Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: February 3, 2011
Publication Date: February 1, 2012
Citation: Klosterman, S.J. 2012. Real-time PCR for the quantification of fungi in planta. In: Bolton, M.D., Thomma, B.P.H.J., editors. Plant Fungal Pathogens: Methods and Protocols. Series: Methods in Molecular Biology. Volume 835. New York, NY: Humana Press. p. 121-131. Technical Abstract: Methods enabling quantification of fungi in planta can be useful for a variety of applications. In combination with information on plant disease severity, indirect quantification of fungi in planta offers an additional tool in the screening of plants that are resistant to fungal diseases. In this chapter, a method is described for the quantification of DNA from a fungus in plant leaves using real-time PCR (qPCR). Although the method described entails quantification of the fungus Verticillium dahliae in lettuce leaves, the methodology described would be useful for other pathosystems as well. The method utilizes primers that are specific for amplification of a b-tubulin sequence from V. dahliae and a lettuce actin gene sequence as a reference for normalization. This approach enabled quantification of V. dahliae in the amount of 2.5 fg/ng of lettuce leaf DNA at 21 days following plant inoculation.