CONTROL OF VIRAL DISEASES TRANSMITTED IN A PERSISTENT MANNER BY THRIPS IN PEPPER (TOMATO SPOTTED WILT VIRUS).

Tomato spotted wilt disease is caused by Tomato Spotted Wilt Virus (TSWV) (Tospovirus, Bunyaviridae), a virus that severely damages and reduces the yield of many economically important plants worldwide and actually it is a major disease affecting the production of tomato and pepper in Italy. Due to the non-predictive nature of its outbreaks combined with the lack of forecasting, adoption of preventive measures have not always been practical, in fact the disease cycle has proven to be extremely difficult to break because of the wide and often overlapping host range of both the virus and the thrips vectors, which transmit the virus in a persistent, circulative, and propagative manner. Moreover recently, resistance breaking (RB) isolates of TSWV that overcome the resistance conferred by the Tsw gene in different pepper hybrids have been recovered in different locations in Italy and also in Brazil, USA, Spain and Australia, and this occurrence raises the question on the importance of a new approach of integrated pest management for TSWV management, including both control of its insect vector and the induction of the plant's resistance against viral infection. In this perspective, a study was performed in 2012 and 2013 with the purpose of evaluating the efficacy of the insecticide Cyantraniliprole alone or combined with Acibenzolar-S-Methyl (ASM), inducer of systemic acquired resistance, in the control of tomato spotted wilt disease in pepper. The experiment was performed in laboratory, in a thermo-conditioned greenhouse, into separate insect-proof cages and consisted of 5 treatments and 2 applications (plus a pre-transplant application for treatments were ASM was used. Variables were the mode of application of ASM in pre-transplant (by foliar or by drench) and the duration of the exposure time of the treated plants to viruliferous insects. Pepper cv. Corno di Toro, devoid of any resistance to TSWV, was used. Plants were observed daily to record any symptom induced by TSWV and/or of phytotoxicity. The possible TSWV infection was verified by DAS-ELISA using antisera supplied by Agdia Biofords. It was shown that the combination of ASM and Cyantraniliprole allowed the best control of TSWV transmission by thrips and of the development of the infection in treated pepper plants, regardless of the exposure time to thrips infestation. The application via drench of the combination seems to be the most effective. The efficacy of Cyantraniliprole used alone was comparable to that obtained with its combination with ASM only when plants were exposed to viruliferous insects for 24 h after product application, and the effectiveness of protection decreased with increasing time of exposure to viruliferous thrips. Treatment with ASM alone determined a good protection to plants regardless of the length of the exposure timing to viruliferous insects, with a smaller number of infected plants and milder symptoms compared to the check. Results of our current experiments raise the interesting possibility to combine in one product an insecticide and a non-specific (to various biotic and abiotic stresses) resistance inducer, particularly against diseases caused by viruses, and offer great opportunity for the integrated pest management (IPM) strategy for the control of pepper diseases.

Resistance breaking tomato spotted wilt virus isolates on resistant pepper varieties in Italy.

In spring 2012, resistance breaking (RB) isolates of tomato spotted wilt virus (TSWV) that overcome the resistance conferred by the Tsw gene in different pepper hybrids have been recovered in different locations in southern Italy (Campania and Apulia regions) in protected cultivation, about one month after transplant. The percentage of symptomatic plants was 5-10% and, only in particular cases of advanced stage of cultivation, it reached 30-50% at the end of cycle. All TSWV isolates induced similar systemic symptoms in all resistant infected pepper hybrids: yellowing or browning of apical leaves, which later become necrotic, long necrotic streakson stems, extending to the terminal shoots, complete necrosis of younger fruits and large necrotic streaks and spots on fruits formed after infection. On ripe fruits, yellow spots with concentric rings or necrotic streaks could be observed. Leaf extracts of these samples were tested in ELISA for the detection of TSWV, Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Impatiens necrotic spot virus (INSV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Pepper mild mottle virus (PMMoV) and Pepper Mottle Virus (PepMoV). Only TSWV was detected in all the field samples tested. The correspondent virus isolates were inoculated mechanically and by Frankliniella occidentalis on to a set of different pepper and tomato hybrids, as well as on some herbaceous test plants, in order to investigate for their ability to overcome the resistance genes Tsw and Sw5, respectively. Tomato hybrids carrying the Sw5 gene were uninfected by all RB isolates, whereas all resistant pepper hybrids became systemically infected. RB isolates did not differ noticeably in transmission efficiency when they were tested with the thrips F. occidentalis. Obtained results demonstrate that evolved strains of TSWV have emerged, that they are able to overcome the Tsw resistance gene in pepper plants experimentally inoculated both by mechanical infection and by the vector F. accidentalis. Moreover, evolved strains, here indicated as TSWV-RB-PI (Pepper, Italy), are not able to infect tomato plants carrying the Sw5 gene, confirming that distinct viral gene products control the outcome of infection in plants carrying Sw5 and Tsw. The simultaneous presence of evolved TSWV strains, showing the same biological characteristics (such as host range and symptomatology on different hosts) in two geographic areas of southern Italy, very far each other, let us hypothesize a unique focus of infection. Only subsequently, infected plants, through trade of plant material, have been diffused in locations so far apart. Further molecular studies are underway to assess the possible correlation between the different isolates in different geographical areas.

New report of Phoma glomerata on Coriandrum sativum L.

In 2011, coriander plants (Coriandrum sativum L.), showing symptoms of greyish to hell brown lesions with dark brown border between discoloured and asymptomatic tissues, were observed in Bulgaria. Pycnidia with small unicellular conidia appeared in the pale centre of the lesions. In this work, the identification of the causal agent of this disease was made applying Koch's postulates. Moreover, for a rapid and unambiguous identification of the fungal species, the internal transcribed spacers and the 5.8 rDNA gene (ITS1-5.8-ITS2) were amplified by PCR from DNA extracted from the isolated colonies. The amplicon was sequenced and analyzed using BLASTn, and showed a homology of 100% with a corresponding sequence of Phoma glomerata (accession number DQ093699). The fungus isolated, after the morphological and molecular characterization, was ascribed to the species Phoma glomerata (Corda) Wollenweber and Hochapfel. This is the first report of P. glomerata as agent of stem rot of coriander in Bulgaria and elsewhere.

Evaluation of efficacy and effect of application timing of a new herbicide, a.i. propoxy-carbazone + iodosulfuron + mefenpyr on Triticum durum.

A study was performed from February to May 2010 by Bioagritest according to EPPO guidelines and Principles of Good Experimental Practice (GEP), in the land of Altamura (BA), in the core of Murgia, Apulia Region (Italy). The purpose of the study was to evaluate the efficacy and effect of two different application times of the herbicide SIT90 (propoxycarbazone + iodosulfuron + mefenpyr) on Triticum durum for weeds' control. Sit90 was applied alone or in combination with the herbicide Dicuran (a.i. chlortolorun) in early post-emergence, and in late post emergence once more alone or in combination with the adjuvant Biopower or with the commercial formulate Atlantis WG (mesosulfuron+ iodosulfuron+ mefenpyr) + Biopower. T. durum cultivar was "Iride". The study has given sufficient results on the use of SIT90, in the conditions foreseen by the protocol and in consideration of the present weeds, which represented the species mostly diffused in the Murgia, mainly Avena ludoviciana and Papaver rhoaes. Treatments with the SIT90 alone (even in combination with Chortoluron), applied in early or in late post-emergence, were very ineffective on grass weeds and dicotyledonous, highlighting the importance of the adjuvant Biopower to enhance the expression of the herbicide. In fact, the presence of the adjuvant allowed the expression of a clear and good overall herbicide activity of SIT90 for the control of P. rhoaes and other dicotyledonous, but also a discrete activity against grasses. More reliable was the treatment with SIT90 mixed with Atlantis, against the entire community of grass weeds and on F. officinalis. The performance of all the treatments was insufficient on P. rhoaes, because of the clear resistance shown by the weed.

Chlorantraniliprole/lambda-cyhalothrin, a new insecticide mixture to control Tuta absoluta and Spodoptera littoralis in tomato.

A study was performed in the period May-July 2011 by Bioagritest test facility according to EPPO guidelines and Principles of Good Experimental Practice (GEP), in the land of Eboli (SA), southern Italy, with the purpose to test a new insecticide mixture in the defense strategies of processing tomato against Tuta absoluta and Spodoptera littoralis. The insecticide mixture Chlorantraniliprole 100 g/lambda-Cyhalothrin 50 g/l 0,4 It/ha was applied in a tomato field in four (A-B-C-D) application timings (a.t.) alone (treatment 2) and in two a.t. (A-B) combined with emamectin benzoate 0.5%, 1,5 Kg/ha (a.t. C-D) (treatment 3). Its effect in containing insect populations was compared with that exerted by emamectin benzoate 0.5%, 1,5 Kg/ha (a.t. A-B-C-D) (treatment 4) and the combination of chlorantraniliprole 35%, 0.115 Kg/ha (a.t. A-B) and indoxacarb 30% 0.125 Kg/ha (a.t. C-D) (treatment 5). Application of treatments (trt.) and mode of assessment (2 assessments), recording and measurements followed the guidelines foreseen by EPPO Standards PP1/150 and PP1/275. The lepidoptera infestation in the tomato crop was very high on both the leaves and fruits, and this enabled us to evaluate the efficacy of the tested products in conditions of severe pests infestation. If we consider the leaf damage, either as number of mines by T. absoluta or as % of leaf erosion by S. litteralis, all the treatments values were statistically lower than control, but with no difference among them. But when using as an infestation threshold the percentage of damaged fruits (much more relevant in economic terms), we observed statistically significant differences among treatments. Actually, treatment with chlorantraniliprole-lambda-cyhalothrin mixture was significantly more effective than the one with emamectin benzoate in reducing the attack of T. absoluta on tomato fruits, in both dates of assessments. As concerns damage by S. littoralis on the fruits, at the first assessment, trt. 2 and trt. 5 significantly reduced the percentage of infested berries in comparison with the control, whereas the infestation was not significantly reduced by trt. 3 and trt. 4. At the second assessment, no statistically significant difference between the four different treatments was observed. Since the infestation of tomato fruits by S. littoralis was much lower than the one caused by T. obsoluta, the overall performance of the four insecticide treatments was mainly due to the control effects towards T. obsoluta.

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications.

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5µm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.

Relationship between the particle size distribution of commercial fully porous and superficially porous high-performance liquid chromatography column packings and their chromatographic performance.

The separation efficiency and kinetics of several commercial HPLC particle types (both fully porous and superficially porous) have been investigated using a pharmaceutical weakly basic N-containing compound as a test molecule. A strong trend between the particle size distribution (PSD) of the particles and the typically employed "goodness of packing"-parameters was observed. The relative standard deviation of the PSD of the tested particles ranged between 0.05 and 0.2, and in this range, a near linear relationship between the A-term constant, the h(min)-value and the minimal separation impedance was found. The experimental findings hence confirm the recent observations regarding the relationship between the narrow PSD of the recently commercialized porous-shell particles and their superior efficiency and kinetic performance. The outcome also suggests that the performance of the current generation of fully porous particle columns could be significantly improved if the PSD of these particles could be reduced.

Evaluation of the efficacy and selectivity of Oberon (Spiromesifen) for the control of Tetranychus urticae on strawberry.

A study was performed in the period June to July 2009 by Bioagritest test facility according to EPPO guidelines and Principles of Good Experimental Practice (GEP), in the campaign of Tursi (MT), southern Italy, in order to evaluate the efficacy and selectivity of Spiromesifen on strawberry for the control of Tetranyichus urticae. Two different dosages of OBERON (a.i. Spiromesifen)--45 and 60 ml/hl--were compared with a unique dosage of two commercial formulates: VERTIMEC (a.i. Abamectine, Syngenta Crop Protection), 60 ml/hl, and MAGISTER (a.i. Fenazaquin, Dow AgroSciences), 60 ml/hl. The study has achieved the purpose of evaluating/measuring with a single application the activity of Spiromesifen (Oberon) to control T. urticae on strawberry and its selectivity on phytoseiids. All the treatments differed significantly from the checks and showed high ability to control mites, on all stages of the population. About selectivity on phytoseiids, only Spiromesifen (at both doses) has demonstrated a good selectivity, while Abamectine (in part) and Fenazaquin (totally), have limited the population of the predator. The study confirmed the usefulness, indeed the need for the new compounds, to get confirmation of their selectivity against the useful entomofauna. The experiment has allowed to demonstrate the suitability of Spiromesifen to be included in strategies for strawberry integrated pest management.

Evaluation of the efficacy of Oberon (Spiromesifen), to contain infestations of mites and whiteflies on Capsicum annuum L.

Following the EPPO guidelines and Principles of Good Experimental Practice (GEP), an experiment was realised in autumn 2008 for evaluating the efficacy of Oberon applied by foliar treatments to contain infestations of mites and whiteflies on Capsicum annuum L.. Two different dosages of OBERON (a.i. Spiromesifen)--45 and 60 g/hl--were compared with a unique dosage of two commercial formulates: VERTIMEC (a.i. Abamectine, Syngenta Crop Protection), 60 g/hl, and MAGISTER (a.i. Fenazaquin, Dow AgroSciences), 110 g/hl. Oberon resulted very effective in the control of phytopathogenic mites at both doses of 45 and 60 g/hl. Its effectiveness demonstrated to be remarkable for approximately one month after application. By contrast, Vertimec and Magister have proven their effectiveness for a much lower period of time (about the first 15 days post application). About the efficacy against whiteflies, even 36 days after the foliar application Oberon showed a strong containment of the populations of aleurodides. There were no phenomena of phytotoxicity nor on leaves nor on flowers and fruits, in none of the treatments. About the phytotoxicity on the useful entomofauna, the assessments made on the different treatments have highlighted the lack of harmful effects on predators and on parasitoids of insects and mites.

First record of tomato yellow leaf curl Sardinia virus (TYLCSV) on pepper in Italy.

During a survey in summer 2007, a disease of pepper (Capsicum annuum) under plastic tunnels was observed in Policoro (Matera), on the Ionic coast of Basilicata Region, with a disease incidence in some cases of more than 50%. Affected cultivars were Eppo and Almund (S Et G). The diseased plants exhibited light mosaic or mottling, leaf distortion, interveinal and marginal leaf chlorosis, upward curling of leaf margins of older leaves. The causal pathogen was suspected to be a begomovirus due to the large population of the whitefly Bemisia tabaci observed on the crop. Detection assays for Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV) were used. In DAS-ELISA, positive results (178 plants resulted positive over 200 symptomatic plants assayed) were obtained using a "broad-spectrum" reagent combination (distributed by Bioreba AG) detecting TYLCV, TYLCSV, and other begamoviruses. A couple of synthetic oligonucleotides allowing the amplification of the whole coat protein (CP) gene of TYLCSV and TYLCV was used for PCR of ELISA positive samples in order to perform the molecular characterisation of the viral isolate responsible of the disease. RFLP analysis performed on the PCR product, 1008 bp long, showed the presence of only TYLCSV in the infected pepper plants. The same couple of primers allowed the detection of the virus also in symptomless pepper plants. To test whitefly transmission, adults of B. tabaci allowed to feed on naturally infected pepper plants were transferred on 10 healthy Eppo pepper seedlings (15 whiteflies/plant). Insects were killed 2 days later using an insecticide. Twenty days post exposition 10 plants/10 resulted positive in ELISA, and showed the same symptoms observed in natural infection. TYLCSV was not reported before on pepper in the surveyed area, but it was recorded with severe outbreaks on tomato, both in protected and in open field crops. This species was probably the primary source of infection from which subsequent diffusion by way of the vector B. tabaci followed on pepper. To our knowledge this is the first time that a natural infection of TYLCSV on pepper is recorded in Italy, with serious implications for the epidemiology of TYLCSV in our country.

Emamectin benzoate: new insecticide against Helicoverpa armigera.

Emamectin benzoate is a new insecticide of Syngenta Crop Protection, with a new mechanism of action and a strong activity against Lepidoptera as well as with and a high selectivity on useful organisms. This molecule acts if swallowed and has some contact action. It penetrates leaf tissues (translaminar activity) and forms a reservoir within the leaf. The mechanism of action is unique in the panorama of insecticides. In facts, it inhibits muscle contraction, causing a continuous flow of chlorine ions in the GABA and H-Glutamate receptor sites. During 2006 and 2007, experimentation was performed by the Bioagritest test facility, according to EPPO guidelines and Principles of Good Experimental Practice (GEP), aiming at establishing the biological efficacy and the selectivity of Emamectin benzoate on industry tomato against Helicoverpa armigera (Lepidoptera: Noctuidoe). The study was performed in Tursi-Policoro (Matera), southern Italy. Experimental design consisted in random blocks, in 4 repetitions. A dosage of 1.5 Kg/ha of the formulate was compared with two commercial formulates: Spinosad 0.2 kg/ha (Laser, Dow Agrosciences Italia) and Indoxacarb 0.125 kg/ha (Steward EC insecticide, Dupont). Three foliage applications were applied every 8 days. The severity of damage induced by H. armigera was evaluated on fruits. Eventual phytotoxic effects were also evaluated. Climatic conditions were optimal for Lepidoptera development, so that the percentage of fruits attacked in 2007 at the first scouting was 68.28%. Emamectin benzoate has shown, in two years of testing, a high control of H. armigera if compared with the standards Indoxacarb and Spinosad. No effect of phytotoxicity was noticed on fruits.

Integrated management of TYLCV/TYLCSV on greenhouse hydroponic tomatoes in Southern Italy.

Tomato yellow leaf curl (TYLC) caused by Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), vectored by the whitefly Bemisia tabaci, is a major disease of tomato in Sardinia and Sicily, and is becoming a serious threat in Southern Italy too. TYLCSV was first reported in Calabria region in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Later, during the 2003-2004 winter, a serious epidemic was observed in protected tomato crops in Castrovillari, Cosenza province. TYLCV was first described in Sicily in 2003 and during 2004 in continental Italy. Both viruses were detected in winter 2005-2006 on the Basilicata Ionic coast, in the Metapontum area, both in protected and in open field tomato crops. Experiments were conducted in Calabria Region, Southern Italy, under controlled conditions in a group of greenhouses where several tomato crops were grown hydroponically to determine the separate and integrated effects of UV-reflective mulch (UVRM), Acibenzolar-S-methyl (Actigard) and the two insecticides Imidacloprid (ADMIRE 2F) and Thiamethoxam (ACTARA 25WG). Highly UV-reflective mulch covered plots were treated with Actigard and insecticides, both alone or in combination. TYLC disease incidence was determined from late August 2005 to late January 2006. The highly UVRM alone was effective in reducing disease incidence of about 28.6% at the end of October, and of 31.7% at the end of January. However, Actigard with UVRM significantly reduced TYLC disease incidence to 70% and 48.5%, in 2 months and 5 months after the first treatment, respectively. The insecticides with UVRM, resulted in a moderate reduction of disease incidence (22.5%) at the end of October. At the end of January a reduction in disease incidence due to insecticide applications was not significant. The use of Actigard combined with the insecticides on UVRM reduced the disease incidence (63.4% with Admire and 56.1% with Actara) at the end of January. Actigard alone or with insecticides on UVRM was effective in reducing disease incidences. Highly UVRM and Actigard were effective in reducing the primary spread of TYLCV/TYLCSV in greenhouse hydroponic tomatoes. Comparative analysis of their effects at different periods post-treatment suggests that multiple applications of Actigard may be necessary to reduce progress of this disease.

Potato virus Y CFH, a putative recombinant isolate from Capsicum chinense cv. Habanero.

Ornamental plants of Chili pepper, Capsicum chinense cv. Habanero, with symptoms of leaf mosaic, necrotic rings on fruits and necrotic stems were observed in June 2003 in a private garden in the province of Naples (Italy). Preliminary serological characterisation allowed the association of these symptoms with infections by Potato virus Y (PVY). The virus was isolated on Nicotiana tabacum cv. Xanthi and characterised by mechanical inoculation on herbaceous hosts and molecular characterisation of the P1 and the coat protein (CP) genes. Symptoms produced on indicator plants were generally consistent with those described for PVY. The identity of PVY was further confirmed by reaction with PVYN, PVYC and PVYO specific monoclonal antibodies: the isolate reacted only with the PVYC specific Mab. Immuno capture reverse transcription polymerase chain reaction (IC-RT-PCR) was performed on extracts of PVY-CFH infected N. tabacum cv. Xanthi plants, using two couples of primers specifically designed out of the P1 and the CP coding regions of the so far fully sequenced PVY isolates. PCR products were then cloned into pCRII-TOPO vector using TOPO-TA cloning kit (Invitrogen) and sequenced. Sequence analysis suggests that PVY-CFH originated from a recombination event involving a virus of the PVYO type and another parental virus, maybe resembling the PVYNP isolates, given the reasonably high similarity shared by PVY-CFH and, respectively, non potato PVY isolates in the CP coding region, PVYO isolates in the P1 coding region. Evidence for the existence of such a recombination comes, apart from similarity analysis, by the different locations of CFH within phylogenetic trees constructed from P1 and CP genomic regions.

Characterisation of Potato virus Y nnp strain inducing veinal necrosis in pepper: a naturally occurring recombinant strain of PVY.

The full-length genome of Potato virus Y (PVY) nnp strain, recovered from pepper showing veinal necrosis of leaves, was cloned and sequenced, finding an organisation typical for PVY species. It consists of 9699 nucleotides (nt) excluding the 3' terminal poly(A) tail and contains an open reading frame of 9186 nt, encoding the putative polyprotein of 3061 amino acids. In ELISA, the isolate reacted with a monoclonal antibody specific for PVY(C) but not with antibodies against PVY(N) or PVY(O). Sequence analysis strongly suggests that PVY-nnp originated from a recombination event involving a virus of the PVY(O) type and another parental virus, maybe resembling the PVY(NP) isolates, given the reasonably high similarity shared by PVY-nnp and Lye84.2 and Son41 isolates. The recombination event involved a breakpoint near the middle of the P1 gene, around position 603 of the viral genome. Proof for the existence of such a recombination comes from several lines of evidence, including similarity analysis, recombination analysis using six different methods and the different locations of nnp within phylogenetic trees constructed from genomic regions on either side of the identified recombination breakpoint.

Severe outbreaks of tomato yellow leaf curl Sardinia virus in Calabria, Southern Italy.

During the winter 2003--2004 a serious disease was observed in protected tomato crops in Castrovillari, Reggio Calabria province, Southern Italy. Symptoms consisted in marginal leaf yellowing, leaf curling, plant stunting, flower abortion. The disease was detected in a group of greenhouses (about 10ha) where several tomato cultivars were grown hydroponically. The highest incidence of infection (60-100%) was observed in tomatoes grafted on Beaufort DRS tomato rootstock. Since the symptoms were similar to those described for Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), detection assays for these viruses were used. In DAS-ELISA positive results were obtained with a abroad-spectrums reagent combination (distributed by Bioreba AG) detecting TYLCV, TYLCSV, and other begomoviruses. When DNA probes were used in tissue print assays, positive reactions were obtained for TYLCSV, but not for TYLCV. The two probes consisted of digoxigenin-labelled DNAs representing the coat protein gene of either TYLCSV or TYLCV. Attempts to isolate the viral agent by mechanical inoculation failed, except in few cases where Potato virus Y and Tobacco mosaic virus were identified following transmission from symptomatic plants to herbaceous indicatorpplants. By contrast, grafting onto tomato seedlings always successfully transmitted the disease. In the Castrovillari area TYLCSV was not reported before. The rootstocks that nurseries used for grafting were obtained from Sicily, where the disease is endemic and both TYLCSV and TYLCV are widespread. Probably the grafted plantlets represented the primary source of infection from which subsequent diffusion by way of the vector Bemisia tabaci followed. In fact the vector had previously been detected in both the glasshouse-grown and open field tomato crops in Calabria region. TYLCV was previously reported in a different area of Calabria in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Since in the Castrovillari area surveyed in the present study tomato is grown throughtout the year in protected crops, the whitefly vector of the virus is present, and some natural hosts of the virus are found, it is feared that TYLCSV may become endemic, as already happened in Sicily, Sardinia, and Spain several years ago. In Spain and Sicily TYLCV, together with TYLCSV, was reported as the causal agent of very severe tomato crop losses. Therefore the danger exists that also TYLCV will reach this area, furthermore complicating the management of tomato crops.

The complete nucleotide sequence of Plum pox virus isolates from sweet (PPV-SwC) and sour (PPV-SoC) cherry and their taxonomic relationships within the species.

Plum pox virus (PPV) sweet (SwC) and sour (SoC) cherry isolates were the first PPV isolates to be recovered from natural infection in sweet and sour cherry plants, respectively. Their complete nucleotide sequences have been determined finding a deduced genome organisation typical for PPV species. Both genomes are 9795 nucleotides long, excluding the 3' terminal poly(A) tail, and contain an open reading frame of 9432 nt, encoding a polyprotein of 3143 amino acids. The nucleotide and predicted amino acid sequences of PPV-SwC and SoC have been pairwise compared with available sequences of different PPV strains. Although a very high similarity exists between the whole genomes and polyproteins of the two cherry isolates, high levels of divergence have been calculated with sequences of PPV-M, D and EA isolates. In particular, the most considerable divergence has been found in part of 5' non coding region, in regions encoding P1, P3 + 6K1, 6K2 and NIa-VPg proteins as well as in the N-terminal domain of the coat protein. Phylogenetic analysis have been undertaken in order to establish the taxonomic localisation of SwC and SoC isolates within PPV species, showing that they are always clustered together and separated from the rest of PPV strains, being clearly the most distant.

A silicon-based biosensor for real-time toxicity testing in normal versus cancer liver cells.

The potentiometric alternating biosensor (PAB) system has been utilized to monitor the effects on normal versus cancer cells of Mitoxantrone, a potent inhibitor of nucleic acid synthesis commonly utilized in the clinical treatment of hepatocarcinomas. In this first series of toxicity tests, we have utilized a primary culture of rat hepatocytes and a stabilized line named HepG2 from a human hepatoma. Primary cultures of hepatocytes represent a unique tool in toxicology/pharmacology as shown in previous works. A suitable microvolume flow chamber has been designed and produced. The chamber is equipped with inlet and outlet circuits and with a fixed transducer (Si/SiO2/Si3N4 chip) facing a cover slip on which cells grow. The PAB system is here utilized only in the pH-sensitive configuration with a single measuring spot, warranting an accurate determination of the change in the extracellular acidifcation rate resulting from drug administration. We have also observed the changes in the acidification rate of 3T6 mouse fibroblasts, induced by a treatment with Ara-C, a well-known DNA antimetabolite. New insights can be obtained from these studies into the drug response of normal versus cancer cells and into the feasibility of real-time PAB monitoring of drug toxicity and efficacy, towards effective human cancer treatment. The effect of increasing drug concentrations on cellular metabolism is here compared with the results obtained from conventional tests (optical microscopy, Neutral Red and Trypan Blue assays).