Discussion
There is a growing interest in the use of beneficial microbial inoculants such as AMF, Trichoderma spp., and PGPR in horticulture as they have multiple beneficial effects on crops (López-Bucio et al., 2015; Rouphael et al., 2015). In the present study, we inoculated pepper plants with the AMF species Rhizoglomus irregularis and Funneliformis mosseae. Trichoderma koningiienhanced mycorrhizal root colonisation whilst Trichodermarhizosphere population accelerated and increased total crop yield by 24% relative to uninoculated plants (Table 1). The increase in pepper yield was attributed to gains in fruit weight and/or number. Colla et al. (2014) reported that compared with uninoculated field-grown zucchini plants, those supplied with live AMF G. intraradices and T. atroviride inocula presented with greater early and total yields. The beneficial fungi act as phytostimulants and improve foliar nutrient content. The phytostimulatory efficacy of beneficial fungi is explained by complex signal exchange and crosstalk between the host plants and the microorganisms affecting phytohormone balance and plant metabolism (Sbrana, Turrini, & Giovannetti, 2017). Metabolomics helps elucidate the metabolic pathways and processes involved in plant-microbe interactions. Growth stage has a hierarchically strong effect on the leaf metabolome. Nevertheless, microbial biostimulants significantly alter the metabolome such that it is readily distinguishable from the control (Figure 1).
The microbial treatments elicited several processes related to plant secondary metabolism. Microbial-based biostimulants promote the accumulation of carotenoids and other terpenes, saponins, phenolic compounds, and phospholipids (Figures 3 and 4).
Plant responses to microbial-based biostimulants involve modulations of the phytohormone network. Treatments with beneficial fungi alter auxins, cytokinins, and gibberellins. Modification of the hormone profile may have been associated with the observed yield increases. Several studies demonstrated that microbial biostimulants promote yield by changing the phytohormone balance, increasing nutrient availability and uptake, and enhancing abiotic stress tolerance (Rouphael et al., 2015; Saia et al., 2019, 2020). Certain putative mechanisms for the biostimulant activity of microbial-based inoculant (AMF + Trichoderma ) in pepper have been proposed. Microbial-based inoculant augments root biomass, length, density, and branching which, in turn, increases macronutrient and micronutrient uptake and boosts crop productivity. It also regulates key phytohormones such as gibberellins, cytokinins, and auxins (Lopez-Bucio et al., 2015; Lucini et al., 2019; Rouphael et al., 2015).
Gibberellins are diterpenoid phytohormones that regulate plant development, flowering, and senescence (Shu, Zhou, Chen, Luo, & Yang, 2018). In response to microbial-based inoculant treatment, gibberellins A81, A36, A37, A12, and A20 increased by 1.3–16× relative to the control at both sampling points. Coordination between gibberellin biosynthesis and oxidation affects pollination and fruit set in tomato (Serrani, Sanjuán, Ruiz-Rivero, Fos, & García-Martínez, 2007). Gibberellin A20 was recently linked to increased maize yield (Tucker et al., 2019).
Here, the microbial-based biostimulant treatment induced the auxins indole-3-acetamide and indole-3-pyruvic acid by 1.7-7.5× relative to the control. Auxins upregulate the genes encoding oxidases regulating gibberellin metabolism (Frigerio et al., 2006). Auxins and gibberellins overlap in terms of root growth and fruit set regulation (Bermejo et al., 2018). Microbial-based biostimulant also increased the accumulation of the cytokinin trans -zeatin by 2.2-5.1× in pepper leaves compared with the control. Cytokinins interact with auxins to fine-tune root and shoot development. Trans -zeatin modulates meristem activity and mediates plant responses to variable extrinsic factors such as abiotic stress (Werner & Schmülling, 2009). We postulate that coordinated auxin, gibberellin, and cytokinin recruitment may have contributed to the enhanced pepper fruit yield observed here. Earlier studies reported that beneficial microbes promote plant growth and productivity by altering phytohormone status (Bhattacharyya & Jha, 2012).
Modulation of plant signalling compounds in response to the microbial-based biostimulant treatment also involved membrane lipids. Phospholipids are plasma membrane components that play important roles in cell signalling, membrane trafficking, and apoptosis (Xue, Chen, & Mei, 2009). The microbial-based biostimulant treatment changed the phospholipids profile. It altered twenty foliar metabolites at the first sampling (vegetative stage) and 31 foliar metabolites in the second sampling (reproductive stage). Accumulation of phosphatidylethanolamines (PE(P-16:0/20:5)), phosphatidic acid (PA(15:0/22:6), PA(O-18:020:3)), phosphatidylinositol (PIM4(18:1/14:0)), and phosphatidylserine (PS(P-16:013:0)) increased by 1.5–30× in the biostimulant-treated plants compared to the control. Relative to the untreated leaves, lysophospholipids (PA(P-16:0e18:2)) increased by 6.5× in biostimulant-treated leaves from the second sampling (reproductive stage). Lysophospholipids release calcium from the endoplasmic reticulum, promote cell division, and inhibit apoptosis (Hou, Ufer, & Bartels, 2016; Ye, 2008).
The microbial treatment also modulated the biosynthesis of carotenoids, saponins, phenolic compounds, and purines. Secondary metabolism is often altered in response to plant interactions with the ambient environment including agronomic practices and plant-microbe interactions (Yang et al., 2018). Here, metabolomics identified substantial alterations in secondary metabolism. Hence, plant responses to microbial biostimulants entails the coordinated modulation of several unrelated pathways.
Compared to the control, by the second sampling date, foliar vitamin A and α-carotene were 1.5× and 8.5× higher, respectively, following microbial biostimulant treatment. Carotenoids absorb light energy, participate in photosynthesis, protect plants against oxidative damage, and are precursors of visual pigment chromophores and volatile apocarotenoids that attract pollinators (Heath, Cipollini, & Stireman, 2013; Sun, et al., 2018). Moreover, they are involved in plant responses to abiotic stresses and plant-microbe interactions (Felemban, Braguy, Zurbriggen, & Al-Babili, 2019). Blumenols comprise a class of apocarotenoids or cyclohexanone derivatives of carotenoid cleavage. They also accumulated in the biostimulant-treated plants. Blumenols accumulate in the roots and shoots of mycorrhized plants and have been proposed as arbuscular mycorrhizal fungi colonisation markers (Wang et al., 2018). Relative to the control, blumenol B was 2× and 2.5× higher at the first and second sampling dates, respectively, after biostimulant application. Their functions in processes other than allelopathy are unknown. However, their levels are strongly correlated with the degree of mycorrhization (Fester et al., 2002). Here, foliar saponins were 1.5–10× higher in plants treated with biostimulant than the untreated control. Saponins are produced constitutively in plants and comprise part of plant defence. They have both antifungal and antifeedant activity. Though they are generally associated with pathogenesis, it was recently reported that saponins may participate in mutualistic relationships among plants, rhizobacteria, and mycorrhizae (Szakiel, Pączkowski, & Henry, 2011).
Compared with the control, plants subjected to the microbial treatments presented with higher levels of phenolic compounds. Phenolic metabolites are essential for lignin and pigment biosynthesis and participate in plant responses to pathogens and external stimuli (Bhattacharya, Sood, & Citovsky, 2010). Mycorrhizae elicit phenolic biosynthesis in other plant species (Baslam & Goicoechea, 2012; Jugran et al., 2015). They also trigger plant defence against abiotic and biotic stresses and improve nutrient availability and use efficiency (Bernardo et al., 2019; Sharma & Sharma, 2017). Here, irrespective of growth stage, skullcapflavone I, pelargonidin-3-o-glucoside, kaempferol, genistein, apiin, and myricatomentoside I accumulated to levels 3–87× higher in the biostimulant-treated plants than the control. Phenolics are associated with plant defence mechanisms. Flavones may protect plants from both biotic and abiotic stress (Martinez et al., 2016). Lignans have high antioxidant activity (Durazzo, Turfani, Azzini, Maiani, & Carcea, 2013; Hu, Yuan, & Kitts, 2007). Compared with the uninoculated, the gibbilimbol B level was 1.5× and 4.2× higher at the first and second sampling dates, respectively, in the inoculated plants. Gibbilimbol B was reported to have fungicidal activity against Fusarium oxysporum f. sp. dianthi . Coumarin upregulation is related to iron nutrition (Curie & Mari, 2017), allelochemistry (Niro et al., 2016), and abiotic stress tolerance (Saleh & Madany, 2015) in plants. Plant coumarins may influence the shape of the root microbiome (Voges, Bai, Schulze-Lefert, & Sattely, 2019).
Relative to the control, the levels of several purines were altered in the plants treated with the microbial biostimulant here. Several studies have focused on the effects of increased levels of adenosine and purines. These compounds are recycled by the so-called “salvage pathway” (Ashihara, Stasolla, Fujimura, & Crozier, 2018). At the first and second samplings, we observed sharp increases in the guanosine (2.7× and 8.7×, respectively) and N6-threonylcarbamoyladenosine (3× and 7.8×, respectively) levels after microbial biostimulant inoculation. Nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) are reducing equivalent exchange cofactors that participate in several redox reactions. At both the first and second samplings, NAD and FAD had increased by 1.5–4.4× in the biostimulant-treated plants relative to the control.
Our metabolomics study revealed that microbial biostimulant treatment had two major effects on pepper. First, the biostimulant modulated the phytohormone profile and phospholipid signalling in the plants. Next, it altered various secondary metabolic processes involving saponins, blumenols, carotenoids, and phenolic compounds. Phytohormones and biochemical messengers are associated with various metabolic processes (Ashihara et al., 2018) and might account for the observed biostimulant-mediated increases in crop productivity. The secondary metabolites modulated by biostimulant treatment have numerous positive influences on plant productivity such as the enhancement of nutrient uptake and assimilation and biotic and abiotic stress tolerance. The elicitation of secondary metabolism by plant beneficial microbes merits further investigation in terms of abiotic stress tolerance and induced systemic response (ISR) induction. Carotenoids and phenolics improve quality and promote health in many fruits including pepper. Thus, the microbial biostimulant treatments applied here could have nutritional implications as well.