Ecole Nationale Supérieure Agronomique de Montpellier ... - CIAM

sensitive technique can be adapted to the objectives, through the design of different primer
sets. For routine diagnosis and experiments, one frequently uses the PCR primers fU5/rU3
(Lorenz et al., 1995) that were designed to detect all the phytoplasmas. However, these
universal primers can match portions of the genome of some epi- or endophytic bacteria:
some plants can shelter on their surface Acholeplasma sp. (Tully et al., 1994) or other bacteria
that cross-react with the universal primers, generating false positives (Baric and Dalla-Via,
2004; Skrzeczkowski et al., 2001). Moreover, several other phytoplasmas have been detected
in Prunus, some of which with a significant prevalence. Peach trees (P. persicae) clearly
exemplify this situation: in the orchard, some symptoms of ESFY, western X-disease, and
peach yellow leaf roll (PYLR) are similar (Kison et al., 1997). Furthermore, peach trees
naturally infected by ‘Ca. P. phoenicium’ (Abou-Jawdah et al., 2002) or by ‘Ca. P. asteris’
(Anfoka and Fattash, 2004) also showed yellows symptoms. Such yellows symptoms,
typically displayed by phytoplasma-infected plants, can be confounded with ESFY symptoms
and thus result in an erroneous diagnosis. The same identification problems occur with the
insects: of course they show no ‘Ca. P. prunorum’-specific symptom, and they contain a
wealth of endogenous prokaryotes. Indeed, they carry gut and cuticle bacteria, symbionts (at
least one species), and parasites (potentially including other phytoplasmas) that should not be
detected by primers designed for ‘Ca. P. prunorum’ phytoplasma. Thus, a more specific
method is often required for the detection and specific identification of ‘Ca. P. prunorum’ in
plants and insects. Two group-specific primers (detecting subclades within the genus ‘Ca.
Phytoplasma’) are also used: fO1/rO1 (Lorenz et al., 1995) and R16(X)F1/R1 (Lee et al.,
1995), but they amplify several phytoplasmas, including the phytoplasma associated with
PYLR. Thus, a classical approach to the specific detection of ‘Ca. P. prunorum’ relies on the
amplification of a 16S rDNA fragment with generic primers followed by time- and resourceconsuming
additional enzymatic digestions (Lorenz et al., 1995). A primer pair targeting a
genomic DNA sequence has also been designed to detect ‘Ca. P. prunorum’ (Jarausch et al.,
1998), but the specific detection was obtained at the expense of sensitivity. Highly specific
and sensitive primers would therefore be useful to detect ‘Ca. P. prunorum’ in plants and
insects.
Quantitative real-time PCR (Q-PCR) can be used either as a very sensitive and specific
detection tool or, of course, as a way to quantify the phytoplasma in plants or insects. This
method has already been developed for several phytoplasmas in plants (Baric and Dalla-Via,
2004; Christensen et al., 2004; Wei et al., 2004) and insects (Jarausch et al., 2004). However,
no protocol was available to quantify ‘Ca. P. prunorum’ in its vector, although this would
allow considerable insights into the (spatio-) temporal dynamics of this phytoplasma within
C. pruni.
In this paper, we describe a new set of complementary tools for the specific detection
and/or quantification of ‘Ca. P. prunorum’ in its plant hosts and insect vector. These tools
consist in (i) a specific primer pair that can be used either for the detection of this
phytoplasma for conventional PCR or for its quantification by real-time PCR with the SYBR
Green device, and (ii) a set of TaqMan primers and probes for the absolute quantification of
‘Ca. P. prunorum’. Primers and probe were available for the quantification of C. pruni DNA
as an internal standard with these two chemistry.
2. Materials and methods
2.1. Sources of phytoplasma
Healthy and ESFY-infected Prunus rootstocks (P. marianna GF 8-1, P. armeniaca cv.
Manicot, P. persicae cv. Montclar) were grown in an insect-proof greenhouse. Several French
ESFY isolates were used. The other phytoplasmas (fig.1A) were maintained on the
experimental host Catharanthus roseus and were kindly provided by X. Foissac (INRA,
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