Extraction of Verticillium microsclerotia from soil

General introduction

In this project we develop an alternative, fast and more reliable method to detect, identify and quantify Verticillium microsclerotia in soil. Verticillium is a plant pathogenic fungus and typically forms microsclerotia in soil. Microsclerotia are survival structures that are a primary inoculum for the plant. Currently, wet-sieving of soil samples, followed by plating on semi-selective medium and microscopic analysis, is the most commonly used technique to quantify microsclerotia-forming Verticillium species in soil. However, the method (1) is restricted to small soil samples, (2) does not allow easy differentiation between species, and (3) takes at least 4 weeks to complete. With our new method we want to counteract these disadvantages. Specifically, we want to be able to differentiate among the three species possibly present in soil, because each species has a different host range.

Research approach

Our goal is twofold. On the one hand, we develop real-time PCR assays allowing  differentiation between the different Verticillium species (V. tricorpus, V. longisporum, & V. dahliae). On the other hand, we develop a sucrose flotation-based method enabling us to analyse high amounts of soil (100 g).

Relevance/Valorisation

The new technique, sucrose flotation combined with real-time PCR, is able to detect the presence, species and amount of Verticillium microsclerotia in a more specific, more sensitive and faster way than the widely used wet-sieving plating method. The new technique was published in a scientific journal as: " Debode, J., Van Poucke, K., França, S. C., Maes, M., Höfte, M., and Heungens, K. 2011. Detection of multiple Verticillium species in soil using density flotation and real-time polymerase chain reaction. Plant Dis. 95:1571-1580” and implemented in the ILVO diagnostic centrum for plants (www.ilvodiagnosecentrumvoorplanten.be). This enables ILVO to give faster and more specific advice to the growers (in what degree a specific Verticillium species has contaminated one field).