Chemical and spectroscopic analyses ((13)C cross-polarization-magic angle spinning NMR and attenuated total reflection Fourier transform infrared spectroscopies) were carried out on the wood of Vitis vinifera cv. Sangiovese with brown-red discoloration and black streaks caused by esca disease. The analyses of the brown-red wood revealed the destruction of hemicelluloses and noncrystalline cellulose as well as modifications in the pectic and ligninic wood fractions. The pectic fraction consisted of carbohydrates associated with polyphenols. The lignin fraction exhibited only a few changes in the aromatic systems and a partial demethylation, and it appeared to be associated with condensed phenolic components probably arising from response polyphenols. The degradation of hemicelluloses and noncrystalline cellulose in brown-red wood, where the pathogens Phaeoacremonium aleophilum and Phaeomoniella chlamydospora prevail with respect to the other fungus Fomitiporia mediterranea, was consistent with reports on the degradative activity of such fungi in vitro carried out on model substrates. The observed alterations could also be attributed to the radical oxidation process caused by the oxidative response of defense itself triggered by infection, as suggested by the accumulation of postinfectional compounds. The analyses of wood tissue with black streaks showed less marked deterioration; here, an increase in pectic and phenolic substances, which probably accumulate in the xylem vessels as a response to the infection, was observed.
A simple and rapid HPLC method, using a high-density C18 column, has been developed for the quantitative analysis of fusaric and dehydrofusaric acids and their methyl esters in the methanol extract of lyophilised culture filtrates of species of Fusarium. The method has been used to determine the content of these metabolites in two strains of Fusarium oxysporum and in strains of F. nygamai and F. udum. Fusaric acid has been isolated and identified from a strain of F. udum for the first time.
The influence of different combinations of illumination and shaking on the growth dynamics, pathogenicity and toxin production of the fungus Fusarium oxysporum f. sp. orthoceras, a biocontrol agent of Orobanche cumana, was studied. The fastest biomass accumulation was obtained under shaking, with or without illumination, with the highest biomass obtained after 3-4 weeks of growth. The biological activity of chloroform extracts of the culture filtrate was characterised: it contained at least two main toxic metabolites that caused necrosis and wilting of various plants and led to mortality of germinating seeds of O. cernua, O. aegyptiaca, O. ramosa and O. cumana. The highest toxic activity of the chloroform extract was obtained under illumination without shaking after 3-4 weeks of growth. The two toxic metabolites were purified and identified as fusaric acid (FA) and 9,10-dehydrofusaric acid (DFA). Both FA and DFA production began in the first week of growth, increasing gradually to their maxima after 4 weeks. The highest level of pathogenic activity of the fungus was obtained after three or more weeks of fungal growth. It can be concluded that in order to produce high levels of toxin and pathogenic activity, the fungus should be grown under illumination without shaking for 4 weeks
Systemic acquired resistance elicitors, BTH and BABA, reduce rust penetration in pea through phytoalexins pathway but differing in their mode of action. It has been previously shown that rust (Uromyces pisi) infection can be reduced in pea (Pisum sativum) by exogenous applications of systemic acquired resistance elicitors such as BTH and BABA. This protection is known to be related with the induction of the phenolic pathway but the particular metabolites involved have not been determined yet. In this work, we tackled the changes induced in phytoalexin content by BTH and BABA treatments in the context of the resistance responses to pea rust. Detailed analysis through high-performance liquid chromatography (HPLC) showed qualitative and quantitative differences in the content, as well as in the distribution of phytoalexins. Thus, following BTH treatment, we observed an increase in scopoletin, pisatin and medicarpin contents in all, excreted, soluble and cell wall-bound fraction. This suggests fungal growth impairment by both direct toxic effect as well as plant cell wall reinforcement. The response mediated by BTH was genotype-dependent, since coumarin accumulation was observed only in the resistant genotype whereas treatment by BABA primed phytoalexin accumulation in both genotypes equally. Exogenous application to the leaves of scopoletin, medicarpin and pisatin lead to a reduction of the different fungal growth stages, confirming a role for these phytoalexins in BTH- and BABA-induced resistance against U. pisi hampering pre- and postpenetration fungal stages.
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