Page 50 - Volume 1, Issue 1, January 2008
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K. Elena & A. Grigoriou
48
were digested with the restriction enzymes
Alu
I,
Msp
I and
Taq
I to generate characteris-
tic banding patterns (Restriction Fragment
Length Polymorphism, RFLP analysis).
Macrogen DNA Sequencing Service
(Macrogen Korea, 10F Meridian Center, 60-
24 Kasan-dong, Kumchun-ku Seoul, Korea
153-023) carried out direct sequencing of
PCR products for the isolate P1 using ITS6
and ITS4 primers.
Pathogenicity tests
To complete Koch’s postulates, 35-day-
old parsley plants were artificially inoculat-
ed with the tested fungus. To determine
the host range of the pathogen additional
35-day-old plants of other vegetable spe-
cies, cultivated in the Marathon area, such
as tomato (
Lycopersicon
esculentum
L.) hy-
brid Belladona, and the winter crops: let-
tuce (
Lactuca sativa
L.) cultivar Paris Is-
land Cos,
cauliflower (
Brassica oleracea
var.
botrytis
L.
)
cultivar Alpha, broccoli (
Brassica
oleracea
var.
asparagoides
L.) cultivar Ramo-
so Calabrese, red cabbage (
Brassica olera-
cea
var.
capitata
L. f.
alba
) cultivar Langedijk
autumn, white cabbage (
Brassica oleracea
var.
capitata
L. f.
tubra
) cultivar Merveille
d’ Octobre, leek (
Allium porrum
L.)
cultivar
Blue solaise, Brussels sprout [
Brassica oler-
acea
L. var.
gemmifera
(DC) Thell] cultivar
Groninger, carrot (
Daucus carota
L.) cultivar
Nantes, and primula [
Primula acaulis
(L.)
Hill, syn.
P. vulgaris
Hudson] were inoculat-
ed with P1, P2, P3 isolates of
P. primulae
.
Parsleyplantswerealsoinoculatedwith
two isolates of
P. cryptogea
(BPIC1189 and
BPIC1191) and
P. citrophthora
(BPIC1133 and
BPIC1185), one isolate of
P. porri
(BPIC1985)
and one isolate of
P. nicotianae
(BPIC2000),
derived from the Benaki Phytopathologi-
cal Institute Culture Collection (BPIC).
All the plants were grown separately in
10 cm pots filled with moist compost (Klass-
man Potground). Three mycelial discs 10
mm in diameter taken from the edge of a
12–day-old colony on lima-bean agar were
inserted around the crown and roots of the
plants, 1 cmunder themoist soil surface and
wounded prior to inoculation, according
to Sitepu and Bumbieris (15). The control
plants were inoculated with Lima-bean-
agar discs. Soil moisture was maintained
by placing each pot in a plastic contain-
er filled continually with distilled water.
For each plant species and
Phytophthora
strain fifteen replications were used. The
air temperature in the greenhouse fluctu-
ated 15-20
0
C with a 12 h photoperiod.
Apple fruits (
Malus domestica
Borkh.),
potato tubers (
Solanum tuberosum
L) and
onion bulbs (
Allium cepa
L.) were artifi-
cially inoculated with
P. primulae
isolate
P1
according to Tomlinson method (18).
Three mycelial discs 10 mm in diameter
taken from the edge of a 12–days-old col-
ony on lima-bean agar were inserted in
three points on each fruit under the rind.
Ten replications of fruits, tubers or bulbs
were used and in respective controls wa-
ter-agar plugs were inserted.
The experiments were repeated once.
Results
Pathogen morphology and physiology
The causal organism, isolated from
diseased parsley plants, was a slow-grow-
ing species of the genus
Phytophthora
. The
pathogen developed on CMA sparse aerial
mycelium. Microscopic examination revealed
coiled hyphae (Fig. 1), and hyphal swellings.
On soil extract medium, at laborato-
ry temperature (18-22
0
C), abundant num-
ber of sporangia were formed within 24 h.
Persistent semipapillate sporangia, simple
and compound, variable in shape and size
were formed on undifferentiated sporan-
giophores. Simple sporangia were ovoid
or ellipsoid, limoniform, very often elon-
gated with several constrictions (Fig. 2B).
The dimensions of these sporangia were
42.5-75 x 22.5-43.75 (average 63 x 31) μm.
