First Report of Stolbur Phytoplasma (16SrXII-A) on Strawberry in Northern Italy

Strawberry (Fragaria × ananassa Duch.) is one of the most important small-fruit crops in northern Italy. During the autumn of 2003, in nurseries located in Ravenna Province (Emilia-Romagna Region), a disease characterized by pronounced stunting and a very poor root system was observed in plants of the cv. Tethis. Older leaves of diseased plants were rolled upward and displayed a marked premature purple discoloration; new leaves showed size reduction, shortened petioles, chlorosis, and were generally cupped. Some of these plants were potted and kept in greenhouse conditions; the following spring, they exhibited typical floral abnormalities as virescent and phylloid petals. Flowers were fully or partly sterile, producing small and deformed fruits; new foliage was dwarfed, asymmetrical, and pale green with chlorotic margins. Later, the affected plants expressed a quick decline consisting of growth cessation, bronzing of mature leaves, wilting, and death. This strawberry yellows-type disease was suggestive of a phytoplasmal infection. Symptoms were identical to "marginal chlorosis", a stolbur-associated disease occurring in France (4). To acquire more information, field inspections were extended to the 2004 and 2005 seasons. Additional cultivars (Alba, Aromas, Camarosa, Gemma, Maya, NF 20, Queen Elisa, Roxana, and Selva) affected by a similar disorder were identified in strawberry nurseries and production fields from different sites of Ravenna and Forlì-Cesena provinces. Total DNA extracted from collected plants was tested using nested polymerase chain reaction (nPCR) performed with universal phytoplasma primers P1/P7, followed by phytoplasma-specific primer pair R16F2/R2 or group 16SrI and 16SrXII-specific primer pair R16(I)F1/R1 (1,2). Results from nPCR revealed that 21 of 23 diseased nursery plants were infected by a phytoplasma. On the contrary, no positive reaction was obtained with diseased strawberry plants collected from production fields. Subsequent restriction fragment length polymorphism analysis of the nPCR-amplified product R16(I)F1/R1 with enzyme MseI indicated that all diseased plants contained the same phytoplasma belonging to the phytoplasma subgroup 16SrXII-A. Subsequently, these results were confirmed by nPCR using group 16SrXII specific primer pair fSTOL/rSTOL (1). The fragments amplified from three samples were sequenced (GenBank Accession Nos. DQ350615-DQ350617) and showed 99.6 to 99.8% nucleotide sequence identity with a grapevine stolbur isolate (GenBank Accession No. AJ964960). In addition, all samples were assayed using nPCR with primer pair fTuf1/rTuf1 and primers fTufAy/rTufAy, specific for groups 16SrI and 16SrXII (1). Results showed the presence of an expected 945-bp product from infected samples. Sequencing of five amplicons (GenBank Accession Nos. DQ418456-DQ418460) shared 99.4 to 99.9% nucleotide sequence homology with a periwinkle stolbur isolate (GenBank Accession No. L46370). Before now, stolbur phytoplasma has been found to be associated with a strawberry plant showing phyllody symptoms in southern Italy (3). Our report is a wider demonstration of this pathogen infecting strawberry in major cultivations areas of northern Italy. References: (1) M. Langer et al. Extended abstracts ICVG 14:66, 2003. (2) I. M. Lee et al. Phytopathology 84:559, 1994. (3) M. Pastore et al. J. Plant. Pathol. 85:314, 2003. (4) L. Zreik et al. Acta Hortic. 551:101, 2001.