Location and characteristics of the study area

The research was carried out in the Valle de Ameca Forest Nursery ("Vivero Forestal Valle de Ameca S. P. R. de R. L."), located at 20°33' N and 104°3' W, at an altitude of 1 235 m, in Ameca, Jalisco, Mexico. The climate is semi-warm subhumid, with summer rains, with a medium humidity, temperatures ranging from 16 to 24 °C, and an annual precipitation of 800 to 1 100 mm (^{Inegi, 1999}). The experiment was performed simultaneously for P. douglasiana and P. devoniana.

The biological material utilized for starting the evaluation consisted of P. douglasiana and P. devoniana plants aged 12 months, produced in polystyrene trays with 60 cavities of 0.165 L each, from the Valle de Ameca forest nursery. The morphological characteristics and foliar mineral content of the plants at the beginning of the experiment (Table 1), were determined at the Laboratorio Forestal of the Departamento de Madera, Celulosa y Papel of the Universidad de Guadalajara and at the Laboratorio de Nutrición Vegetal of the Colegio de Postgraduados, Campus Montecillo, respectively. The culture conditions were established in the protocols of the same nursery for the production of these conifers.

Table 1 Morphological characteristics and foliar mineral content of the Pinus douglasiana Martínez and Pinus devoniana Lindl. species at the beginning of the evaluation. 

The value indicated between parentheses corresponds to the standard error of three repetitions; Dg = P. douglasiana; Dv = P. devoniana.

Black polypropylene (1 and 5 L) containers, which have a special design for the development of conifers (semi-conical, with inner vertical ribs and with the base covered only with a gridded mesh), were utilized. The 1 L container has 18 mm thick walls, a length of 18.5 cm, 10.7 cm of higher diameter, and 8.2 cm of lower diameter. The dimensions of the 5 L container are: 152 mm of wall thickness, 45 cm of length, 17.8 cm of higher diameter and 14.5 cm of lower diameter.

The substrate consisted of a mixture of peat moss (30 %), pine bark (70 %) and Multicote™ 24-12-6-(4) (6 kg m^-3), and was characterized at the Laboratorio de Física de Suelos y Nutrición Vegetal del Colegio de Postgraduados; the results indicate a total porosity of 89 %, an aeriation porosity of 1 %, a capacity for water retention of 78 %, a particle size (weighted mean diameter) of 2 to 3.36 mm and an apparent density of 0.18 g cm^-3. The chemical analysis is shown in Table 2. A broad spectrum fungicide (BUSAN 30 WB, TCMTB-ticino methylthio benzotriazol) was preventively applied in order to avert damage by pathogens.

Table 2 Chemical characterization of the substrate utilized for transplanting into the containers. 

EC = Eletric conductivity; CEC = Cation exchange capacity; OM = Organic matter; TN = Total nitrogen; P= Phosphorus; K= Potassium; Mg= Magnesium; Ca= Calcium; Na = Sodium; Cu= Copper; Fe= Iron; Mn= Manganese.

Experiment establishment and follow-up

The plants of both species produced in trays were transplanted to 1 and 5 L containers; for this purpose, 500 plants per taxon were selected at random. After three days, Raizone-plus rooting powder (40g in 25 L of water) was sprayed with a manual pump. The transplanted plants were kept during one month under a 50 % shade cloth and watered every day in a, even, localized manner. Subsequently, they were transferred to the growth area of the same nursery (out in the open), under the Double-Transplanting system (^{Salcedo et al., 2012}).

After a month, the four treatments were established for each species, considering the effect of the two container sizes (1 and 5 L) and the two fertilization factors (with and without fertilizer). 125 individuals were used for each one. A solution consisting of magnesium nitrate (40 g), calcium nitrate (40 g), monopotassium phosphate (50 g) nitro-potassium (50 g), urea (40 g) and Gro-gree^® (20-30-10 + EM) (40 g), dissolved in 25 L water, was added twice a week to the fertilized plants and applied to a surface area of 527 m². Furthermore, 10 g of Haifa^® Multi-micro were administered once a week to each individual. The management of the fertilizers was carried out within the procedures typically used at the nursery.

During the five months of the evaluation, all the plants were manually watered on a daily basis in an even and localized manner until the substrate reached the saturation point (1.16 L sec^-1 hour).

Experimental design and treatments

The treatments for each species were distributed in a random experimental block design, with a 2² factorial arrangement, in which factor A represented the container size (1 and 5 L), and factor B, fertilization (with and without fertilizer). Each treatment consisted of three repetitions, with sets of five trees as experimental units (15 plants per treatment). Four treatments for each taxon, classified as follows: Dg1NF (1 L container without fertilizer), DgIF (1 L container with fertilizer), Dg5NF (5 L container without fertilizer) and Dg5F (5 L container with fertilizer), for P. douglasiana, and Dv1NF (1 L container without fertilizer), Dv1F (1 L container with fertilizer), Dv5NF (5 L container without fertilizer) and Dv5F (5 L container with fertilizer), for P. devoniana.

Evaluated variables

The morphological variables and the mineral content of each species were evaluated through a destructive sampling (15 plants per treatment). The plants were retired from the container removing the substrate from the root, and the aerial part was separated from the root. The data were recorded six months after the plants were transplanted. The assessed variables were: height from the stem base to the stem apex (cm), the stem diameter at root collar (mm), as well as aerial and root biomass. The aerial and root volumes (cm³⁾ were determined by the water displacement method (^{Harrington et al., 1994}).

The samples of the aerial and root parts were placed separately in paper bags and dried in a rustic drying chamber at 70 °C during 72 hours, until a constant weight (72 h) was registered. The dry aerial (g) and root (g) weights were determined in an analytical scale (Sartorius, MP6 model). These data were used to estimate: the ratio between the aerial and the root parts, Dickson's quality index and the index of robustness (^{Dickson, et al., 1960}; ^{Thompson,1985}). The root container index (ICR) was also determined, and this is an additional contribution of this study. The ICR represents the quotient between the volume of the container (cm³⁾ and the root volume (cm³⁾. It is an indicator that makes it possible to predict the quality of the plant in that container volume and, therefore, the survival rate in field.

The dried and ground needle samples (model SK100 Retsch mill) were used to estimate the foliar mineral content at the Laboratorio de Física de Suelos y Nutrición Vegetal del Colegio de Postgraduados. The semi-microkjedahl method was used for the total nitrogen, with previous acid digestion of the samples, while for phosphorus, potassium, calcium, magnesium, sulfur, iron, copper, zinc, manganese, boron and molybdenum, a humid digestion was utilized. Subsequently, the extracts were analyzed in a Varian™ Liberty II ICP-AES induction spectrophotometer with coupled plasma (^{Alcántar and Sandoval, 1999}).

Statistical analysis

The data were organized using the Microsoft Office 2007 Excel software package and applying a normalcy test (Chi Square and Shapiro's and Wilk's W statistic). Subsequently, a variance analysis (ANOVA) was carried out following the 2² factorial model, using Statgraphics Centurion XV.II, version 15.2.06. Where differences between treatments (p≤0.05) were observed, means were compared using the Least Significant Difference (LSD) and, finally, a Pearson's correlation.

Morphological variables and quality indices for Pinus douglasiana

The variance analysis evidenced significant differences (p≤0.05) in all the variables, except height, as an effect of the container volume (Table 3), while the use of fertilizers significantly affected the aerial volume and both the aerial and root weights. The interaction between the container size and the use of fertilizers was significant only for the aerial volume and root weight variables.

Table 3 P-value of the ANOVA for height, root neck diameter and biomass of Pinus douglasiana Martínez with or without foliar fertilization in two container volumes. 

(*) Significance with a 95.0 % confidence level; gl = Degrees of freedom.

Figure 1 shows the average increases in the morphological variables of plants subjected to the various treatments in relation to the baseline values recorded in Table 1; a clear difference may be observed between the plants in the 1 L and 5 L containers, except for the height. According to the statistical analysis, the Dg5NF had the highest values for aerial volume and aerial and root weight (Table 1). In this sense, ^{Becerra et al. (2013}) recorded survival rates of up to 90 % in plants aged 4 years belonging to six forest species and produced in 10 L containers, assessed two years after their establishment in the field.

Dg1NF = 1 L container without fertilizer; Dg1F = 1 L container with fertilizer; Dg5NF = 5 L container without fertilizer); Dg5F = 5 L container with fertilizer. (*) Significant treatment based on ANOVA, n=15, P<0.05.

Figure 1 Increase in the values of morphological values in relation to the baseline values for Pinus douglasiana Martínez. 

Figure 2 shows the value of the quality indices for P. douglasiana. The plants grown in the larger containers showed a significant difference in Dickson's quality index (QI) and in the root container index (ICR); however, just for the QI there were differences in relation to the use of fertilizers, with a higher value in the 5 L treatment without fertilizer (5.03) within the range of the optimal values established by Dickson et al. (1960) and ^{Sáenz et al. (2010)}.

Treatments: Dg1NF = 1 L container without fertilizer; Dg1F = 1 L container with fertilizer; Dg5NF = 5 L container without fertilizer); Dg5F = 5 L container with fertilizer There are no significant differences between means followed by the same letter (n=15, P<0.05, LSD).

Figure 2 Response to the treatments in terms of the quality indices for Pinus douglasiana Martínez. 

Based on the results obtained, the QI turned out to be the best index for determining the plant quality for P. douglasiana. According to ^{Quiroz et al. (2014)}, the QI is highly correlated with the root volume, and this, in turn, is related to the container volume. Therefore, an increase in the value of the QI is associated with a better plant quality, as a result of a better balance between the aerial and root biomasses (^{Reyes et al., 2005}, Saénz et al., 2010). In this respect, we may point out that the P. douglasiana plants transplanted from trays to large containers and kept under nursery conditions during six months will have a better development without any need to apply fertilizers, and therefore, will have better survival rates in field.

Nutrient content of Pinus douglasiana

The minerals that showed variation due exclusively to the size of the container were nitrogen, iron, copper and manganese, with a tendency to have a higher content in individuals produced in a 5 L container, except for copper (Table 4); this means that the concentration of nutrients in the needles increases with the volume of the container (^{Jeong et al., 2010}).

Table 4 Nutrient content in dry needle samples of Pinus douglasiana Martínez. 

Dg1NF = 1 L container without fertilizer; Dg1F = 1 L container with fertilizer; Dg5NF = 5 L container without fertilizer; Dg5F = 5 L container with fertilizer There are no significant differences between means followed by the same letter in a column (n=15, P<0.05, LSD). (*) Proposed by ^{Landis et al. (1989)}.

The results of the analyses (Table 4) show that the container volume also had an effect on the availability and absorption of some nutrients; the treatments were grouped according to container size, according to the values obtained for the 5 L containers, which, except for copper, are within the range of the foliar nutrient content recommended by ^{Landis et al. (1989)}. In this regard, these elements are considered to be the most important in the photosynthesis and breathing processes (^{Alcántar et al., 2012}); therefore, they are decisive for the fast growing conifer species, as quoted by ^{Nambiar and Sands (1993)}, who pointed out that this botanical group has a higher growth rate with high nitrogen contents.

The Dg1F treatment registered a higher nitrogen concentration due to the restriction of biomass development by the container. This is because smaller containers restrict plant growth, and therefore reduce the availability of water and nutrients (^{Domínguez et al., 2006}).

Although nutrient content levels were higher in 5 L containers, the effects of the treatment with and without fertilizer (Dg5F and Dg5NF, respectively) were the opposite of those obtained with 1 L containers. This may be because larger spaces allow greater development of root biomass in fast growing species like P. douglasiana, and when these seek to meet their nutritional needs, the addition of fertilizers causes an adverse effect on the absorption of minerals (^{Rodríguez, 1982}). Thus the growth of the morphological variables is affected, as documented by ^{Domínguez et al. (2006)}, who point out that the increase in the nitrogen input (250 mg L^-1, in solution) has a negative impact on root growth and results in a lower number of new roots. For example, according to ^{Aldana and Aguilera (2002)}, fast growing conifers require the addition of 50 to 75 ppm of nitrogen.

Within this context, the application of 6 kg of fertilizer (Multicote™) to each cubic meter of substrate is sufficient for the production of P. douglasiana in 5 L containers and under the conditions of this study; however, for 1 L containers, additional fertilizers must be applied.

Correlation between the morphological variables and the foliar nutrient content in Pinus douglasiana

The iron concentration in the needles evidenced a positive relation with the tree height (r=0.99*), as this element accelerates the transportation of electrons for the photosynthetic process and is involved in the synthesis of chlorophyll, as well as in the functioning and structure of the chloroplast (^{Terry and Abadía, 1986}; ^{Alcántar et al., 2012}).

A positive association was found to exist between manganese and stem diameter (r=0.97*), aerial volume (r=0.99*) and aerial weight (r=0.98*). Although the action mechanisms of this element are not known in detail, we do know that it is involved in ion absorption, photosynthesis, breathing and protein synthesis (^{Alcántar et al., 2012}). Therefore, the results indicate that manganese must be regarded as an essential nutrient for the growth of aerial biomass in Pinus douglasiana plants during the initial stages of their development.

Morphological variables and quality indices in Pinus devoniana

Table 5 shows that all the morphological variables had a significant effect (P<=0.05) as a result of the container size, while the use fertilizers produced significant effects only on the aerial and root volumes. The interaction had no significant effects on any of the variables.

Table 5 P-value of the ANOVA for height, stem diameter and biomass of Pinus devoniana Lindl. with or without foliar fertilizer with two different container volumes. 

(*)Significance with a 95.0 % confidence index; gl = Degrees of freedom.

The information generated agrees with the findings of ^{Domínguez (2006)} and ^{Ortega et al. (2006)}, who registered a greater effect on the performance of plants in the nursery due to the container volume than to the use of fertilizers. According to ^{Jeong et al. (2010)}, the application of foliar fertilizers had a significant effect on the growth of P. densiflora and P. thunbergii in larger containers; this was reflected on the biomass production. In addition, a larger container volume admittedly allows the development of a larger root volume in conifer species, whereby the absorption of water and nutrients is favored. The trees will therefore have a higher growth potential and a larger biomass production (^{Landis et al., 1994}; ^{NeSmith and Duval, 1998}; ^{Cañellas et al., 1999}; ^{Hess and De Kroon, 2007}; ^{Prieto et al., 2007}). ^{South et al. (2004)} and ^{Domínguez et al. (2006)} mention that container type and size determine the time period during which the plants can be kept in the nursery without the occurrence of any damage such as strangling of the root collar or limited access to water and nutrients, compared to plants grown in larger containers.

Based on this, it may be pointed out that, despite the use of larger containers, the benefits of Double-Transplanting system in economic and operational terms are unquestionable. The results and advantages obtained with it in the long term may render it an attractive alternative for establishing reforestations in sites with special or adverse edaphoclimatic conditions (^{Salcedo et al., 2012}).

Figure 3 shows that the treatments with fertilizers in both container sizes produced greater increases in most variables, unlike treatments without fertilizers -which is the opposite effect to that obtained for P. douglasiana. The favorable response of the plants to the use of fertilizers, regardless of the container size, indicates that the nutrients that were part of the substrate were insufficient to meet all the nutritional needs of the plants during the initial stages of their growth. Finally, the difference of behavior between species as an effect of the use of fertilizers clearly shows that each of the two species has specific nutritional needs. The current research considers that these differences are related to the contrasting growth habits of the utilized taxa.

Dv1NF = 1 L container without fertilizer; Dv1F = 1 L container with fertilizer; Dv5NF = 5 L container without fertilizer); Dv5F = 5 L container with fertilizer; (*) Significant treatment based on ANOVA, n=15, P<0.05.

Figure 3 Increases in the morphological variables in relation to the baseline evaluation in Pinus devoniana Lindl., as an effect of the various treatments. 

In the case of P. devoniana, the treatments were not clearly grouped by the effect of the container volume but by the effect of the use of fertilizers, although this was different for 1 L and 5 L containers. In general, the effect of the application of fertilizers reflected an increase in the content of nutrients in 1 L containers (Dv1F), and a decrease in 5 L containers (Dv5F).

As for the quality indices, both Dickson's and the root container index showed a significant effect of the container size and the use of fertilizers (Figure 4).

Dv1NF = 1 L container without fertilizer; Dv1F = 1 L container with fertilizer; Dv5NF = 5 L container without fertilizer); Dv5F = 5 L container with fertilizer; (*) Significant treatment based on ANOVA, n=15, P<0.05.Dv1NF = 1 L container without fertilizer; Dv1F = 1 L container with fertilizer; Dv5NF = 5 L container without fertilize; Dv5F = 5 L container with fertilizer. There are no significant differences between measures followed by the same letter (n=15, P<0.05, LSD).

Figure 4 Response of the quality indices of Pinus devoniana Lindl. to the treatments. 

The quality indices (Figure 4) in all the treatments turned out to be adequate for Dickson's index (>0.5) (^{Dickson et al., 1960}; ^{Sáenz et al., 2010}); however, the highest values were determined for treatments Dv5NF and Dv5F, with 8.55 and 7.32, respectively. The root container index (ICR) showed differences between treatments, with a tendency to attain higher values with the Dv5NF and Dv5F treatments (1.16 and 162.00, respectively). Based on this, ICR is said to be a good quality indicator for plants to be transplanted to the field. In this respect, a study by ^{Bernaola (2012)} on Pinus hartwegii Lindl. in nursery conditions pointed out that higher ICRs (27.5 and 125.0) result in higher survival rates (13 and 94 %, respectively) two years after the plants have been established in the field.

Nutrient content of Pinus devoniana

Table 6 summarizes the effects of the treatments on the mineral content. Only the nitrogen content was affected by the container size, with a tendency toward a higher concentration in the 5 L containers, but without significant differences between the treatments with and without fertilizers (1.0 and 0.92). The rest of the minerals (calcium, iron and copper) attained higher values in the 1 L containers; however, only the calcium and iron contents increased in the fertilized plants.

Table 6 Nutrient content of dry Pinus devoniana Lindl. needles. 

Dv1NF = 1 L container without fertilizer; Dv1F = 1 L container with fertilizer; Dv5NF = 5 L container without fertilize; Dv5F = 5 L container with fertilizer. There are no significant differences between measures followed by the same letter (n=15, P<0.05, LSD). (*) Proposed by ^{Landis et al. (1989)}.

The content of nitrogen obtained with the treatments in 5 L containers (with and without fertilizer) is due to the fact that the amount of nitrogen in the substrate was sufficient to meet the nitrogen demand of the trees; these results agree with the assessment by ^{Soriano (2011)} in P. devoniana and P. patula Schiede ex Schltdl. et Cham.; according to Soriano, the effect of nitrogen on the variables height, diameter, dry foliage weight, total biomass and Dickson's quality index was highly significant. ^{Gough et al. (2004)} point out that the use of fertilizers has a direct effect on the photosynthetic capacity, better growth and higher biomass production in P. taeda L. On the other hand, low nitrogen contents in 1 L treatments are due to the smaller amount of substrate and of fertilizer used in them.

Given the importance of nitrogen-rich fertilizers for P. devoniana, according to ^{Aldana and Aguilera (2002)} a daily dose of 75 ppm of nitrogen promotes greater growth in height, diameter and biomass accumulation in P. devoniana; this agrees with the treatment with fertilizers containing a higher dose of nitrogen.

Lower calcium contents in the needles of plants grown in 5 L containers are due to the fact that the calcium was meant to contribute to the structural functions of the plants, rather than for its accumulation in the needles; this is reflected in higher increases in all the growth variables with these treatments, compared with the 1 L treatments, in which the high calcium content is associated to its scarce mobility. Therefore, calcium was accumulated in the needles and the plants showed lower increases of the said variables (^{Alcántar et al., 2012}).

Correlation between the morphological variables and the foliar nutrient content in Pinus devoniana

The correlation analysis between the morphological variables and the foliar nutrient content evidenced that the concentration of nitrogen and copper in the needles had a positive effect on the growth in height (r=0.97*). This is because nitrogen is a major component of proteins and nucleic acids. Furthermore, it is involved in the photosynthesis and breathing of the plant. Copper is part of the metabolism of secondary compounds and favors the development of biomass in pine trees (^{Jeong et al., 2010}; ^{Alcántar et al., 2012}). Manganese had a favorable effect on the increases in aerial weight and root container index (r=0.96*); however, its values are not shown in the results table because they had no statistical difference (ANOVA >0.05). Manganese is also involved in the photosynthesis, breathing and synthesis of proteins (^{Alcántar et al., 2012}).