Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions

Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaic virus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significant economic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventing the viruses from spreading and improving their control. In this study, a procedure based on two multiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serial dilutions of positive controls were analysed by this methodology, and the results were compared with those obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assays were able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA. The multiplex versions were able to detect composite samples containing different concentrations of specific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected in different tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCR and commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, with a higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming the presence of these seven virus species in ItalyPanno, S.; Davino, S.; Rubio, L.; Rangel, E.; Davino, M.; García Hernández, J.; Olmos Castelló, A. (2012). Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions. Journal of Virological Methods. 186(1-2):152-156. doi:10.1016/j.jviromet.2012.08.003S1521561861-

Estimation of vector propensity for Lettuce mosaic virus based on viral detection in single aphids

Lettuce mosaic virus (LMV) is transmitted by aphids nonpersistently causing severe disease outbreaks in commercial lettuce crops. New strategies to control plant viruses have arisen based on molecular techniques, which analyze plantvirus- vector interactions. In this work, two PCR-based methods with a previous immunocapture phase, have been developed to detect LMV in single aphids. Detection rates using a RT-nested-PCR method in single aphids and transmission efficiency of Myzus persicae (vector species) and Nasonovia ribisnigri (nonvector species) were compared. Although the percentage of viruliferous aphids for N. ribisnigri (45.8 ± 2.3) was higher than for M. persicae (39.2 ± 3.5) after the same acquisition access period, N. ribisnigri was unable to transmit the virus while M. persicae proved to be an efficient vector (with a transmission rate per single aphid of 10.4 ± 0.8). A method was proposed to estimate vector propensity for nonpersistent viruses based on the relationship between the percentage of viruliferous aphids and their transmission ability. This methodology could be applied to decision-making and implementing control strategies to prevent virus spreading.Lettuce mosaic virus (LMV) es transmitido de forma no persistente por pulgones causando epidemias severas en cultivos comerciales de lechuga. En el control de virus vegetales se han desarrollado nuevas estrategias para el análisis de las interacciones planta-virus-vector basadas en técnicas moleculares. En este trabajo se ha llevado a cabo la detección de LMV en pulgones individuales mediante dos métodos basados en la reacción en cadena de la polimerasa (PCR) con una fase de inmunocaptura previa. Se compararon la tasa de detección en un único pulgón obtenida mediante RT-nested-PCR y la eficacia de transmisión de Myzus persicae (especie vectora) y Nasonovia ribisnigri (especie no vectora). Aunque el porcentaje de pulgones virulíferos para N. ribisnigri (45,8 ± 2,3) fue mayor que para M. persicae (39,2 ± 3,5) tras el mismo periodo de adquisición, N. ribisnigri fue incapaz de transmitir el virus, mientras que M. persicae resultó ser un eficiente vector (con una tasa de transmisión de 10,4 ± 0,8 con un único pulgón). Se propone un método para la estimación de la propensión vectorial en el caso de virus no persistentes basado en la relación entre el porcentaje de pulgones virulíferos y su capacidad de transmisión del patógeno. Esta metodología podría ser empleada en la implementación y aplicación de estrategias de control para prevenir la dispersión viral en el cultivo