Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.     

Thermal biology of Tuta absoluta: demographic parameters and facultative diapause

Journal of Pest Science - The South American tomato pinworm, Tuta absoluta, (SATP) is now a devastating pest worldwide of crops in the family Solanaceae. Most prior studies of SATP’s thermal...     

Functional investigation of a QTL affecting resistance to Haemonchus contortus in sheep

This study reports a functional characterization of a limited segment (QTL) of sheep chromosome 12 associated with resistance to the abomasal nematode Haemonchus contortus. The first objective was to validate the identified QTL through the comparison of genetically susceptible (N) and resistant (R) sheep produced from Martinik × Romane back-cross sheep. The R and N genotype groups were then experimentally infected with 10 000 H. contortus larvae and measured for FEC (every three days from 18 to 30 days post-challenge), haematocrit, worm burden and fertility. Significant differences in FEC and haematocrit drop were found between R and N sheep. In addition, the female worms recovered from R sheep were less fecund. The second step of the characterization was to investigate functional mechanisms associated with the QTL, thanks to a gene expression analysis performed on the abomasal mucosa and the abomasal lymph node. The gene expression level of a candidate gene lying within the QTL region (PAPP-A2) was measured. In addition, putative interactions between the chromosome segment under study and the top ten differentially expressed genes between resistant MBB and susceptible RMN sheep highlighted in a previous microarray experiment were investigated. We found an induction of Th-2 related cytokine genes expression in the abomasal mucosa of R sheep. Down-regulation of the PAPP-A2 gene expression was observed between naïve and challenged sheep although no differential expression was recorded between challenged R and N sheep. The genotyping of this limited region should contribute to the ability to predict the intrinsic resistance level of sheep.     

Automation in stock-culture maintenance and juvenile separation of the mysid Mysidopsis bahia (molenock)

A batch system with subgravel filter for stock-culture maintenance of Mysidopsis bahia in artificial sea water is described. The feeding of mysids on Artemia is optimized through the cyclic pumping of brine shrimp instar I nauplii from a refrigerator to the stock-cultures. An easily constructed incubator-separator apparatus is described for the standardized harvesting of mysid juveniles to be used as test organisms.     

Surrogate Correlations and Near-Infrared Diffuse Reflectance Sensing of Trace Metal Content in Soils

Near-infrared diffuse reflectance sensing (NIRS) of soils has been the object of considerable interest and research in the last few years. This has been motivated by the prospect that this method seems to provide a cheap, convenient alternative to conventional, time-consuming methods for the measurement of a wide range of soil parameters. In particular, various authors have advocated that NIRS could be used to measure rapidly and non-destructively the concentration of trace metals in surface soils. Correlation analyses between NIRS spectra and trace metal concentration have yielded inconclusive results to date, suggesting that trace metal concentration may belong to a class of “tertiary” soil parameters, linked to NIRS spectra through “surrogate”, or indirect, correlations, involving some other primary or secondary parameter like clay or organic matter content, to which NIRS spectra are very sensitive. To assess the validity of this surrogate correlation hypothesis in the case of trace metals, experiments were carried out with soil samples varying only in the amount of trace metals they contain. Field-aged Hudson and Arkport soil pots spiked with Cu and Zn, freshly spiked samples of the same soils, and samples of a metalliferous peat soil from Western New York naturally rich in Cd and Zn were subjected to NIRS under laboratory conditions. Detailed analysis indicates that the NIR spectrum is sensitive to sample handling, including the orientation of the samples in the NIRS instrument, but that, at the same time, there is no discernable effect of the presence of trace metals on any part of the NIR spectrum. These results provide strong experimental support to the hypothesis of “surrogate” correlation for trace metals, and indicate that trace metals, even in severely contaminated soils, should not interfere with the NIR sensing of primary or secondary parameters, like organic matter content. Further work is needed to determine if this feature of NIR spectra extends to other soil chemical parameters.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Fungal biodegradation of a phenylurea herbicide,diuron : structure and toxicity of metabolites

Microbial degradation, organic synthesis and ecotoxicology were used to investigate the fate of diuron after spreading on soils. Quantitative biodegradation assays were performed with fungal strains, showing that diuron was degraded but not entirely mineralized. The modifications observed consisted in demethylation of the terminal nitrogen atom. The identified metabolites were synthesized in sufficient amounts to confirm their structures and determine their non‐target toxicity using four biotests. The two metabolites exhibited higher effects than parent diuron. This limited biodegradability and potential aquatic toxicity suggest that diuron is of higher environmental concern than previously recognized. © 2000 Society of Chemical Industry