Study area

The experiment was developed from the third week of September, 2013, until the third week of July, 2014, at the GUMAIR forest nursery, located at the geographical coordinates 20° 09’ 08” latitude north and 98° 13’ 12” longitude west, in the municipality of Acaxochitlán, Hidalgo State, at an altitude of 2 400 masl. The area has a temperate subhumid weather with rains in the summer, a mean temperature of 15.1 °C and a mean annual precipitation of 915.5 mm (^{Conagua, 2014}).

The production area of the nursery has a 5 m high shade house-like metallic structure covered with black 50 % shade mesh; 1.5 m wide and 80 cm tall metal tables for trays, anti-weed plastic mesh (ground cover) on the floor surface, fixed micro sprinkler irrigation system, hydraulic dispenser of agrochemicals, and a deep well and a cistern for water storage.

Utilized material inputs. 77 expanded polystyrene trays with a capacity of 160 cm³ , a planting density of 360 plants m^-2 . Pinus pseudostrobus seeds collected in forests of the municipality of Chignahuapan, Puebla State. The substrate treatments were S1 = weathered pine sawdust (60 %), composted pine bark (15 %), peat moss (15 %) and vermiculite (10 %), and S2 = fresh pine sawdust (70 %), composted pine bark (15 %), peat moss (15 %) and vermiculite (10 %). Two controlled release fertilizers with two different nutrient release periods were applied: 8 and 4 month Multicote^®, with the formula 18N - 6P₂ O₅ - 12K₂ O + 2M_gO + micro nutrients (Haifa Chemicals Ltd.), and 8 to 9 month and 5 to 6 month Osmocote Plus^®, with the formula 15N - 9P₂ O₅ - 12K₂ O + micro nutrients (Everris NA, Inc.). The label of their comercial packaging warns that the periods indicated in months are considered as occurring at a constant temperature of the soil or the substrate of 25 °C. They also clarify that the temperatura goes down, in average, to 15 °C during one month, and up to 26 to 27 °C during one month.

In addition, a slow release fertilizer (without polymer coating) made up by micro nutrients -named Micromax^® (Everris NA, Inc.)- was utilized to prevent nutritional deficiencies in the plants.

Experimental design. With the combination of substrates and fertilizers, 12 treatments were generated (six for each substrate and six for each commercial fertilizer brand). In all of them, a single dose of 8 g L^-1 and 1 g L^-1 of the Micromax^® fertilizer was applied to the substrate (Table 1).

Table 1 Treatments for the production of Pinus pseudostrobus Lindl. plants in polystyrene trays. 

T = Tratamiento; S = Sustrato; S1 = Aserrín de pino intemperizado, orteza de pino compostada, turba de musgo y vermiculita, 60, 15, 15 y 10 %; S2 = Aserrín fresco de pino, corteza compostada de pino, turba de musgo y vermiculita, 60, 15, 15 y 10 %; M = Periodo de liberación de nutrimentos en meses.

Each treatment included four repetitions for a total of 48 trays, which were placed at random on one of the tables at the center of the production area in order to minimize edge effects. The total number of plants produced was 3 696.

Production management

Chemical pruning of the side roots previously to the filling of the trays with the substrates was promoted through the impregnation of the inner walls of the cavities with a 7 % copper hydroxide solution, which has been used at the nursery since 2010. Particles with a diameter of over 10 mm were eliminated from the substrates. The seeds were soaked in water during 8 hours, disinfected in a commercial 5 % hydrogen peroxide solution for 20 min, and impregnated with a dose of 3 g kg^-1 of Bactiva^® (Basilus spp. and Trichoderma spp.) in order to prevent the development in the plants of fungal pathogens such as Fusarium spp., Phytophtora spp. and Phytium spp.

Two seeds were planted in each cavity in the third week of September, 2013. The 50 % shade mesh cover was kept spread over the production area in order to protect the plants from the moment of planting to the month of April, 2014. During the development of the plant, no damages caused by pathogens or meteorological phenomena were registered.

Assessed material inputs and variables

Substrates. The physical and chemical properties of each substrate were assessed at the Soil Physics Laboratory of the Colegio de Postgraduados, with three repetitions for the following properties: mean diameter of the particles (MDP), estimated by sifting 500 g samples of the substrate during 3 min in a mechanical stirrer with 0.3, 0.5, 1.0, 2.0, 3.4, 4.8, 6.4 and 10 mm diameter sieves; apparent density (AD), obtained by dividing the dry weight of 1 L substrate samples (dried in a Riossa OHF-125 electric oven at 70 °C during 24 h) by their apparent volume; total porosity (TP), aeration porosity (AP) and water retention porosity (WRP), with the 1 L container procedure described by ^{Landis et al. (1990)}; pH and electrical conductivity (EC) in 100 mL samples of substrate dissolved in 100 mL of distilled water, with 24 h repose; organic matter contents (OM), C, N and the C:N ratio, were determined according to the procedures described in the Mexican Official Norm NOM-021-SEMARNAT-2000 (^{Semarnat, 2002}).

Morphological characteristics of the plant

The evaluation of the plants was carried out during the third week of July, 2014, ten months after planting (Figure 1).

Figure 1 Production area, treatments and repetitions with Pinus pseudostrobus Lindl. plants ten months after planting. 

20 plants were extracted at random from the central part of each tray, adding up to a total of 80 individuals per treatment. Their following variables were determined: stem diameter (D); height of the aerial part (A); dry root weight (DRW) and dry aerial weight (DAW). Using a (OHAUS, Galaxy^® 200) analytical balance after having dehydrated the plants in a (Riossa OHF-125) electric oven at 70 °C for 72 h, the total dry weight (TDW), the DAW/DRW ratio, and Dickson’s quality index (QI) were determined using the equation (^{Dickson et al., 1960}):

[ TDW / (A / D) + (DAW / DRW) ]

Experimental design and statistical analysis

A wholly random experimental design with a 2 × 2 × 3, factorial arrangement, consisting of two substrates, two CRFs and three combinations of release periods for each fertilizer, was used. The following model was utilized

Where:

A = Substrate

B = Fertilizer

C = The combinations of release periods of the fertilizers

Ɛ = Experimental error

The response values were subjected to a variance analysis and a Tukey’s mean comparison (P ≤ 0.05); furthermore, the effect of the interactions between the substrates, the fertilizers and the combinations of nutrient release periods were determined using the 2016 version of the InfoStat ^® statistical software (Di Rienzo et al., 2016).

Physical and chemical properties of the substrates

The estimated values for the physical and chemical properties of the substrates are within the intervals suggested by various authors for plant production in trays (^{Landis et al.,1990}; ^{Mathers et al., 2007}; ^{Prieto et al., 2009}), with the exception of the C:N ratio, which had high values in the two substrates, particularly in S2 made with fresh pine sawdust (Table 2).

Table 2 Physical-chemical characteristics of the substrates utilized in the production of Pinus pseudostrobus Lindl. plants in polystyrene trays. 

Substrates similar to S2 as to the predominance of fresh pine sawdust over the other material input have proven successful with forest species like Cedrela odorata L. (^{Mateo et al., 2011}) and Pinus greggii Engelm. (^{Maldonado et al., 2011}). Particularly prominent is a study of Pinus montezumae Lamb. plant production with nine substrates (^{Hernández et al., 2014}), in which the best quality was obtained using 60, 20 and 20 % peat moss, perlite and vermiculite (a mixture traditionally used in nurseries); 70, 10, 10 and 10 % fresh pine sawdust, composted pine bark, perlite and vermiculite, and 70, 10, 10 and 10 % fresh pine sawdust, peat moss, perlite and vermiculite. The third mixture has been utilized since 2003 in various nurseries of the state of Puebla and in other states of central Mexico.

Morphological variables

The average values of the assessed morphological variables were statistically equal in both substrates, with the exception of the DRW variable, for which the S2 substrate (with fresh sawdust) had a higher value (of p<0.0451) than S1 (with weathered sawdust), partly because a larger amount of micorrhizae was observed in S2. Treatments with the Osmocote Plus^® fertilizer had higher values than with Multicote^® for all the variables, except height (H), in which Multicote^® surpassed Osmocote Plus^®; this may be ascribed to the fact that Multicote^® contains 3 % more nitrogen than Osmocote Plus^®. The higher effectiveness of Osmocote Plus^® in most of the measured variables is due to the fact that this fertilizer includes better balanced contents of N, P and K, more in keeping with the needs of the species. In particular, it contains 3 % more phosphorus than Multicote^®, as stated in a study on root growth potential in Pinus halepensis Miller (^{Oliet et al., 2003}), in which three Osmocote Plus^® formulas were tried: (9N - 13P₂O₅ - 18K₂O; 17N - 10P₂O₅ - 10K₂O and 15N - 8P₂O₅ - 11K₂O). Of these, the first showed significant differences in the emission of new roots, which surpassed the emission of roots with low phosphorus formulas by 50 %.

Treatments 1, 2, 4, 5, 7, 8, 10 and 1, in which fertilizers with different nutrient release periods (I and II) were combined, evidenced significant differences (p<0.0001) in the QI and DRW and DAW/DRW variables with respect to treatments 3, 6, 9 and 12, in which a single nutrient release period (III) was used. The combination of CRFs with different release periods made it possible to increase the availability of nutrients, which resulted in plants with better balanced morphological variables than those produced with CRFs with a single nutrient release period. The best values were, in descending order, for fertilizer combinations II, I and III, respectively. In average, treatment 1 showed the most appropriate values, and the lowest were obtained with treatments 9 and 12 (Table 3).

The two treatments produced plants with a mean diameter above 5 mm and a height of 22 to 25.5 cm (Table 3). These data are within the intervals established for Pinus pseudostrobus in the Norma Mexicana para la Certificación de la Operación de Viveros Forestales (Mexican Official Norm for the Operational Certification of Forest Nurseries) NMX-AA-170-SCFI-2014 (^{SE, 2014}), which establishes a minimum diameter of 4 mm and a height of 20 to 25 cm. Therefore, it can be concluded that good quality plants were obtained with all the treatments.

Table 3 Average values per treatment for the morphological variables of Pinus pseudostrobus Lindl. plants, ten months after planting. 

T = Treatment; S = Substrate; F = Fertilizer; C = Combined nutrient release periods d; D = Stem diameter; H = Height of the plant; DAW = Dry aerial weight; DRW = Dry root weight; DAW / DRW = Dry aerial weight / dry root weight ratio; QI = Dickson’s quality index; S1 = 60, 15, 15 and 10 % weathered pine sawdust, composted pine bark, peat moss and vermiculite; S2 = 60, 15, 15 and 10 % fresh pine sawdust, composted pine bark, peat moss and vermiculite.

In another rating system, ^{Prieto and Sáenz (2011)} propose a set of parameters to rate the plant quality of the native pines of the Sierra Madre Occidental, including Pinus pseudostrobus and other non-cespitous species. The authors consider plants with morphological variables within the following intervals as high-quality plants for reforestation purposes: height, 15 to 25 cm; diameter, ≥ 4 mm; dry aerial weight / dry root weight ratio of 1.5 to 2.5; ≥ 0.5 Dickson’s quality index. In the present study, the variables have these values, with the exception of the values estimated for DAW / DRW, which were higher in all the treatments, reaching figures of 2.7 to 3.7.

The apparently high values for the DAW / DRW variable can be accounted for by the fact that the plant was produced in polystyrene trays with cavities impregnated with copper salts, which contributed to generate root systems with a central axis and thin primary and secondary roots with a diameter of less than 0.5 mm and a length of 3 cm, with a low dry weight (Figure 2).

Figure 2 Conformation of the root with chemical side pruning in Pinus pseudostrobus Lindl. plants produced in polystyrene trays. 

When the root ball of the plants is longitudinally sectioned, the architecture of the root resembles the spine of a fish, without enveloping or ascending roots, unlike individuals of other pine taxa produced in trays with cavities without treatment or not designed to propitiate chemical or aerial root pruning. Three plant quality assessment studies carried out in six forest nurseries in the states of Jalisco and Michoacán (^{Rueda et al., 2010}; ^{Sáenz et al., 2010}; ^{Sáenz et al., 2014}) yielded average values of 2.9 to 4.9 for the DAW / DRW variable in Pinus pseudostrobus plants grown in polystyrene trays with 160 cm³ cavities impregnated with copper salts. ^{Sword et al. (2011)} assessed the effect of chemical pruning and of the size of the cavities of the polystyrene trays on production (Pinus palustris Mill.), and found a reduction of 18 % in the dry weight of the root of plants produced in trays with 160 cm³ cavities and chemical pruning, compared to the same size cavities but without chemical pruning.

Based on the results described above, it may be said that the seemingly low values for the dry weight of the plant roots are a normal condition due to the chemical pruning of the primary and secondary roots; however, this characteristic is not indicative of low quality of the plants’ root system.