Stimulation of bikaverin production by sucrose and by salt starvation in Fusarium fujikuroi

The fungus Fusarium fujikuroi (Gibberella fujikuroi mating group C) exhibits a rich secondary metabolism that includes the synthesis of compounds of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. The effect of the carbon source on their production was checked using a two-phase incubation protocol, in which nine different sugars were added upon transfer of the fungus from repressed to appropriate inducing conditions, i.e., nitrogen starvation for gibberellins and bikaverin and illumination for carotenoids production. Most of the carbon sources allowed the synthesis of these metabolites in significant amounts. However, bikaverin production was strongly increased by the presence of sucrose in comparison to other carbon sources, an effect not exhibited for the production of gibberellins and carotenoids. The bikaverin inducing effect was enhanced in the absence of phosphate and/or sulfate. Similar results were also observed in carotenoid-overproducing strains known to be altered in bikaverin production. The induction by salt starvation, but not by sucrose, correlated with an increase in messenger RNA levels of gene bik1, encoding a polyketide synthase of the bikaverin pathway.     

Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.     

Nitrogen availability and production of bikaverin and gibberellins in Gibberella fujikuroi

Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the nitrogen source, but not after depletion of phosphate. Despite this similarity, the regulation of both pathways by nitrogen involved two different mechanisms. This conclusion was supported by the fact that the production of bikaverin, in contrast to the gibberellins, was not inhibited by nitrate in a mutant that could not utilize it. The different regulation of both pathways was clearly demonstrated by a mutant that overproduced bikaverin but lacked gibberellins. An optimal bikaverin production required a low pH, with a sharp drop at about pH 5. The syntheses of fungal secondary metabolites, such as bikaverin and gibberellins, are not subject to common regulation, but respond to various combinations of signals, such as nitrogen availability, nitrate and the pH of the medium.     

Functional analysis of all nonribosomal peptide synthetases in Cochliobolus heterostrophus reveals a factor, NPS6, involved in virulence and resistance to oxidative stress

Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.     

GAC1, a gene encoding a putative GTPase-activating protein, regulates bikaverin biosynthesis in Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GACI locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wildtype GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

T‐DNA insertion,plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium‐mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T‐DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T‐DNA right border (RB)–genomic DNA junctions in Escherichia coli. Fifty‐one Agrobacterium‐transformed fungal strains, picked up at random from a larger collection of T‐DNA tagged strains (about 500), were analysed. Sixty‐nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T‐DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T‐DNA borders indicating non‐homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium‐mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.     

Bikaverin,an antibiotic fromGibberella fujikuroi,effective againstLeishmania brasiliensis

The red quinone C₂₀H₁₄O₈ (mp 322–324), named bikaverin was isolated from mycelium ofGibberella fujikuroi. Bikaverin is specifically effective againstLeishmania brasiliensis while is not effective against other protozoa, bacteria and fungi. For production of bikaverin in a submerged culture the most convenient media are those with a natural source of nitrogen (corn-steep liquor, soyabean meal) in which the content of bikaverin ranged from 1.0 to 1.7% of dry weight of mycelium.     

Phylogenetically Diverse Cultivable Fungal Community and Polyketide Synthase (PKS), Non-ribosomal Peptide Synthase (NRPS) Genes Associated with the South China Sea Sponges

Compared with sponge-associated bacteria, the phylogenetic diversity of fungi in sponge and the association of sponge fungi remain largely unknown. Meanwhile, no detection of polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) genes in sponge-associated fungi has been attempted. In this study, diverse and novel cultivable fungi including 10 genera (Aspergillus, Ascomycete, Fusarium, Isaria, Penicillium, Plectosphaerella, Pseudonectria, Simplicillium, Trichoderma, and Volutella) in four orders (Eurotiales, Hypocreales, Microascales, and Phyllachorales) of phylum Ascomycota were isolated from 10 species marine sponges in the South China Sea. Eurotiales and Hypocreales fungi were suggested as sponge generalists. The predominant isolates were Penicillium and Aspergillus in Eurotiales followed by Volutella in Hypocreales. Based on the conserved Beta-ketosynthase of PKS and A domain of NRPS, 15 polyketide synthases, and four non-ribosomal peptides synthesis genes, including non-reducing and reducing PKSs and hybrid PKS–NRPS, were detected in these fungal isolates. A lateral gene transfer event was indicated in the comparison between the phylogenetic diversity of 18S rRNA genes and β-ketoacyl synthase domain sequences. Some fungi, especially those with PKS or NRPS genes, showed antimicrobial activity against P. fluorescens, S. aureus and B. subtilis. It was the first time to investigate PKS and NRPS genes in sponge-associated fungi. Based on the detected antibiotics biosynthesis-related PKS and NRPS genes and antimicrobial activity, the potential ecological role of sponge-associated fungi in the chemical defense for sponge host was suggested. This study extended our knowledge of sponge-associated fungal phylogenetic diversity and their potential roles in the chemical defense.     

Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal

The ability of the fungus Fusarium oxysporum to solubilize lignite was found to depend on the presence of a specific carbon source. When grown on glucose or another carbohydrate, the fungus is unable to solubilize coal but it produces the red dye bikaverin. In the coal-solubilizing state, which can be induced by cultivation in the presence of glutamate or gluconate, the fungus does not produce bikaverin. The presence or absence of the pigment can therefore be taken as an indicator of the ability of the fungus to solubilize coal. Addition of extracted and purified bikaverin to F. oxysporum growing on glutamate or gluconate inhibits coal solubilization. Hence, F. oxysporum offers a suitable system for investigating the mechanism of microbial coal degradation by comparing the two growth-substrate-controlled physiological states.     

Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.     

Redirection of pigment biosynthesis to isocoumarins in Fusarium

Colonies of Fusarium species often appear red due to production of pigments, such as aurofusarin or bikaverin. The primary compounds in these biosynthetic pathways are YWA1 and pre-bikaverin, respectively, catalyzed by two multidomain polyketide synthases (PKSs), which both have a claisen-type cyclase domain (CLC) in their N terminal. Disruption of the CLC domains has been shown to result in formation of the lactones citreoisocoumarin and SMA93 instead of YWA1 and pre-bikaverin. In the present study we have discovered a medium with low nitrogen content which partially redirects the aurofusarin and bikaverin pathways to produce citreoisocoumarin and SMA93, respectively. This is first time that SMA93 is identified in a fungus and we suggest that it is renamed bikisocoumarin, as it is derived from the bikaverin pathway. The redirection of the aurofusarin and bikaverin biosynthetic pathways was reverted by adding inorganic nitrogen to the medium, whereas organic nitrogen in form of arginine or glutamine stimulated isocoumarin production. This suggests that nitrogen source can influence isocoumarin production. Production of isocoumarin was also repressed by alkaline conditions, which suggests that nitrogen supply is not the sole regulatory factor in the pathway. The redirection was observed in all producers of aurofusarin (6) and bikaverin (2), suggesting the presence of a conserved regulatory mechanism.     

A secreted serine protease of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> and its interactions with fungal proteins

Background  

Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.