Ines Hilke

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

BACKGROUND AND AIMS Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated. METHODS Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma-optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes. KEY RESULTS The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes. CONCLUSIONS The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.

Transgenic upregulation of the condensed tannin pathway in poplar leads to a dramatic shift in leaf palatability for two tree-feeding Lepidoptera

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levels were predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects.

A novel single-run dual temperature combustion (SRDTC) method for the determination of organic, in-organic and total carbon in soil samples

The quantification of organic (OC) and inorganic carbon (IC) in soils provides an essential tool for understanding biogeochemical processes. Examples of its potential application are the assessment of the humification degree of soil organic matter, the calculation of carbon fluxes and budgets in terrestrial systems on a regional and global scale and the investigation of the carbon storage potential of soils. The verification of changes in carbon stocks requires an extensive number of samples as well as precise and reliable analyses. Due to the wide variation in the concentrations of the two forms of carbon in solid samples, the exact distinction is very difficult. We present the advantages of a single-run dual temperature combustion method (SRDTC) at 515 degrees C for OC and 925 degrees C for IC, which allows the determination of OC, IC and total carbon (TC) within one single analytical run. The three parameters are analyzed in less than 30 min. Additionally, the method is characterized by a significantly reduced variability and low operator bias, as there is no need of chemical sample pre-treatment. It is applicable to a broad range of varying OC and IC contents, which is demonstrated by the use of numerous synthetic soil mixtures that have been analyzed. Furthermore, SRDTC indicates the presence of thermally instable carbonates, like magnesite, in the sample. Use of silver boats as a catalytic agent results in an improved distinction between OC and IC in this case. To examine the accuracy and reliability of the SRDTC method, it was compared to other techniques frequently used for carbon determination in soil samples: total combustion by elemental analysis to determine TC and acidification of the sample prior to combustion to determine OC. We will show that the rugged SRDTC method offers a substantial progress for both the reliable and rapid OC and IC determination in soil samples where elemental carbon is negligible.

Thiosulfate- and hydrogen-driven autotrophic denitrification by a microbial consortium enriched from groundwater of an oligotrophic limestone aquifer

ABSTRACT Despite its potentially high relevance for nitrate removal in freshwater environments limited in organic carbon, chemolithoautotrophic denitrification has rarely been studied in oligotrophic groundwater. Using thiosulfate and H2 as electron donors, we established a chemolithoautotrophic enrichment culture from groundwater of a carbonate‐rock aquifer to get more insight into the metabolic repertoire, substrate turnover, and transcriptional activity of subsurface denitrifying consortia. The enriched consortium was dominated by representatives of the genus Thiobacillus along with denitrifiers related to Sulfuritalea hydrogenivorans, Sulfuricella denitrificans, Dechloromonas sp. and Hydrogenophaga sp., representing the consortiums capacity to use multiple inorganic electron donors. Microcosm experiments coupled with Raman gas spectroscopy demonstrated complete denitrification driven by reduced sulfur compounds and hydrogen without formation of N2O. The initial nitrate/thiosulfate ratio had a strong effect on nosZ transcriptional activity and on N2 formation, suggesting similar patterns of the regulation of gene expression as in heterotrophic denitrifiers. Sequence analysis targeting nirS and nosZ transcripts identified Thiobacillus denitrificans‐related organisms as the dominant active nirS‐type denitrifiers in the consortium. An additional assessment of the nirS‐type denitrifier community in the groundwaterclearly confirmed the potential for sulfur‐ and hydrogen‐dependent chemolithoautotrophic denitrification as important metabolic feature widely spread among subsurface denitrifiers at the Hainich Critical Zone Exploratory.

Determination of trace metals in woodlice and their skins with particular emphasis on quality control

AI, Fe, Mn and Zn were determined in two ditTerent species of woodlouse: Porcellio scaber and Porcellio dilalalus. Both species were cultivated under standardized conditions in a climatic chamber. Moreover, skins of the cultivated animals were collected and analyzed separately to examine a possible way of decontamination by moulting. To obtain enough sample material for each species, IS animals of the same age and size were pooled. For skin analysis, 10–12 skins were collected and pooled. The animals and their skins were dried, ground and digested in pure concentrated nitric acid using multiwave-assisted high-pressure digestion and. finally, analyzed by ICP-OES. Special emphasis was given to quality control: the reference materials Dorm-2, Dogfish Muscle (Squalus acanthias) and SRM 1577b Bovine Liver, were used to evaluate the whole analytical process including sample digestion. These reference materials of animal origin were selected to match the matrix of the samples as closely as possible. While concentrations of Fe. Mn and Zn were traced to both reference materials, the concentration of AI was checked by Dorm-2 only. Analyses of the elements in the reference materials were carried out using four ditTerent wavelengths for each element simultaneously. aiming at the determination of the best suited wavelength for each element. Analyses of woodlouse samples and their skins were finally carried out using the wavelengths with the highest sensitivities after absence of spectral interferences had been demonstrated.

Determination of trace amounts of nitrate in potassium chloride-extracts by HPLC with ultra-violet-detection

Abstract A method is presented that allows the determination of trace amounts of nitrate in 1 M potassium chloride extracts of environmental soil samples from Northern Europe. To avoid the interference of bromine ions on the evaluation of the nitrate peak, 50 µL of a 50 mg/L nitrate standard solution are added to a 5 mL soil extract. This makes sure that the overall nitrate concentration in the spike is >500 µg/L which allows an exact and reliable determination of the nitrate concentration. Analyses of the soil extracts are carried out by anion-exchange HPLC with ultra-violet detection at λ = 210 nm using 0.1 M KCl as eluent. Concentrations down to 25 µg/L of nitrate can be analyzed.