Metabolites from the plant pathogen Alternaria brassicicola: in vitro and in planta production and biosynthesis of brassicicolin A
The phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire together with A. brassicae cause Alternaria black spot (also called dark leaf spot) in Brassica species. During infection, A. brassicicola produces various secondary metabolites to facilitate its colonization and invasion. A systematic investigation of the secondary metabolites produced by A. brassicicola was carried out. In addition to previously reported metabolites, four metabolites, α-acetylorcinol (210), tyrosol (219), depudecin (151) and dihydrobrassicicolin A (220) were isolated and characterized from culture extracts of A. brassicicola for the first time. Chemical synthesis of α-acetylorcinol (210) was accomplished for the first time with an overall yield of 36%. Neither α-acetylorcinol (210) nor tyrosol (219) caused necrotic lesions on the leaves of or displayed elicitor activity in Brassica juncea, B. napus, or Sinapis alba. A. brassicicola was incubated under different sets of culture conditions, and the culture filtrates were extracted and analyzed by HPLC-DAD. The production of brassicicolin A (1), depudecin (151) and α-acetylorcinol (210) was increased in low concentrations of KNO3 or NaNO3, or in the absence of nitrates. These results suggest that nitrate inhibits the biosynthesis of brassicicolin A (1), depudecin (151), and α-acetylorcinol (210). In the iron deficient conditions, A. brassicicola produced larger amounts of siderophores than in control cultures (ferric citrate 2 µM) or in high concentration of ferric citrate (200 µM). Moreover, spore germination fluids (SGFs) collected from detached leaves of B. juncea, B. napus, and S. alba contained fungal metabolites (i.e., siderophores and phomapyrone A (25)). These results suggest that siderophores may play a role in fungal colonization by obtaining iron from host plants. In addition, HPLC analyses of extracts of the detached leaves of B. juncea, B. napus, and S. alba infected with A. brassicicola revealed that phomapyrone A (25), a fungal metabolite and non-phytotoxin, was detected and quantified for the first time. Brassicicolin A (1) is a host-selective phytotoxin produced by A. brassicicola. The biosynthetic precursors of brassicicolin A (1) were investigated using the following isotopically labeled compounds: D-[U-13C6]glucose, L-[15N]valine, and L-[2H8]valine. The labeled brassicicolin A (1e, 1j or 1m) was obtained from the extracts of culture filtrates of A. brassicicola obtained from incubation of cultures with labeled precursors. The 13C NMR and INADEQUATE spectra of 13C-enriched brassicicolin A (1e) obtained showed 13C-13C coupling resulting from adjacent 13C carbons. Enhancement of the isocyanide carbon resonances of the 13C-enriched brassicicolin A (1e) suggested that the isocyanide carbons are labeled. Furthermore, spectroscopic data including 1H NMR, 15N NMR and HR-ESI-MS of 2H-enriched (1j) or 15N-enriched brassicicolin A (1m) indicated that both 2-hydroxy-3-methylbutanoyl and isocyanoisovaleryl units are derived from valine.
α-acetylorcinol, Alternaria brassicicola, HDAC inhibitors, host-selective, phomapyrone A, phytotoxins, secondary metabolites, siderophores, spore germination fluids, tyrosol
Doctor of Philosophy (Ph.D.)