JXB Advance Access originally published online on April 25, 2005
Journal of Experimental Botany 2005 56(417):1779-1784; doi:10.1093/jxb/eri166
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
P-deficiency increases the O2 uptake per N2 reduced in alfalfa
1Institut für Agrikulturchemie der Georg-August-Universität Göttingen, Carl-Sprengel-Weg 1, D-37075 Göttingen, Germany
2INRA-ENSAM Sols Symbioses Environnement, Place Viala 1, F-34060 Montpellier cedex, France
* To whom correspondence should be addressed. Fax: +49 551 39 55 70. E-mail: jschulz2{at}gwdg.de
Received 22 October 2004; Accepted 16 March 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nodulated alfalfa (Medicago sativa L. cv. Saranac) plants were grown in hydroponics at P-sufficient and P-deficient supply levels. After 5 weeks of growth, dry matter accumulation, nodulation, total N and P accumulation, as well as 15N2 uptake, were measured. Moreover, the response of nodule O2-uptake to raising external pO2 was determined in an open-flow measurement system and nodule permeability was calculated. Plants in the P-deficient supply treatment had a lower P concentration in all organs. In both treatments the highest P concentration was found in nodules. In the P-deficient supply treatment plants formed less dry matter, had a lower shoot/root ratio, less nodulation, decreased total N accumulation, and lower 15N2 uptake per dry matter nodule. Nodules in the P-deficient treatment were, on average, smaller and had a higher O2 uptake per N2 reduced, coinciding with increased nodule permeability and conductance. Thus increased oxygen uptake appears to be a mechanism to adjust nodule metabolism to P deficiency in indeterminate N2-fixing nodules such as in alfalfa, as has previously been shown for determinate nodule forms.
Key words: Alfalfa, nitrogen fixation, nodule conductance, oxygen diffusion, phosphorus, respiration, rhizobia, symbiosis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
P-availability can limit legume productivity in the fields by a negative impact on nitrogen fixation. Römer and Lehne (2004)
showed that in a German loess (CAL-P=5 mg kg–1 soil) after long-term organic farming with no P fertilization, P was the principal limiting factor for broad-bean (Vicia faba L.) growth through strongly reduced N2 fixation giving reduced nitrogen availability for a subsequent crop. In tropical soils, P availability is one major restriction to legume crop productivity (Andrew and Robins, 1969). Growing N2-fixing root-nodules are strong phosphorus (P) sinks in legumes. P concentrations in nodules can reach up to 3-fold those of other plant parts (Sa and Israel, 1991). Thus the fast and positive growth reaction of legumes to P supply becomes understandable (Hoch-Jensen et al., 2002).
The metabolic functions of P in nodules are probably multifold and mostly related to intensive carbon and energy turnover. Nodule O2 permeability is thought to be involved in the regulation of nitrogen fixation (Hunt and Layzell, 1993). Restricted O2 supply to the infected zone functions as a widespread stress response of nodules (Denison, 1998). By contrast, P deficiency has been shown to increase nodule O2 conductance and uptake in soybean (Ribet and Drevon, 1995a) and common bean (Vadez et al., 1996).
The objective of the present study was to clarify whether an increased O2 conductance of nodules as a response to deficient P supply also occurs in the pasture legume alfalfa.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Biological material and growth conditions
Seeds of Medicago sativa L. cv. Saranac were surface-sterilized with 70% ethanol for 10 min, washed in sterile water, and germinated on water-saturated vermiculite. At 7 d after emergence (DAE) plants were transferred to glass cylinders (h=600 mm, inner diameter=20 mm) with an air-tight sealed cover (Fig. 1). Plants were held at their stem bases on the open side of the cylinder with sterilized cotton wool leaving roots in the nutrient solution. Plants were inoculated with 1 ml Sinorhizobium meliloti (102F51) inoculum at transfer, and were reinoculated during the first 10 d at each solution change. Inoculum was prepared by growing the bacteria in YEM at 28 °C for 3 d at an approximate cell density of 109 ml–1. The first nodules became visible 5–7 d after inoculation. Inoculation resulted in intensive nodulation and effective N2 fixation while an uninoculated control remained nodule free.
|
The cylinder contained 180 ml of an N and P-free basic nutrient solution consisting of 1 mM MgSO4, 0.7 mM K2SO4, 1.65 mM CaCl2, 16 µM Fe (as Fe-EDTA), 4 µM MnCl2, 22 µM H3BO3, 0.4 µM ZnSO4, 0.05 µM NaMoO4, and 1.6 µM CuSO4 buffered with 2 mM MES [2-(N-morpholino) ethane-sulphonic acid]. Urea to a concentration of 0.5 mM N was added to the nutrient solution during the initial 10 d of growth to avoid N-deficiency during nodule development. The solution was intensly aerated by an airflow of normal air of about 1 vol. min–1 during the experiment.
Two P treatments were applied as follows: sufficient P received KH2PO4 to the nutrient solution in the cylinder to a final concentration of 20 µM while the P concentration in deficient P was 5 µM. An appropriate amount of potassium as K2SO4 was added to the low-P solution to ensure equal potassium supply. In all cases the pH was adjusted to 6.0. The nutrient solution was renewed twice a day at 08.00 h and 04.00 h. Preliminary experiments showed that with the given experimental procedure plant growth appeared to be optimal at 15–20 µM P while above a concentration of about 25 µM severe P-toxicity developed. At a concentration of below 2–3 µM P, shoot development was strongly inhibited and deficiency symptoms occurred.
Plants were grown in a growth chamber at 26/18 °C day/night temperatures and 70–80% humidity with a 14 h photoperiod (06.00–08.00 h). Photosynthetic flux density was 665 µmol m–2 s–1 at shoot height. Plants were arranged in a fully randomized block design and the glass cylinders were wrapped in aluminium foil to ensure a dark rooting environment. Solution that had evaporated or passed through the plant was replaced by deionized water one to four times a day.
O2-uptake measurement
At 33 DAE additional gas inlets and outlets were placed in the cylinder and the open top was sealed with an approximately 10 mm layer of non plant-toxic silicon rubber (‘Tacosil 170’, Tauer and Co AG, Dresden, Germany). An additional layer of water on the silicon rubber ensured air-tight conditions and indicated possible leaks (Fig. 1). The nutrient solution was lowered to 1/3 of its original volume 24 h prior to any measurement to allow the plants to acclimate. The air volume in the cylinder was 115 ml and an airstream (20 kPa O2) of 15 ml min–1 was pumped through the root/nodule compartment. The airflow was kept constant by a flow controller (MKS instruments).
At 34 and 35 DAE the root compartments were connected to an open-flow gas exchange measurement system to measure root/nodule O2 uptake. Data for O2 (Oxynos 100, Rosamount) in the outflowing air were taken and O2 uptake could be calculated from the difference in the O2 content in the in- and out-flowing air and the flow rate (Fig. 1). The flow rate for the open flow measurement of O2-uptake was selected to have at least a difference of 0.1 kPa between the in- and out-flowing air, which could convienently be measured. Following the measurement at 20 kPa, the O2 pressure in the airstream was altered to 15 kPa (85/15, N2/O2, v/v). The airflow for the root/nodule was taken from a larger flow in which O2 pressure was regulated by changing the N2 and O2 mixture of the flow (Fig. 1). In this way a constant flow through the root/nodule compartment could be maintained. Five to eight minutes after changing the O2 content of the inflowing air, a new constant O2 content of the outflowing air was reached. Thus measurements of O2 in the outflowing air were, in all cases, taken at 15 min after switching to a new mixture. Preliminary experiments had shown that the O2 content in the outflowing air remained stable for at least 1 h at all O2 levels once the equilibrium was reached. Accordingly, measurements were made after changing to 25, 30, 40, and 50 kPa O2, in that sequence. Subsequently, the root compartment was disconnected, filled with fresh nutrient solution, and connected to an airstream of 1 vol. min–1.
15N2 uptake measurement
Forty-eight hours after the O2-uptake measurements, the root/nodule compartments were disconnected from the airstream and totally filled with fresh nutrient solution for the 15N2 uptake measurements of all replicates. Subsequently, 75% of the nutrient solution was replaced by a mixture of 15N2 (98 at%exc.)/O2 (80/20, v/v) so that all nodules were above the nutrient solution and the root/nodule compartment was sealed for 1 h. The 15N2 uptake was terminated by replacing the 15N2 containing atmosphere with water. This was followed by immediate harvest and drying of the plants.
Biomass parameter and statistical analysis
At harvest, plants were separated into nodules, roots, and shoots. Roots and shoots were immediately dried at 70 °C to a constant weight, while nodules were previously counted and classified according to their length (0.5–2 mm, 2–3 mm, above 3 mm). Nodule surface area (S) was calculated assuming that all alfalfa nodules are cylinders as 
where ni, di, and hi are the nodule number, mean diameter, and mean length, respectively, in each class. Based on sample measurements, nodule diameter was assumed to be 1 mm for nodules shorter than 2 mm and 1.5 mm for nodules longer than 2 mm.
After drying, all plant material was weighed and ground to a fine powder. N and 15N was determined with a combination of an elemental analyser (Vario EL, Firma Elementar Analysen GmbH, Hanau) and an emission spectrometer (NOI 7, Fischer Analysentechnik, Leipzig). P was determined according to Murphy and Riley (1962) after ashing the plant dry material.
All data were subjected to analysis of variance and mean values of the two P treatments were compared by the t-test. Regression analysis was performed for the dependence of root/nodule O2 uptake as a function of pO2. In all cases the Sigmastat analytical software was used.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant growth and nodulation
The level of P-deficiency applied in this work induced a significant growth reduction (Table 1). Total growth and the shoot/root ratio were significantly lower under P deficiency. Moreover, the slope of a linear regression of total dry matter accumulation as a function of total plant P was increased in the P-deficient treatment (Fig. 2).
|
|
Nodule dry matter was higher in the P-sufficient treatment (164%) (Table 1). This difference was only due to the larger number of nodules in the class of greatest length (Fig. 3), whereas nodule number per plant did not differ between treatments (Table 1). Thus, overall surface and dry matter of nodules per plant were higher as a result of larger individual nodule size (Fig. 3) and dry matter (Table 1) under P sufficiency.
|
By contrast, neither the shoot per nodule nor the total plant dry matter per nodule ratios differed between both P treatments (Table 1). The linear regression of total dry matter formation as a function of nodule dry matter revealed no difference between treatments (Fig. 4).
|
P distribution and total P-uptake
Whatever the P treatment, P was preferentially transported into nodules since, even under P deficiency, P concentration (mg g–1) was higher in nodules than in roots or shoots. Under P deficiency, nodules had only about half the P concentration of those in the P-sufficient treatment (Fig. 5).
|
Under P sufficiency, nodule P concentration was also twice as high as that in roots, and in shoots to a lesser extent, and P concentration was much higher in all plant organs (Fig. 5). Thus, total P-uptake was increased by 374% when compared to the P-deficient treatment.
N assimilation and 15N2-uptake
Total N uptake calculated from the N concentration and plant dry matter was increased in the P-sufficient treatment by 225% when compared to the P-deficient treatment (Table 2). Since N-concentration in the P-deficient treatment was lower in all plant organs, the difference in N accumulation was even stronger than that in dry matter. Total N uptake per individual nodule was increased in the P-sufficient treatment whereas total N uptake per total plant P was decreased (Table 2).
|
In order to evaluate the nodule nitrogenase activity at one stage of the growth curve, 15N2-uptake was measured per individual intact plant. The corresponding values in the P-sufficient treatment were increased by 274% or 167%, as expressed per plant or nodule dry matter, respectively. However, nitrogen fixation per nodule P was significantly increased in the deficient P treatment (Table 2).
O2 uptake linked to nitrogen fixation
Since nitrogenase activity depends upon nodule respiration, the nodulated-root O2 uptake per plant was measured on intact plants, the day before the above nitrogen fixation measurement. The corresponding values were significantly higher by 13% in the P-sufficient treatment at ambient oxygen pressure when compared to the P-deficient treatment (Table 3).
|
In order to assess the effect of P supply on nodule permeability, and the subsequent nitrogenase-linked respiration, the response of nodulated-root O2 uptake to variation of rhizospheric pO2 was measured following the principles described in detail in Jebara and Drevon (2001)
. The O2 uptake to pO2 showed a typical saturation curve indicating that nitrogenase activity was oxygen limited at ambient oxygen pressure (Fig. 6). In both treatments the increase in O2 uptake appeared to be linear between 20 kPa and 30 kPa O2. Thus, nodule permeability was caculated as the slope of the linear response of O2 uptake as a function of pO2 in the 20–30 kPa O2 interval. From data in Fig. 6, P deficiency decreased the overall permeability of the nodule population from a mean value of 2133±325 mm3 h–1 compared with 3339±332 for P sufficiency.
|
The nodule conductance could be calculated by dividing the above permeability values per individual plant by the nodule area calculated from nodule number per plant in Table 1. P deficiency significantly increased the nodule conductance with a mean value of 17.3±1.9 µm s–1 compared with 13.2±1.5 µm s–1 under P sufficiency. Consequently, nitrogenase-linked respiration per unit nodule in the P-deficient treatment was 123% of that in the P-sufficient treatment (Table 3). Thus nodules consumed about twice as much O2 per unit fixed N2 in the P-deficient treatment when compared with the P-sufficient treatment.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Increased nodule O2 permeability (Table 3) confirms, with undeterminate nodules of alfalfa, earlier similar reports on determinate nodules of soybean (Ribet and Drevon, 1995a
) and common bean (Vadez et al., 1996). In this work, the methodical approach was developed further by using 15N2 uptake to measure nitrogenase activity, thus avoiding problems of the flow-through acetylene reduction or H2 evolution assays (Minchin et al., 1983; King and Layzell, 1991; Ribet and Drevon, 1995b), and by using a differential, open-flow O2 uptake measurement for the nodulated-root compartments. The nodules developed under submerged conditions, but did not show visible differences to those grown in soil or sand. The measurement was subsequently done in a gaseous environment after lowering the nutrient solution. It cannot be totally excluded that this somehow influenced nodule permeability, although the plants were allowed to adapt for 24 h.
The physiological importance of increased O2 permeability as a response to P-deficient supply is not yet understood. In P-deficient nodules the adenylate charge, at least of the plant fraction, apears to be decreased (Sa and Israel, 1991). Thus the high O2 uptake might contribute to maintaining a sufficient adenylate charge for high N2 fixation rates. In addition, alternative oxidases are increasingly expressed in P-starved tissues (Rychter et al., 1992). A similar increase in nodule tissues where alternative oxidases are expressed (Millar et al., 1997), could contribute to changes in respiratory costs of nitrogen fixation (Schulze et al., 2000; Adgo and Schulze, 2002). Higher O2 consumption would create an O2 sink which would then, in turn, induce the observed increase in nodule O2 permeability. Increases in nodule permeability were previously associated with nodule cortex-cell expansion (Drevon et al., 1998). A triggering mechanism for such changes involving a low P status in those cells has not yet been elucidated. Inorganic P accumulation in the nodule cortex during nodule growth was shown with 31P-NMR studies (Rolin et al., 1989) and might be involved in osmoregulatory changes in cell size. However, in the case of these experiments the proportion of P in nodules to plant total P did not differ between both treatments, although P concentration in nodules was much higher compared with roots or shoots in both P treatments. Moreover, during recovery from P deficiency, nodules are the primary P sink (Israel, 1993). If the legume adaptation to deficient P comprises a preferential allocation of P to nodules, a relatively higher flow of P into nodules might occur at more severe P restrictions than in this study's experiment. In this work, the most apparent morphological adaptation of the plants grown at deficient P was a smaller individual nodule size, although nodule number per plant did not differ (Table 1).
The nodulated-root compartment had to be as small as possible to allow a differential O2 measurement against a large O2 background pressure. The chosen size of 180 ml for this compartment required the nutrient solution containing 20 µM P to be changed twice a day for a sufficient P supply, since concentrations above 25 µM P would induce P toxicity (Bell et al., 1990; Tang et al. 2001). At 5 µM P for the P-deficient supply, N2 fixation had been particularly affected compared with other growth processes (Table 2), although nitrogenase activity per unit nodule dry matter is maintained for much longer (Almeida et al., 2000; Hoch-Jensen et al., 2002). The increased N/P ratio under deficient P supply (Table 2), consistent with the results of Almeida et al. (2000) and Hoch-Jensen et al. (2002), may indicate an N-feedback effect. In P-stressed tissue, RNA levels are reduced (Nurnberger et al., 1990), resulting in impaired protein synthesis and free amino acid accumulation (Johnson et al., 1996), in particular asparagine in nodules (Almeida et al., 2000). However, total N concentration in the P-deficient nodules was much lower, which might have been the result of less infected tissue in this treatment. The way by which a putative amino acid accumulation would exert its feedback effect is largely unknown (Schulze, 2003), although Neo and Layzell (1997) demonstrated that oxygen diffusion into the nodule is involved, but the data from this study indicate that a P-deficient supply increases rather than decreases nodule O2 permeability.
In conclusion, alfalfa adapt by forming smaller nodules with higher O2 permeability and O2 consumption per unit fixed nitrogen as well as O2 consumption per nodule P. The primary metabolic effect, if any, of P status on nitrogenase functioning remains obscure, and it is unclear in which way it would translate into nodule permeability. Addressing these questions appears to be worthwhile since there is considerable variability in the tolerance of legumes to P deficiency in which the adjustability of nodule O2 permeability is involved (Vadez et al., 1996; Vadez and Drevon, 2001).
|
Acknowledgements |
|---|
We thank Carroll P Vance (University of Minnesota) for providing the rhizobial strain and plant material. Joachim Schulze is indebted to the ‘Deutsche Akademie der Naturforscher, Leopoldina’ for providing a fellowship (FKZ BMBF-LPD 9801-19) that allowed him to complete this study.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Adgo E, Schulze J. 2002. Nitrogen fixation and assimilation efficiency in Ethiopian and German pea varieties. Plant and Soil 239, 291–299.[CrossRef]
Almeida JPF, Hartwig UA, Frehner M, Nösberger J, Lüscher A. 2000. Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.). Journal of Experimental Botany 51, 1289–1297.
Andrew CS, Robins MF. 1969. The effect of phosphorus on the growth and chemical composition of some tropical pasture legumes. II. Nitrogen, calcium, potassium and sodium contents. Australian Journal of Agricultural Research 20, 275–285.
Bell RW, Edwards DG, Asher CJ. 1990. Growth and nodulation of tropical food legumes in dilute solution culture. Plant and Soil 122, 249–258.[CrossRef]
Denison FR. 1998. Decreased oxygen permeability: a universal stress response in legume nodules. Botanica Acta 111, 191–192.[ISI]
Drevon JJ, Frangne N, Fleurat-Lessard P, Payre H, Ribet J, Vadez V, Serraj R. 1998. Is nitrogenase-linked respiration regulated by osmocontractile cells in legume nodules? In: Elmerich C, Kondorosi A, Newton, W, eds. Biological nitrogen fixation for the 21st century. Dordrecht, The Netherlands: Kluwer Academic Publishers, 465–466.
Hoch-Jensen H, Schjoerring JK, Soussana JF. 2002. The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants. Annals of Botany 90, 745–753.
Hunt S, Layzell DB. 1993. Gas exchange of legume nodules and the regulation of nitrogenase activity in soybean. Annual Review of Plant Physiology and Plant Molecular Biology 44, 483–511.[CrossRef][ISI]
Israel DW. 1993. Symbiotic dinitrogen fixation and host-plant growth during development of and recovery from phosphorus deficiency. Physiologia Plantarum 88, 294–300.[CrossRef]
Jebara M, Drevon JJ. 2001. Genotypic variation in nodule conductance to the oxygen diffusion in common bean (Phaseolus vulgaris). Agronomie 21, 667–674.[CrossRef]
Johnson JF, Allan DL, Vance CP, Weiblen G. 1996. Root carbon dioxide fixation by phosphorus-deficient Lupinus albus. Contribution to organic acid exudation by proteoid roots. Plant Physiology 112, 19–30.[Abstract]
King BJ, Layzell DB. 1991. Effect of increases in oxygen concentration during the argon-induced decline in nitrogenase activity in root nodules of soybean. Plant Physiology 96, 376–381.
Millar AH, Finnegan PM, Whelan J, Drevon JJ, Day DA. 1997. Expression and kinetics of the mitochondrial alternative oxidase in nitrogen-fixing nodules of soybean roots. Plant, Cell and Environment 20, 1273–1282.[CrossRef]
Minchin FR, Witty JF, Sheehy JE, Muller M. 1983. A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. Journal of Experimental Botany 34, 641–649.
Murphy J, Riley JP. 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31–36.[CrossRef]
Neo HH, Layzell DB. 1997. Phloem glutamine and the regulation of O2 diffusion in legume nodules. Plant Physiology 113, 259–267.[Abstract]
Nurnberger T, Abel S, Jost W, Glund K. 1990. Induction of an extracellular ribonuclease in cultured tomato cell upon phosphate starvation. Plant Physiology 92, 970–976.
Ribet J, Drevon JJ. 1995a. Increase in permeability to oxygen and in oxygen uptake of soybean nodules under deficient phosphorus nutrition. Physiologia Plantarum 94, 298–304.[CrossRef]
Ribet J, Drevon JJ. 1995b. Phosphorus deficiency increases the acetylene induced decline of nitrogenase activity in soybean (Glycine max L. Merr.). Journal of Experimental Botany 46, 1479–1486.
Rolin DB, Pfeffer E, Boswell T, Schmidt JH, Tu SI. 1989. In vivo 31P NMR spectroscopic studies of soybean–Bradyrhizobium symbiosis. FEBS Letters 254, 203–206.[CrossRef]
Römer W, Lehne P. 2004. Vernachlässigte Phosphor- und Kaliumdüngung im ökologischen Landbau senkt die biologische Stickstoffixierung bei Rotklee und den Kornertrag bei nachfolgendem Hafer. Journal of Plant Nutrition and Soil Science 167, 106–113.[CrossRef]
Rychter AM, Chauveau M, Bomsel JL, Lance C. 1992. The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots. Physiologia Plantarum 84, 80–86.[CrossRef]
Sa T, Israel DW. 1991. Energy status and functioning of phosphorus-deficient soybean nodules. Plant Physiology 97, 928–935.
Schulze J. 2003. Source–sink manipulations suggest an N-feedback mechanism for the drop in N2 fixation during pod-filling in pea and broad bean. Journal of Plant Physiology 160, 531–537.[CrossRef][ISI][Medline]
Schulze J, Beschow H, Adgo E, Merbach W. 2000. Efficiency of N2 fixation in Vicia faba L. in combination with different Rhizobium leguminosarum strains. Journal of Plant Nutrition and Soil Science 163, 367–373.[CrossRef]
Tang C, Hinsinger P, Drevon JJ, Jaillard B. 2001. Phosphorus deficiency impairs early nodule functioning and enhances proton release in roots of Medicago truncatula L. Annals of Botany 88, 131–138.
Vadez V, Drevon JJ. 2001. Genotypic variability in phosphorus use efficiency for symbiotic N2 fixation in common bean (Phaseolus vulgaris). Agronomie 21, 691–699.[CrossRef]
Vadez V, Rodier F, Payre H, Drevon JJ. 1996. Nodule permeability to O2 and nitrogenase-linked respiration in bean genotypes varying in the tolerance of N2 fixation to P deficiency. Plant Physiology and Biochemistry 34, 871–878.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. SCHULZE, G. TEMPLE, S. J. TEMPLE, H. BESCHOW, and C. P. VANCE Nitrogen Fixation by White Lupin under Phosphorus Deficiency Ann. Bot., October 1, 2006; 98(4): 731 - 740. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||