The phenomenon of degeneration of industrial Trichoderma reesei strains.

BACKGROUND: Even if the loss of production capacity of a microorganism is said to be a serious problem in various biotechnology industries, reports in literature are rather rare. Strains of the genera Trichoderma reesei are used for large-scale production of cellulases, which are needed in food and feed, textile, paper industries and biofuel production. RESULTS: Here, we describe the phenomenon of spontaneous degeneration of T. reesei strains during large-scale cultivation. The phenotype of the degenerated population is characterized most importantly by a loss of any cellulase formation. Interestingly, promoter regions of relevant genes had a more compact chromatin in the (cel -) strains compared to productive strains. For a systematic investigation of the phenomenon a protocol for artificially induced and lab-scaled strain degeneration was developed. This workflow allows to determine the degeneration rate and thus, to compare the occurrence of a degenerated population in differently productive strains on the one hand, and to monitor the success of any strategies to prevent or decrease the degeneration on the other hand. While highly productive strains have higher degeneration rates compared to moderate producers, the degeneration can hardly be triggered in moderate producers. The observed (cel -) phenotype is not caused by a mutation in the gene encoding the essential transactivator Xyr1. The development of a non-producing population is also not triggered by any compounds released by either producing or non-producing cells. CONCLUSIONS: The extent of the occurrence of a degenerated strain population relates to the production capacity of the strain and goes along with chromatin condensation in relevant promoter regions.

Erythritol as sweetener-wherefrom and whereto?

Erythritol is a naturally abundant sweetener gaining more and more importance especially within the food industry. It is widely used as sweetener in calorie-reduced food, candies, or bakery products. In research focusing on sugar alternatives, erythritol is a key issue due to its, compared to other polyols, challenging production. It cannot be chemically synthesized in a commercially worthwhile way resulting in a switch to biotechnological production. In this area, research efforts have been made to improve concentration, productivity, and yield. This mini review will give an overview on the attempts to improve erythritol production as well as their development over time.

Multiparametric monitoring of microbial faecal pollution reveals the dominance of human contamination along the whole Danube River.

The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.

Occurrence of human-associated Bacteroidetes genetic source tracking markers in raw and treated wastewater of municipal and domestic origin and comparison to standard and alternative indicators of faecal pollution.

This was a detailed investigation of the seasonal occurrence, dynamics, removal and resistance of human-associated genetic Bacteroidetes faecal markers (GeBaM) compared with ISO-based standard faecal indicator bacteria (SFIB), human-specific viral faecal markers and one human-associated Bacteroidetes phage in raw and treated wastewater of municipal and domestic origin. Characteristics of the selected activated sludge wastewater treatment plants (WWTPs) from Austria and Germany were studied in detail (WWTPs, n = 13, connected populations from 3 to 49000 individuals), supported by volume-proportional automated 24-h sampling and chemical water quality analysis. GeBaM were consistently detected in high concentrations in raw (median log10 8.6 marker equivalents (ME) 100 ml(-1)) and biologically treated wastewater samples (median log10 6.2-6.5 ME 100 ml(-1)), irrespective of plant size, type and time of the season (n = 53-65). GeBaM, Escherichia coli, and enterococci concentrations revealed the same range of statistical variability for raw (multiplicative standard deviations s* = 2.3-3.0) and treated wastewater (s* = 3.7-4.5), with increased variability after treatment. Clostridium perfringens spores revealed the lowest variability for raw wastewater (s* = 1.5). In raw wastewater correlations among microbiological parameters were only detectable between GeBaM, C. perfringens and JC polyomaviruses. Statistical associations amongst microbial parameters increased during wastewater treatment. Two plants with advanced treatment were also investigated, revealing a minimum log10 5.0 (10th percentile) reduction of GeBaM in the activated sludge membrane bioreactor, but no reduction of the genetic markers during UV irradiation (254 nm). This study highlights the potential of human-associated GeBaM to complement wastewater impact monitoring based on the determination of SFIB. In addition, human-specific JC polyomaviruses and adenoviruses seem to be a valuable support if highly specific markers are needed.

Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems.

Automated Sampling Procedures Supported by High Persistence of Bacterial Fecal Indicators and Bacteroidetes Genetic Microbial Source Tracking Markers in Municipal Wastewater during Short-Term Storage at 5°C.

Because of high diurnal water quality fluctuations in raw municipal wastewater, the use of proportional autosampling over a period of 24 h at municipal wastewater treatment plants (WWTPs) to evaluate carbon, nitrogen, and phosphorus removal has become a standard in many countries. Microbial removal or load estimation at municipal WWTPs, however, is still based on manually recovered grab samples. The goal of this study was to establish basic knowledge regarding the persistence of standard bacterial fecal indicators and Bacteroidetes genetic microbial source tracking markers in municipal wastewater in order to evaluate their suitability for automated sampling, as the potential lack of persistence is the main argument against such procedures. Raw and secondary treated wastewater of municipal origin from representative and well-characterized biological WWTPs without disinfection (organic carbon and nutrient removal) was investigated in microcosm experiments at 5 and 21°C with a total storage time of 32 h (including a 24-h autosampling component and an 8-h postsampling phase). Vegetative Escherichia coli and enterococci, as well as Clostridium perfringens spores, were selected as indicators for cultivation-based standard enumeration. Molecular analysis focused on total (AllBac) and human-associated genetic Bacteroidetes (BacHum-UCD, HF183 TaqMan) markers by using quantitative PCR, as well as 16S rRNA gene-based next-generation sequencing. The microbial parameters showed high persistence in both raw and treated wastewater at 5°C under the storage conditions used. Surprisingly, and in contrast to results obtained with treated wastewater, persistence of the microbial markers in raw wastewater was also high at 21°C. On the basis of our results, 24-h autosampling procedures with 5°C storage conditions can be recommended for the investigation of fecal indicators or Bacteroidetes genetic markers at municipal WWTPs. Such autosampling procedures will contribute to better understanding and monitoring of municipal WWTPs as sources of fecal pollution in water resources.

A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources.

The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ß-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ß-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed.

Clostridium perfringens is not suitable for the indication of fecal pollution from ruminant wildlife but is associated with excreta from nonherbivorous animals and human sewage.

During a 3-year study, Clostridium perfringens was investigated in defined fecal sources from a temperate alluvial backwater area of a large river system. The results reveal that using C. perfringens as a conservative water quality indicator for total fecal pollution monitoring is no longer justified but suggest that it can be used as a tracer for excreta from nonherbivorous wildlife and human sewage.

Hypothesis-driven approach for the identification of fecal pollution sources in water resources.

Water resource management must strive to link catchment information with water quality monitoring. The present study attempted this for the field of microbial fecal source tracking (MST). A fecal pollution source profile based on catchment data (e.g., prevalence of fecal sources) was used to formulate a hypothesis about the dominant sources of pollution in an Austrian mountainous karst spring catchment. This allowed a statistical definition of methodical requirements necessary for an informed choice of MST methods. The hypothesis was tested in a 17-month investigation of spring water quality. The study followed a nested sampling design in order to cover the hydrological and pollution dynamics of the spring and to assess effects such as differential persistence between parameters. Genetic markers for the potential fecal sources as well as microbiological, hydrological, and chemo-physical parameters were measured. The hypothesis that ruminant animals were the dominant sources of fecal pollution in the catchment was clearly confirmed. It was also shown that the concentration of ruminant markers in feces was equally distributed in different ruminant source groups. The developed approach provides a tool for careful decision-making in MST study design and might be applied on various types of catchments and pollution situations.

The spectral absorption coefficient at 254 nm as a real-time early warning proxy for detecting faecal pollution events at alpine karst water resources.

Because spring water quality from alpine karst aquifers can change very rapidly during event situations, water abstraction management has to be performed in near real-time. Four summer events (2005-2008) at alpine karst springs were investigated in detail in order to evaluate the spectral absorption coefficient at 254 nm (SAC254) as a real-time early warning proxy for faecal pollution. For the investigation Low-Earth-Orbit (LEO) Satellite-based data communication between portable hydrometeorological measuring stations and an automated microbiological sampling device was used. The method for event triggered microbial sampling and analyzing was already established and described in a previous paper. Data analysis including on-line event characterisation (i.e. precipitation, discharge, turbidity, SAC254) and comprehensive E. coli determination (n>800) indicated that SAC254 is a useful early warning proxy. Irrespective of the studied event situations SAC254 always increased 3 to 6 hours earlier than the onset of faecal pollution, featuring different correlation phases. Furthermore, it seems also possible to use SAC254 as a real-time proxy parameter for estimating the extent of faecal pollution after establishing specific spring and event-type calibrations that take into consideration the variability of the occurrence and the transferability of faecal material It should be highlighted that diffuse faecal pollution from wildlife and live stock sources was responsible for spring water contamination at the investigated catchments. In this respect, the SAC254 can also provide useful information to support microbial source tracking efforts where different situations of infiltration have to be investigated.

Escherichia coli and enterococci are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources.

AIMS: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. METHODS AND RESULTS: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7-7·0CFU g(-1) ) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0-8·4CFU g(-1) ). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3-7·7 CFUg(-1) . To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi-parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment (Reischer et al. 2008; Environ Microbiol 10:2598-2608). CONCLUSION: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.

Microbiological monitoring and automated event sampling at karst springs using LEO-satellites.

Data communication via Low-Earth-Orbit (LEO) Satellites between portable hydrometeorological measuring stations is the backbone of our system. This networking allows automated event sampling with short time increments also for E. coli field analysis. All activities of the course of the event-sampling can be observed on an internet platform based on a Linux-Server. Conventionally taken samples compared with the auto-sampling procedure revealed corresponding results and were in agreement with the ISO 9308-1 reference method. E. coli concentrations were individually corrected by event specific inactivation coefficients (0.10-0.14 day(-1)), compensating losses due to sample storage at spring temperature in the auto sampler.Two large summer events in 2005/2006 at an important alpine karst spring (LKAS2) were monitored including detailed analysis of E. coli dynamics (n = 271) together with comprehensive hydrological characterisations. High-resolution time series demonstrated a sudden increase of E. coli concentrations in spring water (approximately 2 log10 units) with a specific time delay after the beginning of the event. Statistical analysis suggested the spectral absorption coefficient measured at 254 nm (SAC254) as an early warning surrogate for real time monitoring of faecal input. Together with the LEO-satellite based system it is a helpful tool for early-warning systems in the field of drinking water protection.

Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamics.

The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design.

Development of a Scorpion probe-based real-time PCR for the sensitive quantification of Bacteroides sp. ribosomal DNA from human and cattle origin and evaluation in spring water matrices.

Spring water from alpine catchments are important water resources but they can be vulnerable against faecal contamination. Potential faecal contamination sources are wildlife populations, pasturing activities, or alpine tourism. Unfortunately, no faecal source tracking method is available to date which is sensitive enough for appropriate spring water monitoring and source allocation. Our purpose was to develop a Duplex Scorpion real-time PCR approach for the specific and sensitive quantification of Bacteroides sp. 16S rDNA fragments from human and cattle origin. By the developed approach, detection of plasmids, carrying the respective biomarker sequence, was possible over a range of more than seven orders of magnitudes down to six copy numbers per PCR assay. Furthermore, the Duplex Scorpion real-time PCR allowed the specific quantification down to 50 targets in plasmid spiked spring water matrices. Results indicate that microbial source tracking appears feasible in spring water habitats by probe-based real-time PCR technologies. However, preliminary testing of the established approach on faecal samples collected from a representative alpine habitat did not allow unambiguous source allocation in all cases. In the future, the available sequence database has thus to be widened to allow reliable source tracking in alpine spring watersheds and even expand this approach to other potential faecal sources.

Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water.

AIMS: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water METHODS AND RESULTS: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338/EUB338-II/EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77-96%) bacteria and 11% (range 3-22%) identified as Archaea. CONCLUSIONS: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.

A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area.

AIMS: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. METHODS AND RESULTS: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. CONCLUSIONS: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.

Development of a novel amplified fragment length polymorphism (AFLP) typing method for enterococci isolates from cattle faeces and evaluation of the single versus pooled faecal sampling approach.

A novel and highly reproducible amplified fragment length polymorphism (AFLP) typing approach was developed for typing of Enterococcus strains from the environment. Pooled and corresponding single faecal sample isolates were analysed to test the efficiency and coverage of dominant isolates for future sampling procedures. AFLP development was based on the selection of appropriate restriction enzymes and the design of adaptors and primers which was supported by in silico optimisation of selective bases using Enterococcus spp. genome data. Three optimal combinations of selective bases at the 3' end of the designed primers (i.e., CC, GG, CG) could be determined. AFLP fragment analysis using a capillary sequencer and intralane standardisation resulted in excellent methodical stability (> or =98% similarity for GG and > or =94% similarity for CC). Furthermore, the developed typing method was evaluated on 16 type trains of the genera Enterococcus and Streptococcus and 398 faecal isolates of cow pats from five alpine pastures in a karstic catchment area. Statistical analysis revealed a discrimination capacity of DI > or =0.95 (Simpson Diversity Index) and a reproducibility level of > or =94% similarity indicating the methods high typing capacity and robustness. Results of the comparative analysis of single and pooled faecal samples indicate that for a "strain to strain" based faecal source tracking, pooled faecal samples rather than single faecal samples are likely to be the most efficient sampling strategy for collecting the abundant corresponding strains.

Phylogenetic relationship of Fusarium langsethiae to Fusarium poae and Fusarium sporotrichioides as inferred by IGS, ITS, beta-tubulin sequences and UP-PCR hybridization analysis.

Fusarium langsethiae was recently described to accommodate "powdery" isolates of Fusarium poae, which morphologically resemble F. poae, but whose metabolite profile is similar to that of Fusarium sporotrichioides. In order to investigate the phylogenetic relationship of F. langsethiae to closely related species, we sequenced the internal transcribed spacer (ITS) regions 1 and 2 and part of the intergenic spacer (IGS) region of the rDNA cluster and part of the beta-tubulin gene from 109 strains of F. poae, F. sporotrichioides, F. langsethiae and Fusarium kyushuense from different geographic origin. Sequence analysis of ITS1 and 2 was unable to separate all F. sporotrichioides strains from F. langsethiae strains. Sequence analysis of beta-tubulin distinguished all four species, but it did not resolve the phylogenetic relationship between these two species. Sequence analysis of the IGS region distinguished the four species and led to a higher number of subgroups of the individual species, of which that of F. sporotrichioides var. minus isolates was even better supported than that of F. poae and F. langsethiae. Neighbor-joining and POY analyses of all combined sequences reliably separated all species studied, including F. langsethiae, clearly from F. sporotrichioides. The high intraspecific variability of the IGS sequences were found useful to group isolates according to their geographic origin. These results are in accordance with the results of the UP-PCR hybridization analysis. In summary, our data offer molecular support for the description of F. langsethiae as a new species in section Sporotrichiella.

Specific detection of Fusarium langsethiae and related species by DGGE and ARMS-PCR of a beta-tubulin (tub1) gene fragment.

Fusarium langsethiae was recently described as a new toxigenic Fusarium species, which morphologically resembles Fusarium poae, but exhibits a mycotoxin pattern related to Fusarium sporotrichioides. To develop tools for early and specific detection of F. langsethiae and distinguishing it from related species of section Sporotrichiella and Discolor (F. poae, F. sporotrichioides, Fusarium kyushuense, Fusarium robustum, Fusarium sambucinum and Fusarium tumidum) sequence variations in their beta-tubulin-encoding (tub1) gene were employed to design two PCR-based methods, denaturing gradient gel electrophoresis (DGGE) and amplification refractory mutation system (ARMS)-PCR. DGGE reliably separated all these strains, even from mixtures and in the presence of DNA from their natural hosts Zea mais, Triticum aestivum and Avena sativa. In addition, a tetraprimer ARMS-PCR, which employs two primer pairs to amplify, respectively, two different fragments of tub1 in a single PCR reaction resulted in rapid differentiation between F. langsethiae, F. sporotrichioides and F. poae according to the number of amplicons (four, two and one, respectively). These two methods will thus be worthwhile tools in the specific detection of F. langsethiae in infected crops.

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets.

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.