M. Marrakchi

Genetic variability and population structure of the wheat foot rot fungus, Fusarium culmorum, in Tunisia

The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic variability and population structure of Fusarium culmorum isolated from wheat stem bases. A total of 108 isolates, representing seven geographically distinct populations, was collected from five climatic regions in Tunisia. Pseudo-allelic frequencies were estimated at each of the 25 putative RAPD loci analyzed by scoring for the presence or absence of amplified fragments; 92 haplotypes were found among the 108 strains. The analysis of the population structure did not reveal any trend with regard to geographic origin. Total gene diversity (HT* = 0.318) was mostly attributable to diversity within populations (HS* = 0.308). Analysis of molecular variance confirmed that most of the genetic variability was within populations. Genetic differentiation among populations was low to moderate (GST* ranged from 0 to 0.190 and averaged 0.041 over all loci). Cluster analysis with UPGMA using genetic distances did not reveal any spatial clustering of the isolates collected from the different geographic regions. Based on these results, we conclude that the F. culmorum isolates recovered from different regions in Tunisia might be part of a single population pool.

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.

Use of Optimised PCR Methods for the Detection of GLRaV3: A Closterovirus Associated with Grapevine Leafroll in Tunisian Grapevine Plants

We report a modification and optimisation of a previously published procedure (Minafra and Hadidi, 1994) for the detection of GLRaV3 in infected grapevine plants. GLRaV3 RNA was successfully detected not only in total crude nucleic acid extracts of infected grapevine tissues but also in viruliferous mealybug extracts by IC-RT-PCR. This detection was rapid, sensitive and specific without occurrence of any background. A comparative ELISA, RT-PCR and IC-RT-PCR assays were carried out and revealed the greater sensitivity and specificity of PCR techniques.

RNA oligoprobe capture RT-PCR, a sensitive method for the detection ofGrapevine fanleaf virus in Tunisian grapevines

A simple, sensitive and specific RNA capture method is described for the detection of Grapevine fanleaf virus (GFLV) in infected grapevines. This method consists of hybridizing GFLV-RNAs to oligoprobes immobilized on nylon membranes, followed by RT-PCR amplification of targeted viral sequences. The RNA oligoprobe capture RT-PCR method is 10-fold more sensitive than IC-RT-PCR. The efficiency of the RNA oligoprobe capture RT-PCR and the reuse of immobilized oligoprobe membranes without loss of efficiency could make this procedure suitable for the routine diagnosis of GFLV in grapevines.

Peach latent mosaic viroid detected for the first time on almond trees in Tunisia.

Almond (Prunus dulcis Mill) is an important crop in countries of the Mediterranean area. Until now, among viroids, only Hop stunt viroid (HSVd) is known to infect cultivated almond trees (2). In 2004, a survey of almond trees was carried out in orchards in different regions of Tunisia, a major producing and exporting country of almond. Symptoms such as mosaic and necrotic lesions, potentially caused by the Peach latent mosaic viroid (PLMVd), were observed on leaves of cultivated almond trees. Since PLMVd was recently detected in peach and pear trees in Tunisia (4), the presence of this viroid in almond trees was studied. The detection method on the basis of one-tube reverse transcription-polymerase chain reaction (RT-PCR) assays was previously described and validated for the detection of this viroid in fruit trees (4). Amplification products were obtained by using previously reported primer pairs of PLMVd (1). Positive controls included RNA preparations of twigs of PLMVd-infected GF 305 peach seedlings. These materials, provided by B. Pradier (Station de Quarantaine des Ligneux, Lempdes, France), were positive as revealed by chip budding on peach seedling indicator plants grown under greenhouse conditions. RT-PCR analysis of nucleic acid preparations from leaves of almond showed specific amplification products with the expected size of 337 bp for two almond trees among 17 trees tested. Nucleotide sequence analyses of cloned amplification products obtained with the PLMVd primers confirmed a size of 337 bp and revealed a sequence similar to sequences from other PLMVd isolates previously characterized. The sequences shared 94 to 98% identity with the reference isolates of PLMVd from peach (EMBL Accession No. M83545, AF170511, AF170514, and AY685181). The two infected almond trees are proximal to each other and peach trees infected with PLMVd. This suggests that one tree may have served as a source of inoculum for the other through agronomic practices such as pruning or the aphid Myzus percicae (3). Alternatively, PLMVd may have originated in an unknown host and was then transmitted to almond trees. Our investigation shows that almond is a new host for PLMVd. References: (1) N. Astruc. Eur. J. Plant Pathol. 102:837, 1996. (2) M. C. Cañizares et al. Eur. J. Plant Pathol. 105:553, 1999. (3) J. C. Desvignes et al. Phytoma 444:70, 1992. (4) I. Fekih Hassen et al. Plant Dis. 88:1164, 2004.

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.

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.