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ISHS Commission Sustainability tbrough Integrated and Organic Horticulture

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L

1

t-

Editors

I. Mourilo

ESAPL/IPYC, Portugal U. Aksoy

Ege University, Turkey

Editorial Board

A. Hodder,

FAO,Italy

A. Alonso, CItulED, Spain F.P. Weibel,

FiBL,

Switzerland H. Willer,

FiBL,

Switzerland I. Mour6o, ESAPL/PVC,

Portugal

K. Thorup Kristensen, UA/FAS, Denmark

L.M.

Brito, ESAPL/IPVC,

Portugal M. Mota

ISA/UTL,

Portugal M.

Raviv, MA/ARO,

Israel M.

Ben-Kheder,

CMB,

Tunisia N. Halberg, ICROFS, Denmark

R.K. Prange, AAFC/AAC, Canada,

Chair

ISHS Commission Sustainability through Integrated and Otganic Horticulture

Sang

Mok

Sohn, DKU, Republic of Koreo U. Kripke, ISOFTR, Germany

U. Aksoy, Ege University, Turkey

-j

I,IIIT OFCONTENTS Fccsord

Ptcficc

LfotofCmtents Lirr

of Aulhors

lrvltld

Lectures

Tb

Global Extent and Expansion of Organic Horticulture Production

H. Ytllq, D.

Granatstein and E.

Kirby

Agoecological Diversification Strategies to Enhance Biological Pest Regulation in

Horticultural

Systems

Ml. Altieri

^and C.I. Nicholls

PreHawest, Harvest and Post-Harvest Sfrategies for Organic Production

of Fruis

andVegetables

P..K- Prange

Phnt

Breeding and Propagation

Material

Traditional and Commercial Tomato Cultivars Evaluation for Organic Horticulture in

Two

Regions

of

Spain, Caceres and Madrid

H. Sdnchez-Girdldez, M. Ramos, E. Zambrana, J.L. Tenorio, C. de lo Cuadra and

I.

Martfn

Agronomic and Quality Evaluation

of

Spanish Melon Landraces Grown under Organic Farming in Extremadura

J. Gragera- F acundo, C, G. G il-To rralvo, J.

M.

Gu ti err ez- P erer a,

J.M- Cano-Sudrez, C. Avila-Lozono and A. Esteban-Perdig6n Melon Cultivars Evaluation for Organic Horticultural Production

in

Extremadura

H. Sdnchez-Girdldez, M. Ramos.and C. de

la

Cuadra

Local Landraces

of

Dry Beans (Pftaseolus vulgans

L.):

^a Valuable Resource for Organic Production in Greece

I. Papadopoulos,

F.

Papathanasiou, C. Vakali, E. Tamoutsidis and

Y. Kazoglou

Evaluation of Spanish Onion Landraces for Incorporation into Organic Farming

J.M Gutiinez-Perera,

J. Gragera-Facando, C.G.

Gil-Tonalvo

and F. Acero-Ghmez

Soil

Fertility

and

Nutrient

Management

-

Vegetable Crops

Effects of Compost Maturation and Time of Application on the Growth and Nutrient Accumulation by Organic Cabbage

I.

Mourdo, A.L. Amaro, L.M.

Brito andJ.

Coutinho

Compost Based Growing Media

for

Organic Melon Seedlings Production H.M. Abdelrohman, F.G. Ceglie, F.G. Erriquens, V. Verrastro, C.M. Rivera and

F. Tittarelli

6 7 9

t7

23

35

43

53

61

69

75

83

9l

99

Ed dsme l-cgrme

Cover Crops and Organic Fertilizer on Petiole

ftir Com,

Productivity and Fruit Composition

of

'Thompson

br'Crlcvines aE Riz*

fti d lliare

Management Strategies

-

^Vcgetable

Crops

HftregEm€nt in

^{Organic Onion} Production: Optimizing Cultivation

lli5gc

md Mechanical Weed Control to Reduce Hand Labour

n ffla

^@d

A.

^Yieweger

mttiveness

of Soil Solarization and Biofumigation for the

Contol of

Cdy Roc

md Root-Knot Nematode

Meloidogne

spp. on Tomato

L fuq I.

Queiroz,I. ^Mourdo, L.M.

Brito

andJ. Duclos

fhhht'itive

^Effects

of

Garlic

Bulb

Crude Extract

onFulviafulvaof

T6 f-T- YeL ZH.

_{Cheng, Q.} Mo and L. Han

Arifimgal Activity of

Spices Extracts against Sclerotium

rolfsii EA

A&segun, O.S. Adebayo and A.K.

Akintolun

lrcction

of Eggplant and

Chilli

from Bacterial

Wilt'(Ralstonia

furcemn)

with Antagonistic Bacteria

f- Atttiyanto,

^Y.S. Maryudani and S.D. Nurcahyanti

Ilbfimigation

with Brassica juncea Pellets and Leek Material in Carrot

Crq

Rotations

K

^Gtevsen

Cqarative

^{Field Effrcacy of Various} Entomopathogenic Fungi against

I7riN

tabaci: Prospects for Organic Production of Onion

in

India PN-

Gorya

Yisalaluhy and A. Krishnamoorthy

Biodegndable Mulch

Film

ⁱⁿ a Broccoli Production System

J-

Iipa-Marin,

A. Gonzalez, J.A. Ferndndez, J.L. Pablos and C. Abrusci

?ci

end I)isease Management Strategies

- Fruit

^Crops

Clolmd

Beetles (Carabidae) Are Affected by Mulch in Organic ^Highbush Blueberries

J-M

Renkzna, S.J. Wolde, D.H. Lynch, G.C. Cutler and K. MacKenzie

Fwtional

Biodiversity and Farrning Techniques: How to Measure

hpecb?

C.L

Costa, M.C. Godinho, S. Duarte, C. Mateus, E. Figueiredo and

L Llqia

$Ec4tibility

^of Japanese Plum Cultivars to Traraschelia pntni-spinosae

uder

Organic and Conventional Management

in

Southern Spain

H

^Castej6n, F.T.

Anoyo,

P.A. Garcia-Galavis, C.

Santamaia

and A. Daza

407

415 399

447

45s 38r

391

463

l3

433

439

i i

i

-i<-

Protection of Eggplant and Chilli from Bacterial Wilt (Ralstonia

so

lanoeear urn) with Antagonistie Bacteria

T. Arwiyanto, Y.S.

Maryrdani

and S.D. NUrcahyanti

Faculrybf

agriculture,'Gad

jahMaFDfiiver-ilifl --

Jl Flora

No I

Bulaksumur, Yogyakarta, 55281 Indonesia

Keywords:

biological control, Pseudomonas

WtidaPf-2U,

^R. solanqcearum Rs-127

Abstract

Bacterial wilt

caused

by

Ralstonia solanacearum is ^one

of the limiting factors in

eggplant end

chilli production in

Indonesla.

Control

of the disease is

difficult with the

available means.

Biological control

based on antegonism was

therefore

chosen as one

of

the

control

methods.

A

fluorescent pseudomonad (Pseudomonas

putidarPf-20 strain) isolated from the rhizosphere of Mimosa inuisa proved

successful

in

suppressing

R.

solanacearum

in vitro, reducing

^disease

intensity in

greenhouse and

field

experiments. However,

the

degree

of protection

was low.

The

use

of

more

than one

species

of microorganisms to control plant pathogens likely

enhances

the protection level. An avirulent-bacteriocin producing strain of R.

solanacearum (Rs-1.27) was chosen as a companion of Pf-20

to control

the disease. Rs-127

inhibited the growth of other R

solanacearnrr isolate,

with an inhibition

zone

of

12 mmo and as

much

as 867o

of other

isolates were

inhibited.

Rs-127 did not

inhibit

the

growth of Pf-20 in YPGA and

CPG

medium.Ilowever,

Pf-20

inhibited

the

growth

of Rs-127

in King's B

medium

with

an

inhibition

zone

of

15

mm, but did not work in YPGA

and

CPG

medium.

The

greenhouse test showed

that dipping

the

root

system of eggplant

and chilli in bacterial suspension of Rs-127 and Pf-20, both solely and in combination,

were able to suppress the disease and

prolong

the

incubation period. In the control plots, plants

began

wilting

one

week after transplanting. In the

plots

treated ryith Pf-20 and

Rs-127,

the

diseased

plants were first

observed

at two

and

five

weeks

after transplanting, with

a disease

index of

¹³ and 16,

respectivelg while

in the control plots, the

disease

index at that time had already

reached 56.

When Pf-20

and Rs-127

were

combinedn

the

disease

was first

observed

at 5

weeks

after transplanting with

^a disease index of 6.

INTRODUCTION

Bacterial

wilt

caused

by

Ralstonia solanacearum

is difiicult to confiol with

^the available means, especially the one caused by Race

I in

lowland areas (Hayward, 2005).

The ^great

variability of

the pathogens and the complexity of portal entrance into the host

tissue make the

disease

and the

pathogens

diffrcult to conhol (Goto, 1994).

Soil solarization

is not

effective and crop rotation is

not

feasible due

to

the high number

of

host plants that can be attacked

by

the pathogen. Resistant plants are

difficult to

obtain and could be broken

by

the pathogen.

Until

now there

is

no chemical that is effective

to

control the pathogen in the field. Biological control was therefore chosen as an altemative

control

method.

Many

authors report the use

of

antagonistic bacteria

to

control disease.

However, most

of them

used

a

single antagonist instead

of multi

antagonists (Smith, 2005). The use

of

more than one antagonist to control plant pathogen

likely

gives better protection than single application. We report the use

of two

antagonistic bacteria against R. solanacearrm in

viho.

MATERHLSAI\D METHODS

Stain

Pf-20

of

Pseudomonas putida was isolated from the rhizosphere of Mimosa

iwisa rn

1996

(Arwiyanto,

1997).

The

bacteria

was

restored

from stock

kept under mineral oil by streaking on the King's B medium before trse. Ralstonia solanacearum was isolated

from

several host plants on

YPGA

medium (yeast extract

5 g,

peptone

10

^g,

Proc. XXVIII$ IHC - IS on Organic Horticulture:

hoductivity and Susainability Eds.: I. Mour5o and U. Aksoy Acta Hort. 933, ISHS 2012

421

glucose 10 g,

distilled

water 1000 ml,

pH

6.8). Virulent colony type

of

R. solanacearum was transferred to YPGA slant medium.

Each isolate

of

R. solanacearam was tested against each other

for

antagonism _on

CPG

medium

(Arwiyanto et

al., 1993).

A

selected

avirulent

shain

of

R. solonqcearum

producing

bacteriocin

was

tested

for its

antagonistic

activity

against

Pf-20 on

CPG

medium

and

the

antagonistic

activity of

Pf-20 against

R.

solanacearum ^'w&s tested _on

King's

B medium (Arwiyanto and Nurcahyanti, 2007).

Local cultivars

of

^eggplant

('Mustang')

and

chilli ('Lokal')

were used. The seeds were sown in sterile soil and used at 35 days after sowing'

Seedling Treatment

The

root

systems

of

the seedlings were dipped

in

the water-bacterial suspension

for 15

minutes.

A final

concenfiation

of

¹⁰⁰

ppm of

^Tween-2O

was

added

into

the suspension before treafinents. As a control, root systems

of

^the seedlings were dipped for 15 minutes

in

sterile distilled water

with

^{Tween-20 (100 ppm}

final

concentation).

After

dipping, the seedlings were planted in pots filled

with

soil infected

with

virulent isolate

of

R.'

i

olinacearum. fllre treafinents consisted of:

1.

Root dipping in sterile distilled water followed by planting in soil

infected with R. solanacearum.

2.

Root dipping

in

a mixture of Pf-20 and _Rs-127 water suspension

with a l:lratio

(v/v),

with

a lirial Sacterial concentation

of

108 cfir/ml.

3.

Root dipping

in

a mixture of Pf-20 and _Bs-127 water suspension

with

^a 1:1 ratio (v/v),

with

a

liialEacterial

concentration

of

lOe cfrr/hl.

4.

Root dipping in a water suspension

of

Rs-127 (108

cfir/ml)

followed by planting in soil infected with,l?. solanacearum.

5.

Root dipping in a water suspension

of

Rs-127 (l0e

cfir/ml)

followed by planting in soil infected with,R. solanacearum.

6.

Root dipping

in

a water suspension

of

^{Pf-20 (108} cfrr/ml) followed by

in soil

infected

with

^R. solqnacearum.

7.

Root dipping

in

a water suspension

of

Pf-20 (lOe

cfir/ml)

followed by planting

in

soil infected with R. solanacearum.

Disease development, expressed as disease

index

(Winstead and

Kelman,

1952) was recorded for each treatment at specific times after the challenge inoculation.

RJSULTS A}[D

DISCUSSION

As much as 161 isolates of R. solanacearum

from

several host plants were tested against each other

for

their antagonistic activity on CPG medium. Isolate number 127

of

R. solanacearurn inhibited the growth 860lo relative to other isolates tested

with

a zone

of inhibition of

L2.6 mm

in

average (Table 1). This isolate was therefore chosen

for

fttrther experiments because

of

its ability to produce inhibition substance more successfully than other isolates.

The avirulent fomr of isolate Rs-127 (Rs-127

avir)

was generated spontaneously

in YPG

broth medium

after

one week

in still

culture

at room

temperature.

The

avirulent

form

was obtained by streaking ttre culture on YPGA and incubating for 48 hours at room temperature.

The

ability of

Rs-127

avir to

produce

inhibition

substance against

the

virulent isolate of R. solanacearam (Rs

T)

was the same as its parental

wild

type (Table 2). This means that the isolate could be used as a biological control agent.

Since the isolate

of

Rs-127

avir

was used as a companion

of

Pf-20

for contolling the virulent form of

R. solanacearum.

the

antagonistic

activity of

these

control

agents against each other was tested in

vitro.

Pf-20 inhibited the growth

of

Rs-127

avir

in

vitro with

a resulting

inhibition

zone

of

15

mm.

The mechanism

of inhibition

was bacterio- static. However, Rs-127 avir could not suppress the growth of Pf-20 in

vino

^(Table 3).

Rs-127

could

suppress disease development caused

by the virulent form of

R. solanaceorwm. Disease development

in

the plot treated with Rs-127 was slower and the

422

+

-

disease index

was

constant around 10,

from 3

weeks after inoculation onwards. There were

slig[t

differences between plots treated with Rs-127 at a concentration

of

lOe

cfi:/ml

and at 10"

cfir/ml.

On the contrary,

in

the unheated plots, disease development was rapid

and the

disease

index

reached around

70 at the end of the

experiment.

Pf-20

^also srypressed

the

developmertt

of

eggplant bacterial

wilt

^although the degree

of

^protection was lower than the one offered by Rs-127 (Fig.

l).

The disease development

in

the plot freated

with

Pf-20 was constantly low

until

5 after inoculation. The disease index then moved nrosressivelv un rrn{il fhe end o{

-fhe

disease development

in

the plot freated

witl weels

after inoculation. The disease index then moved

weelrs atter inoculatton. f he disease index then moved progressively up

until

the end

of

experiments. However, it was

still

lower than in the untreated control plot.

When

the

antagonistic bacteria were combined to protect eggplant from bacterial

wil!

significant protection was obseryed. The

wilt

^{symptoms in} the treated plot were not observqd

wtil2

weeks after inoculation. When the concenftation

of

antagonistic bacteria was lOe cfrt/ml,

wilting

symptoms went almost undetected until 5 weeks a'fter inoculation.

At

^the end

of

the experiment, the disease index was very low, i.e., at less than 10. On the confrary, disease index

in

the untreated conhol

plot

was more

than l0

at 3 weeks after inoculation and

rapidly

increased to more than 70 at the end of the experiment.

A

similar

resrlt

was obtained when

the

bacteria were used

for contolling chilli

bacterial

wilt (Fie.2).

The

niche of

Pf-20

is in

the rhizosphere,

while

Rs-127 tends

to

invade the host

plants through wounds. Therefore, when they are applied together as

antagonistic bacteria, they do

not

reside

in

the same place, meaning antagonism between them could not occur. This hypothesis,

howevel

needs clarification through firrther experiments.

CONCLUSIONS

R

solanacearum protected

P.

putida

strain

Pf-20

and an avirulent bacteriocin producing strain (Rs-127)

of

Tqcearum protected essDlant and

chilli

from bacterial

wilt

caused bv vinrlent form

R

solanacearum protected eggplant and

chilli

from

of R. solqnaceqrum

in

greenhouse condidons.

ial

wilt

caused by virulent form

Uterature Cited

Anriyanto, T., Goto, M. and Takikawa, Y. 1993.

Characterization

of

bacteriocins produced by Pseudomonas solanacearum.Ann. Phytopath. Soc. Japan 59:114-122.

Arwiyanto,

T.

1997. Biolocal control of tobacco bacterial

wilt: l.

Isolation of antagonistic bacteria. Journal of Indonesian Plant Protection 3 :54-60.

Arwiyanto,

vryanro,

T. r. and Nurchayanti, S.D. ans

Nurcnayanu,

>,D.

2007.

zwt.

AntagonismsAnEgomsms

among strain

sualn

of

Ralstonia solanacearum and between strain

Pf-20

of Pseudomonas

putida.

Paper

solanacearum and between strain Pt--20 of Pseudomonas

putida.

Paper presented at the International Conference on Plant Pathology and Mycology. Hyderbbad, India^

lo,

M.

1994. Fundamental of Bacterial Plant Pathology. Academic Press. New York.

Hayward,

A.C.

2005. Research on Bacterial

Wilt A

Perspective on lnternational Linkages andAccess

to

the

Literature.p.l-8. In:

C.

Allen,

P. Prior

andA.C.

Hayward (edi.), Bacterial

Wilt

Disease and the Rolstonia solanacearann Species Complex, APS Press.

Minnesota.

Goto,

M.

1994. Fundamental of Bacterial Plant

Smith, J.J.,

Murimi, 2.K., Offord,L.C.,

Clayton, S., Mienie,

N.,

Gouws, N.R., Priou, S., Olanya,

M.,

Simmonds, S. and Saddler. GS. 2005. Prosresses

in

the develooment of a

v.v., vr

Olanya,

M.,

Simmonds, S. and Saddler, 5. Progresses

in

the development of a

biocontrol agent

against

bacterial wilt. p.6l-72. In: C. Allen, P.

Prior-

and

A.C.

Hayward Hayward

(eds.). Bacterial

(eds.),

Wilt Wilt

Disease Disease and and

the the

Ralstonia solanacearunr Ralstonia solanacearurn SnecidsSpecies Complex, APS Press. Minnesota.

Winstead,

N.N.

and Kelman,

A. lgsz.Inoculation

tecbniques

for

evaluating resistance to Pseudomonqs solanqceararn. Phvtooatholow 42:628-634.

Pseudomonqs solanqceararn. Phytopathology 42:

423

Tebles

Table

l.

Selected isolates of Ralstonia solanaceatam which inhibited other isolates in

vito.

No Isolate number Zone of inhibition

(mm)*

Inhibition intensity (%)

I

2 3 4 5 6 7 8 9 10

ll

t2

13

t4 l5

16

I7

18

t9

20 59 66 70 104 108

lt4 il5

116 t17 119

t20

122

t24 t25

126 t27 129

l3l

12.27 11.04 t2.57 t0.76 10.28 10.66 t0.22 10.48 10.68 11.29

|.27

9.47 I 1.13 13.51 11.09 t2.13 12.62 7.34 10.10

62.t2

55.22 47.69 76.12 79.10 69.70 66.67 78.79 78.79 81.82 82.81 81.25 82.81 81.25 85.94 81.25 86.1 5 78.46 72.31

* _{average ofthree} replicates.

Table 2. Growth inhibition of a virulent R. solanacearun (Rs T) by antagonistic bacteria.

Medium Inhibition zone (mm)*

Rs'127 against Rs

T

Rs-I27 avir against Rs

T

Pf-20 asainst Rs

T

King's

B

CPG YPGA

0 7.7s 7.7s

0 7.00 7.OO

t2

0 0

*

_{average ofthree} replicates.

Table 3. Antagonistic test between Pf-20 and

Rs-l27.

Inhibition zone (mm)*

Medium _Pf-20

against

Rs-127

against

Pf-20 against

Rs-127 Pf-20

^{Rs-127 avir} Rs-127 avir against Pf-20

0 0 0

015 00 00

t2

0 0 King's

B

CPG YPGA

* _{average ofthree} replicates.

424

Eggplant

90 80

€oo *7o E5s

$oo Eso

6zo

10 0

l-C"nttt l -

!-+-Combination 10E8

i

,

Combinalion 10E9

-x--Avir

^10E8

-+-Avir

10E9

i-+--Pf-20

loEB

t=rr;eleEl--

Development of eggplant bacterial

wilt

treatedwith antagonistic bacteria.

Development of

chilli

bacterial

wilt

treated with antagonistic bacteria.

chilri

90 80

"70

too 5so ftoo Eso

5zo

10 0

f c*a'6

I

--r-

Combination ^'t 0E8

I

Combination 10E9 l--+i-Avir 10E8

-x-Avir

^10E9

--pi-zo

^toEa

--r-Pf-2010E9

2345678

Weeks

afur

lnoculaton

425