Grapevine fanleaf virus: detection and distribution

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.

Grapevine Viroids as Possible Factors in Disease, Clonal Variation and Wine Quality

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.

Meristem tip culturing for the elimination grapevine viruses

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.

Minimizing Fertilizer Inputs for Variegated Leafhopper Management in Grapes

This research investigated the relationship between host plant (i.e,, grapevine) nitrogen status and population dynamics of the variegated leafhopper (VLH) in a commercia1 vineyard for three years. During each year, replicated fieli plots received the following treatments: 1) control (no fertilizer added), .’) 75 lbs. N / acre (synthetic ammonium nitrate), 3) 150 lbs. N / acre (synthetic ammonium nitrate), and 4) two tons of compost / acre ( = 38 lbs. N / acre). The response of VLH to these treatments was investigated by monitoring nymph densities and conducting oviposition (egg-laying) and nymphal development rate tests. During all three years, VLH nymph populations reached a seasonal peak during the first generation. Nymph densities during the second and third generations were especially low in 1989 and 1990, and reached a moderate level during the second generation of 1991. Oviposition tests conducted in July 1989 and June 1990 demonstrated greater VLH egg-laying in the synthetic fertilizer plots. However, oviposistion during the July 1991 test was greatest in the compost plots. No significant differences in VLH nymphal development rates were observed among treatments.

Production of Antisera to Grapevine Viruses

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.

Spiders as Beneficials in Grape Agro-ecosystems

Spiders represent an entirely predacious group of organisms whose potential importance as beneficials in agro-ecosystems has generally been overlooked. The goal of this research was to investigate the potential importance of spiders as a mortality factor for grape insect pests. Spider populations were sampled at frequent intervals in a number of Fresno County vineyards. Except for one site, all vineyards utilized “soft” pesticide alternatives. Furthermore, the sites represented a range of environments including vineyards near and distant from riparian habitats. Spiders were abundant in vineyards managed with the use of “soft” pesticides (e.g., B.t. & soaps) in the Fresno County area. Three species in the families Agelenidae and Clubionidae were particularly common. Species in the family Salticidae were less abundant, although consistently present. Spider populations “built” in the canopy and persisted through the growing season with little fluctuation over short time intervals. The relative abundance of spiders in vineyard canopies suggests that they are of considerable potential importance as biological control agents in vineyards, having been grossly overlooked. It is suggested that the negative impact of pesticide use on spider populations may be extensive. Our findings indicate the need for additional research directed at defining the impact that spiders have on specific insect pests of vineyards. In addition, studies are needed to investigate the effects of ground cover management, pesticide use, and other cultural factors on spider population dynamics.

Use of Prune Refuges and Cultural Practices for Enhancing the Biological Control of the Grape Leafhopper

1. Impact of Prune refuges. Prune refuge management. Results of the analysis from WSFS indicate that significantly greater numbers of leafhopper eggs were laid on the modified pruning treatment during the season. The densities of eggs between treatments were highly variable early in the season, but favored the modified treatments during the later half of the season. We suspect the differences may be a response to the greater leaf densities in the modified treatments which may provide a larger number of potential oppositional sites and possibly a more favorable microhabitat. The results suggest the use of modified pruning practices may allow for greater population densities to be supported within a prune refuge as compared to conventional practices. The initial results of the irrigation study indicated no significant differences in nymph densities among the irrigation treatments, but significant differences were detected for sample date with no significant interaction among the factors. The analysis was repeated using a single factor analysis of variance for each sample date of the season (Table 1). On two dates, 19 August and 16 September, significant differences were found among the treatment means. Comparison of treatment means found trees in the highest irrigation regime had the largest nymph population (P < 0.05). We interpret these results as an indication that irrigation management practices may have an effect on the density of leafhopper nymphs supported by prune trees. The two dates where significant differences were detected correspond to the two sampling dates following cessation of irrigation treatments. Differences were probably not detected on prior dates due to insufficient time for water stress to have an effect on the leafhopper population. The lack of differences during the last sample date may be the result of water stress equally effecting all trees in the study. Longer term effects of irrigation management may be required to impact leafhopper abundance. Effect of irrigation-nitrogen management on the attractiveness of prune leaves to the prune leafhopper. This study addressed the optimum nitrogen and irrigation rates necessary to maximize the production of prune leafhoppers and Anagrus parasites. We evaluated combinations of four nitrogen rates and three irrigation rates. One year old twigs were pruned from each tree and brought to the laboratory and placed in sleeve cages containing adult prune leafhoppers. In the laboratory, the feeding preferences of the leafhoppers were recorded for each of 16 replications. Although the analyses are not yet completed, preliminary results suggest that the leafhoppers are more attracted to leaves having higher concentrations of water or nitrogen. We have not yet determined which factor has the greatest impact. Cultural practices involving cover crops. Effect of cover crops on grape yield. Results of yield comparisons among the three seasons found no differences in berry yield among the cover crop treatments. Overall berry yield varied significantly from year to year, but no significant interaction was detected between year and cover treatment. These results suggest the presence of a cover crop over several seasons does not appear to have a significant effect on yield. Effect of cover crops on Anagrus parasite and leafhopper populations. The presence of a cover crop was examined primarily for its effect on the density of variegated leafhopper (VLH) eggs and the proportion of these eggs which were parasitized. Results from the WSFS and Kearney vineyards suggest the presence of a cover crop had no effect on VLH egg densities as compared to the the non-cover treatment. Furthermore, no differences were detected in the proportion of these eggs which were parasitized. Results on the effect of the cover crop treatments on trap captures of adult insects suggests the presence of a cover resulted in lower parasite captures at WSFS and KAC. Furthermore, no significant differences in the number of grape leafhopper captures were detected among the treatments at Kearney or at WSFS. The effect on variegated leafhoppers were mixed, no differences were detected at the Kearney site while significantly greater numbers were found on vines associated with the non-cover crop treatment at WSFS. These results suggest the presence of cover crops in vine rows can have a significant effect on the number of adult parasites captured on sticky traps, although these differences do not appear to translate into differential parasitization rates among the cover crop treatments.