Penicillium digitatum Metabolites on Synthetic Media and Citrus Fruits MARTA R. ARIZA, ² THOMAS O. LARSEN, ‡ BENT O. PETERSEN, § JENS Ø. DUUS, § AND ALEJANDRO F. BARRERO* ,² Department of Organic Chemistry, University of Granada, Avdn. Fuentenueva 18071, Spain; Mycology Group, BioCentrum-DTU, Søltofts Plads, Technical University of Denmark, 2800 Lyngby, Denmark; and Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark Penicillium digitatum has been cultured on citrus fruits and yeast extract sucrose agar media (YES). Cultivation of fungal cultures on solid medium allowed the isolation of two novel tryptoquivaline-like metabolites, tryptoquialanine A (1) and tryptoquialanine B (2), also biosynthesized on citrus fruits. Their structural elucidation is described on the basis of their spectroscopic data, including those from 2D NMR experiments. The analysis of the biomass sterols led to the identification of 8-12. Fungal infection on the natural substrates induced the release of citrus monoterpenes together with fungal volatiles. The host-pathogen interaction in nature and the possible biological role of citrus volatiles are also discussed. KEYWORDS: Citrus fruits; Penicillium digitatum; quivaline metabolites; tryptoquivaline; P. digitatum sterols; phenylacetic acid derivatives; volatile metabolites INTRODUCTION P. digitatum grows on the surface of postharvested citrus fruits producing a characteristic powdery olive-colored conidia and is commonly known as green-mold. This pathogen is of main concern as it is responsible for 90% of citrus losses due to diseases occurring during the storage period, and it causes serious damages in commerce (1). Most of the research works on P. digitatum focus on treatments against the infection symptoms, and literature about fungal metabolites is scarce (2, 3). To extend the study of P. digitatum metabolites and their possible role in the toxigenesis, as well as to know more about medium-pathogen relation, the microorganism has been cul- tured on natural and synthetic media. Several metabolites have been isolated and studied by NMR. MATERIALS AND METHODS General Experimental Procedures. NMR spectra were recorded in DMSO on a Bruker DRX 600, at 600.13 MHz for 1 H and 150.92 MHz for 13 C according to Larsen et al. (4). The chemical shifts are given relative to DMSO, 2.50 ppm for H 1 and 39.5 ppm for C 13 (Table 1). The circular dichroism (CD) spectra were measured on a JASCO J-710 spectropolarimeter and the UV spectra were measured on a Hewlett-Packard 8452A diode array spectrophotometer. Analytical HPLC conditions were similar to those given by Smedsgaard (5), and retention indices (RI) of fungal metabolites were calculated according to Frisvad and Thrane (6). EIMS for the study of citrus volatiles were obtained from a Hewlett-Packard 5972A mass spectrometer using an ionizing voltage of 70 eV, coupled to a Hewlett-Packard 5890A gas chromatograph. Preparation of TMS Derivatives and Analysis by GC-MS. TMS derivatives were obtained according to the procedure previously described (7). The samples (1 µL) were injected onto a 25 m × 0.32 mm i.d. HP-1 methylsilicone capillary column with a He flow rate of 0.6 mL min -1 . The temperature program for the study of sterols and oxygenated triterpenes went from 120 °C to 220 °C at 5 °C min -1 , from 220 °C to 280 °C at 3 °C min -1 , and 10-min hold at 280 °C. Identification of the sterols and oxygenated triterpenes from P. digitatum biomass has been made by interpretation of the characteristic mode of fragmentation of their TMS-ethers when subjected to GC-MS (7-9), and by comparison of RI and mass spectra with those from standards. Fungal Material and Fermentation. P. digitatum IBT 10051 and IBT 21830 isolates, (ex Mandarin), were obtained from the fungal culture collection (IBT) at BioCentrum-DTU, at The Technical University of Denmark. P. digitatum Cultured on YES Liquid Medium. IBT 10051 was cultured on YES liquid medium, (20 g of yeast extract and 150 g of sucrose in 1000 mL of distilled water). The fungus was inoculated into 50 500-mL Erlenmeyer flasks (100 mL of YES medium per flask) and grown in the dark with shaking at 24 °C for 15 days. P. digitatum Cultured on YES Solid Medium. IBT 21830 was cultured for 14 days at 25 °C in the dark, as three-point mass inoculation on 200 YES agar plates (2). P. digitatum Cultured on Citrus Fruits. IBT 21830 was cultured on citrus fruits (Valencia oranges) for production of both volatile and nonvolatile metabolites. The citrus fruits were inoculated at several points and cultivated at 25 °C. Collection of Volatile Metabolites. Volatile metabolites were collected by diffusive sampling onto Tenax TA and analyzed by GC- * To whom correspondence should be addressed. Phone: 958 24 33 20. Fax: 958 24 33 20. E-mail: afbarre@goliat.ugr.es. ² University of Granada. ‡ University of Denmark. § Carlsberg Research Laboratory. J. Agric. Food Chem. 2002, 50, 6361-6365 6361 10.1021/jf020398d CCC: $22.00 © 2002 American Chemical Society Published on Web 09/20/2002