Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi

The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon 1-1 for 30 d at 25°C. The water activity (a(w)) of the media ranged from 0.925 to 0.998. Growth of M. anisopliae and P. farinosus was stimulated between 0.975 and 0.995 a(w) on glucose media and that of P. farinosus at 0. 975 a(w) on glycerol media. At < 0.970 a(w), growth of each fungal species was significantly reduced (P < 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1 for B. bassiana, 77.3 and 90.3 mg g-1 for M. anisopliae, and 26.7 and 76.1 mg g-1 for P. farinosus. The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1 when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon 1-1 than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1 on glucose media with 142.7 g added carbon 1-1, depending on species. Conversely, conidia of B. bassiana and P. farinosus contained maximum amounts of trehalose ( ≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon l-1, and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon 1-1; conidia of B. bassiana contained up to 154.0 mg glycerol plus erythritol g-1. hen B. bassiana and P. farinosus were grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliae contained < 17.0 mg trehalose g-1 under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.

An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01–100 μM) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 μM. Flow cytometry analysis showed that ENN B exposure (0.1–25 μM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100 μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p < 0.05) by ENN B (10 μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10 μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.

A review on completing arsenic biogeochemical cycle: Microbial volatilization of arsines in environment

Arsenic (As) is ubiquitous in the environment in the carcinogenic inorganic forms, posing risks to human health in many parts of the world. Many microorganisms have evolved a series of mechanisms to cope with inorganic arsenic in their growth media such as transforming As compounds into volatile derivatives. Bio-volatilization of As has been suggested to play an important role in global As biogeochemical cycling, and can also be explored as a potential method for arsenic bioremediation. This review aims to provide an overview of the quality and quantity of As volatilization by fungi, bacteria, microalga and protozoans. Arsenic bio-volatilization is influenced by both biotic and abiotic factors that can be manipulated/elucidated for the purpose of As bioremediation. Since As bio-volatilization is a resurgent topic for both biogeochemistry and environmental health, our review serves as a concept paper for future research directions.

Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

Toluene Dioxygenase Catalysed synthesis and reactions of cis-diol metabolites derived from 2 and 3-methoxy phenols

Using toluene dioxygenase as biocatalyst, enantiopure cisdihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork−Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228–259, 2015a, doi:10.​1016/​j.​copbio.​2015.​02.​010; Ball and Hallsworth in Phys Chem Chem Phys 17:8297–8305, 2015, doi:10.​1039/​C4CP04564E; Cray et al. in Environ Microbiol 15:287–296, 2013a, doi:10.​1111/​1462-2920.​12018). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight−1. Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0–85.7 kJ kg−1). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as ‘halophile’ and ‘polyextremophile’; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Ericoid mycorrhiza:A partnership that exploits harsh edaphic conditions

Plants that form ericoid mycorrhizal associations are widespread in harsh habitats. Ericoid mycorrhizal fungal endophytes are a genetically diverse group, and they appear to be able to alleviate certain environmental stresses and so facilitate the establishment and survival of Ericaceae. Some of the fungal taxa that form ericoid mycorrhizas, or at least closely related strains, also form associations with other plant hosts (trees and leafy liverworts). The functional significance of these associations and putative mycelial links between Ericaceae and other plant taxa, however, remain unclear. Evidence from environments that are contaminated by toxic metals indicates that ericoid mycorrhizal fungal endophytes, and in some instances their plant hosts, can evolve resistance to these metals. The apparent ability of these endophytes to develop resistance enables ericoid mycorrhizal plants to colonize polluted soil. This seems to be a major factor in the success of ericoid mycorrhizal taxa in a range of harsh environments.

Calluna vulgaris root cells show increased capacity for amino acid uptake when colonized with the mycorrhizal fungus Hymenoscyphus ericae

Ericoid mycorrhizas are believed to improve N nutrition of many ericaceous plant species that typically occur in habitats with impoverished nutrient status, by releasing amino acids from organic N forms. Despite the ubiquity of mycorrhizal formation the mechanisms and regulation of nutrient transport in mycorrhizal associations are poorly understood. We used an electrophysiological approach to study how amino acid transport characteristics of Calluna vulgaris were affected by colonization with the ericoid mycorrhiza fungus Hymenoscyphus ericae. Both the V_max and K_m parameters of amino acid uptake were affected by fungal colonization in a manner consistent with an increased availability of amino acid to the plant. The ecophysiological significance of altered amino acid transport in colonized root cells of C. vulgaris is discussed. © New Phytologist (2002).

Intraspecific variation in nitrogen source utilisation by isolates of the ericoid mycorrhizal fungus Hymenoscyphus Ericae (Read) Korf and Kernan

Variation in the abilities of 35 isolates of the ericoid mycorrhizal fungal endophyte Hymenoscyphus ericae from two field sites to utilise inorganic and organic nitrogen sources in axenic culture has been investigated. While most isolates showed a preference for NH₄/^- as a sole nitrogen source, considerable variation was observed in the abilities of isolates to utilise amino acids and protein (BSA). In particular, large intraspecific variation was observed for glutamine and BSA utilisation, with some isolates thriving on these substrates while others produced little growth. The data suggest that individual isolates of H. ericae may vary considerably in their abilities to supply their host plants with nitrogen from different substrates in soil. (C) 2000 Elsevier Science Ltd.

Concentration effects of 1,2-dichlorobenzene on soil microbiology

The effect of increasing concentrations (65, 130, 325, 1,300, and 3,250 μg/g soil dry weight) of 1,2-dichlorobenzene (1,2-DCB) on the microbial biomass, metabolic potential, and diversity of culturable bacteria was investigated using soil microcosms. All doses caused a significant (p < 0.05) decrease in viable hyphal fungal length. Bacteria were more tolerant, only direct total counts in soils exposed to 3,250 μg/g were significantly (p < 0.05) lower than untreated controls, and estimates of culturable bacteria showed no response. Pseudomonads counts were stimulated by 1,2-DCB concentrations of up to 325 μg/g; above this level counts were similar to controls. Fatty acid methyl ester analysis of taxonomic bacterial composition reflected the differential response of specific genera to increasing 1,2-DCB concentrations, especially the tolerance of Bacillus to the highest concentrations. The shifts in community composition were reflected in estimates of metabolic potential assessed by carbon assimilation (Biolog) ability. Significantly fewer (p < 0.05) carbon sources were utilized by communities exposed to 1,2-DCB concentrations greater than 130 μg/g (<64 carbon sources utilized) than control soils (83); the ability to assimilate individual carbohydrates sources was especially compromised. The results of this study demonstrate that community diversity and metabolic potential can be used as effective bioindicators of pollution stress and concentration effects.

Response of soil microbial communities to single and multiple doses of an organic pollutant

The effect of 100 μg 1,2-dichlorobenzene (1,2-DCB) g^-1 dry weight (dw) of soil introduced either as a single dose or multiple (10 fortnightly) doses of 10 μg g^-1 dw, on the microbial biomass, diversity of culturable bacterial community and the rate of 1,2-DCB mineralisation, were compared. After 22 weeks exposure both application regimes significantly reduced total bacterial counts and viable fungal hyphal length. The single dose had the greatest overall inhibitory effect, although the extent of inhibition varied throughout the study. Total culturable bacterial counts, determined after 22 weeks exposure showed little response to 1,2-DCB, but pseudomonad counts in single and multiple treatments were reduced to 9.7 and 0.147%, respectively, of the numbers detected in the control soil. The effect of 1,2-DCB application on the taxonomic composition of the culturable bacteria community was determined by fatty acid methyl ester (FAME) analysis. Compared to control soils, the single dose treatment had a lower percentage of Arthrobacter and Micrococcus. Multiple applications had a significant effect upon pseudomonad abundance, which represented only 2% of the identified community, compared to 45.6% in the control. The multi-dosed soils contained a high percentage of bacilli (> 25%). The effects of 1,2-DCB applications on the metabolic potential of the soil microbial community was determined by BIOLOG profiling. The number of carbon compounds utilised by the community in the multi-dosed soils (49 positives) was significantly less (P < 0.05) than detected in the single dose treatment (76) and control (66). The rate of 1,2-DCB mineralisation, determined by ¹⁴CO₂ production from radiolabelled [UL-¹⁴C] 1,2-DCB, declined throughout the study, and after 22 weeks was slightly but significantly (P < 0.05) lower in the multiply- than the singly-dosed soils. The differential response to 1,2-DCB treatments was attributed to its reduced bioavailability in soils after a single exposure, compared to multiple applications.

Toxic interactions of metal ions (Cd2+, Pb2+, Zn2+ and Sb3- on in vitro biomass production of ectomycorrhizal fungi

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd^2-, Pb²⁺, Zn²⁺ and Sb^3-, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd²⁺ and Zn²⁺ was recorded, while Pb²⁺ and Sb^3- individually had little effect. The presence of Pb²⁺ and Sb^3- in the media did however, ameliorate Cd²⁺ and Zn²⁺ toxicity in some circumstances. Interactions between Cd²⁺ and Zn²⁺ were investigated further over a range of concentrations. Zn²⁺ was found to significantly ameliorate the toxicity of Cd²⁺ to three of the four isolates tested. The influence of Zn²⁺ varied between ECM species and with the concentrations of metals tested.

Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, rivers, waters, and ground waters. The assay utilized decline in luminescence of lux- marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range and is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

Determination of the Mycotoxin Content in Distiller's Dried Grain with Solubles Using a Multianalyte UHPLC-MS/MS Method

There are more than 300 potential mycotoxins that can contaminate food and feed and cause adverse effects in humans and animals. The data on the co-occurrence of mycotoxins in novel animal feed materials, such as distiller's dried grain with solubles (DDGS), are limited. Thus, a UHPLC-MS/MS method for the quantitation of 77 mycotoxins and other fungal metabolites was used to analyze 169 DDGS samples produced from wheat, maize, and barley and 61 grain samples. All DDGS samples analyzed were contaminated with 13-34 different mycotoxins. Fumonisins were present in all 52 maize DDGS samples (81.0-6890 μg/kg for fumonisin B1), and deoxynivalenol was present in all 99 wheat DDGS samples (39.3-1120 μg/kg). A number of co-occurring mycotoxins were also identified. Due to the high co-occurrence of mycotoxins, routine screening of the animal feed ingredients is highly recommended to allow the highlighted risks to be effectively managed.