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Detection of fungal pathogens using the polymerase chain reaction

a technology of polymerase chain reaction and detection of fungal pathogens, which is applied in the field of primers in polymerase chain reaction assays, can solve the problems of shortfall in the nutritional provision of local populations, economic deprivation of farmers, and crop loss

Inactive Publication Date: 2006-07-27
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] 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.

Problems solved by technology

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.
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.
The same pathogens that cause economic problems in almond orchard management also affect other crops.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Fungal Isolates and Genomic Fungal DNA Extraction

[0041] See Table 1 for a listing of the fungal isolates used and their source. 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 protocol of Lee and Taylor (1990; In: PCR Protocols: A Guide to Methods and Applications; Eds.: Innes et al.; pages 282-287).

TABLE 1Source of Test IsolatesIsolateSourceIsolationGeographic OriginColletotrichum acutatum26255ATCC1TomatoNew ZealandColletotrichum acutatum42373ATCC1MangoAustraliaColletotrichum acutatum60468ATCC1VacciniumNew ZealandColletotrichum acutatum66367ATCC1StrawberryIndiana, USAColletotrichum acutatum38689ATCC1Pinus—f.sp. pinearadiataColletotrichum44228ATCC1StylosanthesAustraliagloeosporioideshamata cv.Colletotrichum38237ATCC1Mango—Cladosporium carpophilum52935ATCC1PeachGeorgia, USACladosporium carpophilumBS-1Jones2PeachMichigan, USACladosporium carpo...

example 2

DNA Extraction from Almond Tissues

[0042] DNA was extracted from almond leaves by using a bulk maceration method with a modified version of the CTAB extraction buffer (Wang et al., 1993, “PCR amplification from single seeds, facilitating DNA marker-assisted breeding,” Nucleic Acids Res. 21:2527). The bulk maceration method was used to isolate DNA from several naturally infected tissues from the field. The potential concentration ranges of the buffer ingredients of the modified CTAB extraction buffer are as follows:

[0043] approximately 100 mM Tris, pH 8.0;

[0044] 0.2-2.0 M NaCl;

[0045] 1-200 mM ethylenediaminetetraacetic acid (EDTA);

[0046] 0.1-5% w / v hexadecyltrimethylammonium (CTAB);

[0047] 0.1-5% w / v polyvinylpyrolidine (PVP); and

[0048] 0.01-2% w / v ascorbic acid.

[0049] In other embodiments of the invention, the DNA extraction buffer comprises 100 mM Tris, pH 8.0; or comprises 1.4 M NaCl; or comprises 20 mM EDTA; or comprises 2% w / v CTAB; or comprises 2% w / v PVP; or comprises 0....

example 3

Polymerase Chain Reaction Amplification

[0075] Polymerase chain reactions are performed with the GeneAmp Kit from Perkin-Elmer (Foster City, Calif.; part no. N8O8-0009) using 50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl, pH8.3, containing 200 μM of each dTTP, dATP, dCTP, and dGTP in either 25 or 50 μL reactions containing 50 μM each primer, 0.25 U / μL of Taq polymerase and 0.2 ng / μL of genomic DNA. Reactions are run for 30-40 cycles of 15 s at 94° C., 15 s at 50° C.-70° C., and 45 s at 72° C. in a Perkin-Elmer Model 9600 or 9700 thermal cycler. The products are analyzed by loading 10 μl of each PCR sample on a 1.0% agarose gel and electrophoresing

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Abstract

The present invention relates to the use of primers in polymerase chain reaction assays for the detection of fungal pathogens Colletotrichum acutatum, Ateniaria spp., and Cladosporium carpophilum. Specific primers are identified as being useful for the identification of fungal isolates using PCR based techniques. Also described are novel extraction buffer solutions for use in isolating DNA from an organism, methods of extracting DNA from tissue, and methods of performing PCR analysis on DNA extracted from tissue.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the use of primers in polymerase chain reaction assays for the detection of stone fruit and nut, in particular almond pathogens Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations. The present invention also relates to novel extraction buffer solutions, methods of extracting DNA from tissue, and methods of performing PCR analysis on DNA extracted from tissue. BACKGROUND OF THE INVENTION [0002] 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04G01N33/50C12N15/09G01N33/53G01N33/569
CPCC12Q1/6895
Inventor BARNETT, CHARLESBECK, JAMESPERRY, CHRISTY
Owner SYNGENTA PARTICIPATIONS AG
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