Diagnostics is important in the development and implementation of pest management strategies. The virus diagnostic capabilities of several plant pathology collaborators within the Integrated Pest Management Collaborative Research Support Program (IPM CRSP) host countries were evaluated with the aid of a survey. Very few plant disease diagnostic clinics had funds to cover daily operations despite over half of the responding clinics receiving an operational budget. Academically and government affiliated clinics within the developing host countries had little access to molecular tools and equipment, relying mostly on biological and serological methods. Clinics affiliated with private companies and within the USA relied more upon molecular assays. Ten CMV isolates identified by tissue blot immunoassay (TBIA) were collected from a garden at the Historic Smithfield Plantation on the Virginia Tech campus, and from Painter, Virginia on the Eastern Shore. Three CMV isolates from Smithfield were biologically compared to six early CMV isolates stored since the 1970s, while all isolates were compared serologically and molecularly. Sequences obtained after reverse transcription-polymerase chain reaction (RT-PCR) assigned the CMV isolates into subgroups, with eleven to subgroup 1A and three to subgroup 2. The subgroup assignments were confirmed by TBIA using CMV subgroup-specific monoclonal antibodies (Agdia Inc). At Smithfield Plantation, another virus, Turnip mosaic virus (TuMV) was identified from Dame's Rocket (Hesperis matronalis L.). This is the first report of TuMV in Virginia.  In TBIA virus-infected plant samples are blotted onto nitrocellulose membranes, dried, and processed. Membranes can be stored for long periods of time and transported safely across borders without risk of introducing viruses into new environments, but virus remains immunologically active for several months. Methods were developed with CMV and three potyviruses, using the same membranes, for detecting viral RNA by RT-PCR and direct sequencing of PCR products.. Amplification by RT-PCR  was possible after membrane storage for up to 15 months. The membranes also performed well with samples sent from IPM CRSP host countries and within the USA. This method should improve molecular diagnostic capabilities in developing countries, as samples can be blotted to membranes and sent to a centralized molecular laboratory for analysis. / Ph. D.

The topic of this thesis is focused on the testing of resistance of selected Brassica species to the black rot infection and viral mosaics caused by economically important pathogens of Brassicaceae family. The theoretical part describes characteristics of causal pathogens - Xanthomonas campestris pv. campestris (Xcc), Turnip mosaic virus (TuMV) and Turnip yellow mosaic virus (TYMV), and summarize the current state of a resistance study of these pathogens in the Brassicaceae family. The thesis also describes modern molecular methods used for the detection of bacterial and viral pathogens. In the experimental part, the detections of Xcc, TuMV and TYMV pathogens were optimized by PCR and RT-PCR. For bacterium Xcc, the Real-time PCR targeting a part of the zur gene sequence was designed using a TaqMan® probe. This detection system was subsequently processed in the form of a certified methodology for use in diagnostics. To increase the specificity, Real-time PCR targeting zur gene was involved in the Multiplex Real time PCR reaction. Then the dynamics of the Xcc infection was monitored in 6 hybrid cabbage cultivars. The testing of resistance to the black rot disease was optimized by the procedure including artificial inoculations using the suspension of the Xcc isolates HRIW 3811, 3971A and 1279A and the SU1 isolate originated from the Czech Republic. In a four-year experiment, the total of 42 homozygous breeding lines and 4 hybrid cultivars were tested, where 5 lines were recommended for breeding for resistance to the black rot disease. For the detection of TuMV and TYMV viruses, Real-time RT-PCR approaches based on the TaqMan® probe and SYBR Green dye were tested. The target region of both detections was the coat protein. The TuMV detection has been optimized for SYBR Green approach; for the TYMV detection, the use of the TaqMan® probe has been recommended. Detection systems were used to evaluate artificial inoculations of 6 cabbage cultivars by individual viruses. The tested plants did not show visual symptoms of infection therefore the presence of viruses was evaluated by Real-time RT PCR. The system designed for TYMV detected the presence of virus in all tested samples, TuMV was detected only in two samples. Negative detection results are probably in connection with the absence of TuMV symptoms which indicates unsuccesful plant inoculation. For both detection systems, it was recommended the verification on a wider range of viral isolates prior to standard use in diagnostics

Les plantes sont des organismes immobiles qui doivent répondre et s’adapter à des contraintes abiotiques et biotiques. Parmi les stress biotiques, les maladies virales, établies ou émergentes, peuvent être responsables de pertes de rendement majeures aux conséquences économiques importantes. Face aux phytovirus la lutte génétique constitue le moyen de lutte le plus efficace, le plus respectueux de l’environnement et du consommateur. Comprendre l’interaction entre les plantes et les virus reste indispensable pour rechercher de nouvelles sources de résistances. Ce travail de thèse s’intéresse à l’étude du pathosystème naturel Arabidopis thaliana/Turnip mosaic virus (TuMV). Les essais ont été menés majoritairement en conditions extérieures permettant une analyse de l’interaction dans un environnement multistress. La réponse d’A. thaliana a été explorée par l’étude de traits liés à la maladie et par la variation en métabolites primaires et secondaires. Ce travail a permis i) de caractériser de façon fine la réponse d’A. thaliana au TuMV en conditionsmultistress en exploitant la diversité naturelle d’une population mondiale et française ii) de déterminer l’architecture génétique de cette interaction par des approches de génétique d’association et de QTL mapping. Plusieurs nouveaux loci potentiellement impliqués dans la réponse ont été identifiés iii) de montrer l’intérêt du phénotypage métabolique pour discriminer les accessions en fonction de leur sensibilité au TuMV. La multidisciplinarité des approches constitue la richesse de ce travail de thèse qui contribue à une meilleure caractérisation et compréhension de la réponse des plantes lors d’une infection virale. / Plants are immobile organisms which have to adapt to abiotic and biotic constraints. Among bioticstress, established or emerging viral diseases, may be responsible for major yield losses withsignificant consequences. Genetic control is the most effective, environmentally and consumerfriendlyway to control viral infections. Understanding plant/virus interactions remains essential tosearch for new sources of resistance. This work, focuses on the study of the natural pathosystemArabidopsis thaliana/Turnip mosaic virus (TuMV). Most of the trials were conducted in commongarden conditions allowing the analysis of the interaction in a multistress environment. A. thaliana’sresponse was explored through the study of disease-related traits and the variations in primary andsecondary metabolites. This work allows i) the fine characterization of A. thaliana’s response toTuMV in multistress conditions through the exploration of the natural diversity of a world and Frenchpopulation ii) to determine the genetic architecture of this interaction by genome wide associationsand QTL mapping. Several new loci potentially involved in the response have been identified iii) tohighlight the interest of metabolic phenotyping to discriminate accessions according to theirsusceptibility to TuMV. The multidisciplinary approaches contribute to a better characterization andunderstanding of plant-virus interaction.