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Parietaria mottle virus (PMoV) is an emerging virus in Mediterranean countries, responsible for severe disease in tomato and pepper crops in the field and protected cultivation. The principal wild reservoir of PMoV is Parietaria officinalis, and only few additional wild plants have been described as natural reservoirs of the virus. During field survey in southern Italy, several plants of Phytolacca americana showing virus-like symptoms were collected. Serological and molecular assays showed that these plants were infected by PMoV. Sequence comparison of the movement protein gene of the PMoV isolate from P. americana showed the greatest similarity to the corresponding sequence from tomato plants growing nearby. These results indicate that P. ameriacana is a new natural host of PMoV, and further investigation is warranted to establish the potential of this host as reservoir of the virus in the field.

Chayote (Sechium edule (Jacq.) Sw.) is a vigorous perennial and climbing cucurbits, native to Mesoamerica, and cultivated for alimentary purposes in the American continent, Australia, New Zealand, South Europe, Asia and Africa. During spring 2019, some chayote plants showing bright yellow vein banding rings and lines were observed in a private garden in South Italy (Campania region). Symptoms coalesced in some leaves, covering almost the whole foliar area. Double-stranded RNAs were extracted from symptomatic leaves of a single chayote plant and reverse-transcribed, randomly amplified, and submitted to Illumina sequencing (Marais et al., 2018). Reads were assembled using CLC Genomics Workbench 11.1 (http://www.clcbio.com). Contigs were then annotated by Blastn and Blastx comparison with the Genbank database, which allowed the identification of eight contigs of between 380 and 980 nucleotides sharing significant identity with alfalfa mosaic virus (AMV) genomic RNAs. No other viral contigs were identified. Mapping of reads on AMV genomic RNAs identified 4,209 AMV reads (1.26% of total reads) and allowed the scaffolding of the contigs into three scaffolds corresponding to the three AMV genomic RNAs. To complete the sequence of the AMV chayote isolate genome (named See-1), primers were designed from the contig sequences and used to amplify RACE PCR products spanning the 5' and 3' terminal regions of the three genomic RNAs using the SMARTer™ RACE cDNA Amplification Kit (Clontech, China). All amplicons were cloned into the pGEM-T vector (Promega, USA) and sequenced (three clones for each amplicon) by Microsynth Seqlab (Microsynth AG, Switzerland). Finally, the complete genomic sequences of the three RNAs were assembled by MacVector 17.5 (MacVector Inc., USA). The RNA1, RNA2 and RNA3 of See-1 are 3,643, 2,593 and 2,037 nt respectively (GenBank accession Nos. MT093209 to MT093211), and share the highest nt sequence identity with the RNA1 and RNA3 of AMV isolate (HZ) from tobacco (99.5% for RNA1, HQ316635; 98.7% for RNA3, HQ316637) and with the RNA2 of isolate AMV-Gym from Gynostemma pentaphyllum (98.1%, MH332898), both from China. AMV isolate See-1 was classified as belonging to subgroup I based on the presence of a BamH I and two AvaII sites in the CP ORF (Parrella et al., 2000). Reverse transcription polymerase chain reaction, using primers targeting the CP gene (Parrella et al., 2000), confirmed AMV infection in three symptomatic cayote plants including that used for Illumina sequencing, with 100% of nt sequence identity of amplicons. Three plants each of Chenopodium amaranticolor, Nicotiana benthamiana and Solanum lycopersicon were mechanically inoculated with sap from isolate See-1 infected plant, leading to the appearance of typical AMV symptoms in all three hosts ten days post-inoculation (Jaspars & Bos, 1980). This note describes the first detection of AMV in cayote in Italy and, to the best of our knowledge, in the world. In some areas of Southern Italy, climatic conditions are favorable enough to allow chayote development in the wild. Further studies would be desirable to determine the distribution and incidence of AMV in chayote and to understand the possibility that this species may play a role in AMV epidemiology, representing a threat to other susceptible crops.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting tomato cultivation on the Indian subcontinent. Recently, however, a new strain of the virus, named ToLCNDV-ES, has spread to Mediterranean countries such as Spain, Italy, and Tunisia, and occurred in Cucurbita crops, causing economic damage. Although ToLCNDV is spread by the sweet potato whitefly (Bemisia tabaci), like other begomoviruses, it has not been clear how ToLCNDV suddenly spread from the Indian subcontinent to the Mediterranean region. In 2017, ToLCNDV was diagnosed in young seedlings germinated naturally from fruits fallen in the prior year on a farm located in Giugliano in Campania, Naples, Italy, suggesting a possible role of the seeds in vertical transmission of the virus. Because sweet potato whiteflies were widespread naturally in that region, it was necessary to verify that in an artificial insect vector-free condition. Seeds were harvested from two ToLCNDV-infected zucchini squash cultivars in Naples in 2017 and 2018 to examine whether ToLCNDV can be transmitted from zucchini squash seeds to young plants. Viral DNA was amplified from these seeds and 1- to 3-week-old seedlings germinated from them with a ToLCNDV-specific primer set. According to PCR results, viral contamination was confirmed from all harvested seeds and dissemination was proven from 61.36% of tested seedling samples. Mechanical transmission from seed-borne virus-infected seedlings to healthy zucchini squash plants was also succesful, demonstrating that seedlings from ToLCNDV-infected seeds did act as inoculum. This is the first report demonstrating that ToLCNDV is a seed-transmissible virus in zucchini squash plants in Italy.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (genus Begomovirus, family Geminiviridae), was originally described in tomato in India in 1995 (Padidam et al. 1995). The virus apparently remained confined to the Asian continent for about 20 years and has only recently been discovered in Europe. In Italy, ToLCNDV was detected for the first time in 2015 in zucchini squash in Sicily (Panno et al. 2016) and later on in other regions of continental Italy (Panno et al. 2019). Recently, epiphytotics of ToLCNDV were reported in central and south Italy, associated with yellowing and leaf curling symptoms in pepper crops (Luigi et al. 2019). In 2016, a large-scale survey was conducted to assess the distribution and the genetic diversity of the viral isolates spreading in Italy. Samples collected at that time included cucurbits and solanaceous plants, including five leaf samples from five distinct eggplant (Solanum melongena L.) plants of cultivar Violetta di Napoli that showed yellowing and light curling of the apical leaves and noticed in a cultivation located in Campania region (Castel Volturno municipality). A few Bemisia tabaci individuals were noticed associated to the cultivation. All five leaf samples tested positive for ToLCNDV with ImmunoStrip (Agdia, Elkhart, IN). These results were confirmed by PCR using ToLCNDV-specific primers TLCNDVCP1/TLCNDVCP2 (Parrella et al. 2018) on the total DNA extracted from each sample using an E.Z.N.A. Plant DNA kit (Omega Bio-tek, Norcross, GA). Amplicons of the expected size (∼1.0 kb) were obtained only from the five symptomatic plants, and the nucleotide sequences of these isolates were identical. One representative sample (Som-166/16) was selected for full-length amplification of the genome (DNA-A and DNA-B-like sequences) using the rolling circle amplification (RCA) method with an Illustra TempliPhi amplification kit (GE Healthcare, Piscataway, NJ), in accordance with the manufacturer’s instructions. RCA products were digested with different restriction endonucleases to obtain a 2.8-kb linear DNA fragment. Among the different enzymes tested, BamHI resulted in a maximum DNA fragment length of 2.8 kb, which was cloned into a BamHI-linearized pUC19 plasmid. The ligated products were transformed into a competent DH5α strain of Escherichia coli, and the positive clones were sequenced in both orientations at Microsynth Seqlab (Gottingen, Germany). The obtained full-length DNA-A (2,738 nt; GenBank MN782303) and DNA-B (2,686 nt; GenBank MN782304) sequences showed the highest percentage of nucleotide identity with the ToLCNDV Italian isolates Caa-164/16 (GenBank MK732932) for the DNA-A (99.82%) and Cum-45/16 (GenBank MF688671) for the DNA-B (99.48%). Eggplant leaf yellowing caused by a ToLCNDV variant has been previously described in India (HQ264185 for DNA-A and HQ264186 for DNA-B) (Pratap et al. 2011), but the Italian and the Indian isolates exhibited only 90.20 and 79.00% nucleotide identity with respect to the nucleotide sequences of DNA-A and DNA-B. The Som-166/16 isolate was very similar at the molecular level to previously reported isolates from Mediterranean countries and belonged to the European strain ToLCNDV-ES, because the percentage of nucleotide identity was 98.83% for DNA-A with the ToLCNDV-ES strain A-MU.13.ME/4.3 (GenBank MH577751) and 97.88% for DNA-B with the ToLCNDV-ES strain B-AL.15.ZU/2.1 (GenBank MH577658) (Moriones et al. 2017; Panno et al. 2019). The ToLCNDV-ES strain evolved from ToLCNDV isolates of Asian origin and adapted to infect cucurbits (Moriones et al. 2017). Nevertheless, this report presents further evidence, in addition to a previous report (Luigi et al. 2019), that ToLCNDV-ES isolates can potentially pose a threat not only to cucurbits but also to solanaceous crops as well, because this is the first finding of ToLCNDV in eggplant in Italy. Control measures against ToLCNDV are limited and mainly based on vector control, cultivation in protected environments, and the prompt elimination of infected materials.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) identified for the first time in 1995 in Asia, from where it spread into several countries of the Mediterranean basin. ToLCNDV was found in Spain in 2012, and subsequently in Tunisia and Italy. The first outbreak in Italy occurred at the end of 2015 in Trapani province (Sicily) on zucchini squashes. Then in 2016, ToLCNDV was found in infected zucchini plants in Campania, Lazio and Sardinia regions, and in 2017 in Calabria. This study addressed the dispersion and genetic diversity of ToLCNDV isolates in Italy. A total of 1400 plants were analysed. Phylogenetic analysis showed low variability among the Italian isolates, probably as a consequence of the recent introduction and rapid spread of this virus in Italy. Two statistically significant clusters were reported: one grouping only Italian isolates and the other grouping Italian, Spanish, Tunisian and Moroccan isolates. Furthermore, the highest incidence of ToLCNDV was observed in Sicily, although the disease also appears to be critical in other Italian regions. In this work, a high efficiency of ToLCNDV mechanical transmission into Cucurbita pepo, Cucumis melo inodorus and Cucumis melo cantalupensis has been demonstrated. The rapid spread of ToLCNDV in the Mediterranean basin represents a threat for horticultural production, thus it is very important to develop suitable crop management practices, applying genetic resistance strategies and more restrictive phytosanitary measures.

In July 2019, disease outbreaks were observed in two tomato fields in South Sardinia. The first field located in Samassi was established with tomato spotted wilt virus (TSWV)-resistant cultivars Taylor and Docet. The second field located in Serramanna was established with TSWV-resistant cultivars Dask and SV051, and the TSWV-susceptible cultivar Creso. The two fields were about 4.0 km away from each other. Symptoms resembling those elicited by TSWV, consisting in leaf and stem necrosis, and necrotic rings and patches on the fruits, were noticed on most of the plants in the two fields. Preliminary screening of two symptomatic plants of each cultivar with a specific TSWV Lateral Flow (LF) assay (Forsite Diagnostics, UK) showed that none of the samples, with the exception of Creso, reacted positively to TSWV. Then RT-PCR was conducted with total RNA extracted from non-necrotic tissue of the same plants tested in LF using the E.Z.N.A.® Plant RNA kit (Omega Bio-tek, USA) and orthotospovirus universal primers BR60/BR65 and primers PMoVMP1a/PMoVMP2b specific to parietaria mottle virus (PMoV) (Parrella et al. 2016). Amplicons of the expected size (916 bp) for PMoV were obtained from all symptomatic leaf samples tested, whereas TSWV amplicons (453 bp) were obtained only from the Creso samples. No amplicons were obtained from asymptomatic samples or healthy samples used as controls. The PMoV PCR products from three tomato plants were cloned and sequenced. Sequences of the PMoV isolates were identical (GenBank accession number MN782302) and shared 96% nucleotide identity with the Spanish isolate RAMS1 from tomato (AM182748). Further, PMoV was detected in symptomatic leaf samples by dot-blot hybridization assay using a PMoV-specific riboprobe (Parrella 2002). Using this assay, the PMoV incidence was around 80% in both tomato fields (52 of 67 plants were positive in the first field and 41 of 51 plants were positive in the second field). PMoV has been previously reported in protected and open field tomato cultivation in Spain only with low incidence (Soler et al. 2010). This is the first report of a significant outbreak of PMoV in tomato in the Mediterranean basin

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.

During 2016, a survey was carried out to investigate the presence and distribution of Tomato leaf curl New Delhi virus (ToLCNDV) in Campania region (Southern Italy). In three fields in the Castel Volturno and Giugliano in Campania municipalities (Caserta and Napoli provinces respectively), an unusual disease was noted affecting pumpkin (Cucurbita moschata) plants of the variety “Lunga di Napoli”, a traditional local ecotype. Symptoms consisted of stunting and severe foliar yellow mosaic, which, in some cases, resulted in completely yellow leaves. The affected plants produced less flowers and fruits. The incidence of the disease ranged from 80 to 100% of the plants. The insect vector of ToLCNDV, Bemisia tabaci, was found in each sampled field and the genotype of twenty specimens for each population was identified as previously described (Parrella et al., 2012 and 2014). The Q1 and Q2 variants of the Mediterranean species were identified with a percentage ranging from 11-14% and 86-89% respectively. Four sy...

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C3 and C4 plant carbonic anhydrases (CAs) are zinc-enzymes that catalyze the reversible hydration of CO2. They are sub-divided in three classes: α, β and γ, being distributed between both photosynthetic subtypes. The C4 dicotyledon species Flaveria bidentis (L.) “Kuntze” contains a small gene family encoding three distinct β-CAs, named FbiCA1, FbiCA2 and FbiCA3. We have expressed and purified recombinant FbiCA1, which is localized in the chloroplast where it is thought to play a role in lipid biosynthesis and antioxidant activity, and biochemically characterized it by spectroscopic and inhibition experiments. FbiCA1 is a compact octameric protein that is moderately inhibited by carboxylate molecules. Surprisingly, pyruvate, but not lactate, did not inhibit FbiCA1 at concentrations up to 10 mM, suggesting that its capacity to tolerate high pyruvate concentration reflects the high concentration of pyruvate in the chloroplasts of bundle-sheath and mesophyll cells involved in C4 photosynthesis.

During spring-summer 2012, 2013, and 2015, 10 samples of bell pepper (Capsicum annuum) plants showing basal bright rings and mosaic of the leaves with some necrotic patches, necrotic stems, and fruits with brown patches and corky rings, were identified in the Campania region (southern Italy), in a family garden (four plants in 2012 and three plants in 2015 of the Friariello ecotype) and in a commercial greenhouse (two plants of the Papaccella ecotype in 2012 and one plant of the Eppo hybrid in 2013). Symptomatic samples were tested at the Institute for Sustainable Plant Protection in Portici (Napoli, Italy). Screening of leaf and fruit symptomatic tissues by double antibody sandwich (DAS)-ELISA with commercial kits of antisera (Bioreba AG, Switzerland) to Cucumber mosaic virus(CMV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus(ToMV), Pepper mild mottle virus (PMMoV), and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (potygroup test), gave negative results, whereas all samples tested positive in antigen-coated-plate (ACP)-ELISA with antisera, kindly provided by Dr. P. Roggero (Plant Virology Institute, Torino, Italy), produced against a tomato isolate (namely TI1 isolate) of Parietaria mottle virus (PMoV). The results from indirect ELISA were subsequently confirmed with reverse transcription (RT)-PCR using PMoV specific primers designed to amplify the entire gene of the movement protein (MP): PMoVMP1a (5?-AGGGTTTGAAATGGCATTTGTTC-3?) and PMoVMP2b (5?-CTTAGTGGACATTCACGAATATC-3?). A healthy pepper plant was used as a negative control both in ELISA and RT-PCR assays. RT-PCR products of 916 bp, which covered approximately 40.4% of PMoV RNA3 (isolate T32 as reference, accession number KT005245), were obtained in all symptomatic samples and one amplicon for each pepper ecotype and year of sampling was cloned and sequenced. The nucleotide sequences of the MP gene of three Italian PMoV isolates were deposited in GenBank under accession numbers LT160068 (isolate Fri-1, from the Friariello ecotype, in 2012), LT160069 (isolate Pap-1, from the Papaccella ecotype, in 2012), and LT160070 (isolate Fri-2, from the Friariello ecotype, in 2015). Isolates Fri-1 and Pap-1 showed 98.4 and 97.6% identity with the Spanish PMoV isolate JBT1 from tomato (accession number AM182747), respectively, while Fri-2 showed 96.7% identity with the Spanish PMoV isolate AC1 from tomato (accession number AM182743). To our knowledge, this is the first report of PMoV infecting bell pepper in Italy. In 2004, PMoV was identified in pepper crops in Spain (Janssen et al. 2005). The virus is also known to infect economically important plants as tomato in Italy, France, Spain, and Greece (Aramburu 2001;Marchoux et al. 1999; Roggero et al. 2000), as well as the ornamental Mirabilis jalapa (Parrella 2002). This new finding of PMoV in pepper in Italy confirms the tendency of the virus to broaden its range of natural hosts, particularly concerning species of economic interest. The symptoms induced by PMoV on pepper can be visually confused with those caused by other viruses infecting pepper (e.g., TSWV, CMV). Therefore, it is necessary to monitor regularly for the presence of PMoV in pepper crops in order to know the real incidence of the disease in the field with the aim of developing efficient and durable disease control strategies.