Understanding Vitis vinifera/Esca

Interaction:

A Metabolomic Approach

Marta R. M. Lima*, Alberto C. P. Dias

CITAB-UM, Universidade do Minho , Portugal (acpdias@bio.uminho.pt)

*Present address: CBQF/ESB - Universidade Católica Portuguesa

Email: lima.marta.r.m@gmail.com

GTDs in Portugal

Grapevine Trunk Diseases in Portugal

� Botryosphaeriacea-related infections

� Young grapevine decline

o Petri disease

o Black foot disease of grapevine

� Excoriosis

� Eutypiosis – very low relevance in Portugal

Esca in the Vinho Verde Demarcated Region (RDVV)

� The RDVV occupies ca. 10% of

continental Portugal

� Approximately 90% of the

farmers in this region produce

Vinho Verde

� No completetly effective

treatment is available for esca

� Economical importance

Esca in the Vinho Verde Demarcated Region (RDVV)

Felgueiras, M.Sc. Thesis, Universidade do Minho, 2006

A metabolomic approach – WHY?

Esca:

� Aetiology and epidemiology are not fully

understood

� Complex disease believed to result from the

association of several fungi

� Dramatic upsurge in the last years

Metabolomic approach:

� Broader overview of the disease effects

� Suggestion possible defense mechanisms

� Potential discovery of early infection biomarkers

and mechanisms to be exploited for disease

treatment

A metabolomic approach – HOW?

Proton Nuclear Magnetic

Resonance (1H NMR)

• Powerful technique to

analyse plant extracts,

given the universal

abundance of protons in

organic metabolites

• Sample preparation simple

and rapid

• Measurement times are

short and automated

Principal Components

Analysis (PCA)

• Multivariate statistics analysis

technique that reduces de

dimensionality of multivariate

data while preserving the

variance within it

• Useful for observing

groupings within large

datasets (scores plot)

• Detects and displays the

spectral areas responsible for

the separation in the data

(loadings plot)

A metabolomic approach – HOW?

V. vinifera

‘Alvarinho’

Lyophilization

d4- Methanol extraction

1D and 2D 1H NMR

PCA

Healthy leavesAsymptomatic cordon

Diseased leavesSymptomatic cordon

Experimental design

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

Healthy leaves (hl)

Diseased leaves (dl)

Typical 1D 1H NMR spectra

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

-0.1

-0.05

0

0.05

0.1

-0.2 -0.1 0 0.1 0.2PC

2 (

8%

)

PC1(82%)

PCA whole spectra

hl

dl

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.52.54.56.58.5

PC

1 l

oa

din

gs

Chemical shift (ppm)

Methanol

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

-0.2

-0.1

0

0.1

0.2

-0.4 -0.2 0 0.2 0.4PC

2(2

1%

)

PC1(55%)

PCA aliphatic region

hl

dl

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.511.522.53

PC

1 l

oa

din

gs

Chemical shift (ppm)

Un(1.35)

- 28%

Un(0.82-1.15)

+ 88%

Alanine

+ 26%

Un(1.81)

+ 214%

Shikimic acid

- 6%

Acetic acid

- 9%

Un(1.90)

+ 131%

Pyruvic acid

+ 66%

GABA

+ 215%

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

-0.2 -0.1 0 0.1 0.2PC

2(6

%)

PC1(87%)

PCA sugar region

hl

dl

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

3.23.74.24.75.25.7

PC

1 l

oa

din

gs

Chemical shift (ppm)

β-Glucose

- 19%

Methanol

+29%

α-Glucose

- 17%Fructose

- 31%

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

-0.5 0 0.5 1

PC

2(1

6%

)

PC1(74%)

PCA aromatic region

hl

dl

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

66.577.588.5

PC

1 l

oa

din

gs

Chemical shift (ppm)

Quercetin-3-O-glucoside

- 53%

Caffeic acid

Un(6.77)

Un(6.32)

+ 1164%

Un(6.52)

+ 689%

Un(6.89-7.01)

+ 417%

Un(7.09)

+ 392%

Un(7.58)

+ 334%

Un(7.70)

+ 248%

Un(8.05)

+926%

A metabolomic approach – RESULTS

Lima et al., J. Exp. Bot 61(14):4033-4042, 2010

A metabolomic approach – CONCLUSIONS

� The NMR/PCA approach shows that Esca impacts multiple

metabolic pathways in leaves

� Decrease of glucose and fructose accompanied by

increase in phenylpropanoid compounds → deviaKon of

carbon and energy from primary to secondary metabolism

in order to produce defensive compounds

� Other metabolic alterations include increased levels of

alanine, GABA, pyruvate and methanol → might be

involved in other defensive strategies

� The analytical approach used was very effective

discriminating the metabolomic profiles of healthy and

diseased leaves → great potenKal to study esca

Universidade do Minho

• Mafalda Felgueiras

ACKOWLEDGEMENTS

Universidade de Aveiro

• Ana M. Gil

• Gonçalo Graça

• João E. A. Rodrigues

• António Barros

Direcção Regional de Agricultura e Pescas do Norte

• Gisela Chicau

Instituto Superior de Agronomia

• Cecília Rego

Funding

Fundação para a Ciência e a Tecnologia is acknowledged for funding through

the doctoral scholarships SFRH/BD/17944/2004, SFRH/BD/32159/2006,

SFRH/BD/31056/2006 andSFRH/BD/41869/2007

Thank you for your attention