Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

Separation of natural colorants from the fermented broth of filamentous fungi using colloidal gas aphrons

There is a worldwide interest in the development of processes for producing colorants from natural sources. Microorganisms provide an alternative source of natural colorants produced by cultivation technology and extracted from the fermented broth. The aim of the present work was to study the recovery of red colorants from the fermented broth of Talaromyces amestolkiae using the technique of colloidal gas aphrons (CGA) comprising surfactant-stabilized microbubbles. Preliminary experiments were performed to evaluate the red colorants’ solubility in different organic solvents, octanol/water partitioning, and their stability in surfactant solutions, namely hexadecyl trimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and polyoxyethylenesorbitan monolaurate (Tween 20), which are cationic, anionic and nonionic surfactants, respectively. The first recovery experiments were carried out using CGA generated by these surfactants at different volumetric ratios (VR, 3–18). Subsequently, two different approaches to generate CGA were investigated at VR values of 6 and 12: the first involved the use of CTAB at pH 6.9–10.0, and the second involved the use of Tween 20 using red colorants partially dissolved in ethanol and Tween 20. The characterization results showed that red colorants have a hydrophilic nature. The highest recoveries were obtained with Tween 20 (78%) and CTAB (70%). These results demonstrated that the recovery of the colorants was driven by both electrostatic and hydrophobic interactions. The VR was found to be an important operating parameter and at VR 12 with CTAB (at pH 9) maximum recovery, partitioning coefficient (K = 5.39) and selectivity in relation to protein and sugar (SP = 3.75 and SS = 7.20 respectively) were achieved. Furthermore, with Tween 20, the separation was driven mainly by hydrophobic interactions. Overall CGA show promise for the recovery of red colorants from a fermented broth. Although better results were obtained with CTAB than with Tween 20 the latter may be more suitable for some application due to its lower toxicity.

Fungi, mycotoxins and phytoalexin in peanut varieties, during plant growth in the field

The aim of the present study was to analyze the mycobiota, occurrence of mycotoxins (aflatoxins and cyclopiazonic acid), and production of phytoalexin (trans-resveratrol) in two peanut varieties (Runner IAC 886 and Caiapo) during plant growth in the field. Climatic factors (rainfall, relative humidity and temperature) and water activity were also evaluated. The results showed a predominance of Fusarium spp. in kernels and pods, followed by Penicillium spp. and Aspergillus flavus. Aflatoxins were detected in 20% and 10% of samples of the IAC 886 and Caiapo varieties, respectively. Analysis showed that 65% of kernel samples of the IAC 886 variety and 25% of the Caiapo variety were contaminated with cyclopiazonic acid. trans-Resveratrol was detected in 6.7% of kernel samples of the IAC 886 variety and in 20% of the Caiapo variety. However, trans-resveratrol was found in 73.3% of leaf samples in the two varieties studied. (C) 2011 Published by Elsevier Ltd.

Distribution of fungi and aflatoxins in a stored peanut variety

The objective of the present study was to evaluate the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels) from Tupa, State of Sao Paulo, Brazil. The samples were analyzed monthly over a period of one year. The results showed a predominance of Fusarium spp. (67.7% in hulls and 25.8% in kernels) and Aspergillus spp. (10.3% in hulls and 21.8% in kernels), and the presence of five other genera. The growth of Aspergillus flavus was mainly influenced by temperature and relative humidity. Analysis of hulls showed that 6.7% of the samples were contaminated with AFB(1) (mean levels 15-23.9 mu g/kg) and AFB(2) (mean levels = 3.3-5.6 mu g/kg); in kernels, 33.3% of the samples were contaminated with AFB(1) (mean levels 7.0-116 mu g/kg) and 28.3% were contaminated with AFB(2) (mean levels = 3.3-45.5 mu g/kg). Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB(1) and AFB(2). mu 2007 Elsevier Ltd. All rights reserved.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (""ghosts"") can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Dihydroisocoumarin from Xyris pterygoblephara active against dermatophyte fungi

The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (113 and 2D NMR, IR, MS and UV) as (3R,4R)-(-)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of I was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 mu g/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm +/- s.d.: 4.5 +/- 0.8), Trichophyton mentagrophytes (4.8 +/- 0.4) and Trichophyton rubrum (10.2 +/- 0.8) revealed similar inhibition zones to the positive control amphotericin B (32 mu g/disc; 5.0 +/- 0.2; 5.0 +/- 0.6 and 8.8 +/- 1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis. (C) 2007 Elsevier Ltd. All rights reserved.

Isolation of Brazilian marine fungi capable of growing on DDD pesticide

The fungi Aspergillus sydowii Ce15, Aspergillus sydowii Ce19, Aspergillus sydowii Gc12, Bionectria sp. Ce5, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 and Trichoderma sp. Gc1, isolated from marine sponges Geodia corticostylifera and Chelonaplysylla erecta, were evaluated for their ability to grow in the presence of DDD pesticide. Increasing concentrations of DDD pesticide, i.e., 5.0 mg (1.56 x 10(-12) mmol), 10.0 mg (3.12 x 10(-2) mmol) and 15.0 mg (4.68 x 10(-2) mmol) in solid and liquid culture media were tested. The fungi Trichoderma sp. Gc1 and Penicillium miczynskii Gc5 were able to grow in the presence of up to 15.0 mg of DDD, suggesting their potential for biodegradation. A 100% degradation of DDD was attained in liquid culture medium when Trichoderma sp. Gc1 was previously cultivated for 5 days and supplemented with 5.0 mg of DDD in the presence of hydrogen peroxide. However, the quantitative analysis showed that DDD was accumulated on mycelium and biodegradation level reached a maximum value of 58% after 14 days.

Bioreduction of alpha-chloroacetophenone by whole cells of marine fungi

The asymmetric reduction of 2-chloro-1-phenylethanone (1) by seven strains of marine fungi was evaluated and afforded (S)-(-)-2-chloro-1-phenylethanol with, in the best case, an enantiomeric excess of 50% and an isolated yield of 60%. The ability of marine fungi to catalyse the reduction was directly dependent on growth in artificial sea water-based medium containing a high concentration of Cl(-) (1.2 M). When fungi were grown in the absence of artificial sea water, no reduction of 1 by whole cells was observed. The biocatalytic reduction of 1 was more efficient at neutral rather than acidic pH values and in the absence of glucose as co-substrate.

Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions

Biogenic aerosols play important roles in atmospheric chemistry physics, the biosphere, climate, and public health. Here, we show that fungi which actively discharge their spores with liquids into the air, in particular actively wet spore discharging Ascomycota (AAM) and actively wet spore discharging Basidiomycota (ABM), are a major source of primary biogenic aerosol particles and components. We present the first estimates for the global average emission rates of fungal spores.

Measurement results and budget calculations based on investigations in Amazonia (Balbina, Brazil, July 2001) indicate that the spores of AAM and ABM may account for a large proportion of coarse particulate matter in tropical rainforest regions during the wet season (0.7–2.3 μg m⁻³). For the particle diameter range of 1–10 μm, the estimated proportions are ~25% during day-time, ~45% at night, and ~35% on average. For the sugar alcohol mannitol, the budget calculations indicate that it is suitable for use as a molecular tracer for actively wet discharged basidiospores (ABS). ABM emissions seem to account for most of the atmospheric abundance of mannitol (10–68 ng m⁻³), and can explain the observed diurnal cycle (higher abundance at night). ABM emissions of hexose carbohydrates might also account for a significant proportion of glucose and fructose in air particulate matter (7–49 ng m⁻³), but the literature-derived ratios are not consistent with the observed diurnal cycle (lower abundance at night). AAM emissions appear to account for a large proportion of potassium in air particulate matter over tropical rainforest regions during the wet season (17–43 ng m⁻³), and they can also explain the observed diurnal cycle (higher abundance at night). The results of our investigations and budget calculations for tropical rainforest aerosols are consistent with measurements performed at other locations.

Based on the average abundance of mannitol reported for extratropical continental boundary layer air (~25 ng m⁻³), we have also calculated a value of ~17 Tg yr⁻¹ as a first estimate for the global average emission rate of ABS over land surfaces, which is consistent with the typically observed concentrations of ABS (~10³–10⁴ m⁻³; ~0.1^–1 μg m⁻³). The global average atmospheric abundance and emission rate of total fungal spores, including wet and dry discharged species, are estimated to be higher by a factor of about three, i.e. 1 μg m⁻³ and ~50 Tg yr⁻¹. Comparisons with estimated rates of emission and formation of other major types of organic aerosol (~47 Tg yr⁻¹ of anthropogenic primary organic aerosol; 12–70 Tg yr⁻¹ of secondary organic aerosol) indicate that emissions from fungi should be taken into account as a significant global source of organic aerosol. The effects of fungal spores and related chemical components might be particularly important in tropical regions, where both physicochemical processes in the atmosphere and biological activity at the Earth's surface are particularly intense, and where the abundance of fungal spores and related chemical compounds are typically higher than in extratropical regions.

Contribution of fungi to primary biogenic aerosols in the atmosphere : active discharge of spores, carbohydrates, and inorganic ions by Asco- and Basidiomycota

Spores and related chemical compounds from actively spore-discharging Ascomycota (AAM) and actively spore-discharging Basidiomycota (ABM) are primary biogenic components of air particulate matter (characteristic size range 1–10 μm). Measurement results and budget calculations based on investigations in Amazonia (Balbina, Brazil, July 2001) indicate that the forcible discharge of fungal spores may account for a large proportion of coarse air particulate matter in tropical rainforest regions during the wet season. For the particle diameter range of 1–10 μm, the estimated proportions are ~25% during day-time, ~45% at night, and ~35% on average. For the sugar alcohol, mannitol, the budget calculations indicate that it is suitable for use as a molecular tracer for actively discharged basidiospores (ABS), and that the literature-derived emission ratio of about 5 pg per ABS may be taken as a representative average. ABM emissions may account for most of the atmospheric abundance of mannitol, and can explain the observed diurnal cycle (higher abundance at night). ABM emissions of hexose carbohydrates might also account for a significant proportion of glucose and fructose in air particulate matter, but the literature-derived ratios are not consistent with the observed diurnal cycle (lower abundance at night). AAM emissions appear to account for a large proportion of potassium in air particulate matter over tropical rainforest regions during the wet season, and they can also explain the observed diurnal cycle (higher abundance at night). The results of our investigations and budget calculations for tropical rainforest aerosols are consistent with measurements performed at other locations.

Based on the average abundance of mannitol in particulate matter, which is consistent with the above emission ratio and the observed abundance of ABS, we have also calculated a value of ~17 Tg yr−1 as a first estimate for the global average emission rate of ABS over land surfaces. Comparisons with estimated rates of emission and formation of other major types of organic aerosol (~47 Tg yr−1 of anthropogenic primary organic aerosol; 12–70 Tg yr−1 of secondary organic aerosol) indicate that emissions from actively spore-discharging fungi should be taken into account as a significant source of organic aerosol. Their effects might be particularly important in tropical regions, where both physicochemical processes in the atmosphere and biological activity at the Earth's surface are particularly intense, and where the abundance of fungal spores and related chemical compounds are typically higher than in extratropical regions.

Fungi associated with Asphondylia (Diptera : Cecidomyiidae) galls on Sarcocornia quinqueflora and Tecticornia arbuscula (Chenopodiaceae)

Galls induced by the gall-forming midges Asphondylia floriformis and A. sarcocorniae on Sarcocornia quinqueflora, and A. tecticorniae and A. peelei on Tecticornia arbuscula, were collected from two sites nearMelbourne, Victoria. Microfungi belonging to a broad range of families were found to be associated with external surfaces of galls and Journal Articles of Sarcocornia quinqueflora. However, only Botryosphaeria dothidea was isolated from the fungal mycelium lining gall-midge larval chambers of all four Asphondylia species, on both host plants