An antifungal and plant growth promoting metabolite from a sterile dark ectotrophic fungus Hyun-Ju Kim a , Francesco Vinale b , Emilio L. Ghisalberti c, * , Carol M. Worth d , Krishnapillai Sivasithamparam d , Brian W. Skelton c , Allan H. White c a Faculty of Natural Resources and Life Science, Dong-A University, Busan 604-714, South Korea b Dipartimento di Arboricultura, Botanica e Patologia Vegetale, Universita degli Studi di Napoli ‘Federico II’, Portici, Naples, Italy c School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia d School of Earth and Geographical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Received 12 June 2006; received in revised form 18 July 2006 Available online 30 August 2006 Abstract A sterile dark ectotrophic fungus isolated from roots of an Australian native grass, Neurachne alopecuroidea produces compound 1 in liquid cultures. The structure of the metabolite was determined by spectroscopic and X-ray diffraction studies. The metabolite shows activity against phytopathogens and plant growth promoting activity, properties that are also expressed in vivo by the ectotrophic fungus. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Ectotrophic fungus; Neurachne alopecuroidea; Antifungal; Gaeumannomyces graminis var. tritici; Plant growth promoter 1. Introduction Take-all, the most important root disease of wheat worldwide, is caused by the fungus Gaeumannomyces gra- minis var. tritici (Ggt) (Freeman and Ward, 2004). A num- ber of fungi with ectotrophic growth habits on roots, such as Phialophora spp., have been reported to confer cross- protection to cereals against Ggt (Deacon, 1981; Wong, 1981; Dewan and Sivasithamparam, 1988). Moreover, var- ious sterile fungi isolated from roots and rhizosphere of grasses and cereals have been shown to be beneficial asso- ciates of some plant species (Dewan and Sivasithamparam, 1989; Newsham, 1999). In continuation of our work on the identification of fungi with potential as biocontrol agents, we have investigated the metabolites produced by a strain (SDEF 678) that occurs with high frequency in the roots of an Australian native grass, Neurachne alopecuroidea. This isolate inhibited growth of Ggt in dual plate antago- nism tests and significantly increased the root growth of wheat seedlings grown in natural soil (Worth, 2002). We now report the isolation and structural determination of a metabolite produced in liquid cultures of SDEF 678 that exhibits antifungal and plant growth promoting activity. 2. Results and discussion Extraction of liquid cultures of SDEF 678 with ethyl acetate yielded a dark brown oily residue. The extract was partitioned by flash chromatography (silica gel) with gradient elution into 15 fractions. One of the less polar fractions (F4) afforded a crystalline material that was homogeneous on tlc and which was assigned structure 1 on the following evidence. The HR-FABMS spectrum of 1 showed the [M + H] + ion at m/z 191.069283 correspond- ing to a molecular formula of C 11 H 10 O 3 . The presence of a cyclohex-2,3-enone group was suggested from the 13 C NMR spectrum (d C 189.7, s; 126.7, d; 144.0, d) and the 1 H NMR spectrum (6.03, d, J = 10.5 Hz; 6.65, ddd, J = 10.5, 4.7, 2.6 Hz). The proton resonating at d H 6.65 showed coupling (4.7 Hz) to an oxymethine proton at d H 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.07.022 * Corresponding author. Tel.: +61 08 6488 3174; fax: +61 08 6488 1105. E-mail address: elg@chem.uwa.edu.au (E.L. Ghisalberti). www.elsevier.com/locate/phytochem Phytochemistry 67 (2006) 2277–2280 PHYTOCHEMISTRY