Ecole Nationale Supérieure Agronomique de Montpellier ... - CIAM

the same climatic and altitude conditions. Only a few plum and cherry trees are planted in
private gardens in the vicinity of the site.
The chosen hedge of blackthorns (P. spinosa) was about 50 m long and 2 to 5 m wide, on
the border of a cultivated field. Several other clumps or hedges of P. spinosa are spreaded all
around.
A sample of C. pruni had been taken one year before in this area to verify that some
ESFY infected individuals could be found.
Location of Overwintering Sites
Several individuals of C. pruni were found on conifers (Abies, Picea and Pinus), in what
seem overwintering sites of the psyllid. The first site was 25 km on the West at a 700 m
altitude (Séranne). The second site was 35 km on the North-West at a 1300 m altitude (Lingas
mountain).
Collected Samples of C. pruni
Samples of C. pruni (200 individuals when possible) were collected with a beating tray.
The reimmigrants (adults coming from their overwintering site at the end of the winter and
morphologically distinguishable by their dark color) were collected each week from the end
of February to the end of May. In May and June, 4 samples of adults of the new generation
(adults coming from eggs laid on the P. spinosa plants by the reimmigrants, distinguishable
by their light color) were collected. When possible, 40 C. pruni of each sample were tested
individually.
Collected Samples of P. spinosa
58 regularly spaced P. spinosa plants were labelled along the border of the hedge. One
shoot per plant was then collected to be tested by PCR. The plants detected infected were cut
into distinct parts according to their morphology and each part was tested by PCR to obtain an
assessment of the distribution of the phytoplasma in the plant.
Detection method of ESFY Phytoplasma
ESFY-P was detected by simple and nested-PCR both for plants and insects. DNA was
extracted from plants (0.3-0.5 g of phloem) or individual psyllid using a CTAB method as
described by Maixner et al. (1995). A nested-PCR using universal phytoplasma primers P1/P7
(Schneider et al., 1995) then U3/U5 (Lorenz et al.,1995) was used to detect phytoplasma in
psyllid or plant extracts. DNA amplification was carried out in a Biometra T1 thermocycler.
For P1/P7 a denaturation step of 4 min at 94°C was followed by 40 cycles of 1 min at 94°C, 1
min at 55°C, 1 min at 72°C and ended by a final step of 4 min at 72°C. Then, the product was
diluted 1:30 th and 1 µl was used in a second reaction with U3/U5. A first denaturation step of
1 min at 92°C was followed by 35 cycles of 30 sec at 92°C, 30 sec at 55°C, 45 sec at 72°C
and a final step of 4 min at 72°C. Amplification products (8µl) were analyzed by 1% agarose
gel electrophoresis. DNA was stained with ethydium bromide and visualized on a UV
transilluminator.
A simple PCR was run on positive samples with a pair of ESFY specific primers
(protocole to be published) to confirm the identity of ESFY. An additional confirmation was
done on a sample of positive plants and psyllids by sequencing the internal spacer of 16S-23S
rRNA genes.
RESULTS - DISCUSSION
P. spinosa Plants
Two plants out of 58 were detected infected. One plant was entirely infected (14/14
infected parts). Only one part (1/24) was detected infected for the second plant. This indicates
that only a small proportion of the hedge could provide an ESFY source for C. pruni.
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