Testing for infestation of rapeseed and other cruciferae by the

Details Testing for infestation of rapeseed and other cruciferae by the Testing for infestation of rapeseed and other cruciferae by the

1995-08-29

1999-06-29

Myers, Carla J.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 435810, 536 237, 536 2432, 536 2433, 536 254, 536 2541, 935 6, 935 8, 935 77, 935 78, C12Q 168, C12P 1934, C07H 2104

Patent

active

059167440

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

I. Field of the Invention
This invention relates to tests for blackleg contamination of Cruciferae, particularly Brassica spp., and especially oilseed rape or canola. More particularly, the invention relates to the testing of seeds and other products or tissues of such Cruciferae, particularly rapeseed, for contamination by virulent strains of the fungus responsible for causing the disease.
II. Description of the Prior Art
In the central region of the western Canadian province of Saskatchewan, the disease of blackleg of oilseed rape (Brassica napus and Brassica rapa) has spread from three widely spaced fields in 1975 to almost 90% of the land cultivated with this crop in 1988 and the disease represents a loss in crop yield worth millions of dollars per year. While blackleg infestation can be spread by infested crop residue or plants, the disease is commonly seed-borne and this fact places a considerable responsibility on seed growers to test their seed for absence of the blackleg fungus in order to prevent the spread of the disease to currently uninfected areas.
The introduction of more tolerant varieties of rape has greatly reduced the incidence of the disease in Europe. However, for cold winter climates, such species are not suitably viable and crop rotation is still the most effective means of controlling the disease, so preventing the introduction of the fungus into rapeseed growing areas has primary importance.
The disease is caused by Leptosphaeria maculans (Desm.) Ces. et de Not. blackleg fungus exists in western North America in two forms: weakly virulent (non-aggressive) strains and virulent (severe or highly virulent, aggressive) strains. It is the virulent strains that cause substantial damage to rapeseed (canola) crops and other Cruciferae. The weakly virulent strains cause only mild disease symptoms with no substantial yield loss and their presence in seed is not a matter of serious concern. These strains look similar in culture and can only be differentiated by specialized tests. For example, the 2,4-D blotter method recommended by the International Seed Testing Association cannot differentiate between the weakly and highly virulent isolates. G. A. Petrie has developed a test for differentiating the strains of the fungus (see "The rapid differentiation of virulent and weakly virulent strains of Leptosphaera maculans (blackleg or stem canker) and related pycnidial fungi from Brassica seeds and stems", Canadian Journal of Plant Pathology, 10:188-190, 1988), but this test is based on differences in germ tube length after incubation and is not very convenient.
There is therefore an increasing need for a relatively simple and reliable test for detecting infestations of blackleg of oilseed rape and other Cruciferae, and particularly one which can distinguish the highly virulent strains from the weakly virulent strains.


OBJECTS OF THE INVENTION

An object of the present invention is therefore to simplify testing for the blackleg fungus.
Another object of the invention is to provide a test for the blackleg fungus that can distinguish between the weakly virulent and highly virulent strains of the fungus.
Yet another object of the invention is to develop a test for the blackleg fungus that can be carried out relatively quickly and easily using relatively simple equipment.
A still further object of the invention is to provide a diagnostic test kit for testing for infestations of the blackleg fungus.


SUMMARY OF THE INVENTION

In a primary aspect; the invention relates to a method of testing for infestation with a virulent strain of the fungus Leptosphaeria maculans (L. maculans) of tissue of rape or other Cruciferae. The method comprises isolating DNA of a virulent strain of L. maculans from the issue; subjecting the isolated DNA to amplification by polymerase chain reaction accession number M77515), a repetitive element of L. maculans specific to virulent strains of the fungus, to form a product containing amplified L. maculans DNA in sufficient quantity for detection; a

REFERENCES:
Taylor et al. Current Genetics. 19:263-277, 1991.
Xue et al. Physiological and Molecular Pathology. 41: 179-188, Sep. 1992.
Wostemeyer et al. Advances in Molecular Genetics. 5: 227-240, Dec. 1992.
Goodwin et al. Applied and Environmental Microbiology. 57:2482-2486, Sep. 1991.

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