Pemanfaatan Bakteri Rizosfer untuk Proteksi Cabai terhadap ...
Transcript of Pemanfaatan Bakteri Rizosfer untuk Proteksi Cabai terhadap ...
Seminar Setengah Hari PERMI Seminar Setengah Hari PERMI Bogor 4/03/2010Bogor 4/03/2010
TRI ASMIRA DAMAYANTINISA R. MUBARIKHENDRA PARDEDETRIAS KATERINA
DEPARTEMEN PROTEKSI TANAMANFAKULTAS PERTANIAN
INSTITUT PERTANIAN BOGOR
PEMANFAATAN BAKTERI RIZOSFER UNTUK PROTEKSI CABAITERHADAP INFEKSI GANDA VIRUS
(Hayati, 2007 Vol.14, hal 105-109 & J. ISSAAS, 2008 Vol.14, hal 92-100)
PLANT VIRUSPLANT VIRUS
SubmicroscopicSubmicroscopic particlesparticlesContain Contain RNARNA or or DNADNA ((ssss or or dsds))Nucleic acid protected by coat protein form Nucleic acid protected by coat protein form virionvirionDo not have organelle cellsDo not have organelle cellsObligate parasiteObligate parasite (only living in live cells)(only living in live cells)Mainly replicate in Mainly replicate in viroplasmviroplasm/cytoplasm /cytoplasm
Healthy ChiVMV Mix CMV TMV
Symptoms of virus infection
SYMPTOM AND VIRAL PARTICLES
Tobacco Mosaic Virus Cucumber Mosaic Virus Chilli Veinal Mottle Virus
[Particle Source; CPC 2005]
Tobamovirus Cucumovirus Potyvirus
SYMPTOM OF MIX INFECTION TMV & ChiVMV
Management of virus diseases:- Resistant varieties - Cultural practices - Eradication of vectors- Genetically engineered crops - Cross protection
Root colonizing bacteria ?
Objectives: To utilize the potential PGPR isolates to protect hot pepper against multiple infection of virus
Root colonizing bacteria - Rhizobacteria
Abundantly present in rhizosphereLive from plant root secretionStimulate plant growth,
referred as :
Plant-Growth Promoting Rhizobacteria (PGPR)
The roles of PGPR
Nitrogen fixationPromoting plant growthProtecting plants from infection by pathogen (antibiosis, ISR etc)
Large-scale application of PGPR reduce the use of chemicalfertilizer and pesticides; and increase crop yield
Induced Systemic resistance (ISR)
ISR an increased resistance to disease that develops systemically throughout plants after appropriate stimulation (Hammerschmidt and Kuc, 1995)
HOW IS PGPR SUPPRESS THE DISEASE?
PGPR as stimulant
Seed treatment, Soil drench, Foliar spray, Combination
Challenge inoculation of pathogens
Elicits Plant’s defense response
Decrease disease incidence, severity, symptom expressions
Alters host physiology and metabolic responses, fortifies plant cell wall strengthAntibiosisIncreased SA PRs gene, chitinase etcIncreased Jasmonate Acid and ethylene, peroxidase, phytoalexin, enhance ability to lignifySiderophores (pyoverdine, pyoceline, SA)Competition for iron
MECHANISMS
Rhiszosfersoil
100 ml 0.85% NaCl
Vortex 15 min
24hr shaking
Heating at 60 oC, 10 minShaking 24 hr
Pour on TSA
0,1 ml
Single Colonies
RHIZOBACTERIA ISOLATION
1 g
Streak on TSA
METHODS
SeedsTM-999
Bacteria Suspension1 x 109 cfu/ml
Ssoaked for 3 hr
2 Weeks afterPot
Sterile Soil : cow-dung = 1:1Placed in greenhouse
Seedling + a drop bact. suspensionSeed treatment and cultivation
Methods: Methods: Evaluation of plant growth characteristicsEvaluation of plant growth characteristics
Plant height at 1 day priorPlant height at 1 day prior-- and at 2, and 4 and at 2, and 4 weeks postweeks post--virus inoculation (virus inoculation (wpiwpi))Number of LeavesNumber of LeavesNumber of flowers and fruitsNumber of flowers and fruits
Plant fresh weightPlant fresh weight
2 WPI = 6 WAP
Infected leaves Grind +bufer fosfat pH 7
[1:10 (b/v)]
Observation
Inoculum
Plants at 4 WAP
Inoculation
Method : Viral InoculationMethod : Viral Inoculation
I = ΣnN
X 100%
I = disease incidence (%)n = number of infected plantsN = total number of inoculated
plants
1. Disease incidence (%)
Methods: Disease assessments
2. Disease severity rating made at 2 wpi and 4 wpi.It is performed with mock inoculated plants as standard.
Disease severity rating scales0 = no symptom2 = leaves with mild mosaic symptom4 = leaves with severe mosaic symptoms6 = leaves with mosaic and deformation8 = leaves with severe mosaic, deformation and yellowing along
veins10 = leaves with severe mosaic, deformation, yellowing along veins
and abrupt growth reduction
3. Detection of Viral Protein by ELISA
((Enzyme Linked-Immunosorbent Assay))
Y Y
Y Y
Y Y
E E
Coating 1 st AB
Washing 4-8 times
Antigen bound to the1st AB
Washing 4-8 times
2nd AB conjugatedwith enzyme
Washing 4-8 times
Substrates addition(yellow)
ELISA Reader at OD 405 nm
Y Y
4. PEROXIDASE ENZYME ACTIVITY
Measured by Spectrophotometer at 470 nm wavelength; every 30 seconds for 3 minutes
5. ETHYLANE PRODUCTION
Gas Chromatography methods at Balai Besar Pasca-Panen Cimanggu, Bogor
Samples measured at 5 days post viral inoculation
RHIZOBACTERIA ISOLATES
55 isolates obtained; 17 are gram positive and 38 are gram negative (14 isolates are pathogenic, 5 isolates were unable to re-cultured)
36 isolates were tested for inducing seed germination
Bacterized-seeds showed comparable germination rate, but better seedling vigor and fitness than untreated control
7 isolates were evaluated their ability to protect pepper against 3 viruses
TinggiTinggi TanamanTanaman 1 HSI1 HSI
c b
a
b b
a
b
b
HSI – Hari Sebelum Inokulasi Virus
0
2
4
6
8
10
12
14
C I-2 I-16 I-25 I-30 I-35 II-7 II-10
Treatment
Plan
t Hei
ght (
cm)
cb
a
b ba
b b
TinggiTinggi TanamanTanaman PadaPada 8 MST8 MST
MST MST –– MingguMinggu SetelahSetelah TanamTanam
c
ba
b b
a ae
0
10
20
30
40
50
60
70
C I-2 I-16 I-25 I-30 I-35 II-7 II-10
Treatment
Plan
t Hei
ght (
cm)
Healthy Infected
e
ca
ab
d
a bc ab
c
ba
b b
a a a
TINGGI TANAMAN PADA 12 MSTTINGGI TANAMAN PADA 12 MST
020406080
100120140
C I-2 I-16 I-25 I-30 I-35 II-7 II-10
Treatment
Plan
t Hei
ght (
cm)Healthy Infected
b aa a
ba a a
c
ab b ab ba ab a
CI-2II-10
Pembibitan
12 MST
TANAMAN SEHAT
CI-2II-10
c I-16 I-35 I-16I-35 c
Pembibitan 8 MST
TANAMAN SEHAT
C- +
I-2- +
C
- +
II-10
- +
6 MST
+ : Sakit- : Sehat
+ : Sakit- : Sehat
I-25c-- + +
I-30c-- + +
II-7c-- + + 6 WAP
I-16 c-- + +
8 MST
I-35 c-- + + + : Sakit ; - : Sehat
JumlahJumlah DaunDaun
0
50
100
150
200
250
C I-2 I-16 I-25 I-30 I-35 II-7 II-10
Treatments
Leaf
Num
bers
Healthy Infected
e
ab
cdbc
de
abcbc
a
e
bcb
cdd
a
b b
aa
a
0
50
100
150
200
250
C I-2 I-16 I-25 I-30 I-35 II-7 II-10Treatments
Flow
ers/
Frui
ts N
umbe
rHealthy Infected
b
a a a
d
ab
d
c
c
bc
aba
a
a
a a
JumlahJumlah Bunga/BuahBunga/Buah
BobotBobot BasahBasah
0
20
40
60
80
100
120
140
C I-2 I-16 I-25 I-30 I-35 II-7 II-10Treatments
Fres
h W
eigh
t (g)
Healthy Infected
bab
bab
b
a
babd
bc bc bcc c
b
a
Disease AssessmentsDisease Assessments
Positive if EAV = 2 x C (-) (Orange type)
A. Nilai Absorban ELISA
0.3410.1871.770 1.925II.10
0.3580.1951.660 1.955II.7
0.0650.0891.751 2.039I.35
0.3600.2341.712 1.810I.30
0.4100.2211.769 2.100I.25
0.0740.0801.765 2.198I.16
0.3860.1871.942 1.889I.2
0.4720.3261.8951.624C (+)
0.0610.0610.195 0.195C (-)
4 MSI2 MSI4 MSI2 MSITreatment
ChiVMVTMV
B. Disease Incidence and SeverityB. Disease Incidence and Severity
Disease DiseaseIncidence (%) Severity
C (-)C (+) 100 5.60 aI.2 100 2.00 bcI.16 100 1.67 cI.25 100 3.33 bI.30 100 2.67 bcI.35 100 1.67 cII.7 100 3.33 bII.10 100 2.67 bc
Treatment
PO. Enzyme Activity(U/mg/min)
Ethylene Prod.(umol/gr)
Healthy HealthyInfected Infected
Treatments
C
I - 2
I - 16
I - 25
I - 30
I - 35
II - 7
II - 10
0.76
2.55
5.40
4.20
4.04
4.10
3.30
2.70
3.42
5.70
3.60
6.60
7.74
9.40
3.50
1.40
0.14
0.10
0.12
0.32
0.25
0.08
0.19
0.02
0.16
0.18
0.16
0.20
0.33
0.16
0.19
0.30
C. PEROXIDASE ENZYME ACTIVITY AND ETHYLENE PRODUCTION
Identification of Identification of RhizobacteriaRhizobacteria
Code Species Accession No.
I – 2I - 25I - 35I – 16I – 30*II -7II –10
Bacillus cereusB. cereusB. cereusBrevibacterium sanguinisB. maceransAcinetobacter sp II -7Stenotrophomonas sp II-10
Morphological Characters & 16S rRNA sequences
AB288105AB288105AB288105AB288106
-AB288107AB288108
* Based on morphological characters & Microbact-Kit test only
All tested isolates could enhance plant All tested isolates could enhance plant growth characters and could suppressgrowth characters and could suppressthe severity, even infected by virusesthe severity, even infected by viruses
Bacillus cereusBacillus cereus (I(I--35) and 35) and StenotrophomonasStenotrophomonas sp IIsp II-- 10 were the 10 were the most potential PGPR which able to most potential PGPR which able to protect hot pepper against multiple protect hot pepper against multiple infection of virusinfection of virus
SEAMEO BIOTROP & ITSF SEAMEO BIOTROP & ITSF PHK B Dept. of Plant Protection, IPBPHK B Dept. of Plant Protection, IPBProf. Prof. TetsuroTetsuro OkunoOkuno & Dr. Kazuyuki & Dr. Kazuyuki MiseMiseMr. Edi Mr. Edi SupardiSupardi -- TeknisiTeknisi