Detection of fungal pathogens using the polymerase chain...

Details Detection of fungal pathogens using the polymerase chain... Detection of fungal pathogens using the polymerase chain...

2001-08-24

2003-11-11

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091500, C536S023100, C536S024300

Reexamination Certificate

active

06645720

ABSTRACT:

FIELD OF THE INVENTION
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
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).
Commercial almond growers are faced with a number of fungi that infect their crops in diverse ways. The impact of these pathogens on tree growth depends on a number of factors, and the cause is not immediately apparent from the symptoms a diseased tree may present. Vascular pathogens invade and plug the xylem vessels, thereby halting movement of water and nutrients up from roots (Integrated Pest Management for Almonds, U. California Division of Agriculture and Natural resources publication 3308 (1985)). Symptoms often reflect a partial or complete cut-off from food or water as vascular tissues are closed. Thus, symptoms of crown and root rots of almond, as well as wilts caused by pathogens infecting leaf and branch tissues, may appear very similar to one another; and similar to the consequences of environmental conditions such as the availability of nutrients and water and factors affecting their uptake.
Some pathogens cause infections that are limited to branches, foliage, and fruit. Several of the fungi and bacteria that cause disease in almonds have been found to infect orchards during the fall months and over-winter in host tissues. During the spring, they produce localized lesions or larger infections that become evident after environmental conditions such as a recent rain or irrigation promote the growth of the pathogens.
The same pathogens that cause economic problems in almond orchard management also affect other crops. For instance,
Cladosporium carpophilum
has a host range that extends over stone fruits. For example, in addition to the scabs it causes in almond crops,
C. carpophilum
also causes scabs in peaches, nectarines, apricots, plums, and cherries (
Compendium of Stone Fruit Diseases,
Ogawa, J. M.; Zehr, E. I.; Bird, G. W.; Ritchie, D. F.; Uriu, K.; Uyemoto, J. K. eds., p. 11 (1995 APS Press, St. Paul, Minn.)).
Alternaria spp. has been documented as causing infection on many economically important crops. To date, it has been reported to cause alternaria blight of peas, and alternaria leaf spots have been reported on peanuts, almonds, corn, cotton, and in all soybean growing areas of the world (
Compendium of Soybean Diseases,
4
th
Ed. Hartman, G. L.; Sinclair, J. B.; Rupe, J. C. eds., pp. 12-13 (1999, APS Press, St. Paul, Minn.)); (
Compendium of Cotton Diseases,
Watkins, G. M. ed., p. 28 (1981, APS Press, St. Paul, Minn.);
Compendium of Pea Diseases,
Hagedorn, D. J., ed., p. 15 (1984, APS Press, St. Paul, Minn.)); (
Compendium of Peanut Diseases,
Porter, D. M.; Smith, D. H.; Rodriguez-Kabana, R. eds. Pp. 13 (1984, APS Press, St. Paul, Minn.); (
Compendium of Stone Fruit Diseases,
Ogawa, J. M.; Zehr, E. I.; Bird, G. W.; Ritchie, D. F.; Uriu, K.; Uyemoto, J. K. eds., p. 11 (1995 APS Press, St. Paul, Minn.)); and
Compendium of Corn Diseases,
3
rd
Ed. White, D. G., ed., p. 25 (1999, APS Press, St. Paul, Minn.)). Alternaria spp. is responsible for widespread leaf and pod spots on beans grown in Brazil, Cananda, Costa Rica, Colombia, Chile, East Africa, England, Mexico, the United States, and Venezuela (
Compendium of Bean Diseases,
Hall, R. ed., p. 14 (1991, APS Press, St. Paul, Minn.)). Alternaria spp. also form lesions on potatoes and the leaves of other solanaceous crops; (
Compendium of Potato Diseases,
Hooker, W. J. ed., p. 44 (1981, APS Press, St. Paul, Minn.)) and appear as secondary infections on sugar beet leaves (
Compendium of Beet Diseases and Insects,
Whitney, E. D., Duffus, J. E. eds., p. 11 (1991 APS Press, St. Paul, Minn.)). It causes grain molds in sorghum (
Compendium of Sorghum Diseases,
Frederiksen, R. A. ed., p. 36 (1986 APS Press, St. Paul, Minn.)) and is one cause of black mold rot of tomato (
Compendium of Tomato Diseases,
Jones, J. B.; Jones, J. P.; Stall, R. E.; Zitter, T. A., eds., p. 46 (1991 APS Press, St. Paul, Minn.)). Alternaria spp. also causes fruit spot of papaya, a major disease in orchards located in dry areas and in mangos (
Compendium of Tropical Fruit Diseases,
Ploetz, R. C.; Zentmyer, G. A.; Nishijima, W. T.; Rohrbach, K. G.; Ohr, H. D., eds., pp. 34 and 58 (1994 APS Press, St. Paul, Minn.)).
Colletotrichum acutatum
has been reported to infect a large number of fruit crops including avocado, strawberry, almond, apple, and peach.
C. acutatum
causes post-harvest fruit diseases in avocado and mango.
C. acutatum
is also known to cause both post-bloom fruit drop and key lime anthracnose in citrus crops (see Freeman, S. et al., 1998,
Plant Disease
Vol. 82, No. 6, pp. 596-605).
In view of the above, there is a real need for the development of technology that will allow the identification of specific races of pathogenic 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
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 pathotyp

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