Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity

• Published in: Applied and Environmental Microbiology 2003-05, Vol.69 (5), p.2595-2602
• Main Authors: Cheng, Y, McNally, D.J, Labbe, C, Voyer, N, Belzile, F, Belanger, R.R
• Format: Article
• Language: English
• Subjects: Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; antifungal properties; Biological and medical sciences; biological control agents; Botrytis cinerea; cellobiose; Cellobiose - analogs & derivatives; Cellobiose - chemistry; Cellobiose - isolation & purification; Cellobiose - pharmacology; Deoxyribonucleic acid; DNA; Fundamental and applied biological sciences. Psychology; fungal antagonists; Fungi; Fusarium oxysporum f. sp. radicis-lycopersici; genetic transformation; glycolipids; Glycolipids - chemistry; Glycolipids - isolation & purification; Glycolipids - pharmacology; Idriella bolleyi; insertional mutagenesis; Lipids; Lipids - chemistry; Lipids - isolation & purification; Lipids - pharmacology; Microbiology; Molecular Structure; Mutagenesis, Insertional; mutants; Mutation; Mycology; Nuclear Magnetic Resonance, Biomolecular; Pest Control, Biological; Phomopsis; Phytophthora infestans; plant pathogenic fungi; Pythium aphanidermatum; Transformation, Genetic; Ustilaginales; Ustilaginales - chemistry; Ustilaginales - genetics
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.69.5.2595-2602.2003

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.