Materials and methods

Study area, soil sampling and collection

The study was conducted at the Palma Agricultural Center of the Federal University of Pelotas (UFPel) (31.8031° S, 52.5082° W), located in Capão do Leão, Rio Grande do Sul, Brazil (Fig. 1). The climate is humid subtropical, characterized by hot summers and cold winters (Kuinchtner and Buriol 2001). The study area is situated in a transitional zone between the Pampa Biome and the Atlantic Forest, being characterized by a mosaic landscape composed of herbaceous-shrubby grasslands of the Coastal Plain and remnants of Seasonal Semideciduous Forest distributed over the slopes of the Sul-Riograndense Shield. Wetlands and marshes are also common elements of the regional landscape (IBGE 2019; Venzke 2012).

Fig. 1: Geographic location of the study area. The map shows the location of Rio Grande do Sul in southern Brazil and a zoomed view of the experimental olive orchard at Centro Agropecuário da Palma, Universidade Federal de Pelotas, where the olive cultivars sampled in this study are located

The experimental olive orchard was nine years old and consisted of several olive cultivars grown under the same environmental conditions, with each planting row corresponding to a single cultivar. Three cultivars, namely Arbequina, Coratina, and Picual, were selected for this study because they are among the most representative olive cultivars cultivated in Rio Grande do Sul, occurring in 97%, 30%, and 75% of orchards, respectively (Oliveira et al. 2022), and because they exhibit contrasting agronomic and biochemical characteristics (Coutinho et al. 2009; Grompone and Villamil 2013; Miho et al. 2021). The orchard has been managed under a rainfed, low-input system for the past two years, with no application of synthetic fertilizers, herbicides, or pesticides during this period. At the time of sampling, the trees were in the vegetative growth stage, and spontaneous vegetation, predominantly composed of grasses (Poaceae), was present between rows.

For each cultivar, four trees were randomly selected, totaling twelve sampled trees. To minimize edge effects, only the central trees of each row were considered for sampling. Rhizospheric soil was collected from each tree at a depth of 0–20 cm. Three subsamples were taken at equidistant points around the root zone of each tree after removing the litter layer and were subsequently homogenized to form one composite sample per tree, resulting in four independent composite samples for each cultivar (n = 4), totaling 12 experimental samples. From each composite sample, 100 g of soil were used for spore extraction. Since the three cultivars were grown within the same cultivation system, the remaining soil from the twelve composite samples was pooled and homogenized to obtain a representative sample of the experimental orchard, which was used for the determination of soil macro- and micronutrient contents (Table 1).

Category | Nutrient/Parameter | Value | Unit
pH | pH in water (1:1) | 5.4 | 
Macronutrients | Calcium (Ca) | 3.1 | cmolc/dm3
Magnesium (Mg) | 1.1 | cmolc/dm3
Potassium (K) | 0.15 | cmolc/dm3
Phosphorus (P–Mehlich) | 9.3 | mg/dm3
Sulfur (S–SO4) | 6.9 | mg/dm3
Organic matter (OM) | 1.66 | %
Micronutrients | Zinc (Zn) | 1.2 | mg/dm3
Copper (Cu) | 1.2 | mg/dm3
Manganese (Mn) | 59 | mg/dm3
Sodium (Na) | 9 | mg/dm3
Iron (Fe) | 0.16 | %

Spore extraction and identification

Arbuscular mycorrhizal fungi spores were extracted from each sample using the wet sieving method (Gerdemann and Nicolson 1963), by agitating 100 g of soil in 2 L of water, followed by decantation and passage through 0.84 mm, 0.42 mm, and 0.149 mm sieves, repeating the process three times. The material retained on the 0.149 mm sieve was transferred to 50 mL Falcon tubes and centrifuged at 2,000 rpm for 3 min. The supernatant was discarded, and the sediment was resuspended in a 50% (w/v) sucrose solution, followed by a second centrifugation at 2,000 rpm for 2 min. The supernatant, containing the floating spores, was immediately passed through a 0.149 mm sieve and washed with distilled water. Spores were transferred to Petri dishes and counted under a stereomicroscope. Then, the spores were carefully separated into morphotypes based on size and color and mounted on slides using PVLG (polyvinyl-lactoglycerol) and PVLG mixed with Melzer's reagent (1:1), as described by Koske and Tessier (1983). The slides were kept at room temperature for about a week and subsequently dried in an oven at 35 °C for 3 days. The slides were inspected under a light microscope and identified to genus and/or species level based on the reaction of the spore wall structure to Melzer's reagent, and compared with descriptions from the pages of the International Collection of Arbuscular Mycorrhizal Fungi (INVAM) at the University of Kansas, USA (INVAM 2024).