Development of PCR test system for Pyrenophora tritici-repensis detection
DOI:
https://doi.org/10.26577/eb-2019-3-b11Abstract
In recent years, there has been a noticeable expansion of the range and severity of the tan spot in Kazakhstan. The high adaptive ability of the fungus contributes to the wide spread and increased harmfulness of the pathogen. It is quite difficult to identify the species of fungus by the symptoms of the manifestation of the disease on the leaves of plants and by the morphological features of conidia. The creation of domestic diagnostic tools to identify the causative agent of wheat tan spot Pyrenophora tritici-repensis is an urgent task. Among molecular methods, the most convenient and operational method is PCR diagnostics. PCR makes it possible to detect fungal DNA even before physiological symptoms become visible on plant tissue.
The aim of this work is to develop a PCR test system for the diagnosis of wheat tan spot.
As a result of the studies, species-specific primers were selected and synthesized to identify the causative agent of wheat tan spot, the conditions for PCR formulation were optimized: the composition of the reaction mixture and the temperature and time regime. The specificity and sensitivity of the test system was determined. The developed test system showed rather high specificity and allows detection of the pathogen nucleic acid in the samples under study in the amount of 2,6 pg. This test system can be used for the rapid detection of pathogen DNA in samples.
Keywords: tan spot, PCR, specificity, sensitivity, diagnosis.
References
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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..
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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].
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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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How to Cite
Isabek, A. U., Tajlakova, J. T., Kozhabergenov, N. S., Strochkov, V. M., Sultankulova, K. T., & Rsaliev, A. S. (2020). Development of PCR test system for Pyrenophora tritici-repensis detection. Experimental Biology, 80(3), 117–125. https://doi.org/10.26577/eb-2019-3-b11
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МOLECULAR BIOLOGY AND GENETICS
