Plant Growth Promoting Rhizobacteria: Biofertilization and Biocontrol Agents Aris Tri Wahyudi, PhD (http://aristri-wahyudi.weebly.com) BOGOR AGRICULTURAL UNIVERSITY 2010 BOGOR AGRICULTURAL UNIVERSITY 2010 Plant Growth Promoting Rhizobacteria: Biofertilization and Biocontrol Agents
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Plant Growth Promoting Rhizobacteria: Biofertilization and
Biocontrol Agents
Aris Tri Wahyudi, PhD(http://aristri-wahyudi.weebly.com)
BOGOR AGRICULTURAL UNIVERSITY2010
BOGOR AGRICULTURAL UNIVERSITY2010
Plant Growth Promoting Rhizobacteria: Biofertilization and
Biocontrol Agents
Rhizosphere and Rhizobacteria
The thin layer of soil (~1-5 mm thick) surrounding crop root and the volume of soil occupied by roots known as the rhizosphere
One group of microorganism which are beneficial to crop is bacteria that colonize roots or rhizosphere soil of crop plants: Rhizobacteria
(ISR)
Plant Growth Promoting Rhizobacteria : PGPR
Root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant growth and development via direct or indirect mechanisms.
PGPR play a vital role in growth promotion, crop protection, and improvement of soil health.
Prospect : Bioferlilizer and Biocontrol Agents.
Antimicrobial Compounds/ antibiotics Production
Production of
Siderophore
Non-Symbiotic
N2 Fixation
Plant Growth-Promoting
Rhizobacteria
Phosphate Solubilization
HCN
Production of ACC Deaminase
Phytohormone IAA(Indole Acetic Acid)
(PGPR)
Production of
Chitinase/ Glucanase
Induce Systemic Resistance (ISR)
Direct Effect
Production of phytohormone Indole Acetic Acid (IAA)Phosphate solubilizationProduction of ACC DeaminaseNon-symbiotic N2-fixation (Azospirillum sp, Azotobacter sp, etc)
Mechanisms of PGPR‐Mediated Enhancement of Plant Growth
Production of antimicrobial compounds/ antibiotics: antifungi/antibacteria/antivirusProduction of iron chelating agents: siderophoreProduction of glucanase and chitinaseProduction of HCNInduction of systemic resistance of plant
Indirect Effect
Mechanisms of PGPR-Mediated Enhancement of Plant Growth
Plant Growth Promoting Rhizobacteria (PGPR)
“Many Rhizobacterial Spesies are able to promote plant growth and biocontrol of phytopatogen”
Fig . Performance of Bacillus sp on Nutrient Agar Plate (A) andPseudomonas sp on King’S B agar plate incubated for 48 h (B),Gram Positive cells of Bacillus sp (C), and Gram Negative cells of Pseudomonas sp (D).
B
C D
Rhizobacteria fromthe Rhizosphere
PGPR from Soybean Rhizosphere: Non-Pathogen
Isolate Number IAA Producer*
Pseudomonas sp
Bacillus sp
115 81 (~70%)
113 91 (~80%)
* Non-Pathogen
Nicotiana tabacum2-3 months
Hypersensitivity Test
24 h after inoculation (Negative Reaction)
1 week after inoculation (Negative Reaction)
24 after inoculation (Positive Reaction)
1 week after inoculation (Positive Reaction)
H2O
H2O
Biosynthesis of IAABiosynthesis of IAA
IAM IPA TAM
IPA dekarboksilase (ipdC)
Triptofan (Trp)
Indole-3-pyruvic acid (IPA)
Indole-3-asetaldehid (IAId)
Indole-3-acetic acid (IAA)
Trp transaminase
IAId dehidrogenase
Indole-3-acetamid (IAM)
Triptamin (TAM)
Trp monooksigenase
IAM hidrolase
Trp dekarboksilase
Amin oksidase
(Bartei 1997)
IAA Production : Pseudomonas sp
Kurva produksi IAA isolat BL27
-10
0
10
20
30
40
50
60
70
0 4 8 12 16 20 24 28 32 36 40 44 48 60
waktu inkubasi (jam)
kons
entra
si ia
a (p
pm)
Con
cent
ratio
n of
IAA
(ppm
)
Time (hour)
CRB17
IAA Production : Bacillus sp
CR64
IAA
Production of IAA
Pseudomonas spIAA (ppm)
Bacillus spIAA (ppm)
CRB100 0.33
Isolate Isolate
CR32 0.30
CRB4 9.46
CRB17 16.02
CR36 13.03
CR4 80.82
1
81
1
91
Growth Promotion Assay
Dilution with 1 ml NB (~1010 cells/ml)
Dey et al. (2004)
Soybean Seed(Surface
Sterilization)
Water agar (1 %) medium
Incubation: 5 daysParameters Observed: root length of primary root, number of lateral root, and shoot length.
Statistical Analysis
3 replicates
Bacterial suspension
Control A
Control B
Performance of soybean seedling inoculated by Pseudomonas sp CRB95 (A) significantly promote plant growth
Performance of soybean seedling inoculated by Bacillus sp CR71 (B) significantly promote plant growth
Mechanisms of Plant Growth Promotion
Auxin may stimulate certain proteins in plant’s cell membrane to pump hydrogen ion into the cell wall, lowering the pH within the wall. The low pH activates enzymes that separate cross-linking molecules from larger cellulose molecules in the wall.The cell then swells with water and elongates because its weakened wall no longer resists the cell’s tendency to take up water osmotically.After this initial elongation caused by the uptake of water, the cell sustains the growth by synthesizing more cell wall material and cytoplasm.
How does Auxin make plant cell elongate?
Growth Promotion
Pseudomonas sp : 14 Isolates
IsolateGrowth Promotion
Length of Primary Root Length of Stem No Lateral Root
Identification of 14 16S rRNA Genes: Pseudomonas sp. (BlastN)
P seudom onas sp. Crb 3
P seudom onas sp. Crb 17
P seudom onas sp. Crb 82
P seudom onas sp. Crb 94
P seudom onas s p. Crb 74
P seudom onas fluorescens CHA 0
P seudom onas fluorescens P f-5
P seudom onas putida T7-5
P seudom onas s tutz eri
P seudom onas sp. Crb 16
P seudom onas sp. Crb 44
P seudom onas sp. Crb 60
P seudom onas sp. Crb 8
P seudom onas sp. Crb 1
P seudom onas sp. Crb 15
P seudom onas sp. Crb 93
P seudom onas s p. Crb 84
P seudom onas s p. Crb 95100100
74100
44
64
99
77
29
63
38
11
45
9
55
0.02
Dendrogram Phylogenetic of PGPR Pseudomonas sp.
ReferenceStrains
16S rRNA Gene : Bacillus sp.
~1300 bp
10.000
5.000
1.500
1.000
500
250
(bp)M 24 28 31 33 44 64 66 67 68 69 71
Identification of 11 16S rRNA Genes: Bacillus sp. (BlastN)
Isolate Homology Similarity (%)
Cr 24 Bacillus sp. 1Re28 85
Cr 28 Bacillus pumilus strain S2 91
Cr 31 Bacillus pumilus strain S6-05 98
Cr 33 Bacillus sp. NS-2 95
Cr 44 Bacillus subtilis strain CICC10166 92
Cr 64 Bacillus sphaericus isolae NUC-5 85
Cr 66 Bacillus cereus strain SS-07 94
Cr 67 Bacillus pumilus strain:M1-9-1 94
Cr 68 Bacillus cereus strain KU206-3 94
Cr 69 Bacillus cereus isolate AD2 97
Cr 71 Bacillus shandongensis strain SD 99
Dendrogram Phylogenetic PGPR Bacillus sp.
Figure . Amplification of antibiotics gene from Pseudomonas sp. 1. DAPG from CRB16; 2-6 Phenazin from CRB102; and 7-8 Pyoluteorin from CRB3. M: marker 1 kb DNA Ladder. Gel Agarose 1%
M 1 2 3 4 5 6 7 8 10
1.00.750.50
1.5
3.0
6.0
(Kb)
DAPG(CRB 16)
~700 bp
Phenazin(CRB 102)
Pyoluteorin(CRB 3)
~745 bp
~1600 bp
Pseudomonas sp.
Antibiotics Genes : DAPGOrganized in OPERON
Fig. Biosynthesis genes of DAPG and factors influencing its expression
M 7 8 9 10 M 11 12 13 14
M 1 2 3 4 5 6
10
0.5
1.0
2.0
(Kb)
Bacillomicin(CR 64) ~1100 bp
Zwittermicin A(CR 64) ~900 bp
Iturin (CR 64)~ 2000 bp
Bacillomicin(CR 66) ~ 900 bp
Zwittermicin A(CR 66) ~ 900 bp
Figure . Amplification of antibiotics gene from Bacllus sp. 1-3. Bacillomicin from CR64; 4-6. Zwittermicin from CR64; 7-8. Iturin from CR64; 9-10. Bacillomicin from CR66; 11-14. Zwittermicin from CR66. M: marker 1 kb DNA Ladder. Gel Agarose 1%.
SequenceAlignment
Bacillus sp.
TA Cloning
Sequencing
BioinformaticAnalysis
Antibiotic Gene : Zwittermycin AOrganized in OPERON
Fig. Biosynthetic genes cluster of Zwittermycin A and its function
Figure . Sequence alignment between Zwittermicin A from Bacillus sp CR64 withZwitter A from Bacillus cereus (97 % Identity) (Milner et al. 1996;J. Bacteriol).
ZmaR ORF 1127 bp
ATG TGA951 bp
Fig. The strategy for inverse PCR
EcoRVEcoRV
primer1
primer2
DNA amplifiedDNA amplified
IS (O)
EcoRV digest
DNA genomeof mutant
Ligation(Circularization)
IPCRAmplification
primer2
primer1EcoRV
EcoRV
Primer 1 :
5'-XXXXXXXXXXXXXXXXXXXXX-3'
Primer 2 :
5'-XXXXXXXXXXXXXXXXXXXXX-3'
ZmaR
Inverse PCR Product
Gene Cloning
Expression and StudyOf Its Function
No Primer Primer 5’-3’ Sekuens DNA Jenis Antibiotik1 Phl2a 5’-GAG GAC GTC GAA GAC CAC CA-3’ DAPG