4th Asian PGPR Conference Hanoi, Vietnam

Metabolic and gene expression profile underlying the concurrence of P-solubilizing and biocontrol traits in Pseudomonas aeruginosa P4 in response to P-limitation

Presented by- Aditi Buch, PhD P D Patel Institute of Applied Sciences Charotar University of Science & Technology Changa (CHARUSAT), Changa, Dist, Anand Gujarat, India

Multiple plant-protection and plant growth promotion mechanisms prevalent in soil

Potential modes of action of plant growth promoting microorganisms (PGPM) and biological control agents (BCA) with primary and secondary beneficial effects on plants. Solid lines, primary effect; dash lines, secondary effect. (Adopted from: Avis et al., Soil Biology & Biochemistry 40 (2008) 1733–1740)

Direct Mechanism: Phytohormones; improvement nutrient status

Indirect Mechanism: Reducing the incidences of plant diseases

Catabolic versatility confers great ecological advantage to fluorescent pseduomonads as PGPR

1 • To colonize new habitats including those toxic for most microbes

(J Sorensen, LE Jensen ,2001).

2

• To acquire and develop the specific mechanism for resistance to harmful compounds and adaptations against metal stresses (Schleissner et al., 1997; Hamel et al, 1999).

3

• To promote plant growth (Haas and Defago 2005).

4 • Control plant pathogens by secretion of several antibiotics and

antifungal molecules (Preston, 2004, Haas and Defago 2005).

Adopted from Couillerot et al., Letters in Applied Microbiology 48 (2009) 505–512 and Haas & Defago; Nature Reviews, (2005), 1-13

Plant growth promoting and biocontrol metabolites produced by fluorescent pseudomonads

Fluorescent pseudomonads have the ability to produce metabolites involved in both direct and indirect mechanism of plant growth promotion

Importance of multifaceted rhizosphere microorganisms on plant health and productivity

Plant growth promoting microorganisms (PGPM) and biological control agents (BCA) are shown to possess secondary beneficial effects that would increase their usefulness as bio-inoculants, regardless of the need for their primary function (Avis et al., Soil Biology & Biochemistry 40 (2008) 1733–1740)

This could allow the release of their full potential as multifaceted beneficial bio-inoculants for improved growth and health of plants.

In this regard, fluorescent pseudomonads with an efficient P-solubilizing ability co-existing with a strong biocontrol ability could offer an attractive option in developing multifaceted bio-inoculants.

Regulatory effects on production of plant growth promoting and biocontrol metabolites

Glucose from plant root exudates

Gluconic acid

PQQ-GDH

Solubilizes mineral P

Lowers pH

Can itself act as antifungal agent

Insoluble P

Rhizosphere Secondary metabolites 2,4 DAPG

Fe2+ starvation

Pyoluteorin

Pyrrolnitrin

Salicylic acid

pyochelin

pyoverdin

+

-

+

+

-

Regulated by glucose

-

pyocyanin HCN

+

-

In P. fluorescens CHA0, gluconic acid production represses the production of its most potent antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin (de Werra et al., Appl. And Environ. Microbiol. (2009), p. 4162–4174)

P. aeruginosa P4 – A model organism

P-solubilizing ability of P. aeruginosa P4

P. aeruginosa P4 is known to be one of the highly efficient P-solubilizing pseudomonads (Buch.et al.,2008).

a: Growth of P. aeruginosa P4 on pseudomonas agar

b,c: Zone of solubilization on Pikovaskya’s agar

d,e: Zone of acidification on buffered-RP medium

P. aeruginosa P4P. aeruginosa P4

P. fluorescens 13525P. fluorescens 13525

Siderophore production (a) CAS agar plate (Dave et al., 2006) (b) Characteristic fluorescence in broth

Standard succinate medium (after 48 hours)

(A) King B broth (after 24 hours)

Other direct mechanisms of plant growth promotion of P. aeruginosa P4

Standard SSM an d King’s B broth (Meyer and Abdallah, 1978

IAA production Culture supernatants of LB grown P. aeruginsa P4 was used to measure IAA production n presesnce and absence of 500µg/ml tryptophan

Antifungal activity of P. aeruginosa P4 against Fusarium oxysporum.

P. aeruginosa P4 was co-inoculated with F.

oxysporum on (A) King A agar (B) Nutrient agar with glycine, and the zone of clearance was monitored after 24 hours of incubation at 30 °C.

(A) (B)

Biocontrol activity of P. aeruginosa P4

Growth and HCN production by P. aeruginosaP4 on nutrient agar with 4.4 g/L glycine after 24 hours at 30°C.

A B C

Growth HCN production after 24h Uninoculated control

Pyocyanin production on King’s A brotth (48h)

Seed treatment Radicle length (cm)

Fresh Weight (gm)

Control (n=20) 2.39 ± 1.13 1.08 ± 0.19

Bacterized (n=20) 3.36 ± 1.96 1.28 ± 0.24

p-value 0.028 0.001

Fold change 1.4↑ 1.2 ↑

Root colonization & plant growth promotion by P. aeruginosa P4

Seed treatment Seedling Vigour Index I [(RL+SL)*% Germination]

Bacterial root colonization

Control (n=29) 1198.61 ---

Bacterized (n=29) 1569.66 1x106 CFU/ per cm of root tip

Fold change 1.3↑ ---

Bacterized

Control

Control Bacterized Control Bacterized

ns

n=19

After 10 days of growth

**

***

ns

n=29

Control Bacterized Control Bacterized

After 10 days of growth

n=20

Concurrence of P-solubilizaton and biocontrol phenotypes in P. aeruginosa P4

P-deficient P-sufficient

Growth and pH profile of P. aeruginosa P4 under P-sufficient and P-deficient conditions

Tris-Cl (pH=8.0) buffered minimal medium 100mM Glucose 10mM KH2PO4 : P-sufficient conditions 0.01 mM KH2PO4 :P-deficient conditions.

Media acidification to pH<5 was attributed to high organic acid secretion by P. aeruginosa P4

(c)

Production of plant growth promoting metabolites by P. aeruginosa P4 under P-deficient conditions

0 day 4-7 days

P-sufficient P-deficient

Pyocyanin production increased by about 7fold under P-deficiency. These results are in accordance with the earlier reports that pyocyanin production in P. aeruginosa was stimulated by low P concentrations (Cox, 1986; Turner and Messenger 1986; Tjeerd van Rij et al., 2012)

(c)

Production of plant growth promoting metabolites by P. aeruginosa P4 under P-deficient conditions

‡

***

n=4-7

P-sufficient and -deficient medium with glucose as carbon source with and without tryptophan supplementation.

IAA production Total siderophore production

Pyoverdine production under P-deficiency

***

0100200300400500600700800

400 500 600 700

wavelength (nm)

RFU

/0.1

ml

P-deficient supernatant

P-sufficient supernatant

0

1000

2000

3000

4000

5000

6000

7000

8000

1P-sufficient P-deficient P-sufficient P-deficient

RFU

/OD

* ***

pH=~7

pH<5

Pyoverdin was measured by monitoring characteristic fluorescence emission at 460 nm when excited at 400 nm (Cox and Adams, 1985)

These results are in accordance with earlier reports that high phosphate is important for pyoverdin production (Fallahzadeh et al., 2010)

Ps Pd Ps Pd KB

pH=4 pH=7

Control

Culture supernatants of P. aeruginosa P4 grown under P-sufficient (24h) and –deficient (72h) conditions were acidified and extracted using ethyl acetate and subjected to TLC (CHCl3:acetic acid:water;50:5:2.5).

Rf=0.6

Pch

Pch

Pch

P-deficient

King’s B standard

P-sufficient

0

200000

400000

600000

800000

1000000

1200000

1P-sufficient P-deficient

AU

Pyochelin production under P-deficiency

Mechanisms underlying the concurrence of P-solubilizaton and

biocontrol phenotypes

Pentose phosphate pathway

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1P-sufficient P-deficient

Glu

coni

c ac

id p

rodu

ctio

n (u

g/m

l/OD)

Gluconic acid production increased b ~4 folds

GDH activity increases G-6-PDH activity decreases PYC activity decreases

Metabolic profile under P-deficiency (Buch et al., 2008, Res. Microbiol. 159)

*

Pathways of secondary metabolism producing precursors for synthesis of IAA, pyochelin, pyocyanin in Pseudomonas aeruginosa (Gaille et al., 2003).

Gene

Size of the amplicon

Primer Sequence Encoded Protein

rpoD

200 bp FP: GGGCGAAGAAGGAAATGGTC RP: CAGGTGGCGTAGGTGGAGAA

RNA Polymerase σ factor (house keeping gene control)

proC 200 bp FP:CAGGCCGGGCAGTTGCTGTC RP: GGTCAGGCGCGAGGCTGTCT

Pyroline-5-Carboxylate reductase (positive control)

AroC 300 bp FP: GACCCGGACAAGGTGCCGGAG RP:GGAGATCCCGCCGAGGATGC

Chorismate synthase

PchA 321 bp FP: CTGCCTGTACTGGGAACAGC RP: CAGGGCCAGGGCATCTTCG

Isochorismate synthase

PchE 288 bp FP: GGAGATGGCCCTCAGCCTG RP: GACCACCAGGTCCGGCAC

Pyochelin synthatase

PchG 313 bp FP: CAGGACCTGCTGCGAGC RP: GCCGCCAGTTCGTTGAGC

Pyochelin biosynthetic protein

PvdS 176bp FP: 5’ ACCGTACGATCCTGGTGAAG 3’ RP: 5’ CGACGCGCGACAGCTTGC 3’

Alternate sigma factor, Regulatory gene for pvd synthesis

PvdQ 310bp FP: 5’ ATGCTCCTGGCCAACCCG 3’ RP: 5’ GTAGTGATCGATGGCCAGGT 3’

For conversion of PvdIq to PvdIp

PvdH 381 bp FP: 5’ TTCATCGATTGCCTGGCCGG 3’ RP: 5’ CCCTGGCTCATGCCGTGG 3’

Diamino butyrate 2-oxo-glutarate amino transferase

Semi-quantitative expression analysis of genes involved in biosynthesis of biocontrol metabolites

Exact IAA biosynthesis pathway in P. aerugnosa P4 is unknown hence gene expression profile remains unclear

Relative gene expression of at of selected genes involved in bosythesis of biocontrol metabolites

Gene proC aroC pvdS pvdQ pchA pchE pchG

Fold change in expression

8.3 1.4

7.51 1.04 1.1 0.47 2.23 Pc

h E

rpoD was used as internal control

pchE

Summary

P-solubilzing phenotype in P. aeruginosa P4 co-exists with other important plant growth promoting and biocontrol abilities like production of siderophores, IAA and antifungal metabolites. Under P-deficiency, metabolic profile shifts predominantly to the direct oxidative pathway producing gluconic acid, while reducing the key enzyme activities of phosphorylative pathway. Simultaneously P. aeruginosa P4 demonstrates increased production of metabolites like IAA, pyoverdine, pyochelin and pyocyanin involved in biocontrol. P. aeruginosa P4 offers an interesting model to understand the metabolic and regulatory mechanisms that allow concurrence of P-solubilizing and biocontrol traits, an ability which could be more relevant for identifying the potential PGPR.

Acknowledgements

Vaishnawi Gupta Disha Patel

Prof G Naresh Kumar Department of Biochemistry, M S Universtiy of Baroda, Vadodara, Gujarat

Charotar University of Science and Technology (CHARUSAT)

P D Patel Institute of Applied Sciences

Thank You!