Разработка ПЦР тест-системы для выявления Pyrenophora tritici-repensis
DOI:
https://doi.org/10.26577/eb-2019-3-b11Аннотация
В последние годы наблюдается заметное расширение ареала и вредоносности желтой пятнистости листьев в Казахстане. Широкому распространению и повышенной вредоносности патогена способствует высокая адаптивная способность гриба. Идентифицировать вид гриба по симптомам проявления болезни на листьях растений и по морфологическим признакам конидий довольно сложно. Создание отечественных диагностических средств для выявления возбудителя желтой пятнистости пшеницы Pyrenophora tritici-repensis является актуальной задачей. Среди молекулярных методов наиболее удобным и оперативным является метод ПЦР диагностики. ПЦР позволяет обнаруживать грибковую ДНК еще до того, как физиологические симптомы становятся видимыми на растительной ткани.
Целью данной работы является разработка ПЦР тест-системы для диагностики желтой пятнистости пшеницы.
В результате проведенных исследований, были подобраны и синтезированы видоспецифичные праймеры для выявления возбудителя желтой пятнистости пшеницы, оптимизированы условия постановки ПЦР: состав реакционной смеси и температурно-временной режим. Определена специфичность и чувствительность тест-системы. Разработанная тест-система показала довольно высокую специфичность и позволяет выявлять нуклеиновую кислоту патогена в исследуемых образцах в количестве 2,6 пг. Данная тест-система может быть использована для оперативного обнаружения ДНК патогена в пробах.
Ключевые слова: желтая пятнистость, ПЦР, специфичность, чувствительность, диагностика.
Библиографиялық сілтемелер
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2. Gurung, S., Short, D.P.G., Adhirkari, T.B. (2013). Global population structure and migration patterns suggest significant population differentiation among isolates of Pyrenophora tritici-repentis. // Fungal Genetics and Biology. 52: 32-41.
3. Manning, V.A., Pandelova, I., Dhillon, B., Wilhelm, L.J., Goodwin, S.B., Berlin, A.M., Figueroa, M., Freitag, M., Hane, J.K., Henrissat, B., Holman, W.H., Kodira, C.D., Martin, J., Oliver, R.P., Robbertse, B., Schackwitz, W., Schwartz, D.C., Spatafora, J.W., Turgeon, B.G., Yandava, C., Young, S., Zhou, S., Zeng, Q., Grigoriev, I.V., Ma, L.J., Ciuffetti, L.M. (2013). Comparative genomics of a plant-pathogenic fungus, Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence. // G3: Genes. Genomes. Genetics. 3: 41-63.
4. Rees R.G., Platz G.J., Mayer R.J. (1988) Susceptibility of Australian wheats to Pyrenophora tritici-repentis. // Aust J Agric Res. Vol. 39, p. 141-151.
5. Hosford R.M. Tan spot of wheat and related disease workshop. /R.M. Hosford (ed.). North Dacota State University, Fargo, 1982.
6. Weiss MV: Compendium of Wheat Diseases, ed 2. St. Paul, APS Press, 1987.
7. Oerke E.C. (2016) Crop losses to pests. // J Agric Sci. vol. 144, p. 31-43.
8. Friesen T.L., Stukenbrock E.H., Liu Z., Meinhardt S., Ling H., Faris J.D., Rasmussen J.B., Solomon P.S., McDonald B.A., Oliver R.P. (2006) Emergence of a new disease as a result of interspecific virulence gene transfer. // Nature Genetics. Vol.38, P.953–956.
9. Antoni E.A., Rybak K., Tucker M.P., Hane J.K., Solomon P.S., Drenth A., Shankar M., Oliver R.P. (2010) Ubiquity of ToxA and absence of ToxB in Australian populations of Pyrenophora tritici-repentis // Australasian Plant Pathology. Vol.39, P.63–68..
10. Mikhailova L.A., Ternuk I.G., Mironenko N.V. (2007) Structure of Pyrenophora tritici-repentis populations from European part of Russia by virulence. // Mikologiya I Fitopatologiya. Vol.41, P. 269–275.
11. Kojshybaev M. (2010) Rasprostranenie i razvitie zheltoj pjatnistosti pshenicy v Kazahstane [The distribution and development of yellow spot of wheat in Kazakhstan] // Mikologija i fitopatologija. Vol.45, p. 177-186 [in Russian].
12. Kokhmetova A., Kremneva O., Volkova G., Atishova M., Sapakhova Z. (2017) Evaluation of wheat cultivars growing in Kazakhstan and Russia for resistance to tan spot // Journal of Plant Pathology. Vol. 99 (1), Р. 161-167.
13. Hudcovicova M., Matusinsky P., Gubis J., Leisova-Svobodova L., Heinonen U., Ondreickova K., Mihalik D., Gubisova M., Majeska M., Jalli M. (2015) DNA markers for identification of Pyrenophora tritici-repentis and detection of genetic diversity among its isolates.// Romanian agricultural research. Vol.32, P.1-10.
14. Abdullah A.S., Turo C., Moffat C.S., Lopez-Ruiz F.J., Gibberd M.R., Hamblin J., Zerihun A. (2018) Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat // Front. Plant Sci. – Vol.9, p. 1086-1089.
15. Mihajlova L.A., Mironenko N.V., Kovalenko N.M. (2012) Zheltaja pjatnistost' pshenicy [Tan spot of wheat] // Metodicheskie ukazanija po izucheniju populjacij vozbuditelja zheltoj pjatnistosti Pyrenophora tritici-repentis i ustojchivosti sortov. – Sankt-Peterburg, 56 s [in Russian].
16. Lamari, L., Bernier, C.C. (1991). Genetics of tan necrosis and extensive chlorosis in tan spot of wheat caused by Pyrenophora tritici-repentis.// Phytopathology. Vol. 81, p. 1092-1095.
17. White T.J., Bruns T., Lee S. Tailor J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. // New York, Academic Press Inc, pp 315-322.
18. Bellemain E., Carlsen T., Brochmann C., Coissac E., Taberlet P., Kauserud H. (2010) ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. // BMC Microbiol., р. 189-198.
19. Kulik T., Fordoński G., Pszczółkowska A., Płodzień K., Łapiński M. (2004) Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides // FEMS Microbiol Lett., р. 181-186.
20. Edwards S.G., O'Callaghan J., Dobson A.D.W. (2002) PCR-based detection and quantification of mycotoxigenic fungi. // Mycol Res., Vol. 106, p. 1005-1025.
21. Wang X., Zhao J., Han Q., Huang L., Kang Z. (2008) The development of a PCR-based method for detecting Puccinia striiformis latent infections in wheat leaves. // Eur J Plant Pathol Vol. 120, p. 241-247.
22. Zhao J., Wang X.J., Chen C.Q., Huang L.L., Kang Z.S. (2007) A PCR-based assay for detection of Puccinia striiformis f. sp. tritici in wheat. // Plant Dis., Vol. 91, p. 1669-1674.
23. Ma Z, Yoshimura M, Holtz B, Michailides T.J. (2005) Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California. // Pest Manag Sci, Vol. 61, p. 449-457.
24. Schena L., Nicosia M.G., Li D., Sanzani S.M., Faedda R., Ippolito A., et al. (2013) Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. // J Plant Pathol., р. 7-24.
25. Sanoubar R., Bauer A., Seigner L. (2015) Detection, identification and quantification of Fusarium graminearum and Fusarium culmorum in wheat kernels by PCR techniques. // J Plant Pathol Microbiol., р. 286-287.
2. Gurung, S., Short, D.P.G., Adhirkari, T.B. (2013). Global population structure and migration patterns suggest significant population differentiation among isolates of Pyrenophora tritici-repentis. // Fungal Genetics and Biology. 52: 32-41.
3. Manning, V.A., Pandelova, I., Dhillon, B., Wilhelm, L.J., Goodwin, S.B., Berlin, A.M., Figueroa, M., Freitag, M., Hane, J.K., Henrissat, B., Holman, W.H., Kodira, C.D., Martin, J., Oliver, R.P., Robbertse, B., Schackwitz, W., Schwartz, D.C., Spatafora, J.W., Turgeon, B.G., Yandava, C., Young, S., Zhou, S., Zeng, Q., Grigoriev, I.V., Ma, L.J., Ciuffetti, L.M. (2013). Comparative genomics of a plant-pathogenic fungus, Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence. // G3: Genes. Genomes. Genetics. 3: 41-63.
4. Rees R.G., Platz G.J., Mayer R.J. (1988) Susceptibility of Australian wheats to Pyrenophora tritici-repentis. // Aust J Agric Res. Vol. 39, p. 141-151.
5. Hosford R.M. Tan spot of wheat and related disease workshop. /R.M. Hosford (ed.). North Dacota State University, Fargo, 1982.
6. Weiss MV: Compendium of Wheat Diseases, ed 2. St. Paul, APS Press, 1987.
7. Oerke E.C. (2016) Crop losses to pests. // J Agric Sci. vol. 144, p. 31-43.
8. Friesen T.L., Stukenbrock E.H., Liu Z., Meinhardt S., Ling H., Faris J.D., Rasmussen J.B., Solomon P.S., McDonald B.A., Oliver R.P. (2006) Emergence of a new disease as a result of interspecific virulence gene transfer. // Nature Genetics. Vol.38, P.953–956.
9. Antoni E.A., Rybak K., Tucker M.P., Hane J.K., Solomon P.S., Drenth A., Shankar M., Oliver R.P. (2010) Ubiquity of ToxA and absence of ToxB in Australian populations of Pyrenophora tritici-repentis // Australasian Plant Pathology. Vol.39, P.63–68..
10. Mikhailova L.A., Ternuk I.G., Mironenko N.V. (2007) Structure of Pyrenophora tritici-repentis populations from European part of Russia by virulence. // Mikologiya I Fitopatologiya. Vol.41, P. 269–275.
11. Kojshybaev M. (2010) Rasprostranenie i razvitie zheltoj pjatnistosti pshenicy v Kazahstane [The distribution and development of yellow spot of wheat in Kazakhstan] // Mikologija i fitopatologija. Vol.45, p. 177-186 [in Russian].
12. Kokhmetova A., Kremneva O., Volkova G., Atishova M., Sapakhova Z. (2017) Evaluation of wheat cultivars growing in Kazakhstan and Russia for resistance to tan spot // Journal of Plant Pathology. Vol. 99 (1), Р. 161-167.
13. Hudcovicova M., Matusinsky P., Gubis J., Leisova-Svobodova L., Heinonen U., Ondreickova K., Mihalik D., Gubisova M., Majeska M., Jalli M. (2015) DNA markers for identification of Pyrenophora tritici-repentis and detection of genetic diversity among its isolates.// Romanian agricultural research. Vol.32, P.1-10.
14. Abdullah A.S., Turo C., Moffat C.S., Lopez-Ruiz F.J., Gibberd M.R., Hamblin J., Zerihun A. (2018) Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat // Front. Plant Sci. – Vol.9, p. 1086-1089.
15. Mihajlova L.A., Mironenko N.V., Kovalenko N.M. (2012) Zheltaja pjatnistost' pshenicy [Tan spot of wheat] // Metodicheskie ukazanija po izucheniju populjacij vozbuditelja zheltoj pjatnistosti Pyrenophora tritici-repentis i ustojchivosti sortov. – Sankt-Peterburg, 56 s [in Russian].
16. Lamari, L., Bernier, C.C. (1991). Genetics of tan necrosis and extensive chlorosis in tan spot of wheat caused by Pyrenophora tritici-repentis.// Phytopathology. Vol. 81, p. 1092-1095.
17. White T.J., Bruns T., Lee S. Tailor J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. // New York, Academic Press Inc, pp 315-322.
18. Bellemain E., Carlsen T., Brochmann C., Coissac E., Taberlet P., Kauserud H. (2010) ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. // BMC Microbiol., р. 189-198.
19. Kulik T., Fordoński G., Pszczółkowska A., Płodzień K., Łapiński M. (2004) Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides // FEMS Microbiol Lett., р. 181-186.
20. Edwards S.G., O'Callaghan J., Dobson A.D.W. (2002) PCR-based detection and quantification of mycotoxigenic fungi. // Mycol Res., Vol. 106, p. 1005-1025.
21. Wang X., Zhao J., Han Q., Huang L., Kang Z. (2008) The development of a PCR-based method for detecting Puccinia striiformis latent infections in wheat leaves. // Eur J Plant Pathol Vol. 120, p. 241-247.
22. Zhao J., Wang X.J., Chen C.Q., Huang L.L., Kang Z.S. (2007) A PCR-based assay for detection of Puccinia striiformis f. sp. tritici in wheat. // Plant Dis., Vol. 91, p. 1669-1674.
23. Ma Z, Yoshimura M, Holtz B, Michailides T.J. (2005) Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California. // Pest Manag Sci, Vol. 61, p. 449-457.
24. Schena L., Nicosia M.G., Li D., Sanzani S.M., Faedda R., Ippolito A., et al. (2013) Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. // J Plant Pathol., р. 7-24.
25. Sanoubar R., Bauer A., Seigner L. (2015) Detection, identification and quantification of Fusarium graminearum and Fusarium culmorum in wheat kernels by PCR techniques. // J Plant Pathol Microbiol., р. 286-287.
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Как цитировать
Isabek, A. U., Tajlakova, J. T., Kozhabergenov, N. S., Strochkov, V. M., Sultankulova, K. T., & Rsaliev, A. S. (2020). Разработка ПЦР тест-системы для выявления Pyrenophora tritici-repensis. ҚазҰУ Хабаршысы. Биология сериясы, 80(3), 117–125. https://doi.org/10.26577/eb-2019-3-b11
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