Multiple gene analyses identify distinct "bois noir" phytoplasma genotypes in the Republic of Macedonia

Abstract

"Bois noir" (BN) is a grapevine yellows disease, associated with phytoplasma strains related to 'Candidatus Phytoplasma solani', that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA, tuf, vmp1, and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf (tuf-type a, tuf-type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies.

Headnote

Summary. "Bois noir" (BN) is a grapevine yellows disease, associated with phytoplasma strains related to 'Candidatus Phytoplasma solani', that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA, tuf, vmp1, and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf (tuf-type a, tuf-type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies.

Key words: grapevine yellows, stolbur, multiple gene analyses, membrane proteins.

Introduction

"Bois noir" (BN) is a phytoplasma-associated grapevine yellows (GY) disease that induces severe crop losses in almost all grapevine varieties used for wine production in the Euro-Mediterranean area (Belli et al.,. 2010; Foissac et al., 2013) and in other continents (Botti and Bertaccini, 2006; G ajar do et al., 2009; Karimi et al., 2009; Duduk et al., 2010). BN produces typical GY symptoms, including berry shrivel, desiccation of inflorescences, colour alterations and curling of the leaves, reduction of growth, and irregular ripening of wood (Belli et al., 2010).

Phytoplasmas are cell wall-less obligate intracellular parasites belonging to the Mollicutes class (Lee et al., 2000). As preliminary data on phytoplasma cultivation in cell-free medium have been reported only recently (Contaldo et al., 2013), their differentiation and classification is based on nucleotide sequence analysis of housekeeping genes (IRPCM, 2004; Martini et al., 2007; Hodgetts et al., 2008; Lee et al., 2010; Valiunas et al., 2013). On the basis of multiple gene (.16S rRNA, tuf, rplV-rpsC, secY) sequence analysis, the etiological agent of BN has been attributed to phytoplasmas related to 'Candidatus Phytoplasma solani' (Quaglino et ah, 2013), subgroup 16SrXII-A (Lee et ah, 1998). BN phytoplasmas are transmitted from plant-to-plant by Hyalesthes obsoletus Signoret (Homoptera: Cixiide), a polyphagous vector living preferentially on weeds inside and/or around vineyards (Sforza et al., 1998; Alma et al., 2002; Langer and Maixner, 2004; Berger et al., 2009). The wide plant host range of H. obsoletus suggests that BN may exist in different ecosystems (Johannesen et al., 2012). Furthermore, in vine-growing areas where H. obsoletus is absent, the presence of BN implies the existence of alternative vectors. Recently, Reptalus panzeri has been reported as natural vector of BN in Serbian vineyards (Cvrkovic et al., 2014). Multiple gene analyses of BN phytoplasmas, based on RFLP-mapping and sequencing of tuf (Langer and Maixner, 2004), secY, vmp1 (Fialová et al., 2009) and stamp (Fabre et al., 2011) genes have highlighted the presence of three tuf, 39 secY, 23 vmp1, and 56 stamp genotypes characterized by different distributions and prevalence in the Euro-Mediterranean basin (Foissac et al., 2013).

Even though GY symptoms in vineyards of the Republic of Macedonia were observed from 1975 (Filip Pejcinovski, unpublished data), the first investigations of GY-associated phytoplasmas have been carried out only during the last few years. BN phytoplasmas (tuf-type b) were identified in all grapegrowing areas of the Country (Seruga et al., 2003; Mitrev et al., 2007; Mitrev and Kostadinovska, 2013). A multiple gene typing analysis was carried therefore out on 16S rRNA, tuf, vmp1, and stamp genes to verify the presence of genetic variability among BN phytoplasmas in diverse regions of the Republic of Macedonia, in order to increase knowledge about BN epidemiology in this country.

Materials and methods

Sample collection and DNA extraction

During surveys carried out from mid-August to mid-September 2012, leaf samples were collected from 17 symptomatic plants of white grape variety Chardonnay and red varieties Vranec and Stanusina in vineyards of seven localities throughout the Republic of Macedonia. One Cuscuta spp. plant was also sampled in the investigated area (Table 1). Grapevine leaf veins, separated from laminas by a sterile razor, and all parts of the Cuscuta spp. plant, were stored at -80°C. Total nucleic acids were extracted from 1 g of frozen plant tissues by cetyltrimethylammonium bromide (CTAB) extraction procedure (Angelini et al., 2001).

Molecular identification of bois noir phytoplasmas

Phytoplasma detection was carried out by means of amplification of 16S rDNA in nested PCR assays primed by P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by primer pair R16F1/R16R0 (Lee et al., 1995), and subsequent AluI-, BfaI-, BstUI-, and MseI-RFLP assays on the amplicons obtained. PCR and RFLP reaction conditions were as previously described (Lee et al., 1998; Quaglino et al., 2009). PCRs were performed by using Taq polymerase (Promega) in an automated thermal cycler (MasterCycler Gradient, Eppendorf). PCR and enzymatic digestion products were electrophoresed through 1% and 3% agarose gel, respectively, in TBE buffer, stained with Midori Green Advance (Biosigma) and visualized under UV transilluminator. DNAs from periwinkle [Catharanthus roseus (L.) G. Don] plants infected by phytoplasma strains EY1 ('Ca. P. ulmi', subgroup 16SrV-A), STOL ('Ca. P. solani', subgroup 16SrXIIA), and AY1 ('Ca. P. asteris', subgroup 16SrI-B) were used as reference controls.

Characterization of bois noir phytoplasmas through MLST

Molecular characterization of phytoplasma strains was performed by nested PCR/RFLP-based assays of three phytoplasma genomic portions, including tuf, vmp1, and stamp genes. Reaction mixtures and PCR-RFLP conditions used for amplifying and digesting the genomic segments of tuf (Schneider et al., 1997; Langer and Maixner, 2004), vmp1 (Pacifico et al., 2009), and stamp (Fabre et al., 2011) genes were as previously described. In detail, in the case of the stamp gene, full nucleotide sequences of 'Ca. P. solani' phytoplasmas described by Cimerman et al. (2009) were retrieved from GenBank, compiled in FASTA format, and searched for single nucleotide polymorphisms (SNPs) in recognition sites for restriction enzymes by virtual RFLP analyses using the software pDRAW32 (www.acaclone.com). The enzyme Hpy188I, able to distinguish 'Ca. P. solani' strains producing comprehensive restriction profiles, was selected for performing the digestion of stamp gene amplicons. Phytoplasma reference controls and visualization of PCR products and RFLP profiles were as described above for 16S rDNA.

PCR products of vmp1 and stamp genes, amplified from BN phytoplasmas representative of the RFLP profiles obtained (Table 1), were sequenced in both senses by a commercial service (Primm) to achieve at least 4× coverage per base position. Nucleotide sequence data were assembled by employing the Contig Assembling program of the software BioEdit version 7.0.5 (www.mbio.ncsu.edu/bioedit/bioedit. html). Assembled sequences were deposited in the GenBank database (www.ncbi.nlm.nih.gov) under the accession numbers listed in Table 1.

Phylogenetic analysis

Nucleotide sequences of vmp1 and stamp genes obtained in the present study (Table 1), and of previously described 'Ca. P. solani' strains (Cimerman et al., 2009; Murolo et al., 2010, 2013; Johannesen et al., 2012; Cvrkovic et al., 2014) retrieved from NCBI GenBank, were employed for phylogenetic analyses. Vmp1 and stamp nucleotide sequences were compiled in FASTA format. Vmp1 sequences were trimmed to approx. 1300-nt fragments (TYPH10F/ TYPH10R fragments; Fialová et al., 2009), and stamp sequences to 550-nt fragments (StampFl / StampRl fragments; Fabre et al., 2011), and aligned using the "ClustalW Multiple Alignment" application of the software BioEdit version 7.0.5. Minimum evolution analysis was carried out using the neighbour-joining method and bootstrap replicated 1000 times with the software MEGA5 (Tamura et al., 2011).

Results

Grapevine yellows symptoms

Typical GY symptoms were observed on Chardonnay and in Vranec grapevine varieties. Chardonnay showed leaf yellowing, while Vranec showed leaf reddening (Figure 1A and 1B). The autochthon red variety Stanusina, cultivated in the region Krnjevo, exhibited leaf yellowing (Figure 1C).

Molecular identification of bois noir phytoplasmas

PCR-based amplification of 16S rRNA gene showed that all the examined samples were infected by phytoplasmas. DNA amplification was obtained from periwinkles infected by phytoplasma reference strains, while no amplification was present in healthy periwinkle and negative controls (PCR mixture devoid of DNA). AluI, BfaI, BstUI, and MseI digestion analysis showed that all the identified phytoplasma strains belong to subgroup 16SrXII-A, since their restriction patterns were indistinguishable from one another and from the patterns characteristic of the STOL (16SrXII-A) reference strain (MseI RFLP patterns are shown in Figure 2A; AluI, BfaI, BstUI RFLP profiles are not presented). Moreover, PCR-positivity of all the grapevine samples analyzed confirmed the association between specific GY disease symptoms and phytoplasma presence in the examined vineyards.

Characterization of bois noir phytoplasmas through MLST

Collective RFLP patterns, obtained by multiple gene analyses, revealed the presence of seven distinct BN phytoplasma genotypes, designated as Mac1 to Mac7 (Table 1). RFLP-based results confirmed that these genotypes are distinguishable by non-ribosomal gene sequence analyses (tuf, innpl, and stamp). Two, three and five restriction patterns were evidenced based on tuf, stamp, and innpl genes, respectively (Table 1; Figure 2B, 2C, 2D). In detail, it was possible to identify: (i) HpaII RFLP profiles associated with tuf-type a (two strains) and tuf-type b (16 strains), formerly named VK-I and VK-II (Langer and Maixner, 2004) (Table 1; Figure 2B); (ii) RsaI RFLP profiles associated with innpl patterns V2-TA (three strains), V3 (three strains), V4 (five strains), V18 (one strains), and V14 (six strains) described in previous studies (Murolo et al., 2010,2013; Cvrkovic et al., 2014) (Table 1; Figure 2C); and (iii) / /pi/1881 RFLP profiles S1 (stamp1) (15 strains), S2 (three strains), and S3 (two strains) described for the first time in this study (Table 1; Figure 2D). Attribution of innpl profiles was confirmed by pDRAW32 virtual restriction analyses of innpl nucleotide sequences of BN phytoplasmas representative of innpl (V) RFLP profiles obtained in the present study and comparison with restriction patterns from previous studies (Figure 3).

Based on RsaI RFLP digestions of innpl gene amplicons, the profiles V4 (associated with tuf type-b/ S1 genotype) and V14 (associated with tuf-type b /S1 and tuf-type b/S3 genotypes) were prevalent among the analyzed BN phytoplasmas (Table 1). S2 (stamp) patterns were found exclusively associated with V3 (innpl) profiles, and S3 exclusively with V14 profiles. Pattern S1 was identified in BN phytoplasmas characterized by patterns V2-TA, V4, V18, and V14 (Table 1).

Considering the BN phytoplasma genotypes determined by collective RFLP patterns, Mac1 (tuf-type b/V14/S1), Mac2 (tuf-type b/V4/S1), and Mac3 (tuf-type b/V2-TA/S1) were identified in the majority of the samples (12/18) throughout the examined viticultural regions (Table 1; Figure 4). On the other hand, Mac5 (tuf-type b/V3/S2) and Mac6 (tuf-type b/V14/S3) were found each in two plants. Mac4 (tuf-type a/V3/S2) was identified in only one BNdiseased Vranec plant, and Mac7 (tuf-type a/V18/ S1) was identified only in one diseased Stanusina plant (Table 1; Figure 4).

Phylogenetic analyses

The innpl phylogenetic tree confirmed that BN phytoplasmas identified in the present study and selected as representative of RsaI RFLP profiles V2-TA (strain MK29), V3 (MK44), V4 (MK28), V18 (MK94), and V14 (MK19), clustered with previously reported BN phytoplasmas having the same RsaI RFLP profiles (Figure 5).

The stamp phylogenetic tree highlighted that BN phytoplasmas (tuf-type a) carrying / Ipyl 881 RFLP profiles S2 (strain MK44) and S1 (MK94) were positioned within the tuf-type a cluster, while the BN phytoplasma MK66 (tuf-type b) carrying / Ipyl 881 RFLP profiles S3, grouped within the tuf-type b cluster, including BN phytoplasmas from Balkan and Eastern parts of Mediterranean basin (Greece, Republic of Serbia, Romania, Russia, Lebanon, and Azerbajian) (Figure 6).

Discussion

In previous years, typical GY symptoms were observed in Chardonnay and in Vranec grapevine varieties in vineyards of the Republic of Macedonia (Mitrev et al., 2007). Typically, white grape varieties exhibit leaf yellowing, while red varieties show leaf reddening (Belli et al., 2010). In the present study, however, symptoms associated with white varieties (leaf yellowing) were observed in the red variety Stanusina. In the same geographic area, other red varieties (e.g. Plovdina) showed leaf yellowing when infected by phytoplasmas associated with "flavescence dorée" disease (Kuzmanovic et ah, 2006). To our knowledge, the behaviour of such phytoplasma-infected red varieties, showing leaf yellowing instead of reddening, is unique and seems to be present only in these geographic areas. The characteristic leaf colour alteration associated with phytoplasma infection is caused by the accumulation of flavonoids (mainly flavonols, anthocyanins, and proanthocyanidins) which are different in white and red varieties (Bogs et ah, 2005; Hren et ah, 2009). Comparative studies on physiology and transcriptome / proteome of healthy and phytoplasma-infected plants of these red grape varieties, carried out as in previous studies (Hren et ah, 2009; Margaria and Palmano, 2011), could provide useful data to assist understanding this phenomenon.

Multiple gene analysis has been proposed and employed to distinguish ecologically separated phytoplasma populations (Lee et ah, 2004; Seemüller and Schneider, 2004; Malembic-Maher et ah, 2011; Davis et ah, 2013; Quaglino et ah, 2013). This approach has also been applied for investigating genetic diversity among phytoplasmas associated with several dis- eases in order to identify strain-specific molecular markers useful for improving knowledge of complex phytoplasma ecologies (Arnaud et al., 2007; Casati et al., 2010, 2011; Adkar-Purushothama et al., 2011; Durante et al., 2012). In order to gain an insight into the genetic diversity among BN phytoplasmas in the Republic of Macedonia, the PCR-RFLP based analysis was performed on a gene (tuf) coding for the translational elongation factor Tu (EF-Tu), whose sequence mutations were reported to be associated with different weed hosts of the BN vector H. obsoletus (Langer and Maixner, 2004), and on two genes (vmp1 and stamp) coding for membrane proteins putatively involved in the interaction of BN phytoplasma with its hosts (Cimerman et al., 2009; Fabre et al., 2011), whose sequence mutations seem to be associated with the geographic distribution and host range of this phytoplasma (Murolo et al., 2010, 2013; Johannesen et al., 2012; Foissac et al., 2013; Cvrkovic et al., 2014).

Previous research showed that only the BN tuftype b, prevalent in south-eastern and central Europe (Foissac et al., 2013), was detected in the Republic of Macedonia (Seruga et al., 2003), suggesting a key role of H. obsoletus haplotypes feeding preferentially on bindweed (Convolvulus arvensis L.) (Johannesen et al., 2012) in the diffusion of BN phytoplasmas in this country. Here, identification of the tuf-type a could support the idea that nettle (Urtica dioica L.) is involved also in BN epidemiology in the investigated geographic regions.

Based on RsaI RFLP digestions of vmp1 gene amplicons, the profiles V4 (associated with tuf-type b/S1 genotype) and V14 (associated with tuf-type b/S1 and tuf-type b/S3 genotypes) were prevalent among the analyzed BN phytoplasmas (Table 1). These data confirmed the widespread presence of pattern V4 throughout the Euro-Mediterranean basin and the specific association of pattern V14 with East Europe (Foissac et al., 2013). Moreover, profile V3 was found in association with both tuftypes a and b, in disagreement with previous evidence indicating the exclusive association of V3 and tuf-type a (Foissac et al., 2013). In addition, BN phytoplasmas from three grapevines exhibited the RsaI pattern V2-TA, previously reported only in grapevines and in the insects Reptalus panzeri and R. quinquecostatus in Serbia (Cvrkovic et al., 2014). The identification of V2-TA profile in the vineyards examined in the present study, and the proven R. panzeri vectorship of BN phytoplasmas to grapevine in Serbia suggest a possible role of R. panzeri as BN vector also in the Republic of Macedonia. Moreover, strain MK44 clustering along with phytoplasma strains from European nettles reinforced that this weed can also play a role in the BN diffusion in the Republic of Macedonia. Moreover, strains MK19 and MK29 were grouped in clusters including BN phytoplasmas identified in Serbian R. panzeri insects (Cvrkovic et al., 2014), able to transmit BN phytoplasmas from grapevine and maize to grapevine, and in Italian grapevines (Murolo et al., 2010, 2013). This suggests a new scenario about the possible existence of R. panzeri-related BN diffusion, specific for the strains showing V2-TA and V14 RsaI patterns.

The identification of two distinct BN genotypes (Mac3 and Mac7) in the red grape variety Stanusina suggests that no specific BN phytoplasmas could be related to the symptoms occurring in white varieties. Moreover, the identification of the genotype Mac1 (frequently reported in grapevine) in the Cuscuta spp. plant (Table 1) suggests that it may also be a reservoir of BN phytoplasmas.

Preliminary results showed that BN phytoplasmas from the Republic of Macedonia belonged only to cluster II of tuf-type b, including BN phytoplasmas from Central Europe and Balkan (Germany, Czech Republic, Hungary, Croatia, and Bulgaria) (Foissac et al., 2013; Cvrkovic et al., 2014). Based on information from the present and previous studies, we conclude that BN in the Republic of Macedonia is associated with phytoplasmas strongly related with those reported from Central Europe to the Eastern part of Mediterranean basin and from Balkan regions (Foissac et al., 2013).

Acknowledgements

Field surveys, sample collection and DNA extraction were performed at Universities of Stip, Republic of Macedonia. Phytoplasma detection and molecular characterization were carried out during study by Dr. Kostadinovska at the Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy, University of Milan, Italy, as part of the Student Mobility for Studies (SMS) (Student Academic Year 2012-2013) of the ERASMUS Grant Agreement HEI.

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Accepted for publication: June 21, 2014

Published online: December 22, 2014