An investigation of the shelf-life (storage) of Bacillus isolates on seeds

Changes in bacterial population during storage of treated seeds of maize (Zea mays L.), bean (Phaseolus vulgaris L.), lettuce (Lactuca sativa L.) and cucumber (Cucumis sativus L.) coated with Bacillus sp. were investigated over a 12-month period. The isolates designated as B9, B11, B69, B77 and B81 were grown in Tryptone Soy Broth (TSB) synthetic medium for 72 h at 30 °C (100 rpm) in a shaker (model Sterilab). Cells were harvested at 10 000 rpm for 15 min (Beckman Centrifuge JA 20) and washed with sterile distilled water. Final viable cell concentration of pellets was determined by serial dilutions on Tryptone Soy Agar (TSA). The isolates were each applied to each seed lot using a seed sticker, Pelgel®, according to the manufacturer's (Nitragen [Lymphatec]) specifications. The seeds were coated with approximately 9 log10 CFU/ml of the bacterium, air-dried in a laminar flow chamber and placed at ambient (shelf-life) temperature. For enumeration of attached cells, 1 g of each seed lot was placed in a 9 ml quarter-strength Ringer's solution, vortexed for 2 min, followed by plating of the serial dilutions on TSA. The initial bacterial populations, which adhered to the spermosphere of the seed samples, ranged between 6 and 7 log10 CFU/g seed. There was an initial slight upward trend from month 1 to 5 by 1-2 log CFU/g for maize and bean. For maize, population stabilized between 6.91 and 7.54 log CFU/g in months 5 and 6. For bean, the highest population was obtained in month 4 between 6.52 and 7.95 log CFU/g. Populations stabilized in months 5 and 6 ranging from 6.23 to 6.98 log CFU/g. Thereafter, there was a downward trend from month 7. For cucumber, populations increased slightly up to month 4 in the order of magnitude of 1-2 before stabilizing between 6.11 and 6.78 log CFU/g in months 5 and 6. However, populations on lettuce decreased slightly up to month 2 before increasing slightly in months 3 and 4. Populations stabilized in months 5 and 6 ranging between 6.8 and 8.6. In all the samples, populations decreased from month 7 to the last sampling month. There was a 5% level of significant difference between the months for each of the seeds, but treatments within the seeds were not significant for maize, bean and cucumber.