Detection of viruses infecting Lilium spp. by RT-PCR and Real-Time PCR

In order to enhance the Italian lily bulb production, a breeding program was carried out at CRA-VIV in Pescia (PT – Italy) during the last years. Asiatic hybrids (Lilium × elegans Thunb.), lily cultivars and other native species were involved in the program. The obtained lily lines, which presented interesting traits, were preserved in a collection. After years of vegetative propagation, some of the new selections showed symptoms referable to viral infections. Virus diseases represent some of the most dangerous threats of Lilium, so the application of fast and effective diagnostic techniques for early detection is very important. The aim of the present study, in the frame of a phytosanitary survey of the lily collection, is to investigate the presence and incidence of Cucumber mosaic virus (CMV), Lilium symptomless virus (LSV), Lily mottle virus (LMoV) and two tospoviruses (Impatiens necrotic spot virus, INSV, and Tomato spotted wilt virus, TSWV). Among the 60 samples object of this study, infections by LSV and CMV were frequently observed. Also LMoV was detected in a smaller amount of samples. All of the samples were negative to INSV and TSWV

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties

Lily mottle virus isolate 207/1 coat protein gene, partial cds.

Genbank accession number: JQ65510

Lily mottle virus isolate 117/3 coat protein gene, partial cds.

Genbank accession number: JQ65510

Phytosanitary survey on pome fruit autochthonous germoplasm in Tuscany

During 2010, a phytosanitary survey was conducted, concerning the occurrence of some of the main virus, viroid and phytoplasma pathogens affecting Malus and Pyrus autochthon germplasm from Tuscany. Samples, consisting of leaves, shoots and phloematic tissues, were collected during spring 2010. In detail, a total number of 97 plants, 73 of which belonging to 29 Malus varieties, and 24 belonging to 13 Pyrus varieties, were tested by RT-PCR and nested PCR for the subsequent pathogens: Candidatus Phytoplasma mali (Ca. P. mali), Candidatus Phytoplasma pyri (Ca. P. pyri), Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV), Apple dimple fruit viroid (ADFVd) e Apple scar skin viroid (ASSVd). Among the eight pathogens investigated, ACLSV and ASPV proved to be the most widespread (both present in 67.0% of tested samples), while ASGV was present in 14.4% of surveyed plants. In detail, ACLSV was detected on 59 Malus and 6 Pyrus plants, while ASPV was found on 54 and 13 accessions of Malus and Pyrus, respectively. Concerning ASGV, infections by this virus have been detected on 13 Malus plants and 1 of Pyrus. The remaining pathogens (ApMV, ADFVd, ASSVd, Ca. P. mali e Ca. P. pyri) were never detected in the surveyed plant

Utilizzo dei piccoli mammiferi quali indicatori di contaminazione ambientale da ceppi batterici antibiotico resistenti

Numerose specie animali, soprattutto piccoli mammiferi, vengono spesso considerate "sentinelle ecologiche ambientali" ed utilizzate in indagini eco-tossicologiche. La nostra ricerca ha preso spunto dalla Direttiva 2003/99/CE "Misure di sorveglianza delle zoonosi e degli agenti zoonotici che riporta come, per il controllo del crescente fenomeno della resistenza agli antibiotici, potrebbe rivelarsi opportuno monitorare microrganismi indicatori quali possibili reservoire di geni di resistenza. Tale principio \ue8 tanto pi\uf9 attuale quanto pi\uf9 ci si occupa di animali selvatici non soggetti a trattamenti antimicrobici e che quindi assumono questi geni di resistenza dall'ambiente in cui vivono. Abbiamo ritenuto interessante quindi indagare in tal senso, analizzando ceppi commensali di Escherichia coli isolati da feci di piccoli mammiferi catturati nell'ambito di un piano di monitoraggio per la proposta di estensione di ZPS presso il Parco delle Orobie Bergamasche. Il campionamento \ue8 stato effettuato per mezzo di line transect, di lunghezza 100 m, disposti per fasce altitudinali (I 000-1 600 m s.l.m.) e tipologie ambientali differenti (bosco di conifere, bosco di latifoglie, bosco misto, prateria, roccia, arbusteto). Il monitoraggio \ue8 stato effettuato in VaI Seriana (Bg), in quattro sessioni di cattura (giugno, luglio, agosto e settembre)durante i quali sono state sistemate in loco live-traps,modello Sherman, per 3 notti consecutive. In totale sono stati raccolti 253 campioni fecali: 105 da Apodemus flavicollis, 68 da Apodemus sylvaticus, 63 da Myodes glareolus e 15 da Eliomys quercinus. Il campione fecale veniva consegnato in laboratorio entro le 24ore, seminato in Brain Heart Broth (Oxoid), incubato a 37\ub0C per 24 ore in aerobiosi, trapiantato su Mc Conkey Agar e le colonie con caratteristiche tipiche venivano successivamente identificate con il sistema RapID ONE System (Remel, USA). I 33 campioni identificati come E. coli, sono stati testati per valutare la sensibilit\ue0 agli antibiotici secondo le specifiche previste da EUCAST; in totale sono stati utilizzati 27 antibiotici appartenenti alle famiglie degli Aminoglicosidi, 13-lattamici, Cefalosporine, Chinoloni, Fluorochinoloni, Cicline, Fenicoli, Polimixine e Carbapemeni. Sui ceppi isolati risultano molto attivi i Carbapemeni e i B-lattamici, attivi i Fenicoli, i Fluorochinoloni e le Cicline, mediamente attive le Cefalosporine e poco attivi gli Amminoglicosidi, i Chinoloni e le Polimixine. La resistenza agli antibiotici \ue8 un problema sempre pi\uf9 sentito in medicina umana e in veterinaria e la sua gestione \ue8 oggetto di discussione costante. Gli sforzi per ridurre la resistenza dei microrganismi agli antimicrobici si basano sul presupposto che tale caratteristica sia sviluppata e mantenuta nelle popolazioni batteriche a seguito di una massiccia o scorretta esposizione agli antimicrobici stessi e che la limitazione dell'uso di tali molecole dovrebbe bastare a porre dei limiti alla diffusione delle resistenze. Contrariamente a quanto riportato in bibliografia, sembrerebbe che anche la microflora di piccoli mammiferi selvatici, come quelli da noi campionati, i quali non subiscono la pressione selettiva di una somministrazione eccessiva di antibiotici, possa avere un ruolo nella diffusione di ceppi batterici resistenti agli antibiotici. L'origine di tale fenomeno e i meccanismi di selezione responsabili del mantenimento di un'alta prevalenza di resistenza sono spesso il risultato di un naturale scambio di materiale genetico tra specie batteriche anche differenti tra loro e che originano da diversi ecosistemi. Sicuramente il contatto con animali domestici, che possono essere stati sottoposti a terapie, agevola il fenomeno del trasferimento di geni di resistenza dalla microflora di questi a quella dei piccoli mammiferi selvatici che condividono lo stesso ambiente e le stesse risorse. Occorrerebbe quindi indagare maggiormente l'interazione animale domestico-selvatico-uomo. Il nostro lavoro non ha la pretesa di dare delle informazioni definitive ma sicuramente pu\uf2 essere un piccolo contributo ed uno stimolo a continuare ad indagare sull'ecologia batterica in ambienti naturali e sulla loro interazione con gli animali anche in zone dove si pensa che l'elevata naturalit\ue0 sia sufficiente a mantenere incontaminato l'ambiente

Further data on the distribution of grapevine yellows in Tuscany

Grapevine yellows (GY) are an important disease of Vitis vinifera caused by phytoplasmas. In Italy, GY are mostly represented by Flavescence dorée (FD) and Bois noir (BN), respectively associated with infection by 16SrV and 16SrXII phytoplasma ribosomal groups. In the frame of a regional strategy aimed to monitor GY presence, an extensive survey was carried out in late summer 2010, collecting 629 leaf samples from the inspected commercial vineyards. Samples were preferably collected from plants showing symptoms referable to GY diseases. Each sampled plant was marked and localised by “Global Positioning System - GPS”, in order to allow tracking of FD positive samples for uprooting. DNA extracts were tested with a Real-time PCR assay for the diagnosis of BN and FD phytoplasmas. The results confirmed that BN is largely the most important GY in Tuscany, with an ubiquitous distribution. In fact, it was present in almost every surveyed vineyard with an overall incidence of 53,6% (337 out of 629 plants tested). The presence of FD was recorded in 39 samples distributed in 4 provinces including: Massa-Carrara, Florence, Pistoia and Lucca. An alarming situation was observed in this latter province, where 23 out of 74 plants (31,1%) were infected by FD. On the basis of these results, further investigations on insect vectors, natural reservoirs and molecular characterization of grapevine infecting phytoplasmas are needed in order to monitor and understand the epidemiology of GY diseases in Tuscany

Preliminary results on distribution and diversity of grapevine yellows in Tuscany.

Grapevine yellows (GY) are an important threat to grapevine industry in Europe. In Italy, GY are mostly represented by Flavescence dor\ue9e (FD) and Bois noir (BN), respectively associated with infection by 16SrV and 16SrXII phytoplasma ribosomal groups. In the frame of a regional strategy aimed to monitor GY presence, an extensive survey was carried out covering the ten provinces of the Tuscany region, and collecting 585 leaf-samples in the commercial vineyards inspected in summer 2009. Preference was given to samples showing discoloration, downward rolling and symptoms referable to GY diseases. In order to allow tracking of FD positive samples for uprooting, all the plants were marked and localised by \u201cGlobal Positioning System-GPS\u201d. DNA was extracted and amplified, firstly in a classical nested-PCR reaction and successively with dual-labelled probes specific to FD, BN and grapevine chaperonin gene in a Real-time PCR reaction. Results indicated that BN is largely the most important GY in Tuscany with an ubiquitous distribution; it was present in almost all surveyed vineyard with an overall incidence of 70% (418 out of 585 sample tested). Alarming presence of FD was recorded in 23 samples distributed in 5 provinces including: Arezzo, Massa-Carrara, Florence, Siena and Lucca. Molecular characterization performed with PCR/RFLP analysis on 16S ribosomal and on SecY genes confirmed the presence of either FD-C and FD-D subgroups, this latter is reported for the first time in Tuscany and it is identical to the strain described earlier in France and in Veneto region. On the basis of this results, further investigations on insect vectors, natural reservoirs and molecular characterization of grapevine infecting phytoplasma are needed in order to monitor and understand the epidemiology GY diseases in Tuscany

Detection and tuf-type characterization of Bois Noir phytoplasma in Tuscany by an improved real-time assay or nested PCR

In agreement with a regional strategy aimed to monitor grapevine yellows (GY) disease, an extensive survey was carried out covering the ten provinces of the Tuscany region. Symptomatic leaf-samples were collected from commercial vineyards and nurseries during 2009 and 2010. Laboratory analyses performed by nested PCR, and by Real-Time PCR (Angelini et al., 2007) both on 16S ribosomal gene, confirmed that ‘bois noir’ (BN) is largely the most important GY in Tuscany with ubiquitous distribution. Molecular characterization of BN phytoplasmas relied on genetic diversity studied through multiple step amplification process of one or more genes followed by RFLP analysis. In recent times the latter technique was flanked by sequencing of amplicons and/or cloned amplicons followed by in silico RFLP analyses. This procedure enable to find in short time a wider BN genetic diversity through the identification of several single nucleotide polymorphism (SNP) lineages. Nevertheless, little is known about the ecology of these newly defined subgroups and their putative role in the epidemiology of BN. Consequently, the model proposed by Langer and Maixner (2004) permitting the distinction of three biologically differentiable sequence variants (tuf type-I, tuf type-II and tuf type-III) is still widely adopted to study BN epidemiology. Recently, a newly TaqMan allelic discrimination assay was developed and proposed for the distinction of tuf type-I and tuf type-II (Berger et al., 2009). The objective of this study was to develop a real time PCR assay for the rapid and specific detection tuf type-I and tuf type-II and to compare it with nested PCR assays carried out on the same gene. For this purpose, two newly designed BHQplus probes (Biosearch Tech, CA, USA) were used in an allelic discrimination test to distinguish between tuf type-I and tuf type-II among BN positive samples identified in grapevine in Tuscany. Real-time PCR analysis was performed in duplicates in 12 μl-reactions, containing 6 μl IQ Powermix (Bio-Rad Laboratories, USA), 500 nM of each primer, 100 nM of FAM-VKI probe, 100 nM of CalFluor orange 560-VKII probe, and 1 μl of sample DNA. Assays were carried out on Rotor-Gene Q (Qiagen, Germany). Standard curves traced on serial dilution of synthetic oligonucleotide permitted to calculate the reaction efficiency. The slopes of the linear fits reached -3.329 for the green channel indicating an efficiency of 99.70% (R2= 0.992) for the tuf type-I assay. Similar results were obtained with the tuf type-II probe (Efficiency = 98.64 %, R2= 0.996). When applied to grapevine samples, results were automatically generated through the allelic discrimination option of the Rotor-Gene Q software (version 2.0.2, Qiagen). According to preliminary results, the newly designed assay for the simultaneous detection of tuf type-I and tuf type-II types resulted to be very efficient. A number of BN positive samples are still under characterization process with both methods real-time and nested-PCR to establish the presence of tuf type-I and tuf type-II and to trace a distribution map in Tuscany