PCR-based detection and quantification of Tapesia yallundae and Tapesia acuformis

FIELD OF THE INVENTION

The present invention relates to the use of primers and probes in TaqMan™ quantitative PCR assays for the detection of

Tapesia yallundae

(syn.

Pseudocercosporella herpotrichoides

W-type) and

Tapesia acuformis

(syn.

Pseudocercosporella herpotrichoides

R-type). The use of these assays enables the detection of specific fungal pathogens and their quantification in plant populations. The invention also relates to the use of primers and probes in TaqMan™ quantitative PCR assays for the detection of host wheat DNA for use as an endogenous reaction control.

BACKGROUND OF THE INVENTION

Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, 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).

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 that 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), Pseudocercosporella (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 worldwide and represent a major fraction of world food production. Although yield loss is caused by many pathogens, the necrotizing pathogens Septoria and Pseudocercosporella 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.

Eyespot of wheat is caused by the pathogens

Tapesia acuformis

and

Tapesia yallundae.

These have previously been considered varieties of the same species

Pseudocercosporella herpotrichoides

(Fron) Deighton. 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 that are also virulent on other cereals. The R-strain of the fungus (

Tapesia acuformis

), for example, has also been isolated from rye and grows more slowly on wheat than the W-strain (

Tapesia yallundae

) which has been isolated from wheat. Eyespot is restricted to the basal culm of the plant and can kill tillers or plants outright; however, 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 internodes, as well as fungicide treatment. However, the differing susceptibility of cultivars to different strains of the fungus render the predictive efficacy of fungicide treatments difficult.

In view of the above, there is a real need for the development of technology that will allow the identification of specific races of pathogen fungi 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.

TaqMan™ chemistry and the ABI7700 (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.) provide a means of creating precise, reproducible quantitative assays of DNA and RNA. The foundation of TaqMan™ chemistry is the polymerase chain reaction (PCR). In conventional PCR assays, oligonucleotide primers are designed complementary to the 5′ and 3′ ends of a DNA sequence of interest. During thermal cycling, DNA is first heat denatured. The sample is then brought to annealing and extension temperatures in which the primers bind their specific complements and are extended by the addition of nucleotide tri-phosphates by Taq polymerase. With repeated thermal cycling, the amount of template DNA is amplified.

In TaqMan™ chemistry, an oligonucleotide probe is designed that is complementary to the sequence region between the primers within the PCR amplicon. The probe contains a fluorescent reporter dye at its 5′ end and a quencher dye at its 3′ end. When the probe is intact, its fluorescent emissions are quenched by the phenomena of fluorescent resonance energy transfer (FRET). During thermal cycling, the probe hybridizes to the target DNA downstream of one of the primers. TaqMan™ chemistry relics on the 5′ exonuclease activity of Taq polymerase to cleave the fluorescent dye from the probe. As PCR product accumulates, fluorescent signal is increased. By measuring this signal, the amplified product can be quantified. This method allows the quantitation of disease pressure by targeting pathogen DNA. In combination with the PCR primers, the probe provides another level of specificity in assays to differentiate pathogens.

SUMMARY OF THE INVENTION

The present invention is drawn to methods of identification and quantification of different species of plant pathogenic fungi. The invention provides primer and probe DNA sequences useful in TaqMan™ quantitative PCR assays. Such DNA sequences are useful in the method of the invention as they are used in polymerase chain reaction (PCR) and TaqMan™-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathogens. In combination with the hybridization of the TaqMan™ probe, they can be used to detect and quantify the specific pathogens in host plant material before the onset of disease symptoms.

In a preferred embodiment, the invention provides ITS-derived diagnostic primers and TaqMan™ probes for the detection of

Tapesia yallundae

(syn.

Pseudocercosporella herpotrichoides

W-type) and

Tapesia acuformis

(syn.

Pseudocercosporella herpotrichoides

R-type).

This invention provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse ararmory of fungicides that is available. Furthermore, the invention can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas. The invention provides a method of quantification of disease pressure on a given crop.

Kits useful in the practice of the invention are also provided. The kits find particular use in the identification and quantification of the fungal pathogens

Tapesia yallundae

and

Tapesia acuformis.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NOs:1-34 are the following oligonucleotide probes and primers useful for PCR-based detection of the fungal pathogens

Tapesia yallundae

and

Tapesia acuformis:

SEQ ID NO:

Oligo

Target

Oligo Sequence (5′->3′)

SEQ ID NO:1

ITS1

Fungal 18S rDNA

tccgtaggtgaacctgcgg

SEQ ID NO:2

ITS4

Fungal 25S rDNA

tcctccgcttattgatatgc

SEQ ID NO:3

J103W

Tapesia yallundae

(W)

ggctaccctacttggtag

SEQ ID NO:4

J104W

Tapesia yallundae

(W)

cctgggggctaccctacttg

SEQ ID NO:5

J105W

Tapesia yallundae

(W)

gggggctaccctacttggtag

SEQ ID NO:6

J106W

Tapesia yallundae

(W)

tgggggctaccctacttggtag

SEQ ID NO:7

J107W

Tapesia yallundae

(W)

(FAM)-tttagagtcgtcaggcctctcggagaagc-(TAMRA)

SEQ ID NO:8

J108W

Tapesia yallundae

(W)

atttattcaagggtggaggtcctga

SEQ ID NO:9

J109W

Tapesia yallundae

(W)

aagggtggaggtctgaaccag

SEQ ID NO:10

J110W

Tapesia yallundae

(W)

aagggtggaggtctgaacca

SEQ ID NO:11

J111W

Tapesia yallundae

(W)

caagggtggaggtctgaacc

SEQ ID NO:12

J112R

Tapesia acuformis

(R)

tcaagggtggaggtctgaacc

SEQ ID NO:13

J100R

Tapesia acuformis

(R)

gggccaccctacttcggtaa

SEQ ID NO:14

J101R

Tapesia acuformis

(R)

gaaatcctgggggccaccctacttc

SEQ ID NO:15

J102R

Tapesia acuformis

(R)

cctgggggccaccctact

SEQ ID NO:16

J113R

Tapesia acuformis

(R)

gccaccctacttcggtaaggtt

SEQ ID NO:17

J114R

Tapesia acuformis

(R)

caccctacttcggtaaggtttagagtc

SEQ ID NO:18

J115R

Tapesia acuformis

(R)

aggtaatttattcaagggtggaggt

SEQ ID NO:19

J116R

Tapesia acuformis

(R)

aggtaatttattcaagggtggaggtc

SEQ ID NO:20

J117R

Tapesia acuformis

(R)

aaggtaatttattcaagggtggaggt

SEQ ID NO:21

J118R

Tapesia acuformis

(R)

ttattcaagggtggaggtctgg

SEQ ID NO:22

J119R

Tapesia acuformis

(R)

tattcaagggtggaggtctgga

SEQ ID NO:23

J120R

Tapesia acuformis

(R)

cctgccaaagcaacaaaggta

SEQ ID NO:24

J121R

Tapesia acuformis

(R)

(FAM)-cgggcctctcggagaagcctgg-(TAMRA)

SEQ ID NO:25

J122R

Tapesia acuformis

(R)

cctacttcggtaaggtttagagtcgt

SEQ ID NO:26

J123R

Tapesia acuformis

(R)

tctccgagaggcccgac

SEQ ID NO:27

J124R

Tapesia acuformis

(R)

(FAM)-aagcctggtccagacctccaccc-(TAMRA)

SEQ ID NO:28

J125R

Tapesia acuformis

(R)

aaggatcattaatagagcaatggatagac

SEQ ID NO:29

J126R

Tapesia acuformis

(R)

(FAM)-cgccccgggagaaatcctgg-(TAMRA)

SEQ ID NO:30

J127R

Tapesia acuformis

(R)

tgggggccaccctacttc

SEQ ID NO:31

JB537

Tapesia yallundae

(W)

gggggctaccctacttggtag

SEQ ID NO:32

JB541

Tapesia yallundae

(W)

ccactgattttagaggccgcgag

SEQ ID NO:33

JB540

Tapesia acuformis

(R)

gggggccaccctacttcggtaa

SEQ ID NO:34

JB542

Tapesia acuformis

(R)

ccactgattttagaggccgcgaa

SEQ ID NO:35 is a forward sequencing primer.

SEQ ID NO:36 is a reverse sequencing primer.

SEQ ID NO:37 is a DNA sequence for the Internal Transcribed Spacer of

Tapesia acuformis

(syn.

P. herpotrichoides

R-type), NRRL accession no. B-21234, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-263), 5.8 S rRNA gene (nucleotides 264-419), Internal Transcribed Spacer 2 (nucleotides 420-570), and 5′ end of the large subunit rRNA gene (nucleotides 571-627).

SEQ ID NO:38 is a DNA sequence for the Internal Transcribed Spacer of

Tapesia yallundae

(syn.

P. herpotrichoides

W-type), NRRL accession no. B-21231, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-262), 5.8 S rRNA gene (nucleotides 263-418), Internal Transcribed Spacer 2 (nucleotides 419-569), and 5′ end of the large subunit rRNA gene (nucleotides 570-626).

SEQ ID NO:39 is a consensus DNA sequence of the partial ITS region PCR-amplified from wheat extracts from three different locations (Barton, Elmdon, Teversham) infected with

Tapesia acuformis,

comprising in the 5′ to 3′ direction: partial Internal Transcribed Spacer 1 sequence, 5.8 S rRNA gene, and partial Internal Transcribed Spacer 2 sequence.

SEQ ID NO:40 is a consensus DNA sequence of the partial ITS region PCR-amplified from wheat extracts from three different locations (Barton, Elmdon, Teversham) infected with

Tapesia yallundae,

comprising in the 5′ to 3′ direction: partial Internal Transcribed Spacer 1 sequence, 5.8 S rRNA gene, and partial Internal Transcribed Spacer 2 sequence.

SEQ ID NO:41 is the nucleotide sequence of the gene for cytochrome b-559 in wheat chloroplast DNA (Hird, et al.,

Mol. Gen. Genet.

203: 95-100 (1986)).

SEQ ID NOs:42-44 are the following oligonucleotide primers and probe useful for PCR-based detection of wheat chloroplast DNA:

SEQ ID NO:

Oligo

Primer

Oligo Sequence (5′->3′)

SEQ ID NO:42

Forward Primer

WCP2

cagtgcgatggctggctatt

SEQ ID NO:43

Reverse Primer

WCP3

cgttggatgaactgcattgct

SEQ ID NO:44

TaqMan ™ Probe

WCP1

(VIC)-acggactagctgtacctactgtttttttcttgggatc-(TAMRA)

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides unique DNA sequences that are useful in identifying and quantifying different pathotypes of plant pathogenic fungi. Particularly, the DNA sequences can be used as primers in TaqMan™ PCR-based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include primers and probes derived from Internal Transcribed Spacer (ITS) sequences of the ribosomal RNA gene regions of particular fungal pathogens, which are capable of identifying the particular pathogen. The ITS DNA sequences from different pathotypes within a pathogen species or genus, which vary between the different members of the species or genus, 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. U.S. Pat. No. 5,585,238 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Septoria, Pseudocercosporella, and Mycosphaerella and their use in the identification of these fungal isolates using PCR-based techniques. In addition, WO 95/29260 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No. 5,800,997 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.

Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Despite the high conservation between mature rRNA sequences, the non-transcribed and transcribed spacer sequences are usually poorly conserved and are thus suitable as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS1 and ITS2, of around 300 bp (White et aL, 1990; In: PCR Protocols; Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). The ITS and NTS sequences are particularly suitable for the detection of specific pathotypes of different fungal pathogens.

The DNA sequences of the invention are from the Internal Transcribed Spacer sequences of the ribosomal RNA gene region of particular plant pathogens. The ITS DNA sequences from different pathotypes within a pathogen species or genus vary among the different members of the species or genus. Once having determined the ITS sequences of a pathogen, these sequences can be aligned with other ITS sequences. In this manner, primers can be derived from the ITS sequences. That is, primers can be designed based on regions within the ITS sequences that contain the greatest differences in sequence among the fungal pathotypes. These sequences and primers based on these sequences can be used to identify specific pathogens.

Sequences of representative oligonucleotide primers derived from ITS sequences are disclosed in SEQ ID NOs:1-34. The sequences find use in TaqMan™ quantitative PCR-based identification of the pathogens of interest.

Methods for the use of the primer sequences of the invention in PCR analysis are well known in the art. For example, see U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Schlesser et al. (1991)

Applied and Environ. Microbiol.

57:553-556. See also, Nazar et al. (1991;

Physiol. and Molec. Plant Pathol.

39: 1-11), which used PCR amplification to exploit differences in the ITS regions of

Verticillium albo

-

atrum

and

Verticillium dahliae

and therefore distinguish between the two species; and Johanson and Jeger (1993;

Mycol. Res.

97:670-674), who used similar techniques to distinguish the banana pathogens

Mycosphaerella fijiensis

and

Mycospharella musicola.

The TaqMan™ methodology has recently been used in medical research for the quantitative detection of herpes simplex virus (HSV) DNA in clinical samples (

J. Clin. Microbial.

37(6): 1941-7 (June, 1999)) in veterinary medicine for the detection of parasitic microbes in host animals (

J. Clin. Microbiol.

37(5): 1329-31 (May, 1999)), and has been shown to be useful in the screening of ground beef for bacterial pathogens (

Appl. Envir. Micro.

62(4): 1347-1353 (April, 1996)). Only recently has the TaqMan™ method been used for the identification and/or quantification of fungal pathogens in crop plants (

Phytopathology

89(9): 796-804 (1999)).

The ITS DNA sequences of the invention can be cloned from fungal pathogens by methods known in the art. In general, the methods for the isolation of DNA from fungal isolates are known. See, Raeder & Broda (1985)

Letters in Applied Microbiology

2:17-20; Lee et al. (1990)

Fungal Genetics Newsletter

35:23-24; and Lee and Taylor (1990) In:

PCR Protocols: A Guide to Methods and Applications,

Innes et al. (Eds.); pages 282-287.

The ITS sequences are compared within each pathogen group to locate divergences that might be useful to test in TaqMan™ PCR assays to distinguish the different species and/or strains. From the identification of divergences, numerous primers are synthesized for each probe and tested in TaqMan™ assays. Templates used for TaqMan™ assays are firstly purified pathogen DNA, and subsequently DNA isolated from infected host plant tissue. Thus, it is possible to identify probe-primer combinations that are diagnostic, i.e. that identify one particular pathogen species or strain but not another species or strain of the same pathogen.

Preferred primer-probe combinations are able to distinguish between the different species or strains in infected host tissue, i.e. host tissue that has previously been infected with a specific pathogen species or strain. This invention provides numerous primer-probe combinations that fulfill this criterion for

Tapesia yallundae

and

Tapesia acuformis.

The primers and probes of the invention are designed based on sequence differences among the fungal ITS regions. A minimum of one base pair difference between sequences can permit design of a discriminatory primer or probe. Primer-probe combinations designed to a specific fungal pathogen's ITS region can be used in combination with a primer or probe made to a conserved sequence region within the ribosomal gene's coding region to detect amplification of species-specific PCR fragments. In general, primers should have a theoretical melting temperature (T

M

) near 59° C. to achieve good sensitivity and should be void of significant secondary structure and 3′ overlaps between primer combinations. Primer pairs' T

M

s are typically within 2° C. of one another. Primers generally have sequence identity with at least about 5-10 contiguous nucleotide bases of ITS1 or ITS2. In preferred embodiments, primers are anywhere from approximately 5-30 nucleotide bases long. Probes are generally designed to have a T

M

10° C. higher than that of the primers.

All wheat extractions contain the host wheat DNA as well as any fungal pathogen DNA present. Thus, an endogenous control assay targeting the wheat DNA can be run on extracts to account for any differences among sample extractions. The present invention describes a control assay targeting the cytochrome b-559 gene. The cytochrome b-559 gene is a conserved gene among wheat varieties, necessary for the life of the host plant. These control assays provide a control against false negatives. That is, a negative result for fungal DNA that could be attributed to inhibition of the PCR reaction is verified by an endogenous control assay. These control assays also provide a target against which the fungal DNA quantity is normalized for sample to sample comparison. The present invention describes the use of these control assays in reactions separate from the fungal pathogen assays and in multiplexed reactions. 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, such as tubes or vials. One of the containers 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 containers may contain one or more enzymes or reagents to be utilized in TaqMan™ 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, and other consumables of the like.

The examples below show typical experimental protocols that can be used in the selection of suitable primer and probe sequences, the testing of primers and probes for selective and diagnostic efficacy, and the use of such primers and probes for disease and fungal isolate detection and quantification. 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 Fungal Genomic DNA Extraction

Table 1 provides a listing of the fungal test isolates used and their source. Fungi are grown in 150 ml potato dextrose broth inoculated with mycelial fragments from PDA (Potato Dextrose Agar) cultures. Cultures are incubated on an orbital shaker at 28° C. for 7-11 days. Alternatively, mycelia are isolated directly from a PDA plate. Mycelia are pelleted by centrifugation and then ground in liquid nitrogen, and total genomic DNA is extracted using the protocol of Lee and Taylor (1990; In:

PCR Protocols: A Guide to Methods and Applications;

Eds.: Innes et al.; pages 282-287).

TABLE 1

Source of Test Isolates

Isolate

Organism

Source

Origin

358

Tapesia acuformis

Novartis

1

—

308

Tapesia acuformis

Novartis

1

—

44643

Tapesia yallundae

ATCC

2

Germany

44614

Tapesia yallundae

ATCC

2

Ireland

60973

Tapesia acuformis

ATCC

2

Germany

42040

Pseudocercosporella

ATCC

2

—

herpotrichoides

var.

herpotrichoides

62012

Pseudocercosporella

ATCC

2

Germany

aestiva

24425

Septoria nodorum

ATCC

2

Montana

26517

Septoria tritici

ATCC

2

Minnesota

38699

Septoria glycines

ATCC

2

Illinois

22585

Septoria passerini

ATCC

2

Minnesota

26380

Septoria avenae f. sp.

Bergstrom/Ueng

3

Minnesota

triticea

52182

Ceratobasidium cereale

ATCC

2

Ohio

11404

Drechslera sorokiniana

ATCC

2

Minnesota

R-5391

Fusarium culmorum

Nelson

4

Germany

4551

Fusarium moniliforme

Novartis

1

Indiana

R-8637

Fusarium graminearum

Nelson

4

Morocco

T-534

Fusarium poae

Nelson

4

Pennsylvania

18222

Gerlachia nivalis

ATCC

2

United Kingdom

093

Microdochium nivale

Novartis

1

—

var.

majus

1

Novartis Agribusiness Biotechnology Research, Inc., Research Triangle Park, NC, USA

2

American Type Culture Collection, Rockville, Maryland, USA

3

Dr. Gary Bergstrom, Cornell University, and Dr. Peter Ueng, USDA-ARS, Beltsville, Maryland.

4

Dr. Paul Nelson, Penn State University, State College, Pennsylvania

Example 2

DNA Extraction from Wheat Stem Tissue DNA is extracted from wheat stem tissues (identified in Table 2) as follows:

(1) Up to 25 wheat samples are placed on a clean surface. A sterile scalpel is used to cut the stem just above the first tiller or root. Another cut is made 4 cm above this cut. This 4 cm section constitutes the stem tissue sample which is pooled with the additional wheat samples for bulk maceration.

(2) The stem sample is placed in a Bioreba (Reinach, Switzerland) heavy duty plastic bag (cat#490100). The plant tissue is weighed, plastic bag, with sample minus the tare (weight of the plastic bag,).

(3) An equal volume (mL) of Muller Extraction Buffer (0.1% w/v Tween-80; 0.040 M Tris base; 0.15 M Sodium chloride; 0.1% w/v Bovine serum albumin (Pentex Fraction V); 0.01% w/v Sodium azide; 0.20 M EDTA; pH to 7.7, Store at 4° C.) is added per weight (g) of wheat tissue. Tissue is macerated using a Bioreba Homex 6 homogenizer set at 70. The tissue is ground until fibrous.

(4) Extraction juice is aliquoted into eppendorf tubes on ice.

(a) Extracts are boiled for 5 minutes.

(b) Boiled extracts are kept on ice. The boiled extract is microfuged for 5 minutes at 12,000×G.

(c) 1:20 dilutions of the supernatant are made from the microfuged extract in dH

2

0.

(d) The diluted extracts are stored on ice until ready to use.

TABLE 2

Origin of Wheat Samples Used in Primer and Probe Development

Sample

Description

Origin

W(Barton)

Eyespot infected wheat

United Kingdom

W(Elmdon)

Eyespot infected wheat

United Kingdom

W(Teversham)

Eyespot infected wheat

United Kingdom

R(Barton)

Eyespot infected wheat

United Kingdom

R(Elmdon)

Eyespot infected wheat

United Kingdom

R(Teversham)

Eyespot infected wheat

United Kingdom

Example 3

Isolation and Sequencing of the Internal Transcribed Spacer (ITS) Region DNA from

Tapesia yallundae

and

Tapesia acuformis

Infected Wheat Samples

Approximately 420-bp truncated ITS region fragments are PCR-amplified from wheat extracts identified in Table 2 infected with

Tapesia yallundae

using the

Tapesia yalludae

-specific primers JB537 (SEQ ID NO:31) and JB541 (SEQ ID NO:32). Similarly, the

Tapesia acuformis

truncated ITS fragments are amplified from

Tapesia acuformis

-infected wheat extracts using

Tapesia acuformis

-specific primers JB540 (SEQ ID NO:33) and JB542 (SEQ ID NO:34). 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, pH 8.3, containing 200 μM of each dTTP, dATP, dCTP, and dGTP, 50 pmol each primer, 2.5 units of Taq polymerase and 1 μl 1:10 diluted wheat extract in a final volume of 50 μl. Reactions are run at 94° C. for 15 s and 1 min. at 75° C. for 35 cycles in a Perkin-Elmer Model 9700 thermal cycler.

The PCR products are cloned into the pCR®2.1-TOPO TA-cloning vector using the TOPO-TA Cloning Kit (Invitrogen, Carlsbad, Calif.; part no. K4550-40) according to manufacturer's directions. Clones containing the ITS fragment inserts are sequenced using the TA cloning vector's FORWARD (5′-gtaaaacgacggccagt-3′; SEQ ID NO:35) and REVERSE (5′-caggaaacagctatgac-3′; SEQ ID NO:36) primers. Sequencing is performed on an ABI PRISM 377™ DNA sequencer (Perkin Elmer Applied Biosystems, Foster City, Calif.).

Example 4

Synthesis and Purification of Oligonucleotides

Oligonucleotides and TaqMan™ probes (primers and probes) are synthesized and purified by, for example, either Integrated DNA Technologies (Coralville, Iowa) or Midland Certified Reagent Company (Midland, Tex.).

Example 5

Selection of Species-Specific Primers and Probes

A multiple sequence alignment is made of ITS region consensus sequences of

Tapesia yallundae

(SEQ ID NO:40) and

Tapesia acuformis

(SEQ ID NO:39) obtained from infected wheat tissue as described in Example 3. Also included in the alignment are ITS region sequences from

Tapesia yallundae

and

Tapesia acuformis

fungal DNAs referenced in U.S. Pat. No. 5,585,238 (SEQ ID NO:37 and SEQ ID NO:38, respectively). PCR primers and TaqMan™ probes are designed to the regions that contain the greatest differences in sequence between the fungal species. This produces primers and probes designed to be specific to either

Tapesia acuformis

or

Tapesia yallundae.

The oligonucleotide primers and probes shown below in Tables 4 and 5 are synthesized according to Example 4. The previously described (U.S. Pat. No. 5,585,238)

Tapesia yallundae

-specific primers JB537 (SEQ ID NO:31) and JB541 (SEQ ID NO:32), and

Tapesia acuformis

-specific primers JB540 (SEQ ID NO:33) and JB542 (SEQ ID NO:34) are also synthesized. In addition, the ribosomal gene-specific primers ITS1 (SEQ ID NO:1) and ITS4 (SEQ ID NO:2) published by White et al. (1990: In: PCR Protocols; Eds.: Innes et al. Pages 315-322) are synthesized for testing in combination with the primers specific for the ITS regions.

TABLE 4

Primers and Probes for TaqMan ™ Amplification of

Tapesia acuformis

DNA

SEQ ID NO:

Oligo

Target

Oligo Sequence (5′->3′)

SEQ ID NO:1

ITS1

Fungal 18S rDNA

tccgtaggtgaacctgcgg

SEQ ID NO:2

ITS4

Fungal 25S rDNA

tcctccgcttattgatatgc

SEQ ID NO:12

J112R

Tapesia acuformis

(R)

tcaagggtggaggtctgaacc

SEQ ID NO:13

J100R

Tapesia acuformis

(R)

gggccaccctacttcggtaa

SEQ ID NO:14

J101R

Tapesia acuformis

(R)

gaaatcctgggggccaccctacttc

SEQ ID NO:15

J102R

Tapesia acuformis

(R)

cctgggggccaccctact

SEQ ID NO:16

J113R

Tapesia acuformis

(R)

gccaccctacttcggtaaggtt

SEQ ID NO:17

J114R

Tapesia acuformis

(R)

caccctacttcggtaaggtttagagtc

SEQ ID NO:18

J115R

Tapesia acuformis

(R)

aggtaatttattcaagggtggaggt

SEQ ID NO:19

J116R

Tapesia acuformis

(R)

aggtaatttattcaagggtggaggtc

SEQ ID NO:20

J117R

Tapesia acuformis

(R)

aaggtaatttattcaagggtggaggt

SEQ ID NO:21

J118R

Tapesia acuformis

(R)

ttattcaagggtggaggtctgg

SEQ ID NO:22

J119R

Tapesia acuformis

(R)

tattcaagggtggaggtctgga

SEQ ID NO:23

J120R

Tapesia acuformis

(R)

cctgccaaagcaacaaaggta

SEQ ID NO:24

J121R

Tapesia acuformis

(R)

(FAM)-cgggcctctcggagaagcctgg-(TAMRA)

SEQ ID NO:25

J122R

Tapesia acuformis

(R)

cctacttcggtaaggtttagagtcgt

SEQ ID NO:26

J123R

Tapesia acuformis

(R)

tctccgagaggcccgac

SEQ ID NO:27

J124R

Tapesia acuformis

(R)

(FAM)-aagcctggtccagacctccaccc-(TAMRA)

SEQ ID NO:28

J125R

Tapesia acuformis

(R)

aaggatcattaatagagcaatggatagac

SEQ ID NO:29

J126R

Tapesia acuformis

(R)

(FAM)-cgccccgggagaaatcctgg-(TAMRA)

SEQ ID NO:30

J127R

Tapesia acuformis

(R)

tgggggccaccctacttc

SEQ ID NO:33

JB540

Tapesia acuformis

(R)

gggggccaccctacttcggtaa

SEQ ID NO:34

JB542

Tapesia acuformis

(R)

ccactgattttagaggccgcgaa

Example 6

Initial Screening of the Primer-Probe Library

The species-specific primer libraries designed in Example 5 are tested in initial TaqMan™ screens. Primer and probe combinations are tested for their ability to amplify from the target pathogen's DNA. All other reaction conditions are held constant (1× TaqMan™ Universal Master Mix (Perkin Elmer, Norwalk, Conn.; part no. N430-4447), 200 nM each primer, 100 nM probe, 0.04 ng/μL 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). Pathogen-specific primers and probes are determined by identifying those that best amplify the targeted DNA.

Example 7

TaqMan™ Primer Optimization

Once a primer pair specific for the targeted pathogen's DNA has been identified, the primer concentrations are optimized in a single TaqMan™ run. A matrix of different concentrations of the forward primer are run against those of the reverse primer with all other reaction conditions held constant (1× TaqMan™ Universal Master Mix (Perkin Elmer), 100 nM probe, 0.4 ng/μL 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).

Example 8

TaqMan™ Probe Optimization

Once optimal primer concentrations are determined as in Example 7, the probe concentration is optimized. With primers at their optimal concentrations, different concentrations of probe are run in a typical TaqMan™ run. The probe concentration that gives the best signal in reporting the PCR amplification is chosen. The optimal primers and probe for quantification of

Tapesia acuformis

and

Tapesia yallundae

are recorded along with their optimal reaction concentrations (Tables 6 and 7, respectively). The

T. acuformis

and

T. yallundae

assays are established with an annealing temperature of 60° C. over 35 cycles.

TABLE 6

Primer and Probe Combinations Specific for

Tapesia acuformis

.

Optimized

Sequence

Primer

Concentration

Target

Oligo

Identifier

Name

(nM)

Tapesia

Forward Primer

SEQ ID NO:

J101R

50

acuformis

(R)

14

Reverse Primer

SEQ ID NO:

J115R

900

18

TaqMan ™

SEQ ID NO:

J121R

700

Probe

24

Example 9

Determination of TaqMan™ Assay Specificity to Fungal Genomic DNA

The TaqMan™ assay is validated against a panel of DNA from other cereal pathogens for cross-reactivity (Table 1). TaqMan™ reactions are prepared using the optimal primer and probe concentrations as determined in Examples 7 and 8 and tested against 0.2 ng/μL of the genomic DNA from the cereal pathogens as prepared in Example 1. Depending on the results, changes are made to the thermal cycling parameters to make the assay more stringent. These include changing the annealing/extension temperature or the number of cycles in the run. A successful TaqMan™ assay is sensitive to sub-picogram amounts of target DNA without any cross-reactivity to the panel of cereal pathogens or the plant DNA. In Table 8 results of the

Tapesia acuformis

(R-type) and

Tapesia yallundae

(W-type) assays documented under Example 8 are shown. C

T

values are used to show amplification among isolates screened. Those isolates with a C

T

value of 35 give no amplification with the assays.

TABLE 8

Results of

Tapesia acuformis

TaqMan ™ Assay on

Fungal Genomic DNA Samples

C

τ

Value

C

τ

Value

Isolate

Organism

R-type assay

W-type assay

358

Tapesia acuformis

18.52

35

308

Tapesia acuformis

18.65

35

44643

Tapesia yallundae

35

44614

Tapesia yallundae

35

17.18

60973

Tapesia acuformis

31.36

35

42040

Pseudocercosporella herpo

-

35

18.7

trichoides

var.

herpotrichoides

62012

Pseudocercosporella aestiva

35

35

24425

Septoria nodorum

35

35

26517

Septoria tritici

35

35

38699

Septoria glycines

35

35

22585

Septoria passerini

35

35

26380

Septoria avenae f. sp. triticea

35

35

52182

Ceratobasidium cereale

35

35

11404

Drechslera sorokiniana

35

35

R-5391

Fusarium culmorum

35

35

4551

Fusarium moniliforme

35

35

R-8637

Fusarium graminearum

35

35

T-534

Fusarium poae

35

35

18222

Gerlachia nivalis

35

35

093

Microdochium nivale

var.

majus

35

35

Note:

C

τ

value or threshold cycle, represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected.

The Sequence Detection software generates a Standard Curve of C

τ

vs. (LogN) Starting Copy Number for all standards and then determines the starting copy number of unknowns by interpolation.

Example 10

Determination of TaqMan™ Assay Specificity to Pathogen in Infected Wheat

Wheat samples are identified as

Tapesia acuformis

and/or

Tapesia yallundae

infected based on analysis using the assays described in Example 3. Wheat samples are also tested using the primer combinations listed in Table 6 and the PCR conditions in Example 8. Using Sequence Detection Systems software (Perkin Elmer-Applied Biosciences), the amplification of pathogen DNA from the wheat samples is quantified against a standard curve of the fungal target's genomic DNA (Table 9). Results for the

Tapesia acuformis

specific assay are presented in Table 10. DNA from

Tapesia acuformis

is detected and quantified in all infected samples. Results for the

Tapesia yallundae

specific assay are presented in Table 11. DNA from

Tapesia yallundae

is detected and quantified in all infected samples. No cross-reactivity is observed in uninfected wheat tissue for either assay.

TABLE 9

Standard Curve of

Tapesia acuformis

and

T. yallundae

Genomic

DNAs Run in Duplicate Against the R-type and W-type Assays,

Respectively.

R-type Assay

W-type Assay

Tapesia acuformis

Tapesia yallundae

#308 DNA

C

T

Value

#42040 DNA

C

T

Value

5 ng

18.57

5 ng

18.13

18.38

17.92

500 pg

21.3

500 pg

21.83

21.35

22.02

50 pg

23.57

50 pg

25.26

24.27

25.37

5 pg

27.82

5 pg

29.53

27.89

29.88

500 fg

31.47

500 fg

33.32

31.17

35

50 fg

34.13

No Template Control

35

34.01

35

No Template Control

35

35

TABLE 10

Results of the

Tapesia acuformis

TaqMan ™ Assay on Wheat Extractions. Samples Are

Run in Duplicate and are Documented with Results of Conventional PCR Assays

TaqMan ™ Results

PCR Testing Results

for

Tapesia acuformis

assay

(0 to +5 scale)

Sample

Cτ

Template

Standard

Mean

T.

Number

Value

(pg)

Deviation

(pg)

acuformis

T. yallundae

H

35

0

0

0

−

−

35

0

0

0

6

35

2.50E−02

0

0.02

−

−

35

2.50E−02

0

0.02

57

31.07

4.60E−01

0.03

0.44

+

−

31.20

4.20E−01

0.03

0.44

47

31.13

4.40E−01

0.14

0.54

+

−

30.62

6.40E−01

0.14

0.54

84

33.68

7.00E−02

0.01

0.06

+

−

33.96

5.70E−02

0.01

0.06

23

29.42

1.50E+00

0.28

1.71

++

−

29.10

1.90E+00

0.28

1.71

46

28.67

2.60E+00

0.44

2.90

++

−

28.37

3.20E+00

0.44

2.90

73

30.54

6.70E−01

0.06

0.72

++

−

30.37

7.60E−01

0.06

0.72

21

27.34

6.80E+00

2.28

5.15

+++

−

28.24

3.50E+00

2.28

5.15

38

30.04

9.70E−01

0.71

1.47

+++

−

29.05

2.00E+00

0.71

1.47

43

26.12

1.60E+01

0.97

16.94

+++

−

26.01

1.80E+01

0.97

16.94

41

24.07

7.20E+01

19.75

57.57

++++

−

24.75

4.40E+01

19.75

57.57

72

28.01

4.20E+00

0.29

3.96

++++

−

28.16

3.80E+00

0.29

3.96

74

26.01

1.80E+01

3.03

19.75

++++

−

25.71

2.20E+01

3.03

19.75

5

26.72

1.10E+01

1.50

9.51

+++++

−

27.03

8.50E+00

1.50

9.51

82

26.74

1.00E+01

1.29

9.51

+++++

−

27.01

8.60E+00

1.29

9.51

93

26.05

1.70E+01

2.12

18.68

+++++

+

25.82

2.00E+01

2.12

18.68

96

24.07

7.10E+01

3.75

68.50

+++++

++

24.18

6.60E+01

3.75

68.50

TABLE 11

Results of the

Tapesia yallundae

TaqMan ™ Assay on Wheat Extractions. Samples Are

Run in Duplicate and are Documented with Results of Conventional PCR Assays

TaqMan ™ Results

PCR Testing Results

for

Tapesia acuformis

assay

(0 to +5 scale)

Sample

Cτ

Template

Standard

Mean

T.

Number

Value

(pg)

Deviation

(pg)

acuformis

T. yallundae

H

35

0

0

0

−

−

35

0

0

0

6

35

0

0

0

−

−

35

0

0

0

82

33.41

4.5E−01

0.07

0.40

+++++

−

33.78

3.6E−01

94

33.29

5.2E−01

0.21

0.37

+

+

34.68

2.2E−01

108

34.41

2.6E−01

0

0.26

+++

+

34.40

2.7E−01

111

33.21

5.4E−01

0.02

0.53

++

+

33.28

5.2E−01

33

24.67

9.1E+01

37.45

64.30

++

++

26.13

3.8E+01

54

28.09

1.2E+01

6.31

16.10

+++

++

27.14

2.1E+01

80

26.43

3.1E+01

3.62

34.03

++++

++

26.18

3.7E+01

95

29.98

3.8E+00

0.08

3.7

−

++

30.03

3.6E+00

100

27.16

2.0E+01

1.40

21.32

+++

+++

27.01

2.2E+01

8

25.63

5.1E+01

9.96

57.91

+

+++

25.22

6.5E+01

10

22.36

3.6E+02

79.1

418.46

++

+++

21.91

4.7E+02

16

23.77

1.6E+02

6.18

150.78

++

++++

23.87

1.5E+02

56

25.14

6.8E+01

2.26

66.56

++++

++++

25.22

6.5E+01

88

24.48

1.0E+02

21.89

85.90

++

++++

25.09

7.0E+01

89

23.87

1.5E+02

16.48

157.85

++++

+++++

23.63

1.7E+02

Example 11

An Endogenous Control to be Used with the Fungal Pathogen TaqMan™ Assays

All wheat extractions contain the host wheat DNA as well as any fungal pathogen DNA present. Thus, an endogenous control assay targeting the wheat DNA is run on extracts to account for any differences among sample extractions. These assays provide a control against false negatives. That is, a negative result for fungal DNA that could be attributed to inhibition of the PCR reaction is verified by this endogenous control assay. These assays also provide a target against which the fungal DNA quantity is normalized for sample to sample comparison.

Example 12

Selection of Endogenous Control Primers and Probes

Primers and probes for the amplification and detection of wheat chloroplast DNA are drawn to the coding sequence of the cytochrome b-599 gene (SEQ ID NO:41). Selection of primer and probe sequences is performed using the ABI Primer Express program (PE Applied Biosystems, Foster City, Calif., USA) according to manufacturer's instructions. This program selects TaqMan™ primer and probe sets optimized by melting temperature, secondary structure, base composition, and amplicon length. From the sets chosen by the software, a best set is selected by manually finding primers with the fewest number of thermodynamically stable bases at the 3′ end. The primer/probe set chosen for the amplification of wheat DNA as an endogenous control is documented in Table 12. These are synthesized as in Example 4.

TABLE 12

Primer And Probe Combinations For An Endogenous Control Reaction Targeting

Wheat (

Triticum aestivum

) Chloroplast DNA.

Oligo

SEQ ID NO:

Primer

Oligo Sequence (5′->3′)

Forward Primer

SEQ ID NO:42

WCP2

cagtgcgatggctggctatt

Reverse Primer

SEQ ID NO:43

WCP3

cgffggatgaactgcattgct

TaqManTM Probe

SEQ ID NO:44

WCP1

(VIC)-acggactagctgtacctactgtttttttcttgggatc-(TAMRA)

Example 13

Use of a TaqMan™ Assay to Quantify Wheat DNA in Wheat Extractions

Extractions of wheat tissue are made as in Example 2. The assay presented in Example 11 is run against these tissues as follows: Reactions are prepared in thin-walled optical grade PCR tubes (PE Applied Biosystems, Foster City, Calif., USA). Reaction mixtures are made by bringing forward and reverse primer concentrations to 900 nM and probe concentration to 250 nM in a 1× solution of TaqMan™ Universal Master Mix (PE Applied Biosystems, Foster City, Calif., USA). One microliter of 1:20 diluted wheat extract is added. Additionally, cross-reactivity with fungal DNA is tested by adding 1 μL of 5 ng/μL fungal DNA preparation. The reactions are carried out in a ABI 7700 instrument (PE Applied Biosystems, Foster City, Calif., USA), 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). The ABI 7700 software determines the CT value at which the fluoresence of each reaction reaches a threshold value of 0.4. This data is presented in Table 13. The CT values presented correspond inversely with the amount of wheat target DNA present in each sample. Samples in which a CT of 40 are reported show no amplification. Table 13 shows that the endogenous control assay detects the cytochrome b-559 gene in multiple varieties of wheat. The TaqMan™ assay for wheat chloroplast DNA also shows that different amounts of host DNA are present in each sample. By using dilutions of target DNA, a standard curve can be generated as described in Example 10 against which the wheat DNA can be quantified.

TABLE 13

CT Values Reported For A TaqMan ™ Assay Targeting

Wheat Chloroplast DNA In Wheat And Fungal DNA Extractions.

Sample

Wheat

CT

Number

Variety

Value

6

Madsen

17.17

57

Madsen

19.48

73

Lambert

20.71

21

Brundage

18.9

41

Eltan

20.23

13

Mixed

19.99

5

Madsen

19.19

5 ng

Tapesia acuformis

40

DNA #308

NTC

40

Example 14

Multiplexing of TaqMan™ Assays for Fungal Pathogens and Control Assay for Host DNA

The reaction presented in Example 13 is multiplexed with reactions for quantification of fungal DNA such that both tests take place in the same reaction tube. The probe and primers for

Tapesia acuformis

documented in Table 6 at their optimized concentrations are added to the reactions described in Example 13. These reactions are run as described on infected wheat tissue. The data presented here show that TaqMan™ fungal pathogen assays may be run in the same reaction tube as an endogenous control reaction for the wheat tissue.

TABLE 14

Cτ Values Reported For A TaqMan ™ Assay Targeting Wheat

Chloroplast DNA In Wheat DNA Extractions.

R-type Assay

Wheat

Calculated

PCR Testing Results

Sample

assay

Cτ

Concentration

(0 to +5 scale)

Number

Cτ Value

Value

(pg)

T. acuformis

T. yallundae

6

17.09

40

0

−

−

41

27.70

20.65

24.3

++++

−

13

30.9

19.99

3.69

+++++

+

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.

44

1

19

DNA

Artificial Sequence

Description of Artificial SequenceITS1

1
tccgtaggtg aacctgcgg 19

2

20

DNA

Artificial Sequence

Description of Artificial SequenceITS2

2
tcctccgctt attgatatgc 20

3

18

DNA

Artificial Sequence

Description of Artificial SequenceJ103W

3
ggctacccta cttggtag 18

4

20

DNA

Artificial Sequence

Description of Artificial SequenceJ104W

4
cctgggggct accctacttg 20

5

21

DNA

Artificial Sequence

Description of Artificial SequenceJ105W

5
gggggctacc ctacttggta g 21

6

22

DNA

Artificial Sequence

Description of Artificial SequenceJ106W

6
tgggggctac cctacttggt ag 22

7

29

DNA

Artificial Sequence

Description of Artificial SequenceJ107W

7
tttagagtcg tcaggcctct cggagaagc 29

8

25

DNA

Artificial Sequence

Description of Artificial SequenceJ108W

8
atttattcaa gggtggaggt cctga 25

9

21

DNA

Artificial Sequence

Description of Artificial SequenceJ109W

9
aagggtggag gtctgaacca g 21

10

20

DNA

Artificial Sequence

Description of Artificial SequenceJ110W

10
aagggtggag gtctgaacca 20

11

20

DNA

Artificial Sequence

Description of Artificial SequenceJ111W

11
caagggtgga ggtctgaacc 20

12

21

DNA

Artificial Sequence

Description of Artificial SequenceJ112R

12
tcaagggtgg aggtctgaac c 21

13

20

DNA

Artificial Sequence

Description of Artificial SequenceJ100R

13
gggccaccct acttcggtaa 20

14

25

DNA

Artificial Sequence

Description of Artificial SequenceJ101R

14
gaaatcctgg gggccaccct acttc 25

15

18

DNA

Artificial Sequence

Description of Artificial SequenceJ102R

15
cctgggggcc accctact 18

16

22

DNA

Artificial Sequence

Description of Artificial SequenceJ113R

16
gccaccctac ttcggtaagg tt 22

17

27

DNA

Artificial Sequence

Description of Artificial SequenceJ114R

17
caccctactt cggtaaggtt tagagtc 27

18

25

DNA

Artificial Sequence

Description of Artificial SequenceJ115R

18
aggtaattta ttcaagggtg gaggt 25

19

26

DNA

Artificial Sequence

Description of Artificial SequenceJ116R

19
aggtaattta ttcaagggtg gaggtc 26

20

26

DNA

Artificial Sequence

Description of Artificial SequenceJ117R

20
aaggtaattt attcaagggt ggaggt 26

21

22

DNA

Artificial Sequence

Description of Artificial SequenceJ118R

21
ttattcaagg gtggaggtct gg 22

22

22

DNA

Artificial Sequence

Description of Artificial SequenceJ119r

22
tattcaaggg tggaggtctg ga 22

23

21

DNA

Artificial Sequence

Description of Artificial SequenceJ120R

23
cctgccaaag caacaaaggt a 21

24

22

DNA

Artificial Sequence

Description of Artificial SequenceJ121R

24
cgggcctctc ggagaagcct gg 22

25

26

DNA

Artificial Sequence

Description of Artificial SequenceJ122R

25
cctacttcgg taaggtttag agtcgt 26

26

17

DNA

Artificial Sequence

Description of Artificial SequenceJ123R

26
tctccgagag gcccgac 17

27

23

DNA

Artificial Sequence

Description of Artificial SequenceJ124R

27
aagcctggtc cagacctcca ccc 23

28

29

DNA

Artificial Sequence

Description of Artificial SequenceJ125R

28
aaggatcatt aatagagcaa tggatagac 29

29

20

DNA

Artificial Sequence

Description of Artificial SequenceJ126R

29
cgccccggga gaaatcctgg 20

30

18

DNA

Artificial Sequence

Description of Artificial SequenceJ127R

30
tgggggccac cctacttc 18

31

21

DNA

Artificial Sequence

Description of Artificial SequenceJB537

31
gggggctacc ctacttggta g 21

32

23

DNA

Artificial Sequence

Description of Artificial SequenceJB541

32
ccactgattt tagaggccgc gag 23

33

22

DNA

Artificial Sequence

Description of Artificial SequenceJB540

33
gggggccacc ctacttcggt aa 22

34

23

DNA

Artificial Sequence

Description of Artificial SequenceJB542

34
ccactgattt tagaggccgc gaa 23

35

17

DNA

Artificial Sequence

Description of Artificial Sequenceforward
sequencing primer

35
gtaaaacgac ggccagt 17

36

17

DNA

Artificial Sequence

Description of Artificial Sequencereverse
sequencing primer

36
caggaaacag ctatgac 17

37

627

DNA

Tapesia acuformis

37
tccgtaggtg aacctgcgga aggatcatta atagagcaat ggatagacag cgccccggga 60
gaaatcctgg gggccaccct acttcggtaa ggtttagagt cgtcgggcct ctcggagaag 120
cctggtccag acctccaccc ttgaataaat tacctttgtt gctttggcag ggcgcctcgc 180
gccagcggct tcggctgttg agtacctgcc agaggaccac aactcttgtt tttagtgatg 240
tctgagtact atataatagt taaaactttc aacaacggat ctcttggttc tggcatcgat 300
gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga atcatcgaat 360
ctttgaacgc acattgcgcc ctctggtatt ccggggggca tgcctgttcg agcgtcatta 420
taaccactca agctctcgct tggtattggg gttcgcgtct tcgcggcctc taaaatcagt 480
ggcggtgcct gtcggctcta cgcgtagtaa tactcctcgc gattgagtcc ggtaggttta 540
cttgccagca acccccaatt ttttacaggt tgacctcgga tcaggtaggg atacccgctg 600
aacttaagca tatcaataag cggagga 627

38

626

DNA

Tapesia yallundae

38
tccgtaggtg aacctgcgga aggatcatta atagagcaat gaacagacag cgccccggga 60
gaaatcctgg gggctaccct acttggtagg gtttagagtc gtcaggccgc tcggagaagc 120
ctggttcaga cctccaccct tgaataaatt acctttgttg ctttggcagg gcgcctcgcg 180
ccagcggctt cggctgttga gtacctgcca gaggaccaca actcttgttt ttagtgatgt 240
ctgagtacta tataatagtt aaaactttca acaacggatc tcttggttct ggcatcgatg 300
aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc 360
tttgaacgca cattgcgccc tctggtattc cggggggcat gcctgttcga gcgtcattat 420
aaccactcaa gctctcgctt ggtattgggg ttcgcgtcct cgcggcctct aaaatcagtg 480
gcggtgcctg tcggctctac gcgtagtaat actcctcgcg attgagtccg gtaggtttac 540
ttgccagtaa cccccaattt tttacaggtt gacctcggat caggtaggga tacccgctga 600
acttaagcat atcaataagc ggagga 626

39

415

DNA

Tapesia acuformis

39
gggggccacc ctacttcggt aaggtttaga gtcgtcgggc ctctcggaga agcctggtcc 60
agacctccac ccttgaataa attacctttg ttgctttggc agggcgcctc gcgccagcgg 120
cttcggctgt tgagtacctg ccagaggacc acaactcttg tttttagtga tgtctgagta 180
ctatataata gttaaaactt tcaacaacgg atctcttggt tctggcatcg atgaagaacg 240
cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac 300
gcacattgcg ccctctggta ttccgggggg catgcctgtt cgagcgtcat tataaccact 360
caagctctcg cttggtattg gggttcgcgt cttcgcgggc ctctaaaatc agtgg 415

40

415

DNA

Tapesia yallundae

40
gggggctacc cctacttggt agggtttaga gtcgtcaggc ctctcggaga agcctggttc 60
agacctccca cccttgaata aattaccttt gttgctttgg cagggcgcct cgcgccagcg 120
gcttcggctg ttgagtacct gccagaggac cacaactctt gtttttagtg atgtctgagt 180
actatataat agttaaaact ttcaacaacg gatctcttgg ttctggcatc gatgaagaac 240
gcagcgaaat gcgataagta atgtgaattg cagaattcag tgaatcatcg aatctttgaa 300
cgcacattgc gccctctggt attccggggg gcatgcctgt tcgagcgtca ttataaccac 360
tcaagctctc gcttggtatt ggggttcgcg tcctcgcggc ctctaaaatc agtgg 415

41

554

DNA

Triticum aestivum

misc_feature

(104)..(355)

cytochrome b-559 coding sequence

41
tctcacaagg aatgaaatat cagtaatttt ctatttactg gtcgatccca tcttttacgg 60
aatcaattcc tttttgaatg tacaaaaatt ttgggagttc agcatgtctg gaagcacggg 120
agaacgttct tttgctgata ttattaccag tattcgatac tgggttattc atagcattac 180
tataccttcc ctattcattg cgggttggtt atttgtcagt acgggtttag cttatgacgt 240
gtttggaagt cctaggccaa acgagtattt cacggaaagc cgacaaggaa ttccgttaat 300
aaccgaccgt tttgattctt tagaacaact cgatgaattt agtagatcct tttaggaggc 360
cctcaatgac catagatcga acctatccta tttttacagt gcgatggctg gctattcacg 420
gactagctgt acctactgtt tttttcttgg gatcaatatc agcaatgcag ttcatccaac 480
gataaaccaa attccaacta tagaactatg acacaatcaa acccgaatga acaaaatgtt 540
gaattgaatc gtag 554

42

20

DNA

Artificial Sequence

Description of Artificial Sequence WCP2

42
cagtgcgatg gctggctatt 20

43

21

DNA

Artificial Sequence

Description of Artificial Sequence WCP3

43
cgttggatga actgcattgc t 21

44

37

DNA

Artificial Sequence

Description of Artificial Sequence WCP1

44
acggactagc tgtacctact gtttttttct tgggatc 37

Number	Name	Date	Kind
5585238	Ligon et al.	Dec 1996
5814453	Beck	Sep 1998

	Number	Date	Country
	60/168326	Dec 1999	US
	60/287548	Aug 1999	US

PCR-based detection and quantification of Tapesia yallundae and Tapesia acuformis

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Parent Case Info

US Referenced Citations (2)

Non-Patent Literature Citations (2)

Provisional Applications (2)

Entry
Livak et al. PCR Methods and Applications. 4:357-362, 1995.*
Poupard et al. Plant Pathology (1993) 42, 873-881.