Detection of wheat and barley fungal pathogens which are resistant to certain fungicides using the polymerase chain reaction

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

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. & 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' 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 & 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]

TABLE 1
Identification of Test Isolates
Fungal	Fungal	Subtype		Fungicide Sensitivity1	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

[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 MgCl2, 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'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 MgCl2, 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'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.

Claims

1. A nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NOS: 3-13 or 14.

2. A nucleic acid molecule having sequence identity with at least 10 contiguous nucleotides of SEQ ID NOS: 2-13 or 14.

3. A nucleic acid molecule comprising a nucleotide sequence of SEQ ID NOs: 3-76 or 77.

4. A pair of oligonucleotide primers wherein at least one primer consists of the nucleotide sequence of SEQ ID NOS: 3-76 or 77.

5. A pair of oligonucleotide primers comprising: 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).

6. 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 (d) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.

7. The method of claim 6, wherein the fungal pathogen is Tapesia yallundae, Tapesia acuformis.

8. The method of claim 7, wherein the Tapesia yallundae is subtype Ic., Tapesia acuformis subtypes IIs or IIp.

9. The method of claim 6, wherein at least one primer having the nucleotide sequence of SEQ ID NOS: 3-76 or 77.

10. 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.

11. The method of claim 10, wherein the fungal pathogen is Tapesia yallundae, Tapesia acuformis.

12. The method of claim 11, wherein the Tapesia yallundae is subtype Ic., Tapesia acuformis subtypes IIs or IIp.

13. The method of claim 10, wherein at least one primer having the nucleotide sequence of SEQ ID NOS: 3-76 or 77.

14. The method of claim 10, wherein a 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).

15. 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 claim 1.

16. The diagnostic kit of claim 15, wherein at least one primer comprises SEQ ID NO: 3-76 or 77.

17. The diagnostic kit of claim 15, wherein 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).