Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

46
Aggressiveness of Phaeosphaeria nodorum Isolates and
Their In Vitro Secretion of Cell-Wall-Degrading Enzymes
P. Halama, a F. Lalaoui, a V. Dumortier, a and B. Paul b
a Institut Supérieur d’Agriculture, Université Catholique de Lille, Lille, France
b Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Université de Bourgogne, Dijon, France
Abstract
The relationship between the in vitro production of cell-wall-degrading enzymes and the aggressiveness of three
Phaeosphaeria nodorum isolates was studied. When grown in liquid medium containing 1% cell wall from wheat leaves as
the only carbon source, the isolates secreted xylanase, α-arabinosidase, β-xylosidase, polygalacturonase, β-galactosidase,
cellulase, β-1.3-glucanase, β-glucosidase, acetyl-esterase, and butyrate-esterase. Time course experiments showed different
levels of enzyme production and different kinetics between isolates. Xylanase, cellulase, polygalacturonase, and butyrateesterase
were positively correlated with isolate aggressiveness. The most aggressive isolate produced a higher proportion of
xylanase than the other two isolates, suggesting the role of this enzyme in the pathogenesis of P. nodorum.
Studies have revealed that
Phaeosphaeria nodorum populations
have significant variability in
pathogenicity that has been
measured by the ability of isolates
to cause symptoms on wheat
(Griffith and Ao, 1980; Allinghan
and Jackson, 1981;Yang and
Hughes, 1986; Krupinsky, 1997).
As other plant-pathogenic
fungi, P. nodorum produces a range
of cell-wall-degrading enzymes
(CWDE) that enables it to penetrate
and infect host tissues (Lehtinen,
1993; Magro, 1984). Very little is
known about the variability of
enzyme production in P. nodorum
populations; there is, however, a
need to assess the diversity of these
wall-degrading enzymes and, in
particular, the extent to which this
diversity could lead to differences
in pathogenicity.
The objective of this study was
to compare enzymatic variation
between two wild isolates and one
mutant isolate of P. nodorum by
examining CWDEs, and to
establish the possible relationship
between enzymes associated to
pathogenesis and the
aggressiveness of fungal strains.
Materials and Methods
Fungal strains
Two wild strains (A/5 and 6/T)
that differed significantly in their
pathogenic behavior were isolated
as described by Rapilly et al. (1992).
The other isolate (300.2) is a
carbendazim (MBC) resistant strain
that was obtained using the
protocol described in a previous
study (Halama et al., 1999).
Growth conditions and
preparation of fungal
extracts
The pycniospores used for
inoculation were produced on
synthetic (S) medium (Halama and
Lacoste, 1992). For enzyme
production, isolates of S. nodorum
were grown in liquid medium
(Lehtinen, 1993) in which sucrose
was replaced by 1% wheat cell
walls. Wheat cell wall material was
taken from 1-week-old seedlings of
cv. ‘Soissons’ and prepared
according to Lehtinen’s modified
procedure (Lehtinen, 1993). After
incubating cultures at 24°C under
constant shaking (72 rpm), liquid
filtrates were centrifuged at 8000 g
for 15 min at 4°C. Supernatants were
used for enzyme activity assays, and
each culture experiment was
replicated three times.
Enzyme assays
Cell-wall-degrading enzymes of
P. nodorum were assayed from the
crude culture filtrates using
different methods. The
dinitrosalicylic acid (DNS) modified
method, described by Miller (1959)
for determining the reducing group,
was used for the endo-enzyme
assay. Xylanase, endopolygalacturonase,
cellulase, and β-
1.3-glucanase activities were
measured respectively on oat spelts
xylan, galacturonic acid,
carboxymethyl cellulose, and
laminarin used as substrates.
Absorbances were measured at 540
nm. The reference sugar was
glucose.
Glycosidase activities were
measured using p-nitrophenyl
glycoside as a substrate (Poutanen,
1988). β-glucosidase, β-