Metabolomic Evaluation of Tissue-Specific Defense Responses in Tomato Plants Modulated by PGPR-Priming against Phytophthora capsici Infection.

Plant growth-promoting rhizobacteria (PGPR) can stimulate disease suppression through the induction of an enhanced state of defense readiness. Here, untargeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and targeted ultra-high performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ-MS) were used to investigate metabolic reprogramming in tomato plant tissues in response to priming by Pseudomonas fluorescens N04 and Paenibacillus alvei T22 against Phytophthora capsici. Roots were treated with the two PGPR strains prior to stem inoculation with Ph. capsici. Metabolites were methanol-extracted from roots, stems and leaves at two-eight days post-inoculation. Targeted analysis by UHPLC-QqQ-MS allowed quantification of aromatic amino acids and phytohormones. For untargeted analysis, UHPLC-MS data were chemometrically processed to determine signatory biomarkers related to priming against Ph. capsici. The aromatic amino acid content was differentially reprogrammed in Ps. fluorescens and Pa. alvei primed plants responding to Ph. capsici. Furthermore, abscisic acid and methyl salicylic acid were found to be major signaling molecules in the tripartite interaction. LC-MS metabolomics analysis showed time-dependent metabolic changes in the primed-unchallenged vs. primed-challenged tissues. The annotated metabolites included phenylpropanoids, benzoic acids, glycoalkaloids, flavonoids, amino acids, organic acids, as well as oxygenated fatty acids. Tissue-specific reprogramming across diverse metabolic networks in roots, stems and leaves was also observed, which demonstrated that PGPR priming resulted in modulation of the defense response to Ph. capsici infection.

Concurrent Metabolic Profiling and Quantification of Aromatic Amino Acids and Phytohormones in Solanum lycopersicum Plants Responding to Phytophthora capsici.

Pathogenic microorganisms account for large production losses in the agricultural sector. Phytophthora capsici is an oomycete that causes blight and fruit rot in important crops, especially those in the Solanaceae family. P. capsici infection is difficult to control due to genetic diversity, arising from sexual reproduction, and resistant spores that remain dormant in soil. In this study, the metabolomics of tomato plants responding to infection by P. capsici were investigated. Non-targeted metabolomics, based on liquid chromatography coupled to mass spectrometry (LC-MS), were used with multivariate data analyses to investigate time-dependent metabolic reprogramming in the roots, stems, and leaves of stem-infected plants, over an 8 day period. In addition, phytohormones and amino acids were determined using quantitative LC-MS. Methyl salicylate and 1-aminocyclopropane-1-carboxylate were detected as major signalling molecules in the defensive response to P. capsici. As aromatic amino acid precursors of secondary metabolic pathways, both phenylalanine and tryptophan showed a continuous increase over time in all tissues, whereas tyrosine peaked at day 4. Non-targeted metabolomic analysis revealed phenylpropanoids, benzoic acids, glycoalkaloids, flavonoids, amino acids, organic acids, and fatty acids as the major classes of reprogrammed metabolites. Correlation analysis showed that metabolites derived from the same pathway, or synthesised by different pathways, could either have a positive or negative correlation. Furthermore, roots, stems, and leaves showed contrasting time-dependent metabolic reprogramming, possibly related to the biotrophic vs. necrotrophic life-stages of the pathogen, and overlapping biotic and abiotic stress signaling. As such, the targeted and untargeted approaches complemented each other, to provide a detailed view of key time-dependent metabolic changes, occurring in both the asymptomatic and symptomatic stages of infection.

Metabolic Profiling of PGPR-Treated Tomato Plants Reveal Priming-Related Adaptations of Secondary Metabolites and Aromatic Amino Acids.

Plant growth-promoting rhizobacteria (PGPR) are beneficial microbes in the rhizosphere that can directly or indirectly stimulate plant growth. In addition, some can prime plants for enhanced defense against a broad range of pathogens and insect herbivores. In this study, four PGPR strains (Pseudomonas fluorescens N04, P. koreensis N19, Paenibacillus alvei T19, and Lysinibacillus sphaericus T22) were used to induce priming in Solanum lycopersicum (cv. Moneymaker) plants. Plants were inoculated with each of the four PGPRs, and plant tissues (roots, stems, and leaves) were harvested at 24 h and 48 h post-inoculation. Methanol-extracted metabolites were analyzed by ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). Chemometric methods were applied to mine the data and characterize the differential metabolic profiles induced by the PGPR. The results revealed that all four strains induced defense-related metabolic reprogramming in the plants, characterized by dynamic changes to the metabolomes involving hydroxycinnamates, benzoates, flavonoids, and glycoalkaloids. In addition, targeted analysis of aromatic amino acids indicated differential quantitative increases or decreases over a two-day period in response to the four PGPR strains. The metabolic alterations point to an altered or preconditioned state that renders the plants primed for enhanced defense responses. The results contribute to ongoing efforts in investigating and unraveling the biochemical processes that define the PGPR priming phenomenon.

Rhizobacteria-induced systemic tolerance against drought stress in Sorghum bicolor (L.) Moench.

Induction of systemic tolerance in sorghum [Sorghum bicolor (L.) Moench] against drought stress was studied by screening a large collection of rhizobacterial isolates for their potential to exhibit this essential plant growth-promoting trait. This was done by means of a greenhouse assay that measured the relative change in both plant height and -biomass (roots and shoots) between rhizobacteria-primed versus non-primed (naïve) plants under drought stress conditions. In order to elucidate the metabolomic changes in S. bicolor that conferred the drought stress tolerance after treatment (priming) with selected isolates, untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS)-based metabolomics was carried out. Intracellular metabolites were methanol-extracted from rhizobacteria-primed and naïve S. bicolor roots and shoots. Extracts were analysed on a UHPLC-HDMS system and the generated data were chemometrically mined to determine signatory metabolic profiles and bio-markers related to induced systemic tolerance. The metabolomic results showed significant treatment-related differential metabolic reprogramming between rhizobacteria-primed and naïve plants, correlating to the ability of the selected isolates to protect S. bicolor against drought stress. The selected isolates, identified by means of 16S rRNA gene sequencing as members of the genera Bacillus and Pseudomonas, were screened for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity by means of an in vitro assay and the presence of the acdS gene was subsequently confirmed by PCR for strain N66 (Pseudomonas sp.). The underlying key metabolic changes in the enhanced drought stress tolerance observed in rhizobacteria-primed S. bicolor plants included (1) augmented antioxidant capacity; (2) growth promotion and root architecture modification as a result of the upregulation of the hormones gibberellic acid, indole acetic acid and cytokinin; (3) the early activation of induce systemic tolerance through the signalling hormones brassinolides, salicylic acid and jasmonic acid and signalling molecules sphingosine and psychosine; (4) the production of the osmolytes proline, glutamic acid and choline; (5) the production of the epicuticular wax docosanoic acid and (6) ACC deaminase activity resulting in lowered ethylene levels. These results unravelled key molecular details underlying the PGPR-induced systemic tolerance in sorghum plants, providing insights for the plant priming for abiotic stress.

Unravelling the Metabolic Reconfiguration of the Post-Challenge Primed State in Sorghum bicolor Responding to Colletotrichum sublineolum Infection.

Priming is a natural phenomenon that pre-conditions plants for enhanced defence against a wide range of pathogens. It represents a complementary strategy, or sustainable alternative that can provide protection against disease. However, a comprehensive functional and mechanistic understanding of the various layers of priming events is still limited. A non-targeted metabolomics approach was used to investigate metabolic changes in plant growth-promoting rhizobacteria (PGPR)-primed Sorghum bicolor seedlings infected with the anthracnose-causing fungal pathogen, Colletotrichum sublineolum, with a focus on the post-challenge primed state phase. At the 4-leaf growth stage, the plants were treated with a strain of Paenibacillus alvei at 108 cfu mL-1. Following a 24 h PGPR application, the plants were inoculated with a C. sublineolum spore suspension (106 spores mL-1), and the infection monitored over time: 1, 3, 5, 7 and 9 days post-inoculation. Non-infected plants served as negative controls. Intracellular metabolites from both inoculated and non-inoculated plants were extracted with 80% methanol-water. The extracts were chromatographically and spectrometrically analysed on an ultra-high performance liquid chromatography (UHPLC) system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for chemometric modelling. The computed models indicated time-related metabolic perturbations that reflect primed responses to the fungal infection. Evaluation of orthogonal projection to latent structure-discriminant analysis (OPLS-DA) loading shared and unique structures (SUS)-plots uncovered the differential stronger defence responses against the fungal infection observed in primed plants. These involved enhanced levels of amino acids (tyrosine, tryptophan), phytohormones (jasmonic acid and salicylic acid conjugates, and zeatin), and defence-related components of the lipidome. Furthermore, other defence responses in both naïve and primed plants were characterised by a complex mobilisation of phenolic compounds and de novo biosynthesis of the flavones, apigenin and luteolin and the 3-deoxyanthocyanidin phytoalexins, apigeninidin and luteolinidin, as well as some related conjugates.

Differential Metabolic Reprogramming in Paenibacillus alvei-Primed Sorghum bicolor Seedlings in Response to Fusarium pseudograminearum Infection.

Metabolic changes in sorghum seedlings in response to Paenibacillus alvei (NAS-6G6)-induced systemic resistance against Fusarium pseudograminearum crown rot were investigated by means of untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS). Treatment of seedlings with the plant growth-promoting rhizobacterium P. alvei at a concentration of 1 × 108 colony forming units mL-1 prior to inoculation with F. pseudograminearum lowered crown rot disease severity significantly at the highest inoculum dose of 1 × 106 spores mL-1. Intracellular metabolites were subsequently methanol-extracted from treated and untreated sorghum roots, stems and leaves at 1, 4 and 7 days post inoculation (d.p.i.) with F. pseudograminearum. The extracts were analysed on an UHPLC-HDMS platform, and the data chemometrically processed to determine metabolic profiles and signatures related to priming and induced resistance. Significant treatment-related differences in primary and secondary metabolism post inoculation with F. pseudograminearum were observed between P. alvei-primed versus naïve S. bicolor seedlings. The differential metabolic reprogramming in primed plants comprised of a quicker and/or enhanced upregulation of amino acid-, phytohormone-, phenylpropanoid-, flavonoid- and lipid metabolites in response to inoculation with F. pseudograminearum.

The Chemistry of Plant-Microbe Interactions in the Rhizosphere and the Potential for Metabolomics to Reveal Signaling Related to Defense Priming and Induced Systemic Resistance.

Plant roots communicate with microbes in a sophisticated manner through chemical communication within the rhizosphere, thereby leading to biofilm formation of beneficial microbes and, in the case of plant growth-promoting rhizomicrobes/-bacteria (PGPR), resulting in priming of defense, or induced resistance in the plant host. The knowledge of plant-plant and plant-microbe interactions have been greatly extended over recent years; however, the chemical communication leading to priming is far from being well understood. Furthermore, linkage between below- and above-ground plant physiological processes adds to the complexity. In metabolomics studies, the main aim is to profile and annotate all exo- and endo-metabolites in a biological system that drive and participate in physiological processes. Recent advances in this field has enabled researchers to analyze 100s of compounds in one sample over a short time period. Here, from a metabolomics viewpoint, we review the interactions within the rhizosphere and subsequent above-ground 'signalomics', and emphasize the contributions that mass spectrometric-based metabolomic approaches can bring to the study of plant-beneficial - and priming events.

Effect of Endophytic Fusarium oxysporum on Host Preference of Radopholus similis to Tissue Culture Banana Plants.

The burrowing nematode Radopholus similis is one of the major constraints to banana (Musa spp.) production worldwide. Resource-poor farmers can potentially manage R. similis by using naturally occurring banana endophytes, such as nonpathogenic Fusarium oxysporum, that are inoculated into tissue culture banana plantlets. At present, it is unclear at what stage in the R. similis infection process the endophytes are most effective. In this study, the effect of three endophytic F. oxysporum isolates (V5w2, Eny1.31i and Eny7.11o) on R. similis host preference of either endophyte-treated or untreated banana plants was investigated. No differences were observed between the proportion of nematodes attracted to either root segments excised from endophyte-treated or untreated plants, or in experiments using endophyte-treated and untreated tissue culture banana plantlets. These results imply that the early processes of banana plant host recognition by R. similis are not affected by endophyte infection.