Microalgae are attracting the interest of agrochemical industries and farmers, due to their biostimulant and biofertiliser properties. Microalgal biostimulants (MBS) and biofertilisers (MBF) might be used in crop production to increase agricultural sustainability. Biostimulants are products derived from organic material that, applied in small quantities, are able to stimulate the growth and development of several crops under both optimal and stressful conditions. Biofertilisers are products containing living microorganisms or natural substances that are able to improve chemical and biological soil properties, stimulating plant growth, and restoring soil fertility. This review is aimed at reporting developments in the processing of MBS and MBF, summarising the biologically-active compounds, and examining the researches supporting the use of MBS and MBF for managing productivity and abiotic stresses in crop productions. Microalgae are used in agriculture in different applications, such as amendment, foliar application, and seed priming. MBS and MBF might be applied as an alternative technique, or used in conjunction with synthetic fertilisers, crop protection products and plant growth regulators, generating multiple benefits, such as enhanced rooting, higher crop yields and quality and tolerance to drought and salt. Worldwide, MBS and MBF remain largely unexploited, such that this study highlights some of the current researches and future development priorities.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of b-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, b-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

The antimicrobial activity of saponins from Medicago sativa, M. arborea and M. arabica against a selection of medically important yeasts, Gram-positive and -negative bacteria was investigated. Structure-activity growth inhibitory effects of related prosapogenins and sapogenins are also described. Increasing antibiotic activity was observed going from the saponin extracts to the sapogenin samples, suggesting that the sugar moiety is not important for the antimicrobial efficacy. Activity was especially high against Gram-positive bacteria (Bacillus cereus, B. subtilis, Staphylococcus aureus and Enterococcus faecalis) with M. arabica being the species showing a broader spectrum of action. Discrete antifungal activity was also observed, mainly against Saccharomyces cerevisiae. The observed antimicrobial properties of M. sativa and M. arborea were related to the content of medicagenic acid, while hederagenin seems to contribute to the bioactivity of M. arabica total sapogenins.

Jasmonates (JAs) are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of multiple plant processes, encompassing development, growth and defense. Across the plant kingdom, JAs act as elicitors of the production of bioactive secondary metabolites that serve in the defense against attackers 1-3 . Knowledge on the conserved JA perception and early signaling machineries is increasing 3-6 but the downstream mechanisms that regulate defense metabolism remain largely unknown.Here we show that in the model legume Medicago truncatula JA recruits the endoplasmic reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols 7-9 . An ERAD-type RING membrane-anchor (RMA) E3 ubiquitin (Ub)-ligase is co-expressed with saponin synthesis enzymes to control 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thereby unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that employs distinct E3 Ub-ligases, of the HMGR Degradation 1 (HRD1)-type, to direct destruction of HMGR 10-13 . Hence, the general principles for management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues. 3To identify regulators of plant triterpene synthesis, we monitored the transcriptome of suspension-cultured M. truncatula cells, known to accumulate saponins following elicitation with JAs 14 . The expression of 8,462 transcripts was visualized by cDNA-AFLP transcript profiling and 282 Methyl JA (MeJA)-responsive tags were identified. The comparable MeJAinduced expression pattern of the genes encoding HMGR, squalene synthase, squalene epoxidase, β-amyrin synthase (BAS) and CYP93E2, enzymes catalyzing steps in triterpene saponin biosynthesis 7-9 , indicated co-regulation ( Fig. 1a and Extended Data Fig. 1-2). Several genes corresponding to potential regulatory factors had maximal transcriptional upregulation prior to or concurrent with that of the triterpene saponin genes, including a MYC-like bHLH protein and the JAZ repressor proteins, known elements of the core JA signaling module 4-6 , as well as a gene (MT067) corresponding to an RMA-like E3 Ub-ligase 10 , denominated MAKIBISHI1 (MKB1) (Extended Data Fig. 3). The early MeJA response of MKB1 was confirmed in the M. truncatula Gene Expression Atlas (MtGEA; http://bioinfo.noble.org/geneatlas/) 15 (Fig. 1b).To assess MKB1 function, we generated transgenic M. truncatula hairy roots in which MKB1 was overexpressed (MKB1 OE roots) or silenced (MKB1 KD roots) (Extended Data Fig. 4a). MKB1 KD roots showed a striking phenotype, in particular when transferred to liquid medium, which caused 'dissociation' of the MKB1 KD roots into 'c...

Naturally occurring saponins are a large group of triterpene and steroid glycosides characterized by several biological and pharmacological properties. The Medicago genus represents a valuable source of saponins which have been extensively investigated. This review summarizes the chemical features of saponins from Medicago species and their biological activity, with particular attention to their antimicrobial, insecticidal, allelopathic and cytotoxic effects. Influence of saponins on animal metabolism is also reported.

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

Silymarin, a natural acknowledged hepatoprotector used in humans to treat liver diseases, has been tested in dairy cows during peripartum, a period during which animals are subject to subclinical fatty liver. Ten grams of silymarin (76% pure extract consisting in flavonolignans, taxifolin, and other trace compounds) per day, was administered as a water suspension by an oral drench to 15 cows from d 10 before expected calving to 15 d after calving. Milk production was measured, and colostrum, milk, and blood samples were analyzed during the experimental period. Treated animals showed the peak of milk production at 55 +/- 1.85 d after calving, 1 wk before the control group (62 +/- 3.27 d); the average peak production was 41.6 +/- 1.05 kg for the treated group vs. 39.1 +/- 1.44 kg for the control; the treated animals maintained a greater milk production than control cows throughout lactation (9922.1 +/- 215.7 vs. 9597.8 +/- 225.4 kg). Milk composition was unaffected by treatment. No silymarin residues were detected in colostrum and all milk samples. After calving, body condition score (BCS) decrease was greater for control compared with treated cows. Glucose, urea, triglycerides (TG), total cholesterol, beta-hydroxibutyrate (BHBA), and gamma-glutamyl transferase (GGT) in plasma were unaffected by treatment. Plasma nonesterified fatty acids (NEFA) on d-7 were higher in treated cows compared with the control group (741 vs. 181 micromol/L). From this evidence, it is possible to conclude that silymarin beneficially affected lactation performances and body condition of treated animals. Blood and milk parameters do not indicate any adverse effects of feeding this natural compound.

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.