*2.6. Root Adherence*

To determine bacterial adherence to young roots, exponential phase cultures were exposed to germinating soy seeds. Seeds were sterilized as described previously [24]. Briefly, seeds were sterilized by washing in 30% of concentrated bleach for 5 min while shaking gently, followed by three consecutive washes with sterilized deionized water under shaking. Seeds were suspended in 70% ethanol while being shaken gently for 20 min, followed by seven consecutive washes with sterilize deionized water under shaking. Following a final 20 min soak in sterile deionized water, seven seeds were placed onto each R2A agar plate (Difco) and left in the dark at 30 ◦C for 9 d. R2A comprised of yeast extract 0.5 g/L, proteose peptone No. 3 0.5 g/L, casamino acids 0.5 g/L, dextrose 0.5 g/L, soluble starch 0.5 g/L, sodium pyruvate 0.3 g/L, dipotassium phosphate 0.3 g/L, magnesium sulfate 0.05 g/L and agar 15 g/L. The plates were inspected every 2 d, and plates with seedlings showing microbial outgrowth on the agar were discarded. By day 9, roots varied from 8 to 12 cm in length.

The exponential phase cultures were prepared as described above and diluted in fresh medium to absorbance of 0.100 at 600 nm. Aliquots were taken, and the culturable count was determined using the droplet plate count technique (Lindsay and VonHoly, 1999). Briefly, samples were serially diluted and 20 μL volumes spotted onto R2A plates. After incubation at 30 ◦C for 4 d, colonies were counted. Five seedlings were added to each 250 mL flask containing 50 mL of exponential phase cultures (50 mL) prepared in either PSY or SESOM as outlined above, left for 60 min while shaking gently. Seeds were removed aseptically using sterile tweezers, washed twice with sterilize deionized water and transferred to 50 mL conical tubes containing 50 mL PBS with 0.02% Tween 20 [24]. Tubes were immersed in a sonicator bath (Fisher FS20) and exposed to ultrasound for 30 s, and then transferred to ice water for 30 s to prevent undue heating. This sonication and cooling cycle was repeated 4 times. One mL of treated sample was drawn to perform serial dilutions and triplicate 20 μL volumes were plated on R2A plates. After incubation at 30 ◦C for 4 d, colonies were counted.
