A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

A Comparative Transcriptome Analysis, Conserved Regulatory Elements and Associated Transcription Factors Related to Accumulation of Fusariotoxins in Grain of Rye (Secale cereale L.) Hybrids.

Detoxification of fusariotoxin is a type V Fusarium head blight (FHB) resistance and is considered a component of type II resistance, which is related to the spread of infection within spikes. Understanding this type of resistance is vital for FHB resistance, but to date, nothing is known about candidate genes that confer this resistance in rye due to scarce genomic resources. In this study, we generated a transcriptomic resource. The molecular response was mined through a comprehensive transcriptomic analysis of two rye hybrids differing in the build-up of fusariotoxin contents in grain upon pathogen infection. Gene mining identified candidate genes and pathways contributing to the detoxification of fusariotoxins in rye. Moreover, we found cis regulatory elements in the promoters of identified genes and linked them to transcription factors. In the fusariotoxin analysis, we found that grain from the Nordic seed rye hybrid "Helltop" accumulated 4 times higher concentrations of deoxynivalenol (DON), 9 times higher nivalenol (NIV), and 28 times higher of zearalenone (ZEN) than that of the hybrid "DH372" after artificial inoculation under field conditions. In the transcriptome analysis, we identified 6675 and 5151 differentially expressed genes (DEGs) in DH372 and Helltop, respectively, compared to non-inoculated control plants. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs were associated with glycolysis and the mechanistic target of rapamycin (mTOR) signaling pathway in Helltop, whereas carbon fixation in photosynthesis organisms were represented in DH372. The gene ontology (GO) enrichment and gene set enrichment analysis (GSEA) of DEGs lead to identification of the metabolic and biosynthetic processes of peptides and amides in DH372, whereas photosynthesis, negative regulation of catalytic activity, and protein-chromophore linkage were the significant pathways in Helltop. In the process of gene mining, we found four genes that were known to be involved in FHB resistance in wheat and that were differentially expressed after infection only in DH372 but not in Helltop. Based on our results, we assume that DH372 employed a specific response to pathogen infection that led to detoxification of fusariotoxin and prevented their accumulation in grain. Our results indicate that DH372 might resist the accumulation of fusariotoxin through activation of the glycolysis and drug metabolism via cytochrome P450. The identified genes in DH372 might be regulated by the WRKY family transcription factors as associated cis regulatory elements found in the in silico analysis. The results of this study will help rye breeders to develop strategies against type V FHB.

Fusarium infection alters the m6A-modified transcript landscape in the cornea.

N6-methyladenosine (m6A) is the most common post-transcriptional modification of RNA in eukaryotes that regulates the post-transcriptional expression level of genes without changing the base sequence. The role of m6A in fungal keratitis has not yet been elucidated. Here, we aimed to identify m6A modification changes and their potential roles in fungal keratitis. The murine model of fungal keratitis was established by inoculating mice with Fusarium solani (F. solani). The overall m6A level was detected via an m6A RNA methylation assay kit. The expression levels of key m6A modification-related genes were estimated by quantitative real-time polymerase chain reaction (PCR). The expression and localization of METTL (methyltransferase like)3, the key component of the m6A methyltransferase complex, was determined by immunostaining and Western blotting (WB). Immunoprecipitation methylation microarray was used to describe the changes in m6A modification in F. solani-infected corneal tissue. The overall m6A level in corneal tissue on the 5th day in the F. solani-treated group was upregulated compared with that in the control group. The demethylase levels were unaltered, but the level of the methylase METTL3 was increased significantly after fungal infection. Additionally, differences were found in m6A modifications in 1137 mRNAs, of which 780 were hypermethylated and 357 were hypomethylated. To the best of our knowledge, the present work is the first investigation on the m6A modification profiles in experimental fungal keratitis, and it may provide a potential therapeutic target.

The T2 Toxin Produced by Fusarium spp. Impacts Porcine Duodenal Nitric Oxide Synthase (nNOS)-Positive Nervous Structures-The Preliminary Study.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS-used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

Combined De Novo Transcriptome and Metabolome Analysis of Common Bean Response to Fusarium oxysporum f. sp. phaseoli Infection.

Molecular changes elicited by common bean (Phaseolus vulgaris L.) in response to Fusarium oxysproum f. sp. Phaseoli (FOP) remain elusive. We studied the changes in root metabolism during common bean-FOP interactions using a combined de novo transcriptome and metabolome approach. Our results demonstrated alterations of transcript levels and metabolite concentrations in common bean roots 24 h post infection as compared to control. The transcriptome and metabolome responses in common bean roots revealed significant changes in structural defense i.e., cell-wall loosening and weakening characterized by hyper accumulation of cell-wall loosening and degradation related transcripts. The levels of pathogenesis related genes were significantly higher upon FOP inoculation. Interestingly, we found the involvement of glycosylphosphatidylinositol- anchored proteins (GPI-APs) in signal transduction in response to FOP infection. Our results confirmed that hormones have strong role in signaling pathways i.e., salicylic acid, jasmonate, and ethylene pathways. FOP induced energy metabolism and nitrogen mobilization in infected common bean roots as compared to control. Importantly, the flavonoid biosynthesis pathway was the most significantly enriched pathway in response to FOP infection as revealed by the combined transcriptome and metabolome analysis. Overall, the observed modulations in the transcriptome and metabolome flux as outcome of several orchestrated molecular events are determinant of host's role in common bean-FOP interactions.

Genetic diversity of Fusarium mexicanum, causal agent of mango and big-leaf mahogany malformation in Mexico.

In Mexico, Fusarium mexicanum has been reported causing mango malformation disease and big-leaf mahogany malformation disease. Our objective was to determine the genetic diversity of F. mexicanum isolates obtained from malformed big-leaf mahogany and mango trees, using an internal simple sequence repeat (ISSR) analysis. A total of 61 isolates of F. mexicanum, 32 from mango and 29 from big-leaf mahogany, were initially genotyped using fourteen ISSR primers. Data from five primers that produced the highest number of polymorphic bands were selected for further analysis. The primers generated 49 polymorphic bands (85.96%) from a total of 57 fragments ranging in size from 250 to 2800 bp, with an average of 11.4 bands per primer. An analysis of molecular variance (AMOVA) indicated that the variation within populations, isolates grouped by host and geographic origin, was significant (43%), followed by the variation between the big-leaf mahogany versus mango isolates (34%), while among populations the variation was the lowest (22%). The genetic fingerprints suggested that genetic variability of F. mexicanum populations are structured by the host of origin rather than the geographic region.

Role of the Fusarium oxysporum metallothionein Mt1 in resistance to metal toxicity and virulence.

Soil organisms exhibit high tolerance to heavy metals, probably acquired through evolutionary adaptation to contaminated environments. Essentially, metal tolerance in fungi involves several specific and non-specific mechanisms that include metal efflux, metal binding to cell walls, extracellular and intracellular sequestration and complexation with proteins. However, fungi have adopted different strategies to detoxify heavy metals, although species differ in the mechanisms used. In this complex molecular framework, metallothioneins (MTs) are becoming increasingly relevant in metal homeostasis, even though little is known about their role in metal adaptation and virulence in fungal pathogens. With the aim to decipher the function of metallothioneins in the opportunistic fungus Fusarium oxysporum, we have carried out an in silico analysis that revealed the presence of a hypothetical metallothionein (mt1) that has multiple metal responsive elements in its promoter region and conserved cysteine motifs in its coding sequence. Characterization of strain Δmt1 deficient in the mt1 gene revealed higher sensitivity of this mutant to copper, cadmium and zinc compared to the wild type strain (wt). Expression analyses revealed that Zn specifically activates mt1, but the lack of this gene did not lead to a transcriptional up-regulation of genes gapdh and prx, associated with the oxidative stress response. The lack of mt1 did not alter the pathogenic capacity of the fungus, either in tomato plant or in a murine model of systemic infection. Nevertheless, Δmt1 displayed lower resistance to macrophage killing, suggesting a connection between the absence of mt1 and impaired defence capacity against copper and reactive oxygen species.

Primary metabolism changes triggered in soybean leaves by Fusarium tucumaniae infection.

Sudden death syndrome (SDS) of soybean can be caused by at least four distinct Fusarium species, with F. tucumaniae being the main causal agent in Argentina. The fungus is a soil-borne pathogen that is largely confined to the roots, but damage also reaches aerial part of the plant and interveinal chlorosis and necrosis, followed by premature defoliation can be observed. In this study, two genetically diverse soybean cultivars, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae or kept uninoculated. Leaf samples at 7, 10, 14 and 25 days post-inoculation (dpi) were chosen for analysis. With the aim of detecting early markers that could potentially discriminate the cultivar response to SDS, gas chromatography-mass spectrometry (GC-MS) analyses and biochemical studies were performed. Metabolic analyses show higher levels of several amino acids in the inoculated than in the uninoculated susceptible cultivar starting at 10 dpi. Biochemical studies indicate that pigment contents and Rubisco level were reduced while class III peroxidase activity was increased in the inoculated susceptible plant at 10 dpi. Taken together, our results indicate that the pathogen induced an accumulation of amino acids, a decrease of the photosynthetic activity, and an increase of plant-specific peroxidase activity in the susceptible cultivar before differences of visible foliar symptoms between genotypes could be observed, thus suggesting that metabolic and biochemical approaches may contribute to a rapid characterization of the cultivar response to SDS.

Antimicrobial efficacy of corneal cross-linking in vitro and in vivo for Fusarium solani: a potential new treatment for fungal keratitis.

BACKGROUND: Fungal keratitis is one of the major causes of visual impairment worldwide. However, the effectiveness of corneal collagen cross-linking (CXL) for fungal keratitis remains controversial. In this study, we developed an in vitro and an in vivo models to assess the efficacy of CXL for Fusarium keratitis. METHODS: The effect of in vitro CXL fungicidal was evaluated on the cultures of Fusarium solani which were exposed to irradiation for different durations. Viability of fungal was appraised under four conditions: no treatment (control); CXL: UVA (365 nm)/riboflavin; riboflavin and UVA (365 nm). Each batch of sterile plate culture was irradiated for different CXL durations. The in vivo Therapeutic effect was studied on a mouse keratitis model. The animals were divided randomly into three groups: group A with no treatment (control); Group B with CXL treatment for two minutes and group C with CXL treatment for three minutes. The CXL procedure was performed 24 h post inoculation in each group. All mice with corneal involvement were scored daily for 7 days and 10 days after infection. Corneals were extracted at various time points for quantitative fungal recovery. Histological evaluations were conducted to calculate the number of polymorphonuclear cells. RESULTS: Viability of fungal decreased significantly in CXL group with 30-min irradiation compared with that in control, riboflavin and UVA groups (P < 0.01). The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with CXL treatment (P < 0.05). Clinical scores, corneal lesion, corneal opacity, neovascularization and the depth of ulceration scores in group B and group C were remarkably lower than that in group A (P < 0.05, P = 0.001, P = 0.001, P = 0.034 and P = 0.025 respectively). Scores of group C were much lower than that in group B. Histological revealed that destruction of corneal collagen fibers and infiltration of inflammatory cells into corneal tissue in group B and group C were much lower than that in group A. CONCLUSIONS: We believe that CXL treatment may be applied to fungal keratitis, therapeutic efficacy will improve with longer treatment duration.

TOR signaling downregulation increases resistance to the cereal killer Fusarium graminearum.

TOR is the master regulator of growth and development that senses energy availability. Biotic stress perturbs metabolic and energy homeostasis, making TOR a good candidate to participate in the plant response. Fusarium graminearum (Fusarium) produces important losses in many crops all over the world. To date, the role of TOR in Fusarium infection has remained unexplored. Here, we show that the resistance to the pathogen increases in different Arabidopsis mutants impaired in TOR complex or in wild-type plants treated with a TOR inhibitor. We conclude that TOR signaling is involved in plant defense against Fusarium.

Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani.

Purpose: Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human ß-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani-induced modulation of AMP expression in vitro. Methods: Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results: We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions: These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells.

Dectin-1-Mediated Pathway Contributes to Fusarium proliferatum-Induced CXCL-8 Release from Human Respiratory Epithelial Cells.

Fusarium species are causative agents of human respiratory disorders and are distributed widely in our environment. Little is known of their interaction with human respiratory epithelial cells, which may contribute to allergic airway responses. In this study, we report on the release of C-X-C motif chemokine ligand 8 (CXCL-8) from human bronchial epithelial BEAS-2B cells upon stimulation with Fusarium proliferatum extracts. F. proliferatum-induced cytokine release from BEAS-2B cells was determined by cytokine array and CXCL-8 enzyme-linked immunosorbent assay (ELISA) kits. Blocking antibodies and signaling pathway inhibitors were employed to delineate cell surface receptors and signaling pathways participating in CXCL-8 release. F. proliferatum extracts induced the release of CXCL-8 in a time-dependent manner. The dectin-1 receptor ligands, curdlan and laminarin, reduced CXCL-8 release. Cells pre-treated with anti-Dectin-1 antibodies (2 µg/mL) decreased CXCL-8 release by 24%. Furthermore, F. proliferatum-stimulated CXCL-8 release was reduced by 32%, 53%-81%, 40% and 26% after BEAS-2B cells were pretreated with activation inhibitors of spleen tyrosine kinase (Syk)-piceatannol-, mitogen-activated protein kinases (MAPKs)-PD98059, U0126, SB202190, SP600125-, phosphatidylinositol-3-kinase (PI3K)-LY294002-and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-BAY117082-, respectively. These results suggest that Dectin-1-mediated activation of the Syk, MAPKs, PI3K and NF-κB signaling pathways contributes to F. proliferatum-stimulated CXCL-8 release from BEAS-2B cells and provides an important basis for developing novel therapeutic strategies in clinical allergy.

Flow cytometry based rapid duplexed immunoassay for fusarium mycotoxins.

At small food processing facilities, the most frequently used test to determine if grain-derived mycotoxin concentrations are compliant with legal limits is the enzyme-linked immunosorbent assay (ELISA). Each kit is designed to detect one of the six dangerous mycotoxins. With the increasing occurrence of coinfection of grain with multiple-mycotoxins in the field and/or during storage, ELISA is no longer a cost effective best assay option. With ELISA, each species of mycotoxin requires different sample preparation/extraction and a 45 min incubation. The alternative multiplexed assay presented here, the competitive fluorescent microsphere immunoassay (CFIA), follows current food safety standards. It handles several toxins simultaneously with a single universal extraction protocol. The authors' objective was to modify an existing commercial CFIA kit developed for bench top flow cytometry and extend its utility for point-of-need (PON) applications. The accelerated protocol offers over 60% reduction in total processing time and it detects dual mycotoxin contamination simultaneously. The observed enhanced binding kinetics equations reported here utilizing suspended solid phase particles in liquid phase, are also supported by published theoretical calculations. In the near future portable cytometry may bring rapid multiplexed PON testing to assure the safety of small food processing installations. © 2016 International Society for Advancement of Cytometry.

Identification, transcriptional and functional analysis of heat-shock protein 90s in banana (Musa acuminata L.) highlight their novel role in melatonin-mediated plant response to Fusarium wilt.

As one popular fresh fruit, banana (Musa acuminata) is cultivated in the world's subtropical and tropical areas. In recent years, pathogen Fusarium oxysporum f. sp. cubense (Foc) has been widely and rapidly spread to banana cultivated areas, causing substantial yield loss. However, the molecular mechanism of banana response to Foc remains unclear, and functional identification of disease-related genes is also very limited. In this study, nine 90 kDa heat-shock proteins (HSP90s) were genomewide identified. Moreover, the expression profile of them in different organs, developmental stages, and in response to abiotic and fungal pathogen Foc were systematically analyzed. Notably, we found that the transcripts of 9 MaHSP90s were commonly regulated by melatonin (N-acetyl-5-methoxytryptamine) and Foc infection. Further studies showed that exogenous application of melatonin improved banana resistance to Fusarium wilt, but the effect was lost when cotreated with HSP90 inhibitor (geldanamycin, GDA). Moreover, melatonin and GDA had opposite effect on auxin level in response to Foc4, while melatonin and GDA cotreated plants had no significant effect, suggesting the involvement of MaHSP90s in the cross talk of melatonin and auxin in response to fungal infection. Taken together, this study demonstrated that MaHSP90s are essential for melatonin-mediated plant response to Fusarium wilt, which extends our understanding the putative roles of MaHSP90s as well as melatonin in the biological control of banana Fusarium wilt.

WAX INDUCER1 (HvWIN1) transcription factor regulates free fatty acid biosynthetic genes to reinforce cuticle to resist Fusarium head blight in barley spikelets.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating diseases of wheat and barley. Resistance to FHB is highly complex and quantitative in nature, and is most often classified as resistance to spikelet infection and resistance to spread of pathogen through the rachis. In the present study, a resistant (CI9831) and a susceptible (H106-371) two-row barley genotypes, with contrasting levels of spikelet resistance to FHB, pathogen or mock-inoculated, were profiled for metabolites based on liquid chromatography and high resolution mass spectrometry. The key resistance-related (RR) metabolites belonging to fatty acids, phenylpropanoids, flavonoids and terpenoid biosynthetic pathways were identified. The free fatty acids (FFAs) linoleic and palmitic acids were among the highest fold change RR induced (RRI) metabolites. These FFAs are deposited as cutin monomers and oligomers to reinforce the cuticle, which acts as a barrier to pathogen entry. Quantitative real-time PCR studies revealed higher expressions of KAS2, CYP86A2, CYP89A2, LACS2 and WAX INDUCER1 (HvWIN1) transcription factor in the pathogen-inoculated resistant genotype than in the susceptible genotype. Knockdown of HvWIN1 by virus-induced genes silencing (VIGS) in resistant genotype upon pathogen inoculation increased the disease severity and fungal biomass, and decreased the abundance of FFAs like linoleic and palmitic acids. Notably, the expression of CYP86A2, CYP89A2 and LAC2 genes was also suppressed, proving the link of HvWIN1 in regulating these genes in cuticle biosynthesis as a defense response.

Metabolic Biomarker Panels of Response to Fusarium Head Blight Infection in Different Wheat Varieties.

Metabolic changes in spikelets of wheat varieties FL62R1, Stettler, Muchmore and Sumai3 following Fusarium graminearum infection were explored using NMR analysis. Extensive 1D and 2D 1H NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. In addition, metabolic changes that are observed in all studied varieties as well as wheat variety specific changes have been determined and discussed. A new method for metabolite quantification from NMR data that automatically aligns spectra of standards and samples prior to quantification using multivariate linear regression optimization of spectra of assigned metabolites to samples' 1D spectra is described and utilized. Fusarium infection-induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance.

Wildly Growing Asparagus (Asparagus officinalis L.) Hosts Pathogenic Fusarium Species and Accumulates Their Mycotoxins.

Asparagus officinalis L. is an important crop in many European countries, likely infected by a number of Fusarium species. Most of them produce mycotoxins in plant tissues, thus affecting the physiology of the host plant. However, there is lack of information on Fusarium communities in wild asparagus, where they would definitely have considerable environmental significance. Therefore, the main scientific aim of this study was to identify the Fusarium species and quantify their typical mycotoxins present in wild asparagus plants collected at four time points of the season. Forty-four Fusarium strains of eight species--Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium oxysporum, Fusarium proliferatum, Fusarium sporotrichioides, and Fusarium tricinctum--were isolated from nine wild asparagus plants in 2013 season. It is the first report of F. sporotrichioides isolated from this particular host. Fumonisin B1 was the most abundant mycotoxin, and the highest concentrations of fumonisins B1-B3 and beauvericin were found in the spears collected in May. Moniliformin and enniatins were quantified at lower concentrations. Mycotoxins synthesized by individual strains obtained from infected asparagus tissues were assessed using in vitro cultures on sterile rice grain. Most of the F. sporotrichioides strains synthesized HT-2 toxin and F. equiseti strains were found to be effective zearalenone producers.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

Antifungal activity and computational study of constituents from Piper divaricatum essential oil against Fusarium infection in black pepper.

Fusarium disease causes considerable losses in the cultivation of Piper nigrum, the black pepper used in the culinary world. Brazil was the largest producer of black pepper, but in recent years has lost this hegemony, with a significant reduction in its production, due to the ravages produced by the Fusarium solani f. sp. piperis, the fungus which causes this disease. Scientific research seeks new alternatives for the control and the existence of other Piper species in the Brazilian Amazon, resistant to disease, are being considered in this context. The main constituents of the oil of Piper divaricatum are methyleugenol (75.0%) and eugenol (10.0%). The oil and these two main constituents were tested individually at concentrations of 0.25 to 2.5 mg/mL against F. solani f. sp. piperis, exhibiting strong antifungal index, from 18.0% to 100.0%. The 3D structure of the ß-glucosidase from Fusarium solani f. sp. piperis, obtained by homology modeling, was used for molecular docking and molecular electrostatic potential calculations in order to determine the binding energy of the natural substrates glucose, methyleugenol and eugenol. The results showed that ß-glucosidase (Asp45, Arg113, Lys146, Tyr193, Asp225, Trp226 and Leu99) residues play an important role in the interactions that occur between the protein-substrate and the engenol and methyleugenol inhibitors, justifying the antifungal action of these two phenylpropenes against Fusarium solani f. sp. piperis.

Use of a bio-wipe kit to detect fumonisin B1 in faecal materials.

Fusarium toxins with reference to fumonisin B1 (FB1) have long been regarded as contaminants of maize and maize-based related products. However, when consumed they can cause intoxication, especially in humans. Therefore, effective quantitative methods for assessing the dietary exposure of this toxic fungal metabolite are required. The objective of this investigation was to evaluate the effect on the use of a bio-wipe kit, which is a faecal material collection kit, to detect the presence of FB1. Faecal materials were collected from a rural farming community in Gauteng Province, South Africa. In total, 200 samples of faecal material were analysed for Fusarium species using a serial dilution method, while FB1 was further analysed and quantified by reversed-phase TLC and HPLC. The study showed the presence of 11 different Fusarium species grown on potato dextrose agar culture medium of which F. verticillioides and F. proliferatum, producers of FB1, and F. oxysporum were the dominant species. Fumonisin B1 was recorded at an incidence rate of 65% of the total using TLC. Results from HPLC showed that 84% were positive at different ranges of concentration for FB1. This study supports the use of a bio-wipe as a rapid method to determine human exposure to FB1.