Sphaceloma coryli: A Reemerging Pathogen Causing Heavy Losses on Hazelnut in Southern Italy.

In 2013 to 2014, a disease causing severe crop losses was observed in several growing areas of the Campania region in southern Italy. Disease symptoms consisted of necrotic spots on leaves, bracts, and shells along with vein necrosis. Nuts were necrotic or aborted and symptomatic fruit dropped prematurely. A fungus was consistently isolated from symptomatic tissues and morphologically identified as Sphaceloma coryli. The fungus was first reported in France and Italy (Campania) over 30 years ago and was not subsequently documented until 2006 in the Latium region of Italy. When artificially inoculated on healthy hazelnut plants, disease symptoms were reproduced. The teleomorph of S. coryli was recorded for the first time on overwintering tissues of hazelnut trees. It was also induced in vitro. The morphology of asci and ascospores together with phylogenetic analysis based on internal transcribed spacer sequences indicated that the teleomorph is an undescribed species within the genus Elsinoë for which the name of Elsinoë coryli is proposed. Optimum temperatures for ascospore and conidium germination and mycelium growth were determined in vitro. The inhibition effect of some commercial fungicides on S. coryli was also evaluated in vitro.

Viroid-like RNAs from cherry trees affected by leaf scorch disease: further data supporting their association with mycoviral double-stranded RNAs.

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88%), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

Viroid-like RNAs from cherry trees affected by leaf scorch disease: further data supporting their association with mycoviral double-stranded RNAs.

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88 %), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

First Report of Tomato spotted wilt virus on Tobacco in Campania, Italy.

During the spring and summer of 2010, a survey for viruses was conducted in two tobacco field trials at the Agricultural Research and Experimentation Council in Scafati, Campania, Italy. A total of 1,392 symptomatic and asymptomatic tobacco plants (cv. Burley) were sampled, and leaves were analyzed by double-antibody sandwich-ELISA using polyclonal antisera against five tobacco-infecting viruses: Tobacco mosaic virus, Potato virus Y, Cucumber mosaic virus, Alfalfa mosaic virus, and Tomato spotted wilt virus (TSWV) (Loewe, Munich, Germany). Only one plant was positive to TSWV. Symptoms on this plant were severe necroses on the stem with a few chlorotic/necrotic leaflets on the top of the plant. This result was subsequently confirmed by reverse transcription (RT)-PCR. The primers (5'-ATGTCTAAGGTTAAGCTC-3' forward and 5'-TTAAGCAAGTTCTGTGAG-3' reverse) targeted the nucleocapsid gene of TSWV and amplified the expected product of approximately 800 bp (2). The resultant sequence (GenBank Accession No. JF290419) was aligned and edited using BlastN, displaying 99.9% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets, which confirmed the presence of TSWV in tobacco. Leaf homogenate from the tobacco symptomatic plants was inoculated onto three plants of Nicotiana benthamiana, N. glutinosa, and Datura metel. All plants developed a systemic necrosis after 7 days. Inoculation from N. glutinosa back to cv. White Burley tobacco produced symptoms similar to those observed in the field. Two plants from each species were used as noninoculated controls, which remained asymptomatic. TSWV infection has been responsible for severe epidemics on tobacco throughout the United States and Greece, with losses estimated as much as 85% (1,3). The presence of TSWV in Italy could therefore represent a serious threat for tobacco in the region, especially considering that it is prevalent in other crops in the area and vectors are widespread. References: (1) E. K. Chatzivassiliou. Plant Dis. 92:1012, 2008. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) B. Mandal et al. Ann. Appl. Biol. 151:67, 2007.

First Report of Peach latent mosaic viroid Infecting Peach in Egypt.

Peach latent mosaic viroid (PLMVd) is a widespread pathogen of stone fruit trees in some European and Mediterranean countries and also in North America. To access the presence of the viroid in Egypt, a survey was conducted that covered five commercial peach orchards in the El Khatatba Region in Al Minufiya Governorate. During 2003 and 2004, 73 peach trees (cv. Florida grafted on Nemagard rootstock) were visually inspected and sampled. No symptoms characteristic of PLMVd infection, such as mosaic, delayed growth, or fruit suture cracking, were observed. All samples were tested for the presence of PLMVd using dot-blot hybridization and reverse transcription (RT)-PCR. Aliquots (5 µl) of total nucleic acids extracted from approximately 2 mg of leaf tissue were spotted onto positively charged nylon membranes and hybridized under stringent conditions with a digoxigenin-labeled riboprobe (2). The extracts (1 µl) also were used in RT-PCR as described (1). Only 1 of the 73 peach trees was positive for PLMVd using these detection techniques. The RT-PCR product was of the size expected for PLVMd and was cloned and sequenced. The 339 nucleotide sequence was deposited in GenBank as Accession No. DQ839564. The sequence of this Egyptian PLMVd isolate was 94% identical to the reference PLMVd variant (GenBank Accession No. M83545) and most closely (95%) related to Canadian isolate variant 16 (GenBank Accession No. AJ550911). Such a low incidence compared with other countries may be because the survey was restricted to a limited number of samples, conducted on newly reclaimed lands where no sources of infection existed before, and material with relatively low PLMVd incidence might have been used for planting. Although the incidence of PLMVd was low in this survey, the occurrence represents a threat to the stone fruit tree industry in this country and regular screening of PLMVd in certification programs is suggested. To our knowledge, this is the first report of PLMVd on peach in Egypt. References: (1) S. Loreti et al. EPPO Bull. 29:433, 1999. (2) A. M. Shamloul et al. Acta Hortic. 386:522, 1995.

First Report and High Prevalence in Noncherry Host of Cherry virus A in Italy.

Cherry virus A (CVA) has been reported to naturally infect sweet and sour cherry, apricot, peach, and plum but has not been associated with any symptoms. In the spring and summer of 2005, samples were collected in Prunus spp. germplasm collections in Campania (Italy) and analyzed by a polyvalent reverse transcription-polymerase chain reaction (RT-PCR) assay (1). Amplicons were sequenced, revealing CVA infection in seven apricot cultivars (Prunus armeniaca L.), one plum (Prunus domestica L.), and one cherry (Prunus avium L.). CVA sequences (GenBank Accession Nos. DQ445275 to DQ445292) compared among themselves and with databank sequences showed pairwise nucleotide sequence identity levels of 80.3 to 99.6% (86.8 to 100% for amino acid sequences), significantly extending the known variability range of CVA. The presence of CVA was confirmed by hybridization with a CVA-specific probe (P39) (2), targeting region different from that amplified in the polymerase chain reaction assay. All samples containing CVA were found to be in mixed infections with Apple chlorotic leaf spot virus (ACLSV) and Apricot pseudo-chlorotic leaf spot virus (plum, apricot), or ACLSV plus Cherry green ring mottle virus (cherry) so that CVA symptomatology could not be evaluated. To our knowledge, these results constitute the first report of the occurrence of CVA in Italy. They confirm the ability of CVA to naturally infect noncherry Prunus spp. hosts with surprisingly high prevalence levels (6 of 14 and 1 of 6 tested apricot and plum cultivars, respectively). References: (1) X. Foissac et al. Phytopathology 95:617, 2005. (2) W. Jelkmann J. Gen. Virol. 76:2015, 1995.

Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees.

ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.

A Survey of Citrus Viroids in Campania (Southern Italy).

Within a certification program of citrus, a survey covering 14 citrus fields not previously screened were randomly selected in seven areas of the Campania Region of southern Italy to evaluate the presence and distribution of citrus viroids in the region. During the last 3 years, 112 trees were visually inspected and sampled including 14 'Navelina', 8 'Washington navel', 13 'Biondo Commune', 10 'Tarocco', 6 'Valencia late', 1 'Sanguinello', and 1 'Vanilla' sweet oranges (Citrus sinensis), four sour oranges (C. aurantium), 21 'Commune' and 7 'Monreale' clementines (C. clementina), 8 'Common' and 2 'Avana' mandarins (C. reticulata), 8 'Ovale di Sorrento' and 6 'Zagara' lemons (C. lemon), 2 Tangelos (C. reticulata × C. paradisi), and 1 grapefruit (C. paradisi). Nearly all (37 of 38) mandarin and clementine trees showed cachexia-like symptoms. Severe bark scaling that is characteristic of exocortis disease was observed on a single 'Ovale di Sorrento' lemon, the only tree incidentally found to be grafted onto the exocortis-sensitive trifoliate orange (Poncirus trifoliata) rootstock, since sour orange (C. aurantium) is the main rootstock used in Campania. Samples (104 of 112) that had been graft-inoculated into Arizona 861-S1 'Etrog citron' (C. medica L.) on rough lemon (C. jambhiri Lush.) rootstock showed typical viroid symptoms. Viroid infection was confirmed by sequential polyacrylamide gel electrophoresis analysis (2) and molecular hybridization of imprinted membranes (1). Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III) were the most frequently detected, accounting respectively, for 67.9, 86.6, and 84.8% of the infected samples. Citrus viroid IV (CVd-IV) and Citrus bent leaf viroid (CBLVd) were found in 24.1 and 13.4% of the sources. CBLVd was detected in sweet orange ('Tarocco' and 'Biondo Commune') and lemon ('Ovale di Sorrento' and 'Zagara'), while CVd-IV was detected in sweet orange ('Tarocco', 'Biondo Commune', and 'Washington navel'), lemon ('Ovale di Sorrento' and 'Zagara'), clementine ('Commune'), and sour orange. Most trees (82.1%) were infected with more than one viroid species. The most frequent viroid combinations were CEVd + HSVd + CVd-III (40.2%), HSVd + CVd-III (16.1%), and CEVd + CBLVd + HSVd + CVd-III + CVd-IV (12.5%). To our knowledge, this is the first report of CEVd, CBLVd, HSVd, CVd-III, and CVd-IV in the Campania Region and the first report of CBLVd and CVd-IV in Italy. References: (1) A. Palacio et al. Eur. J. Plant Pathol.105:897, 2000. (2) R. F. Rivera-Bustamante et al. Anal. Biochem. 156:91, 1986.

Variability among turnip mosaic potyvirus isolates.

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.

Apple dimple fruit viroid: Fulfillment of Koch's Postulates and Symptom Characteristics.

The previously reported properties of a small circular RNA isolated from plants of cultivar Starking Delicious with symptoms similar to those of the dapple apple disease induced by Apple scar skin viroid (ASSVd), strongly suggested that it is a distinct viroid species of genus Apscaviroid to which ASSVd also belongs. Nevertheless, the autonomous replication of this novel RNA, termed Apple dimple fruit viroid (ADFVd), and the nature of the symptoms incited when inoculated free of other pathogens that might be present in the original source, were not determined. We addressed these questions by mechanically inoculating purified ADFVd into young apple seedlings (Golden). Dot blot hybridization of leaf extracts collected ten months after inoculation showed the presence of ADFVd in most of the inoculated seedlings, thus proving the ability of this RNA to replicate independently. Material from these ADFVd-infected seedlings grafted onto the ASSVd indicator Starkrimson incited the appearance on the fruit skin of discolored spots more or less depressed which were particularly frequent around the calyx cavity. Apple cultivars such as Gala, Pink Lady, and Braeburn reacted to ADFVd with similar symptoms, occasionally accompanied by scar skin in Braeburn, whereas ADFVd replicated without eliciting any visible symptoms in other apple cultivars such as Golden, or in the pear indicator Fieud 37. No interference in symptom expression or viroid accumulation were observed in plants inoculated first with ASSVd and then with ADFVd.

Tratamiento con urea como alternativa al tolvaptán en el síndrome de secreción inadecuada de la hormona antidiurética / Treatment with urea as an alternative to tolvaptan for the syndrome of inappropriate secretion of antidiuretic hormone

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[Treatment with urea as an alternative to tolvaptan for the syndrome of inappropriate secretion of antidiuretic hormone]. / Tratamiento con urea como alternativa al tolvaptán en el síndrome de secreción inadecuada de la hormona antidiurética.

El síndrome de secreción inadecuada de la hormona antidiurética (SIADH) es una afectación que cursa con hiponatremia entre otros síntomas. Actualmente la familia de fármacos de elección para este síndrome son los vaptanes, entre ellos el tolvaptán, antagonistas de la vasopresina. La urea actúa como diurético osmótico produciendo la retención de sodio. Se presenta un caso en el que el cambio desde el tratamiento con tolvaptán a urea en el SIADH permitió mantener la respuesta, a un coste muy inferior. La paciente tenía 83 años, ingresó en nuestro Hospital al diagnosticarle una hiponatremia grave (108 mEq/L) durante una exploración por una caída en su casa. Durante el ingreso ante la sospecha de SIADH se le prescribió sucesivamente tres tipos de tratamientos: Restricción hídrica y suero salino, seguido de tolvaptán 15 mg y para finalizar se le prescribió 15 gr de urea cada 12 horas. Durante el primer tratamiento los niveles de sodio sérico no aumentaron lo suficiente, por lo que se cambió al tolvaptán, durante el cual los niveles de sodio sérico subieron a rangos normales. Al considerar que el tratamiento iba a ser de larga duración se decidió cambiar a la urea, durante el mismo los niveles de sodio sérico tuvo un ligero aumento. La paciente es dada de alta con la pauta de urea de forma indefinida. El coste diario del tratamiento de tolvaptán es de 66,91 euros; el de urea supone 0,30 euros. En casos como el descrito, el uso de urea preparada como fórmula magistral por el área de farmacotécnia del Servicio de Farmacia, es una alternativa al tolvaptán con un coste marcadamente inferior.

Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease.

A new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (ASSVd) has been found. The new viroid, tentatively termed apple dimple fruit viroid (ADFVd), is a circular RNA of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. It contains the core nucleotides of the central conserved region (CCR) of the ASSVd group as well as the terminal conserved region (TCR) present in all members of the ASSVd and potato spindle tuber viroid (PSTVd) monophyletic groups. ADFVd has the highest sequence similarity with ASSVd and the 294 nucleotide citrus viroid CVd-IIIb sharing with the latter an almost identical left terminal domain. The right- and left-hand termini of ADFVd are formed by almost perfect duplications of sequences found in the CCR upper and lower strands, respectively, of PSTVd and closely related viroids.

Variability of the coat protein gene of Citrus psorosis virus in Campania, southern Italy.

Variability of the Coat protein (CP) gene of Citrus psorosis virus (CPsV) was assessed serologically, and by sequence analyses of two genomic regions located in the 3' (region C) and 5' (region V) halves of the gene. Analysis of 53 psorosis field sources from Campania, Italy, with 23 monoclonal antibodies revealed nine serogroups and at least ten different epitopes. Sequence analysis of 19 of these sources showed limited nucleotide diversity of the CP gene in the population. Diversity was slightly higher in region V than in region C. Phylogenetic analysis of the V and C regions of the CP showed that the Campania sources of CPsV were clearly separated from the CPsV-4 isolate from Florida. For C region, most of the CPsV sources clustered together, whereas two clusters were observed for region V. The ratio between nonsynonymous and synonymous substitutions for regions C (0.083) and V (0.345) indicated negative selective pressure for amino acid changes, more intense in the C region. No correlation was found between serogroups and specific aminoacid sequences, field location or citrus cultivar.