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

Details PCR-based detection and quantification of Tapesia yallundae... PCR-based detection and quantification of Tapesia yallundae...

2000-08-10

2001-11-20

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024300, C536S023100, C536S024100

Reexamination Certificate

active

06319673

ABSTRACT:

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

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