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.