Determination of fungal pathogens by using a polymerization chain

 

(57) Abstract:

The invention relates to Phytopathology, in particular to a method of determining fungal pathogens using specific primers in the polymerization reaction. The method of detection of phytopathogenic fungus involves the following stages: isolation of DNA from plant leaf infected with a pathogen, the amplification of part of intron transcarbamylase region of the pathogen using selected DNA as template using polymerase chain reaction with specific primers, the visualization of the amplified part of the intron transcarbamylase area. We determined the structure of several DNA molecules coding intron transcribers the sequence used to design oligonucleotide primers for detection of phytopathogenic fungus. Designed primers for determination of pathogenic fungi. We developed a quantitative colorimetrically analysis detection of phytopathogenic fungus. The method allows to detect such phytopathogenic fungi as microdochium nivate, Septoria nodorum, Septoria tritici, Pseudocercosporella herpotrichoides, Mycospharella fijiensis, Mycosphaerella musicola, Fusarium culmorum, Fusarium frost, Fusarium moniliform. 6 C. and 12 C.p. f-crystals, 5 tab., 3 Il.

Region technicaj polymerase-cableway reaction for the detection of pathogenic fungi. The use of such primers can detect certain isolates of pathogenic fungi and to monitor disease development in plant populations.

Background of the invention

Plant diseases annually cause significant yield losses, causing economic loss to farmers and, in addition, bringing in many parts of the world for lack of food resources for the local population. The widespread use of fungicides gave significant protection from damage by phytopathogens. However, despite the fact that the cost of fungicides in the world amount to $ 1 billion, global yield losses according to 1981 (James, 1981; Seed Sci. & Technol. 9:679-685) was approximately 10%.

The severity of disease caused by destructive process depends on the aggressiveness of the pathogen and the response of the host. One of the goals of most plant breeding programmes is to increase the resistance to disease of host plants. Usually the different races of pathogens interact differently with different varieties of the same species of cultivated plants, and many of the mechanisms contributing to resistance of the host, promote the protection only from certain races pathogenfree impact on yield. According to Jones and Clifford (1983; Cereal Diseases, John Wiley), one should expect the emergence of virulent forms of the pathogen in the population of the pathogen in response to the introduction of resistance in the cultivated varieties of host plants and therefore it is necessary to monitor populations of pathogens. In addition, there are several recorded cases of developing fungal strains resistance to certain fungicides. Back in 1981, Fletcher and Wolfe (1981; Proc. 1981 Brit. Crop Prot. Conf.) found that 24% of the populations of powdery mildew on spring barley and 53% on winter barley showed significant variability in sensitivity to the fungicide triadimenol and that the distribution of these populations between varieties differed from the most sensitive varieties, leading to more frequent occurrence types with reduced sensitivity. Such differences in the sensitivity of fungi to fungicides registered for powdery mildew of wheat (also triadimenol), Botrytis (to benomyl), Pyrenophora (organomercury fungicides), Pseudocercosporella (fungicides MVS-type) and Mycosphaerella fijiensis to triazolam, as said above authors (Jones and Clifford Cereal Diseases, John Wiley, 1983).

Crops grown around the world, and they form the bulk of the world about the ocercosporella are the most important in the main areas of cultivation of grain crops in Europe and North America (Jones and Clifford Cereal Diseases, John Wiley, 1983). In particular, various symptoms caused by different isolates and species of these fungi, lead to the fact that it is difficult to accurately predict potential losses due to disease. Therefore, for practitioners in the field of plant protection is significant value in the development of improved diagnostic methods for rapid and accurate detection of specific pathogens.

Four species R. Septoria parasitize on fine-grained cereals. Septoria tritici is the causative agent bordered spots and has virulence to wheat, but also affects triticale and rye. Usually this pathogen causes necrosis of leaves. Septoria nodorum is the causative agent of Septoria Koloskova scales and affects wheat, triticale, rye and barley and, although the disease is largely confined to koloskovyy scales, it also extends to the leaf blade and leaf sheath. Septoria avenae parasitizes on oats, wheat and triticale, a Septoria passerinii only on barley. Diseases caused by pathogens R. Septoria, lead to loss in economically significant scale in all fields of wheat. Various diseases caused by pathogens R. Septoria, often compete on the fields, zanatflex". Usually the most commonly encountered species such as S. tritici and S. nodorum. According to Wiese (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. pages 42-45) currently, due to the defeat septoriosis complex annually loses about 2% of the world's wheat crop, and these yield losses mainly due to lack of grain filling. Fungicide treatment can save up to 20% of the harvest in cases of serious infection by pathogens R. Septoria, but often at the initial stage of infection is difficult to recognize various kinds of plants, making it difficult to decide whether or not to apply fungicides, because different varieties of cultivated plants show different degrees of resistance to different species of Septoria.

Cercosporella cereal grasses caused by the fungus Pseudocercosporella herpotrichoides and restricted to the basal part of the stalk of grass. Wheat, rye, oats and other cereals sensitive cercosporella, which occurs in cool, wet climates and is prevalent in Europe, North and South America, Africa and Australia. Wheat is most susceptible to this pathogen views grain, however, identified isolates that are virulent for other cereals. For example, from rye was also selected R-strain which can lead to the death of shoots or whole plants, most often it leads to lodging and/or reduces the size and number of grains. Yield losses from cercosporella are even more substantial in comparison with those caused by Septoria tritici and Septoria nodorum. The usual ways of dealing with cercosporella include treatment with plant growth regulators to strengthen internodes and treated with fungicides. However, the sensitivity of the cultivar to different strains of the fungus complicates the prediction of the expected effectiveness of fungicide treatments.

Leaf spot in banana type sigatoka occurs in two forms, each of which is caused by various fungi. The causative agent is important in economic terms 'drive from sigatoka black is Mycosphaerella fijiensis, while the pathogen is less important economically yellow' drive from sigatoka is Mycosphaerella musicola (Johanson and Movement, 1993; Mycol. Res. 97: 670-674). Black sigatoka is a major problem in the production of bananas, causing serious yield losses of 30% and above. Due to the presence of Mycosphaerella fijiensis resistance to fungicides should limit the application of fungicides to prevent further development of resistance. Therefore, the design of sresty without undue risk of further development of resistance.

Thus, there is a real need to develop ways to identify specific race pathogenic fungi at an early stage of the infection process. By identifying specific races of the pathogen before symptoms become apparent in relation to the standing crops, agronomist can assess the possible impact of further development of the pathogen on a variety of plants, on which it was discovered, and can choose the appropriate fungicide if such processing is necessary.

Summary of the invention

The present invention relates to methods of identification of different pathotypes phytopathogenic fungi. The invention relates to DNA sequences that can detect the variability of different pathotypes mushrooms. Such DNA sequences are suitable for the method according to the invention, because they can be used to generate primers suitable for diagnostic methods based on polymerase-cableway reaction (PCR). These primers generate unique fragments in PCR reactions, in which the DNA template get from certain pathotypes mushrooms and so it can be used to identify the disease.

The present invention provides an opportunity to assess the potential damage to specific varieties of cultivated plants associated with this pathogenic strain, and reasonable to apply different available range of fungicides. In addition, the invention can be used to obtain detailed information on the development and distribution of specific races of the pathogen in wide geographical areas. The invention proposes a method of detection, especially suitable for diseases with long latency periods, such as diseases of wheat caused by Septoria nodorum or Septoria tritici, and diseases of banana, caused by Mycosphaerella fijiensis.

Also designed the sets, suitable for the practical implementation of the invention. The data sets are primarily suitable for the identification of pathogens p.p. Septoria, Pseudocercosporella, Fusarium and Mycosphaerella.

Brief description of drawings

In Fig. 1 presents the comparison of the sequences of internal transcribers spacers of Septoria tritici, Septoria nodorum, the W strain of Pseudocercosporella herpotrichoides (two versions), R-strain of Pseudocercosporella herpotrichoides, Mycosphaerella fijiensis and Mycosphaerella musicola.

In Fig. 2 presents the comparison of the sequences of internal transcribers spacers from Sep spacers from Fusarium frost, Fusarium culmorum, Fusarium moniliforme and Microdochium nivale.

Detailed description of the invention

The present invention relates to unique DNA sequences that are suitable for identification of different pathotypes phytopathogenic fungi. In particular, DNA sequences can be used as primers in the analyses based on the use of PCR to identify pathotypes mushrooms. DNA sequences according to the invention contain internal transcribers spacer (ITS) ribosomal RNA gene specific pathogenic fungi, as well as primers derived from these areas, which allow us to identify the specific pathogen. These sequence ITS DNA from different pathotypes species or genera of pathogens that can be different for different representatives of the species or genera, can be used to identify these specific representatives.

Experts in the biomedical field for several years used with some success methods based on PCR for the detection of pathogens in infected animal tissues. However, only at the present time this method was applied for the detection of phytopathogens. For example, using PCR posledovatel the military wheat (Schlesser and others, 1991; Applied and Environ. Environ. 57:553-556) and markers random amplified polymorphic DNA (i.e., SAPD) proved to be suitable for recognizing the many races of Gremmeniella abietina, the causative agent of cancer scleroderris in conifers.

Ribosomal genes suitable for use as molecular probes to target because they have a large number of copies. Despite the high degree of conservatism in the Mature rRNA sequences, retranscribing and transcribers spacer elements of the sequence are usually a little conservative and, therefore, suitable as sequences of target for the detection of recent evolutionary divergence. Genes rRNA mushrooms are organized into units, each of which encodes three Mature subunit 18S, the 5.8 S and 28S, respectively. These subunits are separated by two internal transcribability the spacers ITS1 and its2 sequences, length of approximately 300 base pairs (White and others, 1990; in PCR Protocols; ed Innes and others, pp. 315-322). In addition, the transcribed units divided netransliruemymi spacer elements sequences (NTS). ITS and NTS sequences are primarily suitable for specific detection of pathotypes various pathogenic fungi.

You'll find the s ribosomal RNA of various pathogenic fungi. The sequence of ITS DNA from different pathotypes species or genera of pathogens can be different for different representatives of the species or genera. If you define the ITS sequence of a pathogen, these sequences can be mapped to other ITS-sequences. Thus, from ITS sequences can be derived primers. This means that on the basis of data on the areas within ITS areas can be designed primers that have the most distinguished sequence among pathotypes mushrooms. These sequences and primers based on these sequences, can be used to identify specific members of pathogens.

Of specific interest are DNA sequences include the sequence of ITS DNA mushrooms R. Septoria, in particular Septoria nodorum and Septoria tritici; p. Mycosphaerella, in particular Mycosphaerella fijiensis and Mycosphaerella musicola; p. Pseudocercosporella, in particular Pseudocercosporella herpotrichoides, first of all W-strain and the R-strain of Pseudocercosporella herpotrichoides; p. Fusarium, in particular F. frost, F. culmorum, F. moniliforme and Microdochium nivale. Such the ITS DNA sequence, and primers of interest shown in the sequences SEQ ID NO: 1-47 and SEQ ID NO: 50-86. These sequences H2">

How to use sequences of primers according to the invention in PCR assays well known in the art. See, for example, the U.S. patents 4683195 and 4683202, and Schlesser and others, (1991) Applied and Environ. Environ. 57: 553-556. CM. also Nazar and others (1991; Physiol. and Molec. Plant Pathol. 39:1-11), who used PCR amplification to detect differences in the ITS-regions Verticillium alboatrum and Verticillium dahliae and, therefore, to separate these two species; and Johanson and Movement (1993; Mycol. Res. 97: 670-674), who used similar methods for separating banana pathogens Mycosphaerella fijiensis and Mycosphaerella musicola.

Sequence ITS DNA according to the invention can be isolated from pathogenic fungi and cloned by methods known in the art. In General, the methods of DNA extraction from isolates of fungi known. See, for example, RV & Broda (1985) Letters in Applied Microbiology 2:17-20; Lee and others, (1990) Fungal Genetics Newsletter 35: 23-24; and Lee and Taylor (1990) in PCR Protocols: A Guide to Methods and Applications, Ed. Innes and others; pp. 282-287.

In another embodiment of interest, the ITS region can be determined using PCR amplification. Primers for amplification of the complete ITS region was designed similarly described by White and others, (1990; in PCR Protocols, edited Innes and others; pp. 315-322) and amplificatoare ITS-sequence subclone Ronni ITS (its2 sequences), and located in the center of the subunit of the 5.8 S rRNA gene. This was done to Septoria nodorum and Septoria tritici, numerous isolates of Pseudocercosporella and Mycosphaerella fijiensis, Mycosphaerella musicola, Septoria avenae triticea, F. frost, F. culmorum, F. moniliforme and Microdochium nivale.

Determined ITS sequence and the compared sequence within each group of the pathogen to identify the location of the divergence, which may be acceptable when tested by PCR to separate the different species and/or strains. Sequences of the ITS regions, which were identified, are represented as sequences of SEQ ID NO: 1-6, SEQ ID NO: 47 and SEQ ID NO: 82-86, as also shown in Fig. 1, 2 and 3. Based on the identification of divergences synthesized numerous primers and tested them using PCR amplification. The matrix used for testing using PCR amplification, first was a purified DNA of the pathogen, and then DNA extracted from infected tissue of the host plant. Thus, it was possible to identify pairs of primers, which were diagnostic, i.e., that allowed to identify one specific type or strain of the pathogen, and not another species or strain of the same pathogen. Predpochtite the I-master, i.e. in host tissue, which had previously been infected with a particular species or strains of the pathogen.

The invention includes numerous combinations of primers for different species of Septoria, Mycosphaerella and Fusarium and various strains of Pseudocercosporella that satisfy this criterion. The primers according to the invention is designed on the basis of differences in sequence between the ITS-regions mushrooms. The difference between sequences of at least one pair of bases allows to construct discriminating primer. For amplification of species-specific PCR fragments primers designed for specific ITS-region DNA of the fungus can be used in combination with a primer created for the conserved region of the sequence within the coding region of ribosomal DNA. Usually to achieve a good sensitivity primers should have a theoretical melting temperature between approximately 60oC and 70oC and should be deprived of a significant secondary structures and overlaps the 3'ends of the combinations of primers. Primers usually have at least from about 5 to about 10 nucleotides.

The suitability of the cloned ITS sequences for the selection of primers for the purpose of di is you W-type and R-type pathogen Pseudocercosporella herpotrichoides have divergent ITS sequences, of which were obtained diagnostic primers. However, the rapid divergence within ITS sequence is based on the observation that there were identified two different versions of the sequence W-type. The identities of the sequences within W-type were 99.4%, while between W - and R-types, it amounted to 98.6%, suggesting a closer evolutionary relationship between the two W-variants as compared to that found between the W - and R-types. This closer relationship is also visible from similar pathogenicity to host two isolates with divergent ITS sequences.

Besides the primers of the sequences with the origin of ITS, for PCR diagnostics of isolates of fungi, the invention also includes the identification of primers from the library of primers SAPD that, when used in PCR allow to distinguish between Septoria nodorum and Septoria tritici. The selected primers are commercially available and are supplied Oregon Technologies Incorporated (Alameda, California). By screening genomic DNA Septoria selected two primers that can detect only S. tritici, and three primers that can detect only S. nodorum.

And, finally, a set can contain all the additional elements required for the implementation of the method according to the invention, such as buffers, extraction reagents, enzymes, pipettes, tablets, nucleic acids, nucleosidases, filter paper, materials for the production of gel materials for transfer, materials for autoradiography etc.

The following examples illustrate, without limiting the scope of the invention, a typical experimental protocols that can be used to highlight the ITS sequences, selection will stifle efficiency, and the use of such primers for detection of diseases and isolate of the fungus. These examples are provided to illustrate and not limit the scope of the invention.

EXAMPLES

Example 1: Isolates of fungi and extraction of genomic DNA

Viable isolates of fungi S. nodorum, S. tritici, S. passerini, S. glycines, Pseudocercosporella herpotrichoides, Pseudocercosporella aestiva, Mycosphaerella citri, Mycosphaerella graminicola, Mycosphaerella fijiensis and Mycosphaerella musicola was obtained from the American type culture collection. Isolates of Fusarium culmorum and Fusarium frost were obtained from Dr. Paul Nelson from the Pennsylvania state University. Isolate Microdochium nivale (synonym Fusarium nivale) was obtained from the company Ciba (Basel), and the isolate of Fusarium moniliforme was obtained from Dr. Loral Castor. Mushrooms were grown in 150 ml of broth potato dextrose inoculated mineralnymi fragments of crops grown in KDA (agar potato dextrose). Cultures were incubated on an orbital shaker at 28oC for 7-11 days. Mycelium was palletizable by centrifugation, then immersed in liquid nitrogen and full genomic DNA was extracted according to the Protocol of Lee and Taylor (1990; in PCR Protocols: A Guide to Methods and Applications; ed Innes and others, pp. 282-287).

Dr. Bruce McDonald from the University of Texas weste Chris Caten from the University of Birmingham has provided the purified DNA of six isolates of the fungus Septoria nodorum. Purified genomic DNA from 12 isolates of Pseudocercosporella herpotrichoides were obtained from Dr. Paul Nicholson center John Inna, Norvik, UK. Six of these isolates were W-type, and the other six isolates were R-type. Types of these isolates was determined on the basis of studying the pathogenicity and polymorphism of the lengths of restriction fragments (RFLP). Andrea Johanson from the Institute of natural resources has provided the genomic DNA of six isolates of M (see Tab. 1), musicola, six isolates of M. fijiensis and one isolate Mycosphaerella musae. Purified genomic DNA Septoria avenae f.sp. triticea ATSS N 26380 was provided by Dr. Peter Ueng from the Ministry of agriculture, U.S. (USDA), Beltsville, Maryland.

Example 2: selecting areas of internal transcribers spacer regions (ITS)

Fragments of internal transcarbamoylase of spacer length of approximately 550 base pairs amplified by PCR from 25 ng of genomic DNA isolated from S. nodorum (ATSS N24425), S. tritici (ATSS N26517), R1-, R2-, W2 -, and W-isolates of Pseudocercosporella herpotrichoides, M. fijiensis (ATSS N22116) and M. musicola (ATSS N22115) using 50 pmoles primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'; SEQ ID NO: 38) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; SEQ ID NO: 41). PCR was carried out according to the method of example 4, except that the reaction was performed in 100 µl and annealing was carried out at 50and others; pages 461-462). DNA resuspendable 50 ál DN2Oh and cloned using a kit for cloning TA Cloning Kit (part N K-01) by Invitrogen Corporation (San Diego, California), using a cloning vector pCRII. DNA sequences from the ITS regions were determined using dideoxy method using an automatic sequencing machine model A Applied Biosystems (Foster City, CA) with primers ITSI (see above sequence), its2 sequences (5'-GCTGCGTTCTTCATCGATGC-3'; SEQ ID NO: 39), ITS4 (see above sequence) and with primer M13 universal-20 (5'-GTAAAACGACGGCCAGT-3'; SEQ ID NO: 48) and reverting primer (5'-AACAGCTATGACCATG-3'; SEQ ID NO: 49). The ITS primers ITS1 (SEQ ID NO: 38), its2 sequences (SEQ ID NO: 39), ITS3 (SEQ ID NO: 40) and ITS4 (SEQ ID NO: 41), used for cloning of the ITS regions, are covered in detail by White and others (1990; in PCR Protocols; ed Innes and others, pp. 315-322).

In addition, internal transcribers spacer regions amplified by PCR from 25 ng of genomic DNA isolated from S. avenae f. sp. triticea, M. nivale, F. moniliforme (N4551), isolates of F. frost R-8417, R-8546 and R-8422 and isolates of F. culmorum R-5126, R-5106 and R-5146. PCR products were purified using the kit for DNA purification company Promega Wizard (Madison, Wisconsin). DNA sequences of the ITS regions was determined as described above using the primers ITS1 (SEQ ID NO: 38), its2 sequences (SEQ ID NO: 39) the matching sequence for F. culmorum and F. frost.

Example 3: Extraction of DNA from leaves of wheat and banana

From the leaves of wheat DNA was extracted using a modified version of the Protocol rapid extraction of DNA of the company MicroProbe Corporation (Garden Grove, California), using the kit for the extraction of nucleic acids IsoQuick (catalog Mat-020-100). Normal outputs were 5-10 µg total DNA from 0.2 g of leaf tissue. Each PCR analysis was used approximately 100 ng of total DNA.

A modified technique of rapid extraction of DNA

First, before applying the set of all the contents of the reagent 2A (20-fold concentration of the dye) was added to the reagent 2 (matrix for extraction).

(1) Approximately 0.2 g of sample leaves were added to the Eppendorf tube of 1.5 ml containing 50 μl of sample buffer and 50 ál 25 lisanova solution of N1. Leaf samples were ground with a pestle and mortar Conte.

(2) Reagent 2 (matrix for extraction) was intensively shaken. To the sample lysate was added 350 μl of reagent 2.

(3) To the sample was added to 200 μl of reagent 3 (buffer for extraction). The sample was intensively stirred for 20 sec.

(4) was Centrifuged at microcentrifuge at 12000xg for 5 minutes

(5 200 ál.

(6) To the aqueous phase of the sample was added to 0.1-fold volume of the aqueous phase reagent 4 (sodium acetate).

(7) To the aqueous phase of the sample was added the same volume of isopropanol and then intensively stirred.

(8) was Centrifuged at microcentrifuge at 12000xg for 10 minutes

(9) the Supernatant was discarded without disturbing the debris nucleic acid. The debris was added 0.5 ml of 70% at -20oC ethanol. The tube was intensively stirred until complete mixing.

(10) was Centrifuged at microcentrifuge at 12000xg for 5 minutes

(11) the Supernatant was discarded and the debris was allowed to dry.

(12) Debris nucleic acid was dissolved in 50 µl of reagent 5 (waterless Mcasa).

Example 4: Amplification-based polymerase-cableway reaction

Polymerase-apevia reaction was performed using the kit gene amplification type GeneAmp company Perkin-Elmer/Cetus (Norwalk, CT; part N N808-0009), using a buffer composed of 50 mm KCl, 2.5 mm MgCl2, 10 mm Tris-HCI, pH of 8.3, containing 100 μm of each of TTF, dATP, dCTP and DSTF, 50 PM primer, 2.5 units Taq polymerase, and 25 ng genomic DNA in a final volume of 50 µl. Reactions were carried out in 30 cycles of 15 s at 94oC, 15 s at 50was jiroveci using gel electrophoresis, loading 20 μl of the sample PCR product from 1.1 to 1.2% agarose gel.

Example 5: Synthesis and purification of oligonucleotides

Oligonucleotides (primers) were synthesized on a DNA synthesizer type Applied Biosystems 380A, using B-cyanocarbonimidate chemistry.

Example 6: the Selection of species-specific markers

Comparing ITS sequences of S. nodorum, S. tritici, R - and W-strains of P. herpotrichoides, M. fijiensis and M. musicola (Fig. 1). Comparing ITS sequences of S. nodorum, S. avenae (Fig. 2). We also carried out a comparison of ITS sequences from F. frost, F. culmorum, F. moniliforme and M. nivale (Fig. 3). Sets of primers were synthesized in accordance with example 5, based on the analysis of mapped sequences. Designed primers to areas with the greatest differences in the sequences for species of fungi, shown in Fig. 1 and 2. In accordance with Fig. 3 designed primers to areas of greatest homologically within ITS for mushrooms R. Fusarium. In addition, for testing in combination with primers specific for the ITS region, synthesized described by White and others, 1990; in PCR Protocols; ed Innes and others, pages 315-322, ribosomal gene-specific primers ITS1 (SEQ ID NO: 38), its2 sequences (SEQ ID NO: 39), ITS3 (SEQ ID NO: 40) and ITS4 (SEQ ID NO: 41) (see tab. 2).

Example 7: what Umarov (sets b and E), consisting of twenty oligonucleotides each, were obtained from Oregob Technologies Incorporated (Alameda, California). The primers were tested on their ability to distinguish purified genomic DNA of S. nodorum, S. tritici, M. fijiensis and M. musicola. The PCR conditions were practically the same as in example 4, except that the number of PCR cycles was increased to 35, the annealing temperature was 30oC and was used only 5 pmole each primer. Identified five SPD-primers, which allowed to distinguish purified genomic DNA of S. nodorum, S. tritici, M. fijiensis and M. musicola. Primers RIA-12 and PR-6 produced a single fragment by amplification from genomic DNA of S. tritici. Primers'OR-12, RIA-19 and PR-15 produced fragments by PCR from genomic DNA of S. nodorum. Under the influence of primers RIA-12 and ER-6 has not received any amplified products of genomic DNA from S. nodorum, M. fijiensis and M. musicola. Primers'OR-12, RIA-19 and PR-15 no no amplified fragments of genomic DNA of S. tritici, M. fijiensis or M. musicola (Cm.table. 3).

Example 8: determination of the specificity of the primer for purified genomic DNA of the fungus

In order to amplify a separate species-specific fragment, PCR was performed in accordance with example 4, using skonstruirovannyh of the ITS region between the 18S and 25S ribosomal subunits of DNA for each interest strain of the fungus (see table. 4).

Example 9: determination of the specificity of the primers for plant tissues, infected mushroom

In accordance with the Protocol described in example 3, was isolated complete genomic DNA from healthy leaves of wheat, from the leaves of wheat infected with S. nodorum leaf of wheat infected with S. tritici, and leaves of wheat infected with the S. nodorum and S. tritici. PCR was performed according to the method of example 4 testing the combination of primers listed in example 8, in relation to DNA from leaves of wheat.

Specific for S. tritici primers JB446 (SEQ ID NO: 12) and ITS1 (SEQ ID NO: 38)(JB410) amplified fragment length 345 base pairs from purified DNA of S. tritici from infected S. tritici leaf tissue of wheat and from infected and S. tritici and S. nodorum sample leaf tissue of wheat. The set of primers is not amplified diagnostic fragment from a healthy leaf tissue of wheat, or of tissue of wheat infected with S. nodorum. Similarly, specific for S. tritici primers JB445 (SEQ ID NO: 11) and ITS4 (SEQ ID NO: 41)(JB415) amplified fragment length 407 base pairs of the same material as the combination of primers JB446 (SEQ ID NO: 12) and ITS1 (SEQ ID NO: 38)(JB410), and also appeared to be suitable for diagnosis.

Similar results regarding diagnosis were obtained for specifics infected S. nodorum tissue of wheat, from a sample of leaf tissue of wheat infected and S. nodorum and S. tritici, as well as from purified genomic DNA of S. nodorum. The combination of primers JB433 (SEQ ID NO: 7) and JB434 (SEQ ID NO: 8) there is no amplified fragments from healthy tissues of the wheat from the tissue of wheat infected with S. tritici, or from purified genomic DNA of S. tritici. Specific for S. nodorum primers JB527 (SEQ ID NO: 10) and JB525 (SEQ ID NO: 9) amplified fragment length 458 base pairs from the same genomic DNA and tissues of wheat, and that the combination of primers JB433 (SEQ ID NO: 7) and JB434 (SEQ ID NO: 8).

Combinations of primers P. herpotrichoides listed in example 8, were tested using PCR in relation to extracts from the stems of wheat, obtained as described in example 12. PCR was performed according to the method of example 4 with the following changes: 35 cycles were performed at 94oC for 15 s and at 70oC for 45 s, using 1.5-2.5 mm MgCl2and 200 μm of each dNTP. For each PCR used 1 µl of the extract of wheat.

The combination of primers JB537 (SEQ ID NO: 15) and JB541 (SEQ ID NO: 19) amplified fragment length 413 base pairs from an extract of wheat infected with W-type putative P. herpotrichoides. No amplification products were not received during the amplification or extract healthy (SEQ ID NO: 17) and JB544 (SEQ ID NO: 22) amplified fragment length 487 base pairs, but a combination of primers JB540 (SEQ ID NO: 18) and JB542 (SEQ ID NO: 20) amplified fragment length 413 base pairs from wheat infected with R-type, but not from healthy wheat or wheat infected with W-type.

Complete genomic DNA was also isolated from healthy leaves of banana and banana leaves infected with M. fijiensis using the Protocol described in example 3. PCR was performed according to the method of example 4 testing the combination of primers M. fijiensis listed in example 8, in relation to DNA from the leaves of the banana.

Specific for M. fijiensis primers JB549 (SEQ ID NO: 29) and ITS1 (SEQ ID NO: 38) (JB410) amplified fragment length 489 base pairs from purified DNA of M. fijiensis and from infected M. fijiensis leaf tissue of banana. The set of primers is not amplified diagnostic fragment from healthy leaf tissue of banana. Specific for M. fijiensis combination of primers JB443 (SEQ ID NO: 26)/ITS4 (SEQ ID NO: 41)(JB415) and ITS1 (SEQ ID NO: 38)(JB410)/JB444 (SEQ ID NO: 30) amplified fragment length 418 base pairs in the fragment length 482 base pairs, respectively, from the same genomic DNA and leaf tissue of banana, and a combination of primers JB549 (SEQ ID NO: 29) and ITS1 (SEQ ID NO: 38) (JB410).

Example 10: Determination of cross-reactivity with species-specific primers with other species and isolates

the 4, using species-specific primers. Tested DNA from other species and isolates of fungi on the ability of species-specific primers to give them a cross-reaction.

Specific for S. tritici primers JB446 (SEQ ID NO: 12) and ITS1 (SEQ ID NO:38)(JB410) amplified fragment length 345 base pairs from all isolates of S. tritici listed in example 1. No cross-reactivity was found with purified genomic DNA of S. nodorum, S. glycines or S. passermi. None of these species of fungi did not give amplificatory product specific for S. tritici primers.

Fragment length 448 base pairs amplified from all isolates of S. nodorum listed in example 1, using specific to S. nodorum primers JB433 (SEQ ID NO: 7) and JB434 (SEQ ID NO: 8). Similarly, specific for S. nodorum primers JB527 (SEQ ID NO: 10) and JB525 (SEQ ID NO: 9) amplified fragment length 458 base pairs from all isolates of S. nodorum listed in example 1. S. tritici, S. glycines and S. passerini did not give any amplification products in the study as with sets specific for S. nodorum primers JB433 (SEQ ID NO: 7) and JB434 (SEQ ID NO: 8), and JB527 (SEQ ID NO: 10) and JB525 (SEQ ID NO: 9).

PCR in relation to the DNA of other species and isolates of the fungi listed in example 1 was carried out using the conditions, the Combinations of primers JB537 (SEQ ID NO: 15) and JB541 (SEQ ID NO: 19) gave the fragment length 413 base pairs of isolates W-type R. herpotrichoides only when studied in relation to isolates of P. herpotrichoides and the following pathogens of cereal crops: P. aestiva, S. cereale, P. sorokiniana, S. tritici and S. nodorum. Combinations of primers JB539 (SEQ ID NO: 17) and JB544 (SEQ ID NO: 22) gave the fragment length 487 base pairs of isolates R-type P. herpotrichoides only when studied in the same DNA. Combinations of primers JB540 (SEQ ID NO: 18) and JB542 (SEQ ID NO: 20) gave the fragment length 413 base pairs of isolates R-type P. herpotrichoides only when studied in the same DNA.

Example 11: the Sources of contaminated Pseudocercosporella herpotrichoides wheat

The stalks of wheat infected with cercosporella, got on stage 1C program fungicidal screening of fungicides company Ciba (Basel). Wheat plants 8 days of age were infected P. herpotrichoides by spraying the base of the stalks of wheat suspension of conidia (5105conidia/ml) in 0.2% Tween20. After inoculation the plants were covered with plastic and incubated for 1 day at 20oC and a relative humidity of 95-100%. Plants transferred into the chamber for cultivation, where they were incubated for 4 weeks at 12oC and a relative humidity of 60%. After this incubation, the plants transferred to the greenhouse and incubated p is s, was taken on 8-9 week after infection, whereas plants infected with strain 308 pathogen R-type, collected at 9-10 week after infection.

Example 12: Extraction of DNA from stalks of wheat to determine P. herpotrichoides

DNA was extracted from stalks of wheat, using the Protocol described by Klimyuk and others (The Plant Journal 3(3):493-494), with some modifications. A piece of the stalk of wheat with a length of 2 cm, cut at a distance of 0.5 cm from the stem base, was placed in 160 μl of 0.25 M NaOH and mixed with a pestle and mortar Conte to complete maceration. The sample was boiled for 30 sec. To the sample was added 160 μl of 0.25 M HCl and 80 μl of 0.5 M Tris-Cl buffer, pH 8.0/0,25% (volume/volume) Nonider P-40. The additional samples were boiled for 2 minutes, then placed in a bath of ice water. For PCR analysis used 1 µl of the extract.

Example 13: the Inclusion of diagnostic tests in the format of quantitative colorimetric analysis

Coulometrically analysis was performed according to the method described by Nikiforov and other PCR Methods and Applications 3:285-291), with the following changes:

1) 30 μl of PCR product from R-type and a mixture of 3 M NaCl/20 mm add was added in the hole with primer grip. In the reaction of hybridization used 50 μl of PCR product from the W-type and a mixture of 3 M NaCl/20 mm etc.

3) Used a dilution of 1:1000 monoclonal antibodies against Biotin peroxidase from horseradish (HRP).

4) After 2 min of incubation with O-phenylenedimethylene (OPD) as substrate to each well was added 50 μl of 3H HCl. 96-well tablets were read at 492 nm and correlated with reading at 570 nm, using a conventional tablet reader used for enzyme-linked immunosorbent assay (ELISA).

Listed in the table. 5 primers synthesized according to the method of example 5 for testing as primers capture when the colorimetric analysis.

Diagnostic for S. nodorum primers JB527 (SEQ ID NO: 10) and JB525 (SEQ ID NO: 9) were combined in a format for quantitative colorimetric analysis. Primer JB527 (SEQ ID NO: 10) was synthesized using the Midland Certified Reagent Complany (Midland, Texas) so that it contained bitenova the label and to the 5'-end had four vnutrikletochnye thiophosphate communication. PCR amplification carried out according to the method of example 4 using the modified primers JB527 (SEQ ID NO: 10) and JB525 (SEQ ID NO: 9) from healthy wheat and wheat varieties with low, medium and high risk of infection of S. nodorum, was not given at all or gave low, medium and high values of A492sootvetstvenno the CR-product.

5'-Primers JB539 (SEQ ID NO: 17) and JB540 (SEQ ID NO: 18), specific for R-type P. herpotrichoides, and 5'-primer JB537 (SEQ ID NO: 15), specific for W-type P. herpotrichoides, also modified so that they contain bitenova label and four vnutrikletochnye thiophosphate communication. Option colorimetric evaluation of PCR analysis for R-type P. herpotrichoides was developed using a modified primer JB540 (SEQ ID NO: 18), primer JB542 (SEQ ID NO: 20) and primer capture JB539'15. The products resulting from amplification of wheat infected with R-type, and in the amplification of genomic DNA R-type when using modified primers JB540 (SEQ ID NO: 18) and primer JB542 (SEQ ID NO: 20), gave a positive colorimetric values in the study by colorimetric method. Positive colorimetric values were also obtained when the colorimetric analysis of PCR products resulting from amplification using a modified primer JB537 (SEQ ID NO: 15) and specific for W-type primer JB541 (SEQ ID NO: 19) of wheat infected with W-type, and genomic DNA W-type, when the primer grip used JB538'15. In addition, the intensity of colorimetric signal corresponded to the intensity of the fragment PCR-protem research microscope and identification of different strains of Pseudocercosporella was only possible with the help of pathological tests. Similarly, a clear identification of Mycosphaerella musicola and Mycosphaerella fijiensis was difficult and even selection of Mature perithecia are not always allowed to make an exact identification (Pons, 1990; Sigatoka Leaf Spot Deceases of Banana, editor R. A. Fullerton and R. H. Stover, International Network for the Improvement of Banana and plantain trunks, France). Currently, for the identification of S. tritici and S. nodorum, Pseudocercosporella herpotrichoides and other pathogens commonly used immunodiagnostics sets, based on the use of ELISA method, however this method does not have the necessary accuracy is limited by the resolution and does not allow to distinguish between different isolates of the present invention. Therefore, the test based on DNA for rapid identification of various strains of these fungi gives real benefits not only for specialists in the systematics of fungi, but also for disease control and field application specific fungicide.

Although the present invention is described with reference to specific examples of its implementation, it is obvious that various changes, modifications and other examples of implementation, and thus the m of the present invention.

Pick

Pick up the following crops have been conducted on March 28, 1994 in culture Collections for the purposes of patenting procedures (NRRL) the agricultural research service, Northern regional research center, 1815 North University Street, Peoria, Illinois 61604, USA:

1. HB101 DH5d (pCRW2-1;SEQ ID NO: 3) Reg.number NRRL B-21231.

2. HB101 DH5d (pCRW5-1;SEQ ID NO: 47) Reg.number NRRL B-21232.

3. E. coli DH5d (pCRSTRIT1;SEQ ID NO: 1) Reg.number NRRL B-21233.

4. E. coli DH5d (pCRR1-21;SEQ ID NO: 4) Reg.number NRRL B-21234.

5. E. coli DH5d (pCRSNOD31;SEQ ID NO: 2) Reg.number NRRL B-21235.

1. The DNA molecule encoding intron transcribers sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 47, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, designed to design oligonucleotide primers for detection of phytopathogenic fungus.

2. Oligonucleotide primer for detection of phytopathogenic fungus other than primernih molecules 5'-GGG CTA CCC TAC TTG GTA G3' and 5'- GGG CCA CCC TAC TTC GGT AA-3', where the primer is selected from the group consisting of sequences SEQ ID NO: 7-37, SEQ ID NO: 42-46 and SEQ ID nos: 50-65.

3. A pair of oligonucleotide primers for detection of phytopathogenic fungi, in which a pair of at least one primer selected from the group consisting of sequences SEQ ID NO: 7-37 and SEQ ID nos: 50-65.

5. A pair of oligonucleotide primers under item 4, in which one primer selected from the group consisting of sequences SEQ ID NO:77-37 and SEQ ID nos: 50-65, and the other primer is selected from the group consisting of sequences SEQ ID nos: 38-41.

6. A pair of oligonucleotide primers under item 4, in which a couple is chosen from the group consisting of the pairs listed in table 4.

7. A pair of oligonucleotide primers under item 4, in which a couple is chosen from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

8. A pair of oligonucleotide primers under item 5, in which a couple is chosen from the group consisting of (a) SEQ ID NO: 10 and SEQ ID NO: 9; (b) SEQ ID NO: 12 and SEQ ID NO: 38; (C) SEQ ID NO: 11 and SEQ ID NO: 41; (r) SEQ ID NO: 29 and SEQ ID NO: 38; (d) SEQ ID NO: 7 and SEQ ID NO: 41; (e) SEQ ID NO: 30 and SEQ ID NO: 38; (g) SEQ ID NO: 15 and SEQ ID NO: 19; (C) SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 18 and SEQ ID NO: 20 and (K) SEQ ID NO: 26 and SEQ ID NO: 41.

9. The method of detection of phytopathogenic fungus comprising the stage of: (a) isolation of DNA from plant leaf infected with a pathogen; (b) amplification of part of intron transcarbamylase region of the pathogen using a given DNA as template using polymerase chain reaction with at least one primer under item 2; (C) visualization amplificare is the Bohm PP.3 - 8 use in stage B.

11. The method according to p. 10, in which the pathogenic fungus selected from S. nodorum, S. tritici, P. herpotrichoides. M. fijiensis, M. musicola, F. culmorum, F. frost, Microdochium nivate and F. moniliforme.

12. The method according to p. 11, in which P. herpotrichoides choose from the W-strain and the R-strain.

13. Kit for detection of phytopathogenic fungus containing media, divided into compartments, one of which contains a primer on p. 2 or a pair of primers according to any one of paragraphs.3 - 8.

14. Quantitative colorimetric analysis for detection of phytopathogenic fungus comprising the stage of: (a) isolation of DNA from plant leaf infected with a pathogen; (b) amplification of the DNA of the pathogen by polymerase chain reaction and (b) visualization of the amplified part of the intron transcarbamylase region, and (I) DNA extracted at the stage (a), used as a DNA template in the amplification of stage (b); (II) the amplification is carried out in the presence of a couple of diagnostic primers for p. 3 and (III) amplified DNA visualize using one of the primers capture, presented in table 5.

15. Analysis under item 14 in which a pair of primers according to any one of paragraphs.3 - 8 used in stage (II).

16. Analysis under item 15, where diag is 2">

17. Analysis under item 15, in which the diagnostic primers are selected from SEQ ID NO: 18 and SEQ ID NO: 20, and the capture primer has the sequence SEQ ID NO: 71.

18. Analysis under item 15, in which the diagnostic primers are selected from SEQ ID NO: 15 and SEQ ID NO: 19, and the capture primer has the sequence SEQ ID NO: 71.

 

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