H. Fakhfakh

Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y.

Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7 kb PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immuno-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.

Incidence of potato viruses and characterisation of Potato virus Y variability in late season planted potato crops in Northern Tunisia

Surveys were conducted of symptomatic potato plants in late season crops, from the major potato production regions in Northern Tunisia, for infection with six common potato viruses. The presence of Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) was confirmed serologically with virus infection levels up to 5.4, 90.2, 4.3, 3.8, 7.1 and 4.8%, respectively. As PVY was prevalent in all seven surveyed regions, further biological, serological and molecular typing of 32 PVY isolates was undertaken. Only one isolate was shown to induce PVYO-type symptoms following transmission to tobacco and to react only against anti-PVYO-C antibodies. Typical vein necrosis symptoms were obtained from 31 samples, six of which reacted against both anti-PVYN and anti-PVYO-C antibodies showing they contained mixed isolates, while 25 of them reacted only with anti-PVYN antibodies. An immunocapture RT-PCR molecular test using a PVYNTN specific primer pair set in the 5’NTR/P1 genomic region and examination of recombinant points in three genomic regions (HC-Pro/P3, CI/NIa and CP/3’NTR) showed that all 25 serotype-N PVY isolates were PVYNTN variants with similar recombinations to the standard PVYNTN-H isolate. This is the first report of the occurrence of the PVYNTN variant and its high incidence in late season potatoes in Tunisia.

Sequencing of Australian Grapevine Viroid and Yellow Speckle Viroid isolated from a Tunisian grapevine without passage in an indicator plant

We report the nucleotide sequences of Australian Grapevine Viroid and Grapevine Yellow Speckle Viroid (type 1) isolated from grapevine trees in a Tunisian vineyard. Our data confirm the worldwide spread of these viroids and record their occurrence in Africa. This is the first description of Australian Grapevine Viroid sequences isolated from its natural host. Moreover, the sequences of these new natural variants suggests that the previous use of an indicator plant to amplify a viroid does not alter its nucleotide composition. In addition, we also present several new features of these two distinct quasi-species.

Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars

Understanding plant response to salinity, one of the major abiotic stresses, provides insights into the improvement of tomato salt tolerance. This work focuses on the responses of tomato cultivars to salt stress. Genotypes, representative of content and enzyme activities. QPCR analysis of WRKY, ERF, LeNHX and HKT genes was also performed. A high K+, Ca2+ and proline accumulation as well as a decrease in Na+ concentration mediated salt tolerance. Concomitant with a pattern of high antioxidant enzyme activities, tolerant genotypes also displayed differential patterns of gene expression.

Interaction patterns between potato virus Y and eIF4E-mediated recessive resistance in the Solanaceae.

ABSTRACT The structural pattern of infectivity matrices, which contains infection data resulting from inoculations of a set of hosts by a set of parasites, is a key parameter for our understanding of biological interactions and their evolution. This pattern determines the evolution of parasite pathogenicity and host resistance, the spatiotemporal distribution of host and parasite genotypes, and the efficiency of disease control strategies. Two major patterns have been proposed for plant-virus genotype infectivity matrices. In the gene-for-gene model, infectivity matrices show a nested pattern, where the host ranges of specialist virus genotypes are subsets of the host ranges of less specialized viruses. In contrast, in the matching-allele (MA) model, each virus genotype is specialized to infect one (or a small set of) host genotype(s). The corresponding infectivity matrix shows a modular pattern where infection is frequent for plants and viruses belonging to the same module but rare for those belonging to different modules. We analyzed the structure of infectivity matrices between Potato virus Y (PVY) and plant genotypes in the family Solanaceae carrying different eukaryotic initiation factor 4E (eIF4E)-coding alleles conferring recessive resistance. Whereas this system corresponds mechanistically to an MA model, the expected modular pattern was rejected based on our experimental data. This was mostly because PVY mutations involved in adaptation to a particular plant genotype displayed frequent pleiotropic effects, conferring simultaneously an adaptation to additional plant genotypes with different eIF4E alleles. Such effects should be taken into account for the design of strategies of sustainable control of PVY through plant varietal mixtures or rotations. IMPORTANCE The interaction pattern between host and virus genotypes has important consequences on their respective evolution and on issues regarding the application of disease control strategies. We found that the structure of the interaction between Potato virus Y (PVY) variants and host plants in the family Solanaceae departs significantly from the current model of interaction considered for these organisms because of frequent pleiotropic effects of virus mutations. These mutational effects allow the virus to expand rapidly its range of host plant genotypes, make it very difficult to predict the effects of mutations in PVY infectivity factors, and raise concerns about strategies of sustainable management of plant genetic resistance to viruses.

Incidence and Characterization of Potato virus Y in Seed Potatoes in Tunisia

Surveys during five consecutive years in four main seed potato-growing areas of Tunisia revealed large differences in Potato virus Y (PVY) incidence. Infection rates at harvest ranged from up to 19% in Cap Bon and in Jendouba, to 31% in Kairouan and 48% in Manouba. However, infection rates were very low across the country in 2003 (1–5%), except in Kairouan, and rather low in 2004 (4–10%). Secondary infections in plants growing from imported seed potatoes were assessed to be 1–6% depending on region and year. Serological analysis of a first set of 90 samples collected in 2004–2005 revealed dominance of the PVYN group (90% of the total number of PVY positives). A second set of 44 isolates from samples collected in 2006 was further analyzed with a combination of serotyping, indexing on tobacco, and RT-PCR tests targeting four genomic regions (5′Ntr/P1, HcPro/P3, CI/NIa, CP). Thirty-five of these 44 isolates were typical PVYNTN isolates, with three recombination junctions in HcPro/P3, CI/NIa, and CP regions, respectively, whereas no recombination junction was identified in the genome of five isolates belonging to the PVYN group. One additional recombinant PVYNTN isolate was recovered from a mixed infection with a PVYO isolate. Only three PVYO isolates were recovered from the samples analyzed.

Molecular characterization of Bemisia tabaci populations in Tunisia: genetic structure and evidence for multiple acquisition of secondary symbionts.

ABSTRACT A survey was conducted during 2009–2010 seasons to identify the distribution of Bemisia tabaci (Gennadius) biotypes in Tunisia. The genetic affiliation of collected populations was determined by polymerase chain reaction (PCR)-restriction fragment-length polymorphism (TaqI) of the mitochondrial cytochrom oxidase I (mtCOI) gene. Results, validated by sequencing and phylogenetic analysis, allowed the clustering of sampled sweetpotato whiteflies into B and Q biotypes. As B. tabaci harbors the obligatory bacterium Portiera aleyrodidarum, and a diverse array of secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Cardinium, Arsenophonus, and Fritschea, we report here the infectious status of Tunisian populations by secondary symbionts to find out a correlation between bacterial composition to biotype. The genetic variability and structure of B. tabaci populations in Tunisia was driven by analysis of molecular variance (AMOVA) and the hypothesis of isolation by distance was explored. Selective neutrality and genetic haplotype network tests suggested that Tunisian sweetpotato whiteflies have been undergoing a potential expansion followed by gene flow restriction.

Some Biological and Molecular Properties of Pepper Veinal Mottle Virus Isolates Occurring in Tunisia

The potyviruses, PVY (potato virus Y) and PVMV (pepper veinal mottle virus) were identified by serological and symptom diagnosis in pepper (Capsicum annuum L.) in Tunisia during 1995–1997. Both were known to be major constraints to pepper production in North Africa. Pepper veinal mottle virus (PVMV) has not been reported previously in North Africa, but PVMV was found to be widely spread in peppers throughout Tunisia. By using reverse transcription followed by polymerase chain reaction (RT-PCR), we have now amplified the PVMV coat protein gene of two Tunisian isolates. DNA polymorphism, determined by restriction fragment length polymorphism (RFLP) of the coat protein gene, revealed two patterns distinguishing PVMV from PVY. Within PVMV isolates, molecular and biological properties showed no polymorphism suggesting that all isolates are representative of one PVMV strain occurring in Tunisia.

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

Sequence analysis of three citrus viroids infecting a single Tunisian citrus tree (Citrus, reticulata, Clementine)

We report the nucleotide sequences of three citrus viroids belonging to three different genera: Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus viroid-III (CVd-III) isolated from a single natural infected Citrus reticulata var. Clementine tree growing in a tree nursery in Manouba (near Tunis Capital). We describe the sequence variability of these viroids from their natural host without using an alternative passage by an indicator host or an artificial inoculation. This work confirms that naturally occurring viroid infections contain a mixture of sequence variants. These are the first sequences of citrus viroids from Africa.