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Ulmus L. and Zelkova Spach are two species of the familyUlmaceaeoccuring in Europe. Ulmus(elm) com-prises about 45 species that show a range of susceptibili-ties to Elm Yellows (EY), the most widespread and seri-ous phytoplasma disease for these hosts. Japanese elm (Zelkova spp.) is known for its resistance to a wide range of diseases and pests, although Zelkova Yellows (ZY) has recently been reported on Z. serrata. Since June 2006, we have carried out surveys to identify the causal agent of the decline of Ulmusspp. and Z. serrata in the Marche Region of Italy. Twenty-five leaf samples were collected from pre-bonsai and bonsai elms (U. parvifolia and Ulmus sp.), and Z. serrata, either symp-tomless or showing typical EY symptoms, and these were analysed by nested-PCR and RFLP based on the phytoplasma 16S rRNA gene. These analyses detected a single infection of ‘Candidatus Phytoplasma ulmi’ (16SrV-A) in all of the symptomatic leaf samples. Se-quence analysis revealed 99% nucleotide homology with the EY reference strain EY-627 (Genbank acces-sion no. AY197658). This appears to be the first report of ‘Ca. Phytoplasma ulmi’ associated with EY in Ulmus spp. and Z. serratathat have been trained as bonsais.

Key words: diagnosis, PCR, PCR-RFLP, sequencing, yellows.

The family Ulmaceaeincludes 18 genera and 150-200 species of trees and shrubs distributed in the temperate zones of the two hemispheres. The genera Ulmus L. and Zelkova Spach, subfamilyUlmoideae, and Celtis L., sub-family Celtidoideae, are the only European representa-tives of the Ulmaceae (Gellini and Grossoni, 1997). Both Ulmusspp. and Z. serratacan be trained as bonsai. One of the most widespread and serious diseases of the elm is Elm Yellows (EY), caused by Candidatus

Phy-Corresponding author: G. Romanazzi Fax: +39.071.2204856


toplasma ulmi (16SrV-A) (Lee et al., 2004). EY shows a range of severity, and is much more epidemic and lethal in native American elms than in European elms. First described by Swinge (1938), EY was reported as being responsible for a severe decline in U. americanaand U. rubra (Braun and Sinclair, 1979; Matteoni and Sinclair, 1985; Griffiths et al., 1999). Since the 1950s, EY has been recorded in Italy (Goidanich, 1951), the Czech Re-public, France and Germany (Mittempergher, 2000). In Italy, EY infections have been detected in U. parvifolia, U. pumila, U. chenmouyi and U. minor Mill (syn. U. carpinifolia Gled.). After the first reports, EY was found in several areas where elms were grown, including Emil-ia Romagna (Pisi et al., 1981; Lee et al., 1995), Tuscany (Lee et al., 1995; Sfalanga et al., 2002), Lombardy (Mit-tempergher et al., 1990), southern Italy (Marcone et al., 1997), and Friuli-Venezia-Giulia (Carraro et al., 2004). Recently, Romanazzi and Murolo (2008) reported infec-tions of Ca. Phytoplasma ulmi in symptomatic Z. serrata (Japanese elm) in the Marche region.

The aim of the present study was to investigate the possible role of phytoplasmas in the decline of U. parvi-folia, Ulmus sp. and Z. serrata pre-bonsai and bonsai plants in the Marche Region, and to identify these phy-toplasmas using molecular tools.

From June 2006, surveys were carried out on pre-bonsai plants showing phytoplasma symptoms, includ-ing chlorosis involvinclud-ing the entire plant or particular branches, foliar reddening on one or more branches, at-tenuation of apical shoots, slow growth and stunting of the entire plant, and witches’ brooms (Fig. 1). Twenty-five leaf samples from symptomless and symptomatic plants of Ulmus sp. and Zelkova serrata (Table 1) were processed, and their total nucleic acids were extracted using the DNeasy plant mini-kit (Qiagen, Germany), with some modifications. One g of fresh plant tissue (leaf) was ground in liquid nitrogen and homogenized in 2% CTAB buffer (6 ml; 200 mM Tris-HCl, pH 8.0, 50 mM EDTA, 2.2 M NaCl, 2% CTAB, 0.06% sodium meta-bisulfite). The ethanol-precipitated nucleic acids derived from each pellet were resuspended in 50 to 100 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8) con-taining RNAse (Promega, USA), and incubated at 37 °C for 1 h.

Journal of Plant Pathology (2008), 90(2), 345-349 Edizioni ETS Pisa, 2008 345











sp. AND



S. Murolo and G. Romanazzi

Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy


The amount of DNA in the extracts was measured using a Versa FluorTMFluorimeter (Bio-Rad, USA) and stored at -20 °C before further use. The DNA samples used as templates for PCR were first diluted to 25 ng/µl with sterile deionized water. The final reaction volume was 50 µl, containing 50 ng of DNA template, 0.4 µM of each primer, 125 µM of each dNTP (Promega, USA), 1 U of Taq DNA polymerase (Promega, USA), and the standard 1×PCR buffer with 1.5 mM MgCl2. The PCR was performed for 35 cycles in a programmable Bio-Rad Cycler using the universal phytoplasma rRNA primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995). These primers amplify almost the entire 16S rRNA gene, the 16-23S rRNA spacer region, and the 5’-end of the 23S rRNA gene.

EY1 (Ca.Phytoplasma ulmi, subgroup 16SrV-A) and STOL (Stolbur phytoplasma, subgroup 16SrXII-A) were kindly provided by Dr. C. Marzachì (IVV-CNR, Turin, Italy), AY1 (Ca. Phytoplasma asteris, subgroup 16SrI-B) was kindly supplied by Prof. P.A. Bianco (Plant Pathology Institute, University of Milan, Italy), and GVX (Green Valley X-disease, subgroup 16SrIII-A) was kindly provided by Prof. A. Bertaccini (Plant Pathology, University of Bologna, Italy), and they were used as reference strains. Samples from healthy

seedlings were used as negative controls.

The products resulting from the P1/P7 amplification were diluted 1:100 with sterile deionized water and 2 µl of each dilution were used as template in the nested PCR, with the primer pairs R16(I)F1/R1, R16(V)F1/R1, and R16(III)F1/R2 (Lee et al., 1994). Ten-µl aliquots of each PCR reaction were evaluated by electrophoresis through 1.2% agarose (Sigma-Aldrich, USA) gels using 1×TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) as running buffer. The gels, stained with ethidium bromide (0.5 µg ml-1), were visualized under UV light at 312 nm using a transilluminator, and images were captured with a Canon Power Shot A95 digital camera. The expected lengths of the amplified DNA fragments were estimated by compar-ison with a 100-bp DNA ladder (Invitrogen, USA).

Restriction fragment length polymorphism (RFLP) analysis of the nested-PCR products was used for iden-tification of the putative phytoplasma. The PCR prod-ucts (5 µl), purified by Spin Column Wizard SV Gel and PCR Clean-Up System (Promega, USA), were di-gested singly with 3 U of the restriction endonuclease BfaI (New England BioLabs, USA) overnight at 37°C. Digestions were separated by electrophoresis through 5% non-denaturing polyacrylamide gels using 1×TBE (90 mM Tris-borate, 2 mM EDTA, pH 8.3) as running Fig. 1.Samples showing attenuation of apical shoots, slow growth, and stunting of the entire plant, witches’ brooms, chlorosis in-volving the entire plant or particular branches, and foliar reddening on one or more branches on Ulmus sp. (A) and Zelkova serra-ta (B) pre-bonsai plants and Z. serratatrained as bonsai (C).


buffer. The restriction products in the gels were stained, visualized and recorded as described above.

Phytoplasmas were identified following sample clean-up through the Spin Column System (Promega, USA) and sequencing in both directions using Applied Biosystems ABI3730XL (BMR Genomics, Padova, Italy) of 5 R16(V)F1/R1 amplicons obtained from phy-toplasma-infected U. parvifolia (Op7, Op9), Ulmus sp. (O4, O3), and Z. serrata(Z1A).

The possible identity of the isolates was considered by comparing their ITS sequences with those in the Gen-Bank database (NCBI, US National Institutes of Health, Bethesda, http:/ Multi-ple alignments were made using CLUSTAL X version 1.8 (Thompson et al., 1997), and in silicodigestions of the se-quences were conducted using the NTI vector version 9.0 software (Invitrogen, USA). MEGA3 (Kumar et al., 2004) was used to calculate the phylogenetic relationship, according to the neighbour-joining method (Saitou and Nei, 1987), with 1000 bootstrap replicates. In the phylo-genetic analysis, ‘Ca. Phytoplasma ulmi’ (accession No. AY197656 and AY197658), Peach Yellows (AY197660), ‘Ca. Phytoplasma ziziphi’ (DQ919060), Rubus stunt (AY197649), Flavescence dorée (AJ548787 and EF581166), and Spartium witches’ broom phytoplasma sequences (AY197652), were included as representing the 16SrV subgroups (A, B, C, D and E). Stolbur (AF248959) and ‘Ca. Phytoplasma mali’ (AF248958) were considered as outgroups.

None of the samples analysed gave PCR products us-ing the universal primer pair P1/P7. In contrast, in all 20 extracts from symptomatic samples (7 from U. parvi-folia, 6 from Ulmussp., and 7 from Z. serrata) a product of 1,050 bp, specific for members of the EY group, was obtained after the nested PCR with the R16(V)F1/R1 primers (Table 1). No products were detected using the other group-specific primer pairs R16(I)F1/R1 and R16(III)F1/R2. All 5 samples from symptomless plants tested negative to phytoplasmas, although in other stud-ies, 16SrI-B and 16SrXII-A phytoplasmas were detected on EY-infected elms (Lee et al., 1993, 1994, 1995; Mar-cone et al., 1997; Griffiths et al., 1999).

RFLP analysis using the restriction enzyme BfaI

yielded a profile typical of the 16SrV-A subgroup from all amplified products obtained with EY group specific primers (Fig. 2). Sequence analysis and in silico diges-tion confirmed that BfaI was able to produce two frag-ments of 450 bp and 650 bp.

Comparison of the nucleotide sequences of isolates O3F, O4F, Op7, Op9 and Z1AF, showed 100% nu-cleotide homology within the 16S ribosomal RNA gene. Moreover, they had a high sequence homology (99%) in the same region with the EY phytoplasma strain EY-627, available in the GenBank database (AY197658). In the phylogenetic tree, the sequences O3F, O4F, Op7, Op9 and Z1AF clustered in one group with the two EY reference strains (AY197658 and AY197656) included in the analysis (Fig. 3).

Although the phytoplasma detected in Z. serrata ap-peared to be identical molecularly to those involved in major EY outbreaks worldwide, properties such as pathogenicity and virulence have not yet been consid-ered. Phytoplasma strains can also be characterized by sequencing DNA fragments that are less conserved than the 16S ribosomal gene. Indeed, Lee et al. (2004)

Journal of Plant Pathology (2008), 90(2), 345-349 Murolo and Romanazzi 347

Table 1. Samples analyzed by PCR using the phytoplasma universal primers P1/P7 and the group-specific primers R16(V)F1/R1, R16(I)F1/R1, and R16(III)F1/R2. All samples from symptomless plants tested negative to phytoplasma.

No. of samples No. of positive samples

Species S+a S–a P1/P7 16Sr(V) 16Sr(I) 16Sr(III) Ulmus parvifolia 7 1 0 7 0 0 Ulmus sp. 6 1 0 6 0 0 Zelkova serrata 7 3 0 7 0 0 Total 20 5 0 20 0 0

aS+ (symptoms present) and S– (symptoms absent).

Fig. 2.Electrophoresis through 5% polyacrylamide gels and vi-sualization of the restriction patterns obtained using the en-zyme BfaI on nested-PCR products amplified using R16(V)F1/R from Ulmus parvifolia(Op7 and Op9) Ulmussp. (O3 and O4), Zelkova serrata (Z1A, Z2, Z3) and phytoplasma reference strain EY1 (16SrV-A). M: 100-bp marker ladder. 022_JPP_181SC_345_colore 21-07-2008 12:06 Pagina 347


showed that RFLP analysis of 16Sr RNA gene se-quences differentiate the EY group into 5 subgroups. Moreover, 12 RFLP subgroups were differentiated on the basis of ribosomal protein and 13 were distin-guished using secY gene sequences. Differences in the secYsequence were observed in ‘Ca. Phytoplasma ulmi’ isolates infecting the same ancient historical elm (U. mi-nor) that had 99% homology in the 16S rRNA region (Quaglino et al., 2005).

Plants of U. parvifolia, Ulmus sp. and Z. serrata showed clear to faint symptoms of witches’ broom, yel-lowing and small epinastic leaves. No differences in symptom expression among the elm species were seen. European and Asiatic elm species and their hybrids gen-erally suffer little damage and rarely die from EY. How-ever, this “little damage” becomes important and has economic disadvantages when considering bonsai elms. If, on the other hand, the stunting and small leaves asso-ciated with EY were to be appreasso-ciated in bonsai art, the yellows symptom and its localization to a few branches would give an undesirable suffering aspect to the plants.

From first observations, ‘Ca. Phytoplasma ulmi’ ap-pears to be transmissible by cuttings (Caudwell et al., 1994), a common means to obtain bonsai starting mate-rial, even if no specific percentages of transmission have been estimated. However, hot water treatment of dor-mant elm material, applied in autumn, and followed in spring by forcing (Boudon-Padieu et al., 2004) is an effi-cient and secure technique for producing phytoplasma-free material for planting and long distance exchange.

In the USA, EY is efficiently transmitted by Scapho-ideus luteolus(Baker, 1949); this vector does not occur in Europe, but its role is assumed by the common insect Macropsis mendax (Ribaut, 1952; Carraro et al., 2004).

The data reported here further highlight the need for a large-scale survey to determine the spread of this phy-toplasma in bonsai elms imported from other countries, and for the monitoring of the presence of other poten-tial vectors involved in EY transmission. The recent oc-currence of EY in Europe (Boudon-Padieu et al., 2004; Carraro et al., 2004) confirms that the frequency of EY is underestimated, and it could thus be dangerous to ex-change cuttings of clonal material, either of indigenous elms or of hybrids between European and Asian elm species, which might be highly sensitive (Mittem-pergher, 2000). Overall, the present study is the first at-tempt to detect ‘Ca. Phytoplasma ulmi’ in pre-bonsai and bonsai elms and in Z. serrata.


This work was carried out within the project “Inves-tigations on some diseases of ornamental plants in the Region” funded by Marche Polytechnic University. The authors are grateful to Lucia Biagiarelli for her excellent assistance in the molecular analyses, and to Dr Davide Streccioni for his help during surveys. Thanks are also expressed to Dr Silvia Zitti for her help in species iden-tifications.

Fig. 3.Dendrogram constructed by the neighbour-joining method with 1,000 bootstrap replicates, showing genetic relationships among the 16S ribosomal regions of the ‘CandidatusPhytoplasma ulmi’ isolates sequenced in this study (with the prefix Op, O, Z) and with the reference strains available in Genbank. Stolbur and ‘Ca. Phytoplasma mali’ were included as outgroups.



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Received January 24, 2008 Accepted March 10, 2008






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