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

a technology of polymerase chain reaction and detection method, which is applied in the field of detection of wheat and barley fungal pathogens which are resistant to certain fungicides using the polymerase chain reaction, can solve the problems of shortfall in the nutritional provision of local populations, economic deprivation of farmers, and crop loss

Inactive Publication Date: 2006-02-02
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[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

Problems solved by technology

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.
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.
In particular, the differential symptomology caused by different isolates and species of these fungi make the accurate predictive determination of potential disease loss difficult.
Although eyespot may kill tillers or plants outright, it more usually causes lodging and / or results in a reduction in kernel size and number.
However, the differing susceptibility of cultivars to different strains of the fungus render the predictive efficacy of fungicide treatments difficult.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Fungal Isolates and Genomic Fungal DNA Extraction

[0045] 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. [0046] 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). [0047] 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. [0048] 3. Add 400 μl chlorophorm:isoamylalcohol (24.1, v:v) shake for 15 min [0049] 4. Centrifuge for 10 min (16000 g) [0050] 5. Transfer the aque...

example 2

Amplification of RAPD Products

[0062] 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 OPJ01-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. N8O8-0009) 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, Fos...

example 3

Selection, Cloning, and Sequencing of Subtype-Specific RAPD-PCR Products

[0064] 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...

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PUM

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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

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 369,796 filed Apr. 3, 2002, which is incorporated by reference in its entirety.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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04C12P19/34
CPCC12Q1/6811C12Q2600/156C12Q1/6895C12Q1/686
Inventor BARNETT, CHARLESBECK, JAMES
Owner SYNGENTA PARTICIPATIONS AG