Studies on the phloroglucinol–HCl reactive material produced by squash fruit elicited with pectinase: Isolation using hydrolytic enzymes and release of p -coumaryl aldehyde by water reflux

Pathological tissues from a variety of plants turn red when treated with the histochemical reagent phloroglucinol (PG)–HCl. This induced PG–HCl reacting material has been termed both wound gum and, more recently, induced lignin or lignin-like material. We are exploring alternative approaches to better identify this induced material. In this report, we describe methods for purifying the PG–HCl reactive material from solvent-washed tissue preparations and measuring the amount of p -coumaryl aldehyde released from the purified material by boiling in water. Acorn squash fruit wall tissue was sliced, sprayed with pectinase to elicit the formation of PG–HCl reactive material and incubated up to 72 h at 27°C. Sampled tissue was washed extensively with water and organic solvents. Insoluble residues were treated with cell wall degrading enzymes to remove cell wall materials, and dimethyl sulfoxide was used to dissolve starch. Yields of residual material increased from 7.5% in the time zero samples to 29.7% after 72 h. Refluxing the purified material in water released <6 μ g p -coumaryl aldehyde g⁻¹ from preparations of tissue at time zero and 8100 μ g p -coumaryl aldehyde g⁻¹ from samples incubated for 48 h. These results suggest that the function of the material is to sequester phytoalexins, but are not consistent with the material being lignin. Published by Elsevier Science Ltd.     

Influence of agronomic factors on yield and quality of hemp (Cannabis sativa L.) fibre and implication for an innovative production system

Research and development of an innovative production system for hemp (Cannabis sativa L.) fibre for textile use requires the integration of multidisciplinary knowledge from cultivation technique to realization of end products. Research was carried out to study the effect of the agronomic factors cultivation year (2003–2004), genotype (Futura 75 and Tiborszallasi), plant population (120, 240 and 360 plants m⁻²) and harvesting time (beginning and full flowering) on fibre yield and quality in the whole hemp stem, and in the basal and apical stem portions separately. The study of separate stem portions was done to determine the effect on fibre quality of an innovative harvesting and processing system in which hemp stems are cut in two portions of approximately 1 m at harvest to enable processing on modern flax scutching lines.     

Molecular diversity in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates from common bean fields in Brazil

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State – UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli.     

A PCR-based assay for the detection and identification of <Emphasis Type="Italic">Pyrenochaeta lycopersici</Emphasis>

The isolation of Pyrenochaeta lycopersici, causal agent of corky root of tomato, is difficult because of its slow growth and poor sporulation. Identification is complicated due the existence of two morphologically similar forms, Types 1 and 2, that differ in several physiological and molecular features. For the rapid and unambiguous identification of isolates, two oligonucleotide primer pairs were designed using ITS region sequences. Specific PCR products of 147 and 209 bp were obtained for isolates of Type 1 and Type 2, respectively. Specificity of both primer pairs was verified using several fungal and bacterial species. As little as 0.7 pg of target DNA could be detected with the protocol. A nested PCR procedure was necessary for the detection of the fungus in plant tissue. This technique will be of use in epidemiological studies and in the implementation of control strategies.     

Relationships between biological and physico-chemical soil characteristics in a mediterranean agricultural area

Fourteen agricultural soils from various areas of Tuscany were characterized by a range of measurements indicative of soil biological activity. The objective of our research was to identify soil parameters suitable as indicators for evaluating their quality. In general, enzyme activities were found to vary widely, with the highest activity for each enzyme being distributed among only five of the 14 soils studied. The narrowest range (14-fold) in enzyme activities for the various soils was observed for catalase and the widest range (577-fold) for g -glucosidase. Biomass C and, among the measured enzyme activities, amylase, were well correlated with total organic carbon, total N, cation and anion exchange capacity. Positive correlations were found between the maximum water holding capacity and dehydrogenase, amylase, biomass C, FDA hydrolytic activity, the biological index of fertility and the enzyme activity number, so showing that soil moisture may play an important role in affecting soil biological characteristics. No significant correlations were observed among the soil enzymes themselves. The FDA hydrolytic activity appeared to be the index most related with the other biological characteristics tested in this study and, for this reason, can be considered the most effective index for putting in evidence relationships existing between the different biological characteristics in the soils investigated.     

Molecular characterization of a compost and its water-soluble fractions

A sequential chemical fractionation was applied to a compost, with its dissolved organic matter (DOM) extracted in water and separated in hydrophilic (HiDOM) and hydrophobic (HoDOM) components and a water extract, following oxidation of compost suspension with an oxygen flux (TEA). The components sequentially isolated by mild extractions and hydrolyses as structurally unbound (SU), weakly bound (WB), and strongly bound (SB) to the matrix of the bulk compost and its water-soluble fractions were identified in their molecular structure. The bulk compost was rich with components derived from both aromatic (phenolic compounds) and aliphatic (long-chain fatty acids, hydroxy acids, diacids, and alcohols) structures of suberins, whereas components derived from cutins were especially extracted from TEA, HoDOM, and HiDOM. The TEA sample also yielded a significant amount of oxidized products that was dominated by dehydroabietic acids. The fractionation sequence highlighted the different intermolecular interactions that bound the isolated molecular components to the compost complex matrix. While a significant part of the bulk compost was still present as a solid residue at the end of the sequential fractionation, all water-soluble fractions were almost completely hydrolyzed. These results indicate that the water-soluble components of compost may be readily separated from the compost matrix and contribute to the environmental dynamics of natural organic matter.     

Hydrolysis of wine aroma precursors during malolactic fermentation with four commercial starter cultures of Oenococcus oeni

The ability of four commercial preparations of Oenococcus oeni lactic acid bacteria (EQ 54, Lalvin OSU, Uvaferm Alpha, and Lalvin 31) to hydrolyze wine aroma precursors was evaluated by measuring the concentration of free and bound aroma compounds at the end of malolactic fermentation carried out in model wines containing a mixture of glycosides extracted from Muscat wine. At pH 3.4 there was a decrease in glycosylated compounds matched by a concomitant increase in free forms in all starter cultures tested. When malolactic fermentation was carried out at pH 3.2, a significant decrease in the ability to hydrolyze aroma precursors was observed for two of the cultures tested (Uvaferm Alpha and Lalvin 31). Large differences in the extent of hydrolysis and in the specificity of this activity toward specific aroma precursors were observed and appeared to be related to the chemical structure of the aglycon as well as to individual characteristics of each starter culture. The amounts of glycosylated aroma compounds released during malolactic fermentation suggest that O. oeni can alter the sensory characteristics of wine through the hydrolysis of aroma precursors.     

Evaluation of ‘di Romagna’ and ‘Frenchgrey’ shallots for quality characters connected to bulb colourand volatile oil content and composition

Local landraces of shallot from the Romagna region(Northern Italy) and North-western France, called Scalogno di Romagna and French grey,respectively, have recently been evaluated for morphological,biochemical and molecular characters. These populations appeared very different from common shallots and onions, so were re-classified as Allium oschaninii O.Fedtsch., whereas almost no variability was observed within these types. Four di Romagna and three French accessions were grown in Italy, and further evaluated for quality characters such asbulb colour and volatile oil content and composition. Colour was measured by means of a tristimulus colorimeter. Volatile oil was extracted by hydrodistillation and its components separated and identified by GC/MS. Colour parameters differentiated thedi Romagna from the French greyaccessions. Essential oil composition differed from what has been reported for most other Alliums, by a lower amount of1-propenyl- substituents. No individual oil component allowed a good differentiation of the two geographic origins.However, the French and di Romagna accessions were perfectly separated by the discriminant analysis of oil composition.Minor 1-propenyl- components seemed more critical for discrimination. The shallot types examined seemed biochemically differentiatedfrom Allium cepa. Variation in the examined quality characters was detected both between the two provenances and among the accessions of common geographic origin.     

Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2

Dietary phenolic antioxidants have been shown to prevent LDL modifications mediated by several physiologic oxidants including peroxynitrite. However, more recent data demonstrated that CO(2) affected the fate of peroxynitrite in biological fluids and significantly reduced peroxynitrite scavenging by polyphenols, raising doubts concerning their antioxidant activity. We found that the oxidation of LDL lipids mediated by peroxynitrite decreased in the presence of bicarbonate, while Trp oxidation and 3-nitroTyr formation increased, suggesting a redirection of peroxynitrite reactivity toward the protein moiety. We therefore evaluated the protective activity of some phenolic antioxidants (quercetin, oleuropein, resveratrol, (+)-catechin, (-)-epicatechin, tyrosol, alpha- and gamma-tocopherol, ascorbate) on peroxynitrite-mediated oxidation of LDL aromatic residues. Some of these phenols protected LDL Trp from oxidation better than ascorbate or alpha-tocopherol, although protection at 100 microM did not exceed 30-40%. However, the same phenolic antioxidants were more active in inhibiting 3-nitroTyr formation and those with a catechin structure provided significant protection (IC(50%) 40-50 microM). Red wine, a polyphenol-rich beverage, showed a protective effect comparable to that of the most active phenolic antioxidants. Direct EPR studies showed that bicarbonate significantly increased the peroxynitrite-dependent formation of O-semiquinone radicals in red wine, supporting the hypothesis that polyphenols are efficient scavengers of radicals formed by peroxynitrite/CO(2). Ascorbate was a poor inhibitor of peroxynitrite/CO(2)-induced LDL tyrosine nitration, but the simultaneous addition to the most active polyphenols halved their IC(50%). In conclusion, although cooperation with other antioxidants can further decrease the IC(50%) of polyphenolics, as demonstrated for ascorbate, their antioxidant activity appears to occur at concentrations at least 1 order of magnitude higher than their bioavailability.     

Retention of coumaric acid by soil and its colloidal components

In this study of the behavior of coumaric acid added to soil, the disappearance of the acid was found to be due to adsorption plus microbial degradation, and was influenced by the concentration and contact time. Adsorption experiments set up with soils varying widely in their chemical and physical properties, showed that the Freundlich isotherm fits the data quite well and that among the different soil factors, only the pH was closely and negatively correlated with coumaric acid adsorption. Approximately at pH > 7 no adsorption occurred, perhaps due to the repulsion between the negatively charged soil colloids and the dissociated acidic groups of coumaric acid. Adsorption experiments carried out with different adsorbents showed that the hydroxy-Fe compound was the most effective in retaining coumaric acid, followed by humic acid, illite, kaolinite and vermiculite, in this order.