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The use of microbial biostimulants in agriculture is recognized as a sustainable approach to promoting crop productivity and quality due to improved nutrient uptake, enhanced stress tolerance, and improved ability to cope with non-optimal environments. The present paper aimed to comparatively investigate the effect of seven different commercial mycorrhizal-based treatments in terms of yield, phytochemical components, and technological traits of Malvasia di Candia Aromatica grape (Vitis vinifera L.) plants. Metabolomic analysis and photosynthetic performance were first investigated in leaves to point out biochemical differences related to plant growth. Higher photosynthetic efficiency and better PSII functioning were found in biostimulant-treated vines, reflecting an overall decrease in photoinhibition compared to untreated plants. Untargeted metabolomics followed by multivariate statistics highlighted a robust reprogramming of primary (lipids) and secondary (alkaloids and terpenoids) metabolites in treated plants. The analysis of berry yield and chemical components exhibited significant differences depending on the biostimulant product. Generally, berries obtained from treated plants displayed improved contents of polyphenols and sugars, while yield remained unchanged. These results elucidated the significant role of microbial biostimulants in determining the quality of grape berries and eliciting biochemical changes in vines.

The application of microbial biostimulants to plants has revealed positive effects related to nutrients uptake, stress tolerance, root development and phenological growth. However, little information is available exploiting the potential synergistic biostimulant action of microbes on the functional quality of the yields. The current research elucidated the effect of single or coupled action of biostimulants, associated with either optimal or reduced nitrogen application, on the functional quality of tomato fruits. Chemical assays and untargeted metabolomics were applied to investigate Rhizoglomus irregulare and Funneliformis mosseae administration (both being arbuscular mycorrhiza, AMF), under optimal or low N input conditions, alone or coupled to Trichoderma atroviride application. The coupling of AMF and Trichoderma fungal inoculations resulted in a synergistic biostimulant effect on tomato fruits under sub-optimal fertility, revealing improved concentrations of carotenoid compounds-B-carotene (0.647 +/- 0.243 mg/100 g), Z-carotene (0.021 +/- 0.021 mg/100 g), 13-z-lycopene (0.145 +/- 0.052 mg/100 g) and all-trans-lycopene (12.586 +/- 1.511 mg/100 g), and increased values for total phenolic content (12.9 +/- 2.9 mgGAE/g), total antioxidant activity (phosphomolybdenum, 0.9 +/- 0.2 mmolTE/g), radical scavenging activity (DPPH, 3.4 +/- 3.7 mgTE/g), reducing power (FRAP, 23.6 +/- 6.3 mgTE/g and CUPRAC, 37.4 +/- 7.6 mg TE/g), and enzyme inhibitory activity (AChE, 2.4 +/- 0.1 mg GALAE/g), when compared to control. However, evidence of carotenoid and bioactive compounds were exclusively observed under the sub-optimal fertility and no significant differences could be observed between the biostimulant treatment and control under optimal fertility.

Plant phenomics is becoming a common tool employed to characterize the mode of action of biostimulants. A combination of this technique with other omics such as metabolomics can offer a deeper understanding of a biostimulant effect in planta. However, the most challenging part then is the data analysis and the interpretation of the omics datasets. In this work, we present an example of how different tools, based on multivariate statistical analysis, can help to simplify the omics data and extract the relevant information. We demonstrate this by studying the effect of protein hydrolysate (PH)-based biostimulants derived from different natural sources in lettuce and tomato plants grown in controlled conditions and under salinity. The biostimulants induced different phenotypic and metabolomic responses in both crops. In general, they improved growth and photosynthesis performance under control and salt stress conditions, with better performance in lettuce. To identify the most significant traits for each treatment, a random forest classifier was used. Using this approach, we found out that, in lettuce, biomass-related parameters were the most relevant traits to evaluate the biostimulant mode of action, with a better response mainly connected to plant hormone regulation. However, in tomatoes, the relevant traits were related to chlorophyll fluorescence parameters in combination with certain antistress metabolites that benefit the electron transport chain, such as 4-hydroxycoumarin and vitamin K1 (phylloquinone). Altogether, we show that to go further in the understanding of the use of biostimulants as plant growth promotors and/or stress alleviators, it is highly beneficial to integrate more advanced statistical tools to deal with the huge datasets obtained from the -omics to extract the relevant information.

Co-inoculation of arbuscular mycorrhizal fungi (AMF) and bacteria can synergically and potentially increase nitrogen use efficiency (NUE) in plants, thus, reducing nitrogen (N) fertilizers use and their environmental impact. However, limited research is available on AMF-bacteria interaction, and the definition of synergisms or antagonistic effects is unexplored. In this study, we adopted a response surface methodology (RSM) to assess the optimal combination of AMF (Rhizoglomus irregulare and Funneliformis mosseae) and Bacillus megaterium (a PGPR—plant growth promoting rhizobacteria) formulations to maximize agronomical and chemical parameters linked to N utilization in maize (Zea mays L.). The fitted mathematical models, and also 3D response surface and contour plots, allowed us to determine the optimal AMF and bacterial doses, which are approximately accorded to 2.1 kg ha–1 of both formulations. These levels provided the maximum values of SPAD, aspartate, and glutamate. On the contrary, agronomic parameters were not affected, except for the nitrogen harvest index (NHI), which was slightly affected (p-value of < 0.10) and indicated a higher N accumulation in grain following inoculation with 4.1 and 0.1 kg ha–1 of AMF and B. megaterium, respectively. Nonetheless, the identification of the saddle points for asparagine and the tendency to differently allocate N when AMF or PGPR were used alone, pointed out the complexity of microorganism interaction and suggests the need for further investigations aimed at unraveling the mechanisms underlying this symbiosis.

Seed inoculation with beneficial microorganisms has gained importance as it has been proven to show biostimulant activity in plants, especially in terms of abiotic/biotic stress tolerance and plant growth promotion, representing a sustainable way to ensure yield stability under low input sustainable agriculture. Nevertheless, limited knowledge is available concerning the molecular and physiological processes underlying the root-inoculant symbiosis or plant response at the root system level. Our work aimed to integrate the interrelationship between agronomic traits, rhizosphere microbial population and metabolic processes in roots, following seed treatment with either arbuscular mycorrhizal fungi (AMF) or Plant Growth-Promoting Rhizobacteria (PGPR). To this aim, maize was grown under open field conditions with either optimal or reduced nitrogen availability. Both seed treatments increased nitrogen uptake efficiency under reduced nitrogen supply revealed some microbial community changes among treatments at root microbiome level and limited yield increases, while significant changes could be observed at metabolome level. Amino acid, lipid, flavone, lignan, and phenylpropanoid concentrations were mostly modulated. Integrative analysis of multi-omics datasets (Multiple Co-Inertia Analysis) highlighted a strong correlation between the metagenomics and the untargeted metabolomics datasets, suggesting a coordinate modulation of root physiological traits.