UPLC-QTOF-MS metabolomics analysis revealed the contributions of metabolites to the pathogenesis of <i>Rhizoctonia solani</i> strain AG-1-IA

<div><p>To explore the pathogenesis of <i>Rhizoctonia solani</i> and its phytotoxin phenylacetic acid (PAA) on maize leaves and sheaths, treated leaf and sheath tissues were analyzed and interpreted by ultra-performance liquid chromatography-mass spectrometry combined with chemometrics. The PAA treatment had similar effects to those of <i>R</i>. <i>solani</i> on maize leaves regarding the metabolism of traumatin, phytosphingosine, vitexin 2'' O-beta-D-glucoside, rutin and DIBOA-glucoside, which were up-regulated, while the synthesis of OPC-8:0 and 12-OPDA, precursors for the synthesis of jasmonic acid, a plant defense signaling molecule, was down-regulated under both treatments. However, there were also discrepancies in the influences exhibited by <i>R</i>. <i>solani</i> and PAA as the metabolic concentration of zeaxanthin diglucoside in the <i>R</i>. <i>solani</i> infected leaf group decreased. Conversely, in the PAA-treated leaf group, the synthesis of zeaxanthin diglucoside was enhanced. Moreover, although the synthesis of 12 metabolites were suppressed in both the <i>R</i>. <i>solani</i>- and PAA-treated leaf tissues, the inhibitory effect of <i>R</i>. <i>solani</i> was stronger than that of PAA. An increased expression of quercitrin and quercetin 3-O-glucoside was observed in maize sheaths treated by <i>R</i>. <i>solani</i>, while their concentrations were not changed significantly in the PAA-treated sheaths. Furthermore, a significant decrease in the concentration of L-Glutamate, which plays important roles in plant resistance to necrotrophic pathogens, only occurred in the <i>R</i>. <i>solani-</i>treated sheath tissues. The differentiated metabolite levels may be the partial reason of why maize sheaths were more susceptible to <i>R</i>. <i>solani</i> than leaves and may explain the underlying mechanisms of <i>R</i>. <i>solani</i> pathogenesis.</p></div

Score plots for the PLS-DA results.

<p>The score plots based on the first two components derived from the PLS-DA results for the leaf tissues analyzed in positive and negative ion modes were presented as (A) and (B), respectively. The score plots based on the first two components derived from the PLS-DA analysis results for the sheath tissues analyzed in positive and negative ion modes were presented as (C) and (D), respectively. The first two components in the PLS-DA models (A), (B), (C) and (D) explained 50.6%, 43.1%, 35.1% and 38.9% of the total variances, respectively. Samples bF 1–5, pF 1–5 and rF 1–5 were from the control, the phenylacetic acid and <i>R</i>. <i>solani</i> treated leaf tissues, respectively. Samples bS 1–5, pS 1–5 and rS 1–5 were from the control, the phenylacetic acid and <i>R</i>. <i>solani</i> treated sheath tissues, respectively.</p

Scatter plots of the metabolites significantly changed in sheath tissues.

<p>(A), the scatter plot of the metabolites that significantly changed in the <i>R</i>. <i>solani</i> infected group. (B), the scatter plot of the metabolites that significantly changed in the phenylacetic acid treated group. The fold change value for each metabolite was Log2 transformed, and the corresponding p-value was -Log10 transformed.</p

Scatter plots of the metabolites significantly changed in leaf tissues.

<p>(A), the scatter plot of metabolites that significantly changed in the <i>R</i>. <i>solani</i> infected group. (B), the scatter plot of the metabolites that significantly changed in the phenylacetic acid treated group. The fold change value for each metabolite was Log2 transformed, and the corresponding p-value was -Log10 transformed.</p

Score plots for the principal component analysis.

<p>(A) and (B), the score plots based on the first two components derived from the PCA analysis of the leaf tissues analyzed in positive and negative ion modes, respectively. (C) and (D), the score plots based on the first two components derived from the PCA analysis of the sheath tissues analyzed in positive and negative ion modes, respectively. In the PCA results (A), (B), (C) and (D), the first two components explained 49.7%, 48.0%, 41.4% and 44.1% of the total variances, respectively. Samples bF 1–5, pF 1–5 and rF 1–5 were collected from the control, the phenylacetic acid and <i>R</i>. <i>solani</i> treated leaf tissues, respectively. Samples bS 1–5, pS 1–5 and rS 1–5 were collected from the control, phenylacetic acid and <i>R</i>. <i>solani</i> treated sheath tissues, respectively.</p

Symptoms induced in the maize sheaths inoculated by <i>Rhizoctonia solani</i> and phenylacetic acid.

<p>(A), filter paper (0.9 cm in diameters) with sterilized water. (B), a PDA plug (0.9 cm in diameters) cut from the growing edge of <i>R</i>. <i>solani</i> AG-1IA colonies. (C), filter paper (0.9 cm in diameters) with phenylacetic acid solution (7.34 mM, 200 μL, filtered by a 0.22 μm filter).</p

Identification of the metabolites.

<p>Identification of the metabolites.</p

Metabolites contributing to <i>Rhizoctonia solani</i> AG-1-IA maturation and sclerotial differentiation revealed by UPLC-QTOF-MS metabolomics

<div><p><i>Rhizoctonia solani</i> is a causative agent of sheath blight, which results in huge economic losses every year. During its life cycle, the formation of sclerotia helps <i>Rhizoctonia solani</i> withstand a variety of unfavorable factors. Oxidative stress is a key factor that induces sclerotium formation. The differentiated and undifferentiated phenotypes of <i>R</i>. <i>solani</i> AG-1-IA were obtained by controlling aerial conditions. Metabolomics based on the mass spectrometry technique combined with multivariate and univariate analyses was used to investigate the metabolic variation in vegetative, differentiated and undifferentiated mycelia. Our results revealed that during maturation, the metabolic levels of N2-acetyl-L-ornithine, 3,1'-(OH)2-Gamma-carotene, (5Z,7E)-(1S,3R)-24,24-difluoro-24a-homo-9,10-seco-5,7,10(19)-cholestatrien-1,3,25-triol, stoloniferone O, PA(O-18:0/12:0), PA(P-16:0/14:0), PA(P-16:0/16:(19Z)) and PA(P-16:0/17:2(9Z,12Z)) were suppressed in both differentiated and undifferentiated mycelia. The concentrations of PE(20:1(11Z)/14:1(9Z)), PE(P-16:0/20:4(5Z,8Z,11Z,13E)(15OH[S])) and PS(12:0/18:1(9Z)) were increased in the differentiated group, while increased levels of N(gamma)-nitro-L-arginine, tenuazonic acid and 9S,10S,11R-trihydroxy-12Z,15Z-octadecadienoic acid were found in the undifferentiated group. Our results suggest that different levels of these metabolites may act as biomarkers for the developmental stages of <i>R</i>. <i>solani</i> AG-1-IA. Moreover, the mechanisms of sclerotium formation and mycelium differentiation were elucidated at the metabolic level.</p></div

Phenotypes of vegetative (G1), undifferentiated (G2) and differentiated (G3) <i>Rhizoctonia solani</i> AG-1-IA in potato dextrose agar plates.

<p>G1: Culturing 36 hours post-inoculation while sealing the plate with a layer of preservative film. G2: Culturing 60 hours post-inoculation while sealing the plate with a layer of preservative film continuously. G3: Culturing 60 hours post-inoculation without sealing the plate beginning from 48 hours to 60 hours (the preservative film was removed at 48 hours). Differentiated sclerotia were formed in G3 due to unlimited aeration. After 48 hours of growth, the mycelia reached the edge of the Petri dish, but at 60 hours, the mycelia remained undifferentiated in G2 due to isolated aeration.</p

Concentration ratio of the same metabolite compared among mycelium groups.

<p>Concentration ratio of the same metabolite compared among mycelium groups.</p