Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hinf I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62-92%) from each site were identified as Hymenoscyphus ericae, but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.

Arbuscular mycorrhizal fungal hyphae reduce soil erosion by surface water flow in a greenhouse experiment

The role of arbuscular mycorrhizal fungi (AMF) in resisting surface flow soil erosion has never been tested experimentally. We set up a full factorial greenhouse experiment using Achillea millefolium with treatments consisting of addition of AMF inoculum and non-microbial filtrate, non-AMF inoculum and microbial filtrate, AMF inoculum and microbial filtrate, and non-AMF inoculum and non-microbial filtrate (control) which were subjected to a constant shear stress in the form of surface water flow to quantify the soil detachment rate through time. We found that soil loss can be explained by the combined effect of roots and AMF extraradical hyphae and we could disentangle the unique effect of AMF hyphal length, which significantly reduced soil loss, highlighting their potential importance in riparian systems.

Back-trajectories Show Export of Airborne Fungal Spores (Ganoderma sp.) From Forests to Agricultural and Urban Areas in England

We propose here the hypothesis that all of United Kingdom (UK) is likely to be affected by Ganoderma sp. spores, an important plant pathogen. We suggest that the main sources of this pathogen, which acts as a bioaerosol, are the widely scattered woodlands in the country, although remote sources must not be neglected. The hypothesis is based on related studies on bioaerosols and supported by new observations from a non-forest site and model calculations to support our hypothesis. Hourly concentrations of Ganoderma sp. spores were measured from 2006 to 2010 using a 7-day volumetric spore trap at the city of Worcester. The concentrations peak during the night and early in the morning. This suggests that the main spore sources are located a few hours away with respect to air masses transport and reach urban areas thanks to air masses transport. The back-trajectory analysis was applied to determine the location of Ganoderma sp. spore sources. The analysis of back-trajectories demonstrated that 78% of the air masses reached Worcester from a 180° arc direction from the East to West. Three episodes were selected for detailed investigation and they revealed that during the episodes air masses always passed main UK woodlands before the arrival in Worcester, independently of their origin, but the long distance transport under certain conditions might be possible. Our studies suggest that the sources of UK Ganoderma sp. spores are mainly to be found in UK. Hence our studies suggest that research and mitigation strategies in UK should give their main attention to national sources, without neglecting the contribution from long distance transport.

Potential Impact of Climate Change on Fungal Distributions: Analysis of 2 Years of Contrasting Weather in the UK

The impact of climate change on fungal growth and spore production is less well documented than for allergenic pollen grains, although similar implications for respiratory tract diseases in humans occur. Fungal spores are commonly described as either “dry” or “wet” according to the type of weather associated with their occurrence in the air. This study examined the distribution of selected fungal spores (Alternaria spp., Cladosporium spp., Didymella spp., Epicoccum spp., Leptosphaeria spp. and rusts) occurring in the West Midlands of UK during 2 years of contrasting weather. Spore specimens were collected using a 7-day volumetric air sampler and then analysed with the aid of light microscopy. Distributions of spores were then studied using normality tests and Mann–Whitney U test, while relationships with meteorological parameters were investigated using Spearman’s rank test and angular-linear correlation for wind direction analysis. Our results showed that so-called wet spores were more sensitive to the weather changes showing statistically significant differences between the 2 years of study, in contrast to “dry” spores. We predict that in following years we will observe accelerated levels in allergenic fungal spore production as well as changes in species diversity. This study could be a starting point to revise the grouping system of fungal spores as either “dry” or “wet” types and their response to climate change

Investigating and Predicting Atmospheric Concentrations of Allergenic Fungal Spores (Alternaria, Cladosporium, Didymella and Ganoderma)

Air quality is an increasing concern of the European Union, local authorities, scientists and most of all inhabitants that become more aware of the quality of the surrounding environment. Bioaerosols may be consisted of various elements, and the most important are pollen grains, fungal spores, bacteria, viruses. More than 100 genera of fungal spores have been identified as potential allergens that cause immunological response in susceptible individuals. Alternaria and Cladosporium have been recognised as the most important fungal species responsible for respiratory tract diseases, such as asthma, eczema, rhinitis and chronic sinusitis. While a lot of attention has been given to these fungal species, a limited number of studies can be found on Didymella and Ganoderma, although their allergenic properties were proved clinically. Monitoring of allergenic fungal spore concentration in the air is therefore very important, and in particular at densely populated areas like Worcester, UK. In this thesis a five year spore data set was presented, which was collected using a 7-day volumetric spore trap, analysed with the aid of light microscopy, statistical tests and geographic information system techniques. Although Kruskal-Wallis test detected statistically significant differences between annual concentrations of all examined fungal spore types, specific patterns in their distribution were also found. Alternaria spores were present in the air between mid-May/mid-June until September-October with peak occurring in August. Cladosporium sporulated between mid-May and October, with maximum concentration recorded in July. Didymella spores were seen from June/July up to September, while peaks were found in August. Ganoderma produced spores for 6 months (May-October), and maximum concentration could be found in September. With respect to diurnal fluctuations, Alternaria peaked between 22:00h and 23:00h, Cladosporium 13:00-15:00h, Didymella 04:00-05:00h and 22:00h-23:00h and Ganoderma from 03:00h to 06:00h. Spatial analysis showed that sources of all fungal species were located in England, and there was no evidence for a long distance transport from the continent. The maximum concentration of spores was found several hours delayed in comparison to the approximate time of the spore release from the crops. This was in agreement with diurnal profiles of the spore concentration recorded in Worcester, UK. Spores of Alternaria, Didymella and Ganoderma revealed a regional origin, in contrast to Cladosporium, which sources were situated locally. Hence, the weather conditions registered locally did not exhibit strong statistically significant correlations with fungal spore concentrations. This has had also an impact on the performance of the forecasting models. The best model was obtained for Cladosporium with 66% of the accuracy.

Comparisons of Fungal Spore Distributions Using Air Sampling at Worcester, England (2006-2010)

This study determined annual and monthly fluctuations in concentration of 20 fungal genera. The selection of taxa was made based upon their high frequency in the air as well as their well-known allergenic properties. Air samples were collected using a spore trap of Hirst design at an urban site where the trap continuously worked throughout a 5-year survey. Weather data were acquired from a meteorological station co-located with the air sampler. Influence of several meteorological parameters was then examined to reveal species–environment interactions and the potential location of fungal spore sources within the urban area. The maximum monthly sum of mean daily spore concentration varied between genera, and the earliest peaks were recorded for Pleospora sp. in April and Ustilago sp. in June. However, the majority of investigated spore types occurred in the greatest concentrations between August and September. Out of the 20 studied taxa, the most dominant genus was Cladosporium sp., which exceeded an allergenic threshold of 3000 s m-3 40 times during very rainy years and twice as much during dry years. A Spearman’s rank test showed that statistically significant (p B 0.05) relationships between spore concentration and weather parameters were mainly rs B 0.50. Potential sources of spores at Worcester were likely to be localised outside the city area.

Fungal contamination in two Portuguese wastewater treatment plants

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Characterizing the fungal and bacterial microflora and concentrations in fitness centres

Fitness centres are special places where conditions for microbiological proliferation should be considered. Moisture due to human perspiration and water condensation as a result of human physical activities are prevalent in this type of buildings. Exposure to microbial contaminants is clinically associated with respiratory disorders and people who work out in polluted environments would be susceptible to contaminants. This work studied the indoor air contamination in three gymnasiums in Lisbon. The sampling was performed at two periods: at the opening (morning) and closing (night) of the three gymnasiums. The airborne bacterial and fungal populations were sampled by impaction directly onto Tryptic Soy Agar (for bacteria) and Malt Extract Agar (for fungi) plates, using a Merck MAS-100 air sampler. Higher bacterial concentrations were found at night as compared to the morning but the same behaviour was not found for fungal concentrations. Gram-negative catalase positive cocci were the dominant bacteria in indoor air samples of the studied gymnasiums. In this study, 21 genera/species of fungal colonies were identified. Chrysosporium sp., Chrysonilia sp., Neoscytalidium hialinum, Sepedonium sp. and Penicillium sp. were the most prevalent species identified in the morning, while Cladosporium sp., Penicillium sp., Chrysosporium sp., Acremonium sp. and Chrysonilia sp. were more prevalent at night. A well-designed sanitation and maintenance program for gymnasiums is needed to ensure healthier space for indoor physical activity.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

Fungal communities in archives: assessment strategies and impact on paper conservation and human health

The main results presented in this PhD Dissertation have been published in interna-tional journals included in the Science Citation Index (SCI)

Pupal cocoons affect sanitary brood care and limit fungal infections in ant colonies.

BACKGROUND: The brood of ants and other social insects is highly susceptible to pathogens, particularly those that penetrate the soft larval and pupal cuticle. We here test whether the presence of a pupal cocoon, which occurs in some ant species but not in others, affects the sanitary brood care and fungal infection patterns after exposure to the entomopathogenic fungus Metarhizium brunneum. We use a) a comparative approach analysing four species with either naked or cocooned pupae and b) a within-species analysis of a single ant species, in which both pupal types co-exist in the same colony. RESULTS: We found that the presence of a cocoon did not compromise fungal pathogen detection by the ants and that species with cocooned pupae increased brood grooming after pathogen exposure. All tested ant species further removed brood from their nests, which was predominantly expressed towards larvae and naked pupae treated with the live fungal pathogen. In contrast, cocooned pupae exposed to live fungus were not removed at higher rates than cocooned pupae exposed to dead fungus or a sham control. Consistent with this, exposure to the live fungus caused high numbers of infections and fungal outgrowth in larvae and naked pupae, but not in cocooned pupae. Moreover, the ants consistently removed the brood prior to fungal outgrowth, ensuring a clean brood chamber. CONCLUSION: Our study suggests that the pupal cocoon has a protective effect against fungal infection, causing an adaptive change in sanitary behaviours by the ants. It further demonstrates that brood removal-originally described for honeybees as "hygienic behaviour"-is a widespread sanitary behaviour in ants, which likely has important implications on disease dynamics in social insect colonies.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.

BACKGROUND: Invasive fungal diseases are important causes of morbidity and mortality. Clarity and uniformity in defining these infections are important factors in improving the quality of clinical studies. A standard set of definitions strengthens the consistency and reproducibility of such studies. METHODS: After the introduction of the original European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions, advances in diagnostic technology and the recognition of areas in need of improvement led to a revision of this document. The revision process started with a meeting of participants in 2003, to decide on the process and to draft the proposal. This was followed by several rounds of consultation until a final draft was approved in 2005. This was made available for 6 months to allow public comment, and then the manuscript was prepared and approved. RESULTS: The revised definitions retain the original classifications of "proven," "probable," and "possible" invasive fungal disease, but the definition of "probable" has been expanded, whereas the scope of the category "possible" has been diminished. The category of proven invasive fungal disease can apply to any patient, regardless of whether the patient is immunocompromised, whereas the probable and possible categories are proposed for immunocompromised patients only. CONCLUSIONS: These revised definitions of invasive fungal disease are intended to advance clinical and epidemiological research and may serve as a useful model for defining other infections in high-risk patients.

Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

An investigation into fungal metabolism of halogenated and related steroids

Fungal metabolism of halogenated and related steroids was investigated. The fungi Aspergillus niger ATCC 9142, Curvularia lunata NRRL 2380 and Rhizopus stolonifer ATCC6227b were studied in this regard. 2l-Fluoro-, 2l-chloro, 2l-bromo- and 2l-methyl-pregn-4-ene-3,20diones were prepared and incubated with ~ niger (a C-2l-hydroxylator) in order to observe the effect of the C-2l substituent on the metabolism of these substrates. In all four cases, the C-2l substituent prevented any significant metabolism of these substrates. llB-Fluoropregn-4-ene-3,20-dione was prepared and incubated with C. lunata (an llB-hydroxylator) and ~ stolonifer (an lla-hydroxylator). With ~ lunata, the ll-fluoro- substituent prevent hydroxylation at the 11 position, but diverted it to a site remote from the fluorine atom. In contrast, with ~ stolonifer the llB-fluoro- substituent, although slowing the apparent rate of hydroxylation, did not prevent its occurrence at the 11a- position. llB-Hydroxypregn-4-ene-3,20-dione was also incubated with R. stolonifer. The llB-hydroxy-;group did not appear to have any significant effect on hydroxylation at the lla- position. The incubation of a substrate, unsaturated at a favoured site of hydroxylation with Rhizopus arrhizus ATCC 11145 provided a complex mixture of products; among them were both the a and S epoxides. The formation of these products is rationalized as arising because of the lack of regio- and stereospecificity of the hydroxylase enzyme(s) involved.