First detection of tomato leaf curl New Delhi virus in melon and zucchini squash in southern Italy

In September 2017, severe symptoms and heavy infestations of aleyrodids were reported on cucurbit crops grown in open fields at the border between Apulia and Basilicata regions (southern Italy). In zucchini squash symptoms consisted in severe curling and brittle fracture of the leaves. Melon plants showed bright yellow mosaic on leaves and necrotic streaks along the stems, flower stalks and fruits whereas squash plants displayed severe yellow mosaic and leaf blade deformation. Disease incidence in the three crops was close to 100%. Symptoms resembled those described recently for infections of tomato leaf curl New Delhi virus (ToLCNDV) (Panno et al., 2016) and watermelon mosaic virus (WMV) (Finetti- Sialer et al., 2012). DNA and RNA preparations from two plants for each species were tested by PCR, respectively, with primers For-5’CCCTTGTAAAGTGCAGTCCT3’ and Rev- 5’GGATTTGATGCGTGAGTACA3’ for the AV1 gene of ToLCNDV DNA-A and with primers For-5’AAACTGGG CAGGGTAGCA3’ and Rev-5’TAACCTGCTGTTAA YCCCGCG3’ for the WMV coat protein gene. Samples of melon and zucchini proved positive for ToLCNDV whereas WMV was detected in squash. No amplification products were obtained with primers for squash leaf curl virus, watermelon chlorotic spot virus, cucumber mosaic virus, cucumber vein yellowing virus and zucchini yellow mosaic virus. Amplicon identities were confirmed by sequencing. Those from zucchini and melon showed 100% identity with ToLCNDV from Spain (KF749224) and Sicily (KU145141) whereas those from squash were 99% and 94% identical to WMV from Belgium (KP980663) and Italy (FJ8231229), respectively. The sequence of a 507 bp fragment of ToLCNDV was deposited in GenBank under the accession number MG269826. This is the first report of ToLCNDV in melon and in the continental part of the Country

Integrative diagnosis, biological observations, and histopathology of the fig cyst nematode Heterodera fici Kirjanova (1954) associated with Ficus carica L. in southern Italy

Morpho-biological notes and histopathology, based on LM and SEM observations, of the fig cyst nematode Heterodera fici isolated from Ficus carica roots, collected in home and public gardens of Apulia region, southern Italy, are described and illustrated. Seventy-five localities throughout the Apulia region were sampled and one-quarter of the sampled localities had fig roots infested with H. fici, with population densities ranging from 44 to 180 cysts/100 ml of soil. All attempts to detect H. fici on ornamental Ficus spp. as well as on imported bonsai in Italy were unsuccessful. Morphometric characters of the Italian population conform to those of the type and re-description populations reported for H. fici. Molecular analysis using ITS, D2–D3 expansion domains of the 28S rRNA, and the partial 18S rRNA sequences of H. fici newly obtained in this study matched well with the corresponding sequences of H. fici present in the GenBank database. Phylogenetic trees confirmed and supported the grouping of H. fici in the Humuli group. Heterodera fici completes its embryogenic development in 14–16 days at 25 °C. Post-invasion development and maturity in the roots of F. carica seedlings is completed in 64–68 days at 25–28 °C with juveniles and adults showing different parasitic habits, being endoparasitic and semi-endoparasitic respectively. The establishment of permanent feeding sites that consist of the formation of large syncytia causes anatomical modification of vascular elements and general disorder in the root stelar structures. Syncytia structures associated with mature females showed different degrees of vacuolisation, numbers of syncytial cells, and contained nuclei and nucleoli which were constantly hypertrophied

Interlaboratory performance of a Real-Time PCR method for detection of Ceratocystis platani, the agent of canker stain of Platanus spp

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes

Integrative diagnosis and parasitic habits of Cryphodera brinkmani a non-cyst forming heteroderid nematode intercepted on Japanese white pine bonsai trees imported into Italy

The non-cyst forming heteroderid nematode Cryphodera brinkmani was detected in Italy parasitizing roots of Japanese white pine bonsai (Pinus parviflora) trees imported from Japan. Morphology and morphometrical traits of the intercepted population on this new host for C. brinkmani were in agreement with the original description, except for some minor differences on male morphology. Integrative molecular data for this species were obtained using D2-D3 expansion regions of 28S rDNA, ITS1-rDNA, the partial 18S rDNA, and the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI). The phylogenetic relationships of this species with other representatives of non-cyst and cyst-forming Heteroderidae using ITS1 are presented and indicated that C. brinkmani clustered together with other Cryphodera spp. and with Meloidodera alni suggesting a monophyletic origin of non-cyst forming nematodes (Heteroderinae sensu Luc et al. 1978), which have been considered close to the ancestor of most species of Heteroderidae. Histological observations of P. parviflora feeder roots infected by C. brinkmani indicated that nematode females induce similar anatomical alterations to those reported for C. kalesari, consisting of formation of a single uninucleate giant cell (nurse cell) with hypertrophied nucleus, prominenet nucleolus, thickened cell wall and expanding into the stele and in contact of xylem, vacuum cambium and phloem. These findings are in agreement with the results of the phylogenetic analysis and indicate a close relationship in the plant responses induced by Cryphodera nematode females with those caused by the genetically related Meloidodera spp., which also induce formation of a uninucletate giant cell.The present work was supported with funds provided by the Italian Ministry of Economy and Finance to the National Research Council for the project “Innovazione e Svi-luppo del Mezzogiorno-Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare-Legge n.191/2009.Peer Reviewe

PLATO: A Predictive Drug Discovery Web Platform for Efficient Target Fishing and Bioactivity Profiling of Small Molecules

PLATO (Polypharmacology pLATform predictiOn) is an easy-to-use drug discovery web platform, which has been designed with a two-fold objective: to fish putative protein drug targets and to compute bioactivity values of small molecules. Predictions are based on the similarity principle, through a reverse ligand-based screening, based on a collection of 632,119 compounds known to be experimentally active on 6004 protein targets. An efficient backend implementation allows to speed-up the process that returns results for query in less than 20 s. The graphical user interface is intuitive to give practitioners easy input and transparent output, which is available as a standard report in portable document format. PLATO has been validated on thousands of external data, with performances better than those of other parallel approaches. PLATO is available free of charge (http://plato.uniba.it/ accessed on 13 April 2022)

Anatomical changes induced by two soil-borne pathogens (Plasmodiophora brassicae and Meloidogyne javanica) in cabbage

Stunted growth of large patches of cabbage cv. Lupini, associated with severe soil infestations by the root-knot nema- tode Meloidogyne javanica and the protist Plasmodiophora brassicae , the casual agent of clubroot disease, was observed in several fields at Castellaneta, province of Taranto, in southern Italy. The host-parasite responses of cabbage roots to parasitism by t he two soil-borne pathogens was studied and compared. In roots infected by P. brassicae , the plasmodia were present in cortex and peri- cycle cells, causing hypertrophy and hyperplasia, and developed into resting spores within host tissues. Parasitism of M. javanica was characterized by the establishment of distinct permanent feeding sites with giant cells in the cortex, endodermis and vascu lar parenchyma, which limit water and nutrient translocation.Peer Reviewe

De Novo Drug Design of Targeted Chemical Libraries Based on Artificial Intelligence and Pair-Based Multiobjective Optimization

Artificial intelligence and multiobjective optimization represent promising solutions to bridge chemical and biological landscapes by addressing the automated de novo design of compounds as a result of a humanlike creative process. In the present study, we conceived a novel pair-based multiobjective approach implemented in an adapted SMILES generative algorithm based on recurrent neural networks for the automated de novo design of new molecules whose overall features are optimized by finding the best trade-offs among relevant physicochemical properties (MW, logP, HBA, HBD) and additional similarity-based constraints biasing specific biological targets. In this respect, we carried out the de novo design of chemical libraries targeting neuraminidase, acetylcholinesterase, and the main protease of severe acute respiratory syndrome coronavirus 2. Several quality metrics were employed to assess drug-likeness, chemical feasibility, diversity content, and validity. Molecular docking was finally carried out to better evaluate the scoring and posing of the de novo generated molecules with respect to X-ray cognate ligands of the corresponding molecular counterparts. Our results indicate that artificial intelligence and multiobjective optimization allow us to capture the latent links joining chemical and biological aspects, thus providing easy-to-use options for customizable design strategies, which are especially effective for both lead generation and lead optimization. The algorithm is freely downloadable at https://github.com/alberdom88/moo-denovo and all of the data are available as Supporting Information

Redescription and molecular characterisation of Xiphinema barense Lamberti et al., 1986 (Nematoda: Longidoridae) from wild olive trees in southern Italy

A population of Xiphinema barense from wild olive trees in Torre Pozzella, Brindisi province, southern Italy, is described using both morphological and molecular studies and compared with the description of the type specimens. The wild olive nematode population agrees very well with all morphometrics provided in the original description. However, detailed observations of the lumen of the tubular portion of the uterus in paratypes and specimens of the new population revealed a clear pseudo-Z-organ with small granules mixed with crystalloid bodies which were previously undetected. Photomicrographs of adult paratypes, which were lacking in the original description, and of specimens of the new population from wild olive trees are provided. The results of the phylogenetic analyses based on the sequences of the D2-D3 expansion regions of the 28S rRNA gene and ITS rRNA genes confirm the species differentiation and indicate the phylogenetic position of X. barense and its relationship with closely related species.The present research was partially funded by the grant 219262 ArimNET_ERANET FP7 2012-2015 Project PESTOLIVE “Contribution of olive history for the management of soilborne parasites in the Mediterranean basin”; and grant AGR-136 from ‘Consejería de Economía, Innvovación y Ciencia’ from Junta de Andalucía, and Union Europea, Fondo Europeo de Desarrollo regional, “Una manera de hacer Europa”.Peer Reviewe

Detection of the camellia root-knot nematode Meloidogyne camelliae Golden in Japanese camellia bonsai imported into Italy: integrative diagnosis, parasitic habits andmolecular phylogeny

The camellia root-knot nematode Meloidogyne camelliae was detected in Italy parasitizing roots of Japanese camellia (Camellia sasanqua) flowering bonsai trees imported from Japan, and it represents a new record for Europe.Morphology and morphometrical traits analysis of the intercepted population of M. camelliae were in agreement with those of the original description of the species, except for some minor differences in second-stage juvenile stylet length and c ratio, spicules and gubernaculuminmales, which may be a result of intraspecific variability. Integrative molecular data for this species were obtained using D2¿D3 expansion segments of 28S rDNA, ITS1¿rDNA, and the protein¿coding mitochondrial gene COI. The phylogenetic relationships of this species with other representatives of Meloidogyne spp. using D2-D3 expansion segments and ITS1 indicated that M. camelliae clustered together with M. artiellia and M. baetica. Histological observations of C. sasanqua feeder roots infected by M. camelliae reveal slight swellings of the root feeding sites, and most egg¿laying females protruding from the root. The anatomical alterations at feeding sites consist of 3¿8 hypertrophied, polinucleate giant cells/female, but no hyperplasia was induced unlike what commonly occurs for most members of the genus.Peer Reviewe

Pathogenicity and Host-Parasite Relationships of Heterodera cruciferae in Cabbage

Stunted cabbage ("eLupini") associated with severe soil infestations by a cyst-forming nematode were observed in large patches of open fields in Castellaneta, province of Taranto, southern Italy. Morphological traits based on mature cysts, males, and second-stage juveniles (J2s) and molecular analysis of ribosomal DNA (D2 to D3 expansion segments of 28S and internal transcribed spacer [ITS]1 region) were used to identify the species. ITS1 sequence information supported the identity of Heterodera cruciferae, also showing a high degree of similarity to other species of the Heterodera Goettingiana group, including H. goettingiana, H. carotae, and H. urticae. Nematodes successfully established permanent feeding sites in cabbage roots which caused cellular alterations in the root cortex, endodermis, pericycle, and vascular cylinder by inducing typical multinucleate syncytia. Syncytial cytoplasm was granular and dense, with variously sized vacuoles and hypertrophied nuclei with nucleoli. Cabbage plant growth was also reduced in pathogenicity tests. The relationship between the initial nematode population density in soil and shoot plant weight was well described by the Seinhorst¿fs equation. Tolerance limits with respect to shoot plant weight of cabbage to H. cruciferae was estimated as 1.50 units of eggs plus J2s/cm3 of soil. The minimum relative value (m) for plant height was 0.71 at an initial nematode population density of (Pi) . 64 units of eggs plus J2s/cm3 of soil. The maximum nematode reproduction rate (Pf/Pi) was 4.6 times that of the initial population density of 8 units of eggs plus J2s/cm3 of soil.Peer Reviewe