DNA: a programmable force sensor

Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNA duplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single-base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.     

Experiences with Novel Approaches in Earthworm Testing Alternatives (7 pp)

Goal, Scope and Background   The earthworm avoidance test is a sensitive screening test. Currently, two test designs, a two-chamber system and a six-chamber system, are under standardization. In the scope of the present study, the two test systems are compared. To assess the results, two procedures are applied, which are based on a threshold value and a statistical method. Moreover, the sensitivity of the avoidance test is compared with the sensitivity of the reproduction test. Methods   The avoidance behaviour of E. fetida towards five chemicals (Cd, Cu, PCP, TBT, TNT) was tested in a sandy and a loamy soil. The ecotoxicological test was performed according to the draft guideline ISO/DIS 17512-1. The results were compared with the number of offspring determined in the reproduction test carried out according to ISO 11268-2. Results and Discussion   The results demonstrate that the avoidance behaviour towards organic chemicals and heavy metals is a suitable screening method showing first tendencies of a chemical's effects on the habitat function of soils. Effects caused by chemical substances become visible at low concentrations and within short test periods. The sensitivity of the reproduction test and the avoidance test is principally comparable; in some cases, the avoidance test showed more sensitive reactions. The dose-response-relationships were more pronounced in the two-chamber test than in the six-chamber-system. Recommendation and Outlook   The two-chamber-system proved to be more feasible than the six-chamber-system. As the sensitivity of the avoidance test and the reproduction test is comparable, the avoidance test can be considered as a suitable screening test as well. A possible field of application may be the selection of soil samples for which the reproduction assay seems necessary. To assess contaminated or remediated soils, the earthworm reproduction test is recommended as an indicator for the habitat function of the soils. To reduce costs, the labour intensive reproduction test could be largely replaced by the avoidance test in performing the earth-worm reproduction test only for relevant samples. Before the Draft International Standard of the guideline will be adopted as an ISO Guideline, there will be sufficient time left to gain further international experience required to finalize the method.     

Effect of essential oils on the growth of Fusarium verticillioides and fumonisin contamination in corn

Essential oils extracted by hydrodistillation from local plants in Benin, western Africa, and oil from seeds of the neem tree (Azadirachta indica) were evaluated in vitro and in vivo for their efficacy against Fusarium verticillioides infection and fumonisin contamination. Fumonisin in corn was quantified using a fluorometer and the Vicam method. Oils from Cymbopogon citratus, Ocimum basilicum, and Ocimum gratissimum were the most effective in vitro, completely inhibiting the growth of F. verticillioides at lower concentrations over 21 days of incubation. These oils reduced the incidence of F. verticillioides in corn and totally inhibited fungal growth at concentrations of 8, 6.4, and 4.8 microL/g, respectively, over 21 days. At the concentration of 4.8 microL/g, these oils did not affect significantly fumonisin production. However, a marked reduction of fumonisin level was observed in corn stored in closed conditions. The oils adversely affected kernel germination at 4.8 microL/g and therefore cannot be recommended for controlling F. verticillioides on stored corn used as seeds, when used at this concentration. The oil of neem seeds showed no inhibitory effect but rather accelerated the growth of F. verticillioides.     

Leaf Waxes of Cassava (Manihot Esculenta Crantz) in Relation to Ecozone and Resistance to Xanthomonas Blight

Summary To elucidate the role of leaf surface structures as first barriers to confer resistance to bacterial blight, leaf stomata and their occlusion with leaf waxes were studied in cassava genotypes. For the first time, cassava leaf waxes were quantitatively and qualitatively analysed. Comparing the susceptible and resistant standard genotypes BEN86052 and TMS30572, respectively, the total quantity of triterpenes was significantly higher in the resistant genotype, grown in three ecozones of Benin. In cuticular leaf waxes of seven cassava genotypes the triterpenes beta amyrins, epi-taraxerol, taraxerone and taraxerol were dominant constituents across genotypes, and alkanes (C₂₅-C₃₃) and acids (C₃₀ and C₃₂) occurred in minor concentrations. Comparing seven genotypes, no clear relation between resistance or ecozones and total quantities of the major wax constituents was observed. Only the highly resistant genotype TMS30572 showed high triterpene levels irrespective of ecozone. Scanning electron-microscopy revealed a regular distribution of waxes at the abaxial leaf surface, covering and occluding stomatal pores of susceptible and resistant genotypes, while on the adaxial leaf surface waxes were in form of crystalloids and did not occlude the stomata. The number of stomata on the abaxial surfaces was about 7–11 fold higher than on the adaxial surfaces, where stomata were located along the midrib and major veins. No significant differences in stomata number were observed between genotypes varying in resistance to bacterial blight. It is suggested, that stomata on the adaxial surface might be portals of entry for the bacteria.     

Could EST-based markers be used for the marker-assisted selection of drought tolerant barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) lines?

To improve our knowledge on the genetic control of drought tolerance, the Oregon Wolfe Barleys (OWB), considered as a reference population in genetic mapping, were subjected to various types of water deficit. Overall, when investigating numerous environments and replications, 40 QTLs were identified in three developmental stages. Based on these loci five QTL clusters were separated, which affect various drought-related traits in at least two developmental stages. Several candidate genes were identified for each QTL cluster using an expressed sequence tag (EST)-based map with high marker density. The putative role of the candidates in drought tolerance is discussed. The phenotypic effect of three of the five candidate genes was also tested on 39 barley landraces and cultivars and a significant relationship was found between the allelic composition of these genes and yield production under stress conditions. This study presents a relevant example of the use of reliable QTL data in the candidate gene approach, while also demonstrating how the results could be practically utilized in marker-assisted selection (MAS).