Multifarious Elicitors: Invoking Biosynthesis of Various Bioactive Secondary Metabolite in Fungi.

Natural products are considered to be the lifeline treatment for several diseases where their structural complexity makes them a source of potential lead molecules. As a producer of antibiotics, food colorants, enzymes, and nutritious food, fungi are beneficial to humans. Fungi, as a source of novel natural products, draw attention of scientists. However, redundant isolation of metabolite retards the rate of discovery. So, apart from the standard conditions for the production of secondary metabolites, certain induction strategies are used to trigger biosynthetic genes in fungi. Advancement in the computational tools helps in connecting gene clusters and their metabolite production. Therefore, modern analytical tools and the genomic era in hand leads to the identification of manifold of cryptic metabolites. The cryptic biosynthetic gene cluster (BGC) has become a treasure hunt for new metabolites representing biosynthetic pathways, regulatory mechanisms, and other factors. This review includes the use of chemical inducers/epigenetic modifiers and co-culture (species interaction) techniques to induce these BGCs. Furthermore, it cites a detailed representation of molecules isolated using these strategies. Since the induction occurs on the genomic molecular DNA and histones, this together brings a significant exploration of the biosynthetic pathways.Graphical Abstract.

Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development, stress resistance and longevity of Thermomyces lanuginosus.

The ATP-dependent Lon enzyme is a highly conserved protease with multiple roles in diverse species such as fungi; however, there are few reports on Lon enzymes in filamentous fungi. Thermomyces lanuginosus, a typical thermophilic fungus, has been widely studied in physiology and cell biology; thus, studies on Thermomyces Lons are important. Two Lons were bioinformatically deduced in T. lanuginosus. Subcellular localization analysis showed that one is present in mitochondria (MLon), while the other is found in peroxisomes (PLon). Although both Lon enzymes were activated by H2O2, they were not induced by heat shock; instead, they were induced by low temperatures. Two single-deletion Lon mutants (ΔMLon and ΔPLon) were generated. Biological analysis demonstrated that ΔMLon decreased the production of conidia but increased the growth of mycelia. By contrast, ΔPLon increased the production of conidia but decreased the growth of mycelia. The lifespan was measured in time and in length of continuous growth. The wild-type strain showed continuous linear growth for 60days, whereas growth was impeded at 30 and 50days for ΔPLon and ΔMLon mutants, respectively, suggesting that PLon is more important for longevity than MLon. Interestingly, ΔPLon, which accumulated larger amount of H2O2 was not only more sensitive to exogenous H2O2 but also much more sensitive to other selected stressors. Taken together, our data indicate that mitochondrial and peroxisomal Lons play opposite roles in controlling growth and development, but exhibit synergistic effects on the normal states of vegetative growth, asexual development, stress resistance and longevity in T. lanuginosus.

Water-, pH- and temperature relations of germination for the extreme xerophiles Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ) and Eurotium halophilicum (FRR 2471).

Water activity, temperature and pH are determinants for biotic activity of cellular systems, biosphere function and, indeed, for all life processes. This study was carried out at high concentrations of glycerol, which concurrently reduces water activity and acts as a stress protectant, to characterize the biophysical capabilities of the most extremely xerophilic organisms known. These were the fungal xerophiles: Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ) and Eurotium halophilicum (FRR 2471). High-glycerol spores were produced and germination was determined using 38 media in the 0.995-0.637 water activity range, 33 media in the 2.80-9.80 pH range and 10 incubation temperatures, from 2 to 50°C. Water activity was modified by supplementing media with glycerol+sucrose, glycerol+NaCl and glycerol+NaCl+sucrose which are known to be biologically permissive for X. bisporus, A. penicillioides and E. halophilicum respectively. The windows and rates for spore germination were quantified for water activity, pH and temperature; symmetry/asymmetry of the germination profiles were then determined in relation to supra- and sub-optimal conditions; and pH- and temperature optima for extreme xerophilicity were quantified. The windows for spore germination were ~1 to 0.637 water activity, pH 2.80-9.80 and > 10 and < 44°C, depending on strain. Germination profiles in relation to water activity and temperature were asymmetrical because conditions known to entropically disorder cellular macromolecules, i.e. supra-optimal water activity and high temperatures, were severely inhibitory. Implications of these processes were considered in relation to the in-situ ecology of extreme conditions and environments; the study also raises a number of unanswered questions which suggest the need for new lines of experimentation.

Thermophilic fungi in an aridland ecosystem.

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45-50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.

The extreme xerophilic mould Xeromyces bisporus--growth and competition at various water activities.

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity (a(w)) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 °C on solid agar media containing a mixture of glucose and fructose to reduce a(w) to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium inops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a(w) and 30°C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a(w) (0.48 mm/day). C. inops and C. xerophilum were more tolerant of high a(w) than X. bisporus, and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures ≥ 30 °C and a(w) ≥ 0.94 (c.f.≤ 25 °C and ~0.88 a(w) for C. inops); and its ability to grow at 0.66 a(w), which is the lowest a(w) reported to date for this species. M. eremophilus grew slowly (max. 0.4mm/day) even in its optimal conditions of ~0.88 a(w) and 25 °C. To investigate the competitive characteristics of X. bisporus at low a(w), both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. inops, C. xerophilum and Eurotium chevalieri, on glucose-fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a(w) and at 25 °C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. inops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species, A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a(w). E. chevalieri was often dominant due to its rapid growth over the entire a(w) range. At a(w)<0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a(w).

Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes.

AIMS: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied. METHODS AND RESULTS: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration. CONCLUSIONS: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The success of these chemical treatments might depend on the mycobiota composition of grapes.

Use of a MS-electronic nose for prediction of early fungal spoilage of bakery products.

A MS-based electronic nose was used to detect fungal spoilage (measured as ergosterol concentration) in samples of bakery products. Bakery products were inoculated with different Eurotium, Aspergillus and Penicillium species, incubated in sealed vials and their headspace sampled after 2, 4 and 7 days. Once the headspace was sampled, ergosterol content was determined in each sample. Different electronic nose signals were recorded depending on incubation time. Both the e-nose signals and ergosterol levels were used to build models for prediction of ergosterol content using e-nose measurements. Accuracy on prediction of those models was between 87 and 96%, except for samples inoculated with Penicillium corylophilum where the best predictions only reached 46%.

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5.

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.

An attempt to optimize potassium sorbate use to preserve low pH (4.5-5.5) intermediate moisture bakery products by modelling Eurotium spp., Aspergillus spp. and Penicillium corylophilum growth.

Mould growth was modelled on fermented bakery product analogues (FBPA) of two different pH (4.5 and 5.5), different water activity (a(w)) levels (0.80-0.90) and potassium sorbate concentrations (0-0.3%) by using seven moulds commonly causing spoilage of bakery products (Eurotium spp., Aspergillus spp. and Penicillium corylophilum). For the description of fungal growth (growth rates) as a function of a(w), potassium sorbate concentration and pH, 10-terms polynomial models were developed. Modelling enables prediction of spoilage during storage as a function of the factors affecting fungal growth. At pH 4.5 the concentration of potassium sorbate could be reduced to some extent only at low levels of a(w), whereas at pH 5.5 fungal growth was observed even by adding 0.3% of potassium sorbate. However, this preservative could be a valuable alternative as antifungal in such bakery product, of slightly acidic pH, if a long shelf life has not to be achieved.

Culture media and sources of nitrogen promoting the formation of stromata and ascocarps in Petromyces alliaceus (Aspergillus section Flavi).

Petromyces alliaceus Malloch and Cain is the only known sexually reproducing fungus classified in Aspergillus section Flavi. The goal of this research was to identify culture media and sources of nitrogen that best support the formation of stromata with ascocarps. Three cultures of P. alliaceus isolated from crop field soils were grown on selected agar media in Petri dishes for 7 months at 30 degrees C in darkness. The largest numbers of stromata were recorded for cultures grown on Czapek's agar (CZA) and a mixed cereal agar (MCA), while the percentage of stromata containing ascocarps was greatest (P

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum.

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw.. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Early detection of fungal growth in bakery products by use of an electronic nose based on mass spectrometry.

This paper presents the design, optimization, and evaluation of a mass spectrometry-based electronic nose (MS e-nose) for early detection of unwanted fungal growth in bakery products. Seven fungal species (Aspergillus flavus, Aspergillus niger, Eurotium amstelodami, Eurotium herbariorum, Eurotium rubrum, Eurotium repens, and Penicillium corylophillum) were isolated from bakery products and used for the study. Two sampling headspace techniques were tested: static headspace (SH) and solid-phase microextraction (SPME). Cross-validated models based on principal component analysis (PCA), coupled to discriminant function analysis (DFA) and fuzzy ARTMAP, were used as data treatment. When attempting to discriminate between inoculated and blank control vials or between genera or species of in vitro growing cultures, sampling based on SPME showed better results than those based on static headspace. The SPME-MS-based e-nose was able to predict fungal growth with 88% success after 24 h of inoculation and 98% success after 48 h when changes were monitored in the headspace of fungal cultures growing on bakery product analogues. Prediction of the right fungal genus reached 78% and 88% after 24 and 96 h, respectively.

Efficacy of sorbates on the control of the growth of Eurotium species in bakery products with near neutral pH.

The effects of sorbic acid and potassium sorbate on growth of different Eurotium isolates when added to a bakery product analogue were tested under different environmental conditions. Water activity of the products was adjusted to values in the range of 0.75-0.90, and storage temperatures were in the range of 15-30 degrees C. Preservatives were added in concentrations ranging from 0.025% to 0.2%. It was observed that 0.025% and 0.05% concentrations always enhanced the isolates growth, while 0.1% had little preservative effect. Finally, even the highest concentration (0.2%) was not suitable as it only controlled fungal growth under certain water activity and temperature levels. It was concluded that these weak-acid preservatives are not useful when added to bakery products with near to neutral pH.

Kinetics of the solid state fermentation of sugarcane bagasse by Thermoascus aurantiacus for the production of xylanase.

Xylanase was produced by solid-state fermentation using Thermoascus aurantiacus. Maximum production (500 U g(-1) bagasse) was achieved on the sixth day of cultivation on solid sugarcane bagasse medium supplemented with 15% (v/w) rice bran extract. The fungal biomass, determined from its glucosamine content, reached 28 mg g(-1) on the 8th day of cultivation. The cell yield against O2 (Y(x/o) = 0.18 g(cell)/g(O2)) and maintenance coefficient (m0 = 0.013 g(O2)/g(cell)h) were determined with the low Y(x/o) value for T. aurantiacus agreeing with the calculated value.

New filamentous fungus Sagenomella chlamydospora responsible for a disseminated infection in a dog.

A filamentous fungus that caused a fatal systemic infection in a dog has been identified as the new species Sagenomella chlamydospora. When the case was initially reported, the fungus was identified as Paecilomyces sp. This study emphasizes how difficult can be the identification of the causative agent of an infection when an uncommon microorganism is involved. This is the first time that this genus has been involved in animal infections, including humans.