WO1994026929A1 - Nucleotide sequences characteristic of erwinia carotovora - Google Patents

Abstract

The present invention relates to nucleotide sequences characteristic of the genome of bacteria of the species in Erwinia carotovora, which are responsible for pathologies such as black leg of the potato. The invention also relates to the utilisation of said nucleotide sequences for implementing methods for the identification and detection of Erwinia carotovora and diagnosis of pathologies caused by said bacteria.

Description

CHARACTERISTIC NUCLEOTIDE SEQUENCES OF ERWINIA CAROTOVORA.

The subject of the present invention is nucleotide sequences derived from the genome of bacteria of the species Erwinia carotovora, and more particularly Erwinia carotovora pathogenic in certain plants, these sequences being usable, in particular as probes and primers, for the detection of these bacteria in hosts likely to be infected with them, or in any environment where these bacteria are likely to survive.

A 1988 study indicated that global losses from plant diseases commonly amount to $ 60 billion a year. This shows the importance of rapid and specific detection of phytopathogenic agents, which should condition cultural practices and the choice of phytosanitary products, their dosage, the time of their application, and to avoid the aggression of the environment.

Prophylactic measures, therefore requiring rapid, sensitive and reliable detection, still constitute one of the most effective and least costly means, both financially and in terms of the environment, of combating diseases, and in particular against bacteriosis for which few means of fight are available, the use of antibiotics being prohibited in the majority of countries. These prophylactic measures essentially consist in producing material, in particular of propagation, healthy, and possibly in destroying the contaminated batches, in sterilization or any other disinfection process of all the materials, premises and culture substrates likely to be in contact with plants and seeds, choosing healthy production locations.

Screening for pathologies of bacterial origin in plants has however not been satisfactory so far, and significant losses of mono- and di-cotyledonous plants are frequently observed in the field, in greenhouses or during their storage.

One of the main causes of this problem is the use of seeds, in the broad sense, apparently healthy, but which are in reality carriers of bacteria likely to be at the origin of the future development of pathologies in plants obtained from these seeds. We will then speak of bacteriosis in the latent phase of infection. Many plant pathologies are characterized by a latent phase of infection. During this phase, the level of infection is generally lower than that which could be detected by visual inspection (absence of apparent symptoms) or by current techniques currently available (isolation and microbiological characterization, indexing, immunology, etc.).

Another important source of plant contamination would also come from the natural or artificial environment, including soil and water.

It should also be noted that isolation and microbiological characterization are generally long and laborious techniques, which may be too costly to consider applying them on a large scale, while immunological methods do not always present the specificity and sensitivity desired.

Pectinolytic bacteria of the genus Erwinia (also referred to below as erwinias) are responsible for a large number of pathologies of this type, one of the symptoms of which is the maceration of tissues in a large number of plants and seeds. These bacteria are particularly capable of infecting potatoes, corn, tomatoes, sugar beets, cabbage, chicory, etc., as well as greenhouse plants, such as ornamental plants which are commonly sold. between countries.

Among these erwinias, one can cite mainly on the one hand Erwinia carotovora, and more particularly Erwinia carotovora subsp. carotovora (Ecc), Erwinia carotovora subsp. atroseptica (Eca), Erwinia carotovora subsp. odorifera (Eco), Erwinia carotovora subsp. betavasculorum (Ecb), and Erwinia carotovora subsp. wasabiae, and, on the other hand, Erwinia chrysanthemi (Ech),

.Erwinia cactecida ...

These bacteria produce several extracellular enzymes capable of attacking the components of the cell wall; pectinases, cellulases, hemicellulases and proteases (for a detailed review, see the article by A. Kotoujansky published in Ann. Rev. Phytopathol. 1987, 25: 405-30).

Erwinia carotovora has been particularly studied because of its pathogenic power on several plants, and especially in potatoes (Pérombelon, 1989). The Erwinia carotovora (Ec) species is currently divided into five subspecies: atroseptica (Eca), carotovora (Ecc), odorifera (Eco) and wasabiae (Ecw) previously mentioned, as well as the betavasculorum subspecies (Ecb).

This classification was carried out on the basis of physiological and biochemical characteristics, as well as their pathogenic power. Eca and Ecb appear relatively close taxonomically. Ecb is responsible for beet rot, known as root necrosis (Thomson et al., 1981). The action of Eca is generally limited to the potato in cool temperate climates, at temperatures between about 18 ° C and about 20 ° C (Pérombelon, 1980). Some strains, not isolated from the potato, have been described as "atypical" Eca due to the fact that they have some particular physiological characteristics, such as their capacity to develop at 37 ° C (Lopez et al., 1989). A new subspecies, called odorifera (Eco) has recently been described as representing "atypical" strains which are pathogenic at least on endive and produce volatile odorous metabolites (Gallois et al., 1992). Other "atypical" Eca strains have been identified as Ecc strains on the basis of phenotypic and genotypic characteristics (S. Priou, ENSAR thesis, Rennes, 1992). Conversely, Ecc is widely distributed in the world and has a large specificity of hosts (Pérombelon and Kelman 1987,

Smith and Bartz 1990).

The main pathologies of potato caused by these bacteria, and more particularly by Eca, are as follows: failure to emerge, seedling loss, black leg, aerial rot of the stem, drying and rot of the tubers, and aerial wilt (Pérombelon and Kelman

1987). Pathologies corresponding to non-emergence and black leg occur mainly from mother tubers and can cause significant losses in the fields. Eca is considered the typical agent of the black leg in Europe because of its pathogenicity at low temperatures, and its ability to induce the disease early in vegetation, which increases losses. Most Ecc strains cannot produce typical black leg symptoms at low temperatures. Ecc is seen as an opportunistic agent rather than a primary causative agent of the disease. However, Ecc has an increased pathogenicity at temperatures greater than or equal to about 25 ° C and is the primary causative agent of the black leg of the potato in Arizona and Colorado (Stanghellini and Meneley, 1975).

As we have seen previously, the control of these bacterial pathologies is essentially based on prophylactic measures, and the means of detection are therefore particularly important (Lelliott and Stead,

1987). The aim of the present invention is to provide nucleotide sequences making it possible to differentiate the strains (or groups of strains) of the Erwinia carotovora species between them.

Another object of the invention is to provide nucleotide sequences capable of differentiating the Erwinia carotovora subspecies from each other.

Another object of the present invention is to differentiate the pathogenic strains of Erwinia carotovora from those which are not pathogenic, whether or not these strains belong to the same subspecies of Erwinia carotovora. The present invention also aims to provide reliable methods for specific determination of the presence of pathogenic Erwinia carotovora strains in a host (in particular plants or seeds) capable of carrying such bacteria, or even in the soil and the soil. 'water.

The present invention aims to provide a reliable method for specific determination of the presence of Erwinia carotovora subsp. atroseptica (Eca), and / or Erwinia carotovora subsp. betavasculorum (Ecb), in a host as described above.

The present invention more specifically aims to make available to potential users, reliable methods of early diagnosis of pathologies caused by bacteria of the species Erwinia carotovora, and more particularly of the black leg of the potato caused. by Eca, or root necrosis caused by Ecb, namely methods making it possible to diagnose the presence of these bacteria and therefore the risk of occurrence of these pathologies, or to identify the bacterial strains responsible for these pathologies. The object of the invention is more precisely to provide new nucleotide sequences which can be used as probes or primers for the implementation of these diagnostic methods, in particular by the techniques proceeding by hybridization between nucleic acids (RNA and / or DNA), optionally followed by amplification of the number of copies of target sequences to be detected, for example according to gene amplification techniques called PCR (Polymerase

Chain Reaction), LCR (Ligase Chain Reaction) and 3SR (Self-Sustained Sequence Replication).

The invention also aims to make available to potential users, diagnostic kits for implementing the above-mentioned diagnostic and identification methods.

The invention is illustrated with the aid of FIGS. 1 to 6 respectively representing the nucleotide sequences A to F described below. The subject of the present invention is nucleotide sequences comprising all or part of the sequences as obtained by genomic subtraction between two different strains of Erwinia carotovora, or comprising all or part of the sequences bordering the latter on the genome of the Erwinia strain carotovora from which the sequences obtained by genomic subtraction are derived, said nucleotide sequences being capable of hybridizing with the genome (or more precisely part of the genome) of at least one bacterial strain belonging to the species Erwinia carotovora, and not being able to not hybridize with the genome of at least one strain belonging to this same species.

The abovementioned bordering sequences are advantageously obtained from the sequences described above themselves obtained by genomic subtraction (for example using the sequences A to F according to the invention), in particular according to the methods described below. A more particular subject of the invention is the nucleotide sequences as described above, characterized in that they are capable of hybridizing with the genome of at least one bacterial strain belonging to a determined subspecies of Erwinia carotovora, but not with that of at least one strain belonging to a subspecies of Erwinia carotovora different from the previous one, or capable of hybridizing with the genome of at least one strain belonging to a given subspecies d 'Erwinia carotovora, but not with that of at least one strain belonging to this same subspecies.

The invention also relates to the nucleotide sequences as described above, characterized in that they are capable of hybridizing with the genome of at least one pathogenic bacterial strain in a given plant, but not with that of at minus a non-pathogenic strain in this same plant, this non-pathogenic strain belonging or not belonging to the same subspecies of Erwinia carotovora as the previous pathogenic strain.

It should be noted that the term non-pathogenic strain is understood, in the foregoing and what follows, as opposed to a pathogenic strain, any strain not being capable of inducing the appearance of a pathology, and more particularly of a type of rot, in a given plant, under conventional conditions (and the current state of knowledge in this field), in particular climatic and geographic, in which said aforementioned pathogenic strain is capable of inducing the appearance of pathology in this plant.

For example, the subspecies Eca can be characterized, in the current state of knowledge, by its ability to cause a symptom known as black leg on potato stem, between 18 and 22 ° C, and in all cases at a temperature below 25 ° C, the ambient relative humidity being sufficient.

The genomic subtraction described above between the two strains of Erwinia carotovora is carried out as follows: - the DNA of a first strain of Erwinia carotovora, or target DNA, is cut into small fragments, of approximately 100 at around 1,000 nucleotides, in particular by the action of restriction enzymes,

- the DNA of a second strain of Erwinia carotovora, or trap DNA, is cut into fragments, of approximately 1,000 to approximately 10,000 nucleotides, in particular by sonication, these fragments being subsequently labeled,

the abovementioned fragments of target DNA and of trap DNA are mixed in proportions such that the trap DNA is in excess relative to the target DNA,

the target DNA and trap fragments thus mixed are denatured by heating, which leads to the formation of target DNA fragments and single strand trap,

- then the single-stranded DNA fragments obtained in the previous step are placed under hybridization conditions such that they allow the formation of double-stranded DNA fragments consisting of two strands of target DNA (target homoduplex), or of two strands of trap DNA (homoduplex trap), or of a strand of target DNA and of a strand of trap DNA (heteroduplex),

the double-stranded DNA fragments obtained in the previous hybridization step are then placed in the presence of a reagent having an affinity for the abovementioned marker, this reagent being immobilized on a solid support so that the homoduplex traps and the labeled heteroduplexes remain fixed on the solid support by bonding between the marker used to mark the trap DNA and the abovementioned reagent,

- recovery, in particular by filtration or sedimentation of the solid support, of the unlabeled target homoduplexes, and therefore remaining in solution, constituting the nucleotide sequences contained in the genome of the first strain of Erwinia carotovora, but not in the genome of the second strain , and therefore likely to hybridize with the genome of the first strain, but not with that of the second strain.

According to one embodiment of the genomic subtraction described above, the first strain of Erwinia carotovora is a pathogenic strain in a given plant, while the second strain of Erwinia carotovora is not pathogenic, more particularly in the same conditions (of culture, climatic, etc.) than for the previous strain, in this same plant.

According to another embodiment of the aforementioned genomic subtraction method, the pathogenic strain of Erwinia carotovora is a strain of Erwinia carotovora subsp. atroseptica (Eca) responsible for rots in certain plants, in particular the black leg of the potato, and the non-pathogenic Erwinia carotovora strain is a strain of Erwinia carotovora subsp. carotovora (Ecc) does not induce rots (and more particularly of the characteristic black leg type) in these same plants. A more particular subject of the invention is the nucleotide sequences as described above, and characterized in that they correspond to one of the nucleotide sequences A (SEQ ID NO 1), B (SEQ ID NO 2), C (SEQ ID NO 4), D (SEQ ID NO 5), E (SEQ ID NO 6), F (SEQ ID NO 7), shown in Figures 1 to 6 respectively, as well as the sequence B 'represented by SEQ ID NO 3, or their complementary sequences, or fragments of these sequences, or any of the above-mentioned sequences modified by substitution and / or addition and / or deletion of one or more nucleotides, said modified fragments or sequences being capable, like the sequences A to F mentioned above, to hybridize with the genome of at least one strain of Erwinia carotovora, in particular a pathogenic strain, and which cannot hybridize with the genome of at least one strain of Erwinia carotovora, including a non-pathogenic strain.

The invention relates more particularly to the sequences corresponding to the nucleotide sequences B and B 'represented respectively by SEQ ID NO 2 and SEQ ID NO 3, or to their complementary sequences, or to fragments of these sequences, or to all of the above-mentioned sequences modified by substitution and / or addition and / or deletion of one or more nucleotides, said fragments or modified sequences being capable, like the sequences B and B 'above, of remaining hybridized: - with the genome of the strains of Eca, in particular pathogenic , as well as with the genome of Erwinia carotovora subsp. betavasculorum (Ecb), in particular the pathogenic strains of Ecb, in particular under the hybridization conditions defined below: hybridization in 6 SSC, 0.1% SDS and 0.01% of skimmed milk at 65 ° C overnight , followed more particularly by two washes of 30 minutes at 65 ° C, 0.3 SSC, 0.5% SDS,

- only with the genome of Erwinia carotovora subsp. atroseptica (Eca), in particular the pathogenic strains of Eca, in particular under the following hybridization conditions: hybridization in 6 SSC, 0.1% SDS and 0.01% skimmed milk at 65 ° C overnight, followed more particularly by two 30-minute washes at 68 ° C, 0.1 SSC, 0.5% SDS.

By sequences bordering those resulting from the implementation of a genomic subtraction process in the above, is meant all sequences as obtained by:

1) cloning the genome of the bacterial strain used in the genomic subtraction method, and from which the sequences obtained by genomic subtraction as described above are derived, in particular from the pathogenic strain, the cloning being carried out by fragmentation of the genome of this strain, followed by the introduction of the fragments thus obtained into suitable cloning vectors (such as plasmids), the latter being cultured in an appropriate host cell (in particular in bacteria such as Escherichia coli),

2) recovery of the host cells thus transformed, lysis of these cells, and denaturation of the DNA of these cells,

3) hybridization, in particular under the conditions indicated above, of the abovementioned DNA thus denatured with a labeled probe, in particular in the manner indicated below, this probe consisting of all or part of a denatured nucleotide sequence obtained by genomic subtraction as described above, in particular with all or part of the sequences A to F, and more particularly of the sequences B and B 'mentioned above,

4) selection of the host cells whose denatured DNA hybridizes with said probe, lysis of these cells, recovery of the DNA fragments of the bacterial strain whose genome was cloned in step 1) and introduced into their genome by the aforementioned vectors, in particular using appropriate restriction enzymes, and purification of these DNA fragments,

5) recovery of the DNA fragments obtained in step 4) above, or parts of these DNA fragments, which do not hybridize with the genome of the strain used in the genomic subtraction method and do not contain the sequences obtained by genomic subtraction as described above, in particular with the genome of the non-pathogenic strain, and hybridizing with the genome of the strain from which said sequences obtained by genomic subtraction originate, these DNA fragments representing a bordering sequence of the nucleotide sequence obtained by genomic subtraction and used as a probe during the above-mentioned hybridization step 2) with the denatured DNA of the above-mentioned host cells,

6) repeating the previous steps using as a probe during the aforementioned hybridization step 2) with the denatured DNA of the above host cells, the bordering sequence obtained after each of these repetitions of steps, until DNA fragments hybridizing with the genome of the two bacterial strains used in the aforementioned genomic subtraction process are obtained, these latter fragments of DNA no longer representing bordering sequences but sequences common to the two abovementioned strains.

Such a method of obtaining bordering sequences according to the invention is also designated by the expression "walking on the chromosome" used conventionally in this technical field. The bordering sequences according to the invention can also be obtained by lengthening the sequences A to F, or those mentioned above obtained by genomic subtraction, after hybridization of the latter to DNA fragments obtained during step 1) of cloning the aforementioned method, this extension being carried out by nucleotide synthesis (from the 3 ′ side and / or from the 5 ′ side of these sequences) proceeding by pairing of the bases complementary to those appearing on the strands of said genome fragments, with the latter.

By way of illustration, the term “modified sequence” in the foregoing, and below, means in particular any sequence comprising nucleotides such as uracil, inosine, 7 deaza 2 ′ deoxyguanosine triphosphate (cdGTP) etc., and corresponding therefore either to a DNA or to a modified RNA.

Preferably, the modified sequences according to the invention have at least about 60% identity with all or part of the above-mentioned nucleotide sequences obtained by genomic subtraction, and more particularly with sequences B and B ′, or with all or part of the bordering sequences mentioned above.

The invention relates more particularly to the abovementioned nucleotide sequences in marked form, in particular radioactive, enzymatically, by a fluorescent compound, by a bond to an antigenic molecule (capable of being recognized by antibodies) or to any other molecule capable of '' be directly or indirectly detected using reagents (or even radiation), this linkage possibly being possible via a spacer arm, in particular via a nucleotide sequence consisting of approximately 5 at 100 nucleotides located at least at one of the ends of these sequences. As examples of directly or indirectly detectable molecules linked to the abovementioned nucleotide sequences, mention may be made of acetylaminofluorene, biotin (detected using avidin, streptavidin) or even digoxigenin. The nucleotide sequences according to the invention are advantageously used as nucleotide probes, where appropriate labeled, in particular in the manner indicated above, more particularly for the detection of strains of pathogenic Erwinia carotovora, and more particularly strains of Eca responsible for the black leg, or Ecb strains responsible for the root necrosis of sugar beet. Like the nucleotide sequences mentioned above, the probes of the invention are capable of hybridizing (in particular under the specific washing conditions defined above) with nucleotide sequences contained in the genome of pathogenic Erwinia carotovora, and more particularly Eca and pathogenic Ecb (the latter sequences being also designated in the foregoing and that which follows, by the expression "specific nucleotide sequences" or "specific sequences"), these specific nucleotide sequences not being contained in the genome of Erwinia carotovora not pathogens, and more particularly in the genome of Erwinia carotovora other than Eca or

Pathogenic Ecb.

Advantageously, the probes of the invention consist of sequences of approximately 7 to approximately 300 nucleotides, chosen from within the nucleotide sequences B and B 'mentioned above. Fragments of the abovementioned nucleotide sequences of the invention can advantageously be used in pairs to form what will be called in the remainder of this text pairs of primers, these pairs of primers being used for the amplification of the number of copies of the abovementioned nucleotide sequences, or of the specific nucleotide sequences contained in the genome of the pathogenic strains of Erwinia carotovora, and more particularly of the strains of Eca responsible for the black leg, or of the strains of Ecb responsible for root necrosis.

These primers allow the amplification (or even the multiplication) of the number of copies of all or part of the specific nucleotide sequences of the genomes of pathogenic Erwinia carotovora, in particular according to the technique

PCR which will be detailed below, these primers being where appropriate marked, in particular in the manner indicated above.

Advantageously, the primers according to the invention consist of approximately 5 to approximately 100 nucleotides, and preferably of approximately 15 to approximately 30 nucleotides, and are capable of hybridizing with a region of a nucleotide sequence according to the invention , or a nucleotide sequence according to the invention, or a nucleotide sequence specific for the genome of Erwinia carotovora, and more particularly of Eca and Ecb, this region being different according to the primers within the same pair, so as to allow the amplification of all or part of the above-mentioned nucleotide sequences. These primers can for example be either common to Eca and Ecb, or different depending on the subspecies. These primers advantageously consist of approximately 7 to 30 nucleotides derived from the abovementioned nucleotide sequences of the invention, and more particularly from the sequences B and B 'described above, and in particular allow the amplification of fragments from approximately 100 to approximately 300 nucleotides contained in specific nucleotide sequences of the Eca responsible for the black leg.

The above-mentioned pairs of primers are chosen so that they hybridize on the 3 ′ side of each of the strands of the abovementioned nucleotide sequences, the 3 ′ ends of each of the primers being directed towards the inside of the sequences to be amplified ( according to the classic PCR technique). A pair of primers from the above-mentioned sequence B, and particularly preferred in the context of the present invention, is such that the two primers constituting it correspond to the following formulas: B105 5 'TAAGGCAGCATTGAATCGCCACAGAGAGTA 3' (SEQ ID NO 8) B106 5 'ACACGCATTTATCGCTATCAGGCACAACAA 3' (SEQ ID NO 9) The primers B105 and B 106 are capable of specifically hybridizing with the genome of the Eca and / or Ecb mentioned above, for example according to the following steps:

- predenaturation by heating for 2 min at 94 ° C, followed by 35 cycles of the following three stages:. heating for 30 s at 92 ° C,

. heating for 30 s at 65 ° C (hybridization temperature),. heating for 45 s at 72 ° C. We thus obtain the amplification of a fragment of 151 base pairs of the genome of Eca and Ecb. Other pairs of primers from the above-mentioned sequence B, which can be used in the context of the present invention, are the following:

- Bl (SEQ ID NO 10) / B44 (SEQ ID NO 11),

- B201 (SEQ ID NO 12) / B202 (SEQ ID NO 13),

- B203 (SEQ ID NO 14) / B204 (SEQ ID NO 15). The subject of the invention is also any method of detecting and identifying strains of pathogenic Erwinia carotovora possibly present in soil or water, or even in a host, in particular in plants and / or seeds, capable of be a seemingly healthy carrier of such bacteria and being in the latent infection phase, this process comprising the following stages:

- the treatment of a sample taken from the soil or water or from this host so as to make the genome of these Erwinia carotovora accessible to the probes and / or primers defined above (that is to say so that the DNA of these bacteria is single-stranded), and where appropriate to any DNA or RNA polymerase making it possible to replicate the two strands of genomic DNA or the RNA derived therefrom, this treatment being in particular carried out by boiling, maceration (in a liquid such as water), grinding, sonication, in particular in the presence of detergent (for example laurylsulfate, Sodium Dodecyl Sulfate,

Triton X100), antioxidant, chelators, alkali, and / or compounds eliminating inhibitors such as polyphenols (polyvinylpyrrolidone for example), or polysaccharides (cetyltrimethylammonium bromide for example), - contacting the sample thus treated with probes, and / or with primers according to the invention,

- if necessary, the amplification using pairs of primers according to the invention, of the number of copies of the nucleotide sequences specific for pathogenic Erwinia carotovora, and contained in the genome of these bacteria likely to be present in this biological sample, and with which the abovementioned probes or primers are likely to hybridize, and / or the amplification of the number of copies of probes according to the invention,

- detecting the possible presence of said specific nucleotide sequences contained in the genomes of pathogenic Erwinia carotovora, and therefore the presence of pathogenic Erwinia carotovora responsible for this pathology in the sample studied.

When the method described above is carried out without amplification of the number of nucleotide sequences specific for pathogenic Erwinia carotovora, the above-mentioned detection step can be carried out using the aforementioned probes, advantageously labeled, said specific sequences being hybridized with the probes mentioned above.

When the method described above is carried out with amplification of the number of said specific sequences, the above-mentioned detection step can be carried out either directly by visualization of the amplification of said specific sequences (for example by visualization of a large mass on gel of electrophoresis corresponding to the amplification carried out, as detailed below), when the primers used are marked, either by hybridization of said sequences thus amplified using the aforementioned labeled probes.

A more particular subject of the invention is any method of identifying and detecting pathogenic Eca and / or Ecb which may be present in a host, in particular in plants and seeds, this method being carried out in the manner indicated above, using all or part of a probe consisting of the nucleotide sequence B or B 'mentioned above, optionally labeled, in particular as indicated above, and / or using the pair of primers B105 / B106 above, where appropriate marked, in particular in the manner indicated above.

Advantageously, the specific detection of Eca is carried out by hybridization, in particular on the membrane, of said specific sequences, amplified or not, with all or part of the probe B or B 'under the following hybridization conditions: hybridization in 6 SSC, 0.1 % SDS and 0.01% skimmed milk at 65 ° C overnight, followed more particularly by two washes of 30 minutes at 68 ° C, 0.1 SSC, 0.5% SDS.

The specific detection of Eca and / or Ecb is carried out either by hybridization of said specific sequences, amplified or not, with all or part of the probe B or B 'under the following hybridization conditions: hybridization in 6 SSC, 0, 1% SDS and 0.01% skimmed milk at 65 ° C overnight, followed more particularly by two washes of 30 minutes at 65 ° C, 0.3 SSC, 0.5% SDS, either directly by viewing the amplification of said specific sequences using primers B105 and B106 labeled.

The identification and detection methods of the invention can be carried out from a host showing pathological signs, or advantageously from a host capable of being an apparently healthy carrier of such bacteria and being in phase latent infection.

The methods for identifying and detecting Erwinia carotovora, and more particularly Eca or Ecb, according to the invention can also be carried out from any medium in which these bacteria are capable of surviving, in particular in water or soil .

Preferably, the identification methods according to the invention comprise a step of amplifying the number of copies of all or part of the specific nucleotide sequences contained in the genomes of the pathogenic Erwinia carotovora, or of the corresponding RNAs.

This gene amplification step can be carried out in vitro or in vivo by any method using conventional techniques of enzymatic amplification of DNA or TARN, such as the LCR (Ligase Chain Reaction) technique. described in particular in the journal PNAS, USA, 88, pp. 189-193 (1991), or the "Qβ Replicase" technique described in the journal Biotechnology (Vol. 6, October 1988), or advantageously the PCR technique as described in particular in the European patent applications of Cetus (n ° 200 362 , 201 184, 229 701 and 258 017), or the 3SR technique described by Fahy et al. in the PCR review

Meth Appl 1, 25-33 (1991), or techniques derived therefrom and any method for amplifying nucleic acids in vitro.

According to a preferred embodiment of the method described above according to the invention, the amplification by PCR technique of the number of copies of all or part of the nucleotide sequences contained in the genomes of Erwinia carotovora and / or the amplification of the number of copies of all or part of the probes defined above, includes the following steps:

- the predenaturation of double-stranded DNA into single-stranded DNA, preferably in a buffer consisting of 10 mM Tris-HCl pH 8.3, 50 raM KC1, 1.5 mM MgCl2, 0.01% gelatin, cofactors of DNA (or RNA) - polymerase, in particular of Mg2 + and K ⁺ ions, of the 4 deoxynucleotides constituting DNA (dCTP, dATP, dGTP, dTTP), or RNA (dCTP, dUTP, dGTP, dTTP), and pairs of primers as defined above, by heating between approximately 80 ° C. and approximately 110 ° C., advantageously at 100 ° C., - the amplification proper by addition to the medium obtained in the preceding stage of Heat-resistant DNA or RNA polymerase or heat-resistant ligase (in the case where these enzymes were not initially present in the aforementioned predenaturation buffer), and

* heating (in particular for approximately 1 minute) to approximately 94 ° C., which corresponds to the denaturation stage proper,

* then heating (in particular for approximately 1 minute) between approximately 35 ° C. and approximately 76 ° C., which corresponds to the stage of hybridization of the pairs of primers with the abovementioned sequences or probes,

* and finally heating (in particular for approximately 1 minute) between approximately 50 ° C. and approximately 76 ° C., and advantageously at 72 ° C., which corresponds to the step of elongation (or elongation) of the primers, hybridized to the previous step, one towards the other, thus producing nucleotide sequences complementary to all or part of the genomic sequences of the Erwinia carotovora or of the abovementioned probes, these sequences or probes being delimited by the nucleotides hybridizing with the primers above,

- the repetition of the previous amplification step between approximately 20 and approximately 50 times, advantageously between 25 and 40 times, the entire procedure can be repeated one or more times from all or part of the medium obtained in the previous step.

According to a preferred embodiment of the invention, the sensitivity of the detection can be improved by continuing the method described above by an amplification of the "nested PCR" type, described in "PCR A Practical Approach" edited by MJ McPherson, P. Quirke, GR Taylor, (1991) Oxford University Press, using primers internal to the previously amplified fragment, which leads to the amplification of the copy number of a DNA or RNA fragment shorter than that whose number of copies was initially increased. It goes without saying that all the time intervals specified in the methods detailed above, can vary among others depending on the apparatus (called cycler or thermocycler) used.

It also goes without saying that if other amplification methods than that by PCR and / or other enzymes are used, the appropriate amplification conditions (buffer, temperature, etc.) will be chosen.

In the case where the identification and detection method of the invention is carried out simply using probes as described above, the detection of the specific nucleotide sequences is advantageously carried out as follows: - fixation on a solid support (such as a membrane or wells of microtitration plates such as those commonly used in the so-called ELIS A technique, and adapted or not to nucleic acids) of a nucleotide sequence according to the invention, designated below " trap sequence ", with which the above specific nucleotide sequences, or fragments of these sequences, are capable of hybridizing, in particular under the conditions defined above, this fixation being carried out according to usual techniques described in particular by Maniatis et al, 1982 , and by Ausubel et al., 1987,

- rinsing of the solid support,

saturation of the free reactive sites of the support, incubation of the trapped nucleotide sequences, thus fixed, with the genomic DNA or RNA originating from the biological sample previously treated in the manner indicated above, under conditions allowing the hybridization of the specific nucleotide sequences with said trap sequences, in particular under the hybridization conditions defined above, - rinsing of the solid support, optionally using 3 x SSC at 65 ° C (or

0.1 x SSC if necessary, at 68 ° C. for 15 ran twice, or even under less stringent conditions depending on the length of the oligonucleotides used as probes, incubation of the specific sequences hybridized in the preceding step with the trap sequences, with one or more probes according to the invention, under conditions allowing the hybridization of the specific sequences mentioned above with said probes, in particular under the hybridization conditions defined more top, these probes being chosen so that they are not complementary to the abovementioned trap sequences,

- rinsing of the solid support, in particular using 3 x SSC (or 0.1 x SSC if necessary, at 68 ° C) at 65 ° C for 15 min twice, or even under less stringent conditions depending the length of the oligonucleotides used as probes,

- Detection of any probes having remained fixed on the solid support after the previous step, thus testifying to the presence of specific sequences, or fragments of these sequences in the biological sample, this detection being carried out either directly when the aforementioned probes are radioactively or fluorescently labeled, either indirectly by means of reagents themselves capable of recognizing these probes, in particular using the antibodies or other reagents described above, in the case where these probes are labeled with antigens or molecules capable of being recognized by these antibodies or reagents, respectively. The detection of specific nucleotide sequences which may be present in the sample studied can also be carried out in the following manner:

- fixation on a solid support of the genomic DNA or RNA coming from the sample previously treated in the manner indicated above, - rinsing of the solid support,

- saturation of the free reactive sites of the support,

incubation of the DNA or RNA thus fixed with a probe, according to the invention, this incubation being carried out under conditions allowing the hybridization of the probes with the genomic material fixed on the solid support, in particular under the defined hybridization conditions upper,

- rinsing of the solid support, in particular using 3xSSC (or 0, lxSSC if necessary at 68 ° C) at 65 ° C for 15 min twice,

- detection of any probes having remained fixed on the solid support after the previous step, thus testifying to the presence of specific sequences, or fragments of these sequences in the biological sample, this detection being carried out either directly when the above-mentioned probes are marked in a radioactive or fluorescent manner, either indirectly by means of reagents themselves capable of recognizing these probes, in particular using antibodies or other reagents described above, in the case where these probes are labeled with antigens or molecules capable of being recognized respectively by these antibodies or these reagents.

In the case where the identification and detection method of the invention comprises a step of gene amplification using the primers described above, the detection of the specific nucleotide sequences or fragments of these sequences, can be carried out by gel electrophoresis of all or part of the reaction medium in which the amplification was carried out. The revelation by visualization of a large (more or less individualized) cluster of sequences at a specific point of the gel, and corresponding to the number of copies of all or part of the specific nucleotide sequences thus amplified, is correlable to the presence of these specific sequences in the sample studied.

Advantageously, the amplification of all or part of the aforementioned specific sequences is capable of being detected using probes as described above, in particular according to one of the methods described above.

Other detection or assay methods, in solid or liquid phase, can of course be envisaged in the context of the present invention.

These methods are described in particular in the article by Bloch, 1991, which deals with the PCR technique in general and with a co-amplification assay method, these methods being able to be applied to the quantification of pectinolytic erwinias present in the sample studied, or in the article by Barany, 1991, dealing with LCR and PCR, or also in the article by Gilliland et al. , 1990, dealing with a competitive PCR method for the quantification of mRNA also applicable in the context of the present invention.

A more particular subject of the invention is the application of the above-mentioned identification and detection methods to the diagnosis of the possible appearance of a pathology caused by Erwinia carotovora, in particular of the black leg caused by Eca, in particular in potato, or root necrosis caused by Ecb, in particular in sugar beet, in a host as defined above.

The result of these diagnostic methods mentioned above makes it possible to assess the risk presented by a host likely to be a carrier of Erwinia carotovora, and more particularly of Eca or of Ecb, to develop and if necessary, to transmit, or no, a pathology caused by these bacteria, and more particularly the black leg, or root necrosis. By Erwinia carotovora, and more particularly Eca and Ecb, detected within the framework of the above-mentioned diagnostic and detection methods, is meant both the natural strains of these bacteria, as well as the modified strains, in particular by genetic engineering. The diagnostic methods according to the invention are carried out by detecting Erwinia carotovora responsible for pathologies, in particular E z responsible for the black leg in Europe, or Ecb responsible for root necrosis in Europe, or any species or sub -species derived later by modification of the current taxonomy. The polymorphism of the nucleotide sequences specific for pathogenic Erwinia carotovora, or fragments of these sequences, may be representative of different groups within these erwinias, such as ecotypes, pathotypes (or pathovars) or biotypes of Erwinia carotovora, the polymorphism being in particular demonstrated either by the action of restriction enzymes, by electrophoresis in temperature gradient or denaturing agents, or after hybridization with an RNA fragment by action with RNase A, or by any other method aimed to demonstrate the polymorphism of nucleic acids in solid or liquid phase, in particular by hybridization with probes as described above. The invention also relates to kits for the implementation of a diagnostic method such as described above, characterized in that they comprise at least one probe as defined above, and / or at least a pair of primers such as d written above.

Advantageously, the kits according to the invention comprise: a solid support, such as a microtiter plate on which is fixed a denatured capture probe represented by an oligonucleotide preferably of about 30 nucleotides, and resulting from a sequence according to the invention,

- a heat-resistant DNA or RNA polymerase,

a reaction medium which advantageously consists, in the case of the use of the PCR technique, of 10 mM Tris-HCl buffer, pH 8.3, 50 mM

KCl, 1.5 mM MgCl2, 0.01% gelatin, DNA cofactors or RNA polymerase, in particular of Mg2 + and K ⁺ ions, and of the 4 constitutive DNA deoxynucleotides (dCTP, dATP, dGTP, dTTP) or RNAs (dCTP, dATP, dGTP, dUTP), - one (or more) labeled revelation oligonucleotide (s) advantageously consisting of one (or more) denatured sequence (s) d '' about 30 nucleotides from a sequence according to the invention. The kits according to the invention can also include:

- one or more restriction enzymes, and, where appropriate, reference restriction fragments characteristic of each of the ecotypes, pathotypes (or pathovars) or biotypes of subspecies of Erwinia carotovora, in particular of Eca or Ecb ,

- and / or a ligase and one (or more) pair (s) of oligonucleotide sequences capable of hybridizing on either side of one (or more) nucleotide (s) located in genes or fragment of genes characteristic (s) of a strain of Erwinia carotovora, and advantageously one (or more) oligonucleotide probe (s) specific (s) to a characteristic region of a determined strain of Erwinia carotovora , in the current state of taxonomy.

The invention also relates to the use of the nucleotide sequences defined above for the implementation of a detection method, where appropriate for diagnostic purposes, in a host or in a medium as defined above, of any microorganism or any cell, into the genomes of which the nucleotide sequences according to the invention, have been introduced transiently or definitively. Such a process is advantageously carried out according to one of the methods described above. As such, the invention also relates to the use of the nucleotide sequences defined above, as markers of cells or micro¬ organisms genetically modified by the introduction or not into their genome of these nucleotide sequences, or of hosts cells infected (or transformed) by such microorganisms, in particular for monitoring the evolution of these cells, microorganisms and cellular hosts in a determined environment.

The invention relates in particular to plant cells (for example protoplasts) transformed with a vector chosen from those derived from the plasmids Ti and Ri of Agrobacterium tumefaciens and Agrobacterium rhizogenes respectively, these plasmids containing in one of their non-sites essential for their replication and the transformation of plant cells, a nucleotide sequence according to the invention.

The invention also relates to plants regenerated from these transformed cells. The invention also relates to animal cells transformed with a vector derived for example from retroviruses and containing a nucleotide sequence of the invention. The invention also relates to any process for obtaining the abovementioned nucleotide sequences.

The preparation of these nucleotide sequences can be carried out either by a chemical process, or by other processes, such as amplification by LCR or PCR or other method of gene amplification.

An appropriate mode of preparation of these nucleotide sequences (comprising a maximum of 200 nucleotides, or base pairs when it is a question of double-stranded nucleic acids) of the invention by chemical route (in the current state of the art in this field ) comprises the following steps: - DNA synthesis using for example the automated method β-cyanethyl phosphoramidite described in Bioorganic Chemistry 4; 274-325, 1986,

- Cloning of the DNA thus obtained into an appropriate vector (plasmid, phage or cosmid) and recovery of the DNA by hybridization with an appropriate probe, or by electrophoresis, or by chromatography, generally after restriction.

A method of preparation, by chemical means, of nucleic acids of length greater than 200 nucleotides - or base pairs (in the case of double-stranded nucleic acids) comprises the following steps:

- The assembly, for example by abutment (or ligation, or ligation, by action of a ligase), of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, according for example to the principle described in Proc. Nat. Acad. Sci. USA, 80 ,; 7461-7465, 1983,

the cloning of the DNAs thus obtained in an appropriate vector (plasmids, phages or cosmids), and the recovery of the DNAs by hybridization with an appropriate probe, or by electrophoresis, or by chromatography, generally after restriction.

The preparation of the nucleic acids of the invention can also be carried out by biological means, in particular after cloning of cellular hosts whose genome is likely to contain these nucleotide sequences, if necessary, after transformation of the genome of these hosts using suitable vectors containing these nucleotide sequences.

As such, the present invention relates to any vector, in particular plasmid, containing in one of its sites not essential for its replication, a nucleotide sequence according to the invention. The invention also relates to any cellular host transformed by a vector according to the invention, and in which said vector, or the nucleotide sequence according to the invention, integrated into the genome of the host, is capable of replicating. The invention relates more particularly to the DH5α strain of Escherichia coli transformed by the plasmid pPMV176 itself obtained by insertion of the nucleotide sequence B represented in FIG. 2, into the BamΑl site of the plasmid pTZ19R described in the article by Mead and al., (1985). The present invention thus relates to a process for obtaining the abovementioned nucleotide sequences, carried out by:

- cultivation of a cell host as described above, transformed with a vector according to the invention,

- recovery of the vectors from this cell host, in particular by an appropriate cell lysis treatment,

recovery of the nucleotide sequences of the invention by treatment of the above-mentioned vectors with the aid of appropriate restriction enzymes (using, for example, BamΑl in the case of the aforementioned plasmid pPMN176),

- purification of the nucleotide sequences thus obtained, in particular by gel or capillary electrophoresis, or by chromatography 1.

Another particularly advantageous process for obtaining the nucleotide sequences according to the invention comprises the following steps:

- bringing together vectors containing the above-mentioned nucleotide sequences, with pairs of primers as described above, under conditions allowing the amplification of the number of copies of these nucleotide sequences, in particular under the conditions described above,

- purification of the nucleotide sequences thus obtained, in particular by gel or capillary electrophoresis, or by chromatography.

More generally, the subject of the invention is the use of the nucleotide sequences according to the invention, and more particularly the sequences A, B, B ', C, D, E and F mentioned above, for the implementation the process of specific identification of strains (or groups of strains) or possible pathovars within the different strains of Erwinia carotovora existing. The invention also relates to the use of the above-mentioned sequences for the implementation of the process for the specific identification of Erwinia carotovora subspecies, in particular Eca or Ecb, (or groups of subspecies such as the group formed by Eca and Ecb), within the species Erwinia carotovora.

The aforementioned specific identification methods are advantageously carried out in an identical manner to that described above for the identification methods of pathogenic Erwinia carotovora.

As such, the invention more particularly relates to the use of probes B or B 'and / or primers B105 / B106 for the implementation of methods as described above, for specific identification of the strains of Ecα and / or Ecb whether pathogenic or not, or of the subspecies Eca and / or Ecb within the Erwinia carotovora.

A more particular subject of the invention is also the above-mentioned sequences Ε and F, as well as their use for the implementation of a method of specific identification of different strains or pathovars of Eca, in particular according to the techniques described above. .

The present invention will be further illustrated with the aid of the detailed description which follows of obtaining the nucleotide sequences A to F according to the invention, and of their specificity with respect to the strains of Erwinia carotovora.

I - MATERIALS AND METHODS

a) Bacterial strains and plasmids The bacterial strains and plasmids used for the implementation of the genomic subtraction method used to obtain the sequence B are described in Table 1. The microorganisms tested in the context of dot-blot hybridization experiments (spot deposits) with the probes obtained are presented in table 2. The strains of Erwinia and Escherichia coli were cultivated on Luria medium (Miller 1972) at 30 ° C. and 37 ° C respectively. Antibiotics were used at the following concentrations: ampicillin 50 μg / ml and streptomycin 100 μg / ml. X-gal (O-nitrophenyl-βD- galactopyranoside, Sigma) was used at a concentration of 40 μg / ml.

b. DNA preparation

Total genomic DNA was extracted and purified by the method of Klotz and Zimm (1972).

The target DNA was digested with Sau3Al according to the manufacturer's recommendations (Boehringer Mannheim). After digestion, the DNA was extracted once with the phenol-chloroform mixture, precipitated, washed and resuspended at 0.1 μg / μl in EE buffer pH 8.0 [10 mM N- (2-hydroxyethyl) -piperazine -N '- (3-propanesulfonic acid), 1 mM EDTA].

Two hundred μg of trap DNA were fragmented by sonication in 3 ml of EE buffer. The average size of the DNA fragments is 1000 bp. The sonicated DNA is concentrated with 7.5 ml of 2-butanol, precipitated with absolute ethanol (2 volumes), washed and resuspended at 1 μg / μl in water.

The concentrations of genomic DNA used for the subtraction are measured very precisely with a spectrophotometer (at 260 nm, 1 DO = 50 μg / ml DNA), and by comparison of intensity of the bands against a standard after electrophoresis on 0.8% agarose gel for 1 hour 30 minutes at 75 V in TBE buffer (Tris Borate EDTA), and bromide staining d 'ethidium (Maniatis and α /., 1982). The genomic DNA for the dot-blot tests was extracted as described above with volumes reduced by 10 times. Each DNA was checked by UV spectrophotometry and electrophoresis after digestion with EcoRI (Boehringer Mannheim).

Large-scale preparations of plasmid DNA were made from clear lysates followed by two density gradient centrifugations of cesium chloride with ethidium bromide (Maniatis et al, 1982).

Small-scale preparations were made using the rapid method of Holmes and Quigley (1981).

c) DNA labeling with (32_ ^ _d ATP

The DNA was labeled using the "random primer" DNA labeling kit from Boehringer Mannheim according to the manufacturer's instructions, 4 hours at room temperature.

The elimination of the non-incorporated tri-phosphate deoxyribonucleotides was carried out by chromatography on 0.5 ml of a Sepharose CL-6B column.

(Pharmacia). 50 ng of DNA were used per probe, while 1 μg was labeled to follow the total genomic DNA.

d) Biotinylation of DNA The reaction is carried out on microtitration plates placed on ice, in a dark room. 50 μl of sonicated DNA, at the concentration of 1 μg / μl are mixed with 50 μl of photobiotin acetate (Sigma) at the concentration of 2 μg / μl per well. The mixture is photoactivated by a UN lamp, 10 min at 360 go. After addition of Tris 1M, pH 9 for a final concentration of 100 mM, the biotinylated ADΝ is extracted four times with 1-butanol (saturated with water), precipitated with ethanol, washed and resuspended at the concentration of 2 , 5 μg / μl in 2.5 EE buffer, pH 8.

e) Genomic Subtraction The nucleotide sequences A to F shown respectively in FIGS. 1 to 6, are obtained by genomic subtraction between two strains of Erwinia carotovora, one of these two strains being pathogenic, while the other is not pathogenic (at least under the conditions, in particular climatic conditions, where the previous strain is pathogenic).

The pathogenic Erwinia carotovora strain is a strain of Erwinia carotovora subsp. atroseptica (Eca) responsible for the black leg in certain plants (in particular in the potato) in the climates where prevails, at least for a certain time, a temperature of approximately 15 ° C to approximately 25 ° C, and the strain non-pathogenic Erwinia carotovora, at least under the temperature conditions mentioned above, is a strain of Erwinia carotovora subsp. carotovora (Ecc). The aforementioned genomic subtraction between the two strains of Erwinia carotovora is carried out according to the method of Straus and Ausubel described in European patent application No. 372,524 filed on December 6, 1989. This genomic subtraction method is preferably carried out in the manner following: the DNA of the Ecα strain, or target DNA, is cut into small fragments, of approximately 100 to approximately 1,000 nucleotides, by the action of restriction enzymes,

- the DNA of the strain of Ecc, or trap DNA, is cut into fragments, of approximately 1,000 to approximately 10,000 nucleotides, by sonication, these fragments being subsequently labeled, for example with biotin or other molecule allowing the detection and recovery of nucleic acids (for example by trapping),

the above-mentioned fragments of target DNA and of trap DNA are mixed in proportions such that the trap DNA is in excess relative to the target DNA, in particular in a range of trap DNA / target DNA ratios of approximately 2 at 100, and advantageously in a ratio of approximately 40, - the target and trap DNA fragments thus mixed are denatured by heating, in particular at a temperature of approximately 100 ° C., which leads to the formation of fragments of Target DNA and single strand trap,

- then the single-stranded DNA fragments obtained in the previous step are placed in hybridization conditions such that they allow the formation of double-stranded DNA fragments consisting of two strands of target DNA

(target homoduplex), or of two strands of trap DNA (homoduplex trap), or of a strand of target DNA and of a strand of trap DNA (heteroduplex), in particular under relatively few hybridization conditions stringent (not very drastic) at a temperature of around 65 ° C in the presence of 1M NaCl, - the double-stranded DNA fragments obtained in the preceding hybridization step, are then placed in the presence of a reagent having an affinity for the abovementioned marker, for example avidin or streptavidin having an affinity for biotin (dissociation constant of approximately 10 "^ M), this reagent being immobilized on a solid support (such as beads), so that the labeled homoduplexes and heteroduplexes, in particular biotinylated, remain fixed on the solid support by bonding between the marker used to mark the trap DNA and the abovementioned reagent, in particular on the beads coupled to avidin or to streptavidin,

recovery, in particular by filtration or sedimentation of the solid support, of unlabeled target homoduplexes constituting the specific nucleotide sequences of the pathogenic Erwinia carotovora strain,

advantageously, repetition of the preceding stages between approximately 3 and 5 times, starting from the solution obtained in the preceding stage as a target DNA solution, so as to eliminate the trap homoduplex and heteroduplex likely to be present in said solution,

- if necessary, ligation of oligonucleotide adapters, of known sequences, on either side of the specific nucleotide sequences (target homoduplex), this ligation being carried out in particular by the action of an enzyme of the ligase type, and amplification of the number of these specific nucleotide sequences by implementing a method of amplification (or multiplication or replication) of the number of copies of said specific nucleotide sequences, in particular according to techniques PCR (Polymerase Chain Reaction), 3SR (Self-Sustained Replication Sequence) or any other technique using hybridization between nucleic acids and making it possible to detect an initially low number of nucleic acids determined by multiplying the number of copies of these.

The aforementioned amplification method can be carried out according to the PCR and 3SR procedures described below. By way of illustration, the adapters used can have the following nucleotide sequence: 5 'CACTCTCGAGACATCACCG 3'.

The homoduplex fragments of target DNA obtained and amplified by the above methods can be isolated and multiplied in pure form, in particular by cloning in an appropriate vector. the strain of Eca is the strain 86.20 deposited at the National Collection of Culture of Microorganisms (CNCM) of the Institut Pasteur in Paris on March 30, 1993 under the number 11295, and the strain of Ecc is the strain CH26 deposited at the CNCM on March 30, 1993 under the number 11294. To carry out this genomic subtraction, 10 μg of biotinylated trap DNA and 250 ng of digested target DNA were used for the first cycle of genomic subtraction according to the protocol of Straus and Ausubel . The procedure was not changed except that the screening DNA and yeast tRNA were not added to the reaction mixture. The binding of biotinylated DNA was carried out with a 5% streptavidin solution (Dynabeads ™ M-280, Dynal AS, Oslo, Norway). At each successive hybridization cycle, 10 μg of trap biotinylated DNA were added to the samples. At the end of the last cycle, the resulting samples were precipitated with ethanol and resuspended in 5 μl of TE pH 8 buffer (Tris EDTA, Maniatis et al, 1982).

Protocol: Ten μg of biotinylated trap DNA and 250 ng of digested target DNA are mixed with 3 ml of 10 EE buffer, pH 8.0. The whole is brought to a boil for 1 min. The whole is then dried by evaporation, and resuspended in

4 ml sterile distilled water. One μl of 5 M NaCl solution is added, mixed and centrifuged in pulses. The sample is then covered with 20 μl of mineral oil (Sigma) and placed in a thermal incubator at 65 ° C for 17 hours.

The hybridization mixture is then taken up in 100 μl of EEN buffer, pH 8.0 (0.5 M NaCl, 10 mM N- (2-hydroxyethyl) -piρérazine-N '- (3-propane sulfonic acid) (EPPS ), 1 mM EDTA) and vigorously mixed (Nortex). The mineral oil is gently removed, and 100 μl of a 5% solution of magnetic beads covered with streptavidin, washed and resuspended in EEΝ buffer (Dynabeads, Dynalabs), are added. The mixture is left to incubate for 30 min at room temperature.

The mixture is then placed on a filter suitable for the Eppendorf tube, and filtered by centrifugation 6,000 g, 15 s. The hybridization tube is rinsed with 200 μl of EEΝ buffer, and the rinsing liquid is placed on the filter and centrifuged under the same conditions. The whole of the filtrate is precipitated with 1 ml of absolute ethanol, 5 min at -80 ° C., centrifuged for 30 min at 10,000 g. The (invisible) pellet is rinsed with absolute ethanol and dried. The pellet is then resuspended in 4 μl of biotinylated trap DNA (10 μg).

The resuspension must be thorough and be carried out all along the wall of the tube (outside side, centrifugal force). The resuspended DNA is brought to the boil for 1 min, and 1 μl of 5 M NaCl is added. The mixture is placed at 65 ° C for 17 hours for a new cycle of subtractive hybridization. After four cycles of subtraction, the precipitated DNA is resuspended in

5 ml of sterile distilled water. ) Ligation of adapters for PCR amplification

The following oligonucleotide primer:

5 'GAC ACTCTCG AGAC ATC ACCGTCC ³ ', and the additional phosphorylated primer in 5 '5' GATCGG ACGGTG ATGTCTCGAG AGTG ³ 'are mixed in equal proportion, 100 ng / μl of each, brought to the boil for 1 minute in a water bath . The primers are left to hybridize in the water bath until it has returned to room temperature.

Two and a half μl of the DNA resulting from the genomic subtraction are mixed with 50 ng (2 μl of the preceding mixture) of the adapter solution, with

1 μl of ligation buffer, 1 μl of ligase (Stratagene) and 3.5 μl of sterile distilled water. The reaction mixture is placed at 16 ° C for 12 hours.

After ligation, the mixture is taken up in 20 μl of TE buffer pH 8.0 (Tris EDTA) and filtered through mini-column of sepharose CL-6B by centrifugation for 5 min at 2000 g. The filtrate is extracted once with phenol-chloroform and precipitated with absolute ethanol, the dried pellet is resuspended in 5 μl of TE buffer.

gl Amplification of the subtractive library by PCR

All of the DNA linked to the adapters is mixed with 5 ml of PCR buffer (Boehringer Mannheim), 100 pmol of the primer

⁵ 'CACTCTCGAGACATCACCG ³ ', 200 mM from each dNTP, with 2.5 units of Taq polymerase (Boehringer Mannheim) in a volume made up to 50 μl with sterile distilled water. The mixture is covered with 50 μl of mineral oil. The amplification is carried out in a thermocycler (Pharmacia): 1 min at 94 ° C, 1 min at 65 ° C and 1 min 30 at 72 ° C, for 25 cycles. An aliquot of

5 μl is taken, after these 25 cycles, and amplified again 25 times under identical conditions.

Ten μl per sample are analyzed by electrophoresis in 6% acrylamide gel (Ausubel et al. 1987), 200 V for 1 h, and staining with ethidium bromide (2 mg / 1).

h) Cloning of fragments from the PCR

The plasmids used for cloning are those described in Table 1.

The ligation procedure is the same as described above, with a 1: 1 ratio. The DH5α strain of Escherichia coli was transformed by electroporation (Ausubel et al. 1987).

Cloning of the mixture of fragments and hybridization protocol on colonies. A first cloning of all of the fragments was carried out in the plasmid pUBS-3 carrying resistance to ampicillin. The plasmid is opened by restriction with the enzyme BamΗI, generating cohesive ends compatible with the palindromic sequences of the SauiAl site. The different fragments from the PCR are freed from their adapters by digestion with the enzyme Sau3Al. This provides prominent Sau3Al ends. The plasmid and the fragments are mixed in equal quantity (200 ng of DNA in total) and ligated according to the above protocol in 10 μl of reaction volume. The mixture is used to transform the DH5α strain of Escherichia coli.

One hundred and fifty μl of a suspension of competent cells of Escherichia coli (20 ml of culture in exponential growth phase in medium L, at 37 ° C., with shaking, ie at OD 0.5 to 600 min, are incubated 15 min on ice, washed in sterile distilled water at 4 ° C, centrifuged at 4,000 g for 20 min and incubated for 10 min on an ice bed, twice, and the final residue is resuspended in 0.5 ml d distilled water sterile at 4 ° C) are mixed with 2.5 μl of the ligation mixture (50 ng of DNA), in an electroporation tank. Immediately, the tank is placed in the device and the solution subjected to an electric current (set to 2.5 KV, 25 mF and 400 W). One ml of SOC medium (0.5% yeast extract, 2% tryptone, 2 mM NaCl, 2.5 mM KCl, 10 mM KgCl2, 10 mM MgSθ4, 20 mM glucose) is added immediately, the mixture transferred to an Eppendorf tube 1.5 ml and incubated for 30 min at 37 ° C. Four transformations are thus carried out.

Ten to 100 μl of the transformation mixtures are spread on L agar medium containing 100 μg / ml of ampicillin. The dishes are incubated at 37 ° C overnight. The rest of the mixture is stored at 4 ° C.

Colonies resistant to the antibiotic are counted, and dilutions are made on the mixtures to obtain a concentration of 300 bacteria per 100 μl, knowing that approximately half of the bacteria will be dead during their storage at 4 ° C. All the processing mixes are spread out,

100 μl per Petri dish on agar medium containing the antibiotic and the dishes incubated at 37 ° C, 24 to 48 hours (to obtain colonies of 1 mm in diameter).

Nylon membranes (Hybond N ⁺ , Amersham), cut to the size of the dishes, are gently applied to the colonies, and membrane / dish position marks are marked. The membranes are then carefully removed and placed on blotting paper impregnated with lysis solution (Tris HC1 50 mM pH 8.0, sucrose 25%, EDTA 10 mM, lysozyme 1.5 μg / ml), 5 min, side in contact with bacteria up. The membranes are then transferred to a blotting paper impregnated with denaturing solution (NaOH 0.5 N, 1.5 M NaCl, Triton X100 0.2%), 5 min, then on neutralizing solution, twice 5 min (Tris HC1 0 , 5 M pH 8.0, NaCl 2.5 M). The membranes are then dried and baked for 1 hour in an oven at 80 ° C under vacuum, to fix the DNA. The boxes containing the bacteria are stored at 4 ° C.

The membranes are prehybridized and hybridized, using as probe labeled with α ^ p all the fragments from PCR. The radioactive labeling and hybridization protocols are described below. After autoradiography of the membranes, the colonies presenting a hybridization signal are identified by the orientation of the membranes, autoradiographs and culture dishes. These colonies are removed with a toothpick, inoculated with 10 ml of medium L, with ampicillin and incubated at 37 ° C. with shaking overnight. The cultures are centrifuged for 10 min at 6000 g. The pellet is included in

50 ml of 50 mM Tris buffer pH 8.0, containing 25% sucrose. A volume of 400 ml of MSTET (5% sucrose, 5% Triton X100, 50 mM EDTA, 50 mM Tris HC1 pH 8.0), added with 7 μl of lysozyme at 40 mg / ml are added and the whole is worn at boil 45 s and immersed in ice. The mixture is centrifuged at 4 ° C, 30 min at 10,000 g. The supernatant is then gently removed, the DNA extracted once with the phenol-chloroform-isoamyl alcohol mixture, precipitated and resuspended in 20 μl of TE buffer pH 8.0.

Five μl of each preparation are digested in the presence of 1 μg of boiled RNAse A, the restrictions are analyzed by acrylamide gel electrophoresis. Clones containing an insert are identified by comparison of the electrophoretic profiles with the starting plasmid.

Cloning of the prepurified fragments by electroelution and selection of the clones on X-gsΛ medium.

Fragments from PCR, stripped of their adapters (100 ng of each fragment), are separated by electrophoresis on acrylamide gel. Each strip is delicately cut with a scalpel and recovered (some close strips cannot be separated and are taken together). Each strip of acrylamide is placed in a dialysis rod (Spectrapore) in distilled water. The tubes are placed in a horizontal electrophoresis tank containing TBE buffer diluted by half. The acrylamide strips are oriented in the lengthwise direction of the rod, anode side. Two hundred volts are applied for 1 hour. The current is then reversed for 10 s, to detach the DNA from the dialysis membrane. We verify under UV lamp that the DNA is well out of acrylamide. The solution is then homogenized, recovered and the DNA extracted once with phenol-chloroform-isoamyl alcohol, precipitated and resuspended in 5 μl of distilled water.

The DNA purified by electroelution are mixed with an equal amount of vector (50 ng of each). In this case, it is the plasmid pTZ19R, carrying the resistance to ampicillin and the α fragment of the β-galactosidase gene (lacZ). The plasmid is opened by restriction with the enzyme BamΑl, the site of which is located in lacZ. Ligation and transformation are carried out as above. The bacteria are then spread on medium containing ampicillin (100 μg / ml), IPTG (Isopropylthiogalactoside; inducer) and the substrate of the enzyme, X-gal (O-nitrophenyl-βD-galactopyranoside, 100 μg / ml). The colonies containing a plasmid are selected by ampicillin, the colonies containing a plasmid with insert are identified by the absence of β-galactosidase activity: the white colonies are mutated by insertion into lacZ, the blue colonies have a β- system functional galactosidase. The white colonies are selected and analyzed as above.

he Hybridizations

Labeling of probes with cχ32p by hybridization of random primers or "Random priming".

Fifty ng of insert purified from the cloning plasmid by digestion, acrylamide gel electrophoresis and electroelution are used as probe. The labeling is carried out with the "Random primed DNA labeling kit" (Boehringer Mannheim). Fifty ng of DNA are mixed with 2 μl of buffer containing hexanucleotide primers, 1 μl of each dNTP

(except dATP). The volume is made up to 14 μl with sterile distilled water, brought to the boil for 3 min, and the tube immersed in ice. One μl of polymerase (Klenow fragment) and 5 μl of (α ^ P) dATP (50 μCi) are added. The mixture is left to incubate for 4 hours at room temperature. The labeled probe is then purified (stripped of the non-incorporated labeled nucleotides and of short non-specific oligonucleotides) by filtration on Sepharose CL-6B, in a 0.5 ml Eppendorf tube pierced, placed in a 1.5 ml tube, in centrifuging for 5 min at 2,000 g. The effectiveness of the marking is checked with the β-ray detector. The probe is denatured just before hybridization by boiling for 3 min, and the tube immersed in ice.

DNA hybridization in "dot-blot" or deposit in spots: The DNA of different microorganisms (Table 2) were denatured and fixed by the alkaline method proposed for Hybond N + membranes (Amersham). The genomic DNA for the Southern method was treated using several endonucleases according to the manufacturer's instructions (Boehringer, Mannheim). The reaction mixtures were run on 0.8% agarose gel using 1 μg of size marker (1 kb of BRL ladder). The Southern method was carried out on Hybond N + membranes according to the manufacturer's instructions. Preparation of membranes.

- membranes for "Dot Mot" hybridization

Two μg of DNA per sample, in a volume of 1.5 μl are mixed with an equal volume of a sodium hydroxide solution (0.4 M NaOH, 1 mM EDTA) and deposited on a nylon membrane (Hybond N ⁺ , Amersham). When all the samples have been deposited, the membrane is placed, DNA side up, for 5 minutes on blotting paper impregnated with a denaturation solution (0.5 M NaOH, 1.5 M NaCl), then 1 minute on a neutralization solution (Tris HC1 0.5 M pH 7.2, NaCl 1.5 M, EDTA 1 mM), 20 min on a fixing solution (NaOH 0.4

M) and rinsed for 1 minute in 2X SSC (0.03 M sodium citrate, 3M NaCl). The membrane is then dried and can be stored at room temperature.

- membrane for Southern hybridization.

Two to three μg of DNA per sample are digested with restriction enzymes and separated by electrophoresis on a large 0.8% agarose gel (300 ml). After reading, the gel is treated to transfer the DNA to a nylon membrane. The gel is treated twice 15 min in 0.25 M HCl, rinsed with distilled water, incubated twice 15 min in denaturing solution (1 M NaOH, 1.5 M NaCl), rinsed with distilled water and 30 min in neutralizing solution (Tris HC1 0.5 M, NaCl 1.5 M pH 7.5).

The following assembly is then carried out: 6 sheets of Whatmann 3M paper cut to the exact size of the gel and soaked in 20X SSC (0.3 M sodium citrate, 3M NaCl) are placed at the bottom of a tray. The electrophoresis gel is then superimposed on these sheets, then covered with the membrane cut to the size of the gel. A sheet of Whatmann paper and a stack of dry "cloths" (15 to

20 cm thick), at the gel size also are placed on top. Finally a glass plate and a weight not exceeding one kg are added. The DNA is transferred by capillary action for at least 6 hours. The membrane is then recovered, dried and baked in an oven at 80 ° C under vacuum. - Hybridization and washes.

The membranes are prehybridized, at 65 ° C., with stirring, at least one hour in a solution of 6X SSC, 0.5% SDS, 0.01% skimmed milk. The hybridization is carried out in a volume of 10 ml, in a sealed plastic bag, in a solution of 6X SSC, 0.1% SDS, 0.01% skim milk containing the denatured probe, 17 hours at 65 ° C.

After hybridization, the membrane is washed once in 3X SSC, 0.5% SDS, at 65 ° C, twice in 0.3X SSC, 0.5% SDS at 65 ° C, and for stringent conditions twice in 0.1X SSC, SDS 0.5% at 68 ° C.

The membrane is then slightly wiped and then placed in plastic film.

The membrane, depending on the number of strokes / min observed on the β-ray counter, is placed for a longer or shorter time in the presence of a self-radiography film in a cassette at -80 ° C. Autoradiography is then developed.

j) Direct sequencing on plasmid

Sequencing is carried out directly on double-stranded plasmid with two primers chosen from the lacZ gene sequence 150 nucleotides away from the cloning site. The sequence reaction is carried out by the "dideoxychain termination" method of Sanger et al. (1977), using the SequenaseT kit (United States Biochemical Corporation). For each sample, the direct primer and the reverse primer are used separately. For each sample, a first elongation step starting from the primer is carried out by DNA polymerase (sequenase) in the presence of a limiting amount of dNTP and (α ^ P) dATP for 5 min at room temperature. The reaction mixture is then distributed, for each sample, into four wells containing dNTP in non-limiting quantity and for each of these wells one of the dideoxynucleotides (ddATP, ddCTP, ddGTP or ddTTP). This reaction is carried out for 5 min at 37 ° C. and is then stopped by adding EDTA. The samples can be stored at -20 ° C and will be brought to the boil for 2 minutes just before being deposited on gel, their deposition being always carried out in the order of ddATP, ddCTP, ddGTP and ddTTP. The different fragments are separated by electrophoresis in acrylamide gel (6%, urea 460 g / 1 in TBE buffer), 80 cm long, at a constant power of 100 W (duration 3-6 h).

The gel is then removed from the mold and fixed in a 10% methanol, 10% acetic acid mixture, transferred to Whatmann 3M paper, covered with plastic film, and the whole is dried by heat and under vacuum in a gel dryer. The gel is then placed with an autoradiography film in a cassette, for a variable duration, according to the number of strokes / min at the β-ray counter. The sequences are then read and entered on a computer for analysis. The programs used are those of the GCG (University of

Wisconsin, Genetics computer Group, Devereux et al. 1984). Sequence homology searches are carried out in the international Genbank and EMBL databases.

II) Results

a ^ Determination of the conditions of subtraction The conditions for DNA biotinylation and for binding to streptavidin were tested at the same time: 1 μg of DNA of the 3937 strain of Erwinia chrysanthemi cut marked with α ³ ^ P were mixed with 49 μg of DNA from strain 3937 cut. The mixture was biotinylated as described previously in the chapter on materials and methods. Ten μg of biotinylated DNA were incubated with different concentrations of beads coated with streptavidin (0.5%, 1%, 2.5% and 5%). For solutions of 0.5, 1 and 2.5%, some radioactivity remains in the free fraction. The 5% solution was determined to represent the most effective binding condition.

The number of subtraction cycles was determined using the following two criteria: binding of homologous sequences and enrichment in non-homologous sequences. Two libraries were obtained with the following pairs: (i) 100 ng of fragment Ω digested with SaύiAl and labeled with α ³² P, 2 kb of the DNA fragment Sm ^r / Spc ^r (Prentki and Krisch 1984), were mixed with 150 ng of the genomic DNA of the strain 3937 digested with Sau Al, and subtracted with the biotinylated DNA of the strain 3937, (ii) 250 ng of DNA of the strain 3937 digested with Sau3Al and labeled with α ³² P was subtracted with the biotinylated DNA from 3937. The recovery at each cycle of non-homologous sequences (fragment Ω of control (i)) in the free fraction and the binding of homologous sequences (DNA of 3937 of control (ii) ) were estimated by counting the distribution of radioactivity in the free and bound fractions. After four cycles of subtraction, the enrichment factor (ratio between the isolated heterologous sequences and the losses in homologous sequences) is 14 and the fixation of homologous sequences is 99%. However, some losses occurred in each cycle for heterologous sequences (approximately 40%), and after 4 cycles the losses are 80

%. For this reason, 4 cycles were estimated to be sufficient to allow enrichment in heterologous sequences and to avoid excessive losses. > Genomic subtraction

Four cycles of subtraction hybridization were carried out for each of the following pairs (target DNA / trap DNA): (i) positive control strain PMV 4071 / strain 3937 (omega insertion fragment (Ω) of the mutant 4071 in the strain Ech 3937 as target DNA and strain 3937 as trap DNA), (ii) the library subtracted between 8620 and CH26 (strain 8620 of Ecα as source of probes and CH26 of Ecc as DNA trap) and (ii) a negative CH26-CH26 control. The results of the subtraction are shown in Figure 1. The positive control shows two amplified fragments of 230 and 300 bp approximately. The restriction enzyme treatment is Sau3Al of the Ω fragment generated eight main fragments of 460, 300, 230, 190, 150, 140, 80 and 60 bp. Among all these fragments, only a part (33% of the fragment) was re-isolated by genomic subtraction. The differential library between Eca and Ecc presents 6 main DNA fragments (190, 200, 250, 300, 400 and

410 bp) and a double band of around 450 bp less clear than the others. No amplified fragment is present either in the negative subtraction control or in the negative PCR contamination control. In all genomic subtraction samples, cut DNA was observed on electrophoresis gels.

c ^ Cloning of the probes from the subtracted library The samples coming from the PCR were treated with Sau3Al before cloning in order to remove the adapters and to have compatible BamΑl ends. First, the entire library was used for global cloning with pUBS-3 treated with 2-temHI as a vector. Colonization hybridization (Maniatis et al., 1982) after cloning of the entire library, made it possible to isolate two inserts containing clones using the entire library as a probe. A second approach consisted in cloning, after separation of the library into four fractions on 6% polyacrylamide gel and purification by electroelution (Maniatis et al, 1982). The cloning vector used in this case was pTZ-19R treated with -Bα HI, the white colonies of transformed E. coli DH5α were selected on X-gal medium. This second strategy led to the production of 4 clones containing inserts. The six clones were named A, B, C, D, Ε and F respectively in order of decreasing size (see Figure 2, and Table 1). d) hybridization experiments

Hybridizations were carried out with the six probes. For most strains, the hybridizations were carried out at least twice on separate membranes. Table 2 illustrates the results. The E and F probes give a signal only for a limited number of Ecα strains. Probe A gives a positive signal for most of the strains tested, including the Ecc strains, and is absent in certain strains and in that of the trap DNA. The results obtained with probe B are shown in FIG. 3. Under stringent washing conditions, only the typical Ecα strains are hybridized, and under less stringent washing conditions some cross hybridizations have been observed with strains of Ecb. The B probe is therefore very specific for Eca, as is the B 'probe (which differs from the B probe only by one nucleotide less), the latter making it possible to find the same sequence of 200 nucleotides in strains 88.33, 15.26, CH3 and 88.1 of Ecα. For the C probe, all the typical strains of Ecα present a positive signal and a few strains of Ecc also. Concerning probe D, all the strains of Ecα ("atypical and typical") and some strains of Ecc were hybridized. Ecα strain 86.20 DNA was digested with several endonucleases

(-BαmHl, Clal and Sau3Al) and transferred after electrophoresis on membranes (so-called Southern technique). The hybridization results with the six probes are presented in FIG. 4. Different DNA fragments obtained with different endonucleases were hybridized with the 6 probes, thus indicating that the probes are not grouped in the genomic DNA (cluster) of Ecû strain 86.20. In addition, the absence of a hybridization signal with the DNA of Ecc CH26 and the presence of this signal with the DNA of Ecα 86.20 indicates that the probes correspond to the subtraction library.

e) DNA sequencing

The sequences of probes A, B, C, D, Ε and F were obtained. The results are shown in FIG. 5. For each probe, homologies were sought on the Genbank and ΕMBL databases. No significant homology was observed for the sequences with the exception of probe A, which exhibits significant homology with the sequence of the put? of Salmonella typhimurium and E. coli. This gene codes respectively for a proline permease (73.8% homology) and a proline transporter (73.2% homology). TABLE 1: Bacterial and plasmid strains

source or reference characteristics

E. coli

DH5 endAl hsdR17 (rγ_- mγ_ ⁺ ) supE44 Bethesda Research thi-1 λ "recAl gyrA §80dlacZAM15 Laboratories A (lacZYA-argF) U169

Eca

86.20 strain isolated from B. Jouan apple (Earth station (France, 1986) Plant Pathology, INRA, Le Rheu, France)

Ecc

CH26 strain isolated from apple from O. Cazelles terre (Switzerland, 1985) (Changins, Switzerland)

Ech

3937 strain isolated from Saintpaulia Kotoujansky et al. ionantha (1982)

PMV 3937 peïEr, fragment Ω Sm ^r -Spc ^r M. Boccara

4071 of R100.1 in the restriction site (Laboratory of

Bgia Plant Pathology, INRA INA PG, Paris, France)

TABLE 1 (continued): Bacterial strains and plasmids

source or reference characteristics

pUCS-3 plasmids pUC derivative containing G. Murphy (IPS, pBLUESCRIPT Norwich, UK) pHP45Ω pUC derivative containing a Prentki and Krisch fragment

Smr-Spcr from R100.l (1984) pTZ19R derivative pUC with a promoter from Mead et al. (1985) the T7 RNA polymerase pPMV174 derived pUBS-3 containing the A Collection probe from the BαmHl site (Ap ^r ) INRA laboratory (Plant Pathology, Paris) pPMV176 derived pTZ19R containing the probe

B in the BαmHl site (Ap ^r ) pPM V 177 derivative pTZ 19R containing the probe

C in the BamΑl site (Ap ^{1 *} ) pPMV175 derivative pUBS-3 containing the probe D in the site -Bα Hl (Ap ^r ) pPMV 178 derivative pTZ 19R containing the probe

E in the BamΑl site (Ap ^r ) pPMV179 derivative pTZ19R containing the probe

F in the site -BαmHl (Ap ^r )

TABLE 2. Hybridization results

Host strains country results of year hybridization with: probes

A B C D E F

Eca

88.33 French apple, + + + + + - earth 19881

88.45 'France, + + + + + - 19881

88.1 'France, + + + + - - 19881

88.22a France, + + + + + + + 19881

88.24 France, + + + + - - 19881

88.30a 'France, + + + + + - 19881

87.7 'France, + + + + - - 19871

87.13 'France, + + + + + + + 19871

87.16a 'France, + + + + + - 19871

87.16b France, + + + + + - 19871

86.14.11 France, + + + + - + 19861

86.20 'France, + + + + + + + 19861

511 'France, + + + + +

1964 ²

SFl. l • Germany3 + + + + + 161 Holland ⁴ + + + + + + +

Cipll4 'Peru, + + + + + + + 19805

Cipl25 'Peru, + + + + - - 1980 ⁵

Cipl31 'Peru, + + + + - - 19805

Cip026 'Peru, + + + + + + + 1980 ⁵

SH164.4 'Reunion, + + + + + + 1988 ³

CH3 'Switzerland, + + + + - - 1985 ⁶ TABLE 2 (continued 1)

CH5 Switzerland, + + + + -

1985 ⁶

CH6 Switzerland, + + --- + + -

1985 ⁶

SF18.296 Switzerland, + + + + + +

1958 ³

1329 UK, 19672 + + + + _ +

1330 UK, 1967 ² + + + + - -

SCRI1043 UK, 1985 ⁷ + + + + + +

1526 UK, 1957 ² + + + + - +

1527 USA, 1973 ² + + + + + +

1525 USA, 1969 ² + + + + + +

1453 tomato France, + + + + -

1973 ²

1546 France, + + + + - -

1973 ²

89.19 * Argentine apple, + - - --- - - from 19891 earth

1H * water Spain, + - - + - -

1989 ⁸

40H * "Spain, + - - --- - -

1989 ⁸

Ecb

2121 beet USA, 1972 ² +

2122 USA, 1972 ² + - - - - -

1520 sunflower Mexico ² . - -. . .

Ecc

SH230.134 banana Cuba ³ + - -

CM1 Malawi cabbage, +

1986 ³

798 carrot USA ² - - - -

(ATCC 495)

CH15 Swiss celery, - - - + - -

19886

1489 chrys- France, - - - -. . anthem 1971 ²

1458 USA, 1971 ² + - + - - -

SH230.115 Cuba ³ corn + - - - - -

1350- Italy ² + - + - - - combre

1285 cycla- Greece ² + - - - - leads

SΕ99.1 chicory France,

19851 TABLE 2 (continued 2)

1488 iris France, -

1973 ²

SB89.7 leek France, -

1982 ³

2046 ^τ apple Denmark + - - - from 1952 ² earth

88.22c France, -

19881

88.29al France, + - - -

19881

88.44 "France, -

19881

87.25 "France, -

19871

86.14.51 France, -

19861

S99 "France, - - - +

19771

S101 France, -

19771

76.26 "France, -

19761

PM2 Malawi, + - - +

1986 ³

194 "Morocco, -

1963 ²

Cip360 Peru, + - + -

1984 ⁵

Cip361 Peru, + - + -

19845

Cip009 Peru, + -

19775

CH24 Switzerland,. . . .

19876

CH26 "Switzerland, -..

1985 ⁶

SCRI193 USA ⁷ + - - -

1336 UK, 1967 ² - -

Si82.1 Vietnam, --- - - -

1989 ³

SG162.6 sunflower France, -

1987 ³ TABLE 2 (continuation 3) 1403 Yougo- - - - slavie,

1969 ²

797 tobacco USA, 1951 ² + - + -. .

SG39.1? Reunion, 4- - -

1987 ³

The meeting,

SG39.3? 1987 ³ + - + - - -

Eco

1893 celery France, - - -

1976 ²

CHU Switzerland, + - -

1985 ⁶

2155 endive France, +

1983 ²

2154 France, + - - - -

19822

1892 France, + - -

1981 ²

1959 France, 4-

1980 ²

1878 France, - - -

1979 ²

1879 France, 4-

1979 ²

1880 France, - - - - -

1979 ²

1646.2 leek France, 4-

1980 ³

1654 France, 4- - - - - 4-

1980 ³

CH4 Swiss lettuce, - - _ - - -

1986 ⁶

Ech

3716 kalen- France, 4- choe 19789

1596 corn France, 4- -. . . -

1978 ²

EP2 ² philo¬ Martinique 4- -. . . - dendron _1,987 10

1271 Egyptian corn. - -. . .

1961 ²

SH230-C94 Cuba ³ tobacco - -. . .

3665 diffenba- France 4- - - - - chia 19749 TABLE 2 (continuation 4) 3937 saint¬ France 4- paulia 1977 ⁹

1499 corn France 1973 ²

1888 France 4- potato 1978 ² CH29 Swiss potato 1988 ⁶

2267 potato Australia 1978 ² 1871 banana Côte 4- d'Ivoire

1976 ²

CH36 Swiss potato 19876

1275 USA eyelet 4-

1971 ²

3805 philo¬ France 4- dendron 1976 ⁹

2015 France 4- potato 1975 ²

1236 parth- USA 4- enium 1945 ²

B374 pelar- Comoros 4- gonium i960 ²

2013 dalhia France 4-

1974 ²

2051 diffenba- USA 4- chia 1957 ²

Erwinia hawthorn France 11 amylovora [1] Erwinia non France 11 4- herbicola [1] pathogen

Erwinia rhubarb Suisse6 4- rhapontici [l] Pseudomonas fluorinated garlic 1976 ³ pv. lomagnae [1] Pseudomonas apple from USA, 1970 ³ marginalis [1] earth Pseudomonas apple from Costa Rica ³ solanacearum [1] earth TABLE 2 (continued 5)

Pseudomonas France 11 syringae pv. phaseolico- / α [l]

Pseudomonas Netherlands ⁴ 4- sp. [2]

Swiss chicory pseudomonas6 viridiflava

[1]

Rhizobium France 11 meliloti [2]

Azorhizobium Sesbania Senegal 10 caulinodens rostrata

[1]

Klebsiella FrancelO pneumoniae

[1]

Agrobacterium France 11 tumefaciens

[1]

Xanthomonas France 11 campestris

[2]

Clavibacter apple USA, michiganensis de terre 1976 ³

[1]

Brucella France 11 melitensis

[1]

Yarrovia France 1 lipolytica

[1]

Yersinia salmo France, ruckeri [1] 1982H

Yersinia patho¬ ATCC- 4- pseudogenes 23207 human tuberculosis -29833

[1] Spain apple bacteria ⁸ earth saprophytes

[8]

Escherichia coli [1]

Bacillus apple France, polymixa de terre 1979 ³

[1] (.) Not done. (*) Atypical Eca strain, recently identified as an Ecc strain by their phenotypic and genotypic characteristics. (}) Type strain of the species, [n] Number of strains tested. (1) Bernard Jouan, National Institute for Agronomic Research, Rennes, France, personal collection. ( ² ) CFBP, French Collection of Phytopathogenic Bacteria, INRA, Angers, France. ( ³ ) Régine Samson, National Institute for Agronomic Research, Angers, France. ( ⁴ ) IPO, Research Institute for Plant Protection, Wageningen, Netherlands. (5) CIP, International Potato Center, Lima, Peru. (6) Olivier Cazelles, Federal Station of Agronomic Research, Changins,

Switzerland, personal collection. ( ⁷ ) SCRI, Scottish Crop Research Institute, UK. ( ⁸ ) Maria Lopez, Instituto Valenciano De Investigaciones Agrarias, Spain, personal collection. (9) Monique Lemattre, National Institute for Agronomic Research, Versailles, France, personal collection. (10) Claudine Elmerich, Institut Pasteur, Paris, France, personal collection. (H) CFISM,

French Computerized Collection of Microbial Strains, National Institute of Agronomic Research, France.

BIBLIOGRAPHY

Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 1987. Current protocols in molecular biology. Greene / Wiley, New York.

Barany, 1991, Biochemistry, vol. 30, No. ll, 2735.

Bloch, 1991, Proc. Natl. Acad. Sci. USA, vol. 88, 189-193.

Devereux, J., Haeberli, P., and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research. 12: 387-

395.

Welsh, A., and Samson, R. 1992. Erwinia carotovora subsp. odorifera subsp. nov., associated with odorous soft rot of chicory, Cichorium intybus L. International Journal of Systematic Bacteriology. 42: 582-588.

Gilliland et al., 1990, PCR Protocols. A guide to Methods and Applications. Edited by Innis, Gelfand, Sninsky, White. Académie Press Inc. Holmes, DS, and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Analytical biochemistry. 114: 193-197.

Klotz, L., and Zimm, B.H. 1972. Size of DNA determined by viscoelastic measurments: Results on bacteriophages, Bacillus subtilis and Escherichia coli. J. Mol. Biol. 72: 779-800.

Kotoujansky, A., Lemattre, M., and Boistard, P. 1982. Utilization of a thermosensitive episome bearing transposon Tn 10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi. J. Bacteriol. 150: 122-131.

Lelliott, R.A., and Dickey, R.S. 1984. Genus VII: Erwinia. Bergey's Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore, MD. 469-473.

Lelliott, R.A., and Stead, D.E. 1987. Methods for the diagnosis of bacterial diseases of plants. British Society for Plant, Blackwell Scientific publications.

Maniatis, T., Fritsch, E.F., Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

545 pp.

Marti, R., Lopez, M.M., Morente, C, and Alarçon, B. 1989. Incidence of erwinia-causing soft rots in irrigation water in Valencia (Spain). In Pro. 7th Int. conf. Plant Path. Bact., Budapest, Hungary, Eds: Z. Klement. 755-760.

Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 466 pp.

Pérombelon, M. CM. 1989. Ecology and pathogenicity of soft rot erwinias: an overview. in. Proc. 7th Int. Conf. Plant Path. Bact., Budapest, Hungary, Eds: Z. Klement. 745-751.

Pérombelon, M. CM., And Kelman, A. 1987. Blackleg and other potatoes diseases caused by soft rot erwinias: proposai for revision of terminology. Plant diseases. 71: 283-285. Pérombelon, MM 1980. Ecology of the soft rot erwinias. Ann. Rev. Phytopathol. 18: 361-387.

Prentki, P., and Krisch, H. M. 1984. In Vitro insertional mutagenesis with a selectable DNA fragment. Uncomfortable. 29: 303-309.

Smith, C, and Bartz, J.A. 1990. Variation in the Pathogenicity and aggressiveness of strains of Erwinia carotovora subsp. carotovora isolated from different host. Plant disease. 74: 505-509.

Stanghellini, M.E. 1982, and Meneley, J.C. 1975. Identification of soft rot Erwinia associated with blackleg of potato in Arizona. Phytopathology. 65: 86-87.

Thomson, SV, Hildebrand, DC, and Schroth, MN 1981. Identification and nutritional differentiation of the erwinia sugar beet pathogen from members of Erwinia carotovora and Erwinia chrysanthemi. Phytopathology. 71: 1037-1042.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: NATIONAL INSTITUTE OF AGRONOMIC RESEARCH

(INRA)

(B) STREET: 147 RUE DE L'UNIVERSITE (C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75338 CEDEX 07

(A) NAME: PARIS-GRIGNON NATIONAL AGRONOMIC INSTITUTE (INA P-G)

(B) STREET: 16 RUE CLAUDE BERNARD

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75231 CEDEX 05

(ii) TITLE OF THE INVENTION: CHARACTERISTIC NUCLEOTIDE SEQUENCES OF ERWINIA CAROTOVORA, AND THEIR USES FOR THE DIAGNOSIS OF PATHOLOGIES CAUSED BY THESE BACTERIA (iii) NUMBER OF SEQUENCES: 15

(iv) COMPUTER-READABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)

(vi) DATA FROM THE PREVIOUS APPLICATION: (A) DEPOSIT NUMBER: FR 9306072 (B) DEPOSIT DATE: 19-MAY-1993

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 404 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

GATCATACCC AGCAGTGCGC CGTTGCGGTC ATACGCGGCC ATAGCAGAGA AATCAAAATC 60

ACCGGTCCGA AGGCGGCACC GAAACCTGCC CAAGCATAGC TCACCAGACC CAGTACACGG 120 TTTTCAGGGT TCAGCGACAG CGCAATAGCA ATGATAGCGA CTAATAGCAC CATCGTGCGG 180

CCCACCCACA CCAATTCTTT CTGACTGGCA TTTTTGCGCA GGAATGGCTT GTACAAATCT 240 TCGGTGATGG CACTGGAGCA CACCAGCAAT TGGCAGCTCA GCGTACTCAT GACAGCAGCT 300

AAAATCGCAG ACAGCAGAAT GCCGGCAATC CACGGATTGA ACAGCAGCAT AGATAGCTCG 360 ATAAAGACGC GCTCGCTATT TTGCGCCACG TTACCCGCCT GATC 404

(2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 303 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

GATCGCTCAT TAGATTTCAT ACATCAGAAA TTGAGCAATA TATCCTTAGC AACATAAGGC 60

AGCATTGAAT CGCCACAGAG AGTACAGTTG TCACAATGAA TCCCTACTTA ACACGGAACG 120 CCCGCTGTTC GCTCATTACG GCCTGCCAAA TTTGATTGTA TTCTGTCCGC GAAAATTGTT 180

GTGCCTGATA GCGATAAATG CGTGTTCGAT GGTTTCCAGA TAGGCTTCAG CCCTTTCTTC 240

ATTAAAATGC CGCAGGCATA AAGATAAATG TCTTCCACAC TCTCGAGACA TCACCGTCCG 300

ATC 303

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 302 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

GATCGCTCAT TAGATTTCAT ACATCAGAAA TTGAGCAATA TATCCTTAGC AACATAAGGC 60

AGCATTGAAT CGCCACAGAG AGTACAGTTG TCACAATGAA TCCCTACTTA ACACGGAACG 120

CCGCTGTTCG CTCATTACGG CCTGCCAAAT TTGATTGTAT TCTGTCCGCG AAAATTGTTG 180

TGCCTGATAG CGATAAATGC GTGTTCGATG GTTTCCAGAT AGGCTTCAGC CCTTTCTTCA 240 TTAAAATGCC GCAGGCATAA AGATAAATGT CTTCCACACT CTCGAGACAT CACCGTCCGA 300

TC 302 (2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 290 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

GATCCCGTAT GGTGAATATG TTACATACGG AACCGTAGCC AAAGATGTTG CGGCTGTTAT 60 GGGTAAAACC AGCATGTCAG CGCAGGCTAC CGGTGGGGCG GTTGGTCACA ACCCAATATC 120

TATTATCATT CCTTGCCACC GGGTCATAGG CACCAATAAC AGCTTGACCG GGTATGCTGG 180

AGGTATCGCA AAAAAAATTC AGCTACTTGA ACTAGAAGGT GTTAACACCG TGAATATGAG 240

CGTCCCTATC AAAGGGACGG CTTTATGATA ATAATCGTCT CTAGCCGATC 290

(2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 233 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

GATCACGGCG CTCAGCGGCC TGACTGCCGC ATTGGAAACA GAGCCCCAGC GACCGATACG 60

CAGCCTATCT GTGCTACCGG AATCAGAACG TCAACAATTG CTGATTGGCT TTAACGCCAC 120

CGATACGGAT AGCCTGCCGC ACGCGCTGAT TCACGAGCGC ATCGAACACG TGGCGAGCCA 180

GACGCCGGAT GCCGTTGCGG TTATATTCGG TAAGCACACC CTCAGTTACG ATC 233

(2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 212 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

GATCAGTAAA GATGAGTGGT TGTTACTTCA ATCTGACCAA AGCAAATCCT ACGCGAGGAC 60 GCCGTAGCAC TGATAATCCT TATAGTGGTC TATTACGCTG TGGATGCGGT GGTGCATTAA 120

TAAAAAGAAA GAGCACGGTA AGAGGGAAAC TGTACGTTTA TCATGGTTGC CTAAACGCCA 180

AAGATGGTCG TTGTTCTCAA AATCTCTCGA TC 212

(2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 183 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GATCGCTTCC GGCTTTTATT AACTTAAACA GTCAATGAGC AATGCATCTA ATTCAATCCA 60

AGCGTCTTCT AATCTGAATG CGATTTCAGG CTTATTTTCT TTTCCATCAA AACTCACATA 120

TGCATTTCGT ACCATTTGTG GAATGGTATA TAAATAAAGC GCCAACTTCT TATCAATAAG 180

ATC 183

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

TAAGGCAGCA TTGAATCGCC ACAGAGAGTA 30

(2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACACGCATTT ATCGCTATCA GGCACAACAA 30

(2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

GATCGCTCAT TAGATTTCAT ACAT 24

(2) INFORMATION FOR SEQ ID NO: 11:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

TGGAAGACAT TTATCTTTAT GCCT 24

(2) INFORMATION FOR SEQ ID NO: 12;

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: CGCTCATTAG ATTTCATA 18 (2) INFORMATION FOR SEQ ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATCGGACGGT GATGTCTC 18

(2) INFORMATION FOR SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:

GCAACATAAG GCAGCATTGA 20

(2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomitis)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CGAGAGTGTG GAAGACATTT 20

Claims

1. Nucleotide sequences comprising all or part of the sequences as obtained by genomic subtraction between two different strains of Erwinia carotovora, or comprising all or part of the sequences bordering the latter on the genome of the Erwinia carotovora strain from which the sequences obtained by genomic subtraction, said nucleotide sequences being capable of hybridizing with the genome of at least one bacterial strain belonging to the species Erwinia carotovora, and being unable to hybridize with the genome of at least one strain belonging to this same species.

2. Nucleotide sequences according to claim 1, characterized in that they are capable of hybridizing with the genome of at least one bacterial strain belonging to a determined subspecies of Erwinia carotovora, but not with that of at least at least one strain belonging to a subspecies of Erwinia carotovora different from the previous one, or capable of hybridizing with the genome of at least one strain belonging to a specific subspecies of Erwinia carotovora, but not with that of '' at least one strain belonging to this same subspecies.

3. Nucleotide sequences according to claim 1 or claim 2, characterized in that they are capable of hybridizing with the genome of at least one pathogenic bacterial strain in a given plant, but not with that of at least one non-pathogenic strain in this same plant, this non-pathogenic strain belonging or not belonging to the same subspecies of Erwinia carotovora as the previous pathogenic strain.

4. Nucleotide sequences according to one of claims 1 to 3, characterized in that the genomic subtraction between the two strains of Erwinia carotovora is carried out as follows:

the DNA of a first strain of Erwinia carotovora, or target DNA, is cut into small fragments, of approximately 100 to approximately 1000 nucleotides, in particular by the action of restriction enzymes,

the DNA of a second strain of Erwinia carotovora, or trap DNA, is cut into fragments, of approximately 1,000 to approximately 10,000 nucleotides, in particular by sonication, these fragments being subsequently labeled, the abovementioned fragments of target DNA and of trap DNA are mixed in proportions such that the trap DNA is in excess relative to the target DNA,

the target DNA and trap fragments thus mixed are denatured by heating, which leads to the formation of target DNA fragments and single strand trap,

- then the single-stranded DNA fragments obtained in the previous step are placed under hybridization conditions such that they allow the formation of double-stranded DNA fragments consisting of two strands of target DNA (target homoduplex), or of two strands of trap DNA (homoduplex trap), or of a strand of target DNA and of a strand of trap DNA (heteroduplex),

the double-stranded DNA fragments obtained in the previous hybridization step are then placed in the presence of a reagent having an affinity for the abovementioned marker, this reagent being immobilized on a solid support so that the homoduplex traps and the labeled heteroduplexes remain fixed on the solid support by bonding between the marker used to mark the trap DNA and the abovementioned reagent,

recovery, in particular by filtration or sedimentation of the solid support, of the unlabeled target homoduplexes therefore remaining in solution and constituting the nucleotide sequences contained in the genome of the first strain of Erwinia carotovora, but not in the genome of the second strain, and therefore likely to hybridize with the genome of the first strain, but not with that of the second strain.

5. Nucleotide sequences according to claim 4, characterized in that the first strain of Erwinia carotovora is a pathogenic strain in a given plant, while the second strain of Erwinia carotovora is not pathogenic, under the same conditions (from culture, climatic, etc.) than for the previous strain, in this same plant.

6. Nucleotide sequences according to claim 5, characterized in that the strain of pathogenic Erwinia carotovora is a strain of Erwinia carotovora subsp. atroseptica (Eca) responsible for rots in certain plants, in particular the black leg of the potato, and the non-pathogenic strain of Erwinia carotovora is a strain of Erwinia carotovora subsp. carotovora (Ecc) does not induce rots in these same plants.

7. Nucleotide sequences according to one of claims 1 to 6, characterized in that they correspond to one of the nucleotide sequences A to F shown in Figures 1 to 6 respectively, or to their complementary sequences, or to fragments of these sequences, or to any of the above-mentioned sequences modified by substitution and / or addition and / or deletion of one or more nucleotides, said modified fragments or sequences being capable, like the sequences A to F mentioned above, of hybridizing with the genome of pathogenic Erwinia carotovora strains.

8. Nucleotide sequences according to one of claims 1 to 7, characterized in that they correspond to the nucleotide sequence B represented in FIG. 2, or to its complementary sequence, or to fragments of these sequences, or to all sequences aforementioned modified by substitution and / or addition and / or deletion of one or more nucleotides, said modified fragments or sequences being capable, like the sequence

B mentioned above, to remain hybridized with the genome of the strains of Erwinia carotovora subsp. atroseptica (Eca) pathogens, in particular under the hybridization conditions defined below: hybridization in 6 SSC, 0.1% SDS and 0.01% skimmed milk at 65 ° C overnight, followed more particularly by two washes 30 minutes at 68 ° C, 0.1 SSC, 0.5% SDS.

9. Nucleotide sequences according to claim 8, characterized in that they are capable of remaining hybridized with the genome of the pathogenic strains of Eca, as well as with the genome of the strains of Erwinia carotovora subsp. betavasculorum (Ecb) pathogens, in particular under the hybridization conditions defined below: hybridization in 6 SSC, 0.1% SDS and 0.01% skimmed milk at 65 ° C overnight, followed more particularly by two washes 30 minutes at 65 ° C, 0.3 SSC, 0.5% SDS.

10. Nucleotide sequences according to one of claims 1 to 9, characterized in that they are marked, in particular radioactively, enzymatically, by a fluorescent compound, by a bond to an antigenic molecule (capable of being recognized by antibody) or to any other molecule capable of being directly or indirectly detected using reagents, this linkage possibly being carried out via a spacer arm, in particular via a nucleotide sequence consisting of '' approximately 5 to 100 nucleotides located at least at one of the ends of these sequences.

11. Nucleotide sequences according to one of claims 1 to 10, characterized in that they are used as nucleotide probes for the detection of strains of pathogenic Erwinia carotovora.

12. Primer pairs used for the amplification of the copy number of the nucleotide sequences according to one of claims 1 to 11, or of the specific nucleotide sequences contained in the genome of the pathogenic strains of Erwinia carotovora, with which said nucleotide sequences according to one of claims 1 to 11, are capable of hybridizing, these primers advantageously consisting of approximately 7 to approximately 30 nucleotides derived from the nucleotide sequences according to one of claims 1 to 10, and being optionally labeled in particular as indicated in claim 10.

13. A pair of primers according to claim 12, characterized in that the two primers correspond to the following formulas: B105 5 'TAAGGCAGCATTGAATCGCCACAGAGAGTA 3' (SEQ ID NO 8) B106 5 'ACACGCATTTATCGCTATCAGGCACAACAA 3' (SEQ ID NO 9)

14. Method for detecting and identifying strains of pathogenic Erwinia carotovora possibly present in soil or water, or in a host, in particular in plants and / or seeds, likely to be an apparently healthy carrier of such bacteria and being in the latent phase of infection, this process comprising the following stages:

- the treatment of a sample taken from the soil or water or from this host so as to make the genome of these Erwinia carotovora accessible to the probes and / or the primers defined in claims 11 to 13, and if necessary to any DNA or RNA polymerase making it possible to replicate the two strands of genomic DNA or the RNA derived therefrom,

bringing the sample thus treated into contact with probes according to claim 11, and / or with primers according to claim 12 or 13,

- where appropriate, amplification using pairs of primers according to claim 12 or 13, of the number of copies of the nucleotide sequences specific for pathogenic Erwinia carotovora, and contained in the genome of these bacteria likely to be present in this biological sample, and with which the probes or primers are likely to hybridize mentioned above, and / or the amplification of the number of copies of probes according to claim 11,

- detecting the possible presence of said specific nucleotide sequences contained in the genomes of pathogenic Erwinia carotovora, in particular using the aforementioned advantageously labeled probes, said sequences being hybridized with the aforementioned probes, and therefore the presence of the responsible pathogenic Erwinia carotovora of this pathology in the sample studied.

15. Method according to claim 14, characterized in that it is carried out using all or part of the probe consisting of the nucleotide sequence B shown in FIG. 2, optionally marked, in particular in the manner indicated in claim 10, and / or using the pair of primers according to claim 13, where appropriate marked, in particular as indicated in claim 10.

16. Method according to claim 14 or claim 15, characterized in that it is applied to the diagnosis of the possible appearance of a pathology caused by Erwinia carotovora, in a host as defined in claim 14.

17. Method according to claim 15 and claim 16, characterized in that it is applied to the diagnosis of the possible appearance of the black leg caused by the Eca, in particular in the potato, or of the root necrosis caused by Ecb, in particular in sugar beet, in a host as defined in claim 14.

18. Kit for implementing a method according to one of claims 14 to 17, characterized in that it comprises at least one probe according to claim 11, and / or at least a pair of primers such as described in claim 12 or 13.

19. Kit according to claim 18, characterized in that it comprises:

- a heat-resistant DNA or RNA polymerase, - a reaction medium advantageously constituted, in the case of the PCR technique, of 10 mM Tris-HCl buffer, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, cofactors of DNA polymerase, in particular of Mg2 + and K ⁺ ions, and of the 4 deoxynucleotides constituting DNA (dCTP, dATP, dGTP, dTTP) or RNA (dCTP, dATP, dGTP, dUTP).

20. Vector, in particular plasmid, containing at one of its non-essential sites for its replication, a nucleotide sequence according to one of claims 1 to 10.

21. A cell host transformed by a vector according to claim 20, and in which said vector, or the nucleotide sequence according to one of claims 1 to 10, integrated into the genome of the host, is capable of replicating.

22. Method for obtaining a nucleotide sequence according to one of claims 1 to 10, comprising the following steps:

culture of a cell host according to claim 21, transformed with a vector according to claim 19,

- recovery of the vectors from this cell host, in particular by an appropriate cell lysis treatment,

recovery of the abovementioned nucleotide sequence by treatment of the vectors recovered in the preceding step, using appropriate restriction enzymes,

- Purification of the nucleotide sequence thus obtained, in particular by gel electrophoresis.