Naphthoquinone Production by Fusariuaa Solan I from Blighted Citrus Trees: Quantity, Incidence and Toxicity1

Diseased fibrous roots of blighted citrus trees were collected from 12 locations throughout the Central Florida citrus growing area. Sixty-five isolates of Fusarium solani (Mart.) Appel & Wr. emend. Snyd. & Hans, cultured from these roots were examined for their ability to produce phytotoxic compounds. When grown as shake cultures on a mineral salts-glucose medium with NH4NO3 as the N source, all isolates produced varying quantities of cisand trans-dihydrofusarubin and fusarubin. These naphthoquinone com pounds were highly toxic to radish roots, inhibiting growth by 97-99% at 100 ppm. A number of isolates also produced other naphthoquinone derivatives, including marticin, isomarticin, and bostrycoidin. Among the isolates studied, there was no apparent geographical trend in total naphthoquinone production or type of compounds produced. A number of studies have suggested that the soilborne fungus Fusarium solani, when favored by certain soil condi tions or cultural practices, contributes to citrus blight symp toms (12, 15, 16). Severe root pruning, resulting in wilting and stunting, occurs when citrus seedlings are inoculated with F. solani (14). F. solani infection in the field is gen erally restricted to fibrous feeder roots, and it is rarely found in larger living roots (15). Although it does occur as a colonizer of rotted pioneer roots, the presence of these decayed larger roots is not considered essential to blight (13). Therefore, if F. solani is involved in blight, aboveground symptoms such as vessel plugging, zinc accumula tion, or leaf deficiency patterns may be a result of phytotoxins elaborated by the pathogen. Selected isolates of F. solani obtained from roots of blighted citrus trees have been studied in detail for their ability to produce toxins (1, 2). Preliminary examination of a small number of isolates revealed a large variation in the capacity for toxin synthesis. Several of these isolates produced a variety of naphthoquinone derivatives in rela tively high yield, some of which were phytotoxic when tested on citrus or radish roots (2). Eleven naphthoquinones have now been identified in culture filtrates of F. solani "^Mention o£ a trademark or proprietary product is for identification only and does not imply an endorsement or warranty of the product over other products which may also be suitable. Proc. Fla. State Hort. Soc. 96: 1983. from citrus (17). Variation in toxicogenic potential has been invoked as a possible explanation of differing pathogenicity among isolates tested on citrus seedlings (15). This survey was undertaken to determine the frequency with which F. solani isolates from blighted trees synthesized naphthoquinones on a standard culture medium, and the amount of specific toxins produced. Isolates obtained from different locations were examined for variations in total toxin synthesis and for yield of specific toxins. Materials and Methods Samples of fibrous roots were collected from blighted trees in 12 locations of the Central Florida citrus area. Collections were made from trees which exhibited moderate to severe symptoms, had no visible evidence of foot rot, and which were in groves where blight was prevalent. Steles of diseased fibrous roots, from which cortical tissue had sloughed naturally, were plated on a modified Komada's medium for isolation of F. solani (9). Cultures of emerging colonies were maintained on potato-dextrose agar (PDA) slants. Isolates were evaluated for toxin production by grow ing them on a previously described mineral salts-glucose liquid medium containing NH4NO3 as the N source (2). Flasks (200 ml of medium/500 ml Erlenmeyer) were inocu lated with spore suspensions obtained by releasing spores grown on PDA slants with sterile distilled water. Cultures were grown for 3 days at 27°C on a Junior Orbital shaker operating at 150 rpm. Cultures were then filtered through cheesecloth and the filtrate extracted twice with equal vol umes of ethyl acetate. The combined extracts were dried with sodium sulfate, reduced to approximately 8 ml on a vacuum rotary evaporator, made up to 10 ml with ethyl acetate in a volumetric flask, and stored at 4°C. Total naphthoquinone content and dihydrofusarubin content were determined with an Aminco DW-2a spectrophotometer. With the exception of bostrycoidin and anhydrofusarubin, all naphthoquinones identified from F. solani absorb at or near 304 nm (17). Absorption at this wave length was therefore taken as a measure of total naphtho quinone content, when compared to absorption o£ a known standard of crystalline naphthoquinones. Absorption at 394 nm was used to measure levels of dihydrofusarubins, since these are the only naphthoquinones from F. solani which absorb at this wavelength. Absorptions were compared to a standard curve prepared with pure Jrans-dihydrofusarubin. Fusarubin and marticin levels were determined by thin-layer chromatography (TLC) of extracts on 250/xm silica gel GF plates. Plates were developed in benzene-nitromethane-acetic acid (150-50-4) and compared to similarly run dilution series