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Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1–3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 for phytoplasma diagnostic methods).

Nel biennio 2010-2011 sono state eseguite specifiche ricerche riguardanti le infezioni da fitoplasmi su ortensia, sia in coltivazione presso aziende floricole della Liguria, sia su esemplari a dimora in aree urbane del Lazio, e precisamente a Bolsena (Viterbo). Da queste ricerche è emerso che negli ultimi anni il “quadro” sanitario è sensibilmente mutato, sia per quanto riguarda i fitoplasmi coinvolti, sia per quanto concerne importanti aspetti sintomatologici (alterazioni morfologiche e cromatiche sulle infiorescenze) ed epidemiologici (trasmissione in natura tramite vettori animati). Alla luce dei risultati e considerata la pericolosità di questi microrganismi patogeni, ci sembra opportuno effettuare un aggiornamento sulle conoscenze delle fitoplasmosi dell’ortensia in modo da contribuire al miglioramento degli interventi di difesa e profilassi a disposizione sia dei vivaisti e produttori di piante in vaso, sia di degli operatori e manutentori del “verde” pubblico.

The presence of ‘Candidatus Phytoplasma asteris’ in oilseed rape already reported in Italy was studied to verify its epidemiological aspects using three cultivars: PR45D01, Excalibur and Catalina. In particular results of analyses carried out to verify germination ability for this phytoplasmas/plant species show that seeds from infected plants have 77.3%, while seeds from asymptomatic plants 89.8% and commercial seeds 93.9%. Seedlings from infected mother plants show malformation, stem thickening, leaf yellowing, however no significant differenced were reported between these plants and seedlings from healthy mother plants in both weight and amount of vegetation. Molecular analyses carried out during the vegetation period show that 40% of seedling from infected mother plants was positive to ‘Ca. P. asteris’. Seedlings from asymptomatic mother plants was positive in 22% of the cases to phytoplasmas allowing to speculate an field-insect transmission or a latent infection in mother plants. Among the insect species collected in infected fields Macrosteles quadripunctulatus, Psammottetix alienus, P. confinis, P. striatus and Toya propinqua, resulted to carry 16SrI phytoplasmas. Phytoplasma infected plants are able to produce seed, seedlings produced were highly infected with same phytoplasma. This can explain the geographic spreading of the disease in non infected areas and the presence of numerous potential insect vector can be the way to further disseminate the disease producing eventually serious epidemic. Research are in progress to verify the roles of both transmission ways in the epidemiology of winter oil seed rape phytoplasmas disease epidemiology.

The recognition that certain plant diseases were associated with mycoplasmalike structures in the sieve-tubes was first reported by Doi and coworkers in 1967 who extended this observation by confirming possible disease causation, and their similarity to mycoplasmas, by showing that disease symptoms could be ameliorated by treatment with tetracycline (Ishiie et al., 1967). Hitherto, these diseases - referred to as the aster yellows group - had been thought to be caused by viruses. In animals and humans, mycoplasma infections had similarly often been regarded as viral in nature due partly to their filterability through membranes of small pore size but also due to the inability to culture the infectious organism in common bacteriological media. Many workers, mainly plant pathologists rather than mycoplasmologists, took up the challenge to culture these organisms. An early outcome of this effort was the culture of spiroplasmas – microorganisms derived from insects capable of causing plant diseases following infection of the sieve tubes. This distinct group of organisms grew readily in mycoplasma media formulations whereas the mycoplasma-like objects (phytoplasmas) did not and a possible reason was that a closer alignment of media to sieve-tube sap composition was required. Morphologically the two groups were quite distinct, spiroplasmas having a spiral structure as the name suggests whereas phytoplasmas were generally pleomorphic. Some reports of successful phytoplasma culture were published (Lombardo and Pignattelli, 1970; Lin et al., 1970; Giannotti and Vago, 1971; Ghosh et al., 1975) but none proved repeatable and as the years of failure increased, hope of cultivation decreased to such an extent that some plant workers came to consider that phytoplasmas were indeed uncultivable (reviewed in Maramorosch, 2011). Over forty years had elapsed since Doi’s publication but there were workers who believed culture to be possible and a collaboration was initiated in 2008 between Bologna University and Mycoplasma Experience. Early success was definitely not anticipated and a lengthy process of medium modification/alignment to sieve-tube sap composition was envisaged.

Phytoplasmas of stolbur group affect a wide range of wild and cultivated plants, and are mainly transmitted by polyphagous planthoppers of the Cixiidae family. In vineyards, grapevine-affecting stolbur phytoplasmas are associated with “bois noir” (BN) disease and are transmitted by Hyalesthes obsoletus Signoret, but in wine-growing areas where H. obsoletus is absent, the presence of stolbur phytoplasma may entail the existence of alternative vectors. Based on actual and virtual RFLP analyses of PCR-amplified 16S rDNA sequences a number of subgroups had been described in the stolbur group, and molecular characterization on a number of its genes show growing evidences of variability related with strain differentiation. Biological complexity of stolbur phytoplasmas is also proved by the existence of numerous herbaceous hosts and diverse insect vectors. The question about host specificity is still open for stolbur as well as for the majority of described phytoplasmas. Characterization of BN phytoplasma populations was performed by multilocus sequence analysis and intriguingly, distribution patterns indicated a diverse prevalence of different populations in the geographic areas investigated (Contaldo et al., 2009; 2011; 2012). Selected BN strains were therefore compared with a number of stolbur strains from other plant host species as well as from H. obsoletus on secY and (st)amp genes in order to verify strains differentiation behaviours.

“Flavescence dorée” (FD) is a disease still of relevant importance considering the ability of phytoplasmas associated with it to differentiate new strains in short periods of time. The first finding of FD in Tuscany dates back to 2003 and concerns a vineyard sited at Montignoso, in Massa Carrara province. In the last decade the monitoring of phytoplasma has highlighted a fairly widespread presence of the disease, only in the wine-growing areas of the provinces of Massa Carrara and Lucca. In the context of the program EUPHRESCO GRAFDEPI, in 2012 surveys were carried out mainly in vineyards in the province of Massa Carrara. The vineyard reported was monitored in 2009 as well, it is made of six varieties, four native (‘Albarola’, ‘Ciliegiolo’, ‘Pollera Pontremolese’ and ‘Vermentino’) plus ‘Chardonnay’ and ‘Merlot’, all grafted on SO4 rootstock. The flora of the border is made up by chestnut plants and also several shrubs including, blackberries, hawthorn, plum, elderberry and especially greybeard. A total of 30 samples of leaves and branches were collected from symptomatic vines (nine in 2009 and 21 in 2012) and a total of six samples of Clematis vitalba were collected from border flora in 2012. All grapevine samples positive to phytoplasmas (7/9 in 2009 and 14/21 in 2012) resulted infected by FD-C. Regarding to the greybeard a sample resulted positive and infected by 16SrXII-A (“stolbur”) phytoplasmas. Molecular characterization was carried out on FD strains identify that all the samples were infected by FD-D phytoplasmas, however the fine characterization on the SecY gene by RFLP analyses showed that all strains but one have a profile indistinguishable from the one typically described in FD-C strains. One sample showed profile mixed between typical FD-C and FD-D strains. This result indicates that also in Tuscany FD-D phytoplasma is producing strains as already reported in other Italian FD-D epidemic as well as in one strain from France. This finding is quite important since the possibility to early identify new FD strains allows to better manage the disease before these strains become epidemically relevant.

Surveys were carried out in order to verify and monitor the presence of Hyalesthes obsoletus Signoret (Homoptera Cixiidae) populations in Serbia under different environmental conditions. H. obsoletus was present in all the localities investigated and different population dynamics were observed in relationship to the host plant. The development cycle of the vector and population density collected on Convolvulus arvensis L. was earlier and lower than those on the population collected on Urtica dioica L. In Udovice region, both nymphs and adults were observed on Artemisia vulgaris L. Since many larvae were found on the root apparatus of A. vulgaris, H. obsoletus appears to be adapted to the species and able to reproduce as well. Moreover, transmission trials were carried out with H. obsoletus population collected on nettle near corn field, allow verifying the ability of the cixiid to transmit the corn reddening disease. Amplification of phytoplasma DNA was obtained after nested PCR assays from corn samples tested after 40 days from insect caging and from batches of H. obsoletus collected on nettle from the same population used for cage-transmission. RFLP analyses allow identification of the detected phytoplasmas as “stolbur” in both, corn experimentally infected and H. obsoletus specimens tested.

The presence of phytoplasmas in potted cyclamen plants showing malformations, phyllody and virescence of the flowers was detected by PCR assays with primers amplifying phytoplasma 16Sr, GroEL, secY, rp, tuf and amp genes. RFLP analyses allowed to identify the detected phytoplasmas in the aster yellows subgroup 16SrI-B. These phytoplasmas were identical to each other and to reference strains belonging to 16SrI-B subgroups on all studied genes except amp gene, and were further classified as rpI-B, SecYI-B and GroELI-III groups. This multigene characterization of a ‘Candidatus Phytoplasma asteris’-related strain infecting cyclamen in Italy allows its distinction from reference strains by RFLP analyses of the amp gene.

Current phytoplasma detection and identification methods are primarily based on nested polymerase chain reaction followed by restriction fragment length polymorphism analysis and gel electrophoresis. These methods can potentially detect and differentiate all phytoplasmas including those previously not described. The present protocol describes the application of this method for identification of phytoplasmas at 16S rRNA (16Sr) group and 16Sr subgroup levels.

Phytoplasma identification has proved difficult due to the pathogen’s lack of morphological traits and their inability to be maintained in vitro. DNA bar-coding is an identification method based on comparison of a short DNA sequence with known sequences from a database. A DNA bar-coding tool was developed for phytoplasma identification. While other sequence-based methods may be well adapted to identification of particular strains of phytoplasmas, they not always can be used for identification of phytoplasmas from different groups simultaneously. This phytoplasma DNA bar-coding protocol based on Tuf and 16Sr genes allows identification of the following phytoplasma groups at group and in several cases subgroup levels: 16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX, 16SrXI, 16SrXII, 16SrXII, 16SrXIV, 16SrXV, 16SrXVIII, 16SrXX, 16SrXXI.

Introduction ‘Flavescence dorée’ (FD) is a quarantine phytoplasma in EU and inspite the reduction of its impact in affected European viticultural areas, it is still of relevant importance, considering the ability of phytoplasmas associated with this disease to differentiate new strains in short periods of time. Therefore knowledge about FD strains differentiation is of major relevance towards the correct disease management. Strains were differentiated on 16S ribosomal gene and on other molecular markers (Martini et al., 2002; Botti and Bertaccini, 2007; Arnauld et al., 2007). In this work molecular characterization of a number of FD strains from diverse grapevine growing areas was performed on secY (traslocase) and tuf (elongation factor Tu) genes. Materials and Methods During 2011 grapevine samples were collected in Emilia-Romagna region (North Italy) in areas where FD epidemic was increasing. As reference strains in periwinkle elm yellows, strain EY1 (‘Candidatus Phytoplasma ulmi’, 16SrV-A) and FD strain FD-AS (16SrV-C) were used. Reference strains in grapevine were FD Veneto 8/08 and Emilia Mo2/08 (16SrV-D) and Tuscany 6, REV2, REV7, and Serbia 86/09 (16SrV-C). After total nucleic acid extraction PCR/RFLP analyses on 16S ribosomal gene plus spacer region using primers B5/P7 (Padovan et al., 1995; Schneider et al., 1995) in seminested and M1/V1731 (Martini et al., 1999) in nested reactions on P1/P7 amplicons were carried out. To distinguish between 16S ribosomal subgroups TaqI (Fast, Fermentas, Lithuania) at 65°C for 10 minutes was employed on 300 ng of amplicon. The FD-D strains were further examined by RFLP analyses on SecY and tuf genes (Angelini et al., 2001; Contaldo et al., 2011) using TaqI and Tsp509I and AlfI respectively. Results and Discussion A total of 26 FD-D infected samples were selected after preliminary screening for further molecular characterization on the SecY and tuf genes. The RFLP analyses on SecY gene was carried out on 23 samples since 3 resulted not amplifiable on this gene. Two different profiles with Tsp509I and TaqI restriction enzymes were detected (Fig. 1) of which one is undistinguishable from reference strain Veneto 8/08 (profile I, Bertaccini et al., 2009) and from FD-88, the FD-D strain representative of the epidemic widespread in France and Northern Italy since 1990. Among the 23 samples examined 9 showed a profile that was clearly differentiable (profile II, Bertaccini et al., 2009). These results confirm the successful spreading of the FD-D strain identified in 2009 in the Lambrusco variety (Bertaccini et al., 2009) and confirm its distribution still restricted to Modena and Reggio Emilia provinces and to the same Lambrusco variety. The RFLP analyses on tuf gene was carried out on 21 FD-D infected samples. It is interesting to underline that the samples non amplified on this gene were not corresponding to those non amplified on SecY gene except in one case. RFLP analyses with AlfI on tuf amplicons from grapevine and reference strains substantially confirmed the differentiation of FD-C and FD-D phytoplasmas in 16S rDNA gene (Martini et al., 1999), however in some cases alternative grouping was observed. In particular strain FD-AS, 16SrV-C (in collection since 1970) showed profile identical to 16SrV-D strains; strain Tuscany 6, 16SrV-C was identical to reference strains EY1, 16SrV-A and finally Serbian strain 86/09, 16SrV-C was undistinguishable from strains REV2 and REV7, 16SrV-C but also from strain Re5 and Ra3 affiliated to subgroup 16SrV-D. These latter information indicates possibility of genetic rearrangement in the tuf gene of field collected FD strains as one of the mechanism involved in ‘flavescence dorée’ strain differentiation.

The presence of phytoplasmas in strawberry showing malformation of the fruits together with the typical green petals symptoms was detected in some North Western Italy cultivations. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rDNA and GroEL sequences specific for phytoplasmas. Bands of respectively 1.2 and 1.4 kb were obtained after nested-PCR assays and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows subgroup 16SrI-C, the GroELI grouping confirms that all the strains from strawberry were identical to each other and are affiliated to GroELI-VII group. This is the first GroEL gene characterization of strawberry green petals phytoplasmas.

Phytoplasmas are bacteria lacking cell wall that are located in the phloem of plants and in the hemolymph of insect vectors. The phytoplasma classification scheme is based on PCR/RFLP analyses of 16S rDNA: a reliable tool for the differentiation that has become the most comprehensive and widely accepted classification system. On the other hand, the ‘Candidatus Phytoplasma’ species description, recently under adoption, refers to 16S rRNA gene sequence with a threshold

The phytoplasmas associated with bois noir (BN) tuf-type a are transmitted to grapevines by means of Hyalesthes obsoletus Signoret using Urtica dioica L. as the inoculum source. In the period 2003–2008, a research was carried out in six vineyards of northern Italy where a large amount of nettles in the ditches surrounding vineyards was detected. The aim was to establish the nettle control effects on the presence of H. obsoletus and new symptomatic grapevines. PCR and RFLP analyses showed the presence of the BN tuf-type a phytoplasmas in symptomatic grapevines of all vineyards and in the vector. Weed control strategies along ditches were always associated with decreases in nettle coverage and H. obsoletus numbers. A reduction in the percentage of new symptomatic grapevines occurred in all vineyards except for one vineyard where the least reduction in nettle and H. obsoletus was observed. The decrease in H. obsoletus population was significantly correlated with a decrease in nettles along ditches, and the reduction in H. obsoletus population was correlated with a decrease in incidence of new symptomatic grapevines compared with the incidence before the application of weed control measures. Nettle weeding can reduce the incidence of BN tuf-type a, but weed control costs and impacts need to be considered.

Grapevine yellows is one of the major diseases affecting the vineyards in Europe as well as in the majority of grapevine cultivation areas worldwide. Phytoplasma presence seriously affect quality and quantity of production therefore accurate and sensitive pathogen detection and identification are relevant for the disease management. Phytoplasmas within single plants are populations of individuals; routine techniques such as PCR followed by RFLP or sequencing of uncloned or cloned products do not show the diversity of these populations. These techniques only allow identification of the most frequent genotypes in the samples and not mixed phytoplasma or pathogen infection. A number of grapevine samples in which single or mixed phytoplasma infection was detected by routine methods were employed for deep amplicon sequencing on the Roche Genome Sequencer FLX system to compare the two detection systems. Materials and Methods Samples employed were selected from those that are routinely processed during surveys for phytoplasma detection and identification in Northern Italy where both ‘flavescence dorée’ (FD) and ‘bois noir’ (BN) diseases are widespread or epidemic (Botti and Bertaccini, 2007). Total DNA was extracted from 1 g of mid-vein leaf tissue following the procedure of Angelini et al., (2001). Phytoplasma detection was carried out by direct PCR on ribosomal gene and spacer region (Martini et al., 2002) followed by nested amplification with R16(I)F1/R1 (Lee et al., 1994) and 16R758F/V1730 (Martini et al., 1999) primer pairs. RFLP analyses with TruI on the first amplicons and TaqI on the second one allow identification of BN and FD phytoplasmas. Samples showing single and mixed phytoplasma infection were selected for deep amplicon sequencing. Tagged primers were used for generating pyrosequencing samples following described PCR protocol (Nicolaisen et al., 2011). PCR products were pooled in equimolar amounts, run on an agarose gel and a band of the correct size was excised from the gel and purified using QIAquick gel extraction kit from QIAGEN. Two pools of 13 and 11 samples respectively were sequenced on a GS FLX plate at Eurofins MWG. Tag-sorted sequences were quality filtered using CLOTU software at the Bioportal webportal (http://www.bioportal.uio.no/). To minimize sequencing errors, only the first ~200 nucleotides of each sequence were used. Accepted sequences were clustered using CD-HIT with a 99% similarity threshold, and singleton sequences were discarded. To identify sequences, these were aligned together with reference sequences from GenBank using MEGA and phylogenetic trees were constructed. Results and Discussion A total of 35521 sequences were generated from the first pool of 13 field collected samples of phytoplasma infected grapevine in which single infection was detected by routine procedures. Four of these samples were tested twice to verify result consistency (table 1). These data overall confirmed RFLP results. Table 1. Number of sequences determined in each sample with single phytoplasma infection by routine methods. FD-C 46 FD-C 51 FD-D 53 FD-D 64 FD-D 66 FD-D 54 BN 33 BN 43 BN 59 FD-C 56 FD-C 63 FD-C 68 FD-C 71 FD-C 46 FD-D 53 BN 33 FD-C 56 16SrV-C/D 2109 1709 3032 885 1559 42 19 15 10 2040 1876 1520 1356 2126 2240 0 2540 16SrXII-A 0 0 0 0 24 0 2213 2730 4716 3 1 0 0 0 0 2756 0 Table 2. Number of sequences determined in each sample with mixed phytoplasma infection determined by routine methods. FD+BN 63 FD+BN 71 FD+BN 73 FD+BN 60 FD+BN 72 BN 67 FD+BN 76 FD TV1 FD+BN 112 FD+BN 120 FD+BN 80 16SrV-C/D 3009 414 54 612 359 2158 130 201 914 3846 33 16SrXII-A 361 5447 4442 1424 3964 1019 9727 4574 698 0 6823 16SrX-B 189 0 0 0 0 109 0 0 278 0 0 Chimeras 39 17 16 8 26 0 11 0 24 0 0 Total 3598 5878 4512 2044 4349 3286 9868 4775 1914 3846 6856 A total of 50,926 sequences were generated from the pool of 11 field collected samples in which mixed BN and FD phytoplasma infection was ...

The plant pathogens that cause significant economic damage to agricultural crops are subject to phytosanitary regulations aimed at protecting the sanitary and quality of propagative material and production. The availability of official diagnostic protocols, therefore, is necessary for the uniformity of results obtained in different laboratories and to give them a legal value, essential for the application of the phytosanitary measures. Moreover, the entire international community is moving in this direction in order to standardize the diagnosis. The aim of the Italian Project ARNADIA, funded by the Ministry of Agriculture Food and Forestry, was to produce diagnostic protocols officially recognized at a national level for the identification of major plant pathogens. One of the sub-tasks of the ARNADIA Project planned the production of reference and validated protocols for the diagnosis of ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma prunorum’. For this purpose a working group was established, including the main Italian scientific institutions which are involved in the study of both phytoplasmas Several diagnostic protocols and reagents were compared in five different laboratories, using the same target and no-target reference samples. Protocols of PCR and real time PCR were selected and developed to determine the performance characteristics for validation under the standard UNI CEI EN ISO/IEC 17025 and EPPO PM7/84 and PM7/98. The results showed that the accuracy and analytical sensitivity of PCR performed with specific primers and nested PCR are comparable, whereas real time PCR results were more reliable. A ringtest among seven Italian laboratories of Phytosanitary Regional Services is currently to evaluate the concordance parameter of all selected protocols.

Phytoplasmas within single plants can be considered as populations of individuals and as phytoplasmas are obligate parasites of plants, single clones cannot be obtained. For identification of phytoplasmas, PCR followed by RFLP, sequencing PCR products directly or sequencing cloned PCR products are standard procedures. These procedures will, however, not show the diversity of populations within single plants, as only the most frequent genotypes will be detected and identified. A number of grapevine samples, in which mixed phytoplasma infections were detected by a nested PCR, were used for deep amplicon sequencing on the Roche Genome Sequencer FLX system. 50,926 phytoplasma sequences from the 5’end of the 16Sr DNA gene were obtained from 16 phytoplasma-infected grapevine samples. After clustering and alignment to phytoplasma reference sequences it was shown that the phytoplasmas in the grapevine plants belonged to diverse 16Sr groups, mainly the same groups that were detected using nested/PCR/RFLP. Furthermore, a high number of single nucleotide polymorphisms were present in individual samples.

Apricot trees are affected by apricot chlorotic leafroll disease in all major cultivation areas in Europe: the disease is killing the plants few years and it widely associated with European stone fruit yellows (ESFY) ‘Candidatus Phytoplasma prunorum’ belonging to 16SrX-B. Samples from apricot trees showing typical chlorotic leafroll symptoms were collected in June/July 2010 in Serbia and Italy: 10 symptomatic and two asymptomatic plants were tested in total. In both Countries the disease was present in scattered distribution in 7 and 8 year old orchards. Phytoplasma detection was achieved by polymerase chain reaction and restriction fragment length polymorphism analyses using nested-PCR assays. Primers employed were P1/P7 followed by F1/B6 or R16F2n/R2. RFLP analyses on F1/B6 amplicons indicated the presence of 16SrXII in one of the samples from Serbia and of 16SrX-B in one of the samples from Italy. RFLP analyses with Tru1I, RsaI, HinfI, HhaI, and HpaII on R16F2n/R2 amplicons showed the presence of 16SrX-B phytoplasmas in 4 samples, two from the Italian orchard and two from the Serbian one. In the remaining three Serbian apricot samples a mixed infection of 16SrX-B and 16SrXII and a mixed infection of 16SrX-B, 16SrXII and 16SrI phytoplasmas were identified; one of the samples was negative. In two of the remaining samples from the Italian orchard a 16SrX-B and 16SrXII mixed infection was detected, while in the last sample 16SrX-B and 16SrIX-C phytoplasmas were identified. The use of nested PCR with PA2 primers on P1/P7 allow to detect 16rXII phytoplasma in the last sample from Serbia. In all the experiments carried out the two asymptomatic plants were negative. This is the first report of presence in symptomatic apricot of phytoplasmas different from ESFY, in particular the 16SrIX-C phytoplasma is reported for the fist time in this species. Work to verify the epidemic relevance of these phytoplasmas in the apricot chlorotic leafroll disease is in progress.

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. BackgroundPhytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.Methodology/Principal FindingsWe designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.Conclusions/SignificanceThis study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.

Phytoplasmas found in Chilean grapevines showing yellows symptoms were identified as belonging to the ribosomal subgroups 16SrI-B and 16SrI-C (‘Candidatus Phytoplasma asteris’), 16SrIII-J (X-disease group), 16SrV-A (‘Ca. P. ulmi’), 16SrVII-A (‘Ca. P. fraxini’), 16SrXII-A (stolbur or “bois noir”) (Gajardo et al., 2009; González et al., 2010). The presence of these pathogens in the plants depends on both propagation of infected plants and spreading by different insect species which feed on grapevine and also on the weeds growing near and/or in vineyards. In infected vineyards, several insects belonging to the family Cicadellidae positives to phytoplasmas were found. The most common was Paratanus exitiosus (Beamer) in which phytoplasmas of 16SrI-B, 16SrIII-J, 16SrVII-A, and 16SrXII-A subgroups were detected. In the present work, we verified the phytoplasma transmission ability of the leafhopper P. exitiosus. Materials and Methods During 2011 (since September till December) and 2012 (since January till May) leafhopper survey was carried out in Chilean vineyards infected by phytoplasmas, two located in Metropolitana Region (1 and 2) and one in Valparaiso Region (3), to know biological characteristics of P. exitiosus and its ability to transmit phytoplasmas. The insects were captured by sweeping with an entomological net. During the sampling period adults of P. exitiosus captured have been released into entomological cages to let them feed on three plants of periwinkle (Catharanthus roseus (L.) G. Don) grown from seed and previously tested to ascertain the absence of phytoplasmas. A total of 81 plants were used. Periwinkle plants were tested starting five months after transmission trials, while dead insects were then preserved in 70% ethanol. Insects and periwinkles were tested in order to identify the phytoplasma presence. Total nucleic acids (TNAs) were extracted with chloroform/phenol methods, dissolved in Tris-EDTA pH 8.0 buffer, and maintained at 4°C; 20 ng/µl of nucleic acid were used for amplification. After direct PCR with primer pair P1/P7, nested PCR with R16F2n/R2 primers (Gundersen and Lee, 1996) was performed. PCR and nested PCR reactions were carried out following published protocol (Schaff et al., 1992). P1/P7 amplicons were purified using Concert Rapid PCR Purification System and DNA fragments were cloned. Putative recombinant clones were analyzed by colony PCR. Selected fragments from cloned DNAs were sequenced in both directions using the BIG DYE sequencing terminator kit. The sequences were then aligned with BLAST engine for local alignment (version Blast N 2.2.12). Identification was done using in silico restriction fragment length polymorphism (RFLP) analyses on sequences amplified with primer pair R16F2n/R2 with BstU1 and HhaI restriction enzymes (Wei et al., 2007). Results and Discussion In the vineyard 1 the P. exitiosus capture rate remained constant during all months, with a decrease in October 2011 and April 2012. In the vineyard 2 the highest number of individuals was obtained in December 2011 and January 2012. In the vineyard 3 P. exitiosus was less abundant in September 2011, remained constant during the other months and increased only during February 2012. These results indicate that P. exitiosus tends to be more abundant during summer time. Three out of 81 periwinkles used for transmission trials were positive to phytoplasmas. Two (VC28C and VC31C) correspond to the transmission trials carried out with insects captured in the vineyard 1 in two different months (November and December 2011 respectively), the third (VC33A) was infected from insects captured in the vineyard 3 during December 2011. Cloned P1/P7 fragments were sequenced and there was no sequence difference between the cloned fragments from the three periwinkles (1,819 bp). The similarity percentages of VC28C, VC31C, and VC33A phytoplasmas, showed a close correlation (99.6, 99.7, 99.6 % respectively) with the strain Ch10 (AF147706...

During survey carried out in 2008 to verify phytoplasma presence in fruit trees in Serbia, samples were collected from two single peach plants (30-Niš and 103-Radmilovac) showing yellows disease and phytoplasma detection was carried out using PCR assays with primer pair P1/P7 in direct reaction, followed by nested PCR with F1/B6, R16F2n/R2, and R16(I)F1/R1. RFLP analyses carried out on R16F2n/R2 amplicons with Tru1I, TaqI, Tsp509I, AluI, and BfaI restriction enzymes showed that in peach 30, a mixed phytoplasma infection was present. Tentatively identification of 16SrII and 16SrXII phytoplasmas was confirmed by RFLP analyses carried out on amplicons obtained with primers R16(I)F1/R1 with Tru1I and Tsp509I restriction enzymes. RFLP analyses on F1/B6 and R16F2n/R2 amplicons with Tru1I showed that 16SrII group phytoplasmas were present also in peach 103. F1/B6 and R16F2n/R2 amplified products (about 1700 bp and 1200 bp respectively) of both peach samples were purified using Qiagen PCR Purification Kit (Qiagen GmbH, cloned in DH5 alfa and a number of clones was screened by PCR with M13 primers followed in nested PCR by F1/B6 and R6F2n/R2 primers (according with amplicons). RFLP analyses with Tru1I on R16F2n/R2 amplicons of seven M13 clones obtained from peach 30 show the presence of 6 profiles referable to 16SrXII and two referable to 16SrII phytoplasmas. AluI restriction enzyme did not show polymorphisms and distinguished between groups 16SrII and 16SrXII while Tsp509I showed different profiles between the two 16SrII group phytoplasmas. Similar results were obtained with Tru1I and AluI restriction enzymes on 3 clones amplified with R16(I)F1/R1. Peach 103 clones were all identical after RFLP analyses. This is the first report of 16SrII phytoplasmas in peach and the 16Sr DNA variability detected in both 16SrXII and 16SrII phytoplasmas is an indication of phytoplasma population presence in the infected plant.

Phytoplasmas infect a high number of plant species. Based on the 16S ribosomal gene sequence, over 20 RFLP groups have been established within the ‘Candidatus Phytoplasma’ taxon, some of which are of quarantine status. To date, no protocol for in vitro cultivation of phytoplasma has been developed, making classical microbiological identification procedures impossible. The basic phytoplasma identification methods is based on RFLP analysis of a 1.2 kbp region of the 16S rDNA, however, this method is not easy to set up and it only considers a part of the available phytoplasma sequence information. It has been suggested that universally amplified, short, and highly variable DNA barcodes may help to rapidly identify organisms. As part of QBOL project a universal DNA barcoding based tool for phytoplasma identification was developed. Two sets of primers amplifying a fragment of the Tuf gene and a fragment of the 16S rDNA gene, respectively, were designed and the potential of these fragments as DNA barcodes was verified. Successful amplification and sequencing of more than 150 phytoplasma strains, and ability to separate various phytoplasma strains to ‘Candidatus species’ level, 16S ribosomal group and sub-group level suggest that these barcodes are efficient phytoplasma identification tools

In this study the spread of ‘bois noir’ (BN) was monitored from 2005 to 2010 in three organic vineyards of cv. Sangiovese established in 2001 and 2002 in the province of Florence, Tuscany (central Italy). Maps with locations of all symptomatic and non-symptomatic vines, a complete census of binary data, were made for each plot and year of assessment. Three indexes (annual incidence, infection and recovery rates) were calculated from the original data sets to describe changes in the epidemic with time. Moreover with the purpose of determining the spatial pattern of the disease (random vs. non random or clustered), for each year of assessment, field data on annual incidence were analyzed at three spatial levels (hierarchies). The values of annual incidence, infection and recovery rates in each year were different at each study site but with a similar trend over time. In general symptomatic plants showed the phenomenon of recovery in a relatively short time meanwhile the infection rate decreased rapidly after the second year of assessments. Results of spatial pattern analysis were consistent at all levels considered and suggested that the arrangement of diseased vines was random across all disease assessment dates in two out of three study sites. On the other hand in the third vineyard the distribution of the disease was always non random although an intensification of clustering over time never became evident. Since the epidemics were different in different environments, a survey on the herbaceous vineyard weeds was carried out in 2010 to study how organic vineyard management practices may influence disease spread. Symptoms of yellowing or reddening were recorded on some species meanwhile others showed symptoms typical for those associated with phytoplasma infections like dwarfing, virescence, and phyllody. Total DNA was isolated from root and or leaf tissues of about 200 samples and nested PCR was carried out using P1/P7 followed by R16F2/R2 primers. RFLP analyses carried out with TruI restriction enzyme allow to identify the following species infected by 16SrXII phytoplasmas (stolbur): Pichiris hieracioides, Daucus carota, Lactuca saligna, Lactuca sp., Bupleurum tenuissimum, Cichorium intibus, Convolvolus spp., Cuscuta spp., Mercurialis annua, Linaria vulgaris, and Medicago lupulina. In some of these species further polymorphisms in R16F2/R2 amplicons were detected after MboII restriction, and in one case also with Hpy188I; the same samples digested with AluI, did not show any polymorphism. The different profiles observed could be referred to some of those identified in grapevine from diverse geographical areas including Tuscany (Contaldo et al., 2011). Further studies to verify the relevance of these polymorphisms in the BN epidemiology are in progress.

Some Auchenorrhyncha specimens were captured, identified and tested to verify phytoplasma presence in Chilean vineyards; many of them belong to the subfamily Delthocephalinae and Agalliinae (family Cicadellidae) and to the families Cixiidae and Delphacidae, all known as potential phytoplasma vectors. Several individuals were positives to the phytoplasmas, in particular Amplicephalus curtulus Linnavuori & De Long, in which were detected the phytoplasmas belonging to subgroup16SrI-B and 16SrXII-A, and Paratanus exitiosus (Beamer) positive to phytoplasmas of the subgroups 16SrI-B, 16SrVII-A and 16SrXII-A. Phytoplasmas belonging to subgroup 16SrI-B and 16SrVII-A were identified in Convolvulus arvensis L. and Polygonum avicu-lare L.; to subgroup 16SrXII-A in C. arvensis; and to subgroup16SrVII-A in Galega officinalis L. In three cases the grapevine samples, weeds and insects collected in the same vineyard were positives to phytoplasmas of the same subgroup.

During a survey carried out in Serbia in black poplar trees typical symptoms of phytoplasma presence were observed: yellowing and undersize of the leaves, witches broom and decline. Survey for phytoplasma presence in Belgrade and in its surrounding al-low to identify phytoplasmas belonging to ‘Candidatus Phytoplasma asteris’ group, in particular to two 16SrI subgroups 16SrI-P and 16SrI-A. Blast search of obtained strain in subgroup 16SrI-P of the 16SrDNA sequence showed 100% homology with aster yellows phytoplasma strain from poplar from Croatia.

In campioni di actinidia prelevati in aree con epidemie conclamate di cancro batterico sono stati individuati fi toplasmi afferibili ai giallumi dell’astro e dell’olmo e allo stolbur

Surveys carried out to identify ‘bois noir’ (BN) phytoplasmas in grapevine yellows outbreaks in vineyards located in Serbia and in several Italian regions were carried out from 2008 to 2010. The presence of BN phytoplasmas was preliminarily demonstrated by RFLP analyses with TruI restriction enzyme on R16F2/R2 amplicons. About 60 samples were selected for further molecular characterization. Reference strains maintained in periwinkle were STOL (from Serbia), STOLC and STOL-PO (from France). RFLP analyses on R16F2/R2 amplicons with Hpy188I, Hpy8I, MboI, MboII, TruI, RsaI, BstUI, AluI, and Tsp509I restriction enzymes were carried out. Tsp509I was only differentiating one samples from Veneto region in Italy, while different profiles were observed in several of the examined samples when MboII, Hpy188I, and AluI were used. In particular, three different profiles were observed with AluI in samples from Veneto and Tuscany (Italy), and Serbia. Using MboII four different profiles (a-b-c-d) were observed in samples from Veneto, Tuscany and Emilia, while only one of them (profile a) was observed in the samples from Serbia. RFLP profiles of reference strains were a for STOL, b for STOL-PO and c for STOLC. Considering that the profile c is referring to an amplicons longer than the expected one, the presence of interoperon heterogeneity and/or of mixed BN strain infection could be hypnotized for this profile. Some of the samples having profile c when digested with Hpy188I showed further polymorphism. The reference strains plus 7 field collected BN strains chosen among those showing the above described polymorphisms were sequenced on the full 16S gene. The sequences were assembled using DNA STAR software, and compared with selected sequences of phytoplasmas in GenBank database using Blast N 2.2.18. Obtained aligned sequences ranged from 1,300 to 1,500 bp, all showing 99% homology among them self and with several of the 16SrXII-related strains deposited in Genbank. Virtual RFLP analyses on R16F2/R2 amplicons were carried out, using pDRAW32 program (AcaClone Software) and it was possible to confirm the variability detected in real RFLP analyses in the majority of BN sequenced strains. The comparison between real and virtual RFLP analyses showed in some cases different profiles when digested with MboII. Strain STOLC showed in this case a b profile confirming that profile c is formed by mixed infection or interoperon heterogeneity. On the other hand the presence of a d profile was detected in one of the sequenced strains from Serbia showing an a profile in RFLP analyses. BN strains differentiation on 16S rDNA on field collected uncloned amplicons indicates that the detected variability could be related with different epidemiological behaviours.

‘Bois noir’ (BN) - associated phytoplasmas were detected in Hungary since long time (Kolber et al., 1997), recently the presence of polymorphisms in some of the genes used as markers for strain differentiation was also reported (Contaldo et al., 2009). To further verify the consistence of molecular variability BN infected samples were collected in 2008-2010 from symptomatic grapevine plants from vineyards of variety Zweigelt located in Sopron area (near to the Austrian border), varieties Riesling-Sylvaner and Chardonnay from Etyek (near Budapest), unknown variety from Southeast Hungary, and from a variety Chardonnay from Eger. PCR/RFLP characterization was carried out on tuf gene (Langer and Maixner, 2004) using nested-PCR procedures, and restriction with HpaII enzyme showed that all the strains belong to tuf type-b (VK-II). Further molecular characterization on 16S rDNA gene, spacer region and beginning of 23S gene were carried out on 12 selected samples by RFLP analyses with TruI, BstUI, Hpy188I, and MboII restriction enzymes on diverse length amplicons. RFLP analyses on R16F2/R2 by BstUI and Hpy188I did not show differences among BN samples. The same amplicons digested with MboII show variability among BN strains: in particular three different profiles (a, b, and d) were observed. While the a profile was present in 4 samples, profile d was observed in 2 samples, and the c profile was observed in all the others. This last profile is very likely derived from a presence of a mixed infection by two BN strains recently differentiated on reference strains STOL and STOL-C (Contaldo et al., 2009, 2011). Selected grapevine samples were sequenced on 16S ribosomal region (about 1,500 bp), and virtual RFLP analyses carried out on R16F2/R2 amplicons showed in some cases differences between real and virtual RFLP profiles confirming the presence of mixed infection in samples with profile c. After Genbank search similar analyses on deposited BN strains showed identical Hpy188I profiles among a sample from Hungary and BN samples from Canada and Spain (EU086529 and AJ964960). These results clearly indicate that among BN strains belonging to tuf type-b there is a strain differentiation on 16S gene achievable using selected restriction enzymes. Further studies will clarify the epidemiological relevance of these strains in BN epidemics.

Cherry is an important fruit crop for which severe decline symptoms associated with the presence of phytoplasmas has been reported in the past years. A cherry decline, termed “leptonecrosis,” was first reported in Emilia region of northern Italy in the early 1930s. An outbreak of a lethal yellows disease on Chinese cherry in the Sichuan province of China in 1989 caused severe losses to the local cherry industry. Electron microscopy study of diseased tissues and disease remission resulting from tetracycline treatment of infected plants suggested that the cherry lethal yellows (CLY) disease may be caused by a phytoplasma . Based on the phylogenetic analysis of the 16S rRNA gene, RFLP analysis of ribosomal protein gene sequences and Southern hybridization analysis of total genomic DNA using selected DNA probes derived from the Elm Yellows (EY) phytoplasma (group 16SrV, subgroup 16SrV-A), it was proposed that the CLY phytoplasma belongs to the elm yellows phytoplasma group (16SrV), and represents a new subgroup, 16SrV-B, distinct from other members of 16S rRNA group V or 16SrV. A recent study showed the presence of 16SrV-B phytoplasmas in severely declining cherry trees in northern Italy. More recently phytoplasmas belonging to two ribosomal groups (stolbur, 16SrXII-A and clover yellow edge, 16SrIII-B, respectively) were identified in cherry plants showing a quick dieback before the harvesting season, this disease is named decline. The symptoms associated with the diverse phytoplasmas in Italy do not differs from each other and are quite similar to the CLY described in China.

To evaluate the seed transmission of phytoplasmas in three herbaceous hosts material from different geographical origin but all collected from infected or symptomatic mother plants was used. Almost 1,000 seeds were germinated under controlled conditions and 652 seedlings were obtained. Samples were grouped in 214 samples and 74 of them resulted positive to phytoplasma presence after three to 90 days from germination by nested PCR assays on 16S ribosomal gene. All the tested species i.e. winter oilseed rape, tomato and corn resulted carrying phytoplasmas belonging to the ribosomal groups retrieved in the infected mother plants.

The usefulness of RFLP analyses on a 435 bp fragment of the tuf gene for preliminary identification of phytoplasmas from a number of phytoplasma ribosomal groups and/or ‘Candidatus Phytoplasma’ was verified. The strains employed belong to 13 16Sr DNA groups and to 22 different subgroups and were both from periwinkle infected plants and from naturally infected plants. The combined RFLP patterns obtained with the three restriction enzymes employed allow the distinction of a total of 18 differ-ent profiles, however no discrimination is provided for some of the ribosomal group for which sequencing remains the main tool for phytoplasma identification.

Turnera ulmifolia, the yellow alder, is a widely distributed species in Brazil where, besides ornamental, it is used as tea for the treatment of gastric diseases. In plants showing yellowing and witches’ broom symptoms phytoplasmas were detected by molecular analyses. The prokaryote characterization on 16S rDNA gene by RFLP analyses allows preliminary classification of these phytoplasmas in the 16SrXIII ribosomal group, that is firstly reported in the present work as occurring in Brazil

Bois noir (BN) phytoplasmas were associated with grapevine yellows diseases in Bosnia and Herzegovina (Delic and Lolic, 2010). The disease is widely to moderate distributed in vineyards with both domestic and imported grapevine cultivars. Molecular characterisation of selected strains of BN phytoplasmas detected in heavily BN-infected vineyards in was done to verify the presence of genetic variability. Thirty-five DNA samples of grapevines that were shown to be positive by PCR assays on the 16S rDNA were selected for the study. Polymorphisms were studied in the 16S rDNA, Tuf and ribosomal protein genes of the selected samples. Tuf1f/r, TufAYf/r, and TufINT1f/TufINT4r primer pairs were used in nested PCR for amplification of Tuf genes; RFLP analyses with HpaII showed the presence of the tuf type-b (VK-II) in all the tested samples. The BN infected samples were further amplified with rp/STOL primers and PCR products were digested with TaiI and SspI restriction enzymes. Finally 16S rDNA genes were amplified with P1/P7 primers in direct PCR and with R16F2n/R16R2 primers in nested PCR assays: both types of amplicons obtained were restricted with MboII and RsaI, MboII, Hpy188I enzymes, respectively. Restriction profiles obtained after RFLP analyses on 16S rDNA and ribosomal protein amplicons weren't uniform. Some samples showed profiles comparable with some of those recently published (Contaldo et al., 2009), while some of the profiles of other samples were totally different from any of those reported in literature. Therefore, polymorphism of these regions should be further studied to better understand BN epidemiology in Bosnia and Herzegovina.

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 (Botti et al., 2005), 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 a, tuf-type b and tuf-type c) 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 a and tuf-type b stolbur phytoplasmas (Berger et al., 2009). A real time PCR assay for the rapid and specific detection tuf-type a and tuf- type b was developed and applied in alternative to 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 a and tuf-type b among selected 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 a and tuf-type b types resulted to be very efficient. A number of BN positive samples are under characterization with both methods real-time or nested-PCR to establish the presence of tuf-type a and tuf-type b and to trace a distribution map in Tuscany.

Interferon gamma (IFN-g) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-g in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-g concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-g mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-g in samples incubated with peptides decreased and the expression of IFN-gmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a ‘‘pre-activated’’ immune response). These cats responded to ‘‘avirulent’’ or ‘‘mixed’’ peptides by increasing the concentration of IFN-g and the expression of IFN-g mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-g especially in cats with a ‘‘pre-activated’’ cell mediated response.

Since the nineteen’s, when the first outbreak of grapevine yellows (GY) in Tuscany was reported, field inspections and laboratory analysis were conducted. However, regular surveys of Flavescence dorée (FD), Bois doir (BN) presence were carried out in Tuscany since 2003. Several regional and non regional public institutions were involved in a control strategy aimed to the containment of GY spreading in the region. During the last years and in strength collaborations among the different institutions, activities ranging from: (i) annual survey through extensive field inspections in vineyards from the ten provinces of the Tuscany region; (ii) collection of samples showing symptoms referable to GY diseases; (iii) application and implementation of molecular techniques for detection and characterization of phytoplasma ribosomal subgroups associated with the disease were carried out. Nested-PCR, PCR/RLFP and Real-time PCR were applied as main laboratory techniques. Often, particularly interesting samples were exchanged among partners and analyzed with different techniques for a final phytoplasma identification and strain characterization. Samples collected and tested with molecular analyses were 3,453, among them 2,304 (66,7%) resulted positive to phytoplasma associated with GY. In details, BN was largely the prevalent GY in Tuscany with an overall incidence of 95.7% (2,205 out of 2,304). The presence of FD was verified in 99 samples distributed in five provinces: Massa-Carrara (59), Florence (12), Lucca (11), Siena (10) and Arezzo (7).

The clear differentiation among ‘Candidatus Phytoplasma asteris’-related strains using Amp-N1/C1 primers amplifying the amp gene was observed in samples classified into different 16SrI subgroups (16SrI-A, I-B, I-C, I-L, and I-M). Samples were maintained in periwinkle or collected from field infected plants and insects; in 27 out of the 31 strains employed the full amp gene was also sequenced. Carrot with yellows symptoms and Macrosteles sexnotatus from the same field in Serbia (16SrI-A, 16SrI-B, and 16Sr-I subgroup not identified); poplar with witches’ broom collected in Serbia (16SrI-P); corn and oil palm samples (16SrI-B) from Colombia showing respectively stunt and decline symptoms were also employed. The amp gene could not be amplified in 16SrI-F and I-P strains; in 16SrI-B, I-L, I-M and in the strain from carrot in a not identified subgroup a 740 bp amplicon was obtained; strains in subgroups 16SrI-A, and I-C produced a 650 bp amplicon. RFLP analyses with TruI and Tsp509I enzymes distinguished up to four different profiles in amplicons from16SrI-B, I-L I-M strains, two profiles in 16SrI-A, while an unique profile was present in 16SrI-C strains. Among the field collected samples the M. sexnotatus sample showed a profile different from those obtained in strains from carrots, but referable to one of those of 16SrI-B phytoplasmas in periwinkle. RFLP profiles of corn and oil palm strains were identical to each others and to the one of 16Sr-I strain carrot. The full sequence of amp gene obtained was also studied with translated nucleotide query using NCBI/BLAST/blastx program. Preliminary results indicate that strains in subgroups 16SrI-A, I-C and I-F and strains DAY, AVUT and PrG in 16SrI-B are the less homologous to strains available in GenBank. The phylogenetic analyses of translated sequences confirmed these results, and showed a further differentiation of strains GLAWC, and PRIVA from the majority of 16SrI-B strains sequenced, and the I-P strain from populus was very different from all the others. Further research could clarify possible relationship between amp gene, plant host and insect vector towards molecular epidemiology study of ‘Ca. P asteris’-related phytoplasmas.

Grapevine yellows (GY) are an important threat to grapevine industry in Europe. 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 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 “Global Positioning System-GPS”. 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.

During 2007/2009 a survey was carried out in 25 vineyards of a grape-growing area in Northern Italy to detect and identify the presence of possible insect vectors of the ‘bois noir’ phytoplasmas (stolbur group) in Lambrusco variety. The leaf/planthopper population was monitored weekly by sticky traps, and sweep nets on herbaceous vegetation and “frappage” on grapevine carried out in the middle and on the border of vineyards from June to September. Captured insects were identified and analyzed with molecular analyses to detect the presence of stolbur phytoplasmas. Identified Cixiidae were Hyalesthes, Cixius and Reptalus. The most abundant species were Hyalesthes obsoletus Signoret (98% of the 761 Hyalesthes genus captured), Cixius cunicularis L. (over 93% of the 273 Cixius captured) and Reptalus panzeri Löw (65% of all the 177 Reptalus captured). Scattered specimens of H. luteipes Fieber, C. nervosus L., C. caledonicus China and R. quinquecostatus Dufour where also identified. The best way to catch the insects was using the sweep net for Hyalesthes and Reptalus (over 97% of H. obsoletus and 78% of Reptalus captured) whereas the “frappage” was the best method for Cixius (64% of all captures). Most of the insects were captured in the vineyard borders (95% Hyalesthes, 72% Reptalus and 60% Cixius). Phytoplasma presence as determined by nested-PCR/RFLP analyses on 16S ribosomal and tuf genes allow identifying stolbur phytoplasmas in different percentages according with species.

In addition to the grapevine flavescence dorée phytoplasmas (ribosomal subgroups 16SrV-C and -D), other closely related members of group 16SrV infect Alnus spp. (16SrV-C), Clematis vitalba (16SrV-C), Spartium junceum (16SrV-C) and Rubus spp. (16SrV-E) in Europe. To investigate which phytoplasmas constitute discrete, species-level taxa, a high number of strains, mainly from grapevine and clematis, were analysed comparing their 16S rDNA and a set of two to five house-keeping genes. Whereas 16Sr DNA identities were well above 97.5% the threshold aimed to distinguish two ‘Candidatus Phytoplasma’ species, phylogenetic analysis of the combined sequences of tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genes showed that two discrete phylogenetic clusters can clearly be distinguished. The first cluster grouped flavescence dorée (FD), alder yellows (AldY), clematis and the Palatinate grapevine yellows (PGY) phytoplasmas, and the second cluster was constituted by Rubus stunt phytoplasmas. High genetic diversity was present among phytoplasma strains belonging to the first cluster and several subclusters could be recognized; whereas among Rubus stunt phytoplasmas all analyzed gene sequences resulted highly conserved. Studies based on sequencing, phylogenetic and RFLP analyses of 16S rDNA, rplV-rpsC and rpl15-secY gene sequences carried out mainly on flavescence dorée phytoplasma strains from Italy, France and Serbia confirmed the presence of genetic variability in phytoplasmas from both Flavescence dorée ribosomal subgroups. These subgroups were only differentiated for the presence of a SNP corresponding to a TaqI restriction site, however this difference enable to distinguish their geographical and epidemic distribution. Analyses on rplV-rpsC and rpl15-secY gene reinforced the clear strain differentiation between flavescence dorée phytoplasma strains of 16SrV-C and –D subgroups. Moreover it was proved the presence of about 10 different flavescence dorée phytoplasma strains in 16SrV-C subgroup, and of 3 different phytoplasma strains in 16SrV-D. In addition to their specificity of insect vector (Scaphoideus titanus and Oncopsis alni for phytoplasmas of the first cluster and Macropsis fuscula for Rubus stunt phytoplasma in the second cluster), the genomes of the phytoplasma strains belonging to the two distinct clusters above mentioned are differentiated enough to propose two novel putative taxa, ‘Candidatus Phytoplasma caudwellii’ for FD, AldY, PGY and clematis phytoplasmas, and ‘Candidatus Phytoplasma rubi’ for rubus stunt phytoplasmas. However, if the rules for the taxonomy of uncultured bacteria were to be strictly applied, this proposition would be not completely acceptable. Indeed, at the 16S rDNA level Rubus stunt phytoplasma can be described as a new species with several specific nucleotide polymorphisms in its 16S rDNA respect to 16SrV-C/D phytoplasmas, in addition to the clear speciation on the five non ribosomal genetic markers. On the contrary, the members of subgroup 16SrV-C and D (FD, AldY, Clematis vitalba and PGY phytoplasmas) could not be described as a new species because no specific oligonucleotide polymorphism can be found in their 16S rDNA. Therefore the entire rules as they stand could not be fulfilled for flavescence dorée and closely related phytoplasma isolate, except if a new rule more based on the genomo-species concept was to be established. This is quite relevant, especially considering that FD is a quarantine-regulated pathogen in Europe.

Cucumber (Cucumis sativus L.) is widely cultivated in Italy in about 1.200 ha (ISTAT, 2008). In Veneto region it is grown in about 90 ha under screen mainly in Verona and Venezia provinces for fresh consumption. Different infectious pathogens are affecting cucumber, fungi: Erisiphe cichoracearum, Pseudoperonospora cubensis and Botrytis cinerea. Among pests: Frankliniella occidentalis, Aphis gossypii, Heliothis armigera, Udea ferrugalis, Liriomyza spp. and Tetranychus urticae are affecting cucumber. Cucumber mosaic virus (CMV), Tobacco Mosaic Virus (TMV) and Zucchini Yellow Mosaic Virus (ZYMV) are also severely affecting cucumber. During 2009 in Verona and Oppeno (Verona province) cucumber plants showing symptoms referred to possible phytoplasma presence were observed such as stunting and abnormal growth, distortion of leaves that have small size and chlorotic borders; a reduced number of malformed flowers were produced and fruits were stunted and malformed. In July samples were collected from symptomatic and asymptomatic plants and nucleic acid extraction using a chloroform/phenol method (Prince et al., 1993) was carried out from shoots, midribs and petioles. Phytoplasma identification was obtained with PCR using primers R16F2/R16R2 specific for phytoplasma 16S rRNA (Lee et al., 1995), followed by RFLP analyses with TruI, BfaI, and HhaI and by nested-PCR with primers R16(I)F1/R1 specific for some of the described phytoplasma ribosomal groups. In amplicons from symptomatic samples identical RFLP profiles were observed in all cucumber samples and these profiles were indistinguishable from the one of strain STOL (stolbur from pepper from Serbia, 16SrXII-A), employed as reference strain. To our knowledge this is the first report of stolbur in cucumber, however the same species was recently reported to be infected by 16SrII phytoplasmas in Iran when phyllody symptoms were present (Tazehkand et al., 2010). The plants studied were produced in different nurseries and belong to the ginoic cultivars Jazzer and Sargon. Disease incidence in field was of 4-6% randomly distributed in the infected plots. Further studies on phytoplasma characterization and on its epidemiological spreading in cucumber are in progress.

Liquidambar stiracyflua, introduced to Bogota 10 years ago as a street plant, is showing symptoms possibly associated to phytoplasmas. Symptoms include changes in the architecture of the crown due to unevenly spaced internodes, deliquescent branching, and presence of small and large leaves in branches. Liquidambar trees are located in the same streets in which Fraxinus with yellows phytoplasma, have been described. DNA samples extracted from leaf midribs of four plants were tested to verify phytoplasma presence by amplification of the16SrDNA gene. Direct PCR assays with primers P1/P7 did not provided amplification from any sample. Nested-PCR assays with primers R16F2/R2 produced bands of the expected length (1.2 kb) from all samples tested. RFLP analyses of amplicons with selected restriction enzymes provided restriction profiles referable to Ash yellows phytoplasmas (‘Candidatus Phytoplasma fraxini’ 16SrVII-A), while in two samples L2 and L4 beside the profiles referable to Ash yellows phytoplasmas profiles corresponding to 16SrI and 16SrXII-A ribosomal groups were detected, respectively indicating the presence of multiple phytoplasma infection. Further amplification with primers R16mF1/R16mR2 on P1/P7 amplicons of samples L1 and L3 allowed amplification of nearly full 16S ribosomal gene (about 1,400 nt). Direct sequencing with R16mF1, M1, F3 and R16mR2 primers generated two fragments of 1,240 and 1,078 nt, respectively (GU810150 and GU810151) showing 100% homology between them for overlapping part (937 bp); this part was also identical those of several ‘Ca. P. fraxini’ strains available in Genbank (16SrVII-A subgroup). Some SNPs of both L1 and L3 strains with ‘Ca. P. fraxini’ strains were observed in the non overlapping parts of the sequences.

Periwinkle shoots infected with stolbur phytoplasma strain CH1 (group 16SrXII), dodder transmitted from grapevine infected with “bois noir” disease in Italy, were employed for phytoplasma growth trials in axenic medium. Sterile 1-2 mm stems from two different shoots of two years in vitro growing cultures, were cut longitudinally with razor blades under sterile conditions, immersed in 2 tubes each containing 2 ml Mycoplasma Experience liquid medium (a medium suitable for a wide range of mycoplasmas containing pig serum, a broth base and yeast extract) and maintained in an incubator at 26°C for 7 days. About 300 µl of the medium containing the cut shoots were then transferred to new tubes containing 2 ml of fresh medium. Twenty days after the transfer, PCR assays were carried out using 1 µl medium, denaturated by boiling, as template. The R16F2/R2 primers specific for phytoplasma 16S ribosomal gene were employed for 35 cycles at 47°C annealing temperature. RFLP analyses with TruI carried out on the 4 amplicons of the expected 1.2 kb length obtained with the 4 tubes inoculated showed the presence of 16SrXII phytoplasma DNA. The tubes of the first transfer were then incubated under the same conditions for 9 months when acid colour-changes were seen. Aliquots of 100 µl from the 4 tubes were transferred to 4 tubes of fresh medium which all gave acid colour changes after incubation for 3 days, but PCR testing for phytoplasmas was negative. However, after 10 days’ incubation, PCR testing was positive for the presence of phytoplasma DNA. PCR amplification was carried out using as template 1 µl of the pellets obtained from full speed centrifugation of 100 µl medium, resuspended in 10 µl of sterile distilled water. Primers employed for these latter amplifications were R16(I)F1/R1, specific for phytoplasma groups 16SrI, II and XII, and were used under published conditions; RFLP analyses confirmed that 16SrXII phytoplasmas were present in the broth media after transfers. Amplification of about 600 bp in the 16Sr of phytoplasmas followed by direct sequencing confirmed RFLP results. Further experiments are in progress.

Barcode regions are used to identify living organisms and the requirements are: polymorphisms to discriminate close relatives; conserved regions for primer design; ideally short 500-700 bp regions. Phytoplasma identification is carried out in a new project funded by EU FP7 to generate barcode sequences from a selected set of genetic regions and for the relevant/quarantine phytoplasmas listed below. – Elm phloem necrosis (ribosomal subgroup 16SrV-A, strain EY) – Peach rosette (ribosomal group 16SrIII) – Peach X (ribosomal subgroup 16SrIII-A, strains CX and WX) – Peach yellows (ribosomal group 16SrIII) – Strawberry witches’ broom (ribosomal subgroup 16SrI-C) – Apple proliferation ’Candidatus Phytoplasma mali’ (ribosomal subgroup 16SrX-A, strains AP, AT) – Apricot chlorotic leafroll ’Candidatus Phytoplasma prunorum’ (rib. subgroup 16SrX-B, ESFY) – Pear decline ’Candidatus Phytoplasma pyri’ (ribosomal subgroup 16SrX-C, PD) – Palm lethal yellowing (ribosomal group 16SrIV) – ’Witches broom’ on Citrus ’Candidatus Phytoplasma aurantifolia’ (ribosomal subgroup 16SrII-B) – Grapevine flavescence doreé (ribosomal subgroups 16SrV-C and 16SrV-D) – Potato stolbur (ribosomal group 16SrXII) – Potato purple top wilt (ribosomal groups 16SrI, 16SrVI, 16SrXVIIII) Phytoplasma ‘barcoding’ has been performed for many years, particularly using the 16S rDNA, but also other genes such as secY, secA, tuf and ribosomal proteins; however most of these regions span more than 1 kb and/or primers are not generic, which make them impractical for routine barcoding of phytoplasmas. Available sequences of elongation factor Tu (Tuf) and 16S genes were explored for selecting regions suitable for phytoplasma DNA barcoding to develop robust markers of a size that can easily be sequenced. A number of phytoplasma strains (about 60) maintained in periwinkle and field collected were used for PCR amplification with newly developed primers for Tuf and 16S regions and then sequenced. The 5’ end of the Tuf and the 5’ end of 16S genes were used for barcoding. Sequences of approximately 450 bp for Tuf and 625 bp for the 16S gene were obtained from 60 and 40 phytoplasma strains respectively, belonging to 12 different 16Sr groups. Using these sequences as barcodes it was possible to identify the phytoplasmas into ‘Candidatus species’ or into 12 of the described 16Sr groups.

Coffee (Coffea arabica L.) is among the most important cultivated crops in Colombia, where it is frequently shaded with nogal cafetero [Cordia alliodora (Ruiz & Pavón) Oken], a tree species also used for timber and reforestation programs. Since 1998 severe cases of phytoplasma-related symptoms were observed in coffee plantations, and starting on 2002 also in nogal cafetero; both epidemics are concentrated to limited areas dispersed in the main coffee growing region, but with incidences and severities above 90%, resulting in total yield losses. Coffee crispiness was recently associated to 16SrIII phytoplasma infection, and PCR analyses allow the identification of phytoplasmas of the same group in nogal cafetero trees showing witches broom, branch malformation and abnormal shoot sprouting. Further molecular characterization was therefore performed towards epidemiological studies to prevent disease spreading. One phytoplasma strain from coffee and one from nogal cafetero were amplified and sequenced on the 16Sr gene and/or spacer region obtaining sequences of 1,356 and 1,487 bp respectively. The two sequences showed 99% of homology between them and to several 16SrIII strains in the GenBank. In particular only two SNPs were present between sequences from coffee and nogal cafetero strains. Phylogenetic analyses and real and virtual RFLP profiles obtained on R16F2/R2 amplicons (1,240 bp) with HhaI and HpaII allow to tentatively classify this phytoplasma in a new subgroup 16SrIII-U. However the real and virtual RFLP profiles obtained with TruI did not match to each other indicating a possible presence of mixed infection in phytoplasmas detected in coffee and in some other nogal cafetero samples. Ribosomal protein (rp) genes of (about 1,100 bp) of some nogal cafetero samples were sequenced after nested amplification and comparison of real and virtual RFLP profiles obtained with TruI, and Tsp509I indicates the presence of two rp differentiable strains in the coffee sample, one of which is the prevalent in nogal cafetero samples. These results indicate a complex disease aetiology involving more phytoplasma strains in the two studied plant species.

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/ restriction fragment length polymorphism (PCR/ RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to Candidatus Phytoplasma japonicum. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.

A research of vector(s) of the phytoplasmas responsible for apricot chlorotic leaf roll disease was carried out in a field plot where European Stone Fruit Yellows Phytoplasma (ESFYP), now named 'candidates Phytoplasma prunorum' ('Ca. P. prunorum'), had heavily infected an apricot collection in 1993. The plants were uprooted in May 1994 and, in 1997, the field was planted again with apricot trees. This report was elaborated with the data collected from February 2003 through July 2007. The only insect species found was Empoasca decedens Paoli with 5,085 insects collected. These insects were tested by direct and nested PCR to verify the presence of the phytoplasma. Individuals of E. decedens, present in the field each month in each year, resulted to be hosts of 'Ca. P. prunorum' in ten months out of twelve, but not of every year. The absence of phytoplasma in the two plants infected by 'Ca. P. prunorum' with E. decendens, four years after the acquisition of the pathogen, was also recorded.

Climate change, expansion of the EU and the increase of international trade may facilitate the spread of phytoplasma associated diseases, therefore a quick and handily system for correct plant pathogen identification is in great demand. DNA barcoding has arisen as a robust and standardised approach to species identification. QBOL, a new project funded by EU FP7 aims to create a barcode database for all quarantine plant pathogens and to make it available for plant health diagnosticians. Phytoplasma ‘barcoding’ has been performed for many years, particularly using the 16S rDNA, but also other genes such as secY, secA, tuf and ribosomal proteins; however most of these regions span more than 1 kb and/or primers are not generic, which make them impractical for routine barcoding of unknown phytoplasmas. Available sequences of elongation factor Tu (Tuf) and 16S genes were explored for selecting regions suitable for phytoplasma DNA barcoding to develop robust markers of a size that can easily be sequenced (400-600 bp) and that can be obtained from most, if not all phytoplasma ribosomal groups and/or ‘Candidatus Phytoplasma’ species using generic primers. A number of phytoplasma strains maintained in periwinkle and field collected were used for PCR amplification with newly developed primers for Tuf and 16S regions and then sequenced. The 5’ end of the Tuf and the 5’ end of 16S genes were used for barcoding. Sequences of approximately 450 bp for Tuf and 625 bp for the 16S gene were obtained from 60 and 40 phytoplasma strains respectively, belonging to 10 different 16Sr groups. Using these sequences as barcodes it was possible to identify the phytoplasmas into ‘Candidatus species’ or 16Sr groups. The obtained sequences will be available in the newly developed QBOL database.

During five years two trials were carried out to assess the plum susceptibility to infection by European stone fruit yellows phytoplasmas. One trial was carried out on commercial varieties /cultivars and new selections grafted on Myrabolan 29C. Plants were evaluated in more than one plot of four plants. Yearly monitoring by visual inspection and PCR/RFLP identification of phytoplasmas allowed verifying an increasing phytoplasma presence in both symptomatic and asymptomatic plants. Eight selections from Japanese plums showed ESFY symptoms or pathogen presence in the 50% of plants and nine selections showed 20% of infection. Only nine selections showed absence of both symptoms and pathogen. Although the European selections/cultivars were not symptomatic, plants belonging to six cultivars were positive to phytoplasma presence. The scond trial was carried out to evaluate cultivar/rootstock combinations; phytoplasma symptoms and presence were observed from the first year after plantation. Two of the rootstocks appeared to induce a delay in symptoms appearance and cultivar T.C. Sun was the most susceptible to the disease.

In questi ultimi anni nelle regioni nord-est italiane, il legno nero della vite (LN) è in forte e continua espansione ed in molte regioni italiane è la fitoplasmosi della vite maggiormente presente (Borgo et al., 2005; Bondavalli et al., 2005). Oltre che nel suo vettore naturale Hyalesthes obsoletus Signoret, il fitoplasma agente della malattia è stato rinvenuto in altre cicaline appartenenti alla famiglia dei cixiidi, Reptalus quinquecostatus (Dufour) e R. panzeri (Löw) (Trivellone et al., 2006; Jovic et al., 2007), che per la loro potenziale capacità di trasmissione potrebbero essere coinvolte nella epidemiologia di questo giallume (Maixner, 2006). Al fine di rilevare ed identificare la presenza di altri potenziali vettori della fitoplasmosi nell’areale di coltivazione del Lambrusco sono stati condotti monitoraggi in 25 vigneti affetti da LN siti nelle provincie di Modena e Reggio Emilia. Nel triennio 2007-2009, settimanalmente da giugno a settembre, è stata verificata sulle viti e sulle aree circostanti i vigneti la presenza dei cixiidi con strumenti di cattura diretta (retino da sfalcio e ombrello entomologico) e indiretta (trappole cromotropiche). Gli insetti catturati sono stati identificati ed analizzati con indagini molecolari per verificare la presenza del fitoplasma con analisi nested-PCR/RFLP sui geni ribosomici 16S e Tuf. Sono stati catturati ed identificati individui appartenenti ai generi Hyalesthes, Cixius e Reptalus; le specie più abbondanti sono risultate H. obsoletus (99,4% dei 1.234 individui del genere Hyalesthes catturati), Cixius cunicularis (L.) (94,9% dei 275 Cixius sp. catturati) e R. panzeri (65% dei 177 Reptalus sp. catturati). Inoltre, in quantità molto limitate, sono stati identificati individui di H. luteipes Fieber, C. nervosus (L.), e R. quinquecostatus. Lo sfalcio della vegetazione erbacea è risultato il metodo più efficace per il monitoraggio di H. obsoletus e Reptalus spp. (rispettivamente il 97,7% e il 78,0% di tutti gli esemplari catturati), mentre il “frappage” delle viti è risultato un valido strumento per studiare la presenza ed l’ecologia del genere Cixius (62,9% di tutti gli individui catturati). La maggior parte degli insetti (88,7% Hyalesthes sp., 70,8% Reptalus sp. and 66,1% Cixius sp.) sono stati catturati sulla vegetazione spontanea presente sui bordi dei vigneti. Alle analisi molecolari sono risultati positivi al fitoplasma stolbur associato a LN H. obsoletus (52% su 58 campioni analizzati), Cixius spp (13,6% su 66 campioni analizzati ), Reptalus spp (15,6% su 51 campioni analizzati); entrambi i tipi tuf individuati in vite sono stati riscontrati. Interessante notare che il 12,1% dei campioni di Cixius spp analizzati è risultato positivi a fitoplasmi del gruppo 16SrI-B (aster yellows), individuati in percentuali crescenti in alcuni dei vigneti studiati.