Environmental conditions and treatments

The study was carried out in the field in the three seasons of the year that prevail in Tabasco: the Nortes season (prolonged low intensity rains and lowest temperatures of the year) from September 2014 to February 2015, dry season from March to May from March to May (<90 mm/month of rain) and Rainy season from June to July 2015 (>200 mm/month of rain), in the KARIGA SPR Ranch of RL, with coordinates 93º28’19.34’’ west longitude and 17º41’31.59’’ north latitude. Soil is acidic (pH= 4.7), high in organic matter (5%), low in nitrogen (0.17%), phosphorus (5.38 mg kg ^-1 ) and potassium (0.11 cmol kg^-1) and sandy clay loam texture. This soil is considered to be transitional to Savanna and is classified as cutaneous hyperdistrictic cutaneous endocritic acrisol (^{Salgado-García et al., 2010}). The precipitation and temperature data of the evaluation period were provided by the CONAGUA Tabasco.

The ^{Salgado-García et al., 2010} meadow was established in 2005 for herding cattle. Within this prairie, the experimental site was selected in a homogeneous area, where plots of 4 m long x 1.5 m wide were formed. The treatments consisted of plots of Humidicola in monoculture (H), Humidicola in monoculture+fertilization (HF), Humidicola+Stylo (HS) and Humidicola+Stylo+fertilization (HSF). Each treatment had four repetitions. Prior to the beginning of the experiment, the plots were cut to 5 cm in height. Later, in the treatments conformed by the association B. humidicola - Stylosanthes guianensis (Stylo), the legume was planted, in the plots of Humidicola, by seed and “chorrillo” in doses of 6 kg ha^-1 and at distances of 30 cm between rows. For phosphatic fertilization, triple superphosphate was used with 46% P₂O₅ in doses of 100 kg ha^-1 (^{Pastrana 1994}). Given the low solubility of phosphorus, fertilization was divided into two equal parts, applying 50 kg ha^-1 on August 20, 2014 and the remaining 50 kg on February 8, 2015; fertilization was manual.

Cut dates and response variables

The cut of uniformity in the 16 plots was made on September 1, 2014 and from this date the cuts, sampling and data collection was made every 35 days. Therefore, the first sampling was on October 6, 2014 and the last on July 13, 2015. The useful plot was located in the center of each plot and represented an area of 2 m². The response variables were: Dry matter yield (RMS, t ha^-1) and phosphorus concentration (g kg^-1MS). In the treatments formed by the associations (HS and HSF), the RMS and the phosphorus concentration were also determined in the biomass of the Humidicola and the Stylo individually. The RMS was determined with the quadrant method with an area of 1 m². For this, the quadrant was placed in the center of each experimental plot and the total biomass within the quadrant was harvested at a height of 5 cm for the H and HF treatments. For the HS and HSF treatments, the Humidicola was cut at a height of 5 cm and the Stylo for being a semi-shrub plant at a height of 20 cm. The fresh weight of the total biomass was obtained from each plot, and from this biomass a subsample of 200 g was taken to determine the dry weight and perform the P analysis.

The subsamples per treatment were placed in paper bags and dried in a forced air oven for 72 h at 60 °C. To calculate the RMS, the data of the fresh weight of the quadrant and the dry weight of the 200 g in the formula were used: RMS= (PF*PS/pf)/100, where RMS: Dry matter yield (t ha^-1), PF: fresh weight of the sample of m² (g of green matter, MV), pf: fresh weight of the subsample (g MV) and PS: dry weight of the subsample (g of MS). In parallel, a second subsample of 200 g was taken in the treatments of the associations to separate the grass from the legume, and analysis of P individually. The different components were weighed and dried in a forced air oven for 72 h at 60 °C. The determination of phosphorus (g kg^-1 MS) in Humidicola in monoculture, of the associations, and of each species within the associations, was by the Olsen method, using wet digestion with nitric-perchloric acid mixture, and the extract analyzed by spectrometry (NOM-021-RECNAT (2000).

Root weight and number of nodules of Stylosanthes guianensis

At the end of each season of the year, roots of S. guianensis were collected in the treatments in association (HS and HSF). The roots were extracted along linear 0.3 m and at a depth of 25 cm, with the help of a straight shovel, taking care not to damage the nodules. From the total of extracted plants, 10 plants of Stylo were selected at random, to obtain the total dry weight of roots and number of nodules. The total dry weight of the roots by treatment and repetition was obtained after drying them in a forced air oven, for 72 h at 60 °C. From the fresh weight and dry root data, the dry matter concentration of roots was calculated in g kg^-1 MS.

Experimental design

The experiment was analyzed as repeated measures by PROC MIXED of the SAS (^{SAS, 2010}). The four treatments were compared using a unifactorial structure of the treatments, with the cutoff date being the repeated measurement factor. In the analysis of variance for each response variable, a model was used that included the effects of treatment, cutoff date and their interactions. The randomization scheme of the treatments on the experimental units was through a completely randomized design with four repetitions.

Environmental conditions during the study period

In September (the final stage of the rainy season) and the beginning of autumn, an average temperature of 28.5 °C and 208 mm of rainfall was recorded. Subsequently, from October to February (Nortes season), which includes autumn and winter, there was a decrease in the average temperature, from 28.5 to 22 °C, registering the highest rainfall in the study period in the months of October (476 mm) and January (335 mm), being the month of December the least rainy (26 mm). In this northern season (Oct-Feb), the accumulated rainfall was 1 300 mm. In spring, during the months of March, April and May (dry season) the average temperature rose, going from 22 ºC (Nortes time) to 30 ºC. The cumulative rainfall was 220 mm, with the highest rainfall (148 mm) and the least rainy month (15.9 mm) in March. These climatic variations will affect the growth of the forage species under study, giving the seasonality of the dry matter production indicated by ^{Jones and Hu (2006)} and observing the positive effect of the complementarity of the grass-legume association shown in other studies (^{Zuppinger-Dingley et al., 2014}; ^{Prieto et al., 2015}).

Dry matter yield (RMS)

Differences in RMS occurred between cut-off dates and between treatments and the interaction treatment x cut was highly significant (Table 1). The chronological pattern of RMS was similar between treatments and decreased from October 7 to December 16 in all treatments. This decrease was greater in Humidicola in monoculture (H and HF), 1.16 t ha^-1, whereas in this same period the associations (HS and HSF), decreased only in 0.63 and 0.5 t ha^-1, respectively. This meant a reduction rate of the RMS of 16.47 kg ha^-1 d^-1, average of the two treatments of Humidicola in monoculture, and of 9 kg and 7 kg ha^-1 d^-1 for HS and HSF, respectively. The decrease in RMS coincided with the average temperature decrease of 5 °C during this period of Nortes.

Table 1 Yield of dry matter and phosphorus concentration of humidicola in monoculture, humidicola in monoculture+fertilization, humidicola+stylo and humidicola+stylo+fertilization, during growth periods of 35 days. 

^*** p< 0.0001, promedios con letras minúsculas distintas difieren significativamente (p< 0.05) entre fechas de cosechas dentro de un tratamiento. Promedios con letras mayúsculas distintas difieren significativamente (p< 0.05) entre tratamientos dentro de una misma fecha de cosecha; dosis de fósforo= 100 kg ha^-1.

The RMS was also not different between treatments within each cutoff date (Table 1). The average RMS of the four treatments in this period was 0.805 t ha^-1. From February 24 to March 31, all treatments increased their RMS (which coincided with an increase of 3 °C in the ambient temperature), except Humidicola in monoculture without fertilization (H) which remained stable. These increases were of the order of 0.6, 0.462 and 0.625 t ha^-1, with respect to HF, HS and HSF (Table 1). Subsequently, during the dry season of the year (April and May), all treatments again decrease their RMS, going from 1.3 t ha^-1 in March (average of the four treatments) to 0.77, 1.05, 0.66 and 1.17 t ha^-1 (H, HF, HS and HSF, respectively) (Table 1), with HSF being the treatment with the highest RMS in this period.

The associations (HS, HSF) were the ones that showed greater stability in the RMS, which coincides with ^{Prieto et al. (2015)}. These authors demonstrated that the diversity of species in a meadow regulates their productivity and stability by the induction of complementary effects. Subsequently, from December 16 to February 24, the four treatments recorded the lowest RMS of this time of Nortes, with average values of 0.74, 0.78, 0.65 and 0.9 t ha^-1 (H, HF, HS and HSF, respectively).

The RMS was also not different between treatments within each cutoff date (Table 1). The average RMS of the four treatments in this period was 0.805 t ha^-1. From February 24 to March 31, all treatments increased their RMS (which coincided with an increase of 3 °C in the ambient temperature), except Humidicola in monoculture without fertilization (H) which remained stable. These increases were of the order of 0.6, 0.462 and 0.625 t ha^-1, with respect to HF, HS and HSF (Table 1). Subsequently, during the dry season of the year (April and May), all treatments again decrease their RMS, going from 1.3 t ha^-1 in March (average of the four treatments) to 0.77, 1.05, 0.66 and 1.17 t ha^-1 (H, HF, HS and HSF, respectively) (Table 1), with HSF being the treatment with the highest RMS in this period.

In this way, in the driest month (April), HSF registered 0.52 t ha^-1 more than RMS with respect to the average of the three remaining treatments. The highest RMS of the HSF association in periods of environmental stress coincided with the highest RMS of the legume in this period (Table 2).

Table 2 Dry matter yield (t ha^-1) of Brachiaria humidicola and Stylosanthes guianensis growing in association with and without phosphate fertilization (100 kg of P₂O₅ ha^-1) from October 2014 to July 2015. 

Promedios con letras distintas sobre la misma hilera indican diferencias significativas (p< 0.05). 1= 7 de octubre, 2= 11 de noviembre, 3= 16 de diciembre, 4= 20 de enero, 5= 24 de febrero, 6= 31 de marzo, 7= 5 de mayo, 8= 9 de junio, 9= 14 de julio. H-HS= humidicola en la asociación HS; S-HS= stylo en la asociación HS; H-HSF= humidicola en la asociación HSF; S-HSF= stylo en la asociación HSF.

These results indicate that phosphate fertilization had a greater effect in the legume than in the grass, even Stylo has been reported with high adaptation to acid soils and ability to take advantage of phosphorus in these soils (^{Yang and Yan, 1998}; ^{Du et al., 2009}). In general, in the dry season of the year (March 31 to June 9), the fertilized treatments HF and HSF, had higher average RMS with 1.05 and 1.18 t ha^-1, respectively, while in the treatments without fertilizing (H and HS), was 0.77 and 0.66 t ha^-1, in order. In the rainy season, due to improved environmental humidity conditions, all treatments increased their RMS. The Humidicola treatments in monoculture (H, HF) registered higher RMS, averaging 2.01 t ha^-1, while in the associations the records were 0.87 and 1.44 t ha^-1 with respect to HS and HSF (Table 2). In other studies, this rapid recovery of Humidicola in monoculture (H, HF) has been observed by placing it in favorable environmental conditions for its growth (^{Saito, 2004}). The total RMS of the 280 study days was similar between treatments H, HF and HSF with an average value of 10.5, which contrasts with the 7.07 t ha^-1 of the HS treatment.

The lower average yield of HS (0.78 t ha^-1) compared to monoculture (1.1 t ha^-1), differs from other studies (^{Sleugh et al., 2000}; ^{Gierus et al. 2012}; ^{Rassmussen et al., 2012}; ^{Albayrak and Turk, 2013}) in which it has been observed that the grass-legume associations present a higher RMS, with respect to grasses in monoculture. In our study, the low RMS of HS could be due to the differences in cutting heights applied to the species within the associations, which should have represented an advantage for the legume in the collection of light and nutrients, and a shade in the Humidicola causing a lower RMS of the associated Humidicola (Table 3) and consequently a lower RMS of the plot (Table 1). Gierus et al. (2012) indicate that the stability of the composition of the species in grass-legume associated grasslands and their result in terms of RMS, are a product of the competition for the resources between the grass and the legume; that is, of their ability to coexist.

Table 3 Phosphorus concentration (g kg-1 MS) of B. humidicola and S. guianensis growing in association with and without phosphate fertilization (100 kg of P2O5 ha-1), in 35-day growth periods from October 2014 to July 2015. 

Promedios con letras distintas sobre la misma hilera indican diferencias significativas (p< 0.05). 1= 7 de octubre, 2= 11 de noviembre, 3= 16 de diciembre, 4= 20 de enero, 5= 24 de febrero, 6= 31 de marzo, 7= 5 de mayo, 8= 9 de junio, 9= 14 de julio. H-HS= humidicola en la asociación HS; S-HS= stylo en la asociación HS; H-HSF= humidicola en la asociación HSF; S-HSF= stylo en la asociación HSF.

In the HS and HSF treatments, the legume maintains a greater proportion throughout the cuts, except in the last two of June 9 and July 14 (Table 2). In HS, the proportion of the legume is higher than 50% of the RMS in most of the cuts, and higher than 70% in HSF. In the HS association, the legume maintains its yield stability throughout the evaluation period, except on June 9 where it records its lowest RMS. On the other hand, Humidicola, within the association Humidicola-Stylo, shows greater variation by decreasing its RMS from December 16 to February 24 (Nortes time), to subsequently increase it again.

In HSF, the legume has lower RMS from January 20 and February 24 and June 9; however, in seven of the nine courts (except June 9 and July 14), it records more than 70% of RMS with respect to Humidicola. Therefore, the Stylo recorded 3.23 and 1.32 t ha^-1 more accumulated RMS, at the end of the evaluation period, which Humidicola within HSF and HS associations, respectively. The greater stability of production of the legume between the three seasons of the year (Table 2) leads to a complementary effect of the RMS of the grass within an association, mainly in times of difficult growth of the plant, as has been observed in other studies. (^{Sleugh et al., 2000}; ^{Tilman et al., 2006}; ^{Prieto et al., 2015}).

The Stylo RMS within the HSF association was 2.47 t ha^-1 higher than its RMS registered in HS (Table 2). However, the number of nodules per plant varied due to fertilization, with 18.8 in HSF treatment and 13.9 in HS, on average for the three seasons. Therefore, the greater vigorousness of the fertilized Stylo, expressed through its high RMS, could be due to the greater number of registered nodules, with respect to the Stylo without fertilizing, which coincides with the results of ^{Lopes et al. (2011)}. At times, the greatest number of nodules was registered in Nortes (28.1 nodules pl^-1), decreasing in the dry season (13.6 nodules pl^-1) and rainfall (7.8 nodules pl^-1).

Phosphorus concentration (P)

There were differences in concentration of P between treatments and between cuts, and the interaction treatment x cut was significant (Table 1). The chronological pattern of the phosphorus concentration during the evaluation period was similar between treatments. As an average of the nine cuts, the phosphorus concentration levels were separated into two groups: the fertilized treatments with the highest concentration of P and the unfertilized treatments with the lowest concentration. Within each group the treatments did not differ. HF and HSF had higher phosphorus concentration averaging 1.62 g kg^-1 MS, followed by H and HS with 0.96 g kg^-1 MS on average. The increase of P in the fertilized treatments, derives from an increase in the availability of P in the soil as a result of the application of this element, given that the absorption of a nutrient by the plant is proportional with the availability of this nutrient in the soil (^{Kulik, 2009}). The higher concentration of P in legumes than in grasses, has been reported in other studies (^{Ramírez-Orduna et al., 2005}; ^{Dasci et al., 2010}).

Fluctuations in the concentration of P in the plant were evident through the cuts, maintaining higher concentrations HF and HSF in each and every one of the cuts (Table 1), with an increase in the four treatments from December 16 to March 31, to subsequently decrease and maintain a concentration similar to the months of October and November. In Nortes, the average concentration of P in the HF and HSF treatments was 1.9 vs 1.38 g kg^-1 MS of the dry and rainy seasons, respectively. The treatments without fertilizing (H, HS) registered a concentration of 1.2 g kg^-1 MS in Nortes vs 0.8 g in dry and rain, on average.

In the present study, a higher concentration of P is always registered in Stylo than in Humidicola (Table 3). This difference increases with phosphate fertilization, showing Stylo 0.43 g kg^-1 MS more than phosphorus fertilized Humidicola (Table 3), while the difference between Stylo and Humidicola unfertilized is 0.12 g kg^-1 MS. The above indicates that Stylo is more efficient in taking phosphorus than the Humidicola because of phosphate fertilization. Similar results were presented by ^{Tomei et al. (2005)}, who observed that Stylosanthes guianensis increased its concentration of P with phosphate fertilization, with higher to higher fertilization dose. However, even when the concentration of P in fertilized treatments is increased, the highest concentration of P was 1.62 g kg^-1 MS, on average, which does not replace the critical maintenance levels of P for ruminants that are 2.1 g kg^-1 MS, according to ^{NRC (2000)}. Therefore, supplement livestock with P-rich mineral salts in this area, will still be necessary with the use of associated meadows fertilized with this element.

In general, the greater stability of MS production of the associated meadow could lead to a stability of the animal’s body condition throughout the year, which would mean improving the production of meat, milk or calf, as well as reducing the amount of food supplement with high phosphorus content for livestock, as legumes are rich in this element. Different studies including the animal should be carried out to better understand the impact of the grass-leguminous association on the sustainability of the grassland and the animal production system, in acid soils in the humid tropical region of our country.