Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-20
2002-03-19
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024320, C536S024330, C536S023100
Reexamination Certificate
active
06358680
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of primers in polymerase chain reaction assays for the detection of fungal pathogens in wheat and barley. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations.
BACKGROUND OF THE INVENTION
Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, 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).
Wheat is currently the most important agricultural commodity in international markets and occupies about 20% of the world's farmed land (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. page 1). Eightly percent of the world's supply of wheat is grown in North America, Europe, China, and the Soviet Union. Approximately 20% of the worldwide production of wheat is lost to disease annually.
Pyrenophora tritici
-
repentis
(Died.) Drechs. (syn.
P. trichostoma
(Fr.) Fckl.), anamorph Drechslera tritici-repentis (Died.) Shoem. (syn.
Helminthosporium tritici
-
repentis
Died.), causes tan spot also known as yellow spot of wheat worldwide (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. page 42). It has resulted in wheat yield losses from 3 to 50 % in Australia, South America, and North America and has been recently considered the most important foliar wheat disease in North Dakota (Zhang et al., 1997; Phytopathology. Vol.87:154-160). It can also contribute to leaf-spotting complexes with other foliar pathogens. Current disease control measures include fungicide application and cultural practices including destroying wheat stubble, using pathogen-free seed and wide row plant spacing to reduce foliage density and relative humidity in the wheat canopy.
Pyrenophora teres
Drechs., anamorph
Drechslera teres
(Sacc.) Shoem. (syn.
Helminthosporium teres
Sacc.) causes net blotch primarily in barley; however, sporadic infections also occur in wheat (Jones and Clifford; Cereal Diseases, John Wiley, 1983). Typical yield losses due to net blotch are between 10 to 40%. Yield losses can approach 100% in fields containing susceptible cultivars (1982; Compendium of Barley Diseases, Amer. Phytopath. Soc. page 22). In addition to affecting overall grain yield and weight, the disease also reduces the carbohydrate content. This reduces malt extract yield and therefore lowers the brewing quality of the grain.
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.
SUMMARY OF THE INVENTION
In view of the above, a primary object of the invention is to provide a method for the identification of specific races of pathogen fungi early in the infection process. The invention therefore provides Internal Transcribed Spacer (ITS) DNA sequences that 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.
In a preferred embodiment, the invention provides novel ITS1 and ITS2 DNA sequences for the fungal pathogen
Pyrenophora tritici
-
repentis.
In another preferred embodiment, the invention provides ITS-derived diagnostic primers for the detection of
Pyrenophora tritici
-
repentis.
In an additional preferred embodiment, the invention provides novel ITS-derived diagnostic primers that are useful for the detection of not only
Pyrenophora tritici
-
repentis,
but also, surprisingly,
Pyrenophora teres
and
Drechslera sorokiniana.
The present invention therefore addresses a long-felt but unfulfilled need to identify different pathotypes of plant pathogenic fungi, especially those that cause tan spot in wheat.
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 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 detection that is especially suitable for diseases with a long latent phase.
Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of the fungal pathogen
Pyrenophora tritici
-
repentis.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO:1 Oligonucleotide Primer ITS1.
SEQ ID NO:2 Oligonucleotide Primer ITS2.
SEQ ID NO:3 Oligonucleotide Primer ITS3.
SEQ ID NO:4 Oligonucleotide Primer ITS4.
SEQ ID NO:5 M13 Universal-20 Primer.
SEQ ID NO:6 Reverse Primer used in Example 2.
SEQ ID NO:7 Oligonucleotide Primer JB629.
SEQ ID NO:8 Oligonucleotide Primer JB630.
SEQ ID NO:9 Oligonucleotide Primer JB631.
SEQ ID NO:10 Oligonucleotide Primer JB632.
SEQ ID NO:11 Oligonucleotide Primer JB633.
SEQ ID NO:12 Oligonucleotide Primer JB634.
SEQ ID NO:13 Oligonucleotide Primer JB635.
SEQ ID NO:14 Oligonucleotide Primer JB636.
SEQ ID NO:15 Oligonucleotide Primer JB637.
SEQ ID NO:16 Oligonucleotide Primer JB638.
SEQ ID NO:17 Oligonucleotide Primer JB651.
SEQ ID NO:18 Oligonucleotide Primer JB652.
SEQ ID NO:19 Oligonucleotide Primer JB653.
SEQ ID NO:20 Oligonucleotide Primer JB654.
SEQ ID NO:21 Oligonucleotide Primer JB655.
SEQ ID NO:22 Oligonucleotide Pri
Kakefuda Mary
Meigs J. Timothy
Myers Carla J.
Syngenta Participations (AG)
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