European Journal of Plant Pathology 105: 733–741, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. Colletotrichum acutatum and C. gloeosporioides cause anthracnose on olives M.P. Mart´ ın 1 and F. Garc´ ıa-Figueres 2 1 Departament de Biologia Vegetal (Bot` anica), Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain; 2 Unitat Sanitat Vegetal, Servei de Sanitat Agr` aria. Via Circulaci´ o Nord, Tram VI, Cant. C/3, Zona Franca, 08040 Barcelona, Spain Accepted 21 July 1999 Key words: Colletotrichum, Olea europaea, anthracnose, RFLP-ITS rDNA, sequences Abstract Morphological and cultural features and restriction fragment length polymorphism analysis of ITS regions, including 5.8S rDNA, from 26 isolates of Colletotrichum species revealed that isolates from olive fruits, previously identified as C. gloeosporioides, belong to two taxa: C. acutatum and C. gloeosporioides. Comparison of both ITS sequence data with reference isolates confirmed the presence of both species in olives affected by anthracnose disease. Introduction Olive trees (Olea europaea L.) in the south of Catalonia (Spain), are affected by anthracnose disease. The causal agent of this disease has been described as Gloeospo- rium olivarum Alm. However, von Arx (1970) and Sutton (1980, 1992) included it in the species complex Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., anamorph of Glomerella cingulata (Ston.) Sp. & Schr., which has been considered the cause of anthracnose on a wide range of plants. Based on conidial morphology and cultural characters, Garc´ ıa et al. (1996) established two groups from the isolates of Colletotrichum from olives in Spain: type N (strains with grey to brown cul- tures and fast growth) and type T (strains with whitish to salmon cultures and slow growth). These two groups cause the same anthracnose symptoms. The main objective of this study was to establish an easy and reliable method to identify the causal agents of anthracnose in olives. To achieve this, isolates from olive fruits, previously identified as C. gloeosporioides, were compared with those derived from different hosts, using not only morphological and cultural characters, but also molecular tools (RFLP and sequencing of ITS rDNA). Molecular techniques, such as arbitrarily primed PCR (Freeman and Rodriguez, 1995), RFLPs and sequence analyses, have been used to resolve the systematic problems in other Colletotrichum species (Sherriff et al., 1995). Materials and methods Fungal strains Field-collected material was cultured on potato dex- trose agar (PDA, ADSA micro, Ref. 1– 438) prior to DNA isolation to confirm the identity of the taxa. Orig- inal identification, host and source of the isolates exam- ined are given in Table 1. In pathogenicity tests all the isolates from olive were able to cause anthracnose symptoms on olive fruits (Garc´ ıa, 1994). Reference iso- lates of C. acutatum, C. fragariae and C. gloeospori- oides from the ‘Colecci ´ on Espa˜ nola de Cultivos Tipos’ (CECT) were also included in this study. Mycelium of each isolate was grown on three aqueous media at 28 ◦ C on casein hydrolysis medium (CHM) prepared according to Paterson and Bridge (1994) (this medium become opaque when cold) for 72 h to determine pro- tease activity and on PDA and Czapek-Dox medium (BIOLIFE S.r.l., code 1360) to assess growth rate.