Study area

The plantation was established in the Otinapa ejido of the town with the same name, at Durango municipality, in the state of Durango, Mexico; it is located between 24°03'52.2" N and 105°01'31.1" W, at an altitude of 2 350 m.

The average annual temperature is 14.6 °C, with a minimum of 6.1 °C and a maximum of 21.1 °C; the mean annual rainfall was 667 mm. The predominant vegetation is composed of various species of the Pinus genus, among which are P. engelmannii, P. cooperi Blanco, P. leiophylla Schltdl. et Cham., P. teocote Schiede ex Schltdl., as well as Juniperus sp. (^{Prieto et al., 2018}; ^{González, 2019}). The soil has a sandy-loam texture (54 % sand, 28 % silt and 18 % clay), with an average pH of 6.9, electrical conductivity of 0.41 mS cm^-1 and organic matter of 1.11 %.

Plant production

The vegetative material was reproduced in the “Praxedis Guerrero” forest nursery, of the Department of Natural Resources and Environment of the Government of the State of Durango, Mexico; It is located at kilometer 12.5 of the Durango-El Mezquital highway (23°56'58.3" N and 104°34'07.4" W), at an altitude of 1 890 m.

The plant was produced in black rigid polyethylene tubes with internal root guides, with a 4 cm upper diameter, 21 cm long and 165 mL. As a substrate, a mixture based on peat (48 %), composted pine bark (22 %), vermiculite (16 %) and agrolite (14 %) was used. During the preparation of the substrate, 4 kg m^-3 of Multicote 8^®, a slow release granulated fertilizer (8-9 months), with the composition 11N-28P₂O₅-11K₂O + micronutrients were added. The seed came from a seed stand, located at 2 465 m altitude, in San Miguel de Cruces, San Dimas, Durango.

Site preparation and planting

The selected site was five years without tree cover, due to an extreme drought in 2012 and subsequent damage in 2013 by bark cutters, which caused tree mortality. Three years before planting, the soil was plowed with an agricultural tractor, through the passage of a subsoiler, with which lines 40 cm deep were marked in the center of the planting line and 3 m apart with a Truper^® mod. TP20ME of 20 m measuring tape. In each line, with a Truper^® mod. 18631 talacho, vines of 25 to 30 cm in diameter and depth were made, separated at 1.0 m (this measurement was made with an Anvil^® mod. 96408 tape measure). The plantation covered an area of 600 m². The 11-month-old plant was established in the field on August 11^th, 2018, with an average diameter of 6.5 mm and height of 8 cm, acceptable morphological characteristics for P. engelmannii, based on the NMX-AA-170-SCFI-2016 standard (^{Secretaría de Economía, 2016}). Measurements of these variables were made with a Truper^® model 14388 calibrator and a Pilot^® model RAS-30 graduated ruler, respectively.

Treatments and experimental design

Six treatments were evaluated, with three inoculants and two fertilization conditions (Table 1). In the mycorrhization factor, one month before planting, the vegetative material was inoculated with three commercial products: A) Bio Bravo^®, B) Ecto-Myc^® and C) MycorTree Ecto-injectable^®, the first two are of national origin, while the third is imported; all three products contained spores. Inoculants A and B were applied 10 mL plant^-1, while for C, 2 mL plant^-1 were added; the doses applied were based on the recommendations of the assessed products. In relation to fertilization, two conditions were established: planted with 7 g of fertilizer and without fertilizer; Granulated fertilizer Multicote 8^® was applied with the 11N-28P₂O₅-11K₂O + micronutrients composition, placing it at the bottom of the strain and covering it with a layer of 5 cm of soil, to avoid direct contact with the roots.

The distribution of the plants was carried out in an experimental design of complete random blocks. Each treatment had four replications and 10 plants per experimental unit.

Assessed variables and statistical analysis

Survival and the cause of death were evaluated every three months for one year, under the following categories: A) Drought, complete plants and no physical damage; B) Grazing, plants broken by trampling or browsing; C) Noxious fauna, partial or total disappearance of the plant coupled with the appearance of a mound of earth in the place where it was planted. In addition, the diameter at the base of the stem was recorded with a Truper^® digital vernier and the height of the plants, measured with a conventional graduated ruler. An analysis of variance was performed for each variable and in the cases in which there were statistical differences, Duncan's means comparison tests (p≤0.05) were performed using the statistical program SAS^TM version 9.2 (^{SAS, 2009}). The percentage values of survival and cause of death were transformed with the arcsine function and square root.

Survival and mortality

Survival at three months ranged from 92.5 to 100 %, at six months from 85 to 100 %, at nine months from 77 to 97 %, and at 12 months from 58 to 83 %; however, only statistical differences (p≤0.05) were found between treatments at nine and 12 months. At nine months, the IA, IC and IC + F treatments were located in the upper statistical group with more than 94 % survival, while in the final evaluation, the IC was better and the IA + F treatment obtained the lowest value (Table 2).

After nine months, there was a general decrease in survival, with lower percentages in the IA + F and IB + F treatments, which coincided with the dry season, normally from February to May. The highest survival, 12 months after planting, occurred in the IC and IB treatments, which lacked fertilization and were inoculated with ectomycorrhizae; in these treatments, mycorrhization could have given the plant a greater chance of survival, as the fungi improve plant growth and favors greater tolerance to drought stress (^{Kipfer et al., 2010}).

According to ^{Mejía et al. (2015)}, forest plantations go through a critical stage after planting, where survival is affected in the first years. These results showed that the survival achieved was greater than the national average (59 %) at one year of planting (^{Conafor, 2019}), but less than that obtained by ^{González (2019)} in a P. engelmannii plantation in a neighboring site, who obtained an average survival of 85 % a year after planting.

At a site located less than 3 km from this study, ^{Prieto et al. (2018)} evaluated survival and growth in P. engelmannii. In this investigation, they handled plants with two initial diameters, 6.5 and 5.0 mm (similar to the diameter of the plants used in this essay), fertilized with slow release material (composition 18N-6P2O5-12K2O+2MgO+ micronutrients), applied in doses of 7 g plant^-1. At 13 months after being planted, survival for the larger diameter specimens was 67.5 %. In this case, the IC treatment obtained a higher survival percentage (82.5 %), in which inoculation without fertilization could have influenced this result (^{López-Gutiérrez et al., 2018}).

In regard to the dead saplings (30 %) one year after being planted, three causes associated with this fact were identified: drought (80 %), grazing (18 %) and noxious fauna (2 %); the higher mortality can be explained by the lack of moisture in the soil during the dry season, which, as it has a sandy-loam texture, it limits water retention, coupled with the low content of organic matter. It should be added that the habitat for the species under study is not ideal as it is a transition zone, with rains in summer and an annual rainfall of 667 mm, which is near the lower limit defined by ^{Martínez (1992)}, from 500 to 1400 mm.

The factors associated with mortality had a different impact on the treatments (Figure 1). In the case of IB and IC treatments, a greater resistance to drought damage is observed, which can be explained by an effective ectomycorrhizal symbiosis, since it is the component they share and it is one of the functions that have been attributed to them (^{Gómez-Romero et al., 2015}; ^{Barroetaveña et al., 2016}). In addition to this, the causes of mortality registered coincide with those reported by ^{Prieto et al. (2016b)} at the national level, of which drought is the most important.

Tratamiento = Treatment; Mortalidad = Mortality; Sequía = Drought; Pastoreo = Grazing; Fauna nociva = Noxious fauna.

Figure 1 Pinus engelmannii Carr. mortality at 12 months after planting based on the use of inoculants in the preconditioning and fertilization stage at the time of planting. 

Although it has been documented that the Pinus genus predominantly establishes ectomycorrhizal symbiosis (^{Bücking et al., 2012}), reference has also been made to the use of arbuscular mycorrhiza on P. engelmannii with which favorable reactions were obtained in the growth of the plant (^{Montes-Rivera et al., 2001}). The results of the present study for the IA treatment may indicate that an effective symbiosis was not established, because the time contemplated for the formation of the mycorrhizal association was possibly insufficient, especially in the case of a slow-growing species, compared to species shrubby or herbaceous, in which the formation of arbuscular mycorrhiza is faster (^{Brundett et al., 1996}).

In fertilized treatments (IA + F, IB + F and IC + F), a possible effect of inhibition of mycorrhiza formation by fertilization is also observed. In this regard, it has been pointed out that increasing this procedure can decrease mycorrhizal colonization (^{Brundett et al., 1996}; ^{Salgado et al., 2009}), as occurred in Pinus engelmannii, where plants inoculated in the nursery with native fungi (Amanita rubescens Pers., Amanita sp., Lactarius indigo [Schwein] Fr., Ramaria sp. and Boletus sp.) plus 3 g L^-1 of controlled release fertilizer, had a higher Dickson quality index and a higher percentage of mycorrhizal colonization, in relation to a plant fertilized with a dose of 6 g L^-1 (^{Salcido-Ruiz et al., 2020}).

The causes of mortality external to the components of each treatment, such as grazing and noxious fauna, had an impact on 20 %. According to ^{Prieto et al. (2016b)}, physical damage to plants by the presence of livestock can be fatal due to trampling or browsing; in this experiment, the affected specimens were trampled and failed to recover. On the other hand, the damage caused by Pappogeomys castanops Baird (1852) (gopher) was also fatal, as ^{Gunn et al. (2016)} stated they are burrowing rodents that consume plants or parts of them, which, being of human interest, are classified as a pest that requires control. However, the importance of the dynamics of gophers has been demonstrated for the conservation of other species that use their gallery system as refuge sites (^{Rosas-Espinoza et al., 2014}), so their benefit or harm is in terms of the study object.

Stem diameter

Statistical differences (p≤0.05) were found between treatments in the four evaluations carried out, with the treatments with the mycorrhizal condition plus fertilization (IA + F, IB + F and IC + F) presenting the best results (Table 3). The final average increase in the treatments with fertilization was 10 mm, while without fertilization (IA, IB and IC) was 6.9 mm.

These results were superior to those of ^{Mejía et al. (2015)}, who evaluated forest plantations in the state of Durango, and recorded an average diameter of 13 mm for a three-year-old P. engelmannii plantation. All treatments evaluated one year after planting exceed this reference, with the exception of treatment IB. However, for ^{Prieto et al. (2018)} and ^{González (2019)} the diameter increases were higher (11.2 to 14.3 mm and from 15.6 to 17.9 mm, respectively) in 13 and 12 months old P. engelmannii plantations in sites close to the one of this study, since those that were fertilized with Multicote^® in doses of 7 and 10 g plant^-1 at the time of planting.

Diameter has been recorded as a good predictor of plant survival in the field (^{Prieto et al., 2018}), hence the NMX-AA-170-SCFI-2016 stipulates minimum values required for each species (^{Secretaría de Economía, 2016}). However, in this investigation, the treatments that produced more outstanding diameters did not correspond to the highest survival rates; this could be explained by the arguments of ^{Grossnickle and Macdonald (2017)}, who indicate that when the diameter is not related to survival in the field, it may be due to the stressful conditions of the site, where the importance of mycorrhizal symbiosis stands out.

Height

The results showed statistical differences (p≤0.05) between some treatments in the four evaluations. In all cases the plants with the highest data were obtained in some of the treatments with ectomycorrhizal inoculant (IB or IC) plus fertilization (F) (Table 4).

The final average increase in the treatments with fertilization was 1.8 cm, while in individuals without fertilization (IA, IB and IC) it was 1.4 cm. ^{González (2019)} recorded a final average increase of 5.8 cm for P. engelmannii seedlings at 12 months of planting, in which slow release fertilizer was also used, only at four months and in higher doses (10 g plant^-1).

The NMX-AA-170-SCFI-2016 standard does not consider the minimum height in the plant quality evaluation of P. engelmannii (^{Secretaría de Economía, 2016}), because it is a species that in its initial stage is characterized by production abundant needles that can measure more than 30 cm, so their growth is cespitose and this causes a similar increase in height (^{Ávila-Flores et al., 2014}; ^{Rosales et al., 2015}).