Abstract
Blackleg is a devastating disease of winter oil seed rape (WOSR) caused by the two species Leptosphaeria maculans and L. biglobosa “brassicae”. In this study, Loop-Mediated Isothermal Amplification (LAMP) assays were developed using two primer sets, SirP and PKS5, to identify and monitor L. maculans “brassicae”, and one primer set PKS21 to monitor L. biglobosa “brassicae”. The three primer sets were tested in real-time LAMP with DNA templates from 38 Leptosphaeria spp. reference isolates. The SirP and PKS5 primer sets exclusively amplified DNA from L. maculans isolates whereas, the PKS21 primers amplified DNA solely from L. biglobosa. Further specificity tests with DNA templates from other ascomycetes showed that both PKS primers were highly specific to Leptosphaeria. The SirP primer set produced in a few cases non-specific amplifications, but they were easily differentiated from the positive reactions by the differences in melting curve temperatures. The detection limits of LAMP assays with PKS21, PKS5 and SirP primers were at 100 fg, 30 pg and 180 fg DNA, respectively. DNA from leaf segments with typical L. maculans lesions was successfully amplified using primer set SirP, as was visually indicated by the colour change of phenol red from pink to yellow. This feature makes the method suitable for simple screening of L. maculans in WOSR leaves and in spores collected by spore traps.
Similar content being viewed by others
References
Ammour, M. S., Bilodeau, G. J., Tremblay, D. M., Heyden, H., Yaseen, T., Varvaro, L., & Carisse, O. (2017). Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves. Plant Disease, 101, 1269–1277.
Denschlag, C., Vogel, R. F., & Niessen, L. (2012). Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay. International Journal of Food Microbiology, 156, 189–196.
Duan, Y., Ge, C., Zhang, X., Wang, J., & Zhou, M. (2014). A rapid detection method for the plant pathogen Sclerotinia sclerotiorum based on loop-mediated isothermal amplification (LAMP). Australasian Plant Pathology, 43, 61–66.
Fitt, B. D. L., Evans, N., Howlett, B. J., & Cooke, B. M. (2006). Sustainable strategies for managing Brassica napus (oilseed rape) resistance to Leptosphaeria maculans (Phoma stem canker). Netherlands: Springer ISBN 1–4020–4525-5 (e-book).
Gall, C., Balesdent, M. H., Desthieux, I., Robin, P., & Rouxel, T. (1995). Polymorphism of Tox0Leptosphaeria maculans isolates as revealed by soluble protein and isozyme electrophoresis. Mycological Research, 99, 221–229.
Gardiner, D. M., Cozijnsen, A. J., Wilson, L. M., Pedras, M. S., & Howlett, B. J. (2004). The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans. Molecular Microbiology., 53, 1307–1318.
Gardiner, D. M., Waring, P., & Howlett, B. J. (2005). The epipolythiodioxopiperazine (ETP) class of fungal toxins: Distribution, mode of action, functions and biosynthesis. Microbiology., 151, 1021–1032.
Goto, M., Honda, E., Ogura, A., Nomoto, A., & Hanaki, K. I. (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxyl naphthol blue. Biotechniques., 46, 167–172.
Grandaubert, J., Lowe, R., Soyer, J., Schoch, C., Van de Wouw, A., Fudal, I., Robbertse, B., Lapalu, N., Link, M., Ollivier, B., Linglin, J., Barbe, V., Mangenot, S., Cruaud, C., Borhan, H., Howlett, B., Balesdent, M., & Rouxel, T. (2014). Transposable element-assisted evolution and adaptation to host plant within the Leptosphaeria maculans-Leptosphaeria biglobosa species complex of fungal pathogens. BMC Genomics, 15, 891.
Hansen, Z. R., Knaus, B. J., Tabima, J. F., Press, C. M., Judelson, H. S., Grünwald, N. J., & Smart, C. D. (2016). Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen. Phytophthora infestans. Journal of Applied Microbiology, 120, 1010–1020.
Jędryczka, M., Burzyński, A., Brachaczek, A., Langwiński, W., Song, P., & Kaczmarek, J. (2013). Loop-mediated isothermal amplification as a good tool to study changing Leptosphaeria populations in oilseed rape plants and air samples. ACTA AGROBOTANICA, 66, 93–100.
Kaczmarek, J., Irzykowski, W., Burzyński, A., & Jędryczka, M. (2014). The detection of Plasmodiophora brassicae using Loop-mediated Isothermal DNA Amplification. ACTA AGROBOTANICA, 67, 59–66.
Kuusk, A.-K., Happstadius, I., Zhou, L., Steventon, L. A., Giese, H., & Dixelius, C. (2002). Presence of Leptosphaeria maculans Group A and Group B Isolates in Sweden. Journal of Phytopathology, 150, 349–356.
Leino, M. (2006). Fungal Diseases on Oilseed Rape and Turnip Rape. Swedish board of agriculture ISBN 91–88264–34-3. 48S.
Lindblad, M., Gidlund, A., Sulyok, M., Börjesson, T., Krska, R., Olsen, M., & Fredlund, E. (2013). Deoxynivalenol and other selected Fusarium toxins in Swedish wheat-occurrence and correlation to specific Fusarium species. International Journal of Food Microbiology, 167, 284–291.
Liu, S. Y., Liu, Z., Fitt, B. D. L., Evans, N., Foster, S. J., Huang, Y. J., Latunde-Dada, A. O., & Lucas, J. A. (2006). Resistance to Leptosphaeria maculans (Phoma stem canker) in Brassica napus (oilseed rape) induced by L. biglobosa and chemical defence activators in field and controlled environments. Plant Pathology, 55, 401–412.
Mendes-Pereira, E., Balesdent, M. H., Brun, H., & Rouxel, T. (2003). Molecular phylogeny of the Leptosphaeria maculans-L. biglobosa species complex. Mycological Research, 107, 1287–1304.
Moradi, A., Almasi, M. A., Jafary, H., & Mercado-Blanco, J. (2013). A novel and rapid loop-mediated isothermal amplification assay for the specific detection of Verticillium dahliae. Journal of Applied Microbiology, 116, 942–954.
Niessen, L., & Vogel, R. F. (2010). Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. International Journal of Food Microbiology, 140, 183–191.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, e63.
Parida, M., Sannarangaiah, S., Dash, P. K., Rao, P. V., & Morita, K. (2008). Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Reviews in Medical Virology, 18, 407–421.
Rouxel, T., Grandaubert, J., James, K., Hane, J. K., Hoede, C., Wouw, A. P., Couloux, A., Dominguez, V., Anthouard, V., Bally, P., Bourras, S., Cozijnsen, A. J., Ciuffetti, L., Degrave, L., Dilmaghani, A., Duret, L., Fudal, I., Goodwin, S., Gout, L., Glaser, N., Linglin, J., Kema, G., Lapalu, N., Lawrence, C., May, K., Meyer, M., Ollivier, B., Poulain, J., Schoch, C., Simon, A., Spatafora, J., Stachowiak, A., Turgeon, B., Tyler, B., Vincent, D., Weissenbach, J., Amselem, J., Quesneville, H., Oliver, R., Wincker, P., Balesdent, M.-H., & Howlett, B. (2011). Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat-induced point mutations. Nature Communications, 1–10.
Stachowiak, A., Olechnowicz, J., Jedryczka, J., Rouxel, T., Balesdent, M.-H., Happstadius, I., Gladders, P., Latunde-Dada, A., & Evans, N. (2006). Frequency of avirulence alleles in field populations of Leptosphaeria maculans in Europe. European Journal of Plant Pathology, 114, 67–75.
Tanner, N. A., Zhang, Y., & Evans Jr., T. C. (2015). Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. BioTechniques., 58, 59–68.
Tomlinson, J. A., Dickinson, M. J., & Boonham, N. (2010). Detection of Botrytis cinerea by loop-mediated isothermal amplification. Letters in Applied Microbiology, 51, 650–657.
Toscano-Underwood, C., West, J. S., Fitt, B. D. L., Todd, A. D., & Jedryczka, M. (2001). Development of Phoma lesions on oilseed rape leaves inoculated with ascospores of A-group or B-group Leptosphaeria maculans (stem canker) at different temperatures and wetness durations. Plant Pathology, 50, 28–41.
Wallenhammar, A.-C., Almquist, C., & Jonsson, A. (2012). In-field distribution of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology, 61, 16–28.
Wang, D. G., Brewster, J. D., Paul, & Tomasula, P. M. (2015). Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules., 20, 6048–6059.
Williams, R. H., & Fitt, B. D. L. (1999). Differentiating a and B groups of Leptosphaeria maculans. Causal agent of stem canker (blackleg) of oilseed rape. Plant Pathology, 48, 161–175.
Wouw, A., Howlett, B., & Idnurm, A. (2018). Changes in allele frequencies of avirulence genes in the blackleg fungus. Leptosphaeria maculans. over two decades in Australia. Crop & Pasture Science, 69, 20–29.
Acknowledgments
This research was funded by the Royal Swedish Academy of Agriculture and Forestry (KSLA) and the Carl Tryggers Foundation for Scientific Research. The authors thank Dr. Lydia Bousset, INRA, Le Rheu, France and Dr. Angela Van de Wouw, School of Biosciences, University of Melbourne, Australia for providing Leptosphaeria fungal cultures. The authors also thank Gunilla Berg, Plant protection adviser, Swedish Board of Agriculture, for providing plant samples, Dr. Eva Edin and Dr. Ida Karlsson for providing fungal cultures.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
the authors declare that they have no conflict of interest.
Human and animal rights
this work does not involve research on humans or animals.
All reference isolates were obtained with signed agreements and import permissions from the Swedish Board of Agriculture.
Rights and permissions
About this article
Cite this article
Omer, Z.S., Wallenhammar, AC. Development of loop-mediated isothermal amplification assays for rapid detection of blackleg pathogens in Swedish winter oil seed rape. Eur J Plant Pathol 157, 353–365 (2020). https://doi.org/10.1007/s10658-020-02004-x
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-020-02004-x