Determination Of Fungal Biomass Research Articles

Isoflavaspidic Acid PB Extracted from Dryopteris fragrans (L.) Schott Inhibits Trichophyton rubrum Growth via Membrane Permeability Alternation and Ergosterol Biosynthesis Disruption.

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum (T. rubrum). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20 μg/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC, 1 × MIC, and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14α-demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC. These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use.

Enhancement of biocontrol potential of biocompatible bovine serum albumin (BSA) based protein nanoparticles loaded bacterial chitinase against major plant pathogenic fungi Alternaria alternata

Abstract Recently, principles of nanoscience and nanotechnology has being embraced in the field of agriculture rather than medicine and health care which has the potential to revolutionize modern day agriculture by effectively controlling insect pests and disease causing phytopathogenic microorganisms. In the present study, biocontrol potential of bovine serum albumin nanoparticles (BSA Nps) loaded extra cellular chitinase (BSA Np-CHS) produced by Serratia marcescens SU05 was studied against phytopathogenic fungi Alternaria alternata. Chitinase was extracted from the chitinase production medium that produced by optimizing various nutrient and process conditions adopting Taguchi method (37 °C temperature, 7 pH, 50 rpm and 1.5 inoculum) shows a extended production of enzyme. Extracted enzyme was purified by ammonium sulphate precipitation and column chromatography separation. Purified enzyme thus obtained was loaded with BSA Nps by cocoeravation method. The method for the preparation of BSA Np-CHS conjugate was optimized by various parameters. Nano enzyme conjugate thus prepared using optimal condition reveals spherical nanosphere with the size range of 110–120 nm and changes in the functional group that was studied by Fourier transform infrared spectroscopy (FTIR) analysis. Biocontrol efficacy of the prepared BSA Np-CHS nano enzyme conjugate against tested fungal strain was studied by determination of fungal biomass and fungal hyphal fragments damage under in vitro condition. BSA Np-CHS nano enzyme conjugate brought about effective reduction of fungal biomass and high rate of fungal hyphae fragments damage in all the tested concentration. Further study will helpful to formulate, apply the nanoformulation under field trails for effective control of phytopathogenic fungi.

Comparison of fatty acid methyl ester methods for characterization of microbial communities in forest and arable soil: Phospholipid fraction (PLFA) versus total ester linked fatty acids (EL-FAME)

Phospholipid fatty acid (PLFA) and ester-linked fatty acid methyl ester (EL-FAME) extraction methods have been commonly used for estimating microbial biomass and characterizing microbial community composition in soil. However, to our knowledge, there is currently no comparison of these two methods across different ecosystems. A regression analysis for 8 different soils showed that there were significant linear relationships between PLFA and EL-FAME extracts (r2=0.97, 0.98 and 0.72, respectively) for determining the fungal (18:2ω6,9) and bacterial abundance and fungal-to-bacterial ratio (F:B ratio). However, regression lines of forest soils for fungal abundance and F:B ratio had a slight but significantly higher intercept than those of arable soils, i.e. higher EL-FAME:PLFA ratio for fungi in forests than those in arable soils. This might be due to that EL-FAME extracts may have additional factors such as physiological status of fungi and the inclusion of substantial amount of humic substances that affect quantitative determination of fungal biomass. Overall, EL-FAME method is simple and would produce similar results to PLFA method for bacteria in both quantitative and qualitative assessments when comparing different soils across ecosystems. However, for fungi, PLFA method would be more suitable than EL-FAME method.

Application of a Stir Bar Sorptive Extraction sample preparation method with HPLC for soil fungal biomass determination in soils from a detrital manipulation study

Ergosterol is a sterol found ubiquitously in cell membranes of filamentous fungi. Although concentrations in different fungal species span the range of 2.6 to 42μg/mL of dry mass, many studies have shown a strong correlation between soil ergosterol content and fungal biomass. The analysis of ergosterol in soil therefore could be an effective tool for monitoring changes in fungal biomass under different environmental conditions. Stir Bar Sorptive Extraction (SBSE) is a new sample preparation method to extract and concentrate organic analytes from liquid samples. SBSE was here demonstrated to be a simple, fast, and cost effective method for the quantitative analysis of ergosterol from field-collected soils. Using this method we observed that soil ergosterol as a measure of fungal biomass proved to be a sensitive indicator of soil microbial dynamics that were altered by changes in plant detrital inputs to soils in a long-term field experiment.

4-MUF-NAG for fungal biomass determination: Scope and limitations in the context of biodeterioration studies

Abstract In the field of biodeterioration of cultural heritage by fungi, quantification of fungal growth is a regularly required procedure. Recently, a fluorometric method for detection and estimation of fungal growth using 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (4-MUF-NAG) was proposed. In order to evaluate the potentials and limitations of this method for fungal biomass quantification, different experimental conditions to develop calibration curves were tested, using Aspergillus niger , Cladosporium cladosporioides , Chaetomium globosum and Penicillium chrysogenum fungal strains. The results showed that the correlation between fluorescence and biomass differs according to fungal species and stage of growth. Therefore, a measurement of fluorescence may not be indicative of a specific value of biomass. Also, false positive and negative results for fungal growth can be obtained. C. globosum was the fungal species that showed the most constant correlation between biomass and fluorescence throughout the different incubation periods.

Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

An optimized HPLC method for soil fungal biomass determination and its application to a detritus manipulation study

The goal of this research was to develop modified analytical method for the quantitative analysis of ergosterol, which is highly effective marker for fungal biomass. We suggest that our optimized method for the determination of ergosterol is an effective way to monitor changes in fungal biomass under different environmental conditions.

Use of agar as carrier in solid-state fermentation for Monacolin K production by Monascus: A novel method for direct determination of biomass and accurate comparison with submerged fermentation

For the first time, a novel solid-state fermentation (SSF) process by using agar as carrier was developed and it was feasible for direct determination of fungal biomass and accurate comparison with submerged fermentation (SmF). Specifically, this novel system was successfully applied for the production of Monacolin K by Monascus purpureus. Results showed that the optimal agar concentration, particle size and glycerol concentration were 4%, 4mm×4mm×4mm and 18%, respectively, in which the Monacolin K production reached 2047.03mg/L, 4.47 times as much as that of SmF. The comparative time course study showed that the biomass accumulation, MK biosynthesis and glycerol consumption in SSF were more rapid than that of the SmF.

Biomassebestimmung von Weißfäulepilzen im Feststoffbioreaktor

The solid state fermentation (SSF) is a robust process perfectly suitable for the on-site cultivation of white-rot fungi, where fungi serve as enzyme producers for the treatment of lignocellulosics. The SSF has been known for a few thousand years, but until today process control remains a major problem. Especially the determination of fungal biomass in this system is difficult. This should be simplified and accelerated by means of a new process based on flow cytometry.

Digital image analysis and fractal-based kinetic modelling for fungal biomass determination in solid-state fermentation

This work deals with a non-destructive method involving image analysis and kinetic modelling to determine fungal biomass in solid-state fermentation (SSF). Fractal dimension, quantifying the morphological changes of mycelia-matrix from culture images, showed correlations with Penicillium decumbens biomass on lignocelluloses substrates. Kinetic models were constructed to describe the variation of fractal dimension of mycelia-matrix along with fungal growth. Fermentations on straw substrates with different particle lengths and moisture contents were carried out to validate the proposed models. Relative errors of the models were 0.541–5.221% for biomass and 0.454–3.885‰ for fractal dimension. Parameters δ and η in fractal kinetic models, which indicated the variation rates of fractal dimension, presented significant specificity for the specific growth rate of P. decumbens, thus can be used to predict fungal biomass in SSF. With advantages of low cost, reasonable accuracy and well adjustability, the coupling of dynamic imaging and computational modelling show potential in the on-line determination of fungal biomass in SSF.

Current approaches and utilization of new screening techniques for evaluation of FHB resistance at CIMMYT

Fusarium head blight (FHB) is a major disease of wheat in most wheat growing areas of the world. Resis- tance to FHB is a key trait for CIMMYT and many wheat breeding programs worldwide. New plant pheno- typing techniques such as quantification of fungal biomass using real-time PCR have become available re- cently. CIMMYT’s approach is to test new techniques for their feasibility and to apply them in routine disease screening programs if they prove to be valuable. Two sets of spring wheat genotypes assembled on the basis of low (group 1) and high (group 2) FHB index observed in previous years, were phenotyped and genotyped in CIMMYT’s FHB screening program. Phenotyping consisted of visual disease scoring (FHB index), mycotoxin analysis (DON) and quantification of fungal biomass. Apart from the FHB index, the two groups differed slightly in terms of DON accumulation although no differences were observed for average fungal biomass. This observation combined with the lack of correlation between disease symptoms and amount of fungal biomass suggested that some useful informa- tion may not be considered to discriminate resistant from susceptible genotypes when field selection is solely based on visual scoring results. DON/biomass-ratio was assessed for all genotypes and was found to be higher in the more resistant group 1 contrary to expectiations. An increase in DON production resulting from a stress or from resistance is discussed as a possible hypothesis. The determination of fungal biomass proved to be potentially valuable as a phenotyping tool. Genotyping results also showed that new genotypes harboring moderate levels of resistance and differing from traditional sources of scab resistance become available.

An enzyme-linked immunosorbent assay for monitoring of Aspergillus ochraceus growth in coffee powder, chilli powder and poultry feed

The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate. An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10-160 microg fungal biomass per millilitre extract of coffee (R(2)=0.989), poultry feed (R(2)=0.987) and chilli (R(2)=0.989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9.8 and 11.8 mg g(-1)) followed by coffee beans (6.8 and 11.3 mg g(-1)) and chilli (5.1 and 6.3 mg g(-1)) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 microg g(-1)) followed by coffee beans (8 and 24 microg g(-1)) and chilli (0.2 and 0.45 microg g(-1)) at 20% and 30% moisture after 20 days of incubation. The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition. The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of >or=0.02% (w/w) and can be used for early detection of specific fungal infestation in food commodities.

Contribution of fungi and bacteria to the formation of dissolved organic carbon from decaying common reed (Phragmites australis)

Bacteria and fungi dominate the decomposition of aquatic plants, a major process in the carbon and nutrient cycling in many aquatic systems. Although phylogenetically distant, bacteria and fungi often live in close proximity with each other. Since these microorganisms also have similar ecological functions, interactions have developed between them. This thesis explores the nature of such interactions, and the potential effects on key components of the decomposition process. The thesis includes a critical assessment of the ergosterol method for determination of fungal biomass, a survey of the environmental factors determining the distribution and taxa numbers of litter-decomposing bacteria and fungi in lakes, and a number of experiments on the interactions between bacteria and fungi. In all the experiments performed, fungi responded to bacterial presence through antagonism, although different fungal strains, bacterial communities and substrates were used. The antagonism seemed to be caused by interference competition for substrate. The fungal effect on bacteria was less consistent. Bacterial growth was suppressed, unaffected, or even enhanced by the presence of fungi. Fungi contributed more to extracellular enzyme production than bacteria, and bacteria were probably able to assimilate intermediate decomposition products formed through the activity of extracellular enzymes of fungal origin. Thus, the effect on bacteria from interacting with fungi was determined by the balance between competition and benefit from excreted enzymes. Bacteria and fungi also used different size fractions of the organic matter, according to their different enzymatic capacities. Hence, bacteria appeared to assimilate low-molecular-weight compounds, while high-molecular-weight compounds were utilized primarily by fungi. In brief, the ecological interactions influenced the growth and hence also the biomass development of bacteria and fungi, which affected enzyme activity as well as utilization of dissolved organic matter. Therefore, I suggest that interactions between bacteria and fungi influence degradation of plant litter in aquatic systems.

Estimation of conversion factors for fungal biomass determination in compost using ergosterol and PLFA 18:2ω6,9

Eleven species of common fungi from compost were analysed for their content of ergosterol and phospholipid fatty acids (PLFAs) after growth on agar media. Mean content of ergosterol was 3.1 mg g −1 dw of fungal mycelium (range 1–24 mg g −1 dw). Total amount of PLFAs varied between 2.6 and 43.5 μmol g −1 dw of fungi (mean 14.9 μmol g −1 dw). The most common PLFAs were 16:0, 18:2ω6,9 and 18:1ω9, comprising between 79 and 97 mol% of the total amount of PLFAs. The PLFA 18:2ω6,9, suggested as a marker molecule for fungi, comprised between 36 and 61 mol% of the total PLFAs in the Ascomycetes, between 45 and 57 mol% in the Basidiomycetes and 12–22 mol% in the Zygomycetes. There was a good correlation between the content of the two fungal marker molecules, ergosterol and the PLFA 18:2ω6,9, with a mean content of 1 mg ergosterol being equivalent to 2.1 μmol of 18:2ω6,9. Based on results from the fungal isolates, conversion factors were calculated (5.4 mg ergosterol g −1 biomass C and 11.8 μmol 18:2ω6,9 g −1 biomass C) and applied to compost samples in which both the ergosterol and the PLFA 18:2ω6,9 content had been measured. This resulted in similar estimates of fungal biomass C using the two marker molecules, but was three to five times higher than total microbial biomass C calculated using ATP content in the compost. This could partly be explained by the fact that both of the markers used for fungal biomass are cell membrane constituents. Thus, the ergosterol and the PLFA content were related to the hyphal diameter of the fungi, where fungi with thinner hyphae had higher ergosterol concentrations than fungi with thicker hyphae. This could also partly explain the large interspecific variation in content of the two marker molecules.

Determinination of soil fungal biomass from soil ergosterol analyses

Determination of fungal biomass (FB) and FB-carbon (FB-C) from soil ergosterol concentration is difficult because of unknown ergosterol-to-fungal biomass (E-to-FB) conversion factors and inefficient ergosterol extraction methods. We applied a microwave-assisted extraction (MAE) and high performance liquid chromatographic (HPLC) procedure to measure ergosterol in soil samples. The E-to-FB conversion factors were determined in six species of fungi grown in vitro. The MAE method was fast and extracted up to nine times more soil ergosterol than a classical refluxing saponification method. Soil ergosterol was separated and quantified rapidly (<10 min) by HPLC. Alternaria alternata, Chaetomium globosum, Fusarium oxysporum, Penicillum chrysogenum, Rhizopus stolonifer and Trichoderma harzianum isolated from soil and plant matrices were grown in batch. Ergosterol and biomass content were determined in mycelia harvested during the stationary and exponential phases of growth. Total mycelial ergosterol ranged from 180 to 2178 μg, and total dry biomass ranged from 17 to 595 mg. Total ergosterol and fungal dry biomass were strongly associated ( r 2 = 0.95). The C content in mycelial mats averaged 43% (±1.1, SD), and was similar among fungal species and growth phases. The analyses of variance showed that the E-to-FB ratio was similar among fungal species or growth phase. An average ergosterol concentration of 4 μg mg −1 dry biomass was determined for the six species of fungi, which gave a conversion factor of 250 μg dry biomass μg −1 ergosterol. The MAE method recovered an average of 62% (±11%, SD) of the ergosterol added in mycelial mats to soils prior to extraction, and its recovery was independent of soil properties. The E-to-FB ratio and percent recovery of mycelial ergosterol helped establish for the first time relationships determining soil FB and FB-C from soil ergosterol concentration. The amount of FB ranged from 155 to 4745 μg g −1 and that for FB-C ranged from 67 to 2040 μg g −1 for different soils, and was higher in samples taken from native undisturbed land than in samples taken from adjacent cultivated fields. Measurement of soil ergosterol concentration is a useful estimate content of the living soil FB.

Caffeine degradation in solid state fermentation by Aspergillus tamarii: effects of additional nitrogen sources

The ability of Aspergillus tamarii V12A25 to use caffeine as sole nitrogen source was investigated in solid state fermentation (SSF) using two different supports, polyurethane foam (PUF) and sugarcane bagasse. Caffeine can be used in SSF as the sole nitrogen source, the carbon source being saccharose. If a simpler nitrogen source (ammonium sulphate, urea) is added to the medium containing caffeine, this source will be used first allowing fungal growth. If saccharose is still present when the simple nitrogen source has been degraded, caffeine will be used as the nitrogen source, together with saccharose. Caffeine at a concentration of 8 g/l has no effect on fungal growth when a simple nitrogen source is not limiting. The main difference between the two solid supports was the time required to complete the fermentation. With sugarcane bagasse as the support, substrates (caffeine, ammonium sulphate, urea) were degraded almost twice as fast as with PUF as the support. Theophylline and 3-methylxanthine, the major products from caffeine degradation, disappeared completely from the culture medium shortly after caffeine degradation stopped. The major advantage of using PUF support over sugar cane bagasse is that the former allows determination of fungal biomass which is not possible with the latter.

Optimization of a polyclonal enzyme-linked immunosorbent assay (ELISA) of fungal biomass for use in studies of plant defence responses

A polyclonal, enzyme-linked immunosorbent assay (ELISA) of Phytophthora infestans has been developed for use in the determination of fungal biomass during the early stages of infection of tuber discs of Solanum tuberosum. By optimizing the dilution of sample extracts and the dilution of primary anti-P. infestans antiserum, quantification of the biomass of P. infestans in zoosporeinoculated tuber discs could be achieved by 8-18 h after inoculation. Differences in growth between avirulent and virulent isolates of P. infestans on the resistant potato cv. Kennebec were quantified by 32-48 h after inoculation. Together with a comparison of the growth of the same isolates on the susceptible cv. King Edward, these results comprised an ELISA of the Quadratic Check. On the resistant host, the growth of the avirulent isolate was essentially arrested by 16 h after inoculation, whereas that of the virulent isolate continued throughout the time course. On the susceptible host, however, the avirulent isolate appeared more aggressive than the virulent isolate. These results demonstrate that the ELISA, which is often simpler to perform than other procedures for estimating fungal growth, may be used to complement biochemical studies of rapidly induced plant defence responses.

The determination of fungal biomass using adenosine triphosphate

Growth of Trichoderma reesei in batch culture with glucose as the carbon source was followed by measuring culture absorbance, culture dry weight, mycelial protein, and adenosine triphosphate (ATP) content of the biomass. All methods except extraction of mycelial protein gave measurements which resulted in similar specific growth rates. During exponential growth the ratio of ATP per unit weight was 6.8 μmol ATP/g dry wt. During autolysis the half-life of the ATP content of the biomass was much shorter than the half-life of mycelial protein or culture dry weight. The addition of holocellulose to the culture did not affect the efficiency of the ATP extraction and detection procedures employed, and therefore ATP measurements can be utilized to estimate fungal biomass in cellulose fermentations. However, the presence of a clay caused a considerable decrease in the amount of ATP recovered from fungal biomass.