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.     

Incidence and Distribution of Grapevine Leafroll-associated viruses in Tunisian Vineyards

Surveys were carried out in Tunisian table grape vineyards for assessing the occurrence and distribution of grapevine leafroll-associated viruses (GLRaVs). Leafroll symptoms were commonly observed in most of the surveyed vineyards. Samples were randomly collected from 712 individual vines for laboratory testing. Enzyme-linked immunosorbent assay (ELISA) tests showed that 81.5% of the vines were infected by one (35.7%) or more (45.8%) viruses. GLRaV-3 was the most widespread virus (76.3%), followed by GLRaV-5 (38.5 %), GLRaV-6 (13.2%), GLRaV-1 (9.1%), GLRaV-2 (6.3%), and GLRaV-7 (0.9%). GLRaV-3 and GLRaV–5, two mealybug-transmissible Ampeloviruses, were present in mixture in 35.9% of samples. The highest infection rate was found in Cape Bon region (81.7 %), where cv. Italia had an infection rate of 79.5%. Superior seedless, the main cultivar in Sidi Bouzid, had 75% infection. GLRaV-6 and -7 were detected for the first time in Tunisia.     

Occurrence and Distribution of Grapevine Leafroll-associated Viruses 1, 2, 3 and 7 in Turkey

Grapevines in central Anatolia region of Turkey were surveyed for the prevalence of grapevine leafroll viruses. The field study and collection of samples were conducted in nine major grapevine‐growing areas. Samples collected from 622 vines were tested for Grapevine leafroll‐associated virus 1, 2, 3 and 7 (GLRaV‐1, ‐2, ‐3 and ‐7). According to diagnostic tests and surveys, 27 of 41 cultivars and 95 of 622 samples (15.27%) were found to be infected at least one virus. GLRaV‐1 (8.36%) was found to be the most frequently encountered virus associated with leafroll disease of grapes, followed by GLRaV‐3 (5.78%), GLRAV‐7 (3.86%) and GLRAV‐2 (2.41%).     

First Detection of Stolbur Phytoplasma in Grapevines (Vitis vinifera cv. Merlot) Affected with Grapevine Yellows in Bulgaria

Between 2003 and 2005, a survey was conducted throughout the grape‐growing regions of Bulgaria to identify possible infection with grapevine yellows diseases, especially Flavescence dorée (FD). The samples were checked for phytoplasmas and viruses inducing similar symptoms in the Central Laboratory for Plant Quarantine. To confirm stolbur phytoplasma infection of grapevine, a multiplex nested‐PCR assay for direct detection of FD and stolbur phytoplasmas was used. Infection of grapevine with phytoplasma was detected. The disease is very common disease in Bulgaria on tomatoes, potatoes and other crops. Monitoring is being continued. This is the first report of phytoplasma‐infected grapevine in Bulgaria.     

Effects of Grapevine leafroll‐associated virus 3 on the physiology in asymptomatic plants of Vitis vinifera

Grapevine leafroll disease is one of the most important viral diseases of grapevine (Vitis vinifera) worldwide. Grapevine leafroll‐associated virus 3 (GLRaV‐3) is the most predominant virus species causing this disease. Therefore, it is important to identify GLRaV‐3 effects, especially in plants which do not systematically show visual symptoms. In this study, effects of GLRaV‐3 on grapevine physiology were evaluated in asymptomatic plants of Malvasía de Banyalbufar and Cabernet Sauvignon cvs. Absolute virus quantification was performed in order to determine the level of infection of the treatment. The net carbon dioxide (CO₂) assimilation (A_N) and electron transport rate (J_flux) were the main parameters affected by the virus. The A_N reduction in infected plants was attributed to restrictions in CO₂ diffusion caused by anatomical leaf changes and a reduction of Rubisco activity. Those effects were more evident in Malvasia de Banyalbufar plants. The reduction of A_N leads to a decrease in the total oxygen uptake rate by the activity of the cytochrome oxidase pathway, producing slight differences in plant growth. Therefore, even though no symptoms were expressed in the plants, the effects of the virus compromised the plant vital processes, showing the importance of early detection of the virus in order to fight against the infection.     

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.     

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.     

Brief Report of a New Highly Divergent Variant of Grapevine leafroll‐associated virus 3 (GLRaV‐3)

The alignment of the complete genomes of genetic variants of Grapevine leafroll‐associated virus 3 (GLRaV‐3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA‐dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein (dCP) gene. Cloning and sequencing of the 549‐bp RT‐PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV‐3, which shared only 69.6–74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517‐bp amplicon of the HSP70 gene of GLRaV‐3 generated in RT‐nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3–78.7% nt similarity with other variants of GLRaV‐3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT‐nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards.     

The southern limit of the Mediterranean vegetation in the Sahara during the Holocene

Holocene records from the southern Sahara in Niger allow a reconstruction of the vegetation history and inform us about the former extension of the Mediterranean. Both pollen and charcoal analyses evidenced the direct contact of Sudanian and Saharan savannas during the middle Holocene at about 19°N, whereas at 20°N the transition from the Saharan savanna to the desert was found. In southwestern Libya (26°N) a combination of a Saharan desert vegetation and a semi‐desert Artemisia shrub on the plateaus demonstrated the contact with Mediterranean influenced formations. Regular ash and charcoal layers in middle‐Holocene sediments of the northern Niger prove an early interference of man with the vegetation development. One has to imagine that, in combination with the cattle‐keeping and the later metal production, man could have changed the former northern Sudanian vegetation into the present Sahelian savanna system from the middle Holocene on.     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

Effects of Grapevine Fanleaf and Stem Pitting Viruses on the Photosynthetic Activity of Grapevine Plants Grown in vitro

The effects of viral diseases on the photosynthetic activity of grapevine (Vitis rupestrisvar. Rupestris du Lot) leaves were investigated. The third and sixth leaves used for measurements were obtained from in vitrogrown healthy plants and plants affected by grapevine fanleaf and rupestris stem pitting viruses. The induction curves of prompt and delayed chlorophyll fluorescence, as well as the temperature characteristics of steady-state, prompt, and delayed emissions, were investigated. Age-dependent changes were found, which were related, on the one hand, to the acceleration of electron transport and the enhancement of thylakoid energization and, on the other hand, to a smaller extent of transmembrane H⁺in the younger sixth leaf compared to that in the third leaf. The infected plants characteristically showed faster electron transport, an elevated energetic efficiency of photosynthesis, and the suppression of CO₂fixation owing to a presumable activation of the adenylate metabolism. An analysis of the thermograms of prompt and delayed fluorescence revealed the shifts in the position of the M ₁peak and a half-inhibition temperature T₅₀towards a higher temperature in infected plants, which indicates a certain increase in the thermal tolerance of thylakoid membranes. The data suggest that the viral metabolism affects the functional activity and stability of thylakoid membranes.     

Photoinhibition and Recovery of photosystem 2 in Grapevine (Vitis vinifera L.) Leaves Grown Under Field Conditions

Photoinhibition of photosynthesis was investigated in grapevine (Vitis vinifera L.) exposed to 2 or 4h of high irradiance (HI) (1 700–1 800 mol m^–2 s^–1) leaves under field conditions at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the photochemical efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined, F₀ increased in both 2 (HI2) and 4 h (HI4) HI leaves sampled at midday. When various photosynthetic activities were followed on isolated thylakoids, HI4 leaves showed significantly higher inhibition of whole chain and PS2 activity than the HI2 leaves sampled at midday. Later, the leaves reached maximum PS2 efficiencies similar to those observed early in the morning during sampling at evening. The artificial exogenous electron donor Mn²⁺ failed to restore PS2 activity in both variants of leaves, while DPC and NH²OH significantly restored PS2 activity in HI4 midday leaf samples. Quantification of the PS2 reaction centre protein D1 and 33 kDa protein of water splitting complex following midday exposure of leaves showed pronounced differences between HI2 and HI4 leaves. The marked loss of PS2 activity noticed in midday samples was mainly due to the marked loss of D1 protein in HI2, while in HI4 it was mainly 33-kDa protein.     

The symptomless leaf infection with grapevine leafroll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis

The photosynthetic changes evaluated by oxygen evolution, chlorophyll fluorescence, photoacoustics, and delayed fluorescence (DF) were studied in leaves of grown in vitro for 8 weeks grapevine plants (Vitis vinifera) infected by grapevine leafroll-associated virus 3 (GLRaV-3). The infected leaves were characterized during the viral infection without visible disease symptoms. The symptomless infection led to a decrease in plant biomass. The non-photochemical fluorescence quenching, qN, declined, whereas the photochemical quenching, qP, and the Chl a/b ratio were not significantly affected. Photoacoustic and oxygen evolution measurements showed that the energy storage and oxygen evolution rate decreased in the infected leaves. Enhanced alternative electron sinks during the symptomless viral infection were also estimated. The changes in fluorescence and DF temperature curves demonstrated an enhanced stability of the thylakoid membranes in the infected leaves. This effect was clearly expressed at high actinic light intensities. The viral infected in vitro grown grapevine plants were used in the present study as a simplified model system that allow to avoid the involvement of different environmental factors that could interfere with the GLRaV infection and the virus-grapevine interactions. Thus, the 'pure' impact of the viral infection on photosynthesis could be investigated.     

Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP-3 in the medium which was due neither to alterations in IGFBP-3 mRNA expression nor to extracellular IGFBP-3 protease activity. Incubation of CTSL-deficient fibroblasts with radiolabeled IGFBP-3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP-3 accumulate transiently in endosomal and lysosomal fractions of CTSL-deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP-3 representing a new mechanism to regulate the extracellular concentration of IGFBP-3.     

Treatment of hepatitis C virus core-positive hepatocytes with the transfer of recombinant caspase-3 using the 2~,5~-oligoadenylate synthetase gene promoter

Hepatitis C virus （HCV） infection is a leading cause of liver-related morbidity and mortality throughout the world. There is no vaccine available and current therapy is only partially effective. Since HCV infects only a minority of hepatocytes, we hypothesized that induction of apoptosis might be a promising approach for the treatment of hepatitis C. In the present study, recombinant caspase-3 gene （re-caspase-3） was used because it has the ability to induce apoptosis that is independent of the initiator caspases. An HCV-specific promoter is required to regulate the cytotoxic caspase-3 expression in HCV-infected cells. It has been reported that HCV core protein can specifically activate the 21,5~-oligoadenylate synthetase （OAS） gene promoter in human hepatocytes. Therefore, we constructed an expression vector consisting of the re-caspase-3 under the OAS gene promoter （pGL3-OAS-re-caspase-3） and then investigated its effect on HCV core-positive liver cells. It was found that the pGL3-OAS-re-caspase-3 construct induced apoptosis in HCV core-positive liver ceils, but not in normal liver ceils. These results strongly suggested that the transfer of the re-caspase-3 gene under the OAS promoter was a novel targeting approach for the treatment of HCV infection.     

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua,New Guinea

Summary Melanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta 1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.