Grafting Local Commercial Tomato Cultivars with H-7996 ang Eg-203 to Suppress Bacterial Wilt (Ralstonia solanacearum) in Indonesia

I

rsHs

lnternational Society

for

Horticultural

Science

Soci6t6 lnternationale de la Science Horticole

ACTA

HoRTlcuttuRRf@

is a scientifically reviewed

publi-cation of ISHS. lnformation about Acta Horticulturae@ and ISHS is given at the end of this book.

Check out www.actahort.org for details on our latest titles.

Editorial Advisory Board

ol

Acta Horticulturae@

Yves Desjardins, Faculty of Agriculture, Department of Plant Science, Horticulture Research Centre, Laval University,

Quebec, Canada, Chair of the Editorial Advisory Board

Jorge Retamales, Universidad de Talca, Escuela de Agronomia,

Talca, Chile

Sadanori Sase, National lnstitute for Rural Engineering, Tsukuba, Japan

Paolo lnglese, Dipartimento di Colture Arboree, Universitd

degli Studi di Palermo, Palermo, ltaly

Y0ksel T0zel, Department of Horticulture, Agriculture Faculty, Ege University, lzmir, Turkey

Julian Heyes, institute of Food, Nutrition & Human Health, Massey University, Palmerston North, New Zealand

Janet Cubey, Science Department, Royal Horticultural Society, Wisley, United Kingdom

lsaac Ore Aiyelaagbe, Department of Horticulture, University

ol Agriculture, Abeokuta, Ogun State, Nigeria

Executive Director

ol

ISHS

lr. J. Van Assche

Secretariat of ISHS

PO Box

500

Phone:

+32.16.2?9427

3001 Leuven

1

Fax:

+32.16.2294 50

Belgium

E-mail:

info@ishs.org

PROCEEDINGS OF

THE

IVth

INTERNATIONAL

SYMPOSIUM

ON

TOMATO

DISEASES

Convener

M.L.

Paret

Orlando, Florida,

USA

June 24-27,2013

ISHS Section Vegetables ISHS Commission Plant Protection ISHS Working Group Tomato Diseases

Acta Horticulturae

1069

ISSN 0567-7572

ISBN 97S 94 6261059 0, Acta Horticulturae n". 1069

Price for non-members of ISHS: € 86,-Published by ISHS, February 2015

Executive Director of ISHS: J. Van Assche

Technical Processing: S. Franssens

ISHS Secretariat, PO Box 500,3001 Leuven 1, Belgium

Printed by Drukkerij Geers, Eekhoutdriesstraat 67, 8-9041 Gent (Oostakker), Belgium

o2ttt5 _{by the Intemational Society for Horticultural Science (ISHS). All rights reserved. No part ofthis book} may be reproduced and/or published in any form or by any means, electronic or mechanical, including photocopying, microfilm and recording, or by any information storage and retrieval system, without written

permission from the publishers.

Photographs on the front cover:

l.

Spring 2012Bs2 trial at the Gulf Coast Research and Education Center, University of Florida canied out by John W. Scott. Trial entry on the left is tomato hybrid Fla-8314 containing the pepper Bs2 gene,

conferring Xanthomonas-resistance. The entry on the right is a susceptible tine, VF36. Bacterial spot disease is evident on VF36 plants as defoliation and reduced fruit set. In this and numerous other trials, Bs2-containing plants have negligible defoliation and typically show a doubling ofyields. (by courtesy

of D. Horvath, Two Blades Foundation).

2.

Bacterial wilt disease caused by Ralstonia solancearum leading to severe wilting of the plants (by

courtesy of M. Paret, University of Florida).

3.

Target spot disease caused by Corynespora cassiicola causing large cracks on tomato fruit (by courtesy of M. Paret, University of Florida).

4.

Tomato spotted wilt vla.r affecting tomato fiuit (by courtesy of M. Paret, University of Florida).

5.

Bacterial spot caused by Xanthomonas perforans causing leaf spots on tomato (by courtesy of M. Paret,

University of Florida).

6.

Fresh market tomato production in North Florida (by courtesy of M. Paret, University of Florida).

Editors

M.L.

Paret

Department of Plant Pathologt/NFREC, University of

Florida,

USA

G.E. Vallad

Department of Plant

Pathologt/GcREc,

University of

Florida,

()SA S. Zhang

Department of Plant Pathologt/TREC, University of

Florida,

USA

J.B. Jones

IJniversity of

Florida,

USA

Editorial Board

A.

Crescenzi, Chair of the ISHS Working Group on Tomato Diseases, Universi4t

of

Basilicata,

Italy

S.

Miller,

The Ohio State University, USA

Jaw-Fen Wang, AVRDC

-

The World Vegetable Center, Chinese Taipei

J.B. Jones, University of

Florida,

USA

A.M.

Alvarez, University of

Hawaii,

USA J. Scott, University of

Florida,

USA

M.A. Al-Saleh, King Saud University, Saudi Arabia S.M. Olson, University of

Florida,

USA

Tao Zhou, China

Agricultural

Universitl,, China P.D. Roberts, SWFREC, University of

Florida,

USA

D. Shteinberg, Ministry of Agricultttre and Rural De;-elopment, Israel T. Momol, Departmenl of Plant Pathologt, University of

Florida,

USA J. Freeman, Virginia Tech, USA

LIST

OF

CONTENTS

Preface

List of Contents List of Authors List of Participants

Host-Pathogen

Interaction

Plant-Pathogen-Symbiont Interactions in the Tomato Phyllosphere

N. Potnis, A.H.C. van Bruggen, J.B. Jones,

K.

Cowles and J.D. Barak

Multilocus Sequence Analysis of a Worldwide Collection of Xanthomonas

Strains, Causing Bacterial Leaf Spot

S. Timilsina, J.B. Jones, N. Potnis,

E.

Goss, G.V. Minsavage, J.C. Hong, G.E. Vallad and M. Kebede

Using Proteomics and Genomics to Search for Low Temperature Virulence

Factors in Ralstonia solanacearum

A.M. Bocsanczy, D.J. Norman, J.C. Huguet-Tapia.

U. Achenbach and A. Mangravita-Novo

The Genetic Determinants of Symptoms Induction by Pepino Mosaic Virus in Tomato

B. I{asi6w-Jaroszewska, J. Minicka, N. Borodynko and H. Pospieszny

Tomato Breeding

for

Disease Resistance

The Pepper BsZ Gene Confers Effective Field Resistarlce to Bacteriai

Leaf

Spot and

Yield

Enhancement in Florida Tomatoes

D.M. Horvath, M.H. Pauly, S.F. Hutton, G.E. l'allad, J.l|r. Scott, J.B. Jones, R.E. Stall, D. Dahlbeck, B.J. Staskawicz, D.

Tricoli

and A.V. Deynze

Identification of Tomato Bacterial Spot Race

Tl,T2,

T3, T4, and

Xanthomonas gardneri Resistance _{QTLs Derived} from

PI

I14490 Populations Selected for Race T4

J.ll.

Scott, S.F. Hutton, R. Shekasteband, S.C. Sim and D.M. Francis

Combinations

of

?'y Resistance Genes Generally Provide More Effective Control against Begomoviruses than Do Single Genes

S.F. Hutton, J.L/. Scou, R. Shekasteband,I. Levin and M. Lapidot

Screening Cultivated and

Wild

Tomato Germplasm for Resistance to

Xanthomonas gardneri

D. Liabeu.f, D.M. Francis and S.C. Sim

Selection for Resistanceto Sclerotium

rolfsii

in Accessions of Solanum

lycopersicum var. cerasiforne in Westcrn Mexico

J.L. Martinez-Ramirez, E.R. Guzman, C.M. Duran-Marlinez, J.V. Navaruo, E.L. Alcocer and G.C. Abascal

Interaction between Fusarium

Wilt

Resistance Genes in Tomato and

Fusarium orysporum f- sp. lycopersrci Races 1 and 2

M. Niederheitmann, J. Gonqalves, A. Mat, T. Bentley and

J.

Green

6

'7

l3

l5

23

33

39

41

53

59

65

7t

i

Gaafting Local Commercial Tomato Cultivars

with

H-7996 and Eg-203 to 9rypress Bacterial W

llt

(Ra I s t o n i a s o I anac e aru m) in Indonesia

T.

Antiyanto,

S.D. Nurcahyanti, D. Indradewa and

J.

ytridada

Management of Powdery

Mildew

in Greenhouse Tomato Production with Biorational Products and Fungicides

F- Balsal-Gurel and S.A. tr,Iiller

Preliminary Results on the Effect of Acibenzolar-S-Methyl on

Abiotic

Stresses in Tomato: "Blossom End Rot" Control

-1- Fanigliulo, O. Mancino, A. Crescenzi and P. Cestari

Control of Tomato

Yellow

Leaf Curl Disease in Tomato

-4. Fanigliulo,

A.

Viggiano, A. Crescenzi, E. Zingariello, R.-

Liguori

and R. Senn

.{,bility of Colored Mulches and Growtubes to Deter Thrips and Reduce

lncidence of Tomato Spotted

Wilt

F.G. Foshee

III,

T.A. Monday, M.J. Kellev, J.L E.K. Blythe, J.F. Murphy and J.E. Brown

M. Lollar.

Tolerance Response to Clavibacter michiganensls subsp. michiganensis in Tomato Plants Treated

with

Selenium

S. Gonzdlez-Morales, R. Castillo-Godina, A. Benavides-Mendoza and

LJ.

R[os-Gonzalez

Development of an Innovative

Multi-Level

Knowledge Based Tomato

Disease Factsheets

I{.L.

Lewis lvey ond S.A.

Miller

Evaluation of Disinfectants to Control Mechanical Transmission of Tomato Viruses and Viroids in Greenhouse Tomatoes

R. Li, S.A.

Miller,

F. Baysal-Gurel, K.-5. Ling and C.J. Kurowski

Effect of Aerobic Compost Tea Inputs and Application Methods on

Protecting Tomato from Phytophthora capsici R.ll'. Nicol and P. Burlakoti

Evaluating the Effect of GreenForce S and GreenForce CuCa Products on the Severity of Bacterial Spot and

Wilt,

and Powdery Mildew Diseases on

Tomato

M.L.V. Resende, L.R.R. Vitorino, P.fu(. Ribeiro

Jilnior,

J.A.G. da Silva,

II.H.L.

Renn6 and S.M. Mathioni

Copper Alternatives for Management of Bacterial Spot (Xanthomonas gardneri) and Bacterial Speck (.Pseudomonas syringae pv. tomalo) in

Processing Tomatoes

C.L. Tn":eman

A

Stimulo-Deterrent Method of Thrips and Tonittto Spotted

Wilt

Virus

Management in Tomatoes

K.A. Tyler-Julian, J.E. Funderburk, S.lt[. alson, M.L. Paret,

C.G. Webster and S. Adkins

t79

l8s

t9l

197

203

211

221

229

235

243

Grafting Local

Commercial Tomato

Cultivars with

H-7996 and

Eg-203

to

Suppress

Bacterial

Wilt

(,Ralstonia

solanacearam)

in

Indonesia

T. Arwivantor'u. S.D. Nurcahvantil. D. [ndradewa2 and J. Widada]

]Labo.dtoryof

nlarrrEi6i@

'

Department of Agronomy

'

Department of Agricultural Microbiology Department of Plant Protection

Faculty of Agriculture University of Gadjah Mada

Jl Flora

No

I

Bulaksumur Yogyakarta 55281

Indonesia

Keywords:

bacterial wilt, Rqlslonia solanacearum, grafring, disease control

Abstract

Bacterial

wilt of

tomato

(Ralstonia

solanqcearun)

causes

serious

losses

of

tomato production

in

Indonesia.

Currently, proper cultural practices are the

only

way performed

by

farmers

to

combat

the

disease.

While

this

practice could not

effectively prevent infection

of

R.

solanacearum

to

the plant,

efforts have

been

sought

to

help farmers keep

their

plants away

from

bacterial

wilt.

Grafting of

commercial

cultivars

with

available rootstocks is easy

to perform

and the

effectivity

of

this

method

in

suppressing

tomato

bacterial

wilt

has been reported

in

many

countries but not

in

Indonesia.

The

objective

of this study

was

to

find

out whether

grafting

susceptible

commercial cultivars of tomato

with

resistant

rootstocks could suppress the development of bacterial

wilt

in

Indonesia. The

grafting

was conducted

in Yogyakarta

regiono Indonesia,

in

2011

with

H-7996 and Eg-203 as rootstocks and

three commercial cultivars

of

tomato (tPermata',

'Lentana'. and

6Fortunat)

as

scions.

The

results indicated

that

the

rootstocks

could

afford the

existence

of

population of

pathogen

in

the stem tissue,

exhibit

lower disease index compared

with

mock

grafted and non-grafted plants. The

pathogen

could be

detected

in

the

stem tissue above

soil

surface

at

21

and

28 days

after inoculation

in

plants grafted

rvith H-7996,

but

it

was detected

in

non-grafted and

in

mock

grafted

plants

just in

7 days

after inoculation.

At

35 days

after inoculatio4 the

pathogen

population in the

ster4

tissue

(2 cm

above

ground; varied

from

103

(plairts

grafteil

ivith

ng-ZO:)

to

10e

(mock grafted plants and non-grafted plants)

cfu/S

mm

tissue.

The grafted

plants exhibited

lower

disease index,

better growth

and

yield

compared

with

those

of

mock

grafted

and

non-grafted plants.

The

result

suggests

that

grafting

commercial

cultivar

tomatoes

with

either

H-1996

or

Eg-203

could

suppress

bacterial growth,

bacterial

wilt

incidence, and enhance

fruit

production.

INTRODUCTION

Bacterial

wilt

of

tomato caused by Ralstonia solanacearum is

difficult to

control

due to genetic

variability of

the pathogen and lack

of

durable resistance

in

plants. Losses

of tomato production due to bacterial

wilt

varied from a few plants infected in the

field

to 100%.

In

Indonesia,

it

has been reported that the disease could devastate the crops and cause harvest

failure.

In

Chinese Taipei, the damage caused losses

cf

l5-55o/o, and in India l0-100% (Wang and

Lin,

2005). In South Carolina, the disease incidence was l50%

with

losses

from l-15%

(Elphinstone,2005). Control

ofthe

disease has been conducted primarily by cultural practices such as crop rotation, soil tillage, proper crop management,

and

seed treatment.

While

these practices

could not effectively

prevent

infection

of

R. solanacearum

to

the plant, efforts have been sought to help farmers keep their plants

" tarwiyanto@yahoo.com

Proc. IV'h IS on Tomato Diseases Eds.: M.L. Paret et al.

Acta Hort. 1069. ISHS 2015

-

Th;

_objective

of

_{the present study was}

to

_{evaluate bacterial w.ilt inci.<lence and}

fruit productioir of tomato influenced by grafting,. Local-commercial cultivars of.tomato in

fruit

oroduction of tomato influenced by grafting. Local commercial cultivars of tomato in Indonesia were grafted

with

H-7996 and Eg-203 and planted

in

the

field

inoculated with

highly virulence

of

R.

solanacearum.

The results indicated that the practice

could

sulpiess the development both of the pathogen and the disease'

MATERIALS AND METHODS

A

highly virulent

strain

of

R.

solanacearum

(strain

Te201

l,

rn9"

I

b_iovar 3,

phylotype

li

was

used as

a

challenge pathogen

lgr

the

experiment.

The

isolate wai it.6at 6d on the surface

of

Casamino-acid Peptone Glucose medium (Kelman, 1954) and

incubated for 48 h at room temperature before used. Rootstocks (H-7996 and Eg-203) and

scion

('Lentana', 'Fortuna',

'Permata') were separately sown

in a

sterile m.edium (top

soil:compost:rice-hull:sand

:

2:3:l:l).

The grafting was

conducted according_

to

the

method developed by Black et al. (2003). The grafted plants were transplanted to the field

at

30

days after grafting and inoculated

by

pouring r,r'ith

a

water

suspension

of

R. solanacearum into the stem base (50 ml/plant,

l0'

cfu/ml).

The

experiment

was

arranged

as

a

randomized complete

block

with

three

replications. Eiperimental plots consisted

of

a double

row of

5 m long and 1.4 m width,

with

between

row

spacing

of

0.8 m, 0.4 m between plant, and the distance between plot was

0.5 m The

plants were transplanted

in

each

block

as one treatment consisting

of

24 plants; 12 plants

for

disease index observation and 12 plants

for

destructive samples

for

determination

of

rhizosphere microbes. The dynamic population

of

R. solanacearum

in

tap root

of

tomato

was

determined

every week

by

plating

the

phosphate buffer suspension

of

crushed cutting-root

in

Casamino-acid Peptone Glucose medium. The disease index (Arwiyanto et al.. 1994) was observed every week for 5 weeks.

RESULTS

AND

DISCUSSION

'Permata' tomato is the most tolerant cultivar against R. solanacearum compared

with

other cultivars tested.

In

the plant grafted

with

H-7996 (HP), R. solanacearum was

detected

at 28

days after inoculation

while

in

the mock grafting (grafted against itself,

B])-,

lt

was

dgtected

at

7

days

after

inoculation as

well

as for

non-grafted

plant

(P)

(Table

l).

At

35 days after inoculation however, the population of the pathogen increased regardless the treatments. The pattern

of

population dynamics of R. solanacearum

in

the

stem tissue

of

'Lentana' and 'Fortuna' was similar

with

that

in

'Permata'

(data not

shown). The grafted plants delayed the

initial

development

of

the pathogen

in

their stem

tissue comoared

with

-those

of

mock grafting and

non-grafted plants.

The

pattern

of

population dynamics

of

the pathogen in the stem tissue

of

tomato plants grafted

with

Eg-203 was quite similar compared

to

that

of

grafted

with

H-7996. The distinct difference was

that

the development

of

the disease

in the

plants grafted

wirh

Eg-203 was much

slower than that in the plants grafted

with

H-7996 (Table

l)

may be Eg-203 more tolerant

to bacterial

wilt.

Disease symptoms in grafted plants were observed starting 7 days after inoculation

until

105 days after inoculation. The first symptom was delayed considerably in the plants

grafted

with

H-7996, i.e., at 56 days after inoculation

while in

the non-grafted planls the

lrst

symptom was observed

only

at

2l

days

after

inoculation

(Fig.

l).

Devetopment

of

disease

in

non-grafted plants occurred rigorously and almost

all

plants

wilted ai

63 days

after inoculation. The disease development

in

the plants grafted

with

H-7996 was muih slower compared

with

that

of

non-grafted plants.

In

contrast, the disease Cevelopment in

the tomato.plants grafted

with

_{Fg-203 was} very

low

indicating that Eg-203 has-a higher level ofresistance against R. solqnacearum

(Fig.2).

The number of

fruit

and

fruit

weight produced by plants grafted rvith H-7996 were

1',74

away

from

bacterial

wilt. Grafting

!f

commercial cultivars

with

available rootstocks _is

easy to perform and ihe

effectivifl

of this method in ing tomato bacterial

wilt

has

easv to perlbrm and ihe

ellcctlvlfy oI

tnls metno0 ln suppresslng wmato Daclerlal

wllt

has

been reported

in

many countries (Burleigh et al.,20051 Fresco,2001; Frank et a1.,2010)

but grafting has not been practiced in Indonesia'

greater than

mock

grafting plants. When the plants were

not

grafted, the results varied

irith

different

cultivars

of

tomatoes tested. Tomato

cultivar

'Permata' performed better compared

with

other cultivars

in

term

of

disease suppression;

fruit

weight,

and

fruit

number produced when grafted

with

either H-7996 or Eg-203. However, compared

with

plants grafted

with

Eg-203, there was a significant difference both

in

the number

of fruit

froduced and

in

the weight

of

tomatoes harvested. Tomato

cultivar

'Permata' produced

more

fruit

when grafted

with

Eg-203 followed

by

'Lentana' then 'Fortuna' whereas mock

grafted and

non-grafted

plants

produced

less number

of

fruit.

However,

'Fortuna' froduced more

fruit

when grafted

with

H-7996 compared with those grafted

with

Eg-203. Tomato

cultivar 'Foftuna'

grafted

with

H-7996 produced

fruit with

highest fresh weight,

followed

by

'Lentana' then

'Permata'. However, when the tomato plants were grafted

with Eg-203, 'Permata'

produced more

weight

compared

with

other

tomato cultivars (Figs. 3 and 4).

These results

confirm

that grafting tomato plants

with

either H-7996

or

Eg-203

suppressed

the growth

of

Ralstonia solanaceqrum; bacterial

wilt

of

tomato

was

less dev-elop

in

the field

when the plants were grafted

with

either H-7996

or

Eg-203; and

tomato production was enhanced

by grafting

of

local

commercial cultivars

with

either H-7996 or Eg-203.

ACKNOWLEDGEMENTS

The work was supported by the Directorate General of Higher Education,

Ministry

of

Education

and

Culture

Republic

of

Indonesia;

grant

No.

18O/SP2H/PL/ Dit. litabmas/lY I 20 12, dated Apri I 30, 20 1 2.

Literature Cited

Arwiyanto,

T.,

Goto,

M.

and Tsuyumu,

S.

1994,

Biological control of

bacterial

wilt of

tomato

by

an avirulent strain

of

Pseudomonas solanqcearum. Ann. Phytopathol. Soc. Japan 60:421-430.

Black,

L.L..

Wu, D.L.. Wanq. J.F.. Kalb. T.. Abbas, D. and Chen. J.H. 2003. International Cooperators

Guide. Grafting

Tomatoes

for

Production

in

the

Hot-Wet

Season.

AVRDC

pub #03-551.

Burleigh, J.R,

Black, L.L.,

Mateo,

L.G.,

Cacho,

D.,

Aganon, C.P., Boncato,

T.,

Arida,

I.A.,

Ulrichs, C. and Ledesma, D.R. 2005. Performance

of

grafted tomato

in

Central

Luzon,

Philippines:

a

case study

on the

introduction

of

a new

technology among

resource-limited farmers. Crop Management.

Elphinstone, J.G. 2005. The current bacterial

wilt

sifuation: a giobal overview. p.9-28. In: C.

Allen,

P. Prior and

A.C.

Hayward (eds.), Bacterial

Wilt

Disease and The Ralstonia

solanacearum

Species

Complex.

APS

Press.

The

American

Phytopathological Society. St. Paul. Minnesota, USA.

Fresco,

M.C.O.

2001.

Grafting

effective

in

producing off-season tomato.

BAR

Today

3(2).

Kelman,

A.

1954. The relationship

of

pathogenicity

in

Pseudomonas solanacearum to colony appearance ou a tetrazolium uediurn. Phytopathology 44:693-695.

Louws, F.J., Rivard,

C.L.

and Kubota, C. 2010. Grafting

fruiting

vegetables

to

manage

soilborne pathogens,

foliar

pathogens, arthropods and

weeds

Scientia Horticulturae

127:127 -146.

Wang, J.F. and Chih-Hung

Lin.

2005. Integrated Management

of

Tomato bacterial

wilt.

AVRDC.

Tables

Table

1.

Colonization

of

tap

root by R.

solanacearum

on

grafted and

non-grafted 'Permata' tomato plants. Values

followed by

the same number

in

the same column were not significantly different according Duncan

Multiple

Range Test'

Distance Rootstock

{mml

Log population at days after inoculation

l4

21 28 35

Grafted

with

H-7996 5

l0

15 20

3.60a

6.31a

2.95b

5.68a

2.00c

5.15a

2.00c

5.00a

Grafted

with

Eg-203 3.26a 2.00c 2.00c 2.00c 4.08b 3.80b 3.60bc 3.24c 5

l0

l5

20 Non-grafted 2.00a 2.00a 2.00a 7.00a 5

l0

l5

20

3.65a

6.48a

3.24ab

6.30a

3.07b

5.26b

2.44c

5.21b

7.26d

9.30d

7.18d

9.20d

7.00d

8.92de

6.00e

8.17e

- not detected

Figures

'&pe4

i __

j:v':I1'"1""!!91

Fig.

l.

Bacterial

wilt

development on tomato grafted

with

H-7996

"i'

176

[image:11.595.206.532.133.310.2]

100.00

90.00 80.00

x70.00

€oo.oo c ?so.oo_o

E+o.oo

o

i530.oo

20.00

10.00 0.00

I I

I

I Days after inoculation

Fig. 2. Bacterial

wilt

development on tomato grafted

with

Eg-203.

Fig. 3. Production of tomato from grafted and non-grafted plants in a plot.

177

lGrafted with EG-2O3

trGrafted with H-7996

---EMock grafting

sl Non grafted

[image:12.595.62.405.65.275.2] [image:12.595.61.406.353.558.2]

rGnft"d wilh H-7996

oGnfted wilh Eg-203

Eitck Grafting

rNon Gnfbd

Pemata Lentana

Fig. 4. Number of tomato fruit produced by grafted and non-grafted plants in a plot.

800 700 6@

!

soo

g

t

40o

o !

5 ,oo

z

200

100

0

[image:13.595.240.494.118.300.2]