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Emergence of ‘flavescence dorée’ in Croatia: distinct genetic clusters and new hotspots

Emergence of ‘flavescence dorée’ in Croatia: distinct genetic clusters and new hotspots Jelena Plavec2, Željko Budinšćak2, Ivana Križanac2, Ivana Samaržija1, Dijana Škorić1, Xavier Foissac3, Martina Šeruga Musić*1 1 Department of Biology, Faculty of Science, University of Zagreb, Marulićev trg 9A, Zagreb ; Croatia ; 2 CCAFRA-Plant Protection Institute, Gorice 68B, Zagreb, Croatia 3 UMR- 1332 Biologie du Fruit et Pathologie, INRA, Université de Bordeaux, 71 avenue Edouard Bourleaux - CS20032, F-33882, Villenave d'Ornon, France * Corresponding author: martina.seruga.music@biol.pmf.hr INTRODUCTION Flavescence dorée (FD) phytoplasma comprising ribosomal subgroups 16SrV-C and –D, is one of the most important agents associated with epidemic and severe grapevine yellows (GY) diseases in Euro-Mediterranean region. It is persistently transmitted by the American grapevine leafhopper Scaphoideus titanus Ball. The role of some other insect species as alternative vectors in FD and FD-related phytoplasma epidemiology has been studied (Filippin et al., 2009 ; Mehle et al., 2011 ; Casati et al., 2016). The first identification of FD phytoplasma infecting grapevine in Croatia was recorded in 2009 (Šeruga Musić et al. 2011) in limited areas of continental regions of the country which initiated very intensive efforts in surveillance of all winegrowing regions. Thus, the objectives of this work were to give the overview of a decade of FD monitoring in Croatia with the emphasis on newly emerging hotspots, as well as to evaluate the advantage in using multilocus sequence typing (MLST) approach for tracing the disease emergence and spread. The distribution and prevalence of FD strains together with the finding of potential alternative plant hosts and insect vectors are also discussed. MATERIALS AND METHODS In the period 2008-2017, more than 1200 samples of grapevine, wild herbaceous and woody plants as well as insect vector Scaphoideus titanus and other potential vectors, were collected from different viticultural regions in the continental and coastal parts of Croatia. Phytoplasma detection and identification was performed by triplex real-time PCR (Pelletier et al., 2009) enabling simultaneous detection of FD and ‘bois noir’ phytoplasma. Routine PCR/RFLP of 16S rDNA was also performed as described elsewhere (Plavec et al. 2015). MLST approach was applied for further characterization and genotyping of selected FD phytoplasma isolates by analyses of secY, map and uvrB-degV genes according to Arnaud et al., (2007). All amplicons were sequenced (Macrogen Europe, Amsterdam, the Netherlands), assembled and edited by using SequencherTM 4.7 (http://www.genecodes.com/) and Geneious software (http://www.geneious.com/), and aligned with ClustalX 2.0 (Thompson et al., 1997). Subsequent phylogenetic analyses were performed by using MEGA 7 software (Kumar et al., 2016) and followed by assignment of a comprehensive genotype. RESULTS AND DISCUSSION After the first detection of FD phytoplasma in 2009 from 2 grapevine samples in the central continental Croatia, and the detection of infected S. titanus samples in 2011, the apparent emergence of the disease and the epidemic trend in some of the important winegrowing regions was observed. In 2014, FD phytoplasma was for the first time identified in the northern coastal part of Croatia (Istria), where in the following year very high incidence of FD phytoplasma-infected vines of cultivar ‘Malvasia’ occurred. New fast developing hotspots were observed in north- western continental region of the country with severe disease outbreaks in years 2016 and 2017 (Figure 1). MLST of selected FD phytoplasma isolates originating from grapevine, insect vectors and wild plants revealed the presence of distinct pathosystems and at least 16 different comprehensive genotypes. The greatest diversity of comprehensive genotypes was observed within mapFD2 cluster which was found to be prevalent in the north-western continental and the only one present in severely affected coastal region of the country clearly associated with the epidemic trend (Figure 1). In the eastern part of continental Croatia where no severe outbreaks have been observed, only FD phytoplasma associated with mapFD3 cluster was found (Figure 1). Moreover, the finding of different genotypes belonging to mapFD2 and mapFD3 genetic clusters identified from the leafhopper Phlogotettix cyclops and invasive tree species Ailanthus altissima Mill., respectively, suggests that alternative players might also be involved in FD epidemiology. Fig 1. Geographic distribution of map genetic clusters (mapFD) of flavescence dorée (FD) phytoplasma in Croatia. Marked is the area encompassing new hotspots with severe FD disease outbreaks in years 2016 and 2017. ACKNOWLEDGEMENTS This work was supported by the Croatian Science Foundation grant no. UIP-2014- 09-9744 and by the Ministry of Agriculture (National Survey of Quarantine Organisms Programme). REFERENCES Arnaud G, Malembic-Maher S, Salar P et al., 2007. Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. Applied and Environmental Microbiology 73, 4001–4010. Casati P, Jermini M, Quaglino F et al., 2017. New insights on Flavescence dorée phytoplasma ecology in the vineyard agro-ecosystem in southern Switzerland. Annals of Applied Biology 171, 37– 51. Filippin L, Jović J, Cvrković T et al., 2009. Molecular characteristics of phytoplasmas associated with Flavescence dorée in clematis and grapevine and preliminary results on the role of Dictyophara europaea as a vector. Plant Pathology 58, 826-837. Kumar S, Stecher G, Tamura K, 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0. Molecular Biology and Evolution 33:1870-1874 Mehle N, Rupar M, Seljak G, Ravnikar M, Dermastia M, 2011. Molecular diversity of ‘flavescence dorée’ phytoplasma strains in Slovenia. Bulletin of Insectology 64, S29–30. Pelletier C, Salar P, Gillet J et al., 2009. Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII-A groups with an endogenous analytical control. Vitis 48, 87–95. Plavec J, Križanac I, Budinšćak Ž, Škorić D, Šeruga Musić M (2015) A case study of FD and BN phytoplasma variability in Croatia: multigene sequence analysis approach. European Journal of Plant Patholology 142 (3): 591-601 Šeruga Musić M, Škorić D, Haluška I, Križanac I, Plavec J, Mikec I (2011) First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia. Plant Disease 95 (3): 353 Thompson JD, Gibbson TJ, Plewniak F. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882 http://www.genecodes.com/) http://www.geneious.com/)

grapevine yellows, epidemiology, pathosystem, FD phytoplasma, phylogeny

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