Analysis of a worldwide collection of strains of Trichoderma asperellum sensu lato using multilocus genealogies of four genomic regions (tef1, rpb2, act, ITS1, 2 and 5.8s rRNA), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) of the proteome and classical mycological techniques revealed two morphologically cryptic sister species within T. asperellum, T. asperellum, T. asperelloides sp. nov. and a third closely related but morphologically distinct species. T. yunnanense. Trichoderma asperellum and T. asperelloides have wide sympatric distribution on multiple continents; T. yunnanense is represented by a single strain from China. Several strains reported in the literature or represented in GenBank as T. asperellum are re-identified as T. asperelloides. Four molecular SPD typing patterns (I-IV) were found over a large geographic range. Patterns I-III were produced only by T. asperellum and pattern IV by T. asperelloides and T. yunnanense. Pattern I was found in North America, South America, Africa and Europe and Asia (Saudi Arabia). Pattern III was found in Africa, North America, South America and Asia, not in Europe. Pattern II was found only in Cameroon (central Africa) and Peru. Pattern IV was found in all continents. All SPD II pattern strains formed a strongly supported subclade within the T. asperellum clade in the phylogenetic tree based on rpb2 and MLS (combined multilocus sequence). The diversity of DNA sequences, SPD markers and polypeptides in T. asperellum suggests that further speciation is under way within T. asperellum. MALDI-TOF MS distinguished T. yunnanense from related taxa by UPGMA clustering, but separation between T. asperellum and T. asperelloides was less clear.

In this study we evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of dermatophytes in diagnostic laboratories. First, a spectral database was built with 108 reference strains belonging to 18 species of the anamorphic genera Epidermophyton, Microsporum and Trichophyton. All strains were well characterized by morphological criteria and ITS sequencing (gold standard). The dendrogram resulting from MALDI-TOF mass spectra was almost identical with the phylogenetic tree based on ITS sequencing. Subsequently, MALDI-TOF MS SuperSpectra were created for the identification of Epidermophyton floccosum, Microsporium audouinii, M. canis, M. gypseum (teleomorph: Arthroderma gypseum), M. gypseum (teleomorph: A. incurvatum), M. persicolor, A. benhamiae (Tax. Entity 3 and Am-Eur. race), T. erinacei, T. interdigitale (anthropophilic and zoophilic populations), T. rubrum/T. violaceum, T. tonsurans and T. terrestre. Because T. rubrum and T. violaceum did not present enough mismatches, a SuperSpectrum covering both species was created, and differentiation between them was done by comparison of eight specific peptide masses. In the second part of this study, MALDI-TOF MS with the newly created SuperSpectra was tested using 141 clinical isolates representing nine species. Analyses were done with 3-day-old cultures. Results were compared to morphological identification and ITS sequencing; 135/141 (95.8%) strains were correctly identified by MALDI-TOF MS compared to 128/141 (90.8%) by morphology. Therefore, MALDI-TOF MS has proven to be a useful and rapid identification method for dermatophytes.

d; POLE Pharma Consulting, Breganzona, Switzerland e The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory. D ermatophytes in the genera Epidermophyton, Microsporum, and Trichophyton are usually characterized and identified by cultural and morphological characters and physiological tests or, more recently, by sequencing (1). Morphological identification is time-consuming and complex, usually requiring expert mycological knowledge, while sequencing is comparatively expensive and at least 2 to 3 days elapse before sequencing results are available.Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a reliable technique for the identification and typing of microbial pathogens such as bacteria (2-7), yeasts (8-10), and filamentous fungi, including dermatophytes (7,(11)(12)(13)(14)(15)(16)(17). Recent studies confirm that this technique may be very attractive for dermatophyte identification (18-22). In one study, the rate of correct identification of isolates belonging to the T. mentagrophytes complex was 89% (19), and in others, the overall rates of successful identification of dermatophyte species reached 95.8% (18), 97.8% (21), and 99.3% (20), demonstrating the potential of MALDI-TOF MS to replace classical identification methods. The technique has now also been used for the direct identification of dermatophytes in c...

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.

A real-time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil AbstractAim: To develop a rapid real-time PCR method for the specific detection and quantification of Bacillus thuringiensis var. israelensis (Bti) spores present in the environment. Methods and Results: Seven soil samples as well as one sediment sample obtained from various regions of Switzerland and characterized by different granulometry, pH values, organic matter and carbonate content were artificially inoculated with known amounts of Bti spores. After DNA extraction, DNA templates were amplified using TaqMan real-time PCR targeting the cry4Aa and cry4Ba plasmid genes encoding two insecticidal toxins (d-endotoxins), and quantitative standard curves were created for each sample. Physicochemical characteristics of the samples tested did not influence DNA extraction efficiency. Real-time PCR inhibition because of the presence of co-extracted humic substances from the soil was observed only for undiluted DNA extracts from samples with very high organic matter content (68%). The developed real-time PCR system proved to be sensitive, detecting down to 1 · 10 3 Bti spores per g soil. One-way analysis of variance confirmed the accuracy of the method. Conclusions: Direct extraction of DNA from environmental samples without culturing, followed by a specific real-time PCR allowed for a fast and reliable identification and quantification of Bti spores in soil and sediment. Significance and Impact of the Study: The developed real-time PCR system can be used as a tool for ecological surveys of areas where treatments with Bti are carried out.