RESUMEN
Based on an earlier survey of putative psyllid vectors of apple proliferation (AP), carried out in 2009 and 2010, Cacopsylla picta (Förster) populations infected with 'Candidatus Phytoplasma mali' were detected in at least two commercial apple (Malus domestica Borkh.) orchards in southern Finland (1). To establish the presence of 'Ca. P. mali' in apple trees, a survey was conducted in 17 commercial apple orchards in August 2012. Phytosanitary inspectors tracked the source of the 'Ca. P. mali' by collecting 33 leaf samples from trees showing probable symptoms. Typical symptoms, including elongated stipules and witches' broom, were rare. Total DNA was extracted from leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and screened for 'Ca. P. mali' with real-time PCR (2) and the commercial Apple Proliferation Group - complete PCR reaction kit (Loewe Biochemica GmbH, Sauerlach, Germany). Two samples tested positive and results were confirmed with TaqMan PCR and conventional PCR assays and DNA sequencing in the Food and Environment Research Agency (Fera), in the United Kingdom. One positive sample was taken from an orchard in Lohja, southern Finland, where high 'Ca. P. mali' incidence in overwintered C. picta was observed in 2010 (1). 'Ca. P. mali' was found in a >40-year-old 'Red Melba' tree with witches' broom but without elongated stipule symptoms. The other positive sample was collected from an orchard in the Aland Islands, where the infected 'Lobo' tree showed symptoms of elongated stipules. This orchard was not monitored for AP vectors. No small fruit symptoms were noted by inspectors or growers in either of the orchards. The positive samples were further analyzed for subtypes using PCR/RFLP and primers AP13/AP10 (3). The amplicons (776 bp) were sequenced and digested with HincII and BspHI (New England BioLabs Inc., Ipswich, MA) following manufacturer's instructions. Both samples proved to be apple proliferation subtypes AT-1 on the basis of RFLP and the sequenced 776-bp region. Sequences of the 776-bp amplicon of the Lohja and Aland isolates showed 100% and 99% identity, respectively, with sequences of apple proliferation isolates (accession nos. L22217.1 and CU469464.1) in GenBank. Both suspected psyllid vectors of 'Ca. P. mali' C. picta and C. melanoneura (Förster) occur in Finland, but their distribution, abundance, and transmission specificity is inadequately documented. The next step to evaluate the risk of spread of apple proliferation in commercial orchards is an extensive survey of the occurrence of Cacopsylla species infected with 'Ca. P. mali'. References: (1) A. Lemmetty et al. B. Insectol. 64:257, 2011. (2) P. Nikolic et al. Mol. Cell. Probes. 24:303, 2010. (3) W. Jarausch et al. Mol. Cell. Probes 14:17, 2000.
RESUMEN
In July 2009, the occurrence of pale yellow, bottle-shaped greenhouse cucumber (Cucumis sativus L.) fruits was reported by a horticultural adviser from Kannus in western Finland. The grower had observed the first symptoms in greenhouse cucumber cv. Rapides in May. The most distinctive symptoms were found in fruits, but also flowers were crumpled. Symptoms had spread along plant rows. Estimated yield loss by the grower was 2 to 3%. Fruit and flower symptoms were typical of cucumber pale fruit disease (3) caused by a strain of Hop stunt viroid (HSVd) (2). The original samples were collected by a phytosanitary inspector and the farmer from approximately 10 symptomatic plants growing in the same greenhouse. Testing two samples, one a cucumber leaf and the other a cucumber fruit, by return-polyacrylamide gel electrophoresis gave clear electrophoretic bands at the same position. However, the position of the bands differed slightly from the positive control (Potato spindle tuber viroid) and two other pospiviroids tested on the same gel, indicating the presence of a different viroid with a shorter length than those from the genus Pospiviroid. This observation of size combined with symptoms on cucumber gave a strong indication of the presence of a viroid, likely to be HSVd. RNA was extracted from two subsamples of fruit and leaf samples with a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Extracted RNA was examined with primer pair HSVdF1/R1 designed to detect HSVd from citrus (1). The PCR reactions were performed using a reverse transcription-PCR protocol developed for Pospiviroids (4). Both fruit and leaf samples gave a PCR amplicon of the same size. Sequencing of the amplicon of approximately 300 bp revealed 99% similarity with 11 GenBank citrus isolates of HSVd and 98% similarity with two cucumber isolates of HSVd (Accession Nos. X07405 and X00524) On the basis of these results, the viroid of these cucumber plants was identified as HSVd. To our knowledge, this is the first finding of this viroid in Finland. Although we could determine the causal agent, we could not find the origin of the infection. The seedling plants had been grown in the same greenhouse where the infection was detected. Even though HSVd is not known to be seed transmitted (3) the other production places that had used the same seed lot were inspected and found to be free of the viroid. Very strict phytosanitary measures were taken to eradicate the infection. Since the viroid is easily sap transmissible, there is a certain risk of spreading of HSVd via human action, e.g., visitors, staff, and the use of common packing facilities. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) T. Sano et al. Nucleic Acids Res. 12:3427, 1984. (3) H. J. M. van Dorst and D. Peters. Neth. J. Plant Pathol. 80:85, 1974. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.
RESUMEN
Carrot (Daucus carota) plants with symptoms resembling those of carrot psyllid (Trioza apicalis) damage (3,4) were observed in 14 commercial fields in southern Finland in August 2008; all cultivars grown were affected at approximately 5 to 35% symptomatic plants per field. T. apicalis, a pest of carrots in northern and central Europe, can cause up to 100% crop loss (3,4). Symptoms on affected plants included leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (3,4). Given recent association of liberibacter with several annual crops affected by psyllids (1,2), an investigation on whether this bacterium is associated with symptoms of psyllid damage on carrots was conducted. Total DNA was extracted from petiole tissue of 20 symptomatic and 18 asymptomatic plants (cv. Maestro, Nanda, Nipomo, Nerac, and Fontana) sampled from 10 psyllid-infested fields in southern Finland, as well as 15 plants (cv. Primecut, Cheyenne, and Triple Play) grown from seed in an insect-free greenhouse, with the cetyltrimethylammoniumbromide (CTAB) method (2). DNA was also extracted from 10 carrot roots (cv. Nantura) of plants continuously exposed to field-collected carrot psyllid colonies in the laboratory. DNA samples were tested by PCR using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of "Candidatus Liberibacter solanacearum" (1,2). A 1,168 bp 16S rDNA fragment was detected in DNA from 1 asymptomatic and 16 symptomatic plants and a 669 bp rplJ/rplL fragment was amplified from DNA from 19 symptomatic and 6 asymptomatic plants, indicating presence of liberibacter. DNA from all 10 root samples yielded similar amplicons with both primer pairs. DNA from all the greenhouse carrot plants yielded no amplicon. Amplicons from DNA from three petioles and three roots with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the 12 amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequences from petiole and root tissues (GenBank Accession Nos. GU373049 and GU373048, respectively) showed 99.9% identity to those of "Ca. L. solanacearum" amplified from Capsicum annuum (FJ957896) and Solanum lycopersicum (FJ957897) from Mexico, and "Ca. L. psyllaurous" from potato psyllids (EU812559). The rplJ/rplL consensus sequences from petioles and roots (GenBank Accession Nos. GU373051 and GU373050, respectively) were 97.9% identical to the analogous rplJ/rplL "Ca. L. solanacearum" ribosomal protein gene sequence from solanaceous crops in New Zealand (EU834131) and to "Ca. Liberibacter" sp. sequence from zebra chip-affected potatoes in California (FJ498803). To our knowledge, this is the first report of "Ca. L. solanacearum" associated with a nonsolanaceous species and the first report of this pathogen outside of North and Central America and New Zealand (1,2). References: (1) L. W. Liefting et al. Plant Dis. 93:208, 2009. (2) J. E. Munyaneza et al. Plant Dis. 93:552, 2009. (3) G. Nehlin et al. J. Chem. Ecol. 20:771, 1994. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.
RESUMEN
During June and July of 2004, several diseased plants in winter wheat (Triticum aestivum L.) were reported by agricultural advisers in the southern and southwestern coastal area of Finland. The plants showed extreme dwarfing, various yellowing symptoms, and reduced or no heading. The damage varied considerably. Yield loss estimates in direct-drilled winter wheat fields were approximately 20 to 40% and in worst cases as much as 100%. A few leafhoppers (Psammotettix alienus Dahlb.) were collected from the field with sweep nets and yellow traps. Roots and symptomatic leaves of winter wheat and the leafhoppers were first tested using a commercial polyclonal antibody (DSMZ, Braunschweig, Germany) specific for Wheat dwarf virus (WDV). For the leaf and root samples, routine double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) procedures were used. Five leafhoppers per sample were homogenized with the extraction buffer provided. The homogenate was centrifuged and the extract was evaluated using DAS-ELISA (2). The highest absorbance values were obtained from leafhoppers suspected to be viruliferous. The mean values varied from 1.002 to 1.990 after incubation in the substrate for 2 h. The absorbance values of several leaf samples exceeded the virus-positive threshold but were lower than those of the viruliferous leafhoppers. The virus was not detected in roots. Low absorbance values of virus-positive plants were confirmed using polymerase chain reaction (PCR) with primers specific for WDV (1). Total DNA extracts (DNeasy Plant Mini Kit; Qiagen, Hilden, Germany) from symptomatic leaves were tested using puRe Taq Ready-To-Go PCR beads (Amersham Biosciences, Buckinghamshire, UK). The PCR amplicon was the expected size (1,201 bp). The high absorbance value of the leafhoppers showed that the leafhoppers were carriers of the virus. These results confirmed that the causal agent of dwarfing and yellowing symptoms in winter wheat was WDV (genus Mastrevirus, family Geminiviridae). References: (1) A. Kvarnheden et al. Arch Virol 147:205, 2002. (2) J. Vacke and R. Cibulka. Plant Prot. Sci. 35:41, 2000.
RESUMEN
Blackcurrant reversion virus (BRV) belongs in the subgroup c of nepoviruses. The 3' NTRs of RNAs 1 and 2 of BRV are 1360 and 1363 nucleotides long, respectively, and highly similar (94.8%). In this study we have compared the sequences of the 3' NTRs of ten BRV isolates, originating from different geographic regions or hosts. All deduced sequences were 94.1-98.8% identical with each other, and with the previously deduced 3' NTR sequences of RNAsl and 2 of the type isolate. The proceeding 480 nucleotides of the CP coding region were 86.9-97.9% identical between the same isolates.
Asunto(s)
Regiones no Traducidas 3' , Secuencia Conservada , Nepovirus/genética , Secuencia de Bases , Proteínas de la Cápside/genética , Datos de Secuencia Molecular , Nepovirus/aislamiento & purificación , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M(r) of 44 220. The coding region was bordered by a 5' leader sequence of 25 nt and a 3'-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5'-terminal consensus sequence and a 40 nt motif (located at positions 264-303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs.
Asunto(s)
Nepovirus/genética , Satélite de ARN/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Satélite de ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
ABSTRACT Black currant reversion is a virus-like disease whose causal agent has not been identified. In rooted cuttings of a black currant plant affected with the severe form of the disease, pronounced chlorotic line patterns and ringspots developed in newly emerging leaves. From such symptom-bearing leaves, a virus was mechanically transmitted with difficulty to Chenopodium quinoa and, from this host, to other herbaceous test plants. The virus was purified and partially characterized, and the purified viri-ons were used for antiserum production. Virus particles were isometric, approximately 27 nm in diameter, and sedimented as two nucleoprotein components. They contained a protein species with a molecular mass of 55 kDa, which was readily degraded into a 54-kDa protein and two major RNA components of about 6,700 and 7,700 nucleotides (nt), each with a poly(A) tail. Most of these properties are shared by nepoviruses, but the virus was serologically unrelated to 14 nepoviruses or putative nepovi-ruses tested. However, the deduced sequence of 1,260 nt at the 3' end of one of the viral RNA species was distinct from any known viral sequence, except that it contained short regions of homology to the 3' terminal sequences of RNAs of seven other nepoviruses and two comovi-ruses. To detect this virus in Ribes plants, primers were designed from the known sequence to amplify a 210-nt region of the cDNA of the virus RNA using an immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) protocol. Using this assay for the virus, we associated its presence with two recognized forms of black currant reversion disease occurring in Finland, Scotland, or New Zealand. We also detected the virus in vector gall mites from reverted plants and in black currant plants on which such vector mites had fed. However, the virus was not detected by IC-RT-PCR in known healthy Ribes plants; in Ribes plants free from reversion, but affected by three other distinct virus-like diseases of Ribes; or in plants infected with arabis mosaic, strawberry latent ringspot, or raspberry ringspot nepoviruses. These data suggest that this virus may be the causal agent of reversion disease, and it is tentatively called black currant reversion associated virus.
RESUMEN
In this prospective follow-up investigation the results in 65 patients treated by the Putti-Platt operation for recurrent dislocation of the glenohumeral joint were analysed. The data was collected at follow-up examinations performed one year after operation and from the replies to a questionnaire distributed to the patients 2--6 years postoperatively. Analysis of the data showed that 95% of the patients considered the condition of the shoulder joint as good or at least as better than preoperatively. During the period of follow-up 23 redislocations occurred and 25% of the patients experienced postoperative pain in the operated shoulder joint. Postoperative limitation of movement of the joint was in general slight and did not hinder normal activities. Owing to the high frequency of redislocation the Putti-Platt operation is not recommended in any event for active sportsmen, since redislocation occurred to a statistically significant extent more often in this group than in other patients.