novel insights into the life cycle and epidemiology of

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.


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


3


‘Verticillium stem striping’

4 Microsclerotia


5 MPP, 17(7), 1004-16, 2016


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?


-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


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


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



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


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


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


-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



Proliferation

after flowering


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


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


Erucic

C22:1


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


Yield & quality, spring OSR

Spring oilseed rape field exp. Weende 2016 destroyed by pollen beetle


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


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


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).


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


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


21


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?


VL lineages - host range

Non-Brassicaceae Brassicaceae

Novakazi et al., Phytopathology 105: 662-673, 2015 23


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


Isolate Pathotype Origin

Crop Country/Region

IPP 0109 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)


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

novel insights into the life cycle and epidemiology of

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