Purpose
Basically we have two main purposes for this science project:
Purpose #1
We wanted to test the desiccant tolerance of the current Bradyrhizobium japonicum strain that is commercially used and to see if we can get some mutants that are more desiccation-tolerant than the parent strain. Desiccation tolerant strains are welcome by the inoculant industry.
Purpose #2
We are also trying to test if Darwin’s Theory of Evolution does indeed apply to rhizobia. Since the theory involves natural selection pressure, we wanted to see if rhizobia could adapt to desiccation conditions by natural mutation plus the selection pressure (long time storage in dry place).
Hypothesis
Our hypothesis is that the rhizobia will adapt to its new and desiccant environment. If the rhizobia that has gone through the desiccation period has a higher survival rate than the ones that haven’t gone through it, then natural selection pressure does exist is correct.
Procedure
We took advantage of Becker Underwood Incorporation’s pre-treated soybean seed to isolate 532C mutant. It is routine work in that company to test its inoculants’ ability of survival on soybean seed. When we started this project, we simply took one soy seed sample that had been treated with 532C culture and stored at room temperature for 2 months. Because the seed itself is quite dry, the rhizobia cells were subjected to a very desiccation environment for long enough time.
Prepare the phosphate buffer (pH 7.0); allocate 100 ml to a Nalgene™ bottle and then autoclave at 121 oC for 20 minutes.
Phosphate Buffer:
K2HPO4 1.2g/L
KH2PO4: 0.34g/L
Universal Peptone 1.0g/L
Tween 80 (Detergent): 1ml/L
Remove the phosphate buffer from the autoclave and let it cool for 15-20 minutes.
Take approximately 10 grams of soybean seed that were coated with rhizobial culture and put them into the phosphate buffer, shake for 5 minutes to wash off the rhizobia cells from the seed. Then further dilute it to 10-1 and 10-2. (Take 1 ml of liquid from the Nalgene bottle and mix it with 9 ml of buffer; that’s a 10-1 dilution. Take 1 ml from the 10-1 dilution and mix it again with 9 ml of buffer. That’s a 10-2 dilution)
Spread 100 µl (0.1 ml) of the diluted rhizobial culture onto 6 MBA plates; 6 replications are needed to insure that we get some growth of rhizobium. Set the plates in an incubator. Only the surviving rhizobia will be able to grow on the medium. This will allow us to select those desiccation-tolerant mutants, if any, which have survived under completely dry conditions for 28 days.
MBA Broth:
K2HPO4 0.55g/L
Mannitol 10g/L
Yeast Extract 0.3g/L
MgSO4 0.2g/L
NaCl 0.1g/L
It takes 6 days for 532C strain to form a 2-3 mm sized colony. Pick 2 well-grown colonies from the MBA plates by an inoculum loop and aseptically streak on an MBA-containing vial. Incubate at 28 oC for 5 days. These are the stock cultures of the mutant strain of 532C. Two colonies were picked for further studies: 532C d1 and 532C d2.
Grow 532C, 532C d1 and 532C d2 culture in special medium:
To ensure all the strains reach their maximal potential of survival long on soybean seed, a special nutrient broth that is provided by Becker Underwood Incorporation was used. Rhizobia grown in this special broth could survive on seed much longer than that grown in any other commonly used broths. We didn’t make this medium, thus the recipe is not available.
In flowhood, disinfect the inoculation loop and take 1 loop full of stock culture to the flask containing the growth medium. 532C, 532C d1 and 532C d2 were inoculated in separate flasks. Each strain was inoculated into two flasks. Set the flasks in a rotary shaker and shake at 28 ºC, 150 rpm for 7 days.
Count the viable cell numbers of the above cultures by plating method. (Plating method: Transfer 1 ml of rhizobial culture from flask and mix with 9 ml of phosphate buffer in test tube. And conduct the serial transfer till 8th tube. Take 0.1 ml out of the 6th, 7th and 8th tube onto MBA plates. Evenly spread the droplet liquid by a L-form glass rod. Incubate the plates in an incubator for 5 days to allow the colony formation. It is assumed that one colony is arisen from one viable bacterium. After incubating 5 to 6 days count the colonies on the plates and calculate cell numbers per ml in the original culture)
Measure the optical density (OD600) of the above cultures with a photo spectrometer. Generally the higher cell numbers per ml the higher OD600 value.
Test the desiccation tolerance of rhizobial cultures:
· Apply 0.688 ml of culture to 250 grams soy seed in Ziploc bags. Mix liquid culture with seed immediately to make sure that rhizobia were evenly coated on seed.
Just after coating soy seed with liquid culture, take 50 seed out of Ziploc bag and put in 100 ml of phosphate buffer solution in a Nalgene bottle. Shake for 5 minutes to wash off rhizobia from seed. Make serial dilution to 10-4. Take 0.1 ml of liquid from 10-4 dilution tube spread onto 3 MBA plates. Incubate these plates at 28 °C. Count colony number after 6 days’ incubation. These will tell us how many rhizobia were added to soy seed.
Repeat the above process and count the viable Rhizobia on seed weekly.
If the mutants demonstrate higher survival rates on dry seed, we can conclude that natural mutations do happen and our selection process does successfully serve as a selection pressure to choose the well-adapted offspring. That proves the Theory of Evolution does apply to microorganisms. The mutants will be subjected to nodulation tests to confirm their capability of nodulation and fixing nitrogen in soy plants. If possible, these mutants should be tested their abilities of increasing soybean yields in field trials or pot experiments. If these natural mutants demonstrate the desiccation tolerance and increase soybean yield, then they can be used in commercial production of soybean inoculants.