The National Wine and Grape Industry Centre is a research centre within Charles Sturt University in alliance with the

Department of Primary Industry NSW and the NSW Wine Industry Australiawww.csu.edu.au/nwgic

Investigations on the spore dispersal patterns of eutypa and botryosphaeria dieback pathogens in

in Australian vineyards

R. Billones-Baaijens, S. Savocchia, M. Ayres, and M. Sosnowski

Eutypa dieback (ED) and Botryosphaeria dieback (BD)

• Ranked in top five priority diseases of Australian wine grape industry

• Ascospores and/or conidia are dispersed by rain splash and wind

• Spores infect pruning wounds leading to cankers, dieback and death

• Spore dispersal patterns in Australian vineyards unknown

• Investigate spore dispersal patterns of ED and BD

• Determine climatic conditions required for spore release

• Identify high risk infection periods in Australian vineyards

Objectives

Barossa Valley

Coonawarra

Griffith

Spore trapping(2013-2016)

Hunter Valley

Spore tapes replaced every 4-5 weeks

Burkard spore trap

Spore drum

DNA extraction from spore tapes

• DNA extracted from ½ of 2-day samples (1.9 1 cm)

• Controls – Inoculated and non-inoculated tapes

• Initially tested for presence of BD-ED by nested PCR

• Positive samples – analysed by qPCR

• Spore numbers – plotted with rainfall and maximum temperatures from each region

Diatrypaceae qPCR using multi-species primers

• Eutypa lata

• E. leptoplaca

• Cryptovalsa ampelina

• C. rabenhorstii

• Eutypella citricola

• Eu. microtheca

• Eu. cryptovalsoidea

• Diatrypella vulgaris

Photos by W. Pitt

18 S 28 S5.8 S

DIA

89RDIA 16F

DITS-1RDITS-1F

SYBR Green

125 bp

Photos by M. Sosnowski

• Diplodia seriata

• D. mutila

• Neofusicoccum parvum

• N. luteum

• N. australe

• Botryosphaeria dothidea

• Lasiodiplodia theobromae

• Spencermartinsia viticola

• Dothiorella iberica

Botryosphaeria qPCR using

multi-species primers

Photos by W. Pitt

Bot BtF1 Bot BtR1

β-tubulin nuclear gene

Bot BtF1

Bot BtR1

R QBot Btub

135 bp

Hydrolysis probe (Taqman)

Bot BtF1

0

200

400

600

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1000

1200

0

5

10

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25

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35

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50

Sep-13 Oct-13 Nov-13 Dec-13 Jan-14 Feb-14 Mar-14 Apr-14 May-14 Jun-14 Jul-14 Aug-14

No

. of

spo

res/

2-d

ay p

erio

d

Tota

l rai

nfal

l (m

m)

and

Max

Tem

pera

ture

(°C

)

Barossa Valley, SA Rain (mm) BD ED Temp (Max)

WinterAutumnSpring Summer

• 179 samples analysed, 19% positive to either ED or BD spores

• BD spores were trapped more frequently (23x) than ED spores (10x)

• ED spores, less in spring

• BD spores greater in winter and spring

96 mm

0

200

400

600

800

1000

1200

1400

1600

1800

2000

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5

10

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45

Dec-14 Jan-15 Feb-15 Mar-15 Apr-15 May-15 Jun-15 Jul-15 Aug-15 Sep-15 Oct-15 Nov-15

No.

of s

pore

s/2-

day

perio

d

Tota

l Rai

nfa

ll (m

m)

and

Max

Tem

per

atu

re (

°C)

Coonawarra, SA Rain (mm) ED BD Temp (Max)

SpringWinterAutumnSummer

54002010

• 172 samples analysed, 33% positive to either ED or BD spores

• BD spores and ED spores – trapped similar frequency

• ED spores – up to 5400 spores, greater in winter (54%)

• BD spores greater in winter (48%)

0

200

400

600

800

1,000

1,200

1,400

0

5

10

15

20

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45

Jun-14 Jul-14 Aug-14 Sep-14 Oct-14 Nov-14 Dec-14 Jan-15 Feb-15 Mar-15 Apr-15 May-15

No. o

f sp

ore

s/2

-da

y p

erio

d

To

tal ra

infa

ll (m

m)

an

d M

ax T

em

pe

ratu

re (°C

)

Griffith, NSW Rain BD ED Temp (Max)

SpringWinter AutumnSummer

2730

• 182 samples analysed, 36% positive to either ED or BD spores

• BD spores and ED spores –similar frequency

• ED spore release – similar across 4 seasons

• BD spores greater in summer (54%), only 14% in winter

0

250

500

750

1000

1250

1500

1750

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2250

2500

0

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10

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Jun-14 Jul-14 Aug-14 Sep-14 Oct-14 Nov-14 Dec-14 Jan-15 Feb-15 Mar-15 Apr-15 May-15

No.

of spore

s/2

-day p

eriod

Tota

l ra

infa

ll (m

m)

and

Max T

em

pera

ture

(°C

)

Hunter Valley, NSW Rain BD ED Temp (max)

SpringWinter AutumnSummer

• 195 samples analysed, 39% positive to either ED or BD spores

• ED spores more frequent (21%) than BD (18%) - Eutypella microtheca, not Eutypa lata.

• ED spores – less in winter (19%), compared to other seasons (27%)

• BD spores greater in autumn (34%)

4980 spores7500 spores

211 mm

Summary of results

• ED and BD spores released sporadically and occasionally both detected in same period/sample

• Seasonal release and no. of spores differed between regions and pathogens SA - higher spores in winter NSW - higher spores in summer Differences in regions - mainly attributed to rainfall

• Rainfall - primary factor in spore release 0.2 mm rain resulted in spore release Not all rain events resulted in spore release Few occasions, spores released a week after rainfall

– dew or relative humidity also a factor?

Differences between pathogens

• ED spores - up to 7,500 spores in a 2-day sample

• BD spores – up to 1,300 in a 2-day sample, mostly only up to 300

spores

• Differences - due to the size and mode of discharge

• ED pathogens - actively eject their ascospores – travel long distances

• BD spores - released in slimy masses – less airborne, more rain-

splashed

• ED ascospores are smaller than BD spores – lighter for wind dispersal

Photos: F. Trouillas, W. Pitt

Collaborators:

Jose Urbez-Torres (SuRDC)

Eileen Scott (Uni of Adelaide)

Chris Steel, Gavin Ash (NWGIC)

Trevor Wicks (ex. SARDI)

RESEARCH TEAM

SARDI Plant Research Centre

University of Adelaide

NWGIC

Regina Billones

Baaijens

Sandra Savocchia

(NWGIC)

Matthew Ayres,

Mark Sosnowski

(SARDI)

Practical Management of Grapevine Trunk Diseases(2013-2016)

Clare Region WGGA

Acknowledgements