Detection and molecular characterization of Egyptian isolates of grapevine viruses.

Selected commercial and/or local vineyards and nurseries in three different governorates of Egypt (Alexandria, El-Beheira and El-Menofia) were surveyed for symptoms indicative of infection by grapevine viruses. Leaf samples from red-fruited and white-fruited Vitis vinefera were tested for grapevine leafroll associated viruses (GLRaV-1, GLRaV-2, and GLRaV-3), grapevine viruses A and B (GVA, GVB), grapevine rupestris stem pitting virus (GRSPaV), grapevine fanleaf virus (GFLV), and grapevine fleck virus (GFKV) from early April to late October 2010. Incidence of these viruses was assessed by RT-PCR in 60 different samples. Selected amplicons were sequenced. While GVA was the most wide spread (30%), GLRaV-1, GVB, GFLV, and GFKV were not detected during the survey. However, GVA, GLRaV-2, GLRaV-3, and GRSPaV were detected in the form of single infection or in mixed infections of 2 to 4 viruses. Phylogenetic analysis was performed on all Egyptian isolates of GLRaV-2 (4), GLRaV-3 (7), GVA (3), and GRSPaV (6). GRSPaV was detected for the first time in Egypt. Phylogenetic analysis provided insights into the evolutionary relationship between the reported Egyptian isolates and other previously reported isolates.

First Report of a γ 3-Proteobacterium Associated with Diseased Strawberries in Italy.

During the fall seasons of 2005 and 2006, diseased strawberry plants (Fragaria × ananassa Duch.) were observed in nurseries and production fields in Ferrara, Forli-Cesena, and Ravenna provinces (Emilia-Romagna region, northern Italy). Symptoms consisted of a conspicuous plant stunting with a poor root system. Older leaves rolled upward and displayed a marked premature purplish discoloration, while young leaves were cupped, chlorotic, generally reduced in size, and had shortened petioles. This strawberry disorder was similar to "marginal chlorosis", an infectious disease occurring in France that can be induced by two different phloem-limited uncultured bacteria: the γ 3-proteobacterium 'Candidatus Phlomobacter fragariae' and the stolbur phytoplasma (16SrXII-A). In strawberry production fields, 'Ca. P. fragariae' is reported as being the prevalent agent of this disease (1). Sixty-seven diseased plants were collected from production fields and nurseries for testing for 'Ca. P. fragariae'. Leaf samples were analyzed by 4',6-diamidine-2-phenylindole staining and PCR. Forty samples showed fluorescent DNA in the phloem, whereas no fluorescence was observed in symptomless strawberries. When tested by PCR with primers Fra4/Fra5, which amplify a 550-bp fragment of the 16S rDNA region of 'Ca. P. fragariae' (1), 13 of 36 strawberries from production fields and 1 of 31 nursery plants gave a positive reaction. On the other hand, 21 samples from nurseries and 5 from production fields tested positive for stolbur phytoplasma (3). No amplification was obtained with DNA from symptomless or healthy strawberry plants. Sequencing Fra4/Fra5 amplicons from three samples (GenBank Accession Nos. DQ362916-DQ362918) showed a 98.1 to 98.6% and a 98.3 to 98.8% identity with the published sequences of the French isolate "LG2001" (GenBank Accession No. AM110766) and the Japanese isolate J-B (GenBank Accession No. AB246669) of 'Ca. P. fragariae', respectively. Higher homology (99.2 to 99.8%) was found with another bacterium-like organism (BLO) of the γ 3-proteobacteria subgroup (GenBank Accession No. AY057392) associated with the syndrome "basses richesses" of sugar beet (SBR). Furthermore, PCR assays performed with primers Pfr1/Pfr4, specific for spoT gene of 'Ca. P. fragariae', did not show any amplification with DNA from the 14 diseased strawberry plants tested. This is in agreement with the SBR BLO identification (2). To better characterize the Italian isolates, the full-length 16S rDNA gene was analyzed with primers fd1/Fra4 and Fra5/rp1, which amplify the 5' and 3' region of 16S rDNA gene of the proteobacteria, respectively (2). PCR products from eight isolates were sequenced, and the 16S rDNA sequences obtained (GenBank Accession Nos. DQ538372-DQ538379) showed a 96.4 to 97.3% identity with the known 'Ca. P. fragariae' isolates, while a higher homology (99.4 to 99.9%) was again found with the SBR BLO. To our knowledge, this is the first report of a γ 3-proteobacterium affecting strawberry in Italy. In the genome region analyzed, our isolates are more similar to the SBR BLO than to 'Ca. P. fragariae'. Further work is in progress to investigate incidence, geographical distribution, epidemiology, and host range of this pathogen in Italy. References: (1) J. L. Danet et al. Phytopathology 93:644, 2003. (2) O. Semetey et al. Phytopathology 97:72, 2007. (3) F. Terlizzi et al. Plant Dis. 90:831, 2006.

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.