PROPAGATION AND RETESTING OF WALNUT ROOTSTOCK GENOTYPES PUTATIVELY RESISTANT TO PESTS AND DISEASES Wesley P. Hackett, Gale McGranahan, Bruce D. Lampinen, Chuck Leslie, Diego Bujazha, and Soussan Hirbod ABSTRACT Over a period of three years this project, in collaboration with plant pathologists, nematologists, and molecular biologists, has demonstrated the feasibility of clonally propagating walnut rootstock genotypes selected or designed for resistance or tolerance to rootstock pests and diseases. We have clonally propagated and grown over 11000 plantlets of over 20 genotypes. Many of these plantlets have been used by Greg Browne and Mike McKenry in replicated disease and pest resistance re-test trials. Over 1500 have been grown in two nurseries to a size large enough for grafting in one growing season. Some of these genotypes have a degree of resistance or tolerance to root lesion nematode, Phytophthora citricola, Agrobacterium tumefacians and cherry leaf roll virus (blackline). These will be tested further in field trials with farm advisors for pest and disease resistance and horticultural characteristics. We have developed protocols for in vitro tissue culture micropropagation and hardwood cutting propagation for many Paradox and a few black walnut rootstock genotypes as outlined in detail in Walnut Research Reports of 2001, 2002, and 2003. To date we have not been as successful as we would like with all genotypes. With microshoots and hardwood stem cuttings, the main limiting factor for some genotypes is the induction and expression of adventitious roots. We are trying several approaches including ex vitro rooting of microshoots and use of root cuttings to try to over come this limitation. For rooted microshoots of most genotypes, we obtain high survival by acclimatizing them in fog chambers that we designed and built. They are then grown in small tree tube containers on greenhouse benches on a year round basis under long days. Plantlets large enough for planting in the nursery row can be produced in three months. They are then chilled artificially or naturally and planted in the nursery row as dormant plantlets. To try to alleviate the problem with induction and expression of adventitious roots in some genotypes, we have done some preliminary experiments using grafted root cuttings based on modification of a technique used in Argentina. These preliminary experiments show that root cuttings are much easier to root than hardwood cuttings from the same genotypes. OBJECTIVES The overall goal of this research is to identify and develop methods for producing disease and pest resistant walnut rootstock clones. This goal has been pursued through the following objectives: 1. Establishment of a field stock block and tissue culture microshoot bank of putatively disease and pest resistant genotypes for re-testing. 2. Development of protocols for tissue culture multiplication, rooting, acclimatization and growth of these genotypes for re-testing.
3. Development of protocols for cuttage propagation and container production of putatively disease resistant genotypes for re-testing. 4. Re-testing of own-rooted individual genotypes for disease resistance in the greenhouse and small field plots in sufficient numbers to verify their resistance. 5. Propagation of sufficient numbers of re-tested clones for orchard rootstock trials to evaluate horticultural and disease resistance characteristics. PROCEDURES AND RESULTS Objective 1 During year three, we completed establishment of the field stock block for the original set of putatively disease and pest resistant genotypes by planting of own-rooted trees of 5 WIP genotypes tolerant to cherry leaf roll virus (CLRV). We are replacing original genotypes that were grafted onto black walnut seedlings and adding newly selected candidate genotypes as own-rooted trees as clonal methods of propagation are developed (see objectives 2 and 3). Genotype 87-32-1 (WIP5) which is tolerant to CLRV and a wingnut genotype (as a resistant control for Phytophthora screening) were added to the tissue culture microshoot bank. Objective 2 We have continued to micropropagate the putatively disease and pest resistant genotypes for retesting for resistance or tolerance and for establishment of field trials by farm advisors (see objectives 4 and 5) using protocols devised during years one and two of the project. Induction and expression of adventitious roots on microshoots continues to be the main limiting factor for efficient propagation of some genotypes. Ex vitro rooting of newly multiplied microshoots shows promise but is erratic and successful ex vitro rooting of microshoots may be dependent on the quality of the microshoots in terms of size, substrate levels and/or degree of succulence (lignfication). During year three, we modified acclimatization and growing protocols by: (1) adding intermittent fog to the greenhouse growing bench and (2) discontinuing use of Promalin treatment which isn't necessary if plantlets are put through a dormancy inducing(50f)-dormancy breaking (42F) treatment. From November 1, 2003 through October 2004 we have produced an additional 4500 plantlets of 20 genotypes for replicated re-testing of disease and pest resistance and tolerance and field trials by farm advisors (Table 1). Overall, survival of rooted microshoots during year three was lower than in year two mainly because more difficult- to- root genotypes with less developed root systems were propagated in 2004 than 2003. Objective 3 Experiments to try to improve hardwood stem cutting propagation techniques for black, Paradox and English-Paradox backcross (blackline tolerant) genotypes were carried out on bottom-heated beds outdoors during January and February, 2004. A total of over 5000 cuttings from 12 genotypes of coppiced and tree-formed trees were stuck in Oasis solid rooting medium at 80-85F. The main experiment compared the use of standard Oasis Rootcubes (High moisture retention) with Oasis Horticubes (Lower moisture retention) as rooting media and the use of 8000mg/l potassium indolebutyric acid (KIBA) with 16000mg/l KIBA to induce adventitious
roots. To try to alleviate die back from the top cut surface of cuttings during and after rooting, we treated the cut surface of all cuttings with an asphalt emulsion at the time cuttings were stuck to try prevent drying. (However, the treatment with asphalt emulsion did not prevent cutting dieback). Most of the cuttings were stuck during the first 15 days of January but some were stuck the first 10 days of February. Data was taken eight to ten weeks after sticking. The results indicated that rooting percentage was much better using Rootcubes (High moisture retention) than with Horticubes (Lower moisture retention). This was probably because Horticubes dried out too much when managing irrigation frequency for Rootcubes (High moisture retention). Rooting percentage was somewhat better with 16000mg/l KIBA than with 8000mg/l KIBA and bud outgrowth was not inhibited by the higher concentration KIBA. In general rooting percentage was lower in 2004 than in 2003. As was the case in 2003, rooting percentage in 2004 was greatly dependent on genotype and varied from 0 to 100% using Oasis Rootcubes and 16000mg/l KIBA. The Paradox genotypes generally had higher rooting percentages than the Paradox-English backcrosses with VX211, Vlach and PX1 having rooting percentages as high as 45, 80, and 100% respectively, but AZ025 and UX022 had essentially 0 % rooting. For PX1 and Vlach rooting percentages were much higher when cuttings were stuck February 10 than when stuck January 15. The highest rooting percentages for Paradox-English backcrosses were for 87-50-1 (WIP6) and for 87-27-4 (WIP3) with 68 and 56% rooting, respectively. However, backcrosses 87-26-4, 87-27-6 and 87-32-1 rooted at or near 0%. The one black walnut genotype in the experiment (AW269) had a rooting percentage just barely above 0. In general rooting percentages for the various genotypes followed a similar pattern in 2003 and 2004 with the exception of Paradox-English backcross 87-32-1(WIP5) which rooted at nearly 100% in 2003 and near 0% in 2004. There was some indication from the 2003 and 2004 rooting data that stock plant cutting source may influence rooting percentage. We have no explanation for the drastic difference in rooting percentage for 87-32-1 from year to year or the possible influence of stock plant cutting source on rooting percentage. Because the success in rooting hardwood stem cuttings varies so greatly from genotype to genotype, we have done some preliminary experiments testing the ease of rooting of root cuttings of the various genotypes. This approach is based on the idea that if root cuttings are easy to root, we can self-graft them with stem scions to obtain whole plants for disease and pest resistance tests or graft nut cultivar scions onto root cuttings to obtain trees for orchard tests. Our preliminary experiments with rooting of root cuttings show that they are easier to root than hardwood stem cuttings of the same genotypes. Rooting percentages varied with genotype and quality of root cutting material from 33 to 95% three weeks after treatment with 3000mg/l KIBA (near optimal concentration) and sticking in vermiculite in a bottom heated fog chamber. Even root cuttings of Chandler, an English clone that is very difficult to root from stem cuttings, are quite easy to root (40 to 95% rooting after three weeks). An experiment with RX1, a Paradox genotype, indicates that root cuttings treated with 3000mg/l KIBA and T-budded with Chandler scions will root in vermiculite, with the bud union callusing simultaneously, in three weeks in a fog chamber with 80F bottom heat. Protecting the scion union from free water with a Kaput culture tube closure was important for successful bud union callusing.
Objective 4 Early in 2004 we provided 48 plantlets of VX211 and AX1 to Mike McKenry for re-testing for lesion nematode tolerance (see Mike McKenry's report). Late in 2004 we provided 778 additional plantlets of 17 genotypes to Greg Browne for re-testing of Phytophthora resistance (Table 2 and see Greg Browne's report). Objective 5 Early in 2004 we provided Joe Grant with approximately 7 plants each of 16 clones that had been previously tested for Phytophthora response by Greg Browne. Both tolerant and susceptible clones were included. These were planted in a completely randomized block at Gotelli s near Jenny Lind in a field infested with P. cinnamomi. Wingnut and paradox seedlings were planted as controls. Ten of the blackline tolerant trees were also provided to Joe Grant for planting at Taylor s Anderson Barngrover site. Also early in 2004 over 1800 dormant plantlets of 20 clones were planted by David Bonilla at his nursery site on Ellenwood (Table 3). These ranged in height from about three to 30 cm at planting. At the end of the season 66% of the plants were of graftable size (over 10mm). The average diameter was 25 mm. Clones differed visually in amount of branching, leaf color and leaflet width. These trees will be distributed bareroot to interested farm advisors early in 2005. We owe a great deal of gratitude to David Bonilla for being willing to experiment and take the time and labor to grow these trees. Stuke s Nursery also cooperated by growing 120 plantlets each of three clones at two spacings: 8 and 12. At the end of the season 81% were graftable and the mean diameter was 20 mm (Table 3). It was concluded that tighter spacing would be appropriate. Two of the clones (Phytophthora tolerant selections AZ2 and RX1) will be grafted in place and grown in an experimental plot at Stuke s in 2006. We thank Stuke nursery and especially Leslie Nerli for her interest, time and effort. TABLE 1. SURVIVAL AND GROWTH OF LAB ROOTED AND EX-VITRO ROOTED PDS AND WIP GENOTYPES FROM NOVEMBER 2003 THROUGH OCTOBER 2004. Genotype Type Total planted Alive Dead Survival % AX1 Lab rooted plants 303 224 79 74 Ex-v plants 46 34 12 74 Total 349 258 91 74 AZ025 Lab rooted plants 223 97 126 43 Ex-v plants 133 59 74 44 Total 356 156 200 44 AZ1 Lab rooted plants 96 84 12 87 Ex-v plants 1 0 1 0 Total 97 84 13 87 AZ2 Lab rooted plants 100 66 34 66 Ex-v plants 2 0 2 0 Total 102 66 36 65 AZ3 Lab rooted plants 107 51 56 48 Ex-v plants 40 11 29 27 Total 147 62 85 42
CW1 Lab rooted plants 218 137 81 63 Total 218 137 81 63 GZ1 Lab rooted plants 142 93 49 65 Ex-v plants 19 10 9 53 Total 161 103 58 64 GZ2 Lab rooted plants 444 386 58 87 Ex-v plants 107 70 37 65 551 456 95 83 JX2 Lab rooted plants 221 151 70 68 Ex-v plants 28 23 5 82 Total 249 174 75 70 MCG 42-1-1 Lab rooted plants 424 126 298 30 Ex-v plants 23 2 21 9 Total 447 128 319 29 NZ1 Lab rooted plants 99 55 44 56 Total 99 55 44 56 Px1 Lab rooted plants 1045 511 534 49 162 87 75 54 Total 1207 598 609 50 RX1 Lab rooted plants 517 446 71 86 Ex-v plants 37 17 20 46 Total 554 463 91 84 UX022 Lab rooted plants 344 150 194 44 Ex-v plants 37 5 32 14 Total 381 155 226 41 UX1 Lab rooted plants 295 125 170 42 Ex-v plants 8 0 8 0 Total 303 125 178 41 UX2 Lab rooted plants 213 164 49 77 Ex-v plants 23 5 18 22 Total 236 169 67 72 VLACH Lab rooted plants 363 260 103 72 Ex-v plants 44 26 18 59 Total 407 286 121 70 VX211 Lab rooted plants 149 116 33 78 Ex-v plants 42 40 2 95 Total 191 156 35 82 WIP2 Lab rooted plants 453 161 292 36 Ex-v plants 31 4 27 13 Total 484 165 319 34 WIP3 Lab rooted plants 999 704 295 70 Ex-v plants 50 16 34 32 Total 1049 720 329 69 Type Total planted Alive Dead Survival % Lab rooted plants 6755 4107 2648 61 Ex-v plants 833 409 424 49 Total 7588 4516 3072 60
TABLE 2. INVENTORY OF CLONAL WALNUT The first column shows ungrafted bareroot rootstocks from Bonilla s Nursery ready for field planting now; the second column shows dormant rootstocks ready to be planted in a nursery; and the third column shows dormant rootstocks distributed for greenhouse retesting for crown gall (DK) or Phytophthora (GB). Nematode retests are already underway. Clone Field trials 2005 Plant in nursery 2005 Pest and disease tests 2005 NOTES Crowngall AZ025 121 DK Most to Kluepfel for retesting MCG 42-1-1 52 DK Gene silenced construct. All to Kluepfel MCG 117-2-1 2 DK Gene silenced construct. All to Kluepfel Px1b 14 DK Control for above. All to Kluepfel Nematodes VX211* 87 78 60 GB Moderately tolerant to P. citricola. Selected for tolerance to nematodes. Most promising. Phytophthora AZ2* 150 14 48 GB Moderately tolerant to P. citricola. AZ3* 28 17 44 GB Moderately tolerant to P. citricola NZ1* 110 6 42 GB Moderately tolerant to P. citricola JX2* 195 149 60 GB Moderately tolerant to P. citricola RX1 78 194 60 GB Moderately tolerant to P. citricola. AX1 86 120 60 GB Susceptible to P. citricola. Control GZ1 82 67 60 GB Susceptible to P. citricola. Control Px1 154 795 60 GB Susceptible to P. citricola. Control AZ1* 37 14 51 GB In process of being retested for P. citricola UX1 23 39 57 GB In process of being retested for P. citricola GZ2 41 293 60 GB In process of being retested for P. citricola UX2 49 51 GB In process of being retested for P. citricola Blackline tolerant WIP3 66 798 60 GB Tolerant to CLRV. Susceptible to P. citricola WIP2 6 75 57 GB Tolerant to CLRV WIP6 8 Vigorous control UX022 59 72 58 GB Selected for crown gall resistance, but retested and found susceptible. Nice trees. Vlach 96 60 GB English Vina 10 11 Sunland 19 23 TOTALS 1231 2918 1137 * Nursery source. May have ownership issues since not all nursery agreements have been turned in to Tech Transfer Center.
TABLE 3. CLONES GROWN AT BONILLA NURSERY - 2004 Planted Graftable Graftable Diameter (mm) Clone N N % Mean SD Range CV Nematodes VX211 106 87 82 31 4.9 21-44 12.6 Phytophthora AZ2 230 151 66 26 5 13-38 19.2 AZ3 49 24 49 25 6.7 11-37 26.8 NZ1 172 111 64 26 4.4 10-39 16.9 JX2 246 191 78 29 4.1 13-39 14.1 RX1 104 78 75 18 1.6 14-22 8.8 AX1 163 86 53 27 4.3 14-40 15.9 GZ1 108 83 77 26 5.4 13-40 20.8 Px1 247 154 62 26 4.6 12-40 17.7 AZ1 52 38 73 30 4.4 22-43 14.7 UX1 27 23 85 25 4 15-30 16 GZ2 47 38 81 26 4.5 15-33 17.3 Blackline WIP3 158 66 42 26 5 12-35 19.2 WIP2 10 6 60 25 2.3 23-29 9.2 Control UX022 71 59 83 23 3.7 14-29 16.1 English Vina 14 10 71 18 3.7 13-24 20.5 Sunland 64 20 31 26 3.8 18-31 14.6 Totals 1868 1225 66 25 TABLE 4. CLONES GROWN AT STUKE NURSERY - 2004 Planted Graftable Graftable Diameter (mm) Clone N N % Mean SD Range CV AX1 120 107 89 19 4.6 10-30 26 AZ2 120 102 85 21 4.7 10-31 22 RX1 120 84 70 19 3.2 10-27 17 Totals 360 293 81 20