Ecole Nationale Supérieure Agronomique de Montpellier ... - CIAM

A toolbox for the specific detection and quantification of the phytopathogenic
agent ‘Candidatus Phytoplasma prunorum’ in plants and insects
Michel Yvon 1* , Gaël Thébaud 1* , Rémi Alary 2 and Gérard Labonne 1
1 Institut National de la Recherche Agronomique (INRA), UMR BGPI, CIRAD TA 41/K,
Campus international de Baillarguet, 34398 Montpellier Cedex 5, France; 2 INRA, UMR PIA,
2 Place Viala, 34060 Montpellier Cedex 1, France.
Abstract
‘Candidatus Phytoplasma prunorum’ is the wall-less bacterium associated with European
stone fruit yellows (ESFY), a severe disease of Prunus (mainly apricot and Japanese plum
trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected
material. The availability of PCR-based methods allowing a sensitive and specific detection
of ‘Ca. P. prunorum’ is crucial for this phytoplasma because, at present, it is uncultured and it
cannot be detected serologically. In contrast to the existing detection tools, we developed a
PCR test allowing both a sensitive and specific detection of ‘Ca. P. prunorum’ in plants and
insects. The sensitivity of this test was assessed by serial dilutions and its specificity was
demonstrated both in silico and experimentally against several phytoplasmas, included the
closely related ‘Ca. P. pyri’ (pear decline) and ‘Ca. P. mali’ (apple proliferation). This test
can also be used semi-quantitatively using real-time PCR. For studies requiring an absolute
quantification of the phytoplasma load in C. pruni (thus including an internal standard for C.
pruni), we developed quantitative real-time PCR assays for the two available chemistries.
Keywords: diagnosis; epidemiology; primer; probe; Prunus armeniaca; Prunus salicina; quantitative PCR.
1. Introduction
Phytoplasmas are uncultivated plant pathogenic bacteria belonging to the class
Mollicutes. These wall-less bacteria are linked with more than 100 plant diseases (Seemüller
et al., 1998). The ‘Candidatus Phytoplasma’ genus is presently subdivided into 15 groups on
the basis of their 16S ribosomal DNA (rDNA) sequence (Firrao et al., 2004), and ‘Candidatus
Phytoplasma prunorum’ is a member of the 16SrX group. It is associated with European stone
fruit yellows (ESFY) (Lorenz et al., 1994; Seemüller and Schneider, 2004), a disease
affecting most of wild and cultivated Prunus species (Carraro et al., 2001; Jarausch et al.,
1998). This disease – previously known as ‘apricot chlorotic leaf roll’ or ‘plum leptonecrosis’
– is widespread in Europe and causes substantial economic loss because of the decline and
death of the infected trees (mainly apricot and Japanese plum trees). Only one insect species,
Cacopsylla pruni, was identified as a vector of this phytoplasma (Carraro et al., 1998), which
can also be transmitted by the grafting of infected buds (Morvan, 1957) and the trade of
planting material. On cultivated stone fruit trees, ESFY generally induces yellows, decline,
and vegetative disorders with typical symptoms, such as early budbreak and leaf rolling
(Lorenz et al., 1994). Nevertheless, the nature and intensity of these symptoms can differ
depending on the season, the host plant, and the isolate of ‘Ca. P. prunorum’ (Jarausch et al.,
2000; Kison and Seemüller, 2001); some infected plants can even be asymptomatic (Carraro
et al., 2002; Torres et al., 2004). Thus, for epidemiological studies on ESFY and disease
management by plant protection services, more objective methods should be available to
detect and quantify ‘Ca. P. prunorum’ plants and in insects, either in experimental,
commercial, or natural conditions.
As no serological test is available and the phytoplasmas are yet uncultured, PCR
amplification is the main detection procedure, at present. The level of specificity of this
* These authors contributed equally to this work.
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