Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

Methylxanthines Modulate Circadian Period Length Independently of the Action of Phosphodiesterase.

In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.

The Neurospora crassa chr-1 gene is up-regulated by chromate and its encoded CHR-1 protein causes chromate sensitivity and chromium accumulation.

The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.

Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa.

The development of production processes that can reduce the environmental impact, offer waste reduction and increase energy efficiency is an important step in the field of application of nanotechnology. In this work the filamentous fungus Neurospora crassa was screened and found to be successful for the production of mono and bimetallic Au/Ag nanoparticles (NPs). Analysis by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM) confirmed the biosynthesis of NPs by the fungus. The shape of NPs was found to be mainly spherical with average diameter of 11nm for silver and 32nm for gold, when the fungus was exposed to the aqueous solutions of 10(-3)M of AgNO(3) and HAuCl(4), respectively. EDS analysis also confirmed the formation of alloy-type Au/Ag bimetallic NPs when three different ratios of AgNO(3)/HAuCl(4) were used. TEM images of thin sections of N. crassa cells confirmed the intracellular formation of silver and gold NPs. The results obtained indicate that N. crassa can be a potential "nanofactory" for the synthesis of metallic NPs. The use of this organism will offer several advantages since it is considered as a non-pathogenic organism, has a fast growth rate, rapid capacity of metallic ions reduction, NPs stabilization and facile and economical biomass handling.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents.

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 microg/ml inhibited radial growth more than fluconazole at 5.0 microg/ml while amphotericin B at 0.05 microg/ml was more effective than nystatin at 0.05 microg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, alpha = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment/through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.

The transcription of the gene for iso-orotate decarboxylase (IDCase), an enzyme of the thymidine salvage pathway, is downregulated in the pregc mutant strain of Neurospora crassa grown under phosphate starvation.

The preg gene encodes a cyclin-like protein that is implicated in the derepression of nucleases and phosphatases that scavenge phosphate from the environment. To better understand the regulatory role of the preg gene product, the differential display reverse transcriptase - polymerase chain reaction was used to isolate transcripts differentially expressed in the pregc mutant strain of the mold Neurospora crassa grown under phosphate starvation, at pH 7.8. Two transcripts, whose differential expressions were confirmed by Northern blotting, were downregulated in a strain of N. crassa carrying a loss-of-function mutation in the preg gene (preg(c) allele). These transcripts revealed genes coding for enzymes involved in the thymidine salvage pathway (iso-orotate decarboxylase) and in the biosynthesis of coenzyme Q (ubiquinone C-methyltransferase), which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.

Antifungal properties of novel N- and alpha,beta-substituted succinimides against dermatophytes.

The synthesis and antifungal properties of a series of new N-aryl alpha,beta-substituted succinimides against a panel of dermatophytes of clinical relevance are reported. Among those compounds possessing a N-phenyl substituent, 7-thia-2-azabicyclo[2,2,1]hept-2-en-3-amine[5,6-c]succinimide was the better inhibitor of Trichophyton rubrum, the major ethiological agent of all infections produced by dermatophytes. In contrast, succinimides containing a N-(p-sulfonylphenyl) substituent, only inhibited Epidermophyton floccosum, all active compounds possessing an oxabicyclo group in positions alpha,beta of the imide. Substituents on the oxabicyclo group were important for the activity. Regarding the mechanism of action, N-(p-N'-4-methoxyphenylsulfamoylphenyl)-8-oxabicyclo[2,2,1]hept-4-en-3- methyl[5,6-c]succinimide produced a mottled inhibition halo in the Neurospora crassa assay, showing that it would act by inhibiting the synthesis or assembly of the fungal cell wall.

In vitro antifungal evaluation and studies on the mode of action of xanthoxyline derivatives.

This study describes the fungistatic effect of xanthoxyline (CAS 90-24-4) and its derivatives against a panel of yeasts, filamentous fungi and dermatophytes, by using the agar dilution method. Results indicated that simple structural modifications led to more potent derivatives, especially in relation with dermatophytes. The most active compound tested (10), which is a benzenesulphonyl derivative, was 12-fold more potent than xanthoxyline itself against Trichophyton rubrum. The evaluation of the mode of action with the whole cell Neurospora crassa assay, suggested that some selected compounds may be acting by the inhibition of fungal cell-wall polymers synthesis or assembly.

In vitro studies on mode of action of antifungal 8.O.4'-neolignans occurring in certain species of Virola and related genera of Myristicaceae.

Neutral racemic antifungal alcohols of 8.O.4'-neolignan type, were evaluated for inhibitory activity towards the fungal cell wall, using the whole cell Neurospora crassa hyphal growth inhibition assay. Results strongly suggested that these compounds could act by inhibiting cell wall polymer synthesis or assembly. Active compounds were tested for their inhibitory activities against (1,3)-beta-glucan synthase, an enzyme that catalyzes the synthesis of the major wall polymer (1,3)-beta-glucan. Although these compounds were found to be inhibitors of the enzyme (inhibition ranging between 2 and 72% at 250 micro/ml), comparison of these results with those from agar dilution assays, allow us to infer that these compounds do not act via the inhibition of glucan synthase. In addition, ketones with same pattern of substitution as alcohols, which have no antifungal properties in agar dilution assays, still displayed similar glucan synthase inhibition.

A dominant negative effect of eth-1r, a mutant allele of the Neurospora crassa S-adenosylmethionine synthetase-encoding gene conferring resistance to the methionine toxic analogue ethionine.

eth-1r, a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48-->N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1+/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1+/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1+ and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1+/ eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1+/eth-1r AdoMet synthetase molecules.

[In situ studies of myoinositol-1-P synthase in wild and inos- strains of Neurospora crassa]. / Estudios "in situ" de la mioinositol 1-P sintasa en cepas silvestre e inos- de Neurospora crassa.

The biosynthetic pathway for myo-inositol consist of two enzymatic steps: first, the cycloaldolization of glucose-6P to L-myo-inositol-IP followed by its hydrolysis to form free myo-inositol. The former reaction is catalyzed by myo-inositol-IP synthase (MIPS) while, a phosphatase is responsible for the hydrolysis step. Depending on its degree of purification and storage age, MIPS activity us to be, from partial to fully, dependent on added NAD. Therefore, we decided to study the kinetic properties of the enzyme within the cell, specially its requirements for free NAD. To this purpose, a method was designed for the assay of MIPS-activity in situ, using toluene permeabilized mycelia. MIPS-activity "in situ" was fully displayed in the absence of added NAD; on the contrary, the purified enzyme showed only 33% of that activity displayed when NAD was included in the assay. Thus, it seems that the native enzyme contains tightly bound NAD, instrumental for its activity, and that during purification or storage, the coenzyme is progressively lost, rendering the NAD-dependent enzyme, as was previously envisage. In addition, the in situ assay method for MIP-Synthase was applied to several mutants of N. crassa having the inosphenotype. Our results showed that only in 3 of 14 cases analyzed the phenotype could be clearly associated to the lack of MIP-synthase activity. Indeed most of the mutants analyzed showed significant levels (from 5 to 21%) of MIP-synthase, when compared to the activity shown by the RL-21 WT strain. Finally, all the mutants and WT strains were zymographically analyzed for phosphatase activity and showed close to equal strong reaction levels.

[Synthesis and structure of substituted bromo and nitrobenzyl benzylidene imidazolidinediones and thiazolidinediones]. / Synthèse et structure des bromo et nitrobenzyl benzylidène imidazolidinediones et thiazolidinediones substituées.

Synthesis and physico-chemical properties of five bromobenzyl-benzylidene-imidazolidinediones and five nitrobenzyl- or benzyl-benzylidene-thiazolidinediones are described. The microbiological activity of bromobenzyl-benzylidene-imidazolidinediones against microorganisms such as Candida albicans, Neurospora crassa and Mycobacterium smegmatis are evaluated.

[Synthesis and antifungal activity of chlorobenzyl benzylidene thiazolidinediones and substituted of imidazolidinediones]. / Synthèse et activité antifongique des chlorobenzyl benzylidène thiazolidinediones et imidazolidinediones substituées.

The synthesis of six benzylidene thiazolidinediones and four benzylidene imidazolidinediones is described. In order to investigate their antifungal activity, they are evaluated against microorganism such as Candida albicans, Neurospora crassa, Staphylococcus aureus and Escherichia coli.

Genes responsive to the alteration of polyamine biosynthesis in neurospora crassa.

Wild-type Neurospora crassa grown in minimal medium was exposed to -difluormethyl ornithine (DFMO), a specific inhibitor of ornithine-decarboxylase (ODC-ase) activity. Protein-synthesis rates impaired by DFMO were restored by the addition of spermidine. The pattern on SDS-acrylamide gels displayed three newly synthesized polypeptides, p27, p31 and p99 after DFMO action in the absence of exogenous polyamine. The ODC-ase mutant (spe-1) grown in spermidine-supplemented medium did not show an induced polypeptide pattern. The lack of ODC-ase activity promotes the expression of p27- and p31-coding genes in both strains but transcription of p31 gene is shut-off after spermidine addition. Both transcripts are also accumulated after exposure to low cycloheximide doses or nutrient starvation. Another cycloheximide-inducible gene coding for p70 is also expressed under DFMO-treatment.

Early response and induced tolerance to cycloheximide in Neurospora crassa.

Incubation of Neurospora crassa mycelia with low doses of cycloheximide induces the expression of several genes. After 6 h in the presence of cycloheximide, mycelia become tolerant to further additions of the drug and the rate of protein synthesis exhibits a lower sensitivity to it. The polypeptide pattern is indicative of a stress situation.

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.

Cyclic AMP-dependent, constitutive thermotolerance in the adenylate cyclase-deficient cr-1 (crisp) mutant of Neurospora crassa.

We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of Neurospora crassa. This strain was observed to be much more resistant to a lethal temperature of 50 degrees C than the wild type. This constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic AMP (cAMP, 2.5 mM) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 degrees C, a temperature which fails to induce thermotolerance in the wild-type strain. Two other crisp-like Neurospora mutants, cr-2 and cr-3 which, in contrast to cr-1, contain normal levels of cAMP, did not exhibit the thermotolerance phenomenon observed for cr-1. A cr-1, pe, fl (crisp-microconidial) strain also lacked the ability to tolerate a lethal heat treatment. Our results demonstrate that the adenylate cyclase deficient cr-1 mutant of Neurospora crassa expresses a constitutive thermotolerant phenotype as a consequence of its primary genetic defect: low levels of cAMP.

[Plant extracts with cytostatic properties growing in Cuba. II]. / Extractos de plants con propiedades citostáticas que crecen en Cuba. Parte II.

The study of the cytostatic activity of aqueous, alcoholic and ketonic extracts from 18 parts of 9 species of superior plants of the families Araceae, Borraginacease, Burseraceae, Cesalpinaceae, Meliaceae, Compositae, Rebiaceae, Cruciferaceae and Verbenaceae using the microbiologic method of described by Kubas in 1972 is pursued. The best results were obtained from Hamelia patens. Lippia alba, Lepidium virginicum, Cassia ligustrina, Bursera simaruba and Heliotropium campechianum extracts.

[Plant extracts with cytostatic properties growing in Cuba. I]. / Extractos de plantas con propiedades citostáticas que crecen en Cuba. Parte I.

The cytostatic activity of aqueous, alcoholic and ketonic extracts of 9 species of superior plants of the families Fitolacaceae, Compositae, Moraceae, Zingiberaceae, Martiniaceae, Mirtaceae, Verbenaceae and Annonaceae was assessed. The Kubas microbiologic method and the fungus Ascomiceto Neurospora crassa were used in the assessment. The fungus growth was measured in millimeters. Inhibition percentages for every case regarding control are reported. The best results were obtained from Annona muricata, Costus spiralis, Cecropia peltata, Xanthium chinense and Pluchea adorata extracts.