Andreja Dobrovin-Pennington

Sexuality and Genetic Identity in the Agaricus Section Arvenses

ABSTRACT Twelve wild collections and one commercial strain were used to characterize breeding systems and to develop molecular identities in the Arvenses section of the genus Agaricus, which includes the “horse mushroom” A. arvensis. Two morphotypes were identified based on macro- and micromorphological features. However, not all collections could be delimited by conventional taxonomic characters. Sequencing of the small subunit intergenic spacer (ITS) region (368 to 370 bp) of the rRNA genes clearly resolved the 13 collections into two clusters consistent with the identified morphotypes. Single-spore progenies and mating type testers were established and used to test intra- and interstock compatibility. The two compatibility groups identified were consistent with ITS clusters. Compatibility group I stocks readily interbred within the constraints of a unifactorial heterothallic system with a multiallelic mating type factor. Compatibility group II had a more restricted breeding pattern, and interactions were difficult to predict on the basis of mating type. Morphological data, ITS sequences, and the ability to interbreed suggest that these collections are part of a complex of interrelated species. Single-spore, homokaryotic isolates from both compatibility groups were able to fruit in compost culture, and two of the collections may represent natural homokaryotic fruiting. We conclude that species from the section Arvenses have versatile unifactorial heterothallic life cycles that permit both interbreeding and homokaryotic fruiting.

Primordia initiation of mushroom (Agaricus bisporus) strains on axenic casing materials

The mushroom (Agaricus bisporus) has a requirement for a casing layer that has specific physical, chemical and microbiological properties which stimulate and promote the initiation of primordia. Some of these primordia then may develop further into sporophores, involving differentiation of tissue. Wild and commercial strains of A. bisporus were cultured in axenic and nonaxenic microcosms, using a rye grain substrate covered by a range of organic and inorganic casing materials. In axenic culture, A. bisporus (commercial strain A15) was capable of producing primordia and mature sporophores on charcoal (wood and activated), anthracite coal, lignite and zeolite, but not on bark, coir, peat, rockwool, silica or vermiculite. Of six strains tested, only the developmental variant mutant, B430, produced rudimentary primordia on axenic peat-based casing material. However, none of these rudimentary primordia developed differentiated tissues or beyond 4 mm diameter, either on axenic casing material in the microcosms or in larger-scale culture. In larger-scale, nonaxenic culture, strain B430 produced severely malformed but mature sporophores in similar numbers to those of other strains. Typically, 3-6% of primordia developed into mature sporophores, but significant differences in this proportion, as well as in the numbers of primordia produced, were recorded between 12 A. bisporus strains.

Volatile C8 compounds and pseudomonads influence primordium formation of Agaricus bisporus

Primordium formation of Agaricus bisporus depends on the presence of a casing layer containing stimulatory bacteria and on sufficient air exchange. The influence of specific pseudomonad populations and volatile organic compounds (VOC) on primordium formation of A. bisporus was studied in microcosm cultures. VOC produced by A. bisporus mycelium were predominantly C8 compounds, some of which could inhibit primordium formation, with 1-octen-3-ol being most inhibitory. A VOC produced by the rye grain substrate, 2-ethyl-1-hexanol, on which A. bisporus was grown also inhibited primordium formation. 2-Ethyl-1-hexanol and 1-octen-3-ol were metabolized by pseudomonad populations and adsorbed by activated charcoal, with both modes of removal enabling primordium formation in the casing. Removal of VOC by ventilation also enabled primordium formation to occur under axenic conditions. The presence of 2-ethyl-1-hexanol and 1-octen-3-ol in the microcosms resulted in higher total bacterial and pseudomonad populations in the casing. The stimulatory effects of the casing and its microbiota and air exchange on primordium formation of A. bisporus at least partly are due to the removal of inhibitory C8 compounds produced by the mycelium and its substrate. Monitoring and controlling the levels of these inhibitory VOC in mushroom culture should enable primordium formation of A. bisporus to be more efficiently and precisely controlled.

Influence of straw types and nitrogen sources on mushroom composting emissions and compost productivity

The effects of different straw types and organic and inorganic nitrogen (N) sources on the chemical composition and odor concentration (OC) of mushroom composting emissions, compost parameters, and mushroom yield were examined using bench-scale and large-scale (windrows and aerated tunnels) composting systems. There were close correlations between the butanol or combined H2S+dimethyl sulfide (DMS) concentration and OC of air samples taken from different composting ingredients (r=0.83 and 0.76–0.87, P<0.01, for loge-transformed data). Differences in N availability, in terms of NH3 and N losses during composting, were found between different N sources. Materials in which the N was less available (chipboard and digester wastes, cocoa shells, ammonium sulfate) produced lower mushroom yields than materials in which the N was more readily available (poultry manure, urea, brewers grains, hop and molasses wastes, cocoa meal). Replacement of poultry manure with the other N sources at 50–100% or wheat straw with rape, bean, or linseed straw in aerated tunnel or windrow composts reduced the OC and emissions of odorous sulfur-containing compounds, but also reduced yield. Urea and cocoa meal may be suitable for “low odor” prewetting of straw, with addition of poultry manure immediately before aerated tunnel composting. Rape straw in compost reduces the formation of anaerobic zones and resulting odorous emissions, since it maintains its structure and porosity better than wheat straw. Journal of Industrial Microbiology & Biotechnology (2002) 29, 99–110 doi:10.1038/sj.jim.7000292

Properties of peat-based casing soils and their influence on the water relations and growth of the mushroom (Agaricus bisporus)

Different combinations of peat and chalk or lime sources with differing moisture contents were used to determine how specific physical and chemical properties of the casing soil relate to the growth and water relations of the mushroom. The peat types varied in terms of decomposition and extraction method; the lime addition varied in terms of rate and type (chalk or sugar beet lime). During the colonisation of the casing soil before fruiting, the extension growth rate of mushroom mycelium was most closely correlated (negatively) with the volumetric moisture content of the casing soil. Scanning electron microscopy showed that mycelium growing at a lower casing soil matric potential (Ψm) had a much finer and branched structure than mycelium growing at a higher Ψm. Across all the peat and lime source treatments, a relationship was found between the mean Ψm of the casing soil and mushroom yield, with an optimum Ψm of -7.9 to -9.4 kPa. Mushrooms are produced in ‘flushes’ at about 8-day intervals and during the development of each flush of mushrooms, there was a significant decrease in casing soil Ψm . This decrease (to below -40 kPa) was greatest in the second flush, which was the highest yielding. There were no relationships between mushroom yield and casing soil osmotic potential Ψπ within the range -93 to -154 kPa or any of the other chemical properties and water and air holding characteristics of the casing soils which were determined. Across different casing soil treatments, mushroom dry matter content was negatively correlated with mushroom yield and positively correlated with mushroom tissue osmotic potential.

Characterization of recycled mushroom compost leachate by chemical analysis and thermogravimetry-mass spectrometry.

Recycled compost leachate (RCL or euphemistically named goody water) can be a potent source of foul odor on mushroom substrate production sites and contributes to composting smells. A complex mixture of sulfur compounds, fatty acids, and nitrogen containing compounds is responsible for odor production. Fifty samples, collected from 14 compost production sites in Ireland and the U.K. over a 2 year period, were analyzed for chemical properties and by thermogravimetry-mass spectrometry (TG-MS) for compositional differences. Results indicated that aerated samples had lower values of electrical conductivity, redox potential, and dry matter content than nonaerated samples and that the higher thermal stability of aerated samples measured by TGA could be attributed to greater mineralization of the substrate due to aerobic processes. The lower temperatures noted for peak evolution of methane, water, and carbon dioxide from TG-MS analysis suggested that a more energetic process had occurred in aerated RCL storage facilities, producing greater decomposition of macromolecules that volatilized at lower temperatures. Chemical composition, thermal stability of the freeze-dried leachate, pyrolysis profiles, and relative amounts of pyrolysis products were all markers of as to how effective control measures could influence RCL quality.

Indicator organisms for assessing sanitization during composting of plant wastes

The potential for using plant pathogens and seeds as indicator organisms for assessing sanitization of plant wastes during composting was tested in bench-scale flask and large-scale systems. Plasmodiophora brassicae was unsuitable due to high temperature tolerance in dry to moist composts, and detection of viable inoculum post-composting using bioassay plants not corresponding with that using TaqMan® PCR, possibly due to preservation of nucleic acids at elevated temperatures. Several other plant pathogens (Sclerotinia sclerotiorum, Microdochium nivale, Phytophthora cinnamomi and Phytophthora nicotianae) were unsuitable due their low temperature tolerance. Fusarium oxysporum f.sp. cepae and f.sp. radicis-lycopersici chlamydospores and tomato seeds were suitable indicators due to their moderate temperature tolerance and ease of viability testing post-composting. Abutilon seeds were more tolerant than tomato seeds of compost temperatures ≥52°C but more prone to degradation at lower temperatures and therefore less suitable as indicators. Relationships between compost temperature during exposures of 2-10 days and subsequent viability of the above chlamydospores or seeds enabled the sanitizing effect of composting processes to be predicted within 2-6 days. Plant waste type (woody or vegetable) had a small but significant effect on the relationship for tomato seeds but not for F. oxysporum chlamydospores.

Aerating recycled water on mushroom composting sites affects its chemical analysis and the characteristics of odor emissions

Recycled water (RW) containing compost leachate can be a potent source of foul odor on mushroom composting sites. Samples of RW were repeatedly collected from storage tanks and pits of 14 mushroom composting sites in England and Ireland. Relationships between the effects of submerged aeration of the RW, the chemical and microbial characteristics of the RW, and the odors emitted were investigated. Recycled water samples were analyzed for pH, electrical conductivity (EC), redox potential, and dissolved oxygen concentration after 7 to 14 d cold storage. Freeze-dried material from the RW samples was chemically profiled by pyrolysis gas chromatography-mass spectrometry (GC-MS), and the content of odor precursor compounds was determined by the ninhydrin colorimetric method. The headspace air of containerized RW samples was analyzed by thermal desorption GC-MS and with gas detection tubes and assessed for odor intensity (OI) and concentration by panelists and serial dilution olfactometry. The predominant odorants in the headspace and freeze-dried residues of RW samples were volatile sulfur-containing compounds and carboxylic acids. The headspace OI, EC, dry matter content, and redox potential of RW were interrelated. The headspace OI and combined concentration of hydrogen sulfide (H(2)S) + dimethyl sulfide of RW were correlated (R(2) = 0.635; P < 0.001). Prediction of the OI of RW by measuring RW EC and the concentration of headspace sulfides using gas detector tubes enables rapid and low-cost monitoring of RW from mushroom composting sites. Submerged aeration of RW in storage tanks or pits reduced the RW headspace air OI and the combined H(2)S + dimethyl sulfide concentration by 88%.

Improving biocontrol of black vine weevil ( Otiorhynchus sulcatus ) with entomopathogenic fungi in growing media by incorporating spent mushroom compost

Amending a peat-based growing medium with 10%xa0v/v spent mushroom compost, a source of fungal chitin and other nutrients, prolonged the persistence of entomopathogenic fungi (Metarhizium brunneum Petsch and Beauveria bassiana (Balsamo) Vuillemin; Hypocreales: Clavicipitaceae). This resulted in improved efficacy of M. brunneum against black vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) larvae compared with using inoculum without spent mushroom compost. B. bassiana only controlled larvae when used in combination with spent mushroom compost (75u2009±u20097% reduction in live larvae). Mixing entomopathogenic fungal inoculum with spent mushroom compost and growing medium was as effective in controlling black vine weevil larvae as using spent mushroom compost colonised with M. brunneum or B. bassiana in the growing medium (80u2009±u200912% reduction in live larvae). The former method is preferable since it does not require production and storage of colonised spent mushroom compost, or registration of new substrate formulations of M. brunneum or B. bassiana.