Abstract:
Primers specific for races of pathogenic fungi which are resistant to certain fungicides are used in polymerase chain reaction assays for the detection of fungal pathogens. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations. The invention includes DNA sequences which show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they can be used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and can thus be used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.

Description:
[0001]    This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/369,796 filed Apr. 3, 2002, which is incorporated by reference in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to the use of primers specific for races of pathogenic fungi which are resistant to certain fungicides in polymerase chain reaction assays for the detection of fungal pathogens. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations.  
         BACKGROUND OF THE INVENTION  
         [0003]    Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and additionally in many parts of the world to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981;  Seed Sci.  &amp;  Technol.  9: 679-685).  
           [0004]    The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains which are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981;  Proc.  1981  Brit. Crop Prot. Conf. ) contended that 24% of the powdery mildew populations from spring barley, and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Tapesia (to MBC-type fungicides) and  Mycosphaerella fijiensis  to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).  
           [0005]    Cereal species are grown world-wide and represent a major fraction of world food production. Although yield loss is caused by many pathogens, the necrotizing pathogens Septoria and Tapesia are particularly important in the major cereal growing areas of Europe and North America (Jones and Clifford; Cereal Diseases, John Wiley, 1983). In particular, the differential symptomology caused by different isolates and species of these fungi make the accurate predictive determination of potential disease loss difficult. Consequently, the availability of improved diagnostic techniques for the rapid and accurate identification of specific pathogens will be of considerable use to field pathologists.  
           [0006]    The eyespot disease of cereals is caused by the fungi  Tapesia yallundae  and  Tapesia acuformis  is restricted to the basal culm of the plant. The two causal pathogens were previously classified as two subspecies of  Pseudocercosporella herpotrichoides  (Fron) Deighton (anamorph).  T. yallundae  refers to the variety herpotrichoides and the SF-,L-,I- or W-types.  T acuformis  corresponds to the variety acuformis and the FE-, N-, II- or R-types (Leroux and Gredt, 1997; 51:321-327). Wheat, rye, oats and other grasses are susceptible to the eyespot disease which occurs in cool, moist climates and is prevalent in Europe, North and South America, Africa and Australia. Wheat is the most susceptible cereal species, but isolates have been identified which are also virulent on other cereals. The R-strain ( T. acuformis ) of the fungus, for example, has also been isolated from rye and grows more slowly on wheat than the W-strain ( T. yallundae ) which has been isolated from wheat. Although eyespot may kill tillers or plants outright, it more usually causes lodging and/or results in a reduction in kernel size and number. Yield losses associated with eyespot are of even greater magnitude than those associated with  Septoria tritici  and  Septoria nodorum.  Typical control measures for eyespot include treatment with growth regulators to strengthen intemodes, and fungicide treatment. However, the differing susceptibility of cultivars to different strains of the fungus render the predictive efficacy of fungicide treatments difficult. In addition, both Leroux et al (1997;  Pesticide Science,  51:321-327) and Dyer et al (2000;  Appl. and Environ. Microbiol.  66:4599-4604) have reported on isolates of  T. yallundae  with reduced sensitivity to the imidazole DMI fungicide prochloraz (1-[N-propyl-N-[2-92,4,6-trichlorophenoxy)ethyl]carbamoyl]-imidazole). Following heavy treatments of benzimidazole fungicides such as benomyl, carbendazim and thiabendazole, acquired resistance to this class of fungicides was determined in both  T. acuformis  and  T. yallundae  (Leroux and Cavelier, 1983;  Phytoma  351:40) and (Cavelier et al, 1985;  Bull. OEPP  85:495).  
           [0007]    Thus, there is a real need for the development of technology which will allow the identification of specific races of pathogen fungi which are resistance to certain fungicides early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.  
         SUMMARY OF THE INVENTION  
         [0008]    The present invention relates to the use of primers specific for races of pathogenic fungi which are resistant to certain fungicides in polymerase chain reaction assays for the detection of fungal pathogens. The invention provides DNA sequences which show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they can be used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and can thus be used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.  
           [0009]    This invention provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides which is available. Furthermore, it can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas.  
           [0010]    Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of Tapesia pathogens.  
           [0011]    The present invention provides a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NOS: 3-13 or 14. In a more preferred embodiment, the nucleic acid molecule has sequence identity with at least 10 contiguous nucleotides of SEQ ID NOS: 2-13 or 14. In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NOs: 3-76 or 77.  
           [0012]    The invention also provides a pair of oligonucleotide primers wherein at least one primer consists of the nucleotide sequence of SEQ ID NOS: 3-76 or 77. In a preferred embodiment, the pair of oligonucleotide primers comprises:  
           [0013]    JB944 (SEQ ID NO: 59) and JB943 (SEQ ID NO: 58;  
           [0014]    JB944 (SEQ ID NO: 59) and JB945 (SEQ ID NO: 60);  
           [0015]    JB934 (SEQ ID NO: 49) and JB935 (SEQ ID NO: 50); and  
           [0016]    JB937 (SEQ ID NO: 52) and JB935 (SEQ ID NO: 50).  
           [0017]    The invention also provides a method for the detection of a fungal pathogen, comprising the steps of:  
           [0018]    (a) isolating DNA from a plant tissue infected with a pathogen;  
           [0019]    (b) subjecting said DNA to polymerase chain reaction amplification using at least one primer having sequence identity with at least 10 contiguous nucleotides of a randomly amplified polymorphic DNA sequence of a Tapesia spp.; and  
           [0020]    (c) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.  
           [0021]    In a preferred embodiment, the fungal pathogen is  Tapesia yallundae, Tapesia acuformis.  More preferably, the  Tapesia yallundae  is subtype Ic., and  Tapesia acuformis  subtypes IIs or IIp. In another preferred embodiment, at least one primer having the nucleotide sequence of SEQ ID NOS: 3-76 or 77.  
           [0022]    The invention further provides a method for the detection of a fungal pathogen, comprising the steps of:  
           [0023]    (a) isolating DNA from a plant tissue infected with a pathogen;  
           [0024]    (b) subjecting said DNA to polymerase chain reaction amplification using at least one primer having sequence identity with at least 10 contiguous nucleotides of a randomly amplified polymorphic DNA from a Tapesia spp.; and  
           [0025]    (c) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.  
           [0026]    In a preferred embodiment, the fungal pathogen is  Tapesia yallundae, Tapesia acuformis.  More preferably, the  Tapesia yallundae  is subtype Ic, and  Tapesia acuformis  subtypes IIs or IIp. In another preferred embodiment, at least one primer having the nucleotide sequence of SEQ ID NOS: 3-76 or 77. In more preferred embodiment, the pair of primers comprises:  
           [0027]    JB944 (SEQ ID NO: 59) and JB943 (SEQ ID NO: 58;  
           [0028]    JB944 (SEQ ID NO: 59) and JB945 (SEQ ID NO: 60);  
           [0029]    JB934 (SEQ ID NO: 49) and JB935 (SEQ ID NO: 50); or  
           [0030]    JB937 (SEQ ID NO: 52) and JB935 (SEQ ID NO: 50).  
           [0031]    The invention also provides a diagnostic kit used in detecting a fungal pathogen comprising at least one primer having at least 10 contiguous nucleotides of a nucleic acid molecule of the nucleic acid molecules described above. In a preferred embodiment, at least one primer comprises SEQ ID NO: 3-76 or 77. In more preferred embodiments, the pair of primers are:  
           [0032]    JB944 (SEQ ID NO: 59) and JB943 (SEQ ID NO: 58;  
           [0033]    JB944 (SEQ ID NO: 59) and JB945 (SEQ ID NO: 60);  
           [0034]    JB934 (SEQ ID NO: 49) and JB935 (SEQ ID NO: 50); or  
           [0035]    JB937 (SEQ ID NO: 52) and JB935 (SEQ ID NO: 50).  
                                             Brief Description of the Sequences in the Sequence Listing                                    SEQ-ID-NO: 1   M13 Sequencing Forward Primer           SEQ-ID-NO: 2   M13 Sequencing Reverse Primer           SEQ-ID-NO: 3   RAPD-PCR Clone Ib1-27           SEQ-ID-NO: 4   RAPD-PCR Clone Ib2-31           SEQ-ID-NO: 5   RAPD-PCR Clone Ib3-33           SEQ-ID-NO: 6   RAPD-PCR Clone Ic1-22           SEQ-ID-NO: 7   RAPD-PCR Clone Ic 020502Ic4and6           SEQ-ID-NO: 8   RAPD-PCR Clone Ic 020602D-20           SEQ-ID-NO: 9   RAPD-PCR Clone Ic 020602D-21           SEQ-ID-NO: 10   RAPD-PCR Clone IIp1-17           SEQ-ID-NO: 11   RAPD-PCR Clone IIp 020602A-11           SEQ-ID-NO: 12   RAPD-PCR Clone IIp 020602B-15           SEQ-ID-NO: 13   RAPD-PCR Clone IIp 020602B-16           SEQ-ID-NO: 14   RAPD-PCR Clone IIs2-39           SEQ-ID-NO: 15   JB900           SEQ-ID-NO: 16   JB901           SEQ-ID-NO: 17   JB902 Probe           SEQ-ID-NO: 18   JB903           SEQ-ID-NO: 19   JB904           SEQ-ID-NO: 20   JB905           SEQ-ID-NO: 21   JB906           SEQ-ID-NO: 22   JB907           SEQ-ID-NO: 23   JB908           SEQ-ID-NO: 24   JB909           SEQ-ID-NO: 25   J8910           SEQ-ID-NO: 26   1B911           SEQ-ID-NO: 27   JB912 Probe           SEQ-ID-NO: 28   JB913           SEQ-ID-NO: 29   JB914           SEQ-ID-NO: 30   JB915           SEQ-ID-NO: 31   JB916           SEQ-ID-NO: 32   J8917 Probe           SEQ-ID-NO: 33   JB918           SEQ-ID-NO: 34   JB919           SEQ-ID-NO: 35   JB920           SEQ-ID-NO: 36   JB921           SEQ-ID-NO: 37   JB922 Probe           SEQ-ID-NO: 38   JB923           SEQ-ID-NO: 39   JB924           SEQ-ID-NO: 40   JB925           SEQ-ID-NO: 41   JB926           SEQ-ID-NO: 42   JB927 Probe           SEQ-ID-NO: 43   JB928           SEQ-ID-NO: 44   JB929           SEQ-ID-NO: 45   JB930           SEQ-ID-NO: 46   JB931           SEQ-ID-NO: 47   JB932           SEQ-ID-NO: 48   JB933           SEQ-ID-NO: 49   JB934           SEQ-ID-NO: 50   JB935           SEQ-ID-NO: 51   JB936           SEQ-ID-NO: 52   JB937           SEQ-ID-NO: 53   JB938           SEQ-ID-NO: 54   JB939           SEQ-ID-NO: 55   JB940           SEQ-ID-NO: 56   JB941           SEQ-ID-NO: 57   JB942           SEQ-ID-NO: 58   JB943           SEQ-ID-NO: 59   JB944           SEQ-ID-NO: 60   JB945           SEQ-ID-NO: 61   JB946           SEQ-ID-NO: 62   JB947           SEQ-ID-NO: 63   JB948           SEQ-ID-NO: 64   JB949           SEQ-ID-NO: 65   JB950           SEQ-ID-NO: 66   JB951           SEQ-ID-NO: 67   JB952           SEQ-ID-NO: 68   JB953           SEQ-ID-NO: 69   JB954           SEQ-ID-NO: 70   JB955           SEQ-ID-NO: 71   JB956           SEQ-ID-NO: 72   JB957           SEQ-ID-NO: 73   JB958           SEQ-ID-NO: 74   JB959           SEQ-ID-NO: 75   JB960           SEQ-ID-NO: 76   JB961           SEQ-ID-NO: 77   JB962                      
 
         DETAILED DESCRIPTION OF THE INVENTION  
         [0036]    The present invention provides unique DNA sequences which are useful in identifying different pathotypes of plant pathogenic fungi. Particularly the DNA sequences can be used as primers in PCR based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include products cloned from RAPD primer analysis of particular fungal pathogens as well as primers which are derived from these regions which are capable of identifying the particular pathogen. These DNA sequences from different pathotypes within a pathogen species or genus which vary between the different members of the species or genus based on different fungicides&#39; susceptibility can be used to identify those specific members.  
           [0037]    Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. Only recently, however, has this technique been applied to detect plant pathogens. The presence of  Gaumannomyces graminis  in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991;  Applied and Environ. Microbiol.  57: 553-556) and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of  Gremmeniella abietina,  the causal agent of scleroderris canker in conifers.  
           [0038]    The DNA sequences of the invention are from randomly amplified polymorphic DNA (RAPD) of different plant pathogens. The RAPD sequences from different pathotypes within a pathogen species or genus vary between the different members of the species or genus. Once having determined the unique RAPD sequences of a pathogen, primers can be derived from the sequences. That is, primers can be designed based on regions within the uniquely identified RAPD fragment sequence among the fungal pathotypes. These sequences and primers based on these sequences can be used to identify specific pathogen members.  
           [0039]    Particular DNA sequences of interest include uniquely identified RAPD sequences from Tapesia, particularly,  Tapesia acuformis  and  Tapesia yallundae,  more particularly for the identification of  T. acuformis  subtypes IIs and IIp and  T. yallundae  subtypes Ia, Ib and Ic. Such DNA sequences as well as primers of interest are given in SEQ ID NO: 3-77. The sequences find use in the PCR-based identification of the pathotypes of interest.  
           [0040]    Sequences from RAPD analysis of uniquely identified fragments include SEQ-ID-NOs: 3-14. The sequences find use in the PCR-based identification of pathogens of interest. In a preferred embodiment the sequence disclosed as SEQ-ID-NO: 10 is useful in the development of primers for differentiating  T. acuformis  subtypes IIs and IIp. In another preferred embodiment the sequence disclosed as SEQ-ID-NO: 8 is useful in the development of primers for the detection of  T. yallundae  subtype Ic.  
           [0041]    Sequences from oligonucleotide primers derived from the uniquely identified RAPD analysis fragments are disclosed as SEQ-ID-Nos: 15-77. In a preferred embodiment, the pair of oligonucleotide primers consists of SEQ-ID-NO: 59 and SEQ-ID-NO: 58 is used for the detection of  T. yallundae  Ic. In another preferred embodiment, the pair of oligonucleotide primers consists of SEQ-ID-NO: 59 and SEQ-ID-NO: 60 is used for the detection of  T. yallundae  Ic. In yet other embodiments,  T. acuformis  subtype IIs can be differentiated from  T. acuformis  subtype IIp using the primer combination consisting of oligonucleotide primers with SEQ-ID-NO: 49 and SEQ-ID-NO: 50 and the primer combination consisting of oligonucleotide primers with SEQ-ID-NO: 52 and SEQ-ID-NO: 50.  
           [0042]    The present invention lends itself readily to the preparation of “kits” containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means, such as tubes or vials. One of said container means may contain unlabeled or detectably labeled DNA primers. The labeled DNA primers may be present in lyophilized form, or in an appropriate buffer as necessary. One or more container means may contain one or more enzymes or reagents to be utilized in PCR reactions. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers.  
           [0043]    Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.  
           [0044]    The examples below show, without limitation, typical experimental protocols which can be used in the isolation of unique RAPD sequences, the selection of suitable primer sequences, the testing of primers for selective and diagnostic efficacy, and the use of such primers for disease and fungal isolate detection. Such examples are provided by way of illustration and not by way of limitation. 
       
    
    
     EXAMPLES  
       [0045]    Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by J. Sambrook, E. F. Fritsch and T. Maniatis,  Molecular Cloning: A Laboratory manual,  Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,  Experiments with Gene Fusions,  Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al.,  Current Protocols in Molecular Biology,  pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).  
       Example 1  
       [0046]    Fungal Isolates and Genomic Fungal DNA Extraction  
         [0047]    See Table 1 for a listing of the fungal isolates used. Isolates for which fungicide sensitivity was characterized were obtained from Institut National de la Recherche Agronomique (INRA, Le Rheu, France). Fungi are grown on PDA (Potato Dextrose Agar) plates. Cultures are incubated for up to 10 days at 28° C. Mycelia are ground in liquid nitrogen, and total genomic DNA is extracted using the following modified CTAB protocol.  
         [0048]    1. Freeze-dried mycelium was homogenized in 1.5 ml Eppendorf tubes (two tungsten carbide 3 mm beads were added) using a Retsch mill (MM200, Retsch GmbH &amp; Co., Haan, Germany).  
         [0049]    2. Add 600 μl extraction buffer (700 mM NaCl, 50 mM Tris HCl, 10 mM EDTA, 1% β-mercapthoethanol, 1% CTAB) and incubate for 1 hr at 65° C., vortexing every 10-20 minute interval.  
         [0050]    3. Add 400 μl chlorophorm:isoamylalcohol (24.1, v:v) shake for 15 min  
         [0051]    4. Centrifuge for 10 min (16000 g)  
         [0052]    5. Transfer the aqueous phase to a new tube and add 400 μl extraction buffer and 400 μl chloroform:isoamylalcool  
         [0053]    6. Shake for 15 min  
         [0054]    7. Centrifuge 10 min  
         [0055]    8. Transfer aqueous phase to a new tube  
         [0056]    9. Add 0.6× volumes of isopropanol  
         [0057]    10. Shake for 5 min  
         [0058]    11. Centrifuge for 5 min  
         [0059]    12. Discard supernatant and dry pellet  
         [0060]    13. Wash pellet with cold EtOH 70%  
         [0061]    14. Centrifuge 2 min  
         [0062]    15. Dry pellet  
         [0063]    16. Resuspend in 50 μl TE  
         [0064]    [0064]                                                                       TABLE 1                           Identification of Test Isolates            Fungal   Fungal   Subtype       Fungicide Sensitivity 1     Cyprodinil            Species   Isolate   Identifier   Triadimenol   Prochloraz   Carbendazim   (Unix)                 Tapesia yallundae     627 N   Ia   S   S   R   S         Tapesia yallundae     646 N   Ib   R   S   R   S         Tapesia yallundae     572 N   Ic   R   R   R   S         Tapesia yallundae     618 N   Ic   R   R   R   S         Tapesia acuformis     634 L   IIs   R   S   S   S         Tapesia acuformis     643 L   IIs   R   S   S   S         Tapesia acuformis     567 L   IIp   R   R   R   S         Tapesia acuformis     617 L   IIp   R   R   R   S         Tapesia acuformis     626 L   IIp   R   R   R   S                                    
       Example 2  
       [0065]    Amplification of RAPD Products  
         [0066]    Polymerase chain reactions are performed to obtain Randomly Amplified Polymorphic DNA (RAPD) profiles for each of the Tapesia spp. subtypes. Forty different RAPD 10-mer primers from Qiagen Operon (Operon Technologies Inc., Alameda, Calif., USA) kits AA and J identified individually as OPAA-01-OPAA-20 and OPJ-01-OPJ-20 are used in amplifications to find RAPD products specific to subtypes Ic and IIp. A single 10-mer RAPD primer is used in RAPD-PCR reactions. Reactions are prepared using the GeneAmp Kit from Perkin-Elmer (Foster City, Calif.; part no. N808-0009) using 50 mM KCl, 2.0 mM MgCl 2 , 10 mM Tris-HCl, pH8.3 and containing 100 μM of each dTTP, dATP, dCTP, and dGTP in 25 μL reactions. In each reaction, 25 pmol of RAPD primer is used with 0.5 Units of AmpliTaq Polymerase. Approximately 25 ng of genomic DNA from the subtypes listed in Example 1 are used as template. Reactions are run in a GeneAmp Model 9700 thermal cycler (Applied Biosystems, Foster City, Calif.). Thermal cycling is run for 45 cycles of 30 s at 94° C., 30 s at 34° C., and 60 s at 72° C. and is proceeded by a hold at 94° C. for one minute and followed by a final hold at 72° C. for ten minutes before being stored at 4° C. The products are analyzed by loading 10 μl of each PCR sample with loading buffer on a 1.0% agarose gel and electrophoresing.  
         [0067]    The gel is stained with ethidium bromide and separated RAPD-PCR bands are observed under ultraviolet light.  
       Example 3  
       [0068]    Selection, Cloning, and Sequencing of Subtype-Specific RAPD-PCR Products  
         [0069]    RAPD-PCR products for each Tapesia spp. subtype are compared. Bands that appear to be specific to a certain subtype are selected for further analysis by DNA sequencing. These bands are cut from the agarose gel by a sterile scalpel. The RAPD-PCR product is purified from the agarose using GenElute Minus EtBr Spin Columns (Product Code 5-6501, Sigma-Aldrich, St. Louis, Mo., USA). The purified product is cloned into the pCR4-TOPO vector and transformed into One Shot chemically compentent bacterial cells using the TOPO TA Cloning Kit for Sequencing (Invitrogen Corporation, Carlsbad, Calif., USA) under manufacturer&#39;s protocol. Transformed cells containing the vector plus RAPD-PCR product insert are identified by endonuclease digestion of minipreped DNA of isolated colonies. Minipreps of vector DNA containing the RAPD-PCR product are sequenced. Sequencing is performed on an ABI PRISM 377™ DNA sequencer (Applied Biosystems, Foster City, Calif.) using primers in the pCR4-TOPO cloning vector: FORWARD (5′-gtaaaacgacggccagt-3′; SEQ ID NO: 1) and REVERSE (5′-caggaaacagctatgac-3′; SEQ ID NO:2). Sequences obtained for each Tapesia spp. subtype are identified in Table 2.  
                                 TABLE 2                           Tapesia spp. subtype-specific RAPD-PCR product sequences                        RAPD-PCR       Sequence       Subtype and   Sequence       Identifier   Fungal Species   Sequence ID   Length               SEQ-ID-NO: 3     Tapesia yallundae     Ib1-27   525       SEQ-ID-NO: 4     Tapesia yallundae     Ib2-31   551       SEQ-ID-NO: 5     Tapesia yallundae     Ib3-33   520       SEQ-ID-NO: 6     Tapesia yallundae     Ic1-22   456       SEQ-ID-NO: 7     Tapesia yallundae     Ic 020502Ic4and6   711       SEQ-ID-NO: 8     Tapesia yallundae     Ic 020602D-20   555       SEQ-ID-NO: 9     Tapesia yallundae     Ic 020602D-21   625       SEQ-ID-NO: 10     Tapesia acuformis     IIp1-17   455       SEQ-ID-NO: 11     Tapesia acuformis     IIp 020602A-11   498       SEQ-ID-NO: 12     Tapesia acuformis     IIp 020602B-15   702       SEQ-ID-NO: 13     Tapesia acuformis     IIp 020602B-16   503       SEQ-ID-NO: 14     Tapesia acuformis     IIs2-39   513                  
 
       Example 4  
       [0070]    Design of Subtype-Specific Primers  
         [0071]    PCR Primers are designed to amplify within the sequences of RAPD-PCR products obtained according to Example 3. Primers are designed to be used either in conventional PCR reactions or with an oligonucleotide probe in TaqMan PCR reactions. Multiple primers are developed to target each RAPD-PCR Tapesia spp. subtype-specific sequence. These primers are listed in Table 3.  
                                                 TABLE 3                           Tapesia spp. subtype-specific primer sequences                Sequence           Target DNA   RAPD-PCR               Identifier   Name   Species   Subtype Product   Oligo Sequence (5′-3′)               SEQ-ID-NO: 15   JB900     Tapesia yallundae     Ib   Ib1-27/Ib2-31   TGCGGTAGGGCGAAGAAAC                   SEQ-ID-NO: 16   JB901     Tapesia yallundae     Ib   Ib1-27/Ib2-31   CATCCTCCACCAACCAATACG               SEQ-ID-NO: 17   JB902 Probe     Tapesia yallundae     Ib   Ib1-27/Ib2-31   AACACCAAAGCGGCTTCGCGAGA               SEQ-ID-NO: 18   JB903     Tapesia yallundae     Ib   Ib1-27/Ib2-31   CAGCCGATTGATCCGGTCTA               SEQ-ID-NO: 19   JB904     Tapesia yallundae     Ib   Ib1-27/Ib2-31   GGCAACGCTGATTCGACTCTA               SEQ-ID-NO: 20   JB905     Tapesia yallundae     Ib   Ib1-27/Ib2-31   GGTTCGATCCCGTCATCCT               SEQ-ID-NO: 21   JB906     Tapesia yallundae     Ib   Ib1-27/Ib2-31   GTCGGTTCGATCCCGTCAT               SEQ-ID-NO: 22   JB907     Tapesia yallundae     Ib   Ib1-27/Ib2-31   CAATACGCTCTGCGGTAGGGCGAA               SEQ-ID-NO: 23   JB908     Tapesia yallundae     Ib   Ib1-27/Ib2-31   GCCGCTTTGGTGTTGGTTT               SEQ-ID-NO: 24   JB909     Tapesia yallundae     Ib   Ib1-27/Ib2-31   CAGCCGATTGATCCGGTCTAT               SEQ-ID-NO: 25   JB910     Tapesia yallundae     Ib   Ib3-33   AATTCGCCCTTGGACCTCTT               SEQ-ID-NO: 26   JB911     Tapesia yallundae     Ib   Ib3-33   TTCGCCCTTGGACCTCTTG               SEQ-ID-NO: 27   JB912 Probe     Tapesia yallundae     Ib   Ib3-33   AGTAACACGCCCCACGGACGGAT               SEQ-ID-NO: 28   JB913     Tapesia yallundae     Ib   Ib3-33   CTGCGGAGTCCTTGCTAGCT               SEQ-ID-NO: 29   JB914     Tapesia yallundae     Ib   Ib3-33   GACCTGCGGAGTCCTTGCT               SEQ-ID-NO: 30   JB915     Tapesia yallundae     Ic   Ic1-22   TTACACTGTATTTGTCTGGTGATTGC               SEQ-ID-NO: 31   JB916     Tapesia yallundae     Ic   Ic1-22   GAGCCTCTCATATCTGGATCTCTAAATC               SEQ-ID-NO: 32   JB917 Probe     Tapesia yallundae     Ic   Ic1-22   TTAACTAGCAGTCATCTGTCCTGTGCCAAGG               SEQ-ID-NO: 33   JB918     Tapesia yallundae     Ic   Ic1-22   GACAAACTCTACCAAGGAGAGACAAAA               SEQ-ID-NO: 34   JB919     Tapesia yallundae     Ic   Ic1-22   CTACCAAGGAGAGACAAAACACAAAA               SEQ-ID-NO: 35   JB920     Tapesia acuformis     IIp   IIp1-17   TCTTGTGAGACTGCATGGACTAGAGT               SEQ-ID-NO: 36   JB921     Tapesia acuformis     IIp   IIp1-17   GATCTTGTGAGACTGCATGGACTAG               SEQ-ID-NO: 37   JB922 Probe     Tapesia acuformis     IIp   IIp1-17   CATGCGAGAATTAAAGAGCTATAGTTGCGTGC               SEQ-ID-NO: 38   JB923     Tapesia acuformis     IIp   IIp1-17   CGCAATCCTTTCTCGACTTCTAA               SEQ-ID-NO: 39   JB924     Tapesia acuformis     IIp   IIp1-17   GTTTCGCAATCCTTTCTCGACTT               SEQ-ID-NO: 40   JB925     Tapesia acuformis     IIs   IIs2-39   GCAGAATTCGCCCTTAAGTCG               SEQ-ID-NO: 41   JB926     Tapesia acuformis     IIs   IIs2-39   TCTGCAGAATTCGCCCTTAAG               SEQ-ID-NO: 42   JB927 Probe     Tapesia acuformis     IIs   IIs2-39   AAGGTAGCCGTATCGGAAGGTGCGG               SEQ-ID-NO: 43   JB928     Tapesia acuformis     IIs   IIs2-39   CCAGAACGGAGGTGATCCAG               SEQ-ID-NO: 44   JB929     Tapesia acuformis     IIs   IIs2-39   TTCCAGAACGGAGGTGATCC               SEQ-ID-NO: 45   JB930     Tapesia yallundae     Ic   Ic1-22   ATATTCTTGCTGAATTGGTC               SEQ-ID-NO: 46   JB931     Tapesia yallundae     Ic   Ic1-22   CAAAATTATTTCATCCTTGGCACAG               SEQ-ID-NO: 47   JB932     Tapesia yallundae     Ic   Ic1-22   AAATTATTTCATCCTTGGCACAGG               SEQ-ID-NO: 48   JB933     Tapesia yallundae     Ic   Ic1-22   ATATTCTTGCTGAATTGGTC               SEQ-ID-NO: 49   JB934     Tapesia acuformis     IIp   IIp1-17   AGATGGGCAGAGTGTAGATCTTGTG               SEQ-ID-NO: 50   JB935     Tapesia acuformis     IIp   IIp1-17   GGAACCGAGAGAGTAGCAACAGAAC               SEQ-ID-NO: 51   JB936     Tapesia acuformis     IIp   IIp1-17   CAGGAACCGAGAGAGTAGCAACAG               SEQ-ID-NO: 52   JB937     Tapesia acuformis     IIp   IIp1-17   GCGTTCGGCTTGAAGTCATG               SEQ-ID-NO: 53   JB938     Tapesia acuformis     Ic   0205021c4and6   CCTTTGGTCGGGTGGGAGAA               SEQ-ID-NO: 54   JB939     Tapesia acuformis     Ic   0205021c4and6   GCCAGGCTGAATCTTGGGAA               SEQ-ID-NO: 55   JB940     Tapesia acuformis     Ic   0205021c4and6   CCAGGCTGAATCTTGGGAAA               SEQ-ID-NO: 56   JB941     Tapesia acuformis     Ic   0205021c4and6   CCAAGTACGCATCTCGGATG               SEQ-ID-NO: 57   JB942     Tapesia acuformis     Ic   020602D-20   GAAGTGTTTACTCTTTGCCG               SEQ-ID-NO: 58   JB943     Tapesia acuformis     Ic   020602D-20   AATATTGGTTCTTGATCCTG               SEQ-ID-NO: 59   JB944     Tapesia acuformis     Ic   020602D-20   TCGAGACAATAGAGATTTTC               SEQ-ID-NO: 60   JB945     Tapesia acuformis     Ic   020602D-20   GTGTGTCATTTTGGAAGATT               SEQ-ID-NO: 61   JB946     Tapesia acuformis     Ic   020602D-21   ACATACCATCTTGTAAATAGCC               SEQ-ID-NO: 62   JB947     Tapesia acuformis     Ic   020602D-21   CATAGTCAATCCAAGCTTTC               SEQ-ID-NO: 63   JB948     Tapesia acuformis     Ic   020602D-21   ATACCATCTTGTAAATAGCC               SEQ-ID-NO: 64   JB949     Tapesia acuformis     Ic   020602D-21   TATGCTTCTGGTCTTTGTTT               SEQ-ID-NO: 65   JB950     Tapesia yallundae     IIp   020602A-11   AATCAATGTCATGCGGTTCG               SEQ-ID-NO: 66   JB951     Tapesia yallundae     IIp   020602A-11   CACTTCCACGGCAGTGATAA               SEQ-ID-NO: 67   JB952     Tapesia yallundae     IIp   020602A-11   TTGTCTCTTGGGTAATCATG               SEQ-ID-NO: 68   JB953     Tapesia yallundae     IIp   020602A-11   GTGCCAAAAGGAACTGATTG               SEQ-ID-NO: 69   JB954     Tapesia yallundae     IIp   020602B-16   TGAGATTCCGGACTGCATTT               SEQ-ID-NO: 70   JB955     Tapesia yallundae     IIp   020602B-16   CAAACTGAGATTTCTCAACG               SEQ-ID-NO: 71   JB956     Tapesia yallundae     IIp   020602B-15   CCTTACCCGACCTGCCATGT               SEQ-ID-NO: 72   JB957     Tapesia yallundae     IIp   020602B-15   CTGGCGGCCATATCGACTTC               SEQ-ID-NO: 73   JB958     Tapesia yallundae     IIp   020602B-15   ATTAGCAACTGGAATGCACA               SEQ-ID-NO: 74   JB959     Tapesia yallundae     IIp   020602B-15   AAGCCAGCTGCATGATGTTC               SEQ-ID-NO: 75   JB960     Tapesia yallundae     IIp   020602B-16   CGCCCTAGCACATCATCAAA               SEQ-ID-NO: 76   JB961     Tapesia yallundae     IIp   020602B-16   CCTAGCACATCATCAAAAGA               SEQ-ID-NO: 77   JB962     Tapesia yallundae     IIp   020602B-16   GGAGCATGGAAGCACTCGTA                  
 
       Example 5  
       [0072]    Synthesis and Purification of Oligonucleotides  
         [0073]    Oligonucleotides (primers) are synthesized by, for example, either Integrated DNA Technologies (Coralville, Iowa) or Midland Certified Reagent Company (Midland, Tex.). Primer sequences labeled as “probe” are synthesized with a fluorescent reporter group attached at the 5′ end for example 6-carboxy-fluorescein or “FAM” and a fluorescence quenching group attached at the 3′ end for example 6-carboxy-tetramethul-rhodamine or “TAMRA” or for example a dark quencher such as the proprietary Black Hole Quencher or “BHQ™” from Biosearch Technologies (Novato, Calif.).  
       Example 6  
       [0074]    Polymerase Chain Reaction Amplification  
         [0075]    Polymerase chain reactions are performed with the GeneAmp Kit from Perkin-Elmer (Foster City, Calif.; part no. N808-0009) using 50 mM KCl, 2.5 mM MgCl 2 , 10 mM Tris-HCl, pH8.3, containing 200 μM of each dTTP, dATP, dCTP, and dGTP in 25 μL reactions containing 50 μM each primer, 0.25 U/μL of Taq polymerase and approximately 25 ng of genomic DNA per reaction. Reactions are run for 30-35 cycles of 15 s at 94° C., 15 s at 50° C.-70° C., and 45 s at 72° C. in a Perkin-Elmer Model 9600 or 9700 thermal cycler. The products are analyzed by loading 10 μl of each PCR sample on a 1.0% agarose gel and electrophoresing. The gel is stained with ethidium bromide and products are visualized under ultraviolet light.  
       Example 7  
       [0076]    Determination of Primer Specificity to Purified Fungal Genomic DNA  
         [0077]    PCRs are performed according to Example 6 using different primer combinations (Table 4) in an attempt to amplify single specific fragments. Specific PCR amplification products are produced from primers designed from RAPD-PCR product sequences of each Tapesia spp. subtype.  
                                     TABLE 4                           Possible combinations of PCR primers for the specific       amplification of Tapesia spp. subtypes Ic and IIp            Target species subtype       Sequence       Sequence       (RAPD-PCR Product)   Primer 1   Identifier   Primer 2   Identifier                 Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB900   SEQ-ID-NO: 15   JB903   SEQ-ID-NO: 18         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB900   SEQ-ID-NO: 15   JB904   SEQ-ID-NO: 19         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB901   SEQ-ID-NO: 16   JB903   SEQ-ID-NO: 18         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB901   SEQ-ID-NO: 16   JB904   SEQ-ID-NO: 19         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB905   SEQ-ID-NO: 20   JB908   SEQ-ID-NO: 23         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB905   SEQ-ID-NO: 20   JB909   SEQ-ID-NO: 24         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB906   SEQ-ID-NO: 21   JB908   SEQ-ID-NO: 23         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB906   SEQ-ID-NO: 21   JB909   SEQ-ID-NO: 24         Tapesia yallundae  Ib (Ib3-33)   JB910   SEQ-ID-NO: 25   JB913   SEQ-ID-NO: 28         Tapesia yallundae  Ib (Ib3-33)   JB910   SEQ-ID-NO: 25   JB914   SEQ-ID-NO: 29         Tapesia yallundae  Ib (Ib3-33)   JB911   SEQ-ID-NO: 26   JB913   SEQ-ID-NO: 28         Tapesia yallundae  Ib (Ib3-33)   JB911   SEQ-ID-NO: 26   JB914   SEQ-ID-NO: 29         Tapesia yallundae  Ic (1c1-22)   JB915   SEQ-ID-NO: 30   JB918   SEQ-ID-NO: 33         Tapesia yallundae  Ic (1c1-22)   JB915   SEQ-ID-NO: 30   JB919   SEQ-ID-NO: 34         Tapesia yallundae  Ic (1c1-22)   JB916   SEQ-ID-NO: 31   JB918   SEQ-ID-NO: 33         Tapesia yallundae  Ic (1c1-22)   JB916   SEQ-ID-NO: 31   JB919   SEQ-ID-NO: 34         Tapesia yallundae  Ic (1c1-22)   JB920   SEQ-ID-NO: 35   JB923   SEQ-ID-NO: 38         Tapesia yallundae  Ic (Ic1-22)   JB920   SEQ-ID-NO: 35   JB924   SEQ-ID-NO: 39         Tapesia yallundae  Ic (1c1-22)   JB921   SEQ-ID-NO: 36   JB923   SEQ-ID-NO: 38         Tapesia yallundae  Ic (1c1-22)   JB921   SEQ-ID-NO: 36   JB924   SEQ-ID-NO: 39         Tapesia acuformis  IIs (IIs2-39)   JB925   SEQ-ID-NO: 40   JB928   SEQ-ID-NO: 43         Tapesia acuformis  IIs (IIs2-39)   J8925   SEQ-ID-NO: 40   JB929   SEQ-ID-NO: 44         Tapesia acuformis  IIs (IIs2-39)   JB926   SEQ-ID-NO: 41   JB928   SEQ-ID-NO: 43         Tapesia acuformis  IIs (IIs2-39)   JB926   SEQ-ID-NO: 41   JB929   SEQ-ID-NO: 44         Tapesia yallundae  Ic (Ic1-22)   JB930   SEQ-ID-NO: 45   JB931   SEQ-ID-NO: 46         Tapesia yallundae  Ic (Ic1-22)   JB930   SEQ-ID-NO: 45   JB932   SEQ-ID-NO: 47         Tapesia yallundae  Ic (Ic1-22)   JB933   SEQ-ID-NO: 48   JB931   SEQ-ID-NO: 46         Tapesia yallundae  Ic (Ic1-22)   JB933   SEQ-ID-NO: 48   JB932   SEQ-ID-NO: 47         Tapesia acuformis  IIs/IIp (IIp1-17)   JB934   SEQ-ID-NO: 49   JB935   SEQ-ID-NO: 50         Tapesia acuformis  IIp (IIp1-17)   JB934   SEQ-ID-NO: 49   JB936   SEQ-ID-NO: 51         Tapesia acuformis  IIs/IIp (IIp1-17)   JB937   SEQ-ID-NO: 52   JB935   SEQ-ID-NO: 50         Tapesia acuformis  IIp (IIp1-17)   JB937   SEQ-ID-NO: 52   JB936   SEQ-ID-NO: 51         Tapesia yallundae  Ic (0205021c4and6)   JB938   SEQ-ID-NO: 53   JB939   SEQ-ID-NO: 54         Tapesia yallundae  Ic (0205021c4and6)   JB938   SEQ-ID-NO: 53   JB940   SEQ-ID-NO: 55         Tapesia yallundae  Ic (0205021c4and6)   JB941   SEQ-ID-NO: 56   JB939   SEQ-ID-NO: 54         Tapesia yallundae  Ic (0205021c4and6)   JB941   SEQ-ID-NO: 56   JB940   SEQ-ID-NO: 55         Tapesia yallundae  Ic (020602D-20)   JB942   SEQ-ID-NO: 57   JB943   SEQ-ID-NO: 58         Tapesia yallundae  Ic (020602D-20)   JB942   SEQ-ID-NO: 57   JB945   SEQ-ID-NO: 60         Tapesia yallundae  Ic (020602D-20)   JB944   SEQ-ID-NO: 59   JB943   SEQ-ID-NO: 58         Tapesia yallundae  Ic (020602D-20)   JB944   SEQ-ID-NO: 59   JB945   SEQ-ID-NO: 60         Tapesia yallundae  Ic (020602D-21)   JB946   SEQ-ID-NO: 61   JB947   SEQ-ID-NO: 62         Tapesia yallundae  Ic (020602D-21)   JB946   SEQ-ID-NO: 61   JB949   SEQ-ID-NO: 64         Tapesia yallundae  Ic (020602D-21)   JB948   SEQ-ID-NO: 63   JB947   SEQ-ID-NO: 62         Tapesia yallundae  Ic (020602D-21)   JB948   SEQ-ID-NO: 63   JB949   SEQ-ID-NO: 64         Tapesia acuformis  IIp (020602A-11)   JB952   SEQ-ID-NO: 67   JB950   SEQ-ID-NO: 65         Tapesia acuformis  IIp (020602A-11)   JB952   SEQ-ID-NO: 67   JB951   SEQ-ID-NO: 66         Tapesia acuformis  IIp (020602A-11)   JB953   SEQ-ID-NO: 68   JB950   SEQ-ID-NO: 65         Tapesia acuformis  IIp (020602A-11)   JB953   SEQ-ID-NO: 68   JB9SI   SEQ-ID-NO: 66         Tapesia acuformis  IIp (020602B-16)   JB954   SEQ-ID-NO: 69   JB955   SEQ-ID-NO: 70         Tapesia acuformis  IIp (020602B-16)   JB954   SEQ-ID-NO: 69   JB960   SEQ-ID-NO: 75         Tapesia acuformis  IIp (020602B-16)   JB954   SEQ-ID-NO: 69   JB961   SEQ-ID-NO: 76         Tapesia acuformis  IIp (020602B-16)   JB962   SEQ-ID-NO: 77   JB955   SEQ-ID-NO: 70         Tapesia acuformis  IIp (020602B-16)   JB962   SEQ-ID-NO: 77   JB960   SEQ-ID-NO: 75         Tapesia acuformis  IIp (020602B-16)   JB962   SEQ-ID-NO: 77   JB961   SEQ-ID-NO: 76         Tapesia acuformis  IIp (020602B-15)   JB956   SEQ-ID-NO: 71   JB957   SEQ-ID-NO: 72         Tapesia acuformis  IIp (020602B-15)   JB956   SEQ-ID-NO: 71   JB959   SEQ-ID-NO: 74         Tapesia acuformis  IIp (020602B-15)   JB958   SEQ-ID-NO: 73   JB957   SEQ-ID-NO: 72         Tapesia acuformis  IIp (020602B-15)   JB958   SEQ-ID-NO: 73   JB959   SEQ-ID-NO: 74                  
 
         [0078]    In an initial screen for specificity, PCR reaction mixtures are made according to Example 6 for each of the primer combinations in Table 4. These are run against a negative control (no DNA added) and approximately 25 ng of fungal DNA for each of the Tapesia spp. subtypes listed in Table 1 prepared as described in example 1.  
         [0079]    When visualized on an ethidium bromide stained gel several primer pairs give satisfactory results: good amplification of target DNA from multiple isolates of the target species subtype with all other reactions (negative control and other fungal DNAs) free of both specific and nonspecific reaction products. Some give unsatisfactory results including nonspecific amplification, no amplification of target DNA, and amplification of DNAs from fungal species other that the target. The primer pairs that give good specific amplification for  T. yallundae  subtype Ic target DNA with no cross-amplification are summarized in Table 5.  
                                     TABLE 5                           PCR primer pairs providing specific and sensitive amplification       of target DNAs for  Tapesia yallundae  subtype Ic.            Target species subtype       Sequence       Sequence       (RAPD-PCR Product)   Primer 1   Identifier   Primer 2   Identifier                 Tapesia yallundae  Ic (020602D-20)   JB944   SEQ-ID-NO: 59   JB943   SEQ-ID-NO: 58         Tapesia yallundae  Ic (020602D-20)   JB944   SEQ-ID-NO: 59   JB945   SEQ-ID-NO: 60                  
 
         [0080]    When primers JB944 and JB943 or primers JB944 and JB945 are run against DNA preparations of the eyespot subtypes listed in Table 1 the following results are recorded (Table 6).  
                                                                             TABLE 6                           Results of primer pairs JB944 and JB943 and JB944       and JB945 on test isolates of Tapesia spp.            Fungal   Fungal   Subtype   PCR Results (+/−)            Species   Isolate   Identifier   JB944/JB943   JB944/JB945                      Tapesia yallundae     627 N   Ia   −   −         Tapesia yallundae     646 N   Ib   −   −         Tapesia yallundae     572 N   Ic   +   +         Tapesia yallundae     618 N   Ic   +   +         Tapesia acuformis     634 L   Iis   −   −         Tapesia acuformis     643 L   Iis   −   −         Tapesia acuformis     567 L   Iip   −   −         Tapesia acuformis     617 L   Iip   −   −         Tapesia acuformis     626 L   Iip   −   −                  
 
         [0081]    Thus, primer pairs JB944 and JB943 and primers JB944 and JB945 are useful in the differentiation of subtypes within the  Tapesia yallundae  species.  
         [0082]    For specificity to  T. acuformis  subtypes IIs and IIp primer pairs were selected that amplify from both  T. acuformis  subtypes but with different sized PCR products that allow differentiation of the subtype by product size. Again, primers were selected based on good amplification of target DNA from multiple isolates of the target species subtype with all other reactions (negative control and other fungal DNAs) free of both specific and nonspecific reaction products. Some give unsatisfactory results including nonspecific amplification, no amplification of target DNA, and amplification of DNAs from fungal species other that the target. The primer pairs that give good specific amplification for  T. acuformis  subtypes IIs and IIp with different sized products for each subtype with no cross-amplification are summarized in Table 7.  
                                     TABLE 7                           PCR primer pairs providing specific and sensitive amplification of target DNAs       for  Tapesia acuformis  subtypes IIs and IIp.            Target species subtype       Sequence       Sequence       (RAPD-PCR Product)   Primer 1   Identifier   Primer 2   Identifier                 Tapesia acuformis  IIs/IIp (IIp1-17)   JB934   SEQ-ID-NO: 49   JB935   SEQ-ID-NO: 50         Tapesia acuformis  IIs/IIp (IIp1-17)   JB937   SEQ-ID-NO: 52   JB935   SEQ-ID-NO: 50                  
 
         [0083]    Primers JB934 and JB935 are run against DNA preparations of the eyespot subtypes listed in Table 1. The results are presented in Table 8).  
                                                       TABLE 8                           Results of primer pairs JB934 and JB935 on       test isolates of Tapesia spp.                        PCR Results (+/−)            Fungal   Fungal   Subtype       Amplification       Species   Isolate   Identifier   (+/−)   Product Size                 Tapesia yallundae     627 N   Ia   −   −         Tapesia yallundae     646 N   Ib   −   −         Tapesia yallundae     572 N   Ic   −   −         Tapesia yallundae     618 N   Ic   −   −         Tapesia acuformis     634 L   IIs   +   ˜600         Tapesia acuformis     643 L   IIs   +   ˜600         Tapesia acuformis     567 L   IIp   +   ˜400         Tapesia acuformis     617 L   IIp   +   ˜400         Tapesia acuformis     626 L   IIp   +   ˜400                  
 
         [0084]    Thus, primer pairs JB934 and JB935 are specific to Tapesia acuformis at the species level and provide differently sized PCR products that are useful in the differentiation of IIs and IIp subtypes within the  Tapesia acuformis  species.  
       Example 8  
       [0085]    TaqMan Based Detection of Tapesia spp. Subtypes  
         [0086]    Some of the primers detailed in Table in 3 were designed for the additional possible use in TaqMan reactions for detection of specific Tapesia spp. subtypes. Possible primer combinations for these reactions are listed in Table 9.  
                                             TABLE 9                            Possible combinations of TaqMan primers and probes for the specific       amplification of Tapesia spp. subtypes            Target species subtype       Sequence       Sequence       Sequence       (RAPD-PCR Product)   Primer 1   Identifier   Probe   Identifier   Primer 2   Identifier                 Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB900   SEQ-ID-NO: 15   JB902   SEQ-ID-NO: 17   JB903   SEQ-ID-NO: 18         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB900   SEQ-ID-NO: 15   JB902   SEQ-ID-NO: 17   JB904   SEQ-ID-NO: 19         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB901   SEQ-ID-NO: 16   JB902   SEQ-ID-NO: 17   JB903   SEQ-ID-NO: 18         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB901   SEQ-ID-NO: 16   JB902   SEQ-ID-NO: 17   JB904   SEQ-ID-NO: 19         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB905   SEQ-ID-NO: 20   JB907   SEQ-ID-NO: 22   JB908   SEQ-ID-NO: 23         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB905   SEQ-ID-NO: 20   JB907   SEQ-ID-NO: 22   JB909   SEQ-ID-NO: 24         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB906   SEQ-ID-NO: 21   JB907   SEQ-ID-NO: 22   JB908   SEQ-ID-NO: 23         Tapesia yallundae  Ib (Ib1-27/Ib2-31)   JB906   SEQ-ID-NO: 21   JB907   SEQ-ID-NO: 22   JB909   SEQ-ID-NO: 24         Tapesia yallundae  Ib (Ib3-33)   JB910   SEQ-ID-NO: 25   JB912   SEQ-ID-NO: 27   JB913   SEQ-ID-NO: 28         Tapesia yallundae  Ib (Ib3-33)   JB910   SEQ-ID-NO: 25   JB912   SEQ-ID-NO: 27   JB914   SEQ-ID-NO: 29         Tapesia yallundae  Ib (Ib3-33)   JB911   SEQ-ID-NO: 26   JB912   SEQ-ID-NO: 27   JB913   SEQ-ID-NO: 28         Tapesia yallundae  Ib (Ib3-33)   JB911   SEQ-ID-NO: 26   JB912   SEQ-ID-NO: 27   JB914   SEQ-ID-NO: 29         Tapesia yallundae  Ic (lc1-22)   JB915   SEQ-ID-NO: 30   JB917   SEQ-ID-NO: 32   JB918   SEQ-ID-NO: 33         Tapesia yallundae  Ic (lc1-22)   JB915   SEQ-ID-NO: 30   JB917   SEQ-ID-NO: 32   JB919   SEQ-ID-NO: 34         Tapesia yallundae  Ic (lc1-22)   JB916   SEQ-ID-NO: 31   JB917   SEQ-ID-NO: 32   JB918   SEQ-ID-NO: 33         Tapesia yallundae  Ic (lc1-22)   JB916   SEQ-ID-NO: 31   JB917   SEQ-ID-NO: 32   JB919   SEQ-ID-NO: 34         Tapesia yallundae  Ic (lc1-22)   JB920   SEQ-ID-NO: 35   JB922   SEQ-ID-NO: 37   JB923   SEQ-ID-NO: 38         Tapesia yallundae  Ic (lc1-22)   JB920   SEQ-ID-NO: 35   JB922   SEQ-ID-NO: 37   JB924   SEQ-ID-NO: 39         Tapesia yallundae  Ic (lc1-22)   JB921   SEQ-ID-NO: 36   JB922   SEQ-ID-NO: 37   JB923   SEQ-ID-NO: 38         Tapesia yallundae  Ic (lc1-22)   JB921   SEQ-ID-NO: 36   JB922   SEQ-ID-NO: 37   JB924   SEQ-ID-NO: 39         Tapesia acuformis  IIs (IIs2-39)   JB925   SEQ-ID-NO: 40   JB927   SEQ-ID-NO: 42   JB928   SEQ-ID-NO: 43         Tapesia acuformis  IIs (IIs2-39)   JB925   SEQ-ID-NO: 40   JB927   SEQ-ID-NO: 42   JB929   SEQ-ID-NO: 44         Tapesia acuformis  IIs (IIs2-39)   JB926   SEQ-ID-NO: 41   JB927   SEQ-ID-NO: 42   JB928   SEQ-ID-NO: 43         Tapesia acuformis  IIs (IIs2-39)   JB926   SEQ-ID-NO: 41   JB927   SEQ-ID-NO: 42   JB929   SEQ-ID-NO: 44                  
 
         [0087]    Primer and probe combinations are tested for their ability to amplify from the target subtypes&#39;s DNA. Reaction conditions are held constant (1× TaqMan™ Universal Master Mix (Perkin Elmer, Norwalk, Conn.; part no. N430-4447), 300 nM each primer, 200 nM probe, approximately 25 ng pre reaction of fungal target genomic DNA, thermal cycling: 50° C. for 2 min., 95° C. for 10 min., 40 cycles of 95° C. for 15 s, 60° C. for 60 s). In initial screens for specificity under these conditions no primer/probe combination provides absolute specificity. It is prophetic that further experimentation with reaction conditions will provide subtype specific tests using these primers that are designed for specificity.  
         [0088]    This invention also provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides which is available. Furthermore, it can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas.  
         [0089]    Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of Tapesia pathogens.  
         [0090]    While the present invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and further embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the scope of the present invention.  
         [0091]    Numerous patents, applications and references are discussed or cited within this specification, and all are incorporated by reference in their entireties.  
     
       
       
         1 
         
           
             77  
           
           
             1  
             17  
             DNA  
             artificial sequence  
             
               primer  
             
           
            1 

gtaaaacgac ggccagt                                                    17 

 
           
             2  
             17  
             DNA  
             artificial sequence  
             
               primer  
             
           
            2 

caggaaacag ctatgac                                                    17 

 
           
             3  
             525  
             DNA  
             Tapesia yallundae  
           
            3 

tgggccctct agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc     60 

ccttaagtcg taacaaggta gccgtatcgg aaggtgcggc tggatcacct cctttctgga    120 

aaacagcatt caatattgaa cgcccacact tatcggttgt tggaagaagt cggtgctaac    180 

cgacatgggt ctgtagctca gctggttaga gcaccgtctt gataaggcgg gggtcgttgg    240 

ttcgagccca actagaccca ccaaatcttc cgaacataag atgcgaggat cagtggggga    300 

ttagctcagc tgggagagca cctgctttgc aagcaggggg tcgtcggttc gatcccgtca    360 

tcctccacca accaatacgc tctgcggtag ggcgaagaaa ccaacaccaa agcggcttcg    420 

cgagaggcct ctttgttgtt ggtccggtat agaccggatc aatcggctgt tctttaaaaa    480 

ttcatagagt cgaatcagcg ttgccggcgg aaagcaggaa actgc                    525 

 
           
             4  
             551  
             DNA  
             Tapesia yallundae  
           
            4 

gggccctcta gatgcatgct cgagcggccg ccagtgtgat ggatatctgc agaattcgcc     60 

cttaagtcgt aacaaggtag ccgtatcgga aggtgcggct ggatcacctc ctttctggaa    120 

aacagcattc aatattgaac gcccacactt atcggttgtt ggaagaagtc ggtgctaacc    180 

gacatgggtc tgtagctcag ctggttagag caccgtcttg ataaggcggg ggtcgttggt    240 

tcgagcccaa ctagacccac caaatcttcc gaacataaga tgcgaggatc agtgggggat    300 

tagctcagct gggagagcac ctgctttgca agcagggggt cgtcggttcg atcccgtcat    360 

cctccaccaa ccaatacgct ctgcggtagg gcgaagaaac caacaccaaa gcggcttcgc    420 

gagaggcctc tttgttgttg gtccggtata gaccggatca atcggctgtt ctttaaaaat    480 

tcatagagtc gaatcagcgt tgcccggcgg aaagcaggaa actgcacccg tgccgccggt    540 

gacaaaaatt t                                                         551 

 
           
             5  
             520  
             DNA  
             Tapesia yallundae  
           
            5 

gggccctcta gatgcatgct cgagcggccg ccagtgtgat ggatatctgc agaattcgcc     60 

cttggacctc ttggagtaac acgccccacg gacggatcag ctagcaagga ctccgcaggt    120 

ctacttgtag taagccctgg tatggactcg gcattttaca aagtaaattc tcatgaaata    180 

tgttttgggc tcggctaatg attgtagggc tttggtctca acattagtaa cgagctggta    240 

aaagtgaagg cgcgaatgct acctgcaccg accctgttgt tcaagggcaa taagccagat    300 

aaggtgcagg atagcttggg actggggtac aagggaacgt ggaacctatc agatatcaca    360 

ttttactcgc ccggtaggcc atctgacgaa accgaatacc tcgaactagg gttcatcaag    420 

atgggaaacg tctccgacac ggacatagac acatttgcaa accagcttca tctagacctg    480 

aacaagtatg gtatcacacc caatcacaac aagaaggaca                          520 

 
           
             6  
             456  
             DNA  
             Talesia yallundae  
           
            6 

ttcgcccttt ggtcgggtgc aaatatgtat ttagaattgt tatatattct tgctgaattg     60 

gtctctttaa tattatataa cgactttctt agtccttttt tttttttaac tgtttttaga    120 

tggttttttc tcttgccatt tttaggattc actcttcact ttgacttcag acagtctgat    180 

tataatgttg ccatggtgga gaccttttta cactgtattt gtctggtgat tgctgagcct    240 

ctcatatctg gatctctaaa tctcttaact agcagtcatc tgtcctgtgc caaggatgaa    300 

ataattttgt gttttgtctc tccttggtag agtttgtcac ccccaagtaa gaactaaggc    360 

tctgagaaaa taagttttgc ccaacctaag tgtagtatgt tcaagtcaga tatttaaacc    420 

taatccaaca tttcttgccc caaccatctc tataag                              456 

 
           
             7  
             711  
             DNA  
             Tapesia yallundae  
           
            7 

ctaaagggac tagtcctgca ggtttaaacg aattcgccct ttggtcgggt gggagaacct     60 

tcgtgctgag attacagaac gatcggaaaa ccaagtacgc atctcggatg tagacttcgt    120 

cctcgctgtc ctcggaatcc atgccttcgt ctattgtact ctccgggggc gtttcccctt    180 

ccgcatatcg aggggagggt tgtgggttct tcagctgtgt aaccatggtt gatccatctc    240 

ccatttgcga atcgtcgaag ctctttcgat gttcgaggtc cttgagcgta agttttggcc    300 

catcattgtc ggattggact tcagaggcca cactttcctc cacggcttct tccacctcgg    360 

tgtccccatt ggcgtcgggc gatgcatcga ccgtgaaaga actagtattt cccccgttct    420 

tctcgagttt cccaagattc agcctggcct ctttcatgtg tattcgtgtc ttgacccttt    480 

cgaacactgt cccaaccatc tgtgtcaaag tcccctgcgc aacctgctgg ttcgcactgc    540 

ttttcgaaag aaggaacaca ttgtagacct gcctgactgc cttcaatagt ccagcgccgt    600 

gtaccacaat tttgtcattc agcacagctg cgagtaacga ctttacaatc tgtaactgga    660 

tttccaccgg cgtagtctcc ccctgaaagc agtcgcatat tgtattgata g             711 

 
           
             8  
             555  
             DNA  
             Tapesia yallundae  
           
            8 

gtgcgagacg atgcgatcgt cgatcagaga agtgtttact ctttgccgaa gctataacag     60 

gacaacagtg aggaagtgta ctcgagtcga gacaatagag attttcaacg aagaacttaa    120 

gctccaacag cagaggggct ttcttttgat ctgcctgaag atggacagcc tcatgttggg    180 

ccgaattcat cgtcagacta cacagccttg ctgcccaatt gacgcccggc tgctgagcat    240 

cacgtgttac cacttttcca gctcagctca gcttagtctg atcttcttcc atttcaagtc    300 

aagacaatct tcgaggtccg tggcaaaact gatttcgata ctttcttctg ttggatatcc    360 

atctttgtac tcgggggcag gatcaagaac caatattcca acccgacgag tgatcccatc    420 

tcgtgtctca agtactaggc cgtgtcgcga gctagcccaa tcttccaaaa tgacacactc    480 

aaacaaatct tgatcgaaaa cttccgacct ctggtcagtc ttacggtctt gtcggttgtc    540 

ggggtagaat agaca                                                     555 

 
           
             9  
             625  
             DNA  
             Tapesia yallundae  
           
            9 

caagggaatg atacacgtga ctgggtctgg atgagtccaa aaatagaaga acataccatc     60 

ttgtaaatag cctcaacaat agccagcatc tcgtcgtagc tgatctttcc gtctccatcg    120 

atatcataga gttggaaggc ccagtctagt ttgtcctcca tcttccccct gctggtgacg    180 

ctcaatgcgc agataaattc cttgaaatcg attgagcctg atttgtcgct atcgaataca    240 

ttgaagacgt aatctgcgaa tgatgatgga tctccgaacg ggaagaattg gcggtaaatc    300 

ttctggaact cctccttggt aagcatgccc gaaggacagt ccttcaagaa gcctggtcat    360 

attagcttat gtttgagaaa agaccaggaa gtccataccc ttgtaccatt gttgcaactc    420 

cttcttgtca aagtgtgttg atcgctgcaa ttcgctgagt tgctcttgcg aaagcttgga    480 

ttgactatga agtaaaatta acttatagtc cgaaacaaag accagaagca taggaataga    540 

agcccttacg attttcccat cttgaagttc tggttatgtg gttgaggagg gttgcgtatt    600 

ctatcgtagc agagagggag actcg                                          625 

 
           
             10  
             455  
             DNA  
             Tapesia acuformis  
           
            10 

cgcgaattcg cccttagatg ggcagagtgt agatcttgtg agactgcatg gactagagtg     60 

ctgggaagtg atgtttttgt aagaggtgcg ttcggcttga agtcatgcga gaattaaaga    120 

gctatagttg cgtgcaatta gaagtcgaga aaggattgcg aaacttcaat atgacaatcg    180 

cccattcaaa agctatcaaa gagtgtgaga tattctagga tatctttttt taaataagtg    240 

aaggctcgtc ggctagcaaa actagctcgc ctcgactttt gttcaaattc attgtcacga    300 

atgtacgtgc cttagttgcc tgcagaactt ctcctttaga tgtttttaca ttgaatgcct    360 

catcataact cgaatgatat cgaaagttct ctctgaaatg atagtaatct gtgtagtgtt    420 

ctgttgctac tctctcggtt cctgaagatg tgctc                               455 

 
           
             11  
             498  
             DNA  
             Tapesia acuformis  
           
            11 

ctcaccaatt tcaggaaatt atttcattgc attctttttg tctcttgggt aatcatgcaa     60 

gtagttcttt ccatgcttga caaatcgata agattacagg tgaaccccca gagtagtgcc    120 

aaaaggaact gattgactgt gattgtcaac aaccctatat tcaggaatat ttcctaactt    180 

gaagctgccc attctctccg tccgtgcgta gtcattcatc ctccgatcct caaaacttca    240 

ccaccgattc atcaacccat gtttaatcat aaatttctgg agctgtcaag cttcactttg    300 

acaggtttca actggctcat cctatccggc ttcgcttcgc ttgagtgact ggctggacct    360 

gccggattgg gacctagagc acgtactgga tccctgacta cgacttatca ctgccgtgga    420 

agtgggatgg agatacaaac gggcggcgaa ccgcatgaca ttgattagtt gattgaatat    480 

atatatcata cattcact                                                  498 

 
           
             12  
             702  
             DNA  
             Tapesia acuformis  
           
            12 

aagggactag tcctgcaggt ttaaacgaat tcgcccttac ccgacctgcc atgtgaccat     60 

gtaatgcttg aagtgtcctc caactgccat cttaaactcg tacaaatcgt caaccattag    120 

caactggaat gcacaactcg agattgcaat caagctctct ctcttacccc caattaccaa    180 

ccatgttttc accgagataa gagtgtcctc cgggtccaga atgaatccca gtccattcaa    240 

catcctgcaa gccttctcat cacctcaaag gagtcaccct cttccatctc aaggctggag    300 

aaagtctcat gatcgtagat ttggcagcct tcaaatatca tccgaacatt gtccaagggt    360 

gtagaaagat attccgcagc caccttccgg agtctaccaa actcggtttg aggcccgatg    420 

cggaagtcga tatggccgcc agtagataac cttgccgtga cgatgacttg acgtgaaccg    480 

gcagtgcctt gactgatggg aacatcatgc agctggcttg cgggtggagc aagtgcacga    540 

ggtgctttga taggatgggt cagaaagaac gacttgtaaa tgccctccat cgcagcgagt    600 

ctcttgcttc tcgattccgc ctcctcgtgg ctggctccct caggtcgggt aagggcgaat    660 

tcgcggccgc taaattcaat tcgccctata gtgagtcgta tt                       702 

 
           
             13  
             503  
             DNA  
             Tapesia acuformis  
           
            13 

tatagggcga attgaattta gcggccgcga attcgccctt acccgacctg agattccgga     60 

ctgcattttg tttggtgttg atgattttcg cgtacttgac gaagatggat tacccagaaa    120 

gagggacaga gatattcaga ggagcatgga agcactcgta aagctcagga tcatatgctg    180 

gcggggtaaa ataggcttgg cacggcggag cgaggggcag tgaggttccg ccagcaagaa    240 

gtccttaagc aatgtctata agtaccgtta tactatttgt cgcatcctaa gagtatcata    300 

actcgaataa agtaaagtaa agtcctgcat cgtttcaaga ctttgatatc atttcatgcg    360 

ttgagaaatc tcagtttgcc catcttttga tgatgtgcta gggcggagct ccactccact    420 

caagctctcc cgcgcaggtc gggtaagggc gaattcgttt aaacctgcag gactagtccc    480 

tttaggaggg ttaattctga gct                                            503 

 
           
             14  
             513  
             DNA  
             Tapesia acuformis  
           
            14 

tgggccctct agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc     60 

ccttaagtcg taacaaggta gccgtatcgg aaggtgcggc tggatcacct ccgttctgga    120 

aaactgcatt caatattgaa cgcccacact tatcggttgt tggaagagtc ggtctgaccg    180 

acatgggtct gtagctcagc tggttagagc accgtcttga taaggcgggg gtcgttggtt    240 

cgagcccaac tagacccacc aaatcttcca aacatcagat gcgaggatca ttgggggatt    300 

agctcagctg ggagagcacc tgctttgcaa gcagggggtc gtcggttcga tcccgtcatc    360 

ctccaccaac caatcggtat caatgcaaca ccaaagaggc tttgaaaaag gcttctttgt    420 

tgttgatcga gattactcag atcaatcggc tgttctttaa aaattcatag agtcgaatca    480 

gcgttgctga tggaaactgc acattcgtaa agg                                 513 

 
           
             15  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            15 

tgcggtaggg cgaagaaac                                                  19 

 
           
             16  
             21  
             DNA  
             artificial sequence  
             
               primer  
             
           
            16 

catcctccac caaccaatac g                                               21 

 
           
             17  
             23  
             DNA  
             artificial sequence  
             
               probe  
             
           
            17 

aacaccaaag cggcttcgcg aga                                             23 

 
           
             18  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            18 

cagccgattg atccggtcta                                                 20 

 
           
             19  
             21  
             DNA  
             artificial sequence  
             
               primer  
             
           
            19 

ggcaacgctg attcgactct a                                               21 

 
           
             20  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            20 

ggttcgatcc cgtcatcct                                                  19 

 
           
             21  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            21 

ggttcgatcc cgtcatcct                                                  19 

 
           
             22  
             24  
             DNA  
             artificial sequence  
             
               primer  
             
           
            22 

caatacgctc tgcggtaggg cgaa                                            24 

 
           
             23  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            23 

gccgctttgg tgttggttt                                                  19 

 
           
             24  
             21  
             DNA  
             artificial sequence  
             
               primer  
             
           
            24 

cagccgattg atccggtcta t                                               21 

 
           
             25  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            25 

aattcgccct tggacctctt                                                 20 

 
           
             26  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            26 

ttcgcccttg gacctcttg                                                  19 

 
           
             27  
             23  
             DNA  
             artificial sequence  
             
               probe  
             
           
            27 

agtaacacgc cccacggacg gat                                             23 

 
           
             28  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            28 

ctgcggagtc cttgctagct                                                 20 

 
           
             29  
             19  
             DNA  
             artificial sequence  
             
               primer  
             
           
            29 

gacctgcgga gtccttgct                                                  19 

 
           
             30  
             26  
             DNA  
             artificial sequence  
             
               primer  
             
           
            30 

ttacactgta tttgtctggt gattgc                                          26 

 
           
             31  
             28  
             DNA  
             artificial sequence  
             
               primer  
             
           
            31 

gagcctctca tatctggatc tctaaatc                                        28 

 
           
             32  
             31  
             DNA  
             artificial sequence  
             
               probe  
             
           
            32 

ttaactagca gtcatctgtc ctgtgccaag g                                    31 

 
           
             33  
             27  
             DNA  
             artificial sequence  
             
               primer  
             
           
            33 

gacaaactct accaaggaga gacaaaa                                         27 

 
           
             34  
             26  
             DNA  
             artificial sequence  
             
               primer  
             
           
            34 

ctaccaagga gagacaaaac acaaaa                                          26 

 
           
             35  
             26  
             DNA  
             artificial sequence  
             
               primer  
             
           
            35 

tcttgtgaga ctgcatggac tagagt                                          26 

 
           
             36  
             25  
             DNA  
             artificial sequence  
             
               primer  
             
           
            36 

gatcttgtga gactgcatgg actag                                           25 

 
           
             37  
             32  
             DNA  
             artificial sequence  
             
               primer  
             
           
            37 

catgcgagaa ttaaagagct atagttgcgt gc                                   32 

 
           
             38  
             23  
             DNA  
             artificial sequence  
             
               primer  
             
           
            38 

cgcaatcctt tctcgacttc taa                                             23 

 
           
             39  
             23  
             DNA  
             artificial sequence  
             
               primer  
             
           
            39 

gtttcgcaat cctttctcga ctt                                             23 

 
           
             40  
             21  
             DNA  
             artificial sequence  
             
               primer  
             
           
            40 

gcagaattcg cccttaagtc g                                               21 

 
           
             41  
             21  
             DNA  
             artificial sequence  
             
               primer  
             
           
            41 

tctgcagaat tcgcccttaa g                                               21 

 
           
             42  
             25  
             DNA  
             artificial sequence  
             
               primer  
             
           
            42 

aaggtagccg tatcggaagg tgcgg                                           25 

 
           
             43  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            43 

ccagaacgga ggtgatccag                                                 20 

 
           
             44  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            44 

ttccagaacg gaggtgatcc                                                 20 

 
           
             45  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            45 

atattcttgc tgaattggtc                                                 20 

 
           
             46  
             25  
             DNA  
             artificial sequence  
             
               primer  
             
           
            46 

caaaattatt tcatccttgg cacag                                           25 

 
           
             47  
             24  
             DNA  
             artificial sequence  
             
               primer  
             
           
            47 

aaattatttc atccttggca cagg                                            24 

 
           
             48  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            48 

atattcttgc tgaattggtc                                                 20 

 
           
             49  
             25  
             DNA  
             artificial sequence  
             
               primer  
             
           
            49 

agatgggcag agtgtagatc ttgtg                                           25 

 
           
             50  
             25  
             DNA  
             artificial sequence  
             
               primer  
             
           
            50 

ggaaccgaga gagtagcaac agaac                                           25 

 
           
             51  
             24  
             DNA  
             artificial sequence  
             
               primer  
             
           
            51 

caggaaccga gagagtagca acag                                            24 

 
           
             52  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            52 

gcgttcggct tgaagtcatg                                                 20 

 
           
             53  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            53 

cctttggtcg ggtgggagaa                                                 20 

 
           
             54  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            54 

gccaggctga atcttgggaa                                                 20 

 
           
             55  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            55 

ccaggctgaa tcttgggaaa                                                 20 

 
           
             56  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            56 

ccaagtacgc atctcggatg                                                 20 

 
           
             57  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            57 

gaagtgttta ctctttgccg                                                 20 

 
           
             58  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            58 

aatattggtt cttgatcctg                                                 20 

 
           
             59  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            59 

tcgagacaat agagattttc                                                 20 

 
           
             60  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            60 

tcgagacaat agagattttc                                                 20 

 
           
             61  
             22  
             DNA  
             artificial sequence  
             
               primer  
             
           
            61 

acataccatc ttgtaaatag cc                                              22 

 
           
             62  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            62 

catagtcaat ccaagctttc                                                 20 

 
           
             63  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            63 

ataccatctt gtaaatagcc                                                 20 

 
           
             64  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            64 

tatgcttctg gtctttgttt                                                 20 

 
           
             65  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            65 

aatcaatgtc atgcggttcg                                                 20 

 
           
             66  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            66 

cacttccacg gcagtgataa                                                 20 

 
           
             67  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            67 

ttgtctcttg ggtaatcatg                                                 20 

 
           
             68  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            68 

gtgccaaaag gaactgattg                                                 20 

 
           
             69  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            69 

tgagattccg gactgcattt                                                 20 

 
           
             70  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            70 

caaactgaga tttctcaacg                                                 20 

 
           
             71  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            71 

ccttacccga cctgccatgt                                                 20 

 
           
             72  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            72 

ctggcggcca tatcgacttc                                                 20 

 
           
             73  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            73 

attagcaact ggaatgcaca                                                 20 

 
           
             74  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            74 

aagccagctg catgatgttc                                                 20 

 
           
             75  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            75 

cgccctagca catcatcaaa                                                 20 

 
           
             76  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            76 

cctagcacat catcaaaaga                                                 20 

 
           
             77  
             20  
             DNA  
             artificial sequence  
             
               primer  
             
           
            77 

ggagcatgga agcactcgta                                                 20