Biodynamic agriculture differs from organic management in the use of specific preparations, applied on crops or soil in very small amounts. These preparations are claimed to stimulate soil nutrient cycle, photosynthesis in plants and optimal evolution of compost, enhancing both soil and crop quality . Research on biodynamic viticulture revealed similarity with the organic system concerning soil characteristics, plant growth and yield, resource utilization and biodiversity . The use of preparations had minor influences on growth and yield and did not affect the final quality of the grape berries .
The application of horn-silica, preparation 501, to the aerial part of the plants is one of the guidelines given by Steiner to accomplish the biodynamic farming. This work was aimed to discover evidences on the effects of 501 distributions in Vitis vinifera cv Garganega plants.
Samples of leaves and berries were collected from plants grown in two separate vineyards closely located, but differentiated by type of soil, both in structure and in plant nutrient contents. The leaves samples were collected at two times, six hours after the 501-treatment (sampling 1, May 10) and 11 days later (sampling 2, May 21) to elucidate whether 501-treatment could induce immediate change in leaves contents or a long-lasting effect could be detectable 11 days after the application. Also, analysis was performed in berries sampled at harvest time.
To assess the variations induced by the application of 501, we first examined the leaf chlorophyll content, commonly regarded as a marker of the nutritional status of the plants. The lack of difference on the level of pigments observed between the leaves of control and 501-treated plants confirms the findings of a previous study on biodynamic treated grapevine .
Metabolomics is successfully applied in plant science [21, 22], and the technical advances and the possibility to acquire large number of raw data, due to high resolution mass spectrometry coupled with liquid chromatography, allow to achieve inclusive phytochemical profile in plants, opening new research opportunities [23–25]. Multivariate statistical analysis approach is a tool to investigate metabolic alteration in complex samples as plant tissues. Principal Component Analysis (PCA) is one of the most used techniques in multivariate analysis, aimed at reducing a dataset to its main components and visualizing similarities. PCA has been used in several studies to distinguish varietal and/or geographical origin of grape juice and wines . Other type of data elaboration, as PLS-DA and its implemented version OPLS-DA, may give the advantage of an easier interpretation of the models. Most “omics” experimental setups aim at the comparison of samples between a control and a case group (e.g. disease or treatment). The goal of such differential analysis is therefore to build up a model able to distinguish the classes of observations and to provide a meaningful interpretation of the observed differences .
We studied the metabolite profiles in leaves and berries by UPLC-QTOF-MS analysis using an untargeted metabolomics approach. Unlike the targeted methods, where most of the metabolites in the matrix are ignored, in untargeted metabolomics the aim is to achieve the widest possible metabolic coverage in an unsupervised manner, including unknown compounds. Consequently, the measured metabolites are not pre-defined and method development and validation follow a workflow different from the targeted analysis .
The PCA for leaf metabolites of both the vineyards showed clusterization only due to sampling time, suggesting limited variation in the leaves composition caused by the 501-treatment compared to time-induced changes. However, further elaboration by supervised methods allowed to detect changes due to treatment, avoiding any bias related to sampling time and location. The separate elaboration of the Paiele and Roncaie plants gives indication on possible common changes due to treatment in the leaves of the plants at both locations. These results are, up to our knowledge, the first demonstration of metabolomic changes related to biodynamic 501 applications on grape leaves.
The metabolites changes in leaves of Paiele and Roncaie plants indicated that secondary metabolism was influenced by 501 biodymanic treatment. The pathway of shikimate leading to the formation of phenylpropanoids, stilbenoids and flavonoids appears to be triggered in leaves of 501-treated Paiele plants, while the boost of caftaric and caffeic acid in Roncaie plants indicates that mostly the phenylpropanoid biosynthesis is influenced in the treated plants. The great increase in caffeic and caftaric acids could be counterbalanced by the drop of the level of resveratrol and epicatechin in Roncaie leaves. It is known that higher plant tolerance to abiotic stresses is related to increased synthesis of polyphenols, such as phenolic acids and flavonoids . Our results can suggest that the application of preparation 501 may trigger the biosynthesis of antioxidants beneficial to enhance stress tolerance in grapevine plants, although the different variations in the leaf metabolite levels recorded in the plants of the two vineyards may be attributable to a site effect.
Recently, the total amino-acid content was found to increase in healthy grape berries during maturation under biodynamic management compared to integrated treatment . In our study, the total amount of free amino-acids in berries did not vary, although 501 induced significant variations in some amino-acids. However, the lack of consistency in changes between the berries of the two vineyards leads to exclude a common effect of 501 on amino-acids biosynthesis. It is known that the amount of individual amino-acids in berries could vary with variety, location, age, cultural practices, and method of analysis .
The berries of the 501-treated plants in both the vineyards showed superimposable pattern of metabolic changes, mostly being identified as phenolics and carotenoids. Observed metabolic changes in berries varied from those found in leaf tissues, and this can be related to the metabolic specificity of the two plant organs. Phenolic compounds are important in grapes due to their protective function against environmental stress and fungal infection . In 501-treated plants of both sites the level of berry phenolic constituents raised, but only violaxanthin increased significantly. Carotenoids are well known photo-protectors in plant tissues and may interfere during the ripening process in grape . Berries metabolically respond to the light by augmenting the level of compounds like polyphenols that have direct antioxidant and “sun-screening” abilities . The rise of epigallocatechin, although only in Roncaie berries, and violaxantin can be seen as a beneficial effect of the biodynamic treatment in terms of enhanced response to oxidative stress. The grape berry polyphenols are extracted during wine making and could influence colour as well as the sensory perception of wine .
Previous paper investigated the phenolics composition in grape berry of cv Pignoletto and cv Sangiovese, comparing conventional, organic and biodynamic management . Catechin and epigallocatechin had comparable content and rutin decreased in biodynamic treatment compared to conventional and organic management in Pignoletto berries. Even Parpinello et al. (2019) evidenced no significant differences between organic and biodynamic Sangiovese wines, although the latter presented higher concentrations of malvidin-3-glucoside and catechin .
Our results revealed that catechin and epigallocatechin increased in berries of Paiele and Roncaie plants (Table 5), suggesting a homogeneous trend under biodynamic treatment, independent from the location. The 501 treatment promoted a general increase of phenolic compounds, except for kaempferol-glucoside, in berries of both the vineyards. These results are in agreement with Reeve et al. (2005) who reported increased polyphenols in wine obtained from grapes under biodynamic management . The increment of polyphenols in berries of biodynamically grown plants can confirm the hypothesis of an upregulation of metabolites assumed to induce stress resistance [18, 19]. Higher levels of flavonoids and anthocyanins, with the consequent higher antioxidant potential, were associated to a lower plant vigour, a feature often reported under biodynamic management .
Overall, our data indicate a stimulation of the biosynthetic pathways of phenolics in leaves and berries due to the application of 501.