A SPECTOS INSTITUCIONALES

Four isolates resulted in significant increases in MDW in comparison with the Nil control; Ba28 (3.37 g pot-1_{), Wi28 (3.40 g pot}-1_{), EE131 (3.42 g pot}-1_{) and Wh15 (3.44 g pot}-1_{) vs Nil control (3.04 g pot}-1₎

(Figure 2.3.4, Table 2.3.6). Isolates EE127, EE132, Ba40 and Ha185 also increased growth of ryegrass but at a level that was not statistically significant at the 5% level (Figure 2.3.4). Two isolates (EN101 and Ha200) reduced the overall MDW of ryegrass but this difference was not statistically significant (Table 2.3.6, Figure 2.3.4).

Figure 2.3.4 Glasshouse pot trial of the ten EPS inoculants. The mean dry weight of ryegrass is recorded as gram per pot (n = 14). The non-inoculated negative control is indicated as the Nil treatment. Error bar denotes LSD at 5 % level (LSD = 0.30). Red line indicates MDW of the Nil treatment (3.04 g pot-1_).

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Table 2.3.5 Soluble phosphate detected and organic acids released by the ten EPS strains after 3 and 7 days incubated in HSU HydroxP medium Organic acid concentration (mM)₤

3 Days

P Conc

EN101 8.28 ± 1.33 24.61 ± 1.08 2.55 ± 1.21 nd nd nd nd EE131 16.50 ± 0.57 29.51± 0.14 12.28 ± 0.36 nd nd nd nd EE132 17.04 ± 0.50 nd 51.03 ± 1.83 9.83 ± 1.92 2.66 ± 0.28 nd nd EE127 9.00 ± 0.38 12.46 ± 1.69 4.64 ± 0.65 nd nd nd nd Ha200 18.75 ± 0.52 25.04 ± 1.05 25.26 ± 2.06 nd 6.33 ± 1.42 nd nd Ha185 19.01 ± 0.80 47.36 ± 0.54 nd nd nd nd 0.78 ± 0.11 Wh15 12.05 ± 1.13 25.12 ± 1.36 6.28 ± 3.60 nd nd nd nd Wi28 10.09 ± 0.67 nd 0.41 ± 0.04 nd 4.53 ± 0.45 nd 2.78 ± 0.51 Ba28 4.90 ± 0.19 nd nd nd 0.40 ± 0.15 nd nd Ba40 7.84 ± 0.22 0.60 ± 0.30 1.17 ± 0.08 nd 0.96 ± 0.18 1.16 ± 0.06 nd 7 Days EN101 0.99 ± 0.80* 0.99 3.07 15.12 0.59 18.77 16.64 2.32 9.79 6.79 8.89 5.92 ± 1.42* _{2.47 ± 1.45 nd nd nd nd} EE131 3.07 ± 0.90* _{6.55 ± 0.79}* _{10.34 ± 2.41 nd 1.15 ± 0.74 nd nd} EE132 15.12 ± 2.27 nd 47.69 ± 13.12 nd* _{nd nd nd} EE127 0.59 ± 0.38* _nd* _{nd nd nd nd nd} Ha200 18.77 ± 0.91 23.63 ± 2.05 23.71 ± 1.13 nd 5.15 ± 2.58 nd nd Ha185 16.64 ±0.85* _{36.40 ± 1.88}* _{nd nd nd nd nd}* Wh15 2.32 ± 0.91* _{4.61 ± 1.83}* _{nd nd 4.69 ± 2.86 nd nd} Wi28 9.79 ± 0.41 nd 0.29 ± 0.02 nd 4.11 ± 0.55 nd 1.21 ± 0.37 Ba28 6.79 ± 0.22 nd nd nd nd nd nd Ba40 8.89 ± 0.17 0.83 ± 0.05 nd nd 1.11 ± 0.42 nd* _nd

ⱡ Soluble P released from HSU HydroxP medium determined by Murphy and Riley’s colourimetric assay.

₤ Value represents mean ± standard error of mean (n=3); nd, not detected. § Unknown organic acid presented in arbitrary units (AU)

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Table 2.3.6 Combined data - PGP traits and pot-trial results for the EPS strains

Glasshouse pot-trial

EPS P releasedⱡ _IAA€ _Hyp-S# _ACC₵ _Na-Phy₸ _SiderophoreΩ _MDW¥ % Weight _{increased Mean P}§ % P _{increased Mean Ca}§ % Ca _increased

Unit (mM) (µg IAA/mL) g/pot % mg/Pot % mg/pot %

Nil 3.04abc _{0 10.43 0 30.64 0} EN101 8.28 ± 1.33 0.69 ± 0.19 - + ++ +++ 2.74a _{-9.86 9.69 -7.05 33.32 8.74} Ha200 18.75 ± 0.52 3.51 ± 0.69 - - ++ +++ 2.98ab _{-2.04 10.37 -0.54 30.78 0.43} EE127 9.00 ± 0.38 0.83 ± 0.07 - + +++ +++ 3.10bcd _{1.98 10.38 -0.40 29.83 -2.67} EE132 17.04 ± 0.50 3.72 ± 0.70 - + +++ ++ 3.16bcde _{4.08 11.27 8.11 33.25 8.49} Ba40 7.84 ± 0.22 8.20 ± 2.23 + + ++ ++ 3.26bcde _{7.21 11.12 6.63 28.10 -8.31} Ha185 19.01 ± 0.80 1.23 ± 0.19 - + ++ + 3.34cde _{9.85 10.84 3.97 32.80 7.03} Ba28 4.90 ± 00.19 5.21 ± 1.11 + + ++ ++ 3.37de _{10.87 11.51 10.36 34.23 11.69} Wi28 10.09 ± 0.67 3.82 ± 0.67 - + ++ - 3.40de _{12.05 11.78 12.98 34.59 12.86} EE131 16.50 ± 0.57 2.35 ± 0.74 - + +++ +++ 3.42e _{12.55 11.69 12.16 32.63 6.49} Wh15 12.05 ± 1.13 3.58 ± 0.44 - - +++ +++ 3.44e _{13.20 11.84 13.52 29.02 5.30*}

ⱡ Soluble P released from HSU HydroxP medium at 3 days determined by Murphy and Riley’s colourimetric assay.

ⱡ, € Value represents mean ± standard error of mean (n = 3).

# Isolates able to solubilise HydroxP by utilising sucrose indicated as “+” or “-” which indicates no solubilisation.

₵ Isolates produce ACC deaminase indicated as “+” or “-” which does not produce the enzyme.

₸Halo size ≥ 8 mm indicated as +++ and size between 5 – 7 mm, ++.

Ω No colour changes indicated as -; orange halo size < 20 mm, +; 20 – 30 mm, ++; > 30 mm. +++. - The symbol “–“ in the % Weight increased, P and Ca increased column indicates a % decrease.

¥ Rows sorted by ascending mean dry weight for isolate treatment from the glasshouse pot-trial (n = 14). § Total P and calcium (Ca) analysed by ICP-MS.

Grouping information using Tukey’s Method and groups which do not have a letter in common differ statistically significantly at the 5% level (LSD = 0.31). Nil represents no inoculant for the pot-trial.

Through generalised linear modelling, the correlation between P increases in ryegrass shoots and shoot biomass is depicted in Figure 2.3.5. In the pot trial, inoculation of ryegrass with isolate EE132 resulted in an 8.11 % of increase in P in the plant biomass, while making little contribution to plant growth (4.08 % increase). In contrast, inoculation with isolate Ha185 resulted in 9.85 % increase in MDW, but the mean P per pot (plant biomass) increased by only 3.97 %; a number outside the 95% confidence interval of expected outcomes derived across the rest of the dataset (Figure 2.3.5). In a strictly P limited system there would be a strong expectation for increased P to relate to plant growth increase and this was observed across the data. Isolate Ha185 was able to increase P uptake for ryegrass. Isolates Ha200 and EN101 did not significantly increase MDW, despite the fact that these isolates were shown to release P from HydroxP, phytate mineralisation and siderophore (Table 2.3.6). Both Ba28 and Wi28 increased shoot calcium by 11.69 and 12.86 % respectively, indicating that additional calcium was released from HydroxP (Ca5(OH)(PO4)3) by these isolates and was taken

up by ryegrass. However, there was no correlation between calcium uptake and ryegrass growth (Table 2.3.6). Four isolates Wh15, EE131, Wi28, and Ba28 (circled in green, Figure 2.3.5), increased ryegrass growth significantly in comparison with the Nil treatment control (Table 2.3.6) and the % increase in plant growth was positively correlated with the % increase in mean P per pot within 95 % confidence (Figure 2.3.5). These isolates were considered as PSB-PGPB, highlighted in green circle in Figure 2.3.5.

Figure 2.3.5 Glasshouse pot trials of ten EPS isolates showed a significant correlation between dry shoot weight and total phosphorus. Each full circle (●) denotes different bacterial

treatment and the 95% confidence interval is indicated by black dotted lines (●●●●●●)(R2 = 0.8742).

Isolates within green shade increased ryegrass growth and isolates within green circle are PSB- PGPB which MDW of ryegrass increased significantly compared to Nil treatment control.

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Through 16S rRNA analysis the PSB-PGPB isolate Ba28 was identified to be Serraria grimesii with 99 % 16S rRNA similarity (Table 2.3.1). Based on this the isolate was designated Serratia grimesii Ba28. Although this isolate is the least effective HydroxP solubiliser out of the 10 EPS strains using glucose as substrate, it gave a 10.36 % of the total P in ryegrass shoot and an 11% increase in dry weight (Figure 2.3.5). The best performing PSB-PGPB isolate among all EPS strains was found to be Wh15, an isolate that shared 95 % similarity to the 16S rRNA of Pseudomonas costantinii (Table 2.3.1), hereafter named Pseudomonas sp. Wh15. This isolate was shown to increase ryegrass growth by 13.2 % compared to the Nil treatment. It was found that Pseudomonas sp. Wh15 was capable of releasing high amount of P from the HSU HydroxP medium, produced IAA, siderophores, and could also mineralise phytate (Table 2.4.1). However, this isolate was unable to release ACC deaminase. This indicated that plant growth promotion was not due to decreased concentration of ACC and ethylene of ryegrass roots alone.