m. fructicola

Моnilinia fructicola – a potential threat for sweet and sour cherry production in

Serbia

Brankica Tanović and Jovana Hrustić

Institute of Pesticides and Environmental Protection Belgrade, Serbia

[email protected]

30% fresh or frozen

70% processed

Stone fruit production

0

5

10

15

20

25

30

35

40

45

2000. godina 2011. godina

Mill

ion

tons

0 5 10 15 20 25

Breskva

Šljiva

Kajsija

Trešnja

Višnja

Million tons *FAOSTAT, 2011 *FAOSTAT, 2011

Stone fruit production in the world

2010 2011

Peach

Plum

Apricot

Sweet cherry

Sour cherry

Stone fruits 61%

Nuts 2%

Pome fruits 27%

Berry fruits 11%

Stone fruit production in Serbia

Breskva

Šljiva

Kajsija

Trešnja

Višnja

0 100000 200000 300000 400000 500000 600000

75.233

58.1874

32.656

28.557

90.596

Plum

Apricot

Sweet cherry

Sour cherry

Peach

Blumeriella jaapii, Glomerella cingulata, Monilinia spp., Stigmina carpophila, Apiognomonia erythrostoma, Armillaria mellea, Pythium spp., Rhizopus stolonifer, Chondrostereum purpureum, Tranzschelia pruni-spinosae, Verticillium spp.

Monilinia laxa Blumeriella jaapii Apiognomonia erythrostoma

Glomerella cingulata

Disease symptoms

Stigmina carpophila

http://www.google.rs/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&docid=THF-4zo9eOZxSM&tbnid=-286IaSVUN40BM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.forst.tu-muenchen.de%2FEXT%2FLST%2FBOTAN%2FLEHRE%2FPATHO%2FPRUNUS%2Fapioeryt.htm&ei=6YtwU_CWJoi0PIu2gPAP&bvm=bv.66330100,d.d2k&psig=AFQjCNEWa9SPPTUZYd1D3JYwjzVH31N7BQ&ust=1399969635738730

Monilinia spp.

Monilinia spp. – causal agents of blossom and twig blight and fruit rot of stone fruits

Worldwide distribution in all major fruit growing areas

Cause one of the most important pre- and postharvest fungal diseases of stone fruits

Three species of this genus are economically important

pome fruits stone fruits fruit rot blossom, twig and branch blight plum

economically the most important blossom blight twig and branch blight fruit rot stone fruits pome fruits apricot cherry peach sour cherry plum

the most destructive species of the genus Monilinia fruit rot including latent infection peach and nectarine

Monilinia laxa

Monilinia fructigena

Monilinia fructicola

Distribution of Monilinia spp. in the world

M. fructigena M. fructicola

M. laxa

M. laxa M. fructigena M. fructicola

Distribution of Monilinia fructicola in Europe

detected not known absence confirmed

Separation of species of the genus Monilinia

Morphological and cultural characteristics

Symptoms

Molecular identification

1

2

3

Fungicides registered:

Monilinia spp. control

• Breeding for resistance, • Cultural practice, • Sanitation, • Biological control, • Chemical control.

azoxistrobin, bitertalon, ciram, dichlofluanid, dikoran, dithianon, fenhexamid, fludioxonil, fluopyram, fluoroimid, imibenconazole, captan, mepanipyrim, oksokonazol fumarate, propioneb, triadimefon, triflumizole, thiram, triforine

Tasks

1

2

3

4

To establish presence and distribution of Monilinia spp. on stone fruits in Serbia

To complete identification procedure to the species level

To determine fitness components of the isolates of all found species of the genus Monilinia in order to assess the risk of spreading of quarantine pathogen M. fructicola in stone fruit orchards:

growth rate at different temperatures temperature range of the growth growth rate at different media pH range of the growth sporulation virulence sensitivity to fungicides

To analyze potential ways of introduction and spreading of M. fructicola

Sampling

2010 - 2013

119 locations

15 regions

6 hosts

362 samples

Samples

Origin of the isolates Number of isolates mature fruit 232 mummified fruit 45 twigs 11 total 288

Isolation and pathogenicity test

Artificial inoculation

wounded detached fruits unwounded detached fruits

Isolation

1

2

3

Identification

•Pathogenic characteristics

•Sequencing of the ITS rDNA region

•Morphological, ecological and cultural characteristics

Key for identification of Monilinia spp.: colony colour colony margin rosetting sporulation presence of sporulation in concentric rings presence of black arcs quantitative growth rate

shape and size of the conidia germ tube length and morphology

Morphological characteristics

Macroscopic

Microscopic

- light to dark gray - lobed margins - absent sporulation - absent concentric rings of spores

- creamy to yellow - entire margins - rare sporulation - concentric rings of spores - black arcs

- hazel zonate - even margins - abundant sporulation - concentric rings of spores

I group II group III group C

olon

y ch

arac

teri

stic

s 257 isolates 8 isolates 23 isolates

11.2-15.4 μm x 8.2-11.1 μm 19.2-23.5 μm x 11.9-13.5 μm 16.0-16.9 μm x 9.9-10.7 μm

40 µm 197 µm 365 µm

Con

idia

cha

ract

eris

tics

I group II group III group

I group II group III group 257 isolates 8 isolates 23 isolates Id

entif

icat

ion

Population structure 2010-2013

2011

one isolate M. fructicola on green market nectarine

2012

(217 samples) two isolates M. fructicola on green market

no isolates in the field nectarine

2013

20 isolates M. fructicola in three independent orchards

nectarine, peach, apricot, plum

Tasks

1

2

3

4


To complete identification procedure to the species level.




16 isolates PDA medium Seven days at 23oC ANOVA, software Statistika

Growth rate of the isolates Monilinia spp.


M. laxa


M. laxa

Growth rate at different temperatures

Temperatures

16 isolates PDA 18-30oC

Seven days ANOVA, software Statistika


M. laxa

Growth rate at extreme temperatures

Temperatures 16 isolates PDA Temperatures 0-4oC Temperatures 31-35oC

Seven days ANOVA, software Statistika

0

5

10

15

20

25

30

35

40

45

31°C 32°C 33°C 34°C 35°C

Gro

wth

rat

e (m

m/7

days

)

TPGRŠMLEsŠPSVTPBNŠMLEVGRSDBPSVBMLEsTPALVGRSEVPBGTPGOŠPPRNPGMNPUD1NPUD2

-2

0

2

4

6

8

10

12

14

0°C 1°C 2°C 3°C 4°C

Gro

wth

rat

e (m

m/7

days

) TPGRŠMLEsŠPSVTPBNŠMLEVGRSDBPSVBMLEsTPALVGRSEVPBGTPGOŠPPRNPGMNPUD1NPUD2

Potato-dextrose agar – PDA Water agar - WA Czapek agar – CzA Malt agar - MA Vegetable-8 juice agar - V8A

12 isolates Seven days ANOVA, software Statistika

Media


M. laxa

Growth rate on different media


M. laxa

Growth rate on different pH

12 isolates PDA pH 2 -pH 12

Seven days at 24°C ANOVA, software Statistika

Sporulation

11,2-15,4 μm x 8,2-11,1 μm 19,2-23,5 μm x 11,9-13,5 μm 16,0-16,9 μm x 9,9-10,7 μm

40 µm 197 µm 365 µm

0.29 x 104/cm2 1.54 x 104/cm2 2.7 x 104/cm2

Conidia production


M. laxa

Sporulation

Stenley

Mildora

Control


Wounded mature plum fruits

Čačanska rodna

Čačanska lepotica

Isolates M. laxa M. fructigena M. fructicola

Sporulation


Stenley

Mildora

Control Wounded immature plum fruits

Čačanska rodna

Čačanska lepotica

Isolates M. laxa M. fructigena M. fructicola

M. laxa

Virulence test

Nine isolates: four isolates M. laxa four isolates M. fructigena one isolate M. fructicola Artificial inoculation of wounded mature stone fruits: peach, apricot, nectarine, plum, sweet cherry, sour cherry Total: 15 fruits/species/isolate Incubation in moisture chamber at 24oC ANOVA, software Statistika

0

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20

30

40

50

60

Nectarine Peach Plum Apricot

lesi

on e

xpan

sion

rat

e (m

m/3

day

s)

TPGR

ŠPSV

VGRSE

BMLEs

TPGO

ŠPPR

VPBG

BŠPBA

NPGM

Virulence


M. laxa

Virulence

0

5

10

15

20

25

Sweet cherry Sour cherry

lesi

on e

xpan

sion

rat

e (m

m/2

day

s)

BMLEsVPBGNPGM


M. laxa

Product Company Active substance Chemical group A.S.

content Concentration range

(mg/l)

Bravo 720 SC Syngenta Chlorothalonil Chloronitrile 720 g/l 2; 1; 0,5; 0,25; 0,125 i 0,06

Kidan EC Bayer CropScience Iprodione Dicarboximides 255 g/kg 10; 5; 2,5; 1,25; 0,6; 0,5; 0,3;

0,25; 0,125; 0,06 i 0,03

Spartak 450-EC Sinochem Ningbo Prochloraz Imidazoles 500 g/kg 0,05; 0,025; 0,0125; 0,006 i

0,003

Quadris Syngenta Azoxystrobin Strobilurins 250 g/kg 100; 25; 10; 5; 1; 0,1; 0,01; 0,001

Akord Galenika Fitofarmacija Tebuconazole Triazole 250 g/kg 0,5; 0,25; 0,125; 0,06; 0,05;

0,03; 0,025; 0,0125 i 0,006

Cantus BASF Boscalid Carboxamide 500 g/kg 2000; 1500; 1000; 750; 500; 0,1; 0,05; 0,025; 0,0125 i 0,006

Luna Privilege Bayer CropScience

Fluopyram Pyridinyl-ethyl-benzamides 500 g/l 1000; 500; 250; 125; 62,5; 2; 1;

0,5; 0,25; 0,125 i 0,06

Sensitivity of Monilinia spp. isolates to fungicides from different chemical groups

7 fungicides

12 isolates

Parameters: EC-50 (mg/l) b (slope of regression line)

Probit analysis

Sensitivity of Monilinia spp. isolates to fungicides from different chemical groups

Active substance EC50 (mg/l)

Chlorothalonil 0.12-1.06

Iprodione 0.13-3.32

Prochloraz 0.006-0.023

Azoxystrobin 0.51-8.83

Tebuconazole 0.01-0.06

Boscalid 0.04-0.09 250 464

Fluopyram 0.11-10.78 5612 386


M. laxa

Monilinia fructicola: Sensitivity of NPGM isolate on iprodione


M. laxa


M. laxa



Tasks Tasks

4

3

1

2


To complete identification procedure to the species level.


Tasks Tasks Tasks

4

3 To determine fitness components of the isolates of all found species of the genus Monilinia in order to assess the risk of spreading of quarantine pathogen M. fructicola in stone fruit orchards:



Protocol: 95oC / 15 min

94oC / 40 s 50oC / 90 s 72oC / 90 s

60oC / 30 min

35 cycles

Termocycler (SensoQuest) ABI PRISM®3100 DNK capillary sequencer

(Applied Biosystems)

SSR marker analysis of M. fructicola isolates from Serbia

Three isolates M. fructicola originating from Serbia

Multiplex PCR kit

Isolate CHMFc5 CHMFc1 CHMFc4 CHML5 CHMFc12 Haplotype according to Jansch et al. (2012)

NPUD1 86 bp 173 bp 216 bp 220 bp 160 bp US50

NPUD2 86 bp 173 bp 216 bp 220 bp 160 bp US50

NPGM 86 bp 173 bp 223 bp 220 bp 160 bp ITA01

Allele (in bp) composition of M. fructicola haplotypes found in Serbian samples

SSR marker analysis of M. fructicola isolates

Belgrade

Vojvodina

Šumadija and Western Serbia

Southern and Eastern Serbia

Kosovo and Metohija

0

20000

40000

60000

Belgrade Vojvodina Sumadija andWestern

Serbia

Southern andEasternSerbia

Kosovo andMetohija

Prod

uctio

n ar

ea (h

a)

plums peach/nectarine apricots sour cherries

Projects: III 46008 COST FA 1104

m. fructicola

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