Viridepyronone, a New Antifungal 6-Substituted 2H-Pyran-2-one Produced by Trichoderma viride ANTONIO EVIDENTE,* ,† ANNALISA CABRAS, ‡ LUCIA MADDAU, ‡ SALVATORICA SERRA, ‡ ANNA ANDOLFI, † AND ANDREA MOTTA § Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Universita ` di Napoli Federico II, 80055 Portici, Italy, Dipartimento di Protezione delle Piante, Universita ` di Sassari, 07100 Sassari, Italy, and Istituto di Chimica Biomolecolare del CNR, 80078 Pozzuoli, Italy A new antifungal 6-substituted 2H-pyran-2-one, named viridepyronone, has been isolated from a cultural filtrate of a strain of Trichoderma viride showing antagonistic activity in vitro toward Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridepyronone was characterized as 6-(4-oxopentyl)-2H-pyran-2-one 2 with spectroscopic methods. Bioassays showed that viridepy- ronone had a good antifungal activity against S. rolfsii, and its minimum inhibitory concentration (over 90% inhibition) was found to be 196 µg/mL. This is the first report of viridepyronone produced by any species of fungi. KEYWORDS: Trichoderma viride; pyran-2-one; viridepyronone; antifungal activity; Sclerotium rolfsii; crown and stem rot; biocontrol INTRODUCTION Seeking fungi suitable for the biological control of soil-borne plant pathogens, a strain of Trichoderma Viride was found that showed antagonistic activity, in Vitro and in ViVo, toward Sclerotium rolfsii, the causal agent of crown and stem rot of artichoke (1, 2). The antagonistic activity exhibited Tricho- derma spp. strains may in part be explained by the production of different classes of bioactive metabolites, including antibiot- ics, which are inhibitors of fungal growth and enzymes (3-5). We have previously isolated and characterized isoharziandi- one, a new tetracyclic diterpene, from the culture filtrates of strain IPVS 1817 of T. Viride able to inhibit fungal growth of S. rolfsii (6). Here we report on a new metabolite produced in liquid cultures by the above T. Viride strain and on the isolation and chemical characterization of viridepyronone, structurally related to 6-n-pentyl-2H-pyran-2-one. This paper is the first report on viridepyronone produced by fungi. MATERIALS AND METHODS Fungal Strains. Trichoderma Viride was isolated from forest soil collected in Sardinia (Italy) and deposited at the collection of the Dipartimento di Protezione delle Piante, Universita ` degli Studi di Sassari, Italy, as IPVS 1817. Slant cultures on potato dextrose agar (PDA) of the fungus were stored in a refrigerator at 4 °C. Sclerotium rolfsii was originally isolated from infected plants of artichoke in Sardinia, and was maintained on PDA in 9-cm-diameter Petri dishes under ambient conditions. Fermentation. Conidial suspensions (2 mL) of T. Viride were inoculated into 50 Roux flasks each containing 150 mL of Czapek medium fortified with 5% yeast extract (pH 5.9). The stationary cultures were incubated for 21 days at 25 °C in the dark. The cultures were filtered under vacuum through filter paper (Whatman N. 1), and the filtrates stored at -20 °C until used for chemical analysis. Extraction and Purification of Antifungal Metabolites. The combined culture filtrate (5 L) was concentrated under reduced pressure to approximately one-quarter of its original volume, acidified to pH 5.0 with 2 N HCl, and extracted exhaustively with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure at 30 °C to give a red-brown oily residue (900 mg). The antifungal activity of the extract was determined at a concentration of 10 mg/mL against S. rolfsii by its direct (40 µL) application to the paper disk surface. The ethyl acetate extract was found to be active against S. rolfsii, and was then submitted to bioassay-guided fraction- ation through column (80 × 3 cm) chromatography on silica gel (100 g), eluted with a gradient of CHCl3-i-PrOH (100:1 f 1:2). The collected fractions (15 mL each) were monitored by TLC analysis and the resulting homogeneous fractions were combined into 10 groups, T 1-T10. All the fractions were screened for their antifungal activity against S. rolfsii as described below. The fractions T4,T5, and T7 were found the most active against S. rolfsii. Purification of fractions T4 (50 mg) and T5 (120 mg) by a com- bination of column chromatography and preparative silica gel TLC gave two known compounds, isoharziandione (6 mg/L) and 6-pentyl-R- pyrone (1, 11 mg/L), respectively, which showed chromatographic and specrtoscopic properties identical with those of standard samples (6, 8). The residue (30 mg) left from fraction T 7 was purified by two successive steps of preparative silica gel TLC eluted by CHCl3-i-PrOH (20:1 and 95:5 v/v, respectively) and yielded 4 mg of viridepyronone (2, 0.8 mg/L) as a homogeneous oil resistant to crystallization [R f 0.28, 0.31, and 0.68, by silica gel and reversed-phase TLC, eluent systems CHCl 3-i-PrOH (95:5), EtOAc-n-hexane (6:4), and EtOH-H2O (1:1), respectively]. * To whom correspondence should be addressed. Phone: +39 081 2539178. Fax: +39 081 2539186. E-mail: evidente@unina.it. † Universita ` di Napoli Federico II. ‡ Universita ` di Sassari. § Istituto di Chimica Biomolecolare del CNR. J. Agric. Food Chem. 2003, 51, 6957-6960 6957 10.1021/jf034708j CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003