Download - 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
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
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 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
16 isolates PDA medium Seven days at 23oC ANOVA, software Statistika
Growth rate of the isolates Monilinia spp.
M. fructigena M. fructicola
M. laxa
M. fructigena M. fructicola
M. laxa
Growth rate at different temperatures
Temperatures
16 isolates PDA 18-30oC
Seven days ANOVA, software Statistika
M. fructigena M. fructicola
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. fructigena M. fructicola
M. laxa
Growth rate on different media
M. fructigena M. fructicola
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. fructigena M. fructicola
M. laxa
Sporulation
Stenley
Mildora
Control
Artificial inoculation
Wounded mature plum fruits
Čačanska rodna
Čačanska lepotica
Isolates M. laxa M. fructigena M. fructicola
Sporulation
Artificial inoculation
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
10
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. fructigena M. fructicola
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. fructigena M. fructicola
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. fructigena M. fructicola
M. laxa
Monilinia fructicola: Sensitivity of NPGM isolate on iprodione
M. fructigena M. fructicola
M. laxa
M. fructigena M. fructicola
M. laxa
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
Tasks Tasks
4
3
1
2
To establish presence and distribution of Monilinia spp. on stone fruits in Serbia
To complete identification procedure to the species level.
To analyze potential ways of introduction and spreading of M. fructicola
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:
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
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