EP2601306A1 - A sequence specific for phytoplasma causing bois noir (bn), uses thereof and bn diagnostic kits - Google Patents

Abstract

The present invention relates to a new sequence specific for the phytoplasma causing Bois noir (BN) in vines, to a method for the diagnosis of BN in vines and parts thereof including rootstocks, and to a kit for the detection of the phytoplasma agents of BN from plant samples.

Description

A SEQUENCE SPECIFIC FOR PHYTOPLASMA CAUSING BOIS NOIR (BN), USES THEREOF AND BN DIAGNOSTIC KITS

The present invention relates to a new sequence specific for the phytoplasma causing Bois noir (BN) in vines, to a method for the diagnosis of BN in vines and parts thereof including rootstocks and to a kit for the detection of the phytoplasma agents of BN from plant samples.

STATE OF THE ART

Grapevine yellows (GY), a complex of diseases that were originally thought to be caused by viruses, are now known to have a phytoplasma aetiology. The first disease described for Vitis vinifera, and the most widely known among GY diseases, is undoubtedly Flavescence doree (FD), which was reported for the fist time in south-west France in the 1950's (Caudwell, 1957), and then spread to other viticultural districts of France, northern Italy and neighbouring European countries. Bois noir (BN), whose symptoms are indistinguishable from those of FD, was also first reported from France, and then from the most important viticultural areas of Europe.

Almost identical symptoms of the GY syndrome are caused by different phytoplasmas and appear on leaves, shoots and clusters of grapevine. Typical symptoms include discoloration and necrosis of leaf veins and leaf blades, downward curling of leaves, lack or incomplete lignifications of shoots, stunting and necrosis of shoots, abortion of inflorescences and shrivelling of berries. Those symptoms are related to callose deposition at the sieve plates and subsequent degeneration of the phloem. Although no resistant cultivars of Vitis vinifera or rootstocks are known so far, the various grape varieties differ considerably as far as symptom severity is concerned. It ranges from fast decline and death in highly susceptible cultivars to tolerant rootstocks as symptomless carriers of the pathogen.

Phytoplasmas are microscopic plant pathogens, similar to bacteria, but much smaller than bacteria. They are phloem-limited, non-helical and wall-less prokaryotes (Firrao, G., et al, 2004.). Differently form viruses, phytoplasmas have their own metabolism, a highly reduced metabolism, and some of the molecules essential for their survival are acquired from the host. They represent a monophyletic clade within the class Mollicutes, which is currently divided into at least 15 subgroups on the basis of sequence analyses of various conserved genes. A new taxon, 'Candidatus Phytoplasma' has been established and various groups or subgroups have been described recently as 'Ca. Phytoplasma' species.

The full genomes of four phytoplasmas are sequenced, providing new information on their biology and their interaction with plant and insect hosts. ("Ca. Phytoplasma asteris" strain AY-WB NC_007716 and OY NC_005303 "Ca. Phytoplasma mali" NC_01 1047, "Ca. P. australiense" NC_010544).

The available data confirm their lack of genes for autonomous metabolism and their reliance on intracellular parasitism. Phytoplasmas of several groups have been found to be consistently associated with GY. They propagate through a vectoring insect; also the propagation of infected grapevines made by man contributes to GY spreading over long distance. This implies a high risk of dissemination of GY to regions where new outbreaks could be provoked if competent vectors existed or would be introduced, too.

GY, however, have different phytoplasma species as causal agent, as well as different insect vectors, which are either leafhoppers or plant-hoppers (Homoptera: Auchenorrhyncha) that feed specifically or just occasionally on the vines. It is worth noting that two or more different phytoplasma species may infect simultaneously individual grapevines, thus causing mixed infections.

Although phytoplasmas are non culturable micro-organisms and, in the case of GY, Koch's postulates have not yet been fulfilled, when phytoplasmas of a specific group or subgroup are found consistently associated with a specific grape disease, they are regarded as being its causal agents. In fact, the identification of phytoplasmas as the cause of GY was made possible over the last decades by molecular detection methods, which allow to distinguish each other the phytoplasma species involved in each single disease.

The classification of phytoplasmas, which are uncultivable and currently described under the provisional genus "Candidatus Phytoplasma," is mainly based on 16S rRNA gene phylogeny, genomic diversity, and plant and insect host ranges.

Phytoplasmas associated with grapevine yellows

In 2003 Boudon-Padieu (Boudon-Padieu, E., 2003) listed several phytoplasmas that were found to be consistently associated with GY. Stolbur, phytoplasma (16SrXII-A), the causal agent of Bois noir (BN), was reported from grapevine in Chile and phytoplasmas of the same group, but also of 16Srl (aster yellows) group were identified in cultivar Syrah in France. BN has now been reported also in Canada. Stolbur phytoplasma was also identified in GY diseased vines in Ukraine, and its presence in Serbia could be confirmed. Recently, new and repeated outbreaks of BN caused problems in various viticultural regions in Europe. These data confirm that BN is an endemic disease in Europe, Asia Minor and the Mediterranean. Aster yellows (16Srl-B), clover phyllody (16Srl-C) and elm yellows (16Sr-V) phytoplasmas were found sporadically in GY diseased grapevines in Italy, and infection of grapevine by aster yellows (16Srl-B) phytoplasma was reported from Tunisia. Infection of papaya and grapevine by "Ca. Phytoplasma australiense" was reported from Israel; however, confusion with stolbur phytoplasma which is also present in this region is possible. A survey in Italy revealed the presence of GY in 80% of the inspected vineyards. BN is widespread all over the country whereas FD is mainly restricted to the northern regions. Among the most common ribosomal groups defined for phytoplasmas are: aster yellows (16Srl group), X-disease (16Srlll group), elm yellows (16SrV group) and solani also called potato stolbur phytoplasma (16SrXII group).

As already indicated BN is the most widespread GY in Europe and in the Mediterranean basin and it is caused by phytoplasmas belonging to stolbur group.

The disease is present in almost all European vine areas as well as in Lebanon and Israel. The disease was firstly signalled in 1986 and the disease has assumed an endemic behaviour due to the principal vector of BN vector transmitting the disease that is Cixiidae planthopper Hyalesthes obsoletus. However, other Cixiidae and Cicadellidae species are known or suspected vectors of stolbur phytoplasmas, too. Some additional species like Goniognathus guttulinervus in Sardinia and Reptalus panzeri in Hungary and Italy were found recently to carry solani (or stolbur) phytoplasma beside the already known species, although their ability to transmit the phytoplasma is still not proved.

The BN vector does not feed only on vines and several alternative host plants of stolbur phytoplasmas play a vital role in the epidemiology of BN. Several herbaceous plants have been surveyed for the presence of 16Sr Xll-A phytoplasma infection and colonization by H. obsoletus and the host can hence transmit the disease from other plants to vines.

Among the plants hosting BN phytoplasma are Urtica dioica and Convolvulus arvensis (that appear to be the major host plants of BN phytoplasma with high epidemic significance), Vitex agnus-castus, whose function as source of the phytoplasma is not yet clear, Taraxum officinalis harbouring the phytoplasma and Ranunculus, Solanum and Lavandula that also could serve as a source of inoculum.

Vine appears to be a final host that does not function as source for the inoculation of the BN phytoplasmas. The multiplicity of hosts for this phytoplasma has practical effects that cannot be ignored, eradication of the diseased vines provides no help for the control or stop of the disease diffusion: it is therefore crucial to clearly identify a BN infection among other possible phytoplasma infections that can be confused with this disease in order to follow the best steps for the treatment of the vineyards, of the vineyards surrounding areas and or of the plant nurseries and garden centres. The symptoms caused by BN are practically the same as those of Flavescence doree, and the name refers to the blackening of non-lignified shoots in winter, which is a symptom of FD.

While Flavescence doree (FD) phytoplasma is strongly epidemic and it is recognized by the European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest (EPPO/CABI, 2003), BN phytoplasma is not included in the quarantine pest list. Accordingly, the winegrower is not forced to eliminate the plants infected. However, it is important to identify the pathogen because the winegrowers can perform preventive strategies to limit the spread of disease.

Practically, each phytoplasma affection requires a different treatment of the infected plants and areas: for BN, by way of example, at present, several control measures have been tried.

Due to the complex epidemic cycle of BN that includes alternative host plants as sources of inoculum and a non-ampleophagous vector whose life history puts it out of reach from insecticides, control of BN is considerably more difficult and less efficient than control of FD although less drastic. FD requires quarantine and often elimination of the affected vines. This drastic measure, as already indicated above, seems to be useless for BN. Chemical control failed to decrease BN whereas control of host plants within affected vineyards and in adjacent areas appears to be a valid measure to decrease infection pressure. However, weed control should not be carried out during the flight of adult vectors as they would be forced to move onto grapevine. Since H. obsoletus is attracted by sparse vegetation on open soil, a well managed green cover reduces the attractiveness of vineyards and the risk of contamination of grapevine. Although not yet tested, it appears that the susceptibility of H. obsoletus to the entomopathogenic fungus Metarhizium anisopliae could be taken into account for a biological control of the disease. Other control strategies, such as planting around the vineyard plants attractive for the vector, seem to be interesting, although the increased vector population in the area of interest renders the belted vineyards constantly at risk for infection.

At present, several methods for BN detection have been developed; they include conventional PCR/RFLP methods based on analysis of ribosomal genes (Lee et al., 1994) and more recently real-time PCR assays (also RT PCR) have been developed (Galetto et al., 2005 ; Angelini et al., 2007 ; Hren et al., 2007; Margaria et al., 2009). However, as known by the skilled person, PCR/RFLP analysis is relatively time consuming and requires several analytical steps involving samples cross-contamination risks. Also some RT PCR assays are available.

The recently developed RT PCR assays are based on single nucleotide polymorphisms (SNPs) which identify BN phytoplasma. However, they target nucleotidic sequences (Angelini et al.2007; Hren et al., 2007) other than those of rpl22 and rps3 genes described in the present invention.

Given above reasons, and the peculiar characteristic of BN vector, that render useless a quarantine or a drastic control on the vine plants themselves, it is evident that a diagnostic test and/or tool allowing a unambiguous and specific identification of the presence or of the absence of BN would be very useful to control phytoplasmic infection, and could avoid drastic measures and unnecessary losses of precious vine plants.

DESCRIPTION

The present description provides a new nucleotide sequence of SEQ ID NO 1 of coding for the gene rpl22 (ribosomal protein rpl22 gene) of phytoplasma "Ca. Phytoplasma solani" (Stolbur) causing BN, and SEQ ID NO 7 coding of the rps3 gene of phytoplasma "Ca. Phytoplasma solani" (Stolbur) causing BN. Both sequences are present in SEQ ID NO 1 , wherein rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095. Both the rpl22 and rps3 genes code for ribosomal proteins.

The rpL22 protein binds specifically to 23S rRNA during the early stages of 50S assembly and it makes contact with all 6 domains of the 23S rRNA in the assembled 50S subunit and ribosome; rpS3 forms a complex with S10 and S14 and it binds the lower part of the 30S subunit head and the mRNA in the complete ribosome to position it for translation.

The newly provided sequences allow for the recognition of different phytoplasmas belonging to said subgroups and provide a new tool for a better understanding and recognition of these microorganisms.

The present sequences (i.e. SEQ ID NO 5, 7 and/or 1 ) hence provide a useful tool for the specific detection of the presence of the BN related phytoplasma in a vine sample.

The rpl22 and rps3 gene sequences specific for the BN related phytoplasma are depicted respectively in SEQ ID NO 5 and SEQ ID NO 7, and code for the ribosomal proteins rpL22 and rpS3 (respectively, SEQ ID NO 6 and SEQ ID NO 8) specific for the phytoplasma associated with BN that differs for single-nucleotide polymorphisms (SNPs) from other phytoplasmas such as "Ca. P. australiense," "Ca. P. asteris", "Ca. P. vitis".

The sequences of these phytoplasmas have been included in the alignment because they show the same symptoms caused by BN phytoplasmas, and they also infect grapevine. Therefore it is necessary a diagnostic tool, specific for BN phytoplasma detection. The knowledge of the presence of BN in a vineyard is an important information for the growers, as they can take phytosanitary measures adequate for BN control.

The present description also provides a sequence of SEQ ID NO 12 that corresponds to SEQ ID NO 1 wherein all SNPs nucleotides have been marked as "n" nucleotides and wherein n nucleotides can be any nucleotide including the one present in SEQ ID NO 1 , hence, a very specific embodiment of SEQ ID NO 12 is represented by SEQ ID NO 1 .

These sequences are workable as a diagnostic tool as they comprise various SNPs specific for BN. Each of said BN-specific SNP is identified in Table 1 where the BN specific nucleotide is indicated, as well as its position in SEQ ID NO 1 . Hence, "nucleotides of Table 1 " are the BN specific nucleotides whereas "nucleotides in the position indicated in Table 1 " may represent in an embodiment of the nucleotides of Table 1 but may also represent the "n" nucleotides of SEQ ID NO 12.

Due to the SNPs identified by the authors of the present invention, it is now possible, reading the teachings of the present description, to design an easy and feasible diagnostic assay that specifically identifies the phytoplasma associated with BN.

The present invention hence provides a nucleotide sequence of SEQ ID NO

1 or the sequence complementary to SEQ ID NO 1 or fragments thereof, said fragments comprising one or more of the nucleotides (or of their complementary nucleotides) of Table 1 , or said fragments being suitable for the amplification of a fragment of SEQ ID NO 1 , or the sequence complementary to SEQ ID NO 1 , comprising one or more nucleotides (or of their complementary nucleotides) of Table 1 , or of their complementary nucleotides, wherein said sequence or fragments thereof are specific for the plant phytoplasma strains associated with the vine disease Bois Noir; the present invention also provides a nucleotide vector comprising a nucleotide sequence of SEQ ID NO 1 , 5, 7 or 12 or the sequence complementary to SEQ ID NO 1 , 5, 7 or 12 or fragments thereof, said fragments comprising one or more nucleotides in the position indicated in Table 1 or of their complementary nucleotides, and a method for the diagnosis of the vine disease Bois Noir comprising the step of

a) identifying one or more of the nucleotides in the position indicated in Table 1 in SEQ ID NO 12 or a fragment thereof, and/or one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12 or a fragment thereof, in a plant sample comprising phloem; wherein the presence of one or more of the residues of Table 1 in the respective position indicated in Table 1 in SEQ ID NO 12 and/or of its complement in the sequence complementary to SEQ ID NO 12 indicates the presence of the phytoplasma strain associated with the vine disease Bois Noir in the sample; and a kit for the diagnosis of the vine disease Bois Noir comprising reagents for the identification one or more of said nucleotides in SEQ ID NO 12 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 12) in a plant phloem.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 represents an alignment of the rpl22 and rps3 genes of four phytoplasmas including "Ca. P. solani", i.e, "Ca. P. australiense", "Ca. P. asteris" and "Ca. P. Vitis". The alignment clearly demonstrates 66 SNPs in the positions reported below and in Table 1 below, each allowing the specific identification of phytoplasmas associated with BN.

Figure 2. Amplification plot of BN65 RT PCR assay. Specific detection of BN phytoplasma. The figure shows the amplification signal in positive control and in grapevine sample infected by BN. No amplification signal was detected in negative controls.

DETAILED DESCRIPTION OF THE SEQUENCES SEQ ID NO 1 nucleotide sequence of "Ca. Phytoplasma solani" (Stolbur) S10-spc operon comprising both rpl22 and rps3 nucleotide sequences

atgactatgg aaactaaaaa cgctaaagcg attgttcgta aagtttcaat cgcccctcga aaatcccgtt tagtgattga cttaattaga ggaaaaaata tcgatcaagc tcaagccatt ttaactttta ctcctaaagt agctgctccc gttattttaa aacttttaaa tagtgctgtt gcaaatgctt taaataattt aaaattacaa cgtgaacaac tttttgtcaa agaagttttt gtcaacgaag gcatacgttt aaagcgtatg tttccgagag ctaaagggtc tggtgatatg attaaaaaaa gaactagtca catcacttta gtagtggctg ctaaaaactt gcaaacatca aaggaggcga acagtgggtc aaaaaactaa tcctaatggt ttaagattag gtattattag aacttgggat tctcaatggt gtgtcaatga caaagaaata ccagctttaa ttaaagaaga atttttaatt cgcaaaataa ttaatcaatt tggtaaaaaa agcgctatta gtcaaattga aatacagcgt ttaaaagaaa aaaccaaaaa tcggatcaca atttcaattc ataccgccaa accaggaatg attatcggta aagagggtga aactcgcaat aaaatagtag cgcaattaaa aacattaact ggcaaagaca ttaacttaaa tattatagaa gttaaaatcc cagataaaat agctttatta gtagcacaaa atattgctga acaattagaa aaccgaatga aatttcgtcg tgttcaaaaa atggcgatcc aaagagccct taaatctggc gctaaaggga ttaaaatttt aatttctggt cgtcttaatg gcaatgaaat agcgcgtagc gaaggaaatg ctgaaggtcg tgttcctttg cacactttaa gagcagatat tgattatgct gcctacgaag cacacaccac ttatggtgtt ttagggatca aagtctggat ttttcacggt gaagttttat ccggtcaaac catcctagac actcgtaaac cttttgtttc gcaaaataaa tttgccaaac accctcgtca ctttaaagga ggtaaataat tatgttaatg ccaaaaagaa ca SEQ ID NO 2 forward primer

tcaattcata ccgccaaacca SEQ ID NO 3 reverse primer

atgattatcg gtaaagag

SEQ ID NO 4 probe BN65

tgcgctacta ttttattgcg agtt

SEQ ID NO 5 "Ca. Phytoplasma solani" (Stolbur) nucleotide sequence of rpl22 gene atg act atg gaa act aaa aac get aaa gcg att gtt cgt aaa gtt tea ate gcc cct cga aaa tec cgt tta gtg att gac tta att aga gga aaa aat ate gat caa get caa gcc att tta act ttt act cct aaa gta get get ccc gtt att tta aaa ctt tta aat agt get gtt gca aat get tta aat aat tta aaa tta caa cgt gaa caa ctt ttt gtc aaa gaa gtt ttt gtc aac gaa ggc ata cgt tta aag cgt atg ttt ccg aga get aaa ggg tct ggt gat atg att aaa aaa aga act agt cac ate act tta gta gtg get get aaa aac ttg caa aca tea aag gag gcg aac agt ggg tea aaa aac taa

SEQ ID NO 6 "Ca. Phytoplasma solani" (Stolbur) amino acid sequence of rpl22 protein

Met Thr Met Glu Thr Lys Asn Ala Lys Ala lie Val Arg Lys Val Ser lie Ala Pro Arg Lys Ser Arg Leu Val lie Asp Leu lie Arg Gly Lys Asn lie Asp Gin Ala Gin Ala lie Leu Thr Phe Thr Pro Lys Val Ala Ala Pro Val lie Leu Lys Leu Leu Asn Ser Ala Val Ala Asn Ala Leu Asn Asn Leu Lys Leu Gin Arg Glu Gin Leu Phe Val Lys Glu Val Phe Val Asn Glu Gly lie Arg Leu Lys Arg Met Phe Pro Arg Ala Lys Gly Ser Gly Asp Met lie Lys Lys Arg Thr Ser His lie Thr Leu Val Val Ala Lys Asn Leu Gin Thr Ser Lys Glu Ala Asn Ser Gly Ser Lys Asn

SE ID NO 7 "Ca. Phytoplasma solani" (Stolbur) nucleotide sequence of rps3 gene gtg ggt caa aaa act aat cct aat ggt tta aga tta ggt att att aga act tgg gat tct caa tgg tgt gtc aat gac aaa gaa ata cca get tta att aaa gaa gaa ttt tta att cgc aaa ata att aat caa ttt ggt aaa aaa age get att agt caa att gaa ata cag cgt tta aaa gaa aaa acc aaa aat egg ate aca att tea att cat acc gcc aaa cca gga atg att ate ggt aaa gag ggt gaa act cgc aat aaa ata gta gcg caa tta aaa aca tta act ggc aaa gac att aac tta aat att ata gaa gtt aaa ate cca gat aaa ata get tta tta gta gca caa aat att get gaa caa tta gaa aac cga atg aaa ttt cgt cgt gtt caa aaa atg gcg ate caa aga gcc ctt aaa tct ggc get aaa ggg att aaa att tta att tct ggt cgt ctt aat ggc aat gaa ata gcg cgt age gaa gga aat get gaa ggt cgt gtt cct ttg cac act tta aga gca gat att gat tat get gcc tac gaa gca cac acc act tat ggt gtt tta ggg ate aaa gtc tgg att ttt cac ggt gaa gtt tta tec ggt caa acc ate eta gac act cgt aaa cct ttt gtt teg caa aat aaa ttt gcc aaa cac cct cgt cac ttt aaa gga ggt aaa taa

SEQ ID NO 8 "Ca. Phytoplasma solani" (Stolbur) amino acid sequence of rps3 protein

Val Gly Gin Lys Thr Asn Pro Asn Gly Leu Arg Leu Gly lie lie Arg Thr Trp Asp Ser Gin Trp Cys Val Asn Asp Lys Glu lie Pro Ala Leu lie Lys Glu Glu Phe Leu lie Arg Lys lie lie Asn Gin Phe Gly Lys Lys Ser Ala lie Ser Gin lie Glu lie Gin Arg Leu Lys Glu Lys Thr Lys Asn Arg lie Thr lie Ser lie His Thr Ala Lys Pro Gly Met lie lie Gly Lys Glu Gly Glu Thr Arg Asn Lys lie Val Ala Gin Leu Lys Thr Leu Thr Gly Lys Asp lie Asn Leu Asn lie lie Glu Val Lys lie Pro Asp Lys lie Ala Leu Leu Val Ala Gin Asn lie Ala Glu Gin Leu Glu Asn Arg Met Lys Phe Arg Arg Val Gin Lys Met Ala lie Gin Arg Ala Leu Lys Ser Gly Ala Lys Gly lie Lys lie Leu lie Ser Gly Arg Leu Asn Gly Asn Glu lie Ala Arg Ser Glu Gly Asn Ala Glu Gly Arg Val Pro Leu His Thr Leu Arg Ala Asp lie Asp Tyr Ala Ala Tyr Glu Ala His Thr Thr Tyr Gly Val Leu Gly lie Lys Val Trp lie Phe His Gly Glu Val Leu Ser Gly Gin Thr lie Leu Asp Thr Arg Lys Pro Phe Val Ser Gin Asn Lys Phe Ala Lys His Pro Arg His Phe Lys Gly Gly Lys

SEQ ID NO 9 Ca. P. australiense as from figure 1

ggaaattaaa aatgctaaag caattgttcg caaagttcca attgctcctc gaaaagttcg tttagttatt gatttaatta ggggtaaaaa aatcgaccaa gcgcaagcaa ttttaacttt tactcctaac gcttctgcac ttattgtttt aaaactttta aatagtgctg ttgctaatgc tttaaataat ttaaaattaa aacgtgaaca actttacgtc aaagaagttt ttgttaacga aggattaaga ttaaaacgaa tgtttccaag agctaaaggt tctggcgata tgatcaaaaa aagaactagc cacatcactt tagtagtgca aacattaaag gaggaggaaa gtgggtcaaa aaactaatcc taatggctta agattaggaa ttattcgaac ttgggattcc caatggtgcg ttaacgacaa agaaatacca gctttaatta aagaagattt tttaattcgt caattaatta ataattttag caaaagaaac tctattagtc aaattgaaat tcaaagatta aaagaaaaaa ctaaaaaccg cattacaatt acgatccaca ctgccaaacc aggaattatt attggcaaag atggtgaaac tcgtaataaa attcaggccc aattaaaaaa attaactcaa aaagacatta atttaaacat tttggaagtt aaaaatcctg acaaaaccgc tgtattagtg gctcaaaaca tggccgaaca attagaaaat cgtatgaaat tccgtcgcgt tcaaaaaatg gccatccaaa aagcttttaa agcaggcgct aaaggaatta aagtgttgat ttctggtcgt cttaatggcg atgaaatagc acgaagcgaa agacatgatg aaggacgtgt tccgttacat actttgagag cagatattga ttacgccgct ttagaagctc acaccactta cggagtttta ggaattaaa gtttggatttt tcatggagaa gttttaccag gtcaaactat tttagacact agaaaacc ttttgtttctca aaataaattt attaaacgtc ctcgttattt taaaggaggt aaaaata

SEQ ID NO 10

Ca. P. Asteris as from figure 1

aataactatg gaaaccaaaa acgccaaagc gattgctaga aaagtttcaa tcgcccctcg aaaagcacgt ttagttgttg atttaattcg aggaaaaaat attgcacaag ctcaagccat tttaactttt acccctaaag tagctgctcc cgttatttta aaacttttaa acagtgctgt ttccaatgct gttaataatt taaaattaaa ccgcgaacaa ctttatgtta aagaagtttt tgtcaacgaa ggtttgcgtt taaaacgtat gtttccaaga gctaaaggtt ctggtgatat gattaaaaaa agaaccagcc acattacttt agtaataact tctagcacaa acttgcaaac atcaaaggag gaagaacaaa gtgggtcaaa aaactaatcc taacggctta agattaggca ttattagaac ttgggaatct caatggtgtg ttaatgataa agaaattcct aatttaatta aagaagattt tttaattcgt aaactaatca ataattttac taaaaaaagt gctatcagtc aaattgacat tgaacgcnta aaagaaaaaa ataaaaaccg tatcactatt tctgtccaca ccgctaaacc aggcgttatt attggaaaag atggcgatac acgcaacaaa ttagttgcca aactcaaaga acttacccaa aaagacgtta atcttaacgt gttagaagtt aaaaactctg ataaaatcgc tttattaatt gctcaaaata tggctgaaca actagaaaat cgtatgtttt tccgccgtgt tcaaaaaatg gcaatccaaa aagccctaaa agctggtgcc aaaggagtaa aaactttaat ttctggtcgt ttgggtggtg ctgaaatagc tcgtagcgaa ggacatgccg aaggcagagt tcctctacac actctaagag cagacatcga ttacgctgct gttgaagctc acactactta tggagtttta ggaattaaag tatggatttt ccacggtgaa gttttaccag gacaaaccat tctagacact agaaaaccgt ttgcttccca atcttctaac actcctaaca gacgccctcg caatttcaaa ggaggcaaca ataa

SEQ ID NO 11

Ca. P. Vitis as from figure 1

tttaaagaag gtatttatat gaaagttaaa gctgtaacaa gtccaatacc tattactcct agaaaagcac gtttagttgc tgatttaata cgtggtaagc atgttaaaga agcagaagct attttaatgt ttacttctaa atcgtctgct cctgttattt ttaaattatt aaaaagtgct gttgcaaatg ctgttcataa ttttaatttt aataaagatg atttatttgt taaagaaata tttgtcgatg aaggtttgcg tttacctcgt ttatttccga gagctaaagg taaaacagat aaaagaaaaa aaagaatgag tcgtgtaaaa atatttcttt cttcatttaa aaaagaaatt caggagatgc aagtaaatgg gtcaaaagag taatcctaat ggtttgagat taggaataat taggacttgg gaatctaaat ggtatgatgt tgataaaaaa gttccttttt tagtcggtga agattttaaa attagaactt taattaaaaa tcattatcct aaatcaacta tttctcaaat agaaatcaaa cgtttaaaaa aatcaaatga tgaatttatt gaaatagatt tatatacttc aaaaataggt atcattcaag gtccggaaaa taagaataaa aatagtttaa ttaataaaat agaaaaatta attaataaaa aagttcaaat taatattttc gaagtaaaag caattaataa aattgctgtt ttagttgctc aaaatatcgc tatgcaattg caacaaagag ctttttataa agctgtttta aaatcagcta ttcaaaaagc tttaaaaagc ggtattaaag gtattaagat tattattaca ggtcgtttag gtggagctga aaaagctaga agagattcta tttctatggg agttgttcct ttgaatactt taagagctga tattgattac gcttttgaag aagcccatac tacttatggt gttttaggtg ttaaagtaat tattaatcat ggtgaggttt tacctaataa aaccatcgca gatactagac aaatattttc ttctcaatat gaaaataaaa aaaataataa taaaagacat tttgttgata agaaaaattt taaaaaaagc acgtcttaat ataatcaaaa gaggggtaca taatatatta tgttaatgcc

SEQ ID NO 12 nucleotide sequence ofS10-spc operon comprising both rpl22 and rps3 nucleotide sequences

atgactatgg aaactaaaaa cgctaaagcg attgttcgta aagtttcaat cgcccctcga aaancccgtt tagtnattga nttaattaga ggaaaaaata tcgancaagc tcaagccatt ttaactttta ctcctaaagt agctgctccc gttattttaa aacttttaaa tagtgctgtt gcaaatgctt taaataattt aaaattanaa cgtgaacaac tttttgtcaa agaagttttt gtcaacgaag gnntacgttt aaancgtatg tttccgagag ctaaaggntc tggtgatatg attaaaaaaa gaactagtca catcacttta gtagtggctg ctaaaaactt gcaaacatca aaggaggngn acagtgggtc aaaaaactaa tcctaatggt ttaagattag gnattattag aacttgggat tctcaatggt gtgtnaatga caaagaaata ccagctttaa ttaaagaaga ntttttaatt cgcaaantaa ttaatnaatt tggtaaaaaa agcgctatta gtcaaattga aatncagcgt ttaaaagaaa aaacnaaaaa ncgnatcaca atttcaatnc ataccgccaa accaggaatn attatnggta aagagggtga aactcgcaat aaaatagtng cncaattaaa aanattaact nnnaaagaca ttaanttaaa tattntagaa gttaaaancc cngataaaat ngctttatta gtngcncaaa atatngctga acaattagaa aaccgaatga aatttcgtcg tgttcaaaaa atggcgatcc aaanagccct taaanctggc gctaaaggna ttaaaatttt aatttctggt cgtcttaatg gcnatgaaat agcncgtagc gaaggaaatg ctgaaggncg tgttcctttg cacactttaa gagcagatat tgattangct gcctangaag cncacaccac ttatggtgtt ttaggnatna aagtntggat ttttcacggt gaagttttan cnggtcaaac catcctagac actngnaaac cttttgtttc ncaaaataaa tttnncaaac accctcgtca ntttaaagga ggtaaataat tatgttaatg ccaaaaagaa ca 1 122, wherein when nucleotide 64 is T, nucleotide 66 is C, nucleotide 75 is G, nucleotide 81 is C, nucleotide 105 is T, nucleotide 208 is C, nucleotide 252 is C, nucleotide 264 is G, nucleotide 288 is G, nucleotide 368 is C, nucleotide 370 is A, nucleotide 372 is C, nucleotide 412 is T, nucleotide 445 is C, nucleotide 481 is A, nucleotide 493 is C, nucleotide 497 is A, nucleotide 506 is C, nucleotide 512 is G, nucleotide 544 is A, nucleotide 547 is G, nucleotide 565 is C, nucleotide 571 is T, nucleotide 574 is G, nucleotide 589 is T, nucleotide 610 is G, nucleotide 616 is C, nucleotide 649 is A, nucleotide 652 is G, nucleotide 663 is C, nucleotide 671 is G, nucleotide 672 is G, nucleotide 673 is C, nucleotide 685 is C, nucleotide 685 is A, nucleotide 708 is T, nucleotide 712 is A, nucleotide 721 is A, nucleotide 733 is A, nucleotide 736 is A, nucleotide 745 is T, nucleotide 804 is G, nucleotide 815 is T, nucleotide 829 is G, nucleotide 863 is A, nucleotide 874 is G, nucleotide 888 is T, nucleotide 937 is T, nucleotide 943 is C, nucleotide 946 is C, nucleotide 952 is A, nucleotide 976 is G, nucleotide 979 is C, nucleotide 985 is Cwhen nucleotide 1010 is T, nucleotide 1012 is C, nucleotide 1034 is C, nucleotide 1036 is T, nucleotide 1051 is G, nucleotide 1064 is G, nucleotide 1065 is C, nucleotide 1081 is C the sequence is specific for the phytoplasma sp associated with BN.

DETAILED DESCRIPTION OF THE INVENTION

The present description provides a new nucleotide sequence of SEQ ID NO 5 coding for the gene rpl22 (ribosomal protein rpl22 gene) of phytoplasma Ca. Solani (Stolbur) causing BN, and SEQ ID NO 7 coding of the rps3 gene of phytoplasma "Ca. P. solani "(Stolbur) causing BN.

Both sequences are present in SEQ ID NO 1 , wherein rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095.

Several SNPs specific for the BN associated phytoplasma have been identified in said sequence and are summarised in Table 1 herein below.

Table 1

Nucleotide specific for SNP Position SNP

BN associated phytoplasma SNP in Identification Number

SEQ ID NO 1 and 12; ( SNP ID)

T 64 1

c 66 2

G 75 3

C 81 4

T 105 5 c 208 6

c 252 7

A 253 8

G 264 9

G 288 10 c 368 1 1

A 370 12

C 372 13

T 412 14 c 445 15

A 481 16

C 493 17

A 497 18

C 506 19

G 512 20

A 544 21

G 547 22

C 565 23

T 571 24

G 574 25

A 586 26

T 589 27

G 610 28

C 616 29

G 625 30

A 649 31

G 652 32

C 663 33

G 671 34

G 672 35

C 673 36

C 685 37

A 695 38

T 708 39

A 712 40

A 721 41

A 733 42

A 736 43

T 745 44 c 763 45

G 804 46

T 815 47

G 829 48

A 863 49

G 874 50

T 898 51 τ 937 52 c 943 53 c 946 54

A 952 55

G 976 56

C 979 57

C 985 58

T 1010 59 c 1012 60 c 1034 61

T 1036 62

G 1051 63

G 1064 64

C 1065 65

C 1081 66

SEQ ID NO 1 comprises both rpl22 and rps3 nucleotide sequences and in particular, rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095. Therefore, the position of each of the single SNPs above defined can be easily identified within the SEQ ID NO 5 (coding rpl22) and the SEQ ID NO 7

(coding rps3) by the skilled person without use of inventive skill. So, by way of example, for the SNPs associated to rpl22 gene, the SNP defined as T in position 64 of SEQ ID NO 1 corresponds to the presence of nucleotide T in position 64 of SEQ ID NO 5; the SNP defined as C in position 252 of SEQ ID NO 1 corresponds to the presence of nucleotide C in position 252 of SEQ ID NO 5; for the SNPs associated to rps3 gene, the SNP defined as T in position 745 of SEQ ID NO 1 corresponds to the presence of T in position 371 of SEQ ID NO 7; the SNP defined as T in position 1010 of SEQ ID NO 1 corresponds to the presence of T in position 637 of SEQ ID NO 7.

Each SNPs and the related nucleotide indicated in Table 1 is specific for the BN associated phytoplasma.

For the sake of simplicity and conciseness all the SNPs and specific nucleotide indicated in table 1 above are also defined as "SNPs of Table 1 " and "nucleotides of Table 1 ", wherein the nucleotides of Table 1 are the ones specific for the BN associated phytoplasma per each identified SNP.

Furthermore, the SNPs disclosed in Table 1 are also numbered and can be identified in the present specification as "SNP 1 ", "2", "3" and so on, and the SNP position as well as the nucleotide specific for the BN associated phytoplasma will be the corresponding ones in the same line of the Table. So the description provides also a nucleotide sequence of SEQ ID NO 1 or the sequence complementary to SEQ ID NO 1 or fragments thereof, said fragments comprising one or more nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or said fragments being suitable for the amplification of a fragment of SEQ ID NO 1 or of the sequence complementary to SEQ ID NO 1 comprising one or more of the nucleotides of Table 1 .

As SEQ ID NO 1 comprises both SEQ ID NO 5 and 7, said sequences can be also defined (and are also defined in the present description) as "fragments of SEQ ID NO 1 comprising one or more of the nucleotides of Table 1 ".

According to the above, hence, the presence of a T in position 64 of SEQ ID NO 1 (or of A in the corresponding position in the complementary strand), and/or of a C in position 66 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a G in position 75 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 81 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a T in position 105 of SEQ ID NO 1 (or of a A in the corresponding position in the complementary strand), and/or of a C in position 208 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 252 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 253 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a G in position 264 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a G in position 288 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 368 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 370 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a C in position 372 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a T in position 412 of SEQ ID NO 1 (or of A in the corresponding position in the complementary strand), and/or of a C in position 445 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 481 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a C in position 493 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 497 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a C in position 506 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a G in position 512 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or A in position 544 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a G in position 547 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 565 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a T in position 571 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a G in position 574 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a T in position 589 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a G in position 610 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 616 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 616 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 649 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a G in position 652 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 663 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a G in position 671 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a G in position 672 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 673 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 685 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 695 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a T in position 708 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of an A in position 712 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of an A in position 721 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of an A in position 733 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of an A in position 736 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a T in position 745 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a G in position 804 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a T in position 815 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a G in position 829 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of an A in position 863 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand) , and/or of a G in position 874 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a T in position 898 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a T in position 937 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a C in position 946 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of an A in position 952 of SEQ ID NO 1 (or of a T in the corresponding position in the complementary strand), and/or of a G in position 976 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 979 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 985 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a T in position 1010 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a C in position 1012 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 1034 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a T in position 1036 of SEQ ID NO 1 (or of an A in the corresponding position in the complementary strand), and/or of a G in position 1051 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a G in position 1064 of SEQ ID NO 1 (or of a C in the corresponding position in the complementary strand), and/or of a C in position 1065 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), and/or of a C in position 1081 of SEQ ID NO 1 (or of a G in the corresponding position in the complementary strand), is specific for the phytoplasma associated with BN vine disease

All the SNPs and specific nucleotides indicated above are also defined as SNPs of Table 1 and nucleotides of Table 1 , wherein the nucleotides of Table 1 are the ones specific for the BN associated phytoplasma per each identified SNP.

The present description also provides an amino acid sequence of SEQ ID NO 6 coding for the rpl22 ribosomal protein and an amino acid sequence of SEQ ID NO 8 coding for the rps3 ribosomal protein, both of said proteins are specific for the BN phytoplasma belonging to the 16SrXII-A subgroup. Given the alignment provided in Figure 1 it has been possible to design a general sequence, namely SEQ ID NO 12, wherein the nucleotides corresponding to the 66 SNPs allowing the BN phytoplasma identification listed in Table 1 are defined as "n" and can be any nucleotide. The corresponding amino acid general sequence can be derived automatically from the nucleotide sequence.

Hence, the present description provides a nucleotide sequence of SEQ ID NO 1 or the sequence complementary to SEQ ID NO 1 or fragments thereof, said fragments comprising one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or said fragments being suitable for amplification of a fragment of SEQ ID NO 1 , or the sequence complementary to SEQ ID NO 1 comprising one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ) wherein said sequence or fragments thereof are specific for the plant phytoplasma strains associated with the vine disease BN.

The sequence allowing the specific detection of the plant phytoplasma strains associated with the vine disease BN can hence be SEQ ID NO 1 or the sequence complementary to it, and the fragments can be fragments of SEQ ID NO 1 or fragments of the sequence complementary to it, provided that they comprise one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or that they are suitable for the amplification of a fragment comprising one or more of said nucleotides, as said particular nucleotides represents SNPs that renders SEQ ID NO 1 specific for the plant phytoplasma strains associated with the vine disease Bois Noir and allow, each alone or in combination with one or more of the others, a distinction of said phytoplasma from highly similar phytoplasmas that are not associated with the BN infection.

The fragments should be fragments of a dimension suitable to be used for the detection of the phytoplasma strains associated with the vine disease BN with any common technique known for SNPs detection in the art. As already said, the fragments can also be represented by SEQ ID NO 5 and/or 7.

Normally, the skilled person would know the suitable size for the fragments that could be from about 10, 20, 30, 40, 50, 60, 100, 150, 200, nucleotides to the full sequence of the gene rpl22 and/or rps3 for identification of the BN related phytoplasma.

The fragments of the present description can be used as probes for PCR, as probes for microarray detection of SNPs, or can be the products of the techniques used for the SNPs identification. Hence, according to the technique used in a method for identification one or more of the nucleotides in the positions indicated in Table 1 , the fragments can be upstream or downstream fragments with respect to one or more of said nucleotides allowing amplification of a sequence comprising one or more of the nucleotides in the positions indicated in Table 1 or can be fragments comprising one or more of the aforementioned nucleotides. The skilled person is well aware that, when detection of a SNP is involved, the identification of the nucleotide(s) of interest can be carried on either strand of the nucleotide sequence, hence, the fragments of the invention can be designed either to identify one or more of nucleotides in the positions indicated in Table 1 , and/or to identify one or more of their complementary nucleotides that are implicitly disclosed by the disclosure of SEQ ID NO 1 , 5, 7 and 12. The following description will clarify in more detail the fragments of the invention, all of them being characterised by the fact that they can be used for the identification of the aforementioned SNPs.

SEQ ID NO 12 represents a generic sequence wherein the SNPs nucleotides of the alignment showed in figure 1 are indicated as "n".

On the other hand, the nucleotide sequence of SEQ ID No 1 or fragments thereof (also such as SEQ ID NO 5 and 7) herein disclosed are specific the phytoplasma associated with BN.

Hence, SEQ ID NOs 1 , 5, and 7 represent each, one of the possible embodiments of SEQ ID NO 12 or fragments thereof, in particular the embodiment wherein the nucleotides in the positions indicated in Table 1 correspond to the nucleotides of Table 1 .

As already indicated in the present specification, the sequences or fragments thereof herein described are applicable as diagnostic tools for the identification, in a grapevine sample, of the phytoplasma associated to the vine disease Bois noir.

The invention also encompasses vectors such as cloning vectors or expression vectors comprising a nucleotide sequence of SEQ ID NO 12, 1 , 5 or 7 or fragments thereof, said fragments comprising one or more of nucleotides in the positions listed in Table 1.

The vector of the invention can be used either as a positive or negative control in a diagnostic method for the detection of BN associated phytoplasma in a vine phloem sample depending on the nucleotides detected in the positions indicated in Table 1 (the exact nucleotides of Table 1 , will provide a positive control whether different nucleotides in the positions indicated in table 1 will provide a negative control) or it could also be used as research tool for the development of antibodies and/or for the study of possible therapies for curing the disease of the plant. Hence the vector above can be represents, per se, a control for the method and the kit herein disclosed.

The vector can also be used for cloning the genes of interest, for the production of the protein(s) coded, for the development of specific antibodies and in methods and studies for the development of therapies for the infected vines against BN.

A further object of the invention is a method for the diagnosis of the vine disease Bois Noir comprising the step of

a) identifying one or more of the nucleotides in the position indicated in Table 1 in SEQ ID NO 12 or a fragment thereof, and/or one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12 or a fragment thereof, in a plant sample comprising phloem;

wherein the presence of one or more of the residues of Table 1 in the respective position indicated in Table 1 in SEQ ID NO 12 and/or of its complement in the sequence complementary to SEQ ID NO 12 indicates the presence of the phytoplasma strain associated with the vine disease Bois Noir in the sample.

The sample according to the present description can be any sample obtainable from a vine plant, provided that the sample comprises phloem of the plant due to the presence of the phytoplasmas being limited to this plant fluid.

The skilled person would know how to obtain such sample; when the sample is taken from a plant it is possible to obtain the fluid directly from the leaf veins.

Phloem samples are also available from vine shoots or from the fruits or from the seeds or from the rootstock. Protocols for the collection of phloem-comprising samples are available to the skilled person without need of further details in the present description (Pasquini et al., 2001 ).

In the method of the invention the phloem obtainable by standard methods from the sample can be used as such, or an extraction of the total nucleic acids

(TNA) can be carried out with standard methods (by way of example the method described in Angelini E, et al (2001 ) Flavescence doree in France and Italy: occurrence of closely related phytoplasma isolates and their near relationships to

Palatinate grapevine yellows and an alder yellows phytoplasma. Vitis 40:79-86.

(Page 80 column 2) or by Seruga Music et al 2008, page 121.

The identification of one or more nucleotides of the SNPs form SNP ID 1 to

66 (i.e. the nucleotides in the positions indicated in table 1 ) can be carried out with any known technique suitable for the detection of the presence, in a given position, of one or more of the nucleotides of Table 1 in SEQ ID NO 12 and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12. The presence of one or more of the nucleotides of Table 1 in the sample analysed indicates the presence in said sample of the phytoplasma associated with the BN vine disease. By way of example, the identification of step a) may comprise a first step of PCR amplification of one or more fragments comprising one or more of the SNPs from SNP ID 1 to 66 and a subsequent analysis of the amplified fragment either by assessing the mass of the amplified fragment(s) and hence defining the base pair present in the position of interest or by merely sequencing the fragment or by using the amplified fragment for other detection techniques as some of the techniques exemplified below.

If the SNPs are amplified in a different fragment each a different amplicon size can be selected for the fragments in order to easy the detection.

In an embodiment of the invention, the identification step a) may comprise a first step wherein a RT PCR, Ligation, Allele Specific Hybridization, primer extension, invasive cleavage or sequencing reaction is carried out and a second step wherein the product obtained in the first step is detected by monitoring the light emitted by said product, by measuring the mass of said products, by monitoring the radioactivity emitted by said product or by sequencing said product.

It will be readily understood by the skilled person that any other suitable protocol and technique known for the identification of a SNP in a nucleotide sequence can be applied to the method herein described.

Several techniques for SNPs identification known in the art and suitable for carrying out the diagnostic method herein described are summarised in Kwok 2001 .

Numerous techniques are known in the art for the identification of a given SNP in a nucleic acid, several of them are applicable to a solid support technology such as microtiters or microarrays and are thus suitable for the screening of a high number of samples. When a screening in a vineyard or in a vine nursery has to be carried out, methods for the detection of the SNP of interest that are easily applicable to a large amount of samples are convenient.

When RT-PCR is used, it is possible to apply a classic RT PCR protocol available in RT-PCR manuals and in laboratory protocols. The methods used to verify the identity of the amplicon(s) produced in RT PCR are sufficiently powerful to detect small variations between sequences. Variations in sequence, including SNPs have been successfully identified in RT PCR assays. One common approach to the detection of sequence variation is to compare melting curves. In general, the effect of base substitutions on the melting kinetics of PCR products is too small to be detected reliably; however, heteroduplexes of relatively long amplicons differing by a SNP can be distinguished from the homoduplexes on the basis of their melting curves.

The melting curves of short fluorescent probes can be used to distinguish between amplicons. This method is sensitive to SNPs, which usually cause a shift in the melting peak of several degrees. The design of primers suitable for RT PCR is easily achievable with suitable programs available to the skilled person (a possible primer pair is depicted in SEQ ID NO 2 and 4) and the disclosure of SEQ ID NO 12, 1 , 5, and 7 and of all SNPs associated with BN phytoplasma, each of them reported in table 1 , is sufficient for the skilled person to readily design the RT PCR assay without use of inventive skill or cumbersome experimentation. The skilled person will easily design primers and oligonucleotides for the amplification and detection of one o more nucleotides (SNPs) in the positions indicated in table 1 or for the amplification and detection of a fragment comprising one o more of said nucleotides or SNPs.

RT PCR may involve the use of fluorescently labelled nucleic acid probes or primers, or DNA-binding fluorescent dyes such as SYBR® Green and others mentioned in the present description, to detect and quantify a PCR product at each cycle during the amplification. If desired, different fluorescent dies can be used for the different SNPs.

Hence, according to the present description, the amplified product(s) or the probes can be labelled with a fluorophore following the manufacturer's instructions when melting curve of short fluorescent probes is analysed.

Many RT-PCR approaches employ two different fluorescent reporters and rely on the energy transfer from one reporter (the energy donor) to a second reporter (the energy acceptor) when the reporters are in close proximity. The second reporter can be a quencher or a fluor. A quencher will absorb the energy from the first reporter and emit it as heat rather than light, leading to a decrease in the fluorescent signal. A fluor will absorb the energy and emit it at another wavelength through fluorescence resonance energy transfer (FRET, reviewed in 2), resulting in decreased fluorescence of the energy donor and increased fluorescence of the energy acceptor. The change in fluorescence is proportional to the accumulation of PCR product.

A common alternative to the melting curve approach is to use hydrolysis (such as TaqMan) probes.

Hydrolysis probes are labelled with a fluorescent dye at the 5'-end and a quencher at the 3'-end, and because the two reporters are in close proximity, the fluorescent signal is quenched. During the annealing step, the probe hybridizes to PCR product synthesized in previous amplification cycles. The resulting probe: target hybrid is a substrate for the 5'→3' exonuclease activity of Taq DNA polymerase, which degrades the annealed probe (3) and liberates the fluor. The fluor is freed from the effects of the quencher, and the fluorescence increases. In an embodiment the identification of the SNP of interest can be carried out by RT-PCR using suitable primer pairs and a suitable allele specific oligonucleotide probe (TaqMan ® probe), e.g. an MGB (Minor Groove Binding) probe. The allele-specific "TaqMan probe" may be designed based on the SNP information described above. The 5' end of TaqMan probe is labelled with fluorescence reporter dye R (e.g. FAM or VIC), and at the same time, the 3' end thereof is labelled with quencher Q (quenching substance). Thus, under these conditions, fluorescence is not detectable since the quencher absorbs fluorescence energy. Since the 3' end of TaqMan probe is phosphorylated, no extension reaction occurs from TaqMan probe during PCR reaction.

In an embodiment of the invention, the TaqMan® MGB probes can be labelled with 6-carboxyfluorescein (FAM) at the 5' end and with a non-fluorescent quencher (NFQ) with minor groove binder (MGB) at the 3' end.

MGB probes in the present invention disclosed a higher melting temperature (Tm) and increased specificity and were MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

All RT-PCR reactions may be carried out on an ABI PRISM® 7300 Sequence Detection System (Applied Biosystems) in optical 96-well plates with optical adhesive covers (both Applied Biosystems) using the following cycling conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 61 ,5°C, which allowed running of all reactions on the same plate. RT-PCR can be performed in a final reaction volume of 25 μΙ_ containing 5 μΙ_ of sample DNA, 300 nm primers, 250 nm probe and 1 * TaqMan® Universal PCR Master Mix (Applied Biosystems), which includes ROX as a passive reference dye.

Suitable primers and probes can be designed with various algorithms and programs available also on the web or the design thereof can be commissioned via commercially available services. In a non limitative way suitable probes could be a forward primer of 21 nucleotides in length provided herein as SEQ ID NO 2, a TaqMan ® probe of 18 nucleotides in length provided herein as SEQ ID NO 3 labelled e.g. with 6-FAM at the 5' end, and a reverse primer of 24 nucleotides in length, provided herein as SEQ ID NO 4.

When SEQ ID NOs 2-4 are used in this assay, the suitable annealing temperature is is 60 °C. Assays using TaqMan ® are well known in the art and full protocols are available to the skilled person, in principle. The 5'-nuclease allelic discrimination assay, or TaqMan assay, is a PCR-based assay for genotyping SNPs. The region flanking the SNP is amplified in the presence of two allele-specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3' end. The presence of two probes allows the detection of both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post-PCR processing.

Techniques suitable for carrying out the method herein disclosed comprise also Allele Specific Hybridization also known as ASO (allele specific oligonucleotide hybridization). This protocol relies on distinguishing between two DNA molecules differing by one base (i. e. the SNP of interest) by hybridization. The TNA obtained by the phloem sample is amplified with suitable PCR primers designed in order to amplify a region of SEQ ID NO 12 comprising one or more of the nucleotides in the positions indicated in Table 1 (or of their complementary nucleotides in a sequence complementary to SEQ ID NO 12) or in other words of the SNPs of Table 1 , in a convenient embodiment, the PCR amplicons are fluorescence labelled. The fragments obtained by PCR are than applied to immobilized oligonucleotide fragments of SEQ ID No 1 comprising the SNP or the SNPs of interest. After suitable hybridization and washing conditions, fluorescence intensity is measured for each SNP oligonucleotide. Due to the fact that a SNP is analysed, stringent hybridization and washing conditions will have to be used, where by stringent the skilled person will understand conditions that will allow only 100% matched hybridised sequences to remain in the double strand form. Typical means for regulating stringency of a hybridisation protocol are salt concentration (the highest the concentration the lowest the stringency) and/or formamide concentration (the highest the %, the highest the stringency) and/or temperature (the highest the temperature, the highest the stringency). Stringency conditions suitable for identification of SNPs by RT-PCR are known in the art and published on standard protocols for this reaction.

This kind of identification can be carried out also in a solid phase, i.e. on a solid support (defined herein after) such as microtiter, microarray chip and the like, allowing the screening of a large number of samples.

In a particular embodiment, microarrays will be used. The microarray slide is a very powerful diagnostic tool capable to identify, in a single experiment, the presence/absence of a high number of targets. The chips take advantage of an important DNA property, which is the complementary bases match (the T matches with the A and the G with the C) in its structure. The diagnostic technique consists of a luminous signal (emitted by a fluorophore at different wave lengths) in correspondence to the hybridization between the target fragment labelled with the fluorophore and the corresponding probe bound to the microarray slide. This binding, with subsequent light emission, shows that in the group of analysed probes a DNA fragment complementary to the probe that is "lighted" is present and, consequently, allows to know the identity of the target fragment. There are several well known techniques for labelling the nucleic acid analysed (direct, indirect) through the creation of link with fluorophores emitting emit light at different wave lengths (usually in the red and in the green). The probe on the microarray slide is not bound to the fluorophore, therefore the microarray slide that is not hybridised, when observed under a confocal scanner does not show any luminous signal.

Prior to the hybridisation on the microarray a PCR amplification of the target DNA, is carried out in order to both increase the target DNA amount which will easy the detection of the signal, and in order to label the target DNA for the detection on the microarray. The labelling can be carried out with any labelling molecule commonly used for microarrays detection.

On the array, several copies of the probe comprising a fragment of SEQ ID NO 1 , 5, and/or 7 wherein one or more nucleotides of table 1 , (and/or one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 , 5 and/or 7) can be placed per spot in order to increase the signal on the microarray when the phytoplasma associated to BN is present in the sample analysed and amplified.

It is evident that the region amplified will have to comprise at least one nucleotide corresponding to one or more of the nucleotides in the positions indicated in table 1 , i.e. the discriminating SNPs, in the amplicon(s). The region amplified hence can comprise one or more SNPs of interest and also more amplifications providing fragments comprising, each one of the SNPs of interest can be carried out.

In the present description, fragments comprising one or more of the nucleotides in the positions indicated in Table 1 (and/or one or more of their complementary nucleotides) are fragments wherein the nucleotides upstream and downstream said nucleotide(s) perfectly match with the nucleotides upstream and downstream the said nucleotide in SEQ ID NO 12 or in SEQ ID No 1 or in their complementary sequences when the complementary nucleotides are considered. By perfectly match it is intended that a 100% alignment between SEQ ID NO 12 or SEQ ID No 1 or its complementary sequence and the fragment according to the present description is present. The design of the primers for the amplification can be carried out with any suitable program available to the skilled person (non limiting examples: Primer Premier, that can be used to design primers for single templates, alignments, degenerate primer design, restriction enzyme analysis, contig analysis and design of sequencing primers; AllelelD and Beacon Designer can design primers and oligonucleotide probes for complex detection assays such as multiplex assays, cross species primer design, species specific primer design and primer design to reduce the cost of experimentation; PrimerPlex is a software that can design ASPE (Allele specific Primer Extension) primers and capture probes for multiplex SNP genotyping using suspension array systems such as Luminex xMAP® and BioRad Bioplex, Primer Express ) or by commercially available services for oligo design.

Starting from SEQ ID NO 12 or from SEQ ID No 1 herein disclosed and knowing that the fragment to be hybridised on the microarray will need to comprise one or more nucleotides in the positions indicated in Table 1 , the skilled person will easily know how to provide suitable primers for the amplification. A nucleotide strand complementary to the amplified sequence will be spotted on the microarray for the subsequent hybridisation. On the microarray, control fragments can be spotted in order to verify the efficiency of the method used.

In all the methods for carrying out the identification of the SNPs, when SEQ ID NO 12 is mentioned it is evident that the same methods apply automatically for those embodiments to SEQ ID NO 1 as SEQ ID NO 1 is comprised in SEQ ID NO 12 and to SEQ ID NO 5 and 7 as they are fragments of SEQ ID NO 1. The skilled person can hence read SEQ ID NO 5 instead of SEQ ID NO 1 for all the embodiments of this specification relating to the identification of BN associated phytoplasma.

Suitable primers for the amplification of the fragment comprising the SNPs in position 610, 161 and 625 to be hybridised on the array are also the primers having SEQ ID NO 2 and SEQ ID NO 4 herein provided as exemplifying primers.

The design of primers for the amplification of a fragment comprising other SNP or SNPs in the positions indicated in Table 1 is easily achievable for the skilled person starting from SEQ ID NO 1 or 12.

The hybridisation conditions for the allele specific hybridisation technique are available in the art, an example of suitable conditions can be 16 hours of hybridisation at 48°C (25% of formamide).

By way of example, the microarray hybridisation could be carried out following a standard protocol as follows, wherein some step may be omitted, modified or added by the skilled person without the use of inventive skill: Microarray hybridization and wash conditions:

Prehybridisation:

1 . Prehybridisation buffer: 5x SSC, 0.1 % SDS and 1 % BSA. Heat to about 50°C while stirring; 2. Slides to be analyzed are placed in a staining jar; prehybridisation buffer is added, and incubation is carried out at about 48°C for 45- 60 min while stirring; 3. The slides are washed by dipping up and down approximately 10 times in two different staining jars of deionised water. Excess water is removed by shaking the slide rack up and down two times; 4. The slides are then dipped in an up and down motion approximately 10 times at room-temperature in isopropanol and spun dried. The slides are used immediately after prehybridisation (less than 1 hr) as hybridization efficiency decreases rapidly if the slides are allowed to dry for more than that time.

Hybridization:

5. 2X hybridization buffer: 50% formamide, 10X SSC and 0.2% SDS. Incubate the solution until it reaches 48°C; 6. The labelled mixtures are re- suspended in 9 μΙ water, and heated to 95°C for 3 min to denature, and are centrifuged at maximum angular velocity for 1 min.; 7. The following is added to each tube in order to block non-specific hybridization. Make a master-mix with the following ingredients for each tube:

· Calf Thymus DNA (1 g L) 8μΙ

• poly(A)-DNA (10mg/ml_) 2μΙ

• yeast tRNA (4mg/ml_) 2μΙ

8. Then, about 21 μΙ 2X hybridization buffer that has been pre-heated to 48°C is added to the target mixture, mixed well, and centrifuged. The samples are kept at 48°C until placed on the slide; 9. The labelled target is applied to a pre- hybridized microarray slide and covered with a 22 x 60 mm glass cover slip; 10. The slide is placed in a sealed hybridization chamber (Corning. Acton, MA), and 12 μΙ water is added to the small reservoirs at each end of the chamber; 1 1. The sealed chamber is placed in a 48°C water bath and incubated for 40-60hr (2).

Post-hybridization washes:

12. The array is removed from the hybridization chamber with care taken not to disturb the coverslip; 13. The slide is placed in a rack for a staining dish containing 1 X SSC, 0.1 % SDS, and 0.1 mM DTT at about 48°C; 14. The coverslip is gently removed while the slide is in solution and agitated for 15 min.; 15. The slides are transferred to a staining dish containing 0.1 X SSC, 0.1 % SDS, and 0.1 mM DTT at about 48°C and agitated for 5 min.; 16. Repeat step 15 two more times; 17. The slides are transferred to a staining dish containing 0.1 X SSC and 0.1 mM DTT at room-temperature and agitated for 5 min.; 18. Repeat step 17 an additional time; 19. Slides are spun dried.

The exemplified conditions above are a standard protocol based on which the skilled person will be capable to apply all suitable modifications for carrying out the method of the invention without use of inventive skill.

Suitable labelling for the examples above, and for all the following examples where fluorescence is used for the detection, can be carried out, e.g. with Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes or any other suitable commercial dye following the manufacturer's instructions.

Another possible technique for carrying out the method of the invention is by the single base extension (SBE) approach.

According to this technique the target region is amplified by PCR followed by a single base sequencing reaction using a primer that anneals one base inside of the polymorphic site. Several detection methods have been described. One can label the primer and apply the extension products to gel electrophoresis. Or the single base extension product can be broken down into smaller pieces and measured by Mass Spectrometry. The most popular detection method involves fluorescence labeled, dideoxynucleotide terminators that stop the chain extension.

As known by the skilled person, primer extension is a very robust allelic discrimination mechanism. It is highly flexible and requires the smallest number of primers/probes. Probe design and optimization of the assay are usually very straightforward. As for the method described above, the skilled person, starting from the sequence and the SNPs herein disclosed can easily and readily design suitable probes merely using commonly available software for probe design. Given the shortness of SEQ ID NO 5 and 1 and the one or maximum two SNPs to be identified, the design of the suitable probes will not be particularly difficult or toilsome and will not require the use of inventive skill, being the mere application of standard procedures or even commercially available services for probe design sufficient. There are numerous variations in the primer extension approach that are based on the ability of DNA polymerase to incorporate specific deoxyribonucleosides complementary to the sequence of the template DNA. However, they can be grouped into two categories: the first is a sequencing (allele- specific nucleotide incorporation) approach where the identity of the polymorphic base in the target DNA is determined; the second is an allele-specific PCR approach where the DNA polymerase is used to amplify the target DNA only if the PCR primers are perfectly complementary to the target DNA sequence. In the sequencing approach, one can either determine the sequence of amplified target DNA directly by mass spectrometry or perform primer extension reactions with amplified target DNA as a template and analyze the products to determine the identity of the base(s) incorporated at the polymorphic site (allele specific nucleotide incorporation). Various ways have been devised in the art for primer extension product analysis in homogeneous assays.

In the allele-specific PCR approach, one relies on the DNA polymerase to extend a primer only when its 3' end is perfectly complementary to the template.

When this condition is met, a PCR product is produced. By determining whether a PCR product is produced or not, one can infer the allele found on the target DNA. Several approaches have been utilized to detect the formation of specific PCR products in homogeneous assays, e.g. based on melting curve analysis, or based on hybridization of target specific probes. A variation of this approach is the allele-specific primer extension. Here, the PCR product containing the polymorphic site serves as template, and the 3' end of the primer extension probe consists of the allelic base. The primer is extended only if the 3' base complements the allele present in the target DNA. Monitoring the primer extension event, therefore, allows one to infer the allele(s) found in the DNA sample.

SBE can also be easily carried out on microarrays using the well known SBE-TAG technique. Protocols suitable for carrying out this embodiment of the invention are described in manuals such as, by way of example, in "DNA microarrays: a molecular cloning manual" by David Bowtell, Joseph Sambrook, Protocol 6, pages 403-420, herein incorporated by reference. In protocol 6 of the above mentioned manual all the teachings that are necessary to the skilled person for the design of this embodiment of the invention, from primer selection and design to buffers, is described in detail. The skilled person can hence easily carry out this SBE-tag embodiment of the invention starting from the teachings of the present description without use on inventive skill and without cumbersome preparations. Another protocol suitable for a SBE identification of the SNP of interest on microarrays is the Affymetrix tag array, the protocol being also available on manuals such as the manual mentioned herein above by Bowtell and Sambrook, described in detail in Protocol 7, pages 421 -428 herein incorporated by reference.

A further suitable technique for the identification of the SNP(s) of interest is the ligation technique. In the Allele Specific Oligonucleotide Ligation, by designing oligonucleotides complementary to the target sequence, with the allele-specific base at its 3'-end or 5-'end, one can determine the genotype of the PCR amplified target sequence by determining whether an oligonucleotide complementary to the DNA sequencing adjoining the polymorphic site is ligated to the allele-specific oligonucleotide or not. This assay relies on the fact that DNA ligase is an enzyme that is highly specific in repairing nicks in the DNA molecule. When two adjacent oligonucleotides are annealed to a DNA template, they are ligated together only if the oligonucleotides perfectly match the template at the junction.

Allele-specific oligonucleotides can, therefore, interrogate the nature of the base at the polymorphic site. One can infer the allele(s) present in the target DNA by determining whether ligation has occurred. Ligation has the highest level of specificity and it is the easiest to optimize among all allelic discrimination mechanisms, but it is the slowest reaction and requires the largest number of modified probes. However, ligation as a mechanism has the potential of genotyping without prior target amplification by PCR. This can be accomplished either by the ligation chain reaction (LCR) or by the use of ligation (padlock) probes that are first circularized by DNA ligase followed by rolling circle signal amplification.

Also the ligation technique is applicable on microarrays, in this case, the technique is called more specifically Ligation Detection Reaction - Universal Array (LDR-UA) and allows the detection of Single Nucleotide Polymorphisms (SNPs) on DNA molecules. The LDR-UA technique takes advantage of two different probes, called Common Probe (CP) and Discriminating Probe (DP), which are designed to anneal juxtaposed on target single strand DNA. DP anneals on the DNA at the 5' end of CP. The two probes can be ligated by a thermo-stable ligase such as the Pfu Ligase. If the complementarity between the 3' end of the DP and the target DNA is not perfect, the ligation reaction is compromised. For this reason the last base at the 3' end of the DP is also called discriminating base.

The system requires that the probes carry the following modifications:

Each DP must be labelled with a distinct fluorophore at its 5' end. The CP must be phosphorylated at its 5' end and must be extended at its 3' end with a "cZIP Code" sequence, which is the complementary and inverse of the "Zip Code" sequence spotted on the Universal Array. Every CP corresponds to a different "ZIP Code".

The ligation product is hybridized to the UA. Each UA spot is composed of different "Zip Code" DNA sequences that capture the corresponding "cZIP Code" sequences at the CP 3' end. The CP-DP ligation event positions the fluorophore at the 5' end of the DP on the corresponding UA spot that is visualized by the subsequent scanning of the UA. The signal from a spot therefore indicates the perfect match between the DP and the target DNA.

Again, for the ligation techniques, the design of probes starting from SEQ ID NO 12 or 1 knowing that the SNPs of interest lie in positions indicated in Table 1 , allows for an easy selection of the primers simple for the skilled person. CP and DP (the term extended also to the ligation technique when carried out not on solid supports) can be ordered to commercially available services specialised for the design and construction of this kind of probes. For the aims of the method herein described, it will not even be necessary to design a DP for each allelic form of the SNPs, as the sole aim is the identification of a the BN specific nucleotide (or of its complement) and there is no need for the identification of other possible bases in the position of interest.

In a yet further embodiment, the identification of the SNP(s) in the positions indicated in Table 1 can be carried out by the invasive cleavage technique as described, e.g. by Wilkins Stevens P. et al 2001 .

As indicated above, also this technique is known to the skilled person as a suitable approach for the identification of SNPs.

The invasive cleavage assay is a probe cycling, signal amplification reaction used for detection of single nucleotide polymorphisms (SNPs). The reaction requires two synthetic oligonucleotides, called the 'upstream oligonucleotide' and 'probe', that anneal to the target sequence with an overlap of 1 nt. This creates a bifurcated overlapping complex that resembles a structure generated during strand displacement DNA synthesis. Structure-specific 5'-nucleases, whose primary cell function is believed to be processing of Okazaki fragments, cleave the bifurcated substrate at the site of the overlap, releasing the 5'-arm and one base paired nucleotide of the probe. The cleaved 5'-arm serves as a signal indicating the presence of target in an analyzed sample. By performing the invasive reaction at the probe melting temperature (Tm), multiple cleavage events can be achieved for each target. Typically, an invasive signal amplification reaction generates 30-50 cleaved probes/target/min, resulting in 103-104-fold signal amplification in a 1-3 h reaction. The unique ability of 5'-nucleases to specifically cleave the overlapping substrate can be utilized for detection of single base mutations. To detect a particular nucleotide in the target sequence, the upstream oligonucleotide and probe are designed to create overlap at this nucleotide, ensuring efficient cleavage of the probe. A substitution at this nucleotide position eliminates the overlap and dramatically reduces the cleavage rate, resulting in mutation discrimination of at least 300:1. Such discriminatory power makes the invasive cleavage assay an excellent tool for identification of SNPs.

Signal detection can be carried out by electrophoresis, microplate (microtiter) enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry methods, and fluorescence resonance energy transfer (FRET) methodology.

The invasive cleavage assay, as the other techniques mentioned above, is adaptable to a solid phase format presenting the possibility of analyzing multiple SNPs or multiple samples for a single SNP in parallel. SNP detection using the invasive cleavage reaction can be performed in 96-well microplates with nanogram amounts of DNA per SNP.

Furthermore, the SNP can be identified by the SniPer method, which allows discriminating alleles by examining the presence or absence of amplification by RCA (rolling circle amplification). Briefly, the DNA to be used as a template is linearized. Then, a probe is hybridized to this linearized DNA. When the probe sequence and the sequence of the linearized DNA as a template are complementary to each other and form a complementary strand, the genomic DNA can be converted into a circular DNA through ligation reaction. As a result, RCA of the circular DNA proceeds. On the other hand, when the ends of the probe do not match with the genomic DNA, the DNA is not ligated to become a circular DNA. Thus, RCA reaction does not proceed. Therefore, in Sniper method, a single-stranded probe which anneals with the genomic DNA and is circularizable is designed. This single- stranded probe is called a padlock probe. The sequences of the two ends of this padlock probe are designed so that they correspond to the SNP to be detected. Then, this padlock probe and the genomic DNA are mixed for ligation. If the two ends of the padlock probe and the SNP site of the genomic DNA are complementary to each other, the two ends of the padlock probe are joined by ligation, yielding a circular probe. If the two ends of the padlock probe and the SNP site of the genomic DNA are not complementary to each other, the probe does not become circular. Therefore, only those padlock probes which are complementary to the SNP to be detected become circular and are amplified by DNA polymerase. By detecting the presence or absence of this amplification, SNP may be detected. For the detection, synthetic oligonucleotides which have a fluorescent dye and a quencher at their respective ends and also have a hairpin structure are used.

Finally, a further technique applicable to the diagnostic method herein disclosed, is the amplification of a fragment comprising one or more of the nucleotides in the positions indicated in Table 1 and sequencing of the same with direct identification of one or more said nucleotides (and/or of their complementary nucleotides).

The sequencing can be readily and easily performed with automated sequencers. When a fragment sequence is carried out, the amplification primers of SEQ ID NO 2 and 4 are suitable also for this technique. It is evident that any primer pair suitable for the specific amplification of all or part of SEQ ID NO 12 or 1 provided that one or more of nucleotides in the positions indicated in Table 1 are comprised in the amplicon obtained (and/or one or more of the complementary nucleotides), or any probe overlapping with one or more of the nucleotides in the positions indicated in Table 1 (and/or one or more of the complementary nucleotides) can be used for carrying out the method of the invention, and that the invention is not limited to primers of SEQ ID NO 2-4.

In all the embodiments described above, where the techniques used allows for the distinction within the two different alleles of the SNP(s) of interest, a control could be carried out by detecting also the allele carrying the non BN specific allele. It is evident that in a kit for carrying out the diagnostic method herein described suitable probes can be provided also for the detection of the non BN specific allele.

As stated above, the diagnostic method herein disclosed, can be carried out on solid supports. Suitable solid supports can be a latex bead, a glass slide, a silicon chip, or the walls of a microtiter well. In some cases, marker specific oligonucleotides are placed on the solid support, and the allelic discrimination reaction is done on the support whereas in other cases, generic oligonucleotides are placed on the solid support, and they are used to capture complementary sequence tags conjugated to marker specific probes. When marker specific oligonucleotides are placed on the solid support, the oligonucleotide arrays act as a collection of reactors where the target DNA molecules find their counterparts, and the allelic discrimination step for numerous markers proceeds in parallel. When generic oligonucleotides are placed on the solid support, the arrayed oligonucleotides are used to sort the products of the allelic discrimination reactions (also done in parallel) performed in homogeneous solution. In both cases, the identity of an oligonucleotide on a latex bead or at a particular location on the microarray (on a glass slide or silicon chip) is known, and the genotypes are inferred by determining which immobilized oligonucleotide is associated with a positive signal. The clear advantage of performing genotyping reactions on solid supports is that many markers or, in the present case many samples, can be interrogated at the same time. Besides saving time and reagents, performing numerous reactions in parallel also decreases the probability of sample/result mix-ups.

As previously mentioned, the detection of the product obtained by the first step of the identification step a), e.g. the product obtained by the aforementioned techniques, can be performed by monitoring the light emission, by measuring of the mass, by monitoring of the radioactivity or by sequencing of the said product.

According to the present description, said monitoring of the light emission can be carried out by monitoring fluorescence, luminescence, time resolved fluorescence, fluorescence resonance energy transfer, or fluorescence polarisation, said measuring of the mass can be carried out by mass spectrometry and said monitoring of the radioactivity can be carried out with radioactivity sensitive tools.

Monitoring light emission is the most widely used detection modality in genotyping, this can be done by measuring or detecting Luminescence, fluorescence, time resolved fluorescence, fluorescence resonance energy transfer (FRET), and fluorescence polarization (FP).

Luminescence is emitted in an ATP-dependent luciferase reaction. When ATP production is coupled with a primer extension reaction, luminescence is observed every time a deoxyribonucleoside is added in the primer extension reaction. Luminescence can be measured with suitable commercial analysers following the manufacturer's instructions (e.g. Applied Biosystems 1700 Chemiluminescent Microarray Analyzer).

Fluorescence can be measured using commercially available fluorescence sensitive imaging devices or measuring devices known in the art and labelled probes or amplified products according to the selected technology for carrying out the method herein described. Suitable fluorophores for labelling the nucleic acids of interest can be selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes.

Detection can be carried out following the manufactures instructions. The skilled person is well aware that all techniques indicated above can all be carried out with fluorophores following conventional protocols.

Detection by Time-resolved fluorescence can be made with dyes having a long half life (such as Lanthanides), the fluorescence reading is done sufficiently long after excitation, such that autofluorescence (which has a very short half-life) is not observed. As lanthanides are inorganic compounds that cannot be used to label nucleic acids directly an organic chelator conjugated to the probe must be used to bind the lanthanides in the reaction. Protocols are available for this kind of detection.

Also for this kind of detection standard protocols and manufacturers protocols are available to the skilled person.

Fluorescence resonance energy transfer (FRET) occurs when two conditions are met. First, the emission spectrum of the fluorescent donor dye must overlap with the excitation wavelength of the acceptor dye. Second, the two dyes must be in close proximity to each other because energy transfer drops off quickly with distance. The proximity requirement is what makes FRET a good detection method for a number of allelic discrimination mechanisms. Basically, any reaction that brings together or separates two dyes can use FRET as its detection method. FRET detection has, therefore, been used in primer extension and ligation reactions where the two labels are brought into close proximity to each other. It has also been used in the 50 nuclease reaction, the molecular beacon reaction, and the invasive cleavage reactions where the neighbouring donor/acceptor pair is separated by cleavage or disruption of the stem-loop structure that holds them together. Dyes pairs suitable for FRET detection are known in the art.

FP can be used in several SNPs detection techniques, commercial systems such as the Perkin Elmer AcycloPrime™-FP SNP Detection System are available to the skilled person.

According to the present description also systems that make no use of fluorescent dyes can be used for the detection of the SNPs, a commonly used system is the mass spectrometry (MS) where the molecular weight of the products formed is measured. In MS detection no labels are needed. High resolution MS can easily distinguish between DNA molecules that differ by only one base in times of milliseconds to analyze each sample. MS can be measured with commercially available mass spectrometers following standard protocols.

A further method requires the use of radiolabels instead of fluorescent labels and in this case radioactivity of the sample is measured in standard ways well known in the art.

Another embodiment of the present invention is a diagnostic kit for the diagnosis of the vine disease Bois noir comprising reagents for the identification of one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 12 and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12 in a plant phloem.

The kit of the present invention comprises any and all components enzymes or components necessary (suitable) for an intended assay. Examples of such components include, but are not limited to, labelled and/or non labelled oligonucleotides, polymerases (e.g. Taq polymerase), buffers (e.g. Tris buffer), dNTPs labelled or non labelled, control reagents (e.g. tissue samples, target oligonucleotides for positive and negative controls, etc.), labelling and/or detection reagents (fluorescent dyes such as VIC, FAM), solid supports, manual, illustrative diagrams and/or product information, inhibitors, and packing environment adjusting agents (e.g. ice, desiccating agents). The kit of the present invention may be a partial kit which comprises only a part of the necessary components. In this case, users may provide the remaining components. The kit of the present invention may comprise two or more separate containers, each containing a part of the components to be used. For example, the kit may comprise a first container containing an enzyme and a second container containing an oligonucleotide. Specific examples of the enzyme include also a structure-specific cleaving enzyme, ligases or other enzymes for use in the techniques for identification and detection described above contained in an appropriate storage buffer or a container. Specific examples of the oligonucleotide include nucleotides for the RT-PCR, nucleotides for the extension technique described above, nucleotides the ligation technique described above, oligonucleotides for the LDR-UA technique described above, oligonucleotides for the invader technique described above, probe oligonucleotides for the hybridisation technique described above, target oligonucleotides for use as control, and the like.

The oligonucleotides can be labelled according to the technique selected.

Alternatively, ore or more reaction components may be provided in such a manner that they are pre-divided into portions of a specific amount. Selected reaction components may also be mixed and divided into portions of a specific amount. It is preferred that reaction components should be pre-divided into portions and contained in a reactor. Specific examples of the reactor include, but are not limited to, reaction tubes or wells, or microtiter plates. It is especially preferable that the pre- divided reaction component should be kept dry in a reactor by means of, for example, dehydration or freeze drying.

The kit of the invention may further comprise solid supports wherein oligonucleotides for the identification of one or more of the nucleotides the positions indicated in Table 1 in SEQ ID NO 12 and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID N012 are anchored in known positions on said support in one or more copy per position.

The anchorage of the oligonucleotides/probes as described above in the section related to the techniques suitable for carrying out the method will be anchored to the solid support by standard techniques selected depending on the support chosen.

The kit of the invention may comprise labelled probes corresponding and/or complementary to a region of SEQ ID NO 12 upstream and/or downstream of one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 12 and/or overlapping a region comprising one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 1. Upstream and downstream oligonucleotides may be directly adjacent to one of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 1 or to its complementary nucleotide in the sequence complementary to SEQ ID NO 1 e.g. in case of kits for identification by ligation or by other techniques as exemplified above requiring probes directly adjacent to the SNP; or may be non directly adjacent to the SNP nucleotide when the kits are for identification with techniques as exemplified above that to not require probes directly adjacent to the SNP.

All the probes and oligonucleotides of the invention (e.g. probes described for the method and for the kit) fall within the definition of fragments of SEQ ID NO 1 or of its complementary sequence and will be of a dimension comprised between about 8 to about 100 nucleotides, normally of a dimension comprised between about 15 to about 50 nucleotides. When the fragments and probes do not comprise one or more the SNPs of interest but allow their identification (e.g. when the fragments are amplification fragments) they also fall within the definition of fragments of SEQ ID NO 1 or 12 or of their complementary sequence and will be of a dimension comprised between about 8 to about 100 nucleotides, normally of a dimension comprised between about 15 to about 50 nucleotides. The fragments, on the other hand, include also fragments that are larger than the oligonucleotides and probes, could be represented e.g. by amplicons and can span from the dimensions of the oligonucleotides and probes indicated above, to a length equal to the length of SEQ ID NO 1 or 12 minus 1 nucleotide.

According to a previously exemplified embodiment of the invention, the probes and oligonucleotides of the kit may be labelled with a fluorophore, e.g. with a fluorophore selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes.

The skilled person will understand that selection of further dies for labelling the probes and oligonucleotides falls within the normal skills of a technician skilled in molecular genetics.

The kit may also comprise the vector of the invention either as a the positive or negative control as described above, or may comprise both a positive and a negative control i.e. the positive control will comprise BN associated a vector encoding a sequence comprising phytoplasma's specific SNP and/or SNPs disclosed in the present description (i.e. from SEQ ID NO 1 , 5 or 7) and the negative control will comprise at the disclosed SNP or SNPs site/s a sequence of SEQ ID NO 12 wherein said sequence is not SEQ ID NO 1.

These controls will allow verifying the correct functioning of the kit or of the method herein described without further need of phytoplasma nucleic acids as controls.

In a further embodiment of the invention, monoclonal or polyclonal antibodies specifically recognising SEQ ID No 6 and/or 8 can be prepared according to standard techniques and even purchased by companies specialised in antibody preparation starting from SEQ ID No 6 and/or 8. The antibody can be produced by standard techniques The antibody can be produced according to any known standard technique such as described also in the monoclonal and polyclonal production related chapters in the manual "Basic Methods in Antibody Production and Characterization" edited by G.C. Howard and D.R. Bethell, ed., CRC Press, 2001 or in other commonly used laboratory manual and the identification of BN associated phytoplasma can be carried out by immunological assays on vine phloem samples where cell lysis has been carried out to expose rpl22-rps3 encoded ribosomal proteins of SEQ ID No 6 or 8 expressed inside the infected cell.

The present invention hence also encompasses a method for the detection of BN associated phytoplasma in a vine sample wherein a primary antibody specifically recognising SEQ ID NO 6 and/or 8 and a labelled secondary antibody specifically recognising the primary antibody are used in an immunodetection assay on a vine phloem sample wherein cell lysis has been carried out.

EXAMPLES

rp!22 and rps3 isolation

Total nucleic acids (TNA) were extracted as previously described (Angelini et al., 2001 ) from 1 g of fresh leaf of 6 grapevine samples collected in Oltrepo Pavese (PV). Detection of 'Ca. Phytoplasma solani' was carried out by means of amplification of 16S rDNA in nested polymerase chain reactions (PCRs) primed by primer pair P1/P7 (Deng & Hiruki, 1991 ) followed by 16Srl groupspecific primer pair R16F1/R16R1 (Lee et al., 1994). And subsequent Msel-restriction fragment length polymorphism (RFLP) assay of amplicons as previously described.

(Lee et al., 1998). 6 positive samples were used to obtain the nucleotide sequence of ribosomal genes rpl22-rps3 of 'Ca. Phytoplasma solani'. The amplification of rpl22-rps3 was carried out in nested polymerase chain reactions ) primed by primer pair rpL2F3/rp(l)R1A (Martini et al., 2007) followed by rpStolF2/rpStolR (Martini et al., 2007) Presence of PCR amplicons was verified by electrophoresis through 1 % agarose gels; DNAs extracted from phytoplasma

strains EY1 ('Ca. Phytoplasma ulmi', subgroup 16SrV-A), STOL (stolbur group, subgroup 16SrXII-A), and AY1

('Ca. Phytoplasma asteris', subgroup 16Srl-B) served as reference strains. These phytoplasmas were maintained in Madagascar periwinkle (Catharanthus roseus (L.) G. Don). Reaction mixtures containing DNA from healthy Madagascar periwinkle plants and reaction mixture without DNA template were used as negative controls., Amplicons from nested PCRs, of one BN phytoplasma sample were cloned in plasmid vector pCRIITOPO (Invitrogen, Carlsbad, CA, USA) and propagated in Escherichia coli as described (Shuman, 1994). Both strands of cloned inserts were sequenced to achieve at least 4_coverage per base position. DNA sequencing was performed in an ABI PRISM 377 automated DNA sequenze (Applied Biosystems). The nucleotide sequence data were assembled by employing the Contig Assembling program of the sequence analysis software BIOEDIT, version 7.0.0 (http://www.mbio.ncsu.edu/Bioedit/bioedit.html).

The size of the nucleotide fragment was 1 122bp and included rpl22 gene sequence and partial sequence of rps3 gene.

Diagnostic assay design

Sequenced amplicons were then compared with each other present in

GenBank in order to identify sequence variation that would allow identifying regions suitable for design of diagnostic assays.

In detail, we have identified one fragment of gene rps3 suitable to design an assay specific for BN on base of several SNPs identified. This nucleotide fragment was submitted in Primer Express software to design the Taqman assay.

We have identified one nucleotide fragment of 71 bp witch includes two primers and one probe suitable to detect BN phytoplasma (Tab2).

Table 2. List of primers and TaqMan® probe sequences used in RT PCR assay

Primer Nucleotide sequence Size (Bp) Tm °C

SEQ ID NO 2 F-701 TCAATTCATACCGCCAAACCA 21 59.8

SEQ ID NO 4 R-771 TGCGCTACTA I I I I ATTGCGAGTT 24 58.3

Probe Nucleotide sequence Size (Bp) Tm °C

SEQ ID NO 3 BN65 ATGATTATCGGTAAAGAG 18 65

Assay optimization Specificity and Sensitivity

Taqman PCR amplification was done in a 25 μΙ final volume including 5 μΙ of purified DNA as template. The PCR Master Mix included BN-701 (TaqMan® forward primer), BN-65 (TaqMan® probe) and BN-771 (TaqMan® reverse primer) and amplification conditions as follows: 94 °C for 10 min, 94 °C for 30 s, 60 °C for 1 min, and amplification of 40 cycles.

The optimal condition for primers and probe concentration was respectively 900nM (both primers) and 250 nM (probe).

We also tested the same assay with primers at 300nM and 600nM, nevertheless we have observed more sensibility of the assay when the concentration of primers was 900nM.

The verify the specificity of the Taqman® assay to "Ca. P. solani", DNA extracted from periwinkle infected by "Ca. P. asteris", "Ca. P.vitis" and "Ca. P. mali", and from healthy grapevine were used as negative controls.

In Fig. 2 is showed an amplification plot example.

We have compared BN65 RT PCR assay with PCR-endpoint/RFLP analysis. The PCR-endpoint was performed with primers pair R16P1/P7 (Deng and Hiruki, 1991 ) and R16(I)F1 -R1 (Lee et al., 1994), respectively used for direct and nested- PCR; to identify the taxonomic subgroup of Bois noir phytoplasma the amplicon R16(I)F1 -R1 (Lee et al., 1994) was digested by Msel. In Table 3 the results obtained have been reported.

Table 3. Comparison between BN65 RT PCR assays conducted using DNA template diluted 1 :10 (A) and diluted 1 :30 (B), PCR/RFLP analyses on leaf samples collected from diseased and healthy vines. (Ct-average represented the average between Ct value of A and B) B. PCR-endpoint/RFLP was performed on 16SrDNA.

Ct value

Sample

Type of sample n° A B Ct-average PCR /RFLP code

1 187 28,33 27,15 28 16SrXII-A

2 191 22,90 23,12 23 16SrXII-A

3 201 30,73 30,79 31 16SrXII-A

4 222 25,38 25,62 26 16SrXII-A

5 223 26,29 26,54 26 16SrXII-A

6 224 25,46 26,84 26 16SrXII-A

Grapevine leaf 7 225 27,71 31 ,31 30 16SrXII-A

8 226 26,47 28,70 28 16SrXII-A

9 227 26,02 28,19 27 16SrXII-A

10 228 26,48 28,75 28 16SrXII-A

1 1 229 27,74 31 ,06 29 16SrXII-A

13 250 24,80 26,51 26 16SrXII-A

14 270 29,52 32,02 31 16SrXII-A

Positive control STOL 18,00 16,00 17 16SrXII-A

Negative control AY negative negative negative 16Srl-A FD negative negative negative negative

AP negative negative negative negative

Hybridisation on microarray

The amplified genomic DNA is labelled with the "BioPrime® Total Genomic Labeling System" kit (INVITROGEN - 18097-012) following the manufacturer's instructions. The labelled sample is precipitated by Spin - Vacuum Savant and is subsequently re suspended in the hybridisation solution.

Microarray hybridisation:

Probes comprising one or more the SNPs region of interest have been blocked on the slide for the detection of the phytoplasma SNP(s) associated with BN disclosed in the present description, in 16 copies per probe. Before the hybridisation reaction, the activation of the slide shall be carried out (glass surface chemistry: EPOXY Surface Coating Slides; spotting buffer: Scott-Nexterion spotting buffer; probe's concentration: 30μΜ), by the use of a blocking solution (10x Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulfate (SDS), 0.066 Sodium Tetrahydridoborate (NaBH₄), H₂0 up to 50ml). The slide is treated with the blocking solution for 20 minutes to 42°C. Washings are carried out: twice (Sodium Saline Citrate 1 x for 5 minutes at room temperature), twice (Sodium Saline Citrate 0.1 x for 5 minutes at room temperature).

Prehybridisation:

A solution consisting of: 5x Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulfate, 200μg Salmon Sperm DNA, 5x Denhardt's Solution (5g Ficoll, 5g Polyvinyl pyrrolidone, 5g albumin of bovine serum, fraction V, H₂0 up to 500ml), H₂0 up to 2ml is prepared. The solution must be filtered by 0.2μηι filters. The slide area comprising the probes is delimited with a "frame" (Gene Frame 21x22mm and cover slips - AB1043 CELBIO). 1 10μΙ of prehybridisation solution are placed within this area and the slide is covered with the cover slip. The slide with the prehybridisation solution is incubated at 42°C for 2 hours.

Hybridisation:

A solution consisting of: 5X Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulfate, 25% Formamide, 200μg Salmon Sperm DNA, H₂0 up to 2ml is prepared. The solution must be filtered by 0.2μηι filters and preheated at 42°C. The sample of the amplified and labelled DNA is re suspended in about 1 10μΙ of hybridisation solution. The DNA sample, resuspended in hybridisation solution, is denatured at 95°C. The hybridisation solution is placed in the centre of the "frame" that defines the area comprising the target probes, and the cover slip is placed on this area. The slide is hence incubated to 42°C for 16 hours. Post-hybridisation washing:

1 ^st WASHING - in a volume of 50ml solution: 1 x Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulphate (SDS) for 5 minutes to 42°C;

2^nd WASHING - in a volume of 50 ml solution: 0.2x Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulphate (SDS) for 5 minutes to 42°C;

3^rd WASHING - in a volume of 50ml solution: 0.2x Sodium Saline Citrate (SSC) for 5 minutes to 42°C;

4^th WASHING - in a volume of 50ml solution: 0.2x Sodium Saline Citrate (SSC) for 5 minutes to 42°C.

The slide has been dried by centrifugation to 800 rpm for 5 minutes.

BIBLIOGRAPHY

- ANGELINI, E., D. CLAIR, M. BORGO, A. BERTACCINI, E. BOUDON- PADIEU, 2001 "Flavescence doree in France and Italy: occurrence of closely related phytoplasma isolates and their near relationships to palatinate grapevine yellows and an alder yellows phytoplasma." Vitis, 40, 79-86

- ANGELINI, E., BIANCHI G.L., FILIPPIN L, MORASSUTTI C, BORGO M.. 2007 "A new Taqman method for the identification of phytoplasmas associated to grapevine yellows by real time PCR assay." Journal of Microbiological Methods 6 8, 613-622.

- BOUDON-PADIEU, E., 2003. The situation of grapevine yellows and current research directions: Distribution, diversity, vectors, diffusion and control. Extended Abstracts 14th Meeting of the ICVG, September 12-17,2003 Locorotondo (Bari), Italy, 47-53.

- CAUDWELL A., 1957. Deux annees d'etudes sur la Flavescence doree, nouvelle maladie grave de la vigne. Annates d'Amelioration des Plantes 12: 359-393

- FIRRAO, G., et al., 2004. 'Candidatus Phytoplasma', a taxon for the wall- less, non-helical prokaryotes that colonize plant phloem and insects. International Journal of Systematic and Evolutionary Microbiology, 54, 1243-1255.

- GALETTO, L, BOSCO D., MARZACHI C, 2005. "Universal and group- specific real-time PCR diagnosis of flavescence doree (16Sr-V), bois noir (16Sr-XI I) and apple proliferation (16Sr-X) phytoplasmas from fieldcollected plant hosts and insect vectors" Annals of Applied Biology, 147, 191 -201

- HREN, M., et al 2007. Real-time PCR detection systems for Flavescence doree and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics. Plant Pathology 56, 785-796.

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- LEE I.M.; MARTINI M., MARCONE C, ZHU S.F. 2004. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of 'Candidatus Phytoplasma ulmi' for the phytoplasma associated with elm yellows". International Journal of Systematic and Evolutionary Microbiology, 54, 337-347.

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Microbiol., 54, 221 -255. - MARGARIA P., TURINA M. and. PALMANO S., 2009. Detection of Flavescence doree e and Bois noir phytoplasmas, Grapevine leafroll associated virus-1 and -3 and Grapevine virus A from the same crude extract by reverse transcription-RealTime Taqman assays. Plant pathology 5 8, 838-845.

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Claims

1 . A nucleotide sequence of SEQ ID NO 1 , or the sequence complementary to SEQ ID NO 1 , comprising SEQ ID NO 5 and/or SEQ ID NO 7, or their complementary sequences, wherein SEQ ID NO 5 and SEQ ID NO 7 coding, respectively, for the rpl22 and rps3 genes of the phytoplasma associated with the Bois Noir vine disease.

2. A nucleotide sequence of SEQ ID NO 1 or the sequence complementary to SEQ ID NO 1 , or fragments thereof, said fragments comprising one or more of the nucleotides of Table 1 or of their complementary nucleotides, or said fragments being suitable for amplification of a fragment of SEQ ID NO 1 , or the sequence complementary to SEQ ID NO 1 comprising one or more of the nucleotides of Table 1 , or of their complementary nucleotides, wherein said sequence or fragments thereof enable for a specific identification for the plant phytoplasma strains associated with the Bois Noir vine disease.

3. An amino acid sequence of SEQ ID NO 6 or 8 coding for one or both of the rpl22 and rps3 genes of the phytoplasma associated with the Bois Noir vine disease.

4. The nucleotide sequence or fragments thereof of claims 1 or 2 or SEQ ID NO 12 as a diagnostic tool for the identification of the presence in a vine sample, of the phytoplasma associated with the vine disease Bois Noir.

5. A vector comprising a nucleotide sequence of SEQ ID NO 1 , 5, 7 or 12 or the sequence complementary to SEQ ID NO 1 , 5, 7 or 12 or fragments thereof, said fragments comprising one or more nucleotides in the position indicated in Table 1 or of their complementary nucleotides.

6. A method for the diagnosis of the vine disease Bois Noir comprising the step of

a) identifying one or more of the nucleotides in the position indicated in Table 1 in SEQ ID NO 12 or a fragment thereof, and/or one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12 or a fragment thereof, in a plant sample comprising phloem;

wherein the presence of one or more of the residues of Table 1 in the respective position indicated in Table 1 in SEQ ID NO 12 and/or of its complement in the sequence complementary to SEQ ID NO 12 indicates the presence of the phytoplasma strain associated with the vine disease Bois Noir in the sample.

7. The method of claim 6 wherein the identification of step a) comprises a first step wherein a PCR, Real Time PCR, Ligation, Allele Specific Hybridization, primer extension, invasive cleavage or sequencing reaction is carried out and a second step wherein the product obtained in the first step is detected by monitoring the light emitted by said product, or measuring the mass of said products or monitoring the radioactivity emitted by said product or by sequencing said product.

8. The method of any one of claims 6 or 7 wherein said monitoring of the light emission is carried out by monitoring fluorescence, luminescence, time resolved fluorescence, fluorescence resonance energy transfer, or fluorescence polarisation, said measuring of the mass is carried out by mass spectrometry or heteroduplex analysis and said monitoring of the radioactivity is carried out with radioactivity sensitive tools.

9. The method of any one of claims 6-8 wherein said method is carried out on solid phase format such as microarray or microplate.

10. The method of any one of claims 6-8 wherein said identification is carried out with a RT-PCR using a forward primer of SEQ ID NO 2, an allele specific probe of SEQ ID NO 3 and a reverse primer of SEQ ID NO 4.

1 1 . A kit for the diagnosis of the vine disease Bois Noir comprising reagents for the identification of one or more of the nucleotides of Table 1 in SEQ ID NO 1 or 12 and/or in a fragment thereof and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 or 12 and/or in a fragment thereof in a vine plant phloem.

12. The kit of claim 1 1 further comprising solid supports wherein oligonucleotides for the identification of one or more of the nucleotides of Table 1 in SEQ ID NO 1 or 12 and/or in a fragment thereof and/or one or more of their complementary nucleotide in the sequence complementary to SEQ ID NO 1 or 12 and/or in a fragment thereof are anchored in known positions on said support in one or more copy per position.

13. The kit of claim 12 further comprising labelled probes and/or oligonucleotides corresponding and/or complementary to a region upstream and/or downstream of one or more of the nucleotides of Table 1 in SEQ ID NO 1 or 12 and/or in a fragment thereof or and/or overlapping a region comprising one or more of the nucleotides of Table 1 in SEQ ID NO 1 or 12 and/or in a fragment thereof.

14. The kit of claim 13 wherein one or more of said probes and/or oligonucleotides are labelled.