Molecular diagnosis of Papaya meleira virus (PMeV) from leaf samples of Carica papaya L. using conventional and real-time RT-PCR

Abstract

Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease. This study describes two methods for molecular diagnosis of PMeV using conventional and real-time PCR. These methods were shown to be more efficient than current methods of viral detection using extraction of PMeV dsRNA and observation of symptoms in the field. The methods described here were used to evaluate the effect of inoculation of papaya plants with purified PMeV dsRNA on the progress of PMeV infection. A single inoculation with PMeV dsRNA was observed to delay the progress of the virus infection by several weeks. The possibility of vertical transmission of PMeV was also investigated. No evidence was found for PMeV transmission through seeds collected from diseased fruit. The implications of these results for the epidemiology of PMeV and the management of papaya sticky disease are discussed.

Early diagnosis of a Mexican variant of Papaya meleira virus (PMeV-Mx) by RT-PCR

“…The LOD of primer pair CB38/CB39 was 1 fg in the latex of fruits and 1 pg in the leaves of seedlings, indicating that PMeV-Mx is more abundant in the latex of fruits than in the leaves. These results are consistent with previous reports for PMeV-Br (Zambolim et al, 2003;Tavares et al, 2004;Abreu et al, 2012). An early diagnostic assay for PMeV-Mx will be useful for farmers and local authorities, enabling the certification of seeds and seedlings prior to transplantation in the field and for the detection of disease in the field prior to fruit production.…”

“…Maradol) growing in an orchard in Conkal, Yucatan, Mexico, were harvested, frozen in liquid nitrogen, and stored at -80°C until use. For the seedling assays, seeds were germinated in peat moss substrate and incubated at 27°C under a 16/8-h light/dark photoperiod; hypocotyls were injected with 20 μL diseased latex diluted to 50% in phosphate buffer, pH 7.0 (Abreu et al, 2012). The first 2 true leaves were collected at 12 days post-inoculation, frozen, and stored at -80°C until use.…”

“…B. Schematic representation of the 669-bp sequence from PMeV-Br (Araújo et al, 2007) targeted by some of the PMeV-Mx Illumina reads. The binding sites of internal PMeVconv Fw and PMeVconv Rv primers (Abreu et al, 2012) are also indicated.…”

“…The removal and destruction of diseased plants is the only method available for controlling Papaya meleira disease (Lima et al, 2001;Ventura et al, 2004). Although the transmission mechanism has been examined in previous studies, it is not well understood (Rodrigues et al, 2009;Abreu et al, 2012).…”

“…However, because symptoms typically do not appear until the papaya plant is flowering and producing fruits, the plants can only be diagnosed when they are approximately 6-9 months old. In 2007, a reverse transcription-polymerase chain reaction (RT-PCR) method based on the 669-bp partial sequence of PMeV was described (Araújo et al, 2007); however, this method is only suitable for detecting the virus in the latex of fruits (Abreu et al, 2012). Recently, internal primers directed against this sequence were designed, enabling the detection of the virus on leaves using conventional and quantitative RT-PCR (Abreu et al, 2012).…”