Introduction
Three major current global challenges are food security, environmental degradation, and climate change. The first may be augmented and the latter two diminished by improving nutrient (nitrogen, phosphorus) use efficiency in agricultural crop production and stabilising yield by practicing sustainable agriculture (Searchinger, Waite, Hanson, Ranganathan, & Dumas, 2018). The application of plant biostimulants such as beneficial microbes (arbuscular mycorrhizal fungi [AMF],Trichoderma spp., plant growth-promoting rhizobacteria [PGPR]), and bioactive substances (humic and fulvic acids, macroalgae and microalgae, protein hydrolysates, and silicon) used either separately or in combination may help crops contend with the aforementioned challenges (Rouphael & Colla, 2020).
Plant biostimulants were recently defined in the Regulations of the European Parliament and Council (Regulation EU 2019/1009) as “…EU fertilising product(s) able to stimulate plant nutrition processes independently of the product’s nutrient content with the sole aim of improving one or more of the following characteristics of the plant or the plant rhizosphere: 1) nutrient use efficiency, 2) tolerance to abiotic stress, 3) quality traits, or 4) availability of confined nutrients in the soil or rhizosphere ”. AMF comprise a very important category of biostimulants (Rouphael et al., 2015). They are members of the Glomeromycotina family and establish mutualistic relationships with 74% of all terrestrial plant species (Spatafora et al., 2016). AMF boost productivity and enhance tolerance to abiotic stress (high temperature, drought, and salinity) in crops. AMF inoculation enhances the growth and vigour of the root apparatus in terms of biomass, length, density, and branching. It improves macronutrient (N, P, and Fe) and micronutrient (Mn and Zn) uptake and assimilation. It ameliorates water relations and photosynthetic activity, upregulates secondary metabolism, and releases low- and high-molecular-weight organic compounds such as amino acids, phenolics, organic acids, and proteins into the rhizosphere. It also modulates phytohormone signalling (Bernardo et al., 2018; Lucini et al., 2019; Rouphael and Colla, 2018; Rouphael et al., 2015; Yakhin, Lubyanov, Yakhin, & Brown, 2017). The indirect and direct mechanisms of AMF influence shoot and root function and augment crop agronomic performance. Other plant beneficial endophytic fungi includeTrichoderma spp. Several of them are registered as microbial biological control agents (López-Bucio, Pelagio-Flores, & Herrera-Estrella, 2015). However, several studies reported that certainTrichoderma spp. including T. atroviride , T. koningii , T. harzianum, and T. virens are also plant biostimulants that boost crop performance and nutrient use efficiency and/or endue plants with abiotic stress tolerance (Colla, Rouphael, Di Mattia, El‐Nakhel, & Cardarelli, 2015; Saia et al., 2019). The direct and indirect mechanisms of the biostimulant action of Trichodermastrains include i) improvement of lateral root development, ii) induction of plant mitogen-activated protein 6, and iii) production and rhizosphere excretion of auxins and secondary metabolites such as volatile and non-volatile substances that stimulate various plant responses and enhance crop nutrient uptake, resilience, and productivity (Lopez-Bucio et al., 2015).
The beneficial effects of combinations of AMF and Trichoderma on vegetable crops were previously demonstrated under both optimal and suboptimal conditions (Colla et al., 2015; Saia et al., 2019). However, the physiological and molecular mechanisms underlining biostimulant action have not been fully elucidated. One strategy to clarify biostimulant efficacy is to analyse metabolic profiling. In turn, this process serves as a basis for subsequent transcriptomic analyses. Metabolomic phytochemical characterisation could identify numerous physiological processes and metabolic pathways modulated by biostimulants (Yakhin et al., 2017).
It has been hypothesised that AMF and Trichoderma can induce an enhance fruit yield by modulating the hormonal balance and secondary metabolic pathways.
In the present study, then, an untargeted metabolomics approach was conducted on greenhouse pepper. The objectives were to illuminate metabolomic reprogramming by microbial biostimulants in leaf tissue at the vegetative and reproductive phenological stages, elucidate biostimulant regulation of key phytohormones, and correlate these molecular-level biostimulant-promoted changes to observed fruit yield and quality variations.