Screening RAPD primers for molecular taxonomy and cultivar fingerprinting in the genus Actinidia

Summary Eighty ten-base long arbitrary primers were tested for PCR-based DNA amplification of three species of the genus Actinidia (A. deliciosa the kiwifruit, A. chinensis, and A. kolomikta), with the aim of screening species-specific and genotype-specific markers.Of the 80 primers tested, 30 gave an average of 3.5 bands which were monomorphic within one or two species and absent in the remaining one(s), thus resulting in useful markers for taxonomic and phylogenetic purposes. None of the primers tested produced bands linked to sex. Twenty primers out of the twenty-five selected from a preliminary screening showed high levels of polymorphism, producing two to eleven patterns each from the 13 kiwifruit cultivars examined.We found the Stoffel fragment and the Taq polymerase were both suitable for RAPD analysis, the most noticeable difference being the smaller size of fragments (0.4–1.2 kb) produced by the former in comparison to the latter (1.0–3.4 kb). We tested also three different annealing temperatures (35, 37, and 39° C) and found the intermediate one best for number of amplified bands and reproducibility of results.Abbreviations 2-BE 2-butoxyethanol - CTAB hexadecyltrimethylammonium bromide - MAS Marker-Assisted-Selection - PCR Polymerase Chain Reaction - RAPD Random Amplified Polymorphic DNA - RFLPs Restriction Fragment Length Polymorphisms     

Molecular characerization of the interaction between the fungal pathogen Cladosporium fulvum and tomato

The fungus Cladosprium fulvum is a biotrophic leaf pathogen of tomato. The fungus develops in the intercellular space without forming specialized feeding structures and does not affect the leaf tissue. The outcome of the C. fulvum-tomato interaction can be described by the gene-for-gene model. Failure of infection is expressed by a hypersensitive response. Two fungal proteins, ECP1 and ECP2, have been isolated and their corresponding genes have been cloned. In a compatible interaction including many physiological races ECP1 and ECP2 are highly produced and a role in pathogenicity is suggestive. The ecp1 gene shows some homology with tumor necrosis factor receptors (TNFRs) while the ecp2 gene shows no homology with sequences known in data bases. However, disruption of one of the two genes showed no reduced pathogenicity of the fungus. Two race-specific elicitors, AVR4 and AVR9, have been isolated and their corresponding genes have been cloned. The avirulence genes Avr4 and Avr9 are only present in C. fulvum avirulent on Cf-4 and Cf-9 cultivars, respectively. The expression of these two genes is, like the expression of the ecp genes, highly induced when the fungus grows in planta. Disruption of the Avr9 gene in wild type avirulent races leads to virulence on tomato genotypes carrying the complementary resistance gene Cf-9. A single base-pair change in the avirulence gene Avr4 leads to virulence on tomato genotypes carrying the Cf-4 resistance gene. Isolation, characterization and possible function of ECP1, ECP2, AVR4, and AVR9 will be discussed.     

Vitamin C,provitamin A carotenoids,and other carotenoids in high-pressurized orange juice during refrigerated storage

Vitamin C, provitamin A carotenoids, and other carotenoids were measured in freshly squeezed juices from oranges (Citrus sinensis L. var. Valencia late) that were subjected to high-pressure (HP) treatment. Also, the stability of these compounds was studied during refrigerated storage at 4 degrees C. HP treatment is an alternative to heat preservation methods for foods; therefore, it is essential to assess the impact of HP on bioactive compounds. Several processes that combine HP treatment with heat treatment for various time periods were assayed: T0, fresh juice (without treatment); T1, 100 MPa/60 degrees C/5 min; T2, 350 MPa/30 degrees C/2.5 min; T3, 400 MPa/40 degrees C/1 min. Fresh and treated samples were kept refrigerated (4 degrees C) over 10 days. After application of HP and during the refrigeration period, the qualitative and quantitative determination of vitamin C, provitamin A carotenoids (beta- and alpha-carotene; beta- and alpha-cryptoxanthin), and the xanthophylls zeaxanthin and lutein was achieved by high-performance liquid chromatography. T1 and T3 juices showed a decrease in ascorbic acid and total vitamin C just after HP treatment (D0) compared with T0 juices. On the contrary, T2 juices, just after HP treatment (D0), had the same levels of both compounds compared to untreated juices. T1, T2, and T3 treatments led to an increase in the extraction of carotenoids and provitamin A carotenoids. Total carotenoid content after the 10-day refrigerated storage period resulted in no significant quantitative changes in T1 juices, whereas in T2 and T3 juices small losses were found at the end of the storage period (20.56 and 9.16%, respectively). These losses could be influenced by the depleted protection of vitamin C toward carotenoid oxidation during the same period. A similar trend was found in provitamin A carotenoids for the different treated juices.     

Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.)

Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fractions of wheat grain. It was found that the aleurone content of these fractions was highly correlated with the antioxidant capacity of the fractions (r = 0.96, p < 0.0001). Ferulic acid appeared to be the major contributor to the antioxidant capacity in fractions with higher antioxidant capacity. The contribution of protein was rather limited. It was concluded that the antioxidant potency of wheat grain fractions is predominantly determined by aleurone content, which can be attributed to the presence of relatively large amounts of phenolic compounds, primarily ferulic acid.     

Bioactive constituent production in St. John's Wort in vitro hairy roots. Regenerated plant lines

A wild strain of Agrobacterium rhizogenes was used to regenerate twelve in vitro plant lines from different hairy roots of H. perforatum (St. John's Wort). The production of the main bioactive constituents was observed even though their yields varied in the different plant lines. Two lines were selected for the hyperoside production (4.9-4.6 mg/gdw) while nine were characterized by significant yields of chlorogenic acid (ranged from 0.47 to 1.09 mg/gdw). Furthermore, one out of twelve lines showed a 10-fold higher hypericin content (0.25 mg/gdw) than that reported for the in vitro shoots in the literature. Morphological and phytochemical features were determined in order to select H. perforatum genotypes enriched in valuable bioactive compounds.     

In vitro bioaccessibility of soil-bound polycyclic aromatic hydrocarbons in successive digestive compartments in cows

Ruminants, which have a central place in the food chain, ingest soil that may contain pollutants. The bioaccessibility of three different polycyclic aromatic hydrocarbon compounds from soil was studied using an in vitro model based on the digestive tract of cows. For this purpose, pasture soil was spiked with (14)C radio-labeled compounds, aged, and then exposed to conditions which simulated the digestive compartments of the rumen, abomasum, and intestines. Our results show that aging generally reduced the bioaccessibility of all the compounds tested. Total bioaccessibilty in the first digestive compartment, i.e., the rumen, depended on the considered compound: elevated for phenanthrene (17-24%), moderate for pyrene (6.6-8.1%), and low for benzo[ a]pyrene (2.3-3.6%). Bioaccessibility was very low in abomasal acidity (generally <2%) and intestinal colloids (<8%). The liquid phases of intestinal medium successfully extracted compounds from freshly contaminated soil (25-28%), but the bioaccessibilty dropped markedly after aging (17% for phenanthrene and <9% for the more lipophylic compounds). Total bioaccessibilty in this in vitro model ranged from 11% for benzo[ a]pyrene in aged soil to 58% for phenanthrene in freshly contaminated soil, and the bioaccessibility of this latter compound was always higher compared to pyrene or benzo[ a]pyrene. Residual soil contained around half of the initial load, the highest residual levels being of benzo[ a]pyrene, which confirms the observed bioaccessibility.     

Treatment of cereal products with a tailored preparation of trichoderma enzymes increases the amount of soluble dietary fiber

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.     

Oxidative degradation of lipids during mashing

Although hardly any polyunsaturated fatty acids (PUFAs) are present in the endproduct, the ingredients used for the production of beer contain a high concentration of PUFAs, such as linolic and linolenic acid. These compounds are readily oxidized, resulting in the formation of lipid-derived products that reduce the taste and quality of beer enormously. During mashing relatively high amounts of PUFAs are exposed to atmospheric oxygen at a relatively high temperature. This makes mashing a critical step in the brewing process with regard to the formation of lipid-derived off-taste products. F1 phytoprostane (PPF1) changes in antioxidant capacity and monohydroxy fatty acids (OH-FAs) were used as markers for the detection of oxidative damage to fatty acids during mashing. The pattern of OH-FA formation indicates that enzymatic oxidation of PUFAs is more important than nonenzymatic oxidation during the mashing process. Nevertheless, substantial nonenzymatic radical formation is evident from the increase of specific OH-FAs and PPF1s. It was found that a low oxygen tension reduces oxidative damage and gives a high antioxidant capacity of the mashing mixture. This indicates that mashing should be done under low oxygen pressure.     

Effects of pig slurry application on soils and soil humic acids

The effect of three annually consecutive additions of pig slurry at two rates (90 and 150 m3 x ha(-1) x year(-1) on soils and soil humic acids (HAs) was investigated in a field experiment under semiarid conditions. Soils and pig slurries were analyzed by standard methods. The HAs were isolated from soils and pig slurry by a conventional procedure based on alkaline extraction, acidic precipitation to pH 1, purification by repeated alkaline dissolutions and acidic precipitations, water washing, dialysis, and final freeze-drying. The HAs obtained were analyzed for elemental (C, H, N, S, and O) and acidic functional group (carboxylic and phenolic) composition, and by UV-vis, FT-IR, fluorescence, and ESR spectroscopies. With respect to the control soil, the pig slurry amended soils had greater pH and electrical conductivity, slightly larger total N content, and smaller values of C/N ratio. A decrease of total organic C was observed only in soils amended for 2 and 3 years at the higher slurry rate. With respect to control soil HA, pig slurry HA was characterized by larger contents of S- and N-containing groups, smaller acidic functional group and organic free radical contents, a prevalent aliphatic character, extended molecular heterogeneity, and smaller aromatic polycondensation and humification degrees. Amendment with pig slurry HA determines a number of modifications in soil HAs, including increase of C, S, and COOH contents, C/N ratios, and aliphaticity and decrease of extraction yields and N, O, phenolic OH, and organic free radical contents. These effects are generally more evident after the first year of slurry application and tend to disappear with increasing number of treatments. Most probably, over the years the slightly humified slurry HA is mineralized through extended microbial oxidation, whereas only the most recalcitrant components, such as S-containing, phenolic, and aliphatic structures, are partially accumulated by incorporation into soil HA.