Real time PCR is a method that allows quantitative detection of fungal pathogens in plant host tissue. This method exploits the 5´nuclease activity of the AmpliTaq polymerase to cleave a Taq Man probe during PCR. The Taq Man probe contains a reporter and quencher dyes. During the reaction, the reporter dye and quencher dye become separated, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reported dye. The initial amount of target DNA in the reaction can be related to a cycle threshold (Ct) defined as the cycle number at which there is a statistically significant increase in fluorescence. A great advantage of real time PCR is its specificity. It is given by primers and Taq Man probes design accuracy. We evaluated primers and Taq Man probes for quantitative detection of Pyrenophora teres f. sp. teres, P. teres f.sp. maculata and of Fusarium culmorum. In all cases primers are strictly species specific. In Pyrenophora experiment we pointed at detection of fungal pathogen – its spot and net form - in barley leaf tissues. Fusarium project was about detection of F. culmorum in wheat and barley seeds. There are two possibilities of real time PCR configuration. In the Pyrenophora project we preferred multiplex configuration that enabled detection of fungal and barley DNAs in one sample. Taq Man probe for fungi was labelled with fam fluorescent dye. Taq Man probe for barley was based on RacB gene sequence data and it was labelled with vic fluorescent dye. Both configurations: multiplex and uniplex were tested and the same results were obtained. In the Fusarium case, we chose the uniplex configuration because it has been relatively easy to get additional data like for example DON content that is rated to be the marker of Fusarium infection measure. It is necessary to check the reliability of the results obtained using real time PCR by testing a correlation relationship with the other data. Ct values obtained in Pyrenophora experiment were compared with the size of symptoms caused by Pyrenophora teres on barley leaves measured using image analysis LUCIA. A good correlation was found. In Fusarium project Ct values were compared with DON contents. Especially in barley case, we found R2 = 0,96 correlation between Ct values and DON contents. In wheat case we found a complicated correlation relationship between Ct values and DON contents. Further study showed that these differences were caused by different course of infection by studied varieties. The quantification of the fungal pathogens using real time PCR could be useful in phytopathological praxis; especially where the classical methods do not correlate well with infection measure. This project is supported by the National Academy of Agricultural Research (project no. QC1361).
Potyviruses of Vanilla : A diagnostic puzzle
Karin Farreyrol1, Michael Pearson1*, Michel Grisoni2 and Laurence Rassaby2
1 School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand, 2 CIRAD-3P, UMR-PVBMT, Ligne Paradis, 97410 Saint Pierre, Reunion Island
Among the limiting factors for the successful cultivation of vanilla are diseases caused by potyviruses, with effects ranging from relatively mild to vine death. To date, three potyviruses, Vanilla mosaic virus (VanMV), Watermelon mosaic virus (syn. Vanilla necrosis virus), and Bean common mosaic virus have been definitively characterised from vanilla, but strain variability creates additional complications for diagnosis, as illustrated by VanMV. Coat protein gene sequence data indicates the existence of at least two distinct strains of VanMV, one from French Polynesia (VanMV-FP) and one from the Cook Islands (VanMV-CI), with distinctive features that suggest different biological properties. In addition, we have detected as yet unidentified potyviruses, including a Bean yellow mosaic-like potyvirus from Reunion Island. The diversity of potyviruses in vanilla makes simple, specific diagnosis difficult. Polyclonal antibodies have proved unreliable for specific virus identification, and although sequencing of PCR products, obtained using degenerate potyvirus primers, has been successfully used to identify some potyviruses in vanilla, it is not convenient for routine diagnosis. Consequently, there is a need for a broad-spectrum diagnostic procedure combined with identification of potyviruses to the species level. Microarray technologies may offer a solution to this problem.
Detection of bymo- and furoviruses on wheat by RT-PCR in Belgium
C. Vaïanopoulos, A. Legrève1, A. Barbier, S. Steyer2, H. Maraite1 & C. Bragard1
Unité de phytopathologie, Université catholique de Louvain, UCL1 & Département Lutte biologique et Ressources phytogénétiques, Centre Wallon de Recherches Agronomiques, CRA-W 2
Several viruses belonging to Bymo- and Furovirus are causing mosaic symptoms on wheat. They are transmitted by Polymyxa graminis, a common root-infecting parasite surviving in soil as resting spores that ensure the persistence of viral particles for several years. These viruses, present in Europe, have never been reported in Belgium. In order to assess the occurrence of these viruses in the country, the first step was to develop an RT-PCR method adapted to detect a wide range of bymo- and furoviruses. Eight primers pairs were designed to target different regions of the genomes of the viruses responsible of wheat mosaics in Europe: three specific for the Wheat spindle streak mosaic virus (WSSMV, a Bymovirus), one for the Soil-borne wheat mosaic virus and one for the Soil-borne rye mosaic virus (SBWMV and SBRMV, two furoviruses) and three universal for the furoviruses (the Soil-borne cereal mosaic virus (SBCMV), the European wheat mosaic virus (EWMV), the SBWMV and the SBRMV). The RT-PCR detections were validated on virus isolates from European origins. A hundred soils, mainly collected in the cereal producing area of the southern part of Belgium (Walloon region), in fields selected because of their potential contamination by barley mosaics, were then screened. RT-PCR tests performed on plant material (susceptible wheat cv. Cezanne and Savannah) grown on the collected soils, under adapted controlled conditions, revealed the presence of WSSMV and furoviruses in respectively 32 and 30 % of the soils. During the spring 2004, symptoms of mosaics were also observed on wheat plants in fields located in five Belgian provinces: Anvers (Malines), Brabant Wallon (Thines, Corroy-le-Grand), Hainaut occidental (Comines) and Namur (Flavion, Lonzée). The presence of WSSMV and furoviruses in these plants