PROPAGATION AND RETESTING OF WALNUT ROOTSTOCK GENOTYPES PUTATIVELY RESISTANT TO PESTS AND DISEASES

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1 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 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 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.

2 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 (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 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, 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

3 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 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 (WIP6) and for (WIP3) with 68 and 56% rooting, respectively. However, backcrosses , and 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 (WIP5) which rooted at nearly 100% in 2003 and near 0% in 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 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.

4 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 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 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 Genotype Type Total planted Alive Dead Survival % AX1 Lab rooted plants Ex-v plants Total AZ025 Lab rooted plants Ex-v plants Total AZ1 Lab rooted plants Ex-v plants Total AZ2 Lab rooted plants Ex-v plants Total AZ3 Lab rooted plants Ex-v plants Total

5 CW1 Lab rooted plants Total GZ1 Lab rooted plants Ex-v plants Total GZ2 Lab rooted plants Ex-v plants JX2 Lab rooted plants Ex-v plants Total MCG Lab rooted plants Ex-v plants Total NZ1 Lab rooted plants Total Px1 Lab rooted plants Total RX1 Lab rooted plants Ex-v plants Total UX022 Lab rooted plants Ex-v plants Total UX1 Lab rooted plants Ex-v plants Total UX2 Lab rooted plants Ex-v plants Total VLACH Lab rooted plants Ex-v plants Total VX211 Lab rooted plants Ex-v plants Total WIP2 Lab rooted plants Ex-v plants Total WIP3 Lab rooted plants Ex-v plants Total Type Total planted Alive Dead Survival % Lab rooted plants Ex-v plants Total

6 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 AZ DK Most to Kluepfel for retesting MCG DK Gene silenced construct. All to Kluepfel MCG DK Gene silenced construct. All to Kluepfel Px1b 14 DK Control for above. All to Kluepfel Nematodes VX211* GB Moderately tolerant to P. citricola. Selected for tolerance to nematodes. Most promising. Phytophthora AZ2* GB Moderately tolerant to P. citricola. AZ3* GB Moderately tolerant to P. citricola NZ1* GB Moderately tolerant to P. citricola JX2* GB Moderately tolerant to P. citricola RX GB Moderately tolerant to P. citricola. AX GB Susceptible to P. citricola. Control GZ GB Susceptible to P. citricola. Control Px GB Susceptible to P. citricola. Control AZ1* GB In process of being retested for P. citricola UX GB In process of being retested for P. citricola GZ GB In process of being retested for P. citricola UX GB In process of being retested for P. citricola Blackline tolerant WIP GB Tolerant to CLRV. Susceptible to P. citricola WIP GB Tolerant to CLRV WIP6 8 Vigorous control UX GB Selected for crown gall resistance, but retested and found susceptible. Nice trees. Vlach GB English Vina Sunland TOTALS * Nursery source. May have ownership issues since not all nursery agreements have been turned in to Tech Transfer Center.

7 TABLE 3. CLONES GROWN AT BONILLA NURSERY Planted Graftable Graftable Diameter (mm) Clone N N % Mean SD Range CV Nematodes VX Phytophthora AZ AZ NZ JX RX AX GZ Px AZ UX GZ Blackline WIP WIP Control UX English Vina Sunland Totals TABLE 4. CLONES GROWN AT STUKE NURSERY Planted Graftable Graftable Diameter (mm) Clone N N % Mean SD Range CV AX AZ RX Totals

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