Study Area

The study was carried out in the ejido Llano Grande, Chignahuapan, Puebla. According to the current silvicultural management program, the ejido's forest area has an average altitude of 2 800 m, undulating and rugged topography with slopes of 5 to 60 % and deep Andosol humic type soils. The area's climate is C(w₁)(w) temperate sub-humid with summer rains, with average annual temperature and precipitation of 14.8 °C and 1 000 mm, respectively; the rainy season is from June to October and the predominant winds are from northeast to southwest. The tree vegetation is made up of pure stands of P. patula and Pinus montezumae Lindl. and associations of Pinus-Abies and Pinus-Quercus in which P. patula is the predominant species.

Inputs used

The nine-month-old P. patula seedlings (October 2016 to July 2017) were obtained from a production experiment, developed for the present research, in the nursery of the ejido Pueblo Nuevo, Chignahuapan, Puebla. Four containers with cylindrical cavities and with the following characteristics were used in production: a) PS-77, a 35 × 60 × 15.2 cm expanded polystyrene tray with 77 cavities of 170 cc, density of 366 cavities per m², top diameter of 4.3 cm and length of 15.2 cm (Aislantes y Empaques, S.A. de C.V., Guadalajara, Jalisco, México); b) P-54, 30.8 × 50.0 × 13.8 cm black plastic tray with 54 cavities of 170 cc, density of 348 cavities per m², top diameter of 4.8 cm, length of 13.8 cm; c) RT-42, plastic grid (37.3 × 37.5 × 23.4 cm) of four vertical supports with 42 individual black plastic containers of 170 cc, density 298 cavities per m², top diameter 4. 8 cm and length of 13.8 cm; d) RTa-42, container with design and dimensions equal to the RT-42 container, with three surrounding openings of 0.5 cm width distributed equidistantly along each tube to encourage aerial pruning of the RL. The last three containers are manufactured by Innovaciones Industriales y Forestales, S. A. de C. V., Azcapotzalco, Estado de México.

To promote chemical root pruning (CRP) in seedlings, half of the P-54, PS-77 and RT-42 containers were manually treated with a plastic solution of 7.0 % Cu(OH)₂) hydroxide, consisting of 1 kg of 5 x 1 vinyl-acrylic sealant (Comex^®), 0.2 kg of tap water and 0.09 kg of commercial Cu hydroxide (Hidromet^®). ^{Aldana and Aguilera (2003)} recommend such concentration of commercial Cu hydroxide for treatment of polystyrene trays in the production of forest species; it is also reported as suitable to promote CRP in the production of Pinus montezumae Lamb. (^{Aguilera-Rodríguez et al., 2016a}) and Pinus pseudostrobus Lindl. plants (^{Aguilera-Rodríguez, Aldrete, Martínez-Trinidad, & Ordaz-Chaparro, 2016b}).

Seedling production treatments were defined by the type of container and type of root pruning: treatments 1, 2 and 3 corresponded to the containers (P-54, PS-77 and RT-42) without Cu and treatments 4, 5 and 6 to the same containers treated with Cu. The alternate treatment (T7) was defined by the RTa-42 container with openings in the walls of the cavities, to promote aerial pruning of the lateral roots.

Four trays were used for each treatment, these trays were filled with substrate composed of fresh pine sawdust (uncomposted), perlite, vermiculite and peat moss in proportions of 60, 20, 10 and 10 % by volume, combined with 8 g·L^-1 of controlled release fertilizer Osmocote^® Plus 8-9M (15-9-12) and 1 gL^-1 of micronutrient fertilizer (Micromax^®). The trays were sowed using seed from the ejido Llano Grande. From the time of sowing until May 2017, seedlings grew protected with black shade cover (50%) and from June they grew outdoors. Irrigation was applied every two or three days.

During the first week of July 2017, at nine months of age, 72 seedlings were randomly extracted from each set of trays per treatment, which were ordered in packages of 12 plants each, for transfer to the planting areas in the ejido Llano Grande.

Field experiment

The planting experiment was established in two clear cut areas (CA1 and CA2) harvested in 2017, corresponding to the fourth year of the ejido's forest management program. The center of CA1 is located at 19° 41' 59.83" N - 98° 09' 26.87" W and CA2 at 19° 41' 58.31" N - 98° 10' 42.33" W. After removing the forest products, two blocks (I and II) of 17.5 × 35.5 m in CA1, and four (III, IV, V, and VI) in CA2 were delimited; the blocks were located in sites representative of the topographic, soil, and exposure conditions of the areas.

Three soil samples were taken from each block at a depth of 25 cm and these were mixed to compose the mixture to determine the characteristics of the planting sites.

The planting was carried out in the first week of July 2017 undera randomized complete block design with six replications. The treatments were randomly distributed in each block and were planted using the “common hole” system in two continuous rows of six plants each, with a spacing of 2.5 m between plants and rows.

Maintenance of the experiment

The logging areas have had fire breaks and permanent surveillance to prevent fires and grazing. After planting, the areas were monitored every two months for weed control, recordof dead plants and identification of causal agents. Weeds were manually removed during the months of September 2017, April and September 2018, and April 2019, allowing plants to grow freely. From the first assessment in September 2017, dead plants were identified due to total root eating by Phyllophaga (May beetle), the greatest impact was recorded in blocks of CA2, adjacent to agricultural areas.

Variables evaluated

At the time of planting, the diameter at the base of the stem (D0) and the height of the plants (A0) were measured. In the first week of July 2019, two years after planting (Figure 1), the stem diameter was measured with digital vernier (TRUPER^® ) around 3 cm above the soil surface (D1), and the height was measured with a 2 m graduated ruler (A1). The dead plant was quantified by treatment and block, to calculate the survival. For each treatment, the annual relative growth rates (Tcra) of diameter and height were estimated with the formula Tcra = (ln Y₂ - ln Y₁) / (T₂ - T₁), where Y₁ = D0, A0; Y₂ = D1, A1; T₁ = age of the plant at the beginning of the test; T₂ = age of the plant at the end of the test (^{Villar et al., 2008}).

Figure 1 Pinus patula saplings, two years after planting (July 2019), in Block III of the logging area 2 (CA2) 

Statistical Analysis

T1 to T6 treatments were evaluated in a completely randomized block experimental design with a 3 × 2 factorial arrangement (three types of containers and two chemical root pruning conditions [with and without]) and six replications. The alternative treatment with aerial root pruning was compared with the RT-42 treatments with and without Cu coating. The differences between the variables evaluated were identified by ANOVA and Tukey's mean comparison (P ≤ 0.05) with the SAS® statistical software version 9.0 (^{SAS Institute Inc., 2002}).

Effect of container and root pruning on P. patula growth

According to the results of the ANOVA, shown in Table 2, the significance (P < 0.05) of the final sizes, survival and relative growth rate were influenced by the block, which is attributed to the contrasting soil and topographical conditions of the planting areas. The influence of root pruning was more significant than the container, while the interaction container*root pruning had no significant effect (P < 0.05) on the variables evaluated.

Table 3 shows the growth and survival results of P. patula plants. Under nursery conditions, seedlings grown in PS-77 trays developed significantly higher diameter and height values (P ≤ 0.05) than the set of seedlings grown in P-54 and RT-42 trays. Under field conditions, the values of relative growth rates of diameter and height and survival were significantly higher (P ≤ 0.05) in plants grown in RT-42 and PS-77 containers. Plants in PS-77 trays also developed larger sizes in final diameter, while in final height there were no significant differences per container.

According to the hypothesis, the group of plants with root pruning developed values of final height, relative growth rates after outplanting (TrcaD, TrcaA), and significantly higher survival rates (P ≤ 0.05) compared to the group of unpruned plants, despite the fact that the latter showed significantly larger sizes at the time of planting.

The higher seedling growth in polystyrene trays is attributable to their physical characteristics (insulating raw material, white color, wall thickness greater than 5 mm), which contribute to maintaining the temperature of the substrate more stable and, therefore, seedlings can develop greater root and aerial growth compared to those grown in plastic trays (^{Landis et al., 2014}). At the nursery stage, lower growth of seedlings with chemical root pruning is attributable to the development of short and thin lateral roots, while the unpruned seedlings developed thick and long lateral roots (^{Aguilera-Rodríguez et al., 2016b}).

Under field conditions, the effect of root pruning may not be significant in the growth of the plants during the first years, because in this period the plants invest more resources in root growth than in aerial growth; however, in this study significant differences in height were recorded in only two years, probably because the species is fast growing, it was established in a natural distribution site where soil is deep and fertile. It is also possible that during the first two years there are no significant differences due to the effect of root pruning, particularly when the plant is grown in cavities smaller than 150 cc, the planting is done in very cold sites or with poor soils and grass stage species, as it happened in the studies cited below.

In a planting experiment with Pinus palustris Mill. (a grass stage species from the south of the United States), ^{South et al. (2005)} used seedlings grown in polystyrene trays (Supperblock^®) immersed with a Cu solution (Spin Out^®), and plastic trays (Hiko V-93) without Cu coating, both with 93 cc cavities; two years later, plants with and without pruning, established in four sites, did not show significant differences (P > 0.05) in diameter (3.7 vs. 3.7 cm), height (23 vs. 21 cm), survival (78 vs. 75 %) and rupture of the grass stage (63 vs. 60 %). In contrast, ^{Haywood, Sung, and Sword (2012)} produced the same species in 60, 108, and 164 cc Cu-treated polystyrene trays and, five years after planting, found that plants with and without pruning showed significant differences (P ≤ 0.05) in diameter (7.0 vs. 6.5 cm) and height (200 vs. 170 cm), although not in survival (94 vs. 91 %).

In another experiment conducted by ^{Regan et al. (2015)} with Pinus monticola Douglas ex D. Don (a slow-growing subalpine species), seedlings were grown in polystyrene trays with 80 and 130 cc cavities treated with Cu. After 5.5 years from outplanting, plants with and without root pruning did not show significant differences (P > 0.05) in survival (59 vs. 52 %), but did show significant differences in diameter (3.5 vs. 3.2 cm) and height (134 vs. 121 cm).

When planting is done in sites with dry climate, differences may be less evident during the first two years, as is the case of Pinus halepensis Mill. ^{Tsakaldimi and Ganatsas (2006)} grew this species in plastic trays with cavities of 650 cc treated with a plastic solution of 3.3 % of basic Cu carbonate (CuCO₃.Cu(OH)₃) and established in a Mediterranean climate site with an average annual rainfall of 581 mm. Two years after planting, trees with and without root pruning showed no significant differences (P > 0.05) in diameter (1.0 vs. 1.16 cm), height (72.0 vs. 82.0) and survival (95.0 vs. 98.3 %).

Effect of the type of pruning (chemical vs. aerial) on the growth of P. patula

Seedlings grown in containers designed for aerial root pruning (RTa-42) was compared with its two similar RT-42s (with and without cavities treated with Cu). According to Table 4, the initial diameter and height were significant (P < 0.05) due to the effect of root pruning; the unpruned seedlings developed larger sizes compared to those with root pruning. After two years from outplanting, the plant with aerial root pruning had a higher annual relative growth rate in height (TcraA) compared to the plants with chemical root pruning and without pruning; in the rest of the variables evaluated there were no significant differences, despite the fact that, at the beginning of the plantation, the plants with aerial and chemical pruning had significantly smaller sizes than the unpruned seedlings.

According to outplanting studies, plants with aerial root pruning can be as efficient as those grown with chemical root pruning and have greater growth than unpruned plants. ^{Cambell, Kiskiila, Philip, Zwiazek, and Jones (2006)} grew Pinus contorta Dougl. var. latifolia Engelm. in three types of trays (Copperblock®, Airblock®, and Superblock®) with 80 cc cavities, with treatment and design for chemical and aerial root pruning, and without root pruning. Plants were established in a subalpine site with average annual rainfall of 780 mm and average daily temperature of -0.3 °C. At the end of the test (1.5 years), plants with aerial pruning developed significantly smaller sizes than plants with chemical pruning and without pruning, with the advantage that root dry weight and root dry weight/air dry weight were not statistically different (P ≤ 0.05).

In the case of pine trees with grass stage growth, Sung and Haywood (2016) grew P. palustris in plastic trays (Rigi-pots^®) with 110 cc cavities, with and without a design to encourage aerial root pruning. After 24 months of planting, plants with aerial root pruning and without pruning showed no significant differences (P ≤ 0.05) in diameter (16.0 vs. 16.2 mm), height (6.0 vs. 6.1 cm) and survival (96 vs. 93 %); in contrast, the number of thick lateral roots (≥0.9 mm) emitted with enveloping growth was statistically different (2.1 vs. 4.5). Based on this root behavior, the authors concluded that plants with aerial root pruning could present greater growth and aerial stability than plants without pruning in subsequent years.

Regarding Mexican species grown with aerial root pruning, we only have the antecedent of a field study carried out by ^{Sánchez-Vásquez et al. (2017)}, who produced Pinus greggii Engelm. var. australis seedlings in 245 cc black and white plastic containers and grid containers with and without surrounding lateral openings, with three types of substrate and two levels of fertilization. The trees were established in a site with precipitation and average temperature of 1 215 mm and 15 °C, respectively. One year later, the plants with aerial root pruning and without pruning did not have significant differences in diameter (5.2 vs. 5.3 mm), height (45.1 vs. 45.3 cm) and survival (90 vs. 89 %), despite the fact that the initial diameter of the plants with aerial root pruning was significantly lower than those of unpruned seedlings (2.9 vs. 3.1 mm).

Production of seedlings in containers designed for aerial root pruning has the advantage of not contaminating the soil or the environment with Cu leachates, as happens in chemical root pruning, although requires greater irrigation frequency and fertilizers during the nursery stage. To reduce water and fertilizer loss, ^{Landis (2005)} recommends several management practices, including separate containers with and without openings, the use of white containers in areas with high temperatures or at the ends of the growing tables, elaboration of a specific irrigation program, and the use of controlled-release fertilizers to reduce leaching losses.

Seedling survival of P. patula

Seedling survival was affected by the factors: block, container and chemical root pruning (P < 0.05), but not by the interaction container*root pruning (Table 2). Outplanting survival of plants grown in P-54 trays was significantly lower than those grown in polystyrene trays (PS-77) and in grid with plastic containers (RT-42) (Table 3); among other causes, this may be attributable to the fact that, under nursery conditions, seedlings grown in these types of trays developed the lowest sizes.

In CA1, mortality was caused by frost in December 2017 and January 2018, with survivals of 86 and 100 % for blocks I and II, respectively, and 99, 97 and 99 % for unpruned plants, with CRP and ARP, respectively. In CA2, Phyllofaga (May beetle) affected from September 2017 to November 2018, with survival of 78, 76, 74 and 88 % for blocks III, IV, V and VI, respectively, and 82, 91 and 89 % for unpruned plants, with CRP and with ARP, respectively. It should be noted that CA2 borders on agricultural areas, where the presence of this type of insect is common; in this regard, ^{Cibrian (2016)} points out that insects of the genus Phyllofaga predominate in temperate climates and are one of the main pests of newly established forest plantations, especially on land that was once used for agricultural activities.

Greater affectation in plants without root pruning can be attributed to the fact that this type of seedlings develop roots with enveloping growth under nursery conditions, which persist after outplanting during the first years (^{South et al., 2001}; Sung & Dumroese, 2013); in contrast, plants with root pruning emit horizontal lateral roots faster than unpruned plants, which gives them greater resistance to damage by pathogens and to extreme natural phenomena (^{Landis et al., 2014}).