Salinity Stress Alters the Secondary Metabolic Profile of M. sativa, M. arborea and Their Hybrid (Alborea).

Increased soil salinity, and therefore accumulation of ions, is one of the major abiotic stresses of cultivated plants that negatively affect their growth and yield. Among Medicago species, only Medicago truncatula, which is a model plant, has been extensively studied, while research regarding salinity responses of two important forage legumes of Medicago sativa (M. sativa) and Medicago arborea (M. arborea) has been limited. In the present work, differences between M. arborea, M. sativa and their hybrid Alborea were studied regarding growth parameters and metabolomic responses. The entries were subjected to three different treatments: (1) no NaCl application (control plants), (2) continuous application of 100 mM NaCl (acute stress) and (3) gradual application of NaCl at concentrations of 50-75-150 mM by increasing NaCl concentration every 10 days. According to the results, M. arborea maintained steady growth in all three treatments and appeared to be more resistant to salinity. Furthermore, results clearly demonstrated that M. arborea presented a different metabolic profile from that of M. sativa and their hybrid. In general, it was found that under acute and gradual stress, M. sativa overexpressed saponins in the shoots while M. arborea overexpressed saponins in the roots, which is the part of the plant where most of the saponins are produced and overexpressed. Alborea did not perform well, as more metabolites were downregulated than upregulated when subjected to salinity stress. Finally, saponins and hydroxycinnamic acids were key players of increased salinity tolerance.

Historic exposure to herbivores, not constitutive traits, explains plant tolerance to herbivory in the case of two Medicago species (Fabaceae).

Mechanisms that allow plants to survive and reproduce after herbivory are considered to play a key role in plant evolution. In this study, we evaluated how tolerance varies in species with different historic exposure to herbivores considering ontogeny. We exposed the range-restricted species Medicago citrina and its closely related and widespread species M. arborea to one and two herbivory simulations (80 % aerial biomass loss). Physiological and growth parameters related to tolerance capacity were assessed to evaluate constitutive values (without herbivory) and induced tolerance after damage. Constitutive traits were not always related to greater tolerance, and each species compensated for herbivory through different traits. Herbivory damage only led to mortality in M. citrina; adults exhibited root biomass loss and increased oxidative stress after damage, but also compensated aerial biomass. Despite seedlings showed a lower death percentage than adults after herbivory in M. citrina, they showed less capacity to recover control values than adults. Moderate tolerance to M. arborea herbivory and low tolerance to M. citrina is found. Thus, although the constitutive characteristics are maintained in the lineage, the tolerance of plants decreases in M. citrina. That represents how plants respond to the lack of pressure from herbivores in their habitat.

The impact of intra-specific diversity in the rhizobia-legume symbiosis.

Rhizobia - nitrogen-fixing, root-nodulating bacteria - play a critical role in both plant ecosystems and sustainable agriculture. Rhizobia form intracellular infections within legumes roots where they produce plant accessible nitrogen from atmospheric nitrogen and thus reduce the reliance on industrial inputs. The rhizobia-legume symbiosis is often treated as a pairwise relationship between single genotypes, both in research and in the production of rhizobial inoculants. However in nature individual plants are infected by a high diversity of rhizobia symbionts. How this diversity affects productivity within the symbiosis is unclear. Here, we use a powerful statistical approach to assess the impact of diversity within the Rhizobium leguminosarum - clover symbiosis using a biodiversity-ecosystem function framework. Statistically, we found no significant impact of rhizobium diversity. However this relationship was weakly positive - rather than negative - indicating that there is no significant cost to increasing inoculant diversity. Productivity was influenced by the identity of the strains within an inoculant; strains with the highest individual performance showed a significant positive contribution within mixed inoculants. Overall, inoculant effectiveness was best predicted by the individual performance of the best inoculant member, and only weakly predicted by the worst performing member. Collectively, our data suggest that the Rhizobium leguminosarum - clover symbiosis displays a weak diversity-function relationship, but that inoculant performance can be improved through the inclusion of high performing strains. Given the wide environmental dependence of rhizobial inoculant quality, multi-strain inoculants could be highly successful as they increase the likelihood of including a strain well adapted to local conditions across different environments.

Complete Substitution of Clover Hay and its Impact on Performance of Barki Sheep in Arid and Semi-arid Regions.

BACKGROUND AND OBJECTIVE: Barley straw is a farm by product that may be useful in animal feeding but its use is limited due to its low nutritional value and high fiber content. Therefore, this study aimed to improving the nutritive value of barley straw by bacterial treatment with Condensed Molasses Soluble (CMS) in the ration of Barki lambs. MATERIALS AND METHODS: In Experiment 1, 12 Barki rams, which were divided into 3 equal groups (4 each), were given rations which consisted of 50% CFM+50%, either clover hay (C) or bacterial treated barley straw (T1) or T1+ CMS (T2). In Experiment 2, 10 Barki lambs were divided into 2 groups, one (C) and T2 as descripted in Experiment 1. Data analyzed by using one way ANOVA model. RESULTS: Digestibility of Dry Matter (DM), Organic Matter (OM), Crude Fiber (CF) and Ether Extract (EE) were insignificantly affected among all rations, while Digestible Crude Protein (DCP) recorded a higher value in T2 compared to T1. Changes in Total Digestible Nutrients (TDN) values were insignificant. Values of DCP were higher for T2 compared to C and T1. The Body Weight Gain (BWG) and Feed Conversion Ratio (FCR) of growing lambs didn't statistically differ among the groups. CONCLUSION: Finally, complete replacement of clover hay by treated barley straw plus CMS may be considered good feeding strategies in the feeding of Barki lambs in Arid and semi-arid regions.

Iron deficient Medicago scutellata grown in nutrient solution at high pH accumulates and secretes large amounts of flavins.

Flavin synthesis and secretion is an integral part of the toolbox of root-borne Fe facilitators used by Strategy I species upon Fe deficiency. The Fe-deficiency responses of the wild legume Medicago scutellata grown in nutrient solution have been studied at two different pH values (5.5 and 7.5). Parameters studied include leaf chlorophyll, nutrient solution pH, concentrations and contents of micronutrients, flavin accumulation in roots, flavin export to the medium, and root ferric chelate reductase and acidification activities. Results show that M. scutellata behaves upon Fe deficiency as a Strategy I species, with a marked capacity for synthesizing flavins (riboflavin and three hydroxylated riboflavin derivatives), which becomes more intense at high pH. Results also show that this species is capable of exporting a large amount of flavins to the external medium, both at pH 5.5 and 7.5. This is the first report of a species having a major flavin secretion at pH 7.5, in contrast with the very low flavin secretion found in other flavin-producing species such as Beta vulgaris and M. truncatula. These results provide further support to the hypothesis that flavin secretion is relevant for Fe acquisition at high pH, and open the possibility to improve the Fe-efficiency responses in legumes of agronomic interest.

Genetic Diversity and Differentiation of Eleven Medicago Species from Campania Region Revealed by Nuclear and Chloroplast Microsatellites Markers.

The species belonging to the genus Medicago are considered a very important genetic resource at global level both for planet's food security and for sustainable rangelands management. The checklist of the Italian flora (2021) includes a total number of 40 Medicago species for Italy, and 27 for Campania region, with a number of doubtful records or related to species no more found in the wild. In this study, 10 Medicago species native to Campania region, and one archaeophyte (M. sativa), identified by means of morphological diagnostic characters, were analyzed in a blind test to assay the efficacy of nine microsatellite markers (five cp-SSRs and four n-SSRs). A total number of 33 individuals from 6 locations were sampled and genotyped. All markers were polymorphic, 40 alleles were obtained with n-SSRs ranging from 8-12 alleles per locus with an average of 10 alleles per marker, PIC values ranged from 0.672 to 0.847, and the most polymorphic SSR was MTIC 564. The cp-SSRs markers were highly polymorphic too; PIC values ranged from 0.644 to 0.891 with an average of 0.776, the most polymorphic cp-SSR was CCMP10. 56 alleles were obtained with cp-SSRs ranging from 7 to 17 alleles per locus with an average of 11. AMOVA analysis with n-SSR markers highlighted a great level of genetic differentiation among the 11 species, with a statistically significant fixation index (FST). UPGMA clustering and Bayesian-based population structure analysis assigned these 11 species to two main clusters, but the distribution of species within clusters was not the same for the two analyses. In conclusion, our results demonstrated that the combination of the used SSRs well distinguished the 11 Medicago species. Moreover, our results demonstrated that the use of a limited number of SSRs might be considered for further genetic studies on other Medicago species.

Cover crop mixture expression is influenced by nitrogen availability and growing degree days.

Cover crop mixtures can provide multiple ecosystem services but provisioning of these services is contingent upon the expression of component species in the mixture. From the same seed mixture, cover crop mixture expression varied greatly across farms and we hypothesized that this variation was correlated with soil inorganic nitrogen (N) concentrations and growing degree days. We measured fall and spring biomass of a standard five-species mixture of canola (Brassica napus L.), Austrian winter pea (Pisum sativum L), triticale (x Triticosecale Wittm.), red clover (Trifolium pratense L.) and crimson clover (Trifolium incarnatum L.) seeded at a research station and on 8 farms across Pennsylvania and New York in two consecutive years. At the research station, soil inorganic N (soil iN) availablity and cumulative fall growing degree days (GDD) were experimentally manipulated through fertilizer additions and planting date. Farmers seeded the standard mixture and a "farm-tuned" mixture of the same five species with component seeding rates adjusted to achieve farmer-desired services. We used Structural Equation Modeling to parse out the effects of soil iN and GDD on cover crop mixture expression. When soil iN and fall GDD were high, canola dominated the mixture, especially in the fall. Low soil iN favored legume species while a shorter growing season favored triticale. Changes in seeding rates influenced mixture composition in fall and spring but interacted with GDD to determine the final expression of the mixture. Our results show that when soil iN availability is high at the time of cover crop planting, highly competitive species can dominate mixtures which could potentially decrease services provided by other species, especially legumes. Early planting dates can exacerbate the dominance of aggressive species. Managers should choose cover crop species and seeding rates according to their soil iN and GDD to ensure the provision of desired services.

Enhancing performance of berseem clover genotypes with better harvesting management through farmers' participatory research at smallholder farms in Punjab.

A field study was conducted on smallholder farmer fields between 2012 to 2014 to evaluate the performance of cv. Agaitti Berseem-2002, against local landraces exchanged between farmers (LBF1) or available from local markets (LBM1). The effects of genotype and harvesting regimen on forage production, quality and seed production were evaluated. Significant differences (P < 0.05) among genotypes and cutting treatments were recorded for forage and seed yields, and forage quality across all research sites in both years. Maximum cumulative fresh forage (89.7 t/ha) and dry matter (DM; 13.4 t/ha) yields were obtained with Agaitti Berseem-2002 when harvesting occurred five times over the season. However, maximum seed yield (1048 kg/ha) with higher 1000-seed weight (3.63 g) were obtained if forage was only harvested three times and the crop then left for seed set. Agaitti Berseem-2002 also produced forage with the higher crude protein content (27%), DM digestibility (69%), digestible organic matter (DM basis; 65%) and metabolizable energy content (10%) compared to the local landraces (LBF1 and LBM1). Therefore, the harvesting regimen for greatest economic return which produced optimum fresh and DM forage yields of highest nutritive values and maximum seed yield, were comprised of taking three forage cuts (at 65, 110 and 150 days after sowing) prior to seed harvest.

Species-dependent effect of cover cropping on trace elements and nutrients in vineyard soil and Vitis.

BACKGROUND: The research focused on the evaluation of the impact of cover cropping on trace metals (Fe, Mn, Cu, Zn, Pb, Co and Cd) and nutrients in vineyard soils and Vitis vinifera L. For this purpose, two types of cover crops (Lolium perenne L. and Medicago sativa L.) and their mixture were planted between vine rows of Muscat white in the vineyard in South Crimea. Trace elements, nutrients and other parameters were analyzed in the soil layers, leaves and grapevines of control and cover cropped plots. RESULTS: The effect of cover cropping was dependent on applied plant species. Ryegrass (L. perenne L.) seems to compete with V. vinifera L. for nutrients - these were lower in the soil and vines of the treated plot. In parallel, lead (Pb) bioconcentration in grapevines was reduced. In contrast, under lucerne (M. sativa L.), nitrogen in the soil and vines, and trace metal bioconcentration (Fe, Pb and Co) were higher. CONCLUSIONS: Our results indicate that cover cropping can influence the chemical composition of soil and vines. This should be considered when selecting cover crops. © 2019 Society of Chemical Industry.

Intercropped relationship change the developmental pattern of apple and white clover.

Intercropping can introduce greater plant diversity and functional complementarity in an arable crop system but inter- and intracompetition can between intercropped crops. The rhizo-box was established of apple-white clover intercropped system to examine the competitive relationship between intercropped crops on the Loess Plateau. The results showed that the competitive relationship between intercropped crops was dynamic and changed with the crop competitiveness. Crop competitiveness was characterized by root development, although intercropping inhibited the development and nutrient accumulation aboveground of apple trunks and branches, intercropped apples still maintained a larger root system than under monoculture and the root morphology of intercropped apples changed significantly. White clover had lower competitiveness than apple at the beginning of the year, which was reflected in the inhibited development in May. However in July and October, intercropped white clover had more biomass and nutrient accumulation than under monoculture.

Foliar Roundup application has minor effects on the compositional and functional diversity of soil microorganisms in a short-term greenhouse experiment.

The herbicide Roundup (and glyphosate, its active ingredient) is extensively used for weed control on a worldwide scale. It is absorbed after foliar application and quickly translocated inside the plant. In this study, we investigated the effects of Roundup speed, a commercial glyphosate formulation, on the structural composition (dominance of microbial groups, phospholipid fatty acid analysis - PLFA) and functional diversity (use of carbon sources, Multiple Substrate Induced Respiration - MSIR) of soil microorganisms. We specifically aimed at understanding the potential impact of biotic interactions on herbicide effects and included plants, earthworms, and endomycorrhizal fungi in the experimental setup. For this, we grew clover (Trifolium repens) in the greenhouse and added mycorrhizal inoculum (Glomus mosseae) and earthworms (Lumbricus terrestris) to the pots. Two weeks after foliar Roundup application and subsequent plant death, the pots were destructively sampled. The application resulted in a significant increase of microbial respiration (SIR) by approximately 30%. A multivariate analysis of the MSIR data exhibited small but significant differences between the microbial communities of treated and untreated pots, while no significant difference was apparent for the PLFA data. Bacterial PLFAs generally decreased following herbicide application, while mycorrhizal and fungal PLFAs were not affected. We did not find a consistent difference between the fatty acid markers of gram negative and gram positive bacteria. For all investigated parameters, there were highly significant differences between the upper (0-5 cm depth) and lower (5-10 cm) soil layers. The fact that rooting density differed by a factor of 3.5 between the two layers indicated that herbicide effects were especially pronounced in the clover rhizosphere and were likely due to changes in root exudate composition. We found significant, though very small, interactions between Roundup and other experimental factors (especially mycorrhizal inoculum).

A SOC1-like gene MtSOC1a promotes flowering and primary stem elongation in Medicago.

Medicago flowering, like that of Arabidopsis, is promoted by vernalization and long days, but alternative mechanisms are predicted because Medicago lacks the key regulators CO and FLC. Three Medicago SOC1-like genes, including MtSOC1a, were previously implicated in flowering control, but no legume soc1 mutants with altered flowering were reported. Here, reverse transciption-quantitative PCR (RT-qPCR) indicated that the timing and magnitude of MtSOC1a expression was regulated by the flowering promoter FTa1, while in situ hybridization indicated that MtSOC1a expression increased in the shoot apical meristem during the floral transition. A Mtsoc1a mutant showed delayed flowering and short primary stems. Overexpression of MtSOC1a partially rescued the flowering of Mtsoc1a, but caused a dramatic increase in primary stem height, well before the transition to flowering. Internode cell length correlated with stem height, indicating that MtSOC1a promotes cell elongation in the primary stem. However, application of gibberellin (GA3) caused stem elongation in both the wild type and Mtsoc1a, indicating that the mutant was not defective in gibberellin responsiveness. These results indicate that MtSOC1a may function as a floral integrator gene and promotes primary stem elongation. Overall, this study suggests that apart from some conservation with the Arabidopsis flowering network, MtSOC1a has a novel role in regulating aspects of shoot architecture.

A comparison of lateral root patterning among dicot and monocot plants.

Lateral root branching along the primary root involves complex gene regulatory networks in model plant Arabidopsis. However, it is largely unclarified whether different plant species share a common mechanism to pattern the lateral root along the primary axis. In this study, we assessed the development pattern of lateral root among several dicot and monocot plants, including Arabidopsis, tomato, Medicago, Nicotiana, rice, and ryegrass by using an agar-gel culture system. Our results reveal a regular-spaced distribution pattern of lateral roots along the primary root axis of both dicot and monocot plants. Meanwhile, the root patterning is tightly controlled by root bending and the plant hormone auxin. However, nitrogen and phosphate starvations trigger distinguished root growth patterns among different plant species. Our studies strongly suggest a partially shared signaling pathway underlying root patterning of various plant species, and also provide a foundation for further identification of genes associated with root development.

MtNRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase upregulated during nodulation in Medicago truncatula.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil.

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated for the first time a functional assay validation of metagenomic and metatranscriptomic datasets by utilizing the clover and Rhizobium leguminosarum sv. trifolii mutualism. The results show that the Most Probable Number results corroborate the results of the 'omics approaches and gives confidence to the study of other biological systems where such a cross-check is not available.

[The plant growth-promoting rhizobacterium Arthrobacter agilis UMCV2 endophytically colonizes Medicago truncatula]. / La rizobacteria promotora del crecimiento vegetal Arthrobacter agilis UMCV2 coloniza endofíticamente a Medicago truncatula.

Arthrobacter agilis UMCV2 is a rhizosphere bacterium that promotes legume growth by solubilization of iron, which is supplied to the plant. A second growth promotion mechanism produces volatile compounds that stimulate iron uptake activities. Additionally, A. agilis UMCV2 is capable of inhibiting the growth of phytopathogens. A combination of quantitative polymerase chain reaction and fluorescence in situ hybridization techniques were used here to detect and quantify the presence of the bacterium in the internal tissues of the legume Medicago truncatula. Our results demonstrate that A. agilis UMCV2 behaves as an endophytic bacterium of M. truncatula, particularly in environments where iron is available.

Historical nectar assessment reveals the fall and rise of floral resources in Britain.

There is considerable concern over declines in insect pollinator communities and potential impacts on the pollination of crops and wildflowers. Among the multiple pressures facing pollinators, decreasing floral resources due to habitat loss and degradation has been suggested as a key contributing factor. However, a lack of quantitative data has hampered testing for historical changes in floral resources. Here we show that overall floral rewards can be estimated at a national scale by combining vegetation surveys and direct nectar measurements. We find evidence for substantial losses in nectar resources in England and Wales between the 1930s and 1970s; however, total nectar provision in Great Britain as a whole had stabilized by 1978, and increased from 1998 to 2007. These findings concur with trends in pollinator diversity, which declined in the mid-twentieth century but stabilized more recently. The diversity of nectar sources declined from 1978 to 1990 and thereafter in some habitats, with four plant species accounting for over 50% of national nectar provision in 2007. Calcareous grassland, broadleaved woodland and neutral grassland are the habitats that produce the greatest amount of nectar per unit area from the most diverse sources, whereas arable land is the poorest with respect to amount of nectar per unit area and diversity of nectar sources. Although agri-environment schemes add resources to arable landscapes, their national contribution is low. Owing to their large area, improved grasslands could add substantially to national nectar provision if they were managed to increase floral resource provision. This national-scale assessment of floral resource provision affords new insights into the links between plant and pollinator declines, and offers considerable opportunities for conservation.

Phytotoxicity of citric acid and Tween® 80 for potential use as soil amendments in enhanced phytoremediation.

Enhanced phytoremediation adding biodegradable amendments like low molecular weight organic acids and surfactants is an interesting area of current research to overcome the limitation that represents low bioavailability of pollutants in soils. However, prior to their use in assisted phytoremediation, it is necessary to test if amendments per se exert any toxic effect to plants and to optimize their application mode. In this context, the present study assessed the effects of citric acid and Tween® 80 (polyethylene glycol sorbitan monooleate) on the development of alfalfa (Medicago sativa) plants, as influenced by their concentration and frequency of application, in order to evaluate the feasibility for their future use in enhanced phytoremediation of multi-contaminated soils. The results showed that citric acid negatively affected plant germination, while it did not have any significant effect on biomass or chlorophyll content. In turn, Tween® 80 did not affect plant germination and showed a trend to increase biomass, as well as it did not have any significant effect on chlorophyll levels. M. sativa appeared to tolerate citric acid and Tween® 80 at the tested concentrations, applied weekly. Consequently, citric acid and Tween® 80 could potentially be utilized to assist phytoremediation of contaminated soils vegetated with M. sativa.

Rhizobial symbiosis effect on the growth, metal uptake, and antioxidant responses of Medicago lupulina under copper stress.

The effects of rhizobial symbiosis on the growth, metal uptake, and antioxidant responses of Medicago lupulina in the presence of 200 mg kg(-1) Cu(2+) throughout different stages of symbiosis development were studied. The symbiosis with Sinorhizobium meliloti CCNWSX0020 induced an increase in plant growth and nitrogen content irrespective of the presence of Cu(2+). The total amount of Cu uptake of inoculated plants significantly increased by 34.0 and 120.4% in shoots and roots, respectively, compared with non-inoculated plants. However, although the rhizobial symbiosis promoted Cu accumulation both in shoots and roots, the increase in roots was much higher than in shoots, thus decreasing the translocation factor and helping Cu phytostabilization. The rate of lipid peroxidation was significantly decreased in both shoots and roots of inoculated vs. non-inoculated plants when measured either 8, 13, or 18 days post-inoculation. In comparison with non-inoculated plants, the activities of superoxide dismutase and ascorbate peroxidase of shoots of inoculated plants exposed to excess Cu were significantly elevated at different stages of symbiosis development; similar increases occurred in the activities of superoxide dismutase, catalase, and glutathione reductase of inoculated roots. The symbiosis with S. meliloti CCNWSX0020 also upregulated the corresponding genes involved in antioxidant responses in the plants treated with excess Cu. The results indicated that the rhizobial symbiosis with S. meliloti CCNWSX0020 not only enhanced plant growth and metal uptake but also improved the responses of plant antioxidant defense to excess Cu stress.