SHORT COMMUNICATION MONITORING GRAPEVINE VIRUSES BY ELISA AND RT-PCR THROUGHOUT THE YEAR

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1 029_JPP456SCFiore_ :53 Pagina 489 Journal of Plant Pathology (2009), 91 (2), Edizioni ETS Pisa, SHORT COMMUNICATION MONITORING GRAPEVINE VIRUSES BY ELISA AND RT-PCR THROUGHOUT THE YEAR N. Fiore, S. Prodan and A.M. Pino Facultad de Ciencias Agronómicas, Universidad de Chile, Santiago, Chile SUMMARY Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1, 2, and 3 (GLRaV-1,-2, and -3), Grapevine virus A (GVA) and Grapevine fleck virus (GFkV) were monitored monthly throughout a year in naturally infected field-grown vines by ELISA and RT- PCR. The organs tested were: opening buds in September, tips or unfolded leaves in September and October, leaf petioles from October to April, completely expanded leaves from November to April, green phloem tissues from October to February and cortical scrapings from December to August. Phloem of lignified canes, when available, is the best source for all viruses tested, allowing 100% detection by ELISA and RT-PCR. This is the first study carried out in South America to establish the best plant material and sampling times to optimize grapevine virus detection by ELISA and RT-PCR. Key words: Grapevine viruses, detection, ELISA, RT- PCR, South America. Grapevine (Vitis vinifera L.) is widely grown in Chile as vineyards cover about 176,000 ha in six regions. The relatively rapid expansion of the acreage given over to grapes and a limited certification program have resulted in the wide spreading of various diseases of viral aetiology. A previous report (Fiore et al., 2008), identified the viruses that decrease grapevine marketable productions as Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1, 2, and 3 (GLRaV-1, -2, and -3), Grapevine virus A (GVA) and Grapevine fleck virus (GFkV). However, grapevine virus testing at different moments of the year was necessary for certification and clean-stock program requirements. Limits to detection are posed by the erratic distribution of viruses within the vines, the type and age of tissue used for analysis, the season, and the environmental factors, which influence virus concentration (Minafra et al., 1992; Rowhani Corresponding author: N. Fiore Fax: nfiore@uchile.cl et al., 1992, 1993, 1997; Minafra and Hadidi, 1994; Monis and Bestwick, 1997). Another important intrinsic character of the grapevines is the high content of polyphenolic and polysaccharide compounds, believed to interfere with RT-PCR (Newbury and Possingham, 1977; Rezaian and Krake, 1987; Demeke and Adams, 1992; John, 1992, Minafra and Hadidi, 1994; Staub et al., 1995), whose levels vary in different tissues, in different phenological stages and at different times of the year. Considering the six viruses mentioned above as the most frequent and dangerous for Chilean grapevines, a one-year study was conducted to optimize their detection. Ability to determine virus presence by ELISA and RT-PCR was investigated on a monthly basis for different kind of tissues throughout the year, to establish which tissue and method is the most fit to be used at a certain time of the year. Samples were collected from infected grapes in commercial vineyards of the Region Metropolitana de Santiago which had been previously checked by ELISA. Three plants with single or multiple infections were used for each of the six viruses. Infected grape varieties tested were Cabernet Sauvignon, Chardonnay, Thompson Seedless, and Petit Syrah (Table 1). Vines were sampled monthly, from September 2003 to August To account for the uneven distribution of viruses within the plant, collections were made from all branches of the same plant to form the sample for analysis. Tested organs were: opening buds in September, tips of emerging shoots or unfolded leaves in September and October, leaf petioles from October to April, completely expanded leaves from November to April, phloem scrapings from green canes from October to February, and phloem scrapings from lignified canes from December to August. ELISA (Clark and Adams, 1977) was carried out using Agritest (Italy) kits: DAS-ELISA for GFLV, GVA, GLRaV-1, -2, -3, and DASI-ELISA for GFkV. Testing was done according to the manufacturer s instructions, except that the minimal absorbance value considered for positiveness was two rather than three times that of the healthy control (Fiore et al., 2008). Total nucleic acids (TNA) extraction was using the silica capture method (MacKenzie et al.,1997; Malinovski, 1997). Fifteen µl aliquots of TNA were primed with

2 029_JPP456SCFiore_ :53 Pagina Monitoring grapevine viruses in Chile Journal of Plant Pathology (2009), 91 (2), Table 1. Samples used for ELISA and RT-PCR assay. The vines used were naturally infected by the viruses listed in the second column. Grapevine variety Viruses infecting the plant Viruses monitored by ELISA and RT-PCR Petit Syrah GFLV, GVA, GFkV GVA Chardonnay GLRaV-1, GVA, GLRaV-3 GLRaV-1, GVA Chardonnay GLRaV-1, GVA, GLRaV-3 GLRaV-1, GVA, GLRaV-3 Chardonnay GLRaV-1, GVA, GLRaV-3 GLRaV-1, GLRaV-3 Chardonnay GLRaV-3 GLRaV-3 Thompson Seedless GFkV, GLRaV-2 GFkV, GLRaV-2 Thompson Seedless GFkV, GLRaV-2 GFkV, GLRaV-2 Thompson Seedless GFkV, GLRaV-2 GFkV, GLRaV-2 Cabernet Sauvignon GFLV GFLV Cabernet Sauvignon GFLV GFLV Cabernet Sauvignon GFLV GFLV GFLV: Grapevine fanleaf virus; GLRaV-1, -2, -3: Grapevine leafroll associated virus 1, 2, 3; GVA: Grapevine virus A; GFkV: Grapevine fleck virus. DNA random hexanucleotids (Roche, Switzerland) and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (M-MLV-RT, Promega, USA). DNA amplification was done using Invitrogen Taq DNA polymerase (Brazil) and the following target specific primers: C3310/H2999 for GFLV (McKenzie et al., 1997); LEV1C447m/LQV1H47m for GLRaV-1 (designed by R. Johnson - AgriCanada - and modified by Fiore et al., 2008); LRaV-2(2)/LRaV-2(1) for GLRaV-2 (Abou-Ghanem et al., 1998); LC2/LC1 for GLRaV-3 (Boscia et al., 2001); C995r/H587f for GVA (Minafra and Hadidi, 1994); GFkV-L630/GFkV-U279 for GFkV (Shi et al., 2000). Positive controls for ELISA and RT-PCR consisted of fresh or lyophilized grapevine cortical scrapings from infected plants. ELISA and RT-PCR results are presented in Table 2 and 3, respectively. The last four months are not included, since they were always tested positive using phloem of lignified canes (Ph.L) with both techniques. Since the sensitivity of both detection methods varied for the six viruses, and also for the different types of tissue, to evaluate detection reliability for all viruses assayed in a certain type of tissue, ratios of positives versus all analyzed samples throughout the year are presented in Table 4. As shown in Table 2, GFLV was 100% detected in most of the organs at all growing stages, except for the leaf petioles, the green phloem tissues and completely Table 2. ELISA detection a from September 2003 to April 2004 b. Virus September 03 October 03 November 03 December 03 B TL LP TL G.Ph LP L G.Ph LP L G.Ph Ph.L GFLV 3/3 3/3 1/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 GLRaV-1 0/3 0/3 0/3 0/3 0/3 1/3 0/3 1/3 3/3 3/3 3/3 n.a. GLRaV-2 0/3 n.a. 0/3 0/3 3/3 3/3 1/3 3/3 3/3 0/3 3/3 n.a. GLRaV-3 0/3 0/3 0/3 0/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 n.a. GVA 0/3 0/3 0/3 0/3 3/3 3/3 0/3 3/3 3/3 3/3 3/3 n.a. GFkV 3/3 n.a. 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 n.a. Total 6/18 3/12 4/18 6/18 15/18 16/18 10/18 16/18 18/18 15/18 18/18 3/3 % Virus January 04 February 04 March 04 April 04 LP L G.Ph Ph.L LP L G.Ph Ph.L LP L Ph.L LP L Ph.L GFLV 3/3 3/3 1/3 3/3 3/3 3/3 n.a. 3/3 3/3 3/3 3/3 3/3 2/3 3/3 GLRaV-1 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 GLRaV-2 3/3 1/3 3/3 n.a. 3/3 0/3 3/3 3/3 3/3 1/3 3/3 3/3 0/3 3/3 GLRaV-3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 GVA 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 2/3 3/3 3/3 1/3 3/3 GFkV 3/3 3/3 3/3 n.a. 2/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 TotaL 18/18 16/18 16/18 12/12 17/18 14/18 15/15 18/18 17/18 15/18 18/18 18/18 12/18 18/18 % a Number of positives versus number of tested samples. b From May to August 2004 the only available tissue for analysis was the phloem of lignified canes (Ph.L), in which ELISA results were always positive. GFLV: Grapevine fanleaf virus; GLRaV-1, -2, -3: Grapevine leafroll associated virus 1, 2, 3; GVA: Grapevine virus A; GFkV: Grapevine fleck virus; B: opening buds; TL: tips or unfolded leaves; LP: leaf petioles; L: completely expanded leaves; G.Ph: green phloem tissues; Ph.L: phloem of lignified canes; n.a.: material not available at the precise moment of sampling. Total: ratio of positives versus all analysed samples for all viruses in the indicated tissue, presented also as a percentage (%).

3 029_JPP456SCFiore_ :53 Pagina 491 Journal of Plant Pathology (2009), 91 (2), Fiore et al. 491 Table 3. RT-PCR detection a from September 2003 to April 2004 b. Virus September 03 October 03 November 03 December 03 B TL LP TL G.Ph LP L G.Ph LP L G.Ph Ph.L GFLV 3/3 3/3 2/3 3/3 3/3 2/3 3/3 3/3 1/3 2/3 2/3 3/3 GLRaV-1 2/3 2/3 3/3 3/3 1/3 3/3 2/3 2/3 2/3 1/3 0/3 n.a. GLRaV-2 2/3 n.a. 2/3 3/3 3/3 3/3 2/3 2/3 2/3 0/3 0/3 n.a. GLRaV-3 3/3 3/3 3/3 3/3 0/3 3/3 2/3 2/3 3/3 2/3 0/3 n.a. GVA 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 n.a. GFkV 3/3 n.a. 3/3 3/3 2/3 3/3 2/3 1/3 2/3 1/3 0/3 n.a. Total 16/18 11/12 16/18 18/18 12/18 17/18 14/18 13/18 13/18 9/18 4/18 3/3 % Virus January 04 February 04 March 04 April 04 LP L G.Ph Ph.L LP L G.Ph Ph.L LP L Ph.L LP L Ph.L GFLV 0/3 1/3 1/3 3/3 1/3 1/3 n.a. 3/3 2/3 3/3 3/3 3/3 1/3 3/3 GLRaV-1 1/3 1/3 1/3 3/3 1/3 1/3 2/3 3/3 2/3 2/3 3/3 2/3 1/3 3/3 GLRaV-2 3/3 2/3 1/3 n.a. 2/3 1/3 2/3 3/3 2/3 1/3 3/3 3/3 1/3 3/3 GLRaV-3 0/3 2/3 1/3 3/3 1/3 2/3 3/3 3/3 2/3 2/3 3/3 0/3 2/3 3/3 GVA 2/3 3/3 2/3 3/3 2/3 2/3 2/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 GFkV 0/3 1/3 0/3 n.a. 1/3 1/3 1/3 3/3 2/3 3/3 3/3 1/3 3/3 3/3 TotaL 6/18 10/18 6/18 12/12 8/18 8/18 10/15 18/18 13/18 14/18 18/18 11/18 11/18 18/18 % a Number of positives versus number of tested samples. b From May to August 2004 the only available tissue for analysis was the phloem of lignified canes (Ph.L), in which RT-PCR results were always positive. GFLV: Grapevine fanleaf virus; GLRaV-1, -2, -3: Grapevine leafroll associated virus 1, 2, 3; GVA: Grapevine virus A; GFkV: Grapevine fleck virus; B: opening buds; TL: tips or unfolded leaves; LP: leaf petioles; L: completely expanded leaves; G.Ph: green phloem tissues; Ph.L: phloem of lignified canes; n.a.: material not available at the precise moment of sampling. Total: ratio of positives versus all analysed samples for all viruses in the indicated tissue, presented also as a percentage (%). expanded leaves in October, January and April, respectively. GLRaV-1 was not detected in developing organs in the first two months. However, detection was positive from two samples in November, to become 100% from December to August in all organs assayed. GLRaV-2, - 3, and GVA were not detectable in September and October in opening buds, shoot tips or unfolded leaves and leaf petioles, but only in the green phloem tissues. From November to August, GLRaV-2 and GVA detection in fully expanded leaves was incomplete, except for GVA in leaf tissue in December and January; whereas detection was 100% in other organs. GFkV was consistently tested positive in all organs over the year, except the petioles of late summer leaves (February and March). Based on monthly detection of each of the six viruses in a certain type of tissue (Table 4), ELISA yielded 100% positive results only for GFLV and GFkV in opening buds in September and in shoot tips or unfolded leaves in September and October, whereas the other viruses could not be detected in these organs. As to leaf petioles and completely expanded leaves, the overall detection level of all viruses was 86% and 76%, respectively. Green cane phloem appeared to be a reliable tissue for GLRaV-2, -3, GVA, and GFkV detection, less so for GFLV and GLRaV-1. The overall detection rate of the six viruses was 92%. Detection by RT-PCR during the whole year for every type of tissue is reported in Table 3. GFLV was successfully detected in September to November, except for two cases in petioles. From December to February, detection became lower in the leaves, petioles and in green cane phloem. In March the detection was low only for leaf petioles, the same as in the already senescent April leaves. GLRaV-1 was partially detectable in opening buds and shoot tips or unfolded leaves in September, and it was 100% detected only in petioles and leaves collected in October. From November to April, RT-PCR generated less positive results from all tissues tested, except for leaf petioles in November and phloem scrapings from mature canes in all months. From September to December GLRaV-2 was consistently found (100%) only in young leaves and green phloem tissues (October), and leaf petioles (November). From January to April the detection was lower in all organs, with occasional exceptions (leaf petioles in January and April), until phloem of lignified canes became available in February, when detection rate went up again to 100%. GLRaV-3, GVA, and GFkV detection was good in September and October from opening buds, shoot tips or unfolded leaves, and leaf petioles, not from phloem of unlignified canes in October, in which detection rates of GLRaV-3 and GFkV were 0% and 66.6% respectively. During November and December, GVA was successfully found in all organs, except for green phloem tissues in December. GLRaV-3 and GFkV were consistently detected in leaf petioles, but GFkV only in November. From January to April, detection rates in the

4 029_JPP456SCFiore_ :53 Pagina Monitoring grapevine viruses in Chile Journal of Plant Pathology (2009), 91 (2), leaves, petioles and green cane phloem were lower than 100% and varied according to the virus. GLRaV-3 was found in 100% of the green canes only in February. GVA showed a good detection in completely expanded leaves, except for February but, in March, detection was 100% in leaf petioles. In January and February GFkV was partially or not detected in the phloem of non lignified cane tissues, in leaf petioles and in the leaves. In March and April, leaf tissue was again appropriate for molecular detection. With reference to RT-PCR assays in certain organs (Table 4), opening buds and shoot tips or unfolded leaves were reliable for detection in September and October (89% and 97% overall detection, respectively), even if GLRaV-1 and GLRaV-2 were not found in 100% of the cases. By contrast, petioles, completely expanded leaves and green cane phloem, especially between December and April appeared not to be good sources of target RNA since detection rate of the six viruses was variable. It decreased in green tissue in late spring and summer, in agreement with what reported for Grapevine rupestris stem pitting associated virus (GRSPaV) (Stewart and Nassuth, 2001). The selection of infected plants used for this study was based on previous ELISA results. Therefore, the negative RT-PCR and ELISA responses registered in some months, depended primarily on the temporary low concentration of viruses in the sampled organs and, for RT-PCR, perhaps also on the presence of inhibitory compounds. The highest detection percentages throughout the year were registered for GFkV and GFLV (98% and 94%, respectively) with ELISA, and for GVA (92%) and GFLV (77%) with RT-PCR (Table 4). Thus, ELISA appeared to be more sensitive than RT-PCR (84% versus 75%), which is clearly misleading, because these data include also those gathered during the period in which RT-PCR was negatively affected by the high level of polyphenols and polysaccharids in the assayed samples. In fact, using cortical scrapings form lignified canes (the best source material), RT-PCR proved always more sensitive than ELISA (data not shown), in agreement with the results by Minafra et al. (1992), Rowhani et al. (1993), Acheche et al. (1999) and Bertazzon and Angelini (2004). With this study, we confirmed that phloem scrapings from mature canes proved to be the optimal tissue for virus detection (100% reliability) using ELISA and RT- PCR (Table 4), in agreement with previous results (Kolber et al., 1985; Monis and Bestwick, 1997), although in July and August, absorbance value and intensity of amplified DNA fragment decreased (data not shown). However, when phloem from mature canes is not available, it is advisable to carry out the analysis using only Table 4. Annual virus detection from each tissue, using ELISA and RT-PCR. ELISA Virus B TL LP L G.Ph Ph.L Total (%) GFLV 3/3 6/6 19/21 17/18 10/12 27/27 82/87 (94) GLRaV-1 0/3 0/6 16/21 15/18 10/15 24/24 65/87 (75) GLRaV-2 0/3 0/3 18/21 3/18 15/15 21/21 57/81 (70) GLRaV-3 0/3 0/6 18/21 18/18 15/15 24/24 75/87 (86) GVA 0/3 0/6 18/21 11/18 15/15 24/24 68/87 (78) GFkV 3/3 3/3 19/21 18/18 15/15 21/21 79/81 (98) Overall detection 6/18 9/30 108/126 82/108 80/87 141/ /510 % RT-PCR Virus B TL LP L G.Ph Ph.L Total (%) GFLV 3/3 6/6 11/21 11/18 9/12 27/27 67/87 (77) GLRaV-1 2/3 5/6 14/21 8/18 6/15 24/24 59/87 (68) GLRaV-2 2/3 3/3 17/21 7/18 8/15 21/21 58/81 (72) GLRaV-3 3/3 6/6 12/21 12/18 6/15 24/24 63/87 (72) GVA 3/3 6/6 18/21 17/18 12/15 24/24 80/87 (92) GFkV 3/3 3/3 12/21 11/18 4/15 21/21 54/81 (67) Overall detection 16/18 29/30 84/126 66/108 45/87 141/ /510 % : Number of positives versus number of tested samples. GFLV: Grapevine fanleaf virus; GLRaV-1, -2, -3: Grapevine leafroll associated-virus 1, 2, 3; GVA: Grapevine virus A; GFkV: Grapevine fleck virus; B: opening buds; TL: tips or unfolded leaves; LP: leaf petioles; L: completely expanded leaves; G.Ph: green phloem tissues; Ph.L: phloem of lignified canes; Overall detection and %: ratio of positives versus all analysed samples for all viruses in the indicated tissue, also presented as a percentage (%).

5 029_JPP456SCFiore_ :53 Pagina 493 Journal of Plant Pathology (2009), 91 (2), Fiore et al. 493 symptomatic samples to confirm visual diagnosis. To our knowledge, this is the first study performed in South America on detection of grapevine viruses in naturally infected field-grown vines, comparing ELISA and RT-PCR in different source materials and sampling times. These results could be the basis for improving detection protocols used to test grapevine propagative material in Chile. ACKNOWLEDGEMENTS Grateful thanks are expressed to Professor G.P. Martelli for his helpful suggestions and critical reading of the manuscript. This work was financed by Fundación para la Innovación Agraria (FIA), project BIOT- BID-PI-C A-013. REFERENCES Abou-Ghanem N., Sabanadzovic S., Minafra A., Saldarelli P., Martelli G.P., Some properties of Grapevine leafrollassociated virus 2 and molecular organization of the 3 region of the viral genome. Journal of Plant Pathology 80: Acheche H., Fattouch S.M., Hirsi S., Marzouki N., Marrakchi M., Use of optimized PCR methods for the detection of GLRaV-3: a closterovirus associated with grapevine leafroll in Tunisian grapevine plants. Plant Molecular Biology Reporter 17: Bertazzon N., Angelini E., Advances in the detection of Grapevine leafroll-associated virus 2 variants. Journal of Plant Pathology 86: Boscia D., Digiaro M., Garau R., Loconsole G., Potere O., Prota V.A., Saldarelli P., Vovlas N., Protocolli per gli accertamenti sanitari degli organismi patogeni di qualità della vite: virus ed agenti virus simili. In: Savino V., Amenduni A., Bazzoni A., Boscia D., Pollastro S., Saponari M. (ed.). Atti progetto POM A32, pp Tipolitografia Vito Rodio, Putignano, Bari, Italy. Clark M., Adams A., Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34: Demeke T., Adams R.P., The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12: Fiore N., Prodan S., Montealegre J., Aballay E., Pino A.M., Zamorano A., Survey of grapevine viruses in Chile. Journal of Plant Pathology 90: John M.E., An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Research 20: Kolber M., Beczner L., Pacsa S., Lehoczky J., Detection of Grapevine chrome mosaic virus in field grown vines by ELISA. Phytopathologia Mediterranea 24: MacKenzie D.J., McLean M.A., Murkerji S., Green M., Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription polymerase chain reaction. Plant Disease 81: Malinovski T., Silicacapture-reverse transcription-polymerase chain reaction (SC-RT-PCR): Application for the detection of several plant viruses. In: Dehne H.W. (ed.) Diagnosis and Identification of Plant Pathogens, pp Kluwer Academic Publishers, Dordrecht, The Netherlands. Minafra A., Hadidi A., Sensitive detection of Grapevine virus A, B or leafroll associated-iii from viruliferous mealybugs and infected tissue. Journal of Virological Methods 47: Minafra A., Hadidi A., Martelli G.P., Detection of grapevine closterovirus A in infected tissue by reverse transcription-polymerase chaín reaction. Vitis 31: Monis J., Bestwick R.K., Serological detection of grapevine associated closteroviruses in infected grapevine cultivars. Plant Disease 81: Newbury H.J., Possingham J.V., Factors affecting the extraction of intact ribonucleic acid from plant tissues containing interfering phenolic compounds. Plant Physiology 60: Rezaian M.A., Krake L.R., Nucleic acid extraction and virus detection in grapevine. Journal of Virological Methods 17: Rowhani A., Chay C., Golino D.A., Falk B.W., Development of a polymerase chain reaction technique for the detection of grapevine fanleaf virus in grapevine tissue. Phytopathology 83: Rowhani A., Uyemoto J.K., Golino D.A., A comparison between serological and biolopgical assays in detecting grapevine leafroll-associated viruses. Plant Disease 81: Rowhani A., Walker M.A., Rokni S., Sampling strategies for the detection of grapevine fanleaf virus and the grapevine strain of tomato ringspot virus. Vitis 31: Shi B.J., Habili N., Symons R.H., Grapevine fleck virus: large sequence variation in a small region of the genome. Extended Abstracts 13 th Meeting of ICVG, Adelaide 2000: 78. Staub U., Polivka H., Gross H.J., Two rapid microscale procedures for isolation of total RNA from leaves rich in polyphenols and polysaccharides: application for sensitive detection of grapevine viroida. Journal of Virological Methods 52: Stewart S., Nassuth, A., RT-PCR based detection of Rupestris stem pitting associated virus within field- grown grapevine throughout the year. Plant Disease 85: Received December 1, 2008 Accepted March 18, 2009

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Epidemiology of Grapevine leafroll associated virus-3 and regional management

Epidemiology of Grapevine leafroll associated virus-3 and regional management Acknowledgements Funding sources: American Vineyard Foundation (AVF) California Grape Rootstock Improvement Commission California