Induction of a Biopolyester Hydrolase (Cutinase) by Low Levels of Cutin Monomers in Fusarium solani f. sp. pisi

Abstract

Cutin hydrolysate induced the production of an extracellular cutinase by glucose-grown Fusarium solani f. sp. pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80 ,ug/ml, and saturation was attained at this level. Glucose was found to be a repressor of cutinase production. A radial immunodiffusion assay for cutinase was developed, and the induction of cutinase by cutin hydrolysate was confirmed by this direct assay. When cutinase was induced by cutin hydrolysate, exogenous labeled phenylalanine was incorporated into cutinase, which was shown to be the major (>70%) protein in the extracellular fluid. Induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and was stimulated (=100%) by cordycepin. Addition of cycloheximide with the inducer, or up to 12 h after the addition of the inducer, resulted in a nearly immediate cessation of cutinase production. Deoxyglucose, an inhibitor of proten glycosylation, inhibited the induction of cutinase by cutin hydrolysate. w-Hydroxy fatty acids were more effective in inducing cutinase than any of the other more polar acids of cutin. Experiments with derivatives and analogues of o-hydroxy C16 acid indicated that a free hydroxyl group at the wposition was the most important factor determining the cutinase-inducing activity. n-Aliphatic primary alcohols with 14 or more carbon atoms induced cutinase, and n-C16 was the most effective inducer. These results strongly suggest that the monomers function as the chemical signal which induces the extracellular hydrolase.

Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani f. sp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively)

“…Earlier studies with F. solani f. sp. pisi showed that induction of cutinase occurred in cultures supplied with 0.25 to 0.5 g l 31 powdered apple cutin [4] or smaller amounts (0.08 g l 31 ) of a hydrolysate prepared from the apple cutin or cutin monomers and their analogues such as a C 16 alcohol [19]. In our work we found that the C 16 alcohol, hexadecanol, was not as e¡ective as an inducer of cutinolytic activity in MPV strains as it was in MPVI strains.…”

“…The mating behaviour of the isolates was previously established by mating with known`tester' strains [18]. Although we were primarily interested in the two cucurbit pathogens (MPI and MPV), strains of the pea pathogen MPVI were included to provide a comparison since most of the knowledge on cutinase activity in N. haematococca has come from a study of that group [5,14,15,19]. A total of 11 MPI, four MPV and six MPVI isolates was studied.…”

“…Roux £asks containing 100 ml mineral medium (MM) [19] were inoculated with approximately 5U10 7 conidia obtained from 10^14-day-old PDA cultures grown at 25³C. As required, the culture medium was amended with glucose (1 g l 31 ), a C 16 alcohol (hexadecanol) (801 00 mg l 31 ) analogue of cutin fractions [19] or powdered cutin (80 mg l 31 to 2.5 g l 31 ). Flasks were incubated without shaking at 25³C for 10^13 days.…”

Effects of Commercial Fatty Acids on Cutinase Release by Ascochyta pisi

“…In the case oi F. solani f. sp. pisi, cutinase was also induced by cutin fatty acids (LIN and KOLATTUKUDY 1978, WoLOSHUK and KOLATTUKUDY 1986, PODILA et al 1988. In the present work, we used commercial fatty acids as carbon source for A. pisi, and showed that juniperic acid is a good inducer of the in vitro release of esterase activity by this fungus.…”

“…Cutinase is known to be induced in fungi by using cutin as sole source of carbon. Fungal cutinase was also induced by low concentrations of fatty acids extracted from hydrolyzed cutin (LIN and KOLATTUKUDY 1978, WOEOSHUK and KOLATTUKUDY 1986, PODILA et al 1988; its activity was severely inhibited by reagents directed towards active serine, such as diisopropyl fluorophosphate (DFPJ (PuRDY and KOLATTUKUDY 1975).…”