Discrimination of Solanaceae taxa and quantification of scopolamine and hyoscyamine by ATR-FTIR spectroscopy.

Plant species of the Solanaceae family (nightshades) contain pharmacologically active anticholinergic tropane alkaloids, e.g., scopolamine and hyoscyamine. Tropane alkaloids are of special interest, either as active principles or as starting materials for semisynthetic production of other substances. For genetic evaluation, domestication, cultivation, harvest and post-harvest treatments, quantification of the individual active principles is necessary to monitor industrial processes and the resulting finished products. Up to now, frequently applied methods for quantification are based on high performance liquid chromatography and gas chromatography optionally combined with mass spectrometry. However, alternative analytical methods have the potential to replace the established standard methods partly. In this context, attenuated total reflection-Fourier transform infrared spectroscopy enabled chemotaxonomical classification of the Solanaceae Atropa belladonna, Datura stramonium, Hyoscyamus niger, Solanum dulcamara, and Duboisia in combination with cluster analysis. Also discrimination of genotypes within species was achieved to some extent. The most characteristic scopolamine bands could be identified in attenuated total reflection-Fourier transform infrared spectra of Solanaceae leaves, which allow a fast characterisation of plants with high scopolamine content. Applying a partial least square algorithm, very good calibration statistics were obtained for the prediction of the scopolamine content (residual prediction deviation = 7.67), and moderate prediction quality could be achieved for the hyoscyamine content (residual prediction deviation = 2.48).

A novel procedure for strain classification of fungal mycelium by cluster and artificial neural network analysis of Fourier transform infrared (FTIR) spectra.

Fourier transform infrared spectroscopy (FTIR) was used to discriminate important wood-destroying fungi. Mycelia of 26 fungal strains belonging to 24 different species were grown on agar plates and subjected to FTIR attenuated total reflection (ATR) measurements. To classify the FTIR spectra, cluster analysis--an unsupervised multivariate data analysis method--was compared with artificial neural network (ANN) analysis--a supervised approach. By internal validation, both methods classified 99% of the spectra correctly. External validation with independent test set spectra resulted in 95% correctly classified spectra, demonstrating the high potential of this method for fungal strain identification.

Characterization of essential oil distribution in the root cross-section of Valeriana officinalis L. s.l. by using histological imaging techniques.

BACKGROUND: The essential oil is an important compound of the root and rhizome of medicinally used valerian (Valeriana officinalis L. s.l.), with a stated minimum content in the European pharmacopoeia. The essential oil is located in droplets, of which the position and distribution in the total root cross-section of different valerian varieties, root thicknesses and root horizons are determined in this study using an adapted fluorescence-microscopy and automatic imaging analysis method. The study was initiated by the following facts:A probable negative correlation between essential oil content and root thickness in selected single plants (elites), observed during the breeding of coarsely rooted valerian with high oil content.Higher essential oil content after careful hand-harvest and processing of the roots. RESULTS: In preliminary tests, the existence of oil containing droplets in the outer and inner regions of the valerian roots was confirmed by histological techniques and light-microscopy, as well as Fourier-transform infrared spectroscopy. Based on this, fluorescence-microscopy followed by image analysis of entire root cross-sections, showed that a large number of oil droplets (on average 43% of total oil droplets) are located close to the root surface. The remaining oil droplets are located in the inner regions (parenchyma) and showed varying density gradients from the inner to the outer regions depending on genotype, root thickness and harvesting depth. CONCLUSIONS: Fluorescence-microscopy is suitable to evaluate prevalence and distribution of essential oil droplets of valerian in entire root cross-sections. The oil droplet density gradient varies among genotypes. Genotypes with a linear rather than an exponential increase of oil droplet density from the inner to the outer parenchyma can be chosen for better stability during post-harvest processing. The negative correlation of essential oil content and root thickness as observed in our breeding material can be counteracted through a selection towards generally high oil droplet density levels, and large oil droplet sizes independent of root thickness.

Genetic Variation of the Host Plant Species Matters for Interactions with Above- and Belowground Herbivores.

Plants are challenged by both above- and belowground herbivores which may indirectly interact with each other via herbivore-induced changes in plant traits; however, little is known about how genetic variation of the host plant shapes such interactions. We used two genotypes (M4 and E9) of Solanum dulcamara (Solanaceae) with or without previous experience of aboveground herbivory by Spodoptera exigua (Noctuidae) to quantify its effects on subsequent root herbivory by Agriotes spp. (Elateridae). In the genotype M4, due to the aboveground herbivory, shoot and root biomass was significantly decreased, roots had a lower C/N ratio and contained significantly higher levels of proteins, while the genotype E9 was not affected. However, aboveground herbivory had no effects on weight gain or mortality of the belowground herbivores. Root herbivory by Agriotes increased the nitrogen concentration in the roots of M4 plants leading to a higher weight gain of conspecific larvae. Also, in feeding bioassays, Agriotes larvae tended to prefer roots of M4 over E9, irrespective of the aboveground herbivore treatment. Fourier-Transform Infrared Spectroscopy (FT-IR) documented differences in metabolic profiles of the two plant genotypes and of the roots of M4 plants after aboveground herbivory. Together, these results demonstrate that previous aboveground herbivory can have genotype-specific effects on quantitative and qualitative root traits. This may have consequences for belowground interactions, although generalist root herbivores might not be affected when the root biomass offered is still sufficient for growth and survival.

Ectomycorrhizal identification in environmental samples of tree roots by Fourier-transform infrared (FTIR) spectroscopy.

Roots of forest trees are associated with various ectomycorrhizal (ECM) fungal species that are involved in nutrient exchange between host plant and the soil compartment. The identification of ECM fungi in small environmental samples is difficult. The present study tested the feasibility of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy followed by hierarchical cluster analysis (HCA) to discriminate in situ collected ECM fungal species. Root tips colonized by distinct ECM fungal species, i.e., Amanita rubescens, Cenococcum geophilum, Lactarius subdulcis, Russula ochroleuca, and Xerocomus pruinatus were collected in mono-specific beech (Fagus sylvatica) and mixed deciduous forests in different geographic areas to investigate the environmental variability of the ECM FTIR signatures. A clear HCA discrimination was obtained for ECM fungal species independent of individual provenance. Environmental variability neither limited the discrimination between fungal species nor provided sufficient resolution to discern species sub-clusters for different sites. However, the de-convoluted FTIR spectra contained site-related spectral information for fungi with wide nutrient ranges, but not for Lactarius subdulcis, a fungus residing only in the litter layer. Specific markers for distinct ECM were identified in spectral regions associated with carbohydrates (i.e., mannans), lipids, and secondary protein structures. The present results support that FTIR spectroscopy coupled with multivariate analysis is a reliable and fast method to identify ECM fungal species in minute environmental samples. Moreover, our data suggest that the FTIR spectral signatures contain information on physiological and functional traits of ECM fungi.

Incorporation of Listeria monocytogenes strains in raw milk biofilms.

Biofilms develop successively on devices of milk production without sufficient cleaning and originate from the microbial community of raw milk. The established biofilm matrices enable incorporation of pathogens like Listeria monocytogenes, which can cause a continuous contamination of food processing plants. L. monocytogenes is frequently found in raw milk and non-pasteurized raw milk products and as part of a biofilm community in milk meters and bulk milk tanks. The aim of this study was to analyze whether different L. monocytogenes strains are interacting with the microbial community of raw milk in terms of biofilm formation in the same manner, and to identify at which stage of biofilm formation a selected L. monocytogenes strain settles best. Bacterial community structure and composition of biofilms were analyzed by a cloning and sequencing approach and terminal restriction fragment length polymorphism analysis (T-RFLP) based on the bacterial 16S rRNA gene. The chemical composition of biofilms was analyzed by Fourier transform infrared spectroscopy (FTIR), while settled L. monocytogenes cells were quantified by fluorescence in situ hybridization (FISH). Addition of individual L. monocytogenes strains to raw milk caused significant shifts in the biofilm biomass, in the chemical as well as in the bacterial community composition. Biofilm formation and attachment of L. monocytogenes cells were not serotype but strain specific. However, the added L. monocytogenes strains were not abundant since mainly members of the genera Citrobacter and Lactococcus dominated the bacterial biofilm community. Overall, added L. monocytogenes strains led to a highly competitive interaction with the raw milk community and triggered alterations in biofilm formation.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Fourier transform infrared microscopy and imaging: detection of fungi in wood.

FTIR microscopy was used to detect and discriminate the two wood decaying fungi Trametes versicolor and Schizophyllum commune in experimentally infected beech wood blocks. The distribution of fungal mycelium in wood was locally resolved and semiquantitatively recorded using FTIR microscopy combined with a focal plane array detector and image analysis. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibers and empty vessel lumina and spectra from mycelium of both fungal species, irrespective of whether the fungi were grown on the surface of wood or inside vessel lumina. Species-specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to discriminate among fungal species decaying wood.