The main objectives of this study were to determine the
effect of certain environmental conditions on the quantity of poly-galacturonase (PG) produced by V. albo-atrum, and to partially
purify and characterize the PGs produced by this fungus in culture
and in infected tomato plants.
V. albo-atrum produced twenty times as much PG when
sodium polypectate was the carbon source as when grown on pectin.
Optimum PG production occurred 6-8 days after inoculation of the
medium. Less pectinmethyesterase (PME) was produced when
V. albo-atrum was grown on polypectate as the carbon source than
when grown on pectin. PME production was observed early in the
growth of the fungus. No PME activity could be detected from
culture filtrates after eight days when either pectin or sodium
polypectate was the carbon source.
Three elution schedules were used to purify PGs from a
DEAE-cellulose column. Step-wise elution produced residual
peaks of PG activity, and gradient elution gave poor separation of
the PGs. Flushing the DEAE-cellulose column with distilled water
and NaC1 gave the best separation of the two endo-PGs. PME was
associated with all fractions containing PG activity.
Assays of PG activity in fractions separated by column
chromatography suggested that V. albo-atrum produced at least
two endo-PGs in culture. One endo-PG preferentially hydrolyzed
sodium polypectate to large fragments; however, the fragments
were eventually hydrolyzed to monogalacturonic acid. The second
endo-PG preferentially hydrolyzed small fragments from the substrate.
Fractions containing the endo-PG that preferentially
hydrolyzed the substrate to large fragments gave ratios of viscosity
reduction to reducing group liberation at least five times as
high as those obtained for fractions containing the endo-PG that
preferentially hydrolyzed the substrate to small fragments.
Culture filtrates from cultures incubated at room temperature
but not on a shaker produced less PG than cultures grown on
a shaker at room temperature. When culture filtrates of the still cultures were eluted from the DEAE-cellulose column, fractions
from the first PG peak eluted from the column hydrolyzed trigalacturonic
acid slowly to monogalacturonic acid.
Cultures grown at 30°C produced only trace amounts of PG
activity. Growth of the fungus at 30°C was similar to cultures
grown at lower temperatures. However, the cultures grown at
lower temperatures produced relatively high PG titers.
The crude enzyme preparation of V. albo-atrum from infected
tomato plants yielded pectic fragments as well as monogalacturonic
acid. This indicated the presence of an endo-PG that hydrolyzed
small fragments from the substrate. Column chromatography of
this enzyme on DEAE-cellulose using a step-wise elution yielded a
PG fraction that preferentially hydrolyzed the substrate into small
fragments similar to fractions obtained by a step-wise elution of
culture filtrates.
Evidence was obtained that V. albo-atrum produced trace
amounts of pectintranseliminase in culture and in infected plants.
Additional studies will be required to understand the significance
of this enzyme in the Verticillium wilt syndrome