John N. Hedger

Management influences on soil microbial communities and their function in botanically diverse haymeadows of northern England and Wales.

The effects of management intensification on the size, activity and structure of soil microbial communities in botanically diverse haymeadows were examined. Paired traditionally managed and intensively managed haymeadows, at three submontane regions in northern England and north Wales, were sampled over four seasons. Management intensification had no significant effect on soil nutrient status, soil microbial biomass and soil microbial activity. Management intensification did influence soil microbial community structure, resulting in a significant reduction in soil fungal biomass, measured as soil ergosterol content, and a decline in the proportion of fungi relative to bacteria in the soil microbial community. Fungi of the genera Fusarium, Mucor, Absidia, Cladosporium, Trichoderma, Acremonium, Zygorhynchus, Phoma and Paecilomyces were commonly isolated from litter and soil of both the traditionally and intensively managed haymeadows of the site tested. Management had a significant effect on the relative isolation frequency of these fungi at this site. All commonly isolated species had proteolytic and urease activity and approximately half had cellulolytic and lignolytic activities. These findings were taken to suggest that although management improvements to submontane haymeadows will induce changes in the size and composition of the fungal community, they do not necessarily influence the functioning of the soil microbial community with respect to soil ecosystem-level processes of organic matter decomposition and nutrient cycling. We suggest that changes in soil microbial communities are related primarily to changes in plant productivity and composition or the form and quantity of fertiliser applied to the site.

Witches' brooms and frosty pods: two major pathogens of cacao

Abstract The agaric Crinipellis perniciosa (Tricholomataceae) is a hemibiotrophic pathogen which causes witches’ broom disease of cacao and has recently decimated the Brazilian cacao industry. In addition to the pathogenic cacao (C‐) biotype, other biotypes are found in association with unrelated plant taxa, notably bignoniaceous lianas (L‐biotype), solanaceous hosts (S ‐biotype), and the shrub Heteropterys acutifolia (H‐biotype). The C‐and S‐biotypes are non‐outcrossing and form broom symptoms on hosts, whereas the L‐biotype is outcrossing and asymptomatic. Phylogenetic analysis of several regions of the rRNA locus revealed near identity between C‐ and S‐biotype isolates from diverse locations, with the L‐ and H‐biotypes forming separate groupings. Preliminary analysis of sequence data from Moniliophthora roreri, causal agent of frosty pod disease, indicates that this morphologically distinct pathogen may be closely related to C. perniciosa. Similarities in host infection between C. perniciosa and M. roreri have previously been noted but it is difficult to reconcile the gross morphological differences. Pairings between C. perniciosa and M. roreri gave rise to a clamped dikaryotic mycelium suggestive of a hybridisation event.

Real-time monitoring of the accretion of Rhizopus oligosporus biomass during the solid-substrate tempe fermentation.

We describe a novel method for the real-time estimation of the accretion of blomass during the solid-substrate tempe fermentation of soy beans, lupins and quinoa by Rhizopus oligosporus Salto. The method is based on measurements of the dielectric permittivity at radio-frequencies, using a four-terminal instrument (the Bugmeter). In all cases, excellent Ilnearity is observed during the growth phase between the dielectric permittivity and the hyphal length as determined microscopically.

Metabolite profiles of interacting mycelial fronts differ for pairings of the wood decay basidiomycete fungus, Stereum hirsutum with its competitors Coprinus micaceus and Coprinus disseminatus

The paper presents the first proof-of principle study of metabolite profiles of the interacting mycelial fronts of a wood decomposer basidiomycete, Stereum hirsutum, paired with two competitor basidiomycetes, Coprinus disseminatus and C. micaceus, using TLC and GC-TOF-MS profiling. GC-TOF-MS profiles were information rich, with a total of 190 metabolite peaks detected and more than 120 metabolite peaks detected per sample. The metabolite profiles were able to discriminate between the interactions of S. hirsutum with the two species of Coprinus. In confrontation with C. micaceus, where S. hirsutum mycelial fronts always overgrew those of C. micaceus, there were down-regulations of metabolites in the interaction zone, compared to monocultures of both S. hirsutum and C. micaceus. In contrast, in pairings with C. disseminatus, whose mycelia overgrew those of S. hirsutum, there were some up-regulations compared with monoculture controls, the majority of the metabolites being characteristic of the S. hirsutum monoculture profile. These differences indicate that up-regulation of metabolites in the mycelia of S. hirsutum may be connected to a defensive role or to stress. The results also show proof of principle for the employment of metabolic profiling for biological discovery studies of metabolites produced by fungi that could be applied to natural product screening programmes.

A novel method for producing basidiocarps of the cocoa pathogen Crinipellis perniciosa using a bran-vermiculite medium

A novel method for the production of basidiocarps from the mycelia ofCrinipellis perniciosa is described. This involved the colonization of a bran-vermiculite medium with pure culture of the fungus, prior to application of a peat-based casing. Basidiocarp production was induced by hanging the cultures in a cabinet where they were subjected to a daily cycle of wetting and drying. The method was successfully and reproducibly used to fruit isolates of all four known biotypes of the fungus within 10–16 weeks of inoculation.

Degradation of nitrocellulose by fungi.

AbstractThree lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei andChaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus andTrichoderma reesei, only 10%–22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.

Stimulation by potassium ions of the growth of Rhizopus oligosporus during liquid-and solid-substrate fermentations.

Soya beans and several other beans and cereals have been used as substrates for tempe fermentation with the fungus Rhizopus oligosporus Saito. Except for the presence of alkaloids, the chemical composition of lupins (Lupinus mutabilis Sweet) is similar to that of soya beans. Therefore the potential of lupins for tempe production in regions with a long tradition of lupin consumption is promising. The preparation of the fermentation substrate when using bitter lupins (which contain significan quantities of alkaloids) as starting material includes a debittering stage to remove the alkaloids. However, we found that the debittering process yielded lupins that did not support the mycelial growth required in the tempe fermentation. We discovered that potassium is preferentially leached out during the debittering process. The effect of potassium on fungal biomass formation was monitored using a computerized system that determines biomass accretion by measurement of the electrical capacitance at radio frequencies. The importance of potassium for the growth of R. oligosporus was confirmed in liquid cultures. A linear relationship was found between biomass yield and K+ concentration in the range of 1 to 10 mg/l. The present report represents one of the few demonstrations of a mineral deficiency during the growth of a fungus on a natural, solid substrate.

Dual culture ofCrinipellis perniciosa and potato callus

Inoculation of potato callus cultures with basidiospores of the cocoa pathogenCrinipellis perniciosa resulted in the development of a mycelium which was morphologically identical to that found in green cocoa brooms. These dual cultures could be maintained for periods of several months. The nuclear condition of this mycelium was found to be variable, in contrast to previous reports. Basidiospores of the L-biotype ofC. perniciosa were also able to form dual cultures, although a biotrophic phase in its life cycle has yet to be demonstratedin vivo. Attempts to form stable heterokaryons between genetically distinct biotrophic mycelia were unsuccessful.

Effects of a range of procyanidins on the cocoa pathogen Crinipellis perniciosa

Abstract Basidiospore germination and germ-tube elongation in Crinipellis perniciosa, a fungal pathogen of cocoa, were inhibited by a range of procyanidins (condensed tannins) and monomeric flavan-3-ols. Dose/response studies revealed a strong trend of increasing anti-fungal potency with increasing molecular weight of the procyanidins tested. The non-ionic detergent Tween 20 inhibited the anti-fungal effects of procyanidin, suggesting that non-covalent complexes with fungal macromolecules may be responsible for these effects.

The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride.

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.