Method for Recovering and Counting Viable Cells from Maize Seeds Inoculated with Azospirillum brasilense

The inoculation of seeds with nitrogen-fixing and plant-growth promoting bacteria is a well-established agricultural practice that has been increasingly adopted worldwide, decreasing costs and environmental impacts of food production. Most of the globally commercialized inoculants are for the soybean crop, and a method for recovery of Bradyrhizobium cells from inoculated soybean seeds for subsequent counting has been adopted by several laboratories of South America, especially to investigate the bacterial survival on seeds treated with pesticides. However, the use of inoculants containing Azospirillum brasilense in cereal crops has exponentially increased, requiring investigation about the recovery and counting of cells from inoculated seeds. We first verified that the method used for recovery and counting of viable cells of Bradyrhizobium from soybean seeds was not applicable for maize seeds inoculated with A. brasilense. We then modified several steps of the method, aiming at succeeding in recovering Azospirillum viable cells. The main limitation was identified in the nature of the seed tegument, dry and poor in nutrients, resulting in A. brasilense cell aggregation. Pre-hydration of seeds for 2 h in sterile distilled water, followed by shaking for 30 min in sterile distilled water with Tween 80 allowed proper counting of A. brasilense cells recovered from maize seeds. The method was successfully applied to count Azospirillum cells recovered from pre-inoculated maize seeds, and to estimate the impact of seed treatment with pesticides on cell survival.