Development of a DNA-based real-time PCR assay to quantify Allorhizobium vitis overtime in grapevine plantlets

(1)

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https://hal.univ-reims.fr/hal-03122636

Submitted on 27 Jan 2021

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Development of a DNA-based real-time PCR assay to quantify Allorhizobium vitis overtime in grapevine

plantlets

Nguyen Ht, Doré J, Essaid Ait Barka, Lavire C, Clement C, Vial L, Sanchez L

To cite this version:

Nguyen Ht, Doré J, Essaid Ait Barka, Lavire C, Clement C, et al.. Development of a DNA-based real-time PCR assay to quantify Allorhizobium vitis overtime in grapevine plantlets. 14 ème rencontre Plante-Bactérie, Jan 2019, AUSSOIS, France. �hal-03122636�

(2)

DEVELOPMENT OF A DNA-BASED REAL-TIME PCR ASSAY TO QUANTIFY ALLORHIZOBIUM VITIS IN GRAPEVINE PLANTLETS

Nguyen-Huu Trong ¹ , Doré Jeanne ² , Ait Barka Essaid ¹ , Lavire Céline ² , Clément Christophe ¹ , Vial Ludovic ² , Sanchez Lisa ¹

1 Unité EA 4707 Résistance Induite et Bioprotection des Plantes (RIBP), URCA, Reims, France

2 UMR Ecologie Microbienne, CNRS, INRA, VetAgro Sup, UCBL, Université de Lyon, Lyon, France Contact: huu-trong.nguyen@univ-reims.fr

Methods Background

Results

Acknowledgement

• Burr, T. J., Bazzi, C., Süle, S., and Otten, L. 1998. Crown gall of grape: Biology of Agrobacterium vitis and the development of disease control strategies. Plant Dis. 82:1288–1297.

• Kuzmanović, N., Puławska, J., Hao, L., and Burr, T. J. 2018. The ecology of Agrobacterium vitis and management of crown gall disease in vineyards. Curr. Top. Microbiol. Immunol. :15–53.

• Mousavi, S. A., Österman, J., Wahlberg, N., Nesme, X., Lavire, C., Vial, L., et al. 2014. Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

Syst. Appl. Microbiol. 37:208–215.

• Ross, A., and Somssich, I. E. 2016. A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana. Plant Methods. 12.

• Schroth, M. N., McCain, A. H., Foott, J. H., and Huisman, O. C. 1988. Reduction in yield and vigor of grapevine caused by crown gall disease. Plant Dis. 72:241–246.

References

PLATE COUNTING Objectives

Develop DNA-based qPCR as an alternative method for  detection and  quantification of A. vitis in grapevine plantlets by comparing and validating with plate counting assays

2

1 Microorganisms Plant material

I

II Primer sensitivity

III Quantification of A. vitis S4 populations infected from different quantities

Conclusion

qPCR as the alternative method for quantifying A. vitis S4 in grapevine

plantlets thanks to its accuracy, rapidity, flexibility, sensitivity and specificity over plate counting assay.

• Crown gall: important disease of grapevine

worldwide, causing yield decrease and even plant death (Burr et al. 1998; Schroth et al. 1988)

• Causal agent: Allorhizobium vitis (previously named Agrobacterium vitis, Mousavi et al. 2014 )

• Risk of spreading and persisting in vineyard

(Kuzmanović et al. 2018)  need of detection of pathogen

• Study the interaction host-pathogen  need of quantification of bacterial population

 Simple

 Reliable

 Quantify living bacterial cells

× Non-storable samples

× Contamination

× Technical repetitive mistakes

× Risk of false-negative

× Time-consuming

× Intensive labor

DNA-BASED qPCR

 Storable samples

 Sensitive

 Accurate, specific

 Rapid

× No distinction between living/dead cells

× Risk of overestimation

Mannitol-Glutamate medium, 180 rpm,

28°C, overnight A. vitis strain S4

Genomic DNA extraction

5 weeks old in vitro

Grapevine Vitis vinifera cv. Chardonnay 1 week

Ex vitro

3 Primer pairs

Avi_1889

Avi_7106

virD3 virD59

virD3cons Avi_5072

Primer specificity

A. vitis strains Non-A. vitis spp.

A. vitis S4

A. vitis BT2-2

A. vitis BT3-1

A. vitis ET2-10

A. vitis KT1-1

A. fabrum C58

A. rhizogenes K84

B. thailandensis E. coli P. brassicacearum NFM 421

S. meliloti 1021

Avi_1889      × × × × × ×

Avi_5072      × × × × × ×

Avi_7106  × ×  × × × × × × ×

virD3  × × × × × × × × × ×

virD59  × × × × × × × × × ×

AvS4 gDNA

virD3

S4 Vv+S4

➢ Primers can quantify different levels of biomass of A. vitis S4.

➢ Their activity is not influenced by plant gDNA matrix.

R² = 0.9993

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

log (AvS4 CFU/mg FW)

Avi_1889

R² = 0.9982

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

Avi_7106

R² = 0.9864

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

virD3

R² = 0.9832

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

virD59

R² = 0.9933

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

virD3cons

R² = 0.9986

-12 -10 -8 -6 -4 -2 0 2

2 3 4 5

ΔCq

Avi_5072

➢ Primers can detect until 300 A. vitis S4 cells per mg grapevine stem fresh weight.

10

⁹

10

⁷

10

⁵

CFU/ml

R² = 0.963

5.5 5.7 5.9 6.1

3e6 3e4 3e2

initial AvS4 CFU inoculated

PLATE COUNTING at 7 days post infection

1 cm

IV Monitoring A. vitis S4 populations in grapevine plantlets

c

a a

b b

2 3 4 5 6 7

1 3 7 14 21

dpi

PLATE COUNTING

1 dpi 3 dpi 7 dpi 14 dpi 21 dpi

5 mm 5 mm 5 mm 5 mm 5 mm

c

a a

ab

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - virD59

R² = 0.9941

-6 -3 0 3 6

2 3 4 5 6

ΔCq

PLOTTING virD59

c

a a

a

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - Avi_5072

R² = 0.9821

-6 -3 0 3 6

2 3 4 5 6

ΔCq

PLOTTING Avi_5072

c

a a

a

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - Avi_7106

R² = 0.9869

-6 -3 0 3 6

2 3 4 5 6

ΔCq

c

a a

a

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - virD3

R² = 0.9913

-6 -3 0 3 6

2 3 4 5 6

ΔCq

PLOTTING virD3

c

a a

a

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - Avi_1889

R² = 0.981

-6 -3 0 3 6

2 3 4 5 6

ΔCq

c

a a

ab

b

-6 -3 0 3 6

1 3 7 14 21

ΔCq

dpi

qPCR - virD3cons

R² = 0.9934

-6 -3 0 3 6

2 3 4 5 6

ΔCq

PLOTTING virD3cons

*ΔCq = Cq (housekeeping gene EF1α) – Cq (interested gene). Same letters indicate no significant difference (Tukey’s test, p < 0.05)

➢ qPCR method gave similar results as plate counting assays in quantifying A. vitis S4 populations in grapevine plantlets over time.

IV III

I II

(Ross and Somssich 2016)

R² = 0.9806

2.5 3 3.5 4 4.5

3e6 3e4 3e2

ΔCq*

qPCR - Avi_1889

R² = 0.9859

1.5 2 2.5 3 3.5 4

3e6 3e4 3e2

ΔCq

qPCR - virD3cons

R² = 0.9506

2 2.5 3 3.5 4

3e6 3e4 3e2

ΔCq

qPCR - virD3

R² = 0.9022

1.5 2 2.5 3 3.5

3e6 3e4 3e2

ΔCq

qPCR - virD59

R² = 0.966

2.5 3 3.5 4 4.5

3e6 3e4 3e2

ΔCq

qPCR - Avi_7106

R² = 0.9733

2.5 3 3.5 4 4.5

3e6 3e4 3e2

ΔCq

qPCR - Avi_5072

*ΔCq = Cq (housekeeping gene EF1α) –Cq (interested gene)

Development of a DNA-based real-time PCR assay to quantify Allorhizobium vitis overtime in grapevine plantlets

Development of a DNA-based real-time PCR assay to quantify Allorhizobium vitis overtime in grapevine

plantlets

DEVELOPMENT OF A DNA-BASED REAL-TIME PCR ASSAY TO QUANTIFY ALLORHIZOBIUM VITIS IN GRAPEVINE PLANTLETS

Nguyen-Huu Trong 1 , Doré Jeanne 2 , Ait Barka Essaid 1 , Lavire Céline 2 , Clément Christophe 1 , Vial Ludovic 2 , Sanchez Lisa 1

1 Unité EA 4707 Résistance Induite et Bioprotection des Plantes (RIBP), URCA, Reims, France

2 UMR Ecologie Microbienne, CNRS, INRA, VetAgro Sup, UCBL, Université de Lyon, Lyon, France Contact: huu-trong.nguyen@univ-reims.fr

Methods Background

Results

Acknowledgement

• Burr, T. J., Bazzi, C., Süle, S., and Otten, L. 1998. Crown gall of grape: Biology of Agrobacterium vitis and the development of disease control strategies. Plant Dis. 82:1288–1297.

• Kuzmanović, N., Puławska, J., Hao, L., and Burr, T. J. 2018. The ecology of Agrobacterium vitis and management of crown gall disease in vineyards. Curr. Top. Microbiol. Immunol. :15–53.

• Mousavi, S. A., Österman, J., Wahlberg, N., Nesme, X., Lavire, C., Vial, L., et al. 2014. Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

Syst. Appl. Microbiol. 37:208–215.

• Ross, A., and Somssich, I. E. 2016. A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana. Plant Methods. 12.

• Schroth, M. N., McCain, A. H., Foott, J. H., and Huisman, O. C. 1988. Reduction in yield and vigor of grapevine caused by crown gall disease. Plant Dis. 72:241–246.

References

PLATE COUNTING Objectives

Develop DNA-based qPCR as an alternative method for  detection and  quantification of A. vitis in grapevine plantlets by comparing and validating with plate counting assays

2

1

Microorganisms Plant material

I

II Primer sensitivity

III Quantification of A. vitis S4 populations infected from different quantities

Conclusion

qPCR as the alternative method for quantifying A. vitis S4 in grapevine

plantlets thanks to its accuracy, rapidity, flexibility, sensitivity and specificity over plate counting assay.

• Crown gall: important disease of grapevine

worldwide, causing yield decrease and even plant death (Burr et al. 1998; Schroth et al. 1988)

• Causal agent: Allorhizobium vitis (previously named Agrobacterium vitis, Mousavi et al. 2014 )

• Risk of spreading and persisting in vineyard

(Kuzmanović et al. 2018)  need of detection of pathogen

• Study the interaction host-pathogen  need of quantification of bacterial population

 Simple

 Reliable

 Quantify living bacterial cells

× Non-storable samples

× Contamination

× Technical repetitive mistakes

× Risk of false-negative

× Time-consuming

× Intensive labor

DNA-BASED qPCR

 Storable samples

 Sensitive

 Accurate, specific

 Rapid

× No distinction between living/dead cells

× Risk of overestimation

Mannitol-Glutamate medium, 180 rpm,

28°C, overnight A. vitis strain S4

Genomic DNA extraction

5 weeks old in vitro

Grapevine Vitis vinifera cv. Chardonnay 1 week

Ex vitro

3

Primer pairs

Avi_1889

Avi_7106

virD3 virD59

virD3cons Avi_5072

Primer specificity

A. vitis strains Non-A. vitis spp.

A. vitis S4

A. vitis BT2-2

A. vitis BT3-1

A. vitis ET2-10

A. vitis KT1-1

A. fabrum C58

A. rhizogenes K84

B.

thailandensis E. coli P. brassicacearum NFM 421

S. meliloti 1021

Avi_1889      × × × × × ×

Avi_5072      × × × × × ×

Avi_7106  × ×  × × × × × × ×

virD3  × × × × × × × × × ×

virD59  × × × × × × × × × ×

virD3cons      × × × × × ×

Nguyen-Huu Trong ¹ , Doré Jeanne ² , Ait Barka Essaid ¹ , Lavire Céline ² , Clément Christophe ¹ , Vial Ludovic ² , Sanchez Lisa ¹