Evaluation of the effect of arbuscular mycorrhization on qualitative and quantitative production of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni is well known because it accumulates, in its leaves, ent-kaurene diterpenoid glycosides, namely steviol glycosides (SVglys), that are non-caloric natural sweeteners, possessing a 250-300 times higher sweetening property than sucrose. Furthermore, stevia leaves contain others important compounds, such are sterols, triterpenoids, essential oils, polyphenols (flavonoids and hydroxycinnamic acids), vitamins, alkaloids, water-soluble chlorophylls, xanthophylls, that can explicate antioxidant and biological activities with potential beneficial effects on human health. In the European market, the introduction of SVglys as food additives, with a purity of more than 95%, was given only at the end of 2011, thanks to the positive opinion issued by EFSA, on their safety, raising the acceptable daily intake (ADI) of 4 mg/kg BW/day, expressed as steviol equivalents (Reg. EU 1131/2011).Whereas dried stevia leaves approval in Europe must pass through the legislative procedure of the regulation on novel foods (Reg. EU 2283/2015). Arbuscolar mycorrhizal fungi (AMF) are soil-borne mutualistic fungi belonging to the phylum of Glomeromycota, that can improve plants growth rate, pathogens resistance, water and mineral nutrient uptake. Thus, these soil-dwelling symbiotic microorganisms are useful for agriculture and for cultivation of medicinal plants, stevia included. There is a crescent interesting for AMF, as agro-system providers able to sustain crop productivity and quality. In fact they are efficient when nutrient availability is low and when nutrients are bound to organic matter and soil particles. Especially for phosphorus uptake in low P soil and plants under stressed environments. This thesis aims to evaluate the effect of AMF symbiosis and phosphorus fertilization on stevia plants. Six treatments have been compared in a pot trial, testing three phosphorus doses (0, 25 and 50 mg P2O5/kg of soil) in plants with and without AMF. Rhizoglomus intraradices has been used as inoculum. Aboveground and belowground samplings have been done at 69, 89 and 123 days after transplanting (DAT) for quantifying root colonization by AMA, the main biometric and productive characteristics, N and P uptake at 123 DAT, SVglys yield and characterization, phenols and flavonoids content, the antioxidant activity assessed by different assay. The results achieved showed as stevia can be successfully colonized by R. intraradices and how both AMF and P fertilization can enhanced the yield and improve phytochemical profile of the stevia leaf extract. AMF symbiosis could represent a valid biofertilization strategy to improve biomass in stevia, even if it was not possible to define a clear trend for all the analysed parameters

Crop and Steviol Glycoside Improvement in Stevia by Breeding

Worldwide demand for purified steviol glycosides (SVglys) from Stevia rebaudiana Bertoni leaves is steadily increasing. In the future, it is expected that the agricultural production capacity will be lower than the market demand. To improve the competitiveness of stevia production it is important to produce higher-performing crops in terms of yield and quality, resource use efficiency, greater resistance and resilience against drought stress, extreme weather conditions and a range of biotic stresses. Therefore, the development of new varieties/cultivars of S. rebaudiana with higher leaf and SVglys yields with greater resistance to abiotic and biotic stresses is the topmost priority and the primary aim for plant breeders. Since genotypic and phenotypic variation in the characters, in which we are interested, was reportedly quite high, researchers have attempted to enhance knowledge about this variability, and to develop breeding strategies, starting to study the feasibility and relevance of the characters being measured under different environmental and agro-climatic conditions. The chapter, aims to provide up-to-date scientific information regarding the breeding and selection programs carried out on stevia, describing both conventional plant breeding approaches and new biotechnological approaches, in order to provide new varieties/cultivars with improving quality traits

Effects of Arbuscular mycorrhizal fungi and phosphorus fertilization on the antioxidant activity of Stevia rebaudiana

The purpose of this study has been to evaluate the effect of mycorrhization and phosphorus fertilization on the antioxidant properties of S. rebaudiana (Bertoni) plants in a pot trial established in Pisa, Italy. Different agronomic treatments were performed in the crop plantation: Three phosphorus doses (0, 25 and 50 mg of P2O5/kg of soil as triple superphosphate) were tested in plants with or without Arbuscular Mycorrhizal Fungi (AMF) inoculation, using Glomus intraradices as inoculum. In turn, leaves were harvested at 2 different periods: 89 and 123 days after transplanting (DAT). Dried leaves aqueous extracts were analyzed for the total phenolic content through Folin-Ciocalteu method. The antioxidant capacity was evaluated by means of the oxygen radical absorbance capacity (ORAC) method, and the cellular antioxidant activity (CAA) assay. It was shown that the higher total phenolic content is obtained during the first harvest in which the plants were in the full phase of vegetative growth; the treatment with 50 mg P showed the highest polyphenols content (66.45 mg GAE/g). This research indicated that the supplementation with 25 mg of P gives the highest ORAC values, being in average 2182.15 μmol trolox equivalents/g stevia dry leaves. Similar results were observed in plants subjected to combined treatment with 25 mg P + AMF inoculation. In general, the ORAC values depended upon the harvest period, being higher in the 123 DAT plants when compared to 89 DAT plants. No significant correlation was found between ORAC values from the first and second harvest. The CAA results for the analyzed treatments with and without AMF inoculation are not significantly different from each other (p > 0.05), ranging in average from 9.0 to 12.1 μmol quercetin equivalents/g stevia dry leaves, depending upon the phosphorus treatment. A strong significantly correlation was observed for CAA values between different harvest times (p< 0.001). The highest CAA mean value was obtained for the plants harvested 89 DAT (mean of all treatments: 13.2 μmol quercetin equivalents/g stevia dry leaves). Between the P2O5-supplemented plants, the treatment with 50 mg, gave the higher antioxidant potential by means of CAA (12.0 μmol quercetin equivalent/g stevia). This result is in agreement with that obtained with the combined treatment with 50 mg P2O5 supplementation + AMF inoculation (12.1 μmol quercetin equivalents/g stevia dry leaves)

Influence of arbuscular mycorrhizal fungi and phosphorus nutrition on secondary metabolite production and antioxidant activity of Stevia rebaudiana Bert.

The European demand for stevia-derived products is steadily increasing and the possibility to grow stevia organically represents an aspect of pivotal importance in obtaining sustainable and safe products. Arbuscular Mycorrhizal Fungi (AMF) can be relevant for organic agriculture since they can act as natural fertilizers, with interesting effects on crop yield and secondary metabolites. Consequently, the aim of this study was to evaluate the effect of AMF symbiosis and phosphorus fertilization on the antioxidant properties of Stevia rebaudiana Bertoni leaves. Six treatments were compared in a pot trial established in Pisa (central Italy), testing three phosphorus (P) levels (0, 25 and 50 mg P2O5/kg of soil as triple superphosphate) in plants grown with and without AMF inoculation. Rhizoglomus intraradices has been used as inoculum. Sampling has been carried out before flowering, 123 days after transplanting. The antioxidant properties of aqueous leaf extracts were investigated, in terms of total phenols (Folin-Ciocalteu method), total flavonoids (aluminum-chloride method), and antioxidant activity, assessed both in vitro (ORAC assay) and at cellular level (CAA assay). The obtained results showed that AMF symbiosis and P level were able to affect the investigated parameters. In particular, AMF-treated plants were characterized by the lowest content of total phenols and flavonoids as well the lowest ORAC values. No significant differences (P > 0.05) were recorded for cellular antioxidant activity (7.94 μmol quercetin equivalents/g stevia dry leaves, as mean value). In addition, this research highlighted that the addition of 25 mg of P2O5 to the soil significantly increased the total phenolic and flavonoid content, together with the antioxidant activity (measured by ORAC assay). Conversely, the highest CAA values were recorded with 50 mg of P2O5. Definitively, this study provides useful information on stevia response to AMF symbiosis and phosphorus fertilization, highlighting that the antioxidant properties of stevia leaves can strongly vary as a function of AMF, P and AMFxP interaction

Stevia response to salt stress: agronomic, physiological and biochemical evaluation using a floating system technique

Today, there is a need to increase the European area under stevia cultivation, due to the rapid progress of stevia market size. It has been estimated that, in Europe, soil salinity affects 1 million hectares, mainly in the Mediterranean regions, where salinity in irrigation water and in soil represents one of the most limiting abiotic factors affecting crop productivity. Consequently, expanding knowledge on basic and applied aspects of stevia productivity under salt stress conditions is of pivotal importance. For this reason, an experimental trial in a floating system under controlled conditions was carried out to evaluate the effect of salt (NaCl) on the quality and quantity of the stevia production. From May to the end of June 2016, four NaCl concentrations (0, 12.5, 25 and 50 mM), each with three replications, have been compared, using a modified Hoagland nutrient solution. Leaf area, plant height, branch number, as well as leaf, stem, root and total dry biomass were measured at the beginning and at the end of the experiment. At the same time, a growth analysis was performed, and steviol glycosides (SVglys) content and profile were evaluated. The results obtained underlined that in stevia, salinity at 12.5 mM onwards negatively affected the main biometric and productive parameters, lowering plant height, root biomass and leaf and stem dry yields. Similarly, stressed plants showed decreased relative growth rate (RGR) and leaf area expansion. The RGR was reduced by 10.5 %, 19.0 % and 26.7 % in the 12.5, 25 and 50 mM treatments, respectively. Net assimilation ratio (NAR) remained stable until 25 mM NaCl treatment, and significantly decreased at 50 mM. At the same time, due to a limited leaf expansion, significant reductions of leaf area ratio (LAR) and specific leaf area (SLA) in stressed plants were observed, with the lowest values for 50 mM-treated plants. On the other hand, no significant differences in leaf weight ratio (LWR) were detected. Regarding the SVglys content and profile, significant differences were observed for stevioside (Stev), rebaudioside A (RebA), RebA/Stev ratio and Stev + RebA content. Stev and Stev + RebA content significantly decreased in 50 mM-treated plants, while both the RebA and RebA/Stev ratio significantly increased at 25 mM and then decreased in 50 mM-treated plants. These results confirm that in stevia, similar to several other plant species, the higher salinity level resulted in lower crop yield and growth, suggesting a moderate NaCl tolerance

Human kynurenine aminotransferase II: reactivity with substrates and inhibitors

Kynurenine aminotransferase (KAT) is a pyridoxal 5â-phosphate- dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild-type KATII and a variant carrying the active site mutation Tyr142âPhe. The transamination and the β-lytic activity of KATII towards the substrates kynurenine and α-aminoadipate, the substrate analog β-chloroalanine and the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high-throughput KATII inhibitor screenings, an endpoint assay suitable for 96-well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination. Kynurenine aminotransferase (KAT) is a pyridoxal 5â-phosphate-dependent enzyme catalyzing the formation of kynurenic acid, an antagonist of excitatory receptors in the CNS. KATII is a potential drug target for the treatment of schizophrenia. KATII spectroscopy, transamination and β-lytic activity were characterized with substrate and substrate analogs using novel continuous and high-throughput assays

Plant growth, steviol glycosides and nutrient uptake as affected by arbuscular mycorrhizal fungi and phosphorous fertilization in Stevia rebaudiana Bert

The increasing demand for products based from Stevia rebaudiana Bertoni (both leaves and purified steviol glycosides) makes of interest the research on sustainable production systems, in order to guarantee secure availability and high quality of agricultural raw materials. Arbuscular mycorrhizal fungi (AMF) symbiosis represents an interesting tool for increasing crop production and quality, especially thanks to improved nutrient absorption, particularly phosphorus (P). In the present study, Stevia rebaudiana Bert. plants were exposed to different levels of P fertilization (0, 25 and 50 mg P2O5kgâ\u88\u921soil) with or without Rhizoglomus irregulare inoculation, in order to evaluate root colonization, plant growth and productive parameters, steviol glycosides (SVglys) yield, as well as nitrogen (N) and P concentrations and uptake. A nutrient balance was also carried out and the nutrient use efficiency was evaluated. Stevia roots were highly colonized by Rhizoglomus irregulare, especially in the absence of P fertilization. During the whole vegetative growth, the AMF symbiosis, in association with the P supply, benefitted stevia growth, especially with regard to leaf dry biomass production and SVglys yield. Arbuscular mycorrhizal fungi symbiosis was able to modify the growth habit of stevia plants, with increased branching and a reduced plant height. At the end of the vegetative growth, mycorrhizal plants reached the highest leaf dry yield, together with the highest SVglys production. The application of 25 mg P2O5kgâ\u88\u921soil in association with AMF symbiosis seemed to be the most effective treatment in improving stevia SVglys yield and P uptake together with P nutrient use efficiency

CAN STEVIA LEAF EXTRACTS PROTECT BETA CELLS AGAINST GLUCO- OR LIPOTOXICITY?

In the present study, the effects of leaf extracts of two Stevia rebaudiana Bertoni biotypes (namely ST2 and ST3) on beta cells exposed to gluco- or lipotoxic conditions have been evaluated. Leaf extracts were obtained through innovative and eco-friendly extraction technologies, able to obtain enriched fractions of desired bioactive compounds. Leaf phytochemical composition was spectrophotometrically assessed, in terms of total phenols and flavonoids as well as for their anti-radical and anti-oxidant activity, evaluated by DPPH radical-scavenging assay and ferric reducing antioxidant power (FRAP), respectively. Furthermore, the content and profile of the main steviol glycosides was analysed by HPLC, using an HILIC column under isocratic conditions, and quantified through standard curves of pure standard mixtures. MTT assay was used in order to evaluate the protection effect of stevia leaf extracts on beta cells exposed to gluco- or lipotoxicity conditions. INS-1E cells were exposed for 4 days to 30 mM glucose or 24h to 0.5 mM palmitate with or without increasing concentrations of stevia leaf extracts.The phytochemical characterisation revealed that ST2 had higher total phenolic and flavonoid content and antioxidant activity, in comparison with ST3. Conversely, ST3 showed higher (p<0.05) concentration of stevioside than ST2. Chronic exposure to high glucose concentration reduced INS-1E cells viability (-40%), that was completely prevented by exposure to 50 or 200 μg/mL ST2 and partially, but significantly, in the presence of ST3 genotype. Similarly, 24h exposure to palmitate resulted in a decrease of INS-1E viability (-20%), that was counteracted by both ST2 and ST3. The study shows that Stevia rebaudiana extracts have beneficial effects on viability of beta cells exposed to gluco- or lipotoxic conditions, possibly depending on their phytochemical composition and related antioxidant capacity