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Influence of late-season iron, nitrogen, and seaweed extract on fall color retention and cold tolerance of four bermudagrass cultivars.

COMMON and hybrid bermudagrass [Cynodon dactylon (L.) Pers. and C. dactylon x C. transvaalensis Burtt Davy, respectively] are frequently used in the transition zone where they can provide an excellent surface for golf course fairways and athletic fields. These species, however, have a great tendency to be injured or killed by winter temperatures in the transition zone.

Although there are conflicting reports (Schmidt and Blaser, 1969; Beard, 1973) regarding the effect of fall fertilization of bermudagrass, recent research suggests that earlier reports of negative effects of late-season N applications on bermudagrass cold tolerance may not be accurate. Goatley et al. (1994) found that late-season N improved fall and spring color and had little effect on total nonstructural carbohydrate (TNC) levels. Schmidt and Chalmers (1993) reported that the positive effects of late-season N applications (better color, longer color retention) occurred without any negative effects on post dormancy recovery in the spring.

Richardson (2001) found that late-season N fertility had no influence on seeded bermudagrass winter survival. Richardson (2002) also found that late-season N applications on Tifway bermudagrass increased fall color, enhanced spring greenup, and had no effect on rhizome cold tolerance. Although many studies have examined the effects of late-season N on TNC concentrations, virtually no work has examined the influence on other physiological parameters.

Iron may be another nutrient that prolongs fall color without negatively affecting cold tolerance. Tests have shown that Fe applications in conjunction with moderate (24 kg [ha.sup.-1] [mo.sup.-1]) summer N applications can improve the performance of bermudagrass during the fall and improve recovery in the spring (White and Schmidt, 1990). White and Schmidt (1989) also report that Fe maintained the aesthetic quality of cold-sensitive and cold-tolerant bermudagrass cultivars after a chilling period and assisted in C[O.sub.2] exchange rate (CER) recovery.

Certain natural products such as seaweed (Ascophyllum nodosum Jol.) extract contain high levels of cytokinins and auxins as well as moderate levels of other hormones (Mooney and Van Staden, 1986; Crouch et al., 1992). Verkleij (1992) stated that the efficacy of these products appeared to be due mainly to cytokinins but may also have been due to trace nutrients found in the products. A recent study by Zhang and Ervin (2004) with creeping bentgrass (Agrostis stolonifera L.) also indicates that beneficial effects of SWE applications such as increased levels of antioxidants and photochemical efficiency during drought may be due to increased endogenous levels of cytokinins.

Cytokinins can act to inhibit senescence in leaves by counteracting the effects of ethylene or abscisic acid (Arteca, 1996; Buchanan et al., 2000). Cytokinins may also maintain membrane integrity by reducing lipase and lipoxygenase activity, processes involved in membrane breakdown (Mok, 1994). Goatley and Schmidt (1990) reported antisenescence responses in excised Kentucky bluegrass (Poa pratensis L.) leaves after treatment with the synthetic cytokinin benzyladenine. However, White and Schmidt (1990) found that treating bermudagrass with benzyladenine did not consistently affect fall color or quality and did not influence carbohydrate levels. Similarly, Nakamae and Nakamura (1982) did not see an increase in leaf chlorophyll content of Manila grass [Zoysia rnatrella (L.) Mere. var. matrella] during autumn after applications of 6-benzyladenine.

Plant membranes must be kept in a fluid state to maintain function. When membranes become less fluid or gel-like, their protein components become impaired or nonfunctional (Samala et al., 1998; Taiz and Zeiger, 1998). Plants maintain membrane fluidity at lower temperatures by increasing the unsaturation of lipids in the phospholipid bilayer. Studies examining fatty acids in [C.sub.4] grasses have shown that during cold acclimation, there is an increase in the unsaturated/saturated ratio (Samala et al., 1998; Cyril et al., 2001, 2002). This increase occurred to a greater extent in cold-tolerant cultivars than cold-sensitive cultivars.

During cold stress, plants accumulate sucrose and other simple sugars as well as proline and glycine betaine, which have been reported to help stabilize membranes and act as osmolytes that maintain water balance within the cell (Nilsen and Orcutt, 1996; Holmstrom et al., 2000). Proline may have many functions in stress tolerance, including osmotic adjustment, protein and membrane stabilization, gene induction, reactive oxygen species scavenging, N and C source, and a reduction equivalent source during stress recovery (Rudolph et al., 1986; Delauney and Verma, 1993; Saradhi et al., 1995; Hare and Cress, 1997; Iyer and Caplan, 1998; Brugiere et al., 1999). Proline levels have been shown to increase during cold acclimation, decrease during de-acclimation, and increase to a greater extent in cold-hardy species (Levitt, 1980). As extracellular ice crystals form, water moves from inside the cell to enlarge these extracellular ice crystals. By increasing solute concentration, cell osmotic potential is decreased, making water movement out of the cell less likely. This reduction would prevent the enlargement of ice crystals and maintain cell hydration (Rossi, 1997).

This study was designed to examine the effects of late-season N, Fe, and SWE applications on fall through spring aesthetic responses, fatty acid saturation levels, and proline concentrations in the stolon tissues of four bermudagrass cultivars. An understanding of these factors will help to better define the effects that late-season nutrient applications have on physiological processes occurring during acclimation and de-acclimation. The objectives of this study were to (i) determine the effects of late-season N, Fe, and SWE applications on bermudagrass fall visual quality, spring greenup, lipid saturation, and proline concentration; (ii) determine if these treatments were associated with changes in freezing tolerance; and (iii) determine biochemical and cold tolerance differences in four bermudagrass cultivars.

MATERIALS AND METHODS

Plant Material and Establishment

A field study was conducted at the Virginia Tech Turfgrass Research Center in Blacksburg, VA. Plots were established on a Groseclose silt loam (fine, kaolinitic, mesic typic Hapludult) with a pH of 6.8 and a K level of 59 mg [kg.sup.-1]. Plots measuring 3.1 by 9.1 m were established 20 June 2001 using four bermudagrass cultivars: Tifway, Midiron, Princess-77, and Riviera. Princess-77 seed was supplied by Dr. Charles Rodgers (Seeds West Inc., Maricopa, AZ), and Riviera seed was supplied by Dr. Charles Taliaferro (Oklahoma State University, Stillwater, OK). Midiron and Tifway sprigs were supplied by the Virginia Tech Turfgrass Research Center. Seeding rates were 48.8 kg pure live seed (PLS) [ha.sup.-1], and sprigging rates were 1800 bu [ha.sup.-1].

Seeded plots were planted under a geotextile fabric to discourage seed movement as well as enhance germination and plant development. Plots were mowed three times per week with a reel mower set at 1.91 cm. Nitrogen was applied in the form of N[H.sub.4]N[O.sub.3] (34-0-0) once monthly at a rate of 48.8 kg N [ha.sup.-1] beginning at establishment and ending 15 August. A complete fertilizer (10N-4.4P-8.3K) was applied the following spring (25 May) at a rate of 48.8 kg N [ha.sup.-1], and monthly N applications (from 34-04)) began again on 15 June 2002. Irrigation was supplied as needed to prevent visual moisture stress.

After bermudagrass establishment, three chemical treatments were applied during the summer to autumn period leading to bermudagrass shoot senescence. A nontreated control was also included within each bermudagrass cultivar.

Fall Chemical Treatments

Fall chemical treatments began 15 August in 2001 and 2002 and continued on a 3-wk schedule until apparent dormancy (80-100% canopy browning). Final chemical treatment dates were 17 Oct. 2001 and 31 Oct. 2002. Seaweed extract was applied at a rate of 0.54 kg [ha.sup.-1] (Zhang et al., 2002); N was applied at a rate of 48.8 kg N [ha.sup.-1] as N[H.sub.4]N[O.sub.3] (White and Schmidt, 1990); and Fe was applied at a rate of 1 kg Fe [ha.sup.-1] as FeS[O.sub.4] (Schmidt and Chalmers, 1993). Chemical subplots measured 3.1 by 1.8 m.

The experimental design was a split plot arrangement of treatments in a randomized complete block with repeated measures and four replications. Cultivar was considered whole plot factor, while chemical treatment was considered subplot factor. Data were analyzed using the mixed procedure of the Statistical Analysis System (SAS) software (SAS, Cary, NC) (SAS, 2003). Appropriate main effects and interactions were separated using Fisher's Protected LSD test at an [alpha] level of 0.05. The study was conducted during the 2001 through 2002 growing season and repeated during the 2002 through 2003 growing season. This design holds true for all response variables with the exception of controlled freezing.

Fall Quality and Spring Greenup

Visual turfgrass quality ratings were taken (using a 1 to 9 scale, where 1 = completely brown, dormant or dead turf, and 9 = lush green turf) monthly during autumn. As plots were well established by late autumn, color was the primary parameter of interest during quality ratings. Spring greenup was visually estimated as the percentage of green ground cover present.

Controlled Freezing

Freeze chamber analyses were performed on acclimating (fall) and acclimated (winter) stolon tissues in 2001-2002 and 2002-2003. Additional analysis was conducted on samples collected in summer 2003. Control samples only were collected during the acclimating period of both years. This was done as the freezing process is time consuming and the possibility of physiological differences occurring in samples between the first and last tests of each sampling period existed. Samples were analyzed from all plots in winter each year and in summer 2003. The experimental design was a split-split plot arrangement of treatments in a randomized complete block with repeated measures and four replications. Cultivar was considered whole plot factor, chemical treatment was considered subplot factor, and temperature regime was considered subsubplot factor. Data were analyzed using the mixed procedure of SAS (SAS, 2003). Appropriate main effects and interactions were separated using Fisher's Protected LSD test at an [alpha] level of 0.05. A 10.2-cm (diameter) cup cutter sample was removed from each plot, cleaned of soil by washing, and divided into four equal subsamples. One of the subsamples was placed in a refrigerator and held at 4.0[degrees]C to act as a "control." The other three subsamples were placed in a freeze chamber that was programmed to ramp from 8.0 to 1.0[degrees]C overnight. The temperature then ramped to -2.8[degrees]C over a 2-h period, stayed at this temperature for 0.5 h, and a subsample was removed. This process continued with ramping to -5.0 and then -7.2[degrees]C over the next 5 h. After removal, subsamples were held over night at 4.0[degrees]C and then placed in a sand-filled mist bench in the glasshouse at 22 [+ or -] 2[degrees]C. Temperatures were verified with a Watchdog 400 data logger (Spectrum Technologies, Inc., Plainfield, IL). Regrowth was visually estimated as the percentage of the sample exhibiting shoot regrowth or appearing green approximately 4 wk after freezing (Schmidt and Chalmers, 1993).

Total Lipid Extraction and Fatty Acid Quantification

Tissue samples were removed from the field during acclimation (November), when acclimated (January-February), and when nonacclimated (July) and stored at -80[degrees]C until analyzed. For each analysis, 1 g of stolon tissue was ground with a mortar and pestle in liquid [N.sub.2]. This ground tissue was then transferred to a centrifuge tube for total lipid extraction using 3 mL of a buffer containing chloroform/methanol/water (1:2:0.8). After soaking for 1 h at room temperature, 1 mL 1% NaCl and 3 mL chloroform were added and centrifugation occurred at 1200 g for 10 min. The lower chloroform layer containing the lipids was transferred to a test tube, and the chloroform addition, centrifugation, and chloroform layer transfer were repeated two more times (Cyril et al., 2001).

In a method described by Goyal (2000) and modified by Shang et al. (2005), the chloroform layer was then evaporated under a stream of [N.sub.2]. After evaporation, 5 mL of 2% NaOH in 90% methanol was added, and tubes were placed in a water bath at 75 to 80[degrees]C for 30 min. Tubes had an air-cool reflux (small funnels placed in tubes) during this process to facilitate hydrolysis. At the end of 30 min, the mouths of the tubes were left open to allow for evaporation of the methanol under a fume hood. Next, 2 mL of distilled-deionized (DD) [H.sub.2]O were added to facilitate dissolution, and the contents were transferred to a 10-mL screw-cap tube. The residue was washed twice more with 2 mL DD [H.sub.2]O, and contents were transferred to a new tube. To this new tube was added 300 [micro]L of 6 M [H.sub.2]S[O.sub.4] to precipitate Na salts out of the fatty acids. The acid form of the fatty acids was recovered by adding 1 mL hexane and centrifuging at 150 g for 5 min. After centrifugation, the hexane layer containing the free fatty acids was transferred to a new 10-mL screw-cap tube and the process was repeated two more times. After the final centrifugation, the hexane volume was reduced to 100 [micro]L under a gentle stream of [N.sub.2] gas, and 100 [micro]L [alpha]-bromoacetephenone (10 mg [mL.sup.-1] acetone) and 100 [micro]L triethylamine (TEA) (10 mg [mL.sup.-1] acetone) were added and caps were tightly fastened. Tubes were placed into a water bath at 100[degrees]C for 15 min. Free fatty acids react with [alpha]-bromoacetophenone in the presence of TEA and produce a derivative that is UV sensitive and can be quantified with a UV detector after HPLC separation. Tubes were then allowed to cool, and 140 [micro]L acetic acid (2 mg [mL.sup.-1] acetone) was added and the tubes were placed back in the 100[degrees]C water bath for 5 min. After cooling a second time, the content was dried under a stream of [N.sub.2] to inactivate the remaining reagents. The residue was dissolved with 500 [micro]L acetonitrile, and the solution was filtered with a 0.2-[micro]m membrane before injection into HPLC.

HPLC Procedures

Chromatographic analyses were performed on an Agilent (Agilent Technologies, Palo Alto, CA) 1100 series HPLC system with a photodiode array detector. An Ultrasphere-C8 (250 by 4.6 mm, 5 [micro]m) (Beckman Coulter, Inc., Fullerton, CA) analytical column with a C-8 guard column (7.5 by 4.6 mm) (Alltech Associates, Deerfield, IL) was used for chromatographic separation. The mobile phase was 90% acetonitrile in water. Samples were eluted at a gradient rate: from 1 mL [min.sup.-1] up to 2 mL [min.sup.-1] within the first 2 min, at an isocratic elution of 2 mL [min.sup.-1] for 10 min, and then down to 1 mL [min.sup.-1] within the next 8 min. Total elution time per sample was 20 min. The injection volume was 20 [micro]L. The fatty acid derivatives were quantified at a wavelength of 214 nm. The retention times (minutes) at the described conditions were 4.6, 5.8, 7.3 and 10.8 for linolenic, linoleic, palmitic and stearic acids, respectively. The limit of identification for the above procedure is 0.07 to 0.6 [micro]mol per injection using three times the standard deviation for one-half of the lowest standard.

Fatty acid standards were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). External standards prepared from the commercial standards were used for calibration. Two reference samples, which were preweighed from a composite tissue sample, were included in each analysis set.

Proline Determination

Additional stolon samples were removed at the same time as the controlled freezing tests (fall, winter, and summer), placed in liquid [N.sub.2] to halt respiration and then placed in a -80[degrees]C freezer for later analysis. Stolons were ground with a mortar and pestle in liquid [N.sub.2] and approximately 0.20 g stolon material was homogenized in 10 mL of 3% sulfosalicylic acid, and the homogenate was filtered through Whatman no. 2 filter paper. Two milliliters of acid ninhydrin and 2 mL glacial acetic acid were added to 2 mL of the filtrate and incubated at 100[degrees]C for 1 h. The reaction was terminated by placing test tubes in an ice bath. The mixture was extracted with 4 mL toluene and vortexed for 15 to 20 s. The chromophore containing toluene was warmed to room temperature and absorbance read at 520 nm using toluene as the blank. Proline concentration was determined from a standard curve (Syvertsen and Smith, 1983).

RESULTS AND DISCUSSION

Turfgrass Quality

As year was not significant, data were pooled across years. Visual ratings commenced shortly after the initial late-summer treatment application. Cultivar differences with respect to visual quality were noticeable in August, with Riviera having better quality than all other cultivars (Table 1). Riviera normally has medium texture and density, and exhibits a darker green color than Midiron (Morris, 1997, 2002). Midiron had similar quality to Tifway, which had better quality than Princess-77. Princess-77, although normally having superior density and texture to the other cultivars tested, was slower in establishing than the other cultivars resulting in poorer density. In September and October there were no differences between Midiron, Riviera, and Tifway, but all had better quality than Princess-77. By November, however, Princess-77 had better quality than all other cultivars. The better color observed in November overrode the issue of density. These findings are in agreement with a statement by Beard (1973) who generalized that cold-tolerant bermudagrass cultivars discolor earlier in the fall than cold-susceptible cultivars. The response observed here may be due to differences in rate of establishment in 2001 and the fact that Princess-77 plots were heavily damaged by winterkill during the first winter (2001-2002) and were still recovering in fall 2002. Seeded cultivars produce smaller stolons during the establishment year and lack rhizome production (Hensler et al., 1999; Munshaw et al., 2001). As a result, the first winter after seeding can be extremely stressful on bermudagrass and can sometimes result in the loss of turf, regardless of planting date. Quality for all cultivars decreased over the fall, but to varying degrees (Table 1). Midiron, Riviera, and Tifway all had quality reductions of over 50% between August and November, whereas Princess-77, which had lower visual quality at the outset, showed only a 36% reduction over the same period.

As the fall progressed, color became the most important aspect of turfgrass quality. In both years of the study, color was not different across cultivars before the first application of the fall chemical treatments in August (Table 2). In September, N resulted in improved color, which corresponded to a slight increase in turfgrass quality over the control. In October and November, N only improved turfgrass quality over the control. As would be expected, overall quality for all treatments decreased as the fall progressed due to color loss (Table 2). All treatments had the same quality in August, but SWE and control plots had greater quality reductions by the end of the observation period in November.

The ability to prolong fall color of bermudagrass with fall chemical treatments is important in non-overseeded bermudagrass, as the longer period of green color may also increase turf functionality. Although the differences between N treated and the control were not large in November, low-budget athletic fields may benefit from this improved color retention by allowing activities to continue on somewhat greener grass. As has been reported previously (Reeves et al., 1970; White and Schmidt, 1990; Schmidt and Chalmers, 1993; Goatley et al., 1994; Richardson, 2001,2002), late-season N applications prolonged green color.

Iron did not significantly improve fall color during the evaluation period. White and Schmidt (1990) did not observe any differences with the application of Fe until air temperatures reached freezing. Schmidt and Chalmers (1993) found a 20% improvement in October turfgrass color due to Fe, but only when no N was applied in September. Rodgers (2003) suggests that the new seeded bermudagrasses use Fe more efficiently than older seeded cultivars. Although no older seeded cultivars were examined in this study, the interaction between cultivar and chemical treatment was not observed. The effect of Fe was minimal on all cultivars. A higher rate of FeS[O.sub.4] or a chelated form of Fe may have resulted in a greater response through the fall due to a greater chance of Fe uptake.

Seaweed extract applications did not have any effect on bermudagrass color retention, as quality ratings were very similar to the control in both years of the study. This finding is in agreement with White and Schmidt (1990), who found that synthetic cytokinin applications did not have a consistent effect on late-season bermudagrass quality or color. Seaweed extracts also contain cytokinins and the response on bermudagrass appears to be very similar with the two products.

Controlled Freezing

As the freeze chamber temperature decreased from 4.0 to -7.2[degrees]C, bermudagrass survival generally decreased (Tables 3 and 4). Further, at all winter sampling dates, Midiron typically had the highest amount of survival and regrowth following freezing, especially following exposure to the colder temperatures. Riviera normally had the next highest amount of survival, followed by Tifway and finally Princess-77. These results are in agreement with previous work where Midiron was described as cold tolerant. Midiron is followed by Riviera with moderate cold tolerance, Tifway somewhat cold susceptible, and Princess-77 having poor cold tolerance (Shashikumar and Nus, 1993: Anderson et al., 2003).

Samples collected after fall 2001 and held at 4.0[degrees]C had varying levels of survival, likely due to air temperatures in the field rather than the moderate refrigerator temperature during storage. The first killing frost in 2001 occurred on 8 October and there were many subsequent days with temperatures dropping below 0[degrees]C (weather data not shown). However, with unseasonably warm temperatures occurring in November of 2001 (22.7[degrees]C on the 2 November sampling date), a high degree of color retention remained for all cultivars. Acclimation may have begun in October 2001, but with warmer temperatures in late October and early November, the acclimation process may have slowed. Although samples had been subjected to some freezing temperatures in the month of October, these temperature events may only have been enough to partially harden field stolons and allow adequate survival at the moderately cold freeze-chamber temperatures of 4.0 and -2.8[degrees]C. This is shown by high amounts of survival at both temperatures. If the samples were not completely hardened by the November 2001 sampling date, the colder freeze-chamber temperatures of -5.0 and -7.2[degrees]C should have had a much greater effect on survival than the warmer temperatures. This was in fact what was found after freezing to -5.0 and -7.2[degrees]C.

In 2002, the first killing frost did not occur until 2 November, and temperatures generally remained cool until the fall sampling date. This 3-wk period of temperatures dropping below freezing may have been enough to harden samples to a level not attained in 2001. The amount of survival after freezing to -5.0 and -7.2[degrees]C in fall 2002 was much higher than in fall 2001. This indicates that the combination of the 3-wk period in early November and a gradual decline in temperatures throughout the fall were sufficient for acclimation. Gatschet et al. (1994) found that lethal temperatures were lowered 5[degrees]C by acclimating bermudagrass for 4 wk at 8/2[degrees]C day/night temperatures. The 3-wk period in November had a mean temperature of 6.7[degrees]C, with a high of 20 and many lows below 0[degrees]C.

Survival at lower temperatures was generally greater in winter than in fall of each year (Table 3 and 4). Clearly, acclimation was still occurring after the fall sampling dates. Anderson and Taliaferro (1995) subjected bermudagrass to freezing temperatures at monthly intervals throughout the fall and winter and found survival was higher in midwinter than in November. There were generally no differences in survival between cultivars in winter 2002 and 2003 (Table 4). Air temperatures leading up to the winter 2002 sampling date had reached as low as -14.4[degrees]C, which may have caused regrowth after 4.0[degrees]C to be less than 100%. Temperatures leading up to the winter 2003 sampling date had reached as low as -16.9[degrees]C, but due to the insulating effects of snow cover, soil temperatures (data not shown) were actually warmer in 2003 than in 2002, allowing for similar amounts of regrowth for both years.

Sampling stolons from the field in summer 2003 and storage at 4.0[degrees]C indicated that chilling temperatures had differing effects on survival of unacclimated samples, with Riviera having more regrowth than Midiron (Table 4). Although seeded bermudagrasses are normally slow to establish, subsequent years after sowing show much greater stolon and rhizome development. This increased density in the summer may have allowed Riviera to become more efficient photosynthetically and to allow the storage of more TNC. Hensler et al. (1999) suggested and Munshaw et al. (2001) reported that increased stolon diameter results in higher TNC levels. Although stolon diameters and TNC were not measured in the current study, they may play an important role in freezing survival. None of the summer 2003 sampled cultivars survived even the most moderate of freezing temperatures. Anderson et al. (1988) found that freeze tests conducted on bermudagrass cultivars in June resulted in much less survival than during the winter.

The effects of sampling date, cultivar, and fall chemical treatment interacted with respect to postfreeze regrowth (Table 5). As explained above, fall treatment effects were not tested in November of either year. In general, there was little effect of chemical treatment on bermudagrass survival across all temperatures and sampling dates, and no chemical treatment consistently reduced survival relative to the control. Turfgrass managers growing bermudagrass typically discontinue N use in late summer with the belief that late-season-applied N will increase succulence and winter injury. Controlled freezing tests on cold-acclimated bermudagrass samples in this study indicated that, across all temperatures, N treatment did not affect freezing tolerance of any cultivar. Richardson (2002) froze late-season N-treated bermudagrass rhizomes to similar temperatures and also did not find any negative effects on freezing tolerance. Further, Schmidt and Chalmers (1993) evaluated regrowth after controlled freezing and did not find any negative effects of late-season N applications.

In this study, Fe-treated bermudagrass exhibited no differences in terms of postfreeze regrowth from the control, with the exception of Midiron in winter 2002 and Tifway in winter 2003 (Table 5). Schmidt and Chalmers (1993) reported a 50 to 100% increase in postfreeze regrowth with Fe treatment relative to the control. Although rates of Fe treatment were the same in both studies, differing Fe sources (Fe DTPA) were used. This may play a role in bermudagrass cold tolerance as a chelated form would have a greater chance of being taken up by the plant instead of precipitating out of the soil (Yust et al., 1984).

Schmidt and Chalmers (1993) observed improved bermudagrass postfreeze regrowth following applications of SWE fortified with humic acid and thiamine. In the present study, SWE alone did not influence postfreeze regrowth (with the exception of Tifway in winter 2002 [[alpha] = 0.10]), indicating that the positive results seen by Schmidt and Chalmers may have been due to humic acid, thiamine, or interactions of the three compounds.

Fatty Acid Analysis

The absolute amounts of key fatty acids in total polar lipids (mainly phospholipids) for each cultivar are shown in Table 6. With the exception of fall 2001, individual fatty acid levels were generally fairly constant. Palmitic and stearic acids have been previously reported (Samala et al., 1998; Cyril et al., 2002) to remain somewhat constant during cold acclimation. In the current study, it was found that there were variations in these fatty acids during different times of the year. However, trends were not consistent and did not hold for all cultivars.

It has also been reported (Samala et al., 1998) that linoleic acid levels decrease and linolenic acid levels increase in bermudagrass during cold acclimation. Increases in linolenic acid levels during acclimation may at least partially explain differences in cold tolerance between different cultivars (Cyril et al., 2001, 2002). In the current study, only one sampling date occurred during the "acclimation" period each fall and thus differences over time during hardening in these two fatty acids cannot be corroborated. However, there did not appear to be reductions in linoleic acid levels at other times of the year when linolenic acid levels were high, which may indicate that triunsaturated fatty acids may not accumulate at the expense of diunsaturated fatty acids. However, as linolenic acid has been shown in the literature to be very important in cold hardiness, fatty acid levels will be presented in terms of the percentage of linolenic acid to all fatty acids measured, or the fraction of linolenic acid to total polar lipids.

In the current study, N, SWE, and Fe linolenic acid levels were not different from the control even though N increased visual quality (Table 2). This suggests that these treatments did not have an effect on bermudagrass cold tolerance via a lipid-mediated mechanism.

There was a sampling date x cultivar interaction for percentage of linolenic acid to total polar lipids (Table 7). Cultivar differences in percentage of linolenic acid showed that cold-tolerant cultivars generally had higher levels than cold-sensitive cultivars. Although linolenic acid level differences were not always significant, Midiron consistently had the highest levels, followed by Riviera, Princess-77, and Tifway, respectively. Previous studies have shown that linolenic acid levels increase during cold acclimation and increase to a greater degree in cold-tolerant than cold-sensitive cultivars (Samala et al., 1998; Cyril et al., 2001, 2002). Linolenic acid levels were high in fall 2001 but dropped in winter 2002. Although it was expected that samples would have as high or higher levels of linolenic acid during this time, air temperature changes in January may at least partially explain this difference. Air temperatures leading up to the sampling date were cool, averaging around 0 to 5[degrees]C. Several days before sampling, however, the air temperature increased to 10 to 15[degrees]C and may have had a short-term effect on the degree of lipid saturation. Further, Beard (1973) explains that grasses experience their highest level of hardiness in early winter and may experience a dehardening period in mid- to late winter. Another possible explanation for high linolenic acid levels in fall 2001 is that samples had much better color retention relative to the fall 2002 sample period. Although quality was not rated in the winter months, plots of all cultivars were completely brown. A greater amount of color would equate to greater amounts of chlorophyll in bermudagrass stolons. Salisbury and Ross (1992) explain that chloroplast pigments encompass 50% of the thylakoid membrane and the fatty acid portion of this membrane has high levels of both linolenic and linoleic acid. Thus, greater amounts of stolon chloroplasts in November 2001 could have resulted in higher linolenic acid levels.

Linolenic acid levels were higher in winter 2003 than in fall 2002 (Table 7). However, postfreeze survival was generally not different in winter and fall (Table 5). Bermudagrass samples removed from the field in winter would naturally be more acclimatized than samples removed in fall and thus should have higher levels of survival. Temperature data shows that a low temperature of -3.5[degrees]C occurred before sampling in fall, while samples removed in winter had been subjected to nearly -17[degrees]C. Even with winter samples being more acclimated and having greater amounts of linolenic acid than fall samples, -17[degrees]C is very cold for bermudagrass and likely reduced survival levels and offset some of the benefits of full acclimation.

High levels of linolenic acid did not necessarily result in greater amounts of survival following controlled freezing. Although it was found that cold-tolerant cultivars had higher levels of linolenic acid than cold-sensitive cultivars, differences within cultivars and between dates could not be correlated with survival. Riviera, Tifway, and Princess-77 all had higher levels of linolenic acid in fall 2001 than fall 2002, however, there were no differences in survival (across all temperatures) between these dates (Table 5 and 7). In winter 2003, a significant regrowth x percentage of linolenic acid correlation (r = 0.28, P = 0.01) existed at 4[degrees]C. Percentage of linolenic acid (r = 0.39, P = 0.01) correlation at -2.8[degrees]C was significant. Percentage of linolenic acid correlation at -5.0[degrees]C (r = 0.37, P = 0.01) and -7.2[degrees]C (r = 0.20, P = 0.05) were also significant. But, as can be seen, lipid unsaturation only explains 4 to 15% of regrowth. Obviously, many other factors are involved in bermudagrass cold hardiness.

The high linolenic acid levels in summer were unexpected (Table 7). As previous work in bermudagrass has shown an increase in unsaturation during the fall cold acclimation period, pre-acclimation levels were expected to be low. However, as air temperatures and light intensity during the summer were conducive to bermudagrass growth, chloroplast pigments were most likely at higher concentrations than in other times of the growing season. As was explained above, thylakoid membranes contain high levels of linolenic and linoleic acids (Salisbury and Ross, 1992). Although chloroplasts were not examined per se in the present study, they would have contributed significantly to sampled tissues mainly in the summer sampling period. The presence of the unsaturated fatty acids in the thylakoid membrane causes a higher level of fluidity (Salisbury and Ross, 1992).

Proline Concentration

Generally, proline concentration varied with cultivar (Table 8). Midiron normally had the highest levels, followed by Riviera, Tifway, and Princess-77. This finding is significant as proline concentration among cultivars follows the same trend as was shown in postfreeze regrowth (Table 5). Previous literature has examined late-season fertility affects on bermudagrass carbohydrate concentrations and has drawn conclusions on cold tolerance based on these data. There are, however, many likely physiological differences in cold-tolerant and cold-sensitive bermudagrasses including lipid unsaturation (Samala et al., 1998) and cellular proline concentration (Munshaw et al., 2004). In cold-hardiness research where proline was measured the results have indicated that cold-tolerant cultivars possess higher levels than cold-sensitive cultivars. As was shown in maize (Zea mays L.), cellular proline concentrations increase during cold acclimation (Chen and Li, 2002). As Rossi (1997) points out, an increase in cellular proline concentrations can have a large impact on osmotic adjustment during freezing events, decreasing the possibility of cytoplasm dehydration, extracellular ice formation, and cell rupture.

Increases in proline content should allow cells to remain intact during cold stress and continue functioning. During midwinter controlled freeze tests on bermudagrass samples, a high concentration of proline was correlated with amount of regrowth after freezing. This result agrees with Munshaw et al. (2004), who showed that more regrowth was evident after freezing when high proline levels were present. Chen and Li (2002) also found that increased proline levels in maize improved tolerance to chilling stress.

Samples tested in fall 2002 showed a significant correlation between proline concentration and regrowth (r = 0.46, P = 0.01) at -7.2[degrees]C. In winter 2003, there were significant correlations between proline concentration and regrowth (r = 0.29, P = 0.01) at -2.8[degrees]C as well as at -5.0[degrees]C (r = 0.36, P = 0.01). There was also a significant correlation between proline concentration and regrowth (r = 0.38, P = 0.01) at -7.2[degrees]C.

In the present study, proline concentration was not consistently or significantly affected by Fe, SWE, or N (Table 8). There were, however, differences in terms of proline concentration between samples that were assumed to be nonacclimated (fall) and acclimated (winter). Munshaw et al. (2004) found that increasing proline concentrations in bermudagrass due to moderate salt applications was correlated with increased regrowth after freezing. Because no previous research has examined the effect of N, SWE, and Fe treatments on proline levels in bermudagrass, relationships must be drawn to the studies that have found very similar results showing no effects of these treatments on carbohydrate concentrations (White and Schmidt, 1990: Goatley et al., 1994: Richardson, 2002). Although fall quality may be affected by these chemical treatments, they appear to have no influence on physiological parameters measured in this, or other studies.

Of interest during the second season was that proline concentrations were much reduced in summer for all cultivar and chemical treatment combinations over samples taken in fall or winter (Table 8). There is likely a reduction in water uptake during periods of cool soil temperatures, resulting in a form of physiological drought. The osmotic stress that may occur during this period could result in an increase in plant proline levels. Postfreeze survival and proline concentrations showed similar trends during the summer as survival was also low for all cultivars (Table 5). It appears that high levels of lipid unsaturation are required during all times of the year in order for cellular membranes to remain fluid. However, because high levels of lipid unsaturation alone do not fully explain differences in cold tolerance, other factors such as proline concentration, TNC level, and perhaps many other physiological processes must act in concert to condition tissues for cold tolerance. This is demonstrated by findings from samples collected in July that had poor freeze tolerance (Table 5), high lipid unsaturation (Table 7), and low proline concentration (Table 8).

Spring Greenup

Percentage of greenup was rated on three dates in both 2002 and 2003. As the spring progressed, greenup increased (Table 9). Midiron and Riviera had greater amounts of greenup early in the spring and across all observation dates than Tifway and Princess-77. In 2002, Riviera and Midiron had 20 to 25% more greenup than Tifway and 55 to 75% more greenup than Princess-77 on 28 April and 15 May (Table 9). On 1 June, Riviera, Midiron, and Tifway all had higher percentage of greenup than Princess-77.

In 2003, Riviera had statistically more greenup at the first observation date (28 April) than all other cultivars (Table 9), and Midiron had more than Tifway and Princess-77. Midiron and Riviera had higher amounts of greenup on 15 May and 1 June than Tifway and Princess-77, and Tifway had more greenup than Princess-77.

Comparison of means within years revealed that all cultivars had higher amounts of greenup at all observation dates in 2002 than in 2003. Daily mean temperatures in the winter of 2003 were 5[degrees]C cooler on average than in 2002. Temperatures in general were cooler during the winter of 2003 and were likely a factor in greenup differences between years. The previous statement can be made with some confidence as the cold-sensitive cultivars were affected by the colder temperatures to a greater degree than the cold-tolerant cultivars and thus were much slower to greenup. Midiron showed a reduction of 36% in April 2003, while Riviera was only reduced 20%. Tifway (somewhat cold sensitive) showed a 79% reduction in greenup in 2003, and Princess-77 (cold sensitive) was reduced 98%.

In both years of the study, the cold-tolerant cultivars Midiron and Riviera rejuvenated earlier and faster than the cold-sensitive cultivars Tifway and Princess-77. Clearly, a large portion of Princess-77 was lost to winterkill in both years of the study. Munshaw and Ervin (2003) reported that, in the transition zone, Riviera had the best spring greenup of all cultivars tested in the 1997 NTEP trials. The NTEP trial also showed that Tifway had significantly worse greenup than Riviera, and Princess-77 was slower to greenup than Tifway (Morris, 2002). These results are identical to the findings in the current study as Midiron and Riviera had better greenup than Tifway and Princess-77.

There was a significant year x chemical treatment interaction (Table 10). In 2002 the June rating showed that the N treatment has significantly less greenup than the control. This response was not consistent throughout the 2002 spring rating period, nonexistent in 2003 and slight in June, thus it is likely not biologically significant. The trends of greater amounts of greenup in 2002 were consistent when considering treatments, as all were higher in 2002 than in 2003. In both years, a positive response of the treatments was not evident during greenup. Goatley et al. (1994) did not see an effect on spring greenup with late-season-applied Fe, even with rates in excess of 4 kg [ha.sup.-1]. White and Schmidt (1990), however, saw enhanced recovery after dormancy with fall-applied Fe at 1.2 kg [ha.sup.-1]. Richardson (2002), as well as Schmidt and Chalmers (1993), found that fall N applications promoted early spring greenup. In the current study, survival after controlled freezing was not affected by N treatment during either winter (Table 5).

Chemical treatment did not affect greenup (P = 0.5) of Midiron, Riviera, and Tifway (data not shown). Princess-77 control plots had greater amounts of greenup than N-treated plots at one date in 2002. These data suggest that greenup, in general, is also genetically controlled. Cultivars better able to prepare for winter than others by increasing lipid unsaturation and/or proline concentration, are the cultivars that begin greenup earlier in the spring and are less affected by winterkill.

CONCLUSIONS

This study provides strong evidence that late-season N applications can have a beneficial effect on bermudagrass color in the fall without negatively affecting cold tolerance using controlled freezing tests. This finding, partially contradicts conventional textbook recommendations (Beard, 1973; Duble, 1989). Proline concentration and fatty acid unsaturation levels were also unaffected by late-season N applications, again indicating that N fertilization may not cause weaker plants that are more prone to winter injury. No beneficial effect on spring greenup was observed due to late-season N applications.

We were unable to increase the duration of late-season growth with seaweed extract. This treatment did not result in improved turfgrass quality late in the growing season. There were also no consistent effects of SWE on postfreeze regrowth, proline concentration, lipid unsaturation, or spring greenup.

Iron treatments resulted in increased turfgrass quality over the control but only late in the growing season. This is a beneficial response, since late-season color retention and quality was a desired outcome. Late-season Fe treatments had no consistent effect on postfreeze regrowth, proline concentration, or lipid unsaturation.

Bermudagrass cultivars vary tremendously in terms of quality, recuperative capacity, and cold tolerance. Although previous research has shown differences among these cultivars in terms of cold tolerance, little physiological explanations have been offered. These results show that cultivars that are known to be cold tolerant produce higher levels of linolenic acid and proline during fall and winter months. Results from 2002 through 2003 showed significant correlations between levels of linolenic acid and proline and regrowth after freezing. This suggests the importance of enhanced levels of these compounds during the winter. However, based on the chemical treatments examined in this study, it appears that proline and lipid unsaturation are genetically controlled and are generally only affected by the environment and not by late-season inputs of N, Fe, or SWE. Cold-tolerant cultivars also showed earlier and more rapid greenup than cold-sensitive cultivars in the spring. It seems logical to conclude that cultivars less affected by stress during fall and winter break dormancy in much better physiological condition than cold-sensitive cultivars.

Recommendations for turfgrass managers in the transition zone based on data generated in this study are to use cold-tolerant cultivars such as Midiron or Riviera that exhibit good quality during the summer and fall while maximizing the likelihood of winter survival. However, because Rivera is propagated by seed, it represents an alternative establishment method for bermudagrass. Seeded cultivars can be more convienient to establish than vegetative cultivars as transportation and/ or storage of vegetative planting stock is not necessary. Also, judicious N applications throughout the entire growing season along with maintaining sufficient soil K levels can prolong the growing season (Gilbert and Davis, 1971). Lengthening the period of greenness through N applications may encourage additional late-season use and improve potential revenue for golf courses and athletic fields.

Abbreviations: CER, C[O.sub.2] exchange rate; SWE, seaweed extract; TNC, total nonstructural carbohydrate.

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G. C. Munshaw, * E. H. Ervin, C. Shang, S. D. Askew, X. Zhang, and R. W. Lemus

G.C. Munshaw, Dep. of Plant and Soil Sci., Mississippi State Univ., Mississippi State, MS 39762; E.H. Ervin, C. Shang, S.D. Askew, and X. Zhang, Crop and Soil Environ. Sci. Dep., Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061; R. Lemus, Dep. of Agric. Sci., Texas A&M Univ.-Commerce, Commerce, TX 75429. The authors wish to acknowledge P.D. Gerard, Dep. of Ag. Info. Sci. and Ed., Mississippi State Univ., Mississippi State, MS 39762 for statistical assistance. Received 25 Jan. 2005. * Corresponding author (gmunshaw @pss.msstate.edu).

doi:10.2135/cropsci2005.0078
Table 1. Observation date x cultivar interaction for visual quality
across all chemical treatments and both years (2001 and 2002).

                               Observation date

Cultivar                 August    September    October

                               Visual quality (1-9)

Midiron                   8.0         7.8         6.6
Riviera                   8.4         7.7         6.6
Tifway                    8.1         7.7         6.6
Princess-77               6.6         6.5         6.1
LSD ([double dagger])     0.3         0.3         0.3

                         Observation
                            date

Cultivar                  November      LSD ([dagger])

                            Visual quality (1-9)

Midiron                    3.8               0.2
Riviera                    3.8               0.2
Tifway                     3.8               0.2
Princess-77                4.2               0.2
LSD ([double dagger])      0.3               --

([dagger]) LSD = comparison of dates within cultivars.

([double dagger]) LSD = comparison of cultivars within dates.

Table 2. Observation date x fall chemical treatment interaction
for visual qulaity across all cultiObservation  date
2002).

                                Observation date
Chemical
treatment                August    September    October

                              Visual quality (1-9)

N                         7.8         7.6         6.7
SWE ([double dagger])     7.8         7.4         6.4
Fe                        7.8         7.4         6.4
NT ([section])            7.8         7.4         6.3
LSD ([paragraph])          ns         0.2          ns

                         Observation
                            date
Chemical
treatment                 November      LSD ([dagger])

                              Visual quality (1-9)

N                            4.1             0.2
SWE ([double dagger])        3.8             0.2
Fe                           3.9             0.2
NT ([section])               3.8             0.2
LSD ([paragraph])            0.2              --

([dagger]) LSD = comparison of dates within chemical treatments.

([double dagger]) SWE, seaweed extract.

([section]) Non-treated control.

([paragraph]) LSD = comparison of chemical treatments within dates.

Table 3. Mean postfreeze regrowth of four bermudagrasses as
a function of freezing chamber temperature and sampling date.

                                              [degrees]C

Sampling Date    Cultivar                   4.0       -2.8

                       Regrowth (%)

Fall 2001        Midiron                  100.0 *    60.0
                 Riviera                  100.0      43.8
                 Tifway                   100.0 *    33.8
                 Princess-77              100.0 *    17.5
                 LSD ([double dagger])      ns       18.1
Fall 2002        Midiron                   62.5      47.5
                 Riviera                   85.0      72.5 *
                 Tifway                    77.5      57.5 *
                 Princess-77               72.5      65.0 *
                 LSD ([double dagger])     18.1      18.1

                                             [degrees]C

Sampling Date    Cultivar                 -5.0       -7.2

                      Regrowth (%)

Fall 2001        Midiron                  29.0       3.8
                 Riviera                   1.3       0.0
                 Tifway                    1.8       0.0
                 Princess-77               0.0       0.0
                 LSD ([double dagger])    18.1        ns
Fall 2002        Midiron                  28.8      22.0 *
                 Riviera                  32.5 *     1.3
                 Tifway                   20.0 *     0.0
                 Princess-77              14.0       0.0
                 LSD ([double dagger])    18.1      18.1

                                            [degrees]C

Sampling Date    Cultivar                 LSD ([dagger])

                     Regrowth (%)

Fall 2001        Midiron                       17.8
                 Riviera                       17.8
                 Tifway                        17.8
                 Princess-77                   17.8
                 LSD ([double dagger])         --
Fall 2002        Midiron                       17.8
                 Riviera                       17.8
                 Tifway                        17.8
                 Princess-77                   17.8
                 LSD ([double dagger])         --

* Values followed by an asterisk are significantly higher within
cultivars and between dates (P < 0.05).

([double dagger]) LSD = comparison of temperature within a cultivars
and sampling date.

([dagger]) LSD = comparison of cultivar within a temperatures and
sampling date.

Table 4. Mean postfreeze regrowth of four bermudagrasses as a
function of freezing chamber temperature and sampling date.

                                             [degrees] C

Sampling Date      Cultivar                   4.0    -2.8

                                             Regrowth (%)

Winter 2002        Midiron                   73.1    59.4
                   Riviera                   80.3    60.9
                   Tifway                    54.7    32.2
                   Princess-77               36.3    25.6
                   LSD ([double dagger])     23.2    23.2
Winter 2003        Midiron                   74.1    68.1
                   Riviera                   79.4    55.0
                   Tifway                    55.6    19.7
                   Princess-77               13.1    12.8
                   LSD ([double dagger])     14.3    14.3
Summer 2003        Midiron                   72.2     0.0
                   Riviera                   97.5     0.0
                   Tifway                    96.9     0.0
                   Princess-77               75.0     0.0
                   LSD ([double dagger])      7.8     ns
Temporal           Midiron                    ns     11.6
LSD ([section])    Riviera                   11.6    11.6
                   Tifway                    11.6    11.6
                   Princess-77               11.6    11.6

                                             [degrees] C

Sampling Date      Cultivar                  -5.0    -7.2

                                             Regrowth (%)

Winter 2002        Midiron                   54.4    41.6
                   Riviera                   28.6     8.1
                   Tifway                    10.1     0.3
                   Princess-77                1.6     0.0
                   LSD ([double dagger])     23.2    23.2
Winter 2003        Midiron                   40.6    21.3
                   Riviera                   27.8     4.2
                   Tifway                     5.4     0.0
                   Princess-77                0.9     0.0
                   LSD ([double dagger])     14.3    14.3
Summer 2003        Midiron                    0.0     0.0
                   Riviera                    0.0     0.0
                   Tifway                     0.0     0.0
                   Princess-77                0.0     0.0
                   LSD ([double dagger])      ns      ns
Temporal           Midiron                   11.6    11.6
LSD ([section])    Riviera                   11.6     ns
                   Tifway                     ns      ns
                   Princess-77                ns      ns

                                              [degrees] C

Sampling Date      Cultivar                  LSD ([dagger])

                                              Regrowth (%)

Winter 2002        Midiron                        11.9
                   Riviera                        11.9
                   Tifway                         11.9
                   Princess-77                    11.9
                   LSD ([double dagger])           --
Winter 2003        Midiron                         9.9
                   Riviera                         9.9
                   Tifway                          9.9
                   Princess-77                     9.9
                   LSD ([double dagger])           --
Summer 2003        Midiron                         0.0
                   Riviera                         0.0
                   Tifway                          0.0
                   Princess-77                     0.0
                   LSD ([double dagger])           ns
Temporal           Midiron
LSD ([section])    Riviera
                   Tifway
                   Princess-77

([dagger]) LSD = comparison of temperature within a cultivars and
sampling date.

([double dagger]) LSD = comparison of cultivar within a temperatures
and sampling date.

([section]) LSD = comparison of cultivars and temperatures across all
dates.

Table 5. Mean postfreeze regrowth of four bermudagrasses as a
function of chemical treatment and sampling date.

                       Chemical treatment

                                                 SWE
Sampling Date    Cultivar              N      ([dagger])     Fe

                          Regrowth (%)

Winter 2002      Midiron              61.6       69.4       38.1
                 Riviera              53.0       35.0       46.9
                 Tifway               22.5       26.8       27.5
                 Princess-77          17.5       18.8       13.1
                 LSD ([paragraph])    27.0       27.0       27.0
Winter 2003      Midiron              57.8       40.3       55.3
                 Riviera              37.2       41.9       48.8
                 Tifway               18.1       26.6       11.8
                 Princess-77          5.3        7.8        8.1
                 LSD ([paragraph])    14.9       14.9       14.9
Summer 2003      Midiron              17.5       18.4       17.5
                 Riviera              25.0       25.0       24.4
                 Tifway               25.0       25.0       23.8
                 Princess-77          17.5       20.0       18.1
                 LSD ([paragraph])     ns         ns         ns
Temporal         Midiron              11.6       11.6       11.6
LSD#             Riviera              11.6        ns        11.6
                 Tifway                ns         ns        11.6
                 Princess-77          11.6        ns         ns

                          Chemical treatment

                                             NT                LSD
Sampling Date    Cultivar             ([double dagger])    ([section])

                             Regrowth (%)

Winter 2002      Midiron                    59.4              18.9
                 Riviera                    43.1               ns
                 Tifway                     20.5               ns
                 Princess-77                14.1               ns
                 LSD ([paragraph])          27.0               --
Winter 2003      Midiron                    50.6               ns
                 Riviera                    38.5               ns
                 Tifway                     24.1              10.6
                 Princess-77                 5.6               ns
                 LSD ([paragraph])          14.9               --
Summer 2003      Midiron                    18.8               ns
                 Riviera                    23.1               ns
                 Tifway                     23.1               ns
                 Princess-77                19.4               ns
                 LSD ([paragraph])           ns                ns
Temporal         Midiron                    11.6
LSD#             Riviera                    11.6
                 Tifway                      ns
                 Princess-77                 ns

([dagger]) SWE, seaweed extract.

([double dagger]) Non-treated control.

([section]) LSD = Comparison of chemical treatment within a cultivar
and sampling date.

([paragraph]) LSD = Comparison of cultivar within a chemical treatment
and sampling date.

(#) LSD = Comparison of cultivars and temperatures across all dates.

Table 6. Summary of the absolute amounts of the four major fatty
acids in total polar lipids of untreated Midiron, Riviera, Tifway,
and Princess-77 at all sampling dates.

          Fatty acid ([micro]g [g.sup.-1]) ([dagger])

Cultivar       Sampling date    Linolenic    Linoleic    Palmitic

Midiron        Fall 2001        115.2 a      204.0 a     329.4 a
               Winter 2002       60.6 ab     135.0 ab    198.4 b
               Fall 2002         39.4 b       85.5 b     124.96
               Winter 2003       83.5 ab     149.7 ab    168.9 b
               Summer 2003       63.3 ab     123.7 ab    116.7 b
Riviera        Fall 2001        109.3 a      222.1 a     309.5 a
               Winter 2002       33.0 b       96.7 b     126.5 be
               Fall 2002         35.4 b       82.5 b     172.1 b
               Winter 2003       30.5 b       68.8 b      85.0 c
               Summer 2003       34.5 b       84.2 b     107.5 be
Tifway         Fall 2001         44.6 a      113.1 a     160.8 a
               Winter 2002       25.2 a      100.9 a     140.3 a
               Fall 2002         24.4 a      107.8 a     245.6 a
               Winter 2003       40.6 a      107.9 a     131.1 a
               Summer 2003       31.7 a       95.9 a     175.7 a
Princess-77    Fall 2001         86.4 a      186.6 a     299.9 a
               Winter 2002       34.4 b       64.0 b     104.3 d
               Fall 2002         27.0 b       87.3 b     187.4 be
               Winter 2003       42.4 b      102.1 b     224.6 b
               Summer 2003       37.5 b       79.9 b     141.2 cd

Fatty acid ([micro]g [g.sup.-1]) ([dagger])

Cultivar       Sampling date    Stearic

Midiron        Fall 2001        47.7 a
               Winter 2002      40.5 a
               Fall 2002        19.8 a
               Winter 2003      25.0 a
               Summer 2003      27.3 a
Riviera        Fall 2001        53.2 a
               Winter 2002      32.4 b
               Fall 2002        19.8 be
               Winter 2003      12.6 c
               Summer 2003      13.0 c
Tifway         Fall 2001        39.4 a
               Winter 2002      36.1 ab
               Fall 2002        25.0 abc
               Winter 2003      17.3 c
               Summer 2003      22.7 cb
Princess-77    Fall 2001        51.6 a
               Winter 2002      21.6 b
               Fall 2002        26.6 b
               Winter 2003      23.7 b
               Summer 2003      19.5 b

([dagger]) Values followed by the same letter within the same column
and cultivar 0.05). are not significantly different (P < 0.05).

Table 7. Mean percentage of linolenic acid of four bermudagrasses
as a function of sampling date.

                                       Sampling date

                         Fall    Winter    Fall    Winter    Summer
Cultivar                 2001     2002     2002     2003      2003

Linolenic acid (Percentage of total)

Midiron                  17.4     12.8     13.6     18.7      18.1
Riviera                  14.0     12.2      9.9     14.2      15.7
Tifway                   12.8     10.6      7.0     21.1      11.7
Princess-77              15.4     13.4      9.3     12.2      14.8
LSD ([double dagger])     2.5      2.5      2.5      2.5       2.5

             Sampling date

Cultivar                 LSD ([dagger])

Linolenic acid (Percentage of total)

Midiron                       2.5
Riviera                       2.5
Tifway                        2.5
Princess-77                   2.5
LSD ([double dagger])         2.5

([dagger]) LSD = comparison of date within cultivar.

([double dagger]) LSD = comparison of cultivar within date.

Table 8. Mean proline concentration of four bermudagrass cultivars
as a function of chemical treatment and sampling date.

                         Chemical treatment

                                                   SWE
Sampling date    Cultivar               N       ([dagger])      Fe

             Proline concentration ([micro]g [g.sup.-1])

Fall 2001        Midiron               168.4      1438.8      1224.1
                 Riviera               456.9       342.5       220.4
                 Tifway                565.1       934.0       413.6
                 Princess-77           243.0       541.5       298.4
                 LSD ([paragraph])     362.2       362.2       362.2
Winter 2002      Midiron               416.5       402.2      1521.0
                 Riviera               103.7       323.4        54.3
                 Tifway                 47.9        49.0       360.5
                 Princess-77            82.2        85.6       116.1
                 LSD ([paragraph])     362.2        ns         362.2
Fall 2002        Midiron               591.7       624.9       524.8
                 Riviera               286.0       381.2       411.8
                 Tifway                141.3       160.5       146.4
                 Princess-77           146.4        76.1       140.8
                 LSD ([paragraph])     362.2       362.2       362.2
Winter 2003      Midiron               325.2       504.6       878.0
                 Riviera              1303.7       139.8       404.3
                 Tifway                451.8       122.5       340.9
                 Princess-77           155.1       364.8       175.0
                 LSD ([paragraph])     362.2       362.2       362.2
Summer 2003      Midiron               105.0        66.0        82.2
                 Riviera                67.0        72.3        62.5
                 Tifway                 96.5        71.3        97.8
                 Princess-77            71.6       132.1        92.5
                 LSD ([paragraph])      ns          ns          ns
Temporal         Midiron               362.2       362.2       362.2
LSD (#)          Riviera               362.2        ns          ns
                 Tifway                362.2       362.2        ns
                 Princess-77            ns         362.2        ns

                          Chemical treatment

                                             NT                LSD
Sampling date    Cultivar             ([double dagger])    ([section])

              Proline concentration ([micro]g [g.sup.-1])

Fall 2001        Midiron                    693.3             362.2
                 Riviera                    550.5              ns
                 Tifway                     219.4              ns
                 Princess-77                183.8              ns
                 LSD ([paragraph])          362.2              --
Winter 2002      Midiron                    167.9             362.2
                 Riviera                     43.2              ns
                 Tifway                      36.3              ns
                 Princess-77                 19.3              ns
                 LSD ([paragraph])           ns                --
Fall 2002        Midiron                    623.3              ns
                 Riviera                    444.4              ns
                 Tifway                     222.3              ns
                 Princess-77                 73.4              ns
                 LSD ([paragraph])          362.2              --
Winter 2003      Midiron                   1204.2             362.2
                 Riviera                    652.5             362.2
                 Tifway                     214.3              ns
                 Princess-77                136.6              ns
                 LSD ([paragraph])          362.2              --
Summer 2003      Midiron                     91.2              ns
                 Riviera                     73.9              ns
                 Tifway                      97.8              ns
                 Princess-77                 89.9              ns
                 LSD ([paragraph])           ns                --
Temporal         Midiron                    362.2
LSD (#)          Riviera                    362.2
                 Tifway                      ns
                 Princess-77                 ns

([dagger]) SWE, seaweed extract.

([double dagger]) Non-treated control.

([section]) LSD = Comparison of chemical treatment within cultivar and
sampling date.

([paragraph]) LSD = Comparison of cultivar within chemical treatment
and sampling date.

(#) LSD = Comparison of cultivars and chemical treatments across all
dates.

Table 9. Mean percentage of greenup of four bermudagrasses
as a function of rating date.

                                       Observation date

Year    Cultivar                 28 April    15 May     1 June

Greenup (%)

2002    Midirou                   65.3 *     100.0 *    100.0
        Riviera                   72.5 *     100.0 *    100.0
        Tifway                    45.0 *      74.1 *     95.6 *
        Princess-77                9.9 *      24.8 *     56.3 *
        LSD ([double dagger])     10.4        10.4       10.4
2003    Midiron                   41.6        89.4       95.9
        Riviera                   58.1        87.0       95.0
        Tifway                     9.4        49.6       63.1
        Princess-77                0.2        12.5       14.8
        LSD ([double dagger])     10.4        10.4       10.4

                                 Observation date

Year    Cultivar                  LSD ([dagger])

Greenup (%)

2002    Midirou                        4.1
        Riviera                        4.1
        Tifway                         4.1
        Princess-77                    4.1
        LSD ([double dagger])           --
2003    Midiron                        4.1
        Riviera                        4.1
        Tifway                         4.1
        Princess-77                    4.1
        LSD ([double dagger])           --

* Values followed by an asterisk are significantly higher within
cultivar and between years (P < 0.05).

([dagger]) LSD = comparison of observation date within cultivar and
year.

([double dagger]) LSD = comparison of cultivar within observation date
and year.

Table 10. Mean percentage of greenup of four bermudagrasses
as a function of chemical treatment and rating date.

                                       Observation date

Year    Treatment                28 April    15 May    1 June

                        Greenup (%)

2002    N                         47.4 *     73.5 *    85.0 *
        SWE ([double dagger])     49.6 *     75.0 *    87.8 *
        Fe                        47.4 *     75.6 *    88.4 *
        NT ([section])            48.3 *     74.7 *    90.6 *
        LSD ([paragrapgh])          ns         ns       5.2
2003    N                         26.3       60.0      66.8
        SWE                       27.0       57.8      65.8
        Fe                        30.5       60.6      69.1
        NT                        25.5       60.1      67.2
        LSD ([paragrapgh])          ns         ns        ns

Year    Treatment                LSD ([double dagger])

                      Greenup (%)

2002    N                                 4.1
        SWE ([double dagger])             4.1
        Fe                                4.1
        NT ([section])                    4.1
        LSD ([paragrapgh])                --
2003    N                                 4.1
        SWE                               4.1
        Fe                                4.1
        NT                                4.1
        LSD ([paragrapgh])

* Values followed by an asterisk are significantly higher within
chemical treatments and between years (P < 0.05).

([dagger]) LSD = comparison of observation date within chemical
treatment and year.

([double dagger]) SWE, seaweed extract.

([section]) Non-treated control.

([paragraph]) LSD = comparison of chemical treatment within observation
date and year.
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Article Details
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Author:Munshaw, G.C.; Ervin, E.H.; Shang, C.; Askew, S.D.; Zhang, X.; Lemus, R.W.
Publication:Crop Science
Geographic Code:1USA
Date:Jan 1, 2006
Words:11248
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