Skip to main content

Use of the polymerase chain reaction to help determine the presence of blackpatch (Rhizoctonia leguminicola) in inoculated red clover leaves

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.


Rhizoctonia leguminicola, the traditional name for the causal agent of blackpatch of red clover (Trifolium pratense) and other legumes, produces alkaloids, one of which causes livestock to salivate excessively. Fungal presence is generally confirmed by microscopy, disappearance of symptoms in livestock after removal of suspect forage, and chromatography of the alkaloid slaframine, in legume tissue. Use of the polymerase chain reaction (PCR) to amplify a pathogen-specific DNA fragment would complement the other methods of pathogen identification. Primers were designed to the R. leguminicola ITS region, sequence provided by another laboratory. Two separate primer pairs each amplified a different fragment–one ~250 bp long (expected length 249 bp), and the other 300 to 400 bp long (expected length 368 bp)–in DNA extracted from cultures of R. leguminicola. Under the experimental conditions, the primers to the larger fragment amplified a stronger band, and a minimum of 0.1 ng DNA per reaction was needed to produce a detectable band. With the primers to the 368-bp fragment, a band 300 to 400 bp long was also amplified in DNA extracted from red clover (cultivar Kenland) inoculated with R. leguminicola and harvested 70 h post inoculation. No amplification with this primer set occurred in DNA extracted from mock-inoculated red clover plants, supporting the likelihood that the primers amplified R. leguminicola DNA extracted from inoculated red clover. This primer set did not amplify DNA extracted from a red clover isolate of the legume pathogen Stemphylium sarcinaeforme, or DNA extracted from two isolates of the legume pathogen Colletotrichum trifolii, indicating specificity for R. leguminicola DNA. Lack of amplification of alfalfa DNA indicated that the R. leguminicola primers will be useful for testing for the presence of blackpatch in alfalfa.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  • Alhawatema, M. S., Sanogo, S., Baucom, D. L., & Creamer, R. (2015). A search for the phylogenetic relationship of the ascomycete Rhizoctonia leguminicola using genetic analysis. Mycopathologia, 179, 381–389.

    Article  PubMed  Google Scholar 

  • Andersen, T. F., & Stalpers, J. A. (1994). A check-list of Rhizoctonia epithets. Mycotaxon, 51, 437–457.

    Google Scholar 

  • Aust, S. D., Broquist, H. P., & Rinehart Jr., K. L. (1968). Slaframine: a parasympathomimetric from Rhizoctonia leguminicola. Biotechnology and Bioengineering, 10, 403–412.

    Article  CAS  Google Scholar 

  • Berkenkamp, B. (1977). Blackpatch of forage legumes. Canadian Plant Disease Survey, 57, 65–67.

    Google Scholar 

  • Borges, A. S., Oliveira-Filho, J. P., Palumbo, M. I. P., Simon, J. J., & Imerman, P. M. (2012). Slaframine toxicosis in Brazilian horses causing excessive salivation. Equine Veterinary Education, 24, 279–283.

    Article  Google Scholar 

  • Cook, D., Ralphs, M. H., Welch, K. D., & Stegelmeier, B. L. (2009). Locoweed poisoning in livestock. Rangelands, 31, 16–21.

    Article  Google Scholar 

  • Cook, D., Gardner, D. R., & Pfister, J. A. (2014). Swainsonine-containing plants and their relationship to endophytic fungi. Journal of Agricultural and Food Chemistry, 62, 7326–7334.

    Article  CAS  PubMed  Google Scholar 

  • Croom Jr., W. J., Hagler, W. M., Froetschel, M. A., & Johnson, A. D. (1995). The involvement of slaframine and swainsonine in slobbers syndrome: a review. Journal of Animal Science, 73, 1499–1508.

    Article  CAS  PubMed  Google Scholar 

  • Farr, D.F., & Rossman, A.Y. (2015). Fungal databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved September 2015 from

  • Fonteque, J. H., Souza, A. H., Schade, J., Muller, T. R., & Weinert, N. C. (2015). Slaframine intoxication in horse in Santa Catarina state. Veterinaria e Zootecnia, 22, 375–379.

    Google Scholar 

  • Hagler, W. M., & Behlow, R. F. (1981). Salivary syndrome in horses: identification of slaframine in red clover hay. Applied and Environmental Microbiology, 42, 1067–1073.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hoeksema, J. D., Hernandez, J. V., Rogers, D. L., Luna Mendoza, L., & Thompson, J. N. (2012). Geographic divergence in a species-rich symbiosis: interactions between Monterey pines and ectomycorrhizal fungi. Ecology, 93, 2274–2285.

    Article  PubMed  Google Scholar 

  • Leach, J. G., & Elliott, E. S. (1951). The blackpatch disease of red clover and other legumes in West Virginia. Phytopathology, 41, 1041–1049.

    Google Scholar 

  • O’Dell, B. L., Regan, W. O., & Beach, T. J. (1959). A study of the toxic principle in red clover. University of Missouri Research Bulletin, 702, 1–12.

    Google Scholar 

  • Riet-Correa, F., Rivero, R., Odriozola, E., de Lourdes Adrien, M., Medeiros, R. M. T., & Schild, A. L. (2013). Mycotoxicoses of ruminants and horses. Journal of Veterinary Diagnostic Investigation, 25, 692–708.

    Article  PubMed  Google Scholar 

  • Sanderson, J.M. (1985). Rhizoctonia leguminicola: studies on the production of slaframine and swainsonine in the field, host plant susceptibility, and isolate variability. M.S. Thesis. University of Wisconsin-Madison.

  • Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences USA, 109, 6241–6246.

    Article  CAS  Google Scholar 

  • Smith, O. F. (1937). A leaf spot disease of red and white clovers. Journal of Agricultural Research, 54, 591–599.

    Google Scholar 

  • Sockett, D. C., Baker, J. C., & Stowe, C. M. (1982). Slaframine (Rhizoctonia leguminicola) intoxication in horses. Journal of the American Veterinary Medical Association, 181, 606.

    CAS  PubMed  Google Scholar 

  • Taylor, N. L., & Quesenberry, K. H. (1996). Red clover science. Dordrecht: Kluwer Academic Publishers.

    Book  Google Scholar 

  • Wijnberg, I. D., van der Ven, P. J., & Fink-Gremmels Gehrmann, J. (2009). Outbreak of salivary syndrome on several horse farms in the Netherlands. Veterinary Record, 164, 595–597.

    Article  CAS  PubMed  Google Scholar 

Download references


I thank the R. Creamer laboratory for providing the sequences of GenBank accessions KM376908, KM376909, and KM376910 prior to release; R. Dinkins for designing primers and providing helpful suggestions; and D. Samac for providing DNA from Colletotrichum trifolii and Medicago sativa, as well as helpful suggestions. This project was funded by the United States Department of Agriculture.

USDA is an equal opportunity employer and provider.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Isabelle A. Kagan.

Ethics declarations


Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kagan, I.A. Use of the polymerase chain reaction to help determine the presence of blackpatch (Rhizoctonia leguminicola) in inoculated red clover leaves. Eur J Plant Pathol 147, 1–6 (2017).

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: