Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Nitrogen fixation of lucerne (Medicago sativa) in an acid soil: The use of rhizotrons as a model system to simulate field conditions

The effect of pelleting seeds of lucerne with lime was studied in an acid sandy soil. In pot experiments, the fraction of seedlings with crown nodules, i.e. nodules on the upper 10 mm of the taproot, increased from 26% to 71%. In rhizotrons, the application of CaCO₃ resulted in an even stronger response.An agar-contact method was used to study pH changes in the rhizosphere during a period up to 12 days. Application of 1.0 µmol of CaCO₃, in drops of 12 µL volume, resulted in an initial soil pH of 6.1 and yielded 75% crown nodulation. In the absence of CaCO₃, roots induced a pH increase from 5.1 (day 0) to 5.7 (day 12). However, this did not increase nodulation (5%). Obviously, this type of alkalinization does not overcome the acid-sensitive step of the nodulation process.     

Effect of lime-pelleting on the nodulation of lucerne (Medicago sativa L.) in an acid soil: A comparative study carried out in the field,in pots and in rhizotrons

The nodulation of lucerne was studied in soil (pH-H₂O 5.2) with seeds either inoculated with Rhizobium meliloti (R), or inoculated and pelleted with lime (RP). For comparison, experiments were done in the field and in two types of micro-cosmos: pots and rhizotrons. In the field experiments, lime-pelleting improved the establishment of seedlings and augmented the nitrogen yield of the first harvest. These positive responses in plant growth were the consequence of a better nodulation on the upper 10 mm of the seedling tap root. The number of seedlings carrying crown nodules increased from 18% (R) to 56% (RP) at 26 days after sowing.In both, pots and rhizotrons, lime-pelleting also increased crown nodulation: in pots from 32% (R) to 60% (RP), and in rhizotrons from 5% (R) to 90% (RP). Rhizotrons, made of plastic petri dishes, allowed for continuously following of early root developments and nodule formation. Crown nodulation could already be measured after 14 days. Based on these experiments, it was concluded (i) that crown nodulation is an adequate parameter to quantify the benefit of lime-pelleting, and (ii) that rhizotrons, because of the more pronounced effects and shorter incubation time, are more suitable to study the nodulation responses in the soil caused by the addition of rhizobia and lime.     

Microgametophytic selection in two alfalfa (Medicago sativa L.) clones

Summary Microgametophytic selection was investigated using two ecologically diverse autotetraploid clones of alfalfa. Several selection pressures (drying, aging, freezing, and high and low temperatures) were applied to microgametophytes at three stages of the life cycle, 1) during microsporogenesis, 2) post-anthesis, and 3) pollen tube growth. Pollen aging produced a progeny population with a greater mean plant size and a lower coefficient of variation than the control progeny. High temperature (29.5 °C) applied both during microsporogenesis and pollen tube growth resulted in progeny populations which were significantly taller and, in one case, had a larger leaf number than the control populations. In contrast, air dried pollen resulted in a progeny population which had significantly smaller character means and larger coefficients of variation than the control population. Also, low temperature (15 °C) during pollen tube growth yielded progeny with reduced branch number and a larger coefficient of variation than the control progeny. In cases where progeny derived from selected microgametophytes were found to differ from the control offspring, corresponding shifts in the reciprocal cross were not observed. For the temperature stress treatments, the lack of reciprocal differences may be related to the different temperature adaptations of the two ecotypes. These results suggest that microgametophytic selection can be effective in shifting the mean of the progeny generation; however, the results obtained will vary depending upon the selection pressure, stage of selection, and the parents used.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Simplified measurement of soil pH using an agar-contact technique

A method for the indirect measurement of soil-pH is described. This method allows the spatial arrangement of soil and rhizosphere to be conserved. The soil is brought into contact with a layer of agar, containing bromocresol purple. A nylon gauze is placed between soil and agar. For quantitative pH measurements, a micro-electrode is inserted into the agar after three hours of contact between soil and agar.The validity of the method was checked by comparing its results with those obtained by standard procedures. At different pH-levels (pH 5.0 to 7.0) in either a sandy or a clay soil, a high correlation (r²=0.98) was found between the two methods. However, in the case of the clay soil, the agar-pH was significantly lower than the standard-pH. In the sandy soil, in the range pH 5.0 to 6.0, the results of both methods agreed very well. The agar method was used to measure the pH dynamics in the rhizosphere of lucerne seedlings, grown in rhizotrons.     

A comparison between the acetylene reduction method,the isotope dilution method and the total nitrogen difference method for measuring nitrogen fixation in lucerne (Medicago sativa L.)

Summary In view of the increasing need for the exact estimation of the input of nitrogen in agroecosystems, an application of the acetylene-reduction technique was developed. The technique, consisting of a plastic bag incubation system, using propane as an internal standard of the apparent volume, made it possible to carry out repeated incubations on the same plant system. The evaluated technique included studies of the diffusion of ethylene and propane in a soil column, as well as studies of the optimal substrate concentration and grade of purity of the substrate. In addition, the conversion factor between amounts of reduced acetylene as compared wtih reduced dinitrogen was determined by¹⁵N₂ incubations to be 4.41. The developedin situ acetylene-reduction technique was compared with an isotope dilution method and a total nitrogen difference method. By comparing the derived total nitrogen fixation values from each with the value derived from the acetylene-reduction method; it was shown that the values differed significantly. The acetylene-reduction method gave the highest nitrogen fixation values, the isotope dilution the lowest values and the total nitrogen difference method was intermediate. No statistical significant difference existed between the two different reference crops used in the isotope dilution method.     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.     

Ecological genetics of Rhizobium meliloti: symbiotic plasmid transfer in the Medicago sativa rhizosphere

A potentially important aspect of the ecological genetics of fast-growing rhizobia is lateral transfer of symbiosis-related genes between strains in the host-legume rhizosphere. We have therefore characterized transfer of the Rhizobium leguminosarum symbiotic (Sym) plasmids pJB5JI and pSymR1897 to strain WL113, a non-attaching, non-nodulating Rhizobium meliloti recipient, in the rhizosphere of its legume host Medicago sativa. Interspecific transfer of pJB5JI generated symbiotically proficient transconjugants of WL113, demonstrating that a mechanism exists for genetic flux in indigenous R. meliloti populations in response to selection pressure provided by the host legume. This could generate fluid groupings of R. meliloti rather than discrete and stable strains.     

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones

Summary Alfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Alfalffa,Medicago sativa L., in highly weathered,acid soils

Summary Alfalfa plants,Medicago sativa L., were selected from the Florida 66 cultivar for vigor in an acid (pH 4.4, Al≥.4 meq/100 g) and a limed, fertilized (pH 6.5, Al=0 meq/100 g, P and K added) Cecil topsoil. The selected plants were intermated by selection condition to achieve two germplasms, acid selected (A-1) and limed, fertile selected (L-1). ARhizobium meliloti strain (79-4s) was isolated from a high acetylene reducing nodule from a plant in a similar acid soil. The germplasms and the Rhizobium strain were then tested in greenhouse pots for agronomic performance under a variety of soil pH and fertility conditions. The 79-4s inoculum, as well as commercially prepared inoculum, gave better plant yield and acetylene reduction (N₂-fixation) at all harvests when compared to a sterile peat control, but the commercial inoculum was the best inoculum treatment. The A-1 germplasm produced higher shoot dry weight at the final harvest than did the L-1 germplasm at all soil pH’s when P and K were applied at the highest rates. The A-1 germplasm also had better root weight (mainly fibrous roots) and acetylene reduction in these soil conditions. The two germplasms appear to be genetically distinct and respond differently depending on soil pH and fertility conditions.     

Genotypes of lucerne (Medicago sativa L.) show differential tolerance to manganese deficiency and toxicity when grown in bauxite residue sand

The physical and chemical characteristics of bauxite residue sand (BRS) affect the availability of a number of nutrients to plants, especially manganese (Mn). Lucerne (Medicago sativa L.) has been chosen as a BRS revegetation species because of its deep-rooting habit and tendency to tolerate moderately alkaline and saline soils, even though it is still prone to Mn deficiency stress. Sixteen commercially available lucerne genotypes were grown in BRS after addition of 5, 50 or 500 g Mn g^–1 BRS in a glasshouse. Manganese deficiency and toxicity symptoms were observed in 5 and 500 g g^–1 treatments, respectively. Symptom expression varied in severity between genotypes. Relative tolerance to Mn deficiency was defined by shoot dry weight at 5 g Mn g^–1 as a percentage of shoot dry weight at 50 g Mn g^–1. Salado, a genotype tolerant to Mn deficiency, and Sirosal, a genotype intolerant to Mn deficiency, were then grown with 0, 10, 20, 50, 100, 200 or 800 g Mn g^–1 BRS and found to have critical shoot Mn concentrations of 17.7 (Salado) and 26.6 g g^–1 (Sirosal). The use of genotypes with high relative Mn deficiency tolerance is recommended to help improve sustainability of BRS revegetation as well as to improve productivity on Mn-fixing agricultural soils.