RESUMEN
In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jovic et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.
RESUMEN
Severe plant stunting, chlorosis, and extensive root galling were observed on sunflower (Helianthus annus Pioneer Hi-bred PR64LE19, Dupont) in a commercial field at Agios Athanasios, Drama Province, northeastern Greece at the end of May 2013. Disease symptoms were observed about 1.5 months after planting, and were distributed in patches that covered approximately 2% of the whole cultivated area. Examination of the soil and root samples from selected infected plants revealed the presence of abundant root-knot nematodes. Juveniles, males, and females were extracted by sieving, decanting, and root dissection for identification using morphological traits. Nematode population densities ranging from 100 to 150 J2s per 100 cm3 of soil, and 150 to 3,000 eggs per g of fresh sunflower roots were observed. Identification was confirmed by perineal patterns of females and by sequencing of the D2-D3 expansion segments of 28S ribosomal RNA gene (1,3,4). All identification methods were consistent with typical Meloidogyne hispanica. Morphology of perineal patterns of females and measurements of the second-stage juveniles (J2s) matched those of the original description of M. hispanica (3). Alignment indicated that the D2-D3 sequence (GenBank Accession No. KF501128) was 99% homologous to other sequences of M. hispanica deposited in GenBank from Brazil, Portugal, and Spain (EU443606, EU443608, and GQ375158, respectively), differing in only one nucleotide. Phylogenetic analyses using maximum likelihood of this sequence placed the Meloidogyne sp. in a highly supported (100%) clade that included all M. hispanica sequences available from the GenBank database (4). Root-knot nematodes in general have been reported to cause economic losses in sunflower in Europe (2), but there are no reports of M. hispanica. M. hispanica was first found in Seville Province, southern Spain, infecting rootstocks of Prunus spp. (3). Its distribution has been confirmed worldwide on different agricultural crops. However, to our knowledge, this is the first report of M. hispanica infecting sunflower in Europe and the first report of this species on any crop for Greece. The identification of M. hispanica in sunflower is relevant because it may represent a threat for sunflower production in Greece. Research to develop sunflower varieties resistant to root-knot nematodes should now also consider M. hispanica along with other species of Meloidogyne. References: (1) K. R. Barker. Page 19 in: An Advanced Treatise on Meloidogyne. Vol. II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh, NC, 1985. (2) M. Di Vito et al. Nematol. Mediterr. 24:109, 1996. (3) H. Hirschmann. J. Nematol. 18:520, 1986. (4) B. B. Landa et al. Plant Dis. 92:1104, 2008.