RESUMEN
IMPORTANCE: In addition to being considered a biocontrol agent, the fungus Trichoderma atroviride is a relevant model for studying mechanisms of response to injury conserved in plants and animals that opens a new landscape in relation to regeneration and cell differentiation mechanisms. Here, we reveal the co-functionality of a lipoxygenase and a patatin-like phospholipase co-expressed in response to wounding in fungi. This pair of enzymes produces oxidized lipids that can function as signaling molecules or oxidative stress signals that, in ascomycetes, induce asexual development. Furthermore, we determined that both genes participate in the regulation of the synthesis of 13-HODE and the establishment of the physiological responses necessary for the formation of reproductive aerial mycelium ultimately leading to asexual development. Our results suggest an injury-induced pathway to produce oxylipins and uncovered physiological mechanisms regulated by LOX1 and PLP1 to induce conidiation, opening new hypotheses for the novo regeneration mechanisms of filamentous fungi.
Asunto(s)
Trichoderma , Animales , Trichoderma/genética , Transducción de Señal , Micelio , Reproducción , Estrés Oxidativo , Regulación Fúngica de la Expresión Génica , Esporas Fúngicas/metabolismoRESUMEN
Bacillus subtilis species complex is known as lipopeptide-producer with biotechnological potential for pharmaceutical developments. This study aimed to identify lipopeptides from a bacterial isolate and evaluate their antifungal effects. Here, we isolated and identified a lipopeptide-producing bacterium as a species of Bacillus subtilis complex (strain UL-1). Twenty lipopeptides (six iturins, six fengycins, and eight surfactins) were identified in the crude extract (CE) and fractions (F1, F2, F3, and F4), and the highest content of total lipopeptides was observed in CE and F2. The chemical quantification data corroborate with the hemolytic and antifungal activities that CE and F2 were the most hemolytic and inhibited the fungal growth at lower concentrations against Fusarium spp. In addition, they caused morphological changes such as shortening and/or atypical branching of hyphae and induction of chlamydospore-like structure formation, especially in Fusarium solani. CE was the most effective in inhibiting the biofilm formation and in disrupting the mature biofilm of F. solani reducing the total biomass and the metabolic activity at concentrations ≥ 2 µg/mL. Moreover, CE significantly inhibited the adherence of F. solani conidia on contact lenses and nails as well as disrupted the pre-formed biofilms on nails. CE at 100 mg/kg was nontoxic on Galleria mellonella larvae, and it reduced the fungal burden in larvae previously infected by F. solani. Taken together, the lipopeptides obtained from strain UL-1 demonstrated a potent anti-Fusarium effect inducing morphological alterations and antibiofilm activities. Our data open further studies for the biotechnological application of these lipopeptides as potential antifungal agents. KEY POINTS: ⢠Lipopeptides inhibit Fusarium growth and induce chlamydospore-like structures. ⢠Lipopeptides hamper the adherence of conidia and biofilms of Fusarium solani. ⢠Iturins, fengycins, and surfactins were associated with antifungal effects.
Asunto(s)
Antifúngicos , Bacillus subtilis , Bacillus subtilis/metabolismo , Antifúngicos/química , Esporas Fúngicas/metabolismo , Biopelículas , Lipopéptidos/metabolismo , Péptidos Cíclicos/metabolismo , Enfermedades de las Plantas/microbiologíaRESUMEN
In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (â³bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The â³bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.
Asunto(s)
Neurospora crassa , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , GTP Fosfohidrolasas/metabolismo , HifaRESUMEN
Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.
Asunto(s)
Aspergillus fumigatus , Águilas , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Caspofungina/farmacología , Águilas/metabolismo , Equinocandinas/metabolismo , Equinocandinas/farmacología , Proteínas Fúngicas/metabolismo , Pruebas de Sensibilidad Microbiana , Esporas Fúngicas/metabolismoRESUMEN
Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.
Asunto(s)
Aspergillus fumigatus/genética , Quinazolinas/metabolismo , Aspergillus fumigatus/metabolismo , Pared Celular/genética , Proteínas Fúngicas/genética , Expresión Génica , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fagocitosis/fisiología , Transducción de Señal , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Transcripción GenéticaRESUMEN
Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.
Asunto(s)
Aflatoxinas/biosíntesis , Antifúngicos/síntesis química , Antifúngicos/farmacología , Aspergillus flavus/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/síntesis química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Aspergillus flavus/genética , Aspergillus flavus/crecimiento & desarrollo , Aspergillus flavus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Metabolismo Secundario , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismoRESUMEN
The chitinases have extensive biotechnological potential but have been little exploited commercially due to the low number of good chitinolytic microorganisms. The purpose of this study was to identify a chitinolytic fungal and optimize its production using solid state fermentation (SSF) and agroindustry substrate, to evaluate different chitin sources for chitinase production, to evaluate different solvents for the extraction of enzymes produced during fermentation process, and to determine the nematicide effect of enzymatic extract and biological control of Meloidogyne javanica and Meloidogyne incognita nematodes. The fungus was previously isolated from bedbugs of Tibraca limbativentris Stal (Hemiptera: Pentatomidae) and selected among 51 isolated fungal as the largest producer of chitinolytic enzymes in SSF. The isolate UFSMQ40 has been identified as Trichoderma koningiopsis by the amplification of tef1 gene fragments. The greatest chitinase production (10.76 U gds-1) occurred with wheat bran substrate at 55% moisture, 15% colloidal chitin, 100% of corn steep liquor, and two discs of inoculum at 30 °C for 72 h. Considering the enzymatic inducers, the best chitinase production by the isolated fungus was achieved using chitin in colloidal, powder, and flakes. The usage of 1:15 g/mL of sodium citrate-phosphate buffer was the best ratio for chitinase extraction of SSF. The Trichoderma koningiopsis UFSMQ40 showed high mortality of M. javanica and M. incognita when applied to treatments with enzymatic filtrated and the suspension of conidia.
Asunto(s)
Quitina/metabolismo , Quitinasas/biosíntesis , Fermentación , Hypocreales/enzimología , Animales , Chinches/microbiología , Agentes de Control Biológico , Biotecnología , Fibras de la Dieta , Nematodos/efectos de los fármacos , Esporas Fúngicas/metabolismo , Temperatura , Zea maysRESUMEN
Commercial products based on Trichoderma are obtained mainly from solid-state fermentation. Submerged liquid fermentation is the most appropriate method compared to the solid medium for large-scale production of Trichoderma spp. The present study aimed to optimize the combination of key variables that influence the liquid fermentation process of Trichoderma asperelloides LQC-96 for conidial production coupled with its efficiency in the control of Sclerotinia sclerotiorum. In addition, we verified whether the optimized culture conditions can be used for the conidial production of Trichoderma erinaceum T-12 and T-18 and Trichoderma harzianum T-15. Fermentation studies were performed in shake flasks following a planned experimental design to reduce the number of tests and consumable costs. The effect of temperature, pH, photoperiod, carbon:nitrogen ratio and water activity on conidial production were assessed, which of pH was the only meaningful factor contributing to increased conidial production of T. asperelloides LQC-96. From the five variables studied initially, pH and C:N ratio were further used in the second design (rotational central composite design-RCCD). Hence, the best conditions for the production of T. asperelloides LQC-96 conidia by liquid fermentation consisted of initial pH of 3.5, C:N ratio of 200:1 at 30 °C, without glycerol, and under 24 h photoperiod. The highest conidial concentration was observed after seven days of fermentation. Under these optimal conditions, T. erinaceum T-12 and T-18, and T. harzianum T-15 were also cultivated, but only LQC-96 efficiently parasitized S. sclerotiorum, precluding sclerotium myceliogenic germination. Our findings propose optimal fermentation conditions that maximize conidial production of T. asperelloides as a potential biofungicide against S. sclerotiorum.
Asunto(s)
Ascomicetos/metabolismo , Fermentación , Hypocreales/metabolismo , Reactores Biológicos , Carbono/metabolismo , Medios de Cultivo/química , Concentración de Iones de Hidrógeno , Nitrógeno/metabolismo , Fotoperiodo , Esporas Fúngicas/metabolismo , TemperaturaRESUMEN
The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5 mM uracil restores their growth and conidia production, and 1 mg ml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.
Asunto(s)
Proteínas Fúngicas/genética , Hypocreales , Orotidina-5'-Fosfato Descarboxilasa , Transformación Genética , Biomarcadores/metabolismo , Genes Fúngicos , Hypocreales/genética , Hypocreales/crecimiento & desarrollo , Hypocreales/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/genética , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Esporas Fúngicas/metabolismoRESUMEN
Aim: The encapsulation of Trichoderma asperellum BRM-29104 using Ca-alginate matrix was evaluated.Methods: Intact and freeze-dried beads containing submerged conidia and microsclerotia (MS) of T. asperellum grown in liquid culture were prepared and characterised. Beads were stored at 8, 25, and 35 °C over 120 days.Results: The mean sizes of beads before and after freeze-drying were 2.5 ± 0.2 mm and 1.5 × 1.1 mm (± 0.1 mm), respectively. Freeze-dried beads stored at 8 °C were more effective in maintaining conidia concentration, while MS concentrations yielded 102 MS/g for both beads at 8 and 25 °C. The concentration of viable cells in freeze-dried beads stored at 8 °C attained 3.0 × 108 CFU/g after 120 days. FIRT analysis showed an interaction between the alginate and the cell wall of the fungus.Conclusion: These findings support the use of alginate beads followed by freeze drying and cold storage for maintenance of viability of T. asperellum.
Asunto(s)
Alginatos/química , Hypocreales , Viabilidad Microbiana , Esporas Fúngicas , Liofilización , Hypocreales/química , Hypocreales/metabolismo , Esporas Fúngicas/química , Esporas Fúngicas/metabolismoRESUMEN
Numerous postharvest diseases have been reported that cause substantial losses of citrus fruits worldwide. Penicillium digitatum is responsible for up to 90% of production losses, and represent a problem for worldwide economy. In order to control phytopathogens, chemical fungicides have been extensively used. Yet, the use of some artificial fungicides cause concerns about environmental risks and fungal resistance. Therefore, studies focusing on new approaches, such as the use of natural products, are getting attention. Co-culture strategy can be applied to discover new bioactive compounds and to understand microbial ecology. Mass Spectrometry Imaging (MSI) was used to screen for potential antifungal metabolites involved in the interaction between Penicillium digitatum and Penicillium citrinum. MSI revealed a chemical warfare between the fungi: two tetrapeptides, deoxycitrinadin A, citrinadin A, chrysogenamide A and tryptoquialanines are produced in the fungi confrontation zone. Antimicrobial assays confirmed the antifungal activity of the investigated metabolites. Also, tryptoquialanines inhibited sporulation of P. citrinum. The fungal metabolites reported here were never described as antimicrobials until this date, demonstrating that co-cultures involving phytopathogens that compete for the same host is a positive strategy to discover new antifungal agents. However, the use of these natural products on the environment, as a safer strategy, needs further investigation. This paper aimed to contribute to the protection of agriculture, considering health and ecological risks.
Asunto(s)
Citrus/crecimiento & desarrollo , Proteínas Fúngicas/genética , Penicillium/metabolismo , Enfermedades de las Plantas/microbiología , Antifúngicos/metabolismo , Citrus/genética , Citrus/parasitología , Frutas/microbiología , Proteínas Fúngicas/metabolismo , Hongos/efectos de los fármacos , Hongos/patogenicidad , Fungicidas Industriales/farmacología , Espectrometría de Masas , Penicillium/patogenicidad , Enfermedades de las Plantas/genética , Esporas Fúngicas/metabolismoRESUMEN
Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1ß, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.
Asunto(s)
Mucor/patogenicidad , Mucormicosis/sangre , Suero/microbiología , Esporas Fúngicas/metabolismo , Animales , Citocinas/metabolismo , Diabetes Mellitus Experimental , Humanos , Peróxido de Hidrógeno , Hifa/crecimiento & desarrollo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Pulmón , Macrófagos/microbiología , Masculino , Ratones , ARN Mensajero/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , VirulenciaRESUMEN
Positive feedback between arbuscular mycorrhizal fungal (AMF) and vascular plants can contribute to plant species establishment, but how this feedback affects plant invasion by Prosopis juliflora SW. (DC.), or resistance to invasion by Mimosa tenuiflora (Willd.) Poir in Brazilian semi-arid region is not well known. In this work, we tested how modified and native AMF communities affect the establishment of P. juliflora and M. tenuiflora plants. We examined the effects of inoculation with modified and native AMF communities on number of AMF spores, root colonization, number of N-fixing nodules, plant dry biomass, plant phosphorous concentration, and plant responsiveness to mycorrhizas of P. juliflora and M. tenuiflora. We found that the modified AMF community enhanced the root colonization, plant dry biomass, and plant phosphorous concentration of invasive P. juliflora, whereas native AMF enhanced M. tenuiflora. Our results demonstrate that the invasive P. juliflora alters soil AMF community composition, and this change generates positive feedback to the invasive P. juliflora itself and decreases AMF associations with native M. tenuiflora.
Asunto(s)
Fabaceae/microbiología , Hongos/aislamiento & purificación , Mimosa/microbiología , Micorrizas/aislamiento & purificación , Biodiversidad , Brasil , Fabaceae/crecimiento & desarrollo , Hongos/clasificación , Hongos/genética , Hongos/metabolismo , Especies Introducidas , Mimosa/crecimiento & desarrollo , Micorrizas/clasificación , Micorrizas/genética , Micorrizas/metabolismo , Fósforo/metabolismo , Raíces de Plantas/microbiología , Suelo/química , Microbiología del Suelo , Esporas Fúngicas/clasificación , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/aislamiento & purificación , Esporas Fúngicas/metabolismoRESUMEN
AIM: To describe a new approach in which production of conidia of an entomopathogenic fungus takes place on the surface of an unstirred shallow liquid culture kept in nonabsorbent wells distributed in plastic sheets resembling a honeycomb. METHODS AND RESULTS: First, liquid incubation time and medium composition for production of Beauveria bassiana aerial conidia were optimized. Wells inoculated with Sabouraud dextrose yeast extract produced 2·2 × 108 conidia per cm2 of liquid surface following 5 days of incubation. Finally, tests were carried out in a prototype comprised of stacked plastic sheets in a cylindrical container. Conidia production on liquid culture surface varied from 1·2 to 1·6 × 109 conidia per ml of fermented broth. Germination rates and insect activity towards Tenebrio molitor larvae were not negatively affected when compared to conidia produced on solid medium. CONCLUSIONS: The two-stage fermentation process here described, based on a simple nonabsorbent inert support, has potential for the application in the production of aerial conidia of B. bassiana and other fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerial conidia are the most extensive propagule type used in commercial mycopesticides, traditionally produced by solid-state fermentation (SSF). The industrial applications and other important benefits of the two-stage fermentation process here described may overcome some hurdles inherent to SSF aiming for the production of aerial conidia. Additionally, production consistency is increased by the use of chemically defined medium, and the better control of the environmental conditions could allow for more reproducible industrial batches.
Asunto(s)
Beauveria/crecimiento & desarrollo , Microbiología Industrial/métodos , Esporas Fúngicas/crecimiento & desarrollo , Animales , Beauveria/química , Beauveria/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Fermentación , Microbiología Industrial/instrumentación , Larva/microbiología , Esporas Fúngicas/química , Esporas Fúngicas/metabolismo , Tensión Superficial , Tenebrio/microbiologíaRESUMEN
Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.
Asunto(s)
Acremonium/genética , Electroporación/métodos , Transformación Genética , Acremonium/efectos de los fármacos , Acremonium/metabolismo , Cefalosporinas/biosíntesis , Farmacorresistencia Bacteriana , Viabilidad Microbiana , Micelio/efectos de los fármacos , Micelio/genética , Micelio/metabolismo , Fleomicinas/farmacología , Protoplastos/fisiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismoRESUMEN
The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.
Asunto(s)
Alcanos/metabolismo , Beauveria/crecimiento & desarrollo , Beauveria/patogenicidad , Entomophthorales/crecimiento & desarrollo , Entomophthorales/patogenicidad , Estrés Oxidativo , Animales , Beauveria/metabolismo , Membrana Celular/ultraestructura , Proliferación Celular , Entomophthorales/metabolismo , Hifa/metabolismo , Insectos/microbiología , Larva/microbiología , Peroxisomas/fisiología , Esporas Fúngicas/metabolismo , VirulenciaRESUMEN
AIMS: Nitrogen is a critical element in industrial fermentation media. This study investigated the influence of various nitrogen sources on blastospore production, desiccation tolerance and storage stability using two strains of the cosmopolitan insect-pathogenic fungus Beauveria bassiana. METHODS AND RESULTS: Complex organic sources of nitrogen such as soy flour, autolysed yeast and cottonseed flour induced great numbers of blastospores after 2-3 days of fermentation, which also survived drying and remained viable (32-56% survival) after 9 months storage at 4°C, although variations were found between strains. Nitrogen availability in the form of free amino acids directly influenced blastospore production and resistance to desiccation. Increasing glucose and nitrogen concentrations up to 120 and 30 g l-1 , respectively, did not improve blastospore production but enhanced desiccation tolerance. Cell viability after drying and upon fast-rehydration was increased when ≥25 g acid-hydrolysed casein per litre was supplemented in the liquid culture medium. CONCLUSIONS: These findings indicate that low-cost complex nitrogen compounds are suitable to enhance yeast-like growth by B. bassiana with good desiccation tolerance and therefore support its further scale-up production as a mycoinsecticide. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen is the most expensive nutrient in liquid media composition, but this study underscores the feasibility of using low-cost nitrogen compounds composed mainly of agro-industrial by-products for rapid production of desiccation-tolerant B. bassiana blastospores by liquid culture fermentation.
Asunto(s)
Beauveria/metabolismo , Nitrógeno/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Beauveria/química , Beauveria/crecimiento & desarrollo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Desecación , Fermentación , Glucosa/metabolismo , Preservación Biológica , Esporas Fúngicas/química , Esporas Fúngicas/metabolismo , Agua/análisis , Agua/metabolismoRESUMEN
The candidiasis caused by C. albicans is a public health problem. The abuse of antifungals has contributed to the development of resistance. B. morelensis has demonstrated antibacterial and antifungal activities. In this work the activity of the essential oil of B. morelensis was evaluated and for its two pure compounds with analysis of the different mechanisms of pathogenesis important for C. albicans. The essential oil was obtained by the hydro-distillation method and analyzed using GC-MS. The anti-Candida activity was compared between to essential oil, α-Pinene and γ-Terpinene. GC-MS of the essential oil demonstrated the presence of 13 compounds. The essential oil showed antifungal activity against four C. albicans strains. The most sensitive strain was C. albicans 14065 (MFC 2.0 mg/mL and MIC50 0.125 mg/mL) with α-Pinene and γ-Terpinene having MFCs of 4.0 and 16.0 mg/mL respectively. The essential oil inhibited the growth of the germ tube in 87.94% (8.0 mg/mL). Furthermore, it was observed that the essential oil diminishes the transcription of the gene INT1. This work provides evidence that confirms the anti-Candida activity of the B. morelensis essential oil and its effect on the growth of the germ tube and transcription of the gene INT1.
Asunto(s)
Antifúngicos/farmacología , Bursera/química , Candida/efectos de los fármacos , Monoterpenos/farmacología , Esporas Fúngicas/efectos de los fármacos , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Monoterpenos Bicíclicos , Candida/genética , Candida/crecimiento & desarrollo , Candida/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Monoterpenos Ciclohexánicos , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Expresión Génica , Pruebas de Sensibilidad Microbiana , Monoterpenos/aislamiento & purificación , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Aceites Volátiles/farmacología , Extractos Vegetales/química , Aceites de Plantas/química , Aceites de Plantas/aislamiento & purificación , Aceites de Plantas/farmacología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismoRESUMEN
The aim of this study was to determine the antifungal activity of Drimenol (1) and its synthetic derivatives, nordrimenone (2), drimenyl acetate (3), and drimenyl-epoxy-acetate (4), and to establish a possible mechanism of action for drimenol. For that, the effect of each compound on mycelial growth of Botrytis cinerea was assessed. Our results showed that compounds 1, 2, 3 and 4 are able to affect Botrytis cinerea growth with EC50 values of 80, 92, 80 and 314ppm, respectively. These values suggest that the activity of these compounds is mainly determined by presence of the double bond between carbons 7 and 8 of the drimane ring. In addition, germination of B. cinerea in presence of 40 and 80ppm of drimenol is reduced almost to a half of the control value. Finally, in order to elucidate a possible mechanism by which drimenol is affecting B. cinerea, the determination of membrane integrity, reactive oxygen species production and gene expression studies of specific genes were performed.
Asunto(s)
Antifúngicos/farmacología , Botrytis/efectos de los fármacos , Fungicidas Industriales/farmacología , Esporas Fúngicas/efectos de los fármacos , Terpenos/farmacología , Antifúngicos/química , Botrytis/metabolismo , Fungicidas Industriales/química , Sesquiterpenos Policíclicos , Especies Reactivas de Oxígeno/metabolismo , Esporas Fúngicas/metabolismo , Terpenos/químicaRESUMEN
BACKGROUND: Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches' broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. RESULTS: A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. CONCLUSIONS: In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease.