Comparison of methods for detecting fungi in Silybum marianum (L.) Gaertn. seeds

Agnieszka Rosińska 1 , Magdalena Jarosz 1 , Dorota Szopińska 1 , Hanna Dorna 1 ,  and Krystyna Tylkowska 1
  • 1 Department of Seed Science and Technology Faculty of Horticulture and Landscape Architecture, , 62-081, Przeźmierowo, Poland


Milk thistle (Silybum marianum (L.) Gaertn.) is one of the most important medicinal plants. The fungi infesting its seeds may negatively influence their germination and health-promoting properties. However, there is no standard method for S. marianum seed health testing. The aim of this study was to find a suitable method for the detection and identification of fungi in/on milk thistle seeds. The following tests were used: deep-freeze blotter test, blotter test with an addition of mannitol, blotter test with an addition of polyethylene glycol, agar test on potato-dextrose agar (PDA), agar test on PDA after seed disinfection, agar test on reduced PDA and an agar test on reduced PDA after seed disinfection. Seeds were incubated for 10 and 14 days. The most prevalent fungi were: Alternaria alternata, Botrytis cinerea, Cladosporium spp., Fusarium spp., Penicillium spp., Rhizopus nigricans and Verticillium spp. A deep-freeze blotter test followed by a blotter test with an addition of mannitol and a blotter test with an addition of polyethylene glycol performed for 14 days could be recommended for the further study of milk thistle seed health testing, as they favoured the growth of the most important fungi.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • Agarval V.K., Sinclair J.B., 1987. Principles of seed pathology. Vol. II. CRC Press, Inc., Boca Raton, Florida, USA.

  • Arroyo-Manzanares N., Garcia-Campaña A.M., Gámiz- Gracia L., 2013. Multiclass mycotoxin analysis in Silybum marianum by ultra high performance liquid chromatography-tandem mass spectrometry using a procedure based on QuEChERS and dispersive liquid-liquid microextraction. J. Chromatogr. A 1282: 11-19. Available online at http://www.ncbi.nlm.nih. gov/pubmed/23415469; cited on 25.08.2013.

  • Aziz N.H., Youssef Y.A., El-Fouly M.Z., Moussa L.A., 1998. Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins. Bot. Bull. Acad. Sin. 39: 279-285.

  • Cwalina-Ambroziak B., Wierzbowska J., Damszel M., Bowszys T., 2012. The effect of mineral fertilization on achenes yield and fungal communities isolated from the stems of milk thistle Silybum marianum (L.) Gaertner. Acta Sci. Pol., Hortorum Cultus 11(4): 157-168.

  • Ellis M.B., 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England.

  • Ellis M.B., 1976. More dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England.

  • Falleiro B.A.S., Almeida P.B.A., Cautinho W.M., Suassuna N.D., Kobayasti L., 2010. Use of osmotic solutions for inhibition of sunflower seed germination in blotter test. Trop. Plant Pathol. 35(6): 343-350. Available online at; cited on 25.08.2013.

  • Ghavani N., Ramin A.A., 2007. Salinity and temperature effects on seed germination of milk thistle. Commun. Soil Sci. Plan. 38: 2681-2691.

  • ISTA, 2012. International Rules for Seed Testing. Annexe to Chapter 7 Seed Health Testing, Seed Health Testing Methods. The International Seed Testing Association, Bassersdorf, Switzerland.

  • Kędzia B., 2000. Drogi zanieczyszczenia surowców zielarskich drobnoustrojami. Herba Polonica 56(1): 35-51. (In Polish) Logrieco A., Moretti A., Solfrizzo M., 2009. Alternaria toxins and plant diseases: an overview of origin, occurrence and risks. World Mycotoxin J. 2(2): 129-140.

  • Machado J.C., Cautinho W.M., Guimarães R.M., Vieira M.G.G.C., Ferreira D.F., 2008. Use of osmotic solutes to control seed germination of rice and common bean in seed health blotter tests. Seed Sci. Technol. 36(1): 66-75.

  • Machado J.C., Guimaraes R.M., Vieira M.G.G.C., Souza R.M., Pozza E.A., 2004. Use of water restriction technique in seed pathology. Seed Testing International 128: 14-18.

  • Machado J.C., Oliveira J.A., Vieira M.G.G.C., Alves M.C., 2003. Control of soybean seed germination in seed health tests using water restriction technique. Rev. Bras. Sementes 25(2): 77-81. Available online at; cited on 25.08.2013. (In Portuguese)

  • Machowicz-Stefaniak Z., Zimowska B., 2000. Grzyby przenoszone przez materiał siewny roślin zielarskich. Acta Agrobot. 53(2): 25-38. (In Polish) Malone J.P., Muskett A.E., 1964. Seed-borne fungi. Description of 77 fungus species. Proceeding of the International Seed Testing Association, 29 (2): 179-384.

  • Mathur S.B., Kongsdal O., 2003. Common laboratory seed health testing methods for detecting fungi. International Seed Testing Association, Basserdorf, Switzerland.

  • Maude R.B., 1980. Disease control. In: The biology of Botrytis. J.R. Colley-Smith, K. Verhoeff, W.R. Jarvis (eds), Academic Press Inc., London Ltd.

  • Maude R.B., 1996. Seed-borne diseases and their control. Principles and practice. CAB International, Wallingford, UK.

  • Morazonni P., Bombardelli E., 1995. Silybum marianum (Carduus marianus). Fitoterapia 66: 3-42.

  • Niaz I., Dawar S., 2009. Detection of seed-borne mycoflora in maize (Zea mays L.). Pak. J. Bot. 41(1): 443-451.

  • Sadowska K., 2006. Owoce ostropestu plamistego jako prozdrowotny dodatek do pieczywa. Food Sci.

  • Technol. Quality 2(47): 290-296. (In Polish) Sauer D.B., Burroughs R., 1986. Disinfection of seed surfaces with sodium hypochlorite. Phytopathology 76: 745-749.

  • Scheffer R., 1992. Ecological and evolutionary roles of the toxins from Alternaria species pathogenic to plants. In: Alternaria. Biology, plant diseases and metabolites. J. Chełkowski, A. Visconti (eds). Elsevier, Amsterdam: 101-122.

  • Seidler-Łożykowsk a K., 2009. Medicinal plant seeds as an element of increasing biodiversity of grassland. SALVERE - Regional Workshop in Poland: 45-47.

  • Sharfun-Nahar, Mushata q M., Hashmi M.H., 2005. Seed-borne mycoflora of sunflower (Helianthus annus L.). Pak. J. Bot. 37(2): 451-457.

  • Sultana N., Ghaffar A., 2007. Seed-borne fungi associated with bitter-gourd (Momordica charantia Linn.). Pak. J. Bot. 39(6): 2121-2125.

  • Szopińsk a D., Tylkowska K., Deng Ch.J., Gao Y., 2012.C omparison of modified blotter and agar incubation methods for detecting fungi in Zinnia elegans seeds. Seed. Sci. Technol. 40: 32-42.

  • Tournas V.H., Sapp C., Trucksess M.W., 2012. Occurrence of aflatoxins in milk thistle herbal supplements. Food Addit. Contam. 29(6): 994-999.

  • Wata nabe T., 2002. Pictorial atlas of soil and seed fungi: Morphologies of cultured fungi and key to species. Second Edition. CRC Press, Boca Raton, London, New York, Washington D.C.


Journal + Issues