Abdessalem Tahiri

Monitoring the dynamics of emergence of a non-canonical recombinant of Tomato yellow leaf curl virus and displacement of its parental viruses in tomato.

Recombinant viruses are increasingly being reported but the dynamics of their emergence is rarely documented. A new recombinant Tomato yellow leaf curl virus (TYLCV-IS76) was detected for the first time in 2010 in Southern Morocco (Souss). An original diagnostic tool was needed to fit its unusual recombination profile. Although IS76 was detected following the appearance of Tylc symptoms on tolerant tomato plants, symptoms could not be associated to IS76 or to a synergy with criniviruses. According to infection profiles of Tylc-associated viruses determined on 879 plant samples collected between 1998 and 2014 and a Bayesian inference applied to genomic sequences of representatives of TYLCV, IS76 emerged in Southern Morocco at the end of the 1990s, replaced the parental viruses between 2004 and 2012 in Souss and is spreading towards the North of Morocco. The emergence of IS76 coincides with the increasing use of tolerant cultivars in the 2000s.

The non-canonical tomato yellow leaf curl virus recombinant that displaced its parental viruses in southern Morocco exhibits a high selective advantage in experimental conditions

Recombination events are frequently inferred from the increasing number of sequenced viral genomes, but their impact on natural viral populations has rarely been evidenced. TYLCV-IS76 is a recombinant (Begomovirus,Geminiviridae) between the Israel strain of tomato yellow leaf curl virus (TYLCV-IL) and the Spanish strain of tomato yellow leaf curl Sardinia virus (TYLCSV-ES) that was generated most probably in the late 1990s in southern Morocco (Souss). Its emergence in the 2000s coincided with the increasing use of resistant tomato cultivars bearing the Ty-1 gene, and led eventually to the entire displacement of both parental viruses in the Souss. Here, we provide compelling evidence that this viral population shift was associated with selection of TYLCV-IS76 viruses in tomato plants and particularly in Ty-1-bearing cultivars. Real-time quantitative PCR (qPCR) monitoring revealed that TYLCV-IS76 DNA accumulation in Ty-1-bearing plants was significantly higher than that of representatives of the parental virus species in single infection or competition assays. This advantage of the recombinant in Ty-1-bearing plants was not associated with a fitness cost in a susceptible, nearly isogenic, cultivar. In competition assays in the resistant cultivar, the DNA accumulation of the TYLCV-IL clone - the parent less affected by the Ty-1 gene in single infection - dropped below the qPCR detection level at 120 days post-infection (p.i.) and below the whitefly vector (Bemisia tabaci) transmissibility level at 60 days p.i. The molecular basis of the selective advantage of TYLCV-IS76 is discussed in relation to its non-canonical recombination pattern, and the RNA-dependent RNA polymerase encoded by the Ty-1 gene.

The typical RB76 recombination breakpoint of the invasive recombinant tomato yellow leaf curl virus of Morocco can be generated experimentally but is not positively selected in tomato

TYLCV-IS76 is an unusual recombinant between the highly recombinogenic tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV), two Mediterranean begomoviruses (Geminiviridae). In contrast with the previously reported TYLCV/TYLCSV recombinants, it has a TYLCSV derived fragment of only 76 nucleotides, and has replaced its parental viruses in natural conditions (Morocco, Souss region). The viral population shift coincided with the deployment of the popular Ty-1 resistant tomato cultivars, and according to experimental studies, has been driven by a strong positive selection in such resistant plants. However, although Ty-1 cultivars were extensively used in Mediterranean countries, TYLCV-IS76 was not reported outside Morocco. This, in combination with its unusual recombination pattern suggests that it was generated through a rare and possibly multistep process. The potential generation of a recombination breakpoint (RB) at locus 76 (RB76) was investigated over time in 10 Ty-1 resistant and 10 nearly isogenic susceptible tomato plants co-inoculated with TYLCV and TYLCSV clones. RB76 could not be detected in the recombinant progeny using the standard PCR/sequencing approach that was previously designed to monitor the emergence of TYLCV-IS76 in Morocco. Using a more sensitive PCR test, RB76 was detected in one resistant and five susceptible plants. The results are consistent with a very low intra-plant frequency of RB76 bearing recombinants throughout the test and support the hypothesis of a rare emergence of TYLCV-IS76. More generally, RBs were more scattered in resistant than in susceptible plants and an unusual RB at position 141 (RB141) was positively selected in the resistant cultivar; interestingly, RB141 bearing recombinants were detected in resistant tomato plants from the field. Scenarios of TYLCV-IS76 pre-emergence are proposed.

Comparison of RNA extraction methods for the detection of BNYVV rhizomania virus from roots of sugar beet

Rhizomania is one of serious threat to sugar beet production in Morocco and in several parts of the world. This disease led to a statistically significant decrease in the quality and yield of sugar beet plantations. Therefore, this study aimed at comparing the efficacy of six commonly used RNA extraction methods for the detection, recovery of RNA of beet necrotic yellow vein virus (BNYVV) and removal of amplification inhibitors by reverse transcription-polymerase chain reaction (RT-PCR). The efficiency of these extraction methods was then compared to that of a commercial isolation kit with high content of phenolic compounds. The results showed that the extraction with the lithium chloride technique, the commercial kit, and direct and membrane spotting crude extract methods were found effective in yielding a higher purity and a higher concentration of RNA when compared to the other tested methods. Extraction with the lithium chloride technique and the Qiagen kit (RNeasy Plant Mini Kit) allowed the most intense band, whereas the CTAB method has generated the least intense band. Furthermore, the silica capture extraction method did not yield any RNA after extraction and electrophoresis. Consequently, it was concluded that, of these six methods, the lithium chloride technique and the Qiagen kit are the most appropriate for the extraction of viral RNA from sugar beet samples prior to RT-PCR for detecting BNYVV.