Phosphate solubilization test

Prepare phosphate solubilization media

[[isolation-pgp-microbes/media/Pikovskaya-agar]] Note: The addition of 0.1% Bromo Cresol Green pH indicator can aid in observation of clearing.

Quantify phosphate solubilizing bacteria in soil samples

Gram negative populations:

1. 5 g of processed soil is weighed out into a plastic weigh-boat.
   • Record the weight of the soil sample and empty weigh-boat.
2. Place the dry soil into 50 ml cold sterile DI-H~2~O
3. Place flask on orbital shaker at ~150 rpm for 1 h.
   • NOTE: It is best to shake the soil solution in the cold.
     • If a refrigerated orbital shaker is not available, the flask can be shaken for 20 min, then refrigerated for 30 mins, and repeat until soil solution has been shaken for 1 h 20 mins.
       • Note: This shake-refrigerate method requires longer total shaking time.
4. Once shaking is finished, serial dilute 10-fold soil solution up to 5 times, for a final 10^-5^ dilution.
5. Plate 100 ul of dilutions 10^-5^, 10^-4^, 10^-3^ on to Pikovskaya's agar.

Gram-positive and spore forming populations:

1. 5 g of processed soil is weighed out into a plastic weigh-boat.
   • Record the weight of the soil sample and empty weigh-boat.
2. Place the soil sample in the chemical fume hood, covered by sterile cheese cloth, for 36 h to dry.
3. Place the dry soil into 50 ml cold sterile DI-H~2~O
4. Place flask on orbital shaker at ~150 rpm for 1 h.
   • NOTE: It is best to shake the soil solution in the cold.
     • If a refrigerated orbital shaker is not available, the flask can be shaken for 20 min, then refrigerated for 30 mins, and repeat until soil solution has been shaken for 1 h 20 mins.
       • Note: This shake-refrigerate method requires longer total shaking time.
5. Once shaking is finished, serial dilute 10-fold soil solution up to 5 times, for a final 10^-5^ dilution.
6. Plate 100 ul of dilutions 10^-5^, 10^-4^, 10^-3^ on to Pikovskaya's agar.

Quantify phosphate solubilizing for individual isolate

1. Inoculate a single isolated colony to isolate appropriate liquid rich media.
2. Incubate for 48 h at 25^o^C with shaking.
3. After incubation adjust culture to OD~600nm~ 0.1
4. Place 4-5 ul drops of adjusted culture in four quadrants of PKA plate, spaced equidistant from each other.
   • NOTE: Unless unavoidable, do not inoculate different isolates onto the same plate.
5. Plates are then incubated at 28^o^C for 7 days
6. After 7 days, observe plates for presence of clearing zone around colonies.
7. If clearing zone is observed, measure diameter of zone and record.
8. Continue incubation for an additional 7 days (14 days total).
9. At 14 days, observed colonies and measures the diameter of clearing zone and record data.
10. Dispose of plates

General notes:

• A positive phenotype is a zone of clearing around the colonies.
  • If a colony is positive for P-sol then the zone diameter will be measured.
  • The clearing is measured at two perpendicular right angles.
• This method can be repeated for potassium and zinc solubilization, only the media needs to be changed.
• See potassium and zinc solubilization media recipes:
  • Potassium
    • Nguyen et al 1992; Prajapati et al 2012; Hu et al 2006
  • Zinc
    • Gandhi and Muralidharan 2016; Edi-Premono et al 1996; Saravanan et al 2011

References:

• Liang et al 2020. Novel phosphate-solubilizing bacteria enhance soil phosphorus cycling following ecological restoration of land degraded by mining
• Rodriguez and Fraga 1999. Phosphate solubilizing bacteria and their role in plant growth promotion
• Liu et al 2015. Characterization of phosphate-solubilizing bacteria isolated from calcareous soils
• Chen et al 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities
• Zaidi et al 2009. Plant growth promotion by phosphate solubilizing bacteria