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  Detection of fungal pathogens using the polymerase chain reactionUSPTO Application #: 20060166246Title: Detection of fungal pathogens using the polymerase chain reaction 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. (end of abstract) Agent: Syngenta Biotechnology, Inc. Patent Department - Research Triangle Park, NC, US Inventors: Charles Jason Barnett, James Joseph Beck, Christy Violet Perry USPTO Applicaton #: 20060166246 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060166246. Brief Patent Description - Full Patent Description - Patent Application Claims
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 fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981; Seed Sci. & Technol. 9: 679-685). [0003] 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 As been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrerpophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Myccasphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983). [0004] 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. [0005] 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. [0006] 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.)). [0007] 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.sup.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.sup.rd Ed. White, D. G., ed., p. 25 (1999, APS Press, St. Paul, Minn.)). Altenzaria 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, MN)). 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, N)) 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.)). [0008] 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). [0009] 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 [0010] 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. [0011] In a preferred embodiment, the invention provides ITS-derived diagnostic primers for the detection of Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. [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. [0013] Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. [0014] The invention also provides methods for preparing an extract of DNA from tissue using novel DNA extraction buffer. The invention further provides for methods for performing PCR analysis on DNA extracted from tissue using the novel DNA extraction buffer and methods of preparing an extract of DNA. BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING SEQ-ID-NO: 1 Oligonucleotide Primer ITS1 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 Oligonucleotide Primer FORWARD SEQ-ID-NO: 6 Oligonucleotide Primer REVERSE Continue reading... 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