Real-Time PCR for the Quantification of Fungi In Planta

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 β-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.