Suoli sottoposti a tecniche di agricoltura conservativa: studio dei parametri chimico-biologici.

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Biostimulant Effects of Seed-Applied Sedaxane Fungicide: Morphological and Physiological Changes in Maize Seedlings

Most crops are routinely protected against seed-born and soil-borne fungal pathogens through seed-applied fungicides. The recently released succinate dehydrogenase inhibitor (SDHI), sedaxane\uae, is a broad-spectrum fungicide, used particularly to control Rhizoctonia spp., but also has documented growth-enhancement effects on wheat. This study investigates the potential biostimulant effects of sedaxane and related physiological changes in disease-free maize seedlings (3-leaf stage) at increasing application doses (25, 75 and 150 \u3bcg a.i. seed-1) under controlled sterilized conditions. We show sedaxane to have significant auxin-like and gibberellin-like effects, which effect marked morphological and physiological changes according to an approximate saturation dose-response model. Maximum benefits were attained at the intermediate dose, which significantly increased root length (+60%), area (+45%) and forks (+51%), and reduced root diameter as compared to untreated controls. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation, particularly in the above-ground compartment, while glutamate synthase (GOGAT) activity remained almost unchanged. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids), mainly represented by p-coumaric and caffeic acids. We conclude that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages

Biostimulant Effects of Seed-Applied Sedaxane Fungicide: Morphological and Physiological Changes in Maize Seedlings

Most crops are routinely protected against seed-born and soil-borne fungal pathogens through seed-applied fungicides. The recently released succinate dehydrogenase inhibitor (SDHI), sedaxane®, is a broad-spectrum fungicide, used particularly to control Rhizoctonia spp., but also has documented growth-enhancement effects on wheat. This study investigates the potential biostimulant effects of sedaxane and related physiological changes in disease-free maize seedlings (3-leaf stage) at increasing application doses (25, 75 and 150 μg a.i. seed-1) under controlled sterilized conditions. We show sedaxane to have significant auxin-like and gibberellin-like effects, which effect marked morphological and physiological changes according to an approximate saturation dose-response model. Maximum benefits were attained at the intermediate dose, which significantly increased root length (+60%), area (+45%) and forks (+51%), and reduced root diameter as compared to untreated controls. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation, particularly in the above-ground compartment, while glutamate synthase (GOGAT) activity remained almost unchanged. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids), mainly represented by p-coumaric and caffeic acids. We conclude that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages

Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity. Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins. Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division. Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses

Biostimulant effects of different products on the metabolism of agro-food plants

Sustainable and environmentally friendly agriculture represents the main strategy to meet rising food needs and to contribute to a stable planet. To achieve these goals, one of the most promising solutions is the adoption of plant biostimulants. Biostimulants are natural compounds which, at low concentrations, are able to enhance plant nutrient uptake, nutrient efficiency, tolerance to abiotic/biotic stresses, and crop yield. Humic substances (HS) fall within the biostimulants definition, as they have positive effects on the uptake of macro and micronutrients that considerably improve the metabolism, the growth and yields of relevant agricultural crops. With this background, the aims of this Ph.D. project were to i) evaluate metabolic and biochemical status of plants treated with HS to elucidate the action mechanisms of plant biostimulants. This might permit the development of a second generation of biostimulants where synergies and complementary mechanism can be functionally designed. ii) investigate potential biostimulant effects of a fungicide compound. In manuscript I Arabidopsis thaliana plants were treated with HS for eight hours. Root proteins were studied by MS spectrometry coupled with iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. 902 protein spots were identified for HS treated roots, where 291 proteins were differentially expressed. Bioinformatic tools such as DAVID, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of the network amongst the identified proteins. From this analysis, it was possible to evaluate that most of the differentially expressed proteins were functionally classified into response to inorganic substances, redox homeostasis, energy metabolism, protein synthesis, cell trafficking and division. With the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Moreover, from the analysis of interactomes and DAVID clusters it was possible to observe previously undiscovered HS effects, i.e. on the Ubiquitin and RACK1A interactome subnetworks. In manuscript II Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analysed by 1H NMR an FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrates contents, and leaf amino acids from protein hydrolysis were identified and quantify by gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC/MS) techniques. Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters. EF treatment had the highest influence on metabolites profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants resulted not significant different from the control. A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity. In manuscript III the biostimulant activity of HS extracted from four leonardites is analysed on maize seedlings. After 48 h of treatment with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L−1) of HS, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analyzed. HS from different leonardites had similar spectroscopic pattern, with small differences. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols. This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. In the manuscript IV it was investigated the potential biostimulant side-effects of sedaxane. Physiological changes in disease-free maize seedlings at increasing application doses (25, 75 and 150 μg a.i. seed-1) under controlled sterilised conditions were analysed. Sedaxane had significant auxin-like and gibberellin-like effects, with maximum benefits attained at the intermediate dose. Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids). It is concluded that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages. As a general conclusion, even if HS were extracted from different sources were applied on different plant species, they displayed similar biological activities. Proteomics and metabolomics studies confirmed that “–omics” techniques are essential tools to have a ‘panoramic’ view on metabolic changes happening inside an organism after a positive or negative external perturbation.L'agricoltura conservativa rappresenta la strategia principale per soddisfare le crescenti esigenze alimentari e contribuire a un pianeta stabile. Per raggiungere questi obiettivi, una delle soluzioni più promettenti è l'adozione dei biostimolanti. I biostimolanti sono composti naturali che, a basse concentrazioni, sono in grado di migliorare l'assorbimento dei nutrienti delle piante, l'efficienza dell’attività dei nutrienti, la tolleranza agli stress abiotici / biotici e la resa delle colture. Le sostanze umiche (HS) sono considerate una classe di biostimolanti, in quanto hanno effetti positivi sull'assorbimento di macro e micronutrienti, migliorano considerevolmente il metabolismo, la crescita e le rese delle colture agricole. Con questo background, gli obiettivi di questo progetto di dottorato sono stati di i) valutare lo stato metabolico e biochimico delle piante trattate con HS per chiarire i meccanismi di azione dei biostimolanti. Ciò potrebbe consentire lo sviluppo di una seconda generazione di biostimolanti in cui meccanismi di azione sinergici e complementari possono essere considerati. ii) studiare potenziali effetti biostimolanti di un composto fungicida. Nel primo articolo le piante di Arabidopsis thaliana sono state trattate con HS per otto ore. Le proteine delle radici sono state studiate mediante spettrometria MS accoppiata con la tecnica iTRAQ (Isobaric Tag for Relative and Absolute Quantification). Per le radici trattate con HS sono stati identificate 902 proteine, dove 291 proteine erano espresse in modo differenziale. Strumenti bioinformatici come DAVID, KEGG, IIS e Cytoscape sono stati utilizzati per interpretare le funzioni biologiche, l'analisi e la visualizzazione delle proteine connesse identificate. Da questa analisi, è stato possibile valutare che la maggior parte delle proteine differenzialmente espresse sono state funzionalmente classificate in risposta alle sostanze inorganiche, redox omeostasi, metabolismo energetico, sintesi proteica, traffico e divisione cellulare. Con il presente studio è stata fornita una panoramica sulle vie metaboliche più influenzate dall'attività biologica delle HS. Inoltre, dall'analisi degli interattomi e dei cluster di DAVID è stato possibile osservare nuovi effetti di HS, come per esempio sugli interattomi dell’Ubiquitina e del RACK1A. Nel secondo articolo, le piantine di Arabidopsis thaliana, coltivate in condizioni idroponiche, sono state trattate per 8 ore con acido indol-3-acetico (IAA), HS da International Humic Substances Society (IHSS) e HS da feci di lombrico (EF), rispettivamente. Le caratteristiche strutturali delle sostanze umiche sono state analizzate mediante spettroscopie 1H NMR e FT-IR. Nelle foglie e nelle radici, mediante gascromatografia-spettrometria di massa (GC / MS) e tecniche di cromatografia liquida-spettrometria di massa (LC / MS) sono stati identificati e quantificati gli amminoacidi, zuccheri e il contenuto di carboidrati liberi, e gli amminoacidi fogliari ottenuti dall'idrolisi proteica. L'analisi discriminante canonica (CDA) è stata utilizzata per valutare l'influenza dei trattamenti sui parametri studiati. Il trattamento con EF ha avuto la massima influenza sui profili dei metaboliti rispetto al controllo, IAA e IHSS. L'analisi del CDA ha evidenziato una chiara distinzione tra le risposte fisiologiche delle piante trattate con EF e IHSS, in base alle diverse proprietà chimiche e strutturali delle HS. Le piante trattate con IAA non sono invece risultate significativamente differenti dal controllo. Una migliore comprensione degli effetti specifici delle diverse HS, anche in relazione alle loro caratteristiche chimiche, potrebbe servire come base per l'identificazione di composti marcatori per la bioattività delle HS. Nel terzo articolo, l'attività biostimolante delle HS estratte da quattro leonarditi è stata analizzata su piantine di mais. Dopo 48 ore di trattamento con 5 concentrazioni (0, 0,1, 0,5, 1 e 10 mg CL-1) di HS, sono stati analizzati la crescita e la morfologia delle radici, l’attività della glutammina sintetasi (GS), l’attività del glutammato sintasi (GOGAT), il contenuto proteico totale, il contenuto di zuccheri solubili, l'attività della fenilalanina ammonia-liasi (PAL), i fenoli solubili e gli acidi fenolici liberi. Le HS estratte da diverse leonarditi avevano un pattern spettroscopico simile, ma con piccole differenze. HS_USA ha indotto una maggior crescita delle radici, della superficie radicale e della proliferazione delle radici secondarie. HS_4, HS_USA e HS_B hanno determinato una miglior efficienza dell'uso dei nutrienti delle piante, incrementando l'attività degli enzimi GS e GOGAT e la produzione totale di proteine. Le quattro HS hanno stimolato anche l'attività dell'enzima PAL, seguita da una maggiore produzione di fenoli solubili totali. Questo studio ha rilevato che, sebbene l'attività delle HS dipendesse dall'origine della leonardite, questi composti possono essere attribuiti ai prodotti biostimolanti, poiché stimolano la crescita delle piante, il metabolismo dell'azoto e l'accumulo di sostanze fenoliche. Nel quarto articolo è stato studiata l’azione biostimolante del sedaxane, intesa come attività secondaria. Sono stati analizzati i cambiamenti fisiologici in piantine di mais all'aumentare delle dosi di applicazione (25, 75 e 150 μg a.i. seme-1) in condizioni sterili controllate (piante non infette). Sedaxane ha dimostrato avere effetti di tipo auxino- e gibberellino-simili significativi, con i massimi benefici raggiunti alla dose intermedia. La lunghezza della radice (+ 60% rispetto ai controlli non trattati), l'area (+ 45%) e le biforcazioni (+ 51%) sono stati significativamente influenzati. Sedaxane ha incrementato l'attività della glutammina sintetasi (GS) delle foglie e delle radici con conseguente maggiore accumulo di proteine. Sedaxane ha anche incrementato l'attività della fenilalanina ammonia-liasi (PAL) nelle foglie, che potrebbe essere responsabile dell'aumento dell'attività antiossidante (acidi fenolici). Si è concluso che, oltre al suo effetto protettivo, il sedaxane può facilitare lo sviluppo delle radici e intensificare il metabolismo dell'azoto e dei fenilpropanoidi nelle giovani piante di mais e può essere utile per superare gli stress biotici e abiotici nelle prime fasi di crescita. Come conclusione generale, anche se le HS sono state estratte da diverse matrici e applicate su diverse specie di piante, queste hanno mostrato attività biologiche simili. Studi di proteomica e metabolomica hanno confermato che le tecniche "-omics" sono strumenti essenziali per avere una visione "panoramica" sui cambiamenti metabolici che avvengono all'interno di un organismo dopo una perturbazione esterna positiva o negativa

Biostimulant effects of different products on the metabolism of agro-food plants

Sustainable and environmentally friendly agriculture represents the main strategy to meet rising food needs and to contribute to a stable planet. To achieve these goals, one of the most promising solutions is the adoption of plant biostimulants. Biostimulants are natural compounds which, at low concentrations, are able to enhance plant nutrient uptake, nutrient efficiency, tolerance to abiotic/biotic stresses, and crop yield. Humic substances (HS) fall within the biostimulants definition, as they have positive effects on the uptake of macro and micronutrients that considerably improve the metabolism, the growth and yields of relevant agricultural crops. With this background, the aims of this Ph.D. project were to i) evaluate metabolic and biochemical status of plants treated with HS to elucidate the action mechanisms of plant biostimulants. This might permit the development of a second generation of biostimulants where synergies and complementary mechanism can be functionally designed. ii) investigate potential biostimulant effects of a fungicide compound. In manuscript I Arabidopsis thaliana plants were treated with HS for eight hours. Root proteins were studied by MS spectrometry coupled with iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. 902 protein spots were identified for HS treated roots, where 291 proteins were differentially expressed. Bioinformatic tools such as DAVID, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of the network amongst the identified proteins. From this analysis, it was possible to evaluate that most of the differentially expressed proteins were functionally classified into response to inorganic substances, redox homeostasis, energy metabolism, protein synthesis, cell trafficking and division. With the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Moreover, from the analysis of interactomes and DAVID clusters it was possible to observe previously undiscovered HS effects, i.e. on the Ubiquitin and RACK1A interactome subnetworks. In manuscript II Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analysed by 1H NMR an FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrates contents, and leaf amino acids from protein hydrolysis were identified and quantify by gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC/MS) techniques. Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters. EF treatment had the highest influence on metabolites profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants resulted not significant different from the control. A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity. In manuscript III the biostimulant activity of HS extracted from four leonardites is analysed on maize seedlings. After 48 h of treatment with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L−1) of HS, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analyzed. HS from different leonardites had similar spectroscopic pattern, with small differences. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols. This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. In the manuscript IV it was investigated the potential biostimulant side-effects of sedaxane. Physiological changes in disease-free maize seedlings at increasing application doses (25, 75 and 150 μg a.i. seed-1) under controlled sterilised conditions were analysed. Sedaxane had significant auxin-like and gibberellin-like effects, with maximum benefits attained at the intermediate dose. Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids). It is concluded that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages. As a general conclusion, even if HS were extracted from different sources were applied on different plant species, they displayed similar biological activities. Proteomics and metabolomics studies confirmed that “–omics” techniques are essential tools to have a ‘panoramic’ view on metabolic changes happening inside an organism after a positive or negative external perturbation

Effects of humic substances and indole-3-acetic acid on Arabidopsis sugar and amino acid metabolic profile

Background and aim. Humic substances (HS) are known to influence plant physiological processes, enhancing crop yield, plant growth and nutrient uptake. The present study sought to gain a better understanding of the specific effects of HS application on the abundance of metabolites in plant tissues, using mass spectrometry analyses. Methods. Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analyzed by 1HNMRan FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrate contents, and leaf amino acids from protein hydrolysis were identified and quantified by gas chromatographymass spectrometry (GC/MS), and liquid chromatographymass spectrometry (LC/MS). Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters. Results. EF treatment had the highest influence on metabolite profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants showed no significant difference from the control. Conclusions A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity.

Biostimulant activity of humic substances extracted from leonardites

Background and aims: Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions. Methods: HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L 121) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed. Results: HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid. Conclusion: This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. \ua9 2017 Springer International Publishing A