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Bruchus pisorum L., commonly known as the pea weevil, is a cosmopolitan insect pest of pea. Currently, its genetic diversity and population structure are still not well understood. Therefore, an examination of the pea weevil population diversity and evolutionary history will provide insight into efficient pest management strategies. In this study, we sequenced and analysed a 393 bp fragment of the mitochondrial Cytochrome C Oxidase subunit I (COI) gene from 29 samples of B. pisorum collected from the major pea-growing regions in northern Tunisia. Analysis of these sequences in addition to 18 supplementary sequences derived from six countries; Australia, China, Ethiopia, Germany, Italy and U.S.A.; led to the identification of 15 haplotypes among which nine were found in Tunisian samples, and among these, six were specific to Tunisia. The FST values reflected a weak population genetic diversity of B. pisorum in Tunisia. The haplotype network and phylogenetic analysis, including all sequences, supported the low genetic structuring of Tunisian B. pisorum and further revealed the presence of widely shared haplotypes. The presence of common haplotypes in geographically distant areas is mainly related to human intervention that facilitated the worldwide expansion of this pest, through the transportation of infested seeds, suggesting that local B. pisorum populations have different origins and may have resulted from multiple introductions.

Plant microRNAs are crucial for post-transcriptional regulation of gene expression, with some of them particularly involved in regulating several disease resistance (R) genes. This study is a computational screening for microRNAs potentially involved in the regulation of disease resistance in bread wheat Triticum aestivum, through the genome and transcriptome of this crop species. We used plant miRNAs of the superfamily miR-482/2118 to find complementarities with the expressed sequence tags (ESTs) coding NBS-LRR proteins of the bread wheat. This analysis revealed a vast recognition potential, highlighting highly variable miRNA-mRNA hybridization schemes. Precursors of miR-2118/482 sequences that showed a potential for recognizing wheat NBS-LRR ESTs were used as input for similarity search in the T. aestivum transcriptome shotgun assemblies (TSA) database, enabling the identification of a couple of wheat TSA sequences (JV952948.1 and JV851699.1) that were predicted to adopt a stable miRNA/miRNA* duplex structure. The genomic regions corresponding to both predicted tae-miR-2118 genes were determined on wheat chromosomes 2DL and 5AS. The findings of the present study will contribute to a better understanding of R gene expression regulation in wheat.

Mariner -like elements (MLEs) are Class-II transposons that are widely present in diverse organisms and encode a D,D34D transposase motif. MLE sequences from two coleopteran species, Bruchus pisorum and B. rufimanus were obtained using the terminal-inverted repeats (TIRs) of mariner elements belonging to the mauritiana subfamily as primer. The characterized elements were between 1073 and 1302 bp in length and are likely to be inactive, based on the presence of multiple stop codons and/or frameshifts. A single consensus of MLE was detected in B. pisorum and was named Bpmar1 . This element exhibited several conserved amino acid blocks as well as the specific D,D(34)D signature. As for B. rufimanus , two MLE consensuses, designated Brmar1 and Brmar2, were isolated , both containing deletions overlapping the internal region of the transposase. Structural and phylogenetic analysis of these sequences suggested a relatively recent origin of Bpmar1 versus a more ancient invasion of Brmar1 and Brmar2 in their respective host genomes. Given that MLEs are potential mediators of insect resistance and have been used as vectors to transfer genes into host genomes, the MLEs characterized in this study will have valuable implications for selecting appropriate transposable elements in transgenesis. https://doi.org/10.2298/ABS160407111D Received: April 7, 2016; Revised: July 23, 2016; Accepted: July 26, 2016; Published online: November 9, 2016 How to cite this article: Djebbi S, Ben Amara W, Makni Hanem, Makni M, Mezghani-Khemakhem M. Molecular characterization of mariner -like elements in Bruchus pisorum and Bruchus rufimanus (Coleoptera: Bruchidae). Arch Biol Sci. 2017;69(2):353-60.

The bird cherry-oat aphid (Rhopalosiphum padi L.) is a devastating cereal pest that develops high resistance to organophosphate and carbamate insecticides. Because acetylcholinesterase (ACE) is the target of carbamate and organophosphate insecticides, the resistance mechanism usually involves mutations occurring in ACE-encoding genes, Ace1 and Ace2. Here, we describe a novel polymerase chain reaction (PCR)-based method for the diagnosis of resistance cases associated with the point mutation F368L in the Ace2 gene. We amplified a 127 bp DNA fragment from Ace2 gene using a modified reverse primer, and digested the amplification product using SmaI endonuclease. This procedure enabled a simple and rapid distinction between resistant and susceptible genotypes for F368L mutation. Subsequently, we screened 152 R. padi samples, and found that F368L mutation occurred at low frequency, in both the homozygous (R/R) and heterozygous (R/S) states. Based upon the results of this study, we believe that molecular...

In this study, a systematic analysis of Nucleotide-Binding Site (NBS) disease resistance (R) gene family in the barley, Hordeum vulgare L. cv. Bowman, genome was performed. Using multiple computational analyses, we could identify 96 regular NBS-encoding genes and characterize them on the bases of structural diversity, conserved protein signatures, genomic distribution, gene duplications, differential expression, selection pressure, codon usage, regulation by microRNAs and phylogenetic relationships. Depending on the presence or absence of CC and LRR domains; the identified NBS genes were assigned to four distinct groups; NBS–LRR (53.1%), CC-NBS–LRR (14.6%), NBS (26%), and CC-NBS (6.3%). NBS-associated domain analysis revealed the presence of signal peptides, zinc fingers, diverse kinases, and other structural features. Eighty-five of the identified NBS-encoding genes were mapped onto the seven barley chromosomes, revealing that 50% of them were located on chromosomes 7H, 2H, and 3H, with a tendency of NBS genes to be clustered in the distal telomeric regions of the barley chromosomes. Nine gene clusters, representing 22.35% of total mapped barley NBS-encoding genes, were found, suggesting that tandem duplication stands for an important mechanism in the expansion of this gene family in barley. Phylogenetic analysis determined 31 HvNBS orthologs from rice and Brachypodium. 87 out of 96 HvNBSs were supported by expression evidence, exhibiting various and quantitatively uneven expression patterns across distinct tissues, organs, and development stages. Fourteen potential miRNA-R gene target pairs were further identified, providing insight into the regulation of NBS genes expression. These findings offer candidate target genes to engineer disease-resistant barley genotypes, and promote our understanding of the evolution of NBS-encoding genes in Poaceae crops. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1478-6) contains supplementary material, which is available to authorized users.

Hessian fly, Mayetiola destructor (Say), is an endemic pest in Tunisia and is among the most destructive biotic stresses of commercial cereals. Genes in wheat that confer resistance to this insect provide the most efficient and economical means of crop protection. Yet, attempts to breed for resistant cereal genotypes are hampered by the periodic emergence of virulent pest biotypes that are unaffected by the plant's resistance factors in use. Therefore, it is crucial to undertake permanent surveys in order to establish baseline information on the status of germplasm resources. A survey of infestation was conducted during the growing season 2011/2012, over the four most important cereal-growing provinces of Tunisia (Beja, Jendouba, Kef and Siliana), which highlighted variation in the incidence and severity of attacks between regions and periods of the year. We recorded two intense outbreaks occurring in early March and late April, suggesting that eradication should be applied in the month of February to hin...

The pea aphid, Acyrthosiphon pisum (Hemiptera: Aphidae), and the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), are two major pests that conjunct in their virulence pathways against hosts by settling a gene-for-gene interaction model. The most effective controlling method of these pests is the deployment of host resistance (R) genes, which strongly depends on both host and insect genotypes. MicroRNAs (miRs) are noncoding RNAs of ∼22 nucleotides inducing sequence-specific post-transcriptional gene silencing. The identification and analysis of miRs is nowadays a useful reverse genetics tool for studying insect virulence and host resistance pathways. in the present study, we utilized 15 virulence-regulatory miRs of the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), to search for homologies across 103 A. pisum mature miRs and 86 D. citri miR-like expressed sequence tags (ESTs), mined from several databases. Our results showed that reference miRs from M. destructor could be grouped with their most similar microRNA counterparts from A. pisum and D. citri within eight phylogenetic clades, and that seven of A. pisum miRs clustered with M. destructor ones at a similarity threshold of 90 %. Such structural similarities strongly suggest that different avirulence (Avr) genes from M. destructor, A. pisum and D. citri would be tightly regulated by a regulatory network including similar miRs. Our study offers a promising way to further explore the structural similarity between miRs toward their potential use in Integrated Pest Management (IPM).

The black bean aphid, Aphis fabae Scopoli is a polyphagous aphid causing significant damage to several cultivated and uncultivated plants. In the present work, we have used COI barcoding to distinguish between three subspecies of A. fabae, frequently found in Tunisia, namely A. f. fabae, A. f. solanella and A. f. cirsiiacanthoidis. Results of our study revealed that COI barcoding was efficient to generate haplotypes discriminating A. f. fabae from the two remaining subspecies. Indeed, among seven haplotypes, identified and named H1–H7, five were found exclusively in A. f. fabae, one represented exclusively A. f. cirsiiacanthoidis, and one was shared by A. f. cirsiiacanthoidis and A. f. solanella. However, the differentiation of Aphis f. fabae from A. f. solanella and A. f. cirsiiacanthoidis was not further supported by a phylogenetic distinction, as it was revealed by neighbour-joining and maximum parsimony phylogenies. Such a coherent genetic structure of the A. fabae complex, not governed by the subspecies factor, as inferred from our present mitochondrial (mt)DNA data, is in agreement with previous analyses based on genomic DNA. Therefore, we hypothesize that the phenotypical and physiological processes having led to differentiation between subspecies would have arisen from weak selection pressures, thus resulting in little genetic change between subspecies. In combination with additional still-to-come data from additional markers, results of this study will be important to constitute a groundwork for pest integrated management.

Pea (Pisum sativum) is one of the most cultivated le-gumes in the world, and its yield and seed quality are affected by a variety of pathogens. In plants, NBS-LRR (nucleotide binding site-leucine-rich repeat) is the main class of disease resistance genes. Using degenerate primers deduced from conserved motifs in the NBS domain of known resistance genes, we identified 10 NBS sequences in three varieties of P. sativum. The deduced amino acid sequences of the iden-tified resistance gene analogues (RGAs) exhibited the typical motifs of the NBS domain (P-loop, kinase-2, kinase-3a, and the hydrophobic domain, GLPL) present in the majority of plant proteins belonging to the NBS-LRR class. Phylogenetic analysis showed that seven RGAs belonged to the non-TIR-NBS-LRR subclass and three to the TIR-NBS-LRR subclass. The results of this study provide insights into the structure of this class of resistance genes in the pea, and their evolution-ary relationships with those of other plant species.

Host resistance is the most economical, effective and ecologically sustainable method of controlling diseases in crop plants. In bread wheat, despite the high number of resistance loci that have been cataloged to date, only few have been cloned, underlying the need for genomics-guided investigations capable of providing a prompt and acute knowledge on the identity of effective resistance genes that can be used in breeding programs. Proteins with a nucleotide-binding site (NBS) encoded by the major plant disease resistance (R) genes play an important role in the responses of plants to various pathogens. In this study, a comprehensive analysis of NBS-encoding genes within the whole wheat genome was performed, and the genome scale characterization of this gene family was established. From the recently published wheat genome sequence, we used a data mining and automatic prediction pipeline to identify 580 complete ORF candidate NBS-encoding genes and 1,099 partial-ORF ones. Among complete gene models, 464 were longer than 200 aa, among them 436 had less than 70 % of sequence identity to each other. This gene models set was deeply characterized. (1) First, we have analyzed domain architecture and identified, in addition to typical domain combinations, the presence of particular domains like signal peptides, zinc fingers, kinases, heavy-metal-associated and WRKY DNA-binding domains. (2) Functional and expression annotation via homology searches in protein and transcript databases, based on sufficient criteria, enabled identifying similar proteins for 60 % of the studied gene models and expression evidence for 13 % of them. (3) Shared orthologous groups were defined using NBS-domain proteins of rice and Brachypodium distachyon. (4) Finally, alignment of the 436 NBS-containing gene models to the full set of scaffolds from the IWGSC’s wheat chromosome survey sequence enabled high-stringence anchoring to chromosome arms. The distribution of the R genes was found balanced on the three wheat sub-genomes. In contrast, at chromosome scale, 50 % of members of this gene family were localized on 6 of the 21 wheat chromosomes and ~22 % of them were localized on homeologous group 7. The results of this study provide a detailed analysis of the largest family of plant disease resistance genes in allohexaploid wheat. Some structural traits reported had not been previously identified and the genome-derived data were confronted with those stored in databases outlining the functional specialization of members of this family. The large reservoir of NBS-type genes presented and discussed will, firstly, form an important framework for marker-assisted improvement of resistance in wheat, and, secondly, open up new perspectives for a better understanding of the evolution dynamics of this gene family in grass species and in polyploid systems.

International audience; The tomato leafminer, Tuta absoluta (Meyrick), (Lepidoptera: Gelechiidae), is a devastating pest of tomato that has recently become a major threat to tomato production in many countries in the Mediterranean Basin, Africa and Asia. Mariner-like elements (MLEs) areclass-II transposons with short inverted terminal repeats (ITR) flanking a single open reading frame (ORF) coding for a transposase. In the present study, we have isolated eight full-length MLEs from the tomato leaf miner by PCR amplification, using a single primerdesigned from the ITR of Hyalophora cecropia L. Mariner signature sequence D,D(34)D, as well as the conserved amino acid motifs, WVPHEL and YSPDLAP(T)D, were identified. However, sequences analysis revealed defective elements with the presence of several kindsof mutations generating stop codons, frameshifts, and non-functional transposase. Based on predicted transposase sequences, T. absoluta MLEs (Tamar1) belongs to the cecropia subfamily and clustered closely with MLEs from other Lepidoptera members suggestingvertical genetic transmission from a common ancestor. The identification of mariner-like elements in this invasive species will be useful to understand the genome dynamics in Lepidoptera.

In crop improvement, the isolation, cloning and transfer of disease resistance genes (R-genes) is an ultimate goal usually starting from tentative R-gene analogs (RGAs) that are identified on the basis of their structure. For bread wheat, recent advances in genome sequencing are supporting the efforts of wheat geneticists worldwide. Among wheat R-genes, nucleotide-binding site (NBS)-encoding ones represent a major class. In this study, we have used a polymerase chain reaction-based approach to amplify and clone NBS-type RGAs from a bread wheat cultivar, 'Salambo 80.' Four novel complete ORF sequences showing similarities to previously reported R-genes/RGAs were used for in silico analyses. In a first step, where analyses were focused on the NBS domain, these sequences were phylogenetically assigned to two distinct groups: a first group close to leaf rust Lr21 resistance proteins; and a second one similar to cyst nematode resistance proteins. In a second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure enabled identifying 8 loci ranging in size between 2,115 and 7,653 bp. Ab initio gene prediction identified 8 gene models, among which two had complete ORFs. While GenBank survey confirmed the belonging of sequences to two groups, subsequent characterization using IWGSC genomic and proteomic data showed that the 8 gene models, reported in this study, were unique and their loci matched scaffolds on chromosome arms 1AS, 1BS, 4BS and 1DS. The gene model located on 1DS is a pseudo-Lr21 that was shown to have an NBS-LRR domain structure, while the potential association of the RGAs, here reported, is discussed. This study has produced novel R-gene-like loci and models in the wheat genome and provides the first steps toward further elucidation of their role in wheat disease resistance.

The Arabian rhinoceros beetle, Oryctes agamemnon arabicus (Burmeister, 1847), is an invasive species, introduced to Tunisia from the United Arab Emirates and causing serious damage to date palm trees in southern Tunisia. Considering the economic importance of this pest, it is necessary to understand the genetic diversity within and among its populations and investigate its dispersal mode. In the present study, the mitochondrial gene encoding the cytochrome oxidase subunit I (COI) was used to analyse the extent of genetic variation between samples of O. agamemnon arabicus, collected from geographically distant locations in Tunisia. A 505 bp DNA fragment, resulting from PCR amplification, was sequenced. Representative sequences were aligned, revealing nine polymorphic sites that identified eight haplotypes. Haplotype diversity (Hd) and Tajima’s D neutral test were 0.707 and –0.043, respectively, indicating a low level of mtDNA variability. The calculated gene flow value was Nm = 0.47, indicating a high amount of gene flow occurring between populations from Tozeur and those from Kebili, although both departments are separated by the Chott-El-Jerid salt lake. The presence of common haplotypes between Tozeur and Kebili as well as the clustering pattern obtained by the NJ method confirmed this result. Based on these facts and considering the limited flight of O. agamemnon arabicus and its slow reproduction, it is likely that its expansion in Tunisia was facilitated mainly by human transportation of infected offshoots.

International audience; Genomic variation among species is commonly driven by transposable element (TE) invasion; thus, the pattern of TEs in a genome allows drawing an evolutionary history of the studied species. This paper reports in vitro and in silico detection and characterization of irritans mariner-like elements (MLEs) in the genome and transcriptome of Bactrocera oleae (Rossi) (Diptera: Tephritidae). Eleven irritans MLE sequences have been isolated in vitro using terminal inverted repeats (TIRs) as primers, and 215 have been extracted in silico from the sequenced genome of B. oleae. Additionally, the sequenced genomes of Bactrocera tryoni (Froggatt) and Bactrocera cucurbitae (Diptera: Tephritidae) have been explored to identify irritans MLEs. A total of 129 sequences from B. tryoni have been extracted, while the genome of B. cucurbitae appears probably devoid of irritans MLEs. All detected irritans MLEs are defective due to several mutations and are clustered together in a monophyletic group suggesting a common ancestor. The evolutionary history and dynamics of these TEs are discussed in relation with the phylogenetic distribution of their hosts. The knowledge on the structure, distribution, dynamic, and evolution of irritans MLEs in Bactrocera species contributes to the understanding of both their evolutionary history and the invasion history of their hosts. This could also be the basis for genetic control strategies using transposable elements.

Mutation in the gene encoding the enzyme acetylcholinesterase (AChE) of the potato aphid, Macrosiphum euphorbiae , associated with resistance to a carbamate insecticide has been characterized. One point mutation producing modification in the predicted amino acid sequence of the product of the M. euphorbiae ace1 gene in resistant vs. susceptible strains has been identified. This modification occurs at one site that is identical to mutation previously described for another aphid species Aphis gossypii . The results obtained here also suggest that the widespread use of carbamate insecticides will likely lead to a predictable acquisition of resistance in wild populations of M. euphorbiae as well as other pest species. For surveys of M. euphorbiae populations that may develop resistance, diagnostic tests using PCR-RFLP based methods for detecting the presence of this mutation in individuals are described.

Using an enriched library method, seven polymorphic microsatellite loci were isolated from the barley stem gall midge, Mayetiola hordei. Polymorphism at loci was surveyed on 57 individual midges collected on barley in Tunisia. Across loci, polymorphism ranged from two to six alleles per locus. The observed heterozygosity varied between 0.070 and 0.877. Based on the number of alleles detected and the associated levels of heterozygosity, we believe that these loci will prove useful for population genetic studies on M. hordei.

The greenbug Schizaphis graminum, is a major pest of wheat worldwide. Biotype screening of this pest is essential to develop pest management programs. In this research, eight greenbug clones, collected on wheat in the cereal-growing region of Béja (north Tunisia), were used to determine their damage on six reference wheat cultivars. All tested clones shared a unique biotypic profile, similar to biotype C. Moreover, DNA from the tested clones and that from seven reference clones of biotypes C, E, F, G, H, I and K, was analyzed, using 5 RAPD-PCR primers. The UPGMA method clustered samples into two distinct clades: a first one (I) included clones from north Tunisia, which were clearly associated to agricultural biotypes C, E, I and K, while a second clade (II) included non agricultural biotypes F, G and H. Results reported in this paper suggest that resistance genes Gb2, Gb3, Gb4, Gb5 and Gb6 in wheat would be the most efficient if used in wheat improvement programs for resistance against greenbug in Tunisia.

The tomato leaf miner Tuta absoluta Meyrick has invaded tomato (Solanum lycopersicum L.) crop in Tunisia since 2008 and is representing today a major threat to the production of this crop. In this study, we used the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) technology to assess the genetic variability within and among seven populations of T. absoluta, collected on tomato from different regions in Tunisia. Using five RAPD-PCR primers and 108 individuals, 140 polymorphic fragments were recorded. From 335 different RAPD phenotypes generated, 71 were redundant and 264 unique to a specific population. The genetic structure of T. absoluta was investigated using analysis of molecular variance (AMOVA), genetic distances (Fst) and multidimensional scaling (MDS). We detected a high genetic diversity within and among populations in conjunction with a significant differentiation between populations, suggesting that different founder genotypes would have been responsible of the introduction of T. absoluta in Tunisia. The presence of overlapping phenotypes probably indicates migration events between populations, mainly through infested plant material carried by humans.El minador de hojas de tomate Tuta absoluta Meyrick ha invadido el cultivo del tomate (Solanum lycopersicum L.) en Túnez desde 2008 y actualmente representa una importante amenaza para su producción. En este estudio usamos la tecnología de ADN polimórfico amplificado al azar-reacción de cadena polimerasa (RAPD-PCR) para evaluar la variabilidad genética dentro y entre siete poblaciones de T. absoluta, colectadas desde tomate en diferentes regiones de Túnez. Usando cinco primers RAPD-PCR y 108 individuos, se registraron 140 fragmentos polimórficos. Se generaron 335 fenotipos RAPD diferentes, entre los cuales 71 fueron redundantes y 264 únicos para una población específica. La estructura genética de T. absoluta se investigó usando análisis de varianza molecular (AMOVA), distancias genéticas (Fst) y escalamiento multidimensional (MDS). Detectamos una alta diversidad genética dentro y entre poblaciones en conjunto con una diferenciación significativa entre poblaciones, sugiriendo que los genotipos fundadores podrían haber sido responsables de la introducción de T. absoluta en Túnez. La presencia de fenotipos superpuestos probablemente indica eventos de migración entre poblaciones, principalmente a través de material vegetal infestado transportado por humanos.

The potato aphid Macrosiphum euphorbiae (Thomas) is a major pest of several economic crops in Tunisia. Using 10 microsatellites, we analyzed five populations of M. euphorbiae sampled during the 2004–2005 solanaceous and cucurbit season (April through September) from five geographic origins. From 235 aphids, 61 different multilocus genotypes were identified of which three genotypes MLG1, MLG2, and MLG42 were predominant on all host plants and regions. MLG1 and MLG2 genotypes were detected in 2004 and did not reappear in spring 2005, while the genotype MLG42 was detected only in 2005. All populations showed significant deviation from Hardy—Weinberg equilibrium even in data sets including one individual per genotype, χ 2 independence tests and analysis of molecular variance showed no significant differentiation among populations collected on different host plant and from different geographic origin, but differences between populations from two successive years were significant. Factorial component a...

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova) showed that the genetic structure was not associated with geographic location or year of collection (P = 0.70 and 0.34, respectively); however, the host-plant had a significant effect on the partitioning of the total genetic diversity (P

Hessian fly, Mayetiola destructor (Say), is a destructive pest of wheat worldwide and an endemic pest in Tunisia. Two natural populations of this insect from the North of Tunisia were evaluated, in the field, for their virulence, based on response developed by bread wheat (Triticum aestivum L.) cultivars carrying H3, H5, H6, H7H8, H11, H13 and H16 resistance genes. H11, H13 and H16 showed a high effectiveness against both populations; therefore, their implication in Hessian fly breeding programs would be of interest. The level of infestation, as well as the yield loss, was assessed, based on the percentage of infested plants and variation in growth parameters due to infestation. The percentage of infested plants, over a 2-yr period in Mateur, averaged 18.82% for durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.) and 32.50% for bread wheat. For the improved durum wheat cv. Karim used as reference, the plant height, number of internodes, number of productive tillers per plant, and 100-seed weight were negatively affected by infestation, while the number of tillers per plant was positively affected. Aiming to update information about the annual number of the fly generations occurring on wheat, we surveyed infestation in Jédéida. At least three Hessian fly generations were detected on bread wheat and durum wheat. Continued regular surveying of Hessian fly populations in terms of virulence, impact on yield and annual generations is required for optimal deployment of resistance genes and integrated management of Hessian fly across all wheat production areas.La mosca de Hess, Mayetiola destructor (Say), es una plaga mundial destructiva del trigo y endémica en Túnez. Se evaluaron dos poblaciones naturales de este insecto desde el Norte de Túnez, en el campo, por su virulencia, basado en la respuesta desarrollada por cultivares de trigo panadero (Triticum aestivum L.) portando los genes de resistencia H3, H5, H6, H7H8, H11, H13 y H16. H11, H13 and H16 mostraron una alta efectividad contra ambas poblaciones; por lo tanto, su implicancia en programas de producción de mosca de Hess podría ser de interés. Se evaluaron el nivel de infestación y la pérdida de rendimiento, basado en el porcentaje de plantas infestadas y variación en parámetros de crecimiento debidos a infestación. El porcentaje de plantas infestadas, en un período de 2 años en Mateur, promedió 18,82% para trigo candeal (Triticum turgidum L. subsp. durum (Desf.) Husn.) y 32,50% para trigo panadero. Para el trigo candeal mejorado cv. Karim usado como referencia, altura de planta, número de entrenudos, número de cañas productivas por planta, y peso de 100 semillas fueron negativamente afectados por infestación, mientras el número de cañas por planta fue positivamente afectado. Con el fin de actualizar la información sobre el número anual de las generaciones de mosca en trigo, investigamos infestación en Jédeida. Al menos tres generaciones de mosca de Hess se detectaron en trigo panadero y candeal. Se requieren ensayos regulares de virulencia, impacto en rendimiento, y generaciones anuales de poblaciones de mosca de Hess para un óptimo despliegue de genes de resistencia y manejo integrado de la mosca por todas las áreas productoras de trigo.

In this study, the genetic structure of 15 populations of the potato aphid, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), from Tunisia was characterized using RAPD markers. Five decameric random primers revealed 113 polymorphic loci that were used to estimate population structure with an average of 22.6 polymorphic bands per primer. Analysis of molecular variance (AMOVA) showed significant (P < 0.001) genetic diversity among populations (78.16 %) rather than within populations (21.84 %) that has been confirmed by Fst and Nm values. A cluster analysis based on genetic distance revealed that the 15 populations were divided into three groups. Populations from the south were differentiated into one group irrespective of host plant, whereas the other populations were distributed between two groups independently of the geographic origin and host plant. These results represent the first evidence for genetic structuring in M. euphorbiae.

In the present study, we have screened 71, 713, 525, 119 and 241 mature miRNA variants from Hordeum vulgare , Oryza sativa , Brachypodium distachyon , Triticum aestivum , and Sorghum bicolor , respectively, and classified them with respect to their conservation status and expression levels. These Poaceae non-redundant miRNA species (1,669) were distributed over a total of 625 MIR families, among which only 54 were conserved across two or more plant species, confirming the relatively recent evolutionary differentiation of miRNAs in grasses. On the other hand, we have used 257 H. vulgare , 286 T. aestivum , 119 B. distachyon , 269 O. sativa , and 139 S. bicolor NBS domains, which were either mined directly from the annotated proteomes, or predicted from whole genome sequence assemblies. The hybridization potential between miRNAs and their putative NBS genes targets was analyzed, revealing that at least 454 NBS genes from all five Poaceae were potentially regulated by 265 distinct miRNA species, most of them expressed in leaves and predominantly co-expressed in additional tissues. Based on gene ontology, we could assign these probable miRNA target genes to 16 functional groups, among which three conferring resistance to bacteria (Rpm1, Xa1 and Rps2), and 13 groups of resistance to fungi (Rpp8,13, Rp3, Tsn1, Lr10, Rps1-k-1, Pm3, Rpg5, and MLA1,6,10,12,13). The results of the present analysis provide a large-scale platform for a better understanding of biological control strategies of disease resistance genes in Poaceae, and will serve as an important starting point for enhancing crop disease resistance improvement by means of transgenic lines with artificial miRNAs.

Inter-simple sequence repeats (ISSR) polymorphism was used to reveal genetic variability and phylogenetic relationships within and between three haplotypes of Mayetiola species . A set of 14 ISSR primers were screened representing di-, tri, tetra and penta-nucleotide repeats out of which 10 generated scorable bands and three were able to distinguish one of three haplotypes. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered the two Mayetiola species according to their mitochondrial haplotype. Moreover, genetic diversity estimated by the coefficient of variation indicates a high intra and inter-haplotypes polymorphism. Our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships of Mayetiola haplotypes. African Journal of Biotechnology Vol. 4 (7), pp. 601-606, 2005

Backround The melon fruit fly Bactrocera cucurbitae and the peach fruit fly Bactrocera zonata are serious pests, native to Asia, that have recently invaded Egypt. In Tunisia, no report of these species has yet been made, but pest risk data suggest that both Bactrocera species are likely to establish in other countries of the Mediterranean region. Results A quick method, based on a PCR-RFLP of the mitochondrial COI gene, has been developed to enhance species identification for quarantine purposes. The restriction enzymes DdeI and XmnI were used to digest COI PCR products, enabling a rapid diagnosis of B. cucurbitae and B. zonata and their distinction from Tunisia's most devastating fruit pest, Ceratitis capitata. Conclusion The simplicity and relatively low cost of this molecular approach will replace the need to rear immature stages through to adults for identification and will facilitate rapid quarantine decisions providing greater plant protection.

Barley yellow dwarf disease is a worldwide ubiquitous virus disease of cereal crops. In order to characterize the B/CYDV isolates occurring in Tunisia, 240 barley leaves were randomly sampled from 6 fields following a North-South trend and analyzed by serological and molecular tests. DAS-ELISA results showed 40 positive samples with a prevalence of barley yellow dwarf virus (BYDV)-PAV (77.5%), followed by cereal yellow dwarf virus (CYDV)-RPV (25%) and BYDV-MAV (15%). Studies of the geographic distribution showed a high incidence of B/CYDV in the Tunisian Southern provinces. RT-PCR assays were performed to amplify the viral coat protein gene (CP) and sequence analyses revealed six BYDV-PAV haplotypes named PAV-TN1 to PAV-TN6. Phylogenetic analysis showed that the six Tunisian haplotypes were close to BYDV-PAV-II subspecies and had a strong similarity with Moroccan, Czech, French and German haplotypes. Although PAV-TN2 and PAV-TN5 showed up to 10% divergence from BYDV-PAV-II at the amino acid level, it seems to belong to the same subspecies but in a separated cluster. Our results will be important in developing appropriate control measures against BYDV disease in Tunisia.

International audience; Mariner-like elements (MLEs) are Class II transposons present in all eukaryotic genomes in which MLEs have been searched for. This article reports the detection of MLEs in seven of the main fruit tree aphid species out of eight species studied. Deleted MLE sequences of 916–919 bp were characterized, using the terminal-inverted repeats (TIRs) of mariner elements belonging to the mauritiana Subfamily as primers. All the sequences detected were deleted copies of full-length elements that included the 3′- and 5′-TIRs but displayed internal deletions affecting Mos1 activity. Networks based on the mtDNA cytochrome oxidase subunit-I (CO-I) and MLE sequences were incongruent, suggesting that mutations in transposon sequences had accumulated before speciation of tree aphid species occurred, and that they have been maintained in this species via vertical transmissions. This is the first evidence of the widespread occurrence of MLEs in aphids.

Proteins containing nucleotide binding sites (NBS) encoded by plant resistance genes play an important role in the response of plants to a wide array of pathogens. In this paper, an in silico search was conducted in order to identify and characterize members of NBS-encoding gene family in the tribe of Triticeae. A final dataset of 199 sequences was obtained by four search methods. Motif analysis confirmed the general structural organization of the NBS domain in cereals, characterized by the presence of the six commonly conserved motifs: P-loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C and GLPL. We revealed the existence of 11 distinct distribution patterns of these motifs along the NBS domain. Four additional conserved motifs were shown to be significantly present in all 199 sequences. Phylogenetic analyses, based on genetic distance and parsimony, revealed a significant overlap between Triticeae sequences and Coiled coil-Nucleotide binding site-Leucine rich repeat (CNL)-type functional genes from monocotyledons. Furthermore, several Triticeae sequences belonged to clades containing functional homologs from non Triticeae species, which has allowed for these sequences to be functionally assigned. The findings reported, in this study, will provide a strong groundwork for the isolation of candidate R-genes in Triticeae crops and the understanding of their evolution.

The tomato leaf miner Tuta absoluta Meyrick has invaded tomato ( Solanum lycopersicum L.) crop in Tunisia since 2008 and is representing today a major threat to the production of this crop. In this study, we used the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) technology to assess the genetic variability within and among seven populations of T. absoluta, collected on tomato from different regions in Tunisia. Using five RAPD-PCR primers and 108 individuals, 140 polymorphic fragments were recorded. From 335 different RAPD phenotypes generated, 71 were redundant and 264 unique to a specific population. The genetic structure of T. absoluta was investigated using analysis of molecular variance (AMOVA), genetic distances (Fst) and multidimensional scaling (MDS). We detected a high genetic diversity within and among populations in conjunction with a significant differentiation between populations, suggesting that different founder genotypes would have been responsible of the introduction of T. absoluta in Tunisia. The presence of overlapping phenotypes probably indicates migration events between populations, mainly through infested plant material carried by humans.

The root borer (Rhinoceros beetle), Oryctes agamemnon (Burmeister, 1847) is an invasive coleopteran pest of date palm trees in southwestern Tunisia introduced accidentally in “Mrah Lahwar” (Department of Tozeur), spread out into “Rjim Maatoug” (Department of Kebili), then into most oases of Tozeur and Kebili departments, where it is now causing a serious damage. We used 101 samples of O. agamemnon collected from 7 oases and 2 date palm varieties, in order to assess several invasion parameters (e.g. level and distribution of genetic diversity, degree of gene flow between populations and number of introduction events). Analysis of molecular variance (AMOVA) of the random amplified polymorphic DNA (RAPD) markers exhibited no significant genetic differentiation in relation with departments (Tozeur vs. Kebili) or host plant varieties (Deglet Nour vs. Kenta), raising the hypothesis that a limited number of founder genotypes would have been at the origin of invasion. A substantial gene flow was revealed among populations, suggesting that the expansion of O. agamemnon, in Tunisian southwestern oases, has been most likely facilitated by human agency, through the propagation of date palm. These preliminary results would provide a framework for more detailed studies on introduced populations of O. agamemnon, in order to help the development of optimized management methods of this insect. Key words: Insecta, Coleoptera, Oryctes agamemnon, introduced populations, gene flow, molecular markers.

Ten microsatellites were isolated from the barley stem gall midge, Mayetiola hordei. Polymorphism at each locus was tested on 40 individual midges, among which 34 were collected on barley and six on wheat crops in Tunisia. Six loci were polymorphic with the number of alleles ranging from two to seven. The observed heterozygosity varied between 0.025 and 0.2. These microsatellite loci revealed a strong effect of host plant on the population genetic structure of M. hordei.

International audience; Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)(10) (GT)(10) and (GATA)(4). Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.

Sugarcane yellow leaf virus (ScYLV) causes severe leaf symptoms in sugarcane (Saccharum spp.). It is a single-stranded RNA virus assigned to the genus Polerovirus, family Luteoviridae (1). ScYLV is transmitted by two aphid species, Melanaphis sacchari and Rhopalosiphum maidis. Although barley (Hordeum vulgare), oats (Avena sativa), and wheat (Triticum spp.) are susceptible to ScYLV when experimentally inoculated (3), this virus, related serologically to Barley yellow dwarf virus (BYDV)-RPV (4), has never been detected naturally in these cereals. In this study, 240 barley leaves were randomly collected from six fields in Tunisia following a north-south trend during the high infestation periods (March/April) in the 2013 growing season. Samples were tested by DAS-ELISA, using three antibodies (Bioreba AG, Switzerland), two of them, BYDV-B and BYDV-F, specific to luteoviruses corresponding to BYDV-PAV and BYDV-MAV, respectively, and the third one, BYDV-RPV, specific to the polerovirus synonymous to Cereal yellow dwarf virus (CYDV)-RPV. Based on DAS-ELISA, 30 samples were found positive for B/CYDV infection; 17 out of the 30 infected samples contained a single serotype, BYDV-PAV, and 13 out of the 30 infected samples contained two serotypes, PAV and RPV. Total RNA was extracted from all positive samples, and RT-PCR of the viral CP gene was performed with Lu1/Lu4 primers (2). A product of 531 bp was cloned and sequenced. The identities among the sequences determined varied between 80 to 100%, and from the 17 samples containing BYDV-PAV, six distinct BYDV-PAV sequences were revealed and named PAV-TN1 to PAV-TN6 (GenBank Accession No. JX402453 to JX402457 and KF271792). Fortuitously, all 13 positive samples corresponding to the serotypes PAV-RPV exhibited 98.7 to 99.3% identity with ScYLV isolates. These 13 samples contained three distinct sequences that were named ScYLV-Tun1 to ScYLV-Tun3 (GenBank Accession No. KF836888 to KF836890). Of the 17 PAV-positive samples collected, six were infected with PAV-TN1, four with PAV-TN2, four with PAV-TN3, one with PAV-TN4, one with PAV-TN5, and the last one with PAV-TN6. Of the 13 ScYLV-positive samples, seven were infected with ScYLV-Tun1, four with ScYLV-Tun2, and two with ScYLV-Tun3. Phylogenetic analysis showed that PAV-TN sequences formed a very tight cluster (>98%) corresponding to BYDV subspecies PAV-II, whereas all three Tunisian ScYLV sequences were clustered together. This study provides the first report of ScYLV isolates infecting barley crops in Tunisia, and confirms serological cross-reactivity between ScYLV and BYDV-RPV when commercial antibodies against BYDV-RPV are used. References: (1) C. J. D'Arcy and L. L. Domier. Page 891 in: Virus Taxonomy, 8th Report of the ICTV. C. M. Fauquet et al., eds. Springer-Verlag, New York, 2005. (2) N. L. Robertson and R. French. J. Gen. Virol. 72:1473, 1991. (3) S. Schenck and A. T. Lehrer. Plant Dis. 84:1085, 2000. (4) J. Vega et al. Plant Dis. 81:21, 1997.

In Morocco, two sympatric species of Mayetiola (destructor and hordei) have been identified on the basis of microscopic characters. Barley is almost exclusively colonized by M. hordei and presents distinct stem swellings (galls), whereas wheat is almost exclusively colonized by M. destructor and has no gall. In Tunisia, previous studies have shown that Mayetiola infestations of wheat or barley never produce galls in the northern region, whereas galls are frequent on barley in the center and the rule in the south. In the present study, we have investigated the electrophoretic polymorphism at four loci of Mayetiola collected on field plants presenting or lacking galls from northern and central Tunisia. We showed that, in central Tunisia, two reproductively isolated groups were present in all samples from wheat, barley and oat whether these plants carried gall or not. In contrast, only one of these groups was found in samples from wheat and barley lacking galls from the north. On the basis of a comparison with Moroccan strains of the two species, these groups were identified as M. hordei and M. destructor which can easily be characterized by diagnostic alleles at two loci, Pgi and Mdh2.

Hessian fly, Mayetiola destructor (Say), is a destructive pest of wheat worldwide and an endemic pest in Tunisia. Two natural populations of this insect from the North of Tunisia were evaluated, in the field, for their virulence, based on response developed by bread wheat (Triticum aestivum L.) cultivars carrying H3, H5, H6, H7H8, H11, H13 and H16 resistance genes. H11, H13 and H16 showed a high effectiveness against both populations; therefore, their implication in Hessian fly breeding programs would be of interest. The level of infestation, as well as the yield loss, was assessed, based on the percentage of infested plants and variation in growth parameters due to infestation. The percentage of infested plants, over a 2-yr period in Mateur, averaged 18.82% for durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.) and 32.50% for bread wheat. For the improved durum wheat cv. Karim used as reference, the plant height, number of internodes, number of productive tillers per plant, and 100-seed weight were negatively affected by infestation, while the number of tillers per plant was positively affected. Aiming to update information about the annual number of the fly generations occurring on wheat, we surveyed infestation in Jédéida. At least three Hessian fly generations were detected on bread wheat and durum wheat. Continued regular surveying of Hessian fly populations in terms of virulence, impact on yield and annual generations is required for optimal deployment of resistance genes and integrated management of Hessian fly across all wheat production areas.