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This study evaluated the effects of the exogenous application of ethylenediaminetetraacetic acid (EDTA), indole-3-acetic acid (IAA) and iron sulfate (FeSO

Context Iron (Fe) is considered as a major cause of rural Tunisian soil contamination. Developing strategies for the cultivation of accumulator plants with permissible iron (Fe) concentrations is an urgent challenge. Aims In this study, the effects of FeSO4 concentration (0, 50, 500 and 1000 mg L−1) on fenugreek morpho-biochemical parameters were investigated. Methods The use of fenugreek as a phytoremediation strategy to control not only the uptake of Fe but also its safe consumption after treatments was evaluated. Key results Results showed that elevated Fe concentrations did not affect the germination rate, but, rather, decreased the radicle length and amylase activity. The elemental analysis showed that Fe uptake was higher in shoots than in roots, but lower in harvested seeds. The translocation factor was higher than 1, suggesting a safe use of fenugreek as an accumulator. Moreover, the increase in Fe concentrations reduced the chlorophyll content and enhanced the production of lipid peroxidation, hydrogen peroxide and superoxide dismutase more frequently on fenugreek shoots than on their roots. In turn, the maximum concentrations of flavonoids and phenols were recorded under the Fe concentration of 50 mg L−1. High-performance liquid chromatography analysis showed that the gallic and syringic acids were the major phenols produced under Fe stress in shoots and that 50 mg L−1 of added Fe concentration induced their elevation. However, the quercetin was detected only in roots and was reduced under the increasing Fe concentrations. Conclusions These results showed that fenugreek is an accumulator plant with admissible concentrations of Fe accumulation, which deploys multiple mechanisms to adapt to Fe stress.

Lead stands as a food contaminant through its accumulation in consumed plants. In this study, the effects of lead (II) chloride (PbCl

Lead stands as a food contaminant through its accumulation in consumed plants. In this study, the effects of lead (II) chloride (PbCl2) and its levels of uptake on morphological and phytochemical responses of fenugreek were assessed to evaluate its tolerance and safety for human consumption. Results revealed that PbCl2 (50–2000 mg L−1) did not affect the germination rate, but it decreased the radicle length and amylase activity. After three months of Pb treatments, the elemental analysis showed that Pb accumulation was greater in roots than shoots, and it was not present in harvested seeds. The bioaccumulation factor > 1 and the translocation factor << 1 observed for 1000 mg L−1 PbCl2 suggested appropriateness of fenugreek as a phytostabilizer. Additionally, increased lipid peroxidation, hydrogen peroxide, flavonoid levels and catalase activity were observed in Pb-treated fenugreek. Meanwhile, decreased chlorophyll content was detected under these conditions. In turn, the total phenol was correlated with Pb treatment only in roots. HPLC analysis proved that under Pb stress, gallic acid was the most produced compound in treated roots compared to shoots, followed by quercetin. Syringic and chlorogenic acids were more produced in shoots. In conclusion, fenugreek can be used for Pb phytoremediation and is safe for consumption after Pb treatments in the traditional medicine system.

As a tree of considerable importance in arid regions&mdash, date palm, Phoenix dactylifera L. survival in contaminated areas of Sfax city has drawn our attention. Leaf samples of the plants grown in the study area showed high levels of cadmium (Cd), copper (Cu), and chromium (Cr). On the basis of this finding, the cellular mechanisms that explain these metal accumulations were investigated in controlled conditions. After four months of exposure to Cd, Cr, or Cu, high bioconcentration and translocation factor (TF&gt, 1) have been shown for date palm plantlets exposed to Cd and low TF values were obtained for plantlets treated with Cr and Cu. Moreover, accumulation of oxidants and antioxidant enzyme activities occurred in exposed roots to Cu and Cd. Secondary metabolites, such as polyphenols and flavonoids, were enhanced in plants exposed at low metal concentrations and declined thereafter. Accumulation of flavonoids in cells may be correlated with the expression of the gene encoding Pdmate5, responsible for the transport of secondary metabolites, especially flavonoids. Other transporter genes responded positively to metal incorporation, especially Pdhma2, but also Pdabcc and Pdnramp6. The latter would be a new candidate gene sensitive to metallic stress in plants. Expressions of gene coding metal chelators were also investigated. Pdpcs1 and Pdmt3 exhibited a strong induction in plants exposed to Cr. These modifications of the expression of some biochemical and molecular based-markers in date palm helped to better understand the ability of the plant to tolerate metals. They could be useful in assessing heavy metal contaminations in polluted soils and may improve accumulation capacity of other plants.

Date palm (Phoenix dactylifera) seeds were exposed to different copper (Cu) solutions to examine plant stress responses. Low Cu concentrations (0.02 and 0.2 mM) caused an increase of seed germination, whereas higher Cu amounts (2 mM) significantly inhibited seed germination, delayed hypocotyl elongation, increased seedling mortality, and reduced the germination index by more than 90%. Metal-related toxicity symptoms appeared after 15 d of 2 mM of Cu exposure. Biochemical activities such as amylase activity and redox balance elements were examined to study the relationship between external Cu amount and internal plant response. The present study showed that amylolytic activity was dose- and time-dependent. Likewise, H2 O2 production increased after exposure to Cu, which was correlated with thiobarbituric acid reactive substance (TBARS) accumulation. Furthermore at low Cu concentrations, superoxide dismutase (SOD) and catalase (CAT) activities increased, suggesting that date palm seed stimulated its metal homeostasis networks. However, the highest cupric ion amounts increased cell oxidant accumulation and reduced enzyme production. Gene expression level measures of P. dactylifera phytochelatin synthase (Pdpcs) and P. dactylifera metallothionein (Pdmt) encoding genes have been carried out to investigate the implication of PdPCS and PdMT proteins in Cu homeostasis and/or its sequestration. Phoenix dactylifera metallothionein induction reached a peak after 30 d of exposure to 0.2 mM of Cu. However, it was down-regulated in plants exposed to higher Cu concentrations. In the same conditions, Pdpcs was overexpressed during 1 mo of exposure before it decreased thereafter. These observations provide a new insight into date palm cell response to Cu, a metal that can be toxic but that is also an essential element. Environ Toxicol Chem 2018;37:797-806. © 2017 SETAC.

Date palm in Tunisia is of major economic importance but are also factors of social, environmental and economic stability. An annotated assembly of the transcriptome of cultivar Deglet Nour was reported. RNA was isolated from plant Cd-contaminated leaves, and 37,049 unique Illumina RNA-seq reads were used in a transcriptome assembly. The draft transcriptome assembly consists of 6789 contigs and 17.285 singletons with a means length of 858 bp and 1.042 bp, respectively. The final assembly was functionally annotated using Blast2GO software, allowing the identification of putative genes controlling important agronomic traits. The annotated transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes pathways. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. A prediction of the genes interaction network was proposed by selecting corresponding functionally similar genes from Arabidopsis datasets, downloaded by GeneMANIA version 2.1. Several Cd-responsive genes expression was monitored in in vitro isolated explant of Cd stressed Deglet Nour. Some chelators encoding genes were upregulated confirming in silico findings. Genes encoding HMs transporters in date palm showed expression enhancement more pronounced after 20 days of exposure. P. dactylifera transcriptome provides a valuable resource for future functional analysis of candidate genes involved in metal stress response. - 2019 Elsevier Inc. Scopus

Iron (Fe) is an essential micronutrient for almost all living organisms and represents one of the most versatile metals in biology.

Phytochelatin synthase and metallothionein gene expressions were monitored via qPCR in order to investigate the molecular mechanisms involved in Cd and Cr detoxification in date palm (Phoenix dactylifera). A specific reference gene validation procedure using BestKeeper, NormFinder and geNorm programs allowed selection of the three most stable reference genes in a context of Cd or Cr contamination among six reference gene candidates, namely elongation factor α1, actin, aldehyde dehydrogenase, SAND family, tubulin 6 and TaTa box binding protein. Phytochelatin synthase (pcs) and metallothionein (mt) encoding gene expression were induced from the first days of exposure. At low Cd stress (0.02 mM), genes were still up-regulated until 60th day of exposure. At the highest metal concentrations, however, pcs and mt gene expressions decreased. pcs encoding gene was significantly up-regulated under Cr exposure, and was more responsive to increasing Cr concentration than mt encoding gene. Moreover, exposure to Cd or Cr influenced clearly seed germination and hypocotyls elongation. Thus, the results have proved that both analyzed genes participate in metal detoxification and their expression is regulated at transcriptional level in date palm subjected to Cr and Cd stress. Consequently, variations of expression of mt and pcs genes may serve as early-warning biomarkers of metal stress in this species.

A somatic embryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.

Date palm (Phoenix dactylifera) seeds were exposed to different copper (Cu) solutions to examine plant stress responses. Low Cu concentrations (0.02 and 0.2 mM) caused an increase of seed germination, whereas higher Cu amounts (2 mM) significantly inhibited seed germination, delayed hypocotyl elongation, increased seedling mortality, and reduced the germination index by more than 90%. Metal-related toxicity symptoms appeared after 15 d of 2 mM of Cu exposure. Biochemical activities such as amylase activity and redox balance elements were examined to study the relationship between external Cu amount and internal plant response. The present study showed that amylolytic activity was dose- and time-dependent. Likewise, H

This chapter describes major viroids and viroid diseases reported from at least 22 countries in Africa and the Middle East.

We tested citrus samples from Tunisia using reverse transcription-polymerase chain reaction (RT-PCR), and for the first time, Citrus viroid V (CVd-V) was reported in North Africa. Fourteen of 38 tested citrus trees were infected by CVd-V including the majority of varieties grown in Tunisia. Some RT-PCR results were also supported by biological indexing. After sequencing the RT-PCR products, three new CVd-V variants were identified, showing 80–91 % nucleotide sequence identity with those reported previously. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups were identified. Furthermore, construction of a genetic network of the detected haplotypes using the same sequences shows a clear geographical structuring of Tunisian CVd-V variants.

The effects of copper on germination and growth of fenugreek (Trigonella foenum-graecum) was investigated separately using different concentrations of CuSO4. The germination percentage and radical length had different responses to cupric ions: the root growth increased with increasing copper concentration up to 1 mM Cu 2 + and was inhibited thereafter. In contrast, the germination percentage was largely unaffected by concentrations of copper below 10 mM. The reduction in root growth may have been due to inhibition of hydrolytic enzymes such as amylase. Indeed, the average total amylolytic activity decreased from the first day of treatment with [ Cu 2 + ] greater than 1 mM. Furthermore, copper affected various plant growth parameters. Copper accumulation was markedly higher in roots as compared to shoots. While both showed a gradual decrease in growth, this was more pronounced in roots than in leaves and in stems. Excess copper induced an increase in the rate of hydrogen peroxide (H2O2) production and lipid peroxidation in all plant parts, indicating oxidative stress. This redox stress affected leaf chlorophyll and carotenoid content which decreased in response to augmented Cu levels. Additionally, the activities of proteins involved in reactive oxygen species (ROS) detoxification were affected. Cu stress elevated the ascorbate peroxidase (APX) activity more than two times at 10 mM CuSO4. In contrast, superoxide dismutase (SOD) and catalase (CAT) levels showed only minor variations, only at 1 mM Cu 2 + . Likewise, total phenol and flavonoid contents were strongly induced by low concentrations of copper, consistent with the role of these potent antioxidants in scavenging ROS such as H2O2, but returned to control levels or below at high [ Cu 2 + ]. Taken together, these results indicate a fundamental shift in the plant response to copper toxicity at low versus high concentrations.

Endophytic flora plays a vital role in the colonization and survival of host plants, especially in harsh environments, such as arid regions. This flora may, however, contain pathogenic species responsible for various troublesome host diseases. The present study is aimed at investigating the diversity of both cultivable and non-cultivable endophytic fungal floras in the internal tissues (roots and leaves) of Tunisian date palm trees (Phoenix dactylifera). Accordingly, 13 isolates from both root and leaf samples, exhibiting distinct colony morphology, were selected from potato dextrose agar (PDA) medium and identified by a sequence match search wherein their 18S-28S internal transcribed spacer (ITS) sequences were compared to those available in public databases. These findings revealed that the cultivable root and leaf isolates fell into two groups, namely Nectriaceae and Pleosporaceae. Additionally, total DNA from palm roots and leaves was further extracted and ITS fragments were amplified. Restriction fragment length polymorphism (RFLP) analysis of the ITS from 200 fungal clones (leaves: 100; roots: 100) using HaeIII restriction enzyme revealed 13 distinct patterns that were further sequenced and led to the identification of Alternaria, Cladosporium, Davidiella (Cladosporium teleomorph), Pythium, Curvularia, and uncharacterized fungal endophytes. Both approaches confirmed that while the roots were predominantly colonized by Fusaria (members of the Nectriaceae family), the leaves were essentially colonized by Alternaria (members of the Pleosporaceae family). Overall, the findings of the present study constitute, to the authors' knowledge, the first extensive report on the diversity of endophytic fungal flora associated with date palm trees (P. dactylifera).

We report here the isolation and characterizations of three somatic embryogenesis receptor kinase (PhSERK) genes from palm date by a rapid amplification of cDNA ends (RACE) approach. PhSERKs belong to a small family of receptor kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. Sequence analysis of these genes revealed the sequence size of 11051 pb (PhSERK1), 7981 pb (PhSERK2) and 10510 pb (PhSERK3). The open reading frames of PhSERK1, PhSERK2 and PhSERK3 are 1914 pb, 1797 pb and 1719 pb respectively. PhSERKs belongs to the LRR-type cell surface RLKs, which possess a number of characteristic domains. These include an extracellular domain (EX) containing a variable number of LRR units, signal pepetide (SP) immediately followed by a single transmembrane domain (TM) and an intracellular kinase domain. The phylogenetic tree shows that the protein PhSERK1, PhSERK2 and PhSERK3 clustered within monocots SERKs proteins groups. We also predicted the secondary and tertiary with ligand binding sites structure of the protein PhSERKs.

Wastewater reuse in agriculture may help mitigate water scarcity. This may be reached if high quality treatments removing harmful pollutants are applied. The aim of the present study was to compare the effect of untreated (UTW) and treated wastewater (TW) on germination and seedlings development of alfalfa (Medicago sativa L.), fescue (Festuca arundinacea Schreb.) and sorghum (Sorghum bicolor (L.) Moench). UTW presented high turbidity (130 NTU), chemical and biological oxygen demand (COD, 719mgL-1, BOD5, 291mgL-1) and metal concentrations. These levels caused mortality (18% for fescue), decreased germination speed in seeds (37.5% for alfalfa) and reductions of root and stem length in seedlings (80% and 22% respectively for alfalfa). Adverse effects on seeds germination were reflected at the biochemical level by increased H2O2 levels (6 times for sorghum after 5days) and by increased Malondialdehyde (MDA) levels (more than 600 times for sorghum roots) during seedlings development. When TW was used, these parameters were close to control seeds ones. They were also dependent on plant species and developmental stage. Therefore, for efficient reclaimed wastewater reuse in irrigation, suitable crops, displaying wide tolerance to toxic contents during germination and later seedling development stages have to be selected.

Viroids are small infectious RNA molecules that replicate in plants via RNA-RNA replication processes. The molecular mechanism responsible for this replication has attracted great interest, and studies on this topic have yielded interesting biological findings on the processes in which RNA is involved. Viroids belonging to the Avsunviroidae family replicate in the chloroplasts of infected hosts. It has by now been established that chloroplasts and cyanobacteria share a common have ancestor. In view of this phylogenetic relationship, we investigated whether a member of the Avsunviroidae family could be replicated in a cyanobacterium. The results obtained here show that Avocado Sunblotch Viroid (ASBVd) RNA is able to replicate in the filamentous cyanobacterium Nostoc PCC 7120. Indeed, Northern blot hybridization showed that linear forms of “minus” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of positive “polarity”, and that linear forms of “negative” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of “positive” polarity. ASBVd replication does not impair the growth of Nostoc. These results provide the first evidence that a prokaryotic cell possesses all the machinery required to sustain the process of RNA-RNA replication. The data obtained here are of great importance, since they might shed light on the evolution of the cellular factors on which RNA replication processes depend.

The rpoA gene coding for the α-subunit of DNA-dependent RNA polymerase located in the chloroplastic genome of date palm has been characterized from the elite cultivar Deglet Nour by cloning and sequencing of the PCR amplification product. The full length of rpoA-Pd (Phoenix dactylifera) gene was 1014 bp. The comparison in Genbank showed that the rpoA gene has a 100% homology with the Khalas cultivar of date palm and a strong homology with Oil Palm (99%). The deduced protein full length is 337 amino acid corresponding to 38,692 Da polypeptide. It contained an Alpha N-terminal domain (alpha-NTD) between 1 to 233 (aa) and Alpha C-terminal domain (alpha-CTD) between 266 to 337 (aa). Initially, we have compared the sequences of the full-length DNA rpoA gene from Date Palm and Oil palm, 15 mutations have been detected, 4 do not affect amino acid sequences. A multiple alignment of protein sequences of Date Palm and other plants shows 6 mutations specific for palms family and one is specific to monocots species. A multiple alignment of 35 nucleotide sequences from different plant species shows 3 SNPs specific to Date Palm, 6 SNPs specific to Palms family and 6 other to monocot species. The phylogenetic analysis performed in this work shows a strong similarity between Pd-rpoA and rpoA genes from other plant species, but it shows a great divergence with the rpoA of E. coli. To explain whether the separation of the two clades was due to selection pressure. We calculate the ratio Ka/Ks for different species. A synteny analyses of rpoA genes was effected, a high genomic synteny is observed for the ropA in all the species included in this study.

Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) in nucleic acid extracts of infected fig leaf tissues. RT-PCR results were confirmed by cDNA sequencing. This is the first report of CEVd and HSVd infecting fig in Tunisia. Alignments and phylogenetic analysis classify the HSVd Tunisian isolates in the plum-type group. CEVd Tunisian isolates are very closely related to the CEVd tomato hybrid callus isolate and to CEVd Gynura isolate from the USA.

Background DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities. Methods The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program. Results The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn2+ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species. Conclusions A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn2+ and dAMP). Electronic supplementary material The online version of this article (doi:10.1186/s12976-015-0013-2) contains supplementary material, which is available to authorized users.

The aim of our study was to isolate the alcohol dehydrogenase (ADH) mRNA from Phoenix dactifera, and examine the molecular evolutionary history of this nuclear gene with others ADH genes from palms and other plants species. The DnADH gene has been isolated in silico by BLAST2GO from a cDNA library of date palm cv Deglet Nour. The prediction of candidate’s mRNA and protein for ADH gene from khalas were performed in silico from whole genome shotgun sequence (ACYX02009373.1) using FGENESH prediction program. Nucleotide polymorphism using DnaSPv5 was examined in four palm ADH mRNA sequences across the entire 1.098 kb length of ADH mRNA. A primary conclusion of the present study is that nucleotide diversity for ADH between palm species is very low. In order to assess selective pressure, we calculated the ratio of non-synonymous to synonymous substitutions. We conclude that ADH palms genes appear to be under very different selective constraints. Phylogenetic analyses using PHYLIP and Notung 2.8 programs suggest that ADH genes of some plants species resulted from relatively ancient duplication events. In this study, we present for the first time a molecular characterization of ADH protein of P. dactylifera L cv Deglet nour and a phylogeny analysis between plants ADH. Keys word: Alcohol dehydrogenase, palms species, evolution, duplication.

Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of Citrus exocortis viroid (CEVd) in total nucleic acids from infected citrus indicator hosts or directly from field trees. Grapevine yellow speckle viroid-1 (GYSVd-1) and Australian grapevine viroid (AGVd) were also detected by this technique in total nucleic acids from infected grapevine plants. Three protocols for nucleic acid extraction were tested: a phenol-based method, a protocol using a commercial reagent (Trizol) and a method in which extraction with a high-salt buffer was combined with partial purification using non-ionic cellulose. The third protocol, coupled to RT-PCR, seems to be the most suitable, allowing detection of the three viroids in Tunisian citrus and grapevine samples. This method should be used in a testing programme to produce certified viroid-free planting material.

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

Besides hop, Hop stunt viroid (HpSVd) infects many woody species including grapevine, citrus, peach, plum, apricot, almond, pomegranate, mulberry and jujube. Here, we report the first detection of HpSVd in pistachio (Pistacia vera L.). Samples corresponding to 16 pistachio cultivars were obtained from a nearby almond collection. From these samples, low molecular weight RNAs were extracted for double polyacrylamide gel electrophoresis, northern-blot analysis and reverse transcription polymerase chain reaction assays. HpSVd was detected in 4 of the 16 pistachio cultivars in the first year and in 6 in the second, being also detected in the almond collection. Examination of the nucleotide sequences of pistachio and almond isolates revealed 13 new sequence variants. Sequences from pistachio shared 92–96 % similarity with the first reported HpSVd sequence (GenBank X00009), and multiple alignment and phylogenetic analyses showed that one pistachio isolate (HpSVdPis67Jabari) clustered with the plum group, whereas all the others clustered with the hop, and the recombinants plum-citrus and plum-Hop/cit3 groups. By identifying pistachio as a new natural host, we confirm that HpSVd is an ubiquitous and genetically variable viroid that infects many different fruit trees cultivated worldwide.

Molecular diversity within six viroid species and different molecular variants, in each species infecting fruit trees was first estimated by the single-strand conformation polymorphism (SSCP) technique and then by direct sequencing analysis. The different variants studied are to three Australian grapevine viroids(AGVd), four citrus dwarfing viroids (CDVd), eleven grapevine yellow speckle viroids type-1 (GYSVd-1), four hop stunt viroids (HSVd), seven peach latent mosaic viroids (PLMVd), and eight pear blister canker viroids (PBCVd). Polyacrylamide gel electrophoresis (PAGE) conditions were compared and optimized to improve the sensitivity of the existing SSCP parameters. The relationships among the various SSCP profiles observed and the variation in nucleotide sequences was studied. The results indicate that the variations of some parameters of electrophoresis for each species allowed higher resolution and hence detection of single nucleotide variations among clones initially clustered into the same group.

Hop stunt viroid (HSVd) variants from cachexia symptomatic citrus tree were subjected to retrotranscription and DNA amplification (RT-PCR), cloning and sequencing. Here we report genetic diversity and phylogenetic analysis of HSVd Tunisian isolate. Our study revealed obvious polymorphism within Tunisian isolates and high similarity with Japanese variants. Neighbor-joining analysis was carried out on the new HSVd-citrus sequences together with 44 previously described HSVd isolates from citrus and one from grapevine. The phylogenetic analysis showed that Tunisian isolates were clustered into 4 different groups (CVd-IIa, b, and c and grapevine group). Furthermore, the predicted secondary structure was scrutinized to be more understanding on how the nucleotide change affects variable (V) and pathogenicity domain (P).

Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA share some properties that include small size, circularity and replication through a rolling-circle mechanism. Replication occurs in different cell compartments (nucleus, chloroplast and membrane-associated cytoplasmatic vesicles) and has three steps: RNA polymerization, cleavage and ligation. The first step generates oligomeric RNAs that result from the reiterative transcription of the circular templates of one or both polarities, and is catalyzed by either the RNA-dependent RNA polymerase of the helper virus on which viroid-like satellite RNAs are functionally dependent, or by host DNA-dependent RNA polymerases that, remarkably, viroids and HDV redirect to transcribe RNA templates. Cleavage is mediated by host enzymes in certain viroids and viroid-like satellite RNAs, while in others and in HDV is mediated by cis-acting ribozymes of three classes. Ligation appears to be catalyzed mainly by host enzymes. Replication most likely also involves many other non-catalytic proteins of host origin and, in HDV, the single virus-encoded protein., This work was supported by the Ministerio de Ciencia e Innovación of Spain (grant BFU2008-03154/BMC) and by the Generalidad Valenciana (ACOMP/2010/278) to R.F, by the National Science Foundation of the USA (grant IRFP-0602042) to D.G, and by the Agencia Española de Cooperación Internacional (A/022313/08) to A.E. Due to space limitations we have been unable to refer to the original work of many authors and, instead, we have recurred to reviews. We apologize for any inconvenience this may have caused.

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