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Growth Performance and Metabolic Changes in Susceptible Mung Bean [Vigna radiata (L.) Wilczek] during Interaction with Rhizoctonia solani and Trichoderma virens
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First Online 12-11-2022|
Methods: This study investigated growth performance and metabolic changes in mung bean seedlings during interaction with R. solani and Trichoderma virens using Gas Chromatography-Mass Spectrometry (GC-MS).
Result: Mung bean infected by R. solani caused root rot and wilting. T. virens treatment reduced the disease severity in infected seedlings and promoted mung bean growth. Seventy-eight metabolites were identified in root extracts and dominated by sugars and fatty acids. The sugars, fatty acids and organic acids were significant metabolite groups that changed in response to pathogen infection and/or T. virens treatment. Five metabolic pathways particularly pyruvate metabolism, glyoxylate and dicarboxylate metabolism, sulfur metabolism, citrate cycle (TCA cycle) and phenylalanine, tyrosine and tryptophan biosynthesis altered significantly based on a metabolic pathway analysis. Acetic acid and aconitine had important roles in mung bean response to R. solani infection and/or T. virens treatment.
Plant-pathogen-Trichoderma interactions are complex and specific systems. Recently, a metabolomics approach has been intensively studied to clarify the mechanisms underlying interactions between beneficial microbes and plant pathogens. The metabolomics is able to describe plant metabolic changes as a response to pathogen infections and biological control agent applications (Allwood et al., 2010; Hu et al., 2017). Rojas et al., (2014) reported that primary and secondary metabolites involve in crop resistance.
An interaction of Trichoderma sp. with R. solani in Phaseolus vulgaris has been thoroughly investigated from genetic to protein levels (Mayo-Prieto et al., 2019). However, information of metabolic studies on legumes is still limited. Pathogen infection and/or Trichoderma sp. trigger the changes of metabolic expression in bean plants (Mayo-Prieto et al., 2019). Evidence of changes in mung bean metabolites caused by R. solani infection and bio-control agent interaction remains unexplored. Therefore, this current study aimed to observe the growth performance and metabolic changes in mung bean seedlings during R. solani infection and/or T. virens treatment.
MATERIALS AND METHODS
Metabolite analysis using gas chromatography-mass spectrometry (GC-MS)
Samples for metabolic analysis were prepared in accordance with Lisec et al., (2006) with modifications. Centrifugation at 11,000 g for 10 minutes was conducted to separate the extracts. The supernatant was evaporated using nitrogen flow. Two steps of derivatization using 40 μL methoxyamine hydrochloride in pyridine and 40 μL N,O-bis(trimethylsilyl) trifluoroacetamide in 5% trimethylchlorosilane were performed. The derivatized samples were analyzed using gas chromatography-mass spectrometry (Trace 1310-ISQ LT, Thermo Scientific).
Data obtained from GC-MS were evaluated using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/home. xhtml). To determine the function of metabolites, openly available metabolite libraries including plant metabolic network (PNM) database (https://plantcyc.org), PubChem (https://pubchem.ncbi.nlm.nih.gov/) and KEEG pathway database (https://www.genome.jp/kegg/pathway.html) were used.
RESULTS AND DISCUSSION
Plant-pathogen-Trichoderma 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) where Trichoderma reduced the occurrence of moth bean root rot and significantly increased grain yield.
T. virens 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
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).
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).
R. solani 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.
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.
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.
Conflict of interest
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