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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)