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