Effective phosphate solubiliser strain selection

Isolates were screened for PGP traits of ACC deaminase, and ability to solubilise phytate, TCaP, HydroxP, and FeP. The screening was carried out using agar plate assays using a 96 pronged colony replicator. At this stage of the screening, there was no attempt to quantify cell numbers and reactions on agar plates were scored visually, using the scoring systems described below. A strain PSB85 previously shown to solubilise inorganic P from the REA plate where a zone of clearance could be observed was used as a positive reference strain for every plate assay. This enabled both validation of media composition and a reference from which to define a relative increase or decrease in halo size of other patched bacteria. Raw data from the screening assays are in Appendix D.

2.2.1.1

In vitro phosphate solubilisation

Previously, the ability of the 105 isolates to solubilise P was assessed by Dr. Carolyn Mander in the MBIE programme “Microbes for reduced P inputs”. The culture medium consisted of various sugars (listed below) at concentrations that reflected the composition of ryegrass root exudates (Mander et al., 2012). Each litre of culture broth contained 5.0 g fructose, 2.5 g sucrose, 1.5 g glucose, 0.5 g inositol, 0.5 g mannitol and 10 mL of trace elements solution (0.4 g NH4NO3, 0.4 g MgSO4.7H2O, 0.3 g

KCl, 0.2 g NaCl, and 0.04 g FeSO4 in 1 L). The pH of the culture broth was adjusted to pH 7.0 prior to

autoclaving for 15 min at 1.1 kg/cm2_{, 121°C, and 20 mL of culture broth was placed in a 50 mL conical}

flask with 30 mg of dry-autoclaved insoluble DCaP (CaHPO4). Bacteria were grown overnight at 25°C

in LB broth, and 100 µL was used as a starter culture. Each conical flask was incubated for 7 days (25°C, 250 rpm) prior to a colourimetric assay developed by Murphy and Riley (1962) which measures the amount of soluble P present in the culture medium. This assay is based on the reaction of acid ammonium molybdate with orthophosphate to form phosphomolybdenum complexes. These complexes are then reduced to molybdenum blue by sulphuric acid with a resultant blue colour change as shown in Figure 2.2.1. The molybdenum blue reagent contained 51 mM ammonium molybdate, 259 mM ferrous sulphate, 6.4 mL of 10% sulphuric acid, and the total volume was

adjusted to 20 mL with dsH2O (Appendix A.1.5). Each reaction was performed by incubating equal

volumes of sample supernatant with the molybdenum blue reagent for 30 min at room temperature, and colour changes were measured at 850 nm using the FLUOstar Omega microplate reader (BGM Labtech, Ortenberg, Germany). The negative control (blank) comprised a flask of culture medium that had not been inoculated with bacteria which was incubated under the same conditions as the treatments. The standard curve was made by dipotassium hydrogen orthophosphate (K2HPO4)

solutions at 200, 400, 600, 800 and 1000 µM concentrations (Figure 2.2.1). Each sample was diluted using dsH2O to appropriate dilutions within the range of the standard curve and the value from the

negative control (uninoculated culture medium, blank) was subtracted before calculations.

2.2.1.2

Tricalcium phosphate solubilisation by PSB

To increase the possibility of finding more effective PSBs, β-tricalcium phosphate (TCaP) was used in agar medium instead of DCaP. The TCaP plate assay was based on a method described by Richardson & Hadobas (1997) for a phytate plate with slight modifications using TCaP instead of sodium phytate. This minimal medium contained glucose for bacterial growth, various salts to provide essential elements, and TCaP as the only P source for bacterial growth. The medium contained 55.5 mM glucose, 62.5 mM NH4NO3, 6.7 mM KCl, 2.0 mM MgSO4, 0.006 mM MnSO4, 0.04 mM FeSO4, 6 g of β-

Figure 2.2.1 Microtitre plate of soluble phosphate concentration measured by Murphy and Riley’s colourimetric method. Colourimetric change from colourless to blue corresponding to the concentration of soluble phosphate. The amount of phosphate released from the in vitro

phosphate solubilising assay after 7 days incubation (25°C) by ten isolates, and was determined after 20-fold dilution. The positive standard controls were 0, 200, 400, 600, 800, 1000 μM of

dipotassium hydrogen orthophosphate (K2HPO4) on the top row, and the absorbance was

tricalcium phosphate (β-Ca3(PO4)2) (Sigma-Aldrich, USA, Cat. 21218), and 15 g of agarose gel per litre

of medium. The mixture was adjusted to pH 7 and autoclaved. Prior to pouring the agar plates, 100 µL of 10 x trace element solution was added and mixed (original recipe from Hoagland’s solution, Appendix A.1.7, Hoagland (1950). Each isolate was then patched on the TCaP plate and the strain PSB85 used as a reference strain (Figure 2.3.1). The plates were incubated at 25°C for 3 days. The ability of each isolate to solubilise TCaP was then defined by the halo size. Isolates scored as “3”

produced a large halo with diameter ≥ 8 mm, “2” produced a medium sized halo of 5 – 7 mm, “1” a

small halo of ≤ 4 mm, and “0” where no halo was observed.

2.2.1.3

Detection of bacterial acidification of medium during tricalcium phosphate

solubilisation

To detect organic acid production by PSBs, methyl red was used as an indicator dye in the agar-based screening system. This indicator dye changes from yellow to red as pH decreases from 6.2 to 4.4, indicating organic acid production by the bacterial isolate (Divya et al., 2008). The agar plates were made by adding 1% methyl red into the TCaP agar mixture after autoclaving and mixing well before being poured into petri dishes. The final TCaP plate containing methyl red was designated as Me-Red. Each isolate was patched onto Me-Red plates and strain PSB85 was included on plates as a reference strain. The plates were incubated at 25°C for 3 days. Isolates that produced large red circles around the colony after 7 days were scored as “3”, pink circles were scored “2”, faint pink colour changes were scored a “1” and colonies were scored as “0” when no colour change was detected in the agar.

2.2.1.4

Hydroxyapatite solubilisation by PSB using glucose or sucrose as the sole carbon

source

Ability of isolates to solubilise hydroxyapatite (HydroxP) was also assessed. Many different carbon sources can be found in root exudates; in addition to glucose, sucrose is one of the sugars most commonly found (Jaeger et al., 1999, Paterson et al., 2007). For this reason the ability of isolates to solubilise P in the presence of glucose or sucrose was compared. Hydroxyapatite (HydroxP) medium was based on the TCaP plate medium described above, but contained HydroxP as the P substrate instead of TCaP. The medium contained 6 g of HydroxP (Ca5(OH)(PO4)3) (Sigma-Aldrich, USA, Cat.

04238), and 55.5 mM of either glucose or sucrose (referred to as Hyp-G or Hyp-S, respectively). The mixture was adjusted to pH 7 and autoclaved. Prior to pouring the plates, 100 µL of 10 x sterile trace element solution (Appendix A.1.4) was added and mixed. Isolates were then patched on either Hyp-G or Hyp-S plate and allowed to grow for 3 days (25°C). PSB85 was used as a reference strain. Isolates

were scored as “1” if there is a visible halo around the colony, and “0” if no halo was observed (Figure 2.3.1). This scoring system was used instead of comparing the halo size, as the majority of isolates screened in the assay exhibited no obvious HydroxP solubilisation using either glucose or sucrose as a carbon source.

2.2.1.5

Iron phosphate solubilisation by PSB

Isolates were patched onto iron phosphate plates (FePA, FePO4·2H2O), prepared as for TCaP plates

described in Section 2.2.1.2 but with a slight modification. It is known that Fe-P precipitates are found in acidic soil, so the agar plates were made to be acidic. The pH was adjusted to pH 4 before FePO4 was added to the mixture, and the composition of other elements remained the same as for

TCaP, as described above. Isolates were then patched onto FePA plates and allowed to grow for 3 days at 25°C. Using this medium, visually different halo sizes were produced around the colonies. A

large halo size (≥ 8 mm) was scored as “3”, medium size halo (5 – 7 mm) as “2”, small halo size (≤ 4

mm) was scored as “1” and “0” where no halo was observed (Figure 2.3.1).

2.2.1.6

ACC deaminase production by PSB

Each isolate was patched onto ACC deaminase agar plates to screen for isolates capable of utilising ACC as the sole nitrogen source i.e. ability to produce ACC deaminase. The plates were prepared as described by Li et al. (2000) using a combination of Dworkin and Foster (DF) salt minimal medium (Dworkin and Foster, 1958) with addition of 3 mM of ACC. DF salt medium consisted of 29.4 mM KH2PO4, 42.3 mM Na2HPO4, 0.8 mM Mg2SO4.7H2O, 11 mM glucose, 10.2 mM gluconic acid, 10.4 mm

citric acid, and 100 µL of 10 x trace element solution (Appendix A.1.4). The solution was adjusted to pH 7.2 prior to adding ACC to a final concentration of 3 mM. The mixture was autoclaved before being poured into sterile petri dishes. Isolates were then patched onto the ACC plates and allowed to grow for 3 days at 25°C. Given that ACC was the sole nitrogen source, isolates that had grown by this time were able to utilise ACC and were considered as ACC deaminase-producing bacteria. Bacteria that grew on these plates were scored as “1” while isolates that failed to grow were scored “0”

2.2.1.7

Phytase mineralisation by PSB

Ability of strains to solubilise phytate was measured on Sodium phytate plates (Na-Phy) using the method described in Richardson & Hadobas (1997) with slight modifications. The Na-Phy medium contained 62.5 mM NH4NO3, 18 mM CaCl2, 6.7 mM KCl, 4.2 mM MgSO4, 0.006 mM MnSO4, 0.04 mM

CAS 14306-25-3), and 15g of agarose gel per litre of medium. The mixture was adjusted to pH 7 and autoclaved. Prior to pouring the agar plates, 10 mL of 20% glucose, 100 µL of 10 x trace element solution (Appendix A.1.4) and 200 µL of thiamine (10 mg/mL) was added and mixed well using a magnetic stirrer. Isolates were then patched onto the Na-Phy plates. The plates were incubated at 25°C for 3 days and the ability of each isolate to utilise sodium phytate was defined by the halo size

and scored as three sizes, “3” as large halo (≥ 8 mm), “2” as medium size halo (5 – 7 mm), “1” as

small halo (≤ 4 mm), and “0” as no halo observed (Figure 2.3.1).