Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

Abstract

Parietaria mottle virus (PMoV-T) infecting tomatoes from different commercial fields in Catalonia, Spain was detected by dot-blot and tissue-printing hybridization and one-step reverse transcriptase PCR (RT-PCR). Specific cDNA was amplified by RT-PCR from mechanically infected Chenopodium quinoa plants and cloned into a plasmid vector. A digoxigenin-labelled riboprobe was synthesized for molecular hybridization, and a specific set of primers (PMoV-1F/ PMoV-1R) were designed for one-step RT-PCR assays. Material from different parts of PMoV-T-infected tomato plants was used for the analysis. The virus was detected on necrotic apical shoots, fruits with symptoms, and symptomless shoots grown from old necrotic branches, but not on the old necrotic branches themselves. Both molecular hybridization and one-step RT-PCR procedures were highly specific, and no cross-reactions were observed with other related ilarviruses. PMoV-T was detected successfully by molecular techniques on all infected tomato plants collected from commercial fields, although one-step RT-PCR was less robust than molecular hybridization. The observed reliability of tissue-printing hybridization could greatly facilitate both the large-scale analysis and epidemiological studies of this important pathogen.

Egg detection in females of the polyphagous predator Macrolophus pygmaeus (Heteroptera: Miridae) by serological techniques

“…The antiserum was tested by ELISA (Clark and Adams 1977) on polystyrene plates (Nunc Roskilde, Denmark) following the indirect protocol described by Galipienso et al (2005). The sample extract was prepared by homogenizing one female abdomen in 100 ll of carbonate buffer pH 9.6 (CB) using a micropestle.…”

Characterization of potato virus Y isolates from tomato crops in northeast Spain

“…The same extracts were also analysed by indirect DAS ELISA using PA for PVY (Plantprint Lts., Valencia, Spain) following manufacturer's instructions. They were also tested by indirect ELISA using PA for parietaria mottle virus (PMoV), kindly provided by P. Roggero, and following the method described by Galipienso et al (2005). Serological characterization was achieved by DAS-ELISA using MAbs for PVY N , PVY O/C and PVY C (Adgen Lts., Scotland, UK) according to manufacturer's instructions.…”

Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado)

“…Este método permite la evaluación del estado fitosanitario del tomate y presenta una clara ventaja por el ahorro de tiempo y la simplificación en el manejo y procesado de las muestras de campo Galipienso et al, 2005). Simultáneamente, se muestrearon y analizaron en 2007, especies de la flora arvense que se encontraban habitualmente en los invernaderos de tomate de Murcia, Gran…”

“…This technique also saves time and simplifies the handling procedure in largescale testing Galipienso et al, 2005). Furthermore, the MH allows the simultaneous detection of different viruses affecting tomato crops by either mixing the corresponding riboprobes in the same hybridization solution (Saldarelli et al, 1996) or using polyprobes .…”

“…Thus, some of the symptomatic (s) and necrosis samples (n) were infected with other tomato viruses which commonly induce necrosis, such as TSWV, PVY, PMoV, CMV, ToCV and TYLCV. PMoV, which induced brown patches on tomato fruits and necrotic mosaic on leaves that progressed to apical stem necrosis, is easily confused with CMV, TSWV (Galipienso et al, 2005) or ToTV symptoms . Given the high percentage of ToTV-infected tomato plants and the lack of an available commercial antibody, we explored alternative detection techniques to the standard serological ELISA method to be used to screen a large amount of samples.…”