Study area

“Tetela del Volcán” forest nursery depends of the Secretaría de Desarrollo Sustentable (SDS) (Department of Sustainable Development) of the Gobierno del Estado de Morelos (Morelos State Government) and is located at the municipality of Tetela del Volcán, between the geographic coordinates 18°52’27.25” N and 98°42’54.27” W (Figure 1).

Figure 1 Location of Tetela del Volcán municipality, Morelos State. 

There are four kinds of climate in the municipality, according to altitude. The area where the actual study took place is located at 3 200 masl, has a sub-humid semi-cold climate with summer rains, average annual temperature from 16 to 17 °C and 1 073.2 mm average annual precipitation (^{Inegi, 2009}).

Plant production

The plant used for the present work was cultivated in trays of 49 cavities with 150 ml capacity per container. The substrate was a mixture of moss peat (42 %), vermiculite (31 %), agrolite (27 %) and 4 kg m-3 of Osmocote^™ 15-9-12 granular fertilizer. At the establishment stage, Initial Ultrasol^™ fertilizer 15-30-15, 112 ppm N, 99 ppm P and 93 ppm K was applied. In the growth stage, Ultrasol^™ 20-10-20 fertilizer was applied, with 144 ppm N, 32 ppm P and 120 ppm K. For the hardening step, the Peters fertilizer finer 4-25-35, with 26 ppm N, 70 ppm P and 186 ppm K was applied. Mycorrhizal inoculum (Glumix^™) was applied to the sowing and to seedlings 20 days later, with 15 g per cavity in each application. As seedlings were not planted, they remained 22 months in the nursery.

Plant evaluation at the nursery

The evaluated plants were randomly selected from the nursery platforms. According to ^{Rojas (2002}), the suggested sampling intensity is 0.5 % for populations greater than 50 000 plants. However, he recommends lowering this intensity when it comes to technically-grown nurseries with homogeneous production. In this work a sample of 0.33 % was obtained, and thus 200 plants of the 60 000 that were produced were selected. The height (cm), the diameter of the neck (mm) were measured; the percentage of mycorrhization was estimated, assigning a scale value of 0 to 100, where zero equals the apparent absence of mycorrhizae in the root ball and 100 to be completely filled with mycorrhizae.

Aboveground and radical architecture was also evaluated by observation. A value of 3 was given if there was no damage, 2 if 30 % of the seedlings had any, and 1 if more than 50 % of the plant had. The plants were sent to the reforestation site.

Plant evaluation in the laboratory

In this evaluation a destructible random sample of 0.16 % of the total plants produced was handled. 96 seedlings were taken to the Laboratorio de Semillas Forestales (Forest Seeds Laboratory) of the División de Ciencias Forestales (Forestry Division) of the Universidad Autónoma Chapingo (Chapingo Autonomous University) to measure them.

The same measurement procedure was performed as in the nursery. In addition, the root mass was removed to clean the root, measure its length (cm) and count the secondary roots > 1 cm.

To continue with the evaluation, the needles were separated from the stem, the stem from the root, and each part was weighed separately. They were then pocketed and labeled. The bags were placed inside the drying kilns of the laboratory, at a constant temperature of 75 °C. After 5 to 7 days, weight became constant. Each of the samples was then weighed to obtain total dry weight, as well as that from foliage, stem and root per plant with an Ohaus Navigator scale.

Plant quality indicators

Most of these indicators, and their models, were taken from ^{Duryea (1985b}) and ^{Landis et al. (2010}). The following attributes were considered: plant height, plant neck diameter, percentage of mycorrhizae, shoot and root architecture. In addition, the following indicators of the destructible sample were obtained: main root length and number of secondary roots > 1 cm

To determine the relation between aboveground and root dry weight (A/S), the following model was used:

A/S = PSA/PSS

Where:

PSA= Dry weight of the aboveground part

PSS = Dry weight of the root

The relative weight of the aboveground part (PR_A) was calculated as follows:

PR _A = PSA/PST

Where:

PSA= Dry weight of the aboveground part

PST = Total dry weight

The relative weight or of the root or the underground part (PR_R) was determined with the following model:

PR _R = PSS/PST

Where:

PSS = Dry weight of the root

PST = Total dry weight

The Slenderness Coefficient (CE) was calculated with the following model:

CE= (A/D)

Where:

A = Height of the plant

D = Diameter of the plant

For Dickson’s Index (ID) the following model was used:

ID = PST / ((PSA/PSS) + (A/D))

Where:

PST = Total dry weight

PSA = Dry weight of the aboveground part

PSS = Dry weight of the root

A/D = Slenderness coefficient

With the data that were obtained in this phase, a Pearson’s Correlation was made, by means of the SAS program (^{SAS Institute, 2012}). In addition to evaluating the former indicators, also the following tests for plant quality were made.

Potential root growth test. Four plastic pots of 4 l each were used for this test, which were filled with a mixture of substrate made up by 1/3 agrolite, 1/3 vermiculite and 1/3 peat moss. Afterwards, three seedlings were put into each one. The pots were left inside the greenhouse, where they were watered every two days (at a temperature of 23-28 °C, with a relative humidity of 60-75 %). After that month, they were taken out of the container, with the aim to count the roots larger >1 cm. Such new roots were identified as they were white and turgid (^{Landis et al., 2010}).

Water stress test. The Scholander chamber (model 1000 PMS Instrument) was used for this test. Ten plants were used, from which was taken as a simple the high part of their stem with a crosscutting procedure. The sample was inserted in the tap of the chamber, which closes another in which the sample is placed, and to which a N gas pressure was applied. In this way the necessary pressure is made to start the extraction of water from the sample (^{Landis et al., 2010}). The first assessment was made to five plants after watering and to the other five, seven days later they were measured, without any more watering during that period, which led to water stress. From the night before they were measured, all the plants were kept in a fresh dark room. For the statistical analysis of this test, a Student’s t test was performed in order to compare the means of the plants under the two applied treatments.

Reforestation assessment

The plantation was established in August 2013 and its evaluation was carried out in June 2015, in a period of 22 months. The height, diameter, and vitality condition of each of them (living or dead) were recorded.

Plots were located in the upper part (3 260 masl) and in the low one (3 170 masl) of a hill, in forest soils, of volcanic origin, in north and south exposure, three in each combination of altitude and exposure, i. e. 12 plots in total. It was a 2 × 2 factorial experiment. Each plot consisted of a plantation frame of 7 × 7 plants, to evaluate only the central part, 5 × 5 (= 25 plants) to avoid the possible border effect. Planting density was 1 200 ha^-1. Therefore, 12 plots with 25 plants each (300 plants in total) were measured; the data were subjected to an analysis of variance using the SAS program (^{SAS, 2012}).

Plant evaluation in the laboratory

Although it is a plant that spent almost two years in the nursery, it exceeds the basal diameter standards established for the species, from 6 to 11.5 mm (^{Bautista et al. 2005}; ^{Hernández et al. 2014}) or for P. devoniana, with up to 13.5 mm (^{Sáenz et al., 2014}; ^{Bernaola et al., 2015}); both species remained in the nursery for a year in similar containers, ranging from 160 to 220 cm³.

The calculated PSA / PSS ratio was rather high, equal to 3. However, such a value was lower than those obtained in other investigations, such as the interval reported by ^{Aguilera et al. (2016}), from 3.1 to 4.6 in different fertilization treatments, although they used larger containers, 10 cm³ than in this work. It should be noted that, of the studies cited in this section, only that of ^{Bautista et al. (2005}) considers establishment of plantation in the field, like the actual work.

Another indicator, the Dickson index, yielded high values, although the recommendation of ^{Sáenz et al., (2014}) for another cespitose species, P. devoniana, is ≥ 1.2. The laboratory data are in Table 2.

Pearson's Correlations of the data from the 11 variables evaluated in the laboratory (Table 3) only indicate those that were significant among the assessed variables with p <0.05 and with the correlation coefficient ≥ 0.4. They emphasize simple variables of measure, like height and diameter, but in particular total dry weight.

Potential root growth test

A vigorous plant response was observed for the production of new roots (mean of 55.3, maximum, 86 and minimum, 29), which is a good indicator of plant quality, since the seedling will have greater facility to acclimatize to the site and therefore their development will be better and their survival rate increased (^{Ritchie and Landis, 2003}).

It is considered that for the pine species studied, an average of 55 new roots (minimum 29) generated in the potential root growth test, implies the average survival obtained in reforestation areas, with 88.6 % for northern exposures and 83.3 % for the southern, under the experimental conditions described. It is worth mentioning that in other studies, a smaller number of new roots are mentioned in similar periods, such as ^{Simpson and Vyse (1995}), who with more than 10 new roots determined a survival in the field greater than 70 % in Picea glauca (Moench.) Voss and Pinus contorta Douglas.

In favorable environments and with species that are good producers of new roots, the CPR test provides its best results (^{Ritchie and Landis, 2003}). The number of new roots referred in the previous paragraph for the three species compared, suggests that P. montezumae has that ability. The fact that the plantation environment was not limiting, which shows the good survival obtained, indicates that, for this species and the selected locality, CPR was satisfactory. This procedure must be carried out under conditions similar to the plantation site to have a result that is closer to reality (^{Folk and Grossnickle, 1997}), as it was done in the present work.

Water stress test

Student's t test showed differences between plant readings with and without water stress (p = 0.029). The plants irrigated the day before this test, had an average of 0.71 MPa, while those that remained without irrigation for a week reached 1.36 MPa. According to ^{Landis et al. (1989}), the first figure is within the recommended range for the hardening period, when growing forest species in containers. The second one overcomes the stress that must be submitted to the plants in the nursery, even during the hardening stage, since it should not exceed 1 MPa ever, as this affects growth and increases the probability of damage and mortality. On the other hand, this test shows that every day that passes without watering, the plant increases the tension by 0.19 MPa.

Reforestation assessment

According to the comparison of nursery and field evaluation, during the planting time, the seedlings had a growth of the order of 1 cm in height and 1.5 to 2.5 mm in diameter, under the different conditions of the experiment (Table 4). Although survival is high, the low growth in height is due to the fact that it is a cespitose species (^{Perry, 1991}).

No significant differences were detected by any individual factor, nor by their interaction, neither for height nor for diameter. In the case of survival, there was an effect of exposure (P = 0.0222), with the north (88.7%) surpassing the south (83.3%) (Table 5). Data from the Tetela del Volcán weather station indicate that the years 2013 and 2014 were wet, with 1 293.5 and 1 179.6 mm, respectively. So abundant precipitation could contribute to the fact that the moisture difference between exposures was smaller, with a small difference in survival and non- significant differences in diameter. Survival on the southern slope was similar to that reported by ^{Bautista et al. (2005}) (83.8%) for the same species, at slightly higher (3 368 masl) and flat (2 % slope) sites in central Mexico. This plant was established with diameters at the base > 6 mm, but survival on the northern slope exceeded this last figure, possibly because it is wetter than flat terrain.