The fruit soft and juicy structure and low enzyme concentration make difficult the histochemical revelation of Polygalacturonase (EC 3.2.1.15),one of the enzymes involved in fruit softening. The detection of the enzyme activity become easier in grapevine berries at the first stages of Botrytis cinerea Pers. infection, due to the strong activity of fungal enzyme. Grapevine berries healthy and infected by Botrytis cinerea were examined using ethylene glycol cryostabilization followed by inclusion in hydrophilic resin (GMA: Technovit 7100, Kulzer). Slices of 2.5 micron thickness were incubated in a 0.5% solution of polygalacturonic acid at room temperature; enzyme activity was revealed by the reduction of tetrazolium salt(TNBT). PG activity was also quantified in grape extracts measuring the diffusion of the enzyme by staining with ruthenium red in agar plates containing Polygalacturonic acid. In healthy berries, the histological assay showed enzymatic activity only in the first cell layers of the pericarp, very near to cell wall. In infected berries, PG activity was more diffused and evident mainly in the areas infected by fungal hyphae. Moreover in this way it was also possible to identify the areas of fungal penetration. The observations by LM were confirmed by quantitative determination: juice extracted from healthy berries showed very low activity, lower than 0.05 mg/ml, while in infected berries the activity was higher than 5.0 mg/ml. The histochemical data obtained using the fluorochrome Nile Red confirmed the lytic activity of Botrytis cinerea hyphae on the wax layer of the berry.
Localization and quantitative determination of enzymatic activity in grapevine berries, healty and infected by Botrytis cinerea Pers.
Vallania R
2003
Abstract
The fruit soft and juicy structure and low enzyme concentration make difficult the histochemical revelation of Polygalacturonase (EC 3.2.1.15),one of the enzymes involved in fruit softening. The detection of the enzyme activity become easier in grapevine berries at the first stages of Botrytis cinerea Pers. infection, due to the strong activity of fungal enzyme. Grapevine berries healthy and infected by Botrytis cinerea were examined using ethylene glycol cryostabilization followed by inclusion in hydrophilic resin (GMA: Technovit 7100, Kulzer). Slices of 2.5 micron thickness were incubated in a 0.5% solution of polygalacturonic acid at room temperature; enzyme activity was revealed by the reduction of tetrazolium salt(TNBT). PG activity was also quantified in grape extracts measuring the diffusion of the enzyme by staining with ruthenium red in agar plates containing Polygalacturonic acid. In healthy berries, the histological assay showed enzymatic activity only in the first cell layers of the pericarp, very near to cell wall. In infected berries, PG activity was more diffused and evident mainly in the areas infected by fungal hyphae. Moreover in this way it was also possible to identify the areas of fungal penetration. The observations by LM were confirmed by quantitative determination: juice extracted from healthy berries showed very low activity, lower than 0.05 mg/ml, while in infected berries the activity was higher than 5.0 mg/ml. The histochemical data obtained using the fluorochrome Nile Red confirmed the lytic activity of Botrytis cinerea hyphae on the wax layer of the berry.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
