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

Abstract

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.

Seed pre‐inoculation with Bradyrhizobium as time‐optimizing option for large‐scale soybean cropping systems

“…The recovery of viable Bradyrhizobium cells from inoculated seeds and counting followed the methodology previously described (Araujo et al., 2017; Penna, Massa, Olivieri, Gutkimd, & Cassán, 2011; Santos et al., 2020), also included in the Brazilian legislation (Mapa, 2010). Five treatments were evaluated, and they are described in detail in the item of field experiments.…”

Compatibility of Azospirillum brasilense with Pesticides Used for Treatment of Maize Seeds

“…The number of A. brasilense cells recovered from maize seeds significantly decreased after 24 h of inoculation for both strains, with or without pesticide ( Figure 1 ). In addition, strain Ab-V5, but not Ab-V6, was significantly affected by the seed treatment with the pesticide after 24 h, with a drastic decrease from 4.56 × 10 5 CFU seed −1 in the day of inoculation to 4.37 × 10 2 CFU seed −1 after 24 h. Santos et al [ 14 ] also reported drastic cell mortality of A. brasilense after 24 h of exposure to another pesticide (carbendazim + thiram). Pereira et al [ 20 ] observed similar results when exposing these same strains to pesticide-coated (metalaxyl-m + fludioxonil + thiamethoxam + abamectin + Peridiam) maize seeds; after 12 h of exposure, the authors observed a cell survival rate of 65.87% in seeds without pesticide and of 13.56% when treated with the pesticide.…”

“…The recovery of A. brasilense strains Ab-V5 and Ab-V6 from maize seeds treated with the pesticide was based on the method described by Santos et al [ 14 ]. The inoculum was prepared in the modified liquid DYGS medium (composed of glucose, 2 g; malic acid, 2 g; Bacto peptone, 1.5 g; yeast extract, 2 g; K 2 HPO 4 , 0.5 g; MgSO 4 ·7H 2 O, 0.5 g; and glutamic acid, 1.5 g; pH 6.8), incubated at 28°C, and shook at 120 rpm until the cell concentration of 10 8 CFU·mL −1 .…”

Combining microorganisms in inoculants is agronomically important but industrially challenging: case study of a composite inoculant containing Bradyrhizobium and Azospirillum for the soybean crop

“…Strains SEMIA 5079 and SEMIA 5080 of Bradyrhizobium spp. were grown in modified-YM broth (Hungria et al 2016 ), and A. brasilense strains Ab-V5 and Ab-V6 in modified-RC ( Rojo Congo ) broth (Santos et al 2020 ), without Congo red, in 100 mL bottles. The samples were incubated at 28 ℃ ± 2 ℃, for 5 days and 3 days, respectively, on a rotary shaker at 120 rpm.…”

“…Cultures were grown on a rotary shaker at 120 rpm, at 28 ℃ ± 2 ℃. The number of colony forming units (CFU) present in each medium was determined from aliquots aseptically removed from the cultures at different intervals during growth, by means of the spread-plate technique (O´Hara et al 2016 ), in modified-YMA solid medium (Hungria et al 2016 ) for Bradyrhizobium and RC solid medium (Santos et al 2020 ) for Azospirillum . In addition, O.D.…”