NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors.

The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

The Control of Fusarium solani f. sp. phaseoli by Fungicide Mixtures

Six fungicides were used either alone or in binary combination to control Fusarium solani f. sp. phaseoli. Of the three methods of application used, seed treatments and soil drenches were the best, although phytotoxicity was evident in some instances. Application as a seed soak for 1 h proved to be the worst method with most treatments showing signs of phytotoxicity. The results are discussed with reference to the use of fungicide mixtures to control foot rot of beans.     

An extracellular enzyme from Fusarium solani f. sp. phaseoli which catalyses hydration of the isoflavonoid phytoalexin, phaseollidin

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) are the prenylated isoflavonoids kievitone and phaseollidin. Two enzyme activities, kievitone hydratase and phaseollidin hydratase, occur in culture filtrates of the bean pathogen, Fusarium solani f. sp. phaseoli, and catalyse similar hydration reactions on the dimethylallyl moieties of the phytoalexins. The enzymes nearly co-purified during hydroxyapatite chromatography followed by preparative native gel electrophoresis. Eluates from successive slices taken from the native gel were assayed for both activities. Although they were not completely separated in the native gel, the activity profiles indicated that the two activities were distinct. The Km of phaseollidin hydratase for phaseollidin was approximately 7 microM.     

Formation of trichothecenes by Fusarium solani var. coeruleum and Fusarium sambucinum in potatoes.

Fusarium solani var. coeruleum can form deoxynivalenol in potato tubers and in liquid medium, although concentrations observed in the rot were highly variable; acetyldeoxynivalenol and HT-2 toxin were detected in 1 to 3 tubers only (of 57). Trichothecenes were also detected in a very few (3 of 20) cultures of Fusarium sambucinum in potato tubers.     

Development and characterization of microsatellite markers for Fusarium virguliforme and their utility within clade 2 of the Fusarium solani species complex

Clade 2 of the Fusarium solani species complex contains plant pathogens including Fusarium virguliforme and closely related species Fusarium brasiliense, Fusarium crassistipitatum, Fusarium tucumaniae, which are the primary causal agents of soybean sudden death syndrome (SDS), a significant threat to soybean production. In this study, we developed microsatellite markers from a F. virguliforme genome sequence and applied them to a F. virguliforme population collection of 38 isolates from Michigan and four reference strains from other locations. Of the 225 detected microsatellite loci, 108 loci were suitable for primer design, and 12 of the microsatellite markers were determined to be highly polymorphic, amplifying on average 5.7 alleles per locus. Using these markers, F. virguliforme isolates were partitioned into three distinct clusters, but isolates were not grouped based on relatedness of sampling sites. In addition, 11 out of 12 markers were demonstrated to be highly transferrable to other closely related species.     

Invasion of the French paleolithic painted cave of Lascaux by members of the Fusarium solani species complex

A major fungal invasion was discovered in the prehistoric painted cave of Lascaux in France in Sep 2001. At least three species of the Fusarium solani complex were isolated and identified with a portion of the translation elongation factor 1alpha gene (EF-1alpha), a portion of the nuclear large subunit rDNA (LSU) and nuclear ribosomal intergenic spacer region (ITS). This study represents the first time that Fusarium species have been reported from a cave containing prehistoric paintings. Significant interspecific molecular variability was observed, suggesting that there might have been repeated introduction of the species, possibly carried by water from soils above the cave.     

The pathogenicities of Cylindrocarpon tonkinense and Fusarium solani in the rabbit cornea

The pathogenicity of Cylindrocarpon tonkinense in the cornea was evaluated and compared with that of Fusarium solani in rabbits. F. solani was inoculated into the right eyes of 14 rabbits and C. tonkinense was into the left eyes of same rabbits. The corneal lesions of both eyes were examined carefully by slit lamp every day for three weeks and the severity of infections were compared each other. For histopathologic study, several eyes were enucleated periodically. C. tonkinense has a pathogenicity equally strong as F. solani in this inculum size (10⁴ microconidia per cornea) and produced severe infection in rabbit eyes.     

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein-protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.     

Synthesis of cellulase by Fusarium sp. in different culture conditions.

The mechanism of the synthesis of cellulases by Fusarium sp. strain was studied. It was found that a significant role in determination of cellulases is played by the adsorption of these enzymes on cellulose. The aeration level of the culture media had a significant effect on the synthesis as well as on the enzymatic complexity of cellulases.     

Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5

Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l⁻¹ in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l⁻¹ or 8 mg l⁻¹) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.     

The loss of biological activity of the preservative bronopol associated with Fusarium solani

An aqueous antacid formulation normally protected from the growth of microbial contaminants, including opportunist pathogens, with the synthetic preservative Bronopol (2-bromo-2-nitropropane-1,3-diol) became contaminated with Fusarium solani. Contamination with F. solani, at levels below those which would be detected by routine quality control, resulted in the loss of antimicrobial protection. Not only was F. solani relatively resistant to Bronopol but it was able to grow and sporulate in the liquid antacid formulation, in which Bronopol was the only demonstrable source of nitrogen.     

Studies on the disease complex incidence by Meloidogyne incognita and Fusarium solani on Poi,Basella rubra

A survey of Poi crop in Ghaziabad (UP) exhibited a disease complex incidence by Meloidogyne incognita and Fusarium solani causing synergistic effect on the host. Paecilomyces lilacinus was found from the egg masses of M. incognita and Aspergillus niger and Aspergillus terreus from the rhizosphere of root-knot infected Poi crop. Paecilomyces lilacinus parasitised the eggs to a greater extent. The level of parasitism was highest (65%) by P. lilacinus while Aspergillus spp. did not colonise the eggs. Fusarium solani which in the present investigation has been established to be pathogenic to Poi plant.     

Light-driven diurnal zonation in the filamentous fungus Fusarium solani

Zonation in growing mycelia of Fusarium solani was induced by diurnal light/dark cycles. Only those parts of the hyphae that grew in darkness for less than 20 hours developed a zone of conidia after illumination. In continuous darkness, in continuous illumination, or after a transition from light to darkness, a conidiation zone failed to appear. Only light periods exceeding a few seconds but lasting less than 21 hours during a 24 hour light/dark cycle induced zonation. This zonation was not caused by periodic staling of the growth medium.     

Depolymerization of a hydroxy fatty acid biopolymer, cutin, by an extracellular enzyme from Fusarium solani f. pisi: isolation and some properties of the enzyme

Fusarium solani f. pisi was shown to grow on the hydroxy fatty acid biopolymer cutin as the sole carbon source. Such growth conditions induced the production of an extracellular cutin depolymerising enzyme. Analysis of products enzymatically derived from labeled cutin by thin-layer chromatography and radio gas-liquid chromatography showed that the Fusarium enzyme released all classes of cutin monomers. This enzyme preparation also catalyzed hydrolysis of several model ester substrates. It did not hydrolyze triacyl glycerol and pancreatic lipase did not hydrolyze cutin, indicating that the Fusarium enzyme is not a nonspecific lipase. With p-nitrophenyl palmitate as the model substrate the enzyme showed a broad pH optimum near 8.5 and it was stimulated by Triton X-100. Maximal stimulation was obtained at 3.7 mg/ ml of the detergent. Apparent K_m for p-nitrophenyl palmitate was 1.6 × 10^?4m. p-Nitrophenyl esters of C₂–C₁₈ acids gave comparable values for K_m and V revealing no striking specificity. Treatment with diisopropyl fluorophosphate severely inhibited the enzyme while iodoacetamide and p-chloromercuric benzoate did not affect the enzymatic activity, suggesting that the Fusarium enzyme is a serine hydrolase.     

New species from the Fusarium solani species complex derived from perithecia and soil in the old World tropics

A large collection of strains belonging to the Fusarium solani species complex (FSSC) was isolated from soil and perithecia in primary forests in Sri Lanka (from fallen tree bark) and tropical Australia (Queensland, from fallen tree fruits and nuts). Portions of the translation elongation factor 1-alpha (tef1) gene, the nuclear large subunit (NLSU) and internal transcribed spacer regions (ITS) of the nuclear ribosomal RNA genes were sequenced in 52 isolates from soil and perithecia. The FSSC was divided previously into three clades with some biogeographic structure, termed Clades 1, 2 and 3. All Sri Lankan and Australian soil isolates were found to be members of Clade 3, most grouping with the cosmopolitan soil-associated species F. falciforme. All but two Sri Lankan perithecial isolates were associated with a set of five divergent phylogenetic lineages that were associated with Clade 2. Australian perithecial isolates resided in a subclade of Clade 3 where most of the previously defined mating populations of the FSSC reside. Isolates from perithecia and those cultured from soil were always members of different species lineages, even when derived from proximal locations. The previous biogeographic assignment of Clade 2 to South America is now expanded to the worldwide tropics. Sri Lanka appears to be an important center of diversity for the FSSC. Nectria haematococca is epitypified with a collection from the type locality in Sri Lanka; its anamorph is described as a new species, Fusarium haematococcum. Neocosmospora E.F. Smith is adopted as the correct genus for Nectria haematococca. These new species are described: F. kurunegalense/Neo. kurunegalensis, F. rectiphorus/Neo. rectiphora/, F. mahasenii/Neo. mahasenii/, F. kelerajum/Neo. keleraja.