# Validation of methods for Flavescence dorée phytoplasma sensu stricto identification through Test Performance Studies.

Marianne Loiseau, Pascaline Cousseau-Suhard, Isabelle Renaudin, Zala Kogej Zwitter, Natasa Mehle, Kerstin Zikeli, Michael Maixner, Stefano Costanzo, Luca Feretti, Paula Sa-Pereira, et al.

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Marianne Loiseau, Pascaline Cousseau-Suhard, Isabelle Renaudin, Zala Kogej Zwitter, Natasa Mehle, et al.. Validation of methods for Flavescence dorée phytoplasma sensu stricto identification through Test Performance Studies.. Sixth European Bois noir workshop and first international Pro-AECOGY conference, INRAE and University of Bordeaux, May 2024, Bordeaux, France. hal-04700053

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Submitted on 8 Oct 2024

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# Validation of methods for Flavescence dorée phytoplasma sensu stricto identification through Test Performance Studies.

Marianne Loiseau<fnref n="1" />*, Pascaline Cousseau-Suhard<fnref n="1" />, Isabelle Renaudin<fnref n="1" />, Zala Kogej Zwitter<fnref n="2" />, Nataša Mehle<fnref n="2" />, Kerstin Zikeli<fnref n="3" />, Michael Maixner<fnref n="3" />, Stefano Costanzo<fnref n="4" />, Luca Ferretti<fnref n="5" />, Paula Sá-Pereira<fnref n="6" />, Esmeraldina Sousa<fnref n="6" />, Thierry Wetzel<fnref n="7" />, Christophe Debonneville<fnref n="8" />, Michele Della Bartola<fnref n="9" />, Jean-Saïd Bey<fnref n="10" />, Pascal Salar<fnref n="10" />, Sylvie Malembic-Maher<fnref n="10" />, Xavier Foissac<fnref n="10" />

## INTRODUCTION

Flavescence dorée (FD), the most impacting grapevine yellows in Europe, is associated to *Scaphoideus titanus*-transmitted phytoplasmas also called Flavescence dorée phytoplasmas sensu stricto (FDp). FDp variants belong to taxonomic subgroups 16SrV-C and 16SrV-D. Up to current knowledge, the single 16SrV-D map-genotype variant M54 as well as the more than 150 variants of subgroup 16SrV-C are all listed on the EU quarantine list, regardless of their epidemic properties in the vineyard. FD phytoplasma epidemic nature is particularly tied to specific forms of the adhesin-like variable membrane proteins (VMP), determining Vectotypes II and III that are epidemic in European vineyards and Vectotype I (16SrV-C subgroup) that are non-epidemic in the vineyard. Vectotype I of European alders, occasionally transmitted from alder trees to grapevine by *Oncopsis alni*, poses no outbreak risks (Malembic-Maher *et al.*, 2020). Distinguishing between epidemic and non-epidemic isolates is important for an effective and sustainable management. Current diagnostic tests (EPPO, 2016) target 16SrV group without differentiation, while vectotypes determination offer the advantage to directly identify vectotypes. This study aims to validate identification methods capable to distinguish FDp from other 16SrV phytoplasmas.

## MATERIALS AND METHODS

**Design of the study.** Eleven participant laboratories from 9 different countries tested an identical series of double-blind samples following the provided working protocols and using their own reagents and machines.

**Samples.** The tested samples consisted of total nucleic acid (TNA) extracts, instead of fresh plant leaf samples, to avoid problems of homogeneity between laboratory extraction methods and stability over shipping. To evaluate analytical specificity, the samples consisted of 15 target isolates of FDp from different plant hosts, different geographical origins and different genotypes and 15 plants contaminated by other phytoplasmas of 16SrV group or phytoplasmas responsible of grapevine yellows or healthy grapevines. Each sample was tested in duplicate. To evaluate the analytical sensitivity, the repeatability and the reproducibility, a 5-level dilution series of grapevine infected by FDp diluted in healthy grapevine was analyzed 6 time.

**Tested protocols.** After intra-laboratory adaptation and selection, three molecular protocols were submitted to the interlaboratory trial: 1) a nested-PCR 16SrV map adapted from Arnaud *et al.* (2007), followed by sequence analysis (Method1); 2) a nested-PCR VmpA-R1 adapted from Rossi *et al.* (2019)

<footnote n="1">¹ANSES, Plant Health Laboratory, Angers, France;</footnote>

<footnote n="2">²National Institute of Biology, Ljubiana, Slovenia;</footnote>

<footnote n="3">³JKI, Institute for Plant Protection in Fruit Crops and Viticulture, Siebeldingen and Dossenheim, Germany;</footnote>

<footnote n="4">⁴USDA APHIS PPQ, Laurel, USA;</footnote>

<footnote n="5">⁵CREA, Research centre for Plant Protection and Certification, Rome, Italy;</footnote>

<footnote n="6">⁶INIAV, Oeiras, Portugal;</footnote>

<footnote n="7">⁷DLR Rheinpfalz, Institute of plant protection, Neustadt, Germany;</footnote>

<footnote n="8">⁸AGROSCOPE, Nyon, Switzerland;</footnote>

<footnote n="9">⁹Department of Agriculture, Food and the Marine, Plant Health Laboratories, Celbridge, Ireland;</footnote>

<footnote n="10">¹⁰University of Bordeaux, INRAE, Fruit Biology and Pathology, Villenave d'Ornon, France.</footnote>

<footnote>*Corresponding author: marianne.loiseau@anses.fr</footnote>
and Malembic-Maher *et al.* (2020), followed by sequence analysis (Method2); 3) a real-time PCR *Vmp-RK-A23B1* (Foissac et al., this congress) (Method3).

**Processing of the results.** The following parameters were calculated: analytical specificity, representing the ability of the methods to correctly identify the target phytoplasma; analytical sensitivity, representing the last level at 100% positive results; repeatability and reproducibility (adapted from EPPO, 2021).

## RESULTS AND DISCUSSION

Analytical specificity varied across the methods. The real-time PCR *Vmp-RK-A23B1* (Method3) exhibited the highest analytical specificity for FDp (98%), while the nested-PCR 16SrV *map* followed by sequence analysis (Method1) showed the lowest analytical specificity (87%). However, for the *Vmp I* cluster, Method3 generated 32.7 % of false positive results. The best analytical sensitivity is obtained with Method1 and Method3 with 100% detection of FDp at 1x10⁻¹. The repeatability of the evaluated methods ranges from 78 to 100% depending on the laboratory. The best average repeatability is obtained with Method3. The reproducibility also varied across the methods from 81 to 96%, with Method3 demonstrating the highest reproducibility.

The performance characteristics of the real-time PCR are most interesting for the identification of FDp. However, the primers and probe designed for the identification of Vectotype I (phytoplasmas of 16SrV group but not FDp) showed low specificity as they target the poorly variable *vmpB* gene. The *Vmp-RK-A1* simplex qPCR method could hence be carried out separately when confirmation of the presence of a Vectotype I variant is needed.

Less accurate results were obtained for the two nested-PCRs followed by sequencing analysis. As it can be expected for such methods, their reproducibility was also lower than that of the real-time PCR. These methods remain useful as they allowed the distinction between different *map* types and vectotypes (here only based on R1-*vmpA* repeated domain), and the specific identification of the other non-epidemic phytoplasmas that can be detected in grapevine. However, critical points have to be underlined and taken into consideration by users for their proper implementation: risk of micro-contaminations, choose of reliable sequencing service providers, experience and/or training in sequence analysis.

By sharing protocols, conducting test performance studies and aligning diagnostic procedures, laboratories involved in routine diagnosis can ensure the accuracy, reliability and reproducibility of diagnostic methods. And, by enhancing our ability to accurately identify FDp, we can significantly improve disease surveillance and sustainable control measures.

## ACKNOWLEDGEMENTS

This work was carried out in the frame of EUPHRESCO project FLADO VIGILANT. The authors would like to thank Fiona Constable and Bianca Rodrigues Jardim (Australian Government) for providing of DNA extracts of some non-target phytoplasmas.

## REFERENCES

* Arnaud, G., Malembic-Maher, S., Salar, P., et. al. (2007). Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence doree phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. Appl Environ Microbiol, 73(12), 4001-4010. https://doi.org/10.1128/AEM.02323-06

* EPPO. (2016). PM 7/079 (2) Grapevine flavescence dorée phytoplasma. EPPO Bulletin, 46(1), 78-93. https://doi.org/10.1111/epp.12280

* EPPO. (2021). PM 7/98 (5) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. EPPO Bulletin, 51(3), 468-498. https://doi.org/10.1111/epp.12780

* Malembic-Maher, S., Desque, D., Khalil, D., et al. (2020). When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence doree epidemics in Europe. PLoS Pathog, 16(3), e1007967. https://doi.org/10.1371/journal.ppat.1007967

* Rossi, M., Pegoraro, M., Ripamonti, et al. (2019). Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale. Appl Environ Microbiol, 85(10), e03123-03118. https://doi.org/10.1128/aem.03123-18