Developmental Regulation of Invertase Isozymes in Fusarium oxysporum

Fusarium invertase has two isozymes; one is developmentally regulated, whereas the other exists throughout the developmental stages of Fusarium.     

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K_s, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K_s, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K_s, about 30 mM).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil.

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Purification and properties of nitroalkane oxidase from Fusarium oxysporum.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

The cell wall of Fusarium oxysporum.

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Isoverrucarol production by Fusarium oxysporum CJS-12 isolated from corn.

Isoverrucarol (3,15-dihydroxy-12,13-epoxy-trichothec-9-ene) was isolated and purified from wheat cultures of a toxic strain of Fusarium oxysporum CJS-12. The toxin was characterized by thin-layer chromatography, gas chromatography-mass spectrometry, and 1H and 13C nuclear magnetic resonance spectrometry. Isoverrucarol caused toxic effects in rats, including loss of appetite, bodily weakness, severe mucosae of the stomach, and death, when administered orally at 10 and 20 mg/kg of body weight. The toxin also caused a definite dermatitic reaction of epidermis and an edematic-necrotic response of the dermis.     

Comparison of Fusarium oxysporum and Fusarium oxysporum var. redolens by Analyzing the Isozyme and Serological Patterns

Extracts from Fusarium oxysporum (F.o.) and F. oxysporum var. redolens (F.o.r.) isolates were compared by means of electrophoresis and crossed immunoelectrophoresis. The polymorphism of five isozyme systems allowed a distinction between F.o. and F.o.r. isolates. The isozyme patterns of three other isozyme systems did not allow this distinction between F.o. and F.o.r. to be made. Both fungi appeared almost identical serologically. Relative amounts of their corresponding proteins differed but the qualitative patterns of the proteins were nearly the same with the anti-F.o.r. serum, only one specific antigen was detected in the extracts from F.o.r., isolates. Although the results obtained indicate a strong similarity between F.o. and F.o.r., they are not sufficient for an unequivocal statement that the fungi belong to the same species.     

Fusarium oxysporum Forms Heterokaryons with Fusarium redolens

Vegetative compatibility among three isolates of Fusarium oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens from diseased lupins was investigated. Pairings between five mutants originated from each isolate revealed two compatibility groups. The first VCG comprised race 1 of F. oxysporum f. sp. lupini and one isolate of F. oxysporum var. redolens; the second VCG comprised race 2 of F. oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens. Heterokaryon formation was observed in many pairings involving mutants of both taxa. These findings provide evidence of the conspecificity of these two taxa and they support Gordon 's classification (1952) according to which F. redolens is actually F. oxysporum.     

Utilization of Lactic Acid by Fusarium oxysporum var. lini: Regulation of Transport and Metabolism

Lactic acid was transported in Fusarium oxysporum var. lini ATCC 10960 by a saturable transport system that had a half-saturation constant of 56.6 ± 7.5 μM and a maximum velocity of 0.61 ± 0.10 mmol h^-1 g^-1 (dry weight) at 26°C and pH 5.0. This transport system was inducible and was not expressed in the presence of a repressing substrate. Evidence is presented that the anionic form lactate^- was taken up by the cells. Propionic, acetic, pyruvic, and bromoacetic acids but not succinic acid competitively inhibited the transport of lactic acid. Bromoacetic acid, which was not metabolized, was taken up to a steady-state level when intracellular and extracellular concentrations were identical, indicating that the transport system was not accumulative. The enzymatic activity that was physiologically more relevant in the metabolism of lactic acid was lactate: ferricytochrome c oxidase. This enzyme did not exhibit stereospecifity and was induced by lactic acid.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici.

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

Purification and characterization of invertase isozymes from Fusarium oxysporum

Fusarium invertase appears to exist in two forms, mycelial (P-1) and conidial (P-2) types. They were purified and partially characterized, but their specific activities were too low when compared with yeast and Neurospora invertases. Present studies describe a method for isolation of highly purified enzymes and their properties. The enzymes are homogeneous by several criteria. Estimation of molecular weights revealed the subunit structure of each invertase, and the association-dissociation of subunits seem to occur as temperature varies. Amino acid compositions and other properties have been studied. The two invertases are glycoproteins which contain 36% (P-1) and 23% (P-2) carbohydrates (predominantly mannose with smaller percentages of glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine). Comparison with the properties of the previous preparations is also described.