The first three objectives of this study are dependent upon the fourth. That objective has been initiated with the establishment of the plant material in containers and the partial completion of the graft inoculations. We saved time establishing the plant materials by using rooted dormant cuttings supplied by the Department of Viticulture and Enologys field crew. These plants were potted into 1 gal containers and established. We also have ample numbers of additional established plants for re-grafting and to use as uninoculated controls. The majority of the rootstocks have been graft inoculated (see the table below) and we will soon begin another round of chip-budding. We will also use ELISA to assess the GFLV levels in the sample tissues both above and below the site of graft inoculation. The Muscadinia rotundifoliaand the Vitis berlandieri are now being propagated under mist conditions and may be graftable by Fall 1991. We sampled for fanleaf and tomato ringspot virus during the summer and fall of 1990 and found fanleaf widely scattered, but also found tomato ringspot in Napa, Sonoma and San Joaquin counties. We have resampled San Joaquin county this spring and with these results began to conclude this initial survey (the rough draft of a article for submission to California Agriculture article is included in Appendix 1). The incidence of TomRSV was higher than we expected, but does not pose a direct threat to the industry. TomRSV causes grapevine yellow vein disease, a disease which causes substantial yield reductions (on the order of fanleaf degeneration) on the east coast of the US. We suspect that this disease mimics fanleaf in California, but does not cause severe yield reductions. It is important for researchers and California Department of Food and Agriculture inspectors to recognize TomRSVs incidence and symptom expression to avoid confusion with fanleaf degeneration. Dr. Walker and Rowhani have completed a related research project determining which sample tissue produces the highest and most reliable ELISA reading over the course of the growing and dormant season (Appendix 2). Shoot tips and young leaves are the best sample tissue when growth is active, cambial scrapings of young phloem, cambium and young xylem are best after growth stops and before dormancy. During the dormant season actively growing tissues (shoots, callus, roots) forced from canes gave the highest values. We will use these results to best quantify the level of GFLV in the infected rootstocks. Elizabeth Frantz, a graduate student of Dr. Walker’s, is researching sampling strategies for GFLV detection in the vineyard. She completed sampling three 1225 vine plots with varying levels of GFLV incidence (low, medium and high) and is now testing each vine for GFLV with ELISA. We can then take the data and apply sampling strategies to it to determine how to best sample GFLV-infected vineyards.
The general overview of this project is to provide an understanding of the importance of the grapevine viroids to vine growth, productivity, and wine quality. In addition, viroids may provide a novel yet practical approach to “customize” vine growth and development to achieve such goals as the reduction in excessive vegetative vigor. VIROIDS were first identified in the 1970’s as causal agents of plant disease. It has been recognized that in some cases these sub-viral RNA molecules can be readily transmitted into receptive plant species without producing any apparent host plant reaction viewed as an expression of plant disease . Thus, the viroid-RNA can be considered as a transmissible yet non-infectious entity. Because of this condition, it is possible that the biological activity of a viroid may be expressed by altering a normal growth response of a plant. With our observation of a virtual ubiquitous occurrence of 1-3 viroids in all vines in California, the role of the viroids in grapevine tissue has become of interest. This unusual property of the widespread association of viroids with grapevines indicates that eyery vine characteristic including viticultural and enological property is viewed through a viroid background. Many of the accomplishments that have been achieved in this project have been published (enclosure copy of Szychowski et al., Vitis 30, 25-36, 1991) or presented in Sept. 1990 at the 10th Meeting b7 the ICVG (International Council for the Study of Viruses and Virus Diseases of the Grapevine) and at the Annual Meeting of the ASEV in June, 1990. These reports provide information concerning: 1) The first field trial of viroid-free grapevines now in the fourth growing season at the Oakville Experiment Station. 2) The commonality among the grapevine viroids from California and Europe. 3) The production and propagation of viroid-free varietals and rootstocks. 4) The comparative properties of viroids from grapevines which relate to the incidence of yellow speckle and vein banding diseases.
Young grapevines have been successfully propagated from virus infected grapes. Ten different virus isolates have been included in- these experiments. We have found that it is easy to culture plants from explants which are 2-3 mm in size or larger. Although we have cultured plants from explants which are l mm or smaller, the survival rate is low. We hope to improve that survival rate by further refinement of technique. Early data using the ELISA test on the plants which are out of tissue culture and in soil indicate that a significant number of explants have been freed of virus. This is very encouraging data which suggest that the meristem tip culture is successful for virus elimination. However, retesting over the next few years as the vines mature is needed to be sure that the virus is completely eliminated from the plants, not just in low concentrations. A large number of explants have been sucsessfully cultured and transferrsd to soil in the 1991-1992 funding year but due to staff and funding shortages they have not been ELISA tested. Grapevine virus collections essential to further studies of virus elimination techniques have been established and are thriving. Experiments are underway to determine the optimum explant size for grapes infected with leafroll, fanleaf, Rupestris stem pitting and corky bark. The progress which has been made in developing antisera for leafroll and corky bark should facilitate this work. In addition, efforts are being made to use antiviral chemotherapeutics as a step in the tissue culture process. Wood from four leafroll-infected cabernet sauvignon selections from Napa Valley has been collected and the selections are undergoing therapy; these materials will be used for evaluations of the effectiveness of the therapy and for subsequent evaluation of the effects of the virus diseases on clonal characteristics.
A good supply of antisera to two isolates (type II and type III) of the long clostroviruses associated with grapevine leafroll (GLRV) has been produced as well as a low tittered antiserum to the type IV GLRV. Monoclonal antibodies are produced to type III of GLRV and the titer of the antibody is quite high when evaluated in ELISA tests. A clostrovirus was mechanically transmitted from leafroll (type III) infected pinot noir to Nicotiana occidentalis. Investigations revealed that this virus is not associated with grapevine leafroll disease. A high-tittered antiserum to grapevine fanleaf virus (GFLV) has been produced and quantities have been increased. A low-tittered antiserum to tomato ringspot virus (TmRV, causal agent of grapevine yellow vein disease) has been produced and efforts are underway to produce a high-tittered antiserum to this virus. The antisera conditions have been optimized for GFLV and GYW detection in ELISA tests and they are routinely being used to test the foundation stocks at FPMS. The same GFLV antiserum also being used by California Department of Food and Agriculture (CDFA), Pest Exclusion branch for testing the registered grapevine material in their registration and certification program. An antiserum to grapevine corky bark virus (GCBV) has also been produced and conditions for optimizing the reactivity of the antiserum in ELISA tests were done. Another polyclonal antiserum to GCBV has been produced. The reason for producing another antiserum for GCBV was to get a cleaner antiserum (with lower healthy background in ELISA test) by doing more rigorous purification of the virus particles. Evaluation of this antiserum is underway. Some monoclonal antibody lines were produced for GCBV but subsequently lost their ability to produce specific antibodies. Our attempt has failed to purify enough rupestris stempitting associated virus for the production of antiserum. Double stranded RNA has been consistently extracted from a grapevine infected with rupestris stempitting and we are planning to make a cDNA clone from the dsRNA and use it in a cDNA hybridization system as a diagnostic tool. A source for GLRV-type I has been identified. Amounts of virus has been purified and within next few weeks we will start immunizing a rabbit to produce specific polyclonal antibody to this virus.