Ecole Nationale Supérieure Agronomique de Montpellier ... - CIAM

Bordeaux). Plant samples were also collected in orchards or in blackthorn (P. spinosa) hedges
in southeastern France. Petioles of C. roseus or phloem from woody shoots of Prunus (about
0.5 g) were used.
Overwintering adults C. pruni were collected from Prunus trees or from shelter conifers.
Nymphs and adults of the new generation were reared in cages in a climatic chamber from the
eggs laid by the overwintering adults. They were reared on either healthy or infected P.
marianna, to provide healthy or infected insects. Psyllids collected were conserved at –80°C
in 1.5 ml Eppendorf tubes until DNA extraction.
2.2. DNA extraction
Collection of plant material and DNA extraction appeared to be crucial for obtaining
accurate diagnosis without false positives. Preliminary trials had demonstrated that accidental
contamination from fingers, surfaces and tools could happen, particularly with experimental
woody material with a high phytoplasma titer. Thus, the routine protocol included the
disinfection of surfaces and experimental tools between samples and precautions to avoid
touching the tested plant material.
Total DNA from plant material was extracted using CTAB (cetyltrimethylammonium
bromide) as described by Maixner et al. (1995). Total DNA of each insect was extracted by
the same procedure with some modifications, following Marzachi et al. (1998). Each psyllid
was ground in 40 µl of CTAB buffer with 3 µl of proteinase K (20 mg/ml) (Invitrogen) with a
sterile micropestle, completed with 460 µl of CTAB buffer and incubated for 30 min at 65°C.
The extraction was performed with 500 µl of chlorophorm-isoamyl alcohol (24:1). Then, 1 µl
of Glycoblue (15 mg/ml) (Ambion) was added to 350µl of cold isopropanol to improve
precipitation and to dye DNA pellets in blue. Total DNA was washed with 70% ethanol and
dried 30 min at 37°C in an incubator. Finally, plant and insect DNA was eluted in 100 µl and
30 µl of sterile water, respectively and stored at –20°C.
To control the reproducibility of DNA extraction, the amount of DNA extracted from
each of 10 insects was measured with the Quant-iT PicoGreen quantification kit, using λDNA
as a standard and according to the manufacturer’s instructions (Invitrogen). After excitation at
480 nm, the fluorescence emission was measured at 526 nm with a F-2500 spectrofluorimeter
(HITACHI SciencTec).
2.3. Specific PCR
The ESFY-specific primer pair ESFYf/r (Table 1) was based on the primers fAT/rPrus
(Smart et al., 1996), with appropriate modifications (i.e., slight shifts). ESFYf was located in
the 16S rDNA and ESFYr in the adjacent intergenic region of the phytoplasma genome,
generating a 504 bp fragment. Sequence alignment using GenBank database showed that
ESFYr had at least three mismatches with the sequences of the other phytoplasma species of
the 16SrX group (fig.3), allowing the specific identification of ‘Ca. P. prunorum’. Each
reaction was performed in 20 µl including 1X PCR buffer (Invitrogen), 1.5 mM of MgCl2,
125 µM of dNTP, 0.35 µM of each primer (Invitrogen), 1 unit of Taq polymerase
(Invitrogen), 10–100 ng of template DNA. To find a good balance between sensitivity and
specificity, a touch-down PCR was designed with a high annealing temperature during the
first cycles: 1 min of denaturation at 94°C, followed by 20 cycles of 30 s at 94°C, 20 s at
65°C, 45 s at 72°C, and then 20 cycles of 30 s at 94°C, 20 s at 62°C, 45 s at 72°C.
Amplification was carried out both in a T1 thermocycler (Biometra) and in a PT100
thermocycler (MJ Research) applying different ramping rates to check the robustness of the
method. Amplification products (8 µl) were analyzed by electrophoresis on 1.5% agarose gel
in 0.5X TBE buffer and visualized using a UV transilluminator after ethidium bromide
staining.
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