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Final Activity Report Summary - AHUPAF (Stepping stones for the adaptation of human pathogenic fungi resulting from global change)

During the project the study design was followed and finished. Main aims of the reported project were:
1) Establishment of P. boydii as a model organism for the evolution of opportunistic pathogenic fungi by the detailed description of effects of human activities on fungal populations and habitats. The project was focused on anthropogen N- and P-sources and their impact on competition among fungi in an experimental model.
2) Reproduction modes realised by P. boydii should be investigated. P. boydii reproduces by mitotic cell division (Graphium and Scedosporium anamorphs) and meiotic production of ascospores.
3) This project should help to clear systematic-taxonomic questions of P. boydii.
In all of the main objectives we were able to achieve developments from a technical and scientific viewpoint.

Main progress was met in the handling and interpretation of competition experimental approaches, covering the application of solid media, liquid media, different temperatures and incubation times, molecular and morphological analysis methods, and different N- and P-sources. Prior to the competition trials the strain- and compound-dependency of N- and P- tolerance was investigated. Competition trials were carried out with different combinations of opponents and were carried out in two independent series with different strain-sets. One series was analysed by morphological means and another with the help of molecular strain characterisation. The results enabled us to assess the effect on the competition between strains of four common P- and N- sources.

During the molecular analysis, the discriminatory power and reliability of fingerprinting methods was evaluated by comparing them with AFLP methods. Mating among ten selected strains and between subcultures of these strains was carried out. The production of ascomata was observed and the first descending generation was characterised with molecular methods. These experiments were carried out twice (analysis based on ascoma production) and the results were reproducible. Results of those experiments led to a major perception of the evolutionary relevance of sexual reproduction in P. boydii. Mating experiments were also evaluated in a taxonomic perspective and were found to be a helpful tool in some special cases.

Most important results and brief description of their significance:
- Ten strains of the P. boydii complex have been tested for their tolerance to KNO3, KH2PO4, urea and (NH4)2SO4. The highest concentration where growth was observed was referred to as maximum tolerance. Maximum tolerance is strain dependent, except for urea and also depending on the compound provided as single N- or P-source. N- or P-tolerance cannot be related to molecular subgroups in the P. boydii complex.
- High concentrations of N- and P- sources do have a selective effect. Therefore excessive supply with the tested compounds is responsible for a selective pressure and for an influence on the evolution of P. boydii. Selection cannot be correlated to reproduction mode, ecologic preferences, source of the strain or systematic position.
- Selection is not depending on the maximum tolerance: a strain with higher maximum tolerance to a N- or P-source is not necessarily the one which is superior in the trial. Therefore, it is concluded, that high concentrations of the tested N- and P-sources do not cause the selection of the more tolerant strains as long as they don't exceed a toxic level for a strain.
- The outcome of a competition trial is depending on the compound which is applied as N- or P-source. This is of significance, because we now know that not only the maximum tolerance is strain dependent but also the competitive ability of a strain varies with the quality of nutrient supply. If population dynamics will be subject to computed modelling, the role of changing nutrient supply must be considered and not only its quantity.
- Competition among P. boydii strains and two other species was tested on solid complex media at two temperatures (20 and 37 degrees Celsius). The trials were dominated by P. boydii strains during > 60 days. Exhausted media gave a chance for the recurrence of oligotroph opponents like Exophiala dermatitidis. In those competition trials, the superior strain (P. boydii) was promoted by higher temperatures (37 degrees Celsius instead of 20 degrees Celsius). The conclusion is that selective effects due to the nutrition medium are amplified by higher temperatures. This illustrates possible effects on fungal communities in habitats which are affected by warming. Soil in urban environments is warmer than in natural habitats, what is primarily a result of sealed surfaces and will be increased by the general climatic trend towards a higher frequency of warmer periods.
- P. boydii tends to produce ascomata with increased frequency, when it comes into contact with other strains. Those structures mainly are not a result of meiotic reproduction but produce generally more resistant spores. The conclusion is that high densities of P. boydii in a given habitat support the production, the quantity and general resistance of propagules.
- P. boydii is a species complex and not a single species. This finding is based on ITS1 and 2 and partial LSU sequence comparisons as well as on parts of the mating experiments. Sequence analysis revealed seven distinct clusters which may represent species. Recently species names for some molecular subgroups were introduced in another study by Gilgado et al. (2005) and there will be proposed more new species in the next future. In medical mycology experts are facing an enormous variability of clinical manifestations of Pseudallescheriasis. Resistance to antimycotic drugs and clinical manifestations may be better understood.
- During mating experiments the first consecutive generation was found to be almost exclusively formed by one partner. In one case both partners were involved in sexual production of progenies with equal frequency. The significance of this result is, that the strains behave mostly like clones. Therefore recombination as basis for fast evolution and reason for a large molecular variability is probably of minor value. The production of generally more resistant ascomatal structures is more important in this respect. The ascoma production of one partner is hampered in the contact zone. This is even true among strains which are closely related from a molecular-taxonomic viewpoint.
- Evidence for mating types was found in a strain of the PbE-clade of the P. boydii complex. This result was observed in mating experiments as formation of ascomata in the contact zone, while the mated strains alone did not produce ascomata. In this respect, the concept of homothallic reproduction of P. boydii is at least for subgroup PbE debatable. Moreover, it strengthens the position of this clade as a discrete entity. During mating and competition experiments the detection of molecular changes as prove of recombination should be done. With the applied techniques changes were found in banding patterns after the experiments compared to the original strains. For control purposes fingerprinting was evaluated by a comparison with AFLP. It turned out, that slight differences in molecular fingerprints are artefacts. This comparison improved the analysis and interpretation of fingerprints massively.
- Flourescence in situ hybridisation (FISH) was tested in order to develop a tool for the recognition of strains in competition experiments. Several test series with probes targeting the ribosomal DNA were only successful in differentiating the genus Pseudallescheria from some species belonging to the Eurotiales. The results were reproducible to a low extent. Because of this experience and the high costs of FISH the tests were quit.
- With regard to contribution to the progress of the field: The project led to insight into the influence of abiotic factors on strain - strain interactions and selection in fungi. It was possible to enlarge knowledge about the human influence on fungi in an evolutionary context. Parts of the work and preliminary results were already presented in several meetings and stimulated the field by focussing on the role of humans for the enrichment of the environment with opportunists like P. boydii. The project contributes to the control of pathogens by providing data on promoting factors. The increased importance of the field is also reflected by the increasing interest in research on scedosporium / pseudallescheria infections and ecology (please see online) and a cooperation with the CBS and the University of Amsterdam in a Marie Curie (ToK) program, for the development of computed models on the distribution of P. boydii where we function as specialists for experimental applications and ecology of P. boydii. Research on P. boydii was also accepted as part of the official research area 'Environmental Research and Biotechnology' at the University of Innsbruck. Concerning the systematic question of P. boydii the project contributed by recognising P. boydii as a species complex in 2004. In 2005, Gilgado et al. published a study with the description of two new species in P. boydii complex. In 2006, the P. boydii complex was divided into seven distinct clusters as a result of the underlying project. At the ISHAM meeting 2006 (Paris, France), new species were proposed by Gilgado et al. The significance of the systematic-taxonomic work will contribute to a better understanding of the diverse clinical manifestations and drug resistance of P. boydii.
- With regard to breaking of new scientific ground: New scientific ground was entered, with the experimental approach for the investigation of the effects of anthropogenic influences on fungal populations. Experiments on the implication of well defined factors are the basis for possible computed modelling of population dynamics and evolution. Inversely open questions can be tested in a controlled environment. Competition trials as well as mating experiments were followed by molecular strain analysis. This led to conclusions on ecological strategies and reproduction biology of P. boydii and hypothesis with a more general character possibly true for many opportunists. The most burning question is, if the results can be verified in a more complex model like in soil samples.
- With regard to most important development of hypothesis The most important hypotheses developed are:
1) Human activity, especially fertilising and atmospheric N-immission, has an impact on enhanced and changed selection in populations of P. boydii compared to a natural level. Distribution and competitive strength of strains in the natural habitat is affected.
2) Rising temperatures increase the selective effect present in the anthropogenic environment. 3) Even though P. boydii is an ascomycete producing ascomata, it reproduces clonal to a high extent.
4) P. boydii is a species complex.
5) The postulated homothallism is questioned for at least one molecular group of the P. boydii complex.
- With regard to development of new methods During this project we introduced competition experiments among fungi on solid and in liquid complex- and assimilation media for the investigation of abiotic factors on fungal populations. Those methods are now established and should be extended to a natural system like soil in a future project. With the introduced methods it was possible to enlighten reproduction behaviour of an ascomycete with two anamorphs. Methodically a step forward was achieved by the evaluation of fingerprinting methods with AFLP methods and the improved analysis.
- With regard to relevance for other areas of science: Other areas, especially medicine and fungal ecology, profit from this project mainly in the way that promoting factors for the distribution of opportunistic pathogens have been tested and can be tested in future with the introduced experimental settings. Data from this project should be used in a computer model aiming to predict distribution and appendant infection risk. Other scientific areas also profit from the clear up of the reproduction biology and systematic-taxonomic questions.

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