Root-associated bacteria modulate the specialised metabolome of Lithospermum officinale L.

Bacteria influence plant growth and development and therefore are attractive resources for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of bacteria on the modulation of SM production in the medicinal plant Lithospermum officinale (Boraginaceae family) with a focus on the naphthoquinones alkannin/shikonin and their derivatives (A/Sd). The study was conducted in an in vitro cultivation system developed for that purpose, as well as in a greenhouse. Targeted and non-targeted metabolomics were performed, and expression of the gene PGT encoding for a key enzyme in the A/S biosynthesis pathway was evaluated with qPCR. Three strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of A/Sd in L. officinale in both systems, demonstrating the strength of our approach for screening A/Sd-inducing bacteria. The bacterial treatments altered other plant metabolites derived from the shikimate pathway as well. Our results demonstrate that bacteria influence the biosynthesis of A/Sd and interact with different metabolic pathways. This work highlights the potential of bacteria to increase the production of SM in medicinal plants and reveals new patterns in the metabolome regulation of L. officinale.

First report of detection of microcystins in farmed mediterranean mussels Mytilus galloprovincialis in Thermaikos gulf in Greece.

BACKGROUND: Microcystins are emerging marine biotoxins, produced by potentially toxic cyanobacteria. Their presence has been reported in aquatic animals in Greek freshwater, while data are few in marine environments. Since the climate change induces eutrophication and harmful algal blooms in coastal marine ecosystems affecting the public health, further research on microcystins' presence in marine waters is required. The aim of this study was to examine the potential presence of microcystins in mussels Mytilus galloprovincialis in the largest farming areas in Thermaikos gulf, in Northern Greece, and to investigate their temporal and spatial distribution, adding to the knowledge of microcystins presence in Greek Mediterranean mussels. RESULTS: A 4-year microcystins' assessment was conducted from 2013 to 2016, in farmed Mediterranean mussels M. galloprovincialis, in five sampling areas in Thermaikos gulf, in northern Greece, where the 90% of the Greek mussels' farming activities is located. The isolation of potentially toxic cyanobacteria was confirmed by molecular methods. An initial screening was performed with a qualitative and quantitative direct monoclonal (DM) ELISA and results above 1 ng g-1 were confirmed for the occurrence of the most common microcystins-RR, -LR and -YR, by Ultra High Performance Liquid Chromatography (UHPLC) coupled with a high- resolution mass spectrometer (HRMS) (Orbitrap analyzer). Microcystin-RR and microcystin-LR were detected, while the intensity of microcystin-YR was below the method detection limit. Most samples that exhibited concentrations above 1 ng g-1 were detected during the warm seasons of the year and especially in spring. Results indicated an overestimation of the ELISA method, since concentrations ranged between 0.70 ± 0.15 ng g-1 and 53.90 ± 3.18 ng g-1, while the confirmation denoted that the levels of microcystins were 6 to 22 times lower. CONCLUSIONS: Microcystin-RR and microcystin-LR were detected for the first time in mussel M. galloprovincialis, harvested from farms in Thermaikos gulf, in Central Macedonia, Greece. Their presence was linked to potentially toxic cyanobacteria. Bioaccumulation was observed in digestive gland, while the concentrations in muscles were found extremely low. Samples with levels above 1 ng g-1 were observed mostly during spring, confirming the seasonal distribution of microcystins. The comparison of the results by the ELISA and the LC-Orbitrap MS method indicated an overestimation of concentration by the ELISA method.

Psychiatrics and selected metabolites in hospital and urban wastewaters: Occurrence, removal, mass loading, seasonal influence and risk assessment.

The occurrence, removal, mass loading, seasonal influence and environmental risk assessment of nine psychiatric pharmaceuticals and four of their selected metabolites, were studied in one hospital and one urban wastewater treatment plant (WWTP) in Ioannina city, in northwestern Greece, providing information about the efficiency of the plants and their contribution into the final receiver's flow. Samples were collected from the influents and the effluents of the plants in different sampling campaigns, from July to December 2016. Analytical methodology was based on ultra-high performance liquid chromatography-Orbitrap high-resolution mass spectrometry, after solid-phase extraction through Oasis HLB cartridges. Concentrations in both WWTPs ranged between

A modified QuEChERS approach for the analysis of pharmaceuticals in sediments by LC-Orbitrap HRMS.

This work reports on a rapid and sensitive analytical method intended to be used for investigation of 25 multiclass pharmaceuticals in sediments. The method comprises a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction, followed by ultra-high-performance liquid chromatography-high-resolution linear ion trap/Orbitrap mass spectrometry, operated in positive ionization mode. The crucial parameters of both extraction and cleanup as well as those related to liquid chromatography and mass spectrometry were optimized and the method was validated in terms of accuracy, method detection and quantification limits, precision, linearity, and matrix effects. A matrix-matched calibration approach and isotopically labeled internal standards were employed for quantification and correction of matrix effects. In the optimized method, recoveries were calculated to be between 64 and 101% in the intermediate spiking level, with a relative standard deviation below 14%. Method quantification limits ranged from 1.3 to 47 ng g-1. The linearity, expressed as a correlation coefficient, was over 0.998 in all cases, within a range from each compound's method quantification limit to 250 ng g-1. The matrix effect study revealed slight signal suppression that decreased after the cleanup step for 50% of the pharmaceuticals, proving its adequacy to limit matrix interferences, without causing any important loss of relevant analytes. The applicability of the method was successfully tested in sediments from two Greek rivers in northwestern Greece, revealing the presence of paracetamol, risperidone, venlafaxine, citalopram, and carbamazepine and estimating for the first time the potential occurrence of pharmaceuticals in these two aquatic systems.

Trace analysis of pesticide residues in sediments using liquid chromatography-high-resolution Orbitrap mass spectrometry.

The present study describes the optimization and validation of an analytical method based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and purification with dispersive solid-phase extraction (dSPE) before analysis, followed by ultrahigh-performance liquid chromatography-high-resolution linear ion trap/Orbitrap (LTQ Orbitrap) mass spectrometry for the determination of 18 pesticides in sediment. To optimize process efficiency, parameters such as pH, extraction salts, sediment amount, and cleanup sorbents were evaluated. Identification was based on both accurate mass and retention time, and further confirmation was achieved by mass spectrometry fragmentation. The optimized analytical method demonstrated good validation characteristics, such as accuracy (recoveries from 70.8% to 106.2%), method quantification limits (below 10 ng g-1 for 89% of the pesticides selected), linearity (coefficient of determination greater than 0.9921 in all cases), precision (repeatability and reproducibility with standard deviations below 18% and 21%, respectively), and matrix effect (signal suppression was exhibited for almost all analytes). The overall method performance, expressed as process efficiency, ranged from 58.8% to 102.1%. The validated method was successfully applied to real samples collected along two rivers in northwestern Greece, revealing the presence of three selected pesticides but at levels below the method quantification limit.