Purification and characterization of pectic enzymes from two races of Fusarium oxysporum f. sp. ciceri differing in virulence to chickpea ( Cicer arietinum L.)

Abstract

Polygalacturonases (PG) and pectate lyases (PL) produced in vitro by races 0 and 5 of Fusarium oxysporum f. sp. ciceri have been purified by gel filtration followed by anion-exchange chromatography and, in some instances, chromatofocusing. Race 0, the least virulent race, produced three PG forms, designated PG I 0, PG II 0 and PG III 0, and one PL form, designated PL 0, whereas race 5, the more virulent race, produced only one PG form, designated PG 5, and two PL forms, designated PL I 5 and PL II 5. The molecular weights of the enzymes were estimated to be 44000 for PG I 0 and PG II 0 76000 for PG III 0 and PG 5, 25000 for PL 0, and 37000 for PL I 5 and PL II 5. By their mode of action, PG I 0 and PG II 0 were endo-enzymes, PG III 0 and PG 5 were exo-enzymes, and the three lyases were endo-enzymes showing, in contrast to the endo-PGs, very low or null activity on oligogalacturonides shorter than pentamers. With polygalacturonic acid as substrate, the values of pH optimum were 4·5 for all four PG forms, 9·5 for PL 0, and 10·5 for PL I 5 and PL II 5; the PL forms showed an absolute Ca 2+ requirement for activity. PG I 0, PL 0 and PL I 5 were cationic proteins with alkaline isoelectric points, whereas PG II 0, PG III 0, PG 5, and PL II 5 were anionic proteins with acidic isoelectric points. All the purified pectic enzymes were able to degrade chickpea cell walls although the degradative activity of the exo-PG 5 and the anionic endo-PL II 5 was notably lower than that of the remaining PG and PL forms. Degradability of wall preparations without ionically bound proteins was not related to host resistance or susceptibility. In contrast, walls containing ionically bound proteins from the chickpea susceptible cv. PV-24 were more extensively degraded by the various PG and PL forms than were similar preparations from the resistant cv. WR-315.