513 research outputs found
Ricerche sulla identificazione di virus e fitoplasmi fitopatogeni che infettano le piante.
No full textIl progetto proposto si pone come obiettivo la prosecuzione della collaborazione in atto da veri anni in maniera informale fra la prof. Assunta Bertaccini e la dr. Jana Franova in relazione alla presenza di virus e fitoplasmi in piante presenti nella repubblica Ceca. La collaborazione è iniziata nel 1993 con una stage della dr. Franova presso il laboratorio di fitoplasmologia diretto dalla prof. Bertaccini presso l’Università di Bologna. Franova in questa occasione ha appreso alcuni metodi di estrazione degli acidi nucleici da piante sintomatiche ed ha potuto dare inizio a ricerche molecolari per l’individuazione di citoplasmi in materiale vegetale individuato in repubblica Ceca. Nonostante la dr. Franova abbia dovuto assentarsi dal lavoro per maternità due volte nel periodo 1998-2003 la collaborazione è continuata ininterrottamente (vedi risultati della collaborazione). Al momento attuale lo studio di virosi e fitoplasmosi è in espansione presso IPMB-ASCR a C. Budejovice mentre presso il DiSTA di Bologna la metodologia e la ricerca sono a livelli di applicazione di routine. La collaborazione è importante in quanto permetterà alle ricercatrici ceche di diventare autonome nella ricerca e nella identificazione di questi patogeni ed al gruppo italiano di ampliare le conoscenze sulla diffusione geografica di ceppi di patogeni che possono essere seriamente pericolosi per l’agricoltura italiana e della Unione Europea in generale.
Oggetto della collaborazione sarà la risoluzione di alcuni problemi tecnici di laboratorio quali la presenza di falsi positivi o falsi negativi nelle analisi molecolari derivati dall’impiego di PCR o RT-PCR, la scelta dei reagenti (primers) più idonei alle diverse situazioni sperimentali, la identificazione di infezioni miste di citoplasmi e di fitoplasmi e virus.
La collaborazione permetterà inoltre di identificare e caratterizzare fitoplasmi e virus presenti in malattia ad eziologia ancora sconosciuta, l’individuazione di citoplasmi in piante sintomatiche o meno in cui il titolo di tali patogeni e particolarmente ridotto. Le esperienze dei due laboratori potranno essere confrontate e discusse comparativamente onde individuare le migliori modalità di ricerca nel settore
Detection of phytoplasmas in plantlets grown from different batches of seed-potatoes.
No full textDuring 2006 and 2007 eight batches of seed potatoes collected in different locations, and belonging to one cultivar were planted
in spring under greenhouse conditions and tested after 2 months to verify phytoplasma presence. A total of 635 asymptomatic
plantlets were examined. Nucleic acid was extracted from small shoots from either a single plant or from batches of 3 plants each.
Nested PCR on both single and grouped samples with general ribosomal primers, without spacer region allowed specific phytoplasma
detection. Phytoplasmas belonging to diverse ribosomal groups were identified after RFLP analyses according to the batch
tested. Ribosomal subgroups 16SrI-B (related to ‘Candidatus phytoplasma asteris’), 16SrI-C (related to clover phyllody: CPh),
16SrII-D (related to tomato big bud from Australia: TBB), 16SrX-A (related to ‘Ca. P. mali’), and 16SrXII-A (related to stolbur)
were identified in different percentages. After further validation tests, the system can be used to screen high quality seed potatos
for phytoplasmas
Grapevine crown gall: an old, emerging disease.
No full textCrown gall is considered one of the most important and widespread bacterial diseases of grapevine (Vitis vinifera L.) throughout the world. It is known in Europe for more than 150 years and can be still of great phytopathologic significance in the vineyards and nurseries, especially in cold-climate regions. The disease is predominantly caused by tumorigenic strains of Agrobacterium vitis, more rarely by tumorigenic A. tumefaciens and A. rhizogenes. Unlike A. tumefaciens and A. rhizogenes, that are broad-host-range pathogens, A. vitis is specific to grapevine. Crown gall reduces vigor and yield of grapevines and severe disease may cause partial or complete death of infected plants. High losses occur in nurseries where different graft combinations with visible symptoms are unmarketable and must be discarded. Typical symptoms of crown gall are tissue proliferation (tumors) formed mostly on the lower areas of the trunk and on aerial canes. Tumorigenic and nontumorigenic strains of A. vitis are also able to cause specific root decay and it has been hypothesized that both types may be factors involved in the “replant disease” syndrome. Wounds mainly caused by freezing temperatures or grafting serve as a crucial entry points for the pathogen and its complex infection process. During the infection process DNA fragment from the bacterial tumor inducing (Ti) plasmid is transferred and integrated into the plant genome (interkingdom gene transfer). This leads to the overproduction of the phytohormones auxin and cytokinin, resulting in an uncontrolled proliferation of plant cells and tumor formation. A. vitis is unevenly distributed within systemically infected grapevines and able to survive in vineyard soil, particularly in the vicinity of infected plants and in their debris. Another important aspect is the ability of the pathogen to be latently present within the grapevine, providing an important means of spread over short and long distances by asymptomatic propagation material. Management of grapevine crown gall is not easy considering that no effective chemical control measures are available. However, production of A. vitis-free grapevines is an essential prerequisite for an effective prevention of the disease, and great efforts should be done in this direction. For this reason, shoot tip propagation of grapevine and thermotherapy are available as control measures. Planting of crown gall and cold-resistant cultivars and rootstocks would be a good practice when establishing new vineyards. Biological control of crown gall is another promising approach in the control of the disease and several antagonistic bacterial strains have shown a certain level of efficiency in preventing tumor formation. Indexing of grapevines for the endophytic presence of A. vitis is a very important preventive measure.
Differentiation and identification of tumorigenic strains can be rapidly assessed by PCR using primer combinations specific for bacterial Ti plasmid and chromosomal genes. However, a high level of genetic diversity among Agrobacterium strains may limit the efficiency of PCR. In our studies virC, virD, virF, pehA and 23S rRNA gene-specific primers (Bini et al., Vitis 47:181, 2008; Pulawska et al., Syst. Appl. Microbiol. 29:470, 2006; Suzaki et al., J. Gen. Plant Pathol. 70:342, 2004; Szegedi and Bottka, Vitis 41:37, 2002) were reliable in routine detection and identification of a broad range of Agrobacterium strains occurring in grapevine. However, there is necessity for development and standardization of indexing procedures including protocols of analysis and sampling methods. In the EU and many other European countries, A. vitis is not listed as a quarantine pathogen and is considered as a “quality organism” which significantly reduces the value of propagation material. Therefore, the importance of proper phytosanitary measures in grapevine nurseries and on commercial lots should be emphasized
European Stone Fruit Yellows (ESFY).
No full textSince the beginning of the twentieth century symptoms of apricot tree decline were observed in France and Italy: Morvan in 1977 named the disease associated with leptonecrosis (Goidanich, 1934) or with new sprouting in winter "apricot chlorotic leaf rolling" (ACLR). Only since the late 1970ies these symptoms were associated with phytoplasma infections, since electron or fluorescence microscopy (by DAPI-staining) allowed to detect phytoplasmas as single cells in sieve tubes (Fig. 1) and transmission experiments to other stone fruit and indicator plants were successfully carried out (Morvan, 1977; Goidanich et al., 1980; Giunchedi et al., 1982, Pastore et al., 1995).
European stone fruit yellows (acronym: ESFY) has been proposed as the common name for phytoplasma-related diseases in European stone fruits (Kison et al., 1997). Among others it includes the French ECA or ACLR, which is a quarantine organism of EPPO (OEPP/EPPO, 1986), included in the EPPO certification scheme for virus tested fruit trees (OEPP/EPPO, 1991/1992).The presence of ESFY disease has been reported in France, Spain, Italy, Greece, and in Hungary, Romania, Switzerland, Germany, former Yugoslavia, the UK and Austria (Nemeth, 1986; Morvan, 1977, Davies and Adams 2000, Laimer da Câmara Machado et al. 2001a) causing decline and death to apricot, Japanese plum, more rarely to peach (Llacer and Medina, 1988) and to almond, flowering cherry and European plum (Seemüller et al. 1998) http://www.boku.ac.at/iam/pbiotech/phytopath/v_esfy.html). An increasing presence of phytoplasma associated diseases such as leptonecrosis on Japanese plum (Prunus salicina) and chlorotic leaf roll on apricot (Prunus armeniaca) has been observed in commercial orchards in several European regions in the last twenty-five years (Giunchedi et al., 1978; Desvignes and Cornaggia, 1982; Dosba et al., 1991; Bertaccini et al., 1993; Laimer et al., 2001; Torres et al., 2004). Prunus rootstocks are also affected by this disease (Dosba et al. 1991, Jarausch et al. 1998). ESFY phytoplasmas have also been detected in wild Prunus species, e.g. Prunus spinosa and P. cerasifera (Carraro et al. 2002) and cherry (Prunus avium) (Paltrinieri et al., 2001). In recent years ESFY phytoplasma has been detected in other wild plants such as Rosa canina, Celtis australis and Fraxinus excelsior (Jarausch et al., 2001) as well as in grapevine in Hungary (Varga et al., 2000) and in Serbia (Duduk et al., 2004)
Micromosaico. Storia, tecnica, arte del mosaico minuto romano
No full textIl mosaico minuto o micromosaico nacque a Roma nella seconda metà del ’700 e il periodo di maggiore produzione di questo tipo di artigianato artistico è quello che va dalla fine di tale secolo a tutto l’800. Nella seconda metà del ’500 era partito il grande progetto di decorare a mosaico le volte di San Pietro e di tradurre a mosaico opere pittoriche della stessa basilica, utilizzando materiali vetrosi, detti anche smalti, di vario colore. Questi ultimi erano prodotti a Venezia, ma presto si cominciò a prepararli anche a Roma. In tale contesto, si iniziò a produrre, anche all’interno dello Studio Vaticano del Mosaico, il micromosaico fatto con tessere di piccole dimensioni. Il vetro usato veniva filato in sottili bacchette da cui venivano ricavate le minuscole tessere. Il supporto su cui veniva composto il micromosaico poteva essere di metallo, quale una lamina di rame con bordi rialzati, o una lastrina di pietra con un incavo o di altro tipo, in cui venivano assemblate le piccole tessere fissandole con un opportuno legante. Le stesse opere avevano dimensioni ridotte, eseguite con grande precisione e rifinite con altrettanta cura e potevano essere inserite, ad esempio, su manufatti quali tavolini con piano di marmo, tabacchiere, fermacarte, ecc., o addirittura costituivano spille e gioielli, quando venivano applicati in oreficeria
Multilocus analyses on grapevine ‘bois noir’ phytoplasmas from Italy and Serbia.
No full textIntroduction
‘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
Complex phytoplasma infection in declining liquidambar trees in Colombia.
No full textLiquidambar 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
Presence of phytoplasma infections in tomato plants in Mauritius.
No full textPhytoplasmas were detected and identified in some tomato cultivation areas in Mauritius. Symptoms most frequently observed
were abnormal shoot proliferation, stunting, reduced leaf and fruit size and shortened internodes. In field-grown tomatoes the incidence
of abnormalities rarely exceeded 10%, but under hydroponics up to 100% incidence has been recorded. Two different
phytoplasmas were identified by PCR/RFLP analyses. Field-grown tomatoes were infected with a phytoplasma belonging to ribosomal
subgroup 16SrI-C and the hydroponically-grown tomatoes were infected with a phytoplasma belonging to ribosomal group
16SrV. Further studies need to be done in order to determine their occurrence, incidence, characterization, host range and mode of
transmission, so that eventually the most effective method to control phytoplasma diseases will be determined
Identification of phytoplasmas using DNA barcodes of selected genes.
No full textBarcode 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
Preliminary results of axenic growth of phytoplasmas from micropropagated infected periwinkle shoots
No full textPeriwinkle 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
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