Konstantinos A. Aliferis

Metabolomics in pesticide research and development: review and future perspectives

The emerge of metabolomics within functional genomics has provided a new dimension in the study of biological systems. In regards to the study of agroecosystems, metabolomics enables monitoring of metabolic changes in association with biotic or abiotic agents such as agrochemicals. Focusing on crop protection chemicals, a great effort has been given towards the development of crop protection agents safer for consumers and the environment and more efficient than the existing ones. Within this framework, metabolomics has so far been a valuable tool for high-throughput screening of bioactive substances in order to discover those with high selectivity, unique modes-of-action, and acceptable eco-toxicological/toxicological profiles. Here, applications of metabolomics in the investigation of the modes-of-action and ecotoxicological–toxicological risk assessment of bioactive compounds, mining of biological systems for the discovery of bioactive metabolites, and the risk assessment of genetic modified crops are discussed.

Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars.

To isolate and characterize indigenous bacterial endophytes from cultivars of switchgrass and study their antimicrobial and growth promoting potential.

FT-ICR/MS and GC-EI/MS Metabolomics Networking Unravels Global Potato Sprout's Responses to Rhizoctonia solani Infection

The complexity of plant-pathogen interactions makes their dissection a challenging task for metabolomics studies. Here we are reporting on an integrated metabolomics networking approach combining gas chromatography/mass spectrometry (GC/MS) with Fourier transform ion cyclotron resonance/mass spectrometry (FT-ICR/MS) and bioinformatics analyses for the study of interactions in the potato sprout-Rhizoctonia solani pathosystem and the fluctuations in the global metabolome of sprouts. The developed bioanalytical and bioinformatics protocols provided a snapshot of the sprouts global metabolic network and its perturbations as a result of pathogen invasion. Mevalonic acid and deoxy-xylulose pathways were substantially up-regulated leading to the biosynthesis of sesquiterpene alkaloids such as the phytoalexins phytuberin, rishitin, and solavetivone, and steroidal alkaloids having solasodine and solanidine as their common aglycons. Additionally, the perturbation of the sprouts metabolism was depicted in fluctuations of the content of their amino acids pool and that of carboxylic and fatty acids. Components of the systemic acquired resistance (SAR) and hypersensitive reaction (HR) such as azelaic and oxalic acids were detected in increased levels in infected sprouts and strategies of the pathogen to overcome plant defense were proposed. Our metabolic approach has not only greatly expanded the multitude of metabolites previously reported in potato in response to pathogen invasion, but also enabled the identification of bioactive plant-derived metabolites providing valuable information that could be exploited in biotechnology, biomarker-assisted plant breeding, and crop protection for the development of new crop protection agents.

A Metabolic Profiling Strategy for the Dissection of Plant Defense against Fungal Pathogens

Here we present a metabolic profiling strategy employing direct infusion Orbitrap mass spectrometry (MS) and gas chromatography-mass spectrometry (GC/MS) for the monitoring of soybeans (Glycine max L.) global metabolism regulation in response to Rhizoctonia solani infection in a time-course. Key elements in the approach are the construction of a comprehensive metabolite library for soybean, which accelerates the steps of metabolite identification and biological interpretation of results, and bioinformatics tools for the visualization and analysis of its metabolome. The study of metabolic networks revealed that infection results in the mobilization of carbohydrates, disturbance of the amino acid pool, and activation of isoflavonoid, α-linolenate, and phenylpropanoid biosynthetic pathways of the plant. Components of these pathways include phytoalexins, coumarins, flavonoids, signaling molecules, and hormones, many of which exhibit antioxidant properties and bioactivity helping the plant to counterattack the pathogens invasion. Unraveling the biochemical mechanism operating during soybean-Rhizoctonia interaction, in addition to its significance towards the understanding of the plants metabolism regulation under biotic stress, provides valuable insights with potential for applications in biotechnology, crop breeding, and agrochemical and food industries.

Metabolite Composition and Bioactivity of Rhizoctonia solani Sclerotial Exudates

Sclerotia are vegetative structures that play a major role in survival of fungi under adverse conditions. The sclerotia of the plant pathogen Rhizoctonia solani AG2-2 IIIB exude liquid brown droplets that were evaluated for their bioactivity and toxicity against microorganisms and plant species. Also, their metabolic composition was analyzed by integrating Fourier transform ion cyclotron resonance-mass spectrometry (FT-ICR/MS), gas chromatography-MS (GC/MS), and proton nuclear magnetic resonance ((1)H NMR) spectroscopy. The results showed that exudates are complex mixtures composed of phenolics (17.40%), carboxylic acids (12.79%), carbohydrates (6.08%), fatty acids (3.78%), and amino acids (3.47%). The presence of such metabolites contributed to their antifungal and phytotoxic activities. The biological interpretation of the results highly suggests that the exudates not only have multiple roles in fungal physiology but also are a potential bioactive source with moderate toxicity. Our findings show with certainty that the integration of different analytical platforms is a powerful approach for extracting the maximum and reliable information on the metabolic composition of complex biological samples.

Gas chromatography–mass spectrometry metabolite profiling of worker honey bee (Apis mellifera L.) hemolymph for the study of Nosema ceranae infection

Here, we are presenting a gas chromatography-mass spectrometry (GC/MS) approach for the study of infection of the worker honey bee (Apis mellifera L.) by the newly emerged obligate intracellular parasite Nosema ceranae based on metabolite profiling of hemolymph. Because of the severity of the disease, early detection is crucial for its efficient control. Results revealed that the parasite causes a general disturbance of the physiology of the honey bee affecting the mechanisms controlling the mobilization of energy reserves in infected individuals. The imposed nutritional and energetic stress to the host was depicted mainly in the decreased levels of the majority of carbohydrates and amino acids, including metabolites such as fructose, l-proline, and the cryoprotectants sorbitol and glycerol, which are implicated in various biochemical pathways. Interestingly, the level of glucose was detected at significantly higher levels in infected honey bees. Metabolomics analyses were in agreement with those of multiplex quantitative PCR analyses, indicating that it can be used as a complementary tool for the detection and the study of the physiology of the disease.

1H NMR and GC-MS metabolic fingerprinting of developmental stages of Rhizoctonia solani sclerotia

Rhizoctonia solani AG-3 is a soilborne plant pathogen that forms resting vegetative structures called sclerotia. These compact structures are crucial to the pathogen’s survival and pathogenesis. The metabolic changes occurring during sclerotia development were monitored using proton nuclear magnetic resonance (1H NMR) spectroscopy and gas chromatography–mass spectrometry (GC-MS). The validation, discrimination, and the establishment of correlative relationships between metabolite signals were performed by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The results of the analyses suggested that out of the 116 compounds that were simultaneously analyzed and compared using GC-MS, α-α-trehalose, d-glucose, 9-(Z)-octadecenoic and 9,12-octadecadienoic acids, xylitol, and glucitol were key metabolites that were highly dependent on the developmental stage of the sclerotia contributing to their discrimination and classification. Furthermore, the application of 1H NMR and GC-MS metabolic fingerprinting on the same biological sample provided complementary information illustrating the value of this integrated approach in the study of metabolic changes in fungal structures.

Alleviation of Drought Stress and Metabolic Changes in Timothy (Phleum pratense L.) Colonized with Bacillus subtilis B26

Drought is a major limiting factor of crop productivity worldwide and its incidence is predicted to increase under climate change. Drought adaptation of cool-season grasses is thus a major challenge to secure the agricultural productivity under current and future climate conditions. Endophytes are non-pathogenic plant-associated bacteria that can play an important role in conferring resistance and improving plant tolerance to drought. In this study, the effect of inoculation of the bacterial endophyte Bacillus subtilis strain B26 on growth, water status, photosynthetic activity and metabolism of timothy (Phleum pratense L.) subjected to drought stress was investigated under controlled conditions. Under both drought-stress and non-stressed conditions, strain B26 successfully colonized the internal tissues of timothy and had a positive impact on plant growth. Exposure of inoculated plant to a 8-week drought-stress led to significant increase in shoot and root biomass by 26.6 and 63.8%, and in photosynthesis and stomatal conductance by 55.2 and 214.9% respectively, compared to non-inoculated plants grown under similar conditions. There was a significant effect of the endophyte on plant metabolism; higher levels of several sugars, notably sucrose and fructans and an increase of key amino acids such as, asparagine, glutamic acid and glutamine were recorded in shoots and roots of colonized plants compared to non-colonized ones. The accumulation of the non-protein amino acid GABA in shoots of stressed plants and in roots of stressed and unstressed plants was increased in the presence of the endophyte. Taken together, our results indicate that B. subtilis B26 improves timothy growth under drought stress through the modification of osmolyte accumulation in roots and shoots. These results will contribute to the development of a microbial agent to improve the yield of grass species including forage crops and cereals exposed to environmental stresses.

Lemna minor L. as a model organism for ecotoxicological studies performing 1H NMR fingerprinting

A validated method applying (1)H NMR fingerprinting for the study of metabolic changes caused in Lemna minor L. by various phytotoxic substances is presented. (1)H NMR spectra of crude extracts from untreated and treated colonies with the herbicides glyphosate, mesotrione, norflurazon, paraquat and the phytotoxin pyrenophorol were subjected to multivariate analyses for detecting differences between groups of treatments. Partial least squares-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) were carried out in order to discriminate and classify treatments according to the observed changes in the metabolome of the plant. Although the compounds at the concentrations used did not cause macroscopically observable symptoms of phytotoxicity, characteristic metabolic changes were detectable by analyzing (1)H NMR spectra. Analyses results revealed that metabonomics applying (1)H NMR fingerprinting is a potential method for the investigation of toxicological effects of xenobiotics on L. minor, and possibly on other duckweed species, helping in the understanding of such interactions.

Chemotaxonomy of fungi in the Rhizoctonia solani species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.