European Journal of Plant Pathology

, Volume 145, Issue 4, pp 731–742 | Cite as

Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of Aphanomyces cochlioides in naturally infested soil samples

  • Charlotta Almquist
  • Lars Persson
  • Åsa Olsson
  • Jens Sundström
  • Anders Jonsson


Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R2 > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.


Aphanomyces cochlioides Real-time PCR Diagnostics Soil-borne plant pathogen Sugar beet root rot 



The authors wish to thank the Swedish Farmers’ Foundation for Agricultural Research, the Faculty of Natural Resources and Agricultural Sciences at the Swedish University of Agricultural Sciences, Nordic Beet Research foundation and Eurofins Agro Testing Sweden AB for funding. Thanks to Dr. Fredrik Heyman, Dr. Eva Blixt and Prof. Christina Dixelius for kindly providing some of the fungal isolates. Also, thanks to Dr. Katarzyna Marzec-Smith and Kristina Nordström for assistance with the soil DNA extractions and qPCR analyses and thanks to Lotta Eriksson for assistance with the bioassay. Thanks to Dr. Ann-Charlotte Wallenhammar and Prof. Christina Dixelius for critically reading of the manuscript. Finally, thanks to Dr. Björn Gustavsson for providing some of the soil samples.


  1. Amein, T. (2006). Soil-borne pathogens infecting sugar beet in southern Sweden. Plant Pathology Journal, 5, 356–361.CrossRefGoogle Scholar
  2. Atkins, S. D., Clark, I. M., Sosnowska, D., Hirsch, P. R., & Kerry, B. R. (2003). Detection and quantification of Plactosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting. Applied and Environmental Microbiology, 69, 4788–4793.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Atkins, S. D., Clark, I. M., Pande, S., Hirsch, P. R., & Kerry, B. R. (2005). The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus. FEMS Microbiology Ecology, 51, 257–264.CrossRefPubMedGoogle Scholar
  4. Beale, J., Windels, C., & Kinkel, L. (2002). Spatial distribution of Aphanomyces cochlioides and root rot in sugar beet fields. Plant Disease, 86, 547–551.CrossRefGoogle Scholar
  5. Bilodeau, G., Koike, S., Uribe, P., & Martin, F. (2012). Development of an assay for rapid detection and quantification of Verticillium dahliae in soil. Phytopathology, 102, 331–343.CrossRefPubMedGoogle Scholar
  6. Cai, P., Huang, Q., Zhang, X., & Chen, H. (2006). Adsorption of DNA on clay minerals and various colloidal particles from an Alfisol. Soil Biology & Biochemistry, 38, 471–476.CrossRefGoogle Scholar
  7. Cullen, D. W., Lees, A. K., Toth, I. K., & Duncan, J. M. (2002). Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathology, 51, 281–292.CrossRefGoogle Scholar
  8. Debode, J., Van Poucke, K., França, S. C., Maes, M., Höfte, M., & Heungens, K. (2011). Detection of multiple Verticillium species in soil using density flotation and real-time polymerase chain reaction. Plant Disease, 95, 1571–1580.CrossRefGoogle Scholar
  9. Dineen, S. M., Aranda, R., Anders, D. L., & Robertson, J. M. (2010). An evaluation of commercial DANN extraction kits fort he isolation of bacterial spore DNA from soil. Journal of Applied Microbiology, 109, 1836–1896.CrossRefGoogle Scholar
  10. Frostegård, Å., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., & Simonet, P. (1999). Quantification of bias related to the extraction of DANN directly from soils. Applied and Environmental Microbiology, 65, 5409–5420.PubMedPubMedCentralGoogle Scholar
  11. Gandahl, R. (1952). Bestämning av kornstorlek med hydrometer. Geologiska Föreningens i Stockholm Förhandlingar, 74, 497–512.CrossRefGoogle Scholar
  12. Gangneux, C., Cannesan, M.-A., Bressan, M., Castel, L., Moussart, A., Vicré-Bibouin, M., Driouich, A., Trinsoutrot-Gattin, I., & Laval, K. (2014). A sensitive assay for rapid detection and quantification of Aphanomyces euteiches in soil. Phytopathology, 104, 1138–1147.CrossRefPubMedGoogle Scholar
  13. Harveson, R. (2007). Aphanomyces root rot of sugar beet. University of Nebraska-Lincoln Extension. NebGuide. G1407. Accessed 20 April 2015
  14. Heyman, F. (2008). Root Rot of Pea caused by Aphanomyces euteiches; Calcium Dependent Soil Suppressiveness, Molecular Detection and Population Structure. Uppsala, Sweden: Swedish University of Agricultural Sciences, PhD thesis.Google Scholar
  15. Hussain, S., Lees, A., Duncan, J., & Cooke, D. (2005). Development of a species-specific and sensitive detection assay for Phytophthora infestans and ist application for monitoring of inoculum in tubers and soil. Plant Pathology, 54, 373–382.CrossRefGoogle Scholar
  16. Larsson, M., & Gerhardson, B. (1990). Isolates of Phytophthora cryptogea pathogenic to wheat and some other crop plants. Journal of Phytopathology, 129, 303–315.CrossRefGoogle Scholar
  17. Larsson, M., & Olofsson, J. (1994). Prevalence and pathogenicity of spinach root pathogens of the genera Aphanomyces, Phytophthora, Fusarium, Cylindrocarpon, and Rhizoctonia in Sweden. Plant Pathology, 43, 251–260.CrossRefGoogle Scholar
  18. Lees, A. K., Cullen, D. W., & Sullivan, L. (2002). Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil. Plant Pathology, 51, 293–302.CrossRefGoogle Scholar
  19. Moliszewska, E. B., & Piszczek, J. (2008). Occurrence of sugar beet root rot (Aphanomyces cochlioides) in Poland. Phytopathologia Polonica, 47, 21–29.Google Scholar
  20. Nechwatal, J., Leiminger, J., Poschenrieder, G., & Zellner, M. (2012). Evidence for the involvement of Aphanomyces cochlioides and Pythium spp. in 'girth scab' disease of sugar beet in Bavaria. Journal of Plant Diseases and Protection, 119, 85–91.CrossRefGoogle Scholar
  21. Olsson, Å., & Olsson, R. (2004). Geographic distribution of the soil borne fungus Aphanomyces cochlioides in Sweden. Proceedings of the 67th International Insistute for Beet Reasearch Congress. 11–12 February. Brussels, Belgium.Google Scholar
  22. Olsson, Å., & Persson, L. (2014). Liming of different soil types – effect on soil factors and sugar yield. Proceedings of the 74th International Insistute for Beet Reasearch Congress. 1–3 July, Dresden, Germany.Google Scholar
  23. Olsson, Å., Persson, L., & Olsson, S. (2011). Variations in soil characteristics affecting the occurrence of Aphanomyces root rot of sugar beet – Risk evaluation and disease control. Soil Biology & Biochemistry, 43, 316–323.CrossRefGoogle Scholar
  24. Papavizas, G.C., & Ayers, W.A. (1974). Aphanomyces species and their root diseases in pea and sugarbeet. A review. U.S. Department of Agriculture. Agricultural Research Service. Technical Bulletin No. 1485. Washington DC. 158 ppGoogle Scholar
  25. Payne, P. A., Asher, M. J. C., & Kershaw, C. D. (1994). The incidence of Pythium spp. and Aphanomyces cochlioides associated with the sugar-beet growing soils of Britain. Plant Pathology, 43, 300–308.CrossRefGoogle Scholar
  26. Persson, L., Larsson-Wikström, M., & Gerhardson, B. (1999). Assessment of soil suppressiveness to Aphanomyces root rot of pea. Plant Disease, 83, 1108–1112.CrossRefGoogle Scholar
  27. Persson, L., & Olsson, Å. (2014). Liming as a method for integrated control of Aphanomyces in sugar beet. Proceedings of the 74th International Insistute for Beet Reasearch Congress. 1–3 July, Dresden, GermanyGoogle Scholar
  28. Persson, L., & Olsson, S. (2000). Abiotic characteristics of soils suppressive to Aphanomyces root rot. Soil Biology & Biochemistry, 32, 1141–1150.CrossRefGoogle Scholar
  29. Persson, L., & Olsson, Å. (2010). Persistence of inoculum of Aphanomyces cochlioides. Proceedings of the 72nd International Institute for Beet Research Congress. 22–24 June, Copenhagen, DenmarkGoogle Scholar
  30. Piszczek, J. (2004). Occurrence of root rot of sugar beet cultivars. Journal of Plant Protection Research, 44, 341–345.Google Scholar
  31. Ratti, C., Budge, G., & Ward, L. (2004). Detection and relative quantification of soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan (R). Journal of Virological Methods, 122, 95–103.CrossRefPubMedGoogle Scholar
  32. Rowntree, J., & Windels, C. E. (2003). Survival of Aphanomyces cochlioides oospores following preconditioning at different humidities. Sugarbeet Research and Extension Reports, 34, 270–274.Google Scholar
  33. Rush, C. M. (1988). First report of Aphanomyces cochlioides on sugar beet in Texas. Plant Disease, 72, 79.CrossRefGoogle Scholar
  34. Sauvage, H., Moussart, A., Bois, F., Tivoli, B., Barray, S., & Laval, K. (2007). Development of a molecular method to detect and quantify Aphanomyces euteiches in soil. FEMS Microbiology Letters, 273, 64–69.CrossRefPubMedGoogle Scholar
  35. Schroeder, K., Okubara, P., Tambong, J., Lévesque, C., & Paulitz, T. (2006). Identification and quantification of pathogenic Pythium spp. from soils in eastern Washington using real-time polymerase chain reaction. Phytopathology, 96, 637–647.CrossRefPubMedGoogle Scholar
  36. SIS - The Standardising Commission in Sweden. (1993). Soil analysis-extraction and determination of phosphorus, potassium, calcium, magnesium and sodium from soils with ammonium lactate/acetic acid solution (the AL-method). SS 02 83 10. Stockholm, Sweden.Google Scholar
  37. Valsesia, G., Gobbin, D., Patocchi, A., Vecchione, A., Petrot, I., & Gessler, C. (2005). Development of a high-throughput method for quantification of Plasmopara viticola DNA in grapevine leaves by means of quantitative real-time polymerase chain reaction. Phytopathology, 95, 672–678.CrossRefPubMedGoogle Scholar
  38. Vandemark, G. J., Kraft, J. M., Larsen, R. C., Gritsenko, M. A., & Bofe, W. L. (2000). A PCR-based assay by sequence-characterized DNA markers for the identification and detection of Aphanomyces euteiches. Phytopathology, 90, 1137–1144.CrossRefPubMedGoogle Scholar
  39. Wallenhammar, A.-C., Almquist, C., Söderström, M., & Jonsson, A. (2012). In-field distribution of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology, 61, 16–28.CrossRefGoogle Scholar
  40. Wang, Y., Zhang, W., Wang, Y., & Zheng, X. (2006). Rapid and sensitive detection of Phytophthora sojae in soil and infected soybeans by species-specific polymerase chain reaction assays. Phytopathology, 96, 1315–1321.CrossRefPubMedGoogle Scholar
  41. Weiland, J., & Rundsbak, J. (2000). Differentiation and detection of sugar beet fungal pathogens using pCR amplification of actin coding sequences and the ITS region of the rRNA gene. Plant Diesease, 84, 475–482.CrossRefGoogle Scholar
  42. Windels, C. E., Brantner, J. R., Sims, A. L., & Bradley, C. A. (2007). Long-term effects of a single application of spent lime on sugar beet, Aphanomyces root rot, rotation crops, and antagonistic microorganisms. Sugar Beet Research and Extension Reports, 38, 251–262.Google Scholar
  43. Windels, C. (2000). Aphanomyces root rot on sugar beet.. Plant Health Progress: 10.1094/PHP-2000-0720-01-DG. Accessed 28 April 2015.
  44. Windels, C.E., & Lamey, H.A. (1998). Identification and control of seedling diseases, root rot and Rhizomania on sugarbeet. NDSU Extension Service, University of Minnesota Accessed 28 April 2015.
  45. Zang, L., Liu, X., Zhu, S., & Chen, S. (2006). Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay. Biological Control, 36, 316–323.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Authors and Affiliations

  • Charlotta Almquist
    • 1
    • 2
  • Lars Persson
    • 3
  • Åsa Olsson
    • 3
  • Jens Sundström
    • 2
  • Anders Jonsson
    • 4
  1. 1.Eurofins Agro Testing Sweden ABLidköpingSweden
  2. 2.Department of Plant Biology, Uppsala BiocenterSwedish University of Agricultural SciencesUppsalaSweden
  3. 3.NBR Nordic Beet ResearchBjärredSweden
  4. 4.Precision Agriculture and Pedometrics, Department of Soil and EnvironmentSwedish University of Agricultural SciencesSkaraSweden

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