Antifungal testing of filamentous fungi generally suffers from incomparability of results. In order to illustrate this problem, the polyacetylene falcarindiol and the naphthoquinone juglone, two known antifungal natural products, were assayed in various dilution and diffusion bioassays against three selected microfungi, Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum. Broth microdilution, based on the 96‐well microtitre plate format, can be scored by various direct and biochemical methods. As examples, direct observation with image analysis and the fluorescein diacetate scoring method are compared. Of the other methodologies used, results obtained by thin‐layer bioautography and the radial growth rate method clearly deviated. Disk diffusion results, however, matched microdilution. In conclusion, microdilution offers the greatest potential of all bioassays to become the future general standard methodology. Copyright © 2000 John Wiley & Sons, Ltd.

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High acidity, low temperature and extremely low concentration of nutrients form Sphagnum bogs into extreme habitats for organisms. Little is known about the bacteria associated with living Sphagnum plantlets, especially about their function for the host. Therefore, we analysed the endo- and ectophytic bacterial populations associated with two widely distributed Sphagnum species, Sphagnum magellanicum and Sphagnum fallax, by a multiphasic approach. The screening of 1222 isolates for antagonistic activity resulted in 326 active isolates. The bacterial communities harboured a high proportion of antifungal (26%) but a low proportion of antibacterial isolates (0.4%). Members of the genus Burkholderia (38%) were found to be the most dominant group of antagonistic bacteria. The finding that a large proportion (89%) of the antagonistic bacteria produced antifungal compounds may provide an explanation for the well-known antimicrobial activity of certain Sphagnum species. The secondary metabolites of the Sphagnum species themselves were analysed by HPLC-PDA. The different spectra of detected compounds may not only explain the antifungal activity but also the species specificity of the microbial communities. The latter was analysed using cultivation-independent single-stranded conformation polymorphism (SSCP) analysis. Using Burkholderia-specific primers we found a high diversity of Burkholderia isolates in the endophytic and ectophytic habitats of Sphagnum. Furthermore, a high diversity of nitrogen-fixing bacteria was detected by using nifH-specific primers, especially inside Sphagnum mosses. In conclusion, this study provides evidence that both Sphagnum species were colonized by characteristic bacterial populations, which appear to be important for pathogen defence and nitrogen fixation.

Meiosis, the key step of sexual reproduction, persists in facultative apomictic plants functional to some extent. However, it still remains unclear how and why proportions of reproductive pathways vary under different environmental stress conditions. We hypothesized that oxidative stress mediates alterations of developmental pathways. In apomictic plants we expected that megasporogenesis, the stage directly after meiosis, would be more affected than later stages of seed development. To simulate moderate stress conditions we subjected clone-mates of facultative apomictic Ranunculus auricomus to 10 h photoperiods, reflecting natural conditions, and extended ones (16.5 h). Reproduction mode was screened directly after megasporogenesis (microscope) and at seed stage (flow cytometric seed screening). Targeted metabolite profiles were performed with HPLC–DAD to explore if and which metabolic reprogramming was caused by the extended photoperiod. Prolonged photoperiods resulted in increased frequencies of sexual vs. aposporous initials directly after meiosis, but did not affect frequencies of sexual vs. asexual seed formation. Changes in secondary metabolite profiles under extended photoperiods affected all classes of compounds, and c. 20% of these changes separated the two treatments. Unexpectedly, the renowned antioxidant phenylpropanoids and flavonoids added more to clone-mate variation than to treatment differentiation. Among others, chlorophyll degradation products, non-assigned phenolic compounds and more lipophilic metabolites also contributed to the dissimilarity of the metabolic profiles of plants that had been exposed to the two different photoperiods. The hypothesis of moderate light stress effects was supported by increased proportions of sexual megaspore development at the expense of aposporous initial formation. The lack of effects at the seed stage confirms the basic assumption that only meiosis and sporogenesis would be sensitive to light stress. The concomitant change of secondary metabolite profiles, as a systemic response at this early developmental stage, supports the notion that oxidative stress could have affected megasporogenesis by causing the observed metabolic reprogramming. Hypotheses of genotype-specific responses to prolonged photoperiods are rejected.

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-012-0470-0) contains supplementary material, which is available to authorized users.