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Untimely fall harvest affects dry matter yield and root organic reserves in field-grown alfalfa.

THE RECOMMENDATION of not harvesting alfalfa during a critical fall rest period based on calendar dates is often questioned, as the regrowth interval between the last two harvests appears to be a greater determinant of winter survival and spring regrowth than calendar dates (Sheaffer et al., 1986). The accumulation of GDD > 5[degrees]C after the last summer harvest has been proposed as a criterion to estimate the duration of this interval (Belanger et al., 1992). In Atlantic Canada, a minimum interval of 500 GDD between the two last harvests was required to maintain DMY across several years (Belanger et al., 1992, 1999). We also recently documented that, under unheated greenhouse conditions, alfalfa regrowth potential was reduced by a third harvest in the fall, taken at 400 or 500 GDD, as compared with plants harvested only twice in the summer (Dhont et al., 2002).

In spite of the fact that fall harvests often reduce alfalfa persistence and yields, the underlying causes remain unclear. Available field data concerning the effects of fall harvests on organic reserves in taproots and their relationship with persistence and yields remain controversial, and solely take into account the concentrations of carbohydrate reserves (Reynolds, 1971; Edminsten et al., 1988). We have recently reported, using plants grown under environmentally controlled conditions and exposed to simulated winter conditions in an unheated greenhouse, that fall harvests did not consistently affect starch or total nonstructural carbohydrate (TNC) concentrations in alfalfa roots (Dhont et al., 2002). However, the amounts of root carbohydrate reserves were significantly reduced by fall harvests, and they were even more closely related to alfalfa regrowth potential than their concentrations.

Furthermore, the close relationship between root N reserves such as amino acids and vegetative storage proteins (VSPs) and alfalfa regrowth, outlined across the past 10 yr (Hendershot and Volenec, 1993; Volenec et al., 1996; Avice et al., 1997), brought a new perspective to the understanding of the regrowth response of alfalfa submitted to fall harvest management. With plants acclimated to simulated winter conditions in an unheated greenhouse, we recently documented a marked reduction of N reserves in roots of alfalfa harvested in the fall with a positive correlation between both concentrations and amounts of N reserves and spring regrowth of alfalfa (Dhont et al., 2003).

Alfalfa persistence is not only affected by the levels of root organic reserves available for spring regrowth, but also depends on the capacity of the plant to cold acclimate and to maintain its freezing tolerance during fall and winter (Paquin, 1985). The increase in soluble sugars (sucrose, raffinose, and stachyose) observed during fall acclimation has been related to the acquisition of freezing tolerance (Castonguay et al., 1998). Additionally, the accumulation of N reserves (amino acids and cold-induced proteins) in taproots of alfalfa during fall acclimation was also shown to be associated to the acquisition of cold tolerance (McKenzie et al., 1988). In that perspective, an untimely fall harvest could have a significant impact not only on the vigor of spring regrowth, but also on the cold tolerance of the plant.

The effect of a fall harvest on field-grown alfalfa in relation to root carbohydrate and N reserves remains to be determined. Our objective was to characterize root organic reserve accumulation and alfalfa yield response to a fall harvest management based on GDD, in search of a compromise between productivity and longevity of alfalfa stands. We further document changes in DMY and root pools (i.e., amounts) of RFO, sucrose, starch, total amino acids, and soluble proteins of field-grown alfalfa submitted to various GDD regrowth intervals between the last summer harvest and a third harvest in the fall.

MATERIALS AND METHODS

Plant Material

The experiment was conducted from 1996 to 1999 at two sites located in different agroclimatic regions in eastern Canada: Normandin (Labare silty clay; 48[degrees]50' N, 72[degrees]32' W; 1333 GDD, basis 5[degrees]C); Pintendre (Kamouraska clay loam; 46[degrees]45' N, 71[degrees]08' W; 1653 GDD, basis 5[degrees]C). Seeds of alfalfa cultivars AC Caribou and WL 225, recommended for production in Quebec, Canada, were seeded at 12 kg [ha.sup.-1] on 27 May 1996 at Normandin (plot size, 7.5 by 7 m), and on 5 Aug. 1996 at Pintendre (plot size, 7 by 7 m). The choice of the cultivars was based on observations of contrasting persistence under the harsh winter conditions of eastern Canada, with AC Caribou being hardier than WL 225 (R. Michaud, 1996, personal communication). Seeds were inoculated with a commercial inoculant (Liphatec Inc., Milwaukee, WI) of Sinorhizobium meliloti (Zakhia and de Lajudie, 2001). Fertilizers were applied at the time of seeding at both sites (in kg [ha.sup.-1], N: 28, P: 48, K: 46 at Pintendre; N: 22, P: 39, K: 74, B: 9 at Normandin), after the first or the second harvest at Pintendre (in kg [ha.sup.-1], P: 15, K: 56 on 6 Aug. 1997: P: 9, K: 41, B: 3, Mg: 4 on 23 June 1998; P: 25, K: 21 on 5 Aug. 1998), and after each harvest at Normandin (in kg [ha.sup.-1], P: 16, K: 62, B: 5: Table 1). At Pintendre, weed control was achieved by applying Gramoxone (paraquat, 1,l'-dimethyl-4,4'-bipyridinium ion) after the second harvest in 1997 (4 L [ha.sup.-1]; 8 Aug. 1998) and after the first harvest in 1998 (5 L [ha.sup.-1]; 25 June 1998). At Normandin, weed control was achieved by applying Cobutox [4-(2,4-dichlorophenoxy)butyric acid] during the establishment year (2 L [ha.sup.-1], 18 June 1996, 7 July 1996) and after the first and second harvests in 1997 (2 L [ha.sup.-1], 27 June 1997, 31 July 1997), and Gramoxone after the first harvest in 1998 (4 L [ha.sup.-1], 26 June 1998). At Pintendre, snow fences were installed in the falls of 1996 and 1997 to trap snow and ensure sufficient snow cover. Soil temperature at a depth of 2.5 cm was monitored every 30 min by stand-alone dataloggers (RD Temp XT dataloggers with external temperature probe, Omega, Stamford, CT, USA) at each site and in each treatment (four dataloggers per site, i.e., one datalogger per harvest treatment) throughout the three overwintering periods. Daily average temperatures were calculated and used for graphical display of seasonal fluctuations (Fig. 1).

[FIGURE 1 OMITTED]

Fall Harvesting Treatments and Dry Matter Yield

During the year of establishment, no harvest was taken at Pintendre, whereas one summer harvest was taken at Normandin (22 July 1996). During the production years, the first two harvests were taken at the early flowering stage (Table 1). Stubble height after each harvest was [approximately equal to] 7 cm. Four harvest treatments were applied in the falls of 1997 and 1998: no additional harvest (two-harvest system), or a third harvest taken either at 400, 500, or 600 GDD cumulated after the second summer harvest (Table 1). The GDD were calculated by subtracting 5[degrees]C from the average of daily maximum and minimum air temperatures (Belanger et al., 1992). Maximum and minimum temperatures were measured at a nearby weather station and accessed daily through an Environment Canada online service.

At each harvest date, two 1-m wide strips of 7 m in length were harvested from each plot to determine DMY. A subsample of [approximately equal to] 500 g fresh weight (FW) was oven dried at 55[degrees]C until constant weight for determination of percentage dry matter (DM). Plant density was estimated in the falls of 1997 and 1998, and in the springs of 1998 and 1999 by counting the number of live plants in two randomly selected quadrats (0.09 [m.sup.2]) in each plot.

Tissue Sampling

Whole plants (approximately the first 20-cm depth of taproots, and the remaining shoots) were sampled in a randomly selected quadrat (0.09 [m.sup.2]) from each plot in fall 1997, spring and fall 1998, and in spring 1999 (Table 1). At each sampling date, plants were washed free of soil under a stream of cold water. Remaining shoots were removed, and crowns (transition zone between shoots and roots) were separated from roots. The entire 20 cm of the roots were fresh weighed for subsequent root DW determination, and then cut into small segments ([approximately equal to] 4 to 5 mm). A 1-g FW subsample was immediately incubated in 8 mL of methanol-chloroform water (MCW) (12:5:3, v:v:v) at 65[degrees]C for 30 min to inhibit enzymatic activities, and was stored at -20[degrees]C until sugar and amino acid analysis. A 5-g FW subsample was stored at -40[degrees]C until soluble protein analysis. A 5- to 10-g FW subsample was oven dried for 48 h at 55[degrees]C to determine the percentage of DM of the roots. Root DW was calculated by multiplying the percentage of DM by the FW of the roots. Because the number of plants per quadrat varied, the data are presented on a per-plant basis.

Carbohydrate and Amino Acid Determinations

Extractions of soluble sugars and amino acids were performed in 15 mL of MCW (12:5:3, v:v:v) as described previously in Dhont et al. (2002, 2003). A 1-mL subsample of the aqueous phase resulting from phase separation with water and chloroform was evaporated to dryness on a rotary evaporator, then solubilized in ethylene-diaminetetraacetic acid (EDTA; [Na.sup.+], [Ca.sup.2+], 50 mg [L.sup.-1]), and kept frozen at -40[degrees]C until soluble sugar analysis. The remaining aqueous phase was collected and kept frozen at -40[degrees]C until amino acid analysis.

Soluble sugars consisted of the RFO (i.e., raffinose and stachyose), and of sucrose that were separated and quantified by high performance liquid chromatography (HPLC; Dhont et al., 2002). Starch was quantified spectrophotometrically with the method of Blakeney and Mutton (1980) with the nonsoluble residues left after soluble sugar and amino acid extractions. Nineteen amino acids (aspartatic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, [gamma]-amino butyric acid, [alpha]-amino butyric acid, methionine, valine, phenylalanine, leucine, isoleucine, and lysine) were separated and quantified by HPLC, while proline was analyzed by spectrophotometry (Dhont et al., 2003). Total amino acids consisted of the sum of the 20 individual amino acids.

Soluble Protein Determinations

At each sampling date, 20 g FW of root tissue was used for each cultivar x harvest treatment combination, by pooling 5-g FW subsamples from each replicate. A subsample of [approximately equal to] 2-g FW of pooled root tissue were used for soluble protein extraction and analysis as described in Dhont et al. (2003). Total soluble proteins were determined according to the method of Lowry et al. (1951).

Data Analysis

Results from carbohydrate and N reserves were initially calculated on a concentration basis. These values were subsequently expressed on a pool (i.e., amount) basis by multiplying the concentration values by the root DW per plant. Since soluble protein determination was performed on pooled samples, no replicate was available to perform an analysis of variance. In spring 1999, at Normandin, no measurements of DMY nor biochemical determinations were made in plots harvested three times due to high mortality in these plots.

The experiment at each site was a factorial combination of the two alfalfa cultivars and four harvest treatments arranged in a randomized complete block design, with four replications for a total of 32 plots per site. Separate analyses of variance were done on data from each sampling date. A priori contrasts were used for comparison of harvest and cultivar x harvest means at each site. Standard errors of the mean (SEM) were calculated for each sampling date. Statistical significance was postulated at P < 0.05. Statistical analyses were performed by SAS statistical procedures (SAS Institute, 1996).

RESULTS

Soil Temperatures

During the 1997-1998 winter, soil temperatures were generally lower at Normandin (-5 < T < -2[degrees]C) than those measured at Pintendre (-2 < T < -1[degrees]C) (Fig. 1A). Fluctuations in soil temperatures were also more pronounced at Normandin, with several peaks at -7 or -10[degrees]C at the end of December 1997 and early January 1998. At both sites, soil temperatures recorded in plots harvested twice were generally higher than those from plots harvested three times.

Variations in soil temperatures at Pintendre in 1998 to 1999 were comparable with those observed in 1997 to 1998 (Fig. 1B). However, at Normandin, there were noticeable differences between the two winter seasons. In winter 1997-1998, soil temperatures in plots harvested twice during summer were very close to those of plots harvested a third time in the fall. Conversely, during the 1998-1999 winter, soil temperatures remained near the freezing point in the plots harvested only twice, whereas they dropped to much lower temperatures in plots harvested a third time in the fall, with prolonged periods between -8 and -12[degrees]C.

Dry Matter Yield and Plant Densities

Fall harvest treatments were applied for the first time during the first production year in 1997, and seasonal DMY of 1997 resulted from the sum of the DMY of two or three harvests. At both sites, the three-harvest systems produced significantly higher seasonal DMY in 1997 than the two-harvest one (Tables 2 and 3). At Pintendre, alfalfa harvested three times at 400 or 600 GDD produced [approximately equal to] 1.2 or 1.8 Mg [ha.sup.-1] more DMY, respectively, than alfalfa harvested only twice during summer 1997 (Table 2). At Normandin, the gain of DMY with the third harvest in 1997 was [approximately equal to] 1.9 Mg [ha.sup.-1] (Table 3).

At both sites and for both cultivars, a third harvest taken at 400 or 500 GDD in fall 1997 significantly reduced the DMY of the first harvest in the following spring (1998) by 0.32 to 2.02 Mg [ha.sup.-1] as compared with plots harvested only twice during summer 1997 (Tables 2 and 3). Fall harvest treatments appear to have a lesser effect at Pintendre than at Normandin, as indicated by the absence of a difference in DMY between WL 225 harvested at 400 GDD and WL 225 harvested twice at Pintendre (Table 2; two harvests x cultivars interaction). Harvesting alfalfa a third time at 600 GDD in fall 1997 had no significant effect on first harvest DMY of spring 1998 (Tables 2 and 3). At Pintendre, the DMY of the second harvest in spring 1998 was not affected by fall harvests taken in 1997 (Table 2). However, at Normandin, harvesting alfalfa a third time in fall 1997 at 400 or 500 GDD reduced the second harvest DMY of spring 1998 (Table 3).

Seasonal DMY of 1998 resulted from harvest treatments in summer and fall 1998, but also reflected the effects of the first application of the harvest treatments in the previous fall (1997). At both sites, seasonal DMY of 1998 was significantly higher in plots harvested a third time at 500 or 600 GDD in fall 1997, by [approximately equal to] 1.6 to 3.5 Mg [ha.sup.-1] as compared with plots harvested only twice. At Pintendre, seasonal DMY of 1998 of AC Caribou harvested at 400 GDD in fall 1997 did not significantly differ from that of alfalfa harvested twice, whereas WL 225 harvested at 400 GDD produced significantly higher seasonal DMY than WL 225 harvested twice (Table 2). At Normandin, harvesting a third time at 400 GDD reduced seasonal DMY of 1998 for both cultivars as compared with plots harvested only twice (Table 3).

The DMY of the first harvest in 1999 reflected the cumulative effects of the harvest treatments applied previously in the falls of 1997 and 1998. At Pintendre, the DMY in spring 1999 was reduced by a third harvest taken in previous falls, except for AC Caribou harvested at 500 GDD, which did not differ from plots harvested twice (Table 2). At Normandin, no DMY was measured in spring 1999 in plots harvested a third time in previous falls (Table 3) since plant mortality was near 100% for these treatments (Table 4). Only plots harvested twice in 1997 and 1998 survived during the 1998-1999 winter.

At both sites, plant density was not significantly affected by fall harvest treatments, with the exception of the winterkill damages observed in spring 1999 in plots harvested three times at Normandin (Table 4). Plant density declined from fall 1997 to spring 1999, and this decline was more rapid and more pronounced at the northern site of Normandin. At Pintendre, plant density was almost stable until spring 1998 at [approximately equal to] 260 to 290 plants [m.sup.-2]. Plant density declined during the summer and the winter of 1998, reaching about 180 plants [m.sup.-2] in fall 1998, and 120 plants [m.sup.-2] in spring 1999. At Normandin, plant density started to decline during the winter 1997 ([approximately equal to] 215 plants [m.sup.-2] in fall 1997 vs. 160 plants [m.sup.-2] in spring 1998), and continued to decrease to reach 100 plants [m.sup.-2] in fall 1998 (Table 4). No difference between cultivars was observed in the reduction of plant density.

Root Dry Weight

Root DW increased markedly during the second production year (Fig. 2). At Pintendre, root DW of alfalfa harvested twice increased from 0.85 g per plant (200 g [m.sup.-2]) in fall 1997 to 1.90 g per plant (360 g [m.sup.-2]) in fall 1998 (Fig. 2A,B). At Normandin, root DW of alfalfa harvested twice increased by [approximately equal to] 0.95 g per plant (180 g [m.sup.-2]) from fall 1997, reaching [approximately equal to] 3.30 g per plant (325 g [m.sup.-2]) in fall 1998 (Fig. 2C,D).

[FIGURE 2 OMITTED]

For both cultivars at Pintendre, a third harvest taken at 400, 500, or 600 GDD in 1997 significantly reduced root DW in late fall (November 1997) as compared with plants harvested only twice in the summer (Fig. 2A,B; Table 5). In the following spring (April 1998), root DW was significantly reduced only by fall harvests taken at 400 or 500 GDD. A repeated application of the 400 GDD harvest in September 1998 reduced root DW in November (Fig. 2A,B; Table 5). A repeated fall harvest taken at 500 or 600 GDD in 1998 had no significant effect on subsequent root DW.

At Normandin, root DW was significantly higher in AC Caribou than in WL 225 in November 1997 (Fig. 2C,D; Table 5). For both cultivars, root DW was reduced in November 1997 and April 1998 in plants harvested a third time at 400 GDD in September 1997 as compared with plants harvested only twice. Harvesting at 500 or 600 GDD in fall 1997 did not significantly affect root DW measured in November 1997 or in the following spring (April 1998). Even though a repeated application of the harvest treatment in fall 1998 tended to reduce root DW later in November 1998, there was no significant effect of harvest treatments on root DW (Fig. 2C,D; Table 5).

Pools of Root Organic Reserves

As a result of the marked increase in root DW at both sites, pools of RFO, sucrose, starch, total amino acids, and total soluble proteins increased with alfalfa aging from the first production year (1997) to the second one (1998) (Fig. 3 to 7). The greatest increase was observed for amounts of starch (235 mg per plant in November 1997 vs. 1110 mg per plant in April 1998 in alfalfa harvested twice at Normandin, Fig. 5C,D) because of a concomitant increase in starch concentrations (data not shown).

[FIGURES 3-7 OMITTED]

Raffinose Family Oligosaccharides

At both sites, pools of RFO were significantly higher in AC Caribou than in WL 225 in November 1997 (Fig. 3; Table 5). At all other sampling dates, there was no significant difference between the two cultivars. In November 1997 at Pintendre, pools of RFO were reduced by a previous harvest at 400 or 500 GDD in September 1997 as compared with alfalfa harvested twice or a third time at 600 GDD (Fig. 3A,B; Table 5). In the following spring, pools of RFO were reduced by fall harvest regardless of its timing as compared with alfalfa harvested twice. A repeated application of the fall harvest treatments in 1998 reduced RFO pools in plants sampled in November only when the fall harvest was taken at 400 GDD, but did not affect these pools in the following spring (Fig. 3A,B; Table 5).

At Normandin, RFO pools measured in November 1997 were lower in plants harvested a third time at 400 GDD as compared with plants harvested twice (Fig. 3C,D; Table 5). Harvesting at 500 or 600 GDD in fall 1997 did not significantly affect these pools in November 1997. In the following year (April and November 1998), RFO pools were not significantly affected by fall harvests (Fig. 3; Table 5).

Sucrose

In November 1997 and April 1998 at Pintendre, pools of sucrose were reduced by harvesting at 400 or 500 GDD in September 1997 as compared with plants harvested twice or a third time at 600 GDD (Fig. 4A,B; Table 5). In November 1998, pools of sucrose were reduced by a repeated application of the 400 GDD harvest in the fall 1998, the 500 or 600 GDD harvest treatments having no significant effect. Harvest treatments taken in fall 1998 did not significantly affect pools of sucrose the following spring (April 1999) (Fig. 4A,B; Table 5).

At Normandin, pools of sucrose measured in November 1997 and April 1998 were reduced in plants harvested a third time at 400 GDD when compared with those harvested only twice (Fig. 4C,D; Table 5). Harvesting at 500 or 600 GDD in fall 1997 had no significant effect on these pools in November 1997 or in April 1998. Repeated fall harvest treatments in fall 1998 did not significantly affect pools of sucrose assessed in November (Fig. 4C,D; Table 5).

Starch

At Pintendre, harvesting a third time at 400 or 500 GDD in September 1997 strongly reduced pools of starch in November 1997 and April 1998 as compared with plants harvested twice (Fig. 5A,B; Table 5). Harvesting at 600 GDD in October 1997 did not affect starch amounts in November but significantly reduced them in April 1998. In spite of the observed tendency for a reduction of starch pools with a new application of harvest treatments in fall 1998, only the 400 GDD treatment significantly affected starch pools in November 1998. A third harvest in fall 1998, regardless of its timing, markedly reduced the amounts of starch remaining in roots of alfalfa in the following spring (Fig. 5A,B; Table 5).

At Normandin, the fall harvests taken in fall 1997, regardless of their timing, significantly reduced starch pools measured in November 1997 and April 1998, with a lesser effect observed when the third harvest was delayed until 600 GDD (Fig. 5C,D; Table 5). In November 1998, pools of starch were significantly reduced by a fall harvest only when taken at 500 GDD in September 1998.

Total Amino Acids

At Pintendre, harvesting a third time at 400 or 500 GDD in September 1997 significantly reduced pools of total amino acids in November as compared with plants harvested only twice (Fig. 6A,B; Table 5). In April and November 1998, total amino acid pools were reduced by the 400-GDD harvest treatment in both cultivars. These pools were also reduced in April 1998 by the 500and 600-GDD harvest treatments in AC Caribou only [Fig. 6A,B; Cultivars x (2 harvests vs. 500) and Cultivars x (2 harvests vs. 600), Table 5]. In spring 1999, pools of total amino acids were reduced by harvesting at 400 or 500 GDD during the previous fall (Fig 6A,B; Table 5).

At Normandin, a third harvest taken either at 400, 500, or 600 GDD in fall 1997 significantly reduced the pools of total amino acids in November 1997 as compared with plants harvested only twice in the summer (Fig. 6C,D; Table 5). In the following spring (April 1998), these pools were still lower in plants harvested at 400 or 500 GDD than in plants harvested twice. A new application of the harvest treatments in the fall 1998 did not significantly affect total amino acid pools in root samples in November 1998 (Fig. 6C,D; Table 5).

Total Soluble Proteins

At Pintendre in both cultivars, the fall harvests generally reduced pools of soluble proteins when compared with those of plants harvested only twice, with lesser or no effect with the longer regrowth interval between the second and the third harvests (600 GDD) (Fig. 7). In spring 1999, this tendency was not observed in AC Caribou since pools of soluble proteins were strongly reduced by the fall harvest, regardless of the timing. The reduction in the pools of soluble protein by the fall harvests was less pronounced at Normandin, and mostly occurred in the plants harvested at 400 GDD (Fig. 7).

DISCUSSION

Agroclimatic Factors, Longevity, and Productivity

The impact of fall harvest treatments on productivity and longevity of field-grown alfalfa was assessed in two different agroclimatic locations in eastern Canada. Soil temperatures measured at the two experimental sites were lower than those observed in our previous study conducted under unheated greenhouse conditions (Dhont et al., 2002), especially at the northern site of Normandin (Fig. 1). Furthermore, soil temperatures were often lower in plots harvested a third time in the fall. Remaining leaves and shoots in plots harvested twice likely allowed snow entrapment and the development of an insulating cover, maintaining soil temperatures near the freezing point throughout the winter. Leep et al. (2001) recently reported that a 10-cm snow cover provides adequate insulation at the plant level (1 cm) and in the underlying soil layers, protecting alfalfa from cold injury. Conversely, shoot removal in plants harvested in the fall led to prolonged exposure to subfreezing temperatures, especially at Normandin during the winter of 1998-1999 (-7 to -12[degrees]C; Fig. 1).

The impact of lower soil temperatures at the northern site (Normandin) in plots harvested in the fall led to a total destruction of alfalfa stand following the winter of 1998-1999. However, survival and plant density were not affected by the fall harvest treatments at Pintendre, where soil temperatures remained near freezing (Table 4). This is in accordance with the results of Sheaffer et al. (1986) in Minnesota, who did not observe winter damage associated with fall harvest management when alfalfa was not exposed to harsh winter conditions. In Atlantic Canada, Belanger et al. (1999) reported that depending on the locations and thus the severity of winter, an additional harvest taken in the fall could reduce plant density. Therefore, under severe winter conditions, alfalfa submitted to a fall harvest could be more sensitive to winter injury. The incidence of winter plant mortality in winter may be attributable to the synergistic effects of exposure to freezing temperatures and to the impact of fall harvests.

Plant density reported in our study ranged between 96 and 145 plants [m.sup.-2] (Table 4) in the spring of the third production year (1999), which is close to values generally reported in the literature (Gervais and Bilodeau 1985; Sheaffer et al., 1986; Belanger et al., 1992). According to previous reports, plant density declined with stand aging (Belanger et al., 1992). This decline in plant density occurred earlier at the northern location of Normandin than at Pintendre. In Atlantic Canada, Belanger et al. (1999) also observed that the decrease in plant density occurred more rapidly at locations with more severe winters because of extensive winterkill. Differences in the evolution of plant density during winter between the two sites were likely attributable to lower soil temperatures at the northern location (Normandin).

In spite of the decrease in plant density, DMY per hectare within harvest treatments remained stable across the three production years (Tables 2 and 3). Belanger et al. (1992) also observed a stability in DMY, even when alfalfa plant density declined with time. Loss in the number of plants has been shown to be compensated by an increase in the number of stems per plant (Gosse et al., 1988). In fact, although the DMY per hectare was stable, the DMY per plant markedly increased (e.g., 2.1 g per plant in spring 1998 vs. 4.25 g per plant in spring 1999 at Normandin, and 1.4 g per plant in spring 1998 vs. 3.1 g per plant in spring 1999 at Pintendre; data not shown). Therefore, the decline in plant density observed during the summer 1998 was compensated by an increase in root DW and amounts of organic reserves per plant.

The DMY (Mg [ha.sup.-1]) of the first harvest of 1998 was strongly reduced by a fall harvest taken at 400 or 500 GDD, but was not affected by a fall harvest taken at 600 GDD. In the third year of production (1999), DMY of the first harvest was reduced by all fall harvest treatments (Tables 2 and 3). This confirms our previous observations under unheated greenhouse conditions, which indicated the adverse effect of a fall harvest taken at 400 or 500 GDD on alfalfa regrowth in the following spring (Dhont et al., 20(12). Similar results were reported by Belanger et al. (1999) from a field study conducted in Atlantic Canada. Although there was, in most cases, a clear benefit on seasonal DMY by harvesting alfalfa a third time in the fall, there were instances such as the 400 GDD treatment at Normandin in 1998, where seasonal DMY of plants harvested three times were significantly lower than that of plots harvested only twice. Our results agree with those of Gervais and Bilodeau (1985), who reported reduced DMY with the earliness of the fall harvest, and with those of Gervais and Girard (1987), who observed higher DMY when fall harvest was delayed to October. Belanger et al. (1999) also reported that seasonal DMY was higher under the three-harvest system, and increased with a lengthening of interval between the summer and the fall harvests from 400 to 600 GDD; this seasonal yield benefit, however, was reduced across time. Our results confirm that there can be some yield advantages of taking an additional third harvest if it is taken at least 500 GDD after the last summer harvest. However, yield benefits from a fall harvest can be relatively short term, considering the potential negative impacts on stand persistence and regrowth potential.

Root Dry Weight and Organic Reserves

The impact of harvest management and agroclimatic factors on root organic reserves of alfalfa remain largely controversial, especially since field studies historically considered concentrations of TNC as an index of the status of organic reserves. Also, reports in the literature concerning the effect of fall harvests on alfalfa root DW across many growing seasons are very scarce. Our study reassesses the impact of fall harvest management on alfalfa by looking at the root DW and by assessing the amounts of various components of the root carbohydrate and N reserves available for regrowth. The values of root DW reported in the current study and the significant increase in root DW in the second year of production (Fig. 2) are comparable with those recently reported by Bolinder et al. (2002). These authors observed that root DW increase was coming from the uppermost 15-cm soil layer. Therefore, the sampling of the first 20 cm of roots in our study allowed us to obtain a representative sample of root organic reserves mobilizable for alfalfa regrowth. In our study, alfalfa root DW was reduced by fall harvests, especially when the third harvest was taken early in the fall (400 GDD). These results confirmed observations in our previous study conducted under unheated greenhouse conditions where alfalfa root DW was markedly reduced by fall harvests (Dhont et al., 2002).

The range of concentrations of RFO, sucrose, total amino acids, and total soluble proteins remained relatively stable throughout the experiment (data not shown). Consequently, the large increase of the pools of these components in the second year of production was mainly the result of the increase in root DW. Contrastingly, the marked increase in pools of starch was not only due to the changes in root DW, but also reflected a doubling in starch concentrations (data not shown). Concentrations (data not shown) of RFO (5 to 15 mg [g.sup.-1] DW) and sucrose (70 to 150 mg [g.sup.-1] DW) were not affected by fall harvests. Higher accumulations of RFO in crowns of alfalfa have been shown to occur in cultivars of superior winter hardiness (Castonguay et al., 1995). Even though the cultivar response to fall harvest treatments was similar, RFO accumulated to higher levels in roots of the hardier cultivar AC Caribou than in WL 225 in fall 1997. Dhont et al. (2002) previously reported an increase in soluble sugar concentrations but a decrease in their amounts in response to fall harvests in alfalfa acclimated under simulated winter conditions. Thus, field observations support our previous conclusion that fall harvests are not likely to impede cryoprotective mechanisms based on the accumulation of soluble sugars (Castonguay et al., 1998).

In agreement with our recent observations made under simulated winter conditions (Dhont et al., 2002, 2003), we observed that pools of starch, total amino acids, and total soluble proteins were generally reduced by fall harvests (Fig. 5, 6, and 7), especially when taken early at 400 GDD. These pools also tended to increase with a delay of fall harvest from 400 to 600 GDD. The impact of fall harvests on pools of organic reserves was less severe at the northern site of Normandin during the second year of production as compared with the first one. Belanger et al. (1999) suggested that the severity of winter could have a determinant impact on alfalfa persistence, superseding the effect of harvest itself. Previous field studies have indicated that a lengthening of the harvest interval or delaying the last harvest in the fall until October allowed starch to accumulate to levels (concentration basis) similar to that of plants harvested only twice (Gervais and Bilodeau, 1985; Sheaffer et al., 1986; Gervais and Girard, 1987; Brink and Marten, 1989). Even though the relationship between starch concentrations and alfalfa regrowth remains unclear (Habben and Volenec, 1991; Boyce and Volenec, 1992), the conversion of starch into soluble sugars during fall acclimation is part of the cold hardening process (Volenec et al., 1991). Furthermore, the positive correlation between amounts of starch and alfalfa spring regrowth (Dhont et al., 2002), as well as the impact of fall harvest on starch amounts, suggest that this carbohydrate-reserve component might be determinant for alfalfa persistence and spring regrowth.

There is still limited information available in the literature on the impact of a fall harvest on N reserves of alfalfa, particularly under field conditions. Amino acids such as asparagine and aspartic acid, as well as specific soluble proteins of 15, 19, 32, and 57 kDa, harboring VSP characteristics, are thought to be involved in alfalfa regrowth based on their marked mobilization during the first 7 d of regrowth (Hendershot and Volenec, 1993; Cunningham and Volenec, 1996; Avice et al., 1996, 1997). A rapidly increasing body of evidence suggests that N reserves could play an important role with regard to cold acclimation and spring regrowth. Noquet et al. (2001) pointed out a potential role of VSP in cold tolerance based on the induction of their accumulation in the fall in response to decreasing photoperiod and temperatures. We recently documented that specific amino acids (proline, arginine, and histidine) markedly increased during fall and winter (Dhont et al., 2003). The amounts of these amino acids and that of asparagine, the most abundant amino acid in roots of overwintering alfalfa, were reduced by a fall harvest (Dhont et al., 2003). It is still not known if the accumulation of these specific amino acids and VSP is associated with the acquisition of cold tolerance of field-grown alfalfa. Further investigation on the relationship between the accumulation of specific N reserves in response to fall harvest will help to elucidate their link with winter stress tolerance and their involvement in alfalfa productivity and longevity.

In conclusion, even though harvesting alfalfa a third time in the fall reduced DMY at the first harvest the following year, the impact of a fall harvest on seasonal DMY was advantageous as long as an interval of 500 or 600 GDD was kept between the last summer harvest and the fall harvest, and the winter conditions were favorable. Root growth and, therefore, the capacity to accumulate organic reserves in the roots (pools of RFO, sucrose, starch, total amino acids, and total soluble proteins), are strongly limited by a fall harvest, especially when taken at 400 GDD. A fall harvest could also prevent the development of an adequate plant-insulating snow cover and even reduce the ability of stands to overwinter under harsh winter conditions. The cumulative effect of a fall harvest in interaction with agroclimatic factors and pathogens (Couture et al., 2002) could contribute to enhance the risks of winterkill and to reduce the long-term stand persistence.

Abbreviations: DM, dry matter; DMY, dry matter yield; DW, dry weight; GDD, growing degree days; FW, fresh weight; HPLC, high performance liquid chromatography; MCW, methanol-chloroform water; RFO, raffinose family oligosaccharides: TNC, total nonstructural carbohydrate; VSP, vegetative storage protein.
Table 1. Field operations at the two experimental sites during the
three production years from 1997 to 1999. Alfalfa was harvested
either only twice during the summer, or three times with the third
harvest taken in the fall 400, 500, or 600 growing degree days (GDD)
after the second summer harvest.

                                          Production years

                                             Pintendre

                               First          Second          Third

First harvest              2 July 1997     23 June 1998    14 June 1999
Second harvest             6 Aug. 1997     5 Aug. 1998          --
Third harvest at:
  400 GDD           Date   9 Sept. 1997    10 Sept. 1998        --
    ([dagger])               (381 GDD)       (429 GDD)
                             ([double
                             dagger])
  500 GDD           Date   22 Sept. 1997   23 Sept. 1998        --
                             (495 GDD)       (534 GDD)
  600 GDD           Date   14 Oct. 1997    9 Oct. 1998          --
                             (601 GDD)       (616 GDD)
Root sampling              19 Nov. 97      19 Nov. 1998         --
                           16 Apr. 1998    21 Apr. 1999         --

                                           Production years

                                               Normandin

                               First          Second          Third

First harvest              23 July 1997    25 June 1998    21 June 1999
Second harvest             30 July 1997    30 July 1998         --
Third harvest at:
  400 GDD           Date   12 Sept. 1997   4 Sept. 1998         --
    ([dagger])               (414 GDD)       (392 GDD)
  500 GDD           Date   29 Sept. 1997   18 Sept. 1998        --
                             (498 GDD)       (495 GDD)
  600 GDD           Date   21 Oct. 1997    14 Oct. 1998         --
                             (561 GDD)       (581 GDD)
Root sampling              12 Nov. 1997    12 Nov. 1998         --
                           21 Apr. 1998    4 May 1999           --

([dagger]) Targeted intervals for the third harvest expressed
in GDD, basis 5[degrees]C.

([double dagger]) Actual intervals for the third harvest
expressed in GDD, basis 5[degrees]C.

Table 2. First and second harvest, and seasonal dry matter
yields (DMY) of two alfalfa cultivars (WL 225 and AC Caribou) at
Pintendre for the 3 yr of production, from 1997 to 1999. Alfalfa
was harvested either only twice during the summer, or three times
with the third harvest taken in the fall 400, 500, or 600 growing
degree days (GDD) after the second summer harvest. Seasonal DMY
resulted from the sum of the DMY of two or three harvests.

                                                   1997

                   Harvest
                  treatments
Cultivar          ([dagger])            First      Second   Seasonal

                                               Mg [ha.sup.-1]

WL 225       2 Harvests                 2.87        2.43      5.29
             3 Har. 400 GDD             2.71        2.46      6.49
             3 Har. 500 GDD             2.75        2.34      6.85
             3 Har. 600 GDD             2.58        2.44      6.89
AC Caribou   2 Harvests                 2.54        2.57      5.11
             3 Har. 400 GDD             2.63        2.39      6.33
             3 Har. 500 GDD             2.63        2.49      6.90
             3 Har. 600 GDD             2.88        2.54      7.14

        Contrast                         Significance probability

Cultivars                            ns ([double   0.041      ns
                                       dagger])
Harvests                                 ns          ns      <0.001
  2 harvests vs. 400                     ns          ns      <0.001
  2 harvests vs. 500                     ns          ns      <0.001
  2 harvests vs. 600                     ns          ns      <0.001
Cultivars x Harvests                    0.017        ns        ns
  Cultivars x (2 harvests vs. 400)       ns          ns        ns
  Cultivars x (2 harvests vs. 500)       ns          ns        ns
  Cultivars x (2 harvests vs. 600)      0.002        ns        ns

                                          1998

                   Harvest
                  treatments
Cultivar          ([dagger])         First    Second

                                     Mg [ha.sup.-1]

WL 225       2 Harvests               3.56     2.90
             3 Har. 400 GDD           3.40     2.96
             3 Har. 500 GDD           3.24     2.80
             3 Har. 600 GDD           3.59     3.05
AC Caribou   2 Harvests               3.93     2.71
             3 Har. 400 GDD           3.05     2.70
             3 Har. 500 GDD           3.05     2.79
             3 Har. 600 GDD           3.41     2.98

                                      Significance
        Contrast                      probability

Cultivars                              ns       ns
Harvests                              0.003     ns
  2 harvests vs. 400                  0.002     ns
  2 harvests vs. 500                 <0.001     ns
  2 harvests vs. 600                   ns       ns
Cultivars x Harvests                   ns       ns
  Cultivars x (2 harvests vs. 400)    0.025     ns
  Cultivars x (2 harvests vs. 500)     ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns

                                      1998     1999

                   Harvest
                  treatments
Cultivar          ([dagger])         Seasonal   First

                                      Mg [ha.sup.-1]

WL 225       2 Harvests                6.45      3.33
             3 Har. 400 GDD            8.07      2.94
             3 Har. 500 GDD            8.07      2.71
             3 Har. 600 GDD            8.51      2.98
AC Caribou   2 Harvests                6.64      3.24
             3 Har. 400 GDD            7.41      2.90
             3 Har. 500 GDD            8.28      3.13
             3 Har. 600 GDD            8.11      3.04

                                        Significance
        Contrast                        probability

Cultivars                               ns       ns
Harvests                              <0.001    0.006
  2 harvests vs. 400                  <0.001    0.002
  2 harvests vs. 500                  <0.001    0.002
  2 harvests vs. 600                  <0.001    0.015
Cultivars x Harvests                    ns       ns
  Cultivars x (2 harvests vs. 400)     0.038     ns
  Cultivars x (2 harvests vs. 500)      ns      0.024
  Cultivars x (2 harvests vs. 600)      ns       ns

([dagger]) Harvest treatments were: 2nd harvest (6 Aug. 1997,
5 Aug. 1998), additional harvests in the fall 400, 500, or 600
GDD after the second one, on 9 Sept., 22 Sept., or 14 Oct. 1997,
respectively, in the first year of  production, and on 10
Sept., 23 Sept., or 9 Oct. 1998, respectively, in the second
year of production.

([double dagger]) ns, not significant at P < 0.05.

Table 3. First and second harvest, and seasonal dry matter yields
(DMY) of two alfalfa cultivars (WL 225 and AC Caribou) at Normandin
for the 3 yr of production from 1997 to 1999. Alfalfa was harvested
either only twice during the summer, or three times with the third
harvest taken in the fall 400, 500, or 600 growing degree days (GDD)
after the second summer harvest. Seasonal DMY resulted from the sum
of the DMY of two or three harvests.

                                                     1997

                    Harvest
                  treatments
Cultivar          ([dagger])            First       Second   Seasonal

                                                 Mg [ha.sup.-1]

WL 225       2 Harvests                  2.73        2.47      5.20
             3 Har. 400 GDD              2.66        2.71      7.11
             3 Har. 500 GDD              2.50        2.45      6.86
             3 Har. 600 GDD              2.76        2.67      7.44
AC Caribou   2 Harvests                  2.62        2.55      5.17
             3 Har. 400 GDD              2.62        2.54      6.83
             3 Har. 500 GDD              2.75        2.37      7.03
             3 Har. 600 GDD              2.76        2.41      7.11

               Contrast                  Significance probability

Cultivars                            ns ([double      ns        ns
                                       dagger])
Harvests                                  ns          ns      <0.001
  2 harvests vs. 400                      ns          ns      <0.001
  2 harvests vs. 500                      ns          ns      <0.001
  2 harvests vs. 600                      ns          ns      <0.001
Cultivars x Harvests                      ns          ns        ns
  Cultivars x (2 harvests vs. 400)        ns          ns        ns
  Cultivars x (2 harvests vs. 500)        ns          ns        ns
  Cultivars x (2 harvests vs. 600)        ns          ns        ns

                                               1998               1999

                    Harvest
                  treatments
Cultivar          ([dagger])         First    Second   Seasonal   First

                                                Mg [h.sup.-1]

WL 225       2 Harvests                3.67    3.84      7.52     4.70
             3 Har. 400 GDD            1.86    2.82      6.79     Dead
             3 Har. 500 GDD            2.44    3.29      9.04     Dead
             3 Har. 600 GDD            3.21    3.53     10.79     Dead
AC Caribou   2 Harvests                3.87    3.89      7.76     4.61
             3 Har. 400 GDD            1.85    2.90      6.87     Dead
             3 Har. 500 GDD            3.09    3.80     10.60     Dead
             3 Har. 600 GDD            3.60    3.93     11.26     Dead

               Contrast                    Significance probability

Cultivars                              ns      0.025      ns       ns
Harvests                             <0.001   <0.001    <0.001     --
  2 harvests vs. 400                 <0.001   <0.001    <0.001     --
  2 harvests vs. 500                  0.005    0.047    <0.001     --
  2 harvests vs. 600                   ns       ns      <0.001     --
Cultivars x Harvests                   ns       ns        ns       --
  Cultivars x (2 harvests vs. 400)     ns       ns        ns       --
  Cultivars x (2 harvests vs. 500)     ns       ns        ns       --
  Cultivars x (2 harvests vs. 600)     ns       ns        ns       --

([dagger]) Harvest treatments were: 2nd harvest (30 July 1997;
30 July 1998), additional harvests in the fall 400, 500, or 600
GDD after the second one, on 12 Sept., 29 Sept., or 21 Oct. 1997,
respectively, in the first year of production, and on 4 Sept.,
18 Sept., or 14 Oct. 1998, respectively, in the second year of
production.

([double dagger]) ns, not significant at P < 0.05.

Table 4. Plant density in falls of 1997 and 1998, and in springs
of 1998 and 1999 at Pintendre and Normandin for two alfalfa
cultivars, WL 225 and AC Caribou, harvested either only twice
during the summer, or three times with the third harvest taken
in the fall 400, 500, or 600 growing degree days (GDD) after
the second summer harvest.

Cultivars       Harvest       Fall                   Spring
               treatments     1997                    1998

                                                     Plants
    Pintendre ([dagger])                           [m.sup.-2]

WL 225       2 Harvests        251                    268
             3 Har. 400 GDD    285                    326
             3 Har. 500 GDD    249                    299
             3 Har. 600 GDD    276                    245

AC Caribou   2 Harvests        247                    275
             3 Har. 400 GDD    260                    275
             3 Har. 500 GDD    253                    313
             3 Har. 600 GDD    256                    292
                        SEM    23                     22

    Normandin ([section])

WL 225       2 Harvests        217 ([paragraph])      177
             3 Har. 400 GDD    233                    132
             3 Har. 500 GDD    226                    133
             3 Har. 600 GDD    272                    176

AC Caribou   2 Harvests        170                    181
             3 Har. 400 GDD    207                    158
             3 Har. 500 GDD    199                    162
             3 Har. 600 GDD    207                    145
                        SEM     23                     13

Cultivars       Harvest
               treatments     Fall 1998    Spring 1999

                                Plants
    Pintendre ([dagger])      [m.sup.-2]

WL 225       2 Harvests          204         96  ([double dagger])
             3 Har. 400 GDD      193        111
             3 Har. 500 GDD      164         99
             3 Har. 600 GDD      171        125

AC Caribou   2 Harvests          181        125
             3 Har. 400 GDD      178        131
             3 Har. 500 GDD      179        145
             3 Har. 600 GDD      182        127
                        SEM       12         17

    Normandin ([section])

WL 225       2 Harvests          103        103
             3 Har. 400 GDD       73         --
             3 Har. 500 GDD       88         --
             3 Har. 600 GDD      101         --

AC Caribou   2 Harvests           97        118
             3 Har. 400 GDD      114         --
             3 Har. 500 GDD       99         --
             3 Har. 600 GDD      114         --
                        SEM        9         --

([dagger]) At Pintendre, harvest treatments were: 2nd harvest
(6 Aug. 1997, 5 Aug. 1998), additional harvests in the fall
400, 500, or 600 GDD after the second one, on 9 Sept., 22
Sept., or 14 Oct. 1997, respectively, in the first year of
production, and on 10 Sept., 23 Sept., or 9 Oct. 1998,
respectively, in the second year of production.

([double dagger]) Cultivar means averaged across all harvest
treatments were significantly different at Pintendre at this
date (P = 0.048).

([section]) At Normandin, harvest treatments were: 2nd harvest
(30 July 1997, 31) July 1998), additional harvests in the fall
400, 500, or 600 GDD after the second one, on 12 Sept., 29
Sept., or 21 Oct. 1997, respectively, in the first year of
production, and on 4 Sept., 18 Sept., or 14 Oct. 1998,
respectively, in the second year of production.

[paragraph] Cultivar means averaged across all harvest treatments
were significantly different at Normandin at this date (P = 0.030).

Table 5. Analyses of variance and a priori contrasts for the
root dry weight (DW) and amounts of carbohydrate and N reserves
measured at Pintendre and Normandin from fall 1997 to spring
1999 in alfalfa roots.

                                                 P > F

                                            Pintendre ([dagger])

                                      Root        RFO
                                       DW     ([section])   Sucrose

                                              19 Nov. 1997

Cultivars                            ns (#)      0.017       0.016
Harvests                             <0.001      0.006       0.015
  2 harvests vs. 400                 <0.001      0.005       0.023
  2 harvests vs. 500                 <0.001      0.017       0.002
  2 harvests vs. 600                  0.005       ns          ns
Cultivars x harvests                   ns         ns          ns
  Cultivars x (2 harvests vs. 400)     ns         ns          ns
  Cultivars x (2 harvests vs. 500)     ns         ns          ns
  Cultivars x (2 harvests vs. 600)     ns         ns          ns

                                              16 Apr. 1998

Cultivars                              ns         ns          ns
Harvests                              0.001      0.003       0.042
  2 harvests vs. 400                 <0.001     <0.001       0.035
  2 harvests vs. 500                  0.001      0.001       0.043
  2 harvests vs. 600                   ns        0.008        ns
Cultivars x harvests                   ns         ns          ns
  Cultivars x (2 harvests vs. 400)     ns         ns          ns
  Cultivars x (2 harvests vs. 500)     ns         ns          ns
  Cultivars x (2 harvests vs. 600)     ns        0.043        ns

                                              19 Nov. 1998

Cullivars                              ns         ns          ns
Harvests                              0.004      0.001       0.031
  2 harvests vs. 400                  0.019      0.058       0.055
  2 harvests vs. 500                   ns         ns          ns
  2 harvests vs. 600                   ns        0.041        ns
Cultivars x harvests                   ns         ns          ns
  Cultivars x (2 harvests vs. 400)     ns         ns          ns
  Cultivars x (2 harvests vs. 500)     ns         ns          ns
  Cultivars x (2 harvests vs. 600)     ns         ns          ns

                                              21 Apr. 1999

Cultivars                              ns         ns          ns
Harvests                               ns         ns          ns
  2 harvests vs. 400                   ns         ns          ns
  2 harvests vs. 500                   ns         ns          ns
  2 harvests vs. 600                   ns         ns          ns
Cultivars x harvests                   ns         ns          ns
  Cultivars x (2 harvests vs. 400)     ns         ns          ns
  Cultivars x (2 harvests vs. 500)     ns         ns          ns
  Cultivars x (2 harvests vs. 600)     ns         ns          ns

                                              P > F

                                                        Normandin
                                        Pintendre       ([double
                                        ([dagger])      dagger])

                                              Tot. AA
                                              ([para-   Root
                                     Starch   graph])    DW

                                                        12 Nov.
                                       19 Nov. 1997     1997

Cultivars                              ns       ns      0.004
Harvests                             <0.001    <0.001   0.028
  2 harvests vs. 400                 <0.001    <0.001   0.003
  2 harvests vs. 500                 <0.001     0.033    ns
  2 harvests vs. 600                   ns       ns       ns
Cultivars x harvests                   ns       ns       ns
  Cultivars x (2 harvests vs. 400)     ns       ns       ns
  Cultivars x (2 harvests vs. 500)     ns       ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns       ns

                                                        21 Apr.
                                        16 Apr. 1998    1998

Cultivars                              ns       ns       ns
Harvests                             <0.001    <0.001   0.035
  2 harvests vs. 400                 <0.001    <0.001   0.009
  2 harvests vs. 500                 <0.001    <0.001    ns
  2 harvests vs. 600                 <0.001     ns       ns
Cultivars x harvests                   ns       0.057    ns
  Cultivars x (2 harvests vs. 400)     ns       ns       ns
  Cultivars x (2 harvests vs. 500)     ns       0.026    ns
  Cultivars x (2 harvests vs. 600)     ns       0.048    ns

                                                        12 Nov.
                                        19 Nov. 1998    1998

Cullivars                            0.016      0.012    ns
Harvests                             0.005     <0.001    ns
  2 harvests vs. 400                 <0.001    <0.001    ns
  2 harvests vs. 500                   ns       ns       ns
  2 harvests vs. 600                   ns       ns       ns
Cultivars x harvests                   ns       ns       ns
  Cultivars x (2 harvests vs. 400)     ns       ns       ns
  Cultivars x (2 harvests vs. 500)     ns       ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns       ns

                                                        4 May
                                       21 Apr. 1999     1999

Cultivars                              ns       ns       ns
Harvests                             <0.001     ns       --
  2 harvests vs. 400                 <0.001     0.034    --
  2 harvests vs. 500                 <0.001     0.025    --
  2 harvests vs. 600                 <0.001     ns       --
Cultivars x harvests                   ns       ns       --
  Cultivars x (2 harvests vs. 400)     ns       ns       --
  Cultivars x (2 harvests vs. 500)     ns       ns       --
  Cultivars x (2 harvests vs. 600)     ns       ns       --

                                          P > F

                                     Normandin ([double
                                          dagger])

                                      RFO    Sucrose

                                       12 Nov. 1997

Cultivars                            0.005    0.040
Harvests                              ns      0.045
  2 harvests vs. 400                 0.019    0.011
  2 harvests vs. 500                  ns       ns
  2 harvests vs. 600                  ns       ns
Cultivars x harvests                  ns       ns
  Cultivars x (2 harvests vs. 400)    ns       ns
  Cultivars x (2 harvests vs. 500)    ns       ns
  Cultivars x (2 harvests vs. 600)    ns       ns

                                       21 Apr. 1998

Cultivars                             ns       ns
Harvests                              ns      0.001
  2 harvests vs. 400                  ns     <0.001
  2 harvests vs. 500                  ns       ns
  2 harvests vs. 600                  ns       ns
Cultivars x harvests                  ns       ns
  Cultivars x (2 harvests vs. 400)    ns       ns
  Cultivars x (2 harvests vs. 500)    ns       ns
  Cultivars x (2 harvests vs. 600)    ns       ns

                                       12 Nov. 1998

Cullivars                             ns       ns
Harvests                              ns       ns
  2 harvests vs. 400                  ns       ns
  2 harvests vs. 500                  ns       ns
  2 harvests vs. 600                  ns       ns
Cultivars x harvests                  ns       ns
  Cultivars x (2 harvests vs. 400)    ns       ns
  Cultivars x (2 harvests vs. 500)    ns       ns
  Cultivars x (2 harvests vs. 600)    ns       ns

                                        4 May 1999

Cultivars                             ns       ns
Harvests                              --       --
  2 harvests vs. 400                  --       --
  2 harvests vs. 500                  --       --
  2 harvests vs. 600                  --       --
Cultivars x harvests                  --       --
  Cultivars x (2 harvests vs. 400)    --       --
  Cultivars x (2 harvests vs. 500)    --       --
  Cultivars x (2 harvests vs. 600)    --       --

                                           P > F

                                         Normandin
                                     ([double dagger])

                                               Tot.
                                     Starch     AA

                                       12 Nov. 1997

Cultivars                              ns      0.042
Harvests                             <0.001    0.002
  2 harvests vs. 400                 <0.001   <0.001
  2 harvests vs. 500                 <0.001    0.005
  2 harvests vs. 600                  0.002    0.007
Cultivars x harvests                   ns       ns
  Cultivars x (2 harvests vs. 400)     ns       ns
  Cultivars x (2 harvests vs. 500)     ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns

                                       21 Apr. 1998

Cultivars                             0.020     ns
Harvests                             <0.001    0.034
  2 harvests vs. 400                 <0.001    0.015
  2 harvests vs. 500                  0.020    0.016
  2 harvests vs. 600                   ns       ns
Cultivars x harvests                   ns       ns
  Cultivars x (2 harvests vs. 400)     ns       ns
  Cultivars x (2 harvests vs. 500)     ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns

                                       12 Nov. 1998

Cullivars                              ns       ns
Harvests                               ns       ns
  2 harvests vs. 400                   ns       ns
  2 harvests vs. 500                  0.029     ns
  2 harvests vs. 600                   ns       ns
Cultivars x harvests                   ns       ns
  Cultivars x (2 harvests vs. 400)     ns       ns
  Cultivars x (2 harvests vs. 500)     ns       ns
  Cultivars x (2 harvests vs. 600)     ns       ns

                                        4 May 1999

Cultivars                              ns       ns
Harvests                               --       --
  2 harvests vs. 400                   --       --
  2 harvests vs. 500                   --       --
  2 harvests vs. 600                   --       --
Cultivars x harvests                   --       --
  Cultivars x (2 harvests vs. 400)     --       --
  Cultivars x (2 harvests vs. 500)     --       --
  Cultivars x (2 harvests vs. 600)     --       --

([dagger]) At Pintendre, harvest treatments were: 2nd harvest (6 Aug.
1997, 5 Aug. 1998), additional harvests in the fall 400, 500, or 600
growing degree days (GDD) after the second one, on 9 Sept., 22 Sept.,
or 14 Oct. 1997, respectively, in the first year of production, and
on 10 Sept., 23 Sept., or 9 Oct. 1998, respectively, in the second
year of production.

([double dagger]) At Normandin, harvest treatments were: 2nd harvest
(30 July 1997; 30 July 1998), additional harvests in the fall 400,
500, or 600 GDD after the second one, on 12 Sept., 29 Sept., or 21
Oct. 1997, respectively, in the first year of production, and on 4
Sept., 18 Sept., or 14 Oct. 1998, respectively, in the second year
of production.

([section]) RFO, raffinose family oligosaccharides.

([paragraph]) Tot. AA, total amino acids.

(#) ns, not significant at P < 0.05.


ACKNOWLEDGMENTS

The technical assistance of Lucette Chouinard, Annie Tremblay, and Pierre Lechasseur is gratefully acknowledged. Thanks are extended to Jean Goulet, Chantal Beaulieu, and Jean-Noel Bouchard for their care of the plots and their help with sampling.

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Catherine Dhont, Yves Castonguay, * Paul Nadeau, Gilles Belanger, Raynald Drapeau, and Francois-P. Chalifour

C. Dhont and F-P. Chalifour, Dep. de Phytologie, Univ. Laval, Sainte-Foy, QC, Canada, G1K 7P4; Y. Castonguay, P. Nadeau, and G. Belanger, Agriculture et Agroalimentaire Canada, Sainte-Foy, QC, Canada, G1V 2J3: R. Drapeau, Agriculture et Agroalimentaire Canada, Normandin, QC, Canada, G8M 4K3. This research was supported by a Research Grant from the Conseil des Recherches en Peche el en Agroalimentaire du Quebec (CORPAQ) and by a Research Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.-P. Chalifour. Contribution no. 754 of the Sainte-Foy Research Centre. Received 7 Feb. 2003. * Corresponding author (castonguayy@agr.gc.ca).
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Title Annotation:Crop Physiology & Metabolism
Author:Dhont, Catherine; Castonguay, Yves; Nadeau, Paul; Belanger, Gilles; Drapeau, Raynald; Chalifour, Fra
Publication:Crop Science
Date:Jan 1, 2004
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