Study site

The assay was performed in El Bajío Experimental Field, Celaya, Guanajuato, México, located at 20° 34' 44.9" N and 100° 49' 09.5" W, at an altitude of 1754 m. The climate of the region according to ^{García (1987)} is BS1hw(W)(e) q, that is, temperate with rain in summer, scarce winter precipitation, and cool winter. The average annual temperature is 20.6 °C, and annual average precipitation is 597 mm.

Soil sampling

Sampling was collected from soil composed of 22 subsamples of 0-5, 5-15, and 15-30 cm in depth at the end of each cultivation cycle. The samples were dried on the shade and at room temperature. They were sieved in 2-mm mesh, and chemical determinations were made, such as water pH ratio 1:2, electrical conductivity, nitrogen content by Kjeldahl method, extractable P by Olsen, organic matter, K content, exchange bases and minor elements. The laboratory methods used in such analyses were those described by ^{Jackson (1976)}.

Implementing conservation agricultural practices

The plot had a surface of 1 hectare. During spring-summer 2010, soil was prepared with conventional farming practices (ploughing, raking, leveling, and furrowing), and maize variety “Jabalí” (Asgrow, Bayer AG, Leverkusen, DE) was sown to favor homogeneous condition of soil fertility and generate the necessary harvest waste to implement agricultural conservation practices; maize production fluctuated from 12‑14 Mg ha^‑1, adding 13 Mg ha^-1 of harvest waste used as mulch to soil surface. This cultivation system was implemented during the 2010-2011 autumn-winter cultivation cycle, sowing wheat variety Cortazar S94 (^{Solís-Moya et al., 1996}), in 0.8 m double-row furrows; grain production reached 7 Mg ha^-1, and approximately 6 Mg ha^-1 of wheat waste used as mulch were distributed on soil surface. Subsequently, during the 2011 spring-summer cycle, the plot was divided by half: one to establish maize, variety “Jabalí” (Asgrow, Bayer AG, Leverkusen, DE), and the other one to establish bean, variety “Flor de Junio Marcela” (^{Castellanos-Ramos et al., 2003}) to continue with the conservation agriculture system; maize produced 12 Mg ha^-1 and bean 3 Mg ha^-1, adding 11 and 2 Mg ha^-1 of harvest waste on soil surface, respectively.

Experiment description

This study assessed cultivation rotation after implementing agricultural conservation practices during the 2011-2012 autumn-winter cycle. Wheat variety Cortazar S94 (^{Solís-Moya et al., 1996}) was sown again in double-furrow on the harvest waste of previous cycles. Two experiments were assessed by randomized block design with four replicates. The first one assessed the inoculation effect of mycorrhizal fungi (Glomus mosseae) in wheat production, for which six treatments were established as described in Table 1. Innocuousness was performed three hours previous to sowing. The seed was mixed directly with adherent, an inoculant that contained 100 spores g^-1 of soil, and 3 kg ha^-1 were applied based on that reported by ^{Grageda-Cabrera and González-Figueroa (2015)}, who recommended applying one kg of inoculant for each 50 kg of seeds. The second experiment consisted of assessing the effect of two crop rotations: gramineae-gramineae (wheat-maize G-G) and gramineae-legume (wheat-bean G-L), which correspond to the continuity of this study previously explained in implementing conservation agriculture. In each rotation, four levels of nitrogen fertilization (NF) were studied: 0, 100, 150 and 200 units of N ha^-1 (Table 2). The experimental units, in both experiments, corresponded to four furrows 0.76 cm wide by 14 m long, randomly distributed in three blocks.

Statistical analysis

The mycorrhizal fungus inoculation assay was analyzed by a statistically randomized block design with four replicates. Tukey’s (α = 0.05) multiple comparison of means was performed. The crop rotation assay was assessed by a randomized block design experiment with 2 × 4 factorial arrangement: two-crop rotation levels of NF. The grain yield of the second assay was also analyzed by regression to make differences evident in slope and intercept by effect of growing N fertilization doses. The date of sowing was 28 December 2011. Irrigation was performed four times: at 0, 30, 57, and 88 days after sowing. The N-P-K fertilization sources were (NH₄)₂SO₄ - Ca(H₂PO₄)₂ -KCl. N was applied in two fractions, half during sowing and the other half during the second irrigation.

Agronomic parameters

The agronomic parameter assessment was performed starting from sample collection, one per experimental unit, 1 m in length per furrow width (0.76 m) where the following parameters were measured - number of plants and stems; final height of a 20-stem plant, measured from the base of the stem to the ear; grain weight, adjusted to 12% humidity; aerial biomass; 1000-grain weight, and harvest index.

Meteorological conditions

During cultivation development, starting the last week of February, temperatures higher than 30 °C at the shade were frequently recorded (Figure 1). March and April were very warm and the maximum temperatures reached 35 °C. Beyond this level of heat in the wheat flowering stage, it may cause crop damage and affect grain yield. According to ^{Solís-Moya (2007)}, the incidence of high temperatures in El Bajío accelerated the bulking period in the phenological flowering stage, consequently, limiting grain yield, which is more likely in late sowing. On the other hand, precipitation was scarce during the cultivation period; rain added up scarcely 19 mm. Precipitation of more than 8 mm was recorded at the end of the cultivation stage.

Figure 1: Distribution of maximum, minimum temperature and precipitation during cultivation development of wheat assays in El Bajío, Celaya, Guanajuato, Mexico. 2011-2012 autumn-winter cultivation cycle. 

Soil analysis

The study was performed on vertisol-pelic soil according to the classification of the United States Department of Agriculture (USDA) (^{Grageda-Cabrera et al., 2004}). Its texture type is clay with more than 60% of the fraction <2 μm, predominant expandable smectite clay, proper of this type of soils. The pH showed a decrease from slightly alkaline to neutral after the three cultivation cycles previous to this assessment (Table 3). Likewise, an increase was observed in the organic soil reserves in the first 5 cm in depth, specially carbon. The N-NO₃ and N-NH₄ concentration was greater where the legume was cultivated in stratus from 0-5 to 5-15 cm in depth after the third cultivation cycle at the end of the crop rotations gramineae-gramineae and gramineae-leguminosae; this result could have been attributed to the bacterial activity of the genus Rhizobium, which are associated symbiotically and naturally with legumes. The extractable P content was medium.

Arbuscular mycorrhizal fungus inoculation assay

The wheat yield components that evidenced significant differences (P < 0.05) by the effect of the mycorrhizal fungus inoculation in combination with the different N fertilization doses were: plant final height, grain yield, one-thousand grain weight, and aerial biomass. The lowest production level was observed in the treatment where only the mycorrhizal fungus strain was inoculated without applying chemical fertilizers (1) (Table 4). In contrast, the treatment with the application of 200 units of N ha^-1 without inoculation, which corresponded to the control group with only chemical fertilization (2), had a greater production of 64% compared to treatment 1. The previous result implied that the biofertilizer on its own did not promote the capacity of the necessary N supply in the radicle system, which the cultivation required for a greater production. Even though some studies in the State of Guanajuato have shown that mycorrhizal fungus inoculation in wheat cultivation has increased the use of N by plants (^{Báez-Pérez et al., 2012}; ^{Grageda-Cabrera et al., 2018}), it only happens if it is combined with a supplementary chemical fertilizer.

The recommended NF dosage in El Bajío for wheat production, according to ^{Solís-Moya et al. (2013)} is 220 kg ha^-1. The combined application of the biofertilizer with the chemical fertilizer showed a greater response in wheat production, as observed in the following contrast: grain yield in the inoculation treatment with G. mosseae combined with the application of 100 kg of N ha^-1 (3) had 23% more production compared with the treatment where only this fertilization dosage was applied (4). The inoculation treatment with the mycorrhizal fungi and supplied with 150 units ha^-1 of N (5) obtained the highest wheat yield and heaviest grains, double with respect to the treatment with inoculation only (1) and 68% more with respect to the treatment with only the application of this chemical fertilizer (6). Grain production evidently had a nitrogen fertilization dosage response; however, the combination with the arbuscular mycorrhizal fungi made it possible to decrease NF dosage without affecting grain yield, which could constitute production cost savings. These results agree with those reported by ^{Aguilar-Carpio et al. (2017)} and ^{Grageda-Cabrera et al. (2018)} in a similar study.

Crop rotation assay

Statistically significant differences (P < 0.05) were observed by the effect of crop rotation and NF dosage on the factors of prime importance, such as the interaction of both factors of study. Grain production in the rotation assay had different responses in function of the growing NF doses (Table 5). The G-G crop rotation was adjusted to a lineal model (R² = 0.90) (Figure 2), observing a maximum production of 4 Mg ha^-1, when 200 units of N ha^-1 were applied, while the L-G crop rotation had a medium adjustment (R² = 0.72). However, the maximum production was 4.7 Mg ha^-1 with only 150 units of N ha^-1, and it was significantly greater (P < 0.05) than G-G crop rotation (Figure 2). The previous results evidenced that the preceding bean cultivation provided nitrogen waste in the soil through the biological fixation that occurs naturally between bacteria of the genus Rhizobium and legumes, which could have been exploited by wheat cultivation. ^{López-Alcocer et al. (2017)} reported 20 strains of Rhizobium in El Bajío soils. The authors mentioned that symbiosis between bacteria of the genus Rhizobium and the legume, in this case bean, is considered a high efficiency process in atmospheric N biological fixation, that could provide up to 90% of nitrogen needs in plants. These results suggested that implementing crop rotation with legumes in production systems with agricultural conservation practices was feasible to decrease NF doses in cereal cultivation and reduce production cost. Furthermore, it was evident in the quantity of inorganic N (N-NO₃ + NH₄) assessed in soil after concluding the bean crop cycle (Table 3) where the N content was approximately 45% greater than that found in the soil where the G-G crop rotation system was located in the first 30 cm in depth.

Figure 2: Relationship between wheat grain yield and fertilizer dosage in crop rotation assay. 

Soil coverage with harvest waste -basic component of conservation agriculture practices- favors immobilization by the N applied with the chemical fertilizer (^{Grahmann et al., 2013}), specially during the second fertilization when it stays in direct contact with the organic material because tillage is not performed and N cannot stay underground. Thus, microbial biomass uses part of the N supplied for its metabolism during organic matter decomposition. El N waste left after being biologically fixed by bacteria of the genus Rhizobium in the legume rhizosphere is available for the next crop and out of the reach of the microorganisms that act on agricultural harvest found on soil surface.