The degradation of forests has generated large areas with intense and frequent disturbances, which causes fragility to the ecosystems and removes the possibility of the vegetation recovering its original condition by natural means (Vilagrosa et al., 2005). For these reasons, it is essential to apply practices such as afforestation, reforestation and soil conservation (Comisión Nacional Forestal [CONAFOR], 2010).
The result of reforestation at degraded sites depends on site environmental conditions and seedling quality at the time of planting (Duryea, 1985; Grossnickle, 2005). The quality of the plant is the result of genetic, morphological and physiological attributes, and the health condition of the propagules used in reforestation (Birchler, Rose, Royo, & Pardos, 1998).
In the case of nursery production, hardening is the most important phase since the plant decreases growth in height and roots, but increases in diameter (Landis, 2013), which confers resistance to drought and frost events. There are four ways of manipulating the hardening process: reduction in the amount of ammoniacal nitrogen, frequency of watering, photoperiod and exposure of the plants to high and low temperatures (Grossnickle, 2012; Landis, 2013).
The aim of this study was to evaluate the effect of three levels of irrigation in the hardening stage on the quality of Pinus oaxacana Mirov, through morphological response (height, diameter, accumulation of biomass and Dickson quality index) and physiological response (root growth potential) of plants grown under nursery conditions. In this study, it was considered that hardening shows a greater ability to form roots in the field and, consequently, greater capacity to avoid water stress, maximum resistance to environmental stress and functional integrity.
Materials and Methods
The study was carried out inside a greenhouse at Posgrado en Ciencias Forestales of Colegio de Postgraduados-Campus Montecillo, Estado de México (19° 28’ 26” N - 98° 53’ 42.18” W and altitude of 2,240 m). The germplasm bank "El Vergel", located in the city of Puebla, provided the seed of P. oaxacana that was used in the sowing. The seed lot, which had 98 % purity and 80 % germination, was collected in 2010 in Perote, Veracruz. Sowing took place in the second half of October 2011; the substrate was a conventional mixture of peat moss, perlite and vermiculite in a 60:20:20 volume ratio, added with controlled release fertilizer (Osmocote® Plus) at a dose of 7 kg·m-3. Black individual containers with a volume of 220 mL and lateral openings were used, which were placed in a tray with 25 cavities.
Seedlings grew for nine months, before applying the hardening treatments. Plant nursery management included light irrigation (1 to 3 cm depth) per day for six weeks, and then heavy irrigation (to saturation) every two days during the rapid growth stage. In addition to controlled release fertilization, soluble fertilizer (Peters®) was applied once a week in irrigation water. In the fast-growing stage, the formulation 20-20-20 was used in doses of 70 µL·L-1 of nitrogen, 62.5 µL·L-1 of phosphorus and 77.5 µL·L-1 of potassium.
The effect of hardening by reduction of irrigation was evaluated from July 2, 2012 on 1,600 plants. The treatments were three levels of irrigation (frequent, medium and low) and the control treatment. In the treatment of frequent (F) level, irrigation was applied when the weight of the container was reduced 30 % with respect to the weight of saturation; in the treatment of medium (M) irrigation, when the weight of the container was reduced between 40 and 45 %; and in the case of low irrigation, when the container lost 46 to 50 % of the weight; in the control treatment (T), irrigation was applied every 48 to 72 hours, to maintain a moisture content in the substrate close to field capacity. The treatments were maintained in that condition for eight weeks.
The weight of the container at field capacity was determined before starting the experiment. All the trays with the containers were irrigated at field capacity and weighed on a digital scale. With the data obtained, the mean and its 95 % confidence interval were calculated per treatment per replicate. From these data, we estimated the weight range equivalent to the loss of 30 %, 40 to 45 % and 46 to 50 %.
The weight of two trays with the containers, per treatment per replicate, was monitored daily to determine the loss of moisture and to define the moment of irrigation per treatment. During irrigation, the trays with the containers were irrigated and then weighed, and if they did not reach the weight at field capacity, they were watered again. The average weight range of the 25 containers at field capacity was 4.221 to 4.617 g with an average of 4.419 g. The weight of the 25 containers when they lost 30 % ranged from 2,915 g to 3.331 g; when they lost between 40 and 45 % ranged from 2,159 g to 2,769 g; and when the loss was 50 %, the weight ranged from 2,000 g to 2,396 g.
Assessment of the morphological characteristics
After the hardening period, eight weeks after starting the treatments two recovery irrigations were applied. Consisting of watering at field capacity leaving a period of two days to let the plants recover from the stress generated by irrigation from weeks ago. A random sample of 12 plants per treatment per block was selected from the central part of the plots and the total height and diameter of the root collar were measured. In each plant, the substrate was removed from the root using tap water in abundance and being careful not to damage the structure. Then the shoot was separated of the radical part with a cut at the collar of the root. Samples were placed in a drying oven at a temperature of 70 °C for 72 h to determine the dry weight of the shoot and root, and the total dry weight of the plant. With the data collected, the sturdiness quotient (plant height and diameter ratio), shoot/root ratio (RPAR) and the Dickson quality index (DQI) were calculated using the following equation (Dickson, Leaf, & Hosner, 1960):
Height was expressed in centimeters and diameter in millimeters.
Root growth potential
The root growth potential (RGP) test consisted in placing a random sample of plants in a favorable controlled environment to promote rapid root growth. The sample size was 12 plants per treatment. The plants were transplanted in pots with a capacity of 10 L using a substrate of bark and perlite in a ratio of 70:30; at the time of transplantation, all white roots were cut. The plants were kept for 40 days in the greenhouse with daily irrigations. The arrangement of the pots was random. At 40 days, the plants were removed from the pots for careful washing of the roots and their measurement. The variables measured were: total number (TN), total length (TL) and dry weight of new roots (DWNR) in growth. The new roots were identified by the white color and only those that had a length greater than 1 cm were considered.
Experimental design and Statistical analysis
The experiment was set up in randomized complete blocks design, in four growth beds; each bed was considered a block. Each treatment consisted of 100 plants per block, which were placed on four tables. The data was subjected to a variance analysis (ANDEVA) using the PROC GLM procedure with SAS software version 9.0 (Statistical Analysis System [SAS], 2002). The statistical model used for the design of completely randomized blocks with subsampling is as follows:
Significant differences were considered when P ≤ 0.05. The Tukey's test was used to determine the least significant difference among treatments. The sturdiness quotient, shoot/ root ratio and Dickson index were transformed with the sine-arc function for data normalization. In the case of the variables of the RGP, a linear regression was also made to find the trend of the data.
Results and Discussion
Morphology of Pinus oaxacana under different levels of irrigation
Hardening treatments with different levels of irrigation showed significant differences (P ≤ 0.05) in the variables diameter, shoot dry weight and total dry weight (Table 1). The control and frequent irrigation treatments (reduction of 30 % with respect to saturation weight) had the highest values in diameter, shoot and total dry weight. In contrast, the plants with low irrigation treatment had the lowest values of diameter and total dry weight. In all treatments there were values of DQI near 1, which indicates balance between the shoot and radical part of the plant.
|Irrigation level||Height (cm)||Diameter (mm)||Dry weight (g)||SRR||Sturdiness quotient||DQI|
|Frequent||29.4 ± 2.9 a||6.5 ± 0.1 a||7.5 ± 0.6 a||2.3 ± 0.1 a||9.8 ± 0.6 a||3.4 ± 0.2 a||4.6 ± 0.4 a||1.3 ± 0.1 a|
|Medium||26.7 ± 1.4 a||6.1 ± 0.2 bc||7.1 ± 0.3 ab||2.0 ± 0.3 a||9.1 ± 0.6 ab||3.6 ± 0.3 a||4.5 ± 0.3 a||1.2 ± 0.1 a|
|Low||26.4 ± 1.5 a||5.9 ± 0.2 c||6.4 ± 0.4 b||2.0 ± 0.2 a||8.4 ± 0.7 b||3.3 ± 0.3 a||4.6 ± 0.2 a||1.1 ± 0.2 a|
|Control treatment||28.9 ± 3.0 a||6.3 ± 0.2 ab||7.5 ± 0.3 a||2.2 ± 0.2 a||9.6 ± 0.5 a||3.6 ± 0.3 a||4.7 ± 0.6 a||1.2 ± 0.1 a|
The response of the plants to the different levels of irrigation in the nursery was related to the availability of water; to greater amount of water available, greater diameter and biomass, while the growth of the root was not reduced. This contradicts the results of Villar-Salvador, Peñuelas-Rubira, and Jacobs (2013), who report that the plants of Pinus pinea L. reduced the growth of the root, because it was inhibited with water stress by the growth of the shoot. Other species have shown similar results in the shoot and total dry weight as a response to severe water stress, because as it increases there is less production of total biomass. This may be due to the fact that drought affects the elasticity of the cell wall, as in the case of Eucalyptus globulus Labill. (Coopman, Jara, Escobar, Corcuera, & Bravo, 2010; Pita & Pardos, 2001). In contrast, in Pinus halepensis Mill. (Royo, Gil, & Pardos, 2001) and Quercus ilex L. (Planelles-González, Villar-Salvador, Oliet-Palá, & López-Arias, 2004; Villar-Salvador et al., 2004), water stress did not affect the shoot/root ratio; that is, it did not have an effect on the equilibrium of the plant. In P. oaxacana, white roots were generated in the event of water stress, because it is found in regions with dry and cold climates. This species has developed adaptation mechanisms such as the rapid emission of white roots to ensure the absorption of water and nutrients, and the plant may present stomatal closure in the cells of the needles to avoid water loss (Valladares et al., 2008).
Root growth potential of Pinus oaxacana under different levels of irrigation
The RGP test showed no significant difference (P > 0.05) between irrigation treatments. The correlation between the level of irrigation and the production of new roots is high; when the level of irrigation is low, the production of white roots decreases. The variability among the data, as shown in Figure 1, is influenced by the high value of the coefficient of variation in the variables total number of roots (0.10), total root length (10.82) and dry weight of white roots (0.17), therefore, it is not possible to conclude which one is the treatment that promotes a greater regeneration of roots. In general, in all treatments, the number of roots emitted on average was greater than 100 and it is inferred that there was a positive response in P. oaxacana at all levels of irrigation.
In a similar study with Q. ilex, the highest root biomass was present in the control treatment, while in low, medium and severe water stress levels (40, 45 and 50 % weight loss from saturation) there was no difference (P ≤ 0.05) in the number of roots emitted (Villar-Salvador et al., 2004). In some conifers such as P. halepensis, the average root yield between treatments was 30 to 42 per plant, and the length varied between 80 and 100 cm; the response to water stress did not differ (P ≤ 0.05) among treatments, except for the treatment with severe stress (Villar-Salvador et al., 1997). In both cases, the response in the formation of new roots at maximum stress was negative. In contrast to the results obtained from the water stress hardening activity, there is no effect on the ability to regenerate the root system, one of the causes may be the intensity and time of hardening (Grossnickle, 2005). Emission of new roots is sensitive to the history of drought stress experienced by the seedling (Tinus, 1996).
In this test it was expected that at higher RGP, plant development capacity in the field would be higher, due to the correlation of a greater ability to form roots in the field and to avoid water stress. The functions of water absorption in roots and leaves indicate that the plant has an optimal physiological condition, i. e., the plant has functional integrity (Villar et al., 1997). According to the above, we can relate the data and infer that P. oaxacana has the capacity to regenerate the radical system in the event of severe drought.
Hardening with reduced irrigation in P. oaxacana seedlings developed morphological parameters in equilibrium. Reduced irrigation increased the diameter of the root collar without affecting the growth of the root system. Hardening in the months of May to June with eight weeks of duration created an adaptation response to water stress. The species tolerates a level of 50 % in the reduction of irrigation without presenting less regeneration of the root system. A reduction of irrigation between 30 and 45 % in P. oaxacana allows the production of hardened plants with greater possibility of success at the time of transplantation in the field.