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Transposable elements (TEs) are genetically mobile units that move from one site to another within a genome. These units can mediate regulatory changes that can result in massive changes in genes expression. In fact, a precise identification of TEs can allow the detection of the mechanisms involving these elements in gene regulation and genome evolution. In the present study, a genome-wide analysis of the Hemipteran pest Bemisia tabaci was conducted using bioinformatics tools to identify, annotate and estimate the age of TEs, in addition to their insertion sites, within or near of the defensome genes involved in insecticide resistance. Overall, 1,292,393 TE copies were identified in the B. tabaci genome grouped into 4872 lineages. A total of 699 lineages were found to belong to Class I of TEs, 1348 belong to Class II, and 2825 were uncategorized and form the largest part of TEs (28.81%). The TE age estimation revealed that the oldest TEs invasion happened 14 million years ago (MYA) and the most recent occurred 0.2 MYA with the insertion of Class II TE elements. The analysis of TE insertion sites in defensome genes revealed 94 insertions. Six of these TE insertions were found within or near previously identified differentially expressed insecticide resistance genes. These insertions may have a potential role in the observed insecticide resistance in these pests.

International audience; Plants harbor diverse and taxonomically structured communities of protective microorganisms that confer beneficial characteristics in plant growth and protection against biotic and abiotic stresses. The aim of this study is to characterize the composition of endophytic bacterial communities in leaves and rhizobacterial communities of the flood affected and unaffected apricot trees by 16S rRNA genes metabarcoding. Results showed a slight bacterial diversity reduction in flood affected condition compared to the unaffected one. The relative bacterial abundance analysis showed that the bacterial taxa were almost uniformly distributed between the leaves. However, a clear differential taxa abundance was observed in rhizosphere soil which may be associated with apricot tree environmental status. In fact, the flood affected rhizosphere soil showed a significant increase in several bacterial taxa including particularly Bacillusfrigoritolerans and Pseudoarthrobacterphenanthrenivorans which are known to be beneficial to soil. However, some Actinobacteria genera that promote growth of plants, including Nocardioidessp.,Streptomycessp.,BlastococcusandGeodermatophilus were decreased in abundance. This distribution of abundance and diversity of the bacterial community in the flood affected tree can be correlated to abiotic stress. These results provide new insights into rhizosphere-associated microbes that are likely playing a crucial role in plant growth and survival.

The availability of the Whole-Genome Sequence of the wheat pest Mayetiola destructor offers the opportunity to investigate the Transposable Elements (TEs) content and their relationship with the genes involved in the insect virulence. In this study, de novo annotation carried out using REPET pipeline showed that TEs occupy approximately 16% of the genome and are represented by 1038 lineages. Class II elements were the most frequent and most TEs were inactive due to the deletions they have accumulated. The analyses of TEs ages revealed a first burst at 20% of divergence from present that mobilized many TE families including mostly Tc1/mariner and Gypsy superfamilies and a second burst at 2% of divergence, which involved mainly the class II elements suggesting new TEs invasions. Additionally, 86 TEs insertions involving recently transposed elements were identified. Among them, several MITEs and Gypsy retrotransposons were inserted in the vicinity of SSGP and chemosensory genes. The findings represent a valuable resource for more in-depth investigation of the TE impact onto M. destructor genome and their possible influence on the expression of the virulence and chemosensory genes and consequently the behavior of this pest towards its host plants.

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.

Mariner-like elements (MLEs) are widespread Class II transposable elements in insects that are subdivided into several subfamilies. In the current study, we carried out in silico analysis and in vitro experiments to identify MLEs belonging to the irritans subfamily in two cecidomyiid flies, Mayetiola destructor and M. hordei. In silico investigation of M. destructor genome allowed the identification of 25 irritans-like elements, which were mostly defective due to several mutations. These defective forms might be the remnants of active elements that ancestrally invaded the host genome. Structural analyses, including signature motifs and transposase-encoding ORFs, revealed structural heterogeneity and the presence of two full length copies. Five consensuses, reflecting the probable evolutionary groups of these elements, were constructed, based on a similarity matrix. The first consensus (Maymarcons1) belonged to Himar1-like elements reported in other insects, while the remaining four (Maymarcons2 to 5) seemed to be more specific to Cecidomyiidae. Moreover, the presence of elements belonging to the Maymarcons4 group was ascertained by PCR amplification, in both Mayetiola species, and was further identified in the Transcriptome Shotgun Assembly (TSA) of the orange fly, Sitodiplosis mosellana (Cecidomyiidae), suggesting the existence of irritans elements within the Cecidomyiidae, which were derived from an ancestral species by vertical transmission during speciation. On the other hand, consensuses that are specific to M. destructor could be derived from a more recent invasion. This study suggests that both M. destructor and M. hordei genomes have been invaded by irritans elements many times with at least two different evolutionary histories.

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.

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.

Background Although transposons have been identified in almost all organisms, genome-wide information on mariner elements in Aphididae remains unknown. Genomes of Acyrthosiphon pisum, Diuraphis noxia and Myzus persicae belonging to the Macrosiphini tribe, actually available in databases, have been investigated. Results A total of 22 lineages were identified. Classification and phylogenetic analysis indicated that they were subdivided into three monophyletic groups, each of them containing at least one putative complete sequence, and several non-autonomous sublineages corresponding to Miniature Inverted-Repeat Transposable Elements (MITE), probably generated by internal deletions. A high proportion of truncated and dead copies was also detected. The three clusters can be defined from their catalytic site: (i) mariner DD34D, including three subgroups of the irritans subfamily (Macrosiphinimar, Batmar-like elements and Dnomar-like elements); (ii) rosa DD41D, found in A. pisum and D. noxia; (iii) a new clade which differs from rosa through long TIRs and thus designated LTIR-like elements. Based on its catalytic domain, this new clade is subdivided into DD40D and DD41D subgroups. Compared to other Tc1/mariner superfamily sequences, rosa DD41D and LTIR DD40-41D seem more related to maT DD37D family. Conclusion Overall, our results reveal three clades belonging to the irritans subfamily, rosa and new LTIR-like elements. Data on structure and specific distribution of these transposable elements in the Macrosiphini tribe contribute to the understanding of their evolutionary history and to that of their hosts. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3856-6) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

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 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

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.

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.

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.

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.

Computational investigation of a set of publicly available plant microRNAs revealed 19 barley- and other plants-encoded miRNAs and their near-complement reverse sequences (miRNA*) that have potential to bind all B/CYDV open reading frames (ORFs) except ORF0 and ORF6. These miRNAs/miRNAs*, their binding positions and targets are discussed in the context of biological protection of cereals against B/CYDV, based on antiviral silencing.