Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease.

Singleplex and multiplex real-time (TaqMan®) RT-PCR assays were developed to detect seven fig-infecting viruses, i.e., fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig mosaic virus (FMV), fig latent virus 1 (FLV-1), fig cryptic virus 1 (FCV-1) and fig fleck-associated virus (FFkaV). The sensitivity of the newly developed TaqMan® assays was compared with the corresponding conventional RT-PCR (RT-PCR) using 10° to 10-6 serial dilutions of both cDNA and crude fig extracts. The results showed that the Taqman® RT-PCR assays were generally 102 to 103-fold more sensitive than the RT-PCR assays, except in the case of FLV-1 detection, where the two techniques had the same sensitivity. In the multiplex Taqman® RT-PCR, only a maximum of five viruses could be detected simultaneously in naturally infected fig trees, regardless of which combination of the virus-specific probes and primers were used. Both the RT-PCR and Taqman® RT-PCR assays were used in a large-scale survey of 100 field-grown fig trees in Egypt. The results showed the presence of all seven viruses under study, mostly occurring as mixed infections (63 %). The prevalence of infections observed in the tested samples were as follows: FMV (62 %), FFkaV (59 %), FLMaV-2 (32 %), FLV-1 (16 %), FLMaV-1 (14 %), FCV-1 (7%) and FMMaV (4%). FMV was invariably associated with diseased trees that presented mosaic-like symptoms. In the few cases where the mosaic-affected trees were found to be free of FMV, they were found to be infected with a mixture of two or more other viruses.

Development of RT-PCR assays for the detection and the resultant phylogenetic analysis of four grapevine vitiviruses based on the coat protein sequences.

Four sets of primers were designed based on the alignment of the complete coat protein (CP) gene sequences of several isolates of four different vitiviruses, i.e. grapevine virus B (GVB), GVD, GVE and GVF, and tested for their efficiency in RT-PCR assays to detect vitiviruses infections in grapevine. The resultant RT-PCR amplicons were sequenced and analyzed for their genetic variability and phylogenetic studies. The results of the RT-PCR assays showed that these primers were highly efficient in detecting different vitivirus isolates in grapevine material originating from ten different Mediterranean countries. In particular, 76 out of 218 tested samples (ca. 35%) were infected with at least one vitivirus. GVE was the most detected (14.7%), followed by GVF (11.5%), GVB (6.9%), and GVD (2.8%). Nucleotide (nt) sequence analysis of the CP genes from this study and Genbank showed that the sequence identity matrixes among isolates of GVB and GVE were the most variable, with nt identity ranging from 77% to 100%, whereas isolates of GVD and GVF showed more conserved nt identities ranging between 82% to 100% and 86.4% to 99.8%, respectively. The phylogenetic trees constructed based on the CP sequences distinguished two main groups of isolates for each vitivirus species, except for the GVD isolates, which did not show any particular subdivision. In general, the distributions of the isolates in the phylogenetic tree were associated with their geographical origin, thus suggesting limited movement of grapevine materials between the different countries. This study reported for the first time: (i) the development of primers based on the complete CP gene sequences for RT-PCR assays for the universal detection of vitivirus species, (ii) the high genetic variability among Mediterranean isolates of vitiviruses and (iii) the presence of GVD in Jordanian vines, of GVE in grapevines from Hungary, Italy, Jordan, Malta and Palestine, and of GVF in grapevines from Afghanistan, Bulgaria, China, France, Hungary, Italy, Jordan, Lebanon and Malta.