- 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)
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
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
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
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