Alessandra Bonetti

Characterization of Some Italian Common Bean (Phaseolus Vulgaris L.) Landraces by RAPD, Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)3GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.

High-performance liquid chromatographic determination of sulfonylureas in soil and water

Abstract Isocratic and gradient conditions for the separation of four sulfonylurea herbicides, namely chlorsulfuron, metsulfuron, chlorimuron and thifensulfuron, by reversed-phase high-performance liquid chromatography (HPLC) on C6 and C18 columns were established. Liquid-liquid (LL) and solid-phase extraction (SPE) procedures for the extraction and concentration of the herbicides from water and soil samples were tested. LL and SPE recoveries, HPLC detection limits and repeatability and dependence of the capacity factor on mobile phase composition are discussed. Typical chromatograms are shown.

Separation and detection of herbicides in water by micellar electrokinetic capillary chromatography.

The herbicides linuron, metolachlor, atrazine and metsulfuron were analysed using micellar electrokinetic capillary chromatography (MECC) after a 1000-fold concentration step by solid-phase extraction (SPE). Recoveries ranged from 80 to 92%, depending on the concentration and the number of active substances in the sample. Furthermore, the hydrolysis products of metsulfuron were analysed by MECC and by gas chromatography-mass spectrometry (GC-MS). Representative MECC and GC-MS profiles are shown and the structures of the hydrolysis products are proposed on the basis of their chromatographic and mass spectra features. A tentative pathway for the degradation of metsulfuron is proposed.

Separation of sulfonylurea metabolites in water by capillary electrophoresis

Abstract The potential of capillary electrophoresis (CE) for the separation and detection of the metabolites of nine sulfonylurea herbicides in aqueous solution was evaluated. A relationship between the structure of the sulfonylureas tested and the metabolites formed was found: the non-o-benzene-substituted sulfonylurea rimsulfuron gave only one metabolite, whereas the other eight, o-benzene-substituted, sulfonylureas gave 4–6 metabolites. CE was confirmed to be a very efficient separation technique, suitable for the determination of sulfonylurea herbicides and their metabolites formed during hydrolysis.

Evaluation of Bt-toxin uptake by the non-target herbivore, Myzus persicae (Hemiptera: Aphididae), feeding on transgenic oilseed rape.

As consequence of the concern about the biosafety of genetically modified plants, biological and ecological studies are considered crucial for environmental risk assessment. Laboratory experiments were carried out in order to evaluate the transfer of the Cry1Ac Bt-toxin from a transgenic Bt-oilseed rape to a non-target pest, Myzus persicae Sulzer. Cry1Ac protein levels in plants and aphids were determined using a double sandwich enzyme-linked immunosorbent assay. Phloem sap from (Bt+) and (Bt-) oilseed rape plants was collected from leaves using a standard method of extraction in an EDTA buffer. Bt-toxin was present in phloem sap, with a mean concentration of 2.7 +/- 1.46 ppb, corresponding to a 24-fold lower level than in oilseed rape leaves. Toxin was also detected in aphid samples, with a mean concentration in the positive samples of 2.0 +/- 0.8 ppb. The evidence that Bt-toxin remains in herbivores, in this case an aphid, could be useful to clarify functional aspects linked to possible consequences of Bt-crops on food chains involving herbivore-natural enemy trophic systems. Further studies are needed in order to improve the knowledge on the functional aspects linked to the transfer of the Cry1Ac Bt-toxin from GM-oilseed rape to aphids and their possible consequence.

Urinary excretion of kaempferol from common beans (Phaseolus vulgaris L.) in humans.

The aim of this study was to assess kaempferol bioavailability in healthy humans, after bean (Phaseolus vulgaris L.) consumption through the monitoring of the excretion in relation to intake. In seven healthy subjects receiving kaempferol from cooked bean, maximum excretion of hydrolysed flavonol was obtained after 2–8 h. Intersexual variations in urinary excretion were found to be 6.10±5.50% and 5.40±5.40% of the kaempferol dose for male and female subjects, respectively. Although a 6.72-fold inter-individual variation between the highest and lowest excretion concentrations was found, all individuals exhibited similar excretion profiles. Moreover, a direct correlation between the percentage of kaempferol excreted and the body mass index of volunteers was observed with a correlation index equal to 0.80. All except two individuals exhibited a first peak of kaempferol excretion 2 h after ingestion. The study reveals information about inter-individual excretion capacity after kaempferol intake and that kaempferol can be used as a biomarker for flavonol consumption.

Direct determination of aliphatic acids in honey by coelectroosmotic capillary zone electrophoresis

SUMMARY Coelectroosmotic capillary zone electrophoresis has been employed for the rapid analysis of 8 aliphatic acids usually present in honey. Polycationic surfactants (hexadimethrine bromide (HDB), cetyltrimethylamonium bromide (CTAB) and tetradecyltrimethyllammonium bromide (TTAB)) have been added to the electrolyte buffer dynamically coating the inner surface of the capillary and causing a fast anodic electroosmotic flow, known as coelectroosmotic flow. The composition of the running buffer (50 mM sodium phosphate adjusted to pH 8 with 1.0 M sodium hydroxide, 25% 2-propanol and 0.001% HDB) and the instrumental parameters (voltage of −20 kV with a hydrodynamic injection for 15 s and an UV detection at 210 nm) have been optimized. Finally, the applicability of the method has been demonstrated for the determination of the aliphatic acids in several kinds of honey without any sample pretreatment.

Characterization of Italian populations of Lolium spp. resistant and susceptible to diclofop by inter simple sequence repeat

Abstract Three Italian Lolium weed populations, one susceptible and two resistant to diclofop, were characterized by the technique of inter simple sequence repeats (ISSR). The goal of this study was to taxonomically identify these Lolium populations as well as to evaluate evidence for introgression of ISSR fragments from Festuca and the potential role of this introgression in the diclofop response. ISSR analysis confirmed the genomic background of the weed populations to be consistent with that of Lolium. However, the great range of variation in ISSR banding patterns highlighted that the three ryegrass accessions are mixed populations made up of individuals resulting presumably from intrageneric and intergeneric hybridization in the Lolium–Festuca complex. Two Festuca genus-discriminating and 20 Festuca species-discriminating ISSR markers were screened among all the three ryegrass populations. The resistant Tuscania population carried the highest percentage of Festuca genome (16.8%) followed by the resistant Roma (13.6%) and susceptible Vetralla (7.6%) populations. On the basis of these data some influence of Festuca genome in diclofop resistance levels of studied ryegrass populations could be hypothesized. Nomenclature: Diclofop; ryegrass, Lolium LOL; fescue, Festuca FES.

Genetic structure and mating system of Italian Xanthium strumarium complex

Abstract The genetic variation at 12 isozyme loci was investigated in the three species (Xanthium italicum, X. strumarium, and X. orientale) forming a X. strumarium complex in Italy. Very little variation was found within species at the loci studied in contrast to the considerable interspecies genetic differentiation at several loci. The gene differentiation between species was ranged from 61 to 91%. The observed genetic structure of the X. strumarium complex was consistent with that found for predominantly autogamous species. The values of maximum outcrossing rates estimated in original sampling sites and in a field test ranged from 8 to 17%, confirming previous observations that Xanthium species are predominantly self-pollinated. Gene duplications were evident in the three Xanthium species because of their likely polyploid origin. The percentage of duplicate loci exhibiting “fixed heterozygosity” was 25, 25, and 16% in X. italicum, X. strumarium, and X. orientale, respectively. Data presented supported that both mating system and ploidy level were fundamental features in adaptation process of investigated Xanthium species. Some evidence suggested that polyploidization occurred before speciation of X. italicum, X. strumarium, and X. orientale. As a consequence, a common ancestral progenitor could be postulated for the three species. During geographical adaptation, the three species fixed alternative alleles in some loci, and the process was favored by the predominantly autogamous mating system. On the contrary, fixed heterozygosity in duplicated loci allowed maintenance of a sufficient level of gene diversity in the three Xanthium species to ensure wide adaptability in different microhabitats (i.e., abandoned land, roadsides, and field crops) and to avoid the negative effect of inbreeding depression. Nomenclature: Xanthium strumarium L. complex XANST.