Mitochondrial DNA diversity of the emerging fungal pathogen Trichoderma longibrachiatum (original) (raw)
Related papers
Journal of Medical Microbiology, 2006
The genetic diversity of the emerging fungal pathogen Trichoderma longibrachiatum was examined at the level of mitochondrial DNA. The 17 investigated strains, comprising nine clinical and eight non-clinical isolates, exhibited seven and ten different mitochondrial DNA profiles by using the restriction enzymes BsuRI and Hin6I, respectively. The sizes of mitochondrial DNAs varied from 34?9 to 39?5 kb. The discriminatory power of the method was higher than that of internal transcribed spacer sequence analysis and therefore should be more suitable for identification and epidemiological investigations. However, clinical and non-clinical isolates did not form separate clusters on the resulting dendrogram and thus there was no indication of a correlation between genetic structure and pathogenicity of the isolates. Abbreviations: ITS, internal transcribed spacer; RAPD, random amplification of polymorphic DNA. 46292 G 2006 SGM Printed in Great Britain
Clinical Microbiology and Infection, 2006
Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorphisms in 10 clinical and 11 non-clinical Trichoderma isolates. After initial testing of 13 enzyme systems for activity and resolution of bands, 7 proved to be appropriate for identifying the different species. Comparing the different electrophoretic types of both clinical and non-clinical isolates, each of the enzyme systems investigated (glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidase A, B, D, and phosphoglucomutase) proved to be diagnostic for at least one species. Based on the results of isoenzyme analysis, several isolates originally identified as T. pseudokoningii, T. koningii or T. citrinoviride were reidentified as T. longibrachiatum. This was supported by previous data from sequence analysis of the internal transcribed spacer region of the isolates. The application of a quick, cheap and reliable diagnostic tool for the identification of T. longibrachiatum strains would be very important, as most of the clinical Trichoderma isolates belong to T. longibrachiatum, Cellulose-acetate electrophoresis seems to be promising for this purpose. Furthermore, as a great number of enzyme systems are available for this method, molecular markers could also be found for the practical diagnostics of other clinically relevant fungal species.
Medical mycology, 2015
Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of qualitycontrolled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to Irinyi et al. 3 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
Genotypic study of strains belonging to the genusTrichosporon
Medical Mycology, 2007
Trichosporon asahii is an opportunistic fungus considered the leading etiologic agent of trichospornosis, a disease that causes great morbidity/mortality among affected patients. The identification of the etiologic agent is generally obtained through physiological and morphological studies. Molecular investigations, such as species-specific primers (PCR), have recently been developed with the aim of applying a more simple, specific, and faster technology for mycological diagnosis. The genetic material amplification technique using ad-random primers (RAPD: random amplified polymorphic DNA) is an epidemiological tool which enables research on infection by and transmission of suspected agents. In this study, the amplified polymorphic DNA technique was used to determine the intraspecific diversity of 10 Trichosporon asahii strains. Primers OPAO-15 and 1821 were used and these allowed association to 5 and 3 electrophoretic patterns, respectively. The T. asahii molecular identification, which had been previously analyzed by conventional methods, was performed by means of primers TAAF and pITS4. Our results support the use of these techniques for clonality studies of the strains of this fungus as well as for the fast and specific identification of its members in clinical cases.
Journal of Clinical Microbiology, 2001
Molecular genotyping of strains of Trichophyton rubrum and T. mentagrophytes from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of T. rubrum and 11 strains of T. mentagrophytes were obtained from 20 patients (16 patients with T. rubrum, 4 with T. mentagrophytes) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of EcoRI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of T. rubrum. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among T. rubrum, T. mentagrophytes, and T. tonsurans, intraspecific RFLP variation was observed for T. rubrum and T. mentagrophytes strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in Candida species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection.
Characterization of Malaysian Trichoderma isolates using random amplified microsatellites (RAMS)
Trichoderma species are commercially applied as biocontrol agents against numerous plant pathogenic fungi due to their production of antifungal metabolites, competition for nutrients and space, and mycoparasitism. However, currently the identification of Trichoderma species from throughout the world based on micro-morphological descriptions is tedious and prone to error. The correct identification of Trichoderma species is important as several traits are species-specific. The Random Amplified Microsatellites (RAMS) analysis done using five primers in this study showed different degrees of the genetic similarity among 42 isolates of this genus. The genetic similarity values were found to be in the range of 12.50–85.11% based on a total of 76 bands scored in the Trichoderma isolates. Of these 76 bands, 96.05% were polymorphic, 3.95% were monomorphic and 16% were exclusive bands. Two bands (250 bp and 200 bp) produced by primer LR-5 and one band (250 bp) by primer P1A were present in all the Trichoderma isolates collected from healthy and infected oil palm plantation soils. Cluster analysis based on UPGMA of the RAMS marker data showed that T. harzianum, T. virens and T. longibrachiatum isolates were grouped into different clades and lineages. In this study we found that although T. aureoviride isolates were morphologically different when compared to T. harzianum isolates, the UPGMA cluster analysis showed that the majority isolates of T. aureoviride (seven from nine) were closely related to the isolates of T. harzianum.
DNA barcoding of human and animal pathogenic fungi: the ISHAM-ITS database
Microbiology Australia, 2015
Human and animal fungal pathogens are a growing threat worldwide. They lead to emerging infections and create new risks for established ones. As such, there is a growing need for the rapid and accurate identification of mycoses agents to enable early diagnosis and targeted antifungal therapy. An international consortium of medical mycology laboratories was formed in order to establish a quality controlled ITS database under the umbrella of the ISHAM (International Society for Human and Animal Mycology) working group on 'DNA barcoding of human and animal pathogenic fungi'.
European Journal of Clinical Microbiology & Infectious Diseases, 2011
Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.
Randomly Amplified Polymorphic DNA Analysis of Native Trichoderma Isolates
Research Paper, 2017
Analysis of all the Trichoderma spp. isolates through Random Amplified Polymorphic DNA (RAPD), a PCR (Polymerase Chain Reaction) based molecular marker system was performed. The procedure was used to examine the genetic similarity among six native isolates of Trichoderma spp. Genomic DNA extracted from the fungal tissues of different isolates of Trichoderma spp. Quality and quantity of DNA checked and subjected to RAPD analysis. Based on the UPGMA dendogram only two clusters were formed, one consisting of the two strains of T. fasciculatum (TFC-1 and TFC-2), T. viride (TVS-1 and TVS-2) and T. harzianum (THCh-1), whereas, T. atroviride (TACh-1) formed the second cluster. The findings of the present experiment undoubtedly indicated all the isolates were moderately (more or less) similar with each other at molecular level by using RAPD analysis. The observed similarity between TFC-1, TFC-2 (Castor) and TVS-1, TVS-2 (Sugarcane) might be due to these species were isolated from the same host.
Microbiology, 2008
The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. longibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.