Revisiting the host effect on ectomycorrhizal fungal communities: implications from host–fungal associations in relict Pseudotsuga japonica forests

Host identity is among the most important factors in structuring ectomycorrhizal (ECM) fungal communities. Both host–fungal coevolution and host shifts can account for the observed host effect, but their relative significance in ECM fungal communities is not well understood. To investigate these two host-related mechanisms, we used relict forests of Pseudotsuga japonica, which is an endangered endemic species in Japan. As with other Asian Pseudotsuga species, P. japonica has been isolated from North American Pseudotsuga spp. since the Oligocene and has evolved independently as a warm-temperate species. We collected 100 soil samples from four major localities in which P. japonica was mixed with other conifers and broadleaf trees. ECM tips in the soil samples were subjected to molecular analyses to identify both ECM fungi and host species. While 136 ECM fungal species were identified in total, their communities were significantly different between host groups, confirming the existence of the host effect on ECM fungal communities. None of the 68 ECM fungal species found on P. japonica belonged to Pseudotsuga-specific lineages (e.g., Rhizopogon and Suillus subgroups) that are common in North America. Most of ECM fungi on P. japonica were shared with other host fungi or phylogenetically close to known ECM fungi on other hosts in Asia. These results suggest that after migrating, Pseudotsuga-specific fungal lineages may have become extinct in small isolated populations in Japan. Instead, most of the ECM fungal symbionts on P. japonica likely originated from host shifts in the region.     

Where is the unseen fungal diversity hidden? A study of Mortierella reveals a large contribution of reference collections to the identification of fungal environmental sequences

? Estimation of the proportion of undescribed fungal taxa is an issue that has remained unresolved for many decades. Several very different estimates have been published, and the relative contributions of traditional taxonomic and next-generation sequencing (NGS) techniques to species discovery have also been called into question recently. ? Here, we addressed the question of what proportion of hitherto unidentifiable molecular operational taxonomic units (MOTUs) have already been described but not sequenced, and how many of them represent truly undescribed lineages. We accomplished this by modeling the effects of increasing type strain sequencing effort on the number of identifiable MOTUs of the widespread soil fungus Mortierella. ? We found a nearly linear relationship between the number of type strains sequenced and the number of identifiable MOTUs. Using this relationship, we made predictions about the total number of Mortierella species and found that it was very close to the number of described species in Mortierella. ? These results suggest that the unusually high number of unidentifiable MOTUs in environmental sequencing projects can be, at least in some fungal groups, ascribed to a lag in type strain and specimen sequencing rather than to a high number of undescribed species.     

Genetic mining of the “dark matter” in fungal natural products

<正>Filamentous fungi are one of the major sources of natural products (NPs; which are also termed as secondary metabolites (SMs)) with diverse biological activities, which have been widely used in agriculture, industry, and pharmaceuticals. Fungi are the second largest species in nature, and their biodiversity implies genomic diversity, which, in turn,predicts the structural diversity of metabolites. In general,     

A new procedure for the measurement of fungal and bacterial α-amylase

Summary A procedure for the measurement of fungal and bacterial -amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end)p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and -glucosidase, and is absolutely specific for -amylase. The assay procedure is simple, reliable and accurate.     

Bacteria–bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance

Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection.     

Analysis of the potential fungal biodeteriogen effects in the “Doctorate Library” of the University of Perugia,Italy

Historical collections held in ancient archives and libraries constitute a cultural and artistic heritage of inestimable value. These collections contain a series of organic materials which may alter and perish in time, especially if they are conserved in inadequate environmental conditions and without any specific precautionary devices to protect them from damage caused by chemical, biological and physical factors. It is essential to prevent ‘at risk’ situations to conserve library and archive heritage. Early detection of fungal spores and moulds in conservation environments will permit us to intervene at the infection site at the right moment and also to detect the micro‐environments where climatic parameters are not suitable for conservation purposes.

The aim of this study is to examine potentially biodeteriogen fungal micro‐flora in the environment of the “Doctorate Library”, one of the most important buildings of the University of Perugia, which contains frescos and library collection of great value. The study was carried out through quantitative and qualitative analysis of airborne fungal species and through the evaluation of viable aerobiological particles as potential agents of biodeterioration. The airspore monitoring was made using the active method (by Personal Volumetric Air Sampler Spore Trap, Burkard Company Ltd.) and the passive one (by sedimentation technique on Petri dishes). The results obtained by both methods allowed us to detect numerous potentially biodeteriogen species, to determine their quantity and influence and to evaluate the levels of their variability in time.     

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.     

Site-directed mutagenesis of a fungal β-1,4-endoglucanase increases the minimum size required for the substrate

Conserved regions within a family-5 β-1,4-endoglucanase (Egl1), produced by the fungus Macrophomina phaseolina, have been modified through site-directed mutagenesis, resulting in production of an enzyme with novel substrate requirements. The engineered form was generated through mutagenesis of D232A, which lies within the substrate-binding cleft of Egl1. Wild-type Egl1 requires a minimum substrate size of five glucosyl units, while the engineered form requires a minimum of six glucosyl units. Screening was facilitated by the unique ability to obtain functional expression of the fungal endoglucanase in Escherichia coli. Wild-type and mutated Egl1 have equivalent activity on cellohexaose, and both release cellobiose from the reducing end of the cellodextrin. This is the first example of protein engineering of an endoglucanase that results in a novel minimum substrate requirement for cellohexaose. This substrate specificity has not been reported for any native endoglucanases, thus the modified Egl1 may prove useful in applications requiring specific hydrolysis of complex carbohydrates such as β-glucans. Received: 10 November 1996 / Received revision: 22 March 1997 / Accepted: 13 April 1997     

Elsinoë fawcettii and Elsinoë australis: the fungal pathogens causing citrus scab

Elsinoë fawcettii and E. australis are important pathogens of citrus. Both species are known to produce red or orange pigments, called elsinochrome. Elsinochrome is a nonhost‐selective phytotoxin and is required for full fungal virulence and lesion formation. This article discusses the taxonomy, epidemiology, genetics and pathology of the pathogens. It also provides a perspective on the cellular toxicity, biosynthetic regulation and pathological role of elsinochrome phytotoxin. Taxonomy: Elsinoë fawcettii (anamorph: Sphaceloma fawcettii) and E. australis (anamorph: S. australis) are classified in the Phylum Ascomycota, Class Dothideomycetes, Order Myriangiales and Family Elsinoaceae. Host range: Elsinoë fawcettii causes citrus scab (formerly sour orange scab and common scab) on various species and hybrids in the Rutaceae family worldwide, whereas E. australis causes sweet orange scab, primarily on sweet orange and some mandarins, and has a limited geographical distribution. Disease symptoms: Citrus tissues infested with Elsinoë often display erumpent scab pustules with a warty appearance. Toxin production: Elsinochrome and many perylenequinone‐containing phytotoxins of fungal origin are grouped as photosensitizing compounds that are able to absorb light energy, react with oxygen molecules and produce reactive oxygen species, such as superoxide and singlet oxygen. Elsinochrome has been documented to cause peroxidation of cell membranes and to induce rapid electrolyte leakage from citrus tissues. Elsinochrome biosynthesis and conidiation are coordinately regulated in E. fawcettii, and the environmental and physiological inducers commonly involved in both processes have begun to be elucidated.     

Do the costs and benefits of fungal endophyte symbiosis vary with light availability?

? Here, we examined whether fungal endophytes modulated host plant responses to light availability. First, we conducted a literature review to evaluate whether natural frequencies of endophyte symbiosis in grasses from shaded habitats were higher than frequencies in grasses occupying more diverse light environments. Then, in a glasshouse experiment, we assessed how four levels of light and the presence of endophyte symbioses affected the growth of six grass species. ? In our literature survey, endophytes were more commonly present in grasses restricted to shaded habitats than in grasses from diverse light environments. ? In the glasshouse, endophyte symbioses did not mediate plant growth in response to light availability. However, in the host grass, Agrostis perennans, symbiotic plants produced 53% more inflorescences than nonsymbiotic plants at the highest level of shade. In addition, under high shade, symbiotic Poa autumnalis invested more in specific leaf area than symbiont-free plants. Finally, shade increased the density of the endophyte in leaf tissues across all six grass species. ? Our results highlight the potential for symbiosis to alter the plasticity of host physiological traits, demonstrate a novel benefit of endophyte symbiosis under shade stress for one host species, and show a positive association between shade-restricted grass species and fungal endophytes.     

Impact of HIF-1α and hypoxia on fungal growth characteristics and fungal immunity

Human pathogenic fungi are highly adaptable to a changing environment. The ability to adjust to low oxygen conditions is crucial for colonization and infection of the host. Recently, the impact of mammalian hypoxia-inducible factor-1α (HIF-1α) on fungal immunity has emerged. In this review, the role of hypoxia and HIF-1α in fungal infections is discussed regarding the innate immune response.     

Can fungal biopesticides control malaria?

Recent research has raised the prospect of using insect fungal pathogens for the control of vector-borne diseases such as malaria. In the past, microbial control of insect pests in both medical and agricultural sectors has generally had limited success. We propose that it might now be possible to produce a cheap, safe and green tool for the control of malaria, which, in contrast to most chemical insecticides, will not eventually be rendered useless by evolution of resistance. Realizing this potential will require lateral thinking by biologists, technologists and development agencies.     

Are tropical fungal endophytes hyperdiverse?

Fungal endophytes are ubiquitous fungi that inhabit healthy plant tissues without causing disease. Endophytes have been found in every plant species examined to date and may be important, but often overlooked, components of fungal biodiversity. In two sites in a lowland, moist tropical forest of central Panama, we quantified endophyte colonization patterns, richness, host preference, and spatial variation in healthy leaves of two co-occurring, understory tree species [ Heisteria concinna (Olacaceae) and Ouratea lucens (Ochnaceae)]. From 83 leaves, all of which were colonized by endophytes, we isolated 418 endophyte morphospecies (estimated 347 genetically distinct taxa), most of which were represented by only a single isolate (59%). Among morphospecies encountered in more than one leaf (nonsingletons), we found evidence of host preference and spatial heterogeneity using both morphospecies frequencies and presence/absence records. Based on these data, we postulate that tropical endophytes themselves may be hyperdiverse and suggest that extrapolative estimates that exclude them will markedly underestimate fungal species diversity.     

Biodiversity of palm fungi in the tropics: are global fungal diversity estimates realistic?

Two questions are addressed: 'How many species of fungi can occur on a single host palm?' and 'What are the implications of this for global estimates of fungal diversity?' Fungal diversity estimates found in the literature are reviewed. Data on the numbers of fungi occurring on the above-ground tissues of six individual palms in the genus Licuala in Australia and Brunei Darussalam (Borneo) are provided. A total of 189 species of fungi were isolated and/or collected from the six palms. In addition, 53 'morphospecies' of mycelia sterilia were isolated, giving a total of 242 taxa from the 2672 isolates/collections made. The three palms in Australia (sampled once) yielded 100 species (each palm supporting an average of 54.7 taxa), while the three palms in Brunei Darussalam (sampled three times) yielded 172 species in total (approximately 111.3 taxa each). The magnitude of global fungal diversity, estimated at 1.5 million species, is discussed. Our results indicate that 33 to 1 would be a more accurate estimate (than 5.7 to 1) of the ratio of host specific fungal to palm species in the tropics. We therefore propose that global estimates of fungal diversity, based on temperate studies, require revision upwards.     

Does forest liming impact the enzymatic profiles of ectomycorrhizal communities through specialized fungal symbionts?

Liming (Ca–Mg soil amendment) is a forestry practice used to correct soil acidification and restore health and productivity in declining stands. Liming is known to modify tree mineral nutrition beyond the sole Ca and Mg. We hypothesized that liming also modifies the very functioning of the tree absorbing system (that is the ectomycorrhizal fine roots) in a way that facilitates the mobilization of mineral nutrients, particularly those entrapped in soil organic matter. This hypothesis has been tested here in beech and Norway spruce stands in North-Eastern France. In autumn, we compared the ectomycorrhizal community structure and the enzymatic profiles of ectomycorrhizal root tips in limed and untreated plots by measuring the activities of eight enzymes related to the degradation of soil organic matter. The results show that the ectomycorrhizal community responds to the Ca–Mg amendment and to the resulting soil modifications by modified enzyme activity profiles and ability to mobilize nutrients from soil organic matter. The effects of liming on the belowground functioning of the tree stands result essentially from specialized ECM fungal species such as Clavulina cristata (with strong glucuronidase activity), Lactarius subdulcis (with strong laccase activity) or Xerocomus pruinatus (with strong leucine aminopeptidase activity).     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.     

Can fungal biomarkers be used to improve antifungal therapy in the intensive care unit?

Invasive fungal infections have an increasing incidence worldwide, and patients in intensive care settings are particularly vulnerable to them. Candidemia is the third most common bloodstream pathogen in intensive care units (ICUs) in the United States, and a number of reports have recently been published regarding the increasing incidence of invasive aspergillosis in ICUs throughout the world. Because research has shown that early antifungal therapy is associated with better outcomes, clinicians are starting to explore new diagnostic fungal markers, which are more sensitive and faster than the traditional diagnostic standards of culture and histopathology.     

Antioxidants in Festuca rubra L. seeds affected by the fungal symbiont Epichloë festucae

Vertically transmitted fungal endophytes can be beneficial for host grasses. While the alkaloid-mediated mechanism for herbivore resistance has been widely studied, underlying physiological mechanisms for increased tolerance to abiotic stress remain scarcely explored. In this study we used three maternal lines of perennial grass Festuca rubra to examine the role of antioxidants in endophyte-mediated effects on seed viability over long-term storage. Uncolonized plants (E?) were generated by removing the endophyte from ramets of naturally endophyte-colonized (E+) plants. The E?+ and E? ramets were planted in a common garden in Salamanca, Spain. Seeds produced in 2009, 2010 and 2011 were harvested at maturity, dried and stored at 10 °C until 2011 when we tested seed and endophyte viability, and measured antioxidants. Seed viability and α-tocopherol antioxidant were negatively affected by the endophyte in two maternal lines. In these same lines, the endophyte viability was lowest at the longest storage time. In the maternal line that showed the highest negative effect of endophyte on seed viability, the pattern of glutathione was opposite to that observed for tocopherols since it was higher for E?+ than for E? seeds. In all maternal lines, the glutathione half-cell reduction potential (E_GSSG/2GSH) and % glutathione disulphide (GSSG) increased with storage time but there was no clear pattern associated with endophyte symbiosis. Whether these parameters are good predictors of seed and endophyte longevity in storage and natural conditions should be further explored.