Production of polygalacturonase and macerating enzymes by Phoma menthae

The fungus Phoma menthae Strasser causes black lesions and cankers on stems and rhizomes of Mentha piperita L. The purpose of this thesis was to: 1) study the production of polygalacturonase (PG) and macerating enzymes (ME) by P. menthae in vivo and in vitro, 2) study the effects of phenolic compounds on the activity of PG and ME produced by P. menthae in vivo, and in vitro, 3) compare polyphenol oxidase acitvities in healthy and diseased peppermint rhizome tissues. PG activity was measured by both viscosity reduction and reducing groups assays, using sodium polypectate (NaPP) as substrate. Fungal PG was first detected in cultures two days old. PG activity in filtrates was highest in cultures five and 23 days old, as determined by reducing groups assay. PG activity, as determined by viscosity reduction assay, was highest in cultures seven days old and remained the same for 28 more days. Growth (dry wt.) of the fungus was highest after 13 days and then gradually declined. In young cultures (two and three days old) pectin was a better substrate than NaPP for production of PG by P. menthae. In older cultures (five to 13 days old), NaPP was a better substrate than pectin for production of PG. PG activities in both cases were measured by reducing groups assay. The hydrolysis curve of NaPP produced by PG from culture filtrate differed from that producted by diseased rhizomes extract, as determined by viscosity reduction assay. When determined by reducing groups assay, hydrolysis curves of NaPP produced by culture filtrate and diseased rhizomes extract did not differ. Therefore, differences in PG produced in vitro and in vivo were revealed only by the viscosity reduction assay method; the reducing groups assay did not reveal this difference. ME activities were highest in filtrates from cultures ten days old. ME activities were determined by an optical density method, using potato discs as substrate. This method accurately detected differences in potato tissue maceration. Culture filtrate of P. menthae macerated peppermint rhizomes. Young peppermint rhizome sections were more susceptible to maceration than older sections. Colored phenolic compounds, were released from the macerated rhizomes and the amount released was correlated with degree of maceration. Among nine phenolic compounds incubated for 15 hrs with culture filtrate of P. menthae (containing PG and ME), tannic acid, 1,2 naphthoquinone and digallic acid inhibited PG and ME, as determined by viscosity reduction and the action on potato discs, respectively. Inhibition of PG and ME by these phenolics indicates that PG plays an important role in the maceration process. PG activity, as determined by reducing groups assay, was inhibited considerably by tannic acid and activated by phenol, p-benzoquinone, 1,2 napthoquinone, 1,4 naphthoquinone and 2 methyl 1,4 naphthoquinone. When healthy and diseased peppermint rhizomes were homogenized in culture filtrate of P. menthae, PG activity was inhibited, as measured by viscosity reduction. In a similar test, PG was activated by healthy and inhibited by diseased rhizome tissues, as determined by reducing groups assay. Biologically oxidized catechol and polymerized p-benzoquinone inhibited PG activity in culture filtrates, as determined by viscosity reduction assay; they activated PG as determined by reducing groups assay. Both PG and ME reached their highest activities in diseased peppermint rhizomes five days after inoculation with P. menthae. PG was determined by both viscosity reduction and reducing groups assays. Extracts from healthy peppermint rhizomes did not show PG or ME activity. Tannic acid, 1,2 naphthoquinone, p-benzoquinone and digallic acid inhibited PG extracted from inoculated peppermint rhizomes, as determined by both viscosity reduction and reducing groups assays. Among nine phenolic compounds tested on the activity of ME extracted from diseased peppermint rhizomes, only catechol, p-benzoquinone and tannic acid showed strong inhibition, while 1,2 naphthoquinone, 1,4 naphthoquinone, 2 methyl 1,4 napthoquinone, and digallic acid showed a moderate inhibition, as measured by the action on potato discs. The effect of phenolic compounds on PG produced by P. menthae both in vivo and in vitro (as determined by both viscosity reduction and reducing groups assays) suggests that two PG's are being produced. One PG is more active in hydrolyzing NaPP thus releasing large quantities of reducing groups, and the other is more active in hydrolyzing NaPP solutions causing a rapid reduction in their viscosity. Healthy peppermint rhizome tissues had higher activities of polyphenol oxidase than diseased tissues; also, polyphenoi oxidase activities in inoculated peppermint rhizomes decreased as time of incubation increased.