Unravelling host plant resistance in

chrysanthemum using NMR

Suzanne Kos, MSc

Introduction

• Plant pests

• Integrated Pest Management

• Host plant resistance

• Ecometabolomic approach– Bioassays to determine resistance– Metabolomics to identify metabolites– In vitro bioassays for confirmation

• Western flower thrips – Polyphagous pest– Silver and growth damage– Transmission of plant viruses

Introduction

Ecometabolomic approach

• Senecio (Leiss et al. 2009) – PAs jacobine, jaconine N-oxide – The flavanoid kaempferol glucoside

• Tomato (Mirnezad et al. 2009) – Acylsugars

• Carrot (Leiss et al. 2013) – The flavanoid luteolin– The phenylpropanoid sinapic acid – The amino acid beta-alanine

Chrysanthemum

• Important Dutch greenhouse ornamental• Pest problem: western flower thrips, celery

leafminer and two-spotted spider mite • Leiss et al. (2009) identified chlorogenic

acid as resistance factor for thrips

1. In-vivo bioassay with 73 cultivars in greenhouse

2. In-vivo bioassays with 12 cultivars in climate room

3. Identification and quantification of metabolites with NMR

4. Cross reference of resistance by in-vitro bioassay

Ecometabolomic approach

1. In-vivo bioassay with 73 cultivars in greenhouse

• 73 cultivars, 5 replicates each• Five adult thrips per plant released • Scored thrips silver damage after 3 ½ weeks

1. In-vivo bioassay with 73 cultivars in greenhouse

• 5 replicates per cultivar• 5 adult thrips per plant released • Scored thrips silver damage after 3 ½ weeks

F=7.571, df=72, p<0.001

1. In-vivo bioassay with 73 cultivars in greenhouse

• 5 replicates per cultivar• 5 adult thrips per plant released • Scored thrips silver damage after 3 ½ weeks

F=7.571, df=72, p<0.001

2. In-vivo bioassays with 12 cultivars in climate room

ANOVA: F=9.848, df=11,p<0.001

2. In-vivo bioassays with 12 cultivars in climate room

• Twenty unsexed adult thrips per leaf in Petri dish for 24h.

• All larvae that emerged from eggs were counted.

ANOVA: F=3.496, df=8,P=0.005

2. In-vivo bioassays with 12 cultivars in climate room

2. In-vivo bioassays with 12 cultivars in climate room

Pearson correlation: R=0.772, p=0.007

• Different protons in a molecule resonate at slightly different frequencies when placed in a magnetic field depending upon the details of the electron motion in the nearby atoms.

• Structural information about molecules

• Easy sample preparation and highly reproducable

• Quantification

Nuclear Magnetic Resonance

14

Phenolics Carbohydrates, PAs

Amino acids, terpenoids

3. Identification and quantification of metabolites with NMR

15

15

15

16

16

16

1616

18

1818

18

8

8

8

8

8

29

29

29

29

54

5454

54

54

424242

4242

48

48

48

48

4869

69

69

6969

73

73

575757

58 58

58

58

PL

S-D

A c

ompo

nent

2 (

31.6

%)

-10

-8

-6

-4

-2

0

2

4

6

8

-20 -15 -10 -5 0 5 10 15PLS-DA component 1 (41.0%)

10

3. Identification and quantification of metabolites with NMR

Resistant

Susceptible

R2=91,4% Q2=68,5%

w*c

[2]

-0,2

-0,15

-0,1

-0,05

0

0,05

0,1

0,15

0,2

0,25

-0,2 -0,15 -0,1 -0,05 0 0,05 0,1w*c[1]

3. Identification and quantification of metabolites with NMR

Resistant

Susceptible

3. Identification and quantification of metabolites with NMR

3. Identification and quantification of metabolites with NMR

ANOVA: F=4.028, df=11,P=0.001

3. Identification and quantification of metabolites with NMR

Spearman: ρ=-0.538, N=12,P=0.035

1. In-vivo bioassay with 73 cultivars in greenhouse

2. In-vivo bioassays with 12 cultivars in climate room

3. Identification and quantification of metabolites with NMR

4. Cross reference of resistance by in-vitro bioassay

Ecometabolomic approach

4. Cross reference of resistance by in-vitro bioassay

Acknowledgements

C. Hermans

Dr. K.A. LeissProf. P. Klinkhamer

Dr. Y.H. Choi

Plant Ecology and Phytochemistry group: Funding provided by:

In cooperation with: