Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations F. Quaglino1,2, Y. Zhao1, P.A. Bianco2,W.Wei1, P. Casati2, G. Durante2 & R.E. Davis1 1 Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD, USA 2 Istituto di Patologia Vegetale, Universita´ degli Studi, Milan, Italy Keywords Abstract Genetic diversity; phytoplasma classification; restriction fragment length polymorphism; Bois noir (BN) is an insect-transmitted grapevine yellows disease caused by single nucleotide polymorphisms. phytoplasmas belonging to the stolbur subgroup 16SrXII-A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive Correspondence spread of the disease and potential for heavy impacts on the wine industry. In R.E. Davis, Molecular Plant Pathology this study, we investigated the genetic diversity of stolbur phytoplasma strains in Laboratory, USDA-Agricultural Research BN phytoplasma populations. Nucleotide sequences of 16S rRNA genes from Service, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. stolbur phytoplasma strains affecting vineyards in the Lombardy region of Email: [email protected] Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Received: 23 July 2008; revised version Calculation of virtual restriction similarity coefficients revealed the presence accepted: 19 September 2008. of new subgroups in group 16SrXII (stolbur phytoplasma group). Representa- tive strains of confirmed new subgroups 16SrXII-F (XII-F) and XII-G and ten- doi:10.1111/j.1744-7348.2008.00294.x tative new subgroups XII-A1 through XII-A19, XII-H, XII-I, and XII-J as well as known subgroup XII-A were from grapevines; strains representing three additional tentative new subgroups (XII-K, XII-L and XII-M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phyto- plasma populations. The findings open new opportunities for in-depth studies of the distribution of grapevine-associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in dif- ferent geographic areas. heavy impacts on Italian viticulture. BN produces typical Introduction GY symptoms, including desiccation of inflorescences, Bois noir (BN), known as Legno nero (LN) in Italy and reduction of growth, berry shrivel and irregular matura- Vergilbungskrankheit (VK) in Germany, is a grapevine tion of wood. yellows (GY) disease that is caused by phytoplasmas Phytoplasmas are cell-wall-less obligate intracellular in Europe, from Spain to Ukraine (Battle et al., 2000; parasites belonging to the class Mollicutes. To date, no Milkus et al., 2005), where it induces severe crop losses phytoplasma has been cultured in a cell-free medium; in almost all varieties used for wine production (Boudon- thus, differentiation and classification of phytoplasmas Padieu, 2003). Results from a recent survey underscored is based on nucleic-acid-based assay techniques (Lee & the prevalence of stolbur phytoplasmas in vineyards in Davis, 1988; Lee et al., 1992). On the basis of 16S rDNA Italy (Botti & Bertaccini, 2007), suggesting potential for sequence analysis, the presumptive aetiological agent of Ann Appl Biol 154 (2009) 279–289 ª No claim to original US government works 279 Journal compilation ª 2008 Association of Applied Biologists Subgroups and SNP lineages in BN strains F. Quaglino et al. BN was identified as ‘Candidatus Phytoplasma solani’, Bois noir phytoplasma identification and a phytoplasma species belonging to the stolbur subgroup characterization (16SrXII-A) (IRPCM Phytoplasma/Spiroplasma Working Total DNA was extracted from 100 mg of leaf veins per Team – Phytoplasma Taxonomy Group, 2004; according plant using the ‘DNeasy PLANT MINI KIT’ (Qiagen, Hil- to rule 28b of the Bacteriological Code, ‘Candidatus den, Germany) according to manufacturer’s instructions. Phytoplasma solani’ is an incidental citation and does Detection of ‘Ca. Phytoplasma solani’ was carried out by not constitute prior citation). Stolbur phytoplasma is means of amplification of 16S rDNA in nested poly- transmitted by polyphagous planthoppers of the family merase chain reactions (PCRs) primed by primer pair Cixiidae (Weintraub & Beanland, 2006) and affects P1/P7 (Deng & Hiruki, 1991) followed by 16SrI group- a wide range of wild and cultivated plants. In vineyards, specific primer pair R16F1/R16R1 (Lee et al., 1994), and BN phytoplasma is transmitted from plant-to-plant by subsequent MseI-restriction fragment length polymor- Hyalesthes obsoletus Signoret (Maixner, 1994; Sforza et al., phism (RFLP) assay of amplicons as previously described 1998; Alma et al., 2002), but in wine-growing areas (Lee et al., 1998). PCRs were performed by using Taq where H. obsoletus is absent, the presence of stolbur phy- polymerase (Applied Biosystems, Foster City, CA, USA) toplasma implies the existence of alternative vectors in an automated thermal cycler (Bio-Rad, Hercules, CA, (Boudon-Padieu, 2000; Gatineau et al., 2003; Garau USA). Presence of PCR amplicons was verified by elec- et al., 2004; Palermo et al., 2004). trophoresis through 1% agarose gels; restriction frag- The biological complexity of BN disease has stimulated ments were separated by electrophoresis through 5% research on molecular markers of grapevine-affecting stol- polyacrylamide gels. DNAs extracted from phytoplasma bur phytoplasma genetic diversity. Seruga Music et al. strains EY1 (‘Ca. Phytoplasma ulmi’, subgroup 16SrV-A), (2008) reported genetic diversity in grapevine-associated STOL (stolbur group, subgroup 16SrXII-A), and AY1 16SrXII-A subgroup strains on the basis of single-strand (‘Ca. Phytoplasma asteris’, subgroup 16SrI-B) served conformation polymorphism (SSCP) analysis of 16S as reference strains. These phytoplasmas were main- rRNA, tuf (elongation factor Tu), and dnaB genes. tained in Madagascar periwinkle (Catharanthus roseus (L.) Pacifico et al. (2007) found that restriction analysis of the G. Don). Reaction mixtures containing DNA from stol-1H10 gene, encoding a putative membrane protein, healthy Madagascar periwinkle plants and reaction mix- was a useful marker to type BN phytoplasmas in plants tures without DNA template were used as negative and insects. Analysis revealed that three tuf sequence controls. variants (VK-I, VK-II and VK-III) of ‘Ca. Phytoplasma solani’ were present in BN-diseased grapevines as well as in certain weeds (Langer & Maixner, 2004). Intrigu- Cloning and sequencing of phytoplasmal ingly, VK-I and VK-II BN phytoplasma strains are differ- 16S rRNA gene entially distributed in distinct geographic regions in Italy Amplicons from nested PCRs, primed by phytoplasma- (Pasquini et al., 2007; Quaglino et al., 2007; Riolo et al., universal primer pairs P1/P7 and R16F2n/R16R2 2007). (Gundersen & Lee, 1996) from 79 BN phytoplasma In the present study, a high degree of genetic heteroge- strains were cloned in plasmid vector pCRIITOPO (In- neity among 79 BN phytoplasma strains, detected in vitrogen, Carlsbad, CA, USA) and propagated in Escher- north-Italian vineyards from 2004 to 2007, was evidenced ichia coli as described (Shuman, 1994). Both strands of by the presence of two new 16SrXII subgroups (16SrXII-F cloned inserts were sequenced to achieve at least 4Â and 16SrXII-G) and of no less than nine genetically coverage per base position. DNA sequencing was per- distinct 16S rDNA single nucleotide polymorphism formed in an ABI PRISM 377 automated DNA sequencer (SNP) lineages. The findings expand the classification of (Applied Biosystems). The nucleotide sequence data group 16SrXII and open new avenues for researching the were assembled by employing the Contig Assembling complex ecology and epidemiology of BN disease. program of the sequence analysis software BIOEDIT, ver- sion 7.0.0 (http://www.mbio.ncsu.edu/Bioedit/bioedit. html) and deposited in the GenBank database. Materials and methods Sample collection Virtual restriction fragment length polymorphism analysis and calculation of Field surveys on the incidence of GY disease were carried similarity coefficients out from 2004 to 2007 in 21 vineyards located in Lombardy, northern Italy. Leaf samples were collected A total of 99 16S rRNA gene sequences (20 from Gen- from 79 BN-diseased, symptomatic grapevine plants. Bank and 79 obtained in this work) were trimmed to an 280 Ann Appl Biol 154 (2009) 279–289 ª No claim to original US government works Journal compilation ª 2008 Association of Applied Biologists F. Quaglino et al. Subgroups and SNP lineages in BN strains approximately 1.25-kb fragment (delimited by R16F2n Results and discussion and R16R2 primer annealing positions, the F2nR2 frag- Bois noir phytoplasma in grapevines in ment), as previously described (Wei et al., 2007), and ex- Lombardy ported to the program pDRAW32 (AcaClone software, http://www.acaclone.com). Each DNA sequence was an- Primer pair R16F1/R16R1, which is known to prime alysed through an automated in silico restriction assay, amplification of 16S rDNA from phytoplasmas classified and digestion results were plotted on virtual gels as in groups 16SrI and 16SrXII (Lee et al., 1994), primed described by Wei et al. (2007). Briefly, each