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

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

Cloning and molecular analysis of double-stranded RNA associated with grapevine leafroll disease*

Eight major dsRNA species ranging from 1.0 to 19.5 kbp were detected in a low-yielding clone of Sultana (Thompson seedless) grape (Vitis vinifera L., cv. Sultana, clone B4L) affected leafroll disease. Using total dsRNA from this Sultana line as template, a number of cDNA clones were produced. The clones were used as probes for northern blot analysis of dsRNA extracted from Sultana B4L, and from six other grapevine leafroll-infected Sultana sources differing in yield performance. Based on the hybridisation of each probe with dsRNA bands from various Sultanas, the cDNA clones could be divided into three groups. One group of cDNA clones hybridised to high molccular weight dsRNA (19.5 kbp) from two low-yielding Sultanas, another group hybridised to high M_r dsRNA from three low-yielding Sultanas and the third group hybridised to a number of smaller dsRNA species ranging in size between 1.15 and 6.5 kbp. Using the latter cDNA clones, the sequence of 965 nucleotides at the 5′-end of a 1.15 kbp dsRNA (dsRNA 6) of B4L Sultana was determined. This RNA contains an open reading frame encoding a putative protein of M, = 33 441 with no homology to known protein sequences. The sequence of dsRNA 6 was found to overlap larger dsRNAs of sizes between 2.2 to 6.5 kbp. This allowed us to determine the sequence upstream of the 5′-end of the positive strand of dsRNA 6. The nucleotide sequence neighbouring the 5′-end of the positive strand of dsRNA 6 conforms to a consensus sequence proposed as a subgenomic promoter element for the coat protein gene of positive strand RNA plant viruses. The results indicate that more than one virus was present in Sultana B4L and that dsRNA 6 may be a subgenomic species of viral origin.     

Evaluation of biological indexing and dsRNA analysis in grapevine virus elimination

Fragmented shoot apex culture (FSAC) was used to eliminate some diseases caused by viruses and virus-like agents from 24 varieties of imported grapevines. Each cultivar was examined by biological indexing before and after FSAC. Graft indexing revealed that leaf roll, stem pitting and yellow speckle diseases were common before FSAC. A correlation was observed between the incidence of leafroll disease and the presence of specific dsRNA species which were removed after FSAC. One of these species, an RNA of about one kilobase pair was associated with low yielding Sultana clones. Both biological indexing and dsRNA assay indicated no recurrence of the leafroll disease in material regenerated by FSAC even after 10 years in the field. It is concluded that dsRNA assay may be used as a faster and less expensive method than biological indexing in assessing the success of leafroll elimination by FSAC. The test also provides some information on the genome size of the viruses associated with leafroll disease. Graft indexing indicated that yellow speckle disease was resistant to elimination by FSAC, while stem pitting was removed from some of the vines and the grapevine fleck disease was eliminated from most sources.     

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

Summary To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N⁶-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.     

A study of the relict fish fauna of northern Chad,with the first records of a polypterid and a poeciliid in the Sahara desert

Seventeen species and sub-species of fishes belonging to four families (Cyprinidae, Clariidae, Aplocheilidae, Cichlidae) were known to occur in perennial bodies of water in the Sahara desert. The study of fishes collected in Lake Boukou near Ounianga Serir (Borkou, northern Chad) shows, for the first time, the occurrence in the Sahara desert of relict populations of Polypterus senegalus (Polypteridae) and Poropanchax normani (Poeciliidae). The Cichlidae Tilapia zilli was also collected in this lake. With these new records, the relict fish fauna currently known in lakes and gueltas of the Borkou plateaus comprises six species. In the Ennedi Mountains, where the specific status of Barbus populations was unclear, B. macrops was collected in Bachikere guelta. The toad Amietophrynus regularis was collected in Ounianga Kebir.     

马铃薯卷叶病毒的提纯

本文提出了一个应用液氮冷冻,一步提取,蔗糖垫层差速离心,Sephadex G-200柱层析以及蔗糖密度梯度离心法纯化马铃薯卷叶病毒的程序,改进后的马铃薯卷叶病毒提纯方法,使病毒产量达到1.18mg/kg酸浆组织,病毒提取物纯度比差速离心者更高,20％蔗糖垫层差速离心能够更加有效地去除宿主细胞成份,纯化病毒的OD260/280,260/240比值分别达到1.77和1.43。     

Status of the Nile crocodile in the Sahara desert

Middle Holocene remains and rock paintings show that the Nile crocodile (Crocodylus niloticus Laurenti) used to occur across the whole Sahara. It also occurred at the South Mediterranean shores, in swamps and rivers and it may even have been circum-mediterranean. Until the beginning of this century, many permanent waters in the Sahara still housed relict populations. Nowadays, only few specimens survive in pools in few river canyons of the Ennedi plateau (N. Chad), where they are threatened with extinction. Another relict population, in the Tagant hills of Mauretania, was found to be probably extinct in 1996.     

Effect of the rootstock on the occurrence of lime-induced chlorosis of potted Vitis vinifera L. cv. ‘Pinot blanc’

The chlorosis susceptible Vitis vinifera L. cv. Pinot blanc was grafted on two hybrid rootstocks with different iron efficiency, as follows: V. Berlandieri × V. rupestris 140 Ru (iron-efficient) and V. riparia × V. rupestris 101-14 (iron-inefficient). The grafted vines were grown in pots of a calcareous and a non-calcareous soil. The shoot growth was periodically checked and leaves, selected at two different times (at the middle of the annual growing period), were assayed for total chlorophyll, ferrous iron, ash alkalinity, percentage of dry matter and chlorosis score. At the end of the growing cycle the roots were oven-dried and weighed. The most significant findings of the trial were: (a) the soil strongly affected the shoot growth, with canes about twice as long in the non-calcareous soil; (b) the iron-efficient rootstock (140 Ru) did not induce chlorosis when growing on the calcareous soil, while the opposite occurred with the iron-inefficient rootstock (101=14); and (c) a high ash alkalinity occurred in light chlorotic leaves compared to green ones, under the same iron concentration.     

Some peculiar elements in the rotifer fauna of the atlantic sahara and of the atlas mountains

In addition to ubiquitous forms, the Adrar of Mauretania has a number of rare tropicopolitan species, which occur here in relative abundance. In the Moroccon Atlas mountains, West-African forms meet boreo-alpine forms.A population of Hexarthra from Atar, Mauretania, was intermediate between the fennica-group and the jenkinae-group.Contribution n° 28 from project Limnology of the Sahara, under contract n° 2.0009/75 with the Fonds voor Kollektief Fundamenteel Onderzoek, Belgium     

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.