In-vitro Mobilizierung von Kalzium-, Eisen-

und Aluminium- Phosphat durch

Rhizosphärenbakterien der Afrikanischen

Ölpalme Wolfgang Merbach1, Henri Fankem1,2, und Annette Deubel3 1) Martin-Luther-University Halle-Wittenberg, Germany 2) University of Douala, Cameroon 3 ) University of Applied Science Anhalt (Bernburg)

Martin-Luther-University Halle-Wittenberg. Germany Institute of Agricultural and Nutritional Science

adult juvenil

African oil palm Elaeis guineensis - one of the most important oil crops in the world

Aim of investigation:

Looking for rhizosphere microbes,which are able to

mobilize Ca, Al and Fe from soil for oil palm nutrition

Basis for bio-fertilizer?

Question 1

Which properties are included in potentially biofertilizer strains?

Ability to mobilize mineral phosphate

Affinity to plant

Hormonal influence on root morphology and P uptake

Not dangerous for human health

Competition with

native microflora

Antiphytopathological potential

Question 2

Why is ability for phosphate mobilisation important for oil palm ?

Problem • phosphate supply is one of the most important yield-

limiting factors in many tropical and subtropical soils

• high reactivity with Ca oder Fe / Al

• mineral fertilizers hardly available

• mycorrhiza needs 1-4 months to be effective, expensive inoculates

Therefore Examination of the occurrence of phosphate-

mobilising bacteria in the rhizosphere of oil palm

How was determined the phosphate mobilization of mineral phosphate by bacteria?

Question 3

Step 1

•Isolation of bacterial strains from rhizosphere soil (187) and root fragments (90); repeated inoculation for purity and stability

• Fig. Distribution of the investigated areas in humid-forest zones of Cameroon. These areas are located in two main agro-ecological zones of Cameroon: a) the humid forest zone with one rainy season and that includes provinces of Littoral and South west

• b) the humid forest zone with two rainy seasons that consists of Centre, South and East provinces. These areas represent the main oil palm plantations.

Step 2 •Qualitative test on ability to solubilize Ca3(PO4)2, FePO4 und AlPO4 using agarplates with dye(bromo cresol green); evaluation of halo (lysing) zones

1-EMJ5-Fe-P 2-EDJ8-Fe-P

3-EDA2-Al-P 4-DA9-Al-P

5-EA4-Ca-P 6-EMR5-Ca-P

Figure: (1-6): Halo zones surrounding colonies on five days agar plates culture containing FePO4·2H2O (1 - 2), AlPO4·H2O (3 - 4) and Ca3(PO4)2 (5 - 6). Criterion for pre-selection: diameter of colonies and lysing zones

Step 3

•Quantitative determination of P release fom Ca3(PO4)2, FePO4 or AlPO4 in liquid culture. Soluble P was determined by colometry

20 selected strains are proved (for comparison also strains without halo zones)

Ca3(PO4)2

0

50100

150200

250300

Con

trol

BOR

8

LEJ7

(-)

LEJ1

4 (-)

DA9

DR

5

DR

9 (-)

EDJ4

EDJ6

(-)

EDJ8

EDJ1

6

EDA5

(-)

EDA7

(-)

EA4

ER5

SJ9

(-)

SR7

EMJ5

(-)

EMR

3

EMR

5

LR7

P (m

g l-1

)

Third day Fifth day Seventh day

3.5

4.0

4.55.0

5.5

6.0

6.5

pH

• P releasing was correlated –as a rule –with ph decrease, but there were differences between both properties (see arrows)

FePO4 . 2 H2O

0

20

40

60

80

100C

ontro

l

BO

R8

LEJ7

LEJ1

4

DA

9

DR

5

DR

9

ED

J4

ED

J6

ED

J8

ED

J16

ED

A5

ED

A7

EA

4

ER

5

SJ9

SR

7

EM

J5

EM

R3

EM

R5

LR7

P (m

g l-1

)

Third day Fifth day Seventh day

3.54.0

4.55.0

5.56.0

6.57.0

pH

•Only some strains mobilized P, despite strong pH decreases

AlPO4 . H2O

0

20

40

60

80

100

Con

trol

BO

R8

LEJ7

LEJ1

4 (-)

DA

9

DR

5

DR

9 (-)

ED

J4

ED

J6

ED

J8 (-

)

ED

J16

ED

A5

(-)

ED

A7

(-)

EA

4

ER

5 (-)

SJ9

(-)

SR

7

EM

J5 (-

)

EM

R3

EM

R5

(-)

LR7

P (m

g l-1

)

Third day Fifth day Seventh day

3.54.04.55.05.56.06.57.07.5

pH

• All strains released P, but no correlation with pH decrease

Question 4

How does the pH affect the solubility of these phosphates?

To prove this question, the 3 phosphates were extracted with HCL and NaOH of different concentrations

0

50

100

150

200

250

300

2.5 3.5 4.5 5.5 6.5 7.5 pH

µg P

ml-1

Ca3(PO4)2FePO4AlPO4

•CaP solubility increases exponentially with decreasing pH (can explain P release in vitro partly)

•AlP only solubilised at pH levels lower than 3.5 (impossible in bacterial cultures and not useful in plant rhizosphere; could explain P release with some fungal strains)

•Acidification no explanation for FeP solubilisation :not soluble at all pH

Question 5

Are carboxylic acids (partly) responsible for P solubilisation? Which carboxylic acids solubilise P under natural soil-pH conditions and in vitro?

0

50

100

150

200

250

Doublelactate

H2O succinate gluconate citrate oxalate

mg

P / k

g so

il

pH 3.7 pH 7

Soil conditions

(90 min extraction of 5 g soil with 250 ml solution containing 5 g l-1 of different substances, average from 10 different soils) Carboxylic acids were able to more P than double lactate, esp. at pH 3.7

Next step therefore: In vitro examination of different carboxylic acids for solubilization of Ca-, Fe- and Al phosphates

Ca3(PO4)2 pH 7

0.00.20.40.60.81.01.2

0 2 4 6 8 10mM

mM

P

citratemalatelactatetatrategluconatesuccinatetrans-aconitatefumarate

At pH 7: Only citrate increased CaP solubility remarkably

Ca3(PO4)2 pH 4

0

1

2

3

4

5

0 2 4 6 8 10mM

mM

P

citratemalatelactatetatrategluconatesuccinatetrans-aconitatefumarate

At pH 4 (like in bacterial cultures) some acids increase CaP releasing and complete pH effect. But: sparingly soluble CaP occurs in neutral or alkaline soils

FePO4 pH 4

0.0

0.20.4

0.6

0.81.0

1.2

0 2 4 6 8 10mM

mM

P

citratemalatelactatetatrategluconatesuccinatetrans-aconitatefumarate

Iron phosphate was mobilised by citrate and malate, in a smaller extend by tartrate and gluconate

AlPO4 pH 4

0.00.2

0.40.60.8

1.01.2

0 2 4 6 8 10mM

mM

P

citratemalatelactatetatrategluconatesuccinatetrans-aconitatefumarate

AlP by citrate, malate, tartrate > gluconate.

Acidification no explaination for P solubisation

Which carboxylic acids were produced by bacterial strains and influenced the P releasing?

This seems important in case of Al or Fe phosphates (no or low pH influence of pH value)

Question 6

DR 9

0.00.51.01.52.02.53.03.54.04.5

Ca3(PO4)2 FePO4 AlPO4

mM

EDJ 6

0.0

2.0

4.0

6.0

8.0

10.0

Ca3(PO4)2 FePO4 AlPO4

mM

(halo zone) (no halo zone)

mg P/l 320 45 80 7d

42 40 50

oxalate trans-aconitate oxalacetate citratetartrate gluconate xylonate malatesuccinate lactate fumarate

Strains produced lot of carboxylic acids and mobilized P. EDJ 6 = most efficient CaP mobilizer: High citrate production and missing halo zone (precipitation of Ca citrate in agar plate test? Limit for pre-selection at agarplates.Substitution by citrate test?). Citrate, tartrate, gluconate most effective.

Conclusions

• P-solubilizing bacteria occur in the rhizosphere of Elaeis guineensis; role of microbial P mobilization can be assumed

• Ca3(PO4)2 -solubilization in vitro by acidification and different carboxylic acids; at pH 7 (natural soil conditions) mainly by citrate

• FePO4 and AlPO4 solubilization by citrate, malate, tartrate, (gluconate)

• Citrate can prevent halo zones by precipitation of Ca-citrate!

• Screening for citrate production would improve the selection of potential P-mobilizing microorganisms

Dr.Henri Fankem, Cameroon

Thank you to all the colleagues

First of all:

0 500 1000 1500

Bokito (4)Bokito (15)

Yaoundé (3)Yaoundé (14)

Douala (4)Douala (11)

Edea (5)Edea (12)

Ebolowa (4)Ebolowa (12)

Sangmelima (5)Sangm. (12)

Ekona (5)Ekona (13)

Limbé (2)Limbé (29)

not available PNH4F/HCl-soluble P (Bray 1)

P concentration in soils (mg kg-1) pH

6.0 4.8 5.2 4.3 4.0 3.7 4.1 4.0 4.9 4.1 4.5 4.8 5.0 5.5 4.7 7.4

% Fe

9.4 10.6 8.9

11.2 4.6 4.5 3.1 2.6 7.0 2.1 0.8 1.0 5.2 0.9 1.2 1.4

% Al

6.1 5.2 5.3 5.5 5.3 5.4 4.5 4.1 3.5 3.1 1.0 1.4 4.5 1.1 1.3 1.8

0

50

100

150

200

250

Doublelactate

H2O succinate gluconate citrate oxalate

mg

P / k

g so

il

pH 3.7 pH 7

(90 min extraction of 5 g soil with 250 ml solution containing 5 g l-1 of different substances, average from 10 different soils)

Soil conditions

DR 5

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Ca3(PO4)2 FePO4 AlPO4

mM

oxalate trans-aconitate oxalacetate citratetartrate gluconate xylonate malatesuccinate lactate fumarate

LEJ 14

0.01.02.03.04.05.06.07.08.09.0

Ca3(PO4)2 FePO4 AlPO4

mM

oxalate trans-aconitate oxalacetate citratetartrate gluconate xylonate malatesuccinate lactate fumarate

EDJ 6

0.0

2.0

4.0

6.0

8.0

10.0

Ca3(PO4)2 FePO4 AlPO4

mM

SR 7

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Ca3(PO4)2 FePO4 AlPO4

mM

Strain EDJ 6 = most efficient CaP mobiliser: High citrate production and missing halo zone (precipitation of Ca citrate in agar plate test?)

mg P/l 320 45 80

(7 d)

(no halo zone) (halo zone)

oxalate trans-aconitate oxalacetate citratetartrate gluconate xylonate malatesuccinate lactate fumarate

EMJ 5

0.0

1.0

2.0

3.0

4.0

5.0

Ca3(PO4)2 FePO4 AlPO4

mM

LR 7

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Ca3(PO4)2 FePO4 AlPO4

mM

oxalate trans-aconitate oxalacetate citratetartrate gluconate xylonate malatesuccinate lactate fumarate

DR 9

0.00.51.01.52.02.53.03.54.04.5

Ca3(PO4)2 FePO4 AlPO4

mM

EDJ 4

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Ca3(PO4)2 FePO4 AlPO4

mM

(halo zone)

mg P/l 42 40 50

(7 d)

(halo zone)