First report of Tomato leaf curl Sinaloa virus infecting tomato crops in Panama

In April 2011 and September 2012, virus-like symptoms were observed in open field- and greenhouse-grown tomato crops (Solanum lycopersicum) in Chiriqu\ued, the westernmost province of Panama. Samples from symptom-bearing plants (127 in all) were collected and tested for the presence of begomoviruses by polymerase chain reaction (PCR) assays with sets of degenerated primers designed to amplify parts of the DNA-A and DNA-B components (Rojas et al., 1993; Table 1). Products of the expected sizes, obtained with both DNA-A- and DNA-B-specific primers for 49 samples, suggested infection with New World bipartite begomoviruses. This corresponds to an incidence of 26% (8 plants) in open field, and 43% (41 plants) in greenhouse crops. Primers specific for ten tomato-infecting begomoviruses found in Central America (Engel et al., 1998; Nakhla et al., 2005; Table 1) were used to typify the PCR-positive samples

First report of Tomato leaf curl New Delhi virus affecting zucchini squash in an important horticultural area of southern Italy

omato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) which infects species in the families Cucurbitaceae and Solanaceae (Padidam et al., 1995; Mizutani et al., 2011). Begomoviruses are transmitted by the whitefly Bemisia tabaci in a persistent manner (Rosen et al., 2015). In October 2015, severe symptoms not previously reported by growers in the horticultural area of the Province of Trapani (Sicily, Italy) were observed on zucchini squash (Cucurbita pepo) in open fields. The symptoms included yellow mosaic, severe leaf curling, swelling of veins of young leaves, shortening of internodes, roughness of the skin of fruit and reduced fruit size; the symptoms were reminiscent of those caused by begomoviruses. Total DNA was extracted from young leaves of 22 plants by phenol/chloroform extraction and ethanol precipitation. PCR was performed with the A1F/A1R primer pair (Mizutani et al., 2011) for the DNA-A component and the pair described by Ruiz et al. (2015) for the DNA-B component to amplify a ~1200-bp fragment of DNA-A and a ~890 bp fragment of DNA-B, respectively. All 10 samples were positive by PCR with both primer pairs. No amplification products were obtained using primers specific for the monopartite begomoviruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus (Davino et al., 2008). DAS-ELISA analysis for Cucumber mosaic virus, Papaya ring spot virus and Zucchini yellow mosaic virus (Loewe Phytodiagnostica, Germany) yielded negative results

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques

Background: Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods: Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. Results: The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time

First outbreak of Pepper vein yellows virus infecting sweet pepper in Italy

Sweet pepper (Capsicum annum) is an economically important crop worldwide, including Sicily where about 4,000 hectares are grown each year. In October 2015, severe symptoms not previously reported by growers in the horticultural area of the province of Trapani (Sicily, Italy) were observed on sweet pepper plants in eight different greenhouses. Symptoms included upward leaf curling, internodal shortening and interveinal yellowing. Symptoms were more evident in the upper part of the plants. These symptoms were reminiscent of those caused by poleroviruses. In the greenhouse, symptoms were evident in about 35% of the plants. Three samples per greenhouse (24 in total) were collected for analysis

OCCURRENCE OF TOMATO LEAF CURL NEW DELHI VIRUS INFECTING ZUCCHINI IN SARDINIA (ITALY)

Tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) is a bipartite, circular, ssDNA virus, able to infect species within the Cucurbitaceae and Solanaceae. In August 2016, field observations carried out in Sardinia (Italy) highlighted in one location (Decimoputzu, CA) some plants of zucchini squash (Cucurbita pepo L.) showing a systemic disease never observed before, even in a previous survey (end-June 2016) on cucurbit viruses

First report of Southern tomato virus in tomato crops in Italy

Twenty-five tomato plants (Solanum lycopersicum) showing symptoms of viral disease were sampled from different greenhouses in the Ragusa province (Southern Italy) in summer 2015. Plants showed chlorosis on leaves and fruits and deformation and depressed spots of dark colour which later evolved into necrosis (Fig. 1). These symptoms were observed on the entire cluster of fruit making the product unsaleable. Based on these symptoms, samples were analysed for Cucumber mosaic virus, Pepino mosaic virus (PepMV), Potato virus Y (PVY), Tomato mosaic virus and Tomato spotted wilt virus by DAS-ELISA with polyclonal antibodies (Loewe Phytodiagnostica, Germany), and for the emerging Southern tomato virus (STV) by RT-PCR (Candresse et al., 2013). Three of the 25 samples analysed were positive only for PepMV whereas the rest of the samples had mixed infections: fifteen plants were co-infected with PepMV and PVY, and seven with STV, PepMV and PVY. The amplification product (894 bp) obtained from one STV-infected plant was purified using the UltraClean® PCR Clean-Up kit (Mo-Bio, USA) and the consensus nucleotide sequences were determined in both senses using an ABI 3130XL Genetic Analyzer (Life Technologies, USA) and deposited in GenBank under accession number KT948068. The nucleotide identity of the Italian STV isolate was greater than 99% with STV isolates Mexico1 (EF442780), BD-13 (KT634055), CN-12 (KT438549), MS7 (EU413670) and FR (KC333078) from Mexico, Bangladesh, China, USA and France, respectively

Antagonism and Antimicrobial Capacity of Epiphytic and Endophytic Bacteria against the Phytopathogen Xylella fastidiosa

Olive quick decline syndrome (OQDS), which is caused by Xylella fastidiosa, poses a severe threat to the agriculture of Mediterranean countries and causes severe damage to the olive trees in Italy. Since no effective control measures are currently available, the objective of this study was the screening of antagonistic bacteria that are potentially deployable as biocontrol agents against X. fastidiosa. Therefore, two approaches were used, i.e., the evaluation of the antagonistic activity of (i) endophytic bacteria isolated from two different cultivars of olive trees (Leccino and Ogliarola salentina) and (ii) epiphytic bacteria isolated from the phyllospheres of different host plant species of X. fastidiosa. In vitro dual culture tests showed that 12 out of 200 isolates inhibited X. fastidiosa growth, with appearances of clear zones between 4.0 and 38.6 mm. 16S rRNA gene sequencing revealed different species of the genera Paenibacillus, Bacillus, Pantoea, Microbacterium, Stenotrophomonas, Delftia, and Pseudomonas. Furthermore, an investigation for antimicrobial activity identified 5 out of the 12 antagonistic bacteria, Paenibacillus rigui, Bacillus subtilis, Bacillus pumilus, Microbacterium oxydans, and Stenotrophomonas rhizophila, that were able to produce culture filtrates with inhibitory activities. Our results are promising for further investigation to develop an eco-sustainable strategy to control X. fastidiosa using biocontrol agents or their secreted metabolites

First Report of Tomato Leaf Curl New Delhi Virus Causing Yellow Leaf Curl of Pepper in Europe

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (family Geminiviridae) with two circular ssDNA genome components (DNA-A and DNA-B), is transmitted in a circulative nonpropagative manner by the whitefly Bemisia tabaci (Gennadius). Although it was first reported in Asia on tomato and other solanaceous crops such as eggplant, potato, and chilli pepper in the Mediterranean basin, this virus was mainly detected on cucurbits and only sporadically on tomato and on two wild solanaceous species, Datura stramonium L. and Solanum nigrum L. (Juárez et al. 2019). In 2018, separate surveys were carried out in protected cultivations of sweet pepper (Capsicum annuum L.) in two Italian regions: Lazio and Campania. The greenhouses were in areas with high density of B. tabaci and where ToLCNDV outbreaks occurred on cucurbits since 2016 (Panno et al. 2019). Some plants showing symptoms of yellowing and leaf curling were found in both regions, whereas fruit symptoms were neither observed nor reported by farmers. This disease syndrome, known as yellow leaf curl disease (YLCD), can be caused in pepper by several begomoviruses, as reported recently in a review listing the viruses causing YLCD in peppers in Thailand (Chiemsombat et al. 2018). Symptomatic leaves were collected during late summer 2018 from different pepper plants as well as from the neighboring zucchini cultivations, showing the typical symptomatology induced by ToLCNDV. Total DNA was extracted (DNeasy Plant Mini kit, Qiagen, Germany), and the presence of ToLCNDV was ascertained by PCR with the specific primers ToLCNDV-CP1 and ToLCNDV-CP2 (Panno et al. 2019; Parrella et al. 2018). ToLCNDV infection was further ascertained in three symptomatic leaf samples from Campania by using specific ToLCNDV ImmunoStrips (Agdia, Elkhart, IN). Successively, one symptomatic pepper sample from each greenhouse was selected and amplified by rolling circle amplification technique (RCA; Inoue-Nagata et al. 2004). The amplicons were cloned, and the DNA-A and DNA-B were full-length sequenced. The sequences were deposited in GenBank NCBI database (MK732932 DNA-A and MK732933 DNA-B, pepper sample from Campania; MK756106 DNA-A and MK756107 DNA-B, pepper sample from Lazio). The RCA analysis was performed also on a ToLCNDV-infected zucchini sample collected in the same area in Lazio region (MK756108 DNA-A and MK756109 DNA-B). The analysis of the ToLCNDV sequences showed a low level of genetic variability between the two pepper isolates from Lazio and Campania regions (rate of substitutions: 0.016 for DNA-A and 0.023 for DNA-B). A high genetic similarity was recorded between the zucchini isolate and both the pepper isolates from Campania (0.019 for DNA-A and 0.023 for DNA-B) and Lazio (0.003 for both DNA-A and B). The three characterized isolates showed a high sequence homology also with both the DNA-A (MH577751 from a melon isolate) and DNA-B (MH577673 from a zucchini isolate) of the ToLCNDV-ES genotype (Fortes et al. 2016), which differed in 15 and 13 nucleotide substitutions from pepper sample from Lazio, 29 and 51 substitutions from Campania sample, and 10 and 5 substitutions from zucchini sample. High homology was also identified compared with the other Spanish isolates collected since the first appearance of the virus (2014) and to the Tunisian (2015) and Moroccan (2018) isolates, confirming the hypothesis that the Mediterranean population of ToLCNDV is highly conserved (Juárez et al. 2019). To our knowledge, this is the first report of ToLCNDV infection on pepper in Europe and indicates that sweet pepper could also act as a reservoir of the virus for further spread to other solanaceous plants and cucurbits

Survey of five major grapevine viruses infecting Blatina and Žilavka cultivars in Bosnia and Herzegovina

The sanitary status of grapevines has not yet been considered sufficiently in vineyards throughout Bosnia and Herzegovina (BiH). An extensive survey of five major grapevine viruses in the country was carried out in 2019. A total of 630 samples from the two dominant autochthonous cultivars, namedZ? ilavka and Blatina, were tested by DAS-ELISA for the presence of grapevine leafroll-associated viruses (GLRaV-1 and 3), grapevine fleck virus (GFkV), grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV). Eighty-eight %of the samples were positive for at least one virus, and all five viruses were detected, thought with different incidence, i.e. GLRaV-3 (84%), GFLV (43%), GLRaV-1 (14%), GFkV (10%) and ArMV (0.2%). The majority of infected plants (about 75%) were asymptomatic. Specific virus symptoms were observed in the remaining infected plants, together with the reported GLRaV vectors, Planococcus ficus and Parthenolecanium corni, while nematodes of the Xiphinema genus were not found in the GFLV- or ArMV-infected vineyards. The GLRaV-3 CP phylogenetic analyses showed 75-100% nucleotide identity between the BiH and reference isolates, and the BiH isolates clustered into the major group. The dNS/dS ratio indicated a negative selection of the virus population, and the lack of geographical structuring within the population was observed. In addition, putative GLRaV-3 recombinants with breakpoints in the 5' of the CP gene were detected, while no recombinant strains were identified for the other four viruses. The obtained results indicate a deteriorated sanitary status of the cultivated grapevines, the prevalence and intraspecies genetic diversity of GLRaV-3 throughout the country. The establishment of certified grapevine material and adequate virus vector control is therefore of primary importance to prevent further spread of these viruses. This study presents the results of the first molecular characterisation of grapevine viruses in Bosnia and Herzegovina

Time-resolved molecular dynamics of single and double hydrogen migration in ethanol

Being the lightest, most mobile atom that exists, hydrogen plays an important role in the chemistry of hydrocarbons, proteins and peptides and most biomolecules. Hydrogen can undergo transfer, exchange and migration processes, having considerable impact on the chemical behavior of these molecules. Although much has been learned about reaction dynamics involving one hydrogen atom, less is known about those processes where two or more hydrogen atoms participate. Here we show that single and double hydrogen migrations occurring in ethanol cations and dications take place within a few hundred fs to ps, using a 3D imaging and laser pump-probe technique. For double hydrogen migration, the hydrogens are not correlated, with the second hydrogen migration promoting the breakup of the C–O bond. The probability of double hydrogen migration is quite significant, suggesting that double hydrogen migration plays a more important role than generally assumed. The conclusions are supported by state-of-the-art molecular dynamics calculationsThis work was funded by the National Science Foundation under award No. 1700551, the MINECO projects FIS2016-77889-R and CTQ2016- 76061-P, ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (SEV-2016-0686) and ‘María de Maeztu’ Programme for Units of Excellence in R&D (MDM-2014-0377). We acknowledge the generous allocation of computer time at the Centro de Computación Científica at the Universidad Autónoma de Madrid (CCC-UAM). S.D.-T. gratefully acknowledges the “Ramón y Cajal” program (RYC-2010-07019) of the Spanish Ministerio de Educación y Cienci