Embed Size (px)
Citation preview
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”
AzotobacterAzospirillumPseudomonasBacillusBurkholderiaSerratiaAcetobacter
(Glick 1995)
Plant Growth Promoting Rhizobacteria (PGPR)
A
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
CRB-1 10.21CRB-3 2.32CRB-8 7.40CRB-15 13.99CRB-16 4.89CRB-17 16.02CRB-44 3.73CRB-60 8.04CRB-74 7.72CRB-82 6.63CRB-84 14.63CRB-93 7.60CRB-94 1.13CRB-95 7.24
(Significant at level 5%)
IAA (ppm)
Growth Promotion
Bacillus sp : 11 Isolates
CR-22 2.32CR-24 2.79CR-28 1.48CR-31 18.63CR-44 1.52CR-64 7.56CR-66 1.75CR-67 11.13CR-68 4.67CR-69 4.32CR-71 9.63
(Significant at level 5%)
Growth PromotionLength of Primary Root Length of Stem No Lateral Root
Isolate IAA (ppm)
Bacillus sp
Pseudomonas sp
CAS Agar Medium(Siderophore Prod)
Pikovkaya Agar Medium(Phosphate Sol)
Bacillus sp
Pseudomonas sp
Phosphate Solubilisation, Siderophore and Chitinase Production
Chitin Agar Medium(Chitinase Prod)
Bacillus sp
Pseudomonas sp
Phosphate Solubilisation, Siderophore and Chitinase Production
Characters Tested Bacillus sp. Pseudomonassp.
Phosphate Solubilisation
74 42
Siderophore Production
78 45
Chitinase Production 17 23
Pseudomonas sp dan Bacillus sp Producing Bioactive Compounds Against Phytopatho-genic Fungi
CR 55 CR 17CR 45
CRB 16 CRB 3CRB 44
1 2 3
4 5 6Fusarium oxysporum Sclerotium rolfsii Rhizoctonia solani
Pseudomonas sp
Bacillus sp
Bacterial Isolates Antagonist Against Phytopathogenic Fungi
Antagonist against Phytopathogenic
Fungi:
Bacillus sp. Pseudomonassp.
Sclerotium rolfsii 3 12
Fusarium oxysporum 18 14
Rhizoctonia solani 50 43
Molecular Genetics Analysis
B. Genes Encoding Antibiotics Biosynthesis
Genes for Antibiotic :DAPGPhenazinePyoluteorinPyrolnitrin
IturinBacillomycinSurfactinZwittermicin A
Specific Primer
P C R
DNA/PCR Products
DNA Sequence Analysis
A. Genetics Diversity
Gene 16S rRNA Phylogenetic Pseud, Bacill
Genetics Diversity !!!ARDRA of 16S rDNA(Primer 63F; 1387R)
Pseudomonas
Bacillus
(Marchesi et al. 1998)
Denaturation 920C; 30’’
Annealing550C; 30’’
Elongation 750C;1’
40C; ~
PCR of 16S rRNA Gene
PCR Reaction in 50 µl
GC buffer : 25 µl
dNTPs : 8 µl
LA Taq : 0.5 µl
Primer 1 dan 2 : 1 µl (10 pmol)
DNA genome : 5 µl
ddH2O : 10.5 µl
Post PCR 750C; 10’
Primer forward
Primer reverse
DNA template
16S rRNA Gene
Primer for 16S rRNA
63f (5’-CAG GCC TAA CAC ATG CAA GTC-3’)
1387r (5’- GGG CGG WGT GTA CAA GGC-3’)
(Marchesi et al. 1998)
16S rRNA Gene : Pseudomonas sp.
M 3 16 17 44 M 15 60 74 82 84 93 M 94 95
~1300 bp ~1300 bp
Isolate Homology Similarity (%)Crb1 Pseudomonas sp. 12M16 91
Crb3 Pseudomonas monteilii strain R23 95
Crb68 Pseudomonas sp. 12M16 91
Crb15 Pseudomonas sp. 12M16 94
Crb16 Pseudomonas sp. M41 94
Crb17 Pseudomonas plecoglossicida strain NyZ12 99
Crb44 Pseudomonas beteli 99
Crb60 Pseudomonas aeruginosa strain NBRAJG90 92
Crb74 Pseudomonas putida strain HRB-4 86
Crb82 Pseudomonas sp. RW10S1 96
Crb84 Pseudomonas sp. BFF-1 92
Crb93 Pseudomonas sp. isolate R-23174 86
Crb94 Pseudomonas mosselii isolate 164 95
Crb95 Pseudomonas sp. isolate R-20954 81
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
2 Phl2b 5’-ACC GCA GCA TCG TGT ATG AG-3’ DAPG
3 Phen1 5’-CCC CTG TTG ACA ATT AAT CAT CGG-3’ Phenazin
4 Phen2 5’-ACC TTG ACG TTG TAC CAT TCC CAA-3’ Phenazin
5 PHZX 5’-TTT TTT CAT ATG CCT GCT TCG CTT TC-3’ Phenazin
6 PHZY 5’-TTT GGA TCC TTA AGT TGG AAT GCC TCC-3’ Phenazin
7 PRND1 5’-GGG GCG GGC CGT GGT GAT GGA-3’ Pirolnitrin
8 PRND2 5’-YCC CGC SGC CTG YCT GGT CTG-3’ Pirolnitrin
9 prnCf 5’-CCA CAA GCC CGG CCA GGA GC-3’ Pirolnitrin
10 prnCr 5’-GAG AAG AGC GGG TCG ATG AAG CC-3’ Pirolnitrin
11 pltCreg1F 5’-AGG CAA TCA CTA CCA TCC GTG CGC-3’ Pyoluteorin
12 pltCreg2r 5’-ATG AGG AGC AGG AGG TGT CGA GCA C-3’ Pyoluteorin
No Primer Primer 5’-3’ Sekuens DNA Jenis Antibiotik
1 ytpA-F1 5'-ATG TGG ACC TGG AAA GCA G-3' Bacilomycin
2 ytpA-R2 5'-CGG TAC TGT CAA CAC ATG C-3' Bacilomycin
3 Swt 678 F 5’-ATG TGC ACT TGT ATG GGC AG-3’ Zwitermicin
4 Swt 667 R 5’-TAA AGC TCG TCC CTC TTC AG-3’ Zwitermicin
5 yerPF 5’-CGCAGATCTGGAGGATATGATGAACCACG-3’ Surfactin
6 yerPR 5’-GGCTCTAGATTACTCTTCTTCCGTTCCCG-3’ Surfactin
7 Srfkn-1 5’-AGCCGTCCTGTCTGACGACG) Surfactin
8 Srfkn-2 5’-TCTGCTGCCATACCGCATAGTC) Surfactin
9 ITUP1-F 5’-AGC TTA GGG AAC AAT TGT CAT CGG GGC TTC-3’ Iturin
10 ITUP2-R 5’-TCA GAT AGG CCG CCA TAT CGG AAT GAT TCG-3’ Iturin
Isolasi PseudomonasTanah rizosfer asal rizosfer kedelai
Media Egar King’s B
1 ml 1 ml 1 ml 1 ml 1 ml
9 ml 9 ml 9 ml 9 ml 9 ml101 102 103 104 105
Pseudomonas spPengamatan morfologi sel &koloniPengamatan fisiologi/biokimia
Isolasi Bacillus spTanah rizosfer asal rizosfer kedelai 1 gr
Agar Nutrient Broth
1 ml 1 ml 1 ml 1 ml 1 ml
9 ml 9 ml 9 ml 9 ml 9 ml101 102 103 104 105
Bacillus sp
80 oC10 Min
Pengamatan morfologi sel &koloniPengamatan fisiologi/biokimia
Measurement of IAAPatten & Glick 2002
Isolat Pseudomonas Medium King’s B atau Bacillus medium NB
Media King’s Batau NB + L-triptofan 0.5 mM
48 jam/24 jam, suhu ruang, shaker 160 rpm
10.000 x g, 10 menit
1 ml supernatan + Reagen Salkowsky 4 ml
Inkubasi pada suhu ruang 30 menit
Ukur absorbansi pada 520 nm
Hitung konsentrasi IAA (perbandingan dengan kurva standar)
