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.

Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression.

Vitis vinifera cultivars Zinfandel-1A and Mission were found to harbor different grapevine yellow speckle viroid-1 (GYSVd-1) variants and characterized to define the relationship to yellow speckle (YS) and vein-banding (VB) diseases. Products from the left terminal (T1), pathogenic (P), and a portion of the central (C) domains of Zinfandel-1A and Mission displayed distinct single-stranded conformation polymorphism (SSCP) patterns, presumably reflecting nucleotide changes in the P domain. The two selections were shown to contain homogeneous populations of type 1 and type 2 GYSVd-1 variants described in Australia. Symptoms of YS were induced only in vines containing the type 2 variant by treatment at a constant temperature of 32 degreesC in continuous light. SSCP of Pagadebit selections from Italy revealed the nonsymptomatic variant was essentially identical to Zinfandel-1A, whereas symptomatic selections were unlike any other previously described. Nucleotide sequence confirmed that nonsymptomatic selections from Italy contained the GYSVd-1 type 1 variant. A total of 43 changes were spread throughout the T1, C, V, and T2 domains from symptomatic selections. This study establishes the Australian type 1 variant as the non-symptom-inducing form of GYSVd-1 and type 2 as the symptom-inducing variant. The distinct symptom-inducing variant from Italy is proposed as a new type 3 variant of GYSVd-1.

Characterization of Grapevine Rugose Wood Disease Sources from Italy.

A collection of 27 sources of grapevine rugose wood (RW) disease from a viticultural region in northern Italy was analyzed by graft-inoculating vines of three selective Vitis indicators (V. rupestris cv. St. George, V. berlandieri × V. riparia cv. Kober 5BB, and hybrid cv. LN 33). On the basis of stem reactivity, different groups were identified among the selected RW inoculum sources: nine isolates induced pitting only on cv. St. George, whereas four induced grooving only on cv. Kober 5BB. These two groups were classified as isolates of rupestris stem pitting and Kober stem grooving. Three of the remaining isolates induced wood abnormalities on cvs. LN 33 and Kober 5BB, seven induced wood abnormalities on cvs. St. George and Kober 5BB, and four induced symptoms on all three indicators. These groups may represent RW sources with various disease combinations. RW-affected grapevine clones used as inoculum sources also were tested for virus infections by enzyme-linked immunosorbent assay (ELISA). ELISA revealed the presence of grapevine fleck virus, grapevine leafroll-associated closterovirus 1 and 3, and grapevine trichovirus A. These viruses infected most of the selected RW sources. However, eight of the latter were ELISA-negative. The findings are discussed, and the biological and etiological complexity of the RW phenomena in grapevine is confirmed.

Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses.

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive quantification of platelet accumulation and release on indwelling venous catheters.

An important element in the evaluation of biomaterials is quantification of the relationships and the sequence of events between blood elements, blood flow, and the foreign surface. We adapted a qualitative two-dimensional 111In-labeled platelet imaging method to a quantitative noninvasive analysis of platelet uptake/release kinetics for infusion catheters in a canine model. Bilateral femoral vein 6 Fr. Groshong catheters (one treated with a hydroxylated siloxane to improve albumin affinity) were monitored at femoral vein sites with a GE 400T gamma camera, interfaced with a Technicare 560 image acquisition computer. The field of view was sufficiently large that all events below the diaphragm were recorded without having to move the camera. Image acquisition time was 2.5 min; images were obtained every 5-15 min for 3 hrs. Continuous recordings were obtained from bilateral ultrasonic velocity probes, attached distal to the catheter implant sites. A 5 ml blood sample was placed in the field to permit calibration of gamma emissions per pixel in terms of labeled platelet density. Signal compensation for near field capillary perfusion was performed. The two-dimensional platelet distribution was computed and displayed. Local, time dependent platelet accumulation on the catheters and adjacent vessel walls was observed. Platelet accumulation proceeded in irregular steps during the implant period. Loss of local platelet deposits was observed. Downstream reattachment of platelet emboli was inferred from simultaneous reductions and increases in local platelet densities at two catheter positions. Platelet attachment was inversely related to vein blood velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

A chemiluminescent immunoassay for the diagnosis of grapevine closteroviruses on nitrocellulose membrane.

A dot-immunobinding assay was adapted on enhanced chemiluminescence (DIBA-ECL), which employs luminol, a cyclic diacylhydrazide, as substrate for horseradish peroxidase conjugated with a secondary antibody, for the diagnosis of grapevine closteroviruses I and III. The sensitivity of DIBA-ECL was also compared to other immunoenzymatic methods. DIBA-ECL proved to be at least 16 times more sensitive than the dot-immunobinding assay using chloronaphthol/diaminobenzidine mixture as a substrate, which was at least twice as sensitive as DAS-ELISA, DAS-indirect avidin-biotin complex ELISA, and dot-immunobinding assay, using alkaline phosphatase as enzyme. Optimisation of all parameters involved in the DIBA-ECL procedure and its advantages are discussed.