Agronomic and phytochemical evaluation of Stevia rebaudiana Bert. as a novel source of bio-active compounds

In recent years much attention has been paid to the plant species that can provide bio-active constituents to be used in food formulations or in pharmaceutical applications, characterized by nutraceutical and functional power. The nutraceutical compounds may be considered constituents able to provide medical or health benefits including the prevention and/or treatment of several diseases. Some species hitherto neglected and underutilized (NUS) may be revalued or implemented in their use, becoming important resources of bio-active compounds able to promote health. At the same time they may become important to enhance biodiversity and for the exploitation of marginal agrarian environments. Neglected and underutilized crops are domesticated plant species that have been used for centuries or even millennia for their properties, but their importance have been reduced over time owing to particular supply and use constraints. Among NUS, Stevia rebaudiana Bert., used for a long time as a sweetener and herbal remedy by the Guaranì people of Brazil and Paraguay, can be fully included in this kind of plants. This species produces, in its leaves, ent-kaurene diterpenoid glycosides, namely steviol glycosides (SVglys). These compounds are natural no-calorie sweeteners more powerful than sucrose. The interest toward this species as substitute of both sucrose and non-caloric artificial sweeteners (NAS), has strongly increased, due to growing concern regarding important chronic diseases, such as obesity and diabetes. Nowadays the worldwide demand for stevia products is steadily increasing and it is predictable that in the coming years the agricultural production capacity will be lower than the market demand. The positive European trend and the expected increments demand for stevia, is surely due to two important legislative initiatives; the first one, promulgated at the end of 2011, approved purified steviol glycosides as food additive. The second one, more recent, is a revision of stevia in novel food category: from June 2017 there is the possibility to prepare tea, herbal and fruit infusion with stevia leaves (such use(s) is considered as not novel). Despite a successfully introduction of stevia in several European countries and in different environmental conditions, stevia commercial-scale production has not been developed yet except in some very limited cases. Consequently an improvement, using efficient agronomic practices, of its cultivation should be achieved. Moreover, there is an increasing interest to guarantee a secure availability for high-quality, safe and traceable agricultural raw materials in order to meet consumer needs and bio-industry requirements. To achieve these goals is important to optimize several agronomic aspects, such as propagation technique, nutrition, plant response to abiotic stress, weed management, irrigation, etc. Therefore the major objective of the present PhD thesis is to investigate thoroughly the effects of some agro-environmental factors (phosphorus fertilization and bio-fertilisers) and salinity, on stevia leaf and steviol glycosides production. Moreover, the reproductive mechanisms of this species have been deeply investigated by analyzing the effect of genotype and insect pollinators on seed production and quality. The final aim is to provide scientific and applied information regarding the effect of some important agronomic and environmental aspects in order to make stevia cultivation more efficient, in particular under temperate climate. The thesis consists of six chapters. In the first chapter an accurate review of the literature about stevia from a wide temporal interval has been done. The chapters 2 and 3 deal with reproductive mechanisms in stevia, by considering the role of insect pollinators, the reproductive characteristics of different F1 genotypes, and their flower volatile organic compounds, on seed production and quality. In the following experiment (chapters 4 and 5) the effects of arbuscular mycorrhizal fungi (AMF), of phosphorus fertilization and their reciprocal interaction (AMFxP) on stevia main biometric and productive parameters, as well as SVglys yield have been analyzed. Finally in chapter 6 the role of salinity on stevia growth and physiological parameters, have been fully examined in plants grown on hydroponic conditions. Being known that stevia usually shows low seed production/quality and on the light of the positive relationship between pollinators and seed production, in the first experiment (chapter 2 and 3) thirty-six F1 genotypes were compared, grown in pots in open-field conditions, were compared. The aims of this experiment were to evaluate i) morphological and phenological differences among the genotypes, ii) to examine the importance of insect pollinators and harvest time on seed production/quality, and iii) to evaluate the relationship between seed yield and plant reproductive phenology. Finally, in order to better understand flower/insect relation and to contribute on the chemistry of volatiles involved in pollinators attraction, the VOCs profile emitted from the flowers of F1 plants were assessed. The results indicate that earlier flowering genotypes produced higher seed quantity, because of the favorable weather conditions during the reproductive phase. Moreover, cross-pollination and the abundance of insect pollinators, as Apidae and Syrphidae, play a crucial role for enhancing the stevia seed yield and quality. Concerning VOCs, limonene was the most representative organic compound responsible to attract pollinators, while high emissions of δ-elemene and bicyclogermacrene were linked to plants that are less attractive to pollinators. Overall, this experiment adds knowledge on floral phenology and pollinator ecological traits of stevia, allowing a deeper understanding of its chemical ecology and pollination. Considering the limited information available on both nutritional and non-nutritional fertilization effects in stevia, a further experiment (chapter 4 and 5) have been conducted in order to evaluate arbuscular mycorrhizal fungi (AMF) and phosphorus fertilization effect. The experiment was performed exposing the plants, cultivated in pot in open-field conditions, to three different P doses with or without AMF inoculation, in order to study biometric, productive and physiological plant responses. The results showed that stevia roots were highly colonized by the AMF, especially in the absence of P fertilization. During the whole vegetative growth, both AMF symbiosis and P supply showed positive effect on leaf dry biomass production and SVglys yield. AMF was also able to modify the growth and architecture of stevia plants, with increased branching and a reduced plant height. Moreover AMF symbiosis stimulates leaf gas exchanges, total soluble sugars and total monosaccharides in the roots while, both AMF and P fertilization positively affected photosynthesis. The increasing interest to explore the possibilities to cultivate stevia under different agro-environmental conditions, permitted, in the third experiment (chapter 6) to explore the impact of salinity in stevia. The experiment was carried out under controlled conditions, using hydroponic cultivation technique (floating system). Four NaCl concentrations were tested with the aim to evaluate the effect of sodium chloride stress on stevia biometric, productive, physiological, biochemical responses. The experiment underlined that, in stevia, the high salinity level (50 mM) resulted in lower crop yield, growth and SVglys content. Stevia plants are able to put into effect several adaptations to salt stress, both at physiological and biochemical level (antioxidant enzyme activities), in order to tolerate and survive to salinity. On the light of the results concerning growth analysis, gas exchange parameter, antioxidant enzyme activities and antioxidant molecules biosynthesis, it was possible to classified stevia as moderate salt tolerant species for 50 mM NaCl and tolerant for lower salt concentrations. The improved knowledge of the role of different agro-environmental factors on stevia propagation, leaf production and bioactive compounds biosynthesis can improve understanding of the biological, biochemical and physiological processes involved in stevia production, thus optimizing the agronomic management for this emerging and promising new crop for Europe.