Non-additive gene effects for creeping-root in lucerne (Medicago sativa L.)

Summary Both general and specific combining abilities for creeping-rootedness of lucerne were found to be highly significant although there were substantial differences among genotypes for both parameters. These results indicate that both additive and non-additive gene effects are involved in the genetic substrate of creeping-rootedness; hence utilization of heterosis would seem to be the most appropriate procedure for further improvement in this trait.     

Tolerance to manganese toxicity among cultivars of lucerne (Medicago sativa L.)

Two sand culture experiments were carried out to identify commercial cultivars of lucerne or alfalfa (Medicago sativa L.) which contain elite, Mn-tolerant plants for use in a selection programme to increase the acid-soil tolerance of this perennial legume. Differences in Mn tolerance, both within and between cultivars, were observed when a range of cultivars were exposed to regular waterings with dilute nutrient solution containing 20 or 25 mg Mn L^–1. Under these moderately toxic regimes, the winter dormant cultivars Cimmaron and WL 318 were found to contain elite plants that had greater dry matter yields than their mean cultivar yield under non-toxic Mn conditions.Cultivars which contained elite, Mn-tolerant plants could not be identified by phenotypic characteristics such as their height or their toxicity symptom score, nor by their winter dormancy class. Possible reasons for the occurrence of elite plants in these cultivars are discussed. The elite, high yielding Mn-tolerant plants could not be identified from the other plants within their cultivar population by their Mn toxicity symptoms nor by their height.     

Isolation of pathogen-synthesized fraction that elicits phytoalexins in lucerne (Medicago sativa L.)

The production of phytoalexins by lucerne cultivars Dupuits (susceptible to Verticillium wilt) and Maris Kabul (resistant to Verticillum wilt) in response to substances (= elicitors) produced by pathogenic and non-pathogenic isolates of Verticillium albo-atrum was investigated. The elicitors are produced in culture filtrates from log-phase and past log-phase stages. The elicitors were isolated by gel-filtration and found to contain both protein and carbohydrate. They were found to be thermostable and non-dialyzable and proved to be sensitive to protease and sodium hydroxide treatment which suggests that they are glycoproteins. There was no evidence of specificity of the elicitors.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: pH dynamics in the rhizosphere of seedlings growing in rhizotrons

A lime-pellet around seeds of lucerne significantly increased crown nodulation in an acid soil. To investigate whether neutralization or calcium were of importance when lime was supplied, experiments with plants were done either in pots or in rhizotrons. Crown nodulation was used to quantify the effect of these two parameters.For the neutralization of the soil, KOH (in pots) or K₂CO₃ (in rhizotrons) was added. The crown nodulation of pot-grown plants increased from 31% to 53%. In rhizotrons, the number of crown-nodulated seedlings increased from 9% to 53%. If calcium was supplied additionally (as CaCl₂ or CaSO₄), 63% crown nodulation was found in pots, and 68% in rhizotrons. These numbers are close to the crown nodulation with lime (CaCO₃) alone: 70% in pots and 71% in rhizotrons. In the soil studied, the beneficial effect of lime is largely due to neutralization (80%), and only a minor part (20%) is due to the input of calcium.Using rhizotrons, the dynamics of the pH in the rhizosphere of lime-treated and untreated seedlings was followed during a period of 12 days. It was found that, even in the absence of lime, the pH along the taproot increased from 5.1 to 5.7. However, this did not result in the formation of root nodules. Nodulation was obtained only by adding neutralizing chemicals, which increased the pH during the initial 3 days, the acid sensitive period of the process.     

The response of lucerne (Medicago sativa L.) to sodium sulphate and chloride salinity

Sodium and sulphate-dominated salinity is a serious environmental problem occurring in soils and groundwater in many parts of the world. The effect of Na2SO4 and NaCl, at electrical conductivity levels ranging from 2 to 17 dS m-1, on the growth and tissue ion concentrations of 16 lines of lucerne (Medicago sativa L.) was examined in the greenhouse over a 2 month period. Averaged across all lines, plants grown at 17 dS m-1 produced 66% of the dry matter of plants grown at 2 dS m-1. However there were significant differences among lines in relative salt tolerance (as defined by the slope of the reduction in dry matter) versus electrical conductivity. Dry matter production was negatively correlated with shoot concentrations of Na+, Cl- and S2- and generally lines that were more tolerant to salinity had lower concentrations of those ions in the shoots. We conclude that lucerne is moderately tolerant to Na2SO4 -predominated salinity, and that the degree of intraspecific variation that exists within this species will allow more tolerant lines to be selected for establishment in conditions where sulphate salinity is a problem.Collaborator     

Effects of sulphur nutrition on nitrogen and carbon metabolism in lucerne (Medicago sativa L.)

Studies were conducted with one-year-old plants of the perennial legume, lucerne ( Medicago sativa L.), to determine the effects of various levels of S (0.3, 1.5, and 7.5 m M ) on N₂-fixation, photosynthesis, herbage free amino acid pools and protein amino acids, levels of protein, and activities of key enzymes of leaf nitrogen and carbon metabolism. Sulphur deficiencies in the 0.3 m M S treatment, as determined by N:S ratios, did not appear until the second growth cycle. This treatment was severely S-deficient by the end of the third growth cycle. Sulphur deficiencies did not occur in the other two treatments over three cycles of growth. By the middle of the second growth cycle acetylene reduction rates of plants grown with 0.3 m M S were lower than those of other treatments. At the end of the third growth cycle acetylene reduction rates of the 0.3 m M S treatment were ca . 30–35% that of the other treatments. There was no effect of S-deficiency on photosynthesis, levels of leaf NADP-isocitrate dehydrogenase and glutamine synthetase activity, or concentrations of leaf protein over the course of the experiments. Levels of NAD-glutamate dehydrogenase, NAD-malate dehydrogenase, and glutamate oxaloacetate trans-aminase activity and concentrations of free methionine, arginine, urea, and ammonia increased, with S-deficiency. Boron concentrations were very high (ca. 300 μg/g dry wt.) in S-deficient plants. The mole percent methiomne in leaf protein decreased slightly and chlorophyll concentration decreased markedly with S-deficiency. This study suggests that N₂-fixation is affected early during S-deprivation of lucerne and that subsequent effects of S-deficiency may be due to loss of fixed nitrogen.     

Evaluation of CropSyst for studying the effect of mulching with lucerne (Medicago sativa L.) in Austria

Conservation of water under semi-arid conditions is of supreme importance to combat forthcoming water scarcity. Crop residue mulches are the key interventions in this regard. Simulation models are emerging as strong analytical tools to study the effect of agronomic interventions but their use to study effect of mulches in Austria is limited. To bridge this knowledge gap, we planned a study to evaluate the efficacy of CropSyst for studying response of lucerne mulches in succeeding lucerne crop. CropSyst is a user-friendly, conceptually simple but sound multi-year multi-crop daily time step simulation model. Above ground biomass and soil water content from field experiments carried out near the city of Vienna, Austria (2007–2008) were compared with simulated results to determine the suitability of CropSyst in regarding mulch. Adequacy of model to regard mulch is demonstrated while pros and cons of disagreement between experimental and modeling results are discussed with a way forward for future research.     

RAPD polymorphism in the prebreeding material for cultivation of synthetic variations of lucerne (Medicago sativa L.)

Genetic diversity between synthetic cultivars (Syn5, Syn7), inbred lines (D₃, D₅, E_1/2, G_1/1, G_1/2) and ecotypes (E16, E51, E182, E231) of lucerne (Medicago sativa L.) was studied using the RAPD-PCR method. The plants differed in the efficiency of seed set and in the yield of green mass. The ecotypes E182 and E231 and the synthetic population Syn5 showed the highest fertility. Additionally, Syn5 also showed the highest efficiency of seed set and the yield of green mass. Among the inbred lines, D3 was characterised by the highest yield of green mass and E1/2 by the highest fertility. An optimal combination of yield and biomass was observed for the synthetic population Syn5, obtained by crossing the lines D₃, D₅ and G_1/1, as demonstrated using comparative analysis. A total of 338 polymorphic products were generated using 20 arbitrary primers. Cluster analysis using the Unweighted-Pair Group Method with Arithmetic Mean (UPGMA) in the Molecular image Gel Doc? XR (Bio-Rad) software based on the Dice’s coefficient of genetic similarity showed a division of the studied forms into two groups based on genetic similarity. The ecotype E16 formed one of the groups whereas all of the other ecotypes observed in this study clustered into the second group. A high level of polymorphism among the studied lucerne forms was detected indicating an interesting gene pool awaiting future exploration. Analysis of variance also supported a high diversity among the studied forms. This study provides insightful information into the heterosis effect of synthetic populations or hybrids of F1 lucerine by providing correlations between the genetic background of the inbred lines and their ability to produce a specific yield.     

Root hair infection and nodulation in lucerne (Medicago sativa L.) as influenced by salinity and alkalinity

Summary Experiments with lucerne (Medicago sativa L.) under bacteriologically controlled conditions on the effects of chloride, carbonate and bicarbonate of sodium, alkaline pH levels and saline/alkaline soil extracts demonstrated that increasing levels of salinity/alkalinity of the root medium resulted in a root system devoid of root hairs, mucilaginous layer and infection thread formation in spite of the optimum growth of Rhizobium under such conditions. The results point out the existence of alkali sensitive steps in the early phases of nodulation which lead to an elimination of legume rhizosphere as postulated by earlier workers.     

Effects of arbuscular mycorrhiza and phosphorus application on arsenic toxicity in sunflower (Helianthus annuus L.) and on the transformation of arsenic in the rhizosphere

Effects of arbuscular mycorrhiza (AM) and phosphorus (P) application on arsenic (As) toxicity were studied in a rhizobox system with As-contaminated soil collected from Shimane Prefecture, Japan. The treatments consisted of a combination of two levels of AM (Glomus aggregatum) inoculation (−AM and +AM) and two levels of P application (−P and +P at 30 mg P kg⁻¹). Sunflower (Helianthus annuus L.) seedlings were cultured in rhizoboxes for 6 weeks. Rates of root AM infection in +AM treatments were about 40% regardless of P application. AM inoculation as well as P application reduced As toxicity symptoms, most clearly so in the +AM−P treatment. Plant growth was highest in the +AM + P treatment. Shoot As concentrations were slightly reduced by AM inoculation but enhanced by P application. Shoot P concentration in the +AM−P treatment was similar to that of +P treatments and was higher than in −AM−P. Analyses of rhizosphere soils at the end of the cultivation period indicated that P application increased water-soluble As (WS−As) in all compartments while AM inoculation increased WS−As in the central compartment only. Both the WS−arsenite [WS−As^(III)] and the dominant form, arsenate [WS−As^(V)], showed gradients toward the root surface. Dimethylarsine (DMAA) was detected in the +AM treatments only. To our knowledge, this is the first report of the occurrence of DMAA in the mycorrhizosphere. AM inoculation increased WS−P similarly as +P treatments did and promoted acid phosphatase activity in the soil. In conclusion, AM inoculation alleviated the effects of As toxicity by improving P nutrition without increasing As concentrations in the shoots. Moreover, AM appeared to be involved in the transformation of soil inorganic As into less toxic organic forms.     

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.     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.     

Flavonoids enantiomer distribution in different parts of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.) and phacelia (Phacelia tanacetifolia Benth.)

Plant material is a rich source of valuable compounds such as flavanones. Their different forms influence bioavailability and biological activity, causing problems with the selection of plant material for specific purposes. The purpose of this research was to determine selected flavanone (eriodictyol, naringenin, liquiritigenin, and hesperetin) enantiomer contents in free form and bonded to glycosides by an RP‐UHPLC‐ESI‐MS/MS method. Different parts (stems, leaves, and flowers) of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.), and phacelia (Phacelia tanacetifolia Benth.) were used. The highest content of eriodictyol was found in goldenrod flowers (13.1 μg/g), where it occurred mainly as the (S)‐enantiomer, and the greatest proportion of the total amount was bonded to glycosides. The richest source of naringenin was found to be lucerne leaves (4.7 μg/g), where it was mainly bonded to glycosides and with the (S)‐enantiomer as the dominant form. Liquiritigenin was determined only in lucerne, where the flowers contained the highest amount (1.2 μg/g), with the (R)‐enantiomer as dominant aglycone form and the (S)‐enantiomer as the dominant glycosylated form. The highest hesperetin content was determined in phacelia leaves (0.38 μg/g), where it was present in the form of a glycoside and only as the (S)‐enantiomer. A comparison of the different analyte forms occurring in different plant parts was performed for the first time.     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Influence of photosynthetic restriction due to defoliation at flowering on seed abortion in lucerne (Medicago sativa L.)

The seed yield of lucerne is poor, in contrast to its good vegetativegrowth. This is partly due to a lack of pollination under naturalconditions, pod abortion and the production of only a few seedsper pod. In this study, lucerne plants were grown in controlledconditions and about 30% of the inflorescences were pollinatedmanually. There were about 10 ovules per flower, which remainedconstant throughout the flowering period. There were 4.5 seedsper pod, indicating over 50% seed abortion. One group of plantswas defoliated and the lack of recent assimilates at floweringcaused a small but significant decrease in the number of seedsper pod (4.1). The effect of defoliation varied with seed age;the youngest fertile inflorescences were the most affected andthere was no effect at around 400C d^–1 . This stage markedthe end of the lag phase, which was determined separately incontrol plants. Thus, it is assumed that this date is the abortionlimiting stage (ALS) for this species. The long lag phase forlucerne could be a key influence on seed yield. The longer theALS, the greater the risk of vegetative competition, thus reducingreproductive development. Key words: Lucerne, flowering, pollination, abortion limiting stage (ALS), seed production     

Plant growth and ion relations in lucerne (Medicago sativa L.) in response to the combined effects of NaCl and P

The combined effect of NaCl and P on the growth of lucerne was studied in two hydroponic greenhouse experiments. NaCl concentrations were identical in each experiment (0, 50 and 100 mM NaCl) while external P concentrations were low (viz. 0.002, 0.02 and 0.2 mM measured as 0.006, 0.026 and 0.2 mM, respectively) in one experiment and higher (0.5 and 5.0 mM) in the second. Plant biomass was reduced more by the low P levels than by high concentrations of NaCl. A significant NaCl^*P effect was found where external P concentrations were low (0.006–0.2 mM) but there was no difference in plant production between the two P concentrations of 0.5 and 5.0 mM. Shoot and root concentrations of Na and Cl increased significantly with increasing NaCl concentration in both experiments and there were some differences in the concentrations of these ions at different external P levels. At low P, NaCl had no significant effect on shoot concentrations of P; however, root P concentrations tended to decrease with increasing NaCl level. Increasing external P from 0.006 to 0.2 mM led to significant increases in P concentrations in both roots and shoots. At higher P, concentrations of P in both the shoots and the roots did not differ with external NaCl or P conditions. Our results illustrate the complex relationship that exists between NaCl and P at low P levels. We conclude that high or non-limiting concentrations of P (0.2 – 5.0 mM) do not affect lucerne's response to NaCl.     

Development of lucerne (Medicago sativa L.) treated with mineral fertilizer and manure at optimal and water deficit conditions

A study on the effect of different rates of mineral fertilizer and manure on yield parameters of lucerne under optimal and water deficit conditions was carried out. Leached chernozem soil and lucerne cultivar Victoria were used. The soil was treated with ammonium nitrate and fully matured cattle manure. The plants were grown under optimum moisture content of 80% and 40% of field capacity. The water deficit stress decreased top and root biomass by 11-75% and 3-29% at mineral and organic fertilization, respectively. The applied mineral and organic N strongly depressed nodules development. Both mineral fertilizer and organic manure at dose of 210 mg N kg(-1) soil completely inhibited the appearance of nodules. Next to nitrogen, water deficit stress further inhibited the development of nodules. Nitrogen fertilization increased seed productivity in the two experimental moisture conditions. The water deficit stress decreased seed productivity by 18 to 33% as compared to optimum conditions. The plant treatments with manure were much more resistant to water deficit and recovering ability of plants was faster as compared to treatments with mineral fertilizer. The application of manure stimulates development of drought-stress tolerance in lucerne. However, the results obtained can be considered for the soil type and experimental conditions used.     

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture

Aims: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. Methods and Results: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. Conclusions: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan–lignin complexes. Significance and Impact of the Study: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.     

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