Several grapevine pathogens are disseminated by propagating material as systemic, but latent infections. Their detection and identification have a basic importance in the production and handling of propagating stocks. Thus several sensitive and reliable diagnostic protocols mostly based on molecular techniques have been developed. Of these methods... quantitative real-time PCR (q-PCR) has recently got an emerging importance. Here we collected primer data for the detection and identification of grapevine pathogens which are important in the production of propagating stocks by q-PCR. Additional novel techniques that use DNA amplification, hybridization and sequencing are also briefly reviewed.
Polymerase chain reaction driven by sequence specific primers has become the most widely used diagnostic method to detect and identify plant pathogens. The sensitive and cost-effective pathogen detection is exceptionally important in the production of propagating material. In this paper we have collected primer sequence data from the literature fo...r the detection of the most important grapevine pathogens disseminated by propagating stocks by conventional polymerase chain reaction. Basic protocols to obtain template nucleic acids have also been briefly rewieved.
The use of pathogen-free planting stock for new vineyard establishment is a key component in the maintenance and expansion of vine and quality table grape production. The success of the necessary changes in the structure of the grape industry is forced by the globalization process, the climate change, the rediscovery of autochton varieties as well... as breeding of new tolerant and resistant varieties. The renewal of vineyards largely depend on the availability of planting stocks. Serbia and Hungary found a common interest in establishing pathogen-free stock materials from newly breed resistant varieties and clonal selections of varieties which are traditional in the Serbian-Hungarian border area. During a cross-border cooperation program a complex system for the production of pathogen-free grapevine propagating material was established. Using heat therapy, in vitro shoot tip culture and traditional and molecular diagnostic techniques new pathogen-free stock materials were established from 26 varieties. They have been or will be tested for the presence of most important grapevine viruses, phytoplasmas, as well as bacterial and fungal pathogens. The complex system applying green grafting for indexing on grapevine indicators can shorten the duration of the procedure from 4 years to two-three years.
Grapevines are affected by three major bacterial diseases worldwide, such as bacterial blight (Xylophilus ampelinus), Pierce’s disease (Xylella fastidiosa) and crown gall (Agrobacterium vitis). These bacteria grow in the vascular system of their host, thus they invade and colonize the whole plant, independently on symptom development. Latently i...nfected propagating material is a major factor in their spreading. Therefore the use of bacteria-free planting stock has a basic importance in viticulture. Today several innovative diagnostic methods, mostly based on polymerase chain reaction, are available to detect and identify bacterial pathogens of grapevines. For production of bacteria-free plants, the use hot water treatment followed by establishment of in vitro shoot tip cultures is proposed.
In vitro cultures have widely been used in horticulture for rapid multiplication of new varieties and clones as well as to produce pathogen-free stock material. To improve efficient hardening and transfer in vitro grown grapevine plants were multiplied by cutting them into single-node internodes with the whole leaf. Microcuttings including the sho...ot tips were rooted in granulated perlite moisted with tapwater under sterile conditions. After 2-3 weeks the rooted microcuttings were supplied by nutrients and hardened by gradual opening and finally by complete removal of the lids of jars or plastic boxes used for growth. Using this method microcuttings of Vitis vinifera cvs. „Chardonnay", „Cabernet franc", „Riesling" and „Sauvignon blanc" and the rootstock varieties Vitis riparia x Vitis cinerea cv. „Barrier" and Vitis berlandieri x Vitis rupestris cv. „Richter 110" formed new roots and shoots and 100% of the tested plants survived the acclimatization procedure. Similar results were obtained when perlite was replaced with rockwool-, or pit-pot blocks. This method may highly increase the efficiency of producing pathogen-free propagating material and new transgenic lines.
The VirEl protein plays a key role in the transport of VirE2 protein from the bacterium to the plant cell during crown gall tumor induction by Agrobacterium. The virEl gene of A. tutnefaciens pTiA6 was cloned into the plant transformation vector pTd33 yielding pTd93virEl that was introduced into A. tuniefaciens EHA101 and used for tobacco transfor...mation. The presence of the foreign DNA in the putative transgenic plants was confirmed by PCR analysis. Nine of the 41 transformed plants formed only small tumors following infection with the wild-type A. vitis octopine strain AB3. This property was inherited into the T1 generation. The expression of virEl gene in TI plants was demonstrated by Northern blot analysis.