Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum.

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.

The ATP-binding protein FgArb1 is essential for penetration, infectious and normal growth of Fusarium graminearum.

ATP-binding cassette (ABC) transporters act mainly to transport compounds across cellular membranes and are important for diverse biological processes. However, their roles in pathogenesis have not been well-characterized in Fusarium graminearum. Sixty F. graminearum ABC protein genes were functionally characterized. Among them, FgArb1 regulates normal growth and importantly is essential for pathogenicity. Thus, the regulatory mechanisms of FgArb1 in pathogenicity were analyzed in this study. FgArb1 interacts with the mitogen-activated protein kinase (MAPK) FgSte7, and partially modulates plant penetration by regulating the phosphorylation of FgGpmk1 (the downstream kinase of FgSte7). The FgArb1 mutant exhibited dramatically reduced infective growth within wounded host tissues, likely resulting from its increased sensitivity to oxidative stresses, defective cell wall integrity and reduced deoxynivalenol (DON) production. FgArb1 also is important for the production of sexual and asexual spores that are important propagules for plant infection. In addition, FgArb1 is involved in the regulation of protein biosynthesis through impeding nuclear export of small ribosomal subunit. Finally, acetylation modification at sites K28, K65, K341 and K525 in FgArb1 is required for its biological functions. Taken together, results of this study provide a novel insight into functions of the ABC transporter in fungal pathogenesis.

Mechanism of action of AMP-jsa9, a LI-F-type antimicrobial peptide produced by Paenibacillus polymyxa JSa-9, against Fusarium moniliforme.

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32µg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K+, protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries.

Effect of fungicide on Fusarium verticillioides mycelial morphology and fumonisin B1 production

The effect of fludioxonil + metalaxyl-M on the mycelial morphology, sporulation and fumonisin B1 production by Fusarium verticillioides 103 F was evaluated. Scanning electron microscopy analysis showed that the fungicide caused inhibition of hyphal growth and defects on hyphae morphology such as cell wall disruption, withered hyphae, and excessive septation. In addition, extracellular material around the hyphae was rarely observed in the presence of fludioxonil + metalaxyl-M. While promoting the reduction of mycelial growth, the fungicide increased sporulation of F. verticillioides compared to the control, and the highest production occurred on the 14th day in the treatments and on the 10th day in the control cultures. Fumonisin B1 production in the culture media containing the fungicide (treatment) was detected from the 7th day incubation, whereas in cultures without fungicide (control) it was detected on the 10th day. The highest fumonisin B1 production occurred on the 14th day, both for the control and for the treatment. Fludioxonil + metalaxyl - M can interfere in F. verticillioides mycelial morphology and sporulation and increase fumonisin B1 levels. These data indicate the importance of understanding the effects of fungicide to minimize the occurrence of toxigenic fungi and fumonisins.

Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: identification of an RGFRRR motif governing fungal cell entry.

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of ß2 and ß3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.

Transcriptome analyses during fruiting body formation in Fusarium graminearum and Fusarium verticillioides reflect species life history and ecology.

Fusarium graminearum and F. verticillioides are devastating cereal pathogens with very different life history and ecological characteristics. F. graminearum is homothallic, and sexual spores are an important component of its life cycle, responsible for disease initiation. F. verticilloides is heterothallic, and produces only modest numbers of fruiting bodies, which are not a significant source of inoculum. To identify corresponding differences in the transcriptional program underlying fruiting body development in the two species, comparative expression was performed, analyzing six developmental stages. To accompany the transcriptional analysis, detailed morphological characterization of F. verticillioides development was performed and compared to a previous morphological analysis of F. graminearum. Morphological development was similar between the two species, except for the observation of possible trichogynes in F. verticillioides ascogonia, which have not been previously reported for any Fusarium species. Expression of over 9000 orthologous genes were measured for the two species. Functional assignments of highly expressed orthologous genes at each time-point revealed the majority of highly expressed genes fell into the "unclassified proteins" category, reflecting the lack of characterization of genes for sexual development in both species. Simultaneous examination of morphological development and stage-specific gene expression suggests that degeneration of the paraphyses during sexual development is an apoptotic process. Expression of mating type genes in the two species differed, possibly reflecting the divergent roles they play in sexual development. Overall, the differences in gene expression reflect the greater role of fruiting bodies in the life cycle and ecology of F. graminearum versus F. verticillioides.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Different active biomolecules involved in biosynthesis of gold nanoparticles by three fungus species.

In this paper, the intracellular gold nanoparticles were biosynthesized using three fungi including Aureobasidium pullulans (A. pullulans), Fusarium sp. and Fusarium oxysporum (F. oxysporum) after immersion the fungal cells in AuCl4- ions solution. UV-vis and FTIR spectrum, and biochemical compositions analysis of Au nano-fungal cells suggested that active biomolecules of reducing sugar of A. pullulans, and proteins in Fusarium sp. and F. oxysporum were tested positive of providing the function of the reduction of AuCI4- ions and the formation of the gold crystals. SDS-PAGE analysis of purified protein from gold nanoparticles synthesized by three fungi showed that three proteins with molecular weight (WM) about 100 kDa, 25 kDa and 19 kDa were in the gold nanoparticles by Fusarium sp. and two proteins with WM about 25 kDa and 19 kDa were in gold nanoparticles of F oxysporum. Further, three purified fungal proteins with WM about 100 kDa, 25 kDa and 19 kDa from gold nanoparticles by Fusarium sp. identified by LC-MS/MS, named plasma membrane ATPase, 3-glucan binding protein and glyceraldehyde-3-phosphate dehydrogenase, respectively. The Au nano-fungal cells ultrathin sections of Fusarium sp. and F. oxysporum showed that the gold nanoparticles mainly produced in intracellular vacuoles of fungal cells. The growth of gold nanoparticles in three fungal cells indicated the reducing sugar led to the gold nanoparticles in spherical morphology and proteins benefited to the gold aggregates.

Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway.

PURPOSE: To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). METHODS: After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0-24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. RESULTS: Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. CONCLUSIONS: Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway.

In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA.

BACKGROUND: Self-complementary RNA transcripts form a double-stranded RNA (dsRNA) that triggers a sequence-specific mRNA degradation, in a process known as RNA interference (RNAi), leading to gene silencing. In vascular plants, RNAi molecules trafficking occur between cells and systemically throughout the plant. RNAi signals can spread systemically throughout a plant, even across graft junctions from transgenic to non-transgenic stocks. There is also a great interest in applying RNAi to pathogenic fungi. Specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of phytopathogenic filamentous fungi. However, double-stranded (ds)RNA/small interfering (si)RNA silencing effect has not been observed in vivo. RESULTS: This study demonstrates for the first time the in vivo interference phenomenon in the pathogenic fungus Fusarium verticillioides, in which expression of an individual fungal transgene was specifically abolished by inoculating mycelial cells in transgenic tobacco plants engineered to express siRNAs from a dsRNA corresponding to the particular transgene. CONCLUSION: The results provide a powerful tool for further studies on molecular plant-microbe and symbiotic interactions. From a biotechnological perspective, silencing of fungal genes by generating siRNAs in the host provides a novel strategy for the development of broad fungi-resistance strategies in plants and other organisms.

Molecular cloning, structural analysis and modelling of the AcAFP antifungal peptide from Aspergillus clavatus.

An abundantly secreted thermostable peptide (designed AcAFP) with a molecular mass of 5777 Da was isolated and purified in a previous work from a local strain of A. clavatus (VR1). Based on the N-terminal amino acid (aa) sequence of the AcAFP peptide, an oligonucleotide probe was derived and allowed the amplification of the encoding cDNA by RT-PCR. This cDNA fragment encodes a pre-pro-protein of 94 aa which appears to be processed to a mature product of 51 aa cys-rich protein. The deduced aa sequence of the pre-pro-sequence reveals high similarity with ascomycetes antifungal peptide. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of an open reading frame of 282 bp interrupted by two small introns of 89 and 56 bp with conserved splice site. The three-dimensional (3D) structure modeling of AcAFP exhibits a compact structure consisting of five anti-parallel beta barrel stabilized by four internal disulfide bridges. The folding pattern revealed also a cationic site and spatially adjacent hydrophobic stretch. The antifungal mechanism was investigated by transmission and confocal microscopy. AcAFP cause cell wall altering in a dose-dependent manner against the phytopathogenic fungus Fusarium oxysporum.

Ultrastructural alterations in Fusarium sambucinum and Heterobasidion annosum treated with aluminum chloride and sodium metabisulfite.

Aluminum chloride (AlCl(3)) and sodium metabisulfite (Na(2)S(2)O(5)) have received increasing attention as antifungal agents for the control of plant diseases. In an effort to understand their toxic action on fungi, ultrastructural changes and membrane damage in Fusarium sambucinum (Ascomycota) and Heterobasidion annosum (Basidiomycota) in response to salt exposure was investigated using transmission electron microscopy. Conidial membrane damage was quantified using SYTOX Green stain, which only enters altered membranes. The results showed that mortality of the conidia was generally closely associated with SYTOX stain absorption in F. sambucinum treated with Na(2)S(2)O(5) and in H. annosum treated with AlCl(3) or Na(2)S(2)O(5), suggesting that these salts cause membrane alterations. For both fungi, ultrastructural alterations in conidia treated with AlCl(3) and Na(2)S(2)O(5) included membrane retraction, undulation, and invagination. At higher concentrations or exposure periods to the salts, loss of membrane integrity, cytoplasmic leakage, and cell rupture were observed. Ultrastructural alterations and increased SYTOX stain absorption in salt-treated conidia appear consistent with a mode of action where AlCl(3) and Na(2)S(2)O(5) alter membrane integrity and permeability.

Histopathological effects of dietary Fusarium graminearum on rat duodenum.

Microscopic pathology of duodenum in rats exposed to Fusarium graminearum, a fungus infecting small-grain cereals, was investigated. Intestinal haemorrhage was observed macroscopically in one of the rats. Light microscopy demonstrated detachments between the surface epithelium and the lamina propria and severe interstitial oedema in the lamina propria in the test group. Electron microscopy identified epithelial absorptive cells with highly expanded endoplasmic reticulum tubules, abundant cytoplasmic vesicles containing electronlucent materials, swollen mitochondria with spongiform appearance, and prominent cellular swelling. Other observations included opening of junctional complexes between epithelial cells lining the duodenum, highly enlarged intercellular spaces in duodenal epithelium, and numerous eosinophilic granulocytes and mast cells in the lamina propria. These findings indicate that dietary F. graminearum causes epithelial cell and connective tissue damage in rat duodenum. This is the first histopathological study showing that F. graminearum ingestion is associated with duodenal damage.

Removal of n-hexane by Fusarium solani with a gas-phase biofilter.

A gas-phase biofilter inoculated with the fungus Fusarium solani, isolated from a consortium grown on hexane vapors, was used to degrade this compound. The biofilter, packed with perlite and operated with an empty bed residence time of 60 s, was supplied with hexane concentrations between 0.5 gm(-3) and 11 gm(-3). Biofilter performance was evaluated over 100 days of operation. Several strategies for supplying the nutritive mineral medium were assayed to maintain favorable conditions for the fungal growth and activity. The Fusarium system was able to sustain an average elimination capacity of 90 gm(-3)(reactor) h(-1) with a maximum of 130 gm(-3)(reactor) h(-1) . The mass transfer limitations due to high biomass development in the biofilter were confirmed in batch experiments. Bacterial contamination was observed, but experiments in the biofilter and in batch reactors using selective inhibitors and controlled pH confirmed the predominant role of the fungus. Results indicate that fungal biofilters can be an effective alternative to conventional abatement technologies for treating hydrophobic compounds.

Five hydrophobin genes in Fusarium verticillioides include two required for microconidial chain formation.

Five hydrophobin genes have been identified in the fungal corn pathogen Fusarium verticillioides. HYD1, HYD2, and HYD3 encode Class I hydrophobins. The predicted structures of Hyd1p and Hyd2p are 80% similar, while Hyd3p has an unusually small number of amino acids between the third and fourth cysteines. HYD4 and HYD5 encode Class II hydrophobins. Mutants with HYD1-5 individually deleted and a hyd1deltahyd2delta double mutant were similar to wild-type strains in the amount of disease caused in a corn seedling infection assay and in the number of microconidia produced. Microconidial chains were rare in hyd1delta and hyd2delta mutants as microconidia were present almost exclusively as false heads. Transformation of hyd1delta and hyd2delta mutants with HYD1 and HYD2, respectively, restored microconidial chain formation, but transformation with HYD1::AcGFP and HYD2::AcGFP did not complement the mutation. HYD1::AcGFP and HYD2::AcGFP localized to the outside of conidia in false heads and in chains.

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

Skin and nail infections due to Fusarium oxysporum in Tuscany, Italy.

Nine cases of skin and nail infection due to Fusarium oxysporum, diagnosed in Tuscany in the period 1985-97, are described. Two manifested as interdigital intertrigo of the feet and seven as onychomycosis. All were diagnosed on the basis of repeated mycological examination, direct microscope observation and culture, as well as histological examination of biopsy specimens in two cases of intertrigo. Fragments of the fungal colonies were examined by scanning electron microscopy (SEM) for more detailed observation of fungal morphology. All patients had normal immune status and a history of the infection extending several years. Four of the patients with onychomycosis were treated with oral itraconazole, and clinical and mycological recovery was achieved in three cases. Two others were treated with cyclopyrox nail lacquer, successfully in one case. One patient with intertrigo was treated with oral itraconazole and one with oral terbinafine; both were also treated and with topical drugs, however clinical recovery was not confirmed by the mycological results.

Choline- and acetylcholine-induced changes in the morphology of Fusarium graminearum: evidence for the involvement of the choline transport system and acetylcholinesterase.

The response of Fusarium graminearum to choline, acetylcholine and a number of related analogues was investigated and their ability to induce a morphological response quantified. A number of mutants resistant to the alkylating agent nitrogen mustard (nim strains) were generated and found to have lost the ability to transport choline. These mutants were found to be insensitive to choline and acetylcholine but not to betaine, ethanolamine and other analogues. In addition, the non-competitive inhibitor hemicholinium-3 was also found to reduce the morphological effect of choline, proving that transport of choline into the hypha is essential for the morphological response. Acetylcholinesterase inhibitors blocked the morphological response to acetylcholine but had no effect on the response to choline, suggesting the presence of a membrane- or wall-bound acetylcholinesterase that hydrolyses acetylcholine to choline which subsequently induces the morphological response. Studies on the in vivo chitin synthase activity revealed that addition of choline caused a transient 75% increase in chitin synthase activity within 30 s, the rate rapidly returning to that observed before the addition of choline. No such effect was observed with the nim mutants.

Endogenous Fusarium endophthalmitis in a patient with acute lymphocytic leukemia.

Endogenous fungal endophalmitis is an uncommon complication of systemic mycosis. Only a few cases involving Fusarium have been reported, most with unfavorable visual outcomes. We examined a 31-year-old woman with acute lymphocytic leukemia who developed sudden visual loss in her right eye. A dense, white placoid infiltrate was present in the right macula extending into the vitreous. An iris nodule and hypopyon were present in the left eye. A vitreous aspirate of the right eye was positive for Fusarium species. The patient progressively lost vision despite amphotericin B and 5-fluorocytosine therapy. She died from bronchopneumonia, fungemia, and multisystem failure. Histopathologic study disclosed a panophthalmitis with Fusarium organisms invading all the ocular coats in the right eye. Leukemic infiltrates were present in the left iris, anterior chamber, and trabecular meshwork. The ocular destructiveness of Fusarium may be caused by marked mycotic vascular invasion and occlusion with consequent infarction and necrosis of ocular tissues.

Micromorfología de algunas especies de fusarium en PDA modificado III / Micromorphology of some fusarium species in modified PDA III

Se estudia el comportamiento de 20 cepas de Fusarium pertenecientes a 9 especies, aisladas de diversos ambientes (suelo, material clínico, alimentos y vegetales), mediante sus características macroscópicas de cultivo en PDA (standard) y su micromorfología en microcultivos en lámina en PDA modificado con 5 g/l de dextrosa, 4 g/l de KCL y 0,002 g/l de Dichlorán. Esta última metodología permitió observar las principales características microscópicas tales como: clamidio, micro, meso y macroconidios, además de sus celulas conidiógenas (mono, polifiálides y fiálides poliblásticas. Esto permitió la identificación tentativa o final de las cepas estudiadas y la confección de una clave. Las cepas estables en la producción de microestructuras fueron: F. avenaceum, F. chlamydosporum, F. sambucinum y F. solani