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’.