Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.     

Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici

Abstract An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M _r of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 °C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identity to other known polygalacturonases.     

Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.     

Cloning a species-specific DNA probe from Fusarium oxysporum fungus]

A method to obtain genus-specific DNA probes is suggested. It consists of specific amplification of the intergenic spacer between the 18S and 5.8S ribosomal RNA genes, using primers deduced from conservative ribosomal DNA sequences. The utility of the method is demonstrated on isolation of the 209 b.p. spacer fragment from the genomic DNA of a plant pathogenic fungus Fusarium oxysporum.     

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.     

Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).     

Functional characterization of a cellulose binding xylanase from Fusarium oxysporum

Summary An extracellular endoxylanase from Fusarium oxysporum binds onto crystalline cellulose. A small peptide (~ 2kDa) could be isolated after partial proteolysis of the native protein. It consists of 18 amino acids, is located in the C-terminal region of the protein and corresponds functionally to a cellulose binding domain (CBD), the first one to be reported in a fungal xylanase. The amino acid sequence of this peptide shows no homology with any known CBD.     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Purification and characterization of a β-glucosidase from the phytopathogenic fungus Fusarium oxysporum f. sp. melonis

T.M. ALCONADA AND M.J. MARTÍNEZ. 1996. Fusarium oxysporum f. sp. melonis produces cellulase and β-glucosidase activities in a medium with glucose and avicel as carbon source. A β-glucosidase from this crude material was purified by gel filtration and ion exchange chromatography successively. This enzyme is a unique band of protein in SDS-PAGE and isoelectric focussing. It had a molecular weight of 66000 and a pI of 5. Using p -nitrophenyl-β-D-glucopyranoside as substrate β-glucosidase shows a K _m of 210 μmol 1^-1, an optimum pH of 5.5 and an optimum reaction temperature of 60°C, being stable in a pH range of 5–7 for 48 h at room temperature.     

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.     

Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum

Abstract Nitrogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N₂O) or dinitrogen (N₂) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N₂O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrified by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system.     

Anaerobic elemental sulfur reduction by fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H2S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H2S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H2S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O2 consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H2S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O2 respiration with sulfur reduction.     

Carbon and nitrogen requirements of Fusarium oxysporum causing cotton wilt

Summary The present work was carried out to study the nutritional requirements of the cotton wilt-inducing fungus, i.e.Fusarium oxysporum on a synthetic liquid medium with regard to the carbon and nitrogen sources at varying concentrations in terms of the average mycelial dry weights.The optimum carbon requirements of the fungus ranged from 7000–8000 p.p.m. irrespective of the carbon source used in experiment. Carbon utilization was best on sucrose followed by maltose, starch, glucose, fructose and cellulose successively.The optimum nitrogen requirements of the fungus were 300 p.p.m. of nitrogen in the medium; nitrogen utilization was best on using nitrate-nitrogen followed by glycine, glutamic acid, ammonium nitrate, asparagine and ammonium sulphate.Maximum growth of the fungus took place on media containing a C/N ratio ranging between 22.8 and 25.7.Colour formation is correlated with varying either source or concentration of nitrogen and not carbon.