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R16(V)F1/R1 (Lee et al., 1994) specific for 16SrI,. 16SrIII and 16SrV phytoplasma groups, respectively. PCRs were performed with thermocycler PTC-200.
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DETECTION AND CHARACTERIZATION OF PHYTOPLASMAS ASSOCIATED WITH DISEASES OF RUBUS spp. IN POLAND M. Cieslinska Department of Plant Protection, Research Institute of Pomology and Floriculture, 96-100 Skierniewice, Pomologiczna 18, Poland

SUMMARY

Rubus spp. plants with severe symptoms of stunting, short and thin shoots were observed in central Poland in both production fields (raspberry and blackberry) and their natural environments. Nested PCR of DNA extracted from symptomatic plants and healthy raspberry, conducted using phytoplasma universal and groupspecific primer pairs, showed the presence of phytoplasmas in all symptom-showing plants selected for this study. RFLP with HpaII, RsaI, HhaI and BfaI and sequence analysis of the 16S rDNA fragment amplified with universal primers R16F2n/R16R2 revealed that assayed plants were infected by phytoplasmas belonging to three different groups. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ and X disease phytoplasma in Rubus spp. in Poland. Key words: aster yellows, X disease, elm yellows, PCR/RFLP, sequencing.

INTRODUCTION

Phytoplasmal diseases affect wild and cultivated red raspberry (Rubus idaeus L.), black raspberry (Rubus occidentalis L.), blackberry (Rubus fruticosus L., R. laciniatus Willd., R. caesius L.), loganberry (Rubus x loganobaccus) and crosses of these species throughout Europe, north-eastern USA and Turkey (Mäurer and Seemüller, 1994; Converse et al., 1982; Davis et al., 2001; Sertkaya et al., 2004). Infected plants may show a variety of symptoms such as stunting, shoot proliferation, small leaves, short internodes, enlarged sepals, phyllody, flower proliferation and fruit malformations (van der Meer, 1987; Mäurer and Seemüller, 1994). These diseases are associated with infection by phytoplasmas belonging to the following groups: elm yellows (Marani et al., 1977; Schneider et al., 1993; Mäurer and Seemüller, 1994; Bertaccini et al., 1995; Lee et al., 1995; Corresponding author: M. Cieslinska Fax: +48.46.8333228 E-mail: [email protected]

Marcone et al., 1997; Davies, 2000; Vindimian et al., 2004), X disease (Davies, 2000; Davis et al., 2001), aster yellows (Borroto Fernández et al., 2007; Fahmeed et al., 2009; Reeder et al., 2010), and stolbur (Borroto Fernández et al., 2007). Symptoms of rubus stunt were observed in Polish raspberry fields in the 1970’s (Dobrowolska-Wilsz, 1973), whose agent was later identified by Cieslinska (2001). This paper reports the results of a more extensive study on detection and molecular properties of phytoplasmas present in naturally infected Rubus spp. in Poland.

MATERIALS AND METHODS

Plant material. Symptomatic raspberries (Rubus idaeus) of cvs Canby, Polka and Veten, loganberry (Rubus loganobaccus), and tayberry (R. loganobaccus x R. idaeus), grown in commercial and experimental fields, as well as the wild blackberry WB8 (Rubus fructicosus L.) were investigated for the presence of phytoplasmas. Rubus spp. plants showed stunting (Fig. 1A), yellowing, premature reddening and epinasty of the leaves, shortening and proliferation of shoots, phyllody and virescence of flowers and abnormal fruits (Fig. 1B). Loganberry and wild blackberry (WB8) plants showed phyllody and virescence (Fig. 1C). Nucleic acid extraction, PCR/RFLP analysis of 16S rDNA. To verify phytoplasma association with diseases of Rubus spp. plants, molecular identification of the agents was carried out using PCR/RFLP and sequencing analyses. Samples of 0.3 g of fresh leaf midribs collected from cvs Canby, Polka and Veten, tayberry, loganberry and wild blackberry WB8 plants were ground in liquid nitrogen. Total DNA was extracted from all samples using the commercial DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Phytoplasmas detection was by nested PCR. The phytoplasma-universal primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) was used for amplification of a 1.8 kb product representing the almost the complete 16S ribosomal RNA (rRNA) gene,

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Fig. 1. A. Stunting of naturally infected blackberry WB8 (healthy blackberry on the left). B. Malformed fruits of cv. Veten. C. Proliferation, phyllody and virescence of loganberry flowers.

the 16-23S rRNA spacer region, and the 5’-end of the 23S rRNA gene. Products obtained after P1/P7 amplifications were diluted 1:39 with sterile deionized water and used as templates in nested PCRs primed by universal primers R16F2n/R16R2 (Gundersen and Lee, 1996) and primer pairs R16(I)F1/R1, R16(III)F2/R1, R16(V)F1/R1 (Lee et al., 1994) specific for 16SrI, 16SrIII and 16SrV phytoplasma groups, respectively. PCRs were performed with thermocycler PTC-200 (MJ Research, USA) and the amplification products (10 µl) were separated in 1% agarose gel followed by staining in ethidum bromide and visualization of DNA bands using an UVi-Tec transilluminator (Syngen,

USA). The molecular weight of PCR products was estimated by comparison with a 100 bp DNA ladder (Fermentas, Lithuania). Two symptomless (healthy) raspberry plants and sterile deionized water were included in the PCRs as negative controls. C. roseus infected with aster yellows phytoplasma (16SrI-B) from strawberry, and DNA of phytoplasmas: OAY (‘Candidatus Phytoplasma asteris’, subgroup 16SrI-B), CX (X disease phytoplasma, subgroup 16SrIII-A) and RS (‘Candidatus Phytoplasma ulmi’, subgroup 16SrV-E) kindly supplied by Prof. A. Bertaccini (University of Bologna, Italy) were included in this study as positive controls. Nested PCR products primed by R16F2n/R16R2

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were digested singly with restriction endonucleases HpaII, RsaI, HhaI and BfaI according to the manufacturer’s instructions (Fermentas, Lithuania). Restriction patterns were analyzed by electrophoresis in 8% polyacrylamide gels in 1% TBE buffer. The resulting RFLP patterns, after staining in ethidium bromide and visualization, were compared with profiles of positive controls and the restriction patterns of the reference strains of phytoplasmas (Lee et al., 1998, 2004b). Nucleotide sequencing of 16Sr RNA gene fragments Specific DNA fragments amplified with primer pair R16F2n/R16R2 from six symptomatic Rubus spp. plants, were excised from the gel, eluted using QIAquick gel extraction kit (Qiagen, Germany) and sequenced in both directions in the Oncology Center of Maria SklodowskaCurie Institute in Warsaw, using ABI Prism 3 100 Genetic Analyzer (Perkin Elmer, USA). Nucleotide sequences of partial 16S rDNA (1.2 kb nested PCR products amplified with primer pairs R16F2n/R16R2) were compared with phytoplasmal sequences from GenBank using the BLAST algorithm (http://ncbi.nlm.nih.gov/ BLAST/). Multiple alignment of these sequences was made using CLUSTALW of the DNASTAR’s Lasergene software (DNASTAR Inc., USA). Phylogenetic and molecular evolutionary analyses were carried out using the neighbor-joining method implemented in CLUSTALW of the genetic analysis software Molecular Evolutionary Genetic Analysis (MEGA), version 4.02 (Tamura et al., 2007). The data were replicated 1000 times and the

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bootstrap percentage values are given at the nodes of the phylogenetic tree.

RESULTS AND DISCUSSION

PCR/RFLP analysis of 16S rDNA. No PCR products were obtained after amplification 16S rDNA using P1/P7 universal primers. Nested PCR primed by R16F2n/R16R2 yielded products of the expected size (1245 bp) from all six symptomatic Rubus spp. plants and from the infected C. roseus (positive control). It was possible to amplify products from cvs Veten, Canby, Polka’ and tayberry using R16(V)F1/R1 primers specific for the elm yellows phytoplasma group. Nested PCR with the primer pair R16(I)F1/R1 specific for the aster yellows phytoplasma group gave positive result only with sample isolated from wild blackberry WB8. R16(III)F2/R1 primers specific for the X disease phytoplasma group, amplified products of expected size only from loganberry. No products were amplified from DNAs of healthy plants. RFLP patterns obtained after digestion of PCR products using the restriction enzymes HpaII, RsaI, HhaI and BfaI showed that Rubus spp. plants were infected by phytoplasmas belonging to three different groups (Fig. 4). RFLP analysis of 1.2 kb of 16S rDNA fragment from cvs Veten, Canby, Polka and tayberry, yielded patterns indistinguishable from those of strains RuSR19, RuS400, RuS971 and RUS belonging in subgroup E of

Fig. 2. RFLP profiles of 1.2 kb nested PCR amplified with primers R16F2n/R16R2 from six samples of Rubus spp. plants and the phytoplasma control strains. HpaII, RsaI, HhaI and BfaI endonucleases were used for restriction analyses. Lanes M, 100 bp DNA Ladder (Fermentas, Lithuania). Lane 1, cv. Veten; lane 2, cv. Canby; lane 3, loganberry; lane 4, Polka; lane 5, WB8; lane 6, tayberry; lane 7, OAY; lane 8, CX; lane 9, EY.

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Fig. 3. Phylogenetic tree constructed with partial nucleotide sequences of the 16S rDNA from phytoplasmas infecting Rubus plants and phytoplasma reference strains of the aster yellows, X disease and elm yellows groups available in GenBank. Acholeplasma laidlawii (M23932) is included as an outgroup.

the elm yellows group, designed as the novel taxon ‘Candidatus Phytoplasma ulmi’ (Lee et al., 2004b). R16F2n/R16R2 product from loganberry plant digested singly with four endonucleases gave profiles identical to those of CX strain classified in subgroup A of the X-diseases phytoplasma group (16SrIII). Restriction profiles allowed also the preliminary identification of the phytoplasma infecting wild blackberry WB8, as belonging to subgroup B of the aster yellows phytoplasma group denoted ‘Candidatus Phytoplasma asteris’ (Lee et al., 2004a). Nucleotide sequences and phylogenetic analyses. Sequence analysis of the rRNA gene fragment amplified with primers R16F2n/R16R2 confirmed the presence of a phytoplasma of the group 16SrV, ‘Ca. P. ulmi’ in cvs Veten, Canby, Polka and tayberry and showed a 99.7-

99.9% sequence similarity to each other. This phytoplasma was closely related to the rubus stunt isolate (AC: Y16395) and the FD isolate (AC: X76560, probably 16SrV-C). A phytoplasma belonging to group 16SrV (Rubus stunt phytoplasma subgroup) was detected and described in wild and cultivated Rubus spp. in Italy (Mäurer and Seemüller, 1994; Bertaccini et al., 1995; Lee et al., 1995; Marcone et al., 1997; Vindimian et al., 2004), the UK (Davies, 2000) and Poland (Cieslinska, 2001). Multiple alignments revealed that strain WB8 shared high 16S rDNA nucleotide sequence similarity (99.9% and 99.5%, respectively) with strains OAY (AC: M30790) and SAY (AC: M86340). Both of these strains belong to subgroup B of 16SrI group (Aster yellows phytoplasma group, ‘Candidatus Phytoplasma asteris’). Occurrence of aster yellows phytoplasma (16SrI-B) has also been reported from wild raspberry and blackberry

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grown in Austrian forests (Borroto Fernández et al., 2007), from blackberry in Pakistan (Fahmeed et al., 2009) and in the UK (Reeder et al., 2010). The 1148 bp rDNA fragment of the phytoplasma strain from loganberry shared 99.8% and 99.6% sequence similarity, with that of strains CYE (AC: L33766) and BRWB (AC: AF302841), respectively, both belonging in the 16SrIII group (X disease phytoplasma group). X disease phytoplasma was previously identified in loganberry in the UK (Davies, 2000) and in black raspberry (Rubus occidentalis) with Witches’ broom symptoms in Oregon (Davis et al., 2001). Phylogenetic analysis grouped isolates from four samples (Veten, Canby, Polka, tayberry) together with RS strain and close to the other members of the Elm yellows phytoplasma group, ‘Ca. P. ulmi’ (Fig. 5). In the phylogentic tree the WB8 isolate clustered in one group with strains OAY and SAY, the members of subgroup B of aster yellows phytoplasma group, ‘Ca. P. asteris’. The loganberry isolate grouped in the same cluster together with phytoplasma strains CYE and BRWB of the X disease phytoplasma group. The sequences obtained during the study were deposited in GenBank under accession Nos GU125723 (Canby), GU125724 (Polka), GU125725 (Veten), GU125726 (loganberry) and GU125727 (Rubus fruticosus clone WB8). Observations and testing of Rubus spp. plants conducted randomly for several years, indicated that phytoplasma diseases do not seem to be widespread in Polish commercial raspberry and blackberry fields (data not shown). The presence of these agents is mostly connected with their transmission with infected plant material during vegetative propagation. However, wild raspberries and blackberries can become a phytoplasma reservoir as these pathogens were detected in many Rubus spp. plants growing in a natural environment. After detecting phytoplasmas from aster yellows and stolbur groups in wild raspberries and blackberries, Boroto Fernández et al. (2007) suggested that infected plants growing in a natural environment may play an important role in their spreading.

ACKNOWLEDGEMENTS

This work was funded as part of a grant from the Ministry of Science and Higher Education (No. 3 P06 A02422). The author is grateful to Mrs. D. Starzec for her excellent technical assistance.

REFERENCES Bertaccini A., Vibio M., Gennari F., Guerrini S., Benni A., 1995. Detection of mycoplasmalike organisms (phytoplas-

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mas) in Rubus by nested polymerase chain reaction (PCR). Acta Horticulturae 385: 126-131. Borroto Fernández E.G., Calari A., Hanzer V., Katinger H., Bertaccini A., Laimer M., 2007. Phytoplasma infected plants in Austrian forests: role as a reservoir? Bulletin of Insectology 60: 391-392. Cieslinska M., 2001. The preliminary results on detection of phytoplasmas associated with small fruit diseases in Poland. Acta Horticulturae 551: 87-92. Converse R.H., Clarke R.G., Oman P.W., Milbrath G.M., 1982. Witches’ broom disease of black raspberry in Oregon. Plant Disease 66: 949-951. Davies D., 2000. The occurrence of two phytoplasmas associated with stunted Rubus species in the UK. Plant Pathology 49: 86-88. Davis R.E., Dally E.L., Converse R.H., 2001. Molecular identification of a phytoplasma associated with witches’-broom disease of black raspberry in Oregon and its classification in group 16SrIII, new subgroup Q. Plant Disease 85: 1121. Deng S., Hiruki C., 1991. Genetic relatedness between two nonculturable mycoplasmalike organisms revealed by nucleic acid hybridization and polymerase chain reaction. Phytopathology 81: 1475-1479. Dobrowolska-Wilsz K., 1973. Etiologia karlowatosci maliny. PhD thesis. Warsaw University of Life Science, Poland. Fahmeed F., Rosete Y.A., Pérez K.A., Boa E., Lucas J., 2009. First report of ‘Candidatus Phytoplasma asteris’ (group 16SrI) infecting fruits and vegetables in Islamabad, Pakistan. Journal of Phytopathology 157: 639-641. Gundersen D.E., Lee I.-M., 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primers. Phytopathologia Mediterranea 35: 144-151. Lee I.-M., Gundersen D.E., Hammond R.W., Davis R.E., 1994. Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology 84: 559-566. Lee I.-M., Bertaccini A., Vibio M., Gundersen D.E., Davis R.E., Mittempergher L., Conti M., Gennari F., 1995. Detection and cheracterization of phytoplasmas associated with disease in Ulmus and Rubus in northern and central Italy. Phytopathologia Mediterranea 34: 174-183. Lee I.-M., Gundersen-Rindal D.E., Davis R.E., Bartoszczyk I.M., 1998. Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology 48: 1153-1169. Lee I.-M., Gundersen-Rindal D.E., Davis R.E., Bottner K.D., Marcone C., Seemüller E., 2004a. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. International Journal of Systematic and Evolutionary Microbiology 54: 1037-1048. Lee I.-M., Martini M., Marcone C., Zhu S.F., 2004b. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. International Journal of Systematic and Evolutionary Microbiology 54: 337-347.

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Marani F., Pisi A., Bertaccini A., 1977. The association of mycoplasma-like bodies with Rubus stunt disease. Phytopathologia Mediterranea 16: 150-153. Marcone C., Ragozzino A., Seemüller E., 1997. Identification and characterization of the phytoplasma associated with elm yellows in southern Italy and its relatedness to other phytoplasmas of the elm yellows group. European Journal of Forest Pathology 27: 45-54. Mäurer R., Seemüller E., 1994. Nature and genetic relatedness of mycoplasma-like organisms causing rubus stunt in Europe. Plant Patholology 44: 244-249. Reeder R., Kelly P.L., Arocha Y., 2010. ‘Candidatus Phytoplasma asteris’ identified in blackberry (Rubus fruticosus) in the United Kingdom. Plant Patholology 59: 394. Schneider B., Ahrens U., Kirkpatrick B.C., Seemüller E., 1993. Classification of plant- pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA. Journal of General Microbiology 139: 519-527. Schneider B., Seemüller E., Smart C.D., Kirkpatrick B.C., 1995. Phylogenetic classification of plant pathogenic my-

Received May 3, 2010 Accepted August 23, 2010

Journal of Plant Pathology (2011), 93 (1), 51-56 coplasma-like organisms or phytoplasmas. In: Razin S., Tully J.G. (eds). Molecular and Diagnostic Procedures in Mycoplasmology, Vol. 1, pp 369-380. Academic Press, San Diego, CA, USA. Sertkaya G., Osler R., Musetti R., Ermacora P., Martini M., 2004. Detection of phytoplasmas in Rubus spp. by microscopy and molecular techniques in Turkey. Acta Horticulturae 656: 181-186. Tamura K., Dudley J., Nei M., Kumar S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599. Van der Meer F.A., 1987. Virus and viruslike diseases of Rubus (raspberry and blackberry). Leafhopper-borne diseases. Rubus stunt. In: Converse R.H. (ed.). Virus diseases of Small Fruits. Agriculture Handbook No 631, pp 197203. USDA,Washington DC, USA. Vindimian M.E., Grassi A., Ciccotti A., Pollini C.P., Terlizzi F., 2004. Epidemiological studies on Rubus stunt (RS) in blackberry orchards located near Trento (Italy). Acta Horticulturae 656: 177-180.