DE19859971A1 - New DNA sequence from Sirococcus, useful for detecting fungal pathogens in plants, particularly conifers, by amplification or hybridization - Google Patents

Abstract

A DNA sequence (I) of 944 base pairs (bp), fully described in the specification, and its fragments, is new. Independent claims are also included for the following: (1) oligonucleotides (II) derived from (I); (2) primer pair comprising two (II); (3) detecting fungal pathogens by amplification with (II) or hybridization to (I) or (II); and (4) test kits containing (II).

Description

The invention relates to new nucleic acid sequences from the genome of the phytopathogen Sirococcus conigenus, oligonucleotide primers derived from these nucleic acid sequences, Procedure for the qualitative and / or quantitative detection of phytopathogenic pathogens under Use of the sequences and / or primers according to the invention and the use of Sequences and / or primers for the specific detection of phytopathogens on the basis of hybridization and / or amplification methods.

Sirococcus conigenus (also known as S. strobulinus) is the causative agent of the instinct death Spruce is known as an economically and ecologically relevant phytopathogen. But not only Picea pungens, Picea abies and other species of spruce are highly sensitive to attack caused by Sirococcus, Sirococcus infections and related damage described for numerous different conifers during the past decades. So were damage caused by Sicococcus infestation in the U.S., especially the death of new ones Shoots, u. a. observed in various types of pine (e.g. Pinus resinosa, Pinus jeffreyi, Pinus coulteri, Pinus lambertiana, Pinus contorta, Pinus ponderosa and Pinus banksiana), Hemlock fir (e.g. Tsuga heterophylla, Tsuga mertensiana) and spruce (e.g. Picea rubens and Picea sitchensis).  

In addition to the death of shoots, the complex pattern of damage includes fire and / or Brown rot on the branches and trunk areas of ongoing annual growth and that Yellowing or browning of the needles of infected shoots at the needle base, which is usually leads to the death of the entire needle. One infection is probably enough to cause a shoot to die; seem to kill a young tree multiple yeast infections may be required. Trees that do not die become common malformed and overgrown. The lower branches of older trees often die as a result multiple infections.

Large scale damage from Sirococcus has been observed recently, in both Europe and the United States, as there is currently no biological resistance to Sirococcus are available and the large-scale use of fungicides is prohibited, the chain of infection can only be effective in the context of reforestation interrupt if the spread of Sirococcus-infected conifers with the help phytosanitary control mechanisms is prevented. Because of a Sirococcus infection in primarily young shoots and sprouts are affected, especially in nurseries Need for effective control and diagnostic procedures.

A purely optical monitoring of stocks through identification and removal infected plant parts is not entirely inadequate not only because of Sirococcus long undetected and asymptomatic in the host parasitized and Sirococcus damage only at a relatively late point in the course of the infection process further complicated by the fact that Sirococcus-related damage easily with winter and Frost damage can be confused. There are also indications that predisposing factors such as environmental stress favor Sirococcus epidemics.

There is therefore a need for detection methods that are necessary for a specific mass screening damaged or potentially infected locations are suitable to prevent infection to be able to determine qualitatively and / or quantitatively with pathogens.

Sirococcus can be detected using classic microbiological detection techniques based on the Cultivation of the fungus based on infected tissues can be detected. However  these classic cultural processes in terms of space and time are extremely complex and for not suitable for use in mass screening. Also overgrown often secondary pathogens such as bswp. Rhizosphaera kalkhoffii, Lophodermium piceae, Epicoccum purpurascens and Penicillium species are the pathogens to be detected during the Cultivation, which makes the subsequent detection of Sirococcus extremely difficult or makes impossible. In addition, is more conventional for experimental implementation Culture methods usually require special microbiological knowledge, so in the present case the experience of a microbiologist with special knowledge of species. Furthermore are immunulogical detection techniques with fluorescence-labeled antibodies have been developed (Mitchell (1986) Can. J. For. Res. 16: 939-944; Mitchell, Sutherland (1986) Can. J. For. Res. 16, 945-948; Mitchell (1988) Plant Disease 72: 664-667; but also not for are suitable for use as part of a site screening. For one, they are immunological procedures are extremely complex and costly, on the other hand they are not species-specific because the antibody-determinants of the surface proteins of Pathogen factors among close relatives are often very similar, leading to undesirable ones Cross reactions.

There is therefore an urgent need for diagnostic procedures that are specific Enable identification of Sirococcus at the beginning of the infection process, so too a time when infection with the naked eye and the like. U. is not yet recognizable. By early identification of the specific pathogen before symptoms of the disease become visible, it is possible to separate out infected shoots, the further development of the Counteract pathogen infestation through the use of fungicidal agents or others to take appropriate control measures.

It is therefore an object of the invention to provide DNA sequences by means of which or on the basis of which the Sirococcus pathogen is specific, simple and reliable in / on its Hosts can be demonstrated.

This object is achieved by the in claim 1 with reference to SEQ: ID No. 1 specified DNA sequence from the genome of Sirococcus conigenus solved. Preferred embodiments of the invention result from the subclaims.  

The invention is based on the successful isolation of DNA sequences from the genome from Sirococcus conigenus, which is a species-specific construction of oligonucleotide enable primarily for specific amplification methods, in particular PCR methods, with the help of Sirococcus infestation of conifers in their organs and tissues, in particular needles and shoot tips, determined quickly, inexpensively and reliably can be.

It is the first time thanks to the identified species-specific genome areas of Sirococcus possible the presence of Sirococcus species specifically in tissues and in / on organs of conifers using hybridization and / or amplification techniques specifically to prove. Cross reactions with other needle pathogens do not occur saprophytic accompanying fungal flora. This is species-specific proof and Quantification of Sirococcus in its hosts even in the presence of a variety of others Microorganisms possible. The invention takes advantage of the polymerase chain reaction, in particular the specificity of the reaction and the small amount of time required to analyze a enable a large number of samples on a laboratory scale without special knowledge in experimental implementation of the method according to the invention is required.

The in SEQ: ID No. 1 shown sequence or fragments thereof or nucleic acid molecules, those in SEQ: ID No. 1, or parts thereof, represent useful ones Hybridization probes because they react specifically with Sirococcus DNA or RNA.

Furthermore, according to the invention on the basis of the in SEQ: ID No. 1 shown DNA Sequence constructed oligonucleotide primers and pairs of oligonucleotide primers provided a species-specific proof and a species-specific quantification enable fungal nucleic acids using the PCR technique. The fiction Sirococcus-specific primers are also used together with other primers be, e.g. B. primers derived from a conserved sequence region (e.g. ITS- Regions) within the fungal DNA. In general, the Oligonucleotide primers have a theoretical melting temperature of about 60 ° C to about 70 ° C to ensure sufficient sensitivity and as free as possible from significant secondary structures and 3 'overlaps between primer combinations  his. Generally, the primers should be at least about 5 to about 10 in length Nucleotides, preferably of at least 20 nucleotides.

The invention also relates to fragments of those shown in SEQ: ID No. 1 shown sequence, the one have sufficient length to be specific with Sirococcus nucleic acids to be able to hybridize.

The oligonucleotides according to the invention, the species-specific detection and species-specific quantification of Sirococcus enable such oligo include nucleotides, the sequence of which is in single or multiple nucleotides by addition, deletion, Substitution or chemical modifications of that described in SEQ: ID No. 1 shown Sequence immediately derived oligonucleotide sequence differ, as far as a ensures specific hybridization of such primers to template DNA from Sirococcus is. The same naturally applies to those used as PCR primers or as hybridization probes fragments used in SEQ: ID No. 1 sequence shown.

Furthermore, the invention also includes those primer sequences whose nucleotide sequence corresponds to that oligonucleotide primer sequences according to the invention is complementary or that of the correspond to RNA sequences derived from oligonucleotide primers according to the invention.

The invention also relates to those oligonucleotide primer sequences which belong to the oligonucleotide primer sequences according to the invention have a sequence homology of at least 80%, preferably at least 90% and particularly preferably of have at least 95%. The invention further encompasses such oligonucleotide primers Sequences with the in SEQ: ID No. 1 shown DNA sequence or genomic DNA of Sirococcus conigenus under usual hybridization conditions, preferably under stringent hybridization conditions, hybridize. Generally, the invention includes such Oligonucleotide primers that are species-specific hybridization and / or amplification of Sirococcus nucleic acid sequences. The professional who is familiar with Hybridization or amplification methods is familiar, is without difficulty in the Able to recognize whether a species-specific binding of the respective oligonucleotide primer with Sirococcus DNA, or whether it is an unspecific, by which Invention not included binding. While in the case of PCR with a non-specific Amplification unexpected additional reaction products occur or a more diffuse  Nucleic acid "smear" can be observed in the context of hybridization reactions non-specific interactions result in that the hybridization probe at high Temperatures and low salt concentrations (e.g. 0.1 × SSC and 0.5% SDS) from the target replaces.

The DNA sequences and oligonucleotide primer sequences according to the invention can in conventional hybridization reactions as well as in amplification processes, especially in polymerase chain reactions (PCR), for species-specific detection of Sirococcus can be used. Those skilled in the art are known basic methods; they are also common in molecular biology Laboratory manuals are described, for example in Sambrook et al. (1989) Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Griffin and Griffin (1994) PCR Technology - Current Innovations. CRC Press, Boca Raton, Ann Arbor, London, Tokyo.

In addition to conventional PCR techniques, in particular PCR and reverse transcriptase (RT) - PCR, which are usually used for quantitative nucleic acid analysis, can PCR methods have also been used that have been developed or have been further developed and z. T. significant advantages over the first mentioned Techniques. In this context, the so-called "nested" are particularly worth mentioning. PCR and the so-called "real time" quantitative PCR. With the nested PCR, an additional second pair of primers are used, the sequences of which are also derived from the sequence shown in SEQ: ID No. 1 shown sequence are derived and within the region of the target sequence with hybridize this, which lies between the first pair of primers. With real time PCR from the region between the forward and reverse primer that is shown in SEQ: ID No. 1 shown sequence derived an additional fluorescence probe, the light emission of which Quantification of the amplicons allowed (see e.g. Heid et al. (1996) Genome Res., Pp. 986-994; Orlando et al. (1998) Clin. Chem. Lab. Med. 36, 255-269). An overview of suitable ones For example, PCR techniques are offered by Griffin and Griffin (1994) supra.

In general, the polymerase chain reaction has the great advantage that it is extremely sensitive is, so very small amounts of DNA are sufficient to detect Sirococcus-specific DNA can. The person skilled in technology is able to do this without any problems, if necessary using a routine experiment  elements, suitable PCR conditions, in particular temperature conditions and number of cycles, to investigate.

The following PCR conditions are generally suitable for the species-specific detection of Sirococcus:

• - Denatuation of the DNA sample to be examined at 94 ° C for at least 1 min .;
• Annealing of the primers according to the invention to the single-strand template for 1 min. at a temperature that is approx. 2-5 ° C below the thermodynamic primer melting point,
• - Extension reaction at approx. 72 ° C, catalyzed by a thermostable DNA Polymerase for at least 1 min. (approx. 500 base pairs / min),
• 20 to 40 reaction cycles, each consisting of denaturation at 94 ° C. for 30 seconds, followed by annealing at the above annealing temperature for 1 min. and Primer extension at 72 ° C for at least 1 min.

In a preferred embodiment, the following PCR protocol is followed:

• - denaturation at 94 ° C for approx. 3 min.,
• - Annealing of the primers according to SEQ ID No. 2 and SEQ ID No. 3 to the person to be examined single-stranded template DNA at at least 60 ° C, preferably at about 66 ° C and especially preferably at approx. 70 ° C for 1 min.,
• - Extension reaction of the two primers along the DNA template at 72 ° C for 2 min.
• - At least 25 and preferably at least 30 reaction cycles as follows. Denaturation at 94 ° C for 30 sec., Annealing at at least 60 ° C., preferably at approx. 66 ° C. and particularly preferably at approx. 70 ° C for 1 min., Primer extension at 72 ° C for 2 min.,
• - finally an extension reaction at 72 ° C for 5 min.

The Sirocossus-specific DNA resulting from the PCR amplification Using conventional methods, molecules can be detected and visualized and, if desired, be quantified. So the reaction products z. B. gel electrophoretic or detected fluorimetrically using fluorescence-labeled oligonucleotides become. Other detection methods known to the person skilled in the art are of course also possible applicable.  

In an alternative embodiment of the invention, the invention Oligonucleotide primer with the sample to be examined for the presence of Sirococcus brought to hybridization, preferably stringent hybridization conditions can be selected to ensure species-specific hybridization. Under stringent reaction conditions in connection with this invention Hybridization temperature of approx. 5-10 ° C below the respective primer melting point Roger that. The stability of the DNA / DNA or RNA / DNA hybrids should be self low salt concentrations corresponding to 0.1 × SSC / 0.5% SDS can be guaranteed. On this can prevent undesirable cross-reactions from occurring with other species become. The respective temperature conditions can, however, depend on the selected test conditions and depending on the to be examined Nucleic acid samples can be different and can be adapted accordingly by a person skilled in the art and optimized if necessary. The detection of the hybridization product can, for example, by Autoradiography in the case of radioactively labeled primer molecules or by fluorimetry Fluorescence-labeled oligonucleotides are used.

Likewise, of course, those in SEQ ID No. 1 DNA sequence shown or Fragments thereof can be used as Sirococcus-specific hybridization probes.

The Sirococcus DNA to be analyzed can be used in the amplification reaction as well in the hybridization reaction either in extracted form or in form fixed slices on which in situ PCR or hybridization is performed.

The invention further relates to a test kit for the specific detection of Sirococcus can be used and one or more of the invention Oligonucleotide primer. The primer (s) can e.g. B. in lyophilized form or in one suitable buffers are available. Other kit components can be enzymes and reagents, which are usually used in the polymer chain reaction, in particular a DNA polymerase. The enzyme (s) can also be in lyophilized form or in suitable buffers are available. The kit can of course also include any additional, for the detection method according to the invention include necessary elements, such as Reaction buffers, extraction reagents, enzymes, nucleic acids, dNTPs, etc.  The following exemplary embodiments serve to explain the invention.

Embodiments example 1 Sirococcus strains used and controlled Picea abies infection

Sirococcus isolates were kept on 2% malt extract medium and on oat agar. Four strains of S. conigenus were grown by the Centraalbureau voor Schimmelcultures in Baarn, Netherlands, preserved: 489.63 and 113.75 were from Picea pungens (origin Germany), 505.50 from P. abies, (origin Norway), and 265.85 from Picea sp. (Origin Germany), isolated. A fifth strain (S289) was from fruiting bodies on needles from P. abies from the Bavarian Forest, Germany, isolated. All tribes were made for the Construction of Sirococcus conigenus-specific PCR markers used.

Additional cultures were used to test the specificity of Sirococcus-specific primer pairs used: Rhizophaera macrosporum (CBS 208.79, 134.81, and 467.82), R. kalkhoffii (CBS 280.38), Lophodermium macrosporum (CBS 200.38, L236), L. piceae (L249), Naemacycle minor (CBS 496.73), N. niveus (CBS 495.73), Botryodiplodia exelsa (CBS 102.64), Sphaeropsis sapinea (CBS 146.61), Gremeniella abietina (CBS 281.70 and 343.58), Epicoccum purpurascens. (K211) and three isolates from Penicillium sp., Obtained from needles by P. abies.

For the infection of Picea abies, seedlings were grown on a mineral substrate according to the following protocol. Polypropylene tubes were filled with autoclaved mineral substrate and 800 ml of sterile distilled water. 12 g of spruce seeds (corresponds to approximately 1000 seeds), obtained from the State Seed Cycle, Laufen, Bavaria, Germany, origin 84026, were soaked in water overnight, then in 100 ml of 30% H ₂ O ₂ for 30 min. Surface-sterilized with stirring at room temperature. After rinsing with sterile distilled water, approximately 150 spruce seeds were evenly distributed per polypropylene jar. In order to maintain a high level of humidity, transparent acrylic glass boxes were placed over the polypropylene tubes. After 4 weeks of incubation in the climate chamber, the acrylic glass boxes were removed, the seedlings cultivated for a further 2 weeks and then used for artificial infection experiments. Here, Sirococcus was incubated for 4 weeks on oat agar (24 ° C., daylight), after which a single UV light pulse of 30 seconds was applied after 3 weeks. The spores were taken up in 0.1% Tween 20 and diluted to a density of 10 ⁵ / ml to 10 ⁶ / ml. The polypropylene vessels with the spruce seedlings were each sprayed with 30-40 ml spore suspension.

The vessels were then closed again with acrylic glass boxes and in Darkness incubated at 24 ° C for approx. 17 h. Then the open ones were cultivated Boxing for 5 weeks in the climate chamber. After 38 days some of the seedlings were dead, others showed brown discoloration.

Example 2 DNA extraction and RAPD analysis

Fungal DNA was obtained from fungal cultures according to the protocol of Möller et al. (Nucl. Acids. Res. (1992) 20: 6115-6116). Spruce DNA was made from needle crowns after the Bahnweg et al. (Anal. Biochem. (1998) 262: 79-82).

The amplification using randomized 10-mer primers was carried out in a reaction volume of 25 μl, containing 50 mM KCl, 2.0 mM MgCl ₂ , 10 mM Tris / HCl, pH 9.0, 100 μM per dNTP (Pharmacia, Freiburg) , 0.2 µM of a RAPD primer (RAPD = random amplified polymorphic DNA; OPERON Technologies Inc., Alameda, California, USA: primer OPA-1 - OPA-20), 0.4 mg bovine serum albumin (DNase-free, Pharmacia) , 1 unit of Taq DNA polymerase (Pharmacia) and approximately 25 ng of fungal template DNA, carried out in a conventional thermal cycler. The PCR reaction conditions were as follows: initial denaturation at 94 ° C (3 min.), Annealing of the primers at 40 ° C (1 min.) And primer extension at 72 ° C (2 min.), Followed by 35 cycles of 94 ° C (30 sec.), 40 ° C (45 sec.) And 72 ° C (2 min.). A final elongation was carried out for 5 min. performed at 72 ° C to ensure a double-stranded amplicon. The PCR products were analyzed by horizontal agarose gel electrophoresis (1.5%) according to Schulze et al. (Eur. J. For. Path. (1997) 27: 225-239).

Example 3 Cloning and sequencing of RAPD fragments

The agarose gels were analyzed for amplicons found in all Sirococcus isolates occurred, but in a collection of the others mentioned above Needle pathogens were not present. Sirococcus-specific PCR bands were obtained from the Agarose gels using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) isolated according to the manufacturer's protocol. Approximately 300ng of agarose gels Isolated DNA, contained in a volume of 2 ul, were with 1 ul pT7-Blue T vector (= 50 ng; Novagen, sold by AGS GmbH, Heidelberg), 1 µl 10 × ligation buffer (Novagen), 0.5 µl 100 mM DTT (dithiothreitol), 0.5 µl 10 mM ATP (adenosine triphosphate), 4.5 µl nuclease-free water and 0.5 µl (1 unit) T4-DNA ligase (Novagen) mixed and incubated at 16 ° C for 12 h. Competent NovaBlue E. coli K12 cells (Novagen) were with a 1 ul aliquot of the ligation reaction mixture according to the manufacturer's protocol transformed. Recombinant bacterial cells were grown at 37 ° C after overnight cultivation on LB agar plates containing 50 µg / ml ampicillin, 15 µg / ml tetracycline, 35 µl of 50 mg / ml X-Gal (5-bromo-4-chloro-indolyl-β-D-galactoside, dissolved in dimethylformamide) and 20 ul 100 mM IPTG (isopropyl-1-thio-β-D-galactoside), selected.

The DNA of randomly selected recombinant pT7-Blue T plasmid derivatives was extracted liquid bacterial cultures using the Qiagen plasmid mini kit and Qiagen tip 20 columns (Qiagen, Hilden, Germany) cleaned. The plasmids obtained became double-stranded sequenced.

In this way, a Sirococcus-specific 944 bp amplicon with the primer OPA- 2 (5'-TGCCGAGCTG-3 ') can be generated. This fragment was transformed into the vector pT7-Blue T cloned and its nucleotide sequence determined. The DNA sequence obtained is in SEQ: ID No. 1 shown. This PCR fragment could not be detected in any of the approaches Contained template DNA of another needle pathogen.  

Example 4 Amplification of specific DNA fragments and quantitative PCR with specific ones Primers

The specific primers SIR01 (5'-TGC CGA GCT GAC GGG CCT C-3 '; SEQ: ID No. 2) and SIR06 (5'-TGC CGA GCT GTC TAC AGA GAA-3 '; SEQ ID No. 3) were from the Sequence of the RAPD amplicon from Example 3 by extending the RAPD primer via their 3 'ends derived beyond.

Using this primer pair, the 944 bp. Fragment (SEQ ID No. 1) could be PCR using template DNA from all Sirococcus isolates, but not with Target DNA from the remaining needle pathogens tested are amplified. About that In addition, it could be shown in dilution experiments that approx. 1 pg fungal DNA / mg fresh plant material (e.g. needles, wood, bark) for the detection of Sirococcus using Use of the primers according to the invention is sufficient, which is approximately 10 to 40 fungal genomes corresponds. Furthermore, it was found that the presence of 200 ng of spruce DNA in each individual reaction had no influence on the sensitivity of the reaction.

The amplification was carried out in 25 μl reaction batches as described in Example 2 following modifications were carried out: 1) Instead of the RAPD primer, the specific Primer pair SIR01 / SIR06 (each 0.2 µM) used, 2) the amount of template DNA varied between 1 pg and 100 ng per reaction batch and 3) the thermal cycler Conditions were as follows: initial denaturation at 94 ° C (3 min.), Primer annealing at 70 ° C (1 min.) and primer extension at 72 ° C (2 min.), followed by 30 cycles: 94 ° C (30 sec.), 70 ° C (1 min.) And 72 ° C (2 min.). A final extension was made for 5 min. at 72 ° C to ensure a double-stranded amplicon. The PCR products were analyzed by horizontal agarose gel electrophoresis (1.5%). The ethidium bromide-stained gels were created using the ImageMaster® video documentation system (Pharmacia, Freiburg) and related software.

Fig. 1 shows the result of a PCR with the primer pair SIR01 / SIR06 after separation by gel electrophoresis. The specific 944 bp amplicon was only observed when using template DNA from Sirococcus strains (lanes 1-4). The DNA tested comes from the following strains:
Lane 1: Sirococcus strobilinus 265.85 (1)
Lane 2: Sirococcus strobilinus 489.63 (1)
Lane 3: Sirococcus strobilinus 113.75 (1)
Lane 4: Sirococcus strobilinus S289 (2)
Lane 5: Rhizosphaera macrosporum 208.79 (1)
Lane 6: Rhizosphaera macrosporum 134.81 (1)
Lane 7: Rhizosphaera macrosporum 467.82 (1)
Lane 8: Rhizosphaera kalkhoffii 280.28 (1)
Lane 9: Lophodermium macrosporum 200.3 8 (1)
Lane 10: Lophodermium macrosporum L236 (2)
Lane 11: Lophodermium piceae L240 (2)
Lane 12: Naemacyclus minor 496.73 (1)
Lane 13: Naemacyclus niveus 495.73 (1)
Lane 14: Botryodiplodia exelsa 102.64 (1)
Lane 15: Sphaeropsis sapinea 146.61 (1)
Lane 16: Gremeniella abietina 281.70 (1)
Lane 17: Gremeniella abietina 343.58 (1)
Lane 18: Epicoccum purpurascens GB211 (3)
Lane 19: Penicillium sp. GB225 (3)
Lane 20: Penicillium sp. GB226 (3)
Lane 21: 100 bp ladder as size standard

Cultures of (1) = Centraalbureau voor Schimmelcures, Baarn, Netherlands; (2) = Federal Biological Research Center, Braunschweig; (3) GSF Research Center for Environment and Health, Neuherberg.

Example 5 Detection of S. conigenus in plant tissue

Samples (green needles, dead needles, bark, wood) of Sirococcus-infected spruces from a reforestation station near Landshut, Bavaria, were analyzed oligonucleotide primer according to the invention. There were also parts of such trees and shoots that showed no symptoms (yet). The DNA was isolated and in PCR reactions with the primer pair SIR01 / SIR06 were used.

Fig. 2 shows the result of a PCR in which the following DNA samples were examined: brown needles of sick shoots in Lane 2, green needles of sick shoots in Lane 3, green needles of symptom-free shoots from sick trees in Lane 4, bark of sick shoots in lane 5, bark of symptom-free shoots in lane 6, wood from diseased shoots in lane 7, wood from symptom-free shoots in lanes 8 and 9, 100 bp. Head as size marker in lanes 1 and 10, and Sirococcus DNA as positive Check in lane 11.

In the practice of the invention are within the scope of a site screening in the forest or in a tree nursery prefers to collect needle and shoot tip samples and from about each 100 mg of sample material is isolated from total DNA using established protocols. In PCR Approaches with the oligonucleotide primers according to the invention are for the pathogen characteristic DNA fragments (corresponding to the DNA sequence according to SEQ: ID No. 1 or parts thereof) and amplified in agarose gel electrophoresis according to standard Methods proven.  

SEQUENCE LOG

Claims (11)

1. DNA sequence according to SEQ: ID No. 1 and fragments thereof. 2. Nucleic acid molecule comprising the DNA sequence according to claim 1 or Fragments of it. 3. oligonucleotide derived from the DNA sequence of claim 1. 4. Oligonucleotide according to SEQ: ID No. 2 or SEQ: ID No. 3rd 5. oligonucleotide primer pair comprising at least one oligonucleotide primer according to claim 3, in particular according to SEQ: ID No. 2 and / or SEQ: ID No. 3rd 6. A method for the detection of a fungal pathogen, preferably Sirococcus, particularly preferably Sirococcus conigenus, comprising the steps:

• a) isolating nucleic acids from a plant tissue / organ infected with the pathogen or producing a cutting preparation suitable for in situ methods and
• b) amplification of pathogen-specific DNA using the isolated nucleic acids as a template or in situ amplification of pathogen-specific DNA in a polymerase chain reaction with at least one oligonucleotide according to claim 3 or 4 as a primer.

7. A method for the detection of a fungal pathogen, preferably Sirococcus, particularly preferably Sirococcus conigenus, comprising the steps:

• a) isolating nucleic acids from a plant tissue / organ infected with the pathogen or producing a cutting preparation suitable for in situ methods and
• b) hybridizing the isolated nucleic acids or in situ hybridizing the cut preparation with at least one nucleic acid molecule or oligonucleotide according to one of claims 2 to 4.

8. Test kit comprising at least one oligonucleotide according to claim 3 or 4.   9. A test kit according to claim 8, comprising the oligonucleotide primer pair according to SEQ: ID. 2 and SEQ: ID No. 3rd 10. Use of a nucleic acid molecule or an oligonucleotide according to one of claims 2 to 4 in one based on amplification and / or hybridization Detection method for fungal nucleic acids. 11. Use of the DNA sequence according to claim 1 for the construction of Oligonucleotides containing nucleic acids from Sirococcus, especially genomic DNA of Sirococcus, hybridize specifically.