Search

Understanding the mechanisms underlying the responses to inorganic phosphate (Pi) deficiency in alfalfa will help enhance Pi acquisition efficiency and the sustainable use of phosphorous resources. Integrated global metabolomic and transcriptomic analyses of mid-vegetative alfalfa seedlings under 12-day Pi deficiency were conducted. Limited seedling growth were found, including 13.24%, 16.85% and 33.36% decreases in height, root length and photosynthesis, and a 24.10% increase in root-to-shoot ratio on day 12. A total of 322 and 448 differentially abundant metabolites and 1199 and 1061 differentially expressed genes were identified in roots and shoots. Increased (>3.68-fold) inorganic phosphate transporter 1;4 and SPX proteins levels in the roots (>2.15-fold) and shoots (>2.50-fold) were related to Pi absorption and translocation. The levels of phospholipids and Pi-binding carbohydrates and nucleosides were decreased, while those of phosphatases and pyrophosphatases in whole seedlings were induced under reduced Pi. In addition, nitrogen assimilation was affected by inhibiting high-affinity nitrate transporters (NRT2.1 and NRT3.1), and nitrate reductase. Increased delphinidin-3-glucoside might contribute to the gray-green leaves induced by Pi limitation. Stress-induced MYB, WRKY and ERF transcription factors were identified. The responses of alfalfa to Pi deficiency were summarized as local systemic signaling pathways, including root growth, stress-related responses consisting of enzymatic and nonenzymatic systems, and hormone signaling and systemic signaling pathways including Pi recycling and Pi sensing in the whole plant, as well as Pi recovery, and nitrate and metal absorption in the roots. This study provides important information on the molecular mechanism of the response to Pi deficiency in alfalfa.

The effects of exogenous ascorbic acid (AsA) on the growth parameters, nitrogen metabolism, energy status, and photosynthetic gas exchange in alfalfa under high-nitrate stress were studied. The seedlings treated with the control, 200 mmol L−1 nitrates (HN) or 200 mmol L−1 nitrate + 0.1 mmol L−1 AsA (HN + AsA), were sampled on days 0 and 10 after treatments. AsA was sprayed on the leaves, while HN was conducted by watering. Both of them were performed once every other day for three times in total. The results revealed that in the HN treatment, the growth parameters were the lowest; total phosphorus (TP), nitrogen-related enzyme activities, soluble protein (SP), adenosine triphosphate (ATP), and energy charge (EC) were reduced; and photosynthetic rate (Photo), conductance to H2O (Cond), transpiration rate (Trmmol), instantaneous water use efficiency (WUE), and apparent CO2 use efficiency (CUE) were also inhibited; and total nitrogen (TN), nitrate-nitrogen (NO3¯-N), ammonium-nitrogen (NH4+-N), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and intercellular CO2 concentration (Ci) were increased compared with the control. However, these parameters changed conversely in the HN + AsA treatment. In addition, there was a good curve regression equation relationship between TN and NO3¯-N, TN and NH4+-N, NO3¯-N and NH4+-N, respectively. It indicates that AsA improves the growth parameters, nitrogen-related enzyme activities, energy metabolism, and photosynthesis, whereas it inhibits the toxicity of excess NO3¯-N and NH4+-N accumulations, thereby promoting the growth of alfalfa under high-nitrate stress. These metabolisms are closely related to each other during the regulatory process in alfalfa. Hence, AsA has potential to be applied to improve the growth of alfalfa under high-nitrate stress.

New Lactobacillus strains, Lacticaseibacillus paracasei L1a and Lactiplantibacillus plantarum S1a R were isolated from bovine feces were used as bioconservants for Medicago sativa (alfalfa) silage after 16S rRNA identification. After 60 days the fungal content in the silage was defined by the method of qPCR using primers based on the ?-actin gene of fungi. L1a and S1a R were able to reduce fungal DNA after ensilaging by 93.7% and 95.3% respectively in comparison to control. Total lactobacillus content in the silage was also determined and results showed a high DNA content of lactic acid bacteria in L1a samples (0.00468 ng) followed by S1a R samples (0.00245 ng) when compared to control samples (0.00176 ng). The ability of these strains to reduce fungi population and the same time increase Lactic acid bacteria is an indicator for the post-conservation of silage and can be used for bio preparations.

Silicon (Si) application to crops is a promising way for the deployment of sustainable agriculture. Here, the effects of Si on salt stress tolerance were investigated in alfalfa (Medicago sativa L.)-rhizobia symbioses. Two Moroccan, Ouad-Lmaleh (OL) and Demnate-201 (Dm), and one European, NS-Mediana-ZMS-V (NS-Med), alfalfa varieties were associated to Ensifer meliloti Rm41 rhizobial strain. One-month-old alfalfa plants were exposed to 120 mM NaCl for five weeks with or without 3 mM of Si. The plants subjected to salt stress showed reduced biomass, chlorophyll (Chl) contents and relative water content (RWC) in comparison to the controls. The alfalfa-rhizobia symbiosis was also impaired under stress as reflected by less root nodulation and lower nitrogen (N) content and nitrogen content index (NCI). Added Si significantly increased plant biomass, nodules number, N content, NCI, Chl contents and RWC under salt stress. Results showed that salt-stressed alfalfa increased malonyldialdehyde (MDA), hydrogen peroxide (H2O2) and electrolyte leakage (EL). However, Si incorporation in the cultured media reduced oxidative damages under salt-stress particularly in NS-Med variety by 26%, 70% and 70% for MDA, H2O2 and EL respectively. The lower amount of MDA, H2O2 and EL in the Si-treated plants seems to be related to its capacity to modulate superoxide dismutase and polyphenol oxidase activities and increase total polyphenol, flavonoid and carotenoid contents. Besides, compatible osmolytes, such as proline, glycine betaine and soluble sugars were found increased particularly in Si-treated OL plants by 46%, 33% and 26% respectively in comparison to Si-untreated plants. Alfalfa varieties reacted differently to Si treatment. Sodium concentration in alfalfa plants increased under salinity and reduced by Si treatment with an increase in the potassium content. Our findings showed that exogenous Si application could be a promising way to mitigate the toxic effect of salt and could improve alfalfa growth and its rhizobial symbiosis when grown in salt-affected soils.

Alfalfa (Medicago sativa L.) is one of the most valuable forage crops having an intermediate salt-tolerant level. In this fact, twelve selected alfalfa from the south Tunisia oasis were subjected to salt stress looking for their adaptability against these abiotic constraints. Hence, this work reports the sodium chloride effect (150 mM) on the basis of biomass yield reduction. For this, some cultural, physiological, and biochemical parameters were monitored. Indeed, the relative water content, the stem and leaves aspects, chlorophyll content, protein amount, free proline amount, K+ and Na+ concentrations, protease and α-amylase activities, superoxide dismutase, and catalase ones were monitored. The findings give rise to no significant salinity effect on stem length and leaf number. However, there was a significant difference in leaf color for plants under salt stress. In addition, salinity decreases both fresh and dry weight of all cultivars. NaCl treatment significantly reduced chlorophyll content and the K+ ion accumulations. Also, under salt stress, there was higher proline accumulation, Na+, and proteins in all alfalfa cultivars than under control. Additionally, these findings noticed the increase of enzymatic scavenging system related to the activity of superoxide dismutase, catalase, and protease under salt stress condition. In contrast, the α-amylase activity decreased under salt treatment. It seems from the studied parameters that our 12 alfalfa oasis cultivars are tolerant to salt stress under oasis conditions.

Alfalfa (Medicago sativa L.) is the most widely cultivated perennial leguminous forage crop woldwide. MicroRNA156 (miR156) precursor genes from dicotyledonous species are reportedly useful for improving alfalfa plant architecture and abiotic stress resistance. However, there has been no report on whether a miR156 precursor gene from a monocotyledonous species functions in alfalfa. We introduced two tandem precursor genes of miR156, rice Osa-MIR156b and Osa-MIR156c (Osa-MIR156bc), into alfalfa. The expression of miR156 in the transgenic (TG) alfalfa was significantly elevated. Compared to wild-type plants, the TG plants overexpressing miR156 had more branches and leaves and showed improved salt and drought tolerance. Overexpression of miR156 slightly reduced plant height, but the biomass yield of TG plants grown in flowerpots was still increased. Forage quality of TG plants was markedly improved by reduction of acid detergent lignin (ADL) content and increase in crude protein content. The expression of the putative miR156 target genes MsSPL6, MsSPL12, and MsSPL13 in TG plants was repressed by miR156 overexpression, and that of all tested MsSPLs would be sharply increased under drought or salt stress. RNA sequencing revealed that overexpression of miR156 affected the expression of genes associated with abiotic stress resistance and plant development in multiple pathways. This first report of overexpression of monocot miR156 precursors in alfalfa sheds light on the function of miRNA156 precursors from the monocot species rice that could be used for genetic improvement of the dicot forage crop alfalfa.

Low availability of phosphorus (P) is a key limiting factor for the growth of many crops. Selenium (Se) is a nutrient for humans that is acquired predominantly from plants. Localised P and Se supply may affect P- and Se-uptake efficiency. Our aim was to examine the mechanisms of alfalfa (Medicago sativa L.) to acquire P and Se when the elements are heterogeneously or homogeneously distributed in soil, and how P and Se supply affect plant growth and uptake of P and Se. We conducted a split-root experiment growing alfalfa in a loess soil with two distribution patterns (i.e. heterogeneous and homogeneous) of P and Se. The application rates of P (KH2PO4) and Se (Na2SeO3) were 0 and 20 mg P kg−1, and 0 and 1 mg Se kg−1, respectively. Our results showed that plants absorbed more Se when both P and Se were supplied homogeneously than when supplied heterogeneously. Supplying Se had a positive effect on plant P content. Localised P supply resulted in the exudation of more carboxylates by roots than homogeneous P supply did. Soil microbial biomass P was significantly greater when P was supplied homogeneously. Shoot-to-root translocation of Se had a positive effect on P-uptake efficiency. These results indicated that, compared with homogeneous P supply, localised P supply promoted P and Se uptake by increasing the amount of rhizosheath carboxylates and weakening the competition between roots and microbes. Translocation of Se within plant organs was promoted by the application of P, thus enhancing the P-uptake efficiency of alfalfa.

Carboxylate release by roots has been considered a strategy for mobilisation and acquisition of phosphorus (P). However, recently, it was argued that carboxylate release may be a way to discharge surplus carbon produced under conditions that limit plant growth. Plant P status may not be the main factor driving carboxylate release by roots. Instead, plant nitrogen (N) status and/or N:P ratio of the soil or plant may play a more important role in enhancing carboxylate release. A greenhouse pot experiment was performed to grow alfalfa in a P-deficient soil, supplied with two rates of P (0 and 20 mg kg−1) in combination with four forms of nitrogen (N) at five rates (0, 25, 50, 75, and 100 mg kg−1), to explore the effects of P rate, N form, N rate, and their interactions on plant growth, P and N status, and carboxylate release, and to determine the factors driving carboxylate release. Nitrogen addition weakened the positive effect of P addition on plant growth, and increased plant N and P concentrations; P addition increased plant P concentration, but weakened the effect of N addition on plant N concentration. The amount of tartrate increased dramatically with increasing N rate, which decreased shoot growth, depending on N form. At high P supply, tartrate exudation correlated negatively with shoot biomass. Nitrogen addition to P-deficient alfalfa decreased shoot growth and enhanced the release of tartrate, likely to discharge surplus carbon; and the effects varied with N form.

This study evaluated the effects of two organic acids on the fermentation of alfalfa silages at different dry matter (DM) contents. Alfalfa was wilted to DM contents of 30% (moderately low) and 38% (normal) and ensiled without additives (control) or treated with 0.6% fresh matter DL-malic acid (MA) or 0.6% fresh matter citric acid (CA) for 60 days. After ensiling, silages with a normal DM were higher in pH, water-soluble carbohydrates (WSC) and DM loss (p

Alfalfa (Medicago sativa L.) has the advantages of high yield and nutritional value as a perennial forage. However, one of the drawbacks of alfalfa is its susceptibility to drought conditions, which is a global problem in agriculture. The purpose of this study was to reveal the effects of exogenous PopW, a harpin protein from Ralstonia solanacearum, treatment on growth parameters, physiological and biochemical mechanism of alfalfa under drought-stress conditions. Growth parameters, relative water content, free proline, leaf area, total chlorophyll, antioxidative enzymes, endogenous hormones including ABA, CTK, GA, JA, SA and IAA were determined in response to exogenous PopW treatment under drought stress in alfalfa cultivar (Victoria). Moreover, relative gene expressions of drought-related and leaf senescence genes were determined. Under drought stress, alfalfa plants had lower shoot dry weight, shoot length, relative water content, leaf area, and total chlorophyll content, compared to control (non-stressed). However, Exogenous PopW treatment significantly increased growth values, relative water content, free proline, leaf area, total chlorophyll content, catalase, glutathione reductase and superoxide dismutase under drought conditions, compared to control and drought stress alone. Moreover, exogenous PopW treatment significantly increased ABA, GA, JA, SA, IAA contents, up-regulated auxin- and drought-responsive genes, down-regulated leaf senescence genes. Exogenous PopW treatment enhanced drought stress tolerance of alfalfa due to changes of endogenous hormone contents and expression levels of drought stress and leaf senescence genes. The results of the study show that PopW treatment could be used to increase the forage yield of alfalfa on areas having drought problem.

This study aimed to analyze the nutritional characteristics of Medicago sativa (Alfalfa) hay for the feeding of ruminants in the central zone of the Sierra Region. The study was conducted in the Laboratory of the Faculty of Pecaurias Sciences of the Higher Polytechnic School of Chimborazo. Cutting was carried out for 60 days in different areas of the central Sierra region. Data were analyzed using descriptive statistics. Results of the bromatological analysis showed that the average total humidity was 11.12 ± 0.20% in (Rp-00542) while the minimum was 7.79 ± 0.05% in (Rp-00224). Values for dry matter ranged between 91.25 ± 0.03% (Rp336) and 88.53 ± 0.09% (Rp492); dry base ash ranged between 12.51 ± 0.08% (Rp500) and 9.16 ± 0.08% (Rp521); dry organic matter ranged between 90.84 ± 0.13% (Rp521) and 87.49 ± 0.08% (Rp521); and dry crude protein ranged between 22.44 ± 0.26% (Rp492) and 18.73 ± 0.08% (Rp336). The following values were also obtained: 1.94 ± 0.93% dry ethereal extract; 32.06 ± 2.11% dry nitrogen free extract; 46.96 ± 3.78% dry neutral detergent fiber; 40.67 ± 3.36% dry acid detergent fiber; and 8.23 ± 0.64% dry lignin detergent acid. Therefore, it can be determined that forage mixture provides all of the nutrients to the species of zootechnical interest. Keywords: Medicago sativa, alfalfa, sierra centro, rumiants. RESUMEN En el Laboratorio de la Facultad de Ciencias Pecaurias de la Escuela Superior Politécnica de Chimborazo, se realizó la Caracterización Nutricional de henos de Medicago sativa (Alfalfa) para la alimentación de Rumiantes en la zona centro de la Región Sierra; Mediante estadística descriptiva se obtuvo los siguientes resultados del análisis bromatológico a los 60 días de corte en las diferentes zonas de la región Sierra - Centro, se puede observar que el mayor promedio de la humedad total fue 11,12 ± 0,20% en (Rp-00542) mientras que el menos fue 7,79 ± 0,05% en (Rp-00224); para Materia Seca-Total los valores se obtuvieron entre 91,25 ± 0,03% (Rp336) y 88,53 ± 0,09% (Rp492); Cenizas base seca 12,51 ± 0,08% (Rp500) y 9,16 ± 0,08% (Rp521); el mayor valor para % Materia Orgánica Base seca fue de 90,84 ± 0,13% (Rp521) y el menor con 87,49 ± 0,08% (Rp521); para % Proteína cruda base seca la mejor respuesta fue 22,44 ± 0,26 (Rp492) % y el menor 18,73 ± 0,08 (Rp336), Fibra cruda base seca; 1,94 ± 0,93% Extracto etéreo base seca; 32,06 ± 2,11% Extracto Libre de Nitrógeno base seca; 46,96 ± 3,78% Fibra Detergente Neutro base seca; 40,67 ± 3,36% Fibra Detergente Acida base seca; 8,23 ± 0,64% Lignina Detergente Acida base seca. Por lo que se puede determinar que la mezcla forrajera aporta todos los nutrientes a todas las especies de interés zootécnico. Palabras clave: Medicago sativa, alfalfa, sierra centro, rumiantes.

Medicago sativa is an important worldwide forage species containing characteristic secondary metabolites such as saponin which are reported to be biologically active. The objective of this study wa...

Background: Salinity is a major abiotic stress that limits plant growth and development. Salinity affects several physiological and biochemical characteristics adversely, which results in oxidative stress in plant species. Plants change the protein pattern to cope with salinity stress. The identified salt-responsive proteins in alfalfa are involved in energy and metabolism, photosynthesis, regulation of carbohydrates, transcription/translation, signal transduction, stress/redox homeostasis, ion binding, and stress and defense with ROS scavenging and detoxification. Objective: The present research aimed to study the response of two contrasting alfalfa varieties to salt stress, and to identify the altered leaf proteins by two-dimensional gel electrophoresis and MALDI-TOF/TOF/MS spectrometry. Methods: Salinity stress significantly decreased shoot fresh and dry weights in both Synthetic II (salt-tolerant) and Khajeh (salt-sensitive) varieties, and K+/Na+ ratio in Khajeh, while it significantly increased K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity in Synthetic II, and peroxidase activity in Khajeh. Conclusions: Salinity stress reduced the yield of alfalfa, but the reduction was more pronounced in the sensitive variety of Khajeh. The salinity-tolerant variety, Synthetic II, responded better to salinity stress in terms of K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity as compared to Khajeh. Proteome analysis showed that the proteins involved in energy metabolism, transcription/- translation, photosynthesis, electron transfer, and defense were more important than other functional categories under salinity stress. These proteins mainly increased in the salt-tolerant variety, whereas they decreased in the salt-sensitive variety.

The mass of leaves and the chlorophyll and selenium content of alfalfa can be increased by the foliar spraying of selenite. To better understand the relationship between changes in the expression of specific proteins and the various metabolic and regulatory pathways affected by selenium treatment, labeling with Tandem Mass Tags (TMT) was used as a proteomics technique to compare control leaves with those enriched with Se. A total of 8,411 proteins were identified, the expression levels of 195 of which were significantly modified, 67 significantly up-regulated and 128 significantly down-regulated. Using gene functional classification and metabolic pathway annotation, selenium treatment was found to have a significant impact on metabolic processes. The energy and substances produced by the metabolic processes of a variety of carbohydrates, lipids, and amino acids, and the metabolism of carbon may be responsible for increasing the yield of alfalfa leaves. Administration of selenium substantially influenced Se-responsive proteins, including ABC transporter G family member 36, Probable glutathione S-transferase and cysteine tRNA ligase. Selenium treatment may also enhance photosynthesis and the defense response of cells. Furthermore, protein ubiquitination also played an important role in the selenium response of alfalfa leaves. In summary, a basic analysis of the selenium response pathway in alfalfa leaves at the proteomics level was conducted, which may assist in a more detailed elucidation of selenium enrichment in alfalfa in the future.

Drought and salt are main environmental factors that affect the growth, development, productivity and distribution of plants of plants. Alfalfa has a strong ability of early and salt resistance. In this work, the varieties Xinjiang Daye was used as material, the effects of drought (simulated with PEG, polyethylene glycol-6000) and salt stress (with NaCl solution) on the antioxidant capacity of alfalfa seedlings and stress resistance genes was studied, to select alfalfa varieties with strong resistance and study its functional principle of resistance related genes. The results showed that with the increase of drought stress, the contents of H2O2, O2−, MDA increased by 323, 247 and 235 (15% PEG treatment). The activities of SOD, CAT and APX increased by 18.01, 15.56 and 587% (15% PEG treatment), respectively. The expression of drought resistance genes increased significantly. With the increase in NaCl stress, the activities of SOD, cat, pod and APX increased by 132.14, 315.60, 102.78 and 27.61%, respectively. The expression of two genes related to salt stress increased significantly. In conclusion, alfalfa leaves have good survival ability under high stress, and the activities of antioxidant enzymes and the expression of related genes have adaptive changes under drought and salt stress.

The use of citric acid (CA) chelator to facilitate metal bioavailability is a promising approach for the phytoextraction of heavy metal contaminants. However, the role of the CA chelator associated with arbuscular mycorrhizal fungi (AMF) inoculation on phytoextraction of vanadium (V) has not been studied. Therefore, in this study, a greenhouse pot experiment was conducted to evaluate the combined effect of CA chelator and AMF inoculation on growth performance and V phytoextraction of plants in V-contaminated soil. The experiment was performed via CA (at 0, 5, and 10 mM kg-1 soil levels) application alone or in combination with AMF inoculation by Medicago sativa Linn. (M. sativa). Plant biomass, root mycorrhizal colonization, P and V accumulation, antioxidant enzyme activity in plants, and soil chemical speciation of V were evaluated. Results depicted (1) a marked decline in plant biomass and root mycorrhizal colonization in 5- and 10-mM CA treatments which were accompanied by a significant increased V accumulation in plant tissues. The effects could be attributed to the enhanced acid-soluble V fraction transferring from the reducible fraction. (2) The presence of CA significantly enhanced P acquisition while the P/V concentration ratio in plant shoots and roots decreased, owing to the increased V translocation from soil to plant. (3) In both CA-treated soil, AMF-plant symbiosis significantly improved dry weight (31.4-73.3%) and P content (37.3-122.5%) in shoots and roots of M. sativa. The combined treatments also showed markedly contribution in reduction of malondialdehyde (MDA) content (12.8-16.2%) and higher antioxidants (SOD, POD, and CAT) activities in the leaves. This suggests their combination could promote growth performance and stimulate antioxidant response to alleviate V stress induced by CA chelator. (4) Taken together, 10 mM kg-1 CA application and AMF inoculation combination exhibited a higher amount of extracted V both in plant shoots and roots. Thus, citric acid-AMF-plant symbiosis provides a novel remediation strategy for in situ V phytoextraction by M. sativa in V-contaminated soil.

Pot experiments with alfalfa, milkvetch root and swamp morning glory were conducted to elucidate the effect of soil vanadium (V) on plant growth and to evaluate their phytoremediation potential under V(V) exposure. Based on biomass analysis, swamp morning glory showed higher tolerance than alfalfa and milkvetch root in response to different soil V(V) levels. The accumulation of V in plants increased with the increasing soil V and the V concentration in roots was 1.95-4.31 times that in shoots. After planting, soil total V, V(V), bioavailable V and water-soluble V all reduced, and the decreases in bioavailable V and V(V) showed significant. The decreased percentage of V(V) in total V in soils demonstrated that the planting process may stimulate the mechanism of V(V) reduction to V(IV). Therefore, the three tested plants, particularly swamp morning glory can be promising phytostabilizers applied to V phytoremediation practices.

Summary The objectives of this study were to investigate the adaptation and competition of Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts using single‐molecule real‐time (SMRT) sequencing technology. Before ensiling, alfalfa was inoculated with L. plantarum (Lp), P. pentosaceus (Pp), E. faecalis (Ef) or their combinations (LpPp, LpEf, LpPpEf) and sampled at 1, 3, 7, 14 and 60 days. After 60‐days fermentation, the Lp‐, Pp‐ and LpPp‐inoculated silages had lower pH but greater concentrations of lactic acid were observed in Pp, LpEf and LpPpEf‐inoculated silages. The inoculants altered the keystone taxa and the bacterial community dynamics in different manners, where L. plantarum, Weissella cibaria and L. pentosaceus dominated the bacterial communities after 14 days‐fermentation in all treatments. The silages with better fermentation quality had simplified bacterial correlation structures. Moreover, different inoculants dramatically changed the carbohydrate, amino acid, energy, nucleotide and vitamin metabolism of bacterial communities during ensiling. Results of the current study indicate that effect of different inoculants on alfalfa silage fermentation was implemented by modulating the succession of bacterial community, their interactions and metabolic pathways as well during ensiling., This study were investigated the adaptation and competition of Lactobacillus plantarum, Pediococcus pentosaceus, and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts of alfalfa silage. The inoculants altered the keystone taxa, dynamics of bacterial community and their functions in different manners, and the silages with better fermentation quality had simplified bacterial correlation structures.

Background The objective of this study was to evaluate the chemical compositions and microbial communities of salt-tolerant alfalfa silage. Salt-tolerant alfalfa was ensiled with no additive control, and cellulase for 30 and 60 to 90 days. In this study, the dry matter (DM) content of the raw material was 29.9% DM, and the crude protein (CP) content of the alfalfa was 21.9% CP. Results After 30 days of fermentation, the DM content with the cellulase treatment was reduced by 3.6%, and the CP content was reduced by 12.7%. After 60 days of fermentation, compared with alfalfa raw material, the DM content in the control group (CK) was reduced by 1%, the CP content was reduced by 9.5%, and the WSC (water-soluble carbohydrates) content was reduced by 22.6%. With the cellulase, the lactic acid content of the 30- and 60-day silages was 2.66% DM and 3.48% DM. The content of Firmicutes in salinized alfalfa raw material was less than 0.1% of the total bacterial content. Before and after ensiling, the microbes had similar composition at the phylum level, and were composed of Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria. The abundance of Pantoea was dominant in fresh alfalfa. In the absence of additives, after 30 days and 60 days of silage, the dominant lactic acid bacteria species became Lactococcus and Enterococcus. Conclusions The results showed that LAB (Lactobacillus, Lactococcus, Enterococcus, and Pediococcus) played a major role in the fermentation of saline alfalfa silage. It also can better preserve the nutrients of saline alfalfa silage. The use of cellulase enhances the reproduction of Lactobacillus. The fermentation time would also change the microbial community of silage fermentation.

Bioassay-guided fractionation of the organic extracts of the endophyte Daldinia eschscholtzii strain GsE13 led to the isolation of several phytotoxic compounds, including two chromenone and two chromanone derivatives: 5-hydroxy-8-methoxy-2-methyl-4H-chromen-4-one, 1; 5-hydroxy-2-methyl-4H-chromen-4-one, 2; 5-methoxy-2-methyl-chroman-4-one, 3; and 5-methoxy-2-methyl-chroman-4-ol, 4; as well as other aromatic compounds: 4,8-dihydroxy-1-tetralone, 5; 1,8-dimethoxynaphthalene, 6; and 4,9-dihydroxy-1,2,11,12-tetrahydroperyl-ene-3,10-quinone, 7. Compounds 1, 4, and 7 were isolated for the first time from D. eschscholtzii. The phytotoxicity of all the compounds was determined on germination, root growth, and oxygen uptake in seedlings of a monocotyledonous (Panicum miliaceum) and three dicotyledonous plants (Medicago sativa, Trifolium pratense, and Amaranthus hypochondriacus). In general, root growth was the most affected process in all four weeds, and chromenones 1 and 2 were the most phytotoxic compounds. Phytotoxins 1-4 inhibited basal oxygen consumption rate in isolated mitochondria from M. sativa seedlings and also caused serious damage to their membrane potential (ΔΨm) in percentages greater than 50% at concentrations lower than 2 mM. Based on these results, compounds 1-4 of endophytic origin could be promising for the development of new herbicides potentially useful in agriculture or for the synthesis of promising new molecules. KEY POINTS: • Endophytic fungus Daldinia eschscholtzii produces phytotoxic compounds. • Phytotoxins inhibit basal oxygen consumption rate in isolated M. sativa mitochondria. • Phytotoxins altered the mitochondrial membrane potential.

The symbiotic association of alfalfa (Medigaco sativa) with Sinorhizobium melilloti contributes to the production of inoculum to improve plant growth and increase yield. The competitiveness of the introduced strains is a very important factor in the performance of an inoculum which is based on the use of rhizobial strains. This competitiveness is due to the production of anti-microbial substances, among them, bacteriocines. In this study, the totals of 30 rhizobial strains were isolated from M. sativa nodules sampled from sa-line soils of Algeria. Twenty strains were nodulated the host plant M. sativa. The antimicrobial activity producing strains SW16 and SW15 were identified as Sinorhizobium melliloti based on 16S rRNA sequance analysis. Partial puri-fication of this inhibitorys substances by ammonium sulfate were precipitat-ed with 75% saturation. These antimicrobial activitys showed the maximum activity against the strain SW10 estimated at 1800 with the strain SW16 and 2500 AU/ml for the strain SW15. The physicochemical properties of these antimicrobial activitys reveals thier thermoresistant nature and sensitive to protease, but theire were resistant to a wide range of pH and also at differ-ent concentrations of Nacl. This inhibitory agent display primary metabolite kinetics as its maximal production was detected in the growth phase. Antimi-crobial substances produced by sinorhizobium mielilloti SW15 and SW16 exhibited inhibitory activity against the phytopathogen (Aspergillus sp., Fusarium sp., Cladosporium sp. and Alternaria altterna).

The use of herbal supplements that promise to improve immune health has gained popularity among dermatology patients. However, there is little to no evidence that herbal supplements improve dermatological conditions. Several in vitro and in vivo studes have shown that Spirulina platensis, Aphanizomenon flos-aqua, Chlorella, Echinacea, and alfalfa activate immune cells, via certain cytokines and chemokines. Case reports suggest the association of ingesting immunostimulatory herbs and the clinical onset or flares of diseases characterized by an exaggerated immune response such as lupus erythematosus, dermatomyositis and autoimmune blistering disorders. Therefore, it is imperative to investigate the prevalence of herbal supplement use in this patient population. In addition, in vitro studies should examine the underlying mechanisms by which herbs stimulate immune pathways that are already overactive in autoimmune patients. CAPSULE SUMMARY: How does this article integrate into what was already known? Spirulina, Aphanizomenon flos-aqua, Chlorella, Echinacea and alfalfa have been shown to stimulate the immune system. The use of these herbs may be associated with the precipitation or flare of autoimmune skin diseases. How does it change practice? Patients with autoimmune diseases should be screened for supplement use and should be advised to avoid these immunostimulatory herbs.

Perfluorooctanoic acid (PFOA) is a perfluorinated alkyl substance used as a surfactant in a wide variety of industrial and consumer products. Over the past decade, concern has increased over the presence of PFOA in biosolids from wastewater treatment plants used as fertilizer on agricultural lands because of the potential for PFOA to enter the food chain. In this study, the uptake of 14C-PFOA from soil by alfalfa and 14C-PFOA bioavailability from consumption of this alfalfa was evaluated in Sprague-Dawley rats. Alfalfa leaves accumulated 14C-PFOA up to 4 to 5 μg/g of dry leaf, approximately 10 times higher than accumulation in the stem. Alfalfa was ground for feeding to 15 female Sprague-Dawley rats (175 to 200 g). Animals within metabolism cages were fed 10 g of feed (6 g of alfalfa plus 4 g of ground rat chow) twice daily for 14 days (equivalent to 50 μg of 14C-PFOA per kg per day). At the end of the feeding period, three rats were euthanized for sample collection on each of withdrawal days 0, 3, 7, 11, and 14. During the feeding and withdrawal phases, urine and feces were collected daily. At necropsy, blood, liver, kidney, adipose, muscle, skin, brain, heart, adrenal glands, spleen, lungs, and thymus were removed and assayed for 14C-PFOA by combustion and liquid scintillation counting. Rats had eliminated 72.8% ± 3.4% of the total dose via urine at 14 days, but urinary radioactivity fell below the level of detection by day 3 of the withdrawal period. Fecal elimination was 6.5% ± 1.2% of the dose and fell below the level of detection by day 2 of the withdrawal period. The rapid and high elimination via urine indicates that a majority of the dose was absorbed. The uptake of 14C-PFOA into alfalfa was low from soil with a high organic concentration; however, 14C-PFOA was highly bioavailable from the alfalfa when used as a feed component for rats. This study provides data for regulators investigating 14C-PFOA bioavailability and disposition in animals or animal products exposed to contaminated feed. HIGHLIGHTS

Here, we assess the differential impact of drought on root carbon metabolism in the widely cultivated alfalfa (Medicago sativa, Ms) and the model legume Medicago truncatula (Mt). Understanding how carbon allocation is regulated under drought stress conditions is a central issue to improving alfalfa productivity under future climate change scenarios. Alfalfa and Medicago truncatula were compared under water deficit conditions. Root carbon metabolism of the taproot and fibrous roots was analysed. M. truncatula drought tolerance variability was compared to that of alfalfa using six accessions of the Medicago Hapmap project. The prominent taproot is much less developed in M. truncatula than in alfalfa with the former exhibiting an extensive fibrous root system. In both examined Medicago species the taproot contained the major pools of soluble protein, sucrose and pinitol, whereas the major pools of hexoses and carbon metabolism enzymes appeared to be in the fibrous roots. Under water-deficit conditions, the response of M. sativa strongly differed from that of M. truncatula at the root level. Water deficit conditions differentially modulate the root carbon metabolism of M. sativa and M. truncatula. Mt maintained a more active carbon metabolism in the fibRs, as sucrose, myo-inositol and pinitol accumulated to cope with the water deficit (WD). Conversely, the root system of Ms did not accumulate cyclitols and carbon metabolism was more severely affected under water deficit conditions. This differentially exerted control may determine the drought response of these two close relatives.

Alfalfa (Medicago sativa L.) is one of the major forage legumes grown in Oman, but its productivity is significantly affected by salt stress. In this study, 20 local Omani alfalfa landraces were characterized for morphological and biochemical traits, tissue minerals, and genetic diversity. The experiment was conducted in a greenhouse with three salinity levels: control (2 dS m−1), moderate salinity stress (6 dS m−1), and severe salinity stress (10 dS m−1). Different morphological and biochemical traits and tissue minerals showed significant variation in response to varying salinity levels. With an increase in salinity stress levels from 2 to 10 dS m−1, there was a decrease in different morphological traits across the landraces, except for forage fresh weight which increased at both levels of salt stress (6 and 10 dS m−1) compared to control owing to high leaf free proline, total soluble phenolics, catalase activity, and leaf potassium concentration. However, different biochemical traits and sodium and chloride increased with the increase in salt stress levels (6 to 10 dS m−1) and the higher values were recorded at higher salt stress level (10 dS m−1). The principal component analysis (PCA) had 47.8 and 49.2% variability for morphological traits under salt stress levels of 6 and 10 dS m−1, respectively. Under both levels of salt stress, there was 81% variability by the two PCs for different biochemical traits. The total variability contributed by the two PCs for mineral nutrients was 69.2 and 66.2% for salt stress levels of 6 and 10 dS m−1, respectively. At both levels of salt stress, the landraces OMA 5, OMA 21, OMA 161, OMA 6, OMA 95, OMA 220, and OMA 285 were grouped on high morpho-biochemical traits and tissue mineral contents. There was a low to moderate level of genetic diversity (H = 0.3252) in the Omani alfalfa populations. However, the level of genetic diversity for the populations from the four regions was moderate (Dhahira) to high (Batinah, Dakiliya, and Sharqiyah). The substantial variation among the landraces on morpho-biochemical traits and tissue minerals can be used in the breeding programs to develop salt-tolerant alfalfa genotypes. The landraces OMA 6, OMA 161, and OMA 285 were excellent in performance based on morphological and biochemical traits and tissue mineral contents under both levels of salt stress.

In this study, calli of Medicago sativa L. cv. Elçi (alfalfa Elçi) were inoculated in cell suspension culture and analyzed for aggregate assay, cell viability test, total phenolic content assay, DPPH free radical scavenging activity and formononetin assay by means of High-Performance Liquid Chromatography (HPLC). Hypocotyl, cotyledon and apical meristem explants were taken from 15-day-old aseptic seedlings and germinated in MS medium. 10 g calli were grown for each explant and then transferred into cell suspension culture. The highest cell viability rate, which was 75%, and the highest DPPH free radical scavenging activity with 51.36% was measured in 1000 mL cell suspension culture, while the highest total phenolic content, i.e. 40.2 mg/g, was quantified in 250 mL cell suspension culture. In accordance with the findings of the study, the production of formononetin was higher in the calli derived from cell suspension cultures than in herb samples of M. sativa. Moreover, in 1000 mL cell suspension culture, 4.99 mg/g of formononetin concentration was quantified, which scored the highest. In large-scale cell suspension cultures of M. sativa, it was possible to increase the production of formononetin production. Hence, due to its medicinal significance, a method has been tested to obtain higher amounts of this compound.

Alfalfa (Medicago sativa L.) is an important forage legume grown in Kansas, USA and its productivity with cool-season grasses however is unknown. The objective of this study was to determine the dry matter yield (DMY) and forage nutritive value of alfalfa-grass mixtures compared to those of alfalfa and grasses grown in monoculture with and without nitrogen fertilization. Three different alfalfa varieties were planted (reduced-lignin alfalfa, Roundup Ready, and conventional alfalfa) and two kinds of cool-season grasses (smooth brome, Bromus inermis Leyss, and tall fescue, Festuca arundinacea Schreb) were planted as a monoculture or in alfalfa-grass mixtures. Nitrogen fertilizer (urea) was applied at green-up at a rate of 56 kg/ha and after the second cutting at a rate of 56 kg/ha in 2016 and 2017, respectively. and control treatments received no nitrogen. DMY was significantly higher in monoculture alfalfa and alfalfa-grass mixtures than in grass monocultures. Between alfalfa monoculture and alfalfa-grass mixtures, no significant differences in DMY were found. For all treatments, nitrogen application significantly increased DMY compared to the control. In 2016 and 2017, the low-lignin alfalfa monoculture had the lowest acid detergent fiber (ADF) and the grass monocultures had the highest ADF. In 2016 and 2017, neutral detergent fiber (NDF) in smooth bromegrass and tall fescue was higher than in other species treatments. A low-lignin alfalfa monoculture had significantly lower NDF concentration compared to alfalfa-grass mixtures. When averaged over 2016 and 2017, relative feed value (RFV) was highest in low-lignin alfalfa and lowest in the grass monocultures. In both years, nitrogen fertilizer application did not affect nutritive values.

To investigate physiological and biochemical changes of thrips-resistant alfalfa (Medicago sativa L. cv. Gan-nong No. 9), we aimed at clarifying the response mechanisms of alfalfa against thrips. Medicago sativa L. cv. including thrips-resistant Gan-nong No.9 (G9), thrips-susceptible Gan-nong No.3 (G3) and highly thrips-susceptible WL363HQ (363) were infested with different thrips densities (3, 5, 7 and 9-thrips per branch). The quantitative change in specific nutrients, secondary metabolites, defensive and antioxidant enzymes were measured at seedling stage of the three alfalfa cultivars. The results showed that with the increase of thrips densities, the damage indices, SS, Pro, flavonoids, tannin and H(2)O(2) in G9, G3 and 363 were significantly increased, but PPO and SOD significantly reduced, compared with CK. Furthermore, the tannin and lignin contents of G9 were significantly higher compared to 363, but SP content was significantly lower than G3 and H(2)O(2) content which was further significantly less compared to 363. Correlation analysis observed that the damage index of the three alfalfa cultivars showed a significant positive association with SS, Pro, flavone, tannin, and H(2)O(2) (P

Medicago sativa L. (alfalfa) is a perennial leguminous forage crop cultivated worldwide. Unique water-soaked brown spots surrounded by yellow haloes were reported extensively on leaves of alfalfa crop from the farmer fields of Hafizabad and Bahawalpur districts in 2018. Olive gray colonies of a fungus, isolated with 95% frequency from the symptomatic tissues, were identified as Curvularia sp. based on morphocultural characteristics. Thus, representative Curvularia isolate “FMB-ALF2-MS” was subjected to molecular analysis to validate its phylogeny. Four genetic regions, ITS, LSU, TEF1-α, and GAPDH, were directly sequenced. BLASTn search showed 100% homology to C. buchloes strain CBS 246.49. Phylogenetic analysis is also identified and congruent with this isolate as C. buchloes with 100% bootstrap support. Four repeated pathogenicity tests were carried out with C. buchloes, and the etiology of the leaf spots was determined. To our best knowledge, this is the first report of C. buchloes causing leaf spots on alfalfa from Pakistan. The new and emerging foliar disease's occurrence and intensity were documented across the Punjab province during 2018–2019. The highest D.I (57%) with an average D.S of 78.5% was recorded from the Bahawalpur district. Eight different (four contact and four systemic) fungicides were tested against the pathogen under in vitro conditions at 50, 100, 150, and 200 ppm concentration. Among all the tested fungicides, maximum inhibition (95%) was recorded through mancozeb (contact fungicide), followed by 92% pathogen growth inhibition through propiconazole (systemic fungicide) at 200 ppm. Characterized pathogenic fungal isolate was deposited to the Culture Collection (FMB-CC-UAF) with accession number FMB0177.

Vacuole membrane proteins play a critical role in the regulation of plant physiological processes including normal growth and development, and responses to stresses. The killing me slowly 1 (KMS1) gene that encodes a soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE) domain-containing vacuole membrane protein was first reported in Arabidopsis. Currently, the function of KMS1 in other plants under stress is poorly understood. In this study, we report cloning, expression, and characterization of a novel KMS1 gene in alfalfa (Medicago sativa L.), designated MsKMS1 (GenBank accession No. JX467688). The full-length cDNA of MsKMS1 was 1 396 bp and contained a complete open reading frame of 1 257 bp, which encoded a putative protein of 418 amino acids. The BLASTp analysis showed that MsKMS1 shared high amino acid sequence similarities with KMS1 from other plants such as Medicago truncatula (99 %), Cicer arietinum (89 %), Glycine max (77 %), Prunus mume (76 %), Ricinus communis (72 %), Populus euphratica (72 %), Theobroma cacao (72 %), and Arabidopsis thaliana (67 %). Transient transformation of onion (Allium cepa) bulb scale epidermal cells by biolistic bombardment showed that MsKMS1 was localized to the plasma membrane. Quantitative real-time PCR revealed that MsKMS1 expression was upregulated under different abiotic stresses (200 mM NaCl, 20 % (m/v) polyethylene glycol 6000] and 10 mg dm-3 abscisic acid. Transgenic tobacco plants were obtained via Agrobacterium-mediated transformation and treated with 200 mM NaCl. Reverse-transcription PCR data showed that MsKMS1 was successfully transcribed and expressed in the leaves of transgenic plants. The MsKMS1-overexpressors showed a lower malondialdehyde content and maintained a higher relative water content and proline content compared with non-transgenic controls under salt stress. These results indicate that the introduction of the MsKMS1 gene could improve salt stress resistance in tobacco plants. This study reveals the role of MsKMS1 in the regulation of plant responses to abiotic stress and provides evidence for further functional studies of the KMS1 family in alfalfa.

The adverse effects of exposure to microcystins in terrestrial crops have been well documented. However, the retention and bioavailability of microcystin-LR, one of the most prevalent cyanotoxins, from soil to plants, is poorly understood. In the present study, the amount of free microcystin-LR from two soil types, a silty sand and clayey loam, with exposure to three toxin concentrations and time was investigated. Using the two soil types, the effects on Medicago sativa (Alfalfa) growth after microcystin-LR exposure via irrigation with spiked water and pre-spiked soil was investigated and the amount of microcystin-LR taken up by the plant quantified. After 3 weeks of growth, the amount of free microcystin-LR remaining in the two soil types with each treatment was quantified. The results indicated that in clayey loam more microcystin-LR is bound to the soil. However, the growth of Alfalfa was only affected in the clayey loam with microcystin/LR exposure via irrigation. Nevertheless, microcystin-LR was detected in Alfalfa grown in both soil types exposed by both irrigation and via pre-spiked soil. Interestingly, more microcystin-LR remained in the silty sand after 3 weeks; yet, more microcystin-LR was taken up by the Alfalfa grown in the silty sand, with a larger concentration in the roots compared to the shoots. The results indicate that the soil type substantially influences the bioavailability and uptake of microcystin-LR and present some insight into the ecological risk posed by microcystin-LR.

Lucerne seeds (Medicago sativa L.) were germinated at different time-temperature regimes (24, 28, and 32 °C for 24, 48, and 72 h). The effect of germination was evaluated on physicochemical properties, techno-functional characteristics, antinutrients, bioactive constituents, antioxidant activity, and mineral profile of lucerne seeds to ascertain the best time-temperature regime for the germination. High germination capacity was observed in seeds germinated at 28 and 32 °C in comparison to 24 °C, however, lower germination losses were manifested at 28 °C. Time-temperature regime of 28 °C for 48 h was most effective for the germination of lucerne seeds. Germination at aforesaid conditions improved hydration properties, foaming capacity, and gelation ascribed to protein denaturation reflected as a change in the α-helix and β-sheets in ATR-FTIR spectra of germinated flours. Furthermore, reduction in tannins (32.91%), lectin (100%), and trypsin inhibitor activity (67.42%) were observed. Increased DPPH∙ RSA (6.90%), ABTS∙+ RSA (10.51%), metal chelating activity (14.64%), and zinc concentration were also manifested with germinated flour. The optimal balance of techno-functionality, reduced antinutrients, and high antioxidant capacity of germinated flour (28 °C for 48 h) was validated using Principle Component Analysis.

To probe the mechanism of action of NaCl in regulating drought adaptation in Medicago sativa, we pretreated M. sativa with different concentrations (0, 50, 100 and 200 mM) of NaCl, then studied the effects of NaCl pretreatment on water content, chlorophyll content, photosynthetic function, and membrane peroxidation of M. sativa plants subjected to drought stress. Although pretreatment with different concentrations of NaCl significantly increased the Na+ content in the aboveground of M. sativa, it had no significant effect on the activity of PSII and PSI. However, different concentrations of NaCl pretreatment significantly increased the water content of the aboveground of M. sativa under drought stress, alleviated the degree of photoinhibition of PSII and PSI, and maintained the flow and stability of donor and acceptor electrons in PSII. NaCl pretreatment promotes the increase of non-photochemical quenching (NPQ), which relieves oxidative damage in the leaves of M. sativa under drought stress, playing an important role in maintaining the activity of PSII and PSI. In conclusion, pretreatment with a certain concentration of NaCl can help M. sativa leaves maintain relatively high-water content and alleviate the photosynthesis inhibition of PSII and PSI under drought stress, thereby improving the drought resistance of M. sativa.