CZ2011874A3 - Hybridization probes for detection of pathogens, causing wheat and barley leaf blotch - Google Patents

Abstract

The present invention relates to hybridization probes for the detection of pathogens causing foliar and spleen leaf pathogens, which have been designed according to the ribosomal RNA gene sequence, the glyceraldehyde-3-phosphate dehydrogenase gene and the RNA polymerase II large subunit gene. These probes can be used to prepare a microcip capable of detecting about 20 different pathogens.

Description

The solution relates to hybridization probes specific for the detection of pathogens causing leaf spot in wheat and barley, which is important in phytosanitary, plant breeding and molecular biology.

State of the art

Accurate and timely diagnosis of pathogens is of great importance in the field crop protection system. Classical diagnostic methods such as visual evaluation of symptoms, microscopic or culture methods are cheap, but especially in the complex of leaf spots, they have only limited possibilities. Different pathogens, such as Pyrenophora teres, Cochliobolus sativus, Rhynchosporium sativum, Ramularia collocygni and Alternaria alternata, can coexist in a single plant and their symptoms overlap and cannot be visually distinguished. Some species, such as Ramularia collo-cygni, can develop for much of the growing season without visible symptoms. Some symptoms can be very easily mistaken for so-called physiological spots caused by abiotic stressors: UV radiation, frost or over-fertilization with some elements, etc. Microscopic examination usually evaluates the morphology of conidia or spores, which are also characterized by a degree of variability. The microscopic method places particularly high demands on the erudition, experience and taxonomic knowledge of the phytopathologist. Cultivation techniques, in turn, may prefer certain types of pathogens to the chosen nutrient medium, to which the given medium is better suited, and this may be the cause of inaccurate diagnosis, moreover, this method is time consuming. With the discovery of the polymerase chain reaction (PCR) (Mullis and Floona: Methods in Enzymology, 1987, 155: 335-350), molecular methods are gradually being used in the diagnosis of pathogens. DNA microarray technology (Augenlicht and Kobrin: Cancer Research, 1982, 42 (3): 1088-1093) offers the advantage over the PCR reaction of simultaneous detection of all pathogens present in a single sample for which a specific probe has been designed. Thus, several independent PCR reactions for the detection of one particular pathogen are replaced by the determination of all pathogens present in one analysis. Self-diagnosis can be performed by personnel without knowledge of taxonomy, who can only handle standard molecular techniques. Although the identification of pathogens causing leaf spot in wheat and barley, although their identification and detection are very difficult in practice, the DNA microarray method has not yet been used, mainly because specific hybridization probes have not yet been developed.

The essence of the invention

The above-mentioned shortcomings in diagnostics are eliminated by hybridization probes for the detection of pathogens causing leaf spot in wheat and barley, according to the invention, the essence of which is that each hybridization probe is specific

to one species of pathogen of composition Probe designation Probe sequence Pathogen species specific: PG-gpd CCATGGAGTCACAGGAGGCAGCAGGCTAAT Pyrenophora graminea PA-gpd CTATTCGGCCCTGGGAGACTGCAGGCTAAT Pyrenophora avenae PB-gpd CGATGGATCTGCAGGATACAGCAGACTAAC Pyrenophora bromi PTT-gpd CCATGGAGTTACAAGAGACAGCAGGCTAAT Pyrenophora teres f. Teres PTM-gpd CCATGGAGTCACAAGATACAGCAGGCTAAT Pyrenophora teres f. Maculata PTR-gpd CGATGGATCTGCAGGATACAGCAGACTAAT Pyrenophora tritici-repentis CS-gpd CACTGGTATCATTCCTTTGATGGAACTATG Cochliobolus sativus RCC-gpd AACAATGGCCAAGACAATCGCATTTAACAT Ramularia collo-cygni RS2-gpd CTATTCGCCCCTGGGAGCCTGCAGGCTAAT Rhynchosporium secalis BG-gpd ATACCATGGGCCGAAACTGGAGCCGAGTAC Blumeria graminis PUT-gpd CCTTGGAATGCAGGTGCTAACAGGACATTG Puccinia triticina AA-gpd CGCTAGAGCTCCTCTGTGGTCGCAGAATGC Alternaria alternata AT-gpd CTATGACTTCAGTACGCAAGCTAACACACG Alternaria triticina AM-gpd CTACAACTACAGGACGCAGGCTAACACACA Alternaria metáchromatica CH-gpd CATTCACTTGCACCGGTCGCTCCTGCTCAC Cladosporium herbarum CC-gpd GCATATAGCAACATCATCATAGACTCGAGC Cladosporium cladosporoides TR-gpd CCGACGTGTGTGCACTTGAATAGTGGCTAA Trichothecium roseum ST-gpd CCTCAACAGGAAGAGCAGCGACAACGGCGA Septoria tritici SN-gpd CATGGAGCGAGACCGGCGCATACTACGTCG Septoria nodorum

RS1-gpd CCATCATACTGGAGTGCAGTCCTACACGAG TA-gpd ACGGTGGCAGCAGCCTGAACCTGCTTTATC HV-gpd TTACGTTGTGGAGTCCACCGGTGTCTTCAC gpd. AA CGATACAATCTACCAGAGCTGACCGCA gpd. CC CAAGCATATAGCAACATCATCATAGACTCG gpd_CH CCGGTCGCTCCTGCTCACTTTGAC gpd CS CAATCCAGAACCTTTCACCAGAGCAAGC gpd HV CTGTGGATGTGTCAGTTGTTGATCTCACT gpd PAV CTCCAGTCTTCCACTCCATCTGTGTCAA gpd PBR ACTCACTCTTACTGCAATTCGCATATGACA gpd PTM CTGTTCAAGTCTTCATCCGAGCCATCATC gpd PTR CACATGACATGCAAGCTAACCGCC gpd_PTT GTTCAAGTCTTCATCCGAGCCATCATCAT gpd_PUC2 CATTATGCTGTGAGTCCGCCCTTATTCG gpd_RCC CATGAGCATGAATCAGAAGGAGACATTGG gpd. RS2 CTCCAGTCTTCCACTCCATCTGTGTCAA gpd st GAGCATGGAAATGAGGGCATACAAACTGAT gpd. TAE ATGACATCAAGAAGGCTATCAAGGCTGC gpd TR CACCATTGCGTCGTATTGCGTCGTAT PG_gpd CTGTTCAAGTCTTCATCCGAGCCATCATC gpd BG GCTGTGAAGTCGAAGTTGTTGCTGTCA RPB1_AA11 AGGATGTAGATGATGGCAGCGACAAGA RPB1_AA5 GCAAGGAAGTCATCACACCGAAGCAG RPB1_AA6 ATGCAGGACGTAGACGACGGAAGTGA RPB1 CC1 GGACAAGAAGACGAAGAAGGAGACAAGG RPB1_CC3 TGTGGTGACGCAACATCAGTGAATTGG RPB1_CS TGTTCAGGATGTCGAAGAGAGTGGCAA RPB1_HV CCGATAACTCCTATGTTCTTGCTCCTGC RPB1 PF AGAAGAAGAAGGAGGACGATGACGGC RPB1_PG GCCACCAAGACAAAGGACGCAAAGAA RPB1_PT CAAGAATCTCGATGATCTGCGGCTGC RPB1_PTR GCGTGATTTCACATACTGACTGCAACATC RPB1 PUT GCCAAGACCGAGGAGAACAATGACGA RPB1 RCC TGTTATAGTGCTTACAGACTCCTCCTAGCG RPB1_RS3 AGCACAGCCAGAGAAGAGACTCATCAG RPB1_ST AATGTTCAGCCGACCATCCGAAAGGTT RPB1_TR AAGAACACCGACCCAGATAAGAAGACACT ITS_AA CACTCTCTATCAGCAAAGGTCTAGCATCCA ITS_BG ACTTCGTTTCGTGTAGTTGACCCTCCA ITS_CS AAGGACGGTAATCCATCAAGACTCTACA ITS_CC ACTATTCGCTAAAGGGTGTTCGGGAGG ITS CH CGCTAAAGGGTGTTCGGGAGGCTAC ITS_HV GCGTCAAGGAACACTGTGCCTAACC ITS_PAV ACGCAGACTGGGCACCCTCG ITS_PG2 CTACTTGTTTCCTTGGCGGGCTCG ITS_PTR CCTCGCTGCCTTGCCCGTCT ITS_RS CGAAGAAGCCTGGTTCAGACCTCCA ITS_ST CTCTGCGTCGGAGTTTACGAGTAAATCG ITS_TR ATGTGTATCCTCTGAGCGAGCCGAA ITS_RCC GCGTCATTTCACCACTCAAGCCTCG ITS_PTTa AAI IGGACI I I Al ICAAACCI I I I I I IA

Rhynchosporium secalis Triticum aestivum Hordeum vulgare Alternaria alternata, Cladosporium cladosporoides Cladosporium herbarum Cochliobolus sativus Hordeum vulgare, Pyrenophora avenae Pyrenophora bromide Pyrenophora teres f. Maculata Pyrenophora tritici-repentis, Pyrenophora teres f. Teres Puccinia triticina Ramularia collo-cygni Rhynchosporium secalis, Septoria tritici Triticum aestivum Trichothecium roseum Pyrenophora graminea Blumeria graminis Alternaria alternata Alternaria alternata Alternaria alternata, Cladosporium Cladosporium cladosporoides cladosporoides Hordeum vulgare, Cochliobolus sativus, Pyrenophora graminea, Pyrenophora flavispora Pyrenophora tritici Pyrenophora teres-repentis Puccinia triticina Ramularia collo-cygni Rhynchosporium secalis, Septoria tritici Trichothecium roseum Alternaria alternata, Blumeria graminis, Cochliobolus sativus cladosporoides Cladosporium Cladosporium herbarum Poaceae

Pyrenophora avenae Pyrenophora graminea Pyrenophora tritici-repentis Rhynchosporium secalis Septoria tritici Trichothecium roseum Ramularia collo-cygni Pyrenophora teres f. Teres

ITS-PTMa AATTGGACTTTATTCAAACCTTTTTTTTTA Pyrenophora teres f. Maculata PTR1-ITS CCTTATTCAAACCTTTTTTTCAGTTGCAAT Pyrenophora tritici-repentis PTR2-ITS TGGTGTTGGGCGTCTTGTCTCTCTCCCGAG Pyrenophora tritici-repentis PTR3-ITS CG I CCA IGAAGCI ICI I I CAACC lili GAC Pyrenophora tritici-repentis HV1-ITS GAGATGGCTAGCTTGCTGGTCGTCACCTGT Hordeum vulgare HV2-ITS GTAGACGGCATCATGGCCTCGAAACGACCC Hordeum vulgare TA1-ITS GGCATGTCTAGCTTGCTAGCCGTCCCTCGT Triticum aestivum TA2-ITS GCAGCTGGCATTATGACCTTTGAACGACCC Triticum aestivum RCC-ITS CTCGCTTGGTATTGGGCGTCGCGAGTCTCT Ramularia collo-cygni PUT-ITS CAI IAAACAI I I I I IAACI IGTTTAAACTT Puccinia triticina

whereas the selection of DNA loci, i.e. the ribosomal DNA gene including spacers, the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and the RNA polymerase II large subunit (RPB1) gene for sequencing and hybridization probe design, is characterized by the application of these three loci the robustness and specificity of the developed hybridization probes for the detection of pathogens causing leaf spot in wheat and barley is guaranteed.

The hybridization probes of the invention are developed for the detection of pathogens causing leaf spot in wheat and barley using DNA microarray technology. For the efficiency and sensitivity of hybridization probes for DNA microarrays and for the overall robustness of the method, the key is the selection of the DNA locus or better the loci whose sequence is the basis for their design.

Thus, the present invention relates to hybridization probes specific to one pathogen species based on DNA locus sequences: the ribosomal DNA gene, including spacers, the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, and the RNA polymerase II large subunit (RPB1) gene. The hybridization probes of the invention are covalently attached to a solid support (commercially available glass) and the microarray thus formed allows hybridization of DNA pathogens present in a DNA sample extracted from infected wheat or barley tissue and labeled with fluorescently labeled primers or deoxycytidine residues (dCTP) during multiplex PCR. Visualization is performed at 532 and 635 nm at the Cy3 or Cy5 fluorescent labels used. The advantage of applying the DNA microarray method with hybridization probes according to the invention is the fact that it is possible to detect such a number of pathogens in one reaction for which these hybridization probes are prepared and which are actually present in the sample. This fact significantly accelerates the detection and determination of pathogens.

The following is a definition of some of the terms used:

The concept of pathogen

Pathogen is a general term for an organism parasitizing on economically important plant species. The pathogen may be a fungus, bacteria, virus, etc.

The concept of leaf spot

Leaf spot is a complex of cereal diseases, in this case wheat and barley, caused by about 20 species, especially fungal pathogens, the most important of which are: Blumeria graminis, Cochliobolus sativus, Rhynchosporium sativum, Pyrenophora teres, Pyrenophora tritici-repentis, Alternaria alternata and Sepia. They cause necrotic spots on the leaves of plants, which reduce the assimilation area of the leaf and lead to its premature death. This results in significant economic losses due to reduced grain yields.

The concept of DNA

The DNA molecule is adapted to store and copy genetic information. DNA is made up of two strands. In each chain, the five-carbon sugar deoxyribose alternates with the rest of the phosphoric acid (so-called phosphate). A heterocyclic compound, the so-called base, is attached to the deoxyribose on the other side. Four different heterocyclic compounds may be present in the DNA at the base site. Two of them are derived from the structure of purine - adenine (A) and guanine (G) - and two from pyrimidine - thymine (T) and cytosine (C). The deoxyribose-phosphoric acid-base residue, the so-called nucleotide, forms the basic unit (monomer) of DNA. Because four different molecules (A, T, G or C) can occur at the base site, there are also four different basic building blocks (nucleotides) - deoxyadenosine phosphate, deoxythymidine phosphate, deoxyguanosine phosphate and deoxycytidine phosphate. Genetic information is written in DNA in the order of bases.

The concept of DNA locus

DNA locus means a specific section of DNA, either a coding region, i.e. a region of a gene, or a non-coding region.

The concept of primers

These are short oligonucleotides (approximately 20 nucleotides) that pair with the stem DNA at the beginning and end of the amplified fragment, each with a different strand of the original double-stranded DNA molecule. It forms the starting point for the synthesis of the polynucleotide strand. The unique combination of primer nucleotides results in amplification of only the desired DNA sequence.

The concept of amplification and PCR

DNA amplification is the increase in the number of copies of DNA molecules in a laboratory setting. PCR - (Polymerase chain reaction) The polymerase chain reaction is a process in which DNA is amplified. This process takes place in three steps: 1. thermal denaturation of the DNA (hydrogen bonds are released and the double helix of the dsDNA breaks up into two separate strands of ssDNA), 2. annealing or annealing of the primers to the template ssDNA and DNA-dependent DNA polymerases. Hence the name polymerase chain reaction. This three-step process is repeated about 30 to 40 times. Common techniques of molecular biology are explained in the literature, eg Sambrook et al., 2001, Molecular cloning - a laboratory manual I., II., III.

The concept of hybridization

Hybridization is the attachment of a single-stranded DNA molecule to another single-stranded DNA molecule using hydrogen bonds between the respective deoxyribonucleotide pairs based on sequence homology.

Hybridization probe concept

It is a short oligonucleotide (approximately 25 to 30 nucleotides) that hybridizes to the target DNA sequence of the organism for which the probe is prepared. The probe is covalently attached to a solid support - usually glass with an appropriate surface treatment.

The term DNA microarrays

DNA Microchips are a technology in which probes in the form of short sections of DNA are covalently bound to a solid support - usually glass coated with aldehyde residues. Sample - usually a solution containing DNA extracted from the analyzed plant tissue is labeled with the fluorescent label Cy3 or Cy5 using either primers or deoxycytidine residues during the PCR reaction. The sample thus prepared is applied to a solid support with bound probes. During incubation at a given temperature and for a given time, the components of the sample hybridize to the appropriate probes. The fluorescence signal is then detected in a special device using a CCD scanner at wavelengths of 532 or 635 nm, depending on the fluorescence label used. The presence or absence of the signal is evaluated by the appropriate software and is the basis for determining the presence or absence of the monitored component of the sample, in our case a specific type of pathogen.

The following example merely illustrates the invention without limiting it in any way.

Exemplary embodiments

The results of all measurements are summarized in the attached Table 1.

Example 1

Verification of functionality and reliability of hybridization probes on biological material (samples of leaf tissues of wheat and barley of various origins with visible symptoms of leaf spots).

Isolates of fungal pathogens were obtained from leaf blade collections with symptoms of leaf spots from the Czech Republic and Scotland and determined according to morphological characteristics. Isolates were grown on PDA broths in petri dish on cellophane foil. DNA was extracted using a commercial UltraClean Microbial DNA Isolation Kit (Mobio). DNA from wheat and barley leaves was extracted with CTAB detergent according to an optimized protocol. The quality and concentration of the DNA samples were verified electrophoretically on a 0.8% agarose gel. A total of 10 samples were obtained, in which the presence of the most important pathogens was confirmed by PCR reaction with species-specific primers. 7 samples were used to test the DNA microarray, as shown in Table 1 below.

Labeling was performed during the multiplex PCR reaction using labeled primers and labeled dCTP. The reaction mixture with a total volume of 25 μl contained 0.2 μΜ of each forward primer (ITS, gpd, RPB1), 0.2 μΜ of each Cy3-labeled reverse primer (ITS, gpd, RPB1), 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 0.1 mM dCTP, 0.04 mM Cy5 labeled dCTP, 1x buffer, 4 mM MgCl ₂ , 1U Tth polymerase Biotools and 2 μΙ DNA sample at 100 ng / μΙ. Reactions were performed in a Sensoquest cycler (Goettingen, Germany); the reaction cycle consisted of the steps: initial denaturation at 95 ° C for 2 min., 35 cycles: 95 ° C 30 sec, 50 ° C 40 sec. 72 ° C 1 min. and the final step of the synthesis at 72 ° C for 7 min. Amplification control was performed electrophoretically on a 1.6% agarose gel and subsequent visualization with ethidium bromide in UV light compared to a 100 bp Plus size standard (Fermentas, Lithuania).

During spotting, the probes were robotically applied to a glass support in a predetermined order - by the BioRobotics Microgrid Compact spotting robot on glasses with aldehyde modification Super Aldehyde Substrates. 10 μί of each probe was mixed on the source plate with 10 μί of Microspotting Solution Plus and mixed thoroughly. The probes thus prepared were spotted with two needles at 70% humidity. Prior to hybridization, free aldehyde groups on the chip surface were first blocked and unbound probes were washed away. The labeled PCR products were denatured and 4 μl of the denatured PCR product was mixed with 16 μl of hybridization solution and this 20 μΙ was applied to the prepared chip. After application, the chip was covered with a coverslip, glued (preventing drying out) and placed in a hybridization chamber (Takara hybridization chamber) with 300 μί 2x SSC. After sealing, the hybridization chamber with the chip was incubated in a water bath at 50 ° C for 2 hours. After hybridization, unbound DNA was removed from the chip by washing in wash solutions: wash solution 1 (2x SSC with 0.2% SDS) for 15 min, 5 min. in wash solution 2 (2x SSC) and 5 min. in wash solution 3 (0.2x SSC). Finally, the glasses were dried by centrifugation and stored in a dark and dry place.

The fluorescence signal was detected using a GenePi 400a Microarray Scanner at 532 nm and 635 nm using Gene PixPro. The evaluation of whether the signal is positive or negative was done visually using the Spotfinder program. Exact data of the detected radiation intensities were converted into an MS Excel spreadsheet.

Measurements showed good functionality of hybridization probes, which were able to demonstrate the presence of fungal pathogen DNA in infected biological material with detectable pathogen levels in plant tissue and, conversely, did not generate a positive response in the absence of pathogen DNA (Table 1). Multiple species of pathogens were found in each sample, which corresponds to the identification, previous verification and experience of phytopathologists. The hybridization probes according to the invention were verified by the inventors in the laboratories of the Research Institute of Plant Production, v.v.i., Prague, CZ.

Table 1

List of tested biological material DNA microchip and results

Sample 1 2 3 4 5 6 7 Origin CR- CR- CR- CR- CR- CR- VB- Stupice Stupice Stupice Stupice Stupice Stupice Scotland H H T. Η. Η. H. H. vulgare Host vulgar - 'Tolar' vulgare 'Bonus' aestivu m - 'Government' vulgare - vulgare - vulgare 'Heidi' 'Saffron' Tolar ' Pathogen * / Probe ** PTT BG, AA PUT, AA RS, PG RS ^P AA 'AA RCC, PTT, RS Hordeum vulgare + + - + + + + Triticum aestivum - - + - - Pyrenophora teres f. Teres + - - - + Pyrenophora teres f. + maculata - Pyrenophora graminea - - + + - - - Pyrenophora avenae - - - - - Pyrenophora bromi - - - - - Pyrenophora tritici-repentis - - - - - CL > O o Pyrenophora flavispora - - - - - Ramularia collo-cygni - + - - + and Rhynchosporium secalis - - - + + - + E Blumera graminis - + - - - of Alernaria alternata - - + + + + - Q Alternaria triticina - - + - - Alternaria metachromatica - - - + + Cochliobolus sativus - - - - + Puccinia tritici - - + - - Cladosporium + + + cladosporoides + + Cladosporium herbarum - + - + + + Trichodermum roseum - - - + + - Septoria tritici + - - - - Septoria nodorum + - + + +

* list of pathogens determined by a phytopathologist and by PCR with specific primers; pathogen abbreviations: PTT- Pyrenophora teres f. teres, PTM - Pyrenophora teres f. maculata, CS Cochliobolus sativus, RS - Rhynchosporium secalis, BG - Blumeria graminis, RCC Ramularia collo-cygni, AA - Alternaria alternata, PTR - Pyrenophoris repativa tr , PUTPuccinia tritici;

** more probes specific to the pathogen species were merged under the name pathogen Positive result of the reaction characterized by amplification of the target DNA is marked with a + (plus) sign, negative response with a - (minus) sign.

Industrial applicability

The microchip with the mentioned hybridization probes is used in phytosanitary medicine in the diagnosis of the most important pathogens causing leaf spot in wheat and barley, which will enable a targeted choice of effective protection of wheat and barley stands. New hybridization probes for the detection of pathogens causing leaf spot in wheat and barley using the technique of DNA microarrays (DNA microarrays) and the selection of DNA loci that were used for sequencing analysis and preparation of these probes. The DNA microchip with the mentioned hybridization probes will enable the specific detection of pathogens causing leaf spots in the leaf tissues of wheat and barley taken from agricultural production areas of wheat and barley.

PV 2044 - 8 Η

PATENT CLAIMS

Claims (1)

PATENT CLAIMS 1. Hybridization probes for the detection of pathogens causing leaf spot of wheat and barley, characterized by the specificity of each pathogen of the composition Probe designation Probe sequence Pathogen species specific: PG-gpd CCATGGAGTCACAGGAGGCAGCAGGCTAAT Pyrenophora graminea PA-gpd CTATTCGGCCCTGGGAGACTGCAGGCTAAT Pyrenophora avenae PB-gpd CGATGGATCTGCAGGATACAGCAGACTAAC Pyrenophora bromi PTT-gpd CCATGGAGTTACAAGAGACAGCAGGCTAAT Pyrenophora teres f. Teres PTM-gpd CCATGGAGTCACAAGATACAGCAGGCTAAT Pyrenophora teres f. Maculata PTR-gpd CGATGGATCTGCAGGATACAGCAGACTAAT Pyrenophora tritici-repentis CS-gpd CACTGGTATCATTCCTTTGATGGAACTATG Cochliobolus sativus RCC-gpd AACAATGGCCAAGACAATCGCATTTAACAT Ramularia collo-cygni RS2-gpd CTATTCGCCCCTGGGAGCCTGCAGGCTAAT Rhynchosporium secalis BG-gpd ATACCATGGGCCGAAACTGGAGCCGAGTAC Blumeria graminis PUT-gpd CCTTGGAATGCAGGTGCTAACAGGACATTG Puccinia triticina AA-gpd CGCTAGAGCTCCTCTGTGGTCGCAGAATGC Alternaria alternata AT-gpd CTATGACTTCAGTACGCAAGCTAACACACG Alternaria triticina AM-gpd CTACAACTACAGGACGCAGGCTAACACACA Alternaria metachromatica CH-gpd CATTCACTTGCACCGGTCGCTCCTGCTCAC Cladosporium herbarum CC-gpd GCATATAGCAACATCATCATAGACTCGAGC Cladosporium cladosporoides TR-gpd CCGACGTGTGTGCACTTGAATAGTGGCTAA Trichothecium roseum ST-gpd CCTCAACAGGAAGAGCAGCGACAACGGCGA Septoria tritici SN-gpd CATGGAGCGAGACCGGCGCATACTACGTCG Septoria nodorum RS1-gpd CCATCATACTGGAGTGCAGTCCTACACGAG Rhynchosporium secalis TA-gpd ACGGTGGCAGCAGCCTGAACCTGCTTTATC Triticum aestivum HV-gpd TTACGTTGTGGAGTCCACCGGTGTCTTCAC Hordeum vulgare qpd AA CGATACAATCTACCAGAGCTGACCGCA Alternaria alternata gpd_CC CAAGCATATAGCAACATCATCATAGACTCG Cladosporium cladosporoides gpd_CH CCGGTCGCTCCTGCTCACTTTGAC Cladosporium herbarum gpd_CS CAATCCAGAACCTTTCACCAGAGCAAGC Cochliobolus sativus qpd HV CTGTGGATGTGTCAGTTGTTGATCTCACT Hordeum vulgare gpd_PAV CTCCAGTCTTCCACTCCATCTGTGTCAA Pyrenophora avenae qpd PBR ACTCACTCTTACTGCAATTCGCATATGACA Pyrenophora bromi qpd PTM CTGTTCAAGTCTTCATCCGAGCCATCATC Pyrenophora teres f. Maculata gpd_PTR CACATGACATGCAAGCTAACCGCC Pyrenophora tritici-repentis gpd_PTT GTTCAAGTCTTCATCCGAGCCATCATCAT Pyrenophora teres f. Teres gpd PUC2 CATTATGCTGTGAGTCCGCCCTTATTCG Puccinia triticina qpd RCC CATGAGCATGAATCAGAAGGAGACATTGG Ramularia collo-cygni qpd. RS2 CTCCAGTCTTCCACTCCATCTGTGTCAA Rhynchosporium secalis gpd_ST GAGCATGGAAATGAGGGCATACAAACTGAT Septoria tritici qpd TAE ATGACATCAAGAAGGCTATCAAGGCTGC Triticum aestivum gpd_TR CACCATTGCGTCGTATTGCGTCGTAT Trichothecium roseum PG_gpd CTGTTCAAGTCTTCATCCGAGCCATCATC Pyrenophora graminea gpd_BG GCTGTGAAGTCGAAGTTGTTGCTGTCA Blumeria graminis RPB1 AA11 AGGATGTAGATGATGGCAGCGACAAGA Alternaria alternata RPB1 AA5 GCAAGGAAGTCATCACACCGAAGCAG Alternaria alternata RPB1 AA6 ATGCAGGACGTAGACGACGGAAGTGA Alternaria alternata RPB1 CC1 GGACAAGAAGACGAAGAAGGAGACAAGG Cladosporium cladosporoides RPB1_CC3 TGTGGTGACGCAACATCAGTGAATTGG Cladosporium cladosporoides RPB1 CS TGTTCAGGATGTCGAAGAGAGTGGCAA Cochliobolus sativus RPB1 HV CCGATAACTCCTATGTTCTTGCTCCTGC Hordeum vulgare RPB1 PF AGAAGAAGAAGGAGGACGATGACGGC Pyrenophora flavispora RPB1_PG GCCACCAAGACAAAGGACGCAAAGAA Pyrenophora graminea RPB1 PT CAAGAATCTCGATGATCTGCGGCTGC Pyrenophora teres RPB1_PTR GCGTGATTTCACATACTGACTGCAACATC Pyrenophora tritici-repentis RPB1 PUT GCCAAGACCGAGGAGAACAATGACGA Puccinia triticina RPB1 RCC TGTTATAGTGCTTACAGACTCCTCCTAGCG Ramularia collo-cygni RPB1 RS3 AGCACAGCCAGAGAAGAGACTCATCAG Rhynchosporium secalis RPB1 ST AATGTTCAGCCGACCATCCGAAAGGTT Septoria tritici RPB1 TR AAGAACACCGACCCAGATAAGAAGACACT Trichothecium roseum ITS AA CACTCTCTATCAGCAAAGGTCTAGCATCCA Alternaria alternata ITS_BG ACTTCGTTTCGTGTAGTTGACCCTCCA Blumeria graminis ITS CS AAGGACGGTAATCCATCAAGACTCTACA Cochliobolus sativus ITS CC ACTATTCGCTAAAGGGTGTTCGGGAGG Cladosporium cladosporoides ITS_CH CGCTAAAGGGTGTTCGGGAGGCTAC Cladosporium herbarum ITS_HV GCGTCAAGGAACACTGTGCCTAACC Poaceae ITS_PAV ACGCAGACTGGGCACCCTCG Pyrenophora avenae ITS_PG2 CTACTTGTTTCCTTGGCGGGCTCG Pyrenophora graminea ITS_PTR CCTCGCTGCCTTGCCCGTCT Pyrenophora tritici-repentis ITS_RS CGAAGAAGCCTGGTTCAGACCTCCA Rhynchosporium secalis ITS_ST CTCTGCGTCGGAGTTTACGAGTAAATCG Septoria tritici ITS_TR ATGTGTATCCTCTGAGCGAGCCGAA Trichothecium roseum ITS_RCC GCGTCATTTCACCACTCAAGCCTCG Ramularia collo-cygni ITS_PTTa AAI IGGACI I IAI ICAAACCI I I I I I IA Pyrenophora teres f. Teres ITS-PTMa AATTGGACTTTATTCAAACCTTTTTTTTTA Pyrenophora teres f. Maculata PTR1-ITS CCI IAI ICAAACCI lilii ICAGITGCAAT Pyrenophora tritici-repentis PTR2-ITS TGGTGTTGGGCGTCTTGTCTCTCTCCCGAG Pyrenophora tritici-repentis PTR3-ITS CGTCCATGAAGCTTCTTTCAACCTTTTGAC Pyrenophora tritici-repentis HV1-ITS GAGATGGCTAGCTTGCTGGTCGTCACCTGT Hordeum vulgare HV2-ITS GTAGACGGCATCATGGCCTCGAAACGACCC Hordeum vulgare TA1-ITS GGCATGTCTAGCTTGCTAGCCGTCCCTCGT Triticum aestivum TA2-ITS GCAGCTGGCATTATGACCTTTGAACGACCC Triticum aestivum RCC-ITS CTCGCTTGGTATTGGGCGTCGCGAGTCTCT Ramularia collo-cygni PUT-ITS CAI lAAAUAl Illi lAACTTGTTTAAACTT Puccinia triticina, with the selection of DNA loci, ie the ribosomal DNA gene including spacers, the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and the RNA polymerase II large subunit (RPB1) gene for sequencing and hybridization probe design, characterized by the fact that The three loci guarantee the robustness and specificity of the hybridization probes developed for the detection of pathogens causing leaf spot in wheat and barley.