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Introduction
In Mexico, Abies religiosa (Kunth) Schltdl. & Cham. (sacred fir) is distributed in the mountainous areas of Sinaloa, Chihuahua, Coahuila, Nuevo León, Tamaulipas, Hidalgo, Veracruz, State of Mexico, Tlaxcala, Puebla, Morelos, Jalisco, Michoacán, Guerrero and Oaxaca. The sacred fir, known as oyamel in Mexico, is a slow-growing species used as a Christmas tree (Pineda-López et al., 2015); its demand varies from 1.6 to 2.0 million, of which 60 % is destined for export to Canada and the United States of America (Zamora-Martínez, 2007).
Several types of containers are used in the mass production of nursery species, with one of them being plastic bags with soil as substrate (Allen, Harper, Bayer, & Brazee, 2017). In Mexico, they are widely used in government-promoted forest nurseries; in 2016, 2 761 443 plants were produced in these bags (Comisión Nacional Forestal [CONAFOR], 2017). Sacred fir seedlings are produced in these containers and remain in the nurseries for 18 to 24 months before being sent to plantations (CONAFOR, 2017). A disadvantage of plastic bags is that when irrigation is applied, some physical soil properties such as compaction are altered (Angst et al., 2017), which could affect plant survival and growth.
Earthworms have physical effects on soil structure (Angst et al., 2017; Brown et al., 2001; Scheu, 2003). Through their excavations, worms increase soil porosity and aeration (Datta, Singh, Singh, & Singh, 2016) and degrade organic wastes, which improves nutrient utilization by plants (Jansirani, Nivethitha, & Vijay, 2012; Pashanasi, Melendez, Szott, & Lavelle, 1992) and benefits their development (Eriksen-Hamel & Whalen, 2007). In addition, evidence indicates that worms benefit plant growth and productivity in arable or grassland soils (Ortiz-Ceballos & Fragoso, 2004). Pontoscolex corethrurus Müller is one of the endogenous earthworms that has demonstrated its effectiveness in soil improvement and crop productivity; moreover, it is common, easy to use and reproduce (Pashanasi, Lavelle, & Alegre, 1994), and is tolerant to extreme conditions (Cuevas-Vázquez, Vázquez-Luna, Martínez-Hernández, Gómez-López, & Ortíz-Ceballos, 2017).
Based on the above, the use of earthworms in the production of seedlings in plastic bags could increase soil porosity and aeration and improve the nutrient absorption of seedlings. This may be reflected in increased growth or biomass accumulation of the seedlings (dry weight). So far there is no evidence of the use of earthworms for the production of seedlings grown in plastic bags in greenhouses or nurseries. In this context, the objective of this research was to evaluate the effect of the earthworm P. corethrurus on the survival and growth of sacred fir seedlings, as well as on soil compaction. The hypotheses put forth were that the presence of the earthworm will allow for greater plant growth and significantly reduce soil compaction in plastic bags.
Materials and methods
Soil collection and experimental design
Approximately 100 kg of soil were collected from sacred fir forests in the El Conejo ejido, municipality of Perote (19° 31’54.5” N and 97° 09’ 14.8” W), located on the northwest face of Cofre de Perote, an inactive volcano in Cofre de Perote National Park, Veracruz Mexico. Tree density in these forests is 1 711.67 individuals·ha-1 (Pineda-López, Ortega, Sánchez-Velásquez, & Vázquez-Domínguez, 2013). The soil is classified as Andosol, which is susceptible to erosion (Instituto Nacional de Estadística y Geografía [INEGI], 2014). The soil of these sites has pH 5.79 ± 0.11 and total carbon, nitrogen and phosphorus percentages of 8.58 ± 0.84, 0.41 ± 0.037 and 0.27 ± 0.038, respectively (Perroni, Sánchez-Velásquez, Garza-Garza, Rojo-Alboreca, & Pineda-López, 2013). Uribe et al. (2012) indicate that these soil characteristics are appropriate for the growth of the earthworm P. corethrurus.
A composite sample was obtained from three samples extracted from five plots of 50 x 12.5 m (n = 15) at a depth of 30 cm in the rainy season (October 2008). The collected soil was mixed and remnants of roots and litter were excluded; it was then placed in black 250-cc, 400-gauge plastic bags. The size of the plastic bag used in this experiment is the same as that currently used in nurseries for the production of Christmas tree plants.
Open-pollinated seed from 10 cones of 10 trees (n = 100 cones) randomly chosen with a distance greater than 100 m between them was used. Scaleless seeds were deposited in wet river sand at 5 °C for eight days to promote germination (Young & Young, 2009). In the greenhouse, the seeds were placed on trays and covered with 3 mm of soil. The soil remained moist until germination, leaving the seedlings growing until the emergence of the first leaves (20 days). Subsequently, a month-old plant was placed in each plastic bag and the experiment was immediately started, consisting of three treatments: 1) an adult worm (1AW) with a developed clitellum, 2) two juvenile worms (2J) without a developed clitellum, and 3) control without a worm (NW). Juvenile worms reach maturity at approximately 60 days; two juveniles were used with the idea that at least one would reach maturity. Juvenile and adult worms weighed 0.011 ± 0.02 and 0.42 ± 0.05 g, respectively. Twenty-five replicates were used for each treatment; the bags for each treatment were chosen at random. The permanence of the worms in the experiment was observed through the soil removed on the surface of the bags. The earthworms were produced at Universidad Veracruzana’s Institute of Biotechnology and Applied Ecology. No pesticide or fertilizer was applied to the soil or plants. The experiment was carried out under nursery conditions with periodic watering, so that the soil was always kept moist (visual evaluation), which prevented the seedlings and worms from experiencing water stress; that is, the plant remained robust and with erect leaves.
At the end of the study (one year), when the seedlings reached the size to be sent to forest plantations (CONAFOR, 2017), the following records were taken: final height; diameter of average cover [(maximum + minimum) / 2]; number of primary and secondary branches; and dry matter of stem, branches, roots and total (sum of their parts). The dry matter was obtained by drying the plant parts at 60 °C until constant weight (72 h). Soil compaction was measured in each bag, at three randomly selected points, with a 0.635-cm-diameter pocket penetrometer, which is compaction-proof to 0.635 cm deep (Forestry Suppliers Inc. MPN: LR-280; UNSPSC: 41113910).
Data analysis
Survival was compared among treatments using a Chi-square test (χ2); that is, the sum of the squared differences of the observed values (number of individuals that survived in each treatment) minus the expected value (mean of the survivors of the treatments) divided by the expected value. Final height, cover, number of primary and secondary branches, dry biomass (stem, branches, roots and total) and combined variables such as the root:stem ratio and the proportion of biomass allocation to the root and aerial part (dry weight of stem and branches) were analyzed through ANOVAs with the SAS GLM procedure (SAS Institute Inc., 2016). All of them were used as response variables (dependent) and treatments (a factor) as a separate variable with three levels (1AW = one adult worm, 2J = two juvenile worms and NW = no worms). The transformed variables were final height (ln), cover area (sqrt; Zar, 2013), primary and secondary branches (ranks; Seaman, Walls, Wise, & Jaeger, 1994), root dry weight proportion (arcosin √root; Zar, 2013) and stem dry weight proportion (arcosin √stem). The variables dry weight of the stems, root, primary and secondary branches and total dry weight, as well as soil compaction and root:stem ratio did not require transformation. The normality of the data was checked with the Shapiro-Wilk test and the homogeneity of variances was analyzed with the Levene test. Multiple comparisons of means were made with Tukey’s test (P ≤ 0.05) when necessary.
Results and discussion
The initial height of the seedlings (4.42 ± 0.23 cm) was similar among the treatment
groups (F = 1.74, P > 0.18, gl = 2, 72). At the
end of the experiment, survival (86 ± 4.25 %) was similar in treatments with and
without worms (
Table 1 Variables evaluated in Abies religiosa seedlings under Pontoscolex corethrurus treatments (1AW = one adult worm, 2J = two juvenile worms and NW = no worms) under nursery conditions.
| Variables | ANOVA (GLM) | Treatment | Mean ± SE |
|---|---|---|---|
| Final height (cm) | F = 3.62 P = 0.03 gl = 2, 62 | 1AW 2J NW | 23.6 ± 1.60 a 22.0 ± 1.80 ab 17.0 ± 1.60 b |
| Root dry weight (g) | F = 4.11 P = 0.02 gl = 2, 62 | 1AW 2J NW | 0.54 ± 0.08 a 0.43 ± 0.06 ab 0.30 ± 0.04 b |
| Soil compaction (kPa) | F = 13.29 P < 0.0001 gl = 2, 53 | 1AW 2J NW | 63.0 ± 8.00 a 61.0 ± 8.00 a 128.0 ± 15.00 b |
| Stem dry weight (g) | F = 2.37 P = 0.10 gl = 2, 62 | 1AW 2J NW | 1.03 ± 0.12 a 0.82 ± 0.11 a 0.71 ± 0.08 a |
| r:s ratio | F = 2.68 P > 0.07 gl = 2, 62 | 1AW 2J NW | 0.45 ± 0.03 a 0.41 ± 0.02 a 0.36 ± 0.02 a |
| Root dry weight (%) | F = 2.67 P > 0.07 gl = 2, 62 | 1AW 2J NW | 30.32 ± 1.41 a 28.44 ± 1.22 a 26.21 ± 1.11 a |
| Stem dry weight (%) | F = 2.67 P > 0.07 gl = 2, 62 | 1AW 2J NW | 69.67 ± 1.41 a 71.56 ± 1.22 a 73.78 ± 1.11 a |
r = root dry weight, s = stem dry weight (stem + branches). SE: standard error. The same letters in each variable indicate that there were no significant differences among treatments according to Tukey's test (P > 0.05).
Figure 1 Growth of Abies religiosa seedlings in plastic bags with Pontoscolex corethrurus (1AW = one adult worm, 2J = two juvenile worms and NW = no worms). The line at the top of each bar (treatment) indicates the standard error of the mean. Different letters on the bars indicate significant differences among means according to Tukey’s test (P < 0.05).
The first objective in the production of nursery plants is to ensure survival. In this study, no significant differences in plant survival were obtained between the traditional treatment (NW) and the other two treatments applied (AW and 2J). Based on the literature reviewed, no studies have been published on the survival of plants associated with earthworms in forest nurseries using only soil and much less for A. religiosa or other conifers. For this reason, it is not possible to compare the results obtained in the present study; however, the 86 % survival obtained in this study may be acceptable in the production of plants for a period of one year. The second objective is the quality of the plant. Some of the attributes used to define plant quality are height, stem diameter and dry weight or combined variables (Poorter & Garnier, 2007). In this study, although stem diameter was not measured, other variables such as the number of primary and secondary branches and total plant cover area were considered. The variables height and root dry weight had significant differences among treatments (Table 1; Figure 1); the adult worm (AW) treatment reached the highest values. A higher allocation of biomass to the root could be associated with a greater gain in growth in the first years of life (Pike, Warren, & Montgomery, 2016). The year-long research experimentally demonstrated that earthworms favor the allocation of biomass to the root. This could be reflected in greater growth when plants are transplanted in the field for the production of Christmas trees, a situation that will have to be tested experimentally. Avendaño-Yáñez, Ortiz-Ceballos, Sánchez-Velásquez, Pineda-López, and Meave (2014) evaluated P. corethrurus and soil mixed with stubble from Mucuna pruriens var. utilis (Wall. ex Wight) Baker ex Burck or fertilizer versus no earthworms (control) in Quercus insignis M. Martens & Galeotti seedlings; the authors demonstrated that height, diameter, leaf biomass and total biomass were higher in treatments with worms. Other worms with positive effects have been used in crop plants; for example, Dichogaster bolaui Michaelsen in sorghum (Ávila, Bautista, Huerta, & Meléndez, 2010).
A combined variable that is also used to evaluate plant quality is the root:stem ratio, because it expresses how plants respond to the environment, primarily to light; the higher the root:stem ratio, the higher the quality of the seedling (Gregory, 2006; Pike et al., 2016). In this study, no significant differences were found among treatments (P = 0.07), possibly because all plants grew in the same shade level (30 %). In this regard, Saldaña-Acosta, Meave, and Sánchez-Velásquez (2009) demonstrated that seedlings of the same species and the same batch of seeds, grown in different shade levels, allocate different amounts of biomass to the root and stem. This underlines the importance of studying the root:stem ratio in nursery plants.
On the other hand, the presence of worms reduced soil compaction (Table 1; Figure 1). Worm burrows in the soil are associated with a reduction in bulk density and therefore in compaction (Joschko, Diestel, & Larink, 1989). The results agree with the reports of other studies carried out both in the field (Blanchart et al., 1999) and under controlled conditions (Joschko et al., 1989; Langmaack, Schrader, Rapp-Bernhardt, & Kotzke, 1999).
Benavides-Meza et al. (2016) assessed the growth of sacred fir plants from eight provenances (10 months and with the application of fertilizer) in smaller containers (93 cc) under greenhouse conditions and observed that the Cofre de Perote provenance achieved height growth of 22 ± 0.94 cm. Likewise, in the present study, the use of seeds from Cofre de Perote, but in much larger containers (250 cc), with earthworms and without fertilizers, achieved height growth (12 months) of 23.6 ± 1.6 cm. This suggests that the use of earthworms in larger containers has the potential to improve the quality of plants grown in forest nurseries, even without the application of fertilizer.
Conclusions
The presence of Pontoscolex corethrurus earthworms in plastic bags, for one year and under nursery conditions, significantly favors height growth and root dry weight of Abies religiosa plants. Similarly, earthworms reduce soil compaction caused by the application of irrigation to the plastic bag where the plant grows. This biotechnological process can contribute to the production of forest species by accelerating the growth of plants in nurseries, even without the application of fertilizers.
