The microbiome of endophytic, wood colonizing bacteria from pine trees as affected by pine wilt disease.

Pine wilt disease (PWD) is a devastating forest disease present worldwide. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Wood samples from trees with different degrees of PWD disease were collected at two sites (A and M) in Portugal. Endophytic bacteria were characterized based on directly extracted DNA by fingerprinting and barcoding using the 16S rRNA gene as marker. Furthermore, cultivation-based approaches were used to obtain isolates of the major taxa to study their ecophysiology. The endophytic microbiome from P. pinaster trees differed significantly between the two sampling sites. Main bacterial OTUs belonged to the Proteobacteria (39% (site M) - 97% (site A)), and Firmicutes (0.70% (site A) - 44% (site M)). However, consequences of the invasion with the pathogen were comparable. Interestingly diversity of wood endophytic bacteria increased with the severity of the diseases, with highest diversity levels observed in in the most affected trees. Our results suggest that in the first stages of the disease, the defence mechanisms of plants are repressed by the pathogen, resulting in a colonization of the wood interior by soil microorganisms

A fatal forensic intoxication with fenarimol: analysis by HPLC/DAD/MSD

Fenarimol (Rubigan®) is a pyrimidine ergosterol biosynthesis inhibitor used as a systemic fungicide. The authors present a fatal fenarimol intoxication case analysed in the Forensic Toxicology Service of the National Institute of Legal Medicine. The results were used to compare two different HPLC techniques, regarding selectivity and sensitivity: an HPLC system with a diode array detector (DAD) and an HPLC system with a DAD and a mass spectrometry detector (MSD) with an electrospray interface. All biological samples were submitted to a solid-phase extraction procedure. The detection and quantification limits of fenarimol, linearity, precision and accuracy were evaluated. The fenarimol concentration levels determined were of 89.0 mg/ml in gastric contents, 1.9 mg/g in liver and 0.4 mg/g in kidney. Blood was not available at autopsy. No published data related to fenarimol self-poisoning were found, so it was not possible to interpret the results obtained by comparison with toxic/lethal levels.http://www.sciencedirect.com/science/article/B6T6W-488NYVD-R/1/712bf23bb727a7110f22a36460ab32f

Analytical method for the determination of strychnine in tissues by gas chromatography/mass spectrometry: two case reports

This paper describes an analytical method for strychnine determination in biological samples by gas chromatography/mass spectrometry and their application in the investigation of two cases involving strychnine ingestion: A fatal case and a clinical one. The strychnine is isolated from biological samples using a liquid-liquid extraction procedure. The clean-up procedure is performed using an acid solution. Papaverine is used as internal standard in the quantification of strychnine. In the analysed specimens, the limits of quantification were 0.1 [mu]g/ml or 0.1 [mu]g/g. The recovery rate ranged from 75.0% to 98.7% and the coefficients of variation ranged from 4.8% to 10.5%.http://www.sciencedirect.com/science/article/B6T6W-408KCFB-8/1/f8bbeefad9e20909aef50467f67a2e8