Climate data

The monthly averages of outdoor temperature (maximum and minimum) and monthly rainfall during the study period were obtained from the agrometeorological station of the College of Postgraduates (Colegio de Postgraduados), located at a distance of 100 m from the experimental site (Figure 1). The maximum monthly temperature ranged from 22.1 to 30.2 °C, while the minimum temperature was from -2.6 to 11 °C. The highest temperature occurred in spring, registering a maximum of 30.2 °C in April, and the lowest temperature, of -2.6 °C was recorded in December. The accumulated precipitation from March 2012 to April 2013 was 312.3 mm, 70 % of which occurred in June, July, August, and September 2012, accumulating a precipitation of 220 mm.

Figure 1 Average maximum and minimum monthly temperature and accumulated precipitation per month during the study period (March 2012 - April 2013) 

Dry matter accumulation

After uniform grazing, three 0.25 m² squares were cut at a height of 5 cm from the ground in each experimental plot for eight weeks. The forage harvested in each quadrant was washed and dried in labeled paper bags in a forced air oven at 55 °C during 72 h in order to estimate the amount of dry matter per hectare at the various regrowth ages.

Botanical and morphological composition

For the purpose of determining the botanical composition of the forage, a subsample of this was collected for a dry matter yield of approximately 20 %¹¹ and was separated into the following components: dead material, weeds, grasses and white clover. The morphological components of white clover (leaf, petiole, runner and flower) were separated. Each separate component was dried in a labeled paper bag and left in a forced air oven at 55 °C during 72 h in order to estimate its dry weight.

Leaf area index

The leaf area index was calculated by separating the leaves of five stolons were separated for each week’s replicate and placing them in a leaf area integrator (LI 3100 LI-COR Inc.) from which the leaf area readings in cm² were obtained. These readings together with the number of stolons per square meter allowed estimating the leaf area index by means of the following formula:

LAI= LA * SD

Where: LAI= leaf area index; LA=leaf area per stem; and SD= stolon density (m^-2).

Statistical analysis

The data were analyzed by the GLM procedures of SAS¹⁸, for a completely randomized experimental design, where the treatments were the weeks of evaluation with three repetitions per season and a regression analysis for each variable. The means were compared with the Tukey test (α= 0.05).

Dry matter accumulation

Figure 2 shows the results of the accumulated dry matter yield, which increased with regrowth age, reaching its maximum in the eighth week of spring (2,688 kg DM ha^-1; P<0.05), the seventh week of summer (2,241 kg DM ha^-1; P<0.05), the eighth week of fall (1,781.3 kg; P<0.05), and the sixth week of winter (1,643 kg; P<0.05). The biomass accumulated in spring was higher by 20 % (447 kg DM ha^-1), 51 % (907 kg), and 64 % (1,045 kg), compared to summer, autumn and winter, respectively (P<0.05). The leaf for spring, autumn and winter increased as the regrowth weeks increased and it was greater than the petiole, however, in summer there was a higher petiole yield and less leaf. During the evaluation period in winter (March 3, 2013), an intense frost occurred; this coincided with the sixth week of regrowth, which limited the biomass yield for said sampling, drastically increasing the dead material.

Figure 2 White clover growth curves by season and by morphological component during a growth cycle of 8 and 9 weeks 

For their part, in their assessment of white clover in the highlands of Mexico, Gutiérrez et al⁹ mention an accumulation of forage as the age of the plant increased in all seasons, reaching the maximum yield for spring, autumn, and winter in the eighth week, with 2,953, 1,592, and 1,791 kg DM ha^-1, respectively, and in the seventh week for summer with 1,971 kg DM ha^-1 (P<0.05).

When establishing associations of white clover with perennial ryegrass (Lolium perenne) and orchard grass (Dactylis glomerata L.), Moreno et al¹⁵ found a maximum white clover yield of 513 kg DM ha^-1 in their first sampling year. However, Maldonado et al³ registered a significant increase in the yield of white clover in these same associations in their third and fourth year of production, adding up to an average of 7,220 kg DM ha^-1. These same authors mention that white clover dominates over time in the meadow because it is a kind of stoloniferous growth habit that allows rapid growth compared to grasses, which are tufted. In another study¹⁹, they reported that 65% of the annual yield occurred in spring and summer, 23 % in winter, and autumn was the season that exhibited the lowest value, of 12 % (P<0.05).

According to various authors^18,20, white clover requires temperatures of 18 to 30 °C, the optimal being 24 °C, and precipitations of 750 mm for best performance. These temperatures were reached in spring (Figure 1), favoring the growth of the meadow as a result of the increase in the leaf area per plant and probably due to the increase in leaf appearance and elongation rates¹⁹. Conversely, the dry matter yield was low in winter; in this regard, various authors ^3,12,21 argue that low temperatures limit growth and forage accumulation, due to their direct influence on a lower leaf appearance and foliar rate.

Botanical and morphological composition

Based on the morphological components and the estimated accumulated biomass yield, the plant morphology was variable (P<0.05). The highest leaf percentage, of 68 %, was produced in the third week in spring; likewise, in summer the highest percentage of leaf, of 46 to 40 %, was produced from the first to the third week, decreasing drastically in the fourth week, to 30 % (P<0.05). In autumn, the highest leaf percentage occurred in the first week (70 %) and remained until the seventh week (59 %), after which it decreased. Finally, in winter, the highest leaf percentage (60 %) occurred in the fifth week (Figure 3).

Figure 3 Botanical and morphological composition of white clover during an 8 and 9 week growth cycle. mm= dead material 

On the other hand, the greatest contribution of the petiole, of 38 %, occurred in summer (P<0.05), while the highest percentage of stolon was reported in spring, being greater only in the first two weeks of growth, when it amounted to 20 % in average (P<0.05). As for pastures, the largest percentage, an average of 15 %, occurred in the summer. The contribution of weeds and flowers was minimal in all seasons and weeks of regrowth, being 3 % in average.

Winter was the season that reported the largest amount of dead material and from the seventh week on there was a drastic increase, of 100 %, since there was a decrease in temperature (Figure 1) frost causing the death of white clover. In this regard, Brock and Tilrock⁸ mention that all plants have an optimal growth temperature and when these surpass it or decrease drastically, there may be cell death, which causes a drastic increase in dead material. On the other hand, as the age of the plant increased, the dead material also built up (P<0.05), due to the maturation of the senescent leaves of the lower strata⁹.

The large proportion of leaves with respect to the petiole and stolon indicates that it is a high-quality forage since this allows it to be more digestible. In addition to this, as observed in all the assessment stages, the content of flower was minimal (P>0.05), which indicates that this forage is not precocious and allows it to increase its nutritional value in the first weeks. As has been shown, the association of this legume with grasses confers it forage value, since it augments the total yield per surface unit to up to 14 t DM ha^-1; however, these qualities can also be affected by the season of the year^19,22.

In addition to this, white clover under grazing conditions is less susceptible to loss of apical meristems due to the horizontal arrangement of leaves and leaf primordia, which allow efficient capture of solar radiation compared to Gramineae^8,14.

Leaf area index

The leaf area index (LAI) allows to estimate the photosynthetic capacity of a plant per unit area and helps to understand its ability to assimilate solar energy and transform it into dry matter yield²³. The highest LAI was reported during spring and autumn, with 3.0 and 2.6, respectively, in week eight, while in summer and winter the highest index was obtained in week five with 2.6 (P<0.05). On the other hand, in all seasons there was a close relationship between the LAI and the regrowth weeks, the highest r², of 0.97, occurring in spring, and the lowest, of 0.83, in summer (Figure 4).

Figure 4 Leaf area index of white clover during a growing season 

The temperature range of 22 to 30 °C during the spring and summer and the higher precipitation in June, July, August, and September resulted in 70 % of the total accumulated precipitation in the experimental period, contributing to the greater growth of the plant, which profited from the biochemical and photosynthesis processes for its optimal development. However, the conditions were not favorable in the fall and winter, when the low temperatures ranged from -2 to 11 °C, causing a reduction of the tissue turnover in winter and thereby affecting the growth and development of the plant, which exhibited the lowest LAI and the lowest yield of accumulated biomass during this season.

In a trial to assess white clover⁹, the highest leaf area index, of 3.0, was observed in the eighth week of spring; later, in the fifth week of summer, it was 1.7, and in the eighth week of autumn and of winter, it had a value of 1.4 and 1.6, respectively ―results that agree with those of this research. In a study directed by Zaragoza et al^1), they reported the highest LAI (P<0.05) for the alfalfa crop in week five of spring (3.5), of summer (2.8), and of autumn (2.0), and in the sixth week of winter(1.9). However, the values were different when evaluating the orchard grass, since the highest LAI (P<0.05) occurred in week six of regrowth in spring(2.3), summer(1.4) and autumn (1.1), and in the seventh week of winter (1.0). Other researches on alfalfa^23,24 reported behaviors similar to those observed in this experiment, since the highest values of LAI (P<0.05), of 3.3 and 4.9, respectively, were recorded in spring-summer.

The LAI varies for each crop and depends on the environmental conditions present. Matthew et al¹⁴ points out that the LAI is optimal when the net forage production is at a maximum point and the highest LAI is reached simultaneously; however, the LAI can be affected indirectly by low temperatures, according to the type of crop and the time of sampling.