Study site

The study was conducted from 2015 to 2018 in Delicias, Chihuahua, Mexico, in the Santa Maria orchard (28° 22’ 88’’ north latitude and 105° 37’ 67’’ west longitude). To do this, 15 'Western' variety trees, from 16 to 19 years old, grafted onto native pecan rootstock (grown from seed) were selected. The orchard was planted at 13 x 13 m, and each tree had a 350 LPH micro-sprinkler (LWP 2450, NETAFIM™, Mexico) for irrigation. Soil characteristics were: sandy loam texture, pH of 7.9, very low organic matter content (0.58 %), 6.0 % CaCO₃ and low salinity (EC of 0.84 dS∙m^-1).

Treatments

The 15 pecan trees selected were homogeneous in terms of trunk cross-sectional area (on average 18 cm) and crown volume (abundant and homogeneous in its four quadrants). Three treatments were established to compare pecan production. The first was conventional foliar Zn fertilization, the second was fertilization with soil-applied chelated Zn, and the third was Zn chelated to the soil plus the addition of Azospirillum brasilense to stimulate Zn solubilization and the mycorrhizal fungus Pisolithus tinctorius (Table 1). The chelated form was chosen because it favors Zn uptake by the roots and translocation by the conducting tissues in the tree (^{Sharma, Patni, Shankhdhar, & Shankhdhar, 2013}). Treatments were applied to the same trees for all four years. Each tree represented one replicate under a completely randomized design.

The initial Zn concentration in the soil was determined by atomic absorption spectrophotometry (Analyst 100, Perkin Elmer®, Mexico) in two samples from the irrigation area, in the 0-30 cm and 30-60 cm strata. Leaf Zn concentration was analyzed in dehydrated tissue by triacid digestion (HNO₃, HClO₄ and H₂SO₄, at a 10:1:0.25 ratio) by means of atomic absorption spectrophotometry using the DTPA method (^{González, Almendros, & Álvarez, 2009}).

Despite finding an adequate concentration of Zn, it was decided to apply chelated Zn twice per growing season, since this Zn is not necessarily available to the tree. This is because the chelated form allows the element to remain available in the soil and water under extreme conditions, such as high pH (> 7.5) and high carbonate concentration (> 6 %).

Vegetative and productive variables

Shoot length was evaluated in June of each year, on one tree shoot per quadrant at a height of 2 m, which was marked in order to always evaluate the same one. Yield (kg of pecans per tree) was determined in October of each year. Foliar Zn analysis (mg∙kg^-1) was carried out on 10 leaflets per tree the first week of August. Leaflets were taken from the external and internal part of the tree, trying to cover the four cardinal points of the canopy, washed with 10 % acetic acid solution in distilled water and subjected to wet digestion with an acid mixture (HNO₃/HClO₄). The measurement was made by atomic absorption spectrometry using the DTPA method (^{González et al., 2009}).

During the years of study, the orchard soil was maintained with a cover of weedy plants with growth controlled by periodic mowing to stimulate the establishment of mycorrhizae and microbial diversity in the soil (^{Madrid-Delgado et al., 2021}). According to ^{Dakora and Phillips (2002)}, the vegetative cover of native weedy plants with limited height causes an increase in the amount of root exudates, as well as in microbial activity and in the chemical transformation of nutrients.

Evaluation of total bacteria and fungi

The concentration of bacteria and fungi (CFU∙g^-1) in the soil was quantified using the techniques established by Official Mexican Standards NOM-111, NOM-092 and NOM-110-SSA-1994 in order to relate the concentrations of the microbiota to the treatments. The ectomycorrhization percentage was determined by observing the roots under a stereoscopic microscope (GmbH 37081, Carl Zeiss microscopy, Germany) with the following equation: colonization percentage = number of mycorrhizae / total number of roots (^{Aguilar-Ulloa, Arce-Acuña, Galiano-Murillo, & Torres-Cruz, 2016}).

Identification of mycorrhizal fungi

The sporocarps found in the orchard were collected for characterization. The surface of the sporocarps was cleaned with 75 % alcohol following the methodology of ^{Zhang, Li, Wu, Ye, and Kang (2019)}. The fungus was isolated from the inner tissue of the central zone of the gleba, and placed on Melin-Norkrans agar. From the growth in the Petri dish, DNA extraction was performed using the commercial Power Soil® kit (QIAGEN) following the manufacturer's protocol. Amplification of the ITS region was carried out by polymerase chain reaction (PCR) using the iTaq Universal SYBR® Green Supermix kit. The concentration of the ITS1/ITS4 primers was 250 nM. The PCR conditions were: initial temperature of 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C for 30 s, an extension at 72 °C for 30 s, denaturation at 95 °C for 15 s, dissociation curve at 60 °C for 1 min and final denaturation at 95 °C for 1 min. Two PCR products from two sporocarps were sequenced using the Sanger method, a quality analysis of the obtained sequences was performed, and the consensus sequence was developed with both sequences. The highest similarity was determined with the NCBI BLASTn tool.

Costs

The cost of each treatment was calculated including the costs of foliar Zn, diesel and payment to the tractor driver. In the second treatment, the cost of chelated Zn per hectare and per production cycle was added. In the third treatment, in addition to chelated Zn, the cost of beneficial microorganisms was added.

Statistical analysis

Analysis of variance of the data was performed according to a completely randomized design, and Tukey's range test (P ≤ 0.05) was also carried out, both with MINITAB 18 statistical software.

Fruiting shoot length

Although no statistical differences were found between treatments (Table 2), foliar Zn exhibited the greatest shoot length, with an average of 13.99 cm in the four years. ^{Tarango-Rivero et al. (2009)} state that average fruiting shoot growth for adult 'Western' pecan trees is 16 to 20 cm per year. The results of the first two years of the study reflect that shoot size was in the range considered conventional by these authors; however, in 2017 and 2018, the average fruiting shoot length was less than 12.6 cm. The above suggests that in pecan there is physiological variation between years, although the reason is unknown.

Specific leaf area

Regarding specific leaf area (SLA), no significant statistical differences were found between treatments (Table 3).

The SLA obtained with soil Zn and the mycorrhizal fungus P. tinctorius is similar to that obtained by ^{Tarango-Rivero and García-Nevárez (2014)}, who report that mycorrhization induced by P. tinctorius in pecan trees improved Zn uptake, growth, and nut production (from 4.6 to 22.9 kg·tree^-1) from 2004 to 2008.

In addition to Zn, there are other environmental and management factors involved in pecan tree growth that should be considered in future studies; among them are irrigation, pruning, and nitrogen fertilization. According to ^{Lombardini, Restrepo-Diaz, and Volder (2009)}, pecan trees need good canopy management to avoid self-shading and maintain productivity (^{Smith, 2008}).

Foliar Zn concentration

The foliar treatment generated the highest leaf Zn concentration (43.64 mg∙kg^-1) during the four years (Table 4). In general, foliar Zn concentration was low in all treatments except in the 2016 fertilization. ^{Heerema et al. (2017)} report Zn ranges in leaves from 14 to 22 mg∙kg^-1 in 'Wichita' pecan trees, so the results of this study are within normal limits for foliar Zn.

The treatment of soil Zn combined with the mycorrhizal fungus generated an increase in foliar Zn concentration of 24 %, compared to chelated Zn alone (Table 4); in addition, growth was greater. A recent study points out that N fertilization has a significant interaction with Zn concentration; the higher the N concentration, the higher the foliar Zn content in pecan trees (^{Cruz-Álvarez et al., 2020}) and other crops (^{Alloway, 2008}).

Although ^{Pond et al. (2006)} note that the normal foliar Zn concentration in pecan trees should be 86 to 256 mg·kg^-1, ^{Sherman (2018)} proposes lower standards in leaves washed before analysis for semiarid regions (30 to 50 mg∙kg^-1 Zn). Based on the above range, only the control reached an adequate Zn concentration. It has been argued for decades that much of the nutrient that is considered sufficient is actually the residual deposit on the leaf surface and not metabolic Zn (^{Smith & Storey, 1976}); this is how this element is translocated from old leaves to developing organs (^{Alloway, 2008}).

Currently, there is little scientific information on foliar Zn applications in pecan trees (^{Ojeda-Barrios et al., 2014}). ^{Ferrandon and Chamel (1988)} and ^{Zhang and Brown (1999)} report that foliar Zn sprays on Pistacia vera and Pisum sativum showed little mobility and, apparently, little effectiveness, since 10 days after the application of the treatments more than 89 % of the element was still found in the leaf of both crops. Due to the low translocation capacity of Zn, many foliar applications are required (^{Swietlik, 2001}). Foliar uptake and nutrient translocation in pecan are poorly understood physiological processes. Apparently, foliar fertilizers are not designed based on plant physiology. An example of this is soluble Zn salts for foliar fertilization, which can cause leaf burn (^{Drissi, Houssa, Bamouh, & Benbella, 2015}).

Yield

Although the yield of adult pecan trees is closely related to the nutritional reserves of the previous year, the nutritional supply of the current year also affects fruiting (^{Smith, 1991}). In this study, all Zn treatments showed good nut production. Foliar and soil treatments with Zn were statistically similar in production (Table 5). ^{Walworth et al. (2017)} report that nut yield was lower in pecan trees receiving 4.4 kg·ha^-1 of Zn, compared to those receiving 2.2 kg·ha^-1, although without statistical differences. Furthermore, these authors achieved higher nut yields in treatments with chelated Zn applied by fertigation in 'Western' and 'Wichita' pecan trees after the third year of application.

Microbial populations and mycorrhization

The activity of native mycorrhizal communities, where neither P. tinctorius nor soil Zn was applied, allowed the Zn available in the soil to be absorbed by the roots of the control treatment. In this regard, ^{Ge, Brenneman, Bonito, and Smith (2017)} reported that soil-available Zn is sufficient for pecan growth and fruiting in the presence of ectomycorrhizal fungi. Therefore, minimum tillage may favor the natural environment of mycorrhizae in the pecan orchard. Apparently, by not applying Zn to the foliage or soil, tree roots stimulate rhizospheric microbial activity to favor nutrient acquisition from the soil (^{Read & Pérez-Moreno, 2003)}, as occurred in the control treatment of this study.

As expected, the trees that received soil Zn and mycorrhizal fungi presented a high concentration of bacteria and fungi in the soil, and a high percentage of ectomycorrhization (Table 6). ^{Tarango-Rivero et al. (2004)} found similar mycorrhizal percentages in young pecan tree roots. ^{Madrid-Delgado et al. (2021)} point out that there are numerous factors that influence the populations of microorganisms in the rhizosphere.

The identity of the native ectomycorrhiza was confirmed by amplification and sequencing of the ITS gene, where the fungus Pisolithus tinctorius was identified (Table 7). The basidiocarps found in the experimental area presented the typical characteristics of this fungus (Figure 1), and their shape varied from globose, obpyriform to claviform, from 4 to 11 cm in diameter and from 3.0 to 8.5 cm in height, without considering the length of the stipe. The latter was fibrous, yellowish and deeply rooted, and reached 5 cm in diameter by 9 cm in length. The stipe had a fine, smooth, bright yellow to yellowish-brown peridium with dark olive tones. The texture of the basidiocarp was fleshy to slightly viscous in the young stages, and brittle pulvinate in the mature stages. When opened, the gleba and peridioles were white, yellowish brown, reddish brown and olive in tone. Spores were spherical, 7 to 8 μm in diameter, with ornamentations of approximately 0.5 μm. The ectomycorrhizal roots of the pecan trees took the form of the coralloid type (Figure 2).

Figure 1 Fruiting body of the ectomycorrhizal fungus Pisolithus tinctorius. Santa María pecan orchard in Delicias, Chihuahua, Mexico. 

Figure 2 Coralloid-type ectomycorrhizal feeder roots of pecan trees (Carya illinoinensis). Santa María pecan orchard in Delicias, Chihuahua, Mexico.  

Zinc application method

According to the results of the study, soil-applied Zn is effective for pecan trees when the nutrient is concentrated in an application band and surface feeder roots are ectomycorrhizal. ^{Wood (2007)} applied Zn sulfate or oxide in a 10-cm-wide band over underground irrigation lines supplying five-year-old pecan trees in acid soils. This author points out that the application of 264 g of Zn per tree supplies the nutrient needs for up to three or four years, since the fertilizer slowly penetrates the soil and each year that passes the feeder roots have contact with the Zn band.

Costs

The cost analysis is summarized in Table 8. The results show that both treatments have the same cost ($116.00 USD·ha^-1), but if mycorrhizal fungi are added, the cost must be increased by $36.00 USD·ha^-1.

In the conventional production model for pecan trees in Chihuahua, 5,400 g·ha^-1·year^-1 are discharged into the environment, while applying 280 g·tree^-1 of chelated Zn to the soil is equivalent to 2,660 g∙ha^-1, which is enough for the pecan cycle. Tractor diesel consumption for fertilizing with Zn sprayed on foliage could be reduced by 65 %, as well as water, and soil compaction would be avoided, since five or more foliar applications are required.