Franz Hadacek

Testing of antifungal natural products: methodologies, comparability of results and assay choice

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

Secondary Metabolites as Plant Traits: Current Assessment and Future Perspectives

Referee: Dr. Peter B. Kaufman, Dept. of Biology, University of Michigan, Ann Arbor, MI 48109-1048 Traditionally, secondary metabolites in plants have been investigated by phytochemists. Originally classified as waste products, these compounds more recently have been investigated extensively by ecologists and pharmacologists, and many complex biological functions have been discovered. Secondary metabolites occur nearly in all living organisms, within bacteria as well as in mammals, and are especially prominent in those organisms lacking an immune system. Functions of plant secondary metabolites comprise attractants, such as color pigments and scents, repellents such as antifeedants against insects and mammals, or toxins that affect growth and development of animal and microbial predators. Conversely, insects can employ plant-synthesized compounds to their own advantage, such as signals for feeding and oviposition and location of prey. Microbes also use secondary metabolites as carbon sources, and bacteria utilize them for quorum-sensing, an aspect recently discovered. Despite the diversity of recognized functions, the biochemical processes underlying these interactions are few. Primarily, they relate to the ability of these small molecules to bind to receptor regions of various proteins such as keys into locks. This review attempts a summary of current knowledge of secondary plant metabolism with focus on history of discovery, development of analytical techniques, theories of origin and function, signal pathways, biosynthesis, and assessment of biological activities. Outlined is current utilization by, and future perspectives in, different disciplines, such as chemosystematics, chemical ecology, and agricultural biotechnology. Examples illustrate the strong potential of research in secondary metabolism, particularly in comparison to more established disciplines such as developmental biology and physiology.

Chemical differences between seeds and elaiosomes indicate an adaptation to nutritional needs of ants

Ant-dispersed plants usually produce seeds with appendages (elaiosomes) as reward for ants. Plants that produce high-quality elaiosomes benefit because ants preferentially disperse their diaspores. We therefore hypothesized that seeds and elaiosomes differ in chemical composition in ways that make elaiosomes of high nutritional quality for ants, capable of providing essential dietary components that explain the increased fitness and higher gyne production documented for colonies with elaiosome consumption. To test the hypothesis we analysed the content and composition of lipids, amino acids, soluble carbohydrates, proteins and starch in seeds and elaiosomes of 15 central European ant-dispersed plants. After separating the different fractions, total lipids were determined gravimetrically, fatty acids and soluble carbohydrates were detected by gas chromatography (GC) and GC–mass spectrometry, free amino acids by an amino acid analyser while starch and protein were analysed photometrically. Seeds accumulated high molecular weight compounds such as proteins and starch, whereas elaiosomes accumulated more easily digestible low molecular weight compounds such as amino acids and monosaccharides. Analysis of similarities and similarity percentages analysis demonstrated that the composition of fatty acids, free amino acids and carbohydrates differed markedly between elaiosomes and seeds. The most important difference was in total amino acid content, which was on average 7.5 times higher in elaiosomes than in seeds. The difference was especially marked for the nitrogen-rich amino acid histidine. The availability of essential nutrients and, in some species, the higher nitrogen content in elaiosomes suggest that their nutritional value for larvae plays a key role in this interaction.

Sulphur-containing amides from Glycosmis species with different antifungal activity

Leaf extracts from different Sri Lankan provenances of Glycosmis mauritiana and G. pentaphylla have been compared by HPLC analyses and tested for antifungal activity. All collections of G. pentaphylla are uniformly characterized by the known alkaloids arborine, skimmianine and arborinine, whereas the different provenances of G. mauritiana differ by displaying chemical polymorphism of different sulphur-containing amides. Three new amides, named illukumbin B, methylillukumbin B and methylillukumbin A, have been isolated and their structures are described on the basis of spectroscopic evidence. They show different antifungal activities in bioautographic tests and appear to be accumulated mainly in individuals collected from more humid and elevated habitats.

Analysis, isolation and insecticidal activity of linear furanocoumarins and other coumarin derivatives fromPeucedanum (Apiaceae: Apioideae).

Peucedanum arenarium Waldst. & Kit.,P. austriacum (Jacq.) Koch,P. coriaceum Reichenb.,P. longifolium Waldst. & Kit,P. officinale L.,P. oreoselinum (L.) Moench,P. ostruthium L., andP. palustre (L.) Moench accumulate different structural types of coumarins including simple coumarins, linear furanocoumarins, linear dihydropyranocoumarins, angular dihydrofuranocoumarins and angular dihydropyranocoumarins. Linear furanocoumarins, known for various biological activities, include some well-known antifeedants, such as bergapten, isopimpinellin, and xanthotoxin. The aim of this investigation was to screen the diverse coumarins fromPeucedanum for insecticidal activity. LC was used to analyze and isolate coumarins for the bioassays. A growth inhibition bioassay with 17 derivatives, comprising all structural types fromPeucedanum, carried out withSpodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) as test organism, indicated the majority of the linear furanocoumarins and the angular dihydrofuranocoumarin athamantin as active compounds. Oxygenation of the prenyl residue of linear furanocoumarins decreased activity. Further formation of an ester with angelic acid even resulted in complete inactivity. Five active linear furanocoumarins, bergapten, isopimpinellin, xanthotoxin, isoimperatorin, and imperatorin, and two linear furanocoumarins with a substituted furan ring, peucedanin and 8-methoxypeucedanin, were compared in a dietary utilization bioassay. Relative growth rate (RGR) and relative consumption rate (RCR) divided the tested coumarins in three groups of similar activity. Isopimpinellin and peucedanin slightly decreased RGR and RCR of the treated larvae, and xanthotoxin, isoimperatorin, and 8-methoxypeucedanin heavily decreased RGR and RCR. Bergapten and imperatorin differed by the lowest RGR values and rather high RCR values. The effects caused by these two coumarins indicate specific postingestive toxicity. The results obtained in this study add to the reputation of coumarins to be an effective chemical defense, postulating that chemical diversity is a necessary trait for well-defended plants.

Similar diversity of alphaproteobacteria and nitrogenase gene amplicons on two related sphagnum mosses.

Sphagnum mosses represent a main vegetation component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand the extend of host specificity and impact of environment, Sphagnum fallax and Sphagnum angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-single-strand conformation polymorphism of 16S rRNA and nitrogenase-encoding (nifH) genes were highly similar for both Sphagnum species. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH) coupled with confocal laser scanning microscopy (CLSM): Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila, and Phenylobacterium as major genera for both mosses. Nitrogen fixation is an important function of Sphagnum-associated bacteria, which is fulfilled by microbial communities of Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g., Cyanobacteria and Deltaproteobacteria. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological, and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities.

Sulphones derived from methylthiopropenoic acid amides from Glycosmis angustifolia

Abstract Seven new sulphones derived from methylthiopropenoic acid amides have been isolated as dominant components from the methanolic leaf extract of Glycosmis angustifolia collected in Sri Lanka. All derivatives contained substituted phenethylamine moieties with prenyloxy (dambullins) or geranyloxy groups (gerambullins) in the para -position. Additional oxidation of the geranyl side chain leads to a diol (gerambullindiol) and to ketones (methylgerambullone and methylisogerambullone). The major component, methyldambullin, exhibited moderate antifungal activity.

Milieu-Dependent Pro- and Antioxidant Activity of Juglone May Explain Linear and Nonlinear Effects on Seedling Development

Juglone, 5-hydroxy-1,4-naphthoquinone, is known for its wide range of biological activities. It has been suggested that juglone’s excellent redox cycling properties contribute to this reputation. Many biological activities are nonlinear with low concentrations exerting stimulating effects, whereas only higher concentrations cause inhibition. Here, we corroborate studies on the nematode Caenorhabditis elegans that point out hormetic effects by showing that juglone may cause a nonlinear effect on postgerminative shoot and root growth of Sinapis alba. This effect was only significantly visible, however, when seedlings were stressed with methanol. Classic and modified versions of the deoxyribose assay were applied successfully to characterize antioxidative (purposeful generation of hydroxyl radicals) and prooxidative (no purposeful generation of hydroxyl radicals) activities. Variants of the assay with and without the addition of the iron chelator EDTA showed that the antioxidant activity is independent on chelation of iron ions by juglone; by contrast, the strength of the prooxidative activity depended on the chelation of iron ions by juglone. The hormetic effects of lower concentrations on germination of Sinapis alba, thus, may be caused by the antioxidant activities of this compound, which are especially effective when the test organism is subjected to higher oxidative challenge. The present study suggests that pronounced prooxidative activities, which are considerably accelerated by chelation of iron ions, may contribute to the toxic effects of juglone at higher concentrations.

Exploration of pro-oxidant and antioxidant activities of the flavonoid myricetin

Abstract Objectives Flavonoids are ubiquitous phenolic plant metabolites. Many of them are well known for their pro- and antioxidant properties. Myricetin has been reported to be either a potent antioxidant or a pro-oxidant depending on the conditions. The reaction conditions for the pro- and antioxidant activities were therefore investigated using variations of the deoxyribose degradation assay systems. Methods The deoxyribose degradation assay systems were conducted as follows; H2O2/FeIII/ascorbic acid, H2O2/FeIII, FeIII/ascorbic acid, and FeIII. Each system was carried out in two variants, FeCl3 (iron ions added as FeCl3) and FeEDTA (iron added in complex with ethylenediaminetetraacetic acid). Results When ascorbic acid was present, myricetin showed antioxidant properties, especially when it occurred in complex with iron. In ascorbic acid-free systems, pro-oxidant activities prevailed, which where enhanced if iron was in complex with EDTA. Discussion Myricetins antioxidant activity depends on both the reactive oxygen species (ROS) scavenging and iron ions chelation properties. The pro-oxidative properties are caused by reduction of molecular oxygen to ROS and iron(III) to iron(II). Myricetin is able to substitute for ascorbic acid albeit less efficiently.

Iron and its complexation by phenolic cellular metabolites: from oxidative stress to chemical weapons.

Iron is a transition metal that forms chelates and complexes with various organic compounds, also with phenolic plant secondary metabolites. The ligands of iron affect the redox potential of iron. Electrons may be transferred either to hydroxyl radicals, hydrogen peroxide or molecular oxygen. In the first case, oxidative stress is decreased, in the latter two cases, oxidative stress is increased. This milieu-dependent mode of action may explain the non-linear mode of action of juglone and other secondary metabolites. Attention to this phenomenon may help to explain idiosyncratic and often nonlinear effects that result in biological assays. Current chemical assays are discussed that help to explore these aspects of redox chemistry.