Development of Next Generation Rootstocks for California Vineyards

2015 Screening of crosses for salt resistance – chloride exclusion in experimental hybrids – A sampling of individuals from 10 hybrid populations (Table 1) was screened for salt tolerance and chloride exclusion.  These hybrid populations were 1) crosses of wild genotypes earlier found to be strong chloride excluders, 2) crosses of commercial rootstocks, or 3) crosses of rootstocks to strong-excluding wild genotypes. Plants were assayed using components of the rapid screen method: herbaceous cuttings grown in fritted clay media in 1-gallon pots, and with a standard growth period prior to salt exposure. However, a higher (75 mM) NaCl solution was used rather than the standard 25 mM NaCl solution because of anticipated strong chloride exclusion derived from one or both parents and the accompanying need to distinguish between individuals using an unusually high chloride concentration. Parentage of hybrid groups is listed in Table 2.  Harvest date was based on the death of the most susceptible individuals, which in this case took nearly two months of high salt exposure.

Salt stress phenotypes at the time of harvest are presented in Figure 1. Although visual symptoms are less informative than chloride concentration in the leaves, which are currently being assayed, such symptoms do generally correlate with chloride concentration and so provide a rough preliminary estimate of results. Most notable from Figure 1 are the poor performance of all hybrids derived exclusively from commercial rootstocks: Ramsey x St. George and Dog Ridge x St. George, despite St. George being an established strong chloride excluder. This result implies that the seven wild genotypes used in this study are imparting a superior chloride exclusion relative to that found in commercial rootstocks. In support of this result is the very strong performance of V. girdiana x V. arizonica hybrids (Figure 1), several of which were completely asymptomatic. Surprisingly, V. vinifera cv. Thompson Seedless also performed relatively well. Several possibilities can account for this, including higher leaf succulence in Thompson Seedless that could mask the ordinary phenotypic effects of high chloride accumulation in the leaves, but the actual performance will not be known until the leaf chloride concentrations have been determined. It is also possible, though unlikely, that V. vinifera has weak chloride exclusion at low concentrations and strong chloride exclusion at high concentrations, perhaps in response to the high osmotic stress of 75 mM NaCl. Forthcoming data on leaf chloride concentration will provide a clarified and robust ranking of the genotypes in this study, and promises to provide direction for salt tolerance screening of hybrid populations in 2016.

2015 screening of seedling populations for resistance to nematodes and salt – Testing of recent seedling populations for nematode resistance (HarmA and HarmC, ring and dagger nematodes) and salt resistance continues. Progress here was held up a bit with the hiring of Becky Wheeler and departure of Liang Zheng. Liang retired with Howard Ferris last summer and we have been trying to ensure a smooth transition. We have completely revamped the greenhouse space for nematode screening and have well-established populations of three root-knot strains (HarmA, HarmC and Race3) and ring nematodes. We are also building our X. index populations too.

In 2015 we scored 41 populations (662 individuals) that were made to combine broad nematode resistance with salt and drought resistance, for horticultural appearance – lack of brushy growth, long internodes, long canes and good vigor. Of these 662 seedlings 18 were excellent and an additional 54 had good horticultural characters. These seedlings and others that passed horticultural screening this year were moved to rooting studies. Last year we tested 571 seedlings from crosses to combine broad nematode resistance with salt and drought resistance for rooting ability; 60 of these seedlings rooted well and will be advancing to nematode testing and root assays for depth and fibrosity

Development of Next Generation Rootstocks for California Vineyards

2014 Pollinations – The 2014 crosses are presented in Table 1. They focused on combining PD resistant rootstocks with nematode resistance from arizonica forms with XiR1 X. index resistance and the GRN rootstocks; using excellent forms of chloride exclusion from Claire Heinitz’ work in crosses with GRN nematode resistance; using double chloride exclusion (shoot and root exclusion – most forms sequester chloride in the roots but prevent it from moving to the shoots, these prevent chloride from building up in the roots); combining drought resistance with chloride exclusion and GRN nematode resistance; combining deep rooting (Dog Ridge and 14uRu) with GRN nematode resistance; and combine vinifera x rotundifolia (VR) ring nematode resistance and potential for virus tolerance with GRN nematode resistance.

2014 Screening of Crosses for Nematode Resistance – Nina Romero and I walked about 1,100 of the 2010-2012 progeny and scored them for horticultural characteristics (cane length and brushiness and internode lengths. About 20{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of these progeny were advanced to rooting tests with ten 2-3 node cuttings. Those that rooted well and scored highly for horticultural characters were advanced to nematode testing against a combined inoculum of HarmA and HarmC (Harmony and Freedom aggressive root-knot nematode strains) and then against ring nematode. Unfortunately, not all selections were tested for both nematodes, but we have selections that will be tested to confirm either resistance. We also tested these selections for salt tolerance in a quick screen to select those with strong resistance and potential for breeding and selection as rootstocks Table 2 presents the best of the ring nematode resistant selections in comparison to nematode numbers and nemas/g or root obtained for O39-16, our highly resistant control. Plants were propagated by Nina and grown in 4 inch pots for testing. They were inoculated with 1,500 ring or 500 root-knot nematodes and evaluated for population development (ring) or egg masses (root-knot) after 3 months of growth. None were as highly resistant as either of our two standards the rotundifolia-based rootstocks O39-16 and GRN-1, but we will select the best in terms of rooting and root-knot and ring resistance to advance to further nematode testing against citrus and dagger nematodes. Table 3 and 4 present the results of testing with the combined HarmA/HarmC root-knot nematode inoculum. The breeding objective for Table 3 progeny was to improve the rooting of the GRN series (particularly GRN-5) and moderate vigor by crossing with 101-14Mgt. Thirty of these with egg mass / g of root data below 2 will be advanced to further testing. The selections tested in Table 4 were hoped to combine salt tolerance, deeper rooting and broad nematode resistance. Thirty-three of these will be advanced to salt and additional nematode testing. They include a broad range of resistance backgrounds and have good promise. Table 5 presents the parentage and number of selections that survived a severe salt screen that Nina devised. About 300 of the 1,100 we scored for horticultural characters and that rooted at 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and above were tested for salt resistance by submerge them in 150 mM NaCl (about 30{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of seawater) for 2 weeks to eliminate as many selections as possible prior to additional testing – 61 did not develop any salt burn symptoms, although they had reduced root and shoot growth; 2 or 30 V. rupestris selections from Missouri also passed this test. All of these 2 selections will be rested with our established screen to compare the effectiveness of this rapid screen. Selections that include the GRN rootstocks as parents will be advanced to screening against all the nematode strains.

Fanleaf – We continue to make progress on identifying and verifying the function of the Xiphinema index resistance gene from V. arizonica b42-26, and it resistance locus XiR1. Two gene candidates are members of the NB-LRR (nucleotide binding-leucine rich repeat) resistance gene family that control recognition of pests and diseases and the triggering of a defense reaction. These two candidates were transformed into St. George and Thompson Seedless and some lines exhibited reduced susceptibility to X. index (Figure 1), but the transformed plants were still susceptible. There are more lines to test (Table 6) and we are examining gene expression with qPCR and will pursue native promoters to determine if they can increase resistance. Xiaoqing Xie and Cecilia Agüero have been producing green-grafted M. rotundifolia and GFLV infected Chardonnay plants to test resistance to the virus in different cultivars of M. rotundifolia. After initial success with Lucida and Trayshed (Figure 2), following experiments include five additional varieties and O39-16. Xiaoqing has also produced a number of tetraploid VR hybrids that we hope will be better able to hybridize with other rootstocks and allow us to introgress rotundifolia’s remarkable resistance, which is very difficult due to the differences in chromosome number (Table 7). The diploid and tetraploid forms of four VR genotypes have been established in the field for further analysis. Olmo was able to produce some fertile VR hybrids but because these are vinifera x rotundifolia some will susceptible to phylloxera. A new MS student Tarana Shaghazi is testing these to determine which have the best phylloxera and ring nematode resistance. Many of these were used in crosses in 2013, and a few were used in 2014, to provide breeding material if they have good phylloxera resistance. Cecilia Agüero is also conducting pre-bloom hormone treatments on clusters in the field to test the effect of candidate cytokinins on reducing fanleaf expression. These candidates were identified by our earlier studies of xylem constituents from O39-16 and associated with its ability to induce tolerance to fanleaf disease (Figure 3). O9-16’s potential to act as natural nematicide to X. index – Evan Goldman is finished his MS thesis on the ability of O39-16 to eliminate X. index from a vineyard. He sampled X. index numbers in a 22-year-old Oakville vineyard that as planted was a large replicated rootstock trial with 4 row x 50 vine blocks. He sampled over the season to compare X. index populations on O39-16, 110R and 3309C, the later two are susceptible to X. index.

His results are summarized in the abstract below from his MS. I am including Figures 4 and 5 from the June 2014 report. Potential to Eradicate Xiphinema index Using the Bioantagonistic Rootstock‘O39-16’ Evan Goldman MS Abstract. Abstract: Previous reproduction studies of Xiphinema index (the dagger nematode) on the grape rootstock ‘O39-16’ showed that populations decreased over time. In addition, the alternative host range of X. index is limited and does not seem to include many common vineyard weeds. This study was conducted to determine the most effective sampling method to recover X. index and to evaluate the possibility that the nematode can be eradicated over time from vineyards that have been planted with‘O39-16’ rootstock. Two sampling methods (shovel vs. Oakfield tube) were used, and the nematodes were extracted and identified. Pearson’s test determined that there was a poor correlation between the two methods and subsequent sampling used the shovel method. The populations of X. index and X. americanum on ‘O39-16’ were compared with adjacent populations on ‘3309C’ and ‘110R’ rootstocks, both susceptible to X. index feeding. Samples were collected from beneath drip emitters on three dates,and on each date the same drip zones were sampled. Nematodes were extracted and identified. Very few X. index were recovered from ‘O39-16’; most samples were devoid of X. index. Significantly fewer X. index were recovered from ‘O39-16’ than from either ‘3309C’ or ‘110R’. There was a tendency for ‘O39-16’ to have more X. americanum than either ‘3309C’ or ‘110R’, although the differences were usually not significant. To verify the absence of X. index on ‘O39-16’, soil pits were dug alongside previously sampled vines. Samples were collected at 25 cm, 50 cm, and 100 cm and nematodes wereextracted and identified. Although the differences were not significant, there was a trend for fewer 3 nematodes at increasing depths. In conclusion, the likelihood that X. index can be eradicated through the use of ‘O39-16’ is high. However, these results need to be verified in other vineyards, especially those planted solely on ‘O39-16’.



Development of Grape Rootstocks with Multiple Nematode Resistance

Rootstocks currently available for nematode resistance have either inappropriate horticultural characteristics (such as the inducement of excessive vigor in scions leading to poor production and quality) or they have insufficient resistance against aggressive nematode strains and species. Several species of plant-feeding nematodes are present in most vineyards, however few rootstocks have resistance to more than one species. Sources of resistance to root-knot and dagger nematodes have been detected in several Vitis species. Crosses made among these species in 1989, 1993 and 1994 were screened first for rooting characteristics and then sequentially against three nematode species: the root knot nematode {Meloidogyne incognita race 3), a strain of root-knot nematode that overcomes the resistance of Harmony rootstock {Meloidogyne arenaria), and the dagger nematode (Xiphinema index). We have selected several candidate rootstocks with strong resistance to the individual nematode species, and some with broad resistance to two or more of the nematode species. Candidate selections will progress to field trials for testing of resistance durability and horticultural characteristics in the coming year. Screening of candidate selections against other nematode species, and against combinations of nematode species, will proceed in greenhouse trials. Rootstock candidates evaluated in these studies are selected from Dr. Walker’s rootstock breeding program. This report addresses the nematode screening, not the breeding and horticultural components of that program.

Development of New Rootstocks for Use in Napa Valley Fanleaf Sites

Funding for this project is primarily directed at supporting a rootstock trial with BV Winery and Walsh Vineyard Management. Development of fanleaf resistant rootstocks is part of a larger project funded by the California Grape Rootstock Improvement Commission. The plot at BV is now well established and being trained up the stake and onto the cordon wires. This year will produce the first crop and the first yield and pruning weight data. We have sampled 100 of the susceptible control St. George vines for the presence of X. index to make sure the nematode is evenly distributed across the plot. All samples had clear feeding damage and had from 20 to 500 X. index per liter of soil. None of the foliar samples were GFLV positive yet, but it generally takes at least 2 years to detect GFLV once the vines are inoculated. This result gives us confidence that the site is uniformly and evenly infested with nematodes. We have found additional sources of X. index resistance in V. cinerea, V. rufotomentosa and M rotundifolia. These will be used in future crosses. A second generation of rupestris X rotundifolia (Rup X Rot) seedlings has been produced from crosses of resistant X susceptible seedlings. These should have excellent viticultural characteristics (less rotundifolia like) and maintain very high X. index resistance. We have also mapped the resistance trait in this population with AFLP DNA analysis and working towards establishing where resistance resides.

Development of New Rootstocks for Use in Napa Valley Fanleaf Sites

The bench grafted plants of a number of Vitis rupestris x Muscadinia rotundifolia rootstocks with Cabernet Sauvignon scions have been planted in BV#2 block C-2 to evaluate their resistance to fanleaf degeneration. This plot is designed to test a wide range of rupestris x rotundifolia hybrids, including some known to be very susceptible to X. index feeding. The inclusion of such selections will allow us to examine the effect that rotundifolia has on disease expression, when GFLV is present in the scion. 039-16 (yinifera Xrotundifolia) has this effect on scions – feeding damage does not occur, but X. index successfully transmits GFLV to the scion. However, fruit loss does not occur, and it may be that the rotundifolia parentage may induce tolerance to GFLV. The end of this plot will also house a separate experiment that Ed Weber and I are collaborating on – examining the potential of 039-16 to act as a resistant interstock, and limit GFLV expression and movement. I am attempting to make the nematode/GFLV pressure as uniform and intense as possible in this trial, by interplanting the highly susceptible St. George between all test vines. There are 5 single vine replicates of 36 rootstock selections, and 3309C, 039-16 and St. George as controls.