Trichoderma spp.-mediated mitigation of heat, stress, and their combination on the Arabidopsis thaliana holobiont: a metabolomics and metabarcoding approach

IntroductionThe use of substances to increase productivity and resource use efficiency is now essential to face the challenge of feeding the rising global population with the less environmental impact on the ecosystems. Trichoderma-based products have been used as biopesticides, to inhibit pathogenic microorganisms, and as biostimulants for crop growth, nutrient uptake promotion, and resistance to abiotic stresses.MethodsIn this work, plant metabolomics combined with roots and rhizosphere bacterial metabarcoding were exploited to inspect the performance of Trichoderma spp. biostimulants on Arabidopsis thaliana under drought, heat and their combination and its impact on plant holobiont.Results and discussionAn overall modulation of N-containing compounds, phenylpropanoids, terpenes and hormones could be pointed out by metabolomics. Moreover, metabarcoding outlined an impact on alpha and beta-diversity with an abundance of Proteobacteria, Pseudomonadales, Burkholderiales, Enterobacteriales and Azospirillales. A holobiont approach was applied as an integrated analytical strategy to resolve the coordinated and complex dynamic interactions between the plant and its rhizosphere bacteria using Arabidopsis thaliana as a model host species

Use of biostimulants to counteract the stresses related to climate change in plant

Lo scopo della presente tesi di dottorato era valutare l'impatto dello stress combinato (alte temperature e siccità) in Arabidopsis thaliana e confrontare i singoloi stress con la loro combinazione per rivelare possibili effetti e interazioni additive. Gli studi di stress combinati sono raramente affrontati da una prospettiva molecolare nonostante la risposta a stress combinati non corrisponde semplicemente alla somma delle risposte a ciascuno stress che possono avere componenti additive o opposte. Queste risposte includono cambiamenti morfologici, fisiologici, biochimici e molecolari e l'attivazione di vie di segnalazione che coinvolgono fitormoni, trasduttori di segnale e regolatori trascrizionali. Nella seconda parte, è stata anche testata la performance di Trichoderma (biostimolante) in condizioni di siccità, calore, e loro combinazione, e il suo impatto sulle relazioni pianta-microrganismi. L'applicazione di funghi come Trichoderma può essere benefica, in quanto il fungo può stabilire relazioni simbiotiche con una pianta ospite ed influenzarne la crescita, la germinazione dei semi e la fioritura. Il fungo inoltre rilascia metaboliti secondari che inibiscono la crescita dei patogeni, aumentano la tolleranza allo stress e inducono la sintesi e l'accumulo di enzimi, la produzione di molecole come i fitormoni di sostanze con attività simile. Inoltre, i metaboliti secondari prodotti dalle piante, fungono da segnale nelle interazioni pianta-microrganismi, influenzando la composizione del microbioma vegetale. Questa relazione è dinamica e coinvolge diversi scambi bidirezionali: alcuni microrganismi regolano la produzione di metaboliti secondari e queste molecole bioattive modellano il microbioma del suolo. Queste interazioni pianta-microrganismi avvengono durante tutte le fasi del ciclo di vita e insieme all'interazione con l'ambiente e altri fattori biotici modellano la composizione del microbiota vegetale. A tal fine, è stata utilizzata una combinazione di metabolomica delle piante e metabarcoding batterico della radice e della rizosfera per svelare gli effetti a livello di "holobiont" vegetale. La combinazione di diversi approcci offre una visione completa delle interazioni che influenzano i passaggi dal genotipo al fenotipo, fornendo un profilo globale per comunità più diversificate.The aim of the present PhD thesis was to evaluate the impact of combined heat and drought stress in Arabidopsis thaliana and compare the individual stress to their combination to reveal additive effects and interactions. Studies on the co-occurrence of more abiotic stresses are rarely approached from a molecular perspective and evidence suggests that the response to a combined stress does not correspond simply to the sum of the responses to each stress but may have antagonistic or opposing components. These responses include morphological, physiological, biochemical, and molecular changes and the activation of signaling pathways that involves phytohormones, signal transducers and transcriptional regulators. In the second part, was also tested the performance of Trichoderma biostimulants on Arabidopsis thaliana under drought, heat and their combination and its impact on plant holobiont. The application of Trichoderma spp. as biostimulant is well recognized. These fungi can establish symbiotic relationships with a host plant and other than the effect on growth, seed germination and flowering, the fungus releases secondary metabolites that inhibit the growth of plant pathogens, increase the tolerance to stress and induce the synthesis and the accumulation of enzymes, the production of secondary metabolites, signaling molecules i.e phytohormones and the release of substances with auxin-like activity. Indeed, the secondary metabolites produced by plants act as signals in plant-microbes interactions, affecting the composition of the plant microbiome. This relationship is dynamic and involves several bi-directional exchanges: some microorganisms adjust the production of secondary metabolites, and these bioactive molecules shape the soil microbiome. These plants-microbes interactions continue during all the life cycle stages and together with interaction with environment and other biotic inhabitants shape the composition of plant microbiota. For these purposes, a combination of plant metabolomics and root and rhizosphere bacterial metabarcoding were used to unravel effects at the plant holobiont level. Combining different approaches offers a comprehensive view of interactions influencing the steps from genotype to phenotype, providing global profiling for more diversified communities

Untargeted Metabolomics to Evaluate the Stability of Extra-Virgin Olive Oil with Added <i>Lycium barbarum</i> Carotenoids during Storage

A carotenoid-rich extract from Lycium barbarum L. was added to extra virgin olive oil (EVOO), obtaining a carotenoid-enriched oil (EVOOCar). The oxidative stability of EVOO and EVOOCar was evaluated during long-term storage of 28 weeks at room temperature, by measuring some classical parameters (acidity and peroxide values, spectrophotometric coefficients, fatty acid composition) and the content of minor compounds (i.e., &#945;-tocopherol and lutein). At the end of the storage, higher content (p &lt; 0.01) of &#945;-tocopherol in EVOOCar in respect to EVOO were observed. Zeaxanthin dipalmitate, the most abundant carotenoid compound of Goji berries, decreased slightly (p &lt; 0.05) in EVOOCar until the end of the storage. In regard to polyphenols, an ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) using untargeted metabolomics was carried out. This latter approach discriminated the two oil samples during long-term storage, allowing to identify also the phenolic classes most exposed to significant variations during storage (i.e., mainly lignans and flavones). Besides, the addition of Goji carotenoids preserved the stability of tyrosol equivalents in EVOOCar during long-term storage. These results highlighted that the enrichment of EVOO with a carotenoid-rich extract can improve the shelf-life and nutritional value of added-oil, protecting EVOO natural antioxidants during long-term storage

A Phenomics and Metabolomics Investigation on the Modulation of Drought Stress by a Biostimulant Plant Extract in Tomato (<i>Solanum lycopersicum</i>)

Biostimulants are gaining increasing interest because of their ability to provide a green and effective strategy towards sustainable crop production. Nonetheless, their mode of action remains often unknown. The object of this work was to unravel the mechanisms through which 4-Vita, a biostimulant plant extract, can mitigate drought stress in tomato. To this aim, tomato plants were treated with two foliar applications of 4-Vita and drought stress imposed to both treated and control plants. Phenomics investigations were coupled to mass spectrometric untargeted metabolomics, and raw data were elaborated by multivariate statistics and pathway analysis. The biostimulant elicited a broad reprogramming of the tomato’s secondary metabolism, including its phytohormones profile, corroborating an improved ability to cope with drought stress. A series of mechanisms could be identified in response to the biostimulant treatment under drought, pointing to the preservation of photosynthetic machinery functionality. The modulation of thylakoid membrane lipids, the increase in xanthins involved in ROS detoxification, and the modulation of chlorophylls synthesis could also be observed. Overall, a series of coordinated biochemical mechanisms were elicited by the biostimulant treatment, supporting the increased resilience to drought stress in tomato

Extraction Kinetics of Total Polyphenols, Flavonoids, and Condensed Tannins of Lentil Seed Coat: Comparison of Solvent and Extraction Methods

The lentil seed coat is a waste by-product still rich in phenolic compounds, specifically condensed tannins. The effect of different solvents, as well as different processes, namely conventional solid–liquid extraction (CSLE) and ultrasound-assisted extraction (UAE), on the extraction yield of specific phenolic compound classes was studied. Four empirical two-parameter models were examined to select the one that better fit the experimental data obtained under different operating conditions. Additionally, ultra-high-pressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-ESI/QTOF-MS) was employed to profile the phenolic compounds obtained under distinct extraction conditions. In the operative conditions adopted here, the bioactive compounds yield achieved using UAE was lower than that obtained with CSLE. The kinetics of polyphenols, flavonoids, and condensed tannins extraction from the lentil seed coat were successfully fitted to the power-law models, yielding mean values of the root mean square &lt; 5.4%, standard error of estimation &lt; 0.53, and coefficient of determination &gt; 0.8. In addition, the UHPLC-ESI/QTOF-MS of the lentil seed coat extracts allowed the putative recognition of nearly 500 compounds, mainly flavonoids and phenolic acids. View Full-Tex

Lentil seed coat as a source of phenolic compounds: influence of geographical origin and genotype

Lentils (Lens culinaris spp.) are promising legumes whose seed coats, the by-products produced from dehulling, can represent a valuable source of functional components. This work aimed at valorizing lentils seed coat as functional ingredients, by investigating its profile across 11 genotypes grown in two different growing locations in Italy. The total phenolic, flavonoids, and condensed tannins contents (mg/g dry weight,) ranged from 30.60 to 70.37 gallic acid equivalents, 0.86 to 2.21 catechin equivalents, and 22.34 to 77.49 catechin equivalents, respectively. The untargeted phenolic profiling through UHPLC/QTOFMS, followed by multivariate statistics, revealed differences in the quality and quantity of polyphenols. A broad and diverse profile could be highlighted, including 420 compounds, mainly ascribable to flavonoids, phenolic acids, and low-molecularweight phenolic compounds. Unsupervised cluster analysis highlighted that origin is hierarchically more important than genotype in determining the phenolic profiles. Pearson’s correlation analysis showed that color was only partially related to phenolic content, while the geographic location and the genotype were more relevant. The significant accumulation of phenolics in some genotypes suggests that the genetic background should be taken into account, when lentils seed coat is to be included in food design as a sustainable source of phenolic compounds

Impact of Cold versus Hot Brewing on the Phenolic Profile and Antioxidant Capacity of Rooibos (Aspalathus linearis) Herbal Tea

Consumption of rooibos (Aspalathus linearis) as herbal tea is growing in popularity worldwide and its health-promoting attributes are mainly ascribed to its phenolic composition, which may be affected by the brewing conditions used. An aspect so far overlooked is the impact of cold brewing vs regular brewing and microwave boiling on the poly(phenolic) profile and in vitro antioxidant capacity of infusions prepared from red ('fermented', oxidized) and green ('unfermented', unoxidized) rooibos, the purpose of the present study. By using an untargeted metabolomics-based approach (UHPLC-QTOF mass spectrometry), 187 phenolic compounds were putatively annotated in both rooibos types, with flavonoids, tyrosols, and phenolic acids the most represented type of phenolic classes. Multivariate statistics (OPLS-DA) highlighted the phenolic classes most affected by the brewing conditions. Similar antioxidant capacities (ORAC and ABTS assays) were observed between cold- and regular-brewed green rooibos and boiled-brewed red rooibos. However, boiling green and red rooibos delivered infusions with the highest antioxidant capacities and total polyphenol content. The polyphenol content strongly correlated with the in vitro antioxidant capacities, especially for flavonoids and phenolic acids. These results contribute to a better understanding of the impact of the preparation method on the potential health benefits of rooibos tea

Plant biostimulants from seaweeds or vegetal proteins enhance the salinity tolerance in greenhouse lettuce by modulating plant metabolism in a distinctive manner

The use of plant biostimulants such as seaweed extracts (SWE) and protein hydrolysates (PH) has grown in the recent years due to their beneficial effects on yield under both optimal and sub-optimal conditions such as salt stress. The comprehension of the mode of action of these two important categories of biostimulants on plant performance will allow to use them more efficiently under different growth conditions. This study aimed to examine the efficacy of a seaweed and plant-based biostimulants on greenhouse lettuce (Lactuca sativa L.) grown under non-saline (0 mM NaCl) and saline conditions (40 mM NaCl) in terms of growth, yield, SPAD index, leaf mineral composition and metabolomic profiling. Shoot fresh weight of lettuce was reduced by 15.3% under 40 mM of NaCl. Nonetheless, it was boosted by both used biostimulants by 9 and 18%, on average, under 0 and 40 mM NaCl salinity, respectively. Na content under saline conditions was reduced in the presence of the biostimulants treatment, where PH reduced it significantly by 15.6% and SWE by 9.4%. On the other hand, Cl content was significantly reduced only under PH treatment. Both biostimulants elicited a broad metabolic reprogramming, involving the accumulation of stress-related compounds such as glucosinolates, terpenoid phytoalexins, and jasmonates. Interestingly, distinctive metabolomic signatures could be observed following the application of the different biostimulants under salinity conditions. In more detail, PH promoted the accumulation of glucosinolates and phytoalexins precursors, while SWE induced a down accumulation of secondary metabolites. Our findings indicate different processes being modulated by PH and SWE, with possible synergistic effects, thus paving the way towards integrated strategies to alleviate the detrimental effects of salinity in lettuce

Biochemical Insights into the Ability of <i>Lemna minor</i> L. Extract to Counteract Copper Toxicity in Maize

Metal trace elements (MTE) can damage crops if present in excessive amounts in the environment. This research investigated the effect of a plant extract of an aquatic species, Lemna minor L. (duckweed) (LE), on the ability of maize to cope with copper (Cu) toxicity. LE reversed the effects of Cu2+ on photosynthetic activity (Pn), evapotranspiration (E), stomatal conductance (gs), sub-stomatal CO2 concentration (Ci) and biomass which did not differ from the untreated controls. LE did not regulate the amount of copper in maize leaves, but compared to Cu-treated samples, the extract decreased the hydrogen peroxide (H2O2; −26% on average) and malondialdehyde (MDA; −47% on average) content, regardless of the dosage applied. Furthermore, the activity of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) was significantly increased by LE compared to samples treated with Cu alone. Untargeted metabolomic profiling revealed that LE activated maize secondary metabolism, eliciting the content of non-enzymatic antioxidants (flavonoids, glutathione and glutathione-related compounds, tocopherols and tocotrienols) and modulating plant stress-related hormones (brassinosteroids and ABA derivatives). The results of this study are promising and pave the way for using duckweed as a biostimulant to trigger beneficial effects in maize and increase its resistance to MTEs