Molecular Diagnostics in the Mycosphaerella Leaf Spot Disease Complex of Banana and for Radopholus similis M. Arzanlou 1,2 , G.H.J. Kema 3 , C. Waalwijk 3 , J. Carlier 4 , I. de Vries 3 , M. Guzmán 5 , M. Araya Vargas 5 , J. Helder 6 and P.W. Crous 1,2 1 Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands 2 CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 3 Plant Research International BV, P.O. Box 16, 6700 AA Wageningen, The Netherlands 4 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR-BGPI, TA41/K, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France 5 CORBANA S.A., P.O. Box 6504-1000 San José, Costa Rica 6 Laboratory of Nematology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands Keywords: detection, quantification, Sigatoka, Mycosphaerella, Radopholus, real-time PCR Abstract Mycosphaerella leaf spots and nematodes threaten banana cultivation worldwide. The Mycosphaerella disease complex involves three related ascomycetous fungi: Mycosphaerella fijiensis, M. musicola and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, since their symptoms and life cycles are rather similar. Diagnosing these diseases and the respective causal agents is based on the presence of host symptoms and fungal fruiting structures, but is time consuming and not conducive to preventive management. In the present study, we developed rapid and robust species-specific diagnostic tools to detect and quantify M. fijiensis, M. musicola and M. eumusae. Conventional species-specific PCR primers were developed based on the actin gene that detected as little as 100, 1 and 10 pg/µl DNA from, respectively, M. fijiensis, M. musicola and M. eumusae. Furthermore, TaqMan real-time quantitative PCR assays that were developed based on the β-tubulin gene detected quantities as low as 1 pg/µl DNA of each species from pure cultures and 1.6 pg/µl DNA/mg of M. fijiensis from dry leaf tissue. The efficacy of the tests was validated using naturally infected banana leaves. Similar technology has been used to develop a quantitative PCR assay for the banana burrowing nematode, Radopholus similis, which is currently being validated. INTRODUCTION Banana represents a very important staple food commodity in the tropics after wheat, rice and corn (Marin et al., 2003; Ploetz, 2000). Banana plants are susceptible to a variety of devastating diseases, including those in the Mycosphaerella leaf spot complex, caused by three related ascomycetous fungi. Mycosphaerella fijiensis (anamorph = Pseudocercospora fijiensis) causes black leaf streak or black Sigatoka disease, M. musicola (anamorph = Pseudocercospora musae) is responsible for Sigatoka disease (commonly known as Yellow Sigatoka) and M. eumusae (anamorph = Pseudocercospora eumusae) causes eumusae leaf spot disease. The diseases represent a serious threat to banana production worldwide. They cause necrotic leaf lesions, thus reducing the photosynthetic capacity of plants resulting in reduced crop yield and premature fruit ripening (Jones, 2002). Sigatoka leaf spot was first reported from Java early last century, then spread to banana-producing areas in Asia, Africa and South America during the 1940s. In the early 1960s, M. fijiensis was identified in Fiji. It later spread to replace M. musicola as the most important constraint in many banana-producing areas. However, Mycospaerella musicola 237 Proc. IS on Banana Crop Prot., Sust. Prod. & Impr. Livelihoods Eds.: D. Jones and I. Van den Bergh Acta Hort. 828, ISHS 2009