© Benaki Phytopathological Institute
Darras
et al.
72
opersicon esculentum
L.) and strain BPIC2531
of
R. solani
isolated from potato plants (
Sola-
num tuberosum
L.) were used. Both strains
were provided by the Benaki Phytopatho-
logical Institute (Kifissia, Athens, Greece).
Tomato plants at the stage of 4 true
leaves (approx. 30 cm in height) were inoc-
ulated with
F. oxysporum
f. sp.
lycopersici
by
applying a conidial suspension at the basal
stem-end of each plant (Dhingra and Sinclair
1995; Akköprü and Demir, 2005). The fungal
inoculum was prepared as follows: initially
the fungus was cultured on potato dextrose
agar (PDA, Oxoid Ltd., Basingstoke, Hamp-
shire, UK) medium in Petri plates at 26°C in
the dark for 12 d. The conidial suspension,
which consisted of both micro- and mac-
roconidia, was prepared by pouring 20 ml
of sterile distilled water containing 0.01%
Tween 80 (Sigma, St. Louis, USA) in each
plate. The conidia were dislodged by gen-
tly rubbing the fungal colony surface with a
sterile razor blade. The suspension was fil-
tered through two layers of fine, nylon, ster-
ile cheesecloth to remove mycelia. The final
conidial concentration was adjusted to 4.5
x 10
6
conidia/ml using a haemocytometer.
Twenty ml of the conidial suspension were
applied to each plant approx. 3 cm below
the surface of the growing substrate and at
a contact with the stem base using a 5 ml
plastic syringe (i.e. 4 applications around the
plant stem) without wounding the roots (Ak-
köprü and Demir, 2005). Control plants were
treated with 20 ml of sterile distilled water.
Tomato plants at the stage of 4 true
leaves (approx. 30 cm in height) were inoc-
ulated with
R. solani
using mycelium plugs
(Dhingra and Sinclair, 1995). Initially,
R. solani
cultures were prepared by placing mycelium
plugs cut from the edges of 12-d-old cultures
at the centre of PDA (Oxoid Ltd., Basingstoke,
Hampshire, UK) plates. The inoculated plates
were incubated at 25°C for 12 d in the dark.
Mycelium plugs, 5 mm in diameter, were
then cut from the edges of the growing colo-
nies using a cork borer. Inoculation of toma-
to plants was carried out by placing three, 5
mm in diameter, mycelium plugs 3 cm below
the surface of the growing substrate and at
a distance of approximately 1 cm from the
stem base. Control plants were treated with
non-inoculated PDA plugs.
Disease assessments
In both experiments, disease symptoms
were recorded 40 and 60 dpi.
In Experiment
1, disease severity index (DSI) and disease
incidence (DI) on tomato plants inoculated
with
F. oxysporum
f. sp.
lycopersici
were as-
sessed on the root system and stem base.
DSI was determined using the arbitrary
scale of: 0: no symptoms, 1: 1% of roots with
symptoms, 2: >1-5% of roots with symp-
toms, 3: 6-10% of roots with symptoms, and
4: >10% of roots with symptoms. DI was cal-
culated according to the following formula:
Disease incidence (DI) = (number of symp-
tomatic plants/total number of inoculated
plants) x 100 (1)
In Experiment 2, DI, number of cankers
(CN) and average canker diameter (ACD)
were recorded. DI was calculated according
to formula (1) above. Canker diameter was
measured in cm using a digital micrometer
(Stock No. 600-880, Mitutoyo, Japan).
Biomass production, number of flowers
and physiological parameters of toma-
to plants
Plant biomass production was record-
ed 40 and 60 dpi. Prior to assessment, the
growing substrate was completely removed
by gentle washing the root system of the
plants under running tap water. Biomass
was determined by measuring the fresh
weight (FW; gr) of the aerial plant parts (i.e.
stems, leaves and inflorescences) and the
root system using a digital balance (Kern &
Sohn GmbH, Balingen, Germany). Then, the
same plant parts were dried separately in
an oven (Daihan Labtech Co. Ltd, Gagok-ri,
Korea) at 75°C for 72 h and the dry weights
(DW; gr) were also measured. The number of
flowers was recorded once every week (total
of eight counts over the 60 dpi period).
The physiological parameters of CO
2
as-
similation (
A
s
; μmol CO
2
/m
2
/s), stomatal con-
ductance (
g
s
; mmol/m
2
/s) and transpiration
(
E
; mmol/m
2
/s) were recorded 16 and 27 dpi
