Isolation of medically important fungi from Ginkgo biloba leaves and crude Ginkgo supplements

摘要

We observed fungal growth in Ginkgo biloba leaves in the middle of the growth season and isolated Phoma sp. and Curvularia sp. from the affected leaves. We also isolated Aspergillus sp. and Curvularia sp. from brown spots of apparently healthy leaves; Aspergillus sp., Chaetomium sp., Fusarium sp., Penicillium sp. and Aureobasidium sp. from tissue explants from brown spots of Ginkgo tea flakes (from three different suppliers); and Chaetomium sp., Penicillium sp., Aspergillus sp. and Aureobasidium pullulans from cold-brewed Ginkgo flake tea (from one supplier). Fungus was not recovered from any of the eight samples of hot-brewed (at 75°C or 90°C for 5 minutes) Ginkgo tea tested, cold- or hot-brewed powdered Ginkgo tea (from two suppliers) and three different brands of Ginkgo tea bags. This data indicated that G. biloba leaves and crude supplements derived from crushed Ginkgo leaves could be contaminated by fungi known to be opportunistic pathogens. Introduction Ginkgo biloba is among the most ancient surviving trees. The tree is remarkably resistant to microbial infections (Major, 1967; Huang et al., 2000; Mazzanti et al., 2000). While examining genetic diversity of Ginkgo trees introduced to the United States (Kuddus et al., 2002) we observed fungal growth in prematurely dying leaves of some adult Ginkgo trees. We also observed brown spots and mildew-like growth in the leaves of Ginkgo trees (all in the middle of Gingko growth season) and isolated fungi from the affected tissues.Various G. biloba preparations are used in complementary and alternative medicines (CAM) as well as (experimental) conventional medicine (reviewed in DeFeudis, 1998; Sierpina et al., 2003; Kuddus, 2005; Carlson et al., 2007). Ginkgo products come as crude preparations such as Ginkgo tea and capsules or in the form of pharmacologically standardized extracts. Crude supplements such as Ginkgo tea are made simply by crushing dried Ginkgo leaves. Thus microbes (and their toxins) present in leaf tissues may also be present in the supplements. Since G. biloba is known to be highly resistant to microbial infections, infection or intoxication risks from consumption of G. biloba supplements could be underestimated. Use of CAM has increased significantly and G. biloba preparations are among the highest selling CAM (Eisenberg et al., 1998; Jones, 2007). Here we describe isolation of fungi, including some known opportunistic pathogens, from both live and prematurely dying G. biloba leaves and crude G. biloba supplements. Materials and methods Field workTwo urban G. biloba populations, one located at Pittsburgh, Pennsylvania (Lat 40° 21' N, Long 79° 55' W, Alt 382 meter) and another at Orem, Utah (Lat 40° 17' N, Long 111° 41' W, Alt 1448 m) were observed year-round for about two years (June 2003 to May 2006). G. biloba leaves emerge in February-March and become senescent by late October to early November in both regions. Trees with excessive numbers of prematurely dry leaves before August and green leaves with visible fungal growth (before September) were collected and inspected by stereomicroscope. Loose Ginkgo tea flakes (from three suppliers), loose powdered tea (from two suppliers) and individually bagged Ginkgo tea (three brands) were purchased from local health food stores (between June 2005 and June 2007).Tissue Staining for Detection and isolation of fungiAffected leaves, tea flakes and healthy leaves (as control) were cleared of superficial microbes by boiling in 2.5% KOH. The treated leaves were stained with trypan blue and counter-stained with Sudan IV to detect intercellular fungi as described previously (Barrow and Aaltonen, 2004). The treated samples were examined using a Labomed CXR3 microscope at 400-1,000x magnification and photographed. At least 20 fields were examined for each leaf sample. To isolate fungi, leaves or tea flakes were initially cleaned using sodium hypochlorite, distilled water and ethyl alcohol as described by Tuite (1969). Tissue e