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Università degli studi di Catania (2012)

Characterization of Moroccan Citrus Tristeza Virus (CTV) isolates and study of their genomic variability after aphid transmission

Afechtal, Mohamed

Titre : Characterization of Moroccan Citrus Tristeza Virus (CTV) isolates and study of their genomic variability after aphid transmission

Auteur : Afechtal, Mohamed

Université de soutenance  : Università degli studi di Catania

Grade : Tesi di Dottorato 2012

A survey of Citrus tristeza virus (CTV) was carried out in the main Moroccan citrus growing areas (Souss, Gharb, Moulouya, Tadla, Haouz and Loukkos), analyzing by DTBIA a total of 5192 plants. About 20% of the tested trees proved to be CTV-infected in the groves, mainly in Loukkos region with a 60% infection rate. On the contrary, only three CTV infected plants were found in Souss region and one plant in Gharb region. No CTV infection was detected in the surveyed nurseries, plots of budwood sources and varietal collections. CTV vectors were monitored in Gharb and Loukkos regions. Aphis spiraecola (56%) and A. gossypii (32%) were the most abundant aphid species, while no evidence of Toxoptera citricidus was reported. Ten out of 12 selected CTV isolates showed, by coat protein gene sequencing, the highest nucleotide similarity with the T30 mild reference strain (Florida) and the Spanish mild isolate T385, with a sequence homology ranging from 98.6% to 99.4%. The remaining isolates from Loukkos clustered close to the Portuguese 19-21 severe reference strain and to the Puerto Rican Poncirus trifoliata resistance breaking (RB) severe isolate B301 (98.5% to 99.2% homology) ; these two Moroccan isolates induced mild stem pitting symptoms on Duncan grapefruit by biological indexing and reacted positively to the MCA13 monoclonal antibodies. This study confirmed high CTV genetic diversity in some Moroccan citrus groves. The finding of the CTV stem pitting represents a serious threat to the Moroccan citrus industry, mainly if T. citricidus is introduced in the country. Meanwhile, aphid transmission experiments by A. gossypii and A. spiraecola on two local CTV isolates (mild and severe), were carried out in laboratory. The obtained results showed that A. gossypii was able to transmit efficiently the L-Clem2-Mor mild isolate (40%), while less was the transmission rate (13.33%) for the L-Clem1-Mor severe isolate whereas, the mild isolate was transmitted at a rate of 6.67% by A. spiraecola, which was not able to transmit the severe one. The transmission trials showed that the mild CTV isolate was more transmissible than the severe one ; this may explain the prevalence of the mild isolate in the region of Loukkos. The field CTV isolates were compared to their correspondent aphid derived sub-isolates using SSCP and sequencing analysis of the p18, p23 and p25 genes. SSCP analysis showed that the CTV isolates had the same migration patterns as their correspondent aphid derived sub-isolates, whereas, sequencing analysis of the p18, p23 and p25 genes revealed minor changes in the sequences obtained from aphid derived sub-isolates compared to their parental field isolates. Consequently, very limited changes where registered in the corresponding predicted amino acids sequences of the studied genes. Finally, a Tissue Print (TP) real time RT-PCR assay for a rapid differentiation and identification of potential severe strains of CTV was developed and validated for testing a panel of 15 CTV isolates from the Mediterranean area ; the obtained results confirmed their corresponding genotypes. This assay was equally used to test 12 CTV sources from Morocco ; interestingly, the two severe isolates that induced previously mild stem pitting on Duncan grapefruit were assigned as a T36-like genotype through this assay


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