Metabolite fingerprints of maize and sugarcane seedlings: searching for markers after inoculation with plant growth-promoting bacteria in humic acids

BACKGROUND: The negative consequences of industrial agriculture greatly affect human health and the environment. Debating our dietary requirements and challenging the means of food production are necessary. In the first years of transitioning to agroecological production, crop yields normally decrease. Humic acids and beneficial bacteria used as plant growth promoters can be helpful during this stressful time. Metabolite target identification will aid in increasing plant responses to these agents. MATERIALS: We evaluated the metabolite fingerprints of maize and sugarcane seedlings after 5 days of treatment with like-humic acids isolated from vermicompost coupled with a combined Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus application. The hydromethanolic foliar extracts were submitted for ¹H nuclear magnetic resonance analysis, and the data were explored using chemometrics procedures. After the preliminary screening, the extracts were analyzed by gas chromatography coupled to time of flight mass spectrometry to identify metabolite targets. RESULTS: The biostimulant significantly changed the metabolic fingerprints independent of the plant species. The main proton spectral regions changed by biostimulant use were from 0 to 2.5 ppm and 3.5 to 5 ppm, as revealed by a principal component analysis. The main signals corresponded to amino acid, sugar and organic acid chemical shifts. Aspartic acid was the amino acid present in greatest amounts in both leaf extracts. A significant change occurred in the region normally attributed to (CHₙ)-protons bound to electron-withdrawing groups, such as carboxyls from mucic, ribonic and saccharic acids derived from sugars and aromatic structures from shikimic acid, 4-hydroxybenzoate and 3,4-dihydroxycinnamic acid. The main organic acids altered by the biostimulant were representatives of the tricarboxylic acid cycle (citric, isocitric, aconitic, malic and fumaric acids). Linoleic and myristic acids, 1-mono palmitin and tocopherol were the major lipid components found at greater levels in the treated leaf extracts. Compounds from the oxidative end products of ascorbic acid metabolism, like threonic, isothreonic and oxalic acids, are putative biomarkers of the biostimulant as are the cyclic polyol identified as quinic acid and trehalose, a disaccharide involved in plant stress responses. CONCLUSION: The biostimulant induced significant changes in the metabolite fingerprints of maize and sugarcane seedlings as revealed by nuclear magnetic resonance. Both primary and secondary metabolisms were affected, and 22 putative biomarkers associated with the biostimulant-treated plant phenotype were identified. This agrees with previous work indicating that the stimulation of primary and secondary metabolisms was partially responsible for biostimulant effects on non-leguminous plants. Moreover, these metabolite targets could be used to genetically manipulate metabolic pathways to aid Poaceae breeding programs in increasing biostimulative responses.