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:
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/369,796 filed Apr. 3, 2002, which is incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     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 
     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). 
     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). 
     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. 
     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). 
     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 
     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. 
     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. 
     Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of  Tapesia  pathogens. 
     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. 
     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: 
     JB944 (SEQ ID NO:59) and JB943 (SEQ ID NO:58; 
     JB944 (SEQ ID NO:59) and JB945 (SEQ ID NO:60); 
     JB934 (SEQ ID NO:49) and JB935 (SEQ ID NO:50); and 
     JB937 (SEQ ID NO:52) and JB935 (SEQ ID NO:50). 
     The invention also provides a method for the detection of a fungal pathogen, comprising the steps of:
     (a) isolating DNA from a plant tissue infected with a pathogen;   (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   (c) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.
 
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.
   

     The invention further provides a method for the detection of a fungal pathogen, comprising the steps of:
     (a) isolating DNA from a plant tissue infected with a pathogen;   (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   (c) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.
 
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:
   

     JB944 (SEQ ID NO:59) and JB943 (SEQ ID NO:58; 
     JB944 (SEQ ID NO:59) and JB945 (SEQ ID NO:60); 
     JB934 (SEQ ID NO:49) and JB935 (SEQ ID NO:50); or 
     JB937 (SEQ ID NO:52) and JB935 (SEQ ID NO:50). 
     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: 
     JB944 (SEQ ID NO:59) and JB943 (SEQ ID NO:58; 
     JB944 (SEQ ID NO:59) and JB945 (SEQ ID NO:60); 
     JB934 (SEQ ID NO:49) and JB935 (SEQ ID NO:50); or 
     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 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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 
     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 
     Fungal Isolates and Genomic Fungal DNA Extraction 
     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.
     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).   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.   3. Add 400 μl chlorophorm:isoamylalcohol (24.1, v:v) shake for 15 min   4. Centrifuge for 10 min (16000 g)   5. Transfer the aqueous phase to a new tube and add 400 μl extraction buffer and 400 μl chloroform:isoamylalcohol   6. Shake for 15 min   7. Centrifuge 10 min   8. Transfer aqueous phase to a new tube   9. Add 0.6× volumes of isopropanol   10. Shake for 5 min   11. Centrifuge for 5 min   12. Discard supernatant and dry pellet   13. Wash pellet with cold EtOH 70%   14. Centrifuge 2 min   15. Dry pellet   16. Resuspend in 50 μl TE   

     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
               
                   1 S = Sensitive, 
               
               
                 R = Resistant 
               
             
          
         
       
     
     Example 2 
     Amplification of RAPD Products 
     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. 
     The gel is stained with ethidium bromide and separated RAPD-PCR bands are observed under ultraviolet light. 
     Example 3 
     Selection, Cloning, and Sequencing of Subtype-Specific RAPD-PCR Products 
     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 
     Design of Subtype-specific Primers 
     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 
     Synthesis and Purification of Oligonucleotides 
     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 
     Polymerase Chain Reaction Amplification 
     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 
     Determination of Primer Specificity to Purified Fungal Genomic DNA 
     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 
               
               
                   
               
             
          
         
       
     
     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. 
     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                    
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   −   −                    
Thus, primer pairs JB944 and JB943 and primers JB944 and JB945 are useful in the differentiation of subtypes within the  Tapesia yallundae  species.
 
     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                    
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                    
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 
     TaqMan Based Detection of  Tapesia  spp. Subtypes 
     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 
               
               
                   
               
             
          
         
       
     
     The combinations listed in Table 7 are tested in initial TaqMan™ screens for subtype level specificity. 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. 
     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. 
     Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of  Tapesia  pathogens. 
     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. 
     Numerous patents, applications and references are discussed or cited within this specification, and all are incorporated by reference in their entireties.