Growth performance of mung bean seedlings
interaction affected mung bean seedling growth and development. R. solani
infection caused rotten seeds and roots as well as damping-off. Disease incidence was the highest in R. solani
treatment in contrast to T. virens
treatment (Table 1). Low disease severity observed in R. solani
infection and the presence of T. virens
(RsTv) was due to direct antagonistic activity and plant growth promotion of T. virens.
Similar results were reported by Godara and Singh (2021)
reduced the occurrence of moth bean root rot and significantly increased grain yield.
treatment increased plant growth particularly root length and fresh biomass (Table 1). The application of T. virens
increased plant capacity to absorb nutrient through root elongation and biomass allocation at the early phase of mung bean growth. During plant-microorganism interactions, Trichoderma
activate chemical signaling and metabolite regulations that lead to plant systemic defense response as well as growth regulation (Rojas et al., 2014).
Composition of mung bean metabolites
Table 1: Disease incidence and growth parameters of mung bean seedlings treated with R. solani and T. virens.
Seventy-eight metabolites were identified in the mung bean root extracts. Based on their functional groups, those metabolites consisted of sugars (38%), fatty acids (15%), sugar alcohols and alcohols (10%), organic acids (10%), amino acids and their derivatives (4%) and other compounds including phenols, sterols, aldehydes and ketones (23%) (Fig 1).
Fig 1: Metabolite compositions in mung bean roots in different treatments. Rs= Plants infected by R. solani, RsTv= Plants infected by R. solani and treated with T. virens, Tv= Plants treated with T. virens and C= Control without R. solani and T. virens.
Sugar compositions in mung bean infected by R. solani
(Rs) were comparable to those in infected plants and T. virens
treatment (RsTv) and control plants (C). Sugar production is carbon source and energy in plant primary metabolism besides as signaling in hormonal coordination pathways (Rojas et al., 2014).
Sugar is also one of the key compounds in abiotic stress adaptation mechanisms (Yadav and Hemantaranjan, 2017)
. Fatty acids and organic acids in infected and T. virens
treatment (RsTv) were higher than those in control plants (C). Aldehydes, ketones, amino acids and other miscellaneous compounds were higher in mung bean treated with T. virens
(Tv). Fatty acids and lipids are essential for cells, as energy for metabolic process and signals for both intracellular and extracellular to trigger immunity (Walley et al., 2013, Lim et al., 2017).
Amino acids are crucial for growth, development, stress responses and the immune system of plants (Kadotani et al., 2016).
Metabolic profiles and changes of metabolites during plant-pathogen-Trichoderma interaction
A heatmap cluster analysis representing metabolic profiles of mung bean roots grouped the treatments into two main clusters (Fig 2). Pathogen infection and Trichoderma
treatment (RsTv) was grouped into different cluster with other treatments (Rs, Tv and C). The presence of the pathogen in combination with the antagonistic fungi (three-way interaction) induced higher relative plant metabolite expression than that in two-way interactions (plant-pathogen or plant-Trichoderma
Fig 2: Heatmap of metabolite contents in mung bean seedlings infected by R. solani and T. virens treatment. Rs= Plants infected by R. solani, RsTv= Plants infected by R. solani and treated with T.virens, Tv= Plants treated with T. virens and C= Control plants.
infection increased the accumulation of several sugars such as D-ribose, tagatose, allose, lyxose and xylose. In contrast, pathogen infection reduced several compounds such as myo-inositol, acetic acid, D-manitol, D-ribose and D-galactopyranoside. Those accumulated metabolites are pathogenesis-related (PR) metabolites which are produced after pathogen infection and antimicrobial activities (Chahed et al., 2021)
and some responsible for resistance against pests (Reddy et al., 2021).
Higher intensities of fatty acids such as ethanedioic and stearic acids were observed in all treated plants. Ethanedioic acid (syn. oxalic acid) is produced by both plants and fungi (Prasad and Shivay, 2017)
. Fatty acids have important roles in both biochemical and physical mechanisms of plant resistance through the production of guard cells (Nakata, 2015)
Changes of amino acids levels were also observed during the plant-pathogen interaction that affected mung bean growth and resistance to pathogens. Purine was detected in both control and treated plants; however, cystathionine and glycine were only presence in the treated plants. Purine and cystathionine are members of cytokinin which promote plant growth and as substrate for microbial growth and development (Schlapfer et al., 2017).
Experimental treatments and metabolic production relationship
A principal component analysis (PCA) and partial least-square discriminant analysis (PLS-DA) were conducted in determining the relationships between experimental treatments and accumulation of metabolites from un-supervised analysis of GC-MS data. Two principal components (PC1 and PC2) showing three-way interaction of mung bean, R. solani
and T. virens
(RsTv) were the most dominant variable affecting the variability of mung bean metabolic expressions (Fig 3a). Based on the PC value, twelve metabolites were responsible for the interaction of mung bean- R. solani
- T. virens,
namely acetic acid, docosadiynedioic acid, heptanone, pyridine carboxylic acid, benzopyran-4-one, 6-Amino-5-cyano-4-(5-cyano-2,4-dimethyl-1H-pyrrol-3-yl), adenosine, benzoic acid, D-allose, D-mannitol, D-ribose and lyxose.
Variable importance in projection (VIP) values from PLS-DA analysis showed that 13 metabolites had scores higher than 1.5 (Fig 3b). Metabolites of 3-heptanone, aconitine, N-acetylneuraminic acid, ethanedioic acid, lutein and H-Azepine-1-carboxylic acid were detected higher in T. virens
treatment. Meanwhile, D-mannitol, acetic acid and gentamicin were higher in infected mung bean seedlings.
Fig 3: (a) Biplot analysis of PCA and (b) VIP-values of significant metabolites in mung bean roots.
Metabolic pathway analysis was carried out using 23 significant metabolites from the PCA and PLS-DA values. Pathogen infection and/or Trichoderma
treatment (Rs, Tv and RsTv) affected 12 metabolite pathways on mung bean networks (Fig 4). Five metabolic pathways showed significant high impact indicating by darker color and bigger circle in the pathway analysis plot namely, glyoxylate and dicarboxylate metabolism, sulfur metabolism, citrate cycle, pyruvate metabolism and phenylalanine tyrosine and tryptophan biosynthesis.
Fig 4: Metabolic pathway analysis plots depict several metabolic alterations in mung bean. The different color of each circle is based on p-values; darker color and bigger size indicate significant higher changes of metabolites in the corresponding pathway.
Further analysis of each metabolite of the twelve significant pathways showed that several overlapping metabolites performed as a link metabolite that was capable of connecting various pathways. In this study, acetic acid and aconitine which involved in more than one significant pathway were affected by all treatments. Acetic acid involved in three metabolic pathways, namely glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis biosynthesis and sulfur metabolism. In addition, aconitine involved in glyoxylate and dicarboxylate metabolisms as well as TCA cycle. Glyoxylate and dicarboxylate metabolism and glycolysis/gluconeogenesis involve in carbohydrate metabolism (Zhang et al., 2019).
Carbohydrates increase resistance to biotic and abiotic stressors indirectly through supplying energy for growth (Saddhe et al., 2021).
In this study, T. virens
(Tv) triggered the elevation of aconitine compared to that in control, whereas pathogen infection (Rs) increased the level of acetic acid. Both pathogen and Trichoderma
affected the accumulation of important metabolites in carbohydrate biosynthesis. Our study also suggested that sulfur and pyruvate metabolisms performed the highest pathway impact, meanwhile, purine metabolism showed the least affected (Fig 4). Several studies reported that sulfur metabolism has a crucial role in determining crop growth and fitness towards microbial and viral infections as well as plant resistance to environmental stress (Aziz et al., 2016).
This finding showed that acetic acid and aconitine had important roles in mung bean metabolic networks.