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, Greg Browne, Diego Bujazha, and Soussan Hirbod ABSTRACT Establishment of the field stock block of putatively disease and pest resistant rootstock genotypes was completed with the exception of 5 WIP genotypes tolerant to CLRV that will be grafted in 4 to already established California black walnut rootstocks. The tissue culture bank of high priority genotypes has been completed with the exception of (WIP5), a CLRV tolerant genotype. We have continued work to micropropagate the disease and pest resistant genotypes using improvements to the fog chamber acclimatization and greenhouse growing protocols developed during Year 1 of the project. Through October 3, we have micropropagated over 5 plantlets of genotypes in 1½ inch tree tubes. We have shown that we can keep these plantlets growing year round in a greenhouse using a daylength extension with HID lights and a Promalin spray treatment every 2 weeks. We have also shown that plantlets in 1 1/2 inch tree tubes can be greenhouse grown to a size (4-5 inches tall with 3-5 tap roots 6 inches long) large enough for transplanting to a nursery row in 3 months. Three-month-old plants can be can be induced to drop their leaves by placing them in cold storage with low light at 5 F for 3 weeks. These dormant plantlets can then be stored for up to 6 months in a cold room at 42 F. Alternatively, they can be placed outdoors in fall and winter for dormancy induction and breaking. Rooting of microshoots is the main factor that limits successful micropropagation. Some genotypes root very well (-7%) while other root considerably below the average (%). Some genotypes recalcitrant for rooting responded well to higher IBA concentrations than had previously been used while others did not. However, use of higher IBA concentrations appears to be a promising way to obtain better rooting of many of the genotypes. For the period November 2 to October 3, the average survival of all genotypes was 65% of lab-rooted plantlets that were transplanted to 1½ inch tree tubes with considerable variation between genotypes. A total of 13 hardwood cuttings were taken from 1 coppiced or tree form genotypes. Rooting percentage varied from -%. Genotype (WIP5, one which we have trouble micropropagating), a CLRV tolerant clone, rooted at nearly % and VX 211, a nematode tolerant genotype rooted at 87%. Rooting of hardwood cuttings also showed some potential (35-5% rooting) for (WIP6), (WIP3), (WIP4) and AZ25, a crown gall resistant genotype for which we have trouble micropropagating. Using methods referred to above, we now have a total of over 39 trees of 21 genotypes available for field trials in 4. We have supplied 493 plantlets of 7 genotypes in 1½ inch tree tubes to Greg Browne for Phytophthora retests during Year 2 and also have 7 plantlets of 8 genotypes in cold storage for addition Phytophthora retests during 4. Genotype RX1 was relatively resistant to P. citricola in two 3 experiments, confirming previous results with the clone. Imposing a cycle of pre-inoculation chilling, dormancy, and growth resumption on the genotypes before screening them facilitated assessment of their genetic susceptibility to the pathogen.

2 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 retesting. 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 of these genotypes for re-testing. 4. Retesting of own-rooted individual genotypes for Phytophthora resistance in the greenhouse. This year, greatest effort has been placed on Objectives 2 and 4. PROCEDURES AND RESULTS Objective 1 During Year 2, establishment of the field stock block of putatively disease and pest resistant genotypes was completed with the exception of grafting 5 WIP genotypes tolerant to cherry leaf roll virus (CLRV) to already established California black walnut rootstocks. AW 269, a putatively nematode tolerant California black walnut genotype and (WIP2) (WIP4) and (WIP1), CLRV tolerant genotypes, were established in an in vitro tissue culture microshoot bank. Only (WIP5), a CRLV tolerant genotype remains to be established in the tissue culture microshoot bank. Objective 2 We have continued to micropropagate the putatively disease and pest resistant genotypes using improvements to the fog chamber acclimatization and greenhouse container growing protocols devised during Year 1 of the project. Through October 3 we have micropropagated and grown over 5 plantlets of genotypes in 1 ½ inch tree tubes. We have shown that we can keep these plantlets growing year round in a greenhouse using a daylength extension with high intensity discharge (HID) lights and a spray treatment with Promalin (a gibberellin-cytokinin product) every 2 weeks. We have also shown that plantlets in 1½ inch tree tubes can be greenhouse grown to a size (4-5 inches tall with 3-5 tap roots 6 inches long) large enough for transplanting to a nursery row in 3 months. Three-month-old plantlets can be induced to drop their leaves and go dormant by placing them in cold storage with low light at 5F for 3 weeks. These dormant plantlets can be stored for up to 6 months in a cold room at 42F. Alternatively, they can be placed outdoors in the fall and winter for dormancy induction and breaking. Survival of rooted plantlets during acclimatization and growth in the greenhouse is not the same for all of the genotypes with which we have worked. For the period November 2 through October 3 the average survival and growth of lab-rooted plantlets of all genotypes was 65%

3 of those transplanted to 1 ½ inch tree tubes (Table 1). However, as shown in Table 1, survival rate ranges from 9% down to 22% for the plantlets of the 16 genotypes planted. Even for some genotypes such as UX22 which appears to form very good to excellent number and quality of roots, survival (42%) is considerably less than the overall average (65%) and much less than the best survival (9%). We will be working on ways to improve survival of such genotypes in Year 3 of the project. To build up numbers of plants as quickly as possible, we have been rooting microshoots that fail to root in the lab, ex vitro, in the fog chamber. For the same period, these recycled, ex vitro rooted plantlets survived and grew (Table 2) at a much lower rate (44%) than the lab-rooted plantlets. However, they resulted in almost 85 additional plantlets. Rooting of microshoots is the main factor that limits the micropropagation overall. Some genotypes root very well (-7%) using the standard low concentration indolebutyric acid (IBA) root induction protocol but others do not (Table 3). Preliminary experiments during Year 2 have indicated that high IBA concentration treatments can improve rooting percentage of many genotypes including some of the genotypes that are recalcitrant for rooting (Table 3). Our results for rooting microshoots indicate that the protocol required for optimal rooting varies between genotypes. Our goal is to devise protocols that will give at least 7% rooting of microshoots of each genotype. Objective 3 Hardwood cuttings from coppiced (hedged) or tree form stock plants of (WIP5), (WIP6), (WIP2), (WIP4), (WIP3), VX 211, AW 269, AZ 25, UX 22 and Vlach were taken during the period from December 13, 2 to February 7, 3, dipped in or PPM potassium indolebutyric acid (K-IBA), stuck in Oasis rooting cubes and placed on a bottom heated rooting bed in a lath house at to 85F. A total of about 13 cuttings were stuck for the 1 genotypes. As shown in Table 4, rooting varied by genotype from to %. The genotypes with the highest rooting percentage were (WIP5), a CLRV tolerant genotype with 94 to % rooting and VX211, a nematode tolerant genotype with 73 to 87% rooting depending on K-IBA concentration. Other genotypes with moderately good rooting percentages were CLRV tolerant genotype (WIP6) and Vlach with about 5% rooting. CLRV tolerant genotypes (WIP3) and (WIP4) rooted at nearly 5% and AZ25 at about 35 % with the PPM K-IBA treatment. In general the PPM K-IBA treatment gave higher rooting percentages than the PPM K-IBA. However, the PPM K-IBA treatment delayed or inhibited outgrowth of buds. We will test PPM K-IBA during Year 3 to try to overcome this problem. Overall, rooting of hardwood cuttings looks very promising for genotype (WIP5, one for which were having trouble with micropropagation) and VX211. Rooting of hardwood cuttings also shows some promise for (WIP6), (WIP3), (WIP4) and AZ25 (a genotype for which we have trouble with micropropagation). Dieback of the stem from the terminal cut during and after rooting of cuttings is a problem that we will try to overcome during Year 3 by painting the terminal cut with asphalt emulsion.

4 Objective 4 By using the in vitro micropropagation and acclimatization and the hardwood cutting protocols along with the container growing and plantlet storage protocols we have developed during project Years 1 and 2, we now have a total of over 39 trees of 25 genotypes available for field trials of disease and pest resistance and horticultural characteristics in 4 (Table5). These consist of trees in 3-gallon deep containers, plantlets in 1 ½ inch tree tubes and trees growing in a nursery row that will be bare-rooted. The trees in 3-gallon containers have been produced from micropropagated plantlets and rooted hardwood cuttings using a drip irrigation system to deliver half strength Hoagland's solution to provide fertilizer and water as needed. For growth in 3-gallon deep containers, plants need at least 1 square foot each but could be grown in 2-gallon deep containers if drip fertigation is provided. Our plantlets and rooted cutting were transplanted to 3-gallon containers in May and many were ½ to ⅞ inch diameter and 6 to 7 feet tall by August even though the spacing was too close. We have supplied 493 plantlets of 7 genotypes in 1½ inch tree tubes to Greg Browne for Phytophthora screening during Year 2 and also have 7 plantlets of 8 genotypes in cold storage for additional Phytophthora screens during 4 (Table 6). The seven paradox genotypes propagated for retesting their resistance to Phytophthora citricola were used in two screening experiments in 3. The tests were used not only to evaluate the resistance of the selections but also to determine the effect of pre-inoculation chilling on physiological susceptibility of the plants. After rooting and acclimatization, the paradox genotypes have tendency to set a terminal bud and stop growing. This is a concern in screening, because previous greenhouse experience with walnut and other perennial crops suggests that the lack of growth may lessen physiological susceptibility to Phytophthora and complicate assessment of the underlying genetic resistance to the pathogen. Promalin sprays have helped to maintain shoot growth on walnut and other perennials, but repeated use of the hormone induced phytotoxicity on walnut in our greenhouse, and not all paradox clones responded identically to the treatments. On the other hand, taking the plants through a cycle of dormancy by exposing them to low light intensity for several weeks at 5 F, then chilling them for 3 to 6 months at 42 F induced vigorous and sustained shoot growth in all potted walnut rootstocks after they were returned to greenhouse at 7 to 9 F. One set of plants designated for the screen with P. citricola in 3 was placed in a pre-chill environment at 5F on 28 December, moved to a chilling environment (42F) on 1 February and removed May 1, providing 3 months of chilling. After returning the chilled plants to the greenhouse, all plants (chilled and non-chilled) were watered daily and fertilized 1 to 2 times per week with a complete nutrient fertilizer solution. On 3 May and 1 July, plants used in Experiments 1 and 2, respectively, were transplanted into into 2-liter pots of non-infested soil or soil artificially infested with a multiple-isolate mixture of P. citricola ( ml of infested V8 juice-vermiculite-oat substrate per liter of soil). For both experiments, six replicate blocks of two plants per rootstock were inoculated with P. citricola, while six blocks of one plant per treatment served as the control. Starting 2 weeks after transplanting and continuing for 2

5 months, all of the plants were subjected to biweekly soil flooding to facilitate infection. Between and after the floodings, the soil was watered as needed and allowed to drain freely. At the conclusion of Experiments 1 and 2 (3 September and 21 October, respectively) the root systems of the plants were washed free from the soil to determine incidence and severity of disease caused by the pathogen. The percentage of root crown length rotted was determined after measuring the total length of each root crown (from the point where major roots converged with the main plant stem to about 3 cm above the soil line) and the proportion of the length that was decayed. None of the rootstocks developed much root or crown rot in non-infested soil, but small to moderate amounts of root and crown rot developed in soil infested with P. citricola (Figs. 1-3). The cycle of chilling increased susceptibility to P. citricola in the AX1 genotype in both experiments, although this effect did not occur consistently in the other two rootstocks subjected to chilling (Fig. 1). We concluded, nevertheless that a cycle of pre-inoculation chilling, dormancy, and growth resumption should be included in all of our future screening tests with P. citricola. Overall, comparing responses of chilled, non-chilled, and averages of the two pre-inoculation treatments, genotype RX1 was marginally less susceptible than AX1 and PX1 to P. citricola (Figs. 1, 2). In Experiment 1, effects of inoculum x rootstock and inoculum x chilling treatment were significant at P=.3 and.4, respectively, and non-inoculated plants had means of.7 to 4% crown length rotted. In Experiment 2, effects of inoculum x rootstock and inoculum x chilling were significant at P=.2, and non-inoculated plants had means of to 16% crown length rotted. Among the plants that did not go through the cycle of pre-inoculation chilling, AZ2 and RX1 were relatively resistant to P. citricola (Fig. 3). Among the non-chilled plants, however, there was only a relatively weak statistical interaction of inoculum treatment x rootstock (P=.7 and P=.1 in Experiments 1 and 2, respectively). The moderate resistance of RX1 confirms results from our 1 and 2 screens, and the genotype merits continued screening and field testing. On the other hand, genotypes GZ1, JX1, and NZ1 were relatively susceptible to P. citricola in at least one of the 3 experiments, and for this reason they will not be tested further. Clones AX1, PX1, and RX1 will be retained in future screens as standards.

6 Table 1. Survival and growth of lab rooted PDS and WIP genotypes from November 2 through October 3 Genotypes Planted Alive % GZ AX RX JX NZ GZ AZ AZ VX WIP AZ AZ25R PX UX22R WIP UX Total

7 Table 2. Survival and growth of ex-vitro rooted PDS and WIP genotypes from November 2 through October 3 Genotypes Planted Alive % AZ VX JX GZ AX PX RX AZ WIP NZ UX22R AZ GZ WIP AZ25R UX Total Table 3. Percent rooting of PDS and WIP genotypes induced to root on gelled medium containing either 5 or PPM IBA Genotypes 5 IBA IBA RX1 GZ UX AX UX PX WIP2 69 AZ UX WIP3 66 AZ25 53 AZ NZ1 47 VX JX2 33 AZ GZ1 26 WIP6 19

8 Table 4. Rooting of hardwood cuttings of putatively disease resistant rootstock genotypes Genotype Cutting source K-IBA Treatment ( PPM ) Percent Rooting New Stuke Block 12 (9 /75) (WIP2) 29 (22 /75) New Stuke Block 55 (41 /75) (WIP6) 39 (29 /75) Clonal Block 1 (5 /1) 8 (4 /5) New Stuke Block 94 (47 /5) (WIP5) (5 /5 ) New Stuke Block ( /5) (WIP4) ( /5) Clonal Block 12 (3 /25) 48 (12 /25) New Stuke Block (1 /5) (WIP3) 46 (23 /5) AZ25 PDS Stock Block 2 (1 /) 36 (25 /7) UX22 PDS Stock Block ( /43) ( /43) AW269 PDS Stock Block 5 (4 /75) 8 (6 /75) VX211 PDS Stock Block 73 (11 /15) 87 (13 /15) Vlach Burchell 45 (34 /75) 55 (41 /75)

9 Table 5.Total plants available for field trials as of November 3 Selection criteria Genotypes Liners 3 gal pots Bareroot Grand Total Phytophthora AX AX AZ AZ AZ GZ GZ JX NZ PX RX UX Crown gall AZ UX Nematodes AW VX Blackline WIP WIP WIP4 5 5 WIP WIP Sunland 3 5 Vina 8 8 Tulare 1 1 Total

10 Table 6. Plants propagated for Phytophthora retests through November 3 Delivered for Cold Stored for Genotypes Phytophthora tests Future Phytophthora Tests Total AX AZ AZ GZ JX NZ PX RX WIP Total

11 Percent crown length rotted Chilled AX-1 PX-1 RX-1 AX-1 PX-1 RX-1 (Inoculated with P. citricola) (Inoculated with P. citricola) Experiment 1 Experiment 2 Not chilled Chilled Not chilled Figure 1. Relative susceptibility of three paradox clones to Phytophthora citricola and the effect of preinoculation chilling on disease severity, 3 greenhouse screens. In Experiment 1, non-inoculated plants had means of.7 to 4% crown length rotted, and effect of inoculum x rootstock and inoculum x chilling were significant at P=.3 and.4, respectively. In Experiment 2, non-inoculated plants had means of to 16% crown length rotted, and effect of inoculum x rootstock and inoculum x chilling were significant at P=.2. For both experiments, six replicate blocks of two plants per rootstock were inoculated with P. citricola, while six blocks of one plant per treatment served as controls. Vertical bars delimit 95% confidence intervals. Percent crown length rotted Non-inoculated P. citricola Non-inoculated P. citricola (Avg. of chilled and non-chilled plants) (Avg. of chilled and non-chilled plants) Experiment 1 Experiment 2 AX-1 PX-1 RX-1 AX-1 PX-1 RX-1 Figure 2. Relative susceptibility of three paradox clones to Phytophthora citricola, data combined for plants subjected to pre-inoculation chilling and non-chilled plants. In Experiments 1 and 2, effect of inoculum x rootstock significant at P=.3 and P=.2, respectively. For both experiments, six replicate blocks of two plants per rootstock were inoculated with P. citricola, while six blocks of one plant per treatment served as controls. Vertical bars delimit 95% confidence intervals. Percent crown length rotted Non-inoculated P. citricola Non-inoculated P. citricola (Non-chilled plants) (Non-chilled plants) Experiment 1 Experiment 2 AX-1 AZ-2 GZ-1 JX-2 NZ-1 PX-1 RX-1 AX-1 AZ-2 GZ-1 JX-2 NZ-1 PX-1 RX-1 Figure 3. Relative susceptibility of three paradox clones to Phytophthora citricola, data combined for plants subjected to pre-inoculation chilling and non-chilled plants. In Experiments 1 and 2, effect of inculation significant at P=.7 and P=.1, respectively. For both experiments, six replicate blocks of two plants per rootstock were inoculated with P. citricola, while six blocks of one plant per treatment served as controls. Vertical bars delimit 95% confidence intervals.