild type. The mutants were also characterized by poor pod fertility(seed per pod). The nodulation and pod fertility phenotypesare specifically controlled at the root and shoot levels, respectively,as assessed with grafting experiments. Mutant shoots graftedon wild type, nitrogen-fixing roots produced a number of seedssimilar to the wild type despite the expression of the poorpod fertility phenotype. Nitrogen and seed yields of wild typeshoots, grafted on mutant, ineffectively nodulating roots wereseverely reduced when compared with control wild type plantsalthough nitrogen accumulation during vegetative phase was similar. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, grafting, nitrogen assimilation     

The influence of colchicin on root nodule formation inTrifolium Pratense

Summary Addition of colchicin to nutrient solutions supplied to red clover plants had an inhibitory effect on root development and nodulation. At the lowest colchicin concentrations, a small, non-significant increase in the number of nodules was often observed; in a few experiments a barely significant increase in the number of nodules developing was observed up to concentrations of 10 mg per 1.Colchicin did not influence the growth of Rhizobium and growth of the bacteria in the presence of colchicin (10 mg per 1) had no effect on their subsequent infectivity or ability to fix nitrogen.Treatment with colchicin resulted in the formation of increased numbers of polyploid cells of the clover roots. In addition, it induced the development of deformed root-hairs which probably facilitated infection by Rhizobium and the formation of infection threads.The nodules developing at colchicin concentrations of 10 mg per 1 and higher were small, had an abnormal structure and were ineffective in nitrogen fixation.It is concluded that any stimulatory effect of colchicin on nodulation does not necessarily require to be associated with an increase in the number of polyploid cells in the root cortex. The effect of colchicin can be fully accounted for, initially by the greater number of infections resulting from the development of increased numbers of deformed root hairs, and subsequently by the reduced inhibitory action of the ineffective nodules on the formation of further nodules.     

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.     

External Nitrogen and Carbon Source-Mediated Response on Modulation of Root System Architecture and Nitrate Uptake in Wheat Seedlings

Nitrogen uptake efficiency is an important component trait that could be targeted for improving nitrogen use efficiency of crop plants. To understand the responses of different nitrate transport systems and the influence of root system architecture on nitrate uptake under limited nitrate conditions in wheat (Triticum aestivum L.) at the seedling stage, we studied nitrate uptake, root system architecture, and expression of different nitrate transporter genes in induced and non-induced wheat seedlings. Further, effects of inclusion of sucrose and two amino acids (glutamine and asparagine) in induction medium on these parameters were also studied. We observed that the induced wheat root system took up more nitrate as compared to non-induced root system in a dose-dependent manner. Gene expression of both high- and low-affinity nitrate transporter gene showed differential expression in the induced root tissues, as compared to non-induced tissues, depending on the concentration of nitrate present in induction medium. External nutrient media containing sucrose, glutamine, and asparagine reduce nitrate concentration in both root and shoot tissues and also influence the gene expression of these transporters. Our observations indicate that upon induction with milder external nitrate concentrations, the root architecture is modulated by changing overall lateral root size and 1st order lateral root numbers along with activation of nitrate transporters which acquire and transport nitrate in roots and shoots, respectively, depending on the carbon and nitrogen source available to seedlings.

     

Coculture of Pachyrhizus erosus (L.) hairy roots with Rhizobium spp.

Summary We have established an in vitro system for the induction and study of nodulation in Pachyrhizus erosus (jicama) via a hairy root-Rhizobium coculture. In vitro-grown P. erosus plantlets were infected with Agrobacterium rhizogenes (ATCC No. 15834) and two hairy root lines were established. Hairy roots were grown in a split-plate system in which compartment I (CI) contained MS medium with nitrogen and different sucrose levels (0–6%), while CII held MS medium without nitrogen and sucrose. Nodule-like structures developed in transformed roots grown in CI with 2–3% surcose, inoculated with Rhizobium sp. and transferred to CII. Nodule-like structures that developed from hairy roots lacked the rigid protective cover observed in nodules from plants grown in soil. Western blot analysis of nodules from hairy roots and untransformed roots (of greenhouse-grown jicama) showed expression of glutamine synthetase leghemoglobin and nodulins. Leghemoglobin was expressed at low levels in hairy root nodules.     

Effects of Nitrate Level on Nitrogen Metabolism in Winged Bean and Soya Bean

The effects of high (15 mM) and low (0.75 mM) solution nitratelevels on nitrogen metabolism in three genotypes (IL 7A, IL13 and IL 21) of winged beans [Psophocarpus tetragonolobus (L.)DC.] and one genotype (Williams) of soya bean [Glycine max (L.)Merrill] were investigated. Plants were grown for 42 days ina greenhouse in solution culture prior to sampling. The 15 mM nitrate treatment resulted in greater growth of allplant parts except roots. Growth of soya beans was more responsiveto nitrate level than was growth of winged beans. The high nitratelevel inhibited nodulation in all plants. The IL 13 and IL 21winged bean genotypes had similar nitrogenase activity (acetylenereduction per plant) as the soya bean and IL 7A winged beangenotype had lower activity. However, the IL 13 winged beangenotype had higher nitrogenase activity (acetylene reductionper unit nodule mass) than the other three genotypes which allhad similar activity. The 15 mM solution nitrate level stimulatedleaf and root nitrate reductase (NR) activity for all plants.All winged bean genotypes had higher leaf NR activity and higherpercentage reduced- and nitrate-nitrogen contents of leavesand stems compared with soya beans. However, total protein (reducednitrogen) was greater in soya beans when sampled indicatingthat more nitrate had been metabolized by soya beans than bywinged beans during the 42-day growth period. Psophocarpus tetragonolobus (L.) DC., winged bean, Glycine max (L.) Merrill, Soya bean, nitrate reductase, nitrogen fixation, nitrogenase activity, nodulation     

Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation

Host legumes control root nodule numbers by sensing externaland internal cues. A major external cue is soil nitrate, whereasa feedback regulatory system in which earlier formed nodulessuppress further nodulation through shoot–root communicationis an important internal cue. The latter is known as autoregulationof nodulation (AUT), and is believed to consist of two long-distancesignals: a root-derived signal that is generated in infectedroots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In Lotus japonicus, theleucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANTROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibitionof nodulation, and is hypothesized to recognize the root-derivedsignal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1)and LjCLE-RS2 as strong candidates for the root-derived signal.A hairy root transformation study shows that overexpressingLjCLE-RS1 and -RS2 inhibits nodulation systemically and, furthermore,that the systemic suppression depends on HAR1. Moreover, LjCLE-RS2expression is strongly up-regulated in roots by nitrate addition.Based on these findings, we propose a simple model for AUT andnitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2peptides and the HAR1 receptor-like kinase.     

Composition of Amino Compounds Transported in Xylem of Casuarina sp.

Seedlings (180-d-old) of Casuarina cunninghamianaM L., C. equisetifoliaMiq. and C. glauca Sieber inoculated with each of two differentsources of Frankia, were analysed for translocated nitrogenouscompounds in xylem sap. Analyses were also made on sap fromnodulated and non-nodulated plants of C. glauca grown with orwithout a range of levels of combined nitrogen. Xylem exudateswere collected from stems, roots, and individual nodules ofnodulated plants and from stems and roots of non-nodulated plants.While the proportional composition of solutes varied, the samerange of amino compounds was found in xylem sap from the threedifferent symbioses. In C. glauca asparagine was the major aminoacid in the root sap followed by proline, while in symbioticC. cunninghamiana arginine accounted for more than 25% of theamino compounds. Citrulline was the major translocated productfound in the stem exudate of symbiotic C. equisetifolia. Increasingconcentrations of ammonium nitrate in the nutrient solutionresulted in increasing levels of free ammonia and glutaminein xylem sap from stems of nodulated and non-nodulated C. glauca,but there was relatively little change in the prominent solutes,e.g. citrulline, proline, and arginine. The composition of nitrogenoussolutes in stem or root exudates of C. glauca was similar tothat of exudate collected from individual nodules and on thisbasis it was not possible to distinguish specific products ofcurrent N₂ fixation in xylem. The main differences in N solutecomposition between the symbioses were apparently due to hostplant effects rather than nodulation or the levels of combinedN. Also, the data indicate that the use of the proportion ofN in sap as citrulline (or indeed any other organic N solute)could not be used as an index of nitrogen fixation.     

The Influence of Foliar Applications of Urea on the Nodulation Pattern of Certain Leguminous Species

In greenhouse experiments with seven species of legumes, thespraying of urea on to the leaves was shown to affect nodulationadversely, without impairing the growth of the plants. In Phaseolusvulgaris, Vicia sativa, and Pisum sativum three-times-weeklysprays of 1 per cent. aqueous urea either prevented or markedlyreduced nodule development during the 8-week experimental period.In Medicago sativa and Trifolium pratense the urea treatmentresulted in a delay in nodulation so that numbers of nodulesat the first sampling (4 weeks) were reduced, while numbersat later samplings were higher since nodulation had been delayeduntil the root system was larger and provided a greater numberof potential nodule sites. In Trifolium hybridum and T. repensthe urea-treated plants showed reduced nodulation throughoutthe 6-week experimental period. In these experiments the advance effects on nodulation cannotbe due to high concentration of combined nitrogen in the rootingmedium, but it is suggested they derive from a high level ofnitrogen within the plant.     

Effect of Source of Nitrogen on the Growth of Fiskeby Soya Bean: the Carbon Economy of Whole Plants

Fiskeby V soya bean was grown from seed germination to seedmaturation with two contrasting patterns of nitrogen metabolism:either wholly dependent on dinitrogen fixation, or with an abundantsupply of nitrate nitrogen, but lacking root nodules. The carbonand nitrogen economies of the plants were assessed at frequentintervals by measurements of photosynthesis, shoot and rootrespiration, and organic and inorganic nitrogen contents. Plantsfixing atmospheric nitrogen assimilated only 25–30 percent as much nitrogen as equivalent plants given nitrate nitrogen:c. 40 per cent of the nitrogen of ‘nitrate’ plantswas assimilated after dinitrogen fixation had ceased in ‘nodulated’plants. The rates of photosynthesis and respiration of the shootsof soya bean were not markedly affected by source of nitrogen;in contrast, the roots of ‘nodulated’ plants respiredtwice as rapidly during intense dinitrogen fixation as thoseof ‘nitrate’ plants. The magnitude of this respiratoryburden was calculated to increase the daily whole-plant respiratory loss of assimilate by 10–15 per cent over thatof plants receiving abundant nitrate. It is concluded that ‘nodulated’plants grew more slowly than ‘nitrate’ plants inthese experiments for at least two reasons: firstly, the symbioticassociation fixed insufficient nitrogen for optimum growth and,secondly, the assimila tion of the nitrogen which was fixedin the root nodules was more energy-demanding in terms of assimilatethan that of plants which assimilated nitrogen by reducing nitratein their leaves.     

The Development and Significance of the Root Nodules of Casaurina

Young plants of Casuarina cunninghamiana and of C. equisetifoliagrowing in water culture developed macroscopic nodules in 26to 35 days from inoculation, the nodulation being most successfulat pH near to neutrality and falling off much more rapidly atlower pH than in other non-legume nodule-forming genera, confirmingthe distinctiveness of the Casuarina organism. The roots springingfrom the nodule lobes are shown to be characterized by upwardgrowth and in this to resemble remarkably closely the correspondingroots of Myrica. Nodulated plants of Casuarina are able to growvigorously in culture solution free of combined nitrogen, showingthat fixation of atmospheric nitrogen occurs, amounting in C.cunninghamiana to 50 mg. per plant during 6 months of activegrowth. The evidence indicates that the fixation occurs in thenodules, and that these have exactly the same functional significanceas those of legumes.     

Some Effects of Sucrose Concentration on Primordium Development in Excised Primary Roots

The effects of varying the amount of sucrose used to supplementthe culture medium maintaining the growth of excised roots ofPisum sativum L., Vicia faba L., Zea mays L. and Phaseolus vulgarisL., on the rates of primordium initiation and subsequent emergenceas lateral roots and on the duration of the interval betweenprimordium inception and emergence as a secondary root throughthe tissues of the primary have been investigated. Variation in the exogenous concentration of sucrose from 0.5to 8 per cent had little effect on the rate of primordium inceptionin Pisum and Vicia and the rates never reached the values obtainedfor the roots of the corresponding intact plants. Moreover,over the 6 day culture period lateral root emergence did notoccur in any of the excised roots of these two species. In contrast,each of the aspects of primordium development examined in theexcised roots of Zea and Phaseolus was markedly affected bythe amount of sucrose used to supplement the culture medium.In addition, in the presence of about 6 per cent sucrose, primordiumdevelopment in these cultured roots was very similar to thatin roots of the corresponding intact plants. The results indicate either that some factor necessary for primordiumdevelopment is present in adequate amounts in excised rootsof Zea and Phaseolus, but not in those of Pisum and Vicia, orthat the factors controlling such development are differentin the former and latter two species. Vicia faba L., Pisum sativum L., Zea mays L., Phaseolus vulgaris L., broad bean, garden pea, maize, dwarf bean, primordium development, sucrose concentration, cultured roots     

The Carbohydrate Nutrition of Tomato Roots: V. The Promotion and Inhibition of Excised Root Growth by Various Sugars and Sugar Alcohols

Iron is only consistently present in an available form in White'sroot culture medium if the inorganic salts are autoclaved withthe sugar. The substitution of ferric ethylenediamine-tetra-acetatefor the inorganic ferric salt of White's medium ensures ironavailability when the carbon source of the medium is renderedsterile by ether treatment and subsequently added to the remainderof the constituents which have been sterilized by autoclaving. The biological activity of sugars, and particularly of dextroseand laevulose, is altered by autoclaving them in presence ofthe inorganic salt solution of White's medium. The only sugar which supports a considerable growth of excisedtomato roots is sucrose. The activity of this sugar is not affectedby alcohol-precipitation nor is it dependent upon the simultaneouspresence of traces of its constituent mono-saccharides. Dextrose or laevulose or a mixture of the two sugars supporta low but sustained level of excised-root growth. All othersugars and sugar alcohols tested were inactive as carbon sources. The addition of sucrose at low concentration (0–2 percent.) to a medium containing dextrose as the main carbon compounddoes not make possible a level of growth comparable with thatobtained with an adequate sucrose supply. It has not been possibleto enhance the growth-rate of excised roots supplied with dextroseby previous presentation of this sugar under conditions permittingactive growth. Using media containing 'etherized' sucrose anddextrose, no evidence was obtained of any competitive inhibitionof sucrose utilization by dextrose. Certain sugars when added to a medium, containing the optimumconcentration of sucrose₁, markedly inhibited excised root growth.Thus l-sorbose, l- and d-arabinose, and d-xylose caused notless than 80 per cent, inhibition at a concentration of 0-5per cent. d-mannose and d-galactose completely inhibited growthat o-1 per cent. The oligosaccharides, dextrose, laevulose,and the sugar alcohols tested had, by contrast, very low inhibitoryactivity.     

Nitrate Assimilation in Higher Plants with Special Reference to the Cocklebur (Xanthium pennsylvanicum Wallr.)

A soluble NADH-dependent nitrate reductase is described forthe shoot system of Xanthium. Young leaves and immature stemtissues contain high levels of the enzyme. They are relativelyrich in free amino acids and amides but store little free nitrate.The specific activity of the enzyme is lower in fully expandedleaves, although these leaves exhibit higher rates of fixationof carbon in photosynthesis than do younger leaves. Neithernitrate nor free amino acids accumulate in the mesophyll ofthe leaf. Older parts of the stem axis accumulate large amountsof soluble nitrogen, almost entirely as free nitrate. Reservesof nitrate in the shoot and root are rapidly depleted if nitrateis removed from the external medium. Nitrate reductase is apparently absent from roots of Xanthium.This finding is supported by analyses of bleeding sap from nitrate-fedplants which show that 95 per cent of the nitrogen exportedfrom roots is present as free nitrate. However, roots are capableof synthesizing and exporting large amounts of amino nitrogenif supplied with reduced nitrogen such as urea or ammonium. A scheme is presented summarizing the main features of the metabolismof nitrate in Xanthium and this is compared with the situationin nitrate-fed plants of the field pea (Pisum arvense L.), aspecies previously shown to be capable of reducing nitrate inits root system.     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Symbiotic nitrogen fixation and physiological performance of bean (Phaseolus vulgaris L.) plants as affected by Rhizobium inoculum position and bean rugose mosaic virus infection

Bean (Phaseolus vulgaris L. var. Tacarigua) plants were grownin sterilized Leonard jars under controlled conditions. Beforesowing, 1 g of gamma irradiated peat containing the Rhizobiumtropici strain CIAT899 was placed at either 2 or 10 cm belowthe sand surface. Mechanical infection of bean rugose mosaicvirus (BRMV) was carried out in 3-d-old seedlings. Thus, theearly events of nodulation occurred before the arrival of virusparticles to roots. Rhizobium inoculation at 2 cm deep resultedin the formation of nodule clusters close to the crown, in contrastto the homogeneous nodulation along the roots observed in plantsinoculated with Rhizobium at a depth of 10 cm. The uniform arrangementof nodules on the roots enhanced the plant shoot biomass, althoughthe total nodule mass per plant did not differ between Rhizobiuminoculation treatments. Nodules located on deeper roots resultedin higher ureide concentrations in shoots and leaves and inreduced carbohydrate concentrations in leaves. In healthy plants,nodules formed on deeper roots had higher allantoinase activityand a greater carbohydrate concentration when compared to thatof nodules located close to the crown. Deeper nodules had ureideconcentrations similar to those of upper nodules, probably asa consequence of increased translocation of N-compounds to aerialorgans. A similar pattern of nodule formation and response toinoculum position was observed in BRMV-infected plants at allharvests. However, virus infection resulted in reduced totalnodule mass, shoot biomass, total leaf area and induced transitoryalterations in the ureide, -amino-N and carbohydrate concentrationin the different plant compartments. The effect of BRMV infectionon plant parameters was more evident during the vegetative stagesof growth. Nevertheless, the magnitude of the effect was alwaysmore pronounced in plants inoculated with Rhizobium at a depthof 2 cm compared to those Inoculated at 10 cm due to a greateractivity of deeper nodules despite virus infection. Deeper nodulesin BRMV-infected plants showed higher carbohydrate concentrationas well as higher allantoinase and uricase activity than thosedeveloped close to the crown, at all harvests. This observationwas further supported by ultrastructural analysis of virus-infectednodules, since virus replication took place in cells containingbacteroids of upper and lower nodules, but only in the interstitialcells of the latter. BRMV infection did not hinder the allantoinaseactivity and the chlorophyll content of uppermost mature leavesregardless of inoculum position. At the flowering and fruitingstages, healthy and BRMV-infected plants did not differ withregard to any of the tested parameters. Only inoculum positionhad an effect. The nearly normal functioning of the symbioticprocess at these stages of growth was attributed to the formationof a new generation of nodules in BRMV-infected plants subjectedto each of the Rhlzobium inoculation treatments. Key words: Bean rugose mosaic virus, symbiotic nitrogen fixation, bean, Rhizobium inoculum position, nodule ultrastructure