Germplasm and substrate

The germplasm of Pinus ayacahuite var. veitchii was acquired at the seed bank of the Protectora de Bosques del Estado de México (Forest Protection Agency of the State of Mexico) (Probosque); the seeds were collected, on April 3^rd and 4^th, 2014, at La Ciénaga, in Santa Cruz Pueblo Nuevo, Tenango del Valle, State of Mexico, with the batch number 216104914 GC- 2015. The germination percentage once out of the bank was 93.33 to 99.86 %.

The compost was made four months in advance, using straw, weeds, goat manure, and green and dry leaves; the vermicompost consisted of cow dung from a livestock farm located in the city of Tlapa de Comonfort, Guerrero; and the forest soil was collected at a pine-oak forest of Xocoapa, in Tlacoapa, Guerrero, eight days before planting. Copper Block expanded polystyrene containers with 77 cavities, with a volume of 170 cm³, a length of 60 cm, a width of 35 cm, and a height of 16 cm.

Sowing and irrigation

The substrate was irrigated at field capacity 12 hours before the sowing, which was carried out on October 20^th, 2015; for this purpose, a seed was manually placed in each cavity at a depth of 1 cm (^{Musálem and Ramírez, 2003}). The first irrigation was applied on the day of the planting; after the seedling emerged, it was watered every other day (^{Romero-Arenas et al., 2013}), with an aspersion system installed at the nursery.

Experimental design

The experimental design was fully random, with four treatments and a control, with four repetitions of each treatment. Each was applied in a tray with 77 cavities, during the germination phase, for a total of 20 experimental units. The substrate mix for each treatment was formulated based on three components: forest soil (fs); vermicompost (vc) and compost (cp); CT (100 % fs); T1 (50 % fs + 50 % vc); T2 (50 % fs + 50 % cp); T3 (50 % fs + 30 % vc + 20 % cp), and T4 (50 % fs + 20 % vc + 30 % cp). During the growth phase, 120 seedlings per treatment, and 30 per tray were selected at random; a period of 174 days was considered as the lowest survival.

Germination, growth and slenderness ratio

Germination in Pinus ayacahuite var. veitchii had a duration of 30 days; in order to keep better control of the experiment, periodical quantifications were carried out at 5, 10, 15, 20, 25 and 30 days. The growth of the seedlings was assessed on April 11, 2016, i.e. 174 days after the planting. The height was measured in centimeters (cm) with an ACME DR30 aluminium rule calibrated in centimeters from the substrate level to the tip of the plant; the diameter was measured in millimeters (mm) from the stem base with a Mitutoyo CD-6”CSX digital vernier caliper. The slenderness ratio was calculated based on the height quotient in cm divided by the stem diameter in mm (^{Mateo et al., 2011}).

Statistical analysis

Variance analyses were carried out for the variables germination (%), stem diameter (mm), height (cm) and slenderness ratio. When significant differences (p< 0.05) between treatments were observed, a Tukey’s mean comparison test was carried out (^{Steel and Torrie, 1980}). All analyses were performed using the IBM SPSS Statistics software, version 20 (^{SPSS, 2011}).

Germination

In general, P. ayacahuite var. veitchii completed its germination at 30 days. The variance analysis indicated a difference between treatments (p = 0.009) (Table 1). T1 (50 % fs + 50 % vc) registered the highest germination percentage, i.e. 84.73 % (Figure 2); this value is similar to that cited by ^{Villagómez and Carrera (1985)}, of 80 %. The result of ^{Romero-Arenas et al. (2013)}, who used a substrate of 50 % walnut shell compost + 25 % agrolite + 25 % vermiculite for the germination of Pinus patula, was 84.25 %. In contrast, ^{Quiroz et al. (2008)} obtained 73.2 % germination in Pinus pinea L. with the use of composted pine bark.

Germinación = Germination; Tratamiento = Treatment

The vertical lines represent the standard deviation; different letters above the bars correspond to statistical differences (p≤0.05).

Figure 2 Germination of Pinus ayacahuite var. veitchii (Roezl) Shaw at 30 days. 

On the other hand, the CT reached only 54.70 % of germination, a much lower percentage than the high germination values registered by ^{Aparicio et al. (1999)}, with 100 % forest soil, for Pinus patula (92 %), Pinus montezumae Lamb. (82 %) and Pinus pseudostrobus Lindl. (78.2 %). For T2 (50 % forest soil + 50 % compost) had 74.03 % germination, while Jatropha spp. had 95 % (^{Martiñón and Aragón, 2014}).

The variance analysis by time point indicated significant differences only at 5 days (p<0.05), for the rest of the time germination was statistically equal (Table 1); notably, T1 had a higher percentage (16.56 %), above the mean of the other treatments, which attained a mere 6.57 % at 5 days (Figure 3).

Germinación = Germination; Tiempo (días) = Time (days)

Figure 3 Accumulated germination in Pinus ayacahuite var. veitchii (Roezl) Shaw at 5, 10, 15, 20, 25 and 30 days. 

As shown in Figure 3, the most active period in the germination phase occurred at 10, 15 and 20 days, while the growth became stabilized at 25 and 30 days, when the last germinations took place, until the process was completed, at 30 days. The first germinations occurred on the third day, unlike the 5 and 6 days registered by ^{Villagómez and Carrera (1985)} for the first germinations of the same taxon.

T1 remained the best treatment throughout the experimental phase, while CT began slightly above T3, at 5 and 10 days, and was surpassed by T3, at 15, 20, 25 and 30 days; eventually, CT was below all other treatments (Figure 3).

Diameter, Height and Slenderness ratio

Generally, Pinus ayacahuite var. veitchii registered a growth in diameter of 2.07 mm, a height of 5.096 cm and a Slenderness ratio of 1.865; differences between treatments occurred at 174 days for the three variables, according to the variance analysis (Table 2).

In the Tukey mean comparison test, T4 had the largest diameter, of 2.85 mm, followed by T3, with 2.78 mm, while T2, T1 and CT were statistically equal, with diameters of 2.71, 2.66 and 2.58 mm, respectively (Table 4). The values for T4 are similar to those cited by ^{Chávez et al. (2014)}, of 2.9 mm at 10 months in Pinus patula with a composted pine bark substrate; conversely, ^{Altamirano (2002)} registered the highest growth rates (3.46 mm), with 30 % mine sand + 20 % forest soil + 50 % vermicompost, seven months after the transplanting of Pinus ayacahuite. Finally, ^{Sáenz et al. (2010)} registered values of 5.2 to 5.6 mm in annual production cycles (75 % forest soil + 25 % compost) for Pinus ayacahuite; the difference is that fertilization was applied in this research, and the plants were produced in 10 cm × 20 cm black polyethylene bags.

T4 (50 % fs + 20 % vc + 30 % cp) exhibited the largest height, of 5.73 cm; in contrast, the lowest measures, of only 4.82 and 4.58 cm, were for T1 and CT, respectively (Table 4). ^{Altamirano (2002)} documented a height of 15.68 cm, with 30 % mine sand + 20 % forest soil + 50 % vermicompost, seven months after the transplanting of Pinus ayacahuite in 18 × 25 cm black polyethylene bags. In the present study, the CT yielded lower values than those obtained by ^{Altamirano (2002)} when assessing the initial growth of Pinus oaxacana Mirov. and Pinus rudis Endl., of 6 and 5.16 cm, respectively. Values of 20.88 and 24.34 cm in height were cited for the annual production cycle in Pinus ayacahuite, when fertilized every two weeks, from January to May, with 18-46-00 and 46-00-00, each time in doses of 4 kg 200 L^-1 of water (^{Sáenz et al., 2010}).

The Slenderness ratio between treatments ranged between 1.72 and 2.00, with a marked statistical difference in T1 and T4 (Table 3), which suggests a greater size and quality of the seedlings in T4. This is similar to the ratio of 2.01 cited by ^{Sallesses et al. (2015)}, with a 100 % chicken-manure compost substrate, for the hybrid of Eucalyptus grandis × Eucalyptus camaldulensis. In contrast, ^{Sáenz et al. (2010)} cite high values of 4.16 to 4.18 for the annual production of Pinus ayacahuite with fertilizers 18-46-00 and 46-00-00, applied every two weeks.