Abstract - A research program was undertaken to determine whether the root electrolyte leakage testing procedure could provide rapid information on the subsequent performance of interior spruce (Picea glauca (Moench) Voss x Picea engelmannii Parry) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings with a range of root damage. Results indicate that this testing procedure can forecast overall seedling performance over a range of root damage. Each species had their own distinct root damage assessment models. These findings are to be incorporated into species specific root damage assessment models that will help forecast performance of seedlings suspected of having root rot. 1Forest Biotechnology Centre, BCRI, 3650 Wesbrook Mall, Vancouver, B.C. V6S 2L2 2B.C. Ministry of Forests, Green Timbers Nursery, 14255-96th Ave.Surrey, B.C. V3V 7Z2

INTRODUCTION

Root rot continues to cause seedling losses at some conifer nurseries in years which have environmental conditions conducive for disease development. Studies have indicated that Douglas-fir root rot can decrease growth and survival of seedlings on reforestation sites (Axelrood et al. 1995). At times adjudicators have difficulty deciding whether to accept or reject conifer seedlings due to suspected root rot. Fusarium and Cylindrocarpon root colonization levels are similar for seedlings which appear healthy or display root rot symptoms, indicating that isolation of these fungi is not an adequate criteria for accepting or rejecting stock. A stock quality testing procedure is needed to identify seedlings with questionable levels of root rot.

Stock quality tests that measure the functional integrity of seedlings help forecast their survival capability and growth under optimum conditions (Grossnickle and Folk 1993). Functional integrity indicates whether a seedling is, or is not, damaged to the point of limiting primary physiological processes. The intent of these testing approaches is to remove seedlings that do not meet certain minimum physiological performance standards (i.e., the "bad apple concept"). Seedlings that meet minimum standards have a greater capability to survive in all but the most severe of field site environmental conditions (Sutton 1988). Assessing seedlings for potential root damage is the intent of many stock quality assessment procedures (reviewed in Grossnickle and Folk 1993).

The root electrolyte testing procedure is an effective stock quality assessment method for determining damage to root systems (McKay and Mason 1991, McKay 1992, Simpson 1993, Bigras and Calme 1994, McKay 1994). This testing approach has the potential to rapidly, and effectively, determine whether seedlings with questionable levels of root damage will meet minimum performance standards. However, validation of this testing approach in relation to standard growth and survival performance criteria of species in question is required before assessing the performance of seedlings suspected of having root rot.

The objective for phase one of this program was to determine whether the root electrolyte leakage assessment procedure could provide rapid and reliable information on the subsequent short- and long-term performance of interior spruce and Douglas-fir seedlings with damaged root systems. Partial results from phase one of this program are reported in this paper. In the second phase of this program, a root damage assessment model, based upon the root electrolyte leakage assessment procedure, will be tested for it's ability to forecast performance of seedlings suspected of having root rot.

MATERIALS AND METHODS

Plant Material
Interior spruce (Picea glauca (Moench) Voss x Picea engelmannii Parry) seedlings (seedlots # 29130 and 4319) used in this study were grown at two commercial nurseries. Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings (seedlots # 6514 and 6282) used in this study were grown at two commercial nurseries. Seedlings were grown at all nurseries under standard operational cultural procedures for producing a spring-plant 1+0 container stocktype with one exception; 2+0 interior spruce seedlings from seedlot # 29130 were produced at one of the nurseries. Seedlings were removed from the nurseries in the fall of 1994 at times when each species would normally be lifted for placement into frozen storage (interior spruce on Nov. 1st, and Douglas-fir on Dec. 21st).

Seedlings (n= 300 from each seedlot x nursery combination) from both species were screened to ensure morphological uniformity within the experimental population by measuring height and caliper and selecting seedlings within the morphological standards for a 1+0 or 2+0 container stocktype defined by the B.C. Ministry of Forests (Scagel et al. 1993). Seedlings selected from all seedlots appeared visually healthy.

Development of root damage
Root damage was created by using a heat treatment applied only to the root systems. Seedlings were brought from the nursery and placed in large plastic pots (to protect the root systems from drying out) and allowed to acclimate under ambient environmental conditions (20 to 22 ^oC air temperature, 50% relative humidity and a 12h photoperiod at 400 mmol m^-2 s^-1) of the controlled environment room for a 48h period. Seedlings did not have their root systems cleaned of growing media prior to the heat treatment.

Seedling root plugs were placed in an aerated hydroponic system that had a water temperature of 56 to 57 ^oC for interior spruce and 55 to 56 ^oC for Douglas-fir. Seedlings for each species had their root systems exposed to a series of incubation times at elevated water temperatures to create a range of root damage conditions. For interior spruce, root exposure times to the heat treatment were: 0, 3, 6, 10, 15, 20, 25, and 45 minutes. For Douglas-fir, root exposure times to the heat treatment were: 0, 5, 8, 12, 26, 45, 60, 90, and 120 minutes.

Seedlings designated for each exposure time (n= 20) were then removed and potted in 4l pots (4 seedlings per pot) with a growing media of sifted peat. Seedlings were placed in the above described controlled environment room, kept well watered, and monitored with the following tests.

Root electrolyte leakage

Root electrolyte leakage (REL) was determined immediately after application of root temperature treatments. Seedlings (n= 8) from each root temperature treatment were selected and root samples were collected. Root were removed from one-side of the middle third of the root / soil media plug (1.5 cm x 3 cm, 1 cm depth, section).

Roots from each seedling were cut at both ends into 1.0 cm lengths, washed in deionized water and transferred, in random groups of 24, to glass culture tubes containing 10 ml of deionized water. Tubes were then stoppered and placed on a 100 rpm shaker at 20 ^oC for 20-24h. Conductivity of the solution in each tube was measured after incubation (initial conductivity = IC). Tubes were then placed in a 90 ^oC water bath for 15 minutes to induce maximum tissue injury and conductivity was re-measured after an additional 20h on a 100 rpm shaker at 20 ^oC (after boiling conductivity = BC).

Measured REL values were interpreted using the following formula:
REL (%) = IC / BC x 100
Measured REL of near 0% indicated no root damage due to the heat-time treatment. As the time was increased, REL values increased indicating greater root damage. As REL values approached 100%, this indicated total cell membrane damage due to heat-time treatments.

Root growth capacity

Seedlings (n=20) were grown in the above described controlled environment room, under optimum soil moisture conditions, after application of root temperature treatments. Root growth capacity (RGC) was determined by counting the number of new white roots >0.5 cm in length after 14 days (Ritchie 1985).

Survival

Seedlings (n= 20) were grown in the above described controlled environment room, under optimum soil moisture conditions, after application of root temperature treatments. Survival was determined eight weeks after application of root temperature treatments.

Experimental design

The same seedling population (n= 20) from each root temperature treatment was used in all testing protocols. A randomly selected subset (n= 8) of this seedling population, from each root temperature treatment, was tagged and used for REL measurements. All seedlings were tagged for each heat-time treatment combination so that each seedling was related to results obtained from other testing protocols. Seedlings from all root temperature treatments were used in all tests.

After application of root temperature treatments, both interior spruce and Douglas-fir seedlings were placed on separate light tables in the controlled environment room in randomized design containing all of the heat-time temperature treatment combinations (interior spruce = 8 treatments, Douglas-fir = 9 treatments).

Statistical analysis

Regression analysis was used to examine the relationship between RGC or survival (dependent or predicted variables), to REL values (independent or predictor variables). Root damage assessment models were developed combining results of seedling populations from all nursery / seedlot combinations. Regression model selection was determined by including significant (p = 0.05) variables that contributed to the highest r² (Kleinbaum et al. 1988).

RESULTS AND DISCUSSION

Root electrolyte leakage was the stock quality assessment test chosen as the potential physiological parameter to be used in the root damage assessment model. Root electrolyte leakage is a measure of root system integrity (McKay 1992). This testing approach is based on the concept that damaged cell membranes allow cell contents to move from the symplast to the apoplast where the electrolytes can be detected, and measured electrolyte leakage values are high. Undamaged living cells do not have damaged membranes, cell contents remain within the symplast, and measured electrolyte leakage values are low.

From a stock quality assessment perspective, the root electrolyte leakage testing procedure provides: 1) simplicity of testing protocols, 2) a short timeframe with which results can be obtained, 3) testing is conducted on root samples where suspected root rot can cause damage, and 4) low expense of testing equipment. Results from this testing approach could then be used to forecast the influence of root damage on seedling root growth and survival.

Results from the root electrolyte testing procedure were compared to the standard stock quality testing procedure of root growth capacity as well as seedling survival. Root growth capacity is a measure of a seedlings ability to regenerate new roots and an indirect measure of a seedlings overall physiological condition (Stone 1955, Ritchie and Dunlap 1980, Ritchie 1985, Burdett 1987, Ritchie and Tanaka 1990, Sutton 1990). When the capability of interior spruce and Douglas-fir seedlings to generate new roots declines, their potential to survive in the field also decreases (Simpson 1990).

Interior spruce seedlings from all nursery / seedlot combinations responded in a predictable manner to increasing root damage (increasing REL values) (Fig. 1). There were four main characteristics of the root damage assessment model. First, at REL values <0.30, there was no root damage resulting in high RGC and survival capability. Second, at REL values between 0.30 and 0.45, root damage started to occur which is reflected in the reduction in RCG, with survival capability ranging from 100 to 70 percent. Third, at REL values between 0.45 and 0.60, significant root damage had occurred resulting in RGC declining to 0 and seedlings had a decreased survival capability. Fourth, at REL values >0.60, functional integrity of the root was completely destroyed resulting in RGC at 0 and seedlings had no survival capability.

Douglas-fir seedlings from all nursery / seedlot combinations also responded in a predictable manner to increasing root damage (increasing REL values) (Fig. 2). There were two main characteristics of the root damage assessment model. First, at REL values <0.20, there was no root damage resulting in variable RGC and high survival capability. Second, at REL values >0.25, functional integrity of the root was completely destroyed resulting in RGC at 0 and seedlings had no survival capability.

Variability in results from stock quality assessment procedures can occur due to species, genetic variability of seedlots and variations in nursery culture (Grossnickle and Folk 1993). To address these concerns, this research program examined two species (Douglas-fir and interior spruce), from two seedlots with both seedlots grown at separate nurseries. Results from this program found that the root electrolyte leakage procedure could forecast root growth and survival for interior spruce and Douglas-fir seedlings with a range of root damage. Each species had their own distinct root damage assessment models, though within species variability due to seedlot and nursery culture variability was minor.

Phase one of this program determined that the root electrolyte leakage assessment procedure could provide rapid and reliable information on forecasting short-term root growth and subsequent survival of interior spruce and Douglas-fir seedlings with damaged root systems. Phase two of this program will determine whether these root damage assessment models can be used to forecast the performance of seedlings suspected of having root rot. Research is ongoing to determine whether the heat-time root damage results found in this study are representative of damage caused by root rot fungi. Data from one population of fall-lifted Douglas-fir seedlings separated into groups having root rot and those without root rot fit within the expected root growth and survival performance capability defined by the root damage assessment model (unreported data). Further testing of populations suspected of having root rot are being examined to substantiate these findings.

Preliminary results have also indicated that the phenological state of a species may alter relationships between root electrolyte leakage values and measured performance. In addition, absolute values in root electrolyte leakage (McKay 1994) and root growth capability (Burr et al. 1989, Grossnickle et al. 1994) can change during the potential fall lifting window as seedlings go into dormancy. During phase two of the program, changes in the root damage assessment model in relation to the phenological state of interior spruce and Douglas-fir seedlings is being examined across the potential fall lifting window.

The final product from this program are specific root damage assessment models. These models are intended to be a rapid and easy to use physiological testing procedure. Results from these stock quality testing procedures would assist adjudicators with their seedling assessments. This would enable nursery operations to setup physiological based culling criteria during lifting operations in the fall for groups of interior spruce or Douglas-fir seedlings suspected of having root rot or other forms of root damage.

REFERENCES

Axelrood, P., Lam, M. and Radley, R. 1995. Influence of root-rot on Douglas-fir seedlings survival and growth on reforestation sites and assessment of Fusarium and Cylindrocarpon root colonization (Final Report). B.C. Ministry of Forests, File No. 1070-20/BCRES. pp. 62.

Bigras, F.J., and Calme, S. 1994. Viability tests for estimating root cold tolerance of black spruce seedlings. Can. J. For. Res. 24:1039-1048.

Burdett, A.N. 1987. Understanding root growth capacity: theoretical considerations in assessing planting stock quality by means of root growth tests. Can. J. For. Res. 17:768-775.

Burr, K.E., Tinus, R.W., Wallner, S.J. and King, R.M. 1989. Relationships among cold hardiness, root growth potential and bud dormancy in three conifers. Tree Physiol. 5:291-306.

Grossnickle, S.C. and Folk, R.S. 1993. Stock Quality Assessment: Forecasting survival or performance on a reforestation site. Tree Planters' Notes 44:113-121.

_____ Major, J.E. and Folk, R.F. 1993. Interior spruce seedlings compared to emblings produced from somatic embryogenesis. I) Nursery development, fall acclimation and frozen storage. Can. J. For. Res. 24:1385-1396.

Kleinbaum, D.G., Kupper, L.L. and Muller, K.E. 1988. Applied regression analysis and other multivariable methods. 2nd edition, PWS-Kent Publishing Co., Boston.

McKay, H.M. 1992. Electrolyte leakage from fine roots of conifer seedlings: a rapid index for plant vitality following cold storage. Can. J. For. Res. 22:1371-1377.

_____ 1994. Frost hardiness and cold-storage of the root system of Picea sitchensis, Pseudotsuga menziesii, Larix kaempferi and Pinus sylvestris bare-root seedlings. Scand. J. For. Res. 9:203-213.

_____ and Mason, W.L. 1991. Physiological indicators of tolerance to cold storage in Sitka spruce and Douglas- fir seedlings. Can. J. For. Res. 21:890-901.

Ritchie, G.A. 1985. Root growth potential: principles, procedures and predictive ability. In Evaluating Seedling Quality: Principles, Procedures and Predictive Abilities of Major Tests. Ed. Duryea, M.L. Oregon State Univ. For. Res. Lab. Corvallis, OR. pp. 93-106.

_____ and Dunlap, J.R. 1980. Root growth potential: its development and expression in forest tree seedlings. N.Z. J. For. Sci. 10:218-248.

_____ and Tanaka, Y. 1990. Root growth potential and the target seedling. In Target Seedling Symposium: Proceedings of the Western Forest Nursery Associations. Eds. Rose R., Campbell, S.J. and Landis, T.D. USDA For. Serv. Gen. Tech. Rep. RM-200, pp. 37-51.

Scagel, R., Bowden, R. Madill, M. and Kooistra, C. 1993. Provincal seedling stock type selection and ordering guidelines. B.C. Ministry of Forests, Victoria. p. 75

Simpson, D.G. 1990. Frost hardiness, root growth capacity and field performance relationships in interior spruce, lodgepole pine, Douglas-fir and western hemlock seedlings. Can. J. For. Res. 20: 566-572.

_____ 1993. Root cold hardiness of western Canadian conifers. In Proceedings, 12th Annual Meeting of the B.C. Forest Nursery Association, Sept. 29 - Oct. 1, 1992, Penticton. B.C. Ministry of Forests, Victoria. pp.97-105.

Stone, E.C. 1955. Poor survival and the physiological condition of planting stock. For. Sci. 1:89-94.

Sutton, R.F. 1988. Planting stock quality is fitness for purpose. In Taking Stock: The Role of Nursery Practice in Forest Renewal. Eds. Smith, C.R. and Reffle, R.J. Great Lakes Forestry Centre, Can. For. Serv., OFRC Symposium Proc. O-P-16. pp. 39-43.

_____ 1990. Root growth capacity in coniferous forest trees. HortSci. 25:259-266.

Figure 1) Root growth capacity and survival for all nursery / seedlot combinations (mean + SE) of interior spruce seedlings in response to root electrolyte leakage. Greater root electrolyte leakage is equated to greater root damage.

Figure 2) Root growth capacity and survival for all nursery / seedlot combinations (mean + SE) of Douglas-fir seedlings in response to root electrolyte leakage. Greater root electrolyte leakage is equated to greater root damage. No root growth capacity results are presented for REL >0.5 because all seedlings had died before the two week measurement period.

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