Effects of exposure to below-freezing temperatures, soil moisture content and nitrogen application on phyllochron in cool-season grasses

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1 Effects of exposure to below-freezing temperatures, soil moisture content and nitrogen application on phyllochron in cool-season grasses P. W. Bartholomew and R. D. Williams USDA-ARS, Grazinglands Research Laboratory, Langston University, Langston, OK, USA Abstract Accumulated temperature may provide an indicator of the phenology of cool-season grass to assist in the timing of management operations but its value is highly dependent on a reliable measure of phyllochron, i.e. time between the elongation of successive leaves. Field and controlled environment studies with Italian ryegrass (IRG, Lolium multiflorum Lam.) and tall fescue (TF, Festuca arundinacea Schreb.) measured leaf-appearance responses to accumulated temperature with varying conditions of exposure to low temperatures, soil moisture content and nitrogen application. In a controlled environment, soil volumetric water contents below 2% (equivalent to moisture potentials greater than )Æ1 MPa) or more frequent exposure to below-freezing air temperatures increased the phyllochron values on the main tiller of IRG and TF. There was no evidence of an interaction between soil moisture content and cold exposure on phyllochron values. Nitrogen application resulted in only small reduction in phyllochron values. In field studies over 4 years the phyllochron values in IRG during the months of January to March were 184, 18, 18 and 167 accumulated C above C leaf )1, more than double the mean value measured in a controlled environment. A greater understanding of the impact of variable low temperatures and of soil moisture potential on the phyllochron is necessary before accumulated temperature can be used to indicate changes in development stages in grasses in different environments. Keywords: Italian ryegrass, tall fescue, phyllochron, soil moisture, low temperature Correspondence to: P. W. Bartholomew, USDA-ARS, Grazinglands Research Laboratory, PO Box 173, Langston University, Langston, OK 735, USA. pbarthol@luresext.edu Received 9 September 25; revised 18 January 26 Introduction So as to optimize timing of management of cool-season grasses, particularly where these are grown in sequence with warm-season forages, there is a need to monitor their stages of development. Direct appraisal in the field is frequently not possible, because of a constraint of time, or because changes in stage of development are not clearly manifested. A reliable indirect measure of the development of cool-season grasses could be of significant practical value for management. Data on accumulated temperature are widely available and, through their linkage with plant development (Frank and Bauer, 1995; Kirby, 1995; Skinner and Nelson, 1995), may provide a useful aid to the optimum timing of management activities such as application of nitrogen fertilizer or harvesting (Lemaire and Salette, 1982; Kowalenko et al., 1989). By using site-specific data of accumulated temperature, it may be possible to tailor management advice, adapted to an extended geographic area, to match more closely the actual growing conditions at an individual site. Several factors affect the consistency of the relationship between accumulated temperature and leaf appearance in small-grain cereals and forage grasses from year to year or site to site. Higher average daily temperatures have been shown to increase phyllochron, i.e. time between elongation of successive leaves, values in cereals and grasses (Cao and Moss, 1989; Bartholomew and Williams, 25), while in a controlled environment, repeated exposure to belowfreezing minimum temperatures increased phyllochron values in cool-season forage grasses (Bartholomew and Williams, 25). In wheat, N deficiency (Longnecker and Robson, 1994) or moisture stress (Krenzer et al., 1991) can increase phyllochron values, but Frank and Ries (199) showed little effect of soil fertility or soil moisture on phyllochron values in cool-season pasture grasses. This apparent contradiction may be reconciled by observations that responses to N (Kirby, 1995) and moisture (McMaster and Wilhelm, 23) are 146

2 Factors affecting phyllochron in cool-season grasses 147 non-linear, and, therefore, need to reach some threshold level before an impact on rate of development is observed. However, such threshold values have not been identified. Increased soil bulk density can increase leaf appearance interval in wheat (Masle and Passioura, 1987), implying that both type and management of soil may influence phyllochron values. In controlled-environment studies, increased daylength reduced phyllochron values in cereals (Cao and Moss, 1994; Mosaad et al., 1995) but the effect of changing daylength is less evident under field conditions (Cao and Moss, 1994; Kirby, 1995). The change in daylength across years is constant, so it is unlikely to introduce significant variation among years at a given site. The objectives were to evaluate the effects of soil moisture content and exposure to low-temperature, or addition of fertilizer-n to soil on leaf appearance response to accumulated temperature, and to compare values of phyllochron for cool-season grasses grown in a controlled environment and in the field. Materials and methods Development of cool-season grasses was studied in the field during the period from January to March in successive years (22 25), and in a series of experiments in controlled-environment chambers. Development of grasses was assessed through the estimation of leaf appearance rate, and thus leaf appearance interval (phyllochron), on the main tiller or on the daughter tiller, according to a thermal (accumulated temperature) timescale. Accumulated temperature ( C) was calculated as the accumulation of mean daily temperature; daily maximum (T max )+daily minimum (T min )/2 above a base temperature (T base )of C so that accumulated temperature over the period day 1 to day n is P n 1 ½ðT max þ T min Þ=2Š T base. Air temperatures at 13 cm above ground level were collected at a weather station adjacent to the areas where observations of leaf appearance were made in the field. In the controlled environment, daily average maximum and minimum air temperatures were derived from temperatures recorded hourly to a HOBO datalogger (Onset Computer Corporation, Pocasset, MA, USA) from probes sited among seedlings at the soil surface. The period of temperature accumulation was 1 January to 31 March in each year of field observations and over a period which allowed an accumulated temperature above C of 35 C following seedling emergence in all growth chamber experiments. Mean leaf appearance rate was calculated by regression of the average number of visible leaves per measured tiller among replicate seedlings at each date of observation with the accumulated temperature above C on that date. Mean leaf appearance interval (phyllochron, accumulated C above C leaf )1 ) was calculated as the reciprocal of the leaf appearance rate. Field measures of leaf appearance rate in cool-season grasses Italian ryegrass (IRG, Lolium multiflorum Lam.) cv Marshall was established by no-till seeding into dormant warm-season pasture comprised predominantly of little bluestem [Schizachyrium scoparium (Michx.) Nash], sideoats grama [Bouteloua curtipendula (Michx.) Torr.], Indiangrass [Sorghastrum nutans (L.) Nash] and big bluestem (Andropogon gerardii Vitman), or by drilling into cultivated ground in late-september 21 at a field site 6 km south of Langston, OK, USA, at N, W. In three subsequent years, IRG was replanted by no-till seeding in mid-september at new locations within the same field site. A no-till seeder (Land Pride, Salina, KS, USA) was used for planting in all cases, at a seed rate of 3 kg ha )1. Soil was a Coyle series clay loam (fine-loamy siliceous thermic Udic Arguistoll). A post-emergence top-dressing of 25 kg N ha )1 was applied in each year, within 2 weeks of crop emergence. In early January 22, 23, 24 and 25 randomly-selected tillers with one or two leaves were individually identified by placement of numbered plastic bird-rings around the base of each selected tiller. Leaf appearance on each of 1 tillers (22), or 24 tillers (23 25) was subsequently measured by counting the number of visible leaves at approximately weekly intervals until five to six leaves had appeared. Comparison of leaf appearance rates of IRG among years, and between establishment methods in 21, was made by comparison of the slopes of the regressions of cumulative leaf number and accumulated temperature using the procedures of Genstat 7 (Genstat, 23). Leaf appearance in controlled environment In all experiments carried out in the controlled environment, seeds of IRG or tall fescue (TF, Festuca arundinacea Schreb.) cv Kentucky 31 were sown in 155 ml cone-tainers (Stuewe & Sons, Corvallis, OR, USA) containing Coyle clay loam field soil collected from the field site. Unless otherwise specified, cone-tainers were held in open racks during treatment so that the soil column was exposed to ambient temperatures. Following emergence, multiple seedlings were thinned to a single seedling per cone-tainer. Plants were maintained in a growth chamber (Controlled Environments Inc., Pembina, ND, USA) with a 15/ or a 22Æ5/7Æ5 C light/ dark cycle temperature regime in a 13-h light and 11-h dark cycle and with an average photosynthetic photon flux density (PPFD) of 285 lmol m )2 s )1 at canopy height. Light input was monitored with a quantum

3 148 P. W. Bartholomew and R. D. Williams sensor (LI-COR Inc., Lincoln, NE, USA) and adjusted at approximately weekly intervals throughout each growing period. In soil moisture studies, the moisture potential of different soil volumetric water content (VWC) treatments was estimated using a dew-point meter (Decagon devices, Inc., Pullman, WA, USA). Experiment 1. Effect of N addition to field soil Individual seedlings of IRG were grown in field soil and maintained under a 22Æ5/7Æ5 C temperature regime in the light/dark phases of the diurnal cycle. N application treatments of or 5 mg of KNO 3 (equivalent to a field application of approximately 55 kg N ha )1 ) per seedling were initiated prior to emergence of the second leaf. Leaf appearance was measured on three replications of five seedlings per treatment in an initial phase and on two replications of five seedlings per treatment in a repetition of the experiment. Differences in phyllochron values between treatments were assessed by ANOVA (Genstat, 23). Experiment 2. Effect of soil moisture content and frequency of exposure to below-freezing temperatures For all experiments in which soil moisture levels were compared, soil VWC was established by packing conetainers to constant weight and volume of air-dry soil to which an appropriate weight of water was added to provide the required soil moisture content. The total weight of each cone-tainer was monitored daily and water was added as necessary to maintain the weight required for each soil moisture treatment. As seedling weight was negligible compared with soil and conetainer total weight, no adjustment was made for seedling growth. In a preliminary study (Experiment 2a) with seedlings grown in Coyle clay loam, the effects of soil VWC of 45%, 3% or 15%, equivalent to moisture potentials of, )Æ4 and )Æ23 MPa, respectively, on leaf appearance in IRG were measured. Each treatment was replicated over ten seedlings. The effects of soil moisture content treatments on mean rate of leaf appearance were estimated by comparisons of the slopes of the regression lines for each treatment, calculated by Genstat 7 (Genstat, 23). In a follow-up experiment (Experiment 2b) seedlings of IRG and TF were grown in a mixture of Æ75 Coyle field soil and Æ25 fine sand and subjected to soil VWC of 3%, 2% or 1%, i.e. moisture potentials of )Æ4, )Æ1 and )Æ61 MPa respectively. A mixture of field soil and sand was used to facilitate equilibration of soil water contents to the target value for each moisture treatment. Soil moisture treatments were imposed in combination with three cold treatments in which plants were exposed to air temperatures of )5 C for 11 h on two or four successive nights, or were maintained at a constant C overnight temperature. Each cold treatment was made by transferring seedlings to a lowtemperature incubator for 11 h, during the dark phase of the growth chamber diurnal cycle. During cold treatment cone-tainers were placed in a polystyrene seedling block that was modified to allow insertion of 155 ml cone-tainers so that the soil column was surrounded by insulating material. Following cold treatment, seedlings were returned to an open rack in the growth chamber. Multiple seeds were germinated in the soil:sand mixture at a constant 2% VWC. Following emergence, seedlings were thinned to a single plant per cone-tainer and soil moisture was adjusted to VWC of 3%, 2% or 1%. Leaf number of the main tiller on five seedlings per treatment was recorded at 2-day intervals after emergence up to an accumulated temperature above C of 35 C. Cold-exposure treatments were started at an accumulated temperature above C of C after emergence and, at an accumulated temperature above C of 35 C, seedlings were harvested and the dry weight of individual leaves and of total above-surface mass was measured. Following harvest, the soil moisture content was adjusted to 3% VWC in all cones and the seedlings were allowed to regrow. Leaf appearance on the first daughter tiller (tiller T1, following the terminology of Klepper et al., 1982) was recorded up to an accumulated temperature above C of 325 C to assess carry-over effects of the prior cold and moisture treatments on regrowth. In both first-growth and regrowth, the phyllochron value for each treatment was estimated as the mean value for five seedlings. Comparisons of treatment effects on phyllochron were made by analysis of variance of the experiment as a full factorial randomized block experiment (two species three VWC three cold-exposure treatments), using two repeats of the experiment as replication in time. Data were subjected to ANOVA and mean separation tests were made by Fisher s protected least significance difference test (LSD, a ¼ Æ5) using Genstat 7 (Genstat, 23). Results Field measures of leaf appearance rate in Italian ryegrass Accumulated temperature above C at the field site for the period from 1 January to 31 March in is shown in Figure 1 and the frequency of occurrence of low-temperature events for air temperature (13 cm above ground) is summarized in Table 1. The 3-year average accumulated temperature and occurrence of low air temperatures, and accumulated January to March precipitation, for Guthrie, OK, USA

4 Factors affecting phyllochron in cool-season grasses Accumulated C above C Leaves per tiller Day of year Figure 1 Accumulated temperatures from January to March in at the Langston field site ( N, W, altitude 336 m), and 3-year average for January to March at Guthrie, OK, USA ( N, W, altitude 327 m). 22 ( ), 23 ( Æ ), 24 ( ), 25 (ÆÆÆÆÆÆ) and, 3-year average ( ). Table 1 Number of below-freezing air temperature events* (13 cm above surface) and cumulative precipitation from 1 January to 31 March and 3-year averages. Year Threshold value ( C) < <)1 <)2 <)5 <)1 Precipitation (mm) (23) (25) (24) (16) year average (31) Value in parenthesis is the total number of occurrences of air temperature minimum <)5 C post-emergence to the end of leaf appearance observations in 22 and to the end of March in *Freezing-temperature events in recorded at the Langston field site. Precipitation values and 3-year averages for temperature and precipitation taken from Guthrie, OK, USA, approximately 19 km west of the field site (Climatedata, 22; Oklahoma Climatological Survey, 26). (Climatedata, 22; Oklahoma Climatological Survey, 26), approximately 19 km from the experiment site, are also shown in Figure 1 and Table 1 respectively Accumula ted C above C Figure 2 Relationship of accumulated temperature to leaves per tiller in Italian ryegrass grown in the field during January to March 22 (n n), 23 (h h), 24 (mæææææm) and 25 (j Æ j). Equations are: leaves per tiller (22) ¼ Æ54 (Æ56) GDD + 2Æ3; leaves per tiller (23) ¼ Æ55 (Æ29) GDD + 1Æ9; leaves per tiller (24) ¼ Æ56 (Æ57) GDD + 1Æ3; leaves per tiller (25) ¼ Æ6 (Æ47) GDD + 2Æ6, where GDD is accumulated number of C above C. Phyllochron (1/leaf appearance rate) values were 184, 18, 18 and 167 C above C leaf )1 for 22, 23, 24 and 25 respectively. Accumulated temperature above C was greater than the 3-year average until the end of February in all of the study years. Total accumulated temperature at the end of March was similar to the 3-year average in 22 and 23 but was slightly greater than the average in 24 and 25. Rate of leaf appearance in IRG in January to March in all years showed a close linear relationship with accumulated temperature above C from 1 January in each year (Figure 2). Mean phyllochron values were 184, 18, 18 and 167 C above C leaf )1 for respectively. However, comparison of the slopes of regressions from which these values were estimated showed no significant difference (P >Æ5) in rate of leaf appearance among years. Comparison of leaf appearance rate according to establishment method in 21 (by drilling into tilled ground or by no-till drilling) showed no significant difference in the rate of leaf appearance, and these data are pooled in Figure 2. There was a difference (P <Æ5) in intercept between the two treatments, indicating that plants on tilled treatments had an average of Æ23 more leaves visible than those on

5 15 P. W. Bartholomew and R. D. Williams no-till drilled treatments, at the same accumulated temperature post-sowing, equivalent to a difference of 42 C above C leaf )1 and consistent with slightly later emergence on no-till drilled plots. Controlled environment: effect of N addition to field soil (Experiment 1) In the controlled environment in Experiment 1, the application of N had only a small effect on leaf appearance of seedlings grown in Coyle field soil. On average, the phyllochron value in seedlings that received nitrogen was 73 C above C leaf )1 and on seedlings grown in field soil without amendment the phyllochron value was 82 C above C leaf )1 (P ¼ Æ65). Controlled environment: soil moisture content and frequency of below-freezing temperature exposure (Experiment 2) In Experiment 2a, there was no difference in rate of leaf appearance of IRG in seedlings grown in field soil with moisture content of 45% or 3% VWC with a mean phyllochron value of 78 C above C leaf )1, but a lower soil moisture content of 15% VWC decreased leaf appearance rate and correspondingly increased the phyllochron value to 9 C above C leaf )1 compared with the higher soil moisture contents (Figure 3). In Experiment 2b with IRG and TF, the phyllochron of TF was greater at all levels of soil moisture treatment than that of IRG, and there was a significant interaction between species and soil VWC treatment, that increased phyllochron relatively more in TF than in IRG (P <Æ1) as soil VWC was reduced (Figure 4). Over both species of grass the mean phyllochron value increased from 12 C above C leaf )1 with no exposure to below-freezing temperatures, to a value of 117 C above C leaf )1 with four 11-h exposures to )5 C on successive nights. However, the responses of both species to increased exposure to a nighttime temperature of )5 C were similar (Figure 5) and there was no significant (P > Æ5) interaction between cold exposure and moisture potential in their effects on phyllochron values of IRG or TF (Figure 6). The phyllochron on T1 tillers during regrowth under a uniform 3% VWC was not affected by prior cold exposure. In both IRG and TF, however, the phyllochron values during regrowth were less (P < Æ5) in seedlings that had previously been maintained under a moisture regime of 1% VWC (mean 92 C above C leaf )1 ) than in those that were maintained at 2% or 3% VWC (means of 19 and 11 C above C leaf )1 respectively). Weight of main tillers, and weight and number of daughter tillers, and, conse- Leaves on main tiller Accumulated C above C Figure 3 Experiment 2a: effect of soil moisture contents of 45 (m m), 3 (hæææææh) or 15(n Æ n) % VWC on leaves on main tillers of in Italian ryegrass grown using Coyle clay loam soil in a controlled environment. The equations are: leaves on main tiller (15% VWC) ¼ Æ111 (Æ36) GDD + Æ68; leaves on main tiller (3% VWC) ¼ Æ127 (Æ36) GDD + Æ6; leaves on main tiller (45% VWC) ¼ Æ131 (Æ36) GDD + Æ57, where GDD is accumulated number of C above C. Mean phyllochron value at 45% and 3% VWC was 78 C above C leaf )1 and for 15% VWC was 9 C above C leaf )1. Phyllochron, accumulated C above C leaf Soil moisture content (%VWC) Figure 4 Experiment 2b: effect of soil moisture content (% VWC) on phyllochron values of main tillers of seedlings of Italian ryegrass (IRG) j and tall fescue (TF) j grown in field soil in a controlled environment. Mean of three cold-exposure treatments. Least significant difference (P ¼ Æ5) is 1Æ6 for comparisons of means. 9

6 Factors affecting phyllochron in cool-season grasses 151 Phyllochron, accumulated C above C leaf # Successive ( 11 h) exposures to 5 C Figure 5 Experiment 2b: effect of frequency of exposure to below-freezing temperature (minus 5 C for 11 h at each exposure) on phyllochron values from Italian ryegrass (IRG) j and tall fescue (TF) j grown in a controlled environment. Means are of three soil moisture contents. Least significant difference (P ¼ Æ5) is 1Æ6 for comparisons of means. Phyllochron, accumulated C above C leaf quently, total seedling weights at harvest were reduced in both TF and IRG as soil VWC was reduced. Mean total seedling weights were 92, 81 and 27 mg with IRG and 35, 23 and 11 mg with TF at soil VWC contents of # Successive ( 11 h) exposures to 5 C Figure 6 Experiment 2b: effect of frequency of exposure of seedlings to below-freezing temperature (minus 5 C for 11 h at each exposure) at soil moisture contents of: 1%, 2%, 3% on phyllochron values from main tillers (means of Italian ryegrass and tall fescue). Least significant difference (P ¼ Æ5) is 13Æ for comparisons of means. 3%, 2% and 1% respectively. Cold treatment, in contrast, did not significantly affect weight of the main tiller or the number of daughter tillers produced, but the weight of daughter tillers, and hence mean total seedling weight, was reduced in both species with increased frequency of exposure to below-freezing temperature. Mean seedling weights were, respectively, 55, 4 and 39 mg at, 2 and 4 overnight exposures to )5 C. Discussion Field observations showed that in IRG there was a close linear relationship of leaf appearance to accumulated temperature in all years of study. Differences in intercept across years are attributable to different dates of sowing and emergence and demonstrate that, while accumulated temperature can indicate rate of change in phenology, it cannot indicate actual developmental stage at any point in time without prior direct observation to provide a baseline estimate. Year-to-year variation in estimated phyllochron was small, particularly over the 3 years from 22 to 24, and might suggest that accumulated temperature could be used, with a single phyllochron value, to measure phenological development in IRG during the period January to March. However, the value estimated for 25 was Æ92 of the mean of (a difference of 14 C above C leaf )1 ) and, while this difference was not statistically significant (P > Æ5), it may have practical significance, as any divergence from the true phyllochron value will be cumulative over successive leaves. A single phyllochron value applied from year to year may, therefore, not be sufficiently accurate to provide an indication of development stage that is acceptable for practical application. The estimated value of the phyllochron for fieldgrown plants was consistently greater (at an overall mean of 177 C above C leaf )1 ) than growth chamber estimates, for IRG that was not exposed to soil moisture or cold stress, of 69 C above C leaf )1 in potting soil (Bartholomew and Williams, 25) and 72 C above C leaf )1 in field soil (from results reported here). A phyllochron value of 88Æ5 C above C leaf )1 estimated by Ball et al. (1995) for field-grown IRG also differed markedly from field estimates reported here, further demonstrating that accumulated temperature alone is not sufficient to predict leaf appearance in different environments. Field estimates of the phyllochron value in tall fescue, at 198 C above C leaf )1 leaf )1 (P. W. Bartholomew, unpublished data), were also greater than values measured in a controlled environment. The reduction in phyllochron in response to N application reflects other work that has shown increase in leaf emergence rate (Pearse and Wilman, 1984) or reduction in phyllochron (Longnecker and Robson,

7 152 P. W. Bartholomew and R. D. Williams 1994) in grass and cereal crops. Although the effect was not large and was non-significant (P ¼ Æ65), it demonstrates that N status of the soil potentially introduces additional variability into estimation of the phyllochron. It has been previously reported that in controlled environment the phyllochron is increased with increased frequency of exposure to below-freezing temperatures. In earlier work each overnight exposure to air temperature of )5 C increased phyllochron values of IRG and TF by a mean of 3Æ2 C above C leaf )1 (Bartholomew and Williams, 25), compared with an estimated 2Æ7 C above C leaf )1, at a soil VWC of 3%, for each overnight exposure recorded in the experiments reported here. The effect of repeated cold exposure on phyllochron corresponds with results reported by Kirby (1995), and helps to explain inconsistencies in reported phyllochron values for IRG observed in field and controlled environment studies (Ball et al., 1995; Bartholomew and Williams, 25). The increase in phyllochron in response to repeated cold exposure appears inconsistent with results reported by McMaster et al. (23), who showed no effect of soil heating at crown depth in spring-sown wheat. However, soil temperatures in the work reported by McMaster et al. (23) did not fall below 1 C at 1 2 cm below the soil surface, compared with field minima (January to March 22 25) of between )1Æ1 and )2Æ4 C at 2 cm below the soil surface, and a mean cone-tainer minimum of )3Æ6 C in the experiments reported here. The benefit, observed in this study from reduced cold exposure and, therefore, warmer soil, probably arose from the avoidance of low-temperature shock, rather than from an effect of increased accumulated temperature anticipated by McMaster et al. (23). Increased exposure to below-freezing temperatures may contribute to differences between controlledenvironment and field estimates of phyllochron, but extrapolation of the results obtained from controlledenvironment studies, in conjunction with the occurrence of low-temperature events presented in Table 1, does not provide a convincing explanation of variation in phyllochron values between environments. In part, this may be because plants in the field were exposed to air temperatures below )5 C on several occasions whereas in the controlled-environment studies reported here the lowest temperature to which seedlings were exposed was )5 C. Any incremental effect on phyllochron of exposure to temperatures below )5 C cannot be estimated from this study. The results reported here emphasize the limitations of using growth chamber measures to estimate plant responses under field conditions, particularly for early and late season, when field crops may be subjected to repeated and variable low-temperature exposure that cannot be readily simulated in a controlled environment. McMaster and Wilhelm (23) commented that there may be limited cumulative moisture stress in the early stages of growth of wheat. Winter rainfall may further reduce the incidence of moisture-limited development in early season (January to March), and it was notable in the field observations reported here that the estimated phyllochron in IRG was quite stable across seasons with rainfall in January to March that varied from 72 mm (in 23) to 227 mm (in 24). However, controlled-environment experiments showed that moisture stress can reach a point where it will impact significantly on rate of leaf appearance and may need to be considered in the measurement of responses in plant development to accumulated temperature. Phyllochron response to soil VWC was, however, not affected by frequency of exposure to below-freezing temperatures. The effects of soil moisture on phyllochron reflect variable responses in cereals and grasses in earlier experiments (Frank and Ries, 199; Krenzer et al., 1991). Reduction in soil VWC from 45% to 3% or from 3% to 2% increased the mean phyllochron value but the effect was generally small and corresponds with the relatively minor change in soil moisture potential ( to )Æ1 MPa) recorded over these ranges of soil VWC. Reductions to 15% or 1% VWC (moisture potentials of )Æ23 and )Æ61 MPa respectively) had a significant effect on phyllochron, supporting the contention that phyllochron response to soil moisture content is likely to follow a non-linear relationship (McMaster and Wilhelm, 23). Estimates of carry-over effect in regrowth of seedlings exposed to moisture stress indicate that there may be some compensation for reduced leaf appearance rate at low soil moisture contents during a subsequent phase of increased moisture availability, thus the impact on phyllochron of periodic moisture stress in the field may be less severe than the short-term controlled environment studies suggest. Further quantification of the effects of duration of moisture stress on phyllochron is needed. Conclusion An accurate estimate of phyllochron is necessary if accumulated temperature is to provide a useful indicator of changes in stage of development in IRG or TF early in the year. However, the large difference in estimated phyllochron between field and controlled environment studies shows that phyllochron may differ across environments and the extent to which this variation can be predicted remains unclear. Increased exposure to below-freezing temperatures, decreased soil moisture content and limited N availability may all

8 Factors affecting phyllochron in cool-season grasses 153 contribute to increased phyllochron. Further investigation of the extent to which these factors influence variation in phyllochron values under field conditions is warranted. Acknowledgments The authors gratefully acknowledge the valuable technical support provided by Kathie Wynn and Nina Terrell throughout the implementation of these studies. Mention of trademark names does not represent an endorsement over any other products by the USDA-ARS. References Ball D.A., Klepper B. and Rydrych D.J. (1995) Comparative above-ground development rates for several annual grass weeds and cereal grains. Weed Science, 43, Bartholomew P.W. and Williams R.D. (25) Cool-season grass development response to accumulated temperature under a range of temperature regimes. Crop Science, 45, Cao W. and MOSS D.N. (1989) Temperature effect on leaf emergence and phyllochron in wheat and barley. Crop Science, 29, Cao W. and Moss D.N. (1994) Sensitivity of winter wheat phyllochron to environmental changes. Agronomy Journal, 86, Frank A.B. and Bauer A. (1995) Phyllochron differences in wheat, barley and forage grasses. Crop Science, 35, Frank A.B. and Ries R.E. (199) Effect of soil water, nitrogen, and growing degree-days on morphological development of crested and western wheatgrass. Journal of Range Management, 43, GENSTAT (23) Genstat for Windows, Release , 7th edn. Oxford, UK: VSN International Ltd. Kirby E.J.M. (1995) Factors affecting rate of leaf emergence in barley and wheat. Crop Science, 35, Klepper B., Rickman R.W. and Peterson C.M. (1982) Quantitative characterization of vegetative development in small cereal grains. Agronomy Journal, 74, Kowalenko C.G., Freyman S., Bates D.L. and Holbek N.E. (1989) An evaluation of the T-sum method for efficient timing of spring nitrogen applications on forage production in south coastal British Columbia. Canadian Journal of Plant Science, 69, Krenzer E.G. Jr., Nipp T.L. and McNew R.W. (1991) Winter wheat mainstem leaf appearance and tiller formation vs. moisture treatment. Agronomy Journal, 83, Lemaire G. and Salette J. (1982) The effects of temperature and fertilizer nitrogen on the spring growth of tall fescue and cocksfoot. Grass and Forage Science, 37, Longnecker N. and Robson A. (1994) Leaf emergence of spring wheat receiving varying nitrogen supply at different stages of development. Annals of Botany, 74, 1 7. Masle J. and Passioura J.B. (1987) The effect of soil strength on the growth of young wheat plants. Australian Journal of Plant Physiology, 14, McMaster G.S. and Wilhelm W.W. (23) Phenological responses of wheat and barley to water and temperature: improving simulation models. Journal of Agricultural Science, Cambridge, 141, McMaster G.S., Wilhelm W.W., Palic D.B., Porter J.R. and Jamieson P.D. (23) Spring wheat leaf appearance and temperature: extending the paradigm? Annals of Botany, 91, Mosaad M.G., Ortiz-Ferrara G., Mahalakshmi V. and Fischer R.A. (1995) Phyllochron response to vernalization and photoperiod in spring wheat. Crop Science, 35, OKLAHOMA CLIMATOLOGICAL SURVEY (26) Oklahoma Mesonet. Available at: summary [Accessed on 22 January 26]. Pearse P.J. and Wilman D. (1984) Effects of applied nitrogen on grass leaf initiation, development and death in field swards. Journal of Agricultural Science, Cambridge, 13, Skinner R.H. and Nelson C.J. (1995) Elongation of the grass leaf and its relation to the phyllochron. Crop Science, 35, 4 1.