A serological investigation of hyphal growth in Fusarium culmorum

Summary Apical growth of hyphae of Fusarium culmorum was demonstrated using an immunofluorescent labelling technique. An antiserum prepared against hyphal tips contained a series of antibodies, detected by immunodiffusion, not present in antisera against mature hyphae or conidia. Absorption of the tip antiserum with hyphae allowed a specific immunofluorescence reaction with hyphal tips only. The antiserum against mature hyphae gave non-fluorescent tips to the hyphae.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum.

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Transformations of steroid esters by Fusarium culmorum

The course of transformations of the pharmacological steroids: testosterone propionate, 4-chlorotestosterone acetate, 17beta-estradiol diacetate and their parent alcohols in Fusarium culmorum AM282 culture was compared. The results show that this microorganism is capable of regioselective hydrolysis of ester bonds. Only 4-ene-3-oxo steroid esters were hydrolyzed at C-17. 17beta-Estradiol diacetate underwent regioselective hydrolysis at C-3 and as a result, estrone--the main metabolite of estradiol--was absent in the reaction mixture. The alcohols resulting from the hydrolysis underwent oxidation at C-17 and hydroxylation. The same products (6beta- and 15alpha-hydroxy derivatives) as from testosterone were formed by transformation of testosterone propionate, but the quantitative composition of the mixtures obtained after transformations of both substrates showed differences. The 15alpha-hydroxy derivatives were obtained from the ester in considerably higher yield than from the parent alcohol. The presence of the chlorine atom at C-4 markedly reduced 17beta-saponification in 4-chlorotestosterone acetate. Only 3beta,15alpha-dihydroxy-4alpha-chloro-5alpha-androstan-17-one (the main product of transformation of 4-chlorotestosterone) was identified in the reaction mixture. 6beta-Hydroxy-4-chloroandrostenedione, which was formed from 4-chlorotestosterone, was not detected in the extract obtained after conversion of its ester.     

Fine Structure and Spore Germination in Fusarium culmorum

The fine structure of the macroconidium of Fusarium culmorum(W. G. Smith) Sacc. is described. The presence of a mucilaginouscoat around the conidium and its expansion during germinationare confirmed. Studies of germinating conidia have shown changesin the numbers of organelles. The germ tube is shown to be formedfrom an entirely new wall laid down in the conidium, and theemergence of the germ tube is brought about by lysis of theoriginal condial wall.     

Wall Structure and spore Germination in Fusarium culmorum

The conidial and germ-tube walls of Fusarium culmorum (W. G.Smith) Sacc. have been examined by various chemical and electron-microscopetechniques. On the basis of these results and hypothesis isproposed for the organization of these walls. Microchemicaltests indicate the presence of chitin in the walls and suggestthat the mucilaginous layer around the conidium is mainly composedof xylan. Chemical analyses of isolated wall material confirmthe presence of chitin constituents in the wall, and a rylanlayer around the conidium. Furthermore, the wall contains apolypeptide moiety which has a different amino acid compositionfrom the rest of the protein of the cell. Electron microscopestudies of replicas and sections of conidia, germ tubes, andhyphae reveal a layered structure for the wall. The centrallayer is non-microfibrillar and is overlaid on both sides witha layer of randomly orientated microfibrils. The mucilaginouslayer of the conidium obscures the microfibrillar structurebeneath it unless the mucilage is removed by hydrolysis. Theproblem of hyphal growth is discussed on the basis of the structureof germ-tube tips and mature hyphae observed.     

Evaluation of Spring and Winter Wheat Reaction to Fusarium culmorum and Fusarium avenaceum

Winter (37), spring (8) wheat accessions and additionally, 7 double haploid (DH) lines were examined for susceptibility to Fusarium seedling blight after inoculation with F. culmorum and F. avenaceum. Winter accessions exhibited lower susceptibility of about 30% to both pathogens than spring cultivars. Susceptibility of winter cultivars varied from low (22%) to high (97%). Evaluation of the root was found to be more reliable than evaluation of coleoptile necrosis.
F. avenaceum infected mostly root and, to a lesser extent, coleoptile and leaves, with about a three times lower disease score of coleoptile against root. F. culmorum caused a 1.5 higher disease score on root than on coleoptile. Susceptibility of DH lines was different from susceptibility of parental forms. Reaction of individual accessions to F. culmorum and F. avenaceum was different.     

Comparison of Fusarium acuminatum and Fusarium culmorum isolates by means of tandem-crossed immunoelectrophoresis

F. acuminatum and F. culmorum strains were compared by means of tandemcrossed immunoelectrophoresis in order to estimate the possiblities of serological classification in Fusarium sections Gibbosum and Discolor. On the basis of qualitative similarity the two species could be distinguished well. By the use of anti-F. acuminatum serum a similarity of S_SM=0.52 was found between F. acuminatum and F. culmorum, but the S_SM coefficient reached a value of 0.67 when the anti-F. culmorum serum was tested. This asymmetric nature of the qualitative similarity is discussed. In the majority of cases the quantitative differences of the common antigens did not allow differentiation between the species.     

Production and secretion of 5-n-alkylresorcinols by Fusarium culmorum

Fusarium culmorum F1 was found to produce and secrete into the culture medium several of 5-n-alkylresorcinols. The amount of resorcinolic lipids was 5.3 microg/g and 0.9 microg/l in mycelium and in post-culture liquid, respectively. First of all F. culmorum F1 produces saturated homologues with C15 to C25 side chains. The extract from the medium contained only homologues with shorter carbon chains (C13 to C17).     

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79?mg/kg DM of DON and 100?μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2?g) as compared to their inoculated counterparts (15.5?g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.     

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79 mg/kg DM of DON and 100 microg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2 g) as compared to their inoculated counterparts (15.5 g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

Inter-simple sequence repeat and aggressiveness analyses revealed high genetic diversity, recombination and long-range dispersal in Fusarium culmorum

Inter‐simple sequence repeat (ISSR) analysis and aggressiveness assays were used to investigate genetic variability within a global collection of Fusarium culmorum isolates. A set of four ISSR primers were tested, of which three primers amplified a total of 37 bands out of which 30 (81%) were polymorphic. The intraspecific diversity was high, ranging from four to 28 different ISSR genotypes for F. culmorum depending on the primer. The combined analysis of ISSR data revealed 59 different genotypes clustered into seven distinct clades amongst 75 isolates of F. culmorum examined. All the isolates were assayed to test their aggressiveness on a winter wheat cv. ‘Armada’. A significant quantitative variation for aggressiveness was found among the isolates. The ISSR and aggressiveness variation existed on a macro‐ as well as micro‐geographical scale. The data suggested a long‐range dispersal of F. culmorum and indicated that this fungus may have been introduced into Canada from Europe. In addition to the high level of intraspecific diversity observed in F. culmorum, the index of multilocus association calculated using ISSR data indicated that reproduction in F. culmorum cannot be exclusively clonal and recombination is likely to occur.     

Development and relations of Fusarium culmorum and Pseudomonas fluorescens in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence: the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed development of F. culmorum mycelium in soil but stimulated formation of fungal chlamydospores. Decreased mycelial density in the presence of P. fluorescens was more pronounced in unsupplemented soil and less pronounced when glucose or cellulose was intiodaced. F. culmorum had no significant effect on P. fluorescens growth in soil.     

Two-dimensional environmental profiles of growth,deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based substrate

AIMS: To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium. METHODS AND RESULTS: Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production. CONCLUSIONS: This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.