novel insights into the life cycle and epidemiology of
TRANSCRIPT
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Alice B. Eseola, Annette Pfordt, Xiaorong Zheng, Antonia Wilch, Birger Koopmann, Daniel Lopisso,
Andreas von Tiedemann
Plant Pathology and Plant Protection Division, Department of Crop Sciences, Faculty of Agriculture,
Georg-August University of Göttingen, Germany
Novel insights into the life cycle and
epidemiology of Verticillium stem striping
1
GCIRC Technical Meeting, Malmö, Sweden, May 8t – 11th 2017
Novel insights into the life cycle and epidemiology of Verticillium stem striping
Alice Biseola Eseola, Annette Pfordt, Xiaorong Zheng, Antonia Wilch, Sarah Bartsch, Birger Koopmann, Daniel
Lopisso & Andreas von Tiedemann
Plant Pathology and Plant Protection Division, Department of Crop Sciences, Faculty of Agriculture, Georg-August
University of Göttingen, Germany
Verticillium longisporum (VL) is a soilborne vascular pathogen which causes stem striping in European and
Canadian oilseed rape (OSR) and may induce significant yield losses. The epidemiological life cycle of VL is
still not entirely understood, particularly referring to the time course of infection in the field and the transmission
of the pathogen into the following rapeseed crop. We conducted field experiments with microsclerotia inoculated
plots and assayed the colonization of VL during the season in spring and winter sown OSR cultivars contrasting
in resistance to VL. Besides, soil temperature was varied in a miniplot soil heating experiment by +1.6 and
+3.2°C above ambient. Enhancement of infection at increased soil temperature was paralleled with the
accelerated colonization of spring type vs. winter type oilseed rape in the field, the earlier growing into the warm
and the latter into the cold season. A climate chamber study revealed a minimum required soil temperature for
stem invasion of 12°C in a susceptible and of 15°C in a moderately resistant interaction. The extended latency of
VL infection and invasion of winter oilseed rape thus appears to be due to soil temperatures below 12-15°C in
the early crop stages during autumn and winter. However, the further systemic spread of VL in the plant appears
independent from environmental factors and mainly governed by the nutritional conditions in the xylem. In
artificially inoculated winter OSR in the greenhouse, VL was transferred into the seeds to a higher rate in
susceptible than in resistant cultivars. This was demonstrated on surface sterilized seeds using 3% sodium
hypochlorite and Ds-RED labeled transgenic strains of VL. Similar results were obtained with spring OSR.
These results obtained in artificial conditions suggest VL to be seed transmissible, however, the transmission of
the disease from seeds in the field awaits further clarification. As a further pathway into the next season, we
identified transmission by colonization of alternative hosts used as cover crops.
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
First report of Verticillium in oilseed rape, Sweden 1969: Kroeker, G. 1970. Vissnesjuka på rabs och rybs i Skåne orsakad av
Verticillium. In English: Verticillium on oilseed rape and turnip rape in
Scania caused by Verticillium. Svensk Frötidning, 19, 10-13.
Global spread of Verticillium longisporum
1969
1988
2014
1957
First report on V. longisporum (supposedly),
Brussels sprout, England, 1957: Isaac, I. 1957 Verticillium wilt of Brussels sprout.
Ann. Appl. Biol. 45, 276-283.
Spring OSR
Winter OSR
2015
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
3
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
‘Verticillium stem striping’
4 Microsclerotia
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
5 MPP, 17(7), 1004-16, 2016
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
6
V. longisporum – the knowledge gaps
Depotter et al., MPP, 17(7), 1004-16, 2016
? Survival
in soil?
? Signals
guiding root
infection?
? Timepoint
of infection?
? Seed
transmission?
? Alternative
hosts?
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
-0,5
0
0,5
1
1,5
2
2,5
3
3,5
VL
DN
A [
ng
/g D
M]
Falcon Root
Falcon Hypocotyl
Falcon Stem
SEM Root
SEM Hypocotyl
SEM Stem
Date
7
Progress of infection - winter oilseed rape
GS 80
GS 63
GS 55
GS 32
GS 18 GS 14
+/-Std.Dev.
Sowing date:
27 August 2015
- 15 g Inoculum/m2
- 5 plants/plot x 4 replicates
- qPCR, ß-tubulin primer
10,3°C 4,9°C 9,8°C
11,0°C
18,7°C
15,9°C
Soil temperature
No hypocotyl infection!
Proliferation
after flowering
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
8
Effects of temperature on VL infection (climate chamber exp.; 45 dpi)
0
200
400
600
800
1000
1200
1400
6°C 9°C 12°C 15°C 18°C
VL D
NA
[p
g/g
DW
]
FC FVL VC VVL
Inoculation with 20 mg microslerotia per kg soil; F = Falcon (WOSR), V = Visum (SOSR)
Msc thesis Annette Pfordt, 2016
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
0
2
4
6
8
10
12
14
16
18
VL
DN
A [
ng
/g D
M]
Visum Root
Visum Hypocotyl
Visum Stem
OP-DLE 7 Root
OP-DLE 7 Hypocotyl
OP-DLE 7 Stem
Date
- 15 g Inoculum/m2
- 5 plants/plot x 4 replicates
- qPCR, ß-tubulin primer
9
GS 80
GS 65
GS 60
GS 51
GS 14
Sowing date:
7 April 2016
+/-Std.Dev.
13,1°C 18,4°C 21,6°C
18,9°C
19,2°C
Soil temperature
Hypocotyl infection!
Proliferation
after flowering
Progress of infection – spring oilseed rape
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Sowing time
early late
Net
OD
(405 n
m)
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
Stem
Root
Sowing time
LSD p <= 0.05
Stem 2.2
Root 1.5
b
b
a
a
PhD thesis Harald Keunecke, 2009
10
Stem/root colonization with VL (ELISA) Field trial 2006, GS 87, cv. ‚Oase‘, Weende, 15 g inoculum/m2
Early: 12. AUG 2005
Late 25. AUG 2005
?
Cabbage fly
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
AugSepOctNovDecJanFebMarAprMayJunJul
Me
an
da
ily t
em
p.
[°C
]
-5
0
5
10
15
20
25
Aug Sep O
ctNov Dec Ja
nFe
bMar
Apr
May Ju
n Jul
Me
an
da
ily t
em
p.
[°C
]
-5
0
5
10
15
20
25
longterm mean
Dis
ea
se
in
cid
en
ce
[%
]0
20
40
60
80
100
b
a
Laser Lion
Dis
ea
se
in
cid
en
ce
[%
]
0
20
40
60
80
100
GD LSD p <= 0.05
b
a
Me
an
yie
ld [
dt/
ha
]
0
10
20
30
40
50
60
Control
VL-inoculated
(15 g/m2)
aa
Laser
Control VL Control VL
Me
an
yie
ld [
dt/
ha
]
0
10
20
30
40
50b
a
Lion
a
GD LSD p <= 0.05
Laser Lion
-21%
2005/06
2006/07
Impact of winter temperature & cultivar
ØNov-Mar 1.3°C
ØNov-Mar 5.8°C
Ø2005/06 51.2 dt/ha
Ø2006/07 35.3 dt/ha
11
PhD thesis Harald Keunecke, 2009
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
-0,5
0
0,5
1
1,5
2
2,5
3
3,5
VL
DN
A [
ng
/g D
M]
Falcon Root
Falcon Hypocotyl
Falcon Stem
SEM Root
SEM Hypocotyl
SEM Stem
Date
12
Progress of infection - winter oilseed rape
GS 80
GS 63
GS 55
GS 32
GS 18 GS 14
+/-Std.Dev.
Sowing date:
27 August 2015
- 15 g Inoculum/m2
- 5 plants/plot x 4 replicates
- qPCR, ß-tubulin primer
Proliferation
after flowering
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Growth of Verticillium longisporum in Brassica
napus xylem sap is independent from cultivar
resistance but promoted by plant ageing
Daniel T. Lopisso, Jessica Knüfer, Birger Koopmann,
Andreas von Tiedemann
(submitted to Phytopathology)
VL growth in xylem sap
Carbohydrates in xylem vs. plant age
FM14 FM18 FM21 FM28 FVL14 FVL18 FVL21 FVL28 AM14 AM18 AM21 AM28 AVL14 AVL18 AVL21 AVL28
0
20
40
60
80
100
120
Xyl
em s
ap t
ota
l CH
O c
on
ten
t (μg/ml)
Mean Mean±SE Mean±SD
def cdef f
a
cde
cde
ef
a
bcd cde
bcd
ab
cde cde
bc
ab
14 18 21 28 14 18 21 28 14 18 21 28 14 18 21 28
Xylem sap sample collection time points (days post inoculation)
Falcon Aviso
Mock VL43 Mock VL43
Xyle
m s
ap t
ota
l CH
O (
ug
/ml)
y = 0.002x + 0.218 r = 0.58 (P=0.048)
0,0
0,1
0,2
0,3
0,4
0,5
0 20 40 60 80 100F
un
ga
l g
row
th 5
DA
I (O
D 5
80
)
Xylem sap CHO (ug/ml)
Fungal growth vs. CHO
Fungal growth vs. plant age
0,0
0,1
0,2
0,3
0,4
0,5
10 15 20 25 30
Fu
ng
al g
row
th (
OD
58
0)
Plant age (DPI)
Plant age vs fungal growth at 5 DAI
Plant age vs daily fungal growth rate
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
14
0
5
10
15
20
25
30
35
40
Kontrol VL Kontrol VL
Falcon SEM
Yie
ld [
dt/
ha]
a
ab
b b
Oleic
C18:1
[% of total fatty acids]
Linolenic
C18:3
[% of total fatty acids]
Erucic
C22:1
[% of total fatty acids]
Glucosinolates
[µmol/g TM]
Falcon Mock 60,33 10,54 <2 10,82
VL 60,00 10,60 <2 10,95
SEM Mock 63,02 10,32 <2 9,28
VL 63,16 10,35 <2 9,19
41
41,5
42
42,5
43
43,5
44
Kontrol VL Kontrol VL
Falcon SEM
Seed
oil
co
nte
nt
[%] a a
b b
-15,1%
Yield & quality – winter OSR
Field exp. Gö-Weende 2015/16
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Yield & quality, spring OSR
Spring oilseed rape field exp. Weende 2016 destroyed by pollen beetle
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
16
Role of cover crops in VL propagation (greenhouse & miniplot field exps., 2016)
Bachelor thesis Sarah Bartsch, 2016
n. a. – not analysed in the field
Cover crop % plants
with MS
(greenhouse)
MS
detected on
field-grown
plants
Mustard (Sinapis arvense) 100 na
Oil radish (Raphanus sativus) 100 +
Turnip rape (Brassica rapa) 100 na
Tansy phacelia (Phacelia tanacetifolia) 72 +
Winter rye (Secale cereale) 0 na
Italian clover (Trifolium incarnatum) 0 na
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Seeds from greenhouse inoculated plants
DsRed labelled strains of
VL detected on seeds.
0 5 10 15
Visby
Treffer
Falcon
Mock
VL DNA (ng/g pod)
Cu
ltiv
ars
0 5 10 15
Visby
Treffer
Falcon
Mock
VL DNA (ng/g seed)
Cu
ltiv
ars
VL DNA in pods & seeds
Msc thesis Alice Bisola Eseola, 2016
b
a
ab
b
0
5
10
15
20
Mock Falcon Visby Treffer
% Frequency of VL infested seeds
s ms mr
Mock
Visby Treffer
Falcon
Re-isolation of VL from seeds
harvested from inoculated plants.
Mock VL
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Summary
Phacelia, mustard, oil radish and turnip rape are alternative hosts of
VL. Pathotype selection?
‘Verticillium stem striping’ is a growing threat to global oilseed rape
production.
Early root/plant colonization apparently determined by soil
temperature (threshold 10-12°C).
18
Seed transmission proven on greenhouse inoculated plants.
Seed transmission in the field awaits confirmation.
Nutritional changes in the xylem sap may determine subsequent post-
anthesis proliferation of VL into the shoot.
Extended latency in colonization of winter OSR, early spread into
roots/hypocotyls in spring OSR.
Significant yield losses occur at/above 60% DI: 15-20% (WOSR).
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Sarah Christina
Dunker Eynck
Birger
Koopmann
Avinash
Kamble
Jessica
Knüfer
Jutta
Schaper Evelin Vorbeck
Antonia
Wilch
Xiaorong
Zheng
The Verticillium group (2005-2017)
Nadine
Riediger Daniel
Lopisso
PIs, Postdocs
PhD students
Lab technicians
Maggie
Siebold
Bachelor- und Master students Eiko Tjaden, Fluture Novakazi, Sarah Bartsch,
Alice B. Eseola
Harald
Keunecke
19
Isabel
Müller
Annette
Pfordt
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Acknowledgements
20
Cooperation partners
Petr Karlovsky, Univ Göttingen
Heiko Becker & Christian Möllers, Univ Göttingen
Christian Obermeier, Rod Snowdon & Wolfgang Friedt, Univ of Giessen
Patrik Inderbitzin & Krishna Subbarao, UC Davis, USA
Avinash Kamble, University of Pune, India
Funding sources
DFG – German Research Foundation
GFPi – German Association for the Promotion of Plant Innovation
BMEL/BLE Federal Ministry of Food and Agriculture
BMBF Federal Ministry of Education & Research
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
21
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
22 Depotter et al., MPP, 2016
Line-
age
Hosts Countries
A1/D1 OSR, cauliflower,
cabbage, birdrape,
sugar beet
France, Germany,
Sweden, Japan, USA
(only cauliflower);
Canada (OSR)
A1/D2 Horseradish; OSR Illinois, USA,
Sweden, Germany
A1/D3 OSR, cabbage,
horseradish
Germany, Japan,
France
VL lineages = pathotypes?
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
VL lineages - host range
Non-Brassicaceae Brassicaceae
Novakazi et al., Phytopathology 105: 662-673, 2015 23
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Impact of soil temperature
Siebold & Tiedemann (2013) Global Change Biology, 19: 1736-1747.
Heros: r² = 0.45
SEM: r² = 0.77
Falcon: r² = 0.67
Göttingen Soil
Heating Experiment
ambient
+1.6°C
+3.2°C
24
Department of Crop Sciences, Plant Pathology & Crop Protection Division
Verticillium tricorpus 1808
Source: Olive mill waste (OMW) compost/California, USA.
MoA: Unknown yet.
Master thesis project Dima Alnajar, 2016
Vt1808
Verticillium isaaci (Vt305)
Vt305
Source: suppressive soil in cauliflower field in Belgium.
MoA: competition for infection sites and ISR? Tryptophan-derived
metabolites produced by the infected roots (root defence)? (Tyvaert et al.
2014)
Piriformospora indica Order: Sebacinales
Source: Indian Thar desert
Mechanism: Resistance induction: (Lahrmann et al., 2015)
Plant growth promotion: (Lahrmann et al., 2012)
Rhizobium radiobacter F4 Source: Endofungal bacterium in P. indica
MoA: Jasmonate-based ISR (similar to Pi). (Glaeser et al., 2015)
Biocontrol of V. longisporum
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
Isolate Pathotype Origin
Crop Country/Region
IPP 0109 D1 Brassica napus Sweden
IPP 0127 D1 Brassica napus Sweden
IPP 0128 D2 Brassica napus Sweden
IPP 0129 D1 Brassica napus Sweden
IPP 0450 D1 Brassica napus Pöhl, Germany
IPP 0453 D2 Brassica napus Rostock, Germany
IPP 0462 D1 Brassica napus Hohenlieth, Germany
IPP 0467 D1 Brassica napus France
IPP 0793 D1 Brassica napus France
IPP 0794 D1 Brassica napus Göttingen, Germany
Isolates used in multiplex PCR. All isolates were obtained from
the culture collection of the University of Göttingen (Masterarbeit F. Novakazi, 2013)
Plant Pathology & Crop Protection Division, Department of Crop Sciences, Faculty of Agriculture
In vitro and planta analysis of seeds from the field
0
40
80
120
160
200
Dis
ea
se p
ara
me
ters
b b ns ns
A A
a ab
B
ns
ns
B
PH AUDPC VL DNA V T V T V T
PH AUDPC VL DNA V T V T V T
VL-inoculated control Plants from seeds of diseased
plants in the field
V, T: cultivars ‚Visum‘,Treffer‘
AUDPC: area-under-disease-progress-curve
PH: plant height
VL DNA: fungal DNA in plant hypocotyl
Seeds were incubated on
PDA for 28 days at 23oC in
the dark. None of the
analyzed seeds (n=
450/cultivar) were found to
be contaminated or infected
with VL.
Testing seeds from OSR
naturally infected in the
field.
Msc thesis Alice Bisola Eseola, 2016