Genetic Diversity of ‘Candidatus Phytoplasma solani’ Strains Associated with “Bois Noir” Disease in Croatian Vineyards

Jelena Plavec1, Željko Budinšćak1, Ivana Križanac1, Goran Ivančan1, Ivana Samaržija2, Dijana Škorić2, Xavier Foissac3, 2 Martina Šeruga Musić 1Croatian Centre for Agriculture, Food and Rural Affairs (CCAFRA), Institute for Plant Protection, Gorice 68b, 10 000 Zagreb, Croatia 2University of Zagreb, Faculty of Science, Department of Biology, Marulićev trg 9a, 10 000 Zagreb, Croatia 3Institut National de la Recherche Agronomique (INRA), Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, 71 avenue Edouard Bourlaux, CS 20032, F-33882 Villenave d'Ornon, France [email protected]

Abstract-Variability of genotypes involved in “bois noir” (BN) Reptalus panzeri (Cvrković et al. 2014). H. obsoletus can feed pathosystem, disease induced by ‘Candidatus Phytoplasma solani’, on various herbaceous plants, but is most commonly associated was assessed by a multilocus sequence typing (MLST) approach. with Convulvulus arvensis, Urtica dioica and Vitex agnus- Genotyping was performed on almost ninety selected strains castus as hosts for its nymphs and adults (Langer and Maixner et detected in the scope of an eight-year national survey al. 2004; Sharon et al. 2005; Kosovac et al. 2016). Host encompassing grapevine samples, weeds and insects from all Croatian viticultural regions. The extensive tuf, secY, vmp1 and plants/inoculum source for R. panzeri within BN pathosystem stamp genes based MLST analyses revealed the presence of at however, is still to be confirmed. BN involved ‘Ca. P. solani’ least 19 different ‘Ca. P. solani’ comprehensive genotypes. The strains can be tied to different epidemiological cycle’s trough prevalent genotype identified is affiliated to the main vector molecular characterization of several genes. Therefore, along with Hyalesthes obsoletus and widespread in central north-western part of tuf (Schneider et al. 1997) and secY (Fialova et al. 2009) genes, Croatia. In addition to H. obsoleteus, ‘Ca. P. solani’ was also the two variable surface protein genes - vmp1 (Cimerman et al. detected in samples from the genus Cixius that grouped within 2009) and stamp (Fabre et al. 2011) are frequently used to evaluate distinct stamp/comprehensive genotype. These results revealed a ‘Ca. P. solani’ genetic diversity. While surface proteins are considerable genetic variability and the wide geographical especially important for understanding vector specificity to ‘Ca. distribution of ‘Ca. P. solani’ strains in Croatia. Furthermore, considerable number of comprehensive genotypes found also P. solani’ phytoplasma of Cixiidae family (Suzuki et al. 2006), on indicated presence of several independent epidemiological cycles the basis of the tuf gene ‘Ca. P. solani’ strains are linked to included in the BN pathosystem. different natural epidemic cycles in the field, and two main genotypes: tuf-type a and tuf-type b were studied (Aryan et al. Key words - ‘Candidatus Phytoplasma solani’, MLST, “ stolbur”, 2014). Hyalesthes obsoletus, molecular epidemiology The aim of the present study was: i) to assess the variability of I. INTRODUCTION ‘Ca. P. solani’ strains from infected grapevine, wild plants and insects by genotyping of tuf, secY, vmp1 and stamp genes and ii) ‘Candidatus phytoplasma solani’ (ribosomal subgroup to study the prevalence and distribution of ‘Ca. P. solani’ strains 16SrXII-B), also known as “stolbur” is associated with grapevine in Croatian viticultural regions. “bois noir” (BN) disease that is widespread in Euro- Mediterranean area (Quaglino et al. 2013; Langer and Maixner, II. MATERIAL AND METHODS 2004). The ‘Ca. P. solani’ life cycle involving two ecological In the frame of a national survey programme, all Croatian niches – herbaceous plants hosting insect vectors - results in grapevine growing regions were visually inspected for the complex BN pathosystem comprising several, possibly presence of GY symptoms. In the eight year period (2009 – 2017) intermixed epidemiological networks (Bertaccini and Duduk, over 800 samples of grapevine and other wild/reservoir plants, 2009; Kosovac et al. 2016). While different cixiid planthoppers along with insect samples of predominantly H. obsoletus together (Hemiptera: Cixiidae) are reported to transmit ‘Ca. P. solani’ in with C. wagneri and Reptalus sp. were collected countrywide. many herbaceous and woody plants, Hyalesthes obsoletus is Plant total nucleic acids (TNA) were extracted following considered to be the principal vector of ‘Ca. P. solani’ previously described CTAB extraction protocol (Maixner et al. associated with BN in many countries (Maixner et al. 1995). Recently, a role in BN transmission has been proven also for 1995; Šeruga et al. 2003) while TNA from single insects was The prevalent comprehensive genotype ST6-V18-tuf-b2 in obtained using the commercial kit OmniPrep (G-Biosciences) Croatian vineyards is also the one considered as an emerging according to the manufacturer’s instructions. strain massively propagated by high H. obsoletus populations developing on U. dioica in Austria (Aryan et al. 2014). Moreover, Grapevine DNA samples were then analysed by triplex real- several genotypes found in positive H. obsoletus captured on time PCR assay according to Pelletier et al. (2009). Collected grapevine correlated with prevalent genotypes found in grapevine, insects together with different weeds were tested by conventional thus suggesting a key role of this vector in spread of the BN PCR assays with phytoplasma universal primers P1/P7 (Deng and disease in the country. Hiruki, 1991; Smart et al. 1995), followed by R16F2n/R2 (Gundersen and Lee, 1996) in nested PCR. Selected ‘Ca. P. However, wide genetic diversity found in limited number of solani’ strains were characterised by MLST of tuf, secY, ‘Ca. P. solani’ strains and undocumented presence of H. vmp1 and stamp genes (Schneider et al. 1997; Fialova et al. 2009; obsoletus within BN foci in some regions suggests involvement Cimerman et al. 2009; Fabre et al. 2011). Sequencing of of additional insect species as vectors in BN epidemiology. In obtained amplicons was performed by the commercial service light of recent BN epidemiological studies (Cvrković et al. Macrogen Inc. (Amsterdam, Netherlands). PCR products were 2014; Kosovac et al. 2016) it is possible to speculate about the directly sequenced on both strands. Raw nucleotide sequences active role of different, not host-specific vectors or vectors tied to were assembled and edited with SequencherTM4.7 software new host/source plants with different ecological dynamics in (http://www.genecodes.com/) and Geneious diffusion of BN strains. (http://www.geneious.com/) and then aligned with ClustalX 2.0 (Thompson et al.1997). Phylogenetic analyses were performed In addition to H. obsoletus, previous research identified the with MEGA 7 software (Kumar et al. 2016) by using maximum presence of other insects from Cixiidae family in Croatian parsimony with CNI on random trees method. Bootstrap vineyards (Budinšćak 2008). New findings of ‘Ca. P. solani’ analyses were performed (500 replicates) to estimate the stability infected insect from genus Ciixius opens additional questions of nodes and to support the inferred clades. and more importantly stresses the need to conduct new vector-searching oriented studies to address the role of possible III. RESULTS other key players in BN epidemics in Croatia. Out of 1212 grapevine samples analysed by triplex-real time With as many as 11 stamp genotypes identified so far in PCR, 208 samples were BN positive belonging to subgroup Croatia, stamp revealed to be the highly discriminative marker. 16SrXII-A. In addition to C. arvensis and V. agnus-castus, CPs Formation of distinct stamp genotypes could be a result of ‘Ca. was detected also in Ailanthus altissima, Robinia pseudoacacia P. solani’ adaptations to a new vector since adaptation to new and Polygonum aviculare plants. Although the sampling strategy hosts have a significant impact on phytoplasma membrane was concentrated mostly on grapevine yellows distribution in proteins, resulting in number of different genotypes as number of different locations, ‘Ca. P. solani’ was also previously reported by Fabre et al. (2011). identified in 9 H. obsoletus out of 61 and 3 out of 11 sporadically collected samples from insects of the genus Ciixius. The data obtained in the present study reveals considerable genetic variability, epidemiological relevance and the At least 19 different ‘Ca. P. solani’ comprehensive genotypes geographical distribution of ‘Ca. P. solani’ BN involved were detected among the selected 84 isolates, for the most part genotypes in Croatia. analysed on the basis of tuf/secY/vmp1/stamp gene typing. The prevalent genotype was S6-ST6-V18-tuf-b2 infecting almost Table 1: Number of analysed and ‘Ca. P. solani’ positive field one third of the analysed samples and found both in H. obsoletus collected grapevine samples in Croatian vineyards and grapevine samples. Two other comprehensive genotypes Collected grapevine samples analysed/positive for ‘Ca. were also abundant while others were relatively scarce, P. solani’ phytoplasma presence sporadically distributed and represented only by a few samples. Year V. vinifera As expected stamp resulted to be most variable marker with at 2009 38/18 least 11 genotypes identified. Analysis of tuf gene revealed 3 2010 54/19 genotypes (tuf-type a, tuf-type b1 and tuf-type b2) of ‘Ca. P. 2011 54/15 solani’ strains linked to both main natural epidemic cycles 2012 46/16 within BN pathosystem. 2013 56/14 IV. DISCUSSION 2014 27/8 On the basis of tuf/secY/vmp1/stamp gene typing, among 2015 199/35 the analysed samples at least 19 ‘Ca. P. solani’ comprehensive 2016 334/55 genotypes were detected thus revealing tremendous complexity 2017 404/48 of BN epidemiological network in Croatian winegrowing regions. Total 1212/208

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