Characterization of an endopolygalacturonase produced by the apple scab fungus, Venturia inaequalis

Abstract

Secretion of a constitutive endopectinase was detected in cultures of Venturia inaequalis grown in complex and defined media. Pectinylase or pectinesterase activity was not present. The attainable level of extracellular pectinase was present after 5–10 d post inoculation, when only about 5–10% of mycelial mass had developed. Time course culture experiments with conidial and mycelial inoculum indicated that enzyme secretion did not depend on physiological effects within or shortly after conidial germination. Cellulosic substrates did not affect pectinase production. Catabolite repression or repression by glucose was not detected and there was no stimulation of enzyme production in media supplemented with pectic substrates. Inhibition of enzyme activity by galacturonic acid was not detected. Nineteen isolates of V. inaequalis were tested, all of which displayed constitutive pectinase production. All isolates were able to grow in the defined media with galacturonic acid or the pectic substrates as the sole carbon source. Mycelial pectinolytic activity was only detectable in substantial quantities when the fungus was grown in media supplemented with 1% pectin. The pectinase preparations displayed a long-term stability at temperatures up to 20°C and within a pH-range of 5–7·5. Activity was very low at 5 and 10° and there was a distinct increase at 15 and 20°. Enzyme activity was highest at pH 4 and decreased with increasing pH. Time course viscometric assays and hplc analyses of liberated galacturonic acid indicated an endo-type degradation of pectin and polygalacturonic acid. Di- and trigalacturonic acid were released from pectin with high enzyme concentration and long incubation. Pectin with a degree of esterification of 93% showed reduced degradability. Pectinase preparations had a macerating activity on solvent extracted apple leaves and caused galacturonic acid release. Isoelectric focusing followed by a zymogram technique revealed a single activity band at pI 10. The pectinase preparations from culture filtrates or mycelia of 19 isolates did not show any differences in characteristics of degradative activity or the activity band detected after isoelectric focusing.