Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-11
2002-11-26
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06485907
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of species-specific primers in polymerase chain reaction assays for the detection of
Rhizoctonia cerealis
, a fungal pathogen of wheat. The use of these primers enables 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.
Sharp eyespot is caused by
Rhizoctonia cerealis
van der Hoeven (teleomorph
Ceratobasidium cereale
Murray & Burpee) and occurs on wheat, barley, oat and rye (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. Page 50). Some isolates of the pathogen are also capable of infecting turfgrass causing yellow patch. Severe wheat infections cause premature ripening and lodging, thereby effecting yield. There are presently no known sharp eyespot-resistant cultivars.
In view of the above, there is a real need for the development of technology that will allow the identification of specific fungal pathogens early in the infection process. By identifying the specific pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
SUMMARY OF THE INVENTION
The present invention is drawn to methods of identification of different pathotypes of plant pathogenic fungi. The invention 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 ITS1 and ITS2 DNA sequences (e.g., SEQ ID NO:17-26) for the pathogen
Rhizoctonia cerealis
. In another preferred embodiment, the invention provides ITS-derived diagnostic primers (e.g., SEQ ID NO:7-16) for the detection of
Rhizoctonia cerealis.
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
Rhizoctonia cerealis.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO:1 Oligonucleotide Primer ITS 1.
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 JB643.
SEQ ID NO:8 Oligonucleotide Primer JB644.
SEQ ID NO:9 Oligonucleotide Primer JB645.
SEQ ID NO:10 Oligonucleotide Primer JB646.
SEQ ID NO:11 Oligonucleotide Primer JB647.
SEQ ID NO:12 Oligonucleotide Primer JB648.
SEQ ID NO:13 Oligonucleotide Primer JB649.
SEQ ID NO:14 Oligonucleotide Primer JB650.
SEQ ID NO:15 Oligonucleotide Primer JB687.
SEQ ID NO:16 Oligonucleotide Primer JB688.
SEQ ID NO:17 DNA sequence of the ITS region PCR-amplified from
R. cerealis
isolate 44235, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-242), 5.8S rRNA gene (nucleotides 244-395), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).
SEQ ID NO:18 DNA sequence of the ITS region PCR-amplified from
R. cerealis
isolate AGDC57, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-242), 5.8S rRNA gene (nucleotides 243-395), Internal Transcribed Spacer 2 (nucleotides 396-629), and 5′ end of the large subunit rRNA gene (nucleotides 630-686).
SEQ ID NO:19 DNA sequence of the ITS region PCR-amplified from
R. cerealis
isolate CAG1BN1, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-243), 5.8S rRNA gene (nucleotides 244-396), Internal Transcribed Spacer 2 (nucleotides 397-630), and 5′ end of the large subunit rRNA gene (nucleotides 631-687).
SEQ ID NO:20 DNA sequence of the ITS region PCR-amplified from
R. cerealis
isolate AGDC73, comprising in the 5′ to 3′ direction: 3′ end of the small subunit rRNA gene (nucleotides 1-30), Internal Transcribed Spacer 1 (nucleotides 31-241), 5.8S rRNA gene (nucleotides 242-394), Internal Transcribed Spacer 2 (nucleotides 395-628), and 5′ end of the large subunit rRNA gene (nucleotides 629-685).
SEQ ID NO:21 DNA sequence of the ITS region PCR-amplified from
R. cerealis
isolate 52182, comprising in the 5′ to 3′ direction: 3′ end of the small subunit r
Barnett Charles Jason
Beck James Joseph
Jones W. Gary
Kakefuda Mary
Meigs J. Timothy
Souaya Jehanne
Syngenta Participations (AG)
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