2.4. Greenhouse Experiment

A greenhouse experiment was carried out to assess the effects of the pesticide used together with the inoculant on the initial development and root morphology of maize. Plants were grown in modified Leonard jars [17] containing 750 cm³ of sterilized substrate, consisting of a mixture of sand and pulverized coal (3 : 1, v/v), containing sterile nutrient solution with all macronutrients and micronutrients [18]. The experiment consisted of the same four different treatments described before: NPNI, WPNI, NPWI, and WPWI. The experimental design was performed in a completely randomized design (CRD) with treatments arranged in a factorial scheme (2 × 2), involving presence/absence of pesticide and presence/absence of inoculation with A. brasilense, with five replicates.

Maize seeds (Agroceres AG-9010) were surface-sterilized with 70% ethanol and 3% sodium hypochlorite [17]. For the treatments WPNI and WPWI, the seeds were treated with Standak™ Top (2 mL per kg of maize seeds) and left to dry for two hours. For the treatments NPNI and NPWI, this step was not performed. The seeds of the treatments NPWI and WPWI were inoculated with a mixture of Ab-V5 and Ab-V6, ensuring a concentration of 10⁵ cells seed⁻¹. Three seeds were sown and thinned to one plant per jar four days after emergence. Nutrient solution was applied as needed, and the temperature and humidity at the greenhouse were controlled by means of air conditioners (25° ± 5°C and 80 ± 5%, respectively).

Thirty-eight days after emergence, plant height (cm) and culm diameter (mm) were assessed with the aid of a ruler and digital caliper. Plants were harvested and the shoots were separated from the roots. The shoots were oven-dried at 60°C until constant dry weight. The roots were washed with running water until completely clean. Approximately, 1 g of fresh roots from each sample was separated and stained in methylene blue (1%) solution for 1 min and washed again in water and scanned with Epson Perfection V370 Photo^® for further morphological analysis. The remaining roots were oven-dried under the same conditions as the shoot.

The scanned root images were analyzed using GiA Roots^® software to assess specific length (m·g⁻¹), weighted average diameter (mm), tissue density, and volume (cm³). The value determined in each scanned root fragment was estimated for the total fresh mass of the root system.

Approximately, 0.1 g of fresh fine roots was obtained from each sample, stored in FAA solution (90% ethanol 70%, 5% formaldehyde, and 5% acetic acid) and used for assessment of root-hair incidence, root-hair length, and number of root branches. The number of root branches was counted using a stereomicroscope at 30X magnification to estimate the number of lateral roots. Root-hair incidence was determined by the presence or absence of root hairs on 150 fine-root intersections using the gridline method [19]. Root-hair length was assessed measuring 50 root hairs in fine-root segments using a microscope at 100X magnification with an ocular micrometer.

The dataset was first evaluated for normality and variance homogeneity. Means were compared using the analysis of variance (ANOVA) followed by Tukey's test at 5% probability. All analyses were performed in the software RBIO®.