4.2
Research work on the increased occurrence of Verticillium infections
Objective Hop wilt, caused by the soil fungi Verticillium albo-atrum and, less often, Verticillium dahliae, currently poses a major challenge to hop growers and hop researchers alike. The incidence of hop wilt on hop farms in the Hallertau has been increasing since 2005. Even varieties such as Northern Brewer and Perle, previously classified as Verticillium-tolerant, have been affected since then. Virulence tests on Verticillium strains found in the Hallertau, performed via artificial infection (Radišek et al., 2006), and, in particular, molecular biological techniques (Seefelder et al., 2009) have shown that not only mild but also highly aggressive Verticillium strains have now spread to Germany (Maurer et al., 2014). Whereas the Hüll breeding lines are able to tolerate attacks by mild strains of the hop-wilt fungus, these highly aggressive Verticillium strains kill off all the currently available Hüll hop varieties, including their roots (which is why they are often called “lethal” strains). As no plant protective agents are available for combating Verticillium, other solutions must be found to help hop growers in Germany protect their crops from the huge threat posed by Verticillium wilt. One successful approach resulting from systematic research work has been the establishment of a molecular test which enables the fungus to be detected directly from hop bines (“in-planta test”) (Maurer et al., 2013) and with which healthy, Vertcillium-free planting stock can be identified very reliably and relatively quickly. V. albo-atrum and V. dahliae are listed as harmful organisms (Council Directive 2000/29/29) and are regarded worldwide as high-risk pathogens. This molecular detection system is therefore of major importance and has already proved to be a highly successful tool. It is used, for example, to guarantee that planting stock provided by the Hüll Hop Research Centre for further propagation is free of Verticillium. The method is also an integral part of all research activities related to the Verticillium fungus. Material and methods Molecular detection of Verticillium The lower section of hop bines and, in special cases (infection tests, in particular on propagation material), also leaves and lateral stems were tested for Verticillium infection using the molecular in-planta test (Maurer et al. 2013a). The specially developed real-time PCR assay (Maurer et al., 2013) permitted simultaneous detection of Verticillium albo-atrum and Verticillium dahliae. The PCR was preceded by DNA isolation (hop DNA + fungal DNA) directly from hop bines using the Invisorb Spin Plant Mini Kit (Invitek) and a homogeniser (MP Biomedicals). All the plants tested were sampled in duplicate and thus tested twice. In each real-time PCR assay, positive control (I) (Verticillium-DNA) and positive control (II) (in-planta DNA from an infected hop plant) were analysed simultaneously with the sample under test, as shown in Fig. 4.4. A special primer (Seefelder, 2014) developed by the Work Group and endorsed by Dr. Radisek in a personal communication was used to distinguish between mild and aggressive forms of Verticillium.
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