Tunisian Isolates Research Articles

Unlocking the story of resistance to Zymoseptoria tritici in Tunisian old durum wheat germplasm based on population structure analysis

BackgroundSeptoria tritici blotch (STB) remains a significant obstacle to durum wheat cultivation on a global scale. This disease remains a challenge for farmers, researchers, and breeders, who are collectively dedicated to reduce its damage and improve wheat resistance. Tunisian durum wheat landraces have been recognized as valuable genetic ressources that exhibit resistance to biotic and abiotic stresses and therefore play a crucial role in breeding program aimed at creating new wheat varieties resistant to fungal diseases as STB, as well as adapted to climate change constraints.ResultsA total of 366 local durum wheat accessions were assessed for resistance to two virulent Tunisian isolates of Zymoseptoria tritici Tun06 and TM220 under field conditions. Population structure analysis of the durum wheat accessions, performed with 286 polymorphic SNPs (PIC > 0.3) covering the entire genome, identified three genetic subpopulations (GS1, GS2 and GS3) with 22% of admixed genotypes. Interestingly, all of the resistant genotypes were among GS2 or admixed with GS2.ConclusionsThis study revealed the population structure and the genetic distribution of the resistance to Z. tritici in the Tunisian durum wheat landraces. Accessions grouping pattern reflected the geographical origins of the landraces. We suggested that GS2 accessions were mostly derived from eastern Mediterranean populations, unlike GS1 and GS3 that originated from the west. Resistant GS2 accessions belonged to landraces Taganrog, Sbei glabre, Richi, Mekki, Badri, Jneh Khotifa and Azizi. Furthermore, we suggested that admixture contributed to transmit STB resistance from GS2 resistant landraces to initially susceptible landraces such as Mahmoudi (GS1), but also resulted in the loss of resistance in the case of GS2 susceptible Azizi and Jneh Khotifa accessions.

Phylogenetic and phylodynamic analyses of subtype-B metapneumovirus from chickens in Tunisia

Swollen Head Syndrome (SHS) is an economically important viral disease of chickens caused by avian metapneumovirus (aMPV). The virus comprises 6 different subtypes (A,B,C,D, New-1 and New-2). To date, no information was available on the presence of the virus in Tunisian poultry. The present work aims to detect the presence of (aMPV) in broiler chicken in Tunisia, then to characterise the isolates in order to determine their subtype and to estimate their geographic origin of introduction. A total of 289 samples were collected, aMPV detection was detected by real time RT-PCR and molecular characterization was warried out by Sanger sequencing on the glycoprotein (G) gene. Phylogenetic analysis was carried out using Beast 2 software. Out of the 289 samples, 21 were revealed positive to aMPV. Only 2 isolates have been confirmed by sequencing analysis ; one isolate sampled in 2015 and another in 2019. Based on the partial G gene sequence, analysis of these 2 Tunisian isolates showed that they belong to subtype B. The isolate sampled in 2015, appeared to be phylogenetically related to derived vaccine strain. However, the one sampled in 2019 appeared to be a field strain. Phylodynamic analysis provided evidence that this field strain derived from a Spanish strain and probably the virus has been introduced from Spain to North Africa back in 2016. This study is the first that highlighted the circulation of (aMPV) in Tunisia. It is possible that aMPV has been circulating in Tunisia and neighboring countries without being detected. Also, multiple strains could be present and therefore multiple introductions have happened. Through this study, we shed the light on the importance of reinforcing farms biosecurity as well as virological surveillance.

Epidemiological and Phylogeographic Study of Equid Herpesviruses in Tunisia.

Equid herpesvirus (EHV) is a contagious viral disease affecting horses, causing illness characterized by respiratory symptoms, abortion and neurological disorders. It is common worldwide and causes severe economic losses to the equine industry. The present study was aimed at investigating the incidence of EHVs, the genetic characterization of Tunisian isolates and a spatiotemporal study, using 298 collected samples from diseased and clinically healthy horses. The global incidence of EHV infection was found to be about 71.81%. EHV2 and EHV5 were detected in 146 (48.99%) and 159 (53.35%) sampled horses, respectively. EHV1 was detected in 11 samples (3.69%); EHV4 was not detected. Co-infections with EHV1-EHV2, EHV1-EHV5 and EHV2-EHV5 were observed in 0.33%, 1.34% and 31.54% of tested horses, respectively. Phylogenetic analyses showed that gB of EHV2 and EHV5 displays high genetic diversity with a nucleotide sequence identity ranging from 88 to 100% for EHV2 and 97.5 to 100% for EHV5. Phylogeography suggested Iceland and USA as the most likely countries of origin of the Tunisian EHV2 and EHV5 isolates. These viruses detected in Tunisia seemed to be introduced in the 2000s. This first epidemiological and phylogeographic study is important for better knowledge of the evolution of equid herpesvirus infections in Tunisia.

Biological activity and genome composition of a Tunisian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV-Tun2)

BackgroundThe baculovirus Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) is an entomopathogenic virus utilized as a biological control agent of the Egyptian cotton leaf worm, Spodoptera littoralis. Several studies have focused on the identification of different SpliNPV isolates from a biological and molecular point of view, but few of them conducted in-depth analyses of the genomic composition of these isolates.ResultsIdentification of a novel isolate of SpliNPV, termed Tun2, which was purified from infected S. littoralis larvae from Tunisia was reported. This isolate was propagated in vivo and its median lethal concentration (LC50) was determined to be 1.5 × 104 occlusion bodies (OBs)/ml for third instar S. littoralis larvae at 7 days of post-infection. OB production in late fourth instar larvae was estimated to be at least 2.7 × 109 OBs/g larval weight. The completely sequenced genome of SpliNPV-Tun2 was 137,099 bp in length and contained 132 open reading frames (ORF). It showed a 98.2% nucleotide identity to the Egyptian isolate SpliMNPV-AN1956, with some striking differences; between both genomes, insertion and deletion mutations were noticed in 9 baculovirus core genes, and also in the highly conserved polyhedrin gene. The homologs of ORF 106 and ORF 107 of SpliNPV-AN1956 appeared to be fused to a single ORF 106 in SpliNPV-Tun2, similar to the homologous ORF 110 in SpltNPV-G2.ConclusionSpliNPV-Tun2 is proposed as a new variant of SpliNPV and a potential candidate for further evaluation as a biocontrol agent for S. littoralis and probably other Spodoptera species.

First Report of Garlic virus A, Garlic virus B, and Garlic virus C on Garlic (Allium sativum) in Tunisia.

Mite-borne viruses belonging to the genus Allexivirus (family Alphaflexiviridae) commonly occur on garlic in many parts of the world. There are usually asymptomatic and cause small damage to the plants, but often occur in mixed infection with potyviruses and carlaviruses, with synergistic effects reducing crop quality and leading to higher losses (Taglienti et al. 2017). Their occurrence on Tunisian garlic crops was studied here in the same garlic germplasm collection mentioned in a previous first report (Ayed et al. 2019). Leaf samples from a total of 66 garlic accessions were tested by DAS-ELISA using specific antibodies (DSMZ, Germany) against garlic virus A (GarV-A), garlic virus B (GarV-B) and garlic virus C (GarV-C). These serological tests showed individual virus incidence of respectively 56.4%, 67.7% and 10%. Our findings corroborate with the results of Chodorska et al (2012). In order to confirm the presence of these viruses, RT-PCR assays were performed using total RNAs extracted using two silica-capture extraction procedures according to Foissac et al (2005) and specific primers targeting the coat protein genes of the various viruses. These primers, designed for the present study are (GarV-A-F: 5' YCTYTTCTCHYTDGCHTGGACYTG 3' and GarV-A-R: 5' RCCYTTCCTAGACCARTTRGCRGG 3' for GarV-A; GarV-B-F: 5' TGGGCYTGYTACCACAAYGGATC 3' and GarV-B-R 5' TCTGCGCGVGTGGADACCATRTT 3' for GarV-B; GarV-C-F: 5' ARGAYCTYTTYTCMCTYGCRTGGGC 3' and GarV-C-R: 5' GGAGGYTCRTGAATYTGTTGTTG 3' for GarV-C). The viruses were detected by a two-step RT-PCR as described by Marais et al (2015). PCRs consisted of one cycle at 95 °C for 5 min; followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 45 °C for 45 s, and elongation at 72 °C for 45s; and a final extension step at 72 °C for 10 min. Products of the expected size (214 bp for GarV-A, 363 bp for GarV-B and 439 bp for GarV-C) were amplified from 58 (88%), 47 (71%) and 56 (85%) accessions, respectively. Forty three samples (65%) were co-infected by the three viruses. Higher numbers of positives revealed by RT-PCR especially in the case of GarV-C may reflect the higher sensitivity and efficiency of this technique compared to ELISA. Direct sequencing of selected amplicons of the expected size obtained for GarV-A, -B, and -C Tunisian isolates was performed and the sequences submitted to GenBank, validating the specificity of the three RT-PCR assays. The two sequenced GarV-A isolates (MK599147 and MN995836) shared 98% nucleotide (nt) sequence identity with each other, and 93-94% identity with the closest isolate in GenBank, the "G118" isolate from China (MN059320). The three sequenced GarV-B isolates (MN995829 to MN995831) shared 88-98% nt identity with each other. For "GarV-B 18.1" (MN995830) and "GarV-B 36.2" (MN995831) the closest isolate was "1109.1" (JX682828) from Spain (92-93% nt identity). For "GarVB 17.2" (MN995829), the closest isolate was "B-Sp-3" (LC97167) from Spain (90% nt identity). The sequenced GarV-C isolate (MN995834) showed the highest sequence nt identity (93%) with the "GarV-9" isolate (HQ724848) from Spain. To our knowledge this is the first report of the presence of GarV-A, -B and -C in Tunisia. The presence of these allexiviruses may pose a threat to the preservation of the Tunisian garlic germplasm and, more broadly, to garlic production in Tunisia. For this reason, the scrupulous identification of viruses occurring in garlic plants will help to use the appropriate strategy to decrease viral incidence in garlic growing area.

Characterization of Pyrenophora tritici-repentis in Tunisia and Comparison with a Global Pathogen Population.

Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. A collection of P. tritici-repentis isolates from Tunisia, located in one of the main secondary centers of diversification of durum wheat, was tested for phenotypic race classification based on virulence on a host differential set and for the presence of the necrotrophic effector (NE) genes ToxA, ToxB, and toxb by PCR analysis. While races 2, 4, 5, 6, 7, and 8 were identified according to their virulence phenotypes, PCR testing indicated the presence of "atypical" isolates that induced necrosis on the wheat differential 'Glenlea,' but lacked the expected ToxA gene, suggesting the involvement of other NEs in the P. tritici-repentis/wheat interaction. Genetic diversity and the P. tritici-repentis population structure were explored further by examining 59 Tunisian isolates and 35 isolates from Algeria, Azerbaijan, Canada, Iran, and Syria using 24 simple sequence repeat markers. Average genetic diversity, overall gene flow, and percentage polymorphic loci were estimated as 0.58, 2.09, and 87%, respectively. Analysis of molecular variance showed that 81% of the genetic variance occurred within populations and 19% occurred between populations. Cluster analysis by the unweighted pair group method indicated that ToxB- isolates grouped together and were distantly related to ToxB+ isolates. Based on Nei's analysis, the global collection clustered into two distinct groups according to their region of origin. The results suggest that geographic origin and the host specificity imposed by different NEs can lead to differentiation among P. tritici-repentis populations.

Screening and Analysis of Anaplasma marginale Tunisian Isolates Reveal the Diversity of lipA Phylogeographic Marker and the Conservation of OmpA Protein Vaccine Candidate.

Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells. A. marginale DNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multiple A. marginale worldwide strains.

Characterization of Coolia spp. (Gonyaucales, Dinophyceae) from Southern Tunisia: first record of Coolia malayensis in the Mediterranean Sea

This study provides the first report of the presence of Coolia malayensis in the Mediterranean Sea, co-occurring with C. monotis. Isolated strains from the Gulf of Gabès, Tunisia (South-eastern Mediterranean) were identified by morphological characterization and phylogenetic analysis. Examination by light and scanning electron microscopy revealed no significant morphological differences between the Tunisian isolates and other geographically distant strains of C. monotis and C. malayensis. Phylogenetic trees based on ITS1-5.8S-ITS2 and D1‒D3/28S rDNA sequences showed that C. monotis strains clustered with others from the Mediterranean and Atlantic whereas the C. malayensis isolate branched with isolates from the Pacific and the Atlantic, therefore revealing no geographical trend among C. monotis and C. malayensis populations. Ultrastructural analyses by transmission electron microscopy revealed the presence of numerous vesicles containing spirally coiled fibers in both C. malayensis and C. monotis cells, which we speculate to be involved in mucus production.

Phylogenetic analysis of the neuraminidase segment gene of Influenza A/H1N1 strains isolated from Monastir Region (Tunisia) during the 2017-2018 outbreak.

Influenza A/H1N1 is widely considered to be a very evolutionary virus causing major public health problems. Since the pandemic of 2009, there has been a rapid rise in human Influenza virus characterization. However, little data is available in Tunisia regarding its genetic evolution. In light of this fact, our paper aim is to genetically characterize the Neuraminidase, known as the target of antiviral inhibitors, in Tunisian isolates circulating in Monastir region during 2017–2018. In total of 31 positive Influenza A/H1N1 detected by multiplex real-time PCR, RT-PCR of neuraminidase was performed. Among the 31 positive samples, 7 samples representing fatal and most severe cases were conducted for sequencing and genetic analysis. The results thus obtained showed genetic evolution of the A/H1N1 neuraminidase between 2009 and 2010 and 2018–2019 outbreaks. All Tunisian isolates were genetically related to the recommended vaccine strain with a specific evolution. Moreover, the phylogenetic analysis demonstrated that France and especially Italian strains were the major related strains. Interestingly, our results revealed a specific cluster of Tunisian isolates where two intragroup were evolved in correlation with the severity and the fatalities cases. From the outcome of our investigation, this study confirms the genetic evolution of the Influenza A virus circulating in Tunisia and gives a preliminary analysis for a better comprehension of new emerging Tunisian strain’s virulence and thus, a more appropriate monitoring of Influenza virus A/H1N1 during each round of outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11756-021-00723-y.

Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green–based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Clonal dissemination of antibiotic resistance among Tunisian Mycoplasma gallisepticum isolates as revealed by gene-targeted sequencing analysis.

SummaryTo date, very little is known about avian mycoplasma infections in Tunisia. Mycoplasma gallisepticum is one of the most economically significant pathogen for poultry in Tunisia and worldwide. Based on the paucity of data regarding the genetic profiles and antibacterial behavior of M. gallisepticum strains in Tunisia, the present study was conducted. Genetic typing and phylogenetic relationships of 40 M. gallisepticum strains (20 Tunisian isolates, 19 international strains collection, and S6 reference strain) were investigated by gene-targeted sequencing (GTS) using 4 loci ( pvpA , mgc2 , vlhA and the InterGenic Spacer Region (IGSR) between the 16S and the 23S rRNA genes). GTS reveals 12 STs that were found to spread over 2 clonal complexes (CC) and 5 singletons.Emergence of enrofloxacin and spiramycin resistance among M. gallisepticum local isolates have been revealed using the broth microdilution method. Causal mutations have been identified by sequencing the quinolone-resistance determining region (QRDR) and domain II and V of 23S rRNA as well as the rplD and rplV genes for enrofloxacine- and macrolide-resistant isolates, respectively. The emersion of antibiotic resistance to enrofloxacin and spiramycin has been identified as being related to a distinctive clonal complex formed by 4 different STs (ST2, ST3, ST4 and ST5) which would suggest that this phenotype was clonally disseminated.

Newly detected mutations in the Meq oncogene and molecular pathotyping of very virulent Marek\u2019s disease herpesvirus in Tunisia

Marek's disease (MD) is a contagious avian viral disease that is responsible for large economic losses to farmers. The disease is caused by Marek's disease virus (species Gallid alphaherpesvirus 2), which causes neurological lesions, immune suppression, and tumor proliferation of lymphoid cells that invade a large number of organs and tissues. Despite widespread vaccination, Marek's disease virus (MDV), has shown a continuous increase in its virulence and has acquired the ability to overcome immune responses induced by vaccines. In the present study, the oncogenic serotype MDV-1 was detected by real-time PCR in DNA samples extracted from organs developing tumor infiltrations. Identification of the pathotype based on a 132-bp tandem repeat and sequencing and phylogenetic analysis of the Meq gene and its encoded protein allowed classification of the isolated viruses as "very virulent", with two new and unique mutations in the Meq gene resulting in amino acid substitutions. Sequencing of pp38, vIl-8, UL1 and UL44 genes did not reveal any new mutations that were characteristic of the Tunisian isolates or correlated with virulence. These results raised concerns about the ability of HVT and CVI988 vaccines, which are currently used in Tunisia and other countries, to protect chickens against highly virulent virus strains.

Molecular phylogeny and genetic diversity based on msp1a, groEL and gltA genes of Anaplasma ovis Tunisian isolates compared to available worldwide isolates and strains

Anaplasma ovis, the causative agent of ovine anaplasmosis in tropical and subtropical countries, is a tick-borne obligatory intraerythrocytic bacterium of sheep, goats and wild ruminants. In Tunisia, data about the molecular phylogeny and the genetic diversity of A. ovis isolates are limited to the analysis of msp4 and groEL genes. The aim of this study was to genetic characterize 40 A. ovis isolates infecting 28 goats, 10 sheep, one camel and one Rhipicephalus turanicus tick located in different geographic regions of Tunisia on the basis of 3 partial genes (gltA, groEL and msp1a). Sequence analysis revealed 6 and 17 different genotypes in the partial gltA and groEL genes, respectively. Phylogenetic analysis revealed, as expected for the groEL gene, that sequences from small ruminants and their infesting ticks clustered separately from those isolated from camels. The analysis of amino-acid Msp1a sequences identified 18 novel genotypes of Msp1a repeats from 20 A. ovis isolates. These Msp1a repeats were highly variable with 33–47 amino-acids, and the number of repeats is one for 19 isolates infecting 18 goats and one R. turanicus tick, and 4 for a single isolate found in one sheep. Phylogenetic trees based on Msp1a partial sequences revealed that the N-terminal region of Msp1a protein appear to be relatively more informative phylogeographically compared to other markers especially according to countries. The presented data give a more detailed knowledge regarding the molecular phylogeny and the genetic diversity of A. ovis isolates occurring in different animal species and their associated ticks in Tunisia.

Genetic diversity of Grapevine rupestris stem pitting‐associated virus isolates from Tunisian grapevine germplasm

AbstractGrapevine rupestris stem pitting‐associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA‐dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.

Molecular characterization of a unique variant of avian infectious bronchitis virus in Tunisia

Avian infectious bronchitis virus (IBV) is responsible of significant economic losses for poultry industry around the world, through evolution of its pathogenicity, inadequacy of vaccines, and virus evasion. Such evasion is related to the unstable nature of its RNA, in particular the S glycoprotein encoding gene, which raises great challenges with regard to the control of the disease, along with the lack of proof reading mechanisms of the RNA polymerase. The emergence of new variants might be a reason for the endemic outbreaks that are being reported in Tunisia, in addition to poor vaccination techniques and ineffective prophylactic programs.In the present study, partial nucleotide sequences of the S1 glycoprotein gene and the 3′-untranslated region (UTR) of 2 Tunisian isolates, TN1011/16 and TN1012/16, identified in 2016, were determined. Specific mutations were found in S1 gene as well as in 3′UTR region. Phylogenetic analysis of the S1 nucleotide sequences showed that both isolates are closely related to the Algerian strains, and formed a common cluster within the genotype I. In addition, these isolates were non-recombinant ones, confirming that they are unique variants. Based on their S1 gene sequences, TN1011/16 and TN1012/16 strains were distant from the H120 vaccine strain, commercially used in Tunisia along with the variant vaccine 793B type (4/91). A comparison between nucleotide sequences of their 3′UTR region and S1 gene showed a difference in IBV classification. The obtained results have confirmed that the IBVsequence continues to drift and brings valuable information in relation with its evolution, vaccine development and better control of the disease.

Genome characteristics of Bordetella pertussis isolates from Tunisia.

The recent increase in pertussis cases observed in some countries may have several causes, including the evolution of Bordetella pertussis populations towards escape of vaccine-induced immunity. Most genomic studies of B. pertussis isolates performed so far are from countries that use acellular vaccines. The objective was to analyse genomic sequences of isolates collected during the 2014 whooping cough epidemic in Tunisia, a country where whole-cell vaccines are used. Ten Tunisian isolates and four vaccine strains were sequenced and compared to 169 isolates from countries where acellular vaccines are used. Phylogenetic analysis showed that Tunisian isolates are diverse, demonstrating a multi-strain 2014 epidemic peak, and are intermixed with those circulating in other world regions, showing inter-country transmission. Consistently, Tunisian isolates have antigen variant composition observed in other world regions. No pertactin-deficient strain was observed. The Tunisian B. pertussis population appears to be largely connected with populations from other countries.

Viral interference between H9N2-low pathogenic avian influenza virus and avian infectious bronchitis virus vaccine strain H120 in vivo

The interaction between a low pathogenic avian influenza virus (A/CK/TUN/145/2012), a H9N2 Tunisian isolate, and a vaccine strain (H120) of avian infectious bronchitis, administered simultaneously or sequentially three days apart to chicks during 20 days, was evaluated using ELISA antibody levels, quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analyses and histopathology examination. First, the in vivo replication interference of avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated using qRT-PCR to detect accurately either AIV or IBV genomes or viral copy numbers during dual infections. Second, we have determined the amount of specific antibodies in sera of chick’s infected with AIV alone, IBV alone, mixed AIV + IBV, IBV then AIV or AIV IBV 3 days later using an ELISA test. Finally, histopathological analyses of internal organs from inoculated chicks were realized. Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. According to our results, vaccine application was safe and do not interfere with AIV H9N2 infection, and does not enhance such infection. In conclusion, co-infection of chicks with AIV and IBV, simultaneously or sequentially, affected the clinical signs, the virus replication dynamics as well as the internal organ integrity. The results proposed that infection with heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated.

Chickpea chlorotic dwarf virus infecting tomato crop in Tunisia

Chickpea chlorotic dwarf virus (CpCDV) a polyphagous mastrevirus, family Geminiviridae, is reported to infect several economically important hosts, among which tomato, in numerous world areas. NGS studies have recently linked the “hard fruit syndrome” of watermelon to CpCDV infection. In this study, we surveyed for CpCDV infection tomato fields in several Tunisian areas and we report this virus for the first time in tomato in this Mediterranean country. We detected CpCDV by PCR with specific primers and obtained a full-length sequence of a tomato-derived isolate, resulting identical to the Tunisian watermelon-derived isolate, and classifiable as CpCDV strain A. Following tomato agro-inoculation of the CpCDV infectious clone under controlled conditions, we checked infection by stem squash, dot blotting and PCR, and demonstrated the ability of CpCDV to replicate in tomato tissues. The alterations observed in fruits of CpCDV-infected tomatoes were similar to those previously described for watermelon, providing hints about the perturbation of fruit development mediated by this virus.

Emergence of tomato leaf curl New Delhi virus in Italy: estimation of incidence and genetic diversity

Tomato leaf curl New Delhi virus(ToLCNDV) is a bipartite begomovirus (familyGeminiviridae) identified for the first time in 1995 in Asia, from where it spread into several countries of the Mediterranean basin. ToLCNDVwas found in Spain in 2012, and subsequently in Tunisia and Italy. The first outbreak in Italy occurred at the end of 2015 in Trapani province (Sicily) on zucchini squashes. Then in 2016, ToLCNDVwas found in infected zucchini plants in Campania, Lazio and Sardinia regions, and in 2017 in Calabria. This study addressed the dispersion and genetic diversity of ToLCNDVisolates in Italy. A total of 1400 plants were analysed. Phylogenetic analysis showed low variability among the Italian isolates, probably as a consequence of the recent introduction and rapid spread of this virus in Italy. Two statistically significant clusters were reported: one grouping only Italian isolates and the other grouping Italian, Spanish, Tunisian and Moroccan isolates. Furthermore, the highest incidence of ToLCNDVwas observed in Sicily, although the disease also appears to be critical in other Italian regions. In this work, a high efficiency of ToLCNDVmechanical transmission intoCucurbita pepo,Cucumis melo inodorusandCucumis melo cantalupensishas been demonstrated. The rapid spread of ToLCNDVin the Mediterranean basin represents a threat for horticultural production, thus it is very important to develop suitable crop management practices, applying genetic resistance strategies and more restrictive phytosanitary measures.

Effects of pH and Aeration on Sclerotium rolfsii sacc. Mycelial Growth, Sclerotial Production and Germination

Sclerotium rolfsii is one of the devastating soilborne fungus responsible for significant plant losses. The effects of pH and aeration on pathogen mycelial growth, sclerotial production and germination were investigated for three Tunisian isolates. Optimal mycelial growth occurred at pH 6 for Sr2 and Sr3 isolates and at pH 6-7 for Sr1. Dry mycelial growth was optimum at pH values ranging between 4 and 7. Sclerotial initiation started on the 3rd day of incubation at all pH values tested and mature sclerotia were formed after 6 to 12 days. Optimal sclerotial production was noted at pH 5. The dry weight of 100 sclerotia varied depending on isolates and pH and occurred at pH range 4-7. At pH 9, mycelial growth, sclerotial production and dry weight of 100 sclerotia were restricted. The optimum sclerotial germination, noted after 24 h of incubation, varied depending on isolates and pH and occurred at pH 4-9. Mycelial growth was optimum in aerated plates with a significant isolates x aeration treatments interaction. Sclerotial initiation occurred at the 3rd day of incubation and mature sclerotia were observed after 6-9 days. Sclerotial development was very slow in completely sealed plates and dark sclerotia were produced only after 15 days of incubation. The highest sclerotial yields were noted in aerated plates. The highest dry weight of 100 sclerotia for Sr1 isolate was recorded in ½ sealed, no sealed and completely sealed plates, while for Sr2, it was noted in ½ and ⅔ sealed plates. For Sr3, the maximum dry weight of 100 sclerotia was recorded in ½, ⅔ and completely sealed plates. Germination of S. rolfsii sclerotia, after 24 h of incubation, did not vary significantly depending on aeration treatments and ranged from 90 to 100% for all isolates.